INTERCELLULAR COMMUNICATION IN THE NERVOUS SYSTEM
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INTERCELLULAR COMMUNICATION IN THE NERVOUS SYSTEM EDITOR-IN-CHIEF
ROBERT C. MALENKA Department of Psychiatry and Behavioral Sciences Stanford University School of Medicine Palo Alto, CA USA
Amsterdam • Boston • Heidelberg • London • New York • Oxford Paris • San Diego • San Francisco • Singapore • Sydney • Tokyo Academic Press is an imprint of Elsevier
Academic Press is an imprint of Elsevier 32 Jamestown Road, London NW1 7BY, UK 30 Corporate Drive, Suite 400, Burlington, MA 01803, USA 525 B Street, Suite 1900, San Diego, CA 92101-4495, USA Copyright ã 2009 Elsevier Inc. All rights reserved The following articles are US government works in the public domain and are not subject to copyright: AMPA Receptors: Molecular Biology and Pharmacology BDNF in Synaptic Plasticity and Memory Long-Term Depression (LTD): Endocannabinoids and Cerebellar LTD NMDA Receptors, Cell Biology and Trafficking No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means electronic, mechanical, photocopying, recording or otherwise without the prior written permission of the publisher. Material in this work originally appeared in the Encyclopedia of Neuroscience, Ed. L.R. Squire, Elsevier Ltd, 2009. Permissions may be sought directly from Elsevier’s Science & Technology Rights Department in Oxford, UK: phone (+44)(0) 1865 843830; fax (+44) (0) 1865 853333; email:
[email protected]. Alternatively you can submit your request online by visiting the Elsevier web site at (http://elsevier.com/locate/permissions), and selecting Obtaining permissions to use Elsevier material Notice No responsibility is assumed by the publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drug dosages should be made. British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library Library of Congress Catalog Number: 2009929652 ISBN: 978-0-12-375072-3 For information on all Elsevier publications visit our website at books.elsevier.com PRINTED AND BOUND IN SLOVENIA 09 10 11 12 13 10 9 8 7 6 5 4 3 2 1
CONTRIBUTORS
A Adamantidis
G Bernardi
Stanford University School of Medicine, Palo Alto, CA, USA
Universita` di Roma ‘Tor Vergata’, and IRCCS Fondazione Santa Lucia, Rome, Italy
G Aghajanian
T P Blackburn
Yale School of Medicine, New Haven, CT, USA
Helicon Therapeutics Inc., Farmingdale, NY, USA
S P H Alexander
N G Bowery
University of Nottingham Medical School, Nottingham, UK
GlaxoSmithKline, Verona, Italy
N J Allen
University of Rome ‘La Sapienza,’ Rome, Italy
Stanford University School of Medicine, Stanford, CA, USA
G Burnstock
R S Aronstam
University of Missouri – Rolla, Rolla, MO, USA G Aston-Jones
Medical University of South Carolina, Charleston, SC, USA D Atasoy
The University of Texas Southwestern Medical Center, Dallas, TX, USA
V Bruno
Royal Free and University College School of Medicine, London, UK D B Bylund
University of Nebraska Medical Center, Omaha, NE, USA L Cancedda
University of California at Berkeley, Berkeley, CA, USA G Casini
B A Barres
Universita` della Tuscia, Viterbo, Italy
Stanford University School of Medicine, Stanford, CA, USA
D Cervia
Universita` della Tuscia, Viterbo, Italy
G Battaglia
Istituto Neurologico Mediterraneo ‘Neuromed,’ Pozzilli, Italy
J-P Changeux
K L Behar
H Cline
Yale University School of Medicine, New Haven, CT, USA
Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA
M V L Bennett
G L Collingridge
Albert Einstein College of Medicine, New York, NY, USA
University of Bristol, Bristol, UK
F Bergquist
University of California at San Diego, La Jolla, CA, USA
University of Edinburgh, Edinburgh, UK
Institut Pasteur, Paris, France
J M Conner
v
vi
Contributors
A C Cuello
B A Grueter
McGill University, Montreal, QC, Canada
Vanderbilt University School of Medicine, Nashville, TN, USA
T C Cunnane
University of Oxford, Oxford, UK
E D Gundelfinger
M O Cunningham
Leibniz Institute for Neurobiology, Magdeburg, Germany
Newcastle University, Newcastle upon Tyne, UK G W Davis
University of California at San Francisco, San Francisco, CA, USA T M Dawson
The Johns Hopkins University School of Medicine, Baltimore, MD, USA
S Harris
Saint Louis University School of Medicine, St. Louis, MO, USA P G R Hastie
University of Bristol, Bristol, UK V Haucke
Freie Universita¨t Berlin, Berlin, Germany
V L Dawson
The Johns Hopkins University School of Medicine, Baltimore, MD, USA
J M Henley
J S Dittman
A M Holohean
Weill Cornell Medical College, New York, NY, USA
University of Miami School of Medicine, Miami, FL, USA
P D Dodson
Geffen School of Medicine, Los Angles, CA, USA A J Doherty
University of Bristol, Bristol, UK S M Dravid
Emory University, Atlanta, GA, USA
University of Bristol, Bristol, UK
M O Huising
The Salk Institute for Biological Studies, La Jolla, CA, USA Department of Animal Physiology, Radboud University Nijmegen, Nijmegen, The Netherlands K A Jacobson
T M Egan
National Institutes of Health, Bethesda, MD, USA
Saint Louis University School of Medicine, St. Louis, MO, USA
P S Kaeser
B A Eipper
University of Texas Southwestern Medical Center, Dallas, TX, USA
University of Connecticut, Farmington CT, USA E T Kavalali J D Elsworth
Yale University School of Medicine, New Haven, CT, USA S M Fitzjohn
University of Bristol, Bristol, UK M Frerking
Oregon Health and Science University, Beaverton, OR, USA Z-G Gao
National Institutes of Health, Bethesda, MD, USA
The University of Texas Southwestern Medical Center, Dallas, TX, USA B L Kieffer
IGBMC,CNRS/INSERM/ULP, Illkirch, France E Kim
Korea Advanced Institute of Science and Technology, Daejeon, South Korea H-C Kornau
Center for Molecular Neurobiology (ZMNH), University of Hamburg, Hamburg, Germany
J Garthwaite
University College London, London, UK D R Gehlert
Eli Lilly and Company, Indianapolis, IN, USA C Giaume
Colle`ge de France, Paris, France T Goetz
University of Aberdeen, Aberdeen, UK
A C Kreitzer
University of California at San Francisco, San Francisco, CA, USA F E N LeBeau
Newcastle University, Newcastle upon Tyne, UK L de Lecea
Stanford University School of Medicine, Palo Alto, CA, USA
Contributors vii S-H Lee
C A Meijas-Aponte
University of California at San Francisco, San Francisco, CA, USA
National Institutes of Health, Baltimore, MD, USA
Y-I Lee
State University of New York at Stony Brook, Stony Brook, NY, USA
The Johns Hopkins University School of Medicine, Baltimore, MD, USA J Lerma
Instituto de Neurociencias de Alicante Consejo Superior de Investigaciones Cientı´ficas– Universidad Miguel Hernandez, San Juan de Alicante, Spain J Lerma
Instituto de Neurociencias de Alicante Consejo Superior de Investigaciones Cientı´ficas-Universidad Miguel Hernandez, San Juan de Alicante, Spain Z Li
L M Mendell
N B Mercuri
Universita` di Roma ‘Tor Vergata’, and IRCCS Fondazione Santa Lucia, Rome, Italy A Merighi
University of Turin, Turin, Italy M P Meyer
Kings College London, London, UK A C Michael
University of Pittsburgh, Pittsburgh, PA, USA
Saint Louis University School of Medicine, St. Louis, MO, USA
W C Mobley
J Lisman
I Mody
Brandeis University, Waltham, MA, USA
Neuroscience Institute, Stanford, CA, USA
R-J Liu
David Geffen School of Medicine at UCLA, Los Angeles, CA, USA
Yale School of Medicine, New Haven, CT, USA
J-P Mothet
B J Lopresti
University of Pittsburgh, Pittsburgh, PA, USA
Institut National de la sante´ et de la Recherche Me´dicale U862, Bordeaux, France
D M Lovinger
R Narendran
National Institutes of Health, Rockville, MD, USA
University of Pittsburgh, Pittsburgh, PA, USA
B Lu
C C Naus
National Institutes of Health, Bethesda, MD, USA
University of British Columbia, Vancouver, BC, Canada
M Ludwig
University of Edinburgh, Edinburgh, UK B Lutz
Johannes Gutenberg University, Mainz, Germany
F Nicoletti
University of Rome ‘La Sapienza,’ Rome, Italy S H R Oliet
C Lu¨scher
Institut National de la sante´ et de la Recherche Me´dicale U862, Bordeaux, France
University of Geneva, Geneva, Switzerland
T S Otis
P J Magistretti
Geffen School of Medicine, Los Angles, CA, USA
Ecole Polytechnique Fe´de´rale de Lausanne (EPFL) and Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland
Y Paas
K L Magleby
University of Missouri – Rolla, Rolla, MO, USA
University of Miami School of Medicine, Miami, FL, USA
L Pellerin
R E Mains
O Peters
University of Connecticut, Farmington CT, USA
Charite´ University Medicine, Berlin, Germany
G Marsicano
R S Petralia
Johannes Gutenberg University, Mainz, Germany and U862 Centre de Recherche INSERM Franc¸ois Magendie Universite´ Bordeaux 2, Bordeaux, France
National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, MD, USA
Bar-Ilan University, Ramat-Gan, Israel P Patil
Universite´ de Lausanne, Lausanne, Switzerland
viii
Contributors
J C Petruska
A D Smith
State University of New York at Stony Brook, Stony Brook, NY, USA
University of Pittsburgh, Pittsburgh, PA, USA
M-M Poo
Stanford University, Stanford, CA, USA
University of California at Berkeley, Berkeley, CA, USA
J A Sobota
S Raghavachari
Duke University Medical Center, Durham, NC, USA A G Ramage
University College London, London, UK B R Ransom
University of Washington School of Medicine, Seattle, WA, USA L F Reichardt
University of California at San Francisco, San Francisco, CA, USA R J Reimer
Stanford University School of Medicine, Stanford, CA, USA B M Reuss
University of Go¨ttingen, Go¨ttingen, Germany R R Ribchester
University of Edinburgh, Edinburgh, UK J Rizo
University of Texas Southwestern Medical Center, Dallas, TX, USA
S J Smith
University of Connecticut, Farmington CT, USA S Takamori
Tokyo Medical and Dental University, Tokyo, Japan H Teng
Washington University School of Medicine, St. Louis, MO, USA N Teramoto
Kyushu University, Fukuoka, Japan S F Traynelis
Emory University, Atlanta, GA, USA A Triller
INSERM U789, Ecole Normale Supe´rieure, Paris, France R W Tsien
Stanford University Medical Center, Stanford, CA, USA M H Tuszynski
University of California at San Diego, La Jolla, CA, USA W W Vale
E Robles
The Salk Institute for Biological Studies, La Jolla, CA, USA
Stanford University, Stanford, CA, USA
C Vannier
R H Roth
Yale University School of Medicine, New Haven, CT, USA
INSERM U789, Ecole Normale Supe´rieure, Paris, France A Volterra
V C Russo
University of Lausanne, Lausanne, Switzerland
Royal Children’s Hospital and University of Melbourne, Parkville, VIC, Australia
R D Wassall
M Saarma
B Waterhouse
University of Helsinki, Helsinki, Finland
Drexel University College of Medicine, Philadelphia, PA, USA
V Scheuss
Max-Planck-Institute for Neurobiology, Martinsried, Germany C G Schipke
Charite´ University Medicine, Berlin, Germany
University of Oxford, Oxford, UK
R J Wenthold
National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, MD, USA M J Werle
University of Toronto,Toronto, ON, Canada
University of Kansas Medical Center, Kansas City, KS, USA
C R Slater
G A Werther
University of Newcastle upon Tyne, Newcastle upon Tyne, UK
Royal Children’s Hospital and University of Melbourne, Parkville, VIC, Australia
P Seeman
Contributors ix T C Westfall
Neuroscience Institute, Stanford, CA, USA
St. Louis University School of Medicine, St. Louis, MO, USA
P Wulff
University of Aberdeen, Aberdeen, UK
R S Wilkinson
Washington University School of Medicine, St. Louis, MO, USA G Wilson
University College London, London, UK D G Winder
Vanderbilt University School of Medicine, Nashville, TN, USA
Z Ye
University of Washington School of Medicine, Seattle, WA, USA H Yuan
Emory University, Atlanta, GA, USA W Zieglga¨nsberger
W Wisden
Max Planck Institute of Psychiatry, Munich, Germany
University of Aberdeen, Aberdeen, UK
M J Zigmond
N H Woo
University of Pittsburgh, Pittsburgh, PA, USA
National Institutes of Health, Bethesda, MD, USA C Wu
K Zito
University of California at Davis, Davis, CA, USA
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CONTENTS
Contributors Contents Preface
v–ix xi–xv xvii–xviii
SECTION I: BASIC MECHANISMS OF SYNAPTIC TRANSMISSION (SYNAPTIC STRUCTURE AND ORGANIZATION) Synaptic Precursors: Filopodia E Robles, S J Smith, and M P Meyer
3
Presynaptic Development: Functional and Morphological Organization D Atasoy and E T Kavalali
11
Postsynaptic Development: Neuronal Molecular Scaffolds E Kim
19
Dendrite Development, Synapse Formation and Elimination H Cline
27
Synapse Formation: Competition and the Role of Activity L Cancedda and M-M Poo
31
Cell Adhesion Molecules at Synapses L F Reichardt and S-H Lee
38
Glia and Synapse Formation: An Overview N J Allen and B A Barres
46
Active Zone P S Kaeser
52
Calcium Channel Subtypes Involved in Neurotransmitter Release R W Tsien
59
SNAREs J Rizo
67
Synaptic Vesicles S Takamori
76
xi
xii Contents
Endocytosis and Presynaptic Scaffolds V Haucke and E D Gundelfinger
84
Postsynaptic Density/Architecture at Excitatory Synapses H-C Kornau
96
Synaptic Transmission: Models S Raghavachari and J Lisman
103
Glial Influence on Synaptic Transmission C G Schipke and O Peters
112
Neurotransmitter Release from Astrocytes A Volterra
120
Retrograde Transsynaptic Influences G W Davis
126
Synaptic Plasticity: Short-Term Mechanisms J S Dittman and A C Kreitzer
132
SECTION II: NEUROMUSCULAR AND GAP JUNCTIONS Neuromuscular Connections: Vertebrate Patterns of C R Slater
141
Neuromuscular Junction: Synapse Elimination R R Ribchester
150
Presynaptic Events in Neuromuscular Transmission H Teng and R S Wilkinson
158
Neuromuscular Junction (NMJ): Presynaptic Short-Term Plasticity of Neuromuscular Transmission A M Holohean and K L Magleby
168
Neuromuscular Junction (NMJ): Postsynaptic Basal Lamina M J Werle
174
Neuromuscular Junction (NMJ): Postsynaptic Events in Neuromuscular Transmission C R Slater
180
Gap Junction Communication C Giaume and C C Naus
189
Gap Junctions and Electrical Synapses M V L Bennett
193
Gap Junctions and Hemichannels in Glia Z Ye and B R Ransom
213
Gap Junctions and Neuronal Oscillations M O Cunningham and F E N LeBeau
219
SECTION III: AMINO ACID TRANSMITTERS AND RECEPTORS Glutamate S P H Alexander
229
Glial Energy Metabolism: Overview L Pellerin and P J Magistretti
239
Contents xiii
Transporter Proteins in Neurons and Glia T S Otis and P D Dodson
245
Vesicular Neurotransmitter Transporters R J Reimer
253
AMPA Receptors: Molecular Biology and Pharmacology S M Dravid, H Yuan, and S F Traynelis
260
AMPA Receptor Cell Biology/Trafficking P G R Hastie and J M Henley
268
NMDA Receptor Function and Physiological Modulation K Zito and V Scheuss
276
NMDA Receptors, Cell Biology and Trafficking R J Wenthold and R S Petralia
284
Metabotropic Glutamate Receptors (mGluRs): Molecular Biology, Pharmacology and Cell Biology F Nicoletti, V Bruno, and G Battaglia
292
Metabotropic Glutamate Receptors (mGluRs): Functions B A Grueter and D G Winder
302
Kainate Receptors: Molecular and Cell Biology J Lerma
308
Kainate Receptor Functions J Lerma
313
Long-Term Potentiation (LTP): NMDA Receptor Role A J Doherty, S M Fitzjohn, and G L Collingridge
321
Long-Term Depression (LTD): Metabotropic Glutamate Receptor (mGluR) and NMDAR-Dependent Forms C Lu¨scher and M Frerking
327
D-Serine:
From Its Synthesis in Glial Cell to Its Action on Synaptic Transmission and Plasticity S H R Oliet and J-P Mothet
333
GABA Synthesis and Metabolism K L Behar
340
GABAA Receptors: Molecular Biology, Cell Biology, and Pharmacology T Goetz, P Wulff, and W Wisden
347
GABAA Receptor Synaptic Functions I Mody
355
GABAB Receptors: Molecular Biology and Pharmacology N G Bowery
360
GABAB Receptor Function A J Doherty, G L Collingridge, and S M Fitzjohn
368
Glycine Receptors: Molecular and Cell Biology C Vannier and A Triller
373
SECTION IV: AMINES AND ACETYLCHOLINE Dopamine J D Elsworth and R H Roth
383
xiv
Contents
Dopamine Receptors and Antipsychotic Drugs in Health and Disease P Seeman
392
Dopamine: Cellular Actions G Bernardi and N B Mercuri
410
Noradrenaline R D Wassall, N Teramoto, and T C Cunnane
414
Norepinephrine: Adrenergic Receptors D B Bylund
424
Norepinephrine: CNS Pathways and Neurophysiology G Aston-Jones, C A Meijas-Aponte, and B Waterhouse
430
Monoamines: Release Studies A D Smith, A C Michael, B J Lopresti, R Narendran, and M J Zigmond
442
Serotonin (5-Hydroxytryptamine; 5-HT): Neurotransmission and Neuromodulation A G Ramage
450
Serotonin (5-Hydroxytryptamine; 5-HT): Receptors T P Blackburn
456
Serotonin (5-Hydroxytryptamine; 5-HT): CNS Pathways and Neurophysiology G Aghajanian and R-J Liu
470
Cholinergic Neurotransmission in the Autonomic and Somatic Motor Nervous System T C Westfall
478
Cholinergic Pathways in CNS A C Cuello
486
Muscarinic Receptors: Autonomic Neurons R S Aronstam and P Patil
494
Nicotinic Acetylcholine Receptors J-P Changeux and Y Paas
503
SECTION V: NEUROPEPTIDES AND NEUROTROPHIC FACTORS Neuropeptide Synthesis and Storage J A Sobota, B A Eipper, and R E Mains
511
Neuropeptide Release F Bergquist and M Ludwig
519
Neuropeptides and Coexistence A Merighi
525
Opioid Peptides and Receptors B L Kieffer
532
Neuropeptide Y (NPY) and its Receptors D R Gehlert
538
Corticotropin-Releasing Hormone and Urocortins: Binding Proteins and Receptors M O Huising and W W Vale
544
Hypocretin/Orexin and MCH and Receptors A Adamantidis and L de Lecea
551
Peptidergic Receptors G Casini and D Cervia
557
Contents xv
Neuropeptides: Electrophysiology W Zieglga¨nsberger
564
Neurotrophins: Physiology and Pharmacology J M Conner and M H Tuszynski
570
Nerve Growth Factor J C Petruska and L M Mendell
576
Retrograde Neurotrophic Signaling C Wu and W C Mobley
584
BDNF in Synaptic Plasticity and Memory N H Woo and B Lu
590
GFL Neurotrophic Factors: Physiology and Pharmacology M Saarma
599
Insulin-Like Growth Factor Signaling and Actions in Brain V C Russo and G A Werther
609
Glial Growth Factors B M Reuss
617
SECTION VI: ATYPICAL NEUROTRANSMITTERS Adenosine K A Jacobson and Z-G Gao
627
Adenosine Triphosphate (ATP) G Burnstock
639
Purines and Purinoceptors: Molecular Biology Overview G Burnstock
648
P2X Receptors Z Li, S Harris, and T M Egan
658
Endocannabinoid Role in Synaptic Plasticity and Learning B Lutz and G Marsicano
664
Long-Term Depression (LTD): Endocannabinoids and Cerebellar LTD D M Lovinger
677
Nitric Oxide G Wilson and J Garthwaite
684
Role of NO in Neurodegeneration Y-I Lee, T M Dawson, and V L Dawson
690
Index
697
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PREFACE
The brain is often considered an information storage and processing machine. It receives information from the external and internal worlds and processes this information to generate specific thoughts, feelings and behaviors, which are often based on the information stored from past experiences. The performance of these tasks depends entirely on the bewildering complexity of intercellular communication in the nervous system; the ability of millions of individual nerve cells to communicate with one another and process information via countless numbers (billions or trillions) of different types of synapses. Intercellular communication is not random but for any given task that the brain performs occurs in specific neural circuits composed of complex patterns of synaptically interconnected cells. In addition, while the vast majority of intercellular communication in the brain occurs via synapses, we now know that glia play a critical role in shaping intercellular communication and that neural circuit activity can also be influenced by additional factors that are not released at specific synaptic connections in a point to point fashion. This book presents a series of articles that attempts to summarize the key features of the major types of intercellular communication that occur in the mammalian brain. It is composed of articles obtained from the Encyclopedia of Neuroscience, a comprehensive and, at times, overwhelming compendium of 1465 chapters that summarize all aspects of modern neuroscience, from the molecular biology and crystal structures of ion channels to the neural circuit basis of higher cognitive functions such as attention. Our current understanding of the pathophysiology of a wide variety of disease states ranging from Alzheimer’s Diseases to schizophrenia is also covered in the Encyclopedia. Clearly, this is a valuable entity that provides a unique reference library but it is difficult to wade through if the reader wants an update on current understanding of a broad, but relatively defined topic such as intercellular
communication. This book fills this need in what I hope is a logical and comprehensive manner. As the synapse remains the primary point of information processing and communication between nerve cells, the book begins with a summary of current understanding of synapse formation and basic synapse structure and function. Several chapters in this first section address the important topic of the role of glia, which are now known to play important roles in synaptogeneis and synaptic transmission. The second section of the book summarizes current knowledge of the most classic of synapses, the neuromuscular junction as well as gap junctions, that play important roles in controlling neural circuit activity in key brain regions. We then move on, in the third section, to the major types of excitatory and inhibitory synapses, those using the neurotransmitters glutamate and GABA (as well as glycine). The subsection on glutamate is by far the longest in the book because in mammalian brains, >75% of all synapses are excitatory and use glutamate as their neurotransmitter. Glutamatergic synapses are the workhorses of the brain, carrying out virtually all of its critical tasks. They also are highly plastic so that the circuits in which they are embedded can be modified by experience and store these experiences as memories. Inhibitory, GABA-using synapses, on the other hand help shape circuit properties in important ways and thus are also an important topic. In Seciton IV, we move on to forms of intercellular communication that involve what are often called neuromodulators. These include the major amines, dopamine, norepinephrine and serotonin as well as acetycholine. Individual chapters review the molecular biology and pharmacology of the receptors for these neuromodulators, their anatomical distribution and connectivity, as well as their varied cellular physiological effects and nervous system functions. The large topic of neuropeptides and neurotrophic factors is reviewed
xvii
xviii
Preface
in Section V. This includes chapters on neuropeptide processing and release as well as on the receptors and actions of a large number of individual neuropeptides and neurotrophic factors. Current knowledge of the functions of neuropeptides and neurotrophic factors is advancing rapidly so it should not be surprising if the latest advances on certain restricted topics are not fully covered. Nonetheless, for the non-expert, this section will provide a comprehensive overview of this broad research area. The book concludes with so-called Atypical Neurotransmitters including the purines, ATP and adenosine, endocannabinoids and nitric oxide. Several of these are not released in the same manner as the classical neurotransmitters and exert their actions in a larger volume of brain tissue. Clearly, several topics of great relevance to Intercellular Communication in the Nervous System, such as Neuroendocrinology and Neuroimmunology, are
not covered in this book. It was felt that broad and important topics such as these deserve a more comprehensive treatment than could be accomplished herein. Nonetheless, we hope that the reader who reads this text cover to cover will leave with a thorough and current understanding of the detailed mechanisms by which intercellular communication in the brain occurs and how it functions to mediate the amazing tasks the brain accomplishes each and every day. We also hope that it serves as a valuable reference for readers to look up details on the many different highly specific topics that are covered by the 87 chapters. Both the publishers and I (the editor) think this is a compendium that will prove valuable to a broad array of neuroscientists and biologists. After delving into it, we hope you agree. Robert C. Malenka Editor-in-Chief
BASIC MECHANISMS OF SYNAPTIC TRANSMISSION (SYNAPTIC STRUCTURE AND ORGANIZATION) A. Synapse Formation B. Synapse Structure and Function
3 52
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Synaptic Precursors: Filopodia E Robles and S J Smith, Stanford University, Stanford, CA, USA M P Meyer, Kings College London, London, UK ã 2009 Published by Elsevier Ltd.
Filopodia as Synaptic Precursors The vertebrate central nervous system (CNS) contains billions of neurons connected by an even greater number of synapses. Despite this complexity, the CNS develops reliably and precisely. The goal of developmental neurobiologists is to understand the mechanisms that control the accurate formation of synaptic connections in the developing brain. This article will review evidence that axonal and dendritic filopodia are fundamental players in the assembly of functional neural circuits. As will become apparent during the course of this article, the highly motile nature of filopodia and the relationship of filopodia to synaptogenesis are central to their role in generating a precisely ordered nervous system. This article is structured, therefore, around the participation of filopodia in synapse formation, the regulation of filopodial motility, and the way such regulation is fundamental to the genesis of appropriate and ordered synaptic connections. In part one we concentrate on a phenomenological discussion of the role of filopodia in synaptogenesis and growth and branching of axonal and dendritic arbors. This is followed by a discussion of intrinsic and extrinsic factors that may regulate the formation of synaptic contacts by altering filopodial motility in developing neurons. Dendritic Filopodia as Spine Precursors
Dendritic spines were discovered in 1888 by Ramon y Cajal, who noticed that Purkinje cells’ dendrites were decorated with small thorns (‘espinas’). He later proposed that these small, submicrometer protrusions from the parent dendrite were the site of axodendritic contact. Cajal’s hypothesis was proved correct 60 years later, when the electron microscope was employed to show that spines were indeed sites of synaptic contact between axons and dendrites. In fact, more recent estimates suggest that more than 90% of excitatory axodendritic synapses in the mature CNS occur on dendritic spines. Spines must therefore be key elements in neuronal circuitry, and their structure suggests that one fundamental function of spines may be to bridge physical gaps between densely packed dendrites and axons. Hence, the mechanisms governing spine morphogenesis likely play a fundamental role in the selection
of synaptic partners and establishment of functional connectivity. A clue to the developmental origins of spines comes from the observations that prior to the overt presence of spines, many elongated filopodia extend from dendritic shafts. These filopodia are long (2–20 mm), thin (<0.3 mm) structures and lack the bulbous head that is characteristic of spines. The elongated morphology of dendritic filopodia hints at an exploratory role, and further evidence for this has come from time-lapse imaging studies showing that filopodia are highly motile structures. For example, in early postnatal hippocampal slice cultures, filopodia extend and retract rapidly from dendritic shafts and have a mean lifetime of only 10 min or so. At later developmental times, there are progressively fewer filopodia, a trend accompanied by a steady increase in the number of more-stable spinelike structures. The scarcity of filopodia on mature dendrites suggests a mainly developmental role, and the structural similarities and sequential appearance of filopodia and spines suggests that filopodia might be spine precursors. Consistent with this hypothesis, in dissociated hippocampal neurons maintained in vitro, the appearance of spines coincides with an increase in the number of functional synapses and a reduction in the number of motile filopodia. Furthermore, the total number of filopodia formed is more than sufficient to account for the entire spine population. Formation of a contact between a dendritic filopodium and a nearby axon has also been observed directly in hippocampal neurons in vitro. This contact event resulted in the stabilization of the filopodium and the formation of presynaptic bouton at the contact site. These in vitro studies have been complemented by several electron microscopy studies of developing tissues that demonstrate the presence of synapses on filopodia, clearly demonstrating that filopodia also form synapses in vivo. These findings have been incorporated into a filopodial model of synaptogenesis and spine formation: (1) dendritic filopodia on immature dendrites are highly dynamic, facilitating short-range exploration and contact initiation; (2) contact between a filopodium and a neighboring axon may result in the formation of a nascent synapse; and (3) maintenance of the nascent synapse stabilizes the filopodium on which it is located, transforming it into a dendritic spine. In contrast, filopodia that fail to contact an axon, or contact an axon but fail to form a stable synapse would then be rapidly eliminated. Caveats and Alternative Models of Spine Formation
Presented with the evidence above, one could be forgiven for thinking that spinogenesis is essentially a
3
4 Synaptic Precursors: Filopodia
process of trial and error. Highly labile filopodia extend randomly from the dendritic shaft, and those that establish a stable synaptic contact with a presynaptic axon are transformed into a more stable spine. While this may be the case for some neurons, it does not appear to be true for all neuronal cell types. A number of lines of evidence suggest that spine formation can occur independently of a presynaptic cell. For example, in ‘weaver’ mutant mice, granule cells – the presynaptic partners of the vast majority of Purkinje cells – are absent. In these animals, the main Purkinje cell dendrites are abnormal but are nevertheless decorated with spines. Even in the normal cerebellum, the distal dendritic branches of Purkinje neurons develop spines before synaptic contact with parallel fibers’ axons. Thus, the normal developmental sequence of events suggests that Purkinje cell spines can exist in the absence of presynaptic axons. Such uninnervated spines have also been observed in the barrel cortex of adult mice. Time-lapse imaging in vivo has demonstrated the emergence of new spines from layer 5 pyramidal neuron dendrites. Reconstruction of new spines using serial electron microscopy has shown that at least some of them lacked a presynaptic partner. These data suggest a model in which spine formation is an intrinsic property of some neurons and so can occur without interaction with a presynaptic cell. However, given the importance of reciprocal preand postsynaptic interactions during nervous system development, it is unlikely that uninnervated spines are completely normal. Filopodia and Formation of Dendritic Branches
The data described previously suggest that filopodia are important for establishing synaptic contact with nearby axons and that successful filopodia are stabilized as spine precursors. However, synapses are also present on dendritic shafts, and shaft synapses are particularly prevalent at early developmental stages. How are shaft synapses formed? Also, filopodia occur in neurons that do not form spines. What is the function of filopodia in nonspiny neurons? One intriguing idea that could answer both of these questions is that filopodia are important not only for establishing spines and spine synapses but also for development of dendritic branches and the shaft synapses on them. Support for this idea has come from time-lapse imaging studies of nonspiny tectal cell dendrites as they arborized in the tectum of live zebra fish larvae. Engineering zebra fish tectal neurons to express a fluorescent postsynaptic marker has enabled time-lapse imaging to be used to study the relationship between dendritic filopodia, synaptogenesis, and growth of
dendritic arbors in vivo. Growth of tectal dendrites is highly dynamic, involving prolific extension and retraction of filopodia, and the use of a fluorescent synaptic marker has revealed that virtually all tectal synapses form on newly extended filopodia. It is surprising that the majority of these nascent synapses and the filopodia that bore them were transient, lasting only tens of minutes or so, indicating a high turnover rate for both synapses and filopodia. The crucial finding, though, was that the fraction of synapses that were maintained in turn stabilized the filopodia on which they were located, and these stabilized filopodia matured into dendritic branches (see Figure 1). Successive iterations of filopodial extension and stabilization by synapses resulted in growth and branching of the entire dendritic arbor. Thus, in nonspiny tectal dendrites, filopodia are important for establishing synaptic contacts (as they do in spiny neurons), but in these cells, stabilized filopodia go on to form dendritic branches. Thus, the dendrite essentially grows out to meet axons via filopodial stabilization at the point of synaptic contact. It is not yet known whether shaft synapses and dendritic branches form this way in dendrites that go on to form spines. Filopodia and Synaptotropic Growth of Dendrites
The data obtained from zebra fish tectal neurons are interesting for several reasons: first, the high turnover of nascent synapses implies that an important function of filopodial dynamics is to establish many trial synapses with nearby axons. Those synapses which are maintained, and presumably ‘correct,’ in turn stabilize growth of the dendrite in regions of appropriate synaptic partners. Conversely, filopodia which do not form stable synapses are rapidly retracted, and further growth in these regions is prevented. Second, because growth is stabilized only in correct target regions, filopodia that extend from a previously stabilized one are more likely to make contact with correct targets. Thus, once a correct synapse forms, a positive feedback is established that guides growth of the arbor into appropriate target regions (see Figure 1). Synaptogenesis in this context is effectively a growth guidance mechanism that provides a means to match the extent of a dendrite to the set of connections it forms, that is, there are no branches without synapses. This mechanism relies on randomly distributed filopodial extension and a local stochastic process of synapse stabilization. Modeling studies have shown that such a process can generate precise patterns of connectivity independently of innate dendrite branching programs or expression of dendritic guidance molecules. This is potentially very important as it is unlikely that the genome can encode
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b Figure 1 (a) Growth of zebra fish tectal cell dendrites in vivo. Growth occurs by an interactive sequence of selective filopodial stabilization and synapse formation. Still images from time-lapse series, accompanied by a schematic rendering for clarity. Green represents accumulations of the postsynaptic marker PSD-95: green fluorescent protein (GFP), and red areas are newly formed (often transient) branches. Brown in the schematic represents persistent branches. (b) Model of synaptotropic guidance of dendrite arbor growth. A number of filopodia (solid red) extend from a dendritic branch. Those that encounter correct partners and form synaptic connections (green dots) are stabilized as new branches (brown), whereas those that establish inappropriate contacts (blue dots) are retracted (dashed red). Successive rounds of selective stabilization result in arborization within a field of appropriate synaptic connections (dashed green region).
explicit instructions to form every synapse in the nervous system. The ‘synaptotropic hypothesis’ of dendrite growth described above was first proposed based on electron microscopy studies of developing rat spinal cord. The presence of synapses on filopodia and the fact that dendrites did not extend until presynaptic partners had arrived are both consistent with the idea that formation of a synapse could stabilize nascent dendritic branches and bias further growth in that direction. A very similar pattern of dendritic growth has been observed in real time in the zebra fish retina. Similar to tectal cell dendrites, retinal ganglion cell (RGC) dendrites were highly dynamic at early developmental stages and demonstrated little net growth indicative of unguided exploration. However, as the laminar organization of amacrine cell processes (the presynaptic partners of RGC dendrites) began to develop, RGC dendrites became progressively more stable and ramified sequentially in amacrine plexuses. Thus, growth and targeting of prepatterned afferents by RGC dendrites appear to occur by a process of selective stabilization of nascent branches, perhaps by a synaptotropic mechanism.
However, there is ample evidence indicating that many dendrites do not rely on a synaptotropic mechanism. For example, hippocampal neurons form complex dendrites in vitro in the absence of presynaptic partners, and in the drosophila olfactory system, second order projection neurons elaborate dendrites in a highly stereotyped manner prior to the arrival of the olfactory sensory neurons. Therefore, during development, multiple strategies are employed to generate appropriate dendritic arbor morphology. Further studies should allow the relative contribution of each strategy to be tested and the contexts in which a particular strategy predominates to be elucidated. Axonal Filopodia, Synaptogenesis, and Growth of Axonal Arbors
So far this article has focused on the role of dendritic filopodia. However, filopodia also extend from shafts of axonal branches. These filopodia are therefore distinct from the filopodia that emanate from the leading edge of axonal growth cones. While growth cone filopodia mediate axonal navigation, the filopodia that emerge from axonal branches appear to play a
6 Synaptic Precursors: Filopodia
role similar to that of dendritic filopodia, that is, in synaptogenesis and the formation of axon branches. Since the role of axonal filopodia has been investigated less extensively, the data that support this idea will be summarized only briefly. Time-lapse imaging has shown that axonal filopodia are highly motile in vitro and in vivo, consistent with an exploratory role. Most axonal filopodia are transient, lasting only a few minutes or so. Second, motility of axonal filopodia is downregulated as development and synaptogenesis proceed. Third, a number of different techniques have been employed to show that axonal filopodia are able to establish synaptogenic contact in vitro and in vivo. These studies have also indicated that formation of a stable synapse stabilizes the axonal filopodium on which it is located. As is the case with zebra fish tectal cell dendrites, axonal filopodia of retinal axons (the presynaptic partners of tectal cells) that are stabilized by synapses go on to form new axon branches from which further growth can occur. Selective stabilization of axonal branches by synapses has also been observed in the Xenopus retinotectal projection. These data suggest that as is the case with tectal cell dendrites, there is also a synaptotropic component to growth of retinal axonal arbors. Thus, stabilization of filopodia by synapses is important for guiding the formation of retinotectal connectivity from both the pre- and postsynaptic sides. Despite the obvious similarities between axonal and dendritic filopodia, there is one notable difference. Axonal filopodia contain clusters of vesicles that are capable of exo-endocytic recycling, whereas dendritic filopodia do not. Thus axonal filopodia may be sources of neurotransmitter release prior to the formation of obvious synapses. Local release of neurotransmitters (and neurotrophic factors) may have an important role in regulating synapse formation by locally modulating filopodial dynamics.
Regulation of Filopodial Motility Filopodia serve an exploratory function during synaptogenesis, actively probing the extracellular environment for instructive cues located in the extracellular environment and on the surface of other cells. Filopodial motility increases the probability of synaptic contact between pre- and postsynaptic cells simply by increasing the area sampled in search of an appropriate synaptic partner. Filopodial motility also provides a mechanism that allows neurons to form many trial synapses with multiple potential synaptic partners. Because of the complexity of nervous system connectivity, this element of trial and error is indispensable as it allows for the formation of precise
circuitry without the need to explicitly encode every synaptic connection within the genome. In contrast to the trial-and-error nature of synaptogenesis, filopodial motility is highly regulated by both intrinsic and extrinsic signals. While intrinsic signals may function to promote filopodial motility throughout the entire neuron, extrinsic signals may function to guide synapse formation and arbor growth through local regulation of filopodial motility. In the following section we describe well-characterized intrinsic and extrinsic factors that regulate filopodial motility and discuss the potential role of these factors during the process of synaptogenesis. Intrinsic Regulation of Filopodial Motility
One potential explanation for the early prevalence of filopodia in developing neurons is that filopodium formation during this developmental period is increased as a result of an intrinsic genetic program. This idea is supported by evidence that cytoskeletal proteins associated with neurite growth are expressed at high levels during periods of neurite growth and synapse formation and are subsequently downregulated in the mature nervous system. Filopodial protrusions consist primarily of parallel-bundled actin filaments oriented with the growing ‘plus’ end toward the filopodial tip. Filopodial initiation and extension occur through regulated actin monomer addition at the plus ends of preexisting actin filaments. Neuronal actin consists of two distinct isoforms, g- and b-actin, of which b-actin is strongly enriched at sites of dynamic cytoskeletal remodeling, such as filopodia and spines. High expression levels of neuronal b-actin during periods of neurite growth and synaptogenesis are consistent with actin-dependent filopodial dynamics’ being controlled by an intrinsic genetic program. In addition to regulation by protein expression, actin dynamics in developing axons and dendrites could be elevated through upregulation of cytoskeletal regulatory proteins. Several types of signaling proteins have been shown to regulate actin dynamics and neuronal architecture, including actinbinding proteins, Rho family guanosine triphosphatases (GTPases), tyrosine and serine-threonine kinases, and motor proteins. In axonal growth cones, several cytoskeletal regulators have been localized to a filopodial tip complex located at the interface between the distal, growing ends of filopodial actin filaments and the plasma membrane. Recent studies also suggest that local intracellular signals generated within this specialized tip complex, function to direct local actin polymerization and drive filopodial extension. Proteins localized to the tip complex in developing
Synaptic Precursors: Filopodia
neurons include members of the Ena/VASP family of actin-binding proteins, Src-family tyrosine kinases, the Rho family GTPase Cdc42, and the Cdc42 effector protein p21-activated kinase (PAK). Although these mechanisms have been best characterized in axonal filopodia, Cdc42 activation has also been shown to correlate with increased motility of dendritic filopodia. This indicates that, in addition to obvious structural similarities, the motility of axonal and dendritic filopodia is regulated by common signaling pathways. Intrinsic regulation of growth-associated mechanisms may contribute to cellwide filopodial motility in neurons during development. However, a synaptotropic model of synapse formation suggests that filopodial dynamics be locally regulated, implying the involvement of extrinsic regulators of filopodial dynamics. Regulation of Filopodial Dynamics by Brain-Derived Neurotrophic Factor
Activity-dependent secretion of neurotrophins may guide synaptotropic arbor growth by promoting filopodial motility at synapse-rich regions of developing neuronal arbors. Brain-derived neurotrophic factor (BDNF), signaling through its receptors TrkB and p75, may serve this function within the CNS. It is secreted in an activity-dependent manner and has been shown to alter neuronal morphology and synapse density. Furthermore, BDNF application has also been shown to promote filopodial motility in axons and dendrites of several types of neurons in vitro. BDNF stimulation has been shown to modulate filopodial dynamics by stimulating the GTPase activity of Cdc42, which likely acts through downstream targets such as PAKs, which regulate filopodial motility in growth cones and synaptogenesis and spinogenesis in hippocampal neurons. Recent findings have also demonstrated that clusters of TrkB receptors are transported into both axonal and dendritic filopodia, consistent with the idea that filopodia can sense extracellular BDNF. In Xenopus laevis tadpoles, local BDNF administration in the optic tectum has been shown to increase the density of synapses on retinal axons and tectal neuron dendrites. This increased synapse formation correlates with an increase in retinal arbor complexity. Together these studies suggest that activity-dependent secretion of BDNF may promote filopodial motility locally and in so doing promote synaptogenic contact between axons and dendrites. Regulation of Filopodial Motility by Neurotransmitters and Neuronal Activity
It is well established that neuronal activity plays an important role in the accurate formation and refinement of neuronal connections during development.
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Given the central role of filopodia during synaptogenesis, an attractive model is one in which neuronal activity promotes filopodial exploration and stability. Globally, action potential firing may operate in conjunction with intrinsic genetic programs to promote filopodial motility in immature neurons during development. In addition, synaptic transmission and local depolarization of specific dendritic regions may promote synaptotropic growth by promoting filopodial extension in the vicinity of functional synapses. Evidence for this model was first provided by reports that application of the excitatory neurotransmitter glutamate stimulates rapid filopodial growth from hippocampal neuron dendrites (see Figure 2). These findings suggested that neurotransmitter secretion could promote synaptogenic contact by triggering local filopodial growth from potential postsynaptic partners. However, it should be noted that glutamate has been shown to either increase or decrease the motility of axonal and dendritic filopodia, suggesting that its effects are highly context dependent. Synaptically evoked action potential firing has also been shown to promote the formation of motile dendritic filopodia. This suggests a mechanism that promotes dendritic filopodial exploration and subsequent synaptogenesis and arbor growth in neurons receiving strong synaptic inputs. Such a mechanism could locally promote further synaptogenic contact between appropriately matched axonal and dendritic arbors. In support of this model, in vivo imaging has provided evidence that sensory experience promotes the protrusion of dendritic filopodia from pyramidal neurons in the developing rat barrel cortex and Xenopus tectal neurons. In the barrel cortex, reduced filopodial motility induced by whisker trimming correlates with the formation of less refined receptive fields, suggesting that activity-dependent filopodial exploration is necessary for accurate formation of sensory maps during development. Neuronal activity may promote filopodial motility by direct activation of second-messenger signaling cascades or by heightening sensitivity to locally secreted cues such as BDNF. Role of Calcium Signaling in Filopodial Motility
The regulation of filopodial motility by action potential firing and glutamate application suggests that intracellular calcium elevations may mediate observed changes in neuronal structure and motility. This possibility is supported by several reports of neuronal growth and morphology being regulated by calcium-sensitive proteins such as calmodulin, calmodulin-dependent protein kinase II (CaMKII), the intracellular protease calpain, and the phosphatase calcineurin. The b isoform of CaMKII has been shown to specifically regulate the motility of dendritic filopodia and the formation of
8 Synaptic Precursors: Filopodia
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k Figure 2 Rapid modulation of filopodial motility by glutamate and brain-derived neurotrophic factor (BDNF). (a, b) High-magnification view of a hippocampal neuron dendrite at indicated times after local pulatile application of glutamate. Note rapid increase in filopodial number and length. Arrow indicates a newly sprouted filopodium. (c) Measurement of filopodial length as a function of time in response to glutamate application for the filopodium indicated by arrow in (b); time in seconds. (d–g) Time-lapse differential interference contrast (DIC) and fluorescence images of a Xenopus spinal neuron expressing green fluorescent protein (GFP)-dSH2, a fluorescent indicator of tyrosine phosphorylation (PY), at indicated times after bath application of BDNF. Note that many filopodia lack enriched GFP-dSH2 at their tip before BDNF application but accumulate GFP-dSH2 after BDNF stimulation. (j), (k) Time-lapse DIC and GFP-dSH2 fluorescence images of boxed regions indicated in (d)and (f)at indicated times after BDNF application. Time between images is 10 s. Arrows point to one preexisting filopodium that acquires tip PY at 80 s after BDNF stimulation and three newly extended, PY-containing filopodia. (h, i) Kinetics of increased filopodium formation and increased GFP-dSH2 accumulation at filopodial tips in response to BDNF. Scale bar ¼ 5 mm (d).
Synaptic Precursors: Filopodia
excitatory synapses by hippocampal neurons in vitro. The beta isoform of CaMKII, which is the predominant isoform expressed during early development, has also been specifically shown to bind to F-actin in hippocampal dendrites and to localize to dendritic regions undergoing dynamic structural rearrangements. Although the specific mechanism by which CaMKIIb functions to promote filopodial motility is unknown, one possibility is that actin binding functions to localize CaMKIIb to a multimolecular signaling complex. Within such a complex, CaMKIIb activation by intracellular calcium elevation and calmodulin binding may lead to spatially precise activation of downstream signaling proteins that regulate the actin cytoskeleton. Another mechanism by which calcium signaling may regulate local motility of filopodia is through the generation of local calcium transients restricted to small regions of a dendritic or axonal arbor. The function of dendritic spines as discrete calcium signaling compartments has been demonstrated by monitoring intracellular calcium levels in response to synaptic transmission. However, similar observations have been made in axonal and dendritic filopodia, suggesting that calcium signaling may regulate filopodial motility and thereby influence synaptogenesis. Consistent with this possibility, calcium imaging in RGCs has revealed that newly formed dendritic branches also exhibit local neurotransmitter-evoked calcium elevations, and these signals may function to stabilize nascent synaptic contacts. This model for calcium-dependent stabilization of dendritic branches is consistent with calcium imaging studies examining the function of local calcium transients in filopodia of hippocampal neuron dendrites and Xenopus spinal neuron growth cones. In both systems, increased frequencies of local calcium transients correlated with the stabilization of active filopodia, suggesting that extracellular signals that promote the stabilization of synaptic contacts may do so through modulation of intracellular calcium levels. Although calcium-sensitive signaling cascades can control neuronal motility, the way in which calcium signals regulate distinct aspects of filopodial motility such as growth and stabilization remains unclear. Filopodial Stabilization by Adhesion Molecules
During synaptogenesis, filopodial exploration and recognition of a putative synaptic partner lead to filopodial stabilization through an adhesive interaction with the target cell. Initial contact is mediated by the binding of transmembrane adhesion receptors such as members of the cadherin, protocadherin, and SynCAM protein families as well as neuroligin and b-neurexin. Of these adhesion molecules, N-cadherin has been localized to
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early contacts between dendritic filopodia and axons, where it functions to stabilize synapses and promote the formation of mature spine synapses. Following transmembrane engagement, structural stabilization of this contact site arises through direct interaction between n-cadherin and a-catenin, an actin-binding protein that forms a functional linkage between the transmembrane adhesion protein and the actin cytoskeleton. Disruption of either n-cadherin or a-catenin function results in altered spine morphologies characterized by the appearance of filopodia-like motile protrusions extending from the spine head. This suggests that in a system in which filopodia function as spine precursors, synapse stabilization by cadherins and catenins normally inhibits filopodial exploration. In addition to serving a structural function in maintaining physical contact between pre- and postsynaptic cells, transmembrane adhesion proteins can serve a signaling role in directing the assembly of synaptic specializations at the original site of adhesion. The homophilic adhesion molecule SynCAM has been shown to be necessary for the formation of glutamatergic synapses by directing the formation of presynaptic specializations. Similarly, presynaptic neuroligin binding to postsynaptic b-neurexin stimulates the local recruitment of both pre- and postsynaptic components. Although the dynamic localization of SynCAM and b-neurexin– neuroligin during synaptogenesis has not yet been determined, the fact that filopodia are known to make synaptic contacts suggests that these synaptogenic molecules may well be expressed on them. Expression of synaptic cell adhesion molecules on the surface of filopodia may function to halt filopodial exploration on formation of a synaptic contact, thereby allowing differentiation of these early contacts into mature, stable synapses. See also: Cell Adhesion Molecules at Synapses; Postsynaptic Development: Neuronal Molecular Scaffolds.
Further Reading Abe K, Chisaka O, VanRoy F, and Takeichi M (2004) Stability of dendritic spines and synaptic contacts is controlled by alpha Ncatenin. Nature Neuroscience 7: 357–363. Alsina B, Vu T, and Cohen-Cory S (2001) Visualizing synapse formation in arborizing optic axons in vivo: Dynamics and modulation by BDNF. Nature Neuroscience 4: 1093–1101. Biederer T, Sara Y, Mozhayeva M, et al. (2002) SynCAM, a synaptic adhesion molecule that drives synapse assembly. Science 297: 1525–1531. Boda B, Alberi S, Nikonenko I, et al. (2004) The mental retardation protein PAK3 contributes to synapse formation and plasticity in hippocampus. Journal of Neuroscience 24: 10816–10825.
10 Synaptic Precursors: Filopodia Bond JF and Farmer SR (1983) Regulation of tubulin and actin mRNA production in rat brain: Expression of a new beta-tubulin mRNA with development. Molecular & Cellular Biology 3: 1333–1342. Dailey ME and Smith SJ (1996) The dynamics of dendritic structure in developing hippocampal slices. Journal of Neuroscience 16: 2983–2994. Eom T, Antar LN, Singer RH, and Bassell GJ (2003) Localization of a beta-actin messenger ribonucleoprotein complex with zipcode-binding protein modulates the density of dendritic filopodia and filopodial synapses. Journal of Neuroscience 23: 10433–10444. Fiala JC, Feinberg M, Popov V, and Harris KM (1998) Synaptogenesis via dendritic filopodia in developing hippocampal area CA1. Journal of Neuroscience 18: 8900–8911. Fink CC, Bayer KU, Myers JW, Ferrell JE Jr., Schulman H, and Meyer T (2003) Selective regulation of neurite extension and synapse formation by the beta but not the alpha isoform of CaMKII. Neuron 39: 283–297. Gehler S, Gallo G, Veien E, and Letourneau PC (2004) p75 neurotrophin receptor signaling regulates growth cone filopodial dynamics through modulating RhoA activity. Journal of Neuroscience 24: 4363–4372. Gibney J and Zheng JQ (2003) Cytoskeletal dynamics underlying collateral membrane protrusions induced by neurotrophins in cultured Xenopus embryonic neurons. Journal of Neurobiology 54: 393–405. Gomes RA, Hampton C, El-Sabeawy F, Sabo SL, and McAllister AK (2006) The dynamic distribution of TrkB receptors before, during, and after synapse formation between cortical neurons. Journal of Neuroscience 26: 11487–11500. Gomez TM, Robles E, Poo M, and Spitzer NC (2001) Filopodial calcium transients promote substrate-dependent growth cone turning. Science 291: 1983–1987. Graf ER, Zhang X, Jin SX, Linhoff MW, and Craig AM (2004) Neurexins induce differentiation of GABA and glutamate postsynaptic specializations via neuroligins. Cell 119: 1013–1026. Hartmann M, Heumann R, and Lessmann V (2001) Synaptic secretion of BDNF after high-frequency stimulation of glutamatergic synapses. EMBO Journal 20: 5887–5897. Jontes JD and Smith SJ (2000) Filopodia, spines, and the generation of synaptic diversity. Neuron 27: 11–14. Katz LC and Shatz CJ (1996) Synaptic activity and the construction of cortical circuits. Science 274: 1133–1138. Lanier LM, Gates MA, Witke W, et al. (1999) Mena is required for neurulation and commissure formation. Neuron 22: 313–325.
Lendvai B, Stern EA, Chen B, and Svoboda K (2000) Experiencedependent plasticity of dendritic spines in the developing rat barrel cortex in vivo. Nature 404: 876–881. Lohmann C, Finski A, and Bonhoeffer T (2005) Local calcium transients regulate the spontaneous motility of dendritic filopodia. Nature Neuroscience 8: 305–312. Lohmann C, Myhr KL, and Wong RO (2002) Transmitter-evoked local calcium release stabilizes developing dendrites. Nature 418: 177–181. Maletic-Savatic M, Malinow R, and Svoboda K (1999) Rapid dendritic morphogenesis in CA1 hippocampal dendrites induced by synaptic activity. Science 283: 1923–1927. Meyer MP and Smith SJ (2006) Evidence from in vivo imaging that synaptogenesis guides the growth and branching of axonal arbors by two distinct mechanisms. Journal of Neuroscience 26: 3604–3614. Niell CM, Meyer MP, and Smith SJ (2004) In vivo imaging of synapse formation on a growing dendritic arbor. Nature Neuroscience 7: 254–260. Robles E, Woo S, and Gomez TM (2005) Src-dependent tyrosine phosphorylation at the tips of growth cone filopodia promotes extension. Journal of Neuroscience 25: 7669–7681. Sanchez AL, Matthews BJ, Meynard MM, Hu B, Javed S, and CohenCory S (2006) BDNF increases synapse density in dendrites of developing tectal neurons in vivo. Development 133: 2477–2486. Sin WC, Haas K, Ruthazer ES, and Cline HT (2002) Dendrite growth increased by visual activity requires NMDA receptor and Rho GTPases. Nature 419: 475–480. Smith SJ and Jahr CE (1992) Rapid induction of filopodial sprouting by application of glutamate to hippocampal neurons. In: Letourneau PC, Kater SB, and Macagno ER (eds.) The Nerve Growth Cone, pp. 19–26. New York: Raven Press. Togashi H, Abe K, Mizoguchi A, Takaoka K, Chisaka O, and Takeichi M (2002) Cadherin regulates dendritic spine morphogenesis. Neuron 35: 77–89. Waites CL, Craig AM, and Garner CC (2005) Mechanisms of vertebrate synaptogenesis. Annual Review of Neuroscience 28: 251–274. Yuan XB, Jin M, Xu X, et al. (2003) Signalling and crosstalk of Rho GTPases in mediating axon guidance. Nature Cell Biology 5: 38–45. Yuste R and Bonhoeffer T (2004) Genesis of dendritic spines: Insights from ultrastructural and imaging studies. Nature Reviews Neuroscience 5: 24–34. Ziv NE and Smith SJ (1996) Evidence for a role of dendritic filopodia in synaptogenesis and spine formation. Neuron 17: 91–102.
Presynaptic Development: Functional and Morphological Organization D Atasoy and E T Kavalali, The University of Texas Southwestern Medical Center, Dallas, TX, USA ã 2009 Elsevier Ltd. All rights reserved.
Structural Features of Central Synapses Chemical synapses are intercellular junctions critical for information transfer and processing in the nervous system. They consist of two compartments physically juxtaposed within several nanometers of each other: presynaptic terminals and postsynaptic specializations. Presynaptic terminals store and release neurotransmitter substances in membranous organelles named synaptic vesicles, whereas postsynaptic structures contain signaling molecules responsible for generation of neuronal responses to released neurotransmitters. Presynaptic terminals are highly organized subcellular structures. At the electron microscopic level, clusters of synaptic vesicles around the plasma membrane regions called active zones can readily distinguish them from other structures within a neuron. Synaptic vesicle exocytosis is thought to take place exclusively at the active zone, whereas synaptic vesicle endocytosis may occur within the vicinity of this region. Active zones are characterized by enrichment of scaffolding molecules, and enable assembly of proteins required for regulated vesicle fusion and recycling. Juxtaposed to the presynaptic terminal, the postsynaptic site is characterized by electron-dense material called the postsynaptic density. Postsynaptic density is enriched in scaffolding molecules that anchor neurotransmitter receptors and organize signaling in response to second messenger cascades activated by the neurotransmitter receptors. The pre- and postsynaptic sides of the synapse are held together with adhesion molecules spanning the synaptic cleft (Figure 1). The size of the active zone and the number of docked vesicles are critical determinants of the functional responses of a presynaptic terminal. These structural markers are continually modified during synapse maturation. Depending on the type of synapse, presynaptic terminals in a given synapse contain varying number of synaptic vesicles, some of which are physically attached or docked at the plasma membrane. The most striking difference of synapses from other cell–cell junctions is the asymmetry of structures on both sides of the synaptic junction. Such asymmetry implies that two compartments must respond differently to the signal(s) that initiate synaptogenesis.
This asymmetry is partially achieved through differential distribution of synaptic components to axonal and dendritic compartments within a neuron. Asymmetric interaction of cell adhesion molecules can also account for triggering divergent cascades of downstream events and induction of pre- and postsynaptic sites. However, it is important to note that despite this asymmetry the sizes of the structures on both sides of the synaptic cleft are all correlated, suggesting that the structural synapse assembly is significantly coordinated across the cleft. In a mature presynaptic terminal, vesicles can be divided into two pools. The first pool contains a relatively small fraction of vesicles close to release sites. These vesicles can be released by brief Ca2þdependent stimuli or by hypertonic stimulation, which is Ca2þ independent. This release-ready pool of vesicles is referred to as the immediately releasable pool or the readily releasable pool (RRP). RRP vesicles are considered to be in a morphologically docked state, although not all morphologically docked vesicles are necessarily release competent at any given time. A priming step in addition to the morphological docking is required to make vesicles fully release competent. A secondary pool of vesicles, the reserve pool, is spatially distant from the release sites and constantly replaces the vesicles in the RRP that have been exocytosed. The rate of replenishment of RRP vesicles from the reserve pool is a critical parameter that determines the response of synapses to repetitive stimulation. Recent evidence indicates that intrasynaptic Ca2þ can facilitate the rate of replenishment. The number of vesicles contained in the RRP is a critical parameter that regulates the probability of release, which is defined as the probability that a presynaptic action potential can result in an exocytotic event. Therefore, the number of vesicles in the RRP and the rate and pathways by which they are replenished is a crucial determinant of presynaptic efficacy and of several forms of short- and long-term synaptic plasticity. Several lines of evidence support the presence of the non-recycling pool of vesicles in the synapse. Mechanisms that can render this pool functional remain to be determined.
Multiple Stages of Synapse Assembly In the mammalian central nervous system (CNS), synapse formation is a precisely timed process. Synapses appear within days in a given brain region. At later stages of development, synapse proliferation
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12 Presynaptic Development: Functional and Morphological Organization
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200 nm Figure 1 Structural organization of a central synapse. In electron micrographs, synapses can be readily distinguished by the cluster of synaptic vesicles around protein-rich (thus electrondense) regions called the active zone. The size of the active zone is tightly correlated with the size of postsynaptic density, another protein-rich region associated with the postsynaptic dendrite. Synaptic cell adhesion molecules structurally linking the two sides of the synapse span the synaptic cleft between the presynaptic and postsynaptic regions. These molecules are thought to coordinate synapse maturation in pre- and postsynaptic compartments and also enable functional interactions between the two sides of the synapse. PSD, postsynaptic density.
is thought to be balanced by synapse elimination and pruning of synaptic contacts through activitydependent mechanisms. Several lines of evidence suggest that synapse formation per se does not require neuronal activity. Initial events that establish immature synaptic contacts in neuromuscular junctions involve the interaction of axonal growth cones with target muscle membrane. In the central synapses, however, initial synapse formation is thought to take place between the axonal shaft and filopodial processes that extend from the dendrites. This type of interaction results in the formation of en passant synaptic boutons along the axonal shaft, which is a common feature of most CNS synapses. Retraction and stabilization of these filopodial processes together with contacted axonal regions or nascent presynaptic terminals marks the beginning of synapse maturation. Further maturation involves structural modifications that increase the anatomical complexity of the synaptic boutons, including an increase in the number of synaptic vesicles, the size of the synaptic boutons, and, in some cases, the number of active zones. An interesting aspect of synapse maturation is the matching change in the pre- and postsynaptic regions that results in a strong correlation
between the size and complexity of both sides of a synapse. The sequence of events leading to synapse formation has recently been studied in detail using timelapse imaging techniques in dissociated hippocampal cultures. These studies took advantage of action potential-dependent, rapid synaptic vesicle recycling as the earliest indicator of synaptogenesis following the initiation of axo-dendritic contacts. These findings support the scenario that prepackaged presynaptic molecules are rapidly released at sites of axo-dendritic contact, forming functional presynaptic terminals. However, it has been previously shown that isolated synaptic vesicles in axons can also recycle in an activitydependent manner prior to target contact. This immature form of synaptic vesicle recycling proceeds with slower kinetics compared to mature synapses. In some cases, this immature form of synaptic vesicle recycling has been shown to be resistant to tetanus toxin implicating the requirement for a vesicular SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) protein other than synaptobrevin-2 (VAMP-2), which is tetanus toxin sensitive. In the developing neuromuscular junction, unlike mature junctions, synaptic vesicle recycling is highly sensitive to Brefeldin A, which disrupts synaptic vesicle trafficking thorough endosomal intermediates. Maturation of presynaptic terminals occurs in structurally and functionally distinguishable stages (Figure 2). During early synapse maturation, synapses are unresponsive to action potential stimulation or hypertonicity (which normally induces swift fusion of docked vesicles), although they can release neurotransmitters and recycle synaptic vesicles during strong stimulation such as induced by elevated extracellular potassium. This form of vesicle recycling detected in these nascent synapses is mechanistically analogous to the form observed in the absence of critical components of the synaptic SNARE machinery. This is consistent with the observation that synaptic vesicle recycling at this stage is tetanus toxin insensitive. Following this initial stage, synapses undergo a transition to become responsive to action potential stimulation and rapidly recycle synaptic vesicles. Examination of electron micrographs of nascent synapses reveals a strong correlation between this functional switch and the formation of the active zone, leading to the assembly of the RRP. Indeed electron microscopic analysis of immature hippocampal cultures shows that synaptic vesicles are not as closely associated with plasma membrane as they are in mature synapses. These vesicles recycle in a calcium-dependent manner as they travel in the axon, indicating that basic machinery needed for
Presynaptic Development: Functional and Morphological Organization 13
SVs
AZ
Docked pool Synapse maturation
Synapse maturation
Figure 2 Sequence of events leading to maturation of presynaptic terminals during synaptic development. Nascent synapses typically contain synaptic vesicles that recycle only in response to strong stimulation. However, at these early stages of synapse assembly, vesicles are not tightly associated with the plasma membrane, presumably due to the immature state of active zones. These synapses lack a set of readily releasable vesicles; thus, they do not effectively respond to presynaptic action potentials. Formation of an active zone coincides with functional maturation of a presynaptic terminal. This stage may also involve actin cytoskeleton increasing association of the synaptic vesicle clusters with the active zone and the surface membrane release machinery. After these initial stages, synapse maturation involves a gradual increase in the size of the total vesicle pool, which also reflected an increase in the number of vesicles available for release. SV, synaptic vesicle; AZ, active zone.
docking and fusion is distributed loosely along the axon. Nevertheless, these hot spots of glutamate release sites may have an important role during the initial stages of synaptogenesis. The released glutamate can stimulate filopodial motility of both dendrites and axons, thereby increasing the chance of axo-dendritic encounter. However, in more mature cultures, glutamate strongly inhibits filopodial motility and stabilizes connections. These contradictory observations can be reconciled if during development changes in protein expression make filopodia less responsive to glutamate, or, alternatively, there is a concentration threshold for glutamate above which its activity has opposite effects.
Besides glutamate, many other secreted molecules increase the number and motility of dendritic and axonal filopodia prior to contact. These include brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), which have been shown to promote axonal arborization, dendritic growth, and synapse maturation. BDNF-coated beads can increase Ca2þ levels and trigger neurotransmitter release in contacting axons in a protein synthesis-dependent manner. Some members of the fibroblast growth factor (FGF) family of proteins (e.g., FGF22, FGF7, FGF10) can induce presynaptic organization via FGF receptor2. In a recent study, Sanes and colleagues used a combination of chromatographic steps to isolate factors that cause axon branching and vesicle aggregation in chick motorneurons and purified FGF22 as an active component. Secreted Wnt proteins (Wnt-7a, Wnt-3) can also induce remodeling of growth cones and accumulation of synaptic vesicles. Wnt-7 knockout mice show a delay in maturation of multisynaptic glomerular rosettes formed between mossy fibers and granule cells in the cerebellum. Deficiency of a Wnt homolog in Drosophila, Wingless (Wg), causes abnormal preand postsynaptic differentiation indicating possible conservation of function. The peak of synapse formation in the mammalian brain coincides with extensive proliferation of glia, in particular astrocytes. Astrocytic protrusions engulf potential contact sites between axonal and dendritic processes, implicating their possible role in synaptogenesis. Astrocytes release several factors that impact synaptic differentiation. Initial studies by Pfrieger and Barres showed that in the absence of glia the number of synapses formed between retinal ganglion cells in culture are reduced and individual synapses are less efficient in neurotransmitter release. Later work by Pfrieger and colleagues led to isolation of Apolipoprotein E (Apo-E) as the factor upregulated in the presence of glia. Apo-E is a carrier for cholesterol. In these experiments, application of cholesterol to retinal cultures caused a massive increase in spontaneous excitatory postsynaptic currents and the number of presynaptic nerve terminals. In parallel, Barres and colleagues identified thrombospondins 1 and 2 as additional glia-derived synaptogenic molecules, which specifically cause an increase in the number of synapses. Thrombospondins are multidomain extracellular matrix proteins, initially identified in platelet activation.
Role of Synaptic Cell Adhesion Molecules in Synapse Assembly Whereas soluble factors play an important role in mediating the initial stages of vesicle aggregation and
14 Presynaptic Development: Functional and Morphological Organization
priming axons for synapse assembly, synaptic cell adhesion molecules mediate the physical contact and functional communication between axonal and dendritic protrusions leading to the formation of synaptic junctions. These molecules are composed of several large families, which include N-cadherins, protocadherins, neural cell adhesion molecules (NCAMs), nectins, neurexins, and neuroligins. In most cases, extensive alternative splicing and differential glycosylation patterns create enormous variability in the possible repertoire of protein products. This high level of variety in individual protein products and the large number of combinatorial possibilities for intermolecular interactions between these molecules may contribute to the specificity of synaptic connections in the brain. However, the process of synapse formation itself seems to be somewhat promiscuous as evidenced by three observations. First, injury or degeneration in the brain can trigger extensive synaptic rewiring, which leads to formation of ectopic synapses between cells that do not normally make synapses with each other. Second, neurons in dissociated cultures form synapses rather promiscuously, where, in some conditions, cells can even form autapses with themselves regardless of their proper in vivo partners. Finally, axonal contacts onto polylysine-coated glass beads can induce assembly of presynaptic specializations. Taken together, these results indicate that the basic mechanism for synapse formation is inherent to all neurons. Furthermore, these observations suggest that synaptogenesis is not an event triggered by a single molecule, but rather a vast repertoire of molecular interactions that can lead to synaptogenesis between neurons. Activitydependent processes test these connections over time and direct stabilization of the most resilient connections. Neuroligin is the first adhesion molecule identified to be a direct inducer of presynaptic terminal assembly. When expressed in nonneuronal cells, they can cause an accumulation of presynaptic vesicle clusters on contacting axons. These synapses are fully functional as demonstrated by fluorescence imaging of synaptic vesicle recycling and electrophysiological detection of neurotransmitter release. The soluble extracellular domain of neurexin can mask this effect of neuroligin, indicating that neurexin is the mediator of the synapse-inducing activity of neuroligins within the presynaptic axon. An attractive part of the neurexin–neuroligin system is that it satisfies the necessary asymmetry required to induce different signaling events on pre- and postsynaptic sides of the synapse. Neurexins are primarily associated with the presynaptic site and intracellularly bind to calcium/ calmodulin-dependent serine protein kinase (CASK) and syntenin. In contrast, neuroligins are located on
the postsynaptic site and their C-termini interact with PSD-95. SynCAM is the only other molecule shown to be sufficient to induce synapse formation when expressed in nonneuronal cells. It is a member of the immunoglobulin (Ig) superfamily, and mediates homophilic interaction through extracellular Ig domains. Similar to neuroligin, synapses induced in vitro by SynCAM are fully functional. Overexpression of SynCAM in dissociated cultures has a dominant positive effect in the functioning and formation of synapses. In contrast, the overexpression of dominant negative SynCAM lacking extracellular domain impairs presynaptic assembly. Unlike the neurexin– neuroligin interaction, SynCAM mediates a homophilic interaction, indicating that different downstream cues are present on different sides of the synapse (Figure 3). How does the interaction of cell adhesion molecules translate into changes within the cell? Following initial contact, assembly of synapses takes 1–2 h and may occur in either of two ways. First, following contact, each molecule could be captured from the stream of axonal cytoplasm, and a synapse can be built depending on protein–protein interactions. A second model suggests that synaptic molecules are pre-assembled in small units in other parts of the neuron and transported to the axon. The speed of assembly favors a pre-assembled trafficking model. The evidence for the existence of cytoplasmic transport packages came from studies of Garner and colleagues. They have been able to isolate large dense vesicles from developing axons containing active zone proteins such as piccolo, bassoon, and RIM. Pre-assembled packages of active zone vesicles fuse with the axonal plasma membrane to create a scaffolding framework for other components. In addition to the active zone components, synaptic vesicles also assemble as clusters in a unitary fashion. Nevertheless, these two components do not seem to travel together along the axon. There is also evidence that at the end of this initial stage of synapse assembly, synaptic vesicles switch to a docked state and associate more closely with the plasma membrane in a synapsin-dependent manner. Analysis of the intracellular interactions of neurexin and SynCAM reveals CASK as a converging downstream target. CASK is a member of the membrane-associated guanylate kinase family (MAGUK) and strongly interacts with neurexin and SynCAM cytoplasmic tail. Upon interaction with neuroligin, neurexin oligomerizes and recruits CASK. Given the multidomain structure of CASK, it is usually envisaged as a recruiter to the newly formed contact sites for both neurexin and SynCAM. CASK also
Presynaptic Development: Functional and Morphological Organization 15
Piccolo Bassoon
Rab-3 RIM Mint
Veli
munc-18 CASK
liprin
ERC
SNARES
p4.1
N-type Ca2+ channel Lar RTK Neurexin
Syndecan
Neuroligin
SynCAM
SynCAM
Figure 3 Putative molecular interactions that link synaptic cell adhesion molecules to the active zone and to the neurotransmitter release machinery. Cytoplasmic tails of neurexins and SynCAM can interact with a modular adaptor protein CASK which in turn binds another adaptor protein Mint interacting with the release machinery (munc-18 which interacts with syntaxin) as well as the voltage-gated calcium channels. Active zone components such as RIM can interact with synaptic vesicle-associated molecules, which include rab3, thus bridging synaptic junctional scaffold to the synaptic vesicle cluster.
forms a well-conserved tripartite complex with Mint and Veli, multidomain PDZ molecules. This complex is proposed to be responsible for the recruitment of vesicle fusion machinery. CASK has also been shown to interact with liprin, which organizes the presynaptic active zone in Caenorhabditis elegans. By tightly interacting with the active zone proteins RIM and ERC, liprins constitute the insoluble backbone of the active zone. As mentioned above, synaptic vesicles are tethered at the vicinity of the active zone by the actin cytoskeleton. Actin depolymerizing agents have a strong disruptive effect on nascent synapses but not on mature synapses, implying a role for actin during synapse formation. CASK can polymerize actin on the neurexin C-tail and stabilize it by interacting with protein 4.1. In this way, synaptic adhesion molecules neurexin and SynCAM (and possibly other CASKinteracting adhesion molecules such as syndecans) can induce local polymerization of actin at contact sites and trap traveling synaptic components. As discussed above, free-moving vesicle clusters have different cycling properties than mature ones. How maturation changes vesicle identity is not known. It could be achieved through transport of mature vesicles or conversion of the identity of
existing vesicles. One can speculate that these initial synaptic vesicles are still present in mature synapses but in a reduced capacity for synaptic vesicle recycling. This may explain the presence of the enormous number of vesicles at synapses, while only a fraction of them are functional. There are a multitude of possible pathways that can lead to eventual assembly of synaptic terminals. This redundancy can increase the robustness of the synapse assembly process and also contribute to the functional and structural versatility of synapses.
Stabilization of Synapses Synapse formation is rather error prone at the initial stage, and a certain degree of mismatch often occurs. Therefore, initial promiscuous synapses either are usually secured by help of additional and more specific adhesion molecules, or are eliminated. Like previous stages, this process also occurs in a hierarchical manner. The synaptogenic molecules neurexin and neuroligin could be good candidates for helping to achieve this specificity. Neurexins undergoes extensive splicing, and recently Boucard and colleagues demonstrated that they interact according to a splice code. However, several other cell adhesion molecules
16 Presynaptic Development: Functional and Morphological Organization
are also postulated to play a role in late stages of synaptic development. One of the best-characterized synaptic cell adhesion proteins is N-cadherin. Similar to neurexins, they link the extracellular adhesive function to actin cytoskeleton via a- and b-catenins. Even though the function of cadherins at the synapse is not clear, several recent experiments provide significant insights. Overexpression of an N-cadherin construct lacking the extracellular domain while maintaining the ability to bind cytosolic partners markedly reduced the number of presynaptic boutons, indicating the importance of the adhesive function. In another set of experiments, expression of mutant a-N-catenin prevented the interaction of cadherin with the actin cytoskeleton but did not strongly affect presynaptic assembly. These experiments imply that cadherins have a rather adhesive function during early synaptogenesis. Therefore, they could act at an intermediate stage between initial contact and final maturation by prolonging the brief lifetime of axo-dendritic contacts. Disruption of cadherin function in mature synapses, however, does not have a strong effect. This finding raises the possibility that some of the cadherin functions are redundant with protocadherins, a subset of the cadherin superfamily. Protocadherins are composed of nearly 60 members expressed by three gene clusters that are expressed in distinct patterns in the nervous system and undergo extensive splicing.
Molecular Components of the Presynaptic Active Zone and the Cytomatrix Active zones are the principal sites of synaptic vesicle fusion in synapses. The molecular components of the active zone are thought to serve a structural role by clustering synaptic vesicles around the active zone and increasing proximity between molecules on the synaptic vesicle membrane and the plasma membrane. Active zone proteins are also involved in priming the vesicles for release and perhaps in vesicle retrieval after fusion. Proteins, such as Bassoon and Piccolo, are recruited to activate the synapses during synaptogenesis. For instance, in experiments conducted by Garner, Ziv, and colleagues, Bassoon was detected in nascent synapses capable of action-potential-dependent uptake and release of FM dyes. In addition, dense core active zone precursor vesicles contain multiple synaptic proteins, including the active zone proteins, Bassoon and Piccolo. Fusion of these vesicles with the plasma membrane can rapidly assemble active zone. Despite extensive data on their localization, the functional properties of these active zone proteins are still unclear. However, for RIM1 and CASK, there are several wellcharacterized biochemical interactions with multiple
proteins. As discussed above, in the case of CASK, these molecular interactions suggest a central role in the bridging of neurexins to munc-18, a critical component of presynaptic release machinery. Recent mouse knockouts of munc-13 and RIM1 uncovered critical functional roles for these molecules. Synapses deficient in munc-13-1 are severely impaired in their function. The remaining munc-13-2-dependent synaptic transmission displays marked synaptic facilitation. RIM1 knockout mice, on the other hand, have a less severe but significantly altered properties of short- and longterm plasticity. Interestingly, loss of these molecules does not lead to structural alterations in the synapse, which are presumably due to the redundancy of molecular interactions that assemble synapse structure. How do the active zone proteins regulate synaptic function? This regulation is likely achieved by the ability of active zone proteins to recruit the components of fusion machinery, such as SNAREs and munc-18. An important step in the chain of events leading to vesicle fusion is the formation of the SNARE core complex between target membrane SNARE proteins (i.e., syntaxin and SNAP-25) and the synaptic vesicle SNARE, synaptobrevin/VAMP. Active zone proteins can exert significant functional effects by regulating the formation and dissociation of SNARE complexes. Replenishment of vesicles released at the active zone requires SNARE core complex assembly and disassembly. This assembly process in the synapse is much faster than the rates of SNARE core complex assembly in vitro. Therefore, the assembly process is most likely facilitated by protein–protein interactions between the components of the presynaptic active zone and synaptic vesicles. For instance, munc-18, a protein required for fusion, could be recruited to the active zone through its interaction with Mint (munc-18 interacting protein), which in turn binds to CASK.
Functional Maturation of Presynaptic Terminals and the Role of Activity Following the initial assembly of synaptic terminals, a large number of synapses are functionally silent. In some cases, these functionally silent synapses can be rendered operational in response to activity. The most commonly studied models of silent synapses propose a postsynaptic mechanism that underlies this silence. According to this model, a fully functional presynaptic terminal may exist but the postsynaptic site does not possess a-amino-3-hydroxy-5-methyl-4isoxazolepropionic acid (AMPA) receptors although it contains N-methyl-D-aspartate (NMDA) receptors. Activity, in turn, induces the insertion of functional AMPA receptors, making silent synapses functional under physiological conditions. In contrast,
Presynaptic Development: Functional and Morphological Organization 17
studies in dissociated hippocampal cultures have also identified a developmental stage where synapses are presynaptically silent after assembly. This model possesses the same apparent features of an NMDAonly synapse, but the NMDA-only nature of these synapses is explained by a presynaptic mechanism and not by lack of postsynaptic AMPA receptors. According to this scheme, activation of postsynaptic AMPA or NMDA receptors can be determined by the kinetics of fusion pore opening and the release profile of glutamate. In young nerve terminals, neurotransmitter release occurs through a narrow fusion pore leading to exclusive activation of NMDA receptors as they have a higher affinity for glutamate. Synapse maturation in turn leads to an increase in preponderance of full fusion events, thus activating NMDA as well as AMPA receptors. An alternative model suggests that immature synapses do not readily respond to action potential stimulation leading to a full presynaptic neurotransmitter failure due to some inadequacy in fusion competence or localization of synaptic vesicles. This model is consistent with the previously discussed findings on the gradual reorganization of synaptic vesicle clusters after synaptogenesis. In a recent study, Shumin Duan and colleagues showed that a burst of action potentials can rapidly awaken these silent synapses by increasing the availability of synaptic vesicles for fusion through BDNF-triggered presynaptic actin remodeling mediated by the small GTPase Cdc42. In most synapses, the initial structural assembly and functional unsilencing is followed by a gradual maturation process that typically involves alterations in short-term plasticity. Studies conducted in acutely isolated brain slices have described functional alterations that are solely of presynaptic origin. One finding of these experiments was an apparent decrease in release probability during synaptic development. This result is rather surprising, given the prevalent structural observation that the number of vesicles within a synapse increases during maturation implying an increase in synaptic reliability and release. Another interesting observation in cortical as well as hippocampal mossy fiber synapses is target-dependent alterations in short-term and some long-term forms of plasticity during the course of development. Cellular mechanisms underlying these developmental changes in short-term plasticity are postulated to involve altered Ca2þ dependence of fusion and regulation of vesicle mobilization in presynaptic terminals. It is tempting to speculate that synaptic cell adhesion molecules either individually or in combination may regulate these target specific functional alterations in the output of single neurons.
As exemplified by the presynaptic unsilencing process discussed above, several aspects of synaptic functional maturation during early development can be influenced by activity and neuromodulators. Most neuronal networks exhibit spontaneous action potential firing patterns and synaptic potentials in the absence of extrinsic influences. The background activity that arises from the properties of individual neurons and their characteristic synaptic connections has been shown to be critical for the refinement of synaptic connectivity within the nervous system. Most of the signaling cascades that play a role in synapse maturation can be physiologically activated or regulated by the background activity. These include several signal transduction pathways, including Ca2þ-signaling mechanisms and the activation of protein kinase C (PKC) and protein kinase A (PKA). For example, direct involvement of cyclic adenosine monophosphate (cAMP)-dependent signaling in synaptic development was demonstrated in hippocampal slices, as well as at the level of individual synapses in culture. Activation of Ca2þ, cAMP, or diacylglycerol second messenger cascades can be triggered either directly by neuronal activity through Ca2þ influx or indirectly by the release of glutamate and activation of metabotropic glutamate receptors. Recent studies have shown that chronic alterations in spontaneous activity levels modify several synaptic properties including the size of postsynaptic responses, probability of neurotransmitter release, as well as the number of synapses. These experiments strongly support a role for background activity in regulating the proper functional maturation of individual synapses. Although activity is an indispensable component of synaptic development, the mechanism through which it influences synapse maturation and the elimination process is still elusive. See also: Cell Adhesion Molecules at Synapses; Endocytosis and Presynaptic Scaffolds; Postsynaptic Density/ Architecture at Excitatory Synapses; SNAREs; Synaptic Vesicles.
Further Reading Ahmari SE, Buchanan J, and Smith SJ (2000) Assembly of presynaptic active zones from cytoplasmic transport packets. Nature Neuroscience 3: 445–451. Akins MR and Biederer T (2006) Cell–cell interactions in synaptogenesis. Current Opinion in Neurobiology 16: 83–89. Biederer T, Sara Y, Mozhayeva M, et al. (2002) SynCAM, a synaptic adhesion molecule that drives synapse assembly. Science 297: 1525–1531. Craig AM, Graf ER, and Linhoff MW (2006) How to build a central synapse: Clues from cell culture. Trends in Neurosciences 29: 8–20.
18 Presynaptic Development: Functional and Morphological Organization Goda Y and Davis GW (2003) Mechanisms of synapse assembly and disassembly. Neuron 40: 243–264. Mozhayeva MG, Sara Y, Liu X, and Kavalali ET (2002) Development of vesicle pools during maturation of hippocampal synapses. Journal of Neuroscience 22: 654–665. Scheiffele P (2003) Cell–cell signaling during synapse formation in the CNS. Annual Review of Neuroscience 26: 485–508. Shen W, Wu B, Zhang Z, et al. (2006) Activity-induced rapid synaptic maturation mediated by presynaptic cdc42 signaling. Neuron 50: 401–414.
Sudhof TC (2001) The synaptic cleft and synaptic cell adhesion. In: Cowan WMST and Stevens CF (eds.) Synapses, pp. 275–313. Baltimore: The Johns Hopkins University Press. Ziv NE and Garner CC (2004) Cellular and molecular mechanisms of presynaptic assembly. Nature Reviews Neuroscience 5: 385–399.
Postsynaptic Development: Neuronal Molecular Scaffolds E Kim, Korea Advanced Institute of Science and Technology, Daejeon, South Korea ã 2009 Elsevier Ltd. All rights reserved.
Introduction Neuronal synapses generally fall into two groups, excitatory and inhibitory. Excitatory synapses are mostly present in dendritic spines, which are thornlike structures on dendrites. Inhibitory synapses are located on dendritic shafts and the cell body. Excitatory synapses are characterized by the presence of an electron-dense thickening in the postsynaptic side known as the postsynaptic density (PSD). The PSD is formed through the assembly of macromolecular postsynaptic protein complexes containing receptors, scaffolds, and signaling proteins. Postsynaptic development at excitatory synapses is thought to involve an initial axodendritic contact, followed by localization of early postsynaptic proteins and recruitment of additional proteins to permit growth into mature postsynaptic structures. During the past decade, mechanisms underling the assembly and molecular organization of excitatory neuronal synapses have been thoroughly studied. Inhibitory neuronal synapses have received relatively less attention, partly because the wealth of proteins in the PSD has attracted scientific interest. This article focuses on postsynaptic differentiation at excitatory synapses. In particular, it discusses how postsynaptic scaffolds contribute to the assembly, organization, and plasticity of the PSD.
Postsynaptic Scaffolding Proteins Components of the PSD have been identified by various experimental approaches including mass spectrometry and the use of protein interaction traps such as the yeast two-hybrid system. Proteomic approaches have identified hundreds of PSD components, including membrane proteins, scaffolding proteins, signaling proteins, and cytoskeletal proteins. Proteomic studies have further provided information on relative and absolute amounts the PSD proteins and their phosphorylation. Here, postsynaptic scaffolding proteins are defined as proteins that are relatively more abundant than are other PSD proteins and that possess various domains for protein–protein interactions. The presence of such domains strongly implies that the proteins are implicated in the assembly and organization of the PSD. Many postsynaptic scaffolds contain the PDZ
(PSD-95-Dlg-ZO1) domain, a 90-amino-acid-long module that interacts with the C-terminal PDZ-binding motif of other proteins. The PDZ domain is one of the most common protein domains known, being found in approximately 580 proteins encoded by the mouse genome. PDZ proteins exhibit 1–13 tandem arrays of PDZ domains. PDZ domain-binding motifs are found in a wide variety of proteins, including membrane proteins and signaling proteins. PDZ proteins, due to their ability to interact with other proteins and form macromolecular protein complexes, are found mainly in specialized cell-to-cell junctions, including neuronal synapses and tight junctions. A well-known PDZ-containing postsynaptic scaffold is PSD-95, which is a family of proteins with four known members: PSD-95/SAP90, PSD-93/chapsyn-110, SAP97, and SAP102. PSD-95 family proteins contain various domains for protein–protein interactions, including three PDZ domains, one SH3 domain, and one GK domain. Splice variants of PSD-95, PSD-93, and SAP97 (PSD-95b, PSD-93z, and SAP97b) contain an additional domain, L27, at the N-terminus. Through these domains, PSD-95 binds to a wide variety of proteins and is involved in the assembly and molecular organization of the PSD. Proteomic studies have shown that PSD-95 is one of the most abundant proteins in the PSD. The four members of the PSD-95 family seem to differ in function, with PSD-95 and PSD-93 being more important in synapses and SAP97 and SAP102 playing roles in protein trafficking. Functionally, transient overexpression of PSD-95 in dissociated neurons increases the number and size of dendritic spines and a-amino-3-hydroxy-5-methylisoxazole propionic acid (AMPA) glutamate receptormediated synaptic transmission. In hippocampal slices, PSD-95 overexpression drives GluR1 AMPA receptors into synapses, occludes long-term potentiation (LTP), and enhances long-term depression (LTD). Conversely, acute PSD-95 knockdown reduces the ratio of excitatory to inhibitory synapses and suppresses AMPA receptor-mediated synaptic transmission. Transgenic mice with truncated PSD-95 exhibit reduced LTD, enhanced LTP, and impaired spatial learning. These knockout mice show unaltered AMPA receptormediated currents, in contrast to acute PSD-95 knockdown, probably due to functional compensation by other PSD-95 family proteins. Indeed, doubleknockout mice lacking both PSD-95 and PSD-93 show a markedly reduced ratio of AMPA/N-methylD-aspartate (NMDA) excitatory postsynaptic currents. Together, these results implicate PSD-95 in the
19
20 Postsynaptic Development: Neuronal Molecular Scaffolds PSD-95
GK
SH3
PSD-93 SAP97b
GK
SH3
SAP102
GK
SH3
GKAP
GH1
Shank SynGAP
GK
SH3 L27
An k PH
C2
S-SCAM
SH3
SAM
RasGAP WW
GK
GRIP/ABP PICK1
BAR
SPAR CASK/LIN2 IRSp53 GIT1
RapGAP
Act1 CaM kinase RCB
L27 CRIB
ArfGAP A n k
SHD
L27
SH3
Act2
GKBD
GK
SH3 GRKBD
Neurabin
SAM
Spinophilin Densin-180
LRR
Syntenin Homer1b
EVH1
CC
Figure 1 Schematic diagram of PSD proteins. Domain structures of selected PSD proteins. PDZ domains are shown as dark ellipses. Other domains are indicated: Act1, actin regulatory domain 1; Act2, actin regulatory domain 2; Ank, ankyrin repeats; ArfGAP, Arf GTPaseactivating protein; BAR, Bin-Amphiphysin-Rvs domain; C2, calcium/lipid binding domain 2; CaM kinase, Ca2þ/calmodulin-dependent kinase (CaMK)-like domain; CC; coiled coil domain; CRIB, Cdc42/Rac-interactive binding; EVH1, ENA/VASP homology domain 1; GH1, GKAP homology domain 1; GK, guanylate kinase-like domain; GKBD, PSD-95 GK binding domain; GRKBD, GRK2 binding domain; kinase, serine/threonine kinase domain; L27, domain initially found in LIN2 and LIN7; PH, pleckstrin homology domain; RapGAP, Rap GTPase-activating protein; RasGAP, Ras GTPase-activating protein; RCB, Rac binding domain; SAM, sterile a motif; SH3, Src homology 3 domain; SHD, Spa2 homology domain; WW, domain with two conserved Trp (W) residues. Proteins: CASK/LIN2; vertebrate homolog of lin2; GIT1, GRK-interacting protein 1; GKAP, GK-associated protein; GRIP/ABP, glutamate receptor interacting protein/AMPA receptor binding protein; IRSp53, insulin receptor tyrosine kinase substrate p53; PICK1, protein interacting with C-kinase; PSD-93, postsynaptic density protein 93; PSD-95, postsynaptic density protein 95; SAP97, synapse-associated protein 97; SAP102, synapse-associated protein 102; Shank, SH3 and ankyrin repeat-containing protein; SPAR, spine-associated RapGAP; S-SCAM, synaptic scaffolding molecule.
regulation of excitatory synapses, dendritic spines, synaptic strength and plasticity, and learning and memory. Other postsynaptic scaffolds implicated in postsynaptic development are listed in Figure 1. A large number of these scaffolds contain PDZ domains, as does PSD-95, suggesting that PDZ-based interaction is a widespread mechanism for postsynaptic assembly and organization.
Principles Governing Postsynaptic Differentiation An important question in synapse formation concerns which side of the synapse initiates synapse formation. Some published studies indicate that the assembly of functional nerve terminals precedes postsynaptic differentiation. Time-lapse microscopy and retrospective immunostaining on cultured hippocampal neurons (11–14 days in vitro (div)) indicate that
contact-induced formation of functional nerve terminals is followed by clustering of postsynaptic proteins including PSD-95 and glutamate receptors in approximately 45 min. Early stage hippocampal neurons (5–7 div) contain nonsynaptic mobile packets with postsynaptic scaffolds such as PSD-95, GKAP/ SAPAP, and Shank/ProSAP. Some of these packets are fast moving and can be recruited to PSD-95/GKAPnegative nascent synapses apposed to functional nerve terminals, suggesting that the presynaptic side instructs postsynaptic assembly. However, a proportion of these mobile packets move slowly. These packets contain neuroligin-1, and when they contact axons, they induce functional nerve terminals by recruiting mobile synaptophysin clusters. This suggests that a certain degree of postsynaptic assembly also occurs prior to the formation of functional nerve terminals, and that synaptogenesis occurs in a bidirectional manner. Presynaptic assembly is thought to be mediated by vesicular intermediates including synaptic vesicle (SV)
Postsynaptic Development: Neuronal Molecular Scaffolds 21
precursors and specialized dense-core vesicles known as Piccolo/Bassoon transport vesicles (PTVs). These two types of vesicles carry preassembled complexes of SV proteins and active zone components (scaffolds and plasma membrane proteins), respectively. Notably, active zones are assembled from a small number (typically two or three) of PTVs. A related question is whether postsynaptic assembly also occurs in this manner. Early stage (3 or 4 div) cortical neurons exhibit rapidly moving NMDA and AMPA receptor transport packets similar to the mobile packets of early hippocampal neurons (mentioned previously). This suggests that modular transport of postsynaptic proteins may play a role in early synaptogenesis. However, late-stage (8–13 div) hippocampal neurons exhibit a gradual recruitment of postsynaptic proteins, including NMDA receptors, PSD-95, and Shank. Although reasons for this discrepancy remain to be determined, a possible explanation is that neurons at different developmental stages may have distinct mechanisms of postsynaptic protein assembly.
Induction of Postsynaptic Differentiation When the presynaptic side induces postsynaptic differentiation, what might be the initiating signals? Neurotransmitters, released from nerve terminals, are good candidates. Neurotransmitters may affect the morphology of dendritic spines and filopodia (long thin protrusions on dendrites), suggesting that the neurotransmitters may instruct synapse formation. However, transgenic mice that cannot release neurotransmitters, due to the absence of the presynaptic protein Munc13 or Munc18, exhibit normal synapse formation. In addition, blockade of neurotransmitter release in cultured neurons does not inhibit synapse development. These data suggest that neuronal activity is not required for synapse formation. Another possible trigger is transsynaptic adhesion between presynaptic and postsynaptic cell adhesion molecules. Ideally, transsynaptic adhesions should be heterophilic to minimize homophilic adhesions between dendrites and axons. In addition, synaptic adhesion molecules need to permit coupling of adhesion events to induction of synaptic differentiation through the recruitment of various synaptic proteins to the sites of contact. A well-known example of heterophilic and synaptogenic transsynaptic adhesion is that between presynaptic neurexins and postsynaptic neuroligins. Neurexins were originally identified as receptors for a-latrotoxin, a potent neurotoxin from black widow spider venom that induces massive neurotransmitter release from nerve terminals. Subsequently, neuroligin was identified as an endogenous postsynaptic ligand of neurexins. In addition,
neuroligin was found to directly interact with the PDZ domains of PSD-95 through the C-terminal tail, providing a novel mechanism of synapse formation. The neurexin–neuroligin complex promotes synapse formation in a bidirectional manner. Neuroligin expressed in nonneural cells induces presynaptic differentiation in contacting axons of co-cultured neurons. Conversely, neurexin presented on nonneural cells or beads induces the clustering of key postsynaptic proteins in contacting dendrites. Direct aggregation of neuroligins on the surface membrane of dendrites induces similar clustering of postsynaptic proteins. Acute knockdown of neuroligins reduces the number and function of synapses. These results suggest that neuroligin is a key mediator of synapse formation. Interestingly, transgenic mice deficient in three neuroligins (mice with triple knockout mutations of neuroligin-1, -2, and -3) exhibit reductions in synaptic transmission, but their synapse number is not affected. This suggests that neuroligins regulate functional maturation of synapses rather than their formation, although further work in this area is required. An interesting feature of the neuroligin family is that neuroligin isoforms differentially localize to excitatory and inhibitory synapses. Specifically, neuroligin-2 is mainly found at inhibitory synapses, whereas other neuroligin isoforms are detected at excitatory synapses. Consistent with these observations, direct aggregation of neuroligin-2 on dendrites induces the clustering of gephyrin, an inhibitory postsynaptic scaffold. Acute knockdown of neuroligin-1, -2, and -3 in cultured neurons results in a greater reduction in the function of inhibitory synapses than excitatory synapses. In addition, the neuroligin triple-knockout mice show an increase in the ratio of excitatory to inhibitory synapses. These results suggest that neuroligin-2 regulates inhibitory synapse formation and/or function. SynCAM is another family of synaptic cell adhesion molecules implicated in excitatory synaptic differentiation. SynCAM family members, expressed in nonneural cells, induce the formation of functional nerve terminals in contacting axons. These terminals have release properties similar to those seen in regular synapses. The C-terminus of SynCAM contains a PDZ-binding motif and associates with synaptic PDZ proteins, including CASK/LIN2 and syntenin. This, together with data from studies on neuroligin (described previously), suggests that synaptic differentiation is mediated by synaptic scaffolds that couple synaptic adhesion events to the recruitment of various synaptic proteins. When the great diversity of neuronal synapses is considered, synaptogenic adhesion molecules other than neuroligin and SynCAM may act in concert
22 Postsynaptic Development: Neuronal Molecular Scaffolds
with postsynaptic scaffolds. NGL, a family of adhesion molecules, interacts with PSD-95 through the C-terminus in a manner similar to the neuroligin– PSD-95 interaction. The extracellular domain of NGL associates with netrin-G/laminet, a GPI-anchored adhesion molecule. The complex of netrin-G, NGL, and PSD-95 is implicated in the regulation of excitatory synapse formation. In support of this idea, NGL presented on nonneural cells or on beads induces presynaptic differentiation in contacting axons. Direct aggregation of NGL on the dendritic surface induces postsynaptic protein clustering. The fact that both neuroligin and NGL associate with PSD-95 suggests that PSD-95 is one of the key postsynaptic scaffolds involved in adhesion-dependent postsynaptic differentiation. In addition, the dual association of PSD-95 with neuroligin and NGL suggests that these two adhesion molecules may have physical or functional interactions. Extracellular factors capable of inducing aspects of postsynaptic differentiation at excitatory synapses include Narp, a secreted immediate early gene product upregulated by synaptic activity, and ephrin, a ligand of EphB receptor tyrosine kinases. Narp presented on nonneural cells induces the clustering of AMPA receptors, but not of NMDA receptors, in contacting dendrites. Narp interacts with the extracellular N-terminal domain of AMPA receptors. Narp induces AMPA receptor clustering at shaft synapses of aspiny neurons, and this leads to secondary NMDA receptor clustering, probably through stargazin and PSD-95. Ephrin activation of EphB receptor tyrosine kinases induces co-clustering of EphB and NMDA receptors. This interaction is mediated by their extracellular domains and does not require the kinase activity of EphB receptors.
Localization and Organization of Postsynaptic Scaffolds If the clustering of postsynaptic adhesion molecules on dendrites is the beginning of postsynaptic differentiation, how might subsequent postsynaptic protein clustering occur? A possibility is that adhesioninduced primary clustering of neuroligin on dendrites may promote secondary clustering of PSD-95 though the C-terminal PDZ interaction, which would lead to additional recruitment of PSD-95-associated proteins. Against this, however, is the observation that while neuroligin-1 selectively binds to the third PDZ domain of PSD-95, synaptic localization of PSD-95 requires the first two PDZ domains but not the third. Notably, NGL, another adhesion molecule binding to both of the first two PDZ domains of PSD-95 but not to the third, enhances synaptic localization of PSD-95.
PSD-95 occurs in molar excess relative to other postsynaptic proteins. Therefore, instead of depending on its synaptic localization on the interaction with other proteins, PSD-95 might increase its cluster size at synapses by self-multimerization. In support of this notion, PSD-95 forms multimers through both N- and C-terminal domains. The expansion of PSD-95 multimers is more likely to be lateral than vertical because, ultrastructurally, PSD-95 mainly localizes to regions close to the postsynaptic membrane. The lateral expansion might be aided by PSD-95 interaction with membrane proteins and/or palmitoylation (a lipid modification promoting membrane attachment). Indeed, PSD-95 co-expressed with membrane proteins in heterologous cells forms large surface clusters in which both proteins are co-localized. In addition, mutations of PSD-95 that block palmitoylation eliminate membrane protein clustering by PSD-95. PSD-95 is likely to recruit other postsynaptic scaffolds, including GKAP and Shank. The C-terminal GK domain of PSD-95 associates with GKAP, and the C-terminal PDZ-binding motif of GKAP further associates with Shank. GKAP and Shank are relatively abundant in deeper layers of the PSD and contain various domains for protein–protein interactions. Shank further associates with Homer, which is in turn linked to metabotropic glutamate receptors and IP3 receptors. Shank promotes spine maturation by mechanisms requiring synaptic Homer recruitment. In support of a possible role for PSD-95 in the recruitment of GKAP and Shank, a GKAP mutant lacking the ability to bind to PSD-95 induces the aggregation and degradation of Shank.
Synaptic Adhesion Molecules Synaptically localized PSD-95 may reversely promote postsynaptic localization of adhesion molecules such as neuroligins or other postsynaptic adhesion molecules, further stabilizing synapse adhesion and promoting presynaptic differentiation. Indeed, overexpression of PSD-95 in cultured neurons concentrates neuroligin-1 and NGL at excitatory synapses. Importantly, PSD-95 induces the translocation of neuroligin-2 from inhibitory to excitatory synapses. Because neuroligin-2 induces presynaptic differentiation at both excitatory and inhibitory synapses, this translocation is likely to increase the number of excitatory synapses at the expense of inhibitory synapses. Therefore, the relative amounts of neuroligin-2 and PSD-95 in a single neuron may determine the ratio of excitatory and inhibitory synapses. Although PSD-95 concentrates neuroligin at excitatory synapses, this does not seem to involve a direct interaction between the two proteins because a
Postsynaptic Development: Neuronal Molecular Scaffolds 23
neuroligin-1 mutant lacking the PSD-95-binding Cterminus is normally targeted to excitatory synapses. Instead, synaptic neuroligin-1 localization requires a membrane-proximal domain in the cytoplasmic domain. This suggests that neuroligin does not depend on its binding either to PSD-95 or to presynaptic neurexins for synaptic localization, and that there is a mechanism that precedes neuroligin localization for early postsynaptic differentiation.
Postsynaptic Receptors Postsynaptic differentiation at excitatory synapses involves synaptic localization and local trafficking of NMDA and AMPA glutamate receptors. NMDA receptors are targeted to synapses at early stages of development, in contrast to AMPA receptors, thus forming NMDA receptor-only silent synapses. PSD-95 may contribute to synaptic localization of NMDA receptors by interaction with NR2 subunits. In support of this idea, C-terminal casein kinase II phosphorylation of NR2B on Ser1480 within the C-terminal PDZ-binding motif disrupts PSD-95 interaction with NR2B and decreases surface expression of NR2B. PSD-95 coexpression slows the internalization rate of chimeras of Tac (a surface membrane protein) containing the distal tail of NR2B. However, an NR2B mutant that lacks the ability to bind to both PSD-95 and AP2, a clathrin adaptor complex, is retained in the synapse, suggesting that PSD-95 binding may not be important for synaptic localization. In addition, mobile NMDA receptor transport packets in early stage neurons can be recruited to nascent synapses lacking PSD-95. In contrast to NR2B, NR2A does not depend on PSD-95 interaction for synaptic localization, suggesting subunit-specific rules for NMDA receptor trafficking. Postsynaptic adhesion molecules may regulate synaptic NMDA receptor clustering. A mutant neuroligin-1 that lacks PSD-95 binding ability no longer induces PSD-95 clustering in cultured neurons but retains the ability to cluster NMDA receptors, although weaker than that afforded by wild-type neuroligin-1. This suggests that neuroligin is capable of recruiting NMDA receptors through PSD-95-independent mechanisms. SALM, a PSD-95-interacting family of synaptic adhesion-like molecules, exhibits NMDA receptor clustering activity. SALM1, a member of this family, directly associates with the NR1 subunit and promotes dendritic clustering of NMDA receptors through mechanisms requiring the PDZ-binding C-terminus. SALM2 forms a complex with both NMDA and AMPA receptors, and direct aggregation of SALM2 on dendrites induces co-clustering of NMDA and AMPA receptors.
Synaptic localization of AMPA receptors is regulated by stargazin/TARP (transmembrane AMPA receptor regulatory protein), which directly associates with both AMPA receptors and PSD-95. Stargazin traffics AMPA receptors to synapses via two distinct mechanisms. Stargazin induces surface expression of AMPA receptors and also facilitates synaptic docking of the stargazin–AMPA receptor complex. The latter mechanism depends on binding of the stargazin C-terminus to PSD-95. In support of a role for PSD-95 in synaptic localization of stargazin and AMPA receptors, a stargazin mutant that lacks PSD-95 binding rescues surface AMPA receptor responses, but not synaptic AMPA receptor responses, in cerebellar granule cells from stargazin-deficient stargazer mice. Other postsynaptic proteins regulating synaptic localization and trafficking of AMPA receptors include GRIP/ABP, PICK1, and N-ethylmaleimidesensitive factor (NSF). GRIP/ABP, a multi-PDZ protein, is implicated in the stabilization of AMPA receptors at the synaptic surface. Synaptic AMPA receptor localization is also regulated by various GRIP-associated proteins including liprin-a (a multidomain protein), GIT1 (a multidomain protein), and LAR (a receptor tyrosine phosphatase). PICK1, through its PDZ domain, associates with protein kinase Ca (PKCa) in addition to AMPA receptors, and it promotes synaptic delivery of PKCa. PKCa directly binds to GRIP, probably promoting the interaction between PICK1-bound PKCa and its substrate, GRIP-bound GluR2. PKC-dependent phosphorylation of GluR2 on Ser880 within the C-terminal PDZ-binding motif selectively disrupts GluR2 association with GRIP but not GluR2 association with PICK1, facilitating AMPA receptor endocytosis. NSF, an ATPase involved in membrane fusion, binds to a cytoplasmic region of AMPA receptors that is distinct from the GRIP/PICK1 binding site. NSF is implicated in maintaining synaptic AMPA receptors by disassembling the AMPA receptor–PICK1 complex and promoting AMPA receptor recycling/delivery. Interestingly, the NSF binding site in AMPA receptors overlaps with that of AP2, and the AP2–AMPA receptor interaction is required for regulated AMPA receptor endocytosis and LTD. In strong support of these in vitro results, cerebellar LTD is absent in GluR2 and PICK1 knockout mice and in two different strains of GluR2 mutant knockin mice (GluR2D7 and GluR2 K882A). GluR2D7 mice have a deletion of the last seven amino acid residues required for both GRIP and PICK1 binding, and GluR2 K882A mice carry a mutation to block PKC-dependent Ser880 phosphorylation. In addition to promotion of endocytosis, PICK1 and NSF regulate AMPA receptor exocytosis. PICK1 and NSF are required for activity-dependent
24 Postsynaptic Development: Neuronal Molecular Scaffolds
insertion of GluR2 (calcium-impermeable)-containing AMPA receptors in cerebellar granule–stellate cell synapses. In support of these in vitro observations, PICK1 knockout and GluR2D7 knockin mice lack this form of plasticity.
Other Membrane Proteins Synaptic scaffolds concentrate and cluster interacting membrane proteins at the surface membrane. Stability of membrane proteins at the synaptic surface seems to be achieved through the inhibition of their endocytosis and promotion of their exit from intracellular pools and insertion into the plasma membrane. PSD-95 inhibits the endocytosis of NMDA receptors, Kv1.4 potassium channels, and b1adrenergic receptors. In addition, PSD-95 promotes the rate of membrane insertion of NMDA receptors. A Cterminal PDZ-binding motif in NR1-3, a splice variant of NR1, suppresses NR1-3 retention at the endoplasmic reticulum (ER), suggesting that a PDZ protein promotes the ER exit and surface expression of NMDA receptors. However, there are examples of the opposite situation, in which scaffolding proteins enhance endocytosis. S-SCAM/MAGI-2, a synaptic multi-PDZ protein, enhances the endocytosis of b1adrenergic receptors, and PICK1 is involved in AMPA receptor internalization, as described previously. Finally, functional properties of membrane proteins can be directly regulated by their interaction with scaffolds. PSD-95 suppresses single-channel conductance of the Kir2.3 potassium channel and increases the channel opening rate of NMDA receptors.
Signaling Pathways Another key event in postsynaptic development is the establishment of signaling pathways in the PSD. A suggested role for synaptic scaffolds is to couple upstream receptor activations to downstream signaling pathways. PSD-95 associates with neuronal nitric oxide synthase, coupling NMDA receptor activation to nitric oxide generation. Similarly, PSD-95 associates with SynGAP, a neuronal GTPase activating protein (GAP) for Ras and Rap small GTPases. This interaction is not involved in synaptic localization of SynGAP but is implicated in functional coupling between NMDA receptors and the Ras-ERK signaling pathway, which regulates AMPA receptor trafficking and synaptic plasticity. Synaptic scaffolds couple kinases and phosphatases with their specific substrates. PSD-95 and SAP97 associate with AKAP79/150, a neuronal A-kinaseanchoring protein interacting with protein kinase
A (PKA), PKC, and protein phosphatase 2B (calcineurin). This interaction, in the context of SAP97 association with GluR1, promotes the PKAdependent phosphorylation of GluR1 on Ser845, a modification implicated in AMPA receptor function and synaptic plasticity. PSD-95 associates with Fyn, a nonreceptor tyrosine kinase, to promote Fyn-mediated tyrosine phosphorylation of NR2A. PSD-95 binds to another nonreceptor tyrosine kinase, Src, regulating NMDA receptor-dependent synaptic transmission and plasticity. Notably, this interaction is not involved in synaptic recruitment of Src but, rather, in the suppression of Src activity and Src-mediated NMDA receptor upregulation, reminiscent of the direct functional regulation of membrane proteins by scaffolding proteins.
F-Actin F-actin is a key cytoskeletal component both in dendritic filopodia and in spines, and it is implicated in the regulation of spine morphogenesis and synaptic plasticity. F-actin is both physically and functionally associated with PSD components. For instance, stable maintenance of GKAP, Shank, and AMPA receptors at synapses requires F-actin integrity. Conversely, LTP- and LTD-inducing stimuli regulate F-actin polymerization. In this context, it is conceivable that PSD proteins may organize F-actin-regulatory signaling pathways. F-actin polymerization in dendritic spines is regulated by small GTPases, including Rac, Cdc42, Rho, and Rap. Proteins acting upstream and downstream of these small GTPases associate with postsynaptic scaffolds, suggesting that these interactions may constitute related signaling pathways in the PSD. PSD-95 associates with and promotes synaptic localization of kalirin-7, a guanine nucleotide exchange factor (GEF) for Rac1. Shank binds bPIX, a GEF for Rac1 and Cdc42, and promotes synaptic localization of bPIX and bPIX-associated PAK, a kinase downstream of Rac1 and Cdc42 that regulates spine morphogenesis through LIMK-1 and MLC. Both PSD-95 and Shank associate with IRSp53, an abundant postsynaptic protein downstream of Rac1 that regulates spine morphogenesis. Shank associates with Abp1, a-fodrin, and cortactin, proteins that have F-actin binding, bundling, and nucleating activities, respectively. GRIP/ABP associates with EphB receptor tyrosine kinases, and ephrin activation of EphB receptors induces dendritic spine formation through the kalirin–Rac1–PAK pathway and the Cdc42 GEF intersection. Neurabin and spinophilin, which bind protein phosphatase 1 and F-actin and regulate dendritic spines, interact with Lfc, a Rho GEF. In
Postsynaptic Development: Neuronal Molecular Scaffolds 25
addition, NMDA receptors associate with and phosphorylate Tiam1, a Rac1 GEF that mediates NMDA receptor-dependent spine regulation.
Neuronal Transport Synaptic proteins synthesized in the cell body must be transported to their target synapses. Kinesin is a microtubule (MT)-based motor protein implicated in this process. There are approximately 45 kinesin family proteins in the mouse and human genomes. Most kinesins move toward the plus end of MTs. MTs in axons are unidirectionally oriented, with their plus ends pointing toward nerve terminals, whereas dendritic MTs are bidirectionally arranged. Kinesindependent transport has been studied mainly in axons, although evidence supports kinesin involvement in dendritic transport. An important question in neuron motor-dependent transport is how the limited number of kinesin proteins can transport a large number of cargoes. Studies raise the intriguing possibility that molecular scaffolds function as ‘motor receptors,’ linking motors to various cargoes through protein interaction domains. In accordance with this notion, synaptic scaffolds link kinesins to their specific cargoes. KIF1Ba, a kinesin motor, interacts with PSD-95 and S-SCAM, which are in turn linked to various synaptic proteins. KIF17 associates with the LIN2/7/10 PDZ protein complex that is coupled to NMDA receptors. KIF1A associates with liprin-a, a multidomain protein interacting with the GRIP–AMPA receptor complex. KIF5 interacts with GRIP/ABP, a binding partner of both AMPA receptors and EphB receptors, and disruption of the KIF5-GRIP interaction suppresses EphB trafficking and dendritic morphogenesis. Interestingly, GRIP drives KIF5 to dendrites, suggesting that a cargo regulates polarized transport of a motor.
Regulation of Postsynaptic Assembly Synapses are dynamically formed and eliminated during development and plasticity. These processes are likely to involve rapid assembly and disassembly of the PSD. Indeed, neuronal activity regulates synaptic localization of PSD components, changing the overall molecular composition of the PSD. GluR1 and protein phosphatase 1 are delivered to synapses by LTP- and LTD-inducing stimuli, respectively. Phosphorylation of synaptic proteins regulates their synaptic localization by affecting, for example, protein–protein interactions. Phosphorylation of the N-terminal domain of PSD-95 by cyclin-dependent kinase 5 suppresses synaptic clustering of PSD-95, whereas
SAP-97 phosphorylation in the L27 domain by CamKII enhances synaptic SAP97 localization. Phosphorylation of PDZ-binding ligands disrupts their binding to PDZ domains. Lipid modification also plays a role in postsynaptic assembly. Known examples include palmitoylation of PSD-95, PSD-93, GRIP, and AMPA receptors. The lipid addition regulates the trafficking and synaptic localization of these proteins. Synaptic activity depalmitoylates and disperses synaptic PSD-95 clusters. Enzymes that mediate protein palmitoylation (palmitoyl acyl transferases) of neuronal substrates have been identified. Lastly, protein degradation through the ubiquitin–proteasome pathway is involved in postsynaptic assembly. Neuronal activity regulates ubiquitination of key PSD proteins, including GKAP, Shank, and AKAP79/150. Mdm2, an E3 ubiquitin ligase, plays a role in ubiquitinating PSD-95. SNK, a polo-like kinase induced by synaptic activity, phosphorylates SPAR, a PSD-95-associated Rap GAP that regulates F-actin, and this in turn induces PSD-95 degradation and spine loss. See also: Cell Adhesion Molecules at Synapses; Dendrite Development, Synapse Formation and Elimination; Neuromuscular Junction (NMJ): Postsynaptic Events in Neuromuscular Transmission; Postsynaptic Density/ Architecture at Excitatory Synapses; Retrograde Transsynaptic Influences.
Further Reading Akins MR and Biederer T (2006) Cell–cell interactions in synaptogenesis. Current Opinion in Neurobiology 16: 83–89. Barry MF and Ziff EB (2002) Receptor trafficking and the plasticity of excitatory synapses. Current Opinion in Neurobiology 12: 279–286. Craig AM, Graf ER, and Linhoff MW (2006) How to build a central synapse: Clues from cell culture. Trends in Neuroscience 29: 8–20. Funke L, Dakoji S, and Bredt DS (2004) Membrane-associated guanylate kinases regulate adhesion and plasticity at cell junctions. Annual Review of Biochemistry 74: 219–245. Garner CC, Waites CL, and Ziv NE (2006) Synapse development: Still looking for the forest, still lost in the trees. Cell and Tissue Research 326: 249–262. Huang K and El-Husseini A (2005) Modulation of neuronal protein trafficking and function by palmitoylation. Current Opinion in Neurobiology 15: 527–535. Kim E and Sheng M (2004) PDZ domain proteins of synapses. Nature Reviews Neuroscience 5: 771–781. Kornau HC, Seeburg PH, and Kennedy MB (1997) Interaction of ion channels and receptors with PDZ domain proteins. Current Opinion in Neurobiology 7: 368–373. Levinson JN and El-Husseini A (2005) Building excitatory and inhibitory synapses: Balancing neuroligin partnerships. Neuron 48: 171–174. Malinow R and Malenka RC (2002) AMPA receptor trafficking and synaptic plasticity. Annual Review of Neuroscience 25: 103–126.
26 Postsynaptic Development: Neuronal Molecular Scaffolds Nicoll RA, Tomita S, and Bredt DS (2006) Auxiliary subunits assist AMPA-type glutamate receptors. Science 311: 1253–1256. Scannevin RH and Huganir RL (2000) Postsynaptic organization and regulation of excitatory synapses. Nature Reviews Neuroscience 1: 133–141. Scheiffele P (2003) Cell–cell signaling during synapse formation in the CNS. Annual Review of Neuroscience 26: 485–508. Sheng M and Kim MJ (2002) Postsynaptic signaling and plasticity mechanisms. Science 298: 776–780. Sheng M and Sala C (2001) PDZ domains and the organization of supramolecular complexes. Annual Review of Neuroscience 24: 1–29.
Sudhof TC (2001) Alpha-latrotoxin and its receptors: Neurexins and CIRL/latrophilins. Annual Review of Neuroscience 24: 933–962. Tada Tand Sheng M (2006) Molecular mechanisms of dendritic spine morphogenesis. Current Opinion in Neurobiology 16: 95–101. Thomas GM and Huganir RL (2004) MAPK cascade signalling and synaptic plasticity. Nature Reviews Neuroscience 5: 173–183. Wenthold RJ, Prybylowski K, Standley S, Sans N, and Petralia RS (2003) Trafficking of NMDA receptors. Annual Review of Pharmacology and Toxicology 43: 335–358. Yi JJ and Ehlers MD (2005) Ubiquitin and protein turnover in synapse function. Neuron 47: 629–632.
Dendrite Development, Synapse Formation and Elimination H Cline, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA ã 2009 Elsevier Ltd. All rights reserved.
Introduction In the central nervous system of adult animals, many neurons have functionally polarized architecture in which dendrites, which receive information from other neurons, are physically separate from axons, which send information to other neurons. In general, axons and dendrites both have complex treelike structures, called arbors. The area covered by the axons and dendrites and the density of branches within the dendritic and axonal arbors govern the type and number of neurons which are connected within a circuit. Consequently, the mechanisms that control the development and maintenance of neuronal structure critically affect the ability of the neuron to function within the brain circuit. For instance, the importance of neuronal structure in brain function is clear from studies of brains of children with mental retardation, in which neurons are dwarfed in size compared to those in healthy children. The acquisition of mature neuronal structure is classically described as being governed by both ‘intrinsic’ and ‘extrinsic’ factors, although in fact extrinsic factors, such as growth factors and synaptic inputs, affect intrinsic events such as gene transcription and trafficking of guidance molecules to the cell surface. Several recent studies using modern molecular genetic, imaging, and electrophysiological methods now provide strong evidence that excitatory synaptic inputs control the development of neuronal structure in the intact brain.
Synaptogenesis and Synapse Maturation The formation and maturation of synapses can be distinguished into several steps: 1. Establishment of an adhesive contact. Dynamic filopodial processes from growing presynaptic axons and postsynaptic dendrites come into contact and form an initial adhesive contact, possibly mediated by integrins, cadherins, or Wnt/Frizzled cell surface adhesive molecules. 2. Conversion of the adhesive contact to a nascent synapse. In the case of glutamatergic synapses in the vertebrate central nervous system, nascent synapses are characterized by the predominance of N-methyl-D-aspartate (NMDA)-type glutamate
receptors. NMDA receptors (NMDARs) require postsynaptic depolarization at the same time as ligand (i.e., glutamate) binding in order to permit conductance through the channel. As such, the NMDAR acts as a coincidence receptor. In the context of synapse formation, this ensures that transmission at nascent synapses occurs when other inputs to the postsynaptic neuron surpass a threshold synaptic strength. NMDARs are permeable to calcium. Intracellular calcium signaling may be required for maturation of the developing synapse. 3. Synapse maturation. Glutamatergic synapse maturation is characterized by the recruitment of a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-type ionotropic glutamate receptors into synapses. This renders the synapses functional at resting potentials. The assembly of the complex postsynaptic density, including scaffolding proteins and signaling proteins, occurs as glutamate receptors are trafficked into developing synapses.
Features of Nascent and Mature Synapses Studies in many experimental systems indicate that synaptic transmission at newly formed synapses is mediated by the NMDA type of glutamate receptors and that AMPA receptors (AMPARs) are added to synapses as they mature. Synapses with only NMDARs are ‘silent’ at resting potential due to the voltage-dependent block of the NMDAR channel, and the addition of AMPARs to synapses renders them functional at resting potentials. Similarly, the fraction of silent synapses, in which transmission is mediated solely by NMDARs, is high in early stages of synapse formation and decreases as synapses and neurons mature, due to the insertion of AMPARs at synaptic sites. Consequently, the fraction of silent synapses and the ratio of AMPA to NMDA receptormediated transmission can be used as indicators of synaptic maturity (see Figure 1). Although some glutamatergic synapses reportedly develop without NMDARs, a sequence in which transmission at new synapses is mediated principally by NMDARs followed by the addition of functional AMPARs to synaptic sites appears to occur at the majority of glutamatergic synapses. Indeed, trafficking of AMPARs into developing synapses is required for their maturation and is required for synaptic plasticity in adult animals under a variety of conditions, including experience-dependent sensory plasticity and fearconditioned learning. Although these data suggest
27
28 Dendrite Development, Synapse Formation and Elimination
NMDAR AMPAR
Immature neuron Immature synapses
Mature neuron Mature synapses
Figure 1 Synaptogenesis and dendrite development are concurrent. In vivo images of a neuron from the optic tectum of Xenopus laevis tadpoles were collected once a day over 3 days. The dendritic arbor increases in complexity over this time period through the net addition of arbor branches. Images collected over shorter intervals (not shown) demonstrate that net arbor growth occurs as a result of rapid branch addition and retraction. Synapses are located throughout the arbor. New excitatory synapses are mediated principally by N-methyl-D-aspartate (NMDA)-type glutamate receptors, shown as red dots. a-Amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-type receptors, shown as green dots, are added to these synapses as they mature. Even after 3 days of growth, the dendritic arbor continues to show structural changes and to add new (NMDA receptor (NMDAR) only) synapses.
that AMPAR trafficking is the key element required for synapse maturation and synaptic plasticity, AMPAR trafficking into synapses may escort other proteins into the postsynaptic density, and it is possible that these other proteins in combination with AMPARs are required for synaptic plasticity. Developing glutamatergic synapses can be distinguished from mature synapse by several other features. Synapse maturation includes changes in the presynaptic element, including recruitment of presynaptic machinery and proteins into the axon terminal and recruitment of synaptic vesicles. Ultrastructural studies indicate that developing synapses have few, sparsely packed synaptic vesicles, and that the most reliable ultrastructural indicator of synaptic maturation is the density of synaptic vesicles. In vertebrate neurons the subunit composition of synaptic NMDARs switches during development, from receptors containing mostly NR2B subunits to those including NR2A subunits. This change in subunit composition can be detected pharmacologically and by a faster decay of the synaptic responses. Furthermore, immature synapses may show greater spillover of transmitter from the synaptic cleft to activate extrasynaptic receptors, due to relatively poor envelopment of nascent synapses by glia. Synapse maturation and developmental plasticity of the strength of synaptic communication are thought to be regulated by changing the efficacy of transmitter release from the presynaptic terminal and by changing the response of the postsynaptic neuron to the input signal. The mechanisms underlying these changes are similar to those which occur during learning and memory in the adult animal. For instance, calcium influx through NMDARs, Ca2þ/calmodulin-dependent
protein kinase II (CaMKII) activation, and AMPAR trafficking underlie both synapse maturation and changes in synaptic strength associated with learning.
Process of Dendritic Arbor Development In vivo time-lapse imaging experiments have shown that dendrites of central nervous system neurons grow by the highly dynamic addition and retraction of fine branches. Although most newly added branches retract within about 10 min of being added to the arbor, a small fraction of branches is maintained and these extend to become more stable components of the arbor. The newly added branches may sample the local environment for appropriate presynaptic contact sites. Establishment and maintenance of synapses may then confer a longer lifetime on the branches that form stable synapses and permit the dendritic arbor to enlarge over time (see Figure 1). Classical studies using fixed anatomical preparations have led to the idea that axonal and dendritic arbors go through a period of exuberant overgrowth followed by pruning of branches based on competitive mechanisms (see Figure 2). This model of neuronal growth and pruning suggests that neurons in the adult brain have limited capacity to recover following injury, and this in turn has discouraged scientists and clinicians from exploring the potential of the brain to regain function after trauma. Modern in vivo imaging methods demonstrate that neurons in the brain grow very differently than thought from classical studies. Specifically, branch additions and retractions are concurrent, so that the neuron has an ongoing capacity to add new branches and to refine its connections within the
Dendrite Development, Synapse Formation and Elimination 29 Sequential model
Axons
Dendrites Growth
Retraction
Concurrent model Axons
Dendrites Concurrent growth and retraction New branch
Eliminated or transient branch
Stable branch
Figure 2 Models of dendritic and axon arbor elaboration. Dendritic and axon arbor elaboration and pruning are concurrent, not sequential. Top: Diagrams of the patterns of growth of axons and dendrites according to the traditional model, in which neurons undergo an exuberant growth phase followed by a temporally distinct phase of net branch retraction. Bottom: Diagrams of the patterns of growth of axons and dendrites in which branch additions and retractions are concurrent. Adapted from Hua JY and Smith SJ (2004) Neural activity and the dynamics of central nervous system development. Nature Neuroscience 7: 327–332.
circuit (Figure 2). The refinement of the arbor structure occurs in response to signals from the environment and from other neurons in the circuit. These observations provide critical insight into the cellular mechanisms governing dendritic and axonal arbor development and clearly indicate that learning and recovery from trauma can occur even in the adult brain by tapping into the cellular mechanisms that shape the neuronal structure during development of the brain.
in which the maturation of glutamatergic synapses was blocked show that AMPAR trafficking into developing synapses is required for the stabilization of newly added dendritic arbor branches and the cumulative elaboration of the complex dendritic arbor. Together, these data indicate that the iterative process of dendritic arbor development requires the coordinate formation and stabilization of glutamatergic synaptic inputs (Figures 1–3).
Synaptic Inputs Increase Dendrite Arbor Growth
Conclusion
Several time-lapse imaging experiments now provide convincing support for the hypothesis that the formation and maturation of synaptic contacts stabilize dendritic arbor structure (Figure 3). Direct in vivo time-lapse imaging reveals that visual stimulation increases the growth of optic tectal dendritic arbors in vivo by promoting the stabilization of newly added branches. Pharmacological blockade of either AMPA- or NMDA-type glutamate receptors decreases dendritic arbor growth in vivo and blocks visual stimulation-induced dendritic arbor growth. Similarly, deafferentation or blocking inputs within the auditory system, as well as other sensory systems, has severe effects on dendritic arbor development of neurons that receive and process sensory information. In vivo imaging experiments
Synapse formation is characterized by the assembly of a complex protein machine that spans a specialized junction, the synapse, which forms between two neurons. Recent multidisciplinary experiments combining timelapse in vivo imaging, molecular manipulations, and electrophysiological recordings demonstrate that synapse formation and maturation are required for the normal development of neuronal structure, including the axon and dendrite. These experiments have also shown that the development of axons and dendrites occur as a result of simultaneous addition and retraction of branches within the neuron. These observations overturn two previous ideas about brain development, that neuronal growth can occur in the absence of synapse formation and synaptic activity, and that neurons undergo a phase of exuberant growth followed by a separate period of pruning. This modern view of
30 Dendrite Development, Synapse Formation and Elimination
Normal level
Increasing circuit activity
neuron development suggests that brain activity will increase synapse formation as well as the establishment and maintenance of optimal neuronal circuits. This view also lends hope to the idea that brain exercises will aid recovery from trauma even in adults. See also: Cell Adhesion Molecules at Synapses; Postsynaptic Development: Neuronal Molecular Scaffolds; Retrograde Transsynaptic Influences; Synaptic Precursors: Filopodia.
Further Reading
Block NMDAR
Block synapse maturation
Figure 3 Synaptic input drives dendrite development. The normal rate of dendritic arbor development is increased by enhanced synaptic input and is decreased when glutamate receptor activity or synapse maturation is blocked. The extent of dendritic arbor growth under ‘normal’ levels of circuit activity in the brain is depicted at the top. The effect of increasing activity on dendritic arbor development is shown in the second row; the effects of blocking N-methyl-D-aspartate receptor activity or blocking synapse maturation are shown in the third and fourth rows. Based on data from Sin WC, Haas K, Ruthazer ES, et al. (2002) Dendrite growth increased by visual activity requires NMDA receptor and Rho GTPases. Nature 419: 475–480; Rajan I and Cline HT (1998) Glutamate receptor activity is required for normal development of tectal cell dendrites in vivo. Journal of Neuroscience 18: 7836– 7846; and Haas K, Li J, and Cline HT (2006) AMPA receptors regulate experience-dependent dendritic arbor growth in vivo. Proceedings of the National Academy of Sciences of the United States of America 103: 12127–12131.
Cline HT (2001) Dendritic arbor development and synaptogenesis. Current Opinion in Neurobiology 11: 118–126. Haas K, Li J, and Cline HT (2006) AMPA receptors regulate experience-dependent dendritic arbor growth in vivo. Proceedings of the National Academy of Sciences of the United States of America 103: 12127–12131. Hua JY and Smith SJ (2004) Neural activity and the dynamics of central nervous system development. Nature Neuroscience 7: 327–332 Niell CM, Meyer MP, and Smith SJ (2004) In vivo imaging of synapse formation on a growing dendritic arbor. Nature Neuroscience 7: 254–260. Rajan I and Cline HT (1998) Glutamate receptor activity is required for normal development of tectal cell dendrites in vivo. Journal of Neuroscience 18: 7836–7846 Rumpel S, LeDoux J, Zador A, et al. (2005) Postsynaptic receptor trafficking underlying a form of associative learning. Science 308: 83–88. Sin WC, Haas K, Ruthazer ES, et al. (2002) Dendrite growth increased by visual activity requires NMDA receptor and Rho GTPases. Nature 419: 475–480. Takahashi T, Svoboda K, and Malinow R (2003) Experience strengthening transmission by driving AMPA receptors into synapses. Science 299: 1585–1588. Van Aelst L and Cline HT (2004) Rho GTPases and activitydependent dendrite development. Current Opinion in Neurobiology 14: 297–304. Wong RO and Ghosh A (2002) Activity-dependent regulation of dendritic growth and patterning. Nature Reviews Neuroscience 3: 803–812. Wu G-Y and Cline HT (1998) Stabilization of dendritic arbor structure in vivo by CaMKII. Science 279: 222–226.
Synapse Formation: Competition and the Role of Activity L Cancedda and M-M Poo, University of California at Berkeley, Berkeley, CA, USA ã 2009 Elsevier Ltd. All rights reserved.
Introduction Synaptic competition is a cellular process by which the presence of one synapse affects the stability or survival of other synapses on the same postsynaptic cell. Synapse elimination refers to synapse loss due to either a low ‘intrinsic merit’ of the synapse for survival or to its failure in winning synaptic competition with other synapses on the same cell. Such competition and elimination can be driven by either neuronal/ synaptic activity or other activity-independent processes. The idea that the strength of synaptic connections between neurons may be modified can be traced back to Ramon y Cajal, who proposed that such modification serves as a cellular mechanism for learning and memory. That memory formation involves making and breaking existing synaptic connections has been a popular idea over the past century, but solid experimental evidence in support of this idea has been elusive. Recent morphological studies of synapse stability in the adult rodent brain have revealed rather limited synaptic remodeling under normal conditions. In contrast, initial synaptic connections established early in the developing nervous system undergo substantial remodeling, with some connections stabilized and others eliminated, as a result of experience. This developmental remodeling of connectivity often involves cooperative and competitive interactions between converging synapses on the postsynaptic cell and in many cases depends on the pattern of electrical activity. We here summarize the evidence of activitydependent synaptic competition and elimination in various brain regions, together with potential underlying cellular mechanisms.
Visual System Activity-dependent synaptic competition and elimination in the central nervous system was first indicated by work of Hubel and Wiesel on the development of ocular dominance columns (ODCs) in the visual cortex. Cortical neurons preferentially responding to one eye or the other are normally found to be segregated into alternating columns in the primary visual cortex, representing sorting of geniculocortical projections serving two eyes during postnatal development. The sorting process occurs prior to eye opening and is highly sensitive to visual
experience. Depriving visual input to one eye of a newborn cat or monkey by suturing the eyelid during a critical period after birth leads to retraction of geniculocortical projections serving that eye. Interestingly, the consequences of visual deprivation during early postnatal period are much more severe in cats subjected to monocular deprivation than binocular deprivation, suggesting that competition driven by activity between two eyes, rather than the reduced activity in one eye, is responsible for sorting geniculocoritical projections. The ODCs develop by a progressive segregation of initially overlapping geniculocortical projections serving two eyes. While the initial alternating pattern of projections is established by mechanisms independent of the retina activity, pruning of extensive overlapping projections is likely to be driven by activity, including spontaneous or visually evoked retinal activity, because blocking the firing of retinal ganglion cells (RGCs) in both eyes prevents complete ODC formation. Early in development there is also substantial overlap of retinogeniculate projections from the two eyes in the lateral geniculate nucleus (LGN). Segregation of these projections into eye-specific layers occurs before the onset of vision, but at a time when spontaneous activity waves are prominent in the retina. Blocking all activity in both eyes prevents segregation of retinogeniculate projections and can desegregate already existing eye-specific projections later in development, although it is unclear whether the specific pattern of the waves is required for eye-specific segregation. When the activity is altered in one eye, the more active eye acquires more synaptic territory than the less active eye, suggesting activity-mediated competition. Furthermore, even after eye-specific segregation is completed, activity in one eye can further prune retinogeniculate projections by reducing the convergence of RGC inputs onto a single LGN neuron from 12–20 to 1. This within-eye pruning based on activity-dependent competition among RGCs faciliates sharpening of the receptive field of LGN neurons. Such activity-dependent competition may be attributed to the competition in the growth or stability of RGC axonal arbors. In the developing optic tectum of zebra fish, reducing neuronal and synaptic activity in a subset of RGCs leads to suppression of their axon growth and branching and this suppression is relieved by blocking the activity of nearby RGC axons as well. The successive levels of the mammalian visual system are organized into retinotopic maps that preserve an orderly representation of visual inputs at the retina, through topographically precise retinogeniculate,
31
32 Synapse Formation: Competition and the Role of Activity
retinocollicular, and geniculocoritical projections. Formation of these maps requires specific patterns of spontaneous activity in the retina because disrupting these waves affects map formation at all levels. While the initial projections may establish a crude retinotopic map via axon guidance based on molecular cues, interfering with both axon guidance cues and spontaneous wave activity in the same animal results in a dramatic cumulative effect in disrupting the map in the superior colliculus. Finally, the development of retina circuitry itself also depends on activity. Depriving the retinal activity by blocking spontaneous activity or dark rearing blocks both the normal maturational loss of ON–OFF responsive RGCs and the pruning of dendrites at the stratified ON and OFF layers of the inner plexiform layer, although it is unclear whether dendritic pruning in this layer results from competitive interaction among bipolar inputs.
Somatosensory System Somatosensory projections in the brain exhibit a somatotopic map, with axons of peripheral receptor sheets projecting in an orderly manner onto central brain structures. The most intensively studied system is the rodent trigeminal pathway where the patterned array of the whisker is replicated in the patchy distribution of afferents and the modular organization of their postsynaptic counterparts along the pathway from the periphery to the primary somatosensory cortex. The whisker-related patterns are first established in the brain stem nuclei, then in the ventroposteromedial nucleus of the thalamus, and finally in the somatosensory cortex (‘barrels’), where neurons respond best or exclusively to deflection of the corresponding facial whisker. These somatotopic maps emerge during early development in ascending order along the neuroaxis, with a sequence that includes afferent fibers segregation before rearrangement of their target neurons in discrete zones along the pathway. It is generally believed that the initial crude topographic projection of the afferent fibers is independent of sensory experience, but the presence of segregated afferent fibers and postsynaptic glutamate receptor activities is required for the subsequent parcellation of their postsynaptic targets. That cortical map formation depends on a competitive process is suggested by the finding that damage of the branch of the trigeminal nerve supplying the whisker pad prior to birth results in a reduction of the cortical representation of the whiskers and a concomitant increase in the representation of other peripheral receptor surfaces. Whether and how such competition occurs through synaptic competition and elimination
are unknown. Synapse elimination has been observed in the ventral posteromedial thalamic nucleus of young animals, where multiple afferents on each neuron are reduced to one or two afferents as the animal matures. However, this synapse elimination and remodeling occur even in animals deprived of sensory experience from birth, indicating that the process is independent of sensory experience, although spontaneous neuronal activity may still be required. Morphological organization of somatosensory cortical neurons into barrels during development depends on signals conveyed by invading thalamic axons and sensory activity. Indeed, primordial visual cortex transplanted into the neonatal somatosensory cortex forms barrels when invaded by axons from somatosensory thalamic nuclei, and genetic manipulations that interfere with the appropriate segregation of these axons disrupt barrel formation. Moreover, that sensory peripheral sensory signals may be instructive in sculpting the somatosensory cortical map is also evidenced by lesion or genetic studies showing that altering the number of functioning whiskers leads to shrinkage, expansion, or addition of barrels. However, such aberrant barrel formation may result from sprouting and retraction of afferents and dendrites due to neuronal degeneration and alteration in the number of afferents, involving competitive synapse formation, rather than activitydependent synaptic competition. Notably, changes in whisker use drive functional changes in the barrel neurons without affecting anatomical appearance of barrels, suggesting no gross reorganization of synaptic connections. Nevertheless, sensory deprivation results in a specific loss of GABAergic synapses and impairment of secondary dendritic branches in the barrel cortex. Thus, activity may lead to synapse elimination of at least a subset of synapses in the somatosensory cortex, presumably through a competitive process.
Auditory System Sound entering the ear stimulates cochlear hair cells, which make synaptic connection with spiral ganglion cells that give rise to the primary auditory nerve for carrying auditory information into the brain stem. From there the signals converge in the inferior colliculus that sends projections to the thalamus, which further relays auditory signals to the auditory cortex. In these relay areas, neurons are arranged in a topographic manner according to the sound frequencies to which they are most sensitive. Although the topography of these connections is apparent early in development, the precision of the map is refined later in development through an activity-dependent process.
Synapse Formation: Competition and the Role of Activity 33
Developmental pruning of both axonal arbors and dendritic branches has been widely observed in the auditory system. Cochlear nerve axons and their target neurons in nucleus magnocellularis (NM) undergo extensive parallel structural transformations involving pruning of cochlear axonal arbor, massive reduction of dendrites in NM neurons, and elimination of polyneuronal innervation. Since otocyst removal has no effect on the extent, timing, and pattern of dendritic loss, this extensive synapse elimination is independent of the sensory activity. Interestingly, after this early massive remodeling, NM neurons undergo further dendritic growth before maturation in a sensory activity-dependent manner. In contrast to that in the NM, developmental remodeling of axons and dendrites in the nucleus laminaris and superior olivary nucleus (SON) after the onset of hearing appears to be activity dependent. Cochlea removal or blockade of glycinergic transmission impairs remodeling of axonal and dendritic morphology in SON. Since most of the remodeling occurs after the onset of hearing, acoustically evoked activity is likely to be involved, although spontaneous activity may also contribute. Indeed, correlated spontaneous activity is present in the embryonic brain stem and auditory nerve. The spatiotemporal pattern of spontaneous firing could provide developmental cues for the spatial ordering of auditory projections, as suggested by the presence of a systematic relationship between the rate of rhythmic bursting and the tonotopic location in the chick. Such activitydependent remodeling of connectivity may contribute to the tonotopic map refinement at many different levels in the auditory system. After the onset of hearing, the auditory cortex undergoes a transition from a tonotopic map dominated by broadly tuned, high-frequency-selective neurons to the adult tonotopic map consisting of neurons that represent the full spectrum of acoustic inputs. Sensory-evoked activity is responsible for this transition. Early acoustic environment is critical for the maturation of tonotopic maps, because exposing rat pups to pulsed white noise or rearing them in continuous moderate-level noise impairs the emergence of adultlike tonotopic map, whereas exposure to pulsed tones of specific frequencies results in accelerated emergence and expansion of auditory cortex representations of those frequencies. This activity-dependent remodeling of cortical maps is likely to involve synaptic competition and elimination, although whether functional refinement of the map directly reflects structural reorganization of synaptic connectivity remains to be determined.
Olfactory System The olfactory sensory neurons in mammals express only one of about 1000 odorant receptor genes, and neurons expressing a given receptor are randomly dispersed within one of four broad zones in the olfactory epithelium. The axons of these sensory neurons converge upon spatially conserved glomerulus within the olfactory bulb. The topographic mapping between sensory neurons and specific glomeruli may depend on the expression of specific molecules along their projection pathways or in themselves. There is evidence, however, that activity in these olfactory neurons may also play a role. Although the patterns of axon convergence in the bulb are largely intact in mice lacking functional olfactory cyclic nucleotidegated channels, hence no odorant-evoked activity occurs in these neurons, noncorrelated spontaneous activity may still be required for the correct mapping process. Indeed, sensory map is not affected when conditional expression of tetanus toxin light chain inhibits synaptic transmission in the majority of olfactory sensory neurons. However, inhibition of synaptic release in a small subpopulation of neurons expressing the P2 receptor results in correct targeting of the sensory axons initially, but the P2 glomerulus is not maintained and P2 neurons ultimately diminish. Preventing excitation of the neuron has a similar effect on the formation of the olfactory map. Thus, spontaneous neuronal activity may play a role in pruning or stabilization of axon terminals of olfactory neurons on their glomerular target cells, but there is little evidence that synaptic competition is involved in olfactory map formation.
Cerebellum The two main afferent systems in the cerebellar cortex are the climbing fibers (CF) originating from the inferior olivary nucleus and the mossy fibers (MF) from various nuclei in the spinal cord, brain stem, and deep cerebellar nuclei. Each CF directly contacts the proximal dendritic compartment of a single Purkinje cell (PC), whereas the MFs influence PCs indirectly through granule cells, whose axons form the parallel fibers (PFs) that synapse onto the dendrites of many PCs. There is evidence for a complex topographic map of these afferents fibers. For example, cutaneous inputs carried by CFs are topographically organized to form a map of peripheral body, with CF axonal arbors in register with cortical parasagittal bands of chemically heterogeneous PCs. The formation of these precise topographic maps
34 Synapse Formation: Competition and the Role of Activity
involves both activity-independent and dependent steps. First, positional information shared between CFs and PCs during embryonic development provides the molecular code for the formation of coarse-grained maps independent of neuronal activity. Activitydependent mechanisms are later required for the transition to a fine-grained map, by pruning CF terminal arbors on each PC from multiple to single CF innervation. This pruning involves strengthening one CF while weakening all other CFs during the first postnatal week, leading to the elimination of the latter. Moreover, alteration of the temporal pattern of CF activities specifically during development impairs such CF elimination in vivo, suggesting an activity pattern-dependent synaptic competition. In addition to potential competition among homologous CFs, there is also heterologous competition between CFs and PFs that results in their segregation into different dendritic domains of each PC. Weakening of the CF input leads to the reduction of its dendritic territory and concomitant strengthening and expansion of the PF input, and vice versa. Furthermore, elimination of CFs depends on the activity of developing PF–PC synapses, because CF elimination is affected by reducing PF inputs, through granule cell degeneration, impairment of granule cell function, or genetic manipulation of PF–PC synapse formation or efficacy. The similarity in the consequence of reducing CF and PF activities in CF elimination suggests that similar mechanisms may underlie synaptic competition/elimination among homologous versus heterologous inputs.
Autonomic Ganglia Ganglionic cells in the autonomic nervous system are innervated by preganglionic neurons of the spinal cord or brain stem and send projections to target tissues via spinal nerves to control involuntary functions of the body. In mammals, characteristic patterns of sympathetic and organ responses elicited by the activity of individual spinal axons are due to mapping between specific spinal segments and peripheral targets. This mapping requires a stereotyped and selective innervation of ganglion cells by preganglionic axons, with each ganglion cell innervated by one or few axons from specific contiguous spinal cord segments. During development there is a transition from initially exuberant innervation of each ganglion cell by many preganglionic axons to innervation by only one or a few axons. Since the spinal segment responsible for activating the mature and neonatal ganglion cell is the same, developmental synapse elimination involves reduction of preganglionic axons from the same spinal segment, suggesting competition occurs
among axons of nearby spinal neurons. Furthermore, transection of a portion of preganglionic nerve innervating a developing ganglion leads to sprouting of residual preganglionic axons and partial restoration of original multiplicity of innervation. These results are all consistent with the idea that synaptic elimination is not simply due to the intrinsic merit of the input, but involves competition among inputs, presumably for a postsynaptic factor of limited supply, for example, locally secreted trophic factor. The competitive interaction that determines the final number of preganglionic axons converging upon a single ganglion cell depends on the proximity of competing synapses, with each surviving synaptic terminal claiming a certain territory on the dendritic or somatic surface and neurons with more extensive dendritic arbors receiving more axons. This distancedependent synapse competition can be explained by a limited amount of available postsynaptic factors. This synapse competition appears to be activity dependent, because among converging inputs to a single ganglion neuron, strong synapses become further strengthened and weak synapses further weakened during synapse elimination. Moreover, the synaptic strength for each synaptic input of a multiply innervated cell is, on average, weaker than that of a singly innervated cell, suggesting that the total synaptic strength of a postsynaptic ganglion cell is conserved, due to a limited amount of a synapse-related factor.
Neuromuscular Junction Each muscle fiber in the neonatal animal is multiply innervated by axon collaterals of several motor neurons, but becomes singly innervated during early postnatal life. This process depends on synapse elimination involving withdrawal of a subset of nerve terminals of each motor neuron innervating a given muscle (i.e., reduction of the size of motor units) rather than reduction in the number of motor neurons innervating a muscle. Partial denervation of the muscle at birth results in the retention of the large motor unit size without substantial collateral motor axon sprouting. Synapse elimination is competitive rather than a random process of withdrawal, since muscle fiber without a single axon is never observed. The elimination is also activity dependent. Blocking motor neuronal activity prevents elimination, while elevating activity accelerates it. Importantly, when the relative synaptic efficacy of two competing axons at a single neuromuscular junction is impaired by genetic deletion of acetylcholine in one axon, the latter loses the competition, suggesting that the strength of the synapse is predictive of the outcome of the competition.
Synapse Formation: Competition and the Role of Activity 35
Mechanisms of Activity-Driven Synapse Competition The Hebb’s Rule for Synapse Competition
Hebb postulated that strengthening of a synapse might be achieved by repetitive presynaptic activation that leads to postsynaptic firing. This postulate was later transformed into a simple rule – coincident preand postsynaptic activity leads to synapse strengthening and stabilization. To account for the finding of Hubel and Wiesel on activity-dependent remodeling of connectivity in the developing visual system, Stent further extended the Hebb’s rule by assuming that noncoincident pre- and postsynaptic activity leads to synapse weakening and elimination. Modeling studies showed that such correlation-based Hebb’s rule could explain activity-dependent refinement of developing visual circuits. In the past decade, a temporally specific form of Hebb’s rule has been proposed, based on findings of spike-timing-dependent synaptic plasticity in a variety of systems. The temporal order in the spiking of pre- and postsynaptic neurons was shown to be critical for synaptic modification, in addition to the extent of coincidence in spiking: ‘Pre-before-post’ results in strengthening, and ‘post-before-pre’ leads to weakening of the synapse. This spike-timingdependent plasticity offers an element of causality in the activity-induced synaptic competition: inputs that contribute to (and cause) the postsynaptic spiking are advantageous in synaptic competition over those inputs arriving after postsynaptic spiking has just occurred. Importantly, experimental evidence for the validity of various forms of the Hebb’s rule mainly came from studies of activity-induced functional changes of synaptic efficacy, for example, long-term potentiation (LTP) or long-term depression (LTD), rather than changes in the morphological connectivity. Importance of Temporal Pattern of Activity
The importance of the pattern of activity in synapse competition has been demonstrated mainly in the development of ocular dominance and orientation selectivity of the primary visual cortex. Rearing kittens with induced squint (strabismus), which alters the pattern but not the absolute level of activity, results in striking changes in the binocular property of cortical cells, reflecting altered synapse competition of geniculocortical inputs. Artificially imposing synchronous activity on optic nerves from the two eyes prevents segregation of thalamocortical projections into ODCs, whereas asynchronous activity allows segregation. Similarly, synchronous activation of optic nerves blocks the development of topographic maps in the optic tectum and reduces orientation
selectivity in the cortex. Spike-timing-dependent synaptic modification provides a natural basis for such pattern-dependent competition. In the developing Xenopus visual system, spike-timing-dependent induction of LTP and LTD has been demonstrated, but whether such persistent changes in functional efficacy of synapse is causally related to structural changes in connectivity remains unknown. Contribution by GABAergic Inhibition
Activity in the brain depends on a proper balance of excitation and inhibition. It is thus not surprising that GABAergic activity plays a regulatory role in the refinement of developing circuits. In mice lacking one form of the enzyme (GAD65) responsible for GABA synthesis, the ocular dominance plasticity resulting from monocular deprivation is absent, and increasing GABA inhibition in diazepam-treated mutant mice allows the appearance of ocular dominance plasticity. In the developing retinotectal system, a proper level of GABAergic inhibition is required for normal refinement of the retinotopic map in the tectum, both the increase and decrease of inhibition impede map refinement. Interestingly, the role of GABAergic inhibition appears not only to reduce the overall neuronal excitation, but also to sharpen the temporal pattern of the neuronal activity by shortening stimulus-evoked discharges, potentially facilitating spike-timing-dependent refinement of neural circuits. Shunting of specific excitatory inputs may also be achieved by selective distribution of inhibitory synapses on the dendrite. Finally, inhibitory synapses may also undergo competition and refinement as well, because they are integral part of the neural circuit. At present, it is unknown how nascent inhibitory connections, while playing important regulatory roles in refining excitatory connections, can themselves undergo activity-driven refinement and be properly consolidated into the mature circuit. Causal Link between LTP/LTD and Synapse Competition
At some synapses repetitive coactivation of the pre- and postsynaptic cell leads to not only homosynaptic LTP, but also heterosynaptic LTD of non-coactive converging synapses onto the postsynaptic cell, thus providing a potential competitive mechanism for synaptic elimination. The hypothesis that synapse stabilization and elimination are mechanistically linked to or even result from activity-induced LTP and LTD, respectively, remains to be fully tested. Many lines of correlative evidence support this hypothesis. Blocking NMDA receptor activation, which abolishes many forms of LTP/ LTD, impedes refinement of developmental circuits.
36 Synapse Formation: Competition and the Role of Activity
Repetitive visual stimuli that modify developing visual circuits can induce NMDA receptor-dependent LTP/LTD of retinotectal synapses. During the postnatal critical period, the composition of NMDA receptors undergoes experience-dependent developmental regulation in the visual system, and there is a correlation between the susceptibility for LTP/LTD induction and for circuit refinement. However, whether LTP/LTD is relevant or a prelude to structural refinement in the visual system remains unknown. Synapse elimination (structurally) will certainly eliminate the synaptic function, but whether LTD will lead to synapse elimination is unclear. Recent studies in hippocampal slices has provided evidence that LTP/ LTD induction is followed by a swelling/shrinkage of dendritic spines, supporting the linkage between synaptic efficacy and synaptic structure. The Trophic Factor Hypothesis
Purves and Lichtman have proposed that synaptic competition involves the competition between coinnervating presynaptic terminals for a limited amount of ‘trophic factors’ derived from the postsynaptic cell. This hypothesis can be extended to factors in the postsynaptic cytoplasm or plasmalemma, together with a localized retrograde signaling to the presynaptic nerve terminal. Competition for the trophic factor can be regulated by activity. The activity can serve a permissive role for synapse competition by regulating the synthesis and release of trophic factors/retrograde signals, whereas other activity-independent mechanisms determine the competitive advantage of a synapse. For activity to serve an instructive role, the pattern of activity in the co-innervating nerve terminals may determine the outcome of competition by controlling the uptake or the efficacy of the trophic factor at the nerve terminal. The local release of trophic factors may also depend on local synaptic activation, which is in turn driven by the pattern of activity, including the timing of pre- and postsynaptic spiking. The neurotrophin family of proteins, which are known to regulate synaptic function and axon/ dendrite morphology, are attractive candidates for the trophic factor in synapse competition. The expression and secretion of neurotrophins, and their potentiating actions on the synapse, are all activity dependent. Neurotrophins are required for the development of ODC and for the induction of activity-induced LTP in several systems. Furthermore, neurotrophin secretion is activity pattern dependent, and the secreted neurotrophins are likely to act locally by its binding to cell surfaces at the synapse, allowing them to serve as local synaptic modulators. Activity-dependent depletion of available neurotrophins in the local
environment of the synapse may lead to the functional and structural modification underlying synapse elimination.
Concluding Remarks Studies of synapse competition and elimination in the near future are likely to be facilitated greatly by the availability of many new technologies for selective neuronal labeling and optical imaging in the living animal. Transgenic animals with selective populations of fluorescence-tagged neurons, together with in vivo multiphoton imaging, will allow us to directly monitor changes in the morphology of axons and dendrites during the process of synapse competition and refinement over prolonged period in the intact brain. Fluorescent Ca2þ or membrane-voltage sensors will allow us to monitor neuronal activities, and photo-activated probes expressed in selective neuronal populations will allow us to directly manipulate neuronal activity and to examine how activity drives synapse competition and elimination. See also: Neurotrophins: Physiology and Pharmacology.
Further Reading Bi G and Poo M-M (2001) Synaptic modification by correlated activity: Hebb’s postulate revisited. Annual Review of Neuroscience 24: 139–166. Buffelli M, Burgess RW, Feng G, Lobe CG, Lichtman JW, and Sanes JR (2003) Genetic evidence that relative synaptic efficacy biases the outcome of synaptic competition. Nature 424: 430–434. Feldman DE and Brecht M (2005) Map plasticity in somatosensory cortex. Science 310: 810–815. Hua JY, Smear MC, Baier H, Stephen J, and Smith SJ (2005) Regulation of axon growth in vivo by activity-based competition. Nature 434: 1022–1026. Hubel DH, Wiesel TN, and LeVay S (1977) Plasticity of ocular dominance columns in monkey striate cortex. Philosophical Transactions of the Royal Society of London B: Biological Science 278: 377–409. Jansen JK, Van Essen DC, and Brown MC (1976) Formation and elimination of synapses in skeletal muscles of rat. Cold Spring Harbor Symposia on Quantitative Biology 40: 425–434. Katz LC and Shatz CJ (1996) Synaptic activity and the construction of cortical circuits. Science 274: 1133–1138. Lichtman JW and Purves D (1980) The elimination of redundant preganglionic innervation to hamster sympathetic ganglion cells in early post-natal life. Journal of Physiology 301: 213–228. Lohof AM, Delhaye-Bouchaud N, and Mariani J (1996) Synapse elimination in the central nervous system: Functional significance and cellular mechanisms. Reviews of Neuroscience 7: 85–101. Poo M-M (2001) Neurotrophins as synaptic modulators. Nature Reviews Neuroscience 2: 24–32. Purves D and Lichtman JW (1980) Elimination of synapses in the developing nervous system. Science 210: 153–157.
Synapse Formation: Competition and the Role of Activity 37 Rubel EW and Fritzsch B (2002) Auditory system development: Primary auditory neurons and their targets. Annual Review of Neuroscience 25: 51–101. Sanes JR and Lichtman JW (1999) Development of the vertebrate neuromuscular junction. Annual Review of Neuroscience 22: 389–442.
Sotelo C (2004) Cellular and genetic regulation of the development of the cerebellar system. Progress in Neurobiology 72: 295–339. Yu CR, Power J, Barnea G, et al. (2004) Spontaneous neural activity is required for the establishment and maintenance of the olfactory sensory map. Neuron 42: 553–566.
Cell Adhesion Molecules at Synapses L F Reichardt and S-H Lee, University of California at San Francisco, San Francisco, CA, USA ã 2009 Elsevier Ltd. All rights reserved.
Introduction The structures and major intracellular interactions of several different synaptic adhesion molecules believed to play important roles at synapses are shown in Figure 1. One large class is the cadherin superfamily with more than 100 members which are defined by the presence of one or more 110-aminoacid cadherin repeats in their extracellular domains. The cadherin superfamily is subdivided into classical or type I cadherins, atypical or type II cadherins, desmosomal cadherins, protocadherins, seven-pass transmembrane cadherins, Fat cadherins, etc. A second large group of adhesion molecules is the immunoglobulin (Ig) superfamily, members of which contain varying number of extracellular Ig domains. In some Ig superfamily members fibronectin type III (FNIII) repeats are inserted between the Ig and transmembrane domains. Neural cell adhesion molecule (NCAM), synaptic cell adhesion molecule (SynCAM), L1, the nectins, Side kicks (Sdk), synaptogenesis (SYG)/nephrin, dendrite arborization and synaptic maturation1 (Dasm1), synaptic adhesion-like molecule (SALM), leukocyte common antigen-related-receptor proteintyrosine phosphatases (LAR-RPTPs), and many other members of this family have been reported to play roles in synapse development in both vertebrates and invertebrates (Table 1). The neuroligins and neurexins are heterophilic adhesion molecules characterized by the presence of an acetylcholine esterase-like domain in the former and 1–6 LNS (laminin A, neurexin, sex hormone-binding protein) domains in the latter. Multiple genes and extensive splicing generate many isoforms of these proteins that promote differentiation of both excitatory and inhibitory synapses. Interactions mediated through constituents of the extracellular matrix (ECM) are essential for synapse development at the neuromuscular junction (NMJ), and also appear to be important modulators of central nervous system (CNS) synapse development and function. Both classical ECM receptors (including integrins and the dystroglycan complex) and novel receptors (such as the receptor tyrosine kinase MUSK) mediate synapse development through ligation to ECM constituents. Virtually all of the synaptic adhesion molecules described above interact with scaffolds and other proteins within the cytoplasm. These interactions function to localize synaptic adhesion molecules at
38
synapses and to permit efficient activation of intracellular signaling cascades after synaptic adhesion molecule ligation. Many synaptic adhesion molecules contain PDZ (PSD-95/Dlg/ZO-1) domain-binding, polyproline and other motifs which interact with PDZ, SH3, and other domains of scaffold proteins. Scaffold proteins act as key organizers to control localization of SNAREs, receptors, other synaptic proteins and organelles on both sides of the synapse.
Classical Cadherins Classic cadherins are a family of calcium-dependent, homophilic, cell–cell adhesion molecules that have been implicated in many developmental processes. Several studies indicate that N-cadherin functions as a synaptic recognition molecule. In Drosophila, absence of N-cadherin prevents synapse formation by the axons of photoreceptors in the medulla. The axons from the individual photoreceptors fail to establish stable connections with their target neurons in the medulla. Although analyzed in less detail, inhibition of cadherin function in the chick optic tectum also disrupts synapse formation by the axons of retinal ganglion cells. Extrapolating from these observations, it seems highly likely that a large number of cadherin members expressed in the vertebrate CNS also affect synapse formation with many having overlapping and redundant functions. Mutation of cadherin-11 has been shown to result in enhanced synaptic plasticity (long-term potentiation (LTP)) in the hippocampus through unknown mechanisms. Much of the progress in understanding the roles of cadherins in forming adhesive junctions has been made in studies of epithelial adherens junctions that share many molecular components with neuronal synapses. These discoveries have provided important insights into the pathways regulating synapse formation. The ‘core’ cadherin complex is composed of a cadherin bound through its cytoplasmic domain to b-catenin which is bound in turn to a-catenin. In addition, the membrane-proximal portion of the cadherin cytoplasmic domain interacts less strongly with members of the p120 catenin family (p120 catenin, d-catenin, p0071, and ARVCF). p120 catenins have been shown to regulate cadherin surface stability and clustering as well as regulate Rho-family GTPases. Cadherin adhesion regulates organization of the actin cytoskeleton via several pathways through (1) direct interaction of a-catenin with actin or actin-binding proteins, including a-actinin, vinculin, spectrin, afadin,
Cell Adhesion Molecules at Synapses 39
Rabphilin Band4.1 Synaptotagmin
Ca2+channel Fer PTP-μ Cdc42 Rac Rho
Cadherin
Pre
Syntenin
a-neurexin
Lin-7/Veli PI3 kinase LAR S-SCAM GluR6 d-catenin
ERC2/CAST1
CASK/Mint Pre
b-neurexin
Neuroligin Protocadherin AMPAR/stargazin NMDAR
p120 catenin b-catenin a-catenin
Abl Cortactin mGluR1a NR2A Presenilin-1 S-SCAM
Post Formin a-actinin Afadin Spectrin
Post
PSD-95
S-SCAM
β-catenin NMDAR
Fyn
GKAP/Shank → b-Pix, IRSp53, cortactin
Pre Pre
Other known ligands: neurocan, integrins, axonin-1/TAG-1, contactin/F3/F11
Pre Other known ligands: L1, laminin, integrins NCAM
Nectin L1CAM FGFR-PLCg −DAG/IP3-PKC
Neuropilin-1 Post
RPTPα−Fyn Spectrin
Post
ZO-1
Tight junction
Afadin a-catenin
Post Ankyrin Spectrin
Cadherin adhesion
Rap-1 − p120 catenin
Agrin a
Laminin-b2
a ECM b
MUSK
b
a Dystroglycan
Integrin
VDCC
Neuregulin Src AKT/PKB p130Cas Talin FAK PDK1 ILK Vinculin Paxillin PINCH Parvin
b
ErbB2-4
Pre Post
Rapsin
Spectrin
Syntaxin and other vesicleassociated proteins
Utrophin/dystrophin MAGI-1c
Cadherin extracellular domain
Synaptic vesicle
LNS (laminin A, neurexin, sex hormonebinding protein) domain
Cytomatrix active zone vesicle
Immunoglobulin domain
Actin cytoskeleton
Fibronectin III domain PDZ-binding motif
Plasma membrane
Nerve terminal
SC
Muscle fiber
Lipid raft Basal lamina
Acetylcholine receptor
Figure 1 Structures and interactions of synaptic adhesion molecules. Top row of three diagrams illustrate interactions of the classical cadherin complex (left), protocadherin complex (middle), and neuroligin/neurexin complex (right). The second row depicts complexes formed by N(neural)CAM (left), L1-family CAMs (middle), and nectins (right). The bottom row illustrates interactions mediated as a result of integrin–ECM interactions (left), agrin/MUSK/dystroglycan interactions (middle), and laminin b2–calcium channel association (right). A lower power schematic of the NMJ is depicted at the lower right corner of this figure. Agrin, laminin b2, and laminin a4 chain-containing laminin trimers reside in the basal lamina depicted in this figure, while the agrin receptor MUSK is localized to the muscle plasma membrane. The three EGF-like repeats are not depicted in the b-neurexin structure. Pre, presynaptic compartment; Post, postsynaptic compartment; VDCC, voltage-dependent calcium channel; SC, Schwann cells. See text for details.
40 Cell Adhesion Molecules at Synapses Table 1 Roles of adhesion molecules at the synapse Function
Molecule
Class
Place of action
Comment
Target recognition
N-cadherin
Cadherin
Synaptic recognition
Protocadherins Flamingo
Cadherin Cadherin
Chick optic tectum, Drosophila visual system CNS?, spinal cord Drosophila visual system
Neurofascin
Ig
Sidekick
Ig
Mouse GABAergic cerebellar Purkinje cells Mouse retina
Nectin
Ig
Hippocampal pyramidal neurons
SynCAM Dasm, SALM
Ig Ig
Hippocampal pyramidal neurons Hippocampal pyramidal neurons
LAR-RPTP
Ig
Hippocampal pyramidal neurons
Induction and maturation of synapse
Neuroligin/ b-neurexin Laminin b2 Laminin b4 Reelin Syndecan-2 Versican CPG15 (neuritin) Synaptic plasticity
Hippocampal pyramidal neurons ECM ECM Proteoglycan ECM proteoglycan GPI-linked surface protein
Cadherin-11 NCAM, FasII, apCAM
Cadherin Ig
Integrins
ECM receptor
Neuromuscular junction (NMJ) in Drosophila and mouse Mouse cortex and cerebellum Drosophila NMJ, hippocampal pyramidal neurons Chick optic tectum Motor neurons Hippocampal pyramidal neurons Vertebrate CNS, Drosophila NMJ and Aplysia sensory neurons Central excitatory synapse
ZO-1, and formin-1 and through (2) regulation by p120 catenins of Rho GTPases and cortactin. Numerous kinases and phosphatases regulate the properties, stability, and interactions of the cadherin complex, including the protein kinases Cdk5, casein kinase I, Fyn, Yes, and Fer, and the protein tyrosine phosphatases PTP-m, LAR, SHP1/2, and PTP1B. In addition to regulators of the actin cytoskeleton, the proteins in the cadherin complex interact with many additional proteins that control intracellular signaling pathways, includingtranscriptionalregulatorslikeKaisoandT-cell factors (TCFs), scaffold proteins (such as A-kinase anchoring protein (AKAP)), and cytoplasmic enzymes including PI3 kinase, Cdk5, and receptor tyrosine kinases,pluscytoplasmicmotorproteinsandotherproteins including presenillin. In addition, b-catenin and d-catenin interact with PDZ-containing scaffold proteins that also mediate their synaptic functions. Several of the catenins appear to function as effectors of cadherins in controlling synapse development.
Determination of synaptic connectivity Interactions of R-cell growth cones with target neurons Subcellular specification of GABAergic synapses Lamina-specific synaptic connectivity in inner plexiform layer Regulation of axon–dendrite interactions Induction of synaptic differentiation Promotion of surface AMPA receptor expression and synaptic maturation Promotion of surface AMPA receptor expression and synaptic maturation Induction of synaptic differentiation Organization of active zone, alignment and maintenance of NMJ Maturation of CNS synapses Postsynaptic maturation Lamina-specific cue for presynaptic maturation of retinal ganglion axons Regulation of axonal branching and synapse maturation Regulation of hippocampal LTP Regulation of new synapse formation and structural synaptic plasticity Modulation of NMDA-mediated synaptic currents and synaptic plasticity
b-Catenin functions as a scaffold to localize synaptic vesicle pools to synaptic sites. The presence of a-N-catenin is required for normal spine maturation and stability. The absence of either d-catenin or p120 catenin severely perturbs central synapses. Absence of p120 catenin reduces dramatically the density of dendritic spines and synapses. Absence of d-catenin results in gross abnormalities in synaptic function and plasticity. In part, these proteins may function through regulation of the small G-proteins: Rac and Rho. They may also function through stabilization of surface cadherins or through regulation of tyrosine phosphorylation and control of cortactin activity.
Protocadherins Protocadherins are characterized by the presence of four to seven extracellular cadherin (EC) motifs, which are distinguishable from those present in other cadherin members, and absence of the conserved
Cell Adhesion Molecules at Synapses 41
motifs in the classic cadherins that mediate interactions with b-catenin and p120 catenin family members. About 80 different protocadherins have been identified and genes of nearly 60 members are tandemly arrayed in three clusters: the a-, b-, g-protocadherin genes (Pcdh-a, -b, and -g) on a single chromosome in mammals. (a-Protocadherins are also known as CNRs, cadherin-related neuronal receptors.) Recent phylogenetic analysis has characterized several conserved motifs shared by an additional subfamily of protocadherins, the d-protocadherins, whose genes are located on several different chromosomes. These include Pch1, Pch7, Pch8 (arcardlin), Pch9, and Pch11. The Pcdh-a and Pch-g clusters contain two large genomic regions, with tandem arrays of large exons expressing the variable extracellular domains followed by a set of common exons, each encoding a small portion of the shared C-terminal cytoplasmic domain. Pch-b lacks a shared constant region. Protocadherins are highly expressed in the CNS with individual members differentially expressed in subsets of neurons, suggesting that they help generate specific synaptic connections within the CNS. Genetic deletion of the Pch-g locus results in a reduction of inhibitory synapse density in the spinal cord. The mechanisms through which the protocadherins regulate synaptogenesis are not well understood. The strength of homophilic adhesion mediated by the extracellular domains of the protocadherins appears to be weaker than adhesion mediated by classic cadherin interactions. Recent evidence suggests that some protocadherins function through regulation of classic cadherin adhesion. Protocadherins may also have noncadherin proteins as ligands. For example, at least one of the Pch-a isoforms, Pch-a4, interacts through its RGD motif with b1 integrins. Protocadherin-7 interacts with PP1, a protein tyrosine phosphatase that dephosphorylates AMPA receptors and CAM kinase II.
NCAM NCAM was the first Ig superfamily member reported to function at the synapse. NCAM is encoded by a single gene with three isoforms of 180, 140, and 120 kDa produced by alternative splicing. The two larger isoforms are transmembrane proteins, differing in the presence of a differentially spliced exon that encodes an insert in the cytoplasmic domain, whereas NCAM-120 is a glycophosphatidyl inositol (GPI)linked protein that lacks a transmembrane and cytoplasmic domain. All isoforms have five Ig domains followed by two FNIII repeats. The NCAMs are believed to mediate both homophilic and heterophilic interactions. Recently identified interacting partners include the Ig-family cell adhesion molecule L1, the fibroblast growth factor (FGF) receptor, laminin, and
some integrins. Intracellular partners include the protein tyrosine phosphatase RPTPa, the tyrosine kinase p59Fyn, and spectrin. The most remarkable feature of NCAM is that it is virtually the sole acceptor for long linear homopolymers of a2,8-linked sialic acid residues (polysialic acid, PSA). The major phenotypes of the NCAM knockout can be mimicked by the enzymatic removal of PSA, indicating that the major essential function of NCAM is to provide a substrate for PSA addition, which is highly charged, thereby increasing the volume of extracellular space and inhibiting adhesive interactions mediated through other cell adhesion molecules. In vivo and in vitro results have demonstrated that NCAM regulates synaptic maturation at the NMJ and hippocampal synaptic plasticity (LTP), in part through associated PSA homopolymers. The NCAM homologs in invertebrates, Drosophila fasciclin II (FasII) and Aplysia apCAM, are involved in synapse growth, stability, and plasticity, and the expression of each is regulated by neuronal activity. Of particular interest, fasciclin II has been shown to function as both a permissive and restrictive regulator of synapse growth at the Drosophila NMJ, depending upon the comparative pre- and postsynaptic levels of this protein. The ability of fasciclin II to promote new synapse formation requires the presence of the Drosophila orthologs of amyloid precursor protein and the scaffold protein Mint. Fasciclin II forms a complex with each of these proteins, the latter through its PDZ-interaction motif. In Aplysia sensory neurons, downregulation of the NCAM homolog, apCAM, is required for the serotonin-induced enhancement of synaptic varicosity formation that accompanies synaptic facilitation. Normally, apCAM is downregulated following synaptic facilitation as a result of phosphorylation at a putative mitogen activated protein (MAP) kinase site. Overexpression of apCAM in sensory, but not motor neurons, completely suppresses the formation of new varicosities without interfering with the activation by serotonin of CREB. Thus, studies in both Drosophila and Aplysia indicate that NCAM homologs are important effectors of signaling pathways that control synaptic structural plasticity.
L1-Family CAMs The L1-type cell adhesion molecules are glycoproteins with six Ig, five FNIII, a single transmembrane, and a cytoplasmic domain. Four members (L1-CAM, Neurofascin, Nr-CAM, and CHL-1) have been identified in vertebrates while Drosophila has only one homolog, Neuroglian. L1-type CAMs mediate several aspects of neuronal development, including neuronal migration, neurite extension, axon
42 Cell Adhesion Molecules at Synapses
pathfinding, myelination, fasciculation, synapse targeting, and LTP. Among these family members, the role and function of L1 have been particularly intensely studied. In addition to binding L1 molecules present on other cells, L1 associates with additional ligands, including the proteoglycan neurocan, several RGDbinding integrins, and the CAMs axonin-1/TAG, and contactin/F3/F11. L1 also interacts with the axon guidance receptor neurophilin in both cis and trans forms to form a receptor for Semaphorin 3A. Of particular interest, localization of neurofascin at the initial axon segment of cerebellar Purkinje neurons results in specific localization of GABAergic synapses to this same region of the axon. At early stages of development both GABAergic synapses and neurofascin are less well localized. Refinement of neurofascin localization precedes and is required for localization of the GABAergic synapses. Neurofascin binds with high affinity to an isoform of ankyrin, an intracellular adaptor protein which is localized also to the initial axon segment and provides a scaffold for association with b-spectrin and the F-actin cytoskeleton. These interactions are required to localize both neurofascin and the GABAergic synapses. Observations in other CNS regions indicate that other L1 CAM family members are also preferentially localized to subdomains of neurons. It seems likely that they also help localize synapse formation. A number of human brain disorders have been linked to mutations in the L1 gene and L1 knockout mice reproduce several aspects of these phenotypes. It will be interesting to determine whether any aspects of the phenotypes associated with these disorders result from mis-targeting of synapses.
Other Synaptic Ig Superfamily Members SYG-1 is a member of the Ig superfamily identified in a genetic screen for synapse mutants in Caenorhabditis elegans that contains four Ig’s, a single transmembrane, and a cytoplasmic domain with a PDZ interaction motif. SYG-1 regulates the position of presynaptic terminal differentiation by HSNL axons which innervate the vulval muscles. It is related to Drosophila Duf/Kirre and the three vertebrate NEPH proteins that are important for organization of the slit diaphragm in the kidney glomerulus. SYG-1 binds to SYG-2, which has six Ig domains and one FNIII domain and is expressed transiently by primary vulval epithelial cells during synapse formation. SYG-2 provides an intermediate target that focuses accumulation of SYG-1 and synaptic vesicles. The mammalian homolog of SYG-2 is nephrin whose presence in podocytes is also essential for organization of the slit diaphragm in the kidney. The roles of
the NEPH family and nephrin in vertebrate synapses are not yet known, but recent work, stimulated by the observations in C. elegans, has documented their expression patterns in the CNS. In addition, the NEPH proteins have been shown to interact with CASK, a constituent of the CASK/MINT/Veli complex, which is thought to participate in pre- and postsynaptic organization. Since both nephrin and the NEPH proteins are expressed in the CNS, it will not be surprising if they prove to have important functions there. The Sdk CAMS, Sdk-1 and Sdk-2, contain six Ig domains, 13 FNIII repeats, a single transmembrane domain, and a cytoplasmic domain terminated by a PDZ domain-binding motif. Interestingly, in the retina the Sdk-1 and Sdk-2 proteins are found in nonoverlapping subsets of presynaptic (amacrine and bipolar) and postsynaptic (ganglion) cells that project to common inner plexiform (synaptic) sublaminae. Sdk-positive synapses are confined to specific sublaminae, and ectopic expression of Sdk in Sdk-negative cells redirects their arbors to Sdk-positive sublaminae, implying that the Sdk’s may control laminar specificity within the retina. The interactions and signaling mechanisms controlled by the Sdks that regulate synapse location are not known. Dasm1 is a recently reported Ig family member with five Ig-like domains, two FNIII domains, and a cytoplasmic domain with a C-terminal PDZ domain-binding motif. Dasm has been shown to promote dendritic elongation and branching as well as the maturation of excitatory glutamatergic synapses. Dasm interacts with PDZ-containing scaffold proteins, including Shank and S-SCAM. In its absence, a-amino3-hydroxy-5-methylsoxazole propionate (AMPA) receptor-mediated, but not N-methyl-D-aspartate (NMDA) receptor-mediated, synaptic transmission is severely impaired in cultured hippocampal neurons. A Dasm mutant lacking the PDZ-interaction motif does not promote AMPA receptor-mediated synaptic transmission. The extracellular ligands for Dasm have not been identified. The SALMs are another recently characterized family of five synaptic adhesion molecules (SALM1– 5) that contain six leucine-rich repeats plus single Ig and FNIII repeats in their extracellular domains. The SALMs are transmembrane proteins with cytoplasmic domains terminated by a PDZ domainbinding motif. Some members of the SALM family have been shown to promote excitatory synapse formation through interactions with PSD-95 and other cytoplasmic scaffold proteins. Members have also been shown to interact with NMDA and AMPA receptors. It is not clear whether they function as typical cell adhesion molecules by mediating adhesion with other cells.
Cell Adhesion Molecules at Synapses 43
The four nectins (nectin 1–4) are transmembrane proteins containing three extracellular Ig-like repeats and a cytoplasmic domain terminated by a PDZ domain-binding motif. While each of the nectins mediates homophilic adhesion, they promote stronger heterophilic adhesion with other family members. Several nectins also interact with the integrin aVb3. Each of the nectins interacts with the F-actin-binding protein afadin, thereby anchoring the nectins directly to the F-actin cytoskeleton. Through afadin-mediated interactions with a-catenin, the nectins recruit and collaborate with the cadherins to promote formation of adherens junctions in epithelial cells. Afadin also binds the small G-protein Rap1. The Rap1–Afadin complex has been shown to interact with p120 catenin and through p120 catenin to activate cadherinmediated adhesion. Active (GTP-bound) Rap promotes maturation of dendritic spines in the CNS and it seems possible that this is, in part, through regulation of the afadin–nectin–cadherin complex. Nectin-1 is preferentially expressed in axons. Preferential engagement of heterophilic adhesion between axonal nectin1 and dendritic nectin3 may provide a basis for the preferential interactions of axons with dendrites versus other axons. Members of the LAR family of receptor protein tyrosine phosphatases are transmembrane proteins with each containing three Ig and eight FNIII domains plus two cytoplasmic tyrosine phosphatase motifs. The cytoplasmic domains of these proteins interact with liprin-a/SYD-2, which binds to the PDZ-containing scaffold protein GRIP. This interaction promotes the surface expression and clustering of receptors at postsynaptic sites. In Drosophila and C. elegans, LAR and liprin-a have been also implicated in presynaptic terminal differentiation. In Drosophila, LAR-mediated synaptic differentiation is regulated by interactions of LAR with the cell surfaceassociated proteoglycans syndecan and dally-like. In cultured hippocampal neurons, LAR is concentrated at mature excitatory synapses where it promotes their development and maintenance through enhanced spine formation and surface AMPA receptor expression. The four members of the SynCAM family (also known as nectin-like proteins) constitute a small subset of the Ig superfamily, each member of which contains three extracellular Ig domains and a PDZinteracting motif in its short cytoplasmic domain. They were identified in a genomic search for members of the Ig superfamily capable of interacting with PDZ-domain-containing scaffolds. Similar to many other Ig family members, the SynCAMs mediate homophilic cell–cell adhesion. SynCAM1 is highly enriched in both presynaptic and postsynaptic membranes of neurons, whereas SynCAM3 is found in
nonjunctional contact sites of presynaptic nerve terminals, axons, and glia cell processes. Clustering of SynCAM1 is sufficient to trigger differentiation of functional presynaptic terminals. SynCAM1 overexpression also increases synaptic function in immature excitatory neurons. Interactions mediated through association with PDZ-domain-containing scaffolds are essential for its synapse-inducing activities. The PDZ domain-binding motif in SynCAM binds to the PDZ domains present in CASK and syntenin. Finally, another family of Ig superfamily members, named the netrin-G ligands (NGLs), have recently been implicated as regulators of synapse formation. The structure of the NGLs includes leucine-rich repeats plus an Ig domain in their extracellular domains, a single-pass transmembrane domain and a cytoplasmic domain terminated by a PDZ domainbinding motif. Three genes encode members of this family whose diversity is further increased by extensive differential splicing. The NGLs interact with the two members of the netrin-G family, proteins associated with the cell membrane through GPI motifs that are structurally related to, but functionally distinct from, the classical netrins that regulate axon guidance. The NGLs bind PSD-95, and NGL2 forms a complex in the brain with PSD-95 and NMDA receptors. Clustering of NGL2 has been shown to promote postsynaptic differentiation of excitatory, but not inhibitory synapses. NGL2 also induces presynaptic differentiation by contacting axons in vitro and it seems likely, although not proved, that differentiation is mediated through binding to netrin-Gs. Thus, interactions of the NGLs with nectin-G-proteins appear to be able to promote both pre- and postsynaptic differentiation in vitro and are strong candidates to control both the formation and specificity of synapse formation in vivo.
Neurexins and Neuroligins Neuroligins are composed of a cholinesterase-like domain, an O-glycosylation region, a single transmembrane domain and a short C-terminal cytoplasmic tail containing a PDZ-binding motif. Three genes encoding neuroligins are present in the rat and mouse while five are present in the human genome. The first neurexin to be characterized was identified as a receptor for the spider venom constituent a-latrotoxin. There are three neurexin genes, each of which encodes two neurexin isoforms with one promoter initiating transcription of a long mRNA encoding a-neurexin isoforms while a second promoter located within an intron initiates transcription of a shorter mRNA encoding b-neurexin isoforms. The extracellular domain of a-neurexin contains three
44 Cell Adhesion Molecules at Synapses
epidermal growth factor (EGF)-like domains, each of which is flanked by a pair of LNS domains. Instead of six LNS domains, only one is present in the b-neurexins, which lack all of the EGF repeats. The extracellular domains of both a- and b-neurexins are followed by a transmembrane domain and a cytoplasmic domain containing a C-terminal PDZ-interacting site. Differential splicing of the transcripts from each of the neurexin and neuroligin genes generates a tremendous number of variants of each protein family. Recent results have demonstrated that splicing controls the specificity of neurexin–neuroligin interactions as well as their activities in promoting excitatory and inhibitory synapse formation. In addition to binding neuroligins, some, but not all isoforms of neurexins interact with dystroglycan. On cultured hippocampal neurons, clustering of the b-neurexins or neuroligins triggers pre- or postsynaptic differentiation, respectively. In contrast to SynCAM, however, neuroligin overexpression does not result in enhanced synaptic transmission in immature excitatory neurons. Similar to the SynCAMs, the synapse-promoting activities of these proteins are dependent on their interactions with PDZ-containing scaffold proteins. The PDZ-interacting motif in neuroligin interacts with PSD-95 (which recruits AMPA and NMDA receptors) as well as with SSCAM (which interacts with AMPA receptors and b-catenin). b-Neurexin interacts with the CASK/ Mint/Veli complex, which is proposed to act as a scaffold for synaptic vesicle transport and localization. b-Neurexin also interacts with syntenin, which has recently been shown to interact with the presynaptic cytomatrix active zone protein ELKS/ERC2/CAST1, which binds in turn several additional active zone proteins, including piccolo, bassoon, and RIM. Interestingly, neuroligin1 is mainly localized to excitatory, whereas neuroligin2 is localized at inhibitory synapses. Overexpression and knockdown experiments in vitro suggest that the expression levels and localization of different neuroligins may play a crucial role in controlling the ratio of excitatory and inhibitory synapses. Mutations in human neuroligin genes are associated with a small percentage of autistic individuals.
ECM-Mediated Interactions ECM molecules regulate synapse development in both the central and peripheral nervous systems. At the NMJ, several constituents of the basal lamina have been shown to be essential for normal synapse formation. Most importantly, the presence of nervederived agrin in the basal lamina and its receptor, the tyrosine kinase MUSK, in muscle are absolutely necessary for synapse formation and maintenance.
In addition, several subunits of laminin also play important roles. The presence of the laminin b2 subunit in the laminin trimer within the synaptic basal lamina appears to play two distinct roles. First, this subunit prevents invasion of the synaptic cleft by Schwann cells, thereby preserving communication between the motor axon terminal and the muscle surface. Second, this subunit directly binds to N-type calcium channels. In its absence few active zones are seen at NMJs, even at sites where Schwann cells have not invaded the synaptic cleft, and synaptic transmission is severely impaired. In contrast, although synaptic transmission is not impaired in its absence, the laminin a4 subunit functions to ensure proper alignment of active zones in the nerve terminal with folds in the postsynaptic muscle membrane. In laminin a4 mutants, the active zones and folds are no longer aligned with each other. Laminin a4 is largely excluded from the small regions of basal lamina aligned with the active zones and muscle folds, suggesting that it may inhibit the formation of these specializations. In addition to MUSK, the integrins and dystroglycan complex function as receptors for basal lamina proteins at the NMJ. Absence of b1 integrins severely impairs NMJ. Mild deficits are observed in the absence of some constituents of the dystroglycan complex. Although there have been many studies, there are less obvious roles for ECM constituents at synapses in the CNS. The ECM glycoprotein reelin, best known for its role in controlling neuronal migration in the cortex and cerebellum, also promotes maturation of many CNS synapses through the same signaling pathway important for its role in cell migration – Src-family kinase-mediated phosphorylation of Dab, following engagement of the reelin receptors VLDLR and apoER2. Several additional ECM receptors also affect synapse maturation. For example, the cell surface proteoglycan syndecan-2 is enriched in the mammalian hippocampus and promotes morphological maturation of dendritic spines in cooperation with the receptor tyrosine kinase EphB2. Syndecan-2 is a transmembrane glycoprotein with a cytoplasmic PDZ-interacting motif, which interacts with CASK, syntenin, and synbindin. As described previously, syndecan regulates LAR in Drosophila. In Drosophila, elegant studies have demonstrated a crucial role for integrins in memory formation in the mushroom body. In hippocampal neuron cultures, several studies have demonstrated important roles for integrins in promoting synapse maturation. In vivo studies have shown that inhibition of RGD binding or b1 integrin function through genetic or pharmacological techniques impairs long-term plasticity. Ligand engagement by integrins results in activation of several intracellular signaling cascades including those
Cell Adhesion Molecules at Synapses 45
initiated by Src-family, focal adhesion kinase, and integrin-linked kinases, plus recruitment of scaffolds and adaptor proteins, such as talin and paxillin that lead to reorganization of actin cytoskeleton.
Concluding Remarks Much exciting evidence demonstrating important roles of synaptic adhesion molecules has been accumulated recently in both in vivo and in vitro model systems. We anticipate that future studies focusing on the crosstalk between different adhesion molecules will further illuminate our understand of synapse development and function. See also: Dendrite Development, Synapse Formation and Elimination; Postsynaptic Development: Neuronal Molecular Scaffolds; Retrograde Transsynaptic Influences.
Further Reading Berardi N, Pizzorusso T, and Maffei L (2004) Extracellular matrix and visual cortical plasticity: Freeing the synapse. Neuron 44: 905–908. Chih B, Engelman H, and Scheiffele P (2005) Control of excitatory and inhibitory synapse formation by neuroligins. Science 307: 1324–1328. Davis GW (2006) Homeostatis control of neural activity: From phenomenology to molecular design. Annual Review of Neuroscience 29: 307–323.
Dean C and Dresbach T (2006) Neuroligins and neurexins: Linking cell adhesion, synapse formation and cognitive function. Trends in Neurosciences 29: 21–29. Essner JJ, Chen E, and Ekker SC (2006) Syndecan-2. International Journal of Biochemistry and Cell Biology 38: 152–156. Gerrow K and El-Husseini A (2006) Cell adhesion molecules at the synapse. Frontiers in Bioscience 11: 2400–2419. Kim S, Burette A, Chung HS, Kwon S-K, et al. (2006) NGL family PSD-95-interacting adhesion molecules regulate excitatory synapse formation. Nature Neuroscience 9: 1294–1301. Montgomery JM, Zamorano PL, and Garner CC (2004) MAGUKs in synapse assembly and function: An emerging view. Cellular and Molecular Life Sciences 61: 911–929. Rougon G and Hobert O (2003) New insights into the diversity and function of neuronal immunoglobulin superfamily molecules. Annual Review of Neuroscience 26: 207–238. Salinas PC and Price SR (2005) Cadherins and catenins in synapse development. Current Opinion in Neurobiology 15: 73–80. Sanes JR and Lichtman JW (1999) Development of the vertebrate neuromuscular junction. Annual Review of Neuroscience 22: 389–442. Takeichi M and Abe K (2005) Synaptic contact dynamics controlled by cadherin and catenins. Trends in Cell Biology 15: 216–221. VanVactor D, Wall DP, and Johnson KG (2006) Heparan sulfate proteoglycans and the emergence of neuronal connectivity. Current Opinion in Neurobiology 16: 40–51. Waites CL, Craig AM, and Garner CC (2005) Mechanisms of vertebrate synaptogenesis. Annual Review of Neuroscience 28: 251–274. Yamagata M, Sanes JR, and Weiner JA (2003) Synaptic adhesion molecules. Current Opinion in Cell Biology 15: 621–632.
Glia and Synapse Formation: An Overview N J Allen and B A Barres, Stanford University School of Medicine, Stanford, CA, USA ã 2009 Elsevier Ltd. All rights reserved.
Introduction The majority of mature synapses are not just composed of a pre- and postsynaptic neuronal element, but also an astrocytic process that envelops the synapse (Figure 1). This close spatial relationship has led to the term ‘tripartite synapse’ to acknowledge the glial contribution. Their synaptic localization means that glial cells are ideally placed to monitor and respond to synaptic activity. Indeed, one glial cell can contact and ensheathe thousands of synapses formed between many different neurons. This morphological specialization suggests important roles for glia in synaptic development and function, which are reviewed here.
Glial Induction of Synaptogenesis Glial cells are in the right place at the right time to play an active role in neuronal synaptogenesis. For example, there is a temporal correlation between when retinal ganglion cell (RGC) axons reach their target structure, the superior colliculus, and when they form synapses. There is a delay of 1 week between target innervation and synaptogenesis, during which time glia are generated, which suggests that neurons require glial-derived signals to enable them to form synapses. The mechanism by which glial signals act is still unclear. Are they permissive in allowing synapses to form, with the location of synapses determined by neurons, or do they instruct neurons where to form synapses? The first demonstration of a role for glial cells in neuronal synaptogenesis was by Pfrieger and Barres in 1997. They showed that when RGCs were cultured in the presence of astrocytes they possessed significantly more synaptic activity than when cultured alone in serum-free media. This was later shown to be due to astrocytes inducing the formation of new synapses in addition to enhancing the efficacy of existing synapses. This effect was not due to astrocytes enhancing the survival of RGCs, as the same effect was seen when astrocytes were added after most of the neuronal death had occurred and RGC survival factors were present in the media. Contact between astrocytes and neurons was not required, as the same effect was seen if the astrocytes were grown in contact
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with the neurons or placed in a feeding layer above them, thus demonstrating that a soluble signal released from astrocytes increased the number of synapses. Interestingly, astrocytes in culture did not require a neuronal signal to stimulate secretion of synaptogenic factors – they were constitutively released. Addition of media conditioned by astrocytes was equally effective in inducing synapses as a feeding layer of astrocytes. There are a number of lines of evidence demonstrating that the synapses induced by astrocytes are functional (Figure 2). First, electrophysiological recordings from RGCs showed an increase in the frequency and amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) following exposure to astrocyte signals. Second, immunostaining for preand postsynaptic markers showed a sevenfold increase in co-localized pre- and postsynaptic puncta, defined as a synapse, following astrocyte addition. Third, FM dye-uptake studies showed that presynaptic vesicle recycling was enhanced by astrocytes. Fourth, electron microscopy analysis of the synapses induced between RGCs by astrocyte exposure showed them to be ultrastructurally normal, exhibiting presynaptic vesicles and an electron-dense postsynaptic density. Taken together these observations show that the synapses induced to form between neurons by glial cells are fully functional. Glia Induce Synapses in Multiple Neuron Classes
Since this initial study, numerous researchers have demonstrated a role for glial cells in inducing synaptogenesis in multiple classes of neurons. In addition to glutamatergic RGCs, spinal motor neurons, g-aminobutyric acid (GABA)ergic hippocampal neurons, and glycinergic neurons all show enhanced synapse formation in the presence of glial cells. Also, synaptogenic effects are not restricted to astrocytes. Oligodendrocytes and Schwann cells, whose primary function is to myelinate and ensheathe axons, have also been shown to induce neuronal synaptogenesis. Perisynaptic Schwann cells, specialized Schwann cells that are present at the neuromuscular junction synapse, are involved in guiding nerve terminals to the muscle during development. These specialized Schwann cells have been shown to enhance synaptogenesis between cultured motor neurons from rodents and between motor neurons and target muscle in Xenopus. Perisynaptic Schwann cell processes make contact with the muscle first and the nerve terminal uses the processes as a guide to locate and innervate the muscle. Selective ablation of perisynaptic Schwann
Glia and Synapse Formation: An Overview
Figure 1 Electron micrograph illustrating the close relationship of astrocytic processes to synapses in the hippocampus. Astrocytic profiles are illustrated (blue) in the vicinity of 11 synapses (arrows). On this section, three synapses have astrocytic profiles at their perimeters (arrowheads). Scale bar ¼ 1 mm. Reproduced from Ventura R and Harris KM (1999) Three dimensional relationships between hippocampal synapses and antrocytes. Journal of Neuroscience 19(16): 6897–6906, with permission.
cells in Xenopus tadpoles resulted in a decrease in the number of synapses that formed, the retraction of nerve terminals that had already formed synapses, and the loss of postsynaptic acetylcholine receptors. GABAergic hippocampal neurons undergo enhanced synaptogenesis in the presence of astrocytes, and as for RGCs, the astrocytic effect is via a soluble factor. Astrocytes induce an increase in pre- and postsynaptic clusters, increased surface levels of GABAA receptors, and increased frequency of miniature inhibitory postsynaptic currents (mIPSCs). One of the downstream effects of astrocyte signals is to regulate brain-derived neurotropic factor (BDNF) and tyrosine receptor kinase B (TrkB) signaling between neurons to enhance synaptogenesis, specifically the maturation of the postsynaptic site and insertion of GABAA receptors, but the identity of the astrocytic signal is currently unknown. Glia Induce Synaptogenesis via Secreted Factors
Much work has focused on identifying the soluble signals secreted by astrocytes that induce formation of functional synapses. To date two signals have been identified, thrombospondin (TSP) and cholesterol, but there are likely to be many more. TSPs are a family of large extracellular matrix proteins that can mediate both cell–cell and cell–matrix interactions. Addition of TSP to cultured RGCs increased the number of structural synapses that formed, to the same extent as that induced by
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astrocytes. TSP-1 and -2 are expressed in the developing brain during the peak period of synaptogenesis but decrease by adulthood. Mice in which both TSP-1 and -2 had been knocked out formed 30% fewer synapses in their brains. These observations have led to the hypothesis that immature astrocytes provide a developmental window during which synaptogenesis can occur, by producing a permissive environment via the secretion of TSP. Interestingly, the synapses induced by TSP are postsynaptically silent – they lack the a-amino-3hydroxy-5-methyl-4-isoxazole propionic acid subtype of glutamate receptor (AMPAR) but contain extrasynaptic N-methyl-D-aspartate receptors (NMDARs). This suggests that a second astrocyte-derived signal is required to trigger insertion of AMPARs into the postsynaptic membrane and make the synapse fully functional. Another astrocyte-secreted factor suggested to be involved in synaptogenesis is cholesterol bound to apolipoprotein (ApoE). Cholesterol is required for normal neuronal synaptogenesis and can be provided by glia when neuronal cholesterol is lacking. Cholesterol enhances presynaptic function and transmitter release, and contributes to dendrite development, although these effects are strongest in clonal-density cultures where neuronal cholesterol may be lacking. Indeed, it has been demonstrated that if cholesterol is depleted from neurons in culture, then surface AMPARs are reduced due to disruption of lipid rafts. Therefore, multiple astrocyte-derived signals promote both synapse formation and function. There are many signals yet to be identified, and it remains to be seen whether different signals induce the formation of different synapse types or between different classes of neurons. Contact-Mediated Synaptogenesis
Glia can also enhance synaptogenesis by contactmediated signaling. It has been shown that integrinmediated astrocyte–neuron contact was required to initiate synaptogenesis between embryonic hippocampal neurons in culture. In these experiments neurons were encircled by noncontacting astrocytes to provide trophic support, but very few synapses were formed even in the presence of soluble astrocyte factors. However, when an astrocyte was added directly to a neuron and physically contacted it, multiple synapses were formed. In contrast, direct contact by astrocytes was not needed to induce postnatal RGCs to form synapses. This raises the question of whether astrocytic contact during embryonic development can render neurons receptive to secreted factors that can induce synaptogenesis.
48 Glia and Synapse Formation: An Overview Plus glia
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Figure 2 Glia induce functional synapses to form between retinal ganglion cells in culture. (a) Electrophysiological recordings from cultured retinal ganglion cells. In the absence of glia, only a low frequency of small spontaneous synaptic currents is observed, whereas in the presence of glia large spontaneous synaptic currents are frequently observed. (b) Electron micrographs of synapses between retinal ganglion cell neurons cultured in the absence of glia and presence of glia. There is no apparent difference in synaptic ultrastructure between the two conditions. Scale bar ¼ 200 nm. (c) Glia increase the clustering of pre- and postsynaptic proteins and their colocalization. Staining of retinal ganglion cells with an antibody against the presynaptic marker synaptotagmin shows that in the absence of glia presynaptic markers are diffuse (left), whereas in the presence of glia presynaptic markers are discretely clustered (right). Staining of retinal ganglion cells with an antibody to the postsynaptic marker PSD-95 reveals that in the absence of glia there are relatively few PSD-95 puncta, whereas in the presence of glia numerous puncta are apparent. In the absence of glia there is little overlap between pre- and postsynaptic markers, whereas in the presence of glia there is a high degree of overlap. Scale bar ¼ 50 mm. Reproduced from Ullian EM, Sapperstein SK, Christophernon KS, and Barres BA (2001) Control of synapse number by glia. Science 291(5504): 657–661, with permission.
Glia Influence Synapse Elimination During development an excess of synapses are formed, but there is a reduction to the final adult number by a process of synapse elimination. It is hypothesized that elimination can be due to retraction of presynaptic terminals and axons or by degeneration of axons and subsequent phagocytosis of the debris by surrounding glial cells. It has now been suggested that glia are not just scavenging axonal debris, but are actively involved in the pruning process. In the Drosophila nervous system, glial cells were shown to engulf axonal varicosities prior to their degeneration. When the ability of glia to phagocytose was perturbed, axon pruning was significantly inhibited. A separate electron microscopy (EM) study showed
peroxidase from labeled neurons inside neighboring glial cells in lysosomal compartments, suggesting phagocytosis. These findings are not direct evidence, but strongly suggest that glia actively contribute to pruning in Drosophila. A similar process of axon removal by Schwann cells, termed axosome shedding, occurs during synapse elimination at the mammalian neuromuscular junction. This raises the possibility that glia actively contribute to synapse elimination in mammals. A combination of time-lapse imaging and serial EM showed that as axons disappeared they shed small membrane-bound particles termed axosomes, and that these axosomes appeared inside neighboring Schwann cells. It was suggested that this is a novel
Glia and Synapse Formation: An Overview
means of transferring information between the cytoplasmic compartments of different cell types.
Glia Modulate Synaptic Strength In addition to dictating the formation of synapses during development, glial cells are also actively involved in modulating the strength of mature synaptic connections. They do this by releasing factors that alter the number of neurotransmitter receptors present at synapses, and via factors that modulate the release of neurotransmitter and act as cofactors for receptor activation. Glia, particularly microglia, release cytokines, including tumor necrosis factor-a (TNF-a), which has been shown to modulate synaptic strength via effects on the trafficking of AMPA and GABAA receptors. Addition of TNF-a to cultured hippocampal neurons increased the surface levels of AMPA receptors while simultaneously decreasing surface GABAA receptors, thus leading to an overall strengthening of the synapse, a phenomenon known as homeostatic synaptic scaling. Blockade of the actions of endogenous TNF-a led to a decrease in surface AMPA receptors. A glial source for TNF-a was shown by experiments in which TNF-a knockout (KO) glia were cultured with wild-type (WT) neurons and synaptic scaling due to activity blockade was no longer present. There were no deficits in long-term potentiation (LTP) in hippocampal slices from TNF-a KO mice, showing that glial TNF-a is involved in controlling overall synaptic strength but has no role in plasticity. Glial Release of Transmitters and Neuromodulators
What are the effects of astrocytic transmitter release on synaptic transmission? Astrocytes have been shown to release ATP, which acts to inhibit overall neuronal excitability, and glutamate receptor agonists, which enhance overall activity levels. Astrocytic ATP release has been implicated in heterosynaptic depression, a process whereby synapses neighboring those that have undergone LTP are depressed. ATP can exert its effects either as itself or by being converted to adenosine in the extracellular space by ectonucleotidases. In the hippocampus glialderived adenosine has been shown to act on presynaptic A1 receptors, leading to inhibition of calcium channels, a decreased probability of vesicular release, and reduced frequency of sEPSCs. These effects were long-lasting and occurred over a large area – many synapses were inhibited. It is not clear if this inhibition was due to the diffusion of adenosine through the extracellular space or to the release of ATP from
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multiple regions on the astrocyte or multiple astrocytes within a network. It has been suggested that glutamate can be released from astrocytes in an activity-dependent manner. Astrocytic glutamate has been shown to activate extrasynaptic NMDAR on neighboring neurons, which could contribute to excitation of the cell. As there is little evidence that glutamate can be accumulated in glia, it remains uncertain whether these effects are attributable to glial glutamate release or release of some other neuroactive substance. In addition to the release of transmitters that directly activate neurotransmitter receptors, a role for astrocytes in releasing receptor co-agonists has been proposed. D-Serine, which has been suggested to be the endogenous ligand of the glycine binding site of the NMDAR, is expressed in glial cells but not neurons, and is necessary for NMDAR to be activated. There is circumstantial evidence that D-serine can be released from astrocytes in response to neuronal activity and that this release is necessary for hippocampal LTP. When astrocytes were cultured in contact with hippocampal neurons, the neurons were capable of undergoing LTP, whereas neurons grown in media conditioned by astrocytes (but without contact) were not. The addition of exogenous D-serine restored the ability of neurons grown in astrocyteconditioned medium to undergo LTP. These data, and further studies carried out in hippocampal slices, suggest that astrocytes are actively involved in synaptic plasticity by regulating the availability of D-serine.
Glia Influence Synaptic Structure, Stability and Location Studies on cultured neurons have shown that if synapses are induced by astrocytes and then the astrocytes are removed, these initial synaptic connections are lost. While the identity of this maintenance factor is unknown, this demonstrates that a constant astrocytic signal is needed in order to maintain synaptic connections. Studies have shown transient physical coupling between astrocytic processes and synapses via ephrin/ EphR interactions in the hippocampus. Dendritic spines express EphA4, which interacts with ephrin-A3 on astrocytic processes. Activation of EphA4 via ephrin-A3 caused a reduction in the length of dendritic spines by 30% and the collapse of 20% of spines, leading to an overall reduction in spine density. Conversely, inhibition of EphA4 caused an increase in spine length and a more disorganized pattern of spines appeared. EphA4 expression levels decrease during
50 Glia and Synapse Formation: An Overview
development, and it is present in an inactive form in the adult brain. This raises the question of whether developmental contact between astrocytic processes and dendritic spines plays a role in the elimination and localization of synapses. Live confocal imaging studies have produced new information about the motility of astrocytic processes at synapses. Imaging of green fluorescent protein (GFP)-labeled astrocytes in brain slices revealed that astrocytes frequently extend and retract fillipodial processes, on a timescale of minutes. Interestingly, this motility only occurred at synaptic sites, not at regions where astrocytes were contacting neuronal somata or blood vessels, and occurred in numerous brain regions. These observations add to the evidence that astrocytes are actively monitoring the synaptic environment. In the hypothalamo-neurohypophysial system (HNS) astrocytes are capable of fully retracting from synapses, in a reversible manner, under the control of hormonal signals. When oxytocin is released it causes astrocytes to retract from neighboring neurons, which then receive more synaptic inputs. Astrocytes were previously blocking these sites and preventing neuronal innervation. In this way astrocytes can control the location and number of synaptic inputs that a neuron receives, thus influencing the overall activity and output of the neuron. This is a normal physiological process, and a similar phenomenon has been demonstrated in the cerebellum when astrocytic transmitter receptors were altered. Climbing fibers release ectopic vesicles directly onto Bergmann glia (BG) in the cerebellum. Released glutamate activates AMPA receptors on the BG; glutamate spilling over from neighboring synapses would not be at a high enough concentration to do this. BG express calcium-permeable AMPA receptors lacking the GluR2 subunit, which usually confers calcium impermeability to the channel. Expression of GluR2 in BG led to retraction of glial processes from the Purkinje cells (PCs) that they are normally in close association with and caused the PCs to be aberrantly innervated by multiple climbing fibers. Therefore, ectopic vesicular release of glutamate directly onto BG AMPA receptors signals the Bergmann glial cell in a calcium-dependent manner to remain in close apposition with the PC it is surrounding. In the central nervous system it has been hard to interpret the effects of removal of astrocytes on synaptic function in mature animals. Experiments in which astrocytes have been ablated have had effects mainly in the cerebellum, with the loss of BG. This led to widespread death of granule neurons, presumably due to excitotoxicity from glutamate that accumulated, as the BG were no longer removing it from the extracellular space. The more global effects of
astrocyte ablation on neuronal survival make it hard to infer anything about effects on synaptic function. More subtle perturbations in astrocyte function do lead to alterations in synaptic physiology. Mice in which the astrocyte-specific intermediate filament protein glial fibrillary acidic protein (GFAP) has been deleted show altered synaptic plasticity, with enhanced levels of LTP. Deletion of GFAP will probably affect the structure of astrocytic processes and presumably their synaptic apposition, although whether this is responsible for the enhanced LTP remains to be determined. An elegant study using the frog neuromuscular junction as a model system investigated the effects of removing synaptic glia from a mature synaptic contact. Perisynaptic Schwann cells in an intact adult frog were selectively labeled with a monoclonal antibody, exposed to complement, and lysed via the complement cascade. This approach left the presynaptic motor neuron terminal and the postsynaptic muscle cell intact, but removed their synaptic partner, the perisynaptic Schwann cell. Interestingly, this had no effect on the structure or function of the synapse until 1 week after the ablation. At this time presynaptic function decreased by half and there was a tenfold increase in the retraction of presynaptic terminals from the muscle. Therefore, glial cells play a role in stabilizing mature synaptic contacts, both in the central and peripheral nervous systems. The studies discussed here demonstrate that glia are constantly monitoring the synaptic environment and can alter their structure and synaptic association in response to neuronal activity. This is yet another way in which they can control synaptic activity. Glial cells are ideally placed to respond to alterations in neuronal activity and to integrate information from many sources.
Conclusions Glial cells are intimately associated with synapses at all stages of development and adult life, both in the central and peripheral nervous system. Glia induce the formation of synapses via the secretion of synaptogenic substances, and secrete additional signals that regulate both pre- and postsynaptic function. Glia contribute to the maintenance of synaptic structure and arrangement, ensuring that neurons receive the correct pattern of innervation.
Further Reading Allen NJ and Barres BA (2005) Signaling between glia and neurons: Focus on synaptic plasticity. Current Opinion in Neurobiology 15: 542–548.
Glia and Synapse Formation: An Overview Auld DS and Robitaille R (2003) Glial cells and neurotransmission: An inclusive view of synaptic function. Neuron 40: 389–400. Fellin T and Carmignoto G (2004) Neurone-to-astrocyte signalling in the brain represents a distinct multifunctional unit. Journal of Physiology 559: 3–15. Feng Z, Koirala S, and Ko C (2005) Synapse–glia interactions at the vertebrate neuromuscular junction. The Neuroscientist 11: 503–513. Glia – Special issue (2004) Glial control of synaptic function. Glia 47(3): 207–298. Kettenmann H and Ranson BR (eds.) (2005) Neuroglia, 2nd edn. Oxford: Oxford University Press.
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Luo L and O’Leary DD (2005) Axon retraction and degeneration in development and disease. Annual Review of Neuroscience 28: 127–156. Pfrieger FW and Barres BA (1997) Synaptic efficacy enhanced by glial cells in vitro. Science 277: 1684–1687. Ullian EM, Sapperstein SK, Christophernon KS, and Barres BA (2001) Control of synapse number by glia. Science 291(5504): 657–661. Venture R and Harris KM (1999) Three-dimensional relationships between hippocampal synapses and artrocytes. Journal of Neuroscience 277: 6897–6900.
Active Zone P S Kaeser, University of Texas Southwestern Medical Center, Dallas, TX, USA ã 2009 Elsevier Ltd. All rights reserved.
Introduction Synapses are highly specialized contacts between nerve cells that transmit signals from the presynaptic neuron to the postsynaptic cell. They are composed of the presynaptic terminal, the synaptic cleft, and the postsynaptic membrane. The terminal of the presynaptic neuron translates the arriving action potential into a chemical signal, which in turn stimulates a response on the postsynaptic cell. In brief, the action potential induces opening of presynaptic voltagegated calcium channels, and the local increase in calcium in the presynaptic terminal promotes fusion of neurotransmitter filled synaptic vesicles at the presynaptic membrane on a millisecond timescale. Fusing vesicles release their content into the synaptic cleft, and the neurotransmitters induce a response in the target cell upon binding to postsynaptic receptors. This brief overview of synaptic transmission demonstrates the asymmetric nature of synapses, where both synaptic compartments – namely the presynaptic terminal and the postsynaptic membrane – contain highly specialized components. In principle, the presynaptic terminal is an organ of membrane trafficking and the postsynaptic compartment is a signal transduction machinery. The apparatus of postsynaptic reception is the postsynaptic membrane with the receptors and the postsynaptic density (PSD), which consists of signaling proteins, scaffolding proteins, and cytoskeletal proteins that enable the postsynaptic cell to sense the presynaptic release of neurotransmitters and respond appropriately. The fusion of synaptic vesicles at the presynaptic membrane is under tight spatial and temporal control. Synaptic vesicle fusion only occurs at highly specialized hot spots called active zones (AZs).
Definition of Active Zones and Functional Participation in the Synaptic Vesicle Cycle Originally, AZs were described morphologically in seminal studies by Couteaux and Pecot-Dechavassine and Akert et al., and they appear as electron dense material that is tightly attached to the presynaptic membrane (Figure 1). Functionally, AZs are defined as sites where neurotransmitters are released into the synaptic cleft, and biochemically, AZs are composed
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of a network of insoluble proteins, containing AZ-specific and other proteins. In the presynaptic nerve terminal, synaptic vesicles undergo a series of events in order to release their content into the synaptic cleft before being recycled and refilled. Investigators divide the synaptic vesicle cycle into nine steps. Synaptic vesicles are filled with neurotransmitters (step 1), and the vesicles then cluster around the AZ (step 2). They are docked to the AZ (step 3), where they gothrough an ATP-dependent priming reaction (step 4) that enables them to undergo fast, calcium-dependent fusion pore opening (step 5) which is initiated by the arriving action potential in the presynaptic nerve terminal. Upon releasing neurotransmitters into the synaptic cleft, three pathways have been proposed: Vesicles might remain in the docked stage (step 6, also referred to as ‘kiss-and-stay’), or they might be undocked and locally refilled (step 7, called ‘kiss-and-run’). Alternatively, they may be recycled through clathrin-dependent endocytosis of synaptic vesicles (step 8), which leads to either direct refilling or refilling after being passed through the endosome (step 9). It is important to note that only approximately 10–20% of all action potentials that arrive in a nerve terminal induce exocytosis of synaptic vesicles. AZs participate in multiple steps within the synaptic vesicle cycle. Their core function is to bring synaptic vesicles in close proximity to the presynaptic plasma membrane and to presynaptic calcium channels in order to enable the synapse for fast, calciumdependent fusion. In addition, AZs participate in a regulatory manner by modifying the probability of neurotransmitter release. This involvement in synaptic plasticity is mediated through AZ-specific proteins, and in principle it can be achieved by changing the number of either docked or primed vesicles. The molecular events that occur within the AZ in order to dock and prime synaptic vesicles and to change synaptic strength are under intense investigation, and aspects of these events are discussed after a more indepth examination of the structure of AZs.
Morphology of the Active Zone The fine structure of AZs is diverse among different species, and it is also variable among different kinds of synapses. Nevertheless, all AZs share common features. In electron microscopic images, they appear as a dense structure that is tightly attached to the presynaptic membrane (Figure 1). The electron dense nature of AZs reflects the very high protein content, and they consist of a network of highly
Active Zone
Figure 1 Electron micrograph of a central nervous synapse. Synaptic vesicles tether in the presynaptic terminal (pre) around the active zone (AZ), which consists of electron dense material that is tightly associated with the presynaptic membrane. The postsynaptic density (PSD) is precisely opposed to the AZ at the postsynaptic cell (post). Courtesy of Dr. Xinran Liu, University of Texas Southwestern Medical Center, Dallas, TX.
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insoluble proteins that interact with each other and with multiple cellular proteins. AZs are precisely opposed to PSDs, and they are separated from PSDs by the presynaptic plasma membrane, the synaptic cleft, and the neurotransmitter receptor containing postsynaptic plasma membrane. This architecture of precise opposition of the area of release and sensing of neurotransmitters is crucial because it minimizes the distance that neurotransmitters have to diffuse before they bind to the postsynaptic receptors. In mammalian synapses, the variability of this geometric assembly of the AZ and the PSD correlates with the synapse type. Whereas excitatory, glutamatergic synapses are typically asymmetric with a PSD that is thicker than the AZ, inhibitory GABAergic and glycinergic synapses appear more symmetric. In typical central nervous system mammalian synapses, electron microscopy has revealed that AZs consist of a hexagonal grid, and synaptic vesicles are embedded in depressions of the grid close to the presynaptic membrane (Figures 2(a) and 2(b)). The electron
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Figure 2 Fine structure of active zones (AZs) of a mouse central nervous system synapse and the frog neuromuscular junction. (a and b) Phosphotungstic acid (PTA) staining of a central nervous synapse in cross section (a) and as top view from the presynaptic terminal (b). PTA selectively visualizes the proteinacious contents of AZs and postsynaptic densities (PSDs); the synaptic plasma membrane is not stained. Whereas the PSD appears as a regular, dense band, the AZ forms dense projections (DPs) that protrude approximately 50 nm into the presynaptic terminal. The DPs are assembled in a regular, hexagonal grid and are connected with thin fibrils. This regular assortment allows the synaptic vesicles to closely approach the presynaptic plasma membrane in the depressions of the grid. Courtesy of Dr. Xinran Liu, University of Texas Southwestern Medical Center, Dallas, TX. (c) Cross section and a model (d) of a frog neuromuscular junction based on conventional two-dimensional electron microscopy. The AZ material is located between the synaptic vesicles (SVs) forming a regular structure with a central beam, ribs that connect the central beam with the SVs, and pegs that connect the ribs with the synaptic plasma membrane containing the calcium channels. pre, presynaptic membrane. Reprinted by permission from Macmillan Publishers Ltd: Nature (Harlow ML, Ress D, Stoschek A, Marshall RM, McMahan UJ (2001) The architecture of active zone material at the frog’s neuromuscular junction. Nature 409: 479–484), copyright 2001.
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dense protrusions, called dense projections, are relatively small compared to other synapses; they extend for approximately 50 nm from the presynaptic plasma membrane into the presynaptic terminal, and they are connected to each other by thin fibrils. Each dense projection has six immediate neighbors, and they are evenly spaced with a distance of approximately 50–100 nm. This highly organized architecture leaves slots for docking of synaptic vesicles in close proximity to the membrane. Harlow et al. analyzed the fine structure of the AZ at frog neuromuscular junction (NMJ) in detail (Figures 2(c) and 2(d)). At these synapses, the vesicles line up at both sides along an elongated ridge at the presynaptic terminals of motor neurons. Each synaptic vesicle is connected via ribs to a central beam, and each rib contains two connections (pegs) to the presynaptic plasma membrane. It is assumed that these pegs consist of macromolecules that connect the synaptic vesicle anchoring ribs with presynaptic calcium channels. Although this study leaves the molecular identities of ribs, pegs, and beams open, it clearly shows the highly specialized organization of presynaptic AZs. The size and morphology of dense projections vary greatly between different synapses and different species. In contrast to the rib- and beamlike structure at the frog NMJ, the Caenorhabditis elegans NMJ forms plaque-like AZs, and Drosophila NMJs contain T-shaped projections called ‘T-bars.’ At so-called ribbon synapses in the rat retina, long, prominent ribbonlike protrusions extend into the cytoplasm and tether synaptic vesicles around them. Many molecular and morphological studies have been performed on ribbon synapses because they are relatively easy to access and visualize. Another sensory synapse with a very specialized architecture is the hair cell in the auditory pathway, where ovoid or spherical dense projections are surrounded by a halo of synaptic vesicles. Both the photoreceptor synapse and the ribbon synapse are functionally very different because they do not respond to incoming action potentials but, rather, to sensory stimuli that produce graded receptor potentials. Their distinct function presumably led to a highly specialized morphology. Some investigators subdivide AZs morphologically into three compartments: the presynaptic plasma membrane, the cytomatrix immediately attached to the membrane (also referred to as ‘CAZ’ – cytomatrix of the active zone), and the electron dense protrusions into the cytoplasm, called ‘dense projections’ (also referred to as membrane thickenings or AZM – active zone material). Although this subdivision is morphologically correct, AZs are functional units, and many proteins involved in their architecture do not respect these morphological borders.
Molecular Components of the Active Zone AZs consist of a dense, insoluble protein network that is tightly attached to the presynaptic plasma membrane. Although several protein/gene families have been described to be involved in the formation, maintenance, and function of AZs, the molecular characterization has only just begun. Many interactions of proteins involved in AZs have been illustrated, but most of them are poorly understood in terms of their functional importance. In addition, the fact that most AZ proteins are large and highly insoluble makes biochemical characterization difficult, and the results of binding studies that have identified new interactions in vitro are difficult to confirm in vivo. Despite these difficulties, investigators have made significant progress in identifying molecules that participate in AZs. Gene knockdown/out studies in Drosophila, C. elegans, and mice have provided fascinating insights into potential AZ functions. In principle, AZs consist of two classes of molecules: non-AZ-specific and AZ-specific (or enriched) components. Among the non-AZ-specific members are five functional classes of proteins that are involved in different processes. First, there are proteins that are directly involved in synaptic vesicle fusion and associated with the presynaptic plasma membrane, such as the SNAREs SNAP25 and syntaxin, and also Munc18, a protein associated with the presynaptic release machinery that is required for synaptic transmission. Second, although the link between the AZ and the cytoskeleton is only poorly understood, it is clear that cytoskeletal proteins such as actin, spectrin, myosin, and tubulin participate in the structural backbone of the AZ. Third, synaptic scaffolding molecules are tightly associated with both AZ and PSD, and presynaptically CASK, Mints, Velis, and SAP97s have been identified. Fourth, as mentioned previously, the core function of the AZ is to bring together synaptic vesicles and calcium channels at the presynaptic terminal. Thus, voltage-gated calcium channels are an integral part of the AZ, but the molecular link between the intracellular portions of the channel subunits and the dense protein mesh of the AZ is only poorly understood. Finally, cell adhesion molecules play a crucial role during synaptic development and in mature synapses. Several molecules have been shown to reach from the AZ through the presynaptic plasma membrane into the synaptic cleft to form either homophilic or heterophilic interactions with proteins in the synaptic cleft or with proteins that reach through the postsynaptic membrane into the PSD. Such proteins are integrins, cadherins, neurexins, and SynCAMs. Several fascinating studies have shown how these transmembrane molecules induce synapse
Active Zone
formation, and neurexins with their postsynaptic interaction partner neuroligin have been suggested to be crucially involved in the pathogenesis of neurodevelopmental defects such as autism. Six families of AZ-specific proteins have been identified (Figure 3): Munc13s, Rab3A interacting molecules (RIMs), RIM-binding proteins (RBPs or RIM-BPs), a-Liprins, ELKS (proteins rich in E, L, K, and S), and Piccolo/Bassoon. They all consist of large multidomain proteins that are encoded by multiple genes and that are involved in protein– protein interactions. Figure 3 demonstrates that these proteins interact with each other in multiple ways to form the dense material that can be observed in electron microscopic images. Table 1 provides an overview of the members of these proteins, their evolutionary conservation, and their involvement in AZ function. Next, the functional and biochemical involvement of the AZ-specific proteins is discussed. Munc13s were initially identified in C. elegans as phorbol ester (b-PE)/diacylglycerol (DAG)-binding proteins encoded by the unc13 gene, and the phenotype in unc13 mutant worms suggested that these proteins were involved in neurotransmitter release. Since then, data obtained from C. elegans, Drosophila, and mouse models have suggested that synaptic transmission is crucially dependent on Munc13-mediated vesicle priming. In mammals, the four Munc13 genes (Munc13-1 to -4) produce three brain-specific Munc13 isoforms (Munc13-1,
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bMunc13-2, and Munc13-3), the ubiquitous Munc13-2, and the nonneuronal Munc13-4 (Table 1). Munc13s consist of a conserved C-terminal region that contains a C1 domain and two C2 domains flanking the Mun homology domain. The Mun domain is sufficient to rescue the defective synaptic transmission in synapses lacking Munc13. In addition, Munc13-1 and ubMunc13-2 have an N-terminal domain that binds to the RIM zinc finger and calmodulin and that contains a C2 domain (Figure 3). It is interesting to note that different synapses have been shown to express different Munc13 isoforms, and the expression pattern of these isoforms determines parameters of short-term synaptic plasticity. The central C1 domain of Munc13 binds b-PE/DAG, and this interaction mediates b-PE/DAG-induced augmentation in the hippocampus. The conserved C-terminal region of Munc13s has been suggested to bind to the synaptic plasma membrane SNARE syntaxin, two proteins that have been implicated in vesicular trafficking/ exocytosis (DOC2a and msec7-1 ARF-GEF), and a novel spectrin (spectrin b-spIIIS) that potentially links the AZ to the cytoskeleton. Functional experiments in C. elegans and in vitro binding studies have proposed a molecular mechanism by which Munc13s promote the open confirmation of syntaxin, which in turn results in a loose assembly of the SNARE complex, bringing synaptic vesicles into a fusion competent state. However, this model, which involves a direct interaction between Munc13 and syntaxin, remains controversial because nuclear magnetic resonance measurements
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munc homology domain
C
coiled-coil region G GIWA motif
FNIII
3 fibronectin type III repeats
C
C
G ELKS2
Figure 3 Schematic overview showing a representative member of each family of active zone (AZ) proteins. Interactions among AZ proteins are indicated with black arrows. All AZ-specific proteins are large proteins that contain multiple domains. Together, they form an insoluble protein scaffold that brings together synaptic vesicles, the presynaptic plasma membrane, and voltage-gated calcium channels at the presynaptic nerve terminal.
56 Active Zone Table 1 Active zone proteins Protein family
Mammalian protein isoformsa
Drosophila/C. elegans homologs
Proposed functions
Munc13s
Munc13-1 ubMunc13-2, bMunc13-2 Munc13-3 Munc13-4
Dunc13/unc-13
SV priming, scaffolding, synaptic plasticity, cytosceleton anchoring
RIMs
RIM1a RIM2a, RIM2b, RIM2g RIM3g RIM4g
RIM-PA/unc-10
SV docking and priming, short- and long-term plasticity, scaffolding, possibly channel anchoring
ELKS
ELKS1A, ELKS1B ELKS2
bruchpilot (brp)?/ELKS
Scaffolding, NF-kB signaling
Piccolo/ Bassoon
Piccolo/Aczonin Bassoon
Not identified to date
Scaffolding, anchoring, membrane trafficking, endocytosis
a-Liprins
Liprin-a1 Liprin-a2 Liprin-a3 Liprin-a4
Dliprin/syd-2
Scaffolding, receptor anchoring, membrane trafficking
RIM-BPs
RIM-BP1 RIM-BP2
Not identified to date
Scaffolding, channel anchoring
a
Each gene is represented on one line. For example, Munc13s are encoded by four mammalian genes, and the Munc13-2 gene produces two protein isoforms (ubMunc13-2 and bMunc13-2).
have not confirmed a direct interaction between the two proteins. The N-terminal RIM zinc finger forms a tripartite complex with Munc13 and Rab3A, a small GTPase that is located on synaptic vesicles. This biochemical interaction is important because it links Munc13s and RIMs directly to synaptic vesicles. A detailed morphological analysis of C. elegans has shown that RIM is necessary for connecting synaptic vesicles to dense projections but not for bringing them in close proximity to the synaptic plasma membrane. In addition, the RIM C2B domain has been suggested to bind to the calcium sensor synaptotagmin 1 in vitro, providing another potential link between AZs and synaptic vesicles. RIMs contain a number of domains and interaction motifs that are involved in scaffolding at AZs by binding to multiple other AZ proteins (Table 1). With their central PDZ domain, RIMs interact with ELKS, and with the C-terminal C2B domain they bind to a-Liprins (Figure 3). RIMs are a conserved gene family, with RIM-PA in Drosophila, unc10 in C. elegans, and four genes (RIM1–4) in mammals. The RIM2 gene has additional internal promoters to produce shorter isoforms (RIM2b and RIM2g), and multiple sites of alternative splicing enhance the variety of RIM1 and RIM2. The absence of RIM1a in a mouse model leads to a large deficit in multiple parameters of synaptic plasticity, including a
reduction of release probability at excitatory synapses and a lack of presynaptic long-term potentiation. This functional deficit is also reflected in the fact that these animals show severe shortcomings in various tests for learning and memory. Electrophysiological and behavioral analysis of the RIM1a mutant mice demonstrates how AZs are critically involved in modulating synaptic transmission, being an essential component for the brain’s capacity to adapt to environmental inputs. When RIM1a and RIM2a, two of the six major RIM isoforms, are absent, the mice show a drastic calcium-dependent release deficit at the NMJ, and they die immediately after birth. Thus, RIMs are critical not only for modulating the plasticity of synapses but also for normal synaptic release. RIM proteins contain a proline-rich region in the linker between the central and the C-terminal C2 domains that binds to RIM-BPs. RBPs are produced by two genes – RIM-BP1 (also referred to as PRAX-1) and RIM-BP2 (Table 1) – and contain three Sarc homology 3 (SH3) domains and three fibronectin type III repeats (Figure 3). No homologs have been identified in either C. elegans or Drosophila. An in vitro study showed that RIMBPs might bind to voltage-gated calcium channels with their SH3 domains, and thus they can potentially link RIMs and, indirectly, all other AZ-specific proteins to presynaptic calcium channels. In addition,
Active Zone
one study suggested a direct interaction between RIMs and calcium channels. Although these data are very interesting, they have not been confirmed in vivo, and functional data have only been provided for the RIM-BP–calcium channel interaction using a neurosecretory PC12 cell line. The in vivo existence of the potential calcium channel–RIM-RIM-BP complex remains to be elucidated. Liprins have been identified as a family of proteins that interact with LAR transmembrane tyrosine phosphatases, and they consist of two subfamilies – Liprin-a and Liprin-b. Liprin-b proteins are not brain specific, and Liprin-as are encoded by four genes (1–4), of which Liprin-a2 and Liprin-a3 are brain specific (Table 1). Mammalian Liprin-a function has only been addressed in vitro, but it has been found that Liprin-a3 forms a complex at the AZ with ELKS and RIM through its N-terminal coiled-coil regions (Figure 3). In C. elegans, it has been convincingly shown that the absence of the Liprin homolog syd-2 disrupts the regular AZ structure, and that syd2-deficient synapses have a defect in neurotransmitter release but a normal amount of synaptic vesicles. It has been concluded that syd-2 is involved in the structural assembly of AZs, and that it probably functions as an intracellular anchor of LAR transmembrane tyrosine phosphatase signaling in synaptic junctions. The structural involvement of Liprins was later confirmed by analysis of Dliprin-deficient flies, which showed a deficit in synaptic morphogenesis. Importantly, Liprins were the first AZ proteins to provide a molecular link between the presynaptic plasma membrane and the electron dense material at the AZ through their interaction with LAR transmembrane tyrosine phosphatases. In addition, Liprin-as have been shown to bind to GIT (which in turn binds to Piccolo) and the motor protein KIF1a via coiled-coil domains, to CASK and Liprin-b via the SAM domain, and to the scaffolding protein GRIP with its C-terminal PDZ binding motif, supporting the function as a scaffolding protein at AZs. ELKS proteins were originally described as proteins that bind to the IKK complex and take part in NF-kB signaling. Later, they were identified in mammals as Rab6-interacting proteins (Rab6IP2) that are involved in Golgi trafficking, and they were also described as AZ proteins called CAST. They were abbreviated as ERCs (ELKS/Rab6IP2s/CAST). A genetic analysis revealed that mammals have two genes, ELKS1 and ELKS2. The ELKS1 gene produces two main isoforms – a ubiquitously expressed ELKS1A and the AZ protein ELKS1B, which is alternatively spliced at the C-terminus and binds to the RIM PDZ domain. ELKS2 also has the PDZ binding motif at its C-terminus (Figure 3) and binds to RIM.
57
ELKS is conserved in C. elegans, and Liprin-a’s function in synapse assembly requires ELKS in the nematode NMJ. In Drosophila, there is only a distant homolog to ELKS, which has a partially conserved N-terminus but a nonhomologous C-terminus that resembles cytoskeletal proteins and lacks the RIMinteracting C-terminal motif. Investigators called this protein bruchpilot (brp; ‘crash pilot’) due to the unstable flight that these flies demonstrate when brp is absent. brp null mutant analysis suggested that T-bars, the dense projections at the neuromuscular synapse of Drosophila, are absent, calcium channel density is reduced, and synaptic release is deficient. Whether ELKS is equally important at mammalian synapses has to be investigated. Piccolo (also called Aczonin) and Bassoon are the largest AZ proteins (530 and 420 kDa, respectively), and they are exceptional because they are not conserved in C. elegans or flies (Table 1). They contain multiple domains, and 10 regions of homology between Piccolo and Bassoon have been summarized as Piccolo–Bassoon homology domains. The large multidomain structure that includes many protein interaction domains make it likely that Piccolo and Bassoon act as AZ scaffolding proteins. They have the potential to act as calcium sensors with their unusual C2 domains, and they are potentially targeted to the presynaptic plasma membrane via N-terminal myristoylation. Interestingly, in a mouse mutant model that expresses a truncated Bassoon which lacks the central region anchoring Bassoon to the AZ, the dense projections at ribbon synapses are freely floating in presynaptic terminals and synaptic transmission is impaired in these synapses. The delivery of these large proteins to AZs depends on an intact Golgi apparatus, in line with the hypothesis that presynaptic AZs might be preassembled as a complex in the Golgi apparatus and then transported in so-called Piccolo–Bassoon transport vesicles to the presynaptic terminal during synaptogenesis.
Conclusion Presynaptic AZs consist of a highly specialized network of proteins that acts in neurotransmitter release. Although there is wide variability in the morphology of AZs in different synapses and among various species, their function and molecular architecture are largely conserved. We have only begun to understand how the various proteins present in AZs work together, and crucial issues such as the tight membrane association, the link to the cytoskeleton, the docking mechanism of synaptic vesicles, and the clustering of calcium channels at these hot spots of neurotransmitter release are only partially
58 Active Zone
understood. A fascinating question that has not been addressed regards the use-dependent structural plasticity of AZs and how it is involved in processes of memory formation and learning. See also: Synaptic Plasticity: Short-Term Mechanisms; Synaptic Vesicles.
Further Reading Akert K, Moor H, and Pfenninger K (1971) Synaptic fine structure. Advances in Cytopharmacology 1: 273–290. Augustin I, Rosenmund C, Sudhof TC, and Brose N (1999) Munc13-1 is essential for fusion competence of glutamatergic synaptic vesicles. Nature 400: 457–461. Couteaux R and Pecot-Dechavassine M (1970) Synaptic vesicles and pouches at the level of ‘active zones’ of the neuromuscular junction. Comptes Rendus Hebdomadaires des Seances de l’Academie des Sciences 271: 2346–2349. Harlow ML, Ress D, Stoschek A, Marshall RM, and McMahan UJ (2001) The architecture of active zone material at the frog’s neuromuscular junction. Nature 409: 479–484.
Phillips GR, Huang JK, Wang Y, et al. (2001) The presynaptic particle web: Ultrastructure, composition, dissolution, and reconstitution. Neuron 32: 63–77. Schoch S and Gundelfinger ED (2006) Molecular organization of the presynaptic active zone. Cell and Tissue Research 326: 379–391. Sudhof TC (2004) The synaptic vesicle cycle. Annual Review of Neuroscience 27: 509–547. tom Dieck S, Sanmarti-Vila L, Langnaese K, et al. (1998) Bassoon, a novel zinc-finger CAG/glutamine-repeat protein selectively localized at the active zone of presynaptic nerve terminals. Journal of Cell Biology 142: 499–509. Wang Y, Liu X, Biederer T, and Sudhof TC (2002) A family of RIM-binding proteins regulated by alternative splicing: Implications for the genesis of synaptic active zones. Proceedings of the National Academy of Sciences of the United States of America 99: 14464–14469. Zhai RG and Bellen HJ (2004) The architecture of the active zone in the presynaptic nerve terminal. Physiology 19: 262–270. Zhen M and Jin Y (1999) The liprin protein SYD-2 regulates the differentiation of presynaptic termini in C elegans. Nature 401: 371–375.
Calcium Channel Subtypes Involved in Neurotransmitter Release R W Tsien, Stanford University Medical Center, Stanford, CA, USA ã 2009 Elsevier Ltd. All rights reserved.
Ca2+ Channels in Excitation–Response Coupling Voltage-gated calcium channels (Ca2þ channels) are members of a superfamily of voltage-gated ion channels which also includes sodium and potassium channels (Naþ and Kþ channels, respectively). Expressed in a wide variety of cell types, Ca2þ channels transduce changes in membrane potential (depolarization) to increased intracellular [Ca2þ], which in turn triggers diverse cell biological processes, including secretion, contraction, migration, proliferation, and transcription. Such ‘excitation–response coupling’ is exemplified by the link between presynaptic depolarization and the triggering of neurotransmitter release, the main topic of this article. Voltage-gated Ca2þ channels offer many advantages for excitation–response coupling. The opening of a voltage-gated Ca2þ channel is a powerful mechanism for delivering the second messenger (Ca2þ) very quickly. There is no lack of Ca2þ in the external milieu, and membrane transport mechanisms operate to keep cytosolic Ca2þ low, providing an approximately 10 000-fold concentration gradient for supporting a large net influx. Because basal levels of free Ca2þ concentration are low (typically 107 M), influx can cause a rapid and large percentage change in intracellular Ca2þ. Unlike Ca2þ channels in membranes that surround intracellular Ca2þ stores, Ca2þ channels in the plasma membrane have direct access to the transmembrane voltage, a global indicator of cellular activity. A rapid, voltage-dependent closing of the channel allows for dissipation of the message by diffusion and strong, rapid local buffering thereby achieving spatiotemporally precise signaling. To capitalize on all of these advantages, Ca2þ channels must satisfy a number of structural and biochemical requirements. First, they should be highly voltage dependent, making them quick to both open and close in response to changes in membrane potential (e.g., action potentials and excitatory postsynaptic potentials). Second, they must be highly permeable to Ca2þ but not to other ions, such as Naþ or Kþ. Calcium channels must also be properly localized near the relevant targets of Ca2þ regulation, allowing for local increases in Ca2þ concentration. Finally, they must be subject to modulation and diverse
enough to allow particular classes of calcium channels to be selectively regulated. All of these requirements seem to have been fulfilled by the Ca2þ channels found in excitable cells in general and those in nerve terminals in particular. Given the wide range of downstream effects mediated by Ca2þ entry, it is perhaps not surprising that a rich diversity of Ca2þ channels has evolved, with individual channel types specialized for, but not exclusively limited to, specific biological roles.
Steps in Coupling Excitation to Neurotransmitter Release Excitation–secretion coupling at typical glutamatergic nerve terminals can be described by the following steps (Figure 1, top): 1. Action potential in axon leads to electrotonic depolarization of axonal bouton. 2. Depolarization causes opening of Naþ channels and action potential in bouton. 3. Action potential depolarization causes opening of voltage-gated Ca2þ channels. 4. K channels open, repolarizing voltage and increasing the driving force for Ca2þ to pass through Ca2þ channels. 5. Ca2þ rises sharply in a microdomain around the mouth of the Ca2þ channels. 6. Ca2þ binds to Ca2þ sensor for vesicle fusion, synaptotagmin triggering vesicle to fuse to surface membrane. 7. Fusion pore opens, providing passage between one or more vesicles containing the neurotransmitter glutamate and the synaptic cleft. 8. Neurotransmitter (e.g., glutamate) passes through fusion pore and diffuses in the cleft. 9. Neurotransmitter binds to multiple sites on postsynaptic receptors (e.g., AMPA receptors), causing their channels to open. 10. Excitatory postsynaptic current ensues. This provides a general cell physiological framework for understanding the role of specific types of Ca2þ channels. The process of neurosecretion relies on two distinct cycles – the classical Hodgkin cycle for nerve excitation (Figure 1, bottom left) and the Heuser–Reese cycle for the clathrin-mediated retrieval of vesicle membrane components (Figure 1, bottom right). Opening of gates in the Hodgkin cycle controls Ca2þ entry via Ca2þ channels, which in turn regulates vesicle priming and fusion and possibly endocytosis as well.
59
60 Calcium Channel Subtypes Involved in Neurotransmitter Release Action potential in bouton generated by opening of Na+ channels +
+
+
+
+
+
+
Presynaptic + bouton + +
+ + +
Depolarization opens Ca2+ channels + + + + + + + + + Ca2+ + ++ ++
+ + + + + + +
Ca2+ elevation occurs in microdomain
+ + + ++
Glutamate passes through fusion pore and diffuses in the cleft
Ca2+ binds to synaptotagmin causing opening of fusion pore
Ca2+
+ +
+ + +
+
+
Dendritic spine
+ +
+ + +
+ +
+
+
Opening of AMPA channel generates EPSC
ΔVm
Δgates
Ionic flux
Ca2+ flux
AP
Ca2+ channels
Exocytosis/ endocytosis
Figure 1 (Top) Schematic showing steps in the process of chemical synaptic transmission, exemplified by an excitatory synapse with postsynaptic receptors for the fast neurotransmitter glutamate. (Bottom) Ca2þ forms the link between the classical Hodgkin cycle of excitability and the synaptic vesicle cycle of the kind envisioned by Heuser and Reese. AP, action potential at the presynaptic terminal; Vm, membrane potential. (Top) Reproduced from Lisman JE, Raghavachari S, and Tsien RW (2007) The sequence of events that underlie quantal transmission at central glutamatergic synapses. Nature Reviews Neuroscience 8: 597–609, with permission. (Bottom) Reproduced from Tsien RW and Barrett CF (2004) A brief history of calcium channel discovery. In: Zamponi G (ed.) Voltage-Gated Calcium Channels. New York: Kluwer/Plenum, with permission.
Discovery of Multiple Ca2+ Channel Types Ca2þ channel types were initially classified according to their voltage threshold for activation through the work of Susumu Hagiwara and others. High-voltageactivated (HVA) channels require strong depolarization, whereas low-voltage-activated (LVA) channels activate with relatively little change in membrane potential. This distinction was reinforced by the finding of Armstrong and colleagues that LVA channels deactivate (shut off) much more slowly with hyperpolarization than HVA channels. Beginning in 1985 and for approximately the next 10 years, understanding of the multiple types of Ca2þ channels was significantly advanced by analysis of their biophysical, pharmacological, and molecular properties in combination. It is now generally agreed that the HVA channels most important for mammalian
neurotransmission (N-, P/Q-, R-type) are different from the HVA channels that dominate excitation– contraction or excitation–transcription coupling (L-type) or the LVA channels that help govern pacemaker activity (T-type). The presynaptic Ca2þ channels are exemplified by N-type channels, first discovered in recordings from cell bodies of sensory neurons and later found to support neurotransmitter release from sympathetic neurons or cholinergic nerve terminals in the ciliary ganglion. P/Q- and R-type channels were discovered later and also contribute to varying degrees to neurotransmission at various central nervous system (CNS) synapses, including large and relatively accessible synapses discussed later. P/Q-, N-, and R-type channels are generated by pore-forming subunits known as CaV2.1, CaV2.2, and CaV2.3, respectively; each is
Calcium Channel Subtypes Involved in Neurotransmitter Release
the specific target of a blocking toxin (o-AgaIVA, o-CTx-GVIA, and SNX-482, respectively). Auxiliary subunits (a2-d and b) contribute to proper cellular trafficking and function of the pore-forming a1 subunits. The single-letter terminology for Ca2þ channel types (L-, N-, and T-type) reflects initial distinctions based on patch clamp recordings of unitary channel activity. The observed pattern of unitary channel properties – their slope conductances and activation and inactivation properties – did not fit neatly into the LVA/HVA categorization of Hagiwara. Closer examination of whole cell and single channel recordings made it clear that three channel types could be distinguished, then called T-type (LVA) and N- and L-type (both HVA). The L-type channels were so named because they had a large unitary conductance to Ba2þ and supported a long-lasting Ba2þ current (different properties were found with Ca2þ as the charge carrier, but the Ba2þ currents provided the most distinctive channel profiles). Importantly, the L-type channel activity was similar to that found in heart cells and could be distinguished by a profound enhancement with the dihydropyridine drug Bay K8644. The T-type channels generated tiny unitary Ba2þ currents, gave rise to a transient average current, showed characteristically slow deactivation following sudden repolarization, and were also found in heart cells. Finally, the Ntype channels were largely specific to neurons, had an intermediate conductance to Ba2þ, and although they required negative holding potentials to be available for opening, they were activated at high voltages, indicating that they were neither T- nor L-type. The diverse nature of the Ca2þ channel, advanced on the basis of patch clamp recordings, has received ample Patch clamping of neurons (1985) TP HP L-type
N-type
T-type
61
corroboration from molecular biology. Sequence analysis has confirmed and extended the original tripartite classification (Figure 2). The identification of N-type channels presaged the discovery of the CaV2 subfamily of a1 subunits, now known to be critical for neurotransmission. Further experiments in the early 1990s resulted in the identification of two additional members of the subfamily exemplified by N-type channels. Llina´s and colleagues discovered a noninactivating, dihydropyridine-insensitive current in the cell bodies of cerebellar Purkinje neurons, henceforth called P-type. Others demonstrated that P-type channels were exquisitely sensitive to the funnel web spider toxin peptides (e.g., o-Aga-IVA). Cerebellar granule displayed a prominently inactivating current with somewhat different properties, labeled Q-type. Multiple lines of evidence obtained with antibodies, antisense techniques, and gene knockout have firmly established that P- and Q-type currents both arise from the same pore-forming subunit, now known as CaV2.1 or a1A. For example, both current types are completely eliminated in cerebellar Purkinje and granule neurons of a1A/mice. The original distinctions between the P and Q components have been mostly accounted for by splice variation in the a1A gene, hence the currently accepted terminology of ‘P/Q-type’ channel. The actions and specificity of available toxins and pharmacological compounds are sufficient to allow a separation of current components based primarily on pharmacology. A combination of nimodipine (L-type-specific), o-conotoxin-GVIA (N-typespecific), and o-Aga-IVA (P/Q-type-specific) can be used to tease apart the various Ca2þ current types in cerebellar granule neurons. Application of inhibitors Cloning of CaCh a1 subunits (up through 1998) Cav a1 Type 1.1 S L 1.2 C L 1.3 D L 1.4 F L 2.1 A P/Q 2.2 B N 2.3 E R 3.1 G 3.2 H 3.3 I
T T T
40 60 80 100 Percent homology Figure 2 Agreement between channel biophysics studied by patch clamping neurons and molecular cloning of a1 subunits in tripartite Ca2þ channel classification. (Left) Distinctions between three types of Ca2þ channels based on recordings of unitary channel activity. N-type channel activity was separated from L- or T-type activity on the basis of its requirement for strongly negative holding potential (HP) and strongly depolarized test potential (TP), as well as its unresponsiveness to dihydropyridines such as Bay K8644 (not shown). (Right) Ensuing molecular cloning revealed three major subfamilies of pore-forming a1 subunit.
62 Calcium Channel Subtypes Involved in Neurotransmitter Release
of the three known channel types revealed a fourth component with kinetic and pharmacological properties distinct from L-, N-, and P/Q-type channels. Because this residual current was resistant to the known inhibitors, it was tentatively named R-type. This current showed unique properties, including very rapid decay and an unusual sensitivity to block by Ni2þ. Various approaches indicate that R-type current is supported by channels encoded by CaV2.3. In several neuronal systems, R-type current can be blocked by a specific inhibitor – a spider toxin peptide known as SNX-482.
In General, Multiple Types of Ca2+ Channels Contribute to Central Nervous System Neurotransmission Ca2þ channels from the CaV2 subfamily are the primary types responsible for excitation–secretion coupling at fast excitatory and inhibitory synapses in the brain. At most of these CNS synapses, the neurotransmitter release is accomplished through synergistic actions of more than one type of channel, most prominently P/Q- and N-type channels. This seems to be the case for typical, approximately 1 mm CNS nerve terminals as well as large presynaptic structures (e.g., the calyx of Held of the auditory brain stem and the mossy fiber synapse of the hippocampus). R-type channels also participate in triggering exocytosis in those instances in which its potential contribution has been carefully explored. Thus, all three members of the CaV2 subfamily may play a significant role in fast central neurotransmission. The biophysical properties of these channels (e.g., their rapid activation upon strong depolarization and brisk deactivation following repolarization) are well suited to responding to typical presynaptic action potentials, which reach strongly positive overshoots but have a brief duration on the order of 1 ms.
Intimate Relationship between Ca2+ Entry and Neurotransmitter Release Only Ca2+ Channels Close to the Release Machinery Matter
As Ca2þ enters, it can act rapidly because only Ca2þ elevation near the Ca2þ channels is required, rather than a change in the bulk cytoplasm. This is because the Ca2þ sensors that trigger vesicle release are strategically located within a ‘microdomain’ near the Ca2þ channels. The first support for this concept came from work on the giant synapse of the squid and indicated that Ca2þ could rise to 200 mM in the microdomain. An important advance occurred with the development
of structural methods, including electron tomography, that helped localize the intramembranous particles thought to be Ca2þ channels. At the neuromuscular junction, it has been estimated that vesicles are positioned within 20 nm of the Ca2þ channels – close enough that the vesicles may even hinder the diffusion of Ca2þ away from the channel. Transmitter Release is Triggered by Ca2+ Entry, whereas Depolarization per se Exerts No Direct Effect
The effects of a rise in presynaptic Ca2þ and membrane depolarization have been dissected apart by using light-induced release of caged Ca2þ to drive vesicle fusion. In these circumstances, strong depolarizations comparable to presynaptic action potentials failed to increase synaptic responses. This excludes the idea that neurotransmission is jointly dependent on both [Ca2þ] and presynaptic membrane potential, although it remains possible that presynaptic membrane voltage can have important modulatory effects, for example, on signaling by G-proteins. The Ca2+ Sensor for Rapid Vesicle Release Is Synaptotagmin
There is substantial evidence that the Ca2þ sensor for fast neurotransmission is a Ca2þ-binding protein known generically as synaptotagmin. Various isotypes of synaptotagmin, most generally synaptotagmin 1 but also synaptotagmins 2 and 9, can act as a trigger for rapid transmission, as indicated by experiments using knockout mice. There are two main theories about how synaptotagmin works: by unleashing the SNARE machinery by removal of a tonic inhibitory brake or by adding a fusion-promoting influence, derived from Ca2þ/syt 1 binding to membrane phospholipids, to that already provided by preformed SNARE complexes. Transmitter Release is Related to Ca2+ Entry by a Steep Power-Law Function
In a classical study on the frog neuromuscular junction, Dodge and Rahamimoff showed that neurotransmitter release was a steep function of external Ca2þ concentration and, by extension, Ca2þ influx. Later investigators extended the analysis to direct measurements of presynaptic Ca2þ currents, and cytosolic Ca2þ changes, converging on the idea that the Ca2þ sensor for secretion behaves as if it were cooperatively activated by the binding of four or five Ca2þ ions. The basis of this cooperativity remains unclear. Synaptotagmin has five Ca2þ binding sites, suggesting that the cooperative properties of single synaptotagmin molecules might be sufficient to
Calcium Channel Subtypes Involved in Neurotransmitter Release
account for the Ca2þ cooperativity of release. Consistent with this, mutations in synaptotagmin that affect Ca2þ binding reduce this cooperativity. However, additional factors might influence the observed Ca2þ cooperativity. Vesicles contain several synaptotagmin molecules, possibly working together to produce release; cooperativity might be influenced by other molecules and the combined effects of multiple SNARE complexes. Multiple Ca2+ Channels (and Channel Types) Provide Input to a Common Ca2+ Sensor
Pharmacological studies are consistent with regard to a scenario in which a mixed population of Ca2þ channels coexist at single release sites and contribute jointly to the local Ca2þ transient that operates on the Ca2þ sensor to trigger vesicular fusion. The convergence of Ca2þ delivered by different types of channels can be manifested as synergism or redundancy, depending on the experimental conditions (Figure 3).
Remaining fraction of synaptic currents (%)
120
a
Cerebellum i.p.s.cs
120
63
With normal action potentials as the stimulus, block of either P/Q- or N-type (but not L-type) Ca2þ channels can cause a significant diminution of transmitter release (Figure 3(a)). In the illustrated example, the percentage inhibition of neurotransmission for block of P/Q-type channels (90%) and for block of N-type channels (40%) totals more than 100%. This makes sense because both Ca2þ channel types contribute to Ca2þ influx at individual release sites and thus provide additive fluxes to take advantage of the steep power law for neurotransmission. Another way to establish that both channel types coexist at individual release sites is to examine the consequences of a depolarization imposed by application of Kþ-rich solution, a much stronger stimulus for channel activation than a single action potential. In this case, both types of channels must be blocked to appreciably inhibit transmission (Figure 3(b)). The explanation for this redundancy is simple: Provided that the depolarizing stimulus is strong and sustained, Ca2þ influx
Spinal cord i.p.s.cs
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100
100
100
80
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w –Aga–IVA w –CgTx Nicardipine
3.0 Cumulative dopamine release (% total)
2.5 2.0 1.5 1.0 0.5 0.0 10 b
100 [K+] (mM)
Figure 3 Differing perspectives on transmitter release and the role of N- and P/Q-type Ca2þ channels. (a) Contributions of various Ca2þ channels at CNS synapses when the stimulus for Ca2þ is a single action potential. Shown are the proportions of synaptic currents remaining after blockade of P/Q-type channels (o-Aga-IVA present), with N-type channels blocked (o-CgTx present), or with L-type channels blocked (nicardipine). Reproduced from Takahashi T and Momiyama A (1993) Different types of calcium channels mediate central synaptic transmission. Nature 366: 156–158, with permission. (b) Effect of blockade of P/Q- or N-type channels, individually or in combination, on dopamine release from rat striatal synaptosomes. Note that with strong depolarization (external potassium concentration >100 mM), both kinds of channels must be blocked in order to strongly inhibit neurosecretion. Circles, no channel inhibition; triangles, N-type channels blocked; upside down triangles, P/Q-type channels blocked; diamonds, both N- and P/Q-type channels blocked. Reproduced from Turner TJ, Adams ME, and Dunlap K (1993) Multiple Ca2þ channel types coexist to regulate synaptosomal neurotransmitter release. Proceedings of the National Academy of Sciences of the United States of America 90(20): 9518–9522, with permission.
64 Calcium Channel Subtypes Involved in Neurotransmitter Release
through either type of channel is sufficient to drive secretion, and the other type can be regarded as ‘spare channels.’ The synergistic or redundant actions of multiple channel types can be observed in the same experiment (Figure 4). The switch between cooperative and redundant behavior can be accomplished simply by prolongation of presynaptic action potentials with the Kþ channel blocker 4-aminopyridine. Prolongation of the action potential causes more activation of each type of Ca2þ channel, favoring its selfsufficiency.
The detailed composition of Ca2þ channel subtypes that mediate transmission can vary widely among individual nerve terminals.
neuromuscular junction and squid giant synapse, in which P- or P/Q-type channels are overwhelmingly prevalent. At some hippocampal inhibitory synapses, GABA release is mediated entirely by P/Q-type or by N-type Ca2þ channels. Neurotransmission at sympathetic neuron–effector junctions is governed almost completely by N-type channels. Differences in the reliance on various CaV2 family members take on additional significance when synaptic circuits are the target of pharmacological and possibly therapeutic intervention. For example, in the dorsal horn of the spinal cord, the main component in the circuitry of pain transmission/modulation, inhibitory synaptic transmission is dominated by P/Q-type Ca2þ channels; in contrast, N-type channels are more closely associated with glutamate release from the peptidergic primary sensory neurons that convey nociceptive information to the dorsal horn.
Not always Multiple Channel Types
Not always CaV2 Channels
Although neurosecretion is supported by more than one type of channel at most CNS synapses, at some synapses transmission may be heavily dominated by a single channel type. Examples include some preparations historically important for discovery of basic principles of neurotransmission such as mammalian
It is generally believed that Ca2þ channels of the CaV2 class are specialized to support transmitter release. This is true, but it is worth pointing out that in some cases, CaV1 and even CaV3 channels can also participate. For example, ribbon synapses in the retina and cochlea rely on CaV1.3 channels to trigger transmitter
Exceptions to Generalizations about Multiple Ca2+ Channel Types
w -CTx-GVIA (1 μM) w -Aga-IVA (1 μM) 120 4-AP 100 80 60 4-AP 40 Control 20 Control 0 −5 0 5 10 15 20 −5 0 5 10 15 20 25 30 a b
EPSP slope (V/s) c
PQ
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PQ N
PQ N
R
d 4-AP (100 μM)
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180 Time (min)
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Figure 4 Concerted or redundant actions of P/Q- and N-type channels, depending on strength of depolarizing stimulus. (a) Dependence of synaptic transmission on N-type Ca2þ channels changes markedly upon prolongation of the presynaptic action potential with 4-AP. Control graph (open circles) shows approximately 50% inhibition of basal transmission by o-CTx-GVIA. In 4-AP (solid circles), the effect of N-type channel blockade is barely evident. (b) 4-AP also reduces the degree of inhibition produced by P/Q-type channel inhibition with o-Aga-IVA. (c) Example of an experiment in which simultaneous blockade of N-type and P/Q-type Ca2þ channels with o-CTx-GVIA and o-Aga-IVA abolished transmission completely, even in the presence of 4-AP. (d) Schematic representation of a release site surrounded by multiple types of Ca2þ channels. (a–c) Reproduced from Wheeler DB, Randall A, and Tsien RW (1996) Changes in action potential duration alter reliance of excitatory synaptic transmission on multiple types of Ca2þ channels in rat hippocampus. Journal of Neuroscience 16: 2226–2237, with permission. (d) Reproduced from Cao Y, Piedras-Renterı´a ES, Smith GB, Chen G, Harata NC, and Tsien RW (2004) Presynaptic Ca2þ channels compete for channel type-preferring slots in altered neurotransmission arising from Ca2þ channelopathy. Neuron 43: 387–400.
Calcium Channel Subtypes Involved in Neurotransmitter Release
release. Furthermore, CaV2 channels can perform important neuronal functions distinct from excitation– secretion coupling, such as excitation–transcription coupling. Not always the Same Type of Ca2+ Channel throughout Development
At some synapses, N-type channels develop early but are gradually supplanted by late-blooming P/Q-type channels. Mammalian development might be echoing a phylogenetic progression seen at the neuromuscular junction, where lizards and frogs use L- and N-type channels, respectively, but mammals deploy P/Q-type channels.
What Are Multiple Ca2+ Channels Good for? Why are there different kinds of presynaptic Ca channels? Almost certainly it is not because of evolutionary pressure for different biophysical properties. Indeed, basic features such as extremely high Ca2þ selectivity, high open channel flux rate, and steeply voltage-dependent channel gating are very similar for P/Q-, N-, and R-type currents. In all cases, selectivity and permeation are conferred by a pore that is continually occupied by at least one Ca2þ ion, which prevents more abundant Naþ ions from rushing through. Despite overall similarities, there are substantial differences in channel regulation, and this is likely to be the main reason for channel diversity (Table 1). For example, P/Q- and N-type channels appear to differ in their susceptibility to modulation by G-proteins. N-type channels show a greater degree of modulation. On the other hand, P/Q-type channels are more susceptible to progressive increases with Table 1 Differences between P/Q- and N-type channels with regard to their properties and contributions to synaptic transmission P/Q-type channels
N-type channels
Specific blocker
o-AgaIVA
Genetic diseases
Migraine, ataxia, epilepsy Late blooming Similar Weaker
o-CTx-GVIA, o-CTx-MVIIA ¼ Prialt No channelopathies found
Development Voltage gating Modulation by G-protein-coupled receptors Facilitation by repeated stimuli
Stronger
Early prominence Similar Stronger
Weaker if at all
65
use, a phenomenon known as Ca2þ channel facilitation. Because of the steep power-law relationship between Ca2þ entry and secretion, even small increases in P/Q Ca2þ entry can have a profound effect on the strength of transmission. The presynaptic Ca2þ channels constitute a key convergence point for regulation of neurosecretion, either by neurohumoral modulation or by activity-dependent regulation. Much is known about the structural motifs in a1 subunits that support various forms of regulation, for example, by G-proteins and protein kinases.
Further Reading Bollmann JH and Sakmann B (2005) Control of synaptic strength and timing by the release-site Ca2þ signal. Nature Neuroscience 8: 426–434. Cao Y, Piedras-Renterı´a ES, Smith GB, Chen G, Harata NC, and Tsien RW (2004) Presynaptic Ca2þ channels compete for channel type-preferring slots in altered neurotransmission arising from Ca2þ channelopathy. Neuron 43: 387–400. Catterall WA (1996) Molecular properties of sodium and calcium channels. Journal of Bioenergetics and Biomembranes 28: 219–230. Dolphin AC (2006) A short history of voltage-gated calcium channels. British Journal of Pharmacology 147(supplement 1): S56–S62. Dunlap K, Luebke JI, and Turner TJ (1995) Exocytotic Ca2þ channels in mammalian central neurons. Trends in Neurosciences 18: 89–98. Ertel EA, Campbell KP, Harpold MM, et al. (2000) Nomenclature of voltage-gated calcium channels. Neuron 25(3): 533–535. Lisman JE, Raghavachari S, and Tsien RW (2007) The sequence of events that underlie quantal transmission at central glutamatergic synapses. Nature Reviews Neuroscience 8: 597–609. Llina´s R, Sugimori M, Hillman DE, and Cherksey B (1992) Distribution and functional significance of the P-type, voltage-dependent Ca2þ channels in the mammalian central nervous system. Trends in Neurosciences 15: 351–355. Miljanich GP (2004) Ziconotide: Neuronal calcium channel blocker for treating severe chronic pain. Current Medicinal Chemistry 11: 3029–3040. Nowycky MC, Fox AP, and Tsien RW (1985) Three types of neuronal calcium channel with different calcium agonist sensitivity. Nature 316: 440–443. Olivera BM, Miljanich GP, Ramachandran J, and Adams ME (1994) Calcium channel diversity and neurotransmitter release: The o-conotoxins and o-agatoxins. Annual Review of Biochemistry 63: 823–867. Piedras-Renterı´a ES, Barrett CF, Cao Y-Q, and Tsien RW (2007) Voltage-gated calcium channels, calcium signaling and channelopathies. In: Krebs J and Michalak M (eds.) Calcium: A Matter of Life or Death, pp. 127–166. Elsevier: New York. Schneggenburger R and Neher E (2005) Presynaptic calcium and control of vesicle fusion. Current Opinion in Neurobiology 15: 266–274. Takahashi T and Momiyama A (1993) Different types of calcium channels mediate central synaptic transmission. Nature 366: 156–158. Tsien RW and Barrett CF (2004) A brief history of calcium channel discovery. In: Zamponi G (ed.) Voltage-Gated Calcium Channels. New York: Kluwer/Plenum.
66 Calcium Channel Subtypes Involved in Neurotransmitter Release Turner TJ, Adams ME, and Dunlap K (1993) Multiple Ca2þ channel types coexist to regulate synaptosomal neurotransmitter release. Proceedings of the National Academy of Sciences of the United States of America 90(20): 9518–9522.
Wheeler DB, Randall A, and Tsien RW (1996) Changes in action potential duration alter reliance of excitatory synaptic transmission on multiple types of Ca2þ channels in rat hippocampus. Journal of Neuroscience 16: 2226–2237.
SNAREs J Rizo, University of Texas Southwestern Medical Center, Dallas, TX, USA ã 2009 Elsevier Ltd. All rights reserved.
Introduction Chemical synaptic transmission is mediated by neurotransmitters that are released by Ca2þ-triggered synaptic vesicle exocytosis. The exquisite spatial and temporal regulation of this process depends on a highly complex protein machinery that is formed in part by components with homologs in most types of intracellular membrane traffic, including N-ethylmaleimide sensitive factor (NSF), soluble NSF attachment proteins (SNAPs), SNAP receptors (SNAREs), Sec1/Munc18 (SM) proteins, and Rab guanosine triphosphatases (GTPases). Among these components, the SNAREs are believed to be at the heart of a conserved mechanism of membrane fusion by virtue of their ability to form tight SNARE complexes that bring two opposing membranes together. The neuronal SNAREs involved in neurotransmitter release are the synaptic vesicle protein synaptobrevin 2 (also known as vesicle associated protein 2 (VAMP2)) and the plasma membrane proteins syntaxin 1 (which in mammals includes two closely related isoforms, syntaxin 1A and 1B) and SNAP-25 (for synaptosomal associated protein of 25 kDa; no relation to SNAPs). Neurotransmitter release depends also on proteins such as synaptotagmin 1 and complexins that play specialized roles in the tight regulation of this process and are not generally involved in other types of intracellular membrane traffic. Several of these proteins bind to the neuronal SNAREs and probably function in the regulation of SNARE complex assembly and/or in conjunction with the SNARE complex. Hence, synaptobrevin 2, syntaxin 1, and SNAP-25 have unique properties that have been adapted to the regulatory requirements of synaptic exocytosis, in addition to conserved features that are shared with SNAREs from other membrane compartments. This article focuses on the structural, biochemical, and functional properties of the neuronal SNAREs, but it also describes key findings on other SNAREs that have helped us to understand which of these properties are general and which are specialized.
SNARE Structure SNAREs constitute a family of proteins characterized by sequences called SNARE motifs that comprise 60–70 residues and have a high propensity to
form coiled coils. Most SNAREs contain only one SNARE motif that is adjacent to a single C-terminal transmembrane (TM) region (e.g., synaptobrevin 2 and syntaxin 1). Some SNAREs contain two SNARE motifs connected by a long linker and do not have a TM sequence (e.g., SNAP-25) (Figure 1), but are attached to membranes through a posttranslational modification such as palmitoylation. Circular dichroism (CD) and nuclear magnetic resonance (NMR) studies showed that isolated SNARE motifs are generally unstructured and adopt a-helical conformations when they bind to other SNARE motifs to form SNARE complexes. A SNARE complex contains four SNARE motifs that assemble into a parallel four-helix bundle, as first demonstrated for the neuronal SNAREs by electron paramagnetic resonance (EPR) spectroscopy and X-ray crystallography (Figure 2(a)). The neuronal SNARE complex is very stable (it is resistant to sodium dodecyl sulfate (SDS) and requires high temperatures for denaturation). This stability is conferred in large part by contacts between many hydrophobic residues that are arranged in layers, with each SNARE motif contributing one hydrophobic side chain to each layer. However, a polar, or zero layer, in the middle of the bundle is formed by one arginine (from synaptobrevin 2) and three glutamine side chains (one from syntaxin 1 and two from SNAP-25) (Figure 2(b)). These features are highly conserved in four different subfamilies of SNAREs and have led to their classification into Qa, Qb, Qc, and R SNAREs, depending on their homology with the SNARE motifs of syntaxin 1 (Qa), SNAP-25 (Qb and Qc for its N- and C-terminal SNARE motifs, respectively), or synaptobrevin (R). This classification is less ambiguous for SNAREs involved in homotypic membrane fusion than the original classification into v- and t-SNAREs (for vesicular and target membrane SNAREs, respectively). The assembly of the SNARE four-helix bundle is most likely at the center of the mechanism of membrane fusion. The functional importance of the polar layer is currently unclear, but it has been suggested to play a role in aiding SNARE complex disassembly or in dictating the proper register for SNARE complex formation. In addition to a SNARE motif and a TM sequence, many SNAREs have an N-terminal region that spans more than half of its sequence. NMR studies have shown that the N-terminal regions of all SNAREs from the syntaxin subfamily, including syntaxin 1, contain an autonomously folded domain that adopts an antiparallel three-helix bundle structure, known as the Habc domain (Figure 2(a)). This domain is connected to the SNARE motif through a linker region and is preceded by a short N-terminal
67
68 SNAREs
sequence (NTS) (Figure 1). The N-terminal regions of SNAREs from other subfamilies may also contain Habc domains or autonomously folded domains with completely unrelated structures, such as longin domains or PX domains. In the assembled SNARE complex, the Habc domain of syntaxin 1 is flexibly linked to the
Synaptobrevin 2 SNARE motif
TM
Syntaxin 1 NTS
SNARE motif
Habc domain
TM
SNAP-25 SNARE motif
SNARE motif
Figure 1 Domain structures of the neuronal SNAREs. The SNARE motifs, TM regions, and the helices of the Habc domain are represented by cylinders; other sequences are represented by black lines. NTS, N-terminal sequence; SNAP-25, synaptosomal associated protein of 25 kDa; SNARE, soluble N-ethylmaleimidesensitive factor attached protein receptor; TM, transmembrane.
four-helix bundle formed by the SNARE motifs. However, the Habc domain binds intramolecularly to the SNARE motif in isolated syntaxin 1, forming a closed conformation that is incompatible with the SNARE complex. These findings suggested that the Habc domain regulates SNARE complex formation and that syntaxin 1 must undergo a large conformational transition from a closed to an open conformation during exocytosis (Figure 3). However, only a few SNAREs from the syntaxin subfamily adopt a closed conformation, which thus appears to represent a specialization arising from unique regulatory requirements of neurotransmitter release and a few other membrane traffic processes. Conversely, some SNAREs outside the syntaxin family adopt closed conformations involving different types of autonomously folded N-terminal domains, indicating that intramolecular regulation of SNARE complex assembly is not restricted to syntaxins. The closed conformation of syntaxin 1 binds tightly to the neuronal SM protein Munc18–1 and its structure at atomic resolution (Figure 2(c)) was revealed by X-ray analysis of a syntaxin 1–Munc18–1 complex. This mode of interaction is not generally conserved
N c
a
V223 L50 V53 Q226
I171
Q53
R56
I230
Q174 L60
c L57
I178
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N c
Figure 2 Three-dimensional structures of the neuronal SNAREs: (a) illustration of the three-dimensional structures of the syntaxin 1 Habc domain determined by NMR spectroscopy (Fernandez et al.) (left) and the neuronal SNARE complex formed by the SNARE motifs of synaptobrevin 2, syntaxin 1, and SNAP-25 determined by X-ray crystallography (Sutton et al.) (right); (b) stick models of selected layers forming the neuronal SNARE complex; (c) illustration of the structure of the closed conformation of syntaxin 1 bound to Munc18–1 determined by X-ray crystallography. In (a), the dashed curved represents the flexible linker between the Habc domain and SNARE motif of syntaxin 1. The color coding is the same as in Figure 1. In (b). the stick models include the polar layer formed by R56 of synaptobrevin 2, Q226 of syntaxin 1, and Q53 and Q174 of SNAP-25, as well as the preceding and ensuing layers, which are formed exclusively by hydrophobic residues. The backbone atoms are in the same color code as in Figure 1. Side chain atoms are in cyan for carbon, red for oxygen, and blue for nitrogen. In (c), the Habc domain of syntaxin 1, its SNARE motif, and the linker region connecting them are in orange, yellow, and black, respectively. Munc18–1 is in violet (Misura et al.). C, C-terminus; N, N-terminus; NMR, nuclear magnetic resonance; SNAP-25, synaptosomal associated protein of 25 kDa; SNARE, soluble N-ethylmaleimide-sensitive factor attached protein receptor.
SNAREs 69 Plasma membrane
N
Plasma membrane
N
Synaptic vesicle Figure 3 Diagram illustrating that syntaxin 1 must undergo a large conformational change during synaptic vesicle exocytosis. In the SNARE complex (right), the Habc domain of syntaxin 1 is flexibly linked to the four-helix bundle formed by the SNARE motifs of syntaxin 1, SNAP-25, and synaptobrevin 2. In isolated syntaxin 1 (left), the Habc domain binds intramolecularly to part of the SNARE motif, forming a closed conformation that is incompatible with SNARE complex formation. The color coding is the same as in Figure 1. N, N-terminus; SNAP-25, synaptosomal associated protein of 25 kDa; SNARE, soluble N-ethylmaleimide-sensitive factor attached protein receptor.
in other syntaxins and SM proteins, but many SM protein/syntaxin interactions depend on the syntaxin N-terminal region, particularly the NTS. Hence, it is likely that the general function of the syntaxin N-terminal region is related to coupling with SM protein function.
Evolution of Ideas on SNARE Function SNARE proteins have been investigated by a wide variety of techniques, providing a vivid example of the power of interdisciplinary research and a fascinating history with twists and turns that may continue for years to come. The first SNAREs were found in genetic screens for membrane traffic defects in yeast and in molecular cloning analyses of components from synaptic vesicle and synaptosomal membranes that identified syntaxin 1, synaptobrevin 2, and SNAP-25. The first evidence of the functional importance of these proteins for synaptic exocytosis was provided by the discovery in the early 1990s that they are the targets of clostridial neurotoxins, agents that potently inhibit neurotransmitter release. Around the same time, sequence analyses revealed a homology between these neuronal proteins and some of the proteins identified in the studies of membrane traffic defects in yeast. Moreover, NSF/Sec18p and SNAPs/Sec17p were identified as crucial factors for vesicle-mediated transport in yeast and mammalian cells. Homologies were also observed between yeast and neuronal Rab proteins, as well as between SM proteins involved in traffic at different membrane compartments. All these findings led to the now widely held notion that a conserved machinery mediates most types of intracellular membrane traffic. Crucial discoveries in the understanding of SNARE function were also the observations in 1993 that synaptobrevin 2, syntaxin 1, and SNAP-25 form a tight 7S complex (the SNARE complex) that binds to SNAPs and NSF to form a larger, 20S complex, and that adenosine triphosphate (ATP) hydrolysis by NSF
leads to SNARE complex disassembly. These findings, and the belief that NSF was directly involved in membrane fusion, led to the proposal that synaptobrevin 2, syntaxin 1, and SNAP-25 act as receptors for the membrane fusion apparatus and hence to their designation as SNAREs (for SNAP receptors). These results led also to a model of intracellular membrane traffic whereby binding of SNAREs in transport vesicles (v-SNAREs) to SNAREs on target membranes (t-SNAREs) mediates vesicle docking and target specificity (known as the SNARE hypothesis). In this model, the SNAREs were envisaged as binding in an antiparallel fashion and the disassembly of the SNARE complex by NSF/SNAPs was proposed to initiate fusion. The finding that clostridial neurotoxins inhibit release by cleaving SNAREs from the synaptic vesicle and plasma membrane was consistent with the notion that SNAREs are required on opposing membranes for fusion, which was further supported by subsequent studies of yeast vacuolar fusion. However, it was noted that the SNAREs could not play a role in vesicle docking because docking is not affected on SNARE cleavage by the neurotoxins or by genetic ablation of syntaxin 1 in Drosophila (note that very recent data have suggested that syntaxin 1 may actually function in docking; these contradictory conclusions that still need to be resolved probably arise because redundant docking mechanisms may exist and/or because of differences in the methodology used to prepare samples for electron microscopy (EM)). The genetic studies in Drosophila also revealed that syntaxin 1 is critical for release and, together with the neurotoxin data, led to the proposal that SNAREs function downstream of docking. Moreover, studies of exocytosis in PC12 cells and of homotypic vacuolar fusion in yeast showed in 1996 that NSF functions at a prefusion step rather than fusion itself, and the SNARE motifs of synaptobrevin 2 and syntaxin 1 were shown in 1997 to interact in an antiparallel fashion by EM and fluorescence resonance energy transfer (FRET). Because the SNARE motifs are
70 SNAREs
SNAREs and Membrane Fusion The minimal model of SNARE-mediated membrane fusion was based on reconstitution experiments that demonstrated lipid mixing between proteoliposome populations containing synaptobrevin 2 or syntaxin 1/SNAP-25. This model has been accepted by many researchers, but has also been strongly challenged by others for diverse reasons. On one hand, there were
a
b
c Ca2+
– –+ +
– + – +
+ + + +
+
adjacent to the TM regions of synaptobrevin 2 and syntaxin 1, which are anchored on the synaptic vesicle and plasma membrane, respectively, this key discovery led to the proposal that the energy of formation of the SNARE complex could be used to bring the membranes together and initiate membrane fusion (known as the zippering model; Figure 4(a)). In this model, disassembly of the SNARE complex by NSF/SNAPs after membrane fusion recycles the SNAREs for another round of fusion. The zippering model is attractive because of its similarity to the mechanism of viral fusion and has gained wide acceptance. A similar model that in addition postulates that the SNAREs constitute a minimal membrane-fusion machinery was proposed based on reconstitution experiments, but this minimal model has been strongly challenged by subsequent reconstitutions. Further debate about the central role of the SNAREs in membrane fusion predicted by both models emerged from analyses of mice lacking synaptobrevin 2 or SNAP-25. The synaptobrevin knockout revealed a strong impairment of evoked release and a less marked decrease in spontaneous or hypertonic sucrose-induced release; evoked release was also impaired strongly in SNAP-25-knockout mice, but spontaneous release was increased. Although these results reinforced the notion that the SNAREs function downstream of vesicle docking, the persistence of release in these mice contrasts with the total abrogation of any form of release in Munc18–1-knockout mice. It seems likely that the release remaining in the absence of synaptobrevin 2 and SNAP-25 may arise from functional redundancy with other isoforms of these proteins, but this hypothesis remains to be validated experimentally. Despite these ongoing debates, there is currently a general consensus that SNARE function is related to their ability to bring two membranes together and that synaptobrevin 2, syntaxin 1, and SNAP-25 play a critical role in neurotransmitter release. In addition to resolving these debates, recent and ongoing research on SNAREs is focusing on investigating the mechanism of SNARE complex assembly and studying how the roles of other key proteins are coupled to SNARE function.
+ + + +
d Figure 4 Models of SNARE function in membrane fusion: (a) original zippering model of SNARE-mediated membrane fusion based on EM data, showing that the SNARE motifs of syntaxin 1 and synaptobrevin 2 bind in a parallel fashion; (b) model showing how isolated assembling SNARE complexes might diffuse to the center of the space between the membranes, which requires much less energy than inducing membrane fusion; (c) same model as (b) with the diffusion prevented because the assembling SNARE complexes are bound to a bulky protein (in violet); (d) model illustrating the notion that complexin (in pink) may favor full or almost full SNARE complex formation and at the same time inhibit fusion to yield a metastable state. In (a), the model assumes that the SNARE motifs and TM regions of both proteins form continuous helices and that membrane fusion is forced as these continuous helices zipper from the N- to the C-terminus. In (b) and (c), the model assumes that the SNARE motifs and TM regions of synaptobrevin 2 and syntaxin 1 are connected by short flexible linkers. In (d), the metastable state (left) is predicted to be the substrate for synaptotagmin 1 to trigger neurotransmitter release by binding simultaneously to both membranes and to the SNARE complex, displacing complexin, on Ca2þ influx (right). Synaptotagmin 1 is in light blue, except for the C2B domain, which is in dark blue. The – and þ signs illustrate overall features of the surface electrostatic potential of the synaptotagmin 1 C2 domains that may prevent phospholipid binding in the absence of Ca2þ or activate phospholipid binding and membrane fusion upon Ca2þ influx. Positive charges at the C-terminus of the SNARE complex that may also help to bend the membranes to induce membrane fusion are not shown. The N-terminal region of syntaxin 1 is not shown in any of the models. EM, electron microscopy; SNARE, soluble N-ethylmaleimide-sensitive factor attached protein receptor; TM, transmembrane.
multiple technical problems with the initial reconstitution experiments and mixing of the proteoliposome contents without leakiness, a key feature expected for physiological membrane fusion, has never been
SNAREs 71
demonstrated; on the other hand, a fundamental problem from a biological perspective is that the minimal model does not account for the strict requirement of SM proteins for all types of SNARE-dependent membrane traffic, including the essential nature of Munc18–1 for neurotransmitter release. Moreover, from a biophysical point of view, the structural-energetic basis of the minimal model is unclear. A key aspect of the original zippering model was the prediction that the SNARE motifs and TM regions of synaptobrevin 2 and syntaxin 1 could form continuous a-helices, which would therefore exert mechanical force on both membranes to induce fusion (Figure 4(a)). However, the available structural data suggest that the SNARE motifs and TM regions are connected by short flexible linkers, which is expected to hamper transduction of the energy of SNARE complex formation into the energy required to bend the membranes and initiate fusion. Hence, as they assemble, the SNARE complexes could diffuse into the space between the membranes and hinder (rather than facilitate) fusion (Figure 4(b)). In addition, introduction of helix-breaking residues or the insertion of long flexible linkers in the sequences between the SNARE motifs and TM regions had little or only moderate effects on the lipid mixing observed in the initial reconstitution experiments. Because the long linkers should have strongly uncoupled SNARE complex formation from its action on the membranes, the observed lipid mixing probably arose in part from factors beyond SNARE complex formation (e.g., instability of the vesicles). Some of these factors became clear in subsequent reconstitution experiments. Thus, the proteoliposomes used in the initial reconstitutions may indeed have been unstable because they contained extremely high protein-to-lipid ratios (1:20 for synaptobrevin 2, which translates into a protein surface concentration comparable to a 12 mM concentration in bulk solution). Moreover, the proteoliposomes were prepared with a reconstitution method that relies on co-solubilization of proteins and lipids with detergent and yields heterogeneous vesicles (both in terms of size and protein density). Reconstitutions with this method using lower protein densities (e.g., 1:200) yielded less efficient lipid mixing, even though these densities are still comparable to a 1.2 mM protein concentration in bulk solution, and the observed lipid mixing could arise from populations of small vesicles with higher than average protein densities (note that the resulting high curvature destabilizes the vesicles because of membrane stress). Indeed, no significant lipid mixing was generally observed at these proteins densities when vesicles were prepared by a reconstitution method that involves the incorporation of detergent-solubilized SNAREs into preformed liposomes and yields more homogeneous proteoliposomes. Significantly, efficient
SNARE complex formation without lipid mixing was observed using proteoliposomes prepared by this method, as predicted from the structural arguments described in Figure 4(b). From the available reconstitution data, it seems clear that SNARE complex formation is not sufficient for membrane fusion, and lipid mixing can only be induced under conditions in which membranes are destabilized and a large number of SNARE complex force the two membranes to collapse. However, an important notion that emerged from some of the reconstitution experiments is that the TM regions of the SNAREs can destabilize the bilayer, which may contribute to the function of SNAREs in membrane fusion. Moreover, recent reconstitution data obtained with SNAREs, synaptotagmin, and complexin appear to reproduce events that may occur during neurotransmitter release. Hence, the reconstitution approach is providing insights into important features of the release machinery and, with the appropriate methodology, this approach promises to provide a critical tool to understand the mechanism of neurotransmitter release. Note also that, even though the minimal model now appears to be incorrect, the original idea arising from the zippering model that SNARE complex formation causes membrane fusion could still be valid without the requirement that the SNARE motifs and TM regions form continuous helices. Thus, a modified model predicts that the energy of SNARE complex assembly could be used to induce fusion if the SNAREs are kept apart by virtue of interactions with a bulky protein or proteins (Figure 4(c)), which would prevent the undesired diffusion of the SNARE four-helix bundle into the center of the intermembrane space (Figure 4(b)). This model remains to be tested but emphasizes the necessity of understanding the functions of additional factors that are crucial for release in order to fully understand the function of the SNAREs themselves.
SNARE Interactions with Other Conserved Components of the Fusion Machinery A wide variety of proteins have been shown to play a role in synaptic vesicle exocytosis. Elucidating how the functions of these proteins are coupled to SNARE function is thus critical to understanding the mechanism of release. A difficulty in this endeavor has arisen because of the stickiness of the SNAREs, which has led to the identification of many interactions that are probably irrelevant (for instance, more than 40 proteins have been described that bind to syntaxin 1). Nevertheless, the complementary information provided by genetic and electrophysiological experiments, together with structural studies, have helped to unravel
72 SNAREs
the nature of some of these interactions and to support their physiological significance. Among the interactions of the SNAREs with other conserved components of the intracellular membrane fusion machinery, those with SNAPs/NSF have a clear functional role, namely the disassembly of the SNARE complex after fusion to recycle the SNAREs for another round of fusion. Rab proteins, which have been implicated in vesicle docking or tethering, do not appear to interact directly with the SNAREs but have been shown to be functionally coupled to the SNAREs in diverse systems and may indirectly regulate the SNARE complex assembly. The most fascinating and at the same time enigmatic SNARE interactions with the conserved components of the fusion machinery are those with SM proteins. The strict requirement for SM proteins in all types of SNARE-dependent membrane fusion, their localization at sites of fusion, and their diverse interactions with SNAREs suggest that SM proteins may have a direct function in fusion. However, this function has remained elusive, in part because of the diversity of these interactions. Neuronal Munc18–1 was shown to bind tightly to the closed conformation of syntaxin-1, but this interaction hinders SNARE complex formation and hence does not explain the essential nature of Munc18–1 for neurotransmitter release. Conversely, the yeast plasma membrane SM protein Sec1p was found to bind to SNARE complexes instead of to the cognate syntaxin. Moreover, syntaxins from diverse intracellular compartments in yeast and mammals bind to SM proteins through their NTS, in an interaction that is compatible with SNARE complex assembly. Finally, the yeast vacuolar SM protein Vps33p appears to bind to SNARE complexes at least in part through the N-terminal PX domain of the SNAP-25 homolog Vam7p. This diversity of SM protein–SNARE interaction modes has probably emerged from distinct regulatory requirements of traffic in different membrane compartments, but it seems likely that a common binding mechanism between these two conserved protein families must exist. In this context, increasing evidence has suggested that all SM proteins bind to SNARE complexes, and a direct interaction between Munc18–1 and neuronal SNARE complexes was recently identified. Hence, Munc18–1 exhibits at least two different modes of interactions with the SNAREs: one with the syntaxin 1 closed conformation, which probably represents a specialization that evolved for the tight regulatory requirements of neurotransmitter release, and another with the SNARE complex, which may be conserved in all types of intracellular membrane traffic. The former interaction may play a role in stabilizing syntaxin 1 or
preventing this promiscuous protein from binding to other proteins. In addition, syntaxin 1 has been shown to form two different types of four-helix bundles with SNAP-25, containing two copies of syntaxin 1; these complexes represent kinetic traps that hinder SNARE complex formation, and binding of Munc18–1 to the syntaxin 1 closed conformation may prevent the formation of these traps. Because the closed conformation contains a four-helix bundle and Munc18–1 also binds to the SNARE complex, it has been proposed that Munc18–1 may provide a template for assembling the four-helix bundle formed by the SNARE motifs of syntaxin 1, SNAP-25, and synaptobrevin 2 in the same site. In this context, Munc18–1 could play the role of the bulky protein depicted in the model in Figure 4(c), but this model remains to be tested and the precise nature of the Munc18–1–SNARE complex interaction still needs to be defined. Moreover, alternative models can be envisaged for the active role of Munc18–1 in release. For instance, the SNARE motifs of synaptobrevin 2 and syntaxin can bind in an antiparallel fashion, which hinders fusion; Munc18–1 may ensure assembly of the SNARE complex in the proper, parallel orientation. The transition from the Munc18–1–syntaxin 1 complex to the Munc18–1–SNARE complex may be a central event in the priming reaction that makes docked synaptic vesicles readily releasable, but the mechanism of this transition is also unclear. Functional experiments in Caenorhabditis elegans have suggested that this transition may be mediated by Unc13/Munc13s and Unc10/Rab3 interacting molecules (RIMs), which are large proteins from presynaptic active zones with critical roles in release. Unc13/Munc13s were initially thought to bind to syntaxin 1, but recent evidence indicates that they do not form binary complexes with syntaxin 1. Hence, gaining insight into the biochemistry of these proteins, the interactions underlying the conformational transition of syntaxin 1, and the role of the Munc18–1–SNARE complex interaction will be crucial to the understanding of the mechanism of neurotransmitter release. It will also be critical to better understanding which aspects of the coupling mechanism between Munc18–1 and the neuronal SNAREs are conserved in other systems and may reflect the general function of SM proteins in membrane fusion, although it is becoming increasingly clear that this function is somehow coupled to SNARE complex assembly.
SNARE Interactions with Specific Components of the Release Machinery Interactions of the SNAREs with components of the exocytotic machinery that have specialized roles in
SNAREs 73
release also appear to be associated with the regulation of SNARE complex assembly, but may also attract factors that facilitate and/or inhibit membrane fusion to confer the exquisite Ca2þ sensitivity of neurotransmitter release (the fast, synchronous components of release is triggered in less than 100 ms after Ca2þ influx in some synapses). An example of the former category is the interaction of syntaxin 1 and SNAP-25 with tomosyn, a large protein with a SNARE motif homologous to that of synaptobrevin 2. Indeed, the crystal structure of tomosyn bound to syntaxin 1 and SNAP-25 revealed a four-helix bundle analogous to the SNARE complex but with synaptobrevin replaced by the tomosyn SNARE motif (Figure 5(a)). Therefore, formation of this complex is expected to inhibit release by preventing the assembly of functional SNARE complexes containing synaptobrevin 2. This prediction has been confirmed by genetic experiments in C. elegans, although tomosyn may play additional functions through the long sequences preceding the SNARE motif. These experiments revealed at the same time a functional interplay between tomosyn and Unc13/Munc13s that may control the balance between unproductive tomosyn–syntaxin 2–SNAP-25 complexes and productive SNARE complexes containing synaptobrevin 2. Diverse functional data have suggested that the SNAREs are directly or indirectly coupled to Ca2þ sensing during neurotransmitter release, including the
Figure 5 Structural basis for SNARE interactions: (a) with tomosyn; (b) with complexin. Crystal structures of the complex formed by the tomosyn, syntaxin 1, and SNAP-25 SNARE motifs (Pobbati et al.) (a) and the complexin–SNARE complex (Chen et al.) (b). Tomosyn is in salmon, complexin is in pink, and the remaining color coding is as in Figure 1. Note the similarity between the structures of the tomosyn–syntaxin 1–SNAP-25 complex (a) and the SNARE complex (Figure 2(a)), with the tomosyn and synaptobrevin 2 SNARE motifs occupying the same positions in the complexes. Note also that complexin forms an a-helix that binds to a groove between the syntaxin 1 and synaptobrevin 2 SNARE motifs in the SNARE complex (b). C, C-terminus; N, N-terminus; SNAP-25, synaptosomal associated protein of 25 kDa; SNARE, soluble N-ethylmaleimide-sensitive factor attached protein receptor.
alteration of the Ca2þ sensitivity of secretion in chromaffin cells caused by some point mutations in SNAP-25 and the finding that elevated Ca2þ can compensate the inhibition of release caused by botulinum neurotoxin A, which cleaves SNAP-25 close to its C-terminus. Whereas NMR studies showed that the SNARE complex does not contain specific Ca2þ-binding sites that could be directly involved in Ca2þ sensing during release, it has become increasingly clear that interactions of the SNAREs with synaptotagmin 1 and complexins are key for the Ca2þ-triggered step of neurotransmitter release. Synaptotagmin 1 is a synaptic vesicle protein that acts selectively as a Ca2þ sensor in synchronous release. This function depends on Ca2þ-dependent phospholipid binding to the two C2 domains that form most of the synaptotagmin 1 cytoplasmic region (the C2A and C2B domain), with the C2B domain playing a preponderant role. Many studies described interactions of the SNAREs with synaptotagmin 1, but it was unclear which of these interactions might be functionally relevant. Recent data may have yielded key insights into this issue. Thus, Ca2þ-bound synaptotagmin 1 was shown to bind simultaneously to the C-terminus of the SNARE complex and to phospholipids through the C2B domain, and the C2B domain was also found to interact simultaneously with two membranes on Ca2þ binding. Moreover, both the Ca2þ-bound C2B domain and the C-terminus of the SNARE complex are highly positively charged. These observations suggest that the SNARE complex and the C2B domain may cooperate in bringing the membranes together and in bending them to accelerate fusion (Figure 4(d), right). Although these ideas provide an attractive mechanism for coupling synaptotagmin 1 and SNARE function that explains the preponderant role of the C2B domain, their validity remains to be demonstrated and alternative models can be envisaged. For instance, it is also possible that the highly positive electrostatic potential generated by the SNARE C-terminus and synaptotagmin 1 may help to open the fusion pore if the synaptic vesicle and plasma membranes are already partially merged (forming a hemifusion state) before Ca2þ influx. Complexins are small soluble proteins that bind tightly to the neuronal SNARE complex in a Ca2þ independent manner. Knockout of the two major complexin isoforms in mice revealed a selective impairment of the Ca2þ-triggered step of synchronous release, but an excess of complexin can also inhibit release. X-ray crystallography and NMR spectroscopy showed that complexin binds in an antiparallel a-helical conformation to a groove between the SNARE motifs of synaptobrevin 2 and syntaxin 1 (Figure 5(b)), stabilizing the SNARE complex.
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Because SNARE complexes are generally believed to assemble from the membrane-distal N-terminus and zippering toward the C-terminus is expected to be hindered by membrane repulsion, these findings led to a model whereby complexin helps to complete SNARE complex assembly, yielding a metastable state that is critical for the fast speed of synchronous release (Figure 4(d), left). Interestingly, recent reconstitution experiments showed that complexin inhibits SNARE-induced liposome fusion at a hemifusion state and that Ca2þ plus synaptotagmin 1 release the inhibition, resulting in a fast burst of fusion that cannot be observed with the SNAREs alone. In addition, simultaneous binding of the synaptotagmin 1 C2B domain to phospholpids and SNARE complexes was shown to displace a complexin fragment bound to these complexes. Altogether, these data suggest that complexin may indeed play an active role in release by promoting SNARE zippering toward the C-terminus and thus bringing the system closer to fusion (perhaps to hemifusion), but at the same time plays an inhibitory role to prevent full fusion before Ca2þ influx; such inhibition is then released by simultaneous binding of synaptotagmin 1 to Ca2þ, to both membranes, and to the SNARE complex to active release (Figure 4(d)). Clearly, there are alternative models to explain the available data, including the active function of complexin for release, but these data show that there is a fascinating interplay among the SNAREs, complexin, synaptotagmin 1, Ca2þ, and phospholipids that is probably crucial for the exquisite regulation of neurotransmitter release. The relation between Munc18–1, complexin, and synaptotagmin 1 binding to the SNARE complex remains to be studied.
SNARE Specificity The contribution of SNAREs to the specificity of membrane traffic has been another area of strong debate about SNARE function. This issue is important for interpreting the phenotypes of synaptobrevin 2 and SNAP-25 mice because, given the observation that neurotransmitter release can still be observed in these mice, an absolute specificity in SNARE pairing implies that the SNAREs are not essential for membrane fusion. Biochemical assays showed that some SNAREs have a tendency to bind to both cognate and noncognate SNAREs, and hence can be quite promiscuous. Conversely, reconstitution studies suggested a high specificity in SNARE-induced lipid mixing, but the noncognate SNARE pairings identified biochemically were not analyzed. A recent study showed that early endosome fusion is specifically mediated by a set of SNAREs in living cells, whereas the same set of SNAREs promiscuously induce liposome fusion
with SNAREs from the plasma membrane or late endosomes. These results can be interpreted in the framework of sequence analyses and the known structures of SNARE complexes. Because SNARE complex assembly involves protein–protein interactions, assembly must involve some degree of specificity. Moreover, the SNAREs are one of the most diverse among the protein families generally involved in intracellular membrane traffic (25 members in yeast and 36 in humans; only Rab proteins are more diverse). However, because coiled-coil interactions can be considerably promiscuous and the hydrophobic residues involved in SNARE complex formation are highly conserved in each subfamily, it is not surprising that some degree of nonspecificity exists in SNARE pairing. Hence, the picture that emerges is that SNAREs contribute to the specificity of traffic in distinct membrane compartments but that this specificity also involves other proteins. In particular, there is little doubt that an important contribution to specificity arises also from Rab protein interactions. Note also that the contribution of the neuronal SNAREs to specificity in neurotransmitter release most likely also involves interactions with factors specialized for release, such as synaptotagmin 1 and complexins.
Outlook Research on SNARE proteins has provided an emphatic example of the power of interdisciplinary approaches to studying protein function and of the difficulties of exactly pinpointing the specific roles of proteins that act as part of complex macromolecular assemblies. Hence, the divide-and-conquer approach has provided crucial clues to the understanding of the functions of the SNAREs and other components of the release machinery, but increasing evidence illustrates the complexity and cooperativity of this system. Clearly, a true understanding of the mechanism of release requires further studies in which more and more of these components are examined together in the presence of membranes. A critical foundation for these studies has been provided by the astonishing advances in this field made during the past 20 years. See also: Neurotransmitter Release from Astrocytes; Synaptic Vesicles.
Further Reading Brunger AT (2005) Structure and function of SNARE and SNARE-interacting proteins. Quarterly Review of Biophysics 38: 1–47.
SNAREs 75 Dulubova I, Sugita S, Hill S, et al. (1999) A conformational switch in syntaxin during exocytosis: Role of Munc18. EMBO Journal 18: 4372–4382. Hanson PI, Roth R, Morisaki H, Jahn R, and Heuser JE (1997) Structure and conformational changes in NSF and its membrane receptor complexes visualized by quick-freeze/deep-etch electron microscopy. Cell 90: 523–535. Jahn R and Scheller RH (2006) SNAREs – engines for membrane fusion. Nature Reviews Molecular and Cell Biology 7: 631–643. Lin RC and Scheller RH (1997) Structural organization of the synaptic exocytosis core complex. Neuron 19: 1087–1094. Link E, Edelmann L, Chou JH, et al. (1992) Tetanus toxin action: Inhibition of neurotransmitter release linked to synaptobrevin proteolysis. Biochemistry and Biophysics Research Communications 189: 1017–1023. Nichols BJ, Ungermann C, Pelham HR, Wickner WT, and Haas A (1997) Homotypic vacuolar fusion mediated by t- and v-SNAREs. Nature 387: 199–202. Poirier MA, Xiao W, Macosko JC, et al. (1998) The synaptic SNARE complex is a parallel four-stranded helical bundle. Nature Structural Biology 5: 765–769.
Rizo J, Chen X, and Arac D (2006) Unraveling the mechanisms of synaptotagmin and SNARE function in neurotransmitter release. Trends in Cell Biology 16: 339–350. Schiavo G, Benfenati F, Poulain B, et al. (1992) Tetanus and botulinum-B neurotoxins block neurotransmitter release by proteolytic cleavage of synaptobrevin. Nature 359: 832–835. Schoch S, Deak F, Konigstorfer A, et al. (2001) SNARE function analyzed in synaptobrevin/VAMP knockout mice. Science 294: 1117–1122. Schulze KL, Broadie K, Perin MS, and Bellen HJ (1995) Genetic and electrophysiological studies of Drosophila syntaxin-1A demonstrate its role in nonneuronal secretion and neurotransmission. Cell 80: 311–320. Sollner T, Bennett MK, Whiteheart SW, Scheller RH, and Rothman JE (1993) A protein assembly-disassembly pathway in vitro that may correspond to sequential steps of synaptic vesicle docking, activation, and fusion. Cell 75: 409–418. Sollner T, Whiteheart SW, Brunner M, et al. (1993) SNAP receptors implicated in vesicle targeting and fusion. Nature 362: 318–324. Sudhof TC (2004) The synaptic vesicle cycle. Annual Review of Neuroscience 27: 509–547.
Synaptic Vesicles S Takamori, Tokyo Medical and Dental University, Tokyo, Japan ã 2009 Elsevier Ltd. All rights reserved.
Introduction Communication between neurons or from neurons to their target tissues takes place at a specialized structure called the ‘synapse’ (Greek meaning ‘to clasp’). Synapses consist of two functionally and morphologically distinct components: the presynapse, from which neurotransmitter molecules are released, and the postsynapse, where specific receptors for the respective neurotransmitters are localized on the surface and the complex signaling cascades proceed. As such, one neuron can activate or inactivate connected neurons or target tissues, depending on the transmitter molecule they utilize for their signal transmission. Based on electrophysiological experiments pioneered by Katz and colleagues in the 1960s, it has been postulated that neurotransmitters are released from presynaptic terminals in discrete packets termed ‘quanta.’ Electron microscopy revealed that at presynaptic terminals, hundreds of small and round membranous vesicles – synaptic vesicles (SVs) – are clustered, which can be reasonably linked to the quanta (Figure 1(a)). Furthermore, biochemical experiments using isolated vesicles from mammalian brains have proven that SVs exhibit a variety of transport activities for major chemical transmitters. Based on these findings, it is generally believed that SVs store neurotransmitters and the fusion of SV membrane to the plasma membrane elicits the quantal release of neurotransmitters. Because of their important roles in basic neural functions, much effort has been focused on understanding the molecular machinery of SVs. This article focuses on general features of SVs in terms of morphology, biogenesis, and recycling and their overall molecular composition.
General Features of Synaptic Vesicles as an Organelle Clustered at the nerve endings, SVs are one of the most striking morphological hallmarks of presynaptic terminals in electron micrographs. They appear to be roughly homogeneous in size and shape, but it has been determined that some heterogeneity exists among them. Under certain experimental conditions, their shape appeared to be of two types – almost spherical and oval or flat. This difference may be an experimental artifact presumably from the fixation
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process. Interestingly, this difference in shape may be associated with their transmitter content. It was found that most excitatory, asymmetric glutamatergic synapses contain spherical vesicles, and inhibitory, symmetric, GABAergic synapses contain the latter. Regardless of the functional significance, this observation has provided a possible morphological characteristic to distinguish glutamatergic SVs and GABAergic SVs. Due to their uniformity in size and abundance in the brain, it is feasible to isolate SVs with high purity and in large amounts – a prerequisite for biochemical and biophysical experiments (Figure 1(b)). Electron microscopic observations of isolated SVs from rat brains have revealed that the surface is uneven. They are decorated with one or more prominent globular structures and several spiky or amorphous substructures are observable under electron microscope. After proteolytic digestion these structures are removed and the rims of the lipid bilayer become clearly visible, demonstrating that the vesicles are coated with proteins on the surface (Figures 1(c) and 1(d)). As measured by electron microscopy, their size varies substantially: The diameter of the outer bilayer ranges from 35 to 50 nm, with an average peak at 42 nm. The total dry mass of an average SV was deduced from a combination of protein quantitation, lipid quantitation, and SV particle counting, resulting in approximately 30 attograms (ag) per vesicle, which consists of 17 ag of proteins and 12 ag of lipids. Assuming the thickness of a lipid bilayer is 4 nm, the average inner volume can be estimated as approximately 20 10–21 l, which provides enough space for approximately 1800 transmitter molecules at a concentration of 150 mM. Within these physical and molecular constraints, SVs are effectively equipped with a unique set of proteins and lipids necessary for executing the fundamental tasks in neurotransmitter release.
Biogenesis of Synaptic Vesicles Like other proteins of the secretory pathway, most SV proteins are synthesized at the endoplasmic reticulum and processed through the Golgi apparatus for maturation in the cell body of neurons (Figure 2(a)). Conceptually, subdomains which selectively collect the SV proteins bud off from the Golgi apparatus and then travel along the axon to the presynaptic terminals. However, since no vesicles are as small as mature SVs, and some larger nonuniform tubulovesicular structures are seen in the axons, SV constituents are thought to travel along the processes on heterogeneous membranes termed SV precursors.
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Figure 1 Morphology of synapse and synaptic vesicles. (a) A transmission electron micrograph of synapse. The presynaptic terminal (T) contains numerous synaptic vesicles (SVs) and mitochondria (Mit). A small portion of SVs is attached to electron dense structures at the presynaptic plasma membrane termed the active zone (AZ), where exocytosis of SVs takes place. Opposite the AZ, there are electron dense structures beneath the postsynaptic membrane termed postsynaptic density (PSD), where neurotransmitter receptors form signaling complexes. D, dendrite; SC, synaptic cleft. (b) An electron micrograph of synaptic vesicle fraction. SVs were purified from mouse whole brains, negatively stained, and imaged by transmission electron microscopy. Inset shows immunogold labeling of the vesicles with an antibody against an SV marker protein, synaptophysin. Approximately 95% of the membranous structures are labeled. (c, d) SVs before (c) and after (d) proteolysis imaged by cryo-electron microscopy. Scale bar ¼ 1 mm (a), 100 nm (b, inset), 20 nm (c). (a) Adapted from the George E. Palade EM Slide Collection at Yale University School of Medicine. (c, d) Reproduced from Takamori S, Holt M, Stenius K, et al. (2006) Molecular anatomy of a trafficking organelle. Cell 127: 831–846, with permission from Elsevier.
Once formed, the SV precursor needs a pathway to reach its destination. Microtubules are known to serve as the roads for transporting cargo. Motor proteins of the kinesin superfamily (KIFs) bind to both the microtubules and the cargo, and they control the directional transport of intracellular transporting cargo. Among the KIFs, KIF1A and KIF1Bb are known to participate in axonal transport of SV precursors. Gene disruption of either KIF1A or KIF1Bb results in reduction of SV density at the presynaptic terminals and therefore impaired neurotransmission. The KIF1A- and KIF1Bb-carrying SV precursors contain the major SV marker proteins, such as synaptophysin, synaptobrevin, synaptotagmin, and rab3A, but not presynaptic plasma membrane proteins, such as syntaxin 1 and SNAP-25. In addition, proteins that form the cytomatrix at the active zone, such as piccolo and bassoon, are transported to the
axon on distinct cargo, suggesting that segregation of the presynaptic proteins is initiated before they arrive at the terminals. The mechanism by which the SV constituents are selectively recruited into the SV precursor is unknown. It is possible that SV proteins have a specific signal sequence that is assembled and forms microdomains at the exit of the trans-Golgi network, but no such common signal sequence has been found. Furthermore, the molecular mechanism regarding how the constituents of the SV precursors can be recognized by KIF1A/1Bb proteins is poorly understood. When SV precursors arrive at the presynaptic terminal, they fuse with the plasma membrane where the main route for SV biogenesis is initiated. This route, the AP-2-dependent pathway, is mediated by several essential proteins for vesicle endocytosis, namely clathrin, dynamin, and AP-2. An alternative route by
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Golgi apparatus
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b Figure 2 Biosynthesis and recycling of SVs. (a) Biosynthesis of SVs. SV proteins are synthesized in the cell body of neurons and the SV precursors bud off from the trans-Golgi network. The SV precursors are transported along the axon, guided by the microtubules and kinesin motor proteins (KIF1A and KIF1Bb). (b) SV cycle at presynaptic terminal. (1) SVs are filled with neurotransmitter. (2) The SVs are then transported to the active zone and docked to the presynaptic plasma membrane (docking) (3). The docked SVs become fusion competent by molecular events called priming (4). When an action potential arrives at the terminal, calcium influx through voltagedependent calcium channels triggers fusion of SV membrane with the plasma membrane (exocytosis), causing discharge of SV content (5). Exocytosed SVs are regenerated either by clathrin-independent fast endocytosis (6) or by clathrin-dependent slow endocytosis (60 ). The newly regenerated SVs are immediately refilled with neurotransmitters (7) or, in some case, they undergo fusion steps with early endosomes (70 ).
which SVs are generated from early endosomal membranes at the presynaptic terminal is dependent on AP-3. The latter pathway does not seem to account for the majority of SVs since there are no remarkable alterations in SV morphology and numbers in the
mocha mouse, which lacks functional AP-3. How, then, can SV proteins, but not plasma membrane residents, be recruited into newly generated SVs? Several amino acid sequence motifs or molecular determinants for each SV protein have been proposed to
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explain this mechanism. First, a dileucine motif located in the cytoplasmic tails of vesicular transporters (VMAT2, VAChT, and VGLUT1) is important for precise sorting and fast recruitment of those SV proteins. A VAChT mutant which lacks the dileucine motif was trapped at the plasma membrane of the cell body of the differentiated cholinergic SN56 cell lines, and the targeting of the mutant protein to neurites and varicosities (which resemble axon terminals) was dramatically reduced, indicating that the SV precursor cargo carrying VAChT is not directly transported through the axon but is delivered to the plasma membrane of the cell body before being transported along the axon. Second, the intravesicular N-glycosylation of synaptotagmin 1 is essential for its vesicular targeting. Interestingly, synaptotagmin 7, one of the synaptotagmin family members which resides preferentially on the plasma membrane, is targeted to vesicles when the intravesicular portion is replaced with a portion containing the N-glycosylation site of synaptotagmin 1. The mechanistic basis and involvement of other factors is not clear, but the interaction of glycoresidues of synaptotagmin 1 with other proteins at the cell surface may govern the targeting of synaptotagmin 1 to vesicles. It is likely that multiple factors, including protein– protein interactions and glycoresidue–protein interactions, modulate the sorting of SV proteins. The general principle of the segregation of SV proteins from plasma membrane proteins during SV biosynthesis has not been clarified.
The Synaptic Vesicle Cycle At the presynaptic terminals, SVs are not of ‘single use.’ They are regenerated at the terminals independent from protein synthesis in the cell body. The SV cycle can be outlined, with the uptake of neurotransmitter into SVs as a first step (Figure 2(b), step 1). Neurotransmitters in the central nervous system (CNS) are synthesized locally in the cytoplasm of the presynaptic terminals and are actively transported into SVs. Away from the plasma membrane, the majority of neurotransmitter-filled SVs are either diffusively floating in the cytoplasm or tethered with cytoskeleton components such as actin and spectrin (step 2). For exocytosis, SVs must come into physical contact with the plasma membrane (docking; step 3). SVs do not evenly dock to the whole area of the presynaptic plasma membrane but, rather, to a restricted area called the active zone. There, large cytomatrix proteins, such as bassoon, piccolo, and munc13, form huge protein complexes that appear as electron-dense structures on electron micrographs. Docked SVs are then transformed into fusion-competent SVs via a process called priming (step 4). As soon as an electrical stimulus
arrives at the terminal, voltage-dependent calcium channels at the active zone open, resulting in a rapid and local increase in Ca2þ concentration. Ca2þ ions trigger the fusion of the SV membrane with the plasma membrane in less than 100 ms. Since other exocytotic reactions (i.e., hormone secretion from endocrine cells) take much longer (seconds to minutes), there must be unique factors present only in neurons to perform such a rapid membrane fusion reaction. After exocytosis, SV components that are incorporated into the plasma membrane are retrieved to form a new SV by endocytosis. There are at least two kinetically distinct modes of endocytosis. The time constants of the fast and slow phase are approximately 1 and 10 s, respectively. Whereas the molecular machinery for the fast phase is not well understood, the slow phase is mediated by the formation of clathrin-coated pits. In both modes, GTP hydrolysis by the GTPase protein dynamin is indispensable for the fission of the invaginated membranes of newly formed SVs. Although various endocytosis-related proteins, such as AP-2, endophilin, amphiphysin, and synaptojanin, have been identified and implicated in controlling endocytosis, their precise roles are a matter of intense research. The reformed SVs then either recycle back and are refilled with neurotransmitters (step 1) or pass through the early endosomal intermediates before recycling back to step 1. An alternative pathway has been proposed that is similar to the exocytosis of secretory granules; that is, SVs do not fully collapse with the plasma membrane upon fusion but instead form a narrow and transient fusion pore which does not allow a complete discharge of neurotransmitter content. As soon as the pore closes, the half-empty SV can either be immediately engaged in another round of exocytosis or go back to step 1. The existence of such a rapid recycling mode in the CNS, termed the ‘kissand-run’ mechanism, is under debate. The SVs clustering at the presynaptic terminals can be divided into two functional pools. The first pool contains a small fraction of SVs (5–10% of the total SVs at the presynaptic terminals) that can be released rapidly by a brief high-frequency train of action potentials or by stimulation with hypertonic solution. This pool is thought to be release-ready and is therefore referred as to the readily releasable pool (RRP). The second pool, the reserve pool (RP), represents a vesicle fraction that does not immediate participate in exocytosis. Instead of participating in exocytosis, the RP vesicles replenish the RRP pool after the RRP vesicles undergo exocytosis. Both the amount of the RRP and the rate of replenishment of RRP with RP are critical parameters to determine the availability of vesicles for exocytosis, thereby affecting the
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characteristics of short- and long-term plasticity of a given neuron. Classically, the RRP was related to a fraction of morphologically docked vesicles at the plasma membrane and the RP was thought to be spatially distant from the plasma membrane. However, one study suggested that the RRP vesicles do not necessarily correlate with the morphologically docked vesicles but are randomly distributed in the SV cluster at the terminal, indicating that there are no correlations between the anatomical locations of SVs and their functional fusion competence. Mechanisms underlying how the mobility and the fate of an individual SV can be molecularly defined are uncertain.
Molecular Composition of Synaptic Vesicles With respect to SV functions as described previously, SVs should be equipped with two classes of essential protein components: transport proteins involved in neurotransmitter uptake into SVs and membrane trafficking proteins involved in the regulation of vesicle cycle and exo-endocytosis. A large body of work has focused on identifying protein components on SVs, leading to an almost complete identification of SV proteins. The stoichiometry of SV proteins may be flexible because many cytoplasmic proteins are temporarily attached to and detached from SVs depending on the status of a specific SV during recycling. On the other hand, a basic set of essential proteins must be present on all SVs for vesicle functions, and the numbers of individual proteins and the proportions within an SV might proteins within an SV to be maintained (Figure 3(a)). Furthermore, immunohistochemical studies on SV proteins have demonstrated that most of the major SV proteins consist of multiple isoforms whose expressions in the CNS are partially overlapping or mutually segregated, the combinations of which would create heterogeneity in protein composition in each SV. In contrast to the identification of SV proteins which became relatively handy with advancements in mass spectroscopy, understanding the mechanistic function(s) of each protein has been not so trivial, mainly because of technical limitations with regard to manipulating and measuring the intracellular events with high temporal and spatial resolution. The following sections introduce some of the essential and abundant SV proteins, which are stoichiometric components with at least one copy per SV. Proteins for Neurotransmitter Uptake
The accumulation of neurotransmitters in SVs is driven by a proton electrochemical gradient which
is generated by a vacuolar-type proton ATPase. The proton pump consists of at least 13 subunits and is the largest functional protein complex in the SV membrane. It contains two functional units – a larger peripheral protein complex (V1) which catalyzes ATP hydrolysis and an integral membrane protein complex (Vo) which builds up a ring structure in the membrane and mediates proton translocation. The molecular weight of the entire complex is approximately 800 kDa; therefore, a single complex accounts for approximately 10% of the total SV protein. An SV contains a single copy of the proton pump complex, which may be sufficient to energize neurotransmitter uptake into the SVs. With the aid of a proton electrochemical gradient, vesicular transporters specific for neurotransmittertype mediate neurotransmitter uptake into the vesicles. There are four distinct uptake systems for neurotransmitters in the CNS. Three isoforms of vesicular glutamate transporters (VGLUT1–3) transport glutamate into SVs. GABA and glycine share the same transporter, the vesicular inhibitory amino acid transporter (VIAAT; initially called vesicular GABA transporter (VGAT)). Two monoamine transporters (VMAT1 and VMAT2) transport all biogenic amines, with VMAT2 preferentially expressed in the brain. The uptake of acetylcholine is mediated by the vesicular acetylcholine transporter VAChT. All four transporter families belong to the solute carrier protein family, but there are no sequence homologies among the transporters for different neurotransmitter types. There are differences in energetics of the transport; some of them preferentially utilize the membrane potential (VGLUTs) and others use the pH gradient (VMATs and VAChT) as the main driving force. The expression of a particular transporter in the SV membrane is the ultimate determinant for the type of neurotransmitter which is released from a given neuron. Moreover, there are indications that the expression level of the transporter per SV modulates the amount of neurotransmitters accumulated in an SV, thereby regulating quantal size. In addition to the neurotransmitter transporters discussed previously, SVs contain a chloride channel to facilitate the acidification of SVs. The voltagedependent chloride channel ClC-3 was proposed to confer this activity on SVs. Although several channel activities, such as a cation selective channel, have been demonstrated by electrophysiological methods on reconstituted systems, the molecular identities of the activities have been elusive. The SV2 protein family and tetraspan vesicle membrane proteins (synaptophysin, synaptogyrin, and SCAMPs) have been identified as SV-specific proteins, and their predicted protein structures suggest their role as a transporter or a channel.
Synaptic Vesicles 81 16BAC-PAGE kDa
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Figure 3 Molecular composition of SV. (a) Protein composition of SVs visualized by 16-BAC/SDS two-dimensional gel electrophoresis. A total of 500 mg of SV proteins was applied. The spots containing the major SV proteins are circled. (b) Structural model of an average SV. Based on quantitative measurements, a three-dimensional model of an average SV was constructed. An average SV contains 70 copies of synaptobrevin, 30 copies of synaptophysin, 10 copies of neurotransmitter transporter (VGLUT), 8 copies of synapsin, 15 copies of synaptotagmin, 25 copies of Rab GTPase, and 1 or 2 copies of SV2, synaptogyrin, SCAMP, and V-ATPase. The numbers of phospholipids and cholesterol are estimated to be approximately 7000 and 6000, respectively.
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However, no clear transport activities associated with these proteins have been demonstrated. It should be noted that although not related to an intrinsic function of SV2s, they have been shown to function as a protein receptor for botulinum neurotoxin A. Proteins for Membrane Trafficking
Neural exocytosis is a tightly regulated process elicited by a rapid increase in Ca2þ concentration. Since Ca2þ ions induce neurotransmitter release within 100 ms, a series of complex enzymatic reactions could not be expected for exocytosis within the short time frame. Thus, SVs that undergo exocytosis not only should be docked but also primed for membrane fusion, waiting for the final stimulus for exocytosis. Two abundant integral membrane proteins on SVs, synaptobrevin (also referred as to VAMP2) and synaptotagmin 1, have been established as the most important players in Ca2þ-triggered exocytosis. Furthermore, SVs contain two abundant peripheral protein families, rabGTPases and synapsins, which are implicated in the modulation of vesicle pools. Synaptobrevin is a small integral membrane protein that contains a SNARE motif and is thus called a v-SNARE (vesicular SNARE, also referred to as R-SNARE because of a conserved arginine in the middle of the SNARE motif). Synaptobrevin forms a tight four-helix bundle, the SNARE complex, with two other SNARE proteins at the plasma membrane – syntaxin 1 and SNAP-25 (t-SNAREs named after target-SNAREs; also referred as to Q-SNARE, which has a conserved glutamine in the middle of the SNARE motif). The SNARE hypothesis regarding membrane fusion in general proposes that the assembly and tight complex formation of appropriate pairs of SNARE proteins pulls the two membranes close together so that the two membranes become competent for fusion. Analogously, SVs become ‘primed’ when synaptobrevin forms the SNARE complex with syntaxin 1 and SNAP-25. The importance of SNARE proteins in neuronal exocytosis is emphasized by the fact that the clostridial neurotoxins, a family of metalloproteases which specifically cleave the neuronal SNARE proteins, abolish Ca2þ-dependent neurotransmitter release while the docked vesicles remain unchanged by the toxin treatment. The formation of the SNARE complex is affected by other binding factors of the SNARE proteins. For instance, it has been proposed that Munc18 protein binds to syntaxin, keeping the syntaxin molecule in a close conformation and thereby preventing syntaxin from forming the SNARE complex. Biochemical experiments using the SNARE-reconstituted proteoliposomes suggest that the SNARE complex indeed mediates
membrane fusion. However, the liposome fusion with SNAREs alone is slow and spontaneously occurs without any regulation, indicating that other factors might play a role in synchronizing the fusion reaction. Synaptotagmin 1 is known as a vesicular Ca2þ sensor for exocytosis that accomplishes rapid membrane fusion in response to Ca2þ ions. It has an N-terminal highly glycosylated intravesicular domain and two cytoplasmic C2 domains that interact with Ca2þ ions. Ca2þ-triggered exocytosis in synaptotagmin 1-deficient neurons is severely impaired, indicating its role as the Ca2þ sensor for exocytosis. The affinity of C2 domains of synaptotagmin 1 for Ca2þ ions is very low, but it increases dramatically (up to 0.5–5.0 mM) when the C2 domains bind to phospholipids. Its apparent affinity to Ca2þ meets the requirement that it should have a Ca2þ concentration sufficient for triggering exocytosis in vivo (in the calyx of Held or in chromaffin cells). However, investigations of synaptotagmin 1-deficient neurons and chromaffin cells have suggested that synaptotagmin 1 is a Ca2þ sensor only for the fast, synchronous exocytosis and not for the slow, asynchronous component. It is not clear if other synaptotagmin isoforms (at least 15 members in mammals which differ in affinity to Ca2þ and subcellular localizations) function as a Ca2þ sensor for the latter. Rab proteins are known to be involved in a variety of intracellular membrane trafficking events. Among the Rabs, Rab3A, Rab5, and Rab11 are abundant isoforms on SVs. They associate with SVs in the GTP-bound forms, whereas they dissociate from SVs in the GDPbound state. The association and dissociation cycle of Rab3A occurs in parallel with SV exocytosis, although Rab proteins are not directly involved in exocytosis or membrane fusion. In Rab3A-deficient neurons, some forms of synaptic plasticity, such as long-term potentiation in hippocampal mossy fibers, are affected, indicating that Rab3A modulates the efficacy of neurotransmission, probably by changing the vesicle pools available for exocytosis. In addition to Rab proteins, the synapsin family represents abundant peripheral membrane proteins which may interact with SVs in an activity-dependent manner. The synapsin family consists of five isoforms (I/IIA, IIB, IIIA, and IIIB) and they form homo- or heterodimer on the surface of SVs. Originally, it was discovered as a neuron-specific abundant protein substrate for the cyclic AMP-dependent protein kinase (protein kinase A) at presynaptic terminals. Their function in synaptic transmission remains unclear. It has been proposed that synapsins function as an anchoring protein to cytoskeleton components to make a portion of vesicles immobile because they exhibit a binding property to actin, tubulin, and spectrin. Such a view is partially supported by the fact that although
Synaptic Vesicles 83
Ca2þ-triggered exocytosis is intact, short-term synaptic plasticity is impaired in the absence of synapsin 1 and 2. Therefore, synapsins might play a modulating role in controlling the vesicle pools at the terminals.
endocytosis has to be guaranteed by precise processes. More studies are needed to explore the structure– function relationships of each SV constituent to understand the function of this intriguing organelle.
Conclusion
See also: Active Zone; SNAREs; Vesicular Neurotransmitter Transporters.
Although significant progress has been made in understanding molecular mechanisms of neurotransmitter release, a mechanistic understanding of SVs is yet to be achieved. Until recently, even elementary quantitative information about SV components was lacking. Quantitative analyses of SV proteins and lipids have allowed, for the first time, the proposal of a structural model of an average SV shown in Figure 3(b). The model demonstrates that SVs are highly decorated with proteins. Numerous copies of the essential proteins for exocytosis, such as synaptobrevin and synaptotagmin, are present on a vesicle indicating that the surface densities of these proteins are not rate limiting for fusion. Furthermore, SVs contain large numbers of the neurotransmitter transporter, supporting a high-speed refilling of the vesicles upon strong repetitive stimulations. One exception among the essential proteins is the V-ATPase, which is estimated to have one or two copies per vesicle, further indicating that the retrieval of SV constituents by
Further Reading Becherer U and Rettig J (2006) Vesicle pools, docking, priming and release. Cell and Tissue Research 326: 393–407. Bonanomi D, Benfenati F, and Valtorta F (2006) Protein sorting in the synaptic vesicle life cycle. Progress in Neurobiology 80: 177–217. Fernandez-Chacon R and Sudhof TC (1999) Genetics of synaptic vesicle function: Toward the complete functional anatomy of an organelle. Annual Review of Physiology 61: 753–776. Jahn R (2004) Principles of exocytosis and membrane fusion. Annals of the New York Academy of Sciences 1014: 170–178. Montecucco C, Schiavo G, and Pantano S (2005) SNARE complexes and neuroexocytosis: How many, how close? Trends in Biochemical Sciences 30: 367–372. Rissoli SO and Betz WJ (2005) Synaptic vesicle pools. Nature Reviews Neuroscience 6: 57–69. Sudhof TC (2004) The synaptic vesicle cycle. Annual Review of Neuroscience 27: 509–547. Takamori S, Holt M, Stenius K, et al. (2006) Molecular anatomy of a trafficking organelle. Cell 127: 831–846.
Endocytosis and Presynaptic Scaffolds V Haucke, Freie Universita¨t Berlin, Berlin, Germany E D Gundelfinger, Leibniz Institute for Neurobiology, Magdeburg, Germany ã 2009 Elsevier Ltd. All rights reserved.
Introduction Neurons communicate with each other by the temporally and spatially controlled release of secretory molecules (the so-called neurotransmitters) via regulated exocytosis. Following diffusion across the synaptic cleft the released nonpeptide neurotransmitters and neuroactive peptides bind to and activate postsynaptic receptors, which then elicit a response within the postsynaptic cell. In the case of most fast-acting transmitters, such as glutamate, g-aminobutyric acid (GABA), or acetylcholine, signaling is elicited by ligand-gated ion channels, which are clustered at morphologically discernible zones specialized for chemical neurotransmission termed postsynaptic densities (PSDs). The PSD is opposed to a corresponding presynaptic element including the ‘active zone’ at which presynaptic neurotransmitter-bearing vesicles (SVs) are clustered. Presynaptic active zones are characterized by an electron-dense grid of scaffolding proteins interconnected with an actin-rich cytoskeleton, which among other functions helps to maintain a pool of vesicles docked at the presynaptic plasmalemma. To sustain chemical neurotransmission under conditions of high activity and to counter-balance net insertion of membrane by exocytic vesicle fusion, SVs undergo activity-driven cycles of calcium-triggered exocytosis and endocytosis within nerve terminals, commonly referred to as the SV cycle. Cycling of SVs must allow them to retain their specific biochemical identity, including the ability to store neurotransmitter by proton pump-driven neurotransmitter transporters, and to undergo further rounds of calcium-induced fusion with the presynaptic plasmalemma. The observed tight coupling between exocytic neurotransmitter release by vesicle fusion and compensatory endocytosis has resulted in a long and still unresolved debate regarding the precise molecular mechanisms involved in SV cycling. Here, we provide a brief summary of the pathways of SV cycling, the role of clathrin and its partner proteins in maintaining SV pools, and the temporal and spatial cues provided by scaffolding proteins and membrane lipids in maintaining presynaptic exocytic– endocytic membrane traffic.
84
Pathways of SV Cycling Clathrin-Mediated Endocytic Cycling of Presynaptic Vesicles
Landmark studies in the early 1970s by Heuser and Reese showed that following fusion by complete collapse into the plasmalemma, SVs are retrieved by compensatory ‘clathrin-dependent endocytosis’ at specialized endocytic areas just outside the active zone. An overwhelming amount of genetic, morphological, biochemical, and physiological data suggests that clathrin-mediated endocytosis indeed constitutes an essential pathway of SV recycling, at least on the organismic level and over extended periods of time. Mutants in clathrin coat components, including the AP-2 complex or accessory and adaptor proteins such as dynamin, AP180, stoned B (the Drosophila ortholog of mammalian stonins 1 and 2), eps15, synaptojanin, endophilin, amphiphysin, or intersectin/DAP160, all display defects in neurotransmission owing, at least in part, to impaired SV endocytosis (Table 1). The most dramatic phenotypes have been observed following injection of dominant-negative domains or inhibitory peptides into the giant reticulospinal synapse of the lamprey. Following electrical stimulation, distinct endocytic intermediates and vacuolar structures accumulate within and around the active zones in conjunction with a partial or complete depletion of the recycling vesicle pool. Some of these intermediates resemble structures seen in neuromuscular junctions following intense stimulation or after genetic perturbation of protein function in temperature-sensitive alleles. The mechanistic details of this pathway will be discussed later. Kiss-and-Run Mode
Based on an apparent lack of correlation between the number of morphologically distinct stable endocytic intermediates and the synaptic endocytic activity, Ceccarelli and colleagues proposed an alternative model according to which SVs release their contents through the controlled opening of a narrow fusion pore, followed by rapid closure and refilling with neurotransmitter (Figure 1). This ‘kiss-and-run’ mode of regulated secretion has been convincingly demonstrated to occur by combined electrophysiological recordings and membrane capacitance measurements in neuroendocrine cells, which mostly secrete peptide hormones or biogenic amines from large secretory granules (SGs; also termed large dense core vesicles (LDCVs)). In the case of SG exocytosis, flickering of
Table 1 Endocytic proteins involved in presynaptic vesicle cycling and their interaction partners Domains and motifs
Interaction partner(s)
Proposed function(s)
Abp1
ADF homology domain SH3 domain
Linking actin cytoskeleton with endocytosis and CAZ
AP-2 (a, b2, m2, s2 subunits)
Trunk a-Appendage b2-Appendage b2-Hinge C-m2 N-BAR domain PWXXW, LLDLD motifs FXDXF, DXF motifs SH3 domain ANTH domain FXDXF, DXF motifs DLL motifs DnaJ domain DPF, WDW motifs WDW, DLL motifs HC-terminal domain LC GTPase domain GED domain PH domain N-BAR domain SH3 domain
F-actin PRDs of synaptojanin, dynamin, Piccolo, and synapsin1 PI(4,5)P2 FXDXF/DXW, WVXF motif proteins [DE]nX1–2FXX[FL]XXR motif proteins Clathrin terminal domain Yxx and basic motif membrane cargo; PI(4,5)P2 (Acidic) phospholipids; dimerization Clathrin terminal domain AP-2 via a-appendage PRDs of dynamin (& synaptojanin) PI(4,5)P2 AP-2 via a-appendage Clathrin Hsc70 AP-2 via a-appendage Clathrin PWXXW, LLDLD, DLL motif proteins HIP1, calmodulin, Hsc70 GDP/GTP Dynamin-GED PI(4,5)P2 (Acidic) phospholipids; dimerization PRD of dynamin and synaptojanin
Amphiphysins
AP180
Auxilin
Clathrin (HC, LCa/b) Dynamin 1
Endophilin
Plasmalemmal recruitment Coat assembly Cargo selection and coat assembly Scaffolding Membrane cargo selection Membrane curvature induction/sensing; scaffolding Coat assembly Vesicle fission Plasmalemmal recruitment Coat assembly Scaffolding Stimulation of ATPase Coated pit recruitment Assembly of scaffold Diverse Control of membrane fission Self-assembly; stimulation of GTPase activity Plasmalemmal recruitment Membrane curvature induction/sensing Coated pit maturation; fission and uncoating Continued
Endocytosis and Presynaptic Scaffolds 85
Endocytic protein
Endocytic protein
Domains and motifs
Interaction partner(s)
Proposed function(s)
Epsins
ENTH domain DPW motifs LLDLD motifs UIMs EH domains DXF motifs UIMs ANTH domain FXDXF, DXF motifs LLDLD motif EH domains SH3 domains Rho-GEF domain PTB DXF motifs PIP kinase domain WYSPL tail peptide WVXF motifs Stonin-homology domain m-Homology domain SAC1 domain 50 -Phosphatase domain FXDXF, WVXF motifs C2A C2B C2AB BAR SH3
PI(4,5)P2 AP-2 via a-appendage Clathrin terminal domain Ubiquitin (Ub) NPF motif proteins AP-2 via a-appendage Ubiquitin (Ub) PI(4,5)P2 AP-2 via a-appendage Clathrin terminal domain NPF motif proteins PRD domain proteins CDC42 PI(4,5)P2 AP-2 via a-appendage PI(4)P, ATP; Arf6-GTP FERM domain of talin AP-2 via a-appendage ? Synaptotagmin (1,2,9) 40 -phosphate-containing phosphoinositides PI(4,5)P2 AP-2 via a-appendage Ca2þ, acidic phospholipids PI(4,5)P2; AP-2 via subdomain B of m2 Stonin 2 via m-homology domain (Acidic) phospholipids; dimerization PRD of N-WASP and dynamin
Membrane curvature induction; coat assembly
Eps15, Eps15R
HIP1, HIP1R
Intersectin
Numb, Numb-like PIPK type Ig Stonin 2
Synaptojanin
Synaptotagmin
Syndapins
Scaffolding Ub-dependent cargo endoctyosis Endocytic protein network formation Coat assembly (edges of CCPs) Ub-dependent cargo endoctyosis Plasmalemmal recruitment Coat assembly Scaffolding; links actin with endocytosis Endocytic protein network formation CDC42-mediated actin polymerization Plasmalemmal recruitment; coat assembly Localized PI(4,5)P2 formation; regulation of PI(4,5)P2 synthesis at cell adhesion sites Coat assembly; coated pit recruitment ? AP-2-dependent recycling 40 -phosphoinositide phosphate hydrolysis PI(4,5)P2 hydrolysis, CCV uncoating coat assembly; coated pit recruitment Ca2þ-triggered membrane fusion membrane fusion; SV recycling SV recycling Membrane curvature sensing actin and dynamin-mediated fission
86 Endocytosis and Presynaptic Scaffolds
Table 1 Continued
Endocytosis and Presynaptic Scaffolds 87
Figure 1 Pathways of SV recycling. Schematic depiction of various proposed modes of synaptic vesicle (SV) recycling: a fast ‘kiss-and-run’ mechanism, where the vesicle connects only briefly to the plasma membrane without full collapse (‘kiss-and-run’) or a slow clathrin-mediated pathway, which either operates from large vacuolar infoldings (cisternae) or by direct recovery of vesicle membrane from plasmalemmal CCPs (‘clathrin-mediated endocytosis’). SVs may also arise from endosomes. Components of the cytomatrix assembled at the active zone (CAZ) together with actin function as molecular scaffolds in the spatial organization of the active zone. The SV cycle is paralleled by a cycle of phosphorylation and dephosphorylation of phosphoinositides, including PI(4,5)P2, that couple exocytosis and endocytosis.
a transient fusion pore precedes complete degranulation. However, in contrast to small clear SVs that undergo local recycling, SGs need to pass through the trans-Golgi network in order to allow refilling with secretory peptides generated from larger precursor proteins. The recent development of lipophilic fluorescent styryl dyes (FM dyes) that rapidly partition into membranes and exhibit a large increase in fluorescence within this hydrophobic environment and of pH-sensitive fluorescent proteins (so-called ‘pHluorins’; Figure 2) has provided the means to follow exocytic–endocytic cycling of SVs in realtime. FM1–43 dye-based single-vesicle tracking in dissociated hippocampal neurons in culture has revealed the existence of at least two types of release: small-amplitude events that show tightly clustered rate constants of dye release, and larger events with a more scattered distribution. The small-amplitude partial release events have been attributed to a pool of vesicles that undergoes cycling by rapid closure of a narrow, approximately 1 nm diameter, fusion pore. One would therefore have to assume that vesicles are targeted for partial release by specific factors that prevent the dilation and thus the complete opening of the
fusion pore. Whether vesicles undergoing transient opening and closure of the fusion pore remain docked (‘kiss-and-stay’) or undergo local cycling (as depicted in Figure 1) is under debate. The balance between partial kiss-and-run-type and full fusion events that may be followed by clathrin-dependent compensatory endocytosis can be shifted depending on the frequency of stimulation. While kiss-and-run exocytosis may prevail under conditions of low activity, high-frequency stimulation results in predominantly complete fusion events. Membrane capacitance measurements of giant terminals (e.g., goldfish retinal bipolar cells or the calyx of Held) have also provided evidence for two kinetically distinguishable cycling vesicle pools. However, all of these studies suffer from the lack of information on specific factors that target SVs for fast kiss-and-run exocytosis–endocytosis and that allow the application of genetic or biochemical tools to molecularly distinguish the proposed kiss-and-run mode from other pathways of SV endocytosis. Vacuolar Bulk Retrieval and Synaptic Endosomes
Extensive stimulation of the presynaptic neuron results in the massive insertion of SV membrane into
88 Endocytosis and Presynaptic Scaffolds
Figure 2 Real-time measurement of SV cycling using pHluorins. Schematic illustration (A) of how synapto-pHluorin can be used to probe SV cycling. Its fluorescence is quenched in the acidic vesicular lumen, but not when residing at the plasmalemma. SynaptopHluorin signals (B) during firing of action potentials. (a)–(c) synapto-pHluorin (spH) is recycled at boutons. (a) Time course of fluorescence intensity, averaged over 13 boutons expressing spH, following stimulation with a train of 600 action potentials at 20 Hz. The dark bar shows the duration of the stimulus. The decay of fluorescence was fit by a single exponential (solid line) with t ¼ 74 s. (b) Time course of average spH fluorescence in the same boutons as those used for (a) during alkalinization with NH4Cl (dark bar). (c) Time course of fluorescence intensity at the same boutons as in (a, b) during train of 600 action potentials followed by exposure to NH4Cl 30 s after the end of the electrical stimulus. NH4Cl-induced changes are completely reversible. (d) and (e) Endocytosis, not reacidification, is rate limiting during fluorescence decay. (d) Time course of the fluorescence intensity of spH-positive boutons (n ¼ 20) following electrical stimulation (dark bar). Exocytosis of spH causes a rapid increase in fluorescence, followed by a slow decay (solid line is a single exponential fit to the average fluorescence decay, t ¼ 68 s). (e) Time course of fluorescence intensity during brief exposures to acidic solution (hatched bars below trace) before and after electrical stimulation (dark bar). Exposure to acid during resting periods led to decreases in fluorescence (quenching), indicating the presence of a resistant surface pool of spH. The fluorescence after acid quenches was similar before and after electrical stimulation, indicating that most of the newly endocytosed vesicles were rapidly reacidified. Reprinted by permission from Macmillan Publishers Ltd: Nature Cell Biology (Sankaranarayanan S and Ryan TA (2000) Real time measurements of vSNARE recycling in CNS synapses. Nature Cell Biology 2: 197–204), copyright 2000.
Endocytosis and Presynaptic Scaffolds 89
the presynaptic plasmalemma. It therefore may not be surprising that at least in some experimental systems, such as the neuromuscular junction of frogs and snakes, parts of the presynaptic membrane can be internalized via large vacuolar structures and cisternae, in particular after chemical induction of neurotransmitter release by application of high concentrations of Kþ and calcium. Some of these vacuoles may still exhibit a narrow tubular connection with the plasma membrane and are sometimes seen to contain clathrin-coated buds at their cytoplasmic ends (Figure 1). Whether such cisternal invaginations are eventually consumed by clathrin- and/or dynamin-dependent processes remains unclear. Once having undergone fission from the plasmalemma, cisternae could undergo additional budding steps and thereby constitute a form of a specialized presynaptic endosome. In fact, early endosomal markers including the small GTPase Rab5 and the SNARE protein Vti1ab are present on SVs. Rab5 mutations in Drosophila interfere with efficient release during repetitive stimulation, suggesting that presynaptic endosomes could play an important functional role in maintaining SV pools.
Clathrin-Mediated SV Endocytosis Clathrin was first purified by Barbara Pearse more than 30 years ago, using coated vesicles isolated from pig brain. In fact, clathrin is most abundantly expressed in the central nervous system, where it is found to be particularly concentrated in presynaptic nerve terminals. The importance of clathrin for SV recycling is further underscored by the fact that clathrin-coated vesicles (CCVs) isolated from nerve terminals are highly enriched in SV proteins. Clathrin, the heterotetrameric adaptor complex (AP-2), and monomeric adaptors and accessory proteins (including epsin, eps15, AP180, HIP1/HIP1R, amphiphysin, endophilin, stonin 2, etc.) play an early role in coat formation. Recruitment of AP-2 to the plasma membrane is a cooperative and presumably highly regulated process involving interactions with phosphoinositides, membrane cargo, and a variety of AP-2a ear domain-binding partners. Many of these adaptor and accessory proteins also display higher expression levels in brain than in other tissues, perhaps owing to their increased half-lives. In addition, neurons contain endocytic protein isoforms, including splice variants of clathrin light chains and aA-adaptin, AP180, auxillin, intersectin, and dynamin 1. Much of what we know about the mechanism of CCV formation has been learned from nonneuronal systems or from structural studies on clathrin, adaptor, and accessory proteins or
domains thereof. In the following sections, we summarize these data and provide a tentative model for how clathrin, dynamin, and their binding partners could act at nerve terminals. Early Steps of Clathrin-Coated Pit Formation
CCVs are formed by the coordinated assembly of clathrin triskelia built from three tightly linked heavy and associated light chains onto the plasma membrane. The recruitment and polymerization of the outer clathrin layer is assisted by mono- and heterotetrameric adaptor proteins, which simultaneously bind to clathrin, to membrane lipids, and in many cases to transmembrane cargo proteins. In addition, there is a large reservoir of preassembled flat hexagonal clathrin lattices at the plasma membrane that, however, need to undergo a structural transition involving the formation of clathrin pentagons in order to accommodate a curved membrane bud. The most important clathrin adaptor is the heterotetrameric AP-2 complex comprising two large subunits (a and b2), a medium subunit (m2), and a small subunit (s2). The two large subunits together with s2 and the amino-terminal domain of m2 (N-m2) form the trunk or core domain of AP-2, and are joined by extended, flexible ‘hinges’ to the appendage or ear domains of a- and b2-adaptins. Since AP-2 associates with clathrin, a variety of accessory endocytic proteins, phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], and membrane cargo proteins, it has been postulated to serve as a main protein interaction hub during coated pit assembly. Many accessory proteins, such as epsins, AP180/CALM, and amphiphysin, also have an adaptor function by linking clathrin assembly to membrane bud formation. These mono- or dimeric adaptors possess a folded lipid-binding domain linked to a more flexible portion of the protein harboring short clathrin- and AP-2-binding motifs, which may aid stabilization of nascent clathrin-coated pits (CCPs) during the assembly process. During CCP assembly transmembrane cargo proteins are recognized by adaptor proteins, most notably the AP-2 complex, which bind to endocytic sorting motifs within their cytoplasmic tails. These motifs include tyrosine-based Yxx (where is a bulky hydrophobic residue) and acidic cluster di-leucine motifs, which bind directly to distinct sites within the AP-2 core domain. Yxx motifs have been co-crystallized with the carboxyterminal portion of the AP-2 m-subunit (C-m2), to which they bind in an extended conformation. Cargo recognition by AP-2 requires the presence of PI(4,5) P2, which stabilizes the protein in an open conformation that enables cargo recognition by its m2-subunit. Consistent with this, clathrin/AP-2-coated pits were
90 Endocytosis and Presynaptic Scaffolds
shown to become stabilized in living cells upon encounter of cargo receptors, suggesting that the process of AP-2 recruitment and initiation of plasmalemmal CCPs is highly cooperative. Despite intense efforts, evidence regarding the presence of canonical Yxx - or di-leucine-type endocytosis signals within SV protein cytoplasmic domains remains scarce. However, C-m2 harbors a structurally unresolved binding site for basic internalization motifs found in a variety of multimeric membrane proteins, including the presynaptic vesicle protein synaptotagmin, the presumed calcium sensor in neuroexocytosis. Neuronal synaptotagmin isoforms also interact with the AP-2-binding m-homology domain containing adaptor protein stonin 2, which is capable of targeting synaptotagmin for clathrin/ AP-2-dependent internalization in neurons as well as in transfected fibroblasts. In addition to its interaction with the ear-domain of AP-2a, stonin 2 can bind to eps15 and intersectin, thereby linking synaptotagmin with other components of the clathrin-dependent endocytic machinery in neurons. Stonin 2 (and by analogy, stoned B in Drosophila) thus represents the first endocytic adaptor protein identified that is specifically dedicated to the endocytic internalization of a SV protein. Synaptotagmin may thus regulate both the exocytic and endocytic limbs of the SV cycle. In support of this hypothesis, it has been observed that genetic or chemical perturbation of synaptotagmin function by fluorophore-assisted light inactivation in mice, flies, or worms results in recycling defects and a partial depletion of SVs. Whether other SV proteins also interact with specific endocytic adaptor proteins or get co-sorted with synaptotagmin, that is, as part of a membrane microdomain, remains an open question. CCP Maturation and Vesicle Fission
After the clathrin lattice is formed, endophilin, epsin, eps15, amphiphysins, and other proteins are involved in membrane bending and clathrin rearrangements as coated pits progressively invaginate and mature. Partitioning of the amino-terminal amphipathic helix of the ENTH domain protein epsin and perhaps other components (i.e., the small GTPase Arf6) drives the acquisition of membrane curvature. Bin–amphiphysin–Rvs (BAR) domain proteins, such as amphiphysin and endophilin, may aid membrane bending, and function as curvature sensors that signal completion of the process. Through their SH3 domains, both amphiphysin and endophilin also interact with and recruit accessory enzymes, including the large GTPase dynamin and the phosphoinositide phosphatase synaptojanin, to the nascent vesicular bud. Dynamin is required for fission
of endocytic membrane vesicles by mechanochemically constricting (‘pinchase’) the vesicle neck. The eminent role of dynamin for SV recycling is best illustrated by the dramatic phenotype seen in shibirets mutants in Drosophila, which exhibit temperature-sensitive paralysis due to the accumulation of unbudded membrane infoldings and endocytic intermediates. Observation of CCPs dynamics using evanescent wave microscopy indicates that during fission, dynamin recruitment to coated pits is rapidly followed by recruitment of actin. Moreover, perturbation of actin disrupts the endocytic reaction with accumulation of coated pits with wide necks, suggesting a role of actin, actin-binding factors, and dynamin-interacting accessory proteins, such as Abp1 or syndapin, in promoting constriction of the neck and removal of endocytosed vesicles from the membrane. In lamprey, snake, and fly neuromuscular synapse the invagination of the membrane into pits, occurs at distinct ‘endocytic zones’ surrounding the active zones of exocytosis (termed the peri-active zone). FM1–43 photoconversion and serial section electron microscopy analysis revealed that labeled clathrin-coated endocytic vesicles were clustered significantly near active zones, consistent with local exocytic–endocytic recycling vesicle pools at this synapse. Together with the regulated turnover and synthesis of membrane phosphoinositides, actin and actin-binding proteins may thus provide spatial and temporal landmarks for SV endocytosis (see the next section).
Protein Scaffolds as Spatial Regulators of Vesicle Cycling Morphologically and functionally, the active zone can be divided into two parts: the core active zone, where regulated exocytosis (and kiss-and-run-type SV retrieval) takes place; and the peri-active zone, where clathrin-mediated endocytosis occurs (see the preceding section). At the ultrastructural level, the core active zone is characterized by the more or less regular array of electron-dense material, called the presynaptic grid, presynaptic particle web, or ‘cytomatrix assembled at the active zone’ (CAZ). During recent years, multiple molecular components – both CAZ-specific ones and those that are recruited through interaction with CAZ scaffolding proteins – have been identified and characterized. Functionally, the CAZ is thought to define the site of regulated neurotransmitter release by localizing presynaptic membrane proteins, including voltage-gated calcium channels and cell adhesion molecules, to organize steps of the SV cycle, including tethering and priming of SV, and to link the exocytic machinery
Endocytosis and Presynaptic Scaffolds 91
Figure 3 Molecular organization of the cytomatrix at the active zone (CAZ). The scheme depicts observed physical interactions between active zone-specific scaffolding proteins (black), associated proteins with putative structural functions (yellow), effector proteins (blue-green), actin cytoskeletal and associated elements (green), small modulatory molecules (gray), proteins involved in SV exocytosis (blue) and endocytosis (red), as well as presynaptic membrane proteins (pink). Some of the interactions for Piccolo and RIM were discovered in pancreatic b-cells and will have to be confirmed for the CAZ. For further details see Table 2. Note: the diagram neither reflects the relative sizes of the proteins nor their exact topographic localization within the presynaptic bouton. The arrow indicates that neurexins (a-forms) are involved in the localization of calcium channels.
with elements of the endocytic zone and with the surrounding actin cytoskeleton (Figure 3). In addition, CAZ elements turned out to be essential mediators of presynaptic plasticity. Molecular Organization of the CAZ
Relatively few CAZ-specific structural and effector proteins have been identified to date that are believed to constitute the scaffold of the CAZ and to mediate its structural and functional organization (black in Figure 3). These proteins belong to four different protein families: the Rab3-interacting molecules (RIMs), the mammalian Unc13 proteins (Munc13s), the two related giant CAZ scaffolding proteins Bassoon and Piccolo, and the ELKS/CAST proteins (Figure 3; Table 2). RIMs are multidomain proteins that were identified as effectors of Rab3, a small GTPase associated with SVs. In particular, the a-forms of RIM1 and RIM2 are important scaffolding molecules that interact with multiple other presynaptic proteins. These include isoforms of Munc13, a liaison that might be involved in
making SVs fusion-competent, as well as ELKS/CAST and Piccolo. Analysis of RIM1a-deficient mouse mutants revealed that this protein is involved in short- and long-term forms of presynaptic plasticity. For example, long-term potentiation at hippocampal mossy fiber terminals or parallel fiber synapses of the cerebellum involve protein kinase A-dependent phosphorylation of RIM1a and phosphorylationdependent binding of 14-3-3 proteins. Further interactions of aRIMs point to a central role of these proteins in active zone organization. They can bind presynaptic voltage-gated Ca2þ channels, either directly or via their binding to RIM-binding proteins, and the Ca2þ sensor synaptotagmin, which is involved in both exocytosis and endocytosis of SVs. The interaction with a-liprins, originally identified as a cytoplasmic adaptor for the receptor tyrosine phosphatase LAR, might serve the formation and maintenance of active zones, as suggested by work in invertebrates. Munc13 isoforms are involved in SV priming and the regulation of synaptic plasticity. They can link
Protein family Relevant members and synonymous names
Domains and motifs
Interacting protein
Proposed function(s)
RIMs – Rab3-interacting molecules
Zn finger
RIM1a; RIM2a,b,g; RIM3g; RIM4g; UNC10 (C. elegans)
Region between Zn finger and PDZ domain PDZ domain C2A
Rab3 Munc13-1, ubMunc13-2 14-3-3 cAMP-GEFII / Epac2 ELKS / CAST Piccolo N-type calcium channels RIM-BPs a-Liprin, N-type calcium channels synaptotagmin
Link to SV, tethering of SV, presynaptic plasticity SV priming Presynaptic plasticity Insulin secretion in pancreatic b-cells CAZ scaffolding CAZ scaffolding Channel anchoring and/or clustering CAZ scaffolding CAZ scaffolding, Anchoring and/or clustering of Ca channels CAZ scaffolding, organization of Ca sensing
Pro-rich motif btw. C2A & B C2B
RIM-binding proteins RIM-BP1 RIM-BP2
SH3 domains 2 and/or 3
RIMs N-type (Cav2.2) and L-type (Cav1.3) Ca channels
CAZ Scaffolding Channel anchoring and/or clustering
Piccolo–Bassoon family Bassoon and Piccolo
Zn fingers Between CC1 and CC2
PRA1 (prenylated Rab3 acceptor) CtBP/BARS Ribeye/CtBP2 ELKS / CAST Abp1 (actin-binding protein 1) GIT (ARF-GAP) Profilin cAMP-GEFII/Epac2 RIMs Piccolo L-type Ca channel a-RIMs
Link to SV? Membrane trafficking? Scaffolding of synaptic ribbons CAZ scaffolding Link to actin cytoskeleton, endocytosis Membrane trafficking Actin regulation Insulin secretion in pancreatic b-cells CAZ scaffolding, organization of SV cycle Homophilic interaction, scaffolding Channel anchoring in b-cells CAZ scaffolding,
Piccolo/Aczonin only
UNC13 proteins
CC3 N-terminal Q domain Between CC1 and CC2 Pro-rich region btw CC1 and 2 PDZ domain C2A C2A and C2B M13-1 and ubM13-2 N-term.
92 Endocytosis and Presynaptic Scaffolds
Table 2 Protein components of the cytomatrix assembled at the active zone
Munc13-1 bMunc13-2 ubMunc13-2 Munc13-3 Munc13-4 UNC13 (C.e.) Dunc13 (D.m.)
Conserved homology region (MUN)
C-terminus
ELKS/CAST proteins ELKS1A/ERC1A ELKS1B/ERC1B ELKS2/CAST/ERC2
CC regions
a-Type Liprins Liprin-a1–a4 SYD-2 (C.e.)
N-term CC region
C-terminus
C-terminal PDZ binding site
SV priming, control of SNARE complex formation
Bassoon, Piccolo a-Liprins RIMs Syntenin
CAZ scaffolding CAZ scaffolding CAZ scaffolding Active zone organization?
RIMs ELKS/CAST GIT (ARF-GEF)
CAZ scaffolding CAZ scaffolding Regulation of membrane trafficking and actin cytoskeleton Transport Regulation of membrane protein anchoring
Kif1A (kinesin motor) LAR receptor protein tyrosine Phosphatase b-Liprin CASK (MAGuK) MALS/Veli GRIP
Regulation of transsynaptic adhesion? Regulation of transsynaptic adhesion? Receptor transport and clustering/postsynaptic
Endocytosis and Presynaptic Scaffolds 93
SAM domains
Ca2þ-dependent plasticity Anchoring to actin/spectrin cytoskeleton Regulation of actin cytoskeleton ?
Calmodulin Spectrin b-spIIIS msec7-1 ARF-GEF DOC2a (double C2 domain protein) Syntaxin (in debate)
94 Endocytosis and Presynaptic Scaffolds
to the actin-spectrin cytoskeleton via binding to a b-spectrin isoform and to presynaptic membranetrafficking processes via the ARF guanine nucleotide exchange factor msec7-1. ELKS/CAST proteins display a very high content of coiled-coil structures and are considered as a major structural component of the CAZ. They can physically interact with RIMs as well as with Bassoon and Piccolo and thus might interconnect the major scaffolding proteins of the CAZ. Bruchpilot, an ELKS/ CAST-related protein in Drosophila, is responsible for the proper anchoring of particular specializations of the CAZ, so-called T-bars, to the active zone membrane. Via syntenin, ELKS/CAST proteins also connect to neurexins, which are specific cell adhesion molecules of the presynaptic membrane occurring as long a- and short b-forms involved in Ca2þ channel localization and linkage to postsynaptic neuroligins, respectively. Neurexins are additionally anchored to the presynaptic cytomatrix via a trimeric protein complex of CASK, Mint, and MALS/Veli, which in turn links to Ca2þ channels and a-liprins (Figure 3). Bassoon and Piccolo are considered as very large multidomain scaffolding molecules of the CAZ. Most of their interaction partners have still to be discovered. They both bind ELKS/CAST and the small prenylated Rab3 acceptor (PRA1), which potentially links into the SV cycle. In addition, Bassoon has been reported to interact with CtBP/BARS and its homolog RIBEYE, a specific component of synaptic ribbons in retinal photoreceptors and inner ear hair cells. The RIBEYE–Bassoon interaction was suggested to be essential for anchoring of ribbons to the presynaptic plasmalemma. The interaction of Bassoon (and potentially also Piccolo) with CtBP/BARS is of interest, as this protein has been implicated in vesicular fission from the trans-Golgi complex. By analogy, a similar function might be envisioned in the presynapse. Links of the CAZ to the Endocytic Machinery and the Actin-Based Cytoskeleton
While multiple interactions of the previously described CAZ proteins underscore the role of the presynaptic cytomatrix in organizing the apparatus for regulated exocytosis, a link to clathrin-mediated endocytosis is less clear. Specific interaction partners for Piccolo might be of particular interest in this context, as they can provide the basis for the physical linkage of exocytic and endocytic presynaptic processes. On the one hand, Piccolo can bind directly to Ca2þ channels and the guanine nucleotide exchange factor cAMP-GEFII/Epac2, as revealed from studies on pancreatic b-cells. Moreover, the C2A domain of Piccolo is discussed as a candidate low-affinity
Ca2þ sensor. These characteristics argue for a role in active zone organization and exocytosis. On the other hand, Piccolo can bind to the ARF-GTPase-activating protein GIT, which has been implicated in endocytic processes such as receptor internalization. The N-terminus of Piccolo specifically interacts with Abp1, an actin-binding factor directly regulating the GTPase dynamin. Indeed, the N-terminal Q-domain of Piccolo can interfere with endocytic processes in heterologous systems. Yet another link between Piccolo and the actin cytoskeleton is profilin, a small G-actin- and phosphoinositide-binding protein that is involved in local remodeling of the actin cytoskeleton. Thus, Piccolo might indeed be an important mediator between the neurotransmitter release apparatus and the neighboring endocytic machinery.
Regulation of the SV Cycle by Membrane Lipids Phosphoinositide Regulation of SV Cycling
Cycling of presynaptic vesicles requires the precise spatial and temporal regulation of protein–lipid interactions. Many synaptic proteins – including synaptotagmin1, calcium-dependent activator protein for secretion (CAPS), the Munc18-interacting proteins Mint-1 and Mint-2, voltage-gated P/Q-type calcium channels, and a variety of endocytic proteins such as AP-2, AP180, epsin, and dynamin (Table 1) – directly bind to and are regulated by [PI(4,5)P2]. SV cycling thus appears to be nested into a local cycle of phosphoinositide phosphorylation and hydrolysis. Accordingly, PI(4,5)P2 acts at multiple stages of the vesicle cycle. Knockout mice lacking PIP kinase type Ig, the major PI(4,5)P2-synthesizing enzyme at synapses, display defects in neurotransmitter release and endocytic recycling that cause synaptic depression. The activity of PIPK Ig is regulated by a variety of factors, including phosphatidic acid (PA), the small GTPases Arf6 and Rac1, and the actin cytoskeletonassociated adhesion protein talin. Association of PIPK Ig with these factors is dependent on their phosphorylation status, providing a means for the temporal and spatial regulation of phosphoinositide metabolism. PI(4,5)P2 is eventually consumed by synaptojanin-mediated dephosphorylation, resulting either in formation of PI(4)P and perhaps PI, cleavage via phospholipase C, or PI3 kinase-dependent synthesis of PI(3,4,5)P3. At least in neuroendocrine PC12 cells, PI(4,5)P2 appears to be concentrated within cholesterol-enriched microdomains near release sites, where it may aid vesicle docking and/or fusion. The recent observation that SV proteins remain clustered during their exocytic–endocytic journey, together
Endocytosis and Presynaptic Scaffolds 95
with the extremely high cholesterol content of SV membranes, suggests that cholesterol-enriched microdomains could serve to spatially organize the SV cycle, perhaps in part by locally concentrating PI(4,5)P2. At present we can only speculate about the exact mechanism of action of PI(4,5)P2 during vesicle fusion, but its role in clathrin-mediated synaptic vesicle endocytosis is much better understood. PI(4,5)P2 is an important factor in recruiting endocytic adaptor and accessory proteins to the membrane where these form a network of protein–protein interactions. The stability of this network critically depends on the PI(4,5)P2 content of the membrane, as suggested by the observation that CCVs accumulate in nerve terminals of synaptojanin knockout mice. These observations also indicate that PI(4,5)P2-hydrolysis may normally occur concomitantly with or directly after dynamin-mediated membrane fission. CCVs at presynaptic sites of synaptojanin knockout mice become trapped in a meshwork of actin filaments, consistent with the fact that PI(4,5)P2 regulates actin polymerization and drives the formation of actin comet tails that may help to propel endocytic vesicles away from the plasmalemma. Lipids and Membrane Deformation
SV fusion and the subsequent formation of endocytic clathrin-coated buds at the presynaptic peri-active zone involve radical geometric remodeling of the membrane in order to generate areas of different membrane curvature. While lipids with bulky polar headgroups and saturated or single fatty acid tails, including lysolipids and many glycolipids, promote positive curvature, lipids with compact headgroups and space-filling hydrophobic tails, such as PA and diacylglycerol (DAG), favor negative curvature. For example, lysophosphatidic acid (LPA) and PA, which are interconverted by LPA-acyl transferase and phospholipase A2 activities, respectively, favor opposite curvatures. Although differential distribution of distinct types of lipids between the two membrane leaflets may contribute to membrane deformation, it is generally assumed that membrane bending requires the action of proteins. During endocytic SV recycling, the forming bud must adopt a positive curvature at the bud center and negative curvatures at the edges. Endocytic proteins may act by one of several mechanisms to bend membranes.
As stated previously, epsin, a PI-(4,5)P2-binding clathrin accesssory protein, is able to partition into the cytoplasmic leaflet of the plasma membrane via PI (4,5)P2-induced formation of an extra a-helical segment that drives acquisition of positive curvature. Dimeric BAR domain-containing proteins, including amphiphysin or endophilin (Table 1), induce curved membranes by an additional amphipathic helix at their amino-terminal end, largely via electrostatic interactions of their concave surface with negatively charged membrane phospholipids. Endophilin has originally been proposed to be an LPA-acyl transferase, but this activity has recently been called into question. Highly curved membranes may become stabilized in addition by other scaffolding proteins, including clathrin itself, which forms a rigid basket around the emanating vesicular membrane bud. Moreover, the transmembrane domains of synaptic vesicle proteins could provide a barrier that prevents local areas of high curvature from lateral diffusion and thereby contribute to maintaining vesicle identity. Finally, protein–lipid interactions and the formation of microdomains might also underlie the choice between fast and slow modes of SV cycling that would require a tight control of fusion pore expansion and constriction. See also: Synaptic Vesicles.
Further Reading Dresbach T, Qualmann B, Kessels MM, et al. (2001) The presynaptic cytomatrix of brain synapses. Cellular and Molecular Life Sciences 58: 94–116. Galli T and Haucke V (2004) Cycling of synaptic vesicles: How far? How fast! Science Signal Transduction Knowledge Environment 264: re19. Gundelfinger ED, Kessels MM, and Qualmann B (2003) Temporal and spatial coordination of exocytosis and endocytosis. Nature Reviews Molecular Cell Biology 4: 127–139. Murthy VN and De Camilli P (2003) Cell biology of the presynaptic terminal. Annual Review of Neuroscience 26: 701–728. Royle SJ and Lagnado L (2003) Endocytosis at the synaptic terminal. Journal of Physiolology 553: 345–355. Shankaranarayanan S and Ryan TA (2000) Real time measurements of vSNARE recycling in CNS synapses. Nature Cell Biology 2: 197–204. Sudhof TC (2004) The synaptic vesicle cycle. Annual Review of Neuroscience 27: 509–547. Ziv NE and Garner CC (2004) Cellular and molecular mechanisms of presynaptic assembly. Nature Reviews Neuroscience 5: 385–399.
Postsynaptic Density/Architecture at Excitatory Synapses H-C Kornau, Center for Molecular Neurobiology (ZMNH), University of Hamburg, Hamburg, Germany ã 2009 Elsevier Ltd. All rights reserved.
Introduction Excitatory chemical synapses of the central nervous system are highly specialized structures which rapidly convey information from one neuron to the other by means of a soluble neurotransmitter, in most cases glutamate. The neurotransmitter is released from the presynaptic terminal upon action potential-evoked calcium influx, diffuses through the synaptic cleft, and opens ligand-gated ion channels on the surface of the postsynaptic cell, resulting in excitatory postsynaptic currents. Signal reception and processing is achieved in dendrites, which are often contacted by presynaptic terminals of thousands of neurons. Excitatory synapses are mostly located on spines, little mushroom-shaped structures protruding from the dendritic shaft. Dendritic spines form a compartment for processing of the individual synaptic input. In electron micrographs the presynaptic side is recognized by a high density of neurotransmitter-containing vesicles. On the postsynaptic side of the cleft a characteristic electron-dense thickening of the membrane, extending approximately 30 nm into the spine cytosol, is observed (Figure 1); this thickening, a structure that has attracted the interest of neurobiologists since the 1950s, is called the postsynaptic density (PSD). Given that synapses are crucial for information processing in the brain, the importance of the PSD, as the site where neurotransmitter receptors are connected to intracellular structures and where synaptic processing within each spine is initiated, is obvious. Molecular biology and biochemical approaches have identified many proteins of the PSD, including (1) adhesion molecules, receptors, and channels, (2) scaffolding, adaptor, and cytoskeletal proteins, and (3) signaling/regulatory proteins, including kinases, phosphatases, small GTPases, and similar molecules. Moreover, the studies revealed how these proteins physically interact with each other or with the postsynaptic membrane. Thus, we can draw preliminary models containing some of the major components of the PSD to get an idea of the way this macromolecular protein complex may function and how it is rearranged during development and in processes of synaptic plasticity.
central nervous system. PSDs are 25- to 50-nm-thick protein assemblies with a diameter of several hundred nanometers. High-resolution electron microscopy reveals a structured 5-nm-thick sheet, which might form the nucleus of the PSD, as well as filamentous structures which connect it to cytoskeletal elements within the cytoplasm of the dendritic spine and to organelles such as the smooth endoplasmic reticulum at the margin of the PSD. Due to their unique density and their resistance to nonionic detergents, PSDs can be purified as intact, disk-shaped structures by differential centrifugation protocols. These preparations serve as the protein sources for the molecular analysis of PSDs. Initially, individual PSD proteins were discovered by amino acid sequencing of protein bands of such PSD preparations after separation by gel electrophoresis. Alternatively, antibodies were raised against complex PSD preparations and used to screen cDNA expression libraries. Coimmunoprecipitates of known PSD components have been a source for finding new elements of this structure as well. Yeast two-hybrid screening, a genetic screen for interacting proteins, supplied a number of proteins physically binding to glutamate receptors or scaffolding proteins of the PSD. Recently, proteomics, which is generally regarded as a large-scale analysis of proteins in a given sample, involving efficient protein separation and highly sensitive protein identification techniques, has been applied to glutamate receptor complexes and to PSD preparations. The results suggest that the PSD contains several hundred different polypeptides. Mass spectrometry techniques have not only identified these proteins with great sensitivity, they have also allowed quantitative determinations. Thus, for some of the components, we know their stoichiometries. Using scanning transmission electron microscopy on a rat forebrain PSD fraction, the mass of an average PSD was measured and the number of copies of specific PSD proteins derived from their relative mass contributions was determined by quantitative gel electrophoresis (Table 1). Consistent with an independent approach using a green fluorescent protein-based calibration technique, these data suggest that a single PSD contains several hundred copies of each of its major polypeptides.
The Protein Composition of Postsynaptic Densities
How Postsynaptic Densities Are Studied
Glutamate Receptor Complexes
Electron microscopy initially identified PSDs as hallmarks of glutamatergic/Gray type I synapses in the
Glutamate receptors and associated proteins were among the first components identified to be part of
96
Postsynaptic Density/Architecture at Excitatory Synapses 97
Figure 1 The PSD, a structural element of excitatory synapses. The electron micrograph shows a representative excitatory synapse (Gray type I) at a synaptic spine (s) of an adult mouse hippocampus. Major synaptic structures are the presynaptic bouton (b) filled with synaptic vesicles (diameter 50 nm), the PSD (arrowheads), and the synaptic cleft in between. The electron micrograph was a kind gift of Dr. Michaela Schweizer, Central Service Facility of Morphology at the ZMNH, University of Hamburg, Hamburg, Germany.
Table 1 Dimensions of a PSD Mean diametera Depthb Mean massa Number of PSD-95 moleculesa
360 nm 25–50 nm 1.1 GDa 300
a
Data from Chen X, Vinade L, Leapman RD, et al. (2005) Mass of the postsynaptic density and enumeration of three key molecules. Proceedings of the National Academy of Sciences of the United States of America 102: 11551–11556. b Data from Valtschanoff JG and Weinberg RJ (2001) Laminar organization of the NMDA receptor complex within the postsynaptic density. Journal of Neuroscience 21: 1211–1217.
the PSD. Glutamate receptor channels mediate the principal postsynaptic functions – that is, changes in membrane potential upon binding of the excitatory neurotransmitter. The different types of ionotropic glutamate receptors are named for their specific
agonists. a-Amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor channels are opened upon glutamate binding alone, whereas N-methylD-aspartate (NMDA) receptor activation requires simultaneous membrane depolarization (e.g., by activation of neighboring synapses). This depolarization relieves the extracellular magnesium block of the NMDA channel, allowing influx of sodium and, importantly, calcium through the channel pore. Calcium acts as a second messenger within the cell and is important for the induction of synaptic plasticity. A very abundant PSD protein involved in these processes and activated by calcium is calcium/ calmodulin-dependent protein kinase II (CaMKII). It is associated with the cytoplasmic region of the NMDA receptor, allowing it to sense the influx of calcium through this channel directly. Subsequent pathways involve a variety of structural rearrangements within the PSD and lead to a change in synaptic efficacy primarily expressed as an altered number of AMPA receptors in the postsynaptic membrane. Both native AMPA and NMDA receptor complexes are associated with a variety of scaffolding/adaptor proteins (e.g., postsynaptic density protein 95), which link the ionotropic glutamate receptors to other integral membrane proteins, to soluble signaling molecules, and to the spine cytoskeleton, and form the core lattice of the PSD. At the margin of the PSD, metabotropic G-protein-coupled receptors for glutamate (mGluRs) modulate the intraspinal calcium level. Proteomics has unraveled individual protein complexes for NMDA receptors, mGluRs, and AMPA receptors. The NMDA receptor complex is tightly linked to the core of the PSD by a series of adaptor proteins comprising postsynaptic density protein 95 family members (PSD-95; also called synapse-associated protein 90 (SAP90), PSD-93/Chapsyn-110, SAP102, and SAP97), guanylate kinase-associated proteins (GKAPs; also called SAP90/PSD-95-associated proteins, or SAPAPs), and Shanks (also called proline-rich synapse-associated proteins, or ProSAPs) (Figure 2). PSD-95 and its relatives encompass three PDZ domains, an SH3 domain, and a guanylate kinase domain, all of which function as protein–protein interaction domains. They belong to the family of membrane-associated guanylate kinases (MAGUKs), which function primarily at sites of cell-to-cell contact. Employing its PDZ domains, PSD-95 links the NMDA receptor to neuroligins, postsynaptic cell adhesion molecules crucial for synapse formation, and to several signaling molecules, among them neuronal nitric oxide synthase (nNOS) and synaptic GTPase-activating protein (synGAP), an abundant regulator of Ras and Rap GTPases. The GKAPs connect PSD-95 to the Shank
98 Postsynaptic Density/Architecture at Excitatory Synapses Synaptic cleft AMPAR
TARP
NMDAR
mGluR
Plasma membrane PSD-95 protein family
Homer
GKAP
Shank sheet Spine cytoskeleton Figure 2 Simplified scheme of the molecular architecture of the PSD. The different types of glutamate receptors are linked to the Shank scaffold, a two-dimensional sheet, either by the PSD-95 family of proteins and by guanylate kinase-associated protein (GKAP) molecules, or by Homer dimers. The Shank scaffold connects the PSD with the spine actin cytoskeleton. The PSD-95 family proteins form a membrane-proximal layer of scaffolds, whereas the Shanks constitute a second, deeper layer. PDZ domains are depicted as blue circles. The C-terminal domains of the glutamate receptor subunits, the PSD-95 family members, as well as the Shanks, recruit numerous signaling molecules to the PSD. The PSD-95 family members connect the PSD with cell adhesion molecules governing the integrity with the presynaptic specialization. The Homer proteins link the metabotropic glutamate receptors (mGluRs) not only to the Shank scaffold, but also to the inositol 1,4,5-trisphosphate receptor within the smooth endoplasmic reticulum. For simplicity, all of these connections were omitted from the scheme. The figure does not reflect the real stoichiometry of the scaffolding proteins. AMPAR, a-amino-3-hydroxy-5methyl-4-isoxazole propionic acid receptor; TARP, transmembrane AMPA receptor regulating protein; NMDAR, N-methyl-D-aspartate receptor.
core of the PSD by binding to both the guanylate kinase domain of PSD-95, on one hand, and to the PDZ domain of a Shank molecule, on the other hand. Four closely related GKAP family members show distinct spatiotemporal expression patterns in the brain. Shanks (three related proteins) contain a number of protein interaction domains, including ankyrin repeats, an SH3 domain, a PDZ domain, a proline-rich region, and a sterile alpha allowing them to bind to a variety of proteins. Moreover, by self-association of their SAM domains they are able to form large sheets within the PSD. The resulting dense platform is associated with the actin cytoskeleton, the smooth endoplasmic reticulum, the different glutamate receptor complexes, and other channels and receptors. Shanks are also early components during synaptogenesis. The three classes of adaptor proteins play a crucial role within the PSD. They form an axis of scaffolds differing in their distance from the plasma membrane (Figure 2): PSD-95 is located approximately 12 nm cytoplasmic to the extracellular face of the postsynaptic membrane, like the C-termini of the NMDA receptor, whereas GKAP and Shank molecules are buried deeper in the cytoplasm (approximately 24–26 nm from the membrane). They recruit a number of signaling molecules to the PSD and connect the PSD with cytoskeletal elements. Proteomic analysis of the NMDA receptor complex has revealed a complexity of more than 100 proteins, including a variety of protein kinases and phosphatases, small G-proteins
and their regulators, and other signaling molecules, such as nNOS, phosphatidylinositol 3-kinase (PI3K), phospholipase C-g, Citron, and Arg3.1/Arc. In addition, several cell adhesion molecules and associated adaptors, including N-cadherins and b-catenin, which regulate dendritic spine morphogenesis, were found in the NMDA receptor complex. Finally, many cytoskeletal proteins are part of this complex. Type I metabotropic glutamate receptors are located at the margin of the PSD. These G-proteincoupled receptors lead to inositol 1,4,5-trisphosphate (IP3) production, which consequently activates the IP3 receptor and induces the release of calcium from intracellular stores. Key molecules of the mGluR protein complex are the Homer proteins, which are expressed from three genes. They can dimerize by a coiled-coil interaction and these dimers connect mGluRs to the Shank backbone of the PSD on the plasma membrane side, and IP3 receptors in the smooth endoplasmic reticulum to the Shank backbone on the cytoplasmic side, of the PSD. Thus, Homer proteins connect mGluRs and IP3 receptors to the PSD and link mGluRs and IP3 receptors, allowing efficient intracellular calcium release upon mGluR activation. In these assemblies the N-terminal EVH domains of the Homer dimers bind to a PPXXF motif in the receptors and to a proline-rich region in Shanks (Figure 2). (The EVH nomenclature derives from ‘enabled-VASP (vasodilator-stimulated phosphoprotein) homology.’) Interestingly, Homer 1a, an
Postsynaptic Density/Architecture at Excitatory Synapses 99
isoform lacking the coiled-coil domain, which is highly expressed as an immediate-early gene, can compete with the constitutive forms of Homer and thus with the link of mGluRs and IP3 receptors to the PSD. This may enable the cell to limit the release of intracellular calcium if required. The mGluR5 complex has been also subject to proteomic analysis. It provided a number of additional candidate proteins of mGluR complexes and confirmed the link to Homer, Shank, and IP3 receptors delineated herein. AMPA receptors mediate the majority of the fast excitatory neurotransmission in the brain. The efficacy of a synaptic connection is related to the number of AMPA receptors in the postsynaptic membrane. Thus, whereas NMDA receptors are mediating the induction of synaptic plasticity, AMPA receptors are crucial for its expression. The number of AMPA receptors in the postsynaptic membrane is indeed quite variable, ranging from zero to several hundred receptors per synapse, and can rapidly change upon usage of a synapse. AMPA receptors are rapidly cycling between plasma membrane and endosomal compartments. Thus, they need a unique set of associated proteins. The best documented interaction of AMPA receptors is their binding to the integral membrane protein stargazin. Stargazin was the first member of a family of four differentially expressed TARPs (for transmembrane AMPA receptor regulatory proteins), which function as auxiliary subunits of AMPA receptors. By two independent interaction sites, TARPs regulate both the trafficking and the gating of AMPA receptors. A C-terminal interaction of TARPs with PSD-95 connects AMPA receptors with the PSD and governs the synaptic localization of AMPA receptors (Figure 2). The rapid cycling of AMPA receptors requires the interaction with AP2 and clathrin. These and N-ethylmaleimide-sensitive fusion protein NSF, a multimeric ATPase playing an important role in membrane fusion, bind to specific motifs in the center of the short, intracellular C-termini of selected AMPA receptor subunits. Additional proteins interact with the very C-terminal amino acids of AMPA receptor subunits and affect the synaptic localization of AMPA receptors. These include an adaptor protein termed glutamate receptor-interacting protein (GRIP) and a protein originally identified as a protein interacting with C kinase, PICK1. Both of these proteins bind to the C-termini of AMPA subunits by PDZ domains. GRIPs (two related proteins) harbor seven PDZ domains and thereby can connect AMPA receptors to several cytoplasmic and membrane-standing proteins (e.g., the ephrin receptor). The PICK1 interaction with AMPA subunits is essential for the
AMPA receptor endocytosis involved in cerebellar long-term depression. Proteomic approaches to tackle the composition of AMPA receptor complexes identified a limited complexity as compared to NMDA receptor or mGluR complexes. However, AMPA receptor cycling between plasma membrane and endosomal locations may specifically require transient or weak interactions, which may be lost during purification of the receptor complex. Multiple interfaces between the individual glutamate receptor multiprotein complexes appear to exist: Homer (mGluR complex) can bind to Shanks in addition to mGluRs and IP3 receptors, and stargazin (AMPA receptor complex) binds to PSD-95 (NMDA receptor complex). Proteomic studies of PSD proteins have been performed on immunopurifications of glutamate receptors (brain protein complexes purified via peptides derived from the Cterminal sequence of the NMDA receptor subunit 2B, a target of PSD-95 family members) and on purifications of entire PSDs. The data from these different approaches have been compiled as our current view of the PSD composition. They show that in addition to the glutamate receptor complexes connected to the different scaffolding proteins we have to integrate a large number of additional integral membrane proteins, signaling/regulatory proteins, and cytoskeletal elements into our model of the PSD. Although the number of proteins identified in PSD fractions goes into the hundreds, only a few principal types of molecule classes can be covered here. Integral Membrane Proteins
Independent analyses of the PSD proteome revealed a variety of integral membrane proteins aside from glutamate receptors. These include not only other channels and receptors, but also cell adhesion molecules (e.g., neuroligins and N-cadherin) which directly link the PSD with the juxtaposed membrane specialization on the presynaptic side termed the active zone. Cell adhesion molecules govern several steps of synaptogenesis and may keep the active zone and the PSD in register during structural changes of the synapse. Regulatory Proteins
The importance of CaMKII as a very abundant PSD protein for the induction of synaptic plasticity has already been mentioned. However, many other types of protein kinases and phosphatases are found in the PSD as well. This is not surprising since phosphorylation and dephosphorylation events often mediate
100 Postsynaptic Density/Architecture at Excitatory Synapses
functional and structural modulation of central nervous system synapses. Small GTPases of the Ras superfamily, termed Ras and Rap, belong to the most important molecules involved in the integration of biochemical signals in the dendritic spine. Their activity determines the amount of AMPA receptors in the postsynaptic membrane. As GTPases they switch between an active, GTP-bound, and an inactive, GDP-bound, state. Activation requires a specific guanine nucleotide exchange factor (GEF); inactivation requires a GTPase-activating protein (GAP). PSD preparations contain several GEFs and GAPs for Ras and Rap, which themselves differ by the signals required for their activation. Small GTPases of the Rho family, including Rac and cdc42, directly influence the state of the spine cytoskeleton. For example, adhesion molecule-mediated recruitment of specific Rac GEFs activates Rac and leads to changes in actin polymerization. These GTPases have a strong impact on the morphology of dendritic spines and their regulators are found in PSD preparations. Cytoskeletal Proteins
The axis of scaffolds in the PSD (PSD-95 family members, GKAPs, and Shanks) is also a nucleus for interactions with the cytoskeleton in the spine. As already indicated in the section on the NMDA receptor complex, PSD preparations contain a variety of cytoskeletal proteins. They include actin, actinbinding proteins, tubulin, spectrin, cortactin, and many others. The proteomic approaches revealed several hundred different proteins in PSD preparations. However, it should be noted that this does not necessarily reflect the complexity at a single PSD. The protein composition of PSDs in different brain regions as well as in different synapses of a single neuron may considerably differ, and thereby may contribute to the large number of identified proteins.
PDZ Domains Like all macromolecular protein networks, the PSD is connected by modular protein–protein interactions. Analysis of the interacting protein sequences has revealed the presence of specific domains. A domain frequently found at sites of cell-to-cell contact is the PDZ domain (so named from PSD-95/discs-large/ zonula occludens-1), which is present in many different proteins in humans. PDZ domains bind, in most cases, to short peptide motifs at the C-termini of other proteins and are classified according to the
sequences they recognize. In addition, some PDZ domains can heterodimerize. The PDZ domain is also central in connecting different components of the PSD (Figure 2). It was first recognized in the prototype PSD scaffolding protein PSD-95 (hence, the name), which contains three N-terminal PDZ domains and uses two of them to bind to the C-termini of NMDA receptor subunits and the third PDZ and other domains to connect the receptor to a variety of PSD components. PDZ domains are essential for the function of many signaling and scaffolding proteins of the PSD. The scaffolding proteins often contain several PDZ as well as other interaction domains, each with a preferred binding partner, allowing the scaffold to coordinate large protein complexes.
Rearrangements of Postsynaptic Densities Developmental Assembly of Postsynaptic Densities
Most of the synapse formation in the rat central nervous system occurs during the first two postnatal weeks. It is initiated by an interaction between preand postsynaptic cell adhesion molecules within the synaptic cleft, including neurexins/neuroligins, cadherins, synCAMs, receptor tyrosine kinases, and others. Among the earliest proteins at synapses are PSD-95 family members and Shanks, suggesting that they play a major role in the assembly of the PSD. Their interactions with the other scaffolding and signaling molecules are thought to promote the formation of the PSD very rapidly. Simultaneously, NMDA receptors are recruited into the PSD, whereas the integration of AMPA receptors into the postsynaptic membrane requires presynaptic glutamate release. Early in development many synapses in the central nervous system lack AMPA receptors and are therefore called silent synapses. PSDs of postnatal day 2 rats contain already many of the proteins found in adult PSDs. However, during development, specific molecular changes are evident. These include a switch in the expression of NMDA receptor subunits from NR2B to NR2A and, simultaneously, of their interaction partners SAP102 to PSD-95, and an increase of AMPA receptors and CaMKII. Although during synaptogenesis the recruitment of key PSD proteins such as NMDA receptor subunits and Shanks appears to occur in a rather gradual manner, preformed transport packets containing either AMPA or NMDA receptors, or mobile clusters containing PSD-95, GKAP, and Shank, driven by actin transport, have been described. Thus, how the PSD is assembled during synapse formation requires further investigation.
Postsynaptic Density/Architecture at Excitatory Synapses 101 Activity-Dependent Changes at Postsynaptic Densities
The size, protein composition, and structure of the PSD are altered in response to developmental and environmental cues. One cellular model for learning and memory, long-term potentiation (LTP), involves a prolonged strengthening of a synapse in response to a high-frequency input. Calcium influx through NMDA receptors, which are open under these depolarizing conditions, allows activation of a number of signaling molecules of the PSD, including CaMKII, calcineurin, nNOS, and several regulators of small GTPases. The primary result is a change in the phosphorylation pattern of PSD proteins, among them the glutamate receptor subunits. Phosphorylation leads to an altered affinity of binding partners and concomitant stabilization or destabilization of the receptor molecules at the PSD. Activation of small G-proteins can trigger endo- or exocytosis of membrane proteins. As an example, additional AMPA receptor subunits are rapidly inserted into the plasma membrane during LTP, resulting in a direct increase of the postsynaptic response. In contrast, AMPA receptors are removed from the synaptic plasma membrane during long-term depression. Other small GTPases, upon activation, alter the polymerization state of actin in the dendritic spine. Thus, synaptic plasticity involves structural plasticity at the PSD. In addition to changes in the phosphorylation pattern, receptor localization, and cytoskeleton structure, local translation and targeted protein degradation contribute to the high capability of the PSD to rearrange. Activity-dependent translation of AMPA receptor subunits in dendrites may mediate synapsespecific modifications. The presence of mRNAs for other PSD components (e.g., Shanks) in dendrites suggests their local translation as well. Dendritic protein synthesis may contribute significantly to synaptic plasticity processes. Posttranslational modifications aside from phosphorylation affect the PSD structure. As an example, PSD-95 family proteins are differentially palmitoylated at two cysteine residues, with important consequences for their clustering ability. Changes in the PSD structure and composition can also result from altered patterns of transcription and splicing in the nucleus. Alternative splicing events affect the interactions of various PSD proteins by adding/removing sequences encoding specific interaction domains or motifs. For example, two forms of PSD-95, a palmitoylated one (see earlier) and one with an N-terminal L27 domain which enables additional interactions of PSD-95, result from alternative splicing. Rearrangements of the PSD may not only result from alterations in the amount or modification
of scaffolding proteins, but may also be influenced by their propensity to interact as a result of alterations in the ionic environment. Of interest in this context, it has been shown that the zinc concentration can, at least in vitro, directly influence the assembly of Shank fibers, a core element of the PSD.
The Physiological Role of Postsynaptic Densities What may be the function of this complex protein assembly? Based on its localization and content, the clustering, localization, and regulated trafficking of glutamate receptors, the compartmentalization of different glutamate receptor complexes, the spatial organization of signaling cascades, the dynamic regulation of the cytoskeleton, and the stabilization of the synaptic structure may be some of the roles the PSD plays. The ordered array of proteins into microdomains of the PSD governs the efficiency of the signal transduction pathways initiated by activation of the different kinds of glutamate receptors. Importantly, the PSD clusters the glutamate receptors at sites directly adjacent to the presynaptic release sites, which is crucial for fast chemical neurotransmission. Its connection to the receptors and cell adhesion molecules and to the cytoskeleton of the dendritic spine allows the PSD to coordinately respond to changes of presynaptic activity with alterations of the structure of the PSD and the morphology of the spine. Numerous links between the amount of PSD scaffolding proteins and the spine density and size as well as the glutamate receptor content of synapses have been experimentally established. An impaired architecture of the PSD would be expected to affect the balance of excitatory and inhibitory neurotransmission in the central nervous system and, therefore, to cause fatal diseases. However, relatively little is known about the connection between neurological disorders and the dysfunction of the PSD; maybe the high molecular redundancy of scaffolding proteins secures that central synapses will function even if one of the PSD-95 family member, GKAP, or Shank genes is mutated. Nevertheless, the Shank 3 gene is linked to a neurological disorder termed 22q13.3 deletion syndrome, characterized by mental retardation, delayed speech, and dysmorphic features. Mutations and chromosomal rearrangements in loci corresponding to PSD-95 and neuroligin genes are associated with autism. Mental retardation is linked to the genes for several additional PSD components and correlates with an abnormal morphology of dendritic spines and synapses. Therefore, studying the PSD may not only help to understand
102 Postsynaptic Density/Architecture at Excitatory Synapses
mechanisms of synaptic transmission, but may also provide insight into diseases of the central nervous system. See also: AMPA Receptor Cell Biology/Trafficking; LongTerm Potentiation (LTP): NMDA Receptor Role; Metabotropic Glutamate Receptors (mGluRs): Molecular Biology, Pharmacology and Cell Biology; NMDA Receptors, Cell Biology and Trafficking; Postsynaptic Development: Neuronal Molecular Scaffolds.
Further Reading Boeckers TM (2006) The postsynaptic density. Cell and Tissue Research 326: 409–422. Carlin RK, Grab DJ, Cohen RS, et al. (1980) Isolation and characterization of postsynaptic densities from various brain regions: Enrichment of different types of postsynaptic densities. Journal of Cell Biology 86: 831–845. Chen X, Vinade L, Leapman RD, et al. (2005) Mass of the postsynaptic density and enumeration of three key molecules. Proceedings of the National Academy of Sciences of the United States of America 102: 11551–11556. Cheng D, Hoogenraad CC, Rush J, et al. (2006) Relative and absolute quantification of postsynaptic density proteome isolated from rat forebrain and cerebellum. Molecular & Cellular Proteomics 5: 1158–1170. Collins MO, Husi H, Yu L, et al. (2006) Molecular characterization and comparison of the components and multiprotein complexes in the postsynaptic proteome. Journal of Neurochemistry 97 (supplement 1): 16–23. Funke L, Dakoji S, and Bredt DS (2005) Membrane-associated guanylate kinases regulate adhesion and plasticity at cell junctions. Annual Review of Biochemistry 74: 219–245. Gray EG (1959) Axo-somatic and axo-dendritic synapses of the cerebral cortex: An electron microscope study. Journal of Anatomy 93: 420–433. Gundelfinger ED, Boeckers TM, Baron MK, et al. (2006) A role for zinc in postsynaptic density assambly and plasticity? Trends in Biochemical Sciences 31: 366–373.
Harris KM, Jensen FE, and Tsao B (1992) Three-dimensional structure of dendritic spines and synapses in rat hippocampus (CA1) at postnatal day 15 and adult ages: Implications for the maturation of synaptic physiology and long-term potentiation. Journal of Neuroscience 12: 2685–2705. Husi H, Ward MA, Choudhary JS, et al. (2000) Proteomic analysis of NMDA receptor–adhesion protein signaling complexes. Nature Neuroscience 3: 661–669. Kennedy MB (2000) Signal-processing machines at the postsynaptic density. Science 290: 750–754. Kennedy MB, Beale HC, Carlisle HJ, et al. (2005) Integration of biochemical signalling in spines. Nature Reviews Neuroscience 6: 423–434. Kim E and Sheng M (2004) PDZ domain proteins of synapses. Nature Reviews Neuroscience 5: 771–781. Kornau HC, Seeburg PH, and Kennedy MB (1997) Interaction of ion channels and receptors with PDZ domain proteins. Current Opinion in Neurobiology 7: 368–373. Nicoll RA, Tomita S, and Bredt DS (2006) Auxiliary subunits assist AMPA-type glutamate receptors. Science 311: 1253–1256. Palay SL (1958) The morphology of synapses in the central nervous system. Experimental Cell Research 14: 275–293. Peng J, Kim MJ, Cheng D, et al. (2004) Semiquantitative proteomic analysis of rat forebrain postsynaptic density fractions by mass spectrometry. Journal of Biological Chemistry 279: 21003–21011. Scannevin RH and Huganir RL (2000) Postsynaptic organization and regulation of excitatory synapses. Nature Reviews Neuroscience 1: 133–141. Scheiffele P (2003) Cell–cell signaling during synapse formation in the CNS. Annual Review of Neuroscience 26: 485–508. Spacek J and Harris KM (1997) Three-dimensional organization of smooth endoplasmic reticulum in hippocampal CA1 dendrites and dendritic spines of the immature and mature rat. Journal of Neuroscience 17: 190–203. Sugiyama Y, Kawabata I, Sobue K, et al. (2005) Determination of absolute protein numbers in single synapses by a GFP-based calibration technique. Nature Methods 2: 677–684. Valtschanoff JG and Weinberg RJ (2001) Laminar organization of the NMDA receptor complex within the postsynaptic density. Journal of Neuroscience 21: 1211–1217. Ziff EB (1997) Enlightening the postsynaptic density. Neuron 19: 1163–1174.
Synaptic Transmission: Models S Raghavachari, Duke University Medical Center, Durham, NC, USA J Lisman, Brandeis University, Waltham, MA, USA ã 2009 Elsevier Ltd. All rights reserved.
Introduction Chemical signaling at a synapse occurs when a synaptic vesicle fuses with the presynaptic membrane in response to calcium influx through voltage-gated calcium channels. Vesicle fusion results in the formation of a fusion pore through which neurotransmitter packaged in the vesicle can escape into the synaptic cleft. The released neurotransmitter then diffuses through the synaptic cleft and binds to ligand-gated receptors on the postsynaptic membrane, which then lead to current flow across the postsynaptic membrane. To understand synaptic transmission, it is necessary to determine the spatiotemporal pattern of channel opening. However, physical limitations preclude direct observation of the dynamics of neurotransmitter release and receptor activation at single synapses. With the vast amount of biophysical and structural data that are now available, it is possible to make constrained models with sufficient biological realism that can be used to answer the following questions about the generation of synaptic responses: 1. How does the structure of the synapse shape the response? 2. How does the biophysics of receptor activation contribute to the response? 3. What is the influence of the kinetics of release of neurotransmitter and its reuptake or degradation? We first discuss modeling methods that can be used for the study of synapses and then discuss the application of these methods to the study of excitatory transmission at the frog neuromuscular junction and at central glutamatergic synapses.
Modeling Approaches The physical processes involved in chemical synaptic transmission are neurotransmitter diffusion and binding to receptors followed by conformational transitions of receptors, neurotransmitter binding to transporters for re-uptake (at central synapses), and neurotransmitter hydrolysis (in case of the neuromuscular junction).
The average time evolution of the neurotransmitter concentration in the cleft subject to these processes can be succinctly written as @Cðr; tÞ @Cðr; tÞ @Cðr; tÞ þ ¼ @t diffusion @t reaction @t @Cðr; tÞ þ ½1 @t uptake where C(r, t) is the concentration of the neurotransmitter at the spatial location r and time t. The diffusion of neurotransmitter is given by @Cðr; tÞ ¼ D2 Cðr; tÞ ½2 @t diffusion
2
where r is the diffusion operator, and D is the diffusion constant (measured in mm2 s1). These processes can be simulated on computers using two fundamentally different mathematical approaches. In the first, the molecular nature of the neurotransmitter and receptors is ignored. The neurotransmitter concentration and the probability of receptor activation are treated as continuous quantities that vary smoothly in space and time. The time evolution of these quantities is then modeled using analytic approximations or partial differential equations. A second approach respects the discrete, probabilistic nature of neurotransmitter diffusion and receptor interaction. The ligand and receptor molecules are represented individually and undergo random motions (corresponding to Brownian diffusion) and stochastic interactions and transitions (corresponding to the reactions). Continuum Methods
The main advantage of these continuum methods is their rapid implementation and the precise prediction of average quantities such as the time course of neurotransmitter clearance or receptor activation. Whereas early work using continuum models used simplified geometries, newer methods have allowed the incorporation of complex geometries, such as that of the neuromuscular junction. In continuum models, the equations for neurotransmitter diffusion, uptake, and binding are supplemented by equations for the concentrations of receptors in different states. This system of differential equations is subject to the appropriate initial conditions, specifying the concentration of the neurotransmitter and the receptor states at time t ¼ 0 at different spatial locations. The information on the synapse geometry is contained in the boundary conditions that govern the behavior of the neurotransmitter at reaction
103
104 Synaptic Transmission: Models
boundaries as well as the inert surfaces. The latter represent physical barriers that the diffusing neurotransmitter cannot cross. These are mathematically known as zero flux boundaries, where the spatial gradient of the concentration @C/@x ¼ 0. Uptake mechanisms or receptors located on the boundaries of the reaction space are instead modeled as absorbing boundaries where the concentration vanishes-that is, C(r, t) ¼ f(C)jsurface. In the absence of reaction and degradation/uptake terms in eqn [1], closed-form solutions of the diffusion equation are only possible for idealized geometries, which have a high degree of symmetry. Some example geometries include a cube, a cylinder, and a sphere. For example, a thin circular disk, with one face representing the presynaptic terminal and the other the postsynaptic membrane, is a good approximation of small central synapses. The solution of the diffusion equation in cylindrical coordinates is given in the form of Bessel functions, which can then, in principle, be used to study the dynamics of receptor opening. However, simple analytic models do not capture the complexity of the diffusion and reaction processes, and one must resort to numerical techniques for the solution of the full equation, including reaction terms and neurotransmitter uptake/degradation. A number of techniques have been developed for the solution of these equations under various approximations. Those relevant for synaptic transmission are discussed next. Finite difference methods Finite difference (FD) methods are a powerful set of techniques to solve nonlinear reaction-diffusion equations (e.g., eqn [1]). These methods represent the concentration C(x) by its values at discrete points in the reaction volume and approximate the derivatives (in space and time) as differences between adjacent points. This discretization results in a set of algebraic equations for the concentration of the diffusing neurotransmitter that can be solved using standard methods of linear algebra which have been implemented in software packages such as MATLAB. This approach is particularly useful for studying the synaptic current due to receptors with complex multistate kinetics as well as the effects of antagonists and modulators of receptors. Due to the long history of FD methods and their wide application, many optimization techniques are available for the simultaneous solution of the neurotransmitter diffusion, uptake, and receptor binding and even complex receptor kinetics. However, these methods are best suited for simplified geometries, but even here, the amount of discretization places severe restrictions on the accuracy of the method as well as its implementation on readily available personal computers.
Finite element methods Finite element (FE) methods are widely used in engineering applications to study how material structures behave under applied loads. These methods have been extended to a wide variety of partial-differential equations, including nonlinear reaction-diffusion equations (e.g., eqn [1]). In a strict mathematical sense, FE methods find a piecewise approximation to the solution of eqn [1]. This is done by subdividing the arbitrarily complex reaction space into small (finite) elements and decomposing the solution as a linear weighted sum of functions that are defined only over the finite elements. This decomposition then results in a nonlinear matrix equation that must be solved in order to determine the coefficients. In three-dimensional space, these elements are usually trapezoidal (other more complex shapes are possible). The accuracy of the method, as for the FD method, depends on the discretization (with finer subdivisions yielding better accuracy.) Similar to FD methods, the discretization can be adaptive, with high spatial resolution where the concentration of neurotransmitter varies rapidly. The diffusion of acetylcholine (ACh) in complex geometries can be implemented using publicly available FE packages to gain insight into the structurefunction relationship at neuromuscular junctions (NMJs). Similarly, the effect of vesicular release and acetylcholinesterase (AChE) distribution on miniature excitatory postsynaptic current (mEPSC) kinetics has also been studied using such models. The key advantage of the FE method is that the computational cost is proportional to the number of finite elements. Thus, a full endplate current (representing the release of approximately 200–300 quanta in a geometrically complex NMJ) can be simulated. However, the implementation of the reaction terms in FE models is nontrivial. The esterase and the receptors must be represented as spatially delimited sinks resulting in the boundary condition ^ðxÞDCðx; tÞ ¼ kCðx; tÞ n
½3
^(x) is the surface normal, and k is a constant where n that is related to the steady-state constant of ACh to the esterase or receptor. The kinetics of the ACh receptor are relatively simple (the only isomerization step involves the opening of the channel), which makes its implementation possible within the FE formalism. More complex reaction schemes (e.g., glutamate receptors) are much more difficult to represent using this method. This method has been used in a few cases to study the effect of differences in fold geometry on the kinetics of the mEPSC. Ultrastructural observations show that fast-twitch and slowtwitch muscles have different structures. Muscular
Synaptic Transmission: Models
dystrophy is also known to distort NMJ structure, leading to differences in mEPSC kinetics.
Continuum methods such as FD and FE outlined previously predict the average properties of chemical reactions. Such models are accurate (within the limits imposed by stability and accuracy considerations) as long as the number of reactions modeled is large. However, the chemical reactions that underlie neurotransmission at a single central synapse present a challenge to such models. Experiments show that the synaptic current at small central synapses is due to the opening of tens of receptors. This implies that each vesicle only contains a few thousand molecules of neurotransmitter, of which several tens participate in receptor activation. Finally, the amplitude of the individual synaptic response is often variable from trial to trial. These considerations imply that the stochastic or ‘noisy’ nature of chemical reactions must be accounted for in modeling synaptic transmission. The main source of this noise is the ubiquitous ‘thermal noise’ that characterizes the diffusive (Brownian) motion of molecules and conformational transitions of receptor proteins. The Monte Carlo method assumes that the physical processes in synaptic transmission are inherently probabilistic. The dynamics of the individual reactions are then followed by rolling a dice at each time step (hence the name Monte Carlo) to decide how much a given ligand molecule will move and whether a particular reaction will occur or not. Thus, variability is an inherent part of the simulation procedure. The average behavior of the system is obtained by averaging the results of the simulations across multiple trial runs. One strength of the Monte Carlo method is the high level of spatial and temporal detail that can be achieved. The heart of the Monte Carlo method is its approach to modeling the diffusion of neurotransmitter in complex reaction spaces. In general, a diffusing molecule at a point P at time t ¼ 0 has some thermal velocity and undergoes collisions with water molecules on the sub-picosecond timescale at room temperature. After many collisions (over a nanosecond timescale), the molecule may have moved to a new location in a random direction. The distribution of displacements from the original location can be calculated from the diffusion equation (eqn [1]). For diffusion in free space, the solution of this equation is given by Cðr; tÞ ¼
Thus, the fraction of molecules in a spherical shell (of volume 4pr2dr) is
C0 ð4DtÞ
er
2
3=2
=4Dt
½4
1
=4Dt ð4r2 drÞ ½5 ð4DtÞ which is the same as the probability of a radial displacement of a single molecule. Thus, diffusion of a single neurotransmitter molecule is simulated by choosing a random number with the previous probability distribution. A second random number specifies the direction of this displacement in three dimensions. When the diffusing molecule encounters an impenetrable barrier, it is reflected back into the reaction volume by an elastic collision. In this way, the zero flux boundary conditions specified previously are naturally handled. Note that collisions need not occur with nonreactive boundaries but also with reactive boundaries (representing a surface receptor). In this case, the reflection occurs with a probability that is related to the macroscopic on-rate constant of the ligand with its receptor. This probability is given by 1 pt 1=2 ½6 pb ¼ kon 2Nav Atile D
fr ¼
Monte Carlo Approaches
105
er
2
3=2
where kon is the macroscopic on-rate constant for the ligand to bind to the receptor, Nav is Avogadro’s number, and Atile is the area of the surface element occupied by the receptor. Unimolecular reactions, such as conformational changes of receptors, also occur in a probabilistic manner, with the probability of any given transition at a time step given as " ! # X ki t ½7 pu ¼ 1 exp i
where ki is the macroscopic rate constants of the ith possible reaction for a receptor at any given state (e.g., a ligand can unbind from a receptor or the receptor might isomerize to a new state). The choice of the time step is dictated by the fact that the reaction probabilities must be <<1. The power of the method comes from the realization that fixing the time step automatically fixes the spatial resolution of the method. The size of the reactive and nonreactive surfaces, on the other hand, can be specified to a much higher precision (a small multiple of the area of a receptor, 10–20 nm). The Monte Carlo technique is thus a powerful approach to simulating ligand–receptor interactions in arbitrarily complex geometries as well as receptors (and ligands) with complex kinetic properties. However, this power comes at a high computational cost. The advent of modern computers and the powerful program MCell, which includes many optimizations that speed up the calculations necessary to track diffusion and ligand–receptor interactions, makes the use of this approach entirely feasible.
106 Synaptic Transmission: Models
Measurement of synaptic response: Quantal analysis The postsynaptic response to stimulating a single input axon may be quite complex; several synapses may be involved and multiple vesicles may contribute to the response, even at a single synapse. The factors that determine the dispersion of vesicle-release times are complex, and it is desirable to simplify the issue by studying the response generated by a single vesicle. Miniature synaptic currents (mEPSCs) occur spontaneously and are thought to result from the release of a single vesicle. Computational modeling of the synaptic response has thus focused on whether the properties of mEPSCs can be accounted for. These include the rise time, fall time, and variance. The rise time of the mEPSC at the NMJ is approximately 100 ms. It initially appeared that the rise time of the mEPSC at glutamatergic synapses of the hippocampus was considerably longer, but with the development of dendritic recording methods that eliminated distortions due to inadequate voltage clamp, rise times similar to those at the NMJ were found. The decay times are also similar (5–10 ms), but the amplitudes differ enormously: 4 nA in the frog compared to 15pA at hippocampal synapses. Interestingly, the geometries of the two synapses, although having obvious differences, are similar in size. Each presynaptic active zone at the NMJ is organized in a linear array approximately 1 mm long; the largest hippocampal synapses have similar dimensions (the smallest are 0.2 mm in diameter). Information needed for modeling A full consideration of the various numerical values that must be known to model synaptic function is beyond the scope of this article. Here, we list the various constants that must be estimated: 1. The transmitter content of the vesicles is estimated to be approximately 2000–4000 for glutamate and approximately 10 000 for ACh. 2. This transmitter exits the vesicle into the cleft through a fusion pore. There is considerable research on the size of the fusion pore; it may be much smaller during kiss-and-run release than during full fusion. Importantly, several methods are available that can provide at least a rough estimate of the size of the fusion pore. 3. The geometry of the cleft can be determined from electron micrographs. 4. The diffusion constant of transmitter in the cleft has been estimated and shown to be close to the diffusion limit; apparently, occlusion of the cleft by transsynaptic structures is not a major impediment. 5. Transmitter may bind to proteins other than the transmitter channels (e.g., transport proteins). The concentration of these sites is probably sufficiently
low that their influence on the rising edge of the response is negligible. 6. The density of transmitter-activated channels has been measured directly. Interestingly, the density of ACh receptors (the channel at the NMJ) is approximately 104 mm2, which is approximately 10 times higher than that of AMPA channels (the major type of glutamate channels). 7. A kinetic model of the transmitter-activated channel is required. This will specify the on-rate constant for binding, the rate constant for opening and desensitization, and so on. Generally, the development of such models is based on data from systems in which transmitter can be applied rapidly and under controlled conditions.
Modeling Cholinergic Transmission at the Neuromuscular Junction The vertebrate NMJ is one of the best-studied model synapses because of its large size, which makes it easy to obtain electrical recordings. The basic rules of synaptic transmission were worked out in this system, and a large amount of physiological and morphological data are available. The NMJ is composed of three distinct compartments: (1)the presynaptic nerve terminal, which contains a large number of vesicles containing the neurotransmitter ACh-a subgroup of the these vesicles is located directly adjacent to the active zones; (2) an extensively folded postsynaptic membrane; and (3) the intervening 50 nm-wide synaptic cleft. The junctional folds (JFs) have variable depth (0.5–1 mm) and are contiguous with the cleft. The acetylcholine receptor (AChR) is a pentamer (subunit composition) and is distributed on the crests of the JFs with a density of approximately 7000–12 000 mm2, declining by 30–50% at the bottom of the folds. In addition to the receptors, the enzyme AChE, which degrades ACh, is present on the postsynaptic membrane at densities of 2000–3000 mm2 on the primary cleft and the JFs in clusters of 12 (three tetramers). Insights from Modeling
The key challenge in modeling synaptic response due to the release of ACh at the NMJ is to incorporate the intricate geometry of the cleft. The physical properties of the NMJ place strong constraints on the time course and magnitude of the mEPSC. Simulations incorporating the synaptic geometry, receptor properties, and speed of vesicle release led to the concept of a saturated disk-a region on the postsynaptic membrane where nearly all the ACh contained in a single vesicle can be bound to receptors. An important
Synaptic Transmission: Models
implication of this finding is that the synapse has evolved for a near perfect postsynaptic detection of each molecule of ACh release (this is not the case for glutamatergic synapse). The area of this saturated disk, adisk / N/s, where s is the density of AChRs and N is the number of ACh molecules in the vesicle. For typical values of s of approximately 10 000 mm2, the area of the saturated disk is approximately 0.5 mm2. Simulations of the mEPSC in a comblike approximation of the NMJ or a detailed reconstruction demonstrated the existence of this saturated disk. The idea of a saturated disk can be used to estimate the contribution of diffusion, binding, and receptor kinetics to the rise time of the mEPSC. Assuming that the entire content of the vesicle is released instantaneously into the cleft, the time required for this ACh to diffuse over the saturated disk is tdiff /
N D
½8
where D is the diffusion constant of ACh in the cleft, N is the number of ACh molecules, and the numerical factor C arises due to the cleft geometry. For typical values in the NMJ, this corresponds to approximately 50 ms. Similarly, the contribution of binding to the rise time is given by the time taken for a given concentration of ACh in the cleft to bind to the receptor: tbind /
h kþ
½9
where h is the height of the cleft, s is the concentration of AChR in the cleft, and kþ is the binding constant of ACh to the receptor ( 4 107 M s1). This value ( 40 ms) is comparable to the diffusion time tdiff. Since ACh does not leave the cleft over the rise time, binding can keep pace with diffusion and all the ACh can be optimally bound to receptors in a small area, ensuring saturation. The third contribution to the rise time is due to the finite time taken for the receptor to change conformation from the doubly bound state to the conducting state. The relevant time constant for this conformational change is approximately 50 ms. These considerations give a rather straightforward account of the 100 ms rise time of the mEPSC. One assumption in the models is that the concentration of ACh builds up rapidly (nearly instantaneously) in the cleft. Ultrastructural studies on the NMJ have led to a model of neurotransmission in which rapid fusion of a vesicle with the presynaptic membrane releases neurotransmitter into the synaptic cleft. Following fusion, a membrane pore approximately 10–15 nm long forms, connecting the vesicle interior to the cleft. The neurotransmitter then flows down the concentration gradient (200–500 mM in
107
the vesicle and 0 mM in the cleft). The diameter of the pore has not been directly measured. However, AC capacitance measurements have been used to measure the conductance of fusion pores formed by small synaptic vesicles. These typically estimate the pore conductance to be >500 ps. Given this geometry, one can use computational models of diffusion through the fusion pore and within the cleft to determine the time course of ACh within the cleft. One can then determine whether this time course, together with the properties of the ACh channel, can account for the measured 100 ms rise time of the mEPSC. Monte Carlo simulations of the NMJ with realistic geometries have shown that the fast rise time is accounted for by passive diffusion provided that the pore expands in diameter at a rate greater than 25 nm ms1 (i.e., the pore would expand by 2.5 nm over the rise time of the mEPSC), which is well within the working stroke of many molecular motors. The power of the Monte Carlo method is best illustrated when studying the factors governing the variability of the quantal response. The stochastic opening and closing of the channels as well as randomness in the release location lead to a CV of 0.02–0.03, which is much smaller than observed (0.2). What are the additional factors? One factor is the variation in the amount of ACh in a single vesicle. However, experimental measurements suggest that the mEPSC amplitude is uncorrelated with its rise time, whereas the simple considerations discussed previously suggest that large mEPSC (corresponding to larger amounts of ACh in a vesicle) would have a longer rise time. However, simulations using detailed spatial reconstruction of the NMJ have been used to show that variations in the cleft height and branching of the JFs contribute a significant amount of the variability.
Hippocampal Synapses Morphological Constraints
There is generally a single synapse on spines of CA1 pyramidal cells. This synapse has a single presynaptic active zone and a single postsynaptic density. There is a large variation in the size of these synapses. The average nonperforated synapse on mushroom or stubby spines on P15 CA1 pyramidal cells is 0.1 mm2; perforated synapses are larger, whereas synapses on thin spines are considerably smaller (0.05–0.07 mm2 area). Immunogold labeling suggests that glutamate receptors of a-amino-3-hydroxy-5-methylisoxazole4-propionic acid (AMPA) type are fairly uniformly distributed within the synapse, with a density of approximately 1000 mm2. Thus, large mushroom or stubby
108 Synaptic Transmission: Models
spines can contain approximately 100 receptors, whereas thin spines have fewer than 20. On the other hand, N-methyl-D-aspartate type glutamate receptors are found on almost all synapses and their number is weakly correlated with the area of the synapse. The presynaptic bouton typically contains a single active zone whose size is matched to the postsynaptic density. The intervening synaptic cleft is typically 15–20 nm in width. Central synapses lack enzymatic processes analogous to AChE to degrade and thus dilute the neurotransmitter released in the cleft. At central synapses, diffusion is the major mechanism for the clearance of neurotransmitter from the synaptic cleft. Thus, a good estimate of the diffusion constant of glutamate is essential for modeling the efficacy of receptor activation at synapses. Most neurotransmitters are small molecules whose diffusion coefficient in free solution is approximately 1 mm2/ms. However, in the brain, their diffusion is likely to be slower for two reasons: The extracellular space resembles a porous medium (much like a sponge) and diffusion is restricted to small (nanomolar-sized) channels, and the synaptic cleft and the extrasynaptic space are packed with both charged and neutral macromolecules which further impede diffusion by volume exclusion. The hindrance of diffusion due to the porous nature of extracellular space as well as macromolecular crowding can be accounted for by estimating an effective diffusion coefficient, Deff ¼ D/l2, where l > 1 is the tortuosity of the medium, a measure of the hindrance (geometric or otherwise) experienced by the diffusing molecules. Measurements suggest that glutamate diffusion in the cleft is similar to its value in solution (decreased by approximately a factor of 2). In the hippocampus, electron microscopy studies show that most functional synapses seem to have glial processes at the perimeter of the axon-spine interface. At most CA1 synapses, the astrocytic processes only partially surround the synapse, allowing glutamate to freely escape into the extracellular space. The astrocytes make up nearly 10% of the total membrane in CA1 and are lined with glutamate transporters (proteins that move glutamate from the extracellular space into the glial cell) at a density of approximately 10 000 mm2. These transporters also bind glutamate with high affinity (10–20 mM) and serve to either buffer or remove synaptically released glutamate. From a modeling standpoint, it is not correct to incorporate the effect of transporters by slowing the diffusion of glutamate in the extracellular space. This is because the buffering action of the transporters means that glutamate actually binds to them, resulting in a loss of glutamate from the extracellular space. Instead, the effects of transporters should be included
by explicit modeling of the binding and unbinding of glutamate rather than a reduction of its diffusion constant. Kinetic Constraints
AMPA receptors act on a rapid timescale and display weak affinity to glutamate (the dissociation constant, KD 400 mM). In recent years, these channels have been cloned and expressed in heterologous systems in order to study their electrophysiological properties under controlled conditions, leading to a deep understanding of the structure and kinetic properties of AMPA channels. These channels are tetramers composed of several stoichiometric combinations of glutamate receptor subunits (GluR) 1–4. Thus, channels may be homomers composed of identical subunits (GluR14) or heteromers composed of distinct subunits in a strict stoichiometric manner (GluR12– GluR22, GluR22–GluR32, etc.). Depending on the subunit composition, AMPA channels may have distinct kinetic properties that shape channel kinetics and even permeability to different cations (GluR14 are calcium permeable). Each subunit has a binding domain which can bind glutamate independent of whether glutamate is bound to any of the other subunits (i.e., there appears to be no cooperativity in glutamate binding). Structural studies indicate that agonist binding causes a closure of the binding domain and initiates conformational changes that serve to activate the channel. It appears that at least two subunits need to have glutamate bound in order for the channel to conduct ions. Importantly, additional binding greatly speeds channel opening and enhances the single channel conductance (typical values of conductance are 5–8, 7–10, and 12–15 ps, depending on subunit occupancy). However, it is not entirely clear whether these conductance steps occur for channels natively expressed in synapses. Furthermore, it appears that glutamate binding and unbinding can occur from both closed and open states, but binding and unbinding from the open states is slow. A hallmark of glutamate channels is their ability to rapidly desensitize–that is, switch to a nonconducting conformation upon binding glutamate. The exact conformational changes that lead to desensitization are a subject of active research, but it is widely accepted that glutamate binding to the subunit can set in motion conformational changes leading to either activation or desensitization. Additionally, desensitization as well as recovery from desensitization show a concentration dependence that can be best explained by considering that each subunit is capable of desensitization upon binding glutamate (i.e., desensitization is not a cooperative phenomenon). These
Synaptic Transmission: Models
considerations lead to models of AMPA receptor gating that may typically have 40–50 distinct states. Once the state diagram for the receptors is chosen, the transition rates between these states can be determined by fitting the data on channel activation and desensitization produced by the controlled application of glutamate to patches excised from neurons. Many of these rate constants are fixed to be multiples of each other due to the assumption of subunit independence. Further reduction is possible due to the presence of loops in the state diagram (different paths through a loop should be equivalent according to the principle of microscopic reversibility). The model that best fits data from controlled glutamate application to AMPA channels from CA1 pyramidal cells suggests that at low concentrations, desensitization competes effectively with channel opening, whereas at higher concentrations the cooperative process of channel opening makes the opening rate much higher than the desensitization rate. Insights from Modeling
This model of receptor kinetics was used in a geometrically constrained model of a synapse to determine the influence of various parameters on synaptic transmission. Time course and amplitude of the mEPSC At large mushroom spines in CA1, the number of neurotransmitter molecules vastly exceeds the number of receptors, and neurotransmitter diffusion and clearance is rapid. Thus, the loss of neurotransmitter due to receptor binding can be largely neglected. Moreover, the uptake mechanisms at central synapses are located outside the cleft. With these simplifications, eqn [1] can be solved analytically for a cylindrical cleft where glutamate is released at the center of the top face of the disk with absorbing boundary conditions at the cleft edges. Modeling the escape of neurotransmitter from the vesicle through a narrow fusion pore leads to a brief ‘concentration spike’ during which a large amount of neurotransmitter is confined to a small volume of the cleft near the release site. This spike extends approximately 100 nm from the release site and is dissipated within 100 ms (i.e., the concentration declines by 1/e of its peak value). During this spike, the glutamate concentration builds up to nearly 3 mM near the fusion pore. The rapid diffusion of glutamate dilutes this spike. The efficacy of the spike in producing receptor occupancy is made clear by a calculation analogous to the neuromuscular junction. If we take the area of the spine to be the upper limit in the expression for tdiff (0.1 mm2 for larger spines), we obtain a diffusion time of approximately 10 ms, which is much shorter than
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that in the NMJ. Assuming typical values of forward binding rate of 2 107 M s1, tbind is 80 ms. This implies that the rise time at central synapses would almost entirely be determined by the binding of glutamate to the receptors as well as the receptor kinetics. Solving the equations for receptor activation using the concentration of glutamate calculated previously results in a simulated mEPSC with an amplitude of approximately 10–15 pA and a 20–80% rise time of <100 ms, which matches experimental observations. Most of the current (nearly 80%) at the peak of the mEPSC is carried by a small number of channels in a 250-nm hot spot around the release site (25). The multiple glutamate binding sites on the channel and the concentration dependence of desensitization play an important role in determining the size of this hot spot. Near the site of release, the concentration is in the millimolar range, which is required to effectively load the multiple glutamate binding sites on AMPA channels; the channel then opens efficiently because channel opening is greatly favored over desensitization. Outside the hot spot, opening is inefficient because site occupancy is low and the opening rate is therefore slower than the desensitization rate. If desensitization is blocked with cyclothiazide (a drug that inhibits desensitization of AMPA receptors), channels outside the hot spot can open, albeit slowly, and enhance the tail of the mEPSC. Unlike the NMJ, the majority of the glutamate content of a vesicle at hippocampal synapses leaves the cleft without activating receptor channels. Moreover, a single vesicle is only able to activate a fraction of channels at a medium to large synapse. This nonsaturation has been observed experimentally. The implication of this nonsaturation is that hippocampal synapses, despite their low receptor density, can respond in a graded manner, which has consequences for the dynamic range of hippocampal synapses. Origins of quantal variability A critical issue regarding the characteristics of transmission at central synapses is the variability of the mEPSC amplitude. At the NMJ, the distribution of the peak mEPSC is roughly Gaussian, with a CV of approximately 0.2. At CNS neurons (specifically at hippocampal CA1 synapses), early work showed that the mEPSC amplitude distribution exhibited a broad tail. This broad distribution could be due to the variation in the amount of glutamate contained in vesicles. Modeling and experimental studies that have varied the vesicular glutamate content do not show the distinct rise time– amplitude correlation that is observed experimentally. On the other hand, histograms of responses evoked by minimal stimulation (stimulation of a single axon synapsing onto a target cell) often show peaks that
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are narrow, indicative of low quantal variation, whereas the overall distribution is broad and skewed. Simulations using Monte Carlo models show that amplitude histograms with multiple narrow peaks could arise from single synapses releasing multiple vesicles. These simulations show that effective quantal summation can occur at single synapses and that fluctuations in release time and position are not sufficient to strongly smear the peaks. Although summation is nearly linear, there are interactions of the glutamate released by multiple quanta that produce some nonlinearity. These may explain why the spacing between quantal peaks may not be exactly even. Furthermore, the CV of high peaks is not higher than that of small peaks as would be expected if quantal responses were independent, and it may decrease and can even be less than that of small peaks. There is extensive experimental evidence of multivesicular release at hippocampal synapses. These considerations suggest that the broad distribution of mEPSCs could also arise from the spontaneous release of multiple vesicles at single synapses. One implication of these results is that the quantal size can be relatively insensitive to the number of channels at the synapse (except at the smallest synapses, which have the dimension of the hot spot). The main postsynaptic determinant of quantal size is the local density of AMPA channels in the region of the synapse where a vesicle has been released. It is necessary to use the word ‘local’ because of the possibility that density may not be uniform within a synapse, as has been found at GABAergic synapses. The unimportance of total AMPA channel number in determining quantal size is illustrated by mossy fiber inputs to CA3 pyramidal cells. This connection occurs on a single giant spine that contains many synapses. These have a nearly identical AMPA channel density but highly variable size and AMPA receptor content. Consistent with importance of density rather than size, quantal size at these synapses is restricted to a narrow range. Glutamate diffusion in extracellular space Synapses in the CNS are packed very tightly. Ultrastructural studies suggest that a cubic micrometer of cortex contains approximately one or two synapses. Unlike the NMJ, a typical hippocampal synapse contains a small number of receptors (1–200). Given a maximum of four binding sites per receptor, the number of glutamate molecules in small vesicles is far in excess of the number of receptors. If a large number of vesicles are released at different synapses, this excess glutamate will build up, leading to a nonnegligible amount of ambient glutamate available that
can affect synaptic transmission. Since neurotransmitter released at central synapses is not degraded enzymatically, it can escape from the cleft by diffusion and activate receptors at nearby synapses. This heterosynaptic activation would appear as a slowly decaying tail in the AMPA receptor responses. Direct manipulations of glutamate diffusivity by largemolecular-weight polymers dramatically alter the tail of the AMPA response while minimally affecting the peak of the response. The process of glutamate diffusion and receptor activation in a geometrically complex space has been modeled in simplified geometries to show that extrasynaptic spillover plays an important role in excitatory transmission in the neocortex, hippocampus, and the cerebellum.
Further Directions in Modeling of Synaptic Transmission Synaptic transmission involves a large array of molecules that show complex regulation and intricate localization properties. It is becoming increasingly apparent that realistic models of synaptic transmission hold great promise in making quantitative predictions. However, these models are only as good as the input parameters, such as accurately reconstructed diffusion volumes, measurements of receptor densities and kinetics, as well as dynamics of the release process. Many of these technologies are under rapid development. For instance, electron tomography in conjunction with improved sample preparation techniques can generate highly detailed (3–8 nm resolution) three-dimensional structures of synapses. Highly sensitive freeze–fracture replica labeling has been used to determine the density of AMPA receptors in synaptic sites. Finally, new recording techniques allow for the detection of sub-millisecond vesicle fusion events. Numerical algorithms that can integrate this level of structural detail in computer models are also a subject of active research. However, these tend to be scattered across many disciplines (e.g., computational geometry and parallel computing). The main challenge is to combine these disparate approaches into a unified model of synaptic transmission including both presynaptic and postsynaptic aspects to obtain a deep quantitative understanding of synaptic physiology. See also: Active Zone; AMPA Receptors: Molecular Biology and Pharmacology; Cholinergic Neurotransmission in the Autonomic and Somatic Motor Nervous System; Postsynaptic Density/ Architecture at Excitatory Synapses; Synaptic Vesicles.
Synaptic Transmission: Models
Further Reading Barbour B and Hausser M (1997) Intersynaptic diffusion of neurotransmitter. Trends in Neurosciences 20(9): 377–384. Bartol TM, Land BR, Salpeter EE, and Salpeter MM (1991) Monte Carlo simulation of miniature endplate current generation in the vertebrate neuromuscular junction. Biophysical Journal 59(6): 1290–1307. Crank J (1980) Mathematics of Diffusion. Oxford: Clarendon. Faber DS, Young WS, Legendre P, and Korn H (1992) Intrinsic quantal variability due to stochastic properties of receptor– transmitter interactions. Science 258(5087): 1494–1498. Franks KM, Bartol TM, and Sejnowski TJ (2002) A Monte Carlo model reveals independent signaling at central glutamatergic synapses. Biophysical Journal 83(5): 2333–2348. Franks KM, Stevens CF, and Sejnowski TJ (2003) Independent sources of quantal variability at single glutamatergic synapses. Journal of Neuroscience 23(8): 3186–3195. Nielsen TA, DiGregorio DA, and Silver RA (2004) Modulation of glutamate mobility reveals the mechanism underlying slowrising AMPAR EPSCs and the diffusion coefficient in the synaptic cleft. Neuron 42(5): 757–771. Raghavachari S and Lisman JE (2004) Properties of quantal transmission at CA1 synapses. Journal of Neurophysiology 92(4): 2456–2467.
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Smart JL and McCammon JA (1998) Analysis of synaptic transmission in the neuromuscular junction using a continuum finite element model. Biophysical Journal 75(4): 1679–1688. Stiles J and Bartol T (2001) Computational Neuroscience: Realistic Modeling for Experimentalists, pp. 87–127. Boca Raton, FL: CRC Press. Stiles JR, Helden DV, Bartol TM, Salpeter EE, and Salpeter MM (1996) Miniature endplate current rise times less than 100 microseconds from improved dual recordings can be modeled with passive acetylcholine diffusion from a synaptic vesicle. Proceedings of the National Academy of Sciences of the United States of America 93(12): 5747–5752. Wahl LM, Pouzat C, and Stratford KJ (1996) Monte Carlo simulation of fast excitatory synaptic transmission at a hippocampal synapse. Journal of Neurophysiology 75(2): 597–608. Wathey JC, Nass MM, and Lester HA (1979) Numerical reconstruction of the quantal event at nicotinic synapses. Biophysical Journal 27(1): 145–164.
Relevant Website http://mcell.cnl.salk.edu – Salk Institute for Biological Studies; MCell: A Monte Carlo Simulator of Cellular Microphysiology.
Glial Influence on Synaptic Transmission C G Schipke and O Peters, Charite´ University Medicine, Berlin, Germany ã 2009 Elsevier Ltd. All rights reserved.
Introduction Glia cells in the central nervous system are composed of three major cell types – microglia, oligodendrocytes, and astrocytes. Microglia are the innate immune cells of the brain, and oligodendrocytes are the myelin-forming cells; thus, these two cell types, due to their morphology and specialized role in the mammalian brain, do not influence synaptic transmission directly at the synapse. Nevertheless, microglia release cytokines and chemokines and thus influence synaptic transmission in large areas or the whole brain by altering the levels of these neuromodulatory substances. Oligodendrocytes have not been described to be directly involved in the influence on synaptic transmission, although they do play an important role in potassium buffering (along fiber tracts) and therefore contribute to ion homeostasis, another important factor for synaptic transmission. Direct influence on synaptic transmission has only been described for astrocytes that have the appropriate morphological properties and thus comply with the requirements to be directly involved in the functional control of single synapses. During the mid-1990s, the first indications that astrocytes might be involved in the modulation of synaptic transmission came from a number of independent studies of cell cultures, in which neurons and astrocytes were cocultured in a single petri dish. These studies described the general ability of astrocytes to release glutamate in a Ca2þ-dependent manner and to consecutively influence neuronal calcium activity. However, a substantial limitation of studies in co-cultures is the artificial environment and the altered morphology of cells, especially astrocytes in culture. For this reason, this article focuses on the mechanisms of glial influence on synaptic transmission that have been described from experiments using acute tissue preparations of the mammalian central nervous system, with the exception of the section of this article on the gliaderived acetylcholine-binding protein. Apart from the brain, a well-studied model system for neuron– glia and glia–neuron interactions is the mammalian retina with its layered structure. The mammalian retina comprises different types of glial cells and the glial
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influence on synaptic transmission in this preparation has been shown in some pioneering work for both excitatory and inhibitory neuronal synaptic transmission. This work and the emerging concepts are discussed later. As previously mentioned, the astrocyte morphology in the intact brain tissue preparation is an important prerequisite for the ability of astrocytes to directly influence neuronal transmission at the synapse: with their fine appendages, often measuring only a few hundred nanometers in diameter, the astrocytes protrude into the extracellular space surrounding neuronal structures. These fine astrocytic structures seal the synapses from the extracellular space, but they contribute much more than just morphological support. The tight astrocytic coverage of synaptic structures limits the room for diffusion for secreted neurotransmitters, thereby influencing the active concentration of transmitters at the synapse. For the major excitatory neurotransmitter glutamate, astrocytes possess high-affinity transporters, thereby also contributing largely to the termination of the synaptic signaling. Based on the first studies of astrocyte–neuron co-cultures, a completely new concept of synaptic function and plasticity has evolved and is currently well accepted – the concept of the ‘tripartite synapse’ (Figure 1). The traditional view of the functional synapse consisting of the presynaptic terminal releasing the neurotransmitter into the extracellular space and binding to postsynaptic neurotransmitter receptors of the adjacent neuron has been modified by adding a third, functionally important element – the astrocyte. It is now clear that astrocytes can release a number of neurotransmitters and neuromodulatory substances, especially glutamate, ATP, and D-serine, thereby directly influencing neuronal signal transmission at single synapses. Most often, the release of neurotransmitters from astrocytes is accompanied by changes of their intracellular Ca2þ concentration in response to neuronal activity. Thus, the activation of astrocytes by neuronal activity and the regulation and function of calcium excitability in astrocytes are extremely important for any kind of astrocyte–neuron communication. This article focuses on the mechanisms that influence synaptic transmission following initial activation of astrocytes, measured as changes in the intraastrocytic calcium level. It is important to take into consideration that a single hippocampal astrocyte in the CA1 area of the hippocampus reportedly occupies a neuropilar volume
Glial Influence on Synaptic Transmission 113
Astrocyte
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b
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Figure 1 A glutamatergic synapse in the central nervous system. (a) The functional tripartite synapse consists of the presynapse (blue), the postsynapse (yellow), and the astrocytic coverage (orange). The presynapse is loaded with synaptic vesicles containing glutamate. On the postsynapse, different subtypes of glutamate receptors, AMPA- and NMDA-type receptors, are expressed. Also, extrasynaptic glutamate receptors are found on the postsynaptic element. The astrocyte is equipped with highly efficient glutamate transporters. (b) Upon the arrival of an action potential, glutamate is released into the synaptic cleft, binds to postsynaptic glutamate receptors, and excites the postsynapse. The influx of Naþ, Kþ, and Ca2þ ions through glutamate receptors depolarizes the postsynapse. This depolarization is then propagated within the neuron. Glutamate is transported away from the synaptic cleft by the activity of astrocytic glutamate transporters. This transport determines the time course of the neuronal excitation because excitation stops when all glutamate is removed from the synaptic cleft. AMPA, a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid; NMDA, N-methyl-D-aspartate.
of approximately 65 000 mm3, thereby ensheathing thousands of synapses. Values for other brain regions have not been determined, but there is no indication that these values differ substantially. These thousands of astrocyte-ensheathed synapses can be of different kinds in terms of transmitters secreted. Thus, a single astrocyte is in position to locally react to neuronal activity but also to serve as an integrator of synaptic activation. In addition, astrocytes communicate over long distances via the intra-astrocytic propagation of calcium waves. Taken together, astrocytes are in the position to react accordingly and are equipped with the instrumentation to modify synaptic transmission at synapses remote from those where the activity was sensed originally.
Influence of Glia-Derived Cytokines on Synaptic Transmission It is unknown whether glia are involved in the rapid continual maintenance of synaptic strength. Synaptic strength is determined not only by the amount of transmitter released into the synaptic cleft or by the balance of excitation and inhibition but also by the availability of functional neurotransmitter receptors at the postsynapse. An influence on synaptic transmission via controlling the turnover of receptors cannot be exerted directly at the synapse but, rather, via influencing the receptor turnover by interfering with the neuronal function in general. In hippocampal slices, a cytokine produced by glia cells, tumor
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necrosis factor-a (TNF-a), has been shown to be critically involved in the maintenance of synaptic strength by controlling the surface expression of AMPA-type glutamate receptors. Thus, only the continual presence of glia-derived TNF-a secures glutamatergic synaptic transmission at a constant level, thereby also introducing a possible way for glia modulation of synaptic transmission. A different form of influence on synaptic transmission is involved in the modulation by glia-derived interferon-g (IFN-g). The long-term presence of this proinflammatory cytokine influences the receptor clustering of AMPA-type glutamate receptors at the postsynapse, which modulates synaptic efficacy. Thus, by controlling the extracellular level of certain cytokines, glia have a role in the short-term (via TNF-a) and long-term (IFN-g) modulation of synaptic transmission.
Astrocytic Modulation of Synaptic Transmission in the Retina Since the beginning of research on the functional interactions at the tripartite synapse, the retina has been a very valuable preparation to study and describe the different mechanisms in glia–neuronal cross-talk. Numerous mechanisms that were first described in the retina were later demonstrated to also be functional in the brain. Astrocytes first activated by neuronal activity have been shown to be able to signal back and to modulate neuronal activity in this preparation. Three different, independent mechanisms of glial influence on synaptic transmission in this preparation have been demonstrated to be functional: inhibitory modulation of neuronal spiking, excitatory modulation of neuronal spiking, and hyperpolarization of neurons and thus inhibition of spontaneous spiking activity of neurons. The last mechanism has been shown to be dependent on ATP release from glia cells. The first two mechanisms need to be elucidated in detail, but it is clear that glial interaction with neuronal excitatory, as well as inhibitory signal transmission is involved.
Morphological Plasticity of Astrocytes as a Modulator of Synaptic Transmission Although the morphology of astrocytes with regard to coverage at single synapses suggests a role for morphological plasticity in modulating synaptic transmission, there are few experimental paradigms to study this assumption. This is mainly because the size of a single synaptic structure is much smaller than the resolution of a conventional microscope. However, within the female rodent brain a remarkable form of glial morphological plasticity can be observed within a well-defined brain region, the supraoptic
nucleus (SON), a structure within the hypothalamus. The morphological plasticity in this particular brain region is induced by strong secretion of neurohypophysial hormones in lactating animals and includes rapid and reversible changes in the astrocytic coverage of the neurons and their synapses. Consequently, the SON serves as a structure that allows the comparison of synaptic transmission in a specified subgroup of neurons under different conditions of astrocytic neuronal wrapping. As previously mentioned, an important functional role of astrocytes covering synaptic structures is to remove the neurotransmitter glutamate from the synaptic cleft, as well as limit the diffusion of neurotransmitters into the extracellular space. Thereby astrocytes influence glutamatergic neurotransmission. Astrocytes control the intersynaptic cross-talk with their fine processes. Furthermore, they are morphologically positioned to sense communication mediated by extrasynaptic transmission. A number of electrophysiological and histological studies have found that, indeed, astrocytes contribute to synaptic transmission in the SON by controlling the clearance and diffusion of glutamate in the extracellular space. Glutamate which is synaptically released in the SON not only binds to the postsynaptic ionotropic glutamate receptors but also binds to presynaptic modulatory metabotropic glutamate receptors (mGluRs). Activation of mGluRs leads to a reduction of glutamate release from glutamatergic synapse by a negative feedback loop. The activation of presynaptic mGluRs is a tonic action, thereby rendering this mechanism extremely sensitive to changing levels of glutamate. When neuronal signal transmission in virgin and lactating animals is compared, experiments clearly show that in lactating animals more glutamate is available in the extracellular space when astrocytic processes are farther away from the active glutamatergic synapses and thus are less effective in removing glutamate. This phenomenon can be measured as a strong reduction of efficacy at excitatory synapses accompanied by higher glutamate levels, leading to a stronger activation of presynaptic mGluRs, which in turn leads to a reduction of glutamate release. However, it appears that this is not the only influence of glia on synaptic transmission in the SON. In lactating animals, under conditions of reduced glial coverage of neurons and synapses, the intersynaptic cross-talk between glutamatergic and GABAergic synapses is also modulated. Glutamate which is released into the synaptic cleft also binds to presynaptic mGluRs at neighboring GABAergic terminals, leading to a potentiation of transmitter release and to a phenomenon called heterosynaptic depression. Heterosynaptic depression is much more pronounced in lactating animals, in which glutamate can easily diffuse to neighboring synapses and is cleared less
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efficiently, than in virgin animals. Whether this effect is a consequence of less effective glutamate uptake or of facilitated diffusion of glutamate in the extracellular space is not clear. Taken together, these results from SON clearly demonstrate that alterations in the morphological relationship between astrocytes and neurons strongly affect synaptic function.
Release of Neurotransmitters and Neuromodulators from Astrocytes to Modulate Synaptic Efficacy The discovery that astrocytes are capable of releasing neurotransmitters and that this process is a regulated action led to the assumption and, later, experiments clearly demonstrating that astrocytes are capable of modulating synaptic transmission and neuronal excitability via the release of gliotransmitters into active neuronal synapses. These gliotransmitters are not different from neurotransmitters; they are almost identical molecules. The term gliotransmitter is therefore mainly used to emphasize that a given molecule is released from glia cells. Generally, modulation of synaptic transmission via the release of neuroactive substances can be achieved in two ways: 1. Directly via the release of neurotransmitters, thereby either enhancing the stimulus or stimulating modulatory receptors at the active synapse. 2. Via the release of neuromodulatory substances, thereby modulating the function and efficacy of neurotransmitter and neurotransmitter receptors. As mentioned previously, not all gliotransmitters are particular to astrocytes. Astrocytes use two of the most widely distributed transmitters – glutamate and ATP – to exert their influence on synaptic transmission. A third important gliotransmitter is D-serine. This potent modulator of synaptic transmission is exclusively synthesized by and released from astrocytes. It is therefore a special gliotransmitter in that neurons rely on astrocytes for the synthesis and release of this neuromodulatory molecule. D-Serine is a coagonist and potent modulator of N-methyl D-aspartate (NMDA) receptors. NMDA receptors are key players in glutamatergic excitatory synaptic transmission and have been implicated in many physiological processes, including learning and memory, but also in pathophysiological processes. Thus, influencing NMDA receptor function is a very potent way of modulating synaptic transmission with regard to the physiological process implicated in the basis of memory formation and learning. This might explain why a number of studies have demonstrated a direct action of astrocytes on neuronal NMDA receptors, either by activating or by modulating their function.
Release of Glutamate from Astrocytes to Influence Synaptic Transmission
One of the first molecules proposed to be released from astrocytes as a gliotransmitter was glutamate. The regulation and mechanism of its secretion from astrocytes have been well studied. Glutamate is released from vesicles in the same manner as it is released from presynaptic terminals. However, astrocytic release sites usually contain less vesicles and do not have a classical postsynapse as the target counterpart. Nevertheless, glutamate released from astrocytes exerts a number of important functions influencing and synchronizing neuronal activity. After the first results were obtained from in vitro co-culture (astrocyte plus neurons) models, the concept that astrocyte-derived glutamate can modulate synaptic transmission was first substantiated in vivo in the hippocampus. In these experiments, it was demonstrated that GABAergic activation of astrocytes, leading to an increase in the intracellular calcium concentration, can cause an NMDA receptor-dependent increase in miniature inhibitory postsynaptic current frequency detected in pyramidal neurons and a strengthening of certain inhibitory synapses. A number of experiments have shown that glia-derived glutamate acts on extrasynaptic ionotropic glutamate receptors or on presynaptic mGluRs to modulate neuronal transmitter release. A direct activation of neuronal NMDA receptors following glutamate release from astrocytes was first shown in the thalamus, in which spontaneously occurring Ca2þ elevations in astrocytes led to the induction of NMDA receptors-dependent currents in neighboring neurons. This again proves the Ca2þ-dependent glutamate release from astrocytes. Later, this finding was substantiated with comparable findings in the hippocampus, in which the astrocytemediated influence on synaptic transmission was analyzed in more detail. In these studies, a variety of stimuli, each of which led to astrocytic calcium oscillations, all caused NMDA receptor-mediated slow inward currents (SICs) in area CA1 pyramidal neurons. Detailed analysis of the pharmacology and kinetics of these currents and immunoelectron microscopy clearly demonstrates that the glutamate released from astrocytes selectively acts on extrasynaptic NMDA receptors that contain the NR2B subunit. The currents induced in neurons following astrocytic glutamate release are large and can lead to the modulation of neuronal function by influencing intraneuronal signaling pathways, such as modulating the pathways controlled by the cAMP response element binding protein. However, glial glutamate not only may be important for the function of single neurons but also may serve to synchronize neuronal activity as demonstrated in the hippocampus. A single astrocyte
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can evoke simultaneous glutamate-mediated SICs in neighboring neurons. The functional consequences of this concerted excitation of neurons by astrocytes have yet to be determined, but it again proves the integrative role of astrocytes not only at single synapses but also across the multicellular network of the functional brain. Thus, glutamate released from astrocytes onto a number of different neuronal glutamate receptors is a potent way of influencing synaptic transmission either short term via modulating synaptic efficacy, synchronizing neuronal activity, or long term via modulating intraneuronal signal transduction pathways (Figure 2). Release of D-Serine from Astrocytes to Influence Synaptic Transmission D-Serine is emerging as one of the most important gliotransmitters because it strongly influences glutamatergic synaptic transmission and is exclusively released from astrocytes (Figure 3). The discovery and mode of action of D-serine are described elsewhere in this encyclopedia; however, the action of astrocyte-derived D-serine on neuronal NMDA receptors is briefly described here. The NMDA-type glutamate receptors require the binding of cofactors to allow the opening of the ion pore. Glycine is such a molecule, binding to the well-defined glycine binding site of the NMDA receptor as a required cofactor. In the past 15 years, it has become clear that D-serine is also an endogenous ligand at the glycine binding site of the NMDA receptor. D-Serine, the only D-amino acid to exert an important function in the human body, is solely produced in astrocytes. Astrocytic
Astrocyte
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serine racemase converts the proteinogenic L-serine into D-serine. Evidence suggests that in some brain regions D-serine, and not glycine, is the only endogenous cofactor at the glycine binding site of NMDA receptors. Thus, secreted D-serine enables astrocytes to influence synaptic transmission by controlling the availability of D-serine and consequently the opening of NMDA receptors. Indeed, in hippocampal cultures devoid of astrocytes, the addition of D-serine is necessary to enable the induction of NMDA receptormediated synaptic plasticity, and in classical long-term potentiation (LTP) experiments the addition of D-serine can be a way to overcome the impairment of LTP induction in older animals. Interestingly, a role for astrocyte-derived D-serine in controlling metaplasticity has been demonstrated in the SON. In this brain structure, depending on the amount of glial coverage of synapses, the amount of D-serine released onto glutamatergic synapses is controlled. Because D-serine is the only cofactor for NMDA receptors in this brain region, astrocytes are able to determine whether a synapse exhibits LTP or long-term depression (LTD). When the astrocytic processes retract and the level of synaptic D-serine is reduced, LTD is induced, whereas in virgin rats that have a high degree of synaptic coverage, the same stimulus induces LTP. Thus, astrocytes also control forms of metaplasticity, influencing the overall signal integration in whole brain areas. Release of Purines from Astrocytes to Influence Synaptic Transmission
In addition to glutamate, astrocytes also release the neurotransmitter ATP. There are much fewer studies
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Figure 2 Glutamate released from astrocytes influences synaptic transmission. Astrocytes can release glutamate from vesicles in a Ca2+-dependent manner. Following an increase in the cytosolic calcium concentration, glutamate is released and binds to presynaptic metabotropic glutamate receptors (mGluRs) and neuronal extrasynaptic NMDA receptors of the NR2B type. The activation of these receptors leads to modulation of synaptic transmission. Activation of mGluRs modulates the amount of transmitter released, and activation of NR2B receptors leads to activation of second messenger pathways that have long-term consequences in the signal integration in the neuron.
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AMPA Postsynapse
NR2A Receptors
Figure 3 Astrocytes release D-serine. NMDA-type glutamate receptors require the binding of a cofactor to the so-called glycine site. This cofactor allows the receptor to open when glutamate binds. D-Serine is such a cofactor. D-Serine is exclusively synthesized in astrocytes and released into the synaptic cleft in a calcium-dependent manner. In certain brain regions, such as the supraoptic nucleus of the hypothalamus, D-serine is the only cofactor for the glycine site of NMDA receptors. Thus, the release of D-serine from astrocytes determines whether NMDA receptors on the postsynapse can open and transmit a signal when glutamate is released from the postsynapse.
on the release mode, as well as the mode of action of glia-derived ATP than studies on glutamate. Nevertheless, it has become clear, from studies of the retina and also from studies of the hippocampus, that gliaderived ATP very strongly modulates neuronal excitability. Purines in general, released from astrocytes, are emerging as the type of gliotransmitter that exerts its action even on remote synapses, at a distance far from synaptic activity that originally induced astrocytic activity. Purinergic modulation of synaptic transmission is mainly brought about by adenosine, although astrocytes originally release ATP. ATP is then rapidly degraded by different types of ectonucleotidases in the extracellular space to ADP, AMP, and, finally, adenosine. All these substances potentially activate different subtypes of purinergic receptors. ATP might be released in concentrations that are insufficient to activate neuronal P2 receptors but high enough to allow an accumulation of adenosine that will activate neuronal A1 receptors. Direct influence on synaptic transmission has been demonstrated for glia-derived ATP, degraded to adenosine in the hippocampus. It has been known for some time that presynaptically located adenosine A1 receptors cause presynaptic inhibition of transmitter release, a signaling pathway that is implicated in the physiological basis of heterosynaptic depression: active synapses that are potentiated as a consequence of their activation lead to the depression of neighboring synapses, thereby enhancing the contrast between different potentiated and unpotentiated pathways. It is clear that astrocyte-derived ATP, degraded to adenosine
in the extracellular space, mediates this form of neuronal plasticity. A dramatically reduced level of heterosynaptic depression has been measured in the hippocampus of transgenic mice in which astrocytes are impaired in their ability to fuse vesicles at the membrane. Since the addition of ATP or adenosine rescues the effect, it is evident that vesicularly released ATP from astrocytes is the substance allowing the establishment of heterosynaptic depression. Astrocytes are activated by GABA following neuronal activation. GABA acts on astrocytic GABAB receptors and induces a calcium signal. The calcium signal is communicated in between astrocytes via gap junctions. Astrocytes remote from the synapses originally activated then release ATP, which, degraded to adenosine, leads to the presynaptic inhibition of transmitter release. This multicellular network, required for the induction of heterosynaptic depression, includes astrocytes as an important signaling partner; thus, the basis for functional modulation of synapses is not one astrocyte or two neurons but, rather, a network of astrocytes integrated into the neuronal signaling pathways (Figure 4).
Modulation of Synaptic Transmission Mediated by Astrocyte-Derived Acetylcholine-Binding Protein A different way to influence synaptic transmission is to control the availability of active transmitter in the synaptic cleft by the presence of transmitter binding and thus neutralizing molecules. Such a molecule is
118 Glial Influence on Synaptic Transmission
Astrocyte
Pre
Astrocyte
Glutamate transporter Glutamate transporter NR2B NMDA AMPA Postsynapse
NR2A Receptors ATP
Neighboring synapses or at a distance – interconnected via the
Adenosine
astrocyte syncytium A1-receptor
Figure 4 Astrocytes release ATP and influence synaptic transmission at neighboring synapses. Astrocytes release ATP from vesicles and possibly also via other mechanisms. In the extracellular space, ATP is rapidly degraded by ectonucleotidases to ADP, AMP, and, finally, adenosine. Adenosine activates presynaptically located A1 receptors. The activation of these receptors leads to an inhibition of neurotransmitter release from the presynapse. In the hippocampus, this mechanism is the physiological basis of heterosynaptic depression, a phenomenon involved in synaptic plasticity. Following communication through the astrocytic syncytium, synapses distant from the original point of activity can also be modulated.
known for acetylcholine. It is a protein that is secreted and binds acetylcholine. The acetylcholine-binding protein (AChBP) is secreted from glia cells in the central nervous system of the freshwater snail, Lymnaea stagnalis, in which it modulates synaptic transmission. Like the nicotinic acetylcholine receptors, this astrocyte-derived molecule assembles into a heptamer with ligand-binding characteristics typical of a nicotinic receptor. Presynaptic release of acetylcholine induces the secretion of this AChBP from astrocytes, and once in the synaptic cleft, it acts as a molecular trap, binding the transmitter and reducing its availability at the synapse. Evidence for the functionality of this influence on synaptic transmission at mammalian synapses is lacking. Nevertheless, an imbalance of cholinergic signal transmission in a number of diseases, such as Alzheimer’s disease and schizophrenia, is well described. The functionality of the AChBP-mediated pathway of glial influence on synaptic transmission is proposed as
an explanation for the unresolved issue of the profound imbalance of the cholinergic system in these diseases. See also: Adenosine Triphosphate (ATP); D-Serine: From its Synthesis in Glial Cell to its Action on Synaptic Transmission and Plasticity; Glutamate; Neurotransmitter Release from Astrocytes.
Further Reading Auld D and Robitaille R (2003) Glial cells and neurotransmission: An inclusive view of synaptic function. Neuron 40: 389–400. Chadwick DJ and Goode J (eds.) (2006) Purinergic Signalling in Neuron–Glia Interactions, Novartis Foundation Symposia. Chichester, UK: Wiley. Hatton GI and Parpura V (eds.) (2004) Glial–Neuronal Signaling. Amsterdam: Kluwer. Haydon PG and Carmignoto G (2006) Astrocyte control of synaptic transmission and neurovascular coupling. Physiological Reviews 86: 1009–1031.
Glial Influence on Synaptic Transmission 119 Kang J, Jiang L, Goldman SA, et al. (1998) Astrocyte-mediated potentiation of inhibitory synaptic transmission. Nature Neuroscience 1: 683–692. Kettenmann H and Ransom B (eds.) (2004) Neuroglia, 2nd edn. Oxford: Oxford University Press. Newman EA and Zahs KR (1998) Modulation of neuronal activity by glial cells in the retina. Journal of Neuroscience 18: 4022– 4028.
Panatier A, Theodosis DT, Mothet JP, et al. (2006) Glia-derived D-serine controls NMDA receptor activity and synaptic memory. Cell 125: 757–784. Pascual O, Casper KB, Kubera C, et al. (2005) Astrocytic purinergic signaling coordinates synaptic networks. Science 310: 313–316. Volterra A, Magistretti PJ, and Haydon PG (eds.) (2002) The Tripartite Synapse. Oxford: Oxford University Press.
Neurotransmitter Release from Astrocytes A Volterra, University of Lausanne, Lausanne, Switzerland ã 2009 Elsevier Ltd. All rights reserved.
Astrocyte Excitation Leads to Release of Gliotransmitters Astrocytes are ‘excitable’ in the sense that, when activated by internal or external signals, they deliver specific messages to neighboring cells – an activity that has been dubbed ‘gliotransmission.’ However, astrocytes cannot generate action potentials. Their excitation, which is chemically encoded, can be revealed not by electrophysiology, as in neurons, but by assays of [Ca2þ]i transients and oscillations. Two main forms of astrocyte excitation are well documented: one that is generated by chemical signals in neuronal circuits (neuron-dependent excitation) and one that occurs independently of neuronal input (spontaneous excitation). Neuron-dependent excitation of astrocytes has been reported in many brain circuits following nerve fiber stimulation and the release of various transmitters and factors such as glutamate, g-aminobutyric acid (GABA), acetylcholine, noradrenalin, dopamine, adenosine 50 -triphosphate (ATP), nitric oxide, and brain-derived neurotrophic factor (BDNF). The transfer of information from neurons to glia occurs through the spill-over of synaptically released transmitters, and probably also by direct synaptic-like communication in which astrocytes represent atypical postsynaptic cells. The binding of neurotransmitters to specific receptors in astrocytes generates [Ca2þ]i elevations whose properties, including amplitude, frequency, and propagation, are governed by the intrinsic properties of both neuronal inputs and astrocytes. Spontaneous excitation is an unexpected property of astrocytes that has been observed both in acute brain slices and in vivo. It occurs in most astrocytes during development and decreases considerably during the first two postnatal weeks, when synaptic circuit formation occurs. However, spontaneous astrocytic [Ca2þ]i oscillations seem not to fully disappear with adulthood. They are generated by Ca2þ release from internal stores when inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) receptors are activated, with additional influx of extracellular Ca2þ. An important response of astrocytes to their excitation, both by neuronal input and by self-generated stimuli, is the release of gliotransmitters – chemicals that act on adjacent neurons, glial cells, and blood vessels (Figure 1).
120
What Is a Gliotransmitter? Gliotransmission was first revealed in 1994 when increases in [Ca2þ]i in cultured astrocytes were shown to induce glutamate release followed by neuronal activation. However, a precise definition of gliotransmitters is missing. Here, we propose the following criteria for molecules released by astrocytes: (1) synthesis by and/or storage in the astrocytes; (2) regulated release triggered by physiological stimuli; (3) activation of rapid (milliseconds to seconds) responses in neighboring cells; and (4) a role in physiological processes. Over the years, the number of proposed gliotransmitters meeting these criteria has increased, and their properties have, in part, been unraveled (Table 1). In the following we focus on the mechanisms of their release.
Regulated Gliotransmitter Exocytosis Exocytosis – the rapid form of transmitter release that is typical of neurons and neurosecretory cells – consists of the fusion of specific, membrane-bound vesicles with the plasma membrane, followed by the quantal discharge of their content. Whether a process of this type also occurs in astrocytes and accounts, at least in part, for gliotransmission has long been debated. Over the years, three main lines of indirect evidence have accumulated in support of exocytotic release: 1. The identification in astrocytes of crucial molecular components of the exocytotic machinery of secretory cells, in particular of the three proteins forming the core complex for membrane fusion, the soluble NSF-associated protein receptor (SNARE) proteins. In neurons these are (a) the vesicle-associated membrane protein 2 (VAMP2; also called synaptobrevin II), exposed at the vesicle surface and defined as a v-SNARE, (b) the synaptosome-associated protein of 25 kDa (SNAP25), and (c) syntaxin1, with the latter two protruding from the plasmalemma into the cytoplasm and defined as t-(target-)SNAREs. 2. The observation that Ca2þ-dependent transmitter release in astrocytes – notably glutamate release – is sensitive to blockers of neuronal exocytosis. Such blockers include (a) the clostridial toxins, tetanus, and botulinum neurotoxin, which are Zn2þ-dependent endopeptidases that inactivate specifically the SNARE proteins and (b) the macrolide antibiotic Bafilomycin A1, a selective inhibitor of the vesicular proton pump (v-ATPase), which provides the driving
Neurotransmitter Release from Astrocytes 121
Astrocyres
Blood vessels
Eicosanoids Cytokines Glutamate
ATP? Eicosanoids
ATP
Astrocyte
ATP Cytokines Peptides D-serine Adenosine Glutamate
Neurons
ATP Cytokines
Mioroglia
Figure 1 Chemical gliotransmission. Schematic representation of an astrocyte sending chemical signals to various types of cell sitting in its local territory of influence (gliotransmission). As depicted, astrocytes can release a variety of chemical gliotransmitters, some of which act preferentially on a specific cell type. Moreover, astrocytes can release gliotransmitters via several different mechanisms (see text) that probably represent distinct modes of action. Some such modes have been proposed to occur only in pathological conditions, whereas others would underlie physiological regulatory functions of astrocytes, for example, the control of synaptic transmission and of local blood flow.
force for uptake and accumulation of transmitters into synaptic vesicles. 3. The observation that a stimulant of exocytosis from synapses and neurosecretory cells such as PC12, the black widow spider toxin, a-latrotoxin, stimulates glutamate release from the astrocytes. For a long time the preceding indirect evidence was counterbalanced by the lack of direct evidence for a specific vesicular population competent for regulated exocytosis. Recently, however, a clear synapticlike microvesicle (SLMV) compartment, which is equipped for the uptake, storage, and release of glutamate, has been identified in adult hippocampal astrocytes by immuno-electron microscopy, and Ca2þ-dependent exocytotic fusion events have been documented in astrocyte cultures by several independent approaches, including total internal reflection and confocal fluorescence imaging, membrane capacitance, and electron carbon fiber measurements. Morphologically, astrocytic SLMVs in situ appear as clear vesicles resembling in size (30–50 nm) and rounded shape the synaptic vesicles present in glutamatergic nerve terminals. However, astrocytic SLMVs have a much less dense and orderly arrangement. In perisynaptic cell expansions, small, loosely arranged SLMV
groups are often distributed just beneath the plasma membrane, opposite neuronal terminals or dendrites, and sometimes within synaptic distance of neuronal N-methyl-D-aspartate (NMDA) receptors (NMDARs). These astrocytic SLMVs express proteins that govern glutamate exocytosis – that is, the v-SNARE vesicle-associated membrane protein 3 (VAMP3, also known as cellubrevin), an analog of VAMP2/synaptobrevin II, together with vesicular glutamate transporters (VGLUTs). The three known VGLUT isoforms (VGLUT1–3) are all expressed by astrocytes, although not by all astrocytes. This indicates either that there are VGLUT-positive and VGLUT-negative astrocytes or that various VGLUT isoforms are distributed over distinct subpopulations of astrocytes, as has been observed in neurons. VGLUT-bearing SLMVs have also been identified at the electron microscopic level in cultured astrocytes, where they present a more heterogeneous size distribution (between 30 and 80 nm) and express VAMP2/ synaptobrevin II together with VAMP3/cellubrevin. Functionally, astrocytic exocytosis has peculiar properties when compared to exocytosis at neuronal synapses. Although an accurate analysis has not been yet performed, the stimulus-secretion coupling
122 Neurotransmitter Release from Astrocytes Table 1 The repertoire of gliotransmitters Gliotransmitter
Cellular storage site
Regulated release mechanism
Release stimulants and modulators
Site of action (receptors)
Cell targets and effects
Glutamate
SLMV
Ca2+-dependent exocytosis
mGluR AMPAR KainateR NMDAR
Astrocytes, neurons (mostly stimulation)
Cytosol
(activation of channels and/or transporters) Ca2+-dependent exocytosis?
Glutamate, GABA, ATP, PG, TNFa SDF-1a, spontaneous astrocytic excitation ATP, glutamate, dopamine, LPA, thrombin
P2Y; P2X
Astrocytes, microglia, neurons, blood vessel cells? (mostly stimulation)
ATP, glutamate, dopamine, LPA, thrombin
A1, A2
Neurons (mostly inhibition)
Glutamate
NMDAR (glycine site) Eicosanoid receptors
Neurons (stimulation)
ATP
DCG?
Cytosol
Adenosine
Cytosol
D-serine
SLMV?
Eicosanoids (PG; HETE)
Not known to be stored
Cytokines (TNFa)
Cell surface
Proteins and peptides (AchBP; ANP; others?)
DCG
(activation of channels and/or transporters) Ectonuclotidasemediated ATP dephosphorylation (activation of channels and/or transporters?) Ca2+-dependent exocytosis Ca2+-dependent synthesis followed by rapid release Ca2+-dependent, TACE-mediated surface proteolysis Ca2+-dependent exocytosis
Glutamate, ATP, TNFa, SDF-1a, noradrenaline ATP, SDF-1a
Ach (for AchBP)
TNFa receptors
Ach binding; ANP and other peptide receptors
Astrocytes, blood vessel cells (stimulation, vasodilation/contraction) Astrocytes, neurons (stimulation)
Neurons (AchBP: inhibition)
Only the agents responding to the four criteria discussed in the text have been included in this table. Several other agents, including taurine and homocysteic acid, are also considered to be gliotransmitters; however, for them the evidence is less complete. PG, prostaglandin; HETE, 20-hydroxyeicosatetraenoic acid; TNFa, tumor necrosis factor a; AchBP, acetylcholine-binding protein; ANP, atrial natriuretic peptide; SLMV, synaptic-like microvesicle; DCG, dense-core granule; TACE, TNFa-converting enzyme; mGlu-R, metabotropic glutamate receptor; AMPA-R, a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid glutamate receptor; NMDA-R, N-methyl-D-aspartate glutamate receptor; P2X, P2Y, purinergic 2X and 2Y receptors; SDF-1a, stromal-derived factor 1a, a chemokine; LPA, lysophosphatidic acid. Adapted from Volterra and Meldolesi (2005) Astrocytes, from brain glue to communication elements: The revolution continues. Nature Reviews Neuroscience 6: 626–640.
mechanism in astrocytes seems to be significantly slower (tens of milliseconds) and the Ca2þ affinity of the release machinery higher (in the nM range). Gliotransmitter release is triggered not by the action potential-induced opening of specific presynaptic Ca2þ channels, but by the activation of G-proteincoupled receptors (GPCRs), with ensuing Ins(1,4,5) P3-induced Ca2þ release from the stores of the endoplasmic reticulum. Among GPCR-evoked processes in various cell types, exocytosis of SLMVs in astrocytes appears to be the fastest, which suggests that endoplasmic reticulum cisternae are close to the vesicular release sites. Differences between gliotransmitter and neurotransmitter release might also arise from differences in the molecular components of the exocytotic
machinery expressed by neurons and astrocytes, including different isoforms of SNARE proteins. Single-cell polymerase chain reaction after reverse transcription (RT-PCR) studies, performed in situ and in freshly isolated astrocytes, and high-resolution immunocytochemistry have documented the expression of four exocytotic proteins: SNAP23, a synaptic t-SNARE, complexin 2, Munc18a, and synaptotagmin IV. By contrast, SNAP25 and the synaptic vesicle proteins synaptotagmin I, synaptophysin, and synaptic vesicle glycoprotein 2 (SV2) were not found. Functionally, VAMP3 can be a substitute for VAMP2, whose expression in tissue astrocytes remains controversial. SNAP23, instead of SNAP25, could enhance the Ca2þ sensitivity of
Neurotransmitter Release from Astrocytes 123
exocytosis. The role of synaptotagmin IV instead of synaptotagmin I, the Ca2þ sensor for exocytosis of synaptic vesicles in nerve terminals, cannot be easily deciphered. The contribution of synaptotagmin IV to the Ca2þ-sensing mechanism has been recently questioned, but it has been reported that this protein favors a kiss-and-run mechanism – that is, the incomplete exocytotic fusion associated with fast vesicle recycling. Indeed, electron carbon fiber measurements of dopamine-loaded astrocytes suggest that kiss-and-run rather than full fusion could be the main mechanism of astrocytic vesicle fusion in response to physiological stimuli. Each kiss-and-run event would release only a small fraction of the total vesicle content, permitting multiple rounds of evoked releases without vesicle refilling. Together with SLMVs, larger (100–700 nM), heterogeneous organelles are present in cultured astrocytes, some of which contain secretogranin II, a typical marker of neuronal dense-core secretory granules (DCGs). They also contain ATP. Interestingly, genetic manipulations aimed at preventing formation of the SNARE fusion complex in astrocytes in vivo led to reduced extracellular levels of adenosine, a metabolic product of ATP, consistent with the possibility that ATP is released from astrocytes via exocytosis of DCGs and then is rapidly transformed into adenosine by the action of specific extracellular enzymes, the ectonucleotidases. Lysosomes would be a further source of ATP release. Not only nucleotides but also peptides, such as the atrial natriuretic peptide (ANP), could be released from organelles that are distinct and regulated differentially from glutamatergic SLMVs. By contrast, D-serine, a potent co-agonist of glutamate at the NMDAR glycine-binding sites thought to be produced selectively in astrocytes, was recently reported to co-localize with markers of SLMVs, and could, therefore, be co-released from the vesicle population that is responsible for glutamate release. Functionally, this combination could be highly effective in activating NMDARs. These observations, although potentially important in view of the coordinate or antagonistic functional roles of the different gliotransmitters, should be interpreted at the present stage with caution because most of the observations have been made in cell cultures in vitro that only partially reproduce the specific gliotransmission properties of astrocytes in their native tissue environment. For example, VGLUT heterogeneity disappears in vitro, and all cultured astrocytes express multiple isoforms of the transporter together with various secretion-related proteins that are absent from tissue astrocytes. In addition, the levels of some of these proteins change with time in culture. More studies utilizing preparations in situ will therefore be necessary to confirm and clarify the
heterogeneity and specific properties of the regulated exocytotic pathways in astrocytes.
Modulation of Gliotransmitter Exocytosis Exocytotic gliotransmission is probably not a simple consequence of increases in [Ca2þ]i. Indeed, several distinct stimuli that all induce large [Ca2þ]i responses evoke different transmitter release responses. Important regulatory mechanisms seem to exist, and, in some cases, GPCR-induced glutamate release was found to be strongly attenuated by blocking the synthesis of prostaglandins, tumor necrosis factor-a (TNFa), or both. The role of these mediators is unclear. They could contribute to one or several of the following processes: (1) Ca2þ release from the internal stores triggering vesicle fusion; (2) priming of the secretory process itself; and (3) amplification of the whole cascade by autocrine/paracrine loops. Astrocytes lacking TNFa signaling display alterations of GPCR-evoked [Ca2þ]i elevations, including reduced and slower Ca2þ peaks, suggesting that TNFa contributes to the shaping of GPCR-dependent [Ca2+]i elevations, although it is not strictly indispensable for their generation. It remains to be defined whether the release of prostaglandins and TNFa is part of the stimulussecretion coupling mechanism triggered by GPCR stimulation or whether these agents are released tonically and set the appropriate basal conditions for efficient [Ca2þ]i elevations and glutamate release. Evidence for both modes exists. Interestingly, gliotransmitter release can be modified by concomitant activation of distinct receptors, with ensuing positive or negative cooperativity. For example, activation of a-amino-3-hydroxy-5-methyl4-isoxazole propionic acid (AMPA) receptors, which does not stimulate glutamate release per se, was found to potentiate metabotropic glutamate receptor (mGluR)-evoked release and strengthen mGluRdependent prostaglandin E2 (PGE2) production, possibly through transactivation of epidermal growth factor receptors (ErbB receptors). By contrast, the interaction between mGluRs and a1-noradrenergic receptors is an example of negative cooperativity. The [Ca2þ]i elevations induced by the latter fail to stimulate glutamate release, but instead suppress a coincident mGluR-evoked release.
Nonexocytotic Gliotransmitter Release Molecular transport across the plasma membrane through specialized proteins such as channels and transporters represents a second modality by which hydrophilic agents can be released from cells. Three types of large plasma membrane channel have been
124 Neurotransmitter Release from Astrocytes
claimed to participate in the nonexocytotic release of gliotransmitters from astrocytes, in particular of ATP and glutamate: (1) volume-regulated anion channels (VRACs), (2) gap junction hemichannels, and (3) purinergic P2X7 receptors. 1. The existence of specific VRACs was deduced from the observation that anionic amino acids, such as glutamate, aspartate, and taurine, are released in a nonvesicular, Ca2þ-independent fashion when astrocytes swell, notably in hypotonic media. Recent work raises the possibility that GPCR-evoked [Ca2þ]i elevations may also trigger release of anionic amino acids including glutamate through VRACs. One important caveat concerning this release pathway is the fact that the molecular identity of VRACs is still unknown and the existence of such channels is generally inferred via pharmacological studies. However, drugs used as VRAC antagonists are not specific; for instance, they inhibit gap-junction hemichannels (see next listed item). In addition, these drugs affect anion channels in synaptic vesicles and prevent loading of transmitter into the vesicles. Therefore, identification of VRACs based on pure pharmacological criteria is inadequate. An additional, more solid criterion consists of the direct electrophysiological recording of the ion current generated by the passage of anionic gliotransmitters such as glutamate through the plasma membrane channels. Such currents have been recorded, however, only when high glutamate concentrations were added to the patch pipette. Therefore, it remains to be established whether the channels would drive significant glutamate release at the physiological cytosolic concentrations of the amino acid, about 50-fold lower than those used so far. 2. Gap-junction hemichannels, also known as connexons, are large (1–1.2 kDa), nonselective ion channels formed by hexamers of connexin 43 (CX43) that assemble in astrocytes and might function as autonomous permeation pathways instead of being coupled with a hemichannel of an adjacent cell to form a gap junction. The opening probability of the hemichannels, which is very low in astrocytes bathed in physiological media, increases significantly by reducing the concentrations of divalent cations, notably of Ca2þ, and was reported to be accompanied by the release of both ATP and glutamate. The involvement of hemichannels is consistent with the suppression of the release of these gliotransmitters by gap junction channel blockers (but see the following) and, in the case of ATP, by the absence of release in a glial cell line that lacks CX43, with restoration after transfection of the connexin. 3. The purinergic P2X7 receptor, which has been proposed to be an additional source of glutamate and
ATP release, shares several properties with hemichannels, including an increased open probability at low extracellular Ca2þ concentrations. Moreover, recent work has shown that P2X7 receptors (900 Da) are blocked by the so-called gap-junction channel blockers, indicating that use of such agents cannot discriminate between gliotransmitter release via hemichannels and that via P2X7 receptors. This observation calls for a reconsideration of the interpretation of several previous studies. However, the pharmacology of P2X7 receptors has some additional and distinctive features with respect to those of connexin hemichannels: P2X7 is activated by ATP at high concentrations (EC50 ¼ 300 mM) and by 30 -O-(4-benzoylbenzoyl) ATP (BzATP) at lower concentrations and can be blocked by oxidized ATP but not by suramin. It is not clear whether the high ATP concentrations needed for P2X7 gating can be reached extracellularly in the healthy brain. Alternatively, prolonged activation of P2X7 channels in pathological conditions could lead to the release of cytosolic proteins and cell death. Plasma membrane transporters may represent additional sources of gliotransmitter release. Among them are: (1) the ATP-binding cassettes (otherwise known as multidrug resistance transporters); (2) the high-affinity plasma membrane glutamate carriers, working in reverse mode; (3) the cystine/glutamate exchanger; and (4) the equilibrative nucleoside transporters (ENTs). 1. The ATP-binding cassettes have been proposed to account for the swelling-induced release of ATP; however, the evidence is still preliminary. 2. Reversal of high-affinity plasma membrane glutamate carriers is unlikely to occur during normal brain function, but probably is a main source of excitotoxic extracellular glutamate accumulation under ischemic conditions. Although most of the glutamate carriers are expressed in astrocytes, the neuronal carriers seem to strongly contribute to ischemic glutamate release: glutamate levels are higher in the neuronal than in the astrocytic cytosol, and this condition favors functioning in reverse mode. 3. Another source of glutamate release could be by the cystine/glutamate exchanger, a candidate for the cytosolic accumulation of cystine that is necessary for the synthesis of glutathione, one of the main endogenous antioxidants. Whether the extracellular concentrations of endogenous cystine are high enough to trigger the exchanger activity and whether its glutamate release occurs from astrocytes are unclear. 4. ENTs passively transport adenosine through cell membranes in either direction depending on the intraand extracellular adenosine concentration through facilitated diffusion. Isoforms of these transporters
Neurotransmitter Release from Astrocytes 125
are expressed in astrocytes. Although under physiological conditions adenosine is mainly formed extracellularly, upon release of ATP from the astrocytes followed by ecto-nucleotidase-dependent catabolism, and ENTs mainly act as reuptake system for the nucleoside, under hypoxic conditions adenosine is formed inside the astrocytes, probably via the action of cytosolic nucleotidases, and released via a nonexocytotic process that is inversely controlled by [Ca2þ]i, that is, is suppressed by [Ca2þ]i elevation. ENTs might play a role in such a release, although no direct evidence exists at present. In conclusion, current evidence indicates that some gliotransmission might occur through channels and transporters; however, many aspects of these processes need to be clarified. Most importantly, it should be determined whether these processes occur under physiological conditions and, in this case, how relevant they are, especially because no mechanisms that account for their specificity and regulation have been identified. Alternatively, several of these mechanisms could play roles in a variety of pathological conditions. At present, the results of numerous studies in the field are still inconclusive because of poor pharmacology and/or because the available information about the participating transport proteins is insufficient, preventing accurate molecular analysis of localization and function in situ. See also: Adenosine; Adenosine Triphosphate (ATP);
D-Serine: From its Synthesis in Glial Cell to its Action on Synaptic Transmission and Plasticity; Gap Junctions and Hemichannels in Glia; Glutamate.
Further Reading Bezzi P, Carmignoto G, Pasti L, et al. (1998) Prostaglandins stimulate calcium-dependent glutamate release in astrocytes. Nature 391: 281–285. Bezzi P, Gundersen V, Galbete JL, et al. (2004) Astrocytes contain a vesicular compartment that is competent for regulated exocytosis of glutamate. Nature Neuroscience 7: 613–620. Calegari F, Coco S, Taverna E, et al. (1999) A regulated secretory pathway in cultured hippocampal astrocytes. Journal of Biological Chemistry 274: 22539–22547. Chen X, Wang L, Zhou Y, Zheng LH, and Zhou Z (2005) “Kissand-run” glutamate secretion in cultured and freshly isolated rat
hippocampal astrocytes. Journal of Neuroscience 25: 9236–9243. Coco S, Calegari F, Pravettoni E, et al. (2003) Storage and release of ATP from astrocytes in culture. Journal of Biological Chemistry 278: 1354–1362. Cue´nod M, Do KQ, Grandes P, Morino P, and Streit P (1990) Localization and release of homocysteic acid, an excitatory sulfur-containing amino acid. Journal of Histochemistry and Cytochemistry 38: 1713–1715. Domercq M, Brambilla L, Pilati E, et al. (2006) P2Y1 receptorevoked glutamate exocytosis from astrocytes: Control by tumor necrosis factor alpha and prostaglandins. Journal of Biological Chemistry 281: 30684–30696. Duan S, Anderson CM, Keung EC, et al. (2003) P2X7 receptormediated release of excitatory amino acids from astrocytes. Journal of Neuroscience 23: 1320–1328. Jourdain P, Bergersen LH, Bhaukaurally K, et al. (2007) Glutamate exocytosis from astrocytes controls synaptic strength. Nature Neuroscience 10: 331–339. Kimelberg HK, Goderie SK, Higman S, Pang S, and Waniewski RA (1990) Swelling-induced release of glutamate, aspartate, and taurine from astrocyte cultures. Journal of Neuroscience 10: 1583–1591. Kreft M, Stenovec M, Rupnik M, et al. (2004) Properties of Ca2þ-dependent exocytosis in cultured astrocytes. Glia 46: 437–445. Montana V, Ni Y, Sunjara V, Hua X, and Parpura V (2004) Vesicular glutamate transporter-dependent glutamate release from astrocytes. Journal of Neuroscience 24: 2633–2642. Mothet JP, Pollegioni L, Ouanounou G, et al. (2005) Glutamate receptor activation triggers a calcium-dependent and SNARE protein-dependent release of the gliotransmitter D-serine. Proceedings of the National Academy of Sciences of the United States of America. 102: 5606–5611. Parpura V, Basarsky TA, Liu F, et al. (1994) Glutamate-mediated astrocyte-neuron signalling. Nature 369: 744–747. Parpura V, Fang Y, Basarsky T, Jahn R, and Haydon PG (1995) Expression of synaptobrevin II, cellubrevin and syntaxin but not SNAP-25 in cultured astrocytes. FEBS Letters 377: 489–492. Szatkovski M, Barbour B, and Attwell D (1990) Non-vesicular release of glutamate from glial cells by reversed electrogenic glutamate uptake. Nature 348: 443–446. Volterra A and Meldolesi J (2005) Astrocytes, from brain glue to communication elements: The revolution continues. Nature Reviews Neuroscience 6: 626–640. Ye ZC, Wyeth MS, Baltan-Tekkok S, and Ransom BR (2002) Functional hemichannels in astrocytes: A novel mechanism of glutamate release. Journal of Neuroscience 23: 3588–3596. Zhang Q, Pangrsic T, Kreft M, et al. (2004) Fusion-related release of glutamate from astrocytes. Journal of Biological Chemistry 279: 12724–12733. Zhang Z, Chen G, Zhou W, et al. (2007) Regulated ATP release from astrocytes through lysosome exocytosis. Natural cell Biology 9: 945–953.
Retrograde Transsynaptic Influences G W Davis, University of California at San Francisco, San Francisco, CA, USA ã 2009 Elsevier Ltd. All rights reserved.
Introduction More than 50 years ago, work by Victor Hamburger and his contemporaries identified target-derived factors that influence neuronal growth. Since that time it has been demonstrated that target-derived molecules are essential for many aspects of neural development, including dendrite growth, synaptic growth, synaptic stabilization, and the choice of neurotransmitter. In general, these studies have highlighted the action of growth factors, neurotrophins, and morphogen signaling proteins. These signaling proteins and their receptors are potent intercellular signaling molecules utilized repeatedly throughout development. Although studied extensively, new functions for these signaling systems continue to be identified in the context of axon guidance, stem cell maturation in the brain, and neurodegenerative disease. Other retrograde, transsynaptic signaling systems also exist that are essential for the proper development and function of neural circuitry. In many cases, the molecular bases of these retrograde influences are not known. This article covers three fundamental phenomena that seem to require retrograde, transsynaptic signaling. First, retrograde signaling during synapse formation is considered. Retrograde signaling is well established in this context and several molecular players are known, yet the full repertoire of retrograde signaling molecules remains to be defined. This is followed by a discussion of two phenomena that require retrograde signaling of unknown identity. These include the specification of presynaptic release probability during synapse development, and the homeostatic modulation of presynaptic release probability.
Retrograde Signaling during Synapse Formation Synapse formation involves the transformation of a motile growth cone into a stable synapse capable of mediating high-fidelity neurotransmission. This process requires the assembly and precise apposition of pre- and postsynaptic protein assemblies. These protein assemblies are known as the pre- and postsynaptic densities due to their electron-dense appearance when examined using transmission electron microscopy.
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The presynaptic density includes proteins that are necessary for the calcium-dependent release and recycling of synaptic vesicles. The postsynaptic density includes neurotransmitter receptors, signaling molecules, and scaffolding proteins that control the detection of neurotransmitter. Bidirectional communication between the pre- and postsynaptic cells occurs during synapse formation in order to precisely assemble the pre- and postsynaptic element. Insight into the nature of this bidirectional signaling has been achieved at both the neuromuscular junction (NMJ) and at synapses in the central nervous system. Synaptogenesis at the Neuromuscular Junction
At the vertebrate neuromuscular junction, prior to the arrival of the growth cone, cellular specializations exist both pre- and postsynaptically; these set the stage for ensuing synapse formation. Presynaptically, the growth cone is able to release neurotransmitter and other signaling molecules that are required for synapse formation. Postsynaptically, neurotransmitter receptors (acetylcholine receptors; AChRs) are prepatterned into AChR clusters in a loose array that prefigures the site of eventual synapse formation. It remains unknown how many signaling molecules in addition to AChRs are also prepatterned to the site of eventual synapse formation. Upon arrival at a muscle target, the presynaptic growth cone releases agrin, a heparan sulfate proteoglycan, which is deposited into the synaptic basal lamina. Agrin represents an anterograde signal that is necessary for the induction of the postsynaptic density (Figure 1). Agrin signals to the muscle via two highly conserved muscle proteins, muscle-specific kinase (MuSK) and an associated adaptor protein named rapsyn. Genetic knockout experiments demonstrate that each of these proteins is necessary for synapse formation to proceed, identifying agrin– MuSK–rapsyn signaling as an essential anterograde signaling cascade that is required for synaptogenesis. The release of the neurotransmitter acetylcholine (ACh) also participates in the early process of synapse formation. Genetic experiments in mice combining mutations in the ACh synthetic enzyme choline acetyltransferase (ChAT) with mutations in MuSK and agrin support a model in which ACh released onto the muscle actively disperses prepatterned AChRs (mechanism unknown), while agrin stabilizes those receptors that reside directly at the site of synapse formation. The next stage of synapse formation requires a retrograde signal from muscle to nerve that directs the maturation of the presynaptic nerve terminal
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Postsynaptic induction
Agrin Ach
Presynaptic induction Laminin integrin
MuSK rapsyn
Figure 1 Reciprocal signaling during synapse formation at the neuromuscular junction. At left, the motile growth cone releases both agrin and acetylcholine (ACh). ACh disperses prepatterned ACh receptor (AChR) clusters (orange box) that reside at the site of innervation. Agrin, deposited in the basal lamina, counteracts the effects of ACh and stabilizes AChRs by signaling through musclespecific kinase (MuSK) and rapsyn in muscle. This results in AChR clustering and stabilization at the neuromuscular junction (middle diagram). Following the formation of the postsynaptic density, a retrograde signal specifies presynaptic maturation, including the formation of presynaptic dense bodies and clustered presynaptic calcium channels (blue oval) directly opposite the postsynaptic receptors. Molecules implicated in this retrograde signal include laminins and integrins.
(Figure 1). The release of this retrograde signal follows the induction of the postsynaptic specialization. In support of this conclusion, the transformation of a motile growth cone into a fully differentiated presynaptic terminal will not proceed in animals that lack MuSK or rapsyn. Indeed, muscle transplant experiments provide evidence that presynaptic maturation may never occur in the absence of postsynaptic differentiation. Thus, it appears that the agrin-dependent induction of the postsynaptic specialization leads to the production of a retrograde signal that is subsequently required to transform a motile motoneuron growth cone into a stable presynaptic terminal (Figure 1). The retrograde signal that induces the maturation of the presynaptic terminal remains unknown, though several candidates are strongly implicated. Laminins present in the synaptic basal lamina have a wellestablished function in presynaptic maturation. Genetic deletion of laminin-b2 leads to a poorly differentiated presynaptic nerve terminal and impairs the association of the Schwann cell with the presynaptic terminal. Knockout of laminin-a4 leads to misalignment of the presynaptic active zone with postsynaptic specializations, though presynaptic maturation proceeds. Although a role for laminin in presynaptic induction seems clear, the means by which the laminins induce the transformation of a growth cone into a presynaptic nerve terminal are less obvious. One interesting possibility is that laminins interact directly with key presynaptic proteins. Laminin-a4 has been suggested to be a ‘ligand’ for presynaptic calcium channel ‘receptors.’ Presynaptic calcium channels may well constitute an organizing center for the construction of a presynaptic nerve terminal. Other retrograde influences also exist. Recently, integrin-mediated signaling has also been implicated in presynaptic induction. Deletion of integrin-b1 in muscle results in failure of the presynaptic nerve
terminal to fully differentiate, a phenotype resembling the MuSK knockout. Systems amenable to forward genetic analysis of synapse formation hold promise for identification of new genes that participate in the bidirectional signaling necessary for synapse formation. Genes that influence the specificity and speed of synapse formation have been found by forward genetic approaches, several of which are highlighted here. At the Drosophila NMJ, a four-transmembrane-spanning protein (tetraspanin) encoded by a gene termed late bloomer (lbl) is required presynaptically, for the efficient transformation of a motile growth cone into a stable presynaptic nerve terminal. However, as is the case for many synaptogenic proteins (see later), synapse formation ultimately proceeds in the absence of the lbl-encoded protein. In the nematode Caenorhabditis elegans, forward genetic approaches have identified an intercellular signaling system that is necessary for proper synapse formation. These studies demonstrate that two presynaptic scaffolding molecules, SYD-1 and SYD-2, are necessary for the assembly of the presynaptic active zone. In mutants that lack the genes for these proteins, synaptic vesicles and other presynaptic proteins fail to accumulate at the developing active zone. These scaffolding proteins function, genetically, downstream of the intercellular signaling proteins SYG-1 and SYG-2, which are required for the proper placement of motoneuron synapses (synapse specificity). The SYG-1 protein is an immunoglobulin (Ig) domain-containing protein with similarity to vertebrate NEPH1. SYG-2 is present on an epithelial guidepost cell that prefigures synapse placement. SYG-2 binds to SYG-1, which is expressed by the motoneuron. Together, these studies suggest a model in which intercellular signaling mediated by SYG-2 and SYG-1 organizes presynaptic SYD-1 and SYD-2, which, in turn, direct the assembly of the presynaptic active
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zone. Although synapse formation proceeds at inappropriate sites in a syg-1 mutant, this transsynaptic signaling system represents a potent intercellular signaling system capable of directing appropriate synapse formation. Synaptogenesis in the Central Nervous System
In the vertebrate central nervous system, the Wnt family of morphogen signaling molecules are involved in both axon guidance and synapse formation. Data support a model in which Wnt, released from the target cells, causes growth cone stalling and characteristic cytoskeletal rearrangements that are observed during normal synaptogenesis Although synapse formation proceeds in vivo in a Wnt receptor knockout, the process is delayed, suggesting that Wnts can function to promote synapse formation. A similar function has also been attributed to target-derived fibroblast growth factor 22 (FGF-22) during synapse formation in the vertebrate central nervous system. Other proteins have been identified that are sufficient to induce the transformation of a motile growth cone into a presynaptic nerve terminal in vitro. These signaling proteins include neuroligin and the Ig domain-containing protein SynCam. In both cases, expression of these proteins on the surface of a heterologous cell is sufficient to transform a growth cone into a structure that resembles a presynaptic terminal. The presynaptic terminals that form on these heterologous cells share ultrastructural and molecular hallmarks of presynaptic nerve terminals and are capable of vesicular neurotransmitter release. Neuroligin can signal to the presynaptic nerve terminal by binding presynaptic neurexin, which, in turn, can be linked biochemically to an extensive array of presynaptic proteins including presynaptic calcium channels. Thus, a model has emerged that is similar to that proposed in the nematode. Transsynaptic signaling via a receptor–ligand pair induces the formation of pre- and postsynaptic protein scaffolds that serve as a template for the eventual assembly of pre- and postsynaptic densities.
properties of impinging presynaptic nerve terminals. The basis for this phenomenon is the demonstration that a single presynaptic neuron can establish synaptic connections with very different release properties, simultaneously, on different target cells (Figure 2(a)). A common finding is that a presynaptic neuron can simultaneously form low-release probability, facilitating synapses with one target, and high-release probability, depression-prone synapses with a second target (Figure 2). In each example, it is clearly established that differences in release probabilities and short-term dynamics are a consequence of presynaptic differences, rather than reflecting differences in the detection of neurotransmitter by the postsynaptic targets. As a result, it seems likely that the target cell, or a target-associated glial cell, determines the steady-state release properties of impinging presynaptic nerve terminals. If so, this type of retrograde influence may be a general feature of synapse development, even in circumstances wherein presynaptic terminals are formed at only a single target type. The ability of a single presynaptic neuron to form synaptic connections expressing a different release probability at separate targets has profound importance
Target A: Facilitation
a
Target A: Bitufted interneuron Layer 2/3 pyramidal cell
There is now widespread agreement that target-derived retrograde signals participate in the activity-dependent modulation of presynaptic function. Retrograde signals include the endocannabinoids, neurotrophins, and growth factor signaling molecules. Throughout the nervous systems of both vertebrates and invertebrates, evidence has also accumulated demonstrating that target cells specify the steady-state release
Small presynaptic calcium transient Low presynaptic release probability Short-term facilitation Large presynaptic calcium transient High presynaptic release probability Short-term depression
b
Target-Dependent Control of Presynaptic Release Probability
Target B: Depression
Target B: Multipolar interneuron
Figure 2 Retrograde specification of presynaptic release probability. (a) A single neuron is able to make synapses of different types, simultaneously, at two different target neurons. Synaptic terminals at target A have a low probability of release and facilitate during a train of action potentials. Synaptic terminals at target B have a high probability of release and depress during a train of action potentials. (b) In the rat cortex, layer 2/3 pyramidal cells are able to make synaptic connections simultaneously with bitufted and multipolar interneurons. Synapses contacting bitufted interneurons have a small presynaptic calcium transient and lowrelease probability and express facilitation. Synapses with multipolar interneurons have a large presynaptic calcium transient and high-release probability and express short-term depression.
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for neural circuit function and animal behavior. For example, in the leech nervous system, a single sensory neuron makes low-probability, facilitatory synapses with one motoneuron (MN1) and high-probability, depression-prone synapses with a second motoneuron (MN2). In response to noxious stimulation, the sensory neuron first recruits MN2, leading to a contraction of the skin. After a short delay, synaptic facilitation brings MN1 to threshold, leading to a body bend away from the noxious stimulus. In this manner, the sequential emergence of two distinct behaviors is achieved by a single sensory neuron. As another example, in the arthropod, distinct classes of external sensory organs encode information about wind velocity versus acceleration. Acceleration detectors contact one postsynaptic target with low-probability, facilitatory synapses while velocity sensors contact a second target with high-probability, depression-prone synapses. A small number of sensory organs encode both velocity and acceleration and contact both postsynaptic targets, simultaneously making synapses appropriate for each target cell type. Again, it appears that the target cell specifies the release properties of impinging presynaptic nerve terminals, and this is directly relevant to the function of the surrounding neural circuit and animal behavior. Nearly identical phenomena have been observed in the mammalian central nervous system (CNS). For example, layer 2/3 pyramidal neurons in the cortex contact bitufted interneurons with low-probability, facilitating synapses, and simultaneously contact multipolar interneurons with a higher probability, depression-prone synapse. Similar phenomena have been observed in other regions of the mammalian CNS. In each example, target-dependent specification of presynaptic release properties is predicted. Interestingly, in the cortex, target specification of presynaptic release probability changes over the course of development. It is interesting to speculate that stable, steady-state release properties of the presynaptic terminal could be modulated by retrograde signaling systems over time. Ultimately, the retrograde signal that is responsible for specifying presynaptic release probability and associated short-term plasticity is unknown.
Retrograde Signaling and the Homeostatic Modulation of Presynaptic Function Homeostasis refers to the ability of a cell or a system of cells to respond to a perturbation and maintain a constant physiology. Experimental evidence now supports the existence throughout the nervous system of homeostatic signaling systems that control the firing properties of individual cells. Based upon these
data, it is hypothesized that homeostatic signaling systems interface with the mechanisms of neural plasticity in order to maintain stable neural function over time. Experimental support for homeostatic control of neural function has been documented throughout the central and peripheral nervous systems. In each case, (1) homeostatic regulation was observed following an experimental perturbation that altered the steady-state electrical properties of nerve or muscle cells, (2) the cells responded to the experimental perturbation by altering ion channel density, neurotransmitter receptor density, or the probability of neurotransmitter release at impinging presynaptic nerve terminals, and (3) the modulation of these parameters restored cellular activity to baseline levels observed prior to the experimental perturbation. This precise restoration of baseline (set-point) cellular activity is a fundamental property of homeostatic signaling. There are several examples wherein homeostatic regulation of nerve or muscle activity is achieved by a retrograde signal that modulates synaptic transmission. For example, central neurons, both in vitro and in vivo, that experience chronic activity blockade undergo a wide range of compensatory, homeostatic changes (Figure 3). Chronic activity blockade leads to altered ion channel density at the cell surface as well as altered surface expression of both excitatory and inhibitory neurotransmitter receptors. In several cases, the impinging presynaptic nerve terminals are also modified in a homeostatic manner. Presynaptic changes include altered neurotransmitter release, altered active zone size, and altered synaptic vesicle pool size. Some of these effects may be a direct consequence of altered postsynaptic neurotransmitter receptor abundance. However, other changes, such as presynaptic release probability, may require active, homeostatic signaling from the postsynaptic cell to the presynaptic element. The molecular basis of such a transsynaptic, homeostatic signaling system is unknown. Interestingly, a glial-derived intercellular signal mediated by tumor necrosis factor-a (TNF-a) is both necessary and sufficient for the homeostatic modulation of glutamate receptor abundance in the CNS. Whether this signal could mediate altered presynaptic release remains to be determined. The neuromuscular junction of organisms ranging from Drosophila to rodents and humans shows clear evidence of a homeostatic signaling system that requires a retrograde signal from muscle to nerve. For example, at the Drosophila NMJ, chronic impairment of postsynaptic neurotransmitter receptor sensitivity leads to a compensatory increase in presynaptic transmitter release that precisely counteracts the change
130 Retrograde Transsynaptic Influences Activity blockade
CNS
Receptor mutation Increase release NMJ
Figure 3 Homeostatic control of neural function. Top: The activity of a neuron is determined by a balance of synaptic excitation (synapses with red vesicles and red receptors), synaptic inhibition (blue vesicles and blue receptors), and the abundance of ion channels that depolarize the cell (red ovals) or hyperpolarize the cell (blue ovals) in the central nervous system (CNS). In response to pharmacological activity blockade (black arrow), central neurons can change the abundance of ion channels to reestablish baseline neural activity. Chronic activity blockade can also cause homeostatic changes in the surface expression of neurotransmitter receptors, including excitatory (a-amino-3-hydroxy-5-methyl4-isoxazole propionic acid)-, inhibitory (g-aminobutyric acid)-, and N-methyl-D-aspartate-type postsynaptic neurotransmitter receptors. Bottom: At the neuromuscular junction (NMJ), impairment of postsynaptic neurotransmitter receptor sensitivity leads to a compensatory increase in presynaptic neurotransmitter release that precisely offsets the change in receptor function, to achieve normal synaptic depolarization of the muscle. Adapted from Marder E and Prinz AA (2002) Modelling stability in neuron and network function: The role of activity in homeostasis. Bioessays 24: 1145–1154; and Davis GW (2006) Homeostatic control of neural activity: From phenomenology to molecular design. Annual Review of Neuroscience 29: 307–323.
in receptor function to achieve normal muscle depolarization (Figure 3). Several variations on this experiment have been performed. For example, impairing postsynaptic membrane excitability directly, through overexpression of an inward rectifying potassium channel (Kir2.1), initiates a similar retrograde, homeostatic increase in presynaptic transmitter release. Conversely, increasing muscle excitation by superinnervating the muscle leads to a compensatory decrease in presynaptic release probability. Nearly identical experimental results have been obtained at the rodent and human NMJ, demonstrating the existence of a highly conserved, homeostatic, retrograde signaling system. Although the nature of the retrograde signaling systems capable of increasing and decreasing presynaptic release remains unknown, a recent series
of experiments has defined several parameters that specify how this retrograde transsynaptic signaling system may work. First, it was demonstrated that a homeostatic increase in presynaptic release could be achieved in 10 min following application of subblocking concentrations of a postsynaptic glutamate receptor antagonist (philanthotoxin). In these experiments, evidence of altered presynaptic release could be observed in as little as 2 min, suggesting that the homeostatic signaling system was able to detect altered receptor function and initiate a change in presynaptic release in a time frame of seconds to minutes, far more rapidly than previously thought. This study also demonstrated that the induction of homeostatic signaling is independent of new protein synthesis and is independent of neural activity. The experimenters suggested that the spontaneous release of neurotransmitter that persists in the absence of activity-dependent neurotransmitter release is sufficient to induce the retrograde, homeostatic modulation of presynaptic release following postsynaptic receptor inhibition. Another interesting feature of retrograde, homeostatic signaling is that presynaptic transmitter release is precisely (quantitatively) tuned by the retrograde signaling system to correctly offset the magnitude of postsynaptic receptor perturbation. For each synapse analyzed, the decrease in postsynaptic receptor sensitivity, estimated by average spontaneous miniature excitatory postsynaptic potential (mEPSP) amplitude, is precisely offset by a compensatory increase in presynaptic vesicle release such that muscle depolarization (EPSP amplitude) is constant. Thus, the retrograde homeostatic signaling system is not only rapid, but is also sensitive enough to detect small changes in postsynaptic receptor sensitivity. Furthermore, the retrograde signal is able to convert the magnitude of the postsynaptic perturbation into a precise change in presynaptic release. Such a precise modulation of presynaptic release is quite remarkable when compared to other forms of synaptic plasticity. The molecular mechanisms of rapid, precise, homeostatic modulation at the Drosophila NMJ have yet to be clearly defined. However, forward genetic screens are beginning to identify some of the molecular players. It was recently shown that mutations that perturb the functionality of a presynaptic CaV2.1 calcium channel homolog abolish the rapid induction and sustained expression of synaptic homeostasis at the Drosophila NMJ. The involvement of a presynaptic calcium channel fits with the rapid, precise modulation of presynaptic release in this system. The requirement of a presynaptic calcium channel for synaptic homeostasis induced by postsynaptic receptor inhibition also supports the existence of a retrograde, transsynaptic signaling system.
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The nature of the retrograde signal at this synapse remains to be identified. Recent experiments have demonstrated that bone morphogenetic protein (BMP) signaling strongly influences the growth and stability of the Drosophila NMJ. These experiments provided evidence that the BMP ligand, named glassbottom-boat, can be released from muscle and can signal to BMP receptors on the presynaptic motoneuron. The presynaptic receptors and the downstream canonical signaling pathway to the motoneuron nucleus are necessary for normal synaptic growth. Recent experiments demonstrate that the BMPs are necessary for the homeostatic modulation of presynaptic release in response to postsynaptic glutamate receptor blockade. However, because the BMPs are also necessary for both synaptic growth and synapse stability, it remains unclear whether the BMPs represent a homeostatic signal, or whether the NMJ is simply crippled following the loss of BMP signaling. Thus, the precise nature of the retrograde signal at the NMJ remains to be clearly defined.
Conclusions Retrograde signaling during synapse formation and modulation is a common process. Retrograde signals are diverse and include direct transsynaptic linkages, growth factors, morphogens, and endocannabinoids. Despite remarkable advances, many phenomena exist that seem to require retrograde signaling systems of unknown identity. Forward genetic and proteomic approaches to neural development may hold the key to identification of these elusive transsynaptic signaling systems. See also: Cell Adhesion Molecules at Synapses; Postsynaptic Development: Neuronal Molecular Scaffolds; Retrograde Neurotrophic Signaling; Synapse Formation: Competition and the Role of Activity; Synaptic Precursors: Filopodia.
Further Reading Davis GW (2006) Homeostatic control of neural activity: From phenomenology to molecular design. Annual Review of Neuroscience 29: 307–323. Goda Y and Davis GW (2003) Mechanisms of synapse assembly and disassembly. Neuron 40: 243–264. Koester HJ and Johnston D (2005) Target cell-dependent normalization of transmitter release at neocortical synapses. Science 308: 863–865. Kummer TT, Misgeld T, and Sanes JR (2006) Assembly of the postsynaptic membrane at the neuromuscular junction: Paradigm lost. Current Opinion in Neurobiology 16: 74–82. Marder E and Prinz AA (2002) Modelling stability in neuron and network function: The role of activity in homeostasis. Bioessays 24: 1145–1154. Marder E and Goaillard JM (2006) Variability, compensation and homeostasis in neuron and network function. Nature Review Neuroscience 7: 563–574. Marder E and Prinz AA (2003) Current compensation in neuronal homeostasis. Neuron 37(1): 2–4. Perez-Otano I and Ehlers MD (2005) Homeostatic plasticity and NMDA receptor trafficking. Trends in Neuroscience 28: 229–238. Pun S, Santos AF, Saxena S, et al. (2006) Selective vulnerability and pruning of phasic motoneuron axons in motoneuron disease alleviated by CNTF. Nature Neuroscience 9: 408–419. Reyes A, Lujan R, Rozov A, et al. (1998) Target-cell-specific facilitation and depression in neocortical circuits. Nature Neuroscience 1: 279–285. Sanes JR and Lichtman JW (2001) Induction, assembly, maturation and maintenance of a postsynaptic apparatus. Nature Reviews Neuroscience 2: 791–780. Scheiffele P (2003) Cell–cell signaling during synapse formation in the CNS. Annual Review of Neuroscience 26: 485–508. Shen K, Fetter RD, and Bargmann CI (2004) Synaptic specificity is generated by the synaptic guidepost protein SYG-2 and its receptor, SYG-1. Cell 116: 869–881. Stellwagen D and Malenka RC (2006) Synaptic scaling mediated by glial TNF-alpha. Nature 440: 1054–1059. Turrigiano GG and Nelson SB (2004) Homeostatic plasticity in the developing nervous system. Nature Reviews Neuroscience 5: 97–107.
Synaptic Plasticity: Short-Term Mechanisms J S Dittman, Weill Cornell Medical College, New York, NY, USA A C Kreitzer, University of California at San Francisco, San Francisco, CA, USA ã 2009 Elsevier Ltd. All rights reserved.
Introduction One of the fascinating features of chemical synapses is their capacity to alter their output based on past history. This temporal plasticity is thought to contribute to the brain’s faculty for computation, learning, and memory. Short-term synaptic plasticity (STSP) occurs over a range of milliseconds to minutes and involves temporary enhancement or depression of synaptic strength. The molecular mechanisms that underlie STSP involve regulation of neurotransmitter release (presynaptic) and changes in sensitivity to neurotransmitter (postsynaptic). This article focuses on the mechanisms and molecules that underlie presynaptic STSP. After an action potential (or series of action potentials) invades a presynaptic terminal and triggers calcium-dependent fusion of one or more synaptic vesicles (SVs), the likelihood of releasing neurotransmitter in response to subsequent action potentials may either increase or decrease. These increases and decreases in likelihood are referred to as short-term enhancement (STE) and short-term depression (STD), respectively. Historically, STE has been divided into several distinct processes, such as paired-pulse facilitation (PPF), augmentation, and posttetanic potentiation (PTP), based on kinetics and pharmacology. These forms of plasticity are in large part driven by the residual intracellular calcium that accumulates in presynaptic terminals following depolarization. Presynaptic STD may simply reflect a transient depletion of available SVs, although there is evidence for a refractory period during which SVs cannot fuse. Recovery from STD is regulated by residual calcium and therefore represents another type of STSP. The molecular processes that account for presynaptic STSP are only just beginning to be revealed as experimental synaptic preparations are becoming more amenable to genetic perturbations and imaging techniques. Hundreds of proteins play a role in coordinating neurotransmitter release, and mutations in some of these proteins can result in changes in both basal synaptic transmission and STSP. It is difficult to ascribe specific contributions of these proteins solely to STSP because any change in synaptic transmission
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can indirectly affect STSP as well. For instance, many perturbations that raise the probability of transmitter release will concomitantly decrease the amount of PPF or PTP simply because there is less room for further increase in release probability. These changes may incorrectly be interpreted as playing a crucial role in STE when in fact they may be entirely independent of facilitation. However, most of the proteins that coordinate SV docking and priming with calciumtriggered fusion have multiple interactions with other proteins and lipids, and removal of any one of these will likely affect numerous processes involved in both basal synaptic transmission and presynaptic STSP. Thus, the selective removal of a particular form of plasticity without altering basal transmission may be the exception to the rule when analyzing synaptic mutations. Despite these limitations, several molecules have been implicated in STSP and regulation of the SV cycle.
Activity-Dependent Regulation of Neurotransmitter Release The centerpiece of presynaptic function is the SV cycle, which is composed of vesicle biogenesis and trafficking to the plasma membrane, docking and priming steps which determine the SVs that are competent for fusion, calcium-dependent fusion of the SV, retrieval of SV membrane and proteins, and, finally, refilling with neurotransmitter and reentry into the SV cycle. A synaptic varicosity typically contains hundreds of SVs, but only a small number are competent to fuse with the plasma membrane at any particular time. This subset of SVs is called the readily releasable pool (RRP), and the size of the RRP may be a key determinant of synaptic strength. Processes that modulate the RRP are therefore wellsuited to account for various forms of presynaptic STSP. Active Zone Proteins
SVs fuse at a specialized site within the synapse known as the active zone (AZ) which is composed of a variety of protein families, including RIMs, Munc13s, ELKS proteins, Liprin-as, Piccolo, and Bassoon. Munc13-1 and -2 are large multidomain cytoplasmic proteins that play an important role in the priming of SVs in part by facilitating the formation of the ternary SNARE complex (synaptobrevin on SVs, and syntaxin and SNAP-25 on the plasma membrane) which brings SVs in close apposition with the plasma membrane. In addition, Munc13s appear to serve other functions in supporting SV fusion. They interact
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with RIMs, syntaxin, spectrin, the ARF-GEF Msec7-1, diacyl glycerol (DAG), and calmodulin. The relative abundance of Munc13-1 versus Munc13-2 may determine whether a synapse expresses STE or STD when challenged with a high-frequency stimulus. Residual calcium bound to calmodulin has been shown to interact with a conserved region near the N-terminus of Munc13s. This interaction appears to increase STE (Munc13-2) and recovery from STD (Munc13-1). In both of these cases, the underlying mechanism of Munc13-dependent enhancement of release is thought to be increased SV priming. The details of this process are not fully understood but may involve interactions with the plasma membrane SNARE protein, syntaxin 1. Syntaxin 1 can exist in a ‘closed’ conformation which cannot participate in forming complexes with the vesicle SNARE, synaptobrevin. Munc13 interactions may convert syntaxin 1 into an ‘open’ or permissive conformation thereby facilitating a complete SNARE complex formation required for maturation of the SV to a release-ready state. There is also evidence that Munc13s facilitate vesicle fusion at an additional step downstream from open syntaxin. Phorbol esters are known to enhance transmitter release in large part through their interaction with C1 domain-containing proteins such as protein kinase C (PKC) and Munc13s. This protein–lipid interaction is thought to be a pharmacological mimetic of DAG–C1 interactions occurring in vivo to recruit C1 domains during signaling. Thus, local DAG production near AZs may recruit and activate Munc13s, which in turn increase SV priming and release probability. NCS-1/Frequenin
Another molecule implicated in facilitation of neurotransmitter release is NCS-1 (also known as frequenin). NCS-1 is a small cytoplasmic protein possessing an EF-hand domain that binds calcium. This protein localizes to presynaptic terminals and enhances transmitter release when overexpressed. In hippocampal cultures, overexpression of NCS-1 converts the form of STSP from depression to facilitation without altering basal release probability, perhaps by mobilizing additional SVs or activating silent release sites. This observation implies that at least in some cases, a form of STSP can be separated from the core process of synaptic transmission. Interactions with phophatidylinositol 4-kinase and extracellular signal-related kinase (ERK1/2) appear to be important for the function of NCS-1. These interactions may connect residual calcium with lipid signaling, IP3-gated intracellular
calcium stores, small GTPases such as Ras, and the MAP kinase pathway, which have been implicated in STSP in Aplysia and mice. There is also evidence that NCS-1 can affect P/Q-type voltage-dependent calcium channels, which in turn provide the calcium that drives SV fusion. Thus, NCS-1 appears to have a central role in coordinating calcium increases with a variety of intracellular signaling pathways that modulate transmitter release. SNARE Regulation
Interactions between vesicular and plasma membrane SNAREs play a critical role in determining both where an SV can fuse and how likely it is to fuse. Furthermore, after fusion, plasma membrane ternary SNARE complexes must be separated and sorted in order to provide free synaptobrevin upon endocytosis. Several molecules have been suggested to inhibit synaptic transmission via inhibition of SNARE complexes and thus provide a means of modulating synaptic transmission. The transmembrane vesicular protein synaptotagmin IV (SytIV) can act to inhibit SV fusion by direct interaction with SNAREs, perhaps by competing with synaptotagmin I (SytI). SytI and SytIV are members of a family of transmembrane proteins containing tandem C2 domains (C2 domains are Ca2þ-dependent membrane-targeting protein modules). SytI is required for normal calcium-triggered SV fusion and believed to be the primary ‘sensor’ of the calcium that triggers synchronous release by acting on the SNARE complex. Presynaptic expression of SytIV can be rapidly induced in particular brain regions of rodents, and increased SytIV decreases basal synaptic transmission and leads to enhanced PPF and PTP. This inhibition is hypothesized to result from SytIV competing with SytI for SNARE binding. Thus, SytIV can modulate synaptic transmission by inhibiting normal SNARE function. Another molecule which is proposed to inhibit SNARE function is the syntaxin-interacting protein tomosyn. Tomosyn is a large cytoplasmic protein that contains a SNARE motif at its C-terminus. By binding to syntaxin and SNAP-25 in place of synaptobrevin, tomosyn prevents assembly of the ternary SNARE complex. Regulation of tomosyn function would therefore be predicted to modulate the priming of SVs. Overexpression of tomosyn in chromaffin cells and pancreatic b cells inhibits fusion of large densecore vesicles. In Caenorhabditis elegans, removal of tomosyn increases the size of the RRP and prolongs the decay time of evoked EPSCs. Protein kinase A (PKA) has been shown to phosphorylate tomosyn,
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decreasing its affinity for syntaxin. These observations suggest that tomosyn is a critical regulator of SNARE complex formation and may be an important target of PKA-mediated enhancement of synaptic transmission. SNAP-25 has been studied as a regulator of facilitation. In mammals, there are two splice variants of SNAP-25 (SNAP-25a and -b), and their expression changes during development such that there is a shift from SNAP-25a to SNAP-25b. Artificial sustainment of high levels of SNAP-25a results in an enhancement of PPF without altering basal transmission. SNAP-25 has also been shown to be a target of PKC, and blockade of this phosphorylation appears to inhibit SNARE assembly. The isoform and phosphorylation state of one of the SNAREs therefore contributes to STSP in addition to its role as a core component of SV fusion. Finally, recycling of SNAREs may also be an important site of SV regulation. SNAP-29 is a small cytoplasmic protein that binds to plasma membrane ternary SNARE complexes and prevents disassembly by competing with a-SNAP. Inhibition of disassembly ultimately may slow the rate of recovery from STD. Indeed, decreased SNAP-29 levels have been found to correlate with increased recovery from STD, whereas increased SNAP-29 slowed recovery from STD, and this slowing could be reversed by overexpression of a-SNAP. Kinase Pathways
Signaling complexes associated with receptor tyrosine kinases (RTKs) have been proposed to regulate synaptic strength and STSP. In particular, the brainderived neurotropic factor receptor TrkB is complexed with an actin-based motor protein myosin VI (Myo6) and a Myo6-interacting protein GIPC1. Loss of either Myo6 or GIPC1 results in decreased basal synaptic transmission and increased PPF, whereas PTP is decreased. The Abl family nonreceptor tyrosine kinases Abl and Arg have also been implicated in STSP. abl/ and arg/ mice show normal basal synaptic transmission and PTP, whereas PPF is selectively reduced. Since RTKs and non-RTKs such as Abl and Arg will be unlikely to phosphorylate their targets on the rapid timescale of facilitation, it is more likely that loss of these kinases alters the function of many synaptic proteins that require constitutive phosphorylation for optimal function. Other kinases, such as CaMKII, PKA, and PKC, may also contribute to STSP, and it will be important to identify the phosphorylation states of the molecules that regulate
synaptic transmission so that the pleiotropic effects of disrupting kinases and phosphatases can be interpreted in terms of their net effects on critical downstream targets.
G-Protein-Mediated Regulation of Neurotransmitter Release Short-term plasticity of neurotransmitter release is not only triggered by activity-dependent changes within the terminal itself but also can be induced by activation of presynaptic G-protein-coupled receptors (GPCRs). GPCRs represent a large family of seven transmembrane domain receptor proteins that, when activated by a ligand, function as guanine nucleotide exchange factors (GEFs) for heterotrimeric G-proteins. This GEF activity leads to the replacement of bound GDP by GTP on Ga subunits, resulting in the dissociation of the a and b/g subunits and their subsequent interaction with effector molecules. G-protein signaling is terminated by the hydrolysis of a-bound GTP to GDP and the reassociation of a and b/g subunits. Although more than 300 different types of GPCRs have been described, they can be generally classified by their interactions with different types of G-proteins. Gi/o-Coupled Receptors
Presynaptic modulation of neurotransmitter release is best characterized for Gi/o-coupled receptors, which include group II and III metabotropic glutamate receptors (mGluRs), GABAB receptors, adenosine A1 receptors, cannabinoid CB1 receptors, opioid receptors, and muscarinic M2 and M4 receptors. These GPCRs are widely expressed and are often found presynaptically adjacent to active zones. They can be activated through the spillover of neurotransmitter from the active zone or through the actions of modulators released from other neurons, including the postsynaptic cell. Although presynaptic activation of Gi/o-coupled receptors almost invariably leads to inhibition of neurotransmitter release, this may be in large part due to their localization near the release machinery, which would permit certain target interactions, primarily via b/g signaling mechanisms. Signaling through a subunits is also likely to play an important role through modulation of cAMP levels and kinase activity. Additional functions of Ga subunits may include determining the specificity of b/g subunit interactions, as well as the termination of G-protein signaling through reassociation with b/g subunits following GTP hydrolysis.
Synaptic Plasticity: Short-Term Mechanisms 135 Gq/11-Coupled Receptors
Gq/11-coupled receptors, such as group I mGluRs and muscarinic M1 and M3 receptors, are also critical for short-term presynaptic modulation, although the bestdescribed example comes from their postsynaptic role in the production of endocannabinoids. In numerous brain regions, including the cortex, striatum, hippocampus, and cerebellum, group I mGluR-mediated activation of Gq/11 signaling leads to the postsynaptic biosynthesis of endocannabinoids, at least in part due to the activation of phospholipase C beta (PLCb) and DAG production. A similar effect of M1/M3 receptor activation has been observed in the striatum and hippocampus, indicating that activation of PLCb represents a common target for enhancing endocannabinoid release through different Gq/11-coupled receptors. After synthesis and release from postsynaptic neurons, endocannabinoids can bind to presynaptic Gi/o-coupled receptors and inhibit neurotransmitter release for tens of seconds. Direct presynaptic actions of Gq/11-coupled receptors have been more difficult to establish, particularly given the need to rule out endocannabinoid signaling in cases of apparent direct presynaptic inhibition. Because no firm evidence exists for presynaptic Gq-coupled receptor modulation, it is difficult to predict the effects of such signaling. Although it is possible that Gq-coupled receptors could have inhibitory actions via b/g subunit signaling, Gq/11-mediated DAG production via PLCb as well as subsequent PKC activation are predicted to have powerful excitatory presynaptic effects, given the actions of phorbol esters and PKC-mediated phosphorylation on proteins involved in vesicle priming and exocytosis. If presynaptic Gq signaling does occur, its precise nature may depend on the localization of the receptors relative to their possible signaling targets. Gs-Coupled Receptors
Presynaptic Gs-coupled receptors are far less characterized in the central nervous system, but evidence suggests that these receptors can enhance neurotransmitter release, presumably through cAMP-dependent mechanisms. Paradoxically, some of the reported presynaptic actions of Gs-coupled receptors, such as dopamine D1 receptors, are actually inhibitory. For example, presynaptic Gs-coupled dopamine D1 receptors in the nucleus accumbens and cortex have been reported to reduce glutamate release. One explanation for these findings is that b/g subunits released following activation of Gs-coupled receptors can inhibit release through mechanisms described later, although this has never been demonstrated in neurons.
Another peculiar finding is that some metabotropic receptors seem to be able to switch their G-protein coupling in response to different conditions. For example, Gi/o-coupled cannabinoid CB1 receptors may switch to Gs signaling when activated with low agonist concentrations or when associated with dopamine D2 receptors. In the cortex, Gq/11-coupled 5-HT2A receptors appear to also signal through Gi/o when activated by class 5-HT2A agonists that have hallucinogenic properties in humans. Thus, metabotropic receptors may be capable of multiple conformational states that can modulate their coupling to G-protein signaling cascades.
Effectors and Targets of Presynaptic G-Protein Signaling Many of the signaling targets of presynaptic modulation are the same proteins involved in activitydependent short-term plasticity described in previous sections. Indeed, some signaling pathways may require both presynaptic activity and G-protein signaling. Thus, Ga signaling can lead to increases or decreases in important second messengers that control the phosphorylation state and function of presynaptic proteins involved in the docking, priming, and exocytosis of vesicles at the active zone. On more rapid timescales, Gb/g. signaling can cause rapid and transient modulation of voltage-gated conductances as well as the protein complexes involved in SV priming and fusion. Presynaptic Ga Signaling
Among the first signal transduction pathways described was the stimulation (by Gs) and the inhibition (by Gi) of adenylyl cyclase (AC) and cAMP. A striking feature of Gs activation and other similar signaling cascades is the capacity for robust signal amplification. In the case of Gs, only one or two GPCRs is required to stimulate GDP turnover on tens of Gs subunits, and each Gs-activated AC can in turn produce thousands of cAMP molecules. Another important aspect of signaling cascades is the divergence of signaling targets. In the case of the cAMP cascade, signaling can proceed in parallel through different mechanisms, including activation of PKA as well as activation of cAMP-GEFs (Epacs), both of which have been shown to mediate enhancement of neurotransmitter release at central nervous system synapses. PKA can phosphorylate numerous proteins, such as synapsin, snapin, a-SNAP, syntaphilin, tomosyn, CSP, SNAP-25, and RIM. All of these proteins are involved in the SV cycle, and their phosphorylation may alter
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docking, SNARE complex formation, and release of SVs. In addition to these actions, Epac activation of monomeric G-proteins such as Rap and Ras can lead to additional signaling through MAP kinases and other signaling pathways, although the precise presynaptic actions of Epac have yet to be determined. A second major signal transduction cascade in the presynaptic terminal is mediated by Gq/11 activation of PLCb. The same principle of amplification applies for PLC-mediated synthesis of DAG and IP3, although membrane availability of the DAG precursor PIP2 can play a limiting role in this reaction, and PIP2 may be under dynamic control. DAG can activate PKC, which, like PKA, has numerous synaptic protein targets. For example, the syntaxin-binding protein Munc18, which binds to and stabilizes the closed form of syntaxin, is phosphorylated by PKC, leading to a decrease in its affinity for syntaxin and an increase in the probability of SNARE formation. PKC also phosphorylates other key synaptic proteins, including calcium channels and SNAP-25. DAG may also be important for presynaptic signaling independent of PKC via its interactions with Munc13, which were discussed previously. Presynaptic Gb/g. Signaling
Given the critical role of calcium channels in the triggering of neurotransmitter release, it is perhaps not surprising that modulation of calcium channels represents a major target for G-protein signaling. However, what initially surprised investigators was that this inhibition was not mediated by Ga subunits but instead by direct binding of G-protein b/g subunits to calcium channels. In contrast to Ga signaling, no amplification occurs with b/g. signaling, suggesting that such signaling is likely to be more local and also have faster kinetics. Interestingly, Ga subunits may play an important role not only in the localization, specification, and timing of Gb/g. signaling but also through downstream kinase signaling. For example, b/g binding to calcium channels can be blocked by phosphorylation of the channel by PKC, suggesting the possibility of metaplastic control by Gq/11 signaling. G-protein b/g subunits can also directly bind and activate a class of potassium channels known as G-protein-activated inward rectifying K-channels (GIRKs). Although in theory, such a mechanism could reduce calcium influx by speeding action potential repolarization, little evidence for a presynaptic role of GIRKs has been found. It has also been recognized that b/g subunits can exert an inhibitory influence on AC activity, particularly calcium- and calmodulin-dependent AC1 and AC8, suggesting that metabotropic receptor signaling can override activitydependent changes in cAMP. Perhaps the most
intriguing target of Gb/g. is SNAP-25, a critical component of the SNARE complex that binds synaptotagmin and mediates vesicle fusion. Gb/g. subunits can directly bind to SNAP-25 and inhibit release, possibly by disrupting the association of SNARE complexes with synaptotagmin 1, providing a direct mechanism for G-protein-mediated modulation of the release machinery independently of calcium influx. Regulation of G-Protein Signaling
Initially, it was thought that G-protein signaling was activated solely by metabotropic receptors and terminated by the intrinsic GTPase activity of Ga subunits, but the discovery of activators of G-protein signaling (AGS) and regulators of G-protein signaling (RGS) proteins suggests that these concepts need revision. AGS proteins represent a family of receptorindependent G-protein activators that function through either GEF activity or by sequestering Ga or Gb/g. subunits. A number of different AGS family members are expressed in neurons, where they could modulate receptor-mediated signaling in unexpected ways, such as uncoupling metabotropic receptors from Gi/o or Gs pathways or enhancing basal inhibition by Gb/g through sequestration of Ga-GDP. However, their specific roles in presynaptic neuromodulation have yet to be characterized. On the other hand, RGS and related family proteins have already been shown to play important roles in neuronal modulation and presynaptic plasticity, both through their GTPase-accelerating protein (GAP) activity and through other domain–domain interactions. For example, RGS6, -7, -9, and -11 have a G-protein g-subunit-like domain that can bind to Gb5 subunits. Gb5 acts with RGS as an obligate heterodimer to drive GAP activity rather than as a classical b subunit in the b/g complex. RGS12 and -14 contain Ras binding domains (RBDs) as well as Gai binding domains. Interestingly, the RGS14 RBD binds Rap, suggesting a possible interaction between Epac and RGS signaling. Functionally, RGS proteins have been shown to modulate both basal synaptic properties and presynaptic modulation. In cultured hippocampal neurons, RGS2 overexpression gives rise to enhanced release probability and paired-pulse depression, whereas RGS2 knockout mice exhibit decreased release probability and enhanced paired-pulse facilitation, without any alterations in vesicle cycling, indicating that RGS2 is likely modulating basal G-protein inhibition of calcium channels. In the striatum, RGS92 has been reported to reduce modulation by dopamine D2 receptors in both cholinergic and medium spiny neurons, and RGS4 reduces the efficacy of muscarinic M4 autoreceptor inhibition from cholinergic interneurons. Thus, the RGS family likely provides a widespread
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mechanism for the regulation of G-protein signaling in the central nervous system.
Conclusion Short-term plasticity can arise from numerous sources, including presynaptic calcium-dependent signaling pathways, changes in the distributions and associations of presynaptic proteins following vesicle fusion, as well as transient cell surface signaling through G-protein-coupled receptors. However, all of these signaling mechanisms appear to converge onto common targets representing the proteins mediating SV trafficking, docking, priming, fusion, and recovery. In order to fully understand the mechanisms of neurotransmitter release and STSP, a comprehensive list of presynaptic proteins involved in neurotransmitter will have to be determined. Furthermore, threedimensional models will have to be developed that incorporate the massive web of intermolecular associations among these proteins, as well as their regulation by calcium and phosphorylation, in both space and time. Although such a description is many years away, some of the most important proteins and their interactions are beginning to be characterized, giving us a glimpse into the vast complexity underlying regulated neurotransmitter release. See also: Active Zone; Endocytosis and Presynaptic Scaffolds; Neuromuscular Junction (NMJ): Presynaptic Short-Term Plasticity of Neuromuscular Transmission; SNAREs; Synaptic Transmission: Models; Synaptic Vesicles.
Further Reading Baba T, Sakisaka T, Mochida S, and Takai Y (2005) PKA-catalyzed phosphorylation of tomosyn and its implication in calcium-
dependent exocytosis of neurotransmitter. Journal of Cell Biology 170: 1113–1125. Blumer JB, Cismowski MJ, Sato M, and Lanier SM (2005) AGS proteins: Receptor-independent activators of G-protein signaling. Trends in Pharmacological Sciences 26: 470–476. Ferguson GD, Wang H, Herschman HR, and Storm DR (2004) Altered hippocampal short-term plasticity and associative memory in synaptotagmin IV (/) mice. Hippocampus 14: 964–974. Gerachshenko T, Blackmer T, Yoon E-J, et al. (2005) Gb/g. acts at the C terminus of SNAP-25 to mediate presynaptic inhibition. Nature Neuroscience 8: 597–605. Han J, Mark MD, Li X, et al. (2006) RGS2 determines short-term synaptic plasticity in hippocampal neurons by regulating Gi/omediated inhibition of presynaptic calcium channels. Neuron 51: 575–586. Junge HJ, Rhee JS, Jahn O, et al. (2004) Calmodulin and Munc13 form a calcium sensor/effector complex that controls short-term plasticity. Cell 118: 389–401. McCudden CR, Hains MD, Kimple RJ, Siderovski DP, and Willard FS (2005) G-protein signaling: Back to the future. Cellular and Molecular Life Sciences 62: 551–577. McEwen JM, Madison JM, Dybbs M, Kaplan JM, et al. (2006) Antagonistic regulation of synaptic vesicle priming by Tomosyn and UNC-13. Neuron 51: 303–315. Rhee JS, Betz A, Pyott S, et al. (2002) Beta phorbol esterand diacylglycerol-induced augmentation of transmitter release is mediated by Munc13s and not by PKCs. Cell 108: 121–133. Schoch S and Gundelfinger ED (2006) Molecular organization of the presynaptic active zone. Cell Tissue Research 326: 379–391. Sippy T, Cruz-Martin A, Jeromin A, and Schweizer FE (2003) Acute changes in short-term plasticity at synapses with elevated levels of neuronal calcium sensor 1. Nature Neuroscience 6: 1031–1038. Sudhof TC (2004) The synaptic vesicle cycle. Annual Review of Neuroscience 27: 509–547. Takamori S, Holt M, Stenius K, et al. (2006) Molecular anatomy of a trafficking organelle. Cell 127: 831–846. Tedford HW and Zamponi GW (2006) Direct G protein modulation of Cav2 calcium channels. Pharmacological Reviews 58: 837–862. Zucker RS and Regehr WG (2002) Short-term synaptic plasticity. Annual Review of Physiology 64: 355–405.
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NEUROMUSCULAR AND GAP JUNCTIONS A. Neuromuscular Junctions
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B. Gap Junctions
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Neuromuscular Connections: Vertebrate Patterns of C R Slater, University of Newcastle upon Tyne, Newcastle upon Tyne, UK
The rest of this article is limited to a consideration the innervation of skeletal muscles in vertebrates.
ã 2009 Elsevier Ltd. All rights reserved.
Diversity of Vertebrate Skeletal Muscles Neural Control of Muscles Movement is a feature of all animals, and a central function of all nervous systems is to control movement in a way that promotes survival. In most animals, movement results from the neural activation of specialized contractile cells, the muscle fibers. This activation occurs at the neuromuscular junction (NMJ), the interface between nerve and muscle. While many features of muscle innervation and neuromuscular transmission are similar in all vertebrates, there are also many important differences. This article describes this diversity and how patterns of neuromuscular connectivity have changed during vertebrate evolution. There are several distinct types of vertebrate muscles. In all of them, contraction arises from the interaction of the motor protein myosin with the cytoskeletal protein actin. However, myosin and actin are organized in different ways in different types of muscles. In striated muscles, they are present in extended arrays of overlapping filaments that give rise to periodic crossbanding, visible in the large cylindrical cells known as muscle fibers. Striated muscles that are attached to skeletal elements, and are used to move body parts (e.g., limbs, digits, eyes) relative to the external environment, are known as skeletal muscles. They are innervated by motor neurons at highly specialized NMJs. The muscles of the internal organs are controlled by the autonomic nervous system. The cardiac muscle that causes the forceful pumping action of the heart is striated but has a very different cellular organization from skeletal muscle. In the smooth muscles of most internal organs, the actin and myosin arrays are less highly organized and no striations are present. Many cardiac and smooth muscles are intrinsically active, producing myogenic cycles of contraction and relaxation. The effect of the autonomic innervation is to alter the frequency and strength of these cycles. This involves both excitatory and inhibitory effects, mediated by different transmitters. These transmitters are released from beadlike swellings, or varicosities, along the autonomic motor axons. Although the molecular basis of release and transmitter action is similar to that in skeletal muscle, the elaborate NMJs that mediate rapid neuromuscular transmission in skeletal muscles are not present.
The properties of individual skeletal muscles, and of the fibers within them, vary greatly both between and within vertebrate species. A particularly important distinction exists between muscle fibers that are electrically excitable, that is, that generate propagating action potentials when adequately depolarized, and those that are not. Muscle contraction is regulated by the concentration of free calcium in the cytoplasm of the muscle fiber and this, in turn, is controlled by the electric potential across the muscle fiber membrane. In lower vertebrates, as in most invertebrates, inexcitable muscle fibers are relatively common. They are innervated at multiple sites along the muscle fibers where nerve impulses give rise to local depolarizations (see later). The summed effect of these local depolarizations causes what may be very finely graded contractions of the muscle fiber. In general, the speed of contraction of these fibers is relatively slow (see later). This class of muscle fiber has been variously referred to as slow, tonic, and red, depending on the context. An example is found in the frog, where the properties of inexcitable fibers in vertebrates were first studied in detail, and where they were referred to as slow. Unfortunately, slow is also used to distinguish different subclasses of excitable fibers (see later). In this article, electrically inexcitable, multiply innervated muscle fibers are referred to as ‘nonEx-MIF.’ Most muscle fibers, even in lower vertebrates, are electrically excitable. Each action potential in the muscle fiber gives rise to a brief contraction of relatively constant amplitude known as a twitch; fibers excited in this way are called twitch fibers. Within this broad class, there is considerable diversity of functional properties. This has led to definition of fast and slow subtypes of twitch muscle fibers on the basis of contraction speed. In addition, the situation is further complicated by classification of muscle fibers into red and white, based on the appearance of the live muscles. Red fibers tend to contract slowly and to be able to maintain their contraction for long periods of time. Associated with this ability are a predominantly oxidative energy metabolism, a rich vascular supply, and a high concentration of the oxygen-carrying heme protein myoglobin, which gives the fibers their red color. White fibers tend to contract more rapidly but also to fatigue more rapidly. They tend to utilize anaerobic metabolism, to have a less dense vascular supply, and
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to have a much lower concentration of myoglobin, making them uncolored, or ‘white.’ In addition to these two broad classes of muscle fibers, nonEx-MIF and twitch, there are numerous intermediate forms. In several animal groups, fibers exist that are multiply innervated but can also generate action potentials. While white fibers are generally twitch in character, red is used in the literature to describe both nonEx-MIF and slow twitch muscles. This makes for a confusing nomenclature, but it also highlights the important underlying natural diversity of muscle properties. Not surprisingly, this is reflected in a similar diversity in the patterns of muscle innervation.
General Features of the Motor Innervation of Skeletal Muscles Skeletal muscles are innervated by specialized motor neurons. The cell bodies of most vertebrate motor neurons are located in the ventral horn of the spinal cord and in corresponding regions of the brain stem. Each spinal motor neuron has a single axon that leaves the central nervous system via the ventral spinal roots. It then extends unbranched in one of the peripheral nerves until it reaches its one target muscle. There, it branches extensively to make contact with many muscle fibers. In the twitch muscles of higher vertebrates, it is generally the case that every action potential in a motor neuron causes contraction of every muscle fiber that neuron innervates. As a result, each motor neuron, together with the set of muscle fibers it innervates, operates as a functional ‘motor unit.’ The muscle fibers within a motor unit tend to have very similar functional properties, so that the motor unit as a whole is, for example, fast or slow. In lower vertebrates, as in most invertebrates, muscle fibers are often innervated by more than one motor neuron. They may thus be viewed as parts of more than one motor unit. It is likely that these muscle fibers are used in different ways, depending on which of their neuronal inputs is active. The force of contraction of a muscle can be controlled by the nervous system in several ways. One is by regulating the frequency of activation of a given motor unit. Skeletal muscle fibers respond to a single nerve action potential with an all-or-none twitch that is brief, but nonetheless much longer than an action potential. If a second nerve action potential arrives before the first twitch has fully decayed, the second twitch will sum with it and the peak force achieved will be greater than that in a single, isolated twitch. Within a range that differs for different muscles, the
peak force developed by a muscle increases with increasing frequency of neural activation. Many fast motor neurons fire in stereotyped high-frequency bursts, during which the force developed by the muscle increases rapidly to a maximum. In some situations the duration of these bursts, and with it the peak force, are varied as an additional means of regulating force output. A second, very different, type of control can be achieved by varying the number of active motor units. In mammalian muscles, the force generated by activation of a small set of motor units is nearly linearly related to the sum of the forces of the motor units when they are stimulated independently. The fineness with which contraction can be graded by this form of control is determined by the number of motor units in a muscle.
Phylogenetic Variation in Patterns of Skeletal Muscle Innervation During the course of vertebrate evolution, there have been major changes in the organization of muscles and the patterns of their innervation. The appearance of limbs and the increasingly sophisticated control of digits are reflected in the increasingly specific control over small groups of muscle fibers by the central nervous system. This is most developed in humans where pyramidal cells of the motor cortex make direct synaptic input to spinal motor neurons. In what follows, some of the main features of muscles and their innervation in the major groups of vertebrates are described. For each group, the species for which most information is available is indicated. Agnatha (Hagfish, Lamprey)
In primitive vertebrates of the class Agnatha, the arrangement of muscles and their innervation differ from that in other vertebrates. Within the segmental myotomes, the muscle fibers are arranged into ‘muscle units’ in which large-diameter ‘white’ fibers are placed centrally. They are surrounded by a superficial layer of much smaller ‘red’ fibers. Within the muscle units, the large central fibers are typical twitch fibers. Those nearest the abdominal cavity are innervated at their tendinous ends, sometimes by more than one axon. Stimulation of the nerve evokes an all-or-none action potential that propagates along the full length of the muscle fiber and evokes contraction. In lampreys, though probably not in hagfish, the remaining central fibers (those nearest the skin) are apparently not directly innervated but are electrically connected to innervated fibers by gap junctions. This ensures rapidly synchronized contraction
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of the central fibers in each unit and an economy of innervation. However, it appears to be a relatively inflexible arrangement designed for speed, rather than subtlety, of action. The most superficial fibers are not electrically excitable. Both anatomical and electrophysiological observations indicate that they are innervated at multiple sites along their length, often by the axons of more than one motor neuron. Thus they are similar to the nonEx-MIF fibers of other vertebrates. Cartilaginous Fish (Dogfish)
In the myotomes of both cartilaginous and bony fish, two distinct types of muscle fibers, often referred to as white (or fast) and red (or slow), are present. In both types of fish, the white fibers form the central bulk of the muscles, while the red fibers form a superficial layer (Figure 1, dogfish). As in Agnatha, the white fibers in dogfish appear to be innervated mainly at their ends, while the red fibers are innervated at multiple sites along their length. However, so-called aberrant fibers are also present in which innervation occurs at a single centrally placed site, as in many higher vertebrates. Bony Fish (Tench, Zebra Fish)
In many teleosts (bony fish), as in sharks, fast and slow muscle systems exist, with the slow fibers present in a superficial layer. The slow fibers are innervated at multiple sites by branches of several fine motor axons. There is considerable variation in the patterns of innervation of the fast white fibers in different species. In the catfish and eel, the pattern is similar to that in sharks, with nerve–muscle contacts made at the ends of the fast fibers. In most other teleosts that have been examined, the pattern of innervation of the white fibers is similar to that of the red fibers, with several sites of nerve–muscle contact, originating from more than one motor neuron, along the fibers (Figure 1, tench). Nonetheless, the white muscle fibers generate action potentials and twitch contractions, so are not typical nonEx-MIF fibers. In the zebra fish, two distinct populations of motor neurons innervating the fast white muscles have been identified in the spinal cord. Primary motor neurons have large cell bodies and axons and are the first to appear during development. There are three primary motor neurons innervating each segmental myotome on each side of the fish. The primary motor neurons innervate a nonoverlapping set of muscle fibers within the myotome. Their axonal arbors do not cross the boundaries between segments. These motor neurons can thus be recognized unambiguously from one individual to the next.
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In addition to the primary motor neurons, there is an additional class of secondary motor neurons. These are smaller than the primaries and have peripheral fields of innervation that are smaller and less focused than those of the primary motor neurons, often innervating muscle fibers in more than one segment. Individual white muscle fibers receive input from one primary and several secondary motor neurons. These synaptic inputs differ in the strength of their effect on the muscle fibers. Stimulation of the primary neurons evokes a large, short-latency endplate potential, suggesting that they may be used to in escape behavior. In contrast, secondary neurons evoke smaller, longer latency endplate potentials. Because white muscles of fish are both multiply innervated and generate action potentials, they form a group with properties intermediate between nonEx-MIF and twitch fibers as described previously. Amphibia (Salamanders, Frogs)
The evolution of limbs made important new demands on the organization of skeletal muscles and their innervation. In what was to prove to be the beginning of a sustained evolutionary trend, the slow fiber system that plays an important part in normal fish behavior appears to be reduced in importance in amphibia, and greater reliance is placed on focused innervation of individual fast muscle fibers. Urodeles In salamanders and newts, both twitch and nonEx-MIF muscle fibers are present. The twitch muscle fibers are generally innervated by several motor axons (typically one to three), as in fish. The contractile properties of individual motor units vary greatly. Interestingly, individual muscle fibers may belong to motor units with quite different contractile properties. Anura In frogs and toads, most muscle fibers are twitch fibers, and are innervated by a single motor neuron (Figure 1, frog – fast). These twitch fibers vary in their functional and molecular properties, and two or three different types have been defined. NonEx-MIF fibers are also present in many anuran muscles (Figure 1, frog – slow). However, in contrast to fish, they are generally not present as contiguous groups. In addition, some muscle fibers of an intermediate type have been found in frogs (submaxillary muscles) that are electrically excitable, but are innervated by several motor axons. Reptiles (Garter Snake, Lizard, Turtle)
Reptiles, like fish and amphibia, have both nonEx-MIF and twitch muscle fibers. In most reptiles, however, the
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Figure 1 Some common patterns of innervation of vertebrate muscles. In dogfish, multiply innervated slow muscle fibers form a superficial layer surrounding the central fast muscle fibers. The latter are mainly innervated by basketlike endings at the ends of the fibers. Some intermediate forms, with centrally located plaquelike endings, are also present. In the tench, as in many bony fish, both fast and slow muscle fibers are multiply innervated. Reproduced from Bone Q (1964) Patterns of muscular innervation in the lower chordates. International Review of Neurobiology 33: 99–147, with permission from Elsevier. In frogs, fast muscle fibers are generally innervated at a single site by nerve endings whose several branches extend along the muscle fiber. In addition, electrically inexcitable, multiply innervated muscle (nonEx-MIF) fibers are present which are innervated at multiple sites by more diffuse endings. The intrafusal muscle fibers present in the sensory muscle spindles are innervated by branches of the motor neurons that innervate both twitch and nonEx-MIF extrafusal fibers. From Barker D (1968) L’innervation motrice du muscle strie´ de vertebre´s. Actualite´s Neurophysiologiques 8: 23–71. In mammals, most twitch muscle fibers are innervated at a single site, but in some, multiply innervated muscle fibers are present in the extraocular muscles that control the position of the eye (sheep extraocular). Reproduced from Harker DW (1972) The structure and innervation of sheep superior rectus and levator palpebrae extraocular muscle. Investigative Opthamology and Visual Science 11: 956–969, with permission. The contractile intrafusal fibers of the mammalian muscle spindle (cat) receive multiple motor innervation derived from both a- and g-motor neurons. The latter terminate in both compact plaque endings and more diffuse trail endings. Reproduced from Barker D (1970) Fusimotor innervation in the cat. Philosophical Transactions of the Royal Society B 8: 336, with permission from Philosophical Transactions of the Royal Society B.
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twitch fibers are generally innervated by a single motor neuron. Different functional types (fast and slow) of twitch fibers have been identified. In the sheetlike transversus abdominis muscle of the garter snake, fast and slow twitch fibers alternate with nonEx-MIF fibers in a highly regular pattern. Available evidence suggests that this pattern is not determined by the nerve and is therefore assumed to be determined by inherent differences in the muscle fibers. In turtles, nonEx-MIF fibers are present and of particular importance in the neck muscles that support the head. Birds (Chicken)
Chickens also have both multiply innervated nonExMIF and singly innervated muscle fibers. In contrast to nonEx-MIF fibers in frogs, those in chickens are capable of generating regenerative action potentials, at least in laboratory conditions. Whether this occurs during normal use is unclear. One often studied chicken muscle, the anterior latissimus dorsi, is composed predominantly of slow, multiply innervated nonEx-MIF muscle fibers. Mammals (Human, Rat, Mouse, Cat)
In mammals, virtually all muscle fibers are twitch fibers (but see later). Each fiber receives innervation at a single site from a single motor neuron. The number of muscle fibers in a mammalian motor unit varies from less than 10 to 1000 or more. The number of motor units in an individual muscle ranges from about 25 to several hundred, and sets limits on how finely contractions can be graded. There is considerable variability in the properties of mammalian twitch fibers. In particular, they vary in their speed of contraction and in their ability to maintain contraction without fatiguing. Mammalian twitch fibers are generally recognized as being slow twitch (or type I) or fast twitch (or type II). However, the fast-twitch fibers are further subdivided into subtypes (IIA, IIB, IIX) based on their contractile properties and the main subtype of myosin expressed in them. The muscle fibers in a single motor unit are normally all of the same subtype. Like muscle fibers, mammalian motor neurons vary in their properties. For example, within the set of motor neurons innervating an individual muscle, some motor neurons have larger cell bodies than others. Axon diameter, and hence the speed of action potential conduction, are closely correlated with cell body size. In this sense, large motor neurons are also fast. Fast motor axons branch more extensively within the muscle and innervate more muscle fibers than do smaller, slower, ones. As a result, fast motor units tend to generate more force than do slow ones.
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Finally, the muscle fibers innervated by fast motor neurons are themselves functionally fast. Thus the functional properties of the nerve and muscle fibers are closely matched in each motor unit. In contrast to lower vertebrates, there are very few nonEx-MIF muscle fibers in mammals. The extraocular muscles are a notable exception (Figure 1, sheep extraocular). Some of these have a substantial population of nonEx-MIF fibers that are capable of generating finely graded contractions in response to varying frequencies of neural input. It has been suggested that the pattern of innervation of these fibers may be a relic of a primitive anterior ‘vegetative’ region of the vertebrate body. NonEx-MIF fibers of a second group are contained within the sensory muscle spindles. These so-called intrafusal fibers (within the spindle, as distinct from the majority of extrafusal fibers) are primarily innervated by a distinct class of g-motor neurons (Figure 1, cat-spindle). Some of these neurons terminate in plaque endings, similar to those of the a-motor neurons on extrafusal twitch fibers, and others end in more diffuse trail endings, reminiscent of endings on the nonEx-MIF fibers of lower vertebrates. Indeed, in frogs, the motor axons ending in trail endings sometimes also innervate extrafusal nonEx-MIF fibers (Figure 1, frog – spindle). Some intrafusal fibers are of an intermediate type in that they are capable of generating action potentials.
Variations in NMJ Properties Vertebrate NMJs are as diverse as the motor neurons and muscle fibers of which they are composed. At most vertebrate NMJs, the sites of quantal release are small domains 2–5 mm in diameter, known as boutons. Separate boutons are generally connected by fine unmyelinated axon branches, as in fish, snakes, and humans. However, in some species, they may be variably fused into more linear arrays, as in the frog and turtle (Figure 2). In the great majority of mammalian muscles, each muscle fiber is innervated at a single point by the dense plaquelike (‘en plaque’) terminal of a single motor neuron. By contrast in lower vertebrates, and in fish in particular, multiterminal polyaxonal innervation is more common, and individual terminals often consist of a dispersed set of boutons, often described as like a bunch of grapes (‘en grappe’). Associated with the differing patterns of innervation in different muscle types and in different animal groups is a range of the efficacy of neuromuscular transmission. In mammals, at one extreme, each motor nerve impulse leads to a depolarization of the muscle fiber that is several times greater than required
146 Neuromuscular Connections: Vertebrate Patterns of Fish
Frog
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Figure 2 Variations in neuromuscular junction structure in different vertebrates. The top row of drawings shows the typical appearance of individual NMJs in twitch muscle fibers of the species indicated. Many of these consist of distinct boutons which are the sites of transmitter release. In some species these are variably merged so that the nerve terminal becomes a continuous band. In fish, each NMJ has two types of endings, one more continuous than the other. Frogs are one of few species in which the NMJ is greatly elongated in the long axis of the muscle fiber. In snakes and humans, the individual boutons are distinct but much more numerous in the snake.
to trigger an action potential. Two main factors contribute to this high safety factor. On the one hand, the nerve routinely releases more transmitter than is needed to reach threshold. On the other, the threshold for action potential generation in the muscle is lowered in the region of the NMJ. Each of these factors is associated with distinctive structural features of the NMJ. Many studies have shown that the amount of transmitter released from a motor nerve terminal is roughly proportional to its area of contact with the muscle. The transmitter at the NMJ, acetylcholine (ACh), is released in multimolecular packets known as ‘quanta.’ Each quantum corresponds to the ACh contained in a single presynaptic vesicle. The number of quanta released by a single nerve impulse is known as the ‘quantal content’ of release. During lowfrequency stimulation (1 Hz) in vitro, many NMJs release 0.15–0.25 quanta mm 2. In contrast, there are substantial variations in quantal content at NMJs in different species (e.g., frog, 200; human, 20). In many cases, the differences in quantal content are associated with equivalent differences in NMJ size (Figure 3(a)). The effect of the ACh released from the nerve on the membrane potential of the muscle fiber is also influenced by structural factors. For example, the smaller the fiber, the higher its electrical ‘input’ resistance and the greater the depolarization caused by the current flowing through the ion channels opened by a quantum of ACh. In order for the depolarization caused by ACh to trigger an action potential, it must open an adequate number of the voltage-gated sodium channels that underlie the action potential. These channels are concentrated in the postsynaptic
membrane, so a smaller fraction of them needs to open to trigger an action potential than if their density was lower. In many species, particularly of higher vertebrates, the postsynaptic membrane is highly folded (Figure 3(b)). This enhances the effect of the increased concentration of sodium channels in the postsynaptic membrane and has led to the suggestion that an important function of the folds is to act as amplifiers of the effect of the ACh released from the nerve. The NMJs in different animal groups use different balances between pre- and postsynaptic effects to achieve the necessary reliability of neuromuscular transmission (Figure 3(c)). In humans the terminals are small and release relatively few quanta. However, human NMJs have very extensive postsynaptic folds which amplify the effect of the modest amount of ACh released. It is likely that this plays an important part in ensuring an adequate safety factor of transmission. In frogs, the terminals are large and release many quanta but the folding is less extensive than in humans. Rats and mice have intermediate degrees of folding. In fish and chickens, postsynaptic folds are generally lacking and it is possible that the safety factor is relatively low in these species. Finally, in electrically inexcitable fibers, postsynaptic folds are absent. Thus there is a clear correlation between the extent of postsynaptic folding and the tendency to generate an action potential in the muscle cell.
Patterns of Motor Unit Use in Vertebrate Muscles The diversity of functional properties of vertebrate muscle fibers only makes physiological sense because fibers with different properties can be specifically activated by motor neurons which themselves have distinctive properties and patterns of activity. However, the selective innervation of individual muscle fibers with specific properties is a much more prominent feature of higher vertebrates such as mammals than of lower vertebrates such as fish. In fish, the fast white muscle fibers are innervated by both primary and secondary motor neurons (see earlier). The secondary motor neurons activate the relatively low-force, and possibly more finely regulated, contractions needed to power smooth movement. The primary motor neurons trigger the rapid movements that give rise to escape behavior. Since each primary motor neuron innervates a large block of muscle fibers in each segment (see earlier), it is clear that all these fibers are likely to fire simultaneously during rapid escape movements. As most white fibers are innervated by both primary and secondary motor neurons, it is likely that they are used in the generation of both slow and fast movements.
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Frog
Frog
Rat Rat
1 µm
50 µm
Human a
Human
b 8 Human
7
Folding index
6 5 4
Rat, mouse
3 Frog 2 1 0 0
c
50
100 150 Quantal content
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Figure 3 Neuromuscular junctions vary in size and extent of postsynaptic folding. (a) Light microscope views of NMJs in frog, rat, and human, showing region of NMJ in white, muscle fiber in black. (b) Electron microscope views of NMJs in the same species, showing much more extensive folding in humans. (c) Inverse relationship between extent of folding (‘folding index’) and amount of transmitter released (‘quantal content’).
In mammals, individual muscle fibers are innervated by a single motor neuron, and are only active when that neuron fires. Studies of rat leg muscles show that motor neurons innervating type I muscle fibers (slow twitch) fire at about 10–20 Hz, in prolonged trains, which are associated with sustained postural contractions of antigravity muscles. In contrast, motor neurons innervating type II fibers (fast twitch) fire in brief stereotyped bursts of 5–10 action potentials at a frequency of about 50–100 Hz. In the type I fibers, as found in the rat soleus muscle, the natural frequency of firing is the lowest that maintains maximal force output. By contrast type II motor units, which are often used when a rapid acceleration of a body part is required, fire in short bursts of closely spaced impulses that are optimal for the rapid development of force.
The distinctive patterns of activity in different mammalian motor neurons are largely determined by the intrinsic properties of their membranes rather than by the temporal patterns of inputs to them. Following an action potential in a motor neuron, there is generally a period of increased negativity during which generation of another action potential is suppressed. The duration of this afterhyperpolarization (AHP) is greater in motor neurons innervating type I muscle fibers than in those innervating type II muscle fibers. There is a close correlation between the time course of the AHP and of the twitch of the muscle fibers in the motor unit. This illustrates further the effect of matching of the properties of motor neurons to those of the muscle fibers they innervate.
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A second aspect of motor unit use is revealed during graded voluntary contractions in mammals when there is an orderly increase in the number of active motor units. For relatively weak contractions, such as are used to maintain posture, the motor units activated are small and slow. As the force output increases, as during a rapid movement of a limb, increasingly large and fast motor units are recruited. An important effect of this orderly recruitment is that the greater the preexisting force being generated, the greater is the extra force added by each newly recruited motor unit. Thus the fractional increase in force per recruited unit stays roughly constant. This tendency for an orderly recruitment of motor units of increasing size and speed was first emphasized by Henneman and his colleagues and is known as the ‘size principle.’ A simple biophysical feature of motor neurons appears to underlie the size principle. Thus, consistent with Ohm’s law, small motor neurons have a relatively high ‘input resistance.’ As a result, a synaptic current of a given intensity causes a larger depolarization and is more likely to trigger an action potential than it would in a larger, lower resistance, neuron. As the synaptic input, or ‘drive,’ to a pool of motor neurons innervating a muscle increases, the first neurons to be recruited are therefore likely to be those with the highest input resistance and, hence, the smallest size and slowest conducting axons. This relationship assumes that the intrinsic membrane properties of motor neurons of different sizes are similar. Important exceptions to the size principle are known to exist. Thus, during constant activation of the motor neuron pool, some units may abruptly cease firing while others of similar strength begin to fire. This allows individual units to have periods of rest without loss of overall force. There is relatively little information about the natural activity patterns of motor units in freely moving lower vertebrates. It is therefore difficult to interpret the functional consequences of the varied arrangements of muscle fiber types and arrangements seen in those animals. It seems very likely that motor units composed of nonEx-MIF muscle fibers, whether they are distributed throughout a mixed muscle or concentrated into a single muscle, are used to generate slow, maintained contractions that can be very finely graded in strength. A good example may be the extraocular muscles that must position the eye with great accuracy. The ‘stepwise’ increase in tension associated with the recruitment of additional motor units may simply be too coarse for this purpose.
Conclusions It is clear that vertebrate muscles and their innervation are specialized to carry out distinctive sets of tasks. As
the vertebrate body plan has evolved, muscles and their innervation have become adapted to new functional requirements. Several general trends can be identified in this process. One of these is the increasing specificity of control of individual muscle fibers by the nervous system. In Agnatha, many muscle fibers are not even directly innervated, and depend on electrical coupling to innervated fibers for their control. In this situation the nerves exert their control on blocks of muscle fibers. This results in speed of response but limited flexibility of how muscles are used. In fish and some amphibia, while all muscle fibers are innervated, many are innervated by more than one motor neuron. This means that individual muscle fibers are part of several motor units, and are presumably used in somewhat different ways depending on the circumstances. Elucidating the way in which the several inputs to each muscle fiber interact to control overall behavior in these animals remains an important task for the future. By contrast, in higher vertebrates, including mammals, virtually all muscle fibers are innervated at a single point by a single motor neuron whose properties are closely matched to those of the muscle fibers it innervates. This gives the nervous system unambiguous access to highly specialized sets of muscle fibers, allowing appropriate ones to be activated for particular tasks. As with many phylogenetic trends, the loss of polyneuronal innervation of muscles is repeated in the ontogeny of higher vertebrates. A second closely related trend is the loss of importance of the system of multiply innervated muscle fibers. These are present in most muscles of most vertebrates up to birds, but are largely absent in mammals. Thus the pattern of innervation that is predominant in higher vertebrates may be seen to derive from the fast system associated with escape behavior in lower vertebrates. It may be that the fine of control of force associated with the graded activation of nonEx-MIF motor units in lower vertebrates is achieved in other ways in mammals, possibly involving more subtle regulation of antagonistic muscles under the control of the sensory muscle spindles. A third trend is the gradual increase in the efficacy of neuromuscular transmission. In Agnatha and fish, most individual motor nerve terminals are small and release relatively few quanta of ACh. Thus activation of the muscle requires either repetitive firing of the nerve or activation of more than one axon or both. In most vertebrates higher than fish, every nerve impulse causes a sufficient depolarization of the muscle fiber membrane to trigger an action potential. The high safety factor of neuromuscular transmission in these species is either associated with large neuromuscular junctions, a highly folded postsynaptic membrane rich in voltage-gated sodium channels, or both.
Neuromuscular Connections: Vertebrate Patterns of See also: Neuromuscular Junction (NMJ): Presynaptic Short-Term Plasticity of Neuromuscular Transmission; Neuromuscular Junction: Synapse Elimination; Presynaptic Events in Neuromuscular Transmission.
Further Reading Banks RW (1994) The motor innervation of mammalian muscle spindles. Progress in Neurobiology 43: 323–362. Barker D (1968) L’innervation motrice du muscle strie´ de vertebres. Actualite´s Neurphysiologiques 8: 23–7. Barker D (1970) Fusimotor innervation in the cat. Philosophical Transactions of the Royal Society B 8: 336. Bone Q (1964) Patterns of muscular innervation in the lower chordates. International Review of Neurobiology 33: 99–147. Bone Q (1972) The dogfish neuromuscular junction: Dual innervation of vertebrate striated muscle fibres? Journal of Cell Science 10: 657–665. Harker DW (1972) The structure and innervation of sheep superior rectus and levator palpebrae extraocular muscle. Investigative Opthamology and Visual Science 11: 956–969. Hennig R and Lomo T (1985) Firing patterns of motor units in normal rats. Nature 314: 164–166. Lichtman JW and Wilkinson RS (1987) Properties of motor units in the transversus abdominis muscle of the garter snake. Journal of Physiology 393: 355–374.
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Mark RF, von Campenhausen G, and Lischinsky DJ (1966) Nerve– muscle relations in the salamander: Possible relevance to nerve regeneration and muscle specificity. Experimental Neurology 16: 438–449. Morgan DL and Proske U (1984) Vertebrate slow muscle: Its structure, pattern of innervation, and mechanical properties. Physiological Reviews 64: 103–169. Ogata T (1988) Structure of motor endplates in the different fiber types of vertebrate skeletal muscles. Archives of Histology & Cytology 51: 385–424. Rome LC (2002) The design of vertebrate muscular systems: Comparative and integrative approaches. Clinical Orthopaedics and Related Research 403(supplement): S59–S76. Slater CR (2003) Structural determinants of the reliability of synaptic transmission at the vertebrate neuromuscular junction. Journal of Neurocytology 32: 505–522. Walrond JP and Reese TS (1985) Structure of axon terminals and active zones at synapses on lizard twitch and tonic muscle fibers. Journal of Neuroscience 5: 1118–1131. Westerfield M, McMurray JV, and Eisen JS (1986) Identified motoneurons and their innervation of axial muscles in the zebrafish. Journal of Neuroscience 6: 2267–2277. Wilkinson RS and Teng H (2003) The nerve-muscle synapse of the garter snake. Journal of Neurocytology 32: 523–538. Wood SJ and Slater CR (2001) Safety factor at the neuromuscular junction. Progress in Neurobiology 64: 393–429.
Neuromuscular Junction: Synapse Elimination R R Ribchester, University of Edinburgh, Edinburgh, UK ã 2009 Elsevier Ltd. All rights reserved.
Introduction Motor neurons supply the last outposts of the motor system, where volition finally interfaces with and influences the material world. In higher vertebrates, the axon of an adult somatic motor neuron projects to a single anatomically defined muscle target. The motor axon then divides into several tens to hundreds of intramuscular collateral branches, each terminating on a muscle fiber at a neuromuscular junction (NMJ) to form its sole innervation. A high margin of safety for neuromuscular transmission ensures that every action potential generated in the motor neuron reliably triggers an action potential and contraction of all the muscle fibers it supplies. Thus, a motor neuron and its muscle fibers function together in a unitary fashion: The combined set is referred to as a motor unit. The number of muscle fibers in one unit is termed the motor unit size. Motor units are activated in an orderly fashion during voluntary movements, often sequentially by size. The variation in motor unit sizes (more than tenfold in some muscles and more than 1000-fold between other muscles) confers a remarkable dynamic range on the motor system as a whole. All historic and contemporary images of adult NMJs reveal a remarkable and stereotyped pattern of the motor nerve supply to skeletal muscle fibers, found throughout the vertebrate subphylum but particularly well represented in mammals: Each well-defined motor junctional area, also called the ‘endplate,’ is supplied by the axon of one and only one motor neuron. However, this pattern is not formed de novo. Rather, the motor endplates of individual muscle fibers are initially supplied by terminals derived from several motor neurons. This state is called polyneuronal innervation. The transition from polyneuronal to mononeuronal innervation during development occurs by a process commonly known as synapse elimination, the subject of this article. The reduction in motor unit convergence (overlap) has another important consequence: Since in most cases the number of muscle fibers does not increase during synapse elimination, the degree of motor divergence also decreases; that is, synapse elimination also brings about a reduction in motor unit size, therefore ultimately limiting the maximum force a motor unit can produce.
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Three main physiological factors regulate motor unit force production in vertebrate muscle. In addition to motor unit size, these are the frequency of activation and the functional ‘type’ of the muscle fiber. In rodents, where these factors have been studied in greatest detail, they all change during development. It is interesting to note that the three properties appear to be linked: Frequency of activation and intrinsic properties such as motor neuron and muscle fiber type play roles in steering the process of synapse elimination to its conclusion, thus determining the motor unit size. Different rules govern the maturation of innervation patterns in smooth, cardiac, and some specialized types of voluntary striated muscle fibers that contract relatively slowly. Persistent, stable polyneuronal innervation is the norm in these muscle types, and they are therefore not discussed here. Synaptic remodeling also occurs during development of invertebrate muscles, and this plasticity is increasingly studied in the fruit fly, Drosophila melanogaster, mainly because of the power and pace of molecular genetic analysis in this species. However, although pruning of motor axon branches occurs during metamorphosis, neuromuscular synapse elimination comparable to that in mammalian muscles does not normally take place in Drosophila. The present discussion is therefore mainly restricted to mammalian skeletal muscle.
From p to m Neuromuscular synapses form about halfway through gestation in mice and rats. In some muscles, axonal growth cones appear to contact random points on the membrane of the immature muscle fibers (myotubes) and to induce specialized synaptic features in them. In other muscles, it appears that growth cones contact preformed postsynaptic sites. Either way, axons from other motor neurons then add to the first neuromuscular inputs. Thus, by birth, virtually all muscle fibers are innervated by terminals of several motor neurons, that is, polyneuronal innervation (p). The first evidence of polyneuronal innervation in mammalian muscle was obtained in 1916–17 in a study of fetal human tissue by Tello. Contemporaneously, Boeke reported polyneuronal innervation in reinnervated intercostal muscles after nerve injury in hedgehogs. Skeletal muscles of mice and rats are presently the tissues of choice for descriptive and mechanistic studies of neuromuscular synapse elimination both in neonates and in reinnervated muscles in adults. More-recent interest in synapse elimination began with two electrophysiological expose´s, one on neonatal
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rat diaphragm muscle and the other on reinnervated adult rat hind limb muscles. Both studies showed that when the muscle nerves were stimulated with electric pulses of gradually increasing strength, discontinuous, stepwise increments occurred in the amplitude of the endplate potentials recorded with intracellular electrodes from individual muscle fibers. This is in contrast to the one-step response normally observed during graded nerve stimulation in adults. The simplest explanation of these observations is that multiple axons converging on the same endplate, and having distinct electrical excitation thresholds, are progressively recruited as the stimulating current is increased. Polyneuronal innervation was also demonstrated using electrophysiological techniques at about the same time in other immature vertebrates, including tadpoles and chick embryos. These studies were complemented by physiological evidence of polyneuronal innervation in skeletal muscles of neonatal kittens by recording isometric tension. These studies showed that the force produced in response to the simultaneous stimulation of two immature motor axons is usually less than the arithmetic sum of the responses to stimulating both separately. This is also most simply explained by convergent innervation of muscle fibers. More recently, direct histological evidence of polyneuronal innervation and synapse elimination has been obtained by confocal microscopy analysis of three- and four-dimensional image stacks (X,Y,Z þ/ T), for example in preparations immunostained for neurofilament protein in axons (Figure 1) or expressing different fluorescent proteins. Such images have provided unequivocal confirmation of the convergence of several axon terminals on individual motor endplates at birth in rodents and synapse elimination during the first 3 postnatal weeks. One early study has since achieved iconic status in the field: a comprehensive analysis of polyneuronal innervation and synapse elimination carried out by Jansen, Brown, and Van Essen. These investigators brought to bear a powerful combination of tension measurements, intracellular recordings, and histology. All contemporary studies and analysis of polyneuronal innervation and neuromuscular synapse elimination can be traced back to this study. Its main findings and conclusions were the following: . Motor unit size is maximal at birth and declines roughly exponentially during the first 3 postnatal weeks, during which time there is no reduction in the total number of motor neurons. Therefore, synapse elimination is due to withdrawal of motor nerve terminals and pruning of axon collateral branches rather than to complete degeneration of individual motor neurons.
Figure 1 Polyneuronally innervated neuromuscular junctions in an 8-day-old mouse skeletal muscle, stained for neurofilament proteins in axons (green) and acetylcholine receptors on muscle fibers at motor endplates (red). Two axons can be seen projecting in the same motor endplate on several of these muscle fibers.
. The rate of synapse elimination is highest during the first postnatal week. However, virtually all muscle fibers remain innervated by at least two inputs during this period. Mononeuronally innervated muscle fibers emerge rapidly during the second to third postnatal weeks. . Partial denervation at birth leads to reduced synapse loss in the surviving motor units but does not prevent synapse elimination completely. Mononeuronal innervation therefore arises partly by competition between the terminals of different motor neurons converging on the same endplate and partly by an intrinsic tendency of motor neurons to withdraw a proportion of their terminal branches. . Implanting the cut end of a nerve that normally innervates one neonatal muscle into another, at some distance away, can result in stable polyneuronal innervation. The distance between converging synaptic inputs therefore mitigates elimination of polyneuronal connections: The further apart two inputs are, the more likely they are to persist and become stable. A striking feature of the process of synapse elimination at the developing NMJ revealed by these studies is that the great majority of NMJs end up innervated by one and only one motor neuron m. Although the response to partial denervation at birth suggests that there is a limit to the number of supernumerary branches that can be maintained, it is clear that the
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usual result of synapse elimination is not consistent with a random loss of a fraction of the branches formed initially. This would leave some NMJs innervated by several axons and others by none. Rather, the observations point to some sort of local competition at each NMJ that results in a single ‘winner.’ (Whether synapse formation by motor axon branches is initially random is another unresolved matter.) Comparable findings were subsequently reported by several groups using similar combinations of histological and electrophysiological methods in studies of reinnervated muscles in adults. Following certain forms of nerve injury, particularly crushing the nerve so that the surrounding connective tissue sheaths remain intact, the damaged motor axons grow back into the target muscle. Regeneration of damaged axons may restore some or all their original neuromuscular connections, in some cases leading to excellent functional recovery. When axons regenerate into partially denervated muscles, they often polyneuronally innervate endplates already occupied by intact motor nerve terminals. In such cases, a form of competition takes place between the original and the regenerated axons. The many similarities between synapse elimination in neonatal muscles and reinnervated adult muscles led to the working hypothesis that the underlying mechanisms are the same. Synapse Elimination versus Synapse Degeneration
Ultrastructural analysis of NMJs established that the characteristics of synapses and axon collaterals undergoing elimination and withdrawal are quite unlike those of the orthograde (Wallerian) degeneration that occurs when axons are damaged. Wallerian degeneration is rapid and involves radical breakdown of cytoplasmic integrity and organelles within intramuscular axons and motor nerve terminals. By contrast, synapse elimination results in regression of a subset of the collateral branches in a motor neuron’s axonal arbor. It is protracted over several days, and for the most part it involves gradual retraction of synapses and resorption of the affected branches into the parent axon trunk with no overt loss of integrity of the cytoplasm and organelles (e.g., mitochondria and synaptic vesicles appear to remain healthy). Real-Time Visualization of Synapse Elimination In Vivo
An improvement in the basic description of the neonatal remodeling of motor units has followed the development of transgenic mice expressing fluorescent proteins in motor neurons. This enables all the connections made by individual motor neurons to be
visualized at all stages of development. These transgenic mice have been made by inserting variants of the fluorescent jellyfish protein Green Fluorescent Protein into the mouse genome under the control of a chemically modified promoter, thy1.2, that has the effect of selectively driving protein expression mainly in neurons. Collectively, the members of this family of fluorescent transgenic mice are referred to below as XFP mice (X ¼ R for red, Y for yellow, G for green, or C for cyan). Recently, via Cre-Lox floxed-stop tamoxifen induction, variants have been generated that express variable amounts of YFP. In all these cases, expression of the fluorescent proteins in neurons appears to be completely harmless: The transgenic mice are indistinguishable from their littermates until their brains, spinal cords, and peripheral nerves are viewed in a fluorescence microscope. Use of XFP mice has already revealed interesting new insights into the process of synapse elimination and is likely to continue to do so for some time. Is competition local? Visualization of XFP-labeled NMJs during postnatal synapse elimination has confirmed and extended previous interpretations based on the more indirect methods applied for the first time by Jansen, Brown, and Van Essen. Studies of motor nerve terminal size, number, and disposition at different stages in the elimination process show that synapses and collateral branches retract into their parent nerve trunks such that during early stages, a dwindling fraction of each NMJ is occupied by the withdrawing synapses. The local competitions experienced by each of the numerous terminals of an immature motor neuron do not occur simultaneously but by an asynchronous process protracted over several days. At any given time, some of the terminals of a single neuron are ‘victorious,’ fully occupying the endplate, while others show variable fractional occupancies, giving way to or taking over the space occupied by the terminal synapses of other motor neurons. Takeover of endplates during synapse elimination has now been observed directly, by repeated visualization of polyneuronally innervated junctions whose axonal inputs are labeled by selective expression of YFP and CFP in different motor neurons. These direct observations appear to refute the hypothesis previously favored by some, that the main route to mononeuronal innervation was via elimination of acetylcholine receptors and their overlying synaptic boutons, with no takeover by the remainder. The routine takeover of endplates by one terminal, with simultaneous retraction of others, is now a generally accepted account of the normal process leading from polyneuronal to mononeuronal innervation. This view also accords with the progressive recovery
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of synaptic area observed in partially denervated muscles following regeneration of injured axons, as suggested by static images of vital staining of regenerating and intact axons. Expression of different fluorescent proteins (CFP and YFP) in different motor neurons has also been used to study unusual behavior at some NMJ. A few synapses show oscillating ‘flip-flop,’ in which first one of two inputs occupies most of the endplate at the expense of the other, but then the second gains the upper hand and the most fractional occupancy. It is unknown what determines this remarkable dynamic interaction or its ultimate outcome in favor of one of the two inputs, with complete elimination of the other. At first glance, it is tempting to conclude that asynchronous retraction and flip-flop behavior suggest that synapse elimination is driven locally, entirely by competitive interactions between converging terminals. However, experiments show that asynchronous synapse loss also occurs when global properties such as axonal transport are interrupted. For example, axonal injury in adult mutant mice with the ‘Wallerian degeneration, slow’ mutation (WldS), in which orthograde degeneration is delayed, also results in protracted, asynchronous synapse retraction. Thus, observations of asynchronous synapse loss constitute insufficient evidence to conclude that local interactions drive synapse elimination (see below). The fate of the losers Finally, detailed descriptions have been obtained by time-lapse imaging in vivo of the last stages of synaptic retraction. These have confirmed that removal of the losing synapse partly involves retraction of a collateral nerve branch into the parent axon trunk, identified by characteristic end bulbs (retraction bulbs) on the withdrawing axon branch. In addition, combined confocal and ultrastructural (electron microscopic) analysis has also shown that some retracting synapses and axon branches undergo fragmentation, with sequestration of the cellular residues, named axosomes, into Schwann cells and other phagocytic cells in the vicinity of the NMJ. Collectively, the retraction and axosomal fragmentation of synapses undergoing elimination appear remarkably similar to the loss of synapses following axotomy in some forms of neuromuscular pathology. This includes neuromuscular synaptic degeneration in WldS mutant mice after nerve injury; mouse models of some forms of motor neuron disease, such as in the SOD1G93A transgenic mouse model of amyotrophic lateral sclerosis (ALS); and in smn mutant models of spinal muscular atrophy (SMA; see below). However, it remains to be seen whether these remarkable morphologically similarities are caused by similar underlying molecular mechanisms.
Are There Intrinsic Hierarchies Among Motor Neurons? A recent study by Kasthuri and Lichtman seems set to reignite debate over the forces that establish which synapses persist when two motor nerve terminals belonging to different motor neurons vie for exclusive occupancy of the same motor endplates. Transgenic mice expressing YFP in a small subset of motor neurons and CFP in a different subset were examined. Sometimes only two motor neurons supplying the same muscle were labeled, one yellow and the other cyan. Thus, the investigators were able to identify all the polyneuronally innervated motor endplates where both axons contributed a motor nerve terminal. They found that while both axons innervated variable fractions of the endplates they did not share, the same member of the pair nearly always occupied a greater fraction than the other at all endplates that they did share. This suggests that while competitive interactions take place locally at motor endplates, a pecking order or dominance hierarchy may operate within motor neuron pools, intrinsically biasing the outcomes of all their interactions with others during synapse elimination. An important prediction of this hypothesis is that all the branches of the motor neuron with the highest ranking should be ‘winners.’ By contrast, the lowest-ranked motor neurons should lose virtually all competitions and have either all or nearly all their terminals eliminated. The suggestion of a fixed hierarchy among motor neurons needs to be viewed in the context of several previous studies. First, time-lapse observations of flip-flop (made by the same research group) made over several days in vivo suggest that if there is a hierarchy, then either it may be a dynamic one or the differences that determine the hierarchical rank could be very small and subtle. Second, indirect evidence based on isometric tension recordings in the watershed study by Jansen et al. suggests that some motor neurons withdraw their terminals from motor endplates in the absence of competitors, albeit this observation has been challenged by other studies and the issue remains unresolved. Third, axons regenerating into partially denervated muscles fail to displace sprouts from most of the terminals they previously innervated. Fourth, high levels of stable, persistent polyneuronal innervation are found following recovery from muscle paralysis. However, all but the first of these caveats are based on indirect evidence that predated the availability of transgenic XFP mice. It is to be hoped that the general availability of these mice will eventually allow clarification of the possibility of an intrinsic hierarchy among motor neurons.
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The Role of Activity in Synapse Elimination One of the questions examined in the immediate wake of the Jansen, Brown, and Van Essen study was whether activity plays an obligatory and decisive role in synapse elimination. In rats and mice, synapse elimination occurs in hind-limb muscles as the animals are first beginning to use those limbs to bear weight. By analogy in the visual system, use of the eye during a critical period when the precision of adult connectivity is being established plays a vital part in determining the outcome of that process. It therefore seemed plausible that something similar might happen at the NMJ. Several studies have explored the effect of changes in neuromuscular activity on both the timing and the outcome of synapse elimination, either in development or during reinnervation of adult muscle. The various studies have led to the following conclusions: . Reducing activity delays elimination of polyneuronal innervation. . Increasing activity makes synapse elimination occur sooner. . Selectively altering activity levels in motor nerve terminals at NMJs supplied by convergent motor neurons frequently biases the outcome of synapse elimination in favor of the more active axons. . Polyneuronal innervation is not an inherently unstable state: it persists indefinitely in active muscle under some experimental conditions. . Activity is not strictly necessary for synapse elimination since it can occur at some NMJs even when they are experimentally paralyzed. . Activity-dependent competitive vigor correlates with the synaptic strength of convergent inputs, that is, the amount of neurotransmitter release per unit area. The overall conclusion from these studies is that activity is strongly influential but not strictly decisive in determining the outcome of synapse elimination. Other variables likely to mitigate competitiveness include intrinsic limits on the numbers of peripheral connections any one motor neuron can support, sensitivity to neurotrophic factors, and selective recognition or adhesion of motor neurons to specific muscle fibers based on either topographic or histochemical markers.
Muscle Fiber Type Specificity and Selective Synapse Elimination Skeletal muscle fibers are metabolically heterogeneous. For example, some are specialized for brief, faster and others for prolonged, slower contractions.
In addition, many are segmentally or compartmentally organized with axonal inputs constrained to arborize within the segmental or compartmental boundaries. Synapse elimination in development therefore occurs largely within rather than between segments or compartments. Occasionally, however, it appears that mistakes are made and editing of these occurs by axon branch pruning. In rats and mice, some muscle fiber subtypes are already specified at birth: specifically, type I fibers expressing myosin isoforms characteristic of slowtwitch muscles. In contrast, fast-twitch type II muscle fibers develop mainly postnatally. In adults, these subtypes of fibers are selectively innervated by motor neurons whose activity patterns match the muscle fiber type characteristics. There is some evidence for nonselective innervation of muscle fiber types before synapse elimination occurs. Thus in mice in the first week after birth, individual motor units contain both fast and slow muscle fibers. By the time synapse elimination is complete, the homogeneity of the units has increased to nearly its adult level. This suggests that specific matching of motor neuron type to functionally appropriate muscle fiber types plays a role in synapse elimination. However, as yet there are no selective molecular markers for different motor neuron types, so this hypothesis awaits a stringent test.
Molecular Mechanisms of Synapse Elimination and Axonal Pruning Progress toward understanding the molecular mechanisms that induce and execute synapse elimination has been exasperatingly slow. Neuromuscular synapses are virtually inaccessible to systematic biochemical analysis, because they comprise such a small fraction of the volume of skeletal muscles (unlike the brain, where synapses are the main constituent). However, several studies have used the incidence of polyneuronal innervation as a bioassay for the effects of pharmacological blockers, growth factors, and other treatments on synapse elimination. Virtually all these studies show only transient effects and have thus far provided no clear understanding at a molecular level of the causal events leading to mononeuronal innervation. Neurotrophic Influences on the Rate of Synapse Elimination
Neurotrophic factors play a crucial role in maintaining the size of neuronal populations and the number and disposition of their connections. For instance, survival and growth of autonomic and sensory
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neurons depend on maintenance of physiological levels of nerve growth factor. Some cortical neurons show similar requirements for the related neurotrophin, brain-derived neurotrophic factor (BDNF). Members of another class, the neurotrophic cytokines, have potent effects on motor neuron survival. The effects of different families of these cell-survival molecules have been tested on neonatal muscles to see whether they may play similar regulatory roles in synapse elimination. The results have been largely equivocal and/or negative. For example, administration of either BDNF or ciliary neuronotrophic factor delays synapse elimination in mice, but only by about a day. Remarkably, however, transgenic expression of glial cell line-derived neurotrophic factor (GDNF) in mouse muscle delays synapse elimination by about 1–2 weeks. Unfortunately, GDNF is unlikely to be a physiological regulator because many muscle fibers in normal mice do not express it at the right stage of development. Role of Proteases and the Ubiquitin-Proteasome System
Some studies have focused on the possibility that regulated proteolysis of extracellular components of the NMJ might be involved in the elimination of supernumerary nerve terminals. There is some evidence that inhibition of either Ca-activated or serine proteases delays synapse elimination. Likewise, it has been reported that activation of protein kinase C delays synapse loss. At present, however, there is no detailed model of how these effects might operate during normal NMJ development. Recently, there has been interest in the possible role of the ubiquitin-proteasome system (UPS), the mechanism within cells for targeted breakdown of proteins. Proteins tagged with a chain of ubiquitin monomers are recognized by chaperone proteins that convey them to proteasomes, organelles that execute protein degradation. Interest in this system stems partly from the role of the UPS in several forms of neurodegenerative disease, including ALS. There is evidence that the UPS plays a significant role in both Wallerian degeneration and axon pruning. For instance, synaptic degeneration is delayed in WldS mutant mice, in which there is expression of a chimeric protein, part of which contains the N-terminal 70 amino acids of a ubiquitination cofactor, Ube4b. Axotomy induces asynchronous synaptic retraction and fragmentation in these mice. Inhibitors of the UPS also delay axon pruning in Drosophila and axon degeneration in the SOD1G93A mouse model of ALS. However, there are as yet no published data on the role of the UPS in neonatal synapse elimination.
Role of Other Nonneural Cell Types at Immature and Adult NMJs Parts of three cell types are thought to constitute the normal NMJ: muscle fiber, motor nerve terminal, and one or more perisynaptic (terminal) Schwann cells. These structures are bonded to one another by synaptic basal lamina. Nerve injury in adults triggers, after a latent period of about 3 days, proliferation and branching of terminal Schwann cells, which then form a scaffold of bridges between motor endplates. These appear to facilitate reformation of synapses by regenerating motor axons. The average number of Schwann cells per NMJ increases postnatally, but there is no compelling evidence that this process plays a role in selective elimination of inputs during synapse elimination. However, Schwann cells engulf degenerating nerve terminals after axotomy, during Wallerian degeneration, and appear to fulfill a similar function in synapse elimination, with formation of axosomal fragments. Recent availability of transgenic mice expressing fluorescent proteins in these terminal Schwann cells will permit longitudinal studies that should help establish whether they have a role in synapse elimination. NMJs also appear to be a favorable local environment for a population of so-called perisynaptic fibroblasts. These cells have been observed at most NMJs in frogs, chickens, rodents, and human muscle. They lie outside the basal lamina, but although they react by spreading and expression of specific cell adhesion molecules, their precise functions at NMJs remain intriguing but unknown.
Synapse Elimination and Neurodegenerative Disease Most neurodegenerative diseases show an increased likelihood of onset with age. This applies to most adult forms of motor neuron disease (MND), such as ALS. Most adult forms of MND (more than 90%) are sporadic, the remainder inherited. About 20% of the inherited forms (i.e., less than 2% overall) are due to mutations in genes coding for superoxide dismutase (SOD). However, the availability of good animal models of SOD-dependent ALS has led to this form’s being the most widely studied. Overexpression of SOD genes in transgenic mice leads to defective axonal transport and motor neuron degeneration, with clinical signs resembling the human disease. Other forms of MND are neonatal and juvenile. These include SMAs of various types, the most aggressive and earliest onset being type I (Werdnig-Hoffman disease), which is lethal within the first few months of life. In contrast to ALS, most forms of SMA are inherited, and in more
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than 95% of cases, the mutations responsible have been pinpointed to the smn (survival of motor neurons) gene. There are good animal models of these forms of motor neuron disease as well. Recent studies of neuromuscular pathology in mouse models of ALS and SMA, as well as other mutants, such as pmn and wasted, have revealed some remarkable common features. Specifically, neuromuscular synapses within some motor units in all these models degenerate or retract into their parent axons at early stages in the disease process, in advance of axonal or motor neuron cell body degeneration. This has led to three views: first, that these motor neuron disease variants may represent a form of dying-back neuropathy, beginning with primary pathology at neuromuscular synapses; second, that neurodegenerative processes in general may be compartmentalized, with independent mechanisms regulating the degeneration of synapses, axons, and cell bodies; and third, that degeneration of synapses in circumstances like MND may share some common molecular mechanisms with normal forms of synaptic plasticity, including synapse elimination in postnatal development. These views are not universally held and remain open to stringent experimental validation. For instance, although there may be strong morphological and physiological similarities between developmental synapse elimination and MND pathology in SODdependent ALS, recent data suggest that synaptic degeneration in SOD1G93A mouse variants shows loss of synaptic vesicle proteins in advance of degeneration, which synapses undergoing elimination in development do not. Moreover, synapses in SOD1G93A mice degenerate synchronously in some muscles, suggesting a primary axonopathy (consistent with the disruptive effect of the SOD-1 mutation on axonal transport). Synapses are known to be especially vulnerable to defects in axonal integrity, perhaps partly due to the high metabolic and energetic demands placed on them by the exigencies of synaptic transmission. Thus, it is perhaps not surprising that early loss of synapses could lead to the erroneous conclusion that these foci are the primary loci of disease. On the other hand, primary synaptic pathology has not been conclusively ruled out, either for SOD1-related ALS or for any other form of MND. Synapses contain abundant mitochondria, so these foci of oxidative metabolism may be especially vulnerable to direct, primary effects of oxidative stress. Either way, genetic or pharmacological strategies aimed directly at inhibiting synaptic degeneration could prove highly effective in mitigating disease progression. For instance, pmn mutant mice cross-bred with WldS mice showed strong evidence of mitigation of
disease progression. However, transferring the WldS gene to SOD1G93A mice has, at best, only a weak neuroprotective effect. This may be partly explained by the weakening of the protective effect of the WldS gene on synapses as mice age. Screens of mutations induced by ethylnitrosourea are currently under way in a search for novel mutations that may protect synapses as effectively as WldS protects axons. Such mutations could ultimately be exploited so as to mitigate disease progression in several forms of motor neuron disease.
Summary We still do not know precisely what genetic, environmental, or even stochastic factors determine whether a synaptic bouton should persist or be removed from a p-junction or indeed whether there is a single deciding factor. Activity levels and sensitivity to neurotrophic factors, selective recognition between motor neurons and muscle fibers, and other intrinsic properties of motor neurons and the muscle fibers they innervate may all help to establish the size of motor units, shaping their organization and orderly recruitment, as we accumulate repertoires of behavior during development that we carry with us into adulthood. Ultimately, all these potential regulators may individually play influential rather than decisive roles. For example, perhaps inactivity induces and sustains polyneuronal innervation merely by stimulating and maintaining nerve branching, enhancing the opportunities for competitive interactions to occur between synaptic terminals. Intrinsic determinants of synaptic strength, differential sensitivity to neurotrophic factors, and differential adhesion may then bias the outcome of the subsequent interplay as motor terminals advance or retreat over endplate territory defined by the boundaries of postsynaptic receptor clusters they require to mediate their primary function. But the final outcome could also be generated by chance, through some fundamentally stochastic process. By analogy, reliable chemical synaptic transmission is produced by an underlying stochastic mechanism in which action potentials elevate the probability of exocytosis rather than determining precisely how many vesicles fuse with release sites in presynaptic active zones. Perhaps chance and necessity, an apt epithet for the forces regulating molecular evolution, may also prove to be a fitting summary of the interplay between environment andgene expression leading to development of functionally appropriate innervation patterns, not only at NMJs but throughout the developing nervous system, thereby individualizing all voluntary behavior.
Neuromuscular Junction: Synapse Elimination 157 See also: Gap Junctions and Neuronal Oscillations;
Neuromuscular Connections: Vertebrate Patterns of.
Further Reading Barry JA and Ribchester RR (1995) Persistent polyneuronal innervation in partially denervated rat muscle after reinnervation and recovery from prolonged nerve conduction block. Journal of Neuroscience 15: 6327–6339. Bishop DL, Misgeld T, Walsh MK, et al. (2004) Axon branch removal at developing synapses by axosome shedding. Neuron 44: 651–661. Brown MC, Jansen JK, and Van Essen DC (1976) Polyneuronal innervation of skeletal muscle in new-born rats and its elimination during maturation. Journal of Physiology 261: 387–422. Costanzo EM, Barry JA, and Ribchester RR (2000) Competition at silent synapses in reinnervated skeletal muscle. Nature Neuroscience 3: 694–700. Gillingwater TH and Ribchester RR (2003) The relationship of neuromuscular synapse elimination to synaptic degeneration
and pathology: Insights from WldS and other mutant mice. Journal of Neurocytology 32: 863–881. Kasthuri N and Lichtman JW (2003) The role of neuronal identity in synaptic competition. Nature 424: 426–430. Keller-Peck CR, Walsh MK, Gan WB, et al. (2001) Asynchronous synapse elimination in neonatal motor units: Studies using GFP transgenic mice. Neuron 31: 381–394. Sanes JR and Lichtman JW (1999) Development of the vertebrate neuromuscular junction. Annual Reviews of Neuroscience 22: 389–442. Schaefer AM, Sanes JR, and Lichtman JW (2005) A compensatory subpopulation of motor neurons in a mouse model of amyotrophic lateral sclerosis. Journal of Comparative Neurology 490: 209–219. Walsh MK and Lichtman JW (2003) In vivo time-lapse imaging of synaptic takeover associated with naturally occurring synapse elimination. Neuron 37(1): 67–73. Zuo Y, Lubischer JL, Kang H, et al. (2004) Fluorescent proteins expressed in mouse transgenic lines mark subsets of glia, neurons, macrophages, and dendritic cells for vital examination. Journal of Neuroscience 24: 10999–11009.
Presynaptic Events in Neuromuscular Transmission H Teng and R S Wilkinson, Washington University School of Medicine, St. Louis, MO, USA ã 2009 Elsevier Ltd. All rights reserved.
Introduction Because of its accessibility, the neuromuscular junction (NMJ) is the most studied vertebrate synapse. The NMJ is also the simplest synapse – a relay, so called because its function is to relay information, traveling on motor axons originating from within the central nervous system, to the membrane of muscle fibers. The information is digital, in the form of all-or-nothing depolarizing pulses, or action potentials (APs). Each pulse instructs the muscle fibers to contract or (for most fibers) twitch. Thus the NMJ amplifies a weak control signal (current supplied by the axonal AP) into a stronger one capable of depolarizing the muscle fiber, which is much larger than the axon. In contrast, and as described elsewhere in this encyclopedia, synapses within the brain and spinal cord are thought to serve far more complex functions – those that underlie behavior, such as learning, memory, emotion, and addiction. To do so, brain synapses utilize various kinds of use-dependent behavior, such as depression, potentiation, and facilitation. They are also able to integrate, or combine, multiple inputs (APs originating from many neurons) and arrive at a single decision – namely, whether or not to generate an output AP. With such disparate roles, it seems remarkable that the simple NMJ can serve as the model synapse, as it is called, for understanding synaptic function throughout the vertebrate nervous system. There are two reasons. First, many of the characteristics of brain synapses are found at the NMJ, albeit in more rudimentary form, and indeed were discovered there. Second, and most relevant for what follows herein, is the fact that much still remains to be learned. One key aspect of all synapses – how the presynaptic nerve terminal, or presynapse, releases its chemical signal – remains poorly understood. The importance of gaining this knowledge becomes clear if one considers the output side of the synapse, where the postsynaptic receptors convert an increased concentration of the chemical neurotransmitter, in the synaptic cleft, into the depolarization that triggers muscle excitation. Here, a great deal is known. Early information came exclusively from the NMJ, but many classes of receptors at different synapses are now studied in detail. Knowledge of precisely how these receptors work has
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advanced substantially in recent years, and continues to advance. As a result, pharmacological intervention is possible, and many successful drugs have postsynaptic receptors as their target. A similar arsenal of presynaptic drugs, if available, could alter neurotransmitter release directly, providing additional avenues for intervention. Such drugs would have a profound impact on the treatment of disease, as have those that modify synaptic function by acting on receptors. But, unlike receptors, the basic physiology of nerve terminals remains to be elucidated. If history serves, the synapse most likely to reveal these secrets is the simplest and most accessible one, the NMJ.
Anatomy of Motor Nerve Terminals Presynaptic function derives from extreme evolutionary specialization. Cells in early animals communicated with each other via hormones, which are chemicals released by certain cells into the extracellular milieu; these chemical signals are detected by other cells, often some distance away, that express the appropriate receptors. One problem with this scheme is that each signal to be communicated requires a unique hormone. Another is that chemical communication over long distances is slow. Though still extant today (the endocrine system), some hormonal communication evolved into neural communication precisely to avoid these problems. Neural communication occurs only between adjacent cells. The cells are so close (50 nm for NMJs) that only a tiny amount of chemical released by one is sufficient to rapidly influence receptors of the other. After the signal is delivered, the chemical diffuses away and is harmlessly diluted, influencing no other cells. Alternatively, it is degraded (as at the NMJ) or taken up for reuse. The most remarkable aspect of this specialization is that, during development, one cell, the presynaptic neuron, extends a thin process, its axon, over a distance of up to 1 m or so (in humans) and finds its target muscle fibers with precision. Thus a motor neuron in the spinal cord becomes, in effect, adjacent to a muscle cell in the foot (or more than one cell – the axon can branch). The specialization at which the terminal of the axon comes in near-contact with the receptors of the muscle cell is the NMJ. As a result, many muscle cells can be independently and rapidly controlled by their innervating motor neurons, using only one chemical neurotransmitter. In vertebrates, this transmitter is acetylcholine (ACh). A consequence of this system is that each motor nerve terminal undertakes its function without much
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help from the motor neuron’s distant cell body. Although the latter contains the nucleus and proteinmaking machinery, axonal transport of molecules from cell body to the motor terminal of the NMJ, or vice versa, can require up to several months. In particular, the need for independent function requires that motor terminals supply their own energy and continuously resynthesize and package ACh for reuse (see later, vesicle recycling). Nerve Terminals at NMJs of Different Vertebrate Species
The most fundamental, unitary synapse comprises a presynaptic bouton (button), a narrow cleft, and a cluster of membrane-bound postsynaptic receptors (Figure 1). The bouton contains, principally, mitochondria (M), synaptic vesicles (SVs) that contain neurotransmitter, and additional transmitter within the cytoplasm. Active zones (AZs), sites of neurotransmitter release, are located within the presynaptic membrane, the portion of the plasma membrane that apposes postsynaptic receptors. Interestingly, solitary boutons are common within the central and autonomic nervous system, but rarely serve as NMJs. However, the structure of NMJs of various species is essentially a cluster of boutons, each with its own postsynaptic receptor specialization. In amphibians (frog), thousands of boutons are linearly arranged, each containing a single AZ. In reptiles, 60 boutons are clustered, much like a bunch of grapes, and each contains 60 AZs (Figure 2). In mammals, such as mice, the NMJ resembles a tangle of worms. Each worm appears to be formed by the fusion of several reptilian boutons, and
indeed, during development, the not-yet-fused individual boutons are evident. Interestingly, human NMJs retain separate boutons to some extent and thereby resemble those of reptiles more than rodents! Thus the NMJs of different species are homologous, with reptilian (and human) NMJs most resembling the discrete bouton synapses found in the mammalian central nervous system. Anatomical Correlates of Synaptic Strength
The strength, or efficacy of a motor terminal (amount of transmitter released) is reflected in its size, which in turn is matched to the requirements of its target muscle fiber. Thus larger fibers receive larger terminals. So do faster ones, such as those involved in fright-orflight response. These are important for survival and therefore receive more transmitter than is minimally required for initiating contraction (the safety factor). Conversely, smaller fibers, as well as slow, chronically used muscle fibers that are relatively unimportant to acute survival, receive smaller terminals (e.g., eye muscles, anal sphincter). In the snake and probably other species as well, postsynaptic receptors are more efficient in fibers that are larger, faster, or more important for survival. Thus nerve terminal size is not the only determinant of synaptic strength. Boutons in small and large terminals are roughly the same size – it is the number of boutons that changes. The Active Zone
In the 1950s, when Sir Bernard Katz and his colleagues elucidated the basis of synaptic function at the frog NMJ, transmission electron microscopy had recently become available to provide images of
Schwann cell
M
SVs
Fold 500 nm AZ Figure 1 Cross section of typical presynaptic bouton. (Left) Diagrammatic representation showing bouton (yellow) capped by Schwann cell (purple) and innervating the postsynaptic membrane (endplate) of muscle fiber (blue). Mitochondria (M; green) reside toward the back of the bouton, away from the presynaptic membrane. Synaptic vesicles (SVs) are clustered throughout the bouton, but particularly near active zones (AZ; blue). Deep invaginations, or folds, within the postsynaptic muscle membrane precisely appose AZs. (Right) Electron micrograph thin section of a bouton at the snake neuromuscular junction. Contractile proteins in the muscle fiber (seen along the bottom of the micrograph) appear in cross section. Folds are continuous along the postsynaptic surface with active zones spaced along them (see Figure 2). Thus some (or all) folds may lack active zones in a particular electron micrograph thin section.
160 Presynaptic Events in Neuromuscular Transmission
10 µm
Figure 2 Light-level image of snake neuromuscular junction from the single-fiber-thick transversus abdominis muscle. The postsynaptic membrane of the muscle fiber has been stained with a lectin (fluorescein–Vicia villosa agglutinin; green) to reveal the location of acetylcholine receptors (AChRs). Each concave, bowl-like cluster of AChRs corresponds to the dimpling of the endplate membrane where it contacts boutons (blue in diagram of Figure 1). The repeated stripes within each bowl, resembling fingerprints, are postsynaptic folds where AChR density is highest. Boutons are labeled with sulforhodamine 101 (red), a fluorescent marker of endocytosed structures (see Figure 6). Larger structures are endosomes (arrows); small dots are vesicle clusters near active zones. Particularly near the edges of boutons, it can be appreciated that the clusters appose folds. Confocal 3-D reconstruction from 115 image planes.
synapses at high magnification. The numerous small (50 nm) vesicles that filled boutons were thought to be the anatomical correlate of the singular electrical events, or quanta, observed with intracellular electrophysiological recording. Similarly, electron-dense structures located in the presynaptic membrane, precisely opposite infoldings of the postsynaptic membrane and often surrounded by vesicles, were thought to be likely sites of transmitter release. Two more recent advances in electron microscopy (EM) – the freeze-fracture technique used by J Heuser and colleagues and electron tomography used by UJ McMahan and colleagues – have provided additional detail about
the structure of AZs (Figure 3). Freeze-fracture EM replicas reveal that AZs contain arrays of large intramembraneous particles that are thought to be calcium channels, or calcium-dependent potassium channels, or both. The arrangement of particles is in striking quadruple or double rows of various lengths. Associated with the particles are rows of vesicles are evidently captured or docked, awaiting release. The specific arrangement and number of vesicles docked at the AZ (4–40, the latter in frog) depend not only on species (frog, lizard, rat, etc.) but also on fiber type (twitch, tonic) within a particular species. Tomography has revealed further structural components, named pegs, ribs, and beams, whose functions are still unknown. This technique promises to relate high-resolution anatomical images of AZs with structures (e.g., the SNARE complex, so named for the soluble NSF (Nethylmaleimide-sensitive factor) attachment receptor) conceived from the molecular biological study of transmitter release. Eventually, molecules known to participate in docking vesicles for release at AZs will be visible, along with a detailed view of the vesicles. EM provides only a static picture of the AZ and associated vesicles, but (as discussed later), vesicle release is a transient phenomenon. An ingenious technique – rapid, or slam, freezing – was developed by Heuser in an attempt to overcome the static nature of EM. In lieu of chemical fixation, living tissue is instantaneously frozen by slamming it against a cold metal block. By stimulating a frog NMJ at the time of freezing, Heuser hoped to capture a dynamic snapshot of vesicles just as they were released from AZs. This proved unsuccessful, presumably because two very brief yet independent transient events, vesicle release and the freezing of the NMJ, were unlikely to coincide. Only by utilizing a chemical agent to prolong and enhance transmitter release was it possible to capture such a snapshot, albeit under nonphysiological conditions. Although long suspected, actual proof that AZs release transmitter under normal conditions came only recently. Zefirov and colleagues, using three recording electrodes to triangulate the source of synaptic currents at an active frog NMJ, found that the loci of all such currents (spontaneous and evoked) correspond precisely to the anatomical positions of AZs.
Classical (Katz) Theory of Transmitter Release The standard model of transmitter release is based on the work of Katz and his colleagues, mainly over the period 1950–80 that followed the introduction of intracellular recording in 1949. In a series of experiments, they established the following events:
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1. Activation of frog motor terminals by nerve stimulation produced potentials that could be recorded from muscle fibers near the endplate (i.e., endplate potentials, or EPPs). 2. Depolarizing electrical events (miniature endplate potentials, or mEPPs) occurred spontaneously at endplates; mEPPs (0.5 mV) resembled EPPs but were smaller. 3. The amplitude of EPPs evoked by nerve stimulation varied randomly, and the occurrence of spontaneous mEPPs was random in time. 4. The concentration of Ca2þ in the bath determined the range of EPP amplitudes: EPPs were absent when Ca2þ was zero, fluctuated among amplitudes corresponding to 0, 1, 2, 3, 4, etc. mEPPs when Ca2þ was low (0.2 mM), and rose to 20–40 mV at physiological bath Ca2þ levels (2–3 mM). These experiments, together with subsequent ones by AR Martin (who had been Katz’s student), resulted in a series of hypotheses that comprise the classical theory. The Quantal Hypothesis
c
Beams Ribs Pegs Channels
d
Figure 3 Electron microscopic visualization of active zones at the vertebrate neuromuscular junction. (a) Transmission electron micrograph of thin (70 nm) sections of snake neuromuscular junction reveals electron-dense structures (active zones; arrows) surrounded by vesicles. The active zones are within the bouton (note vesicles and mitochondria (M)) and are associated with its presynaptic membrane. Each active zone precisely apposes a fold in the postsynaptic membrane of the muscle fiber. (b) Freeze-fracture electron microscopy separates regions of the presynaptic lipid bilayer membrane into outer and inner leaflets, thereby allowing imaging of intramembraneous proteins. Shown are several active zones (blue) within the presynaptic membrane of a bouton at the snake neuromuscular junction. The orientation is as if the bouton in micrograph a were viewed looking in the direction of the arrows. Each active zone appears as two double rows of particles (Ca2þ channels; rows are colored blue). One active zone (arrow) is shown at higher magnification in the insert (upper right; scale bar applies to both micrographs (a) and (b). (c) Electron tomography produces high-resolution surface models; shown is a portion of one active zone at the frog neuromuscular junction. The upper image shows docked synaptic vesicles (dark blue), presynaptic membrane (pale blue), and active zone material (gold). The active zone material is composed of a network of filamentous macromolecules, many of which contact the docked vesicles and presynaptic membrane. The lower image is of the superficial 15 nm of active zone material, showing beams (arrows), which lie in the midline of the active zone material, and ribs, which extend from the beams to the docked vesicles. (d) Schematic of the frog active zone derived from electron tomography and freeze-fracture electron microscopy. Shown is the
Katz’s observations indicated that motor terminals secrete relatively uniform packages of ACh called quanta. Individual quanta are released randomly, and create mEPPs. Classical experiments conducted by Kuffler and Yoshikami, using the snake NMJ, demonstrated that one quantum does not correspond to a single ACh molecule but rather to 5–10 thousand molecules, synchronously released. This number is consistent with what might be packed into a single vesicle, furthering evidence that the quantum and the synaptic vesicle are one and the same. With nerve stimulation, several quanta are released nearly simultaneously, so that the postsynaptic currents they cause add together into a summed endplate current (the EPC). The EPC, in turn, develops a potential, the EPP, across the muscle fiber’s membrane resistance (Ohm’s law). To activate the muscle fiber, some
presynaptic membrane (pale blue-gray), docked synaptic vesicles (darker blue), and the superficial 15 nm of the active zone material, consisting of beams (brown gold), ribs (yellow-gold), and pegs (orange-gold). Positions of macromolecules, which include calcium channels, in the presynaptic membrane are shown in green (compare to (b)). (a, b) Image provided by J Heuser. (c) Reprinted by permission from MacMillan Publishers Ltd: Nature, Vol. 409, Harlow ML, Ress D, Stoschek A, et al. The architecture of active zone material at the frogs neuromuscular junction, 479–484, Copyright 2007. (d) Reproduced from Ress DB, Harlow ML, Marshall RM, et al., (2004) Methods for generating high resolution structural models from electron microscope tomography data. Structure 12: 1763–1774, with permission from Elsevier.
162 Presynaptic Events in Neuromuscular Transmission
minimum number of quanta is required, either to directly activate contraction in tonic fibers (which we do not consider further) or to drive the fiber’s membrane well above the threshold for action potential initiation (twitch fibers). The actual number of quanta released with each stimulus is in principle random, but it varies little and is statistically quite unlikely to be below threshold (see the section titled ‘Binomial statistics with normal extracellular calcium’). The Calcium Hypothesis
Rise in intracellular Ca2þ is now well understood as a ubiquitous signaling mechanism in animals. Calcium entry (or its release from intracellular stores) triggers hormone secretion, muscle contraction, and fertilization of eggs, as well as transmitter release. Thus Katz’s fundamental observation regarding the dependence of transmitter release on calcium entry into the nerve terminal was seminal far beyond synaptic physiology. At the NMJ, entry of Ca2þ via voltagedependent Ca2þ channels causes a transient increase in the intracellular level of Ca2þ in the bouton, particularly near AZs where the channels are heavily concentrated. The increase triggers the fusion with the plasma membrane of some of the vesicles already poised for release at the AZ. With the vesicle lumens now continuous with the extracellular face of the plasma membrane, their vesicular contents, including ACh, diffuse into the synaptic cleft. In motor terminals, probability of release is a steep (fourth power) function of intracellular Rest
5 Hz stim.
Ca2þ. At rest, low levels of Ca2þ (40 nM) are associated with the release of an occasional vesicle every second or so (mEPPs) from an entire terminal. There is some evidence that mEPPs may serve a trophic or tropic function, or they may simply represent the inability of the system to turn off release completely. With the arrival of an AP from the motor neuron, the voltage-dependent channels open, and extracellular Ca2þ flows passively into the terminal (down both concentration and electrical gradients). In the snake, intracellular levels rise to 300 nM (Figure 4), and hot spots near AZs are probably at micromolar concentrations. Just as rapidly, buffers within the terminal (including mitochondria) return Ca2þ levels to near the resting level. The result is a rapid, transient increase in release probability. A terminal (containing, say, 3000 AZs) releases 50–100 vesicles in the 1 ms during which Ca2þ was elevated, rather than a single vesicle per second – a rise in release probability by a factor of 105! Poisson Statistics with Low Extracellular Calcium
Lowering bath Ca2þ by a factor of 5–10, and/or increasing the concentration of magnesium or other divalent cations that compete with it, will decrease the activity of extracellular Ca2þ and decrease the gradients that support its entry. Instead of releasing 50–100 vesicles per AP, a terminal releases a much smaller number. Katz estimated this number by dividing the EPP amplitudes he observed under these conditions by the mean amplitude of spontaneous mEPPs. The number fluctuated dramatically! For Recovery nM 50 100 150 200 250 300
10 µm Figure 4 Intracellular Ca2þ transient in a snake motor terminal during a 5 Hz, 30 s stimulation. The Ca2þ-sensitive dye fura-2 was loaded into the muscle nerve to permit measurement of bulk intrabouton Ca2þ concentration. Stimulation increased Ca2þ level from 50 nM at rest (left) to 300 nM during activity (center). Levels remained elevated for 30 s after the end of stimulation, after which they rapidly returned to baseline (right).
Presynaptic Events in Neuromuscular Transmission 163
example, a terminal might release on average 5 quanta per stimulus (called the mean quantal content, m). But the actual number varies about that mean with each AP delivered – 8, 4, 1, 7, 0, 10, etc. Katz, and later Martin, showed that the EPPs generated by frog motor terminals with low bath calcium obeyed Poisson statistics. Poisson’s formulation allows one to assess the probability of transmitter release, p, by recording the distribution of EPP amplitudes after many trials. It was found that the distribution fit Poisson’s theory (meaning that quantal events appeared to be independent and therefore randomly distributed about their mean), p was low (0.01), and p increased with increasing bath Ca2þ. Binomial Statistics with Normal Extracellular Calcium
Poisson’s statistical treatment is applicable only for probabilities below 0.02, which was indeed the case in early experiments where bath Ca2þ was deliberately lowered. The more general statistical model of random two-state phenomena (i.e., a vesicle is either released with the arrival of an AP or it is not) is binomial statistics, which must be used under physiological conditions where p is known to be higher (normal bath Ca2þ). Unfortunately, p is not as easily measurable as it is with Poisson statistics. The binomial distribution depends on p plus a second parameter, n, the binomial number of release sites. For binomial statistics to apply, the release sites must be presumed independent; that is, release from one must not influence release from another. The mean quantal content is m ¼ np. Events are said to be simply binomially distributed (and therefore truly random, or independent) if and only if their fluctuations about the mean, m, correspond to those predicted by a particular pair of n and p. The expected fluctuations are given by Nx ¼ Ntot n!px ð1 pÞnx =ðn xÞ!x! where Ntot is the number of stimuli delivered and Nx is the number of responses having the amplitude x ¼ 0, 1, 2, . . ., n quanta (the symbol ! means factorial). For example, if p is 1 (certainty), then each of n sites releases one vesicle and m ¼ n; there are no fluctuations. If p is 1/2 (the same probability as that of obtaining heads in a coin flip), then m ¼ n/2. If a bouton contained, say, four binomial release sites and p were 1/2, on average m ¼ 2 vesicles would be released per stimulus, just as flipping four coins at once would on average produce two heads per flip. But how often would one expect to see exactly two heads? Our equation answers that question, giving the number of trials for which one would expect
0 (all tails), 1, 2, 3, or 4 (all) heads (1, 4, 6, 4, and 1 out of 16 trials, respectively). Note that release of the mean number of quanta (2) is the most probable, but this particular outcome is expected in only 6 of 16, or 38% of the trials. There are several other general features of binomial statistics. Fluctuations about the mean increase with decreasing p, as can be inferred from the preceding comments. The variance (standard deviation squared) is given by np(1 p). Variance is zero if p is 1 and increases until, with p < 0.02, it approaches equaling the mean, m (as it does with Poisson statistics). Fluctuations also depend upon n, but in a different way. If 1000 coins are flipped simultaneously, it is unlikely that exactly 500 will be heads, just as in the preceding example (500 is the most likely single outcome, but there are very many other possibilities). However, it is also unlikely that the distribution will deviate far from the mean. Fluctuations over the range from 480 to 520 heads are feasible, but the probability of 499 heads and only one tail would be akin to winning the lottery. The properties of the parameters p and n as discussed here make difficult the statistical analysis of quantal release at NMJs under physiological conditions. Quantal fluctuations are quite small in normal bath calcium, presumably because n is so large. EPP fluctuations due to thermal ‘noise,’ change in membrane properties, etc. mask EPP fluctuations due to varying quantal release. One way to overcome this difficulty, although it is not completely satisfactory, is to attempt to isolate small portions of the NMJ in order to lower n. Because snake NMJs comprise 60 discrete boutons, about 2% of the nerve terminal is studied by activating a single isolated bouton. At this small synapse, quantal fluctuations are indeed evident in normal Ca2þ bath (Figure 5), and binomial analysis suggests that p is 0.4 and n is 4. Mean quantal content, m, is 1.6 vesicles per bouton. These data are for low-frequency (1–5 Hz) continuous stimulation; m is often higher for the first stimulus or two after rest, and lower after sustained stimulation at high frequency. In other NMJ preparations (human, frog), p has usually been reported in the range from 0.1 to 0.3, measured or estimated using a variety of techniques. The Binomial n and the Countable Number of AZs
If the Katz theory is correct, the binomial n should have an anatomical correlate corresponding to the number of vesicles that are independently available for release upon arrival of an AP. EPPs are generally sufficient to depolarize the muscle fiber above threshold with a safety factor of 2–4, meaning that m is 100. Thus, with p in the range from 0.1 to 0.4,
164 Presynaptic Events in Neuromuscular Transmission Factors Underlying the Binomial p 1 mV 5 ms
Figure 5 Quantal fluctuations recorded from a snake motor bouton in normal Ca2þ concentration bath. Endplate potentials were recorded intracellularly from a muscle fiber endplate, the entire nerve terminal of which, save a single bouton, was removed. Shown superimposed are the responses to five stimuli (negativegoing artifact at left) delivered at 1 s intervals. Either zero, 1–2, or 2–4 quanta were released. Two spontaneous miniature endplate potentials (arrows) are contained in the records, showing approximate quantal size.
n is 250–1000. In contrast, by direct count, an average snake terminal comprising 60 boutons contains 3600 AZs. A similar number of AZs is present at the frog NMJ, and probably at other NMJs as well. These rough estimates ignore important systematic variations among NMJs of various species, and among different types of muscle within one animal. Nevertheless, it seems likely that N, the anatomical number of AZs, exceeds n, the number of release sites calculated from binomial statistics, perhaps by as much as an order of magnitude. Moreover, as already discussed, each AZ contains two or more apparently releasable vesicles. If each such vesicle acts independently (which is unknown), N is still larger. There is at present no conclusive explanation for the disparity between n and N, although several models have been proposed. One is that some AZs have higher p than others, and thus form a functional pool of release sites whose number approximates n, not N. Presumably, these AZs are employed under conditions of normal use, with the others awaiting recruitment when needed. A second model is that the mEPP, or quantum, actually comprises several vesicles that are synchronously released. Thus the anatomical correlate of n is not one AZ, but rather several AZs. A third explanation is that transmitter release viewed at the level of single boutons, not whole NMJs, is partly deterministic, not random. Put another way, the AZs within one bouton act cooperatively, not independently. For example, an AZ that has recently released a vesicle may wait its turn before it releases again. Any sort of systematic cooperation among AZs would reduce the observed variance, and, consequently, the calculated n. There is currently some support, but no conclusive evidence, for each of these models.
The dependence of p on Ca2þ levels indicates that one contributing factor is the opening probability of Ca2þ channels and Kþ channels associated with the AZ. This probability could change dynamically, influencing release from particular AZs. A second factor, operating on a slower time scale, might originate in the postsynapse. Retrograde feedback from the effect of activating receptors apposing an AZ could regulate that AZ’s efficacy. Alternatively, vesicle membrane proteins might regulate an AZ once the proteins are inserted into the plasma membrane – signaling that transmitter release from that AZ has occurred. Two vesicle proteins, SV2 and synaptotagmin, have already been found to interact with other proteins within the synaptic cleft. All of the preceding mechanisms have the potential to add a deterministic component to transmitter release, thereby explaining the disparity between n and N. Both p and n vary systematically with various paradigms of use, although the mechanisms are not fully understood. A recent finding is that use regulates release by influencing the rate at which vesicles are made available for re-release. Also, the response of NMJs to various paradigms of use is highly dependent on species and type of innervated muscle. Of the many factors involved, some potentiate m, while others depress it; the behavior of a particular synapse is the algebraic sum of many genetic and usedependent influences. Asynchronous Transmitter Release
Ca2þ levels, and consequently the probability of vesicle fusion and transmitter release, increase dramatically when a terminal is invaded by an AP, as already discussed. But intracellular Ca2þ does not return to resting levels immediately. When several APs arrive in rapid sequence, residual elevated Ca2þ in the terminal can increase p between APs (as shown in Figure 4) and for a period (seconds to minutes) after the train is completed. Among the consequences is an increase in mEPP frequency. This release is in addition to that ascribed to the quantal content, m. It is evoked, but not synchronized, with the stimulus. At NMJs, the role of asynchronous release is unknown. At certain other synapses, it may be the dominant form of release under certain conditions.
Vesicle Processing The number of vesicles stored within a typical motor terminal is in the range from 105 to 106. There is evidence that not all are releasable; for example, measurements in snake set the releasable number at
Presynaptic Events in Neuromuscular Transmission 165
130 000 (the functional releasable pool). As mentioned previously, motor terminals must rely on their own machinery to remake spent vesicles. The process includes reuptake of vesicular membrane, formation of new vesicles, separation of vesicle proteins from those endogenous to the plasma membrane, reintroduction of these proteins into the vesicular membrane, reuptake of choline (ACh is hydrolyzed into choline and acetate), synthesis of new ACh, packaging of ACh (and other molecules) into vesicles, and, finally, mobilization of reformed vesicles into the releasable pool. Reuptake of Vesicular Membrane by Endocytosis
Endocytosis is used by cells for many purposes – for example, to sample and obtain nutrients from the extracellular milieu. There are several variants, including bulk membrane invagination (phagocytosis and pinocytosis, which mean, respectively, to eat and to drink the contents of the extracellular fluid) and clathrin-mediated endocytosis (CME), which is often associated with specific receptors. At NMJs, both bulk membrane invagination (which we call macroendocytosis) and CME are utilized. The function of endocytosis at nerve terminals (and other secretory cells) is unique, however, and is called compensatory – compensating for vesicular exocytosis and thereby maintaining a relatively constant plasma membrane area. Without endocytosis (as, e.g., in muscles treated with black widow spider venom, nerve terminals swell and are no longer congruent with the postsynaptic membrane. Methods for Observing Endocytosis
Compensatory endocytosis was documented in 1973 at the frog NMJ by two independent groups, led by J Heuser, and B Ceccarelli. Both groups utilized transmission EM and a bath-applied marker (horseradish peroxidase or dextran) which is endocytosed and thereby labels the internalized endocytic structures (see Figure 6). Heuser observed CME (evidenced by a clathrin coat surrounding endocytosed vesicles loaded with horseradish peroxidase) at some distance from AZs, while Ceccarelli observed virtually the opposite: labeled vesicles located near AZs, and lacking clathrin coats. Endosomes (called cisternae), now known to be the products of macroendocytosis (ME), were also labeled. These were initially thought to be formed by fusion of vesicles internalized by CME, just as similar endosomes are formed in nonneuronal cells. An optical method for visualizing endocytosis at NMJs, particularly in living terminals, emerged with the development of sensitive fluorescent endocytic
probes (see Figure 7) by Betz and colleagues in 1992. Use of optical probes, and other recent techniques, has confirmed Heuser’s observations and possibly Ceccarelli’s as well. The current model of endocytosis (Figure 6) includes three different endocytic mechanisms. The first is consistent with Ceccarelli’s model and is called kiss-and-run transmitter release – the brief fusion of a vesicle with the plasma membrane to release its contents, followed by immediate withdrawal. This occurs at the AZ, and clathrin is not required. Kiss-and-run has been observed in central synapses but remains putative at vertebrate NMJs. The second is CME (Heuser’s model), which can occur, depending on the species, either near or away from the AZ. The third is ME, which can also occur both near and away from the AZ. Macroendocytosis directly produces most, if not all, of the endosomes, that were first thought to be created by vesicle fusion. These endosomes in turn dissipate, via a clathrin-dependent process, into vesicles. While the three processes may differ in speed, location, and other details, their product is the same: vesicles of the proper size (50 nm) for refilling with transmitter. Refilling of Vesicles for Reuse
An enzyme within the nerve terminal, choline acetyltransferase (ChAT), resynthesizes ACh from choline that has been taken up from the cleft. Loading of vesicles involves acidification of the vesicle’s lumen via a proton pump, and use of the resulting proton gradient to drive a proton–ACh exchanger, or transporter. Both the pump and the ACh transporter are among the proteins resident in the vesicle membrane. The time required for refilling is 1 min or more in snake (at room temperature), and probably much faster in mammals. Mobilization of Reformed Vesicles to AZs
Pathways for recycling vesicles have been studied extensively at the frog and snake NMJ, each providing a different model. In frog, there are two distinct recycling strategies, which are differentially recruited according to levels of use. Each strategy employs its own vesicle pool, either a small (local) pool associated with a particular AZ, or a global (reserve) pool that occupies much of the bouton (see Figure 6). The first strategy, associated with low levels of use (e.g., brief low-frequency stimulation), comprises exocytosis at the AZ, endocytosis near the AZ (either CME or putative kiss-and-run), mobilization of spent vesicles into the local pool (which is not anatomically distinct, but distributed among vesicles of the reserve pool), and return of the same vesicles to the AZ for preferential reuse. Thus a particular vesicle is locally
166 Presynaptic Events in Neuromuscular Transmission
200 nm a
b
c
RES
RR
AZ3 d
AZ1
AZ2
Figure 6 Current models of vesicle processing at the vertebrate neuromuscular junction. (a–c) Electron micrographs of snake boutons, illustrating structures common to all models. Endocytosed structures are labeled with horseradish peroxidase (a) or photoconverted fluorescent endocytic probe FM1-43 (b, c). Fuzzy clathrin coats are visible on vesicles that have been recently endocytosed by clathrinmediated endocytosis (white arrowheads in (b) and (c) as well as on those still continuous with the presynaptic membrane (yellow arrowheads). A large, recently internalized endosome in a has budded several vesicles, some still clathrin coated (white arrowhead in (a). In (b), note the active zone (white arrow), local clathrin-mediated endocytosis (labeled, coated vesicle just left of the active zone), and macroendocytosis (deep membrane invagination already in the process of budding; red arrowhead). (d) Diagram depicting three types of endocytosis. On the left, macroendocytosis and clathrin-mediated endocytosis occur away from active zone 1 (AZ1); vesicles budded from the membrane, and from the endosome, enter the reserve pool (RES), which then repopulates the readily releasable (RR) pool near AZ1. In the center, clathrin-mediated endocytosis occurs locally near AZ2, and reformed vesicles immediately repopulate the RR pool of AZ2. On the right, a vesicle undergoes partial membrane fusion at AZ3, then returns to the RR pool of AZ3 via kiss-and-run transmitter release.
exo- and endocytosed repeatedly, without involvement of the reserve pool, and without involvement of plasma membrane distal to the AZ. The second strategy, associated with high levels of use (e.g., prolonged high-frequency stimulation), comprises exocytosis at the AZ but endocytosis some distance away, followed by mobilization of the vesicle to the reserve pool, where it remains for some time before it makes its way to the AZ for reuse (possibly via a myosin– actin motor). Endocytosis in this case is via ME (followed by budding of vesicles from the endosome) and probably CME directly from the membrane as well. In effect, mechanisms very similar to those proposed early on by Heuser and Ceccarelli are both used, albeit under different conditions. This two-tiered system of
recycling has been observed at other synapses – for example, the glutamatergic NMJs of most Drosophila muscles. In contrast, snake NMJs utilize the same endocytic strategies as in frog (CME, particularly near the AZ, and ME), but both operate concomitantly at high and low levels of use. Moreover, both strategies supply vesicles exclusively to the reserve pool; local recycling has not been observed at the snake NMJ. Vesicle Processing as a Presynaptic Determinant of Synaptic Strength
Endocytosis, filling with transmitter, and mobilization of vesicles to release sites require time and energy (hydrolysis of ATP or GTP). Consequently, these
Presynaptic Events in Neuromuscular Transmission 167
Figure 7 Snake motor nerve terminals stained with the fluorescent endocytic probe FM1-43. (Left) Typical twitch fiber terminal. (Right) Terminal from same animal treated with black widow spider venom, which inhibits compensatory endocytosis. Note severe swelling of boutons.
processing steps have the potential to limit the availability of reformed vesicles for release, particularly with high levels of use. Recent studies indicate that this is indeed the case. Thus m, the mean quantal content, depends not only on the exocytotic mechanisms discussed herein but on recycling as well. Once viewed as simple housekeeping, vesicle processing can be rate limiting for transmitter release. Vesicle processing is therefore a subject of considerable research interest, particularly as a potential new substrate for the pharmacological regulation of synaptic strength. See also: Neuromuscular Connections: Vertebrate Patterns of; Neuromuscular Junction (NMJ): Postsynaptic Events in Neuromuscular Transmission; Neuromuscular Junction (NMJ): Presynaptic Short-Term Plasticity of Neuromuscular Transmission.
Further Reading Bennett MR and Kearns JL (2000) Statistics of quantal release at nerve terminals. Progress in Neurobiology 60: 546–606. del Castillo J and Katz B (1954) Quantal components of the endplate potential. Journal of Physiology 124: 560–573. Fatt P and Katz B (1951) An analysis of the end-plate potential recorded with an intra-cellular electrode. Journal of Physiology 115: 320–370. Harlow ML, Ress D, Stoschek A, et al. (2001) The architecture of active zone material at the frog’s neuromuscular junction. Nature 409: 479–484. Martin AR (1955) A further study of the statistical composition of the end-plate potential. Journal of Physiology 130: 114–122. Rizzoli SO and Betz WJ (2005) Synaptic vesicle pools. Nature Reviews Neuroscience 6: 57–69. Royle SJ and Lagnado L (2003) Endocytosis at the synaptic terminal. Journal of Physiology 553: 345–355. Zefirov AL, Benish T, Fatkullin N, et al. (1995) Localization of active zones. Nature 376: 393–394.
Neuromuscular Junction (NMJ): Presynaptic Short-Term Plasticity of Neuromuscular Transmission A M Holohean and K L Magleby, University of Miami School of Medicine, Miami, FL, USA ã 2009 Elsevier Ltd. All rights reserved.
Introduction Short-term stimulation-induced changes in transmitter release have been characterized in terms of various underlying components. Facilitation, augmentation, and potentiation are components of increased transmitter release that differ from one another on the basis of their kinetic properties and time courses of decay. Depression is a component of decreased transmitter release. The properties and possible mechanisms of these components of variable synaptic efficacy are described in the following sections.
Techniques of Study Variable synaptic efficacy has been studied extensively at many different synapses, including the neuromuscular junction. The frog neuromuscular junction will form the basis for much of the material presented herein. In a typical experiment the sartorius muscle is placed in a small chamber and bathed with a simple salt solution containing 114 mM NaCl, 2 mM KCl, 0.1–2 mM CaCl2, 0–15 mM MgCl2, and a pH buffer. The motor nerve leading to the muscle is stimulated with a train of impulses, and the resulting change in transmitter release after each impulse is determined from changes in the amplitude of successive postsynaptic responses. Postsynaptic responses are determined either from endplate potential (EPP) amplitudes measured using a microelectrode placed in the muscle cell at the region of an endplate, or by measuring the current that flows through the endplate (endplate current) using a two-microelectrode voltage clamp. For experimental conditions in which the EPP amplitudes exceed threshold for generation of action potentials in the muscle, the postsynaptic receptors can be partially blocked with tubocurare to prevent the muscle from contracting. Since the purpose of these studies is to examine stimulation-induced changes in synaptic efficacy, the response to the first impulse in the train serves as a control for the changes that occur during and after the train. Sufficient time is allowed between each train of impulses so that the synapse returns to its unstimulated (control) state. Stimulation-induced changes in synaptic efficacy have been found to involve a number of different
168
processes or components. To facilitate the study of the individual components, it is advantageous to modify the external media by reducing or increasing the concentration of Ca2þ or Mg2þ. Reducing Ca2þ or increasing Mg2þ decreases the amount of transmitter released so that depression due to depletion of synaptic vesicles no longer masks the processes that act to increase transmitter release. An example of variable synaptic efficacy under conditions of decreased transmitter release is shown in Figure 1. Transmitter release was decreased by reducing the amount of extracellular Ca2þ available for entry into the nerve terminal and by increasing the amount of extracellular Mg2þ which inhibits Ca2þ entry. Each record presents EPPs recorded during 5 s of repetitive stimulation at the indicated stimulation rate. Because of the slow time base, each EPP appears as a vertical line. The amplitudes of the EPPs, which are a good measure of transmitter release under the conditions of this experiment, increase during the trains, with the greatest increase at the higher stimulation rates. The enhancement in transmitter release shown in Figure 1 is due to an increase in the number of synaptic vesicles that release their contents of transmitter from the presynaptic nerve terminal into the synaptic cleft for each impulse. This increased release under conditions of low Ca2þ can be accounted for in terms of four components: potentiation, augmentation, and the first and second components of facilitation. Each component of increased transmitter release is defined in a similar manner, as the fractional increase in transmitter release over the control level in the absence of the other components, such that yðtÞ ¼ ðEPPðtÞ EPP0 Þ=EPP0
½1
where y(t) is the magnitude of the specified component of increased transmitter release at time t, EPP (t) EPP0 is the increase in EPP amplitude at time t due to the component, and EPP0 is the control EPP amplitude when the component equals zero. The components have been distinguished from one another on the basis of widely separated decay rates and differential sensitivities to Sr2þ and Ba2þ. The time courses of decay of the components are determined by placing test impulses at various intervals after one or more conditioning impulses. Since the decay of each component can be approximated by a single exponential, decay rates are characterized in terms of time constants – the time required to decay to 37% (1/e) of the initial magnitude. The properties of each component are summarized in the following sections.
Neuromuscular Junction (NMJ): Presynaptic Short-Term Plasticity of Neuromuscular Transmission 169
During repetitive stimulation, the facilitation added by each release combines with that remaining from previous impulses, so that facilitation builds up during the train, reaching a steady-state level after approximately 1 s of stimulation. The greater the frequency of stimulation, the greater the combined facilitation. Because of their large magnitudes and rapid time courses of decay, stimulation-induced changes in the two components of facilitation can lead to large and rapid changes in transmitter release following step changes in stimulation rate. Facilitation decays to insignificant levels within about 1 s after a conditioning train ends. Replacing Ca2þ in the bathing solution with Sr2þ leads to a selective increase in the magnitude and time constant of the second component of facilitation.
1 s−1
5 s−1
10 s−1
Augmentation of Transmitter Release Each nerve impulse adds an increment of augmentation which increases transmitter release about 1% above the control level. Augmentation then decays with a time constant of about 7 s. The augmentation added by each impulse can be described by
12.5 s−1
aðtÞ ¼ 0:01 et=7 s Figure 1 Variable synaptic efficacy at the frog sartorius neuromuscular junction under conditions of low levels of transmitter release. Each record presents endplate potentials recorded with a surface electrode when the nerve was stimulated at the indicated rate. Each vertical line represents an endplate potential. The amplitude of the endplate potentials is proportional to the amount of transmitter released by each nerve impulse. Transmitter release increased during the trains, and the increased release was greater for higher stimulation rates. Vertical bar ¼ 0.5 mV; horizontal bar ¼ 0.5 s. Reproduced from Magleby KL (1973) The effect of repetitive stimulation on facilitation of transmitter release at the frog neuromuscular junction. Journal of Physiology (London) 234: 327–352, with permission.
½3
where a(t) is augmentation at time t. The increment of augmentation added by each impulse typically increases above the 0.01 level during trains of several hundred impulses. Because of its slow 7 s decay rate and the progressive increase in the increment of augmentation added by each impulse, augmentation can build up to large levels during several hundred impulses in the absence of depression, acting to increase the transmitter released by each nerve impulse several fold in the absence of depression (see later). Addition of small amounts of Ba2þ to the bathing solution leads to a selective increase in the magnitude of augmentation while having little or no effect on its time constant of decay.
Facilitation of Transmitter Release Facilitation includes two components of increased transmitter release. Each nerve impulse adds a first and second component of facilitation. The first component increases transmitter release about 80% above the control level and then decays with a time constant of about 50 ms. The second component increases release about 12% and then decays with a time constant of about 300 ms. The facilitation added by a single nerve impulse can be described by f ðtÞ ¼ 0:8et=50 ms þ 0:12et=300 ms
½2
where f(t) is the fractional increase in transmitter release at time t following the conditioning impulse.
Potentiation and Posttetanic Potentiation of Transmitter Release Each nerve impulse adds an increment of potentiation that increases transmitter release about 1% above the control level. Potentiation then decays with a time constant that ranges from tens of seconds to minutes. The time constant of decay, which is about 60 s after the end of a 300-impulse conditioning train, increases with the number and frequency of conditioning impulses. Potentiation after a train can develop with a delay if depression (see later) occurs during the conditioning train. Under these conditions potentiation is often called posttetanic potentiation (PTP), because
170 Neuromuscular Junction (NMJ): Presynaptic Short-Term Plasticity of Neuromuscular Transmission
it becomes apparent with a delay after the tetanus due to the slow recovery from the masking depression. The potentiation added by each impulse may be approximated by pðtÞ ¼ 0:01 et=60 s
½4
where p(t) is potentiation at time t. Potentiation builds up during repetitive stimulation, often increasing release for each impulse two times the control level after several hundred impulses.
Relationship between the Components of Increased Transmitter Release Potentiation, augmentation, and the two components of facilitation retain their kinetic properties during and following repetitive stimulation. The relationship between these four components is not entirely clear, but their effects do not add linearly to one another. Instead, there appears to be a multiplicative relationship among the components. This is expressed as EPPðtÞ=EPP0 ¼ ðFðtÞ þ 1ÞðAðtÞ þ 1ÞðPðtÞ þ 1Þ
½5
where EPP(t)/EPP0 is the increase in transmitter release at time t over the control level in the absence of repetitive stimulation, and F(t), A(t), and P(t) are the magnitudes of facilitation, augmentation, and potentiation at time t. The combined properties of the four components of increased transmitter release are sufficient to account for stimulation-induced changes in transmitter release under conditions of low quantal release (e.g., low extracellular Ca2þ). The four components build up during repetitive stimulation and decay thereafter, with their characteristic time constants. Under conditions of low quantal release the combined effects of the four components can increase the transmitter released by each impulse an order of magnitude over the control level after several hundred impulses of repetitive stimulation at 10–20 impulses per second.
Comparison of the Components of Increased Transmitter Release at Three Different Preparations Even though the time constants of decay of the four components of increased transmitter release are temperature dependent in reptiles and mammals, the decay rates of the components in mammalian sympathetic ganglion at 34 C and in fascia dentata of rat hippocampus at 35 C are similar to those stated in the previous sections for the frog neuromuscular junction at 20 C. The general kinetic properties of
facilitation, augmentation and potentiation appear to be similar at frog neuromuscular junction and fascia dentata of rat hippocampus. In contrast, the increments of the second component of facilitation and augmentation added by each impulse in the mammalian sympathetic ganglion are about 6 to 10 times larger than at the frog neuromuscular junction.
Mechanisms for the Components of Increased Transmitter Release Depolarization of the presynaptic nerve terminal by an action potential opens voltage-sensitive channels in the nerve terminal that are permeable to Ca2þ. It is the influx of Ca2þ into the nerve terminal through these Ca2þ channels that leads to evoked transmitter release. Since Ca2þ triggers transmitter release, it might be expected that increases in the amount of Ca2þ in the nerve terminal would lead to increases in transmitter release, and it has been suggested that all four components of increased transmitter release are related to increased Ca2þ in the nerve terminal. Such a hypothesis is attractive since Ca2þ does accumulate in the nerve terminal during repetitive stimulation, and the time course of Ca2þ accumulation and removal can be similar to the combined time courses of the components of increased transmitter release. An increase in intracellular Ca2þ during repetitive stimulation could arise from several different mechanisms, including incomplete removal of the Ca2þ entering through Ca2þ channels with each nerve impulse, saturation of the buffering capacity of the nerve terminal, increased Ca2þ entry through the Ca2þ channels, or release of Ca2þ from intracellular stores. While all of these processes may act to increase Ca2þ under some conditions, facilitation appears to be associated with residual Ca2þ remaining from preceding impulses. Augmentation follows a similar time course as Ca2þ decay in the nerve terminal and therefore may be due to delayed extrusion of residual Ca2þ by pumps such as the Naþ/Ca2þ exchanger, where a reduction in the Naþ gradient due to continual stimulation will slow or even reverse the rate of exchange between Naþ and Ca2þ. Potentiation follows a time course similar to the efflux of the Ca2þ accumulated in the mitochondria during prolonged stimulation. On this basis, the different time courses of the components of increased release may reflect the time course of diffusion, buffering, and multicompartment uptake and extrusion of Ca2þ from the nerve. A residual Ca2þ model for the components of increased transmitter release can give an approximate multiplicative relationship between the components if the residual Ca2þ is expressed as a power, such that the transmitter release is proportional to the third or fourth
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power of Ca2þ acting at one or a combination of proteins involved in release. Additionally, the components of increased Ca2þ release could also reflect the time course of binding and release of Ca2þ from Ca2þ binding sites on proteins involved in synaptic release; an example is synaptotagmin, a vesicle membrane protein which has multiple binding sites for Ca2þ. The differential effects of Ba2þ and Sr2þ could reflect different kinetic properties of these ions on one or more of the multiple proteins involved in vesicle release and/or on the various uptake mechanisms for Ca2þ. There are a number of difficulties with a simple residual (free) Ca2þ hypothesis for stimulation-induced increases in transmitter release. The correlation between free Ca2þ and facilitation does not always follow the expected properties for such a hypothesis, and the expected concentrations of residual free Ca2þ calculated from the observed increases in transmitter release predict that the rate of transmitter release between nerve impulses, measured as the frequency of miniature endplate potentials seen, should be many times higher than is observed. However, release evoked by an action potential and residual release from miniature endplate potentials may involve different release sites/mechanisms, so such correspondence might not be expected, and such calculations typically ignore the complexity of a multistep release process. It has also been proposed that one reason why residual free Ca2þ does not produce the predicted high level of transmitter release is because of a hypothesized voltage component that directly enhances release at the time of the action potential in the presynaptic nerve terminal, although effective release models have been developed without such a voltage-dependent component. Residual Ca2þ could alter a number of factors involved in the transmitter release, giving rise to the components of increased transmitter release. Such factors could include an increase in the number of sites with vesicles available for release and an increase in the probability that a primed site will release the contents of its synaptic vesicle. The mechanism for the multiplicative relationship between the components is less clear, but if release is determined by the joint probability of multiple factors, such as sites and probability, then these factors could have a multiplicative relationship. Release would then be given by the product of these factors. Studies of the mechanism of transmitter release suggest that a number of Ca2þ-dependent steps with different affinities for Ca2þ precede exocytosis. On this basis, the time course of decay of some of the components of increased transmitter release may reflect the time course of Ca2þ unbinding associated
with the various Ca-dependent steps, as well as the time course of removal of Ca2þ. At least nine families of proteins associated with synaptic vesicles and the presynaptic membrane, many of which are expressed in multiple isoforms, have been implicated in the release of transmitter. These proteins are involved in synaptic vesicle turnover, docking of the vesicles at the release sites, priming to enable the vesicles to be released, and the release process itself that leads to the exocytosis of the vesicle contents. When the functions of all these proteins are clearly defined, as well as the properties of the systems involved in the uptake, sequestration, and extrusion of Ca2þ, then the precise molecular mechanisms of facilitation, augmentation, and potentiation may also become clear.
Depression of Transmitter Release Depression is a decrease in postsynaptic potential amplitude due to a decrease in the number of synaptic vesicles that release their contents into the synaptic cleft by each nerve impulse. Depression is typically insignificant under conditions of low levels of transmitter release (low Ca2þ) and becomes more pronounced the greater the level of release. Depression typically develops during prolonged or high-frequency repetitive stimulation under physiological conditions. The magnitude of depression, measured as the fractional decrease in transmitter release, increases with higher stimulation rates or increased duration of stimulation. In physiological Ca2þ, the higher the stimulation rate, the greater the resulting depression of transmitter release. Figure 2 presents an example of depression during repetitive stimulation of the rat diaphragm neuromuscular junction (Figure 2(a)) and the frog sartorius neuromuscular junction (Figure 2(b)) under conditions of normal levels of transmitter release. The postsynaptic response recorded in each case was the endplate current (EPC); inward current through the endplate is plotted downward. Shown in Figure 2(a), EPC amplitudes are successively decreased during a short train of 150 stimuli per second. In Figure 2(b), successive EPC amplitudes first increase and then decrease during a train of 100 stimuli per second. The decrease in amplitude, which results from a decrease in transmitter release, is referred to as depression. Depression is typically superimposed on or masks facilitation, augmentation, and potentiation, which also develop during repetitive stimulation, even under conditions of normal or increased quantal release. The increase in EPC amplitudes in Figure 2(b), before depression becomes prominent, is due to the components of increased transmitter release. Depression is pronounced in Figure 2 over what would be
172 Neuromuscular Junction (NMJ): Presynaptic Short-Term Plasticity of Neuromuscular Transmission
a
some synapses, such as those in fascia dentata of rat hippocampus, depression does not appear to be significant, and facilitation, augmentation, and potentiation dominate the response. On the other hand, depression is more prominent at rat diaphragm neuromuscular junctions, and tends to mask augmentation and the onset of potentiation.
Mechanisms of Depression
b Figure 2 Depression of transmitter release at rat diaphragm (a) and frog sartorius (b) neuromuscular junctions under physiological levels of transmitter release. Each record presents endplate currents recorded under voltage clamp from a neuromuscular junction. The nerve was stimulated at 150 impulses per second in (a) and 100 impulses per second in (b). Inward current through the endplate is plotted downward, and the absolute endplate current amplitude gives a measure of the amount of transmitter released. The decrease in transmitter release that occurs after the first impulses in (a) and after 5 impulses in (b) results from depression. The first endplate current amplitude in (a) is 100 nA and the endplate currents were evoked every 6.67 ms. The first endplate current amplitude in (b) is 140 nA and endplate currents occur every 10 ms. Reproduced from Magleby KL, Pallotta BS, and Terrar DA (1981) The effect of (þ)-tubocurarine on neuromuscular transmission during repetitive stimulation in the rat, mouse, and frog. Journal of Physiology (London) 312: 97–113, with permission.
observed under physiological stimulation rates because of the high stimulation rates used in this experiment. Depression can be divided into two components under physiological levels of transmitter release: a fast component depression that decreases transmitter release about 15% below the control level after a single impulse and recovers with a time constant of about 5 s, and a slow component depression that decreases transmitter release about 0.1% for each impulse and recovers with a time constant of tens of seconds to minutes. The slow component depression is only apparent after prolonged stimulation, and its time course of recovery is slowed as the duration of stimulation increases. At the frog neuromuscular junction, the fast component of depression has been shown to conceal an underlying component of augmentation and the slow component of depression masks the onset of potentiation (posttetanic); the components of augmentation and potentiation increase incrementally during successive impulses of stimuli, but under normal levels of transmitter release the components of depression dominate the response. The relative amount of depression that develops under physiological levels of transmitter release depends on the synapse and stimulation pattern. At
The observation that the magnitude of depression is related to the amount of transmitter released from the nerve terminal has led to the suggestion that depression is due to a depletion of the transmitter immediately available for release. On this basis, the fast component depression might reflect a depletion of the synaptic vesicles at the release sites (the readily releasable pool of vesicles), with a 5 s time constant of recovery reflecting the time required for depleted synaptic vesicles to be replaced from the larger pool of synaptic vesicles in the nerve terminal. A number of factors could give rise to the slow component depression: it may reflect a more general depletion of a larger pool of synaptic vesicles; vesicle release and membrane recycling could decrease the number of functional sites available for transmitter release or decrease their release probability; the coupling between Ca2þ channels and release sites may decrease with prolonged release. The depression associated with prolonged, slow (0.5–10 Hz) nerve stimulation in the frog occurs without changes in the entry of Ca2þ into the nerve terminal. Thus, decreases in Ca2þ entry into the nerve terminal with each impulse do not appear to be the major mechanism for depression in the frog. At some synapses, however, a decrease in Ca2þ entry through Ca2þ channels may also contribute to depression. Mobilization is often used to refer to the replacement of depleted synaptic vesicles. The rate of mobilization may be modified by the level of intracellular Ca2þ, the activation of second messengers acting on the mobilization machinery, the size of the mobilization pool, and also the fractional depletion of vesicles at the release sites. If the mobilization process overshoots the original level, then mobilization might lead to an increase in transmitter release, or, conversely, if mobilization cannot keep up with release, the result would be a depression of transmitter release. Second messengers acting presynaptically may also be involved in depression and recovery from depression. For example, transmitter release at the neuromuscular junction can be inhibited by the ATP that is co-released from the nerve terminal with acetylcholine, and also by adenosine, a by-product of ATP hydrolysis. These agents may act at some presynaptic site to induce
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depression without altering Ca2þ entry into the nerve terminal. It is not known whether ATP or adenosine induces depression by slowing mobilization of transmitter or by acting through some other mechanism.
Developing Models to Describe Mechanisms Consistent with Short-Term Plasticity Given the number of components of increased and decreased transmitter release and their overlapping time constants, together with the large numbers of factors that contribute to transmitter release, such as the numbers of release sites, the fraction of release sites with docked vesicles, the fraction of docked vesicles that are primed for release, the changes in release probability based on Ca2þ concentrations at multiple sites, possible cooperativity of multiple binding sites for Ca2þ on proteins involved in release, the localization of Ca2þ channels and their relationship to release sites, the diffusional microdomains of Ca2þ, the intracellular Ca2þ buffers, the uptake and storage of Ca2þ by the mitochondria, the extrusion of Ca2þ by Naþ/Ca2þ exchangers, and the multiple pools of synaptic vesicles with different rates of replenishment and recycling (just to name some of the factors), models of transmitter release must be formulated and tested in terms of solutions of simultaneous differential equations, as the interactions among the various factors make it difficult if not impossible to study a single factor in isolation. Considerable progress has been made using this approach to develop realistic physical models of short-term synaptic plasticity that include many of the complex features of transmitter release.
Short-Term Synaptic Memory Facilitation, augmentation, potentiation, and depression are different components of short-term synaptic memory (synaptic plasticity). These components act to integrate information about previous synaptic activity over time, and the synapse indicates its memory of the previous activity by releasing a different amount of transmitter to subsequent nerve impulses. This short-term synaptic memory provides a ready means for the nervous system to store information about previous synaptic activity for a period of milliseconds to several minutes. The multiplicative relationship between the components would provide a means for low-frequency input to a synapse to modulate higher frequency input. It is not known, however, to what extent the nervous system makes
functional use of this short-term synaptic memory, or whether the nervous system is designed to operate reliably in spite of it, but such short-term synaptic plasticity is present in the central nervous system and has been shown to have profound effects on the efficacy of synaptic transmission. See also: Presynaptic Events in Neuromuscular Transmission; Synaptic Plasticity: Short-Term Mechanisms.
Further Reading Augustine GJ, Santamaria F, and Tanaka K (2003) Local calcium signaling in neurons. Neuron 40: 331–346. Delaney KR, Zucker RS, and Tank DW (1989) Calcium in motor nerve terminals associated with posttetanic potentiation. Journal of Neuroscience 9: 3558–3567. Gandhi SP and Stevens CF (2003) Three modes of synaptic vesicular recycling revealed by single-vesicle imaging. Nature 423: 607–613. Jackson MB and Chapman ER (2006) Fusion pores and fusion machines in Ca2þ-triggered exocytosis. Annual Review of Biophysics and Biomolecular Structure 35: 135–160. Kalkstein JM and Magleby KL (2004) Augmentation increases vesicular release probability in the presence of masking depression at the frog neuromuscular junction. Journal of Neuroscience 24: 11391–11403. Magleby KL (1973) The effect of repetitive stimulation junction. Journal of Physiology (London) 234: 327–352. Magleby KL (1987) Short-term changes in synaptic efficacy. In: Edelman GM, Gall WE, and Cowan WM (eds.) Synaptic Function, 1st edn., pp. 21–56. New York: Wiley. Magleby KL and Zengel JE (1982) Quantitative description of stimulation-induced changes in transmitter release at the frog neuromuscular junction. Journal of General Physiology 80: 613–628. Magleby KL, Pallota BS, Terrar DA (1981) The effect of (þ)-tubocurarine on neuromuscular transmission during repetitive stimulation in the rat, mouse, and frog. Journal of Physiology (London) 312: 97–113. Millar AG, Zucker RS, Ellis-Davies GCR, et al. (2005) Calcium sensitivity of neurotransmitter release differs at phasic and tonic synapses. Journal of Neuroscience 25: 3113–3125. Rizzoli SO and Betz WJ (2005) Synaptic vesicle pools. Nature Reviews Neuroscience 6: 57–69. Schneggenburger R and Neher E (2005) Presynaptic calcium and control of vesicle fusion. Current Opinion in Neurobiology 15: 266–274. Schneggenburger R and Forsythe ID (2006) The calyx of held. Cell and Tissue Research 326: 311–337. Stevens CF and Wesseling JF (1999) Augmentation is a potentiation of the exocytotic process. Neuron 22: 139–146. Su¨dhof TC and Scheller RH (2004) The synaptic vesicle cycle. Annual Review of Neuroscience 27: 509–547. Van Der Kloot Q and Molgo J (1994) Quantal acetylcholine release at the vertebrate neuromuscular junction. Physiological Reviews 74: 899–991. Von Gersdorff H and Borst JGG (2002) Short-term plasticity at the calyx of held. Nature Reviews 3: 53–64. Zucker RS and Regehr WG (2002) Short-term synaptic plasticity. Annual Review of Physiology 64: 355–405.
Neuromuscular Junction (NMJ): Postsynaptic Basal Lamina M J Werle, University of Kansas Medical Center, Kansas City, KS, USA ã 2009 Elsevier Ltd. All rights reserved.
Introduction The primary function of skeletal muscle is to produce force. This force is controlled by the motor neuron that contacts the cell. This cell-to-cell contact was best described by Ramo´n y Cajal as a ‘protoplasmic kiss.’ The synapse between the motor neuron and the skeletal muscle cell has many names, including motor endplate, neuromuscular synapse, and neuromuscular junction. We will refer to this synapse as the neuromuscular junction. There are three cellular components of the neuromuscular junction – the skeletal muscle cell, the motor neuron terminal (NT), and the terminal Schwann cell (SC) (Figure 1). Surrounding the muscle cell is an extracellular matrix. The extracellular matrix also covers the Schwann cell. The focus of this article is the specialized extracellular matrix that is found between the nerve terminal and the skeletal muscle cell; this specialized extracellular matrix is called the synaptic basal lamina. The synaptic basal lamina contains many molecules that are commonly found in all basal laminae throughout the organism, but it also contains many molecules that are unique to the neuromuscular junction. The extracellular matrix is a vital and regulated extension of the cell. The fact that the extracellular matrix is part of the cell is easily understood by the fact that the matrix attaches to a number of membrane-bound structures. These membrane-bound structures in turn attach to the cytoskeleton of the cell. The matrix thus provides mechanical support for the cell. This is particularly important when considering the fact that the muscle fiber is twitching and moving large distances. The motor neuron is very well attached to the surface of the muscle cell that it is innervating. For example, in Figure 1 the motor nerve terminal innervates the muscle cell seen at the bottom of the image. On top of the motor neuron sits the terminal Schwann cell. On top of the Schwann cell is another muscle cell that is regulated by another motor neuron. Thus, the two muscle cells in this image will twitch and move at different times. The extracellular matrix keeps the motor neuron precisely aligned with the muscle cell it innervates. The precision at which the extracellular matrix aligns the motor neuron and muscle cell is astounding. The active zone is the site on the motor nerve
174
terminal, where the synaptic vesicles fuse with the cell membrane to release the contents of the synaptic vesicle. This active zone is also the site where the voltage-gated calcium channels are concentrated in the membrane of the nerve terminal. The active zones are found precisely opposite the junctional folds in the skeletal muscle cell. The junctional folds are the invaginations in the membrane of the muscle cell. The acetylcholine receptors (AChRs) are concentrated on the crests of the junctional folds, immediately adjacent to the active zones from which ACh is released. The molecules in the synaptic basal lamina lie between these two membranes, and undoubtedly are intimately involved in both the mechanical linking of the two cells, and the cell-to-cell signaling that is essential for maintaining the structure and function of this synapse. To provide a basis for understanding the synaptic basal lamina, in the following sections we first discuss the molecules that are found in all basal lamina. An important point to emphasize is that in a functional sense, the synaptic basal lamina extends across the nerve and muscle cell membranes to make contact with the cytoskeletons of both cells, thus accounting for the strong mechanical coupling between them. After considering all the extracellular molecules, we therefore discuss the multimolecular complexes that are found in the cell membrane and how they, in turn, are attached to the intracellular cytoskeleton. Finally, we discuss how the synaptic basal lamina is altered by the activities of proteases that cleave the matrix components. Altogether, these features reveal that the synaptic basal lamina is a dynamic cellular component that plays a critical role in the structure and function of the synapse.
Synaptic Basal Lamina Components Collagen IV
The type IV collagens are critical components of basal laminae throughout the organism, and particularly of the synaptic basal lamina. There are features of collagen IV that are common to all basal laminae, and also specializations that are unique to the synaptic basal lamina. The a chains of collagen IV have a long collagenous tail that has a glycine at every third amino acid. Characteristic of other collagen proteins there is a noncollagenous domain at the C-terminus; this region is called the NC1 domain. There are six genes that code for collagen IV a chains (COL4A1 to COL4A6). These a chains can form either homo- or heterotrimers.
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Figure 1 Electron micrograph of a neuromuscular junction from a mouse diaphragm. The left panel is an electron micrograph of a normal mouse neuromuscular junction. The right panel is the same image with the structures colorized and labeled. There are two muscle cells (red and light red). The muscle cell on the bottom is the cell that is innervated by the nerve terminal (green; NT). There is a separate muscle cell that sits on top of the nerve terminal; on top of the nerve terminal is a Schwann cell (yellow; SC). Also seen are the subsynaptic nucleus of the muscle cell (dark red; N) and the mitochondria (blue) in the muscles and in the nerve terminal. Also within the nerve terminal are clusters of synaptic vesicles (dark green). These synaptic vesicles are focused on the active zones (dark black). The active zones are found opposite the mouths of the junctional folds (JF). The synaptic basal lamina (SBL; red) is found in the area between the nerve terminal and the muscle cell on the bottom.
There could therefore be a large number of different combinations, but the only identified groupings are (two a1 and one a2); (a3, a, a5), and (two a5 and one a6). a1 and a2 are found everywhere in the basal laminae, but a3, a4, a5, and a6 are concentrated at the synaptic basal lamina. The type IV collagens form a meshwork that is critical to the structure of the basal lamina. Three individual collagen IV a chains will combine to form a flexible trimer. The trimer is organized such that the NC1 domains are bundled together at one end, and the N-terminal 7S domains are bundled at the other end. These collagen IV trimers can further associate into larger structures. The N-terminal 7S domains from four trimers will bind to form an X-shaped structure, with the NC1 domains pointed outward (Figure 2). The NC1 domains on collagen IV trimers can then bind head to head with the NC1 domains on a separate collagen IV trimer. The resulting structure is a wellorganized and regular meshwork of collagen IV. The type IV collagens can also associate with other components of the synaptic basal lamina, and with proteins on the surface of the muscle, nerve terminal, and Schwann cell. In the synaptic basal lamina, the most common binding partners are the nidogens, which link the collagen IV meshwork to the laminins. On the cell surfaces, the NC1 domains can interact with integrins, proteoglycans, and the dystroglycan complex. Matrix metalloproteinases can cleave the type IV collagen, and this cleavage results in the release of the NC1 domains. The soluble NC1 domains are known to have potent effects on vascular
Figure 2 Highly schematic diagram of the type IV collagen meshwork in the basal lamina. Each collagen IV protein has an NC1 domain that is depicted as an oval, and a long collagenous tail. Three subunits assemble into a trimer, with the NC1 domains grouped together, and the collagenous tails intertwined. The NC1 domains from two trimers will interact head to head, and the 7S domains from four trimers will interact to form an X-shaped structure. The individual collagen IV proteins are depicted as either red, green, or blue. This diagram is overly simplified to show a single sheet of the collagen IV matrix.
formation, but the role of the NC1 domain at the neuromuscular junction is still largely unknown. Laminins
Laminins are heterotrimeric glycoprotein structures. Each laminin is composed of one a subunit, one b subunit, and one g subunit. There are five genes
176 Neuromuscular Junction (NMJ): Postsynaptic Basal Lamina
that code for a subunits, four genes that code for b subunits, and three genes that code for g subunits. Of the myriad of possible combinations, currently there are 15 identified laminins. The laminins are important components of basal laminae throughout the organism, and play a special role at the neuromuscular junction. The skeletal muscle cell produces the laminins found surrounding the skeletal muscle cell in the extrajunctional regions, and also the laminins found in the synaptic cleft. There are strong differences in the distribution of the various laminin subunits, revealing that the release of the laminins by the muscle cell is controlled. The subunits produced by the skeletal muscle cell are predominantly the a2, a4, or a5, plus the b1, b2, and g1 subunits. Of particular importance to the neuromuscular junction is the b2 subunit. The b2-containing laminins are highly concentrated at the synaptic cleft. Since the skeletal muscle cell produces the laminins, the production and release of the b2 subunit must be tightly controlled. The mechanism is still unknown. The b2-containing laminins do play an important role in the structure and function of the synapse. Mice that lack the b2 subunit have defects in synaptic release. The mice die at an early age due to a combination of problems associated with the loss of synaptic function, and also the loss of kidney function. One of the main features observed is the infiltration of the Schwann cell into the synaptic cleft. Thus one of the cellular mechanisms performed by the b2 subunit may be to stop the Schwann cell from wrapping the nerve terminal. In addition, the b2 laminin null mutants have disrupted active zones. Thus, the b2 subunit plays an important role in both the structure and function of the synapse. The distribution of the a subunit is also controlled by the skeletal muscle cell. The a2 subunit is predominantly found in the extrasynaptic basal lamina. The a5 subunit is found concentrated at the neuromuscular junction, and it extends into the junctional folds. The a4 subunit is found concentrated in a small, specialized region of the synaptic basal lamina. Of particular interest are recent findings that the a4 subunit will bind to the voltage-gated calcium channels on the nerve terminal. The distribution and binding of the a4 subunit reveal that this laminin chain is likely to play an important role in the communication between the muscle and the nerve terminal. Nidogens (Entactins)
There are two nidogens in mammalian species. Nidogen-1 is a 150-kDa glycoprotein. It is a common component of all basal laminae, where it acts as a
link by binding to both type IV collagens and laminin. Nidogen-2 is found in the basal lamina of skeletal muscle and its distribution mirrors that of nidogen-1. Nidogen-2 will bind to perlecan and collagen IV. Nidogen-2 also weakly binds to laminin-1. Interestingly, mice deficient in nidogen-1 have an apparently normal phenotype, and nidogen-2 is upregulated in the skeletal muscles of these animals. At the neuromuscular junction there is a uniquely glycosylated form of entactin. The size (150 kDa) and the activity of this uniquely glycosylated form of entactin are consistent with its identification as nidogen-1. There is also recent evidence revealing that nidogen can play a role in central nervous system (CNS) synaptic release, thus it is likely that the nidogens play a role at the neuromuscular junction. Fibronectin
Fibronectin is a large glycoprotein that plays an important role in linking integrins to the extracellular matrix. Fibronectin will bind to a large number of matrix components, and substrates. The important binding partners in the synaptic basal lamina are the collagens and heparan sulfate proteoglycans (predominantly agrin). Fibronectin will then bind to the integrins on the surface of either the muscle cell or the nerve terminal. Fibronectin binding has been shown to alter synaptic release from the motor nerve terminal in culture, and to influence AChR aggregation in cultured myotubes. This activity has been shown to be dependent upon the activation of protein kinases in the cells. Thus, fibronectin acts as a mechanical link between matrix proteins in the extracellular matrix. It also acts as a mechanical link between the matrix and the cell cytoskeleton via the integrin. The binding to integrin also serves as a cell-signaling ligand that increases protein kinase activity. The protein kinase activity has been shown to work predominantly through protein kinases A and C, but it is likely that other protein kinases are also implicated in this signaling. Heparan Sulfate Proteoglycans
Heparan sulfate proteoglycans (HSPGs) are a family of glycoproteins that share unique features. The molecules contain a protein core with large carbohydrate chains. The carbohydrate chains are sensitive to heparitinase. HSPGs are negatively charged. In their native forms the HSPGs run in bands as an extremely large smear (>200 kDa) when separated by apparent molecular mass in polyacrylamide gels. After heparitinase treatment the core protein is much smaller in size. HSPGs are found in all basal laminae and also associated with the cell surfaces. The three
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major HSPGs found in skeletal muscle extracellular matrix are perlecan, agrin, and collagen XVIII. Perlecan Perlecan has a widespread distribution. It is found in all basal laminae, and plays an important role in the storage and release of growth factors. There are three glycosaminoglycan (GAG) side chains extending form the N-terminal region. Collagen XVIII Collagen XVIII has been recently shown to be a HSPG and to be present in the basal lamina of skeletal muscle. Loss of collagen XVIII in Caenorhabditis elegans has been shown to disrupt neuromuscular junction formation. There are no reports showing that collagen XVIII is concentrated at the vertebrate neuromuscular junction, so it remains unclear whether it has a function there. Agrin Agrin is the most extensively studied HSPG at the neuromuscular junction, and it plays a pivotal role in the structure and function of the neuromuscular junction. Early experiments performed by UJ McMahan and colleagues were instrumental in revealing the fact that cell-signaling molecules were found in the extracellular matrix. When muscle fibers were damaged, in a way that left the extracellular matrix intact, the damaged portions of the muscle cells would be removed. In addition, the nerve terminal could be removed, leaving behind an empty basal lamina sheath. When the muscle cell regenerated by the proliferation and fusion of skeletal muscle satellite cells, the skeletal muscle would reform within the basal lamina sheath. The regenerated muscle cells would aggregate AChRs on their surfaces precisely at the spot that they contacted the previous synaptic site on the basal lamina. These experiments clearly showed that molecules stably bound to the synaptic basal lamina could direct the aggregation of AChRs on the surface of the muscle cell. Subsequent experiments revealed that the protein agrin could direct the aggregation of AChRs in culture. Mice that lack agrin do not develop neuromuscular junctions, and these mice die at birth. Agrin is a multifunctional component of the synaptic basal lamina. The C-terminal region of agrin has been most extensively studied, since this is the region that is sufficient to induce the aggregation of AChRs. The number of other molecules that interact with agrin at the neuromuscular junction is extensive. The N-terminal region of agrin has been shown to bind to laminin. Agrin will also bind to a-dystroglycan and thus helps to link the dystroglycan complex to the extracellular matrix. Agrin will also bind to integrins, and to neural cell adhesion molecules (N-CAMs) on the surface of the motor neuron and muscle cell.
In addition to these interactions, agrin will also bind to nidogen and collagen. Agrin clearly plays an important role in the structure and function of the neuromuscular junction, and this role is both structural (linking other proteins in the intracellular matrix together) and functional (acting as a cell-signaling molecule). AChE
The main form of acetylcholinesterase (AChE) at the neuromuscular junction and the synaptic basal lamina is the collagen-tailed form of AChE, ColQAChE. ColQ-AChE is bound to the heparan sulfate proteoglycan perlecan and will also interact with the muscle-specific tyrosine kinase receptor. Its main function is to remove ACh from the synaptic cleft. In fact, the ACh released from the nerve terminal must first run the gauntlet of AChE that is bound to the synaptic basal lamina, before it can bind to the AChR concentrated on the crests of the junctional folds.
Cell Surface and Membrane Receptors Dystroglycan Complex
The main components of the dystroglycan complex are a- and b-dystroglycan. These two forms are from a single gene, but have very different sizes and properties. The a-dystroglycan is 156 kDa in size; it is an extracellular protein that binds noncovalently to b-dystroglycan in the dystroglycan complex, on the one hand, and to laminin and/or agrin in the extracellular matrix, on the other. b-Dystroglycan is a 43 kDa glycoprotein and is an integral membrane protein. Numerous other proteins associate with the dystroglycans and together they form a large structure called the dystroglycan complex. The other members of the dystroglycan complex are the sarcoglycans (a-, b-, d-, and g-sarcoglycans), a-dystrobrevin, neuronal NO synthase (nNOS), and the syntrophins (a, b1, and b2). In addition, and of critical importance, the dystroglycan complex will bind to dystrophin or utrophin. On the intracellular side, the dystroglycan complex will bind to the proteins dystrophin or utrophin. In turn, dystrophin and utrophin will bind to the actin cytoskeleton. The protein utrophin is shorter than dystrophin, and utrophin is concentrated at the neuromuscular junction. The dystroglycan complex is found throughout the entire length of the skeletal muscle cell and is particularly concentrated at the neuromuscular junction and the myotendinous junction. The dystroglycan complex is greatly reduced in Duchenne muscular dystrophy patients, who suffer from muscle fiber breakdown and degeneration. Thus, the dystroglycan complex plays an important
178 Neuromuscular Junction (NMJ): Postsynaptic Basal Lamina
role in linking the intracellular actin cytoskeleton to the extracellular matrix. This link is undoubtedly important in the structural integrity of the muscle cell membrane and is probably critical to protect the membrane from tears that would result from the contractions of the skeletal muscle cell. At the neuromuscular junction the dystroglycan complex may also play an important role in cell-to-cell signaling. The binding of agrin to dystroglycan may be an important feature of the processes that regulate the induction and maintenance of the postsynaptic apparatus in muscle. The exact role of the agrin/dystroglycan complex interaction in postsynaptic signaling is still unknown. MuSK
The aggregation of AChRs induced by agrin depends absolutely on the presence of a muscle-specific tyrosine kinase (MuSK). MuSK is clearly a downstream mediator of agrin, and MuSK knockout mice have virtually the same phenotype as those lacking agrin. However, the direct binding of agrin to MuSK has not been demonstrated, and the exact choreography of the events that start with agrin and end with the activation of MuSK has not been determined. MuSK has a large extracellular domain and an intracellular kinase domain. More recently it has been found that MuSK is required for the anchoring of the ColQAChE complex to the synaptic site. It is also been shown that the Abl kinases (Abl1 and Abl2) are downstream of activated MuSK. Thus, MuSK plays a central role in the events leading to the proper formation of the neuromuscular junction. Integrins
Integrins are transmembrane protein complexes that form from heterodimers of a and b chains. Both chains are important for ligand binding. There are 18 a subunits and eight b subunits known, and currently 24 unique combinations of these have been identified. In addition, many of the subunits are alternatively spliced so that there are multiple variants of many subunits. Of particular interest is the a7 subunit, since it is found concentrated at the neuromuscular junction. The a7 subunit interacts with the b1 to form the a7b1 integrin. This integrin binds to laminin via the RGD (arginine-glycine-aspartate) domain on the b2 arm of the synaptic laminin. The a7 subunit is alternatively spliced, and there are at least six variants of the a7 integrin. While the binding of the a7b1 integrin to laminin is a structural link between the matrix and the cell, the integrins also participate in intracellular signaling. On the intracellular side the integrins play a key role in linking to the actin cytoskeleton. This linkage is
very similar to the molecular linkage found in a focal adhesion. Namely, the integrin binds to vinculin, a-actinin, and talin. All of these components are found aggregated at the postsynaptic site. In addition, integrins play an important role in regulating the activities of kinases. Of particular interest are the Src kinase family members Abl and Fyn, which have been shown to play a role in the assembly and maintenance of the postsynaptic apparatus. Thus, the integrins play an important structural and signaling role in the postsynaptic apparatus. Integrins also are likely to play a presynaptic role, since treatment of frog muscles with RGD-containing peptides will reduce the increase in miniature endplate potential release upon muscle stretch. In addition to the a7b1 integrins, the a3b1 integrins are found at the presynaptic active zones. The a3b1 integrins also interact with laminins in the synaptic basal lamina and can activate intracellular kinases. The precise role of the a3b1 integrin is still largely unknown, but it is clear, at least in frogs, that the binding of integrin to matrix also has an important presynaptic cell-signaling function. Cadherins
Cadherins are membrane-bound proteins that require calcium for their activity. They play an important role in the early development of the skeletal muscle cell. There are many cadherins, but the main ones produced in adult skeletal muscle are neural (N)-cadherin and muscle (M)-cadherin. Immunolabeling studies have revealed that both N-cadherin and M-cadherin are concentrated at the neuromuscular junction. The role of cadherins at the neuromuscular junction has been largely uninvestigated. N-Cadherin promotes neurite outgrowth in vitro and it is of interest that this effect is blocked by agrin. Both N-cadherin and M-cadherin play an important role in the formation of multinucleated muscle cells. They also may play a role at the synapse. The cadherins will form calcium-dependant bonds with cadherins on adjacent cells. Cadherins also interact on the intracellular surface with catenins. The a-, b-, and d-catenins have been detected at the neuromuscular junction. In other systems it is known that the a- and b-catenins form a link with the actin cytoskeleton. In addition, the liberation of b-catenin from cadherins can result in nucleocytoplasmic shuttling to alter gene transcription via the interaction of b-catenin with T cell factor (TCF). Similarly, d-catenin has also been shown to be present at the neuromuscular junction and interacts with the promoter region of rapsyn. Altogether, these results argue that the cadherin system functions at the neuromuscular junction to regulate cell structure and function.
Neuromuscular Junction (NMJ): Postsynaptic Basal Lamina 179
Proteases at the Neuromuscular Junction Matrix Metalloproteinases
The matrix metalloproteinases (MMPs) are a large family of enzymes whose main function is to degrade the extracellular matrix. There are over 25 metalloproteinases, each with preferred matrix substrates. Most MMPs are released from cells in a pro form and must be first activated by other proteases. While it is clear that the MMPs play a role in matrix development and remodeling, it is clear that they also have an influence on cell-to-cell signaling. For example, cleavage of the NC1 domain from type IV collagens is known to play a major role in the signaling of vascular development and remodeling. The strongest evidence for the involvement of MMPs at the neuromuscular junction is the fact that mice that lack MMP3 have increased junctional folds and have AChR receptors on their surface. This likely results from an accumulation of matrix molecules that would normally be removed by MMP3, particularly agrin. MMP2 and MMP9 have been shown to play an important role in the reinnervation of muscle following nerve damage. The fact that MMPs are present at the neuromuscular junction is not a surprise, since it is clear that the matrix must be remodeled to allow synaptic growth. The mechanisms that control MMP activation at the neuromuscular junction are still unknown. Tissue Inhibitors of Metalloproteinases
Balancing the activity of the MMPs are the tissue inhibitors of metalloproteinases (TIMPs). There are four TIMPs, and one of these, TIMP2, has been shown to be present at the neuromuscular junction. Mice that lack TIMP2 have altered neuromuscular junctions. TIMPs inhibit MMPs, but are also required for the activation of some MMPs. Thus, there is a delicate balance between the activity of MMPs and TIMPs. The control of matrix structure and function is likely to be central to the mechanisms that control synaptic structure and function.
Conclusion The synaptic basal lamina contains molecules that are common to all basal laminae throughout the body, and also a number of unique molecules. The synaptic basal lamina has both a structural role that links
the cytoskeletons of the synaptic components, and a cell-to-cell signaling role. The organization of the synaptic basal lamina is extremely precise, and the maintenance of this structure is undoubtedly important in the organization of the synapse. The synaptic basal lamina is a dynamic structure, and proteases are constantly sculpting this complex matrix. The control of matrix structure and function is likely to be central to the mechanisms that control synaptic structure and function. See also: Neuromuscular Connections: Vertebrate Patterns of; Neuromuscular Junction (NMJ): Postsynaptic Events in Neuromuscular Transmission.
Further Reading Barresi R and Campbell KP (2006) Dystroglycan: From biosynthesis to pathogenesis of human disease. Journal of Cell Science 119(Pt. 2): 199–207. Bixby JL, Baerwald-De la Torre K, Wang C, et al. (2002) A neuronal inhibitory domain in the N-terminal half of agrin. Journal of Neurobiology 50(2): 164–179. Burkin DJ and Kaufman SJ (1999) The alpha7beta1 integrin in muscle development and disease. Cell Tissue Research 296(1): 183–190. Fox MA and Umemori H (2006) Seeking long-term relationship: Axon and target communicate to organize synaptic differentiation. Journal of Neurochemistry 97(5): 1215–1231. Kummer TT, Misgeld T, and Sanes JR (2006) Assembly of the postsynaptic membrane at the neuromuscular junction: Paradigm lost. Current Opinion in Neurobiology 16(1): 74–82. Miner JH and Yurchenco PD (2004) Laminin functions in tissue morphogenesis. Annual Review in Cell and Developmental Biology 20: 255–284. Ortega N and Werb Z (2002) New functional roles for non-collagenous domains of basement membrane collagens. Journal of Cell Science 115(22): 4201–4214. Patton BL (2003) Basal lamina and the organization of neuromuscular synapses. Journal of Neurocytology 32(5–8): 883– 903. Rotundo RL, Rossi SG, Kimbell LM, et al. (2005) Targeting acetylcholinesterase to the neuromuscular synapse. Chemico– Biological Interactions 157–158: 15–21. Strochlic L, Cartaud A, and Cartaud J (2005) The synaptic musclespecific kinase (MuSK) complex: New partners, new functions. BioEssays 27(11): 1129–1135. Werle MJ and VanSaun M (2003) Activity dependent removal of agrin from synaptic basal lamina by matrix metalloproteinase 3. Journal of Neurocytology 32(5–8): 905–913. Yurchenco PD, Amenta PS, and Patton BL (2004) Basement membrane assembly, stability and activities observed through a developmental lens. Matrix Biology 22(7): 521–538.
Neuromuscular Junction (NMJ): Postsynaptic Events in Neuromuscular Transmission C R Slater, University of Newcastle upon Tyne, Newcastle upon Tyne, UK ã 2009 Elsevier Ltd. All rights reserved.
Essentials of Neuromuscular Transmission in Vertebrates The neuromuscular junction (NMJ) is the interface between the central nervous system and the muscles it controls. The contraction of muscle fibers is activated by depolarization of the muscle fiber membrane. When motor neuron firing is triggered, action potentials (APs) are initiated near the nerve cell body and then travel along the motor axon to the NMJ. There, the AP causes the release of neurotransmitter molecules that act locally to depolarize the postsynaptic muscle fiber. The ultimate effect of the transmitter is to make the muscle contract. In most vertebrate muscle fibers, an essential intermediate step in the process of neuromuscular transmission is the generation of an AP in the muscle fiber. This AP travels rapidly along the muscle fiber, activating contraction of the whole fiber at nearly the same time. This article describes how vertebrate muscles respond to transmitter released from the nerve and how the APs that activate muscle contraction are generated. In the invertebrates, most muscles do not generate APs and the details of muscle activation are therefore somewhat different. This is also true of a distinct class of electrically inexcitable muscle fibers in vertebrates, discussed briefly at the end of this article. Most vertebrate twitch muscle fibers are innervated at a single site, often roughly midway along the length of the fiber, by a single motor axon. Following a nerve AP, transmitter released from the nerve causes a local reduction in membrane potential, or depolarization, of the muscle fiber. This brief event (10 ms) is referred to as the endplate potential (EPP; Figure 1), since the NMJ is sometimes called the motor endplate. The EPP is a local event that has its maximum amplitude at the NMJ and declines to a low level within 1–2 mm along the fiber (see later). Since muscle fibers may be more than 10 cm long, some other event must account for the depolarization that activates contraction. This event is the AP (Figure 1(a)), which can propagate rapidly in an all-or-none fashion from the NMJ to the ends of the muscle fiber. APs are generally triggered when the membrane potential of the muscle fiber becomes less negative than about 60 to 55 mV. Thus, from a resting potential of 90 to 70 mV, the membrane must be
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depolarized by at least 25 mV. In most vertebrates, the EPP is substantially larger than required to reach the AP threshold. This results in very reliable and complete activation of the contractile apparatus, leading to an ‘all-or-none’ twitch contraction, in each muscle fiber, by every motor nerve impulse. The postsynaptic events of neuromuscular transmission occur within the highly specialized region of the muscle fiber known as the postsynaptic apparatus. This region contains very high concentrations of the ion channels that mediate the response of the muscle fiber to the transmitter released from the nerve. In many vertebrates, there are also striking structural features of this region, including a highly folded surface membrane and an accumulation of specialized myonuclei with distinctive patterns of gene expression.
AChRs and the Generation of the Endplate Current The neurotransmitter acetylcholine (ACh) is released from the vertebrate motor axon terminal in multimolecular quanta. The ACh causes a depolarization by binding to, and thus opening, cation-selective ion channels in the muscle fiber membrane known as ACh receptors (AChRs; Figures 2(a) and 2(b)). When these channels open, the internal negativity of the muscle fiber drives the net entry of positive ions into the cell. It is this effect that gives rise to the depolarizing phase of the EPP. The AChRs present at the vertebrate NMJ are pentameric complexes of four types of subunits (Figure 2(a)). While the structures of these subunits are broadly similar there are also important differences and each subunit is encoded by a different gene. In the muscles of some lower vertebrates, and in immature or paralyzed mammalian muscles, each AChR molecule has two a-subunits and one each of b-, d-, and g-subunits. In many vertebrates, including mammals, the g-subunit is largely replaced by an e-subunit. Each subunit has a molecular mass of about 50 kDa, so the entire complex has a molecular mass of about 250 kDa. Every AChR molecule has two ACh binding sites, each of which includes part of one a-subunit. When no ACh is bound, the AChR channel is closed and no current flows through it. To open the channel, two ACh molecules must bind, one to each binding site. When this happens the channel opens rapidly (Figure 2 (b)). The open channel has a conductance of about 30–50 pS. In the ionic conditions found in an intact mammal, the entry of positive charge through this
Neuromuscular Junction (NMJ): Postsynaptic Events in Neuromuscular Transmission 181 15
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Figure 1 Electrical events associated with transmitter action at the vertebrate NMJ. (a) Intracellular recordings of endplate potentials (EPP) and action potentials (AP). When the EPP fails to reach the AP threshold (horizontal dashed line) no AP is generated. The smaller EPPs illustrated were recorded during partial block of AChRs by curare. The larger EPPs illustrated were recorded during block of NaV1 channels in the muscle fiber by a cone snail toxin. These EPPs would normally trigger an AP. (b) Miniature endplate currents (mEPC) result from the flow of current into the muscle fiber induced by a single quantum of ACh. The nerve impulse causes the release and action of many quanta, resulting in the much larger endplate current (EPC). The electrical properties of the muscle fiber convert these currents into equivalent changes in membrane potential, the miniature endplate potential (mEPP) and the larger endplate potential (EPP). Note that the potential changes are slower than the currents that give rise to them, a result of the membrane capacitance.
conductance represents an ‘inward’ current of about 2.5–4 pA (Figure 2(c)). After being opened, each channel closes spontaneously and abruptly, on average after about 1 ms (for AChRs containing an e-subunit). However, the duration of open times, even of the same channel on different occasions, varies considerably and randomly, with brief openings being the most common and long open times the least common. At the vertebrate NMJ, each ACh quantum consists of 5000–10 000 molecules. Each quantum causes the opening of 1000–2000 ACh-gated channels, resulting in a brief influx of a current of about 3–5 nA into the
muscle fiber. The current induced by a single quantum is known as a miniature endplate current (mEPC; Figure 1(b)). Although all the channels activated by a single quantum of ACh open almost simultaneously, the variability of the open times means that some close very quickly and others remain open for several milliseconds. As a result, the shape of the mEPC differs from that of the individual channels, having an abrupt rise to a maximum value and a slower exponential decay with a time constant of 1–1.5 ms (Figures 1(b) and 2(d)). In the case of AChRs containing a g-subunit (see earlier), the time constant is severalfold longer.
182 Neuromuscular Junction (NMJ): Postsynaptic Events in Neuromuscular Transmission
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NMJ. The AChR channels opened by a single quantum do so within less than 100 ms. During this time ACh molecules, on average, can diffuse about 0.3 mm. Each quantum thus acts on about 0.25 mm2 of postsynaptic membrane. For most vertebrate NMJs, this is less than 0.1% of the membrane containing AChRs. As a result, the sites of action of individual quanta on the muscle fiber membrane are effectively separated from each other, allowing the effects of the individual quanta to sum to produce the much larger endplate current (EPC; Figure 1(b)). In different vertebrate species, the EPC may vary in amplitude from 200 to 2000 nA. Since the quanta released by a single nerve AP act almost simultaneously on the muscle, the time course of the EPC is usually very similar to that of the mEPCs.
From EPCs to EPPs The passive electrical properties of the muscle fiber determine how the current of the EPC is converted into the depolarization that is the EPP. A brief account of these properties is given in the following sections. Passive Cable Properties of Muscle Fibers
4 6 d 2
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Figure 2 Acetylcholine receptors (AChRs) mediate permeability changes in the postsynaptic membrane. (a) The five subunits of the AChR associate to form a pore that spans the plasma membrane. (b) A model of the open and closed states of the AChR as viewed looking along the pore axis. Small relative movements of the subunits, induced by ACh binding, result in changes in the diameter of the ion pore that have a big impact on permeability. (c) Record of current flowing through a single AChR channel. Each event has an abrupt start and finish. Although the duration of the open states varies, the intensity of the current in different events is very similar. (d) Scheme of how single channel openings sum to give rise to a mEPC. In the upper part, each of six channels is shown to open simultaneously. Each channel stays open for a time that varies randomly about a mean value. Thus the six channels close at different times. In the lower part is shown a model mEPC consisting of the sum of the currents flowing through the six channels. It decays exponentially from the initial peak with a time constant equal to the duration of the mean open time of the individual channels. (b) Diagram provided by Nigel Unwin. (c) Reprinted by permission from Macmillan Publishers Ltd: [Nature] Mishina M, Takai T, Imoto K, et al. (1986) Molecular distinction between fetal and adult forms of muscle acetylcholine receptor. Nature 321: 406–411, copyright (1986).
At the NMJ of vertebrate twitch muscles, between 20 and 200 quanta are typically released by a single nerve AP. In general, the sites of release of these quanta are distributed randomly over the entire
The flow of current in muscle fibers is governed by the so-called cable properties of the cell. These derive from the relatively high electrical conductivity of the cytoplasm and the low conductivity (or high resistivity) of the plasma membrane. Current entering the cell at a point, such as the NMJ, may flow longitudinally in the cytoplasm or transversely through the membrane (Figure 3(a)). Only the latter pathway leads to a change in the membrane potential. The fraction of the current that takes each route depends on the relative resistance each pathway presents. In practice, the conductivity of the cytoplasm is roughly the same in most cells and the resistivity of the membrane is similar in many (but not all) skeletal muscle fibers. As a result, the dominant factor determining the distribution of current flow in different muscle fibers is their diameter. When the diameter is large, current can flow much more easily along the muscle fiber than across the membrane, and there is relatively little voltage change across the membrane at the site of current entry. In contrast, if the diameter is small, the resistance to longitudinal current flow is high and more current crosses the membrane close to the site of entry, giving rise to a larger local change in voltage. The peak amplitude of the potential change caused by a given intensity of current flow entering the cell at a single point is determined by the so-called input resistance of the muscle fiber. Typical vertebrate skeletal muscle fibers have ‘input resistances’ of
Neuromuscular Junction (NMJ): Postsynaptic Events in Neuromuscular Transmission 183 V
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Figure 3 The passive electrical properties of the muscle fiber shape the voltage change caused by current flowing across the membrane. (a) Diagram of a typical experiment. Two electrodes are placed within the muscle fiber, one to pass current into the cell (i ) and the other to measure the resulting change in transmembrane potential (V). Arrows show flow of current. The current across the membrane is greatest near the site of current injection and decreases with distance from that site. (b) Decay of transmembrane potential (Vm) with distance from the site of injection. The space constant (l) is the distance at which the potential has fallen to 36% (1/e) of its maximum (Vo). (c) Changes in membrane potential (upper part) in response to rectangular current pulses. Note the slower change in voltage due to the capacitance of the membrane. Horizontal dashed line shows AP threshold: depolarizations greater than this trigger an AP. (d) Potential changes in response to injection of current with a time course similar to that of the postsynaptic mEPC and EPC that occur during neuromuscular transmission. Note that the passive properties of the membrane convert these currents into potential changes very similar to mEPP and EPP. The action potential threshold is very similar to that observed with rectangular pulses. (c) Reproduced from Wood SJ and Slater CR (1995) Action potential generation in rat slow- and fast-twitch muscles. Journal of Physiology 486: 401–410, with permission from Blackwell Publishing. (d) Reproduced from Wood SJ and Slater CR (1997) The contribution of postsynaptic folds to the safety factor for neuromuscular transmission in rat fast- and slow-twitch muscles. Journal of Physiology 500: 165–176, with permission from Blackwell Publishing.
0.2–1 MO (see later). If the full amplitude of the EPC (e.g., 250 nA) were instantly converted into a depolarization, its value (for a resistance of 0.4 MO) would be 100 mV.
Spatial factors The decay of potential from a site of current injection (e.g., the NMJ) can usually be approximately described by a single exponential (Figure 3(b)). For many vertebrate muscle fibers, the
184 Neuromuscular Junction (NMJ): Postsynaptic Events in Neuromuscular Transmission
space constant of the decay – that is, the distance required for the potential to drop to 37% (1/e) of its value at the origin – is 0.5–2 mm. It is because of this that the ACh released from the nerve has little direct effect on the membrane potential more than a few millimeters away from the NMJ. Effects of membrane capacitance Like all cell membranes, the muscle fiber membrane has electrical capacitance as well as resistance. When positive charge enters the cell through the opened AChRs, it must bring about a reorientation of the charge stored in the molecular dipoles in the membrane before a potential difference develops across the membrane. This takes time. For an instantaneous change in current flow across the membrane of a mammalian muscle fiber, the voltage typically takes 3–5 ms to reach 84% of that, a measure of the so-called time constant of the muscle fiber (Figure 3(c)). For a flow of current which is brief relative to the membrane time constant, such as the mEPC, the capacitance distorts the time course of the resulting voltage change (Figure 3(d)). In addition, because the mEPC is short relative to the time constant of the membrane, the potential never reaches the maximum value that corresponds to the current at the peak of the mEPC. In practice, the distorting effect of the membrane capacitance means that the observed peak amplitude of the EPP is typically about 30–40 mV.
Initiation of the Muscle AP Most vertebrate skeletal muscle fibers, like neurons, have voltage-gated sodium channels in their membranes. The specific forms of these channels present in vertebrate skeletal muscle are of the class designated as NaV1 and are very similar in their structure and function to those in other excitable tissues. However, they differ in detail and are encoded by different genes which are normally expressed only in muscle cells. Each NaV1 channel contains a major poreforming a-subunit with a molecular mass of about 250 kDa and a smaller accessory b-subunit. Like most voltage-gated ion channels, NaV1 channels are largely closed at the normal resting membrane potential and open when the membrane potential becomes sufficiently less negative. In the case of NaV1 channels in mammalian muscle fibers, opening begins at a membrane potential of about 60 mV and is maximal at about 30 mV. In a vertebrate twitch muscle fiber, the action potential is normally initiated at a single point along its length, the NMJ. In order for this to happen, the charge entering at that point, and the depolarization it gives rise to, must open sufficient nearby NaV1
channels so that the inward current entering through them outweighs the flow of longitudinal current away from the NMJ and thus brings the surrounding membrane to threshold. In muscle fibers, this threshold is conventionally measured away from the NMJ using two closely spaced (<100 mm) intracellular electrodes, one to pass current and the other to record the resulting change in membrane potential (Figure 3(a)). In rat skeletal muscle fibers in which the resting potential is maintained at 80 mV, the threshold recorded in this way, using a current pulse that is long relative to the AP, is about 55 mV (Figure 3(c)). In other words, a depolarization of about 25 mV is required to initiate an AP. A similar threshold depolarization is observed when a current pulse which has a time course and amplitude similar to that of an EPC is used (Figure 3(d)). A number of observations in mammalian muscle suggest that the effective threshold at the NMJ is significantly less than in the extrajunctional region. For example, when the amplitudes of the EPP and EPC are progressively decreased by adding the AChR blocker curare, EPPs as small as 12 mV (starting from a resting potential of 80 mV) can just trigger an AP. To understand the basis of this effect, it is necessary first to consider the structure and molecular organization of the postsynaptic region.
Structure of the Postsynaptic Apparatus A striking feature of the postsynaptic region at many, but by no means all, vertebrate NMJs is the presence of infoldings of the muscle fiber membrane (Figure 4). These are typically 0.5–1 mm deep, 0.1 mm wide, and spaced 0.5–2 mm apart. Detailed investigation of mammalian NMJs, where the folds are particularly prominent, has shown that there is a remarkable degree of segregation of ion channels within the postsynaptic membrane. At the crests of the folds, close to the overlying motor nerve terminal, AChRs are clustered at a density of approximately 10 000 mm 2. This means that the individual AChR molecules are virtually in contact with each other in a near-crystalline array. The region of high AChR density typically extends about one-third of the way down the fold, where it ends abruptly. Outside this region, the density of AChRs is generally no more than a few per square micrometer. In contrast to the membrane at the crests of the folds, that in the depths of the folds, and in a perijunctional zone extending about 20–30 mm away from the NMJ, contains a high density of NaV1 channels. While the absolute density of these channels is not known accurately, it is estimated to be about
Neuromuscular Junction (NMJ): Postsynaptic Events in Neuromuscular Transmission 185 Schwann cell
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Figure 4 The postsynaptic apparatus and its amplifying effect. (a) A schematic diagram of a mouse NMJ. Note particularly the highly folded postsynaptic membrane and the nonoverlapping distributions of acetylcholine receptors (AChRs) and NaV1 molecules. AChRs are concentrated near the sites of transmitter release from the nerve while the NaV1 channels are concentrated in the depths of the folds. (b) Current flow in the muscle fiber induced by the action of ACh. Positive charge enters the muscle fiber through opened AChR channels at the crest of the folds. The resulting current flows in the narrow space between the folds into the cytoplasm and then out across the nonsynaptic membrane. (c) Equivalent circuit of an active fold at a rat NMJ. Note that the resistance of the interfold cytoplasm (4.8 MO) is an order of magnitude greater than that of the input resistance of the fiber (0.5 MO). (d) AP threshold during neural activation of a rat muscle fiber. The EPP was reduced in amplitude by partially blocking AChRs with curare so that only occasional EPP are big enough to trigger an AP. The AP threshold determined this way (dashed line, about 60 mV) is substantially less than when determined when current is injected away from the NMJ (solid line, about 50 mV; see Figure 2(a)). This is a result of the combined effects of the high concentration of NaV1 channels and the high interfold resistance.
2000 mm 2. This is some 10 times greater than in the remainder of the muscle fiber membrane. These features of the postsynaptic membrane of the mammalian NMJ have an important impact on
the current flows initiated by ACh released from the nerve. The current flowing through the opened AChRs enters the cell at the crest of the folds (Figure 4(b)). Because the effective resistance of the folded membrane
186 Neuromuscular Junction (NMJ): Postsynaptic Events in Neuromuscular Transmission
is very high, the path of least resistance for this current is through the thin sheet of cytoplasm between the folds. However, the resistance of this pathway is some 10 times greater than the input resistance of the muscle fiber as a whole (Figure 4(c)). As a result, there is a steep potential gradient within the cytoplasm between the top of the folds and that in the bulk of the muscle fiber. This results in a similar spatial gradient of the transmembrane potential. At NMJs in the rat, it has been estimated that a single quantum of ACh depolarizes the membrane at the crest of a fold by more than 10 mV, while in the bulk of the fiber the depolarization recorded by an intracellular electrode is only 0.5–1 mV. The functional significance of this local amplification of the depolarizing effect of ACh is enhanced by the high density of NaV1 channels in the depths of the folds. To initiate an AP, a minimum number of NaV1 channels at the NMJ must be opened. As a result of the high density of these channels in the postsynaptic membrane, a smaller fraction of the channels needs to open to generate an AP than if the density were as low as it is in the extrajunctional region of the fiber. In addition, the amplifying effect of the geometry of the folds means that a larger fraction of those channels is opened by a quantum of ACh than if the membrane was not folded. As a result, the effective threshold for AP generation at the NMJ is about half of that in the extrajunctional region (Figure 4(d)).
Safety Factor for AP Generation The pattern of muscle innervation varies in different species and muscles. In some cases, multiple axons innervate each muscle fiber and no AP is generated (see later). However, in most higher vertebrates, and in mammals in particular, an important principle of the functional organization of muscle innervation is that each AP in a motor neuron causes contraction of every muscle fiber that its axon innervates. This requires that neuromuscular transmission is very reliable, even during intense high-frequency activity when fewer quanta are released per impulse. This reliability results from the release of substantially more quanta of ACh than are necessary to reach the threshold for AP generation in the muscle. Neuromuscular transmission is thus said to have a high ‘safety factor.’ In rat limb muscles, where it has been studied in most detail, the safety factor is about 4. It is clear that the postsynaptic specializations just described make an important contribution to the safety factor in mammalian muscle fibers. By lowering the effective threshold they reduce the number of quanta that must be released from the nerve to ensure reliable transmission.
Termination of ACh Action The action of ACh is terminated by the enzyme acetylcholinesterase (AChE), which is bound to the basal lamina located in the space between the nerve and the muscle. AChE cleaves the ACh into choline and acetate. Although the AChE lies on the diffusion path taken by ACh molecules on the way to their targets, there are many fewer AChE molecules than ACh or AChRs. As a result, most ACh molecules released from the nerve reach the postsynaptic membrane and bind to AChRs. However, when they subsequently dissociate from the AChRs, the ACh molecules are bound and cleaved by the AChE, thus preventing them from rebinding to a second AChR. AChE thus acts to limit both the spatial extent and temporal duration of ACh action.
How Different Species Achieve Reliability Not all vertebrate species achieve an adequate safety factor in the same way. While mammals rely on the amplifying effect of the postsynaptic folds, many fish and birds have few or no folds. Comparison of the quantal content and intensity of folding in several species suggest that there is often a reciprocal relationship between these two properties of the NMJ (Figure 5). In frogs, the nerve terminals are very big and release many quanta, but the folding is less prominent than in mammals. At the other extreme are human NMJs, which are relatively small and release relatively few ACh quanta (20–50 vs. 100–200 in the frog) but have very extensive folding. The NMJs of rats and mice are of intermediate size and intensity of folding. There is little reliable information about the number of quanta released at avian NMJs. It is also possible that the safety factor is not as high in birds as in some other species. It is unclear why some species rely more on presynaptic release and others on postsynaptic amplification to achieve an adequate safety factor. In large animals such as humans, each motor neuron innervates many more muscle fibers than in small animals. It may be that cellular economy dictates that the total volume of nerve terminals supported by a single motor neuron should be kept to a minimum. The postsynaptic specializations that increase the efficiency of transmitter action may have evolved in response to such pressures.
Some Special Cases In the great majority of vertebrate muscle fibers, APs are generated as part of the process by which contraction is activated. However, there are some important exceptions to this rule. In many species, muscle fibers
Neuromuscular Junction (NMJ): Postsynaptic Events in Neuromuscular Transmission 187
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Figure 5 Inverse relationship between the extent of folding and the amount of transmitter release. (a) Light micrographs of NMJs in the indicated species. The muscle fiber is shown in black and the NMJ in white. Note small size of the human NMJs relative to those in frogs. (b) Electron micrographs of NMJs in the same species. Note that human NMJs have much greater postsynaptic folding than do frog NMJs. (c) In these species, the extent of folding (reflected by the folding index) is inversely related to the number of transmitter quanta released from the nerve. This provides the structural basis for the postsynaptic amplification of ACh action that is particularly prominent in humans.
are present that appear to be incapable of generating APs. These fibers tend to have small diameters and very high membrane resistance, and to contract relatively slowly. A common feature of these fibers is that they are innervated at multiple sites along their length, typically at intervals of 1–2 mm. Contraction is activated by the summed effect of the local depolarizations caused by transmitter quanta acting at these sites. These fibers are referred to by a number of terms, the most common being ‘slow’ and ‘tonic.’ Unfortunately, both of these terms are used in a number of different ways, leading to much confusion in the literature. The term ‘nonEx-MIF’ (nonexcitable, multiply innervated fibers), though cumbersome, is more precise.
Frog ‘Slow’ Fibers
In lower vertebrates, nonEx-MIFs are found in many skeletal muscles. In frogs, the vertebrates in which this type of NMJ was first studied in detail, stimulation of the nerve elicits junction potentials, analogous to EPP. Analysis of the properties of these potentials with intracellular electrodes is greatly complicated by the presence of multiple sites of innervation of individual muscle fibers, and the contamination of recordings at one site by events at others. Nonetheless, it seems that AChRs with kinetics and pharmacology similar to those in twitch fibers are concentrated at these junctions and account for the increased
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conductance that underlies the junction potentials. A common feature of electrically inexcitable muscle fibers, both in frogs and in mammals (see later), is that they have a very high input resistance. This is probably a result of a low resting permeability to chloride ions. An important consequence of this is that the depolarization caused by the action of a quantum of ACh is much greater than it would be in a typical twitch fiber. This is of obvious importance in the activation of contraction in the absence of APs.
binding. This current is converted into a depolarization by the passive electrical properties of the muscle fiber. In the case of those species in which there are prominent postsynaptic folds, these focus the depolarizing effect on the folded membrane, in a manner analogous to the isolating effect of the neck of dendritic spines on central neurons. The depolarization acts to open NaV1 channels that are concentrated in the postsynaptic region. When enough of these channels open, a regenerative AP is initiated.
Mammalian Extraocular Fibers
See also: Neuromuscular Connections: Vertebrate Patterns of; Neuromuscular Junction (NMJ): Postsynaptic Basal Lamina.
In higher vertebrates, including mammals, nonEx-MIFs are mainly restricted to a few specialized types of muscles. The extraocular muscles include fibers that receive innervation from several motor neurons. In contrast to the analogous fibers in frogs, these fibers contract very rapidly in response to the graded stimulus provided by their multiple innervations. Many of these fibers appear unable to generate APs, although a subclass shows some degree of excitability in vitro. In these intermediate fibers, the level of excitability seems to vary along the length of the fiber, being greatest in the central region. Very little is known in detail about the quantitative aspects of ACh action or voltage-gated sodium channel activation in these very small diameter muscle fibers. Intrafusal Fibers
The second main class of non-Ex-MIF in higher vertebrates is the intrafusal fibers of the muscle spindles. The motor innervation of these fibers is complex, arising mainly from specialized g-motor neurons, but in some cases from collateral branches of the a-motor neurons that also innervate extrafusal fibers. Very little is known in detail about neuromuscular transmission in these fibers, which are even more challenging to investigate than those of the extraocular muscles.
Conclusions At vertebrate NMJs, the postsynaptic response to ACh quanta released from the nerve is determined primarily by two key ion channel species and the geometry of the postsynaptic region. AChRs mediate the initial influx of excitatory depolarizing current in response to ACh
Further Reading Chiarandini DJ and Davidowitz J (1979) Structure and function of extraocular muscle fibers. Current Topics in Eye Research 1: 91–142. Martin AR (1994) Amplification of neuromuscular transmission by postjunctional folds. Proceedings of the Royal Society of London, Series B 258: 179–187. Mishina M, Takai T, Imoto K, et al. (1986) Molecular distinction between fetal and adult forms of muscle acetylcholine receptor. Nature 321: 406–411. Morgan DL and Proske U (1984) Vertebrate slow muscle: Its structure, pattern of innervation, and mechanical properties. Physiological Reviews 64: 103–169. Salpeter MM (1987) Vertebrate neuromuscular junctions: General morphology, molecular organization, and functional consequences. In: Salpeter MM (ed.) The Vertebrate Neuromuscular Junction, pp. 1–54. New York: Alan R. Liss, Inc. Slater CR (2003) Structural determinants of the reliability of synaptic transmission at the vertebrate neuromuscular junction. Journal of Neurocytology 32: 505–522. Slater CR, Lyons PR, Walls TJ, et al. (1992) Structure and function of neuromuscular junctions in the vastus lateralis of man. A motor point biopsy study of two groups of patients. Brain 115(Pt. 2): 451–478. Vautrin J and Mambrini J (1989) Synaptic current between neuromuscular junction folds. Journal of Theoretical Biology 140: 479–498. Wood SJ and Slater CR (1995) Action potential generation in rat slowand fast-twitch muscles. Journal of Physiology 486: 401–410. Wood SJ and Slater CR (1997) The contribution of postsynaptic folds to the safety factor for neuromuscular transmission in rat fast- and slow-twitch muscles. Journal of Physiology 500: 165–176. Wood SJ and Slater CR (2001) Safety factor at the neuromuscular junction. Progress in Neurobiology 64: 393–429.
Gap Junction Communication C Giaume, Colle`ge de France, Paris, France C C Naus, University of British Columbia, Vancouver, BC, Canada
also involved in extracellular pathways mediating autocrine and paracrine processes.
ã 2009 Elsevier Ltd. All rights reserved.
Brain Connexins: Where and When For a long time, it has been well established that gap junctions provide a direct pathway for cell-to-cell communication. This mode of intercellular communication is widely present in all vertebrate species and also in most tissues and organs including the central nervous system (CNS). Compared to other types of cell interaction, gap junctional communication (GJC) mediates direct cytoplasmic exchanges from one cell to one or several neighbors. These exchanges are characterized by a weak ionic selectivity (electrical coupling) and a permeability for small molecules up to 1.8 kDa (metabolic or biochemical coupling). Such properties indicate that intercellular communication through gap junctions is likely relevant for excitable cells as neurons where they constitute the morphological substrate of electrical synapses. However, GJC also plays an important role in nonexcitable cells such as glial cells since they are permeable to signaling molecules. Connexins (Cxs) are the molecular constituents of gap junction plaques consisting of closely packed hemichannels formed by a hexameric ring of Cxs which align head-to-head between neighboring cells to form functional and regulated intercellular channels. Invertebrates have a different, but functionally equivalent, family of proteins making up gap junctions, the innexins. Moreover, another gene family with functional characteristics similar to the Cxs and innexins has been discovered and named the pannexins, at least two of which are expressed in the mammalian CNS, whereas their function is not well defined. Interestingly, evidence is accumulating that Cxs interact by their cytoplasmic domains with other proteins that may be important for different aspects of their biology. These Cx-interacting partners, which are signaling and/or scaffolding proteins, have been proposed to be components of a dynamic multiprotein complex involved in the intracellular transport of Cxs, hemichannel assembly, full channel formation, junctional plaque assembly and stability, and regulation of channel permeability. In addition to the well-known intercellular function of full gap junction channels, it has been reported that Cx channels can also operate as hemichannels, allowing exchanges of ions and small molecules between the cytoplasm and the extracellular space. Thus, in addition to providing a direct cell-to-cell pathway, Cxs are
Between Neurons
Although coupling persists in many brain regions in the adult, during development there is a progressive decrease in Cx expression and GJC in CNS neurons. The correlation between changes in Cx expression and neuronal differentiation suggests that early GJC permits signaling molecules to diffuse between neurons and contributes to their differentiation. Eight different Cxs have been detected in defined neuronal cell populations (Cx26, Cx32, Cx36, Cx37, Cx40, Cx43, Cx45, and Cx47), mainly in the hypothalamus, striatum, inferior olive, hippocampus, olfactory bulb, retina, cerebral cortex, and cerebellum. Interestingly, in the CNS Cx36 represents the first Cx isoform expressed exclusively in neurons and exhibits typical functional properties compared to other Cxs. In the hippocampus of the adult rat, this Cx predominates and is mainly expressed in interneurons. Between Glial Cells
In the CNS, glia represents the major population of cells coupled through gap junctions, and their connections establish compartments of communicating cells. This expression persists throughout adulthood, with qualitative and quantitative differences between glial classes: (1) each glial cell type expresses a set of specific Cxs, none being specific for glia; (2) the strength of coupling and the level of connexin expression depend on the glial cell type; and (3) not all classes of glial cells are coupled together, which contradicts the initial concept of a glial syncitium. Indeed, astrocytes are organized as a network composed of only subpopulations of glia that are under the control of biological signals, including neurotransmitters. This network organization can be studied in acute brain slices after injecting low-molecular-weight tracers into a single astrocyte that results in the staining of large groups of 10–100 coupled cells. Such large coupling areas have also been described in vivo in the cortex, the hippocampus, and the cerebellum (Bergmann glial cells). Besides minor detection of Cx26, Cx40, and Cx45, two main junctional proteins are expressed in astrocytes: Cx43, which is widely present at both adult and embryonic stages, and Cx30, which is detected in mature gray matter astrocytes. In addition, the relative levels of Cx43 and
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Cx30 expression vary according to the developmental stage and region studied. Oligodendrocytes, the myelin-forming cells of the CNS, are endowed with gap junctions found in almost all areas of their plasma membranes; however, they are not detected between successive layers of myelin. Expression of Cx32 and Cx47 has been shown in oligodendrocytes studied in vitro as well as in situ, and indirect evidence suggests that Cx29 is also expressed in oligodendrocytes. Finally, the presence of Cx43 has been described in microglial cells. In normal tissues, <5% of microglial cells in adult rat cerebral cortex are Cx43 immunoreactive, and in culture only low levels of Cx43 have been detected. However, in vivo after a stab wound or in culture after treatment with cytokines, Cx43 is observed at the interfaces between activated microglia cells that become dye coupled.
functional status of intercellular channels can be regulated by both covalent and noncovalent modifications of Cxs which affect the channel structure, a third form of intercellular language called ‘gating.’ Despite the overall degree of homology, Cxs exhibit in their two major cytoplasmic domains stretches of unique sequences that probably adapt channel properties to the specific tissue requirements. The behavior of Cx channels has been studied extensively in various in vitro expression systems, and these biophysical studies have established that individual Cxs are endowed with unique gating properties, including voltage dependency, pH sensitivity, subconductance states, and residual conductance.
What Goes through Connexin Full and Hemichannels?
The Language of Connexins
Intercellular Communication through Gap Junctions in Neurons and Glia
The molecular complexity of Cx channels bears important consequences on their function. Cxs speak an articulate language and have evolved diverse forms of intercellular communication among cells. First, channels composed of different Cxs are endowed with distinct biophysical and regulatory properties. Although Cxs do not form ion-selective channels in the classical sense, they exhibit a certain degree of selectivity with a preference for cations versus anions. Because gap junctions are permeable to cyclic nucleotides and inositol 1,4,5-trisphosphate (IP3), which are negatively charged at normal intracellular pH, ionic selectivity may allow the discrimination and modulation of the kinetics of intercellular propagation of known second messengers. Size selectivity is also a Cx-specific feature. It had long been thought that connexins were permeable to molecules up to 1800 Da. The use of different fluorescent tracers demonstrated that some connexins can discriminate between molecules of 400 and 600 Da, thus favoring the passage of a distinct set of signals. A second form of intercellular language is based on compatibility – that is, the ability of Cxs to functionally interact with different family members. On the basis of Cx topology relative to the plasma membrane, the docking of two facing hemichannels implicates the recognition between their two extracellular domains. Experimental data have shown that the ability to establish direct communication between adjacent cells is dependent, at least in part, on the ability of Cxs to discriminate between different partners. A consequence of the restricted ability of Cxs for heterotypic interactions is that expression of different Cxs in a group of cells may provide a powerful means to limit or segregate intercellular communication. Finally, the
The initial evidence of gap junction-mediated exchanges of ions was obtained by electrophysiological recordings of electrical coupling between neurons or between glial cells. These pioneering works were then confirmed by a number of studies that established the contribution of gap junctions to electrical coupling between CNS cells and their regulation by several factors, including neurotransmitters, voltage, and certain brain pathologies or lesions. This was further investigated by expressing neuronal or glial Cxs in cell lines lacking endogenous Cxs and by using the double patch clamp technique to establish selectivity profiles for cations versus anions and the number of charges. The development of dual patch clamp recordings from neurons in brain slices permitted functional studies of electrical synapses. Of great interest is the permeability of Cx channels for small molecules since this intercellular exchange supports a biochemical and metabolic coupling that is likely important for signaling between nonexcitable cells. Thus, an important issue concerning the role of gap junctions between glia is the identification of signaling molecules exchanged through this intercellular pathway. Since the permeability profile of Cx channels depends on their molecular constituents, it is expected that the amount and the nature of permeating ions or signals is directly related to their identity. The diversity of Cx expression in CNS cells suggests that intercellular exchanges are likely dependent on several types of gap junction channels made by these Cxs. Although essential, this issue has been very difficult to address in situ due to technical limitations and the expression of several Cxs per cell. For this reason, the situation is much more favorable in vitro for
Gap Junction Communication 191
cultured astrocytes since most gap junction channels are made up of Cx43, as demonstrated by quantitative analysis showing that 95% of the electrical coupling is lacking in astrocytes cultured from Cx43 knockout mouse brain. Although Cx43 channels are poorly selective for ions, double patch clamp recordings have established that they favor the passage of cations versus anions. In addition, there is converging evidence indicating that intercellular exchange of signaling molecules occurs in cultured astrocytes and in glioma cell lines transfected with Cx43. The permeability of astrocyte gap junctions for glucose and its metabolites, including lactate, was initially characterized by using radiolabeled compounds and by investigating their intercellular diffusion by adapting the scrape-loading dye-transfer technique. Moreover, the use of several unrelated gap junction inhibitors has shown that this diffusion occurs through gap junction channels. This has been confirmed by use of a fluorescent glucose molecule, 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-Y1) amino)-2-deoxyglucose. Interestingly, gap junctions were shown to participate in the propagation of intercellular calcium waves. In this context, the demonstration that astrocyte gap junctions are permeable to IP3 has contributed to establish the key role of this second messenger in the propagation process. In addition, transfection of C6 cells with Cx43 allowed the demonstration that several nucleotides, particularly ATP and ADP, can be transferred through Cx43 channels. Finally, radiolabeled glutamate and glutamine have also been shown to permeate Cx43 channels in astrocytes. Whereas in some systems electrical coupling between neurons is strong enough to allow transmission of action potentials, such as in early development, in many cases coupling is weak. In addition, gap junction channels act as a low-pass filter, and gap junctions are generally located primarily on dendrites so that fast action potentials are attenuated. Taking these considerations into account, it is unlikely that coordination of spike propagation through gap junctions is the primary function of coupling between neurons. Although this statement argues for a role of biochemical coupling between coupled neurons, there is very little information about what can cross neuronal gap junctions. Indeed, besides the demonstration that glycine can be exchanged through gap junctions between neurons in the retina, the other evidence for the passage of signaling molecule concerns IP3. In this case, the permeation of IP3 was reported to contribute to the propagation of calcium waves in neuronal domains, whereas an increase in Ca2þ alone was not sufficient.
Connexin Hemichannels: An Alternative Release Pathway
Classical pathways for neurotransmitter release that have been extensively described in neurons and in astrocytes include calcium-dependent vesicular release and the purinergic ionotropic receptor (P2X7). In addition, Cx hemichannels have also been proposed to operate in certain retinal neurons and in astrocytes. The unitary conductance of Cx hemichannels has been recorded in cultured astrocytes and is twofold higher than that recorded for the Cx43 full channel. The activity of these Cx hemichannels is very low in resting conditions but can be increased either after ischemic treatment or metabolic inhibition or in low divalent solution. There is evidence that ATP and glutamate can be released from astrocyte Cx hemichannels under these nonphysiological conditions.
Functional Consequences of Cx-Mediated Communication In addition to its contribution to synaptic transmission, the permeability of gap junction channels for ions may serve to maintain ionic homeostasis between cells, particularly between glial cells, which have for a long time been proposed to play a role in ionic buffering. This is particularly relevant for Kþ since astrocytes mainly exhibit voltage-dependent and passive conductance for this ion. Indeed, several studies have also demonstrated that in cultured astrocytes, GJC is also involved in the intercellular homeostasis of Naþ and Ca2þ. This is particularly relevant when the pharmacological heterogeneity of astrocytes is considered, since not all astrocytes will respond primarily to the stimulation of a membrane receptor agonist. However, the permeability of gap junction channels for calcium ions and IP3 will enable neighboring coupled cells to develop a secondary calcium response. Accordingly, the regulation of GJC may contribute to unmask a pharmacological heterogeneity of astrocytes. In addition, the permeability of Cx43 channels for IP3 is proposed to contribute to the propagation process of intercellular calcium waves, establishing an important functional role for gap junctions in astrocyte physiology and neuroglial interactions. The possibility for astrocytes to exchange energetic compounds provides an intercellular pathway that may participate in metabolic trafficking between the source of energetic substrates and the main site of energy consumption – respectively, the blood circulation and neurons. In this way, gap junctions may contribute to the distribution of cellular metabolites and the maintenance of the energy status over a glial
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cell population. This role for gap junctions is further strengthened by the enriched expression of Cx43 and Cx30 antibodies at contacts between astrocytic endfeet enwrapping blood vessels. This may create local zones of intercellular communication that favor glucose uptake and its spreading into several astrocytes through a metabolic network. The observation that astrocyte gap junctions are permeable to glutamate and glutamine is of particular interest since after release from glutamatergic neurons, the neurotransmitters are rapidly inactivated by cellular uptake, predominantly into astrocytes. Glutamate is then amidated to glutamine by the ATP-consuming reaction catalyzed by glutamine synthetase, an enzyme present in astrocytes but not in neurons. Glutamine is then released by astrocytes and taken up by neurons, where its hydrolysis provides glutamate. Consequently, the regeneration of this neurotransmitter should not solely be considered as the result of the contribution of isolated and independent astrocytes but, rather, as a result of groups of communicating astrocytes that can exchange glutamate and glutamine. Thus, the permeability for this excitatory amino acid may play an important role in the interaction between astrocytes and neurons that are in part mediated by glial release of glutamate. In certain conditions, this release could also be mediated by Cx hemichannels. A similar situation may occur for the release of ATP that was also shown to operate through Cx43 hemichannels in astrocytes. In these situations, Cx hemichannel-mediated communication has been proposed to contribute to the propagation of calcium waves between astrocytes as well as to the modification in neuronal activity. Finally, Cx hemichannels in horizontal cells of the retina exert a feedback-mediated response that affects the activity of calcium channels and subsequently glutamate release controlling the output of the cones.
Conclusions and Perspectives It is clear that the analysis of the functional state of gap junctions in the brain is still limited for at least several reasons. First, it is still difficult to study intercellular coupling in brain slices, especially at adult stages, and only a few studies have been performed in the mature brain. Second, dye-coupling experiments may not be sufficiently informative to identify the role of gap junctions in nonexcitable cells. Indeed, this approach only allows one to follow the intercellular diffusion of small molecules (Lucifer yellow, biocytin, 6-carboxyfluorosceine, sulforhodamine B, etc.) that have no biological relevance and solely diffuse along their concentration gradient. Also, not all Cxs have the same permeability for these intercellular tracers.
Moreover, the morphology, the location of coupling sites, and the electrical properties of astrocytes studied in situ and in cultures are not suitable for optimizing the information obtained by dual patch clamp recordings. Consequently, in the future it will be beneficial to develop and use new molecules or probes that will allow the tracking of biological signals which are relevant to astrocytic gap junctions. Also, many of the experiments performed to demonstrate that gap junctions in the CNS are permeable to signaling molecules used cultured cells. This limits the relevance of these observations, and in future this should be assessed in more integrated systems to weigh the physiological significance of this mode of intercellular communication. Finally, the role and the real contribution of Cx hemichannels is still unclear and needs further investigation to evaluate the conditions under which they are functional and which physiologically relevant molecules cross them. See also: Gap Junctions and Electrical Synapses; Gap Junctions and Hemichannels in Glia; Gap Junctions and Neuronal Oscillations.
Further Reading Bennett MV, Contreras JE, Bukauskas FF, and Saez JC (2003) New roles for astrocytes: Gap junction hemichannels have something to communicate. Trends in Neurosciences 26: 610–617. Bukauskas FF and Verselis VK (2004) Gap junction channel gating. Biochimica Biophysica Acta 1662: 42–60. Giaume C and McCarthy KD (1996) Control of gap-junctional communication in astrocytic networks. Trends in Neurosciences 19: 319–325. Giaume C, Tabernero A, Medina JM, Swanson RA, and Magistretti PJ (1997) Metabolic trafficking through astrocytic gap junctions. Glia 21: 114–123. Hormuzdi SG, Filippov MA, Mitropoulou G, Monyer H, and Bruzzone R (2004) Electrical synapses: A dynamic signaling system that shapes the activity of neuronal networks. Biochimica Biophysica Acta 1662: 113–137. Nagy JI and Rash JE (2000) Connexins and gap junctions of astrocytes and oligodendrocytes in the CNS. Brain Research Reviews 32: 29–44. Nakase T and Naus CC (2004) Gap junctions and neurological disorders of the central nervous system. Biochimica Biophysica Acta 1662: 149–158. Pereda AE, Rash JE, Nagy JI, and Bennett MVL (2004) Dynamics of electrical transmission at club endings on the Mauthner cells. Brain Research Reviews 47: 227–244. Rouach N, Avignone E, Meˆme W, et al. (2002) Gap junctions and connexin expression in the normal and pathological central nervous system. Biology of the Cell 94: 457–475. Scemes E (2000) Components of astrocytic intercellular calcium signaling. Molecular Neurobiology 22: 167–179. Theis M, So¨hl G, Eiberger E, and Willecke K (2005) Emerging complexities in identity and function of glial connexins. Trends in Neuroscience 28: 188–195. Volterra A and Meldolesi J (2005) Astrocytes, from brain glue to communication elements: The revolution continues. Nature Reviews Neuroscience 6: 626–640.
Gap Junctions and Electrical Synapses M V L Bennett, Albert Einstein College of Medicine, New York, NY, USA ã 2009 Elsevier Ltd. All rights reserved.
Introduction Synapses are specialized areas of contact between neurons, between sensory cells and neurons, or between neurons and effector cells. Synapses mediate communication between cells; synaptic transmission from pre- to postsynaptic cell can be mediated by electrical current flowing from the presynaptic cell or by a neurotransmitter molecule secreted by the presynaptic cell. (Neurons are not known to communicate by mechanical means, but some muscles are mechanosensitive, and junctions between them can mediate mechanical transmission.) A key word in the first sentence is ‘specialized’; there may be disagreement about what it means. Electrical and chemical interactions can occur over greater separations than at the canonical synapses. ‘Ephapse’ and ‘ephaptic transmission’ have been used to describe electrical interaction at a fortuitous close apposition of two cells; ‘field effects’ denotes interactions across greater distances and may involve many cells. Analogs for chemical transmission include transmitter ‘spillover,’ in which a high frequency of presynaptic secretory activity causes transmitter to diffuse beyond where its usual receptors are located, and paracrine action, in which transmitter is released and acts at some distance without an obviously specialized relation between secreting and responding cells. Endocrine actions by hormones involve widely separated secretors and targets. The same molecule can be a transmitter at synapses, have paracrine actions, or be a hormone, such as epinephrine. The most common and best characterized electrical synapses are gap junctions between neurons. A gap junction is a cluster of small channels connecting the interiors of adjoining cells (Figure 1(b)). There are also electrical inhibitory synapses, originally described at the Mauthner cell of teleosts and subsequently in the mammalian cerebellum. This type of synapse is uncommon but will be described briefly in the section titled ‘Electrical Inhibitory Synapses’. Some appositions between neuronal cell bodies are specialized in that intervening glia are absent (e.g., hippocampal pyramidal cells, some hypothalamic neurons), but there do not appear to be gap junctions. Electrical interactions are more likely to be found between these cells than between neurons separated
by glial ensheathment. If there are interactions, these relatively unspecialized appositions might well be considered electrical synapses. Since gap junctions provide ultramicroscopic cytoplasmic continuity between cells, their presence could be viewed as contradicting Cajal’s neuron doctrine that each neuron is a distinct cell (although he wrote in his wonderful autobiography, “Neuronal discontinuity . . . could sustain some exceptions”). As will be seen, the properties of gap junctions are reasonably subtle in spite of their relative simplicity; a functional syncytium, as coupled cells have been described, does not necessarily mean that all cells do the same thing.
Properties of Gap Junctions Molecular Composition of Gap Junctions: Connexins and Pannexins
Known gap junctions in vertebrates are composed of connexins, encoded by a gene family in mammals of 20 members. Connexins are commonly named by their predicted molecular mass to the nearest kilodalton with a prefix for species or an additional significant figure where necessary to avoid ambiguity, as in human Cx30, Cx30.2, and Cx30.3, encoded by three distinct genes. Molecular weights range from 25 to 57 kDa. Most connexin genes have the entire coding region in a single exon; the Cx36 gene and several others have the coding region in two exons. In some connexins, alternative splicing in the 50 noncoding region permits use of different tissue-specific promoters. The connexins are tetraspan integral membrane proteins, that is, they cross the membrane four times. The N- and C-termini (NT, CT) are cytoplasmic, and there are four transmembrane domains (TM1–TM4) connected by two highly conserved extracellular loops (E1 and E2) and a cytoplasmic loop (CL). This topology was confirmed for several connexins by antibody labeling and identification of peptides following protease treatment that cleaved at exposed sites. The degree of conservation of sequence differs along the molecule. The N-terminus, membranespanning regions and extracellular loops are well conserved; the cytoplasmic loop and C-terminal domain are much more variable, in length as well as sequence, and length of the CT domain accounts for most of the differences in molecular weight. Conservation of the extracellular loops may reflect the shared function of docking with another hemichannel. Several regions in the CT contain binding sites for protein–protein interaction, including with the cytoskeletal protein ZO-1.
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Figure 1 Gap junction morphology. (a) A gap junction in thin section, an electrical synapse on a Mauthner cell dendrite. A long gap junction is seen between the axon (upper) and dendrite (lower). The axon also contains presynaptic vesicles. In an enlarged view (inset) the gap junction exhibits the typical seven-layer structure. (b) Diagram of a gap junction showing how hexamers of the subunit protein, connexin, in each membrane form a channel crossing the extracellular gap. (c) Immunofluorescence stain of ventricular myocytes with an anticonnexin 43 antibody. Intercalated discs and some lateral appositions are labeled. (d) Immunogold labeling of a cardiac gap junction with the same antibody, electron micrograph. (a, c) From Bennett MVL, Barrio LC, Bargiello TA, Spray DC, Hertzberg E, and Saez JC (1991) Gap junctions: New tools, new answers, new questions. Neuron 6: 305–320. (b) From Makowski L, Caspar DL, Phillips WC, and Goodenaugh DA (1977) Gap junction structures. II. Analysis of the x-ray diffraction data. Journal of Cell Biology 74: 629–645.
Given that the human and mouse genomes have been sequenced, few additional mammalian connexins are likely to be found. Different connexins assemble to form junctions that differ in single-channel conductance, gating, permeability (depending on both size and charge), and temporal and spatial patterns of expression. At a gap junction, each cell membrane provides hemichannels, or connexons, that dock one to one with hemichannels in the other membrane. Hemichannels are hexamers, homomeric if they are comprised of one kind of connexin and heteromeric if they are comprised of more than one kind. Many connexins are expressed in more than one kind of cell, and co-expression of multiple connexins is common in a given cell type. Heteromeric hemichannels do occur, although their prevalence and stoichiometry are poorly known. (Factors controlling oligomerization are not well understood, but assembly does not appear to be random.) Hemichannels of the same kind form homotypic junctions;
hemichannels of different kinds form heterotypic junctions. It is also useful to distinguish homo- and heterocellular junctions, that is, junctions between the same or between different types of cells, and these junctions may be homo- or heterotypic. (The terms ‘homologous’ and ‘heterologous’ are sometimes used for junctions between the same and between different kinds of cells, respectively.) ‘Compatibility’ between connexins, the property that allows the formation of heterotypic junctions, has been determined for only a small fraction of the 190 possible pairwise combinations of homomeric hemichannels (for 20 different connexins), but it is not universal and may be uncommon. For example, Cx32, Cx36, and Cx43 do not form junctions in any of the three pairwise heterotypic combinations. Compatibility in this sense is only one of the factors determining where gap junctions form; cell adhesion molecules and almost certainly other as yet undetermined mechanisms are also operative.
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The connexins found in the nervous system include the following: the most common neuronal connexin is Cx36 (which is also expressed in pancreatic islets). An additional neuronal connexin is Cx45. Retinal horizontal cells are coupled by Cx57 junctions. Other connexins may be expressed by neurons transiently during development. Astrocytes express predominantly Cx43, but also Cx26 and Cx30. Oligodendrocytes express Cx29, Cx32, and Cx47. Distributions may vary. Astrocyte Cx26 and Cx30 can mediate coupling to oligodendrocyte Cx32. Identification of the connexin genes permits gene knockout, antisense or small interfering RNA(siRNA) knockdown, replacement of the coding sequence with a reporter gene, and exogenous expression in cell lines or other tissues. Knowledge of sequence also permits in situ hybridization, polymerase chain reaction, and the raising of antibodies against specific (or common) sequences to be applied to identification and localization of connexin expression. Site-directed mutagenesis can be used to identify residues critical for particular junctional properties. These approaches have led to an explosion of knowledge of function and distribution. Although most mammalian connexins have orthologs in other vertebrates and known vertebrate gap junctions are connexin based, gap junctions in nematodes, mollusks, and arthropods (the protostome line) are formed of a family of proteins known variously as innexins (which they were named when they were thought to be expressed only in invertebrates) or pannexins (when it was demonstrated that the same gene family is found in both major branches of the animal kingdom, protostomes and deuterostomes, which include vertebrates). Cnideria have pannexins, and connexins may have evolved in the deuterostome
lineage. C. elegans has 25 innexins/pannexins; Drosophila has eight. Mouse and human each have three pannexins. Two of the mammalian pannexins are expressed in neocortex, hippocampus, and cerebellum, apparently in association with the postsynaptic density. The mammalian pannexins can form gap junctions when exogenously expressed in Xenopus oocytes but may only form hemichannels in mammals. The many convergences and few differences between connexin gap junctions in deuterostomes and pannexin gap junctions in protostomes will be noted in the section titled ‘Evolution.’ Otherwise, gap junction will refer to connexin-based junctions. Structure
In an early electron microscopic thin section study of gap junctions, the extracellular space between the adjoining cells appeared reduced to a narrow gap (Figure 1(a)), and the channels crossing the gap were not well resolved. Thus, the term ‘gap junction’ derives from a structural characteristic not obviously related to function. The gap distinguishes these junctions from ‘tight junctions’ or ‘zonulae occludentes’ (singular: zonula occludens), in which the intercellular space between adjoining cells appears completely occluded. The gap could be penetrated by small extracellular markers, such as colloidal La(OH)3. Freeze-fracture replicas in the plane of the junction show junctional particles on the protoplasmic face and corresponding pits on the external face (Figures 2 and 3). These structures are often hexagonally packed. With good resolution, the particles show a central depression that presumably is the site of the aqueous channel (Figure 2, inset). Gap junctions can be isolated by several techniques that
PF
EF 50 nm Figure 2 Gap junction between blastomeres of a fish shown by freeze fracture electron microscopy. The protoplasmic leaflet of the plasma membrane (P face, or PF) lies to the upper left; the external leaflet (E face, or EF) lies to the lower left. The gap junction is characterized by an aggregate of P-face particles and complementary E-face pits where the particles were pulled out in the fracture process. The inset shows the particles at higher magnification. Many of them show a small central pit that corresponds to the channel. Reprinted from Ne’eman Z, Spira ME, and Bennett MV (1980) Formation of gap and tight junctions between reaggregated blastomeres of the killfish, Fundulus. American Journal of Anatomy 158: 251–262, with permission of Wiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc. Copyright (1980).
196 Gap Junctions and Electrical Synapses
Figure 3 Freeze fracture replica immunolabeling (FRIL) of gap junctions between cells of the nervous system. Gap junctions, characterized by aggregates of external leaflet face (E-face) pits or plasma membrane face (P-face) particles, are clearly labeled by specific antibodies followed by secondary antibodies with attached gold beads of predetermined diameter. The technique labels only a small fraction of the replicated channels, and the length of the antibodies permits the gold beads to be some distance from the edge of the junctions. (a) A dendrite in the inferior olive of the rat shows two gap junctions as arrays of E-face pits. 1 and 2 are endings on the dendrite; DS is a cross-fractured dendritic spine. The gap junctions are shown enlarged in (b, c), and each is immunolabeled with six 20 nm gold beads. The primary antibody is against Cx36, a neuron-specific connexin. (d) Gap junctions on an inferior olive oligodendrocyte show both P-face particles (Ol P) and E-face pits; the junctions are labeled with an anti-Cx32 primary antibody and 20 nm gold beads; the rightmost junction is enlarged. A gap junction on an astrocytic process in the same replica (lower left, identified by aquaporin square arrays of particles not included in the micrograph) is negative for Cx32, but these junctions can be labeled using Cx43 antibodies. Scale bar ¼ 100 nm (a–c). Reproduced from Rash JE, Staines WA, Yasumura T, et al. (2000) Immunogold evidence that neuronal gap junctions in adult rat brain and spinal cord contain connexin-36 but not connexin-32 or connexin-43. Proceedings of the National Academy of Sciences of the United States of America 97: 7573–7578, with permission from National Academy of Sciences, USA. Copyright (2000).
dissolve away surrounding membrane. Isolated junctions, when negatively stained, usually show domains of hexagonal symmetry (Figure 4). There is a central dot at the presumed site of the channel lumen and a hexagonal lattice surrounding the dots that results from stain in the intercellular gap, which is interrupted by the bridging structures (cf. Figure 1(b)). Atomic force microscopy of separated junctional membranes confirms the general features of the junctions and suggests structural changes associated with gating (Figure 5). Low-dose electron microscopy of isolated junctions reveals four alpha helical regions crossing the membrane, but matching to the amino acid sequences is not yet possible (Figure 6). Scanning cysteine accessibility mutagenesis is revealing channel lining residues, although the data are still somewhat controversial. When hemichannels come together to form a cell– cell channel, one hemichannel is rotated by 30 relative to the other (Figure 7). Antibodies against the junctional protein or connexin can be used to label gap junctions at the light and electron microscopic levels; the intercalated discs of cardiac ventricle are
Figure 4 Isolated liver gap junction from rat, negatively stained with uranyl formate. The junctional channels are hexagonally arrayed. The hexagonal lattice represents stain in the extracellular gap; the central dots mark the location of the channel. From Hertzberg EL and Gilula NB (1979) Isolation and characterization of gap junctions from rat liver. Journal of Biological Chemistry 254: 2138–2147.
illustrated in Figures 1(c) and 1(d)). Freeze fracture followed by immunolabeling of residual protein on the replica allows precise localization of particular connexins in identifiable gap junctions (Figure 3).
Gap Junctions and Electrical Synapses 197
Figure 5 Atomic force microscopy of external face of an isolated gap junction that has been split open mechanically. The same junction in zero Ca2þ (left) shows larger openings than after addition of 0.5 mmol l1 Ca (right). The insets are averages. The (hemi)channel has six subunits. The circles indicate channels that presumably have been damaged. The apparent openings in the channels are wider in zero Ca2þ (left) than in 0.5 mmol l1 Ca2þ, a concentration that closes gap junction channels when in the cytoplasm. From Muller DJ, Hand GM, Engel A, and Sosinsky GE (2002) EMBO Journal 21: 3598–3607.
C
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Figure 6 Molecular organization of a gap junction channel. (a) Side view. (b) Side view showing the channel interior. The approximate boundaries for the membrane bilayers (M), extracellular gap (E), and the cytoplasmic space (C) are indicated to the left. (c) Cross-sections parallel to the membrane at the levels indicated by the arrows in (b). The red contours in (c) define the boundary of the channel. The yellow contours are at increased density and are consistent with four transmembrane alpha helices for each of the six connexins, as predicted from the amino acid sequence. The red asterisk in (b) marks the narrowest part of the channel, where the channel lumen is 1.5 nm in diameter, excluding the contribution of amino acid side chains that were not resolved. Comparison of upper and lower cross sections in (c) shows the 30 rotation in docking the two hemichannels. Note that both are viewed from the same side of the channel and are therefore mirror images. From Figure 2 in Unger VM, Kumar NM, Gilula NB, and Yeager M (1999) Three-dimensional structure of a recombinant gap junction membrane channel. Science 283: 1176–1180.
Distribution
Gap junctions connect many kinds of nonneuronal excitable cells where they mediate propagation of action potentials, such as in heart, smooth muscle, and pancreatic islets. Gap junctions are also found widely in inexcitable tissues, such as liver, epidermis, exocrine pancreas, salivary glands, neuroglia, and
lining of the gut from mouth to anus. Although gap junctions are very common in these tissues, they are absent in many contacts between specific kinds of cells. They are not found between adult vertebrate skeletal muscle fibers or erythrocytes and are rare or absent between neurons and glia and between many kinds of neurons that come into close contact.
198 Gap Junctions and Electrical Synapses
Cytoplasmic surface
Extracellular surface
a
b Figure 7 Fitting of the extracellular surfaces of two hemichannels to form a cell–cell channel. (a) Surface representation of a single hemichannel (connexon) viewed from the cytoplasm, from the membrane side, and from the extracellular side. The structure of the external and cytoplasmic surfaces are quite different. (b) Two hemichannels fit together at their external surfaces by rotating one 30 . From Perkins GA, Goodenough DA, and Sosinsky GE (1998) Formation of the gap junction intercellular channel requires a 30 degree rotation for interdigitating two apposing connexons. Journal of Molecular Biology 277: 171–177.
Electrical Coupling and Circuit Parameters
When a current is passed in one cell to change its potential, a smaller potential change is recorded in cells joined to it by gap junctions (Figure 8). Current flows from cell to cell through the junctional channels just as in electrotonic spread along a core conductor (hence the terms ‘electrotonic junction’ and ‘electrotonic synapse’); the gap junctions act as a conductor, and both hyperpolarizing and depolarizing potentials are recorded in the follower cell. This current is carried largely by Kþ ions, the primary mobile ion in the cytoplasm. Before concluding that the cells are coupled by gap junctions, cytoplasmic continuity must be excluded, which is commonly done with gap junction blockers (see the section titled ‘Pharmacological block of gap junctions’) or by injecting a visualizable molecule that is too large to permeate gap junctions, such as fluorescently labeled dextran (see the section titled ‘Dye coupling and permeability’). Also, recording outside the cells may be necessary to demonstrate that the intracellular potential in the coupled cell is not a result of a voltage drop in the extracellular space. Both electrical and dye (see the section titled ‘Voltage gating of gap junctions’) measurements indicate that there is little leakage from the channels as
they cross the gap and that they constitute a private pathway between the cell interiors that is not accessible from the extracellular space. For steady state potentials, the ratio of pre- to postjunctional potential is termed the coupling coefficient or coupling ratio. In the equivalent circuit of two isopotential cells connected by a gap junction, each cell is represented by its conductance and capacitance in parallel, and the cells are connected by the junctional conductance, gj (Figure 9). (Isopotential means that the cytoplasmic resistance is small compared with the membrane resistance and that all points within the cell are at virtually the same potential. In neuronal processes, i.e., the axon and dendrites, cytoplasmic resistance is no longer negligible, and the potentials in these processes can differ from that in the soma.) For small potentials, junctional and nonjunctional conductances can be treated as constant. The junctional membrane has little capacitance compared with nonjunctional membrane because of its small area and the presence of the gap, which is likely to be at a potential close to that of the surrounding extracellular space. The parameters of steady state transmission (which excludes the effect of cell capacitances) are easily described using the circuit. The
Gap Junctions and Electrical Synapses 199 Bipolar 1 to bipolar 2
Bipolar 2 to bipolar 1
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Figure 8 Electrical coupling mediated by gap junctions between bipolar (inhibitory) interneurons in the visual cortex of the rat. Four superimposed records with current applied in cell 1 in the upper set in (a) and in cell 2 in the upper set in (b); recordings from the follower cell at higher gain in the middle sets of traces. Both hyperpolarization and subthreshold depolarization spread between cells. Larger polarizations in the polarized cell result in larger polarizations in the follower cell, and the relation is essentially linear under these conditions. Impulses, which are evoked by the strongest current, are more attenuated than steady polarizations, and the peak of the ‘spikelet’ in the postsynaptic, nonstimulated cell is delayed compared to the presynaptic spike; superimposed presynaptic spikes and postsynaptic responses are shown in the bottom records at greater time resolution. Pre- and postsynaptic are defined here as the stimulated and follower cells; the synapse is symmetric, and transmission is bidirectional. Reproduced from Venance L, Rozov A, Blatow M, et al. (2000) Connexin expression in electrically coupled postnatal rat brain neurons. Proceedings of the National Academy of Sciences of the United States of America 97: 10260–10265, with permission from National Academy of Sciences, USA. Copyright (2000).
Vpre
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Figure 9 Equivalent circuit of a coupled pair of cells. (a) Isopotential cells: Each of the cell has a nonjunctional conductance (g1, g2) and a membrane capacitance (C1, C2). The cells are joined by a junctional conductance, gj. Current is applied and voltage measured in each of the cells (I1, I2, V1, V2). Capacitance of the junctional membrane is negligible. (b) An axosomatic synapse. The postsynaptic response (electrical excitatory postsynaptic potential, EPSP, Vpre) is attenuated and delayed with respect to the presynaptic impulse, Vpre. The delay introduces a synaptic delay that can be comparable to delays at chemical synapses. gj and gpost, junctional and postsynaptic conductances; c, postsynaptic capacitance. (b) From Bennett MVL (2000) Seeing is relieving: Electrical synapses between visualized neurons. Nature Neuroscience 3: 7–9.
coupling coefficient for a current applied in cell 1 is the ratio of the potential generated in cell 2 to that in cell 1: k12 ¼ V2/V1. If the nonjunctional conductances of the cells are g1 and g2 and the junctional conductance is gj, k12 ¼ gj/(g2 þ gj), and for currents applied in cell 2, k21 ¼ gj/(g1 þ gj). Note that k12 is independent of g1, and k21 is independent of g2.
If k12 is small and gj < g2, g2 I2/V2 and gj g2k12, and similarly for k21. Where the circuit of Figure 9(a) is valid, one can apply current first in one cell and then in the other and from the resulting voltages calculate the cell and junctional conductances precisely, but the equations are somewhat messy. A simpler method to determine gj is
200 Gap Junctions and Electrical Synapses
to voltage clamp the two cells to a common holding potential by means of a voltage clamp and then step the voltage in one cell to a new value. The current applied in the other cell to keep its voltage constant is equal and opposite to the current flowing through the junction at the given transjunctional voltage, Vj ¼ V1 V2 and gj ¼ I2/Vj the minus sign is because the current into cell 2 is opposite in sign from the current flowing through gj); g1 is given by ( I1 I2)/V1. Stepping cell 2 while holding V1 constant measures gj and g2. (If both cells are stepped to the same potential, Vj ¼ 0, g1 ¼ I1/V1, and g2 ¼ I2/V2.) Equality of gj for Vj of either sign is an important validation of the equivalent circuit. If the cells are not isopotential and even if they are part of a large coupled network, one can still calculate effective values for g1, gj, and g2 for any two recording sites. However, the values will not correspond in any simple way to the conductances of the gap junctions coupling the cells, and coupling conductance, or gc, is a more accurate term than gj. Voltage Gating of Gap Junctions
Gap junctions formed of most connexins exhibit varying degrees of sensitivity to Vj. In dual voltage clamp, a large step in cell 1 causes a current in cell 2 that decreases to a new steady state more or less exponentially, and this new value is smaller for larger Vj (Figure 10). (A virtue of dual voltage clamp is that the change in gj is immediately apparent in the I2 record.) In homotypic junctions, the decrease is in most cases symmetric for transjunctional voltages of hCx32
either sign generated by hyperpolarization or depolarization in either cell. This finding indicates that the junctions are insensitive to the potential between the interior of the cells and the external medium. A few connexin-based junctions show a minor degree of dependence on the voltage between the cell interior and the extracellular medium (Vi-o or Vm). The eponymous gap at the junction is accessible to the extracellular medium, so there should be little or no potential change along the gap, and the voltage across the channel walls should be the voltage between the channel lumen and the extracellular space at some distance from the junction. If the voltage differs in the two cells, the voltage is not uniform along the channels. The steady state macroscopic currents for each polarity of Vj can be reasonably described by a Boltzmann relation: gj ¼ ½gimax gjmin =ð1 þ exp½AðV j V 0 ÞÞ þ gjmin where gjmax is the maximum value of gj, which is approached at large values of Vj; A is a constant expressing voltage sensitivity; and V0 is the voltage at which the conductance is halfway between gmax and gmin. The Boltzmann relation describes the equilibrium between two states, in this case high and low conductance, where the energy difference between them is a linear function of Vj V0 and A ¼ nz/(kT), where n is the equivalent number of electron charges, z, moving through the full voltage Vj, and kT has its usual meaning. The time course of changes in macroscopic currents in response to voltage steps
hCx32-220 stop
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Vj Figure 10 Voltage-dependent gating of gap junctions formed by human Cx32 wild-type and a mutant, Cx32-220 stop, one of many mutations that cause Charcot–Marie–Tooth disease, a peripheral neuropathy. The cRNAs were expressed in Xenopus laevis oocytes that were then paired and allowed to form junctions. The upper sets of traces show voltages applied in one cell (Vj) while the other cell was clamped at the holding potential. The lower sets of traces are the currents in the cell clamped at the holding potential and are equal and apposite to the transjunctional currents (Ij) associated with the voltage steps. The graph shows the steady state junctional conductance (gj) as a function of transjunctional voltage (Vj). gj decreases quite symmetrically for increasing Vj of either sign. The decrease approaches a plateau value accounted for by the residual conductance seen in voltage gating at the single-channel level (see Figure 11). The decline is steeper for the mutant (filled circles) than for wild-type (triangles). From Revilla A, Castro C, Barrio LC (1999) Molecular dissection of transjuctional voltage dependence in the connexins-32 and connexins-43 junction. Biophysical Journal 77: 1374–1383.
Gap Junctions and Electrical Synapses 201
is approximately exponential, consistent with a Boltzmann process. Single Channels and Gating
The conductances of single gap junction channels of known composition can be measured in connexindeficient, high-resistance cells after transfection with specific connexins. In well-coupled cells, the number of open channels can be reduced with various blocking agents (see the section titled ‘Pharmacological block of gap junctions’) until single-channel activity is observable (Figure 11). Alternatively, separated cells can be apposed and the formation of junctions observed at the level of the first channels to open. The clear transitions between closed and quite stable open states, and often substates, allow identification of single-channel conductances. These conductances range between 15 pS for Cx36 and 200 pS for Cx37. Two modes of voltage gating, fast and slow, can be identified from single-channel recordings. Fast gating occurs on a millisecond timescale between the fully open state and a residual state with a conductance in the range of 10–30% of the maximal value. Slow gating requires 5 ms or more and occurs between the fully open state and the substate or the fully closed state. It appears to result from poorly resolved transitions between multiple unstable substates. For most connexins studied, both kinds of gate have a high open probability at Vj ¼ 0, and open probability is decreased by increasing Vj. The more rapid rate constants of the fast gating process and its greater voltage sensitivity cause it to dominate the current–voltage relation. In this case gj for either sign
of Vj can be reasonably described by a Boltzmann relation for each of the oppositely oriented series hemichannels; one sign of Vj closes the gate in one hemichannel and has little effect on the gate in the other hemichannel. In this description, probability of being in the fully open state is the dependent variable; macroscopic gj is given by the open probability Po multiplied by the number of channels, N, and single-channel conductance, g. gj ¼ Po Ng The oppositely oriented gates in the two halves of a cell–cell channel are closed by opposite signs of Vj, which accounts for the symmetry of the I/V relation. Symmetry, however, precludes inference of the sign voltage sensitivity of the individual hemichannels from these data. Comparison of heterotypic and homotypic junctions or experimental induction of asymmetry by, for example, unilateral acidification, does allow determination of gating polarity, and it has been shown that different connexins can form hemichannels with opposite signs of gating polarity. Moreover, charged amino acids that constitute part of the voltage sensor have been identified, and charge substitutions in the N-terminal residues can reverse the gating polarity. While only an approximation, and definitely not mechanistically justified for slow gating, Boltzmann relations are a convenient descriptor of the degree of voltage sensitivity of fast gating, as well as of slow gating when fast gating is absent. Voltage sensitivity in terms of charge can approach that of the channels
5 pA
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Figure 11 Single-channel currents showing fast gating between the fully open state and a substate and slow gating between the closed and fully open states. A well-coupled pair of fibroblasts expressing Cx43 were exposed to CO2 equilibrated saline, which blocked the junctional conductance. (a) The upper record shows the last two channels closing in response to cytoplasmic acidification (arrows). The solid lines indicate the conductance level of the two open channels (10, 20). The dotted lines (s) indicate the substate conductance level, and c indicates the closed state. The boxed record below shows at higher gain and faster timescale two sojourns to the substate via fast transitions before the last slow transition to the closed state. The small circles on the records occur at 1 ms intervals. (b) The upper record shows the first two channels to open again after 10 min washing in normal saline (arrows). The second channel opens fully while the first channel is in the substate (right arrow); subsequent fast transitions between open state and substate cannot be unambiguously assigned to one or the other channel. The boxed record below shows the initial slow transition from closed to open state, followed by a fast transition to the substate and a fast transition from substate to open state. From Bukauskas FF and Peracchia C (1997) Two distinct gating mechanisms in gap junction channels: CO2-sensitive and voltage-sensitive. Biophysical Journal 72: 2137–2142.
202 Gap Junctions and Electrical Synapses
of impulse-generating membrane, but the transition rate constants are long, in many cases hundreds of milliseconds to seconds. For a given connexin, the slow gating mechanism may have voltage sensitivity opposite that of the fast gating mechanism.
For most gap junctions, Vj dependence of gj appears too weak and slow to have much physiological significance and appears unsuitable for processing of neuronal impulse activity. For some tissues, such as heart, with its long-duration action potentials, voltage insensitivity could be an advantage.
Heterotypic Channels, Gating Asymmetry, and Rectification
Dye Coupling and Permeability
Hemichannels formed of different connexins can form heterotypic gap junctions that are quite symmetric or very asymmetric. Asymmetry of voltage gating results in junctional conductance that depends on polarity of transjunctional voltage, but the characteristic rates of change in conductance are slow, as described in the preceding section. At one extreme, the hemichannels have opposite polarity of voltage sensitivity referred to the voltage at their cytoplasmic ends, and both hemichannels are closed by the same sign of Vj. The macroscopic current–voltage relation of these junctions is very asymmetric. Another factor in gating asymmetry is single hemichannel conductance. If a low-conductance hemichannel is in series with a high-conductance hemichannel, most of an applied voltage will be developed across the low-conductance (high-resistance) hemichannel. Its sensitivity to an applied Vj will be increased relative to its sensitivity in a homotypic junction. Similarly, the sensitivity of the high-conductance hemichannel will be reduced compared to that in its homotypic junction. In heterotypic junctions, small shifts in resting potential difference between two cells can cause large changes in junctional conductance and may be important in controlling chemical communication. Asymmetric junctions can also show fast rectification (important in some rectifying synapses) in which the open channel rectifies, presumably on a microsecond timescale, and passes more or less current for a given transjunctional voltage, dependent on its sign. Rectifying electrical synapses are morphologically asymmetrical in that they occur between cells of different types and have a preferred direction of transmission. The polarity of rectification determines direction of impulse propagation and which elements are the pre- and postsynaptic ones. The constituent connexins have not yet been identified and may be encoded by different genes, or the same connexin on both sides may be differentially modified posttranslationally. The rectification is ascribed to differences in fixed charge in the constituent hemichannels. Fast rectification in inwardly rectifying K channels is due to channel block by internal polyamines. Similarly, differences in the composition of cytoplasm on the two sides of a gap junction could lead to rectification.
A cell may be injected with (or induced to take up) a probe molecule such as a dye or a radioactive tracer, and contacting cells can then be assayed for appearance of the probe (Figure 12). Measurements of this kind indicate that the channel diameter is 1.4 nm, and molecules of molecular weight of 1 kDa can permeate (as can extended molecules of somewhat larger molecular weight, such as short interfering RNAs). The channel can be traversed by small ions, polypeptides, and nucleotides, including for some connexins cyclic adenosine monophosphate (cAMP), adenosine triphosphate (ATP), and other molecules of intermediary metabolism. This chemical communication may mediate metabolic cooperation in which one cell provides a molecule that another cannot synthesize or where random metabolic differences between cells are compensated for. Some but not all connexins form junctions with significant charge selectivity, and maximum size for permeation varies with connexin (Figure 12). Gap junctions are impermeable to large polypeptides, long nucleic acids, and, of course, subcellular organelles. Dye coupling can be quantified in terms of junctional permeability, which is conveniently normalized to junctional conductance. For probes to which the surface membrane is impermeable, the dye coupling coefficient, defined as steady state ratio of concentration in a recipient cell to that in the source cell, is unity. However, in large networks of cells, the time to reach equilibrium may be longer than the usual measurement time, and postjunctional concentration may not reach its theoretical final value. Gap junctions do not rectify in respect to dye flux; in the absence of an asymmetric driving force, permeation is not polarized. However, dye coupling between a large cell and a small cell can be asymmetric at short times; the same dye flux in one or the other direction would produce a higher concentration in the smaller cell. Differences in nonjunctional membrane permeability could also lead to apparent asymmetry in dye coupling. A further issue in asymmetric systems is degree of binding, which can alter fluorescence of the bound molecules and concentration of the free dye in the cytoplasm. A crude but convenient technique to measure dye coupling is scrape loading. As commonly used,
Gap Junctions and Electrical Synapses 203 Lucifer yellow2− gj = 18 nS
DAPI2+ gj = 20 nS
gj = 9 nS
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Figure 12 Cells joined by gap junctions show spread of dyes from one to another, that is, dye coupling. Dye coupling depends on the connexin, some of which are permeable to larger molecules than others are and some of which are charge selective. The figure shows Neuro-2a cells transfected with Cx46 (upper row), Cx32 (middle row), or Cx43 (lower row), phase micrographs on the left and fluorescence on the right in each pair of images. In each case, cell 1 was injected with the fluorescent dye Lucifer yellow (ion molecular mass 443 Da, two negative charges, left column) and DAPI (40 ,6-diamidino-2-phenylindole, ion molecular mass 279, two positive charges, right column). Cx46 junctions are cation selective and are impermeant to Lucifer yellow but show some permeation by DAPI, which fluoresces when bound to nuclei. Cx32 junctions are anion selective and are permeable to Lucifer yellow but not to DAPI. Cx43 junctions readily transfer both Lucifer yellow and DAPI. Junctional conductance, gj, is shown for each cell pair, and low level of coupling did not account for selectivity. From Trexler EB, Bukauskas FF, Kronengold J, Bargiello TA, Verselis VK (2000) The first extracellular 100p domain is a major determinant of charge selectivity in connexin 46 channels. Biophysical Journal 79: 3036–3051.
a confluent monolayer is cut with a razor blade in the presence of dye. Many of the cells can heal over, trapping dye in their cytoplasm. The dye bathing solution is then washed away, permitting observation of dye coupling to cells at some distance from the cut. Simultaneous application of a larger, junction-impermeant molecule with a different fluorescence allows identification of cells that were cut open, which have both markers, and the recipient cells, which have only the smaller, permeant species. Formation and turnover In addition to the rapid gating mechanisms, specific treatments can induce cells to form gap junctions or to remove them. Most types of connexin hemichannel are assembled in membrane of the endoplasmic reticulum (ER) or post-ER compartments. Cx26 may be an exception and can insert into membranes posttranslationally. Vesicles with hemichannels are transported to the cell surface and inserted into the external membrane
(Figure 13). Hemichannels then diffuse in the plane of the membrane until finding a hemichannel in an apposed membrane. Unapposed hemichannels in the surface in general are not open. The final steps are apposition or docking of intact hemichannels in the two membranes (Figure 7) and interaction with the extracellular loops to form the complete channel in a kind of ligand–receptor interaction in which each is a molecule of the same kind. When the first channel opens, the current through the nonjunctional membrane is unchanged (Figure 14). Thus, the hemichannels forming the first channel are not open to the extracellular space before they open to connect the two cells. (In some conditions, physiological or pathological, hemichannels open without interacting with a hemichannel in an apposed membrane (see the section titled ‘Hemichannels that open’). Gap junctions can grow by accretion of new channels at their periphery because membranes are closely apposed in this region rather than exhibiting the wider gap of nonjunctional appositions. Targeted
204 Gap Junctions and Electrical Synapses
Lumen becomes extracellular
(Enlarged) Insert and diffuse to find partner in an apposed membrane
Golgi
ER
Figure 13 Connexins are synthesized in the endoplasmic reticulum (ER) and assembled into hemichannels in the ER or later compartment. They are then transported to the surface as integral membrane proteins in vesicles, which are inserted. The hemichannels diffuse in the surface to find a hemichannel in an apposed membrane and then join to form a cell–cell channel; each cell contributes a hemichannel to a cell–cell channel. Unpublished diagram by Jorge E. Contreras.
delivery of vesicles with hemichannels to junctional regions has been reported, but membrane insertion can also occur without directional transport. The mechanism by which the separated membranes are pushed close enough together for initial junction formation is unknown. There is evidence that a large number of hemichannels must be clustered before the first cell–cell channel opens. Quantitation of junctional area and number of open channels determined from conductance indicates that only a small fraction of cell–cell channels are open, 10%, a fraction that is likely to be connexin dependent. It is not clear whether a subpopulation of channels opens with a larger probability or whether probability is uniform and small. The atomic force data in Figure 5 suggest that all the channels are changing conformation during gating. Normal connexin turnover time is a few hours in several cell types in culture and in rodent liver and heart. In marked contrast, gap junctions in the lens do not turn over in the lifetime of the organism after maturation of the lens fibers, in which their messenger RNA (mRNA) is lost. In turnover, junction removal apparently proceeds through internalization of the full thickness of the junction by one or the other cell (Figure 15) and may involve an entire junction or just a small region from the center. The hole in each cell that this mechanism requires presumably seals over with negligible leak. Internalization leads to the production of ‘annular’ gap junctions that can be seen in
the subsurface cytoplasm; these structures are the internalized junctions with a little perijunctional membrane, and they contain a small amount of cytoplasm from the ‘loser’ cell. The transfer of cytoplasm from one to the other cell could be important in antigen transfer and presentation. Eventually the internalized junctions are transported to lysosomes for degradation; there is no indication of recycling of hemichannels or connexins that have formed cell–cell channels. However, unapposed hemichannels in the surface membrane can enter the constitutive membrane recycling pathway and be internalized and reinserted. Activation of kinases may accelerate junction formation or removal, depending on the tissue. In some systems, precursor connexin appear to be present, and junction formation can occur spontaneously even when ATP levels are reduced; in other systems, protein must be synthesized. Formation requires cell adhesion, and block of adhesion can block junction formation. Also, specific antibodies to the extracellular loops or peptides corresponding to the extracellular loops can prevent junction formation. If junction turnover is sufficiently rapid, these agents potentially provide a highly specific method of blocking junctional communication. Degree of coupling can be regulated by controls of transcription and mRNA degradation as well as by those of formation and internalization. The cell biology of regulation of coupling is a field of active exploration.
Gap Junctions and Electrical Synapses 205
I1 I2
1
4 pA
2
1s
a V1 = ~ –50 mv
V2 = ~ 10 mV
I1 = g1 V1 + Ij
I2 = g2 V2 – Ij gj
g1
g2
b Figure 14 Hemichannels in cell–cell channels are closed before opening to connect the interiors of the apposed cells. (a) Opening of the first channels after washout of a agent that blocked gap junctions connecting two cardiac myocytes. (b) Equivalent circuit of the cells, where cell 1 and cell 2 each has a voltage, V1 and V2, a current, I1 and I2, and a conductance g1 and g2, and Ij is the current through gj, the junctional conductance. The first cell–cell channel to open caused equal and opposite currents in the two cells. Thus, the nonjunctional currents in the two cells were unchanged, indicating that the hemichannels were not open to the extracellular medium prior to opening to couple the cells. At the arrow, a second channel opened. The dotted lines indicate the substate conductances of the two channels. Similar results are obtained with the first opening of newly formed channels when dissociated cells are pushed together while recording. (a) From Valiunas V, Bukauskas FF, and Weingart R (1997) Conductances and selective permeability of connexin43 gap junction channels examined in neonatal rat heart cells. Circulation Research 80: 708–719.
Pharmacological block of gap junctions An important tool in functional analysis is the use of specific blockers. Although numerous blockers are available, such as heptanol, octanol, halothane, carbenoxolone, glycyrrhetinic acid, flufenamic acid, anandamide, and oleamide, they are not very specific. Octanol and heptanol are widely used but affect many membrane channels. Carbenoxolone and glycyrrhetinic acid are more specific but may also affect excitability properties. Mefloquine is quite selective for Cx36 junctions, common between neurons. Where measured, conductance is reduced to zero by the slow gating mechanism. Low cytoplasmic pH (Figure 11) and high Ca2þ block most if not all gap junctions. The sensitivity to pH varies with the connexin and possibly the tissue. Domains involved in pH sensitivity are being identified.
In some instances, conductance is close to maximum at resting pH, and small degrees of acidification markedly depress it. Here intracellular acidification is a plausible mechanism for cellular control of junctional conductance. In the few cases analyzed quantitatively, the sensitivity to Ca2þ appears to be less than that to Hþ. Calcium ions would, however, act to uncouple from its neighbors a cell whose surface membrane became leaky. ‘Healing over’ of an injury to cardiac muscle is thought to involve a decrease in junctional conductance due to an effect of this kind, although scrape loading and retention of a gap junction-impermeant molecule indicates that ruptured cells can indeed heal over. One can pretty reliably infer that if several gap junction blockers do not block a particular response, then the response is not mediated by gap junctions. At this time, it is much less reliable to infer that gap junctions mediate a response because gap junction blockers reduce or prevent it. So-called connexin(or pannexin-) mimetic peptides with sequences corresponding to regions of the extracellular loops, as well as molecular approaches using siRNAs, should be quite specific in preventing junction formation and will be useful where gap junctions turn over rapidly enough for an effect of loss of function to be observed (see the section titled ‘Formation and turnover’). Connexin knockout by recombinant techniques is and has been extremely useful but suffers from poor time resolution. Phosphorylation Probably most connexins are phosphoproteins. There are consensus sites for phosphorylation in the cytoplasmic C-terminal domain that vary among connexins. Cx43 has at least 14. Phosphorylation may result in an increase or decrease in macroscopic conductance associated with altered single-channel conductance or open time. For example, gap junctions between hepatocytes are increased in conductance by cAMP-increasing agents; conversely, conductance of the junctions between horizontal cells in the retina is decreased by the same treatments. Nitric oxide mediates synaptic modulation of gap junction synapses at several sites. Because these changes are relatively rapid and are likely to involve changes in single-channel properties rather than formation or removal, they may be considered a form of gating. Phosphorylation can also affect insertion and removal. What Do Gap Junctions Do for Neurons?
Transmission between neurons In the equivalent circuit of Figure 9, transmission of small potentials is governed by the steady-state relations described
206 Gap Junctions and Electrical Synapses Cell 1
Cell 2
Cell 1
Cell 2
Gap junction
Intercellular space a
b
Cell 1
Cell 2
Cell 1
The evagination of cell 2 has pinched off c
An evagination of cell 2 with the juntion protrudes into cell 1
d
Cell 2
Cell 1 has endocytosed the evagination of cell 2 and the junction
Figure 15 Removal of gap junction channels involves internalization of both junction membranes by one cell and transfer of a small volume of cytoplasm from one cell to the other. Successive stages of internalization of an entire junction are indicated, but small areas from the center of a junction can also be internalized. Between (b) and (c), the protrusion from cell 2 into cell 1 has pinched off from cell 2, and its membranes have resealed. Between (c) and (d), cell 1 has endocytosed the protrusion from cell 2 and resealed its membranes to include a small volume of extracellular material as well as the content of the protrusion.
above, and gj values can be determined. Generally, voltages in neurons are too small or brief to engage voltage dependence of gj, and the principle neuronal connexin, Cx36, is relatively insensitive to voltage. Because the membrane capacity of the nonjunctional membrane is much greater than that of the junctions, gap junction synapses act like a low pass filter (Figure 9). The postsynaptic potential is slowed because some of the junctional current goes to change the charge on the postsynaptic capacitance. Steady state coupling does not depend on capacity, but coupling is less for faster presynaptic potentials; peak amplitude is reduced, and the peak is delayed (Figures 8 and 9). One important consequence is that the measured synaptic delay can be as great at an electrical synapse as at a chemical synapse; although the electrical synapse starts to transmit without delay, time is required for the postsynaptic potential to reach a level at which it can be detected. The delays tend to be more nearly alike in warm-blooded
animals because the delay at chemical synapses is less at higher temperatures whereas the delay at electrical synapses is not very temperature sensitive. Slowing of the postsynaptic potential is generally an obvious feature of neural signaling across gap junctions. Neuronal action potentials are usually considerably briefer than the input time constant and so are relatively attenuated compared with slower potential changes such as synaptic potentials or applied hyperpolarization (Figures 8 and 9). In coupled cells, presynaptic impulses can produce postsynaptic responses that have been termed spikelets or fast prepotentials. Spikelets are briefer than synaptic potentials, and one hypothesis has been that they result from spikes generated in a dendrite that fail to invade the soma. While this may be true in some cases, dendritic spikes are often longer lasting than somatic spikes and spikelets. A factor that may contribute to the relatively short time course of an electrical postsynaptic potential (PSP)/spikelet is that the
Gap Junctions and Electrical Synapses 207
presynaptic action potential may have an afterhyperpolarization that reverses the current flow across the electrical synapse and shortens the postsynaptic response. In Cajal’s neuron, transmission is polarized. At a gap junction synapse, if g1 g2, then k12 k21 and coupling is much stronger in one direction that the other. Although inefficient, electrical coupling could be made arbitrarily unidirectional by adjusting the cell conductances. Unidirectional impulse propagation can also be achieved where the junctional membrane rectifies (see the section titled ‘Heterotypic channels, gating asymmetry, and rectification,’ above). At electrical axosomatic or axodendritic synapses, either electrical or chemical, impulses need not propagate from axon to postsynaptic cell on a one-to-one basis. Transmission is bidirectional, but coupling is weak, and the postsynaptic cell is excited only when there are enough presynaptic impulses to depolarize the cell to threshold. Impulses in the postsynaptic cell may or may not invade the electrically coupled presynaptic axons. In coupled groups of neurons, a significant part of the input conductance of a single cell can be due to the conductances into the junctions it makes with other cells, and there generally are multiple pathways connecting any two cells. Significant voltage drops can be developed along dendrites connecting the cells. Furthermore, active conductances in the coupling pathways can influence the measured coupling and calculated value of gc. Synchronization Dendrodendritic gap junctions are the most common type of electrical synapse in mammals; these synapses generally synchronize electrical activity of cells of the same kind. In synchronization, current flows from a more positive cell to depolarize a less positive cell, and the first cell is itself made less depolarized by that current flow; thus, the synchronizing function is both excitatory and inhibitory, and pre- and postsynaptic do not have an obvious, fixed meaning where electrical signals can go in either direction. Examples of these coupled systems include numerous types of inhibitory interneurons, such as fast spiking cells, low threshold spiking cells in the neocortex, comparable cells in the hippocampus and striatum, stellate cells in cerebellum, thalamic reticular neurons, and various subclasses of interneurons expressing somatostatin, parvalbumin, or calbindin. Like neurons tend to be coupled, and a coupled network can extend for quite long distances, although electrical spread is measurable only between close neighbors. In these systems, synchronization of the inhibitory neurons is not very precise, but the circuitry is made simpler by allowing reciprocal
connections that transmit not only impulse activity but subthreshold potentials of either sign. Moreover, the cells can excite each other through their gap junctions and inhibit downstream cells as well as each other by chemical synapses, a problematic arrangement for chemical transmission. Synchronization can be increased by mutual inhibition mediated chemically. In a rhythmically discharging pool of neurons that are electrically coupled, firing of one cell tends to fire its neighbors, but depolarization is immediately followed by inhibition, restricting the time at which those cells can fire and also preventing delayed firing so that they are more likely to fire the next time the first cell fires. Generally in these pools of neurons, the influence of one cell on a coupled neighbor is small, but if many cells are active simultaneously, the excitatory effect on coupled cells can be very strong. A number of classes of synchronously active coupled cells have axons that project out of the brain region in which they are coupled. Mitral cells of the olfactory bulb project to olfactory cortex and are coupled in their dendritic regions in the olfactory glomeruli. Olfactory neurons expressing the same receptor project to the same glomerulus, and the innervating mitral cells tend to fire synchronously. Cells of the inferior olive, which give rise to climbing fibers exciting cerebellar Purkinje cells, are coupled. The olive is a relatively large structure and tends to be subdivided into regions that are synchronously firing; the subdivision may be under control of inhibitory inputs to the dendrites that decrease electrical current spread along the dendrites and decouple groups of cells from each other. Neurons of the striatum are coupled inhibitory neurons that project to other brain regions. Neurons of the hypothalamus that release neuropeptides in the median eminence can be coupled with like neurons to facilitate hormone release. Neurons of the locus coeruleus are coupled, fire more or less synchronously, and project to much of the cortex. The suprachiasmatic neurons are coupled and project to the paraventricular nucleus. Many neurons in the retina are coupled, including type II amacrine cells and horizontal cells, where coupling can be both within and between classes. Coupling mediates lateral spread of excitation (rather than synchronization) that then causes recurrent inhibition. Electrical synapses may also involve other types of retinal cell. Rod bipolar cells excite type II amacrine cells that are electrically coupled to each other and to cone bipolar cells that then excite ganglion cells. Pools of neurons connected by gap junctions differ markedly in the degree of coupling. At one extreme, an impulse initiated in one cell propagates to all the
208 Gap Junctions and Electrical Synapses
other cells (not true of mammalian inhibitory interneurons). In less well coupled groups of cells, the synchrony of firing may be rather poor. However, even weak coupling can promote precise synchronization if other inputs are driving the cells to fire at about the same frequency. Synchronization mediated by electrical coupling has been characterized in a number of lower vertebrate and invertebrate species. Examples include neurons controlling electric organ discharge in fishes, which in most species is highly synchronous; motor systems in which speed and synchronization are important, such as those mediating saccadic eye movements, and sonic muscles, in which the fundamental frequency of the emitted sound is the frequency of contraction. Other systems are less well synchronized; examples include neurons controlling cardiac ganglia in arthropods and supramedullary neurons, a group of autonomic effectors found in teleosts. In the highly synchronized systems, the short delay possible at electrical synapses is required for the observed degree of synchronization. In other cases, synchronization could be mediated by drive from a higher center with fewer cells or by feedback mediated by chemical synapses. When a pool of neurons has fired synchronously, additional adaptations are necessary to assure that the effector cells are active synchronously, if distances from the ‘command center’ to effectors are sufficiently different to introduce asynchrony if conduction is ‘as the crow flies’ and of fixed velocity. One solution is to have the pathway to the more distant effectors conduct more rapidly. Others are to have more devious pathways to the nearer effectors or to introduce compensatory delays along the pathways. Escape! Most animals have a fast escape response to escape from predators and other dangers. An example that has been important in the study of synaptic transmission is the Mauthner cell system found in teleost fishes and some urodele amphibians. There are two Mauthner cells, one on either side of the medulla. Their axons decussate and descend the spinal cord to excite the axial motor neurons on the side contralateral to the Mauthner cell body, and firing of one Mauthner cell mediates a tail flip escape response away from the stimulated side. Large myelinated fibers from the sacculus, a hearing organ, form ‘club endings’ on the lateral dendrite of the Mauthner cells that transmit both electrically and chemically; the electrical component is of shorter latency and presumably reduces the latency of the escape response. Excitation of the axial motor neurons by the Mauthner axons is chemically mediated, as is transmission at the peripheral neuromuscular junction. The
Mauthner cells also inhibit each other through interneurons that act electrically (see the section titled ‘Electrical inhibitory synapse’) and then chemically so that firing of one cell rapidly inhibits the other; clearly, it would be counterproductive for swimming if muscles on both sides contracted simultaneously. (Each Mauthner axon also inhibits the contralateral motor neurons through crossed inhibitory interneurons on its side of the cord. Excitation of the inhibitory neurons is electrical so that in a given motor neuron, the chemical excitation from one Mauthner axon and the crossed chemical inhibition from the other have about the same latency, preventing simultaneous contraction of muscles on the two sides, should the Mauthner cells fire at the same time. In contrast, there is a bilateral component of the Mauthner cell escape reaction which closes the mouth and adducts the pectoral fins. Interneurons mediating these actions are excited by both Mauthner cells at chemical synapses and then transmit electrically to the motor neurons.) The lateral giant axons controlling the tail flip of many crustaceans, including lobsters and crayfish, are made up of multiple segments, one neuron per segment coupled together end to end to make in effect a single giant axon. (The gap junctions connecting the segments are presumably pannexin based.) The lateral giant axons conduct impulses at a comparable speed to the medial giant axons, which are true single axons arising from a single pair of cell bodies in the supraesophageal ganglia. One can appeal to developmental factors to account for the structural difference. The medial giants are excited by synapses in the head region, and the lateral giants can be excited in many of the abdominal ganglia. The two systems give somewhat different escape responses. The giant axons of many annelids are segmental and mediate the fast withdrawal. The septa between segments contain gap junctions. The gap junctions in all the invertebrates are presumably pannexin based; the pannexin mediating the escape jump reflex in Drosophila is known. How important is electrical transmission in these systems? Synaptic delays can be less at electrical synapses, particularly at lower temperatures. In an escape response, shorter latency is better, which may account for the high incidence of electrical transmission in escape systems of poikilotherms. There remains an obligatory chemical synapse to the muscle fibers, and one appeals to as yet undefined developmental constraints to account for the lack of electrical neuromuscular junctions. The time for muscle contraction is greater than the delay at a chemical synapse, but a small percentage increase in escape probability could provide powerful selection pressure. However, as an organism gets bigger, conduction times get
Gap Junctions and Electrical Synapses 209
longer, the time required for movement becomes greater, and the saving at an electrical synapse a smaller fraction of the response time. Electrical synapses are is found in the vestibular nuclei and mesencephalic nucleus of the trigeminal nerve of rodents, where it may reduce response latency. In mammals, where transmission in motor systems has been characterized, there are few instances of electrical transmission. However, anatomical studies of Cx36 distribution indicate that it may be much more common that realized hitherto.
decreases to the less frequent and more precisely targeted level seen in the adult. The roles of this early coupling are obscure. It may mediate synchronous firing that helps guide axons to their appropriate targets, or it may mediate chemical communication for differentiation. Mice with targeted deletion of any one of a number of connexins expressed in brain still exhibit relatively normal central nervous system (CNS) structure and function, suggesting either redundancy or relative unimportance.
Spatial and temporal summation If many axons form gap junction synapses on a postsynaptic cell, the postsynaptic response is graded with the number of presynaptic axons activated, and firing of the postsynaptic cell depends on the summation of inputs from the presynaptic axons, just as at a cell receiving many excitatory chemical synapses. In the classic sense, the system shows spatial summation of the excitatory synaptic potentials. A second classical form of shortterm interaction is temporal summation. Electrical as well as chemical PSPs can change the charge on the postsynaptic capacity, which requires time to decay back to the resting value. Electrical synapses can show temporal summation, when the postsynaptic time constant is comparable to or greater than the interval between presynaptic impulses. Furthermore, presynaptic action potentials can be prolonged or decreased in amplitude as a result of repetitive firing, both of which can modulate both electrical and chemical postsynaptic potentials.
As noted, gap junction synapses mediating synchronization are both excitatory and inhibitory. Furthermore the postsynaptic potential generated by a presynaptic action potential with an afterhyperpolarization is biphasic, depolarization followed by hyperpolarization. Depending on the cell, the mean potential can be hyperpolarizing, and the result of a train of action potentials is an initial excitation followed by a prolonged inhibition with small superimposed depolarizations associated with each presynaptic spike. A completely different kind of electrical inhibitory synapse occurs at the initial segment of the Mauthner cell. Interneuron axons converge on a structure termed the axon cap, a specialized region around the initial segment, which is where the impulse arises. Presynaptic action potentials in the inhibitory neurons generate a positivity in the extracellular space of the axon cap, the time course of which is like that of an action potential (Figure 16). It is likely that the axonal membrane acts simply as a fixed battery with a series resistance. The axons may go on to form chemical inhibitory synapses on the nearby soma of the Mauthner cell. The positivity, or ‘extrinsic hyperpolarizing potential,’ causes current to flow inward through the initial segment membrane (hyperpolarizing it) into the cell soma (and axon), along the cell cytoplasm and out through more-distant membrane (causing depolarization of this membrane). Since the conductance of this latter pathway is large compared with that of the initial segment membrane, most of the external potential is developed across the initial segment membrane, and impulse initiation is inhibited. When the Mauthner cell is active, it generates a large negativity in the axon cap region. This current tends to hyperpolarize the excitable membrane in the inhibitory interneurons and inhibit them. The current flows and equivalent circuit during inhibitory neuron and Mauthner cell activity are diagrammed in Figure 16. The electrical inhibition is a specialization for speed. Electrical inhibition reduces the latency of onset of inhibition by one chemical synaptic delay. The electrical component is brief, but it is followed by long-lasting chemical inhibition. As noted (see the
Other forms of plasticity Coupling between neurons can be regulated in a number of ways. Inhibitory synapses along a dendrodendritic path coupling cell bodies can short-circuit coupling between them and allow them to fire independently in response to excitatory inputs closer to their cell bodies. This form of regulation has been proposed for the mammalian inferior olive and for buccal motor neurons in a mollusk. Gap junctions may be regulated by second messengers, as in retinal horizontal cells and amacrine cells. Transmitters, such as dopamine, acting through G-protein-coupled receptors, lead to increase in cytoplasmic cAMP, which is followed by decrease in junctional conductance. It remains to be determined whether phosphorylation sites on the connexins are involved. Long-term potentiation and depression of both electrical and chemical components have been demonstrated at the club endings on the Mauthner cell. As more electrical synapses are examined, more instances of this class of regulation are likely to appear. Coupling of cortical neurons is very widespread and promiscuous in early development but then
Electrical Inhibitory Synapses
210 Gap Junctions and Electrical Synapses
rs M cell
I cell active
M cell active re
– a
+
rt
–
+
I cell M cell + –
rs
M cell active I cell active re + – I cell
rt
b Figure 16 Diagram of an electrical inhibitory synapse at the initial segment of the Mauthner (M) cell. (a) Impulse activity of the inhibitory interneurons (I cell) produces outward current through their axon terminals (rt) that divides to (1) flow across the external resistance (re) of the axon cap or (2) enter the M cell through its impulse initiation site, hyperpolarizing it and inhibiting M-cell firing. The current entering the M cell leaves through the resistance of dendritic (rs) and axonal membrane, but the resulting depolarization does not significantly affect impulse initiation. The circles with impulses drawn in them represent the impulse-generating membranes. Conversely, impulse activity of the M cell causes current that hyperpolarizes and inhibits the impulse-generating membrane of the I cell. Reciprocity of the inhibitory interaction is made clearer by the redrawn equivalent circuit in (b). From Bennett MVL (1977) Electrical transmission: A functional analysis and comparison with chemical transmission. In: Kandel ER (ed.) Cellular Biology of Neurons, pp. 357–416. Baltimore: Williams and Wilkins.
section titled ‘Escape!’) a requirement for an effective escape response is that the Mauthner cells do not fire simultaneously and each inhibits the other. There is evidence for similar electrical inhibition at the axon hillock of mammalian cerebellar Purkinje cells.
Gap Junctions and Genetic Disease Given the size of the connexin gene family (and Murphy’s law that anything that can go wrong, will), one would expect there to be diseases caused by mutations in these genes. The first discovery was that mutations or deletions of Cx32 cause X-linked Charcot–Marie–Tooth disease, a peripheral
neuropathy. Only after positional cloning demonstrated the involvement of Cx32 was it shown that this connexin is expressed by Schwann cells that form myelin. The effects of the mutations on Cx32 junctions may be subtle, such as minor changes in voltage sensitivity or permeability, or marked, leading to perturbed trafficking or complete loss of the protein. Light microscopic immunocytochemistry shows Cx32 at perinodes and Schmidt-Lanterman incisures, but their arrangement has not been clearly resolved in thin section or freeze-fracture electron microscopy. The gap junctions are apparently reflexive, that is, between different parts of the same cell, and are presumed to facilitate the interchange of small molecules between the periaxonal cytoplasm and the outermost part of the Schwann cell. The question remains as to what is communicated from outside to inside or vice versa. And it is unclear why loss of this communication, which has no apparent effect on myelin formation during growth and development, leads eventually to demyelination and degeneration of nerve fibers. Furthermore, there is no obvious pathology in other tissues expressing Cx32, either because Cx32-mediated communication is unimportant in these tissues or because another connexin takes over the function of Cx32. Some minor effects are seen in central nerve conduction and in the Cx32 knockout mouse in the liver and pancreas. Mutations in Cx26 are the most common cause of nonsyndromic deafness (deafness without other defects). Cx26 is expressed in the cochlear epithelium, not including the hair cells, and the pathogenesis may involve impaired communication between supporting cells. In any case, degeneration of the hair cells occurs early in the development of hearing but after the major structures of the inner ear have developed. As for Cx32, few other symptoms are reported, although a minority of patients exhibit defects in skin. Mutations of Cx31, which is expressed in skin, cause erythrokeratodermia, a thickening and reddening of the skin. Cx30 mutations cause deafness or skin disease; differential effects of the mutations at different points in the life cycle of the connexin may account for these differences. Mutations in the lens connexins, Cx46 and Cx50, cause congenital cataract. Mutations in Cx43 cause oculodentodigital dysplasia, a disease involving multiple defects, including in the nervous system. Cx36 is implicated in juvenile myoclonic epilepsy, but mechanisms are obscure. Targeted gene disruption (knockout) in mice reveals other potential genetic diseases involving mutations in connexins. Knockout of Cx32 is quite benign, but the double knockout of Cx32 and Cx47 causes loss of central myelin and early death. Knockout of Cx43 is lethal at birth because of cardiac malformation, and there are subtle abnormalities in cortical layering.
Gap Junctions and Electrical Synapses 211
Knockout of Cx26 is embryonic lethal because of defects in the trophoblast, but conditional knockouts using various strategies to permit knockout of a gene in a specific tissue are becoming important tools for analysis of gene function that will be relevant to human disease.
Evolution Vertebrate connexins have been grouped into alpha and beta (or group II and group I, respectively). Assignment into further divisions, gamma and delta, may be justifiable, but deep branches of the evolutionary tree are less reliable at this time. The zebra fish, Danio rerio, has 37 connexins, almost twice as many as mammals have, probably because teleosts have undergone a whole genome duplication. Connexins are also found in ascidians. The number of connexins in the common ancestor of ascidians and vertebrates may be no more than one. Most vertebrate connexins have a single coding exon, and alternative splicing is limited to noncoding regions. Cx36 is an exception and has two coding exons. Ascidian connexins have several coding exons, as do pannexins/innexins. The ancestral connexin is uncertain. Occurrence in hemichordates has not been investigated. The sea urchin genome has been sequenced and is reported to lack both connexins and pannexins. The anatomical basis of coupling in this group, reported in several studies, is thus unknown. Although there were early inferences of connexins in Cnideria based on antibody labeling and Western blotting, genomic sequencing has yet to demonstrate a connexin. Their gap junctions, coupling, and electrical synapses are likely to be pannexin/innexins based. What is the survival value of having multiple gap junction-forming proteins? Functional differences between connexins in gating, permeability, and trafficking have been demonstrated and provide one explanation for the observed diversity. There are the obvious functional differences in permeability, gating, and posttranscriptional regulation of formation and degradation. Differences in transcriptional and translational control may be more important than the functional differences in the connexins themselves. Convergence between Connexins and Pannexins
Although differences in the gene sequences indicate that connexin-based and pannexin-based gap junctions are separate evolutionary adaptations, there is a remarkable degree of functional convergence, including block by many of the same pharmacological agents, by low cytoplasmic pH, and by high cytoplasmic Ca2þ. Fast Vj gating to a substate and slow gating to the
completely closed state are also found in both classes, although dependence on membrane potential (Vi-o) is more pronounced in at least one pannexin junction. In pannexin junctions, the channel diameter is a little bigger, the gap is a little wider, and the number of channels per unit area is a little lower. Some pannexin junctions under specific uncoupling conditions may split, with each membrane taking its own hemichannels, which can then be reused. Pannexins do mediate coupling when expressed in Xenopus oocytes, but it is not yet clear whether they do so in mammals. Like connexins, pannexins form hemichannels that can open in the surface membrane without contact with a hemichannel in an apposed membrane. Hemichannels That Open
The life history of a connexin molecule includes some time in the membrane as a hemichannel without a partner (Figure 13). Because of the predicted high conductance and lack of permselectivity, it was thought that these hemichannels should not open because they would stress the cells by letting in excess Ca2þ and Naþ and permitting loss of Kþ and metabolites. Furthermore, measurements during junction formation indicated that channels were not open before connecting with hemichannels in an apposed membrane (see Figure 14). This conventional wisdom was confirmed when expression of Cx46, which is expressed only in lens and forms hemichannels when expressed in Xenopus oocytes, killed the oocytes unless Ca2þ in the bathing solution was kept at relatively high levels to keep the hemichannels closed. Now it is clear that hemichannels formed of a number of connexins can open to the extracellular space and can mediate both physiological and pathological responses. The open probability is generally very low, and cells can release significant amounts of signaling molecules, such as ATP, without impact on viability. Conversely, under pathological conditions hemichannels can speed death of the cells expressing them. Also, the molecules they release, such as glutamate from astrocytes, can have a toxic action on neighboring cells, in particular neurons. A number of criteria indicate that connexins can form hemichannels. Different treatments, such as bathing in low Ca2þ solution, cause uptake of dyes to which gap junctions are permeable but normal cell membranes are not, and this uptake requires expression of connexins. The uptake is also blocked by gap junction blockers. Furthermore, single-channel activity can be recorded that has the predicted conductance of about twice that of the cell–cell channel formed by the same connexin. Moreover, their gating properties correspond to those of the cell–cell channels.
212 Gap Junctions and Electrical Synapses
Pannexins in mammals may form only hemichannels, and one mode of opening is through activation of purinergic receptors. The linkage between purinergic activation and pannexin hemichannel opening is unclear. As with connexins, pannexin hemichannel opening may result in release of signaling molecules, such as ATP. See also: Gap Junction Communication; Gap Junctions and Neuronal Oscillations.
Further Reading Bennett MVL (1977) Electrical transmission: A functional analysis and comparison with chemical transmission. In: Kandel ER (ed.) Cellular Biology of Neurons, pp. 357–416. Baltimore: Williams and Wilkins. Bennett MVL (1997) Gap junctions as electrical synapses. Journal of Neurocytology 26: 349–366. Bennett MVL, Barrio LC, Bargiello TA, Spray DC, Hertzberg E, and Saez JC (1991) Gap junctions: New tools, new answers, new questions. Neuron 6: 305–320. Bennett MVL (2000) Seeing is relieving: Electrical synapses between visualized neurons. Nature Neuroscience 3: 7–9. Bennett MVL, Contreras JE, Bukauskas FF, and Saez JC (2003) New roles for astrocytes: Gap junction hemichannels have something to communicate. Trends in Neuroscience 26: 610–617. Bennett MVL and Zukin RS (2004) Electrical coupling and neuronal synchronization in the mammalian brain. Neuron 41: 495–511. Bukauskas FF and Peracchia C (1997) Two distinct gating mechanisms in gap junction channels: CO2-sensitive and voltage-sensitive. Biophysical Journal 72: 2137–2142. Connors BW and Long MA (2004) Electrical synapses in the mammalian brain. Annual Review of Neuroscience 27: 393–418. Cruciani V and Mikalsen SO (2007) Evolutionary selection pressure and family relationships among connexin genes. Biological Chemistry 388: 253–264. Eastman SD, Chen TH, Falk MM, Mendelson TC, and Iovine MK (2006) Phylogenetic analysis of three complete gap junction gene families reveals lineage-specific duplications and highly supported gene classes. Genomics 87: 265–274. Gaietta G, Deerinck TJ, Adams SR, et al. (2002) Multicolor and electron microscopic imaging of connexin trafficking. Science 296: 503–507.
Harris AL (2002) Emerging issues of connexin channels: Biophysics fills the gap. Quarterly Reviews of Biophysics 34: 325–472. Erratum in: Quarterly Reviews of Biophysics (2002) 35:109. Harris AL (2007) Connexin channel permeability to cytoplasmic molecules. Progress in Biophysics and Molecular Biology 94: 120–143. Meier C and Dermietzel R (2006) Electrical synapses: Gap junctions in the brain. Results and Problems in Cell Differentiation 43: 99–128. Perkins GA, Goodenough DA, and Sosinsky GE (1998) Formation of the gap junction intercellular channel requires a 30 degree rotation for interdigitating two apposing connexons. Journal of Molecular Biology 277: 171–177. Revilla A, Castro C, and Barrio LC (1999) Molecular dissection of transjuctional voltage dependence in the connexins-32 and connexins-43 junction. Biophysical Journal 77: 1374–1383. Saez JC, Retamal MA, Basilio D, Bukauskas FF, and Bennett MVL (2005) Connexin-based gap junction hemichannels: Gating mechanisms. Biochimica et Biophysica Acta 1711: 215–224. Sohl G, Maxeiner S, and Willecke K (2005) Expression and functions of neuronal gap junctions. Nature Reviews Neuroscience 6: 191–200. Theis M, Sohl G, Eiberger J, and Willecke K (2005) Emerging complexities in identity and function of glial connexins. Trends in Neuroscience 28: 188–195. Trexler EB, Bukauskas FF, Kronengold J, Bargiello TA, and Verselis VK (2000) The first extracellular 100p domain is a major determinant of charge selectivity in connexin 46 channels. Biophysical Journal 79: 3036–3051. Valiunas V, Bukauskas FF, and Weingart R (1997) Conductances and selective permeability of connexin43 gap junction channels examined in neonatal rat heart cells. Circulation Research 80: 708–719. Venance L, Rozov A, Blatow M, et al. (2000) Connexin expression in electrically coupled postnatal rat brain neurons. Proceedings of the National Academy of Sciences of the United States of America 97: 10260–10265. Yen MR and Saier MH Jr (2007) Gap junctional proteins of animals: The innexin/pannexin superfamily. Progress in Biophysics and Molecular Biology 94: 5–14.
Relevant Website http://www.scholarpedia.org – Eugene M. Izhikevich, Scholarpedia.
Gap Junctions and Hemichannels in Glia Z Ye and B R Ransom, University of Washington School of Medicine, Seattle, WA, USA ã 2009 Elsevier Ltd. All rights reserved.
Introduction Gap junctions (GJ) are unique large channels that connect the cytoplasm of two adjacent cells. Half GJs, called connexons or hemichannels (HC), expressed by individual cells may also be functionally active, and when open would connect a cell’s cytoplasm to extracellular space. Gap junctions mediate a primitive and important form of intercellular communication that is seen across phyla. Discovered in 1952 in heart muscle cells, GJs are expressed in most mammalian cells; mature skeletal muscle cells, spermatozoa and erythrocytes are the only exceptions. Gap junction mediated coupling between glial cells was first noted in the leech central nervous system (CNS) by Stephen Kuffler and colleagues in 1964. In the mammalian CNS, connexins, the proteins that form GJs and HCs, are most abundantly expressed by glial cells. All subtypes of glia express connexins and GJs, with the possible exception of the newly defined NG2 cell. The characteristics and possible functions of GJs and HCs in glial cells are the focus of this review. First, however, some important general principles about these channels and the proteins that make them will be discussed.
Connexins Form Gap Junctions Gap junctions (GJs) are so named based on their appearance in electron micrographs. At GJs, the width of the extracellular space is narrowed to a small ‘gap’ of 2.5 nm (normal width 20 nm; Figure 1). In mammals, GJs are formed from connexins, a protein family with ancient origins and containing >20 members (Figures 1 and 2). The molecular mass of cloned connexins ranges from 25 to 62 kDa, and they are named on this basis (e.g., the 43 kDa connexin is called Cx43; Figure 2). Most cells express at least two connexin subtypes, although one is likely to predominate. Invertebrates form abundant GJs but do not express connexins. Instead, their GJs are composed of proteins called innexins that are analogous, but not strongly homologous, to connexins. Curiously, innexin-like proteins called pannexins have been detected in mammals and may mediate coupling between hippocampal pyramidal neurons. Pannexins, in contrast to connexins, however, are more likely to form hemichannels (HCs) than GJs.
Each GJ is composed of two HCs, one embedded in each of the closely opposed membranes (Figures 1 and 3). Each HC consists of six connexin molecules, and these can be aggregates of six identical connexins or a mixture of different connexins. Functional GJs can form between HCs composed of different connexins. For example, astrocytes and oligodendrocytes form GJs composed of Cx43 and Cx47, respectively. Connexins and related proteins have four transmembrane domains, and the N-terminal (NT) and C-terminal (CT) segments are both intracellular, resulting in two extracellular loops (ELs) and one cytoplasmic loop (CL). When two HCs ‘dock’ to make a GJ, the extracellular loops mesh to form a structural ‘tight seal,’ guaranteeing that the channel will not leak to the extracellular space. Covalent bonds are not involved in the formation of this tight seal. Curiously, connexin proteins have a very high turnover rate; the average halflife of these proteins is several hours. Macroscopic GJs are aggregates of many, usually hundreds, of tightly packed GJ channels. In the mammalian central nervous system (CNS), GJs are most numerous between glial cells, especially between astrocytes. They are also seen between certain populations of neurons, including cortical interneurons and certain brain stem neurons. More detail about the expression of connexins and GJs in glial cells is given in the following sections.
Biophysical Properties of GJs The biophysical properties of GJs can be partly understood based on their structure. Open GJs have a very large limiting pore diameter of 1.5 nm and are permeable to a much wider range of ions and molecules than are typical ion channels (typical pore diameter, 3–5 nm). Consequently, GJ pores can admit molecules up to a molecular mass of about 1 kDa, largely independent of charge. Coupled cells, therefore, are able to exchange a wide range of biologically important molecules, including cyclic nucleotides, small molecules of intermediate metabolism, vitamins, and inorganic ions. Obviously, strongly coupled cells will have a tendency to have similar concentrations of those cytoplasmic constituents that easily pass through GJs. This principle has been conclusively demonstrated for the concentration of intracellular Naþ in astrocytes. GJs composed of different connexins, however, vary considerably in what can pass through them. For example, GJ channels composed of Cx32 and Cx43 show marked differences in permeability to adenosine, which is 12-fold more permeable through Cx32 versus Cx43 channels.
213
214 Gap Junctions and Hemichannels in Glia Cytoplasm Gap junction
Extracellular space
a
20 nm
Cytoplasm
2.5 nm
Hemichannel (connexon)
b
EL1
Extracellular
EL2
M
NT
CL CT
Cytoplasm
Connexin
c
Connexins (vertebrate)
Gap junction protein family
Figure 1 Gap junction and hemichannel structure. (a) Schematic drawing of a gap junction as visualized by electron microscopy. The distance between neighboring cells narrows to about 2 nm in the junction. (b) Gap junctions are formed by two aligned hemichannels or connexons, one in each adjacent cell. Hemichannels are unopposed connexons. This is the only possible configuration when a connexon is inserted into the membrane outside the junction area. (c) Schematic drawing of a hemichannel. Each hemichannel is composed of six connexin molecules. An exploded view of a connexin molecule is shown on the right. Each connexin has four-transmembrane-spanning domains. Both the C-terminal (CT) and N-terminal (NT) portions of the connexin are located within the cell. This creates a single cytoplasmic loop (CL) and two extracellular loops (EL1 and EL2). Modified from Ransom BR and Ye ZC (2005) Gap junctions and hemichannels. In: Kettenmann H and Ransom BR (eds.) Neuroglia, 2nd edn., pp. 177–189. New York: Oxford University Press.
Cx25, Cx26, Cx30, Cx30.3, Cx31, Cx31.1, Cx32 Cx57, Cx59, Cx62 Cx33, Cx37, Cx38, Cx41, Cx43, Cx46 Cx40, Cx42, Cx44, Cx45.6, Cx49, Cx50, Cx56 Cx29, Cx31.3, Cx43.4, Cx44.2, Cx45, Cx47 Cx30.2, Cx31.9, Cx34.7, Cx35, Cx36, Cx40.1 Pannexins (Px1, Px2, Px3; vertebrate) Innexins (invertebrate)
Figure 2 Phylogenetic ‘tree’ of known gap junction proteins in vertebrate (connexin and pannexin) and invertebrate (innexin) animal cells. More than 20 distinctive gap junction proteins have been identified (note that some of the 39 proteins listed here are only slightly different from one another and therefore do not count as distinctive proteins).
Electrophysiological analysis of single GJ channels indicates that, like other ion channels, they abruptly open and close. The permeability of GJ channels is influenced by physiological variables, including membrane potential and the intracellular concentrations of Hþ and Ca2þ. As a general principle, the permeability of GJs is greatest when there is no voltage difference across the junction; this corresponds to the situation when the two coupled cells have the same resting potential. Increases in intracellular concentrations of either Hþ or Ca2þ rapidly decrease
junctional conductance (i.e., increase electrical resistance) and permeability to large molecules. Coupling conductance is sensitive to intracellular pH changes that are within the physiological range of this variable (i.e., pH 6.5 0.5). Because neuronal activity can cause fluctuations of intracellular pH in astrocytes, this variable may be an important modulator of GJ function. Gating by pH depends on the connexin CT. Truncation of the CT eliminates pH sensitivity. It is believed that pH changes induce the CT to interact with a portion of the CL to close the channel, in a
Gap Junctions and Hemichannels in Glia
fashion analogous to a ‘ball and chain.’ The increases in intracellular [Ca2þ] ([Ca2þ]i) that decrease junctional conductance are high (e.g., 10 mM), and this variable is not likely to affect coupling under normal physiological conditions; cell injury causes large increases in [Ca2þ]i that could lead to uncoupling. GJ coupling is decreased by a wide range of drugs, including lipophilic agents such as the higher-order alcohols heptanol and octanol, and the anesthetic halothane. The mechanism of blockade by these agents is not clearly understood.
Hemichannels HCs have not traditionally been thought of as ‘standalone’ ion channels. The presumption was that these channels existed only transiently in membranes before being incorporated into GJs. Moreover, to the extent that unpaired HCs existed, they would predominantly be in the closed state because of high extracellular [Ca2þ] ([Ca2þ]o) (Figure 3). A structural explanation for this was provided by the discovery of a site sensitive to near-millimolar [Ca2þ] that is located on the extracellular portion of the HC pore. This site would not affect the permeability of GJs, however, because it would be inaccessible due to the tight meshing of the extracellular loops necessary to form a GJ. Because of their large pore diameter and presumed high permeability and low level of selectivity, open HCs could dissipate ion and metabolite gradients would seemingly threaten cellular integrity. This reasoning, and the fact that HCs have proved difficult to study for technical reasons, may explain why relatively little attention has been paid to HCs. Their involvement in brain function and pathology remains controversial. Because HCs connect cell cytoplasm to the extracellular space, the roles of HCs and traditional GJs are fundamentally different.
Ca2+ gating ([Ca2+]o ~ 1.5 mM)
Unlike gap junctions, HCs are directly exposed to extracellular ions and thus can be effectively gated by extracellular, as well as intracellular, ion changes. A wide range of factors influence HC gating. For example, Ca2þ-free solution rapidly opens these channels in astrocytes and other cells. Unexpectedly, and in contradistinction to GJs, some HCs may also open in response to modest increases in [Ca2þ]i. Open HCs are detected by uptake of appropriate marker dyes and by the release of molecules such as glutamate and ATP. Most factors that regulate gap junctions, but probably not all, have similar effects on HCs. The ELs of an unopposed HC, for example, are far more exposed than are the same loops of two aligned HCs forming a GJ, making these ELs a prime candidate site for differential modulation (see earlier). For now, however, caution is needed when interpreting results obtained with agents that block gap junctions, because these would probably also block HCs. HCs may also be confused with other membrane ion channels that have very large pores, such as the P2X7 purinergic receptor channel.
Gap Junctions in Glial Cells Connexins are variably expressed by all the main types of mammalian glial cells, with the possible exception of NG2 cells. Even microglial cells, felt at one time to be devoid of connexins, can express Cx43 and form gap junctions. Astrocytes are the most robustly coupled glial cells. Astrocytes of various types (e.g., protoplasmic, fibrous, Bergmann glia) form homotypic gap junctions with other astrocytes and heterotypic junctions with oligodendrocytes and ependymal cells. Astrocytes, as well as other glial cells, express several different connexins (Table 1) that may be regulated during development and affected by injury or cellular environment. Physiological variables
([Ca2+]o < 0.5 mM) Na+
Open hemichannel
Closed hemichannel
• Amino acids • Ions • Metabolites • Second messengers
Astrocyte
215
Glutamate (>1 mM)
[Na+]i
Astrocyte
Figure 3 Schematic diagram of gap junctions and hemichannels in astrocytes. Gap junctions are typically open because they are exposed to low intracellular [Ca2þ]; they mediate diffusion of molecules up to 1000 Da. Hemichannels (i.e., unopposed connexons) are gated closed under normal conditions by high extracellular [Ca2þ] (see text). When opened, hemichannels allow intracellular contents to equilibrate with extracellular fluid (e.g., glutamate leaves and Naþ enters).
216 Gap Junctions and Hemichannels in Glia Table 1 Gap junction coupling in mammalian glial cells Glial cell pair
Relative coupling strengtha
Connexin pairingsb
Astrocytes Astrocyte–oligodendrocyte Oligodendrocytes Schwann cells Microglia Mu¨ller cells Mu¨ller cell–astrocyte Ependymal cells
þþþþ þþ þþ þþ þ þþ þþ þþþ
Cx43–Cx43, CX43–Cx30, Cx30–Cx30, Cx30–Cx26, Cx26–Cx26 Cx43–Cx47, Cx30–Cx32, CX26–Cx32 Cx32–Cx32, Cx29–Cx29 Cx32–Cx32, Cx29–Cx29 Cx43–Cx43 Cx43–Cx43, Cx43–Cx45, Cx45–Cx45 Cx43–Cx43, Cx43–Cx45, Cx45–Cx45 Cx43–Cx43
Relative coupling strength is shown as a semiquantitative score between þ and þþþþ, with þ being the lowest strength of coupling. Connexin pairings are shown in order of decreasing frequency. For example, Cx43–Cx43 is the most frequent connexin pairing for gap junctions between astrocytes. a b
such as glutamate application or depolarization can increase, while inflammatory cytokines decrease, astrocyte GJ coupling. The predominant connexin expressed by these cells is Cx43, but its expression varies by brain region. Not surprisingly, Cx43 is the most abundantly expressed connexin in the CNS. There are intriguing reports indicating that astrocytes form gap junctions with neurons. However, these junctions may be limited to immature cells and occur only during development. Mammalian astrocytes in situ typically show widespread dye coupling with each other. When the GJ-permeable dye Lucifer Yellow is injected into individual cortical astrocytes, as many as 100 adjacent cells are stained. Oligodendrocytes primarily form gap junctions with astrocytes. The coupling between oligodendrocytes and astrocytes is mainly mediated by GJs composed of Cx43 (astrocytes) and Cx47 (oligodendrocytes) (Table 1). Although functional coupling between oligodendrocytes is readily detected in vitro, actual GJs are only rarely seen using electron microscopy on freshly prepared whole brain. Based on the scarcity of morphological GJs, it has been suggested that communication between oligodendrocytes might be mediated by connections through the astrocytic syncytium. ‘Coupling strength’ measured electrically and by dye passage is much greater between astrocytes than between astrocytes and oligodendrocytes. In the peripheral nervous system, proliferating Schwann cells during development, or after peripheral nerve crush injury, primarily express Cx46 and are coupled to one another. When Schwann cells begin myelinating axons, they stop expressing Cx46 but increase expression of Cx32, which is confined to paranodal regions and Schmidt–Lanterman incisures. These so-called reflexic gap junctions connect the paranodal folds, perhaps facilitating ion and small-molecule movements to and from the tight, periaxonal space. The X-linked form of the hereditary neuropathy known as Charcot–Marie–Tooth disease is associated with mutations in the Cx32 gene.
Other specialized glial cells, such as ependymal cells, Mu¨ller cells of the retina, and Bergmann glia of the cerebellum, express connexins and GJs (Table 1). Activated, but not resting, microglia express Cx43 and show dye coupling in vitro. Interestingly, activated microglia can downregulate Cx43 expression and coupling in nearby astrocytes, probably via cytokine secretion, suggesting that astrocyte communication may be modulated by CNS inflammation mediated by microglia.
Functions of GJs and HCs The functions of connexins, and the channels that they form (both GJs and HCs), are still debated and undoubtedly vary by cell type. This uncertainty about function is especially true for GJs and HCs in glial cells. The following comments about function are directed mainly at astrocytes. In general, GJs may serve to coordinate the electrical and metabolic activities of cell populations, act to amplify the consequences of signal transduction, control intrinsic proliferative capacity, and help to orchestrate the complex events of embryonic morphogenesis. Roles of GJs in astrocytes include equilibrating intracellular ions among coupled cells (Figures 3 and 4(a)), regulating cell proliferation, participation in signal dispersion (Figure 4(b)), participation in Ca2þ wave propagation (Figure 4(c)), and involvement in brain injury. Because astrocytes are strongly coupled, they can experience GJ-mediated signal dispersion (or amplification). Consider a cell stimulated to produce an intracellular second-messenger molecule (e.g., cAMP, inositol 1,4,5-trisphosphate, Ca2þ) by binding a specific ligand (Figure 4(b)). If the cell is not connected to its neighbors by gap junctions it will act in isolation. If the cell is widely coupled to other cells, however, the diffusible junction-permeable second messenger has the potential of affecting many cells, effectively amplifying the initial signal. Astrocyte Ca2þ waves are propagated waves of elevated intracellular Ca2þ that may be elicited by
Gap Junctions and Hemichannels in Glia
217
Homeostasis
Mem. Pot. [Na+] [Ca2+ ] Amino acids Metabolites
Mem. Pot. [Na+] [Ca2+ ] Amino acids Metabolites
Mem. Pot. [Na+] [Ca2+ ] Amino acids Metabolites
a Stimulus
Response amplification
Receptor
cAMP IP3 Ca2+
cAMP IP3 Ca2+
Response
Response
cAMP IP3 Ca2+ Response
b Signaling interaction: astrocyte calcium waves
Glutamate receptors Glutamate . .. . . release .. .
PLC
..
PLC
.
..
ATP
. . .. . .. . . .
IP3 [Ca2+]
i
.
. .
. . ... . . . ... . .. . . . . . . .. . .. . . . . .. .... .. . .. . . .. . . Altered .. . . . . excitability . . . .. . . . . . .. . .
Stimulus (e.g., glutamate)
Astrocyte
Ca2+
Ca2+ stores
. ..
.
.
Neuron
c Figure 4 Illustration of possible functions of gap junctions and hemichannels. (a) In strongly coupled cell aggregates, the intracellular concentrations of ions and molecules less than 1000 Da in size (including amino acids, sugars, and second-messenger molecules) will tend to be similar due to free exchange across gap junctions. (b) Glial cells express many receptors for signaling molecules (e.g., neurotransmitters, cytokines). Coupling can amplify the physiological consequences of a chemical signal acting upon a single cell. Such stimuli often evoke production of second-messenger molecules, such as Ca2þ, inositol 1,4,5-trisphosphate (IP3), and cAMP, that can diffuse into adjacent cells via gap junctions; the coupled cells are then recruited to respond. (c) Gap junctions and hemichannels participate in astrocyte Ca2þ waves. Astrocyte Ca2þ waves are provoked in many ways, such as by the application of glutamate. Effective stimuli activate intracellular signaling pathways, such as those involving phospholipase C (PLC) and IP3, which cause Ca2þ release from Ca2þ stores. IP3 and Ca2þ diffuse to adjacent cells through gap junctions, creating a ‘Ca2þ wave’. Astrocytes also release ATP upon activation, probably through hemichannels. In turn, ATP activates purinergic receptors on nearby astrocytes, the predominant mode by which Ca2þ waves propagate. Increase in astrocyte [Ca2þ]i causes glutamate release, which modulates the excitability of neighboring neurons. Reproduced from Ransom BR and Ye ZC (2005) Gap junctions and hemichannels. In: Kettenmann H and Ransom BR (eds.) Neuroglia, 2nd edn., pp. 177–189. New York: Oxford University Press, with permission.
glutamate application, mechanical perturbation, or ischemia (Figure 4(c)). As the wave passes through astrocytes it can elicit glutamate release and alter the behavior of adjacent neurons; in essence, this event is a slow form of signaling (slow compared to action potentials and synaptic transmission in neurons). Ca2þ wave propagation is dependent on both gap junctions and extracellular signaling pathways,
including hemichannel-mediated ATP release; the relative contribution of these two mechanisms may vary in different brain regions. Gap junctions tend to restrict cell proliferation. During neoplastic transformation, gap junctions are usually lost and most malignant cells are not coupled. Most primary brain tumors derive from glial cells, and loss of GJs could plausibly contribute to
218 Gap Junctions and Hemichannels in Glia
CNS neoplasia. How GJs suppress mitotic activity is not clear. Genetic manipulation is one method of assessing the function of connexins. Animals null for Cx43 die at birth because of a cardiac defect. Conditional mutants have been created in which Cx43 is suppressed only in astrocytes. These animals live to adulthood and exhibit subtle neurological changes, including increased exploratory behavior, an anxiolytic-like state, and impaired motor capacities. In humans, mutations in Cx43 cause an autosomaldominant syndrome called oculodentodigital dysplasia (ODDD). At least half of these patients have neurological abnormalities, including spastic paraplegia and neurodegeneration (i.e., marked changes in cortex and white matter on magnetic resonance imaging), in addition to craniofacial and limb dysmorphisms. The exact pathophysiology of this condition remains uncertain and some mutations may cause a toxic gain of function or disturb the normal interactions known to occur between connexin and nonconnexin proteins. The roles of glial HCs are mainly a matter of speculation at this time. Possible roles include (1) release of signaling molecules such as ATP or glutamate, (2) modulation of membrane potential or ion gradients in a manner similar to conventional ion channels, and (3) mediation of cell injury under pathological conditions such as ischemia. HCs can open under pathological conditions, perhaps due to oxidative stress, and may release glutamate, which could contribute to excitotoxic injury.
Conclusions Connexins and the channels they form have been the focus of intense investigation for greater than a half century since their discovery. As more information about these fascinating channels has emerged, more questions have appeared. This is especially so in the brain, where these channels are widely expressed in glial cells. We lack conclusive insight about how they participate in brain function and pathological conditions. We are at a point, however, where critical hypotheses about their function can be formulated and tested using improved research techniques, including regional and cell-type-specific genetic manipulation of connexin expression. See also: Gap Junctions and Electrical Synapses; Gap Junctions and Neuronal Oscillations.
Further Reading Bennett MV, Barrio LC, Bargiello TA, et al. (1991) Gap junctions: New tools, new answers, new questions. Neuron 6: 305–320.
Bruzzone R, Hormuzdi SG, Barbe MT, et al. (2003) Pannexins, a family of gap junction proteins expressed in brain. Proceedings of the National Academy of Sciences of the United States of America 100: 13644–13649. Contreras JE, Sanchez HA, Eugenin EA, et al. (2002) Metabolic inhibition induces opening of unapposed connexin 43 gap junction hemichannels and reduces gap junctional communication in cortical astrocytes in culture. Proceedings of the National Academy of Sciences of the United States of America 99: 495–500. Cotrina ML, Lin JH, Alves-Rodrigues A, et al. (1998) Connexins regulate calcium signaling by controlling ATP release. Proceedings of the National Academy of Sciences of the United States of America 95: 15735–15740. Dermietzel R, Gao Y, Scemes E, et al. (2000) Connexin43 null mice reveal that astrocytes express multiple connexins. Brain Research – Brain Research Reviews 32: 45–56. Froes MM, Correia AH, Garcia-Abreu J, et al. (1999) Gapjunctional coupling between neurons and astrocytes in primary central nervous system cultures. Proceedings of the National Academy of Sciences of the United States of America 96: 7541–7546. Gomez-Hernandez JM, de Miguel M, Larrosa B, et al. (2003) Molecular basis of calcium regulation in connexin-32 hemichannels. Proceedings of the National Academy of Sciences of the United States of America 100: 16030–16035. Goodenough DA, Goliger JA, and Paul DL (1996) Connexins, connexons, and intercellular communication. Annual Review of Biochemistry 65: 475–502. Harris AL (2001) Emerging issues of connexin channels: Biophysics fills the gap. Quarterly Review of Biophysics 34: 325–472. Kettenmann H and Ransom BR (1988) Electrical coupling between astrocytes and between oligodendrocytes studied in mammalian cell cultures. Glia 1: 64–73. Levin M (2002) Isolation and community: A review of the role of gap-junctional communication in embryonic patterning. Journal of Membrane Biology 185: 177–192. Phelan P and Starich TA (2001) Innexins get into the gap. BioEssays 23: 388–396. Ransom BR and Ye ZC (2005) Gap junctions and hemichannels. In: Kettenmann H and Ransom BR (eds.) Neuroglia, 2nd edn., pp. 177–189. New York: Oxford University Press. Rouach N, Glowinski J, and Giaume C (2000) Activity-dependent neuronal control of gap-junctional communication in astrocytes. Journal of Cell Biology 149: 1513–1526. Saez JC, Berthoud VM, Branes MC, et al. (2003) Plasma membrane channels formed by connexins: Their regulation and functions. Physiological Reviews 83: 1359–1400. Saez JC, Contreras JE, Bukauskas FF, et al. (2003) Gap junction hemichannels in astrocytes of the CNS. Acta Physiologica Scandinavika 179: 9–22. Sontheimer H, Minturn JE, Ransom BR, et al. (1991) Cell coupling is restricted to subpopulations of astrocytes cultured from rat hippocampus and optic nerve. Annals of the New York Academy of Sciences 633: 592–596. Spray DC, Ye ZC, and Ransom BR (2006) Functional connexin hemichannels: A critical appraisal. Glia 54(7): 758–773. Unger VM, Kumar NM, Gilula NB, et al. (1999) Three-dimensional structure of a recombinant gap junction membrane channel. Science 283: 1176–1180. Ye ZC, Wyeth MS, Baltan-Tekkok S, et al. (2003) Functional hemichannels in astrocytes: A novel mechanism of glutamate release. Journal of Neuroscience 23: 3588–3596.
Gap Junctions and Neuronal Oscillations M O Cunningham and F E N LeBeau, Newcastle University, Newcastle upon Tyne, UK ã 2009 Elsevier Ltd. All rights reserved.
Introduction In addition to neuronal chemical neurotransmission, electrical transmission via gap junctions, which are protein channels that directly link the cytoplasm of adjacent cells, is now known to constitute an important type of cell-to-cell communication. Although initially thought to be important mainly in development, it is now clear that electrical signaling via gap junctions plays an important role in the adult central nervous system (CNS). Considerable advances have been made in understanding the contribution of electrical signaling, via gap junctions, to neuronal signaling, most notably the role of synchronizing oscillatory or rhythmic activity. Oscillatory neuronal activity underlies the production of the signals recorded with an electroencephalogram (EEG) and reflects the coordinated activity of large populations of neurons. An EEG of the brain reveals rhythmic oscillatory activity in distinct frequency bands that occur during different behavioral states, and can be altered in a number of neurological and psychological diseases. Work from many research groups over the past decade has highlighted the role of electrical signaling via gap junctions in the generation, and/or spread, of synchronous oscillatory network activity. Electrical coupling via gap junctions can occur in both neurons and glial cells throughout the CNS. However, in this article, we focus on electrical coupling in excitatory principal cells and inhibitory interneurons, mainly in the neocortex and hippocampus. We provide an overview of the role of electrical signaling via gap junctions in the generation of oscillatory network activity and discuss briefly the importance of gap junction function in neurological conditions such as epilepsy.
Historical Background The idea that neurons can form an interconnected network via direct cell-to-cell connections is not a novel concept. Proposed by Camillo Golgi in the late nineteenth century, the ‘reticular theory’ postulated that the nervous system could form a syncytium consisting of nerve fibers forming an intricate connected network and that the nerve impulse could propagate along such a diffuse network. However, at the same time, Ramo´n y Cajal’s ‘neuron doctrine’
hypothesized that the nervous system was composed of anatomically and functionally independent units (neurons) with chemical synapses connecting them. Today we understand that in the adult mammalian brain, each of these theories reflects an important aspect of the communication in neuronal networks.
Structure and Properties of Gap Junctions in the CNS Gap junctions form a continuous link between two separate neuronal structures (Figure 1) that allows the direct transmission of electrical signals between neurons. Neurons can be coupled together via gap junctions connecting soma-to-soma, dendrite-to-dendrite, or soma-to-dendrite. More recently, evidence has appeared of gap junctions linking neurons via axonto-axon connections. The major proteins that form gap junctions are termed connexins (Cxs). Different Cx subtypes are defined by their molecular mass (in kilodaltons). A number of different Cx subtypes have now been identified in the vertebrate CNS, including Cx26, Cx32, Cx36, Cx43, and Cx45, but of these only Cx36 forms have been shown to be specific for neurons. A functional channel connecting two different neurons is formed by the apposition of two connexons, one in the plasma membrane of each neuron. Each connexon contains six Cx proteins. A functional gap junction is thus composed of 12 Cx subunits. Diversity in the composition of connexons in a gap junction can arise, as hemichannels can either consist of the same Cx subtype (homomeric) or different Cx subtypes (heteromeric). Recently, a new family of gap junction proteins, the pannexins, has been identified in the CNS. Related to innexins, an invertebrate family of gap junction proteins, the so-called pannexins (Px) have been demonstrated to form functional gap junctions in recordings from paired Xenopus oocytes. Three Px subtypes have been described in the rat and human genome, with Px1 and Px2 expressed solely in the CNS. There are strong expression patterns for Px1 and Px2 in the cortex and hippocampus, but their functional role in network activity is unknown. Gap junctions are not simply inert pores between neurons, as they can be dynamically modulated in a number of ways. Evidence suggests that the number of gap junctions, the pattern of expression of Cx proteins, and conductance properties of the individual channels can all be modified by neurotransmitters, second messengers, calcium ion concentrations, intraand extracellular pH, and transmembrane voltage. This sort of modification occurs during development
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220 Gap Junctions and Neuronal Oscillations Closed
Open
Connexon Connexin monomer
Plasma membranes
Intercellular space 2–4 nm space
Hydrophilic channel
Figure 1 Structure of a gap junction, showing a cluster of gap junctions (yellow) spanning two opposing plasma membranes (blue sheets). The intercellular space narrows at the point of the gap junction, bringing the two membranes into close contact. A gap junction consists of two hemichannels (one in each plasma membrane) that form a continuous channel between the two cells. Each hemichannel contains six connexin proteins, which, as a unit, are called a connexon. Insets show that a conformational change in the connexin proteins allows the gap junctions to open and close in response to modulators such as pH, calcium, and cAMP.
and in response to a variety of factors in the adult. Modulation allows for the complex, connexin-specific regulation of electrical signaling via gap junctions, although to date little is known about the mechanism.
Approaches to Studying Gap Junctions Evidence for electrical coupling mediated via gap junctions in the CNS comes from using a multidisciplinary approach. The CNS consists of excitatory principal cells and inhibitory interneurons, and there is evidence for electrical signaling in both neuronal populations. Electron microscopy and freeze-fracture studies have anatomically demonstrated gap junctions between both interneurons and pyramidal cells in the cortex. Early electrophysiological evidence for electrical signaling via gap junctions was obtained by recording from principal cells in the cortex and hippocampus. These studies also showed that certain dyes (e.g., Lucifer yellow) could pass from one principal neuron to another via gap junctions. In dye coupling experiments a specific dye is injected into one cell, and if that cell is coupled to other cells, dye transfer may be detected in the connected cells. However, dye coupling studies are complicated because different connexins may have different permeabilities to dyes, and dye transfer depends on the time for filling and the locations of the gap junctions. For this reason a lack
of dye coupling does not completely exclude the presence of functional gap junctions. Interneurons represent only 10–20% of the neuronal population and are thus difficult to record ‘blind’ with intracellular sharp electrode techniques. In order to surmount this issue experimentalists have utilized infrared differential interface contrast (IR-DIC) microscopy and whole-cell patch recording techniques. This has made it possible to identify interneurons visually from their basic morphology and thus conduct simultaneous whole-cell patch recordings from two or more interneurons. Using this technique electrical coupling between pairs of interneurons has been demonstrated. When current is injected into one cell to change the membrane potential, a potential change is also seen in the coupled cell (Figure 2). The ratio of the potential change in cell 1/cell 2 is termed the coupling coefficient or coupling ratio. As with the dye transfer studies, the absence of electrical coupling potentials cannot totally exclude the possibility that gap junctions may be present at sites too distant for current transfer to be detected with somatic recordings. Electrically coupled interneurons have now been shown to be widespread in many areas of the vertebrate CNS, including the hippocampus and neocortex. However, elucidating the functional significance of electrical signaling via gap junctions has been made difficult by the lack of specific agents to block gap
Gap Junctions and Neuronal Oscillations 221
Cell 2
Cell 1
2 mV
40 mV
Cell 2
2 mV
Cell 1 20 mV
Figure 2 Electrical coupling potentials, showing electrical coupling between fast-spiking interneurons. Paired whole-cell recordings of two fast-spiking interneurons from the hippocampal dentate gyrus region in the mouse are depicted. Traces show the voltage responses in cell 1 following depolarizing (upper) and hyperpolarizing (lower) current injections; voltage responses were also reflected in cell 2 but were smaller in amplitude.
junctions. Uncoupling agents that are currently used include heptanol, octanol, halothane, carbenoxolone, and 18a-glycyrretinic acid. However, these agents will block all gap junctions (including glial gap junctions), making it impossible to clearly identify the importance of any specific gap junctions. Recently, blockers for Cx36 have been described, including quinine and the more specific mefloquine. However, all of these compounds have been reported to have a range of nonspecific effects that could also affect neuronal activity. New strategies have been developed to study the role of electrical signaling via gap junctions using genetically engineered mice that lack specific connexin proteins (see later).
Evidence for Gap Junctions between Principal Neurons Dye coupling studies show that when one principal cell is injected with dye, often one or two other cells also fill with dye, suggesting direct coupling between the cells. Procedures that increase gap junction conductance (e.g., reduced extracellular calcium) tend to increase the number of dye-coupled neurons. Conversely, the degree of dye coupling is diminished when gap junction conductance is reduced by the application of gap junction blockers. In these and earlier studies, potentials resembling small action potentials (<10 mV) were observed in recordings from the soma of principal cells. Termed fast prepotentials (FPPs), and first observed in vivo, these events were initially
believed to arise from electrogenesis in dendrites. Subsequent experimental studies, however, suggested that FPPs or spikelets (presently the more commonly used term) represent action potentials in electrically coupled neurons. The location of the gap junctions that mediate coupling between principal cells has yet to be conclusively demonstrated. Paired whole-cell patch recording techniques have failed to demonstrate significant electrical coupling between the somas of principal cells. However, recent intracellular recordings with sharp electrodes show high levels of coupling between pyramidal cells in the hippocampus. There is evidence to suggest that electrical signaling via gap junctions could be axo-axonic in principal cells. Computational studies first proposed that the fast kinetics of the spikelets seen in pyramidal cells could only be explained if the gap junctions were located between axonal compartments. If the gap junctions were located at remote sites on the dendrites of pyramidal cells, then the electrical-filtering properties of the dendrites would result in slower kinetic properties of the spikelets. Experimental evidence was subsequently provided when one study, using paired recordings from the soma and axon hillock/axon initial segment of the same cell, showed that following antidromic stimulation of the relevant fiber tract, the evoked spikelet appeared first in the axon hillock/initial segment and then in the soma. Subsequent visualization experiments revealed that the path taken by dye loaded into one cell could be followed in real time, as it was observed first filling in the axon and then subsequently in the soma of a second neuron. One recent electron microscopy study has found ultrastructural evidence for gap junctions between pairs of mossy fiber axons in the CA3 region of the hippocampus. However, the identities of the proteins that compose the putative axo-axonic gap junctions are not fully known, although the pannexins and Cx36 have both been proposed as possible candidates.
Evidence for Gap Junctions between Interneurons Perhaps the greatest advance in our understanding of the role of electrical signaling via gap junctions has come from studies of interneurons. Interneurons in the hippocampus and neocortex consist of a number of distinct subtypes that can be identified on the basis of their morphology, physiological properties, and their expression of specific chemical markers such as peptides or calcium-binding proteins. Early anatomical studies clearly demonstrated evidence for the existence of dendrodendritic or dendrosomatic gap junctions between interneurons in the cortex.
222 Gap Junctions and Neuronal Oscillations
Recent physiological studies have also shown that interneurons can be electrically connected. Electrical coupling between interneuron pairs has been shown to be bidirectional, with current usually flowing equally in both directions. In addition, gap junctions function as low-pass filters in that they preferentially transmit low-frequency signals. Fast signals, such as action potentials, are not transmitted as well and appear in the coupled cell as a truncated spikelet. Nonetheless, spikelets can still facilitate the generation of action potentials in the coupled cell and so enhance the spread of activity. The traditional view of electrical signaling via gap junctions as important for mediating fast excitation is an oversimplification. Gap junctions can transmit depolarizing and hyperpolarizing current equally well. For example, the afterhyperpolarization that follows an action potential is also transmitted to the coupled cell, so a spikelet can be followed by a significant hyperpolarization. Thus electrical signaling can be both excitatory and inhibitory. A key feature of gap junction coupling observed between cortical interneurons is that it is mainly between interneurons of the same class (e.g., fast-spiking basket cells, multipolar bursting cells, neurogliaform cells, and low-threshold spiking cells). As it has long been postulated that different classes of interneurons are involved in different functions, this specificity
1
LS
CB1 IS
2/3
of connectivity leads to the formation of distinct networks of electrically coupled interneurons, each of which could be involved in a separate function. In the neocortex, at least five different interneuron networks of electrically coupled cells have now been identified (Figure 3). Interneurons belonging to the same morphological class exhibit gap junction coupling, often with a high incidence (e.g., 50% of basket cells coupled). However, only a few cases of electrical coupling between different interneuron classes have been reported. Paired whole-cell patch recordings reveal that electrical coupling between all inhibitory interneurons so far studied is mediated by the same connexin (Cx36). In mice (though not in rats), Cx36 is expressed only in interneurons, and mice that lack Cx36 exhibit no functional electrical coupling between pairs of interneurons. As yet we know nothing about the mechanisms that regulate the specificity of interneuron coupling so as to ensure that connections only form between interneurons of the same type. This remains an important issue to address in the future. There is strong evidence for the developmental refinement of the cell-specific distribution of gap junctions. The degree of electrical coupling in both rodent and human brain declines with maturation. This decline in the extent of electrical coupling was one reason that electrical signaling via gap junctions
LS
CB1 IS
MB
MB
PV-CB
PV-CB
4 LTS LTS
5
FS PV
SST
SST FS PV
Trends in Neurosciences 6 Figure 3 Five networks of distinct electrically coupled g-aminobutyric acid (GABAergic) interneurons in the cerebral cortex. Numbers indicate cortical laminae. Pyramidal cells are shown in black. Different colored circles represent different interneuron classes that are electrically coupled (jagged lines). Some interneuron classes are also chemically synaptically coupled, and all form synapses onto the pyramidal cells. CB, calbindin; CB1 IS, cannabinoid receptor-1-expressing irregular-spiking cell; FS, fast-spiking cell; LS, late-spiking cell; LTS, low-threshold spiking cell; MB, multipolar bursting cell; PV, parvalbumin; SST, somatostatin.
Gap Junctions and Neuronal Oscillations 223
was thought to be important mainly in development. However, a number of studies have demonstrated that electrical coupling persists in the adult brain, and computational studies predict that low levels of coupling (e.g., one to two gap junctions per cell) are in fact sufficient to enhance network synchronization.
Gap Junctions and Oscillations of Cognitive Relevance Oscillatory, or synchronized, network activity occurs in the EEG in different frequency bands during specific behavioral states in mammals, including humans. Oscillations can occur at from less than 1 Hz to several hundred hertz. This broad range of activity is tightly linked to higher cognitive function such as memory formation, sensory perception, and integration. Two types of oscillatory activity in which the role of electrical signaling via gap junctions has been particularly well studied are the gamma frequency (20–80 Hz) and ultrafast (>80 Hz) or very fast oscillations (VFOs). Gamma frequency activity can be recorded in many brain areas but is particularly prominent in the neocortex and hippocampus. It is associated with a number of functions such as sensory perception, attention, learning, and memory. Ultrafast oscillations in the hippocampus are also associated with memory functions, particularly the consolidation of declarative memories.
Role of Electrical Signaling via Gap Junctions in Generating Fast Network Oscillations A major methodological advance in the study of gamma frequency oscillations has been the ability to study this activity in vitro. Persistent gamma oscillations can be induced and sustained in brain slices of hippocampus and cortex by the bath application of drugs that can depolarize both pyramidal cells and interneurons such as kainate, muscarinic, or glutamate metabotropic receptor agonists. Gamma frequency activity cannot be generated in the absence of inhibitory neurotransmission, and several studies have identified specific interneuron classes, particularly fast-spiking basket cells, that fire at gamma frequency during the oscillation and so contribute to the generation of this activity. However, in addition to its role in GABAergic inhibition, electrical signaling via gap junctions also appears to be important for the generation of gamma frequency oscillations. Recent work suggests that electrical coupling between both interneurons and pyramidal cells is important for the generation of gamma frequency activity.
It had been known for some time that application of gap junction blockers (halothane, octanol, and carbenoxolone) can abolish gamma frequency oscillations, but these studies could not distinguish the contribution of electrical signaling via gap junctions in interneurons versus pyramidal cells. However, it is possible in vitro to generate a gamma frequency oscillation in which pyramidal cell activity has been blocked – the so-called interneuronal network gamma (ING). Computational modeling studies initially suggested that, along with chemical inhibitory transmission, electrical signaling via dendritic gap junctions between interneurons was important for synchronizing gamma frequency activity. Using the in vitro ING model it was found that blockade of gap junctions markedly reduced the synchrony of the oscillations, although some gamma frequency activity still remained. The contribution of electrical coupling between interneurons to fast network oscillations was tested further using Cx36 knockout mice that specifically lack functional electrical coupling between interneurons. Using the Cx36 mice, in which excitation is intact, it was found that the ability of hippocampal neuronal networks to generate gamma frequency activity was significantly reduced. Reductions in gamma frequency activity were seen both in vitro and in vivo. At a cellular level, recordings from principal cells and interneurons in vitro showed a disruption in the amplitude, rhythmicity, and coherence of inhibitory inputs. Under normal conditions, spikelets in interneurons increase the likelihood of full action potentials being generated in the coupled cells, thus enabling a network of interneurons to fire synchronously. These synchronous inhibitory outputs impinge on the postsynaptic principal cells and control the timing of the spike output, as the principal cells can only fire once the inhibition has decayed. The inability of interneurons in the Cx36 knockout mice to synchronize properly their firing via gap junctions leads to a reduction in the precision of their action potential timing and thus their rhythmic inhibitory output. Behavioral studies have shown that mice lacking Cx36 exhibit learning and memory impairments, an observation consistent with the idea that normal gamma frequency activity is important for these cognitive tasks. Importantly, the loss of Cx36 did not lead to the complete abolition of gamma frequency activity. A residual gamma oscillation was still present both in vivo and in vitro. In vitro it was shown that this remaining gamma frequency oscillation was blocked by the putative gap junction blocking agent, carbenoxolone. This suggested that some other form of
224 Gap Junctions and Neuronal Oscillations
gap junction-mediated signaling is also required for the generation of the gamma frequency activity, and this is now thought to arise from the pyramidal cell population. Electrical signaling via gap junctions between pyramidal cells was initially proposed to underlie the generation of certain types of ultrafast or ‘ripple’ activity. Using an in vitro brain slice model, highfrequency oscillatory activity, or ‘ripples,’ occurring at 80–150 Hz can be generated in the hippocampus in the absence of chemical synaptic transmission when using a nominally calcium-free bath medium. The lack of chemical synaptic transmission in these experiments supports the hypothesis that this activity is dependent on direct electrical connections between neurons. The ‘ripple’ activity is abolished in the presence of pharmacological blockers of gap junctions, such as halothane or carbenoxolone. At the level of individual neurons, intracellular recording from principal cells revealed that, concomitant with the extracellular field potential ‘ripple,’ the cells discharged either a full-action potential or a spikelet. The kinetic properties of these spikelets (as discussed earlier) were too fast to be due to dendrodendritic gap junctions, and so axo-axonal connections were proposed. In addition, in the Cx36 knockout mice, this ultrafast oscillatory activity was normal both in vivo and in vitro, suggesting that interneuronal electrical coupling did not underlie this activity. More recent work has now demonstrated that the ultrafast oscillations coexist with the gamma frequency activity. Ultrafast oscillations can be generated in the axon plexus of pyramidal cells in the hippocampus. It is currently thought that these highfrequency oscillations occur as a result of random ectopic action potentials (action potentials arising from regions other than the axon hillock) in the axon plexus. This random activity spreads throughout the axon plexus, via axo-axonic gap junctions, resulting in a barrage of fast excitatory synaptic potentials that can be recorded in the interneurons, and provides the necessary excitatory drive for the interneurons. The combination of chemical and electrical connections between the interneurons then allows a synchronized output back to the pyramidal cells at gamma frequency. Gap junctions are therefore required between the pyramidal cells to generate the drive to the interneurons, and the gap junctions between the interneurons are needed to temporally coordinate their activity.
Gap Junctions and Epilepsy The EEG is an important test in the diagnostic process of treating epilepsy. Traditionally, this approach
has been noninvasive, using small, metal electrodes attached to the scalp. This approach is satisfactory for the medical evaluation of the seizure activity. However, there are limitations to this approach. The advent of digital recordings and invasive human studies using depth electrode recordings or subdural electrode mats now allows the measurement of electrical signals with frequencies greater than those recordable with conventional EEG equipment. Using these techniques, ultrafast oscillatory activity has been observed in close proximity to seizure onset zones in both animal models and humans. Indeed, it has been suggested that this type of activity is a reliable electrophysiological marker of epileptogenicity. A common feature of focal epileptic seizure activity, in both humans and experimental models, is the presence of ultrafast oscillations. Thus fast ripples associated with interictal spikes currently provide information on localization and epileptogenicity of the tissue involved in their generation. Evidence from developmental studies further supports a role of gap junctions in epilepsy. Thus genetic linkage mapping demonstrates that Cx36 is a high-ranking positional and functional candidate gene for juvenile myoclonic epilepsy. A number of molecular biology studies have demonstrated that in neuronal tissue obtained from animal models and human cases of epilepsy there are obvious alterations in the abundance of connexins. Most of this work concerns the expression of connexins, which is limited to astrocytes. Several studies have examined protein and or mRNA expression, but the results from these studies yield conflicting results, with some reporting increases in connexin expression and others reporting decreases. Differences in a number of the experimental parameters applied could account for these discrepancies. It is obvious that more work is required to determine whether Cxs are modified in epileptic tissue, and if so in what directions. Moreover, if putative axo-axonic gap junctions are composed of pannexins, then attempts should be made to examine the expression and localization of such proteins in both human epileptic tissue and tissue from animal experimental models of epilepsy.
Conclusions Recent advances over the past decade have demonstrated that both chemical signaling and electrical signaling play a central role in cortical communication. It is generally widely accepted that chemical signaling allows for highly complex, specific, and modifiable neuronal communication. However, it is now apparent that electrical signaling mediated via gap junctions can also demonstrate a high degree of functional specificity. Thus, the combination of
Gap Junctions and Neuronal Oscillations 225
multiple coexistent synaptic and electrical networks serves to greatly enrich the processing capabilities on neuronal networks. See also: Gap Junctions and Electrical Synapses.
Further Reading Bennet MLV and Zukin RS (2004) Electrical coupling and neuronal synchronization in the mammalian brain. Neuron 41: 495–511. Buzsaki G and Draguhn A (2004) Neuronal oscillations in cortical networks. Science 304: 1926–1929.
Connors BW and Long MA (2004) Electrical synapses in the mammalian brain. Annual Review of Neuroscience 27: 393–418. Galarreta M and Hestrin S (2001) Electrical synapses between GABA-releasing interneurons. Nature Reviews Neuroscience 2: 425–433. Hestrin S and Galerreta M (2005) Electrical synapses define networks of neocortical GABAergic neurons. Trends in Neuroscience 28: 304–309. Schmitz D, Schuchmann S, Fisahn A, et al. (2001) Axo-axonal coupling: A novel mechanism for ultrafast neuronal communication. Neuron 31: 831–840. Whittington MA and Traub RD (2003) Interneuron diversity series: Inhibitory interneurons and network oscillations in vitro. Trends in Neuroscience 26: 676–682.
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AMINO ACID TRANSMITTERS AND RECEPTORS A. Glutamate
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B. GABA
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C. Glycine
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Glutamate S P H Alexander, University of Nottingham Medical School, Nottingham, UK ã 2009 Elsevier Ltd. All rights reserved.
Introduction Glutamate is easily the most abundant transmitter in the nervous system and is reported to be the transmitter at 40% of all synapses in the brain. Indeed, it has been said that if a neuron fails to respond to glutamate, it is either not a neuron or it is dead. In itself, therefore, this abundance can lead to some issues. For example, a wide distribution of multiple pharmacological targets suggests that agents targeting the glutamate system will either lack target selectivity or impact on multiple bodily functions. However, it has become clear in recent years that the numerous receptors for glutamate and their localized expression have allowed the development of several potentially exciting therapeutic agents, although relatively few are sufficiently advanced as to reach a clinical setting. In common with many other neurotransmitters (g-aminobutyric acid (GABA), acetylcholine, 5-hydroxytryptamine, and adenosine 50 -triphosphate), the action of glutamate is mediated via both rapidly acting transmitter-gated channels (ionotropic receptors, Table 1) and more slowly acting seven-transmembrane (7TM), G-protein-coupled (metabotropic, Table 2) receptors.
Metabolism, Uptake, and Release Since glutamate is a component of every cell, there is no clear synthetic route that sets apart neurotransmitter glutamate. One intriguing aspect of glutamate metabolism, however, is glutamic acid decarboxylase activity. This enzyme performs an elegant switch in emphasis, by catalyzing conversion of the major excitatory neurotransmitter (glutamate) to the major inhibitory neurotransmitter (GABA). Glutamate Transport: Plasma Membrane and Vesicular Mechanisms
Due to the major roles of glutamate as an extracellular signaling molecule and as an intracellular building block for proteins combined with the toxicity associated with elevated extracellular levels, effective mechanisms exist for removal of glutamate from the extracellular fluid and also for concentrating the amino acid in synaptic vesicles. Thus, at least five cell-surface glutamate (and aspartate) transporters
have been defined as members of the solute carrier family 1 (SLC1), while three vesicular transporters (of the SLC17 family) have also been identified. The cell-surface transporters are multisubunit, homo- or heteropentameric structures in which each subunit appears to have 8TM spanning domains and at least one re-entrant loop that ‘dips’ into the plane of the membrane. One of the physiological roles for the avid glial glutamate transport system is to allow glial conversion of glutamate to glutamine, which is then exported via the extracellular space into neurons as a means of regenerating neurotransmitter glutamate. Determination of glutamate uptake in tissue slices or cultured cells is most often accomplished through the use of radiolabeled glutamate, as metabolism to other entities may confound direct assessment of glutamate accumulation. Detection of Glutamate Release
In order to define a role for glutamate as a mediator of a physiological or pathophysiological mechanism, it is fundamental to be able to measure changes in extracellular glutamate in nervous tissues. In vitro, this is commonly accomplished through the use of [3H]-D-aspartate as a surrogate marker, since the D-isomer is less likely to be metabolized. However, there are doubts as to whether [3H]-D-aspartate accumulates in synaptic vesicles and thus the assay may more accurately reflect cytoplasmic glutamate release. It is, therefore, preferable to assess glutamate release directly. One means is to label tissue with [3H]-L-glutamine, with a view to labeling preferentially the neuronal glutamate precursor pool. However, this approach has the drawback that a chromatographic separation of glutamate from glutamine and GABA is required, although it may be useful in some cases to identify simultaneously levels of both excitatory glutamate, as well as inhibitory GABA. A means of directly measuring glutamate release in vitro is through the use of high-performance liquid chromatography (HPLC) separation of extracellular fluid following amino acid derivatization with o-phthaldehyde. This has the potential advantage of allowing levels of glutamate and GABA (as well as other bioactive released amino acids such as glycine, aspartate, and taurine) to be assessed concurrently, although it has the disadvantage that a certain period of accumulation (usually 15–20 min) is required to obtain sufficient material to detect. Two ‘real-time’ in vitro assays for glutamate have been described: an enzyme-coupled spectrophotometric version making use of exogenous glutamate dehydrogenase activity and a glutamateselective electrode.
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230 Glutamate Table 1 Ionotropic glutamate receptors: composition and pharmacology AMPA
Kainate
NMDA
Quisqualate GLUA1, GLUA2, GLUA3, GLUA4 Homo- or hetero-tetramers Naþ, Kþ, (Ca2þ) Polyamines, argiotoxin, Joro toxin
GLUK1, GLUK2, GLUK5, GLUK6, GLUK7 Homo- or hetero-tetramers Naþ, Kþ Polyamines
GLUN1, GLUN2A, GLUN2B, GLUN2C, GLUN2D, GLUN3A, GLUN3B Heterotetramers Naþ, Kþ, Ca2þ Dizolcipine, ketamine, phencyclidine, memantine, amantidine
Endogenous ligand(s)
L-Glutamate
L-Glutamate
L-Glutamate,
Selective agonists
AMPA, (S)-fluorowillardiine
ATPA, (S)-5-iodowillardiine, (2S,4R)-4-methylglutamate, LY339434, domoic acid UBP302
DL-(tetrazol-5-yl)
Other names Subtypes Composition Ions gated Channel blockers
Glutamate site L-Aspartate
Selective antagonist
NBQX, ATPO, LY293558, GYKI53655
In vivo detection of extracellular glutamate has been conducted through the use of microdialy coupled to HPLC separation and o-phthaldehyde detection or alternatively through the use of a glutamate-sensitive electrode.
Receptors and Signaling Twenty-four separate gene products which make up the cell-surface receptors for glutamate have been identified and are divided into two major classes both structurally and functionally; rapidly responding ‘ionotropic’ transmitter-gated channels and slower responding ‘metabotropic’ 7TM receptors (Figure 1). The ionotropic receptors consist of three groups of receptors (yellow highlight in Figure 1), identified by the selective synthetic agonists a-amino-3-hydroxy-5methyl-4-isoxazole proprionic acid (AMPA), kainate, and N-methyl-D-aspartate (NMDA). Metabotropic glutamate (mGlu) receptors also consist of three groups as identified by similarities in primary sequence (blue highlight in Figure 1), known simply as group I, group II, and group III. Ionotropic Glutamate Receptors: AMPA, Kainate, and NMDA Glutamate Receptors
Rapid responses (<1 s) to glutamate in neurons are mediated through cell-surface transmitter-gated channels. These are tetrameric proteins arranged in a radially symmetrical fashion around a central pore, which is permeable to cations (Table 1). Each subunit comprises a large extracellular N-terminus (S1), which interacts with an extracellular loop (S2) to form the agonist binding site. There are three transmembrane segments (TM1, TM3, and TM4) with a single channel
glycine, homoquinolinic acid D-AP5, CGS19755
Glycine site Glycine, D-Serine (þ)HA966
5,7-DCKA, L689560, L701324
lining re-entrant segment (termed MD2 or p-loop) that inserts into the plane of the membrane from the cytoplasmic face (Figure 2) between TM1 and TM3. The cytoplasmic C-terminus is the site for interaction of a number of protein partners (vide infra). Nomenclature and composition As ionotropic glutamate receptors are multi-subunit proteins with few selective pharmacological ligands, nomenclature of the individual subunits is based on physical (i.e., primary sequence) distinctions rather than pharmacological means. Of the eight subunits identified as NMDA receptor components, a minimal configuration in heterologous expression of the NMDA receptor appears to be two pairs of GLUN1 and GLUN2 subunits, although it appears more likely that, physiologically, NMDA receptors are mixtures of all three subunit groups (GLUN1, GLUN2, and GLUN3, encoded by the genes GRIN1, GRIN2A/2B/2C/2D, and GRIN3A/3B). The GLUN3 subunits appear to have a generally inhibitory influence on NMDA receptor function. Many of the genes encoding NMDA receptor subunits undergo alternative splicing, which can generate multiple isoforms with differing pharmacological profiles. AMPA receptors appear to be made up of any permutation of GLUA1, GLUA2, GLUA3, and GLUA4 subunits (derived from the genes GRIA1–4, respectively); although GLUA2 subunits appear to require co-expression with other AMPA receptor subunits for cell-surface localization. A feature of the AMPA receptor subunit GLUA2 is the ability to function as two versions inter-converted following RNA editing (resulting in a switch from a glutamine to arginine residue; a Q/R switch) from a channel highly permeable
Table 2 mGlu receptors: biochemistry, localization and pharmacology
Signalling Group selective ligands Endogenous
Group I
Group II
Group III
Gq/11, PLC-b, Ca2þ
Gi/o, #AC, #cAMP, "Kþ
Gi/o, #AC, #cAMP, "Kþ
L-Glutamate, L-Aspartate
L-Glutamate, L-Aspartate,
L-Glutamate, L-Aspartate, L-Serine-O-phosphate
Synthetic agonists
Quisqualate, 3.5-DHPG
Antagonists Subtypes Protein partners
LY393675 mGlu1 Homer, TRPC1, PIKE
Selective agonists Selective antagonists Allosteric enhancers Allosteric inhibitors Impact of gene disruption
mGlu5 Homer, FMR1, PIKE
NAAG DCG-IV, 2R,4R-APDC, LY354740 EGLU, LY307452 mGlu2 mGlu3
3-MATIDA
CHPG ACDPP
PCCG-4
NAAG
Ro67-4853
DFB
LY487379
CPCCOEt Altered synaptic transmission in the hippocampus and cerebellum; symptoms of cerebellar ataxia; increased pain tolerance
MPEP, MTEP Disruption of hippocampal synaptic transmission & LTP; weakened spatial learning and memory acquisition; reduced cocaine self-administration; a deficit in prepulse inhibition
Reduced hippocampal LTD associated with an absence of agonist-induced inhibition of glutamate release
L-AP4,
(RS)PPG
MAP4, MSOPPE mGlu4
()PHCCC
mGlu6
mGlu7
Homo-AMPA THPG
mGlu8
MPPG AMN082
Disruption of spatial learning and memory acquisition; loss of efficacy of the anti-epileptic agent baclofen
Normal retinal structure but marked impairments in visual receptiveness
A shortened lifespan associated with epileptic seizures
Heightened anxiety in behavioural tests
Glutamate 231
232 Glutamate GRM1 GRM5 GRM2 GRM3 GRM4 GRM8 GRM7 GRM6 GRIA1 GRIA2 GRIA3 GRIA4 GRIK1 GRIK2 GRIK3 GRIK4 GRIK5 GRIN1 GRIN2A GRIN2B GRIN2C GRIN2D GRIN3A GRIN3B Figure 1 Phylogram of the human glutamate receptor subunit peptide sequences. Picked out in yellow highlight are the three families of ionotropic receptor (AMPA, kainate, and NMDA receptors). Blue highlighting identifies the three groups of mGlu receptor.
GLU
Agonist binding domains (S1, S2)
Transmembrane domains
Protein:protein interaction
Figure 2 An ionotropic glutamate receptor subunit. Indicated is an extracellular N-terminus agonist binding site (S1, S2), the transmembrane domains (TM1, TM3, TM4, and re-entrant loop) and the intracellular C-terminus.
to calcium ions, to one much less permeable. All AMPA receptor subunits are subject to alternative splicing, which results in two variants termed ‘flip’ and ‘flop’ with distinct desensitization characteristics. Of the kainate receptors, GLUK1 and GLUK2 subunits (from the genes GRIK1 and GRIK2, respectively) are a subfamily which exhibits high-affinity agonist binding without function and which appear to require co-expression of members of the other kainate receptor subfamily, GLUK5, GLUK6, or GLUK7 subunits (from
GRIK3, GRIK4, and GRIK5 genes, respectively) to show functional activity. In contrast, the latter subfamily of subunits appears capable of generating functional kainate receptors as homotetramers. Pharmacology and signaling The NMDA receptor is primarily located postsynaptically and is only activated in the presence of both glutamate and glycine, this dual requirement being unique amongst transmitter-gated channels. Glutamate acts on GLUN2
Glutamate 233
subunits, while glycine binds to both GLUN1 and GLUN2 subunits, with receptor activation requiring two molecules of each ligand. Under resting conditions, the NMDA receptor ion channel is blocked by Mg2þ ions, which dissociate when the cell is depolarized. The open channel exhibits a relatively high permeability to Ca2þ, along with the monovalent cations Naþ and Kþ. Similar to the GABAA receptor, the NMDA receptor exhibits multiple allosteric modulatory sites, including inhibitory responses to Zn2þ and protons, as well as subunit-selective potentiatory responses to neurosteroids and polyamines. Selective agonists exist for both glutamate- and glycine-binding sites of the NMDA receptor (Table 1 and Figure 3), as well as selective antagonists. However, the most widely employed antagonists function as channel blockers and include dizocilpine (previously MK-801), ketamine (Figure 4), and phencyclidine (Table 1). It is thought that the abuse of the latter two agents (known as ‘special K’ and ‘angel dust’ or PCP, respectively), originally produced as dissociative anesthetics, is mediated at the molecular level through blockade of the NMDA receptor. Antagonists at the strychnine-insensitive glycine co-agonist binding site of the NMDA receptor, such as 5,7-dichlorokynurenate (Figure 4), are effective in vitro and in vivo inhibitors of NMDA responses.
AMPA receptors can be activated by high concentrations of kainate (>30 mM), while activation of kainate receptors by AMPA is dependent on the constituent subunits, with the GLUK2 subunit appearing to confer AMPA sensitivity (Figure 3). The quinoxalinedione antagonists, such as NBQX (Figure 4) and CNQX, show some limited selectivity in distinguishing AMPA from kainate receptors (Table 1). Synthetic allosteric inhibitors of AMPA and kainate receptor function (GYKI5246 and NS3763, respectively) have also been described. Intriguingly, a number of natural toxins (e.g., from Joro spiders, Figure 4) have been described which function as channel blockers for AMPA (and, to a lesser extent, kainate) receptors. Presumably, these have evolved as a means of predators disabling insect prey, since glutamate is the neurotransmitter at the invertebrate neuromuscular junction, acting on postsynaptic AMPA-like receptors. In addition, aniracetam, cyclothiazide, and diazoxide are widely used to interfere with the channel desensitization characteristics of AMPA (and, to a lesser extent, kainate) receptors through an undefined allosteric interaction. Protein partners A key area of regulation of (particularly) AMPA and kainate receptors is desensitization and intracellular trafficking. An intracellular protein
O O
O
HO
O
O HO
OH
HN O
AMPA
Kainate O
O H
NMDA
NH2
O
O
HO
OH O
NH
O
N
HO
OH
OH
NH2
O
OH O
O
HO
NH
O
P
OH
N H
1S,3R-ACPD
HO O
O O HO
HO
OH
OH NH2 L-Glutamate
NH2
LSOP
NAAG
O
OH
O
NH2
L-Aspartate
Figure 3 Glutamate receptor agonists. The top row indicates the eponymous agonists at AMPA, kainate, and NMDA ionotropic glutamate receptors. The middle row indicates mGlu receptor agonists: the nonselective agonist 1S,3R-ACPD, the mGlu3-selective NAAG, and the mGlu4/7/8-selective L-serine-O-phosphate. The bottom row shows the endogenous excitatory amino acids, L-glutamate and L-aspartate.
234 Glutamate O HO
Cl
OH
P
O
OH
O
H N
O OH
NH2
NH O Cl DCKA
D-AP5
Ketamine OH
S NH2 O
O
O
H N
N H
UBP302 HO
O
O
N+ O O
O
HO
O H2N
O N
N O
JSTX3
O
NH2
S NH2 N
N
H O
LY341495
OH
NH
OH NH2
O HN
O
HN
NBQX H N
H N
N H
O
H N
O
Cl
OH
N
O
O
OH
O
P
OH
HO NH2 MTEP
MAP4
Figure 4 Glutamate receptor antagonists. The top row illustrates the three classes of NMDA receptor antagonist: antagonists competing for glutamate (D-AP5) and glycine (DCKA) binding sites, as well as an uncompetitive (open channel blocker, ketamine) antagonist. The middle row presents competitive AMPA (NBQX) and kainate (UBP302) receptor antagonists and a spider venom-derived AMPA channel blocker (JSTX3). The bottom line illustrates a nonselective mGlu receptor competitive antagonist (LY341495) and noncompetitive mGlu5 (MTEP) and competitive mGlu4/6 (MAP4) receptor antagonists.
called glutamate receptor-interacting protein (GRIP) appears to allow interaction between GLUA2 subunits and other proteins, including protein interacting with C kinase (PICK, which recruits protein kinase C) and kinesin (an intracellular molecular motor associated with the cytoskeleton). AMPA receptors also interact with Lyn, a protein kinase, which in turn signals via the extracellular signal-regulated kinase signaling pathway, leading to an increased expression of brainderived neurotrophic factor. This growth factor is associated with neurogenesis in the adult brain and its expression is reduced following exposure to stress. mGlu Receptors
mGlu receptors are members of the family C of 7TM receptors, which also includes GABAB and calciumsensing receptors. This family is made up of long polypeptides (1000 aa) and is characterized by the presence of a large extracellular N-terminus (600 aa, Figure 5). This portion of the receptor contains the agonist binding site and a cysteine-rich domain, which appears to stabilize the three-dimensional structure of the receptor. The 7TM domains bear little sequence homology with the classical biogenic amine receptors (members of the rhodopsin class,
family A), which make up the ligand-binding site in the plane of the plasma membrane. The N-terminus binding site is often described as a ‘Venus fly-trap’ arrangement in which the ligands bind within the hinge region (Figure 5). Conceptually, there is thus an obstacle to overcome as to how ligand binding at some distant extracellular point is able to influence G-protein activity across the hydrophobic plasma membrane. The means by which this appears to be mediated is through dimerization of the receptor, thereby allowing conformational changes in the extracellular region to be transduced through the membrane. A further consequence of the extended structure of the C family of 7TM receptors is the regulation of receptor activity through ligands binding at sites other than the cognate ligand-binding site. Thus, for many of the mGlu receptor subtypes, selective positive and/or negative allosteric modulators have been described which extends the potential for pharmacological/therapeutic exploitation considerably (vide infra). The cytoplasmic C-terminus is also enlarged in comparison with most members of the rhodopsin group of 7TM receptors and this is the site for interaction with a number of intracellular protein partners (vide infra).
Glutamate 235
GLU
GLU
Glutamate binding domains
Cysteinerich regions
Transmembrane domains
Protein:protein interaction
Figure 5 A dimeric mGlu receptor. Indicated are the extracellular N-terminus glutamate binding site, cysteine-rich conformation stabilizing regions, the transmembrane domains (TM1–7) and the intracellular C-terminus site for protein:protein interactions.
Eight subtypes of mGlu receptor have been identified. They are usually classified into three groups on the basis of similarities in primary sequence (Figure 1), signaling characteristics and pharmacology. Thus, group I mGlu receptors (mGlu1 and mGlu5) couple primarily to elevation of calcium, while group II (mGlu2 and mGlu3) and group III (mGlu4, mGlu6, mGlu7, and mGlu8) receptors evoke an inhibition of cAMP. Localization mGlu receptors are located throughout the brain and spinal cord, and are also found in the retina (mGlu6), pancreas (mGlu1, where they appear to subserve a role in the monitoring of nutritional status), and in taste buds (mGlu4, where they mediate sensation to the ‘umami’ taste). In neural tissues, there is evidence for differential neuronal and astrocytic locations of mGlu receptors. mGlu1 receptors appear to be exclusively expressed in neurons, while mGlu5 receptors are found on both astrocytes and neurons. Similarly, mGlu2 receptors are selectively neuronally expressed, while mGlu3 receptors have been identified on both neurons and astrocytes. Of the group III receptors, mGlu6 receptors appear to be exclusively retinal, while the remainder are primarily neuronally located. Pharmacology and Signaling
Although glutamate is an endogenous ligand at all of the mGlu receptors, the potency of glutamate varies widely. mGlu1, mGlu3, and mGlu5 receptors respond to submicromolar glutamate, while mGlu2, mGlu4, mGlu6, and mGlu8 receptors respond to glutamate at concentrations up to 10 mM. In contrast, mGlu7 receptors only respond to concentrations
of glutamate approaching the millimolar range, leading to the suggestion either that glutamate is not the principal endogenous ligand or that the mGlu7 receptor acts presynaptically as a glutamate autoreceptor, sensing synaptic release of glutamate and switching off further release as ‘toxic’ levels are reached. Metabotropic receptors may be activated selectively compared to the ionotropic glutamate receptors by 1S,3R-ACPD (Figure 3), while LY341495 is a useful antagonist with a similar selectivity (Figure 4). Selective agonists and antagonists exist for the three groups of, if not the individual, mGlu receptors (Table 2). Thus, quisqualate is a high potency group I agonist, although it also activates AMPA receptors potently, while LY393675 acts as a group I-selective antagonist. A useful group II agonist is LY354740, while EGLU antagonizes group II receptors, albeit with low affinity. L-AP4 is a nonselective group III agonist, while MAP4 blocks most of these receptors (Figure 4). Some members of the mGlu receptor family also respond to other endogenous ligands, raising the possibility of native activation in the absence of glutamate. Thus, N-acetylaspartylglutamate (also known as spaglumic acid) is a selective mGlu3 receptor agonist, while L-serine-O-phosphate activates group III mGlu receptors (Table 2 and Figure 3). A relatively unusual feature of the family C of 7TM receptors is the regulation of receptor activity through allosteric sites. Thus, both positive and negative allosteric modulators have been described for mGlu1, mGlu2, mGlu4, mGlu5, and mGlu7 receptors. This means (for example) that mGlu5 receptors can be activated by 2-chloro-5-hydroxyphenylglycine, a response which can be enhanced by 3,30 -difluorobenzaldazine,
236 Glutamate
which alone is ineffective. Activation of the mGlu5 receptor can be inhibited either competitively (3-hydroxy-6-methyl-N-(6-methylpyridin-2-yl)pyridine-2carboxamide) or noncompetitively (3-[(2-methyl-1,3thiazol-4-yl)ethynyl]pyridine, Table 2). The group I mGlu receptors, mGlu1 and mGlu5, couple primarily via activation of members of the Gq/11 family of G-proteins to phospholipase C-b and, hence, to elevation of intracellular calcium ion levels. The primary mode of signaling of the remaining mGlu receptors is via pertussis toxin-sensitive Gi/o family members. This results in inhibition of adenylyl cyclase activity and the subsequent reduction in cyclic AMP levels. Arguably of more importance in neuronal cells is the activation of potassium channels and the subsequent hyperpolarization, resulting (typically) in a reduction in transmitter release. The mGlu6 receptor, expressed in ON retinal bipolar cells, couples to activation of PDE6, a cyclic GMP-selective phosphodiesterase, thereby leading to the closure of cGMPactivated cation channels. Protein partners It is apparent that the expanded C-terminus of the mGlu receptors has a role in the interaction with intracellular proteins, beyond the archetypal G-proteins. In particular, the group I mGlu receptors are associated with an immediate early gene product, Homer, which is enriched at the postsynaptic density. Homer appears to play an anchoring role, bringing together elements of the signaling cascade, including mGlu1 and mGlu5 receptors, phospholipase C, cell-surface transient receptor potential canonical (TRPC) calcium channels, intracellular inositol 1,4,5trisphosphate receptors, and the phosphatidylinositol 3-kinase enhancer protein, PIKE. It has been suggested that the major function of Homer is to act as a ‘brake’ on group I mGlu receptor activity through the calcium mobilization pathway and to enhance coupling via PI 3-kinase. These protein partners may also be involved in bringing together mGlu and NMDA receptors, leading to a functional interaction, such that mGlu5 receptor activation enhances NMDA receptor function, while NMDA receptor activation reverses mGlu5 receptor desensitization. Physiological Functions
The primary physiological action of glutamate in the central nervous system (CNS) is as an excitatory transmitter, allowing neuronal pathways to function throughout the brain. At the same time, glutamate is thought to be the major transmitter underlying the acquisition of memory. The molecular correlates of memory acquisition appear to differ according to the
synapse and (possibly) the maturity of the animal. The phenomenon of long-term potentiation (LTP), which has been suggested to occur at every excitatory synapse in the CNS, is demonstrated as an increased efficacy of synaptic transmission following a period of high frequency stimulation. It is well described at synapses of the Schaffer collateral–commisural pathway in the hippocampus, in which high-frequency stimulation of the perforant path input from the entorhinal cortex causes a long-lasting enhancement of low-frequency activation of the CA1 pyramidal cell output pathway. Cerebellar long-term depression (LTD) is a model of synaptic memory in which the efficiency of synaptic transmission (activation of Purkinje neurons) is reduced following high-frequency stimulation (coincident activation of parallel and climbing fiber presynaptic pathways). One explanation of these changes in efficacy of synaptic transmission, which does not hold true at all synapses, is that the underlying mechanisms involve trafficking of AMPA receptors, such that LTP may be associated with an increase in postsynaptic AMPA receptor expression (possibly resulting from recruitment of cryptic receptors), while LTD may result from phosphorylation and internalization of AMPA receptors, reducing postsynaptic receptor numbers. This plasticity, therefore, is consistent with an increased and decreased efficiency of synaptic conductance, respectively. One of the most insightful means of determining physiological roles of particular proteins is though the use of gene disruption models, although a caveat with embryonic knockout models is that the possibility exists for compensatory mechanisms to lessen or alter the true role of the gene and protein in question. Table 2 identifies some of the findings from such gene disruption studies focusing on mGlu receptors. A common theme is an alteration in synaptic plasticity, observed with disruption of mGlu1, mGlu2, mGlu4, and mGlu5 receptors, further underlining the importance of neurotransmitter glutamate in memory acquisition.
Glutamate as a Peripheral Neurotransmitter There is good evidence that glutamate subserves an excitatory transmitter function in the gastrointestinal tract, with indications of glutamate release, ionotropic and mGlu receptors, and glutamate transporters in enteric ganglia. In addition, application of glutamate and its analogs has been shown to regulate gastrointestinal motility and solute secretion. Synapses in the enteric nervous system undergo
Glutamate 237
LTP-type plasticity, and it is thought that group I mGlu receptors mediate this phenomenon, at least in part.
Pathology Stroke, Ischemia, and Neurodegenerative Diseases
An overabundance of extracellular glutamate is associated with excitotoxicity of neurons, which appears to involve activation of NMDA receptors. The mechanism of excitotoxicity is not absolutely defined, but there is a crucial role of a prolonged elevation of intracellular calcium ions, leading to mitochondrial damage and apoptosis. The NMDA receptor antagonist dizocilpine reached an advanced preclinical level as a potential antistroke therapy, failing on the basis of damage to unstressed brain areas due to a narrow therapeutic window. This drug may have been a victim of its own success, in that the high affinity and low reversibility displayed as an open channel blocker allowed little differentiation between positive and harmful effects. The generation of a lower-affinity agent, memantine, which exhibits greater reversibility and an increased tolerability has allowed approval for its use in man. The current indication for memantine is for treatment of Alzheimer’s disease. The rationale for its use is that the prolonged presence of glutamate (albeit at lower concentrations) leads to protracted NMDA receptor activation and excitotoxicity of neurons and a cognitive deficit. By reducing NMDA receptor activation, it is hoped to reduce the excitotoxicity and reduce the cognitive deficit. The accumulation of plaques positive for b-amyloid in Alzheimer’s disease is associated with neurodegeneration and, since processing and secretion of g-secretase (an enzyme which cleaves the amyloid precursor protein) is enhanced by activation of group I mGlu receptors, these receptors have also become a focus for research in this area. Another neurodegenerative disorder in which glutamate overactivity has been implicated is Parkinson’s disease. The glutamatergic pathways involved may be either thalamostriatal or corticostriatial, and so it has been suggested that group II agonists or mGlu5 antagonists might be therapeutically useful by inhibiting glutamate release or action, respectively, at these synapses. Indeed, LY354740 has been observed to display efficacy in animal models of Parkinson’s, although the mGlu5 antagonist 2-methyl-6-(phenylthynyl) pyridine was ineffective. A further potential exploitation of glutamate signaling in combating the effects of neurodegenerative diseases, such as Alzheimer’s and Parkinson’s, is the use of ‘AMPAkines,’ agents which enhance AMPA
receptor function. As described above, a number of agents interfere with AMPA receptor function as allosteric modulators or by impeding desensitization. Aniracetam is an example of such an agent, which has been exploited as a nootropic substance, also known as ‘smart drugs.’ These agents have a positive profile in animal models and those in clinical and ‘street’ use are reported to enhance cognitive powers (concentration, memory, etc.); their use has yet to be approved in the treatment of Alzheimer’s, although piracetam is used to treat the involuntary twitches suffered by Parkinson’s patients. Epilepsy
In vitro and in vivo focal application of glutamate results in epileptiform activity. This action appears to be mediated by the ionotropic receptors, and postmortem examination of the brains of epileptics suggests a neurodegenerative profile consistent with overactivity of these receptors. One potential therapeutic avenue is the exploitation of mGlu4 and mGlu7 receptors, which have been suggested to be glutamate autoreceptors, acting presynaptically to reduce glutamate release at the synapse. As noted in Table 2, disruption of either of these receptors leads to changes in seizure susceptibility further highlighting the potential for exploitation of glutamate signaling in treating this disorder. Anxiety and Depression
In preclinical assessments for leads as anti-anxiety/ anti-depressant action, there are promising results for both mGlu2-selective agonists and mGlu5-selective antagonists. It is hypothesized that the mGlu2 agonists function by reducing frontal cortex transmitter release presynaptically, while the mGlu5 antagonists regulate postsynaptic glutamate excitability. Pain
Several observations suggest that mGlu receptors represent an excellent target for the treatment of pain. Members of all three groups of mGlu receptors are present at the primary afferent-spinal cord synapse or the subsequent ascending pathway. There is particular interest in group I mGlu receptors, partly because of evidence for diminished pain sensitivity in transgenic mice (Table 2), but also because of the efficacy of antagonists in multiple pain models. Schizophrenia
Schizophrenia is suggested to be the consequence of an imbalance of glutamatergic and dopaminergic signaling systems. Phencyclidine, which blocks
238 Glutamate
NMDA receptors (as well as dopamine uptake), induces schizophrenia-like symptoms in man. In animal models, the group II agonist LY354740 was observed to reduce some of the behavioral effects of PCP, with a distinct pattern to traditional neuroleptics. In addition, the observation that mGlu5-knockout mice have an altered behavioral trait observed in schizophrenics (Table 2) has led to heightened interest in the therapeutic exploitation of these receptors for this condition.
See also: AMPA Receptors: Molecular Biology and Pharmacology; Long-Term Depression (LTD): Metabotropic Glutamate Receptor (mGluR) and NMDARDependent Forms; Long-Term Potentiation (LTP): NMDA Receptor Role; Metabotropic Glutamate Receptors (mGluRs): Functions; Metabotropic Glutamate Receptors (mGluRs): Molecular Biology, Pharmacology and Cell Biology.
Addiction
Further Reading
There is extensive interest in the use of group I antagonists and group II agonists as potential therapeutic agents in the treatment of drug addiction. In animal models, these agents reduce the symptoms of withdrawal from nicotine and morphine. In addition, there is evidence for sensitization of group II mGlu receptors in particular brain areas associated with drug dependence, following chronic exposure to morphine.
Alexander SPH, Mathie A, and Peters JA (2006) Guide to receptors and channels, 2nd edn. British Journal of Pharmacology 147 (supplement 3): S1–S183. Chen PE and Wyllie DJ (2006) Pharmacological insights obtained from structure–function studies of ionotropic glutamate receptors. British Journal of Pharmacology 147: 839–853. Dingledine R, Borges K, Bowie D, and Traynelis SF (1999) The glutamate receptor ion channels. Pharmacological Reviews 51: 7–61. Hinoi E, Takarada T, Tsuchihashi Y, and Yoneda Y (2005) Glutamate transporters as drug targets. Current Drug Targets CNS and Neurological Disorders 4: 211–220. Kew JNC and Kemp JA (2005) Ionotropic and metabotropic glutamate receptor structure and pharmacology. Psychopharmacology 179: 4–29. Lujan R, Shigemoto R, and Lopez-Bendito G (2005) Glutamate and GABA receptor signalling in the developing brain. Neuroscience 130: 567–580. Madden DR (2002) The structure and function of glutamate receptor ion channels. Nature Reviews Neuroscience 3: 91–101. Matute C, Domercq M, and Sanchez-Gomez MV (2006) Glutamate-mediated glial injury: Mechanisms and clinical importance. Glia 53: 212–224. Niswender CM, Jones CK, and Conn PJ (2005) New therapeutic frontiers for metabotropic glutamate receptors. Current Topics in Medicinal Chemistry 5: 847–857. Robbins TW and Murphy ER (2006) Behavioural pharmacology: 40þ years of progress, with a focus on glutamate receptors and cognition. Trends in Pharmacological Sciences 27: 141–148. Swanson CJ, Bures M, Johnson MP, et al. (2005) Metabotropic glutamate receptors as novel targets for anxiety and stress disorders. Nature Reviews Drug Discovery 4: 131–144. Watkins JC and Jane DE (2006) The glutamate story. British Journal of Pharmacology 147(supplement 1): S100–S108.
Cerebellar Ataxia
Animal models of disrupted mGlu1 receptors suggest many similarities with cerebellar ataxia, but of arguable greater significance is the observation of auto-antibodies against mGlu1 receptors in patients suffering from paraneoplastic cerebellar degeneration. These antibodies were observed to inhibit cerebellar function in vitro and in vivo arguing for a pivotal role for mGlu1 receptors in this disorder.
Concluding Remarks Although there has been an abundance of research investigating the physiological and pathophysiological roles of glutamate, as well as extensive medicinal chemistry programs to develop selective ligands, there is still an under-representation of therapeutic agents focusing on this pivotal transmitter.
Glial Energy Metabolism: Overview L Pellerin, Universite´ de Lausanne, Lausanne, Switzerland P J Magistretti, Ecole Polytechnique Fe´de´rale de Lausanne (EPFL) and Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland ã 2009 Elsevier Ltd. All rights reserved.
Introduction In the late nineteenth century, neuroanatomists described the morphological features of glial cells and their cytoarchitectural relationships with other elements of the central nervous system. Among glial cells, astrocytes are the most numerous and exhibit a number of striking characteristics. First, they occupy a strategic position, being usually located between blood vessels and neurons. Second, they possess specialized processes called end-feet, and these processes come into close contact with blood vessels, covering a large part of their surface. Third, they also ensheathe numerous synapses. Altogether, these characteristics suggested to those early neuroanatomists, and among them Camillo Golgi, that astrocytes could play a critical role in the allocation of metabolic substrates to neurons. This view was nicely formulated by Andriezen in 1893: The development of a felted sheath of neuroglia fibers in the ground-substance immediately surrounding the blood vessels of the Brain seems therefore . . . to allow the free passage of lymph and metabolic products which enter into the fluid and general metabolism of the nerve cells.
It is only recently, however, that such a theory experienced a revival through a series of experimental demonstrations that benefited from the advances made in the isolation of specific brain cell types and their use in vitro to study their metabolic characteristics. The current understanding of brain energy metabolism is still incomplete, but in recent years insights have been gained into a number of aspects related to glial energy metabolism.
Aerobic Glycolysis: A Versatile Metabolic Feature of Astrocytes to Cope with the Burden of Glutamate Recycling Several studies have demonstrated the large glycolytic capacity of astrocytes that is accompanied by a sizable production and release of lactate despite the sufficient amount of oxygen present and active oxidative phosphorylation. Not only is their basal glycolytic
rate elevated, but astrocytes also possess a large glycolytic reserve that can be mobilized under various conditions. This is the case under hypoxia or if oxidative phosphorylation is inhibited. But more important, this reserve can be used in specific physiological situations. Thus, it has been shown that enhanced sodium entry within an astrocyte, causing an elevation of intracellular sodium concentration, will lead to an increased glycolytic rate. Such a situation occurs notably when astrocytes are exposed to the excitatory neurotransmitter glutamate. Glutamate transport occurs in astrocytes via two glutamate transporters, glutamate aspartate transporter (GLAST) and glutamate transporter 1 (GLT1). Both depend on the Naþ gradient and carry glutamate, together with three Naþ ions, inside the cell. The increase in intracellular Naþ concentration that accompanies glutamate transport will activate Naþ/Kþ adenosine triphosphatase (ATPase), and as a consequence of adenosine triphosphate (ATP) consumption, an activation of glucose utilization will take place, together with enhanced lactate production (Figure 1). Such an activation of aerobic glycolysis in astrocytes appears to be facilitated by a few molecular characteristics. First, it has been shown that astrocytes have a large part of their pyruvate dehydrogenase enzyme present in a phosphorylated and thus inactive form. This characteristic may limit the entry of pyruvate into the tricarboxylic acid (TCA) cycle (Figure 2). Second, astrocytes appear to have very reduced levels of an essential component of the malate–aspartate shuttle called ARALAR 1. As a consequence, the transfer of reducing equivalents from the cytosol to the inner mitochondria is reduced, and cytosolic nicotinamide adenine dinucleotide (NADþ) regeneration is limited. To maintain a high glycolytic rate as observed in astrocytes, which requires a rapid regeneration of NADþ levels, the solution is to convert pyruvate to lactate via the lactate dehydrogenase enzyme. To facilitate this task, astrocytes prominently express the lactate dehydrogenase M isoform that is not inhibited by pyruvate and sustains high enzymatic rates. Moreover, astrocytes were found to express the monocarboxylate transporter (MCT) isoforms MCT1 and MCT4 that are particularly suited for lactate export from glycolytic tissues because of high Km values. An important question is whether such a metabolic response of astrocytes can be triggered by other neuroactive substances. The main inhibitory neurotransmitter, g-aminobutyric acid (GABA), is an interesting case since it is also transported by a Naþ-dependent mechanism in astrocytes and it is the most important neurotransmitter after glutamate. However, it was
239
240 Glial Energy Metabolism: Overview
Astrocyte
Glutamatergic synapse
Capillary
Lactate Glutamine
Synaptic vesicles
ADP ATP
Glucose
Glucose
Glutamate EAAT
G
H+ K+ Na+
Neuronal glutamate receptors
K+
Na/K ATPase
Glycolysis ATP ADP
Figure 1 Schematic representation of the mechanism for glutamate-induced glycolysis in astrocytes during physiological activation. EAAT, excitatory amino acid transporter. Modified from Pellerin L and Magistretti PJ (1994) Glutamate uptake into astrocytes stimulates aerobic glycolysis: A mechanism coupling neuronal activity to glucose utilization. Proceedings of the National Academy of Sciences of the United States of America 91: 1065–10629.
shown that GABA does not cause a similar enhancement of aerobic glycolysis in astrocytes. The explanation resides in a rapid reduction in GABA transport with increasing intracellular Naþ concentration before it reaches the threshold to activate glycolysis, a phenomenon not observed with glutamate transport. Several other neurotransmitters and neuromodulators have been tested, but all failed to activate glycolysis except noradrenaline. However, the mechanism by which noradrenaline stimulates glycolysis in astrocytes is different from that of glutamate but remains unclear. Another intriguing issue is whether the effect of glutamate is linked only to its uptake via glutamate transporters or whether activation of glutamate receptors (GluRs) can also participate in the metabolic effect. Indeed, astrocytes express both ionotropic and metabotropic glutamate receptors. Among ionotropic receptors, a-amino-3-hydroxy-5methyl-4-isoxazole propionic acid (AMPA)/kainate receptors are abundantly expressed, and all four subunit isoforms (GluR1–4) have been detected in astrocytes. Although activation of AMPA/kainate receptors with the specific agonist AMPA leads to a transient increase in intracellular Naþ concentration, this is not sufficient to cause an activation of aerobic glycolysis. In this case, rapid desensitization of the receptors prevents the Naþ influx from activating
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Figure 2 Metabolic pathways of glucose. LDH, lactate dehydrogenase; NADP, oxidized nicotinamide adenine dinucleotide phosphate; NADPH, reduced nicotinamide adenine dinucleotide phosphate; TCA, tricarboxylic acid cycle.
metabolism. Kainate causes a more important intracellular Naþ change and a small but significant stimulation of aerobic glycolysis. This effect could be explained by the fact that kainate, in contrast to
Glial Energy Metabolism: Overview
AMPA, leads to only a partial desensitization of AMPA/kainate receptors. One particularity of AMPA/kainate receptors is that they contain allosteric sites allowing certain agents to modulate their degree of desensitization/deactivation. In recent years, a series of compounds have been developed on the basis of their ability to prevent desensitization/deactivation of AMPA/kainate receptors. It is quite interesting that they were shown to exhibit neuroprotective and cognitive enhancement effects in various models showing impaired cognitive functions. Although the main explanation proposed for these effects is related to their action on neuronal AMPA/kainate receptors, it has also been suggested that a potentiation of glutamate-induced aerobic glycolysis in astrocytes occurs and participates in the beneficial effects of these drugs. Thus, enhancing the glycolytic response of astrocytes might represent a valuable therapeutic target for the future. A new dimension of the role of enhanced aerobic glycolysis in astrocytes was revealed recently. It has been known for a while that astrocytes communicate with each other through Ca2þ waves that propagate in a regenerative manner along the astrocytic network. The specific role of these traveling Ca2þ waves is still not entirely clear. But in parallel with Ca2þ, Naþ waves have been described. These Naþ waves arise from glutamate released by one astrocyte on elevation of intracellular Ca2þ and taken up by its neighbors. As a consequence of this Naþ influx, a metabolic wave of enhanced aerobic glycolysis will ensue among the astrocytic network. It is purported that such a mechanism could be used to coordinate metabolism and provide adequate energy substrate supply to activated neuronal ensembles, irrespective of their neurotransmitter phenotypes. If confirmed in vivo, such a phenomenon would have important implications not only for an understanding of neuroenergetics but also for the analysis of images obtained with brain imaging techniques that rely on metabolic signals.
Glycogen: Helping to Face Increased Energy Demands Glycogen has been viewed traditionally as an energy reserve maintained to face periods of low energy supply. This notion has been inherited from studies in peripheral organs such as the liver and muscle. However, the amount of glycogen contained in the brain is relatively small: approximately 10 times less than in muscle and 100 times less than in liver. Moreover, under physiological conditions, energy supply to the brain is maintained within narrow limits such that the idea of maintaining an emergency reserve for pathological situations appears unlikely. Rather, a series of
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observations argues in favor of a role for glycogen as an energy buffer rather than an energy reserve in the central nervous system. Glycogen turnover was shown to be enhanced in vivo under sustained stimulation. In contrast, glycogen was found to accumulate in the brain under anesthesia. These observations suggest that glycogen utilization is tightly linked to neuronal activity. It is interesting that at the cellular level, glycogen is found almost exclusively in astrocytes. Since it is purported that the vast majority of energy expenditures are the consequence of neuronal activity, it may seem paradoxical that the sole energy reserve is located in a nonneuronal cell type. However, several lines of evidence indicate that astrocytes respond to neuronal signals and mobilize their glycogen stores to release a metabolite for use by neurons as an energy substrate. It has been shown that the brain, and in particular astrocytes, do not possess glucose-6-phosphatase and thus cannot release glucose as a product of glycogenolysis. Rather, it has been demonstrated that lactate constitutes the main substrate formed from glycosyl residues arising from glycogenolysis and released by astrocytes. Among the various neuroactive substances shown to induce glycogenolysis are the neurotransmitters noradrenaline and serotonin, the neuropeptide vasoactive intestinal peptide (VIP), and the neuromodulators adenosine and ATP. It has also been shown that elevated Kþoccurring as a consequence of neuronal activity can efficiently cause glycogenolysis in astrocytes. In addition to glycogenolysis, several of these neuroactive substances promote a delayed but massive resynthesis of glycogen in astrocytes that takes place over several hours. Such resynthesis is critically dependent on the expression of a key protein called protein targeting to glycogen. This protein serves as a scaffold that binds not only glycogen but also all the enzymes involved in the regulation of glycogen synthesis and degradation. It is also massively induced in astrocytes following treatment with noradrenaline or VIP (Figure 3). Although its purpose is still a matter of debate, this mechanism might be essential to replenish glycogen stores and thus ensure adequate levels in order to face subsequent periods of high activity. In the optic nerve, glial glycogen has been shown to play a critical role in maintaining neuronal activity, in particular by providing lactate to meet the energetic demands of the axon.
The Pentose Phosphate Pathway: Energy Fluxes and Neuroprotection ATP is not the only form of metabolic energy, as reducing power is needed in addition to ATP to energize
242 Glial Energy Metabolism: Overview NA
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Figure 3 Regulation of glycogen metabolism by noradrenaline. Similar regulatory mechanisms are activated by vasoactive intestinal peptide and by adenosine. ATP, adenosine triphosphate; cAMP, cyclic adenosine monophosphate; GS, glutamine synthase; mRNA, messenger RNA; NA, nicotinamide adenine; Prot, protein; PTG, protein targeting to glycogen.
several important cellular processes. This reducing power is provided by the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH). The processing of glucose through the pentose phosphate pathway produces NADPH (Figure 2). A critical cellular process for which NADPH is needed is the scavenging of reactive oxygen species (ROS). The superoxide radical anion (O 2 ) hydrogen peroxide (H2O2), and the hydroxy radical (HO.) are three ROS generated by the transfer of single electrons to molecular oxygen as by-products of several physiological cellular processes. A considerable contribution to the generation of ROS is the oxidative metabolism of glucose taking place in the mitochondrial electron transfer chain associated with oxidative phosphorylation see below). Other ROS-generating reactions include the activities of monoamine oxidase, tyrosine hydroxylase, nitric oxide synthase, and the eicosanoid-forming enzymes lipoxygenases and cyclooxygenases. Reactive oxygen species are highly damaging to cells because they can cause DNA disruption and mutations, as well as activation of enzymatic cascades, including proteases and lipases, that can eventually lead to cell death. The coordinated activity of NADPH and glutathione is essential in protecting cells against ROS-mediated damage, or oxidative stress. Scavenging of ROS is ensured by the sequential action of superoxide dismutase (SOD) and glutathione peroxidase. Thus, two superoxide anions are converted by SOD into H2O2, still an ROS. Glutathione peroxidase converts H2O2 into H2O and O2
at the expense of reduced glutathione, which is regenerated by glutathione reductase in the presence of NADPH. Scavenging of ROS provides another example of metabolic cooperation between neurons and astrocytes. Glutathione is a tripeptide (g-L-glutamylL-cysteinylglycine (GSH)) synthesized through the concerted action of two enzymes, g-GluCys synthase, which combines glutamate and cysteine to yield the dipeptide g-Glu Cys, and glutathione synthase, which adds a glycine to the dipeptide to yield GSH. The glutathione content and reducing potential are considerably higher in astrocytes than in neurons; this fact, combined with the much higher oxidative activity of neurons than of astrocytes, makes neurons more vulnerable to oxidative stress as well as highly dependent on astrocytes for protection. Indeed, a cooperativity between astrocytes and neurons appears to exist for glutathione metabolism; astrocytes release GSH, which is cleaved by the ectoenzyme g-glutamyltransferase, releasing CysGly. The dipeptide is transported into neurons (note that neurons cannot take up GSH), providing two precursors for GSH synthesis. Glutamate, the third precursor of GSH, is also provided by astrocytes to neurons in the form of glutamine, from which glutamate is produced through the action of glutaminase. This astrocyte–neuron metabolic cooperation aimed at controlling the damaging effect of ROS generated by cellular activities may be critical for neuroprotection. Indeed, neurons, being highly oxidative cells
Glial Energy Metabolism: Overview
dependent on astrocytes for scavenging ROS, are at high risk for neurodegeneration. Indeed, dysfunction of such neuroprotective mechanisms has been described in certain neurodegenerative diseases, such as a familial form of amyotrophic lateral sclerosis, due to an SOD mutation. Evidence for a decrease in GSH content in the substantia nigra has also been described in Parkinson’s disease.
The TCA Cycle, Glutamate Metabolism, and Anaplerosis The main metabolic pathway for ATP production is the TCA cycle. While this pathway is active in both neurons and astrocytes, neurons, being richer than astrocytes in mitochondria where the TCA cycle occurs, are overall more oxidative than astrocytes. In addition to its central role in ATP production through its coupling to oxidative phosphorylation, the TCA cycle contributes to another critical aspect of astrocyte–neuron metabolic cooperation, related to glutamate metabolism. Synaptically released glutamate is removed rapidly from the extracellular space by a transportermediated reuptake system that is particularly efficient in astrocytes. This mechanism contributes in a crucial manner to the fidelity of glutamate-mediated neurotransmission. Indeed, glutamate levels in the extracellular space are low (<3 mmol1), allowing for optimal glutamate-mediated signaling after depolarization while preventing overactivation of glutamate receptors, which could eventually result in excitotoxic neuronal damage. What is the metabolic fate of glutamate taken up by astrocytes? Glutamate cannot be stored like carbohydrates or lipids. In astrocytes, one pathway involves the TCA cycle, namely, the transfer of the a-amino group of glutamate to oxaloacetate to yield a-ketoglutarate (a-KG) and aspartate in a reaction catalyzed by aspartate amino transferase (AAT). The a-KG generated is an intermediate of the TCA cycle and is therefore oxidized further. Another transamination reaction catalyzed by alanine amino transferase (ALAT) transfers the a-amino group of glutamate to pyruvate, resulting in the formation of alanine and a-KG. Two other pathways exist in astrocytes to metabolize glutamate. First, glutamate can be converted directly to a-KG through an NAD-requiring oxidative deamination catalyzed by glutamate dehydrogenase (GDH). Glutamate, by entering the TCA cycle indirectly (through AAT or ALAT) or directly (through GDH), is an energy substrate for astrocytes. Second, the quantitatively predominant metabolic pathway of glutamate in astrocytes is its amidation to glutamine, an ATP-requiring reaction in which an ammonium
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ion is fixed on glutamate. This reaction is catalyzed by glutamine synthase, an enzyme almost exclusively localized in astrocytes, and provides an efficient means of disposing not only of glutamate but also of ammonium (Figure 1). Glutamine is released by astrocytes and is taken up by neurons, where it is hydrolyzed back to glutamate by the phosphatedependent mitochondrial enzyme glutaminase. This metabolic pathway, often referred to as the glutamate– glutamine shuttle, is a clear example of cooperation between astrocytes and neurons. It allows the removal of potentially toxic excess glutamate from the extracellular space while returning to the neuron a synaptically inert (glutamine does not affect neurotransmission) precursor with which to regenerate the neuronal pool of glutamate. However, not all glutamate is regenerated through the glutamate–glutamine shuttle because some of the glutamate released by neurons enters at the a-KG level, the TCA cycle in astrocytes; therefore, de novo synthesis is required to maintain the neuronal glutamate pool. Glutamate can be synthesized through NADPH-dependent reductive amination of a-KG catalyzed by GDH (note here another role of NADPH, in addition to ROS scavenging, in cooperation with glutathione). For this pathway leading to the synthesis of glutamate, glucose provides the carbon backbone as a-KG does through the TCA cycle, while plasma leucine, taken up by astrocytic end-feet, provides the nitrogen required for net glutamate synthesis from a-KG. Because this reaction, catalyzed by leucine transaminase, takes place in astrocytes to replenish the neuronal glutamate pool, the astrocytes export glutamate as glutamine. As can be appreciated, because a-KG is used for glutamate synthesis, metabolic intermediates downstream of a-KG must be available to maintain a sustained flux through the TCA cycle in astrocytes. This need is met by the activity of the enzyme pyruvate carboxylase, which fixes CO2 on pyruvate to generate oxaloacetate, which, by condensing with acetyl-CoA, maintains the flux through the TCA cycle. The carboxylation of pyruvate to oxaloacetate is referred to as an anaplerotic (Greek for ‘fill up’) reaction. It is interesting that like glutamine synthase, pyruvate carboxylase is selectively localized in astrocytes. The fact that these two enzymes are localized in astrocytes in conjunction with the existence of a glutamate– glutamine shuttle stresses that astrocytes are essential for maintaining the neuronal glutamate pool used for neurotransmission.
The Astrocyte–Neuron Metabolic Unit In conclusion, the axon terminals at glutamatergic synapses, which represent more than 80% of all
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synapses in the cerebral cortex, and the astrocytic processes that surround them should be viewed as a metabolic unit in which the neuron furnishes the activation signal (glutamate) to the astrocyte, and the astrocyte provides, not only the precursors needed to maintain the neurotransmitter pool (glutamine), but also the energy substrate (lactate) and a neuroprotective agent (glutathione) to scavenge ROS formed by the active oxidative phosphorylation of neurons. Thus the efficacy of the predominant excitatory synapse in the brain, the glutamatergic synapse, cannot be maintained without a close astrocyte–neuron interaction.
Further Reading Beal FM (2005) Mitochondria take center stage in aging and neurodegeneration. Annals of Neurology 58: 495–505. Bernardinelli Y, Magistretti PJ, and Chatton J-Y (2004) Astrocytes generate Naþ-mediated metabolic waves. Proceedings of the National Academy of Sciences of the United States of America 101: 14937–14942. Brown AM (2004) Brain glycogen reawakened. Journal of Neurochemistry 89: 537–552.
Dringen R (2000) Metabolism and functions of glutathione in brain. Progress in Neurobiology 62: 649–671. Erecinska M and Silver IA (1990) Metabolism and role of glutamate in mammalian brain. Progress in Neurobiology 35: 245–296. Hyder F, Patel AB, Gjedde A, Rothman DL, Behar KL, and Shulman RG (2006) Neuronal-glial glucose oxidation and glutamatergic-GABAergic function. Journal of Cerebral Blood Flow and Metabolism 26: 865–877. Magistretti PJ (2003) Brain energy metabolism. In: Squire L (ed.) Fundamental Neuroscience, 2nd edn., pp. 339–360. San Diego: Academic Press. Magistretti PJ (2006) Neuron-glia metabolic coupling and plasticity. Journal of Experimental Biology 209: 2304–2311. Magistretti PJ, Pellerin L, Rothman DL, and Shulman RG (1999) Energy on demand. Science 283: 496–497. McEwen BS and Reagan LP (2004) Glucose transporter expression in the central nervous system: Relationship to synaptic function. European Journal of Pharmacology 490: 13–24. Pellerin L and Magistretti PJ (1994) Glutamate uptake into astrocytes stimulates aerobic glycolysis: A mechanism coupling neuronal activity to glucose utilization. Proceedings of the National Academy of Sciences of the United States of America 91: 1065– 10629. Tsacopoulos M and Magistretti PJ (1996) Metabolic coupling between glia and neurons. Journal of Neuroscience 16: 877–885.
Transporter Proteins in Neurons and Glia T S Otis and P D Dodson, Geffen School of Medicine, Los Angles, CA, USA ã 2009 Elsevier Ltd. All rights reserved.
Nerve cells communicate with one another primarily by chemical means, and such chemical messages are carried by neurotransmitters released at specialized contacts called synapses. In order to produce fast transmission between neurons, the chemical signal must be rapidly terminated. One way in which the signal can be terminated is by the passive diffusion of the neurotransmitter. However, at most synapses in the brain and peripheral nervous system, the termination of chemical messages is also aided by active processes, either enzymatic degradation or removal of the neurotransmitter from the extracellular space. At synapses between motor neurons and skeletal muscles, the excitatory neurotransmitter acetylcholine is rapidly degraded by an enzyme located within the synaptic cleft (see Figure 1). This enzyme, acetylcholinesterase, is one of the most avid enzymes described; a single acetylcholinesterase protein is capable of degrading more than 10 000 molecules of acetylcholine each second. Such capacity reduces the time period in which acetylcholine must act, and it also limits the spatial extent over which it signals. In the brain, fast excitatory synaptic signals are mediated primarily by the excitatory amino acid glutamate. Neurotransmitter clearance at glutamatergic synapses is quite different from the neuromuscular synapse. Here the actions of glutamate are terminated in part by a special class of neurotransmitter transporter, called excitatory amino acid transporters (EAATs), located on the plasma membranes of glial cells and neurons (Figure 1). EAAT proteins are responsible for capturing glutamate from the extracellular space, undergoing a conformational change which transports the glutamate through the plasma membrane, and then releasing it into the cytoplasm. In general, neurotransmitter transport can serve many purposes. First, it is thought to act as part of a recycling mechanism that allows for the renewal of neurotransmitter pools. Second, it can limit the temporal actions of the neurotransmitter. Third, it can restrict the spatial extent of neurotransmitter action and, in so doing, can sometimes determine which pools of receptors are activated. Fourth, it can help set resting levels of neurotransmitter. This is significant because distinct high-affinity classes of receptors are often found outside synaptic specializations (called extrasynaptic receptors) and such extrasynaptic receptors typically respond to low levels of neurotransmitter.
Still other types of receptors desensitize when exposed to very low levels of neurotransmitter, much lower than those required to fully open the receptor. In these ways, even low resting levels of the neurotransmitter can serve as signals of their own or as negative influences on surrounding receptors.
A Family of Excitatory Amino Acid Transporters Responsible for Clearing the Major Excitatory Neurotransmitter Glutamate Molecular cloning techniques have identified EAATs in a wide variety of species: prokaryotes, insects, amphibians, fish, and mammals. Mammals possess a family of EAATs consisting of five genes. Three of these genes were initially identified in 1992 and named GLAST (glutamate aspartate transporter), GLT1 (glutamate transporter 1), and EAAC1 (excitatory amino acid carrier 1). Soon after, corresponding sequences for these genes were cloned from the human motor cortex and their homologs were named EAAT1 (GLAST), EAAT2 (GLT1), and EAAT3 (EAAC1). Subsequently EAAT4 and EAAT5 were also identified. For the sake of simplicity, we hereafter refer to each subtype by its human (i.e., EAAT) designation. Molecular Structure of Excitatory Amino Acid Transporters
Our understanding of the molecular structure of EAATs has benefited enormously from the recently determined X-ray crystal structure of a prokaryotic EAAT homolog. This protein, found in the thermophilic archaebacterium Pyrococcus horikoshii, has approximately 35% sequence homology to its mammalian counterparts. Figure 2 summarizes the key features of the structure. The transporter exists as a homotrimer with three wedge-shaped subunits forming the sides a large bowl that dips into the membrane and faces the extracellular space (Figure 2(b)). Each subunit exhibits a complicated transmembrane topology (Figure 2(a)), with eight complete membranespanning segments and two reentrant loops. The reentrant loops are situated at the bottom of the bowl, where the tips of the three wedges contributed by each subunit come together. This is a key functional region of the transporter protein; the reentrant loops had been identified in prior studies as the likely spot at which glutamate binds. Indeed, the crystal structure shows electron densities characteristic of single glutamate molecules bound to each subunit at the base of the bowl, suggesting that three glutamate molecules bind at these locations before being
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Glutamate transporters Presynaptic terminal
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Figure 1 Degradative enzymes: (a) in the vertebrate neuromuscular junction synapses; (b) in the extracellular space at most excitatory synapses within the brain. In (a), following release the neurotransmitter acetylcholine (ACh) is degraded rapidly by acetylcholinesterase (ACh esterase), an enzyme situated within the synaptic cleft. In (b), there is no degradative enzyme; rather, high-affinity excitatory amino acid transporters (EAATs) remove glutamate from the extracellular space into intracellular compartments in neurons and glia.
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Figure 2 Transmembrane topology of a prokaryotic excitatory amino acid transporter (EAAT) from the archaebacterium Pyrococcus horikoshii: (a) structure of the transporter subunits; (b) side-on cross-sectional view of the transporter structure. In (a), each subunit has eight membrane-spanning segments and two reentrant loops, termed hairpin 1 (HP1) and hairpin 2 (HP2). The N- and C-termini are located on the intracellular side of the membrane. In (b), the large bowl-shaped structure formed by three subunits is oriented with the bowl facing extracellularly (top). Glutamate binding and coupled ion binding are believed to occur at the base of the bowl. Reproduced by permission from Macmillan Publishers Ltd.: Nature, MP Kavanaugh, Accessing a transporter structure, 431: 752–753, copyright 2004.
transported into the cytoplasm. Although it is not certain, these features of the structure suggest that each subunit may transport glutamate independently. Energetics of Excitatory Amino Acid Transporters
The energy for transport comes from the coupling of the movement of glutamate to the cotransport and the countertransport of sodium, potassium, and hydrogen ions down their respective electrochemical gradients. Flux measurements have established the stoichiometry for a complete cycle of glutamate transport to be three Naþ and one Hþ cotransported and one Kþ countertransported per glutamate molecule. This elaborate coupling mechanism has a clear benefit; it allows EAATs to accumulate glutamate to a huge concentration gradient. With typical physiological
ion gradients, resting potentials, and the coupling stoichiometry just mentioned, glutamate can be taken up and concentrated inside the cell to well more than a millionfold at equilibrium. Given that cytoplasmic glutamate concentrations are thought to be in the millimolar range in some cells (e.g., neurons), these energetics allow for extracellular glutamate levels to be maintained at submicromolar, and possibly even nanomolar, levels. Of course, even at steady state, resting glutamate levels may not be near this theoretical limit. This is because, in addition to the ionic gradients, the amount of glutamate released, the density of EAATs, and the size and shape of the extracellular volume all influence the degree to which transporters can reduce the extracellular glutamate concentration.
Transporter Proteins in Neurons and Glia Uncoupled Ionic Conductances of Excitatory Amino Acid Transporters
The stoichiometric movement of ions during the glutamate transport cycle provides the energy to accumulate glutamate. Because of the net charge flux of þ2 charges moving inward per cycle (one negative glutamate, three positive sodium ions, and one proton in and one positive potassium ion out) transport gives rise to very small transmembrane ionic currents. However, to varying degrees, all five EAATs allow another ionic current to flow. This current is carried by chloride ions under physiological circumstances, and this movement of anions is not coupled energetically to glutamate transport. In simple terms, we might imagine that at a particular point in the cycle of transport the EAAT protein adopts a conformation contains an anion-selective pore across the membrane. The anions flowing through EAATs move according to their own electrochemical gradient, and this anion flux is neither affected by nor affects the cycling of glutamate, sodium, potassium, and hydrogen ions. The relative size of the anion component of the total current varies characteristically among different EAAT isoforms. EAAT4 has the largest anion current; it dominates the total ionic flux under physiological circumstances. In contrast, EAAT2 has the smallest anion conductance. Several of the EAATs also exhibit a noticeable anion leak under resting conditions (i.e., in the absence of glutamate), and this leak is most prominent in EAAT4. Physiological roles for the uncoupled anion fluxes are not known, but in principle such conductances could allow EAATs to act as ligand-gated inhibitory channels in addition to their glutamate clearance role. In cerebellar Purkinje neurons, in which EAAT4 is expressed at high levels, this does not appear to have a major influence on excitability, but in retinal ON-bipolar cells it has been argued that EAAT-mediated anion currents can couple decreases in photoreceptor glutamate release with the excitation of the ON-bipolar cell.
Cellular Localization of Excitatory Amino Acid Transporters EAATs are expressed at the highest levels in the nervous system, but are also found in peripheral tissues such as the kidney, enteric nervous system, pancreas, placenta, mammary glands, and possibly bone. The expression of particular EAATs within the brain occurs in overlapping patterns and is largely cell-type specific. Two of the five transporters are found predominantly in the glia, two are found only in certain types of neurons, and one is expressed only in the retina in both neurons and the glia.
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The Glial Transporters EAAT1 and EAAT2
EAAT1 is the predominant glial glutamate transporter in cerebellum. It is also expressed at relatively high levels in glial cells of the inner ear and in Mu¨ller glial cells of the retina. EAAT1 protein does not appear to be present in oligodendrocytes or in neurons. In cerebellar Bergmann glia, EAAT1 is expressed at extremely high densities (4700 m2 of plasma membrane). It is thought to be the dominant transporter in these cells; consistent with this view, animals in which the EAAT1 gene has been disrupted have cerebellar behavioral deficits and exacerbated responses to cerebellar injuries. However, EAAT2 is also expressed at approximately sixfold lower levels in the cerebellar cortex, and thus functional transporters may exist as heteromers. At a subcellular level, EAAT1 is not found to be distributed uniformly along the plasma membrane of astrocytes. Rather, hot spots of EAAT1 are observed and the highest densities of EAAT1 are seen where astrocytic membranes oppose neuronal membranes (see Figure 3(a)). In the cerebellar cortex, this arrangement could be a mechanism for helping to limit glutamate spillover from active synapse to their neighbors. Like EAAT1, EAAT2 is also concentrated on fine astrocytic processes that surround excitatory synapses (see Figure 3(b)). Depending on the brain region, these EAAT2-rich astrocytic processes are located at different distances from sites of glutamate release. At one extreme, they can encapsulate pre- and postsynaptic elements (as EAAT1/EAAT2-studded Bergmann glial processes do in the cerebellar cortex). At the other extreme, such as at Schaffer collateral synapses on to CA1 pyramidal neurons of the hippocampus, EAAT2containing processes are located at some distance from postsynaptic densitities and sites of glutamate release. Finally, in certain brain regions such as the superoptic nucleus of the hypothalamus, the arrangement of astrocytic processes varies according to behavioral state, thereby influencing excitatory synaptic transmission in numerous ways by permitting glutamate to reach or limiting it from reaching specific pools of receptors. The Neuronal Transporters EAAT3 and EAAT4
An unexpected feature of the localization patterns of the neuronal EAATs is that they are found on dendritic, but not on axon terminal, membranes. EAAT3 is the more widely expressed of the neuronal EAATs, showing localization on excitatory and inhibitory neuronal dendrites in the hippocampus, cerebellum, basal ganglia, cortex, and thalamus. In those neurons that express it, EAAT3 protein is present at relatively high densities on intracellular membranes as well as
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Glutamatecontaining vesicles VGLUT GLAST (EAAT1) GLT (EAAT2) EAAC (EAAT3) EAAT4 Unidentified
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b Figure 3 Localization of excitatory amino acid transporters (EAATs) at different types of glutamate synapses: (a) parallel fiber to Purkinje neuron synapses in the cerebellar cortex; (b) Schaffer collateral synapse in hippocampus. In (a), the synapses are completely surrounded by astroglial membranes containing the glial isoforms EAAT1 and EAAT2 at average densities of 4700 and 740 molecules mm2, respectively. The neuronal transporter EAAT4 is concentrated opposite glia-covered parts of the membranes of Purkinje dendrites and is highest in the spines and thinner dendrites. EAAT4 is expressed at varying density in different zones of the cerebellar cortex, but has an average density of 1800 molecules mm2. EAAT3 is present in Purkinje neurons membrane as well as the cytoplasm, but quantitative data on the precise subcellular distribution are currently unavailable. In (b), the synapses are only contacted, not surrounded, by astroglia. EAAT1 and EAAT2 are present in the astroglial membranes at average densities of 2300 and 8500 molecules mm2, respectively, with highest concentrations toward the neuropil. Quantitative information is not available for EAAT3, nor is information available for the putative glutamate transporter on the nerve terminal. EAAC, excitatory amino acid carrier; GLAST, glutamate aspartate transporter; GLT, glutamate transporter; VGLUT, vesicular glutamate transporter. Reproduced from Danbolt N (2001) Glutamate transporters. Progress in Neurobiology 65: 1–105, with permission from Elsevier.
on the plasma membrane. That said, the density of EAAT3 is believed to be quite low and it is unclear whether it plays a prominent role in clearing glutamate into these neurons.
EAAT4, found almost exclusively in cerebellar Purkine neurons, is present at extremely high densities on dendritic shafts and spines, estimated to be on average (1800 m2 of plasma membrane). However, this expression shows a large-scale patchiness, with zones of high expression and low expression occurring at periodic spatial intervals throughout the cerebellar cortex. The resulting zebra-striped pattern matches that of several other gene products (most notably the protein Zebrin). The reasons for this nonuniform expression pattern are not understood, but recent work suggests that in regions of low EAAT4 expression it is easier for certain types of glutamate receptors (GluRs; e.g., metabotropic GluRs (mGluRs)) to be stimulated, whereas in regions with high EAAT4 expression certain GluRs appear to be shielded from released glutamate. Because mGluRs are responsible for initiating particular forms of synaptic plasticity, the variability in EAAT4 may be a mechanism for region specific control of synaptic plasticity. At the subcellular level, both EAAT3 and EAAT4 have been reported to be excluded from the postsynaptic density but to be concentrated at the edges of these densities, a pattern termed perisynaptic. Such a pattern of localization seems ideally suited to aiding in preventing glutamate escape from synaptic clefts at active synapses (spillover) and in preventing glutamate from gaining access to the postsynaptic densities of inactive synapses (cross-talk). These phenomena are illustrated in Figure 4; physiological evidence suggesting that they occur is discussed in latter sections. The Retinal Transporter EAAT5
EAAT5 has been reported to be expressed in various neurons (photoreceptors, bipolar cells, and amacrine cells) and Mu¨ller glial cells of the retina. Functional evidence suggests that EAAT5 may be a major glutamate transporter on bipolar cell terminals; this work is also notable because it serves as the only direct evidence to date that an EAAT is involved in glutamate uptake into a presynaptic terminal.
Synaptic Functions of Excitatory Amino Acid Transporters Recycling Glutamate
Constant vesicular release of glutamate from terminals in the brain requires a concurrent replenishment of neurotransmitter stores. It is likely that this happens via an indirect pathway because there is little evidence for the presence of glutamate transporters located on presynaptic terminals. Rather than being directly reabsorbed into the nerve terminal, EAATs in surrounding glia may take up the bulk of glutamate.
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Figure 4 Transporters limit (a) spillover of glutamate; and (b) of synaptic cross-talk. In (a), during moderate levels of activity (left), glutamate transporters take up glutamate and limit its escape from the cleft (red crosses). During high levels of activity, the transporters become saturated, so the transmitter spills over (arrow) to activate extrasynaptic receptors. In (b), during high levels of activity (left), transporters are saturated and glutamate escapes the synaptic cleft. Transporters surrounding adjacent synapses take up this glutamate (red cross), limiting cross-talk between synapses. AMPAR, a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor; EAAT, excitatory amino acid transporter; mGluR, metabolic glutamate receptor.
Transporter Proteins in Neurons and Glia
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250 Transporter Proteins in Neurons and Glia
Within glia, glutamate is converted to glutamine by the enzyme glutamine synthetase. Glutamine is an inactive amino acid and thus can be returned to the presynaptic terminal via the extracellular space; once it is back in the presynaptic terminal, it can be made back into glutamate (or g-aminobutyric acid (GABA)). There is also indirect evidence that EAATs on inhibitory synaptic terminals take up glutamate to be used for the synthesis of GABA. In these studies, inhibiting glutamate uptake leads to a reduction in GABA release. This result might also explain why many GABAergic neurons express EAAT3 and EAAT4, although, if such proteins are present on the terminals of these neurons, they are, for unknown reasons, immunohistochemically silent. Maintaining Low Resting Glutamate Concentrations
In order to preserve the sensitivity of postsynaptic receptors, steady-state concentrations of glutamate in the synaptic cleft must remain submicromolar. For example, extrasynaptic receptors such as mGluRs and N-methyl-D-aspartate (NMDA) receptors have median effective concentrations (EC50s) in the micromolar range, whereas a-amino-3-hydroxy-5-methyl-4isoxazole propionic acid (AMPA) receptors desensitize to similarly low concentrations of glutamate. Because the rate at which glutamate can leave the synaptic cleft is proportional to its concentration gradient, in order to rapidly terminate the signal a large concentration gradient must be established. Several experiments have demonstrated that EAATs control the resting levels of glutamate. The strongest evidence is based on the measurement of tonic activation of GluR currents, typically NMDA currents. For example, during cortical and cerebellar development many migrating neurons receive tonic nonsynaptic signals mediated by NMDA receptors. Such signals, and the rate of migration of these neurons, can be altered by inhibiting EAATs. In adults, EAATs also appear to play a role in controlling steady-state levels of glutamate. Experiments in cultured hippocampal slices show that large tonic NMDA currents appear within seconds after the inhibition of transporters. Synaptic signals mediated by AMPA receptors are sometimes altered by inhibiting glutamate uptake, but this seems to depend on the microstructure of the synapse. Three categories of effects are observed when EAATs are inhibited. At some synapses that are not enveloped by glia, as in the hippocampus (schematized in Figure 3(a)), inhibiting glutamate uptake has no detectable effect. This stands in contrast to the slowing of the decay of the excitatory postsynaptic current (EPSC) in response to single stimuli seen at
other types of synapses, such as the calyceal synapses or synapses, which are made on postsynaptic spines with extensive glial wrapping as in the cerebellar cortex (schematized in Figure 3(b)). Finally, at several glial-enveloped synapses that are made on dendritic shafts, blocking transport causes a slowing of the decay but only in response to trains of presynaptic stimuli; a good example is the parallel fiber to stellate cell synapse in the cerebellum. Ensuring Synapse Specificity
In order to ensure independent signaling, glutamate must be prevented from diffusing from one synaptic cleft into neighboring synaptic clefts. The escape of glutamate from the synaptic cleft and the subsequent activation of extrasynaptic AMPA or NMDA receptors have been termed spillover; the breakdown of synaptic independence caused by invading glutamate into an inactive synaptic cleft has been termed crosstalk (see Figure 4). Given the abundant evidence that glutamate escapes the synaptic cleft at significant concentrations and can activate targets at considerable distances, it would seem that these two phenomena are common. Close examination of excitatory transmission at several synapses yields clear evidence for spillover and tantalizing suggestions of cross-talk. Where it has been examined, glutamate uptake plays a pivotal role in minimizing or shaping these unconventional signals. Limiting cross-talk between synapses An early indication that cross-talk might occur came from a comparison of the relative variabilities of NMDA and AMPA receptor-mediated EPSCs in the hippocampus. Due to the stochastic properties of vesicular release (release probability is not 1.0), EPSCs vary in peak amplitude from trial to trial. Recordings of dual component EPSCs in CA1 pyramidal cells showed that NMDA-mediated EPSCs are consistently less variable than those mediated by AMPA receptors. These results have been interpreted to mean that NMDA receptors detect a larger number of quanta than do AMPA receptors. But where do these extra quanta come from? NMDA receptors have approximately 100-fold higher affinity than AMPA receptors, and it has been hypothesized that this allows NMDA receptors to detect release from neighboring presynaptic terminals not directly apposed to the postsynaptic density in which they reside. A key piece of evidence supporting this idea is that, at higher temperatures, the difference in relative variability between NMDA and AMPA receptors is reduced. Glutamate transport is known to be very temperature sensitive, and consistent with a role for glutamate uptake in this puzzle,
Transporter Proteins in Neurons and Glia
inhibitors of glutamate transport erased the effects of increased temperature, making the NMDA EPSCs relatively more variable than AMPA EPSCs. These results imply that cross-talk to NMDA receptors is occurring at room temperature but that at more physiological temperatures glutamate transporters operate more efficiently and are able to prevent glutamate from indirectly activating the higher-affinity NMDA receptors. Perhaps the most straightforward evidence of cross-talk has been observed at parallel fiber inputs to stellate cells in the cerebellum, Here, slowly rising AMPA- and NMDA-mediated synaptic responses can be observed under certain conditions in response to trains of presynaptic stimuli. Inhibiting glutamate uptake increases and prolongs such slow responses, indicating that EAATs at least partially limit the indirect access of glutamate to AMPA receptors. Other pharmacological evidence suggests that these indirect responses result at least in part from glutamate entering inactive, neighboring synapses and binding to AMPA or NMDA receptors. Neuronal EAATs located at the edges of synapses (see Figure 3) seem to be ideally situated to prevent glutamate from entering synaptic clefts from the extracellular space. Is there any evidence that transporters might serve in this capacity? In CA1 pyramidal cells of the hippocampus, experiments suggest that postsynaptic uptake, presumably by EAAT3, can limit the activation of NMDA receptors. Similarly, in cerebellar Purkinje neurons, EAAT4 appears to prevent low concentrations of glutamate from entering inactive synapses. Limiting spillover of neurotransmitter to extrasynaptic receptors Many potential targets for glutamate are located adjacent to the synapse or completely outside of synaptic clefts – on the same neuron, on presynaptic terminals, or on glial cells. In order for these targets to be activated, glutamate must escape a synapse and diffuse for variable distances in the extracellular space. Recently published data not only demonstrate that glutamate can indeed spill out of the synaptic cleft to activate extrasynaptic receptors but also that EAATs play important roles in ensuring that these receptors are activated only under certain circumstances (e.g., during high levels of activity). Spillover to receptors on glia In the hippocampus, where glial cell membranes are typically located at some distance from the edges of synaptic clefts, synaptic signals mediated by GluRs or EAATs can be directly recorded in astrocytes. Based on such
251
measurements, it has been proposed that glial membranes outside the synaptic cleft sense glutamate within 1 ms after release, results consistent with expectation maximization (EM) reconstructions of astrocyte membranes and with the localization data for EAAT2 placing the transporters at 1 mm from synaptic specializations. Likewise, in the cerebellum, AMPA receptor-mediated and EAAT-mediated synaptic currents can be recorded from Bergmann glial cells. These findings suggest that glutamate commonly escapes synaptic clefts at concentrations easily capable of activating most glutamate receptor subtypes. Spillover to extrasynaptic receptors on neurons In various brain regions, including the hippocampus, retina, hypothalamus, and cerebellum, glutamate escaping synaptic clefts can activate NMDA receptors and these signals are often potently regulated by EAATs. Glutamate escaping synapses can also activate various classes of mGluRs. Some of these are located at the very edges of synapses, surrounding the AMPA and NMDA receptor-rich postsynaptic densities. Both cerebellar parallel fiber synapses and Schaffer collateral synapses have postsynaptic mGluRs (group I) that encircle the postsynaptic densities. At cerebellar synapses, high-frequency trains of presynaptic stimuli are required to elicit mGluR-triggered EPSCs and intracellular calcium signals. In both the hippocampus and cerebellum, the activation of these perisynaptic G-protein-coupled receptor subtypes appears to be limited by EAATs (see Fig ure 4). Postsynaptic neuronal EAATs are co-localized with the mGluRs, and in the cerebellum neuronal EAATs seem to be specialized to limit the activation of these perisynaptic mGluRs. Spillover to receptors on presynaptic terminals Another class of distant glutamate targets include group II mGluRs (mGluR2 and mGluR3), which are located on excitatory presynaptic terminals. In hippocampal cultures, the mGluR-mediated presynaptic inhibition of glutamate and GABA release is enhanced by antagonizing glutamate uptake. In acute hippocampal slices, a presynaptic mGluR-mediated inhibition caused by spillover of glutamate to distant heterosynaptic targets is limited by EAAT activity. Thus, glutamate acting at some distance from release sites on presynaptic mGluRs generates a form of presynaptic autoinhibition, and this autoinhibition is tightly controlled by glutamate uptake. Similar findings are obtained for presynaptic mGluRs on GABA-releasing terminals. See also: Glutamate.
252 Transporter Proteins in Neurons and Glia
Further Reading Amara SG and Fontana AC (2002) Excitatory amino acid transporters: Keeping up with glutamate. Neurochemistry International 41: 313–318. Bergles DE, Diamond JS, and Jahr CE (1999) Clearance of glutamate inside the synapse and beyond. Current Opinions in Neurobiology 9: 293–298. Danbolt N (2001) Glutamate transporters. Progress in Neurobiology 65: 1–105. Kavanaugh MP (2004) Accessing a transporter structure. Nature 431: 752–753. Otis TS, Brasnjo G, Dzubay JA, and Pratap M (2004) Interactions between glutamate transporters and metabotropic receptors at excitatory synapses in the cerebellar cortex. Neurochemistry International 45: 537–544. Otis TS, Kavanaugh MP, and Jahr CE (1997) Postsynaptic glutamate transport at the climbing fiber-Purkinje cell synapse. Science 277: 1515–1518. Palmer MJ, Taschenberger H, Hull C, Tremere L, and von Gersdorff H (2003) Synaptic activation of presynaptic
glutamate transporter currents in nerve terminals. Journal of Neuroscience 23: 4831–4841. Robinson MB (2003) Signaling pathways take aim at neurotransmitter transporters. Science Signal Transduction Knowledge Environment 207: pe50 (online). Sattler R and Rothstein JD (2006) Regulation and dysregulation of glutamate transporters. Handbook of Experimental Pharmacology 175: 277–303. Scanziani M (2002) Competing on the edge. Trends in Neuroscience 25: 282–283. Theodosis DT, Piet R, Poulain DA, and Oliet SH (2004) Neuronal, glial and synaptic remodeling in the adult hypothalamus: Functional consequences and role of cell surface and extracellular matrix adhesion molecules. Neurochemistry International 45: 491–501. Yernool D, Boudker O, Jin Y, and Gouaux E (2004) Structure of a glutamate transporter homologue from. Pyrococcus horikoshii. Nature 431: 811–818. Zerangue N and Kavanaugh MP (1996) Flux coupling in a neuronal glutamate transporter. Nature 383: 634–637.
Vesicular Neurotransmitter Transporters R J Reimer, Stanford University School of Medicine, Stanford, CA, USA ã 2009 Elsevier Ltd. All rights reserved.
Introduction A little more than 50 years ago, Katz and colleagues demonstrated that acetylcholine was released at the frog neuromuscular junction in packets containing thousands of molecules. It was later proposed that the neurotransmitter is stored at the nerve terminal in small vesicles and that the fusion of these vesicles with the plasma membrane leads to release of the neurotransmitter. Subsequent physiological, anatomical, biochemical, and molecular studies have established that synaptic transmission, defined by the quantal release of neurotransmitters through exocytosis of synaptic vesicles, is the primary mechanism for information transfer in nervous systems of all organisms. Because classic neurotransmitters such as acetylcholine are synthesized in the cytoplasm, a mechanism is required for their uptake into synaptic vesicles. Storage of neurotransmitter in synaptic vesicles has a number of theoretical advantages. First, it permits the simultaneous release of thousands of neurotransmitter molecules and allows a rapid rise in the synaptic cleft concentration. Second, vesicular storage sequesters neurotransmitter away from the cytoplasm. The maintenance of low cytoplasmic concentrations facilitates the efficient reuptake of released neurotransmitter and, in the case of monoamines, may reduce toxicity. Third, it provides a large pool of releasable neurotransmitter in nonrecycling reserve vesicles. Last, it imparts the potential for the simultaneous co-release of neuromodulators with a neurotransmitter and even co-release of two different classes of neurotransmitters. Biochemical studies defined synaptic vesicle transport activities that mediate the uptake of the classic neurotransmitters. Four unique transport systems have been characterized: one for monoamines, a second for acetylcholine, a third for g-aminobutyric acid (GABA) and glycine, and a fourth for glutamate. The transport processes facilitate the rapid refilling of vesicles and can generate lumen-to-cytoplasm concentration gradients of greater than 104. Recent molecular studies have identified the proteins that catalyze these processes. As expected, separate multitransmembrane domain proteins mediate each of the four biochemically described activities (Table 1 and Figure 1). The characterization of these proteins has generated specific molecular markers for cells that
release a given neurotransmitter and has led to the identification of potential mechanisms for regulating neurotransmitter release.
Vesicular Neurotransmitter Transport Is Driven by a Vesicular Proton Electrochemical Gradient Unlike Naþ-dependent plasma membrane transporters, the vesicular activities couple uptake to a proton electrochemical gradient (DmHþ) across the vesicle membrane (Figure 1). This gradient is generated by a vacuolar-type Hþ-adenosine triphosphatase (ATPase) that couples the hydrolysis of one adenosine triphosphate (ATP) molecule to the inward transmembrane movement of two protons. Recent data suggest that there is a single ATPase on each synaptic vesicle. Each pump is a multimeric complex containing cytoplasmic (V1) and transmembrane (V0) domains, with both domains composed of several polypeptide subunits. The proton electrochemical gradient generated by the Hþ-ATPase has a chemical (DpH) and electrical (Dc) component. Under normal physiological conditions there is a 1–2 pH-unit gradient and a 40–80 mV potential across the synaptic vesicle membrane. Due to the size of synaptic vesicles, a membrane potential (inside positive) is rapidly generated as protons are transported into the vesicle. This creates an energy barrier for the inward movement of protons that cannot be overcome by the Hþ-ATPase and limits the pH gradient that can be achieved. However, anions such as Cl can permeate the synaptic vesicles membrane and reduce Dc. The reduction in membrane potential lowers the energy barrier and facilitates further Hþ-ATPase activity with formation of a larger DpH. It has been suggested that a major regulatory mechanism for acidification of intracellular vesicles, including synaptic vesicles, is the flux of Cl through channels. The primary mediator of the Cl conductance in synaptic vesicles is believed to the ClC3, a member of the ClC family of chloride channels. The dependence of all of the vesicular neurotransmitter transport activities on DmHþ is well established. Bafilomycin, a specific inhibitor of the vacuolar Hþ-ATPase, the proton ionophores carbonyl cyanide m-chlorophenylhydrazone (CCCP), and carbonylcyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) reduce vesicular storage of all neurotransmitters. The dependencies of the different transport systems on the chemical and electrical components of DmHþ have been determined by selectively reducing DpH and Dc. Nigericin, a Hþ/Kþ exchanger, and weak bases such as ammonium sulfate are used to selectively
253
254 Vesicular Neurotransmitter Transporters Table 1 Synaptic vesicle transporters Transporter
SLC designation
Substrate
Affinity
Predicted stoichiometry
Expression pattern
Sample inhibitors
VMAT1
SLC18A1
Monoamines
Low micromolar
SLC18A2
Monoamines including histamine
Low micromolar
VAChT
SLC18A3
Acetylcholine
Low millimolar
2Hþ/1AChþ exchange
VGAT/VIAAT
SLC32A1
Low millimolar
Unknown
VGLUT1
SLC17A7
GABA and glycine Glutamate
Low millimolar
Unknown
Adrenal medulla, enterochromaffin cells Monoaminergic neurons in the brain, sympathetic postganglionic neurons, mast cells Cholinergic neurons in the central, peripheral, and autonomic nervous systems Inhibitory neurons, islet cells Neocortex, cerebellar cortex, hippocampus
Reserpine
VMAT2
2Hþ/1MAþ exchange 2Hþ/1MAþ exchange
VGLUT2
SLC17A6
Glutamate
Low millimolar
Unknown
Thalamus, cerebellar and brain stem nuclei
VGLUT3
SLC17A8
Glutamate
Low millimolar
Unknown
ZnT3 ClC3 Vacuolar HþATPase SV2 (A, B, and C)
SLC30A3
Zinc Chloride
Unknown
Unknown Unknown 2Hþ/cycle
Astrocytes, monaminergic, GABAergic, cholinergic neurons Glutamatergic neurons Broad Broad
0.1 mM Unknown
Reserpine, tetrabenazine
Vesamicol
g-Vinyl GABA Evans blue, rose Bengal, DIDS Evans blue, rose Bengal, DIDS Evans blue, rose Bengal, DIDS DIDS Bafilomycin
All neurons and neuroendocrine cells
ACh, acetylcholine; ATP, adenosine triphosphate; DIDS, 4,40 -diisothiocyanatostilbene-2,20 -disulfonic acid; GABA, g-aminobutyric acid; MA, monoamine; SV2, synaptic vesicle protein 2; VAChT, vesicular acetylcholine transporter; VGAT, vesicular GABA transporter; VGLUT, vesicular glutamate transporter; VIAAT, vesicular inhibitory amino acid transporter; VMAT, vesicular monoamine transporter.
reduce DpH, whereas valinomycin, a Kþ ionophore, and increasing concentrations of permeant anions are used to selectively reduce Dc. With these approaches, it has been shown that the vesicular monoamine and acetylcholine transport systems depend primarily on DpH, GABA transport relies on both DpH and Dc, and glutamate transport relies almost exclusively on Dc (Figure 1). These bioenergetic differences presumably reflect a combination of differences in substrate charge and the stoichiometry of coupling. In addition to its role in proton translocation, the V0 domain has also been implicated in formation of the exocytotic fusion pore. Earlier biochemical studies identified the membranous subunit of the vacuolar Hþ-ATPase as the mediatophore, a membrane protein complex that facilitates the release of acetylcholine at the synaptic cleft. This finding has been complemented by more recent genetic studies with Saccharomyces cerevisiae and Drosophila melanogaster that implicate the V0 domain in formation of membrane fusion pores. To form part of the fusion pore requires the dissociation of V1 and V0. In yeast, regulated dissociation of the two complexes has been
shown to occur, suggesting that a similar process could occur at the synapse. Dissociation of V1 could allow for V0 on synaptic vesicles to interact with other membrane structures such as the plasma membrane, but could also function as a mechanism for limiting the vesicular proton electrochemical gradient and neurotransmitter accumulation.
Vesicular Monoamine Transport Is Mediated by Two Homologous Proteins Studies on chromaffin granules, large intracellular vesicles from chromaffin cells of the adrenal gland, led to the characterization of a transport activity that mediates the vesicular accumulation of monoamines, including dopamine, norepihephrine, epinephrine, and serotonin. The subsequent molecular identification of the vesicular monoamine transporter resulted from a screen for genes from PC12 cells (an adrenal glandderived cell line) that confer resistance to the parkinsonian neurotoxin MPPþ in a sensitive cell line. MPPþ bears a structural similarity to the monoamine neurotransmitters, and the resistance apparently results from
Vesicular Neurotransmitter Transporters 255 2H+ ADP + Pi
NT
NT
ATP nH+
NT
H+
H+ H+
pH 5.5
NT H+
−60 mV
nH+
H+
H+
ΔpH = Δy
ΔpH > Δy MA
nH+ H+
2H+
2H+
ΔpH < Δy Glu–
GABA, gly
ACh
nH+
nH+
nH+
Substrate charge Protons per cycle
+1 2
+1 2
0 1−2
−1 1−2
Net charge per cycle
+1
+1
+1 − 2
+2 − 3
Figure 1 Stoichiometry and bioenergetic dependence of the vesicular neurotransmitter transporters. A vacuolar type Hþ-ATPase on synaptic vesicles generates a proton electrochemical gradient. The gradient consists of a pH gradient and a transmembrane potential of approximately 60 mV (inside positive). The different transporter families have different dependencies on the two components of the gradient – VMATs and VAChT are most dependent on the pH gradient, VGAT can be driven by either the pH or electrical gradient, and VGLUTs are most dependent on the electrical gradient. For monoamine and acetylcholine transporters, the stoichiometries have been determined, but for VGAT and VGLUTs the specifics of energetic coupling remain to be determined. Dc, electrical gradient; ADP, adenosine diphosphate; ACh, acetylcholine; ATP, adenosine triphosphate; GABA, g-aminobutyric acid; Glu, glutamate; gly, glycerin; MA, monoamine; NT, neurotrophin; Pi, inorganic phosphate; VAChT, vesicular acetylcholine transporter; VGAT, vesicular GABA transporter; VGLUT, vesicular glutamate GABA transporter; VMAT, vesicular monoamine transporter.
the sequestration of the toxin inside vesicles, away from its primary site of action in mitochondria. The cloned vesicular monoamine transporter VMAT1 is predicted to have 12 transmembrane domains and is structurally related to the bacterial drug-resistance transporters. In addition to mediating the uptake of MPPþ, VMAT1 also transports dopamine, norepihephrine, epinephrine, and serotonin. VMAT1 is expressed by cells of the adrenal medulla, by neurons in sympathetic ganglia, and by other nonneural cells that release monoamines. In contrast, neuronal populations in the nervous system express the closely related VMAT2. The substrate specificity for the two isoforms is similar, and the apparent affinities for the monoamines are in the low micromolar range for both transporters, though VMAT2 has a slightly higher apparent affinity for all monoamines. In addition, only VMAT2 appears able to transport histamine at physiological concentrations, consistent with its expression by histamine-releasing mast cells. The high affinity of the VMATs may reflect a need to keep cytosolic concentrations of the relatively toxic monamines low. Transport by the VMATs involves the exchange of two lumenal protons for one protonated molecule of transmitter carrying a net single positive charge and,
thus, the outward movement of one net positive charge. For a vesicular pH gradient of 1.5 pH units and membrane potential of 60 mV, this predicts the accumulation of transmitter inside vesicles at concentrations 104–105-fold greater than those in cytoplasm. This is consistent with the micromolar and near-molar catecholamine concentrations measured in the cytosol and vesicles, respectively. Because osmotic forces limit the free transmitter concentration to 150 mM, a fraction of the measured concentration, it has been suggested that the formation of macromolecular complexes consisting of a proteinaceous core, ATP, and the catecholamines reduces the effective osmolarity inside the vesicle. The broad specificity of these transporters may be of particular importance in epinephrine-releasing cells. In these cells, VMATS transport dopamine into secretory vesicles, where the transmitter is converted to norepinephrine by the action of the dopamine b-hydroxylase, an enzyme restricted to the vesicle lumen. Norepinephrine then exits the vesicle, most likely through the action of VMATs, into the cytoplasm where it is converted to epinephrine by the action of phenylethanol-amine-N-methyltransferase. Epinephrine is then transported back into vesicle,
256 Vesicular Neurotransmitter Transporters
again presumably by VMATs. Although these last two steps probably occur through heterologous exchange (e.g., lumenal norepinephrine is exchanged for cytoplasmic epinephrine with no net proton movement), epinephrine released by exocytosis passes through VMATs three times. Two well-characterized inhibitors act on the VMATs: reserpine and tetrabenazine. Both VMATs are irreversibly inhibited by reserpine, which initially showed clinical promise as an antihypertensive medication. The depressive effects of reserpine led to its more limited use but helped to formulate the original monoamine hypothesis of affective disorders. Reserpine appears to interact with the transporters near the site of substrate recognition. Tetrabenazine reversibly inhibits VMAT2, but is markedly less effective as an inhibitor of VMAT1. It has a limited clinical use in the treatment of some movement disorders. Amphetamines and 3,4-methylenedioxy-N-methylamphetamine (MDMA, or extasy) act as weak bases to reduce the vesicular DpH. Loss of this driving force for the vesicular transporters leads to the efflux of the lumenal contents into the cytoplasm and subsequent release through reversal of the plasma membrane transporters. The specificity of their effects (amphetamines lead to dopamine and, to a lesser extent, norepinephrine release, whereas MDMA leads to serotonin release) is thought to be due to selective uptake by the plasma membrane transporters. The association of polymorphisms in the VMAT1 gene with an increased risk for bipolar disorder also suggests a link between vesicular monoamine transport and molecular psychopathology. A major mechanism for the regulation of vesicular neurotransmitter transport appears to involve changes in protein trafficking. VMATs undergo phosphorylation by casein kinase, and this posttranslational modification influences their retrieval from maturing large dense-core vesicles (LDCVs). Because sorting to LDCVs versus synaptic vesicles determines the site and mode of transmitter release, the regulation of transporter trafficking has great potential to influence signaling. In particular, differential trafficking of VMAT2 in midbrain neurons can lead to the release of dopamine from exocytosis at their cell bodies and dendrites, if trafficked to LDCVs, or axon terminals, if trafficked to synaptic vesicles. Regulation of VMAT activity by the heteromeric G-protein Gao2 has also been demonstrated. It appears that intralumenal monoamines activate the G-protein and downregulate uptake. This may be a mechanism to assure consistency in quantal size and may also serve to limit the efflux of the neurotransmitter through reverse transport once a vesicle has been filled.
The Vesicular Acetylcholine Transporter Is Structurally Related to the Vesicular Monoamine Transporters The electric ray organ of Torpedo californica was an early source of synaptic vesicles. Subsequent to the determination that these vesicles are filled with acetylcholine, a specific activity for acetylcholine transport was characterized. The molecular identification of the transporter, however, required genetic studies in Caenorhabditis elegans. A screen for nematode mutants resistant to aldicarb, an inhibitor of acetylcholinesterase, identified unc17, a gene encoding a protein closely related to the VMATs. Based on the similarity to the VMATs, it was predicted that unc17 functions as an acetylcholine transporter and that the resistance phenotype results from the reduced release of acetylcholine. The binding of vesamicol, a known inhibitor of vesicular acetylcholine transport, was also found to be absent in membrane fractions from the unc17 animals. Subsequent studies on the mammalian ortholog demonstrated that the protein is indeed a vesicular acetylcholine transporter. In vertebrates, the vesicular acetylcholine transporter VAChT is expressed in cholinergic neurons in the central, peripheral, and autonomic nervous systems, including the basal forebrain neurons, lower motor neurons, and parasympathetic neurons. In an interesting genomic structure, the VAChT gene lies within the first intron of the gene encoding cholineacetyltransferase, the enzyme that catalyzes the synthesis of acetylcholine from choline and acetyl CoA. This structure, which suggests a spatial component to transcriptional regulation of factors conferring cholinergic characteristics to a neuron, is conserved from C. elegans to humans. Like VMATs, VAChT recognizes a cationic substrate and depends primarily on DpH. VAChT has been postulated to have a similar stoichiometry of one cytosolic AChþ exchanged for two luminal Hþ. In contrast to VMATs, which have substrate affinities in the low micromolar range, the mammalian VAChT exhibits an apparent affinity of approximately 1 mM. The higher affinity of VMATs may reflect a need to keep cytoplasmic levels of potentially toxic monoamine transmitters low. The measured quantal size at the neuromuscular junction ( 10 000 molecules) is approximately fivefold larger than that expected for the 150 mM vesicular acetylcholine concentration predicted by osmotic limits. This suggests that, as with monoamine storage, mechanisms exist to limit the osmotic effects of acetylcholine in synaptic vesicles.
Vesicular Neurotransmitter Transporters 257
The vesicular acetylcholine transport can be inhibited by vesamicol and several related compounds. Vesamicol competitively inhibits transport by binding to a cytoplasmic domain on VAChT with a Kd of approximately 5 nM. Vesamicol binding can be used to estimate transporter number, but neither vesamicol nor its analogs are currently used clinically.
Vesicular GABA and Glycine Transport Activities Are Mediated by a Single Protein The primary excitatory and inhibitory neurotransmitters in the mammalian brain are the amino acids glutamate and GABA. There are no sources of readily purified glutamatergic or GABAergic vesicles, as there are for monoaminergic and cholinergic vesicles (e.g., chromaffin granules and electric organ synaptic vesicles, respectively). Early biochemical studies defining vesicular transport systems for these neurotransmitters were, therefore, carried out on mixed synaptic vesicles purified from either the bovine or rodent brain. As for VMATs and VAChT, molecular characterization of the vesicular glutamate transporters (VGLUTs) and vesicular GABA transporter (VGAT) relied on additional information from molecular genetic studies. The molecular identity of the vesicular VGAT was determined from a screen for C. elegans genes involved in GABAergic synaptic transmission. One gene identified in the screen, unc47, encodes a multitransmembrane domain protein that is localized to synaptic vesicles in GABAergic neurons. The functional characterization of a vertebrate ortholog confirmed that the protein mediated vesicular GABA transport. As predicted from studies with purified synaptic vesicles, VGAT mediates the uptake of GABA with an apparent affinity in the low millimolar range and uptake can be driven by DpH or Dc. VGAT also recognizes glycine as a substrate, but with a lower affinity, and is therefore also referred to as for vesicular inhibitory amino acid transporter (VIAAT). VGAT shows no sequence similarity to VMATs or VAChT. Rather, it belongs to a large family of amino acid transporters. This family includes the proteins responsible for the transport activities biochemically defined as amino acid transport system N and system A. The latter is responsible for much of the active amino acid uptake by mammalian cells. Characterization of the function and cellular and subcellular localizations of these transporters suggests that the system N transporters and system A transporters mediate glial glutamine release and neuronal uptake, respectively, in the glutamine–glutamate cycle. This intercellular metabolic pathway is involved in the
recycling of synaptically released glutamate and, to a lesser extent, GABA. VGAT is expressed in GABAergic and glycinergic neurons as well as in the pancreas and has been reported to be expressed in the glial cells of the pineal gland. The targeted disruption of the VGAT gene in mice leads to a marked reduction in the synaptic release of GABA and glycine. The loss of the transporter leads to embryonic lethality, failures in gut withdrawal, and formation of a cleft palate. It is suggested that these defects are secondary to the loss of inhibitory neurotransmission and resulting paralysis. Vesicular GABA transport can be competitively inhibited by amino acids, including glycine and b-alanine. Transport can also be competitively inhibited by g-vinyl GABA, a derivative of GABA. g-Vinyl GABA has been used in the clinical treatment of epilepsy and is known to inhibit GABA transaminase, an enzyme that metabolizes GABA. The mode of action for g-vinyl GABA as an antiepileptic drug is thought to be through its effects on GABA transaminase, but the inhibition of vesicular GABA transport could have an effect by increasing the nonvesicular release of GABA.
Vesicular Glutamate Transporters Define Glutamatergic Neurons Glutamate is the principal excitatory neurotransmitter in the mammalian central nervous system. Three unique vesicular glutamate transporters (VGLUT1, -2, and -3) have been identified. VGLUT1 was initially isolated in a screen for mRNA transcripts upregulated in cultured neurons by subtoxic doses of the excitotoxin N-methyl-D-aspartate (NMDA). Structural similarity to a class of proteins characterized as inorganic phosphate transporters led to an initial impression that the protein was a Naþ-dependent phosphate transporter and to a temporary designation of the protein as brain-specific Naþ dependent inorganic phosphate transporter (BNPi). Although subsequent studies have demonstrated that the primary function of the protein is synaptic vesicle glutamate transport, some controversy remains regarding the role of the protein in phosphate transport. The expression of VGLUT1 is limited to a subset of glutamatergic neurons in the brain. The highly homologous protein VGLUT2, also initially characterized as a phosphate transporter and named differentiationassociated Naþ-dependent inorganic phosphate transporter (DNPi), has a nearly complementary pattern of expression with one of the two proteins present in all established glutamatergic neurons. In general, VGLUT1 predominates in the neocortex and cerebellar cortex, whereas VGLUT2 predominates in the
258 Vesicular Neurotransmitter Transporters
brain stem nuclei, thalamic nuclei, and cerebellar deep nuclei. The septal nuclei, nuclei of the diagonal band, and hypothalamus also express VGLUT2. Although all cortical layers express VGLUT1, layer IV of frontal and parietal cortex and layers IV and VI of temporal cortex also express VGLUT2. Conversely, VGLUT2 predominates in the thalamus, but certain thalamic nuclei such as the medial habenula express VGLUT1. In the hippocampus, dentate gyrus granule cells express only VGLUT1, whereas pyramidal neurons from CA1 through CA3 express VGLUT1 as well as lower levels of VGLUT2. In the amygdala, the medial and central nuclei express VGLUT2, and the lateral and basolateral nuclei express VGLUT1. The third vesicular glutamate transporter, VGLUT3, is expressed in neurons not classically considered glutamatergic. Immunohistochemical and in situ studies indicate VGLUT3 is expressed in GABAergic, serotonergic, dopaminergic, and cholinergic neurons as well as astrocytes. Recent findings suggest that VGLUT1 and VGLUT2 are expressed in astrocytes as well, but these studies are in isolated and cultured cells, not in intact tissue. Glutamate uptake by the VGLUTs depends primarily on Dc rather than DpH, with apparent affinities in the low millimolar range. Interestingly, the VGLUTs do not appear to recognize aspartate. This is consistent with studies of synaptic vesicles and suggests that aspartate is not readily accumulated in synaptic vesicles through an active transport system. Vesicular glutamate transport shows a biphasic dependence on chloride with an optimum at 2–10 mM, and recent studies with purified VGLUT1 suggest that glutamate transport requires Cl. Like VMAT transport, VGLUT transport also appears to be regulated by Gao2; specifically, Gao2 reduces the chloride dependence of transport. The role of chloride in vesicular glutamate transport is further complicated by the finding that the expression of VGLUT1 appears to increase the vesicular Cl conductance. Although the VGLUTs exhibit similar transport activities, they are expressed in cells with very different properties. For example, compared to VGLUT1containing neurons, VGLUT2 neurons, in general, have a lower firing rate and a higher probability of release. The three isoforms also appear to have different subcellular localizations that could influence glutamate-release characteristics. Most notably, VGLUT3 is expressed in vesicular structures within the dendrites of some neurons in the hippocampus and striatum, suggesting a role in retrograde signaling. Further, two polyproline domains, which are present in the C-terminal cytoplasmic tail of VGLUT1 (but not VGLUT2 or -3), mediate interactions with the endocytic protein endophilin. This interaction may
regulate synaptic vesicle recycling and the mode in which VGLUT1 is internalized. Recent studies indicate that VGLUT1 and VGLUT2 are present in multiple copies on synaptic vesicles. Correcting for the number of vesicles containing the transporters, it is estimated that there are 9 copies of VGLUT1 and 14 copies of VGLUT2 in the synaptic vesicles in which they are expressed. Although not directly demonstrated, a similar stoichiometry probably exists for the other vesicular neurotransmitter transporters. Several compounds that inhibit vesicular glutamate transport have been identified. These include the dyes Evans blue and rose Bengal. In addition, the stilbene derivative 4,40 -diisothiocyanatostilbene-2,20 -disulfonic acid (DIDS), a compound commonly used as an inhibitor of anion channels, inhibits vesicular glutamate transport. Most known inhibitors have a limited utility because they are membrane impermeant, with the exception of rose Bengal. No inhibitors unique for specific isoforms have been identified.
Neuromodulators Are Also Stored in Vesicles and Released through Exocytosis Synaptic vesicles mediate the release of neuropeptides and small molecules other than the classic neurotransmitters. Neuropeptides enter the lumen of the secretory pathway in the endoplasmic reticulum through cotranslational translocation and are sorted to the secretory vesicle pathway, where they undergo processing to form the biologically active species. After release, it is believed that neuropeptides are degraded and not repackaged. Of the synaptically released small-molecule neuromodulators, zinc and ATP are the best characterized. NMDA and GABA receptors contain binding sites for zinc, and zinc exerts a direct effect on excitatory and inhibitory neurotransmission. ATP activates both ionotropic and G-protein-coupled receptors. As with the classical neurotransmitters, the exocytotic release of these compounds requires transport into synaptic vesicles. The multitransmembrane domain protein ZnT3 has been implicated in zinc uptake by synaptic vesicles. ZnT3 belongs to a family of zinc transporters and localizes to synaptic vesicles. Mice deficient in ZnT3 show a loss of zinc staining from hippocampal neurons, and the expression of ZnT3 in PC12 cells increases vesicular zinc staining. Although ZnT3 transport has not been directly demonstrated, these findings strongly support a role for ZnT3 in synaptic vesicle zinc transport. The phenotype of ZnT3-deficient mice is mild, with the most striking abnormality being an increased susceptibility to seizures. Chromaffin granules, platelet dense-core vesicles, and synaptic vesicles contain concentrations of ATP
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many fold higher than cytosolic concentrations, suggesting active vesicular uptake. ATP transport has been demonstrated in chromaffin granules and synaptic vesicles, and the process appears to depend on DmHþ. It has generally been assumed that ATP is costored only with monoamines and acetylcholine, as an anion to balance to cationic charge of those transmitters. However, the extent of ATP storage and release by different neuronal populations remains unknown, and the proteins responsible for ATP uptake by secretory vesicles have not been identified. One of the first synaptic vesicle membrane proteins to be identified was SV2. This protein is a multitransmembrane domain protein with limited structural similarity to the VMATs and VAChT. The protein was identified in synaptic vesicles from T. californica, and three isoforms (A, B, and C) have been identified in mammals. The expression patterns of the vertebrate proteins show partial overlap and together cover essentially all neurons. Targeted disruption of isoform A and both isoforms A and B demonstrates that these proteins are crucial for normal brain function in mice. Although a specific biochemical function was not been defined by these studies, it has been suggested that SV2 might be involved in regulating the size of the readily releaseable pool of vesicles or Ca2þ homeostasis at the nerve terminal. SV2 is thought to be the binding site for botulinum A toxin entry into neurons and to be a binding site for the antiseizure medication levetiracetam. It has also been suggested that the long sugar chains on SV2 serve as a smart-gel that regulates the release of neurotransmitters for the fused vesicle by limiting diffusion (Table 1).
Summary The vesicular uptake of neurotransmitters is a requirement for the quantal release of neurotransmitters. Earlier biochemical studies that identified four primary activities have been complimented by the molecular identification of unique proteins that mediate vesicular neurotransmitter transport activities. The cloned transporters can be divided into three structural families – the VMATs and VAChT; VGAT; and the VGLUTs. All the transporters are driven by the proton electrochemical gradient generated by the vacuolar Hþ-ATPase. Multiple copies of a given transporters are present on a single synaptic vesicle, suggesting that expression levels and regulated trafficking may play roles in modulating synaptic vesicle filling. A greater number of transporters in the vesicle increases the rate of filling and, if there is a significant leak, also leads to an
increase in the steady-state concentration of transmitter achieved. Although the potential for the modulation of the vesicular neurotransmitter transporter activities has been established, the extent to which neurotransmitter accumulation and synaptic transmission are regulated by vesicular transporter density, G-protein-mediated modulation, and the co-storage of other small molecules such as zinc and ATP remains to be determined. See also: Glutamate; Synaptic Vesicles.
Further Reading Bellocchio EE, Reimer RJ, Fremeau RT Jr., and Edwards RH (2000) Uptake of glutamate into synaptic vesicles by an inorganic phosphate transporter. Science 289(5481): 957–960. Brunk I, Holtje M, von Jagow B, et al. (2006) Regulation of vesicular monoamine and glutamate transporters by vesicleassociated trimeric G proteins: New jobs for long-known signal transduction molecules. Handbook of Experimental Pharmacology 175: 305–325. Carlson SS, Wagner JA, and Kelly RB (1978) Purification of synaptic vesicles from elasmobranch electric organ and the use of biophysical criteria to demonstrate purity. Biochemistry 17(7): 1188–1199. Fremeau RT Jr., Voglmaier S, Seal RP, et al. (2004) VGLUTs define subsets of excitatory neurons and suggest novel roles for glutamate. Trends in Neuroscience 27(2): 98–103. Johnson RG Jr. (1988) Accumulation of biological amines into chromaffin granules: A model for hormone and neurotransmitter transport. Physiological Reviews 68(1): 232–307. Lein ES, Hawrylycz MJ, Ao N, et al. (2007) Genome-wide atlas of gene expression in the adult mouse brain. Nature 445(7124): 168–176. Liu Y, Peter D, Roghani A, et al. (1992) A cDNA that suppresses MPPþ toxicity encodes a vesicular amine transporter. Cell 70(4): 539–551. McIntire SL, Reimer RJ, Schuske K, et al. (1997) Identification and characterization of the vesicular GABA transporter. Nature 389(6653): 870–876. Nishi T and Forgac M (2002) The vacuolar (Hþ)-ATPases – nature’s most versatile proton pumps. Nature Reviews Molecular Cell Biology 3(2): 94–103. Reimer RJ and Edwards RH (2004) Organic anion transport is the primary function of the SLC17/type I phosphate transporter family. Pflugers Archiv 447(5): 629–635. Stobrawa SM, Breiderhoff T, Takamori S, et al. (2001) Disruption of ClC-3, a chloride channel expressed on synaptic vesicles, leads to a loss of the hippocampus. Neuron 29(1): 185–196. Takamori S, Holt M, Stenius K, et al. (2006) Molecular anatomy of a trafficking organelle. Cell 127(4): 831–846. Voglmaier SM, Kam K, Yang H, et al. (2006) Distinct endocytic pathways control the rate and extent of synaptic vesicle protein recycling. Neuron 51(1): 71–84. Wojcik SM, Katsurabayashi S, Guillemin I, et al. (2006) A shared vesicular carrier allows synaptic corelease of GABA and glycine. Neuron 50(4): 575–587.
AMPA Receptors: Molecular Biology and Pharmacology S M Dravid, H Yuan, and S F Traynelis, Emory University, Atlanta, GA, USA Published by Elsevier Ltd.
Introduction Glutamate, one of the fundamental amino acid building blocks of proteins, is also a major excitatory neurotransmitter in the central nervous system (CNS). Neurons synthesize and package glutamate into presynaptic vesicles for release into the postsynaptic cleft. Synaptically released glutamate that diffuses across the 30 nm distance encounters a series of transmembrane postsynaptic proteins that comprise the glutamate receptor family. One class of glutamate receptors, metabotropic glutamate receptors, comprises transmembrane proteins that have extracellular clamshell-like domains that bind glutamate. When activated by glutamate binding, these G-protein-coupled receptors shift intracellular concentrations of signaling molecules to control a diverse set of cell properties. A second class of glutamate receptors, ionotropic glutamate receptors, comprises transmembrane proteins that contain an ion conduction path through the plasma membrane, as well as an array of clamshell-like extracellular ligand-binding domains, some of which bind to glutamate. Mammalian ionotropic glutamate receptors are ligand-gated ion channels encoded by 18 genes, and are subdivided into four major families on the basis of agonist pharmacology and sequence homology. These four receptor classes are known as amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), kainate, N-methyl-D-aspartate (NMDA), and d receptors. This article focuses on the structure and function of AMPA receptors. The AMPA receptor family is composed of four genes encoding the GluR1–GluR4 subunits (sometimes called GluRA–GluRD). In humans the chromosomal location of GluR1, GluR2, GluR3, and GluR4 encoding genes is 5q33, 4q32–33, Xq25–26, and 11q22–23, respectively. AMPA receptors were the first class of glutamate receptor cloned by screening a rat brain cDNA library for expression of kainate-activated ion channels in Xenopus laevis oocytes. After initial identification of GluR1, GluR2–GluR4 were rapidly identified by homology screening. There is about 70% sequence homology among different AMPA receptor subunits. A great deal of information now exists about AMPA receptor structure and function, and it could be argued that more is known about the structure of AMPA receptors than any other class of glutamate receptor.
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Expression of AMPA Receptors AMPA receptors are abundant and widely distributed in the central nervous system. Hippocampus, outer layer of cortex, basal ganglia, olfactory regions, lateral septum, and amygdala of the CNS are all enriched with GluR1, GluR2, and GluR3 subunits. In contrast, GluR4 expression is lower in many regions of the CNS except cerebellum, thalamus, and brain stem, where the expression is high. Immunoprecipitation studies have shown that the pyramidal cells of the hippocampus expressed AMPA receptors composed of GluR2 receptor in complex with either GluR1 or GluR3 subunits. GluR1 homomeric receptors, which have unique ion permeation properties, are thought to be expressed in select neuronal populations. The expression of AMPA receptors is developmentally regulated. The GluR2 subunit appears as early as embryonic day 16 in rats whereas other receptors are upregulated later during the development. The GluR2 subunit can also be selectively altered during synaptic plasticity as well as during CNS injury, such as global ischemia. These changes in receptor subunit composition are known to change functional receptor properties. AMPA receptors are present both postsynaptically and presynaptically. AMPA receptors are present on the synaptic membrane; however, 60–70% of AMPA receptors are present intracellularly. Glial cells also express AMPA receptors, which appear to be involved in glutamate-induced cell death. Activation of glial AMPA receptors also leads to release of ATP or nitric oxide.
Topology and Assembly of AMPA Receptor Subunits All of the ionotropic glutamate receptors share a common topology, which consists of an extracellular N-terminal domain, a ligand-binding domain, three transmembrane domains (M1, M3, and M4), a cytoplasm-facing reentrant membrane loop (M2), and an intracellular C-terminal domain (Figure 1(a)). AMPA receptors are composed of approximately 900 amino acids and have a molecular mass of 105 kDa. The location of the N-terminus and Cterminus was first deduced by use of specific antibodies. Because the N- and C-terminal regions were located on the opposite ends of the polypeptide chain, it was proposed that AMPA receptors had an odd number of membrane-spanning domains. Further studies delineated the membrane topology, and showed that the M2 segment is a reentrant loop.
AMPA Receptors: Molecular Biology and Pharmacology 261 Amino terminal domain
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Figure 1 Structure of the AMPA receptor subunit. (a) Transmembrane topology of the AMPA receptor, indicating flip/flop site, Q/R site, R/G site, and phosphorylation sites (PKA, protein kinase A; PKC, protein kinase C; CaMKII, Ca2þ/calmodulin-dependent protein kinase II). (b) Crystal structure of the ligand-binding domain of the GluR2 subunit, with glutamate bound in the cleft formed by clamshells (Protein Data Bank code 1FTJ). The lime-colored structure is domain 1 and the violet-colored structure is domain 2 (TM, transmembrane domain).
This transmembrane topology shares a parallel organization to potassium channels, which are now considered a model for AMPA receptor membrane domain structure. The pore diameter of AMPA receptors is about 0.8 nm and, in contrast to potassium channels, permits the entry of Naþ and, for some subunit combinations, Ca2þ. The N-terminal domain in AMPA represents up to 45% of the mature polypeptide, but its function is poorly understood. Hypothesized functions of this domain include receptor assembly, allosteric modulation of the ion channel (similar to NMDA receptors), and binding of a second ligand. Contradictory to other subunits, the GluR4 subunit can form normally functioning homomeric channels even in the absence of the N-terminal domain. The semiautonomous ligand-binding domain has been studied in detail using X-ray crystallography. The GluR2 agonist-binding domain is composed of two discontinuous peptide segments of approximately 150 amino acid residues. The first segment (S1) is adjacent and N-terminal to the M1 domain, whereas the second segment (S2) is located between M3 and M4 domains (Figure 1(a)). The agonistbinding domains of the AMPA receptors share similarities in sequence and structural arrangement with the ligand-binding site of several bacterial periplasmic amino acid-binding proteins. Like potassium channels, AMPA receptors assemble as tetramers. Studies in recombinant and native receptors suggest that AMPA receptors assemble as
dimer-of-dimers in a two-step manner. First, the monomers interact through the N-terminal domain to form dimers. Next, the dimers combine via the membrane domains to form the tetramer.
AMPA Receptor Function A remarkable step forward in understanding the AMPA receptor structure and function occurred in 1995 when a water-soluble mini-receptor that included only the agonist-binding core was described. This was a fusion protein consisting of the S1 and S2 domains of GluR4 joined together via a short hydrophilic linker peptide that replaces the membranespanning regions. The engineered agonist-binding domain functionally reproduced the AMPA-binding properties of the GluR4 receptor. This concept paved the way for the subsequent production of soluble agonist-binding core and later generation of crystals for X-ray diffraction, which ultimately could be produced by careful refolding of the denatured ligand-binding core. The first crystal structure of the GluR2 ligandbinding domain complexed with kainate revealed a bilobed clamshell-like shape, with agonist bound deep in the cleft formed by the two lobes. Subsequent descriptions of crystal structures of the nonliganded form (apo state) as well as forms complexed to a variety of ligands were obtained. These studies revealed that the clamshell was geometrically opened widest in the apo state. In the glutamate-bound state, the clamshell was 21 more closed than in the apo
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state (Figures 1 and 2). Competitive antagonists of the receptor stabilized the open-cleft state. Glutamate makes a number of hydrogen-bonded contacts within the binding pocket with both upper (D1) and lower (D2) domains. The upper domain of the clamshell-like structure is formed by segment S1 and the C-terminal portion of segment S2. The N-terminus of S2 forms the lower D2 domain. It has been proposed that when glutamate first encounters the ligand-binding pocket, it initially docks or interacts with the D1 domain, which then promotes the rotation of the D2 domain toward D1 and induces closure of the clamshell. The closed conformation is stabilized by glutamate, itself forming a cross-domain bridge, as well as a number of other hydrogen bonds that form between the domains during glutamate binding. Water appears to persist inside of the agonist binding pocket, particularly for some agonists that interact directly with it. This closure of the cleft within the isolated ligand-binding domain is considered to be analogous to domain closure in the full-length native receptors.
Domain closure has been hypothesized to pull the linker that connects the S1 and S2 domains. In native receptors, this pull or strain is considered to be the force responsible for rearrangement of the pore-forming membrane domains, leading to the opening of the transmembrane conduction path. Thus, each subunit can bind glutamate and undergo conformational changes that contribute to dilatation of the pore. In agreement with this view, single-channel analysis of intact receptor shows at least three conductance states that have been proposed to correspond to two, three, or four liganded subunits within a receptor complex. Pore conductance is therefore conceptually related to the fraction of subunit occupancy and activation within each receptor complex. This suggests that subunits can make incremental contributions to pore opening, gradually shifting the unitary conductance through the channel to higher levels as more subunits become activated. The observed concentration dependence of conductance levels supports this idea. This view of fourfold rotational symmetry is also supported by the similarities of the pore-forming
AMPA
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Figure 2 Conformational changes induced by agonist binding to the AMPA receptor. Crystal structure of the ligand-binding domain of the GluR2 subunit shown in nonliganded (apo –state; Protein Data Bank code 1FTO) and liganded (AMPA bound state; Protein Data Bank code 1FTM) states. Agonist binding in the cleft leads to closure of the clamshell (top).
AMPA Receptors: Molecular Biology and Pharmacology 263
AMPA receptor domain to potassium channels. However, several other features of AMPA receptors point to a twofold rotational symmetry for the tetrameric receptor complex. First, the glutamate-binding domains crystallize as dimers. Second, the D1–D1 dimer interface plays a role in channel activation by forming a structural scaffold that leads to the movement of D2. However, excessive tension can trigger rearrangement of the D1–D1 interface, leading to AMPA receptor desensitization. Mutations that affect desensitization lie on the interface. Additionally the AMPA receptor desensitization inhibitor cyclothiazide (see below) also acts on the dimer interface. Third, the reactivity of cysteine-modifying reagents on cysteine residues inserted into the M3 domain by site-directed mutagenesis fits well with a twofold symmetry rather than a fourfold symmetry. However, additional structural information about the full AMPA receptor complex would be needed to ascertain receptor symmetry. Partial agonists are typically considered as ligands that induce a response at the maximally effective concentrations, which is lower than that of the endogenous ligand glutamate. The mechanism of action of partial agonists has been studied in AMPA receptors using a series of 5-substituted willardiines, which show lower efficacy compared to glutamate. The 5-substituted willardiines differ in only a single atom at the same position in the molecule. Structural and functional studies suggest that the degree of domain closure of the agonist binding cleft forms the basis for the partial agonist action at the AMPA receptor. Specifically, a combination of crystallographic and functional data show that the degree of domain closure of the clamshell is correlated with the efficacy of the agonist at an individual subunit such that agonists that induce less domain closure appear less effective in opening the channel. However, other conformational rearrangements of the agonist binding domain can also impact agonist efficacy.
Posttranscriptional Modification of AMPA Receptors Alternative RNA Splicing
All four AMPA subunits undergo alternative RNA splicing in the C-terminal half of the M3–M4 loop, leading to so called flip/flop splice variants. The locations of flip/flop splicing for GluR1, GluR2, GluR3, and GluR4 are 742–793, 736–787, 740–791, and 737–788, respectively. In rats expression of the flip variant predominates up to postnatal day 8, but in adults both forms are expressed to a similar extent in many regions. The flip splice variant endows receptors with a diminished form of desensitization and a
faster rate of recovery from desensitization as compared to the flop splice variant. GluR4-flop has the fastest desensitization (<1 ms at room temperature) among all the studied ionotropic glutamate receptors. GluR2 and GluR4 subunits are also alternatively spliced at their C-terminal ends to generate short or long isoforms. A short isoform for GluR1 has not been reported, whereas GluR3 shows only the short isoform due to absence of splice sites. More than 90% of GluR2 is in the short form while the long form of GluR4 is predominant. The postsynaptic density/disc/zonula occludens-1 (PDZ) motif, which interacts with several proteins necessary for targeting of AMPA receptors to the synapse, is only present in the short form. RNA Editing
One for the most interesting properties of AMPA receptor subunits is the consequence of differential RNA editing. The GluR2 subunit undergoes RNA editing at the so-called Q/R site (Q607) located at the tip of the reentrant loop of the second membrane-associated region. RNA editing leads to conversion of a glutamine codon to an arginine codon. This residue is a major determinant of the ionic selectivity of the pore. Replacement of the glutamine with an arginine leads to low calcium permeability of GluR2 subunit, low single-channel conductance, and a linear current–voltage relationship that reflects lack of block by intracellular polyamines (see later). Conversely, AMPA receptors lacking the GluR2 subunit have higher unitary conductances and show substantial Ca2þ permeability. The latter property may underlie some forms of plasticity at central synapses. Virtually all of the GluR2 subunit RNA is edited with high efficiency, and reduction in editing efficiency can lead to epilepsy and early death in mice. This may reflect the increased Ca2þ permeability of AMPA receptors, since it is well established that intracellular calcium overload can lead to cell death during glutamate-mediated neurotoxicity. Reduction in the efficiency with which GluR2 is edited has been proposed to play a role in schizophrenia, Huntington’s disease, Alzheimer’s disease, epilepsy, and malignant glioma, although more work will be required to evaluate these hypotheses. Another site of RNA editing is the R/G site, where glycine replaces the arginine in GluR2–GluR4 subunits. The locations of the R/G site for GluR2, GluR3, and GluR4 are R764, R769, and R7665, respectively. This editing leads to reduced desensitization and rate of recovery from desensitization in GluR3 and GluR4 subunits. In adult mammals 50–90% of GluR2– GluR4 subunits are edited at the R/G site. GluR1 only has an arginine at the R/G site (R757).
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Posttranslational Modification Phosphorylation of AMPA Receptors
AMPA receptor function and trafficking is posttranslationally regulated by protein kinases and phosphatases. AMPA receptors are closely localized to cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) and calcineurin by the binding of the A-kinase anchoring protein (known as AKAP) to scaffolding proteins within the postsynaptic density. In hippocampal neurons PKA phosphorylation potentiates AMPA receptor function by increasing the open probability, increasing the mean open time of the channel, and facilitating receptor trafficking to the membrane surface. PKA phosphorylates Ser845 residue present at the intracellular C-terminal tail of the GluR1 receptor. Mutation of this residue to alanine (S845A) eliminates the ability of PKA to potentiate GluR1-mediated currents. Phosphorylation and dephosphorylation of the Ser845 site are one important component of the molecular events that underlie long-term potentiation (LTP) and long-term depression (LTD) of synaptic strength, respectively, at certain synapses. AMPA receptors are also phosphorylated by Ca2þ/ calmodulin-dependent protein kinase II (CaMKII), which is a major constituent of postsynaptic densities at the glutamatergic synapses and forms approximately 1% of the brain protein. CaMKII phosphorylates the Ser831 residue present exclusively in the GluR1 subunit among AMPA receptors. CaMKII enhances the current through native AMPA receptors in hippocampal neurons and increases the singlechannel conductance of GluR1 receptors. This change in conductance reflects a shift in the proportion of subconductance to higher levels, rather than creation of new levels. This shift can be accounted for by changes in subunit-dependent gating, in which individual GluR1 subunits that are phosphorylated at Ser831 are more likely to become activated, increasing the probability that the receptor will be in the higher conductance states that require more activated subunits. Protein kinase C (PKC) is a family of heterogeneous protein kinases activated by Ca2þ and/or phospholipids. PKC can be converted to a persistently active form by calpain-mediated proteolysis. GluR1 is phosphorylated by PKC at Ser831, which has similar effects on current response amplitude and conductance to CaMKII phosphorylation at this site. The GluR2 subunit is phosphorylated by PKC at Ser880 and Ser863. PDZ domains are present in a number of proteins that are capable of meditating interaction with the AMPA receptors. The GluR2 Ser880 residue is present in the C-terminal sequence
that is responsible for PDZ domain binding and its phosphorylation leads to decrease in binding to glutamate receptor-interacting protein 1 (GRIP 1), which can synaptically anchor GluR2. GluR2 Ser880 phosphorylation does not affect the binding to other targeting auxiliary proteins, such as PICK 1 (protein interacting with PKC 1). Phosphorylation by PKC at the Ser880 site facilitates the internalization of the GluR2 receptor.
Pharmacology of AMPA Receptors AMPA Receptor Agonists
A number of natural products have been known for decades to have neurological or psychoactive effects, which were later attributed to actions on glutamate receptors. These include ibotenic acid, quisqualic acid, domoic acid, and willardiine (see Figure 3 for structures). Ibotenic acid is derived from the mushroom Amanita muscaria (the fly mushroom, or toadstool). Quisqualic acid is obtained from the seeds and fruit of Quisqualis chinensis, domoic acid is a phycotoxin (algal toxin) associated with certain algal blooms and shellfish poisoning, and willardiine is from Acacia willardiana and Mimosa asperata. Although L-glutamate is the endogenous agonist of AMPA receptors, these receptors show higher selectivity for the synthetic ibotenic acid analog AMPA, thus deriving their name. Kainic acid can also activate AMPA receptors, although with 10- to 20-fold lower potency for GluR1 and GluR2 over subunits within the kainate receptor family, such as GluR5. AMPA receptors have lower affinity for glutamate compared to NMDA receptors. Glutamate and AMPA act as full agonists and induce rapid desensitization, which is thought to involve rearrangement of the dimer formed by the ligand-binding domains. Kainic acid, which is the defining agonist for so-called kainate receptors composed of GluR5–GluR7 and KA1 and KA2, functions as a partial agonist and induces little desensitization on AMPA receptors. 5-Fluorowillardiine is not only more selective for AMPA receptors than AMPA itself (5-fluorowillardiine has a 70- to 150-fold higher affinity for GluR1 and GluR2 over GluR5), but also shows some selectivity for different AMPA receptor subunits. AMPA Receptor Competitive Antagonists
Structurally different classes of competitive AMPA antagonists have been found which bind to the glutamate-binding site of the receptor. The first selective and useful AMPA receptor antagonists were 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 6,7-dinitroquinoxaline-2, 3-dione (DNQX). However,
AMPA Receptors: Molecular Biology and Pharmacology 265 O O
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Figure 3 The structures of AMPA receptor agonists a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), L-glutamate, ibotenic acid, quisqualate, domoic acid, and willardiine; AMPA receptor antagonists 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f ]quinoxaline (NBQX) and GYKI 52466; and AMPA receptor modulator cyclothiazide.
these quinoxaline derivates also have some affinity at the glycine-binding site of NMDA receptors. Newer antagonists have increased potency, higher AMPA receptor specificity, increased water solubility, and a longer duration of action in vivo. Examples of some of these competitive antagonists include 2,3dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline (NBQX), 1,4,7,8,9,10-hexahydro-9-methyl-6-nitropyrido-[3,4-f ]-quinoxaline-2,3-dione (PNQX), [2,3-dioxo-7-(1H-imidazol-1-yl)-6-nitro-1,2,3,4-tetrahydro-1-quinoxaliny]acetic acid (YM872), and [1,2,3,4-tetrahydro-7-morpholinyl-2,3-dioxo-6-(trifluoromethyl)quinoxalin-1-yl]methylphosphonate (ZK200775). Although some of these compounds have entered clinical trials for the treatment of ischemia, side effects and poor water solubility complicated clinical usage. AMPA Receptor Noncompetitive Antagonists
The first noncompetitive AMPA receptor antagonist described was 1-(4-aminophenyl)-4-methyl-7,
8-methylenedioxy-5H-2,3-benzodiazepine hydrochloride (GYKI 52466), which is structurally classified as a benzodiazepine. A related series of selective, noncompetitive antagonists were derived from three classes of compounds: 2, 3-benzodiazepine (GYKI 52466), phthalazine (GYKI 53784/LY303070, GYKI 53773/LY300164), and quinazolinone derivatives (CP-465,022 and CP-526,427). The noncompetitive AMPA antagonists are of interest as potential therapeutic agents because they function effectively even in the presence of high levels of glutamate. Some noncompetitive AMPA receptors antagonists have been evaluated as drug candidates in a number of clinical trials for various neurological disorders. Positive Allosteric Modulators
When AMPA receptors are activated by glutamate they rapidly desensitize in 1 (or a few) ms (Figure 4(a)). This desensitization has been hypothesized to be triggered by domain closure around the agonist, which exerts strain on the ligand-binding dimer
266 AMPA Receptors: Molecular Biology and Pharmacology
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Figure 4 Electrophysiological properties of the AMPA receptor. (a) Simulated traces showing activation of GluR1 homomeric receptors by short (0.5 ms) and long (25 ms) pulses of 10 mM glutamate. The faster decay of current in the red trace activated by short pulse of glutamate represents deactivation. The relatively slower decay of current in green trace represents desensitization. (b) Evoked excitatory postsynaptic currents (eEPSC) from a pyramidal CA1 hippocampal neuron mediated by AMPA receptors in the presence of NMDA and GABA receptor antagonists. The eEPSC in green is in the presence of AMPA receptor desensitization inhibitor cyclothiazide. (a) Model used for simulation from Partin KM, Fleck MW, and Mayer ML (1996) AMPA receptor flip/flop mutants affecting deactivation, desensitization, and modulation by cyclothiazide, aniracetam, and thiocyanate. Journal of Neuroscience 16(21): 6634–6647.
interface. The strain can be relieved by opening of the channel or breakdown of the ligand-binding interface, which triggers desensitization. A number of AMPA receptor-positive allosteric modulators have been identified, sometimes referred to as AMPA receptor potentiators. These allosteric potentiators do not activate the AMPA receptors themselves, but enhance the agonist-evoked current by slowing the rate of desensitization (Figure 4(b)). Drugs that positively regulate AMPA receptors fall into three classes: pyrrolidones (also known as ampakines, including aniracetam and piracetam), the benzothiadiazides (cyclothiazide, diazoxide), and biarylpropylsulfonamides (4-[2-(phenylsulfonylamino)ethylthio]-2,6-difluorophenoxyacetamide (PEPA), N-2-[4-(4-cyanophenyl)phenyl]propyl-2propanesulfonamide (LY404187)). Both aniracetam and cyclothiazide almost completely eliminate desensitization of flip splice variants but only slow the entry into desensitized state for flop splice variant. PEPA potentiates AMPA receptor function by attenuating receptor desensitization without any effect on deactivation; Cyclothiazide and PEPA are approximately 100–500 times more potent compared to aniracetam in potentiating AMPA ion currents. PEPA has varying degrees of selectivity for the flop variants. LY404187 suppresses receptor desensitization with a distinct time dependence in the presence of agonist and shows high potency for flip splice variants. Accumulated evidence suggests that AMPA-positive modulators may act as cognitive enhancers, which could be useful as novel therapeutic agents for treating brain disorders such as Alzheimer’s disease and schizophrenia.
The binding site of compounds that relieve desensitization appears to reside within the ligand-binding dimer interface. Crystallographic studies of aniracetam suggest that binding can stabilize the ligand-binding core dimerization. This supports the interpretation that desensitization involves breakdown of the dimer interface. Pore-Blocking Molecules
The current–voltage relationship for AMPA receptors is controlled by subunit composition, with GluR2 endowing receptors with a linear current–voltage curve that reverses near 0 mV. By contrast, AMPA receptors lacking the GluR2 subunit show inward rectification, a term that means cations can flow through the channels into cells at negative potentials more easily than they can flow out of the channel at positive potentials. Inward rectification occurs due to the presence of intracellular polyamines such as spermine, which can block the flow of current out of the cell by entering and blocking the conduction path. A number of elongated and positively charged molecules such as spermine, spermidine, and polyamine toxins (ArgTX-636, PhTX-343, JSTX-3) are noncompetitive open-channel blockers of cation-conducting channels. Spermine is expressed in many CNS neurons and is implicated in regulation of cell division, protein synthesis, and perhaps specific functions in the nervous system. Polyamine toxins are nonoligomeric, low-molecular-weight compounds isolated from spiders and wasps. The mechanism by which polyamines block AMPA receptors is thought to involve a deep binding site
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See also: AMPA Receptor Cell Biology/Trafficking; Kainate Receptors: Molecular and Cell Biology; Metabotropic Glutamate Receptors (mGluRs): Molecular Biology, Pharmacology and Cell Biology; NMDA Receptors, Cell Biology and Trafficking.
Dingledine R, Borges K, Bowie D, et al. (1999) The glutamate receptor ion channels. Pharmacological Reviews 51: 7–61. Erreger K, Chen PE, Wyllie DJA, et al. (2004) Glutamate receptor gating. Critical Reviews in Neurobiology 16: 187–225. Gouaux E (2004) Structure and function of AMPA receptors. Journal of Physiology 554: 249–253. Kew JN and Kemp JA (2005) Ionotropic and metabotropic glutamate receptor structure and pharmacology. Psychopharmacology (Berlin) 179(1): 4–29. Marenco S and Weinberger DR (2006) Therapeutic potential of positive AMPA receptor modulators in the treatment of neuropsychiatric disorders. CNS Drugs 20: 173–185. Mayer ML and Armstrong N (2004) Structure and function of glutamate receptor ion channels. Annual Reviews in Physiology 66: 161–181. Nicoll RA, Tomita S, and Bredt DS (2006) Auxiliary subunits assist AMPA-type glutamate receptors. Science 311: 1253–1256. Palmer CL, Cotton L, and Henley JM (2005) The molecular pharmacology and cell biology of alpha-amino-3-hydroxy-5-methyl4-isoxazolepropionic acid receptors. Pharmacological Reviews 57(2): 253–277. Partin KM, Fleck MW, and Mayer ML (1996) AMPA receptor flip/ flop mutants affecting deactivation, desensitization, and modulation by cyclothiazide, aniracetam, and thiocyanate. Journal of Neuroscience 16(21): 6634–6647.
Further Reading
Relevant Website
Armstrong N and Gouaux E (2000) Mechanisms for activation and antagonism of an AMPA-sensitive glutamate receptor: Crystal structures of the GluR2 ligand binding core. Neuron 28: 165–181.
http://www.iddb.com – Investigational Drugs Database.
within the receptor pore, the binding to which appears voltage dependent. At positive potentials, polyamine binding is enhanced as the membrane potential favors entry of polyamines from the cell into the channel pore. At sufficiently positive potentials, polyamine block can be relieved as polyamines leave their block site to permeate the channel, joining the outward flow of cations. As described previously, GluR2 subunits that have been edited at the Q/R site at the apex of the reentrant loop have a positively charged arginine at the Q/R site in GluR2, as opposed to glutamine in GluR1, GluR3, and GluR4. The presence of an arginine at this site removes inhibition by intracellular polyamines and like molecules, rendering the current–voltage relationship linear.
AMPA Receptor Cell Biology/Trafficking P G R Hastie and J M Henley, University of Bristol, Bristol, UK ã 2009 Elsevier Ltd. All rights reserved.
a-Amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs) are ligand-gated ion channels that are crucial for normal brain function. They mediate nearly all fast excitatory synaptic transmission in the mammalian central nervous system (CNS). Changes in functional postsynaptic AMPARs mediate the two main forms of synaptic plasticity that are believed to underlie learning and memory. Long-term potentiation (LTP) involves the activity-dependent recruitment of AMPARs to the synapse and a concurrent increase in AMPA-mediated transmission. Conversely, long-term depression (LTD) is a decrease in synaptic AMPAR function. Given their importance, AMPARs are stringently regulated and the mechanisms of this regulation have been, and continue to be, the subject of intense study.
Subunit Composition of AMPARs AMPARs are tetrameric assemblies of combinations of four individual subunits (glutamate receptor (GluR)1–4). Based on the length of the intracellular C-terminal domains of their predominant splice isoforms, GluR2 and GluR3 are classified as short and GluR1 and GluR4 are classified as long subunits. As discussed here, short and long subunits bind to different sets of intracellular proteins and are trafficked differently.
AMPAR Trafficking There are three fundamental ways of targeting proteins in polarized cells: 1. Selective delivery in which cargo is sent directly to the target destination. 2. Selective fusion – exocytic machinery packaged with the cargo fuses with a particular membrane subregion. 3. Selective retention – cargo is removed from inappropriate membranes. In general, dendritic sorting most likely relies on selective delivery because green fluorescent protein (GFP) attached to proteins containing dendritic sorting signals traffics directly to the somatodendritic compartment. Live imaging with GFP-labeled GluR1 and GluR2 suggests that AMPARs can be independently
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transported intracellularly rather than in clusters. This is consistent with a process of selective fusion acting as the regulated step in synaptic delivery. Endoplasmic Reticulum to Synapse Trafficking Pathway
AMPARs are delivered to the synapse via the secretory pathway (Figure 1). AYxxLxxR motif targets the translated proteins to the endoplasmic reticulum (ER) for folding, assembly of dimers, and enzyme modification. GluR1 is able to rapidly exit the ER, but RNA editing in GluR2 delays the exit of this subunit. Therefore, a large pool of GluR2 is held available in the ER, ready to combine with other subunits, and thus most hippocampal AMPARs contain GluR2. An extreme N-terminal signal may further promote GluR1 exit from the ER because truncation mutants lacking these nine amino acids co-localize with ER markers despite forming heteromers with GluR2. It is believed that the assembly of dimers into tetramers (a dimer of dimers) leads to masking of the retention signals, allowing only correctly folded, desensitized AMPARs to exit the ER. AMPARs next enter the trans-Golgi network (TGN) and become fully glycosylated. Passage through this pathway is dependent on the C-terminus of the receptor, a site for interaction with a number of proteins, particularly those containing postsynaptic density protein (PSD-)95/Dlg-A/ZO-1 (PDZ) domains. PDZ domains are protein interaction modules that, depending on the type of PDZ motif, can bind selectively to a range of peptide ligand sequences present on target proteins. Anterograde trafficking of AMPARs occurs along microtubules. One candidate for mediating forward trafficking is mLIN-10/mint1/X11, the Caenorhabditis elegans ortholog of which, LIN-10, is required for the correct localization of GLR-1 receptors. The microtubule-associated kinesin motor protein KIF5 binds to the AMPAR binding protein (ABP) glutamate receptor interacting protein 1 (GRIP1) and is involved in selective dendritic delivery. Further evidence that AMPARs travel along microtubules comes from another GRIP1-interacting partner, liprin-a/SYD2, which links to the scaffold protein GIT1 and to microtubules via KIF1A. Interference with either of these interactions specifically reduces GluR2/3 dendritic clustering. In the case of GluR1, linkage to microtubules can occur through a synapse-associated protein (SAP-)97 interaction with KIF1Ba. AMPAR synaptic delivery to and insertion at dendritic spines are two distinct processes, employing the
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Figure 1 Delivery of AMPARs: (a) AMPAR synaptic delivery and insertion as two distinct processes; (b) differential transport rates of AMPAR subunits; (c) AMPAR synaptic delivery steps. In (a), a dominant negative C-terminal construct of the exocyst component Sec8 prevents the delivery of GluR2-GFP to the distal dendrites (upper panel). A dominant negative construct of another exocyst component, Exo70, prevents the surface insertion of CaMKII phosphorylated GluR1-GFP, causing the accumulation of correctly delivered AMPARs within the spine. In (b), 40 mm segments of cultured hippocampal dendrites expressing either GluR1-GFP or GluR2 were photobleached. Fluorescence recovery from both distal (d) and proximal (p) dendritic ends was greatest for GluR1-GFP, suggesting faster rates of transport and/or greater surface mobility for this subunit. (c) Shows the events in the AMPAR synaptic delivery: (1) individual AMPAR subunits enter the ER and form dimers and then a dimers-of-dimers; Q/R editing in GluR2 prevents GluR2 homomer assembly and thus most AMPARs contain GluR2; (2) ER exit is dependent on the correct assembly of the tetrameric complex, which must be able to enter a desensitized state to exit the ER; TARPs are also required for ER exit; (3) high-mannose sugars on the extracellular surface of the AMPAR are replaced by complex carbohydrates in the Golgi; AMPARs most likely pass through a sorting endosome before being delivered to the surface, with GluR1-containing AMPARs exiting this compartment more slowly than GluR1-lacking AMPARs; (4) anterograde trafficking of AMPARs occurs along microtubules as complexes containing either GluR1-K1F1b-SAP97 or GluR2-KIF5-GRIP; (5) synaptic activity is required for surface delivery of GluR1, which is accompanied by the delivery of slot proteins (e.g., PSD-95) to the synapse; GluR1containing AMPARs are then replaced by constitutively cycling GluR2/3 heteromers; (6) delivery of AMPARs to the synapse is dependent on an interaction between GluR2 and NSF; this interaction prevents internalization due to AP2 and PICK1 binding to the receptor, thus stabilizing AMPARs at the synapse. AMPAR, a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor; a.u., arbitrary units; CaMKII, calmodulin-dependent protein kinase II; ER, endoplasmic reticulum; GFP, green fluorescent protein; GluR, glutamate receptor; NSF, N-ethyl maleimide sensitive factor; PDZ, PSD-95/Dlg-A/ZO-1; PICK1, protein interacting with C kinase 1; PSD-95, postsynaptic density protein 95; Q/R, glutamine to arginine; RFP, red fluorescent protein; TARPS, transmembrane AMPAR interacting proteins. (a, middle and bottom) From Gerges NZ, Backos DS, Rupasinghe CN, Spaller MR, and Esteban JA (2006) Dual role of the exocyst in AMPA receptor targeting and insertion into the postsynaptic membrane. EMBO Journal 25: 1623–1634. (b) Adapted from Perestenko PV and Henley JM (2003) Characterisation of the intracellular transport of GluR1 and GluR2 a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor subunits in hippocampal neurons. Journal of Biological Chemistry 278: 43525–43532.
exocyst, a multisubunit complex required for vectorial targeting of a subset of secretory vesicles. The exocyst complex acts to fuse vesicles to the plasma membrane. Proteins on one end recognize Rab
GTPases on secretory vesicles, whereas proteins on the other end localize to the plasma membrane, probably by binding to Rho GTPases that mediate the activity of the cytoskeleton. The proteins in between
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may act as a structural core to transmit information between the two membranes. A dominant negative C-terminal construct of the exocyst component Sec8 prevents the delivery of GluR2-GFP to distal dendrites. A dominant negative construct of another exocyst component, Exo70, prevents the surface insertion of GluR2-GFP and calmodulin-dependent protein kinase (CaMK)II-phosphorylated GluR1-GFP, causing the accumulation of otherwise correctly delivered AMPARs within the spine.
Roles of Short and Long AMPAR Subunits The long and short AMPAR subunits appear to serve different functions in AMPAR surface expression, and a subunit-specific AMPAR trafficking model has been proposed to explain how LTP is expressed and maintained. To summarize, the forward trafficking characteristics of the long-tail GluR1 subunit are dominant and AMPARs containing this subunit are inserted into the synapse during periods of activity. AMPARs lacking GluR1 default to the shorttail GluR2 subunit characteristics of constitutive cycling. GluR2-mediated cycling replaces synaptic AMPARs without changing synaptic efficacy. Therefore, GluR1-containing AMPARs are inserted during the initial phase of LTP, and these are then replaced by GluR2/3 heteromers. This hypothesis is based on the following experimental observations. Constitutive Cycling of AMPARs Is N-Ethyl Maleimide-Sensitive Factor-Dependent
Plasma membrane insertion of GluR2short is much more rapid than for GluR1long under basal conditions, and the delivery of GluR2 continues when neuronal activity is blocked. However, surface expression of GluR2 is short lived; the blockade of exocytic events leads to a rapid run-down of AMPAR responses. GluR2-containing AMPAR insertion and/or stabilization at the synapse requires the hexameric adenosine triphosphatase (ATPase) N-ethyl maleimide-sensitive factor (NSF). NSF binds the GluR2 C-terminus upstream of the PDZ binding site between residues Lys844 and Gln853. Blocking this interaction with a peptide (pep2m) corresponding to the interaction site on GluR2 causes a decrease in surface GluR2, indicating the rapid internalization and recycling back to the membrane of GluR2-containing receptors. Pep2m also prevents de novo LTD. However, pep2m also blocks an overlapping binding site for the clathrin adaptor protein AP2, which interacts with GluR1–3 subunits and is required for AMPAR endocytosis. A more specific peptide, pep-R845A, which blocks only the GluR2– NSF interaction, leads to the loss of functional AMPARs but has no effect on LTD.
Activity-Dependent Insertion of AMPARs
Insertion of GluR1long-containing AMPARs at the hippocampal synapses requires synaptic activity and occurs following N-methyl-D-aspartate receptor (NMDAR)-dependent LTP induction. Receptor phosphorylation is critical in the process, and in older neurons CaMKII activation is required, whereas protein kinase A (PKA) activation is more important in younger neurons. Although GluR1 is responsible for the majority of activity-dependent insertion in adults, GluR4 and the long C-terminal variant of GluR2 contribute significantly in younger animals when the expression of these subunits is prevalent. In GluR1, the PDZ protein binding region comprises a –TGL motif. This is a type I PDZ ligand and therefore binds a different subset of proteins than the type II –SVKI motif present in short AMPAR subunits. SAP-97 directly interacts with the GluR1 PDZ ligand, and the overexpression of SAP-97 increases AMPAR surface delivery; but truncation mutants eliminating GluR1 binding do not affect AMPAR clustering and mice lacking the –TGL motif have normal levels of synaptic GluR1, suggesting that SAP-97 may be important in relieving intracellular retention interactions at this site. There is close similarity between the signal transduction pathways for LTP and long-tailed AMPAR synaptic insertion. Activation of the Ras GTPase pathway during LTP leads to specific synaptic insertion of long-tailed AMPARs. Low-level activation leads to extracellular signal-regulated kinase kinase (MEK) and p42/44 mitogen-activated protein kinase (MAPK) signaling, leading to delivery of GluR2long. Higher levels of activity stimulate the PI3K and protein kinase B (PKB) pathways leading to GluR1 delivery. This action of GluR2long may explain the persistent LTP in mice lacking the GluR1 PDZ ligand. Maintenance of Increased Synaptic AMPARs Requires Slot Proteins
An important aspect of this model, which depends on an initial increase in surface expression due to long subunits but the maintenance of the increased expression via short subunits, is the need for marker or slot proteins to mark the potentiated synapses. Once trafficked to the PSD, these slot proteins remain there to maintain the increased expression of short subunit AMPARs unless a signal such as LTD induction triggers their removal. They therefore can act as placeholders, defining the number of AMPAR insertion sites at the synapse. If these were not added to the synapse, the internalized GluR1 could not be replaced by GluR2/3 because there would be no scaffolding structure for these new short AMPARs to attach to.
AMPA Receptor Cell Biology/Trafficking
AMPARs Interact with a Complex Network of Scaffold Proteins Cytoskeletal interactions at the synapse are both dynamic and complex, and there is likely to be redundancy between many of the binding partners. The most direct links with the actin cytoskeleton occur through long AMPAR subunits. Binding of protein 4.1N to the C-terminus of GluR1 and GluR4 upstream of the phosphorylation sites controls the surface expression of these subunits, possibly mediated through SAP-97 because the removal of the 4.1N binding site on this protein reduces AMPAR clustering. SAP-97 also links AMPARs to the actin cytoskeleton through myosin VI, although this minus-end-directed motor protein is involved in removal of AMPARs. A further interaction is provided by reversion-induced Lin11/rat Isl-1/Mec3 (LIM) domain-containing protein (RIL). Unusually, it is the LIM domain that specifically binds GluR1, whereas the PDZ domain links to the actin cytoskeleton via a-actinin, increasing the surface accumulation of AMPARs at the spines. PSD-95 Regulates Synaptic AMPAR Content
Postsynaptic density protein of 95 kDa (PSD-95), also known as synaptic-associated protein 90 (SAP-90), represents 2.3% of the protein mass of the PSD (equating to 300 PSD-95 molecules per synapse). This protein is a member of the membrane-associated guanylate kinase (MAGUK) family due to the guanylate kinase (GK) homology domain in the C-terminus. The C-terminus also contains a src homology (SH3) domain. The N-terminus contains a consensus sequence for palmitoylation. Three PDZ domains make PSD-95 an effective scaffold protein. PSD-95 is required for AMPAR synaptic localization, and there is a correlation between PSD-95 and AMPAR accumulation at the synapse. Expression of PSD-95-GFP increases the synaptic delivery of GluR1, thus occluding LTP, and increases the AMPAR-mediated currents in experience-deprived animals. Furthermore, PSD-95 gene transcription is upregulated by neuronal activity. An attractive model is that activity-dependent delivery of PSD-95 occurs prior to, and enables, the recruitment and insertion of GluR1-containing AMPARs and then acts as the slot protein for subsequent GluR2/3 AMPARs. Transmembrane AMPAR Interacting Proteins Are Required to Anchor AMPARs to PSD-95
By definition, the five members of the transmembrane AMPAR interacting protein (TARP) family (g2/stargazin, g3, g4, g7, and g8) bind AMPARs. This
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separates them from closely related proteins such as the g1 calcium channel subunit. Mutations in g2/stargazin lead to ataxia in waggler mice, which lack surface AMPARs on cerebellar granule cells. It has been suggested that TARPs constitute auxillary AMPAR subunits and that a type I PDZ domain at the Cterminus of TARPs enables interaction with PSD-95, SAP-97, and other MAGUKs. TARPs are involved in both AMPAR surface expression and lateral movement of AMPARs in the membrane to the synapse via an interaction with PSD-95. Whereas stargazin/ g2 is highly expressed in the cerebellum, g8 is most highly expressed in the hippocampus and is not mutated in the waggler mice; this explains why they show locomotor but not learning deficiencies. GRIP and ABP Anchor Short AMPAR Subunits
GRIP1 and ABP both contain a maximum of seven PDZ domains and interact with short AMPAR subunits through PDZ domains 4–5 and 3, 5, and 6, respectively. The number of PDZ domains and their ability to form homo- and heterodimers make GRIP1 and ABP ideal candidate scaffolds. Both proteins are enriched in dendritic spines and it is proposed that they anchor AMPARs at the synapse. Ser880Ala mutation in the GluR2 PDZ domain selectively prevents its interaction with GRIP/ABP, leading to a reduction in synaptic accumulation of GluR2 over time. Synaptic targeting was not affected, suggesting GRIP/ABP is specifically required for the synaptic retention of AMPARs. Thus, GRIP and ABP possibly work in tandem with PSD-95 to provide slot proteins for AMPAR insertion and retention at the synapse. Neuronal Activity Regulated Pentraxin-Induced Clustering of AMPARs
The N-terminus of GluR1 is required for ER exit and synaptic targeting. Neuronal activity regulated pentraxin (NARP), an immediate-early gene released from the stimulated hippocampus, interacts at an extracellular site causing the clustering of GluR1–3 in heterologous cells and co-immunoprecipitates with these subunits, as well as clustering GluR1 in cultured spinal neurons. Endogenous NARP action varies between neurons. Hippocampal axons only secrete NARP at contacts with interneurons, and the exogenous application of NARP fails to cause clustering on pyramidal cells. Spinal neurons cluster AMPARs and NMDARs on cultured hippocampal interneurons where contacts are made on the dendritic shaft but not on pyramidal cells where contacts are made at dendritic spines.
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AMPAR Endocytosis The best understood model for internalizing proteins in neurons is clathrin-mediated endocytosis (CME). Hypertonic sucrose inhibits CME and prevents both agonist-induced GluR1 internalization and insulininduced GluR2 internalization. Blocking dynamin activity prevents the internalization of GluR1 and GluR2 and blocks LTD. A peptide corresponding to the SH3 domain of amphiphysin also blocks LTD. Disruptions to specific components of the CME machinery (e.g., CPG2 and huntingtin interacting protein 1 (HIP1)) also caused reduced AMPAR internalization. Increased colocalization and interaction of AP2 with AMPARs following agonist treatment suggested AP2 recruits AMPARs to coated pits. This is supported by evidence that the proteins directly interact through the b-adaptin subunit of AP2. This interaction does not occur with the AP1 subunit responsible for cargo recruitment at intracellular sites. It is not clear if non-CME AMPAR endocytosis pathways exist, although lipid rafts, often associated with caveolar structures, are involved in stabilizing surface AMPARs. Lateral Diffusion to Extrasynaptic Clathrin-Mediated Endocytosis Sites
The development of ecliptic GFP or pHlourins, which fluoresce only at pH > 7.0 allowed the measurement of dynamic changes to the surface expression of proteins. Because intracellular compartments are more acidic than the extracellular environment, super eclipitic phluorin (SEP) fluorescence is suppressed until the protein is surface expressed. This technology was used to show that, in response to NMDAR activation, endocytosis of synaptic GluR2 is preceded by the removal of extrasynaptic receptors (Figure 2). An obvious reason for this is that the endocytosis machinery is located at extrasynaptic sites. Quantum dot-tracking experiments show that the proportion of mobile GluR2 increases following neuronal activity, representative of increased lateral diffusion from synaptic anchors. This suggests that, rather than the regulation of endocytosis itself, the limiting step in generating LTD through internalization is the release of AMPARs from the PSD. Protein Interacting with C Kinase 1 Releases AMPARs from the Synapse
Protein interacting with C kinase (PICK1) interacts with a number of neuronal receptors, channels, and transporters. A single PDZ domain enables an interaction with short AMPARs leading to LTD. This occurs because PICK1 competes for the GRIP/ABP binding site on GluR2, uncoupling the AMPARs
from the synaptic scaffold. This is controlled by protein kinase C (PKC) phosphorylation of GluR2 at Ser880, which prevents GRIP/ABP but not PICK1 binding. Phosphorylation is facilitated by PICK1 itself, which binds PKCa to target the kinase to AMPARs. PKCa binding removes an intramolecular interaction, exposing the BAR domain of PICK1. A second phosphorylation switch with the same function has recently been found at Tyr876, and this residue must be Src phosphorylated in response to drug treatment for internalization to occur. NSF hydrolysis of adenosine triphosphate (ATP) disrupts the PICK1–GluR2 interaction in complexes containing a-SNAP. This stabilizes the AMPARs at the surface. b-SNAP, however, prevents this dissociation and causes receptor internalization. Thus, in addition to actively inserting AMPARs at the synapse, NSF activity prevents their internalization. The presence of a BAR domain in PICK1 suggests this protein can recruit AMPARs to clathrin-coated vesicles (CCVs) in constitutive endocytosis in which AP2 does not seem to be involved. Ca2þ binding to PICK1 increases the affinity of the GluR2 interaction. This has obvious implications for LTD, in which Ca2þ concentration is elevated in spines. The increase in affinity may be another switch in the balance between constitutive cycling and longlasting endocytosis of AMPARs. PICK1 is not the only Ca2þ-sensing molecule implicated in the endocytosis of AMPARs during LTD. The neuronal calcium-sensor hippocalcin binds AP2 in a Ca2þdependent manner, and the Ca2þ-sensing region of hippocalcin is required for the generation of LTD. It is thought that Ca2þ binding, which exposes a myristyl tail in hippocalcin, recruits AP2 to the membrane, enabling AMPAR sorting into CCVs. Hippocalcin also complexes with transferrin receptors but in a Ca2þ-independent manner, suggesting the function of this protein is modified for a specific role in AMPAR endocytosis. Degradation of Synaptic Scaffolds Accompanies AMPAR Internalization
PSD-95 is ubiquitinated following synaptic NMDA activation. Truncated PSD-95 mutants lacking the ubiquitination motif prevent NMDA-induced GluR2 internalization. One function of ubiquitination is as a signal for proteasomal protein degradation, suggesting that the synaptic scaffold may have to be dismantled for AMPAR release. Although this has not yet been shown for the proteasome pathway, calpain cleavage of PSD-95, SAP-97, and GRIP1 has been shown. AMPARs are themselves cleaved at the C-terminus by calpain, allowing their release from the PSD.
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Figure 2 AMPAR internalization and recycling: (a) experiments using recombinant HA-tagged AMPAR subunits; (b) virally expressed pHluorin-GluR2 labeling resting, with NMDA, and after NMDA; (c) main steps in AMPAR recycling. In (a), experiments using recombinant HA-tagged AMPAR subunits demonstrate that they are differentially sorted on internalization. All HA-tagged AMPAR subunits are internalized following the application of 50 mM NMDA for 8 min. All subunits colocalize with the early endosome marker EEA1 after 10 min, but only GluR1 continues to accumulate in this compartment up to 30 min following NMDA treatment. Both GluR2 and GluR3 pass through the syntaxin 13-positive recycling endosome and accumulate in the Lamp1-positive late endosome prior to degradation, whereas GluR1 remains in the recycling endosome. In (b) under resting conditions virally expressed pHluorin-GluR2 labels both punctate spines (red, purple) and diffuse shaft regions (blue). Treatment with 50 mM NMDA causes an immediate loss of the diffuse staining, representative of GluR2 endocytosis. This is followed by the loss of punctate staining after approximately 10 min. (c) Shows a schematic of the main events in AMPAR recycling: (1) AMPARs are released from the PSD by switching binding partners accompanied by degradation of the PSD; (2) receptors exit the spine by lateral diffusion to sites of clathrin endocytic machinery; (3) clathrin-mediated exocytosis transports AMPARs to the early endosome; (4) under all conditions, GluR3 is sent to the late endosome prior to degradation, and GluR2 enters this pathway after NMDA treatment but is sent to the recycling endosome following AMPA treatment; (5) GluR1 constitutively enters the recycling endosome, but its rate of exit from this compartment is increased by NMDA; (6) GluR2 exit from the recycling endosome is promoted by an interaction with NEEP21; (7) GluR2 is rapidly recycled to the synapse following AMPA treatment or in the absence of activity. AMPA, a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid; E, early endosome; EEA1, early endosome marker; GluR, glutamate receptor; HA, peptide derived from human influenza haemagglutinin; L, late endosome; Lamp1, lysosome associated membrane protein 1; NEEP21, neuron-enriched endosomal protein of 21 kDa; NMDA, N-methyl-D-aspartate; PSD, postsynaptic density protein; R, recyclingendosome. (a) from Lee SH, Simonetta A, and Sheng M (2004) Subunit rules governing the sorting of internalized AMPA receptors in hippocampal neurons. Neuron 43: 221–236. (b) Adapted from Ashby MC, De La Rue SA, Ralph GS, Uney J, Collingridge GL, and Henley JM (2004) Removal of AMPA receptors (AMPARs) from synapses is preceded by transient endocytosis of extrasynaptic AMPARs. Journal of Neuroscience 24: 5172–5176.
Small GTPase Signaling Triggers AMPAR Endocytosis in LTD
NMDAR activation of the Rap1–p38MAPK pathway via the NR2B subunit causes internalization of GluR2/3 heteromers. Activation of the Rap2–c-Jun N-terminal kinase (JNK) pathway causes the internalization of GluR1/2 heteromers in depotentiation, which is the resetting of synapses that have undergone LTP back to the baseline. Rab5 overexpression specifically depresses AMPAR-mediated excitatory postsynaptic currents (EPSCs) through the removal of GluR1–3 from dendritic spine surfaces. A dominant negative mutant of Rab5 prevents LTD induction.
Rab5 activation is likely to occur downstream of p38MAPK activation because this regulates its interaction with Rab-GDI. As would be expected from the localization of Rab5 to endosomes, these effects of overexpression occur downstream of Ser880 phosphorylation and release from the PSD. Fate of Internalized AMPARs Is Subunit Specific
GluR1 internalizes rapidly and independently of activity, supporting the LTP model in which GluR1containing subunits are inserted and then replaced by GluR2/3 heteromers. Overexpressed GluR2 and GluR3 are internalized on agonist treatment, leading
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to GluR2 being recycled, but NMDAR activation targets GluR2 to lysosomes. GluR1 is recycled and GluR3 is directed to endosomes independent of drug treatment (Figure 2). These subunit rules appear to be determined by the NSF binding site, with GluR2 being dominant over GluR1. NSF may promote the recycling of the receptors, and this further supports a model in which NSF activity or binding to GluR2 is reduced during LTD, allowing targeting to lysosomes. Overall, these data suggest that NMDAR activation during LTD leads to the degradation of GluR2-containing receptors, consistent with an activity-dependent depletion of AMPARs at the synapse. However, exposure to the sodium-channel blocker tetrodotoxin (TTX), which prevents action potentials and therefore evoked presynaptic glutamate release, reverses the effects of NMDA and AMPA. The physiological relevance of this is not clear, but it does highlight the importance of the prior experience of the synapse in determining the effect of plasticity protocols. It has been suggested that this might represent a form of homeostatic plasticity known as synaptic scaling, in which neuronal sensitivity is altered in line with the overall sensitivity of the network. TTX-treated neuronal cultures have been used to show the role of proteins other than NSF involved in directing AMPARs through the endocytic pathway. Suppression of neuron-enriched endosomal protein of 21 kDa (NEEP21) reduces the rate of GluR1 recycling in response to NMDA. GluR2 internalizes to a NEEP21 positive compartment following NMDA treatment. The presence of Rab4 and syntaxin 13 in this compartment suggests that NEEP21 promotes trafficking to the recycling endosome. In neurons with suppressed NEEP21, AMPAR-mediated EPSCs are decreased, but this effect can be overridden with dynamin mutants to block endocytosis. Because LTP is blocked in the absence of NEEP21, this protein may also have a role in trafficking receptors from intracellular holding pools. The forward trafficking of GluR2 from the early endosome to the recycling endosome is mediated by the formation of a GluR2– GRIP1–NEEP21 complex. Although it is assumed that an as yet unidentified GluR1–NEEP21 complex interacting protein mediates the recycling of GluR1, the lack or low availability of such a protein may explain the retarded recycling of GluR1 to the cell surface.
Summary The mechanisms of AMPAR trafficking, which in turn influence synaptic efficacy, are complex. However, overall patterns of AMPAR forward traffic,
recruitment, and cycling have been established. The insertion of AMPARs at newly formed synapses and at synapses that have received a signal to undergo LTP depends on GluR1. Insertion of these GluR1containing AMPARs is preceded and/or accompanied by an upregulation of slot proteins at the synapse that hold the AMPARs at the PSD until such time as they are replaced by constitutively cycling GluR2/3containing AMPARs. During LTD, AMPARs are released from the synapse by alterations to their scaffold binding partners, which are then degraded. AMPARs are then endocytosed at sites separate from the PSD. The fate of the receptors endocytosed by LTD appears to differ. GluR2/3-containing receptors are degraded, whereas GluR1-containing receptors are slowly recycled to extrasynaptic sites. Although this model can explain many of the features of plasticity, clearly a great deal of work remains to be done to obtain a full mechanistic understanding of how AMPARs are regulated. Given the crucial role of these receptors in brain function and dysfunction, we believe that gaining this information is a goal of fundamental importance. See also: AMPA Receptors: Molecular Biology and Pharmacology; NMDA Receptor Function and Physiological Modulation; NMDA Receptors, Cell Biology and Trafficking; Transporter Proteins in Neurons and Glia.
Further Reading Ashby MC, De La Rue SA, Ralph GS, Uney J, Collingridge GL, and Henley JM (2004) Removal of AMPA receptors (AMPARs) from synapses is preceded by transient endocytosis of extrasynaptic AMPARs. Journal of Neuroscience 24: 5172–5176. Ashby MC, Ibaraki K, and Henley JM (2004) It’s green outside: Tracking cell surface proteins with pH-sensitive GFP. Trends in Neuroscience 27: 257–261. Bredt DS and Nicoll RA (2003) AMPA receptor trafficking at excitatory synapses. Neuron 40: 361–379. Choquet D and Triller A (2003) The role of receptor diffusion in the organization of the postsynaptic membrane. Nature Reviews Neuroscience 4: 251–265. Collingridge GL, Isaac JT, and Wang YT (2004) Receptor trafficking and synaptic plasticity. Nature Reviews Neuroscience 5: 952–962. Dingledine R, Borges K, Bowie D, and Traynelis SF (1999) The glutamate receptor ion channels. Pharmacological Reviews 51: 7–61. Gerges NZ, Backos DS, Rupasinghe CN, Spaller MR, and Esteban JA (2006) Dual role of the exocyst in AMPA receptor targeting and insertion into the postsynaptic membrane. EMBO Journal 25: 1623–1634. Horton AC and Ehlers MD (2003) Neuronal polarity and trafficking. Neuron 40: 277–295. Lee SH, Simonetta A, and Sheng M (2004) Subunit rules governing the sorting of internalized AMPA receptors in hippocampal neurons. Neuron 43: 221–236.
AMPA Receptor Cell Biology/Trafficking Malenka RC and Bear MF (2004) LTP and LTD: An embarrassment of riches. Neuron 44: 5–21. Palmer CL, Cotton L, and Henley JM (2005) The molecular pharmacology and cell biology of alpha-amino-3-hydroxy-5-methyl4-isoxazolepropionic acid receptors. Pharmacological Reviews 57: 253–277.
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Perestenko PV and Henley JM (2003) Characterisation of the intracellular transport of GluR1 and GluR2 a-amino-3hydroxy-5-methyl-4-isoxazole propionic acid receptor subunits in hippocampal neurons. Journal of Biological Chemistry 278: 43525–43532.
NMDA Receptor Function and Physiological Modulation K Zito, University of California at Davis, Davis, CA, USA V Scheuss, Max-Planck-Institute for Neurobiology, Martinsried, Germany ã 2009 Elsevier Ltd. All rights reserved.
Introduction With key roles in essential brain functions ranging from the basics of excitatory neurotransmission to the complexities of learning and memory, the N-methyl-D-aspartate (NMDA) receptor can be considered one of the fundamental neurotransmitter receptors in the brain. Named for its most potent exogenous agonist, the NMDA receptor has been thoroughly characterized via electrophysiological and pharmacological techniques, as well as through molecular manipulations and transgenic knockout strategies. The NMDA receptor belongs to a family of ionotropic receptors for the excitatory amino acid glutamate and is characterized by high affinity for glutamate, a high unitary conductance, high calcium permeability, and a voltage-dependent block by magnesium ions. this article focuses on the basic biophysical properties and physiological functions of NMDA receptors and how these are modulated by various signaling molecules and biochemical cascades under physiological conditions.
Biophysical Properties of NMDA Receptors During excitatory neurotransmission, presynaptic release of glutamate activates glutamate receptors in the postsynaptic membrane, resulting in the generation of an excitatory postsynaptic potential (EPSP). Contributing to the EPSP are two classes of glutamate receptors, the non-NMDA receptors (a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/kainate receptors) and the NMDA receptors (Figure 1). The rise times for NMDA receptor currents (10–50 ms) are much slower than those of non-NMDA receptors (0.2–0.4 ms). NMDA receptors also deactivate with a slower time course (50–500 ms vs. 2 ms for non-NMDA glutamate receptors), much longer than the time course of glutamate in most synaptic clefts (1.2 ms). Therefore, during synaptic transmission, the non-NMDA glutamate receptors provide rapid depolarization in response to neurotransmitter release, and the NMDA receptor kinetics determine the duration of the synaptic current. Activation of NMDA receptors by the neurotransmitter glutamate requires glycine as an essential
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coagonist. Glutamate and glycine molecules bind different subunits of the receptor; two of each are thought to be required for maximum activation of the receptor. Recent experiments have shown that D-serine can also bind at the glycine site and might represent another physiological coagonist. Compared with glutamate receptors of the non-NMDA type, which have low-affinity binding sites for glutamate (EC50 500 mM), NMDA receptors bind glutamate with high affinity (EC50 1 mmol l1). Despite this high affinity for glutamate, NMDA receptors are not saturated during synaptic transmission at synapses of cortical pyramidal neurons. NMDA receptor activation leads to opening of an ion channel that is selective for cations, resulting in the influx of Naþ and Ca2þ ions and efflux of Kþ ions. Although most glutamate receptors are cation selective, few are permeable to calcium ions. Its exceptional calcium permeability is the first of two key properties of the NMDA receptor that form the basis for its regulatory role in synaptic plasticity. Entry of calcium into the postsynapse via the NMDA receptor (Figure 2) permits coupling of electrical synaptic activity to biochemical signaling via activation of Ca2þ-dependent enzymes and downstream signaling pathways. In this way, calcium influx through the NMDA receptor can lead to long-term changes in synaptic strength and other cellular modifications, including alterations in synaptic structure or connectivity. The NMDA receptor has a high single channel conductance (30–50 pS) compared with that of other glutamate receptor types (4–15 pS). The open probability of agonist-bound receptors has been estimated to range between 0.04 and 0.3, and open times can vary from 0.1 to 8 ms. The molecular events that underlie the opening of NMDA receptors are predicted to include two independent agonist-binding steps preceding a single, concerted conformational change that results in channel opening. Channel closing is thought to be controlled both by the unbinding rate of glutamate and by receptor desensitization. In fact, NMDA receptors can enter long-lived desensitized conformations in which glutamate is bound but the channel remains closed. The second key biophysical property of the NMDA receptor, and the one that bestows its proposed role in learning and memory, is that it is blocked by magnesium ions in a voltage-dependent manner. At resting membrane potential, NMDA receptors are blocked by magnesium ions; however, if excitation by synaptic inputs causes sufficient depolarization of the neuron, the Mg2þ block is relieved and those NMDA receptors which have glutamate bound will open (Figure 3).
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Figure 1 NMDA receptor component of excitatory postsynaptic potentials (EPSPs) and excitatory postsynaptic currents (EPSCs). (a) Unitary EPSP (‘EPSC’) recorded in a L2/3 cortical pyramidal neuron. The NMDA receptor-mediated component (subtraction) was obtained by subtracting the non-NMDA receptor-mediated component (a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPAR) component) when NMDA receptors were blocked with the NMDA receptor antagonist AP5. (b) Unitary EPSC (‘EPSC’) recorded in a L2/3 cortical pyramidal neuron. The NMDA receptor-mediated component (subtraction) and the non-NMDA receptor mediated component (AMPAR component) were determined as in (a). Reproduced from Blackwell Publishing: figure 6A and B in Feldmeyer D, Lu¨bke J, Silver RA, et al. (2002) Synaptic connections between layer 4 spiny neuron-layer 2/3 pyramidal cell pairs in juvenile rat barrel cortex: Physiology and anatomy of interlaminar signaling within a cortical column. Journal of Physiology 538(3): 803–822.
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Figure 2 Synaptic activation of NMDA receptors causes calcium entry into spines. (a) Two-photon fluorescence image of a dendritic branch of a hippocampal CA1 pyramidal neuron loaded with the calcium indicator Oregon Green BAPTA. Scale bar ¼ 1 mm. (b) Sequences of line scans in which the vertical dimension corresponds to the line indicated in (a) and the horizontal dimension represents time. Below: Relative change in fluorescence (DF/F(t)) at the bottom spine (averaged over window indicated by bracket) during calcium entry evoked by synaptic stimulation (time indicated by arrowhead). The neuron was voltage clamped at positive potential to isolate NMDA receptor-mediated Ca2þ influx. Scale bars ¼ 250 ms, 50% DF/Fmax. (c) Average EPSC (bottom) and corresponding DF/F(t) (top) at the same spine as in (b). Scale bars ¼ 25% DF/Fmax, 100 pA, 25 ms. Reprinted by permission from MacMillan Publishers Ltd.: Nature (Mainen ZF, Malinow R, and Svoboda K (1999) Synaptic calcium transients in single spines indicate that NMDA receptors are not saturated. Nature 399: 151–155) copyright 1999.
Since opening of the NMDA receptor requires simultaneous activation by glutamate and depolarization to relieve the magnesium block, the NMDA receptor can act as a coincidence detector for pre- and postsynaptic activity. Glutamate and glycine affinities, calcium permeability, ion conductance, channel kinetics, and sensitivity to Mg2þ block of the NMDA receptor are all determined in part molecularly, by alternative splicing and subunit composition. Most of these biophysical properties of NMDA receptors can also be modulated by various ionic and molecular interactions and signaling pathways (described below).
NMDA Receptor Physiological Function The outstanding physiological function of NMDA receptors is the coupling of electrical to biochemical signaling in neurons by mediating calcium influx in response to synaptic activity. However, NMDA receptors also serve important functions in electrical neurotransmission alone. Despite the voltage-dependent magnesium block, NMDA receptors can contribute significantly to the amplitude of unitary evoked postsynaptic potentials. The slow kinetics of NMDA receptor-mediated excitatory postsynaptic currents (EPSCs) facilitates temporal summation and reduces
278 NMDA Receptor Function and Physiological Modulation Control Mg-free
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Figure 3 Magnesium block of NMDA receptors. (a) Voltage dependence of glutamate-induced currents in Mg2þ-free (circles) and Mg2þ-containing solution (500 mM; squares). (b) Voltage and Mg2þ-dependence of NMDA receptor current noise. Reprinted by permission from MacMillan Publishers Ltd: Nature (Nowak L, Bregestovski P, Ascher P, et al. (1984) Magnesium gates glutamate-activated channels in mouse central neurons. Nature 307: 462–465) copyright 1984. (c) Schematic of the mechanism of the magnesium block. At resting potential, the pore of the NMDA receptor channel is blocked by magnesium ions. Upon depolarization, the magnesium ions are removed from the pore, and the channel can pass current.
dendritic filtering of synaptic inputs. In addition, coactivation of multiple synapses can trigger NMDA receptor-dependent dendritic spikes by generating sufficient depolarization to overcome the magnesium block. Such dendritic spikes cause nonlinear synaptic integration, which enhances the computational power of neurons and may allow neurons to detect the synchrony of inputs (Figure 4). The NMDA receptor plays an essential role in brain plasticity, that is, the ability of the brain to change in response to external stimuli, such as during learning and memory. First investigated pharmacologically, chronic blockade of the NMDA receptor by
infusion of an antagonist into the ventricles of rats was shown to impair spatial learning. Since that time, similar behavioral experiments have been performed using knockout mice lacking NMDA receptor subunits from specific subregions of the hippocampus. These experiments have repeatedly provided evidence for the importance of the NMDA receptor in learning and memory processes. The role of the NMDA receptor in learning and memory is attributed to its ability to regulate excitatory synaptic transmission. The NMDA receptor has been shown to play an essential role in both the strengthening of synapses, through long-term
NMDA Receptor Function and Physiological Modulation 279 Paired-pulse stimulation Control APV Individual EPSP Arithmetic sum Combined EPSP
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Figure 4 NMDA receptors in nonlinear synaptic integration. (a) Somatic voltage responses of a layer 5 pyramidal neuron to stimuli from two electrodes (A and B) placed 30 mm apart on a single dendritic branch display nonlinear summation (left panel). With application of the NMDA receptor antagonist, APV, the summation is linear. EPSP, excitatory postsynaptic potential. (b) Expected vs. actual peak somatic responses plotted for different stimulus intensities before (black) and after (blue) application of APV. NMDA receptor blockade linearizes synaptic summation. Reprinted by permission from MacMillan Publishers Ltd: Nature Neuroscience (Polsky A, Mel BW, and Schiller J (1994) Computational subunits in thin dendrites of pyramidal cells. Nature Neuroscience 7: 621–627), copyright 1994.
potentiation (LTP), and the weakening of synapses, through long-term depression (LTD). LTP and LTD are proposed to be cellular mechanisms underlying learning and memory. Via its role as a coincidence detector, the NMDA receptor is capable of signaling coincident pre- and postsynaptic activity. Detailed studies of the relationship between the temporal correlation of pre- and postsynaptic activity and the resulting type of plasticity have led to the discovery of spike-timing dependent plasticity. If the presynaptic action potential (AP; and thus synaptic activity) repetitively precedes the generation of the postsynaptic AP by less than 50 ms, the synapse is potentiated; however, if the postsynaptic AP occurs within 50 ms before the presynaptic AP (and thus independently of presynaptic activity), the synapse is depressed (Figure 5). With respect to the more complex and irregular patterns of neuronal activity that are likely found in vivo, it has been suggested that both rate and timing determine the direction of neuronal plasticity.
Figure 5 Spike-timing-dependent synaptic plasticity. Change in excitatory postsynaptic potential (EPSP) slope induced by repetitively paired pre- and postsynaptic spikes in layer 2/3 of rat visual cortex. Insets depict the sequence of spiking. Pre before postsynaptic spiking leads to potentiation while post before presynaptic spiking leads to depression. Used from The American Physiological Society: figure 1 in Dan Y and Poo MM (2006) Spike timingdependent plasticity: From synapse to perception. Physiological Reviews 86: 1033–1048.
The NMDA receptor also acts as a coincidence detector during development, where it plays a critical role in the maturation of synapses and the activitydependent establishment of topographic maps in the brain. By acting to strengthen neighboring connections of similar activity patterns, the NMDA receptor enforces the principle that ‘cells that fire together, wire together.’ In addition, early in development, many excitatory synapses appear to contain only NMDA receptors, and therefore these synapses are thought to be at first silent (no EPSP in response to synaptic stimulation). It is hypothesized that only through coincident activity sufficiently robust to depolarize the postsynaptic cell and relieve the voltage-dependent Mg2þ block of the NMDA receptors does the insertion of non-NMDA receptors at these synapses occur. While presynaptic NMDA receptors have been detected by immuno-electron microscopy, their function remains far less established compared with their postsynaptic counterparts. As autoreceptors, presynaptic NMDA receptors have been shown to modulate synaptic transmission in the spinal cord, cerebellum, and entorhinal cortex and to play a role in cerebellar and cortical LTD. Calcium influx through the NMDA receptor is thought to be responsible for its roles in LTP and LTD and for both neuroprotective and neurotoxic effects. Although these various effects may at first seem contradictory, the explanation lies in the spatial
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and temporal patterns of calcium influx. Calcium influx at synaptic sites is thought to activate signaling pathways that lead to LTP or LTD, depending on the temporal pattern and amplitude of the calcium transient. Synaptic calcium influx also leads to activation of cyclic adenosine monophosphate response element binding protein (CREB), a transcription factor, resulting in activation of gene expression. One of the genes upregulated in response to activated CREB is brainderived neurotrophic factor (BDNF), which acts to promote prosurvival programs. In contrast, calcium influx through extrasynaptic NMDA receptors is thought to lead to CREB deactivation and inhibition of BDNF expression, which can have deleterious effects on cell health. In fact, overstimulation of NMDA receptors results in neuronal excitotoxicity, which has been implicated in the loss of neurons associated with ischemic stroke; Alzheimer’s, Parkinson’s, and Huntington’s disease; and amyotrophic lateral sclerosis.
Desensitization
As the NMDA receptor is a ligand-gated ion channel, it can display a decrease in conductance despite the continuous presence of agonist, a process referred to as desensitization. Three different types of desensitization have been reported for NMDA receptors, a glycine-sensitive, a glycine-insensitive, and a calciumdependent type. The glycine-sensitive desensitization refers to the transition of the NMDA receptor into a glutamate-bound closed state, reflecting a negative allosteric interaction between the glutamate and the glycine binding sites, which results in a reduced glycine affinity in the presence of high glutamate concentrations. High glycine concentrations can overcome this type of desensitization. Glycineinsensitive desensitization has been observed only in dialyzed cells and excised membrane patches and thus does not appear to be physiologically relevant. The calcium-dependent desensitization (also referred to as calcium-dependent inactivation) provides a negative feedback loop by which calcium entering the cell via NMDA receptors in turn leads to the desensitization of the receptor (Figure 6), although calcium from other sources (voltage-gated calcium channels or release from intracellular stores activated by second messenger cascades) has the same effect. In a current model, calcium influx activates calmodulin to displace a-actinin2 from the NMDA receptor. The resulting dissociation of the NMDA receptor from the cytoskeleton is proposed to cause a conformational change in the receptor, which reduces its open probability.
Physiological Modulation of NMDA Receptors Although glycine and/or D-serine are essential coagonists of the NMDA receptor, and thus, strictly speaking, not modulators, their cytosolic levels nevertheless regulate NMDA receptor activation. One of two amino acids appears to predominate, dependent on the brain region. The cytosolic concentrations are controlled by release and uptake by both neurons and glia. Stimulated release of glycine and D-serine can be induced by depolarization or non-NMDA glutamate receptor activation. In the spinal cord, glycine spillover from inhibitory synapses has been shown to enhance NMDA receptor currents.
Endogenous Allosteric Modulators
NMDA receptor function is fine-tuned by various forms of allosteric modulation involving endogenous extracellular substances such as zinc ions, protons, polyamines,
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Figure 6 Calcium-dependent desensitization of NMDA receptors. Whole cell currents of cultured rat hippocampal neurons (left and right panels) were evoked in low calcium (0.5 mM) solutions by application of NMDA. During a train of seven excitatory postsynaptic currents (EPSCs) evoked in 2.7 mM calcium, the NMDA receptor mediated EPSC was inactivated to 50% of control (middle). The whole cell current was inactivated to a similar degree and recovered within 4 min. Used from The American Physiological Society: figure 3 in Rosenmund C, Feltz A, and Westbrook GL (1995) Calcium-dependent inactivation of synaptic NMDA receptors in hippocampal neurons. Journal of Neurophysiology 73: 427–430.
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and reducing and oxidizing agents (Figure 7). Zinc and polyamines mediate a voltage-dependent block of NMDA receptors, which is weaker but appears to involve the same intrapore residues as block by magnesium. Some of the voltage-independent effects of allosteric modulators are mutually interdependent, and structural evidence suggests convergence by overlapping binding sites or common downstream structural modifications. Zinc, protons, and oxidizing agents inhibit NMDA receptor function, while polyamines and reducing agents cause potentiation by modifying channel open frequency and time or agonist or modulator affinities. These modulations are considered to be physiologically relevant during synaptic transmission, when synaptic activity can cause shifts in pH, co-release of Zn2þ occurs at certain synaptic terminals (e.g., mossy fiber-CA3), and the amine histamine is released from modulatory afferents (e.g., those arising from the anterior hypothalamus). An important aspect of the negative regulation of NMDA receptor function by pH and oxidizing agents is that it can limit or delay cell damage under pathological conditions such as stroke and ischemia. Phosphorylation
NMDA receptors can be phosphorylated by the serine/ threonine kinases protein kinase C (PKC), protein kinase A (PKA) and calcium/calmodulin-dependent protein kinase II (CaMKII), as well as by the tyrosine kinases Src and Fyn. In general, phosphorylation enhances NMDA receptor function (Figure 7).
A significant percentage of NMDA receptor subunits in the brain are estimated to be phosphorylated by PKC or PKA at one or more sites (10–70%, depending on the region of the brain). Phosphorylation by PKC reduces the affinity for extracellular magnesium ions and increases the open probability. Calcium influx through the NMDA receptor itself can enhance the potentiation mediated by PKC. However, in some preparations, phosphorylation by PKC reduced NMDA receptor-mediated responses by preventing NMDA receptor subunit clustering. Less is known about the regulation of NMDA receptors by PKA and CaMKII. Active CaMKII directly associates with the NMDA receptor, and this association is required for some forms of activity-driven synaptic potentiation. PKA activation has been shown to increase the fractional Ca2þ influx through the NMDA receptor; however, some of the effects of PKA activation on NMDA function appear to be indirect. In contrast, fewer NMDA receptor subunits on neural membranes are phosphorylated on tyrosine residues (2–4%). Phosphorylation by Src enhances NMDA receptor function (Figure 8) by reducing the potency of Zn2þ block of recombinant NMDA receptors expressed heterologously; however, evidence is lacking for this mechanism in hippocampal or spinal cord neurons. An alternative view proposes that phosphorylation of NMDA receptor subunits by Src-family kinases could affect downstream signaling proteins, either by recruiting them to the NMDA receptor, by
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Figure 7 Schematic overview of modulators of NMDA receptor functions. Activation of the NMDA receptor requires binding of glutamate (Glu) and glycine (or serine) together with membrane depolarization to release the magnesium block (center). Modulators causing depression of NMDA receptor currents (left) include Hþ, Zn2þ, polyamines, reducing agents, and the protein phosphatases PP1, PP2A, and PP2B/calcineurin. Polyamines and Zn2þ cause a voltage-dependent block similar to that caused by Mg2þ. Modulators causing potentiation of NMDA receptor currents (right) include external polyamines, oxidizing agents, and the protein kinases PKC, PKA, CaMKII, Src, and Fyn. For details see text.
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protecting them against degradation, or through blocking the assembly of signaling complexes. Srcmediated phosphorylation appears to play a role in LTP induction and seems to be modulated by BDNF. Phosphorylation by Fyn may target the NMDA receptor to the plasma membrane by antagonizing its interaction with spectrin. In some cases, Src activity is required for PKCmediated activation of NMDA receptor currents, placing Src downstream of PKC on the same pathway. PKC stimulation leads to the activation of the nonreceptor protein tyrosine kinase, cell adhesion kinase-b, which activates Src by disrupting the intramolecular interactions that maintain Src in a low-activity state. Activation of Src then leads to the potentiation of NMDA receptor currents. The effects of Src on NMDA receptors can be modulated by H-Ras, a small guanosine triphosphate (GTP)-binding protein, which binds Src and inhibits its activity. Hippocampal pyramidal neurons from H-ras homozygous null mice displayed enhanced NMDA receptor synaptic responses. The activation of NMDA receptors by phosphorylation can be reversed by serine and threonine phosphatases 1, 2A, and 2B (calcineurin) and endogenous tyrosine phosphatases. Inhibition of endogenous protein tyrosine phosphatase activity led to potentiation of NMDA receptor currents, indicating that these phosphatases participate in determining the basal phosphorylation level of the NMDA receptor.
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Figure 8 The protein tyrosine kinase pp60c-src potentiates NMDA currents. Whole cell recordings were made from mouse cultured hippocampal neurons. Peak currents evoked by rapid application of NMDA (100 mM) are plotted in the graph on the left. The tyrosine kinase pp60c-src (30 U per milliliter) was actively perfused through the recording electrode. The period of the perfusion is indicated by the horizontal bar on the graph. On the right, representative currents recorded before (Control) and after (Src) intracellular perfusion with pp60c-src are shown. NMDA was applied as indicated by the trace above the currents. Reprinted by permission from MacMillan Publishers Ltd: Nature (Wang YT and Salter MW (1994) Regulation of NMDA receptors by tyrosine kinases and phosphatases. Nature 369: 233–235), copyright 1994.
Calcineurin can be activated by calcium influx through the NMDA receptor itself. Modulatory Signaling Receptors
Modulation of NMDA receptors by kinases and phosphatases occurs downstream of a number of membrane receptors, the largest group of which are the G-protein-coupled receptors (GPCRs). GPCRs are integral membrane proteins which transmit extracellular signals to the cytoplasm by coupling to heterotrimeric GTP-binding proteins. Activation of a subset of GPCRs, including metabotropic glutamate receptors (mGluRs), muscarinic acetylcholine receptors, m opioid receptors, lysophosphatidic acid (LPA) receptors, and the protease-activated receptor PAR1, enhances NMDA receptor currents. Those GPCRs that are coupled to Gq proteins, such as mGluR5, m1 muscarinic receptor, and the LPA receptor, are thought to act through a PKC-Src signaling pathway, and mGluR1 also activates Src, but through a PKCindependent pathway. A second set of GPCRs, those coupled through Gs, are thought to enhance activity of the NMDA receptor through activation of Src-family kinases via PKA and receptor for activated C kinase 1 (RACK1). One such GPCR, the pituitary adenylate cyclase activating polypeptide (PACAP) receptor, is activated by the neuropeptide PACAP(1–38), which has been shown to potentiate NMDA receptor-mediated excitatory postsynaptic field potentials in hippocampal slices. Finally, NMDA receptors have been shown to interact directly with G-protein-coupled dopamine receptors. Depending on the class of dopamine receptor, activation of dopamine receptors has been reported both to enhance and to inhibit NMDA receptor currents. NMDA receptor currents are also influenced by downstream signaling from receptor protein tyrosine kinases, including the EphB receptors, platelet-derived growth factor (PDGF) receptors, and insulin receptors. EphB receptors interact directly with NMDA receptors in cultured neurons, and their activation by ephrinB2 increases NMDA receptor-dependent calcium responses via the activation of Src. Insulin has been shown to enhance NMDA receptor activity in hippocampal slices via a mechanism that requires PKC and tyrosine kinase activity. In contrast, application of PDGF depresses NMDA receptor currents in hippocampal pyramidal neurons via a phospholipase C-IP3 receptor-cyclic AMP-PKA pathway that inhibits Src activity. Cytokine receptor activation has been shown to enhance NMDA receptor currents. One group of cytokines, leptins, enhances NMDA receptor-mediated EPSCs in hippocampal slices via activation of the leptin receptor Ob-Rb. The leptin-mediated potentiation was
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reduced by inhibitors of PI3-kinase, mitogen-activated protein kinase, and Src-family kinases. Another cytokine, interleukin (IL)-1b, enhanced the rise in intracellular calcium following application of NMDA to cultured hippocampal neurons, a response mediated by the IL-1RI receptor. NMDA Receptor Complex
The ability of modulators to influence NMDA receptor currents is dependent on their abundance and proximity to the receptor. Proteomic characterization identified synaptic NMDA receptors as part of a remarkably large macromolecular signaling complex called the NMDA receptor complex. NMDA receptors were found linked to receptors, adhesion molecules, scaffolding proteins, signaling molecules, cytoskeletal proteins, and various novel proteins, in complexes lacking non-NMDA receptors. Among the signaling proteins were kinases, phosphatases, GTPases, and GTPase-activating proteins. Physical linkage of these receptors, signaling molecules, and the cytoskeleton to the NMDA receptor is an important way to facilitate rapid modulation of the receptor in response to synaptic activity. See also: D-Serine: From its Synthesis in Glial Cell to its
Action on Synaptic Transmission and Plasticity; Kainate Receptor Functions; Long-Term Depression (LTD): Metabotropic Glutamate Receptor (mGluR) and NMDARDependent Forms; Long-Term Potentiation (LTP): NMDA Receptor Role; NMDA Receptors, Cell Biology and Trafficking.
Further Reading Cull-Candy SG and Leszkiewicz DN (2004) Role of distinct NMDA receptor subtypes at central synapses. Science’s STKE: Signal Transduction Knowledge Environment 255: re16.
Dan Y and Poo MM (2006) Spike timing-dependent plasticity: From synapse to perception. Physiological Reviews 86: 1033–1048. Dingledine R, Borges K, Bowie D, et al. (1999) The glutamate receptor ion channels. Pharmacological Reviews 51: 7–61. Edmonds B, Gibb AJ, and Colquhoun D (1995) Mechanisms of activation of glutamate receptors and the time course of excitatory synaptic currents. Annual Review of Physiology 57: 495–519. Feldmeyer D, Lu¨bke J, Silver RA, et al. (2002) Synaptic connections between layer 4 spiny neuron-layer 2/3 pyramidal cell pairs in juvenile rat barrel cortex: Physiology and anatomy of interlaminar signaling within a cortical column. Journal of Physiology 538(3): 803–822. Husi H, Ward MA, Choudhary JS, et al. (2000) Proteomic analysis of NMDA receptor-adhesion protein signaling complexes. Nature Neuroscience 3: 661–669. Kotecha SA and MacDonald JF (2003) Signaling molecules and receptor transduction cascades that regulate NMDA receptormediated synaptic transmission. International Review of Neurobiology 54: 51–106. Lester RA and Jahr CE (1992) NMDA channel behavior depends on agonist affinity. Journal of Neuroscience 12: 635–643. Mainen ZF, Malinow R, and Svoboda K (1999) Synaptic calcium transients in single spines indicate that NMDA receptors are not saturated. Nature 399: 151–155. Nowak L, Bregestovski P, Ascher P, et al. (1984) Magnesium gates glutamate-activated channels in mouse central neurons. Nature 307: 462–465. Polsky A, Mel BW, and Schiller J (2004) Computational subunits in thin dendrites of pyramidal cells. Nature Neuroscience 7: 621–627. Rosenmund C, Feltz A, and Westbrook GL (1995) Calciumdependent inactivation of synaptic NMDA receptors in hippocampal neurons. Journal of Neurophysiology 73: 427–430. Salter MW and Kalia LV (2004) Src kinases: A hub for NMDA receptor regulation. Nature Reviews Neuroscience 5: 317–328. Schneggenburger R, Zhou Z, Konnerth A, et al. (1993) Fractional contribution of calcium to the cation current through glutamate receptor channels. Neuron 11: 133–143. Vanhoutte P and Bading H (2003) Opposing roles of synaptic and extrasynaptic NMDA receptors in neuronal calcium signalling and BDNF gene regulation. Current Opinion in Neurobiology 13: 366–371. Wang YT and Salter MW Regulation of NMDA receptors by tyrosine kinases and phosphatases. Nature 369: 233–235.
NMDA Receptors, Cell Biology and Trafficking R J Wenthold and R S Petralia, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, MD, USA Published by Elsevier Ltd.
Introduction The N-methyl-D-aspartate (NMDA) receptor is present at the postsynaptic membrane of nearly all glutamatergic synapses, where it is found with other glutamate receptors, particularly a-amino-3-hydroxy5-methyl-4-isoxazole propionic acid (AMPA) receptors. It is a key player in synaptic plasticity, and its malfunction has been implicated in a wide range of neurological and psychiatric disorders ranging from ischemia to schizophrenia. It is unique among neurotransmitter receptors since it requires activation by two agonists, glutamate and glycine, which interact with the NR2 and NR1 subunits, respectively. The ion channel of the NMDA receptor is normally blocked by magnesium, and the channel opens only after the neuron is depolarized by activation of AMPA receptors, thus allowing the NMDA receptor to serve as a coincidence detector. Finally, the ion channel of the NMDA receptor is permeable to calcium, a universal intracellular signaling molecule. These properties have made the NMDA receptor one of the most intensely studied molecules in the brain. Since neurons are highly compartmentalized, forming synaptic connections with thousands of other neurons, and the NMDA receptor has a restricted distribution within a neuron, trafficking of this receptor is central to its functional regulation. Here we define trafficking as the nonrandom movement of a protein within a neuron to, or from, a site where it is required for function. This process undoubtedly depends on interactions with other molecules, particularly other proteins, lipids, sugars, and small molecules.
Structure and Subunits Three subunits of the NMDA receptor complex have been identified. A single NR1 subunit has eight splice variants. There are four NR2 subunits (NR2A–D) and two NR3 subunits. NR2A and NR2B are the most abundant NR2 subunits, with NR2B being predominant early in development and NR2A appearing later, along with a general decrease in NR2B expression. All subunits have the same topology, with three transmembrane domains, a reentrant loop, an extracellular N-terminus, and a cytoplasmic C-terminus,
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which is unusually long in all NR2 subunits. Supporting several previous functional and biochemical reports, the recent determination of the crystal structure of the extracellular domains of the NMDA receptor indicates a tetrameric protein made up of two NR1 and two NR2 subunits. In a tetramer, the NR2 subunits can be the same, such as two NR2B subunits, or different, such as one NR2B and one NR2A, in this complex. While NR3 can also assemble into complexes with NR1 and NR2, the function of NR3 remains unclear, and it has been reported that NR3 can assemble with NR1 alone to produce a functional glycine receptor. The NMDA receptor subunits interact with a large number of other proteins, which modify their function and influence their trafficking (Table 1). The subunit composition determines the functional properties of the NMDA receptor. This is dependent mostly on the NR2 subunits forming receptors, with kinetics ranging from the slowest (containing NR2D) to the fastest (NR2A). Thus, channels with faster kinetics allow less calcium to enter the neuron, and would probably be less effective in developing plasticity. When NR3 is assembled into a complex with NR1 and NR2, it decreases channel function. NR3A knockout mice are normal, but show enhanced NMDA responses early in development.
NMDA Receptors in the Endoplasmic Reticulum All NR2 subunits and some NR1 subunits, depending on the splice variant, are retained in the endoplasmic reticulum (ER) in their unassembled form and are released after assembly (Figure 1). This is a standard mechanism of quality control to assure that unassembled or misfolded proteins do not reach the cell surface. For the NR1 subunit, ER retention is controlled by an RXR motif (X indicates any nonacidic amino acid) in the C-terminal domain of the major splice variant, NR1-1. NR1 splice variants (NR1-2 and NR1-4) that lack this retention motif are not retained, and others (NR1-3) that contain this motif plus the C20 cassette are also not retained. Thus, only the NR1-1 splice variant is retained in the ER; this is the major splice variant, accounting for about 60% of the total NR1. The C20 cassette causes the subunit to terminate in the amino acid sequence – STVV, and this motif is required for overriding the RXR retention motif. This is a postsynaptic density/disc/zonula occludens-1 (PDZ) binding motif and also a site of interaction for components of the coat protein complex II (COPII),
NMDA Receptors, Cell Biology and Trafficking Table 1 Proteins that bind directly to NR1 and/or NR2 NR1a
NR2
PSD-95 (to NR1-3, NR1-4) SAP102 (to NR1-3, NR1-4) PSD-93 (to NR1-3, NR1-4) SAP97 (to NR1-3, NR1-4) a-Actinin Tubulin Spectrin Myosin regulatory light chain Dopamine D1 receptor CaMKII NADH dehydrogenase subunit 2 (ND2)b Calmodulin Neurofilament-L Yotiao EphB receptors Apolipoprotein E receptor 2 SALM1
PSD-95 SAP102 PSD-93 SAP97 a-Actinin Tubulin Spectrin Myosin regulatory light chain
Sec23/24 of COPII
Dopamine D1 receptor CaMKII NADH dehydrogenase subunit 2 (ND2)b S-SCAM (¼ MAGI-2) CIPP mLin-7 (¼ Veli ¼ Mals) Phospholipase C-g Rack1 m subunit of adaptor protein complexes (AP1–4) a1-Chimerin RasGRF-1 Cyclin-dependent kinase-5 (cdk5)b
a
NR1-3 and NR1-4 are splice variants. cdk5 may bind directly to NR2A, and ND2 appears to bind Src to NMDA receptors, but the exact nature of these interactions has not been determined. b
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which is involved in ER-to-Golgi trafficking. Thus, the PDZ or COPII interaction, or both, may control the ER export of these NR1 splice variants and override the retention that is mediated by the RXR motif. It remains unclear why some NR1 splice variants can exit the ER unassembled, or if they actually do in neurons, since there is no evidence that NR1 homomeric complexes exist in neurons or serve any function. NR1 subunits lacking retention motifs may be more readily exported from the ER assembled with NR2. The mechanism by which NR2 subunits are retained in the ER is more complex than that of NR1 and not well characterized. There appears to be an ER retention signal(s) in the C-terminus although the specific site has not been identified. There is also an additional mechanism for ER retention since deletion of the C-terminus does not allow the remainder of the molecule to reach the cell surface unassembled, as is the case for NR1; however, this C-terminus-deleted construct can assemble with NR1 to produce functional receptors. Equally poorly understood is the mechanism by which the retention signals on NR1 and NR2 are negated to allow the assembled complex to exit the ER. Mutual masking of the retention motifs in the C-terminus, as has been demonstrated for some other proteins, does not
Large excess of NR1 NR1 NR2A NR2B PDZ protein
Rapid degradation (t1/2 = 1–2h) of unassembled NR1 subunits
NR1 splice variants NR1-2, NR1-3, and NR1-4 can exit the ER unassembled
Assembly
ER Exit. NR2A and NR2B require HLFY motif in C-terminus Early PDZ protein association
Figure 1 Assembly of the NMDA receptor in the ER. All NR2 subunits and the NR1-1 splice variant are retained in the endoplasmic reticulum (ER). Assembly negates retention and allows exit from the ER. NR1 subunits are synthesized in excess of NR2, and most are retained in the ER and rapidly degraded. In heterologous cells, some splice variants that lack the ER retention signal present in the C1 cassette, or contain the C20 cassette, can exit the ER unassembled, probably as homodimers. It is not known if this is also the case in neurons or if it is of functional significance. Assembly can lead to receptor complexes with two identical or two different NR2 subunits. Receptors can assemble and be functional in the ER based on MK801 binding, but export requires a four-amino-acid segment (HLFY in NR2A and NR2B) immediately following transmembrane domain 4.
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appear to play a role in the NMDA receptor, since subunits with a deletion of either the NR1 or NR2 C-terminal tails alone can form functional receptors. An interesting finding that further complicates our understanding of the ER retention of NR2 is that the NR2 subunit does not need to be intact to form functional receptors. The NR2A subunit can be divided into two polypeptides in the extracellular loop between transmembrane domains 3 and 4 (TM3 and TM4). Co-expression of these two fragments with NR1 produces functional receptors on the cell surface. A single neuron can contain a variety of NMDA receptor complexes (such as NR1/NR2B, NR1/ NR2A, and NR1/NR2A/NR2B), and the question arises whether or not the formation of these complexes simply reflects the availability of subunits, or is regulated in some way. There is a large excess of NR1 subunits synthesized, which would ensure that most NR2 subunits would find an NR1 binding partner and form a functional receptor. This fits with the fact that the particular NR2 subunit controls the properties of the receptor.
Trafficking of NMDA Receptors in the Dendrite NMDA receptors are concentrated at postsynaptic sites on dendrites while synthesis occurs predominantly in the cell body, requiring a mechanism to transport receptors from the cell body to synapses. This could be done in two ways. Receptors could be delivered to the plasma membrane at the cell body, and the receptors could reach the dendrite by lateral diffusion or through some form of regulated transport while they are in the plasma membrane. Alternatively, receptors can be delivered to dendritic sites through association with intracellular transport vesicles. The latter is assumed to be the major mechanism of delivery, but lateral movement to the synapse is probably also necessary for movement into the synapse after delivery to an extrasynaptic location (Figure 2). NMDA receptors can associate early in the biosynthetic pathway with PDZ proteins, including members of the PSD-95 family of membrane-associated guanylate kinases (MAGUKs), which are transported in the dendrite as a complex with the NMDA receptors. The MAGUK allows the indirect association of the NMDA receptors with multiple other proteins that can influence the trafficking of the NMDA receptor. The exocyst, a complex of eight proteins, associates with the receptor through a PDZ interaction with the PSD-95-family MAGUK, SAP102 (synapse-associated protein), and perhaps with other PDZ proteins; disruption of this interaction will dramatically reduce the
Myosin complex Actin filament
Kinesin KIF1Ba Microtubule
Adaptor complex?
Adaptor complex?
mPins + Ga i Kinesin Exocyst complex
mLin complex Kinesin KIF17
Figure 2 Proposed complexes involved in the transport of NMDA receptors. Two kinesins that indirectly interact with NMDA receptors are KIF17 and KIF1Ba, and additional kinesins may also play a role. Delivery of receptors to spines is likely to involve myosins, which remain to be identified. Both Sec8, probably in a complex with other exocyst proteins, and mPins bind to SAP102 and other related PDZ proteins (MAGUKS) and may target the NMDA receptor/PDZ protein complexes to the plasma membrane. Also, G-protein signaling via the interaction of mPins and Gai may regulate the effect of mPins on NMDA receptor trafficking and postsynaptic spine structure.
surface expression of receptor. Another trafficking molecule, mPins/LGN (mammalian homolog of Drosophila partner of inscuteable) interacts with the receptor complex through the Src homology 3/guanylate kinase (SH3/GK) motifs of the MAGUK and disruption of this interaction also reduces NMDA receptor surface delivery. mPins/LGN interacts with a number of additional proteins, including Gai, suggesting a possible link between G-protein signaling and NMDA receptor trafficking. Transport vesicles carrying NMDA receptors appear to be largely distinct from those carrying AMPA receptors, suggesting that these two receptor populations are packaged into distinct populations of transport vesicles as they exit the trans-Golgi network (TGN). In young neurons transport in dendrites involves multiple rounds of endo- and exocytosis with the plasma membrane and association of the transport vesicles with SAP102. Two motor systems
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have been identified in the transport of NMDA receptors and MAGUKs. The kinesin KIF17 associates indirectly with NMDA receptors through a complex with mLin-7, mLin-2, and mLin-10 and may mediate the transport of NMDA receptors along microtubules. MAGUKs can associate directly with the kinesin KIF1Ba, making it a candidate for transporting NMDA receptors associated with SAP102. Myosin motors, which can mediate transport of proteins along actin filaments, are also likely to play a role. NMDA receptors are regulated by myosin light chain kinase and show a direct interaction with myosin regulatory light chain. The NMDA receptor, therefore, is transported as part of a macromolecular complex. However, unlike the presynaptic active zone, the postsynaptic density, a structure with which the NMDA receptor is associated at synapses, is not transported as a completely preassembled complex. The NMDA receptor complex may remain intact as it is delivered to the synapse, but a more likely scenario is that the addition and removal of proteins are a normal parts of the trafficking process to change parameters as the local environment changes.
Trafficking of NMDA Receptors at the Synapse Synaptic Delivery and PDZ Proteins
The steps involved in delivering NMDA receptors present in transport vesicles in the dendrite to the synapse are largely unknown. This process may involve an intermediate stop in an endosomal compartment, where they may mix with recycling receptors, or direct delivery to the plasma membrane. The site of delivery is also unknown but is likely adjacent to the synapse rather than directly at the synapse. This will require movement within the plasma membrane, and these receptors may represent the extrasynaptic pool of NMDA receptors (discussed later). The number and composition of NMDA receptors at the synapse are relatively stable compared to those of AMPA receptors. However, the mechanism regulating synaptic NMDA receptors is not well understood. The number of receptors is not based on availability, since overexpression of NR2 subunits does not increase the synaptic number of receptors, but does increase the extrasynaptic number, indicating a local mechanism of control for synaptic receptors. The NR2/PSD-95 interaction was the first PDZ interaction identified (subsequent studies have shown that the other three members of the PSD-95 family of MAGUKs, SAP102, SAP97, and PSD-93, also can interact with the NR2 subunits), and it generally has been assumed that synaptic NMDA receptors are tethered to the PSD
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through this PDZ interaction. A number of studies have supported this by showing that the PDZ binding domain is required for an NR2 subunit to be admitted to the synapse. However, recent studies have shown that the NMDA receptor/PSD-95 interaction is not straightforward. Knockout of PSD-95 or its overexpression does not change synaptic NMDA receptors, but overexpression does increase AMPA receptors. Studies have also shown that an NR2A subunit lacking its PDZ binding motif can enter the synapse, although NR2B subunits without the PDZ binding motif cannot. A variety of adhesion proteins are found associated with NMDA receptors at synapses and these proteins may help regulate NMDA receptor distribution and function. Postsynaptic EphB2 receptors are activated by their presynaptic ligand, ephrin-B, and bind directly to the extracellular domain of NR1, affecting NMDA receptor function directly and subsequent dendrite arborization indirectly. SALM1 can bind to the extracellular domain of NR1 and this and some other SALMs also can associate with NMDA receptors via a mutual PDZ-mediated binding to MAGUKs of the PSD-95 family. SALM1 can enhance surface expression of transfected NR2A. A number of other adhesion factors do not bind directly to NMDA receptors but instead form associations with them at the synapse and may affect NMDA receptor trafficking and activation and subsequent neuronal structure and function; examples are neuroligin, cadherin/catenin, and a number of members of the L1/NrCAM and NCAM (neural cell adhesion molecule) families of adhesion proteins. Neuroligin binds to neurexin in the presynaptic terminal and to PSD-95 and other NMDA receptor-associated MAGUKs in the postsynaptic density. Neuroligin localization at synapses may help determine if developing synapses become excitatory or inhibitory. In fact, a number of adhesion proteins such as neuroligin, cadherin, NCAM, and EphB receptors may mediate the earliest stages of synaptogenesis that are required for subsequent trafficking of MAGUKs and NMDA receptors to the nascent postsynaptic membrane. Clathrin-Mediated Internalization and Regulation of NMDA Receptors
The number of molecules of any protein on the cell surface can be regulated by internalization, and several studies have now shown that a number of factors can increase the internalization of NMDA receptors. These include synaptic activity, activation of type 1 metabotropic receptors, and exposure to the agonist glycine. In addition, run down of extrasynaptic receptors appears to depend on internalization.
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YEKLSSIESDV AP2 PDZ NR1/NR2B NR2
PDZ protein
NR2 Y Fyn
Fyn
AP2 complex
P Y
Anchor
a
NR1/NR2B NR2A-containing receptors are enriched at the synapse
NR1/NR2A
NR2B-containing receptors are enriched at the extrasynaptic membrane
NR2B-containing receptors are recycled
b
NR2A-containing receptors are degraded
Figure 3 (a) Model for regulation of NR2B-containing receptors at the synapse. Internalization is mediated by the interaction of AP2 with the YEKL motif in the distal C-terminus of NR2B. At the synapse, Y1472 in this motif is phosphorylated by Fyn kinase and is unable to interact with AP2. Fyn is recruited by PSD-95, which associates with NR2B through its PDZ binding domain. Loss of phosphorylation of Y1472 facilitates the interaction with AP2 and leads to removal from the synapse and internalization. Retention of NMDA receptors at the synapse may involve additional anchors. (b) NR2A- and NR2B-containing receptors are regulated differently at the synapse. Several studies have suggested that NR2A-containing receptors are more abundant at the synapse while NR2B-containing receptors are more abundant at extrasynaptic sites. Several factors could contribute to this differential localization. Receptors can enter the synapse by lateral diffusion in the plasma membrane. A filter may selectively exclude NR2B-containing receptors from the synapse. Alternatively, NR2B-containing receptors may be more likely to be removed from the synapse through internalization (as described in panel (a)). Finally, internalized NR2A-containing receptors are more susceptible to degradation while NR2B-containing receptors are recycled. If they enter the extrasynaptic pool, the recycled receptors would contribute to the enrichment of NR2B-containing receptors in this pool.
NMDA receptors can be removed from the plasma membrane through a clathrin-dependent route involving the adaptor protein AP2 (Figure 3). Clathrindependent internalization requires the recognition of a particular motif in its cytoplasmic domain by the adaptor protein. The NR2B subunit has such a motif, YEKL, near its C-terminus, that has been shown to play a role in the surface stability of the receptor. Interestingly, a similar motif in the NR2A subunit is
not involved in the internalization of this subunit, but rather an upstream dileucine motif (also a substrate for the AP2 adaptor) was shown to play such a role. The differences in the trafficking of these two subunits are highlighted in the different pathways followed by the internalized receptors. While both initially enter early endosomes, NR2B is trafficked to recycling endosomes while NR2A is trafficked to late endosomes. Therefore, NR2B-containing receptors are more likely
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to be recycled and NR2A-containing receptors are more likely to be degraded after internalization. Does internalization play a role in regulating the number of synaptic NMDA receptors? It is unlikely that internalization per se regulates the number of receptors, but the availability of endocytic motifs such as the YEKL motif on NR2B may. This motif contains a tyrosine, Y1472, which is a major substrate for Fyn kinase. In the striatum, synaptic membranes have a high level of phosphorylated Y1472 while intracellular membranes have a low level of phosphorylated Y1472. In cultures of cerebellar granule cells and hippocampal neurons, using transfection of NR2B subunits with mutations in this motif, it was shown that mutations that block the interaction of AP2 increase the number of synaptic receptors. Furthermore, while the PDZ binding domain of NR2B is required for synaptic localization, NR2B subunits that lack this domain and have mutations of the YEKL motif still localize to the synapse. One explanation is that the PDZ interaction may not be critical for anchoring the receptor complex to the postsynaptic protein complex, but rather plays a role in maintaining the phosphorylation of Y1472. PSD-95 has been shown to interact with Fyn kinase, and, at the synapse, PSD-95 may recruit Fyn kinase, thus maintaining the phosphorylation of Y1472 and preventing its interaction with AP2 and its subsequent internalization. The fact that the internalization of NR2A is not affected by mutations in a homologous motif, YKKM, indicates that different factors control the surface stability of NR2A-containing receptors. The YEKL and dileucine motifs near the distal end of the C-terminus of NR2B and NR2A, respectively, are not the only endocytic motifs found on the NMDA receptor. Another tyrosine-containing motif is present in the proximal part of the C-terminus, near the last transmembrane domain. This motif was functionally characterized in receptors with the NR2A subunit, but it is present in all NR2 subunits and the NR1 subunit. Since mutation of neither this motif nor the distal motif can block all internalization, there are likely other motifs present in the NR1 and NR2 C-termini that mediate clathrin-dependent internalization. Nonclathrin mechanisms may also exist. Extrasynaptic Receptors
NMDA receptors are found not only at the postsynaptic membrane but have also been reported on glia, presynaptic terminals, the perisynaptic membrane, and the extrasynaptic membrane. Most extensively studied are perisynaptic and extrasynaptic receptors, which may be functionally important and activated when glutamate that is released at the synapse is not
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sufficiently sequestered and is able to diffuse outside the synapse. While most neurons contain both synaptic and extrasynaptic receptors, in a few specific cases such as the retinal ganglion cell, NMDA receptors are found only outside the synaptic cleft. In other neurons, NR2A-containing receptors are preferred at the synapse and NR2B-containing receptors are preferred at the extrasynaptic membrane, although the separation is not complete. This subunit preference may reflect functional differences between these two pools of receptors. This, however, is difficult to reconcile with studies that show diffusion of receptors from the extrasynaptic plasma membrane into the synapse. In one study, synaptic receptors were blocked with the use-dependent antagonist, MK801, and recovery of function was monitored. The recovery of function was surprisingly rapid, and controls ruled out sources other than the extrasynaptic membrane. The preferential localization of NR2A- and NR2B-containing receptors would be consistent with these findings if there was a filter that favors the entry of NR2A into the synapse or the removal of NR2B from the synapse. NMDA receptors do recycle, although not as fast as AMPA receptors, and the enrichment of NR2B in the extrasynaptic membrane could reflect this. As noted earlier, internalized NR2B receptors are more likely to be recycled and NR2A receptors are more likely to be degraded. If receptors are recycled to the extrasynaptic membrane, the difference in intracellular trafficking could favor NR2B at the extrasynaptic membrane. Such a mechanism would also assume that there is an exchange between the synaptic and extrasynaptic pools of receptors. The functional consequences resulting from activation of these receptors is also an area of intense study. Activation of extrasynaptic receptors promotes cell death. A series of controversial studies has indicated that NR2A-containing receptors mediate longterm potentiation (LTP) and that NR2B-containing receptors mediate long-term depression (LTD). This requires more work, and it will be interesting to determine if position of the receptor – for example, synaptic versus perisynaptic – plays a role. Developmental Changes in NMDA Receptors
During development, most neurons begin expressing predominantly NR2B, which then gradually decreases as NR2A expression increases. The functional implication of this switch is that NMDA receptors in young animals have slower kinetics (due to a preponderance of NR2B), and receptors in older animals are faster (due to a preponderance of NR2A). It has been proposed that this change, in which ‘adult’ NMDA receptors are faster and admit less calcium, may be related
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to the decrease in synaptic plasticity in older animals. While most neurons continue to express NR2B in older animals, and therefore have a mixture of NR2A- and NR2B-containing receptors and perhaps those containing both subunits, cerebellar granule cells completely stop synthesizing NR2B. During development, NR2A and NR2C expression increases in these neurons. Trafficking plays a role in the preferential localization of subunits during development. A series of elegant studies has shown that, within hours, visual experience can cause synaptic NMDA receptors in the visual cortex to become more NR2A-like. Thus, either NR2A-containing receptors are preferentially added or NR2B-containing receptors are preferentially lost, or both. While this could depend somewhat on new protein synthesis, it is unlikely that such a change could be effected in the time period, and a more likely explanation is that NR2A-containing receptors are recruited from extrasynaptic or intracellular sites. Similar transitions from NR2B- to NR2A-containing receptors may be occurring at other synapses during development, or even at mature synapses. Thus, the mechanism underlying this switch is fundamentally important to NMDA receptor trafficking.
Phosphorylation and Trafficking of NMDA Receptors As discussed in the preceding section, Fyn kinase phosphorylation has a major impact on trafficking of the NR2B subunits. The NMDA receptor is a substrate for several other kinases, including other members of the Src family of protein tyrosine kinases, calcium/ calmodulin-dependent protein kinase II (CaMKII), cyclin-dependentkinase-5 (cdk5),cyclic AMP-dependent protein kinase A (PKA), protein kinase C (PKC), casein kinase II, and others. The NMDA receptor directly interacts with CaMKII at the postsynaptic density. This interaction is important for the translocation of CaMKII to the synapse. CaMKII has been shown to interact with all three of the common subunits, NR1, NR2A, and NR2B, through their C-termini, although the affinities of the interactions and the functional implications are very different. A recent study in which the interacting domains on NR2A and NR2B were switched showed that the CaMKII interaction with the NR2B subunit is required for LTP. Phosphorylation of NMDA receptor subunits affects the trafficking of the individual subunits. The subcellular distribution of the NR1 subunit expressed alone in heterologous cells can be regulated by PKC phosphorylation of the serine residues in the C1 domain. These amino acids are near the ER retention signal, and mutations that mimic phosphorylation greatly increased surface expression of NR1/tac chimeric constructs.
Phosphorylation by several other kinases, particularly casein kinase II and PKC, affects the trafficking of the assembled receptor. The NR2B subunit terminates in ESDV, which is a PDZ binding domain. Serine-1480, present in this domain, is a substrate for casein kinase II, and phosphorylation of Ser1480 blocks interaction with PDZ proteins. Therefore, this may be an important mechanism for regulating receptor/PDZ interactions. PKC activation increases the delivery of NMDA receptors to the cell surface.
Degradation of NMDA Receptors While trafficking can regulate local amounts and distributions of a protein effectively, altering either synthesis or degradation can effect global changes in proteins. Like other proteins, the NMDA receptor is continually degraded and, at steady state, replenished by synthesis of new receptors. In cultured cerebellar granule cells, the half-life of assembled NMDA receptor is about 1 day. Unassembled NR1 subunits, which are synthesized in excess compared to NR2, are retained in the ER and degraded rapidly, with a half-life of 1–2 h. As presented previously, there is a developmental change in the relative amounts of NR2A and NR2B, which could be achieved by altering synthesis or degradation. Since mRNA expression often matches protein levels, it suggests that synthesis is the main controlling factor. The degradation of NMDA receptors may not be entirely random, but may depend on the subunit composition and activity of the neuron; degradation, therefore, could change the subunit balance or the total number of receptors at a particular synapse. As noted above, receptors containing NR2A or NR2B follow different pathways after internalization. Recent studies have shown that ubiquitination plays a major role in the removal of postsynaptic proteins, including NMDA receptors. Covalent attachment of ubiquitin can cause internalization of membrane proteins and degradation in the proteasome. Several proteins of the PSD are ubiquinated, including PSD-95. NR1 can be ubiquitinated on its extracellular domain. In this case, activity-dependent NMDA receptor degradation may be mediated by retrotranslocation followed by ubiquitination, although the mechanism is not well understood. See also: Long-Term Depression (LTD): Metabotropic Glutamate Receptor (mGluR) and NMDAR-Dependent Forms; Long-Term Potentiation (LTP): NMDA Receptor Role; NMDA Receptor Function and Physiological Modulation; Postsynaptic Density/Architecture at Excitatory Synapses.
NMDA Receptors, Cell Biology and Trafficking
Further Reading Ali DW and Salter MW (2001) NMDA receptor regulation by Src kinase signaling in excitatory synaptic transmission and plasticity. Current Opinions in Neurobiology 11: 336–342. Barria A and Malinow R (2002) Subunit-specific NMDA receptor trafficking to synapses. Neuron 35: 345–353. Barria A and Malinow R (2005) NMDA receptor subunit composition controls synaptic plasticity by regulating binding to CaMKII. Neuron 48: 289–301. Dunah AW and Standaert DG (2001) Dopamine D1 receptordependent trafficking of striatal NMDA glutamate receptors to the postsynaptic membrane. Journal of Neuroscience 21: 5546–5558. Ehlers MD, Tingley WG, and Huganir RL (1995) Regulated subcellular distribution of the NR1 subunit of the NMDA receptor. Science 269: 1734–1737. Kornau HC, Schenker LT, Kennedy MB, et al. (1995) Domain interaction between NMDA receptor subunits and the postsynaptic density protein PSD-95. Science 269: 1737–1740. Lavezzari G, McCallum J, Dewey CM, et al. (2004) Subunitspecific regulation of NMDA receptor endocytosis. Journal of Neuroscience 24: 6383–6391. Li B, Chen N, Luo T, et al. (2002) Differential regulation of synaptic and extra-synaptic NMDA receptors. Nature Neuroscience 5: 833–834. McIlhinney RAJ, LeBourdelles B, Molnar E, et al. (1998) Assembly intracellular targeting and cell surface expression of the human N-methyl-D-aspartate receptor subunits NR1a and NR2A in transfected cells. Neuropharmacology 37: 1355–1367. Mu Y, Otsuka T, Horton AC, et al. (2003) Activity-dependent mRNA splicing controls ER export and synaptic delivery of NMDA receptors. Neuron 40: 581–594. Nong Y, Huang YQ, Ju W, et al. (2003) Glycine binding primes NMDA receptor internalization. Nature 422: 302–307. Perez-Otano I and Ehlers MD (2005) Homeostatic plasticity and NMDA receptor trafficking. Trends in Neuroscience 28: 229–238. Prybylowski K, Chang K, Sans N, et al. (2005) The synaptic localization of NR2B-containing NMDA receptors is controlled by interactions with PDZ proteins and AP-2. Neuron 47: 845–857.
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Quinlan EM, Philpot BD, Huganir RL, et al. (1999) Rapid, experience-dependent expression of synaptic NMDA receptors in visual cortex in vivo. Nature Neuroscience 2: 352–357. Rao A and Craig AM (1997) Activity regulates the synaptic localization of the NMDA receptor in hippocampal neurons. Neuron 19: 801–812. Roche KW, Standley S, McCallum J, et al. (2001) Molecular determinants of NMDA receptor internalization. Nature Neuroscience 4: 794–802. Rumbaugh G and Vicini S (1999) Distinct synaptic and extrasynaptic NMDA receptors in developing cerebellar granule neurons. Journal of Neuroscience 19: 10603–10610. Sans N, Prybylowski K, Petralia RS, et al. (2003) NMDA receptor trafficking through an interaction between PDZ proteins and the exocyst complex. Nature Cell Biology 5: 520–530. Sans N, Wang PY, Du Q, et al. (2005) mPins, the mammalian homologue of Drosophila Pins, modulates PSD-95 and SAP102 trafficking and influences NMDA receptor surface expression. Nature Cell Biology 7: 1179–1190. Setou M, Nakagawa T, Seog DH, et al. (2000) Kinesin superfamily motor protein KIF17 and mLin-10 in NMDA receptorcontaining vesicle transport. Science 288: 1796–1802. Sprengel R, Suchanek B, Amico C, et al. (1998) Importance of the intracellular domain of NR2 subunits for NMDA receptor function in vivo. Cell 92: 279–289. Standley S, Roche KW, McCallum J, et al. (2000) PDZ-domain suppression of an ER retention signal in NMDA receptor NR1 splice variants. Neuron 28: 887–898. Tovar KR and Westbrook GL (2002) Mobile NMDA receptors at hippocampal synapses. Neuron 34: 255–264. Vissel B, Krupp JJ, Heinemann SF, et al. (2001) A use-dependent tyrosine dephosphorylation of NMDA receptors is independent of ion flux. Nature Neuroscience 4: 587–596. Washbourne P, Bennett JE, and McAllister AK (2002) Rapid recruitment of NMDA receptor transport packets to nascent synapses. Nature Neuroscience 5: 751–759. Wenthold RJ, Prybylowski K, Standley S, et al. (2003) Trafficking of NMDA receptors. Annual Reviews of Pharmacology and Toxicology 43: 335–358.
Metabotropic Glutamate Receptors (mGluRs): Molecular Biology, Pharmacology and Cell Biology F Nicoletti and V Bruno, University of Rome ‘La Sapienza,’ Rome, Italy G Battaglia, Istituto Neurologico Mediterraneo ‘Neuromed,’ Pozzilli, Italy ã 2009 Elsevier Ltd. All rights reserved.
Background The first challenge to the general belief that all glutamate receptors were ligand-gated ion channels was the demonstration by Sladeczek, Pin, Recasens, Bockaert, and Weiss that glutamate stimulates polyphosphoinositide (PI) hydrolysis in cultured striatal neurons. Similar data were obtained in brain slices, where the PI response to glutamate is maximal in the early postnatal life and declines progressively during postnatal development. The term ‘metabotropic glutamate (mGlu) receptors’ was introduced in 1987 by H Sugiyama and colleagues, who examined responses to excitatory amino acids in Xenopus oocytes injected with rat brain mRNA. Cloning of the first mGlu receptor subtype by S Nakanishi and colleagues in 1991 was the milestone for any further development of the field.
Classification of mGlu Receptors mGlu receptors form a family of eight subtypes subdivided into three groups based on their sequence similarities and G-protein coupling.
Homologous desensitization of group I mGlu receptors involves phosphorylation mechanisms mediated by either PKC or G-protein-coupled receptor kinases (GRKs). Different GRK isoforms, including GRK2, GRK4, and GRK5, have been found to desensitize mGlu1 receptors in recombinant cells. GRK4 is also required for desensitization of native mGlu1 receptors expressed by cerebellar Purkinje cells. While desensitization by GRK4 is mediated by receptor phosphorylation, desensitization by GRK2 is independent of phosphorylation and may involve a direct interaction with activated Gaq. The mGlu5 receptor is selectively desensitized by members of the GRK2 family (GRK2 and GRK3) through a mechanism that involves phosphorylation of the Thr840 residue in the C-terminus domain of the receptor. The Regulator of G-protein signaling, RGS4, which acts as effector antagonist by increasing the GTPase activity of Gaq, inhibits mGlu1 and mGlu5 receptor signaling. Group II mGlu Receptors
Group II includes mGlu2 and mGlu3 receptors, which are coupled to Gi/Go proteins (Figure 1). Their activation inhibits cAMP formation, inhibits L- and N-type voltage-sensitive Ca2þ channels, activates Kþ channels, and stimulates the MAPK and PI3K pathways. The latter two effects are presumably mediated by the bg subunits of Gi. Activation of group II mGlu receptors can also amplify the stimulation of cAMP formation by b-adrenergic receptor agonists in cultured astrocytes, and the stimulation of PI hydrolysis by mGlu1/5 receptor agonists in brain slices.
Group I mGlu Receptors
Group III mGlu Receptors
Group I includes mGlu1 (variants: mGlu1a, -1b, -1c, -1d, and -1e) and mGlu5 (variants: mGlu5a and -5b) receptors, which are primarily coupled to Gq/G11 proteins (Figure 1). Secondary coupling involves Gs and the pertussis toxin-sensitive Go. Activation of both subtypes in recombinant cells stimulates PI hydrolysis with an ensuing formation of the two intracellular second messengers, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 releases Ca2þ from intracellular stores, whereas DAG activates protein kinase C (PKC). Activation of group I mGlu receptors can also stimulate cyclic adenosine monophosphate (cAMP) formation, arachidonic acid release, the mitogen-activated protein kinase (MAPK) pathway, and the phosphatidylinositol 3-kinase (PI3K) pathway. The mGlu1 receptor is negatively coupled to a variety of Kþ channels and to the C1 type of transient receptor potential (TRP) channels.
Group III includes mGlu4, mGlu6, mGlu7 (variants: mGlu7a and -7b), and mGlu8 (variants: mGlu8a, -8b, and -8c) receptors, which are all coupled to Gi/Go proteins (Figure 1). Activation of mGlu4, mGlu7, and mGlu8 receptors inhibits cAMP formation and N-type voltage-sensitive Ca2þ channels. In cells expressing mGlu4 receptors, GRK2 attenuates agonist-stimulated MAPK activation without affecting inhibition of adenylyl cyclase. Activation of mGlu6 receptors inhibits cGMP-dependent cation channels in ON bipolar cells of the retina (see later).
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Structure and Mechanisms of Activation of mGlu Receptors mGlu receptors belong to class C G-protein-coupled receptors (GPCRs), which also includes g-aminobutyric acid B (GABAB) receptors, the Ca2þ-sensing receptor
Metabotropic Glutamate Receptors (mGluRs): Molecular Biology, Pharmacology and Cell Biology 293
Groups
Subtypes
Main transduction pathways Glutamate
I
mGlu1a mGlu1b mGlu1c mGlu1d mGlu1e
PtdIns-4,5-P2
PLC
Gq DAG Ins-1,4,5-P3 Ca2+
PKC Ca2+ Glutamate K+ channel
mGlu5a mGlu5b
mGlu2 II
-
Gq
Glutamate
mGlu3 Gi/o mGlu4
-
AC cAMP ATP
III
mGlu7a mGlu7b
Glutamate Ca2+ channel
mGlu8a mGlu8b mGlu8c
-
Gi
Glutamate
III
PLC
mGlu6
PtdIns-4,5-P2
Go DAG Ins-1,4,5-P3 Ca2+
PKC Ca2+
Figure 1 The mGlu receptor family. PtdIns-4,5-P2, phosphatidylinositol 4,5-bisphosphate; Ins-1,4,5-P3, inositol 1,4,5-trisphosphate; DAG, diacylglycerol; PKC, phosphokinase C; PLC, phospholipase C.
expressed parathyroid cells, the taste receptors that detect sweet and umami, and some pheromone receptors. mGlu receptors are formed by (1) a large N-terminus extracellular domain (about 600 amino acids) containing the glutamate binding site and a Cys-rich region, (2) a seven-transmembrane (7TM) domain, and (3) an intracellular C-terminus domain that varies in length depending on the receptor subtype/splice variant. mGlu1a, mGlu5a, and mGlu5b receptors have long C-terminus domains, which favor their interaction with adaptor and scaffolding proteins (see later). In the mGlu1b and -1d variants, the 313 C-terminus amino acids are substituted by 20
(mGlu1b) and 22/26 (mGlu1d human/rat) amino acids. The mGlu1e isoform corresponds to the soluble extracellular domain of the mGlu1 receptor. The mGlu5b variant differs from mGlu5a for the presence of 39 amino acids inserted 49 residues downstream of the seventh TM domain. The existence of truncated forms of mGlu3 and mGlu6 receptors, corresponding to the extracellular portion of the receptors, has been demonstrated in human tissue. In the mGlu7b variant, the last 16 amino acids of mGlu7a are substituted by 23 different residues. The elegant report by Kunishima and colleagues in 2000 has afforded insights into the structure
294 Metabotropic Glutamate Receptors (mGluRs): Molecular Biology, Pharmacology and Cell Biology
and mechanism of activation of mGlu receptors. Glutamate binds to a ‘venus fly trap’ (VFT) region of the N-terminus domain constituted by two globular lobes separated by a hinge. The lobes oscillate between a close/active conformation and an open/ inactive conformation. Agonist binding to the hinge facilitates the closure of the two lobes, whereas binding of competitive antagonists stabilizes the open conformation. Similarly to other class C GPCRs, mGlu receptors function as dimers. Dimerization involves a hydrophobic interaction between lobe I of the VFT in each monomer, and is stabilized by a disulfide bond. The use of receptor chimeras containing the C-terminus domains of type 1 and type 2 GABAB receptors (which form obligatory heterodimers) has allowed to establish that the mGlu receptor dimer can be activated by a single molecule of orthosteric agonist, but that binding of two molecules (one to each monomer) is required for full receptor activation. In contrast, it appears that only one 7TM domain is required for effective G-protein activation. The 7TM domain contains the binding sites for positive allosteric modulators (‘potentiators’ or ‘enhancers’), which amplify receptor activation only in the presence of an orthosteric agonist – that is, in an activity-dependent manner. One enhancer molecule per dimer is needed for full amplification of mGlu receptors. The 7TM domain also bears the binding sites for negative allosteric modulators, which inhibit mGlu receptors independently of the concentrations of ambient glutamate. mGlu receptors interact with a variety of adaptor and scaffolding proteins, which regulate receptor expression and coupling with other receptors or enzymes. Homer proteins, which contain both postsynaptic density/disc/zonula occludens (PDZ) and Ena/ vasodilator-stimulated phosphoprotein homology-1 (EVH-1) domains, bind to a proline-rich motif in the C-terminal region of mGlu1a and mGlu5 receptors. Constitutively expressed, long isoforms of Homer proteins (Homer 1b, -1c, -2, and -3) have a C-terminal coiled-coil domain that mediates self-multimerization and allows the interaction of mGlu1a/5 receptors with other Homer-interacting proteins, such as the IP3 receptor (Figure 2(a)). This interaction is disrupted by the short inducible isoform of Homer (Homer 1a), which lacks the coiled-coil domain. Interestingly, Homer 1a is encoded by an early inducible gene, which is expressed in response to synaptic activation. The interaction between Homer and the long isoform of the PI3K enhancer, PIKE, mediates the stimulation of PI3K by mGlu1 receptor activation. mGlu1 receptors functionally interact with ephrin-B2, a member of the ephrin/Eph receptor family of transmembrane proteins which mediate processes of cell-to-cell interaction
during development and in the adult life. This interaction might have interesting implications for processes of developmental plasticity. Other proteins interacting with group I mGlu receptors are the E3 ubiquitin ligase, Seven in absentia homolog 1a, protein phosphatase 1c, tubulin, and tamalin. Protein phosphatase 2C binds selectively to, and dephosphorylates, mGlu3 receptors. mGlu7 receptors interact with calmodulin, protein interacting with PKC 1 (PICK 1), glutamate receptor-interacting protein 1 (GRIP 1), syntenin, a-tubulin, filamin A, and the catalytic g subunits of protein phosphatase 1C. A simultaneous or exclusive binding to these proteins tightly regulates mGlu7 receptor signaling. mGlu8 receptors interact with sumo1 and other components of the sumoylation cascade, such as ube2a, Pias1, Piasg, and Piasxb.
Cell Biology, Pharmacology, and Implications for Human Pathology The three groups of mGlu receptor ligands (Figure 3) have potential clinical applications, as outlined in Table 1. mGlu1 Receptor
The mGlu1 receptor consists of 1194 amino acids in humans and 1199 amino acids in rats and mice; accession numbers are Q13255 (human), P23385 (rat), and P97772 (mouse). The gene is named GRM1 (human) and Grm1 (rat, mouse); chromosomal locations are 6q24 (human), 1p13 (rat), and 10 A2 (mouse). At a subcellular level, mGlu1 proteins are mostly found in postsynaptic elements at the periphery of the postsynaptic density (PSD). This contrasts with the distribution of N-methyl-D-aspartate (NMDA) and a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors, which are localized in the central region of the PSD. mGlu1 receptors are expressed by Purkinje cells of the cerebellar cortex, where they participate in the induction of longterm depression (LTD) at the parallel fiber–Purkinje cell synapse. LTD is a particular form of synaptic plasticity that, in the cerebellum, underlies motor learning. Mice with genetic deletion of mGlu1 receptors are ataxic and show persistent multiple climbing fiber innervation of cerebellar Purkinje cells. Interestingly, anti-mGlu1 receptor antibodies are found in a subset of patients with Hodgkin’s lymphoma and paraneoplastic ataxia. The mGlu1 receptor is also expressed in the olfactory bulb, pars compacta of the substantia nigra, hippocampus, and thalamic nuclei. In the hippocampus, activation of mGlu1 receptors inhibits GABA release and mGlu1 receptor antagonists are protective against excitotoxic and hypoxic/ischemic neuronal death. Unexpectedly, that
Metabotropic Glutamate Receptors (mGluRs): Molecular Biology, Pharmacology and Cell Biology 295
mGlu1/5
Glut
NMDAR
mGlu1/5
TRPC PSD95 Dynamin3
GKAP
Shank Cortactin IP3R
PIKE L
Ho mer-long
ER
F-actin
Ho mer-short
a
Photoreceptor
mGlu4/7/8 mGlu2/3
Na+
Glut
Glut
cGMP-dependent channel
EAAT
DAG
mGlu5 mGlu3
Calcineurin (PP2B)
Go
Ins-1,4,5-P
+ +
TGFb, NGF
b
mGlu6 PLC
ON-bipolar cell
Glia
TGFb, NGF
c
Figure 2 Synaptic localization of mGlu receptors. (a) mGlu1 and mGlu5 receptors are found at the periphery of postsynaptic elements. mGlu1a, mGlu5a, and mGlu5b receptors interact with scaffolding and adaptor proteins, including Homer. (b) mGlu2/3 and mGlu4/7/8 receptors are predominantly found in presynaptic terminals, where their activation inhibits neurotransmitter release; mGlu3 and mGlu5 receptors are also found in glial cells. (c) Activation of mGlu6 receptors by the glutamate released from photoreceptor cells inhibits the activity of ON bipolar cells. Glut, glutamate; IP3R, inositol 1,4,5-trisphosphate receptor; ER, endoplasmic reticulum; EAAT, excitatory amino acid transporter; PP2B, protein phosphatase 2B; TRPC, transient receptor potential-C. (a) Adapted from Fourgeaud L (2005) Addicted to Holmer? Journal of Neuroscience 25(42): 9555–9556.
ectopic expression of mGlu1 receptors in melanocytes promotes the development of multiple melanomas in mice. Because mGlu1 receptors are found in human melanomas but not in benign nevi, it is possible that receptor activation contributes to neoplastic transformation of melanocytes. There are no selective agonists of mGlu1 receptors. 3,5-dihydroxyphenylglycine (DHPG) activates
both mGlu1 and mGlu5 receptors, although it has no activity at other glutamate receptor subtypes. The following drugs are examples of competitive mGlu1 receptor antagonists (in the rank order of affinity): LY367385 ¼ 4C3HPG > 4CPG > AIDA. Compounds Ro01–6128 and Ro67–7476 are mGlu1 receptor enhancers with nanomolar potency, whereas BAY36–7620 and CPCCOEt are negative allosteric
296 Metabotropic Glutamate Receptors (mGluRs): Molecular Biology, Pharmacology and Cell Biology Orthosteric ligands of mGlu receptors OH
OH
OH
H HO2C
CO2H
HO2C
CO2H Cl
Me
H
H
NH2 H2N
CO2H
H2N
DHPG L-Glutamate (Non-selective, low potency) (mGlu1/5 Ago)
CHPG (mGlu5 Ago)
CO2H
NH
CO2H
H H
2R,4R-APDC (mGlu2/3 Ago)
CO2H H
NH2
NH2
CO2H
H2N
LY379268 (mGlu2/3 Ago)
LY354740 (mGlu2/3 Ago)
LY367385 (mGlu1 Ant)
CO2H
O
H
CO2H
CO2H
H2N
EGlu (mGlu2/3 Ant)
PO3H2
O
PO3H2
PO3H2
H2N
H
O H2N
H H2N
CO2H
H
Me HO2C
CO2H H2N
L-AP4 (mGlu4-8 Ago)
PO(OH)2
HO2C
O CH2
CO2H
H2N
AIDA (mGlu1 Ant)
H
H
HO2C
CO2H
H2N
CO2H
L-SOP (mGlu4-8 Ago)
CO2H
H2N
CO2H
R,S-PPG (mGlu4-8 Ago)
LY341495 (pan mGlu Ant)
MSOP (mGlu4-8 Ant)
Positive allosteric modulators of mGlu receptors
N
O H N
O
O
N F
H N F
DFB (mGlu5)
Ro 67-4853 (mGlu1)
Ro 01-6128 (mGlu1)
OH O
N O
N O
S
N
O O
O
N
N
H2O
S
O
NH2
O
F
O HN
F F
3-MPPTS (mGlu2)
APPES (mGlu2)
(−)-PHCCC (mGlu4)
Negative allosteric modulators of mGlu receptors OH T
N A
S
H N
N
N NH
O
O
O
N
O
Cl
O T
O
CPCCOEt (mGlu1)
N
OEt
BAY36-7620 (mGlu1)
MPEP (mGlu5)
MTEP (mGlu5)
Fenobam (mGlu5)
Figure 3 Selected mGlu receptor ligands. Ago, agonist; Ant, competitive antagonist.
modulators of mGlu1 receptors. (Abbreviations: LY367385, (E)-2-methyl-6-stryrylpyridine(–)-2oxa-4-aminocyclo[3.1.0]exane-4,6-dicarboxylic acid; 4C3HPG, 4-carboxy-3-hydroxyphenylglycine;
4CPG, 4-carboxyphenylglycine; AIDA, (R,S)-1aminoindan-1,5-dicarboxylic acid; Ro 01–6128, ethyl diphenylacetylcarbamate; Ro 67–7476, (S)-2-(4fluorophenyl)-1-(toluene-4-sulfonyl)pyrrolidine;
Metabotropic Glutamate Receptors (mGluRs): Molecular Biology, Pharmacology and Cell Biology 297 Table 1 Potential clinical applications of mGlu receptor ligands Group
Application
Group I
Chronic pain (mGlu1/5, Ant/NAM) Anxiety disorders (mGlu5, Ant/NAM) Drug addiction (mGlu5, Ant/NAM) Fragile X syndrome (mGlu5, Ant/NAM) Parkinsonism (mGlu5, Ant/NAM) Schizophrenia (mGlu5, PAM) Brain ischemia (mGlu1, Ant/NAM) Generalyzed anxiety disorders and panic attack (mGlu2/3, Ago/PAM) Drug addiction (mGlu2/3, Ago/PAM) Schizophrenia (mGlu2/3, Ago/PAM) Chronic pain (mGlu2/3, Ago/PAM; mGlu2 inducers) Epilepsy (mGlu4/7/8, Ago/PAM) Parkinsonism (mGlu4, Ago/PAM) Anxiety (mGlu8, Ago/PAM)
Group II
Group III
Ago, agonist; Ant, competitive antagonist; PAM, positive allosteric modulator; NAM, negative allosteric modulator.
CPCCOEt, 7-(hydroxyimino)cyclopropa[b]chromen1a-carboxylate ethyl ester.) mGlu5 Receptor
The mGlu5 receptor consists of 1212 amino acids in humans and 1203 amino acids in rats; accession numbers are P41594 (human) and P31424 (rat). The gene is named GRM5 (human) and Grm5 (rat, mouse); chromosomal locations are 11q14.2 (human), 1q32 (rat), and 7 D3 (mouse). Activation of recombinantly expressed mGlu5 receptors induces oscillatory increases in intracellular Ca2þ release. This property, which is not shared by mGlu1 receptors, relies on the presence of a Ser residue in the C-terminus domain, which is phosphorylated by PKC, and places the mGlu5 receptor at the core of basic processes in cell biology. Similar to the mGlu1 receptor, the mGlu5 receptor is localized in postsynaptic elements, where it is physically linked to NMDA receptors via a chain of interacting proteins, which include PSD-95, Shank, guanylate kinaseassociated protein (GKAP), and Homer (Figure 2(a)). Activation of mGlu5 receptors facilitates the opening of NMDA-gated ion channels, and activation of NMDA receptors amplifies mGlu5 receptor function by inhibiting receptor desensitization. Because of their functional interaction with NMDA receptors, mGlu5 receptors are involved in the induction of long-term potentiation (LTP), a putative electrophysiological substrate of associative learning. mGlu5 knockout mice show a defective LTP in the hippocampus and an impaired spatial learning. Activation of mGlu5 receptors also mediates a nonNMDA-dependent form of LTD in the hippocampus. mGlu5a and mGlu5b variants show an opposite developmental pattern of expression in the central
nervous system (CNS). mGlu5a receptors are abundantly expressed early after birth, and mediate the robust PI response to excitatory amino acids in the first 2 weeks of postnatal life. Expression of mGlu5b receptors increases with age and is prominent in the adult hippocampus, striatum, and cerebral cortex. mGlu5 receptors are also found in peripheral cells, including thymocytes, hepatocytes, melanocytes, cells of the male germinal line, osteoblasts, and insulinoma cell lines, where their function begins to be explored. Remarkably, mGlu5 receptors are present in both embryonic and neural stem cells, and their activation supports proliferation and self-renewal of these cells. A dysfunction of mGlu5 receptors has been associated with the fragile X syndrome, the most frequent form of inherited mental retardation. This syndrome is caused by the absence of the fragile X mental retardation protein (FMRP), an RNA-binding protein which functions as a negative regulator of protein synthesis. LTD mediated by mGlu5 receptors is enhanced in mice lacking FMRP, and pharmacological blockade of mGlu5 receptors partially corrects neurological and behavioral abnormalities in these mice. mGlu5 receptors are selectively activated by 2-chlorohydroxyphenylglycine (CHPG), which, however, is a relatively weak agonist. No selective competitive antagonists are currently available. 2-Methyl-6-(phenylethynyl)pyridine (MPEP), 6-methyl-2-(phenylazo) pyridin-3-ol (SIB-1757), and (E)-2-methyl-6-stryrylpyridine (SIB-1893) are prototypes of a growing list of negative allosteric modulators of mGlu5 receptors, which includes 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl] pyridine (MTEP) and the anxiolitic drug, fenobam. Compounds 3,3’-difluorobenzaldazine (DFB), S-(4-fluorophenyl)-{3-[3-(4-fluorophenyl)-[1,2,4] oxadiazol-5-yl]-piperidin-1-yl}methanone (AD X47273), 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl) benzamide (CDPPB), and N-{4-chloro-2-[(1,3dioxo-1,3-dihydro-2H-isoindol-2-yl) methyl]phenyl}2-hydroxybenzamide (CPPHA) are example of mGlu5 receptor enhancers which amplify receptor function in an activity-dependent manner. mGlu5 receptor ligands are of potential interests for the treatment of psychiatric and neurological disorders, such as anxiety, schizophrenia, parkinsonism, and neuropathic pain. Systemic administration of MPEP produces anxiolytic effects in a number of tests, including fear-potentiated startle, auditory and contextual fear conditioning, the elevated plus maze, conflict tests, and stress-induced hyperthermia. These effects are mediated by mGlu5 receptor blockade in the hippocampus and in the central nucleus of the amygdala, two regions that are actively involved in the acquisition and expression of fear conditioning. Pharmacological blockade or genetic
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deletion of mGlu5 receptors disrupts prepulse inhibition of the acoustic startle reflex in rodents, thus mimicking the deficit in sensorimotor gating typical of schizophrenic patients. Administration of MPEP also enhances the cognitive deficit induced by the psychotomimetic drug, phencyclidine. Hence, mGlu5 receptor enhancers are currently developed as potential antipsychotic agents. mGlu5 receptors are highly expressed in all stations of the basal ganglia motor circuit, and regulate the ‘indirect pathway’ of motor control by opposing the action of dopamine released from nigrostriatal terminals. The activity of this pathway is negatively regulated by the dopamine released from nigrostriatal fibers, which acts on D2 inhibitory receptors located on medium-size spiny GABAergic neurons that project to the external portion of the globus pallidus (GPe). In striatal projection neurons, mGlu5 receptors physically interact with A2A adenosine receptors, and act synergistically with them in antagonizing the inhibitory control exerted by D2 receptors. In addition, activation of mGlu5 receptors inhibits GPe neurons by a mechanism of cross-desensitization with mGlu1 receptors, and stimulates neurons of the subthalamic nucleus. Pharmacological blockade or genetic deletion of mGlu5 receptors protects mice against nigrostriatal damage produced by the parkinsonian toxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), or the psychostimulant, methamphetamine. This raises the attractive possibility that mGlu5 receptor antagonists/negative modulators behave as antiparkinsonian agents by relieving motor symptoms and reducing the ongoing degeneration of nigrostriatal neurons at the same time. mGlu5 and mGlu1 receptors are implicated in the pathophysiology of chronic pain. Both receptor subtypes are localized on peripheral unmyelinated sensory afferents, and their activation increases the sensitivity to noxious heat, a phenomenon termed ‘thermal hyperalgesia.’ Activation of mGlu5 receptors in peripheral nociceptors stimulates PI hydrolysis and arachidonic acid release from DAG, leading to prostaglandin formation. Autocrine or paracrine stimulation of prostanoid receptors by prostaglandins in nociceptors results into an increased sensitivity of vanilloid TRPV1 receptors, thus inducing thermal hyperalgesia. Group I mGlu receptors also contribute to the regulation of nociceptive transmission and plasticity in the dorsal horns of the spinal cord, and perhaps in other stations of the pain neuraxis. Antagonists/negative modulators of mGlu5 and mGlu1 receptors produce analgesic effects in models of inflammatory and neuropathic pain. Finally, mGlu5 receptors have been implicated in the synaptic mechanisms underlying drug addiction. These receptors positively regulate brain reward
function and drive drug consumption. Blockade of mGlu5 receptors by MPEP elevates the intracranial self-stimulation threshold, decreases cocaine and nicotine self-administration, and attenuates naloxoneinduced somatic signs of morphine withdrawal. mGlu2 and mGlu3 Receptors
The mGlu2 receptor consists of 872 amino acids in humans, rats, and mice; accession numbers are Q14416 (human) and P31421 (rat). The gene isnamed GRM2 (human) and Grm2 (rat, mouse); chromosomal locations are 3q21.31 (human), 8q32 (rat), and 9 (mouse). The mGlu3 receptor consists of 877 amino acids in humans and 879 amino acids in rats and mice; accession numbers are Q14832 (human), P31422 (rat), and Q9QYS2 (mouse). The gene is named GRM3 (human) and Grm3 (rat, mouse); chromosomal locations are 7q21.1–q21.2 (human), 4q32 (rat), and 5A1-h (mouse). mGlu2 and mGlu3 are highly homologous and have a similar functional and pharmacological profile. Both receptors are predominantly localized in the preterminal region of the axon, and, if present in glutamatergic nerve terminals, can only be activated by amounts of glutamate sufficient to escape the clearance mechanisms around the synapses (Figure 2(b)). Activation of group II mGlu receptors attenuates neurotransmitter release as a result of a reduced cAMP formation and inhibition of voltage-sensitive Ca2þ channels. In brain slices, activation of mGlu2/3 receptors fails to stimulate PI hydrolysis per se, but amplifies the stimulation of PI hydrolysis mediated by group I mGlu receptors. The functional significance of this mechanism is unknown. mGlu3 receptors are also found in astrocytes, where their activation stimulates the MAPK and PI3K pathways and leads to the production of tropic factors (Figure 2(b)). Both mGlu2 and mGlu3 receptors are activated by 2R,4R-4-aminopyrrolidine-2,4-dicarboxylic acid (APDC; (a selective agonist with micromolar affinity), and by the cyclopropan derivatives, 2S,20 R,30 R)2-(20 ,30 -dicarboxycyclopropyl)glycine (DCG-IV) and (2S,10 S,20 S)-2-(carboxycyclopropyl)glycine (L-CCG-I). Both drugs activate mGlu2 and mGlu3 receptors in the mid-nanomolar range; however, DCG-IV is also an NMDA receptor agonist, whereas L-CCG-I can also activate group I mGlu receptors. (1S,2S,5R,6S)(þ)-2-Aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid (LY354740) and (–)-2-oxa-4-aminocyclo[3.1.0] hexane-4,6-dicarboxylic acid (LY379268) are conformationally constrained glutamate analogs in which the glutamate backbone is locked into a fully extended state by incorporation into a bicycle[3.1.0]
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hexane ring system. Both compounds are potent and selective mGlu2/3 receptor agonists with affinity in the low nanomolar range, and are systemically active. LY354740 and its prodrugs are currently under clinical development for the treatment of anxiety and panic disorders (see later). (1R,2S,5S,6S)-2-Amino6-fluoro-4-oxobicyclo[3.1.0]hexane-2,6-dicarboxylic acid monohydrate (MGS0028) is another potent and selective agonist of mGlu2/3 receptors. Compounds (2S,10 S,20 S)-2-(9-xanthylmethyl)-2-(20 -carboxycyclopropyl)glycine (LY341495) and (1R,2R, 3R,5R,6R)-2-amino-3-(3,4-dichlorobenzyloxy)-6fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic acid (MGS0039) are mGlu2/3 receptor antagonists with nanomolar affinity. However, LY341495 recruits other mGlu receptor subtypes at micromolar concentrations and is considered as a pan-mGlu receptor antagonist. There are only a few tools that can help discriminate mGlu2 from mGlu3 receptors. No subtype-selective mGlu2 receptor agonists are available. N-Acetyl-aspartyl-glutamate (NAAG) is an endogenous compound that activates mGlu3 receptors and has no activity at mGlu2 receptors. However, NAAG displays a low potency and is also active at NMDA receptors. A growing number of positive allosteric modulators, such as 2,2,2-trifluoro-N-[3-(2-methoxyphenoxy)phenyl]-N-(3-pyridinylmethyl)-ethanesul fonamide (3-MP PTS) and 2,2,2-trifluoro-N-[3(cyclopentyloxy)phenyl]-N-(3-pyridinylmethyl)ethanesulfonamide (cyPPTS), selectively amplify responses mediated by mGlu2 receptors. No mGlu3 receptor enhancers are currently available. The development of mGlu2/3 receptor agonists or enhancers is aimed at the treatment of a number of psychiatric disorders, such as anxiety disorders, schizophrenia, and drug addiction. The agonist LY354740 is effective in all animal models of anxiety, and is also active in humans in preventing CO2-induced anxiety and panic attacks and relieving symptoms of generalized anxiety disorder (GAD). Remarkably, pharmacological activation of mGlu2/3 receptors does not produce adverse effects, such as sedation, ataxia, and dependence, that are typical of benzodiazepines and other CNS depressants, although it may disrupt memory processing. mGlu2 and mGlu3 receptors are highly expressed in limbic regions, and may regulate transmission at the synapses between the basolateral nuclei and the central nucleus of the amygdala. A final effect of LY354740 appears to be an enhanced GABAergic activity within the central nucleus of the amygdala, as shown by an increased Fos protein expression in GABAergic neurons. It appears that both mGlu2 and mGlu3 receptors are necessary for the anxiolytic effect of LY354740, although selective mGlu2 enhancers can also relieve anxiety.
mGlu2/3 receptor agonists or mGlu2 receptor enhancers are active in models predictive of antipsychotic activity, including the disruption of working memory, motor activity, sensory–motor gating (only mGlu2 enhancers), and glutamate efflux induced by phencyclidine or amphetamine. In addition, there is a functional antagonism between mGlu2/3 receptors and serotonin 5-HT2A receptors, which mediate the psychotomimetic action of LSD and other hallucinogenic drugs. Thus, there is a common pathway for the action of mGlu2/3 receptor agonists and atypical antipsychotic drugs, such as clozapine or olanzapine, which act as 5-HT2A receptor antagonists. Although there is no association of polymorphism in the mGlu2 receptor gene with schizophrenia, an increased expression of mGlu2/3 receptors is found in the prefrontal cortex of schizophrenic patients. Thus, one expects that the brain of schizophrenic patients is highly responsive to drugs that activate mGlu2/3 receptors. mGlu3 receptors are present in astrocytes and microglia, where their activation stimulates the synthesis of neurotropic factors, such as transforming growth factor-b (TGF-b), nerve growth factor (NGF), and brain-derived neurotropic factor (BDNF). An increased secretion of TGF-b from astrocytes mediates the neuroprotective activity of group II mGlu receptor agonists in culture. mGlu3 receptors are also found in neural stem cells and human glioma cells, and their blockade inhibits cell proliferation by dampening the stimulation of the MAPK and the PI3K pathways. Group II mGlu receptors have been recently shown to play a role in the synaptic adaptations that occur during the development of drug dependence. Activation of mGlu2/3 receptors negatively regulates brain reward function, as agonists elevate the intracranial self-stimulation threshold and decrease relapse to drug-taking behavior during abstinence, thus regulating the negative affective state observed during withdrawal. Finally, mGlu2/3 receptors mediate analgesic effects via a variety of mechanisms, including a reduced sensitivity of TRPV1 channels in peripheral nociceptors, and a reduced neurotransmitter release at the synapses between primary afferent fibers and second-order neurons in the dorsal horn of the spinal cord. L-Acetylcarnitine, a drug currently used for the treatment of painful neuropathies, induces the expression of mGlu2 receptors in dorsal root ganglia neurons by amplifying the activity of transcription factors of the nuclear factor-kappa B (NF-kB) family. mGlu4, mGlu7, and mGlu8 Receptors
The mGlu4 receptor consists of 912 amino acids in humans, rats, and mice; accession numbers are Q14833 (human), P31423 (rat), and Q68EF4 (mouse). The gene is named GRM4 (human) and
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Grm4 (rat, mouse); chromosomal locations are 6p21.3 (human), 20p12 (rat), and 17 A3.3 (mouse). The mGlu7 receptor consists of 915 amino acids in humans, rats, and mice; accession numbers are Q14831 (human), P35400 (rat), and Q68ED2 (mouse). The gene is named GRM7 (human) and Grm7 (rat, mouse); chromosomal locations are 3p26.1 (human), 4q42 (rat), and 6 E3 (mouse). The mGlu8 receptor consists of 908 amino acids in humans, rats, and mice; accession numbers are O00222 (human), P70579 (rat), and P47743 (mouse). The gene is named GRM8 (human) and Grm8 (rat, mouse); chromosomal locations are 7q31.3–q32 (human), 4q22 (rat), and 6 A3 (mouse). These three receptor subtypes are preferentially, although not exclusively, located in the terminal region of the axons and share the ability to inhibit neurotransmitter release at various synapses in the CNS. Their location in the vicinity of the active zones of neurotransmitter release makes these subtypes as putative glutamate autoreceptors (Figure 2(b)). L-2-Amino-4-phosphonobutanoate (L-AP4) and the endogenous compound L-serine-O-phosphate (L-SOP) are prototypical orthosteric agonists of all group III mGlu receptors. (þ)-4-Phosphonophenylglycine (4-PPG) activates receptors with the following rank order of potency: mGlu8 > mGlu4 >> mGlu7. (S)3,4-Dicarboxyphenylglycine (DCPG) is an agonist with greater affinity for mGlu8 than for mGlu4, whereas (1S,3R,4S)-1-aminocyclopentane-1,3,4-tricarboxylic acid (ACPT-I) activates mGlu4 and mGlu8 receptors with equal potency. Neither DCPG nor ACPT-I has any effect at mGlu7 receptors. (R,S)-aCyclopropyl-4-phosphonophenylglycine (CPPG) and (S)-a-methyl-2-amino-4-phosphonobutanoic acid (M AP-4) are competitive antagonists with higher affinity for mGlu8 receptors. CPPG has no apparent activity at mGlu7 receptors. (–)-N-Phenyl-7-(hydroxyimino) cyclopropa[b]chromen-1a-carboxamide (PHCCC) is an enhancer with high selectivity for mGlu4 receptors. At high concentrations, PHCCC may also recruit mGlu1 receptors because of its similarity with CPCCOEt (see earlier). A selective agonist of mGlu7 receptors, N,N0 -dibenzhydrylethane-1,2diamine dihydrochloride (AM N082), has recently been synthesized. This compound is atypical because it does not bind to the VFT (like all classical mGlu receptor agonists), but to a site located in the 7TM domain of mGlu7 receptors. Activation of mGlu4 receptors is neuroprotective and mGlu4 receptor agonists/enhancers have been proposed as potential therapeutic agents in Parkinson’s disease for their ability to reverse parkinsonian symptoms in animal models. This action appears to be mediated by the inhibition of GABA release in
the GPe, which lies along the ‘indirect pathway’ of the basal ganglia motor circuit. Activation of mGlu4 receptors is also protective against experimental parkinsonism induced by MPTP in mice. mGlu4 receptors are highly expressed by cerebellar granule cells, and are also found in medulloblastoma cells, which derive from granule cell neuroprogenitors. Interestingly, activation of mGlu4 receptors inhibits proliferation of medulloblastoma cells and limits the spontaneous growth of medulloblastomas in the cerebellum of tumor-prone mice. The function of mGlu7 and mGlu8 receptors is only partially known. Both receptors have been proposed as drug targets in treatment of anxiety and stress-related disorders, but there are some caveats. The mGlu7 receptor agonist, AMN082, relieves anxiety, but genetic deletion of mGlu7 receptors does the same. In contrast, mGlu8 knockout mice show an increased level of anxiety, similar to that found in animals subjected to stressful conditions. Finally, mGlu4, mGlu7, and mGlu8 receptors have been considered as potential targets for the experimental therapy of epilepsy. Drugs that activate mGlu4 or mGlu8 receptors reduce generalized seizures in epilepsy-prone mice and rats and chemically induced seizures. In addition, mice lacking mGlu7 or mGlu8 receptors show an increased susceptibility to chemically induced seizures. Expression of mGlu4 receptors is increased in the dentate gyrus of mice with a low susceptibility to seizures and of patients with temporal lobe epilepsy. In contrast, activation of mGlu4 receptors is required for the induction of absence seizures in mice. mGlu6 Receptor
The mGlu6 receptor consists of 877 amino acids in humans and 871 amino acids in rats and mice; accession numbers are O15303 (human), P35349 (rat), and Q5NCH9 (mouse). The gene is named GRM6 (human) and Grm6 (rat, mouse); chromosomal locations are 5q35 (human), 10 (rat), and 11 B1.3 (mouse). This is a peculiar receptor; it is exclusively localized in the dendrites of ON bipolar cells of the retina and responds to the glutamate released from rod and cone photoreceptor cells in the dark. Activation of mGlu6 receptors leads to hyperpolarization of ON bipolar cells by closing cGMP-dependent cation channels. This effect may involve a receptor coupling with Go and activation of the Ca2þ-dependent protein phosphatase, calcineurin (Figure 2(c)). Light reduces glutamate stimulation of mGlu6 receptors, leading to opening of cation channels and depolarization of ON bipolar cells. Mice lacking mGlu6 receptors show a loss of ON response but unchanged responses to light. Interestingly, mutations in the gene encoding
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the mGlu6 receptor are associated with night blindness and abnormal electroretinogram ON responses in humans. The mGlu6 receptor shows the same pharmacological profile of other group III mGlu receptors, being activated by L-AP4, L-SOP, and 4PPG, and antagonized by CPPG, MSOP, and MAP-4. See also: Long-Term Depression (LTD): Metabotropic Glutamate Receptor (mGluR) and NMDAR-Dependent Forms; Metabotropic Glutamate Receptors (mGluRs): Functions.
Further Reading Alexander GM and Godwin DW (2006) Metabotropic glutamate receptors as strategic targets for the treatment of epilepsy. Epilepsy Research 71: 1–22. Bear MF, Huber KM, and Warren ST (2004) The mGluR theory of fragile X mental retardation. Trends in Neurosciences 27: 370–377. Bruno V, Battaglia G, Copani A, et al. (2001) Metabotropic glutamate receptor subtypes as targets for neuroprotective drugs. Journal of Cerebral Blood Flow and Metabolism 21: 1013–1033. Conn PJ, Battaglia G, Marino MJ, et al. (2005) Metabotropic glutamate receptors in the basal ganglia motor circuit. Nature Review Neuroscience 6: 787–798. DeBlasi A, Conn PJ, Pin J, et al. (2001) Molecular determinants of metabotropic glutamate receptor signaling. Trends in Pharmacological Sciences 22: 114–120. Fourgeaud L (2005) Addicted to Holmer? Journal of Neuroscience 25(42): 9555–9556. Kenny PJ and Markou A (2004) The ups and downs of addiction: Role of metabotropic glutamate receptors. Trends in Pharmacological Sciences 25: 265–272.
Kunishima N, Shimada Y, Tsugji Y, et al. (2000) Structural basis of glutamate recognition by a dimeric metabotropic glutamate receptor. Nature 407: 971–977. Pollock PM, Cohen-Solal K, Sood R, et al. (2003) Melanoma mouse model implicates metabotropic glutamate signaling in melanocytic neoplasia. Nature Genetics 34: 108–112. Nakanishi S (1992) Molecular diversity of glutamate receptors and implication for brain function. Science 258: 597–603. Nicoletti F, Wroblewski J, Iadarola MJ, et al. (1986) Excitatory amino acid recognition sites coupled with inositol phospholipid metabolism: Developmental changes and interaction with alpha 1-adrenoceptors. Proceedings of the National Academy of Sciences of the United States of America 83: 1931–1935. Pin JP, Kniazeff J, Liu J, et al. (2004) Allosteric functioning of dimeric class C G-protein-coupled receptors. FEBS Journal 272: 2947–2955. Schoepp DD, Jane DE, and Monn JA (1999) Pharmacological agents acting at subtypes of metabotropic glutamate receptors. Neuropharmacology 38: 1431–1476. Sladeczek F, Pin JP, Recasens M, et al. (1985) Glutamate stimulates inositol phosphate formation in striatal neurones. Nature 317: 717–719. Sugiyama H, Ito I, and Hirono C (1987) A new type of glutamate receptor linked to inositol phospholipid metabolism. Nature 325: 531–533. Swanson CJ, Bures M, Johnson MP, et al. (2005) Metabotropic glutamate receptors as novel targets for anxiety and stress disorders. Nature Review Drug Discovery 4: 131–144. Varney MA and Gereau RW 4th (2002) Metabotropic glutamate receptor involvement in models of acute and persistent pain: Prospects for the development of novel analgesics. Current Drug Targets CNS Neurological Disorders 1: 283–296.
Relevant Website http://www.iuphar-db.org – IUPHAR Receptor Database.
Metabotropic Glutamate Receptors (mGluRs): Functions B A Grueter and D G Winder, Vanderbilt University School of Medicine, Nashville, TN, USA ã 2009 Elsevier Ltd. All rights reserved.
Metabotropic Glutamate Receptors In the mid-1980s, glutamate was shown to stimulate inositol 1,4,5-trisphosphate (IP3) production, indicating the potential existence of a ‘metabotropic,’ or non-ion-channel-forming, glutamate receptor. This led to the distinction between ionotropic and metabotropic glutamate receptors (mGluRs). There are eight subclasses of mGluRs that have been identified and cloned. mGluRs belong to family C of the G-protein-coupled receptor superfamily, which includes calcium-sensing and g-aminobutyric acid B (GABAB) receptors and act to modulate neuronal excitability in many brain regions. Eight separate gene products (mGluRs 1–8) comprise the mGluR family of receptors, several of which can exist as alternatively spliced variants. These subtypes are classified into three groups based on pharmacology, similarities in coupling mechanisms, and sequence homology (Table 1). mGluRs of the same group show about 70% sequence identity, whereas between groups this percentage decreases to about 45%. Group I receptors (mGlu1 and mGlu5) are linked to Gq, whereas, group II (mGlu2 and mGlu3) and group III (mGlu4, mGlu6, mGlu7, and mGlu8) receptors are coupled to Gi/o. Intracellularly, the large C-terminus of mGluRs can interact with a variety of signaling systems. G-Protein-Coupled Receptors
G-protein-coupled receptors (GPCRs) are seventransmembrane-domain (7TM) proteins that interact with G-proteins. Receptor activation leads to associated guanosine triphosphatase (GTPase) activity and ultimately to activation or inhibition of an effector protein. G-protein regulation of effectors, particularly in neurons, occurs through two broad mechanisms. One is direct G-protein binding to ion channels. More traditionally, G-proteins function to activate or inhibit second-messenger-forming enzymes, leading to direct second messenger binding to ion channels, second messenger activation of kinases or phosphatases that phosphorylate/dephosphorylate ion channels, or alterations in protein– protein interactions involving an ion channel. These mechanisms constitute the major routes for mGluR signaling, although it should be noted that these receptors have also been reported to signal through
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G-protein-independent ways through as yet unclear mechanisms. Structure of mGluRs
mGluRs are composed of two main domains, a large extracellular domain (ECD), and a 7TM region, and share very little sequence homology with other GPCRs. Proposed models confirmed by X-ray crystallography suggest the unique ECD contains two globular lobes separated by a cleft which contains the orthosteric ligand-binding site. While glutamate is commonly thought to be the primary ligand at this site, it is important to note that many excitatory amino acids are present in neural tissue, many of which can also activate mGluRs. In addition, allosteric binding sites have also been identified in the 7TM region. The second intracellular loop of mGluRs infers selectivity for G-protein coupling while a highly conserved third loop plays a crucial role in G-protein activation. Interactions among the first loop, third loop, and C-terminal tail are thought to control efficacy of G-protein coupling. The intracellular C-terminal region of mGluRs plays a major role in regulation and trafficking. Regulation of mGluR Signaling
mGluR signaling is tightly regulated by both homologous and heterologous mechanisms. Both agonistinduced (homologous) and heterologous forms of regulation of mGluRs have been observed. Phosphorylation by several different kinases can alter mGluR signaling. For instance, protein kinase C (PKC)dependent phosphorylation of mGluRs, particularly group I mGluRs, is thought to serve as a major mechanism of desensitization. b-Arrestin-induced desensitization of mGluRs has also been described. The bg subunits of G-proteins are thought to target G-protein-coupled receptor kinases (GRKs) to mGluRs, resulting in b-arrestin-mediated sequestration and ultimately desensitization. Group I mGluR expression at the plasma membrane and the functional coupling of group I mGluRs to intracellular signaling molecules are dependent upon scaffolding proteins. The Homer family of proteins is a major example of this. There are three genes encoding Homer proteins. Homer 1a is an immediate-early gene product while Homer 1b/c, Homer 2, and Homer 3 are constitutively expressed. Homer proteins link group I mGluRs through the C-terminal tail to signaling molecules such as the IP3 receptor, and to scaffolding components of the postsynaptic density, such as Shank. It is suggested that Homer
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Table 1 Classification of mGluRs Family receptor
Coupling
Group/subtype-selective pharmacological agentsa
Group I mGluR1
Gq-coupled
mGluR5
Gq-coupled
Agonists: DHPG, ACPD, quisqualate Antagonist: LY393675 Inverse agonist (allosteric antagonist): LY367385 Agonists: DHPG, ACPD, quisqualate, CHPG Inverse agonist (allosteric antagonist): MPEP
Group II mGluR2
Gi/o-coupled
mGluR3
Gi/o-coupled
Group III mGluR4
Gi/o-coupled
mGluR6
Gi/o-coupled
mGluR7
Gi/o-coupled
mGluR8
Gi/o-coupled
Agonists: DCG-IV, LY354730, ACPD Antagonist: LY341495 Agonists: DCG-IV, LY354730, ACPD Antagonist: LY341495 Agonists: L-AP4, L-SOP Antagonists: MSOP, MAP4 Agonists: L-AP4, L-SOP Antagonists: MSOP, MAP4 Agonists: L-AP4, L-SOP Antagonists: MSOP, MAP4 Agonists: L-AP4, L-SOP, DCPG Antagonists: MSOP, MAP4
a ACPD, (1S,3R)-1-amino-1,3-cyclopentanedicarboxylate; L-AP4, L-2-amino-4-phosphonobutyric acid; CHPG, (R,S)-2-chloro-5-hydroxyphenylglycine; DCG-IV, (2S,20 R,30 R)-2-(20 ,30 -dicarboxycyclopropyl)glycine; DCPG, (S)-3,4-dicarboxyphenylglycine; DHPG, 3,5-dihydroxyphenylglycine; MAP4, a-methyl-2-amino-4-phosphonobutyrate; MPEP, 2-methyl-6-(phenylethynyl)pyridine; MSOP, methylserine-O-phosphate; L-SOP, L-serine-O-phosphate.
proteins may regulate the expression and function of group I mGluRs at multiple levels, including targeting, surface expression, clustering, physical linkage to other synaptic and subsynaptic complexes, and modulation of constitutive activity. Interestingly, biochemical evidence suggests the C-terminus of Homer 2a interacts with the Rho family of small GTPases, in a GTP-dependent manner. Homer proteins have also been shown to form complexes with Shank proteins, which act as scaffolding proteins and link group I mGluRs with other proteins in the postsynaptic densities (PSDs), such as Dynamin2, an important molecule implicated in endocytosis, and a-amino-3hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR)–glutamate receptor-interacting protein (GRIP) complexes. Expression of mGluRs
mGluRs have unique but overlapping distributions throughout the central nervous system (CNS). In addition, mGluRs have been reported to function in the peripheral nervous system and elsewhere. For example, mGluRs have been shown to play an important role in nociceptive transmission, as will be described in a later section. Glutamate Sources from Which mGluRs Can Be Activated
mGluRs are often located in the areas adjacent to the synapse. Functionally, their location suggests that they
become activated in situations of repeated stimulation of afferents, which results in considerable glutamate accumulation in the synaptic cleft coupled to spillover to extrasynaptic sites. In addition to the frequency and duration of synaptic activity, the extracellular concentration of glutamate also depends on the efficacy of neuronal and glial uptake, which is dictated by the number of glutamate transporters (GluTs). GluTs, along with passive diffusion, are the primary means of glutamate removal from the extracellular space. The ability of mGluRs to participate in signaling is thought to be tightly regulated by GluT activity. Another potentially important contributor to extracellular glutamate levels is the cystine–glutamate exchanger. The nonsynaptic cystine–glutamate antiporter exchanges extracellular cystine for intracellular glutamate with a minimal contribution of synaptic glutamate release. It has been suggested that one form of cocaine-induced neuroadaptation may be due to a decrease in basal levels of extracellular glutamate in the N-acetylcysteine (NAc) after several weeks of withdrawal from cocaine, an effect attributed to a reduced function of the cystine–glutamate transporter.
mGluR Function Neuromodulation
Neuromodulation is a subtle influence on synaptic efficacy or neuronal excitability. Neuromodulation
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can also involve longer-lasting changes such as alterations in gene expression. The ‘fine tuning’ of neuronal function by neuromodulation makes receptors and pathways attractive therapeutic targets for many diseases and disorders. mGluR activation serves as a major source by which glutamatergic transmission can modulate synaptic efficacy, neuronal excitability, and gene expression. Synaptic Plasticity
Persistent alterations in synaptic efficacy initiated by transient stimuli are referred to as forms of synaptic plasticity. Changes in synaptic efficacy can occur by presynaptic mechanisms such as altered neurotransmitter release as well postsynaptic mechanisms. Activation of mGluRs can play an important role in facilitation of multiple forms of synaptic plasticity. mGluR activation can induce long-term depression (LTD) at many glutamatergic synapses. Further activation, primarily of group I mGluRs at some synapses, can positively modulate N-methyl-D-aspartate receptor (NMDAR)-dependent long-term potentiation (LTP) induction.
Group I mGluRs Group I receptors (mGluR1 and mGluR5) are linked to activation of phospholipase C (PLC), leading to phosphoinositide hydrolysis, calcium release, and PKC activation. Activation of group I mGluRs can also indirectly regulate adenylyl cyclase, leading to increased cyclic adenosine monophosphate (cAMP) formation, as well as cyclic guanosine monophosphate (cGMP) accumulation, phospholipase A, and extracellular signal-regulated kinase (ERK) activation. Although highly homologous, studies suggest mGluR1 and mGluR5 receptors have distinct functions in regulating synaptic transmission. This implies differential regulation of these receptors and potential divergence in effector systems. Conversely, at some synapses only one type of group I mGluR is functionally important: for instance, only mGluR1 has been shown to function in the ventral tegmental area and cerebellum. Synaptic Locus of Group I mGluRs
Mechanisms underlying group I mGluR function vary across brain regions and synapses. Much information on the function of these receptors can be gained by determining the synaptic locus of action of these receptors. Immunohistochemical and electrophysiological results reveal that group I mGluRs localize and function presynaptically, postsynaptically, and on glia. However, group I mGluRs are predominantly
located at the periphery of the postsynaptic density (perisynaptically). Activation of group I mGluRs causes postsynaptic effects such as neuronal depolarization, excitation, and spike frequency adaptation in a number of brain regions. For instance, selective activation of group I mGluRs with the agonist 3,5-dihydroxyphenylglycine (DHPG) increases postsynaptic membrane excitability in hippocampus, cortex, striatum, amygdala, subthalamic nucleus, and hypothalamic nuclei. Increases in neuronal excitability via activation of group I mGluRs are thought to occur in large part through modulation of Kþ channels. Specific activation of group I mGluRs induces net inward currents by inhibiting Kþ channel conductance in neurons in the hippocampus and hypothalamus. For instance, Kþ channels are a major target of mGluRs, and many diverse types of Kþ channels are inhibited following activation of mGluRs. Activation of mGluRs has been shown to block the IAHP (IAHP is an outward, Ca2þ-activated, Kþ current with slowly activating and inactivating (2 s) kinetics) and to thereby reduce accommodation of spike firing in hippocampal CA1 pyramidal cells, CA3 pyramidal cells, the dentate gyrus, cultured cerebellar Purkinje cells, and basolateral amygdaloid nucleus. Activation of group I mGluRs has also been shown to modulate voltage-gated calcium channels (VGCCs). Alterations in VGCCs can occur either presynaptically, consequently changing vesicular release probability, or postsynaptically, leading to changes in intracellular calcium levels. Although mGluR-dependent modulation of channels has been shown to be a consequence of direct G-protein-linked action – for example, inhibition of Ca2þ channels – many other effects occur as a result of activation of intracellular messenger pathways. Group I, in contrast to group II, mGluRs can use several distinct signal transduction pathways to inhibit Ca2þ channels, both Ca2þ intracellular-independent and -dependent mechanisms. In addition to the postsynaptic modulation of synaptic transmission by alteration of neuronal excitability, group I mGluRs have been reported to function presynaptically. One example is in the hippocampus, at the level of the synapses between Schaeffer collaterals and CA1 pyramidal cells. Consistent with presynaptic function, an increase in paired pulse facilitation has been observed upon activation of mGluR5, suggesting a decrease in vesicular release probability. Retrograde Signaling
Cannabinoids Of great interest in the mGluR field was the finding that endocannabinoid synthesis can be
Metabotropic Glutamate Receptors (mGluRs): Functions
triggered by activation of group I mGluRs. Retrograde signaling through the endocannabinoid system helped explain biophysical evidence suggesting a presynaptic change in probability of neurotransmitter release, with immunochemical data suggesting that group I mGluRs are primarily localized to postsynaptic structures. Use of pharmacological tools as well as genetic manipulations has provided evidence for a role of arachidonic acid metabolites – more specifically, the 12-lipoxygenase metabolite of arachidonic acid, 12(S)hydroxyeicosa-5(Z),8(Z),10(E),14(Z)-tetraenoic acid (HETE) – in inducing group I mGluR LTD in the hippocampus. Direct application of this metabolite mimicked and occluded mGluR5-dependent LTD at these synapses; mGluR5-dependent LTD in the hippocampus is absent in mice lacking the ‘leukocyte type’ 12-lipoxygenase, and group I mGluR LTD-inducing stimuli promote 12-lipoxygenase activity. A suggested potential downstream target of 12-lipoxygenase is p38 kinase. p38 kinase, like ERK, is a member of the mitogen-activated protein kinase (MAPK) family and has been implicated in group I mGluR LTD. For instance, in the dentate gyrus, group I mGluR LTD is dependent on p38 kinase signaling and in the CA1 of the hippocampus activation of group I mGluRs results in AMPA receptor endocytosis (which will be discussed in greater detail in a subsequent section) and is dependent on p38 kinase signaling as well as protein synthesis. Kinases Involved in Group I mGluR Function
Group I mGluRs are linked to the activation of multiple kinases. Consistent with coupling of group I mGluRs with Gq and Ca2þ signaling, activation of these receptors can lead to activation of the Ca2þ/diacylglycerol (DAG)-dependent PKC family. mGluR-induced activation of PKC can lead to phosphorylation of a multitude of downstream targets, ultimately resulting in neuronal modifications such as LTP or LTD. One such target of mGluR-stimulated PKC activity is the MAPK/ERK pathway. The ERK pathway is a signaling cascade that plays a crucial role in a variety of cell regulatory events, including cell proliferation, differentiation, and survival, as well as an involvement in long-term synaptic changes and behavior. In neurons, ERK activation has been shown to be involved in processes associated with synaptic remodeling and long-term changes in synaptic efficacy. These processes include protein synthesis, changes in gene expression, dendritic spine stabilization, modulation of ion channels, and receptor insertion. The ERK cascade is activated by a variety of extracellular agents, including growth factors, hormones, and neurotransmitters, and serves as an important
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point of convergence for the PKC and protein kinase A (PKA) pathways. PKC regulates ERK activity through an interaction with either Ras or Raf-1, leading to activation of mitogen-activated protein extracellular kinase (MEK) and consequently ERK. Additionally, it was discovered that DAG, another second messenger product of PLC activity, is capable of activating ERK independent of PKC activation. Consistent with a role for PKA and PKC in some forms of group I mGluR LTD, group I mGluR LTD in the hippocampus, bed nucleus of the stria terminalis (BNST), and cerebellum is dependent on ERK activation. In addition to these kinases, receptor tyrosine phosphatases have also been shown to play an integral role in the group I mGluR-induced removal of AMPARs from the synapse, resulting in LTD of excitatory synaptic transmission. Pharmacology of Group I mGluRs
The recent development of mGluR-selective ligands (e.g., phenylglycine derivatives acting either as agonists or as antagonists) has helped begin the elucidation of the functional roles of mGluRs in brain and behavior. The pharmacological profile of group I mGluRs has been determined in mammalian heterologous expression systems on cloned mGluRs. The rank order of potency of the most common agonists is quisqualate > DHPG ¼ glutamate > (1S,3R)-1amino-1,3-cyclopentanedicarboxylate (ACPD). The specific agonist for group I mGluRs used in the studies presented in the following sections is DHPG, which is devoid of activity at other mGluRs. An agonist for mGluR5 – (R,S)-2-chloro-5-hydroxyphenylglycine (CHPG) – has been reported, although it is not very potent. The first antagonist described for group I mGluRs was a-methyl-4-carboxyphenylglycine (MCPG). However, MCPG also antagonizes group II mGluRs. There are several drugs that selectively inhibit mGluR5; the most widely used is the noncompetitive antagonist 2-methyl-6-(phenylethynyl)pyridine (MPEP), with the caveat that at high concentrations it blocks NMDA receptors. Group I mGluRs and Plasticity
Group I mGluRs have been shown to potentiate either NMDAR- or AMPAR-mediated responses. However, a major function of group I mGluR activation is to produce a persistent weakening of glutamatergic transmission at synapses in the CNS. There appear to be at least two mechanisms through which this is accomplished. One involves the recruitment of endocannabinoid signaling and presynaptic alterations, and has been observed in the dorsal and ventral striatum.
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A second major mechanism is through endocannabinoid-independent signaling mechanisms, and has been heavily studied in the hippocampus and cortical regions. Both presynaptic and postsynaptic mechanisms have been described for endocannabinoidindependent forms of group I mGluR LTD. Group I mGluRs Can Alter iGluR Function
Group I mGluRs have been shown to modulate iGluRs in several ways. Activation of mGluRs can lead to changes in neuronal excitability and therefore influence NMDAR activity. For instance, membrane depolarization via group I mGluR stimulation can enable enhanced NMDAR-mediated synaptic plasticity. Additionally, through activation of kinases/phosphatases, group I mGluRs can alter the signaling properties of iGluRs and can influence trafficking of these receptors into and out of the synapse. PKC phosphorylates NMDA and AMPA receptors. The activation of PKC by mGluRs might therefore modulate the phosphorylation state of these two ionotropic receptors, resulting in a potentiation of the response. Activation of group I mGluRs can lead to a removal of AMPA receptors from the membrane, resulting in a depression of synaptic transmission. More specifically, following activation of group I mGluRs there is a reduction in the density of postsynaptic AMPARs.
mGluR2 and mGluR3, with an EC50 of 1050 nM in the rat cortex, hippocampus, and striatum, and 10 or 30 nM in cells expressing recombinant mGluR2 or mGluR3, respectively. LY341495 ((2S)-2-amino-2-[(1S,2S)-2carboxycycloprop-1-yl]-3-(xanth-9-yl) propanoic acid) is an antagonist of mGluRs, with nanomolar potency against group II mGluRs and micromolar potency against group III mGluRs. LTD of excitatory transmission can be induced by activation of group II mGluRs in several brain regions. For example, group II mGluR LTD has been shown presynaptically in the basolateral amygdala (BLA), the nucleus accumbens, the BNST, and the striatum. In contrast, it was shown that stimulation of thalamic inputs to the lateral nucleus of the amygdala induces a group II mGluR-mediated postsynaptic LTD. Additionally, in the dentate gyrus and the medial PFC it was found that group II mGluR activation induced a postsynaptic LTD that was PKA and PKC dependent. Behavioral studies of mice lacking mGluR2 or mGluR3 support earlier findings of a role of these receptors in anxiety behaviors. Consistent with the role of these receptors in various animal models of anxiety/stress, group II mGluR agonists have been considered promising candidates as therapeutic agents for the treatment of anxiety as well as other brain disorders.
Group III mGluRs Group II mGluRs Group II mGluRs (mGluR2/3) are widely distributed throughout the CNS, where they are moderately to highly expressed in brain regions that are commonly associated with anxiety disorders, including the hippocampus, prefrontal cortex (PFC), and amygdala. mGluR2 is generally localized at the periphery of the presynaptic terminal, suggesting a role for these receptors at instances of spillover of glutamate beyond the synaptic cleft in response to repetitive stimulation. By contrast, mGluR3 is more diversely localized, including both pre- and postsynaptic localization on neurons, as well as relatively heavy localization on glial cells. Group II mGluRs most commonly function presynaptically to decrease the probability of synaptic vesicle release, but at multiple synapses within the CNS postsynaptic mechanisms have been reported. Voltagedependent Ca2þ channels are likely to be involved in the presynaptic inhibition mediated by mGluRs. Additionally, it has been suggested that Kþ channels mediate some of the effects induced by activation of group II or III mGluRs. LY354740 ((1S,2S,5R,6S)-(þ)-2-aminobicyclo[3.1.0] hexane-2,6-dicarboxylic acid) and several related compounds are potent and selective agonists at
Like group II mGluRs, group III mGluRs are coupled to inhibition of cAMP production, and modulation of ion channels. However, in contrast to the other groups, group III mGluRs are primarily located at presynaptic active zones at the axon terminal. As with the group II mGluRs, the group III mGluRs inhibit neurotransmitter release, resulting in a reduction in glutamatergic or GABAergic synaptic transmission. Unfortunately, there are few pharmacological tools with the desired receptor subtype selectivity ideal for testing the significance of the subtypes of group III mGlu receptors. L-2-Amino-4phosphonobutanoate (L-AP4), the most commonly used selective group III agonist, has a high affinity for mGluR4, mGluR6, and mGluR8 and a low affinity for mGluR7. One of the most promising drugs available for group III mGluRs is the positive allosteric modulator of mGluR4, (–)-N-phenyl-7(hydroxyimino)cyclopropa[b]chromen-1a-carboxamide (PHCCC). (S)-3,4-Dicarboxyphenylglycine (DCPG), a more recently described group III mGluR agonist, is a relatively specific mGluR8 agonist. While group III mGluR function has been reported in brain regions such as the BLA, central nucleus of the amygdala, BNST, and hippocampus using an agonist
Metabotropic Glutamate Receptors (mGluRs): Functions
pharmacology approach, behavioral analysis of specific receptor mutant mice has also proved useful. In combination with the limited pharmacology, mutants of mGluR4, mGluR7, and mGluR8 have all been shown to have behavioral phenotypes. For instance, consistent with a dysregulation of the hypothalamic– pituitary–adrenal (HPA) axis, mice with targeted deletion of mGluR7 show changes in animal behavior paradigms predictive of antidepressant and anxiolytic action. They also exhibit a deficit in fear responses and learning paradigms. Interestingly, administration of the mGluR4 allosteric potentiator, PHCCC, also has anxiolytic-like effects, implicating a role for this receptor in anxiety as well. mGluR4 knockout (KO) mice have altered glutamate and GABA release and, interestingly, mGluR4 activation protects against neurodegeneration. mGluR8 has been implicated in conditioned fear, and similar to the other group III mGluRs, mice lacking mGluR8 have an increased anxiety phenotype. Of the mGluRs, mGluR6 has the most restricted expression. These receptors are expressed postsynaptically at ON bipolar cells in both rod and cone systems and function as the main excitatory receptor responsible for relaying synaptic signals in these retinal cells. Consistent with their important functional role in visual processing, genetic targeting of mGluR6 impairs detection of visual contrasts.
Diseases Many psychiatric as well as neurological diseases have been linked to alterations in neuronal excitability via the glutamatergic system. As mGluRs function to regulate a large number of synapses, and are known to play a role in pathophysiological conditions, compounds that act on these receptors are prime targets for a number of therapeutic applications. For instance, drugs targeting mGluRs have been shown to prevent certain forms of pain and anxiety. There are positive potential therapeutic effects of mGluR compounds in treating Parkinson’s disease as well as the conditions of drug dependence and withdrawal. Also, activating or potentiating mGluRs may help in treatment of schizophrenia and Alzheimer’s disease. An interesting model suggests a specific role for group I mGluRs in fragile X mental retardation. Intense investigation reveals a role for mGluRs in nociception both centrally and peripherally. For instance, group I mGluRs have been implicated in the processes of central sensitization and persistent nociception, whereas activation of group II mGluRs is effective against neuropathic or inflammatory pain.
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Pharmacology/Therapeutic Utility of mGluRs An exciting new direction in the pharmacological targeting of mGluRs has been the development of subtype-selective allosteric modulators. An advantage of allosteric modulators over traditional compounds is that allosteric modulators rely on activation by the endogenous transmitter released in an activity-dependent manner. mGluRs are prominent targets as both positive and/or negative allosteric modulators with unique pharmacological properties. Allosteric modulators, as compared to orthosteric ligands, appear to show a unique degree of selectivity. See also: Glutamate; Long-Term Depression (LTD): Metabotropic Glutamate Receptor (mGluR) and NMDARDependent Forms; Long-Term Depression (LTD): Endocannabinoids and Cerebellar LTD; Long-Term Potentiation (LTP): NMDA Receptor Role.
Further Reading Anwyl R (1999) Metabotropic glutamate receptors: Electrophysiological properties and role in plasticity. Brain Research Reviews 29: 83–120. Bordi F and Ugolini A (1999) Group I metabotropic glutamate receptors: Implications for brain diseases. Progress in Neurobiology 59: 55–79. Conn PJ and Pin JP (1997) Pharmacology and functions of metabotropic glutamate receptors. Annual Review of Pharmacology and Toxicology 37: 205–237. DeBlasi A, Conn PJ, Pin J, et al. (2001) Molecular determinants of metabotropic glutamate receptor signaling. Trends in Pharmacological Sciences 22: 114–120. Pin JP and Duvoisin R (1995) The metabotropic glutamate receptors: Structure and functions. Neuropharmacology 34: 1–26. Rouse ST, Marino MJ, Bradley SR, et al. (2000) Distribution and roles of metabotropic glutamate receptors in the basal ganglia motor circuit: Implications for treatment of Parkinson’s disease and related disorders. Pharmacology & Therapeutics 88: 427–435. Schoepp DD (2001) Unveiling the functions of presynaptic metabotropic glutamate receptors in the central nervous system. Journal of Pharmacology and Experimental Therapeutics 299: 12–20. Sladeczek F, Pin JP, Recasens M, et al. (1985) Glutamate stimulates inositol phosphate formation in striatal neurones. Nature 317: 717–719. Sugiyama H, Ito I, and Hirono C (1987) A new type of glutamate receptor linked to inositol phospholipid metabolism. Nature 325: 531–533. Tanabe Y, Masu M, Ishii T, et al. (1992) A family of metabotropic glutamate receptors. Neuron 8: 169–179.
Kainate Receptors: Molecular and Cell Biology J Lerma, Instituto de Neurociencias de Alicante Consejo Superior de Investigaciones Cientı´ficas – Universidad Miguel Hernandez, San Juan de Alicante, Spain ã 2009 Elsevier Ltd. All rights reserved.
It is now well established that there are three types of ionotropic glutamate receptors (GluRs), whose nomenclature reflects their preferred ligands: AMPA (a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors, NMDA (N-methyl-D-aspartate) receptors, and kainate receptors (KARs). The cloning of many GluRs at the beginning of the 1990s and the discovery of their structural relationships confirmed the legitimacy of this pharmacological subdivision. Likewise, their cloning has enabled us to advance our understanding of the biophysical properties and the physiological role of each receptor subtype in the mammalian brain. Indeed, analyzing just a few of these GluR subunits emphasized that true KARs are ion channels with a stronger preference for this agonist rather than for AMPA and that they display rapidly desensitizing responses. Thus, defining the molecular biology of KAR subunits represents a real breakthrough in the study of these receptors, establishing the foundations for us to better understand their physiology.
The Diversity of Kainate Receptors It is now accepted that all ionotropic GluRs share a conserved transmembrane topology and stoichiometry. Like the AMPA receptors (AMPARs) and NMDA receptors (NMDARs), KARs are tetrameric combinations of the GluR5, GluR6, GluR7, KA1, and KA2 subunits. Of these, GluR5/6/7 can form functional homomeric or heteromeric receptors, whereas KA1 and KA2 participate in functional receptors only when accompanied by GluR5/6/7 subunits. In the ionotropic GluRs, each monomer carries its own ligand binding site and it contributes with a specific amino acid stretch to the channel lumen. In addition to this hydrophobic sequence, each subunit has three transmembrane segments (M1, M3, and M4) arranged in such a manner that the N-terminal domain of each protein lies extracellularly and the C-terminal region lies within the cell. As in the case of AMPARs, the glutamate binding site of KARs is formed by residues that are distributed throughout the distal N-terminal domain (called S1) and the loop between M3 and M4 (called S2). Indeed, this latter segment contains specific residues that confer
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the receptor with high or low sensitivity to various agonists (AMPA and kainate), as well as to ions that participate in the channel gating. According to the rules governing subunit assembly and the different arrangements that give rise to functional receptors, a wide diversity of KARs may be expressed in the brain. Of the five KAR subunits that have been cloned so far, some combinations do not render functional channels and others are not specific to kainate. For example, the GluR5 subunit forms receptors that can be activated not only by glutamate but also by kainate, domoate, and AMPA. When activated by kainate these receptor channels desensitize relatively slowly, whereas when they bind glutamate their desensitization is faster and almost complete. In contrast, GluR6 subunits form homomeric channels with fast-desensitizing kinetics when activated by either agonist. Unlike the GluR5 receptors, GluR6 homomeric channels are not sensitive to AMPA or to its derivative ((RS)-2-amino-3(3-hydroxy-5-tert-butylisoxazol-4-yl) propanoic acid (ATPA) (Figure 1(b)). Functional homomeric receptors can also be formed by GluR7 subunits, these displaying a very low affinity for glutamate and complete insensitivity to AMPA and domoate. Each of these three subunits also form heteromeric assemblies with KA1 or KA2, and the resulting heteromeric receptors present slightly different biophysical and pharmacological channel properties. Moreover, GluR5, GluR6, and GluR7 may also combine with one another to form functional receptors. The sequence alignment reveals a homology of 75–80% between GluR5, GluR6, and GluR7, whereas KA1 and KA2 present 68% of similarity. Homology, however, drops to 45% when these two subfamilies of KARs are considered. The homology of KAR subunits with AMPARs is also approximately 40%, although they are not able to form heteromeric receptors.
Kainate Receptor Subunits Are Subject to Alternative Splicing In addition to the inherent variation in KARs, alternative splicing generates a number of different isoforms of KAR subunits (Figure 1(a)). Indeed, two different GluR5 variants have been found, one that contains an extra segment of 15 amino acids in the N-terminal region (GluR5-1) and another that lacks this insert (GluR5-2). Furthermore, the GluR5-2 subunit presents four different C-terminal domains. The C-terminus of the 2a variant is 49 amino acids shorter than the variant originally described (which was then renamed 2b) due to the introduction of a premature stop codon. Variant 2c contains an extra in-frame exon
Kainate Receptors: Molecular and Cell Biology 309 GluR6 N-term
M4
M1 M2 M3 S1 I/V Y/C
K823
S2
Q/R
Glu 1 mM
C-term GluR6a
ATPA 1 mM
54 aa
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15 aa
200 ms GluR5 N-term GluR5-2 GluR5-1
15 aa insert
Q/R Q
-2a
CLS....
..VA875
CLS....
..VA904
29 aas
-2b 20 pA
-2c
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GluR7 N-term
Glu 30 mM M1 M2 M3
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C-term 64 aa
Q
N-term
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M1 M2 M3
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Figure 1 Kainate receptor (KAR) subunits: (a) structure of subunits; (b) electrophysiological recordings of responses to different agonists of the corresponding homomeric receptors recombinantly expressed in HEK293 cells. In (a), the KAR subunits are all made of a chain of approximately 900 amino acids (100 kDa) that spans the membrane three times (hydrophobic segments M1, M3, and M4). Thus, the N-terminal domain of the protein is extracellular and the C-terminal domain lies intracellularly. The M2 hydrophobic sequence does not cross the membrane, but it is thought to dip into the membrane forming a loop and forming the pore of the channel. Two isoforms of GluR6 subunits exist, -a and -b, differing in their C-terminal domain. The GluR5 can be found as two different variants: GluR5-1, which contains 15 extra amino acids in the N-terminal region, and GluR5-2, which lacks this insert. GluR5-2 presents three additional possibilities generated by a splice of the C-terminal domain. GluR5-2a is 49 amino acids shorter than the GluR5-2b because of the introduction of a premature stop codon. GluR5-2c contains an extra in-frame exon that makes it 29 amino acids longer than the 2b isoform. GluR7 can be present as two isoforms with different C-terminals due to a 13-amino-acid insertion (blue segment), which produces a new open reading frame and a completely different C-terminus (red segment). In contrast, no splice variants have been found for the KA1 and KA2 subunits. GluR5 and GluR6 subunits undergo mRNA editing at the M2 (Q/R site), generating a mixture of edited and nonedited subunits. GluR6 presents two additional editing sites in M1. In every case, a single receptor channel is believed to be composed of four subunits. In all KARs, the binding domain is formed by two peptide segments, S1 and S2, which are situated before the M1 (S1) and linking M3 and M4 (S2). In (b), responses of GluR6 are also shown when this subunit heteromerizes with KA2 subunits (bottom). GluR, glutamate receptor. ATPA, (RS)-2-amino-3(3-hydroxy-5-tert-butylisoxazol-4-yl) propanoic acid; Dom, domoic acid.
that makes it 29 amino acids longer than the 2b isoform. Finally, the last 15 amino acids of variant 2d (not shown), from Q824 onward, are completely unrelated to any other variant, and significantly this last isoform was isolated from human tissue. Two splice variants of the murine GluR6 have also been found and, again, these differ in their C-terminal domains. Furthermore, in the rats and humans two splice variants of the GluR7subunit have been discovered. In this case,
the insertion of 40 nucleotides out-of-frame in the C-terminal sequence of GluR7a leads to the production of a shorter protein, GluR7b, which bears no significant homology in this region to any of the known ionotropic GluRs. In contrast, the KA1 and KA2 subunits do not seem to undergo alternative splicing. Despite the existence of this repertoire of isoforms, the role of the different KAR splice variants (e.g., in receptor trafficking) is just starting to be determined.
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Some Kainate Receptor Subunits Undergo mRNA Editing The structural repertoire of KAR subtypes is even larger because the pre-mRNA of the KAR subunits GluR5 and GluR6 can undergo editing at the so-called Q/R site of the second membrane domain (M2). As in AMPARs, the Q to R substitution in homomeric KARs assembled either from GluR5 or GluR6 decreases the permeability of the receptor to calcium. Furthermore, this substitution transforms the rectification properties of these receptors from inwardly rectifying to linear or slightly outwardly rectifying, and it reduces the unitary conductance of the channels. As in AMPARs, inward rectification arises from the blockade of the ion channel by internal polyamines, such that the presence of an arginine (R) residue not only drastically reduces calcium permeability but also abolishes ion channel block by polyamines. In addition, GluR6 alone presents two further sites in the first transmembrane domain (M1) that are susceptible to editing, where I567 and Y571 could be changed to V and C, respectively. Although the role of these M1 editing sites remains unclear, editing within the M2 domain of GluR6 KARs has recently been shown to exert a significant effect on synaptic physiology.
Kainate Receptors Are Widely Distributed throughout the Brain Most of the studies defining the distribution of KARs in the brain come from in situ hybridization analysis of mRNA. KAR subunits are widely expressed throughout the nervous system, although there are a few known cell types that exclusively express a given subunit. For instance, GluR5 and KA2 are the only subunits that have been detected in the dorsal root ganglion (DRG) neurons. GluR5 transcripts are also abundant in the Purkinje cells of the cerebellum as well as in the subiculum, the piriform and cingulate cortices, and the hippocampal and cortical interneurons. On the other hand, GluR6 is the most abundant subunit in cerebellar granule cells, the striatum, the dentate gyrus, and the CA1 and CA3 regions of the hippocampus. The mRNA encoding GluR7 is expressed at low levels throughout the brain, but it is particularly prominent in the deep layers of the cerebral cortex, in the striatum, and in the inhibitory neurons of the molecular layer of the cerebellum. The KA1 subunit is present at low levels in the dentate gyrus, in the amygdala, and in the entorhinal cortex, being more abundant in the CA3 region. In contrast, KA2 mRNA can be found in virtually every part of the nervous system. Although the different KAR subunits are already present in the embryo, most of these expression
patterns emerge during the early postnatal period. Thus, the amount of GluR5 mRNA peaks between P0 and P5, and it then begins to decline toward the levels found in adults by P12. Similarly, the patterns of GluR6 and KA1 expression observed within the hippocampal formation begins to change at around P0 (GluR6) and P12 (KA1). We are still awaiting the development of specific antibodies against various KAR subunits. Although in situ hybridization is informative, it is not capable of revealing the subcellular distribution of a given subunit. Relatively specific antisera against the KAR subunits GluR6/7 and KA2 have been used to detect KARs, and to date, the most convincing studies have been carried out in the rat retina. In this tissue, the localization of these subunits at the synapse has been investigated by electron microscopy and the selective synaptic distribution of KARs in both plexiform layers of the retina has been observed. Whereas GluR6/7 could be found in horizontal cell processes postsynaptic to both rod spherules and cone pedicles, the KA2 subunit was found only in postsynaptic densities of the cone pedicles in the dendrites of off-bipolar cells. Anti-GluR6/7 and less specific anti-GluR5/6/7 antibodies have also been used to study other neurons in the brain. Although the limitation of these antibodies, particularly the latter, must be taken into account when interpreting the data obtained, they appeared to map KARs to fibers and synaptic terminals, as well as to dendrites and postsynaptic membranes. Hence, it seems that KARs may be found in both pre- and postsynaptic locations. In particular, GluR5/6 and KA2 have been found on parallel fibers and GluR5/6 immunoreactivity has been identified in a large population of terminals that form axospinal and axodendritic asymmetric synapses in the monkey striatum.
Regulation of the Surface Expression of Kainate Receptors Although more recent experiments have been directed toward understanding how KARs are targeted to specific membrane domains, the mechanisms controlling surface expression remain unclear. Retention in the endoplasmic reticulum (ER) plays a rate-limiting role in KAR trafficking. As in most GluRs, the residues important for ER retention or exit are proximal to Psd-95, Dlg, and ZO1 (PDZ) binding motifs present in the C-terminal domain. A number of proteins containing PDZ motifs have been shown to interact with KAR subunits, although most of these are promiscuous, also binding to AMPARs and, possibly,
Kainate Receptors: Molecular and Cell Biology 311
NMDARs. These include postsynaptic density protein (PSD-)95/ synapse-associated protein (SAP-)90, CASK, glutamate receptor interacting protein (GRIP), protein interacting with C kinase 1 (PICK1), and syntenin. However, PDZ proteins such as PICK1, GRIP, and PSD-95 do not appear to play a significant role in the exit of GluR5 or GluR6 from the ER but, rather, they have been implicated in the insertion and/or maintenance of AMPARs and KARs at the synapse. Indeed, it was recently shown that the number of KARs in the membrane surface can rapidly be altered through protein–protein interactions. These PDZ proteins appear to regulate kainate and AMPARs in a distinct manner. Disrupting the interaction with GRIP decreases synaptic responses through KARs while concomitantly increasing the AMPAR-mediated component. Similarly, interfering with the interaction of PICK1 with KARs and AMPARs depresses the KAR-mediated excitatory postsynaptic current (EPSC), leaving the current mediated by AMPARs unaltered. It is likely that the synaptic expression of KARs is tightly regulated through interactions with still unknown scaffolding proteins, as well as through proteins that influence the trafficking and targeting of KARs. For instance, the rules governing the polarized targeting of KARs to
GluR6
The Atomic Structure of Kainate Receptor Binding Domain Has Been Resolved One of the most exciting advances in the structure– function relationships of KARs has been the achievement of high-resolution crystal structures for the ligand-binding core of some subunits. This has provided a detailed understanding of agonist recognition not only for KARs but also for other ligand-gated ion channels. Indeed, the crystal structure of the GluR5
GluR5 B
NH2
axons and dendrites remain unknown. The study of mice that fail to express different subunits has not demonstrated a link between subunit composition and the compartment to which KARs are targeted. Nevertheless, a variety of trafficking determinants in kainate receptors may promote either membrane expression or intracellular sequestration. The identification of the proteins involved (chaperones) should shed light on the mechanisms that regulate KAR signaling and function. Indeed, a recent study has identified the co-atomer protein complex I (COPI) vesicle coat as a critical mechanism for the retention of KA2 subunits in the ER, such that assembly of KA2 subunits with other KAR subunits to form heteromeric receptors reduces their association with COPI.
B
NH2 J
J
A C
A
D
K
F
D
K
C
F
I
COOH
COOH G
G H
a
Extracellular
I
To TM1 TM2
Intracellular H
To TM1 TM2 b
Figure 2 Atomic structure of glutamate-bound GluR6 and GluR5 binding domains: (a) ribbon representation of GluR6 and GluR5 crystal structures at resolution 1.65 and 2.1 A˚, respectively; (b) suggested molecular architecture of glutamate ionotropic receptors. In (a), S1, S2, and loops 1 and 2 are colored blue, gold, and green, respectively. The linker shown in gray replaces the TM segments 1 and 2. The C-terminus of GluR5 appears partially disordered (discontinued line) in the crystal. The bound glutamate molecule is illustrated with ball and stick representations. The three ligand binding pocket side chains shown are conserved in all kainate receptors. Letters A through K name the different a-helical structures loops in the molecule. In (b), the architecture is composed of the crystal structures of the mGluR1 ligand-binding domain dimer (green), the GluR5 ligand-binding core dimer (S1, cyan; S2, orange), and two subunits from the KcsA potassium channel tetramer (gray). An intact glutamate receptor is believed to assemble as a dimer of dimers. GluR, glutamate receptor; mGluR, metabolic glutamate receptor. (a) Adapted from Mayer ML (2005) Crystal structures of the GluR5 and GluR6 ligand binding cores: Molecular mechanisms underlying kainate receptor selectivity. Neuron 45: 539–552. (b) Adapted from Mayer ML (2006) Glutamate receptors at atomic resolution. Nature 440: 456–462.
312 Kainate Receptors: Molecular and Cell Biology
and GluR6 KAR ligand binding cores complexed with glutamate and other agonists, such as kainate and quisqualate, have been shown at a resolution of 1.65 ˚ (GluR5) (Figure 2(a)). As in the (GluR6) and 2.1 A AMPARs and NMDARs, the structure of the KAR binding domain revealed a two-domain closed shell motif linked by b strands. Glutamate binds in a cavity that is isolated from the external solution. For example, agonist binding induces shell closure; this evokes a conformational rearrangement, leading to the opening of the channel. Interestingly, the cavity is smaller in GluR6 than in GluR5 but larger than that of the AMPAR subunit GluR2, due to the presence of unique side chains in KAR subunits. Nevertheless, the mechanism by which glutamate binds to GluR5, GluR6, and GluR2 is nearly identical. Like GluR2, the GluR6 domain associates as a dimer with many conserved interdimer contacts. Thus, an examination of the atomic structure explains why some residues are critical for GluR5-selective ligands (e.g., ATPA). Binding of these agonists to GluR6 is prevented by steric occlusion because the cavity in GluR6 is too small to accommodate the agonists that, like ATPA, bind to GluR5. In Figure 2(b), a recently suggested molecular structure for ionotropic glutamate receptors, including KARs, is presented. It seems that ionotropic GluRs are formed by the fusion of discrete segments, whose ancestors could be found as bacterial proteins. The structure has been built by taking the solved crystal structures of both the N-terminal domain of metabotropic GluRs and the agonist binding domain of KARs. So far, the structure of the GluR ion channel is unknown at the atomic resolution. However, based on the fact that the ion channel of ionotropic GluRs presents remarkable sequence homology with bacterial potassium channels, the crystal structures of a bacterial potassium channel, the KcsA potassium channel, has been incorporated as the channel domain. Although it is likely that a KAR presents a similar arrangement, much work remains to finally solve the structure of the GluR channel. It is expected that this rapidly developing area will soon provide the real structure at the atomic resolution. See also: AMPA Receptor Cell Biology/Trafficking; AMPA Receptors: Molecular Biology and Pharmacology; Kainate Receptor Functions; NMDA Receptors, Cell Biology and Trafficking.
Further Reading Bettler B, Boulter J, Hermans-Borgmeyer I, et al. (1990) Cloning of a novel glutamate receptor subunit, GluR5: Expression in the nervous system during development. Neuron 5: 583–595. Jaskolski F, Coussen F, and Mulle C (2005) Subcellular localization and trafficking of kainate receptors. Trends in Pharmacological Science 26: 20–26. Kohler M, Burnashev N, Sakmann B, and Seeburg PH (1993) Determinants of Ca2þ permeability in both TM1 and TM2 of high affinity kainate receptor channels: Diversity by RNA editing. Neuron 10: 491–500. Lerma J, Paternain AV, Naranjo JR, and Mellstro¨m B (1993) Functional kainate selective glutamate receptors in cultured hippocampal neurons. Proceedings of the National Academy of Sciences of the United States of America 90: 11688–11692. Lerma J, Paternain AV, Rodriguez-Moreno A, and Lopez-Garcia JC (2001) Molecular physiology of kainate receptors. Physiological Reviews 81: 971–998. Madden DR (2002) The structure and function of glutamate receptor ion channels. Nature Reviews Neuroscience 3: 91–101. Mayer ML (2005) Crystal structures of the GluR5 and GluR6 ligand binding cores: Molecular mechanisms underlying kainate receptor selectivity. Neuron 45: 539–552. Mayer ML (2006) Glutamate receptors at atomic resolution. Nature 440: 456–462. Paternain AV, Herrera MT, Nieto MA, and Lerma J (2000) GluR5 and GluR6 kainate receptor subunits coexist in hippocampal neurons and coassemble to form functional receptors. Journal of Neuroscience 20: 196–205. Paternain AV, Morales M, and Lerma J (1995) Selective antagonism of AMPA receptors unmasks kainate receptor-mediated responses in hippocampal neurons. Neuron 14: 185–189. Petralia RS, Wang YX, and Wenthold RJ (1994) Histological and ultrastructural localization of the kainate receptor subunits, KA2 and GluR6/7, in the rat nervous system using selective antipeptide antibodies. Journal of Comparative Neurology 349: 85–110. Schiffer HH, Swanson GT, and Heinemann SF (1997) Rat GluR7 and a carboxy terminal splice variant, GluR7b, are functional kainate receptor subunits with a low sensitivity to glutamate. Neuron 19: 1141–1146. Seeburg PH (1996) The role of RNA editing in controlling glutamate receptor channel properties. Journal of Neurochemistry 66: 1–5. Swanson GT, Feldmeyer D, Kaneda M, and Cull-Candy SG (1996) Effect of RNA editing and subunit co-assembly single-channel properties of recombinant kainate receptors. Journal of Physiology 492: 129–142. Wisden W and Seeburg PH (1993) A complex mosaic of highaffinity kainate receptors in rat brain. Journal of Neuroscience 13: 3582–3598.
Relevant Website http://www.ebi.ac.uk – European Bioinformatics Institute.
Kainate Receptor Functions J Lerma, Instituto de Neurociencias de Alicante Consejo Superior de Investigaciones Cientı´ficas-Universidad Miguel Hernandez, San Juan de Alicante, Spain ã 2009 Elsevier Ltd. All rights reserved.
After finding specific kainate receptors (KARs) in central neurons, physiologists suffered many problems trying to elucidate the role of these receptors in the nervous system, principally due to the lack of pharmacological tools to activate or antagonize KARs. Indeed, many of the pharmacological agonists and antagonists that activate KARs also interact with a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs). This lack of pharmacological specificity has hindered our understanding of KAR function for several years. Thus, the discovery that 2,3-benzodiazepines, and particularly GYKI 53655 (LY300168 or the active isomer LY303070), antagonize AMPARs but not KARs represented a significant breakthrough in the field. More recently, other compounds that specifically act on KAR subunits have been developed and mice deficient for KAR subunits have been generated, paving the way for the study of the synaptic physiology of KARs. As a result, KARs have been seen to play a role in synaptic transmission, influencing both neuronal excitability and information transfer in the brain. An interesting characteristic of KARs that has emerged is that they use two forms of signaling: a canonical pathway that involves ion flow and another, more unexpected noncanonical signaling pathway that links KAR activation to G-proteins and second-messenger cascades.
Elementary Properties of Kainate Receptor Channels KARs are nonselective cation channels. Therefore, the single-channel conductance of some KAR assemblies has been determined. According to stationary noise analysis, the single-channel conductance of KARs lies in the picosiemen range with values of 2.9 and 5.4 for homomeric glutamate receptor (GluR)5(Q) or GluR6(Q), respectively. A more detailed study of channel openings revealed three subconductance levels that are approximately two or three times the size of the smallest current. Like AMPARs, the replacement of Q by R in the channel domain as the result of RNA editing reduces the single-channel conductance of KARs by more than one order of magnitude. Furthermore, the conductance is also altered
by heteromerization. For instance, the elementary conductance of GluR5(R) channels is less than 200 fS, but it increases to 950 fS on co-assembly with KA2. Similarly, GluR6(R) homomers have a single-channel conductance between 230 and 260 fS, whereas the value for GluR6(R)/KA2 heteromers lies between 570 and 700 fS. The effect of KA2 on the conductance of unedited homomeric receptors is more subtle, with increases to 4.5 and 7.1 pS, for GluR5 and GluR6, respectively. Interestingly, native receptors display similar values, a conductance of 2–4 pS having been measured in dorsal root ganglion (DRG) cells and three subconductance levels, similar to those found for GluR5(Q) or GluR5(Q)/KA2, have been identified. These findings are in accordance with the abundant expression of GluR5 in DRG neurons. In addition, the conductance of KARs expressed in immature proliferating cerebellar granule neurons has been determined to be 1 and 4 pS, the channels spending most of their open time in the 4 pS state.
Desensitization of Kainate Receptor Currents Rapid desensitization is one of the major characteristic features of KARs. The time course of current decay in the continued presence of an agonist follows a single or double exponential decay. The speed of desensitization is dependent on the receptor subtype and on the cell type being analyzed. GluR6 homomers and native hippocampal KARs have desensitization time constants of 11–13 ms, a value similar to that found for recombinant channels in excised patches or lifted cells. However, KARs recover slowly from the desensitized state, and this recovery is dependent on the nature of the agonist. Indeed, whereas the recovery from desensitization by a pulse of kainate takes over 1 min, the receptor requires only 15 s when glutamate is the agonist. The subunit composition also affects recovery from desensitization; thus, whereas GluR5 homomeric receptors recover in about 1 min, the time constant for the recovery of GluR5/KA2 heteromers is just 12 s. These dramatic differences in the time scale of desensitization and recovery imply that the equilibrium between the two states is strongly displaced toward the inactive state. In other words, the receptors spend most of their time desensitized. This contradicts the fact that low concentrations of kainate and glutamate have a striking effect on native KARs, as seen in neurons in brain slices. There is still no clear explanation for this behavior, which is not predicted from rapid
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314 Kainate Receptor Functions
activation and inactivation kinetics. However, it is possible that unknown proteins interact with native KARs, altering their gating properties, as seen for other receptors and channels.
Pharmacological Aspects As already mentioned, a major difficulty in understanding KAR function has been the lack of a specific pharmacology for these receptors. For instance, kainate, albeit showing a clear preference for KARs, has a significant action on AMPARs at relatively low doses. Indeed, there is only a 5–30 times difference between the apparent affinity of these two receptors for kainate. There has also been some progress concerning selective KARs agonists. Some derivatives of willardiine have been developed as selective KAR agonists (Table 1). Among them, (S)-5-iodowillardiine shows a 130-fold selectivity for KARs (median effective concentration (EC50) ¼ 140 nM). SYM 2081, the (2S-4R) diastereomer of 4-methylglutamate, displays a potency three orders of magnitude higher for KARs than for AMPARs, both in binding and in functional assays, but the selectivity of this molecule for kainate over N-methyl-D-aspartate receptors (NMDARs) is significantly lower. Although its pharmacological profile is incomplete, SYM 2081 does not seem to show the subunit specificity observed for ATPA and for (S)-5-iodowillardiine; it elicits rapidly desensitizing currents on GluR5 and GluR6 homomeric channels. Due to this property, this compound has also been used as a functional antagonist of the KARs because it efficiently desensitizes KARs at low concentrations (Table 2). Similarly, the prototypic non-NMDAR antagonists, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), 6,7dinitroquinoxaline-2,3-dione (DNQX) and 6-nitro-7sulphamoylbenzo[f]quinoxaline-2,3-dione (NBQX), just show poor selectivity between AMPARs and KARs, although it has been shown that low concentrations of NBQX do not have any action on KARs. However, the separation of KAR-mediated responses from the AMPAR-mediated currents is possible by using 2,3-benzodiazepine GYKI 53655 (Lilly’s code LY300168), which is selective for antagonizing AMPARs. Therefore, in the presence of GYKI53655, or its active isomer LY303070, CNQX could be used as a KAR antagonist (Table 2). Several other very useful compounds have been developed lately. In particular, LY382884 ((3S, 4aR, 6S, 8aR)-6-((4-carboxyphenyl) methyl-1,2,3,4,4a,5,6,7,8,8a-decahydro isoquinoline3-carboxylic acid), seems to antagonize KARs at concentrations that do not affect AMPARs or NMDARs. Actually, LY382884 is a selective antagonist at neuronal KARs containing the GluR5 subunit (Table 2).
Table 1 Kainate receptor agonists Compound
Selectivity
ATPA S-5-iodowillardine Kainate Domoate AMPA Me-Glutamate (SYM 2081)
GluR5-contaning; GluR6/KA2 GluR5-containing; GluR6/KA2; GluR7/KA2 All assemblies GluR5- and GluR6-containing. GluR5-containing; GluR6/KA2; GluR7/KA1 GluR5- and GluR6-containing
AMPA, a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid; GluR, glutamate receptor.
Table 2 Kainate receptor antagonists Compound
Selectivity
Mechanism
CNQX
All assemblies; more potent at AMPAR GluR5-containing GluR5-containing Homomeric GluR5 GluR5-containing GluR5-containing and homomeric GluR6 GluR5- and GluR6-containing
Competitive
LY382884 UBP296 NS3763 LY377770 Kynurenate SYM2081
Competitive Competitive Noncompetitive Competitive Competitive Desensitization
AMPAR, a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; GluR, glutamate receptor.
Kainate Receptors Have a Fundamental Role in Controlling Synaptic Neurotransmitter Release The discovery that GYKI 53655 (or LY303070, the active isomer) antagonizes AMPARs but not KARs paved the way for the study of the participation of KARs in synaptic responses. Nowadays, there is considerable evidence suggesting that KARs may be situated on both sides of the synapse. A considerable amount of effort has been devoted to determining the role of presynaptic KARs in controlling transmitter release, particularly in the hippocampus. In this structure, KARs are widely distributed in presynaptic buttons, and they can bidirectionally regulate the release of glutamate at the mossy fibers to CA3 synapses. The mild activation of KARs enhances the release of glutamate, whereas when they are more intensely activated glutamate release is inhibited. As such, a proportion of the high-frequency-dependent facilitation, a hallmark of the mossy fiber synapses, can be attributed to the activation of presynaptic KARs by synaptically released glutamate, as if low concentrations of exogenous kainate had been applied (Figure 1(b)). Therefore, the characteristic short-term plasticity of mossy
Kainate Receptor Functions 315
Dentate gyrus granule cells Mossy fiber
Mossy fibers
b
CA3 neuron
CA3 pyramidal neurons
–GYKI = EPSCAMPA EPSCAMPA +GYKI = EPSCKAR
+KAR antagonist
40 ms
a Without KAR antagonist c
d
40 ms EPSCAMPA
5 pA EPSCKAR
25 ms
e Figure 1 Proposed roles of kainate receptors (KARs) in synaptic transmission: (a) mossy fiber-CA3 synapses; (b) synaptic response of the mossy fibers to CA3 pyramidal cells; (c) presynaptic role; (d) postsynaptic current; (e) postsynaptic responses at the thalamo-cortical synapse. These can be isolated in the presence of APV (to block NMDA receptors) and GYKI53655 (to block AMPA receptors). In all cases, the KAR-mediated synaptic responses are much slower than those observed for AMPA receptors and usually present one-tenth their amplitude (in (d), both responses have been normalized). In (c), presynaptic KARs can enhance or inhibit glutamate release, depending of the degree of activation. The best-established example is the synapse made by mossy fibers on to CA3 pyramidal neurons, where KARs seem to be responsible of a part of the high-frequency facilitation, a characteristic of these synapses. KARs also mediate synaptic responses of Schaffer collateral to interneurons (not shown). In (e), EPSCs mediated exclusively by AMPA or KARs have been isolated by minimal stimulation. AMPA, a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid; EPSC, excitatory postsynaptic current; Glu, glutamate; NMDA, N-methyl-D-aspartate. (d) Adapted from Lerma J (2003) Roles and rules of kainate receptors in synaptic transmission. Nature Reviews Neuroscience 4: 481–495. (e) From Kidd FL and Isaac JT (1999) Developmental and activity-dependent regulation of kainate receptors at thalamocortical synapses. Nature 400: 569–573.
fiber–CA3 synapses is partly mediated by the longlasting activation of a kainate autoreceptor. This mechanism has been shown to impose associative properties on mossy fiber long-term potentiation (LTP) because the activity in neighboring mossy fiber synapses, or even associational/commissural synapses, influences the threshold for inducing LTP. However, higher concentrations of kainate, instead of facilitating mossy fiber synaptic transmission, depress it. Interestingly, this phenomenon is reproduced by synaptic activity, such that a brief conditioning of tetanus on associational-commissural fibers enhances the mossy fiber responses, whereas
prolonged tetanus depresses it. The exact mechanism involved in the presynaptic facilitation of glutamate release is unclear. One explanation for the facilitation of glutamate release is that presynaptic KARs depolarize synaptic terminals to a level at which further release is enhanced. Also, it has been claimed that a Ca2þ-induced Ca2þ release from the intracellular stores is required for this phenomenon to occur. Therefore, such a mechanism remains unclear. How the larger activation of KARs might lead to the inhibition of glutamate release in mossy fiber buttons also remains a matter of debate. It could be the case that the large depolarization of synaptic terminals inactivates
316 Kainate Receptor Functions
the generation of action potentials, leading to a failure in release. However, recent data show that inhibition (but not facilitation) of release by presynaptic KARs depends on a G-protein-dependent mechanism. This indicates that KARs act independent of their ion channel activity. Therefore, it is possible that the threshold for activating one or other KAR signaling pathway could determine the physiological response. It is now widely accepted that presynaptic KARs also control the release of the inhibitory neurotransmitter g-aminobutyric acid (GABA). Although KAR stimulation may enhance the release of GABA at
connections between the hippocampal interneurons, stimulation of these receptors inhibits GABA release at interneuron–pyramidal cell synapses. Thus, data indicate that the spillover of glutamate from adjacent terminals could activate KARs at presynaptic GABA terminals (Figure 2(b)). The specific role that the receptors controlling GABA release play in hippocampal synaptic networks remains unclear. However, the infusion of kainate in vivo reduces the efficiency of recurrent inhibition, producing a state of hyperexcitability that leads to the generation of recurrent epileptic spikes. Accordingly, KAR antagonists should be able to prevent and/or abolish epileptic
IPSCs Control CA1 pyramidal neuron Kainate 0.3 µM
GABAergic terminal
CA1 interneuron
c Stratum oriens single stimulus
Schaffer collaterals
IPSCs
a Glu
Schaffer collateral stimuli d EPSCs +GYKI
Schaffer collateral
IAHP
−GYKI
Control Kainate
b
e
f
40 mV
1s
Figure 2 Role of kainate receptors (KARs) in neurotransmitter synaptic release: (a) CA1 pyramidal neurons receive excitatory inputs from the Schaffer collateral and inhibitory actions from interneurons; (b) illustration of an excitatory and an inhibitory synapse on a CA1 pyramidal cell dendrite; (c) a low concentration of kainate selectively reduces the GABA release, as evidenced by a reduction in the evoked IPSC, under pharmacological isolation of GABAA-mediated responses; (d) glutamate could spill over to activate presynaptic KARs present in GABAergic terminals such that the IPSCs (induced by a single shocks to the stratum oriens) are reduced after a train of stimuli is applied on the Shaffer collateral pathway; (e–f) synaptically released glutamate could activate postsynaptic KARs. Synaptically released glutamate could also activate presynaptic (not shown) KARs. Although the activation of postsynaptic KARs does not induce any synaptic current ((e), þGYKI), the afterhyperpolarization current ((f), IAHP) is depressed. KARs have been shown to exert all these actions by activating G-proteins. EPSC, excitatory postsynaptic current; GABA, g-aminobutyric acid; Glu, glutamate; IPSC, inhibitory postsynaptic current. (d) Adapted from Min MY, Melyan Z, and Kullmann DM (1999) Synaptically released glutamate reduces gamma-aminobutyric acid (GABA)ergic inhibition in the hippocampus via kainate receptors, Proceedings of the National Academy of Sciences of the United States of America 96: 9932–9937. (f) From Lerma J (2006) Kainate receptor physiology. Current Opinion in Pharmacology 6: 89–97.
Kainate Receptor Functions 317
activity. This has indeed been demonstrated when epilepsy is induced in rats by a pilocarpine injection. In this model, KAR antagonists (e.g., LY377770, Table 2) not only abolish the established seizure activity but also prevent the development of seizures when they are applied beforehand. Therefore, these and other data indicate that the excitability of the network may be tightly controlled by the extracellular concentrations of glutamate through the transient and/or tonic activation of KARs. One important issue that has remained a matter of intense debate over the last few years relates to the mechanism by which KARs affect release at these inhibitory synapses. There is evidence indicating that GABA release is inhibited by a G-protein- and protein kinase C (PKC)-dependent mechanism (Figure 2(b)). It is possible that the large barrage of inhibitory postsynaptic current (IPSC) received by pyramidal cells on kainate perfusion may cause some short circuiting, but it is now generally accepted that this is not sufficient to account for such inhibition. Indeed, it should be taken into account that KARs may regulate CA1 GABAergic circuits through two distinct and opposing mechanisms. Whereas the activation of somatodendritic KARs may increase the activity of GABA interneurons, presynaptic KARs may diminish inhibition. These two effects are mediated by receptor populations that are functionally, physically, and pharmacologically distinct, one of which is located in the somatodenritic and/or axonal compartments and the other at the presynaptic terminals directly inhibiting GABA release. Likewise, there is considerable data that point to a prominent role of KARs in the regulation of neuronal excitability through the modulation of the GABAergic system in a variety of brain nuclei, such as the amygdala, the neocortex, the striatum, and the cerebellum.
Kainate Receptors Mediate Part of the Postsynaptic Response at Certain Synapses Synaptic release of glutamate can also activate postsynaptic KARs, as seen primarily in synapses established by mossy fibers on CA3 neurons. Subsequently, KARmediated synaptic responses were demonstrated in Schaffer collaterals–hippocampal CA1 interneurons, at the parallel fiber–Golgi cell synapses, in the basolateral amygdala, in Purkinje cells of the cerebellum, at thalamo-cortical connections on cortical interneurons (Figure 1(e)), in neocortical pyramidal neurons, and in the synapse established by cones on bipolar cells in the retina. Similarly, a synaptic component mediated by KARs has been found in the synapse between afferent sensory fibers and dorsal horn neurons in the spinal
cord. With the exception of excitatory postsynaptic currents (EPSCs) in the retina, the KAR-mediated EPSCs all displayed common characteristics such as a low amplitude (approximately an order of magnitude lower than the component mediated by AMPA receptors) and a longer-lasting time course than the EPSC mediated by the AMPA receptor (Figure 1(d)). What could be the functional significance of a small and long-lasting EPSC for neurons? Although it is not definitively determined, a slow small EPSC, such as that mediated by KARs, offers the possibility of integrating excitatory inputs over a wide time window. Thus, the relative contribution of KARs to synaptic drive could be significant. It has been calculated that KARs elicit sufficient charge transfer so as to exert a substantial impact on the activity of interneurons. Indeed, the kinetics of KAR-mediated EPSPs are sufficiently slow to allow substantial tonic depolarization even during modest presynaptic activity. Such tonic depolarization could account for the observation that single afferent inputs are very effective in driving interneuron spiking. Accordingly, KARs contribute to the ongoing ionotropic glutamatergic transmission in the hippocampus. Therefore, neurons may display unique integration properties in areas where KARs are expressed. Why native and recombinant KARs present different kinetic properties has not been convincingly explained. However, KARs take direct part of the glutamatergic synapse because these receptors are activated by quantal release of glutamate at both mossy fibers–CA3 synapses and excitatory synapses on CA1 interneurons. In addition, there is evidence that AMPARs and KARs can be segregated at different synapses. This seems to be true at least in CA1 interneurons and at thalamo-cortical synapses, although not in individual mossy fiber synapses. These studies allowed the kinetics of elementary KAR-mediated EPSC in CA1 interneurons to be determined, revealing that they are still slower than the pure AMPARmediated EPSC. Therefore, there should be factors such as the dissimilar composition of the synaptic receptors or accessory proteins that may explain the difference between the response kinetics of the native and recombinant receptors studied so far. The retina constitutes one of the systems in which KARs have been shown to display similar properties to those found in recombinant receptors, specifically at the synapse from cones to the off bipolar cells. In this system, the kinetics of the synaptic responses fits well with the activation and desensitization properties of KARs determined in recombinant systems. In these synapses, the slow kinetics of recovery from desensitization of KARs is exploited in processing the afferent signals rather than suffering the constrictions
318 Kainate Receptor Functions
of the faster time course of AMPARs. In the dark, a cone releases sufficient neurotransmitter to desensitize most postsynaptic KARs, leaving just a small persistent postsynaptic current. Because the postsynaptic KARs desensitize rapidly and recover from desensitization slowly, little recovery can occur during the brief hyperpolarizations that are produced in the cones by repetitive light stimuli. Therefore, the large increase in conductance that would saturate the membrane voltage if the rate of transmitter release by the cone was high is prevented. Actually, signaling between the cones and the three morphological types of off bipolar cells occurs through distinct postsynaptic receptors. Whereas two cell types use KARs with distinct properties, the other type uses AMPARs. Each receptor recovers from desensitization with a particular time course, and this property contributes to determining the temporal characteristics of synaptic transmission in the retina (Figure 3). Recent studies have demonstrated that KARs are also involved in long-term synaptic plasticity (LTP). Indeed, mossy fiber LTP may be prevented by the GluR5 antagonist LY382884. This antagonist,
K+ channels
however, is ineffective for preventing LTP induced by tetanic stimulation of associational/commissural fibers in the CA3 region or at CA1 synapses. Therefore, a role for KARs in the induction of LTP seems to be specific for the mossy fiber pathway, although pharmacological data are at odds with results obtained from knockout mice. The use of KAR subunit-deficient mice confirmed that KARs play a critical role in mossy fiber LTP. However, mossy fiber LTP was reduced, but not abolished, in GluR6knockout mice, whereas GluR5-deficient mice exhibited normal LTP at these synapses. Clearly, more investigation is needed to understand the molecular mechanisms by which KARs may play a permissive role at mossy fibers to induce a permanent change in release properties. Also interesting is the fact that at thalamo-cortical synapses in the neonatal barrel cortex, LTP expression is associated with a reduction in the KAR-mediated component of the synaptic response. Thus, as recently confirmed, KAR-mediated synaptic transmission can undergo a form of long-term depression (LTD) that is mechanistically distinct from the LTD seen for AMPARmediated transmission. Kainate receptor
Ca2+ channels
1 Kainate
Na+
N-type Ca2+ current DG Kainate Recovery Control 3
Ca2+
PKC IP3 2
200 pA 20 ms
Intracellular stores
Figure 3 Canonical and noncanonical signaling of kainate receptors (KARs). As ligand-gated ion channels, the activation of KARs evokes an inward current, through which they depolarize the membrane (1). However, they can also set in motion a noncanonical signaling pathway triggered by the activation of a pertussis toxin-sensitive G-protein (G). The signaling pathway involves phospholipase C (PLC), leading to the synthesis of diacylglycerol (DG) and inositol 1,4,5, triphosphate (IP3). Although IP3 could release Ca2þ from intracellular stores, as revealed by confocal Ca2þ imaging (2), DG activates protein kinase C (PKC), which in turn inhibits Ca2þ channels, as illustrated by the direct recording of Ca2þ currents (3). This pathway is also responsible for the inhibition of the Ca2þ-dependent Kþ current, accounting for the hyperpolarization of the membrane after repetitive firing (afterhyperpolarization). These effects of kainate do not seem to require ion channel receptor activity, providing evidence for the existence of a metabotropic KAR that inhibits Ca2þ and Kþ channels. Adapted from Rozas JL, Paternain AV, and Lerma J (2003) Noncanonical signaling by ionotropic kainate receptors. Neuron 39: 543–553.
Kainate Receptor Functions 319
Dual Signaling of Kainate Receptors It was recently demonstrated that KARs and other ionotropic glutamate receptors may transmit signals both through ion flux and by receptor coupling to G-proteins, which in turn may regulate voltagedependent Ca2þ channels (Figure 3). This was initially described in the hippocampus, and it was later confirmed in other tissues. Indeed, activation of KARs in cultured DRG cells, which almost exclusively express GluR5 and KA2 KAR subunits, induces the release of Ca2þ from intracellular stores in a G-protein-dependent manner (Figure 3). Interestingly, the concomitant activation of PKC inhibited voltagedependent N-type calcium channels. This noncanonical signaling is independent of ion channel activity and provides insights into the dual signaling of KARs because both pathways depend on a common ionotropic subunit, GluR5. But the way in which an ion channel becomes coupled to a G-protein is still unclear, although it seems likely that intermediate or linker proteins may assist in the process. Unfortunately, none of the proteins that interacts with KARs identified to date seem to carry out this role, and thus, these intermediate elements remain to be identified. In addition, this property does not seem to be restricted to a given KAR subunit. Postsynaptic KARs can also inhibit hyperpolarizing currents through similar signaling mechanisms (Figure 2(f)), and this seems to involve KA2 subunits rather than GluR6 or GluR5 subunits. There is compelling evidence indicating that synaptically released glutamate could activate postosynaptic KARs in CA1 and CA3 pyramidal neurons that do not involve the generation of inward currents. Rather, these receptors reduce the typical afterhyperpolarization current seen after cell firing (IAHP). Inhibition of after hyperpolarization has a great impact on the neuron excitability, such that the response to a given input is drastically altered. Afterhyperpolarization is induced by the activation of Ca2þ-dependent Kþ channels, curtailing repetitive firing. The reduction of such a current causes the neuron to generate large burst of spikes, largely increasing the neuronal output. Together with the inhibition of GABA release, this represents an example in which KARs may exert a striking effect on neuronal excitability by a noncanonical signaling. Therefore, temporal integration of neuronal inputs may be modulated by KARs not acting as ion channels, just by attenuating hyperpolarizing conductances such as IAHP and GABA receptor-mediated current. Although exogenous kainate could facilitate or inhibit transmitter release, depending on the concentration and type of synapse, cumulative data indicate that the rule is that the inhibitory activity of KARs is linked to this noncanonical signaling. However, the variety
of signals activated by these metabotropic KARs indicates a certain lack of predictability in the coupling of KARs to G-proteins. Hence, much work remains to be done to clarify when KARs may work through classical or noncanonical signaling pathways. Although not without their problems, the availability of specific drugs as well as of knockout mice for each of the KAR subunits allows us to clarify which subunit participates in specific KAR-mediated responses.
Involvement of Kainate Receptors in Neuropathology The implications of KARs for nervous system pathology is being underscored but not without uncertainties. For instance, a contribution of KARs to pain is supported by pharmacological data because the GluR5-selective antagonist LY293558 has demonstrated preclinical and clinical efficacy in models of pain. Indeed, GluR5 subunits are essential for KARmediated responses in DRG neurons and for presynaptic regulation of transmitter release in the spinal dorsal horn. However, nociceptive thresholds are largely unaffected in GluR5- and GluR6-deficient mice. Only one report found decreased response to capsaicin and formalin in GluR5-deficient mice. Such a profile raises the possibility that KAR antagonists could be effective for the treatment of certain forms of pain and acute migraine. Similarly, excitotoxicity is triggered by the enhanced activation of GluRs. Although kainate was demonstrated to be a neurotoxin long ago, there is no compelling evidence for a direct role of KARs in this process. The most sensitive area in the brain to kainate-induced excitotoxicity is the hippocampal CA3, a region rich in high-affinity KARs. However, in GluR6-knockout mice, kainateinduced CA3 toxicity is attenuated but not eliminated. Recently, KARs activation has been linked to the activation of c-jun N-terminal kinase 3 (JNK) and mixed lineage kinase 3 (MLK3) that seem to link GluR6-containing KARs to the cell death pathway activated after ischemic insults. The best-established brain disorder in which KARs are implicated is the excitatory imbalance linked to epilepsy. Certainly, kainate injections have provided an animal model for studying human temporal lobe epilepsy. The inhibition of GABA release leading to recurrent epileptic activity may account for the acute epileptogenic effect of kainate. Recently, it has been observed that the synaptic response mediated by KARs provides a substantial component of the excitatory transmission at functional aberrant synapses formed by sprouting of glutamatergic fibers, a hallmark of human temporal lobe epilepsy. Thus, these
320 Kainate Receptor Functions
results raise the possibility of designing antiepileptic therapies based on KAR antagonists. A number of studies have examined the link between the expression of KARs genes and/or singlenucleotide polymorphisms (SNPs) to disorders with a genetic background with disparate results. No strong associations have been found between the expression of genes coding for KAR subunits (GluR6 and GluR7 (GRIK2 and GRIK3 in human nomenclature)) and obsessive–compulsive disorder patients. Only GRIK2 SNP I867 was transmitted less readily than expected. Interestingly, this variant has also been associated with autism. An allele of the GluR6 gene is associated with the early age of onset of Huntington’s disease; GluR6-mediated excitotoxicity has been implicated in the pathogenesis of Huntington’s disease. Allelic variants of GRIK1 (GluR5), but not GRIK2 (GluR6), appear to contribute a major genetic determinant to the pathogenesis of juvenile absence epilepsy and related phenotypes. Interestingly, the GluR5 gene is located at chromosame 21q22.1, and physical mapping situates it near the familial amyotrophic lateral sclerosis APP and SOD1 regions, making it a possible candidate for familial amyotrophic lateral sclerosis and other diseases. Whether GluR5 influences the phenotypes associated with partial trisomy or monosomy of chromosome 21 remains to be determined. Other recent results have indicated that there is a disequilibrium of GRIK2 (GluR6) transmission in autism and schizophrenia. However, no linkage has been established with GluR5 (GRIK1) SNPs and their haplotypes in schizophrenia. Whether the described SNPs may impact the function and/or trafficking of KARs is yet to be determined. Therefore, a definitive role of KARs in these disorders is possible but not demonstrated. See also: AMPA Receptor Cell Biology/Trafficking; AMPA Receptors: Molecular Biology and Pharmacology; Kainate Receptors: Molecular and Cell Biology; NMDA Receptors, Cell Biology and Trafficking.
Further Reading Castillo PE, Malenka RC, and Nicoll RA (1997) Kainate receptors mediate a slow postsynaptic current in hippocampal CA3 neurons. Nature 388: 182–186. Christensen JK, Paternain AV, Selak S, Ahring PK, and Lerma J (2004) A mosaic of functional kainate receptors in hippocampal interneurons. Journal of Neuroscience 24: 8986–8993. Contractor A, Swanson G, and Heinemann SF (2001) Kainate receptors are involved in short- and long-term plasticity at mossy fiber synapses in the hippocampus. Neuron 29: 209–216. Cossart R, Epsztein J, Tyzio R, et al. (2002) Quantal release of glutamate generates pure kainate and mixed AMPA/kainate EPSCs in hippocampal neurons. Neuron 35: 147–159.
Coussen F, Perrais D, Jaskolski F, et al. (2005) Coassembly of two GluR6 kainate receptor splice variants within a functional protein complex. Neuron 47: 555–566. Epztein J, Represa A, Jorquera I, Ben-Ari Y, and Crepel V (2005) Recurrent mossy fibers establish aberrant kainate receptoroperated synapses on granule cells from epileptic rats. Journal of Neuroscience 25: 8229–8239. Frerking M, Malenka RC, and Nicoll RA (1998) Synaptic activation of kainate receptors on hippocampal interneurons. Nature Neuroscience 1: 479–486. Frerking M and Ohliger-Frerking P (2002) AMPA receptors and kainate receptors encode different features of afferent activity. Journal of Neuroscience 22: 7434–7443. Kidd FL and Isaac JT (1999) Developmental and activitydependent regulation of kainate receptors at thalamocortical synapses. Nature 400: 569–573. Lerma J (2003) Roles and rules of kainate receptors in synaptic transmission. Nature Reviews Neuroscience 4: 481–495. Lerma J (2006) Kainate receptor physiology. Current Opinion in Pharmacology 6: 89–97. Lerma J, Paternain AV, Rodriguez-Moreno A, and Lopez-Garcia JC (2001) Molecular physiology of kainate receptors. Physiological Reviews 81: 971–998. Melyan Z, Lancaster B, and Wheal HV (2004) Metabotropic regulation of intrinsic excitability by synaptic activation of kainate receptors. Journal of Neuroscience 24: 4530–4534. Min MY, Melyan Z, and Kullmann DM (1999) Synaptically released glutamate reduces gamma-aminobutyric acid (GABA) ergic inhibition in the hippocampus via kainate receptors. Proceedings of the National Academy of Sciences of the United States of America 96: 9932–9937. Mulle C, Sailer A, Perez-Otano I, et al. (1998) Altered synaptic physiology and reduced susceptibility to kainate-induced seizures in GluR6-deficient mice. Nature 392: 601–605. Paternain AV, Morales M, and Lerma J (1995) Selective antagonism of AMPA receptors unmasks kainate receptor-mediated responses in hippocampal neurons. Neuron 14: 185–189. Rodriguez-Moreno A, Herreras O, and Lerma J (1997) Kainate receptors presynaptically downregulate GABAergic inhibition in the rat hippocampus. Neuron 19: 893–901. Rodriguez-Moreno A and Lerma J (1998) Kainate receptor modulation of GABA release involves a metabotropic function. Neuron 20: 1211–1218. Rozas JL, Paternain AV, and Lerma J (2003) Noncanonical signaling by ionotropic kainate receptors. Neuron 39: 543–553. Rubinsztein DC, Leggo J, Chiano M, et al. (1997) Genotypes at the GluR6 kainate receptor locus are associated with variation in the age of onset of Huntington disease. Proceedings of the National Academy of Sciences of the United States of America 94: 3872–3876. Schmitz D, Mellor J, Breustedt J, and Nicoll RA (2003) Presynaptic kainate receptors impart an associative property to hippocampal mossy fiber long-term potentiation. Nature Neuroscience 6: 1058–1063. Smolders I, Bortolotto ZA, Clarke VR, et al. (2002) Antagonists of GLU(K5)-containing kainate receptors prevent pilocarpineinduced limbic seizures. Nature Neuroscience 5: 796–804.
Relevant Website http://www.ebi.ac.uk – European Bioinformatics Institute.
Long-Term Potentiation (LTP): NMDA Receptor Role A J Doherty, S M Fitzjohn, and G L Collingridge, University of Bristol, Bristol, UK ã 2009 Elsevier Ltd. All rights reserved.
Introduction The subtype of glutamate receptor known as the N-methyl-D-aspartate (NMDA) receptor is the trigger for both long-term potentiation (LTP) and long-term depression (LTD) at the majority of synapses in the central nervous system (CNS), where long-lasting synaptic plasticity has been observed. The role of NMDA receptors (NMDARs) in synaptic plasticity was first identified, and has been most extensively studied at, the synapses made between CA3 and CA1 pyramidal neurons in the hippocampus, the Schaffer collateral–commissural pathway. While most is known about LTP at these CA1 synapses, it seems that the principles discovered here apply elsewhere in the brain. The following discussions pertain to LTP at CA1 synapses.
Glutamate Receptors and Synaptic Transmission It is commonly considered that a-amino-3-hydroxy-5methyl-4-isoxazole propionic acid receptors (AMPARs) mediate synaptic transmission and NMDARs trigger alterations in synaptic efficiency. However, the reality is more complex. AMPARs do indeed mediate the majority of the response to low-frequency stimulation. However, NMDARs contribute significantly to high-frequency synaptic transmission. Given that in the conscious animal CA3 neurons often discharge in high-frequency bursts, then NMDARs are likely to play a major role in the transmission of synaptic information within the CA1 area. It is also sometimes mistakenly considered that only AMPARs mediate the response that is modified during LTP. While it is true that it is often the AMPAR-mediated response that is studied, it is equally true that the NMDARmediated component of synaptic transmission has the capacity to undergo LTP.
Second, this block is highly voltage dependent. Third, the NMDAR-mediated conductance has slow kinetics, compared with the AMPAR-mediated response. Fourth, the NMDAR not only passes monovalent cations but also has a significant permeability to Ca2þ. During low-frequency transmission there is rapid activation of AMPARs. However, while L-glutamate binds to NMDARs, they are largely blocked by Mg2þ, which is present in the synaptic cleft at around 1 mM (Figure 1). This prevents NMDARs from contributing appreciably to the synaptic response. During high-frequency transmission there is a sustained depolarization, due in part to the temporal summation of AMPARmediated excitatory postsynaptic potentials (EPSPs), and this alleviates the voltage-dependent Mg2þ block to enable NMDARs to contribute to the synaptic response. Since they have a slow decay phase, the NMDAR-mediated EPSPs summate very effectively.
Induction of LTP Induction by High-Frequency Stimulation
Conditions that enable the activation of NMDARs can result in the induction of LTP. The most commonly used induction stimulus is a continuous high-frequency train (tetanus), sometimes delivered more than once. This provides the depolarization to transiently relieve the Mg2þ block of NMDARs. The Ca2þ that then enters the neuron through the NMDARs provides the initial trigger for the plastic change. Although a tetanus typically comprises 100 stimuli or more (e.g., 100 Hz, 1 s), far fewer stimuli can induce LTP, provided that they are appropriately timed. One such protocol is the delivery of brief high-frequency bursts (e.g., four shocks at 100 Hz) with an interburst frequency of around 5 Hz (i.e., an interburst interval of 200 ms). This pattern of activity mimics the physiological activity that occurs during the theta rhythm, a frequency of activation observed in the electroencephalogram (EEG) when animals are exploring their environment. The minimal induction protocol is the delivery of a single priming stimulus followed approximately 200 ms later by a single burst (which could comprise as few as two stimuli, though typically four are used). Thus, under optimal conditions LTP can be induced by three to five stimuli.
NMDAR Properties Govern Their Role in Synaptic Transmission
Induction by Pairing
The NMDAR has unique properties that govern its role in synaptic processing. First, it is strongly antagonized by micromolar concentrations of Mg2þ.
LTP can also be induced by pairing. This is achieved by maintaining low-frequency stimulation but depolarizing the neuron artificially by injecting current through
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322 Long-Term Potentiation (LTP): NMDA Receptor Role
No stimulation
Basal stimulation
High-frequency stimulation AMPAR NMDAR Mg2+
Figure 1 N-Methyl-D-aspartate receptors (NMDARs) are activated by high-frequency stimulation. Under basal conditions, NMDAR activity is blocked due to voltage-dependent binding of Mg2þ ions. A single stimulus results in the release of L-glutamate and activation of a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs) but does not provide sufficient depolarization to repel the Mg2þ ions. High-frequency stimulation results in a more sustained depolarization of the postsynaptic terminal, leading to the release of the Mg2þ block and an increase in cytosolic Ca2þ ions. The rise in Ca2þ concentration may be localized to the activated synapse, ensuring that only the activated synapse undergoes long-term potentiation.
the recording electrode. Typically, approximately 50 pairings of low-frequency stimulation and depolarization are employed. LTP can also be induced by the pairing of appropriately timed presynaptic and postsynaptic action potentials. The common feature of all of these induction protocols is that they enable sufficient depolarization to transiently relieve the Mg2þ block of NMDARs. Once this occurs, it enables Ca2þ to enter the synapse through the NMDARs in sufficient amounts to trigger the biochemical cascades that result in enhanced synaptic transmission. NMDAR Activation without LTP
However, activation of NMDARs does not necessarily induce LTP. Under conditions whereby the LTP process has been saturated (by prior periods of LTP induction), protocols that normally induce LTP result in no change in synaptic strength. In addition, certain patterns of activation can activate NMDARs but induce longterm depression rather than LTP. If a pathway has already undergone LTP, then these same patterns of activation may induce depotentiation (DP) of the potentiated response. Typically, NMDAR-dependent LTD and DP are induced by prolonged periods of low-frequency stimulation (1–3 Hz). Finally, an intermediate frequency (e.g., 5 Hz) will neither induce LTP nor LTD, while still activating NMDARs.
Features of NMDAR-Dependent LTP Input Specificity
NMDAR-dependent LTP has several hallmark features. Input specificity refers to the property that if two inputs are activated and converge on a population of neurons, or indeed a single neuron, but an induction protocol is delivered to just one of the inputs, then only that input will undergo LTP. The other input typically will not change its efficiency of transmission, or under certain circumstances may be depressed via a process known as heterosynaptic depression. Input specificity is an important property. It means that the unit of modification is the activated input, not the entire neuron. This implies that the unit of information storage can be as small as a single synapse. Input specificity is due to the requirement for L-glutamate to activate NMDARs and the fact that the Ca2þ that permeates NMDARs does not spread very far. Therefore, when a neuron is sufficiently depolarized, those synapses that are activated by synaptically released L-glutamate can undergo LTP. Nearby neighbors may also undergo LTP if either the synaptically released L-glutamate ‘spills over’ in sufficient amounts to activate enough NMDARs or if the Ca2þ spreads beyond the activated spine. In its purest form, LTP is restricted to the activated inputs.
Long-Term Potentiation (LTP): NMDA Receptor Role Cooperativity
Cooperativity refers to the need to stimulate multiple afferent fibers to induce LTP. Experimentally this is observed as the failure of a ‘weak tetanus’ to induce LTP, while a ‘strong tetanus’ that activates more fibers is able to induce LTP. Cooperativity can be explained by the need for multiple inputs to be activated around the same time to provide sufficient depolarization to remove enough of the Mg2þ block to enable the induction of LTP. This property provides a threshold of activity that has to be exceeded for synaptic plasticity to occur. CA3 neurons typically fire in brief, synchronous bursts and this is presumably to exceed the cooperativity threshold to enable LTP to occur at CA1 synapses. Associativity
Associativity is an extension of cooperativity, but whereby the ‘strong tetanus’ is delivered to an independent input. There is a critical timing window in which the strong input can be delivered after the weak input and still be effective. Associativity is explained by the weak input providing the necessary L-glutamate but insufficient depolarization, whereas the ‘strong input’ provides the (additional) depolarization to sufficiently alleviate the Mg2þ block. The associative depolarization could be provided by a second glutamatergic input or by a different neurotransmitter system that provides or enables depolarization at the critical time. Experimentally, the depolarization can be provided by the recording electrode (in a pairing experiment) and this can permit a single input onto a single neuron to undergo LTP. Thus the requirement for synchronous activity to induce LTP is to provide sufficient depolarization. In conclusion, the Mg2þ block and Ca2þ dependence of the NMDAR explain the properties of input specificity, cooperativity, and associativity.
Role of g-Aminobutyric Acid Inhibition in LTP Role of Postsynaptic g-Aminobutyric Acid Receptors
g-Aminobutyric acid (GABA) inhibition plays a pivotal role in LTP. When several afferents are activated at a low frequency the EPSP is mediated essentially exclusively by AMPARs. This is only because of synaptic inhibition, which is also activated via the excitation of feed-forward interneurons. If GABA receptors are blocked pharmacologically, then the AMPA-mediated EPSP is prolonged and NMDARs contribute much more substantially to the later part of a composite EPSP. Thus, it is the hyperpolarization by GABA to maintain, and indeed intensify, the Mg2þ block that
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prevents NMDARs contributing to any appreciable extent during low-frequency transmission. Thus, blocking GABA inhibition can facilitate the induction of LTP. However, the inhibition of GABA is not just some pharmacological curiosity. During the more physiological patterns of induction (priming and theta) there is an activity-dependent depression of GABA inhibition that is required to enable sufficient activation of NMDARs. This activity-dependent depression is mediated largely by the activation of a presynaptic GABAB autoreceptor. Role of GABAB Autoreceptors
During low-frequency stimulation, GABA is released and evokes a biphasic inhibitory postsynaptic potential (IPSP), mediated by GABAA and GABAB receptors, to restrict the synaptic activation of NMDARs (Figure 2). Some of the GABA that is released activates a presynaptic GABAB autoreceptor, which inhibits subsequent GABA release. During low-frequency transmission this has no effect on GABA transmission, since the effect fully recovers within a second or so. But during high-frequency transmission the autoreceptor mechanism leads to a depression in GABA inhibition. The maximum effect occurs after around 200 ms, which explains the effectiveness of this interval for theta and priming-induced LTP. The GABAB autoreceptor mechanism is essential for the induction of LTP under these conditions, since antagonizing this receptor completely blocks priming-induced LTP. However, the depression of synaptic inhibition only occurs for a few stimuli as the synapse undergoes an activity-dependent facilitation, and, when sufficient stimuli are delivered, a depolarizing potential is generated. Therefore when more artificial induction protocols are used, such as tetanus, blocking GABAB autoreceptors has no effect on the induction of LTP. Given that in the exploring animal there are brief periods of synchronized high-frequency activity occurring at the theta frequency, it is likely that GABAB autoreceptors are critically involved in the induction of LTP in situ.
Signaling Mechanisms Involved in LTP The induction of LTP requires an elevation of postsynaptic Ca2þ. This Ca2þ enters the synapse via the activated NMDARs, where the Ca2þ signal may be greatly magnified by Ca2þ-induced Ca2þ release from intracellular stores. It then activates various kinases. Several protein kinases have been implicated in the induction of LTP. These include Ca2þ/calmodulindependent protein kinase II (CaMKII), protein kinases A and C (PKA, PKC), protein tyrosine kinase (PTK), mitogen-activated protein kinase (MAPK), and
324 Long-Term Potentiation (LTP): NMDA Receptor Role
Excitatory terminal (glutamatergic)
AMPAR NMDAR
Inhibitory interneuron (GABAergic)
GABAAR GABABR
EPSP
a
Excitatory terminal (glutamatergic)
AMPAR NMDAR Inhibitory interneuron (GABAergic)
GABAAR GABABR
EPSP
b Figure 2 Role of g-aminobutyric acid (GABA ergic) inhibition in the induction of long-term potentiation. (a) Activation of glutamatergic inputs onto CA1 hippocampal neurons is accompanied by the activation of inhibitory GABAergic interneurons. These in turn provide an inhibitory input onto the pyramidal cells. GABA release is delayed with respect to glutamate release, as there are two synapses for the signal to pass through, rather than one. This results in a rapid depolarization due to a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) activation followed by a rapid inhibition due to GABAA receptor (GABAAR) activation, ensuring the maintenance of the Mg2þ block. Sustained inhibition is then maintained by activation of postsynaptic GABABRs. Simultaneously, activation of presynaptic GABAB autoreceptors results in a temporary inhibition of GABA release (EPSP, excitatory postsynaptic potential). (b) A burst of high-frequency stimulation while GABA release is reduced (200 ms after the priming stimulus) results in sustained postsynaptic depolarization, overcoming the weakened inhibitory inputs. This sustained depolarization results in the release of Mg2þ from the N-methyl-D-aspartate receptor (NMDAR) and the influx of Ca2þ ions, leading to the induction of long-term potentiation.
phosphatidylinositol 3-kinase (PI3K). Why the need for multiple kinases? It seems that different kinases may be important at different developmental stages and for different phases of LTP. A full understanding of how kinases lead to persistent alterations in synaptic strength during LTP is far from complete.
Expression Mechanisms Involved in LTP Phases of LTP
Numerous studies have attempted to determine how AMPAR-mediated synaptic transmission is enhanced in LTP, and multiple mechanisms have been identified.
Long-Term Potentiation (LTP): NMDA Receptor Role
It is clear that different mechanisms occur at different stages of the LTP process. For example, the earliest phase of LTP, often referred to as short-term potentiation (STP) and most prevalent when a high-frequency tetanus is used to induce the process, probably involves an increase in L-glutamate release. The next phase, commonly referred to as early-phase LTP (E-LTP), most probably involves an alteration in AMPAR number or properties. A similar alteration in AMPAR function may also account for the next, protein synthesisdependent phase of LTP, known as late-phase LTP (L-LTP). However, the expression mechanisms of LTP seem also to be developmentally regulated, with presynaptic mechanisms appearing to play a more important role in E-LTP within the first week of life. Mechanisms of LTP Expression
Two ways in which AMPARs function at synapses may be directly altered during LTP have been identified. There can be an increase in the number of receptors or an increase of the efficiency of the receptors (Figure 3). In the latter case, the mechanism involves an increase in the single-channel conductance of the receptors, so that they pass more current in unit time. The extent to which this results from a modification of receptors already inserted in the synapse – for example, by phosphorylation, or by the exchange of high- for low-conductance receptors – is not yet known. One idea is that AMPARs lacking the GluR2 subunit can be transiently inserted during LTP. This has the effect of both increasing singlechannel conductance and providing an activitydependent Ca2þ signal, by permeation through the now Ca2þ-permeable AMPARs. This Ca2þ signal
Baseline response
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might then drive the insertion of additional Ca2þimpermeable AMPARs. A number of proteins that interact directly with AMPARs have been found to affect their targeting and stabilization at synapses, or their functional properties. These include two membrane-associated guanylate kinases (MAGUKs) – synapse-associated protein 97 (SAP97) and postsynaptic density-95 protein (PSD95), the latter of which binds to an accessory AMPAR protein known as a transmembrane AMPA receptor protein (TARP). Another protein that binds to AMPARs and may be involved in LTP is PICK1 (‘protein interacting with C kinase 1’). Elucidating how these and other interacting proteins, together with small guanosine triphosphatases (GTPases) and protein kinases, function to regulate AMPARs during LTP is the focus of much current interest.
Metaplasticity Metaplasticity is a term that is used to refer to the plasticity of synaptic plasticity. With respect to NMDAR-dependent LTP this can be manifest in several ways. For example, a stimulus protocol (e.g., continuous low-frequency stimulation) that has no effect on baseline transmission can cause LTD of synaptic transmission following the induction of LTP. When the depression is conditional on prior LTP it is referred to as depotentiation. Two forms of depotentiation have been identified; one is dependent on the activation of NMDARs and the other is dependent on activation of metabotropic glutamate receptors (mGluRs). Similarly, two forms of LTD of baseline transmission (sometimes referred to as de novo LTD)
Increase in receptor number
Increase in single channel conductance AMPAR NMDAR Mg2+ Phosphorylation
Figure 3 Expression of long-term potentiation. There are multiple mechanisms that result in the long-term increase in synaptic strength that come under the umbrella of long-term potentiation. The number of a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs) present in the postsynaptic membrane may be increased by exocytosis from an intracellular pool, or the activity of those AMPARs already present may be modified by, for example, phosphorylation. Additional mechanisms include the increase in glutamate release (NMDAR, N-methyl-D-aspartate receptor).
326 Long-Term Potentiation (LTP): NMDA Receptor Role
have been identified that are dependent on activation of NMDARs or mGluRs. The induction of LTP, while enabling depotentiation, can also inhibit the induction of NMDAR-dependent de novo LTD. The induction of NMDAR-dependent LTP can be affected by the prior history of synaptic activity. For example, prior activity at NMDARs can result in inhibition of subsequent LTP, while prior activity of mGluRs can result in facilitation of LTP. Finally, it should be noted that the NMDARmediated component of synaptic transmission is itself plastic – both LTP and LTD have been demonstrated. See also: AMPA Receptors: Molecular Biology and Pharmacology; Long-Term Depression (LTD): Metabotropic Glutamate Receptor (mGluR) and NMDARDependent Forms; Metabotropic Glutamate Receptors (mGluRs): Functions; Metabotropic Glutamate Receptors (mGluRs): Molecular Biology, Pharmacology and Cell Biology; NMDA Receptor Function and Physiological Modulation; NMDA Receptors, Cell Biology and Trafficking.
Further Reading Alford S, Frenguelli BG, Schofield JG, et al. (1993) Characterization of Ca2þ signals induced in hippocampal CA1 neurones by the synaptic activation of NMDA receptors. Journal of Physiology 469: 693. Ault B, Evans RH, Francis AA, et al. (1980) Selective depression of excitatory amino acid induced depolarizations by magnesium ions in isolated spinal cord preparations. Journal of Physiology 307: 413–428.
Bliss TV and Collingridge GL (1993) A synaptic model of memory: Long-term potentiation in the hippocampus. Nature 361: 31–39. Bliss TVP, Collingridge GL, and Morris RGM (2007) Synaptic plasticity in the hippocampus. In: Andersen P, Morris RGM, Amaral DG, et al. (eds.) The Hippocampus, pp. 343–474. Oxford, UK: Oxford University Press. Collingridge GL, Kehl SJ, and McLennan H (1983) Excitatory amino acids in synaptic transmission in the Schaffer collateralcommissural pathway of the rat hippocampus. Journal of Physiology 334: 33–46. Collingridge GL, Isaac JT, and Wang YT (2004) Receptor trafficking and synaptic plasticity. Nature Reviews Neuroscience 5: 952–962. Dale N and Roberts A (1985) Dual-component amino-acidmediated synaptic potentials: Excitatory drive for swimming in Xenopus embryos. Journal of Physiology 363: 35–59. Davies CH, Starkey SJ, Pozza MF, et al. (1991) GABA autoreceptors regulate the induction of LTP. Nature 349: 609. Derkach VA, Oh MC, Guire ES, et al. (2007) Regulatory mechanisms of AMPA receptors in synaptic plasticity. Nature Reviews Neuroscience 8(2): 101–113. Herron CE, Lester RA, Coan EJ, et al. (1986) Frequency-dependent involvement of NMDA receptors in the hippocampus: A novel synaptic mechanism. Nature 322: 265–268. MacDermott AB, Mayer ML, Westbrook GL, et al. (1986) NMDAreceptor activation increases cytoplasmic calcium concentration in cultured spinal cord neurones. Nature 321: 519–522. Nowak L, Bregestovski P, Ascher P, et al. (1984) Magnesium gates glutamate-activated channels in mouse central neurones. Nature 307: 462–465.
Relevant Website http://www.bris.ac.uk – University of Bristol, Medical Research Council Centre for Synaptic Plasticity.
Long-Term Depression (LTD): Metabotropic Glutamate Receptor (mGluR) and NMDAR-Dependent Forms C Lu¨scher, University of Geneva, Geneva, Switzerland M Frerking, Oregon Health and Science University, Beaverton, OR, USA ã 2009 Elsevier Ltd. All rights reserved.
How Is LTD Induced? Although N-methyl-D-aspartate receptor (NMDAR-) and metabotropic glutamate receptor (mGluR-) longterm depression (LTD) can both be elicited in vivo, the mechanisms of LTD have been best studied in the acute brain-slice preparation, where it can be induced with a number of protocols. At most synapses, NMDAR-LTD is induced by a modest activation of NMDARs, whereas stronger activation leads to longterm potentiation (LTP). Exceptions to this rule may apply to the parallel fiber (PF)–Purkinje cell synapse in the cerebellum. Activity-Dependent Induction
For NMDAR-LTD, prolonged low-frequency afferent stimulation (e.g., 900 stimuli at 1–5 Hz) has proven to be very efficient. Brief low-frequency afferent stimulation coupled to weak postsynaptic depolarization to –40 mV (to promote a modest relief of the Mg block of the NMDAR) can also efficiently induce NMDARLTD. In addition, NMDAR-LTD can be induced by the correlated activation of the pre- and postsynaptic neuron in many systems in which the firing of the postsynaptic neurons precedes the presynaptic action potential (AP) that releases the glutamate. The proposed mechanism to explain this spike timing-dependent LTD is that the AP in the postsynaptic cell backpropagates to the synapse, relieving the Mg block of the NMDAR. If the back-propagating AP precedes the release of glutamate, a low level of NMDAR activation is generated that leads to LTD. To induce mGluR-LTD, a wide variety of stimulation protocols have been used. Typically mGluR-LTD requires a higher stimulation frequency than NMDAR-LTD (up to 300 Hz) or burst firing (e.g., several repetitions of five stimuli at 66 Hz), most likely due to the extrasynaptic location of mGluRs. In the hippocampus, a typical induction protocols consists of stimulation at 5 Hz for 3 min or the delivery of pairs of presynaptic APs in rapid succession. Chemical Induction
Because activity-dependent LTD is generally specific to the small fraction of synapses that receive that activity,
there has also been interest in chemical induction of LTD with receptor-selective agonists. This approach is particularly useful for studying the mechanisms underlying both NMDAR-LTD and mGluR-LTD because such treatment uniformly changes transmission in the majority of synapses. NMDAR-LTD can be chemically induced by the exposure of a slice with NMDA for a few minutes. This chemically induced NMDAR-LTD occludes with NMDAR-LTD that is induced by synaptic stimulation, suggesting that the two overlap substantially. Similarly, mGluR-LTD can be induced by bath application of the drug dihydroxyphenylglycol (DHPG), a selective agonist of group I mGluRs. As with NMDARLTD, mGluR-LTD induced chemically occludes with mGluR-LTD induced by synaptic activity.
What Receptors Are Involved in the Induction of LTD? An interesting idea, introduced about 6 years ago, is that the subunit composition of activated NMDARs determines whether LTD or LTP is elicited. Based on the use of subtype-selective NMDAR antagonists, it has been reported that the NR2A-containing NMDARs selectively elicit LTP but not LTD, whereas NR2B-containing receptors selectively elicit LTD but not LTP in both the hippocampus and cortex. However, results from genetic manipulations suggest that NR2B can contribute to LTP. Several studies have also failed to replicate the reported NMDAR-subtype dependence of LTP or LTD in the hippocampus. LTD is, similarly, not obviously subunit-dependent in the anterior cingulate cortex. mGluR-LTD requires the activation of mGluRs, but there are several mGluR subtypes (mGluR1–8) and the particular mGluR leading to LTD appears to depend on the preparation in which it is induced. DHPG, a selective agonist of group I mGluRs (mGluR1 and mGluR5) elicits mGluR-LTD, which occludes with synaptically induced plasticity in many different systems, suggesting that in most cases mGluR-LTD is mediated by mGluR1, mGluR5, or both. The selective mGluR1 antagonist CPCOOEt blocks synaptically induced mGluR-LTD in the ventral tegemental area (VTA), and mGluR1 is also implicated in LTD in the cerebellum and neostriatum. mGluR5, on the other hand, has been shown to be the principal receptor responsible for mGluR-LTD in the hippocampus, cortex, and nucleus accumbens, although recent data in knockout mice indicate that the two
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328 Long-Term Depression (LTD): Metabotropic Glutamate Receptor (mGluR) and NMDAR-Dependent Forms
receptors may work in synergy. The activation of group II mGluRs (mGluR2 and mGluR3) has also been implicated in the CA3 and dentate gyrus of the hippocampus, as well as some regions of the cortex and the amygdala.
Intracellular Events Triggered by the Induction of LTD The crucial event triggered by NMDAR activation during NMDAR-LTD is calcium influx (see Figure 1). The requirement for modest, but not strong, NMDAR activation suggests that a modest increase in calcium can lead to NMDAR-LTD, whereas strong increases give rise to NMDAR-LTP. This notion is supported by manipulations of intracellular calcium in which weak and prolonged calcium stimuli lead to LTD, whereas rapid and robust calcium stimuli lead to LTP. The exact determinants of the time course and spatial extent of the calcium signal still have to be worked out, and it remains unclear whether [Ca] levels alone are sufficient to determine whether LTP or LTD is induced. However, an attractive explanation for the bidirectional effects of calcium is that the calcium-dependent effectors that are required for NMDAR-LTD are very sensitive to calcium, whereas the effectors for NMDAR-LTP are less so.
mGluR1/5
NMDAR
GluR2
GluR1
Ca2+
Gq PICK1 PKCa
+
Kinase
+
Calcineurin
ser-880 NSF
GRIP/ABP
−
PKA
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ser-845
PICK1 Endocytosis
PSD-95 SAP-97
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Conductance recycling
Figure 1 Schematic of intracellular signaling pathways involved in postsynaptic NMDAR-LTD and mGluR-LTD. Both forms of LTD involve the phosphorylation of ser-880 on the C-terminal tail of GluR2, which eventually increases endocytosis of mobile AMPARs. Mobility is conferred by exchanging GRIP/ABP for PICK1. NMDAR-LTD is, in addition, associated with a dephosphorylation of ser-845 on the C-terminal tail of GluR1, leading to decreased conductance and recycling. AKAP regulates the balance between constitutive PKA phosphorylation and activitydriven dephosphorylation of ser-845. AKAP, A-kinase associating protein; GRIP/ABP, glutamate receptor interacting protein/AMPA receptor binding protein; mGluR, metabotropic glutamate receptor; NMDAR, N-methyl-D-aspartate; receptor; NSF, N-ethylmaleimide sensitive factor; PICK1, protein interacting with C kinase 1; PKA, protein kinase A; PKC, protein kinase C; PSD, postsynaptic density protein; SAP, synapse-associated protein; ser, serine.
Consistent with this line of reasoning, NMDARLTD requires the calcium/calmodulin-dependent phosphatase calcineurin, which has a very high affinity for calcium-bound calmodulin (100 pM). The protein phophatase 1 (PP1) is also required for at least some forms of NMDAR-LTD. NMDAR-LTD leads to a dephosphorylation of ser-845 of the a-amino-3hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) subunit GluR1. Thus, NMDAR activation leads to the activation of PPs, which leads to AMPA receptor dephosphorylation. A caveat, however, is that there is much yet to be understood regarding how phosphatase activity is required for NMDAR-LTD; activity-dependent NMDAR-LTD requires both calcineurin and PP1, but chemically induced NMDARLTD does not appear to require PP1. Moreover, the phosphatase that actually directly targets GluR1 has not yet been identified. mGluR-LTD depends on the activation of G-proteins of the pertussis-toxin-insensitive Gq and Ga11 family (see Figure 1). Group I mGluRs couple to phosphoinositide signaling via Gq. Gq seems to predominate in mGluR-LTD at most synapses, including in area CA1 of the hippocampus and at cerebellar PF synapses, where LTD was strongly reduced in Gq/ mice, whereas in Ga11/ mice the reduction was much smaller. The events that happen downstream of Gq activation, however, are less clear. Gq is coupled to phospholipase C (PLC), which triggers the synthesis of inositol triphosphate (IP3) and protein kinase C (PKC). This ultimately leads to release of calcium from intracellular stores. PKC is clearly involved in mGluR-LTD in the cerebellum and the VTA, but in the hippocampus mGluR-LTD seems not to require PKC or even PLC. The involvement of several other signaling pathways has been suggested, including p38 mitogen-activated protein (MAP) kinase, the extracellular signal-regulated kinase, tyrosine phosphatases, and phosphoinositide-3-kinase. Further study to define the intracellular cascades activated by mGluRs in the hippocampus will be of interest.
Expression Mechanisms Underlying NMDAR- and mGluR-Dependent LTD There is a general agreement that at least some forms of NMDAR-LTD and mGluR-LTD are expressed as a postsynaptic reduction in AMPAR function, and evidence suggests that both forms of LTD act primarily via the clathrin-mediated endocytosis of AMPARs. However, there are several differences between these two types of LTD, and a number of significant questions remain to be addressed.
Long-Term Depression (LTD): Metabotropic Glutamate Receptor (mGluR) and NMDAR-Dependent Forms 329 AMPAR Trafficking
AMPAR Subunits and LTD
The first evidence that NMDAR-LTD was due to a withdrawal of AMPARs from the cell surface came from a series of studies demonstrating that NMDAR activation could lead to the internalization of AMPARs in cultured hippocampal neurons and that NMDAR-LTD in hippocampal slices was blocked by disrupting interactions between key components of the endocytic machinery (the proteins amphiphysin and dynamin). LTD is abolished by blocking the interactions between AMPAR subunits and the AP2 complex, which links to clathrin and is involved in the formation of endocytic-coated pits. In addition, LTD is impaired by viral transfections of the dominant-negative form of the small GTPase Rab5, which is thought to be involved in the trafficking of vesicles between the plasma membrane and endosomal compartments. Thus, NMDAR-LTD requires several components of endocytotic machinery at different stages of the endocytic pathway, and NMDAR activation leads to the withdrawal of AMPARs from the cell surface. Some evidence also implicates receptor endocytosis in mGluR-LTD, although the issue has received less direct experimental attention. In the cerebellum, the postsynaptic infusion of peptides that interfere with the amphphysin–dynamin interaction blocks mGluRdependent LTD. Activation of group I mGluRs has also been found to cause a reduction in AMPAR clustering at synapses in hippocampal cell cultures that is, again, blocked by interfering with the amphiphysin – dynamin interaction. Thus, mGluR-LTD appears to lead to loss of synaptic AMPARs via receptor internalization. However, not all data are readily compatible with this model. NMDAR-LTD leads to a reduction in postsynaptic responsiveness to exogenous agonists, as does mGluR-LTD in the cerebellum. Surprisingly, however, mGluR-LTD induced by DHPG application in the hippocampus does not. One possible explanation is that in the hippocampus, mGluR-LTD leads to a selective internalization or dispersion of synaptic AMPARs while leaving extrasynaptic AMPARs unaffected. In fact, with both types of LTD the time course and location of endo- and exocytosis remains to be fully identified. In hippocampal neuronal cultures, NMDAR activation induces a rapid lateral movement that precedes internalization at extrasynaptic sites. Several studies also implicate a presynaptic reduction of transmitter release probability in mGluRLTD, particularly in hippocampus. It remains unclear whether mGluR-LTD has both presynaptic and postsynaptic components that operate in tandem, or whether AMPAR loss preferentially affects synapses which have a high release probability.
Because AMPARs are heteromultimeric complexes that can be composed of any of four different subunits, GluR1–4, there has been considerable emphasis on the possible differential involvement of specific AMPAR subunits in the expression of LTD. GluR2 The extreme C-terminus of GluR2 and GluR3 contains a postsynaptic density protein (PSD-) 95, Dlg, and ZO1 (PDZ) ligand sequence that is recognized by several proteins with PDZ domains (the glutamate receptor interacting protein (GRIP), the AMPA receptor binding protein (ABP), and the protein interacting with C kinase 1 (PICK1)). The binding of GluR2 to GRIP/ABP appears to be important in stabilizing AMPARs at the synapse and may also be involved in retaining AMPARs in intracellular pools. Blocking the interaction between GluR2 and PICK1 blocks the removal of GluR2 from the plasma membrane and recent data suggest a model in which the binding of PICK1 allows the mobility of AMPARs. The GluR2/ PICK1 complex can be disassembled by ATPase activity of the N-ethylmaleimide sensitive factor (NSF), which binds to GluR2 (but not GluR3) at a site upstream of the PDZ ligand. In addition to binding with GluR2, PICK1 also binds to the a isoform of PKC and to GRIP/ABP. This complex of PICK1, PKC, and GRIP/ABP recruits PKCa to synapses. GluR2 has a PKC phosphorylation site (serine 880 (ser-880)) that is near the PDZ ligand sequence, and the phosphorylation of GluR2 at ser880 disrupts the interaction between GluR2 and GRIP/ABP but not the interaction between GluR2 and PICK1. Thus, a simple model is one in which LTD induction activates PKCa, leading to the PICK1-mediated targeting of PKCa to synapses. This would promote phophorylation of ser-880 on GluR2, causing GluR2 to release GRIP/ABP and bind to PICK1, promoting lateral diffusion and ultimately internalization. An elegant series of studies suggest that just such a mechanism accounts for the mGluR-dependent LTD in the cerebellum. Cerebellar LTD requires PKC, and in particular PKCa. LTD induction leads to the phosphorylation of ser-880, and genetic manipulations that disrupt the PKC consensus site on GluR2, as do disruptions of the PDZ ligand site on PKCa that mediates its interaction with PICK1. PDZ domain-mediated interactions among GluR2, PICK1, and PKCa also appear to be required for cerebellar LTD. Peptides that disrupt the PDZ interactions between GluR2 and PICK1 attenuate cerebellar LTD without affecting basal transmission. Cerebellar LTD is also abolished in knockout mice lacking either GluR2
330 Long-Term Depression (LTD): Metabotropic Glutamate Receptor (mGluR) and NMDAR-Dependent Forms
or PICK1. More specific evidence of the necessity for GluR2–PICK1 interaction is provided by the observation that cerebellar LTD is also impaired by genetic manipulations that disrupt the PDZ ligand on GluR2 or the PDZ domain on PICK1. Finally, interactions between NSF and GluR2 appear to be essential for the synaptic incorporation of AMPARs that are competent to undergo cerebellar LTD. Transfection of GluR3 into Purkinje cells from GluR2 knockout mice does not rescue cerebellar LTD, nor is the transfected GluR3 incorporated into synapses. However, a mutated version of GluR3 that contains the NSF binding site of GluR2 is incorporated into Purkinje cell synapses from GluR2 knockout mice, and these synaptically incorporated AMPARs are competent to undergo cerebellar LTD. Do similar mechanisms account for mGluR-LTD observed at other synapses or for NMDAR-LTD? It is clear that at least some synapses express a form of mGluR-LTD that cannot occur via a loss of GluR2containing AMPARs – at excitatory synapses on to dopamine cells in the VTA, for example, mGluR-LTD causes a shift in the biophysical properties of the AMPAR excitatory postsynaptic current (EPSC) that suggests a selective loss of AMPARs that lack the GluR2 subunit. At first glance, NMDAR-LTD also seems unlikely to act via similar mechanisms because NMDAR-LTD does not require PKC activity. However, NMDARLTD can lead to PKC-independent phosphorylation of ser-880 through an as yet unidentified kinase, raising the possibility that a similar mechanism can be engaged by the same phosphorylation mediated by a different kinase. Moreover, peptides that interfere with the interaction between GluR2 and PDZ domain-containing proteins do impair LTD, although there is disagreement about whether GRIP/ABP or PICK1 is the essential mediator of these effects. Peptides that disrupt the interaction between GluR2 and NSF have also been observed to cause a rundown of synaptic transmission and a concomitant impairment of LTD. However, a few notes of caution are in order. The NSF binding site on GluR2 overlaps substantially with a binding site for the AP-2 adaptor complex that links to clathrin and is involved in the formation of endocytic-coated pits. This AP-2 binding site is also present in GluR1 and GluR3, and the peptides used in initial studies of the NSF–GluR2 interaction also disrupt the interaction between AMPARs and AP-2. Experiments with more selective peptides suggest that AP-2 interactions with AMPARs are required for NMDAR-LTD, whereas NSF–GluR2 interactions are required for the rundown of basal transmission. Mutated versions of GluR2 in which the interactions with GRIP, ABP, and PICK1 are abolished can still
undergo normal NMDA-induced internalization in hippocampal cultures, and a mutated version of GluR2 in which the phosphorylation of ser-880 is disrupted is still competent to undergo LTD in hippocampal slices, albeit less efficiently than wild-type GluR2. More decisively, NMDAR-LTD is unaffected in GluR2 knockout mice, and it is actually enhanced in GluR2–GluR3 double-knockout mice. This makes it difficult to argue that either GluR2 or GluR3 is a critical component of NMDAR-LTD. One possible explanation is that the peptides used to selectively interfere with GluR2 function are not as specific as previously thought. This possibility is supported by the difficulties in distinguishing between NSF and AP-2 interactions with GluR2 and also by the observation that the peptides that interfere with GluR2– PDZ domain interactions can also impair PDZ domain interactions with distantly related kainate receptor subunits. GluR1 An alternative subunit-selective mechanism for NMDAR-LTD is suggested by the observations that the ser-845 residue on GluR1 is dephosphorylated by exogenous NMDA in cell culture or by more conventional LTD induction in hippocampal slices. Ser-845 is constitutively phosphorylated by protein kinase A (PKA), and PKA activation has little effect on AMPAR function under basal conditions, presumably because of basal phosphorylation. However, the inhibition of PKA can depress AMPAR-mediated synaptic transmission and occlude LTD. PKA and calcineurin are colocalized by the A-kinase associating proteins (AKAPs), which are targeted to synapses by interactions with the scaffolding proteins PSD-95 and synapse-associated protein (SAP-)97. An accumulating body of evidence suggests indicates that AKAPs are dynamically regulated during LTD to shift the equilibrium between PKA and calcineurin localization at synapses. This determines the phosphorylation state of ser-845, and manipulations that interfere with PKA binding to AKAPs cause a depression of AMPAR-mediated transmission that occludes LTD. Further experiments to selectively examine the role of ser-845 in NMDAR-LTD will be of interest. How might ser-845 dephosphorylation be involved in LTD? Phosphorylation of ser-845 can enhance AMPAR function by increasing the peak open probability of the channel, so calcineurin-mediated dephosphorylation of GluR1 could directly contribute to LTD expression even in the absence of changes in receptor trafficking. However, dephosphorylation of ser-845 appears to be downstream of receptor internalization and prevents the recycling of AMPARs after they have been internalized. Thus, current evidence suggests that the dephosphorylation of GluR1 is a
Long-Term Depression (LTD): Metabotropic Glutamate Receptor (mGluR) and NMDAR-Dependent Forms 331
fairly late event in the mechanisms underlying LTD that may have more to do with preventing AMPAR reinsertion than with the withdrawal of AMPARs from the synapse. Maintenance of LTD
The previous section focused on the mechanisms by which AMPAR function is depressed during LTD. We now turn our attention to the mechanisms that allow the depression to persist long after the induction event is over. The answers to this question are still largely unknown, although the available data are sufficient in some cases to draw some surprising conclusions that deserve mention. In the case of NMDAR-LTD, some evidence is consistent with the idea that LTD is maintained by the change in phosphorylation of the AMPAR itself. As discussed previously, dephosphorylation at ser-845 on GluR1 prevents internalized AMPARs from recycling back to the synapse, suggesting that the phosphorylation state of this site may determine whether LTD persists; consistent with this idea, PKA activation can lead to a reversal of LTD following the stable induction of LTD, as can synaptic activity. Synaptically induced de-depression can be reduced by the inhibition of PKA, providing further evidence for dephosphorylation of PKA targets as a mechanism that is responsible for the maintenance of NMDAR-LTD. However, it need not be the case that NMDARLTD is maintained by the duration of modifications to AMPARs. An alternative is that the number of AMPARs at the synapse is limited by proteins that tether the AMPAR at the postsynaptic density; a decrease in the amount of this tether would preclude AMPAR reinsertion at synapses following LTD induction even if the AMPARs were recycled to the cell surface because there would be no mechanism to hold them at the synapse. The protein PSD-95 has many properties that are consistent with such a tether. Manipulations that increase or decrease PSD-95 expression cause parallel changes in the synaptic AMPAR-mediated response. This may be relevant to the maintenance of NMDAR-LTD, because NMDA applied to hippocampal neurons in culture leads to the ubiquitination and subsequent proteosomal degradation of PSD-95. This loss of PSD-95 is dependent on calcineurin and blocked by PKA activation, similar to LTD; moreover, the inhibition of proteosome function prevents the NMDA-induced reduction in surface expression of GluR1 and GluR2. NMDA also actively recruits proteosomes to dendritic spines. Further characterization of this potential mechanism for LTD maintenance will be of interest. In the case of mGluR-LTD, it has long been known that the LTD induced by DHPG can be reversed
long after stabilization by the application of group I mGluR antagonists. This indicates, surprisingly, that the expression mechanisms underlying mGluR-LTD are maintained by constitutive activation of an mGluR following LTD induction; in the absence of mGluR activity, the depression is quickly reversed. An alternative model that has some experimental support is that mGluR-LTD might be maintained by the synthesis of new proteins that stabilize LTD expression. Both LTD and the internalization of AMPAR subunits are blocked by inhibitors of mRNA translation. This potential link between translation of new proteins and mGluR-LTD became a source of considerable excitement when it was found that mice lacking the fragile X mental retardation protein (FMRP) have enhanced mGluR-LTD in both the hippocampus and cerebellum. The loss of function of FMRP is responsible for the fragile X syndrome in humans, an X-linked form of mental retardation. FMRP is an RNA-binding protein, although it remains a topic of debate whether FMRP represses or stimulates translation. Regardless of the precise action of FMRP on translation, the observation that FMRP enhances mGluR-LTD seemed at first to clearly indicate a role for changes in protein synthesis as an essential part of mGluR-LTD. Surprisingly, however, subsequent analysis of FMRP knockout mice indicated that mGluR-LTD in these mice is not only enhanced but also independent of protein synthesis. Similarly, the inhibition of proteosome function has recently been found to block mGluR-LTD in wildtype mice but not in FMRP knockout mice. These results are difficult to reconcile with an integral role for protein synthesis or degradation in mGluR-LTD, and further study will be of interest. See also: Long-Term Potentiation (LTP): NMDA Receptor Role; Metabotropic Glutamate Receptors (mGluRs): Functions; Metabotropic Glutamate Receptors (mGluRs): Molecular Biology, Pharmacology and Cell Biology; NMDA Receptor Function and Physiological Modulation; NMDA Receptors, Cell Biology and Trafficking.
Further Reading Anwyl R (2006) Induction and expression mechanisms of postsynaptic NMDA receptor-independent homosynaptic long-term depression. Progress in Neurobiology 78: 17–37. Bear MF (2003) Bidirectional synaptic plasticity: From theory to reality. Philosophical Transactions of the Royal Society of London. B. Biological Sciences 358: 649–655. Bear MF, Huber KM, and Warren ST (2004) The mGluR theory of fragile X mental retardation. Trends in Neuroscience 27: 370–377. Bellone C and Lu¨scher C (2005) mGluRs induce a long-term depression in the ventral tegmental area that involves a switch of the subunit composition of AMPA receptors. European Journal of Neuroscience 21: 1280–1288.
332 Long-Term Depression (LTD): Metabotropic Glutamate Receptor (mGluR) and NMDAR-Dependent Forms Carroll RC, Beattie EC, vonZastrow M, and Malenka RC (2001) Role of AMPA receptor endocytosis in synaptic plasticity. Nature Reviews Neuroscience 2: 315–324. Cull-Candy S, Kelly L, and Farrant M (2006) Regulation of Ca2þpermeable AMPA receptors: Synaptic plasticity and beyond. Current Opinion in Neurobiology 16: 288–297. Dan Y and Poo MM (2006) Spike timing-dependent plasticity: From synapse to perception. Physiological Reviews 86: 1033–1048. HanleyJG(2006)MolecularmechanismsforregulationofAMPARtraffickingbyPICK1.Biochemical Society Transactions 34:931–935. Jorntell H and Hansel C (2006) Synaptic memories upside down: Bidirectional plasticity at cerebellar parallel fiber-Purkinje cell synapses. Neuron 52(2): 227–238. Lu¨scher C, Nicoll RA, Malenka RC, and Muller D (2000) Synaptic plasticity and dynamic modulation of the postsynaptic membrane. Nature Neuroscience 3: 545–550.
Malenka RC and Bear MF (2004) LTP and LTD: An embarrassment of riches. Neuron 44: 5–21. Malinow R and Malenka RC (2002) AMPA receptor trafficking and synaptic plasticity. Annual Review of Neuroscience 25: 103–126. Meng Y, Zhang Y, and Jia Z (2003) Synaptic transmission and plasticity in the absence of AMPA glutamate receptor GluR2 and GluR3. Neuron 39(1): 163–176. Oliet SH, Malenka RC, and Nicoll RA (1997) Two distinct forms of long-term depression coexist in CA1 hippocampal pyramidal cells. Neuron 18: 969–982. Segal M (2005) Dendritic spines and long-term plasticity. Nature Reviews Neuroscience 6(4): 277–284.
D-Serine:
From Its Synthesis in Glial Cell to Its Action on Synaptic Transmission and Plasticity
S H R Oliet and J-P Mothet, Institut National de la sante´ et de la Recherche Me´dicale U862, Bordeaux, France ã 2009 Elsevier Ltd. All rights reserved.
Introduction When thinking about brain signaling, neurophysiologists traditionally refer to the transfer of information from one neuron to another. Accordingly, the chemical synapse is viewed as the structure in which such communication occurs between the pre- and postsynaptic elements and where neurotransmitters are released from the presynaptic bouton to activate specific receptors localized on the postsynaptic neuron. This model, however, needs to be revised in view of the findings describing the role of glial cells as the third element of the chemical synapse. At the anatomical level, astrocytic processes enwrap up to 60% of the synaptic volume. Despite such an intimate structural relationship, astrocytes were believed to ensure only a housekeeping function at synapses. There is now accumulating evidence indicating that glial cells, and particularly astrocytes, contribute actively to synapse development, synaptic transmission, and neuronal excitability. These results fuel the emerging concept of the tripartite synapse, which considers astrocytes as an integral part of central and peripheral synapses. According to this model, glial cells first sense synaptic activity through a broad variety of ion channels, transporters, and receptors expressed at their surface. Synaptic activation of glial cells then triggers intracellular second-messenger pathways, including Ca2þ increases that vary according to the synaptic inputs solicited and the glial receptors involved. In turn, activation of these second-messenger pathways induces the release of active substances termed gliotransmitters (by analogy to neurotransmitters). These gliotransmitters mediating astrocyte-to-neuron signaling include glutamate, taurine, and adenosine triphosphate (ATP), which can be hydrolyzed in adenosine. Another major gliotransmitter which could have a major role in brain signaling is D-serine. The discovery of this amino acid in the brain has forced us to reconsider the dogma that only L-isomers of amino acids occur in mammals. Organic molecules, and therefore biological molecules such as D-amino acids, are based on the chemistry of the carbon atom. Carbon atoms can have up to four bonded groups attached to them in three-dimensional space,
forming a tetrahedron. Because of this structure, carbon-containing molecules can have the same four constituents, yet can differ in their structure by the location of the four groups in space. This property of carbon-based molecules is known as chirality. As an example, our hands are mirror images of one another, but they cannot be superimposed on one another. Similarly, carbon atoms with four different groups attached occur in two forms, known as enantiomers, that are not superimposable mirror images of one another (see Figure 1). Although the chemical and physical properties of L-amino acids and D-amino acids are extremely similar, only L-amino acids seemed to have been selected from the origin of life on the primitive Earth. In this chemical evolutionary step, D-amino acids were eliminated, and it was thought that all living organisms were composed only of L-amino acids. This asymmetry in biology is assumed to be a feature of fundamental physics because it turns out that natural L-amino acids are more stable than their ‘unnatural’ mirror images, D-amino acids. Until the last 30 years, it was believed that D-amino acids were excluded from living systems except for D-amino acids in the cell walls of microorganisms. Now, biologists have discovered that nature can deal with at least two D-amino acids, D-serine and D-aspartic acid, in higher organisms. Of these two atypical amino acids, D-serine is potentially very important for the nervous system because it is likely be the major endogenous ligand for the strychnineinsensitive glycine-binding site of N-methyl-D-aspartate receptors (NMDARs), key receptors for excitatory transmission and cognitive functions. Since the late 1980s, it has been established that activation of NMDARs required glutamate and a co-agonist that was first identified as glycine. Yet significant levels of D-serine are present in rodent brain areas enriched in NMDARs, suggesting that D-serine might be an alternative to glycine for NMDAR activation. D-Serine and serine racemase (SR), an enzyme that synthesizes D-serine from L-serine, are mainly, if not exclusively, localized to astrocytes. That glial-derived D-serine is an endogenous ligand of NMDARs and was unambiguously demonstrated by the use of D-amino acid oxidase (DAAO), an enzyme that selectively degrades D-serine but not glycine. Under conditions in which D-serine levels are dramatically reduced with DAAO, NMDAR activity is markedly impaired in different brain regions. It is now obvious that glial cells, through the release of D-serine, contribute actively to NMDAR function in the mammalian brain.
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D-Serine:
From Its Synthesis in Glial Cell to Its Action on Synaptic Transmission and Plasticity
COOH
H
C
R
NH2
COOH
R
C
H
NH2
Figure 1 The chirality in your hands. The term chiral (from the Greek word for hand) is used to describe an object which is not superimposable on its mirror image, for example, our two hands. This property is based on the fact that carbon atoms (C) bind four groups – COOH, NH2, H, or any other radical (R) – in a threedimensional space. Such a tetrahedral hybridized carbon with four different groups always forms a chiral center. Conversely, a substrate carbon atom is not chiral if two out of four of its groups are identical, as it is in the case of the amino acid glycine.
Localization of D-Serine D-Serine is present in significant amounts in the brain of rodents and humans, where its levels (500 mM) are up to one-third of the total free serine pool. Its distribution is heterogeneous with the highest concentrations in the telencephalon and the developing cerebellum. This pattern of distribution throughout the rat brain resembles that of NMDARs. In contrast, glycine immunoreactivity does not correspond to NMDAR distribution excepted for some regions where D-serine is also present. Detailed analysis of D-serine staining shows that it is mostly present in astrocytes that ensheath synapses. In the developing cerebellum, D-serine is localized to Bergmann glia, whereas in adults it declines to negligible levels. Recent investigations have found that microglia cells as well as Schwann cells contain significant amount of D-serine and SR, which are also found in Mu¨ller glial cells in the retina and in the supporting cell of the vestibular sensory epithelium. Whether D-serine and SR are also present in other types of glial cells, such as oligodendrocytes, pituicytes, tanycytes, or ependymal cells, remains unknown. Although at very low levels, neuronal immunoreactivity for D-serine has been still observed in the neurons of the cerebral cortex, the brain stem, and the olfactory bulb.
Synthesis and Degradation of D-Serine The idea that D-amino acids and particularly D-serine serves specific roles in the brain was strengthened by
the discovery of its metabolic pathway. High levels of D-serine in the mammalian brain are generated by the activity of a pyridoxal 50 -phosphate (vitamin B6)-dependent enzyme, SR. This enzyme not only converts L- into D-serine, but also converts D- into L-serine, with a lower affinity. The enzymatic-catalyzed racemization of L-serine proceeds by removal of a proton from the asymmetric C–H bond of the amino acid to form a carbanion intermediate. The trigonal carbon atom of the carbanion, having lost the original asymmetry, then recombines with a proton to regenerate as an inverted tetrahedral structure corresponding to D-serine. The distribution of SR closely resembles that for D-serine, with the highest expression in the hippocampus and corpus callosum and very low levels in the amygdala, subthalamic nuclei, and the thalamus. Regarding cellular distribution, detailed analysis indicates that SR, like D-serine, is mostly confined to astrocytes, although substantial expression of SR is present in some neurons. However, due to the numeric preponderance of astroglia cells over neurons, glial cells remain the principal source of D-serine in the brain. SR activity can be negatively regulated by a series of cellular compounds. Thus, glycine and a series of metabolites related to L-aspartic acid competitively inhibit the enzyme. Because glycine concentrations in astrocytes are approximately 3–6 mM, glycine would constitutively inhibit SR activity unless glycine and SR show different compartmentalizations within the astrocyte cytosol. The metabolic pathway for D-serine degradation remains more elusive. Mammalian D-serine can be metabolized by the peroxisomal flavoprotein DAAO, an enzyme highly present in astrocytes of the hindbrain and cerebellum. Adult DAAO-deficient mice display increased D-serine levels, especially in areas where it normally occurs at low levels. However, although DAAO protein is present in the forebrain, an area enriched in D-serine, levels of the D-amino acid appear relatively unchanged in this region in DAAOdeficient mice. This result suggests that other mechanisms are probably implicated in regulating D-serine concentrations in this brain area. SR and DAAO may not work in isolation because SR and DAAO activities are controlled in opposite ways by nitric oxide (NO). Although NO enhances SR activity, it decreases that of DAAO, thus downregulating the intracellular levels of D-serine. In turn, D-serine inhibits NO synthase in glial cells and stimulates the production of NO in neurons. Neuronal NO could thus represent an inhibitory feedback mechanism tightly regulating D-serine metabolism in astrocytes and thereby preventing its overproduction and excessive stimulation of NMDARs.
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Release of D-Serine Glial cells detect changes in their environment through a broad variety of ion channels, transporters, and receptors expressed at their surface. Specific activation of metabotropic and non-NMDA glutamate receptors on astrocytes induces the efflux of D-serine from these cells in culture (Figure 2). Whether activating these glutamate receptors is the only stimulatory pathway for D-serine release remains to be determined. Similarly, the conditions during which the activation of glial glutamate receptors, and, consequently, D-serine release occur in response to afferent synaptic activity are unknown. Interestingly, release of glutamate from glial cells also occurs when metabotropic and non-NMDA glutamate receptors are stimulated. An important issue for D-serine release, and for gliotransmission in general, is the identification of the molecular pathway responsible for the efflux of active substances from cells that were long thought to be passive and/or unexcitable. Astrocytes, like all eukaryotic cells, use secretory lysosomes to transport new membrane and proteins to the plasma membrane during constitutive exocytosis. Most cell types also possess a pathway of regulated exocytosis in which secretory vesicles undergo
Astrocyte
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AMPAR KainateR mGluR D-Serine
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Figure 2 Induction of D-serine release at glutamatergic synapses. Synaptically released glutamate diffuses locally to neighboring glial processes and binds to AMPA (AMPAR), kainate (kainaiteR) and/or metabotropic (mGluR) receptors. This triggers the release of D-serine within the synaptic cleft, enabling NMDAR activation on the postsynaptic neuron. AMPA, a-amino-3-hydroxy5-methyl-4-isoxazole propionic acid; AMPAR, AMPA receptor; kainaiteR, kainaite receptor; mGluR, metabotropic glutamate receptor; NMDAR, N-methyl-D-aspartate receptor.
Ca2þ-dependent fusion with the plasma membrane. Until recently, Ca2þ-regulated exocytosis was considered as a hallmark of neurons in the nervous system. Both constitutive and regulated secretory pathways require specialized proteins to bring together the membranes of the vesicles with the plasma membrane. The soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptors (SNAREs) are leading candidates for mediating membrane fusion and most of them are present in glial cells. In addition, glial cells contain also synaptic-like microvesicles (SLMV) and large dense-core vesicles (LDCV), as revealed by electron microscopy and confocal microscopy analyses. Results obtained over the last decade using electrophysiological, biochemical, and imaging techniques suggest that astrocytes use a Ca2þregulated SNARE-dependent exocytosis to release glutamate from SLMVs but also ATP and peptides from LDCVs. In cultured astrocytes, activation of metabotropic and non-NMDA receptors effectively triggers a Ca2þ- and SNARE-dependent release of D-serine. In this experimental model, modifying extracellular or intracellular Ca2þ concentrations or depleting thapsigargin-sensitive Ca2þ stores, considerably impairs D-serine release. These results reveal that activation of non-NMDARs is the major source of extracellular Ca2þ and that mobilization of Ca2þ from internal stores is necessary for D-serine release. This is consistent with the observation that both inositol triphosphate (IP3)- and caffeine/ryanodinesensitive Ca2þ stores control the release from glial cells of glutamate, another gliotransmitter. Furthermore, inhibiting exocytosis with clostridial toxins or preventing amino acid uptake into vesicles with concanamycin or bafilomycin, impairs the release of D-serine from astrocytes. These results are thus consistent with a vesicular storage and release of the D-amino acid. Interestingly, astrocytic glutamate also appears to be stored in, and released from, vesicles. If glutamate and D-serine were co-stored in the same vesicles, this would be the perfect gliotransmitter cocktail to activate NMDARs. This hypothesis is indeed supported by the observation that some D-serine immunoreactivity is found in vesicles expressing the vesicular transporter for glutamate. The final proof that D-serine and glutamate are co-stored and co-released, however, awaits further investigations. The existence of a vesicular pathway does not exclude the possibility that other routes for D-serine release coexist in glial cells. Such nonvesicular pathways could involve processes that have been linked already to gliotransmitter release such as hemichannels, P2X7 receptors, volume-sensitive channels, and reversal of transporters (Figure 3).
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D
D
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b
Figure 3 Concentration of D-serine in the synaptic cleft and the number of NMDARs available: (a) control, (b) with glial withdrawal in lactating rats. In (a), the concentration of D-serine is sufficient to make most NMDARs on magnocellular neurons available for activation by synaptically released glutamate. In (b), glial withdrawal causes a reduction of synaptic D-serine levels, thereby diminishing the number of NMDARs available. NMDAR, N-methyl-D-aspartate receptor.
Another important issue regarding D-serine release, and more generally gliotransmission, is to know whether specialized sites for its release exist in astrocytes. Regarding D-serine, it is not clear whether its release is restricted to the fine processes apposed to NMDARs or whether it can occur from any compartment of the glial cell. Interestingly, astroglial cells have functionally distinct compartments, known as microdomains, where localized high Ca2þ increases occur and which tightly enwrap synapses. Furthermore, electron microscopy analysis has revealed that glutamate-containing vesicles are found in glial processes that contact dentritic spines bearing NMDARs. It is thus tempting to speculate that D-serine release occurs from such localized glial microdomains in close vicinity to the synapses and to NMDARs. This hypothesis is supported by electrophysiological recordings obtained in the hippocampus and in the hypothalamus demonstrating that the level of occupancy of the NMDAR glycine site is higher at synaptic than at extrasynaptic receptors. Although this could simply reflect the proximity of synaptically released glutamate, which is the putative trigger for D-serine release, it could also indicate the existence of microdomains for gliotransmitter release localized at the synapses. D-Serine
its hydrolysis by specific ectonucleotidases located in the extracellular compartment, and/or through cellular uptake as occurring for glutamate. Injection of D-serine into the lateral ventricle results in a preferential accumulation of the amino acid in glial cells, as expected if glial uptake is the main process involved in the clearance of the D-amino acid. Several putative candidates for D-serine transport have been identified on the membrane of glial cells but also on neurons. Glial cells express a Naþ-dependent transporter with low affinity for D- and L-serine and with characteristics similar to those of the alanine–serine–cysteine transporter (ASCT) system, which carries D-serine in cultured astrocytes as well as in isolated retina. Another neutral amino acid transporter, the ASC-1, has also been identified in neurons, and its cellular localization on axon terminals and dendrites suggests that it could contribute to the synaptic clearance of þ D-serine. Finally, a novel Na /Cl sensitive transporter has been described in rat brain synaptosomes which, in contrast to the ASCT system, has limited affinity for other neutral amino acids, including cysteine and alanine. It is thus conceivable that multiple transport systems, some of which may still be awaiting identification, contribute concomitantly to the regulation of D-serine concentrations in the extracellular space.
Clearance
Like for most neurotransmitters, the signaling action of D-serine should be terminated by its clearance from the extracellular space. This could occur through metabolic breakdown, as it is the case with ATP and
D-Serine Contribution to Synaptic Transmission and Plasticity
NMDARs are peculiar ionotropic receptors in the sense that they require not one but two different
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agonists to be activated: glutamate and glycine. NMDARs are tetramers formed by the association of two NR1 and two NR2 subunits. On the NR1 subunits, there is a strychnine-insensitive binding site for glycine. This so-called glycine site can be activated not only by glycine but also by D-serine, with a higher potency according to the subunit composition of NMDARs. The discovery that D-serine occurs naturally in the mammal brain has thus completely changed our view regarding the identity of the endogenous ligand for NMDAR glycine-binding site in the brain. Although glycine was first identified as such, it is obvious today that in several brain areas the endogenous ligand of the NMDAR glycine site is D-serine and not glycine. And because D-serine is released from glial cells, these cells are likely to play an active, if not essential, role in regulating NMDARdependent processes, including synaptic transmission, synaptic plasticity, rhythmic activity, activation of second messenger pathways, gene expression, and pathophysiological phenomenon such as excitoxicity and neurodegeneration. The first indication that D-serine could play a role in NMDAR function came from the observation that the D-amino acid was present in astrocytes ensheathing neurons bearing NMDARs. In vitro studies then revealed that D-serine was released from astrocytes on activation of their glutamatergic receptors. These findings strongly suggested that, in some regions of the brain, glutamate released from nerve terminals triggered D-serine release from glial cells, which, in turn, could modulate NMDARs localized on adjacent neurons. The functional role of D-serine was first studied in the hippocampus, where high densities of D-serine and NMDARs occur in the subiculum as well as in the CA1 and CA3 regions. In hippocampal neurons co-cultured with atrocytes, specific enzymatic degradation of D-serine with exogenous DAAO considerably reduces agonist-evoked and spontaneous NMDAR-mediated currents. Because DAAO does not affect glycine levels, this result unambiguously demonstrates that endogenous D-serine is required for NMDAR activity in this brain area. Similar results were obtained in the retina, where Mu¨ller glial cells appear to control NMDAR-mediated neurotransmission through the release of D-serine, as revealed by the inhibitory effect of DAAO on the NMDAR-mediated responses evoked by an agonist or light in retinal ganglia cells. The ability of D-serine to control NMDARdependent synaptic transmission has been confirmed through the use of a naturally occurring mouse strain lacking DAAO activity. In DAAO-deficient mice, the levels of D-serine are very high in the brain stem and spinal cord. As expected, NMDAR-mediated excitatory postsynaptic currents recorded from dorsal horn
neurons in the spinal cord are significantly augmented in these animals. Knockout mice for the transporter ASC-1 provides another experimental model for studying the relevance of D-serine in glutamatergic neurotransmission; these mice display NMDAR-dependent hyperexcitability, presumably resulting from elevated extracellular D-serine associated with a deficient clearance of the amino acid. Because in many brain areas NMDARs are responsible for the induction of long-term potentiation (LTP) and long-term depression (LTD), the cellular substrates for learning and memory in the mammalian brain, it is then important to know whether D-serine, and thus astrocytes, contributes to such forms of synaptic plasticity in the brain. The contribution of D-serine in LTP was shown in hippocampal cell cultures and brain slices where reducing D-serine levels with DAAO dramatically compromised the ability of high-frequency stimulation to induce LTP in CA1 pyramidal cells. This result confirms that D-serine, rather than glycine, is the endogenous ligand of NMDARs in this brain region, not only during basal transmission but also when synaptic inputs are stimulated intensively. Interestingly, it is commonly believed that senescence is associated with impaired NMDAR-dependent synaptic plasticity and notably LTP. In agreement with this hypothesis, a deficient LTP is observed in the hippocampus of a senescenceaccelerated mouse strain. This senescent-related deficiency in synaptic plasticity appears to be due to an impaired D-serine metabolism, as indicated by the reduced production of the D-amino acid measured in the hippocampi of senescent rats. In agreement with this observation, the hippocampal LTP is completely rescued when the D-serine is supplied to the tissue. The key role of D-serine in governing NMDAR activity in the hippocampus is further supported by the demonstration that it is the major, if not the only, endogenous ligand involved in NMDAR-mediated neurotoxicity in this structure. Neuronal death is indeed prevented when slices are treated with serine deaminase, another enzyme that degrades D-serine, whereas supplying the slices with glycine oxidase (GO), an enzyme that degrades specifically glycine, does not affect NMDAR-dependent neurotoxicity. Taken together, these findings suggest that astrocytic D-serine modulates NMDAR-dependent neurotransmission and synaptic plasticity in different brain regions. Because D-serine is synthesized and released from astrocytes, its action in these different regions depends on the astrocytic environment of neurons. Indeed, it is now accepted that glial coverage of neurons is not static and that it undergoes profound reversible anatomical remodeling in different
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areas under different physiological and/or pathological conditions. The hypothalamo-neurohypophysial system (HNS) constitutes certainly the most striking example of such anatomical remodeling that can be observed physiologically because it occurs during lactation, parturition, and chronic dehydration. This system is made of magnocellular neurons, localized in the hypothalamic supraoptic and paraventricular nuclei, whose axons project to the neurohypophysis where their hormonal content, namely oxytocin and vasopressin, can be directly released in the bloodstream. The morphological plasticity of the HNS is characterized by a pronounced reduction in astrocytic coverage of oxytocin-secreting magnocellular neurons, which is entirely reversible on the cessation of the stimulation. Both SR and D-serine are found at high levels in this brain structure, and their expression is strictly restricted to astrocytes. The HNS thus provides a remarkable model for studying the physiological impact of glial-derived D-serine in the context of synaptic transmission and synaptic plasticity. As reported for the hippocampus and the retina, treating hypothalamic slices with the highly specific enzyme DAAO dramatically reduced NMDARmediated synaptic responses recorded in magnocellular neurons, whereas degrading specifically glycine with GO has no effect, thereby indicating that D-serine is the endogenous co-agonist of NMDAR in the HNS. Accordingly, D-serine levels within the synaptic cleft, and thus the level of occupancy of the glycine site of synaptic NMDA receptors, is reduced under conditions in which the astrocytic coverage of neurons is diminished. This results in NMDAR-mediated synaptic responses of smaller amplitude in lactating than in control animals, a difference that disappears when saturating concentrations of D-serine are supplemented. Because the reduction of D-serine concentrations within the synaptic cleft caused by glial withdrawal lowers the number of synaptic NMDARs available for activation, the activity-dependence of phenomena such as LTP and LTD, whose induction depends on NMDAR activation, is modified. The neuron–glia remodeling causes a shift of the activity-dependence of long-term synaptic changes toward higher activity values, similar to what can be observed when NMDARs are partially blocked with pharmacological agents. Therefore, the glial environment of neurons, through its capacity to provide D-serine, has a profound impact not only on NMDAR-mediated synaptic transmission but also on the direction and magnitude of NMDAR-dependent long-term synaptic plasticity. Such astrocyte-mediated metaplasticity is likely to exist at all synapses where endogenous D-serine is involved in regulating NMDARs.
Conclusion It is now clear that D-serine fulfils all criteria that identify it as the leader of a new class of brain messengers, the D-amino acids. Glial-derived D-serine plays essential roles in the mammalian brain. By modulating NMDARs, this amino acid contributes actively to the transfer and storage of information. Because of this role in governing NMDAR function, glial cells may become prime targets in pathological events that cause neurons to degenerate. It is well documented that over- or downstimulation of NMDARs are implicated in a large number of acute and chronic degenerative disorders, including stroke, epilepsy, peripheral neuropathies, Parkinson’s disease, Alzheimer’s disease, Huntington’s disease, and in psychiatric disorders such as schizophrenia. Whereas previous therapeutic approaches centered directly on NMDARs have been associated with deleterious side effects, the discovery of D-serine as the predominant endogenous ligand of the NMDARs thus provides new strategies for the development of drugs that target NMDAR function indirectly through the modulation of D-serine metabolism, for example, by inhibiting SR or DAAO. See also: Glial Influence on Synaptic Transmission; Glutamate; Glycine Receptors: Molecular and Cell Biology; Long-Term Depression (LTD): Metabotropic Glutamate Receptor (mGluR) and NMDAR-Dependent Forms; Long-Term Potentiation (LTP): NMDA Receptor Role; NMDA Receptors, Cell Biology and Trafficking.
Further Reading Araque A, Parpura V, Sanzgiri RP, and Haydon PG (1999) Tripartite synapses: Glia, the unacknowledged partner. Trends in Neuroscience 22(5): 208–215. Boehning D and Snyder SH (2003) Novel neural modulators. Annual Review of Neuroscience 26: 105–131. Fields RD and Stevens-Graham B (2002) New insights into neuronglia communication. Science 298(5593): 556–562. Fujii N (2002) D-Amino acids in living higher organisms. Origins of Life and Evolution of Biospheres 32(2): 103–127. Haydon PG and Carmignoto G (2006) Astrocyte control of synaptic transmission and neurovascular coupling. Physiological Review 86(3): 1009–1031. Hirrlinger J, Hulsmann S, and Kirchhoff F (2004) Astroglial processes show spontaneous motility at active synaptic terminals in situ. European Journal of Neuroscience 20(8): 2235–2239. Johnson JW and Ascher P (1987) Glycine potentiates the NMDA response in cultured mouse brain neurons. Nature 325(6104): 529–531. Laming PR, Kimelberg H, Robinson S, et al. (2000) Neuronal-glial interactions and behaviour. Neuroscience and Biobehavioral Reviews 24(3): 295–340. Mothet JP, Parent AT, Wolosker H, et al. (2000) D-Serine is an endogenous ligand for the glycine site of the N-methyl-D-
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aspartate receptor. Proceedings of the National Academy of Sciences of the United States of America 97(9): 4926–4931. Mothet JP, Pollegioni L, Ouanounou G, Martineau M, Fossier P, and Baux G (2005) Glutamate receptor activation triggers a calcium-dependent and SNARE protein-dependent release of the gliotransmitter D-serine. Proceedings of the National Academy of Sciences of the United States of America 102(15): 5606–5611. Panatier A, Theodosis DT, Mothet JP, et al. (2006) Glia-derived D-serine controls NMDA receptor activity and synaptic memory. Cell 125(4): 775–784.
Shleper M, Kartvelishvily E, and Wolosker H (2005) D-Serine is the dominant endogenous coagonist for NMDA receptor neurotoxicity in organotypic hippocampal slices. Journal of Neuroscience 25(41): 9413–9417. Theodosis DT (2002) Oxytocin-secreting neurons: A physiological model of morphological neuronal and glial plasticity in the adult hypothalamus. Frontiers in Neuroendocrinology 23(1): 101–135. Volterra A and Meldolesi J (2005) Astrocytes, from brain glue to communication elements: The revolution continues. Nature Reviews Neuroscience 6(8): 626–640.
GABA Synthesis and Metabolism K L Behar, Yale University School of Medicine, New Haven, CT, USA ã 2009 Elsevier Ltd. All rights reserved.
Introduction g-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in mammalian brain and is found widely throughout the central and peripheral nervous systems. In the neocortex GABAergic neurons are plentiful, constituting 15–30% of all neurons. GABA serves both metabolic and trophic functions, in addition to its role as a neurotransmitter, influencing the migration of neurons and astroglia to their target locations in the cortex. During early brain development GABA elicits excitatory (depolarizing) rather than inhibitory (hyperpolarizing) postsynaptic responses. Later in development GABA influences the synaptic organization and fine-tuning of local circuits. Because of GABA’s many important roles – as neurotransmitter, neuromodulator, trophic factor, and cellular metabolite – GABA metabolism may impact many aspects of brain function. Brain tissue is highly heterogeneous, and compartmentation of the enzymes of the GABA shunt between neurons and astroglia plays an important role in the function of GABA as a neurotransmitter. The discovery of metabolic compartmentation in the early 1960s to the late 1970s laid the conceptual foundation for our current understanding of brain glutamate and GABA metabolism. Studies using 14C- and 15N-labeled substrates revealed that brain glutamate metabolism was not homogeneous and could be separated kinetically into distinct pools – a large pool labeled from [14C]glucose and a small, rapidly turning over pool labeled from [14C]acetate, [14C]butyrate, and [14C] bicarbonate. The subsequent discoveries that glutamine synthetase (GS; glutamate–ammonia ligase, EC 6.3.1.2) and pyruvate carboxylase (PC; EC 6.4.1.1) were astroglial enzymes provided key information that the large and small pools of glutamate corresponded to metabolism in neurons and astroglia, respectively. Glutamatergic and GABAergic neurons are included within the definition of the ‘large pool,’ although glutamate levels in the respective neurons differ greatly. Most of the 8–12 mmol g1 of glutamate measured in brain tissue is present in glutamatergic neurons (80%), with GABA neurons (2–10%) and astroglia (10%) containing much less. Astroglia play a critical role in glutamatergic and GABAergic function by maintaining low synaptic and interstitial levels of glutamate and GABA through active uptake
340
and metabolism. Astroglia also provide glutamine, and potentially other precursors, necessary for the resynthesis of these neurotransmitters. The study of brain glutamate and GABA metabolism has been advanced significantly in recent years through the use of nuclear magnetic resonance (NMR) spectroscopy in conjunction with 13C- and 15N-labeled substrates. The use of isotopically labeled substrates in these studies allows metabolic pathways of GABA and glutamate synthesis and neuron/glial substrate trafficking to be examined noninvasively and in unprecedented detail, revealing new insights into these pathways and their functional interactions.
GABA Synthesis and Degradation in the GABA Shunt GABA synthesis and degradation occur at the level of the tricarboxylic acid (TCA) cycle. GABA, a product of glucose metabolism, is synthesized almost exclusively from glutamate, a product of the TCA cycle intermediate, a-ketoglutarate, in an a-decarboxylation catalyzed by the pyridoxal-50 -phosphatedependent enzyme, glutamate decarboxylase (GAD; EC 4.1.1.15). GABA is degraded to succinate by the concerted actions of two enzymes: pyridoxal-50 phosphate-dependent GABA transaminase (GABA-T; 4-aminobutyrate transaminase, EC 2.6.1.19) and nicotinamide adenine dinucleotide (NAD)-dependent succinate-semialdehyde dehydrogenase (SSADH; EC 1.2.1.24). In the first step of catabolism, GABA nitrogen is transferred to a-ketoglutarate by transamination, forming succinic-semialdehyde and glutamate. In the second step succinic-semialdehyde is oxidized to succinate with reduction of NADþ to NADH2; succinic-semialdehyde is a short-lived intermediate and is present at low cellular concentrations. The pathway of GABA metabolism from a-ketoglutarate to succinate forms a metabolic shunt, by passing two enzymes of the TCA cycle, a-ketoglutarate dehydrogenase and succinyl-CoA synthetase (Figure 1). Glucose oxidation through the GABA shunt is slightly (3%) less energy efficient than through the complete TCA cycle, resulting in one less molecule of guanosine triphosphate (GTP) for each molecule of a-ketoglutarate traversing the shunt. GABA synthesis occurs only in neurons, whereas GABA catabolism occurs both in neurons and glia. Thus, the resynthesis of glutamate from a-ketoglutarate during transamination with GABA does not occur totally in the same cells in which GABA was formed. Because GABAergic and other neurons are incapable of de novo synthesis of glutamate precursors, due to
GABA Synthesis and Metabolism 341 Glucose Lactate
Pyruvate Acetyl-CoA
Citrate
TCA cycle Succinate
NADH2 SSADH
a-Ketoglutarate GTP NADH2 GABA-T
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Succinic semialdehyde
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histidine) and g-hydroxybutyrate (GHB). Homocarnosine is synthesized from GABA and histidine by homocarnosine synthetase, and hydrolysis of this dipeptide by a serum carnosinase regenerates GABA and histidine. GHB is synthesized from succinic-semialdehyde by a specific NADPH-dependent succinic-semialdehyde reductase, and oxidation back to succinic-semialdehyde is catalyzed by GHB dehydrogenase. Tissue levels of GABA, homocarnosine, and GHB can be elevated substantially by pharmacologic inhibitors or gene deficiencies in GABA-T and SSADH.
CO2 NAD+
Figure 1 Metabolic pathway depicting the synthesis and catabolism of g-aminobutyric acid (GABA) in the GABA shunt and its relationship to glucose metabolism and the tricarboxylic acid (TCA) cycle. The GABA shunt bypasses two enzymatic steps of the TCA cycle, resulting in one less molecule of guanosine triphosphate (GTP) formed for each molecule of a-ketoglutarate metabolized to succinate through the shunt. GABA synthesis occurs in GABAergic neurons but GABA catabolism occurs in neurons and astroglia. Astroglial glutamate precursors (e.g., glutamine) are required to replenish GABA removed and metabolized by astroglia. GAD, glutamic acid decarboxylase; GABA-T, GABA transaminase; SSADH, succinic semialdehyde dehydrogenase; NAD, nicotinamide dinucleotide; CoA, coenzyme A.
the absence of the necessary anaplerotic enzymes, glutamate precursors must be supplied by astroglia to maintain TCA cycle intermediates (and GABA) at constant levels. The concentration of GABA in brain tissue ranges from 1 to 5 mmol g1, with the variation in concentration reflecting the density of GABA neurons and their terminals. GABA levels are very low in the extracellular fluid and in cells which do not express GAD. The GABA concentration within GABAergic terminals may reach 50–100 mM, although much of this is contained in synaptic vesicles and is inaccessible to GABA-T. The concentration of GABA in the cytoplasm and the mitochondrial intermembrane space where GABA-T is located is not well known, but levels are likely to be high and saturating for GABA-T, which has a relatively low Km (1 mM). Thus, in neurons GABA levels are likely to be regulated by changes in synthesis and GAD activity rather than catabolism. In contrast to neurons, in astroglia where GABA levels are low and nonsaturating for the glial transaminase, GABA level is likely to regulate the rate of catabolism. The GABA shunt also generates precursors for synthesis of a limited number of other compounds, the most studied of which are the neuromodulator/ neurotransmitters, homocarnosine (g-aminobutyryl
GABA Transporters Are Expressed in GABAergic Neurons and Astroglia GABA is transported across the plasma membranes of neurons and astroglia by electrogenic GABA transporters (GATs), which facilitate the reversible symport of GABA with two Naþ ions and one Cl ion. These transporters, like the electrogenic glutamate transporters, require the energy of ATP to maintain the Naþ ion gradient that provides the driving force and direction of GABA transport. GATs operate at much slower rates than do glutamate transporters, so that efficient clearance of GABA from the synapse requires relatively high transporter densities. The rate of GABA transport is determined by the number of functional GATs residing on the cell surface which are recruited from the cytoplasm. The distribution of GATs between cell surface and cytoplasm is regulated by extracellular GABA levels, protein kinase C, and ligands of G-protein-coupled receptors.
Pathways of GABA Clearance Following Release: Neuronal Reuptake and Glial Uptake GABA clearance following release into the synaptic cleft occurs both by reuptake into presynaptic terminals and by transport into surrounding astroglia. Isotopic studies which follow the labeling of GABA from different precursors cannot distinguish between the different uptake pathways because GABA metabolism in neurons and glia leads to the same labeling patterns. Thus, the relative quantitative importance of these two pathways to total GABA clearance in vivo is unknown and this issue remains a vexing problem. Studies using cell cultures have shown that GABA transport capacity and uptake are greater in neurons than in glia, supporting a predominant role for reuptake in GABA clearance from the synapse. Although reuptake has been suggested to be more energetically efficient because new GABA
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synthesis is not required, the activity of GABAergic neurons and vesicular loading of GABA has been shown to involve new GABA synthesis, thus weakening a rationale for reuptake. GATs are reversible and can function in GABA release as well as uptake. GATs in interneurons operate closer to their equilibrium potentials than do glutamate transporters, providing more favorable conditions for calcium-independent, nonvesicular transport of GABA in either direction. Under depolarizing conditions GABA transport reversal can lead to loss of GABA from the cell, but it has not been shown whether the recovery of GABA levels during repolarization involves reuptake or new synthesis from glial precursors. GABAergic synapses are closely surrounded by glial end-processes possessing high densities of GATs, and astroglia possess all the enzymes needed to degrade GABA. Studies of isolated glial fractions, astrocyte cultures, and intracellular recordings of glial GABA transporter currents in tissue slices show that glia have a substantial capacity to transport GABA. In situ levels of extracellular GABA and the occupancy of postsynaptic GABA receptors are regulated by astroglial transporters. Inhibitors of astroglial transporters show greater antiepileptic efficacy than do neuronal transport inhibitors of GABA, suggesting that glial clearance is quantitatively significant. These observations are consistent with the high rate of GABA synthesis from glutamine (23% of total glutamate plus GABA cycling) measured in rat cortex in vivo using 13 C NMR. Whether glial GABA transport is more important in paracrine signaling or synaptic transmission is unclear at present, but such differences may be related to the functions of the two isoforms of GAD, as discussed in a later section.
Precursors of GABA Synthesis
glutamate and ammonia by phosphate-activated glutaminase (PAG; EC 3.5.1.2), a highly regulated enzyme, present on the outer surface of the inner mitochondrial membrane, which provides glutamate for GABA synthesis to recharge cytoplasmic and vesicular pools. Release of GABA from neurons followed by uptake and metabolism to succinate in the astroglia completes the GABA shunt while regenerating glutamate (by transamination of a-ketoglutarate with GABA). Thus, de novo synthesis of a-ketoglutarate by anaplerosis is not required to replenish the GABA catabolized in astroglia. However, GABA catabolism requires an equivalent flow of acetyl-CoA from glucose-derived pyruvate or other substrates (e.g., acetate) to maintain the availability of a-ketoglutarate for transamination with GABA. The glutamate generated by transamination (GABA-T) can be converted to glutamine by GS and is released for uptake and replenishment of glutamate and GABA in neurons. In contrast to GABA, which requires oxidative metabolism in the TCA cycle, much of the glutamate removed by glia (70–80%) following release from glutamatergic neurons is converted directly to glutamine. A schematic of GABA and glutamate neurotransmitter cycling between GABAergic and glutamatergic neurons and astroglia is shown in Figure 2. Although glutamine has been shown to be a major glial precursor of GABA in vivo and in vitro, studies of isolated nerve terminal preparations indicate that glial TCA cycle intermediates (e.g., a-ketoglutarate or malate) might also contribute to GABA synthesis. In addition, the role of phosphate-activated glutaminase in GABA synthesis has been challenged; immunohistochemical findings in situ have shown that many neocortical interneurons which co-express certain peptides do not stain positively for PAG, suggesting that a substrate other than glutamine (e.g., a-ketoglutarate) might be utilized for GABA synthesis in these neurons.
The Glutamate/GABA–Glutamine Cycle
GABA Synthesis from Extracellular Glutamate
Glutamate precursors for GABA synthesis can be produced directly from the metabolism of glucose in neurons. However, glucose metabolism cannot replace glutamate and GABA carbon lost from neurons during activity due to the absence of the necessary anaplerotic enzymes (e.g., PC), which are expressed only in the astroglia. GABAergic neurons import the needed precursors from astroglia mainly in the form of glutamine. The astroglia which surround GABAergic (and glutamatergic) neuronal processes express high densities of the N-type electroneutral glutamine transporter (SN1), while GABAergic neurons express both of the A-type electrogenic glutamine transporters (SA1 and SA2). Glutamine is hydrolyzed to
In addition to glial precursors, extracellular glutamate can enter some GABA neurons through the EAAC1 glutamate transporter, where it can be used in GABA synthesis. This pathway has been observed in the hippocampus and thalamus but not in the cortex. Regional differences in GABA precursors might be related to the apparent heterogeneity seen in PAG immunostaining for different populations of GABAergic neurons. For interneurons using extracellular glutamate in GABA synthesis, glial precursors must ultimately replenish this glutamate in an extended loop, whereby glutamine is redirected first through the glutamatergic neuron and released as glutamate before uptake and conversion to GABA.
GABA Synthesis and Metabolism 343
Pyr
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Figure 2 Depiction of GABA and glutamate cycling with glutamine between GABAergic and glutamatergic neurons with astroglia via extracellular fluid (ECF). (a) GABAergic neuron and astroglial trafficking in the glutamate/GABA–glutamine cycle. (b) Glutamatergic neuron and astroglial trafficking in the glutamate–glutamine cycle. Enzymes and their catalyzed fluxes: Ac, acetate; AcCoA, acetyl-coenzyme A; aKG, a-ketoglutarate; Cit, citrate; GAD, glutamic acid decarboxylase; Glc, glucose; Gln, glutamine; Glu, glutamic acid; GS, glutamine synthetase; lac, lactate; Mal, malate; OAA, oxaloacetic acid; PAG, phosphate-activated glutaminase; PC, pyruvate carboxylase; PDH, pyruvate dehydrogenase; Pyr, pyruvate; Suc, succinate; VGAD, rate of GABA synthesis; Vshunt, rate of GABA shunt in GABAergic neurons; Vcyc(Gab/Gln), rate of glutamate/GABA–glutamine cycling; Vgln, rate of glutamine synthesis; Veff, rate of glutamine efflux; Vpdh(Gab) and Vpdh(a), rate of acetyl-CoA formation from pyruvate in GABAergic neurons and astroglia, respectively; VAc, rate of acetyl-CoA formation from acetate.
GABA Synthesis from Polyamines
Alternative but quantitatively minor pathways of GABA synthesis from putrescine and other polyamines have been described, but these pathways account for only a small fraction (1%) of synthesis in the mature brain. Polyamines may play important but largely unknown roles in GABA metabolism in the developing brain and retina. Putrescine and other
polyamines increase in response to stress, but a potential link to GABA synthesis has not been reported.
Nitrogen Homeostasis in Brain Glutamate and GABA Metabolism The operation of the glutamate/GABA–glutamine cycle requires the efficient transfer of nitrogen as well as
344 GABA Synthesis and Metabolism
carbon between neurons and astroglia. Nitrogen fixed as glutamine and released from astroglia for the synthesis of glutamate and GABA in neurons must ultimately return to the glia to permit continuous operation of the neurotransmitter cycles. Ammonia is freely diffusible through cell membranes, and the ammonia generated by PAG in the neurons could provide, in principle, the amide-N needed for glutamine synthesis from glutamate, part of which is acquired through uptake of extracellular glutamate or is produced by transamination with GABA. However, astroglia also undergo significant de novo synthesis of glutamate (anaplerosis) to replace glutamate and GABA lost by oxidation and diffusion, which may approach 18–30% of total glutamine synthesis, thus requiring a source of amine-N. Glutamate dehydrogenase (GDH; EC 1.4.1.2) is highly expressed in astroglia but is not believed to be sufficiently active in the direction of glutamate formation under normal physiological conditions. Recent studies suggest instead that amine-N for de novo glutamate (and glutamine) synthesis from a-ketoglutarate occurs by transamination with alanine or a branched-chain amino acid (BCAA), such as leucine. Two different nitrogen carrier cycles have been proposed – one involving an exchange of alanine for glutamine in the glia with the product of transamination, pyruvate, returning to the neuron as lactate (alanine/lactate shuttle), and the other involving an exchange of a BCAA (leucine) for glutamine with the product of transamination, branched-chain keto acid (a-ketoisocaproic), returning to the neuron. Alanine and leucine are regenerated in the neurons by transamination with glutamate, the latter formed by GDH and the ammonia from the PAG reaction. Both neurons and astroglia express the necessary transaminases needed to support the operation of these cycles, alanine aminotransaminase (AAT; EC 2.6.1.2) and branched-chain-amino-acid transaminase (BCAT; EC 2.6.1.42). Distinct cytosolic (neuronal) and mitochondrial (astroglial) isoforms of BCAT have been found. Studies of the BCAAs and their transaminases, together with findings of significant 15N labeling of brain glutamate in rodents during intravenous infusion of [15N]leucine in vivo, indicate that as much as 25% of brain glutamate nitrogen may be derived from leucine. While studies of nitrogen shuttling between neurons and astroglia have focused mainly on glutamatergic neurons, leucine is likely to be a source of glutamate nitrogen in some GABAergic neurons. GABAergic Purkinje neurons in the cerebellum and basket neurons in the hippocampus express BCATc in their cell bodies. In Purkinje neurons, PAG expression is low, suggesting that GABA synthesis involves substrates other than glutamine. In these neurons and others potentially not expressing PAG,
leucine transamination of a-ketoglutarate derived from astroglia during GABA catabolism or de novo synthesis could supply glutamate needed for GABA synthesis. Whether the GABA synthetic pathways operating in the many different classes of interneurons (e.g., chandelier, double bouquet, Martinotti neurons) differ from one another is largely unknown, but such differences could play an important role in their function.
Relationship of Cytoplasmic and Vesicular GABA to Different Release Pathways Studies of cultured neocortical neurons enriched in the GABAergic phenotype demonstrate the presence of cytoplasmic and vesicular GABA pools; one is released by agonists of the glutamate N-methyl-D-aspartate (NMDA) receptor and is calcium independent (cytoplasmic GABA), and the other is released by 55 mM potassium and is calcium dependent (vesicular GABA). The vesicular and cytoplasmic GABA produced from glutamine can be distinguished metabolically when neurons are depolarized in a manner related to the pathway of GABA release. Vesicular GABA produced from glutamine involves metabolic processing in the TCA cycle and equilibration of the glutamate carbon skeleton with endogenous mitochondrial glutamate. In contrast, glutamate produced directly from glutamine appears to be a better precursor of GABA released through reversal of membrane transporters (i.e., cytoplasmic GABA). Neuronal firing patterns may also play an important role in the mode of GABA release and thus the functional expression of the GAD isoforms, as discussed in the following section.
Relationship of the GAD Isoforms to Cytoplasmic and Vesicular GABA Synthesis GAD is expressed as two major isoforms, of 65 and 67 kDa, which are the products of two different genes. The two isoforms of GAD are distributed differently within GABAergic neurons: GAD67 is present throughout the cytoplasm, with more in the cell bodies, whereas GAD65 is highly enriched in axon terminals and is associated with synaptic vesicles. GAD67 appears to play the major role in basal GABA synthesis, and knockout of the gene for this isoform results in a major loss of tissue GABA. GAD67 expression is regulated both by transcriptional and by posttranscriptional mechanisms, whereas GAD65 expression is regulated at the transcriptional level and by short-term (kinetic) mechanisms. GAD65 knockout mice display electrophysiological features consistent with reduced vesicular release of GABA. GAD65 binding to synaptic vesicles was shown recently to involve a multiprotein
GABA Synthesis and Metabolism 345 GABAergic neuron Glucose
Astroglia
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suc
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GABA GABA Figure 3 An idealized depiction of the relationships between cytoplasmic and vesicular g-aminobutyric acid (GABA) synthesis, GABA release pathways, and glutamic acid decarboxylase (GAD) isoforms. Cytoplasmic GABA (GABAcyto) synthesis is provided mainly by GAD67, and this pool/isoform is associated with the majority of neuronal GABA shunt flux. Forward and reverse movements of GABA through GATs lead to uptake or release of GABA to the extracellular fluid (GABAecf). Vesicular GABA synthesis is catalyzed by GAD65, and GABA not returned to the terminals by reuptake is metabolized in the astroglia to succinate (suc) and oxidized in the mitochondrial TCA. Transamination of GABA in the glia by GABA-T generates glutamate from a-ketoglutarate (a-KG), which is used in the synthesis of glutamine by glutamine synthetase (GS). Phosphate-activated glutaminase (PAG) provides glutamate precursors for GABA synthesis. In some GABA neurons not expressing PAG, a-ketoglutarate could serve as a GABA precursor following its uptake and transamination to glutamate in the terminals, possibly by leucine or alanine.
complex of vesicle-associated proteins, the vesicular GABA/Hþ antiporter and Hþ-ATPase, and Ca2þ/calmodulin protein kinase II, which together facilitate vesicular loading of GABA driven by the hydrogen ion gradient. An idealized depiction of the relationship between the two GAD isoforms and the synthesis of the vesicular and cytoplasmic GABA pools is shown in Figure 3. GAD65 is more strongly regulated by the cofactor, pyridoxal phosphate, than is GAD67. The majority of GAD in brain is present as inactive apoenzyme (GAD without bound cofactor), which serves as a reservoir of inactive GAD that can be activated when demand for GABA increases. GAD65 binds pyridoxal phosphate less tightly than does GAD67 and accounts for the majority of the apoGAD in rat brain. In contrast, pyridoxal phosphate is more tightly bound to GAD67, which explains the importance of this isoform in basal synthesis. The interconversion between pyridoxal phosphate bound (active) and free (inactive) GAD involves a complex cycle of reactions catalyzed by GAD. This cycle is strongly regulated by nucleoside triphosphates (ATP) and inorganic phosphate,
which could serve as important regulators to increase GABA synthesis in response to increased demand. The activities of both GAD isoforms also appear to be regulated in opposing directions by protein phosphorylation.
Inherited Disorders of GABA Metabolism Rare inherited disorders of GABA metabolism have been described involving deficiencies in enzymes in the pathways of GABA catabolism (GABA-T, SSADH, and serum carnosinase) and a cofactor (pyridoxine) required in GABA synthesis. High levels of GABA accumulate in brain and cerebrospinal fluid of individuals deficient in GABA-T and SSADH. In SSADH deficiency, the buildup of the product of GABA-T, succinic semialdehyde, promotes increased synthesis and accumulation of GHB. Homocarnosine levels are elevated in brain and cerebrospinal fluid of individuals with homocarnosinosis, a condition resulting from deficiency of serum carnosinase, a dipeptidase which hydrolyzes homocarnosine and carnosine. Pyridoxinedependent epilepsy, a rare autosomal recessive inherited
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trait characterized by intractable seizures, is treated by administration of pyridoxine (vitamin B6) at pharmacologic doses. Although pyridoxine is a required cofactor of GABA synthesis, and reduced pyridoxine interaction with GAD is suspected in this disorder, a definitive mechanism has not been established. See also: GABAA Receptor Synaptic Functions; GABAA
Receptors: Molecular Biology, Cell Biology and Pharmacology; GABAB Receptor Function; GABAB Receptors: Molecular Biology and Pharmacology.
Further Reading Asada H, Kawamura Y, Maruyama K, et al. (1996) Mice lacking the 65 kDa isoform of glutamic acid decarboxylase (GAD65) maintain normal levels of GAD67 and GABA in their brains but are susceptible to seizures. Biochemical and Biophysical Research Communications 229: 891–895. Asada H, Kawamura Y, Maruyama K, et al. (1997) Cleft palate and decreased brain gamma-aminobutyric acid in mice lacking the 67-kDa isoform of glutamic acid decarboxylase. Proceedings of the National Academy of Sciences of the United States of America 94: 6496–6499. Bak LK, Schousboe A, and Waagepetersen HS (2006) The glutamate/GABA-glutamine cycle: Aspects of transport, neurotransmitter homeostasis and ammonia transfer. Journal of Neurochemistry 98: 641–653. Battaglioli G, Liu H, and Martin DL (2003) Kinetic differences between the isoforms of glutamate decarboxylase: Implications for the regulation of GABA synthesis. Journal of Neurochemistry 86: 879–887. Beckman ML, Bernstein EM, and Quick MW (1999) Multiple G protein-coupled receptors initiate protein kinase C redistribution of GABA transporters in hippocampal neurons. Journal of Neuroscience 19: RC9 1–6. Belhage B, Hansen GH, and Schousboe A (1993) Depolarization by Kþ and glutamate activates different neurotransmitter release mechanisms in GABAergic neurons: Vesicular versus non-vesicular release of GABA. Neuroscience 54: 1019–1034. Berl S and Clarke DD (1969) Compartmentation of amino acid metabolism. In: Lajtha A (ed.) Handbook of Neurochemistry, vol. 2, pp. 447–472. New York: Plenum. Chaudhry FA, Schmitz D, Reimer RJ, et al. (2002) Glutamine uptake by neurons: Interaction of protons with system a transporters. Journal of Neuroscience 22: 62–72. Conti F, Minelli A, and Melone M (2004) GABA transporters in the mammalian cerebral cortex: Localization, development and pathological implications. Brain Research: Brain Research Reviews 45: 196–212. Fonnum F and Walberg F (1973) An estimation of the concentration of g-aminobutyric acid and glutamate decarboxylase in the inhibitory Purkinje axon terminals in the cat. Brain Research 54: 115–127. Hutson SM, Berkich D, Drown P, et al. (1998) Role of branched-chain aminotransferase isoenzymes and gabapentin
in neurotransmitter metabolism. Journal of Neurochemistry 71: 863–874. Jin H, Wu H, Osterhaus G, et al. (2003) Demonstration of functional coupling between gamma-aminobutyric acid (GABA) synthesis and vesicular GABA transport into synaptic vesicles. Proceedings of the National Academy of Sciences of the United States America 100: 4293–4298. Kanamori K, Ross BD, and Kondrat RW (1998) Rate of glutamate synthesis from leucine in rat brain measured in vivo by 15 N NMR. Journal of Neurochemistry 70: 1304–1315. Kaufman DL, Houser CR, and Tobin AJ (1991) Two forms of the gamma-aminobutyric acid synthetic enzyme glutamate decarboxylase have distinct intraneuronal distributions and cofactor interactions. Journal of Neurochemistry 56: 720–723. Lieth E, LaNoue KF, Berkich DA, et al. (2001) Nitrogen shuttling between neurons and glial cells during glutamate synthesis. Journal of Neurochemistry 76: 1712–1723. Martin DL and Tobin AJ (2000) Mechanisms controlling GABA synthesis and degradation in the brain. In: Martin DL and Olsen RW (eds.) GABA in the Nervous System: The View at Fifty Years, pp. 25–41. Philadelphia: Lippincott Williams & Wilkins. Patel AB, de Graaf RA, Martin DL, et al. (2005) Evidence that GAD65 mediates increased GABA synthesis during intense neuronal activity in vivo. Journal of Neurochemistry 97: 385–396. Patel AB, de Graaf RA, Mason GF, et al. (2005) The contribution of GABA to glutamate/glutamine cycling and energy metabolism in the rat cortex in vivo. Proceedings of the National Academy of Sciences of the United States of America 102: 5588–5593. Pearl PL and Gibson KM (2004) Clinical aspects of the disorders of GABA metabolism in children. Current Opinion in Neurology 17: 107–113. Richerson GB and Wu Y (2003) Dynamic equilibrium of neurotransmitter transporters: Not just for reuptake anymore. Journal of Neurophysiology 90: 1363–1374. Seiler N (1980) On the role of GABA in vertebrate polyamine metabolism. Physiological Chemistry and Physics 12: 411–429. Sepkuty JP, Cohen AS, Eccles C, et al. (2002) A neuronal glutamate transporter contributes to neurotransmitter GABA synthesis and epilepsy. Journal of Neuroscience 22: 6372–6379. Shank RP, Bennett GS, Freytag SO, et al. (1985) Pyruvate carboxylase: An astrocyte-specific enzyme implicated in the replenishment of amino acid neurotransmitter pools. Brain Research 329: 364–367. Soghomonian JJ and Martin DL (1998) Two isoforms of glutamate decarboxylase: Why? Trends in Pharmacological Science 19: 500–505. Waagepetersen HS, Sonnewald U, Gegelashvili G, et al. (2001) Metabolic distinction between vesicular and cytosolic GABA in cultured GABAergic neurons using 13C magnetic resonance spectroscopy. Journal of Neuroscience Research 63: 347–355. Wei J, Davis KM, Wu H, et al. (2004) Protein phosphorylation of human brain glutamic acid decarboxylase GAD65 and GAD67 and its physiological implications. Biochemistry 43: 6182–6189.
GABAA Receptors: Molecular Biology, Cell Biology, and Pharmacology T Goetz, P Wulff, and W Wisden, University of Aberdeen, Aberdeen, UK ã 2009 Elsevier Ltd. All rights reserved.
Introduction If nervous systems possessed only excitatory neurons, there would be no brake on any signal, excitation would generate further excitation, and a chain of neurons would produce amplifying cascades of excitation, making the system incapable of doing anything useful. By contrast, networks made from both excitatory and inhibitory neurons can self-organize and generate complex properties. Inhibition is essential for every operation performed by any neuronal circuit in any brain region. In vertebrate brains, the inhibitory agent used most often by neurons is g-aminobutyric acid (GABA), which after release from the presynaptic terminals diffuses across the synaptic cleft to bind to receptor molecules – the so-called GABAA receptors. As for all other ligandgated channels, GABAA receptors convert chemical messages into electrical signals. In less than a millisecond, the binding of two (tiny) molecules of GABA induces a conformational change in the (giant) receptor oligomer that opens the central chloride ion channel. This remarkable process is called ‘gating.’ Cl diffuses through the receptor pore, down the electrochemical gradient, entering the cell and hyperpolarizing it. This results in fast synaptic inhibition (on the millisecond timescale) of a domain of the postsynaptic cell (Figure 1). Strictly, GABAA receptors are actually GABA-gated anion-permeable channels, with a HCO 3 /Cl permeability ratio of approximately 0.2–0.4. In adult cells, Cl ions usually move into the cell to produce strong inhibitory hyperpolarization, as the reversal potential for Cl is 15–20 mV more negative than the resting membrane potential. The Cl gradient is maintained by K-Cl cotransporters. HCO 3 ions move out of the cells through the GABAA receptor channel (Figure 1); the HCO 3 efflux is mildly depohas a reversal potential of 12 mV), larizing (HCO 3 but this is normally offset by the Cl hyperpolarization. Depending on the local internal Cl concentration, Cl ions can also move out after GABAA receptor activation and depolarize the cell; this happens especially during embryonic and postnatal neuronal development.
Molecular Biology of GABAA Receptors In mammals, GABAA receptors form as heteropentameric assemblies from a family of 19 subunits encoded by distinct genes (a1–a6, b1–b3, g1–g3, d, e, y, p, and r1–r3). Depending on the subunit composition, GABAA receptors differ in their biophysical properties and affinity for GABA, their pharmacology, and their location on the cell. GABAA receptors were cloned by the combined efforts of the Eric Barnard and Peter Seeburg groups in 1987 by the classical method: Peptide sequences obtained from purified (bovine brain) receptors were used to construct synthetic DNA probes to screen brain complementary DNA (cDNA) libraries. This was the starting point. By 1990, this now historical technique of screening cDNA libraries had revealed most of the gene family, all the a1–6, b1–3, g1–3 subunits and one d subunit; over the remaining decade, a few more subunits, such as e, y, and p, were characterized. With the completion of the human genome database, an in silico hybridization method was used to screen for further mammalian GABAA receptor genes, but it found none. Most of the subunit gene family members are in clusters, suggesting gene and then cluster duplication during the evolutionary origin of vertebrates: b2, a6, a1, g2 form a cluster in that order on human chromosome 5q34; the b3, a5, g3 genes cluster in that order on human chromosome 15q13; the g1, a2, a4, b1 genes cluster in that order on chromosome 4p12; the e, a3, y genes in that order on X q28; the r1 and r2 genes, 40 kbp apart on 6 q15; and the p, r3, and d subunit genes are isolated on human chromosomes 5q35.1, 3q12.1, and 1p36.3, respectively. As determined by both in situ hybridization (mRNA localization) with gene-specific probes (Figure 2) and immunocytochemistry (protein localization) with subunit-specific antibodies, the expression of the individual subunit genes is age and region specific. Does the clustering of the GABAA receptor subunit genes imply they are coregulated? The a1 and b2 genes do indeed share identical transcription patterns; thus, these two genes may share regulatory elements. All the other subunit genes have sometimes common, sometimes divergent expression patterns, with no correlation with which gene is in which gene cluster.
GABAA Receptor Structure The GABAA receptor belongs to a superfamily of ligand-gated ion channels (‘Cys-loop receptors’) that
347
348 GABAA Receptors: Molecular Biology, Cell Biology, and Pharmacology 2 Na+ GABA
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Figure 1 The GABAergic synapse in the mammalian brain. GABA is synthesized from glutamate by glutamic acid decarboxylase (GAD) and transported into vesicles by the vesicular GABA transporter (vGAT). When the presynaptic terminal is depolarized sufficiently, GABA vesicles are released into the cleft and diffuse rapidly to activate the postsynaptic GABAA receptors. GABA is salvaged from the synaptic cleft by, for example, the GAT-1 transporter on presynaptic terminals. The GABAA receptor is a GABA-gated Cl and HCO 3 channel.
a3 dg CPu
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in vertebrates includes the nicotinic acetylcholine receptors, the 5-hydroxytryptamine type 3 (5HT3) receptors, the zinc-activated ion channel, and the glycine receptors. No direct structural information is available for GABAA receptors. So in imagining how the GABAA receptor must look, we can do no better than quote Unwin for his empirical observations on the Torpedo nicotinic acetylcholine receptor: The receptor (a large 290 kDa glycoprotein) is composed of elongated subunits, which associate with their long axes approximately normal to the membrane, creating a continuous wall around the central ion-conducting path. The whole assembly presents a rounded, nearly 5-fold symmetric assembly when viewed from the synaptic cleft, but is wedge-shaped when viewed parallel with the membrane plane. The subunits of the receptor all have a similar size 30 A 40 A 160 A and the same three-dimensional fold. Each subunit is a three-domain protein and so portions the channel naturally into its ligand-binding, membrane-spanning and intracellular parts.
In GABAA receptors, the arrangement of subunits around the channel is probably gbaba or dbaba counterclockwise when viewed from the extracellular space (Figure 3(a)). For those cells in which they are expressed, e and p subunits probably replace
a5 Cbm
gr a6 Figure 2 Expression of the GABAA receptor a subunit genes in adult rat brain (sagittal sections) as detected by in situ hybridization. BS, brain stem; CA3, subdomain of hippocampus; Cbm, cerebellum; CPu, caudate-putamen; Ctx, neocortex; DCN, deep cerebellar nuclei; dg, dentate granule cells; GP, globus pallidus; gr, cerebellar granule cells; H, hippocampus; T, thalamus. Roman numerals mark neocortical layers.
the g and d subunit within the pentamer, whereas the y subunit might replace a b subunit. As for all members of the nicotinic receptor superfamily, all GABAA receptor subunits contain a large extracellular N-terminal domain of approximately 200 amino acids shaped by a cysteine disulfide bridge (the socalled Cys-loop, Figure 3(b)). For GABAA subunits, the amino acid consensus sequence of the Cys-loop is C******F/YP*D***C*****S (where * is a degenerate residue).
GABAA Receptors: Molecular Biology, Cell Biology, and Pharmacology 349
Subunit Assembly Rules for GABAA Receptors
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GABAA receptor subunit combinations are partly governed by which cell types express which genes and partly by preferential partnering of subunits within a given cell; for example, the a4 and a6 subunits assemble preferentially with the d subunit (Figure 4(a)). The majority of mammalian brain GABAA receptors are probably abg2 combinations. The subunit ratio is probably 2a/2b/1g. Some receptors also contain different a and b subunits, such as a1a2b2g2. The a1bg2 combination is the most abundant GABAA receptor subtype in the brain (60% of total; Figure 4(b)).
C C Out TM4
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In b Figure 3 (a) Subunit arrangement in g2-containing (synaptic) and d-containing (extrasynaptic) GABAA receptors; (b) schematic of an individual subunit topology. C, cysteine; N, N-terminus of the protein TM, transmembrane domain. The pore lining domain, TM2, is shown in blue. Pink triangles represent GABA; blue circle represents a BZ ligand.
Each subunit contains four predicted transmembrane spanning domains (TM1–TM4) of about 20 amino acids and a large intracellular loop between TM3 and TM4 (TM3–TM4 loop) (Figure 3(b)). Many GABAA receptor subunits have the amino acid sequence (TTVLTMTT) in the TM2 domain. Five of these eight amino acids probably line the ion channel. TM1, TM3, and TM4 segregate TM2 from membrane lipid. The selectivity filter and gate lies at the intracellular end of the TM2 domains and includes part of the TM1–TM2 loop. In the 5-HT3 and nicotinic receptors, residues in the TM3–TM4 loop region influence single channel conductance. The TM3–TM4 loop contributes key sites for attaching anchor and regulatory proteins involved in locating the receptor at synapses and in governing the activity of GABAA receptor. In the extracellular domain of a typical GABAA receptor consisting of two a, two b, and one g2 subunit, the binding pocket for GABA forms at the interface between the a and the b subunit, and the binding pocket for benzodiazepines (BZs) lies at the interface of the a and the g subunit (Figure 3).
Synaptic GABAA Receptors: abg Subunit Combinations and Anchoring Role of the g2 Subunit and Gephyrin Placing GABAA receptors at synapses requires specific proteins that interact directly or indirectly with the g subunits. For example, targeting some GABAA receptor subtypes to GABAergic terminals involves the widely expressed microtubule-binding protein gephyrin. Gephyrin either helps convey some GABAA receptor subtypes to the synapse or anchors them there – this requires the g2 subunit. Without the g2 subunit, no GABAA receptors are found in synapses in the developing or adult hippocampus, and without gephyrin, much-reduced numbers of some synaptic GABAA receptor subtypes, especially those containing a2, are found; some receptor clusters, especially those containing the a1 subunit, persist in hippocampal gephyrin knockout neurons. Other g subunits can replace synaptic targeting function of g2; in g2 knockout mice, GABAA receptors can be restored to hippocampal synapses by expressing the g3 subunit by transgenic rescue. Some conserved sequence identity in the large TM3–TM4 intracellular loops of the g subunits may indicate binding sites for parts of the synapse anchoring mechanism. Distributed cysteine residues are conserved in the g subunit large intracellular loops but are absent from the b and d subunits. Palmitoylation of these cysteine residues via a thioester bond plays some role in targeting g subunit-containing receptors to the synapse. A surprising finding is that the TM4 region of the g2 subunit is also involved in synaptic targeting, possibly by interacting with lipid rafts occurring in the synapse or by other membrane proteins. The g2 TM4 is necessary and sufficient for postsynaptic clustering of GABAA receptors, whereas the cytoplasmic g2 subunit domains are dispensable. In contrast, both the TM3–TM4 loop and the TM4 domain of the g2 subunit contribute to efficient recruitment of
350 GABAA Receptors: Molecular Biology, Cell Biology, and Pharmacology a4
b2
d b2 a4
d
b2
a4 T
a 4b 2d
a a1
OB
b2
g2 b2 a1
g2 H a1
Cb
b2
a 1b 2g 2
b Figure 4 Some GABAA receptor subunit combinations found in adult rat brain: (a) a4b2d; (b) a1b2g2. The gene expression is visualized by in situ hybridization (adult rat brain, horizontal sections). Cb, cerebellum; H, hippocampus; OB, olfactory bulb; T, thalamus. The a4b2d receptors are particularly prominent in the thalamus; the a1b2g2 subtype is widespread.
gephyrin to postsynaptic receptor clusters. Thus, the g2 subunit TM3–TM4 cytoplasmic loop might be needed for inserting receptors into the plasma membrane but is dispensable for delivery of receptors to subsynaptic dendritic sites. Gephyrin does not bind the g2 receptor subunit directly. The identity of the missing link(s) between gephyrin and GABAA receptor subunits is unknown.
GABAA Receptor Occupancy at Synapses Is Dynamic GABAA receptor expression on the surface of neurons is dynamic; receptors rapidly recycle and leave from or insert into the synapse by lateral diffusion and/or endo/exocytosis. GABAA receptors diffuse into a synaptic zone and are transiently ‘captured’ by the anchoring complex. However, for some inhibitory hippocampal synapses, a direct relationship exists between the number of synaptic GABAA receptors and the strength of the synapse, but it is not clear what mechanisms maintain fixed numbers of GABAA receptors long-term at specific synapses. As for glutamate receptors at excitatory synapses, neurons probably recycle GABAA receptors as a strategy for setting their degree of excitability. GABAA receptors constitutively internalize by clathrin-dependent endocytosis; this requires interactions between the b and g2 subunits and the AP2 adaptin complex.
Different Synaptic GABAA Receptor Subtypes Can Be Enriched on Different Domains of the Same Neuron As mentioned above, considerable GABAA receptor complexity arises from differential subunit gene expression and specific subunit assembly rules. In addition, there are undefined determinants specifying that particular abg2 subunit combinations are enriched at different postsynaptic locations on the same cell. An example of GABAA receptor complexity on a single cell type is provided by a hippocampal pyramidal cell (Figure 5). A pyramidal cell is covered with GABAergic terminals; a typical rat CA1 pyramidal cell receives around 1700 GABAergic synapses, with the highest density on the perisomatic region. Inhibition on different domains affects different aspects of pyramidal cell function. GABAA receptor-mediated inhibitory postsynaptic currents can be generated along the whole somatodendritic domain and on the axon-initial segment (AIS) of CA1 pyramidal cells. The apical dendritic trunk has a high density of GABAergic terminals relative to the rest of the dendrite; strong GABAergic stimulation onto this apical trunk region will isolate the dendritic compartment from the cell body. The inhibition on the more distal dendrites controls Ca2þ spike propagation. Inhibiting the AIS will powerfully clamp down the pyramidal cell’s activity as this is where action potentials initiate – a typical rat CA1 pyramidal cell has more
GABAA Receptors: Molecular Biology, Cell Biology, and Pharmacology 351
Extrasynaptic GABAA Receptors: a4bd and a6bd Subtypes Dendrite
Soma
Parv CCK Axon
GABA Parv Basket cell GABA Parv
Basket cell GABA CCK
Axon hillock
Axo-axonic cell
= a 1b g 2 = a 2b g 2 Figure 5 Enrichment of a1- and a2-subunit-containing abg2 GABAA receptors opposite different classes of GABAergic terminal (cholecystokinin (CCK)- or parvalbumin (Parv)-containing) on a hippocampal pyramidal cell. Note that for clarity, only a small subset of GABAergic interneurons that project onto any given pyramidal cell are shown.
than 25 GABAergic terminals per 50 mm of the AIS. Thus, GABAA receptors in the AIS deliver inhibition which controls the overall level of output activity of pyramidal cells (Figure 5). GABAA receptors show differential subunit distribution at different synapses of the same pyramidal cell. On hippocampal pyramidal cells, the a2 subunit is enriched in the AIS but is present at only a minority of cell body synapses and synapses on dendrites. The AIS synapse also contains the a1 subunit and possibly the a4 and a5 subunits. GABAA receptors at the AIS are positioned to exert strong inhibitory influence over action potential generation. Synapses formed by two types of presynaptic interneuron cells (GABAergic basket cells) on the soma of CA1 pyramidal cells contain distinct GABAA receptor subtypes: Only those synapses coming from parvalbumin-negative/CCK-positive basket cell interneurons contain the a2 subunit; synapses from parvalbumin-positive basket cells are often a2immunonegative. This is a preferential targeting or enrichment of the a2 in a particular type of synapse – not an absolute selectivity. Both synapse types contain the same level of immunoreactivity for the b2/3 subunits; both types of synapse made by basket cells onto pyramidal cell somata also contain the a1 and the g2 subunits. However, the a1 subunit is less abundant in the parvalbumin-negative pyramidal cell synapses than in the parvalbumin-positive ones (Figure 5).
Besides mediating precisely timed synaptic pointto-point inhibition (phasic inhibition) via g2 subunitcontaining receptors, GABAA receptors can convey less-time-locked signals. Low GABA concentrations in the extracellular space, resulting from GABA diffusing from the synapse, can tonically activate extrasynaptic GABAA receptors. This ‘tonic inhibition’ is temporally uncoupled from the fast synaptic events, causing a continually present background inhibitory conductance. Such conductances alter the input resistance of the cell and thus influence synaptic efficacy and integration; tonic extrasynaptic conductances, by increasing the electrical leakiness of the dendritic membrane, substantially and indiscriminately diminish the size of excitatory signals in dendrites (Figure 6). Receptors with the d subunit, a4bd in forebrain and a6bd in cerebellar granule cells, are extrasynaptic; d subunits are perisynaptic (annular), localized around the edge of synapses in hippocampal dentate granule cells, and totally extrasynaptic on cerebellar granule cells. In all regions so far tested (cerebellar granule cells, hippocampal dentate granule cells, thalamic relay nuclei), d subunits contribute to GABAA receptors that provide an extrasynaptic tonic conductance. For GABAA receptors containing a4bd and a6bd subunits, their key properties are high affinity for neurotransmitter and limited desensitization, enabling them to contribute to tonic background conductances (Figure 6).
GABAA Receptor Agonists, Antagonists, and Allosteric Modulators GABAA receptors display a rich pharmacology. Generic GABAA receptors are selectively activated by the GABA agonist muscimol and blocked competitively by the GABA antagonists bicuculline and SR95531 (receptors assembled with r subunits are bicuculline and barbiturate insensitive, having their own unique pharmacology). Picrotoxin blocks GABAA receptors noncompetitively, probably by binding to a site in the channel. Many drugs bind at sites on the GABAA receptor distinct from the GABA binding site; these drugs change the shape of the receptor oligomer so that the efficacy of GABA at opening the channel is either increased (positive allosteric agonists, e.g., BZs such as diazepam) or decreased (negative allosteric agonists, e.g., b-carbolines). A few allosteric modulators occur naturally in the brain (e.g., Zn2þ, neurosteroids). Generally, positive allosteric agonists are used widely in medicine (e.g., for the induction and maintenance of general anesthesia or to treat anxiety disorders, states of agitation, epilepsy, or
352 GABAA Receptors: Molecular Biology, Cell Biology, and Pharmacology
GABA
a 4b d
a 1b g 2 Phasic
Tonic Gaboxadol
BZ
pA
pA s
Gaboxadol
ms GABA GABA + BZ
Figure 6 Synaptic and extrasynaptic GABAA receptors conferring tonic (a4bd or a4bd) and phasic (g2) inhibition, respectively. Synaptic (g2-containing) GABAA receptors can be activated by BZs and BZ-like ligands (e.g., zolpidem). BZs act to increase the peak amplitude and slow the rate of decay of the Cl current (red trace). Synaptic transmission is on the millisecond timescale. Extrasynaptic (d-containing) receptors can be selectively activated by Gaboxadol (red). Note the longer timescale.
sleep disorders), and there is scope to develop these drugs further to produce receptor subtype-selective drugs with fewer side effects; however, negative allosteric agonists also have potential clinical applications; for example, the drug L-655 708 works selectively at a5bg2 receptors (a subtype mainly expressed in the hippocampus), and by decreasing GABA’s action there, it acts as a cognition enhancer. A feature of all allosteric modulators is that they usually work only when GABA is at submaximal activating concentrations (below 1 mmol l1), and they do not work in the absence of GABA (with the exception of some intravenous anesthetics). Nevertheless, some modulators (e.g., BZs) also strongly influence the deactivation rate of the receptors, even at peak synaptic GABA concentrations, and this may be how some of their in vivo effects originate. In the following sections, the drugs that act on particular GABAA receptor subunit combinations are considered in more detail. GABAA Receptors: Allosteric Modulation by BZs and Related Ligands
The main effects of BZs are sedation, anxiolysis, suppression of seizures, and muscle relaxation. These drugs require abg2-type receptors (a1b2g2, a2b2g2, a3b3g2, and a5bg2) with the drug-binding site located between the a and g2 subunits (Figure 3(a)). Note that a4bg2- and a6bg2-type receptors are insensitive to most BZ drugs, as are any receptors that contain the
d subunit. The substances that act at the BZ binding site include the classical BZs like diazepam or flunitrazepam, as well as chemically different substances, such as the imidazopyridine zolpidem (relatively selective for a1bg2-type receptors), which is used as a sleeping pill. The BZ site is situated at the interface between the a and the g subunit. In the a1, a2, a3, and a5 subunits, a mutation from histidine to arginine at position 101 abolishes binding of classic agonists like diazepam. The diazepam-insensitive a4 (or a6) subunits naturally contain an arginine residue at the homologous position, and so a4bg2 or a6bg2 receptors are insensitive to most BZ ligands. In a beautiful series of studies, mice with specific mutations in the key H101 coding position affecting BZ sensitivity were generated in the a1, a2, a3, and a5 subunit genes, and the behavioral effects of diazepam were tested. These mice have normal GABAA receptors, but in a1H101R mice, for example, only the a2bg2-, a3bg2-, and a5bg2-type GABAA receptors are diazepam sensitive. Thus by a process of subtraction, it can be deduced how different a1bg2, a2bg2, a3bg2, and a5bg2 subtypes contribute to the diverse in vivo pharmacological effects of diazepam and other ligands requiring the H101 site. Thus, a1H101R mice no longer become sleepy when given diazepam, and so the a1bg2 receptors are required for the sedative effects of diazepam, whereas the a2 (and a3) mediates diazepam’s anxiolytic effects (under the influence of diazepam, a2H101R mice do not venture more into threatening areas, whereas their wild-type littermates do). A different set of studies using a3 selective agonists and inverse agonists also showed a significant contribution of the a3 subunit in anxiogenesis and anxiolysis. The muscle relaxant activity of diazepam is mediated by the a2 and a3 subunits, probably because these subunits are expressed in spinal motor neurons. New Subtype-Selective Drugs for GABAA Receptors That Work at the BZ Site
GABAA receptors have always been fertile ground for drug companies. BZs, although for many years the mainstay of clinical treatments for anxiety disorders, fell out of favor to selective serotonin reuptake inhibitors (SSRIs) because of side effects like sedation, cognitive impairment, and abuse liability. But SSRIs are too slow acting for some situations, requiring several weeks to work. Thus there is a medical need for fast-acting anxiolytics with few or no side effects. The knock-in mouse studies mentioned above suggest that a2-containing GABAA receptors would be a prominent target for developing drugs which would be specifically anxiolytic.
GABAA Receptors: Molecular Biology, Cell Biology, and Pharmacology 353 GABAA Receptors: Allosteric Modulation by Intravenous Anesthetics
At clinically relevant concentrations, general anesthetics modulate the activity of various ion channels. Whereas volatile anesthetics (e.g., halothane, enflurane, or isoflurane) are positive modulators of recombinant GABAA receptors, the intravenous anesthetics (e.g., barbiturates, steroidal anesthetics, propofol, and etomidate) can modulate GABA’s action at the receptor but can also activate the receptor directly in the absence of GABA at higher concentrations. Based on the analysis of knock-in mouse lines with propofoland etomidate-insensitive b subunits (see below), propofol and etomidate exert nearly all their anesthetic actions entirely through GABAA receptors, whereas volatile anesthetics produce their actions through many diverse ion channel targets. The action of etomidate and propofol requires residues in TM2 and TM3 in the b2 or b3 subunits. A mutation of asparagine to methionine at position 265 (N265M) in the second transmembrane domain of the b3 subunit abolishes the modulatory and direct effects of etomidate and propofol in recombinant receptors. A mutation of aspargine at the same position in the b2 subunit also abolishes the action of etomidate on the GABAA receptor. In b3(N265M) mice, propofol and etomidate do not suppress noxious-evoked movements and show a strongly decreased duration of the loss of righting reflex, two different endpoints of anesthesia. These results suggest that propofol and etomidate act mainly via the GABAA receptor and the b3 subunit in particular to induce deep anesthesia. The remaining effects of propofol and etomidate could be mediated by b2 subunit-containing receptors. Studies on b2(N265S) mice have suggested that the b2 subunit mediates the sedative effects of etomidate, whereas the b3 subunit is required for etomidate to induce a loss of consciousness. A highly interesting issue is the location in the brain where etomidate and propofol exert their anesthetic effects. Is the modulation of GABAA receptors in specific nuclei required to induce anesthesia or do these drugs produce global effects at many GABAA receptors in all brain circuits? GABAA Receptors: Allosteric Modulation by Neurosteroids
Neuroactive steroids modulate GABAA receptor function in many brain regions. Naturally occurring steroid metabolites form locally in the brain: 5a-reductase transforms progesterone to 5a-DPH, which in turn is reduced by 3a-hydroxysteroid oxidoreductase to allopregnanalone. Allopregnanalone potently activates
GABAA receptors. No absolute specificity of neurosteroids for particular GABAA receptor subunit combinations exists. Many GABAA receptors are sensitive to the steroid THDOC, but receptors with the d subunit are particularly sensitive. Thus, endogenous allopregnanolone may act on extrasynaptic a4bd and a6bd GABAA receptors to increase basal levels of inhibition. New Sleep-Promoting Ligands Acting as GABA Mimetics on Extrasynaptic a4bd Receptors
Modulating extrasynaptic GABAA receptors can produce profound effects on the nervous system (Figure 6). Many people suffer from insomnia. This is often treated with ligands, such as zolpidem, with a preferential affinity for the BZ site on synaptic a1bg2 receptors. By contrast, Gaboxadol is a selective extrasynaptic GABAA receptor agonist that has its greatest efficacy at a4bd and a6bd GABAA receptors, that is, BZ-insensitive receptors that contribute to tonic inhibitory conductances rather than synaptic inhibitory postsynaptic currents. It is important to emphasize that Gaboxadol, unlike BZs or related ligands, is an agonist at the GABA binding site itself – that is, it is a GABA mimetic, opening the receptor directly. Drugs which promote sleep acting through the BZ biding site of a1bg2-type receptors can cause various side effects such as disturbances in memory, rebound insomnia on drug withdrawal, and drug dependence. Gaboxadol acts on a4bd occurring exclusively extrasynaptically and enriched in particular neuronal pathways (e.g., thalamus – Figures 4(a) and 6), and thus has a different side effect profile. Sleep induced by increasing specifically the activity of extrasynaptic GABAA receptors may be of higher quality.
Conclusions Many subtypes of GABAA receptor are used by the nervous system to impart inhibition. These GABAA receptors differ in their affinity for neurotransmitter and allosteric modulators, activation rate, desensitization rate, channel conductance, and location on the cell. But exactly what selective advantages are conferred by so many receptor subtypes to brain function is unclear and remains a fascinating research issue. In particular, the large number of abg subunit-containing synaptic receptor subtypes, often co-expressed on a single cell type such as a cortical pyramidal cell (Figures 2 and 5), might enhance the computational potential of the cell. Receptors at different synapses might have properties (e.g., deactivation rates) fine-tuned for individual inputs. In principle, evolutionary processes selected these subunit combinations
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to be optimal, or at least to provide a reasonable compromise, for the inhibitory function required at each type of location. In some cases, however, multiple genes may exist because of the need to have complex transcriptional regulation that would have been difficult to organize from one gene promoter, rather than from the need to have different receptor properties. If one could engineer it, the hippocampus, or even the entire brain, might work perfectly well with just one type of GABAA receptor, such as an a1b2g2 combination. In practice, the GABAA receptor system remains an important target for new therapeutic approaches. See also: GABA Synthesis and Metabolism; GABAA
Receptor Synaptic Functions; GABAB Receptor Function; GABAB Receptors: Molecular Biology and Pharmacology.
Further Reading Belelli D and Lambert JJ (2005) Neurosteroids: Endogenous regulators of the GABAA receptor. Nature Reviews Neuroscience 6: 565–575. Buzsaki G (2006) Cycle 3: Diversity of cortical functions is provided by inhibition. In: Buzaki G (ed.) Rhythms of the Brain, pp. 61–79. New York: Oxford University Press. Darlison MG, Pahal I, and Thode C (2005) Consequences of the evolution of the GABAA receptor gene family. Cellular and Molecular Neurobiology 25: 607–624. Ernst M, Bruckner S, Boresch S, and Sieghart W (2005) Comparative models of GABAA receptor extracellular and transmembrane domains: Important insights in pharmacology and function. Molecular Pharmacology 68: 1291–1300. Fang C, Dent L, Keller CA, et al. (2006) GODZ-mediated palmitoylation of GABAA receptors is required for normal assembly and function of GABAergic inhibitory synapses. Journal of Neuroscience 26: 12758–12768. Farrant M and Nusser Z (2005) Variations on an inhibitory theme: Phasic and tonic activation of GABAA receptors. Nature Reviews Neuroscience 6: 215–229. Fritschy JM and Brunig I (2003) Formation and plasticity of GABAergic synapses: Physiological mechanisms and
pathophysiological implications. Pharmacology & Therapeutics 98: 299–323. Hammond C (2001) The Ionotropic GABAA receptor. In: Hammond C (ed.) Cellular and Molecular Neurobiology, 2nd edn., pp. 227–250. San Diego, CA: Academic Press. Korpi ER and Sinkkonen ST (2006) GABAA receptor subtypes as targets for neuropsychiatric drug development. Pharmacology & Therapeutics 109: 12–32. Olsen RW and Betz H (2006) GABA and glycine. In: Siegel GJ, Albers RW, Brady ST, and Price DL (eds.) Basic Neurochemistry: Molecular, Cellular and Medical Aspects, 7th edn., pp. 291–301. Amsterdam: Academic Press. Rudloph U and Mohler H (2004) Analysis of GABAA receptor function and dissection of the pharmacology of benzodiazepines and general anesthetics through mouse genetics. Annual Review of Pharmacology and Toxicology 44: 475–498. Schofield PR, Darlison MG, Fujita N, et al. (1987) Sequence and functional expression of the GABAA receptor shows a ligand-gated receptor super-family. Nature 328: 221–227. Seeburg PH, Wisden W, Verdoorn TA, et al. (1990) The GABAA receptor family: Molecular and functional diversity. Cold Spring Harbor Symposia on Quantitative Biology 55: 29–40. Siegel E, Baur R, Boulineau N, and Minier F (2006) Impact of subunit positioning on GABAA receptor function. Biochemical Society Transactions 34: 868–871. Simon J, Wakimoto H, Fujita N, Lalande M, and Barnard EA (2004) Analysis of the set of GABAA receptor genes in the human genome. Journal of Biological Chemistry 279: 41422–41435. Somogyi P and Klausberger T (2005) Defined types of cortical interneurone structure space and spike timing in the hippocampus. Journal of Physiology 562: 9–26. Unwin N (2005) Refined structure of the nicotinic acetylcholine receptor at 4A resolution. Journal of Molecular Biology 346: 967–989. Wafford KA and Ebert B (2006) Gaboxadol: A new awakening in sleep. Current Opinion in Pharmacology 6: 30–36. Whiting PJ (2006) GABAA receptors: A viable target for novel anxiolytics? Current Opinion in Pharmacology 6: 24–29.
Relevant Websites http://www.ionchannels.org – Ionchannels.org. http://www.ebi.ac.uk – Ligand-Gated Ion Channel Database. http://www.nature.com – Nature: Supplement: Insight: Ion Channels. http://en.wikipedia.org – Wikipedia articles on benzodiazepine and GABA.
GABAA Receptor Synaptic Functions I Mody, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA ã 2009 Elsevier Ltd. All rights reserved.
Introduction The amino acid neurotransmitter g-aminobutyric acid (GABA) is responsible for mediating most of chemical inhibition in the central nervous system (CNS). The ionotropic GABA receptors (GABAARs) are members of the cysteine-loop ligand-gated ion channel family and form a Cl- and HCO 3 -permeable ion pore assembled from five (heteropentameric) subunits selected from the following subunits: a1–6, b1–3, g1–3, d, E, y1–3, p, and r1–3. Hundreds of thousands of different combinations are possible, yet no more than a few dozen receptor combinations are likely to exist in the mammalian brain. This is thought to result in part from precise rules of subunit assembly (e.g., two a and two b subunits assemble with either one g or one d subunit) and in part from certain rules of specific subunit partnerships that also appear to define their subcellular localization inside and outside of synapses. Loss-of-function mutations in some of these subunits are associated with various rare genetic forms of epilepsy in humans, indicating their importance in subduing neuronal hyperactivity. Over the years GABAARs have become synonymous with inhibition, but we now know that depending on a variety of conditions, their activation may excite neurons or may synchronize them, resulting in physiological oscillations or pathological synchrony leading to seizures. In 1963, on behalf of the Nobel Committee upon awarding the Prize to Sir John C Eccles, Ragnar Granit stated: ‘‘If the arriving impulse is connected to excitatory synapses the response of the cell is yes, i.e., excitability increases; vice versa the inhibitory synapses make the cell respond with a no, a diminution of excitability.’’ Forty-three years later it is clear that in response to GABAergic ‘inhibition’ many cells have expanded their vocabulary to ‘maybe’ and even to ‘yes.’
GABAARs at Synapses In the mammalian brain GABA synapses constitute a minority. Only about every sixth synaptic terminal is GABAergic and only every fifth neuron utilizes GABA as a transmitter. Excitation clearly outnumbers inhibition. Yet, in spite of this overpowering excitatory drive, most of the time the GABAergic
system manages to keep excitability in check. It does this through a spatially and temporally selective activation of the GABAergic system. The pioneering Golgi impregnation studies of Ramo´n y Cajal, Lorente de No, and Ja´nos Szenta´gothai have distinguished a marked feature of cortical interneurons: they have a highly selective axonal arborization, innervating distinct spatial domains of the principal cells. GABA synapses can be found on cell bodies, dendritic shafts and branch points, spine necks, and axon initial segments, in far more diverse places than excitatory synapses. The strategic positioning of GABA synapses on the target cells can annihilate a specific input or the entire output of the cell. A good example of such selective control of a given input is the hippocampal oriens-lacunosum moleculare (OLM) cell that precisely innervates the most distal dendrites of pyramidal cells. This is precisely the region where the entorhinal cortical excitatory input lands on the principal cells. Since OLM cells are activated by massive intrahippocampal excitatory events, this spatial arrangement of their inhibitory output ensures that the entorhinal input does not have a say during this time. In contrast, the cortical chandelier or axo-axonic cells do not synapse onto a cellular region receiving a specific input. Instead, they control the output of pyramidal cells, by innervating the axon initial segment, where the ultimate action potential output of the target neuron is generated. According to the latest findings however, some of these axo-axonic inhibitory interneurons may elicit firing at the axon initial segment (see later), in stark contradiction to the function implied by their ‘inhibitory’ name. The location specificity of inhibitory synapses is complemented by a highly specific assembly of the GABAARs that depends on the type of inhibitory interneuron that forms the synapse. There is little known about the mechanisms that sustain this specificity at a given synapse over the lifetime of the neuron. Of the thousands of possible heteropentameric GABAAR combinations that can be formed through assembly from nearly 20 subunits, only a few dozen combinations occur in the brain. These combinations do not appear to be randomly inserted at synapses. The types of subunits present at a given synapse tend to be specified by the GABA neuron that provides the presynaptic bouton of the synapse. The axo-axonic chandelier cells, and the pyramidal cell soma innervating cholecystokinin (CCK)-positive basket cells, are known to form synapses on predominantly a2 subunit-containing receptors. The parvalbumincontaining basket cells synapse onto a1-containing
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GABAARs, while other combinations are found at synapses made by other interneuron types. The functional significance of this highly specific arrangement is only beginning to emerge. Different GABA receptor subunit assemblies have very different kinetic and desensitization properties when studied in expression systems. Accordingly, one would expect the various synaptic currents generated by diverse GABAARs also to be highly variable. However, paired recordings from two interneurons and their target cells usually show similar inhibitory postsynaptic current (IPSC) kinetics, in spite of known differences of GABAAR assemblies at the two synapses. Even if the kinetic differences observed in expression systems do not hold at GABAARs assembled at synapses in the brain, the different compositions and locations of GABAARs have undoubtedly evolved to participate in widely different control functions of neuronal excitability. Insight into these functions has come from cleverly engineered mice expressing various a subunits insensitive to benzodiazepines (BZs), a class of potent allosteric GABAAR modulators. Two a, two b, and a single g subunit assemble to form the most commonly found GABAARs in the brain. The function of these receptors is enhanced by BZs as long as the a subunits are either a1, a2, a3, or a5, but not a4 or a6. There is a single amino acid in a critical position (His) that differs between the BZsensitive and BZ-insensitive a subunits (Arg in the latter). Mice have been genetically engineered to have BZ-insensitive a1, a2, a3, or a5 subunit-containing GABAARs. Studies in these mice conclusively demonstrated that the a1 subunit-containing GABAARs are involved in the sedative–hypnotic actions of BZs, while the receptors containing a2 subunits mediate anxiolytic effects of BZs. Does this mean that the function of the synapses predominantly containing a2 subunits, such as those found at the axon initial segment or those apposed to the terminals of CCK-containing basket cells, are mainly related to the control of anxiety? At this time it is still too early to say, as there might be specific brain regions rich in a2 subunits that could play a significant role in the control of anxiety. Nevertheless, it is clear that specific GABAAR subunit assemblies innervated by specific interneurons play highly specialized roles in the development of neuronal circuitry and in mediating specific higher nervous system functions.
Activation of GABAARs at Synapses – Phasic Inhibition The GABAARs usually found at synapses are most likely to contain g2 subunits. GABA synapses are ‘symmetrical’ synapses; called such because on electron
micrographs they are devoid of a wide electron-dense postsynaptic density (PSD) which would make the appearance of the synapse ‘asymmetric.’ Therefore, compared to the PSDs of excitatory glutamatergic synapses containing a stable assembly of over 70 different receptor, scaffold, and signaling proteins, there is a lackluster showing of postsynaptic proteins at the poorly developed PSDs of GABA synapses. Glutamate receptors at synapses associate with the subsynaptic cytoskeleton. GABAARs do not. There are, however, some proteins that seem to play a role in the anchoring, insertion, and removal of GABAARs at synapses. Gephyrin links up with the subsynaptic microtubule and actin cytoskeleton. It interacts with several microfilament-regulating proteins, including collybistin, profilins, Mena/VASP proteins, and dynein light chain (Dlc)/myosin-Va. However, the role of these protein– protein interactions for the shaping of GABA synapses is largely a mystery. Some GABA synapses express the dystrophin–glycoprotein complex, better known as a connecting element between the extracellular and cytoskeletal matrices of muscle cells. Other proteins are implicated in the trafficking of GABAARs: AP2, BIG2, GABARAP, GODZ, Plic-1, and PRIP1/2. To make things more complicated, trafficking of GABAAR subunits seems to depend on the phosphorylation state of the subunit. Various kinases and phosphatases are at hand, as they bind directly to the subunits or are linked to specific GABAARs through highly specific adaptor proteins. As molecular biologists, biochemists, and anatomists continue to pursue the means of GABAAR anchoring and targeting to synapses, physiologists and biophysicist have focused on the means of activation of GABAARs at synaptic junctions. The kinetic profile of synaptic GABAARs consists of a moderate to low affinity for GABA, some desensitization, and a rapid activation/deactivation. The time course of GABA in the synaptic cleft has been estimated to be less than 500 ms, and the peak concentration reaching the receptors is probably in the low millimolar range. This means that at many synapses where usually no more than tens of GABAARs are present, the number of GABA molecules is in excess of the available binding sites, thus providing for a high occupancy of the available receptors. However, this may not be the case at all GABA synapses, as in cerebellar basket/stellate cells, where synapses with hundreds of receptors and synapses with tens of receptors coexist, only the latter appear to be saturated by the released transmitter. In general, the relatively low number of postsynaptic GABA receptors compared to the amount of GABA released into the cleft renders the process regulating the number of receptors an important aspect of GABAAR plasticity. The kinetic properties of the
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subunits assembled at the synapses should also affect the properties of inhibition, but thus far there are consistent reports only on the speedy decay of a1 subunit-containing synaptic receptors, while the variety of the biophysical receptor properties seen in expression systems has yet to be demonstrated at synapses. Desensitization is also an important mechanism to consider for regulating the decay of IPSCs, but prolonging the synaptic GABA transient by using uptake blockers does not always prolong the duration of synaptic events as it would be expected from a significant role of desensitization. It appears, however that the duration of the GABA transient in the cleft may itself regulate the decay of synaptic events, which in turn might make it difficult to discern differences that are simply due to underlying channel kinetics. The various brain-state-altering effects of BZs that act to prolong the duration of IPSCs predominantly affecting g2 subunit-containing synaptic receptors show the importance of regulating the decay phase of IPSCs. Like at any central synapse, presynaptic mechanisms can also effectively determine how GABA synapses are activated. Again, the most interesting presynaptic aspect of GABA synapses is their specificity with regard to the cell type supplying the presynaptic terminal. For example, there are clear differences in the way GABA is released from parvalbumin- and CCK-containing basket cells, and this may have important functional consequences for the operation of neuronal networks. Other GABA synapses are depressed most of the time by ambient levels of endocannabinoids, and do not become active unless the presynaptic cell fires at high frequencies. Different presynaptic Ca2þ channels are responsible for activating GABA release in different interneurons. Moreover, there is also accumulating evidence that the intricate vesicular release machinery may be different between glutamate- and GABA-containing terminals.
Activation of Peri- and Extrasynaptic GABA Receptors: Tonic Inhibition As is the case for many ligand-gated ion channels, synapses are not the only place GABAARs are likely to show up on a cell’s surface. But perhaps for more than any other ligand-gated channel, GABAARs found outside the synapses are quite special. Although many different combinations of GABAARs are scattered outside of synapses, there are only two types of combinations that have been shown to actually respond to the ambient GABA concentrations of less than a few micromoles per liter, as found in the interstitial space of neurons. These two combinations
are the d subunit-containing and the a5 subunitcontaining GABAARs. The d subunits combine with a4, a6, and most likely other a subunits to form, together with b2 or b3 subunits, a GABAAR combination that is well suited to be activated by low levels of ambient GABA. These receptors have a high affinity for GABA, they do not desensitize, and are preferentially found extra- or perisynaptically. Their modulation mainly occurs by increasing the efficacy of GABA rather than its potency, as the natural agonist is not very efficacious at these receptors. The other GABAAR combination shown to be activated by ambient GABA is the a5 subunit-containing receptors. The a5 subunits are not exclusively extrasynaptic; they probably combine with other a subunits in synaptic receptors, but in the cells where they are present they readily mediate tonic inhibition. The tonic inhibition, when present, is a significant force in controlling the excitability of the neurons. Charge movement through tonically active GABA channels outweighs 3:1 to 5:1 the charge going through the synaptic receptors, even when the frequency of the synaptic activity is quite high (40– 60 Hz). Such a tonically active conductance globally is well suited to regulate the input–output function of the cells. The d subunit-containing GABAARs mediating tonic inhibition are also an important site of action for various endogenous and exogenous allosteric modulators. Neurosteroids, brain-synthesized metabolites of various sex and stress steroid hormones, specifically act upon these receptors to enhance their activity. Ethanol, at concentrations relevant to impairing sobriety, also appears to target the d subunitcontaining GABAARs, which may justify them lately being referred to as the ‘ethanol receptor.’ The receptors mediating tonic inhibition show a remarkable plasticity, both during physiological (ovarian cycle) and pathological (epilepsy) alterations in neuronal excitability. Selectively eliminating tonic inhibition in hippocampal CA1 pyramidal cells facilitates certain forms of learning, presumably by increasing the sensitivity of pyramidal cells to incoming stimuli. These recent developments in the physiology and pharmacology of extrasynaptic tonically active GABAARs will lead to exciting insights into their complex roles in the control of neuronal excitability.
Excitation and Synchrony through ‘Inhibitory’ Synapses There is nothing mysterious about ‘inhibitory’ synapses extending their vocabulary to ‘maybe’ and ‘yes.’ The relationship between the time-averaged membrane potential of the neuron and the GABA
358 GABAA Receptor Synaptic Functions
equilibrium potential, which is a function of the equilibrium potentials of the two permeant ions, Cl and HCO3, determines whether activation of GABAARs will depolarize or hyperpolarize a cell. In fact, ‘yes’ is the first word uttered by an activated GABAAR. Early on during brain development, when cells are loaded with Cl, the activation of GABAAR channels leads to efflux of negatively charged Cl ions, thus depolarizing the cell. This makes GABA one of the first depolarizing signaling molecules during development. The depolarization helps bring the membrane potential to the threshold of activation of voltagegated Ca2þ entry, a cation that, once inside the cells, is critical for the activation of a myriad of important developmental signaling cascades. Eventually, later during development, Cl is extruded from the cells by various transport mechanisms. Inhibition can thus start to learn to say ‘no.’ Yet, depolarizing GABAARmediated events continue to play an important role in the adult nervous system. The survival of newly generated neurons in the mammalian brain depends largely on the activation of depolarizing, tonically active GABAARs. Local peaks in the GABA reversal potential can trigger depolarizing inhibitory postsynaptic potentials (IPSPs) that can passively propagate along the neuron to elevate excitability, or can outright trigger action potentials at the axon initial segment, as we have recently learned from the potentially ‘excitatory’ function of axo-axonic cells in the neocortex. Regulated by several exchangers and transporters, the GABA reversal potential can itself become a dynamic force in the control of neuronal excitability. One of the key players is a Kþ/Cl cotransporter called KCC2. This molecule is critical in extruding Cl during development, but reducing its function during spike timing-dependent plasticity of GABA synapses, high-frequency firing of the cells, or neuronal injury leads to an intracellular accumulation of Cl and thus the conversion of a hyperpolarizing event into one that will depolarize the affected neuron. An altered KCC2 function is most likely at work to cause the pathological alteration of the GABA reversal potential in subicular pyramidal neurons recorded in tissue samples obtained from temporal lobe epilepsy patients, and causes the conversion to a GABA-induced depolarization of spinal cord neurons during neuropathic pain. Even if depolarizing GABA-mediated events do not push the membrane potential all the way past the threshold of activation of voltagedependent Naþ or Ca2þ conductances, such events can passively propagate through the cell, thus increasing excitability and promoting neuronal synchrony.
Depolarizing neurons is not the only means by which activation of inhibitory synapses can promote synchrony. If the neurons are equipped with a hyperpolarization-activated excitatory conductance (e.g., Ih) or a low-threshold (T-type) Ca2þ current, as most neurons are, then a hyperpolarization induced in several neurons at the same time can lead to a rebound excitation following the cessation of the hyperpolarizing event. Hundreds or thousands of principal cells innervated by a single inhibitory interneuron, or by a few interneurons activated in unison, can thus synchronize their firing without requiring any synchronous excitatory drive. This type of synchronization may specifically spring into action when diffusely acting neurotransmitters, such as acetylcholine (ACh) or glutamate, enhance GABA release by acting at once on the terminals of several inhibitory cells through actions at nicotinic ACh or N-methyl-D-aspartate (NMDA) receptors. In summary, phasic and tonic GABAergic inhibitions exert a substantial control over neuronal excitability. The incredibly diverse GABAergic cells of the brain are the real puppet masters of the principal cells, by controlling their activity both at the level of different single neuronal compartments and at the level of intricately connected neuronal networks. For certain, the diversity and specificity of GABAARs, GABAergic neurons, and their functions will keep neuroscientists busy for a while. It may have taken some time, but now we know that it is much easier for the ‘inhibitory’ GABAergic system to say ‘maybe’ and ‘yes’ than it is for excitatory glutamate synapses to learn to say ‘no.’ See also: GABA Synthesis and Metabolism; GABAA
Receptors: Molecular Biology, Cell Biology and Pharmacology; GABAB Receptor Function; GABAB Receptors: Molecular Biology and Pharmacology.
Further Reading Ben-Ari Y (2002) Excitatory actions of GABA during development: The nature of the nurture. Nature Reviews in Neuroscience 3: 728–739. Buzsa´ki G (2006) Rhythms of the Brain. New York: Oxford University Press. Buzsa´ki G, Geisler C, Henze DA, et al. (2004) Interneuron diversity series: Circuit complexity and axon wiring economy of cortical interneurons. Trends in Neuroscience 27: 186–193. Chen ZW and Olsen RW (2007) GABA receptor associated proteins: A key factor regulating GABA receptor function. Journal of Neurochemistry 100(2): 279–294. Cobb SR, Buhl EH, Halasy K, et al. (1995) Synchronization of neuronal activity in hippocampus by individual GABAergic interneurons. Nature 378: 75–78.
GABAA Receptor Synaptic Functions 359 Cohen I, Navarro V, Clemenceau S, et al. (2002) On the origin of interictal activity in human temporal lobe epilepsy in vitro. Science 298: 1418–1421. Coull JA, Beggs S, Boudreau D, et al. (2005) BDNF from microglia causes the shift in neuronal anion gradient underlying neuropathic pain. Nature 438: 1017–1021. Farrant M and Nusser Z (2005) Variations on an inhibitory theme: Phasic and tonic activation of GABAA receptors. Nature Reviews Neuroscience 6: 215–229. Fiumelli H, Cancedda L, and Poo MM (2005) Modulation of GABAergic transmission by activity via postsynaptic Ca2þdependent regulation of KCC2 function. Neuron 48: 773–786. Freund TF and Buzsa´ki G (1996) Interneurons of the hippocampus. Hippocampus 6: 347–470. Gaiarsa JL, Caillard O, and Ben-Ari Y (2002) Long-term plasticity at GABAergic and glycinergic synapses: Mechanisms and functional significance. Trends in Neuroscience 25: 564–570. Hanchar HJ, Wallner M, and Olsen RW (2004) Alcohol effects on gamma-aminobutyric acid type A receptors: Are extrasynaptic receptors the answer? Life Sciences 76: 1–8. Hefft S and Jonas P (2005) Asynchronous GABA release generates long-lasting inhibition at a hippocampal interneuron–principal neuron synapse. Nature Neuroscience 8: 1319–1328. Hevers W and Lu¨ddens H (1998) The diversity of GABAA receptors. Pharmacological and electrophysiological properties of GABAA channel subtypes. Molecular Neurobiology 18: 35–86. Lien CC, Mu Y, Vargas-Caballero M, et al. (2006) Visual stimuliinduced LTD of GABAergic synapses mediated by presynaptic NMDA receptors. Nature Neuroscience 9: 372–380. Lu¨scher B and Fritschy JM (2001) Subcellular localization and regulation of GABAA receptors and associated proteins. International Review of Neurobiology 48: 31–64. Lu¨scher B and Keller CA (2004) Regulation of GABAA receptor trafficking, channel activity, and functional plasticity of inhibitory synapses. Pharmacology and Therapeutics 102: 195–221. Macdonald RL, Gallagher MJ, Feng HJ, et al. (2004) GABAA receptor epilepsy mutations. Biochemical Pharmacology 68: 1497–1506. Maguire JL, Stell BM, Rafizadeh M, et al. (2005) Ovarian cyclelinked changes in GABAA receptors mediating tonic inhibition alter seizure susceptibility and anxiety. Nature Neuroscience 8: 797–804.
Mody I and Pearce RA (2004) Diversity of inhibitory neurotransmission through GABA(A) receptors. Trends in Neuroscience 27(9): 569–575. Mozrzymas JW (2004) Dynamism of GABAA receptor activation shapes the ‘personality’ of inhibitory synapses. Neuropharmacology 47: 945–960. Rivera C, Voipio J, Thomas-Crusells J, et al. (2004) Mechanism of activity-dependent downregulation of the neuron-specific K–Cl cotransporter KCC2. Journal of Neuroscience 24: 4683–4691. Rudolph U and Mo¨hler H (2004) Analysis of GABAA receptor function and dissection of the pharmacology of benzodiazepines and general anesthetics through mouse genetics. Annual Reviews of Pharmacology and Toxicology 44: 475–498. Sieghart W and Sperk G (2002) Subunit composition, distribution and function of GABAA receptor subtypes. Current Topics in Medicinal Chemistry 2: 795–816. Somogyi P and Klausberger T (2005) Defined types of cortical interneurone structure space and spike timing in the hippocampus. Journal of Physiology 562(1): 9–26. Somogyi P, Tamas G, Lujan R, et al. (1998) Salient features of synaptic organisation in the cerebral cortex. Brain Research: Brain Research Reviews 26: 113–135. Soltesz I (2005) Diversity in the Neuronal Machine: Order and Variability in Interneuronal Microcircuits. New York: Oxford University Press. Stell BM, Brickley SG, Tang CY, et al. (2003) Neuroactive steroids reduce neuronal excitability by selectively enhancing tonic inhibition mediated by delta subunit-containing GABAA receptors. Proceeding of the National Academy of Sciences United States of America 100: 14439–14444. Szabadics J, Varga C, Molnar G, et al. (2006) Excitatory effect of GABAergic axo-axonic cells in cortical microcircuits. Science 311: 233–235. Watts J and Thomson AM (2005) Excitatory and inhibitory connections show selectivity in the neocortex. Journal of Physiology 562: 89–97. Whiting PJ (2003) The GABAA receptor gene family: New opportunities for drug development. Current Opinion in Drug Discovery and Development 6: 648–657.
GABAB Receptors: Molecular Biology and Pharmacology N G Bowery, GlaxoSmithKline, Verona, Italy ã 2009 Elsevier Ltd. All rights reserved.
Introduction g-Aminobutyric acid (GABA) is the single most important inhibitory neurotransmitter within the mammalian brain, in which it is estimated that 40% of all inhibitory synaptic activity is mediated through this neutral amino acid. GABA can activate both ionotropic (GABAA) and metabotropic (GABAB) receptors, and these are present inside and outside the brain of mammals and also evident in lower species. This article focuses on the metabotropic receptor, GABAB. The receptor was originally defined in the 1980s by pharmacological characterization in isolated tissue preparations. The structure of the receptor was identified approximately 18 years later. Unlike GABAA receptors, GABAB sites do not affect Cl membrane conductance in neurons but instead their activation increases Kþ conductance and decreases Ca2þ conductance. These effects tend, although not exclusively, to be site directed such that the decrease in Ca2þ is more associated with presynaptic receptors, whereas Kþ effects are predominantly postsynaptic. As a consequence of these effects, receptor activation can produce neuronal hyperpolarization (Kþ effect) or a decrease in evoked neurotransmitter release (Ca2þ effect). It is this latter effect that was the basis of the first observed characteristic of GABAB receptors. GABA dose dependently reduced the electrically evoked release of tritiated noradrenaline from rat isolated atria. This effect was neither blocked by recognized GABA antagonists nor mimicked by established GABA agonists. Conversely, baclofen (b-chlorophenyl GABA), which is inactive at chloride-dependent GABA receptors, was able to mimic the action of GABA in the atrial preparation and in other GABAB receptor systems. The subsequent development of a membrane receptor binding assay firmly established the existence of the metabotropic GABA receptor in mammalian brain tissue.
activating the G-protein-coupled signaling system. GABAB1 and GABAB2 must remain linked as a dimer after insertion into the cell membrane to fully maintain receptor function. GABAB1 subunits remain associated with the endoplasmic reticulum, and only when they link with GABAB2 is the dimer transported to the cell membrane. Each subunit has a seven transmembrane spanning domain and they are linked via their C-termini through coiled-coil structures. A number of splice variants of the GABAB1 subunit have been identified, namely GABAB1a–1g (Figure 1, inset), but not all have been shown to have a physiological function. Only GABAB1a and GABAB1b have been implicated in the functional receptor, and although GABAB1c has been reported to be functional in vitro, evidence for an in vivo role is equivocal. The 1a and 1b proteins differ significantly in their amino acid sequences within the N-terminal; 1a has a unique sequence of 162 amino acids, whereas 1b has a shorter unique sequence of 47 amino acids. The distribution patterns of these two subunits within the rat brain indicate that they have different roles, although these have yet to be established. For example, within the spinal cord, the level of GABAB1a is reported to be tenfold higher than that of GABAB1b within the primary afferent terminals, and the GABAB1b subunit appears to predominate at postsynaptic sites in the cerebellum. However, elsewhere in the brain, GABAB1b appears to predominate in presynaptic terminals, whereas GABAB1a is associated with postsynaptic sites, for example, within the thalamocortical system. There is no unequivocal evidence to support the existence of variants of the GABAB2 subunit, although two variants of the human GABAB2 subunit have been proposed. However, these were probably artifacts of the isolation process. The expression of GABAB1 and that of GABAB2 appear to be independent of each, even though there is a 1:1 stoichiometric relation between the two proteins. Invariably, GABAB2 is expressed more abundantly than GABAB1 under experimental pathological conditions.
Receptor Structure
Protein–Protein Interactions
GABAB receptors exist naturally as heterodimers and comprise subunits designated GABAB1 and GABAB2 (Figure 1). Although the subunits exhibit 35% homology, they have quite distinct characteristics. Whereas GABAB1 contains the binding domain for GABA in its extracellular N-terminal, the GABAB2 subunit appears to be responsible for engaging and
The possibility of a mismatch between GABAB1 and GABAB2 levels raises the possibility that other proteins might form a dimer with the GABAB subunit to form a functional receptor. Numerous proteins have been described which interact with either GABAB1 or GABAB2, and some of these have such high affinity that they are able to compete with one GABAB
360
GABAB Receptors: Molecular Biology and Pharmacology 361 GABAB1
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Allosteric modulator site N
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Human 1c Rat/human 1a [62 aa deletion] [147 aa]
Rat/human 1b [18 aa]
1 2
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Rat 1c [31 aa addition]
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Rat 1d [25 aa modification]
Figure 1 Diagram of the heterodimer that forms the GABAB receptor. The receptor comprises two subunits, GABAB1 and GABAB2, which are inserted into the plasma membrane after having been coupled intracellularly. GABAB1 contains the GABA binding domain in its extracellular N-terminal chain, whereas GABAB2 does not bind GABA but has a modulatory site within the transmembrane region which appears to be specific to this subunit. This subunit also seems to be responsible for coupling to the second messenger G-protein system. Receptor activation manifests as an increase in Kþ conductance, a decrease in Ca2þ conductance, and/or an inhibition of adenylate cyclase (AC). The inset indicates the primary isoforms of GABAB1. Courtesy of Stefano Tacconi and Rachel Ginham.
subunit for binding to the other subunit. However, no evidence of the formation of a functional receptor from any protein–protein interaction has been reported. Thus, if the interaction is not producing a receptor, what purpose(s) does this protein coupling serve? It might be to provide a directional mechanism to enable the correct site insertion of the dimer into the plasma membrane or merely to act as a scaffold or anchoring device to support the dimer when inserted. Alternatively, because of the high affinity of these proteins, they might regulate the formation of the dimer, limiting the level of functional receptor, or by interacting intracellularly with the formed dimer, they may reduce its cell surface expression. The exact role of these interacting proteins, including CREB2/ATF4, 14-3-3, fibulin-2, CHOP, and Marlin-1, has yet to be established.
Receptor Subtypes Despite the presence of structural variants of the GABAB1 subunits and the possibility of functional modifications of the receptor heterodimer by interacting proteins, there is still no unequivocal evidence for the existence of functional GABAB receptor subtypes. Pharmacological differences between autoreceptors and heteroreceptors as well as between the dual actions of GABAB agonists on adenylyl cyclase activity have been suggested by individual research
groups. However, both a lack of reproducibility and sufficient distinction between the effects and the failure to emulate observations made by other groups have cast doubt on the existence of functional receptor subtypes. In general, the available receptor agonists and antagonists do not distinguish between potential subtypes of the GABAB receptor. Nevertheless, it has been suggested that gabapentin is a GABAB agonist which selectively activates the form of the receptor comprising a combination of GABAB1a and GABAB2 and expressed in isolated Xenopus oocytes. However, this could not be confirmed by others, especially in in vivo systems. Thus, although this was an exciting and potentially important observation, the failure of others to reproduce the original findings has tempered enthusiasm not only for understanding the mechanism of action of this anticonvulsant/analgesic agent but also for the possibility of relating receptor structure to functional diversity. A further lack of support for receptor subtyping derives from studies performed on developmental knockout mice in which either GABAB1 or GABAB2 subunits have been deleted. No supporting evidence for any receptor subtyping was observed in these animals.
Receptor Distribution GABAB receptors are distributed throughout the mammalian system and are not confined to nervous tissue.
362 GABAB Receptors: Molecular Biology and Pharmacology
Although their function is probably of greatest physiological importance within the central nervous system, their presence in peripheral organs is well established. In fact, it was their presence in the heart atrium of the rat that led to their discovery. They are present on autonomic nerve terminals, and when activated by GABA, this decreases the evoked release of transmitter from the terminal. Of course, this is only of physiological importance when the receptor agonist is present, and in the periphery this is unlikely to occur at most sites. However, in the enteric nervous system, in which GABA-releasing neurons are present and impinge on autonomic cholinergic nerve fibers, GABAB receptors probably contribute to the control of intestinal movement and sphincters. Individual GABAB1 and GABAB2 subunits have been detected in peripheral tissue, but their distribution does not always appear to be coincident, for example, in the uterus and spleen. In general, there appears to be a paucity of GABAB2, suggesting that another protein may form a dimer with GABAB1 if the receptor is to function as described elsewhere. There is also a differential distribution of the GABAB1 isoforms such that GABAB1a is in, for example, the adrenals and prostate of the rat, whereas GABAB1b is the form present in the kidney. The distribution of GABAB sites in the mammalian brain is quite wide, although there are regional variations. Receptor autoradiography has shown that the highest densities of the receptor are in the thalamic nuclei, cerebellum (molecular layer), cerebral cortex, interpeduncular nucleus, and dorsal horn of the spinal cord. Moderate to low levels are present elsewhere, but this does not necessarily reflect the physiological importance of the receptor in those regions. For example, in the hippocampus the significance of GABAB receptors in neural transmission may not be matched by the overall density of receptors. The distribution patterns of the individual receptor subunits, as determined by immunocytochemistry, generally match each other, supporting the concept of heterodimer receptors. However, in the caudate putamen GABAB2 is not detectable even though GABAB1 and the native receptor are present, which could support the notion of another, as yet undefined, protein subunit.
Physiological Significance The physiological relevance of the GABAB receptor was first established within the hippocampus, where it was shown to be responsible for generating the late hyperpolarization associated with orthodromic synaptic transmission in Ammon’s horn. Subsequent studies provided evidence for the same pattern of activity within other brain regions, such as the lateral geniculate nucleus.
The mechanism underlying this neuronal hyperpolarization is an increase in membrane Kþ conductance mediated by activation of postsynaptic GABAB receptors. However, this is not the only mechanism through which GABAB activation occurs. The predominant location of the receptors appears to be presynaptic, where they function as autoreceptors and heteroreceptors to limit the release of a variety of neurotransmitters. A reduction in membrane Ca2þ conductance is produced by activation of GABAB sites, which reduces the cytosolic concentration of Ca2þ to inhibit transmitter release. Whereas the physiological significance of autoreceptors seems straightforward because of the local availability of neurotransmitter, the role of heteroreceptors on non-GABAergic terminals is not so obvious because evidence for axo-axonic contacts at nerve terminals is lacking. The only substantiated evidence is at primary afferent terminals in the spinal cord, where GABA interneurons innervate the terminal regions of the primary afferent fibers. However, despite the apparent lack of innervation, there is strong evidence for GABA acting in a ‘paracrine’ manner to activate GABAB heteroreceptors. GABA released from GABAergic terminals ‘washes over’ onto adjacent terminals where GABAB sites are present. The estimated concentration of GABA in the cleft, following synaptic release, is approximately 0.01 M, and because the affinity of GABA for heteroreceptors is approximately 0.000 000 01 M, sufficient GABA should be available to interact with sites in close proximity. Heteroreceptors are therefore of physiological as well as pharmacological importance.
Effector Mechanisms As indicated previously, more than one signaling system mediates the response to GABAB receptor activation. Either an increase in Kþ or a reduction in Ca2þ membrane conductance provides the neuronal response, and both of these events are mediated by G-proteins that are members of the pertussis toxinsensitive family Gia/Goa. However, presynaptically mediated events, which are generally associated with reduced Ca2þ conductance, appear to be less sensitive to pertussis toxin. The predominant calcium channel linked to GABAB sites appears to be the ‘N’ type, although ‘P’ and ‘Q’ type channels are also implicated. Multiple Kþ channel types seem to be associated with postsynaptic GABAB receptors. The third signaling system associated with GABAB sites is adenylyl cyclase, which is normally inhibited by receptor activation but if the enzyme has been activated by Gs-coupled receptor agonists such as isoprenaline, the b-adrenoceptor ligand, GABAB
GABAB Receptors: Molecular Biology and Pharmacology 363
receptor activation enhances the formation of cAMP above the level achieved with isoprenaline alone. This observation prompted a search for receptor subtypes by comparing the abilities of different GABAB receptor agonists to inhibit or enhance cAMP production. However, no clear separation has been established.
GABAB Receptor Ligands Agonists
One of the original criteria for establishing the presence of GABAB receptors was to show that b-[4-chlorophenyl] GABA (baclofen) was a stereospecific agonist. Subsequently, other agonists, such as 3-aminopropylphosphinic acid (3-APPA) and 3-aminopropyl-methylphosphinic acid (3-APMPA), emerged which were approximately tenfold more potent than R-()-baclofen, the active isomer, at GABAB receptor binding sites (Figure 2). In vivo studies have also indicated that 3-APMPA has greater brain penetration than either baclofen or 3-APPA. The agonist CGP44532, which has an affinity comparable to that of () baclofen for GABAB receptors (Figure 2), is reported to be selective for GABAB autoreceptors.
Structures and comparative activities of GABAB receptor ligands Comparative receptor binding in rat brain GABAB receptor agonists membranes (IC50s) R-(–)-baclofen
32 nM
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Receptor activation within the mammalian brain can produce a variety of effects as a consequence of
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Antagonists
The first selective GABAB antagonists to be described were phaclofen, saclofen, and 2-hydroxy saclofen (Figure 2). These have only low affinity for the receptor, with pKi values of 4–5. However, phaclofen was used to great effect in obtaining the first evidence in 1988 for the physiological role of GABAB receptors in synaptic transmission within the rat hippocampus. Subsequent studies led to the introduction of a compound that was able to cross the blood–brain barrier (CGP 35348) and the first orally active agent (CGP 36742). However, both of these compounds have low affinities, as does the chemically distinct antagonist SCH50911 (Figure 2). A major breakthrough was obtained when 3,4-dichlorobenzyl or 3-carboxybenzyl substituents were attached to the existing molecules to produce a variety of compounds with affinities in the low nanomolar range, such as CGP 55845 and many others, which in all cases contain a phosphinic acid moiety. Ultimately, such antagonists, when radiolabeled with 125I, provided photoaffinity ligands suitable for the successful elucidation of the structure of the GABAB receptor.
5 nM
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-
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Figure 2 Selection of GABAB receptor ligands and their comparative potencies at the native receptor in rat brain cerebral cortex membranes. Note the radical increase in potency of the examples of the dichlorobenzyl-substituted antagonists, CGP52432 and CGP55845.
364 GABAB Receptors: Molecular Biology and Pharmacology Table 1 GABAB receptor activation Effect
Site of action
Antinociception Antitussive action Drug addiction suppression Enhanced feeding Fat intake reduction Gastrin/gastric acid secretion altered Generation/exacerbation of absence epilepsy Inhibition of neurotransmitter release Inhibition of cognitive function Insulin/glucagon release Modulation of long-term potentiation Muscle relaxation Smooth muscle relaxation Smooth muscle contraction
Spinal cord, thalamus Cough center in medulla CNS–mesolimbic system Higher centers Higher centers Vagal center
Neuronal hyperpolarization Neutrophil chemotaxis enhanced Respiratory depression Suppression of CRH/MSH release Suppression of panic behavior Vasopressor action
Thalamus/somatosensory cortex CNS and peripheral nerve terminals Higher centers Pancreas CNS, hippocampus
allow for poor brain penetration. This problem has been addressed by the introduction of intrathecal administration using an indwelling pump inserted into the peritoneal cavity. Administration in this way, directly to the site of action within the spinal cord, requires only local concentrations that are too low to appear in the systemic circulation, thus avoiding the production of adverse effects. The reduced extracellular level of the agonist also decreases the possibility of receptor desensitization. The response to systemic administration of baclofen is reduced after chronic treatment, but this does not seem to be the case after chronic intrathecal infusion. Nociception
Spinal cord Lung, bladder, intestine Uterus, oviduct, gall bladder CNS (numerous locations) Leukocytes Brain stem Pituitary Dorsal periaqueductal gray Nucleus tractus solitarius
inhibition of transmitter release and/or postsynaptic neuronal hyperpolarization. Information derived from knockout mice, which exhibit hyperalgesia, seizures, hyperlocomotion, impaired learning, loss of responses to baclofen, and lack of GABAB binding sites throughout the brain, together with the actions of GABAB agonists and antagonists have provided the basis for defining the physiological role of the receptor as well as indicating the potential therapeutic benefits of receptor ligands. Some of the actions of baclofen (in vitro and in vivo) are shown in Table 1, and predominant among the in vivo effects are the muscle relaxant, antinociceptive, anti-drug-craving effects and reduction in cognitive behavior.
Potential Therapeutic Uses of GABAB Receptor Ligands Skeletal Muscle Relaxation
The centrally mediated muscle relaxant properties of baclofen make it the drug of choice for spasticity associated with cerebral palsy, multiple sclerosis, stiff-man syndrome, and tetanus. However, the side effects produced by this drug, which include seizures, nausea, drowsiness, dizziness, hypotension, muscle weakness, hallucinations, and mental confusion, are often poorly tolerated by patients. These are, in part, due to the need for high doses to be administered to
Baclofen and other GABAB receptor agonists have antinociceptive activity in acute pain models, such as the tail flick and hot plate tests in rodents. This occurs at doses below the threshold for muscle relaxation, enabling impairment of locomotor activity to be excluded as a confounding reason for the effect. This antinociceptive action stems, in part, from a reduction in the release of nociceptive transmitter from primary afferent fibers within the dorsal horn of the spinal cord. In addition, action within higher centers, particularly the thalamus, also contributes. In spinal cord slices from control rats, the application of GABAB receptor antagonists produces little or no increase in the evoked release of transmitter, but if the antagonist is applied to a cord slice from a monoarthritic rat, an evoked release of primary afferent transmitter occurs. If the antagonist is administered in vivo to rats with the same lesion, significant hyperalgesia occurs. This contrasts markedly with a lack of effect in control rats. These results suggest that an increase in GABAB innervation to primary afferent terminals occurs during chronic inflammation, and this acts as a pathological antinociceptive process to decrease the enhanced sensory input. The use of baclofen as an analgesic in humans has been very limited, presumably due, in part, to rapid tolerance and adverse effects following systemic administration. As mentioned previously, the spinal cord is not the only site where GABAB agonists exert their antinociceptive effect. Focal injections into the ventrobasal complex within the thalamus can suppress nociceptive processing in chronic inflammation. It has also been observed that the antinociceptive effect of the GABA uptake inhibitor tiagabine in rodents, which can be attributed to GABAB receptor activation, is associated with an increase in the extracellular concentration of GABA within the thalamus. In contrast to models of inflammatory pain, the induction of neuropathic pain in rodents does not
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produce an increase in GABA levels within the spinal cord. However, baclofen can produce an antinociceptive effect in models of chronic neuropathy. Support for a role for GABAB receptors in pain mechanisms also derives from developmental knockout studies in mice. In these mice, functional GABAB receptors are not formed because the mice are deficient in either of the individual GABAB1 or GABAB2 subunits. In both forms of null mutant mice, hyperalgesia was exhibited in acute nociceptive tests, suggesting that functional heteromeric GABAB receptors are required to maintain pain thresholds. Cognitive Function
GABAB receptor agonists suppress cognitive behavior in animals, and this action is reversed by GABAB antagonists. Moreover, basal cognitive activity can even be enhanced by GABAB antagonists without prior suppression by an agonist. This raises the possibility that GABAB antagonists might provide a novel opportunity to treat cognitive impairment in humans, and this hypothesis is being tested in clinical trials. Evidence suggests that the site of action of GABAB antagonists in relation to cognition may be the hippocampus, where an increase in long-term potentiation (LTP) has been implicated but the nature of this modification appears to depend on the frequency of stimulation employed to produce LTP. Depression and Anxiety
A role for GABAB receptors in functional depression was first proposed more than 20 years ago. An upregulation in GABAB binding sites occurs in rat frontal cortex after chronic administration of a variety of antidepressant drugs, and although these findings were disputed, there is little doubt that GABAB mechanisms can be associated with depression. Antagonism of GABAB receptors produces a reversal of depressantlike behavior in animal models such as the rodent forced swim test and learned helplessness models. It has been observed that mice lacking GABAB1 or GABAB2 receptor subunits exhibit antidepressant-like behavior but they appear to be more anxious. Therefore, GABAB receptor activation appears to produce anxiolytic activity, whereas a loss or blockade of GABAB receptor function produces antidepressantlike effects. In support of this distinction, GABAB receptor activation in the dorsal periaqueductal gray of rats impairs one-way escape in the elevated T-maze test, which is consistent with an anxiolytic effect. Drug Addiction
Therapeutic treatment of dependence on drugs of abuse is still inadequate and is therefore a major clinical
goal. A number of receptor systems appear to provide potential targets, including dopamine and glutamate receptors, but GABAB receptor activation may provide an additional approach. Baclofen was initially shown to reduce the reinforcing effects of cocaine in rats, but it soon became clear that other drugs of addiction, including nicotine, morphine-related agents, and ethanol, were also sensitive to GABAB agonists, whereas food reinforcement was unaffected. The finding that baclofen reduces craving for a variety of unrelated addictive substances, including heroin, alcohol, and nicotine, suggests that there may be an underlying common mechanism for the GABAB agonist in all cases. The reward center within the mesolimbic system, possibly the ventral tegmental area, would provide the focus for this action where control of the release/action of dopamine is implicated. Raising endogenous GABA levels within the mesolimbic system should have the same effect as administering a GABAB agonist. Thus, if vigabatrin, an inhibitor of GABA metabolism, or the GABA uptake inhibitor NO-711 are administered centrally in rats, they both attenuate heroin and cocaine selfadministration and prevent cocaine-induced increases in dopamine in this brain region. Clinical data indicate that baclofen is also effective against cocaine and alcohol craving in humans. However, the administration of baclofen can produce adverse effects as well as muscle relaxation, and these effects may detract from any potential benefits. Therefore, what might provide an alternative approach? The presence of a positive allosteric modulatory site on the GABAB2 subunit could provide the answer.
Allosteric Modulation of GABAB Receptors The location of the modulator allosteric site appears to be the heptahelical domain of the GABAB2 subunit. Although the GABAB1 subunit has the agonist binding domain, it does not have an allosteric site, whereas the converse appears to be true for GABAB2. Two compounds, CGP7930 and GS39783 (Figure 2), were originally reported to be positive modulators. Neither has any direct agonist activity, but both accentuate the effects of GABA and baclofen. These compounds have been examined in rat models of addiction and both modulators reduced the self-administration of cocaine.
Seizure Generation in Absence Epilepsy Absence seizures have a characteristic EEG waveform of a 3-Hz spike and wave which stem from discharges in the thalamic nuclei. It is believed that the thalamus
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is the site from which these discharges originate, and although an intact thalamocortical network is necessary for generating spike and wave discharges, the origin of these discharges appears to lie outside the thalamus. The site of origin appears to be within the perioral region of the somatosensory cortex. This discharge spreads rapidly across the cortex and initiates a corticothalamic cascade. Injection of a GABAB agonist into the ventrobasal thalamus or reticular nucleus of a rat exacerbates this activity. By contrast, injection of a GABAB antagonist into the same regions suppresses the spike and wave discharges. If the GABAB antagonist is administered systemically, the same effect occurs. This might indicate that interference with the GABAergic innervation from the reticular nucleus disrupts the thalamocortical loop which generates the spike and wave activity. However, microinjection of the same selective antagonist into the somatosensory cortex can also suppress the seizure activity, indicating the involvement of GABABergic mechanisms also at this level. In patients with existing absence seizures, GABAB receptor agonists would likely enhance seizure activity, and any increase in GABA concentration in the vicinity of the thalamus would be expected to enhance seizure activity. Thus, for example, vigabatrin and tiagabine would be, and are, contraindicated in such individuals. The mechanism(s) underlying seizure exacerbation by GABAB agonists is unclear, but involvement of transient Ca2þ T currents seems possible. It has been suggested that g-hydroxybutyrate (GHB), which can produce absence-like seizures, is a weak GABAB receptor agonist. If GHB mimics the effect of the endogenous agonist, GABA, at GABAB receptors, then GABAB receptor antagonists would be expected to block the spike and wave discharges produced by GHB, which they do.
Conclusions The GABAB receptor system is of predominant, but not exclusive, importance within the central nervous system, and it may provide an important drug target in a variety of central nervous system disorders. It is a member of group 3 G-protein-coupled receptors and exists as a heterodimer with various isoforms comprising the GABAB1 subunit. However, lack of demonstrable functional subtyping of the receptor has limited exploitation of any pharmacological specificity. The only receptor agonist in current clinical use is baclofen, which has many limitations, including poor brain penetration. To date, little has emerged which is an improvement over baclofen. However, the discovery of allosteric modulators of the GABAB system may provide the possibility of producing
compounds that will readily gain access to the central nervous system while facilitating receptor function. See also: GABA Synthesis and Metabolism; GABAA
Receptor Synaptic Functions; GABAA Receptors: Molecular Biology, Cell Biology and Pharmacology; GABAB Receptor Function.
Further Reading Bettler B, Kaupman K, Mosbacher J, and Gassmann M (2004) Molecular structure and physiological functions of GABAB receptors. Physiological Reviews 84: 835–867. Binet V, Brajon C, Le Corre L, Acher F, and Pin J-P (2004) The heptahelical domain of GABAB2 is activated directly by CGP7930, a positive allosteric modulator of the GABAB receptor. Journal of Biological Chemistry 279: 29085–29091. Bowery NG, Bettler B, Froestl W, et al. (2002) International Union of Pharmacology. XXXIII: Mammalian g-aminobutyric acidB receptors: Structure and function. Pharmacological Reviews 54(2): 247–264. Cryan JF and Kaupmann K (2005) Don’t worry ‘B’ happy! A role for GABAB receptors in anxiety and depression. Trends in Pharmacological Sciences 26(1): 36–43. Froestl W and Mickel SW (1997) Chemistry of GABAB modulators. In: Enna SJ and Bowery NG (eds.) The GABA Receptors, pp. 271–296. Totowa, NJ: Humana Press. Gassmann M, Shaban H, Vigot R, et al. (2004) Redistribution of GABAB(1) protein and atypical GABAB responses in GABAB(2)deficient mice. Journal of Neuroscience 24: 6086–6097. Ipponi A, Lamberti C, Medica A, Bartolini A, and MalmbergAiello P (1999) Tiagabine antinociception in rodents depends on GABAB receptor activation: Parallel antinociception testing and medial thalamus GABA microdialysis. European Journal of Pharmacology 368: 205–211. Kaupmann K, Malitschek B, Schuler V, et al. (1998) GABAB receptor subtypes assemble into functional heteromeric complexes. Nature 396: 683–687. Lehmann A, Antonsson M, Bremner-Danielsen M, Fla¨rdh M, Hansson-Branden L, and Ka¨rrberg L (1999) Activation of the GABAB receptor inhibits transient lower esophageal sphincter relaxations in dogs. Gastroenterology 117: 1147–1154. Lloyd KG, Thuret F, and Pilc A (1985) Upregulation of gammaaminobutyric acid (GABAB) binding sites in rat frontal cortex: A common action of repeated administration of different classes of antidepressant and electroshock. Journal of Pharmacology and Experimental Therapeutics 235: 191–199. Malcangio M and Bowery NG (1994) Spinal cord SP release and hyperalgesia in monoarthritic rats: Involvement of the GABAB receptor system. British Journal of Pharmacology 113: 1561–1566. Malcangio M and Bowery NG (1996) GABA and its receptors in the spinal cord. Trends in Pharmacological Sciences 17: 457–462. Manning J-P, Richards DA, and Bowery NG (2003) Pharmacology of absence epilepsy. Trends in Pharmacological Sciences 24: 542–549. Prosser HM, Gill CH, Hirst WD, et al. (2001) Epileptogenesis and enhanced prepulse inhibition in GABAB1-deficient mice. Molecular and Cellular Neuroscience 17: 1059–1070. Robbins MJ, Calver AR, Fillipov AK, Couve A, Moss SJ, and Pangalos MN (2001) The GABAB2 subunit is essential for G protein coupling of the GABAB receptor heterodimer. Journal of Neuroscience 21: 8043–8052.
GABAB Receptors: Molecular Biology and Pharmacology 367 Schuler V, Luscher C, Blanchet C, et al. (2001) Epilepsy, hyperalgesia, impaired memory, and loss of pre- and post-synaptic GABA B responses in mice lacking GABAB1. Neuron 31: 47–58. Slattery DA, Markou A, Froestl W, and Cryan JF (2005) The GABAB receptor-positive modulator GS39783 and the GABAB receptor agonist baclofen attenuate the reward-facilitating effects of cocaine: Intracranial self-stimulation studies in the rat. Neuropsychopharmacology 30: 2065–2072. Thuault SJ, Brown JT, Sheardown SA, et al. (2004) The GABAB2 subunit is critical for the trafficking and function of native GABAB receptors. Biochemical Pharmacology 68(8): 1655–1666.
Urwyler S, Mosbacher J, Lingenhoehl K, et al. (2001) Positive allosteric modulation of native and recombinant g-aminobutyric acidB receptors by 2,6-di-tert-butyl-4-(3-hydroxy-2,2dimethyl-propyl)-phenol (CGP7930) and its aldehyde analog CGP13501. Molecular Pharmacology 60: 963–971. Urwyler S, Pozza MF, Lingenhoehl K, et al. (2003) GS39783 (N,N0 dicyclopentyl-2-methylsulfanyl-5-nitro-pyrimidine-4,6-diamine) and structurally related compounds: Novel allosteric enhancers of g-aminobutyric acidB receptor function. Journal of Pharmacology and Experimental Therapeutics 307: 322–330. White JH, Wise A, Main M, et al. (1998) Heterodimerization is required for the formation of a functional GABAB receptor. Nature 396: 679–682.
GABAB Receptor Function A J Doherty, G L Collingridge, and S M Fitzjohn, University of Bristol, Bristol, UK ã 2009 Elsevier Ltd. All rights reserved.
Postsynaptic g-Aminobutyric Acid B-Mediated Inhibitory Postsynaptic Potential Activation of postsynaptic g-aminobutyric acid B (GABAB) receptors produces a hyperpolarization of neuronal membrane potential, accompanied by a decrease in cell input resistance. Synaptic activation of GABA receptors elicits a biphasic inhibitory postsynaptic potential (IPSP), of which the early component is sensitive to GABAA receptor blockade, and inhibitors of GABAB receptors block the slower, late component of the IPSP (Figure 1). This late component typically has a time to peak of 50–250 ms and decay time of 100–500 ms. The difference in time course of these two components is due to the different nature of the two GABA receptors. Thus, GABAA receptors consist of a ligand-gated Cl channel with fast kinetics, whereas the GABAB component is via G-protein activation of a Kþ conductance. The GABAB IPSP is mediated by bg G-protein subunits activating inwardly rectifying Kþ (Kir) channels, with efflux of Kþ causing membrane hyperpolarization (Figure 1). In the hippocampus, GABAB receptors couple to activation of the Kir3.2 subunit, and the GABAB receptor-mediated hyperpolarization of membrane potential is absent in Kir3.2 knockout mice. However, evidence suggests that a small component of the slow IPSP may, in some cases, also be mediated by activation of other Kþ channels, such as small-conductance Ca2þ-activated Kþ (SK) channels. In hippocampal pyramidal neurons, there is a strong co-localization of GABAB receptors and the Kir3.2 subunit in dendritic spines, with both proteins being enriched around glutamate synapses. In the main dendritic shaft, however, the two proteins show a much more segregated distribution. Kir channels are coupled to G-protein-coupled receptors by membrane diffusion of the bg subunit, with effective coupling occurring if the two proteins are located within a 500 nm distance. Thus, it is likely that GABABmediated activation of Kir and the associated membrane hyperpolarization are functionally much more important in dendritic spines than in the main dendritic shaft. GABAergic presynaptic fibers generally synapse onto the main dendritic shaft of hippocampal
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pyramidal neurons, suggesting that the activation of Kir by postsynaptic GABAB receptors is initiated by GABA spillover from nearby GABA terminals. The slow time course of the GABAB IPSP and the localization of GABAB receptors in close vicinity to glutamate synapses have important implications for GABAB receptor function. Being closely located near the excitatory input to neurons, the GABAB-mediated response limits postsynaptic excitation. One function of the GABAB receptor-mediated increase in Kþ conductance and membrane hyperpolarization is to reduce activity of the N-methyl-D-aspartate (NMDA) subtype of ionotropic glutamate receptor (NMDAR). The NMDAR has slow kinetics and also displays a voltage-dependent block of the receptor channel by Mg2þ ions at membrane potentials more negative than 35 mV. As GABA is released from feed-forward interneurons, which receive their synaptic drive from the same glutamatergic fibers that form excitatory synapses onto hippocampal pyramidal neurons, the dual GABAA/GABAB IPSP is perfectly timed to prevent NMDAR activation during basal low-frequency activity. Activation of NMDARs is a key initiator of multiple forms of synaptic plasticity, which means that the GABAB receptor is an important factor in controlling the induction of synaptic plasticity.
Presynaptic Regulation of Neurotransmitter Release At presynaptic sites, GABAB receptors inhibit neurotransmitter release at both GABA synapses (acting as autoreceptors) and other neurotransmitter synapses (acting as heteroreceptors, e.g., at glutamate and noradrenaline synapses). This regulation is predominantly caused by inhibition of N- and P/Q-type voltage-gated Ca2þ channels. The inhibition is mediated by the bg subunits of GABAB receptorcoupled G-proteins. Inhibition of transmitter release brought about by activation of presynaptic Kþ channels may also occur. Presynaptic GABAB receptors produce a powerful effect on transmitter release, being capable of inhibiting release by more than 90%. Autoreceptors
GABAB receptors located on presynaptic GABA terminals act to decrease GABA release when two or more stimuli are delivered to presynaptic axons, leading to paired-pulse depression (PPD) of postsynaptic GABAA receptor-mediated responses (Figure 2). PPD occurs when pairs of stimuli are delivered in the frequency range of 0.1–50 Hz, with maximum PPD
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Excitatory terminal (glutamatergic) AMPAR NMDAR Inhibitory interneuron (GABAergic)
GABAAR
GABABR
Ca2+ K+
Cl−
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Figure 1 Postsynaptic g-aminobutyric acid B (GABAB) receptors produce a slow postsynaptic membrane hyperpolarization. Schematic diagram showing membrane potential changes produced by synaptic stimulation. Release of glutamate from excitatory synapses activates postsynaptic a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs), producing a fast excitatory postsynaptic potential (EPSP). Activation of GABAergic interneurons releases GABA that activates postsynaptic GABAA receptors, leading to Cl efflux and a fast inhibitory postsynaptic potential (IPSP). Spillover of GABA from the synapse activates GABAB receptors, where bg G-protein subunits activate nearby inwardly reactivating Kþ channels, producing a slow IPSP. This combined IPSP curtails the AMPAR-mediated EPSP and prevents activation of N-methyl-D-aspartate receptors (NMDARs). The traces show the change in membrane potential produced by activation of each receptor type. The color of the lines in these traces corresponds to the color of the receptor in the main diagram. The composite EPSP recorded in the postsynaptic cell is also shown.
seen at around 5–10 Hz. Depression of postsynaptic GABAA responses also occurs during trains of stimuli due to a depression of GABA release, although the effect of presynaptic GABAB inhibition decreases as the number of stimuli in the train increases or the frequency of the train increases (beyond around 10 Hz for a train of ten stimuli). With sufficient numbers of stimuli, the postsynaptic GABAA response undergoes activity-dependent facilitation and converts into a depolarizing response. Role of GABAB Autoreceptors in the Induction of NMDAR-Dependent Long-Term Potentiation
The effect of decreasing postsynaptic GABA-mediated inhibition during short trains of activity is important for the induction of long-term potentiation (LTP) of excitatory glutamatergic synaptic transmission, which has been particularly well studied in the CA1 region of the hippocampus. LTP is a well-studied form of synaptic plasticity that occurs in many brain regions and is considered a synaptic model of learning. Many, but not all, forms of LTP rely on the activation of NMDARs to act as a trigger for LTP induction. As discussed above, the postsynaptic GABAB receptormediated IPSP acts to inhibit the induction of LTP during basal, low-frequency synaptic activity
by preventing activation of the NMDAR. However, during short, high-frequency trains of activity, GABA release from presynaptic terminals is reduced by feedback of GABA onto presynaptic autoreceptors. The result is a decrease in postsynaptic GABA inhibition which, coupled to release from glutamate terminals, leads to a more persistent depolarization of the postsynaptic cell, activation of NMDARs, calcium influx, and the subsequent induction of LTP (Figure 2). Multiple protocols exist for inducing LTP in experimental systems, and the role of GABAB receptors in LTP induction varies depending on the protocol used. A typical induction protocol is the delivery of brief high-frequency bursts (e.g., two or more trains of four shocks at 100 Hz) with an interburst frequency of around 5 Hz (i.e., an interburst interval of 200 ms). This is termed a theta burst stimulus train as the 5 Hz pattern of activity mimics the physiological activity that occurs during the theta rhythm, a frequency of activation observed in the EEG when animals are exploring their environment. Such theta burst trains are thus considered potential physiological induction protocols for LTP induction. This frequency corresponds to the near-maximal effect of presynaptic GABAB autoreceptors, and activity at GABAB receptors is essential for induction of LTP. Thus,
370 GABAB Receptor Function
Excitatory terminal (glutamatergic)
AMPAR NMDAR
Inhibitory interneuron (GABAergic)
GABAAR
GABABR
Ca2+ EPSP
a
Excitatory terminal (glutamatergic) AMPAR NMDAR Inhibitory interneuron (GABAergic)
GABAAR GABABR
Ca2+
EPSP
b Figure 2 Activation of presynaptic g-aminobutyric acid B (GABAB) autoreceptors contributes to the induction of N-methyl-D-aspartate (NMDA) receptor-dependent LTP. (a) In response to a single stimulus, activation of postsynaptic GABA receptors produces a membrane hyperpolarization that curtails the excitatory postsynaptic potential (EPSP) and prevents NMDA receptor (NMDAR) activation, which remain blocked by Mg2þ ions (.). (b) In response to a train of stimuli, GABA activates presynaptic GABAB autoreceptors which decrease Ca2þ entry into the presynaptic bouton and thus decrease GABA release. This reduces the postsynaptic GABA-mediated inhibitory postsynaptic potential, which prolongs the AMPA receptor (AMPAR)-mediated membrane depolarization leading to expulsion of Mg2þ ions from the NMDAR and subsequent Ca2þ entry into the postsynaptic neuron. The inset traces show the membrane potential change produced by each constituent receptor and the composite EPSP recorded form the postsynaptic neuron.
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GABAB receptor antagonists block the induction of LTP by theta burst stimuli. Likewise, LTP induced by the delivery of a single priming stimulus followed approximately 200 ms later by a single burst (which could comprise as few as two stimuli, though typically four are used) is blocked by GABAB receptor antagonists. In this case the priming stimulus allows activation of the presynaptic GABAB autoreceptors such that during the subsequent burst of stimuli, GABA release is depressed sufficiently to allow NMDA receptor activation. LTP may also be induced by stronger stimulus trains, typically 100 stimuli delivered at a frequency of 100 Hz. Under these conditions, LTP induction is independent of GABAB receptor activity due to the conversion of the postsynaptic GABAA response into a depolarizing response. Depression of Vesicle Recruitment
At the excitatory calyx of Held synapse in the brain stem, activation of presynaptic GABAB receptors by GABA spillover from nearby inhibitory synapses elicits heterosynaptic depression of glutamate by depression of Ca2þ influx, as seen at other synapses in the brain. However, presynaptic GABAB receptor activation has an additional effect at this synapse in that it slows vesicle recruitment after strong presynaptic stimulation. This effect is mediated by G-protein inhibition of adenylyl cyclase and thus a reduction in cyclic adenosine monophosphate (cAMP) production. Vesicle priming at this synapse is enhanced by cAMP and Ca2þ/calmodulin, and thus a reduction in cAMP levels slows the recruitment of vesicles ready for release.
LTP of Postsynaptic GABAB Responses In addition to being involved in the induction of LTP of excitatory synaptic transmission, postsynaptic GABAB receptors may also undergo LTP. Thus in the hippocampus, NMDAR activation can lead to an enhancement of the slow, GABAB receptor-mediated IPSC in CA1 hippocampal pyramidal neurons. This form of LTP requires influx of Ca2þ through the NMDA receptor and the subsequent activation of Ca2þ/calmodulin dependent protein kinase II, although the precise mechanism of the enhancement of GABAB receptor function is unclear. It remains to be seen whether other forms of plasticity exist to modulate GABAB receptor function.
Coupling of GABAB and Metabotropic Glutamate Receptors In the cerebellar cortex, GABAB receptors are expressed in Purkinje cells (PCs) at the extrasynaptic site of excitatory input from parallel fibers. This extrasynaptic localization is also seen for the metabotropic glutamate
receptor subtype 1 (mGluR1), stimulation of which produces a slow excitatory postsynaptic current and mobilization of Ca2þ from intracellular stores. Activation of postsynaptic GABAB receptors by agonists or synaptic GABA release produces an enhancement of mGluR1-mediated synaptic currents and Ca2þ mobilization, an effect that is dependent on Gi/o G-protein signaling, activation of phospholipase C, and release of Ca2þ from intracellular stores. It is interesting that facilitation of mGluR1 in PCs by GABAB receptors can also occur independently of classical receptor activation. In the absence of GABAB receptor agonists, increases in extracellular Ca2þ elicit a constitutive increase in the sensitivity of mGluR1 to glutamate, facilitating mGluR1 effects. This effect of extracellular Ca2þ is independent of Gi/o activity and thus differs from the enhancement described above. GABAB and mGluR1 receptors appear to exist in a protein complex in PCs, and thus this enhancement may be due to changes in protein complex structure rather than to G-protein signaling.
Therapeutic Potential of GABAB Receptor Ligands Genetic deletion of either of the two GABAB receptor subunits or pharmacological blockade of native GABAB receptors produces spontaneous epileptic seizures, demonstrating the important role that GABAB receptors have in preventing overexcitation of neuronal networks and maintaining normal brain function. As such, it is possible that dysfunction of GABAB receptor activity may be implicated in human epilepsies, and polymorphisms of the GABAB1 receptor subunit are linked to increased susceptibility to temporal lobe epilepsy. In contrast, antagonists of GABAB receptors suppress absence seizures seen in animal models, whereas agonists promote seizures in these animals, suggesting that antagonists may be of potential therapeutic use in human absence seizures. However, in these models, high doses of GABAB receptor antagonists are prone to induce convulsions, highlighting the difficulty in targeting the GABAB receptor to treat this disease, in particular because of the widespread distribution of these receptors and the lack of receptor subtypes. The GABAB receptor agonist baclofen is currently used to treat spasticity and muscle rigidity in patients with multiple sclerosis and spinal cord injury. The effect of baclofen is thought to be due to activation of presynaptic heteroreceptors in the spinal cord, reducing the release of excitatory transmitters. Reduction of transmitter release in the spinal cord (particularly glutamate and substance P) probably also contributes to the antinociceptive effect of baclofen administration, although additional actions in the
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brain, such as in the rostral agranular insular cortex, likely also contribute to it. GABAB receptor subunit knockout animals display heightened sensitivity to painful stimuli such as heat, confirming the role of GABAB receptors in modulating pain pathways. A potential use of GABAB receptor agonists is in the treatment of drug addiction. Thus, agonists such as baclofen can reduce craving seen with drugs such as alcohol, cocaine, morphine, and nicotine. In addition, genetic studies suggest that there is a link between GABAB receptor variants and potential for addiction. This action of GABAB receptor activation in reducing addiction probably relates to activity in the mesolimbic dopamine system, which is part of the reward and reinforcing pathways of the brain. A further postulated use of activating GABAB receptors is to treat anxiety as GABAB receptor subunit knockout mice show increased anxiety in behavioral tests and baclofen has anxiolytic effects. See also: GABA Synthesis and Metabolism; GABAA
Receptor Synaptic Functions; GABAA Receptors: Molecular Biology, Cell Biology and Pharmacology; GABAB Receptors: Molecular Biology and Pharmacology; Long-Term Potentiation (LTP): NMDA Receptor Role; NMDA Receptor Function and Physiological Modulation.
Further Reading Bettler B, Kaupmann K, Mosbacher J, and Gasmann M (2004) Molecular structure and physiological functions of GABAB receptors. Physiological Reviews 84: 835–867. Bowery NG, Hill DR, Hudson AL, et al. (1980) ()Baclofen decreases neurotransmitter release in the mammalian CNS by an action at a novel GABA receptor. Nature 283: 92–94.
Davies CH, Davies SN, and Collingridge GL (1990) Paired-pulse depression of monosynaptic GABA-mediated inhibitory postsynaptic responses in rat hippocampus. Journal of Physiology 424: 513–531. Davies CH, Starkey SJ, Pozza MF, and Collingridge GL (1991) GABA autoreceptors regulate the induction of LTP. Nature 349: 609–611. Dutar P and Nicoll RA (1988) A physiological role for GABAB receptors in the central nervous system. Nature 332: 156–158. Hirono M, Yoshioka T, and Konishi S (2001) GABA(B) receptor activation enhances mGluR-mediated responses at cerebellar excitatory synapses. Nature Neuroscience 4: 1207–1216. Huang CS, Shi SH, Ule J, et al. (2005) Common molecular pathways mediate long-term potentiation of synaptic excitation and slow synaptic inhibition. Cell 123: 105–118. Kornau HC (2006) GABA(B) receptors and synaptic modulation. Cell Tissue Research 326: 517–533. Kulik A, Vida I, Fukazawa Y, et al. (2006) Compartment-dependent colocalization of Kir3.2-containing Kþ channels and GABAB receptors in hippocampal pyramidal cells. Journal of Neuroscience 26: 4289–4297. Newberry NR and Nicoll RA (1984) Direct hyperpolarizing action of baclofen on hippocampal pyramidal cells. Nature 308: 450–452. Sakaba T and Neher E (2003) Direct modulation of synaptic vesicle priming by GABA(B) receptor activation at a glutamatergic synapse. Nature 424: 775–778. Soltesz I, Haby M, Leresche N, and Crunelli V (1988) The GABAB antagonist phaclofen inhibits the late Kþ-dependent IPSP in cat and rat thalamic and hippocampal neurones. Brain Research 448: 351–354. Tabata T, Araishi K, Hashimoto K, et al. (2004) Ca2þ activity at GABAB receptors constitutively promotes metabotropic glutamate signaling in the absence of GABA. Proceedings of the National Academy of Sciences of the United States of America 101: 16952–16957.
Glycine Receptors: Molecular and Cell Biology C Vannier and A Triller, INSERM U789, Ecole Normale Supe´rieure, Paris, France
mediated through gephyrin, but no direct link between GABARs and gephyrin has been demonstrated.
ã 2009 Elsevier Ltd. All rights reserved.
Molecular Biology and Topology of the Glycine Receptor Introduction The amino acid neurotransmitters glycine and g-aminobutyric acid (GABA) mediate inhibition in the vertebrate mature central nervous system by activating chloride conductance. Their ionotropic receptors belong to the canonical nicotinic acetylcholine receptor superfamily mediating fast synaptic transmission. Two features of this family, also known as the Cys-loop receptors, are its members’ heteropentameric structure delineating the channel pore, and their assembly from distinct gene products or subunit splice variants that all possess a strictly conserved disulfide loop in their extracytoplasmic domain. Upon transient neurotransmitter binding, a pore opens in the pentamer that is responsible for the selective movement of ions through the plasma membrane according to their electrochemical gradient. In fully mature neurons, glycine binding to its receptor mediates inhibition by eliciting an influx of chloride ions. Whereas GABAergic transmission is widespread in the nervous system, glycine-mediated inhibition and the glycine receptor (GlyR) are predominantly encountered in the brain stem and spinal cord. In the mid-1980s, GlyR was the first CNS receptor shown, by Triller and co-workers, to accumulate at postsynaptic differentiations (PSDs), in front of presynaptic release sites. Later on, variable subcellular organizations were demonstrated for many other receptors, including GABAA receptors (GABARs), which were analyzed by electron microscopy. Inhibitory synapses are intriguing, as both glycine and GABA open chloride channels and have a similar effect on the postsynaptic membrane. In the spinal cord, as well as in the brain stem and cerebellum, these synapses are either GABAergic, glycinergic, or mixed (i.e., respond to both neurotransmitters). The respective receptors accumulate in microdomains enriched with GABAR, GlyR, or a mixture of both, and it is the identity of the presynaptic element which imposes the composition of the postsynaptic membrane. Another protein, named gephyrin, has been copurified with GlyR on an affinity column. Antisense experiments and work with knockout mice demonstrate that gephyrin is required for the synaptic localization of both GlyR and GABAR. The GABAR g2 subunit is influential in synaptic insertion, since its absence in mouse results in a reduced number of GABA receptor clusters at synapses. The g2 subunit role is
GlyR was the first neurotransmitter receptor to be isolated from the mammalian brain more than two decades ago. The purification of the native molecule was achieved in H Betz’s laboratory, using affinity chromatography and taking advantage of the highaffinity interaction (KD 1–10 nM) of the receptor with its main antagonist, the well-known convulsant plant alkaloid, strychnine. The receptor appeared to be a complex of three proteins of 48 (a), 58 (b), and 93 kDa, in various vertebrate species. Several biochemical studies of purified (or the membrane-anchored) form of GlyR have established that the integral membrane glycoproteins a and b represent the constitutive subunits of the receptor. Cross-linking techniques showed that a and b assemble to build up the channel-containing transmembrane core of GlyR. The size of the complex (250 kDa) logically suggested a pentameric assembly of the subunits, a quaternary structure well established for other Cys-loop receptors (Figure 1). Unlike the a and b subunits, the 93 kDa co-purifying protein, gephyrin, is a nonglycosylated polypeptide able to interact reversibly with the a/b pentamer. Gephyrin is a cytoplasmic extrinsic membrane protein which binds tubulin dimers in vitro in a cooperative interaction of high affinity in the nanomolar range. It can also link brain microtubules to GlyR in a copolymerization assay. This finding underpinned the important notion that gephyrin could be a physical link between microtubules and GlyR in the postsynaptic membrane, as discussed in the following sections. The Glycine Receptor as a Member of the Ligand-Gated Ionotropic Receptor Family
Analysis of the primary structures of the a and b proteins revealed the high homology of GlyR subunits, with amino acid sequence identity close to 50%. The GlyR subunit domain organization is comparable to that of other subunits of the Cys-loop receptor family. Hydropathy analysis of the mature polypeptide predicted a conserved region of four hydrophobic segments (M1–M4) able to cross the membrane bilayer at positions identical to those of transmembrane segments of acetylcholine and GABAR subunits (Figure 1(a)). GlyR subunits are polytopic type I membrane proteins. In contrast to M4, the
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374 Glycine Receptors: Molecular and Cell Biology NH2 a
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Figure 1 Structure of the glycine receptor. (a) Membrane topology of the a subunit, indicating the position of disulfide bridges, including the common Cys-loop delineated by Cys138 and Cys152 (red). Amino acid residues functionally important for agonist binding or modulation of the channel activity are shown. (b) Arrangement of a and b subunits in the mature a2b3 receptor. One a subunit is depicted in cross-section, and one b subunit is rendered transparent to show the four packed M1–M4 transmembrane regions (cylinders). The pore-forming domain M2 of the a subunit is dark brown. (c) The a/b complex viewed from the extracellular space along the fivefold symmetry axis. Shown are the N-terminal regions of the five protomers (dashed lines) the counterclockwise organization of the four transmembrane domains.
transmembrane domains M1–M3 are highly conserved among the various subunits of GlyR. All subunits possess an extended N-terminal domain, bearing potential glycosylation sites, which were detected early by immunocytochemistry in the synaptic cleft. Finally, a large region of significantly lower homology is represented by a hydrophilic loop between the M3 and M4 segments; this loop protrudes into the cytoplasm (M3–M4 loop). Thus GlyR exhibits the molecular design and transmembrane topology of the acetylcholine receptor superfamily, based on four membranespanning domains able to form a helices (M2) and/or b strands (M1, M3, M4), and is assumed to function as an allosteric protein. Our knowledge of this structure arose from extensive biochemical and electron microscopy data on the nicotinic acetylcholine receptor; these findings were recently refined by electron diffraction studies. The tertiary and quaternary structure of the soluble pentameric acetylcholine-binding protein (AChBP), which shares 20–24% and 17% sequence homology with nAChR and GlyR, respectively, has been useful in confirming or determining structure–function parameters for receptors of the family. Aside from an understanding of glycine and strychnine binding (see later), AChBP crystal structure provides a powerful model of the N-terminal domain of the Cys-loop receptor family. It shows that it is mainly a sandwich of antiparallel b sheets positioning conserved residues in order to stabilize the protomers, whereas variable residues are at their interface. The topology of the transmembrane domains (Figure 1) delineates the ion permeation pathway
away from the hydrophobic core of the phospholipid bilayer. For all Cys-loop receptors, the subunit’s a-helical M2 domain lines the central water-filled pore, while M1, M3, and M4 form the interface with the lipids and isolate M2 from a hydrophobic environment. Recent studies on the GlyR a1 subunit have challenged the four-helix model and provide evidence that whereas M2 and M4 are entirely helical, M1 and M3 also contain b strands. Assembly of GlyR
Initial studies of purified and membrane-anchored GlyR had suggested that a1 and b subunits assemble as a pentameric complex of a3b2 stoichiometry (Figures 1(b) and 1(c)). Short amino acid sequences (named assembly boxes), all located in the N-terminal extracytoplasmic domain of the b subunit and corresponding to three diverging motifs in a and b subunits, have been identified as having a role in precisely governing receptor assembly. Replacement of these motifs in the b subunit by the corresponding a1 motifs results in the loss of the subunit ratio in a/b oligomers, suggesting that different amino acid positions are determinants in the early step of subunit–subunit interaction. On the other hand, a1 and a2 co-assemble at variable subunit ratios. Importantly, these residues impose a mutually exclusive mode of assembly, either in complexes of invariant a/b stoichiometry or in homo-oligomers. A different stoichiometry (a2b3) has recently been established using affinity purification of expressed engineered tandem subunits. It confers on the b subunit a dominant role in the agonist binding properties of hetero-oligomeric a1/b GlyR, since ionic
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interactions at the a/b interface are required to stabilize glycine. Such a structure for GlyR is consistent with the concept that neurotransmitter binding in the Cys-loop family requires extracellular segments from two adjacent subunits. Molecular Diversity and Expression of Subunit Isoforms
The initial discovery of glycine receptor subtypes resulted from biochemical and immunochemical studies during rat spinal cord development. A comparison of GlyR immunoreactivities in neonatal and adult membrane extracts revealed a distinct form of GlyR predominating around birth (neonatal receptor, GlyRN) that displays a relatively low strychnine-binding affinity and only one major polypeptide band of 49 kDa. The discovery of functional a2 pentamers predominantly found during the fetal and neonatal stages supported the idea of a developmental switch from GlyRN to the adult form, GlyRA, corresponding to the formation of a1/b heteromers. This switch, taking place within 3 weeks after birth, is not as complete in other regions of the nervous system where mixtures of a1, a2, and b subunits remain throughout adulthood. One current view is that postsynaptic GlyR corresponds to a mixture of a2 pentamers and a2/b heteromers in immature neurons, while a1/b heteromers predominate in mature synapses. A greater diversity of GlyR subunits than anticipated was established and includes not only the original a1 and a2 but also the a3 and a2* subunits. A murine a4 subunit has also been identified which is absent in rat and human. These isoforms are highly homologous, with amino acid sequence identities on the order of 80%, and display a very low degree of interspecies variation (identity close to 99%). These isoforms can form glycine-gated chloride channels of comparable strychnine sensitivity, except a2*, which is more than 99% identical to human a2 but exhibits a 500-fold lower strychnine sensitivity. Further generation of diversity resides in alternative exon usage. For the a1 subunit, a variant mRNA encodes the form denoted ‘a1ins,’ a subunit containing eight additional amino acids. This sequence, inserted in the M3–M4 loop, bears a serine residue, representing a potential phosphorylation site and allowing a functional modulation of the a1 subunit. Alternative splicing was also found for the a2 and a3 subunits. The distribution of individual GlyR subunits was determined using in situ hybridization. However, transcription is not necessarily correlated with surface expression of functional receptors and the identification of the actual composition of GlyR in particular areas is not yet firmly established. The gene encoding
the a1 subunit and that encoding the a3 subunit, though to a lesser extent, are mainly transcribed in spinal cord and brain stem at later postnatal stages. The a3 subunit mRNA is present in the infralimbic system, the hippocampal complex, and the cerebellar granular layer. Expression levels of the a2 subunit, by contrast, are high in the embryonic and perinatal stages but barely detectable in the adult brain, with some expression in higher cortical regions. The a4 subunit, which is expressed at low levels in the adult but forms functional GlyRs, is restricted to the spinal cord and the sympathetic nervous system. The b subunit mRNA is more widely expressed throughout the embryonic and adult nervous system as compared to a subunit transcripts, and is found in brain loci devoid of strychnine-binding sites or GlyR immunoreactivity. The physiological significance of this widespread expression of the b subunit transcript is not understood, as so far there is no evidence that it assembles with a subunits of other receptors.
Gephyrin and Gephyrin-Binding Proteins The Gephyrin Polypeptide
Gephyrin is the best characterized GlyR-interacting protein. Biochemical characterization and heterologous expression have shown that gephyrin binds cooperatively to tubulin dimers with a high affinity, and to GlyR via an 18-amino-acid sequence in the b subunit M3–M4 cytoplasmic loop. It was therefore postulated that gephyrin is a linker of the receptor to microtubules, adapted to anchor GlyR at synapses via the cytoskeleton. Gephyrin exists as several splice variants, as indicated by the characterization of the mouse gephyrin single gene and of its exonic structure, and the cloning of several different full-length coding sequences. Gephyrin variants, differing by the presence of distinct nucleotide sequences termed cassettes, are differentially expressed in numerous tissues, including brain, heart, skeletal muscle, and kidney, liver, lung, spleen, or testis, illustrating the ubiquitous nature of gephyrin. Tertiary and Quaternary Structures of Gephyrin
The gephyrin polypeptide originated during evolution from the fusion of two genes of bacterial origin, respectively encoding the MogA and MoeA proteins involved in the biosynthesis of the molybdenum cofactor in Escherichia coli. These proteins are homologous to the gephyrin N- and C-terminal domains (G- and E-domains), respectively, flanking a 170residue unique linker region. Intriguingly, in gephyrin these domains are enzymatically functional, and
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disruption of the gephyrin gene leads to molybdoenzyme activity deficiency. These results indicate that gephyrin maintains MogA and MoeA activities in various tissues. Gephyrin tertiary and quaternary structures were determined following the crystallization of MogA and MoeA, and of gephyrin G- and E-domains. The G- and E-domains are compact structures forming stable trimers and dimers, respectively, which can be detected in crystals. If conserved in the gephyrin molecule, these oligomerization properties allow the construction of a two-dimensional hexagonal gephyrin lattice. This oligomerization pattern confers on gephyrin its function as a scaffold core protein and its ability to recruit GlyR at postsynaptic localizations. The GlyR-binding activity of gephyrin has been assigned in the E-domain to a pocket adjacent to the dimer interface.
a
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Gephyrin is the core of the inhibitory postsynaptic scaffold (Figure 2). Binding partners for gephyrin other than GlyR have recently been identified, but their exact functions in receptor postsynaptic clustering have not yet been determined. Gephyrin may also be implicated in the modulation of signaling pathways. Gephyrin could thus participate in translational controls by binding to the RAFT1 kinase (a rapamycin and FKB12 target protein). The dynein light chains 1 and 2 (Dlc1/2) were recently identified as gephyrin-binding proteins. Further, gephyrin interaction with collybistin, a GDP/GTP exchange factor for GTPases of the Rho/Rac family, or with proteins such as profilin and Mena/VASP, could be a determinant for microfilament organization. This hypothesis
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Figure 2 Molecular partners of the glycine receptor (GlyR)/gephyrin complex. Gephyrin is the only glycine receptor-interacting protein known so far. At synapses, it could form intermolecular trimers and dimers via its G- and E-domains, respectively. The localization of the receptor at synaptic loci relies on the ability of the former to interact directly with microtubules and indirectly with other proteins (collybistin and actin-binding proteins; yellow) able to modulate the dynamics of microfilaments. Until now the existence at synapses of a complex built up via simultaneous interactions involving all of these proteins has not been demonstrated. Double arrows indicate interactions disclosed by biochemical or two-hybrid strategies. A rapamycin and FKB12 target protein (RAFT1) is a translational activator which could mediate the synthesis of proteins required at synapses (Dlc2, dynein light chain 2; MoCo, molybdenum cofactor). Membrane binding sites for some partner proteins such as collybistin and profilin can be provided by phosphatidylinositol 3,4,5-trisphosphate (PIP3). Functions fulfilled by components of the proposed interaction network are shown by red arrows.
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is consistent with results supporting the role of microfilaments in the control of postsynaptic gephyrin– GlyR co-cluster density. A feature of gephyrin is the presence of sequence stretches predicted to be WW (class IV) domain interaction motifs (ELM database search). A WW domain which is encountered in dystrophin/utrophin polypeptides might mediate phosphorylation-dependent binding to gephyrin. Interestingly, dystrophin, which is co-localized with dystroglycans, is present in a subset of inhibitory GABAergic synapses in the hippocampus, where it could be involved in a link between the extracellular matrix and the cytoskeleton.
Glycine Receptor Dynamics Postsynaptic membrane domains and associated PSDs have long been viewed as stable multimolecular assemblies ensuring that a specific set of functions is performed locally. However, in recent years the notion has emerged that the postsynaptic domain is a highly dynamic protein complex adapted to the mechanisms underlying the plasticity of synaptic contacts. On the one hand, as for cellular multimolecular assemblies, postsynaptic complexes that contain GlyR correspond to the equilibrium between the exo- and endocytic delivery routes for membrane proteins. On the other hand, interactions involving cytoplasmic scaffolding proteins are pivotal in this steady-state balance that can be modified by cell differentiation or receptor activity. Activity and GlyR Stability at Synapses
Receptor stabilization and aggregation, mediated by interactions with scaffold elements, are coupled at synaptic loci. The formation of GlyR clusters retained in the postsynaptic membrane in relation with receptor activation has been studied using an activity blockade by strychnine of cultured spinal cord neurons. This blockade leads to a reversible intracellular redistribution of GlyR which disappears from synaptic loci without alteration of gephyrin. Therefore, aggregation of gephyrin and that of GlyR are governed by distinct mechanisms. GlyR redistribution results from a defect in biosynthetic routing. At the steady state, the spinal cord neurons incorporate only a minor fraction of newly synthesized GlyR in synapses. It turns out that the stability of glycinergic synapses is due to a tight control of the relative rates of receptor synthesis, synaptic anchoring, and degradation. In order to explain selective positioning of active GlyR in front of glycinergic terminals, it has been proposed that glycine, which depolarizes immature neurons, triggers a calcium influx promoting the assembly of the postsynaptic scaffold.
Ubiquitination has recently been proposed to account for the regulation of cell surface GlyR a1 subunit endocytosis and degradation, thus determining postsynaptic receptor density. This hypothesis is based on the demonstration that ubiquitination of homopentameric GlyR occurs almost exclusively at the plasma membrane rather than during intracellular transport of the receptor. Routing of Newly Synthesized GlyR to Synapses
The mechanisms of selective accumulation of receptors at synaptic sites are not fully understood. However, newly synthesized homomeric receptors spontaneously form microaggregates devoid of gephyrin in the neuronal plasma membrane. Although they can also in part remain in the extrasynaptic space, they associate with endogenous synaptic gephyrin according to a mechanism depending on the formation of the inhibitory innnervation. Interestingly, newly synthesized GlyR molecules are inserted in the plasma membrane as stable clusters which initially appear in the extrasynaptic space of the soma and dendritic proximal segments. Subsequently, they are redistributed over the dendritic tree. This occurs by diffusion of cell surface molecules, but not via exocytosis of vesicles routed to dendrites. Therefore, GlyR postsynaptic accumulation results from a diffusion/retention mechanism. This posits the crucial role of the retention signal, and in turn the anchoring role of gephyrin emerges. The complexity of the mechanisms of postsynaptic anchoring process, which relies on the exocytosis site, diffusion velocities, and on the concentration of synaptic binding sites, is illustrated by glutamate receptors of different compositions which accumulate at excitatory synapses with different apparent rates. Gephyrin associates with vesicular compartments of GlyR intracellular trafficking and is a mediator of the insertion of newly synthesized receptor molecules in the plasma membrane, a process dependent on microtubule integrity. It has been further proposed that a nonsynaptic gephyrin/GlyR complex can be recruited to microtubules by dynein for retrograde transport along neurites. The existence of intracellular gephyrin/GlyR complexes indicates a dual function in trafficking and in assembly of postsynaptic scaffolds, as for proteins of excitatory synapses. Local dendritic insertion of newly synthesized receptors is also a means of changing receptor numbers at synapses via mRNA localization. The discovery of the dendritic localization of GlyR a subunit mRNAs supported the view that local synthesis plays a role in synaptic plasticity. Remarkably, a microsecretory apparatus able to participate in the translation of membrane proteins is present in the subsynaptic cytoplasm.
378 Glycine Receptors: Molecular and Cell Biology Diffusion Properties of Cell Surface GlyR
In addition to membrane traffic, lateral diffusion within the plasma membrane has been demonstrated for GlyR. This concept has been extended to almost all excitatory and inhibitory receptors so far studied. The single-particle tracking (SPT) approach showed that receptor movements display interspersed periods of high and low diffusion rates in the extrasynaptic and synaptic membrane, respectively. Whereas periods of fast diffusion correspond to Brownian movement, those of slow mobility mainly result from transient association of GlyR with clusters of gephyrin. This association, which reduces diffusion coefficients from up to 0.5 mm2 s 1 to less than 10 2 mm2 s 1, imposes a restricted area of exploration (confinement) similar to that generated for other proteins by insertion into lipid rafts or by contact with protein fences or obstacles. The fact that the receptors are not irreversibly retained in gephyrin domains, but can also exit from them, is a key finding which sheds light on how receptor clusters are formed or modified during plastic processes. It favors the new notion that any postsynaptic cluster of gephyrin can behave as donor or/ and acceptor of GlyR. Therefore, the dynamic properties of GlyR–gephyrin interactions are also a means whereby the number of synaptic receptors can be regulated. It remains to be seen how the stabilizing scaffold molecules regulate these transitions, both in synaptogenesis and synaptic plasticity. Because of its interaction with scaffold proteins, the cytoskeleton is a component of the postsynaptic differentiation, at least during differentiation or plastic changes. Because gephyrin is thought to link GlyR to microtubules, depolymerization of the latter would disrupt GlyR clusters. Interestingly, microtubule depolymerization alters the number and density of synaptic GlyR/gephyrin clusters, but the same treatment has no effect on GABA receptor/gephyrin clusters. This discrepancy may reflect the structural heterogeneity of inhibitory synapses that likely relies on receptorspecific assemblies of cytoplasmic proteins.
Pathology – Molecular Biology of Spastic Syndromes Defects in glycinergic transmission are the cause of several neurological diseases. Some hereditary motor disorders in humans, mice, horses, and cattle result from the expression of mutated glycine receptor genes. The hallmark of these disorders is an exaggerated startle reflex in response to sudden stimuli, giving rise to rapid generalized responses in the form of hypertonia. Startle syndromes related to distinct
genetic defects in glycine-mediated neurotransmission correspond either to impairment of GlyR agonist binding function or to reduced expression of functional channels. Both mechanisms lead to inefficient glycinergic inhibition and increased muscle tone. Human Hyperexplexia
Hyperexplexia is a disorder in which unexpected sensory stimuli provoke exaggerated startle reflexes. Hereditary hyperexplexia, or Kok’s disease or familial startle hyperexplexia (STHE), is a rare autosomal dominant neurologic disorder characterized by marked and continuous, sometimes fatal, muscle rigidity in infancy which progressively evolves into transient massive muscle contractions, a reaction that persists throughout adulthood. Several missense mutations identified in the a1 subunit of GlyR can be responsible for higher glycine EC50 and/or lower channel conductance. The defective phenotype often results from mutations within, or in the vicinity of, the M2 transmembrane domain. Startle Syndromes in the Mouse
Studies of mutants in the mouse reveal that defects in two genes can cause recessive disorders with clinical and pharmacological features reminiscent of STHE. The inherited spasmodic and spastic phenotypes are autosomal diseases characterized by muscle tremor, hypertonia, and pronounced startle reactions. They are rarely lethal, with the exception of the oscillator phenotype, and during the first 2 weeks after birth homozygous mutant mice have a normal phenotype. The phenotypic similarity of spasmodic (spd) phenotype and STHE prompted the identification of the GlyR a subunit as the defective protein. A missense mutation, A52S, close to the protein N-terminus, decreases glycine sensitivity without changing the affinity for strychnine, in agreement with the normal binding of the antagonist in the spd spinal cord. The mechanism of action of this mutation is unknown, since the region of the mutation is not involved in ligand binding, receptor assembly, or gating. The phenotype of the mutant mouse ‘oscillator’ (spdot) is characterized by a fine motor tremor and muscle spasms. Oscillator is allelic with spasmodic but corresponds to a microdeletion coincident with the cytoplasmic extremity of the M3 transmembrane segment. A translational frameshift generates a truncated form of the a1 subunit lacking the M3–M4 loop and the M4 domain. The highly penetrant recessive ‘spastic’ mutation is associated with a dramatic reduction in the number of expressed GlyRs in the adult spinal cord and brain stem, but residual receptors exhibit normal subunit
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structure and function. The delayed onset of the spasticity coincides with the developmental switch of GlyR, suggesting a selective lack of the adult form, in the absence of transcriptional alteration of the a1 subunit. A mutation affects a b subunit gene allele, which results from the intronic insertion (intron 5) of a LINE-1 transposable element. As a consequence, fulllength b mRNAs are barely detectable, and truncated b subunits are generated by creation of premature stop codons. The general loss of surface receptors in ‘spastic’ animals is consistent with the role of the b subunit, and results from the lack of a gephyrin-binding site in (hetero-)oligomers. However, it should be noted that a reduction in transport to, and insertion in, specific membrane areas may alternatively arise from an altered quaternary structure that does not allow exit from the endoplasmic reticulum and leads to degradation of the misassembled receptor.
Concluding Remarks GlyR, which plays essential roles in inhibitory neurotransmission via a heterogeneous composition, has been the center of numerous studies during recent years. Better understanding has been gained at various levels, from structure–function relationships to the cell biology of synapses, including the associated postsynaptic density built up on gephyrin. Remarkably, pioneering work exploiting GlyR as a model molecule has helped develop new concepts concerning not only the biology of neurotransmitter receptors, but also the characterization of the dynamic equilibrium of GlyR-enriched postsynaptic membrane domains; such equilibrium accounts for the strength of synapses via control of their receptor number. GlyR and gephyrin are central to the assembly and activity of a nanomachine, which exploits the diffusion properties of the receptors in order to participate in the excitation–inhibition balance that is modified during plastic events within neuronal networks.
See also: GABA Synthesis and Metabolism.
Further Reading Aguayo LG, van Zundert B, Tapia JC, et al. (2004) Changes on the properties of glycine receptors during neuronal development. Brain Research: Brain Research Reviews 47: 33–45. Cascio M (2004) Structure and function of the glycine receptor and related nicotinicoid receptors. Journal of Biological Chemistry 279: 19383–19386. Choquet D and Triller A (2003) The role of receptor diffusion in the organization of the postsynaptic membrane. Nature Reviews Neuroscience 4: 251–265. Colquhoun D and Sivilotti LG (2004) Function and structure in glycine receptors and some of their relatives. Trends in Neuroscience 27: 337–344. Dahan M, Levi S, Luccardini C, et al. (2003) Diffusion dynamics of glycine receptors revealed by single-quantum dot tracking. Science 302: 442–445. Hanus C, Vannier C, and Triller A (2004) Intracellular association of glycine receptor with gephyrin increases its plasma membrane accumulation rate. Journal of Neuroscience 24: 1119–1128. Kim EY, Schrader N, Smolinsky B, et al. (2006) Deciphering the structural framework of glycine receptor anchoring by gephyrin. The EMBO Journal 25: 1385–1395. Kneussel M and Betz H (2000) Clustering of inhibitory neurotransmitter receptors at developing postsynaptic sites: The membrane activation model. Trends in Neuroscience 23: 429–435. Laube B, Maksay G, Schemm R, et al. (2002) Modulation of glycine receptor function: A novel approach for therapeutic intervention at inhibitory synapses? Trends in Pharmacological Sciences 23: 519–527. Legendre P (2001) The glycinergic inhibitory synapse. Cellular and Molecular Life Sciences 58: 760–793. Lynch JW (2004) Molecular structure and function of the glycine receptor chloride channel. Physiological Reviews 84: 1051–1095. Moss SJ and Smart TG (2001) Constructing inhibitory synapses. Nature Reviews Neuroscience 2: 240–250. Rosenberg M, Meier J, Triller A, et al. (2001) Dynamics of glycine receptor insertion in the neuronal plasma membrane. Journal of Neuroscience 21: 5036–5044. Triller A and Choquet D (2005) Surface trafficking of receptors between synaptic and extrasynaptic membranes: And yet they do move! Trends in Neuroscience 28: 133–139. Vannier C and Triller A (1997) Biology of the postsynaptic glycine receptor. International Review of Cytology 176: 201–244.
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AMINES AND ACETYLCHOLINE A. Dopamine and Norepinephrine
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Dopamine J D Elsworth and R H Roth, Yale University School of Medicine, New Haven, CT, USA ã 2009 Elsevier Ltd. All rights reserved.
Anatomy Mesencephalon
The majority of central dopaminergic projections arise in the midbrain from the ventral tegmental area (VTA; A10), substantia nigra (SN; A9), and retrorubral area (A8) to innervate three principal sets of targets: the striatum (the caudate and putamen), the limbic cortex (medial prefrontal, cingulate, piriform, and entorhinal areas), and other limbic structures (regions of the septum, olfactory tubercle, nucleus accumbens, and amygdaloid complex). These systems are frequently referred to as the nigrostriatal, mesocortical, and mesolimbic dopamine (DA) projections, respectively. Although the A9 cells preferentially innervate the striatum, and the A10 cells mainly target the limbic and cortical regions, Figure 1 illustrates that the projections of the midbrain DA cells are not restricted to these regions. Many of the SN DA neurons give off collateral branches and ramify within more than one region. The projection fields of the A8, A9, and A10 cell groups are collectively known as the mesotelencephalic DA system. This term has been used to refer to the projections from the midbrain DA neurons (A8, A9, and A10) to the striatum and nucleus accumbens. The A8–A10 groups comprise approximately 20 000–30 000 neurons on each side of the rat brain. Also located in the mesencephalon, the rostral linear nucleus is sometimes considered part of the VTA. DA neurons of the rostral linear nucleus and periaqueductal gray region project locally or to different brain regions, such as the central amygdaloid nucleus, bed nucleus of the stria terminalis, sublenticular extended amygdala, and substantia innominata. It is now known that heterogeneities of DA innervation occur within a region. Thus, the nucleus accumbens can be divided into two cytoarchitectonically, physiologically, and pharmacologically distinct compartments, termed core and shell. The core region and the shell region receive preferential inputs from the A9 and A10 cell groups, respectively. Furthermore, two histochemically distinct compartments within the striatum have been defined, known as ‘patch’ (or ‘striosome’) and ‘matrix,’ which contain different densities of a number of markers for dopaminergic, cholinergic, and peptidergic neurons and
represent another level of processing in the basal ganglia. Evidence suggests that the mesostriatal DA input to these compartments arises from distinguishable subsets or ‘tiers’ of the DA-containing midbrain neurons. Diencephalon
These systems include the A11–A15 cell groups, which are located in the diencephalon, including regions such as the hypothalamus, caudal thalamus, zona incerta, and preoptic region. These neurons project principally within the diencephalon and have been divided into periventricular, incertohypothalamic, and tuberohypophyseal systems. There are approximately 2000–4000 DA neurons on each side of the brain in the rat diencephalic A11–A15 groups. An additional long-length system is the descending projections from the A11 group in the diencephalon to the spinal cord, where a dopaminergic innervation is apparent in the dorsal horn at all levels of the cord. Other Systems
Among the other DA systems are the short-length interplexiform amacrine-like neurons, which link inner and outer plexiform layers of the retina. There is also a population of periglomerular DA neurons in the olfactory bulb, which link mitral cell dendrites in separated adjacent glomeruli.
Function The different locations of the various DA systems in the brain dictate that they have different afferent and efferent connections, which in turn determine the roles they play in brain function. These are reviewed here. It should also be appreciated, however, that it is not possible to completely segregate the motor, motivational, and cognitive behaviors that are often attributed to the nigrostriatal, mesolimbic, and mesocortical DA systems, respectively. Nigrostriatal System
Dopaminergic innervation of the basal ganglia plays an essential role in many aspects of motor control, cognition, and emotion. The striatum is one of the principal input structures of the basal ganglia. The importance of the DA input to the striatum is evident from the motor abnormalities (e.g., bradykinesia, rigidity, and tremor) exhibited by patients with Parkinson’s disease, which is characterized by a marked loss of nigrostriatal DA neurons. DA innervations to limbic and cortical regions are also affected in Parkinson’s disease but to a lesser extent than the
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Figure 1 Schematic diagram illustrating the distribution of the main neuronal pathways containing DA in the central nervous system. The cell groups are named according to the 1965 nomenclature of Dahlstrom and Fuxe. Different colors denote the terminal field projections of the A8, A9, and A10 cell groups. Recent investigations have established that collaterals of ascending DA neurons also provide innervation of the subthalamic nucleus, thalamus, and globus pallidus.
striatal input. DA release in the striatum modulates activity in two striatopallidal circuits (‘direct’ and ‘indirect’), which in turn exert control over thalamocortical circuits essential for voluntary control of movement. The signs of Parkinson’s disease do not appear until a large majority (approximately 80%) of nigrostriatal DA neurons have been lost, as surviving DA neurons efficiently increase their activity to compensate for the damage. It is now apparent that nigrostriatal DA neurons do not merely allow motor behavior to occur but that they also play an important role in the selection and initiation of actions and establishing motor skills and habits. Current treatments for Parkinson’s disease typically involve administration of the DA precursor, dihydroxyphenylalanine (L-dopa), or DA receptor agonists. Future treatments are likely to include restoration of dopaminergic tone by transplantation of fetal DA neurons or stem cells or by gene therapy. Mesolimbic System
The function of the mesolimbic dopaminergic system, particularly the projection from the VTA to the nucleus accumbens, has been strongly implicated in goal-oriented (motivated) behaviors, in addition to reward, attention, and pharmacologically induced locomotion. Enhancement of DA transmission in
this system has been linked with the addicting, reinforcing, and sensitizing effects of repeated exposure to psychostimulant drugs of abuse. Furthermore, the therapeutic effects of antipsychotic drugs used in the treatment of schizophrenia may depend on the inhibition of mesolimbic DA neuron activity that these drugs induce. The ability of antipsychotic drugs to block DA receptors and thereby reduce DA transmission is central to the 40-year-old DA hypothesis of schizophrenia, which posits that the disease is related to excessive central DA activity. Imaging studies in patients have provided support for the existence of disrupted DA transmission in schizophrenia. A revision to the hypothesis is the concept of underactivity in cortically projecting DA neurons together with overactivity in subcortical DA systems. Persuasive evidence implicates developmental, functional, and structural abnormalities in schizophrenia, involving other transmitter systems besides DA, such as glutamate and g-aminobutyric acid (GABA). Mesocortical System
The prefrontal cortex has rich connections with other neocortical regions, limbic regions, and other subcortical regions. The prefrontal cortex has been implicated in a wide variety of cognitive functions, and it particularly appears to be involved in directing
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appropriate attention, prioritizing the significance of stimuli, monitoring the temporal sequence of stimuli, referencing stimuli to internal representations or cues, and devising abstract concepts. The well-defined DA projection to the prefrontal cortex has been suggested to be involved in short-term and ‘working’ memory. It has been hypothesized that the mesoprefrontal DA system is involved in the pathophysiology of schizophrenia and that defects in this system, or its associated neuronal connections, may be responsible for the negative and cognitive deficits that characterize the disorder. Diencephalon Systems
DA neurons in posterior dorsal hypothalamus and periventricular gray of central thalamus (A11) project to spinal cord and appear to have a role in sensory and nociceptive processing and sensory integration. Tuberoinfundibular DA neurons (A12) are located in the arcuate nucleus and periventricular nucleus of mediobasal hypothalamus and project to the external layer of median eminence. They play an important role in inhibiting the release of prolactin from the anterior lobe of the pituitary. Incertohypothalamic DA neurons (A13) reside in the rostral portion of medial zona inserta and terminate in the central nucleus of amygdala, horizontal diagonal band of Broca, and paraventricular nucleus of the hypothalamus. Their functions are not clear but probably involve integration of autonomic and neuroendocrine responses to sensory stimuli. Two subpopulations of A14 neurons have been distinguished. The periventricular– hypophysial (tuberohypophyseal) DA neurons terminate in the intermediate lobe of the posterior pituitary and inhibit secretion of a-melanocytestimulating hormone and pro-opiomelanocortinderived peptides such as b-endorphin. DA neurons also innervate the neural lobe, but their origin is controversial. The other defined group of A14 DA neurons is located in the periventricular hypothalamic nucleus. Fibers of these neurons extend into the medial preoptic nucleus and anterior hypothalamus. Females possess greater number of these neurons than males, and these neurons are believed to play a role in gonadotrophin secretion in females and reproductive behavior in males. DA neurons in the ventrolateral hypothalamic comprise the A15 cell group, and they extend processes to the lateral hypothalamus and caudal supraoptic nucleus, suggesting a role in oxytocin and/or vasopressin secretion. Other Systems
Retinal DA neurons in vertebrates appear to be particularly involved in contrast sensitivity and light adaptation. In 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
(MPTP)-treated parkinsonian monkeys, a loss of retinal DA occurs, along with spatial frequency-dependent abnormalities in both the pattern electroretinogram and the visual evoked potential. In Parkinson’s disease, abnormalities occur in ocular motor control, visual evoked potentials, electroretinograms, color vision, and visual contrast sensitivity. Changes are also possible in the acquisition and perception of visual information. In addition to the DA systems in the brain and pituitary, DA appears to have a restricted role in the periphery. The best defined role for peripheral DA neurons is in renal regulation of sodium homeostasis, and impairment of this function may underlie development of hypertension.
Life Cycle Figure 2 is a schematic model of a dopaminergic nerve terminal illustrating the life cycle of DA and the regulatory mechanisms that modulate the synthesis, storage, and release of this important neurotransmitter. A brief description of this idealized nerve terminal and model synapse will help to summarize the present state of knowledge concerning the life cycle and regulatory control of DA in a typical DA synapse and possible sites at which drugs and chemicals can influence dopaminergic transmission. Synthesis
Blood-borne tyrosine, derived from dietary proteins and from phenylalanine metabolism, enters the brain by a low-affinity amino acid transport system. Tyrosine in brain extracellular fluid is taken up into DA neurons by high- and low-affinity amino acid transporters. The relative circulating levels of tyrosine and phenylalanine can affect central catecholamine metabolism because these amino acids compete for transport into the brain and for transport into the neuron. DA is synthesized from tyrosine in two enzymatic steps. The first reaction is catalyzed by the enzyme tyrosine hydroxylase in the cytosol. Because this step is rate limiting, it sets the pace for the conversion of tyrosine to DA and is the step most susceptible to physiologic regulation and pharmacologic manipulation. Short-term activation of tyrosine hydroxylase involves phosphorylation of the regulatory domain by protein kinases. The activated form of tyrosine hydroxylase is thought to have a lower Km for its pterin cofactor and a higher Ki for DA, which effectively reduces end product inhibition. The activity of the enzyme dihydropteridine reductase is indirectly linked to DA biosynthesis because this enzyme catalyzes the recycling of the
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Figure 2 Schematic model of a dopaminergic nerve terminal illustrating the life cycle of dopamine (DA) and the mechanisms that modulate its synthesis, release, and storage. Invasion of the terminal by a nerve impulse results in the Ca2þ-dependent release of DA. This release process is attenuated by release-modulating autoreceptors. Increased impulse flow also stimulates tyrosine hydroxylation. This appears to involve the phosphorylation of tyrosine hydroxylase (TH), resulting in conversion to an activated form with greater affinity for tetrahydrobiopterin cofactor and reduced affinity for the end product inhibitor DA. The rate of tyrosine hydroxylation can be attenuated by (1) activation of synthesis-modulating autoreceptors, which may function by reversing the kinetic activation of TH, and (2) end product inhibition by intraneuronal DA, which competes with cofactor for a binding site on TH. Release- and synthesis-modulating autoreceptors may represent distinct receptor sites. Alternatively, one site may regulate both functions through distinct transduction mechanisms. The plasma membrane DA transporter (DAT) serves an important physiological role in the inactivation and recycling of DA release into the synaptic cleft. The vesicular monoamine transporter (VMAT) transports cytoplasmic DA into storage vesicles, decreasing the cytoplasmic concentration of DA and preventing metabolism by MAO. VMAT modulates the concentration of free DA in the nerve terminals. Reproduced from Cooper JR, Bloom FE, and Roth RH (2003) The Biochemical Basis of Neuropharmacology, 8th edn. New York: Oxford University Press, with permission from Oxford Press.
quinonoid dihydrobiopterine to tetrahydrobiopterine, which is an essential cofactor of tyrosine hydroxylase. In addition, the synthesis of tetrahydrobiopterine is dependent on the activity of another enzyme, GTPcyclohydrolase-1. The second step is catalyzed by L-dopa decarboxylase in the cytosol. This enzyme is also referred to as aromatic amino acid decarboxylase because it catalyzes the decarboxylation of several endogenous aromatic amino acids. This enzyme so avidly decarboxylates L-dopa that the levels of this amino acid in brain are very low under normal conditions. Metabolism
DA can be metabolized in brain by several enzymatic reactions, which are summarized in Figure 3. The major mammalian enzymes of importance in the metabolic degradation of catecholamines are monoamine oxidase (MAO) and catechol O-methyltransferase (COMT). MAO converts catecholamines to their corresponding aldehydes. This aldehyde intermediate
is rapidly metabolized, usually by oxidation by the enzyme aldehyde dehydrogenase to the corresponding acid. In some circumstances, the aldehyde is reduced by aldehyde reductase. MAO is located on the outer membranes of mitochondria and thus, in brain, is present primarily in nerve terminals and glia. Separate genes encode two isoforms of MAO (types A and B), which can be distinguished by substrate specificity and sensitivity to the irreversible selective inhibitors. In brain, MAO-A is preferentially located in dopaminergic and noradrenergic neurons, whereas MAO-B appears to be the major form present in serotonergic neurons and glia. MAO is a particlebound protein localized largely in the outer membrane of mitochondria. Usually considered to be an interneuronal enzyme, it also occurs in abundance extraneuronally. The second enzyme of importance in catabolism of catecholamines is COMT. This is a relatively nonspecific enzyme that catalyzes the transfer of methyl groups from S-adenosylmethionine to the m-hydroxyl
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CH2
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Figure 3 Main pathways of catabolism of DA. Enzymes involved (shown in italics) are monoamine oxidase (MAO), catechol-Omethyltransferase (COMT), aldehyde dehydrogenase (ALD-D), aldehyde reductase (ALD-R), and alcohol dehydrogenase (ADH). Cofactors for the enzymes are shown in parenthesis; SAM, S-adenosyl-L-methionine. In addition, dopamine (DA) and metabolites are substrates for phenolsulfotransferase; sulfation occurs on the 3-position, indicated on the structure for DA. In brain, DA is metabolized mainly to acidic (DOPAC and HVA) metabolites, with less formation of alcoholic metabolites (DOPET and MOPET); in the periphery, the alcohol metabolites may be metabolized further by alcohol dehydrogenase. In the periphery, glucoronyltransferase is able to form glucoronide conjugates of DA and metabolites.
group of catecholamines and various other catechol compounds. There are two isoforms of COMT, a membrane-bound form and a soluble form. Membrane-bound COMT is the major form found in the central nervous system (CNS), has a higher affinity for catecholamines, and is located principally in neurons. The soluble form has a lower affinity for catecholamines and is the major form expressed in the periphery, but it is present also in CNS glia. The membrane-bound isoform of COMT, having a high affinity for DA, is expressed at neuronal dendritic processes in cortex and striatum, but the expression and activity of COMT are higher in frontal cortex than in striatum. Relevant to this is evidence that in the prefrontal cortex DA transporter (DAT) is rarely expressed within synapses and exerts minimal influence on DA flux in the prefrontal cortex. Thus, COMT activity appears to have a more important function in regulating DA neurotransmission in the frontal cortex than in other regions. In fact, it has been estimated that the flux of DA through the COMT pathway exceeds 60% in the prefrontal cortex but only 15% in the striatum, where DAT is the chief mechanism for terminating the action of DA. The importance of COMT to the actions of DA in the prefrontal cortex is strongly supported by the finding
that compared with wild-type mice, COMT knockout mice have increased DA levels in prefrontal cortex, but not in striatum, and that such mice perform better on prefrontal cortex-dependent behavioral tasks. Another interesting facet to the role of COMT in frontal cortex is the finding that the COMT gene contains a polymorphism (Val158Met) that affects the in vivo activity of the enzyme. Met158 homozygotes have approximately one-third less COMT enzyme activity in prefrontal cortex than Val158 homozygotes. Consistent with its role in modulating prefrontal cortex DA levels, the Val158Met polymorphism is associated with performance on tests of working memory and executive function, which depend on prefrontal cortex function. Thus, the high-activity Val158 allele is linked with relatively poorer performance on such tasks, relative to the Met158 allele, presumably as a result of increased DA metabolism. The COMT polymorphism has been implicated in a number of neuropsychiatric phenotypes. In particular, there is evidence to support an association between COMT allele frequency and the genetic risk of schizophrenia. Working memory is highly dependent on DA function in the prefrontal cortex, and working memory dysfunction is a cardinal feature of schizophrenia.
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The major DA metabolites found in brain differ, depending on the species under study. In general, however, acidic rather than neutral metabolites predominate. In rodents, the primary metabolites of DA found in the CNS are 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) and a small amount of 3-methoxytramine. DOPAC usually predominates. In addition, a considerable proportion of these metabolites in rodent brain are found in the form of conjugates. In both human and nonhuman primates, however, the major DA metabolite is HVA, and only a small amount is found in the conjugate form. It is well documented that in the rat, short-term fluctuations in DOPAC and HVA concentrations in the striatum, nucleus accumbens, and prefrontal cortex provide a useful index, respectively, of alterations in impulse flow in the nigrostriatal, mesolimbic, and mesocortical DA pathways.
Since the original studies on DA uptake, it has been known that DA is a good substrate for the NE transporter. However, not until relatively recently has the importance of this been appreciated. While in regions, such as striatum and nucleus accumbens, in which DA neurons express a high density of DAT and there is a relatively sparse NE innervation, DA uptake depends primarily on DAT. However, in regions such as the prefrontal cortex, in which DA neurons express low levels of DAT and there is a rich NE innervation, DA uptake depends strongly on the norepinephrine transporter. In fact, it has been suggested that DA can be co-released with norepinephrine by norepinephrine neurons as a result of nonspecific uptake of DA by the norepinephrine transporter and/or because DA is a precursor in the biosynthesis of NE.
Transporters
Much effort has been directed toward elucidating the regulatory mechanisms that control the function of the various DA systems because this may lead to an understanding of the vulnerability of certain DA systems to disease and may also enable drugs to be developed that target specific DA projections. Some aspects of regulation were discussed in relation to the life cycle of DA. Here, other important controls of DA neurotransmission are reviewed.
Presynaptic vesicles or granules present in the nerve terminals are specialized for the uptake and storage of DA, thus protecting it from degradation by the enzyme MAO found in mitochondria present in the nerve terminal. The storage granules contain a vesicular monoamine transporter (VMAT2) that has a pharmacology distinct from the plasma membrane DAT found on the nerve terminals. The Naþ- and Cl dependent DAT actively transfers the DA from the synaptic cleft back into the presynaptic cell, where it can be stored and reused. DA is concentrated to approximately 0.1 M in the storage vesicles, 10–1000 times higher than the level in the cytosol. The tuberoinfundibular, tuberohypophysial, olfactory bulb, mesoprefrontal, and mesoamygdala DA neurons appear to differ from the other DA systems described by the absence, or diminished expression, of a high-affinity DA uptake (transport) mechanism. High-affinity DA uptake serves to recapture released DA and limits the concentration and actions of DA in the synapse. Importantly, neurons equipped with a high density of DATs are especially susceptible to drugs that target this site, such as cocaine and the parkinsonian neurotoxins, 6-hydroxydopamine and MPTP. In fact, the toxicity of MPTP is dependent on the function of MAO-B, DAT, and VMAT2. Once inside the brain, MPTP is metabolized by MAO-B to the actual toxic species, MPPþ, which is a substrate for DAT. Whereas DAT is responsible for transport of the toxin into DA neurons, VMAT2 aids in sequestering the toxin and diminishing its detrimental impact on mitochondria in the neuron. Thus, the DAT-to-VMAT2 ratio should be a determinant of the susceptibility of a given brain region to MPTPinduced toxicity.
Regulation
Firing Pattern
Electrophysiological studies have shown that DA cells fire in two different modes, either single spiking or burst firing, which have a marked impact on the release of DA. Phasic levels of DA are mediated primarily by bursting events at the level of the cell body and lead to a much larger DA release (150–400 nM in striatum) than when these neurons fire in a slow, irregular single spike mode associated with tonic levels (5–10 nM in striatum). Because cells are able to switch between these levels, the transition in activity is a mechanism for altering the impact of DA neurotransmission on receptive cells. Bursting activity of DA neurons is thought to represent a key component of reward circuitry, signaling a reward, or indicating to what extent a reward occurs differently than predicted. Alterations in tonic levels of DA efflux occur on a much slower time scale than changes in phasic levels and enable function on a variety of motor, cognitive, and motivational processes, which are deficient in Parkinson’s disease and which can be restored by DA replacement therapy. Autoreceptors
A receptor sensitive to the transmitter secreted from the cell on which the receptor is located is termed an
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‘autoreceptor.’ Autoreceptors exist on most portions of DA cells, including the soma, dendrites, and nerve terminals, so that autoreceptors are responsive to both terminal and dendritic DA release. Stimulation of DA autoreceptors in the somatodendritic region slows the firing rate of DA neurons, whereas stimulation of autoreceptors located on DA nerve terminals results in an inhibition of DA synthesis and release. Somatodendritic autoreceptors may also regulate DA release and synthesis by changing impulse flow. Thus, somatodendritic and nerve terminal autoreceptors work in concert to exert feedback regulatory effects on dopaminergic transmission. Three types of autoreceptors can be defined according to their functional effects: impulse-modulating, release-modulating, and synthesis-modulating autoreceptors. In general, all DA autoreceptors can be classified as D2-like DA receptors. The terminals of all midbrain DA neurons examined to date have been found to possess releasemodulating autoreceptors. This is not the case for synthesis- and impulse-modulating autoreceptors. Whereas most DA neurons located in the SN and many DA neurons in the VTA possess somatodendritic impulse-modulating and nerve terminal synthesismodulating autoreceptors, the DA neurons that project to the prefrontal and cingulate cortices and amygdala appear to have either a greatly diminished number, or none, of these receptors. Some of the differences in responsiveness among various midbrain DA neurons may be explained, in part, by differences in their autoreceptor function. Data suggest distinct differences in the autoreceptor regulation of hypothalamic DA neurons. Thus, in contrast to the incertohypothalamic DA neurons, tuberoinfundibular DA neurons appear to lack synthesis-modulating autoreceptors. The activity of tuberoinfundibular DA neurons is regulated by circulating levels of prolactin.
with nontransmitter proteins, including acetylcholinesterase, vesicular glutamate transporter, protein O-carboxymethyltransferase, cytochrome P450 reductase, and a vitamin D-dependent calcium-binding protein. The functional significance of such co-localization is not clear. DA neurons in which neuropeptides are co-localized may be regulated by peptide autoreceptors in a fashion analogous to DA autoreceptors. Furthermore, because it appears that release mechanisms for DA and co-stored peptides may be dissociated under certain conditions (e.g., dependent on the firing pattern and firing frequency of the neuron), co-localized peptides may be part of a cascade of regulatory features that exist in dopaminergic neurons. For example, nerve terminal autoreceptors in the prefrontal cortex have been shown to exert reciprocal effects on DA and neurotensin release. Stimulation of DA autoreceptors diminishes DA release and enhances neurotensin release, whereas blockade of DA receptors augments DA release and diminishes neurotensin release. Such differential release of DA and neurotensin could conceivably allow the prefrontal cortex DA neurons to differentially modulate the physiological activity of cortical postsynaptic follower cells. It appears likely that co-localization of peptides or nontransmitter proteins and DA will distinguish certain subpopulations of DA neurons. For example, cholecystokinin–DA co-localized neurons of the VTA project to the caudal, but not rostral, nucleus accumbens. Such distinctions may have important implications for the regionally specific function of DA in psychiatric and neurological disorders and also for the response of specific DA systems in these pathological conditions to pharmacological treatment.
Co-localized Peptides and Proteins
The different DA systems also vary in the nature of their interaction with other neurotransmitter systems that impinge on the DA neurons. At the cell body level, afferent inputs into the VTA and SN have been most extensively investigated. Many studies have suggested that these cells can be differentially modulated by afferent inputs. DA neurons are also regulated by neurotransmitter interactions that occur at the level of the nerve terminals. However, a detailed description of such interactions is outside the scope this article.
Another interesting intrinsic feature that may contribute to the variable susceptibility of DA neurons to toxicity is the presence of intracellular calcium-binding proteins, such as calretinin and calbindin. These proteins are preferentially expressed in DA neurons that are spared from degeneration in Parkinson’s disease and MPTP-treated animals, possibly by buffering calcium overload and protecting cells against excitotoxic damage. DA is co-localized in some neurons with neuropeptides and nontransmitter proteins. Certain midbrain DA neurons contain the peptide cholecystokinin, whereas another subpopulation of mesencephalic DA neurons contains the peptide neurotensin, and a third group of DA neurons in the VTA contains cholecystokinin, neurotensin, and DA. Similarly, DA is co-localized in certain mesencephalic neurons
Extrinsic
Postsynaptic Signaling There are known to be at least six different forms of the DA receptor. The D1 class of DA receptor has been divided into D1 and D5 receptor subtypes, and
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the D2 class comprises D2short, D2long, D3, and D4 receptor subtypes. The pharmacological profile and regional distribution in brain of each subtype are different, with the exception of D2short and D2long, which appear to be identical in these respects. Although these different receptors are reviewed elsewhere, certain interesting or novel aspects of DA signaling are discussed here. Neuromodulation
It is now generally accepted that DA is not a classical, fast ionotropic neurotransmitter like glutamate acting at a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) or N-methyl-D-aspartate (NMDA) receptors or GABA acting at GABA-A receptors that elicit excitatory or inhibitory postsynaptic potentials that last a few milliseconds. All the DA receptors are G-protein-coupled receptors that are slow, metabotropic receptors with response times exceeding 100 ms and function to modulate other receptor systems and/or ion channels. Thus, DA is thought of as a neuromodulator and cannot be classified as either an excitatory or an inhibitory neurotransmitter, which may explain why DA does not yield identical effects under all experimental conditions. Volume Transmission
Neurons may operate via two modes of communication, the synaptic mode and the mode of communication involving short- to long-distance diffusion of chemical signals in the extracellular fluid pathways (volume transmission). The process of volume transmission has been defined as a functionally significant association of release and receptor sites via extrasynaptic diffusion. It is now clear that in certain regions DA functions by volume transmission. The retina provides the clearest example of DA-mediated volume transmission. In the SN, DA receptors are located extrasynaptically, indicating that volume transmission is the predominant form of intercellular communication mediated by DA in this region. In the striatum, volume transmission appears not to play a major role in DA transmission since most DA terminals establish synaptic contacts, and the high density of DA terminals express a high density of DAT. Volume transmission may be more important in maintaining dopaminergic signaling in the striatum after partial loss of nigrostriatal DA neurons, as occurs in Parkinson’s disease. With the degeneration of dopaminergic terminals and consequent loss of DAT, DA that is released from the remaining neurons may diffuse in the striatal extracellular fluid to reach supersensitive high-affinity DA receptors.
Somatodendritic Release
There is good evidence that DA can be synthesized and released from dendrites and cell bodies in the SN. The release is vesicular and mediated by exocytosis, as it is in axon terminals. Somatodendritic release is calcium and depolarization dependent in a variety of paradigms. In dendrites, DA appears to be stored both in classical vesicles and in saccules of smooth endoplasmic reticulum, consistent with the expression of VMAT2 in both of these organelles. The function of DA released in the SN in this manner is probably to interact with autoreceptors to control the firing rate of DA neurons, in addition to signaling via DA receptors located on non-DA neurons in the region. Long-Term Potentiation and Long-Term Depression
DA has a critical role in two distinct forms of plasticity, called long-term depression (LTD) and long-term potentiation (LTP). In regions such as the striatum and prefrontal cortex where DA modulates glutamate signaling, combined stimulation of both glutamate and DA receptors is able to induce either enhancement or weakening of synaptic transmission, depending on the parameters of the stimulation and the receptor subtypes that are activated. These mechanisms may lead to more permanent changes, such as the construction of new connections (synapses). Such plastic changes are thought to have an important role in learning and memory (hippocampus and prefrontal cortex) and acquisition of complex motor skills (striatum). Pyramidal Cell Dendritic Spines
DA also plays a critical role in the modulation of pyramidal cell dendritic spine synapses and spine density. Several studies have demonstrated that the loss of DA in the striatum results in decreased dendritic length and spine density. Employing multiphoton imaging, selective elimination of glutamatergic synapses on striatopallidal neurons has been observed in Parkinson disease models. It has clearly been shown that DA depletion leads to a rapid and profound loss of spines and glutamatergic synapses on striatopallidal medium spiny neurons but not on neighboring striatonigral medium spiny neurons. This loss of connectivity is triggered by an interesting mechanism – dysregulation of intraspine Cav1.3 L-type Ca2þ channels. The disconnection of striatopallidal neurons from motor command structures is likely to be a key step in the emergence of pathological activity that is responsible for symptoms in Parkinson’s disease.
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DA modulation of pyramidal cell dendritic spines and synapses has been extended to the prefrontal cortex, where work has shown that selective lesion of the DA innervation of the prefrontal cortex caused a decrease in dendritic spine density in pyramidal cells in the prefrontal cortex. It is noteworthy that atypical antipsychotic drugs which cause a large increase in synaptic DA reverse the DA depletioninduced dendritic spine loss in prefrontal cortical pyramidal neurons. In schizophrenia, the prefrontal cortex DA innervation is compromised and postmortem studies have reported decreased dendritic spine density. Taken together, these data suggest that disruption of dopaminergic signaling may be the proximate cause of dendritic remodeling in the prefrontal cortex in schizophrenia. Excessive DA input may have the opposite effect because chronic cocaine or amphetamine use has been shown to increase spine density in nucleus accumbens and prefrontal cortex and increase synapse number in the prefrontal cortex. See also: Dopamine Receptors and Antipsychotic Drugs in Health and Disease; Dopamine: Cellular Actions.
Further Reading Akil M, Kolachana BS, Rothmond DA, et al. (2003) CatecholO-methyltransferase genotype and dopamine regulation in the human brain. Journal of Neuroscience 23: 2008–2013. Cooper JR, Bloom FE, and Roth RH (2003) The Biochemical Basis of Neuropharmacology, 8th edn. New York: Oxford University Press. Day M, Wang Z, Ding J, et al. (2006) Selective elimination of glutamatergic synapses on striatopallidal neurons in Parkinson disease models. Nature Neuroscience 9: 251–259.
Dunnett SB, Bentivoglio M, Bjorklund A, and Hokfelt T (2005) Handbook of Chemical Neuroanatomy, vol. 21. Elsevier: Amsterdam. Eisenhofer G, Kopin IJ, and Goldstein DS (2004) Catecholamine metabolism: A contemporary view with implications for physiology and medicine. Pharmacological Reviews 56: 331–349. Elsworth JD and Roth RH (1997) Dopamine autoreceptor pharmacology and function: Recent insights. In: Neve KA and Neve RL (eds.) The Dopamine Receptors, pp. 223–265. Humana: Totowa, NJ. Grace AA (2002) Dopamine. In: Davis KL, Charney D, Coyle JT, and Nemeroff C (eds.) Neuropsychopharmacology: The Fifth Generation of Progress, pp. 120–132. Philadelphia: Lippincott Williams & Wilkins. Jay TM (2003) Dopamine: A potential substrate for synaptic plasticity and memory mechanisms. Progress in Neurobiology 69: 375–390. Jentsch JD, Roth RH, and Taylor JR (2000) Role for dopamine in the behavioral functions of the prefrontal corticostriatal system: Implications for mental disorders and psychotropic drug action. Progress in Brain Research 126: 433–453. Joel D and Weiner I (2000) The connections of the dopaminergic system with the striatum in rats and primates: An analysis with respect to the functional and compartmental organization of the striatum. Neuroscience 96: 451–474. Lookingland KJ and Moore KE (2005) Functional neuroanatomy of hypothalamic dopaminergic neuroendocrine systems. Handbook of Chemical Neuroanatomy 21: 435–523. Miller GW, Gainetdinov RR, Levey AI, and Caron MG (1999) Dopamine transporters and neuronal injury. Trends in Pharmacological Sciences 20: 424–429. Rice ME (2000) Distinct regional differences in dopaminemediated volume transmission. Progress in Brain Research 125: 277–290. Seamans JK and Yang CR (2004) The principal features and mechanisms of dopamine modulation in the prefrontal cortex. Progress in Neurobiology 74: 1–58. Tzschentke TM (2001) Pharmacology and behavioral pharmacology of the mesocortical dopamine system. Progress in Neurobiology 63: 241–320.
Dopamine Receptors and Antipsychotic Drugs in Health and Disease P Seeman, University of Toronto,Toronto, ON, Canada ã 2009 Elsevier Ltd. All rights reserved.
Introduction The discovery of dopamine receptors is intertwined with the discovery and development of antipsychotic drugs. The research in this area started with the development of antihistamines after World War II, particularly with H Laborit using such compounds to enhance surgical analgesia. In patients receiving these medications, Laborit noticed a ‘euphoric quietude,’ and that the patients were ‘‘calm and somnolent, with a relaxed and detached expression.’’ Of this series of Rhoˆne-Poulenc compounds, RP4560, now known as chlorpromazine, was the most potent. Chlorpromazine was tested by many French physicians for use in various medical illnesses. Although Sigwald and Bouttier were the first to use it as the sole medication for a psychotic patient, their work was not reported until 1953, after a 1952 report by Delay and colleagues that chlorpromazine alleviated hallucinations and stopped internal ‘voices’ in eight patients. A significant aspect of the action of chlorpromazine was that it was effective within 3 days. This rapid improvement, especially during the first week of antipsychotic treatment, has been observed in many studies and summarized in reviews by S Kapur and O Agid. The clinically successful action of chlorpromazine stimulated the search to identify chlorpromazine’s mode of action. The assumption then, as now, was that the discovery of such a mode of action would open the avenue to uncovering the biochemical cause of psychosis and possibly of schizophrenia.
Early Days: Before Discovery of Dopamine Receptors In searching for the mechanism of chlorpromazine action in the 1960s and 1970s, many types of electrophysiological and biochemical experiments were done. Because high doses of chlorpromazine and other antipsychotics (or ‘neuroleptics,’ as they were then called) also elicited parkinsonism as a side effect, the basic science quickly focused on the action of antipsychotics on dopamine pathways in the brain. The rationale for examining brain dopamine regions was based on the finding by H Ehringer and O Horniekiewicz that the parkinsonism of Parkinson’s disease was associated
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with a massive loss of brain dopamine. It was felt, therefore, that the unwanted side effect of chlorpromazine-induced parkinsonism, as well as the antipsychotic action itself, might arise by antipsychotics interfering with dopamine neurotransmission. The working assumption was that if the brain targets for antipsychotics could be found, then perhaps it could be determined whether these sites were overactive or underactive in psychosis or schizophrenia. A variety of mechanisms were explored for the mode of action of chlorpromazine, including its action on mitochondrial enzymes, sodium–potassiumATPase, and related enzymes, and its membranestabilizing action, such as its strong potency to inhibit membrane action potentials and to stabilize cellular and subcellular membranes from releasing their contents. It also became clear in 1963 that all antipsychotics were surface active, readily explaining their hydrophobic affinity for membranes. Some of these nonreceptor-related findings, such as the surface activities of the antipsychotics, showed an astonishingly excellent correlation with clinical antipsychotic potencies.
Therapeutic Concentrations of Antipsychotics All of the early experiments in the 1960s revealed that the in vitro active concentrations of the antipsychotics were generally between 20 and 1000 nM. These concentrations, however, were found in 1971 to be far in excess of the nanomolar concentrations (e.g., 1–2 nM for haloperidol) that exist in the spinal fluid in patients being successfully treated with these medications. In Vivo Experiments
In parallel with the in vitro experiments, there were many in vivo experiments by F Bloom, by G Aghajanian, and by B Bunney, showing that dopamine agonists can excite or inhibit neurons in the nigrostriatal dopamine pathway. Moreover, other workers (WD Heiss, J Hoyer) showed that direct application of dopamine on neurons also stimulated or inhibited snail neurons, and that haloperidol or fluphenazine could block these actions (H Struyker Boudier). These studies provided evidence for the existence of distinct dopamine receptors on neurons. Additional work in vivo showed that chlorpromazine and haloperidol increased the production of normetanephrine and methoxytyramine, metabolites of epinephrine and dopamine, respectively. To explain the increased production of these metabolites,
Dopamine Receptors and Antipsychotic Drugs in Health and Disease 393
Carlsson and Lindqvist suggested that ‘‘the most likely [mechanism] appears to be that chlorpromazine and haloperidol block monoaminergic receptors in brain; as is well known, they block the effects of accumulated 5-hydroxytryptamine.. . .’’ In other words, they proposed that antipsychotics might block all three types of receptors for noradrenaline, dopamine, and serotonin, but they did not identify which receptor was selectively blocked or how to identify or test any of these receptors directly in vitro. This study in 1963 by Carlsson and Lindqvist is often mistakenly cited as discovering ‘the dopamine receptor’ and that antipsychotics are selectively acting on this receptor. However, N-E Ande´n, a student of A Carlsson, had a different view, and proposed that ‘‘chlorpromazine and haloperidol delay the elimination of the (metabolites).. . .’’ Moreover, 7 years later Ande´n reported that antipsychotics increased the turnover of both dopamine and noradrenaline, but he could not show that the antipsychotics were selective in blocking dopamine; for example, chlorpromazine enhanced the turnover of noradrenaline and dopamine equally. Therefore, it remained for in vitro radio-receptor assays to detect the dopamine receptor directly and to demonstrate antipsychotic selectivity for the dopamine receptor. The Dopamine D1 Receptor
With the advent of assays for adenylate cyclase in the 1960s, J Kebabian found that dopamine stimulated adenylate cyclase in the superior cervical ganglion. This receptor was later named the dopamine D1 receptor, selectively labeled by [3H]SCH23390, and subsequently cloned by three research groups in 1990. The dissociation constants at D1 for dopamine agonists and antagonists of medical therapeutic interest are given in Tables 1 and 2. There is no correlation between the antipsychotic clinical doses and the dissociation constants of the antipsychotic antagonists at D1, as illustrated in Figure 1. These data suggested that D1 was not the major or common target for antipsychotics, in addition to the fact that the antipsychotic molarities at D1 are between 10 and 10 000 nM, far in excess of the therapeutic concentrations in the spinal fluid of treated patients. In addition to the lack of targeting D1 receptors by clinical doses of the common antipsychotics, D1selective compounds have not been found to be effective as antipsychotics (Figure 2). Discovery of the Antipsychotic Dopamine Receptor, or the Dopamine D2 Receptor
In 1974 and 1975, in order to detect and discover the dopamine receptors on which the antipsychotics
presumably acted, it was essential to label a receptor with a ligand, such as radioactive haloperidol, having an affinity (or dissociation constant) of 1 nM, because this was the haloperidol therapeutic concentration found in the spinal fluid or plasma water of treated patients. For this to occur, the specific activity of [3H]haloperidol would have to be at least 10 Ci mmol1. Although the [3H]haloperidol donated in 1971 by Janssen Pharmaceutica (J Heykants, J Brugmans) had a low specific activity of 32–71 mCi mmol1, I R E Belgique (M Winand) custom synthesized [3H]haloperidol at even lower specific activity (10.5 Ci mmol1) for Seeman’s laboratory by June 1974. Specific binding of this new [3H]haloperidol to brain striatal tissue was readily detected in 1975, and the following concentrations of compounds were found to inhibit the binding of [3H]haloperidol by 50%: 2 nM for haloperidol, 20 nM for chlorpromazine, 3 nM for (þ)butaclamol, and 10 000 nM for (–) butaclamol. The stereoselective action of butaclamol and the good correlation between the IC50% values and the clinical doses, as shown in Figure 3, indicated that the ‘antipsychotic receptor’ had finally been discovered. Equally important, of the endogenous compounds tested, dopamine was the most potent in inhibiting the binding of [3H]haloperidol, indicating that the antipsychotic receptor was actually a dopamine receptor (see Table 1). Using sequences related to the b-adrenoceptor, the antipsychotic/dopamine receptor, now named the dopamine D2 receptor, was finally cloned in 1988; its amino acid sequence is shown in Figure 4. Using the cloned D2 receptor, values for the dissociation constants for agonists and antagonists can be determined (Table 1). It is now known that therapeutic levels of antipsychotic drugs occupy 60–80% of brain D2 receptors, as shown by L Farde and colleagues. In fact, using the antagonist dissociation constants in Table 1, the concentrations necessary to occupy 75% of D2 receptors can be calculated and are essentially identical to the free molarities of the antipsychotics found in either the plasma water or the spinal fluid of treated patients, as shown in Figure 5. Because radiolabeled raclopride is less tightly bound to D2 receptors than radiolabeled spiperone, which in turn is less tightly bound to D2 receptors than radiolabeled nemonapride, the dissociation constant for any antagonist at D2 receptors is lowest when using radiolabeled raclopride, but rises when using radiolabeled spiperone, or rises higher still when using radiolabeled nemonapride (see Table 1). This dependence of the dissociation constant on the ligand is also seen in positron emission tomography studies, where it has been found, for example,
D1
D2
D3
D4
D5
Tissue [Ref.]:
Rat striatum or human D1 [f]
Human D2 Long clone [b]
Human D3 clone [b]
Human D4 clone
Human D5 clone
Radioligand:
[3H]SCH 23390
[3H]SCH 23390
[3H]SCH 23390
[3H]spiperone
[3H]SCH 23390
K High (nM )
K50 (nM )
K High (nM )
KLow (nM )
K High (nM )
K50 (nM )
K High (nM )
K50 (nM )
K50 (nM )
Neurotransmitters Dopamine
10.1
2340 [g]
25 I [a] 22 S n.i. n.i. n.i.
228 [g]
700 1700 9700 [g]
25 D 3.9 I [a] n.i. n.i. n.i.
148 [f]
No high No high No high
1400 D 729 R n.i. 805 D 4300 D
28 [f]
Noradrenaline Adrenaline Serotonin
2.4 D 1.4 R;1.3 A 9.8 D No high No high
n.i. n.i. n.i.
2200 [f] n.i. 4180 [f]
12 000 [g] n.i. 3000 [g]
1.2 39 No high No high 3.5 No high No high No high
1816 [g] n.i. 5400 672 [g] 360 [e] 330 [e] 2600 8200
0.14 D 0.4 D 0.5 D 0.8 D 0.75 D 1.3 D 1.7 D 12 D
54 D rat 62 D 1400 D 20 D 140 D 19 I [a] 202 R[d] 2600 D
0.25 D 0.6 D 3.2 D 1.3 D 2.6 D 0.9 D 0.45 R[d] 9D
0.8 n.i. n.i. 7.4 I [a] 73 I [a] 2.3 I [a] 256R [d];50 I [e] 11 I [e]
n.i. n.i. n.i. n.i. 4.1 [f] n.i. n.i. n.i.
4f 47 f n.i. 250 [e] 5 [e] 62 [e] 31 [e] 120 [e]
1136 [g] n.i. n.i. 720 [e] 13 [e] 39 [e] >1000 [g] >1000 [e]
1.1 [c] 10 [c]
380 [c] 4700 [c]
150 S[c] 1. 1 R [d]
8800 S[c] 42 R [d]
No high 0.06 R [d]
5000 [a] 0.2 R [d]
n.i. n.i.
1800 [f] 650 [a]
100 [g] n.i.
Dopamine agonists N-Propyl-norapomorphine-R-() (þ)PHNO ()Quinagolide.HCl, or CV205 502 Bromocriptine.base Apomorphine-R-().HCl Pergolide mesylate [LY 127,809] (–)Quinpirole, or ()-LY 171,555.HCl Pramipexole monohydrate SKF 38393 (þ)-7-OH-dipropylaminotetralin
[a], Sokoloff et al. (1992); [b], Seeman et al. (2005); [c], Seeman and Niznik (1988); [d], Malmberg and Mohell (1995); [e], Millan et al. (2002); [f] P. Seeman; [g], Sunahara et al. (1991); K50 (dissociation constant), C50% (conc. to inhibit 50% of binding)/(1 þ C*/Kd), where C* – concentration of radioligand in competition with agonist, and Kd – dissociation constant obtained by saturation with radioligand (Scatchard analysis); A, [3H]dopamine (Kd ¼ 1.3 nM); D, [3H]domperidone (Ref. b or P. Seeman, unpublished; Kd ¼ 0.43 nM); I, [125I]Iodosulpride (Kd ¼ 0.6 nM); R, [3H]raclopride (Kd ¼ 1.9 nM); S, [3H]spiperone (Kd ¼ 65 pM); rat, rat striatum; no high, no high-affinity state recognized by competing compound; n.i., no information available.
394 Dopamine Receptors and Antipsychotic Drugs in Health and Disease
Table 1 Agonist potencies at dopamine receptors (in 120 mM NaCl)
Dopamine Receptors and Antipsychotic Drugs in Health and Disease 395 Table 2 K values (dissociation constants) Human clone:
M1 nM
D1 nM
[3H]ligand used:
QNB
Sch.
[3H]Amisulpride Amoxapine Aripiprazole Butaclamol-(þ) Chlorpromazine [3H] Chlorpromazine Clozapine [3H]Clozapine Clozapine-iso Cyproheptadine Droperidol Epidepride Flupentixol-cis Flupentixol-trans Fluphenazine Haloperidol [3H]Haloperidol Iloperidone (HP873) Loxapine Loxapine-iso Melperone Molindone Norclozapine Olanzapine [3H]Olanzapine Perphenazine Pimozide Prochlorperazine Quetiapine [3H]Quetiapine Raclopride [3H]Raclopride Remoxipride Risperidone Risperidone-9-OH Sertindole [3H]Sertindole Spiperone [3H]Spiperone Sulpiride-S Trifluperazine Ziprasidone
D2 nM
1.7 49
3.4
16.5
— — — — 0.77
9.5 14.4
90 120
1.8
— 51 — — — — —
794
2.6 55
— —
109
5.6
—
117 203
18 16 148 1558 73 9.2
— — — — — —
0.4
17 2.1
2.7 4.5 470 135
>10 mM >10 mM 400
7.7 290
4900 42
— — — — 104 — 1.9 — —
22
3 1.2 — 0.065
265
2.9 9
— — —
D2 nM
D2 nM
D3 nM
Raclo.
Spip.
Raclo.
1.8 21 1.8 0.14 1.2 —
4.6 56
1.4
—
—
75 — 15 24 0.54 0.036 0.38 151 0.55 0.74 — 5.4
180 — 60
190 — 21
— 10
— 20
9.2 22 152 4.9 180 7.4 — 0.27 1.4 1.7 140 — 1.6 — 67 1.09 1.6 1.9 — 0.018 — 9.9 1.4 2.7
22.7 86 375 15 300 21 — 0.47 0.95 4 680 — 7.1 — 800 4
7.2 18 315 44
2.3 0.06 0.7
0.7
1.2 2.7
0.17 8.8
— — 1.2 — 2 — — — — — — — 0.85 —
5.5 70 9.6 — 22 — 21 2 15 30 2 — 9.6
14 — 0.23
— — — — 1.6 —
240 — 2.9 1.6 960 3.5
— — — — — — —
89 2000 — 2400 — 2400 4.4
3 —
0.06 8 3.8 6
Spip.
8 11 720 3900 120 15 — 32
6.5
—
D4 nM
— —
0.9 4.6
—
D4 nM
0.32 10 0.7 1.5
— 0.85 — 0.086 — — —
11 — — 1000 39 8
Blank cells indicate ‘not done’. Sch., Schering 23390; Raclo., Raclopride; Spip., spiperone.
when monitoring patients with [11C]raclopride, that therapeutic doses of clozapine occupy 50% of D2 receptors but that there is much less occupancy of D2 receptors when using [11C]methylspiperone or [18F]fluorethylspiperone, both of which bind more tightly to D2 receptors than raclopride (see Table 2). The correlations in Figures 3 and 5 remain a cornerstone of the dopamine hypothesis of psychosis or schizophrenia, and the dopamine hypothesis is still
the major contender for an explanatory theory of schizophrenia etiology. Two Classes of Dopamine Receptors
The D1 site and the [3H]haloperidol/dopamine receptor binding site were soon considered as distinct, because B Roufogalis found that the sulpiride antipsychotic did not block dopamine-stimulated adenylate cyclase. Two general classes of dopamine receptors were recognized,
396 Dopamine Receptors and Antipsychotic Drugs in Health and Disease
10−5
D1
K(mol l−1) on 3H-SCH23390 binding
Clebopride
Sulpiride Molindone
10−6
Chlorpromazine
Spiperone Clozapine 10−7
Haloperidol Thioridazine Fluphenazine Trifluperazine Flupenthixol
10−8
0.1
1
10 100 Range and average clinical dose for controlling schizophrenia (mg d−1)
1000
Figure 1 There is no correlation between the clinical antipsychotic doses and the antipsychotic dissociation constants (or concentrations) that inhibit the binding of a D1 ligand ([3H]SCH23390) at dopamine D1 receptors in homogenized striatal tissue. The high concentrations inhibiting the D1 receptor are far higher than those found clinically in the plasma water or spinal fluid. Adapted from Seeman P (1987) Dopamine receptors and the dopamine hypothesis of schizophrenia. Synapse 1: 133–152, with permission from John Wiley & Sons Inc.
Figure 2 Amino acid sequence of the dopamine D1 receptor. The two hydroxyls of dopamine are presumed to be associated with the two serine residues, while the tertiary nitrogen atom is presumed to be associated with the aspartic acid residue (D in transmembrane 3).
Dopamine Receptors and Antipsychotic Drugs in Health and Disease 397 10−7 Promazine Chlorpromazine Trazodone Clozapine Thioridazine Molindone Prochlorperazine Moperone Trifluperazine
IC50 (mol l−1)
10−8
Thiothixene Haloperidol Droperidol Fluphenazine Pimozide Trifluperidol
10−9
Benperidol 10−10 Spiroperidol
0.1 1 10 100 1000 Range and average clinical dose for controlling schizophrenia (mg d−1) Figure 3 The clinical antipsychotic doses correlate with the concentrations that inhibit by 50% the specific binding of [3H]haloperidol in homogenized caudate nucleus tissue (calf). These concentrations are similar to those found in the plasma water or spinal fluid in patients treated with antipsychotic drugs. Adapted from Seeman P, Lee T, Chau-Wong M, et al. (1976) Antipsychotic drug doses and neuroleptic/ dopamine receptors. Nature (London) 261: 717–719, with permission from Nature.
Figure 4 Amino acid sequence of the dopamine D2 receptor. The variants or polymorphisms of D2 include an alanine instead of a valine at position 96 (1% of population), an insertion of a 29-amino-acid polypeptide (D2Long) at the position shown by the lower left arrow, a serine instead of a proline at position 310, and a cysteine instead of a serine at position 311. Dopamine is presumed to be associated with the two serines (S) in transmembrane 5 and the aspartic acid (D) in transmembrane 3 (see Figure 2).
Line for identical values
S-Sulpiride Molindone Olanzapine
cu
pa
nc
y
100
Clozapine Remoxipride
%
oc
10 75
Concentration needed to occupy 75% of D2 (nM)
398 Dopamine Receptors and Antipsychotic Drugs in Health and Disease
1
Chlorpromazine Raclopride Thioridazine Haloperidol cis-Flupentixol Perphenazine
1 10 100 1000 Therapeutic free neuroleptic (nM) in spinal fluid or plasma water Figure 5 The therapeutic antipsychotic concentrations in the spinal fluid or in the plasma water in treated patients are essentially identical to the antipsychotic concentrations that occupy approximately 75% of the D2 receptors in vitro. The concentrations in the plasma water were obtained by correcting for the amount bound to the plasma proteins. The concentrations to occupy 75% of D2 were calculated as being three times higher than the dissociation constant at D2. Adapted from Seeman P (2002) Atypical antipsychotics: Mechanism of action. Canadian Journal of Psychiatry 47: 27–38.
D1 site (= dopamine-sensitive adyenylate cyclase) Dopamine: ∼3000 nM Spiperone: ∼2000 nM
D2 receptor
D3 site
Dopamine:∼5000 nM Spiperone:∼0.3 nM
Dopamine: 3 nM Spiperone: ∼1500 nM
D4 site Dopamine: 3 nM Haloperidol: ∼1 nM
Figure 6 Early version of dopamine receptors before clones of receptors became available. The D1 receptor (dopamine-stimulated adenylate cyclase) was stimulated by 3000 nM dopamine and inhibited by high concentrations of butyrophenones such as 2000 nM spiperone. The D2 receptor, or the [3H]haloperidol binding site, was highly sensitive to spiperone, but required 5000 nM dopamine to inhibit adenylate cyclase. The D3 site was a site labeled by [3H]dopamine and, therefore, very sensitive to dopamine at 3 nM, but required very high concentrations of antipsychotics to be inhibited. The D4 site was defined as being sensitive to both the agonists and the antipsychotic antagonists. While these definitions for the D3 and D4 sites are no longer used, the D1 and D2 properties are still valid for the cloned D1 and D2 receptors, with the additional point being that both D1 and D2 have high- and low-affinity states.
therefore, coupled or uncoupled to adenylate cyclase. These two classes were named D1 and D2 by J Kebabian and D Calne. Nomenclature of Dopamine Receptors
The data for the pattern of binding of [3H]haloperidol identifying the antipsychotic/dopamine D2 receptor
were very different from those for the pattern of [3H] dopamine binding described by studies in the mid1970s. For example, the binding of [3H]haloperidol was inhibited by 5000 nM dopamine, while that of [3H]dopamine was inhibited by 3 nM dopamine, as summarized in Figure 6. For several years, this latter [3H]dopamine binding site was termed the ‘D3 site,’ a
Dopamine Receptors and Antipsychotic Drugs in Health and Disease 399
term which is not to be confused with the later discovery of the dopamine D3 receptor.
Dopamine D2 Receptor Variants As noted in Figure 4, there are several variants of D2, the most important of which are the short form and the long form of D2, the latter having an additional 29 amino acids. There is also a D2Longer form where a dipeptide, valine–glutamine, is inserted into the intracellular loop, as shown in Figure 7. There are at least three polymorphisms in D2 (Figure 4): alanine replaces valine at position 96 in about 0.8–1% of some populations, serine replaces proline at position 310 in 0.4% of people, and cysteine replaces serine at position 311 in approximately 3–4% of the population. The variants at 310 and 311 are markedly less effective in inhibiting the synthesis of cyclic AMP than is the more common form of D2. Silent polymorphisms have also been found in the DNA code for D2, but there is no change in the amino acid (leucine at 141, histidine at 313, and proline at 319). There are also polymorphisms in the noncoding regions of D2, including an A-to-G at position –241, a C insert at position –141, an A-to-G before transmembrane 1 (Taq1B polymorphism), an A-to-G in the intron
within transmembrane 2 (Taq1D polymorphism), an A-to-C in the intron before transmembrane 4, a G-to-A in the intron before transmembrane 6, and an A-to-G situated 10 kb beyond transmembrane 7 (Taq1A polymorphism). A further mutation in D2 occurs in individuals with hereditary autosomal dominant myoclonus dystonia, with valine replaced by isoleucine at position 154 at the beginning of the fourth transmembrane segment of D2 (see Figures 4 and 7).
D2 Function and Distribution A wide variety of psychomotor functions, including biochemical, physiological, and pathological, have been attributed to D2. Specifically, D2 inhibits action potentials by eliciting a prolonged ‘inhibitory postsynaptic potential’ (IPSP), inhibits adenylate cyclase, and inhibits the entry of calcium ions into cells, thereby inhibiting many aspects of stimulus–response coupling in a variety of neurons and cells. D2 receptors are located on presynaptic terminals and, therefore, can readily inhibit the release of dopamine. These presynaptic receptors appear to be predominantly D2Short, while D2Long receptors are mostly postsynaptically located on dendritic spines. The genetic absence of D2 receptors leads to animals that are akinetic and Parkinson-like.
Figure 7 Amino acid sequence of D2Longer. Compared to D2Short and D2Long (Figure 4), D2Longer has an extra valine–glutamine dipeptide (as indicated by the arrow) that is usually spliced out in D2Short and D2Long.
400 Dopamine Receptors and Antipsychotic Drugs in Health and Disease
In alcoholics, it has been reported that the prevalence of the Taq1A polymorphism is about twofold higher than in control subjects. In schizophrenia, it has been found in 27 studies, comprising 3707 patients and 5363 controls, that the serine311cysteine polymorphism was significantly associated with schizophrenia. Furthermore, the number of D2 receptors in the caudate-putamen is elevated in schizophrenia, as measured in vivo (Corripio) or in postmortem samples in vitro (Figure 8). Although Figure 8 shows that the density of D2 receptors in postmortem human schizophrenia tissues is elevated, some of this elevation may have resulted from the antipsychotic administered during the lifetime of the patient. The postmortem tissues from half of the patients with schizophrenia revealed elevated densities of [3H]spiperone-labeled D2 receptors in the caudate-putamen tissue. The other half of the postmortem schizophrenia tissues were normal in D2 density, even though most of the patients were known to have been treated with antipsychotics during their lifetime. Such findings have long been controversial, because the D2 density is not elevated in schizophrenia when using [11C]raclopride. It should be noted, however, that the number of D2 receptors is significantly Control striata 12.9 + − 0.2 pmol g−1
20–88 years
Schizophrenia
elevated in healthy identical co-twins of individuals with schizophrenia, suggesting that an elevation of D2 may be a necessary but not sufficient requirement for schizophrenia. The distribution of D2 receptors within the various brain regions is reflected in the gene expression pattern of D2, as shown in Figure 9. An additional unique feature is that D2 receptors are organized in bands in the normal human temporal cortex, but these bands are not found in brains of patients with Alzheimer’s disease. Finally, the density of D2 is not constant over one’s lifetime, but slowly falls by about 2% per decade, as shown in the postmortem human tissues in Figure 10. There is a sharp transient rise during ages 1–3, but this is followed by a gradual pruning of neurons with D2 receptors.
The Dopamine D3 Receptor Using methods similar to those used for cloning the D2 receptor, the D3 receptor was cloned in 1990; its sequence is shown in Figure 11. D3 has several polymorphisms, including a serine replacing glycine at position 9 in 28% of the population (see Figure 11). In addition, there are nonfunctional forms of D3, where the amino acid chain stops after transmembrane 2, after transmembrane 3, or before transmembrane 6, the latter being found in, but not diagnostic for, Alzheimer’s disease and schizophrenia. Although BP897 is a partial agonist at D3, with a selectivity for D3 of about 100-fold higher than that for D2, this drug (at 10 mg day1) did not appear effective against schizophrenia symptoms. Other drugs moderately selective for D3, such as S33138 and A437,203, are currently being tested in schizophrenia patients. The highly D3-selective drug, FAUC 365, has not yet been tested in this disease. It is possible that the D3-selective compounds may be helpful in treating drug abuse.
The Dopamine D4 Receptor
0
4
8
12 16 20 24 28 D2 density (pmol g−1)
32
36
40
Figure 8 Bimodal pattern of D2 receptors in postmortem striata from individuals who had schizophrenia during life. The control individuals had died of nonneurological disorders. The bimodal pattern was found in the schizophrenia tissues, regardless of whether the individuals had received antipsychotics or not. Each square indicates a different postmortem human brain (caudate nucleus or putamen regions). Adapted from Seeman P and Niznik HB (1990) Dopamine receptors and transporters in Parkinson’s disease and schizophrenia. FASEB Journal 4: 2737–2744, with permission from Federation of American Societies for Experimental Biology.
The dopamine D4 receptor was cloned in 1999; its sequence is shown in Figure 12. The D4 receptor probably has more polymorphisms than any other protein in the body. For example, the intracellular loop contains repeat forms of a 16-amino acid polypeptide, the number of repeats varying from person to person. Most people have four such repeats, but up to ten repeats are known. Moreover, the precise sequence within each repeat usually varies from person to person, with at least 20 different types of repeat units known, thereby resulting in a massive number of polymorphisms in the human population. An unusual polymorphism in D4 occurs at position 194, where glycine replaces valine in 13% of Africans
Dopamine Receptors and Antipsychotic Drugs in Health and Disease 401
Figure 9 Anatomical location for gene expression of dopamine receptor genes in human brain. abbreviations: CX, cerebral cortex; L, lateral ventricles; C, caudate nucleus; P, putamen; G, globus pallidus; AC, nucleus accumbens; O, olfactory tubercle; H, hypothalamus; AM, amygdala; Hipp, hippocampus; VTA, ventral tegmental area; SN, substantia nigra; ICG, Islands of Calleja.
and Caribbeans (Figure 13), but not in Caucasians. This mutant form of D4 markedly reduces its sensitivity to dopamine. One young man in the Caribbean was found to be homozygous for this V194G polymorphism, but no medical abnormalities were found. Another D4 polymorphism occurs when the GASA sequence is missing at position 21. Interestingly, clozapine has a higher affinity at D4 than at D2, as shown in Table 2. Nevertheless, despite clozapine’s selectivity for D4, clozapine occupies the necessary 60–70% of brain D2 receptors at clinical doses (400 mg day1), compatible with the idea that D2 is the therapeutic target for clozapine, as with all the
other antipsychotics. It may be noted that isoclozapine causes catalepsy, in contrast to clozapine, which does not elicit catalepsy. Both drugs have dentical affinity for D4, but isoclozapine has higher affinity for D2 (see Table 2), and, therefore, causes catalepsy. Although the gene expression of D4 was found to be elevated in the frontal cortex of schizophrenia tissues, selective D4 antagonists, such as sonepiprazole and L-745,870, did not have any antipsychotic action. Some evidence suggests that the longer forms of D4, such as D4.7, with seven repeats, or D4.9, are found in hyperactive individuals or in those persons who take unusual risks, but this is controversial.
402 Dopamine Receptors and Antipsychotic Drugs in Health and Disease
D2 control striata (m and f) 2.2% loss per decade (p < 0.002)
D2 density, pmol g−1
30
Upper limit for adults 20
10
0
0 1 2 3 4 5 10
20
30 40 50 Age, years
60
70
80
90 100
Figure 10 Postmortem human D2 densities in the striatum. After an initial rapid growth period in the first 3 years of life, the D2 receptor density is rapidly pruned before age 10, and thereafter decays by 2.2% every 10 years. Adapted from Seeman P, Bzowei NH, Guan H-C et al. (1987) Human brain dopamine receptors in children and aging adults. Synapse 1: 399–404, with permission from John Wiliy & Sons Inc.
Figure 11 Amino acid sequence of the human cloned dopamine D3 receptor. A polymorphism occurs at position 9, where glycine replaces serine. When a frame shift occurs in the cytoplasmic loop, as shown, the receptor is nonfunctional.
The Dopamine D5 Receptor
Regulation of Dopamine Receptors
The dopamine D5 receptor was cloned in 1991 (Sunahara), and its sequence is shown in Figure 14. There are two pseudogenes of D5, where the amino acid sequence stops at position 154. Although D5 is essentially D1-like in sequence and function, the characteristic feature of D5 is that it is more sensitive to dopamine than D1 is, as indicated in Table 1 for the K50 values.
Each of the five dopamine receptors has a state of high affinity and a state of low affinity for dopamine, an example of which is shown in Figure 15 for the D2 receptor in anterior pituitary tissue. Dopamine receptors belong to a group of more than 1000 receptors known to be associated with G-proteins. The binding of an agonist to such a G-linked receptor occurs in two concentration ranges. Low nanomolar
Dopamine Receptors and Antipsychotic Drugs in Health and Disease 403
Figure 12 Amino acid sequence of the human dopamine D4 receptor and its polymorphisms. The cytoplasmic loop has repeat units of 16 amino acid polypeptides. Different humans have different numbers of repeats. Shown are four repeats (D4.4), the most common, and seven repeats (D4.7).
concentrations of the agonist binds to the high-affinity state of the receptor, while high micromolar concentrations bind to the low-affinity state of the receptor. Generally, it is the high-affinity state of the receptor that is the functionally active state of the receptor, because the agonist affinities for the high-affinity state are usually identical to the concentrations that elicit the physiological action of the agonists. This holds for many neurotransmitter receptors, including dopamine D2 receptors, cholinergic muscarinic receptors, a2-adrenoceptors, and b2-adrenoceptors. D2High is the functional state in the anterior pituitary, upon which dopamine and other dopamine-like drugs (bromocriptine) act to inhibit the release of prolactin. D2High is presumably also functional on the terminals of the dopamine-containing terminals, and these receptors are usually referred to as presynaptic receptors. Although it has been reported that 90% of the D2 receptors in brain slices are in the D2High state, the proportion of D2 receptors in the high-affinity
state in homogenized striatum in vitro is generally between 15% and 20%. The D2High state can be quickly converted into the D2Low state by guanine nucleotide, as illustrated in Figure 15 for anterior pituitary tissue and in Figure 16 for the striatum. Furthermore, as shown in Figure 16, the high-affinity state of D2 is most readily detected by dopamine competing with [3H]domperidone, but not [3H]raclopride or [3H]spiperone, which are less sensitive to the competitive action of dopamine. In fact, it is known that the physiological concentration of dopamine in the synaptic space (between neurons) is 2–4 nM, matching the known dissociation constant of 2 nM for dopamine at the D2High receptor. This latter value of 2 nM is obtained from the dopamine/[3H]domperidone competition curve in Figure 16, using the standard Cheng–Prusoff equation to correct for the ligand concentration and the [3H]domperidone Kd. There are at least two views of the physical existence of the high-affinity state. The traditional view is that
404 Dopamine Receptors and Antipsychotic Drugs in Health and Disease D4
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Figure 13 Additional polymorphisms of the human dopamine D4 receptor. Approximately 13% of Africans and Caribbeans have a glycine replacing valine at position 194. Approximately 8% of Italians have an additional ASAG peptide inserted at the position shown.
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Figure 14 Amino acid sequence of the human dopamine D5 receptor. Two polymorphisms of D5 exist where the sequence abruptly stops to create a sequence of 154 amino acids instead of the full-length sequence of 477 amino acids.
the high-affinity state of the receptor exists when the receptor, R, is associated with the G-protein, and the agonist, D, binds to this high-affinity state to form the ‘ternary complex,’ namely DRG. This view
of the receptor proposes that the low-affinity state occurs when the G-protein is not associated with the receptor. However, there are many significant shortcomings with this view of the high-affinity state of the
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receptor in the ternary complex model. For example, the ternary complex suggests that RG should have a transient existence. This is not the case, however, because it has been found that the purified muscarinic RG is stable. Moreover, the purified muscarinic receptor, free of G and GDP, clearly shows high-affinity and low-affinity states. An alternate view of the high-affinity state of the receptor is the ‘cooperativity’ model, as worked out by J Wells and colleagues. The cooperative model proposes that the receptor cooperates with other receptors to form either a dimer, a tetramer, or a larger oligomer. The receptor is in the high-affinity state when it is vacant and unoccupied by the agonist. However, when the agonist binds to the vacant receptor, the occupied receptor interacts or ‘cooperates’ with the other receptors (within the tetramer) such that the affinity of the other receptors for the agonist is markedly reduced. This reduced affinity for the agonist is a result of ‘negative cooperativity’ between the receptors, and corresponds to the low-affinity state of the receptor. In other words, if there is very strong negative cooperativity, then the second, third, and fourth receptors (within the tetramer) would hardly bind the agonist, and only the high-affinity sites would be observed in the competition between, say, dopamine and [3H]domperidone, all taking place at the first receptor. These events are depicted in a diagram in Figure 17.
D2 Interactions with Other Receptors The D2 receptor is known to interact with the D1 receptor as well as with other receptors, such as the adenosine A2 receptor. Many D1/D2 interactions occur at all levels, including the molecular level, where D1 and D2 can form functional heterodimers with one another. Such interaction also occurs at the cellular level, where the block of D1 (by SCH23390) unmasks the high-affinity state of the D2 receptor, D2High, as shown in Figure 18. This experiment shows that D1 normally inhibits or suppresses the high-affinity state of D2.
Psychosis and the D2High Basis of Dopamine Supersensitivity The dopamine hypothesis of psychosis or schizophrenia was first outlined by J. Van Rossum in 1967: The hypothesis that neuroleptic drugs may act by blocking dopamine receptors in the brain has been substantiated by preliminary experiments with a few selective and potent neuroleptic drugs. There is an urgent need for a simple isolated tissue that selectively responds to dopamine so that less-specific neuroleptic drugs can also be studied and the hypothesis further tested. . . . When the hypothesis of dopamine blockade by neuroleptic agents can be further substantiated it may have fargoing consequences for the pathophysiology of schizophrenia. Overstimulation of dopamine receptors could then be part of the aetiology.
406 Dopamine Receptors and Antipsychotic Drugs in Health and Disease D2 clone: D2High detected by [3H]domperidone, but not by [3H]spiperone or [3H]raclopride in 120 mM NaCI +20 0 gua mM nine nuc leot ide
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Dopamine (nM) Figure 16 Low nanomolar concentrations of dopamine readily inhibit the binding of [3H]domperidone at the high-affinity state of dopamine D2 receptors (between 1 and 100 nM), in contrast to [3H]raclopride and [3H]spiperone, which are less sensitive to competition by dopamine. The high-affinity state, D2High, is converted to the low-affinity state, D2Low, by guanine nucleotide. Adapted from Seeman P, Tallerico T, and Ko F (2003) Dopamine displaces [3H]domperidone from high-affinity sites of the dopamine D2 receptor, but not [3H] raclopride or [3H]spiperone in isotonic medium: Implications for human positron emission tomography. Synapse 49: 209–215, with permission from John Wiley & Sons Inc.
As noted earlier, it has not been clearly established that D2 receptors are elevated in psychosis or schizophrenia, although studies using brain imaging of healthy co-twins of schizophrenia individuals, as well as single-photon brain imaging of nonmedicated psychotic patients in at least one study, have shown significant elevations of D2. At present, the most promising direction in this field is to examine the molecular basis of dopamine supersensitivity, because up to 70% of patients are supersensitive to either methylphenidate or amphetamine at doses which do not affect controls. Moreover, a wide variety of brain alterations in animals (lesions, birth injury by C-section, amphetamine or phencyclidine treatment, knockouts of a variety of receptors) all lead to the final common finding of
behavioral dopamine supersensitivity and elevated proportions of D2 receptors in the D2High state in the striatum. For example, repeated administration of amphetamine to animals or humans leads to behavioral dopamine supersensitivity. While the density of D2 receptors in the striatum does not change in such studies, it is remarkable that the density of D2High receptors increases dramatically by several fold, as shown in Figure 19. A similar situation occurs in animals that receive hippocampal lesions neonatally. Such animals, as adults, reveal behavioral dopamine supersensitivity, and the striatum contains a marked increase in the proportion of D2 receptors in the high-affinity state, as shown in Figure 20. Therefore, the molecular control of the high-affinity state of D2 is emerging as a
Dopamine Receptors and Antipsychotic Drugs in Health and Disease 407
Figure 17 (a) The cooperativity model for the dopamine D2 receptor, according to J Wells and colleagues. The receptor is proposed to exist as an oligomer of D2 receptors, such as a tetramer of D2. Each of the vacant receptors is in the high-affinity state, D2High. When dopamine first attaches to one of the vacant D2 receptors in the tetramer, the occupied D2 then interacts with the other three receptors within the tetramer to reduce their affinity for dopamine (i.e., negative cooperativity). (b) One molecular explanation for dopamine supersensitivity is that an unknown factor may reduce the negative interaction between the D2 receptors, thereby allowing more dopamine to occupy more D2High receptors.
Block of D1 reveals high states for D2 in rat striatum Control 2500
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Figure 18 Regulation of D2High by the D1 receptor. (a) Dopamine inhibited the binding of [3H]raclopride to D2 receptors at dopamine concentrations higher than 100 nM. However, in the presence of SCH23390 to block D1 receptors, the binding of [3H]raclopride was readily inhibited by 1–100 nM, corresponding to the presence of D2High. These data suggest that D1 actively suppresses the existence of the functional D2High state. (b) In comparison to 100 nM SCH23390 unmasking the D2High state (a), other drugs do not lead to such unmasking of D2High. L745,870 is a dopamine D4 receptor antagonist. Adapted from Seeman P and Tallerico T (2003) Link between dopamine D1 and D2 receptors in rat and human striatal tissues. Synapse 47: 250–254, with permission from John Wiley & Sons Inc.
408 Dopamine Receptors and Antipsychotic Drugs in Health and Disease
central problem in this field. At present, there is uncertainty as to whether this high-affinity state of D2 is controlled through Go or one of the Gi proteins, because this varies from cell to cell.
According to the negative cooperativity model (Figure 17), the increased number of D2 receptors in the high-affinity state, D2High, found in the striata of supersensitive animals, may be attributed to a reduction in the overall negative cooperativity between the receptors, as illustrated in Figure 17. Thus, in order to determine the molecular mechanism of dopamine supersensitivity, it will be essential to determine the factors that reduce negative cooperativity among the D2 receptors or that alter the association of the receptor with its G-protein. The role of guanine nucleotides in regulating the overall sensitivity of the dopamine D2 receptors would be to alter the extent of the receptor–receptor negative cooperativity.
Current Clinical and Basic Research on Dopamine Receptors
Figure 19 Repeated administration of amphetamine to rats leads to behavioral dopamine supersensitivity. While the total density of D2 receptors was normal in the striata of such supersensitive rats (about 26 pmol g1), the density of D2High receptors was markedly elevated by 355%, from a control value of 2.9 pmol g1 to the elevated level of 10.3 pmol g1. Nonspecific binding of [3H]raclopride was done in the presence of 10 mM S-sulpiride. In the presence of 200 mM guanilylimidodiphosphate (G.N.), all the D2High receptors were converted to D2Low. Adapted from Seeman P, Tallerico T, Ko F, et al. (2002) Amphetaminesensitized animals show a marked increase in dopamine D2High receptors occupied by endogenous dopamine – Even in the absence of acute challenges. Synapse 46: 235–239, with permission from John Wiley & Sons Inc.
Of the five dopamine receptors and their many variants, the D2 receptor and its properties continue to be most actively investigated, because D2 is the main clinical target of antipsychotics and of dopamine agonist treatment of Parkinson’s disease. The D1 receptor, however, also has an important clinical role in treating Parkinson’s disease because the stimulation of D1 synergizes with the stimulation of D2, possibly via D1/D2 heterodimers or cell–cell interactions. A current active area of clinical research on dopamine receptors is to measure the occupancy of D2 receptors both in the striatum and outside the striatum in individuals taking antipsychotic medications. Some researchers find that the same D2 occupancy occurs in both striatal and limbic regions, while others find a lower occupancy in the limbic regions.
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As previously noted, therapeutic doses of antipsychotics occupy 60–80% of the D2 receptors in psychotic patients, while D2 occupancies higher than 80% are associated with elevated serum prolactin and parkinsonism. The new aripiprazole antipsychotic tends to occupy more than 80% of D2 but does not seem to cause parkinsonism at these higher levels. This may be a result of the fact that aripiprazole, like clozapine and quetiapine, quickly desorbs from the D2 receptor (in under 60 s in vitro), minimizing prolactin elevation and parkinsonism. Rapid desorption permits the dopamine neurotransmission to proceed more normally. However, if the antipsychotic drug dose or antipsychotic concentration in the plasma remains high, then the antipsychotic drug will readsorb. In effect, fat-soluble antipsychotics remain adsorbed to D2 in patients for 1 or 2 days or even more, while the plasma concentration falls. Less fat-soluble antipsychotics such as clozapine or quetiapine remain attached to D2 in patients for only 6–12 h, immediately falling in D2 occupancy as the plasma concentration falls. Probably the most central question in determining the basis of psychosis or schizophrenia is to determine the molecular basis of dopamine supersensitivity and to determine which proteins or genes regulate the maintenance of D2 receptors in their high-affinity state. See also: Dopamine; Dopamine: Cellular Actions.
Further Reading Agid O, Kapur S, Arenovich T, et al. (2003) Delayed-onset hypothesis of antipsychotic action: A hypothesis tested and rejected. Archives of General Psychiatry 60: 1228–1235. Baumeister AA and Francis JL (2002) Historical development of the dopamine hypothesis of schizophrenia. Journal of the History of Neurosciences 11: 265–277. Bressan RA, Erlandsson K, Jones HM, et al. (2003) Is regionally selective D2/D3 dopamine occupancy sufficient for atypical antipsychotic effect? An in vivo quantitative [123I]epidepride SPET study of amisulpride-treated patients. American Journal of Psychiatry 160: 1413–1420. Farde L, Nordstrom AL, Wiesel FA, et al. (1992) Positron emission tomographic analysis of central D1 and D2 dopamine receptor occupancy in patients treated with classical neuroleptics and clozapine. Relation to extrapyramidal side effects. Archives of General Psychiatry 49: 538–544.
George SR, Watanabe M, Di Paolo T, et al. (1985) The functional state of the dopamine receptor in the anterior pituitary is in the high affinity form. Endocrinology 117: 690–697. Hagberg G, Gefvert O, Bergstro¨m M, et al. (1998) N-[11C]methylspiperone PET, in contrast to [11C]raclopride, fails to detect D2 receptor occupancy by an atypical neuroleptic. Psychiatry Research 82: 147–160. Hirvonen J, van Erp TG, Huttunen J, et al. (2005) Increased caudate dopamine D2 receptor availability as a genetic marker for schizophrenia. Archives of General Psychiatry 62: 371–378. Kapur S and Seeman P (2001) Does fast dissociation from the dopamine D2 receptor explain the action of atypical antipsychotics? – A new hypothesis. American Journal of Psychiatry 158: 360–369. Seeman P (1987) Dopamine receptors and the dopamine hypothesis of schizophrenia. Synapse 1: 133–152. Seeman P (2002) Atypical antipsychotics: Mechanism of action. Canadian Journal of Psychiatry 47: 27–38. Seeman P, Bzowej NH, Guan H-C, et al. (1987) Human brain dopamine receptors in children and aging adults. Synapse 1: 399–404. Seeman P, Chau-Wong M, Tedesco J, et al. (1975) Brain receptors for antipsychotic drugs and dopamine: Direct binding assays. Proceedings of the National Academy of Sciences of the United States of America 72: 4376–4380. Seeman P, Lee T, Chau-Wong M, et al. (1976) Antipsychotic drug doses and neuroleptic/dopamine receptors. Nature (London) 261: 717–719. Seeman P and Niznik HB (1990) Dopamine receptors and transporters in Parkinson’s disease and schizophrenia. FASEB Journal 4: 2737–2744. Seeman P and Tallerico T (1999) Rapid release of antipsychotic drugs from dopamine D2 receptors: An explanation for low receptor occupancy and early clinical relapse upon drug withdrawal of clozapine or quetiapine. American Journal of Psychiatry 156: 876–884. Seeman P and Tallerico T (2003) Link between dopamine D1 and D2 receptors in rat and human striatal tissues. Synapse 47: 250–254. Seeman P, Tallerico T, and Ko F (2003) Dopamine displaces [3H] domperidone from high-affinity sites of the dopamine D2 receptor, but not [3H]raclopride or [3H]spiperone in isotonic medium: Implications for human positron emission tomography. Synapse 49: 209–215. Seeman P, Tallerico T, Ko F, et al. (2002) Amphetamine-sensitized animals show a marked increase in dopamine D2High receptors occupied by endogenous dopamine – Even in the absence of acute challenges. Synapse 46: 235–239. Seeman P, Weinshenker D, Quirion R, et al. (2005) Dopamine supersensitivity correlates with D2High states, implying many paths to psychosis. Proceedings of the National Academy of Sciences of the United States of America 102: 3513–3518. Wilson AA, McCormick P, Kapur S, et al. (2005) Radiosynthesis and evaluation of [11C]-(þ)-PHNO as a potential radiotracer for in vivo imaging of the dopamine D2 high affinity state with positron emission tomography (PET). Journal of Medicinal Chemistry 48: 4153–4160.
Dopamine: Cellular Actions G Bernardi and N B Mercuri, Universita` di Roma ‘Tor Vergata’, and IRCCS Fondazione Santa Lucia, Rome, Italy ã 2009 Elsevier Ltd. All rights reserved.
There is a plethora of experimental and clinical evidence indicating that dopamine (DA) is a key element in promoting and regulating the reward and motivation processes occurring in the brain. Therefore, the state of pleasure and well-being that one might derive from everyday activities such as playing, eating palatable food, drinking, listening to a melody, smelling a perfume, and having sex certainly rely on the correct operation of this catecholamine at the neuronal level. On the other hand, DA plays a significant role when an altered behavior is instituted. Hence, drug abuse and craving, compulsive acts, and affective problems could depend on maladaptive changes in DA neurotransmission. This catecholamine is synthesized by discrete groups of cells (mesencephalic, ipothalamic, and retinal). It is noteworthy that the main source of DA for the central nervous system arises from the population of neurons originating in the ventral mesencephalon (ventral tegmental area, substantia nigra pars compacta) and mainly terminating in the cerebral cortex, accumbens, and striatum. The diffuse projections of the dopaminergic neurons suggest that DA is able to act in different brain areas to induce motivation and reward. Accordingly, behavioral, pharmacological, and clinical studies have demonstrated that the cerebral regions principally involved in incentive and behavioral activation are the prefrontal cortex, the ventral/dorsal striatum, and the ventral mesencephalon itself. Indeed, these regions are the richest in DA. Therefore, in order to better understand the reward processes, it is of crucial importance to understand the functions of DA at the cellular and synaptic level within the different areas indicated previously. Another important aspect to be considered for the clarification of DA action is that the catecholamine stimulates subtypes of specific receptors that cause distinct effects on neurons. There are two families of G-protein-linked DAergic receptors, D1 and D2. While the D1 family (constituting the D1 and D5 receptors), linked to Gas, stimulates adenylate cyclase, the D2 family (constituting the D2, D3, and D4 receptors), linked to Ga0/i, inhibits adenylate cyclase. Usually, the D1-mediated effects are cyclic adenosine monophosphate (cAMP)and protein kinase A-dependent, while the D2mediated effects depend on the inhibition of cAMP and protein kinase A (PKA), eventually the activation
410
of phospholipase C-inositol triphosphate (IP3), and changes in intracellular Ca2þ. The dichotomy in the formation of cAMP results in opposite consequences on neuronal excitability. In addition, by stimulating either similar or different receptors, DA might exert competing presynaptic and/or postsynaptic actions to modify neuronal responsiveness. In the present article we reexamine the electrophysiological actions of DA on neurons located in those brain areas that are linked to the reward processes.
Dopamine Actions in the Frontal Cortex DA in the frontal cortex has implications in reward, the psychomotor and hedonic effects of drug abuse, drug seeking, and certain forms of memory formation linked to salience. Electrophysiological data have extensively shown a variety of effects caused by DA and DAergic agonists in this structure. Although there is a consensus that DA inhibits cortical neurons, several studies have also reported DA-mediated neuronal excitation. DA might have different effects on the excitability of pyramidal neurons by reducing either persistent voltage-dependent sodium currents or voltage-dependent potassium currents. It has been also reported that the stimulation of D1 receptors can activate firing rate by modulating synaptic strength. In fact, some authors have shown that DA significantly enhances excitatory postsynaptic current (EPSC) amplitudes in the pyramidal cells. Recent data have also suggested that DA potentiates the late postsynaptic responses in an excitatory postsynaptic potential (EPSP) train evoked by a sustained presynaptic stimulation. However, a combined activation of D1 and D2 receptors could also determine a reduction of the efficacy of excitatory synaptic transmission at synapses onto pyramidal cells. Moreover, contrasting effects of DA on neuronal responsiveness to glutamate agonists have been reported. Thus, bath application of Nmethyl-D-aspartate (NMDA), a-amino-3-hydroxy-5methyl-4-isoxazole propionic acid (AMPA), and the D1 agonist SKF 38393 induces concentration-dependent excitability increases, whereas the D2 receptor agonist quinpirole causes a concentration-dependent excitability decrease. Furthermore, opposing D1- or D2mediated actions, depending on DA concentrations, have been shown to modify the inhibitory drive on cortical neurons. As a result, a nanomolar concentration of DA might enhance the inhibitory postsynaptic currents (IPSCs) via D1 receptors while a micromolar concentration could decrease the IPSCs via D2 receptors. In contrast, it has been reported that the stimulation of presynaptic D1-like receptors
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decreases the IPSCs in layer II–III of neocortical pyramidal cells. In addition, DA could induce important changes in the local network connections of pyramidal cells with g-aminobutyric acid (GABA)ergic interneurons. Thus, the activation of D1 receptors reduces the amplitude of the EPSPs or causes a reversible membrane depolarization in the fast-spiking (FS) interneurons. On the other hand, the stimulation of D4 receptors, limiting interneuronal excitability, favors multiple spike discharge of pyramidal cells. Furthermore, DA has been shown to sustain enduring changes of the synaptic signals. Thus, the long-term potentiation (LTP) of the excitatory transmission on pyramidal neurons requires D1 receptor stimulation. This could activate PKA and increase the surface expression of GluR1 in prefrontal cortex neurons. On the other hand, a constant presence of DA in the extracellular space might facilitate long-term depression (LTD). It is supposed that these DAmediated persistent changes in synaptic responses (LTP-LTD) could be important in processes linked to reward.
Dopamine Actions in the Ventral and Dorsal Striatum A sizable body of evidence considers the ventral striatum (nucleus accumbens) as a fundamental station for the DA-regulated reward processes. In fact, accumbens DA modulates motivation and cognitive aspects related to incentives. In addition, DA effects in this structure are particularly important in mediating the activating and addictive effects of psychostimulants. As in the cerebral cortex, accumbal DA might change the activity of medium spiny neurons by involving multiple mechanisms. It has been shown that this catecholamine causes a membrane hyperpolarization that is due to the activation of D1 receptors and is associated with an increase in potassium conductance. However, DA also causes a membrane depolarization that is generated by the stimulation of D2 receptors and sustained by a decrease in potassium conductance. In addition, D2 receptors seem to reduce the direct excitability of medium spiny accumbal neurons by inhibiting evoked Naþ spikes through the involvement of slow A-type Kþ currents, while reported effects of DA on inward rectification and ‘leak’ Kþ currents could favor excitability. D2 receptors could also cause an enhancement of voltage-sensitive sodium currents mediated by the suppression of the cyclic AMP/PKA cascade and the facilitation of intracellular Ca2þ signaling. In addition, D1 receptor stimulation appears to suppress N- and P/Q-type Ca2þ currents by activating a cAMP/
protein kinase A/protein phosphatase signaling system, presumably leading to channel dephosphorylation. Moreover, a coactivation of D1 and D2 receptors and signaling through G-protein bg subunits and PKA could enhance spike firing in nucleus accumbens shell medium spiny neurons. Referring to the DA-induced changes of synaptic functions, there are data supporting an inhibitory role of this catecholamine on the EPSP-IPSP sequences The depression of the EPSP appears to be mainly mediated by a D2 receptor-induced decrease of AMPA currents, while the inhibition of the EPSCs progressively diminishes; the inhibition of the IPSCs seems to be persistent. The resultant effect could cause an increased excitability of these neurons. Activation of D3 receptors is also reported to suppress the efficacy of the inhibitory synaptic transmission in the nucleus accumbens by increasing the phospho-dependent endocytosis of GABA-A receptors. Interestingly, DA also attenuates the neuronal responses to repetitive activation of glutamatergic afferents and thereby could block LTP. On the other hand, the accumbal LTD requires, among other factors, dopamine D1 receptor stimulation and cAMPPKA activation. DA-mediated plastic adaptations of the neural synaptic functions in the nucleus accumbens caused by psychostimulants might control the development and long-term maintenance of sensitization to abused drugs. The increase of extracellular DA in the dorsal striatum of humans (caudoputamen) related to selfreported measures of liking and the feeling of a ‘high’ (euphoria) implies that this structure is an essential element of the circuitry responsible for the control of motivated behavior and reward. However, in spite of the recognized importance of DA in reward-related locomotor activation, the precise nature of the modulation that DA exerts on striatal neurons remains largely elusive. There are early electrophysiological studies demonstrating a predominant inhibitory effect of DA on low-firing medium spiny striatal cells. Thus, the most common responses produced by DA on these cells are the decrease of voltage-dependent inward conductances and the modulation of the corticostriatal synaptic transmission. The DA-mediated inhibition of firing activity seems to be mediated by the activation of D1 receptors. The stimulation of D1-like receptors initiates a cascade of intracellular events, including cAMP formation and activation of cAMP-dependent PKA that reduce excitability and possibly activate the neuronal changes linked to reward. An additional stimulation of D2 receptor reduces glutamate- and GABA-mediated currents. Some authors have also described a reduction of
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AMPA-mediated postsynaptic current caused by D2 receptors in medium-sized striatal neurons. In contrast, the stimulation of D1 receptors could potentiate the NMDA-induced currents and reduce GABAevoked currents by activating a PKA/dopamine- and cAMP-regulated phosphoprotein (DARPP-32)/protein phosphatase 1 signaling cascade. It must also be considered that the cholinergic interneurons are affected by DA. While the stimulation of D1 receptors depolarizes and excites the cells, the stimulation of D2 receptors reduces the GABAergic and cholinergic inputs on these interneurons. With regard to the synaptic function, the two main subclasses of DA receptors contribute in the formation of the long-term changes (LTD/LTP) occurring at the corticostriatal terminals. Therefore, D1-like and D2-like receptors interact synergistically to allow LTD formation. A form of LTD in the striatum is also bidirectionally modulated by D2 receptors and requires the regulation of mGluR-dependent endocannabinoid release. The LTP is blocked by dopamine depletion but is not affected by a D2 antagonist. Furthermore, the use of knockout mice with the ablation of D1 receptors has demonstrated a disrupted corticostriatal LTP, whereas pharmacological blockade of D5 receptors prevented LTD in these animals. Interestingly, DA-dependent stimulation of DARPP-32, a small protein expressed in the spiny neurons which acts as a potent inhibitor of protein phosphatase-1, is necessary for the expression of both corticostriatal LTD and LTP and is required for the full expression of the behavioral response to cocaine and amphetamine. On this basis, a role for neostriatal DA in the formation of a pathological drug habit has been hypothesized.
Dopamine Actions in the Ventral Mesencephalon There is homogeneous experimental material demonstrating that DA, released from the dendrites, has an inhibitory action on the dopaminergic cells located in the ventral tegmental area and substantia nigra pars compacta. This inhibition is D2-mediated and is due to a membrane hyperpolarization depending on a G-protein (Go/i)-activated potassium current (GIRK). Therefore, by modifying firing discharge of the DAergic cells, the catecholamine might encode the salience of stimuli linked to natural and artificial rewards. Although there is evidence that the dopaminergic neurons fire during reward anticipation, encoding errors in reward prediction, the inhibition of firing mediated by locally released DA could play part of the reward processes. Therefore, a delicate balance of activity in different subsets of DA cells could regulate the timing of the various expressions of reward. In addition, DA, by modifying the strength
of the excitatory transmission in the ventral mesencephalon, might contribute to enhancing memory processes linked to salience. Thus, DA could block the induction of LTD in the ventral midbrain via activation of D2-like receptors. This DA-dependent modulation of the synaptic code could be a substrate of memory traces linked to reward. Of note is the fact that the DA-mediated long-term alterations in the strength of the excitatory transmission have been reported to occur after treatments with psychomotor stimulants. Since the development of addictive behaviors shares common features with traditional learning models, these DA-dependent modifications of synaptic plasticity could represent an important substrate for the acquisition of reward-related behaviors.
Conclusions It is believed that the multiple and, in some instances, opposite effects of DA on cerebral neurons could account for the whole range of behavioral modifications linked to reward processes. However, in spite of the large amount of experimental work so far available, we have to admit that the overall picture describing the actions of DA on single cells and groups of cells is rather complex. Undoubtedly, there is a certain unpredictability in the neuronal responses to DA that could likely depend on competing presynaptic, postsynaptic, and nonsynaptic mechanisms, the type of receptor involved, and the type of neuron. Notwithstanding these limitations, future effort in studying the function of DA should be concentrated on individuating and eventually correcting the processes that control reward at the cellular level. See also: Dopamine; Dopamine Receptors and Antipsychotic Drugs in Health and Disease.
Further Reading Bernardi G, Cherubini E, Marciani MG, et al. (1982) Responses of intracellularly recorded cortical neurons to the iontophoretic application of dopamine. Brain Research 245: 267–274. Beurrier C and Malenka RC (2002) Enhanced inhibition of synaptic transmission by dopamine in the nucleus accumbens during behavioral sensitization to cocaine. Journal of Neuroscience 22: 5817–5822. Bonci A, Bernardi G, Grillner P, et al. (2003) The dopaminecontaining neuron: Maestro or simple musician in the orchestra of addiction? Trends in Pharmacological Science 24: 172–177. Centonze D, Grande C, Saulle E, et al. (2003) Distinct roles of D1 and D5 dopamine receptors in motor activity and striatal synaptic plasticity. Journal of Neuroscience 23: 8506–8512. Cepeda C, Colwell CS, Itri JN, et al. (1998) Dopaminergic modulation of NMDA-induced whole cell currents in neostriatal neurons in slices: Contribution of calcium conductances. Journal of Neurophysiology 79: 82–94.
Dopamine: Cellular Actions 413 Chen G, Kittler JT, Moss SJ, et al. (2006) Dopamine D3 receptors regulate GABAA receptor function through a phospho-dependent endocytosis mechanism in nucleus accumbens. Journal of Neuroscience 26: 2513–2521. Kreitzer AC and Malenka RC (2005) Dopamine modulation of state-dependent endocannabinoid release and long-term depression in the striatum. Journal of Neuroscience 25: 10537–10545. Lacey MG, Mercuri NB, and North RA (1987) Dopamine acts on D2 receptors to increase potassium conductance in neurones of the rat substantia nigra zona compacta. Journal of Physiology 392: 397–416. Law-Tho D, Hirsch JC, and Crepel F (1994) Dopamine modulation of synaptic transmission in rat prefrontal cortex: An in vitro electrophysiological study. Neuroscience Research 21: 151–160. Perez MF, White FJ, and Hu XT (2006) Dopamine D(2) receptor modulation of K(þ) channel activity regulates excitability of
nucleus accumbens neurons at different membrane potentials. Journal of Neurophysiology 96: 2217–2228. Schultz W (2002) Getting formal with dopamine and reward. Neuron 36: 241–263. Seamans JK and Yang CR (2004) The principal features and mechanisms of dopamine modulation in the prefrontal cortex. Progress in Neurobiology 74: 1–58. Thomas MJ, Malenka RC, and Bonci A (2000) Modulation of long-term depression by dopamine in the mesolimbic system. Journal of Neuroscience 20: 5581–5586. Uchimura N, Higashi H, and Nishi S (1986) Hyperpolarizing and depolarizing actions of dopamine via D-1 and D-2 receptors on nucleus accumbens neurons. Brain Research 375: 368–372. Wise RA (2004) Dopamine, learning and motivation. Nature Reviews Neuroscience 5: 1–12.
Noradrenaline R D Wassall, University of Oxford, Oxford, UK N Teramoto, Kyushu University, Fukuoka, Japan T C Cunnane, University of Oxford, Oxford, UK ã 2009 Elsevier Ltd. All rights reserved.
Introduction From its humble beginnings, with biochemical analysis of adrenal glands and neurotransmitter release, we have come to realize that sympathetic neurotransmission is not quite as simple as first thought. We are taught that noradrenaline (norepinephrine) is the major neurotransmitter released from sympathetic nerves, and that with adrenaline (epinephrine), it is a key player in the ‘fight-or-flight’ response. Indeed, few people can escape the discussion of the ‘adrenaline-high’ associated with athletes and thrillseekers alike, and few of us will avoid the use of drugs to alter sympathetic signaling for cardiovascular problems, anxiety or depression as we age. We might think that such an important and familiar neurotransmitter system would be well characterized, but it has not always been so. In the past, noradrenaline proved to be an elusive agent to identify when the concepts of ‘neurochemical transmission’ were being formed, and even today, there are a myriad of questions surrounding its co-storage, release, and postjunctional receptor mechanisms. Here, we give a brief overview of some of the facts, and unravel some of the mysteries of this celebrated molecule.
A Brief History of the Discovery of Noradrenaline Galen (AD 130–200), arguably the greatest anatomist and physiologist of antiquity, first described the gross anatomical features of autonomic nerves. He suggested that, being hollow, they allowed the transfer of so-called ‘animal spirits’ between organs, producing the phenomena of ‘sympathy’, which was a vague term used to describe the coordination or cooperation of organs. However, the anatomical division of the parasympathetic and sympathetic nervous systems can be traced to Eustachius in the sixteenth century, who regarded the sympathetic and vagus nerves as separate, although describing incorrectly that the ‘sympathetic nerve’ was a continuation of the abducens (cranial nerve VI). Through the work of Winslow, Whytt, and Bichat two centuries later, an important outline of the sympathetic nervous system was laid out, with the visceral organs controlled by the
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ganglia, whereas voluntary action was concerned with the brain. The first functional studies on true sympathetic nerves began with Bernard, who dilated vessels by sectioning the sympathetic nerves, and Brown-Se´quard, who contracted the vessels by stimulating the cut end of the nerve. Irritation of various neuronal structures had been shown to increase the heart rate and its contractility; however, modern work on the autonomic nervous system and its functional divisions has been based largely on the work of Gaskell and Langley, who defined the essentials of the macroscopic functions of the sympathetic nervous system in the early to mid-1900s. Langley’s student, Elliott, showed that adrenaline, discovered approximately 20 years earlier by Bates, had the same general effect as stimulation of the sympathetic nerves. The chemical nature of sympathetic neurotransmission had been assumed by Elliott and Dale, who showed the sympathetic-like effects of ergot alkaloids, and when Loewi monumentally demonstrated the chemical nature of neurotransmission in a frog’s heart, later found to be due to acetylcholine, it seemed that adrenaline would be an attractive candidate neurochemical in the sympathetic nervous system. The isolation of ‘sympathin’ from sympathetic nerves by Cannon revealed that adrenaline was similar to, but chemically distinct from, the neurotransmitter released, and its identity remained elusive until von Euler revealed that the true neurotransmitter was the nonmethylated derivative of adrenaline, noradrenaline.
Structure and Biosynthesis of Noradrenaline Noradrenaline, a catecholamine, is derived from L-tyrosine, an aromatic amino acid present in the body fluids and taken up by noradrenaline-producing cells. Through various intermediate steps (Figure 1), L-tyrosine is converted to noradrenaline and, finally, to its methylated form, adrenaline, in phenylethanolamine N-methyltransferase-containing cells. The first cytosolic enzyme, tyrosine hydroxylase, is inhibited by excess production of noradrenaline and is the ratelimiting step in the regulation of noradrenaline synthesis. Tyrosine hydroxylase is found only in the cytosol in catecholamine-containing cells and is used as a marker for detection of adrenergic neurons. Dopamine b-hydroxylase is also used as a selective marker for catecholamine-containing cells, but it is located in the secretory vesicles, usually membrane bound; however, a small amount is soluble and released with the vesicle contents upon exocytosis.
Noradrenaline 415 COOH L-tyrosine
NH2
HO
Tyrosine hydroxylase HO
COOH
L-3,4-dihydroxyphenylalanine
NH2
HO
(L-DOPA) DOPA Decarboxylase
HO Dopamine NH2
HO
OH
Dopamine b-Hydroxylase
HO Noradrenaline HO
NH2 OH
HO HO
Phenylethanolamine N-methyltransferase Adrenaline
NH CH3
Figure 1 Biosynthetic pathway of noradrenaline and adrenaline.
Where Is Noradrenaline Found? Noradrenaline is released with adrenaline into the blood from the medullae of the adrenal glands where it can act as a circulating hormone, but it is classically thought of as the main neurotransmitter released from postganglionic sympathetic neurons. Sympathetic fibers project to the heart and along blood vessels, controlling cardiovascular responses to maintain blood pressure, and, with the aid of circulating adrenaline, regulate bronchodilation, glycogen and fat metabolism, thermogenesis, and secretions of hormones and mucus membranes (Table 1). There are also large networks of noradrenergic and adrenergic neurons originating in or close to the locus coeruleus and reticular formation in the brain stem, extensively projecting to the cortex, hippocampus, and cerebellum, and these neurons are involved in baroreceptor and blood pressure reflexes, and also in complex behaviors such as arousal and mood. Many of these tissues are being used to study noradrenaline release and reuptake, but the vas deferens offers the ideal system in which to study sympathetic neuronal release due to its ease of isolation, dense sympathetic innervation, high noradrenaline content, and exquisite sensitivity to pharmacological manipulation. It is perhaps useful at this stage to remind the nonspecialist reader of the anatomy of the autonomic nervous system. As shown by Langley, autonomic nerves typically have both a preganglionic nerve traveling
from the central nervous system and a postganglionic nerve traveling to the effector organ. Parasympathetic fibers either arise from specific cranial nuclei and travel in the occulomotor (III), facial (VII), glossopharyngeal (IX), and vagus (X) cranial nerves or leave the spinal cord at sacral levels S2–S4 before synapsing close to the tissues in collections of cell bodies known as ganglia, and continuing by way of short postganglionic fibers to the effector cells. Sympathetic neurons, however, are generally characterized by short preganglionic fibers, exiting the spinal cord at thoracolumbar levels T1–L3, synapsing in the paravertebral chains, and continuing to the effector organs as long postganglionic fibers (possibly more than 1 m long). The innervation of the rodent vas deferens, a wellcharacterized model system, is unusual in that it is supplied by short postganglionic sympathetic neurons whose cell bodies lie within the hypogastric ganglia situated close to the prostatic end. These postganglionic nerves are mainly nonmyelinated axons (0.2–2 mm in diameter) embedded in Schwann cells, together with a small number of fine myelinated fibers (1 or 2 mm in diameter), which travel through the connective tissue and divide into numerous branches as they enter the prostatic end. Traversing the connective sheath, branches pass into the smooth muscle layers, splitting into smaller bundles of two to eight axons, and become varicose. The varicosities, which are approximately 1 or 2 mm in length and 1–1.5 mm in cross section,
416 Noradrenaline Table 1 Summary of major adrenoceptor signaling and some drugs that can affect it Adrenoceptor subtype
a1
a2
b1
b2
b3
Primary signal transduction
Stimulation of phospholipase C (Gq/11) "IP3, "DAG, and "Ca2þ Stimulation of phospholipase D Vasoconstriction Relaxation of gastrointestinal tract smooth muscle Contraction of seminal tract and uterus Contraction of iris radial muscle Glycogenolysis in liver Kþ release from salivary glands
Inhibition of adenylyl cyclase (Gi/0)
Stimulation of adenylyl cyclase (Gs) "cAMP Stimulation of guanylyl cyclase (Actions mainly on heart) Positive inotropy Positive chronotropy Positive dromotropy Amylase secretion from salivary glands Renin release from juxtaglomerular cells of the kidney
Stimulation of adenylyl cyclase (Gs) "cAMP Stimulation of guanylyl cyclase Vasodilation Bronchodilation Relaxation of gastrointestinal tract smooth muscle Relaxation of seminal tract and uterus Relaxation of ciliary muscle Glycogenolysis in liver and skeletal muscle Increased release of noradrenaline from nerve terminals Salbutamol Salmeterol Butoxamine ICI 118,551
Stimulation of adenylyl cyclase (Gs) "cAMP Stimulation of guanylyl cyclase Lipolysis and thermogenesis in adipose tissue Thermogenesis in skeletal muscle
Secondary signal transduction Major physiological effects
Selective agonists Selective antagonists
Phenylephrine Cirazoline Prazosin Doxazosin
#cAMP Stimulation of adenylyl cyclasea Vasoconstriction Relaxation of gastrointestinal tract smooth muscle Decreased release of acetylcholine and noradrenaline from autonomic nerve terminals Decreased insulin release Platelet aggregation Inhibition of sympathetic outflow in brain stem Clonidine Medetomidine Yohimbine Idazoxan
Dobutamine Xamoterol Atenolol Metoprolol
BRL 37344 ZD 7114 SR 59230A
a The role of stimulating adenylyl cyclase in addition to its inhibition by a2-adrenoceptor activation in some systems remains unknown. For simplicity, subtypes of receptors are not shown. IP3, inositol-1,4,5-trisphosphate; DAG, diacylglycerol; cAMP, 30 -50 -cyclic adenosine monophosphate.
are packed with vesicles and mitochondria and are separated by intervaricose regions approximately 3–5 mm in length. The diameter of the axon spanning the intervaricose regions is only 0.1 mm, and each axon gives rise to between 10 000 and 30 000 varicosities (Figure 2). The conduction velocity in these nerves is less than 1 m s1. In the vas deferens, the varicosities traverse the smooth muscle cells in complex patterns, such that at least one varicosity makes close contact (approximately 20–50 nm) with each smooth muscle cell, with the varicosities being the sites where noradrenaline is synthesized and released.
How Can Noradrenaline Release Be Measured? Early electrophysiological investigations into sympathetic nerves relied on intracellular recording. Basing the technique on the Nobel prize-winning work of Hodgkin, Huxley, and Katz, the nerve cell bodies in ganglia were impaled using glass microelectrodes and electrical changes across the membrane were
measured. As at the neuromuscular junction, electrical signals could be detected in the absence of stimulation (excitatory postsynaptic potentials in nerves and end plate potentials in the motor end plate), as could the resulting action potentials on stimulation. Although this method gave fascinating insights into the release of acetylcholine, the primary neurotransmitter at these synapses, it could not reveal anything about the terminal release of noradrenaline from the postganglionic nerve. Electrophysiological measurements from nerve terminal varicosities proved difficult due to their small size and the large microelectrode diameters; progress remained slow. In an effort to study and localize noradrenaline, a number of methods have been employed, including high-performance liquid chromatography, overflow, radiolabeling, and voltametry techniques. These approaches all indicated a neuronal release of noradrenaline, the most abundant neurotransmitter in sympathetic neurons, and that noradrenaline was stored in the vesicles in preparation for exocytosis. However, fractional release studies showed that
Noradrenaline 417
~0.1 µm 2 µm ~1.5 µm
30 mm 1 cm–1 m
~1 µm
Soma a
b
Axon bundles
c
Varicosities
Figure 2 (a) Schematic diagram of a postganglionic sympathetic neuron showing the difference in size between cell body, axon, and varicosity. (b) A set of axon bundles loaded with the Ca2þ indicator Oregon Green 488 BAPTA-1 dextran. Note that only a single axon is represented in the corresponding box in (a). (c) Image of part of a sympathetic terminal loaded with the Ca2þ indicator Oregon Green 488 BAPTA-1 dextran. Reproduced from Jackson VM and Cunnane TC (2001) Neurotransmitter release mechanisms in sympathetic neurons: Past, present, and future perspectives. Neurochemical Research 26: 875–889, with permission from Springer Science and Business Media.
there was a disparity between the amount of noradrenaline released and the noradrenaline content of a vesicle. Although these methods did not have the temporal or spatial resolution at the time to investigate the release characteristics of noradrenaline from a single varicosity, biochemical analysis of these studies provided two radically different hypotheses to explain the mechanism of noradrenaline release from the ‘average’ varicosity. First, the ‘fractional release’ model suggested that the action potential only released a small fraction of the contents of one vesicle (1–3%) from each varicosity every time the nerve was stimulated, similar to the modern ‘kiss-and-run’ mechanism observed at some synapses in the central nervous system. In this scenario, on average 30–100 action potentials are required to release the equivalent of the neurotransmitter content of one vesicle from each varicosity, and this would mean that there is a constant release of neurotransmitter during neuronal activity providing
an average ‘biophase’ concentration which bathes the smooth muscle and maintains a tonic contraction. With the assumption that the average varicosity has 1000 vesicles, this would mean that only approximately 300–400 molecules of transmitter would be released with each action potential, which should nonetheless be sufficient to reach maximal concentrations for receptor activation in a close contact neuroeffector junction. Today, many textbooks still favor this explanation (as did the early investigators) because it appears to be a more efficient and economical way of transmitter turnover to achieve a given effector response while at the same time reusing the vesicles and allowing for efficient noradrenaline reuptake (approximately 90%). However, this would mean that sympathetic nerves would not behave like all other nerves, where quantal transmission was accepted. An equally radical hypothesis is the ‘intermittent release’ model, which suggested that each action potential activates only 1–3% of all varicosities in the
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tissue. When the release machinery was activated, an individual varicosity discharged a single packet of neurotransmitter equivalent to the neurotransmitter content of a single vesicle. In this case, neurotransmitter release would be highly intermittent at the level of the individual varicosity. This would cause approximately 50-fold higher noradrenaline concentrations in the junction, giving a larger ‘margin of safety’ for transmission, but would demand extreme efficiency of the local noradrenaline reuptake mechanisms to clear noradrenaline and terminate the response before it diffused away. If this theory were true, it would raise more questions about why and how a varicosity would ‘decide’ to release neurotransmitter or not. It was understandable that the investigators favored the previous explanation. If this controversy was to be settled, then techniques with better resolution needed to be developed.
What Can We Learn About Sympathetic Neurotransmitter Release from Electrophysiology? Burnstock and Holman, working in Melbourne, Australia, used one of the most densely innervated sympathetic tissues in the body, the vas deferens, to study neurotransmitter release. Sympathetic nerves in the body typically have low-frequency firing rates, and unlike the overflow studies, which require high frequencies to cause measurable overflow of transmitter, electrophysiology allowed the measurement of release on an impulse-to-impulse basis. Using microelectrode techniques similar to those used in ganglia, they impaled smooth muscle cells of the guinea pig vas deferens and recorded resting membrane potentials. In response to sympathetic nerve stimulation, transient depolarizations were recorded, termed excitatory junction potentials (EJPs). EJPs had a fairly slow time course (0.5–1 s), were graded with stimulus intensity, and increased in amplitude in response to the first few stimuli in a train (i.e., they exhibited facilitation). Perhaps more important, in the absence of stimulation, spontaneous EJPs (SEJPs) were recorded, which were reminiscent of miniature end plate potentials (MEPPs) at the skeletal neuromuscular junction that had famously been used to describe the phenomena surrounding quantal neurotransmission. SEJPs implied that neurotransmitter was released in multimolecular packets; however, unlike MEPPs and end plate potentials in skeletal muscle, EJPs and SEJPs had different time courses and could not be compared directly. The time course differences were probably a reflection of the tight electrical coupling between smooth muscle cells of the vas deferens, which caused rapid dissipation of charge from the point
source, and also that the EJP represented the sum of electrical activity at several simultaneous release sites being activated throughout the muscle. Taken together with the overflow studies, and the difficulty in interpreting the results for a single release site, the electrical signal seemed to support the fractional release model.
High-Resolution Studies of EJPs As the fractional release model enjoyed prime status, new methods were devised to break down EJPs into their component parts. Blakeley and Cunnane noted discontinuities between the rising phases of individual EJPs in the mouse and guinea pig vas deferens, and, on electronically differentiating the rising phases, these discontinuities were extracted as transient peaks in the rate of depolarization of the EJP, termed ‘discrete events’ (Figure 3). On comparing discrete events generated from SEJPs and EJPs, it was found that they matched almost perfectly, suggesting that release was intermittent and monoquantal. Various groups quickly verified the data, and so the fractional release model was moved aside for the intermittent model. Perhaps, in hindsight, it should not have come as a surprise that sympathetic nerves behave like all other nerves in the body and exhibit quantal or packeted neurotransmission.
0.5 V s–1
10 mV 20 ms Figure 3 Intracellular recordings of the membrane potential and its time derivative in single smooth muscle cells of the guinea pig vas deferens. The preparation was stimulated at 0.91 Hz and the transient peaks in the rate of depolarization (the ‘discrete event’) occur intermittently, showing that action potential evoked neurotransmitter release is intermittent. Reproduced from Blakeley AG and Cunnane TC (1979) The packeted release of transmitter from the sympathetic nerves of the guinea-pig vas deferens: An electrophysiological study: Journal of Physiology 296: 85–96; and Jackson VM and Cunnane TC (2001) Neurotransmitter release mechanisms in sympathetic neurons: Past, present, and future perspectives. Neurochemical Research 26: 875–889, with permission from Springer Science and Business Media.
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Mechanisms of Intermittence Sympathetic nerves proved to be difficult to comprehend. If they only released once in every 100 stimuli or so, then how could we use them to study release? We need to understand some of the mechanisms behind intermittence before we can investigate noradrenaline release further. There were two suggestions put forward to explain intermittence: . Intermittent failure of the action potential to propagate throughout the complex nerve terminal network of branching axons and varicosities . A very low probability of neurotransmitter release in the invaded varicosities To resolve this conflict, focal extracellular measurement was developed by Brock and Cunnane. By moving an electrode close to the tissue and applying gentle suction, a small area could be ‘isolated’ from the tissue and drugs applied either inside or outside the electrode, which was perfused with physiological solution. It allowed the simultaneous measurement of the nerve terminal impulse (NTI) and also neurotransmitter release – the excitatory junction current (EJC). In the absence of stimulation, spontaneous EJCs were recorded, and following electrical nerve stimulation it was found that the action potential (NTI) arrived each time, whereas neurotransmitter release was highly intermittent (Figure 4). Application of tetrodotoxin, an inhibitor of voltage-gated Naþ channels, abolished the NTI and evoked neurotransmitter release, but did not abolish spontaneous EJCs. Intermittence of neurotransmitter release was concluded to be due to some failure in depolarization–secretion coupling after the sympathetic nerve terminal is invaded by the action potential rather than action potential propagation failure in the complex terminal arborization of varicosities. One of the more interesting observations occurred when pharmacological agents were used to manipulate neurotransmission. Following blockade of a-adrenoceptors, the major type of receptors for noradrenaline in the vas deferens, there was an increase in the overflow of noradrenaline and also an increase in the electrical signals recorded with microelectrodes in the smooth muscle cells. It transpired that there were receptors located on nerve terminals, a2-adrenoceptors, that were distinct from postjunctional adrenoceptors (mainly a1-adrenoceptors) and limited the release of noradrenaline. This led to the novel concept of a2-adrenoceptor-mediated inhibition of neurotransmitter release in the great majority of sympathetically innervated tissues and added a new dimension to our understanding of the control of sympathetic nerve function. Previously, the centrally
1 Hz 1 Hz 2 Hz
2 Hz
4 Hz 4 Hz 100 µV
NTI
EJCs
10 ms Figure 4 Frequency-dependent facilitation of neurotransmitter release in the guinea pig vas deferens. Simultaneous measurement of the nerve terminal impulse (NTI) and evoked excitatory junctional currents (EJCs) using focal extracellular recordings showed that although the impulses travel through the varicosities every time, EJCs are intermittent. Each panel shows 25 stimuli at 1, 2, and 4 Hz in the top, middle, and bottom panels, respectively. Reproduced from Jackson VM and Cunnane TC (2001) Neurotransmitter release mechanisms in sympathetic neurons: Past, present, and future perspectives. Neurochemical Research 26: 875–889, with permission from Springer Science and Business Media.
determined patterns of nerve firing were primarily thought to control neurotransmitter release, but now it was shown that neurotransmitter release could be controlled locally due to feedback inhibition of the action of released noradrenaline at distinct receptors on nerve terminals producing a brake on further neurotransmitter release. There was no doubt intermittence could be regulated by this local inhibition at the level of the varicosity, and although this idea was largely born in the peripheral nervous system, it is generally accepted that presynaptic receptors exist at every synapse. Indeed, we now know that many nerves have both inhibitory and excitatory receptors located on their nerve terminals, such as the prejunctional excitatory nicotinic receptors and the prejunctional inhibitory muscarinic receptors at the neuromuscular junction, which can increase or decrease acetylcholine release, respectively. The importance of increases in intracellular Ca2þ concentrations as the trigger for exocytosis is wellknown, and so perhaps intermittence was due to the failure of most varicosities to undergo appropriate changes in Ca2þ levels. Developments in confocal microscopy and Ca2þ indicators mean that we can now finally resolve Ca2þ dynamics in intact tissue preparations. By loading the nerve terminals through the cut end of the vas deferens, it has been shown that Ca2þ enters all varicosities when the action potential arrives.
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Figure 5 Discreet and intermittent smooth muscle Ca2þ transients following nerve stimulation. The first six frames show selected images of the same smooth muscle cell taken during 2 Hz stimulation. There is no response to most stimuli (frame 1), whereas some stimuli evoke focal Ca2þ transients in the smooth muscle cell (frames 2–6). The final frame was obtained from a confocal section 3 mm above that of the preceding images, and it shows an overlying nerve terminal varicosity. The white dots denote the location (epicenter) of a single smooth muscle Ca2þ transient occurring at some time during eight sets of recordings. Reproduced from Brain KL, Jackson VM, Trout SJ, and Cunnane TC (2002) Intermittent ATP release from nerve terminals elicits focal smooth muscle Ca2þ transients in mouse vas deferens. Journal of Physiology 541: 849–862.
Through Ca2þ studies on neurons, it was questioned whether it would be possible to resolve neurotransmitter release optically. Occasionally, for indeterminate reasons, some smooth muscle cells were loaded with the Ca2þ indicator, and distinct changes in Ca2þ were noted following nerve stimulation both in the varicosity and directly below in the smooth muscle cell. The focal changes in Ca2þ recorded in the smooth muscle cells were termed neuroeffector Ca2þ transients (NCTs) (Figure 5). By simultaneously monitoring both pre- and postjunctional responses evoked by low-frequency nerve stimulation with cell-permeant Ca2þ indicators, Brain, Jackson, and Cunnane found that these NCTs correlated well with action potentialevoked Ca2þ transients in varicosities and with the release probability of individual varicosities on the same axon. Surprisingly, they found that some varicosities had an apparent release probability as high as 0.09, but there was a population of varicosities that never released and remained ‘silent.’ Finally, there is an optical method that provides a unique approach to detect neurotransmitter release at the level of the individual sympathetic neuroeffector junction (i.e., single varicosities on the same nerve terminal branch) at an unparalleled resolution. Currently, we still do not know the reason for intermittence. However, the ability to demonstrate neurotransmitter release at high resolution surely holds the key to unraveling the mechanisms underlying release modulation at individual varicosities on an impulseto-impulse basis in an intact organ. Autoinhibition, variation in secretory proteins, and discrete changes in Ca2þ in different varicosities are among some of the current hypotheses undergoing evaluation, but we are now in a better position to study neurotransmitter
release from these nerves. However, what is the neurotransmitter? Surely it is noradrenaline.
The Identity Revealed Although there was broad agreement across all techniques that noradrenaline is released on an impulseto-impulse basis from sympathetic nerves, there were a number of observations that did not fit the noradrenergic theory. For example, when the rodent vas deferens was depleted of noradrenaline using reserpine, which irreversibly interferes with the Mg2þand adenosine-50 -triphosphate (ATP)-dependent uptake of biogenic amines into vesicles and thereby depletes noradrenaline from nerves, electrophysiological methods showed that the EJPs were still present and often larger in amplitude. This was due to a prejunctional action rather than a postjunctional sensitization because the size of the SEJPs remained unchanged. Similarly, when yohimbine was applied to block prejunctional autoinhibitory a2-adrenoceptors, EJPs were no longer potentiated. In other words, we have a tissue devoid of noradrenaline, but EJPs, the electrical sign of neurotransmitter release, are still present – a strange paradox. Further investigation using selective antagonists led to the rather revolutionary concept of noradrenaline– ATP co-transmission. Imagine the controversy when Burnstock, Westfall, and others suggested that ATP, Krooll’s ‘universal currency of bioenergy,’ could also be released from sympathetic nerves and that it was ATP, and not noradrenaline, that was responsible for the generation of the EJP. This was not so surprising in retrospect because Douglas and Poisner had shown that ATP was co-stored and co-released
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with adrenaline and noradrenaline in the adrenal medulla approximately 20 years previously. Some people called purinergic transmission the ‘pure imagination’ hypothesis on account of its radical and unbelievable concepts, but it is now generally accepted that ATP is released from almost all nerves and acts as a separate neurotransmitter. Indeed, moving away from Dale’s principle, which states that each neuron can only secrete one type of neurotransmitter, we now know that neurotransmitter release can differ at different synapses of the same cell, both in the class of neurotransmitter and also in the quantities of the cocktails of neurotransmitters released. Yet again, an idea born in the peripheral nervous system is now accepted widely throughout the central nervous system. Work on purinergic transmission spawned the discovery of G-protein-coupled P2Y purinoceptors and of a unique class of ligand-gated ion channels, P2X receptors, which do not follow the structure of any other family of ligand-gated channels. After activation by extracellular ATP, through nonselective cation entry, P2X receptors are responsible for the EJP and SEJP because P2X receptor antagonists and desensitizers abolish both EJPs and SEJPs. NCTs are a measure of the Ca2þ flowing through the nonselective cation pore together with amplification from the release of Ca2þ from intracellular stores, and similarly, these drugs abolish NCTs in the vas deferens, blood vessels, and bladder. In the vas deferens, the purinoceptor subtype P2X1 is particularly important because knockout mice that do not have P2X1 receptor expression lack EJPs, SEJPs, NCTs, and spontaneous NCTs.
It would seem that the electrophysiological and confocal investigations have not been monitoring the effects of released noradrenaline but, rather, measuring how sympathetic nerves release ATP. However, if noradrenaline and ATP are co-stored in sympathetic nerve vesicles, as the biochemical analysis would suggest, then ATP release will also reflect how noradrenaline is being released assuming exocytosis of the entire neurotransmitter content of a vesicle. Indeed, research using amperometry, a direct measurement of the oxidation current of noradrenaline, has shown that noradrenaline can be released intermittently on an impulseto-impulse basis (Figure 6). Finally, overflow studies, optical studies, and electrophysiological methods appear to be converging in order to explain sympathetic neurotransmitter release, be it ATP or noradrenaline.
The Elusive Nature of Noradrenaline – The Paradox Noradrenaline produces its effects through G-proteincoupled receptors (Table 1) and its action is terminated by reuptake into the prejunctional neuronal (uptake 1; inhibited by cocaine, desipramine, amphetamines, and tricyclic antidepressants) and postjunctional nonneuronal (uptake 2; inhibited by corticosterone) cells. Reuptake results in either the metabolism of noradrenaline by monoamine oxidases and catechol-O-methyl transferases within a reaction pathway that finally produces vanillylmandelic acid or 3-methoxy,4-hydroxyphenylglycol, which are excreted in the urine, or the noradrenaline can be recycled and released again from the prejunctional nerve terminal. In the smooth muscle
0.5 pA
1.5 s Figure 6 Intermittent release of noradrenaline from rat tail artery revealed by continuous amperometry. Five stimuli per panel at 0.1 Hz. Reproduced from Jackson VM and Cunnane TC (2001) Neurotransmitter release mechanisms in sympathetic neurons: Past, present, and future perspectives. Neurochemical Research 26: 875–889, with permission from Springer Science and Business Media; and Reproduced from Msghina M, Gonon F, and Stja¨rne L (1993) Intermittent release of noradrenaline by single pulses and release during short trains at high frequencies from sympathetic nerves in rat tail artery. Neuroscience 57: 887–890, with permission from Elsevier.
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of the vas deferens, noradrenaline mediates most of its effects through a1-adrenoceptors, which activate phospholipase C, and with increases in inositol-1,4,5trisphosphate (IP3) and diacylglycerol (DAG), respectively, lead to increases in intracellular Ca2þ and contraction and should be independent of an electrical change. Of course, there are exceptions to this rule, the most notable example being the rat anococcygeus, in which increases in intracellular Ca2þ result in an electrical depolarization by opening a Cl channel, but generally under low-frequency stimulations, most tissues show little or no electrical changes on a1-adrenoceptor activation. It would be expected that IP3-generated Ca2þ waves would be detected using Ca2þ-imaging confocal microscopy. Indeed, slow propagating waves in small arteries following nerve stimulation can be attributed to a1-adrenoceptor stimulation because they are blocked by prazosin, an a1-adrenoceptor antagonist. Similarly, cholinergic stimulation in the bladder results in waves that are blocked by cyclopentolate, a muscarinic receptor antagonist, which are thought to be generated secondary to muscarinic receptor-induced IP3 synthesis. Such waves rarely occur within the longitudinal layer of the vas deferens upon nerve stimulation, but they can be induced by applying exogenous noradrenaline. We therefore have a paradox: exogenously applied noradrenaline causes Ca2þ waves, whereas neuronally released noradrenaline does not readily produce waves, and yet both cause a contraction that is mediated by a1-adrenoceptors. Once again, the remarkable sympathetic nervous system confronts us with a paradox. The knockout mouse for P2X1 purinoceptors provided the ideal opportunity to study noradrenergic transmission in isolation because P2X1 receptor effectswerelost,andoneofthecompensatorymechanisms for the purinergic ‘shortfall’ was the supersensitivity of the tissue to noradrenaline. Using higher frequency nerve stimulation, which is known to favor the noradrenergic component of release, even in the presence of both yohimbine, to antagonize autoinhibitory a2-adrenoceptors to maximize noradrenaline release, and desipramine, to inhibit the reuptake of noradrenaline and maximize its concentration within the neuroeffector cleft, there were still few Ca2þ transientsduetoneuronalnoradrenalinerelease. The preparations still responded well to exogenously added noradrenaline and appeared to have a larger contraction mediated by a1-adrenoceptors to both neuronal and exogenous noradrenaline. Perhaps neuronally released noradrenaline activates a different population of receptors compared to exogenously applied noradrenaline and these receptors have different pathways. Indeed, prazosin
is a non-subtype selective a1-adrenoceptor antagonist, with a1A-adrenoceptors favoring the IP3-mediated increase in intracellular Ca2þ, whereas a1B- and a1D-adrenoceptors may favor the DAG and protein kinase C pathways of contraction (which do not require such large increases in intracellular Ca2þ); this might explain the apparent differences due to prazosin’s nonselectivity. Of course, there are crossovers between the phospholipase C (IP3 and DAG) and D (DAG only) pathways because the receptors are promiscuous and activate many different intracellular signaling cascades, including the Rho-kinase pathway, which allows contraction without any concomitant increase in Ca2þ within smooth muscle cells. How these results can be explained and how the various pathways and cotransmitters interact remain under investigation. Noradrenaline and its effects remain elusive despite the current sophisticated investigative techniques.
Conclusion We began with noradrenaline being the only neurotransmitter released from postganglionic sympathetic nerves, but investigations into the nature of neurotransmitter release have revealed major new insights into the mechanism by which sympathetic nerves function – intermittent neurotransmitter release, autoinhibition through prejunctional/presynaptic receptors, facilitation of release through prejunctional nicotinic receptors, cotransmission, and perhaps the most startling finding of all, that some varicosities do not release neurotransmitter at all. There is a rich diversity of pre- and postjunctional receptors and interactions that remain to be investigated, and we look forward to the advances that studying the sympathetic nervous system will bring. It is fair to say that in the twenty-first century we remain surprisingly ignorant of how nerves release neurotransmitters and the effects that noradrenaline and its cotransmitters have on the innervated organs. See also: Adenosine Triphosphate (ATP).
Further Reading Blakeley AG and Cunnane TC (1979) The packeted release of transmitter from the sympathetic nerves of the guinea-pig vas deferens: An electrophysiological study. Journal of Physiology 296: 85–96. Brain KL, Jackson VM, Trout SJ, and Cunnane TC (2002) Intermittent ATP release from nerve terminals elicits focal smooth muscle Ca2þ transients in mouse vas deferens. Journal of Physiology 541: 849–862. Brock JA and Cunnane TC (1987) Relationship between the nerve action potential and transmitter release from sympathetic postganglionic nerve terminals. Nature 326: 605–607.
Noradrenaline 423 Brock JA and Cunnane TC (1992) Electrophysiology of neuroeffector transmission in smooth muscle. In: Burnstock G and Hoyle CHV (eds.) The Autonomic Nervous System: Autonomic Neuroeffector Mechanisms, pp. 121–213. Chur, Switzerland: Harwood Academic. Goodman LS, Gilman A, Hardman JG, and Limbird LE (2001) Goodman & Gilman’s the Pharmacological Basis of Therapeutics, 10th edn., New York: McGraw-Hill. Hague C, Chen Z, Uberti M, and Minneman KP (2003) a1Adrenergic receptor subtypes: Non-identical triplets with different dancing partners. Life Science 74: 411–418. Jackson VM and Cunnane TC (2001) Neurotransmitter release mechanisms in sympathetic neurons: Past, present, and future perspectives. Neurochemical Research 26: 875–889. Li F, De Godoy M, and Rattan S (2004) Role of adenylate and guanylate cyclases in b1-, b2-, and b3-adrenoceptor-mediated relaxation of internal anal sphincter smooth muscle. Journal
of Pharmacology and Experimental Therapeutics 308: 1111–1120. Msghina M, Gonon F, and Stja¨rne L (1993) Intermittent release of noradrenaline by single pulses and release during short trains at high frequencies from sympathetic nerves in rat tail artery. Neuroscience 57: 887–890. Rang HP, Dale MM, Ritter JM, and Moore PK (2003) Pharmacology, 5th edn., London: Churchill Livingstone. Wenzel-Seifert K, Liu HY, and Seifert R (2002) Similarities and differences in the coupling of human b1- and b2-adrenoceptors to Gsa splice variants. Biochemical Pharmacology 64: 9–20.
Relevant Website http://www.iuphar.org – International Union of Pharmacology.
Norepinephrine: Adrenergic Receptors D B Bylund, University of Nebraska Medical Center, Omaha, NE, USA ã 2009 Elsevier Ltd. All rights reserved.
Introduction Norepinephrine (also called noradrenaline) is a neurotransmitter in both the peripheral and central nervous systems. Norepinephrine produces many effects in the body, the most notable being those associated with the ‘fight-or-flight’ response to perceived danger. The effects of norepinephrine and a related catecholamine, epinephrine (also called adrenaline), are mediated by the family of adrenergic receptors. The chemical structure of norepinephrine, as shown in Figure 1, indicates that it is a catecholamine, because it has both the catechol moiety consisting of a benzene ring (green) with two hydroxyl groups (red) and an amine (blue). Adrenergic receptors (also called adrenoceptors) are widely distributed throughout the body. There are three major adrenergic receptor types: alpha-1, alpha-2, and beta. Each of these three receptor types is further divided into three subtypes. Adrenergic receptors are seven transmembrane receptors which consist of a single polypeptide chain with seven hydrophobic regions that form alpha-helical structures that span or transverse the membrane. Because the mechanism of action of adrenergic receptors includes the activation of guanine nucleotide regulatory binding proteins (G-proteins), they are also called G-protein-coupled receptors. Stimulation of adrenergic receptors by endogenous catecholamines released in response to activation of the sympathetic autonomic nervous system (as well as by exogenous drugs called adrenergic agonists) results in a variety of effects such as increased heart rate, regulation of vascular tone, and bronchodilatation. In the central nervous system (CNS), adrenergic receptors are involved in many functions including memory, learning, alertness, and the response to stress.
History In the late 1940s, Ulf von Euler in Sweden and Holtz in Germany identified norepinephrine as the neurotransmitter of the mammalian sympathetic nerves and soon thereafter also found it to be a normal constituent of mammalian brain. Norepinephrine was subsequently shown to also be a central neurotransmitter and was visualized in the brain by fluorescent and immunohistochemical techniques.
424
Adrenergic receptors were originally divided into two major types, alpha and beta, based on their pharmacological characteristics (i.e., rank order potency of agonists). Subsequently, the beta adrenergic receptors were subdivided into beta-1 and beta-2 subtypes, and more recently a beta-3 subtype was defined. The alpha adrenergic receptors were first subdivided into postsynaptic (alpha-1) and presynaptic (alpha-2) subtypes. After it was realized that not all alpha receptors with alpha-2 pharmacological characteristics were presynaptic, the pharmacological definition was used. The current classification scheme is based on three major types: alpha-1, alpha-2, and beta. Each of these three receptor types is further divided into three subtypes as shown in Figure 2: alpha-1A, alpha-1B, alpha-1D; alpha-2A, alpha-2B, alpha-2C; and beta-1, beta-2, beta-3. Although adrenergic receptors are found throughout the body in tissues innervated by both the peripheral and CNSs, a few locations are of special interest. Alpha-1 adrenergic receptors are found peripherally in some blood vessels, whereas alpha-2 receptors are located on platelets and on nerve terminals in both the peripheral and CNSs. In the heart, beta-1 receptors predominate, where as in the smooth muscles of the lungs, some blood vessels, and the uterus, beta-2 receptors are relatively abundant.
Neurochemistry of Norepinephrine Biosynthesis of Norepinephrine
Norepinephrine is synthesized in neurons starting with the amino acid tyrosine, which is obtained from the diet and can also be synthesized from phenylalanine. Tyrosine is converted to dihydroxyphenylalanine (DOPA) by the enzyme tyrosine hydroxylase, which is the rate-limiting enzyme for norepinephrine biosynthesis. DOPA, in turn, is converted to dopamine in the cytoplasm. Dopamine, which is also a neurotransmitter, is taken up into vesicles and converted to norepinephrine by the enzyme dopamine beta-hydroxylase. In the adrenal medulla and in a few brain regions, norepinephrine is converted to epinephrine by the enzyme phenylethanolamine N-methyltransferase. Storage, Release, Reuptake, and Metabolism of Norepinephrine
Norepinephrine is stored in vesicles (also called storage granules) in the nerve terminals, which concentrates it and protects it from metabolism, until it is released following nerve stimulation. The major mechanism by which the effects of norepinephrine
Norepinephrine: Adrenergic Receptors 425
are terminated is reuptake back into the nerve terminal by a high-affinity transporter. Norepinephrine can also be metabolized to inactive products. Inhibition of either of these processes results in an increase in the synaptic level of norepinephrine and a prolongation of its effects. The reuptake of norepinephrine is mediated by the norepinephrine transporter (NET). Approximately 80% to 90% of the norepinephrine released into many synapses is cleared by this mechanism. NET belongs to the protein superfamily defined as Naþ/Cl-dependent transporters and has 12 putative transmembrane domains. Some inhibitors of NET are antidepressants and are used in the treatment of clinical depression and other affective disorders. They are also sometimes used to treat anxiety disorders, obsessive–compulsive disorder, attentiondeficit/hyperactivity disorder (ADHD), and chronic neuropathic pain. Several classes of antidepressant drugs inhibit NET, including the tricyclic antidepressants (e.g., imipramine, desipramine, doxepin), serotonin-norepinephrine reuptake inhibitors (SNRIs; e.g., venlafaxine, duloxetine), and the norepinephrine reuptake inhibitors (e.g., atomoxetine, reboxetine). Norepinephrine is metabolized by the enzymes monoamine oxidase and catechol-O-methyltransferase
to 3-methoxy-4-hydroxymandelic acid and 3-methoxy4-hydroxyphenylglycol. These inactive degradation products can be quantitated in tissues, blood, and urine as a measure of norepinephrine turnover.
Ontogeny of Norepinephrine in the Brain Cell bodies expressing tyrosine hydroxylase can be detected by 4 weeks of gestation in humans, and norepinephrine itself is detectable by 5–6 weeks. Norepinephrine levels in humans increase throughout the first trimester, especially from about 2 months of gestation forward. Following the initial increase in spinal norepinephrine, a decrease of 30–40% in concentration occurs between 6 months of gestation and early childhood. Noradrenergic neurons differentiate in rats between gestational day 10 and 13. From this point forward there is a steady differentiation and a nearly linear development of markers for noradrenergic neurons in the CNS, increasing approximately 100- to 1000-fold by adulthood. Thus, although the development of norepinephrine occurs fairly early and is reasonably rapid, it is slower than some of the other neurotransmitter systems, particularly the serotonin system.
Effects of Norepinephrine Effects Mediated by the Autonomic Nervous System
OH (R)
NH2
The autonomic nervous system (also called the involuntary nervous system) is divided into two components, the sympathetic nervous system and the parasympathetic nervous system. The final nerves (postganglionic) in the sympathetic system are
HO OH Figure 1 The structure of norepinephrine.
Adrenergic receptors
Alpha-1
Alpha-1A
Alpha-2
Alpha-1B
Alpha-1D
Alpha-2A Figure 2 The classification of the adrenergic receptors.
Alpha-2B
Beta
Beta-1
Alpha-2C
Beta-2
Beta-3
426 Norepinephrine: Adrenergic Receptors
adrenergic and thus release norepinephrine in the various tissues (end organs). The adrenal medulla, which is part of the sympathetic system, releases epinephrine into the circulation. The activation of the sympathetic system, in response to perceived danger, results in the release of large quantities of norepinephrine and epinephrine. Norepinephrine acting at alpha-1 receptors causes vasoconstriction (contraction) of cutaneous blood vessels, whereas epinephrine acting at beta-2 receptors in the blood vessels of the skeletal muscles causes vasodilatation (relaxation), resulting in blood flow being increased in the muscles. Norepinephrine and epinephrine acting at beta-1 receptors increase the force and rate of contraction of the heart, whereas epinephrine acting at beta-2receptors causes bronchodilatation in the lungs and relaxation of the smooth muscle in the uterus. Effects Mediated by the CNS
In the CNS, the cell bodies of noradrenergic neurons are found primarily in the locus coeruleus in the brain stem. These neurons, however, project widely throughout the brain and spinal cord. The locus coeruleus, and hence norepinephrine, is an important regulator of a variety of physiologic functions, including sleep/wake cycles, attention, orientation, mood, memory, and cardiovascular as well as other autonomic and endocrine functions. Although adrenergic receptors are found throughout the brain, the alpha-2 receptors in the CNS are of particular importance because they regulate the release of norepinephrine, as well as many other neurotransmitters.
Alpha-1 Adrenergic Receptors Three genetic and four pharmacological alpha-1 adrenergic receptor subtypes have been defined. The alpha-1A and alpha-1B subtypes were initially defined based on their differential affinity for adrenergic agents such as WB4101 and phentolamine and their differential sensitivities to the site-directed alkylating agent chloroethylclonidine. The alpha-1B subtype was subsequently cloned from the hamster, and the alpha-1A was cloned from bovine brain, although it was originally called the alpha-1c adrenergic receptor. A third subtype, the alpha-1D adrenergic receptor, was subsequently cloned from the rat cerebral cortex, although this clone was originally called the alpha-1a subtype by some investigators. A fourth pharmacological subtype, the alpha-1L, has been identified in vascular tissues from several species but may represent a conformational state of the alpha-1A receptor. The current classification scheme includes
the alpha-1A, the alpha-1B, and the alpha-1D, but there is no alpha-1C (Figure 2). Pharmacological and Molecular Characteristics of Alpha-1 Adrenergic Receptors
In addition to norepinephrine and epinephrine, alpha-1 receptors are activated by agonists as phenylephrine and methoxamine. These agonists are relatively selective for alpha-1 receptors and have lower affinity for alpha-2 and beta receptors. By contrast, they have similar affinities for the three alpha-1 subtypes and are thus non-subtype-selective agonists. Similarly, antagonists including prazosin and tamsulosin are relatively selective for alpha-1 receptors and block alpha-2 and beta receptors only at high concentrations. Several other antagonists such as phentolamine and phenoxybenzamine block both alpha-1 and alpha-2 adrenergic receptors with similar affinities. Alpha-1A-selective antagonists include 5-methylurapidil and niguldipine, whereas cirazoline appears to be a selective alpha-1A agonist. The alpha-1 adrenergic receptors are single polypeptide chains of 446–572-amino-acid residues that span the membrane seven times, with the N-terminus being extracellular and the C-terminus intracellular. Thus, there are three intracellular loops and three extracellular loops. In contrast to the alpha-2 receptors, but similar to the beta receptors, the alpha-1 receptors have a long C-terminal tail (137–179amino-acid residues) and a short third intracellular loop (68–73-amino-acid residues) The C-terminal tails also have multiple sites of phosphorylation which are thought to be important in the desensitization, recycling, and downregulation of the receptor. The human alpha-1 adrenergic receptor genes consist of two exons and a single large intron of at least 20 kbp in the region corresponding to the sixth transmembrane domain. No splice variants are known for the alpha-1B and alpha-1D subtypes. By contrast, at least ten splice variants of human alpha-1A subtype have been reported, but only four produce full-length receptors.
Alpha-2 Adrenergic Receptors Three genetic and four pharmacological alpha-2 adrenergic receptor subtypes have also been defined (Figure 2). The alpha-2A and alpha-2B subtypes were initially defined based on their differential affinity for adrenergic agents such as prazosin and oxymetazoline. These subtypes were subsequently cloned from human, rat, and mouse. A third subtype, alpha-2C, was originally identified in an opossum kidney cell line and
Norepinephrine: Adrenergic Receptors 427
has also been cloned from several species. A fourth pharmacological subtype, the alpha-2D, has been identified in the rat, mouse, and cow. This pharmacological subtype is a species ortholog of the human alpha-2A subtype and thus is not considered to be a separate genetic subtype. Pharmacological and Molecular Characteristics of Alpha-2 Adrenergic Receptors
In addition to norepinephrine and epinephrine, alpha-2 receptors are activated by clonidine and brimonidine. These agonists are relatively selective for alpha-2 receptors and have lower affinity at alpha-1 and beta receptors. Similarly, the antagonist yohimibine is relatively selective for alpha-2 receptors and blocks alpha-1 and beta receptors only at higher concentrations. Antagonists that are at least somewhat selective for one of the alpha-2 subtypes include BRL44408 for the alpha-2A, prazosin and ARC-239 for the alpha-2B (note, however, that these two agents have much higher affinities for alpha-1 receptors), and rauwolscine for the alpha-2C subtype. Ozymetazoline is a partial agonist that has a higher affinity for the alpha-2A subtype as compared to the alpha-2B and alpha-2C subtypes. The alpha-2 adrenergic receptors are single polypeptide chains of 450–462-amino-acid residues. In contrast to the alpha-1 and beta receptors, the alpha-2 receptors tend to have long third intracellular loops (148–179-amino-acid residues) and a short C-terminal tail (20–21-amino-acid residues). The third intracellular loops have multiple sites of phosphorylation which are thought to be important in the desensitization, recycling, and downregulation of the receptor. The alpha-2 adrenergic receptor genes do not contain introns, and thus there are no splice variants.
Pharmacological and Molecular Characteristics of Beta Adrenergic Receptors
Isoproterenol is the prototypic non-subtype-selective beta agonist which has little effect at alpha-1 and alpha-2 receptors. Epinephrine is 10- to 100-fold more potent at the beta-2 receptor as compared to the beta-1 subtype, whereas norepinephrine is more potent than epinephrine at the beta-3 subtype. Many beta-2selective agonists, such as terbutaline and salmeterol, have been developed for the treatment of asthma. Due to their subtype selectivity they have a lower incidence of side effects mediated by the beta-1 receptor. Propranolol is the prototypic non-subtype-selective beta antagonist which has equal affinities at the beta-1 and beta-2 subtypes. Other nonselective beta adrenergic antagonists include timolol, pindolol (which is actually a weak partial agonist), and carvedilol, which is also an alpha-1 antagonist. Several beta-1-selective antagonists have been developed, such as metoprolol and esmolol. The beta adrenergic receptors are single polypeptide chains of 408–477-amino-acid residues. In contrast to the alpha-2 receptors, but similar to the alpha-1 receptors, the beta receptors tend to have longer C-terminal tails (61–97-amino-acid residues) and shorter third intracellular loops (54–80-amino-acid residues). The C-terminal tails have multiple sites of phosphorylation which are thought to be important in the desensitization, recycling, and downregulation of the receptor. The beta-1 and beta-2 adrenergic receptor genes do not contain introns, and thus they have no splice variants. By contrast, the beta-3 receptor has one intron, resulting in two splice variants. However, no functional differences have been found between the two splice variants.
Regulation of Adrenergic Receptors Beta Adrenergic Receptors Three beta adrenergic receptor subtypes have been identified. The beta-1 adrenergic receptor, the dominant receptor in heart and adipose tissue, is equally sensitive to epinephrine and norepinephrine, whereas the beta-2 adrenergic receptor, responsible for relaxation of vascular, uterine, and airway smooth muscle, is less sensitive to norepinephrine than to epinephrine. The beta-3 receptor is insensitive to the commonly used beta adrenergic receptor antagonists and was previously referred to as the ‘atypical’ beta adrenergic receptor. A beta-4 receptor has been postulated; however, definitive evidence of its existence is lacking, and it is now thought to be a ‘state’ of the beta-1 adrenergic receptor.
The processes involved in desensitization and downregulation have been extensively investigated for the beta-2 adrenergic receptor. The other adrenergic receptors, as well as many other G-protein-coupled receptors, appear to behave in a similar, although not identical, manner. Initial uncoupling of the beta-2 receptor from the G-protein after agonist binding is mediated by phosphorylation of specific residues in the carboxyl tail of the receptor. The phosphorylated beta-2 receptor serves as a substrate for the binding of b-arrestin, which not only uncouples the receptor from the signal transduction process but also serves as an adapter protein that mediates the binding of additional signaling proteins and entry into the internalization pathway. The mechanisms of beta-2 adrenergic receptor downregulation appear to involve both
428 Norepinephrine: Adrenergic Receptors
an increase in the rate of degradation of the receptor and a decrease in the levels of beta receptor mRNA.
Adrenergic Receptor Signal Transduction Pathways The alpha-1 adrenergic receptors activate the Gq/11 family of G-proteins, leading to the dissociation of the a and bg subunits and the subsequent stimulation of the enzyme phospholipase C. This enzyme hydrolyzes phosphatidylinositol in the membrane, producing inositol trisphosphate (IP3) and diacylglycerol. These molecules act as second messengers mediating intracellular Ca2þ release via the IP3 receptor and activating protein kinase C. Other signaling pathways that have also been shown to be activated by alpha-1 receptors include Ca2þ influx via voltage-dependent and independent calcium channels, arachidonic acid release, and activation of phospholipase A2, phospholipase D activation, and mitogen-activated protein kinase. The alpha-2 adrenergic receptors activate the Gi/o family of G-proteins and alter (classically inhibit) the activity of the enzyme adenylate cyclase, which in turn, decreases the concentration of the second messenger cyclic AMP. In addition, the stimulation of alpha-2receptors can regulate several other effector systems including the activation of Kþ channels, inhibition or activation of Ca2þ channels, and activation of phospholipase A2, phospholipase C, and Naþ/Hþ exchange. The beta adrenergic receptors activate the Gs family of G-proteins and activate adenylate cyclase, thus increasing in cyclic adenosine monophosphate (AMP) concentrations. Beta adrenergic receptors interact with many other signaling proteins, including the phosphoprotein EBP50 (ezrinradixin-moesin-binding phosphoprotein-50), the Naþ/Hþ exchanger regulatory factor, and cyclic nucleotide ras guanine-nucleotide exchange factor (CNrasGEF). Traditionally it has been assumed that intrinsic efficacy of a ligand for the adrenergic receptors is a property of the ligand. However, recent evidence has shown that certain functionally selective ligands may act as either agonists or antagonists for different functions mediated by the same receptor. This functional selectivity likely involves differences in ligand-induced intermediate conformational states, the diversity of G-proteins, scaffolding and signaling partners, and receptor oligomers.
Adrenergic Receptor Polymorphisms Polymorphisms have been identified in some of the alpha-2 and beta adrenergic receptor subtypes, which may have important clinical implications. A common polymorphism has been identified in the third
intracellular loop of the alpha-2B receptor, which consists of a deletion of three glutamate residues (301–303) and is a risk factor for acute coronary events, but not hypertension. This deletion results in a loss of short-term agonist-induced desensitization. The most common polymorphism associated with the human alpha-2C receptor is the alpha-2C-del (residues 322 to 325 are missing), which occurs in 40% of Blacks and 4% of those from other ethnic backgrounds. As the alpha-2C-del receptor appears to be less efficiently coupled to G-proteins, it is less able to mediate a response to agonists. The consequence of this in humans is an impaired feedback inhibition of catecholamine release that is associated with elevated blood pressure and an increased risk of heart failure. The gene encoding the human beta-1 adrenergic receptor is quite polymorphic, with 18 single nucleotide polymorphisms (SNPs), seven of which cause amino acid substitutions. A total of 13 polymorphisms in the beta-2 adrenergic receptor gene and its transcriptional regulator upstream peptide have been identified. Three closely linked polymorphisms, two coding regions at amino acid positions 16 and 27 and one in the upstream peptide, are common in the general Caucasian population. The glycine-16 receptor exhibits enhanced downregulation in vitro after agonist exposure. In contrast, arginine-16 receptors are more resistant to downregulation. Some studies have suggested a relationship among these polymorphisms, airway responsiveness (e.g., asthma), and the responsiveness to beta adrenergic agonists. A tryptophan-64 to arginine polymorphism has been identified in the beta-3 adrenergic receptor. The allele frequency is approximately 30% in the Japanese population, higher in Pima Indians, and lower in Caucasians. Type 2 diabetic patients with this mutation showed a significantly younger age of onset of diabetes and an increased tendency to obesity, hyperinsulinemia, and hypertension.
Drugs Which Mimic or Block the Effects of Norepinephrine Norepinephrine itself is rarely used as a drug due to its rapid metabolism and many sites of action. Drugs which evoke responses similar to sympathetic nerve stimulation are called sympathomimetic drugs. They produce their effects either directly by stimulating adrenergic receptors (adrenergic receptor agonists) or indirectly by promoting the release of norepinephrine or by blocking its reuptake. Table 1 gives examples of various sympathomimetic drugs with their mechanism of action, the effects they produce, and their therapeutic indications.
Norepinephrine: Adrenergic Receptors 429 Table 1 Drugs which mimic the effects of norepinephrine Receptor
Examples of drugs
Effect
Therapeutic indication
Alpha-1 Alpha-2 Beta-1 Beta-2 (Indirect acting) (Indirect acting) (Indirect acting)
Phenylephrine Clonidine, brimonidine Dobutamine Albuterol, terbutaline, ritodrine Amphetamine Desipramine, tomoxetine Phenelzine
Vasoconstriction Lowers fluid pressure Increases heart rate Relaxes smooth muscle in lung and uterus CNS stimulant Blocks norepinephrine reuptake Inhibits monoamine oxidase inhibitor (MAO)
Hypotension, nasal congestion Hypertension, glaucoma Cardiovascular shock Asthma, premature labor Narcolepsy, hyperactivity Depression Depression
Table 2 Drugs which block the effects of norepinephrine Receptor
Examples of drugs
Effect
Therapeutic indication
Alpha-1 Alpha-2 Beta Beta-1
Prazosin, terazosin Mirtazapine Propranolol, timolol Atenolol, metoprolol
Vasodilatation Antidepressant Decreases heart rate Decreases heart rate
Hypertension, benign prostatic hypertrophy Depression Hypertension, angina, glaucoma Hypertension
Drugs which block the responses to sympathetic nerve stimulation and thus block the effects of norepinephrine are called adrenergic receptor antagonists. They have no direct effects of their own, but act by blocking the effects of either released norepinephrine or an administered adrenergic receptor agonist. Table 2 gives examples of adrenergic receptor antagonists (also called adrenergic blockers) with their receptor selectivity, the effects they produce, and their therapeutic indication. See also: Monoamines: Release Studies; Norepinephrine: CNS Pathways and Neurophysiology.
Further Reading Bylund DB (1988) Subtypes of alpha-2 adrenoceptors: Pharmacological and molecular biological evidence converge. Trends in Pharmacological Science 9: 356–361. Bylund DB, Eikenberg DC, Hieble JP, et al. (1994) International Union of Pharmacology nomenclature of adrenoceptors. Pharmacology Review 46: 121–136. Cooper JR, Bloom FE, and Roth RH (2003) Norepinephrine and epinephrine. In: The Biochemical Basis of Neuropharmacology, 8th edn., pp. 181–223. New York: Oxford University Press. Eisenhofer G, Kopin IJ, and Goldstein DS (2004) Catecholamine metabolism: A contemporary view with implications for physiology and medicine. Pharmacology Reviews 5: 331–349. Ferguson SSG (2001) Evolving concepts in G protein-coupled receptor endocytosis: The role in receptor desensitization and signaling. Pharmacology Reviews 53: 1–24. Frazer A (2000) Norepinephrine involvement in antidepressant action. Journal of Clinical Psychiatry 61(supplement 10): 25–30. Gordon M Autonomic (ANS) pharmacology: Introduction. In: Medical Pharmacology and Disease-Based Integrated,
Instruction, Ch. 4. http://www.pharmacology2000.com/Autono mics/Introduction/Introobj1.htm(accessed April 2007). Hieble JP, Bylund DB, Clarke DE, et al. (1995) International Union of Pharmacology. X: Recommendation for nomenclature of alpha-1 adrenoceptors: Consensus update. Pharmacology Reviews 47: 267–270. Hokfelt T, Johansson O, and Goldstein M (1984) Central catecholamine neurons as revealed by immunohistochemistry with special reference to adrenaline neurons. In: Bjo¨rklund A and Hokfelt T (eds.) Handbook of Chemical Neuroanatomy, Vol. 2: Part 1: Classical Transmitters in the CNS. Amsterdam: Elsevier. Murrin LC, Sanders JD, and Bylund DB (2007) Comparison of the maturation of the adrenergic and serotonergic neurotransmitter systems in the brain: Implications for differential drug effects on juveniles and adults. Biochemical Pharmacology 73: 1225–1236. Neumeister A, Charney DS, Belfer I, et al. (2005) Sympathoneural and adrenomedullary functional effects of alpha-2C adrenoreceptor gene polymorphism in healthy humans. Pharmacogenetics and Genomics 15: 143–149. Rockman HA, Koch WJ, and Lefkowitz RJ (2002) Seventransmembrane-spanning receptors and heart function. Nature 415: 206–212. Small KM, McGraw DW, and Liggett SB (2003) Pharmacology and physiology of human adrenergic receptor polymorphisms. Annual Review of Pharmacology and Toxicology 43: 381–411. Urban JD, Clarke WP, von Zastrow M, et al. (2007) Functional selectivity and classical concepts of quantitative pharmacology. Journal of Pharmacology and Experimental Therapeutics 320: 1–13.
Relevant Websites http://www.adrenoceptor.com – Adrenoceptor Online. http://pubchem.ncbi.nlm.nih.gov – PubChem Compound Summary: PubChem Compound 5814.
Norepinephrine: CNS Pathways and Neurophysiology G Aston-Jones, Medical University of South Carolina, Charleston, SC, USA C A Meijas-Aponte, National Institutes of Health, Baltimore, MD, USA B Waterhouse, Drexel University College of Medicine, Philadelphia, PA, USA ã 2009 Elsevier Ltd. All rights reserved.
Norepinephrine Cell Groups and Pathways Locus Coeruleus
The nucleus locus coeruleus (LC) is a dense cluster of noradrenergic neurons in the dorsorostral pons. This nucleus gained prominence in the 1960s when new anatomical approaches revealed it to be the major source of norepinephrine (NE) in brain with projections throughout most central nervous system (CNS) regions. These findings stimulated a great deal of research into this system, resulting in a wealth of knowledge at the cellular, systems, and behavioral levels. Considerable evidence indicates that these neurons are important in a variety of cognitive, affective, arousal, and other behavioral functions, as well as associated clinical dysfunctions. Locus coeruleus efferent projections As summarized in Table 1, LC NE neurons project throughout the neuraxis, including the cerebral cortex, thalamus, cerebellum, midbrain, and spinal cord. In this regard, it is different from other ‘typical’ brain systems that have very limited projection targets (e.g., thalamocortical projections). Despite this high degree of divergence, LC neurons exhibit substantial regional and laminar specificity in their efferent projections. Notably, brain areas in primates that are associated with attentional processing (e.g., parietal cortex, pulvinar nucleus, and superior colliculus) have particularly dense LC projections and NE receptors. Notable areas that receive little or no input from the LC include the caudate putamen and most of the hypothalamus. Ultrastructural evidence indicates that these neurons often make conventional synaptic appositions with postsynaptic targets, although the frequency of such synaptic contacts is controversial. Extrasynaptic (paracrine) release of NE from LC terminals also appears to be possible. Locus coeruleus afferents A variety of evidence indicates that the LC receives a wealth of different neurotransmitter inputs. This nucleus is densely innervated by fibers that contain opiates, glutamate,
430
g-aminobutyric acid (GABA), serotonin, epinephrine, histamine, and the peptide orexin/hypocretin. The sources of these various inputs have not been fully elucidated, though some major inputs have been identified. The nucleus paragigantocellularis lateralis (PGi) in the ventrolateral rostral medulla, a major input that strongly excites LC neurons, is a source for glutamate, GABA, enkephalin, corticotropin-releasing hormone (CRH), and epinephrine. A strongly inhibitory GABA and enkephalin input originates from the dorsomedial rostral medulla. Excitatory orexin and histamine inputs originate in the hypothalamus. The shell of dendrites surrounding the LC nucleus offers additional extensive targets for afferent termination, and indeed it appears that several areas target these extranuclear dendrites more so than the LC nucleus proper. Thus, projections from the periaqueductal gray matter, parabrachial region, preoptic area, amygdala, and medial prefrontal cortex, among other sites, project primarily to the peri-LC region, with relatively little input to the LC proper. Although some of these projections have been shown to contact LC dendrites, additional ultrastructural studies are needed to examine others. A recent study has extended our understanding of LC afferents to the circuit level. Using transynaptic tracing, lesions, and electrophysiology, Aston-Jones and colleagues determined that the suprachiasmatic nucleus (SCN) indirectly innervates the LC, using the dorsomedial hypothalamus as a relay. Based upon the role of the SCN as the brain’s major circadian pacemaker and the role of the LC in arousal and performance (see later), these findings reveal a possible circuit for circadian regulation of arousal and performance. Recent work by Iba, Aston-Jones, and colleagues reveal that in monkey strong projections to LC originate in cingulate and orbital cortices (Figure 1). Given the roles of these prefrontal areas in conflict monitoring, decision making, and other cognitive functions, these projections may be responsible at least in part for the different modes of activity that occur in LC neurons during cognitive performance (described later). Interestingly, these prefrontal projections to LC appear to be stronger in primates than in rats, in concert with the more highly evolved prefrontal cortex and more prominent role of this structure in regulating behavior in primates. Taken together, these observations indicate that LC is not a relay nucleus for primary sensory or motor information and that it receives highly processed information concerning internal and external sensory stimuli as well as behavioral and affective states. This suggests a highly integrative function for this system.
Norepinephrine: CNS Pathways and Neurophysiology 431 Table 1 Organization of the adrenergic system Fiber density
Forebrain Cortex Hippocampal formation Amygdala Medial Central Basal Basal forebrain/septum Medial septum Medial preoptic area Substatia innominata Lateral septum Bed nucleus stria terminalis Ventral pallidum Zona incerta Subthalamic nucleus Nucleus accumbens Thalamus Sensory Lateral geniculate nu Medial geniculate nu Ventroposterior medial nu Ventroposterior lateral mu Ventrobasal nu Ventromedial nu Reticular nu Motor Ventrolateral nu Ventralanterior nu
Locus coeruleus A4 þ A6
2þ 2–3þ
a a
1þ 3þ 3þ
a a
2þ
3þ 5þ
a a a a a a
Dorsal medulla cluster
A1
C1
A5
A2
C2
C3
a a
a
a
a a
a
a
a a a a a a a
a a a a a a
a a a a
1–3þ
3–4þ 3þ 3–4þ
a a a a a a a
3þ 3þ
a a
Viscero-limbic Anteriormedial nu Anteriodorsal nu Anteriovental Mediodorsal nu Central medial nu Central lateral nu Reuniens nu Parafascicular nu Paraventrivular nu Rhomboid nu
4þ 3þ 2þ 1–2þ 1þ 1–2þ 1þ 1þ2 5þ 2–3þ
a a a a a a a a *
Epithalamus Medial habenulla Lateral habenulla
5þ 4–5þ
* *
Hypothalamus Periventricular nucleus Suprachiamatic nucleus Retrochiasmatic Paraventricular nuclues Supraoptic nuclues Dorsomedial Arcute nuclues Medial eminence Perifornical area Lateral area
5þ 1–2þ 4þ 3–5þ 4þ 3–5þ 3þ 2þ 4þ 3þ
a *
2þ
a
Brain stem Sensory Main sensory trigeminal nu
Ventral pons and medulla cluster
3–4þ 1–2þ
a a a a
a a a a a a a
a
a
a
a
a a a a
A7
a a a a a
a a a a
a
a a a a
a
Continued
432 Norepinephrine: CNS Pathways and Neurophysiology Table 1 Continued Fiber density
Spinal trigeminal nu Mesencephalic trigeminal nu Superior olivary complex Cochlear nu Vestibular nu
2þ 4þ 1þ 2þ 0–1þ
Motor Oculomotor and trochlear nu Motor trigeminal nu Abducens nu Facial nu Hypoglossal nu Nucleus ambiguus Inferior olive Red nucleus Cerebellum
0–1þ 4þ 0–1þ 3–4þ 2–3þ 5þ 5þ 1þ
Sensorimotor Superior colliculus Superficial layers Deep layers Inferior colliculus Visceral Nucleus of the solitary tract including A2 and C2 areas Dorsal motor nucleus of vagus Caudal ventrolateral medulla including A1 area Rostral ventrolateral medulla including C1 area Periaqueductal gray Parabrachial nucleus Area postrema Modulatory systems Substantia nigra, pars compacta Ventral tegmental area Retrorubral field Raphe Central linear Dorsal Magnun Pallidus Obscurus Locus coeruleus A5 area Spinal cord Intermediolateral cell column Dorsal horn Ventral horn
Locus coeruleus A4 þ A6
a a a a a
a a a a
Ventral pons and medulla cluster
Dorsal medulla cluster
A1
A2
C1
a
a
a
a
a a
a
a
a a
a a
2þ5 4þ 1þ
a a a
a a a
a a a
a a
a a a a
a **
a a
a
a
a
a
a
3þ
2þ 2þ 3þ 5þ 1þ 2þ
C3
a
a
a
a a
a
a
5þ 3þ
0–1þ 1þ 1þ
C2
a
a
a
5þ
A7
a
2þ
2þ
A5
a
a a a
a a
a a a a
a
a
a
a
a
a
This table is based on the organization of LC versus non-LC systems as originally reviewed by Moore and Card in 1984 and published in the Handbook of Chemical Neuroanatomy, volume 2. The descriptions of fiber density were obtained from Moore and Card, whereas the information regarding efferent projections was updated based upon a comprehensive survey of research reports available in the literature. The information presented is meant to reflect major efferents of the adrenergic cell groups. Single asterisks indicate structures known to not receive LC afferents but for which the source of adrenergic innervation is unknown. The double asterisk indicates ventral horn innervation by LC, which is dependent upon rat strain.
Norepinephrine release Microdialysis methods have recently provided in vivo confirmation that certain drugs and behavioral conditions increase or decrease the release of norepinephrine from locus
coeruleus axons. For example, reuptake blockers and stress both enhance extracellular NE levels in areas (such as the neocortex) that receive their only NE innervation from the LC. In addition, the
Norepinephrine: CNS Pathways and Neurophysiology 433 A24
A25
CC
A26
A27
CC
a
A22
CC
A23
CC
A24
CC
b Figure 1 Plots of retrogradely labeled neurons in the nucleus accumbens and orbitofrontal cortex after injections of cholera toxin B into monkey locus coeruleus. (a) Accumbens neurons labeled from the monkey locus ceruleus. Lower sections are high-power views containing plotted cells; upper sections are low-power views to give orientation. (b) Orbitofrontal cortex neurons labeled from the monkey locus coeruleus. Sections on the right are high-power views containing plotted cells; sections on the left are low-power views to give orientation. For both panels, labels A22–A27 refer to distances (in millimeters) from the interaural line. CC, corpus callosum. From Aston-Jones G and Cohen JD (2005) Adaptive gain and the role of the locus coeruleus–norepinephrine system in optimal performance. Journal of Comparative Neurology 493: 99–110.
amount of NE release in cortex increases in proportion to activity within LC, even at low frequencies (2–4 Hz). Notably, burst stimulation elicits more NE release per impulse than evenly do spaced stimuli. This indicates that phasic LC responses (see later) may be particularly effective in releasing NE in target areas. Locus coeruleus impulse activity Sleep and waking. Spontaneous LC activity in rats, cats, or monkeys varies with the stage of the sleep–wake cycle, with firing being most rapid during wakefulness, slower during slow-wave sleep, and virtually absent during paradoxical sleep. LC neurons are phasically
activated by conspicuous unconditioned stimuli. Notably, stimuli that elicit large LC responses in either rats or monkeys also typically disrupt sleep or ongoing behavior, and evoke waking or a behavioralorienting response. The same stimuli do not disrupt behavior if they elicit small LC responses. Stress. Other studies have revealed strong activation of LC neurons by stressors. Stimuli such as sciatic nerve activation or other painful events strongly activate LC cells. Other stressors, such as a puff of air in the awake monkey or a variety of environmental or physiological stressors, also activate LC neurons. In addition, LC neurons are activated by corticotropin-releasing hormone, which mediates the response of LC cells to certain physiological stressors such as hypotension. Locus coeruleus activity during cognitive performance. The preceding results suggest that, in addition to a role in sleep and waking, LC neurons may be involved in behavioral responses to sensory stimuli. Aston-Jones and colleagues tested this by recording impulse activity of LC neurons in monkeys performing a target detection task. These studies indicated that the LC exhibits two modes of impulse activity: a tonic mode in which tonic activity is high but phasic responses to targets are low or absent, and a phasic mode in which LC neurons fire tonically more slowly but exhibit consistent activation shortly following target stimuli. Behaviorally, the tonic mode corresponds to poor task performance with many false alarm errors, whereas the phasic mode is associated with nearly error-free performance. These results indicate that these different modes of LC activity may facilitate either selective, focused attention (phasic mode) or flexible behavior and scanning (tonic mode). Each of these LC and behavioral modes is adaptive, depending on the context. LC activation at decision completion. Recent studies recording LC neural activity in monkeys performing a forced-choice task revealed that the aforementioned phasic responses described are neither purely sensory nor motor/premotor in nature. As shown in Figure 2, these LC responses are more closely linked to behavioral responses (lever releases) than to sensory stimuli that trigger the behavioral responses, but do not occur for lever releases elicited outside of the task context. Finally, the onset of these LC responses occurs at about 200 ms prior to the lever releases, at a time that the decision concerning lever release is being made. Aston-Jones and colleagues concluded from these results that these phasic LC responses reflected completion of a decision process, and that the modulatory gain-enhancing effects of the associated NE release (see later) facilitate brain processes and behaviors called upon by the decision just
434 Norepinephrine: CNS Pathways and Neurophysiology
9
Correct trials Incorrect trials
8
Omissions
7
Spikes s−1
6 5 4 3 2 1
Lever release
0
−0.4
−0.2
a
0.0
0.2
0.4
0.6
0.8
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1.2
1.4
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Incorrect trials
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Bar releases not assoc. with stimuli
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6 5 4 3 Stimulus onset
2 1 0
b
−0.6
−0.4
−0.2
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Response Time ( s )
Figure 2 Stimulus- and response-locked population perievent time histograms (PETHs) showing LC responses to cues for trials yielding correct and incorrect behavioral responses. (a) Stimulus-locked population PETHs showing LC response to cues (presented at time 0) for trials yielding correct or incorrect behavioral responses. Note that the LC response peaks sooner and is less prolonged on correct, compared to incorrect, trials in this analysis (17 533 and 1362 trials, respectively). No fluctuation in LC activity was detected on omission trials (dashed line, 1128 trials). Vertical lines indicate the mean behavioral RTs. Curves below PETHs represent the normalized RT distributions for correct and incorrect trials. (b) The difference in the phasic LC response between correct and incorrect trials is no longer evident on response-locked population PETHs. In addition, no LC response occurred prior to or following lever releases not associated with stimulus presentation (dashed line, 3381 trials). Vertical lines indicate the mean stimulus onset times. From Clayton EC, Rajkowski J, Cohen JD, et al. (2004) Phasic activation of monkey locus coeruleus neurons by simple decisions in a forced choice task. Journal of Neuroscience 24: 9914–9920.
made. In this way, the LC phasic response supports the current task/goal and facilitates focused performance in that task. The projections to LC from orbital and cingulate cortices (described previously) may mediate these phasic LC responses associated with decision processes.
Non-LC Norepinephrine and Epinephrine Cell Groups
Neurons in other brain nuclei aside from those in the LC constitute 55% of all CNS NE and epinephrine (E) cells. These neurons are clustered in two
Norepinephrine: CNS Pathways and Neurophysiology 435
rostrocaudally aligned columns within the brain stem. One column is located primarily in the ventral aspect of the pons and medulla and contains 44% of all NE/E neurons, whereas the second is situated in the dorsomedial medulla. The individual clusters of non-LC NE/E neurons within these cell columns are further subdivided on the basis of their specific anatomical location and the ability to synthesize E (Figure 3). Non-LC NE/E projections An analysis of projection patterns of LC versus non-LC NE/E neurons reveals similarities and differences (Table 1). Non-LC NE neurons project exclusively subcortically and make a preponderance of connections onto motor nuclei in the medulla and spinal cord, and onto structures associated with visceral function. Within the brain stem, targets of non-LC NE/E neurons include cranial motor nuclei such as the dorsal motor vagus, motor trigeminal, motor facial, and the hypoglossal nucleus, as well as visceral–sensory nuclei such as the nucleus of the solitary tract, parabrachial complex, and periaqueductal gray. In the forebrain, non-LC cell clusters provide dense projections to visceral-related structures such as the bed nucleus of the stria terminalis (BNST), central nucleus of the amygdala, and paraventricular hypothalamus. Thalamic and epithalamic targets include the paraventricular nucleus, rhomboid nucleus, and habenula. Within the different non-LC adrenergic nuclei, there is a preferential innervation of motor systems by A5 and A7 groups, whereas visceral–sensory areas are primarily innervated by medullary A1 and A2 nuclei. The E-containing fibers from C1 neurons
AQ
Cer
ebe
llum
Q
V4
A7 Po n
LC s
A5
Hind brai n C3 C2
A2
CC
C1 M e d u l l a A1
Figure 3 Localization of norepinephrine (red) and epinephrine (green) nuclei in the brain stem. The cell column in the ventral pons and medulla includes A1, C1, A5, and A7 cell groups, whereas the dorsal medulla column includes A2, C2, and C3 cell groups.
innervate both motor and visceral–sensory structures. C1 neurons send collaterals that innervate both spinal cord and brain stem targets. However, most C1 neurons that project to hypothalamus do not project to spinal cord or brain stem structures. In clear contrast to this pattern of organization, LC projections to the brain stem and spinal cord are directed almost exclusively to relay sites along ascending sensory pathways, including those associated with somatosensory, auditory, gustatory, and visual systems. This sensory-predominant bias of the LC efferent projection continues to the level of the thalamus and also includes the olfactory system. An overlap between LC and non-LC NE efferent projections to motor structures becomes evident in the cerebellar system, and the LC is the sole source of NE fibers to thalamic motor relay nuclei and motor (as well as sensory) processing regions of the cerebral cortex. Non-LC impulse activity LC and non-LC neurons have similar electrophysiological signatures but exhibit differing responses to afferents and neurotransmitter systems. They are slow-firing neurons (<5 Hz) with wide action potentials (lasting more than 2 ms). Pharmacologically, both LC and non-LC neurons are inhibited by a2 adrenoceptor agonists, indicating the presence of a functional a2 autoreceptor. Conversely, sciatic nerve stimulation activates LC neurons, and also to a lesser extent A5 neurons, but not A2 neurons, indicating selectivity in the systems that activate the different adrenergic groups. Moreover, morphine strongly inhibits LC neurons but is reported to excite A5 neurons. The organization of the non-LC adrenergic system and its connection to visceral-related areas prompted many studies of the non-LC adrenergic neurons; the findings indicate a diversity of roles in modulating cardiovascular, respiratory, renal, gastrointestinal, and endocrine functions. For example, the A1 area is involved in hemodynamic regulation by controlling the release of vasopressin, whereas C1 neurons are sensitive to blood pressure and are involved in regulating sodium and water balance. A5 neurons influence the respiratory rhythm generator of the rostral ventrolateral medulla, and A2 cells have a role in the regulation of food intake. Stress Physiological (cardiovascular, osmotic, and immune challenges) as well as psychological (restraint and intermittent footshock) stressors activate both LC and non-LC NE/E neurons. These stressors also activate major targets of the non-LC system, such as the medial and central nuclei of the amygdala, BNST, and paraventricular hypothalamus, suggesting that nonLC adrenergic neurons are part of a functional stress
436 Norepinephrine: CNS Pathways and Neurophysiology
circuit. A significant difference between LC and nonLC cell groups may be habituation to stressors. Under chronic footshock regimens, LC neurons habituate whereas non-LC cells in A1, C1, A2, and C2 exhibit persistent stress responses. Thus, NE/E transmission in response to acute and persistent stressful conditions is differentially distributed across brain structures according to the unique efferent topographies of the LC and non-LC systems. Drugs of abuse have profound effects on the activity of NE neurons throughout the brain. The aversiveness of opiate withdrawal is blocked by antagonizing b adrenoceptors in BNST; this NE input largely originates in A1/A2 neurons. A1/A2 neurons also appear to play a particularly prominent role in relapse to drug-seeking induced by stress. Several studies have shown that selective lesions of ascending A1/A2 NE projections, or antagonism of NE receptors in amygdala or BNST, attenuate relapse to drug-seeking induced by footshock stress. Summary for non-LC systems In summary, several principles of organization emerge from examination of the efferent connections of the central NE/E cell groups. First, the distribution of adrenergic fibers is particularly dense in terminal fields innervated by non-LC adrenergic neurons (Table 1). Second, within the brain stem, the LC preferentially innervates primary sensory pathways while non-LC adrenergic nuclei innervate motor and visceral–sensory systems. Third, non-LC forebrain projections target visceral– sensory and autonomic circuits. Fourth, areas that subserve motor integrative functions such as the cerebellar circuit are mainly innervated by LC, with the exception of the inferior olive, which is mainly innervated by the A5 and A7 areas. Fifth, LC is the only source of NE innervation to the cortical mantle. Sixth, although all NE/E neurons have similar electrophysiological properties, LC and non-LC cells may respond differently to the same afferent inputs and neurotransmitter systems. This analysis leads to an appreciation of the complex anatomical relationships and physiological/behavioral properties of the different brain NE/E systems. Postsynaptic Actions of Norepinephrine
Investigations in the 1960s and 1970s viewed NE as a putative conventional neurotransmitter and set out to determine its ability to increase or decrease the firing rate of target neurons. For the most part, local application of NE by microiontophoresis, or stimulation of LC, suppressed the spontaneous discharge of neurons in cerebellum, cerebral cortex, and elsewhere in the brain. These depressant responses were mimicked and blocked by adrenoceptor agonists and
antagonists, respectively. Taken together, these findings support the idea that NE might function primarily as an inhibitory transmitter at central synapses. Modulatory effects of NE on single neurons In the mid-1970s pioneering studies by Foote et al. in monkey auditory cortex demonstrated a differential depressant effect of microiontophoretically applied NE on single neurons, such that the spontaneous firing rate of recorded cells was suppressed to a greater extent than were stimulus-evoked discharges, thus yielding a net increase in the signal-to-noise ratio. This initial study prompted many other laboratories to investigate the actions of NE in a variety of sensory circuits within the mammalian brain, including olfactory bulb, sensory thalamus, visual, auditory, and somatosensory cortices. In many cases local application of NE was found to enhance extracellular responses of individual sensory neurons to synaptic stimuli; however, mixed effects were observed in other studies, with suppression of stimulus-evoked discharge predominating. Later work by Waterhouse and colleagues examined NE actions at levels that had minimal or no effect on spontaneous discharge of target neurons. Under these conditions iontophoretic NE frequently produced absolute increases in stimulus-evoked discharge with minimal or no change in baseline firing. In other cases local administration of NE revealed robust cellular responses to otherwise subthreshold synaptic stimuli. The demonstration of NE-induced facilitation of cellular responses to direct iontophoretic application of GABA or glutamate under both normal and low-calcium/high-magnesium conditions argued that such effects were mediated postsynaptically. These findings in cerebral cortex and cerebellum of anesthetized and awake, behaving animals supported the general hypothesis that a prominent physiological function of central noradrenergic pathways is to enhance the efficacy of both excitatory and inhibitory synaptic transmission within target neuronal circuits, rather than to directly suppress cell firing. Later work by Waterhouse et al. in cat and rat primary visual cortex showed that NE could alter specific receptive field properties (e.g., receptive field size, direction selectivity, velocity tuning) of visually responsive cells (Figure 4). Other studies showed that NE was capable of modifying the receptive field structure of cells in somatosensory and auditory cortices and cochlear nuclei. As such, these results go beyond the demonstration of simple monoamine-induced changes in the magnitude of synaptically evoked responses and begin to show that such actions can lead to selective alteration of the feature extraction properties
Norepinephrine: CNS Pathways and Neurophysiology 437 Control Shut
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Figure 4 Effects of norepinephrine (NE) on responses of rat visual cortical neurons to visual field stimulation. (a, b) The raster and corresponding perievent histogram records show the extracellularly recorded responses of two single neurons in the rat visual cortex to movements of a vertically oriented (shown on the left) bar of light across the contralateral hemivisual field before, during, and after periods of microiontophoretic administration of NE. For each set of records, the uppermost line depicts the opening (step-up) and closing (step-down) of the shutter controlling the stimulus display. The trapezoid waveform indicates the position of the stimulus in visual space as it moves in the temporal (T) to nasal (N) direction and then back along the same path in the reverse direction. Time is indicated along the horizontal axis of the histogram, and discharge frequency of the cell is indicated along the vertical axis. Stimulus-bound excitatory or inhibitory components of the response were expressed as a percentage increase or decrease, respectively, of background firing rate (dotted line beneath histogram). (a) In the first cell the control response (top) consisted of an excitatory burst (solid bar) preceded by an inhibitory trough as the stimulus moved in the T to N direction, but no significant change in activity as the stimulus moved along the same path in the opposite direction (N to T). The visually evoked excitatory peak represented a 172% increase in firing over the basal level of discharge. Iontophoretic application of NE (5 nA) suppressed basal firing rate but produced an absolute increase in the visually evoked response (middle). Recovery to the control level of response was observed following cessation of NE administration (bottom). Each histogram sums unit activity during 13 trials; bin width ¼ 15 ms. (b) In the second cell, there was no discernible response to the moving bar of light during the control period (top); however, during NE (5 nA) administration (middle) an obvious stimulus-evoked excitation (broken bar beneath histogram) and a stimulus-bound inhibition (dotted line beneath histogram) became evident. After cessation of NE iontophoresis (bottom), the cell gradually lost its sensitivity to the visual stimulus. Control and NE histograms, n ¼ 22 trials; recovery histogram, n ¼ 31 trials. Bin width ¼ 15 ms.
of individual sensory neurons. More recent work by Arnsten, and colleagues indicates that NE and NE agonist drugs can modify the working-memory-related discharge of cells in prefrontal cortex of behaving
monkeys. These latter findings suggest that the noradrenergic actions observed in sensory circuits may be generalized across brain regions that serve many different functions.
438 Norepinephrine: CNS Pathways and Neurophysiology
Additional studies in cortex and elsewhere have determined the pharmacologic specificity and dose dependency of NE-mediated modulatory effects. For example, the magnitude of cellular response to direct glutamate application can be maximally enhanced at a specific level of NE administration, and only moderately or minimally be affected by concentrations above and below this level (Figure 5(a)), thus revealing an inverted-U dose–response relationship for this effect of NE. Furthermore, noradrenergic enhancement of neuronal responses to excitatory synaptic stimuli are mimicked and blocked by a1-receptor agonists (Figure 5(b)) and antagonists,
respectively, whereas the augmentation of inhibitory responses to GABA application (Figure 6) or afferent pathway stimulation involves activation of b receptors. Other studies, particularly those in hippocampus, provide data suggesting the opposite (i.e., NE enhancement of excitatory synaptic transmission is dependent upon b-receptor mechanisms and augmentation of synaptic inhibition is mediated by a1-receptor mechanisms). These disparate results have not been reconciled but may reflect unique receptor distributions or second-messenger linkages among neurons within different brain regions. Overall, these observations indicate that within a specified brain circuit
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Figure 5 Modulatory actions of ionotphoretically applied norepinephrine and a1 agonist (phenylephrine) on glutamate-evoked discharges in rat barrel field cortical neurons. Responses of individual layer V neurons to iontophoretic pulses of suprathreshold levels of glutamate were quantified by analysis of perievent histograms (shown as insets) that were collected before, during, and after continuous microiontophoretic application of norepinephrine. Responses were then expressed as a percentage change from control (i.e., response to glutamate application alone ¼ 100%) for different iontophoretic doses (nA) of norepinephrine. Each histogram and corresponding point on the graph represents the cell’s response to six consecutive glutamate applications. (a) Example of a layer V cortical neuron: its excitatory response to glutamate (28 nA) was progressively increased with increasing levels of norepinephrine (peak at 5 nA) and then progressively suppressed from this optimal value with further increases in norepinephrine ejection current (6–20 nA). (b) Example of a second layer V cortical neuron: its excitatory response to glutamate (40 nA) was likewise progressively increased with increasing levels of phenylephrine (peak at 5 nA) and then progressively suppressed from this peak value with further increases in phenylephrine ejection current (10–30 nA).
Norepinephrine: CNS Pathways and Neurophysiology 439 Control
Control 72%
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88%
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72% 5 5
a
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Figure 6 Actions of norepinephrine (NE) and the b agonist isoproterenol (ISO) on g-aminobutyric acid-induced inhibition of cortical neuron spontaneous discharge. Histogram records show responses of two somatosensory cortical cells (a and b) to regularly spaced, uniform iontophoretic pulses of g-aminobutyric acid applied before, during, and after NE (a) or ISO (b) administration. Numbers above each histogram indicate the percentage of inhibition of spontaneous firing rate produced by g-aminobutyric acid application. Each histogram sums unit activity for an equal number of g-aminobutyric acid pulses, n ¼ 5. Both NE (10 nA) and ISO (15 nA) produced a marked enhancement of the g-aminobutyric acid response relative to a suppression of background firing. Recovery to the control level of response was observed in each case after termination of adrenergic drug application. Calibration: vertical, 5 counts per address; horizontal, 5 s.
an array of noradrenergic modulatory actions is possible, depending upon cellular expression of receptors and levels of NE available for interaction with those receptors. Cellular mechanisms of NE action Intracellular experiments have demonstrated multiple actions of NE on properties of neuronal membranes. For example, NE-induced blockade of a calcium-activated potassium conductance leads to accommodation block and prolongation of evoked excitatory discharges. It is widely accepted that this effect, in conjunction with a membrane hyperpolarizing action of NE, can account for noradrenergic potentiation or signal-to-noise ratio enhancement of threshold level excitatory synaptic responses observed in vivo. McCormick and colleagues have found that NE’s depolarizing action influences a complex interaction between intrinsic membrane currents in thalamic relay neurons. This interplay moves thalamic neurons back and forth between a non-signal-transmitting oscillatory mode during sleep and a single-spike transmission, or ‘relay,’ mode during waking.
Available evidence implicates the b-receptor-linked cyclic AMP system in NE-mediated augmentation of GABA-induced inhibition. For example, in vivo, local application of agents that elevate intracellular levels of cyclic AMP mimic or augment NE-mediated enhancement of GABA-induced inhibition of cerebellar Purkinje neurons. Subsequent studies in acutely dissociated cerebellar Purkinje neurons demonstrated augmentation of GABA membrane currents via intracellular diffusion of 8-bromo-cyclic AMP or the catalytic subunit of protein kinase A. And finally, additional investigations found that subunits of the GABA-A receptor can be phosphorylated by protein kinase A. The working hypothesis is that, upon release, NE initiates a cascade of events via postsynaptic b receptors that results in activation of protein kinase A and phosphorylation of GABA-A receptor subunits. In Purkinje neurons this phosphorylation of the receptor leads to enhanced GABA binding capacity or chloride conductance, thus providing for an overall increase in GABA response. The intracellular events associated with NE-induced enhancement of stimulus-evoked excitatory discharges have not been identified with as much certainty, although work by several laboratories identifies protein kinase A and protein kinase C as candidates for mediating transmitter/modulator effects on synaptically evoked excitation. Elucidating the intracellular events and accompanying physiological actions that underlie NE-induced enhancement of responsiveness to synaptic inputs are major issues for future studies. Effects of locus coeruleus output on neuronal and neural circuit response properties Studies in anesthetized animals show that stimulation of the LC efferent path elicits NE-like modulatory effects on target neurons in sensory circuits. These findings support the general hypothesis that a major function of the central LC–noradrenergic efferent system is to facilitate the transfer of information through sensory circuits following events that phasically activate the LC nucleus. More recent work in waking animals has shown that phasic and tonic activation of the LC efferent path can enhance the probability of stimulus-evoked spiking in both cortical and thalamic neurons according to an inverted-U function. Thus, as described for iontophoretically applied NE, neuronal responses to afferent stimulation are increased to a maximum at intermediate levels of LC stimulation, and to submaximal levels with LC activation levels above and below this level. As reported for iontophoretically applied NE, activation of the LC efferent pathway can also reveal or ‘gate’ cellular responses to otherwise subthreshold synaptic inputs. LC stimulation also decreases mean response latency and reduces
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trial-to-trial variability of response latency for both sensory thalamic and cortical neuron responses to afferent input. Thus, increased output from the LC is capable of altering the magnitude and timing of cellular responses to sensory-driven synaptic input as well as recruiting (gating) additional cells into a response network. These results are important in that they extend observations of LC- and NE-mediated modulatory actions into the waking condition. Further, they show that LC output can simultaneously facilitate stimulusevoked discharge patterns at multiple levels of an ascending sensory pathway. The observed effects of LC stimulation on response threshold, response magnitude, and response latency in individual cells suggests that under certain behavioral conditions synaptically released NE can optimize information processing within sensory circuits and provide for a network-wide enhancement of sensory signal transmission. This idea is further supported by recent multineuron recording studies in which LC activation and NE release have been shown to modulate the representation of sensory information across ensembles of functionally related thalamic neurons. Functional and Clinical Implications
The widespread NE system appears to be functionally multifaceted. On a 24 h timescale it modulates sleep and waking, permitting sleep with inactivity but promoting arousal by becoming active. Recent results for a circuit from the SCN to the LC indicate that this system may contribute to the circadian regulation of arousal and sleep–waking. However, on a much shorter timescale of seconds, this system appears to modulate attention and performance during the waking state. In this domain the LC system regulates behavioral flexibility by promoting either stable selective attention and focused behavioral performance or a scanning type of attention and more flexible behavior. These findings have implications for the involvement of the LC system in clinical disorders. The role proposed for the LC in attention suggests that dysfunction of this system could contribute to the cognitive and sensory signal-processing deficits that are prominent in schizophrenia, attention-deficit/ hyperactivity disorder, and dementia. In regard to dementia, LC neurons have been found to degenerate in both Alzheimer’s and Parkinson’s diseases. Many clinical studies have also implicated this system in depression and anxiety. This is consistent with findings that there are substantial projections of NE neurons throughout the limbic system and that the LC system is engaged by novel and alarming stimuli. The
high responsiveness of LC neurons to acute and chronic stressors is also consistent with a role for this system in depression. Moreover, many of the drugs used successfully to treat anxiety and depression target the noradrenergic system. There is, as well, a growing body of clinical evidence suggesting a role for the LC–NE system in posttraumatic stress disorder. There have also been various proposals for a role of the LC in the etiology of sleep disorders. This is consistent with both the close association of activity of these cells with different stages of the sleep–wake cycle and the disrupted sleep patterns that are a hallmark of depression. Recent results indicate that the role of the LC extends to circadian disorders in sleep and performance as well. See also: Monoamines: Release Studies; Norepinephrine: Adrenergic Receptors.
Further Reading Aston-Jones G, Chen S, Zhu Y, et al. (2001) A neural circuit for circadian regulation of arousal. Nature Neuroscience 4: 732–738. Aston-Jones G and Cohen JD (2005) Adaptive gain and the role of the locus coeruleus–norepinephrine system in optimal performance. Journal of Comparative Neurology 493: 99–110. Aston-Jones G and Cohen JD (2005) An integrative theory of locus coeruleus–norepinephrine function: Adaptive gain and optimal performance. Annual Review of Neuroscience 437: 556–559. Berridge CW and Foote SL (1991) Effects of locus coeruleus activation on electroencephalographic activity in the neocortex and hippocampus. Journal of Neuroscience 11: 3135–3145. Berridge CW and Waterhouse BD (2003) The locus coeruleus– noradrenergic system: Modulation of behavioral state and state-dependent cognitive processes. Brain Research Reviews 42: 33–84. Clayton EC, Rajkowski J, Cohen JD, et al. (2004) Phasic activation of monkey locus coeruleus neurons by simple decisions in a forced choice task. Journal of Neuroscience 24: 9914–9920. Florin-Lechner S, Druhan J, Aston-Jones G, et al. (1996) Enhanced norepinephrine release in prefrontal cortex with burst stimulation of the locus coeruleus. Brain Research 742: 89–97. Foote SL, Bloom FE, and Aston-Jones G (1983) Nucleus locus coeruleus: New evidence of anatomical and physiological specificity. Physiological Reviews 63: 844–914. Guyenet PG (1991) Central noradrenergic neurons: The autonomic connection. Progress in Brain Research 88: 365–380. Hokfelt T, Johansson O, and Goldstein M (1984) Central catecholamine neurons are revealed by immunohistochemistry with special reference to adrenergic neurons. In: Bjorklund A and Hokfelt T (eds.) Classical Transmitters in the CNS. Part I, pp. 157–276. Amsterdam: Elsevier. Moore RY and Card JP (1984) Noradrenaline-containing neuron systems. In: Bjorklund A and Hokfelt T (eds.) Classical Transmitters in the CNS. Part I, pp. 123–156. Amsterdam: Elsevier.
Norepinephrine: CNS Pathways and Neurophysiology 441 Ramos BP and Arnsten AF (2007) Adrenergic pharmacology and cognition: Focus on the prefrontal cortex. Pharmacology & Therapeutics 113: 523–536. Sawchenko PE, Li HY, and Ericsson A (2000) Circuits and mechanisms governing hypothalamic responses to stress: A tale of two paradigms. Progress in Brain Research 122: 61–78. Shaham Y, Shalev U, Lu L, et al. (2003) The reinstatement model of drug relapse: History, methodology and major findings. Psychopharmacology (Berlin) 168: 3–20.
Valentino RJ, Foote SL, and Page ME (1993) The locus coeruleus as a site for integrating corticotropin-releasing factor and noradrenergic mediation of stress responses. Annals of the New York Academy of Sciences 697: 173–188. Woodward D, Moises H, Waterhouse B, et al. (1979) Modulatory actions of norepinephrine in the central nervous system. Federation Proceedings 38: 2109–2116.
Monoamines: Release Studies A D Smith, A C Michael, B J Lopresti, R Narendran, and M J Zigmond, University of Pittsburgh, Pittsburgh, PA, USA ã 2009 Elsevier Ltd. All rights reserved.
Introduction Neurons in the brain act on their targets via the release of chemical transmitters, a process that is generally assumed to occur across a synapse. In the case of monoamines, there is reason to believe that the field of influence can be considerably larger and often involves nonsynaptic or ‘volume transmission.’ Even this larger space between a given monoaminergic neuron and its target is likely to defy direct chemical monitoring, and there is a long history of attempts to overcome this hurdle through a wide variety of indirect approaches. Many of these approaches have been reviewed in the past. This article reexamines the issue from the standpoint of current technology and understanding. Monoamine neurotransmitters, by virtue of their easily oxidized catechol or indole functional groups, can be detected by means of electrochemistry. This has spurred the development of two technologies for monoamine analysis: one comprises electrochemical detectors coupled to chromatography and electrophoresis systems; the other comprises microelectrodes implanted directly into brain tissue for in vivo measurements, so-called in vivo voltammetry. A third approach, the use of positron emission tomography (PET), is based on an entirely different principle, which is that once released, an endogenous monoamine can displace a radioligand that also binds to a receptor on which the monoamine acts. This article reviews each of these three approaches to monitoring monoamine release in the intact brain.
Voltammetry Electrochemistry has revolutionized the capacity to detect monoamine release. Electrochemical detection and voltammetry both use electrochemistry but are otherwise fundamentally different techniques. In the former, the electrode is intended to be nonselective, giving a signal for all oxidizable components of the sample after their separation on a column. It is of great value in studies of fluid samples that can be collected and then analyzed via high-performance liquid chromatography (HPLC). It will be considered at some length in the next section. In contrast, in
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voltammetry, various strategies, including customized potential waveforms and tailored electrode surfaces, are used to render the electrode as selective as practicable for a target monoamine. Absolute selectivity between the monoamines is not possible by electrochemical means alone, so experimental conditions are often chosen carefully in light of this point. For example, in vivo voltammetry is often applied in regions of the brain where a single monoamine is predominant, as in the case of DA within the striatum, to measurements of monoamine release during selective stimulation of pathways; or to measurements of an exogenously applied monoamine. Rather than selectivity, the forte of in vivo voltammetric techniques is their sensitivity, speed of response, and spatial resolution. Indeed, the technology has steadily improved over the course of the last three decades to the point that voltammetry provides access to information not available by other means. Electrode Design
Although a variety of microelectrode designs are described in the literature, the majority of recent in vivo measurements of monoamines have relied on carbon fiber microelectrodes (Figure 1). Carbon fibers, which are available with diameters as small as 5 mm, are usually sealed into a pulled glass capillary with epoxy or sometimes just by virtue of the glass– carbon junction. The fiber protruding from the glass tip can be cut to a desired length, or the tip can be beveled on a polishing wheel so that only the cross section of the fiber is exposed. Although these beveled electrodes are usually called microdisk electrodes, they are actually elliptical in shape. The microdisk geometry is useful when high spatial resolution is needed, such as during the recording of individual exocytotic events, whereas the longer electrodes are suitable when spatial averaging is preferred. The tiny dimensions of carbon fiber microelectrodes offer several benefits to in vivo voltammetry. The electrodes, for example, do very little damage to brain tissue during implantation. Electron microscopy of striatal tissue from anesthetized rats following acute carbon fiber implantation showed that the majority of tissue damage was confined to within approximately 3 mm of the electrode track. This short distance permits faithful recording of in vivo chemical events with minimal diffusional distortion because small molecules need only about 10 ms to diffuse 3 mm through the brain extracellular space. Thus, the minimization of tissue trauma contributes to the rapid speed of response available with in vivo voltammetry.
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Figure 1 Scanning electron micrographs illustrate the features and dimensions of the carbon fiber microelectrodes used in the majority of in vivo voltammetry studies. The fibers in the image have a diameter of 7 mm. The fibers are sealed into a pulled glass pipet with epoxy. The microcylinder electrode protrudes from the pipet, whereas the microdisk is prepared by polishing the tip on a microelectrode beveller.
Fast-Scan Cyclic Voltammetry
Another benefit of the small dimensions of the microelectrodes is the small capacitance of the electrode surface. The small capacitance allows for rapid changes in the potential applied to the electrode, which has enabled the technique called fast-scan cyclic voltammetry. In this technique, the electrode is subjected to linear potential sweeps at rates of 300 V s1 or more. Since monoamines can be detected with potential sweeps spanning about 3 V, individual voltammetric scans are completed in 10 ms or less. The ability to rapidly scan the potential, therefore, is another contributor to the rapid speed of response. Furthermore, as the potential sweep rate increases, voltammetry becomes selective for monoamine neurotransmitters over their acid metabolites and other oxidizable substances, including ascorbate and urate, encountered in brain tissue. This is due to a quirk of the kinetics of the oxidation and reduction reactions of the monoamines, which are substantially faster than those of the potential interfering substances. The fast-scan cyclic technique generates a voltammogram, a plot of the electrode current versus the applied potential, which provides a fingerprint of the detected substance. Recently, as discussed further in the section titled ‘Recording naturally occurring DA release on a subsecond timescale,’ statistical analysis of these fingerprints by means of principal components analysis has greatly advanced the analytical capabilities of in voltammetric recording techniques. While the rapid potential scanning approach has several advantages, they come at a price. Rapidly scanning the potential induces a flow of current called
the electrode charging current. Even though the electrode capacitance is small in an absolute sense, the charging current is massive in comparison to the currents associated with the detection of monoamines. Consequently, the monoamine-associated current appears as a tiny signal component superimposed on a much, much (1000 times) larger background current. For this reason, voltammetry is well suited only to differential measurements, that is, voltammetry can be used to track changes in monoamine concentrations from a starting condition. On the other hand, voltammetry is not able to tell us ‘absolute’ or basal monoamine concentrations. Fortunately, much useful insight into the functional aspects of brain monoamine systems has been derived from differential voltammetric measurements. Constant Potential Amperometry
As an alternative to fast-scan voltammetry, constant potential amperometry has proved a highly useful recording technique. Here, the electrode potential is simply poised at a value appropriate to the oxidation of monoamines, usually near 0.6 V versus Ag/AgCl. This approach provides no selectivity and thus is suitable only in those specialized experimental conditions in which there is no doubt about the identity of the substance being detected. However, several such conditions exist. One example is the use of the clearance of exogenously applied monoamine to evaluate transporter kinetics. Another example is electrical stimulation of the medial forebrain bundle to selectively release DA in the striatum and accumbens. In these experiments, the speed of the recording tends to be of the utmost concern, over and above the selectivity of the response. In the case of constant potential amperometry, the speed of the recording is determined by the data acquisition hardware rather than the features of an applied potential waveform. Characterizing Monoamine Transporter Kinetics
One recent and extensive application of in vivo voltammetry has been to characterize the kinetics of the DA transporter (DAT) in several dopaminergic regions of the rat brain. DAT is responsible for clearing DA from the extracellular space to terminate the actions of DA on pre- and postsynaptic receptors. Two main strategies for evaluating DAT kinetics are described in the literature. One involves recording the clearance of extracellular DA following the electrical stimulation of DA axons in the medial forebrain. Detailed analysis of the descending phase of the stimulus response leads to values of the Vmax and KM of the clearance process. The ability to determine these kinetic parameters has engendered detailed study of
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the factors that regulate them, include drugs and autoreceptor mechanisms. Much attention has also been focused on regional differences in DAT kinetics, a topic of long-standing interest in the field of substance abuse. A second approach to evaluating transporter kinetics is to use in vivo voltammetry to monitor the clearance of exogenously applied substrate. Applications by both pressure ejection and iontophoresis have been described. A point that remains incompletely resolved is that these methods produce substantially smaller kinetic constants than those obtained by monitoring DA clearance after electrical stimulation. There has been speculation that this may be due to the voltage dependency of the transporter that might be affected by stimulation of an as yet unspecified diffusional process during the application of substrate. Nevertheless, the technique reveals modulation of clearance rates by drugs and anesthetics and reveals regional variations in transporter activity. Daws and coworkers have extended this procedure to 5-HT, reporting recently, for example, that the effect of ethanol on 5-HT clearance is enhanced in mice lacking the 5-HT transporter, a finding that sheds new light on the role of 5-HT in alcoholism. At present, however, voltammetry has not been extensively used to monitor endogenous 5-HT release.
A key technical development that enabled this advance was the introduction of the principal components regression analysis of individual voltammograms recorded in the brain of conscious, freely moving rats. The regression algorithm separates the voltammetric signals arising from DA from those arising from pH fluctuations in the brain extracellular space. Fluctuations in pH trigger voltammetric signals at carbon fiber microelectrodes. However, the fingerprint voltammograms associated with pH changes are different from those associated with DA changes, which enables the regression analysis to distinguish pH and DA contributions to the signals. By separating these signal components, which statistically account for the entire signal, voltammetry achieves DA detection limits near 20 nmol l1 and subsecond temporal resolution. According to this voltammetric system, naturally occurring DA release comprises a series of subsecond transients in the nanomolar concentration range. It appears likely, although it remains to be definitively established, that the DA transients are associated with the burst firing of DA neurons. The amplitude, frequency, and duration of the transients are increased by DAT inhibitors, including cocaine and nomifensine, suggesting that the transients may play a key role in the psychostimulant properties of these drugs.
Autoreceptor Control of DA Release
Microdialysis
Considerable use has been made of voltammetry to examine the control of DA release by receptors present on the DA terminal itself, the so-called autoreceptors. In one such experiment by Franc¸ois Gonon and co-workers, amperometric recording was used to examine the time course of the control of evoked DA release by autoreceptors. Their data indicate that the D2 subtype of DA receptors can modulate DA release on a msec timescale and that the duration of the autoreceptor-mediated control of DA release is far longer than the stimulus-evoked elevation in DA itself, suggesting a role for downstream intracellular second messengers. Such studies both illustrate the power of high-speed recordings cases in which the identity of the monoamine is not in question and provide a task to which voltammetry is uniquely suited.
Basis of Approach
Recording Naturally Occurring DA Release on a Subsecond Timescale
Recently, R. Mark Wightman and his colleagues have described the voltammetric recording of naturally occurring DA release on the subsecond timescale. This represents a significant advance in the capabilities of in vivo voltammetry, which previously had been used mainly to monitor electrically evoked DA release.
Measurement of transmitter release via intracerebral dialysis is based on the combination of three separate techniques: (1) a dialysis membrane to collect sample by mass diffusion of small solutes across that membrane, (2) HPLC to separate out the several molecules that are collected in this manner, and (3) a detector to quantify the molecules of interest. In the case of monoamines, that detector is typically an electrochemical detector. Microdialysis has a much slower timescale and provides much less anatomical resolution than voltammetry. On the other hand, microdialysis offers an unambiguous identification of a great many compounds, including the monoamines and their metabolites; provides absolute values; can be used to monitor the substances over many hours; and provides a means for delivering compounds as well as collecting them. Diffusion is initiated when the inner surface and the outer surface of the dialysis membrane are in contact with two aqueous solutions, one containing a higher concentration of a substance of interest than the other. If the aqueous solution in contact with the inner surface of the membrane is continuously flowing and has a significantly lower concentration
Monoamines: Release Studies 445 14 12
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Time (min) Figure 3 Temporal effect of local perfusion of KCl (100 mmol l1) on extracellular DA in the striatum.
Figure 2 Schematic diagram of the dialysis process using a concentric tube microdialysis probe. A drug in the perfusate (squares) can dialyze outward into tissue, while small molecules in the tissue (circles) diffuse inward and are collected with the dialysate for subsequent assay. From Kissinger PT (1996) Electrochemical detection in bioanalysis. Journal of Pharmaceutical and Biomedical Analysis 14: 871–880.
of the substance of interest, a concentration gradient is created resulting in continuous diffusion of the substance from the external medium into the membrane. If the dialysis membrane is attached to an inflow and outflow line, the continuous flow of solution carries the substance to the end of the outflow, line where it can be collected for later analysis (Figure 2). Early Development of Monitoring Techniques
Several sampling procedures preceded intracerebral dialysis as a means of measuring the chemical environment within the brain. The earlier technique of ventricular perfusion did not allow sampling of distinct brain regions, and cortical cup perfusion allowed sampling of only the cortical surface in anesthetized animals. Push–pull cannula perfusion, introduced by John Gaddum in 1961, was the first technique in which extracellular fluid from distinct subcortical
regions could be measured. Two concentric tubes were implanted into the brain, with the tip being within the region of interest. The inner tube was used to deliver the perfusion solution and the outer tube to collect the perfusate for subsequent analysis. Because the perfusion fluid was in direct contact with brain tissue, significant tissue damage was incurred from the fluid flow. By placing a semipermeable bag over the tip of the push–pull cannula to circumvent this problem, Jose Delgado and colleagues introduced the first intracerebral dialysis probe, called the dialytrode. With the advent of highly sensitive methods of detection, such as HPLC coupled with electrochemical detection, Urban Ungerstedt extended this approach, pioneering its use to measure endogenous DA in extracellular fluid in response to drugs such as amphetamine. Soon this was further extended to include stimuli such as KCl (Figure 3) and electrical stimulation and to monitor various transmitter releases during exogenous stimuli and behavior. In vivo microdialysis quickly became the most common means by which in vivo transmitter efflux was monitored, resulting in more than 5000 publications by 2007. Advantages of Microdialysis
Microdialysis has several advantages over earlier in vivo sampling methods. The damage incurred by the surrounding extracellular environment is minimized not only by the presence of the dialysis membrane but also by a decrease in the overall size of the probe. Another advantage of the dialysis membrane is that it acts as a filter against large molecules, such as enzymes, that may act to degrade the substance of interest, and it also purifies the sample for subsequent analysis. Furthermore, because intracerebral dialysis is merely a sampling technique, in conjunction with a
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suitable assay, it can be used to study a broad range of compounds. However, the most studied class of compounds has been the monoamines, DA in particular. Although modern dialysis probes minimize the damage to the surrounding tissue, studies have shown that immediately after implantation of the probe, not all transmitter ‘release’ is either tetrodotoxin (TTX) sensitive or calcium dependent and thus may represent a response to damage. On the other hand, by the next day, release is both TTX sensitive and calcium sensitive, at least in the case of DA, and thus is generally considered to reflect a physiological process. The release of DA is known to occur largely if not entirely via exocytosis, and its actions are terminated by removal from the synapse by a combination of diffusion and high-affinity uptake via DAT. These processes can be exploited to determine the functional state of the DA system in response to various treatments. For example, in a normal intact DA system, addition of a high concentration of Kþ ions to the perfusate increases the concentration of DA sampled by the probe via an increase in exocytotic release of DA. However, infusion of 6-hydroxydopamine, a catecholaminergic toxin, into the nigrostriatal DA pathway decreases potassium-evoked DA release, indicating that the system is not functioning at full capacity. Amphetamine increases extracellular levels of DA by reversal of the DAT, a calcium-independent process. In a normal nigrostriatal DA system, amphetamine induces an approximate 50-fold increase of DA in dialysate. However, in the denervated striatum, amphetamine-induced DA release is attenuated. Monitoring extracellular monoamine levels under basal conditions or during stimulation may provide information about the functional integrity of a given projection. However, care must be taken to extrapolate from extracellular levels to release. Extracellular transmitter concentration is a net result of release and removal of transmitter, and removal in turn is a consequence of diffusion and high-affinity uptake. Likewise, care must be taken before an assumption that extracellular transmitter levels are a simple reflection of the number of nerve terminals. For example, extensive damage to a catecholaminergic system is required to reduce basal extracellular levels of either norepinephrine or DA. In addition, some trophic factors, particularly glial-derived neuronal factor, have been shown to increase the synthesis and release of DA without affecting the apparent number of DA terminals. Problems with Microdialysis
A major disadvantage of microdialysis, because of the need for large amounts of material for detection, has been temporal resolution. For example, even in the
case of extracellular DA, which is present in high concentration compared with other monoamines or even DA in other brain regions, a sampling interval of at least 10 min has typically been required. However, separation techniques such as capillary electrophoresis and microbore columns for HPLC, requiring smaller sample volumes, coupled with more sensitive detection methods (1 ml) have allowed the temporal resolution to be decreased to 1 min. Of course, this resolution still lags far behind that of synaptic events and in vivo voltammetry, which are on the millisecond scale. Another drawback of intracerebral dialysis has been an inability to measure the actual concentration of endogenous substances. At the crux of this problem is a description of the relationship between the dialysate concentration and the actual concentration of substance within the extracellular fluid, or the recovery. However, here again, techniques have been developed which allow relatively quantitative measurement of the extracellular concentration of neurotransmitters independent of the recovery. One method uses a very slow rate, at which the in vivo recovery approaches 100%. However, this method requires long sampling intervals, which dramatically reduces temporal resolution and also introduces problems of sample stability. A second approach to the problem is the difference method (or method of no net flux) developed by Peter Lo¨nnroth. In this technique, concentrations of the compound of interest above and below what is expected to be present in the extracellular fluid are perfused through the probe and the resulting dialysate concentration measured. When the differences between the inflow and outflow concentrations are plotted versus the inflow concentration, the resulting regression yields the extracellular concentration (zero point) and the in vivo recovery. Again, a disadvantage of this technique is the time required for its execution. A third problem with microdialysis is that the probe size, typically 250 mm, precludes fine spatial resolution and also is associated with considerable local tissue damage characterized by acute and chronic histological, physiological, and biochemical changes in adjacent tissue. An area of altered release and uptake exists within the trauma layer, which could effectively result in discrepancies between the extraction fraction and the relative recovery, rendering an extracellular concentration that is over- or underestimated.
Imaging Dopaminergic Transmission The reduction in the availability of D2 receptors as measured with benzamide radioligands (such as 123 I-iodobenzamide (IBZM) and 11C-raclopride) following the administration of psychostimulants known
Monoamines: Release Studies 447 11C-raclopride
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Figure 4 Effect of amphetamine on 11C-raclopride and 11C-()-N-propyl-norapomorphine (NPA) in a nonhuman primate (baboon). Magnetic resonance images (MRIs) and corresponding parametric binding potential (BP) maps shown in the same baboon under four conditions: 11C-raclopride baseline study, 11C-raclopride postamphetamine study (0.5 mg kg1), 11C-NPA baseline study, and 11C-NPA postamphetamine study (0.5 mg kg1). Following the same dose of amphetamine, 0.5 mg kg1, a greater decrease in BP is observed with the agonist radioligand 11C-NPA (46%) than with the antagonist 11C-raclopride (26%). From Narendran R, Hwang DR, Slifstein M, et al. (2004) In vivo vulnerability to competition by endogenous dopamine: Comparison of the D2 receptor agonist radiotracer ()-N-[11C]propyl-norapomorphine ([11C]NPA) with the D2 receptor antagonist radiotracer [11C]-raclopride. Synapse 52: 188–208.
to release DA (e.g., amphetamine or methylphenidate) is a clear example of an interaction between endogenous neurotransmitter and radioligand that has been extensively validated in both nonhuman primates and humans. In baboons, the increase in synaptic DA elicited by amphetamine is associated with a displacement of the binding of the single photon emission computed tomography (SPECT) D2 receptor radiotracer 123I-IBZM and of the PET radiotracer 11C-raclopride (Figure 4). By combining SPECT and microdialysis in baboons, Marc Laruelle and colleagues established that the magnitude of the 123 I-IBZM displacement after administration of amphetamine was related to the magnitude of DA release, and that a linear correlation existed between microdialysis measurements of DA release and radioligand displacement. Over the years, this technique of measuring the reduction in radiotracer binding following DA release via a stimulant challenge has been successfully used to study dopaminergic transmission in the living human brain in both healthy and diseased states such as schizophrenia and addiction. Limitations of Imaging
The capacity to monitor neurotransmitter release in humans as well as experimental animals is a great advantage of in vivo imaging techniques employing radioligands. However, there are several limitations to these approaches. Some are similar to those of voltammetry: Both methods are applicable to a relatively small number of compounds. Indeed, only a few radioligands have been shown to have in vivo binding characteristics sensitive to fluctuations in endogenous neurotransmitter release. Other limitations are similar to those of microdialysis: limited
spatial and temporal resolution. Although modern PET scanners can now provide images of the entire human brain with 2 mm spatial resolution, assessments of neurotransmitter release via these techniques are limited to the tissue level. Other limitations are unique to imaging in general or to the specific radioligand employed. One example is the relatively low sensitivity of measuring endogenous transmitter by imaging its displacement of radiolabeled ligand. A 1% decrement in radiotracer binding corresponds to a 50% increase in extracellular DA as measured by microdialysis. Another limitation is the ‘ceiling effect’ seen when any of the available benzamide radiotracers are used: Only about half of the radiotracer’s specific binding is vulnerable to changes in extracellular DA concentration. Both the low sensitivity and the ceiling effect may be related at least in part to the fact that D2 receptors are configured in interconvertible states of high or low affinity for agonists. This is because benzamide radiotracers such as 123I-IBZM and 11C-raclopride are antagonists that bind with equal affinity to both states, but DA is not expected to compete efficiently with the fraction of the 123I-IBZM or 11Craclopride binding to the low-affinity state. Thus, in contrast to the antagonist, a large fraction of the in vivo binding of an agonist radiotracer to D2 high sites will be available for endogenous competition by DA, thereby making it a more sensitive probe to study stimulant-induced DA release. Recent preclinical PET studies comparing novel agonist radioligands (in anesthetized nonhuman primates and cats) such as [11C]()-N-propyl-norapomorphine (NPA) and 11CPHNO with the reference antagonist radiotracer 11 C-raclopride tend to support this hypothesis. If the
448 Monoamines: Release Studies
increased sensitivity of these agonist radioligands extends to humans, they may provide researchers with a superior probe with which to report on acute changes in synaptic DA concentration. Such probes might also allow researchers to control for baseline affinity states (availability of receptors configured in D2 high) that may differ between clinical populations. One limitation of the SPECT and PET radiotracers 123 I-IBZM and 11C-raclopride is that the affinity of these ligands provides a quantifiable signal only in the striatum where receptors are abundant. However, a major advancement in availability of higher-resolution PET cameras has allowed the study of amphetamineinduced DA release in the functional subdivisions of the striatum with 11C-raclopride, and further advances in instrumentation may provide even higher resolution. Demonstration That Imaging Can Monitor Monoamine Release, Though with Relatively Low Sensitivity
In spite of methodological limitations and limited sample sizes, several reports indicate that imaging can detect DA release in response to cognitive challenges such as yoga, psychosocial stress tasks, and video games, as well as motor challenges such as finger tapping and foot movement. Nonetheless, such stimuli, like psychostimulants, lead to decreases in 11C-raclopride binding that are on the order of 10%, rather than the 50–1, 500% increases in extracellular DA observed with microdialysis. Recent studies that combined PET with microdialysis highlighted the discord between microdialysis and PET as applied to measurement of DA release. For example, one such study showed that a dose of amphetamine that elicited the same degree of inhibition of 11C-raclopride binding as a given dose of methylphenidate resulted in a fourfold greater increase in DA release as measured by microdialysis. To reconcile such differences, it has been hypothesized that the DA release measured with PET radiotracers using endogenous competition techniques may be reflective of fluctuations in intrasynaptic DA, whereas the DA release measured using microdialysis techniques may be reflective of extrasynaptic DA concentrations.
Imaging Serotonergic Transmission Defects in other monoamine systems, particularly serotonin (5-HT), have been implicated in various psychiatric disorders, including major depression, anxiety disorders, and schizophrenia. Efforts to measure endogenous release of 5-HT with PET have been modeled after similar experiments designed to measure
DA release in vivo, whereby a pharmacologic stimulus is used to enhance or deplete extracellular 5-HT and thereby elicit a reproducible change in radioligand binding that correlates with the degree of endogenous neurotransmitter release. It has been demonstrated, for example, that acute administration of the psychostimulant fenfluramine resulted in a two- to 15-fold increase in extracellular 5-HT concentrations in the rat hippocampus and that this endogenous release resulted in dose-dependent decreases in the binding of the 5-HT1A radioligand 18F-MPPF. Other studies conducted in rodents have reported little or no effect of fenfluramine administration on the binding of another 5-HT1A PET radioligand, 11C-WAY-100635 or the 5-HT2A radioligand 11C-MDL-100907. In nonhuman primate brain, acute administration of fenfluramine failed to elicit significant decreases in the binding of 18F-MPPF. Although one study reported modest decreases in the binding of the 5-HT2A radioligand 18F-altanserin in whole human cortex (14–23%) after acute administration of the selective serotonin reuptake inhibitor clomipramine (25 mg), it is unclear whether this effect can be attributed to endogenous competition by 5-HT or to direct competition with clomipramine itself, which has been shown to have a 50–60 nmol l1 affinity for the 5-HT2A receptor. Unfortunately, for reasons that are unclear, attempts to measure endogenous competition by serotonin using PET have been unsuccessful. This may have to do with the fraction of receptors configured in vivo in the highaffinity state for the agonist, cellular localization (intracellular versus extracellular), and their distance from the synaptic cleft.
Conclusions Using such methods as immunohistochemistry, electrophysiology, and genomics, it is now possible to examine the characteristics of single cells in the brain, either in vivo or at least immediately after brain removal. Although the direct monitoring of transmitter release from a single neuron has now been achieved in culture, it is not yet possible within an intact brain. Nonetheless, the judicious application of voltammetry, capillary electrophoresis, microdialysis, and in vivo imaging is allowing us to draw important conclusions about the release of monoaminergic transmitters from groups of neurons, and important observations made with these approaches continue to provide enlightenment about both normal and abnormal brain function. Moreover, the sensitivity and specificity of these methods have improved significantly over the past decade, and these improvements should continue until biochemistry takes its place
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alongside histology, electrophysiology, and molecular biology as a route to the monitoring of a single cell within the living brain. See also: Dopamine; Dopamine Receptors and Antipsychotic Drugs in Health and Disease; Norepinephrine: CNS Pathways and Neurophysiology; Serotonin (5Hydroxytryptamine; 5-HT): CNS Pathways and Neurophysiology; Serotonin (5-Hydroxytryptamine; 5-HT): Receptors.
Further Reading Borland LM and Michael AC (2004) Voltammetric study of the control of striatal dopamine release by glutamate. Journal of Neurochemistry 91: 220–229. Carson RE (2003) Tracer kinetic modeling in PET. In: Valk PE, Dale L, Bailey DL, et al. (eds.) Positron Emission Tomography: Basic Science and Clinical Practice, pp. 147–180. London: Springer. Daws LC, Montanez S, Munn JL, et al. (2006) Ethanol inhibits clearance of brain serotonin by a serotonin transporter independent mechanism. Journal of Neuroscience 26: 6431–6438. Fillenz M (2005) In vivo neurochemical monitoring and the study of behaviour. Neuroscience and Biobehavioral Reviews 29: 949–962. Finlay JF and Zigmond MJ (1995) A critical analysis of neurochemical methods for monitoring transmitter dynamics in brain. In: Bloom FE and Kupfer DJ (eds.) Psychopharmacology: The Fourth Generation of Progress pp. 29–40. New York: Raven Press. Heien MLAV, Khan AS, Ariansen JL, et al. (2005) Real-time measurement of dopamine fluctuations after cocaine in the brain of behaving rats. Proceedings of the National Academy of Sciences of the United States of America 102: 10023–10028. Kissinger PT (1996) Electrochemical detection in bioanalysis. Journal of Pharmaceutical and Biomedical Analysis 14: 871–880. Laruelle M (2000) Imaging synaptic neurotransmission with in vivo binding competition techniques: A critical review. Journal of Cerebral Blood Flow and Metabolism 20: 423–451.
Laruelle M, Abi-Dargham A, van Dyck CH, et al. (1995) SPECT imaging of striatal dopamine release after amphetamine challenge. Journal of Nuclear Medicine 36: 1182–1190. Michael AC and Borland LM (eds.) (2007) Electrochemical Methods for Neuroscience. Boca Raton, FL: CRC Press. Narendran R, Hwang DR, Slifstein M, et al. (2004) In vivo vulnerability to competition by endogenous dopamine: Comparison of the D2 receptor agonist radiotracer ()-N-[11C]propylnorapomorphine ([11C]NPA) with the D2 receptor antagonist radiotracer [11C]-raclopride. Synapse 52: 188–208. Peter JL, Miner LH, Michael AC, and Sesach SR (2004) Ultrastructure at carbon fiber microelectrode implantation sites after acute voltammetric measurement in the striatum of anesthetized rats. Journal of Neuroscience Methods 137: 9–23. Robinson DL, Venton BJ, Heien ML, and Wightman RM (2003) Detecting subsecond dopamine release with fast-scan cyclic voltammetry in vivo. Clinical Chemistry 49: 1763–1773. Rouge´-Pont F, Usiello A, Benoit-Marand M, Gonon F, Piazza PV, and Borrelli E (2002) Changes in extracellular dopamine induced by morphine and cocaine: Crucial control by D2 receptors. Journal of Neuroscience 22: 3293–3301. Sabeti J, Gerhardt GA, and Zahniser NR (2003) Individual differences in cocaine-induced locomotor sensitization in low and high cocaine locomotor-responding rats are associated with differential inhibition of dopamine clearance in nucleus accumbens. Journal of Pharmacology and Experimental Therapeutics 305: 180–190. Smith AD, Olson RJ, and Justice JB Jr. (1992) Quantitative microdialysis of dopamine in the striatum: Effect of circadian variation. Journal of Neuroscience Methods 44: 33–41. Westerink BH (2000) Analysis of biogenic amines in microdialysates of the brain. Journal of Chromatography. B, Biomedical Sciences and Applications 747: 21–32. Wightman RM (2006) Probing cellular chemistry in biological systems with microelectrodes. Science 311: 1570–1574. Wu Q, Reith MEA, Walker QD, Kuhn CM, Carroll FI, and Garris PA (2002) Concurrent autoreceptor-mediated control of dopamine release and uptake during neurotransmission: An in vivo voltammetric study. Journal of Neuroscience 22: 6272–6281. Zhang MYand Beyer CE (2006) Measurement of neurotransmitters from extracellular fluid in brain by in vivo microdialysis and chromatography-mass spectrometry. Journal of Pharmaceutical and Biomedical Analysis 40: 492–499.
Serotonin (5-Hydroxytryptamine; 5-HT): Neurotransmission and Neuromodulation A G Ramage, University College London, London, UK ã 2009 Elsevier Ltd. All rights reserved.
Transmission In this review the nature of 5-hydroxytryptamine (5-HT) transmission will be discussed with particular reference to the control of the cardiovascular system, micronutrition, the gut, and airways (autonomic nervous system) which most reviews fail to cover, concentrating on mood, cognition, perception, sleep, appetite, sex, and thermoregulation, to name but a few! There is direct evidence that 5-HT neurotransmission is involved in the central regulation of autonomic function as autonomic reflexes can be accurately measured and the underlying neuronal pathways have been fairly well delineated. Considering the relatively small number of 5-HT-containing neurons that are found in the brain, it is surprising that 5-HT is involved in so many functions; the total number of neurons in the mammalian brain is in the billions whereas the number of 5-HT-containing neurons is in the thousands. The majority of these 5-HT-containing neurons are located in the brain stem in the raphe nuclei, which are mainly located along the midline. These clusters of 5-HT-containing neurons have been labeled B1–B9. They send dense and divergent projections to all regions of the brain, as might be expected by their involvement in so many functions. Although some of these projections form classical synapses, many do not, indicating they may contribute to paracrine transmission, otherwise known as volume transmission (VT). VT would allow transmitter (information) to reach a variety of targets over a wide area, and thus it is more likely that 5-HTcontaining neurons are involved in the modulation of a series of processes, such as rhythm generation. It should be noted that VT can also involve communication with glial cells. When 5-HT-containing neurons form classical synapses with their target neurons, which are sometimes referred to as ‘private synapses,’ the transmitter has only a very short distance to diffuse within such synapses, and so its action is rapid. Such fast transmission is referred to as ‘wiring transmission’ (WT). VT differs in that it can allow neuronal interaction via the transmitter and additional substances in the extracellular space. The ability of 5-HT to be involved in VT may explain the involvement of 5-HT neurons in such diverse functions. Data in support of this idea, that VT is occurring
450
in a particular system, is the extrasynaptic localization of transmitter uptake transporters. In addition, the large number of receptor subtypes that 5-HT can interact with, which can have opposing actions, gives a vast repertoire of possible responses. For VT, the topographical organization of these different receptor subtypes would be important. More recently, retrograde neural transmission has been demonstrated; however, whether this applies to 5-HT remains to be determined as presumably 5-HT-containing vesicles would be required in the postsynaptic specialization. The question arises as to whether 5-HT is the only transmitter released by 5-HT-containing neurons. In this respect, the term ‘5-hydroxytryptaminergic or serotoninergic’ is often probably inappropriately used to indicate that these effects are mediated by such neurons using that one named transmitter. This is known as ‘Dale’s principle.’ There is, however, much evidence that 5-HTcontaining neurons release more than one transmitter and that cotransmission may be considered the norm. Normally cotransmission means the release of a peptide together with the main transmitter, which is usually associated with higher frequencies of neuronal firing. It is interesting to note that for 5-HT, there is also good evidence that glutamate may be co-released. In this respect, it has been suggested that where 5-HTcontaining neurons form conventional synapses, that is, are involved in WT, glutamate only is released from these synapses, and at unconventional synapses (VT), 5-HT is released. This would in a sense be a return to Dale’s principle in that each terminal (synapse) is releasing only one type of transmitter, although overall the neuron will release more than one type of transmitter depending on the synapse.
The Need for Multiple Receptors The affinity of 5-HT for the different 5-HT receptor subtypes varies. For instance, the affinity that 5-HT has for 5-HT1 receptor subtypes is higher than for most other subtypes, with 5-HT having the lowest affinity for the ligand-gated ion channel receptor, the 5-HT3 receptor. This different affinity enables the target neuron or neurons to respond differentially to different amounts of 5-HT released, with lower concentrations favoring 5-HT1 receptor activation. In addition, receptor desensitization rates vary; thus a slow and regular firing rate, a supposed characteristic of 5-HT-containing neurons, will mainly recruit 5-HT1 and 5-HT2 receptors. However, high firing rates would recruit 5-HT3 and 5-HT4 receptors.
Serotonin (5-Hydroxytryptamine; 5-HT): Neurotransmission and Neuromodulation 451
All these receptors may undergo cycles of desensitization and recovery as firing patterns change. Thus changes in firing patterns would activate different receptors and in turn initiate temporal changes in the responsivity of the target neurons and so on to the next coding sequence. In addition, the fact that 5HT receptors use a large repertoire of second messengers enables 5-HT neurons to have a large capacity to influence cellular events modulated by these second messengers. Thus it would be possible for neurons to have a ‘memory’ of the normal inputs they receive from 5-HT terminals. Overall, there are multiple ways that central 5-HT-containing neurons can modify transmission in other neurons. It should also be noted that 5-HT neurons can modulate their output of transmitter by using dendritic and somatodendritic autoreceptors.
Evidence that 5-HT Transmission Is Involved in Physiological Processes with Reference to Central Control of the Autonomic Nervous System Central Cardiovascular Regulation
Brain stem 5-HT pathways are known to play important roles in cardiovascular regulation, although the precise role of each of the identified 5-HT receptor subtypes in this process remains to be determined. The main receptor subtypes that are known to be in this pathway are 5-HT1A, 5-HT3, and 5-HT7. This has been determined using selective 5-HT receptor antagonists to interfere with defined reflex pathways, that is, inputs, which, when activated, give a well defined and characteristic output. In addition, studies depleting 5-HT have been carried out although it is very hard to know if all the ‘functional’ 5-HT has been removed in such studies. Nevertheless, data indicate that in rats with around only 6% of their total brain stem 5-HT, blood pressure is elevated and baroreceptor gain is attenuated. On the other hand, there is, at present, no evidence that 5-HT receptor antagonists can increase resting blood pressure in rats, suggesting that background tone in the resting condition is not mediated by 5-HT-containing neurons. In this respect, it has been suggested that 5-HT7 receptors are involved in normalizing blood pressure after it has been destabilized by sensory input. This may have implications in cardiovascular changes induced by stress. The main types of receptors that have been identified in controlling sympathetic drive and thus the central maintenance of blood pressure are 5-HT1A (sympathoinhibitory) and 5-HT2 (sympathoexcitatory). Data suggest that it is the A subtype of the 5-HT2 receptor that is primarily
involved. However, blockade of either of these receptor subtypes has little effect on resting blood pressure. (It is interesting to note that it is these receptors which are also believed to be responsible for the hallucinogenic effects of LSD and related compounds.) It is somewhat surprising that 5-HT2A receptor antagonists do not lower blood pressure, as activation of these receptors causes large central increases in sympathetic drive. However, this may be a problem with selectivity of the 5-HT2 receptor antagonists used. Long-term block of 5-HT2 receptors does prevent deoxycorticosterone acetate-salt hypertension, which probably reflects the importance of central 5-HT-containing neurons in the release of vasopressin (antidiuretic hormone). The release of vasopressin, though, involves the activation of 5-HT2A receptors on a central angiotensinergic pathway. Overall, this implicates central 5-HT pathways in the control of blood volume, and in this respect, one of the characteristic unwanted effects of selective serotonin reuptake inhibitors is dilutional hyponatremia due to the release of vasopressin. It is the role of central 5-HT neurotransmission in the control of vagal tone to the heart rather than sympathetic tone to the blood vessels that has been more thoroughly elucidated. The archetypical 5-HT1A receptor agonist 8-OH-DPAT was observed to cause large increases in vagal drive to the heart. Vagal tone to the heart can also be increased by activation of a number of cardiovascular reflexes (cardiopulmonary and baroreceptor reflexes and the chemoreflex), and the afferents for these reflexes run in the IXth and Xth (vagus) cranial nerves and terminate along with other visceral afferents (e.g., gut) in the nucleus tractus solitarius (NTS). Of these reflexes, the cardiopulmonary has been identified in all species tested as involving 5-HT1A receptor-mediated transmission. Those sensory nerve endings located in the pulmonary circulation and in the heart and coronary vessels have 5-HT3 receptors located on them. The sites at which 5-HT transmission can modulate these reflexes in the central nervous system (CNS) are the NTS, the dorsal vagal motor nucleus, and the nucleus ambiguus. These latter two nuclei contain vagal (parasympathetic) preganglionic neurons. In the former, the neurons have unmyelinated axons mainly innervating parasympathetic ganglia (myenteric plexus) in the gut, while the latter have myelinated axons and mainly innervate parasympathetic ganglia in the heart and airways. Their activation could cause the heart to stop or cause severe bronchoconstriction. Central application of the archetypical 5-HT1A receptor antagonist WAY100635 attenuates the cardiopulmonary reflex vagal bradycardia, and this is believed to occur at the level of the nucleus ambiguus. As 5-HT1A receptors are
452 Serotonin (5-Hydroxytryptamine; 5-HT): Neurotransmission and Neuromodulation
usually inhibitory, this excitatory action is probably one of disinhibition (see Figure 1). It has thus been suggested that when NTS neurons activate cardiac vagal preganglionics in the ambiguus, this causes the release of 5-HT, which activates a 5-HT1A receptor inhibiting a tonically active g-aminobutyric acid (GABA)-ergic interneuron. More recently, a 5-HT7 receptor-mediated pathway has been shown to play a global role in the control of vagal tone by all these reflexes. 5-HT7 receptors differ from 5-HT1A receptors in that they are excitatory, increasing the levels of cyclic adenosine monophosphate. The site at which these receptors act is believed to be within the NTS as 5-HT7 receptor blockade affects most of the components of these reflexes. It should be noted that there is overlap in the pharmacology of 5-HT7 and 5-HT1A receptors. In addition, at the level of the NTS, 5-HT3 receptors are also involved in the vagal afferent excitation of NTS neurons. They do this indirectly by causing the release of glutamate, which has also been identified as a crucial transmitter in these reflex
pathways. It is interesting to note that in dealing with the observations that blockade of 5-HT7 receptors or glutamate receptors abolishes the baroreceptor reflex, the question arises as to why both transmitters are seemingly of equal importance. Furthermore, how 5-HT3 receptors fit into such a system remains unclear. The importance of 5-HT3 receptors in the control of these reflex pathways, although seemingly essential for transmission within the NTS, is at present not clear. It is thought that 5-HT3 receptors may be located on glial cells, causing the release of glutamate from glial cells themselves. However, it is surprising that after vagotomy, there is considerable reduction in 5-HT3 receptor density within the NTS, indicating that 5-HT3 receptors are mainly located at the terminals of vagal afferents; see Figure 2 for a proposed functional 5-HT pathway within the NTS. Thus 5-HT transmission plays an important role in cardiovascular afferent integration and in controlling vagal efferent output to the heart. Although 5-HT receptor activation can affect sympathetic outflow
5-HT3
5-HT NTS Proposed inputs involved in the reflex
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Figure 1 A diagrammatic representation of involvement of pathways containing 5-hydroxytryptamine (5-HT) and receptors in the control of the activity of cardiac and bronchoconstrictor (airway) vagal preganglionic neurons. The 5-HT-containing neurons are in green whereas glutamate-containing neurons are in yellow. The vagal preganglionic neurons can be reflexly activated via the nucleus tractus solitarius (NTS) by the cardiopulmonary afferents that run in the vagus (see Figure 2 for fuller details of the role of 5-HT3, 5-HT7, glutamate, and glial cells in activating NTS neurons). It is proposed that the NTS neurons activate (1) a 5-HT-containing and (2) a glutamatergic pathway. The 5-HT pathway inhibits the g-aminobutyric acid (GABA)-mediated (blue) ‘brake,’ allowing the glutamatergic pathway to fully excite the preganglionic vagal neurons, resulting in a bradycardia or bronchoconstriction. It should be noted that the 5-HT1A receptors are located presynaptically, not postsynaptically. In addition, the diagram shows a 5-HT-containing pathway directly innervating vagal preganglionic neurons, which activates 5-HT2A receptors to cause excitation. This pathway is not believed to be involved in the reflex activation of vagal preganglionic neurons; however, it is speculated that the putative 5-HT1A receptors that mediate inhibition of vagal preganglionic neuronal activity could be located on the nerve terminals of this pathway and as such would function as autoreceptors. It should be noted that these inhibitory 5-HT1A receptors could also be located on the terminals of the 5-HT-containing pathway, which inhibits the putative GABAergic brake. This could apply to all bronchial and bladder vagal preganglionic neurons in which 5-HT1A-mediated transmission has been identified. NMDA, N-methyl-D-aspartate. Reproduced from Wang Y and Ramage AG (2001) The role of central 5-HT1A receptors in the control of B-fibre cardiac and bronchoconstrictor vagal preganglionic neurons in anaesthetized cats. Journal of Physiology 536: 753–767, with permission from Blackwell publishing.
Serotonin (5-Hydroxytryptamine; 5-HT): Neurotransmission and Neuromodulation 453
Nodose ganglia
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Figure 2 A diagrammatic representation of the interaction of vagal sensory afferents, involved in the cardiopulmonary reflex, and a 5-HT-containing projection from the raphe on a nucleus tractus solitarius (NTS) neuron. It is proposed that the vagal afferent can contain either 5-HT (green) or glutamate (GLU; yellow). It should be indicated that there is no reason why both transmitters cannot be contained in one afferent fiber. Further, these vagal afferent projections could just release either 5-HT or glutamate. However, for the present it is considered that they release both. In this respect, the 5-HT-containing afferents make apposition to the glial cell (the transmission here could be volume transmission (VT) or wiring transmission (WT); intuitively, VT is preferred). The released 5-HT activates 5-HT3 receptors, which cause the release of glutamate from the glial cell and thus excitation of the NTS neuron via the activation of N-methyl-D-aspartate (NMDA) and non-NMDA receptors. The other afferent releases glutamate directly to activate the NTS neuron. It is proposed that the 5-HTcontaining raphe projection controls the ‘gain’ of this system via activation of 5-HT3 receptors on the sensory afferent input and/or glial cell and 5-HT7 receptors on the NTS neuron (this again could be a mixture of WT and VT). It should be noted that 5-HT terminal autoreceptors would be expected to be found in this system. This proposed system could apply to other afferent processing involving 5-HT.
to blood vessels, a role for 5-HT transmission in sympathetic (blood pressure) control is unclear, but 5-HT does seem to play a role in the regulation of blood volume by the CNS. Airways
The predominant drive to airway smooth muscle is again the vagus, and again data indicate that 5-HT neurotransmission operating through 5-HT1A and probably 5-HT7 receptors is important for their regulation, increasing vagal drive. These bronchoconstrictor preganglionic neurons have a respiratory modulation effect opposite to that of cardiac neurons’ being excited on inspiration. So the effects of 5-HT are unlikely to be due to an action on respiratory drive. Sadly, very little experimentation has been carried out on such neurons despite their ability to close down the airways. Micturition – The Bladder
Storage and elimination of urine are performed by the bladder and urethra. This involves the sympathetic nervous system, which is considered important for storage, that is, increasing the compliance of
the bladder, and the parasympathetic, which causes contraction of the bladder and thus voiding. The somatic nervous system is important in the control of the external urethral sphincter for the control of continence. The CNS seems to control micturition at two levels, one in the midbrain and the other at the sacral level of the spinal cord, where the preganglionic vagal neurons innervating the bladder are found. It has long been thought that 5-HT neurotransmission is inhibitory to micturition. As central regulation of micturition is very complex, it is not surprising that the majority of 5-HT receptors have been implicated; in order of known importance, they are: 5-HT1A, 5-HT7, 5-HT2A/C, 5-HT3, 5-HT4, and 5-HT5A. Again, what is surprising is that both 5-HT1A and 5-HT7 receptor antagonists inhibit micturition, indicating, as above, that although both have opposite effects on adenylyl cyclase, both play an excitatory role in this process, implying that 5-HT-containing pathways can be considered excitatory rather than inhibitory. The 5-HT pathway utilizing 5-HT7 receptors operates only at the level of the brain stem whereas the 5-HT1A pathway operates at both the brain stem and the sacral spinal cord regions involved in micturition.
454 Serotonin (5-Hydroxytryptamine; 5-HT): Neurotransmission and Neuromodulation
A pathway mainly operating through 5-HT2C receptors has also been strongly implicated in the control of micturition, and this pathway is proposed to be inhibitory. As this is an excitatory receptor, again disinhibition has been proposed for these actions, that is, activation of GABAergic interneurons. Indeed, administering selective 5-HT2C receptor agonists does cause the inhibition of micturition, and this occurs at both brain stem and spinal cord levels. Nevertheless, the selective 5-HT2C receptor antagonist SB 242084 did not affect the micturition reflex, indicating that these receptors have no physiological role. However, preliminary experiments suggest that there may be an excitatory role for 5-HT2A receptors. Recently the discovery of a high density of 5-HT5A receptors in Onuf’s nucleus, the site of somatic motor neurons controlling the external urethral sphincter, implies a role for 5-HT transmission in the control of this nucleus, which is also important for fecal continence. Thus, the above data would suggest that depletion of brain 5-HT or destruction of 5-HT neurons would interfere with micturition. This has not been observed so far, although destruction of 5-HT neurons and terminals in the spinal cord by around 75% increased micturition volume, favoring an excitatory action of 5-HT. Another question that arises from the above observations is, where are the 5-HT-containing neurons located that are involved in these actions? Further, do these neurons send axons all the way down to the sacral spinal cord? In the brain stem/pons area, the major structures involved in the control of micturition are the pontine micturition center, periaqueductal grey, and locus coeruleus. In this area of the brain is also located the dorsal raphe nucleus. This nucleus has very large numbers of 5-HT-containing neurons, which mainly project rostrally to the cortex. It is possible that the 5-HT-containing terminals, which presumably innervate these micturition areas, originate from this nucleus, probably operating by activating 5-HT7 receptors to initiate micturition and/or switch from filling to voiding, that is, acting as a ‘flip-flop switch.’ Once more, 5-HT1A receptors, as for cardiac vagal neurons, appear to switch off a tonic GABAergic input, a ‘brake,’ in the pontine micturition center so these neurons can then activate bladder parasympathetic preganglionic neurons to cause contraction of the bladder and voiding. This system could also act at the level of parasympathetic neurons in sacral spinal cord. However, the site these sacral 5-HT terminals come from remains to be determined. Yet again the role of 5-HT neurotransmission in controlling the sympathetic and sensory afferent side of this system remains to be fully determined.
Gut
5-HT is an important neurotransmitter and paracrine signaling molecule in the gut and is released from enterochromaffin cells by local stimulation of the intestinal mucosa. The released 5-HT activates 5-HT3 and 5-HT4 receptors on sensory afferents, initiating the peristaltic reflex. In this respect, treatment with a 5-HT3 receptor antagonist slows down transit time through the gut. Another use of these antagonists is the treatment of vomiting and nausea. This action is believed to occur at two levels, at the level of the sensory nerves in the gut and in the area postrema (chemical triggers zone), sometimes referred to as the vomiting center and one of the circumventricular organs (allowing factors to ‘circumvent’ the blood–brain barrier). It also projects to the NTS, which is found just below it on the floor of the fourth ventricle. The area also receives vagal afferents and is rich in 5-HT3 receptors, which again are mainly on vagal afferent nerve endings, as in the NTS (see above). The data suggest that these vagal afferents again release 5-HT; whether directly or indirectly remains to be determined.
Overview The above data indicate that 5-HT neuronal transmission plays a vital role in the central control of the autonomic nervous system. How this 5-HT innervation integrates between the use of volume transmission and so-called tight private synapses to control these processes remains to be elucidated. In addition, the relationship between the different types of receptors involved also remains to be determined, and the site from which this 5-HT innervation arises is still unknown. See also: Serotonin (5-Hydroxytryptamine; 5-HT): CNS Pathways and Neurophysiology; Serotonin (5Hydroxytryptamine; 5-HT): Receptors.
Further Reading Bunin MA and Wightman RM (1999) Paracrine neurotransmission ion the CNS: Involvement of 5-HT. Trends in Neuroscience 22: 377–382. Crowell MD (2004) Role of serotonin in the pathophysiology of the irritable bowel syndrome. British Journal of Pharmacology 141: 1285–1293. Damaso EL, Bonagamba LGH, Kellett DO, et al. (2007) Involvement of central 5-HT7 receptors in modulation of cardiovascular reflexes in awake rats. Brain Research 1144: 82–90.
Serotonin (5-Hydroxytryptamine; 5-HT): Neurotransmission and Neuromodulation 455 De Groat WC (2006) Integrative control of the lower urinary tract: Preclinical perspective. British Journal of Pharmacology 147: S25–S40. Jacobs BL and Azmitia EC (1992) Structure and function of the brain serotonin system. Physiological Reviews 72: 165–229. Jordan D (2005) Vagal control of the heart: Central serotonergic (5-HT) mechanisms. Experimental Physiology 90: 175–181. Ramage AG (2001) Central cardiovascular regulation and 5-hydroxytryptamine receptors. Brain Research Bulletin 56: 425–439. Ramage AG (2006) The role of central 5-hydroxytryptamine (5-HT, serotonin) receptors in the control of micturition. British Journal of Pharmacology 147: S120–S131.
Silva ALS, Cabral AM, Abreu GR, et al. (2005) Chronic treatment with mianserin prevents DOCA-salt hypertension in rats: Evidence for the involvement of central 5-HT2 receptors. European Journal of Pharmacology 518: 152–157. Sykova´ E (2004) Extrasynaptic volume transmission and diffusion parameters of the extracellular space. Neuroscience 129: 861–876. Trudeau L-E (2004) Glutamate co-transmission as an emerging concept in monoamine neuron function. Reviews Psychiatry Neuroscience 29: 296–310. Uphouse L (1997) Multiple serotonin receptors: Too many, not enough, or just the right number? Neuroscience and Behavioral Reviews 21: 679–698.
Serotonin (5-Hydroxytryptamine; 5-HT): Receptors T P Blackburn, Helicon Therapeutics Inc., Farmingdale, NY, USA ã 2009 Elsevier Ltd. All rights reserved.
Introduction During the past 50 years, since its discovery in 1948, studies of 5-hydroxytryptamine (5-HT; serotonin) have provided considerable insight into the metabolism and physiological functions of this monoaminergic neurotransmitter in the peripheral and central nervous system (CNS). Considerable interest in the biosynthesis, metabolism, and receptor pharmacology of 5-HT (Figure 1) has elevated this simple indolealkylamine into one of the most researched neurotransmitters of the twentieth century. The synthesis of 5-HT begins with the essential amino acid tryptophan, which can be found in foods such as bananas, turkey, and milk. The enzyme tryptophan hydroxylase hydroxylates tryptophan’s benzene ring, to form 5-hydroxytryptophan. Aromatic amino acid decarboxylase then decarboxylates 5-hydroxytryptophan to produce 5-HT. 5-HT is formed in nerve terminals, where it is held in storage granules; when nerve impulses release 5-HT into the synaptic cleft, it acts on the postsynaptic neuron to affect the process of neurotransmission across the synapse. Released 5-HT is inactivated primarily by an active serotonin reuptake transporter (SERT) process that moves 5-HT back into the neuron. Once inside the 5-HT neuron, the neurotransmitter is either repackaged into storage granules or degraded metabolically by monoamine oxidase (MAO). Pre- and postsynaptic 5-HT receptors exist on the 5-HT nerve terminals and cell bodies. Activation of these receptors leads to a disruption in the synthesis and release of serotonin. The role of the presynaptic autoreceptor is to modulate the concentration of serotonin in the synaptic cleft, thereby reducing further release and synthesis of 5-HT (see the sections ‘The 5-HT1A(Gi/Go) receptor’ and ‘The 5-HT1B(Gi/Go) receptor’). It is probable that early work on the synthesis, neurochemistry, and distribution of 5-HT stimulated the expectation that 5-HT might be important in anxiety, depression, and schizophrenia. It is quite remarkable, and a far cry from those early days, that the total sales of drugs modulating central and peripheral 5-HT activity exceeded $20 billion (US) in 2006. This is undoubtedly due to an increasing awareness of the 5-HT receptor subtypes associated with the multitude of disorders treated by drugs
456
modulating 5-HT function; migraine, chemotherapyinduced emesis, depression, anxiety, schizophrenia, and irritable bowel syndrome are examples of such disorders. The promise of further therapeutic opportunities emerging awaits the development of more selective agents (combination agents) and the possibility of discovery of further novel 5-HT receptor subtypes.
Evidence for Multiple Serotonin Receptor Genes The classification and nomenclature of 5-HT receptors in the past 10 years has emerged from criteria established by the International Union of Basic and Clinical Pharmacology (IUPHAR) Subcommittee for the Classification and Nomenclature of 5-HT receptors Guide to Receptors and Channels (GRAC) 2007 (Figure 2; Tables 1–5). To date, 13 distinct subtypes of human serotonin receptor are recognized on the basis of structural, transductional, and operational characteristics. A fourteenth purported receptor subtype (5-ht5B) has been identified in rodents only. The various subtypes are subdivided into seven different classes (5-HT1–7), based on gene encoding, and in some cases only one gene product has been identified. These gene products are referred to by use of lower case (e.g., 5-ht1E, 5-ht1F) and will only be accepted into the 5-HT family and given ‘upper-case’ status when there is unequivocal evidence that the receptor exists as an operational endogenous, physiologically relevant receptor.
G-Protein-Coupled 5-HT(1–7) Receptor Family 5-HT1 serotonin receptors are expressed in a variety of neurons in the peripheral and central nervous systems. The 5-HT1 receptor family – 5-HT1A, 5-HT1B (formerly termed 5-HT1Db), 5-HT1D (formerly 5-HT1Da), 5-ht1E, and 5-ht1F – are all seven-transmembrane (7TM) G-protein-coupled receptors (via Gi or Go; see nomenclature in Figure 2). They are all encoded by intronless genes (of approximately 365–421 amino acids) and are negatively coupled to adenylyl cyclase, and principally cause hyperpolarization. The 5-HT1A(Gi/Go) Receptor
The most studied of the 5-HT1 receptor family is the 5-HT1A receptor, located on human chromosome HTR1/5q11.2–q13, which encodes 421 amino acids.
Serotonin (5-Hydroxytryptamine; 5-HT): Receptors 457
The receptor is found largely on neuronal soma and dendrites and epithelial cells, where it is found on both apical and basolateral membranes. Release of 5-HT in the CNS is under the control of a 5-HT1A autoreceptor (and 5-HT1B autoreceptors, see later). These autoreceptors fall into two categories: cell body autoreceptors and terminal autoreceptors. The former inhibit 5-HT release through inhibition of cell firing; the latter act through direct inhibition of release at the terminal. Cell body (or somatodendritic) autoreceptors belong to the 5-HT1A receptor subtype in all species studied so far. In vivo immunocytochemical experiments have clearly demonstrated that, in striatal medium spiny neurons, the 5-HT1A receptor is restricted to the somatodendritic level, while 5-HT1B receptors are shipped exclusively toward axon terminals. In all systems examined to date, there appears to be a differential sorting of the 5-HT1A and 5-HT1B receptors. This observation is largely based on an in vivo transgenic system and appears to represent the only model that reconstitutes proper sorting of these receptors. 5-HT1A receptors in the raphe nuclei act as somatodendritic autoreceptors and have little effect on extraneuronal 5-HT levels alone, but potentiate the increase seen after selective serotonin reuptake inhibitor (SSRI) administration. It is this effect that has been purported to be the mechanism of action of SSRIs, whereby desensitization of the 5-HT1A autoreceptors may underlie the ability of chronic, but not acute, SSRI administration to increase synaptic cleft levels of 5-HT. Evidence from in vivo studies indicates that activation of the postsynaptic 5-HT1A receptor in the rat
NH2 HO N H Figure 1 5-Hydroxytryptamine structure.
Receptor type:
results in a characteristic 5-HT syndrome consisting of flat body posture, forepaw treading and head weaving, hypothermia, and adrenocorticotropic hormone (ACTH) release. Stimulation of postsynaptic 5-HT1A receptors may also cause anxiogenic-like responses. In contrast, activation of presynaptic 5-HT1A receptors induces both hyperphagia and anxiolytic-like effects in rats and hence may account for the clinical anxiolytic efficacy of the 5-HT1A receptor agonists, buspirone and gepirone. 5-HT1A receptor agonists are also active in animal models of depression, such as the rat forced swimming and mouse tail suspension tests, consistent with their purported antidepressant efficacy. Several agonists show selectivity for the 5-HT1A receptor, particularly R(þ)-8-hydroxy-(di-n-propylamino) tetralin (8-OH-DPAT; Table 1), U92016A, and R(þ)-UH301, which act as high-efficacy agonists in most systems. In contrast, the nonbenzodiazepine anxiolytics (buspirone and gepirone) and other ligands (such as MDL72832) have been shown to be partial agonists (low efficacy) at the 5-HT1A receptor. Several apparent antagonists have also been characterized, such as NAN190, BMY7387, MDL 73005EF, WAY100135, S()-UH301, S()-pindolol, spiroxatrine, spiperone, SDZ216525, and NAD299 (robalzotan). However, all have demonstrated partial agonist properties in studies of somatodendritic autoreceptor function, perhaps due to the much larger receptor reserve associated with these, as opposed to postsynaptic receptors. To date, two selective high-affinity silent antagonists at this receptor have been identified; WAY100635 (pKB 8.7; Table 1) and lecozotan (SRA-333; Ki 1.6 nM). The latter compound is currently in phase I studies for mild to moderate symptoms of Alzheimer’s disease. The 5-HT1A receptor remains an enigma; however, continued great interest in this receptor persists, based on the ability of azapirone partial agonists (e.g., gepirone, isapirone, and zalospirone) to modify 5-HT function by an action on the 5-HT autoreceptor. 5-HT1A agonists in this class include GR127935 and WAY163426, which are reported to
5-HT1
5-HT2
AC
PLC (Gq /11)
5-HT3
5-HT4
5-HT5
5-HT6 5-HT7
Ion Effector:
(G1/G0)
AC
AC
Na+/K+/Ca+ (Gs)
(Gs)
channel
?
Subtypes: 5-HT1A 5-HT1B 5-HT1D 5-HT1E 5-HT1F
Figure 2 Classification of 5-HT receptor subtypes.
5-HT2A
5-HT2B
5-HT2C
5-HT5A 5-HT5B
AC
AC
(Gs) (Gs)
458 Serotonin (5-Hydroxytryptamine; 5-HT): Receptors Table 1 5-HT1A, 5-HT1B, 5-HT1D, and 5-ht1E, receptorsa Feature
Other names Ensemble ID Principal transduction Selective agonists Selective antagonists (pKB) Probes (KD)
Receptor 5-HT1A
5-HT1B
5-HT1D
5-ht1E
– ENSG00000178394 Gi/o
5-HT1Db ENSG00000135321 Gi/o
5-HT1Da ENSG00000179546 Gi/o
ENSG00000168830 Gi/o
8-OH-DPAT, (R)-UH301, U92016A ()WAY100635 (8.7), (S)UH301, NAD299 (robalzotan, 9.2) [3H]WAY100635 (0.3 nM), [3H]8-OH-DPAT, [11C]WAY100635 (PET ligand)
Sumatriptan, L694247, CP93129, CP-122288 SB236057 (8.9), SB224289 (8.5), GR55562 (7.4)
PNU10929, sumatriptan, L694247 SB714786 (pKi 9.1), BRL15572 (7.9)
[3H]Sumatriptan, [125I]GTI, [3H]GR125743 (2.6 nM), [3H]L694247
[3H]Sumatriptan, [125I]GTI, [3H]GR125743 (2.8 nM), [3H]L694247
[3H]5-HT
Overview (see also Tables 2–5) of 5-HT receptors. Modified nomenclature as agreed by the Nomenclature Committee of the International Union of Basic and Clinical Pharmacology (IUPHAR), subcommittee on 5-HT receptors; receptors and subsequently revised types are, with the exception of the ionotropic 5-HT3 class, seven-transmembrane receptors, for which the endogenous agonist is 5-HT. The diversity of 5-HT receptors is increased by alternative splicing that produces isoforms of the 5-HT2A (nonfunctional), 5-HT2C (nonfunctional), 5-HT4, and 5-HT7 receptors. RNA editing produces 5-HT2C receptor isoforms that differ in function, such as in efficiency and specificity of coupling to Gq/11.The 5-HT3 receptor exists as a pentamer of 4TM subunits that form an intrinsic cation selective channel. Three 5-HT3 receptor subunits (5-HT3A, 5-HT3B, and 5-HT3C) have been cloned, but only homo-oligomeric assemblies of 5-HT3A and hetero-oligometric assemblies of 5-HT3A and 5-HT3B subunits have been characterized in detail. Putative HTR3D and HTR3E genes have also been described but there is no evidence to date that they encode functional subunits. Also, a hetero-oligometric receptor that contains two copies of the 5-HT3A subunit and three copies of the 5-HT3B subunit has been described. The 5-HT3B subunit has distinctive biophysical properties upon hetero-oligomeric versus homo-oligomeric (5-HT3A) recombinant receptors but generally has little effect upon the apparent affinity of agonists and antagonists. However, homo- and hetero-oliogomeric 5-HT3 receptors have been reported to differ in their allosteric regulation by some general anesthetic agents. The diversity of 5-HT3 receptors is also further increased by alternative splicing of the 5-HT3A subunit. Evidence to date, indicates that the 5-HT3A subunit appears to be essential for 5-HT3 operational characteristics. Tabulated KD values refer to binding to human 5-HT receptors, with the exception of SB207710 (piglet) and RP62203 (rat). The nomenclature of 5-HT1B/5-HT1D receptors has been revised. Only the nonrodent form of the receptor was previously called 5-HT1Db: the human 5-HT1B receptor (tabulated) displays a pharmacology different from that of the rodent forms of the receptor, due to Thr335 of the human sequence being replaced by Asn in rodent receptors. NAS-181 is a selective antagonist of the rodent 5-HT1B receptor. Fananserin binds with high affinity to dopamine D4, in addition to 5-HT2Areceptors. The human 5-ht5A receptor has been claimed to couple to several signal transduction pathways when stably expressed in C6 glioma cells. The human ortholog of the mouse 5-ht5B receptor is nonfunctional due to interruption of the gene by stop codons. In addition to the receptors listed in the table, an ‘orphan’ receptor, unofficially termed 5-HT1P, has been described in the literature. Abbreviations here and for Tables 2–4 (subtype-selective 5-HT compounds): BIMU8, (endo-N-8-methyl-8-azabicyclo[3.2.1]oct-3-yl)-2,3-dihydro-3-isopropyl-2-oxo-1H-benzimidazol-1-carboxamide hydrochloride; BRL15572, 3-[4-(3-chlorophenyl)piperazin-1-yl]-1,1,-diphenyl-2-propanol; BW723C86, 1-[5(2-thienylmethoxy)-1H-3-indolyl]propan-2-amine hydrochloride; 5-CT, 5-carboxamidotryptamine; 8-OH-DPAT, 8-hydroxy-2-(di-n-propylamino)tetralin; EGIS-7625, 1-benzyl-4-[(2-nitro-4-methyl-5-amino)-phenyl]-piperazine; GR55562, 3-[3-(dimethylamino)propyl]-4-hydroxy-N-[4-(4-pyridinyl)phenyl] benzamide; GR113808, [1–2[(methylsulfonyl)amino]ethyl]-4-piperidinyl]methyl-1-methyl-1H-indole-3-carboxylate; GR125743, n-[4methoxy-3-(4-methyl-1-piperizinyl)phenyl]-3-methyl-4-(4-pyrindinyl)benzamide; CP 93,129 ([3-(1,2,5,6-tetrahydropyr-id-4-yl)pyrrolo[3,2b]pyrid-5-one]), CP-122,288 (5-methyl-aminosulfonylmethyl-3-(N-methylpyrrolidin-2R-yl-methyl)-1H-indole), GTI, [125I]5-hydroxytryptamine-5-O-carboxymethylglycyltyrosinamide, GR65630-(5-methyl-1H-imidazol-4-yl)-1-(1-methyl-H-indol-3-yl)-1-propanone, LY278584, 1 methyl-N-(8-methyl-8-azabicyclo[3.2.1]oct-3-yl)-1H-indazole-3-carboxamide, TMB-8, 8-(diethylamine)octyl-3,4,5-trimethoxybenzoate; GTI, 5-O-carboxamidomethylglycyl[125I]tyrosinamide-tryptamine; PNU109291, (S)-3,4-dihydro-1-[2-[4-(4-methoxyphenyl)-1-piperazinyl] ethyl]-N-methyl-1H-2-benzopyran-6-carboximide; Ro 60–0175, (S)-2-(6-chloro-5-fluroindol-1-yl)-1-methyethylamine; Ro 63–0563, 4amino-N-[2,6-bis(methylamino)pyridin-4-yl]benzenesulfonamide; RP62203, 2-[3-(4-(4-fluorophenyl)-piperazinyl)propyl]naphto[1,8-ca] isothiazole-1,1-dioxide; RS127445, (2-amino-4-(4-fluoronaphthyl-1-yl)-6-isopropylpyrimidine); RS57639, 4-amino-5-chloro-2-methoxybenzoic acid 1-(3-[2,3-dihydrobenzo[1,4]dioxin-6yl)-propyl]-piperidin-4yl methyl ester; RS100235, 1-(8-amino-7-chloro-1,4-benzodioxan-5-yl)-5-((3-(3,4-dimethoxyphenyl)prop-1-yl)piperidin-4-yl)propan-1-one; RS102221, 8-[5-(5-amino 2,4-dimethoxyphenyl) 5-oxopentyl]-1,3,8-triazaspiro[4,5]decane-2,4-dione; SB204070, 1-butyl-4-piperidinylmethyl-8-amino-7-chloro-1–4-benzoioxan-5-carboxylate; SB207710, 1-butyl-4-piperidinylmethyl-8-amino-7-iodo-1,4-benzodioxan-5-carboxylate; SB224289, 10-methyl-5[((20-methyl40-)5-methyl-1,2,4-oxadiazol-3-yl)biphenyl-4-yl]carbonyl-2,3,6,-7-tetrahydrospiro[furo[2,3-f]indole-3,40-piperidine]oxalate; SB236057, 10-ethyl-5-(20-methyl-40-(5-methyl-1,3,4-oxadiazol-2-yl)biphenyl-4-carbonyl)-2,3,6,7-tetrahydrospiro[furo[2,3-f]indol3–3,40-piperidine; SB242084, 6-chloro-5-methyl-1-[2-(2-methylpyridyl-3-oxy)-pyrid-5-yl-carbamoyl] indoline; SB258585, 4-iodo-N-[4-methoxy-3-(4-methylpiperazin-1-yl)-phenyl]-benzenesulfonamide; SB258719, (R)-3,N-dimethyl-N-[1-methyl-3-(4-methylpiperidin-1-yl)propyl]benzene sulfonamide; SB269970, (R)-3-(2-(2-(4-methylpiperidin-1-yl)ethyl)pyrrolidine-1-sulfonyl)phenol; SB271046, 5-chloro-N-(4-methoxy-3-piperazin1-yl-phenyl)-3-methyl-2-benzothiophenesulfonamide; SB357134, N-(2,5-dibromo-3-fluorophenyl)-4-methoxy-3-piperazin-1-yl-benzenesulfonamide; SB656104, 6-((R)-2-[2-[4-(4-chlorophenoxy)-piperidin-1-yl]-ethyl]-pyrrolidine-1-sulfonyl)-1H-indole hydrochloride; SB699551, 3-cyclopentyl-N-[2-(dimethylamino)ethyl]-N-[(40-{[(2-phenylethyl)amino]methyl}-4-biphenylyl)methyl]propanamide dihydrochloride; SB714786, 2-methyl-5-({2-[4-(8-quinolinylmethyl)-1-piperazinyl]ethyl}oxy)quinoline; SR57227, 4-amino-(6-chloro-2-pyridyl)-1-piperidine hydrochloride; UH301, 5-fluoro-8-hydroxy-2-(dipropylamino) tetralin; U92016A, ()-(R)-2-cyano-N,N-dipropyl-8-amino-6,7,8,9-tetrahydro3H-benz[e]indole; WAY100635, N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridyl)-cyclohexanecarboxamide trichloride. a
Serotonin (5-Hydroxytryptamine; 5-HT): Receptors 459 Table 2 5-HT1F, 5-HT2A, 5-HT2B, and 5-HT2C receptorsa Feature
Other names Ensemble ID Principal transduction Selective agonists Selective antagonists (pKB) Probes (KD)
Receptor 5-HT1F
5-HT2A
5-HT2B
5-HT2C
5-HT1Eb, 5-HT6 ENSG00000179097 Gi/o
D, 5-HT2 ENSG00000102468 Gq/11
5-HT2F ENSG00000135914 Gq/11
5-HT1C ENSG00000147246 Gq/11
LY334370, LY334864
DOI
DOI, Ro 60-0175, BW723C86 RS127445 (9.5), EGIS-7625 (9.0)
DOI, Ro 60-0175
[3H]5-HT
[3H]Mesulergine (0.67), [3H]LSD
Ketanserin (8.5–9.5), M100907 (9.4) [3H]LY334370 (0.5 nM), [125I] LSD
[3H]Ketanserin (0.45 nM), [3H]RP62203 (fananserin, 0.13 nM), [11C]M100907 (PET ligand), [18F]altanserin (PET ligand)
SB242084 (9.0), RS102221 (8.6)
See Table 1 footnote for overview and abbreviations.
a
Table 3 5-HT3 receptorsa Nomenclature Other names Ensemble ID Selective agonists Selective antagonists (plCDD) Channel blockers Radioligands (KD)
5-HT3 M 5-HT3A ENSG00000166736, 5-HT3B ENSG00000149305 2-methyl-5-HT (5.3–5.5), 3-chlorophenyl-blguanide (5.4–5.7) granisetron (9.5), ondansetron (9.5), tropisetron (9.2) Diltiazem, TMB-B, picrotoxin [3H]ramosetron (0.15 hM). [3H]granisetron (1.2 nM), [3H]GR65630 (2.6 nM). [3H]LY278584
See Table 1 footnote for overview and abbreviations.
a
Table 4 5-HT4, 5-ht5A, 5-ht5B, and 5-HT8, receptorsa Feature
Other names Ensemble ID Principal transduction Selective agonists Selective antagonists (pKB)
Radioligands (KD)
Receptor for 5-HT4
5-ht5A
5-ht5B
5-HT8
ENSG00000164270 Gs
5-HT5a ENSG00000157219 Gi/Go
ENSMUSG00000050534 None identified
ENSG00000158748 Gs
BIMU8, ML10302, RS67506 GR113808 (9.0–9.5), SB204070 (10.8), RS100235 (11.2) [3H]GR113808 (0.1 nM), [125I] SB207710 (86 pM), [3H]RS57639
SB699551 (8.0)
[3H]5-CT, [125I]LSD
SB271046 (8.7), SB357134 (7.6), Ro 63–0563 (7.9)
[3H]5-CT, [125I]LSD
[125I]SB258585 (1.0 nM), [3H]Ro 63–0563 (5 nM), [3H]5-CT, [125I]LSD
See Table 1 footnote for overview and abbreviations.
a
raise serotonin levels more rapidly than occurs with an SSRI, and which exhibit activity in chronic models of depression that are consistent with a more rapid onset. Two compounds, VPI-013 and PRX-00023,
are also adding to the renewed interest in this area; although they have mixed 5-HT1A receptor antagonist and sigma agonist properties, they are reported to be in phase II studies for depression.
460 Serotonin (5-Hydroxytryptamine; 5-HT): Receptors Table 5 5-HT7 receptor featuresa Other names: 5-HTX, 5-HT1-like Ensemble ID: ENSG00000148680 Principal transduction: Gs Selective agonists: none Selective antagonists (pKB): SB656104 (8.5), SB269970 (8.5), SB258719 (7.2) Radioligands (KD): [3H]SB269970 (1.2 nM), [3H]5-CT, [125I]LSD, [3H]5-HT See Table 1 footnote for overview and abbreviations.
a
The 5-HT1B(Gi/Go) Receptor
5-HT1B receptors are expressed throughout the mammalian central nervous system. These receptors are located on the axon terminals of both serotonergic and nonserotonergic neurons, where they act as inhibitory autoreceptors or heteroreceptors, respectively. 5-HT1B receptors inhibit the release of a range of neurotransmitters, including serotonin, noradrenaline, g-aminobutyric acid (GABA), acetylcholine, and glutamate. In rats and mice, the terminal autoreceptor is known to be a 5-HT1B receptor, whereas in humans, pigs, rabbits, and guinea pigs, the terminal autoreceptor is thought to belong to the 5-HT1D receptor subtype (see later). The 5-HT1B receptor is located on human chromosome HTR1/6q13 and the receptor gene is largely concentrated in the basal ganglia, striatum, and frontal cortex. The receptor was originally defined by its pharmacology and also because of species differences in the binding affinity of key ligands, such as the b-adrenoceptor antagonist, cyanopindolol, which was thought to exist only in rodents. More recently, the amino acid sequence of the receptor has been characterized (humans, 390 amino acids; rodents, 386 amino acids) and found to be 93% identical overall and 96% identical with the transmembrane domains of the 5-HT1Db receptor, a close homolog found in higher species. This has a distribution and function similar to that of the 5HT1B receptor. Indeed, the differences in the pharmacology of these two homologs are now attributed to the mutation of a single amino acid. Only the nonrodent form of the receptor was previously called 5-HT1D: the human 5-HT1B receptor displays a pharmacology different from that of the rodent forms of the receptor due to Thr335 of the human sequence being replaced by Asn in rodent receptors in the transmembranespanning region. In the rodent it is possible to differentiate between the pharmacology of the 5-HT1B and 5-HT1D receptors, as the 5-HT ortholog in these species exhibits a different pharmacology due to an Asp123/Arg123 switch in the rodent receptor. Thus, it has recently been agreed to classify the receptors as species homologs of the same receptor, termed
h5-HT1B (formerly 5-HT1Db) and r5-HT1B, with the h and r prefixes referring to the human and rat species, respectively. Highly selective agonists and antagonists (Table 1) have now been identified to facilitate characterization of this receptor. There has been accumulating evidence, however, that 5-HT1B receptors modulate drug reinforcement, stress sensitivity, mood, anxiety, and aggression. The general results of a number of studies and human genetics suggest that reduced 5-HT1B heteroreceptor activity may increase impulsive behaviors, whereas reduced 5-HT1B autoreceptor activity may have an antidepressant-like effect. A large number of studies have established the 5-HT1B terminal autoreceptor in controlling 5-HT release in rats, guinea pigs, and humans. The notion that a selective 5-HT1B antagonist might prevent 5-HT negative feedback via this site, thereby increasing extraneuronal 5-HT and mimicking SSRI antidepressants, awaits clinical efficacy studies. RU24969 was the first reported full agonist at the 5-HT1B receptor, but, in binding studies, it is only fivefold selective over the 5-HT1A and 5-HT1D receptors. Since the first reports, several other compounds with dubious selectivity have emerged, at best having five-to tenfold selectivity in binding and functional assays (CGS 12066B, anpirtoline trifluoromethylphenylpiperasine (TFM), m-cholorophenylpiperazine (mCPP), MK 464, BW311C90, KSF99101H, and GR46611). The most potent agonist reported is L694247 (pKD 10.0). None of these, however, differentiates between the 5-HT1B and the 5-HT1D receptor subtypes. More recently, selective 5-HT1B receptor agonists (L694247) and antagonists (GR55562, pKB 7.4; SB224289, pKB 8.5; SB236657, pKB8.9; SB236057, pKB8.9) have been reported, generating further excitement around this receptor subtype and promising to shed light on the therapeutic utility of such agents. The first potent and selective 5-HT1B/1D antagonist to be reported, GR127935 (pKD 9.9), decreased extraneuronal 5-HT in the brain when administered systemically to guinea pigs. This may be due to its partial agonist activity at the 5-HT1B receptor or to the presence of 5-HT1D receptors on the raphe acting as somatodendritic autoreceptors, at which GR127935 also has agonist properties. This interpretation is supported by the failure of the recently characterized selective silent 5-HT1B antagonist, SB224289 (pKB 8.5, >60-fold selective over 5-HT1D and other receptors; Table 1), to increase 5-HT release in the frontal cortex. NAS-181 has been reported to be the only selective antagonist of the rodent 5-HT1B receptor and it may add to our knowledge of the operational characteristics of the rat receptor subtype.
Serotonin (5-Hydroxytryptamine; 5-HT): Receptors 461
Complementary studies in 5-HT1B null mutant (‘knockout’) mice have proved unhelpful in understanding the pharmacology of the 5-HT1B receptor, as it has been suggested that a compensatory adaptation to the constitutive loss of 5-HT1B receptors becomes an important determinant of the altered response of 5-HT1B knockout mice to a variety of pharmacological challenges. Altered expression and functions of serotonin 5-HT1A and 5-HT1B receptors in knockout mice lacking the 5-HT transporter (SERT knockout mice) have also been reported. Although the effects on 5-HT release are unclear from 5-HT1B knockout mice, in vivo studies have shown that stimulation of central postsynaptic 5-HT1B receptors in mice, but not rats, causes hyperlocomotion, and penile erection and hypophagia in rats are also reportedly 5-HT1B receptor mediated. Postsynaptic 5-HT1B receptor activation in another species, the guinea pig, is reported to induce hypothermia, while the hypothermic response to 5-HT1B agonists in the rat remains to be fully characterized. The putative 5-HT1B receptor agonist, anpirtoline, has analgesic, cognitive, and antidepressant-like properties in rodents and it is of interest that mutant mice lacking the 5-HT1B receptor are reported to be both highly aggressive and have an increased preference for alcohol. Great interest in 5-HT1B receptor agonists has been largely generated by the highly successful antimigraine drug sumatriptan, a nonselective 5-HT1D and 5-HT1B receptor agonist with low selectivity for other receptors in functional studies (Table 1). This compound may act either via constriction-mediating 5-HT1B receptors on cerebral arteries or by blocking neurogenic inflammation and nociceptive activity with trigeminovascular afferents. This latter action has been argued to be 5HT1B receptor mediated, as protein extravasation induced by trigeminal ganglion stimulation is blocked by sumatriptan, the nonselective 5-HT1B receptor agonist, by CP-93129 (>100-fold selectivity over 5-HT1A, 5-HT1B, and 5-HT2 receptors; Tables 1 and 2), and by the close structural analog of sumatriptan, CP-122288, in wild-type, but not mutant, mice lacking the 5-HT1B receptor. However, while 5-HT1B receptor mRNA has been detected in rats, only 5-HT1D mRNA has been detected in the guinea pig and human trigeminal ganglia. Thus, in humans, the antimigraine properties of sumatriptan may be either 5-HT1B or 5-HT1D receptor mediated. It is therefore abundantly clear that our understanding of the operational and physiological consequences of the 5-HT1B activation across species is still largely in its infancy. The 5-HT1D(Gi/Go) Receptor
The 5-HT1D receptor has 63% overall structural homology with the 5-HT1B receptor (formerly
5-HT1Db) and a 77% amino acid sequence homology in the seven-transmembrane domains. The receptor is located on human chromosome HR1D/1p34.3–p36.3 and contains 377 and 374 amino acids for the human and rodent gene, respectively. Low levels of the 5-HT1D receptor mRNA are found in the rat brain, predominantly in the caudate putamen, nucleus accumbens, hippocampus, and cortex, but also in the dorsal raphe and locus coeruleus. It has been proposed that neurogenic inflammation and nociceptive activity within the trigeminovascular afferents may be 5-HT1D receptor mediated due to the presence of 5-HT1D, but not 5-HT1B, receptor mRNA in the guinea pig and human trigeminal ganglia. Thus antagonism of plasma extravasation, induced by electrical stimulation of the trigeminal nerves, is observed with the antimigraine 5-HT1B/1D receptor agonists sumatriptan, naratriptan, rizatriptan, and zolmitriptan. Some advocates, however, claim that this is a 5-HT1D receptor-mediated response. Several other pharmacological nonselective tool 5-HT1B/1D ligands have been used extensively to characterize the 5-HT1D receptor. These include sumatriptan, PU109291, L694247 (pKD 10.0), CP-122288, MK 464, and BW311C90, and the more brain penetrant agonists SKF 99101H, GR46611, and GR125743. The location of 5-HT1D receptor mRNA in the raphe suggests that, like the 5-HT1B receptor, it too may function as a 5-HT autoreceptor. Indeed, there is electrophysiological, release, and voltammetric evidence to this effect. These data are further substantiated by the use of ketanserin and ritanserin, which, in addition their high affinity for the 5-HT2A site, also have high affinity for the 5-HT1D, but not for the 5-HT1B, site (Table 1). The antagonist SB714786 (pKD 9.1) and BRL-15572 (pKD 7.9) 5-HT1B/D ligands mediate their effects at autoreceptors involved in the local inhibitory control of 5-HT release and may play a role in the pathogenesis of major depressive disorder (MDD) and in the antidepressant effects of the SSRIs in patients. A number of compounds in this class – including GR125743, SB224289, and L694247; AZD8129, a 5-HT1B receptor antagonist; and CP-448187, a 5-HT1B/1D receptor antagonist – are in phase II studies for depression, and the outcome of these studies is eagerly awaited. The 5-HT1E(Gi/Go) Receptor
The 5-HT1E receptor was first characterized in humans as a [3H]-5-HT binding site in the presence of 5-carboxyamidotryptamine (5-CT) that blocked binding to the 5-HT1A and 5-HT1D receptors. Human brain binding studies have reported that 5-HT1E receptors (representing up to 60% of 5-HT1 receptor binding) are concentrated in the caudate putamen,
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with lower levels in the amygdala, frontal cortex, and globus pallidus. This is consistent with the observed distribution of 5-HT1E mRNA. The receptor has been mapped to human chromosome HTR1E/6q14–q15, and the gene encodes a 365-amino-acid protein (Table 1). There are no reported selective or highaffinity ligands for this receptor (except for 5-HT) and its function is currently unknown. However, based on its distribution, one could argue that the amygdala is regarded as the ‘emotional engine,’ and dysfunction of the amygdala (and caudate putamen) has been associated with numerous psychiatric and neurological disorders, ranging from epilepsy to anxiety disorders, attention-deficit/hyperactivity disorder (ADHD), and social phobia, to Alzheimer’s disease. Some schools of thought have even proposed that the amygdala is involved in sociopathic and criminal behaviors. Only time will tell, with the development of selective agents for the 5-HT1E receptor. The 5-HT1F(Gi/Go) Receptor
This receptor subtype is most closely related to the 5-HT1E receptor, with 70% sequence homology across the 7TM domains. The gene is located on chromosome HTR1F/3p11, encoding 366 amino acids across human and rodent species. The mRNA coding for the receptor is concentrated in the dorsal raphe, hippocampus, and cortex of the rat and also in the striatum, thalamus, and hypothalamus of the mouse. 5-HT1F receptor mRNA has been detected in human brain and is also present in the mesentery and uterus. Sumatriptan has almost equal affinity for the 5-HT1F (pKi 7.6) and 5-HT1B/1D receptors (pKi 8.4 and 8.1, respectively; Table 1). Thus, it has been hypothesized that the 5-HT1F receptor might be a target for drugs with antimigraine properties. 5-HT1F mRNA has been detected in the trigeminal ganglia, the stimulation of which leads to plasma extravasation in the dura, a component of neurogenic inflammation that is thought to be a possible cause of migraine. The first 5-HT1F receptor selective agonist, LY334370 (pKD 9.4; see Table 2), with >100-fold separation over the 5-HT1B/ 1D receptors, has been claimed to block the effects of trigeminal nerve stimulation, as does sumatriptan, naratriptan, rizatriptan, and zolmitriptan . LY334370 has also been used successfully as a radioligand (KD 0.5 nM) and demonstrated a reasonable correlation between the receptor protein and mRNA distribution, with the highest binding in the cortical areas, striatum, hippocampus, and olfactory bulb. The 5-HT2(Gq11) Receptor Family
5-HT2 receptors are characterized by having a relatively lower affinity for indolealkylamines, including 5-HT, and are linked to the Gq/phospholipase
C pathway of signal transduction. The structural organization of the 5-HT2 receptor has largely been determined by mutagenesis analyses that have identified a number of primary binding residues located mainly on TMs 3, 5, and 6, which are believed to constitute the purported binding pocket. Other residues identified on TMs 2, 3, 6, and 7 have been implicated in receptor activation and G-protein coupling. The 5-HT2 receptor family mediates a large array of physiological and behavioral functions in humans via three distinct subtypes: 5-HT2A, 5-HT2B, and 5-HT2C. While selective 5-HT2A receptor antagonists have been known for some time, knowledge of the precise effects of 5-HT2C and 5-HT2B receptor antagonists has been hampered by the existence of only mixed receptor antagonists for 5-HT2A, 5-HT2B, and 5-HT2C. However, selective 5-HT2A, 5-HT2C, and 5-HT2B receptor antagonists have emerged over the past 5–10 years. Indeed, several structural classes belonging to the various pharmacophores have been reported. The 5-HT2A Receptor Subtype
The 5-HT2A receptor subtype refers to the classical ‘D’ 5-HT2 receptor as defined by the neuroleptic [3H]spiperone in rat brain frontal cortex. Much of the early pharmacology of the 5-HT2A receptor was pioneered using the selective ligand, [3H]ketanserin, which made an important contribution to the characterization and localization of 5-HT2A receptors. Since the early ligand binding studies, only a few compounds have shown over all selectivity for the 5-HT2A site. Of these compounds, MDL100097 possesses subnanomolar affinity for the 5-HT2A receptor (pKB 9.4), with a minimum 300-fold selectivity for the 5-HT2A site (pKB 8.5–9.5) over other receptors examined, including the 5-HT2C receptor. Other less selective compounds include ICI 169369, ICI 170809, RP62203 (fananserin), and SR46349B (20- to 30-fold selective). The recent introduction of more selective 5-HT2A receptor antagonists (e.g., APD-125) will greatly accelerate the clinic potential of this receptor subtype. However, as yet, no selective agonists exist to provide a more precise classification of this receptor. Only a-methyl-5-HT has been shown to be a useful selective agonist for identifying 5-HT2A/2C/2B receptors (Table 2) Other (nonselective) compounds with agonist properties at 5-HT2 receptors include 1-(2,5-dimethoxy-4-methylphenyl)-2-amino propane (DOM) and its 4-bromophenyl (DOB) and 4-iodophenyl (DOI) congeners. DOI is the preferred radioligand, albeit that it is relatively nonselective. The actions elicited via 5-HT2A receptors comprise mostly those previously considered to be mediated by postsynaptic ‘D’ receptor and include vascular
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smooth muscle contraction in several preparations (e.g., rabbit aorta, rat caudal artery, dog gastrosplenic vein); contraction of bronchial, uterine, bladder, and some gastrointestinal smooth muscle; platelet aggregation; increased capillary permeability; and some behavioral syndromes in rodents, such as head twitch, forepaw treading, and coarse body tremors induced by 5-HT agonists. In humans, hallucinations and vivid dreams have been reported to be induced by 5-HT2A receptor stimulation. The contribution of 5-HT neurotoxins in understanding the role of 5-HT2A receptors and the mechanisms of action of hallucinogenic amphetamine analogs such as 3,4-methylenedioxymethamphetamine (MDMA) have implicated the 5-HT2A/2C receptors in psychiatric disorders. Several nonselective 5-HT2A/5-HT2C antagonists under investigation have failed to show any clinical utility for the treatment of several psychiatric disorders, including schizophrenia, anxiety, neurotic depression, migraine, sleep disorders, and drug abuse. However, a number of compounds have been shown to have therapeutic potential in cardiovascular disorders and migraine prophylaxis. As considered earlier, the striking similarities between 5-HT2A and 5-HT2C receptors with respect to amino acid sequence (70% in 7TM domains) holds true for the signal transduction mechanism, as both receptors stimulate phosphatidylinositol (PI) turnover. However, the receptors have distinct chromosomal localizations (HTR2A/13q14–q21 and HTR2C/Xq24). The characterization of the 5-HT2A receptor-coupled signal transduction system was first studied in human platelets. It is likely that the biochemical events mediated by the 5-HT2A receptor are important for both the shape change and the aggregation of human platelets. Controversy still exists, but a few studies have reported 5-HT-induced platelet aggregation in patients with peripheral cerebrovascular disease. Peripheral arterial vasospasms subsequent to platelet activation appear to be mediated in part by 5-HT and can be effectively reduced by ketanserin. In animal studies, postthrombotic peripheral collateral circulation can be significantly restored by treatment with ketanserin (pKD 8.5–9.5) and MDL100907 (pKD 9.4), and 5-HT-induced reduction of blood supply through the collateral system can be effectively counteracted by pretreatment with ketanserin. In ex vivo human platelet studies, the 5-HT2A/5-HT2C/5-HT2B receptor antagonist ICI 170809 significantly inhibited 5-HT-induced aggregation (minimum effective dose, 0.1 mg kg1 per os), thus lending support for the potential therapeutic role of 5-HT2A receptor antagonists in modulating the vasospastic effects of 5-HT released from platelets during vascular trauma. The clinical significance of selective 5-HT2A (see also
discussions of 5-HT2B and 5-HT2C) receptor antagonists in this vascular disorder and various psychiatric disorders still awaits further conclusive evidence of clinical efficacy with selective agents. In psychiatric disorder studies, two recent 5-HT2A receptor antagonists have shown significant clinical efficacy in schizophrenia. Pimavanserin (ACP-103) significantly improved efficacy scores (positive and negative syndrome scale; PANSS), showing a faster onset and improved side-effect profile, following co-therapy with risperidone. Fananserin, which binds with high affinity to dopamine D4, in addition to 5-HT2A receptors, also showed good clinically efficacy in schizophrenia, with an improved side-effect profile. This therapeutic opportunity is currently been actively pursued by several pharmaceutical companies, and several compounds acting at 5-HT2A and dopamine D2 receptors, are at various stages of clinical development (e.g., asenapine, blonanserin, eplivanserin, pruvanserin, paliperidone, uoperidone, and vabicaserin). Another selective 5-HT2A receptor antagonist, APD-125, is currently being assessed in the clinic for insomnia and sleep maintenance. The 5-HT2B(Gq11) Receptor
The 5-HT2B mRNA transcript has been now been identified in human and nonvertebrates and the receptor has been mapped to human chromosome HTR2B/2q36.3–2q37.1, with the human gene encoding 481 amino acids (Table 2). It was first identified in the stomach of the rat and in the small intestine, kidney, heart, and cerebellum in the mouse. The presence of the transcript in the rat stomach and the pharmacology of the cloned 5-HT2B receptor have led ultimately to defining the rat stomach fundus receptor. In Gaddum and Picarelli’s first classical attempt at subdivision of 5-HT receptors, the D subtype (5-HT2A) preceded the first descriptions of the rat stomach fundus 5-HT receptor by only a few pages in the same issue of the British Journal of Pharmacology, whereas the 5-HT2C receptor was not identified until some 28 years later. The 5-HT2B receptor represents the second member of the 5-HT2 family, but it is still unclear how the clone relates pharmacologically to that 5-HT receptor which mediates contraction of the rat stomach fundus. Evidence indicates that the 5-HT2B contractile receptor in the rat stomach fundus is coupled to calcium influx through voltage-dependent calcium channels, intracellular calcium release, and activation of protein kinase C (PKC). These actions may reflect a novel coupling mechanism unrelated to increases in PI hydrolysis, unlike the other 5-HT2 receptors. In the 1990s a series of potent, selective 5-HT2B receptor antagonists were identified, of which
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SB204741 (pKi 7.8) has proved to be an important tool for characterizing this receptor, along with the less selective 5-HT2B receptor agonists BW723C86 and a-Me-5-HT. Recent additions to the 5-HT2B tool-kit have been the introduction of two highly selective 5-HT2B receptor antagonists, RS127445 (pKi 9.5) and EGIS-7625 (pKi 9.0). The clinical potential of compounds selective for the 5-HT2B receptor site has pointed to an alternative target for initiation of migraine and may account for the continued use of methysergide, pizotifen, and cyproheptadine for migraine prophylaxis. More selective compounds have been developed for this indication, but to date no selective 5-HT2B compound has shown clinical efficacy for migraine. Moreover, the only 5-HT2B compound in current clinical development for the treatment of pulmonary hypertension is PRX-08066, which is surprising, considering the critical importance of the 5-HT2B receptor in mammalian heart development. The 5-HT2C(Gq11) Receptor
The human 5-HT2C receptor gene HTR2C/Xq24 encodes 377 amino acid residues with seven hydrophobic domains of 20–30 residues (Table 2). The rat and mouse sequences suggest the presence of an eighth transmembrane region not seen in the human receptor. The rat and human sequences are otherwise very similar, with a 90% overall homology. They are also positively coupled to phosphoinositide hydrolysis, providing a common secondary messenger system for 5-HT2C and 5-HT2A receptors. It is well established that 5-HT2C receptors display a high degree of constitutive activity, and consequently inverse agonists have a large effect when they bind to the 5-HT2C receptor. Furthermore, the mRNA for the 5-HT2C receptor undergoes mRNA editing, which results in the expression of multiple 5-HT2C receptor isoforms. Correspondingly, INI (unedited) and VSV (a fully edited version) isoforms are abundant in rat brain. The VSV isoform lacks the highaffinity recognition site for 5-HT, which may be caused by low-efficiency coupling to G-proteins. These edited isoforms appear to have reduced constitutive activity and reduced capacity to couple to Gq11 signaling proteins. This suggests that the INI isoform of the 5-HT2C receptor is pharmacologically similar to the VSV form of the 5-HT2C receptor, but that it couples more efficiently to G-proteins. Importantly, the capacity of some cells in the brain to edit the 5-HT2C mRNA is reduced. It has been suggested that this may well be the underlying pathophysiology in schizophrenic patients and that this reduced capacity is evident in brains of patients who had committed suicide. Changes in the RNA-editing capacity of the
brain will result in altered patterns of expression of the edited 5-HT2C isoforms and perhaps in altered responses to drugs. Consequently, it is important to understand the signaling characteristics and mechanism of the edited 5-HT2C receptor isoforms in various psychiatric and neurological conditions, and selective agents are allowing this work to progress. The pharmacological characteristics of the 5-HT2A and 5-HT2C (formally named 5-HT1C) receptors are remarkably similar, with many of the ‘classical’ 5-HT receptor antagonists showing high affinity for both sites. Furthermore, some compounds may have different affinities for human as opposed to rat receptors. The first 5-HT2C selective antagonist SB200646A (50-fold) was reported in the early 1990s; however, few truly selective 5-HT2C receptor agonists have been identified. MK 212 (30-fold) is the most selective CNS active agonist, but is also a 5-HT2A agonist, while a-methyl-5-HT has equal affinity for all 5-HT2 receptor subtypes. Of the various 5-HT2 receptor agonists, (þ)-DOB and m-chlorophenylpiperazine (mCPP) possess approximately tenfold selectivity over other binding sites in the rat but not in humans. However, mCPP is of particular interest because of its use as a clinical probe in the 1990s to study human 5-HT2C receptor function. mCPP is commonly observed to induce anxiety and panic attacks in humans. This mCPP-induced anxiogenesis is inhibited by the 5-HT2C and 5-HT2A receptor antagonist ritanserin, which is consistent with 5-HT2C receptor mediation. If 5-HT2C receptor activation is anxiogenic, the blockade of this receptor might induce anxiolysis, provided the receptors are tonically innervated. This argument is supported by the anxiolytic properties of SB243213, a selective 5-HT2C antagonist, in rat models of anxiety with quite different motivational and aversive bases (the rat social interaction and Geller–Seifter tests). To date this hypothesis awaits clinical testing. In support of this hypothesis, the selective 5-HT reuptake inhibitors (SSRIs) paroxetine, fluoxetine, clomipramine, sertraline, and citalopram, on chronic administration, have all been observed to desensitize behavioral responses to mCPP. The monoamine oxidase inhibitors phenelzine and nialamide, which also raise extraneuronal 5-HT levels, have a similar effect after chronic treatment. These results suggest that SSRIs and MAO inhibition antidepressant treatments may desensitize the 5-HT2C receptor, although there is no direct evidence from receptor binding, mRNA, or secondary messenger systems as yet. Furthermore, unlike other SSRIs, fluoxetine and its metabolite norfluoxetine have weak affinity for the 5-HT2C receptor and are therefore likely to accumulate and directly block 5-HT2C sites.
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Paradoxically, there is an emerging rationale and literature for the use of selective 5-HT2C receptor agonists in psychiatric and eating disorders that has been driven by recently reported compounds that have shown greater selectivity for the 5-HT2C receptor than has mCPP. Early studies with mCPP pointed the way to far more selective agonists in eating and psychiatric disorders. Several reports suggest that the 5-HT2C receptor agonists VER-2692 and WAY16909 are potent and selective agonists, and these are currently being evaluated as antiobesity agents. Early clinical studies with mCPP in bulimic patients induced migraine-like headaches 8–12 h later. This response was correlated with plasma levels of mCPP and was more pronounced in patients with a personal or family history of migraine. Conversely, the nonselective 5-HT2C/5-HT2A receptor antagonists pizotifen, cyproheptadine, and methysergide are all clinically effective as migraine prophylactics. ICI 169369 and sergolexole, which have high affinity for 5-HT2C and 5-HT2A receptors, but little affinity for other sites, have subsequently been found to have modest antimigraine efficacy in an early trial. As these drugs only share a high affinity for the 5-HT2C and 5-HT2A receptors, and as the selective 5-HT2A receptor antagonist ketanserin is ineffective as a migraine prophylactic, it was proposed that blockade of 5-HT2C receptors mediated this effect (although this has recently been challenged on the basis of their affinity for the 5-HT2B receptor). Furthermore, downregulation of 5-HT2C receptors may mediate the antimigraine properties of the antidepressants amitriptyline and fluoxetine; alternatively, the latter compounds may act by direct antagonism of vascular 5-HT2C receptor sites. Other possible therapeutic targets for drugs acting at 5-HT2C receptors include eating disorders, sexual dysfunction, and head trauma, based on the actions of nonselective 5-HT2C/2A agonists and antagonists. It is highly likely that the 5-HT2 family will continue to grow. There is already considerable evidence for a number of other ‘orphan’ receptors which may be adopted into the family. Several orphans are thought to exist, one of which is the endothelial 5-HT receptor, which is present in rabbit and rat jugular vein and pig pulmonary artery and vena cava. The rat jugular vein is now known to represent a 5-HT2B receptor. More recently, a site identified in choroid plexus has been shown to bind the 5-HT2-selective ligand RP62203 with high affinity, but does not possess identity with any of the three established 5-HT2 receptors. However, it would appear that the endothelial receptors do not represent a pharmacologically homogeneous class. While possessing many of the
characteristics of 5-HT2C and 5-HT2B receptors, subtle yet robust differences render any explicit affiliation impossible. These problems are confounded by the need, in many instances, to make comparisons across species. The potential for substantial species differences in pharmacology is highlighted on pharmacological comparison of mouse and rat homologs of the cloned 5-HT2B receptor. Thus the eventual classification of the orphan endothelial 5-HT receptor awaits robust interspecies comparison of 5-HT2A, 5-HT2B, and 5-HT2C receptors. In this regard, in rat jugular vein, the endothelial receptor, which was previously classified as 5-HT2C-like, has now been shown to be more like 5-HT2B receptors, using ligands which discriminate the rodent (rat) 5-HT2 receptor subtypes. Another therapeutic area that has gained prominence recently arose from observations in humans that mCPP reduces total sleep time, sleep efficiency, slow-wave sleep (SWS), and rapid eye movement sleep (REMS). The actions of 5-HT2A/2C receptor antagonists on sleep architecture have since been widely studied. Thus ritanserin has been reported to increase SWS, reduce sleep onset latency, and improve subjective sleep quality in volunteers, while REMS is reduced in some, but not all, studies. Ritanserin has also been observed to be of therapeutic use in insomniacs and in patients suffering from dysthymia. These effects were maintained with chronic treatment. Other nonselective 5-HT2A/5-HT2C receptor antagonists such as mianserin, cyproheptadine, and pizotifen have similar effects. In rats, ritanserin generally increases SWS and reduces REMS, although some studies report that the deepest phase of SWS (SWS2) is increased but total SWS is unaffected. Studies with three other 5-HT2A/5-HT2C receptor antagonists, ICI 169369 and ICI 170809 and SR46349B, report reduced REMS, but little effect on undifferentiated SWS. These results, therefore, suggest that a 5-HT2C receptor antagonist may improve sleep quality (see also agomelatine). Of great interest to pharmaceutical companies is the role of the 5-HT2C receptor in anxiety and depression. Two 5-HT2C receptor inverse agonists, SB243213 and WAY163909, have been reported to be in development for the treatment of depression. The fact that both compounds show a similar pharmacology in several animal models of depression reflects the ability of the 5-HT2C receptor agonists and antagonists/inverse agonists to desensitize the 5-HT2C receptor, as observed following chronic SSRI treatment. Another drug that has recently gained European regulatory approval is agomelatine, which is a melatonergic, MT1 and MT2 receptor agonist/5-HT2C antagonist with
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antidepressant properties and which may be acting through a similar mechanism. It is reported to improve disrupted sleep patterns in depressed patients (which also is likely, due to its 5-HT2C receptor antagonist properties), without affecting daytime vigilance. The reported lack of effect on sexual function, tolerability problems, or discontinuation symptoms offers several advantages over current therapy for the treatment of depression. It is clearly evident that therapeutic opportunities are still to be realized in the 5-HT2 receptor family and will undoubtedly follow with clinical evaluation of selective, nonselective, and combination agents. The Ligand-Gated 5-HT3 Receptor
The original naming of the ‘M’ receptor by Gaddum and Picarelli was based on the ability of morphine to antagonize the serotonin receptor which mediates depolarization of cholinergic nerves in the guinea pig ileum. Morphine was the first pharmacological tool to help characterize this receptor, now referred to as the 5-HT3 receptor. Since the time of these early observations, 5-HT3 receptors have been reported to be present on postganglionic autonomic neurons in the peripheral sympathetic and parasympathetic nervous systems, on enteric neurons, and on sensory neurons in various tissues. The 5-HT3 receptor is a member of the Cys-Cys loop ligand-gated ion channel superfamily and as such is composed of multiple subunits, containing 478 amino acids. Five human genes have been identified on chromosome HTR3/11, which encodes the 5-HT3 subunit (5-HT3A–E) (Table 3). To date, conflicting reports as to the existence of central 5-HT3B subunits have been reported. However, immunohistochemical studies using sections of human temporal lobe demonstrated both 5-HT3A and 5-HT3B immunoreactivity associated with pyramidal neurons within all CA fields of the hippocampus, as well as with large neurons within the hilus. The expression of both h5-HT3A and h5-HT3B subunit mRNAs in human hippocampus would therefore support the presence of both homomeric h5-HT3A and heteromeric h-5-HT3A/3B receptors in human hippocampus. The pathophysiological relevance of central h5-HT3A and h5-HT3B receptors remains to be fully evaluated with selective receptor antagonists in preclinical and clinical studies. The pioneering animal studies in the 1980s with the nonselective antagonist ondansetron clearly point to a role for 5-HT3 receptors in anxiety, depression, and schizophrenia. However, this was not substantiated in human studies with several 5-HT3 receptor antagonists, including, ondansetron, granisetron, BRL46470, and dolasetron.
5-HT is found in both the brain and the gut, but it is now widely understood that 95% of the serotonin in the body resides in the gut. The two leading 5-HT3 receptor antagonists, ondansetron and granisetron, have proved to be highly successful antiemetic agents, and they are widely used in the prophylactic treatment of chemotherapy-induced nausea and vomiting. This discovery arose from carefully reasoned experimental research. It was shown in animal studies that chemotherapeutic agents such as cisplatin released 5-HT in the gut in vast quantities, activating 5-HT3 receptors in this region. Activation of 5-HT3 receptors resulted in the firing of a vagally mediated response resulting in emesis. The discovery of the antiemetic properties of these 5-HT3 receptor antagonists, along with several others that followed, opened the way for this class of compounds to become the gold standard in the clinical treatment of chemotherapy-induced nausea and emesis. A second non-CNS therapeutic target that has gained prominence in recent years has been irritable bowel syndrome (IBS) and its association with the 5-HT3 receptor and 5-HT4 receptor. 5-HT3 receptor antagonists were predicted to be effective in diarrheapredominant IBS based on animal studies. Clinical studies with ondansetron and granisetron showed a reversal in rectal hypersensitivity in IBS, and the longer acting agent alosetron showed significant clinical improvement in IBS symptoms. However, alosetron has had a checkered clinical existence following early withdrawal after reports of rare serious gastrointestinal adverse events among patients taking the drug. Similar drugs (i.e., cilansetron, ramosetron, and DDP-733) are also in clinical studies for IBS, with the hope that these side effects are not class related. The latter compound is a partial agonist that has recently been reported to be active in a phase II IBS study. The complex symptomatology in IBS may be attributed to activation of other 5-HT receptor subtypes in the gastrointestinal tract. It was the identification of 5-HT4 receptors that led many pharmaceutical companies to believe that agents acting as 5-HT4 receptors would be useful in the treatment of this condition (see discussion of the 5-HT4 receptor and Table 4). The 5-HT4(Gs) Receptor
The 5-HT4 receptor was originally identified in primary cell cultures of mouse embryo colliculli, where later it was found to have a broad tissue distribution and to be positively coupled to adenylate cyclase. In early functional assays studies the 5-HT4 receptor was identified in the rat esophageal muscularis mucosae and guinea pig colon, where activation resulted in
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relaxation and contraction, respectively. The cloned human and rodent receptor was shown to consist of 388 and 406 amino acids, respectively. Various isoforms of the 5-HT4 receptor have now been identified (5-HT4b,c and 5-HT4d), encoded by a gene located on chromosome HTR4/5q31–q33 (Table 4). In the brain, 5-HT4 receptors appear to be located on neurons, whereas in the peripheral nervous system they are reported to facilitate the release of acetylcholine in smooth muscle of the intestinal tract. It was these observations on the gastrointestinal tract that led to the notion that 5-HT4 receptors may have a role in the pathophysiology of irritable bowel syndrome and gastroesophageal reflux disease (GERD). The complex symptomatology and pharmacology of IBS led to the development of several highly selective 5-HT4 receptor agonists and antagonists that are now established in clinical practice for this indication. Tegaserod (HTF-919) and purcalopride (R93877) are 5-HT4 receptor partial agonists, and piboserod (SB207266) is an antagonist. All of these medications are reported to relieve the symptoms of constipation, the notion being that the 5-HT3/4 agonists are intended to have prokinetic activity, like the mixed 5-HT4 receptor agonist/5-HT3 receptor agonist, cisapride. Such agents would therefore be beneficial for constipation-predominant IBS. Conversely, the antagonist piboserod would be indicated for diarrhea-predominant IBS. However, the therapeutic potential of piboserod was noted to be limited to a decrease in rectal sensitivity, and the compound was not advanced in further clinical studies. The disappointment with this class of drugs has been their lack of efficacy in IBS-related pain. However, other 5-HT3 agonists (pumosetrag) and 5HT4 agonists (TD-2749 and TD-5108) are also being evaluated in IBS, which may address this important, unmet medical need. The potential CNS efficacy of 5-HT4 receptor antagonists is less well developed, though the 5-HT4 partial agonist SL65.0155 (5-(8-amino-7-chloro2,3dihydro-1,4-benzodioxin-5yl)-3-[1-(2-phenylethyl)4-piperidinyl]1,3,4-oxadiazol-2(3H)-one-monohydro chloride) has been reported to show cognitive enhancing effects in animal models. The high density of 5-HT4 receptors in the nucleus accumbens has suggested that these receptors may be involved in reward mechanisms and may influence self-administration. Recent studies with GR113808 and its effects on reducing alcohol intake in rats would concur with this suggestion. The clinical benefit of selective agents for the 5-HT4 receptors in medical practice and the promise of more developments clearly represent one of many success stories of 5-HT drug development.
The 5-ht5 Receptor (G-Protein Coupling, None Identified)
The 5-HT5 receptor is highly localized in the rodent CNS; its distribution in the hippocampus (CA1, CA3, dentate gyrus), cortex, cerebellum (granular layer), olfactory bulb, habenula, and spinal cord would suggest a role in the pathophysiology of psychiatric and neurological disorders. A human 5-HT5A, but not a 5-ht5B, receptor has been identified; the human 5-HT5A receptor gene, like the rodent gene, contains two codon exons separated by a single large intron. In the case of the human and rodent 5-HT5A receptor, the gene contains the same number of amino acids (357); similarly, the rodent 5ht5b gene contains 387. To elucidate the pathophysiology of the 5-HT5 gene, chromosomal localization studies have identified the receptor on HTR5A/7q36, whereas the 5ht5b gene is localized on htr5b/2q11–q13. The signal transduction mechanism for the 5-HT5 receptor is still a matter for conjecture, as some suggest it may utilize a novel (perhaps ion channel) second-messenger system (Table 4). The physiological function of the 5-HT5A receptor is poorly understood. 5-HT5A null mutant mouse studies suggest a role in exploratory behavior, but the lack of selective pharmacological tools has hampered progress. Localization studies have revealed that 5-HT5A receptors have widespread distribution in the CNS. It has been speculated that, on the basis of their localization, the receptors may be involved in motor control, learning and memory consolidation, and anxiety and depression. Recently a selective 5-HT5A receptor antagonist, SB699551A, has been identified. This compound has been reported to attenuate 5-carboxyamidotryptamine-induced inhibition of raphe neuronal cell firing in vitro and to increase 5-HT levels in prefrontal cortex in vivo, suggesting a role for the 5-HT5A receptor in the modulation of raphe 5-HT neuronal activity. This is another pharmacological opportunity whereby ‘tool’ compounds will expose the receptor subtype operational and functional characteristics in native tissue, as it has been suggested that many of the operational characteristics of the 5-HT1D receptor may be attributed to the 5-HT5A receptor. The 5-HT6(Gs) Receptor
5-HT6 receptors have been identified in areas of the rat and human brain associated with learning and memory: hippocampus, CA1, CA3, dentate gyrus, olfactory tubercles, cerebral cortex, nucleus accumbens, and striatum. The 5-HT6 gene is localized on chromosome HTR6/1p35–p36, and studies to
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determine its linkage to several CNS disorders (memory dysfunction and schizophrenia) are actively being pursued (Table 4). However, the potential of 5-HT6 receptor antagonists in the treatment of cognitive disorders has been an actively researched topic in recent years, and the availability of numerous tool compounds for this receptor, some of which are currently in clinical trial, will advance our understanding of the role of this receptor in cognitive process. The notion that several atypical antipsychotic agents such as clozapine, quetiapine, and olanzapine possess high affinity for the 5-HT6 receptor adds translational creditability to this approach. The leading pharmacophores are indole-related compounds, of which several have emerged, including the recently described aryl sulfonamide indole chemotype, SB271046 (pKi 8.6), with excellent CNS bioavailability and in vivo efficacy, compared to several earlier compounds. More recently, 5-HT6 receptors have been demonstrated to regulate central cholinergic neurotransmission. In this regard, administration of the 5-HT6 receptor-selective antagonist Ro 04–6790 reversed scopolamine-induced rotation in 6-hydroxydopaminelesioned rats. Additionally, 5-HT6 receptor antisense oligonucleotides or 5-HT6 receptor-selective inhibitors enhanced retention by rats of the learned platform position in the Morris water maze. These data suggest that 5-HT6 receptor antagonists might boost cholinergic neurotransmission and reduce the cognitive impairments experienced by patients with dementia or schizophrenia. Intriguingly, it has recently been determined that the 267C allele of the 5-HT6 receptor is a significant risk factor for Alzheimer’s disease. Taken together, these findings indicate that 5-HT6 antagonists might prove useful in treating a number of common illnesses, including dementia and schizophrenia. A major concern in drug discovery is that the cloned mouse receptor is significantly different in rat and human 5-HT6 receptors. In addition to species differences in the binding of drugs to 5-HT6 receptors, differences in the regional expression of 5-HT6 receptors are also apparent. Furthermore, quantitative polymerase chain reaction studies have demonstrated that the mouse 5-HT6 receptor mRNA is at least tenfold less abundant than are the rat and human 5-HT6 receptor mRNAs, in every brain region examined. Surprisingly, whereas 5-HT6 receptor mRNA and radioligand binding activity is enriched in the basal ganglia of rat and human brain, there is no such enrichment in the mouse brain. Finally, using a combination of site-directed mutagenesis and molecular modeling studies, it has been
shown that the peculiar mouse 5-HT6 pharmacology is attributable to two amino acids – Tyr188 (in helix 5, which is Phe188 in rats and humans) and Ser290 (in helix 6, which is Asn290 in rats and humans) – and these account for the bulk of the differences in pharmacology. These findings are a cautionary note but may have important implications for drug discovery. The 5-HT7(Gs) Receptor
Since the identification of the 5-HT7 receptor in the early 1990s, evidence has accumulated supporting a role for 5-HT7 receptors in various physiological functions, including sleep disorders, circadian rhythms, cognition, mood, and sleep (see Table 5). A number of splice variants of the 5-HT7 receptor have been identified and exhibit overlapping mRNA distributions, and all couple to Gs, though no differences in operational characteristics have been shown, which may have led to differentiation of behavioral phenotypes. Work is still ongoing to elucidate the pathophysiology of these splice variants, and chromosomal localization studies have identified the receptor on HTR5A/7q36, encoding 445 and 488 amino acids for the human and rodent receptor, respectively (Table 5). The finding that several drugs used in clinical psychiatric practice had high affinity for 5-HT7 receptor binding sites in the brain initiated an ongoing interest in evaluating the involvement of neuropsychiatric disorders. The distribution of 5-HT7 receptors in areas of the brain associated with neuropsychiatric disorders – hippocampus (CA1, CA2), hypothalamus, thalamus, raphe nuclei, and superior colliculus – supported this notion. In concordance with its distribution in the hippocampus, the selective 5-HT7 receptor antagonist SB269700-Z was shown to attenuate phencyclidine (PCP)-induced cognition dysfunction associated with schizophrenia. The strongest case has been made for the importance of the 5-HT7 receptor in depression. Inactivation or blockade of the receptor leads to an ‘antidepressant’ state in behavioral models of depression. Furthermore, 5-HT7 receptor downregulation following chronic administration of antidepressants in animal studies points to a possible molecular mechanism of action. This hypothesis is supported by recent animal experiments using 5-HT7 receptor knockout mice that clearly show an antidepressantlike profile in the rat forced swim test and mouse tail suspension test. Selective antagonist tool compounds SB258717, SB269970, and SB656104-A have all been shown to exhibit antidepressant-like properties in animal studies and to modulate REM sleep in rats, results that concur with this line of investigation. Several lines
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of evidence have shown that 5-HT-induced hypothermia is mediated by the 5-HT7 receptor, as defined with selective antagonists and in 5-HT7 knockout mice studies. What is interesting, however, is recent work with the endogenous fatty acid oleamide that suggests it may act through an independent mechanism as well as at an allosteric 5-HT7 receptor site to regulate body temperature and perhaps circadian rhythms. Clinical progress in this area has been limited by suboptimal clinical candidates, and clinical efficacy data for selective agents are still awaited.
The Future of 5-HT Research It is now over 50 years since Gaddum first suggested ‘‘that a drug with a specific antagonistic action to 5-HT – might be used in therapeutics.’’ Clearly, drugs acting at the 5-HT receptor subtypes have revolutionized therapeutics during the intervening years. However, it is only now that selective agents are appearing to differentiate many operational and physiological characteristics of 5-HT actions on the various receptor subtypes currently identified. The future therapeutic potential of drugs acting at these 5-HT subtypes and isoforms or in combination with other neurotransmitter receptors will open up next generation of novel serotonergic therapeutic agents. See also: Serotonin (5-Hydroxytryptamine; 5-HT): CNS Pathways and Neurophysiology; Serotonin (5-Hydroxytryptamine; 5-HT): Neurotransmission and Neuromodulation.
Further Reading Alexander SPH, Mathie A, and Peters JA (2008) Guide to receptors and channels. British Journal of Pharmacology 153 http://www. nature.com/bjp/journal/vgrac/ncurrent/index.html (accessed Jul. 2008). Barnes NM and Sharpe T (1999) A review of central 5-HT receptors and their function. Neuropharmacology 38: 1083–1152. Bonasera SJ and Tecott LH (2000) Mouse models of serotonin receptor function: Toward a genetic dissection of serotonin systems. Pharmacology & Therapeutics 88: 133–142. Branchek TA and Blackburn TP (2000) 5-HT6 receptors as emerging targets for drug discovery. Annual Review of Pharmacology and Toxicology 40: 319–334.
Brockaert J, Claeysen S, Compan V, et al. (2004) 5-HT4 receptors. Current Drug Targets – CNS & Neurological Disorders 3: 39–51. Glennon RA (2003) Higher-end serotonin receptors: 5-HT5, 5-HT6, and 5-HT7. Journal of Medical Chemistry 46: 2795–2812. Hartig PR, Hoyer D, Humphrey PP, et al. (1996) Alignment of receptor nomenclature with the human genome: Classification of 5-HT1B and 5-HT1D receptor subtypes. Trends in Pharmacological Sciences 17: 103–105. Hoyer D, Clarke DE, Fozard JR, et al. (1994) International Union of Pharmacology classification of receptors for 5-hydroxytryptamine (serotonin). Pharmacological Reviews 46: 157–203. Hoyer D and Martin G (1997) 5-HT receptor classification and nomenclature: Towards a harmonization with the human genome. Neuropharmacology 36: 419–428. Lanfumey L and Hamon M (2004) 5-HT1 receptors. Current Drug Targets – CNS & Neurological Disorders 3: 1–10. Jones JJ and Blackburn TP (2002) The medical benefit of 5-HT. Pharmacology, Biochemistry and Behavior 71: 555–568. Leysen JE (2004) 5-HT2 receptors. Current Drug Targets – CNS & Neurological Disorders 3: 11–26. Lui M and Gershon MD (2005) Slow excitatory (‘‘5-HT1P’’-like) responses of mouse myenteric neurons to 5-HT: Mediation by heterodimers of 5-HT1B/1D and Drd2 receptors. Gastroenterology 128(4, supplement 2): A87. McLean PG, Borman RA, and Kee K (2007) 5-HT in the enteric nervous system: Gut function and neuropharmacology. Trends in Neurosciences 1: 9–13. Nelson DL (2004) 5-HT5 receptors. Current Drug Targets – CNS & Neurological Disorders 3: 53–58. Pauwels PL (2000) Diverse signalling by 5-hydroxytryptamine (5-HT) receptors. Biochemical Pharmacology 60: 1743–1750. Peters JA, Hales TG, and Lambert JJ (2005) Molecular determinants of single channel conductance and ion selectivity in the Cys-loop transmitter-gated ion channels: Insights from the 5-HT3 receptor. Trends Pharmacological Sciences 26: 587–594. Sanders-Bush E, Fentress H, and Hazelwood L (2003) Serotonin 5-HT2 receptors: Molecular and genomic diversity. Molecular Interventions 3: 319–330. Steward LJ, Ge J, Bentley KR, et al. (2007) Guide to receptors and channels (GRAC), 2nd edition (2007 revision). British Journal of Pharmacology 150(supplement 1): S1–S168. Thomas DR and Hagen JJ (2004) 5-HT7 receptors. Current Drug Targets – CNS & Neurological Disorders 3: 81–90. Woolley ML, Marsden CA, and Fone KC (2004) 5-ht6 receptors. Current Drug Targets – CNS & Neurological Disorders 3: 59–79.
Relevant Website http://www.iuphar-db.org – IUPHAR Receptor Database.
Serotonin (5-Hydroxytryptamine; 5-HT): CNS Pathways and Neurophysiology G Aghajanian and R-J Liu, Yale School of Medicine, New Haven, CT, USA ã 2009 Elsevier Ltd. All rights reserved.
Introduction Serotonin (5-hydroxytryptamine; 5-HT), a biogenic indoleamine derived metabolically from the dietary amino acid tryptophan, was first discovered in peripheral tissues and subsequently found in brain. Shortly afterward, in the early 1950s, a structural relationship was noted between serotonin and d-lysergic acid diethylamide (LSD), the most potent of all known hallucinogenic drugs. Subsequently, it was recognized that a wide range of psychedelic hallucinogens, both indoleamines such as N,N-dimethyltryptame (DMT) and phenethyamines such as mescaline, had structural similarities to LSD (Figure 1). This relationship to powerful psychotropic drugs sparked interest in the idea that serotonin might function as a central ‘neurohumoral’ substance with a profound influence on behavior. However, it was not obvious how to investigate this idea directly until the mid-1960s, when, through the use of a new histochemical method (the Falck– Hillarp method), it was discovered that serotonin was highly concentrated in specific sets of neurons located almost exclusively in the raphe nuclei of the brain stem. Histochemical maps showed that serotonergic projections emanating from the raphe nuclei innervate, to varying degrees, virtually all other parts of the central nervous system, ranging from the cerebral cortex to the spinal cord. Based on these maps, it became possible to study the properties of identified serotonergic neurons and their postsynaptic targets. Although it was its link to hallucinogenic drugs that first brought it to prominence, serotonin has now been implicated in almost every conceivable physiological or behavioral function – affect, aggression, appetite, cognition, emesis, endocrine function, gastrointestinal function, motor function, neurotrophism, perception, sensory function, sex, sleep, respiration, and vascular function. Moreover, many drugs that are currently used for the treatment of psychiatric disorders (e.g., depression, mania, schizophrenia, autism, obsessive–compulsive disorder, anxiety disorders) are thought to act, at least partially, through serotonergic mechanisms. How is it possible for serotonin to be involved in so many different processes? One answer lies in the anatomy of the serotonergic system, where small
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clusters of serotoninergic cell bodies located in the brain stem raphe nuclei are positioned through their vast projections to influence all regions of the neuraxis. Another answer lies in the molecular diversity and differential cellular distribution of the many serotonin receptor subtypes that are expressed differentially in brain and other tissues. For example, the effects of hallucinogenics are mediated via the 5-HT2A subtype of serotonin receptor, which is expressed extensively in the cerebral cortex. In contrast, antidepressant effects are thought to involve members of the 5-HT1 family of receptors located in the brain stem raphe as well as in certain postsynaptic regions. Given this diversity, it is useful to approach serotonin neurophysiology within the context of the neuroanatomical and cellular distribution of serotonin receptor subtypes and their associated transduction pathways.
Serotonergic Neuronal Pathways Serotonergic neurons are clustered in a series of midline nuclei, labeled B1–B9, extending from the midbrain to the medulla oblongata. The dorsal and median raphe nuclei (B7 and B8) comprise the largest of these groupings of brain serotonergic neurons, and together they provide the major serotonergic input to the forebrain; the lower brain stem raphe nuclei supply the main serotonergic projections to the spinal cord. A single serotonergic neuron projects to large numbers of postsynaptic cells and does so in an overlapping fashion with other serotonergic neurons. While the density of this input varies widely, both regionally and between different cell types within a given region, virtually no corner of the CNS is lacking a serotonergic input despite the fact that serotonergic neurons represent far less than 1% of all the neurons in the brain. Given these anatomical considerations, the serotonergic system is well positioned to provide a broad modulatory influence upon diverse functions rather than the precise point-to-point transmission characteristic of traditional sensory, motor, or associational pathways. In view of its widespread projections arising from a relative handful of neurons, is it possible to assign an overarching function to the serotonergic system? An answer to this question may come from the fact that serotonergic neurons display a pattern of firing that is dependent upon behavioral state: they fire most rapidly during alert waking, decelerate considerably during slow-wave sleep, and become almost entirely silent during dream – or rapid-eye-movement
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H5C2 NH2
H5C2
O NC
N CH3
HO N H
N H
5-HT (5-hydroxytryptamine) H3C N
LSD
CH3
N H N,N-dimethyltryptamine
NH2
H3CO
OCH3 OCH3 Mescaline
Figure 1 The structural formulas of serotonin and three related hallucinogenic drugs: LSD, DMT, and mescaline. Note that serotonin, LSD, and DMT have in common an indolethylamine moiety. While it is not an indolethylamine, mescaline shares with LSD a phenethylamine moiety. The structures are drawn so as to emphasize these shared structural features. Provided by GK Aghajanian.
(REM) – sleep. Thus, in a broad sense, one can view serotonergic neurons as constituting a ‘wake-on’ system. In this context, many of the electrophysiological effects of serotonin in postsynaptic regions can be seen as facilitating motor, sensory, and cognitive functions characteristic of the waking state. All of this would be reversed when serotonergic neurons become quiescent during sleep states, when there is an emergence of sleep- and dream-related systems. Such a broad, state-related function for serotonin has been likened to the peripheral sympathetic system where small clusters of noradrenergic neurons project to a myriad of organs including the heart, gut, and skin to influence their function in a concerted manner. Interestingly, a central counterpart to the sympathetic system exists in the brain stem where small clusters of noradrenergic neurons (e.g., in the locus coeruleus) parallel the broad projections of the serotonergic system and similarly have a broad influence on a wide range of wake-related functions.
Regulation of Serotonergic Neuronal Activity Ultimately, the electrophysiological effects of serotonin depend upon the activity of the serotonergic cells of origin in the raphe nuclei. In various mammalian species, serotonergic neurons in waking states have been found to have a slow, tonic pattern of firing
Figure 2 Schematic diagram showing norepinephrine (NE), hypocretin/orexin (hcrt/orexin), and g-aminobutyric acid (GABA) inputs to a 5-HT cell in the brain stem raphe. The NE input originates from nearby sites in the brain stem while the hcrt/orexin input is hypothalamic in origin; both of these are excitatory (þ). Also depicted is a 5-HT collateral input to the 5-HT cell; this input is inhibitory () via somatodendritic 5-HT1A autoreceptors. Finally, an inhibitory input () from a local GABA interneuron is shown; the GABA cell itself receives 5-HT collaterals, which can be excitatory (via 5-HT2A receptors) or inhibitory (via 5-HT1A) receptors. Provided by GK Aghajanian.
(1–4 spikes s1). During slow-wave sleep, there is a deceleration of firing rate, and during REM (or dream) sleep, nearly a total cessation of activity. Thus, serotonergic neurons can be seen as being ‘wake-on’ cells. The maintenance of rhythmic firing during the waking state has suggested that serotonergic neurons possess regulated tonic pacemaker mechanisms. Intracellular and whole cell recordings from dorsal raphe neurons show that spikes arise from gradual depolarizing ramps (pacemaker potentials) rather than synaptic potentials. The regular pacemaker rhythm of serotonergic neurons is shaped by a complex interplay of intrinsic ionic currents; these include a voltage-dependent transient outward potassium current, a low-threshold inward calcium current, and a large calcium-activated outward potassium current. However, the drive behind the pacemaker activity mainly comes from extrinsic excitatory inputs, including brain stem noradrenergic inputs and hypothalamic hypocretin (orexin) inputs (Figure 2). The hypocretin system is essential for maintaining the normal waking state and loss of this system leads to the condition of narcolepsy, characterized by excessive somnolence. Both norepinephrine (via a1-adrenoceptors) and the peptide hypocretin/orexin
472 Serotonin (5-Hydroxytryptamine; 5-HT): CNS Pathways and Neurophysiology
Figure 3 Activation of pacemaker activity in a serotonergic neuron by norepinephrine (NE) or hypocretin in brain slice. Whole cell recording from a serotonergic neuron located in the dorsal raphe nucleus of a midbrain slice. As is typical of serotonergic neurons in brain slice where excitatory afferents are disconnected, this cell shows no basal spiking activity. When NE (top traces) or hypocretin/orexin (hcrt; bottom traces) is applied, there is a slow depolarization leading to tonic pacemaker activity. The expanded traces to the right show the gradual depolarizing ramps leading up to the onset of spiking activity. NE and hcrt/orexin can be seen as restoring the tonic pacemaker activity that is typical of serotonergic neurons in vivo. Provided by R-J Liu and GK Aghajanian.
(via hypocretin receptors 1 and 2), by closing potassium channels and opening nonselective cation channels, accelerate pacemaker activity of serotonergic neurons by inducing a slow depolarization (Figure 3). In addition to neurotransmitter inputs, elevated levels of CO2 also have a strong activating effect on serotonergic as well as noradrenergic neurons. This would provide an alerting signal in response to states of respiratory insufficiency. Serotonergic pacemaker activity is negatively modulated by the autoinhibitory action of serotonin, acting via somatodendritic 5-HT1A autoreceptors. The maintenance of tonic 5-HT1A autoinhibition depends upon the rate of serotonins synthesis, which in turn depends on availability of the initial serotonin precursor L-tryptophan. The activity of tryptophan hydroxylase, the limiting step in serotonin synthesis, is highly regulated by the tryptophan hydroxylase-activating kinases calcium/calmodulin protein kinase II (CaMKII) and protein kinase A (PKA). Increased calcium influx at higher firing rates, by activating tryptophan hydroxylase via CaMKII and PKA, can work together with tryptophan to enhance negative feedback control of the output of the serotonergic system.
Electrophysiology of Serotonin Receptor Subtypes The electrophysiological actions of serotonin are a function of the receptor subtype(s) expressed by a given cell. The Gi/Go-coupled 5-HT1 receptors generally mediate inhibitory effects on neuronal firing through an opening of potassium channels or a closing
of calcium channels. Inhibitions mediated by 5-HT1A and other 5-HT1 receptor subtypes have been observed in postsynaptic neurons located in many regions of the central nervous system. Presynaptic 5-HT1A receptors, located at the somatodendritic region of serotonergic neurons, have been termed ‘autoreceptors,’ mediating negative feedback inhibition via the cell’s own transmitter. The 5-HT2A receptors mediate slow excitatory effects through a decrease in potassium conductance or an increase in nonselective cation conductance. These are merely a few examples of how serotonin, with its widespread projections and large array of receptor subtypes/transduction pathways, is able to modulate neuronal excitability in a complex fashion throughout the neuraxis. While the electrophysiological actions of serotonin may seem quite varied, there is considerable uniformity within each of the major receptor families. Because of their common G-protein coupling, all members of the 5-HT1 receptor family tend to have inhibitory actions either pre- or postsynaptically. Similarly, all members of the 5-HT2 family tend to have excitatory actions. Therefore, the discussion of serotonin electrophysiology in selected regions of brain will be organized according to receptor families and their transduction pathways (Figure 4). 5-HT1 Receptors
Members of the 5-HT1 receptor family are coupled via Gi/Go G proteins and produce their effects through opening of inwardly rectifying Kþ channels or closing of Ca2þ channels. Particularly high levels of 5-HT1A receptors are found in certain regions,
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The somatodendritic autoreceptors of serotonergic neurons in both the dorsal raphe and other raphe nuclei appear to be predominantly of the 5-HT1A subtype, as a variety of drugs with 5-HT1A selectivity (e.g., 8-OH-DPAT) share the ability to potently inhibit raphe cell firing in a dose-dependent manner. Highly selective 5-HT1A antagonists (e.g., WAY 100635) have been found which potently block the direct inhibition of dorsal raphe serotonergic neurons both by serotonin and selective 5-HT1A agonists. As described above, the somatodendritic 5-HT1A autoreceptor is importantly involved in the negative feedback regulation of sertonergic cell activity through the release of endogenous serotonin.
Figure 4 The four major groupings of serotonin receptor families and their transduction pathways. Three of the four groups are coupled through G-proteins: 5-HT1-Gi/Go, 5-HT2-Gq, and 5-HT4,6,7-Gs. The remaining receptor is 5-HT3, which is a ligand-gated channel and is not coupled through a G protein. cAMP, adenosine 30 ,50 -cyclic monophosphate; DAG, diacylglycerol; PKC, protein kinase C; sAHP, slow after hyperpolarization; PLC, phospholipase C. Provided by GK Aghajanian.
including the dorsal raphe nucleus, hippocampus, lateral septum, and certain layers of the cerebral cortex. Studies in these regions and other regions have been useful in delineating the physiological role of this receptor. Raphe nuclei Serotonergic neurons of the raphe nuclei are inhibited by the local (microiontophoretic) application of serotonin to their cell body region. Thus, the receptor mediating this effect has been termed a somatodendritic autoreceptor (as opposed to the prejunctional autoreceptor). Functionally, the somatodendritic serotonin autoreceptor has been shown to mediate collateral inhibition. The ionic basis for the autoreceptor-mediated inhibition, either by serotonin or LSD, is an opening of Kþ channels to produce a hyperpolarization; these channels are characterized by their inwardly rectifying properties. Patch clamp recordings in the cell-attached and outside-out configuration from such acutely isolated dorsal raphe neurons show that the increase in Kþ current results from a greater probability of opening of unitary Kþ channel activity.
Other subcortical regions Inhibitory or hyperpolarizing responses to serotonin have been reported in a wide variety of neurons in the spinal cord, brain stem, and diencephalon. In general, such responses have been attributed to mediation by 5-HT1 receptors. In the nucleus prepositus hypoglossi, focal electrical stimulation evokes inhibitory postsynaptic potentials (IPSPs) that are mediated by 5-HT1A receptors to activate an inwardly rectifying Kþ conductance and a novel outwardly rectifying Kþ conductance. In the midbrain periaqueductal gray, a region known to be involved in pain modulation and fear responses, approximately half the cells are inhibited/hyperpolarized by 8-OH-DPAT, suggesting mediation by 5-HT1A receptors. In the ventromedial hypothalamus and lateral septum, serotonin and 5-HT1A agonists produce inhibitory effects also by activating a potassium conductance. In addition to these postsynaptic effects, serotonin has been shown to suppress glutamatergic synaptic transmission via presynaptic 5-HT1B receptors in various regions including the hypoglossal nucleus and the nucleus accumbens. In the rat laterodorsal tegmental nucleus (LDT), bursting cholinergic neurons are hyperpolarized by serotonin via 5-HT1 receptors. In freely behaving rats, the direct injecton of serotonin into the LDT was found to suppress REM sleep. In unanesthetized cats, a corresponding population of neurons that are active selectively during REM states (REM-on neurons) in the LDT are inhibited by direct application of the 5-HT1A agonist 8-OH-DPAT. It has been proposed that during REM sleep, the removal of a tonic inhibitory serotonin influence from these cholinergic neurons may be responsible for the emergence of an activated EEG during this behavioral state (see later). Hippocampus Pyramidal cells of the CA1 region express high levels of 5-HT1A receptor mRNA and 5-HT1A receptor binding. Serotonin-induced inhibition in both CA1 and CA3 pyramidal cells is mediated
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by the activation of receptors of the 5-HT1A subtype coupled to an opening of Kþ channels. In addition to the above direct effects on pyramidal cells, serotonin has been shown to depress both excitatory and inhibitory synaptic potentials in the hippocampus. Relatively high concentrations of serotonin cause a reduction in electrically evoked excitatory postsynaptic potentials (EPSPs) in CA1 pyramidal cells, an effect that is mimicked by 8-OH-DPAT, suggesting mediation by 5-HT1A receptors. Indirect measures indicate that serotonin acts presynaptically to reduce Ca2þ entry and thereby gluamatergic synaptic transmission. In addition, there is a 5-HT1A-mediated inhibitory effect on putative inhibitory interneurons of the hippocampus. Functionally, the 5-HT1A-mediated inhibition of GABAergic interneurons in the hippocampus leads to a disinhibition of pyramidal cells in CA1. Clearly, the effects of serotonin in the hippocampus are highly complex, involving both pre- and postsynaptic actions which may, to varying degrees, be inhibitory or disinhibitory, facilitatory or disfacilitatory. Cerebral cortex 5-HT1A-induced hyperpolarizing/ inhibitory responses in pyramidal cells of the cerebral cortex have been described in a number of studies. In entorhinal cortex, where there is an especially high density of 5-HT1A receptors (and a low density of 5-HT2A receptors), unopposed 5-HT1A receptormediated hyperpolarizing responses are seen. However, cortical neurons in most other regions typically display mixed inhibitory and excitatory responses to serotonin due to the expression by the same pyramidal cells of multiple serotonin receptor subtypes (e.g., 5-HT1A, and 5-HT2/2C). Hyperpolarizing responses mediated by 5-HT1A receptors are often unmasked or enhanced in the presence of 5-HT2 antagonists, consistent with the idea that there is an interaction between 5-HT1A and 5-HT2A receptors at an individual neuronal level. In addition to these postsynaptic effects, there are various presynaptic effects mediated by 5-HT1 receptors in the cerebral cortex. In cingulate cortex, serotonin, acting upon presynaptic 5-HT1B receptors, reduces the amplitude of electrically evoked EPSPs, including both N-methyl-D-aspartate (NMDA) and non-NMDA components. Similar modulations of EPSPs, mediated by 5-HT1A or 5-HT1B receptors, have been reported for several cortical regions including medial prefrontal and entorhinal cortex. 5-HT2 Receptors
Members of the 5-HT2 family of receptors are coupled through Gq-type G proteins and have excitatory effects through closing of Kþ channels or opening
nonselective cation channels. High concentrations of 5-HT2 are expressed in certain regions of the forebrain such as the neocortex, piriform cortex, claustrum, and olfactory tubercle. With few notable exceptions (e.g., motor nuclei and the nucleus tractus solitarius), relatively low concentrations of 5-HT2 receptors or mRNA expression are found in the brain stem and spinal cord. Studies aimed at examining the physiological role of 5-HT2 receptors in several of these regions are described in the following sections. Motoneurons In spinal cord and brain stem motor nuclei, motoneurons have a high density of 5-HT2 receptor binding sites. Early studies in vivo showed that serotonin applied microiontophoretically does not by itself induce firing in the normally quiescent facial motoneurons but does facilitate the subthreshold and threshold excitatory effects of glutamate. Intracellular recordings from facial motoneurons in vivo or in brain slices in vitro show that serotonin induces a slow, subthreshold depolarization associated with an increase in input resistance, indicating a decrease in a resting Kþ conductance. Selective 5-HT2 antagonists are able to selectively block the excitatory effects of serotonin in facial motoneurons. The facilitation of motoneuron excitability contributes to the role of serotonin as a ‘wake-on’ system to promote activity during the waking state (see later). Other subcortical regions In brain slices of the medial pontine reticular formation, serotonin induces depolarizing responses that have a 5-HT2 pharmacology and are associated with a decrease in membrane conductance resulting from a decrease in an outward Kþ current. In brain slices of the substantia nigra pars reticulata, a majority of neurons are excited by serotonin via 5-HT2 receptors, possibly of the 5-HT2C rather than 5-HT2A subtype. Neurons in the inferior olivary nucleus are excited by serotonin via 5-HT2A receptors, thereby altering the oscillatory frequency of input to cerebellar Purkinje cells. In the nucleus accumbens, the great majority of neurons are depolarized by serotonin, inducing them to fire. This depolarization is associated with an increase in input resistance due to a reduction in an inward rectifier Kþ conductance. In addition, GABAergic neurons within a number of regions (e.g., dorsal raphe nucleus, medial septal nucleus, hippocampus, cerebral cortex) are also excited by serotonin via 5-HT2 receptors, suggesting that in multiple locations within the CNS there are subpopulations of interneurons that are excited by serotonin via 5-HT2 receptors, giving rise to indirect inhibitory effects.
Serotonin (5-Hydroxytryptamine; 5-HT): CNS Pathways and Neurophysiology 475
Cerebral cortex The electrophysiological effects of serotonin have been studied in several corticalregions. In vitro studies in the brain slice preparation have shown that pyramidal cells in various cortical regions respond to serotonin by either a small hyperpolarization, depolarization, or no change in potential. Depending on the region of cortex under study, as described below, the depolarizations appear to be mediated by 5-HT2A or 5-HT2C receptors. In addition to these postsynaptic effects, serotonin induces an increase in ‘spontaneous’ (nonelectrically evoked) postsynaptic potentials or currents (PSPs/PSCs) recorded in brain slices from various cortical regions. These may originate from the activation of intracortical pathways or through interactions with subcortical inputs such as the thalamus. Activation of 5-HT2A receptors also enhances late components of evoked responses in cerebral cortex (Figure 5). These late responses (also termed UP states) are generated by sustained recurrent network activity. It has recently been shown that UP states rely on glutamate spillover onto extrasynaptic NMDA receptors. Interestingly, these UP states are characteristic of alert waking and are enhanced by psychedelic hallucinogens which are
known to be 5-HT2A/2C partial agonists. It has been proposed that the hallucinogenic drugs produce their characteristic aberrations in perception, cognition, and affect by driving cortical UP states beyond normal limits. It is significant that the facilitation of UP states by 5-HT2A receptors is opposed by 5-HT1A receptors which tend to suppress UP states. This damping effect of 5-HT1A receptors on UP states may explain why selective 5-HT2A agonists are hallucinogenic, whereas serotonin, the natural transmitter, is not. This example further illustrates how serotonin, rather than having a monolithic action, exercises numerous checks and balances at a cellular and systems level via its diverse receptor subtypes.
Figure 5 Enhancement of electrically evoked recurrent network activity (prolonged late excitatory postsynaptic current (EPSC) or UP state) in prefrontal brain slice by the hallucinogen LSD. Under basal conditions (top traces), note the fast EPSC, which occurs within a few milliseconds of a local electrical stimulus applied to the brain slice; following the fast EPSC, a low-amplitude late EPSC can be seen. After the application of a low concentration of LSD in the bath, the late EPSC or UP state is greatly enhanced both in amplitude and duration. Whole cell recording from a layer V pyramidal cell in rat prefrontal cortex. Provided by GK Aghajanian.
5-HT4, 5-HT6, 5-HT7
5-HT3 Receptors
Excitatory responses to serotonin have been found in various central neurons that have a rapid onset of action and rapid desensitization, features that are typical of ligand-gated ion channels rather than G-protein-coupled receptor responses. The cloned 5-HT3 receptor homologous with the nicotinic acetylcholine receptor and the b1 subunit of the GABAA receptor indicate that it is a member of the ligandgated ion channel superfamily. Typically members of this superfamily are comprised of multiple subunits; however, only one 5-HT3 receptor subunit and an alternatively spliced variant has been cloned to date. In hippocampal slices, serotonin has been reported to increase spontaneous GABAergic IPSPs, most likely through a 5-HT3 receptor-mediated excitation of inhibitory interneurons; these responses also show fading with time. A similar induction of 5-HT3 receptor-mediated induction of inhibitory postsynaptic currents (IPSCs) has been reported in the neocortex. While fast, rapidly inactivating excitation has generally become accepted as characteristic of 5-HT3 receptors, non-desensitizing responses have also been reported. In dorsal root ganglion cells, a relatively rapid but non-inactivating depolarizing response has been described that has a 5-HT3 pharmacological profile. In neurons of the nucleus tractus solitarius brain slices, there is a postsynaptic depolarizing response to 5-HT3 agonists that is not rapidly desensitizing.
There are three known Gs-coupled serotonin receptors: 5-HT4, 5-HT6, and 5-HT7, all mediating positive coupling of serotonin responses to adenylyl cyclase. The resulting increase in cyclic AMP can produce its electrophysiological effects through interacting directly with ion channels or through the activation of the PKA signal transduction pathway, which can interact
476 Serotonin (5-Hydroxytryptamine; 5-HT): CNS Pathways and Neurophysiology
with a myriad of ion channels, transporters, and other kinases. At this time, electrophysiological studies are available only for the 5-HT4 and 5-HT7 receptors, as described below. 5-HT4 receptors Binding studies using a selective 5-HT4 ligand indicate that 5-HT4 receptors are present in several discrete regions of the mammalian brain including the striatum, substantia nigra, olfactory tubercle, and hippocampus. As these regions also express 5-HT4 receptor mRNA, it appears likely that these receptors function postsynaptically to mediate certain actions of serotonin. The best studied of these regions is the hippocampus where both biochemical and electrophysiological studies have provided detailed picture of the actions of serotonin at 5-HT4 receptors. Electrophysiological studies show that 5-HT4 receptors mediate an inhibition of a calcium-activated potassium current, which is responsible for the generation of a slow afterhyperpolarization (AHP) in hippocampal pyramidal cells of the CA1 region. A suppression of the AHP would enhance the ability of these cells to respond to excitatory inputs with robust spike activity. 5-HT7 receptors The 5-HT7 receptor is positively coupled to adenylate cyclase and can mediate electrophysiological actions through the cyclic AMP pathway. One such effect is through a cyclic nucleotide enhancement of the hyperpolarizing-activated nonselective cationic current Ih. An increase in Ih tends to prevent excessive hyperpolarization and increase neuronal excitability. 5-HT7 receptors can also influence neuronal excitability through cyclic AMPactivated PKAs, leading to suppression of slow AHPs. This occurs in the midline/intralaminar nuclei of the thalamus where the highest expression of 5-HT7 receptors in the brain occurs. As the thalamocortical pathway projecting from the midline/ intralaminar nuclei is the final link in the ascending arousal pathway, this is one way that serotonin exerts its effects as a wake-on system to promote alertness and attention (see later).
Behavioral and Clinical Correlations The diversity of receptors and transduction pathways underlying the varied electrophysiological actions of serotonin, together with the differential expression of these receptors in different neuronal populations, helps explain how one transmitter can be involved in such a large array of behaviors, clinical conditions, and drug actions. Alterations in serotonin function have been linked to affective disorders, anxiety states,
schizophrenia, obsessive–compulsive disorder, eating disorders, migraine, and sleep disorders, including sleep apnea. Drugs that modify serotonergic transmission include antidepressants, atypical antipsychotics, antiemetics, hallucinogens, antimigraine drugs, and appetite suppressants. For example, the most commonly used antidepressant drugs act by blocking serotonin reuptake, which results in increased synaptic availability of this transmitter. Antagonism of 5HT2A receptors has been proposed as contributing to the favorable clinical profile of atypical antipsychotic drugs. Conversely, an agonist action of psychedelic hallucinogens at 5-HT2A receptors is thought to mediate the psychotomimetic effects of these drugs. Because of its ubiquitous distribution and diverse actions in the central nervous system, it is likely that future research will continue to uncover an involvement of serotonin in many pathological as well as normal behavioral processes. It has been hypothesized that activation of serotonin neurons works in concert with other components of the ascending arousal system projecting to the forebrain. For example, noradrenergic neurons, which also constitute a wake-on system, directly activate serotonergic neurons. A further linkage is seen by the fact that both noradrenergic and serotonergic neurons are activated by inputs from hcrt/orexin neurons, which are essential for normal wakefulness. A concerted action is also seen upstream, by the excitatory/facilitatory effects of both serotonin and hcrt upon the relay neurons of the midline/intralaminar nuclei which project to prefrontal cortex. This prefrontal input, which represents the final synapse in the ascending arousal system, is essential for awareness and attention. Within the cortex itself, 5-HT2A receptors enhance UP states, which are closely associated with the alert waking state. However, excessive stimulation of 5-HT2A receptors by hallucinogenic drugs can be pathological by leading to uncontrolled UP states. In conclusion, through its effects on neuronal excitability in diverse regions of the brain and spinal cord, the serotonergic system is positioned to coordinate complex sensory and motor patterns during different behavioral states. More specifically, since the activity of serotonergic neurons is greatest during periods of active waking, reduced during slow-wave sleep, and almost completely absent during REM (dream) sleep, the serotonin system can be seen as promoting neuronal activity in support of the waking state while concomitantly suppressing neuronal activity underlying sleep and dream states. See also: Serotonin (5-Hydroxytryptamine; 5-HT): Neurotransmission and Neuromodulation; Serotonin (5-Hydroxytryptamine; 5-HT): Receptors.
Serotonin (5-Hydroxytryptamine; 5-HT): CNS Pathways and Neurophysiology 477
Further Reading Aghajanian GK and Sanders-Bush E (2002) Serotonin. In: Davis KL, Charney D, Coyle JT, et al. (eds.) Neuropsychopharmacology: The Fifth Generation of Progress, pp. 15–46. New York: Raven Press, Ltd. Jacobs BL and Azmitia EC (1992) Structure and function of the brain serotonin system. Physiological Reviews 72: 165–229. Lambe EK and Aghajanian GK (2006) Electrophysiology of 5HT2A receptors and relevance for hallucinogen and atypical drug actions. In: Roth BL (ed.) The Serotonin Receptors: From Molecular Pharmacology to Human Therapeutics. Totowa, NJ: Humana Press. Lambe EK and Aghajanian GK (2006) Hallucinogen-induced UP states in the brain slice of rat prefrontal cortex: Role of
glutamate spillover and NR2B-NMDA receptors. Neuropsychopharmacology 31: 1682–1689. Liu R-J, Lambe EK, and Aghajanian GK (2005) Somatodendritic autoreceptor regulation of serotonergic neurons: Dependence on L-tryptophan and tryptophan hydroxylase-activating kinases. European Journal of Neuroscience 21: 945–958. Liu R-J, van den Pol AN, and Aghajanian GK (2002) Hypocretins (orexins) regulate serotonin neurons in the dorsal raphe nucleus by excitatory direct and inhibitory indirect actions. Journal of Neuroscience 22: 9453–9464. Richerson GB (2004) Serotonergic neurons as carbon dioxide sensors that maintain pH homeostasis. Nature Reviews Neuroscience 5: 449–461. Whitaker-Azmitia PM (1999) The discovery of serotonin and its role in neuroscience. Neuropsychopharmacology 21: 2S–8S.
Cholinergic Neurotransmission in the Autonomic and Somatic Motor Nervous System T C Westfall, St. Louis University School of Medicine, St. Louis, MO, USA ã 2009 Published by Elsevier Ltd.
Introduction Acetylcholine is the principal neurotransmitter at all autonomic preganglionic nerve terminals (sympathetic and parasympathetic), all postganglionic parasympathetic neuroeffector junctions, a few postganglionic sympathetic neuroeffector junctions (e.g., eccrine sweat glands), the somatic motor nerve neuromuscular junction, and cholinergic synapses in the central nervous system (CNS). The synthesis, storage, release, and inactivation of acetylcholine are similar at all these structures. This is probably also true for nonneuronal cholinergic systems in various parts of the body (e.g., human airway). The neurochemical events involved in the life cycle of acetylcholine are depicted in Figure 1.
Synthesis of Acetylcholine Choline and Choline Transport
Critical to the synthesis of acetylcholine is the availability of choline. There is little de novo synthesis of choline in cholinergic neurons; therefore, choline is provided mostly from the diet. Choline is a dietary component essential for normal function of all cells, not just cholinergic neurons. Choline or its metabolites ensure the structural integrity and signaling functions of all cell membranes; it is the major source of methyl groups in the diet, and it obviously directly affects cholinergic neurotransmission. Cholinergic neurons have especially large requirements for choline because they use choline not only for membrane synthesis but also for acetylcholine synthesis. Choline is taken up from the extracellular space by two transport systems. First is the ubiquitous low-affinity, sodium-independent transport system that is inhibited by hemicholinium 3 with a Ki of about 50 mmol l1. Second is the high-affinity, sodium- and chloride-dependent, hemicholinium 3-sensitive (Ki ¼ 10–100 nmol l1) choline transport system. The latter form is found predominantly in cholinergic neurons and is responsible for providing choline for acetylcholine synthesis. Once acetylcholine is released from cholinergic neurons following the arrival of action potentials, acetylcholine is hydrolyzed by acetylcholinesterase (AChE; see the section titled
478
‘Enzymatic hydrolysis: AChE’) to free acetate and choline. Choline is recycled after reuptake into the nerve terminal of cholinergic cells and utilized for acetylcholine synthesis. Under many circumstances, this reuptake and availability of choline appear to serve as the rate-limiting step in acetylcholine synthesis. Acetylcholine synthesis and release are sustained during persistent and prolonged neuronal stimulation, as long as choline is available and the transporter is functional. The low-affinity choline uptake transporter is used to synthesize membrane phospholipids. These lipids are reservoirs from which choline can also be used by choline neurons for acetylcholine synthesis. Despite the fact that the high-affinity choline uptake system was first described in the1960s, the gene for the high-affinity choline transporter (CHT1) has only recently been cloned from a variety of species: Caenorhabditis elegans, mouse, Torpedo, Limulus, and humans. It is interesting that CHT1 is not homologous to neurotransmitter transporters but is homologous to the Naþ-dependent glucose transporter family. With the molecular identification of the CHT1, it was discovered that it does not predominantly reside at the nerve terminal plasma membrane but rather in intracellular vesicular structures. It has been observed that CHT1 co-localizes with synaptic vesicle markers such as vesicle-associated membrane protein 2 and vesicular acetylcholine transporter (VAChT), at least when expressed in a cholinergic cell line. Although information is still incomplete, it has been hypothesized that arrival of action potentiates in nerve terminals results in exocytosis of synaptic vesicles, release of acetylcholine into the synaptic cleft, and increased trafficking of CHT1 to the plasma membrane, where it functions to take up choline after hydrolysis of acetylcholine. Because choline transport is rate-limiting for acetylcholine synthesis, increased availability of choline via its transport by CHT1 would favor an increase in acetylcholine stores to maintain high levels of transmitter release during neuronal stimulation. This also suggests that the availability of CHT1 at the cell surface is dynamically regulated in a manner very similar to the regulation of the exocytosis of synaptic vesicles. Much recent information has been obtained on how trafficking of CHT1 between the cell surface and subcellular compartments can regulate choline uptake activity. Nevertheless, the precise mechanisms involved in maintaining the distribution of CHT1 predominantly in intracellular vesicles rather than at the terminal surface like other neurotransmitter transporters is unclear.
Cholinergic Neurotransmission in the Autonomic and Somatic Motor Nervous System 479
Hemicholinium AcCoA + choline +
Choline
−
ChAT
Na+
Mitochondrion ACh
−
3Na+
Vesamicol ADP ATP
Ach ACh Co-T Co-T
Cholinergic varicosity Ca2+
Na+, K+ - ATPase 2K+
nAChR
+
mAChR VAMPS Ca2+ − Botulinium toxin
Choline
Co-T ACh
Acetate
SNAPS ACh + +
Effector cell membrane
AChE
mAChR (M1 − M5)
nAChR (NN − NM)
Figure 1 Schematic representation of a cholinergic neuroeffector junction showing features of the synthesis, storage, and release of acetylcholine (ACh) and receptors on which ACh acts. The synthesis of ACh in the varicosity depends on the uptake of choline via a sodium-dependent carrier. This uptake can be blocked by hemicholinium. Choline and the acetyl moiety of acetyl coenzyme A (AcCoA), derived from mitochrondria, form ACh, a process catalyzed by the enzyme choline acetyltransferase (ChAT). ACh is transported into the storage vesicle by another carrier that can be inhibited by vesamicol. ACh is stored in vesicles along with other potential cotransmitters (Co-T) such as adenosine triphosphate (ATP) and vasoactive intestinal polypeptide at certain neuroeffector junctions. Release of ACh and the Co-T occurs on depolarization of the varicosity, which allows the entry of Ca2þ through voltage-dependent Ca2þ channels. Elevated [Ca2þ]in promotes fusion of the vesicular membrane with the cell membrane, and exocytosis of the transmitters occurs. This fusion process involves the interaction of specialized proteins associated with the vesicular membrane, called vesicle-associated membrane proteins (VAMPs) and the membrane of the varicosity, called synaptosome-associated proteins (SNAPs). The exocytotic release of ACh can be blocked by botulinum toxin. Once released, ACh can interact with the muscarinic receptors (mAChRs), which are G-protein-coupled receptors, or nicotinic receptors (nAChRs), which are ligand-gated ion channels, to produce the characteristic response of the effector. ACh can also act on presynaptic mAChR or nAChR to modify its own release. The action of ACh is terminated by metabolism to choline and acetate by the enzyme acetylcholinesterase (AChE), which is associated with synaptic membranes. ADP, adenosine diphosphate; ATPase, adenosine triphosphatase. From Westfall TC and Westfall DP (2006) Neurotransmission: The autonomic and somatic motor nervous systems. In: Brunton LL, Lazo JS, and Parker KL (eds.) Goodman and Gilman’s the Pharmacological Basis of Therapeutics, 11th edn., pp. 137–182. New York: McGraw-Hill.
Choline Acetyltransferase
The synthesis of acetylcholine occurs within the cytoplasm of cholinergic nerve terminals by a reaction in which choline is acetylated with acetyl coenzyme A (CoA) by the enzyme choline acetyltransferase. Acetyl CoA is derived mainly from glycolysis and is ultimately produced by the enzyme pyruvate
dehydrogenase. The synthesis of acetyl CoA occurs at the inner membrane of mitochondria, and it is transported by citrate to the cytoplasm, where citrate is freed by citrate lyase. Choline acetyltransferase is synthesized in the perkaryon and transported along the length of the axon to the terminal by axoplasmic flow.
480 Cholinergic Neurotransmission in the Autonomic and Somatic Motor Nervous System
Storage of Acetylcholine Following its synthesis in the cytoplasm of the nerve terminal, acetylcholine is transported into synaptic vesicles by VAChT, which uses a proton electrochemical gradient to move acetylcholine to the inside of the organelle. Hydrophobicity algorithm prediction of secondary structure of VAChT suggests a protein comprising 12 transmembrane domains, with hydrophilic N- and C-termini in the cytoplasm. Sequence homology places VAChT into a family of transporter proteins that includes two vesicular monoamine transporters. An adenosine triphosphatase that pumps protons into the vesicle provides the energy necessary for the transport. Transport of protons out of the vesicle is coupled to uptake of acetylcholine into the vesicle and against a concentration gradient via an acetylcholine antiporter. Two types of vesicles appear to exist in cholinergic terminals: an electron-luscent vesicle (40–50 nm in diameter) and dense core vesicles (80–150 nm). The vesicle core contains both acetylcholine and adenosine triphosphate (ATP), which are dissolved in the fluid phase with metal ions (Ca2þ and Mg2þ) and a protoglycan vesiculin. In some cholinergic terminals, there are also peptides such as vasoactive intestinal polypeptide (VIP) that act together with ATP and acetylcholine as cotransmitters. The peptides are thought to reside in the larger dense-core vesicles. Vesicular membranes are rich in lipids (cholesterol and phospholipids) as well as proteins. The VAChT allows for the transport of acetylcholine agonist, which has a considerable concentration gradient and is saturable and adenosine triphosphatasedependent. Vesamicol is a drug which inhibits the transport of acetylcholine via targeting the acetylcholine Hþ antiporter channel. Vesamicol leads to a reduction in acetylcholine storage and interferes with neurosecretion. Although not precisely known, the content of synaptic vesicles is estimated to range from 1000 to more than 50 000 molecules per vesicle. There may also be large amounts of acetylcholine in the extravesicular cytoplasm.
Release of Acetylcholine Release of acetylcholine and cotransmitters (e.g., ATP and VIP) occurs on depolarization of the nerve terminals and takes place by exocytosis. Depolarization of the terminals allows the entry of Ca2þ through voltage-gated Ca2þ channels. Elevated Ca2þ concentration promotes fusion of the vesicular membrane with the plasma membrane, allowing exocytosis to occur. The molecular mechanisms involved in the release and regulation of release have been the subject of intense study but are still not completely
understood. As mentioned, acetylcholine, like other neurotransmitters, is stored in vesicles located at special release sites, close to presynaptic membranes and ready for release on the appropriate stimulus. The vesicles initially dock and are primed for release. A multiprotein complex appears to form and attach the vesicle to the plasma membrane close to other signaling elements. The complex involves proteins from the vesicular membrane and the presynaptic neuronal membrane, as well as other components that help link them together. It has been established that these various synaptic proteins, including the plasma membrane protein syntaxin and synaptosomal protein 25 kDa (SNAP-25), and the vesicular membrane protein, or synaptobrevin, form a complex. This complex interacts in an ATP-dependent manner with soluble N-ethylmalemide-sensitive fusion protein and soluble SNAPs. The ability of synaptobrevin, syntaxin, and SNAP-25 to bind SNAPs has led to their designation as SNAP regulators (SNARES). It has been hypothesized that most, if not all, intracellular fusion events are mediated by SNARE interactions. Important evidence supporting the involvement of SNARE proteins in transmitter release comes from the fact that botulinum neurotoxins and tetanus toxin, which block neurotransmitter release, proteolyze these three proteins. Two pools of acetylcholine appear to exist. One pool, the ‘depot’ or ‘readily releasable’ pool, consists of vesicles located near the plasma membrane of the nerve terminals and is filled with newly synthesized transmitter. Depolarization of the terminals causes these vesicles to release acetylcholine rapidly or readily. The other pool, the ‘reserve’ pool, seems to replenish the readily releasable pool and may be required to sustain acetylcholine release during periods of sustained or intense nerve stimulation.
Regulation of Acetylcholine Neurotransmission It is now well accepted that receptors are located on perkaryon, dendrites, and axons of neurons when they may respond to neurotransmitters or neuromodulators released from the same neurons (autoreceptors) or adjacent neurons or cells (heteroreceptors). Cholinergic nerve terminals also contain autoreceptors and heteroreceptors. Acetylcholine neurotransmission is therefore subject to complex regulation by mediators including acetylcholine itself acting on M2 and M4 autoinhibitory receptors and nicotinic acetylcholine excitatory receptors. Acetylcholine-mediated inhibition of acetylcholine release via muscarinic cholinergic receptors M2 and M4 is thought to represent a physiological
Cholinergic Neurotransmission in the Autonomic and Somatic Motor Nervous System 481
negative feedback control mechanism. Acetylcholine released from cholinergic neurons may also alter the release of other neurotransmitters. For instance, at the neuroeffector junction in the myenteric plexus in the gastrointestinal tract or sinoatrial node of the heart, postganglionic sympathetic and parasympathetic terminals lie in juxtaposition to each other. The release of acetylcholine not only produces autoinhibition of its own release but may attenuate the release of norepinephrine via an inhibitory action of muscarinic heteroreceptors located on sympathetic terminals. The reverse is also true: after its release of sympathetic nerve terminals, norepinephrine can inhibit the release of acetylcholine by an action of a2A and a2C heteroreceptors on parasympathetic nerve terminals. Muscarinic and nicotinic auto- and heteroreceptors also represent drug targets, or targets for locally formed autacoids, hormones, or transmitters.
Inactivation of Acetylcholine Enzymatic Hydrolysis: AChE
Subsequent to its release from cholinergic neurons, acetylcholine is subjected to a number of inactivation processes, including (1) diffusion from the site of release, (2) dilution in extracellular fluids, (3) binding to nonspecific sites, and (4) enzymatic destruction. The most important of these by far is the enzymatic destruction of acetylcholine to its hydrolysis products, acetic acid and choline. The reaction is catalyzed by AChE. While AChE is found in cholinergic neurons (dendrites, perikarya, and axons), it is distributed more widely than cholinergic synapses. It is highly concentrated at postsynaptic sites, especially at the neuromuscular junction. AChE exists in red blood cells and the placenta as well as nervous tissue. A second enzyme is butyrylcholinesterase (BuChE). BuChE exists in intestine and skin and abundantly in plasma. AChE can be distinguished from BuChE by selectivity of acetylcholine over butyrylcholine hydrolysis. BuChE shows a wider substrate capacity than AChE does. Almost all the pharmacological effects of AChE drugs are due to the inhibition of AChE, with a consequent accumulation of endogenous acetylcholine in the vicinity of the nerve terminal. Distinct but single genes encode AChE and BuChE in mammals, and the diversity of molecular structure of AChE arises from alternate messenger RNA processing. Complementary DNAs for several species, including Torpedo, Drosophila, nematode, rat, mouse, and human AChE, as well as mouse, rabbit, and human BuAChE, are known. The contact between acetylcholine and the surface of AChE occurs at two places on the protein, One is
the anionic site, which is negatively charged and stereospecific. It attracts the positively charged nitrogen atom of acetylcholine and binds the attracted methyl groups by Van der Waals forces. The other site is the ‘esteratic’ site, which combines the electrophilic carboxy carbon atom of the acetyl group and the serine hydroxyl group of the esteratic site (the nucleophilicity of which is enhanced by the histadine imidazole group). The three stages for the hydrolysis are as follows: (1) equilibrium is established between the enzyme and acetylcholine, forming an enzyme substrate complex; (2) the complex reacts to release choline and leaves an acetylated enzyme; and (3) the acetylated enzyme reacts with water to give acetic acid and regenerated enzyme with a t½ of approximately 40 ms. Although the principal function of AChE is to inactivate acetylcholine at cholinergic neuronal junctions, it has been reported to have multiple biological functions that are not obvious. These include neritogenesis, cell adhesion, synaptogenesis, amyloid fiber assembly, and activation of dopamine receptors, hematopoiesis, and thrombopoiesis, to name but a few.
Cholinergic Receptors Once released from cholinergic neurons, acetylcholine produces its physiological or pharmacological effects by first interacting with specific receptors. The various responses are mediated by the interaction of the transmitter with different types of receptors located in different organs, tissues, or cells. There are two general types of cholinergic receptors: muscarinic and nicotinic. The original classification was based on acetylcholine’s mimicking the effect of the alkaloid muscarine (muscarinic) or nicotine (nicotinic). Multiple subtypes are now known to exist. Originally the subtypes of receptors were defined by the rank order of potency of agonists and the specificity of antagonists (e.g., atropine as an antagonist of muscarinic receptors and hexamethonium or curare as antagonists for nicotinic acetylcholine receptors (nAChRs)). More recently, the various subtypes have been identified by molecular cloning of the genes. nAChRs
The nAChRs are members of a superfamily of ligandgated ion channels (ionotropic receptors). They primarily exist at the neuromuscular junction, autonomic ganglia and adrenal medulla, parasympathetic and sympathetic nerve terminals, and central nervous system. These receptors are the physiologic target of the neurotransmitter acetylcholine as well as naturally occurring alkaloids and synthetic drugs. The nAChR
Receptor (primary receptor subtype)a
Main synaptic location
Membrane response
Molecular mechanism
Agonists
Antagonists
Skeletal muscle (Nm) (a1)2 b1 e d Adult (a1)2 b1 g d Fetal
Skeletal neuromuscular junction (postjunctional)
Excitatory; endplate depolarization; skeletal muscle contraction
Increased cation permeability (Naþ; Kþ)
ACh Nicotine Succinylcholine
Peripheral neuronal (NN) (a3)2 (b4)3
Autonomic ganglia; adrenal medulla
Increased cation permeability (Naþ; Kþ)
ACh Nicotine Epibatidine Dimethylphenylpiperazinum
Central neuronal (CNS) (a4)2 (b4)3 (a-btox-insensitive)
CNS; pre- and postjunctional
Excitatory; depolarization firing of postganglion neuron; depolarization and secretion of catecholamines Pre- and postsynaptic excitation Prejunctional control of transmitter release Pre- and postsynaptic excitation Prejunctional control of transmitter release
Atracurium Vecuronium d-Tubocurarine Pancuronum a-conotoxin a-bungarotoxin Trimethaphan Mecamylamine
Increased cation permeability (Naþ; Kþ)
Cytisine, epibatidine Anatoxin A
Increased permeability (Ca2þ)
Anatoxin A
(a7)5 (a-btox-sensitive)
CNS; pre- and postsynaptic
Mecamylamine Dihydro-b-erythrodine Erysodine Lophotoxin Methllycaconitine a-Bungarotoxin a-Conotoxin IMI
a Nine individual subunits have been identified and cloned in human brain which combine in various confirmations to form individual receptor subtypes. The structure of individual receptors and the subtype composition is incompletely understood, however. Only a finite number of naturally occurring functional nAChRs constructs have been identified to date. a-btox, a-bungarotoxin. Adapted from Westfall TC and Westfall DP (2006) Adrenergic agonists and antagonists. In: Brunton LL, Lazo JS, and Parker KL (eds.) Goodman and Gilman’s the Pharmacological Basis of Therapeutics, 11th edn., pp. 237–296. New York: McGraw-Hill.
482 Cholinergic Neurotransmission in the Autonomic and Somatic Motor Nervous System
Table 1 Characteristics of subtypes of nicotinic acetylcholine receptors
Table 2 Characteristics of muscarinic acetylcholine receptor subtypes Size: chromosome location
Cellular and tissue locationa
Cellular responseb
Functional responsec
M1
460 aa 11q 12–13
CNS; most abundant in cerebral cortex, hippocampus, and striatum Autonomic ganglia Glands (gastric and salivary) Enteric nerves
Increased cognitive function (learning and memory) Increased seizure activity Decreased dopamine release and locomotion Increased depolarization of autonomic ganglia Increased secretions
M2
466 aa 7q 35–36
Widely expressed in CNS, heart, smooth muscle, autonomic nerve terminals
Activation of PLC; " IP3 and " DAG ! " Ca2+ and PKC Depolarization and excitation (" sEPSP) Activation of PLD2 " AA Couples via Gq/11 Inhibition of adenylyl cyclase # cAMP Activation of inwardly rectifying K+ channels Inhibition of voltage-gated Ca2þ channels Hyperpolarization and inhibition Couples via Gi/Go (PTX sensitive)
M3
590 aa Iq 43–44
Widely expressed in CNS (< than other mAChRs) Abundant in smooth muscle and glands Heart
Activation of PLC; " IP3 and " DAG ! " Ca2+ and PKC Depolarization and excitation (" sEPSP) Activation of PLD2 ; PLA2; " AA Couples via Gq/11
M4
479 aa 11p 12–11.2
Preferentially expressed in CNS, particularly forebrain
M5
532 aa 15q 26
Expressed in low levels in CNS and periphery. Predominant mAchR in dopamine neurons in VTA and substantia nigra
Inhibition of adenylyl cyclase #cAMP Activation of inwardly rectifying Kþ channels Inhibition of voltage-gated Ca2þ channels Hyperpolarization and inhibition Couples via Gi/Go (PTX sensitive) Activation of PLC; " IP3 and " DAG ! " Ca2+ and PKC Depolarization and excitation (" sEPSP) Activation of PLD2, PLA2; " AA Couples via Gq/11
a
Heart: SA node: slowed spontaneous depolarization; hyperpolarization #HR AV node: decrease in conduction velocity Atrium: # refractory period, # contraction Ventricle: slight # contraction Smooth muscle: "Contraction Peripheral nerves: Neural inhibition via autoreceptors and heteroreceptor) # Ganglionic transmission. CNS: Neural inhibition " Tremors; hypothermia; analgesia Smooth muscle: " contraction (predominant in some, e.g., bladder) Glands: " secretion (predominant in salivary gland) Increases food intake, body weight fat deposits. Inhibits dopamine release Synthesis of NO Autoreceptor and heteroreceptor mediated inhibition of transmitter release in CNS and periphery. Analgesia; cataleptic activity Facilitation of dopamine release Mediator of dilation in cerebral arteries and arterioles (?) Facilitates dopamine release Augmentation of drug-seeking behavior and reward (e.g., opiates, cocaine)
Most organs, tissues, and cells express multiple mAChRs. M1, M3, and M5 mAChRs appear to couple to the same G-proteins and signal through similar pathways. Likewise, M2 and M4 mAChRs couple through similar G-proteins and signal through similar pathways. c Although multiple subtypes of mAChRs coexist in many tissues, organs, and cells, one subtype may predominate in producing a particular function, or there may be equal predominance. mAChR, muscarinic acetylcholine receptor; PLC, phospholipase C; IP3, inositol-1,4,5-triphosphate; DAG, diacylglycerol; PKC, protein kinase C; EPSP, excitatory postsynaptic potential; PLD2, phospholipase D; AA, arachidonic acid; CNS, central nervous system; PLA, phospholipase A; cAMP, cyclic adenosine monophosphate; SA node, sinoatrial node; AV node, atrioventricular node; HR, heart rate; PTX, pertussis toxin; VTA, ventral tegmental area. Adapted from Westfall TC and Westfall DP (2006) Adrenergic agonists and antagonists. In: Brunton LL, Lazo JS, and Parker KL (eds.) Goodman and Gilman’s the Pharmacological Basis of Therapeutics, 11th edn., pp. 237–296. New York: McGraw-Hill. b
Cholinergic Neurotransmission in the Autonomic and Somatic Motor Nervous System 483
Receptor
484 Cholinergic Neurotransmission in the Autonomic and Somatic Motor Nervous System Table 3 Summary of peripheral cholinergic receptors and responses Organ Eye Sphincter (circular) muscle of iris Iris Ciliary muscle Heart SA node Atria AV node Ventricle Blood vesselsa Arteries Veins Lung Bronchial muscle Bronchial glands Gastrointestinal tract Motility Sphincter Secretion Urinary bladder Detrusor muscle Trigone and sphincter Glands Sweat, salivary, lacrimal, bronchial, etc. Nasopharyngeal Nerves Parasympathetic terminal Sympathetic terminal Autonomic ganglia Skeletal muscle (Neuromuscular junction)
Response
Receptor
Contraction (miosis) Contraction (accommodation)
M3, M2 M3, M2
Decrease in rate (negative chronotropic) Decrease in contractile strength (negative inotropic) Decrease in refractive period Decrease in conduction velocity (negative dromotropic effect) Increase in refractory period Small decrease in contractile strength
M2M3 M2M3 M2M3 M2M3 M2M3
Dilation (via nitric oxide from endothelial cells) Dilation (via nitric oxide from endothelial cells)
M3 M3
Contraction (bronchoconstriction) Stimulation
M2 ¼ M3 M3, M2
Increase Relaxation Stimulation
M3 ( M2) M, M2 M3, M2
Contraction Relaxation
M3 > M2 M3 > M2
Secretion
M3
Decrease in release of acetylcholine Decrease in release of norepinephrine Stimulation
M2, M4 M2, M4 NN or N1
Contraction
NM or N2
a
Most blood vessels are not innervated by parasympathetic nerves but have M3 receptors on endothelial cells.
is a pentamer consisting of five subunits around a central ion channel pore. Several subunits have been identified: a, b, g, d, and e. All these subunits share 35–50% homology with one another. Depending on the location of the receptor, it has a different combination of subunits. At the neuromuscular junction (N1 or Nm), the AChR is composed of two a (a1) subunits; one b (b1); one d; and in embryonic or denervated muscle, one g subunit. In the adult innervated muscle, the g is replaced with one e subunit. Functional neuronal nAChRs (CNS Nn) also exist as pentamers composed of two a and three b subunits. Eight human a subunits (a2–a7, a9, and a10) and three b subunits (b2–b4) have been cloned. Both the muscle and the neuronal nAChR share structural and functional properties with other ionotropic receptors, such as g-aminobutyric acid A receptor, 5-hydroxytryptamine type 3, and glycine receptors. Autonomic ganglia (N2)
and the adrenal medulla form homomeric a7 and heteromeric a3/b4, with (a3)2 (b4)3 being the most prevalent. The a subunits are responsible for binding acetylcholine, and the conformational change in the a subunits is responsible for permitting ion flow through the central pore of the receptor. The pentameric structure of the CNS–neuronal receptor and the large molecular diversity of its subunits (a2–a8, b2–b4) suggest there could be a large number of nAChRs with different physiological properties. They may subserve discrete functions and obviously represent multiple drug targets. The stoichiometry of the nAChR in brain is the subject of intense study. Selective ligands are becoming more and more prominent, but it is not yet possible to make a pharmacological classification based on subtypes. The characteristics of subtypes of nAChR are described in Table 1.
Cholinergic Neurotransmission in the Autonomic and Somatic Motor Nervous System 485 Muscarinic Acetylcholine Receptors
There are five distinct subtypes of muscarinic acetylcholine receptors (mAChRs), M1–M5, each produced by a distinct gene and all members of the family of G-protein-coupled receptors (metabotropic receptors). Like the different nAChRs, M1–M5 have distinct anatomical locations in the periphery and CNS and different chemical specificities. The mAChRs have been shown to be present in virtually all organs, tissues, and cell types, but various subtypes predominate in various tissues and organs (e.g., M2 in the heart, M3 in the detrusor muscle of the bladder). The mAChRs are located in smooth muscle, exocrine and endocrine glands, and the myocardium. They mediate the actions of acetylcholine in organs and tissues innervated by postganglionic parasympathetic nerves and postganglionic sympathetic nerves innervating eccrine sweat glands. They are also present at sites that lack parasympathetic nerves (e.g., most blood vessels). Table 2 summarizes the effects produced by activation of mAChRs. In the CNS, mAChRs are involved in regulating a large number of cognitive, behavioral, motor, and autonomic functions although some of these are also influenced by nAChRs. Effects produced by activating mAChR are coupled through G-protein-induced changes in membrane-bound effectors and production of second-messenger molecules. The M1, M2, and M3 receptor subtypes couple through the pertussis toxininsensitive G11 and G13, resulting in stimulation of phospholipase C activity. This results in the hydrolysis of membrane phosphatidylinositol 4,5 diphosphate to form inositol triphosphate (IP3) and diaceylglycerol. IP3 causes release of intracellular Ca2þ from the endoplasmic reticulum, which results in smooth muscle contraction as well as secretion from the appropriate cell. Diaceylglycerol activates protein kinase C, resulting in phosphorylation of numerous proteins and various physiological responses. Activation of M1, M2, and M3 receptors also results in the activation of phospholipase A2, leading to the release of arachidonic acid and consequent eicosanoid synthesis. Stimulation of the M2/M4 receptors leads to interaction with Gi and Go proteins, which results in inhibition of adenylyl cyclase and the resultant decrease in cyclic adenosine monophosphate, activation of inwardly rectifying Kþ channels, and inhibition of
voltage-gated Ca2þ channels. Functional consequences are hyperpolarization and inhibition of excitable membranes.
Physiological Responses Produced Following Increases in Cholinergic Neurotransmission Table 3 depicts the physiological responses due to increases in cholinergic neurotransmission or stimulation of parasympathetic neurons. See also: Cholinergic Pathways in CNS; Muscarinic Receptors: Autonomic Neurons; Nicotinic Acetylcholine Receptors.
Further Reading DeBiasi M (2002) Nicotinic mechanisms in the autonomic control of organ systems. Journal of Neurobiology 53: 568–579. Dhein S, van Koppen CJ, and Brodde OE (2001) Muscarinic receptors in the mammalian heart. Pharmacological Reviews 44: 161–182. Ferguson SM and Blakely RD (2004) The choline transporter resurfaces: New roles for synaptic vesicles? Molecular Interventions 4: 22–37. Jahn R, Lang T, and Su¨dhof T (2003) Membrane fusion. Cell 112: 519–533. Lindstrom JM (2000) Acetylcholine receptors and myasthenia. Muscle & Nerve 23: 453–477. Ribeiro FM, Black SA, Prado VF, Rylett RJ, Ferguson SS, and Prado MA (2006) The “ins” and “outs” of the highaffinity choline transporter CHT1. Journal of Neurochemistry 97: 1–12. Soreq H and Seidman S (2001) Acetylcholinesterase: New roles for an old actor. Nature Reviews Neuroscience 2: 294–302. Taylor P (2006) Anticholinesterase agents. In: Brunton LL, Lazo JS, and Parker KL (eds.) Goodman and Gilman’s The Pharmacological Basis of Therapeutics, 11th edn., pp. 201–216. New York: McGraw-Hill. van Koppen CJ and Kaiser B (2003) Regulation of muscarinic acetylcholine receptor signaling. Pharmacology & Therapeutics 98: 197–220. Wang H and Sun X (2005) Desensitized nicotinic receptors in brain. Brain Research. Brain Research Reviews 48: 420–437. Wess J (2004) Muscarinic acetylcholine receptor knockout mice: Novel phenotypes and clinical implications. Annual Review of Pharmacology and Toxicology 44: 423–450. Westfall TC and Westfall DP (2006) Neurotransmission: The autonomic and somatic motor nervous systems. In: Brunton LL, Lazo JS, and Parker KL (eds.) Goodman and Gilman’s The Pharmacological Basis of Therapeutics, 11th edn., pp. 137–182. New York: McGraw-Hill.
Cholinergic Pathways in CNS A C Cuello, McGill University, Montreal, QC, Canada ã 2009 Elsevier Ltd. All rights reserved.
Acetylcholine as a Central Nervous System Transmitter Historically, acetylcholine (ACh) was considered to be the most important central nervous system (CNS) transmitter. As early as 1936, Quastel demonstrated that ACh was synthesized in the brain. Indeed, ACh was the first substance to be proposed to act as a CNS transmitter in the pioneering work of Marthe Vogt and Feldberg in the 1940s and 1950s, largely based on prior lessons obtained with this substance in the peripheral nervous system by a number of outstanding pharmacologists, ranging from Otto Loewi to Sir Henry Dale. Later, Fatt, Katz, and collaborators provided evidence for a quantal release of ACh in the neuromuscular junction, laying the foundations for the idea that this transmitter substance was somehow ‘packaged’ in nerve terminals. The realization that this was the case came about with the ultrastructural finding in 1955 of synaptic vesicles in CNS nerve terminals by De Robertis and Bennett, and also Palay and Palade, and the compelling demonstration that isolated synaptic vesicles obtained from subcellular fractionation were enriched in ACh content, as shown by De Robertis in Buenos Aires and Whitaker in Cambridge. These studies not only were important for the realization that ACh was a bona fide CNS transmitter but they also provided strong foundations to the chemical synaptic transmission hypothesis, a concept currently applied to a wide variety of so-called classical (e.g., amines, aminoacids) and nonclassical transmitters (e.g., peptides).
CNS Cholinergic Synapses Contrary to historical expectations, current methods have demonstrated that cholinergic synapses do not account for the majority of transmitter-specific synapses in the CNS. In the cerebral cortex, for example, cholinergic synapses account for about 5–7% of the total synaptic population. However, considerable variations in the number of cholinergic synapses across the CNS should be expected, given the degree of differential concentrations of the ACh-synthesizing enzyme (choline acetyltransferase, ChAT) in diverse brain areas. ChAT is localized mainly in the cytosolic compartment. The CNS cholinergic synapses are endowed with the expected set of organelles (e.g.,
486
mitochondria, synaptic vesicles) and molecules (e.g., classical synaptic proteins), but in addition they contain ‘signature’ proteins such as the high-affinity choline transporter (CHT1) and the vesicular acetylcholine transporter (VAChT). These two proteins endow cholinergic synapses with their physiological specifications. CHT1 plays an essential role in maintaining the required level of ACh synthesis, as it is the availability of choline rather than the ChAT enzymatic activity that is the rate-limiting factor for the synthesis of ACh. As expected, CHT1 is localized in synaptic membranes to facilitate the incorporation of the choline precursor. Dietary levels of choline have been shown to have an important impact on the replenishment of ACh stores in the CNS. Besides its predicted synaptic membrane localization, CHT1 is abundantly present in synaptic vesicles. This is somewhat perplexing, as ChAT is primarily a cytosolic enzyme and the bulk of the ACh synthesis is known to occur in the cytoplasm. This location might be a consequence of the retrieval of synapses after exocytosis, but the vesicular CHT1 might also play a role in mobilizing choline from vesicles to cytoplasm and vice versa. The VAChT is heavily localized in membranes of synaptic vesicles of cholinergic presynaptic endings. Its function is the straightforward uptake of newly synthesized cytoplasmatic ACh into the vesicular stores. The ACh-enriched vesicles are thus ready for release on demand. The newly synthesized ACh appears to be the first to be released, probably from vesicles closer to the synaptic membranes, while more distant vesicles containing ‘older’ ACh pools are seemingly located in the cholinergic presynaptic terminal further away from the point of synaptic contact. The cholinergic synaptic vesicles, from the early De Robertis descriptions, are recognized as being small, clear, and roundish. Figure 1 represents the main features of CNS cholinergic synapses. After the identification of the CNS cholinergic neurons and their projections (see later), it was proposed that their terminations at target sites, particularly in the cerebral cortex, were nonsynaptic in nature. In other words, it was assumed that CNS cholinergic presynaptic boutons release ACh diffusely in a ‘cloudlike’ modality. The idea of ‘volume’ or ‘extrasynaptic’ transmission of acetylcholine in the CNS was born out of the prevalent notion that CNS terminations containing monoamines are apparently nonsynaptic, in a manner reminiscent of the peripheral autonomic axonal terminations. The cholinergic cortical terminations were, therefore, included in the category of nonsynaptic neurons due to the lack of evidence for
Cholinergic Pathways in CNS 487 Acetate Choline ChAT CHT1 VAChT Acetate Choline ACh
AChE
ChAT
AChE Nicotinic receptor
Muscarinic receptors (M1−M5)
Figure 1 Schematic representation of typical CNS cholinergic synapses, as described in the text. ACh, acetylcholine; ChAT, choline acetyltransferase; CHT1, choline transporter 1; VAChT, vesicular acetylcholine transporter; AChE, acetylcholinesterase; M1–M5, muscarinic receptors; M2, presynaptic muscarinic receptor 2; N, nicotinic receptors.
synaptic specialization in ChAT-immunoreactive (IR) boutons when observed by electron microscopy. However, Mesulam and collaborators, using ChAT antibodies, noted that cholinergic boutons in the cerebral cortex of the human brain mainly displayed symmetric synaptic contacts. A more comprehensive ultrastructural study in the rat neocortex, applying improved protocols for tissue preservation and utilizing antibodies against VAChT, revealed that the cortical cholinergic presynaptic boutons in their majority establish classical synaptic contacts with symmetric membrane specializations (parallel membranes of equal thickness in the pre- and postsynaptic sides of contacts). These classical synaptic contacts occur preferentially in dendritic shafts and differ in their termination pattern from the bulk of ‘noncholinergic’ presynaptic boutons. Cholinergic presynaptic boutons are seldom observed contacting neuronal cell bodies and only occasionally contacting dendritic spines. These latter synapses are exclusively of an asymmetric nature (i.e., the postsynaptic membrane is notably thicker than the opposing presynaptic membrane). Figure 2 illustrates the typical ultrastructural characteristics of cholinergic (VAChT-IR) presynaptic sites (boutons) in layer V of the rat parietal cortex.
Main CNS Cholinergic Pathways Although the existence of CNS cholinergic neurons was well accepted, it took nearly half a century for
their unequivocal identification. Early attempts to define CNS systems operating with ACh applied immunohistochemical methods to reveal the presence (activity) of acetylcholinesterase (AChE), the AChinactivating enzyme, a procedure which was pioneered by Shute and Lewis at Cambridge University. This method revealed cell groups that later proved to be indeed cholinergic in nature, but it had the intrinsic problem that it demonstrated both ‘cholinergic’ and ‘cholinoceptive’ neurons. This approach was modified later by Butcher and collaborators when they introduced the so-called pharmaco-histochemical technique. This technique involved the inhibition of AChE, followed, after few hours, by the AChE histochemical reaction, to demonstrate newly generated AChE in cell body groups, thus avoiding the confounding images of AChE-positive neurites in the CNS tissue. While this approach was of some value, the problem remained that it also revealed several AChE-positive cells which were not cholinergic in nature. We now know that there is not a 100% correlation between ChAT-IR and AChEpositive histochemical reactions in CNS neurons. Notable examples are the conspicuous presence of AChE-positive material in dopaminergic substantia nigra and noradrenergic locus coeruleus neurons in the absence of ChAT immunoreaction. The chemical isolation of ChAT allowed the generation of reliable polyclonal and monoclonal antibodies, which opened the door for the reliable demonstration of the main groups of CNS cholinernergic neurons. Thus, a number
488 Cholinergic Pathways in CNS
Figure 2 Electron micrograph images of VAChT-IR boutons displaying synaptic specializations in layer V of the rat parietal cortex. (A) and (B) VAChT-IR boutons (asterisks) establishing symmetric synaptic contacts with dendritic branches (d). Note the aggregation of synaptic vesicles adjacent to the presynaptic membranes. (C) Illustration of a small VAChT-IR varicosity which is occasionally observed in a synaptic contact with a dendritic spine (s); note the synapse is of the asymmetric type. (D) Micrograph of one of the few VAChT-IR boutons establishing a synaptic contact with a cell body (cb), probably belonging to a nonpyramidal neuron. Synaptic contacts are indicated by two arrowheads; g, Golgi complex. Scale bar (for all micrographs) ¼ 0.25 mm. Reprinted from Turrini P, Casu MA, Wong TP, et al. (2001) Cholinergic nerve terminals establish classical synapses in the rat cerebral cortex: Synaptic pattern and age-related atrophy. Neuroscience 105(2): 277–285, with permission from Elsevier.
of reports arose during the early 1980s, applying immunohistochemistry to depict the localization of CNS ChAT-IR cell bodies and fiber tracts. At the time, the Swedish histochemists Fuxe and Dahlstrom introduced a letter and number nomenclature to define the catecholaminergic pathway. This proved popular and was widely accepted. Mesulam and collaborators, following the same principles, applied a Ch and number nomenclature to define CNS cholinergic nuclear organizations, as revealed with anti-ChAT monoclonal antibodies. These studies were the most exacting and comprehensive and provided a useful guide for further investigations, although the Ch classification is not universally applied. Following these developments, a number of lesion and track-tracing experiments revealed the existence of a variety of CNS pathways and local circuit cholinergic neurons. The main cholinergic cell groups and pathways are represented in Figure 3 in a parasaggital view of the rat CNS, where the prominent and
extensively studied basalocortical and septo-hippocampal pathways are highlighted in gray. In summary, these immunohistochemical investigations demonstrated that the ventral telencephalon displays a continuous stream of cholinergic cell bodies, notably in the olfactory tubercle, islands of Calleja, all components of the diagonal band, medial septum, nucleus preopticus magnocellularis, and the innermost region of the globus pallidus. These neurons are usually referred to (as an ensemble) as the basal forebrain cholinergic system. In rodents, the ChAT immunoreactive neurons distributed loosely in the innermost third of the globus pallidus represent the nucleus basalis, which is responsible for the large majority of cholinergic input to the cerebral cortex. The rodent cerebral cortex contains in addition a few homogenously dispersed, local circuit, fusiform cholinergic cell bodies. The organization of this ‘basalocortical’ pathway is very different from that of the
Cholinergic Pathways in CNS 489
C
H OB CP
CG
MH BN
TH
LDT
S AON DB IP
PPT
OT Ar
A Figure 3 Schematic sagittal view of the rat CNS, representing the main cholinergic pathways, as discussed in the text. OB, olfactory bulb; AON, anterior olfactory nucleus; DB, diagonal band; S, septum (cholinergic neurons restricted to medial division); CG, cingulated cortex; CP, large interneurons in the caudate putamen; H, hippocampus; BN, nucleus basalis; A, amygdala; TH, thalamus; Ar, arcuate nucleus; PPT, peduculo-pontine tegmental nucleus; LDT, lateral doral tegmental nucleus; C, cortex; IP, nucleus interpenducularis; MH, medial habenula; OT, olfactory tubercle. For clarity, cholinergic motor neurons and cholinergic preganglionar neurons are not represented. The gray bands represent the major forebrain basalocortical and septal–hippocampal cholinergic pathways. Adapted from Cuello AC and Sofroniew MV (1984) The anatomy of the CNS cholinergic neurons. Trends in Neurosciences 7: 74–78.
human brain. In the human brain, the equivalent cholinergic group is composed of large (ergo ‘magnocellularis’) multipolar neurons located in a flattened nuclear organization lying below the anterior commissure. This organization would correspond to the neuronal group, which is nowadays referred to as the nucleus basalis magnocellularis of Meynert, or simply the nucleus of Meynert. However, it has to be stressed that this nomenclature is only applicable to the human brain and to subhuman primate brains. The rodent structure should be referred to simply as the nucleus basalis. These nuclei, both in rodents and humans, correspond to Ch4 in Mesulam’s classification. A possible explanation of this differential organization of the nucleus basalis in primates and rodents might reside in the fact that in rodents the globus pallidus is split where the medial division of the globus pallidus becomes the entopenducular nucleus (also containing cholinergic neurons), while the external division becomes the globus pallidus. In consequence, in rodents, this component of the basal forebrain cholinergic neurons does not find the obstacle of the anterior commissure barrier, invading the innermost third of the globus pallidus in a diffuse manner. In the rodent, the most caudal portions of the nucleus basalis become a flattened neuronal group located between the optic tract and the last portion of the corpus striatum. Much attention has
been paid to this nuclear organization, as it has been reported to be compromised in Alzheimer’s disease (AD), a disease that, in advanced states, is accompanied by the loss of cortical cholinergic markers (see the section entitled ‘CNS cholinergic neurons and AD’). It is well documented that the nucleus basalis innervates all areas of the neocortex in a topographicspecific manner, that is, more rostral regions of the nucleus basalis will supply cholinergic fibers to the most rostral cortical regions, and the most caudal to the most caudal. While the ‘basalocortical cholinergic pathway’ supplies fibers to the entire neocortex, the cingulate and entorhinal cortex most probably is innervated by cholinergic septal and diagonal band neurons. Figure 4 illustrates the appearance and relative density of cortical cholinergic presynaptic boutons as compared to the overall density of presynaptic boutons as revealed by synaptophysin. The other prominent basal forebrain cholinergic nuclear group is located in two arching bands of neurons meeting at the midline at dorsal aspects of the septal nucleus. This group of neurons is part of a continuum of cholinergic neurons from more rostrally located cholinergic neurons of the diagonal band of Broca. These septal cholinergic neurons project heavily to all regions of the hippocampal formation via the fimbria-fornix; this is referred to as the ‘septal–hippocampal’ cholinergic pathway.
490 Cholinergic Pathways in CNS
a
b
Figure 4 Immunocytochemical demonstration of cholinergic presynaptic boutons in the cerebral cortex of the rat (parietal cortex, lamina (b) as revealed with anti-VAChT antibodies. The pattern and density of the total population of cortical presynaptic boutons, as revealed with antisynaptophysin antibodies, are illustrated in (a). Scale bar ¼ 20 mm.
These two cholinergic nuclei (basalis and medial septum) are frequently referred to in the literature as ‘the’ basal forebrain cholinergic neurons; however, the basal forebrain cholinergic system includes other nuclei forming a continuum in that region of the brain (as previously described). These two cholinergic neuronal groups (basolocortical and septo-hippocampal) are heavily involved in higher CNS functions such as attention, learning, and memory, and are compromised in aging and in AD (see the section entitled ‘CNS cholinergic neurons and AD’). The largest CNS concentration of cholinergic fibers occurs, without any doubt, in the nucleus caudatus in humans and in the caudoputamen complex in rodents. These fibers and nerve terminals originate in very large local circuit neurons distributed throughout this region of the basal ganglia. Indeed, the highest CNS concentration of ACh and cholinergic markers in general occurs in the basal ganglia. The cholinergic system here plays an important role in mechanisms implicated in movement control and there is good physiological and pharmacological evidence for a dopaminergic/ cholinergic balance in this region. Other less conspicuous cholinergic cell groups are found in the thalamus (midline, intralaminar), epithalamus (medial habenula), and hypothalamus (posterior region and arcuate nucleus), with less defined functions than those of the forebrain cholinergic neurons. The medial habenula nuclei project heavily to the nucleus interpeduncularis via the so-called fasciculus retroflexus. In the brain stem, the motor and autonomic preganglionar neurons of cranial nerves are all of cholinergic nature, as expected from classical physiological studies preceding the advent of immunocytochemistry. In addition to these motor neurons, there is a collection of ChAT-IR neurons distributed in various regions of the brain stem. Thus, there is a diffuse reticular system (lateral reticular nucleus, parvicellular) and several nuclei,
including the pedunculo-pontine tegmental (PPT), lateral dorsotegmental (LDT), parabrachial, nucleus trapezoid, superior olive, and the raphe system (nucleus magnus and obscurus). PPT and LDT assembly of cholinergic neurons projects mainly to the thalamus, epithalamus, and tectum, but also sends axons more ventrally to the basal forebrain, lateral hypothalamus, and substantia nigra. Descending branches from the PPT and LDT cholinergic nuclei reach diverse nuclei in the lower brain stem, and long projections from these neurons can reach the spinal cord. In the spinal cord, occasional small cholinergic neurons can be observed in the dorsal horn in addition to the preganglionar nuclei of the lateral horn and the large motor neurons of the ventral horn. Although a number of cholinergic neuronal pathways have been described, there is still an incomplete account of the physiological roles for the diverse CNS cholinergic projections. The PPT–LDT ascending cholinergic pathways, along with forebrain neurons, appear to play an important role in sleep control mechanisms, whereas there is scant information regarding the possible functions of the brain stem– spinal cord descending cholinergic projections. The latter projections are suspected to participate in paincontrol mechanisms.
The Functions and Neurobiology of Basal Forebrain Cholinergic Neurons For several decades, the CNS cholinergic system was suspected to play an important role in memory. The most influential early studies pointing out such CNS cholinergic functions were carried out by Drachman and collaborators in young human subjects in which the muscarinic antagonist scopolamine, in low doses, brought about memory deficits similar to those observed in aged individuals. On the other hand, the application of the muscarinic agonists in young
Cholinergic Pathways in CNS 491
volunteers improved the recall of learned verbal material. These observations on the participation of the cholinergic system in memory tasks were corroborated in subhuman primates by Bartus and collaborators, who elaborated ‘‘the cholinergic hypothesis of geriatry memory dysfunction.’’ Numerous further investigations in rodents supported the concept of a cholinergic involvement in memory and learning. These functions, attributed initially to the so-called ascending reticular pathway, elaborated in the 1940s by Moruzzi and Magoun, had an important impact on developing physiological–anatomical theories for higher CNS functions. This anatomically ill-defined system was described as responsible for the control of wakefulness, sleep patterns, alertness, and memory. The anatomical–neurochemical identification in the brain stem and basal forebrain of neuronal cell groups and ascending pathways of catecholaminergic, serotoninergic, and cholinergic nature in the 1970s and 1980s allowed a more precise and specific understanding of the contribution of these transmitter systems to these functions. Today, the basal forebrain cholinergic system can be considered to be the rostralmost representation of such a system. There is solid experimental evidence that the basal forebrain cholinergic neurons do participate importantly in attentional, learning, and memory functions. These investigations constitute a very rich assembly of pharmacological, anatomical, and selective lesion studies combined with a wide variety of behavioral tasks. In more recent years, some emphasis has been placed on the attentional functions of cortical cholinergic inputs, thus conditioning learning and memory functions. Nevertheless, a dramatic argument in favor of the idea that ACh is necessary for learning and memory has been provided by the restoration of these functions with the cortical grafting of genetically modified cells producing ACh in rats with learning and memory impairments as a consequence of nucleus basalis lesions. The forebrain cholinergic input to cortex and hippocampus might have important modulatory roles in defining activity-dependent synaptic maps. The most provoking evidence in that direction was provided by Kilgard and Merzenich in 1998. This study demonstrated that episodic electrical stimulation of the nucleus basalis, paired with an auditory stimulus, resulted in a progressive reorganization of the primary auditory cortex in the adult rat, similar to that with behavioral training. While these findings suggest that the basal forebrain plays a role in modifying cortical synaptic sensory representation, additional pharmacological data would be necessary to firmly relate this to a cholinergic function.
Trophic Factor Dependency of Forebrain Cholinergic Neurons The embryonic development of the basal forebrain cholinergic system is highly dependent on the expression of nerve growth factor (NGF) and the highaffinity NGF receptor TrkA and, to a lesser extent, on the expression of low-affinity p75LNTR receptors. The low- and high-affinity p75LNTR neurotrophin receptors appear to function, depending on the physiological and/or pathological circumstances, in a cooperative or competitive manner. In early postnatal stages, relatively high levels of NGF are expressed, but this expression substantially decreases after birth. In early ontogenic stages, forebrain cholinergic neurons remain highly sensitive to exogenously administered NGF. The expression and release of NGF are very low after neuronal differentiation in adulthood. However, the NGF dependency of cholinergic neurons in mature and fully differentiated CNS remains. The experimental evidence indicates that in the adult CNS, NGF maintains cholinergic neuronal phenotype. The main supply of NGF is thought to be ‘target derived,’ that is, from the cerebral cortex for cholinergic nucleus basalis neurons, and from the hippocampus for medial septum neurons. Thus, target ablation of the neocortex or hippocampus provokes cholinergic neuronal cell shrinkage but not death. However, transection of the fimbria-fornix can provoke a mixed situation of neuronal atrophy and cell death of cholinergic neurons in the medial septum. The application of exogenous NGF can restore the cholinergic phenotype in both lesion models. Interestingly, NGF application after limited cortical strokelike lesions is sufficient to restore the size and biochemistry of nucleus basalis cholinergic neurons as well as preventing behavioral memory deficits, even when applying NGF after lesions (see Figure 5). Exogenous application of NGF in cortically lesioned adult rats also induces cholinergic synaptogenesis in the remaining, intact neocortex. On the other hand, the blocking of endogenously produced NGF by applying NGF-immunoneutralizing monoclonal antibodies in the cerebral cortex or synthetic TrkA receptor antagonists was shown to remove preexisting cortical cholinergic synapses in vivo. In other words, these observations would indicate that the production of small, baseline endogenous NGF in the cerebral cortex regulates the steady-state number of cholinergic synaptic boutons in the adult CNS. As trophic factors have been shown to be produced and liberated in an activity-dependent fashion, it would be reasonable to assume that the number of cortical
492 Cholinergic Pathways in CNS
cs c o o
a
d
b
e
c
f
n
c
sm CPu
o o
o
RF
o o
o
o
Rt
o
sm
31
o oo
Pir ACo LOT
SCh
Io
Figure 5 The schematic drawing on the left represents the distribution of abundant forebrain neurons intensively immunoreactive to p75 low-affinity NGF receptor, represented as filled circles. The cells in the inner portion of the globus pallidus correspond to cholinergic neurons of the nucleus basalis. Open circles indicate moderately p75 immunoreactive large neurons in the caudate putamen (cholinergic interneurons). The micrographs on the right (a) indicate cholinergic neurons of the nucleus basalis, as revealed by ChAT immunocytochemistry. Note the rich dendritic branching of the cholinergic neurons of the nucleus basalis. The extreme right column illustrates the computer-assisted definition of the cell soma cross-sectional area. The micrographs illustrate the efficacy of NGF to rescue retrogradely shrunken neurons following cortical lesions: (a) and (d) show ChAT-IR nucleus basalis neurons from naive control adult rats; (b) and (e) show gross cell shrinkage of the same neurons after cortical lesions; and (c) and (f) show prevention of cell shrinkage and maintenance of normal phenotype with the administration of microgram amounts of NGF. ACo, anterior cortical olfactory nucleus; c, corpus callosum; CPu, caudate putamen; cs, cingulate striae; lo, lateral olfactory tract; LOT, nucleus of the olfactory tract; n, fimbria fornix; Pir, piriform cortex; RF, rhinal fissure; Rt, reticular nucleus of the thalamus; SCh, nucleus suprachiasmaticus; sm, stria medullaris; 3, third ventricle. Reproduced from Cuello AC (2006) Cholinergic synaptic terminations in the cerebral cortex, trophic factor dependency, and vulnerability to aging and Alzheimer’s pathology. In: Giacobini E and Penney JB (eds.) The Brain Cholinergic System in Health and Disease, figure 3.2, pp. 33–46. Oxon, UK: Informa Healthcare, with permission from Taylor & Francis.
cholinergic synapses changes constantly in the human brain, based on experience and brain activity. Such a concept would be in line with the classical tenet of Hebb that the strength of synaptic connections was linked to a growth process which takes place with synaptic efficacy.
CNS Cholinergic Neurons and AD It is possible that CNS cholinergic neurons are somehow involved in a variety of neurodegenerative disorders. However, up to the present, none of these conditions appears as striking as in the case of
Alzheimer’s disease. In the 1970s, Davis and Maloney and Bowen et al. reported evidence of marked deficits in cortical cholinergic markers (particularly loss of ChAT levels) in AD brain. After the identification of the main CNS cholinergic groups, Whitehouse and collaborators reported losses of ‘magnocellular’ neurons in the the nucleus basalis of Meynert. These findings were coincidental with ‘‘the cholinergic hypothesis of geriatry memory dysfunction’’ (as discussed previously). At the time, evidence that the loss of substantia nigra dopaminergic neurons was mainly responsible for Parkinsonian symptoms, along with the evidence that the administration of dopamine
Cholinergic Pathways in CNS 493
precursors could be an effective transmitter-based therapy, brought about by analogy ‘the cholinergic hypothesis of Alzheimer’s disease.’ This proposal provoked a great deal of experimental and clinical research. We now know that the cholinergic involvement in AD is a component of the neuropathology, most likely secondary to the Ab burden in the cerebral cortex and hippocampus. It is now also well established that the cholinergic depletion occurs at advanced stages of the AD pathology, while at the AD-prodromic stage of mild cognitive impairment (MCI) an upregulation of cholinergic markers takes place instead. Furthermore, transgenic animal models of the amyloid pathology can replicate features of the cholinergic involvement, suggesting a secondary rather than a primary involvement in the disease process. However, as discussed previously, the forebrain cholinergic system is so intimately related to memory processes that it was perceived as a viable therapeutic target. Early attempts at using muscarinic agonists and choline supplements – in the latter case to stimulate ACh synthesis, given (as discussed previously) that the high-affinity choline uptake system is the de facto rate-limiting factor – were without substantive effect on memory or other cognitive parameters. Instead, the application of cholinesterase inhibitors (capable of blocking acetylcholinesterases or butyrilcholinesterases in diverse degrees) to block ACh degradation has been used widely as symptomatic therapy in AD. This approach is by no means capable of halting the pathological process (disease modifying), but has proved effective in delaying cognitive decay in a good number of patients, although the efficacy wanes after a year or two of treatment. The cholinergic system is, however, involved in the disease process. The activation of M1 and M3 receptors has been shown to shift amyloid precursor protein (APP) metabolism to a nonamyloidogenic modality, an aspect that has been confirmed repeatedly in experimental models and in the clinic with the application of anticholinesterases. These observations have provided renewed interest in the development of newer, safer, and more efficaceous muscarinic agonists. Knowing the great trophic dependency of forebrain cholinergic neurons on NGF support, clinical attempts have involved applying mouse NGF in the brain of Alzheimer’s patients, but with disapponting results. In these patients, the large doses applied and the diffusion of NGF to undesirable targets (e.g., sensory nociceptive fibers and hypothalamic nuclei) produced pain and marked weight loss. As NGF can act on the nucleus basalis also in the somatodendritic region in a paracrine fashion (in contrast with target derived), there is current interest in applying grafts of genetically modified NGF-producing cells, implanted
in nucleus basalis neighboring areas of Alzheimer’s patients, and, long range, of regulable lentoviral vectors, allowing the in situ expression of NGF. New therapeutic approaches to correct the cholinergic deficit might emerge from the realization that this is most probably due to the dysregulation of the protease cascade, which is responsible for the maturation of the NGF-precursor protein (ProNGF) into mature and biologically active NGF and for its degradation. See also: Cholinergic Neurotransmission in the Autonomic and Somatic Motor Nervous System; Nicotinic Acetylcholine Receptors.
Further Reading Bartus RT, Dean RL, Beer B, et al. (1982) The cholinergic hypothesis of geriatric memory dysfunction. Science 217: 408–417. Butcher LL and Woolf NJ (2004) Cholinergic neurons and networks revisited. In: Paxinos G (ed.) The Rat Nervous System, pp. 1257–1268. London: Elsevier. Cuello AC (1996) Effects of trophic factors on the CNS cholinergic phenotype. Progress in Brain Research 109: 347–358. Cuello AC (2006) Cholinergic synaptic terminations in the cerebral cortex, trophic factor dependency, and vulnerability to aging and Alzheimer’s pathology. In: Giacobini E and Penney JB (eds.) The Brain Cholinergic System in Health and Disease, pp. 33–46. Oxon, UK: Informa Healthcare. Cuello AC and Sofroniew MV (1984) The anatomy of the CNS cholinergic neurons. Trends in Neurosciences 7: 74–78. Debeir T, Saragovi HU, and Cuello AC (1999) A nerve growth factor mimetic TrkA antagonist causes withdrawal of cortical cholinergic boutons in the adult rat. Proceedings of the National Academy of Sciences of the United States of America 96(7): 4067–4072. Kilgard MP and Merzenich MM (1998) Cortical map reorganization enabled by nucleus basalis activity. Science 279(5357): 1714–1718. Mesulam MM (1990) Human brain cholinergic pathways. Progress in Brain Research 84: 231–241. Mesulam MM, Mufson EJ, Wainer BH, et al. (1983) Central cholinergic pathways in the rat: An overview based on an alternative nomenclature (Ch1–Ch6). Neuroscience 10(4): 1185–1201. Ribeiro FM, Black SA, Prado VF, et al. (2006) The ‘ins’ and ‘outs’ of the high-affinity choline transporter CHT1. Journal of Neurochemistry 97(1): 1–12. Sofroniew MV, Eckenstein F, Thoenen H, et al. (1982) Topography of choline acetyltransferase containing neurons in the forebrain of the rat. Neuroscience Letters 33: 7–12. Sofroniew MV, Galletly NP, Isacson O, et al. (1990) Survival of adult basal forebrain cholinergic neurons after loss of target neurons. Science 247: 338–342. Turrini P, Casu MA, Wong TP, et al. (2001) Cholinergic nerve terminals establish classical synapses in the rat cerebral cortex: Synaptic pattern and age-related atrophy. Neuroscience 105(2): 277–285. Whitehouse PJ, Price DL, Struble RG, et al. (1982) Alzheimer’s disease and senile dementia: Loss of neurons in the basal forebrain. Science 215: 1237–1239. Winkler J, Suhr ST, Gage FH, et al. (1995) Essential role of neocortical acetylcholine in spatial memory. Nature 375(6531): 484–487.
Muscarinic Receptors: Autonomic Neurons R S Aronstam and P Patil, University of Missouri – Rolla, Rolla, MO, USA ã 2009 Elsevier Ltd. All rights reserved.
Introduction Muscarinic receptors recognize the neurotransmitter acetylcholine, translating this recognition into electrical transients and altered cell behavior by activating and suppressing an assortment of signaling pathways. Muscarinic receptors comprise one of the two classes of receptors for the neurotransmitter acetylcholine, with nicotinic receptors comprising the other class. Muscarinic receptors are selectively activated by the alkaloid muscarine from the mushroom Amanita muscaria and are blocked by belladonna alkaloids, such as atropine and scopolamine (Figure 1). Muscarinic receptors are involved in the transduction of cholinergic signals in the central nervous system, autonomic ganglia, smooth muscles, and other parasympathetic end organs. The history of muscarinic systems is intimately associated with the development of receptor theory, pharmacology, and the discovery of neurotransmitter transmission. Muscarinic receptors are members of the superfamily of G-protein-coupled receptors, specifically class A (rhodopsin-like) receptors. Muscarinic receptors are related to the ionotropic nicotinic acetylcholine receptors only insofar as their physiological activator is acetylcholine; muscarinic and nicotinic receptors share little similarity in their structure, physiological functions, or pharmacology (except for a few close analogues of acetylcholine).
Muscarinic Receptor Subtypes The genes for five subtypes of muscarinic receptors, M1–M5, were identified, cloned, and sequenced between 1986 and 1990. These receptor subtypes differ in their primary structure, distribution, pharmacology, and signal transduction activity (Table 1). This heterogeneity presents the possibility of selectively affecting specific muscarinic functions in the brain and other organs; accordingly, the pharmacology of muscarinic receptor subtypes has been the subject of intensive investigation. Unfortunately, the selectivity of antagonists for receptor subtypes rarely exceeds tenfold. In the absence of pharmacological uniqueness, knockout mice are proving exceptionally useful in delineating functions associated with specific receptor subtypes.
494
Our understanding of muscarinic receptor biology is largely dependent on (1) physiological and behavioral analysis of the actions of muscarinic agonists and antagonists (notably, smooth muscle contraction, cardiac function, vascular tone, glandular secretion, arousal, attention, and memory); (2) ligand binding studies employing radiolabeled probes (notably, the antagonists [3H]N-methylscopolamine and [3H]quinuclidinyl benzilate); (3) biochemical characterization of receptor proteins (including primary, secondary, and tertiary protein structure, and patterns of phosphorylation, glycosylation, phosphorylation, and lipid modification, dimerization, nitrosylation, and internalization); (4) measurement of receptor influence on intracellular second messengers (notably, inositol triphosphate (IP3), diacylglycerol, Ca2þ, nitric oxide, cAMP, cGMP, and arachidonic acid); (5) analysis of receptor gene sequence and control; (6) genetic manipulation of receptor expression; (7) genetic manipulation of receptor protein structure; and (8) elimination of specific receptor subtypes using gene knockout technology. The properties of the different subtypes are summarized in Table 1. A schematic diagram of the human M2 receptor is presented in Figure 2, a possible arrangement of the seven transmembrane domains is schematically represented in Figure 3, the amino acid sequence alignment of the five subtypes is presented in Figure 4, and an alignment tree of receptor amino acid sequences suggesting evolutionary relations is shown in Figure 5. The receptors were named on the basis of the order of their discovery, which reflects their relative abundance. Muscarinic receptors have been traditionally divided into two groups based on both their pharmacology and signaling properties: M2 and M4 comprise one group, and M1, M3, and M5 comprise the other (Figure 6).
Muscarinic Receptor Structure Muscarinic receptor proteins are single polypeptides of 460–590 amino acids with an extracellular N-terminus and an intracellular C-terminus (Figures 2 and 3). The N-termini do not possess consensus sites for protein cleavage, and epitope tags applied to the N-terminus provide convenient markers to monitor receptor expression and transport. The coding regions of muscarinic receptor genes are contained within a single exon. Hydropathic analyses of the amino acid sequences reveal seven regions of 20–24 amino acids that are likely to form membrane spanning a-helical structures. The amino acid composition of the membrane-spanning regions is
Muscarinic Receptors: Autonomic Neurons 495 Muscarinic ligands H O
CH3
HO
CH3
H2 C
H3C C
H3C
OCH2CH2
N
CH3
CH3 O
H
Acetylcholine endogenous agonist
+ N
H
CH3
CH3 Muscarine prototypical nonselective agonist
CH3 N + OCH2CH2N(C2H5)3 + OCH2CH2N(C2H5)3
O
O
CH2OH
C
CH
+ OCH2CH2NH(C2H5)3
Atropine nonselective antagonist
Gallamine allosteric ligand
O H
O N
CH C
C
CH3 O
+
N CH3
4-DAMP M3 selective antagonist
N O
H N N
O N
C
N AF-DX 116 M2 selective antagonist
Pirenzepine M1 selective antagonist
O N
N CH3 Figure 1 Structures of muscarinic ligands. Acetylcholine is the physiological agonist. Muscarine and atropine are the prototypical agonist and antagonist which define the receptor class. Gallamine is an allosteric receptor antagonist. Pirenzepine, AF-DX 116, and 4-DAMP are antagonists with a degree of selectivity for the M1, M2, and M3 receptor subtypes, respectively.
highly conserved (90% sequence similarity) among the five subtypes, as it is among the larger family of G-protein-coupled receptors. Between the fifth and sixth membrane-spanning units is a large intracellular loop that is highly variable in composition and size. Several consensus sites for phosphorylation are located on the third intracellular
loop, as well as on the C-terminal chain. A disulfide bridge is formed between a conserved cysteine adjacent to the third transmembrane segment and a cysteine in the middle of the second extracellular loop in all five subtypes. Chemical modification of these cysteine groups decreases agonist binding affinity as well as the ability of the receptor to couple to transducer
Property
Molecular weighta Amino acids Genomic location G-protein coupling Second messengersb Tissuesc
Brain regions
Subtypeselective antagonistsd Functions revealed by analysis of knockout mice
a
Subtype M1
M2
M3
M4
M5
51 240
51 715
66 127
53 058
60 186
460 11q13
466 7q31–q35
590 1q43
479 11p12–p11.2
532 15q26
Gq, G11
Gi, Go
Gq, G11
Gi, Go
Gq, G11
IP3/DAG, NO
cAMP (#)
IP3/DAG, NO
cAMP (#)
IP3/DAG, NO
Brain, autonomic ganglia, secretory glands, vas deferens, sympathetic ganglia Cerebral cortex, hippocampus and dentate gyrus, striatum, olfactory bulb and tubercle, amygdala Pirenzepine, telenzepine, mamba venom toxins
Brain, heart, sympathetic ganglia, lung, ileum, uterus and other smooth muscles Cerebellum, medulla, pons, basal forebrain, olfactory bulb, diencephalon
Brain, secretory glands, smooth muscles
Brain, lung
Cerebral cortex, thalamus, piriform cortex, olfactory bulb, brain stem nuclei
Midbrain
AF-DX-116, methoctramine, gallamine, himbacine
4-DAMP, hexahydro-sila-difenidol
Occipital cortex, caudate and putamen, visual nuclei, olfactory tubercle, hippocampus Tropicamide, himbacine
Agonist-induced seizures, learning, control of locomotor activity; indirect inhibition of N- and L-type calcium channels; M current inhibition in sympathetic ganglion neurons; MAPK pathway activation
Agonist-induced akinesia and central tremor; corticosterone release; analgesia; hypothermia; smooth muscle contraction; bradycardia; inhibition of N- and P/Q-type calcium channels
Papillary and urinary bladder constriction; salivary secretion; smooth muscle contraction; control of dopamine release; maintenance of body mass and food intake
Dopamine release; acetylcholine release (hippocampus); depression of motor control; analgesia
Dilation of cerebral arteries and arterioles; dopamine release
Molecular weights are calculated for the polypeptide contribution only. IP3/DAG, stimulation of phospholipase C to release the second messenger inositol triphosphate (IP3) and diacylglycerol (DAG) from phosphatidylinositol; NO, increase in nitric oxide production. cAMP(#): inhibition of adenylate cyclase, thereby lowering cellular content of the second messenger cAMP. c Only low levels of M5 receptor expression have been detected in tissues. d Antagonist selectivity of the subtypes is relative; antagonists generally differ in their interactions with the different subtypes by a factor of less than 20. b
496 Muscarinic Receptors: Autonomic Neurons
Table 1 Properties of muscarinic receptor subtypes
A
L
N
S
L
S
S
N
N
S
T
N
N
M
G
E C
V
Y
G
T S
D
E
V
T
R
P
Y K
T
F
F V
A
I
L
Y
T
S
M
S I
L
I
S
V
D
N
L
M
F
L A L
L V F
I C F
N R
L
Q
T
L
Y
D
I
S F
N
S
I
I
D R
N
Y
A
M T
G W
K
K
F C V
T
K
P
Y
T
F
I
L
I
L
R
E
S
A T
V
F
K
K
K
K
A
P Q K
T M
K
I
V
T S
V
G
S
S
G
Q
N
G
E
V
T
T
N
T
P
T
C
S
D S
L
G
H
S
K
D
E N
S
K
Q
T
D
Q
T
I
E
D
D
R
E
K
E
S
S
N
D
E
S V
T N
E
T
E N
C
V Q
G
V
P
D
R
P
A
K
G
N
Q
I
K N
D
P
V
S
P
S
L
V
Q
G
R
Q
K
H L
R
M
K
Q
N
I
A
V
K
T K
P
C
C
E
H
T
R
I
M N
S
A
V
D S
T
L G
S D
H
K
G
I
E
Y N I
V D
G A
A
T
R
S
S
A N
F
K
K P
T
K
S P
I
V
K
P
N
N
N
N
M
Figure 2 Schematic diagram of a human M2 muscarinic receptor showing the primary amino acid sequence. Four consensus sites of glycosylation at asparagine residues are indicated on the extracellular N-terminal domain. The aspartate and tyrosine and threonine residues in the membrane-spanning regions that are thought to be involved in agonist binding are highlighted in yellow. Regions of the third intracellular loop involved in G-protein recognition and coupling are indicated by blue shading. Possible sites of threonine phosphorylation in the C-terminal domain are also indicated by highlighting.
Muscarinic Receptors: Autonomic Neurons 497
V
K
E
A
L
C
V
I
D
P
A
C
Y
L P
R
K
T
I
N
K
K
Y
I
N
S
N
K
K
W
L
W
A
T
I
Y G C
T
I
T
V
W
P
S
Y
T
L
S
P
A
P
P
T
R
I N
R
S V
P
V
I
R
A
A
H
T
M
L
L
Y
V
I
M
T
N
I
L
P
V
I
A
I
M
M
W
A
I
A
F
Y
L
T
A
I
A
F
S
L
V A
L
L
V S
W
V T
F
G
L
I S
I
C
N
N
A
A
V A
P
A
S
W
F
L
V
M A
N
V H
A
N
A
Y
K V
D
L
N V
G
L
W
L
Q
P
T
F
F
A
C
F
F
C
Y
Q
I
V V
G
I
V
G
I
P
Y T
L
V G
V
V V
S
L T
V
I S-S
V
G
W E
I
L
P
498 Muscarinic Receptors: Autonomic Neurons
IV
II
V I
III VI
VII
Figure 3 Possible arrangement of muscarinic receptor transmembrane domains, as suggested by structural analysis of rhodopsin. Amino acid moieties involved in the binding of acetylcholine are located approximately one-fourth of the way into the plane of the membrane.
G-proteins. Moreover, nitrosylation of receptor cysteines after reduction of the disulfide bond severely diminishes ligand binding to the receptor. Elimination of either of these cysteines, however, precludes expression of the receptor. A conserved cysteine in the N-terminus may be a site for palmitoylation (thereby creating another intracellular loop) and an N-terminal polybasic region in four of the subtypes (all except the M2 receptor) may be involved in recruitment of adapter proteins in certain signaling pathways. M3 receptors (at least) have been found to form disulfide-linked dimers on the cell surface. Two to four consensus sites for N-glycosylation are present in the extracellular N-terminal domain of each receptor, and 25% of the receptor mass may be contributed by these oligosaccharides. Although this carbohydrate component may affect the processing and orientation of the receptor, it plays no discernible role in ligand binding or signal transduction: enzymatic removal of the carbohydrate has little effect on the binding and signal transduction potential of mature receptors. Chimeric receptors and mutational analysis have revealed sites on the receptor proteins that are specifically involved in ligand binding and coupling to transducer G-proteins. Acetylcholine binds to a site within a pocket formed by the roughly circularly arranged transmembrane domains (Figure 3). An aspartate moiety in the third transmembrane
region participates in an ionic interaction with the quaternary nitrogen of acetylcholine, whereas a series of tyrosine and threonine residues located in the membrane-spanning segments approximately onefourth of the distance through the membrane likely form hydrogen bonds with muscarinic ligands. In agreement with classical pharmacological analysis, the binding site for competitive antagonists is thought to overlap the acetylcholine recognition site but additionally involves contiguous hydrophobic areas of the receptor protein and membrane. Muscarinic receptors also possess a site(s) or region that participates in allosteric regulation by a variety of compounds, including gallamine (Figure 1). These ligands compete with classical muscarinic ligands but slow the dissociation of previously bound probes, indicating the existence of a ternary complex (receptor plus allosteric and orthosteric ligands). Gallamine binding is sensitive to modification of the sixth transmembrane domain as well as the third extracellular loop. A number of regions have been identified that participate in the interactions of muscarinic receptors with transducer G-proteins. In particular, the second intracellular loop and the N- and C-terminal portions of the third intracellular loop are involved in coupling to G-proteins. Desensitization of muscarinic receptors may entail phosphorylation of conserved threonine residues on the C-terminal portion of the receptor, as well as other sites on the third intracellular loop. Prolonged stimulation of muscarinic receptors induces a rapid phosphorylation that is associated with a decrease in agonist affinity and an uncoupling from transducer G-proteins. Agonist-induced phosphorylation involves a G-protein receptor kinase (GIRK) and not kinases activated by Ca2þ, cAMP, cGMP, or phorbol esters. Phosphorylation-dependent internalization of muscarinic receptors appears to be mediated by arrestins. The evolutionary relationship of muscarinic receptor subtypes is suggested by amino acid sequence (Figure 5). M1, M3, and M5 comprise one cluster, with M1 and M3 being somewhat more closely related. M2 and M4 comprise a second cluster. Analysis of nucleotide sequence of the coding regions reveals an essentially similar pattern. Sequence analyses reveal more genomic divergence within the two clusters than between the two groups. The five muscarinic receptor genes are scattered throughout the genome; the only two on the same chromosome (M1 and M4 on chromosome 11) are only distantly related.
Muscarinic Receptor Gene Structure Whereas the coding sequences of the gene for each subtype are uninterrupted or intronless, the genes are
Muscarinic Receptors: Autonomic Neurons 499
M1 M2 M3 M4 M5
10
20
30
40
50 60 70 80 MNTSAPPAVSPNITVLAPGKGPWQVAFIGITTGLLSL MNNSTNSSNNSLALTSPYKTFEVVFIVLVAGSLSL MTLHNNSTTSPLFPNISSSWIHSPSDAGLPPGTVTHFGSYNVSRAAGNFSSPDGTTDDPLGGHTVWQVVFIAFLTGILAL MANFTPVNGSSGNQSVRLVTSSSHNRYETVEMVFIATVTGSLSL MEGDSYHNATTVNGTPVNHQPLERHRLWEVITIAAVTAVVSL .. * . ..*
M1 M2 M3 M4 M5
90 100 110 120 130 140 150 160 ATVTGNLLVLISFKVNTELKTVNNYFLLSLACADLIIGTFSMNLYTTYLLMGHWALGTLACDLWLALDYVASNASVMNLL VTIIGNILVMVSIKVNRHLQTVNNYFLFSLACADLIIGVFSMNLYTLYTVIGYWPLGPVVCDLWLALDYVVSNASVMNLL VTIIGNILVIVSFKVNKQLKTVNNYFLLSLACADLIIGVISMNLFTTYIIMNRWALGNLACDLWLAIDYVASNASVMNLL VTVVGNILVMLSIKVNRQLQTVNNYFLFSLACADLIIGAFSMNLYTVYIIKGYWPLGAVVCDLWLALDYVVSNASVMNLL ITIVGNVLVMISFKVNSQLKTVNNYYLLSLACADLIIGIFSMNLYTTYILMGRWALGSLACDLWLALDYVASNASVMNLL *. **.**..* *** *.*****.* ********** **** .* * . . * ** . ******.*** *********
M1 M2 M3 M4 M5
170 180 190 200 210 220 230 240 LISFDRYFSVTRPLSYRAKRTPRRAALMIGLAWLVSFVLWAPAILFWQYLVGERTVLAGQCYIQFLSQPIITFGTAMAAF IISFDRYFCVTKPLTYPVKRTTKMAGMMIAAAWVLSFILWAPAILFWQFIVGVRTVEDGECYIQFFSNAAVTFGTAIAAF VISFDRYFSITRPLTYRAKRTTKRAGVMIGLAWVISFVLWAPAILFWQYFVGKRTVPPGECFIQFLSEPTITFGTAIAAF IISFDRYFCVTKPLTYPARRTTKMAGLMIAAAWVLSFVLWAPAILFWQFVVGKRTVPDNQCFIQFLSNPAVTFGTAIAAF VISFDRYFSITRPLTYRAKRTPKRAGIMIGLAWLISFILWAPAILCWQYLVGKRTVPLDECQIQFLSEPTITFGTAIAAF .*******..*.**.* .** . * .** **..**.******* **. ** *** .* *** * .*****.***
M1 M2 M3 M4 M5
250 260 270 280 290 300 310 320 YLPVTVMCTLYWRIYRETENRARELAALQ--GSETPGKGGGS----------------SSSS--ERSQPGAE-------YLPVIIMTVLYWHISRASKSRIKKDKKEPVANQDPVSPSLVQG---------------RIVKPNNNNMPSSD-------YMPVTIMTILYWRIYKETEKRTKELAGLQASGTEAETENFVH----------------PTGS--SRSCSSYE-------YLPVVIMTVLYIHISLASRSRVHKHRPEGPKEKKAKTLAFLKS---------------PLMKQSVKKPPPGE-------YIPVSVMTILYCRIYRETEKRTKDLADLQ--GSDSVTKAEKRKPAHRALFRSCLRCPRPTLAQRERNQASWSSSRRSTST *.** .*. ** .* . * .
M1 M2 M3 M4 M5
330 340 350 360 370 380 390 400 GSPETPP-GRCCRCCRAPRLLQAYSWK---EEEEEDEGS------------MESLTSSEG-EEPGSE------VVIKMPDGLEHNKIQNG-KAPRDPVTENCVQGE---EKESSNDSTS-----------VSAVASNMRDDEITQD------------LQQQSMKRSNRRKYGRCHFWFTTKSWKPSSEQMDQDHSSSDSWNNNDAAASLENSASSDE-EDIGSETRAIYSIVLKLPG AAREELR--NG-KLEEAPPPALPPPPR---PVADKDTSNESS----------SGSATQNTKERPATE----------LST TGKPSQATGPSANWAKAEQLTTCSSYP---SSEDEDKPATDP--------VLQVVYKSQGKESPGEE----------FSA . .
M1 M2 M3 M4 M5
410 420 430 440 450 460 470 480 --MVDPEAQAPTK---QPPRSSPNTVKRPTKKGRDRAGK-----GQKPR------------------------------ENTVSTSLGHSKD---ENSKQTCIRIGTKTPKSDSCTPT-----NTTVEVVGSSGQNGD--------------------HSTILNSTKLPSSDNLQVPEEELGMVDLERKADKLQAQKSVDDGGSFPKSFSKLPIQLESAVDTAKTSDVNSSVGKSTAT TEATTPAMPAPPL---QPRALNPASRWSKIQIVTKQTGN---ECVTAIEIVPATPAGMR--------------------EETEETFVKAETE---KSDYDTPNYLLSPAAAHRPKSQK---CVAYKFRLVVKADGNQE---------TNNGCHKVKIMP .
M1 M2 M3 M4 M5
490 500 510 520 530 540 550 560 -----------------GKEQLAKRKTFSLVKEKKAARTLSAILLAFILTWTPYNIMVLVSTFCKDCVPETLWELGYWLC -----EKQNIVARKIVKMTKQPAKKKPPPSR-EKKVTRTILAILLAFIITWAPYNVMVLINTFCAPCIPNTVWTIGYWLC LPLSFKEATLAKRFALKTRSQITKRKRMSLVKEKKAAQTLSAILLAFIITWTPYNIMVLVNTFCDSCIPKTFWNLGYWLC -----PAANVARKFASIARNQVRKKRQMAAR-ERKVTRTIFAILLAFILTWTPYNVMVLVNTFCQSCIPDTVWSIGYWLC CPFPVAKEPSTKGLNPNPSHQMTKRKRVVLVKERKAAQTLSAILLAFIITWTPYNIMVLVSTFCDKCVPVTLWHLGYWLC * *.. *.* ..*. *******.**.***.***. *** *.* * * .*****
M1 M2 M3 M4 M5
570 580 590 600 610 YVNSTINPMCYALCNKAFRDTFRLLLLCRWDKRRWRKIPKRPGSVHRTPSRQC* YINSTINPACYALCNATFKKTFKHLLM* YINSTVNPVCYALCNKTFRTTFKMLLLCQCDKKKRRKQQYQQRQSVIFHKRAPEQAL* YVNSTINPACYALCNATFKKTFRHLLLCQYRNIGTAR* YVNSTVNPICYALCNRTFRKTFKMLLLCRWKKKKVEEKLYWQGNSKLP* *.***.** ****** .*. **. **.
G-Protein binding region ACh binding site Intramolecular disulfide bond Possible site of glycosylation
Palmitoylation site
Transmembrane regions in boxes
Figure 4 Amino acid sequence alignment of the five human muscarinic acetylcholine receptor subtypes. Conserved and semiconserved residues are indicated by the asterisks and dots below the sequences; possible N-terminal glycosylation sites are indicated by yellow shading; the conserved cysteines involved in disulfide bond formation are indicated by blue shading; the N-terminal site of palmitoylation is highlighted; sequences believed to be involved in receptor interactions with G-proteins are indicated by red bars; transmembrane regions are identified by the green shading. Alignment was performed using the ClustalW procedure provided in MacVector.
500 Muscarinic Receptors: Autonomic Neurons 0.198
0.071
0.198 0.236
0.033
0.225 0.225
M4 M2 M5 M3 M1
0.05 Figure 5 UPGMA distance tree illustrating the evolutionary relationship of the muscarinic receptor subtypes revealed by amino acid sequence. M1, M3, and M5 comprise a cluster; M2 and M4 comprise a second cluster. Analysis of nucleotide sequence of the coding regions reveals an essentially similar pattern. The 0.05 scale marker is approximately equivalent to a 5% difference in sequence.
Receptor
M2, M4
M1, M3, M5
Gi
Gq/11
Transducer
− Effector
2nd Messenger
Kinase
+ + Adenylate cyclase
ATP
+ PLC-beta
cAMP
IP3
DAG
PKA
Ca2+
PKC
Figure 6 Muscarinic receptor signaling pathways. The initial stages of signal transduction include receptor, transducer (G-protein), effector (typically an enzyme or ion channel), second messenger production (e.g., Ca2þ, cAMP, cGMP, DAG, and IP3), and protein kinase activation. It has long been appreciated that M2 and M4 receptors inhibit adenylate cyclase through activation of Gi, whereas M1, M3, and M5 receptors stimulate phospholipase C-beta through activation of Gq/11. In fact, muscarinic signaling involves an impressive variety of other pathways. For instance, under the right conditions all receptors can stimulate adenylate cyclase through the activation of Gs. Other pathways are activated directly or secondarily. PKA, protein kinase A; PKC, protein kinase C; IP3, inositol triphosphate; DAG, diacylglycerol.
complex, spanning large distances, and often contain multiple exons that result in multiple splice variants that are under the control of tissue-specific promoters. TATA and CAAT boxes are not involved in the transcriptional control of muscarinic receptor gene expression. The promoter structures of several muscarinic acetylcholine receptor subtypes have been analyzed. Consensus regulatory sequences, including binding sites for SP1, AP-1, and AP-2, as well as tissuespecific silencer elements, have been identified. All muscarinic receptor genes contain a large (10–26 kb) intron close to the 50 end of the translational start site. The relatively simple M1 gene contains an upstream 657 bp exon with a flanking 900 bp promoter region
that contains consensus sites for SP1, NZF-1, AP-1, AP-2, E-box, NF-kB, and Oct-1. The M2 gene spans over 126 kb on chromosome 7, and it contains four exons that result in at least eight different splice variants. The M2 gene has at least three distinct promoters that result in distinct tissue-specific expression, and these promoters are highly conserved between mammalian species. The M3 gene spans at least 285 kb encompassing eight exons (the coding sequence is in exon 8). Multiple AP-2 regulatory sequences are located in the 1.2-bp 50 -flanking promoter region, and the transcription factor AP-2a increases M3 expression. Muscarinic gene sequences tend to be highly conserved; relatively few nonsynonymous polymorphisms have been reported.
Physiological Functions Muscarinic receptors subserve a number of physiological functions. Muscarinic receptors are present in autonomic ganglia and on the target organs of postganglionic parasympathetic fibers. Thus, muscarinic receptors mediate such parasympathetic effects as smooth muscle contraction, blood vessel dilation, decreases in heart rate and cardiac contractility, and glandular secretion. Muscarinic agonists have relatively little use in therapeutics, being limited to stimulating muscles of the gastrointestinal tract and bladder and to lowering intraocular pressure. A great deal of work has focused on developing M1 subtype-specific agonists that might be useful in alleviating the cholinergic losses that characterize Alzheimer’s disease. It has been sporadically proposed that subtype-selective cholinomimetics would be useful in the treatment of psychiatric diseases. Arecoline, an alkaloid found in the betel nuts that are chewed by a significant portion of the world’s population, is undoubtedly the most widely consumed muscarinic agonist. Arecoline causes parasympathetic stimulation (notably increased salivation) and produces euphoria and enhanced cognitive function.
Muscarinic Receptors: Autonomic Neurons 501
Muscarinic antagonists are used clinically to inhibit parasympathetic activity in a variety of situations. For example, antimuscarinic drugs have been used in anesthesia (to reduce secretions), parkinsonism, overactive bladder, bradycardia, and irritable bowel syndrome. In the central nervous system, cholinergic fibers arise in nuclei in the midbrain and pons to diffusely innervate muscarinic receptors on neurons in the thalamus and other diencephalic structures, as well as the reticular formation and other brain stem and cranial nerve nuclei. A second major projection system arises from the basal forebrain cholinergic system (which includes the medial sepal nucleus, diagonal band nuclei, and nucleus basalis) and innervates muscarinic receptors on neurons throughout the telencephalon. Cholinergic interneurons innervating muscarinic receptors are located in several brain structures, including the cerebral cortex, nucleus accumbens, and striatum. Muscarinic receptor density is particularly high in the striatum, cerebral cortex, hippocampus, and other specific nuclei. Muscarinic receptor subtypes display striking regional and laminar heterogeneity (Table 1). Central muscarinic systems are prominently involved in arousal, attention, learning, and memory. Other functions involving central muscarinic actions include movement, analgesia, and temperature regulation. Subsets of M2 and M4 receptors have a presynaptic localization and play a role in the control of neurotransmitter release; the other receptors are predominantly postsynaptic. Analysis of knockout mice lacking expression of specific receptor subtypes has shed considerable light on the physiological functions of receptor subtypes (Table 1). Muscarinic knockout mice are both healthy and fertile; recognition of deficits frequently requires specific focused analysis. These studies reveal that M1 receptors are involved in M current (potassium channel) regulation, agonist-induced seizures mitogen-activated protein kinase (MAPK) activation, certain learning paradigms, and inhibition of a slow, voltage-independent calcium current. M2 receptors are involved in the production of bradycardia; smooth muscle contraction (stomach, urinary bladder, and trachea); and central tremor, analgesia, and hypothermia. M3 receptors are seen to be involved in salivary secretion, pupillary constriction, and bladder detrusor contraction. M4 receptors are seen to be involved in limiting locomotor activity (including modulation of motor responses to dopamine). There is an interesting relationship between M5 receptors and dopamine neurotransmission: M5 receptors facilitate dopamine release, thereby affecting brain reward systems. M5 receptors also appear to play a specific role in the dilation of cerebral arteries and arterioles.
Muscarinic Signal Transduction Muscarinic receptors alter the activity of receptive cells by a number of different signal transduction pathways (Figure 6). The biochemical pathways activated depend on the nature and quantity of the receptor subtype, transducer proteins, effector molecules, and protein kinase substrates expressed in the tissue, as well as the potential for cross-talk between the various transduction pathways. Conventional wisdom holds that the odd-numbered receptor subtypes – M1, M3, and M5 – efficiently couple through pertussis toxin-insensitive G-proteinsofthe Gq/11 family to a stimulation of phospholipase C (b subtype). Phospholipase C releases the second messengers diacylglycerol and IP3 from the membrane phospholipid, phosphatidylinositol-4, 5-bisphosphate. Diacylglycerol activates protein kinase C, whereas IP3 releases Ca2þ from intracellular stores by acting on specific receptors on the endoplasmic reticulum. M1, M3, and M5 also activate the MAP kinase pathway as well as phospholipase D (releasing arachidonic acid). Cross-talk between signaling pathways makes the distinction between proximal and indirect signaling events problematic. M2 and M4, inhibit adenylate cyclase through actions involving Gi (any of the three Gi proteins may be involved). A few, but by no means all, isoforms of mammalian adenylate cyclase are inhibited by Gia subunits (e.g., AC5 and AC6). An even larger number of adenylate cyclase subunits are inhibited by Gbg dimers. Thus, the molecular basis for inhibition of the enzyme is not always obvious. Inactivation of Gi by ADP-ribosylation catalyzed by pertussis toxin uncovers activation of adenylate cyclase by all receptor subtypes. In the case of M3 at least, this activation seems to be mediated by a muscarinic receptor–Gs interaction. Thus, our initial binary classification of muscarinic signaling paradigms (i.e., receptor:transducer:effector ¼ M1/M3/M5:Gs:phospholipase Cb or M2/M4: Gi:adenylate cyclase) has been extended in response to the identification of pathways that involve additional transducer proteins as well as an appreciation of secondary effects and cross-talk between the pathways. Other well-documented muscarinic actions include stimulation of adenylate cyclase, inhibition of phosphodiesterase, stimulation of phospholipase A2 (M1, M3, and M5), and stimulation of phospholipase D (M1 and M3). Muscarinic receptor stimulation activates a number of depolarizing and hyperpolarizing currents through both direct and indirect mechanisms. Prominent effects include (1) stimulation of inwardly rectifying potassium conductances by M2 and M4 receptors; (2) activation of calcium-dependent potassium, chloride,
502 Muscarinic Receptors: Autonomic Neurons
and cation conductances by M1, M3, and M5 receptors; (3) inhibition of the M current, a voltage- and time-dependent potassium conductance, by M1 and M3 receptors; and (4) inhibition of a calcium conductance by M2 and M4 receptors, directly via Go and indirectly via reductions in cAMP. Other novel biochemical effects of muscarinic receptor activation have been described. This signaling heterogeneity may reflect (1) multiple isoforms of signal transduction effectors (e.g., at least 11 isoforms of adenylate cyclase), (2) cross-talk between transduction pathways due to a lack of specificity or the presence of indirect and secondary effects, or (3) cell type-specific differences due to expression of different assortments of signal transduction molecules. Muscarinic receptormediated activation of both phosphodiesterase and nitric oxide synthetase has been reported. Muscarinic receptors have substantial mitogenic potency. The molecular details of muscarinic receptors of the pathways that mediate these effects are incompletely understood. The activities of multiple kinases and phosphatases are affected by muscarinic receptors under specific conditions, including Raf, tyrosine kinases, MAPK, and phosphatidyl inositol-3-kinase. The activities of multiple small GTPases, including Ras and Rho, are also affected by muscarinic receptor activation. Muscarinic signaling is subject to regulation by agonist-induced phosphorylation and downregulation (internalization). Mice deficient in G-protein-coupled receptor kinase-5 display muscarinic supersensitivity. Although domains located in the third intracellular loop are prominently involved in receptor phosphorylation and downregulation, a number of subtypedependent variations in arrestin association and internalization pathways have been described. Muscarinic receptors regulate the expression of a variety of genes, notably a variety of transcription factors and signaling factors. An appreciation of this regulation promises to increase our understanding of muscarinic physiology. See also: Cholinergic Neurotransmission in the
Autonomic and Somatic Motor Nervous System.
Further Reading Ashkenazi A and Peralta EG (1994) Muscarinic acetylcholine receptors. In: Peroutka SJ (ed.) Handbook of Receptors and Channels. G Protein-Coupled Receptors, pp. 1–27. Boca Raton, FL: CRC Press.
Beltran B, Orsi A, Clementi E, and Moncada S (2000) Oxidative stress and S-nitrosylation of proteins in cells. British Journal of Pharmacology 29: 953–960. Birdsall NJ and Lazareno S (2005) Allosterism at muscarinic receptors: Ligands and mechanisms. Mini-Reviews in Medicinal Chemistry 5: 523–543. Brann MR, Klimkowski VJ, and Ellis J (1993) Structure/function relationships of muscarinic acetylcholine receptors. Life Sciences 52: 405–412. Buckley NJ, Bachfischer U, Canut M, et al. (1999) Repression and activation of muscarinic receptor genes. Life Sciences 64: 495–499. Eglen RM (2005) Muscarinic receptor subtype pharmacology and physiology. Progress in Medicinal Chemistry 43: 105–136. Felder CC (1995) Muscarinic acetylcholine receptors: Signal transduction through multiple effectors. FASEB Journal 9: 619–625. Hulme EC, Birdsall NJM, and Buckley NJ (1990) Muscarinic receptor subtypes. Annual Review of Pharmacology and Toxicology 30: 633–673. Ma W, Li BS, Zhang L, and Pant HC (2004) Signaling cascades implicated in muscarinic regulation of proliferation of neural stem and progenitor cells. Drug News and Perspectives 17: 258–266. Nadler LS, Rosoff ML, Hamilton SE, et al. (1999) Molecular analysis of the regulation of muscarinic receptor expression and function. Life Sciences 64: 375–379. Nathanson NM (2000) A multiplicity of muscarinic mechanisms: Enough signaling pathways to take your breath away. Proceedings of the National Academy of Sciences of the United States of America 97: 6245–6247. Saffen D, Mieda M, Okamura M, and Haga T (1999) Control elements of muscarinic receptor gene expression. Life Sciences 64: 479–486. Tobin AB and Budd DC (2003) The anti-apoptotic response of the Gq/11-coupled muscarinic receptor family. Biochemical Society Transactions 31: 1182–1185. von der Kammer H, Demiralay C, Andresen B, et al. (2001) Regulation of gene expression by muscarinic acetylcholine receptors. Biochemical Society Transactions 67: 131–140. Watson S and Arkinstall S (1994) The G-Protein Linked Receptor Facts Book. London: Academic Press. Wess J (1993) Molecular basis of muscarinic acetylcholine receptor function. Trends in Pharmacological Sciences 14: 308–313. Wess J (2004) Muscarinic acetylcholine receptor knockout mice: Novel phenotypes and clinical implications. Annual Review of Pharmacology and Toxicology 44: 423–450.
Relevant Websites http://www.alomone.com – Alomone Labs. http://www.calbiochem.com – Calbiochem. http://www.gpcr.org – GPCRDB: Information system for G-proteincoupled receptors. http://www.scbt.com – Santa Cruz Biotechnology. http://www.sigma-aldrich.com – Sigma–Aldrich. http://stke.sciencemag.org – Signal transduction knowledge environment. http://www.cdna.org – University of Missouri – Rolla cDNA Resource Center.
Nicotinic Acetylcholine Receptors J-P Changeux, Institut Pasteur, Paris, France Y Paas, Bar-Ilan University, Ramat-Gan, Israel ã 2009 Elsevier Ltd. All rights reserved.
Following Claude Bernard’s investigations on the effect of curare, Langley proposed in 1905 that a ‘receptive substance,’ presently designated as a receptor, ‘‘receives the stimulus from the nerve and transmits it to the effector cell.’’ On the basis of the resemblance of acetylcholine (ACh) effects to those of naturally occurring plant alkaloids, Sir Henry Dale distinguished in 1914 between two main classes of receptors for ACh, the muscarinic and the nicotinic receptors. Typically, muscarinic receptors are activated by muscarine and blocked by atropine, whereas nicotinic receptors are activated by nicotine and blocked by curare. Subclasses have also been described. Only nicotinic ACh receptors (nAChRs) are present at the motor endplate of vertebrate skeletal muscles. Muscarinic receptors are found on smooth muscles and gland cells and, together with nicotinic receptors, on autonomic ganglion cells and neurons from the central nervous system.
The nAChR Has an Intrinsic Cationic Channel Binding of ACh to the nAChR causes the opening of a cationic channel that is physically linked to the receptor site (Figure 1). This intrinsic channel is permeable to Naþ, Kþ, and, in many nAChR-subunit combinations, Ca2þ ions. By contrast, muscarinic ACh receptors transduce extracellular signals into the cell by interacting with a G-protein to regulate indirectly effector systems such as adenylate cyclase and ion channels. In 1970, the nAChR became the first neurotransmitter receptor to be isolated and purified and, in 1983, it was the first to be chemically defined by molecular genetic methods. Two factors played a decisive role in this fundamental progress: (1) the presence of very high concentrations of nAChR in the electric organs of the electric fish Electrophorus electricus and Torpedo, and (2) the availability of small polypeptide a-toxins from snake venoms that bind with very high affinity and selectivity to the nAChR from electric organs. The nAChR is an oligomeric glycoprotein of about 300 000 Da. In side views obtained by cryo-electron microscopy, it appears as a transmembrane cylinder of 8 nm diameter and 16 nm length; the long axis being
perpendicular to the membrane plane (Figure 1(a)). In a top view, it looks like a rosette of five subunits organized around a symmetry axis (Figure 1(b)).
Combinatorial Assembly of Subunits Encoded by Homologous Genes Results in a Wide Diversity of nAChRs In the electric organ and muscle, the nAChR oligomer is composed of four different polypeptide chains associated into a pentamer with the a1, g, a1, d, b1 subunits organized in a counterclockwise manner (Figure 1(b)). In nerve cells, combinatorial assembly of up to nine different a subunits (a2 to a10) and three b subunits (b2 to b4) contributes to a wide diversity of pentamers, which may include one, two, or three different types of subunits and possess distinct pharmacological, electrophysiological, and kinetic properties. Each subunit traverses the cell membrane four times and all subunits are in contact with the lipid bilayer. The complete amino acid sequences of the electric organ, muscle, and neuronal subunits in vertebrates and invertebrates have been deduced based on the nucleotide sequence of their cloned cDNA. The primary structure of the nAChR subunits is highly conserved throughout the vertebrate phylum from Torpedo to humans. For instance, the a1 subunit of Torpedo shares 77% sequence identity with the human a1 subunit. Likewise, the human a1 subunit shares 36%, 33%, 30%, and 34% identity with the human b1, g, e, and d subunits, respectively (in mammals, an e subunit replaces the fetal g subunit early in postnatal life). These striking homologies suggest that the various subunits evolved by gene duplication from a common ancestral gene. Tissue-specific expression accounts for the muscle and neuronal subunit subfamilies.
Three Major Domains Arise from the Transmembrane Topology of the nAChR Subunits Each subunit is a polypeptide of 450–700 amino acids that consists of a long extracellular N-terminal segment, four transmembrane hydrophobic segments of about 20 amino acids each (termed M1–M4), a long cytoplasmic segment, and a short extracellular C-terminal tail. The five long extracellular segments fold and assemble into a large extracellular hydrophilic ACh-binding domain (Figure 1). The transmembrane segments form a membrane-embedded domain whose elements are closely organized around
503
504 Nicotinic Acetylcholine Receptors
Figure 1 Structural model of the nACHR from the Torpedo electric ray. Ribbon representations were prepared using coordinates deposited in the research collaboratory for structural bioinformatics (RCSB) protein data bank (PDB) under ID# 2BG9. The ribbon representations are shown on the background of the gray-colored molecular surface. The two facing a1 subunits are colored in orange while the b1, g, and d subunits are shown in cyan, yellow, and green, respectively. (a) Side view, from within the membrane. The thick gray lines delineate the membrane borders. (b) Top view, from the extracellular side. The M2 segments are five a helices closely organized around the axis of ion conduction that is at the center of the molecule and is perpendicular to the viewer. In both panels, the extrcellular end of M2 of the right a1 subunit is labeled with a black asterisk. Note that most of the cytoplasmic domain is missing due to the lack of 3-D structural information.
an axis perpendicular to the membrane plane, delineating thereby an aqueous pore. The five cytoplasmic segments fold into a hydrophilic domain that is thought to associate with components that anchor the receptor to the cytoskeleton, for example, the 43-kDa-Rapsyn protein. The cytoplasmic domain also contains structural elements that modulate channel activity. Notably, the nAChR-subunit topology is common for all other pentameric neurotransmitter receptor channels that are gated by serotonin, glycine, g-aminobutyric acid (GABA), glutamate, or histamine. These ionic channels constitute the Cys-loop receptor superfamily whose members share a conserved disulfide bridge in their extracellular ligandbinding domain. The binding pockets for ACh are formed by the interface between each a subunit and the adjacent subunit. The a subunit contributes, to the ACh-binding pocket, the so-called principal component, which consists of a series of loops carrying highly conserved ACh-binding residues, mainly aromatic but also a cysteine pair. The neighboring subunit contributes a series of complementary ACh-binding residues. Affinity labeling, binding studies, and electrophysiological recordings performed with wild-type (WT) and mutated nAChRs identified ACh-binding pockets at the a/g and a/d interfaces in the case of the electric organ and
muscle nAChRs (Figure 1(b)), a/b interfaces in the case of neuronal heteromeric nAChRs, and a/a interfaces in the case of homomeric neuronal nAChRs. The acetylcholine-binding protein (AChBP) is a glial pentameric water-soluble protein sharing structural homology with the extracellular neurotransmitterbinding domain of the nAChRs’ a subunits. It does not have the transmembrane channel and the cytoplasmic domains (Figure 2). The AChBP is secreted to the synaptic cleft between neurons of the mollusk Lymnaea stagnalis and of Aplysia, where it apparently sequesters ACh, regulating thereby cholinergic transmission. The high-resolution X-ray crystal structure of the AChBP also supports the concept of intersubunit ligand-binding pockets (Figure 2). The five M2 helical segments of the full-length nAChRs line the ion-channel pore (Figure 1), which was initially identified with noncompetitive pore blockers such as chlorpromazine or triphenylmethylphosphonium. Residues belonging to the M2 segments define the activation gate, which acts as a barrier obstructing the flow of ions when the receptor is at rest (i.e., displaying a closed pore). The location of the activation gate is still controversial. On the one hand, three-dimensional (3-D) structural models built on the basis of cryo-electron microscopy suggested that the activation gate is a ‘hydrophobic girdle’
Nicotinic Acetylcholine Receptors 505
Figure 2 X-ray crystal structure of the AChBP from the mollusk Lymnaea stagnalis. Ribbon representations were prepared using coordinates deposited in the PDB under ID# 1UW6. Nicotine molecules are shown at the interface between each two neighboring subunits, as green and blue spheres (carbon and nitrogen atoms, respectively). Note that the five subunits share identical primary, secondary, and tertiary structures. (a) Side view; (b) top view.
located midway between the extracellular and intracellular pore’s vestibules. It was further suggested that the ‘hydrophobic girdle’ breaks apart when the M2 segments rotate, around their own longitudinal axis, upon activation (i.e., upon channel opening). On the other hand, state-dependent accessibility of methanethiosulfonates or zinc ions to cysteines or histidines introduced along the pore revealed that the activation gate is a constriction located close to the intracellular end of the M2 segments, that is, close to the bottom of the pore. These and other structure–function relation studies also indicate that (1) the resting and active states share similar patterns of amino acid accessibility to the pore’s solvent and (2) channel gating predominantly involves rigid tilting motions of the M2 segments, which widen (open) or narrow (close) the bottom-pore constriction. Amino acids located within the M1–M2 connecting segment, close to the intracellular end of the M2 segments, contribute to the cationic (versus anionic) selectivity of the channel. As these residues are also part of the bottom-pore constriction, it is concluded that components of the activation gate act as a selectivity filter upon channel opening.
The nAChR is an Allosteric Protein Cryo-electron microscopy, affinity labeling, binding kinetics of various nicotinic ligands, and rapid kinetic measurements of ion-channel opening and closing events showed that the ACh receptor exhibits properties typical of allosteric proteins. That is, first, the ACh-binding site and the ion channel are spaced far apart (35 A˚). Second, the nAChR can undergo reversible transitions between distinct allosteric conformations even in the absence of an agonist, including rare but detectable spontaneous channel openings. The various allosteric states, which preexist in
reversible equilibrium already before ligand binding, are stabilized not only by agonists or competitive antagonists that bind to the ACh-binding sites, but also by ‘allosteric’ effectors (e.g., Ca2þ ions) that bind to topographically distinct sites. The ion channel of cloned nAChRs expressed in heterologous cell systems opens at relatively high concentration of ACh (KD in the 50–100 mM range), which is close to the concentration of ACh found in the synaptic cleft during transmission. On the other hand, prolonged exposure of the nAChR to the neurotransmitter leads to stabilization of states that display (1) higher affinity for the agonists (KD 1 mM or 5 nM for ACh in case of neuronal or muscle nAChR, respectively) and (2) a closed pore conformation that does not conduct ionic currents. These states are referred to as ‘desensitized,’ and they are stabilized by ACh at equilibrium. Hence, the high-affinity binding states do not take part in the transduction of the physiological signal at the synapse; rather, they play a role in the regulation of the efficacy of signal transmission.
Compartmentalized Expression of Muscle nAChRs Depends on Electric Activity and Release of Neurotrophic Factors In the adult neuromuscular junction, nAChR is localized almost exclusively in the postsynaptic (muscle) membrane of the motor endplate, with a density of 10 000 to 25 000 molecules mm–2 at the crest of the folded membrane. In the embryonic muscle fiber, nAChR molecules are evenly distributed extrasynaptically on the surface of the muscle cell, freely mobile and metabolically labile (half-life of about 18 h). During the formation of the endplate, nAChR molecules aggregate on postsynaptic membrane regions,
506 Nicotinic Acetylcholine Receptors
opposite to the motor-nerve ending, where they become immobile and metabolically stable (half-life of about 11 days). Around birth, in some species, an e subunit replaces the fetal g subunit, giving rise to an adult form with changed channel properties. Meanwhile, the extrasynaptic receptors disappear owing to repression of their synthesis, which takes place as soon as the muscle electric activity starts. In the adult, preventing stimulation of the muscle by sectioning of the motor nerve reactivates the biosynthesis of extrasynaptic nAChR, leading to the so-called phenomenon of ‘denervation hypersensitivity.’ Electrical stimulation of the muscle or its reinnervation by neurons restores the original postsynaptic distribution. Such compartmentalized expression of nAChR genes results, in part, from the regulation of transcription, which is active in most sarcoplasmic nuclei of developing myotubes. In the adult, this process becomes restricted to the subsynaptic ‘fundamental’ nuclei. In the promoter of the receptor genes, distinct elements (N Box vs. E Box) control subsynaptic transcription versus activitydependent extrasynaptic repression. Posttranslational mechanisms include the conformational maturation of the receptor protein, its transit via a specialized Golgi apparatus (in the mature endplate), and its targeting, aggregation, metabolic stabilization, and immobilization in the postsynaptic membrane. Factors of neural origin that are involved in compartmentalizing nAChR gene expression include acetylcholine receptor-inducing activity (ARIA), a factor homologous to human heregulin, and glial growth factor, which binds to tyrosine kinase receptors of the erbB family. Another important component, the acetylcholine receptor-aggregating factor, referred to as AGRIN, elicits formation of nAChR clusters. A cytoskeletal protein 43-kDa-Rapsyn, which is considered to be associated with the nAChR, contributes to AGRIN immobilization and stabilization in the postsynaptic membrane. In the nematode Caenorhabditis elegans, a transmembrane protein, LEV-10, was found to be required for clustering of nAChR molecules in cholinergic neuromuscular junctions. Another transmembrane protein of C. elegans, RIC-3, is involved in enhancing the maturation (subunit folding and assembly) of nAChRs.
nAChRs Play Decisive Physiological and Pathological Roles Activation of nAChRs on the muscle fibers initiates a cascade leading to muscle contraction. Hence, elimination of nAChR molecules from the neuromuscular junction, as occurs in myasthenia gravis, results in severe muscle weakness. The latter is an autoimmune
disease induced by anti-nAChR autoantibodies and aggravated by activation of autoimmune T lymphocytes. Congenital myasthenic syndromes are human disorders resulting from a range of mutations in the muscle nAChR that impair channel activity and may lead to degeneration of the endplate. In the brain, nicotinic receptor genes are expressed exclusively in neurons and display different patterns of expression, from highly restricted to a few nerve cells (e.g., the a2 subunit) to widespread ones (e.g., the b2 subunit). Inactivation of the b2-subunit gene by homologous recombination in mice interferes with their passive avoidance learning and alters a cognitive behavior referred to as ‘exploratory,’ while a more automatic ‘navigatory’ behavior is preserved. nAChRs are responsible for autosomal dominant nocturnal frontal lobe epilepsy and modulate pain transmission, and are also considered to be involved in autism and schizophrenia. Correlations have been reported between cigarette smoking and protection against ulcerative colitis and Parkinson’s disease. Addiction to nicotine involves high-affinity nicotinic receptors associated with midbrain dopaminergic neurons, as demonstrated in laboratory mice lacking the nAChR b2 or a4 subunit. These mice are also useful animal models of attention-deficit activity disorders and sudden infant death syndrome. Nicotinic receptor-binding drugs are considered as potential therapeutic agents in Alzheimer’s disease, Tourette’s syndrome, and anxiety disorders. See also: Cholinergic Pathways in CNS; Cholinergic Neurotransmission in the Autonomic and Somatic Motor Nervous System.
Further Reading Brejc K, van Dijk WJ, Klaassen RV, et al. (2001) Crystal structure of an ACh-binding protein reveals the ligand-binding domain of nicotinic receptors. Nature 411: 269–276. Bourne Y, Talley TT, Hansen SB, et al. (2005) Crystal structure of a Cbtx–AChBP complex reveals essential interactions between snake alpha-neurotoxins and nicotinic receptors. EMBO Journal 24: 1512–1522. Celie PH, van Rossum-Fikkert SE, van Dijk WJ, et al. (2004) Nicotine and carbamylcholine binding to nicotinic acetylcholine receptors as studied in AChBP crystal structures. Neuron 41: 907–914. Changeux JP and Edelstein SJ (2005) Nicotinic Acetylcholine Receptors. New York: Odile Jacob Publishing Corporation. Cymes GD, Ni Y, and Grosman C (2005) Probing ion-channel pores one proton at a time. Nature 438: 975–980. Engel AG, Ohno K, and Sine SM (2003) Neurological diseases: Sleuthing molecular targets for neurological diseases at the neuromuscular junction. Nature Reviews Neuroscience 4: 339–352.
Nicotinic Acetylcholine Receptors 507 Gally C, Eimer S, Richmond JE, et al. (2004) A transmembrane protein required for acetylcholine receptor clustering in Caenorhabditis elegans. Nature 431: 578–582. Halevi S, McKay J, Palfreyman M, et al. (2002) The C. elegans ric-3 gene is required for maturation of nicotinic acetylcholine receptors. EMBO Journal 21: 1012–1020. Hansen SB, Sulzenbacher G, Huxford T, et al. (2005) Structures of Aplysia AChBP complexes with nicotinic agonists and antagonists reveal distinctive binding interfaces and conformations. EMBO Journal 24: 3635–3646. Karlin A (2002) Emerging structure of the nicotinic acetylcholine receptors. Nature Reviews Neuroscience 3: 102–114. Keramidas A, Moorhouse AJ, Schofield PR, et al. (2004) Ligand-gated ion channels: Mechanisms underlying ion selectivity. Progress in Biophysics & Molecular Biology 86: 161–204. Lester HA, Dibas MI, Dahan DS, et al. (2004) Cys-loop receptors: New twists and turns. Trends in Neuroscience 27: 329–336. Lindstrom J (1997) Nicotinic acetylcholine receptors in health and disease. Molecular Neurobiology 15: 193–222. Maskos U, Molles BE, Pons S, et al. (2005) Nicotine reinforcement and cognition restored by targeted expression of nicotinic receptors. Nature 436: 103–107.
Paas Y, Gibor G, Grailhe R, et al. (2005) Pore conformations and gating mechanism of a Cys-loop receptor. Proceedings of the National Academy of Sciences of the United States of America 102: 15877–15882. Sine SM and Engel AG (2006) Recent advances in Cys-loop receptor. Nature 440: 448–455. Sunesen M and Changeux JP (2003) Transcription in neuromuscular junction formation: Who turns on who? Journal of Neurocytology 32: 677–684. Tapper AR, McKinney SL, Nashmi R, et al. (2004) Nicotine activation of alpha4 receptors: Sufficient for reward, tolerance, and sensitization. Science 306: 1029–1032. Taylor P, Osaka H, Molles B, et al. (2000) Contributions of studies of the nicotinic receptor from muscle to defining structural and functional properties of ligand-gated ion channels. In: Clemanti F, Fornasari D, and Gotti C (eds.) Handbook of Experimental Pharmacology: Neuronal Nicotinic Receptors, vol. 144. Berlin: Springer. Unwin N (2005) Refined structure of the nicotinic acetylcholine receptor at 4 A˚ resolution. Journal of Molecular Biology 346: 967–989. Wilson GG and Karlin A (1998) The location of the gate in the acetylcholine receptor channel. Neuron 20: 1269–1281.
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NEUROPEPTIDES AND NEUROTROPHIC FACTORS A. Neuropeptides
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Neuropeptide Synthesis and Storage J A Sobota, B A Eipper, and R E Mains, University of Connecticut, Farmington CT, USA ã 2009 Elsevier Ltd. All rights reserved.
Introduction Small, biologically active peptides are crucial to intercellular communication in most multicellular organisms. Both neuropeptides and classical neurotransmitters are secreted at most nerve terminals. Neurons and endocrine cells have the ability to synthesize bioactive peptides, store them for long periods, and secrete them upon command; many other cells in the body also use peptides for communication, although storage in nonneuroendocrine cells is usually minimal. Peptides are used as neurotransmitters, neuromodulators, neurotoxins, autocrine and paracrine agents acting locally, and growth factors and hormones acting long distance. These peptides range in size from three amino acids (TRH) to more complex, disulfide-linked peptides such as insulin, with its A (21 amino acids) and B (30 amino acids) chains. All bioactive peptides share important common features in their biosynthesis, and these features affect the way in which peptides are stored for secretion in a regulated manner.
Steps in Peptide Biosynthesis Peptides are synthesized as larger inactive precursors with an NH2-terminal signal sequence that guides the insertion of the nascent chain into the lumen of the endoplasmic reticulum (ER), after which the signal sequence is removed (Figure 1). Peptide synthesis always begins in the cell body and requires the ER and the Golgi complex. This is a critical difference from the biosynthesis of small-molecule neurotransmitters such as glutamate, g-aminobutyric acid (GABA), acetylcholine, and norepinephrine, which are synthesized at the nerve terminal and can be replenished locally after depletion during neurotransmission. Some peptide precursors are glycosylated; N-linked oligosaccharide chains are added co-translationally (Figure 1, ER). The NH2-terminal regions of many promolecules contain Cys residues that form intramolecular disulfide bonds; this process occurs in the ER. As intact promolecules proceed through the Golgi complex, N-linked oligosaccharides are modified, O-linked sugar chains can be added, and other modifications such as sulfation and phosphorylation
can occur (Figure 1, Golgi). The same enzymes that carry out these early modifications of peptide precursors are also responsible for modifying integral membrane proteins and other secreted proteins; they are not unique to neuropeptide biosynthesis. Depending on the promolecule and the cell type, the first enzymatic step unique to neuropeptide biosynthesis, endoproteolytic cleavage, may begin to occur at the distal, or exit, side of the Golgi complex in the trans-Golgi network (TGN) (Figure 1). In neurons and endocrine cells, the promolecules and a number of peptide-processing enzymes enter immature secretory granules, where a series of cell-type specific endoproteolytic cleavages proceed more rapidly. As the secretory granules mature, endoproteolytic cleavages continue, along with additional enzymatic modifications of the smaller peptides: COOH-terminal and NH2-terminal exoproteolytic removal of basic amino acids, a-amidation at exposed COOH-terminal Gly residues, conversion of NH2-terminal Gln residues into pyroglutamic acid, and NH2-terminal acetylation. Many of these seemingly minor modifications are essential for biological activity. For many neuropeptides, closely related precursors produce families of related product peptides; examples include the oxytocin and vasopressin precursors; the three precursors for opiate peptides, b-endorphin, methionine-enkephalin, and dynorphin; substance P; and related tachykinins. The information content encoded by neuropeptides is magnified by several factors. Nearly every peptide precursor is expressed in more than one tissue (e.g., procholecystokinin and proenkephalin in the gut and brain), and different sets of product peptides are often found in each tissue. For example, cholecystokinin (CCK-4) is produced in the brain, whereas larger molecules such as CCK-8 and CCK-33 are more prevalent in the gut; methionineenkephalin is produced in the brain, whereas larger molecules such as peptides E and F are found in adrenal chromaffin cells. To add further complexity, several bioactive peptides can be produced from a single precursor. The most widely studied example is proopiomelanocortin (POMC), which can give rise to corticotropin (which stimulates adrenal glucocorticoid production); melanotropins (which cause skin darkening and affect appetite); and b-endorphin, an opiate-active peptide. The anterior pituitary POMC-producing cells (corticotropes) produce corticotropin, but do not make the endoproteolytic cleavages that yield melanotropins or opiate-active peptides. The intermediate pituitary POMC-producing cells cleave corticotropin to
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Ribosomes
mRNA KR
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Q
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cis Oligosaccharide modification
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Figure 1 Steps common to the biosynthesis of neuropeptides. The major steps leading from mRNA encoding a preproneuropeptide to its final products are outlined. The subcellular location of step is indicated. <E, pyroglutamic acid; Ac, acetyl; CPE, carboxypeptidase E/H; ER, endoplasmic reticulum; G, glycine; KR, lysine-arginine; NAT, N-acetyl-transferase; PAM, peptidylglycine a-amidating monooxygenase; PC, propeptide/prohormone convertase; Q, glutamine; QC, glutaminyl cyclase.
produce melanotropin, and POMC neurons in the arcuate nucleus of the hypothalamus produce both b-endorphin and melanotropins. Most of the seemingly minor posttranslational alterations of bioactive peptides are essential for full biological activity. Cholecystokinin is inactive on the gallbladder and pancreas unless a specific Tyr residue is sulfated; opiate peptides are inactivated when their NH2-terminal Tyr residue is N-acetylated. In contrast, NH2-terminal acetylation activates melanotropin, vasopressin is inactive without its COOHterminal a-amide group, and gonadotropin-releasing hormone is inactive without its NH2-terminal pyroglutamic acid residue. Finally, the pattern of precursor processing in a given tissue may be developmentally regulated. Anterior pituitary corticotropes cleave corticotropin to produce melanotropin activity until a time around birth, when adrenocortical function is necessary; at this time, corticotropin cleavage ceases, adrenocorticotropin (ACTH) is produced and glucocorticoid production is stimulated. Transient expression of many peptides and peptide-processing enzymes occurs during development, but the significance of this transient expression is not understood. Diseases associated with peptide biosynthesis include disorders of protein folding within the ER. Unstable and hypofunctional gene products are produced as a result of mutations impairing prohormone folding. Retention and accumulation of mutant proteins within the ER severely challenges its folding capacity and generates an ER stress response. For
example, the Ins2C96Y mutation found in the diabetic Akita mouse prevents the formation of an essential disulfide bond between the A and B chains of insulin, impeding proper folding and the processing of the mutant proinsulin. The misfolded proinsulin is retained in the ER of pancreatic b cells; apoptotic cell death of b cells correlates with the progression of diabetes and is thought to be a consequence of proteotoxicity to the ER. The pathogenesis of the human syndrome hereditary neurogenic diabetes insipidus is similarly thought to be related to ER proteotoxicity, caused by the accumulation of misfolded mutant arginine vasopressin (AVP) in neurons.
Precursor-Processing Enzymes The enzymes responsible for peptide processing fall into two broad classes: those specific for peptide processing and those that are important in the secretory pathway for many other molecules as well as for bioactive peptides. The latter group includes the enzymes responsible for signal peptide cleavage, oligosaccharide attachment and maturation, O-glycosylation, disulfide bond formation, phosphorylation of luminal Ser and Thr residues, and sulfation of Tyr residues. Endoproteolytic cleavage of prohormones frequently occurs at pairs of basic amino acids, although single Arg residues are not uncommon as sites of cleavage. The endoproteases responsible for these cleavages include a set of proteins structurally related to the bacterial subtilisins and to the yeast enzyme
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Kex2. The members of this propeptide/prohormone convertase (PC) family include PC1/3, PC2, PC4, PC5/6A, PC7/8, paired basic amino acid cleaving enzyme (PACE)4, and furin. The subtilisins are all synthesized as inactive precursors with an NH2terminal proregion (Figure 2); the removal of the 80-residue proregion is necessary for catalytic activity and occurs autocatalytically in several cases. PC1/3 and PC2 are enriched in peptide-containing secretory granules, where they cleave prohormones on the C-terminal side of pairs of basic residues (Lys or Arg). PC1/3 and PC2 cleave secretory granule proteins and are not thought to cleave other proteins in the secretory pathway. There is evidence supporting the participation of aspartyl and cysteinyl endoproteases in the biosynthesis of selected peptides. Two enzymes expressed in most neurons and endocrine cells mediate the final events in peptide maturation: carboxypeptidase E/H (CPE) removes the basic residues exposed by the PCs; peptidylglycine a-amidating monooxygenase (PAM) converts COOHterminal Gly residues into a-amide groups. Glutaminyl cyclase (QC), which converts NH2-terminal Gln residues into pyroglutamic acid residues, modifies a small subset of peptides. Much less is known about the N-acetyltransferase (NAT) and aminopeptidase that function in secretory granules. Because they function late in the peptide biosynthetic pathway, these granule enzymes must be active at pH 5–6, the pH of mature secretory granules (Figure 2).
Most of the peptide-processing enzymes require a divalent metal ion for full activity: the PCs are CA2þ-dependent; CPE and QC require zinc; bifunctional PAM requires copper, zinc, iron, and calcium. PAM is unusual in that it requires a cofactor, ascorbic acid, which is oxidized as molecular oxygen is consumed in each reaction cycle. Cytochrome b561, an integral membrane protein, shuttles reducing equivalents from the cytosol into the lumen of the secretory granule, replenishing the luminal supply of reduced ascorbate. Tyrosine sulfotransferase requires adenosine 30 -phosphate,50 -phosphosulfate (PAPS) as the active sulfate donor and Mn2þ. Neuropeptide expression, which varies in response to many stimuli, is generally controlled by regulating the transcription of mRNA encoding the preprohormone. The expression of peptide precursors is usually tightly coordinated with expression of the relevant peptide-processing enzymes. Thus, when the amount of peptide produced is increased, the pattern of postranslational processing is largely unaltered. Secretory rates can temporarily exceed the synthetic rates for peptides, but mRNA levels usually change in response to changes in secretory demand. The activity of each PC is controlled by its proregion, by interactions with specific chaperones, and by the pH within different compartments of the secretory pathway (Figure 2). As for bacterial subtilisin, the proregions of PC1/3 and PC2 bind to their active sites. Autocatalytic cleavage of proPC1/3 occurs in
Autoactivation SAAS
ER pH 7.4
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PHM AscA e−
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QC
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Cu
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AscA Figure 2 Activation of peptide-processing enzymes. Key steps in the activation of PC1 and PC2 are indicated, as are the various divalent metal ions which much be supplied to the PCs and to CPE, PHM, and PAL. Acidification of the luminal compartment is indicated. 7B2 and SAAS are small proteins that serve a mixed chaperone–inhibitor function for the PCs. AscA, ascorbic acid; CD, cytoplasmic domain; CPE, carboxypeptidase E/H; ER, endoplasmic reticulum; PAL, peptidyl-hydroxyglycine a-amidating lyase; PC, propeptide/ prohormone convertase; PHM, peptidylglycine a-hydroxylating monooxygenase; QC, glutaminyl cyclase.
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the ER. The proregion of PC2 is not cleaved until proPC2 reaches the immature secretory granules. The activities of PC1/3 and PC2 are also affected by their interactions with naturally occurring inhibitors, pro-SAAS and 7B2, respectively (Figure 2). In the case of PC2, pro7B2 interacts with proPC2 early in the secretory pathway, facilitating its transport from the ER to the TGN. Whereas the NH2-terminus of 7B2 is essential for PC2 activation, the COOH-terminus of 7B2 inhibits its activity. Like the proregions of PC1 and PC2, the 10 kDa-propeptide fragment of 7B2 acts as an inhibitor, binding at the active site of PC2. Only when these 8–10 kDa peptides are further cleaved do they lose their affinity for their target PC, allowing it to exhibit activity (Figure 2).
Processing Enzyme Knockouts Targeted deletion of processing enzymes in the mouse has increased our knowledge of specific PC–substrate pairs. In some cases, redundant activities were identified via the examination of the peptides present in knockout mice. Disruption of the TGN-localized enzymes furin and PACE4 both result in embryonic lethality, with developmental defects associated with the impaired processing of essential inductive signaling molecules (transforming growth factor (TGF)-b, nodal, and bone morphogenetic protein (BMP) family members). In these cases, the mutant phenotypes partially overlap with, but are less severe than the phenotypes of the substrate knockouts, suggesting that a limited degree of processing enzyme redundancy exists. Mice lacking PC1 are viable, but have a severe block in proinsulin cleavage at the B–C chain junction. Although they are hyperproinsulinemic, they do not show abnormalities in glucose tolerance. Dwarfism occurs due to defective cleavage of the growth hormone-releasing hormone (GHRH) precursor . The PC2-null mice also accumulate proinsulin; cleavage at the A–C junction is impaired. Proglucagon processing is completely blocked, with progressive islet hyperplasia and chronic hypoglycemia. Other PC2 substrates accumulate in the brains of mutant animals: enkephalin, CCK, orphanin FQ/nociceptin, and dynorphin. Homozygous mutants for the amidating enzyme PAM die between embryonic day (E)14.5 and E15.5, with severe generalized edema. This phenotype is strikingly similar to the adrenomedullin (AM) knockout phenotype; the AM gene encodes two amidated peptides which function as cardiovascular regulators, and amidation is likely essential for their full bioactivity. Naturally occurring mutations in processing enzymes are also known. In humans, a syndrome associated with PC1 deficiency has been reported;
this syndrome includes extreme childhood obesity, abnormal glucose homeostasis, hypocortisolism, amenorrhea, and impaired gastrointestional (GI) function. In addition, CPE missense mutations have been reported in patients with type 2 diabetes. In mice, another CPE mutation was found (S202P); the mutant CPE (CPEfat/fat) is inactive and rapidly degraded. These CPEfat/fat mice are infertile, hyperglycemic, and develop late-onset obesity. The phenotype of the true CPE null mice is similar, although more severe than that of the CPEfat/fat mice. Additional carboxypeptidase family members are probably responsible for the retention of reduced peptide processing that prevents lethality in these animals. Carboxypeptidase D (CPD), which is localized to the TGN and immature granules, probably plays a moderate role because it can only complete reactions initiated at the TGN.
Granule Biogenesis and Storage Neuroendocrine cells producing proinsulin or POMC generally secrete equimolar amounts of all of the products derived from the promolecule. This means that product peptides with no clearly defined function (e.g., C-peptide derived from proinsulin) are secreted along with the active peptide(s). However, the separation of product peptides into different granules and differential secretionhas been observed in other cell types. Following the synthesis and translocation of nascent polypeptide chains into the lumen of the ER, secretory proteins are transported through the Golgi complex to the TGN. Once this sorting station is reached, vesicles destined for different subcellular compartments acquire the correct cargo. In all cell types, a constitutive pathway delivers soluble secretory proteins directly to the plasma membrane, where they are released without the need for a stimulus. In addition to the constitutive pathway, a regulated pathway or pathways exist in neurons and endocrine cells; regulated proteins are stored in secretory granules until released in response to an incoming signal. Two models, not mutually exclusive, provide a framework for thinking about the mechanisms by which constitutive and regulated soluble proteins are targeted to their respective pathways for secretion. One model, known as sorting for entry, suggests that granule content proteins bind selectively to a membrane receptor in the TGN (Figure 3). This could involve either direct binding to a membrane component of the TGN such as cholesterol-rich membrane microdomains (i.e., lipid rafts) or interaction with a specific sorting receptor. Studies on PC2 support a role for lipid rafts in its entry into the regulated
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TGN
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Mature granules: responsive to secretagogue
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Unstructured cargo: NPY, ACTH
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Figure 3 Intracellular sorting of neuropeptides and enzymes to different vesicle populations. Although some sorting begins in the ER/ Golgi, most occurs in the TGN and immature secretory granules which undergo significant remodeling. Some cargo is unstructured and mixes with other unstructured cargo; other cargo self-aggregates, excluding other molecules. There is evidence for cargo receptors (proteins and lipid rafts) which steer molecules into regulated granules. A number of proteins present in immature secretory granule membranes must be removed for mature secretory granules to function properly. ACTH, adrenocorticotropin; CgA, chromogranin A; CPE, carboxypeptidase E/H; ER, endoplasmic reticulum; NPY, neuropeptide Y; PC, propeptide/prohormone convertase; PHM, peptide histidine-methionine; TGN, trans-Golgi network; VAMP4, vesicle-associated membrane protein 4.
pathway. It has been proposed that CPE binds prohormone aggregates in the TGN and, through its association with lipid rafts, serves as a sorting receptormediating prohormone entry into regulated granules. Proteins unable to bind to the sorting receptor enter the constitutive pathway by default. A second sorting model, known as sorting by retention, is based on the idea that protein entry into immature granules occurs by bulk flow; the retention of regulated cargo is facilitated by the aggregation and condensation of these proteins. Nonaggregating cargo proteins may be retained through membrane interaction. In professional secretory cells, in which over 10% of the total protein synthesis may be one preprohormone, it is difficult to envision a specific receptor with a large enough capacity to accomplish sorting for entry. Granule trafficking of peptide-processing enzymes with transmembrane domains (PAM, CPD, and PC6) is mediated by signals in their cytoplasmic domains and can be regulated by phosphorylation. Whereas soluble processing enzymes are secreted along with their product peptides, integral membrane-processing enzymes can be retrieved from immature granules during maturation and from the endocytic pathway following exocytosis. Remodeling of the membranes of immature granules is an essential part of the granule maturation process and must occur before secretion can be stimulated. Clathrin is recruited to immature granules by the
adaptor protein Golgi-associated g-ear-containing ADP-ribosylation-factor-binding-protein (GGA), and the selective removal of nongranule proteins occurs via budding of clathrin-coated vesicles. In this manner, the immature granule functions as a more advanced sorting station, succeeding the TGN. Experimental support for this type of remodeling process comes from the progressive removal of transfected exocrine proteins from granules in AtT-20 cells, as well as the transient presence of lysosomal proteins in immature granules. Although a conserved sorting signal does not exist for regulated secretory proteins, sequence and structural similarities have been found in subsets of proteins. An N-terminal sorting signal comprising a hydrophobic domain, characteristic amino acid content, and amphipathic a-helix has been identified. Structural features of the sorting domain, such as N-terminal disulfide-bonded loops, have been proposed to be important for stabilizing its structure and may contribute to the routing of POMC, chromogranin B, and chromogranin A. Although loopmediated homodimerization of chromogranin A is required for its sorting into the regulated secretory pathway, there is disagreement on the importance of the disulfide loops of POMC. A key feature in the sorting of regulated secretory proteins is their ability to selectively aggregate
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(Figure 3). Aggregation may begin as early as the ER and may result in homotypic aggregates of secretory proteins. For pancreatic exocrine proteins, the majority of condensation occurs between the ER and cisGolgi; aggregated atrial natriuretic peptide (ANP) has been identified in all layers of the Golgi complex. Other granule proteins, most notably members of the granin family, are known to aggregate when exposed to the slightly acidic (pH <6.5), high calcium (millimolar) conditions in the TGN. Low pH is also important for the aggregation of CPE, insulin, prolactin, POMC, and proPC2; the aggregation of POMC also requires Ca2þ, and the aggregation of prolactin requires Zn2þ. The aggregation conditions required for each protein may be related to its isoelectric point. Constitutively secreted proteins are excluded from these aggregates. The granins are thought to function as aggregation vehicles and could act by binding or co-aggregating with other secretory proteins. It is in this way that chromogranin A may act as an assembly factor that initiates aggregation. Finally, the ability of some granule content proteins to generate granulelike structures when expressed in cells lacking a regulated secretory pathway is consistent with an important role for aggregation. The mechanisms by which integral membraneprocessing enzymes are targeted to the regulated pathway are fundamentally different from those governing soluble proteins. The cytosolic domains of granule membrane proteins contain information essential for routing. For example, sorting of the vesicular monoamine transporter (VMAT) to secretory granules requires its COOH-terminal extended dileucine motif, suggesting an interaction with adaptor proteins. PAM proteins with a truncated cytoplasmic domain localize to the plasma membrane instead of granules. Cytosolic proteins that interact with PAM and regulate its trafficking include Rho guanosine diphosphate (GDP)/guanosine triphosphate (GTP) exchange factors (kalirin and trio) and a Ser/ Thr kinase (P-CIP2). The cytoplasmic domain of PAM is multiply phosphorylated and cycles of phosphorylation/dephosphorylation are essential for normal trafficking within both the biosynthetic and endocytic pathways. Furin enters immature secretory granules, but is excluded from mature granules, returning to the TGN; furin also cycles between the plasma membrane and endosomes. Both the TGN localization of furin and its retrieval from endosomes are mediated by phosphofurin acidic cluster-sorting protein (PACS)-1, which recognizes furin that has been phosphorylated by casein kinase (CK)II. This interaction facilitates binding of adaptor protein (AP-)1, which mediates the entry of furin into clathrin-coated vesicles. PACS-1 also controls the
endosome to TGN trafficking of CPD. Like furin, CPD cycles between the TGN and cell surface; the binding of adaptor proteins to its 58-residue cytosolic tail is required for trafficking, which is dependent on the phosphorylation of its CKII sites. The dynamic regulation of secretory granule membrane composition also plays a role in the acquisition of regulatability. Secretagogue application does not stimulate the exocytosis of immature granules. The maturation of immature secretory granules involves the removal of vesicle-associated membrane protein (VAMP)4 and synaptotagmin IV; removal is an ADP-ribosylation factor (ARF)-dependent process. Remodeling of granule membranes is inhibited by brefeldin A, causing granules to remain in a stimulusinsensitive state. Mature granules acquire the ability to be stimulus responsive. Removal of synaptotagmin IV, a potential inhibitor of Ca2þ triggered exocytosis, may be the key to establishing competence to undergo regulated exocytosis. Retrieval of VAMP4 requires phosphorylation-dependent recruitment of AP-1 to immature granules, a process mediated by PACS-1, suggesting that the removal of these inhibitory proteins from immature granules is likely to occur via clathrin-coated vesicles.
Peptides in the Nervous System Although most work on the synthesis and storage of neuropeptides has been performed in endocrine and neuroendocrine primary cell cultures and cell lines, the general principles apply to primary neurons. In both the central and peripheral nervous systems, neuropeptides have a widespread distribution and are processed primarily by the endoproteases PC1/3 and PC2. The majority of neurons coexpress peptides along with classical low-molecular-weight neurotransmitters such as glutamate, GABA, acetycholine, and biogenic amines. The two types of neurotransmitter are stored in different subcellular compartments within the same neuron. For neuropeptides, secretory granules (also known as large dense-core vesicles) are the only site of storage. These granules are localized to the neuronal cell soma, axons, and dendrites. In contrast to neuropeptides, small-molecule neurotransmitters are localized to small synaptic vesicles, which are concentrated at axon terminals (Figure 4). One major functional implication of this differential distribution of peptides compared to smallmolecule transmitters is that release is selective. Whereas the release of small-molecule neurotransmitters is triggered by a small localized increase in cytoplasmic Ca2þ, the release of neurotransmitter from large dense-core vesicles requires a greater increase in Ca2þ in the terminal. Low-frequency stimulation may
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LDCV Vasopressin Galanin Nuc
SSV Microtubules and neurofilaments
ms
ms
Figure 4 Peptidergic transmission in neurons. Most neurons contain large dense-core vesicles (secretory granules) and small synaptic vesicles (SSVs). The SSVs can be emptied and refilled at the nerve terminal while the large dense-core vesicles, which contain peptides, cannot be refilled. When two peptide precursors are expressed in one cell, the products can be packaged in the same granules or packaged into separate granules that are trafficked to different parts of the cell. LDCV, large dense-core vesicles; Nuc, nucleus. Adapted from Landry M, Vila-Porcile E, Hokfelt T, and Calas A (2003) Differential routing of coexisting neuropeptides in vasopressin neurons. European Journal of Neuroscience 17: 579–589.
release only small synaptic vesicles, whereas highfrequency stimulation releases a combination of peptides and small-molecule neurotransmitters. Consistent with the widespread distribution of peptide receptors, peptide release can occur at a significant distance from any synapse. Peptides can diffuse a significant distance after release and often mediate longer-lasting responses than classical neurotransmitters. Furthermore, depending on the stimulus, peptide release from secretory granules may be axonal or somatodendritic and may occur in either a coordinated or independent manner. Secretion from axons versus dendrites provides neuropeptides to different target tissues and produces different physiological effects. For example, thyrotropin-releasing hormone (TRH) neurons in the hypothalamic paraventricular nucleus (PVN) project axons to the median eminence, where TRH entry into the hypophysial-portal system stimulates TSH release from the anterior pituitary. Intra-PVN release of TRH from the same neurons may contribute to the regulation of sympathetic outflow during cold stress. In the magnocellular neurons of the supraoptic nucleus (SON), local oxytocin release is stimulated by suckling before a significant peripheral secretion of oxytocin occurs. After osmotic stimulation, the release of oxytocin and AVP within the SON lags behind peripheral secretion.
Neurons often express more than one neuropeptide precursor. The products of the two precursors may be stored in separate granules or may be stored in the same granules; co-storage usually implies corelease and co-transmission. One such example is the primary sensory neurons of the dorsal root and trigeminal ganglia; tachykinins co-localize with calcitonin gene-related peptide (CGRP) in neuronal cell bodies and at the central and peripheral terminals. In this case, there does not appear to be a selective transport mechanism to form different granules to be shipped to functionally distinct processes. CCK and corticotropin-releasing hormone (CRH) are similarly co-stored in the periportal nerve terminals in the rat median eminence. In contrast, in hypothalamic magnocellular neurons, galanin and vasopressin, are only partially co-stored; galanin-containing granules are preferentially targeted to dendrites whereas vasopressin-containing granules are targeted to axons that extend into the posterior pituitary. These neurons have regions of their ER devoted to the synthesis of vasopressin and other regions devoted to galanin synthesis; separation at this early stage may promote packaging into separate granules, which would allow targeting to dendrites versus axons. The bag cell neurons of Aplysia contain granules filled with peptides derived from the N-terminal part of the egg-laying hormone precursor and granules filled with peptides derived from its C-terminal region; cleavage of the egg-laying hormone precursor occurs before exit from the Golgi complex. Granules containing N-terminalderived bag cell peptides are released locally, whereas granules containing C-terminal-derived egg-laying hormone are released into the circulation.
Genes Controlling Expression of Granules and Granule Proteins That a master gene may control secretory granule biogenesis and the expression of granule proteins has been proposed as a mechanism by which these processes are coordinately regulated. Although the idea is controversial, the acidic glycoprotein chromogranin A (CgA) has been put forth as a candidate master molecule responsible for regulating the initiation of secretory granule biogenesis. Antisense RNAmediated reductions of CgA expression in PC12 cells result in proportional granule depletion, with a loss of regulation of other content proteins. Consistent with this, a transgenic mouse line with antisense-mediated reduction in CgA expression resulted in aberrant adrenal chromaffin cell granules. In addition, the expression of CgA in nonendocrine cells induced the formation of granulelike structures. However, analysis of one line of CgA knockout mice revealed no
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structural abnormalities in adrenal chromaffin cells; vesicle size and number were normal, with upregulation of other granin proteins probably compensating for the CgA deficiency. In an independent line of CgA knockout mice, there were significant changes in chromaffin granule number and appearance, along with marked hypertension, but no general endocrine problems that would be expected if CgA were crucial to the function of all large dense-core vesicles. The fact that not all cells with a regulated secretory pathway contain CgA (e.g., pituitary sommatotropes and mammotropes, which have CgB) also argues against an essential role for CgA for all peptide-secreting cells. CgA may serve as an assembly factor; low pH and Ca2þ clearly induce CgA aggregation in the TGN, and aggregated CgA may specifically bind to or co-aggregate with cargo proteins. An additional role for chromogranin A in granule biogenesis may be to protect regulated secretory proteins from degradation by upregulating the expression of protease nexin-1, an endogenous serine protease inhibitor. In Drosophila, the neuropeptidergic phenotype is under the control of the basic helix-loop-helix protein DIMM, a member of the atonal family. DIMM is related to similar molecules in vertebrates such as neuroD, neurogenin, and olig. Recently, dual functionality has been proposed for DIMM. In addition to being a critical regulator of neuropeptide gene expression in differentiating cells, spatiotemporal manipulation of the dimm gene revealed that DIMM regulates the expression of neuropeptides and peptide histidine-methionine (PHM) in mature neurons. This ability to regulate secretory protein expression suggests an effective mechanism by which peptidergic signaling may be maintained and dynamically adjusted in fully differentiated cells. See also: Neuropeptide Release.
Further Reading Arvan P (2004) Secretory protein trafficking: Genetic and biochemical analysis. Cell Biochemistry and Biophysics 40: 169–178. Borgonovo B, Ouwendijk J, and Solimena M (2006) Biogenesis of secretory granules. Current Opinion in Cell Biology 18: 365–370.
Czyzyk TA, Morgan DJ, Peng B, et al. (2003) Targeted mutagenesis of processing enzymes and regulators: Implications for development and physiology. Journal of Neuroscience Research 74: 446–455. Day R and Gorr SU (2003) Secretory granule biogenesis and chromogranin A: Master gene, on/off switch or assembly factor? Trends in Endocrinology and Metabolism 14: 10–13. Eaton BA, Haugwitz M, Lau D, and Moore HP (2000) Biogenesis of regulated exocytotic carriers in neuroendocrine cells. Journal of Neuroscience 20: 7334–7344. Fisher JM, Sossin W, Newcomb R, and Scheller RH (1988) Multiple neuropeptides derived from a common precursor are differentially packaged and transported. Cell 54: 813–822. Fortenberry Y, Hwang JR, Apletalina EV, and Lindberg I (2002) Functional characterization of ProSAAS: Similarities and differences with 7B2. Journal of Biological Chemistry 277: 5175–5186. Fricker LD and Leiter EH (1999) Peptides, enzymes and obesity: New insights from a ‘‘dead’’ enzyme. Trends in Biochemical Sciences 24: 390–393. Hewes RS, Gu T, Brewster JA, Qu C, and Zhao T (2006) Regulation of secretory protein expression in mature cells by DIMM, a basic helix-loop-helix neuroendocrine differentiation factor. Journal of Neuroscience 26: 7860–7869. Landry M, Vila-Porcile E, Hokfelt T, and Calas A (2003) Differential routing of coexisting neuropeptides in vasopressin neurons. European Journal of Neuroscience 17: 579–589. Merighi A (2002) Costorage and coexistence of neuropeptides in the mammalian CNS. Progress in Neurobiology 66: 161–190. Prigge ST, Mains RE, Eipper BA, and Amzel LM (2000) New insights into copper monooxygenases and peptide amidation: Structure, mechanism and function. Cellular and Molecular Life Sciences 57: 1236–1259. Ron D (2002) Proteotoxicity in the endoplasmic reticulum: Lessons from the Akita diabetic mouse. Journal of Clinical Investigations 109: 443–445. Seidah NG and Prat A (2002) Precursor convertases in the secretory pathway, cytosol and extracellular milieu. Essays in Biochemistry 38: 79–94. Taylor NA, Van de Ven WJ, and Creemers JW (2003) Curbing activation: Proprotein convertases in homeostasis and pathology. FASEB Journal 17: 1215–1227. Thiele C and Huttner WB (1998) Protein and lipid sorting from the trans-Golgi network to secretory granules-recent developments. Seminars in Cell and Developmental Biology 9: 511–516. Thomas G (2002) Furin at the cutting edge: From protein traffic to embryogenesis and disease. Nature Reviews Molecular Cell Biology 3: 753–766. Zhu YL, Conway-Campbell B, Waters MJ, and Dannies PS (2002) Prolonged retention after aggregation into secretory granules of human R183H-growth hormone (GH) a mutant that causes autosomal dominant GH deficiency type II. Endocrinology 143: 4243–4248.
Neuropeptide Release F Bergquist and M Ludwig, University of Edinburgh, Edinburgh, UK ã 2009 Elsevier Ltd. All rights reserved.
Introduction A main criterion for interneuronal chemical messengers, including neurotransmitters, neuromodulators, and neurohormones, is that their release be regulated and conditioned by neuronal activity. This article discusses the regulated release of neuropeptides, a large class of neuroactive messengers present in both the central nervous system (CNS) and peripheral nervous system. Other characteristics of neuropeptides that are vital for their function as interneuronal messengers, such as receptor effects, synthesis, storage, and breakdown/inactivation are also highlighted here because it is important to understand how neuropeptide release differs from classical neurotransmitter release. The neuropeptide messenger family is large, and the list of members is still increasing steadily some 70 years after Gaddum and von Euler’s discovery of substance P. More than 100 neuropeptides have established messenger properties. For most of these, the mechanisms of release have not been studied in detail, but there seem to be some common themes regarding activity-dependent neuropeptide release that allow some generalizations. The following section discusses some characteristics that are believed to be common to the release of most neuropeptides.
Neuropeptide Release Compared to Classical Neurotransmitter Release Synthesis
Peptides are synthesized as larger precursors in the cell body but not in axons and axon terminals. The precursors are stored in vesicles where they are broken down by convertases into active peptides, and the vesicles are transported to the release sites. The supply of neuropeptides can, therefore, not be replenished quickly by synthesis at the release site, as is the case with conventional neurotransmitters. Thus there are mechanisms that economize the release of peptidergic vesicles, and transport mechanisms that can deal with changes in demand at particular release sites. Vesicle Types and Release Sites
Releasable neuropeptides are always stored in and released by exocytosis from large dense-core vesicles
(LDCVs). LDCVs differ from the other main class of neurotransmitter vesicles, small synaptic vesicles (SSVs) not only by size and physical density but also by the different composition of membrane proteins involved in the release processes and by a different cellular localization (Figure 1). SSVs are the predominant vehicle for classical neurotransmitter release, although amino acids and acetylcholine are also released from dense-core vesicles to some extent, together with peptides and sometimes amines. As indicated by the name, SSVs are mostly found at synapses, and release from SSVs is restricted to these highly specialized membrane structures (active zones). In contrast, LDCVs are broadly distributed and can release their contents from any part of the neuron, including the cell body and the dendrites. Release of classical transmitters from SSVs is thus restricted spatially, and the action of a classical transmitter is further constrained to the release sites by a highly efficient termination-of-release signal by uptake mechanisms and a fast breakdown. Peptidergic signaling, on the other hand, mostly takes place outside synaptic specializations (Figure 1). Calcium Dependent Release
All activity-dependent peptide release appears to be triggered by an increase in intracellular Ca2þ concentration. The release machinery that mediates neuropeptide release has a higher affinity for Ca2þ than that involved in release of classical transmitters. This is necessary because, unlike SSVs, LDCVs are not positioned close to Ca2þ channels, and the Ca2þ that enters through these channels in response to depolarization is rapidly buffered on its way into the cell. The Ca2þ sensor on SSVs is synaptotagmin I, but the identity of the Ca2þ sensor on LCDVs is not yet known. When neuropeptides are released from dendrites, the release can be triggered not only by influx of Ca2þ via channels in the cell membrane but also by the mobilization of Ca2þ from intracellular stores. Timescale of Neuropeptide Release and the Type of Stimulus Required
The characteristic Ca2þ dependence of LDCV fusion influences the speed at which neuropeptides are released. Classical neurotransmitters in SSVs are typically released with latencies of 0.3–1 ms after a Ca2þ trigger, whereas the latencies of LDCV release range from 30 to 2000 ms. The long LDCV latencies arise partly from the wider distances between LDCV Ca2þ sensors and the sources of Ca2þ entry into the cytosol,
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Figure 1 Characteristic features of the small synaptic vesicle (SSV) and large dense-core vesicle (LDCV) release mechanisms. The SSV exocytosis (upper-left insert) can be evoked by low-frequency firing or single action potentials. The vast majority of docked SSVs are primed and therefore readily releasable. When an action potential reaches the axon terminal, voltage-sensitive Ca2þ channels open and increase the intracellular Ca2þ concentration close to the cell membrane to high micromolar levels that trigger SSV release (intracellular Ca2þ marked as blue dots). SSVs are found in close apposition to the Ca2þ channels and are released with short latencies after the arrival of an action potential. The SSVs can either be integrated into the terminal membrane for later recycling or release their contents by a kiss-and-run mechanism. SSV neurotransmitters are released into a narrow synaptic cleft, where they act on the postsynaptic receptors. Efficient uptake and/or breakdown processes rapidly terminate the signal, and very few transmitter molecules escape the synaptic cleft to act at a distance from the release site. SSVs are refilled by local neurotransmitter synthesis and/or uptake of already released neurotransmitters. The LDCV release of neuropeptides (upper-right insert) can take place from any part of the neuron. LDCVs typically require high-frequency or burst-patterned firing to be released. Compared to SSVs, a much smaller fraction of the docked LDCVs are primed, and only a small fraction of the vesicles present at a release site are available for release. There is no close relationship between Ca2þ channels and LDCVs, and LDCVs have a more sensitive Ca2þ sensor that responds to the lower Ca2þ concentrations that are attained at a distance from the Ca2þ source. The release latencies are long, from tens of milliseconds to seconds. Neuropeptides are released outside synaptic specializations and are not recycled into the releasing neuron but are broken down slowly in the extracellular space. Often the breakdown results in one or more active metabolites that require further breakdown to be inactivated. Consequently neuropeptides have a long range of action. Synthesis and packaging of neuropeptides only take place in the cell body, and all LDCVs need to be transported from there to the release sites. LDCVs in transport are captured into the releasable pool of vesicles or the reserve pool at active release sites. The capture of LDCVs is believed to be upregulated when there is increased demand for release at a particular site. Somatodendritic oxytocin release (lower insert) can be triggered by Ca2þ from stores in the endoplasmatic reticulum (ER) in addition to the inflow of extracellular Ca2þ. In vasopressin and oxytocin neurons, mobilization of intracellular Ca2þ can lead to the sustained enhancement of release by promoting recruitment of LDCVs to the releasable pool. Oxytocin cells can autoregulate the dendritic release by the activation of oxytocin receptors (OTRs) that release intracellular Ca2þ via an inositol-3,4,5-phosphate (IP3)-mediated pathway.
and partly from differences in the release machineries of SSVs and LDCVs. Neuropeptides require strong stimulation, such as high-frequency firing or burstpatterned firing, to be released, whereas classical neurotransmitters respond also to low frequencies and even single action potentials (Figure 1). The requirement of more intensive stimulation for neuropeptide release is probably linked to the long latencies to
release and reflect a need for repeated depolarizations and accumulation of intracellular Ca2þ. The reason why neuropeptides play hard to get in this fashion might be that it is disadvantageous to trigger a prolonged and widespread signal too easily, but also it is time- and energy-demanding to refill the neuropeptide stores. Some neuropeptides act in an autoinhibitory fashion, and the restrictive release of such messengers
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could serve as an emergency brake that prevents cellular excitotoxic damage or could contribute to cyclic activity in neuronal networks. Molecular Release Mechanisms – SNARE Machinery
The molecular mechanisms of exocytosis have been studied in some detail over several decades. Some of the cellular systems that have been most useful for these studies examine mainly LDCV fusion; this is the case in adrenal chromaffin cells, from which release of easily oxidized catecholamines can be monitored with high time resolution using carbon-fiber electrodes, and pancreatic b-cells, in which insulin release is usually monitored as changes in cell capacitance. Detailed information about vesicle kinetics and exocytosis has also been obtained by expressing fluorescent tags directed to vesicle membrane proteins or lumen. Less is known regarding exocytosis in peptidergic neurons, but it is usually assumed that LDCVs in neurons fuse with the cell membrane by a mechanism similar to those of peripheral LDCVs because there are many recurring motifs in peripheral and central exocytosis. Here we only briefly describe the characteristics particular for the release of LDCVs. LDCV exocytosis from endocrine cells occurs from a small fraction of vesicles present near the cell membrane, the releasable pool (1% of vesicles present at the release site). Some of these vesicles are immediately releasable, but a larger portion requires a longer time to be released. The release process involves several steps and includes the formation of soluble N-ethylmaleimide-sensitive factor attached protein receptor (SNARE) complexes consisting of vesicular proteins (vSNARE), cell membrane-bound proteins (target SNARE or tSNARE), and interactions between SNARE complexes and soluble proteins. Before a vesicle can undergo exocytosis, it must be docked to the cell membrane. However, not all docked vesicles are releasable; the docked vesicle needs further preparation, which might involve the binding of Munc13 or Ca2þ-dependent activator protein for secretion (CAPS) in a process called priming. The docking and priming processes ensure that only some of the available vesicles are released and that a reserve pool is retained for subsequent release, minimizing the need for newly synthesized peptides (Figure 1). However, in situations of increased demand, it appears that newly synthesized vesicles are released before the already-present reserve pool. The reason for this is unclear, but it might be a way to update the composition of the vesicle content. SSVs also undergo docking and priming before release, but the priming process is more efficient. For example, nearly all docked vesicles are primed and ready to be released in hippocampal neurons. CAPS
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appears to be essential for the late stages of LDCV exocytosis, but is not necessary for SSV exocytosis, indicating molecular differences in the priming and release processes of the two types of vesicles. The fusion process is believed to be similar, but probably not identical, for SSVs and LDCVs. In principle, the fusion of vesicle and cell membrane can range from being total, including a flattening and integration of the vesicle membrane into the plasma membrane, to being restricted to a short-lived fusion pore, which may or may not allow the vesicle content to escape. Overall, the fusion times appear to be longer for LDCVs than for SSVs, and although fast retrieval of intact vesicles (called kiss-and-run events) occur with LDCVs, quick recycling of intact vesicles is functionally more important for classical neurotransmitter release, in which the vesicle can be quickly refilled or topped up (Figure 1). Termination of the Signal
The termination of a signal is critical for information transfer. The simpler the signal is, the more important this is. If we wanted to use a flashlight to send a signal in the dark, a simple solution might be to switch the flashlight on and off in Morse code. However, if we had a box of matches instead of a flashlight, it would be laborious to create a Morse code signal. A better approach might be to write our message on the wall and illuminate it by lighting a few matches. In the brain, neuropeptides signal like illuminating matches, and the message that they convey is dictated by the pattern of receptor expression, which in this metaphor is the writing on the wall. The signaling mode of classical transmitters resembles the Morse code approach, and to achieve this, the action of classical transmitters is both rapidly turned on and rapidly terminated. Efficient uptake mechanisms and fast breakdowns ensure that the signal can be repeated with high frequencies. By contrast, neuropeptide signals are terminated by the slow breakdown of the peptides by extracellular peptidases. The cerebral half-lives for the neuropeptides vasopressin and oxytocin, for example, are 20 min, whereas fast-acting classical neurotransmitters typically have half-lives of 5 ms. Because neuropeptides display high-affinity interactions with their receptors (typically in low nanomolar ranges), the action can be widespread in both time and space. This kind of transmission, in which molecules act at a distance from the release site, is sometimes referred to as paracrine, indicating that the message indiscriminately reaches other cells near the site of release. Neuropeptides can, in this respect, be thought of as paracrine messengers in the brain, and an important function appears to be to convey persistent modulating or tuning effects on
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neuronal networks rather than to transmit immediate but fugacious messages. The lack of peptide uptake mechanisms has an important bearing on release dynamics; unlike amines and amino acids, a released peptide cannot be reused but must be synthesized de novo whenever it is released. There might be some exceptions; a highaffinity uptake of cholescystokinin has been reported, for example. Neuropeptide Release Is Not Uniform
Variations between different neurons and over time Although the release of different neuropeptides shares characteristics that are related to the type of vesicle used, there are many variations. Even the morphological appearances of LDCVs indicate differences. LDCVs vary in diameter from 60 to 500 nm. This variation occurs between and within neurons of a population, as well as between different neuron populations. However, it is not clear whether different sizes are associated with differences in release mechanisms other than the obvious difference in released quanta. The content of individual vesicles varies not only between neurons but also within them. LDCVs often contain more than one neuropeptide, and in fact many neurons release a mixture of neuropeptides, sometimes referred to as a neuropeptide cocktail. For instance, vasopressin coexists with dynorphin and galanin in magnocellular neurons of the supraoptic nucleus and paraventricular nucleus and with corticotropinreleasing hormone (CRH) in parvocellular neurons of the paraventricular nucleus. On the other hand, oxytocin in the supraoptic nucleus coexists with enkephalin, dynorphin, cocaine- and amphetamine-regulated transcript (CART), and cholecystokinin. Neuropeptides can also be co-released with classical neurotransmitters such as acetylcholine and g-aminobutyric acid (GABA) or neuromodulators such as amines, which provide additional flavors to the cocktail. For example, the neuropeptide Y (NPY) neurons also make the classical inhibitory neurotransmitter GABA. Moreover, most central neuropeptide-releasing neurons appear to have at least one major classical neurotransmitter that sustains fast neurotransmission via SSV release, in addition to the slower effects of neuropeptide release from LDCVs. The amount of co-released and/or co-expressed classical transmitters can also be subject of variation, contributing to the diversity of neuropeptidergic transmission. A dynamic change in neuropeptide expression over time is another factor. Some peptidergic neurons have a relatively stable neuropeptide expression (e.g., some hypothalamic peptides, such as vasopressin, oxytocin, and galanin), but others express neuropeptides only transiently during development or display pronounced
upregulation from low levels in response to appropriate physiological stimuli (e.g., vasoactive intestinal peptide (VIP), galanin, or NPY in primary sensory neurons). It is natural to assume that two or more messengers that are released at the same time from the same axon terminal (or even the same vesicle) interact with one another’s effects. There are, indeed, several examples both of synergism between co-released neuropeptides and classical transmitters (e.g., acetylcholine and VIP in salivary glands) and of antagonism between co-released transmitters (e.g., galanin and noradrenalin in the brain stem). However, co-released neuropeptides might not always serve as co-transmitters. Because the physical and timedependent range of neuropeptide action is so large, it is possible that two neuropeptide transmitters released from the same axon terminal or vesicle have completely independent effects, even involving separate receiving neurons. Neuropeptide release is clearly a multifaceted phenomenon in the brain, and we are only beginning to understand the reasons for this complexity. One suggestion which has been put forward is that the co-release of some neurotransmitters constitutes phylogenetic remnants that are no longer linked to function, and it cannot be ruled out that this is, at least sometimes, the case. However, this is not to suggest that the neurotransmitter in question is without biological action. It might just not have a physiological function in every cell and vesicle where it is present. Variation within neurons – axon terminal versus somatodendritic release Neuropeptides can be differentially released from separate compartments within a neuron. This is the case in hypothalamic magnocellular oxytocin and vasopressin neurons. These neurons release neuropeptides into the bloodstream from axon terminals in the posterior pituitary but also into the CNS from dendrites that are densely packed with oxytocinor vasopressin-containing LDCVs. The particular anatomical arrangement of these neurons makes it possible to separate the release from axon terminals from the release originating in somatodendritic elements. The magnocellular neurons have virtually no axon collaterals that project back to the dendritic region. Consequently, all axon-terminal release takes place in the posterior pituitary, and conversely, in the supraoptic nucleus, all vasopressin and oxytocin release comes from magnocellular somata and dendrites. The peripheral action of vasopressin is to promote water retention and increase blood pressure. When magnocellular neurons are activated by systemically increased osmolarity, they respond by releasing vasopressin (and in the rat, oxytocin) from their terminals in the posterior pituitary but also from the dendrites in the supraoptic nucleus. Interestingly, the release from
Neuropeptide Release
dendrites and terminals does not occur simultaneously, as might be expected. The dendritic release in vivo is delayed by approximately 1 h and persists longer than terminal release. An opposite order of events is observed in lactating rats when oxytocin release in response to suckling is measured in vivo; in this case, dendritic release precedes terminal release. Apparently, axon-terminal and somatodendritic release employ distinct activity-dependent release mechanisms. One way in which dendritic oxytocin release differs from terminal release is by its dependence on mobilization of Ca2þ from intracellular stores (Figure 1). This is illustrated by the effects of a-melanocyte-stimulating hormone (a-MSH), which, when administered to oxytocin cells, triggers intracellular Ca2þ mobilization by activating MC4 receptors. In vivo this leads to dendritic oxytocin release but also to decreased firing of the oxytocinergic neurons and thereby decreased axon-terminal release into the blood. Somatodendritic vasopressin and oxytocin release, furthermore, displays a kind of priming which is not seen with axon-terminal release. Mobilization of intracellular Ca2þ from thapsigargin-sensitive stores of the endoplasmic reticulum leads to enhancement of subsequent release. The consequence is a more pronounced somatodendritic release of the neuropeptides in response to a given stimulus. Interestingly, this enhancement of dendritic release is long-lasting, and it is not a direct effect of the increase in intracellular calcium because it develops some time after the intracellular Ca2þ concentrations have returned to normal. As might be expected, the lasting enhancement of somatodendritic neuropeptide release is associated with an increased number of vesicles that are closely associated with the cell membrane, reflecting a change in the recruitment of vesicles to the readily releasable pool (Figure 1). The described enhancement (priming) of somatodendritic oxytocin and vasopressin release can be evoked by drugs such as thapsigargin and cyclopiazonic acid (Ca2þ mobilizers) but also by endogenous oxytocin. Oxytocin released from the somata and dendrites therefore promotes its own release by a feed-forward mechanism that facilitates recruitment of LDCVs from the reserve pool. As demonstrated by these examples, axon-terminal and somatodendritic neuropeptide release can occur independently, indicating that neuropeptides can have separate physiological functions depending on which compartment of the neuron they are released from. Vasopressin and oxytocin neurons differ from most other neurons that release neuropeptides in the brain by the unusually high concentration of peptide-containing LDCVs in the dendritic compartments. It remains to be investigated whether differential dendritic and terminal release is also found in other
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peptidergic neurons. It is, however, already known that dendritic release is not restricted to peptidergic neurons but is a feature of many, if not all, neurotransmitter systems. Dopaminergic cells also display differences between terminal and somatodendritic release that suggest independent release mechanisms, and accumulating evidence favors the concept that dendritic release is a distinct mode of neurotransmission.
Physiological Effects of Neuropeptide Release We have touched on the characteristics of neuropeptide release that single out this type of transmission from fast classical neurotransmission. The many neuropeptides in the nervous system can have an equally large number of physiological effects. Neurotransmitters generally affect the excitability of other neurons by depolarizing them or by hyperpolarizing them. Peptides have much more diverse effects; among other things, they can affect gene expression, local blood flow, synapotogenesis, and glial cell morphology. Some examples, which illustrate how the characteristic neuropeptide release mechanisms translate into physiological functions, are summarized next. Neuropeptides have well-established roles in a number of physiological systems such as CNS control of food intake, pain and anxiety, stress, learning and memory, and social behavior. They may also have a role in the pathophysiology of addiction, depression, and eating disorders. Common to the action of neuropeptides in these examples is the long timescale. For example, the CNS control of food intake is not concerned with matching intake and expenditure on millisecond, second or even daily basis but, instead, provides a balance over a week and longer. The action of hypocretins/orexins, ghrelin, NPY, a-MSH, and other appetite-regulating neuropeptides obviously does not last that long per se, but they can evidently tune the drive for food intake over time periods of this magnitude. It is easily recognized that fast and spatially precise signals are not needed for this. Even peptides that counteract epileptic seizures, such as galanin and NPY, act on a relatively long timescale. NPY-deficient mice can, in most electrophysiological aspects, not be separated from wild-type mice. When tested with the pharmacological seizure-inducer kainic acid, wild-type and NPY / mice display inseparable latencies to electroencephalographic seizure activity, and partial seizures are also similar in behavioral appearance. However, when seizure activity generalizes, wild-type mice seizures end within 30 s, whereas NPY / seizures last more than twice as long and ultimately progress into status
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epilepticus and death. Thus, it seems that NPY can serve as a brake to stop epileptic seizures. The fact that the major difference between wild-type mice and NPY / is that the latter have problems terminating epileptic activity is consistent with the need for intensive stimulation to evoke neuropeptide release, as well as the long latencies to release. Somatodendritic releases of vasopressin and oxytocin have autoregulatory effects. In the case of vasopressin, this regulation can be either inhibitory or excitatory, depending on the prevailing activity of the neuron. Autoregulation in vasopressin neurons can be amplified by the insertion of k-opioid receptors, which are present in the LDCV membrane and inserted into the cell membrane on exocytosis. Through such insertions of opioid receptors or other neuropeptide receptors, neuropeptidergic neurons can use LDCV exocytosis to transmit a signal to surrounding neurons and at the same time change their own responsiveness. One autoregulatory effect of oxytocin is to promote burst activity. Synchronized burst firing of oxytocin neurons is seen during labor and in response to suckling in lactating females but not otherwise. The transition from the normal continuous unsynchronized behavior of oxytocin neurons into synchronized burst-firing response is mediated by central oxytocinergic transmission. By priming somatodendritic oxytocin release using thapsigargin, it is also possible to generate burst-patterned firing in virgin rats, indicating that this network plasticity is mediated by specific dendritic release mechanisms. This kind of functional rewiring of neural networks through paracrine signaling has recently emerged as a mechanism by which dendritically released neuropeptides can alter behavior. Neurons that release luteinizing hormone-releasing hormone (LHRH) also display dendritic neuropeptide release, in addition to the terminal release into the hypophysial portal circulation. Unlike vasopressin and oxytocin neurons, the LHRH neurons in the preoptic/septal area are scattered throughout a relatively large area. In spite of this, they release LHRH in a synchronized pulsatile manner, and it has been suggested that LHRH autoregulation plays a role in the generation of this pulsatile behavior, which triggers puberty and is necessary for normal ovulatory menstrual cycles in females. Neuropeptide release clearly has widespread physiological effects that stretch beyond the scope of this chapter.
Summary A large number of different neuropeptides are released from neurons in the periphery and in the brain. In most
cases, little is known about the release mechanisms and the regulation of release, but the available information reveals some common themes that distinguish neuropeptide release from the release of classical neurotransmitters. Neuropeptides are always released from LDCVs and have to be synthesized de novo for each release event. An economizing transport and recruitment mechanism is therefore needed to prevent depletion. This is mediated in part by a more restrictive priming process, which only allows a small fraction of the present vesicles to be released in response to depolarization, and in part by a spatial separation of LDCV Ca2þ sensors and voltage-sensitive Ca2þ channels. Typically, neuropeptides are, therefore, released only in response to high-frequency or burst-patterned firing or in response to intercellular messengers that mobilize intracellular Ca2þ stores. On the other hand, the action of neuropeptides is prolonged and extended due to slow inactivation and high-affinity receptors. This slow and wide mode of transmission is reflected in many physiological actions of neuropeptides. See also: Neuropeptide Synthesis and Storage; SNAREs; Synaptic Vesicles.
Further Reading An S and Zenisek D (2004) Regulation of exocytosis in neurons and neuroendocrine cells. Current Opinion in Neurobiology 14(5): 522–530. Baraban SC and Tallent MK (2004) Interneuron diversity series: Interneuronal neuropeptides – endogenous regulators of neuronal excitability. Trends in Neurosciences 27(3): 135–142. Burgoyne RD and Morgan A (2003) Secretory granule exocytosis. Physiological Reviews 83(2): 581–632. Harata NC, Aravanis AM, and Tsien RW (2006) Kiss-and-run and full-collapse fusion as modes of exo-endocytosis in neurosecretion. Journal of Neurochemistry 97(6): 1546–1570. Ho¨kfelt T, Bartfai T, and Bloom F (2003) Neuropeptides: Opportunities for drug discovery. Lancet Neurology 2(8): 463–472. Ho¨kfelt T, Broberger C, Xu ZQ, et al. (2000) Neuropeptides – an overview. Neuropharmacology 39(8): 1337–1356. Kits KS and Mansvelder HD (2000) Regulation of exocytosis in neuroendocrine cells: Spatial organization of channels and vesicles, stimulus-secretion coupling, calcium buffers and modulation. Brain Research Reviews 33(1): 78–94. Landgraf R and Neumann ID (2004) Vasopressin and oxytocin release within the brain: A dynamic concept of multiple and variable modes of neuropeptide communication. Frontiers in Neuroendocrinology 25(3–4): 150–176. Ludwig M (ed.) (2005) Dendritic Neurotransmitter Release. New York: Springer. Ludwig M and Leng G (2006) Dendritic peptide release and peptide-dependent behaviours. Nature Reviews Neuroscience 7(2): 126–136. Martin TF (2003) Tuning exocytosis for speed: Fast and slow modes. Biochimica et Biophysica Acta 1641(2–3): 157–165. Salio C, Lossi L, Ferrini F, and Merighi A (2006) Neuropeptides as synaptic transmitters. Cell and Tissue Research 326(2): 583–598.
Neuropeptides and Coexistence A Merighi, University of Turin, Turin, Italy ã 2009 Elsevier Ltd. All rights reserved.
General Concepts and Definitions The term ‘neuropeptide’ was originally coined to indicate small protein molecules that are contained in neurons and that are composed of 3–100 aminoacid residues. These molecules are usually produced as large, inactive precursors, which are enzymatically cleaved to yield the biologically active peptide. Commonly, precursors contain several molecules of the same neuropeptide and/or more or less structurally related compounds. Neuropeptides exert several different biological effects, such the regulation of gene transcription, local blood flow, synaptogenesis, and glial cell architecture. Notably, most neuropeptides also influence membrane excitability, and, although this should perhaps not be thought of as their main biological action, this has made them very attractive to neurobiologists. When thinking of neuropeptides as being involved in cell-to-cell communication, the first consideration to be made is that they are about 50 times larger than low-molecular-weight ‘classical’ neurotransmitters, such as acetylcholine, biogenic amines, and amino acids. As a consequence, neuropeptides possess several more recognition sites for receptors than do smaller neurotransmitters, and thus a higher receptor-binding affinity (about 1000 times greater, with values in nanomoles/liter vs. micromoles/ liter) and selectivity. For these reasons neuropeptides can elicit their biological effects even when released at lower quantities. Although large neuropeptide molecules can diffuse and bind slowly to receptors, their half-lives in the brain extracellular space are remarkably long: for example, the half-lives of oxytocin and vasopressin have been estimated to be in the order of about 20 min. Neuropeptide receptors are usually seventransmembrane-region G-protein-coupled receptors (GPCRs), which are internalized upon activation as a desensitization mechanism. For example, the neurokinin-1 receptor (the preferred substance P receptor) is internalized 5 min after agonist activation, and is then recycled and restored to the cell membrane within 30 min.
Cellular and Subcellular Sites of Neuropeptide Storage Localization within Large Granular Vesicles
Identification of the subcellular site of storage of neuropeptides remains crucial to the understanding
of their mechanism of action as synaptic (or nonsynaptic) transmitters and of their interaction with other molecules involved in chemical neurotransmission. Neuropeptides are commonly found to be present together with low-molecular-weight classical neurotransmitters in individual nerve cells. However, initial observations indicated that the two classes of transmitter molecules were stored in different subcellular compartments: in large granular vesicles (LGVs) for the neuropeptides, and in small clear synaptic vesicles (SSVs) for the classical transmitters (Figure 1). In the past, the debate regarding such a compartmentalization was left open, since it could not be excluded that, at least in some cases, LGVs also contained some of the more conventional transmitters, although peptides never seemed to be found in small clear vesicles. Nowadays, the concept that LGVs are the sole site of neuropeptide storage is widely established. The histological demonstration of differential subcellular sites of storage for neuropeptides and low-molecular-weight neurotransmitters is consistent with the possibility that they are selectively released upon specific stimuli. On the other hand, coexisting neuropeptides, even when synthesized from different mRNAs, are usually stored together in LGVs. This has a series of functional implications, since co-stored neuropeptides are not differentially released, and a modulation of biological effects is most readily accomplished by regulating their relative proportions of synthesis. Synthesis, Storage, and Targeting to Processes
As occurs for all peptide molecules, neuropeptides are synthesized in precursor form within the rough endoplasmic reticulum (RER) and subsequently move to the Golgi apparatus, where they are packaged into LGVs. The primary sensory neurons are a unique class of pseudounipolar neurons; they are grouped outside the central nervous system (CNS) in the sensory ganglia, associated with certain cranial nerves and in dorsal root ganglia (DRGs), and display an extremely abundant and variegated neuropeptide content. Immediately after emerging from the cell body the single process of these neurons divides into a central branch and a peripheral branch that function, respectively, as an axon and a dendrite. Pioneering work carried out with the use of multiple immunogold labeling methods about 20 years ago led to the localization of different tachykinins and calcitonin gene-related peptide (CGRP) to individual LGVs in neuronal cell bodies and central peripheral and branches of DRG neurons. In these cells, virtually
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Figure 1 Subcellular localization of neuropeptides and presence within large granular vesicles at central synapses. Calcitonin gene-related factor/substance P (a) and brain-derived neurotrophic peptide/substance P (b) dual immunogold labeling (10 and 20 nm gold particles) in central terminals of a dorsal root ganglion neuron within the substantia gelatinosa of spinal cord. A multisynaptic complex (glomerulus) contains small synaptic vesicles (SSVs) and large granular vesicles (LGVs) with a dense core. Note in both images that SSVs are consistently unlabeled, whereas the majority of LGVs are double labeled. Note also that LGVs are not in proximity of synaptic specializations (arrowheads), unlike SSVs (m, mitochondrion). The arrows indicate the LGV that is shown at higher magnification in the corresponding insets at top right of each panel.
all LGVs are multiple/dual-labeled in both central and peripheral branches, whereas double (multiple)labeled LGVs in the cell body relatively rare. These observations show that when multiple peptides are produced by one neuron, they are not selectively packaged into different LGV subpopulations, but rather a cocktail of neuropeptides is consistently found within individual LGVs. Moreover, given that LGVs (and therefore the neuropeptides packed therein) are detected at both central and peripheral branches of DRG neurons, it seems reasonable to hold that no selective transport to functionally different neuronal processes (the axons and the dendrites) and/or to different branches of the same process occurs. In neuronal cell bodies, LGVs containing just one component of the cocktail can be regarded as immature vesicles that will probably incorporate the other peptide(s) before being transported to terminals. Studies on nonneuronal cell regulated secretion suggest that peptides are packed into immature LGVs budding from the trans-Golgi network, and ultimately transform into mature LGVs. Fusion between immature secretory granules has been demonstrated, and it is thought that after granule–granule fusion, soluble contents condense, and excess membrane are removed by vesicle budding. Very recent work has shown that an analogous pattern of co-storage is also apparent in central neurons, and that co-stored neuropeptides are quantitatively detected in LGVs with remarkably constant ratios. The ratio might be regulated at the level of neuropeptide synthesis, before packaging in the trans-Golgi network (Figure 2).
Coexistence Coexistence, the concurrent presence of two or more transmitters in a single neuron, is now regarded as a common feature of central and peripheral neurons. Neuropeptides coexist with other neuropeptides, low-molecular-weight fast-acting neurotransmitters, the gaseous transmitter NO, and certain neurotrophins. When multiple neuropeptides are present in neurons, coexistence equals co-localization, since, as discussed earlier, coexisting peptides are co-stored in LGVs. As a rule, neurons produce a combination of one (or more) low-molecular-weight transmitter(s) and one (or more) high-molecular-weight neuropeptide(s). One remarkable exception seems to be represented by the oxytocin/vasopressin magnocellular neurons that contain a complex cocktail of peptides, but apparently no low-molecular-weight transmitters. When a neuropeptide coexists with a classical transmitter, the latter is generally believed to be the principal messenger, whereas the neuropeptide appears to modulate neuronal response by acting on pre- and/or postsynaptic GPCRs (see the section titled, ‘Functional implications of neuropeptide coexistence’). Neuropeptide Co-Localization
Several areas of the CNS are particularly enriched with neuropeptides. In most circumstances, it is common that individual neurons in these areas produce and store more than a single peptide. Relevant examples are given in the following sections.
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Mature LGVs
Immature LGVs
Immature LGVs
TGN
Early immature granules budding from TGN
Prepropeptides from RER Figure 2 Packaging of neuropeptides in large granular vesicles (LGVs). For simplicity, only two peptides (indicated by the red and yellow circles) are represented, although there is direct evidence that more neuropeptides can be stored in individual LGVs. Prepropeptides reach the Golgi apparatus from the rough endoplasmic reticulum (RER). They are packaged in granules budding from the trans-Golgi network (TGN); the granules subsequently fuse together to give rise to immature LGVs. Immature LGVs are larger and display a more irregular shape compared to their mature counterparts. As maturation of LGVs proceeds, there is a condensation of the granin content, so that the vesicles assume the typical size, shape, and electron density of mature LGVs. When a neuron produces more than a single neuropeptide, it is possible that immature granules budding from the TGN (left) already contain a mixture of cargo molecules. Alternatively, each peptide is singularly packed into individual immature granules (right), which then fuse together to give rise to the mature LGVs. In both cases, the latter contain the cocktail of neuropeptides that are eventually transported to terminals.
Spinal cord The co-localization of tachykinins and CGRP is one of the most widely studied example of peptide co-localization (Figure 1(a)). The cell bodies of the neurons containing these peptides are located in the peripheral nervous system in the DRGs and trigeminal ganglia, and in several autonomic ganglia and intramural plexuses. Galanin is co-localized mainly with enkephalin, but sometimes also with neuropeptide Y (NPY) in some local neurons of the substantia gelatinosa (lamina II) of the dorsal horn. In the same location, the co-localization of tachykinins and enkephalin is also reported. Galanin and substance P are co-localized in neurons of the deeper layers of the dorsal horn. Co-localization of these neuropeptides and neurotensin with galanin has been demonstrated also at the ultrastructural level. Hypothalamus The hypothalamic hormones oxytocin and vasopressin were the first neuropeptides discovered, and an extensive literature exists on the localization, biological actions, and plasticity of these
peptides. The oxytocin- and vasopressin-containing neurons are located in both large (magnocellular) and small (parvocellular) hypothalamic neurons. In the supraoptic nucleus the oxytocin/vasopressin system undergoes a remarkable plasticity under normal and experimental conditions. Although oxytocin and vasopressin neurons usually constitute two separate populations, there are some plasticity inducing conditions, such as parturition and lactation, in which co-localization of the two peptides has been hypothesized on the basis of in situ hybridization and single-cell reverse transcription–polymerase chain reaction studies. Considerable evidence has accumulated that approximately 50% of the corticotropin-releasing hormone (CRH) neurons in the paraventricular nucleus contain vasopressin or its precursors. Moreover, about 30% of the CRH parvocellular neurons in the median eminence contain vasopressin. These neurons are also highly plastic, and the relative proportion of released vasopressin and CRH changes under different physiological and experimental conditions.
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Magnocellular oxytocin neurons contain several opioid peptides of the enkephalin family, and magnocellular vasopressin neurons contain dynorphin. In addition, some oxytocin neurons contain either cholecystokinin (CCK) or CRH. Co-localization of CCK and CRH has been demonstrated in periportal nerve terminals of the rat median eminence. It has also been shown that galanin is co-produced in a subpopulation of luteinizing hormone-releasing hormone (LHRH) neurons. The incidence of LGVs immunolabeled for both neuropeptides is high in the female and low in the male rat. Ovariectomy results in a dramatic decline in the number of LHRH/galanin-co-expressing LGVs, which is reversed by the administration of estradiol.
the arcuate nucleus, basolateral amygdala, and dorsal horn; with substance P in the hypothalamus and retinal ganglion cells; with galanin in the dorsal horn; with somatostatin in the entorhinal cortex; with CRH in the hippocampus; and with vasopressin in the suprachiasmatic nucleus.
Coexistence of Neuropeptides and Fast-Acting Neurotransmitters
Biogenic amines Several different neuropeptides have been found to coexist with biogenic amines such as serotonin (5-hydroxytryptamine; 5-HT), and with noradrenaline. The most widely reported combinations are the coexistence of substance P and 5-HT in caudal medullary neurons projecting to the spinal ventral horn, and in dorsal raphe nuclei neurons, and the coexistence of enkephalin or galanin and 5-HT in the dorsal raphe nuclei. Other combinations have been found with noradrenaline. These include substance P and noradrenaline in the medulla oblongata, NPY and noradrenaline in the hypothalamus, and galanin and noradrenaline in the cerebral cortex and hippocampus. Multiple combinations are possible, and there is strong evidence that more than one neuropeptide may be contained within biogenic amine-synthesizing neurons. Also, these neurons can produce other classical transmitters, such as transmitter amino acids.
Acetylcholine Neuropeptides coexist with acetylcholine in numerous areas of the brain. For example, galanin is found to coexist with acetylcholine in the basal forebrain and hippocampus, where the coexistence of somatostatin and acetylcholine has also been described. Coexistence of acetylcholine and CGRP has been reported in neurons of the vestibular nuclei.
Nitric oxide Certain peptides have been found to be present in nitric oxide-producing neurons (detected after NADPH diaphorase histochemistry or nitric oxide synthase immunocytochemistry). Examples include somatostatin in the hippocampus, vasopressin in the hypothalamus, and galanin in the dorsal raphe nucleus.
Amino acids The coexistence of peptides and aspartate, glutamate, g-aminobutyric acid (GABA), or glycine is a widespread finding in the CNS. Aspartate coexists with substance P in the spinal cord, and with NPY or galanin in the arcuate nucleus. The coexistence of glutamate and peptides is by far more common. Coexistence of enkephalin and glutamate has been shown to occur in the locus coeruleus. Glutamate and PACAP are localized within the same neurons in the retinohypothalamic tract. Glutamate and substance P coexist in the spinal cord. GABA has been found to be present with several different neuropeptides in various areas of the brain: with enkephalin in the cochlea, striatum, and spinal cord; with somatostatin in the visual and entorhinal cortex, suprachiasmatic nucleus, and basolateral amygdala; with cholecystokinin-8 (CCK-8) and/or vasoactive intestinal peptide (VIP) in the hippocampus dentate gyrus, basolateral amygdala, and suprachiasmatic nucleus; with CGRP in the cerebellum; with NPY in
Coexistence of Neuropeptides and Brain-Derived Neurotrophic Factor
Retina A large number of neuropeptides occur in amacrine cells, and to a lesser extent, in the ganglion cells. These include avian pancreatic polypeptide (APP), enkephalin, neurotensin, NPY, pituitary adenylyl cyclase-activating peptide (PACAP), pancreatic polypeptide (PP), somatostatin, and substance P. Many of these neuropeptides are co-localized within individual neurons.
A direct demonstration for the selective storage of brain-derived neurotrophic factor (BDNF) in LGVs has recently been provided by ultrastructural immunocytochemistry (Figure 1(b)), in terminals within the central nucleus of amygdala and spinal cord dorsal horn. These terminals originate from central and peripheral neurons that are capable of synthesizing and anterogradely transporting the neurotrophin. Individual LGVs have been found to contain a cocktail of BDNF, CGRP, and substance P. Such a pattern of cellular storage is compatible with a transmitter function of BDNF in these neuronal populations.
Neurotransmitter Function of Neuropeptides Despite the enormous amount of literature on neuropeptide distribution in mammals, much of our
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knowledge about neuropeptide function comes from studies in invertebrates. Indeed, it is far easier to demonstrate the presence of multiple messengers in neurons than it is to establish their physiological role, or even to show that they have any kind of biological activity at all. Moreover, although an organic framework to describe the function(s) of individual neuropeptides at synapses and/or nonsynaptic sites is now available, relatively little is known about the functional interactions and the control of release of co-stored neuropeptides at central synapses. The existence of synapses can be unequivocally demonstrated only by electron microscopy. Ironically, however, ultrastructural studies have also shown the existence of nonsynaptic transmission. The identification of gases as interneuronal signals or the modulation of neuronal function by lipophilic substances has left no doubt regarding the existence of nonsynaptic information transfer in the CNS. Nonetheless, it is more difficult to accept that vesicle-stored transmitters can be released following quantal mechanisms and operate at distant nonsynaptic sites. This notion applies not only to neuropeptides, but also to certain low-molecular-weight classical neurotransmitters. Neuropeptides as Slow-Acting Neurotransmitters
Neurotransmitters are consistently packaged in two different morphological types of vesicles at synapses. The most abundant SSVs accumulate fast-acting, low-molecular-weight neurotransmitters. The less frequently observed LGVs store the neuropeptides (Figure 1(a)). The distribution of SSVs and LGVs in typical axodendritic central synapses follows a rather stereotyped pattern. SSVs occupy a variable, but usually large, area of the axon terminal. Some are docked at the presynaptic grid, being ready to be released at the active synaptic zone. LGVs, on the other hand, are localized away from the presynaptic membrane, singularly or in clusters. In addition, electron microscopy has shown that release of LGVs can occur at plasma membranes even in the absence of synaptic specializations. The major functional implication of such an arrangement is that fast- and slow-acting transmitters may be selectively released upon activation of different cellular pathways. Early evidence was obtained showing that the release of coexisting peptides and classical neurotransmitters could be differential and dependent on the frequency and pattern of firing. In general terms, neuropeptide release is triggered by a small increase in the intracellular Ca2þ concentration, whereas release of transmitter amino acids from SSVs requires a rise of intracellular Ca2þ concentration in the proximity
of the Ca2þ channels at synapses. Therefore, in terminals with both types of vesicles, a focal increase in Ca2þ at the synaptic membrane leads to a preferential discharge from SSVs, whereas a more general elevation of Ca2þ inside the terminal favors the release of LGVs. The remarkably short delay in response to fast-acting transmitters (about 1–3 ms) is a close reflection of this mode of discharge from SSVs. On the other hand, the spatial independence from Ca2þ channels clustered at synapses explains why neuropeptide release can occur independently from synapses. Among the consequences of the existence of selective mechanisms of release for coexisting peptides and classical transmitters is the possibility that long-lasting intracellular Ca2þ elevation may cause the release of neuropeptides to outlast the duration of electrical activity, thus uncoupling release from spiking. Mode(s) of Release
Even upon prolonged stimulation, not all vesicles at synapses unload their transmitter content. A variable fraction of SSVs and LGVs is readily releasable, but the remaining ones, forming the reserve pool, need further steps to become competent. Two different mechanisms of transmitter emptying have been shown to occur in SSVs and LGVs. These include the slower classical exocytosis, with complete fusion of the vesicle to the plasma membrane, or a faster mechanism whereby vesicles come in close proximity to the membrane and, with the formation of a transient pore, release part of their transmitter content by a process described as ‘kiss and run.’ The transient pore mechanism allows the quick, simultaneous passage of amine transmitters (and perhaps other small molecules, which may be present in LGVs together with neuropeptides) from LGVs into the extracellular fluid. On the other hand, neuropeptides contained within LGVs remain trapped inside the retrievable vesicle, as a consequence of their higher molecular weight. Therefore, release of most neuropeptides from the LGVs is unlikely to occur through kiss and run, for several reasons. These include the larger size of the peptides relative to the transient pore, and the slow emptying of peptide content from LGVs upon exocytosis. In support, simultaneous capacitance measurements and confocal imaging have shown that peptide release by kiss and run is negligible. On the other hand, complete vesicle fusion is usually required, through a mechanism involving a priming step followed by retrieval of the vesicle as a coated vesicle. A divergence in this respect between peptide-containing LGVs and amine-containing LGVs may merely be additional
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to the several differences between these two classes of neurotransmitters. Indeed, neuropeptides do not have a known re-uptake mechanism, as opposed to biogenic amines, so that there is no way locally to refill the peptide-containing LGVs after emptying. Moreover, neuropeptides are synthesized at the rough endoplasmic reticulum in the neuronal perikarya, but not in axon terminals, whereas amines can also be synthesized inside LGVs. When a neuron produces more than a single neuropeptide (as appears to be the rule), several theoretical possibilities arise regarding their release. It is possible that all co-stored neuropeptides can be released all at once, at all processes. Alternatively, or in addition, individual neuropeptides can be theoretically liberated singularly or in different combinations at different processes. In Aplysia, the various neuropeptides derived from a common prohormone seem to be targeted to different neuronal processes. The situation is far less clear in mammals, considering that most studies have been carried out on isolated neurons. Nonetheless, all co-stored neuropeptides can be reasonably assumed to be released together upon exocytosis. For example, the co-release of CGRP and substance P (and other tachykinins) has been demonstrated, and this occurs at both central and peripheral endings of the DRG neurons. Moreover, if the co-release of co-stored neuropeptides is indeed the rule, then it should occur from any neuronal process containing LGVs, although this latter issue also needs further clarification. The major functional implication is that co-released peptides probably act together in determining the response of target cells. A differential release of costored peptides (if indeed this occurs in vivo) would more likely rely on mechanisms different from those that apply to co-stored biogenic amines and/or coexisting low-molecular-weight neurotransmitters. From this perspective, the relative rate of peptide dissolution from the LGV core might be of primary relevance, since this appears to be the critical determinant of the speed of peptide secretion in vitro.
Functional Implications of Neuropeptide Coexistence For an evolutionary perspective, coexistence of multiple neurotransmitters in neurons has led to substantial advantages. In particular, neuropeptides have a wide diversity of direct or modulatory effects on the electrical responses of target cells, in addition to trophic effects. When they are co-released with other neurotransmitters, the wealth of responses of
target neurons increases dramatically. Neurotransmitters produced and released by a single neuron are often defined as cotransmitters. However, it is probably unsafe to consider that cotransmitters must display some kind of interaction, simply because they are coreleased, even though such a co-release occurs under physiological conditions. The common existence of a combination of neuropeptides and classical neurotransmitters in neurons enables fast (2–5 ms) and slow (100–500 ms) synaptic communication to take place. Fast- and slow-acting cotransmitters can act on completely independent targets and, therefore, do not interact at all. However, there is a general consensus that when multiple neurotransmitters are released within the extracellular space, they usually display at least some type of interactive actions, irrespective of the finding that such a release occurs from the same neuron – that is, they are true cotransmitters, or from separate sources. The simplest mode of interaction of two (or more) neurotransmitters occurs when two (or more) distinct receptor complexes are present in the (postsynaptic) membrane of target cells, and a receptor–receptor interaction occurs (Figure 3). When neuropeptides coexist with low-molecular-weight neurotransmitters, the neuropeptide usually acts on GPCRs, whereas the low-molecular-weight transmitter generally opens a ligand-gated ion channel. The low-molecular-weight transmitter is generally the principal messenger, and the neuropeptide interacts with it by altering the ion channel gating properties or its response to further signals. This occurs by direct operation on the receptor complex or by the activation of second-messenger systems that, in turn, act on the receptor complex. Hence, one neurotransmitter may alter the number of receptors or the affinity of the receptor to the other(s) simultaneously released. Interestingly, receptor recruitment from the interior of the cell to the plasma membrane may be an additional and ubiquitous mechanism of modulation of signal transduction, leading to receptor sensitization. The interaction of cotransmitters also occurs through presynaptic regulation. This implies the existence of presynaptic receptors for one or more messengers. In this case, one of the neurotransmitters feeds back on presynaptic receptors and thus affects its own release or the release of the cotransmitter(s). This latter possibility has been demonstrated, for example, in the striatum in which tachykinins presynaptically stimulate the release of dopamine, and in the locus coeruleus in which noradrenergic neurons can be activated by the stimulation of neurokinin-3 receptors.
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Co-storage
Coexistence
b
a
Coexistence
Coexistence
c
d
Figure 3 Functional implications of neuropeptide coexistence. For simplicity, only two peptides (indicated by the red and yellow circles) are represented, and only postsynaptic interactions are considered. In purely peptidergic neurons (a), neuropeptides co-stored within large granular vesicles are released together and affect the activity of their target neurons upon binding to their cognate G-protein-coupled receptors. In neurons producing and releasing a combination of neuropeptides and a fast-acting neurotransmitter (b–d), the coexistence of these neuroactive molecules does not necessarily imply that they are acting as cotransmitters. As depicted, in panel (b) only the neuropeptides affect the target neuron that expresses specific G-protein-coupled receptors, whereas in panel (c) the target neuron binds receptors (ion channels) only for the low-molecular-weight transmitter. Only in panel (d) are the neuropeptides and the low-molecularweight transmitter true cotransmitters. In this case the general rule is that the low-molecular-weight transmitter acts as the principal messenger, whereas the neuropeptides subserve a modulatory role.
See also: Neuropeptide Release; Neuropeptide Synthesis and Storage; Neuropeptides: Electrophysiology.
Further Reading Ho¨kfelt T, Johansson O, Ljungdahl A˚, et al. (1980) Peptidergic neurons. Nature 284: 515–521. Ho¨kfelt T, Broberger C, Xu Z-QD, et al. (2000) Neuropeptides – an overview. Neuropharmacology 39: 1337–1356. Ho¨kfelt T, Pernow B, and Wahren J (2001) Substance P: A pioneer amongst neuropeptides. Journal of Internal Medicine 249: 27–40.
Meldolesi J, Chieragatti E, and Malosio ML (2004) Requirements for the identification of dense-core granules. Trends in Cell Biology 14: 13–19. Merighi A (2002) Costorage and coexistence of neuropeptides in the mammalian CNS. Progress in Neurobiology 66: 161–190. Nusbaum MP, Blitz DM, Swensen AM, et al. (2001) The roles of co-transmission in neural network modulation. Trends in Neuroscience 24: 146–154. Salio C, Lossi L, Ferrini F, et al. (2006) Neuropeptides as synaptic transmitters. Cell and Tissue Research 326(2): 583–598. Salio C, Averill S, Priestley JV, et al. (2007) Costorage of BDNF and neuropeptides within individual dense-core vesicles in central and peripheral neurons. Developmental Neurobiology 67: 326–338.
Opioid Peptides and Receptors B L Kieffer, IGBMC,CNRS/INSERM/ULP, Illkirch, France ã 2009 Elsevier Ltd. All rights reserved.
The Opioid System: A Short History Discovery of the opioid system stems from the use and abuse of opium in ancient history. Opium, extracted from poppy seeds (Papaver somniferum; see Figure 1(a)), has powerful pain-relieving properties and produces euphoria. This substance has been used both medicinally and recreationally for several millennia. Morphine, named after the god Morpheus, is the most active ingredient of opium. The compound was isolated in 1805 and rapidly became the clinical treatment of choice to alleviate severe pain. Heroin was synthesized chemically by morphine diacetylation in the late 1800s and was commercialized as the first nonaddictive opiate to treat cough and asthma. The strong addictive properties of heroin were soon acknowledged, and both heroin and opium were prohibited in 1910. Today morphine remains the best pain-killer in contemporary medicine, despite a wide array of adverse side effects (respiratory depression, constipation, tolerance, and dependence). Heroin is a main illicit drug of abuse, and heroin addiction represents a major public health issue. Because of their extraordinarily potent analgesic and addictive properties, opiates have prompted scientists to seek to understand their mode of action in the brain. In 1973, three independent teams showed that opiates bind to membrane receptors in the brain, and these receptors were named m, d, and k a few years later. In 1975, two pentapeptides, Met- and Leuenkephalin were isolated as the first endogenous ligands for these receptors. Many peptides followed, forming the opioid peptide family. Three distinct genes encoding the peptides were isolated in the late 1970s and early 1980s; three other genes encoding the receptors were cloned a decade later. In the mid 1990s the entire endogenous opioid system was characterized at the molecular level (Figure 1(b)).
Molecular Components of the Opioid System The Peptides
Opioid peptide genes encode large precursor proteins, which are further cleaved into shorter peptides. All opioid peptides share a common N-terminal Tyr-GlyGly-Phe signature sequence, which interacts with
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opioid receptors. The preproenkephalin gene encodes several copies of the pentapeptide Met-enkephalin and one copy of Leu-enkephalin. This gene also encodes longer versions of Met-enkephalin. Notably, one of them, the opioid peptide bam22, activates opioid receptors via its N-terminal sequence while activating another receptor family known as sensory neuron-specific (SNS) receptors via its C-terminal part. The preprodynorphin gene also encodes multiple opioid peptides, including dynorphin A, dynorphin B, and neo-endorphin. The preprodynorphin-derived peptides all contain the Leu-enkephalin N-terminal sequence and basic amino acid residues in their C-terminal end. Finally the preproopioimelanocortin gene encodes a single opioid peptide, b-endorphin, among other peptides with nonopioid biological activity. b-endorphin is the longest opioid peptide (31 amino acids) and shows highly potent and long-lasting analgesic properties. The Receptors
Opioid receptors are membrane receptors with a seventransmembrane topology that belong to the large Gprotein-coupled receptor (GPCR) superfamily. This family comprises several hundred members within the mammalian genome. m-, d-, and k-opioid receptor genes constitute a GPCR subfamily, together with a fourth member encoding the orphanin FQ/nociceptin receptor. The last receptor and its endogenous ligand share high structural homology with the opioid receptors and peptides. However, the receptor does not bind opioids and the peptide orphanin FQ/nociceptin shows no affinity toward m, d, or k receptors. All four receptor genes display similar genomic organization, with conserved intron–exon junctions, suggesting a common ancestor gene. These genes have been characterized throughout vertebrates, but cannot be identified earlier in evolution. The four genes are referred as to Oprm1 (m), Oprd1 (d), Oprk1 (k), and Oprl1 (orphanin FQ/ nociceptin) (genomic information is available on the NCBI website). The encoded receptors are classically named MOR, DOR, KOR, and ORL-1, respectively, throughout the literature. A novel nomenclature MOP (m), DOP (d), KOP (k), and NOP (orphaninFQ/ nociceptin) has recently been proposed by the International Union of Basic and Clinical Pharmacology (IUPHAR) to unify receptor abbreviations. Many single-nucleotide polymorphisms have been identified in both coding and regulatory sequences of human opioid receptor genes, and large genetic investigations are underway to associate haplotypes with neurological or psychiatric diseases. At present, coding
Opioid Peptides and Receptors 533
Opiates CH3 N
HO
O
Opioid receptors
Opioid peptides
OH
Morphine
m d k
Analgesia addiction
rewarding properties and abuse liability, whereas k agonists are strongly dysphoric. The latter activity has hindered the development of centrally acting k agonists for pain control in the clinic. The pharmacology of d agonists has shown slower progress. The notion that d agonists may represent useful analgesics with low abuse liability has been proposed for many years and is currently being explored. Genetics Approach
Preproenkephalin Preprodynorphin Preproopiomelanocortin
a Pain Hedonic status Stress Others b Figure 1 The opioid system: (a) exogenous opiates activate opioid receptors; (b) the endogenous opioid system. In (a), morphine is the main active ingredient of opium and has strong analgesic and addictive properties, which result from a direct interaction of the drug with opioid receptors. In (b), the endogenous opioid system is a neuromodulatory system. The three opioid receptors naturally interact with a family of opioid peptides, produced by proteolytic cleavage of three large precursor proteins. Receptors and peptides are broadly expressed throughout the nervous system and control nociceptive pathways (pain), mood, well-being (hedonic status), and responses to stress. The system also controls respiration, gastroinstestinal motility, and endocrine and immune functions.
polymorphisms as well as silent or noncoding polymorphisms that may influence receptor expression levels in the brain are under study.
Physiology of the Opioid System In Vivo Opioid peptides and receptors are strongly expressed in the limbic system, as well as in nociceptive pathways and along the hypothalamic–pituitary–adrenal axis. In these neural circuits, the opioid system regulates addictive and emotional behaviors, and controls responses to pain or stress. Pharmacology
Three decades of pharmacology have demonstrated an analgesic activity for all three m, d, and/or k agonists. Generally, opioid analgesics used in the clinic are m agonists. These compounds show strongest efficacy in the treatment of severe acute pain, but their activity remains variable in situations of chronic pain. k agonists are potential candidates in the treatment of visceral pain. Significantly, studies of m and k compounds have highlighted opposing activities of m and k agonists on mood. m agonists show potent
For a decade, mice lacking m, d, or k receptors, as well as preproenkephalin, preprodynorphin, or b-endorphin, have been created by gene-targeting (knockout mice). Single mutant mice, or even the triple receptor knockout mice, are viable and fertile and show no obvious developmental deficit, indicating that the opioid system is not essential for survival. These mutant mice have been extensively analyzed either for spontaneous behaviors or in response to opioid and nonopioid drugs. Parallel to the pharmacology, this genetic approach has clarified the specific contribution of each molecular component of the opioid system in opioid-controlled physiology and behaviors in vivo. In particular, the comparative analysis of m, d, and k knockout mice has clearly shown very distinct activity patterns for each receptor in vivo (Figure 2). Similar comparisons are currently underway for peptide knockout mice. Main conclusions from gene knockout mice are the following. m receptors represent the primary molecular target for morphine in vivo and mediate both beneficial and adverse effects of the most broadly used opiate. m receptors also mediate the rewarding properties of nonopioid drugs of abuse including cannabinoids, alcohol, and nicotine or even natural reinforcers such as social interactions. m receptors therefore represent a key molecular trigger for reward, and most likely contribute to the initiation of addictive behaviors. k receptors, as predicted by the pharmacology, mediate dysphoric activities of both k opioids and cannabinoids and oppose m receptors in regulating the hedonic tone. d receptors are less directly involved in hedonic control. Very distinct from m and k receptors, d receptors regulate emotional responses and show anxiolytic and antidepressant activity. This specific function of d receptors is now confirmed both by gene knockout and pharmacological studies using SNC80, the only commercially available highly selective d compound. Further analysis of d knockout mice and the development of more selective compounds will probably reveal other activities of delta receptors. m, d, and k receptor-deficient mice all exhibit enhanced pain sensitivity. This indicates that the three receptors, activated by endogenous opioid peptides, tonically inhibit nociceptive responses. Noticeably,
534 Opioid Peptides and Receptors Morphine
U50488H
SNC80
b-Endorphin
Enkephalins
d
m
Reward
Dynorphins
Emotional reactivity
k
Dysphoria
Analgesia Figure 2 Physiological role of opioid receptors and peptides in vivo. Opioid peptides show some, although weak, selectivity toward m, d, and k receptors. A highly selective exogenous (morphine) or synthetic (SNC80 and U50488H) agonist is indicated for each receptor. The combination of pharmacological studies with gene-knockout approaches (targeted gene inactivation in mice) showed that each receptor controls specific neural responses. m and k receptors produce euphoria or dysphoria, respectively, whereas d receptors control levels of anxiety and depressive-like behaviors (emotional reactivity). The activation of all three receptors inhibits pain (analgesia), but each receptor controls a distinct panel of pain modalities.
the phenotypes of mutant mice differ across pain assays. In models of physiological or acute pain, m receptors modulate mechanical, chemical, and supraspinally controlled thermal nociception, whereas k receptors modulate spinally mediated thermal nociception and visceral pain. Again, d receptors differ from m and k receptors in that there is no obvious regulation of acute pain. In contrast, there is evidence for a role of d receptors in reducing hyperalgesia in situations of inflammatory and neuropathic pain. These data, combined with many pharmacological studies, clearly demonstrate a specific role for each receptor in regulating the broad diversity of pain modalities. Finally, due to the short half-life and structural similarity of the peptides, identifying a specific role for each endogenous opioid in vivo has been difficult. Data from opioid peptide knockout mice do not necessarily follow data from receptor knockouts, consistent with the notion that multiple peptides probably act at each receptor. Although affinity studies and pharmacological approaches suggest a relationship between b-endorphin and m receptors, enkephalin and d receptors, and dynorphin and k receptors (Figure 2), knockout data provide a more complex picture. It is likely that the specific anatomical location of receptors and peptide release largely drive opioidergic synapse functioning.
Receptor Structure Receptor Binding
Overall m, d, and k receptors show a 60% amino acid sequence identity. Closest homology occurs within the seven-transmembrane helical core, which contains the opioid-binding pocket (Figure 3(b)). Extracellular domains, including three extracellular loops and the N-terminal domain, determine m, d, and k selectivity. These domains differ strongly among receptors and probably act as a gate filtering m, d, and k agonists or antagonists entering the binding pocket. Note that synthetic compounds have been developed with high m, d, and k selectivity, whereas endogenous peptides show limited receptor preference (Figure 2). Receptor Signaling
As in all GPCRs, opioid receptors convey extracellular signals within the cell by activating heterotrimeric G-proteins, which interact with cytoplasmic domains of the receptor. Agonist binding modifies helical packing of the receptor, and a rearrangement in the positioning of transmembrane domains 3, 6, and 7 has been proposed to drive the transition between inactive and active conformations of the receptor.
Opioid Peptides and Receptors 535 m, d, k selectivity
Outside Binding pocket Signal transduction Inside
a
Gi/o-protein binding
Regulation
b
c Figure 3 Opioid receptors: (a) receptors belong to the G-protein-coupled receptor superfamily with a seven-transmembrane topology; (b) three-dimensional computer model of the human d-opioid receptor; (c) direct visualization of d receptors in native neurons. As shown in (a), m, d, and k receptors are highly homologous. Each receptor domain contributes to receptor function, as indicated. Regulation occurs immediately after the receptor is activated by an opioid agonist and includes receptor phosphorylation, uncoupling from the G-protein, and internalization. In (b), the seven-transmembrane helices are shown in red, and portions of extrahelical loops are in white. The binding pocket penetrates halfway within the helical bundle, and side chains of amino acid residues forming the main binding site are shown in blue and green. As shown in (c), hippocampal neurons extracted from a genetically engineered mouse endogenously produce fluorescent d-opioid receptors. The receptor is observed at the surface of both cell bodies and processes of the neuron (see continuous green fluorescence on the plasma membrane, top). Exposure to the agonist produces receptor internalization, visible under the form of highly fluorescent dots within the neuron. Note the concomitant fading of surface fluorescence (bottom).
This helical movement modifies the receptor’s intracellular structure, hence the receptor–G-protein interaction. Intracellular loops of the receptor form a large part of the receptor–G-protein interface. These intracellular receptor domains are almost identical across m, d, and k receptors, consistent with the fact that all three receptors interact with inhibitory G-proteins of the Go/Gi type. G-protein subunits dissociate from the activated receptor and, in turn, modulate intracellular effectors and pathways. Several opioid-evoked signaling events have been identified both in transfected cells and native tissues. Opioids inhibit voltage-dependent Ca2þ channels or activate inwardly rectifying potassium channels, thereby diminishing neuronal excitability. Opioids also inhibit the cyclic adenosine monophosphate (cAMP) pathway and activate mitogen-activated protein kinase (MAPK) cascades, both of which affect cytoplasmic events and transcriptional activity of the cell. Overall, opioids inhibit neurons by decreasing
either neuronal firing or neurotransmitter release, depending on the post- or presynaptic localization of the receptors. Finally, opioid receptors are expressed on both excitatory and inhibitory neurons and can therefore exert inhibition or disinhibition within neural circuits. Regulation of Receptor Signaling
The C-terminal domain differs largely across opioid receptors. Together with intracellular loops, this receptor domain contributes to receptor–G-protein coupling. Significantly, both the C-terminus and intracellular loops also interact with other cellular effectors following receptor activation. Effectors include receptor-specific (G-protein-coupled receptor kinase (GRK)) or-nonspecific (protein kinase A (PKA) and protein kinase C (PKC)) kinases, arrestins that act as adaptors between the receptor and the endocytic machinery, and other recently identified proteins.
536 Opioid Peptides and Receptors
These interacting proteins drive the desensitization of receptor signaling, which typically occurs as an adaptive response after receptor stimulation. Desensitization mechanisms include receptor phosphorylation and uncoupling from the G-protein, as well as receptor endocytosis (Figure 3(c)) and redistribution between the cell surface and intracellular compartments. Largely determined by C-terminal epitopes, these dynamic phenomena differ across m, d, and k receptors. Many studies have addressed the mechanisms of opioid receptor trafficking and desensitization in cellular models and, more recently, in vivo. At present, studies on native neurons suggest that agonist-stimulated d receptors are mainly targeted toward degradation pathways after endocytosis, whereas internalized m receptors are resensitized in early endosomes and rapidly recycle back to the cell surface. Understanding the dynamic aspects of receptor trafficking to and from the cell surface has become a major field in GPCR research. Chronic Opiates
Regulatory events that occur at the receptor level, as already described, represent adaptive mechanisms that classically contribute to cellular homeostasis under acute agonist stimulation. Chronic exposure to opiates, however, has long-term consequences, which lead to strong and perhaps irreversible alterations of brain functioning at the cellular, synaptic, and network levels. Behaviorally, these adaptations are manifested by tolerance, defined as a reduced sensitivity to the drug effects, and dependence revealed by drug craving and withdrawal symptoms. Many efforts have been made for several decades to understand the molecular basis of these elaborate responses to chronic opiates. Modifications of opioid receptor-associated ion channels and second-messenger pathways have been demonstrated in several brain areas. Also several protein kinases, including PKA, PKC, and calcium/ calmodulin-dependent protein kinase (CaMK)II; glutamate receptors; cytoskeleton proteins or neurotrophic factors are involved. Finally opposing neurotransmitter systems – called anti-opioid systems – are recruited. Integrating these observations into a coherent explanation for tolerance and dependence in vivo remains a challenge.
Opioid Receptor Heterogeneity The Existence of Opioid Receptor Pharmacological Subtypes
Opioid receptor pharmacology is complex and the existence of multiple m, d, and k receptor types has been proposed since the early 1970s. Gene cloning led
to the characterization of only three receptor genes, and the molecular basis for pharmacological diversity has long remained a matter of debate. Alternative splicing has been reported, but it has been extremely difficult to establish the biological relevance of these alternative transcripts in vivo and to correlate their existence with the multiple opioid receptor subtypes that were described earlier by the pharmacology. Today, it is admitted that the three main Oprm1-, Oprd1-, and Oprk1-encoded receptors are highly dynamic proteins, which may indeed account for the wide diversity of pharmacological subtypes. Three Receptor Proteins, Many More Cellular Responses
There are several ways to explain pharmacological heterogeneity of opioid receptors. First, increasing evidence supports the notion that m, d, and k receptors may adopt multiple active conformations. Mutagenesis data suggest the existence of multiple binding modes for opioids within the binding pocket. Further, signaling studies in cellular models show that receptor activation and subsequent regulations are strongly drug-dependent. Hence the ligand–receptor complex, rather than the receptor itself, determines the ultimate physiological cellular response. A second source of heterogeneity is the direct cellular environment of the receptor. Heterotrimeric G-proteins differ across cell types, and the number of possible G-protein-associated signaling pathways has expanded dramatically. The variable combinations of G-protein subunits and the nature of associated signaling networks necessarily generate neuron-specific, or even neuron compartment-specific, responses. Also, many regulatory proteins directly interact with the receptor C-terminal tail (as already discussed) and potentially influence opioid-receptor pharmacology, as was demonstrated for several other GPCRs. A third potential receptor modulator is another receptor molecule. The possibility that GPCRs exist as dimeric or oligomeric complexes has gained evidence in the recent years. Co-expression data suggest that the physical association of opioid receptors either as homodimers or heterodimers, or even with other GPCRs, creates novel receptor entities with unique pharmacological properties, which increases opioid receptor heterogeneity. Whether receptor dimerization truly modulates opioid pharmacology in vivo remains an important question. In conclusion, molecular approaches have provided a novel view of opioid receptors. The receptor is now considered a dynamic multicomponent unit rather than a single protein entity. It is likely that the complexity of opioid responses will extend beyond the previously reported pharmacological subtypes as in vivo molecular pharmacology develops. Rational design of opioid
Opioid Peptides and Receptors 537
compounds that activate a specific subset of m, d, or k receptor-associated signaling pathways is a possible strategy for developing novel drugs of high therapeutic value and low adverse activities, but this still remains a far distant goal.
Conclusion The last decade in opioid research has led to a better understanding of the genetic basis of opioidcontrolled behaviors and physiology. It has also opened avenues toward the development of novel therapeutic opioids in the field of chronic pain, emotional disorders, and addictive diseases. Recombinant technologies have allowed high-throughput screening for novel opiate compounds, the detailed molecular analysis of receptor structure and function, and the identification of associated proteins regulating receptor signaling. Ongoing studies have returned to analyzing opioid receptor and peptides operating in their physiological environment, using molecular and genetic tools as well as high-resolution imaging techniques. There is a need to identify neural sites where opioid peptide release and subsequent opioid receptor activation controls nociceptive, emotional, and motivational circuits. Also, it is important to characterize signaling pathways that are relevant to specific behavioral or physiological responses to opioids in vivo. In addition, novel insights into brain function will arise from understanding the interactions of the opioid system with other neurotransmitter systems. For example, whether the opioid system interacts with the cannabinoid system or antiopioid systems at the molecular, cellular, or circuit level remains an open question. Last, the elucidation of molecular and network adaptations to chronic opiates will undoubtedly shed light on the general mechanisms of brain plasticity.
Further Reading Akil H, Watson SJ, Young E, et al. (1984) Endogenous opioids: Biology and function. Annual Review of Neuroscience 7: 223–255.
Bailey CP and Connor M (2005) Opioids: cellular mechanisms of tolerance and physical dependence. Current Opinion in Pharmacology 5: 60–68. Brownstein MJ (1993) A brief history of opiates, opioid peptides and opioid receptors. Proceedings of the National Academy of Sciences of the United States of America 90: 5391–5393. Chang KJ, Porreca F, and Woods JH (eds.) (2004) The Delta Receptor. New York: Dekker. Contet C, Kieffer BL, and Befort K (2004) Mu opioid receptor: A gateway to drug addiction. Current Opinion in Neurobiology 14: 370–378. Devi LA (2001) Heterodimerization of G-protein-coupled receptors: Pharmacology, signaling and trafficking. Trends in Pharmacological Sciences 22: 532–537. Dickenson TH and Kieffer BL (2005) Opiates – basic. In: Koltzenburg M, and McMahon SB (eds.) Wall & Melzack’s Textbook of Pain, 5th edn., ch. 27. London: Elsevier. Evans CJ (2004) Secrets of the opium poppy revealed. Neuropharmacology 47(supplement 1): 293–299. Ikeda K, Ide S, Han W, et al. (2005) How individual sensitivity to opiates can be predicted by gene analyses. Trends in Pharmacological Sciences 26: 311–317. Kieffer BL (1997) Molecular aspects of opioid receptors. In: Dickenson A and Besson J-M (eds.) Handbook of Experimental Pharmacology, Vol. 130: The Pharmacology of Pain, pp. 281–303. Berlin: Springer. Kieffer BL and Gaveriaux-Ruff C (2002) Exploring the opioid system by gene knockout. Progress in Neurobiology 66: 285–306. LaForge KS, Yuferov V, and Kreek MJ (2000) Opioid receptor and peptide gene polymorphisms: Potential implications for addictions. European Journal of Pharmacology 410: 249–268. Mansour A, Fox CA, Akil H, et al. (1995) Opioid-receptor mRNA expression in the rat CNS: Anatomical and functional implications. Trends in Neuroscience 18: 22–29. Nestler EJ (1996) Under siege: The brain on opiates. Neuron 16: 897–900. Pierce KL, Premont RT, and Lefkowitz RJ (2002) Seven-transmembrane receptors. Nature Reviews Molecular Cell Biology 3: 639–650. Williams JT, Christie MJ, and Manzoni O (2001) Cellular and synaptic adaptations mediating opioid dependence. Physiological Reviews 81: 299–343.
Relevant Website http://www.ncbi.nlm.nih.gov – NCBI.
Neuropeptide Y (NPY) and its Receptors D R Gehlert, Eli Lilly and Company, Indianapolis, IN, USA ã 2009 Elsevier Ltd. All rights reserved.
Introduction Pancreatic polypeptide (PP) was originally isolated as a contaminant observed during the preparation of insulin. Subsequently, neuropeptide Y (NPY) was isolated from pig brain and named for the presence of C- and N-terminal tyrosines, while peptide YY (PYY) was isolated from pig intestine. While NPY is primarily localized to neurons and PP is found mainly in endocrine pancreas, PYY is found in both endocrine cells and neurons and, therefore, may exhibit neurotransmitter functions as well. While these peptides exhibit diversity in their expression, they share a common three-dimensional structure. Amino acid residues 1–8 form a type II proline helix followed by a loop, after which residues 15–32 form an a-helix, and the four C-terminal residues are in a flexible loop conformation. When comparing the amino acid sequences from a variety of mammals, only 2 of the 36 amino acids of NPY are variable. As such, NPY is one of the most evolutionary conserved peptides known. On the other hand, the PYY sequence exhibits eight variable amino acids between different orders of mammals. PP is one of the least conserved peptides known and appears to have undergone rapid and recent evolution. Despite the limited conservation observed in the PP and PYY amino acid sequences, the general three-dimensional structure (Figure 1) is maintained across a wide variety of species, indicating its potential importance in function.
Expression and Regulation of NPY The NPY gene is composed of four exons and results in the synthesis of a 97-amino-acid preproNPY and is located on human chromosome 7 at the locus 7p15.1. PreproNPY is proteolytically processed by a prohormone convertase into the C-terminal peptide of NPY (CPON) and NPY1-39. This is further processed by carboxypeptidase to NPY1-37. The processing and C-terminal amidation of NPY is accomplished by peptidylglycine a-amidating monooxygenase and is essential for biological activity. NPY can be further processed to NPY3-36 and NPY2-36 by two enzymes, dipeptidyl peptidase IV and aminopeptidase P, respectively. While the functional significance of CPON is
538
unknown, the C-terminal fragments of NPY have some selectivity for the Y2 receptor subtype. In the rat brain, NPY is found in numerous brain regions including the hypothalamus, amygdala, hippocampus, nucleus of the solitary tract, locus coeruleus, nucleus acumbens, and cerebral cortex. NPY is colocalized with norepinephrine, g-aminobutyric acid (GABA), and somatostatin in agouti-related protein (AGRP) containing neurons of the hypothalamus. In addition, NPY is found in peripheral neurons including sympathetic neurons where it is co-stored and co-released with norepinephrine. The adrenal medulla is the primary source of circulating NPY, though it is also expressed in liver, heart, kidney, spleen, pancreas, platelets, and endothelial cells of blood vessels. The human PYY and PP genes are found 10 kb apart on chromosome 17q21.1. Like NPY, the PYY and PP genes are organized into four exons with three introns. The rat preproPYY sequence is 98 amino acids, consisting of a preceding signal peptide sequence and followed by a 31-amino-acid C-terminal extension peptide called CPOP. PYY is found primarily in the L cells of the gastrointestinal tract, with the highest concentrations found in the rectum, followed by the ileum and the colon. Like NPY, PYY is cleaved by dipeptidyl peptidase to the 3-36 fragment that is thought to be the principal active circulating peptide. PYY and PYY3-36 are stored in the gut mucosal endocrine cells, and both are found in the circulation. Rat preproPP has a signal peptide sequence and a 30-residue C-terminal extension peptide. PP is found primarily in the pancreas and is localized to the islets of Langerhans. NPY synthesis and secretion are regulated by a number of hormones and neurotransmitters. At the cellular level, activation of either protein kinase A or protein kinase C will result in increased NPY synthesis and secretion. Catecholamines and muscarinic agonists will increase NPY, while GABA agonists will decrease it. Glucocorticoids, growth hormone, ghrelin, and gonadal steroids are hormones that increase NPY synthesis and secretion in the hypothalamus, while insulin, leptin, and retinoic acid will decrease it. NPY synthesis is also increased by a variety of growth factors such as nerve growth factor, insulinlike growth factor-I and brain-derived neurotrophic factor in several brain regions. Of particular interest is the increase in brain NPY observed following treatment of experimental animals with antidepressants, amphetamines, and lithium, indicating that it may play a role in the clinical response to these agents.
Neuropeptide Y (NPY) and its Receptors 539 36 Tyr Arg
34 Gln
15 Ala
Ala
Glu
Leu Asp
Tyr
Pro
25
Ser Arg Tyr
Ala Leu
His
Asp Glu
Gly 10
Leu Thr Ile 31
Pro
Pro
Pro
Ile
Tyr
20
Ala
Lys
Asn Asp
Arg
Asn
Arg
5
Ser
Tyr 1
Neuropeptide Y Figure 1 Representation of the three-dimensional structure of NPY.
Functional Activities of NPY When administered directly into the brain, NPY produces marked increases in food intake and a preference for carbohydrate-rich food. This effect has been seen in a number of species ranging from fish to rhesus monkey. Exogenous NPY is the most potent and efficacious orexigenic peptide known. Chronic central administration to rats results in increased adiposity and the symptomology of the metabolic syndrome. Centrally administered NPY also decreases energy expenditure to promote adiposity. Less is understood about the role of endogenous NPY in the regulation of feeding. NPY levels are changed in experimental animals exhibiting a disturbed energy balance such as that seen in anorexia, bulimia neurosa, and diabetes. Starvation increases the concentrations of NPY and NPY mRNA in the hypothalamus, and, when administered centrally to rats, antisense oligonucleotides decrease feeding. NPY produces a number of other centrally mediated effects including anticonvulsant activity, modulation of arousal, anxiolysis, and modulation of cognition. Central administration of NPY produces antidepressant-like effects in animal models, and chronic administration of antidepressant drugs affects hippocampal NPY expression. NPY also acts with the central and peripheral nervous systems to decrease neuronal excitability. NPY has been reported to decrease excitability in vitro using hippocampal and dorsal root ganglion preparations. Mice lacking NPY are more susceptible to seizures, and people with temporal lobe epilepsy have increased NPY expression in CA3 regions as well as prominent rearrangements in receptor density and distribution. Furthermore, NPY-deficient mice were found to have an impaired development of neurons in the olfactory epithelium, suggesting a role in
neuronal development. Central NPY also regulates ethanol consumption in rodents. In transgenic mice, an inverse correlation between NPY levels and drinking has been shown. Furthermore, a point mutation (leucine7 to proline) in the preproNPY gene has been associated with higher alcohol consumption in humans and linkage analysis suggesting that dysfunction in the NPY gene locus contributes to ethanol preference in rats. In addition to its role in feeding, hypothalamic NPY is important in the regulation of reproductive function. NPY acts at both the hypothalamic and pituitary levels to regulate reproductive hormone release. In the presence of estrogen, NPY stimulates leutinizing hormone (LH), while with low levels of estrogen, NPY will inhibit LH release. Continuous central infusion of NPY inhibits LH release in both male and female rats. NPY also exhibits a steroid-dependent regulation of gonadotropin-releasing hormone (GnRH) release. As hypothalamic NPY release is regulated by a vast number of neurotransmitters and hormones, NPY may be an important integrator for the multiple signals that regulate reproductive status. NPY is frequently co-localized with noradenaline (NA) in sympathetic nerves and in the peripheral nervous system. Exogenous NPY enhances NA-mediated vasoconstriction, especially upon strong stimulation. In addition, NPY appears to stimulate smooth muscle proliferation and is important in angiogenesis and revascularization of ischemic tissues. Centrally administered NPY reduces arterial blood pressure and heart tone in both rats and dogs. Several cardiovascular dysfunctions as well as some tumor diseases are associated with increased plasma levels of NPY. In addition, NPY can act as an antinociceptive peptide, probably through inhibition of substance P release in the dorsal horn of the spinal cord. PYY and PYY3-36 Function
PYY is the found primarily in the periphery and is released from the gastrointestinal (GI) tract in response to meals. Many of the GI effects described for PP, including inhibition of gall bladder secretion, gut motility, and pancreatic secretion, can also be produced by PYY. Other peripheral effects produced by PYY include the inhibition of fluid and electrolyte secretion in the intestinal tract and direct vasoconstriction. When administered centrally, PYY increases food intake with potency and efficacy similar to that seen with NPY. Via dipeptidyl peptidase IV, PYY is posttranslationally processed to PYY3-36, which appears to be the primary circulating form of the peptide. Peripherally administered PYY3-36 produces a potent decrease in food intake in both human and rodents.
540 Neuropeptide Y (NPY) and its Receptors PP Function
PP is almost exclusively expressed in endocrine pancreas and is released in response to meals. PP produces its biological effects mainly in the GI tract, where it inhibits pancreatic secretion, gall bladder activity, intestinal motility, ileum contractions, and gastric emptying and stimulates colon contractions. In addition, PP affects metabolic functions including glycogenolysis and decreases fatty acid levels. However, binding sites for PP have been found in several rat brain regions, including the interpeduncular nucleus, hypothalamus, and brain stem, suggesting that PP may also have direct effects of brain function. This may explain why intracerebroventricular (ICV) injection of PP has been found to stimulate feeding in several different species of experimental animals. In contrast, peripherally administered PP inhibits food intake in humans and rodents.
Receptors for the NPY Family of Peptides Four functional G-protein-coupled receptors (GPCRs) have been identified that bind the NPY family of peptides, termed Y1, Y2, Y4, and Y5 (Table 1). An additional y6 receptor has been identified as a functional receptor in some species including mice and rabbits; however, it does not encode a functional protein in humans and is absent in rats. Y1, Y2, and Y5 bind NPY and PYY with similar high affinities. PP binds to Y4 with higher affinity than the other peptides. Y1 and Y2 are the predominant brain receptor populations (Figure 2). All the NPY receptors have been shown to couple to Gi and thus mediate inhibition of cAMP synthesis. When expressed in Chinese hamster ovary (CHO) cells, intracellular signaling involves pertussis toxin-sensitive phosphorylation of extracellular signal-regulated protein kinase (ERK)-1 and -2, confirming that these receptors couple to Gi and/or Go. In addition, Y1 activates mitogen-activated protein kinase in gut epithelial cells (IEC-6). Y1, Y2, Y4,
Table 1 Y receptors and their proposed functions Receptor
Agonist actions
Y1
Angiogenesis, anxiolysis, cellular proliferation, circadian rhythm regulation, endocrine regulation, increase feeding, sedative, vasoconstriction Angiogenesis, anticonvulsant, anxiogenesis, cellular proliferation decrease feeding, gastrointestinal motility, and secretion Decrease feeding, gastrointestinal regulation Anticonvulsant, circadian rhythm regulation, increase feeding
Y2
Y4 Y5
and Y5 receptors can couple to phospholipase C to provoke release of Ca2þ from intracellular stores. Y1 Receptor
The gene for Y1 is located in a cluster together with Y2 and Y5 on human chromosome 4q31. Y1 is primarily localized to the brain, peripheral nervous system, and peripheral blood vessels. The highest Y1 levels in the brain are found in the anterior thalamus, cerebral cortex, medial geniculate, and amygdala. In the peripheral nervous system, Y1 mRNA is found in the superior cervical and dorsal root ganglion. In the vasculature, Y1 is localized to intramyocardial, colonic, and renal blood vessels with the expression being localized to the intima as well as the media. Y1 mRNA has been detected in a number of peripheral tissues including the colon, kidney, adrenal gland, heart, and placenta. Within the kidney, Y1 mRNA is detected in the renal collecting ducts, loop of Henle, and the juxtaglomerular apparatus. Y1 receptor is important in the feeding response produced by exogenous NPY. Central injection of Y1-selective agonists increase feeding, while Y1 antagonists can inhibit NPY-induced feeding. Most of the vasoconstriction produced by NPY and PYY is transduced via the Y1 receptor, as shown by the inhibitory affects of various Y1-selective antagonists on NPY-induced vasoconstriction and the absence of effects in Y1-deficient mice. Many of the other central effects of NPY have been attributed to the Y1 receptor, including reduction of blood pressure and heart rate, anxiolysis, and antidepressant activity. Stimulation of brain Y1 receptors inhibits the consumption of ethanol by rodents, inhibits neurogenic inflammation, and is antinociceptive. Y2 Receptor
The Y2 gene is located near the Y1 gene on chromosome 4 and encodes a 381-amino-acid protein. Y2 has more than 90% amino acid identity within mammals, although Y2 is only about 30% identical to the Y1 and Y4 receptors. Y2 is present in the central and peripheral nervous system, the intestine, and certain blood vessels. Brain Y2 is pronounced in the hypothalamus, lateral septum, hippocampus, and amygdala. In the peripheral nervous system, Y2 is localized to the superior cervical ganglion and dorsal root ganglia. Using polymerase chain reaction (PCR) detection, mRNA encoding the Y2 receptor has been found in a variety of regions of the GI tract, including the crypt, villus, colonic epithelium, and nonepithelial jejunum. Y2 is predominantly a presynaptic autoreceptor inhibiting further release of NPY and cotransmitters.
Neuropeptide Y (NPY) and its Receptors 541
Y1
FC
Y2
AON SL
Cl
CA3 LD fi
MG
CA3
Figure 2 Differential autoradiographic localization of Y1 and Y2 receptors in the rat brain. Rat brain sections were labeled with 125 I-Leu31-Pro34-PYY (Y1) and 125I-PYY3–36 (Y2) and pseudocolor representations of the receptor distributions obtained. Abbreviations are frontal cortex (FC), accessory olfactory nucleus (AON), claustrum (Cl), lateral septum (SL), lateral dorsal thalamus (LD), CA3 subregion of the hippocampus (CA3), fimbria (fi), and medial geniculate (MG).
An opposing relationship between Y1 and Y2 is evident for the central effects of NPY on blood pressure, as centrally administered Y2 agonists increase blood pressure while central Y1 stimulation decreases it. Given into the brain, Y2 agonists produce anxiogenic responses, presumably by inhibiting the release of NPYand/or GABA from neurons within the amygdala to limit their postsynaptic actions. In the pig spleen, Y2 stimulation evokes potent vasoconstriction that is inhibited by a Y2-selective antagonist. In addition, Y2 is involved in NPY-induced angiogenesis and NPYinduced effects on circadian rhythms. The phenotype of Y2-deficient mice indicates the potential importance of Y2 in feeding responses and bone formation. Peripherally administered PYY3–36 produces a decrease in food consumption by rodents and humans, presumably by activating Y2 receptors in the hypothalamus. Y4 Receptor
The human Y4 gene is found on chromosome 10 and encodes a protein of 375 amino acids. Of the Y receptors, Y4 is the least conserved across species and is thought to have evolved rather recently.
Considerable differences in peptide pharmacology between the rat and human receptors have been noted. For instance, when position 34 of NPY or PYY is replaced by proline, there is an increase in rat Y4 affinity while the human Y4 affinity is unaffected by this change. In contrast to the Y1 and Y2 receptors, the Y4 receptor exhibits a rather restricted distribution within the brain. While mRNA encoding this receptor exhibits a rather broad distribution, studies using radiolabeled PP indicate a high level expression in the interpeduncular nucleus and locus coeruleus in the nucleus of the solitary tract. A lower level of expression is seen within the hypothalamus and thalamus as well as a number of other brain regions. Interestingly, the binding site labeled by 125 I-PP in the interpeduncular nucleus does not pharmacologically match the Y4 receptor, indicating that this may be an additional PP binding site. Y4 mRNA is also abundant in the human colon with somewhat lower levels in the prostate and pancreas. In addition, mRNA encoding Y4 has been found in skeletal muscle, thyroid gland, heart, stomach, small intestine, adrenal medulla, cerebral cortex, and nasal mucosa.
542 Neuropeptide Y (NPY) and its Receptors
Since PP is likely to be the preferred endogenous ligand for Y4, this receptor may mediate many of the GI effects produced by PP, including rabbit ileum contractions. Intravenously administered PP can also reduce food consumption, though the role of endogenous peptide and the Y4 receptor in appetite and body weight regulation has not been established. Y5 Receptor
Y5 is encoded by a gene located on chromosome 4 encoding a 446-amino-acid protein. The Y5 receptor is highly conserved (88–90% overall amino acid identity) between orders of mammals. While Y5 mRNA receptor has a broad distribution in the central nervous system, only low levels of Y5 receptor protein are found in the brain using receptor autoradiography and immunohistochemistry. In the periphery, Y5 mRNA is been reported to be present in the intestine, ovary, testes, prostate, spleen, pancreas, kidney, skeletal muscle, liver, placenta, and heart. The importance of Y5 in NPY-induced feeding has been confirmed by studies involving antisense knockdown, knockout animals, and Y5-selective agonists. Other effects of NPY that are mediated by the Y5 receptor include reproduction through inhibition of LH release and regulation of brain excitability and seizures. Y5 receptor stimulation inhibits neuronal activity in the suprachiasmatic nucleus without generating a phase shift, indicating that Y5 may be indirectly involved in regulation of circadian rhythms. Regulation of Y Receptors
Internalization and desensitization of NPY family receptors Cell surface expression of Y receptors is regulated primarily by agonist-induced desensitization. This is an important mechanism in vivo since NPY knockout mice have a dramatic increase in Y receptor binding when compared to wild-type controls. After agonist stimulation, approximately 20–30% of Y1 expressed in the neuroblastoma cell line SK-N-MC was internalized after stimulation. Y1 internalization follows an endosome pathway followed by rapid recycling to the cell surface. Y4 and Y5 also exhibit rapid internalization after agonist stimulation, while theY2 receptor appears to internalize at a very slow rate. b-Arrestin 2 is an important protein involved in the initial desensitization and internalization of GPCRs. After agonist stimulation, Y1 and Y5 exhibited a rapid recruitment of b-arrestin 2, while Y2 showed a very weak and slow increase in b-arrestin 2 association. Y4 interacts at a rate intermediate to the other receptors.
Dimerization among NPY family receptors It has been recently established that GPCRs form homoand heterodimers and these complexes have functional implications by altering the signaling, the pharmacology, or the internalization. GPCR dimers can be covalently linked as described for the metabotrophic glutamate receptors or held together by noncovalent interaction like that observed for many of the rhodopsin-like receptors (family A). The Y receptors homodimerize when expressed in HEK293 cells. Y1, Y2, and Y5 homodimers were not affected by agonist treatment, whereas Y4 appears to be assembled as a dimer that dissociates upon agonist stimulation. More recently, Y1 and Y5 were found to heterodimerize when co-expressed. This heterodimer has similar binding properties to what would be expected with a Y1 and Y5 mixture; however, functional coupling and internalization are dramatically altered.
The NPY System and Medication Development Given the robust effects of the NPY family of peptides on feeding behavior and body weight, there has been considerable interest in the discovery and development of molecules that exploit these effects. In the early 1990s there were a number of efforts by pharmaceutical companies to discover centrally active Y1 and Y5 antagonists. In general, it was difficult to obtain Y1 compounds with suitable properties for clinical development. Adequate oral bioavailability and brain penetration were the consistent challenges observed with a number of chemical series. Several compounds were evaluated preclinically and were able to reduce food consumption and body weight. With the discovery of the Y5 receptor in the mid1990s, a number of efforts targeted this receptor for drug discovery. While a number of very nice compounds were identified, the highly selective Y5 antagonists produced very modest, if any, changes to food consumption and body weight in preclinical testing and, with one compound, no effect in clinical testing. To date, alternate clinical applications for this class of compounds have not been identified. In the early 2000s, peripherally injected PYY3–36 was found to decrease food intake through activation of the hypothalamic Y2 receptor. The efficacy was observed in preclinical models as well as several clinical trials. Subsequent efforts have found the potential for improved efficacy using peptides with affinity for both the Y2 and Y4 receptors. Several of these peptides are under clinical development, and an improved understanding of the potential of these peptides to
Neuropeptide Y (NPY) and its Receptors 543
treat obesity and the metabolic syndrome should be forthcoming. In addition to body weight disorders, there are substantial opportunities to discover and develop agonists or antagonists for the treatment of a variety of cardiovascular disorders, epilepsy, immune disorders, psychiatric disorders, and substance abuse disorders. See also: Neuropeptide Release; Neuropeptide Synthesis and Storage.
Further Reading Balasubramaniam A (2003) Neuropeptide Y (NPY) family of hormones: progress in the development of receptor selective agonists and antagonists. Current Pharmaceutical Design 9: 1165–1175. Berglund MM, Hipskind PA, and Gehlert DR (2003) Recent developments in our understanding of the physiological role of PP-fold peptide receptor subtypes. Experimental Biology & Medicine 228: 217–244. Colmers WF and El Bahh B (2003) Neuropeptide Y and epilepsy. Epilepsy Currents 3: 53–58. Gehlert DR and Crawley JN (eds.) (2004) Special Issue on Neuropeptide Y. Neuropeptides 38: 135–267.
Heilig M and Thorsell A (2002) Brain neuropeptide Y (NPY) in stress and alcohol dependence. Reviews in Neuroscience 13: 85–94. Jacques D, Sader S, Perreault C, and Abdel-Samad D (2006) NPY and NPY receptors: Presence, distribution and roles in the regulation of the endocardial endothelium and cardiac function. EXS 95: 77–87. Kalra SP and Kalra PS (2004) NPY – an endearing journey in search of a neurochemical on/off switch for appetite, sex and reproduction. Peptides 25: 465–471. Magni P (2003) Hormonal control of the neuropeptide system. Current Protein and Peptide Science 4: 45–57. Morton GJ and Schwartz MW (2001) The NPY/AgRP neuron and energy homeostasis. International Journal of Obesity and Related Metabolic Disorders 25(Supplement 5): S56–62. Obuchowicz E, Krysiak R, and Herman ZS (2004) Does neuropeptide Y (NPY) mediate the effects of psychotropic drugs? Neuroscience and Biobehavioral Reviews 28: 595–610. Redrobe JP, Dumont Y, and Quirion R (2002) Neuropeptide Y (NPY) and depression: From animal studies to the human condition. Life Sciences 71: 2921–2937. Ubink R, Calza L, and Hokfelt T (2003) ‘Neuro’-peptides in glia: Focus on NPY and galanin. Trends in Neuroscience 26: 604–609. Zukowska Z and Feuerstein G (eds.) (2005) The NPY Family of Peptides in Immune Disorders, Inflammation, Angiogenesis and Cancer. Basel: Birkha¨user Verlag.
Corticotropin-Releasing Hormone and Urocortins: Binding Proteins and Receptors M O Huising, The Salk Institute for Biological Studies, La Jolla, CA, USA and Department of Animal Physiology, Radboud University Nijmegen, Nijmegen, The Netherlands W W Vale, The Salk Institute for Biological Studies, La Jolla, CA, USA
Many of the behavioral effects that are associated with stress, such as increased anxiety, improved cognition, increased depression, and reduced feeding behavior, are often the direct effect of the activation of central CRF circuits.
ã 2009 Elsevier Ltd. All rights reserved.
CRF: Founding Member of a Small Family of Neuropeptides
Discovery of CRF and Its Role in the Stress Response Corticotropin-releasing factor (CRF) was identified in 1981 as the key hypothalamic factor to induce the release of adrenocorticotropic hormone (ACTH) from the anterior pituitary gland. This feat was accomplished by the purification of the peptide from sheep hypothalami. CRF is the most potent known stimulator of ACTH release, although other hypothalamic factors, most notably arginine vasopressin (AVP) and oxytocin, can synergize with CRF to augment the release of ACTH. Nevertheless, the observation that the release of ACTH from the anterior pituitary gland is almost completely blocked by administration of CRF antisera established that CRF is the predominant regulator of pituitary ACTH release. Mature bioactive CRF is a 41-amino-acid neuropeptide that is cleaved from a larger precursor protein lacking intrinsic biological activity (Figure 1). CRF is produced in the parvocellular neurons of the paraventricular nucleus (PVN) of the hypothalamus. These neurons project to the median eminence and release their neuropeptide content into the capillary bed of the hypophysial portal system. This portal system carries CRF to the anterior pituitary corticotropes, which respond by releasing ACTH into the general circulation to induce the release of glucocorticoids from the adrenal cortex. This endocrine axis, referred to as the hypothalamic–pituitary–adrenal (HPA) axis, controls reactions to stress. The stress response is an adaptive response aimed at maintaining or restoring homeostasis in the face of a plethora of potentially adverse stimuli from diverse origins. The glucocorticoids – cortisol in humans and corticosterone in rodents – that are released as a result of HPA axis activation are responsible for many of the hallmarks of the stress response, such as increased energy mobilization via enhanced liver glycogenolysis and the general inhibition of vegetative functions, including digestion, growth, reproduction, and immunity. Importantly, glucocorticoids engage in negative feedback at the level of both the pituitary gland and the hypothalamus to inhibit the synthesis and release of ACTH and CRF, aimed at a timely termination of the stress response. 544
Shortly after the discovery of CRF in sheep, the CRF genes of humans, mice, rats, and other mammals were identified. Urotensin-I (UI), a peptide resembling CRF, was isolated from a fish species (Catastomus commersonii) following the discovery of CRF in mammals. This peptide was initially regarded as the fish ortholog (i.e., the same gene in a different species) of mammalian CRF, until a second peptide also resembling CRF was identified from the same fish species. As this second CRF-like peptide shared much higher similarity with mammalian CRF, it was clear that it represented the actual ortholog of mammalian CRF. The mammalian ortholog of fish UI was identified in 1995 with the isolation and characterization of urocortin 1 (Ucn1). Another peptide with resemblance to the Ucn1 of mammals and the UI of fishes was isolated from the skin of the waxy monkey tree frog (Phylomedusa sauvagei), a species from the Amazonian rain forest. This peptide was named sauvagine and although it is only known from the skin of a single amphibian species, sauvagine has been frequently used in the characterization of many aspects of CRF signaling. In recent years, two additional members of the CRF family of neuropeptides were independently discovered by two groups employing a bioinformatics approach. The current nomenclature for these peptides is urocortin 2 (Ucn2) and urocortin 3 (Ucn3), although one of the identifying groups initially named them stresscopin-related peptide and stresscopin, respectively. All members of the vertebrate CRF family have several characteristics in common with CRF. All are preprohormones that contain at their C-termini mature neuropeptides that are presumed to be liberated by cleavage at a mono- or dibasic cleavage site (Figure 1). In keeping with their discovery via bioinformatics, the existence of mature neuropeptides for Ucn2 and Ucn3 has been predicted, and their bioactivity following de novo synthesis has been established, but neither mature peptide has been directly isolated from an endogenous source.
CRF Family Neuropeptides Signal via One of Two CRF Receptors Two receptors for CRF have been identified and are termed CRFR1 and CRFR2. Both are class B
Corticotropin-Releasing Hormone and Urocortins: Binding Proteins and Receptors 545 Prepropeptide
Mature peptide
Human CRF Mouse CRF Human Ucn1 Mouse Ucn1 Sauvagine Carp UI Human Ucn2 Mouse Ucn2 Human Ucn3 Mouse Ucn3 a
1
11
21
31
41
Human CRF Mouse CRF Human Ucn1 Mouse Ucn1 Sauvagine Carp UI Human Ucn2 Mouse Ucn2 Human Ucn3 Mouse Ucn3 b Figure 1 Corticotropin-releasing factor (CRF) family ligands (Ucn1, Ucn2, and Ucn3; urocortins) are encoded as preprohormones. (a) The C-terminal mature neuropeptides are liberated from the prepropeptide sequence by cleavage at mono- or dibasic cleavage sites. At the amino acid level CRF and urocortins share moderate amino acid similarity. (b) Amino acids with similar biochemical properties are color-coded for clarity.
(secretin-like) seven-helix transmembrane G-proteincoupled receptors and consist of 395 to 440 amino acids. CRFR1 and CRFR2 share approximately 70% amino acid identity, with most of their amino acid differences concentrated in the N-terminal region. As the N-terminal domain (together with the extracellular loops) is responsible for ligand binding, this accounts for the substantial differences in the pharmacology of the receptors. CRFR1 is the principal receptor activating pituitary corticotropes during the stress response and has nanomolar affinity for CRF. CRFR1 binds Ucn1 with a similarly high affinity, but has no appreciable affinity for physiological doses of Ucn2 and Ucn3. The CRFR2 receptor has highest affinity for Ucn1 and also binds Ucn2 and Ucn3 with high affinity. The affinity of CRFR2 for CRF is about 10- to 30-fold lower than its affinity for the urocortins, but is still below 100 nM. This illustrates the redundancy of the CRF system, wherein multiple ligands share, and under certain circumstances may even compete for, two different receptors (Figure 2). A further layer of complexity is added to CRF signaling by the observation that multiple transcripts can be generated from both CRFR genes. The CRFR1 gene (CRHR1, humans; Crhr1, rodents) consists of 14 exons in humans and 13 exons in rodents. More
than one dozen alternative transcripts have been found that result from the skipping of one or several exons during gene transcription. The predicted proteins from some of these splice variants partially or completely lack the extracellular domain (ECD), or lack one or several transmembrane domains. Nevertheless, the mechanism responsible for these CRFR1 splice isoforms is unknown and the physiological roles played by CRFR1 splice variants remain presently unclear; direct evidence for the presence of corresponding CRFR1 proteins is lacking for most of these splice isoforms. A clear example of how alternative splicing can generate functionally different receptor proteins is found in the mammalian gene encoding CRFR2. Two different receptor isoforms, CRFR2a and CRFR2b, are transcribed from the CRFR2 gene by splicing alternative start exons onto a common set of downstream exons. A third isoform, CRFR2g, is produced in a similar fashion in primates only. The resulting receptor proteins differ only at the extreme N-terminus of their ECD. As the majority of the ECD is encoded by exons four, five, and six, which are shared by all CRFR2 isoforms, their pharmacological properties do not differ substantially. Nevertheless, the different CRFR2 isoforms vary markedly in their central and
546 Corticotropin-Releasing Hormone and Urocortins: Binding Proteins and Receptors Ucn1
CRF
Ucn2
Ucn3
CRF-BP
CRFR1
CRFR2
Figure 2 Corticotropin-releasing factor (CRF) and the urocortins (Ucn1, Ucn2, and Ucn3) signal through one of two CRF receptors (CRFR1 and CRFR2). Both CRF receptors differ in the repertoire of ligands that they bind. Furthermore, CRF and Ucn1, and to a lesser extent Ucn2, bind to CRF-binding protein (CRF-BP). The arrows pointing away from each ligand indicate the ligand potential targets. The thickness of the arrows indicates the approximate affinity for each of the ligands.
peripheral tissue distribution, as the transcription of each isoform is driven by its own unique promoter sequence. In rodents, the expression of CRFR2a is largely restricted to the central nervous system (CNS), with expression in the olfactory bulb, ventromedial hypothalamus (VMH), the lateral septum (LS), and the bed nucleus of the stria terminalis (BNST). In contrast, the expression of CRFR2b within the CNS is restricted to the choroid plexus. In the periphery of rodents, the situation is completely reversed. Expression of CRFR2b is widespread, with highest expression levels in skeletal muscle, heart, and skin. Interestingly, CRFR2a is the predominantly expressed isoform in the human periphery. By comparison, CRFR1 is expressed in brain and pituitary as well as in peripheral sites, including adrenal gland, spleen, testis, and skin. Recently, several other splice variants were discovered that are generated from the CRFR2 gene. One particularly interesting splice variant stems from the deletion of exon six, resulting in a premature stop codon caused by a shift in the amino acid reading frame. The resulting 143-amino-acid protein consists of the complete ECD of CRFR2a followed by a unique hydrophilic 38-amino-acid C-terminal tail. The resulting soluble CRFR2a can abrogate the response to CRF and Ucn1 in vitro and may serve as a natural modulator of the actions of CRF and urocortins. A two-step model has been proposed for hormone binding and receptor activation of CRF receptors. First, the C-terminus of the ligand binds the extracellular domain of the receptor, followed by the partial insertion of its N-terminus into the transmembrane region. This results in a conformational change that leads to exchange of guanosine diphosphate (GDP) for guanosine triphosphate (GTP) on the Ga subunit of
the G-protein that is coupled to the third intracellular loop of the receptor. The activated Ga subunit dissociates from the Gbg dimer, and both species can activate various downstream signaling pathways. In many cells, activation of the G-protein activates the adenylyl cyclase–cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway. This pathway results in downstream modulation of target proteins by PKA as well as transcriptional regulation via cAMP response element-binding protein (CREB). Depending on the cell type that expresses CRF receptors, other signaling pathways can be activated downstream of CRF receptors via different G-proteins. These pathways include extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK), protein kinase C (PKC), nuclear factor-kappa B (NF-kB), and several others.
CRF-Binding Protein A unique player in the network of CRF signaling exists in the form of a soluble 37 kDa protein that binds CRF with high affinity. This CRF-binding protein (CRF-BP) was initially discovered in late gestational maternal plasma, where it prevents activation of the stress response via the substantial amounts of CRF from placental origin that circulate prior to parturition. The CRF-BP gene consists of seven exons that encode a protein of 322-amino-acids, unrelated to the CRF receptors. In fact, its primary amino acid sequence bears no resemblance to that of any other known protein. CRF-BP is characterized by ten conserved cysteine residues that form five consecutive disulfide bridges, and a single N-linked glycosylation site. CRF-BP orthologs have been demonstrated for a number of vertebrate species and were recently
Corticotropin-Releasing Hormone and Urocortins: Binding Proteins and Receptors 547
discovered in species of insects. In mammals, CRF-BP binds CRF and Ucn1 with an affinity that is several fold higher than the affinity of CRF receptors for either peptide. Human CRF-BP also binds mouse, but not human, Ucn2 with nanomolar affinity and does not bind Ucn3 (Figure 2). Interestingly, several peptide fragments, such as CRF(6–33) and CRF(9–33), which consist only of the core of the mature peptide hormone, retain the capacity to bind to CRF-BP with high affinity, but have lost the ability to activate CRF receptors. This indicates that different areas of the peptide hormone are involved in binding to the receptor and to the binding protein. The high affinity of CRF-BP for CRF and Ucn1 suggests an antagonistic role by preventing these hormones from activating their receptors. Indeed, CRF-BP is expressed in anterior pituitary corticotropes and is capable of abrogating the CRF-induced release of ACTH from pituitary cells in vitro. CRF-BP is widely expressed within the CNS, including the cerebral cortex, the amygdala, BNST, olfactory bulb, and various nuclei within the brain stem and hypothalamus. Peripherally, complexes of CRF-BP and CRF are rapidly cleared from the circulation, supporting an antagonistic role for CRF-BP.
Central CRF Pathways Are Involved in Anxiety and Depression Although initially discovered as an activator of the HPA axis, CRF is produced at many other sites in the brain outside of the parvocellular section of the PVN. Within the hypothalamus CRF is produced in several other nuclei, including the preoptic, supraoptic, and suprachiasmatic nuclei. Prominent sites of CRF localization outside of the hypothalamus include the cortex, the central nucleus of the amygdala (CeA), the BNST, the locus coeruleus (LC), and the parabrachial nucleus (PB). These nuclei are generally associated with autonomic function. Consensus is that within the CNS, CRF acts as a neurotransmitter by activating downstream neurons. Central CRF pathways are involved in behavioral patterns that are commonly associated with stress, such as changes in arousal and anxiety. Several rodent models have been developed for the measurement and quantification of the behavioral manifestations of arousal and anxiety. Transfer of a rodent from a familiar nonstressful environment, such as its home cage, to a completely new environment is considered an acutely stressful event for the animal. This transition is immediately reflected in a series of behavioral changes that can be measured as a reduction in locomotor activity and reduced exploration of the novel environment, a
reduction in rearing, and an increase in grooming and freezing. Injections of CRF (intracerebroventricularly; directly into the ventricles of the brain) can reproduce this panel of behaviors associated with novelty stress. Mice that lack CRFR1 display a marked reduction in anxiety-related behaviors in addition to blunted HPA activation. This suggests that the majority of the anxiogenic effects of CRF are mediated via CRFR1. Data regarding the role of CRFR2 in anxiety are less clear. Both anxiogenic (anxiety-promoting) and anxiolytic (anxiety-reducing) effects have been attributed to CRFR2, and the direction and magnitude of the effects mediated via CRFR2 may be dependent on the precise anatomical location of CRFR2 expression as well as on confounding factors such as gender. Dysregulation of CRF pathways is implicated in the etiology of depression. CRF concentrations are significantly elevated in the cerebrospinal fluid of depressed patients, and reduction of these levels by electroconvulsive therapy correlates with alleviation of depression symptoms. Several rodent models have been developed that display behavior reflecting some traits associated with depression in human patients. In one of these models a mouse is suspended by the tail and the severity of depression is quantified as the fraction of the time the animal is immobile. In another test mice are subjected to involuntary swimming and depression is measured as a reduction in swimming and climbing behavior and increased immobility. In these animal models for depression, selective CRFR1 antagonists reduce immobility, implicating central CRF pathways that employ CRFR1 in the pathophysiology of depression. Recently, mice were generated that are deficient for the selective CRFR2 agonist Ucn2. One of the major findings in these Ucn2deficient mice is their antidepression-like phenotype, as measured by reduced immobility in the forced swim test and the tail suspension tests. These experiments demonstrate a prominent role for CRFR1 and CRFR2 in the development of depressive disorders.
Feeding, Digestion, and Metabolism Several CRF pathways are involved in the regulation of feeding and digestion. Central circuits employing CRF regulate key aspects of appetite control and feeding behavior, while peripheral CRF receptors relay signals that affect gastrointestinal motor functions. Much of the integration of central metabolic cues with peripheral indicators of nutrient status, such as circulating levels of leptin and insulin, takes place in the hypothalamus. It has long been known that injection of CRF and Ucn1 directly into the brain ventricle produces anorexic effects. Activation of either
548 Corticotropin-Releasing Hormone and Urocortins: Binding Proteins and Receptors
CRF receptor produces anorexic effects, but with different kinetics. Intracerebroventricular injection of CRFR1-specific agonists results in a rapid inhibition of food intake, while the specific activation of CRFR2 results in prolonged anorexia that requires several hours to establish. These findings are supported by studies on CRF-deficient mice: mice lacking CRFR1 are insensitive to short-onset anorexia induced by a general CRF receptor agonist such as Ucn1, while CRFR2-deficient mice fail to mount prolonged hypophagia following Ucn1 administration. The arcuate nucleus has strong CRFR1 expression while the ventromedial hypothalamus features prominent CRFR2 expression. Both nuclei play key roles in the regulation of food intake and metabolism. The VMH receives abundant projections of Ucn3, and, to a lesser extent, Ucn1, while it is only sparsely innervated by CRF. Ucn2 and Ucn3 are also detected in the arcuate nucleus. These observations suggest that many of the anorectic effects of CRF family peptides are mediated via CRFR2 and that the urocortins constitute its endogenous ligands, modulating appetite control and feeding behavior. CRFR2 expression levels in the VMH drop during fasting and are correlated with circulating leptin levels, suggesting that plasma leptin levels directly or indirectly affect the level of CRFR2 expression in this nucleus. Direct stimulation of the VMH by CRFR2 agonist injection or electrical stimulation not only results in reduced food intake, but also activates the sympathetic nervous system. Via this route energy expenditure is increased through the stimulation of brown adipose tissue (BAT) thermogenesis. Thus, feeding behavior is enhanced and BAT thermogenesis is decreased during starvation, when plasma leptin and consequently VMH CRFR2 expression are low. During times of surplus body energy stores, increased leptin concentrations and CRFR2 levels drive increased BAT thermogenesis and inhibit food intake. CRF signaling is involved in the regulation of gastrointestinal motor function. Although the inhibition of gastromotor function that is observed during stress is partially mediated via central CRFR2, CRF and urocortins have direct effects on the gastrointestinal tract. Peripheral administration of urocortins delays gastric emptying and inhibits gut motility. These inhibitory effects on gastromotor functions are blocked by selective CRFR2, but not CRFR1, antagonists. In contrast to the inhibition of gastric motility by central and peripheral CRFR2 activation, colonic motility can be stimulated during stress via CRFR1-mediated activation of the parasympathetic nervous system as well as via CRFR1 present in the colonic myenteric nervous system. The physiological
relevance of the dichotomy in the activation of different stretches of the gastrointestinal tract is explained by adaptive value in times of stress. Inhibitions of gastric and gut activity and colon evacuation are different ways of inhibiting digestion, thus contributing to the maximal allocation of resources toward responding to stress.
Cardiovascular Effects of Urocortins via CRFR2 It has long been known that CRF influences cardiovascular function. During acute stress, heart rate, blood pressure, and cardiac output increase as the result of activation of the sympathetic nervous system. This overall stimulation of the cardiovascular system is an important adaptive response, assisting an animal to actively cope with a stressful event. Central injection of CRF mimics the cardiovascular effects that are normally observed following stress, provided that the sympathetic nervous system is intact. Since coadministration of a CRFR1-specific antagonist attenuates CRF-induced increases in heart rate, the CRF-induced activation of the sympathetic nervous system is, at least in part, mediated via CRFR1. In contrast to the generally stimulatory effects of central CRF injections on the cardiovascular system, CRF delivered peripherally causes vasodilatation and a drop in mean arterial blood pressure. Systemic CRF and Ucn1 also increase cardiac contractility. These direct effects of systemically administered CRF and Ucn1 on the cardiovascular system suggest the presence of local CRF receptors. Indeed, CRFR2 is expressed in significant amounts in cardiac tissue, including myocardium, epicardium, arterioles of atrium and ventricle, and aorta. Its role in the cardiovascular system was elegantly demonstrated by comparing the hemodynamic properties of normal mice with mice lacking CRFR2. The latter mice have an elevated mean arterial blood pressure that is insensitive to systemic Ucn1 administration, whereas normal mice respond to Ucn1 with significant and prolonged (>1 h) hypotension. Ucn1 and Ucn2 are potential endogenous ligand(s) for CRFR2 in the cardiovascular system, as both urocortins are expressed in isolated cardiac myocytes. Alternatively, it is possible that CRF family peptides reach the heart via nerve terminals or through the general circulation to activate cardiac CRFR2. In addition to the effects on blood pressure and cardiac contractility mediated via CRFR2 in vivo, urocortins display cardioprotective effects. The expression of Ucn1 and Ucn2 in cultured cardiomyocytes is increased after hypoxia followed by reoxygenation,
Corticotropin-Releasing Hormone and Urocortins: Binding Proteins and Receptors 549
a model for cardiac ischemia. The presence of urocortin peptides in media reduces the percentage of cultured cardiomyocytes that can enter apoptosis and necrosis following hypoxia. CRFR2 is required for these cardioprotective effects, since Ucn2 and Ucn3 fail to rescue cardiomyocytes isolated from CRFR2-deficient mice from apoptosis. Moreover, the administration of Ucn2 and Ucn3 immediately following experimentally induced ischemia of the intact rat heart results in a substantial reduction of the infarct zone. These cardioprotective effects of urocortins acting through CRFR2 are of particular interest in the context of cardiac ischemia. In particular, the fact that Ucn2 and Ucn3 lack the affinity for CRFR1 required for HPA activation or induction of anxiety makes them promising targets in the quest for therapeutical interventions aimed at alleviation of the adverse outcome of cardiac ischemia in humans.
CRF and Pregnancy CRF and Ucn1 as well as their cognate receptors and CRF-BP are expressed in different tissues of the female reproductive system. CRF plays a remarkably versatile role affecting pregnancy, from blastocyst implantation to parturition. Mammalian reproduction, in which the fetus is carried inside the womb, is an awkward phenomenon from an immunological point of view. The developing fetus is only 50% ‘self’: the paternal contribution to the fetus qualifies it as a foreign body and would as such subject it to attack by the maternal immune system. For obvious reasons, this would be incompatible with successful reproduction. CRF protects the fetus from attack by the maternal immune system and simultaneously prevents the developing fetal immune system from attacking maternal tissues. Around the time of blastocyst implantation into the uterus, CRF is produced in the embryonic trophoblast and the maternal decidua, the contact interface of maternal and fetal tissues. Locally, CRF increases the expression of an important proapoptotic factor, Fas ligand (FasL), via CRFR1. FasL induces apoptosis in cells carrying its cognate binding partner, Fas, which is expressed by many immune cells. As Fas only appears at the cell surface following leukocyte activation, CRF-induced upregulation of FasL at the site of implantation allows the specific deletion of only those immune cells that are activated at the maternal–fetal interface. CRF is expressed in the placenta of primates, but not rodents, and CRF from placental origin is detectable in the maternal circulation beginning approximately halfway through gestation. Circulating CRF levels rise sharply toward the end of pregnancy and,
close to term, reach levels equivalent to peak CRF levels measured in the hypothalamic–pituitary capillary bed during acute HPA activation. Yet, these high levels of CRF do not activate the HPA axis, because they are bound to a surplus of CRF-BP, which is also produced by the placenta. In the fetal circulation, increasing levels of CRF will activate the fetal HPA axis. This results in glucocorticoid release from the fetal adrenal gland, a process that drives the maturation of a number of fetal organs, including the lungs in preparation for delivery. Throughout much of gestation, CRF in the placenta evokes quiescence of uterine smooth muscle cells via the CRFR1-dependent activation of the PKA pathway. In a series of intricate molecular switches that involve the oxytocin-dependent phosphorylation of CRFR1, CRFR1 is coupled to a different G-protein toward parturition. This causes a shift in the intracellular cascade that is activated upon ligand binding to CRFR1 from PKA to PKC. Instead of inducing myometrial relaxation, CRF now can assist in the induction of myometrial contractility and labor.
Summary CRF was initially noted for its potent capacity to activate the stress response. In the decades that followed its discovery, three additional ligands related to CRF, as well as two receptors and a soluble binding protein, have been discovered. While CRF still has strong ties to ‘stress,’ we now know that CRF signaling is involved in a multitude of other central and peripheral processes. These include the regulation of anxiety, arousal, and depression, the modulation of food intake and sympathetic nervous system activation, control of cardiovascular output as well as cardioprotection, and a combination of fascinating roles in the regulation of human reproduction. Many of these processes are at the heart of human well-being, which has sparked widespread interest in CRF. The challenge that lies ahead will be the development of strategies to intervene in isolated CRF pathways without causing widespread activation of CRF receptors throughout the body. See also: Neuropeptide Release; Neuropeptide Synthesis and Storage.
Further Reading Charmandari E, Tsigos C, and Chrousos GP (2005) Endocrinology of the stress response. Annual Review of Physiology 67: 259–284. Chen A, Perrin M, Brar B, et al. (2005) Mouse corticotropinreleasing factor receptor type 2a gene: Isolation, distribution,
550 Corticotropin-Releasing Hormone and Urocortins: Binding Proteins and Receptors pharmacological characterization and regulation by stress and glucocorticoids. Molecular Endocrinology 19: 441–458. Coste SC, Quintos RF, and Stenzel-Poore MP (2002) Corticotropinreleasing hormone-related peptides and receptors: Emergent regulators of cardiovascular adaptations to stress. Trends in Cardiovascular Medicine 12: 176–182. Heinrichs SC and Koob GF (2004) Corticotropin-releasing factor in brain: A role in activation, arousal, and affect regulation. The Journal of Pharmacology and Experimental Therapeutics 311: 427–440. Hillhouse EW and Grammatopoulos DK (2002) Role of stress peptides during human pregnancy and labour. Reproduction 124: 323–329. Hillhouse EW and Grammatopoulos DK (2006) The molecular mechanisms underlying the regulation of the biological activity of corticotropin-releasing hormone receptors: Implications for physiology and pathophysiology. Endocrine Reviews 27: 260–286. King BM (2006) The rise, fall, and resurrection of the ventromedial hypothalamus in the regulation of feeding behavior and body weight. Physiology & Behavior 87: 221–244. Lewis K, Perrin MH, Blount A, et al. (2001) Identification of urocortin III, an additional member of the corticotropin-releasing factor
(CRF) family with high affinity for the CRF2 receptor. Proceedings of the National Academy of Sciences of the United States of America 98: 7570–7575. McLean M and Smith R (2001) Corticotrophin-releasing hormone and human parturition. Reproduction 121: 493–501. Owens MJ and Nemeroff CB (1991) Physiology and pharmacology of corticotropin-releasing factor. Pharmacological Reviews 43: 425–473. Reyes TM, Lewis K, Perrin MH, et al. (2001) Urocortin II: A member of the corticotropin-releasing factor (CRF) neuropeptide family that is selectively bound by type 2 CRF receptors. Proceedings of the National Academy of Sciences of the United States of America 98: 2843–2848. Westphal NJ and Seasholtz AF (2006) CRH-BP: The regulation and function of a phylogenetically conserved binding protein. Frontiers in Bioscience 11: 1878–1891. Winsky-Sommerer R, Boutrel B, and de Lecea L (2005) Stress and arousal: The corticotrophin-releasing factor/hypocretin circuitry. Molecular Neurobiology 32: 285–294. Zorilla EP, Tache´ Y, and Koob GF (2003) Nibbling at CRF receptor control of feeding and gastrocolonic motility. Trends in Pharmacological Sciences 24: 421–427.
Hypocretin/Orexin and MCH and Receptors A Adamantidis and L de Lecea, Stanford University School of Medicine, Palo Alto, CA, USA ã 2009 Elsevier Ltd. All rights reserved.
Discovery The hypocretins (Hcrts) were discovered as part of a subtractive hybridization study to identify messenger RNAs restricted to discrete nuclei within the rat hypothalamus. Among these was a species, named preprohypocretin, whose expression was restricted to a few thousand neurons that were bilaterally distributed within the dorsolateral hypothalamus. The sequence of preprohypocretin suggested that it encoded two putative neuropeptides, named hypocretin-1 (Hcrt1) and Hcrt2, with significant similarity to the incretin peptide family. A large collaborative study to identify endogenous ligands for orphan G-protein-coupled receptors (GPCRs) discovered the peptides independently. This group referred to the peptides as orexins because they stimulated acute food intake when administered to rats during the daytime. In this article, we refer to the peptides by their first-used name, hypocretins (from the sequence similarities with various members of the incretin family and its hypothalamic localization), but both names have been used extensively in the large literature that has grown up around the peptides. Melanin-concentrating hormone (MCH) was originally discovered in salmon pituitaries, in which it acts as a circulating hormone and lightens skin in response to the background color change or during stress reaction. MCH acts antagonistically to a- and b-melanocyte-stimulating hormone (MSH) and induces pigment aggregation of melanin granules in skin melanophores in vitro and in vivo. In fish, MCH is a cyclic neuropeptide of 17 amino acids which is generated along with other peptide (neuropeptide EI (NEI)) by cleavage of a preprohormone. Subsequent to its identification in fish, MCH immunoreactive-like labeling in rat brains led to the identification of this peptide, its gene, and its tissue localization in this species. In vertebrates, MCH-producing neurons are restricted to the lateral hypothalamus (LH) and zona incerta (ZI).
Structure Both Hcrt and MCH peptides are derivatives of a longer precursor of 130 and 165 residues, respectively, with an apparent signal sequence.
The C-terminal 19 residues of these two putative peptides – Hcrt2 (28 residues; RPGPPGLQG RLQRLLQANGNHAAGILTM-amide) and Hcrt1 (33 residues; EPLPDCCRQKTCSCRLYELLHGAGNHAAGILTLamide) – share 13 amino acid identities, suggesting that the peptides have related structures and functions. This region of Hcrt2 contains a seven-amino-acid match with secretin. Hcrt1 contains two intrachain disulfide bonds. Human Hcrt1 is identical to the rodent peptide, whereas human Hcrt2 differs from rodent Hcrt2 at two residues. The nonamidated forms of the peptides are not electrophysiologically active. The MCH intrachain disulfide bond leads to a cyclic peptide (19 residues; DFDMLRCMLGRVY RPCWQV). Strong sequence similarity has been found with the somatostatin peptide family.
Distribution Hcrt and MCH neurons are morphologically very similar, multipolar or fusiform in shape, with two to five primary dendrites that are either smooth or sparsely invested with dendritic spines. In rats (3000 neurons) and humans (50 000–80 000 neurons), cells highly positive for Hcrt mRNA and immunoreactivity are located between the rat fornix and the mammillothalamic tracts (Figure 1). In addition to their close localization to the Hcrt neurons, MCH-producing neurons are also found in the ZI. Transcripts and immunostaining for MCH in a small set of neurons of the olfactory tubercules and pontine tegmentum have also been reported. The projections of Hcrt and MCH cells are widely distributed within the central nervous system (CNS) in accordance with their respective receptors’ transcripts distribution. Sparse anatomical and physiological evidence supports the existence of subpopulations of Hcrt or MCH neurons. Based on c-fos profile expression, Harris and colleagues proposed two subpopulations of Hcrt neurons related to arousal and consummatory behaviors. Subpopulations of MCH neurons have been identified with the presence of the cocaine- and amphetamine-regulated transcript (CART) peptide immunoreactivity and neuroanatomical projections. There are several reports of Hcrt, MCH, and receptors expression in the periphery, including in the enteric nervous system, pancreas, kidney, stomach, and ileum. Hcrt expression has also been detected in the human retina, anterior pituitary, and adrenal gland.
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Figure 1 Neuroanatomy of the hypocretin (Hcrt) and melanin-concentrating hormone (MCH) systems. Schematic drawing of a saggital section through the rat brain showing the organization of the MCH and Hcrt systems. Dots indicate the relative location and abundance of MCH (green)- and Hcrt (red)-expressing cell bodies. Arrows point out some of the more prominent terminal fields. In situ hybridization of both precursor mRNA (ppMCH and ppHcrt) in mice is shown below in a coronal brain section made at the level of the vertical black line. Amy, amygdala; Ctx, cortes; Hipp, hippocampus; LC, locus coeruleus; ME, median eminence; OB, olfactory bulb; OT, olfactory tubercule; PP, posterior; Sp, Ch, Spinal chord; Th, thalamus; VTA, ventro-tegmental area.
Receptors and Signaling Cascades The initial orphan GPCR, Hcrt receptor 1 (Hcrtr1) (also referred to as OX1R), binds Hcrt1 with high affinity, but binds Hcrt2 with 100- to 1000-fold lower affinity. A related GPCR, Hcrtr2 (OX2R), sharing 64% identity with Hcrtr1, was identified by searching database entries with the Hcrtr1 sequence and has a high affinity for both Hcrt2 and Hcrt1. These two receptors are highly conserved (95%) across species. Radioligand-binding studies and Ca2þ flux measurements have shown Hcrt1 to have an equal affinity for Hcrtr1 and Hcrtr2, whereas Hcrt2 has 10-fold greater affinity for Hcrtr2 than Hcrtr1. The mRNAs that encode the two hypocretin receptors and the receptor proteins themselves, detected by immunohistochemistry, are both enriched in the brain and moderately abundant in the hypothalamus. But they have different distributions within the brain. The distribution of Hcrt receptors is largely consistent with Hcrt axon innervation patterns. The composite distribution of the two Hcrt receptors strongly resembles the distribution of MCH receptor 1 (MCHR1). In the locus coeruleus (LC), amygdala, and other brain stem noradrenergic groups, MCHR1 mRNA distribution is similar to that of the Hcrtr1. In regions such as the septum, hypothalamus, and much of the brain stem, the distribution of MCHR1 mRNA resembles that of the Hcrtr2.
Interestingly, in silico analysis of the human genome sequences revealed a second MCH receptor (MCHR2), which shares 38% identity with MCHR1. However, the MCHR2 is only functional or encoded in the dog, ferret, monkey, and human, whereas it is absent or encoding a nonfunctional truncated protein (pseudogene) in the mouse, hamster, rat, guinea pig, and rabbit. In addition to a different gene structure (five exons instead of the two in MCHR1) MCHR2 expression is restricted to the cortex, hippocampus, amygdala, and hypothalamus in the CNS. The Hcrt and MCH receptors are also widely expressed in the periphery, especially in endocrine tissues, including the pituitary, adrenal gland, testis, gastrointestinal tract, pancreas, and pineal gland. Both Hcrt1 and Hcrt2 act through a family of guanosine triphosphate (GTP)-binding proteins (Gq) that activate protein kinase C (PKC) and the mobilization of intracellular Ca2þ. Gq-activated signaling cascades result in phosphorylation of Ca2þ channels, which can increase Ca2þ conductance and neuronal excitability. Probably depending on the tissue considered, the MCHR1 GPCR could be coupled to Gi/o, Ga, Gs, or Gq via different intracellular cascade including mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase ERK(1/2). In contrast, the MCHR2 receptor has been reported to act only through Gq.
Hypocretin/Orexin and MCH and Receptors 553
Activation of specific intracellular pathways by the MCH may rely on the modification of MCHR1 (or MCHR2) binding properties through interactions with cytosolic or membrane protein. Recently, using a yeast two-hybrid approach, the group of Bachner identified several proteins (MCHR1-interacting zinc-finger protein (MIZIP), periplakin, and neurochondrin), which interact with the C-terminus of the MCHR1 receptor. They may be involved in the regulation of MCHR1 signaling and may play a role in modulating MCHmediated physiological functions in vivo.
Input/Output Hcrt and MCH neurons in the LH are highly interconnected with a network of glutamatergic, GABAergic, dopaminergic, and cholinergic neurons (Figure 2). Glutamate, excitatory amino acid transporter (EAAT)3, and the vesicular glutamate transporters VGLUT1 and VGLUT2 are expressed by Hcrt neurons; thus, Hcrt neurons are likely to be glutamatergic. Other proteins detected in Hcrt neurons include dynorphin; GABAA receptor epsilon subunit; serotonin receptor (5-HT-1A); m-opioid receptor; pancreatic polypeptide Y4 receptor; adenosine A1 receptor; leptin receptor; signal transducer and activator of transcription (STAT-)3; and the neuronal pentraxin Narp, implicated in clustering of ionotropic glutamate receptors. In the rat, CART has been show to be coexpressed mainly in the MCH neuron of the rostro-median ZI. Moreover, part of these CART-expressing MCH neurons also express the glutamate decarboxylase enzyme, confirming that part of the MCH neurons are GABAergic. Recently, substance P has been co-localized with few MCH neurons. Part of the MCH neuronal population expresses several membrane proteins, including neurokinin receptor (NK)3, the receptor to the chemokine stromal cell-derived factor-1a (CXCR4), leptin receptor (Ob-R, undefined subtype), glutamate and GABA receptors, adrenoreceptor (a2), muscarinic and serotoninergic receptors, and HcrtR1 and/or -2. Interestingly, neuropeptide Y (NPY) has been shown to inhibit MCH neurons by pre- and postsynaptic mechanisms, whereas the melanocortin agonist MTII and melanocortin antagonist SHU9119 were without effect.
Biological Actions within the Brain Experimental administration of MCH and Hcrt peptides to animals stimulates food intake, affects motivation and rewarding behavior and modifies arousal. It is noteworthy that in mammals MCH and Hcrt follow a diurnal pattern of gene expression
and probably peptide release. In vivo recordings of Hcrt neurons revealed that these cells do not fire tonically but, rather, show bursts of activity at the transitions between sleep and wakefulness and during exploratory or consummatory behavior. Feeding and Metabolism
Sakurai and colleagues found that intracerebroventricular (ICV) administration of either Hcrt1 or Hcrt2 increased short-term food consumption in rats. Furthermore, rats that had been deprived of food for 48 h showed increased concentrations of Hcrt mRNA and peptides in the hypothalamus. Feeding responses can be elicited by the local administration of Hcrt1 to the paraventricular nucleus, the dorsomedial nucleus, the LH, or the perifornical area. ICV administration of Hcrt2 also increases food intake in sheep and goldfish. Many observations leave little doubt that the Hcrt system influences and is influenced by primary nutritional homeostasis circuits. For instance, Hcrt neurons are sensitive to glucose, leptin, and triglyceride concentrations. Hcrt neurons are contacted by NPY-containing neurons in the arcuate nucleus, and much of the food intake increase elicited by Hcrt1 seems to be mediated by NPY. Other findings suggest that the Hcrts are not critical players in feeding activities but, rather, play roles in increasing arousal and motivation levels so that feeding can take place. Continuous administration of Hcrt1 for 7 days in rats does not significantly alter daily food intake, body weight, blood glucose, total cholesterol, or free fatty acid levels, suggesting that many of Hcrt’s effects may be limited to the short-term immediate stimulation of feeding behavior consequent to the increased wakefulness. If Hcrt has a direct role on food intake, we expect a lean phenotype in Hcrt-deficient mice, as has been described for MCH. Hcrt knockout mice show very modest, if significant, differences in food intake. Hcrt ataxin-3 mice, which are genetically depleted of Hcrt neurons and would be expected to be lean, show obesity and hypolocomotion, and this effect appears to be dependent on diet and genetic background. During fasting, Hcrt1 accumulation in the cerebrospinal fluid (CSF) does not exceed concentrations normal for the waking period. Also, Hcrt ataxin-3 mice, unlike wild-type animals, do not show an increase in locomotor activity after fasting. All these data suggest that some of the food-uptake effect may result from arousal rather than direct feeding pressure. Among the putative roles of MCH neurons, their involvement in feeding behavior, energy homeostasis, and body weight are by far the best documented. Chronic intracerebral injection increased food intake
NPY
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554 Hypocretin/Orexin and MCH and Receptors
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Figure 2 Inputs to hypocretin (Hcrt) and melanin-concentrating hormone (MCH) neurons. Schematic showing electrophysiologically demonstrated excitatory (green) and inhibitory (red) inputs on Hcrt and MCH neurons. Note that some transmitters have pre- and postsynaptic mechanisms on glutamatergic and GABAergic interneurons, and therefore they can be excitatory and inhibitory. Hcrt neurons are known to contact and excite MCH cells, and in the reciprocal connection it is likely that MCH project back onto Hcrt neurons. Hcrt1 excites Hcrt neurons via glutamatergic interneurons; direct changes in membrane potential have not been observed.
Hypocretin/Orexin and MCH and Receptors 555
during the hours following injections without modifying the ingested food amount in 24 h or the body weight. Also, both ppMCH mRNA and MCH peptide increase after fasting, as does the MCHR1 mRNA. Mice overexpressing MCH develop mild obesity and hyperphagia and MCH knockout mice are lean but hyperphagic, probably because of their increased nocturnal locomotor activity and consequent increased metabolic rate. Accordingly, transgenic mice depleted of MCH neurons also develop late-onset leanness. Finally, leptin has been shown to depress MCH gene expression in a series of obese leptin-deficient animal models (ob/ob knockout mouse, fa/fa rat, and yellow agouti mice, in which animals develop overfeeding). At the cellular level, glucose dose-dependently enhances the electrical excitability of MCH neurons by inducing depolarization and increasing membrane resistance, but it has the opposite effect on the electrical activity of Hcrt cells. As a result of this participation in food intake behavior and energy homeostasis, a plethora of anorexigenic MCHR1 antagonists has been developed in recent years. Motivation and Addiction
Intracranial self-stimulation (ICSS)isa well-characterized paradigm for quantifying brain reward. Infusion of Hcrt1 in the brain ventricles elevates ICSS thresholds, indicating that the peptide decreases brain reward function in a way that is similar to stress. Hcrt neurons are highly responsive to morphine, and their activation is linked to preferences for cues associated with drug and food reward and also naltrexone-precipitated withdrawal. Moreover, Hcrt reinstates extinguished drug-seeking behavior, an effect blocked by an Hcrt1 antagonist. Hcrt1 infusion into the ventricle or the ventral tegmental area leads to a dose-dependent reinstatement of drug-seeking behavior, and it also increases ICSS threshold. Hcrt-induced reinstatement of cocaine seeking was prevented by the blockade of noradrenergic and corticotropin-releasing hormone systems. Accordingly, Hcrtr1 antagonist blocks footshock-induced reinstatement of previously extinguished cocaine-seeking behavior, leading to the conclusion that Hcrts are a major gate in driving stressmediated drug-seeking behavior. Hcrt knockout mice exhibit dramatically attenuated morphine-withdrawal symptoms. However, the response of these neurons is heterogeneous, suggesting that there might be different subpopulations of Hcrt cells. Expression of peproHcrt increases after precipitated withdrawal. Hcrt neurons express the m-opioid receptor; hence their response may be directly related to morphine and naltrexone. These observations might explain why animals self-administer heroin to the LH. Hcrt neurons
have extensive projections to the mesolimbic dopamine and noradrenergic (LC) pathways, regions well studied for their roles in drug addiction. These neurons also project to and inhibit nucleus accumbens neurons, resulting in a negative regulation of the activity of brain reward circuitries. In contrast, MCH neurons are not activated during acute and chronic morphine treatment or following naltrexone-induced withdrawal. Interestingly, MCH injection in the nucelus accumbens has been shown to induce food intake, whereas an MCHR1 antagonist has the opposite effect. Theses data support the idea that, like Hcrt peptide function, the feeding response relies at least partly on activation of brain reward mechanisms. Furthermore, MCHR1 knockout mice fail to develop behavioral (locomotor) sensitization to cocaine and do not exhibit any sign of cocaine conditioning during a saline challenge. It is speculated that MCHR1 may contribute to the neurobiological mechanisms of conditioned cocaine-induced psychomotor effects, possibly to these underpinning sensitizations, and to a lesser extent to those subserving acute pharmacological cocaine action. Arousal
Rodent models Continuous recording of the behavior of Hcrt gene-deleted mice (Hcrt knockout mice) revealed periods of ataxia, which were especially frequent during the dark period. Electroencephalograph (EEG) recordings showed that these episodes were not related to epilepsy, and that the mice suffered from cataplectic attacks and their EEGs showed episodes of direct transition from wakefulness to rapideye movement (REM) sleep, the hallmarks of narcolepsy. Similar observations were made in rats in which the Hcrt neurons of the LH were inactivated by saporin targeting, although in this model, cataplexy was not observed. Mice with an inactivated Hcrtr2 gene had a milder narcoleptic phenotype than the Hcrt knockouts; Hcrtr1 knockouts exhibited only a sleep-fragmentation phenotype, whereas double Hcrtr1/Hcrtr2 mutants recapitulated the full Hcrt knockout phenotype, suggesting that signaling through both receptors contributes to normal arousal, although the role of Hcrtr2 is greater than that of Hcrtr1. Transgenic rats and mice depleted of Hcrt neurons have been generated by expressing a mutant form of ataxin 3 in Hcrt neurons. These animals show a narcolepsy-like phenotype as well as reduced locomotor activity on fasting, strongly suggesting a key role of Hcrt neurons in linking metabolic needs with arousal. Human narcolepsy Nishino and colleagues studied Hcrt concentrations in the CSF of normal controls
556 Hypocretin/Orexin and MCH and Receptors
and patients with narcolepsy by radioimmunoassay. In control CSF, Hcrt concentrations were highly clustered, suggesting that tight regulation of the substance is important. However, of nine patients with narcolepsy, only one had a Hcrt concentration within the normal range. One patient had a greatly elevated concentration, and seven patients had no detectable circulating Hcrt. In an expanded study, Hcrt was undetectable in 37 of 42 narcoleptics and in a few cases of Guillain–Barre´ syndrome. CSF Hcrt was in the normal range for most neurological diseases, but was low, although detectable, in some cases of CNS infections, brain trauma, and brain tumors. Peyron, Thannikal, and their teams of collaborators found that, in the brains of narcolepsy patients, they could detect few or no Hcrt-producing neurons. Whether the Hcrt neurons are selectively depleted, as is most likely, or only no longer expressing Hcrt is not yet known, although one report showed some indications of gliosis. The codistributed MCH neurons were unaffected. Furthermore, a single patient with a nonhuman leukocyte antigen (HLA)-linked narcolepsy carries a mutation within the hypocretin gene itself. The mutation results in a dominant negative amino acid substitution in the secretion signal sequence that sequesters both the mutant and heterozygous wildtype Hcrt nonproductively to the smooth endoplasmic reticulum. These findings leave no doubt as to the central role of the Hcrt system in narcolepsy. Because most cases are sporadic, mutations in the Hcrt gene or those for its receptors can account for no more than a small subset of the human narcolepsies. The HLA association, loss of neurons with signs of gliosis, and age of disease onset are consistent with autoimmune destruction of the Hcrt neurons accounting for the majority of narcolepsy, although a non-immune-mediated degenerative process has not been ruled out. As mentioned previously, the MCH cell population is still intact in narcoleptic patients, and no mutation in the ppMCH or MCHR1/2 genes has been found or linked to arousal pathology. MCHR1 antibody has been found in vitiligo patients, an autoimmune pathology causing skin depigmentation. On the other hand, it has been proposed that MCH neurons act as the counterpart of Hcrt cells. In this way, Verret and
collaborators showed in rats that, during a REM sleep rebound induced by specific REM sleep deprivation, a large population of MCH neurons were immunoreactive for the c-FOS protein, a marker of neuronal activity. In addition, after ICV infusion of the peptide in rats, REM sleep and non-REM sleep amounts were increased, leading the authors to propose a hypnogenic role for MCH neurons.
Concluding Remarks The anatomical and electrophysiological data suggest that Hcrt neurons integrate complex information about circadian, limbic, and metabolic variables. This information is transmitted into a coherent output that excites arousal centers depending on the physiological demand. In vivo recordings of Hcrt neurons have revealed that these cells are active in anticipation of wakefulness and during consummatory behaviors but are mostly silent. Together the data gathered thus far indicate that the Hcrts provide an alarm signal and stability to arousal centers. In contrast, MCH neurons, which strongly innervate Hcrt cells and regulate Hcrt activity, appear to be modulated mostly by energy demand. The cross-talk between these two peptidergic neurons in the LH provides a balance system to modulate complex behaviors dependent on metabolic needs.
Further Reading Bittencourt JC, Presse F, Arias C, et al. (1992) The melaninconcentrating hormone system of the rat brain: An immunoand hybridization histochemical characterization. Journal of Comparative Neurology 319: 218–245. de Lecea L, Kilduff TS, Peyron C, et al. (1998) The hypocretins: Hypothalamus-specific peptides with neuroexcitatory activity. Proceedings of National Academy of Sciences of the United States of America 95: 322–327. de Lecea L and Sutcliffe JG (eds.) (2005) Hypocretins: Integrators of Physiological Functions. New York: Springer. Sakurai T, Amemiya A, Ishii M, et al. (1998) Orexins and orexin receptors: A family of hypothalamic neuropeptides and G protein-coupled receptors that regulate feeding behavior. Cell 92: 573–585. van den Pol AN, Acuna-Goycolea C, Clark KR, and Ghosh PK (2004) Physiological properties of hypothalamic MCH neurons identified with selective expression of reporter gene after recombinant virus infection. Neuron 42: 635–652.
Peptidergic Receptors G Casini and D Cervia, Universita` della Tuscia, Viterbo, Italy ã 2009 Elsevier Ltd. All rights reserved.
Peptides and Intercellular Communication Neuropeptides are peptides involved in nervous system function. They are synthesized in cells in large precursor proteins, and generally several biologically active peptides are contained in the same precursor molecule. In the 1970s the hunt for new peptides was intense, and an ever-growing list could be compiled. Peptidergic signaling molecules are widely distributed throughout the central and peripheral nervous systems and in the peripheral organs. They may act in a neurocrine, paracrine, autocrine, or endocrine manner; the same peptides may participate in intercellular communications through different modalities. For instance, many peptides expressed in the enteric system belong to a class of agents known as brain–gut peptides, functioning both as neuropeptides and gut hormones. Neuropeptides may be co-stored with other neuropeptides or, alternatively, may coexist with ‘classical’ neurotransmitters in different cellular compartments. Different combinations of transmitters and peptides are found in ganglionic cells of the autonomic nervous system. For instance, the sympathetic postganglionic input to the heart arises in a large part from the stellate ganglion; these neurons commonly contain neuropeptide Y (NPY) in addition to norepinephrine, similar to almost all sympathetic postganglionic neurons innervating the vasculature of the gut and hindlimbs. Other sympathetic postganglionic neurons in the stellate ganglion project to the sweat glands using acetylcholine as their major transmitter (instead of the expected norepinephrine), and also contain at least two other peptides, that is, calcitonin gene-related peptide (CGRP), and vasoactive intestinal polypeptide (VIP). In addition, many postganglionic parasympathetic neurons contain acetylcholine and VIP. Thus, while autonomic neurons are commonly referred to as cholinergic or adrenergic, they may also contain permutations of other neuropeptide transmitters including angiotensin II (Ang II), enkephalin, neurotensin, substance P (SP), somatostatin, CGRP, etc. Functionally, specific permutations of transmitters may comprise a ‘chemical code’ for neurons subserving specific actions. Co-stored peptides
and classical transmitters are released together at all terminals of a given neuron, and they act together in determining the responses of target cells. It is a general rule that when a peptide and a classical transmitter coexist, the first mediates long-lasting responses and the latter short-term synaptic events in the target cells.
Peptidergic Receptors Independent of the modality, peptides act through specific receptors which are generally located on the plasma membrane of target cells, although there is growing evidence that certain receptors are also present on the nuclear membrane. Peptidergic receptors belong to the superfamily of heterotrimeric G-proteincoupled receptors (GPCRs), which are characterized by the presence of seven transmembrane domains. The specialized feature of these receptors is to achieve information transfer via the binding of ligands to a recognition domain and allosterically transmit the presence of that ligand to an intracellular domain that leads to G-protein activation. The human genome may code for more than 5000 GPCRs. Less than 10% are known, of which those activated by peptides represent a large proportion. In particular, mammalian peptidergic receptors are grouped into two families of GPCRs: the rhodopsin-like family (the largest family of GPCRs, also called class A), which contains the majority of the known peptidergic receptors, and the secretin-like family (class B). The ligand peptides for the receptors in the secretin-like family are relatively large polypeptides (27–141-amino-acid residues), and seven of them – glucagon, glucagon-like peptides 1 and 2, gastric inhibitory polypeptide, VIP, pituitary adenylate cyclase-activating peptide (PACAP), and growth hormone-releasing hormone – are structurally related. It has been generally assumed that peptides exert their actions on GPCRs, but there are some exceptions. For instance, FMRFamide (Phe-Met-Arg-Phe-NH2) and related peptides typically exert their action through GPCRs. However, two ionotropic receptors for these peptides have recently been identified which may have functional roles in the central and the peripheral nervous systems. Another example is one of the neurotensin receptors, which is a single transmembrane domain protein that belongs to a recently identified family of sorting receptors. Although there is at least one receptor cloned for almost all of the peptides discovered so far, it still remains to be elucidated how many receptors can be
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found for each of the peptides. In many cases, it does not seem unreasonable to assume that there are several receptors for one peptide. However, their number may not approach those described for the receptors of some classic transmitters, such as the 15 or so receptors for serotonin. At present, many ‘orphan peptides’ (for which there are no known receptors) still exist, and many others may be discovered soon. Recently, MrgX2 receptor has been identified as the cortistatin-specific receptor. In contrast, some peptides have been found via one of the many ‘orphan receptors,’ that is, receptors for which the endogenous ligand is unknown. For instance, orexin-A and -B were initially identified as endogenous ligands for an orphan GPCR. In addition, the endogenous ligand for the growth hormone secretagogue receptor was recently discovered and named ghrelin, while a study focusing on another orphan receptor, the glucocorticoid receptor 3, revealed that a prolactin-releasing peptide in the brain represents an endogenous ligand for this receptor. Also, many naturally occurring peptides exhibit a high degree of promiscuity across GPCRs. The physiological significance of this phenomenon and the degree to which it occurs is not well characterized, but it does not seem an exception in the nervous system. For instance, cortistatin binds to somatostatin receptors with similar high affinity to that of somatostatin itself and it may produce physiological effects similar to those of somatostatin. In addition, the orphan peptide histidine methionine-27 was recently shown to be a potent agonist at the human calcitonin receptors. Functionally, there is a general agreement that peptidergic receptors are coupled to multiple components of transduction pathways including adenylyl cyclase/cyclic AMP, phospholipase C/diacylglycerol/ inositol triphosphate, phospholipase A2/arachidonic acid, guanylyl cyclase, protein kinases and phosphatases, ionic channels and exchanger, reactive oxygen species, metalloproteases, transcription factors, and other intracellular cytoplasmic and nuclear regulators. The diversity of the transduction pathways reflects the pleiotropic actions of peptides. The prevailing mechanisms in any given cell depend on many factors, including receptor distribution and the signaling elements expressed by the cell. Often the situation is further complicated by cross-talk between different pathways. In addition, at the cellular level, many mechanisms are involved in the amplification effects, which occur in the signaling cascade. In particular, posttranslational modification of receptors, different expression of factors interacting with G-proteins, and differences in G-protein repertoire and signaling isoenzymes may affect function in a process which is generally referred to as cross-talk and receptor
trafficking. On the other hand, recent results suggest the existence of multiple ligand-specific receptor conformations, which may differentially couple to the intracellular pathways within the signal transduction network. Indeed, peptidergic receptor reserve, coupling efficiency, and probably receptor trafficking may not only depend on transduction mechanisms, but also on the nature of the ligand and the receptor at play. In addition, the type and the magnitude of the response elicited by a ligand is also dictated by the level of receptor available at the plasma membrane. Endocytosis and recycling of receptors are important for desensitization and resensitization in response to endogenous ligands and drugs. Recent studies of peptidergic receptors have provided exciting insights into specific mechanisms that control endocytosis of receptors from the plasma membrane (internalization) and regulate proteolytic degradation of receptors. For instance, the internalization of the AT1 receptor (a specific receptor for Ang II) occurs via clathrin-coated pits, but there is evidence that, in contrast to the internalization of other GPCRs, it is independent of dynamin and beta-arrestin. In addition, AT1 receptor internalization requires two regions in the cytoplasmic tail of the receptor, but it is independent of G-protein activation. The dependence of AT1 receptor internalization on the presence of a serine–threoninerich region suggests that phosphorylation of the receptor tail may regulate the internalization process. GPCRs have been found to be dysfunctional/dysregulated in a growing number of human diseases and the property of ‘constitutive activity’ which is associated with GPCRs provides a persistent tonic mechanism to fine-tune synaptic transmission during both acute and chronic information transfer. In this respect, GPCRs have been a very successful target for drug discovery and development and almost a third of currently available prescription drugs function as GPCR ligands. In particular, the clinical potential of neuropeptides is well known and, needless to say, the multiplicity of peptidergic receptors offers unique and important openings for the development of specific drugs, which may be useful for treatment of various disorders. In fact, receptor-subtype-selective antagonists and agonists have been developed, and recently an SP-receptor antagonist has been shown to have clinical efficacy in the treatment of major depression and chemotherapy-induced emesis. In addition, the use of somatostatin and its analogs is well established for certain indications, such as acute esophageal variceal bleedings, gastrointestinal disorders, neuroendocrine tumors, and acromegaly as well as the diagnosis and radiotherapy of some tumors. Several other neuropeptide receptor ligands are on clinical trials for various indications.
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Peptidergic Receptors in the Autonomic Nervous System and in Peripheral Organs Multiple peptides and receptor subtypes are expressed in the autonomic nervous system and in peripheral organs, where they are involved in a variety of functions (Table 1). In this section, data from recent scientific reports and review articles have been collected to provide examples of the occurrence and functional actions of peptides and their receptors in the autonomic nervous system and peripheral organs. Modulation of Parasympathetic Neurons
The submandibular ganglion (SMG) is a parasympathetic ganglion, which receives inputs from preganglionic cholinergic neurons and innervates the submandibular salivary gland to control saliva secretion. Neurotransmitters and neuropeptides acting via GPCRs change the functional activity of neurons in this ganglion by modulating voltage-dependent Ca2þ channels. Opioid peptides Endogenous opioid peptides and their receptors are integral components of neuronal circuits modulating pain transmission. They also produce various effects, such as analgesia, inhibition
of diarrhea, respiratory depression, and catalepsy. Opioid peptides act at three distinct types of opioid receptors, namely, m-, d-, and k-opioid receptors. Met-enkephalin causes hyperpolarization, while m-, d-, and k-opioid receptor agonists inhibit currents in voltage-dependent Ca2þ channels in a dosedependent manner. Similar actions of opioid peptides have been reported using different receptor subtype-specific agonists in rat dorsal root ganglion neurons, rat parasympathetic intracardiac neurons, neurons of the rat nodose ganglion, and avian ciliary ganglion. Tachykinin peptides Three types of tachykinin receptors, neurokinin (NK)-1, NK-2, and NK-3, have been cloned and characterized pharmacologically. They preferentially bind the endogenous tachykinins SP, neurokinin A (NKA), and neurokinin B, respectively. SP is a well-known pain-related peptide synthesized and secreted from primary afferent neurons. SP and NKA are reported as neurotransmitters of the SMG neurons. SP, acting at NK-1 receptors, causes depolarization of SMG neurons by inhibiting M-type Kþ channels. In addition, an NK-1 receptor agonist inhibits voltage-dependent Ca2þ channel currents. SP also facilitates ganglionic transmission in sympathetic
Table 1 Examples of the main effects mediated by some of the peptidergic receptors expressed in sympathetic neurons or in sympathoeffector cells Neuropeptides
Receptor subtype
Site of expression
Effect
Calcitonin gene-related peptide Cholecystokinin
CGRP1
Postganglionic vasomotor neurons
Vasodilatation
CCK1
Preganglionic neurons of rostral ventrolateral medulla Preganglionic vasomotor neurons
Inhibition of postganglionic neuron activity Increase of postganglionic neuron activity Inhibition of transmitter release Increase of vasoconstrictor transmitter release/inhibition of vasodilatator transmitters Increase of oxidative stress/increase of vasoconstrictor transmitter release Inhibition of transmitter release Vasoconstriction Inhibition of vasoconstrictor transmitter release Inhibition of transmitter release Increase of neuron excitability/ increase of vasoconstrictor transmitter release/vasodilatation
CCK2 Endocannabinoids Endothelin
CB1 ETA
Preganglionic nerve terminals Postganglionic nerve terminals of vasculature/vascular smooth muscles
ETB
Postganglionic neurons/postganglionic nerve terminals of vasculature
Enkephalins Neuropeptide Y
Opioid d and k Y1 Y2
Somatostatin Tachykinins
sst2 and sst3 NK1
Postganglionic neurons Vascular smooth muscles Postganglionic nerve terminals of vasculature Postganglionic nerve terminals Postganglionic neurons of superior cervical ganglion/spinal preganglionic neurons and postganglionic axons of vasculature/vascular smooth muscles Postganglionic neurons in gastrointestinal tract Postganglionic neurons of superior cervical ganglion Heart and vascular smooth muscles
NK2 Vasoactive intestinal polypeptide/pituitary adenylate cyclase activating peptide
PAC1 VPAC1 and VPAC2
Smooth muscle relaxation Increase of transmitter release Vasodilatation
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ganglia, while it inhibits currents in voltage-dependent Ca2þ channels in a variety of neuronal cells. CGRP CGRP is generated through an alternate splicing of the calcitonin gene primary transcript. CGRP receptors are heterodimeric complexes composed of the heptahelical calcitonin receptor-like receptor and the single transmembrane receptor activity-modifying protein 1. They are widely distributed throughout the nervous system and have been studied in several species. Similar to SP, in SMG neurons, CGRP causes depolarization by inhibition of voltage-insensitive Kþ channels. In addition, CGRP facilitates voltagedependent Ca2þ channels. Ang II Ang II has various physiological effects, including stimulation of increased blood pressure, water and sodium intake, vasopressin secretion, and modulation of baroreflex function. In addition, Ang II acts as a neurotransmitter at two types of receptors (AT1 and AT2 receptors). In SMG neurons, Ang II causes depolarization by the combination of an increase in Naþ conductance and a decrease of Kþ conductance. On the other hand, Ang II inhibits voltage-dependent Ca2þ channel currents via AT1 with transduction mechanisms involving protein kinase C. VIP and PACAP VIP was first isolated from porcine intestine as a peptide capable of inducing vasodilatation. PACAP was initially isolated from ovine hypothalamus for its ability to stimulate pituitary cell membrane adenylyl cyclase and was further shown to exist in two biologically active forms of 27 and 38 amino acids. At least three receptors for these peptides have been identified in mammals: VPAC1, VPAC2, and PAC1. VPAC1 and VPAC2 respond to VIP and PACAP with comparable affinity, while PAC1 binds PACAP with high affinity and VIP with much lower affinity. VIP coexists with acetylcholine in postganglionic SMG neurons, and following stimulation of parasympathetic nerve, it is released from the submandibular gland together with acetylcholine. In SMG neurons, VIP causes depolarization by inhibition of voltage-insensitive Kþ channels. Both VIP and PACAP inhibit voltage-dependent Ca2þ channel currents. The inhibition mediated by VPAC1/VPAC2 receptors involves both protein kinase A and protein kinase C, while the PAC1 receptors inhibit the voltage-dependent Ca2þ channel via the bg subunits of a Gs-protein. VIP and PACAP may also facilitate voltage-dependent Ca2þ channels; however, VIP- and PACAP-induced facilitation is less frequently reported than inhibition.
Functional implications A variety of neuropeptides and their receptors act in SMG neurons. However, such a diversity of signaling substances would presumably not be necessary to only signal neuronal excitation or inhibition. The voltage-dependent Ca2þ channels are essential in the regulation of many neuronal processes including membrane excitability, enzyme activity, gene expression, neuronal outgrowth, neuronal regeneration, differentiation, and proliferation. The G-protein-dependent signals elicited by the action of multiple peptidergic receptors and their interactions may provide the necessary versatility to properly accomplish those neuronal functions. Presynaptic Modulation at Sympathoeffector Junctions
The transmitter release at sympathetic varicosities is modulated by a plethora of different signaling molecules, including classical transmitters, ATP, histamine, prostanoids, and a variety of peptides. Presynaptic receptors located on the sympathetic terminals are activated by transmitters that are released either from the same nerve ending or from a different axon terminal, and the receptors involved are called auto- or heteroreceptors, respectively. The peptidergic receptors involved in this regulation include receptors for NPY as autoreceptors and for Ang II, bradykinin, cannabinoid peptides, endothelin, natriuretic peptide, opioid peptides, somatostatin, and VIP/PACAP as heteroreceptors. NPY Y2 receptors NPY shares a family of common receptors with peptide YY and pancreatic polypeptide, which consists of at least five members. These are named Y1, Y2, and Y4 through Y6 receptors. Selective antagonists have been developed for Y1 and Y2 receptors. NPY is co-stored and co-released together with ATP and norepinephrine in most sympathetic axon terminals. In the vasculature, NPY has been found to induce contractions mediated by Y1 receptors localized on smooth muscle cells. In some cases, postsynaptic effects of NPY may also involve Y2 receptors. In in vitro preparations of noradrenergic postganglionic sympathetic neurons, NPY reduces transmitter release. This inhibitory effect of NPY is typically mimicked by Y2 receptor agonists. In addition, there is evidence that presynaptic Y2 receptors are indeed autoreceptors. However, there are reports that a selective Y2 receptor antagonist has no effect on excitatory junction potentials, indicating a lack of NPY-dependent feedback modulation of sympathoeffector transmission. Thus, it remains to be established under which conditions NPY may mediate an autoinhibition of sympathetic transmitter release.
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Ang II AT1 receptors In a variety of sympathetically innervated tissues obtained from a large number of different species, a facilitation of norepinephrine release by Ang II has been reported. In addition, evidence has been provided that this stimulatory effect is mediated by AT1 receptors. However, in some instances, presynaptic angiotensin effects have been observed to be antagonized by both AT1 and AT2 receptor antagonists, revealing some uncertainty concerning the subtypes of presynaptic angiotensin receptors in sympathetic axonal terminals. Bradykinin B2 receptors Actions of bradykinin are mediated by at least two types of receptors, B1 and B2, and the existence of a B3 receptor has also been suggested. B1 receptors are mainly restricted to the vasculature, whereas B2 receptors have been detected in most types of tissues including the central and peripheral nervous systems. There are reports that bradykinin may modulate sympathetic transmitter release in a bimodal way, causing inhibition via B1 and facilitation via B2 receptors, but in most cases bradykinin enhances stimulation-evoked norepinephrine release via B2 receptors. However, in a considerable number of investigations, these apparently presynaptic effects of bradykinin have been shown to be indirect and to involve Ang II with AT1 receptors, CGRP, or SP. Cannabinoid CB1 receptors At least two types of cannabinoid receptors, CB1 and CB2, have been identified. Aside from their psychoactive and immunomodulatory effects, cannabinoids exert pronounced cardiovascular actions such as vasodilatation, tachycardia, and changes in blood pressure, all mediated most likely by CB1 receptors. Because central sites of action are not involved in these effects, cannabinoids are believed to elicit vasorelaxation through peripheral mechanisms, which may include inhibition of sympathoeffector transmission. Indeed, there is experimental evidence that cannabinoids may cause sympathoinhibition through presynaptic CB1 receptors on sympathetic nerve terminals, the activation of which reduces transmitter release. Endothelin receptors Endothelin-1 was identified originally as an endothelium-derived vasoconstricting peptide, but the family of endothelins (endothelin-1, -2, and -3) has recently been suggested to act as neuropeptides in the central and peripheral nervous systems. The actions of endothelins are mediated by either ETA or ETB endothelin receptors. There are data showing presynaptic facilitatory and inhibitory and postsynaptic effects of endothelins. Thus, it appears that endothelins may modulate sympathetic
neurotransmission via facilitatory and inhibitory presynaptic receptors, but the receptor subtypes involved remain to be unequivocally identified. Natriuretic peptide receptors The family of receptors for natriuretic peptides (atrial natriuretic peptide, brain natriuretic peptide, and C-type natriuretic peptide) comprises two members, the ANPA and the ANPB receptors. Both are membrane receptors with one transmembrane domain and intracellular guanylyl cyclase activity. The ANPA receptor is preferentially activated by atrial and brain natriuretic peptides, whereas C-type natriuretic peptide is the endogenous agonist for ANPB. A role of presynaptic receptors for natriuretic peptides on sympathetic axon terminals remains to be fully elucidated, and the natriuretic peptides may rather be assumed to control the sympathetic nervous system through central sites of action. Opioid receptors Opioid receptor agonists have long been known to modulate transmitter release in the peripheral nervous system. Evidence has been obtained that each of the three known opioid receptor subtypes may be located on sympathetic axon terminals and, upon activation, reduce transmitter release. Somatostatin receptors Somatostatin is widely distributed in the nervous system, and five different G-protein-coupled somatostatin receptors (sst1–sst5) have been characterized by molecular cloning. Inhibitory effects of somatostatin on transmitter release from sympathetic terminals have been documented. In addition, this action of somatostatin may be mimicked by sst2 or sst3 receptor agonists; however, a definite pharmacological characterization of inhibitory presynaptic somatostatin receptors on sympathetic axonal terminals is still missing. VIP/PACAP receptors Various forms of PACAPs and VIPs are present in and released from sympathetic nerve terminals. Nevertheless, autoregulation of sympathetic transmitter release by VIP or PACAP has not been demonstrated. Therefore, the respective receptors are considered presynaptic heteroreceptors. Presynaptic receptors for VIP and/or PACAP may either facilitate or inhibit sympathetic transmitter release, but the receptor subtypes mediating these opposite effects remain to be identified. Regulation of Gastrointestinal Motility
The peptide cholecystokinin Cholecystokinin (CCK) is a potent regulator of gastrointestinal motility. It and the related peptide gastrin belong to the class of brain– gut peptides. The actions of CCK include stimulation
562 Peptidergic Receptors
of exocrine and endocrine secretion, motility and growth in the gastrointestinal tract, and regulation of satiety, anxiety, pain, and behavior in the central and peripheral nervous systems. Alterations in CCK release and tissue responses to the peptide have been implicated in the pathogenesis of irritable bowel syndrome. There are two principal sources of CCK: endocrine cells in the duodenum and peptidergic nerves, both in the enteric and central nervous systems. In the periphery, CCK-containing neurons are found in the myenteric plexus, submucosal plexus and muscle layers of the small intestine and colon, and in the celiac plexus and the vagus nerve. CCK receptors The biological actions of CCK are mediated by CCK receptors that are distributed throughout the gut. They include two distinct types that are the products of two distinct genes and are termed CCK1 and CCK2 receptors. There is identity between the CCK2 receptor and the gastrin receptor. The CCK1 receptor has an approximately 1000-fold greater affinity for CCK than for gastrin, while the CCK2 receptor has the same high affinity for both CCK and gastrin. The activated G-protein leads to activation of phospholipase C, but high agonist concentrations can also activate adenylyl cyclase via a different G-protein. Although the nucleotide sequences of cloned cDNAs of CCK1 receptors from different sources are identical, their affinity states may vary. The available evidence suggests that the CCK1 receptor exists in both high- and low-affinity states and that CCK binding results in the initiation of different intracellular events and consequent biological responses. The CCK analog JMV-180, which is an agonist at the high-affinity CCK1 receptors and an antagonist toward the low-affinity ones, is a useful tool for functionally distinguishing the two receptor states. CCK receptors in the gastrointestinal tract In the colon, the main target of CCK is the myenteric plexus, which has predominantly CCK1 receptors. In addition, CCK1 receptors are also present, at moderate-to-low density, in the longitudinal muscle, while the circular muscle is negative for both CCK1 and CCK2 receptors. Neither CCK1 nor CCK2 receptors are in the blood vessels, lymphoid tissue, mucosa, or muscularis mucosa. These data suggest that CCK affects colonic motility by two fundamentally different pathways: acting on neurons in the myenteric plexus and directly on the smooth muscle cells. A large number of studies implicate vagal afferent mechanisms in the control of several CCK actions such as relaxation of proximal stomach, gallbladder contraction, and pancreatic enzyme secretion.
CNS Vagal afferent
Vagal efferent
CCK Figure 1 CCK released by endocrine cells in the duodenum acts on CCK receptors expressed by vagal afferent fibers originating in the duodenal mucosa. The response carried by vagal efferent fibers stimulates pancreatic secretion.
Expression of both CCK1 and CCK2 receptors has been demonstrated in vagal afferent fibers and in the nodose ganglion. Several studies indicate that CCK acts via an afferent vagal pathway originating in the duodenal mucosa to stimulate pancreatic secretion in rats as well as in humans (Figure 1). Furthermore, activation of these receptors is thought to initiate a vagovagal reflex inhibition of gastric motor function. CCK receptor agonists/antagonists in disease treatment Because CCK is involved in sensory and motor responses to distension in the intestinal tract, it may contribute to the symptoms of constipation, bloating, and abdominal pain that are often characteristic of functional gastrointestinal disorders and irritable bowel syndrome. These dysfunctions are associated with motor abnormalities in the small intestine and colon that may depend on exaggerated and prolonged CCK release. CCK1 receptor antagonists have been evaluated clinically to stimulate transit and treat irritable bowel syndrome. CCK also belongs to the growing list of factors modulating food consumption, and CCK molecules are actually promising targets for anti-obesity drugs. Since the initial discovery of its property as a food-intake inhibitor, CCK was demonstrated to be a short-term, mealreducing signal in most mammalian species including
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humans. The availability of potent and specific CCK agonists and antagonists has allowed the identification of the CCK1 receptor as the receptor type involved in the satiety actions of CCK. Indeed, specific CCK1 receptor agonists potently inhibit food intake, whereas agonists of the CCK2 receptor do not. The CCK1 receptors mediating CCK action on food intake are localized on the vagus nerve, which relays to the hypothalamus via brain stem areas such as the nucleus tractus solitarius and the area postrema. In the brain, CCK contributes to the sensation of satiety, and it may be involved in the mediation of the control of food intake. In addition to the CCK1 receptors, the CCK2 receptor may also be involved in the CCK control of food intake. Indeed, CCK2 receptors are expressed in the vagus nerve brain stem complex that mediates the satiety effect of peripheral CCK, and the CCK2 receptor is the predominant form found in the brain. Consistent with a possible involvement of CCK2 receptors, centrally or peripherally administered CCK2 receptor antagonists have been reported to increase food intake. See also: Neuropeptide Release; Neuropeptides and Coexistence.
Further Reading Boehm S and Kubista H (2002) Fine-tuning of sympathetic transmitter release via ionotropic and metabotropic presynaptic receptors. Physiological Reviews 54: 43–99. Brain SD and Grant AD (2004) Vascular actions of calcitonin generelated peptide and adrenomedullin. Physiological Reviews 84: 903–934. Dakhama A, Larsen GL, and Gelfand EW (2004) Calcitonin generelated peptide: Role in airway homeostasis. Current Opinion in Pharmacology 4: 215–220. Dufresne M, Seva C, and Fourmy D (2006) Cholecystokinin and gastrin receptors. Physiological Reviews 86: 805–847. Dvora´kova´ MC (2005) Cardioprotective role of the VIP signaling system. Drug News and Perspectives 18: 387–391.
Endoh T (2004) Modulation of voltage-dependent calcium channels by neurotransmitters and neuropeptides in parasympathetic submandibular ganglion neurons. Archives of Oral Biology 49: 539–557. Groneberg DA, Rabe KF, and Fischer A (2006) Novel concepts of neuropeptide-based drug therapy: Vasoactive intestinal polypeptide and its receptors. European Journal of Pharmacology 533: 182–194. Harmar AJ (2004) Receptors for gut peptides. Best Practice and Research Clinical Endocrinology and Metabolism 18: 463–475. Huidobro-Toro JP and Donoso MV (2004) Sympathetic cotransmission: The coordinated action of ATP and noradrenaline and their modulation by neuropeptide Y in human vascular neuroeffector junctions. European Journal of Pharmacology 500: 27–35. Leeb-Lundberg LMF, Marceau F, Mu¨ller-Esterl W, Pettibone DJ, and Zuraw BL (2005) International Union of Pharmacology. XLV. Classification of the kinin receptor family: From molecular mechanisms to pathophysiological consequences. Pharmacological Reviews 57: 27–77. Ohki-Hamazaki H, Iwabuchi M, and Maekawa F (2005) Development and function of bombesin-like peptides and their receptors. International Journal of Developmental Biology 49: 293–300. Pedrazzini T (2004) Importance of NPY Y1 receptor-mediated pathways: Assessment using NPY Y1 receptor knockouts. Neuropeptides 38: 267–275. Peeters TL (2003) Central and peripheral mechanisms by which ghrelin regulates gut motility. Journal of Physiology and Pharmacology 54: 95–103. Sternini C (2001) Receptors and transmission in the brain-gut axis: Potential for novel therapies. III: m-Opioid receptors in the enteric nervous system. American Journal of Physiology – Gastrointestinal and Liver Physiology 81: G8–G15.
Relevant Websites http://www.gproteins.com – G Protein Coupled Receptors (GPCR) and G Protein Research Portal. http://www.gpcr.org – Information System for G Protein-Coupled Receptors (GPCRs) (GPCRDB). http://www.iuphar-db.org – Receptor Database (IUPHAR). http://www.signaling-gateway.org – UCSD Nature Signaling Gateway Home.
Neuropeptides: Electrophysiology W Zieglga¨nsberger, Max Planck Institute of Psychiatry, Munich, Germany ã 2009 Elsevier Ltd. All rights reserved.
Neuropeptides versus Classical Neurotransmitters There are several main differences between neuropeptides (3–100 amino acid residues) and classical neurotransmitter molecules such as glutamate, g-aminobutyric acid (GABA), dopamine, noradrenaline, glycine, and acetylcholine with respect to their synthesis, storage, and signaling function. Because classical transmitters can be rapidly synthesized in nerve terminals and stored in small synaptic vesicles they maintain a fairly constant level, whereas neuropeptide transmitters, which are synthesized in the cell body and stored in large dense-core vesicles transported to the terminal, may be more rapidly depleted. Neuropeptides, which are up to 50 times larger than classical neurotransmitters, display a complex threedimensional structure, and some possess several recognition sites with nanomolar affinity for their receptors. Neuropeptides are typically well conserved across species and outnumber the classical neurotransmitters. They are present in numerous neurons, together with one or even two small classical neurotransmitters (coexistence), which may affect their target neurons differentially. In cases of co-localization the peptide transmitters are often released at higher firing rates and particularly under burst-firing patterns from afferent fibers (Figure 1). In general, it is unlikely that neuropeptides participate in rapid integrative functions in a neuronal assembly.
Volume Transmission In contrast to classical neurotransmitters, which interact with receptors linked to elaborate pre- and postsynaptic specializations, neuropeptides, once released, diffuse and may activate receptors far from their release site, a mechanism termed volume transmission (Figure 2). Thus, in contrast to conventional synaptic transmission neuropeptides may influence large ensembles of neurons in a divergent gating function rather than mediating rapid local point-to-point communication. The effective concentrations of neuropeptides needed for this mode of signaling are much lower than for synaptic transmission mediated by classical neurotransmitters, which reach much
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higher concentrations at their receptors, due to the small volume of the synaptic cleft. The diffusion of the neuropetide depends on the size of the extracellular space, its tortuosity, and the degradation encountered. After diffusing, the neuropeptide binds more slowly, but with much higher binding affinity and selectivity, to its receptor than classical transmitters confined to the synaptic cleft. Peptide-induced receptor trafficking and redistribution of heterogeneous extrasynaptic receptors (Figure 3), spontaneous release and specific regional inactivation mechanisms, and information transfer via glia cells add to the complexity of this mode of delocalized signaling.
Neuropeptides: Synthesis, Release, and Degradation Neuropeptides occur in a bewildering range of shapes and sizes in the animal kingdom. Neuropeptides are found in all types of neurons and glia cells of the mammalian central nervous system (CNS). Genes that code for a neuropeptide family are generally not clustered and are spread out all over the mammalian genome. Duplications and point mutations in old primordial genes are probably the origin of the presently known members of the family of a neuropeptide. Because the precursors for neuropeptides are encoded in the genome, their functions can be detailed in transgenic animal models that do not produce or that overexpress the neuropeptide. However, the deletion of one neuropeptide gene often shows no noticeable effect because the action of the coded neuropeptide may be too subtle or is compensated by related neuropeptides. Significantly, even if the gene that encodes the precursor is identified, it is not clear what particular peptide it will create because neuropeptide precursors undergo extensive posttranslational processing. The proneuropeptides are processed into active forms in large dense-core vesicle precursors by the concurrently packaged enzymes. Although certain peptidases may produce a variety of biologically active fragments, most commonly the peptide that is stored in vesicles and then released is considered the transmitter. After trafficking into the presynaptic compartment, they may dock and fuse with the plasma membrane after a specific priming process at sites different from the synaptic vesicles containing classical neurotransmitters. The release from dense-core vesicles is probably regulated by specific protein kinases, and they are replenished after exocytosis by anterograde
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565
6 7
8
G
10
2 SV
1 DCV 5
4
11 6 9
P
G 3 G 3
7
Figure 1 Co-localization and co-release of classical neurotransmitters and neuropeptides. Classical transmitters are synthesized in nerve terminals and stored in small synaptic vesicles (SV), released, and recycled (1 and 2). They interact with subsynaptic receptors, including metabotropic receptors (3). Neuropeptides are synthesized in the cell body and transported to the terminal (4), stored in large dense-core vesicles (DCV; 5) and released at nonsynaptic sites (6). Neuropeptides interact with pre- and postsynaptic receptors (7). Voltage-gated Ca2+ channels control the release of classical transmitter and neuropeptide (8). Activation of a neuropeptide receptor induces, e.g., the phosphorylation (P) of an ionotropic receptor (9). Peptide transmitters are often released at higher firing rates, particularly under burst-firing patterns (10 and 11).
3
Co-release: 2
Volume transmission vs. Point-to-point transmission
1 Figure 2 Point-to-point vs. volume transmission. Neuropeptides, once released (1 and 2), diffuse and activate receptors (3) far from their release site (volume transmission). Classical neurotransmitters mediate local point-to-point communication (2). Co-released neuropeptides participate in signaling at nonsynaptic and synaptic somatic and dendritic sites.
transport of new secretory granules from the transGolgi network. Classical neurotransmitters are packaged into small clear synaptic vesicles that are clustered near release sites at synaptic terminals where they
undergo repeated rounds of exocytosis, endocytosis, and refilling with neurotransmitter. The function of neuropeptide-encoding mRNAs in neuronal processes beyond the perikaryon still remains to be elucidated. Neuropeptides participate in signaling at nonsynaptic and synaptic somatic and dendritic sites of the target neuron, and there is evidence that they also exert trophic actions. The dendritic release of neuropeptides is partially independent from axon terminal release, and the exocytosis machinery at the two sites seems to be different (Figure 4). Action potentials backpropagating from the axon hillock into the dendritic tree probably open high-voltage-activated Ca2þ channels to trigger and modulate dendritic release. Synapses located on dendritic spines are probably important sites for the plastic changes produced by such a retrograde signaling, which questions the classical point of view of synaptic transmission and short-term modifications of excitatory and inhibitory synaptic efficacy. The invasion of the dendritic tree by the backpropagation of action potentials is subtly controlled by, for example, dendritic voltage-sensitive Naþ- and Kþ-conductances, and it appears to be highly dynamic. It remains to be shown whether the dendritic release of neuropeptides and classical transmitters is, in addition, differentially
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7 G Glia cells Extrasynaptic receptors 6
9
Receptor trafficking 5
Dendritic release
4
3 G
8 Postsynaptic density scaffolding proteins
1 SV
G
2
DCV Spine Subsynaptic receptors
G Dendrite
Figure 3 Receptor localization and trafficking on a dendritic spine. Transmitter release from synaptic vesicles (SV; 1); some dense-core vesicles (DCV) carrying receptors for the neuropeptides (2); presynaptic receptors (3) and postsynaptic receptors (7). Receptors moving from extrasynaptic sites to subsynaptic sites to join subsynaptic receptors (receptor trafficking; 4, 5, and 6); postsynaptic density in a dendritic spine (8); dendritic release (9).
8
Dendritic peptide synthesis
5
Volume transmission
Dendritic release
2 3 Backpropagating action potentials
7 Glia release
6 Axon terminal release
4
1 Somatic peptide synthesis
Figure 4 Release sites of neuropeptides. Neuropeptides are synthesized somatically (1) and dendritically (2). Dendritic release (5) is triggered by action potentials backpropagating (3) from the axon hillock (4) into the dendritic tree. Peptides are also released from presynaptic terminals (6) and glia cells (7) and may act in concert on remote receptors (volume transmission; 8).
Neuropeptides: Electrophysiology
regulated by the release of Ca2þ from intracellular pools. New experimental approaches employing novel techniques will soon lead to a better understanding of the dynamics of dendritic computation and its contribution to neuronal signal processing. The release of a neuropeptide is, like that of classical transmitters, Ca2þ-dependent, but requires higher frequencies of discharge in afferent fibers and is considerably slower because neuropeptides cannot diffuse through the diffusion pore. There is evidence that neuropeptides may come into play as messengers mainly when the nervous system is trying to respond to a pathological challenge such as nerve injury, seizure, or stress. Under these conditions, neurons and glia cells change their phenotype in regard to neuropeptides and their endowment with neuropeptide receptors. This phenotypic switch may be seen as an attempt to promote survival and regenerative mechanisms in a neuronal system. After release, peptide transmitters appear to be inactivated enzymatically or by diffusion. With few exceptions (e.g., cholecystokinin), they lack a highaffinity reuptake process that removes the transmitter from the extracellular space. The introduction of in vivo push–pull cannulae and microdialysis has been useful for the measurement of extracellular concentrations of neuropeptides. Another successful in vivo approach has been to insert electrodes coated with antibodies into the specific area of the brain for a short period. The removed probes are then exposed to a radiolabeled peptide, which binds to receptors that are not already occupied by the peptide that was released in the brain tissue. Without these techniques, the release of some neuropeptides may not have been detected because they were present at too low a concentration or only during particular developmental stages.
Neuropeptide Receptors Neuropeptides bind more tightly than classical neurotransmitters to their receptors, which are with few exceptions G-protein-coupled receptors (GPCRs). The receptors for neuropeptides belong to different families (rhodopsin-like, glucagon-vasoactive intestinal peptide/calcitonin). Several GPCRs form homodimers or heterodimers, and most commonly several receptor subtypes exist for a given peptide ligand. Furthermore, there is evidence that so-called receptor activity-modifying proteins can alter the pharmacological profiles of a single receptor. Most neuropeptide receptors are rapidly (5 min) desensitized after agonist activation. The receptor– neuropeptide complexes are internalized, and the receptors are then recycled and restored to the cell membrane within 30 min. It remains to be shown
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to what extent this recycling process is brought about by metalloendopeptidases affecting intracellular degradation of signaling peptides. Conjugation of the toxin saporin to the neuropeptide (e.g., substance P) can be used to selectively destroy neurons involved in pain processing in the dorsal horn of the spinal cord. Animal models show that this selective targeting technology prevents the formation of hyperalgesia and can reverse established neuropathic pain behavior. The action of an endogenously released neuropeptide may depend on the local circuitry as well as the strategic location of the receptor subtypes. In addition to actions at neighboring neurons, neuropeptides can regulate the excitability of the neuron from which they are released via an autoreceptor function. The release of some neuropeptides seems to be regulated in a reciprocal fashion by autoreceptors of coexisting classical neurotransmitters such as dopamine.
Peptidergic Interneurons The interneuronal system, often classified according to neuropeptide content, is crucial for maintaining appropriate brain function under normal and pathophysiological conditions. It is widely accepted that the effects of neuropeptides can be very distinct at the network level and that neuropeptides released from interneurons can trigger diverse Kþ currents such as the voltage-sensitive Kþ current IM or the voltage-insensitive Kþ leak current ILK. Both currents hyperpolarize the neuron and shunt excitatory synaptic potentials and can markedly affect neuronal excitability. The augmentation of the nonselective cation current IH and the reduction of voltage-sensitive N-type Ca2þ channels have been suggested as mechanisms underlying pre- and postsynaptic peptidergic signaling. Electron-microscopy studies have shown neuropeptide-immunoreactive terminals localized to the dendritic shafts and spines of principal neurons. There is evidence from recent studies that dendritic spines undergo an activity-dependent structural remodeling and that memories are created by alterations in glutamate-dependent excitatory synaptic transmission on dendritic spines. In the past, electrophysiological recordings were often limited to short-lasting impalements of the soma of only vaguely identified neurons. The advent of novel recording techniques for synaptic potentials and single-channel activity from dendritic sites remote from the soma under visual control and its combination with extremely targeted stimulation techniques in which the active transmitter is uncaged from a larger molecule by an ultraviolet (UV) laser has opened the possibility that we can detail these processes. Electrophysiological investigations into the acute effects of neuropeptides suggest that
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they interfere not only with short-term alterations in synaptic efficacy but can also affect neuronal plasticity in neuronal circuits in a long-lasting way through indirect effects on release mechanisms of excitatory or inhibitory classical transmitters. The inhibition of glutamate release decreases the frequency of spontaneous glutamate-mediated events and the amplitude of evoked excitatory postsynaptic responses. Such a reduction in glutamate release and co-released GABA could act in concert to reduce the likelihood of neuronal firing and the generation of long-term potentiation. It has been shown for central neurons that the early and the late components of a GABAergic inhibitory postsynaptic potential (IPSP) are mediated by GABAA and GABAB receptors, respectively. Synaptic strength at glutamatergic synapses shows a remarkable degree of use-dependent plasticity. This dynamic regulation of synaptic efficacy plays a crucial role in experience-dependent modification as well as in the formation of neuronal connections in neural circuitry as physiological correlates to learning and memory. Some neuropeptides may be considered as endogenous antiepileptic agents because they reduce excitatory synaptic transmission in the CNS. Their application elicits a long-lasting decrease in evoked excitatory postsynaptic current (EPSC) amplitude and a delayed, long-lasting increase in the amplitude of an evoked monosynaptic inhibitory postsynaptic current (IPSC). Like opioid peptides, substance P and other neuropeptides of the tachykinin family also can indirectly affect pyramidal neurons via an effect on neighboring hippocampal CA1 interneurons. Neurokinin (NK)-1 receptors are located both on presynaptic terminals and at somatodendritic sites of GABAergic neurons. By increasing the excitability of CA1 GABAergic interneurons, tachykinins can powerfully facilitate the inhibitory synaptic input to pyramidal neurons. This indirect inhibition could play a role in regulating short-term and long-term synaptic plasticity, promoting neuronal circuit synchronization or influencing epileptogenesis. Because tachykinins do not alter the frequency or amplitude of miniature inhibitory synaptic currents and are without effect on evoked inhibitory synaptic currents, these neuropeptides probably act at the somatodendritic membrane of GABAergic interneurons rather than at their axon terminals. In the hippocampus, opioid peptides increase the excitability of CA1 pyramidal neurons via a reduction of GABAergic inhibition from neighboring interneurons (disinhibition).
Peptidergic Synaptic Potentials Most of our knowledge about peptidergic transmission and most therapeutically used drugs is based on
exogenous application in which substances may diffuse in the extracellular space and evoke their actions, for example, via the simultaneous activation and/or inhibition of ensembles of neurons or glia cells. Even when highly selective agonists and antagonists are available or transgenic mice expressing antibodies to the neuropeptide under study are combined with state-of-the-art single-unit recordings in in vitro preparations, these studies are still notoriously hard to interpret. Most of the information that has been gathered so far about the actions of neuropeptides in the CNS in vivo derives from extracellular recording of singleunit activity in combination with iontophoretic or pneumatic administration from multibarreled micropipettes in regions containing a high density of specific binding sites. Most of these in vivo studies do not attempt to differentiate a presynaptic site of action from overlapping postsynaptic effects or to characterize the role of single neurons in an ensemble of neurons. There is evidence that endogenously released neuropeptides shift membrane permeability and can directly cause postsynaptic inhibition and elicit peptidemediated IPSPs. These findings indicate that neuropeptides may not just alter the response of a target neuron to classical neurotransmitters without interfering directly with neuronal excitability. However, such consistent postsynaptic effects on neuronal excitability are documented only for few neuropeptides. Such a shift of the membrane to a more hyperpolarized level directly reduces the voltage-dependent glutamatergic transmission mediated by N-methyl-D-aspartate (NMDA) receptors involved, for example, in cognition or epileptogenesis and shift glutamatergic transmission preferentially to a-amino-3-hydroxy-5-methyl-4isoxazole propionic acid (AMPA) receptor-mediated mechanisms in the whole neuron. The generation of excitatory postsynaptic potentials (EPSPs) by neuropeptides has been reported in neurons of the peripheral nervous system and a few neurons in the CNS.
Growth Factors Some of the known growth factors such as brainderived nerve growth factor (BDNF), which are secreted from neurons and glia cells, fulfill the neuropetide criteria and may act like neurotransmitters or get indirectly involved in the interneuronal communication by affecting, for example, ion transporters, which control the intracellular concentration of chloride in neurons. It has recently been shown that BDNF released from activated glia cells reduces the chloride transport out of the target neurons. The increase in intracellular chloride concentration shifts the commonly inhibitory effects of GABAA receptor activation toward excitation in these GABAergic
Neuropeptides: Electrophysiology
neurons, and thus dramatically affects acute functioning and plasticity in these neuronal circuits.
Conclusion Considerable progress has been made in the field of neuropeptide research, and fascinating detailed insights into novel signaling modes in the nervous system have been gained through the advent of new technologies. New neuropeptides were described, and a multitude of receptors for neuropeptides have been characterized and cloned. Nevertheless, there is still only little information available on their role in humans. Because numerous neuropeptides are involved in mammalian brain functions under normal and pathological conditions, it was predicted that many agonistic and antagonistic drugs, as well as peptidase inhibitors preventing peptide breakdown and thus strengthening the peptidergic transmission, would rapidly be developed for therapeutic use. To date, this prediction has not yet materialized. Only substance P has secured a position in the field of psychopharmacology. It was shown that substance P (NK-1) antagonists have an antidepressant activity comparable to the most commonly prescribed serotonin reuptake inhibitors but are associated with less pronounced side effects. There is no doubt that peptidergic
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signaling mechanisms in the human brain will remain important targets for drug development and novel therapeutic algorithms. See also: Neuropeptide Release; Neuropeptide Synthesis and Storage.
Further Reading Baraban SC and Tallent MK (2004) Interneuronal neuropeptides – endogenous regulators of neuronal excitability. Trends in Neuroscience 27: 135–142. deWied D and Burbach JPH (1999) Neuropeptides and behaviour. In: Adelman G and Smith BH (eds.) Encyclopedia of Neuroscience, vol. 2, pp. 1431–1436. Amsterdam: Elsevier. Dodt HU, Eder M, Schierloh A, and Zieglga¨nsberger W (2002) Infrared-guided laser stimulation of neurons in brain slices. Science Signal Transduction Knowledge Environment 120: PL2 [online]. Ho¨kfelt T, Bartfai T, and Bloom F (2003) Neuropeptides: Opportunities for drug discovery. Lancet Neurology 2: 463–472. Ubink R, Calza L, and Ho¨kfelt T (2003) ‘‘Neuro’’-peptides in glia: Focus on NPY and galanin. Trends in Neuroscience 26: 904–909. Zieglga¨nsberger W, Dodt H-U, Deisz R, and Pawelzik H (1991) Peptides in neurotransmission. In: Fuxe K and Agnati L (eds.) Volume Transmission in the Brain: Novel Mechanisms for Neural Transmission, pp. 371–380. New York: Raven Press.
Neurotrophins: Physiology and Pharmacology J M Conner and M H Tuszynski, University of California at San Diego, La Jolla, CA, USA ã 2009 Elsevier Ltd. All rights reserved.
Introduction Nerve growth factor (NGF) was the first neurotrophin family member and the first nervous system growth factor to be identified. NGF was originally isolated from a mouse sarcoma based upon its ability to promote the hypertrophy of, and fiber outgrowth from, peripheral sensory neurons. Subsequently, other neurotrophin family members were identified, including brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT3), and neurotrophin-4 (NT4). Although originally described as a potentially novel growth factor, neutrotrophin-5 was found to be identical to NT4. Two other neurotrophin members, neurotrophin-6 and-7, were identified in fish but do not appear to have corresponding homologs in mammals.
Neurotrophin Production and Release Neurotrophin proteins are synthesized as precursors of approximately 270 amino acids (30–35 kDa) and are subsequently cleaved to yield mature proteins of 120 amino acids (13 kDa) in length. While most physiological actions of neurotrophins have been ascribed to the mature proteins that generally function as soluble, noncovalently linked homodimers, recent studies have identified potentially critical roles for the unprocessed neurotrophin precursors (see later). Elements within the precursor sequence of newly generated neurotrophins presumably direct the intracellular sorting of neurotrophin molecules into one of two distinct pathways, resulting in either constitutive or regulated secretion. This process of intracellular sorting plays a critical role in determining the possible spectrum of physiological actions mediated by a given neurotrophin molecule by determining when and where neurotrophin secretion will take place. While neurotrophins were originally believed to function exclusively as target-derived factors that mediate signaling in a retrograde manner, recent studies have provided substantial evidence that some neurotrophins function partially, or exclusively, as anterograde molecular signals.
Neurotrophin Actions Are Mediated by Distinct Cell Surface Receptors Once neurotrophins are released from the cell of origin, their actions are mediated by binding to
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distinct cell surface receptors. The specificity of neurotrophin action is controlled primarily by the pattern of expression of receptors in distinct populations of neurons located at the site of neurotrophin release. Two distinct classes of neurotrophin receptors have been identified. The p75 neurotrophin receptor (p75NTR) is a member of the tumor necrosis receptor family that binds all of the mature neurotrophins with a similar, nanomolar affinity. Recent studies have indicated that the p75NTR can also bind unprocessed neurotrophin precursors with a high affinity. The second class of neurotrophin receptors is made up of three distinct members of the tropomyosin-related kinase (Trk) receptor family. The various neurotrophins show some degree of specificity with respect to their binding of distinct Trk receptors as shown in Figure 1. Binding of neurotrophins to their respective Trk receptors occurs with an affinity 100-fold greater than binding of mature neurotrophins to the p75NTR. Moreover, the various Trk family members have highly homologous intracellular domains, suggesting they use similar signaling pathways to mediate their effects. Differential splicing of TrkB and TrkC genes can also produce neurotrophin receptors that lack the intracellular kinase domains. The functions of these truncated receptors are unknown, but studies have suggested that they may play a role in regulating the availability or presentation of secreted neurotrophins to full-length and active receptors. The physiological role of the p75NTR has been a subject of considerable study. The p75NTR was initially thought to act as a coreceptor, conferring highaffinity binding of neurotrophins to their respective Trk receptors and promoting retrograde transport of neurotrophins from their targets. However, in the absence of Trk receptor expression, the p75NTR may promote apoptosis via multiple pathways, including an increase in nuclear factor-kappa B (NF-kB) activity and by inducing sphingomyelin hydrolysis into ceramide (Figure 2). Neurotrophin binding to the p75NTR also leads to increased activation of c-Jun N-terminal kinase (JNK), a stress-activated protein kinase that has also been implicated in cell death. Binding of the precursor for NGF to the p75NTR has recently been demonstrated to increase apoptotic death signaling; the consequence of binding other neurotrophin precursor molecules to this receptor remains to be determined. A key factor in determining the fate of neurotrophin signaling through the p75NTR appears to be the extent to which Trk receptors are co-expressed; in the presence of Trk receptors, neurotrophins primarily support cell survival, whereas
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Protease cleavage Unprocessed ligands NGF
BDNF
Processed ligands NT3
NT4
BDNF
NGF
P75
TrkA
NT4
TrkB
NT3
TrkC
Figure 1 Neurotrophins binding to their receptors. All neurotrophin precursors associate with the p75NTR with similar high-affinity binding. Processed (mature) neurotrophin proteins also bind to the p75NTR with a 100-fold lower affinity. Mature neurotrophins bind with high affinity to members of the tropomyosin-related kinase (Trk) family. Interactions with Trk receptors show a high degree of ligand specificity, with nerve growth factor (NGF) binding selectively to TrkA, brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT4) binding to TrkB, and NT3 binding primarily to TrkC. From Segal RA (2003) Selectivity in neurotrophin signaling: Theme and variations. Annual Review of Neuroscience 26: 299–330.
Trk
p75NTR
Ras TRAF6
Cell membrane
SC-1
PI3k NRAGE
Ceramide
NIK
Jun kinase pathway
NRIF RhoA
Cell cycle arrest
Apoptosis Cell death
IKK
Ras-Raf PI3K
Apoptosis mitogenic response
Reduced growth cone motility Ik B
NFk B
Nucleus
Ik B NFk B
Figure 2 Neurotrophin signaling through the p75 neurotrophin receptor (p75NTR). Binding of neurotrophins to the p75NTR leads to alterations in the function of many associated proteins, leading in turn to changes in gene expression and apoptosis. Adapter proteins are shown in red, kinase proteins in green, small G-proteins in blue, and transcription factors in brown. Trk, tropomyosin-related kinase; PI3K, phosphatidylinositol 3-kinase; NIK, NF-kB-inducing kinase; IKK, inhibitor of kB kinase; IkB, inhibitor of kB; NF-kB, nuclear factor-kappa B; TRAF6, tumor necrosis factor receptor-associated kinase-6; SC-1, Schwann cell factor-1; NRAGE, neurotrophin receptor-interacting MAGE (from melanoma antigen); NRIF, neurotrophin receptor-interacting factor. From Huang EJ and Reichardt LF (2001) Neurotrophins: Roles in neuronal development and function. Annual Review of Neuroscience 24: 677–736.
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Cell membrane Src
FRS-2/SNT
Y
490
Y
Shc
Grb2
SOS
Ras
SH-PTP-2 Crk C3G
rapl
FR
Grb2
B-raf IP3 + DAG
MEK1/2
PKCd
Raf
Gab1
Grb2 Y 670 SH2B Y 674 675 Y S P rA
Shp-2
PI3k
S2/ SN T
Endosome
Y 785 Y PLCg
MEK1/2
PDKs CHK Akt1/2
MAPK1/2 p53
3
14-3-
BAD
Ik B
NFk B
Forkhead
Rsk
BAD Bcl-2
CREB
FasL, Bcl-2, Bax Nucleus Figure 3 Neurotrophin signaling through Trk receptors. Binding of mature neurotrophins to Trk receptors leads to the phosphorylation of a wide variety of associated proteins, which ultimately induce alterations in gene expression. Neurotrophin actions through Trk receptors affect neuronal survival, neurite outgrowth, cell morphology, and synaptic efficacy. Adapter proteins are shown in red, kinase proteins in green, small G-proteins in blue, and transcription factors in brown. From Huang EJ and Reichardt LF (2001) Neurotrophins: Roles in neuronal development and function. Annual Review of Neuroscience 24: 677–736.
in the absence of Trk receptors, neurotrophin signaling through p75NTR primarily mediates cell death. Binding of mature neurotrophins to their respective Trk receptors induces dimerization of the receptors and the activation of kinase activity. Increased phosphorylation activity mediated through activated Trk receptors drives cellular responses through three distinct molecular pathways as indicated in Figure 3, resulting ultimately in a variety of transcriptional changes. Neurotrophin signaling through Trk receptors can influence many aspects of cell function, including cell survival, neurite outgrowth, and cellular differentiation.
Neurotrophin Physiology Cell Survival
One of the classic physiological functions attributed to neurotrophins is the regulation of cell survival, specifically within neuronal populations expressing a particular type of neurotrophin receptor. Within the
peripheral nervous system, tightly regulated neurotrophin expression is thought to be responsible for controlling neuron number through the process of programmed cell death, whereby a precisely timed and limited expression of target-derived neurotrophins limits the survival of responsive neurons projecting to a given peripheral target. Data from genetically mutant mice with targeted deletions of specific neurotrophins have provided evidence supporting a role for neurotrophins as target-derived regulators of neuronal survival in the periphery. Consistent with known patterns of neurotrophin and receptor expression, targeted deletion of either specific neurotrophins or their receptors results in profound, yet specific, losses of distinct populations of peripheral neurons during development. For instance, targeted deletion of NGF results in a nearly complete loss of peripheral sympathetic and nocioceptive sensory neurons that express TrkA receptors. Targeted NGF deletion, however, does not significantly reduce the number of TrkC-expressing peripheral sensory neurons.
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Based upon observations made within the peripheral nervous system during development, it was initially postulated that neurons within the central nervous system (CNS) may also rely upon neurotrophin support for their survival, not only during development but also in the adult. In spite of this prediction, there is strikingly little evidence suggesting that neurotrophins play a significant role in promoting cell survival within the CNS, either during development or in the adult. For instance, basal forebrain cholinergic neurons, which possess both p75NTR and TrkA receptors throughout life and show numerous physiological responses to NGF, show minimal deficits in developmental cell survival following targeted deletions of either NGF or its corresponding TrkA receptor. Moreover, deletion of the cellular source of neurotrophins in the adult animal also fails to cause cell death. However, neurotrophins appear to have many other effects upon neurons in the CNS, especially pertaining to cellular differentiation, target innervation, and synaptic plasticity. Cell Differentiation and Morphology
In addition to their well-defined actions on cell survival, neurotrophins have been implicated in numerous aspects of cellular differentiation. Some of the earliest studies with NGF demonstrated that this neurotrophin induced dramatic outgrowth of neurites from developing peripheral neurons. Subsequently, the various members of the neurotrophin family have been implicated in mediating a variety of morphological changes, in a multitude of distinct neuronal populations, including the extension and guidance of axons, hypertrophy of the cell body, and changes in dendritic complexity and dendritic spine density. For example, respective expression of BDNF or NT3 in cortical target regions of extending axons during brain development appears to define zones of axon growth termination in strikingly laminar patterns, helping to establish the detailed topography of mature innervation. In addition to controlling the initial outgrowth of neuronal processes, neurotrophins have been strongly implicated in the process of maintaining target innervation in both central and peripheral targets. One of the most striking effects observed in NGF knockout animals is the failure of NGFsensitive cholinergic neurons to retain appropriate innervation of their central targets. Neurotrophins also play an important role in other aspects of cellular differentiation, helping to determine the ultimate fate and function of certain cells. For example, NGF promotes the differentiation of sympathoadrenal precursors into sympathetic neurons, as opposed to adrenal chromaffin cells. Neurotrophins
also play more subtle roles in neuronal function by regulating the expression of neurotransmitters, ion channels, and receptors, both during development and, in many cases, throughout adulthood. Synaptic Function and Synaptic Plasticity
In addition to regulating neuronal function, neurotrophins have direct actions on synaptic function and synaptic plasticity. Application of neurotrophins at developing and mature synapses can stimulate or modulate neurotransmitter release under many circumstances. The induction of long-term potentiation (LTP), a possible electrophysiological substrate for long-term storage of information in the nervous system, is potently regulated by BDNF. In vitro and in vivo applications of BDNF to the hippocampal formation are capable of inducing long-lasting synaptic potentiation in the absence of any additional excitatory stimulus. Moreover, the ability to induce LTP in the hippocampus with an electrical stimulus is greatly attenuated when BDNF signaling is disrupted. Together, these results suggest that BDNF may play a critical and necessary role in regulating synaptic plasticity in certain populations of neurons. Whether or not the regulation of synaptic plasticity is a selective property of BDNF is unknown at present, but NGF and NT3 have not been demonstrated to influence synaptic function and LTP in a similar manner.
Neurotrophin Pharmacology The potent survival effects of neurotrophins on neurons of the peripheral nervous system originally discovered 50 years ago fostered speculation for many years that neurotrophins may also promote the survival of CNS neurons, thereby representing pharmacological candidates for treating neurodegenerative diseases. The degeneration of specific neuronal populations is a hallmark of the most common neurodegenerative disorders, including losses of midbrain dopaminergic neurons in Parkinson’s disease and the loss of basal forebrain cholinergic neurons as one component of multisystem cell loss in Alzheimer’s disease. The hypothesis that neurotrophins might represent a new class of therapeutic molecules was substantiated by studies published in the mid-1980s, demonstrating that NGF prevented the injuryinduced death of basal forebrain cholinergic neurons in adult rats. NGF neuroprotection of adult cholinergic neurons was also reported after lesions in the brains of adult primates. Other studies reported that spontaneous atrophy and dysfunction of basal forebrain cholinergic neurons in aged rats and primates could also be reversed by NGF administration. Further, NGF treatment significantly ameliorated
574 Neurotrophins: Physiology and Pharmacology Table 1 Potential clinical uses for neurotrophins Neurotrophin
Targeted cell population
Disease
Nerve growth factor
Basal forebrain cholinergic Sensory nociceptive Cortical neurons Spinal motor neurons Midbrain dopaminergic Cortical neurons Sensory proprioceptive
Alzheimer’s disease Peripheral neuropathy Alzheimer’s disease Amyotrophic lateral sclerosis Parkinson’s disease Acute brain injury/stroke Peripheral neuropathy
Brain-derived neurotrophic factor
Neurotrophin-3
age-related memory impairments. Collectively, these findings established a theoretical basis for pursuing clinical trials targeting NGF therapy for the treatment of Alzheimer’s disease, whereby a significant loss of basal forebrain cholinergic neurons is postulated to contribute to ongoing cognitive decline. Analogous experimental paradigms explored the possibility that the neurotrophins could protect cell populations affected in a diverse spectrum of neurodegenerative diseases, including nigral neurons, motor neurons of the spinal cord, and dorsal root ganglion sensory neurons (see Table 1). Clinical Trials with Neurotrophins
The potent actions of neurotrophins in preventing neuronal degeneration and augmenting cell function made them intriguing candidates for clinical testing in human neurological disorders. To date, neurotrophins and other growth factors have been tested in clinical trials in Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, stroke, and peripheral neuropathy. Trials are also under consideration in treating Huntington’s disease and spinal cord injury. In spite of the extensive animal data supporting the potential use of neurotrophins in treating a variety of neurological diseases, no studies to date have yielded clear evidence of efficacy. One of the most important limiting factors in testing the clinical potential of neurotrophins has been the availability of an effective, safe, and sustained method of delivering growth factors to the brain. Because neurotrophins are large and charged proteins, they do not cross the blood– brain barrier after peripheral administration. When infused into the cerebrospinal fluid space of the brain (intraventricular or intrathecal infusions), growth factors usually fail to diffuse to neuronal targets. In the case of NGF, intraventricular or intrathecal infusions broadly disseminate the neurotrophin throughout the subarachnoid space of the brain and spinal cord, inducing the migration of Schwann cells into the CNS, stimulating nociceptive axons of the dorsal root ganglia (DRG) (resulting in pain), eliciting
sympathetic axon sprouting around the cerebral vasculature, and causing weight loss. Thus, clinical testing of neurotrophins requires a delivery method that both achieves adequate growth factor concentrations at target neurons that may lie deep within the brain, and restriction of growth factor delivery to target regions in order to avoid adverse effects of broad growth factor dissemination in the cerebrospinal fluid space. Chronic intraparenchymal growth factor infusion into the brain, or growth factor gene delivery into the brain, may be a means of achieving these goals. Indeed, recent signs of possible growth factor activity in Alzheimer’s disease and Parkinson’s disease have been reported using targeted intraparenchymal delivery methods. In Alzheimer’s disease, a recent phase I clinical trial of NGF gene delivery to cholinergic neurons was reported to increase glucose utilization in the cortex by positron emission scanning, and to elicit new growth of axons toward the NGF source. Potential effects on cognition are being explored in phase II clinical trials of NGF gene delivery in Alzheimer’s disease. A clinical trial of growth factor gene delivery in Parkinson’s disease is also under way (using neurturin, a glial-derived neuronal growth factor). Three recent clinical trials using intraparenchymal infusion of glial-derived neuronal factor (GDNF) have also been conducted in Parkinson’s disease. The development of these targeted, restricted, and sustained growth factor delivery methods raises the possibility that the next few years will definitively establish whether growth factors will prove useful for the treatment of CNS disorders. Neurotrophins have also undergone clinical testing for peripheral neuropathy: NGF is a tropic factor for a subpopulation of nociceptive sensory neurons with cell bodies in the DRG, and NT3 is a tropic factor for large-diameter proprioceptive neurons of the DRG. NGF was tested in patients with diabetes using intermittent peripheral subcutaneous injections, but failed to show efficacy. Once again, problems with potentially subtherapeutic doses, inadequate targeting, and rapid peripheral degradation of NGF limited the
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interpretability of the study. NT3 is also being tested in clinical trials for drug-induced peripheral neuropathy. Solutions to the challenge of targeted, sustained, and safe growth factor delivery for treatment of peripheral nervous system disorders await identification, to determine whether early potential signals of growth factor activity in the CNS may also be achievable in the peripheral nervous system.
Further Reading Chao MV (2003) Neurotrophins and their receptors: A convergence point for many signalling pathways. Nature Reviews Neuroscience 4: 299–309. Dechant G and Barde YA (2002) The neurotrophin receptor p75 (NTR): Novel functions and implications for diseases of the nervous system. Nature Neuroscience 5: 1131–1136. Hefti F (1997) Pharmacology of neurotrophic factors. Annual Review of Pharmacology and Toxicology 37: 239–267. Hempstead BL (2006) Dissecting the diverse actions of pro- and mature neurotrophins. Current Alzheimer Research 3: 19–24.
Huang EJ and Reichardt LF (2001) Neurotrophins: Roles in neuronal development and function. Annual Review of Neuroscience 24: 677–736. Huang EJ and Reichardt LF (2003) Trk receptors: Roles in neuronal signal transduction. Annual Review of Biochemistry 72: 609–642. Lessmann V, Gottmann K, and Malcangio M (2003) Neurotrophin secretion: Current facts and future prospects. Progress in Neurobiology 69: 341–374. McAllister AK, Katz LC, and Lo DC (1999) Neurotrophins and synaptic plasticity. Annual Review of Neuroscience 22: 295–318. Reichardt LF and Mobley WC (2004) Going the distance, or not, with neurotrophin signals. Cell 118: 141–143. Segal RA (2003) Selectivity in neurotrophin signaling: Theme and variations. Annual Review of Neuroscience 26: 299–330. Thoenen H and Sendtner M (2002) Neurotrophins: From enthusiastic expectations through sobering experiences to rational therapeutic approaches. Nature Neuroscience 5(supplement): 1046–1050. Tuszynski MH, Thal L, Pay M, et al. (2005) A phase I clinical trial of nerve growth factor gene therapy for Alzheimer’s disease. Nature Medicine 11: 551–555.
Nerve Growth Factor J C Petruska and L M Mendell, State University of New York at Stony Brook, Stony Brook, NY, USA ã 2009 Elsevier Ltd. All rights reserved.
Early History The discovery of nerve growth factor has its origin in studies of the relationship between the number of sensory and motor neurons and the size of the peripheral tissue to be innervated. In the course of these investigations, studies carried out in culture indicated that a diffusible factor from mouse sarcomas could elicit substantial outgrowth of fibers from sensory and sympathetic ganglia. Further experiments demonstrated that nerve fiber growth-promoting factors were located in snake venom and also in mouse salivary glands, the mammalian homolog of the reptilian glands secreting the active venom. The factor responsible for this effect was called nerve growth factor (NGF). It was eventually purified and found to exist in two forms called NGF 7S (S is sedimentation constant) and NGF 2.5S. These two different forms are the consequence of being composed of different peptide chains: NGF 7S (MW 130 kDa) consists of two copies of each of three chains (a, b, and g). NGF 2.5S (MW 26 kDa) contains only the b chains and is the active form of NGF isolated from mouse salivary glands.
Neurotrophin Family It is now known that NGF is a member of a family of growth factors that collectively are referred to as neurotrophins. Other members of the neurotrophin family are brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT4/5 was so named because in the early days it was known as NT-4 or NT-5). These molecules exhibit a homology of approximately 55% with NGF. A precursor form of neurotrophins (proNGF, proBDNF, and proNT-3) has been identified in neural tissues in vivo. ProNGF has been shown to activate the p75 receptor and thus elicit neural apoptosis.
Neurotrophin Receptors NGF exerts its effects by activating two transmembrane receptors. One of these is the trkA receptor, a member of the tropomyosin receptor kinase (trk) family. The other is the p75 receptor. These are often called the high-affinity and low-affinity receptor, respectively; however, the differences relate more to
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kinetics of binding than to affinity, which is in the nanomolar range for both receptors. There is evidence that trkA and p75 function cooperatively to mediate the cellular effects of NGF, with p75 acting as a chaperone to improve the binding to trkA. Structural studies suggest that the interaction is more likely to take place within the cell via convergence of signaling pathways from trkA and p75. This issue remains to be definitively resolved. However, the different intracellular signaling elicited by activated p75 and trkA suggests that NGF could elicit some effects via independent pathways. Members of the neurotrophin family signal largely through different trk receptors: NGF, trkA; BDNF, trkB; NT-3, trkC; and NT-4/5, trkB. However, they all signal via the p75 receptor. Therefore, it is the presence of specific trk receptor types that determines which neurotrophin can be active in any particular tissue. Trk receptors can also be present in a truncated form with the cytoplasmic end absent. Neurotrophins bind to such receptors but elicit no cellular action, effectively diminishing their functional effect.
Neurotrophin Signaling NGF exerts its trkA-mediated effects by becoming internalized and activating diverse signaling pathways. In many situations, NGF is internalized in the axon terminals and is transported to the cell body where the cell signaling takes place. This requires specialized cellular machinery. It has been suggested that clathrin plays a crucial role in internalizing the NGF/trkA complex into endosomes that are transported to the cell body. Another suggestion is that a novel membrane protein named Pincher is responsible for internalizing NGF/trkA complex via a process known as macroendocytosis which has properties of both macropinocytosis and endocytosis. Reports have also indicated that the neurotrophin NT-3 can signal via trkA with a molecular mechanism different from that mediating NGF/trkA signaling. The NT-3/trkA complex is not internalized, unlike NGF/trkA. It activates local membrane signaling, which is believed to play a role in determining local axonal growth, rather than the cell body signaling elicited by the transported NGF/trkA complex which promotes survival and differentiation. TrkA Signaling
The trkA receptor is a 140 kDa glycoprotein which binds NGF. The NGF-activated trkA receptor undergoes dimerization and autophosphorylation at several tyrosine residues that selectively trigger activity
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in several intracellular signaling pathways via binding of specific effector proteins to phosphorylated docking sites. Phosphorylation at Tyr751 activates PI3kinase and the Akt protein kinase, which has been associated with NGF-induced cell survival. Similarly, phosphorylation at Tyr490 is required for association of the Shc adapter proteins that triggers the Ras–MAP kinase signaling cascade and nuclear transcription factors such as CREB and c-fos. The resulting changes in gene expression elicit differentiation and neurite growth. Phosphorylation at Tyr785 activates phospholipase C-g, causing release of intracellular Ca2þ and activation of diacyl glycerol via IP3. This leads to increased levels of protein kinase C (PKC).
an antibody to NGF results in reduction of cell number in the ganglion. The loss of cells was shown to be limited to small-diameter cells identified as nociceptors because it was largely restricted to cells expressing peptides known to be involved in nociception (substance P (SP) and calcitonin gene-related peptide (CGRP)). Other work has shown that cells dying as a consequence of NGF depletion project to lamina II, the known termination site of nociceptive afferent fibers. In vivo studies suggest that the effect of NGF on DRG cell number in rats lasts until approximately postnatal day 2.
p75 Signaling
NGF also has developmental effects on nociceptors beyond postnatal day 2 since treatment of neonatal rats with an antibody against NGF depletes Ad highthreshold mechanonocieptors (HTMs) in the sensory nervous system. This depletion is most profound when NGF is antagonized between postnatal days 4 and 11, and it is accompanied by an increase in the proportion of Ad low-threshold mechanoreceptors known as D-hairs. The proportion of another population of Ad receptors innervating deep tissue is unaffected, suggesting that the effect of NGF is not to simply kill nociceptors since all remaining populations of cells would be expected to increase in number. Thus, Ad HTMs require NGF available in the skin in order to undergo final differentiation to HTMs. If epidermal NGF is not available, these terminals migrate to the dermis to innervate D-hair follicles. This change has been referred to as a phenotypic switch. The cell loss of sensory neurons related to disrupting the NGF/trkA system during development was recognized to be much greater than would have been expected based on the numbers of neurons expressing trkA and responsive to NGF in adult animals. Subsequent work demonstrated that although trkA is expressed by virtually all small- (and many medium-) diameter sensory neurons up to early postnatal development, by 4 weeks of age this expression pattern changed dramatically such that one-third to one-half of small-diameter neurons replaced trkA expression with components of the glial-derived neurotrophic factor (GDNF) receptor complex (GFR-a and c-Ret). Neurons that convert from trkA to ret expression lack p75 from early postnatal times (Figure 1) and become the nonpeptidergic small-diameter sensory neurons (in that they lack CGRP and SP, although some express somatostatin). These GDNF-sensitive nociceptive neurons have spinal terminations in the inner portion of lamina II, distinct from the superficial portion of lamina II where trkA-positive nociceptors
Activation of p75 leads to the activation of numerous intracellular pathways, notably ones promoting apoptosis and cell survival. This suggests that the balance of activity between these two pathways is of crucial importance. In addition, activation of the p75 receptor also activates the enzyme sphingomyelinase promoting synthesis of ceramide. This pathway may be responsible for an independent p75-mediated action on cell physiology.
NGF and Development Axon Growth
NGF promotes fiber outgrowth of sensory neurons cultured from embryonic mouse. In vitro studies suggest that NGF elicits its effects on axonal growth by activating PI3K in growth cones. This signaling pathway enhances actin polymerization. It also inactivates glycogen synthase kinase 3b, leading to upregulation of adenomatous polyposis coli (APC), a microtubulebinding protein. Blockade of NGF signaling removes APC from growth cones and results in their collapse. Cell Survival
The neurotrophic hypothesis was advanced to describe the effect of neurotrophins on neuronal survival. It states that neurotrophins are required for survival of neurons innervating peripheral tissues but that they are present there in limiting amounts. Sensory, motor, and autonomic cells grow into peripheral tissue and compete for the appropriate trophic molecule. Sensory and sympathetic neurons which express trkA compete for the available NGF. The ‘winners’ survive; the ‘losers’ die via apoptosis. This is believed to be an important mechanism for matching the number of central neurons to the size of the periphery to be innervated. Early studies in the dorsal root ganglion (DRG) demonstrated that prenatal depletion of NGF using
Postnatal Development
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Figure 1 Section of P10 rat lumbar DRG demonstrating the distribution of different receptors using immunohistochemistry; trkA (green; a, d), p75 (red; b), and TRPV1 (purple; e). Bottom panels show the combined labels; trkA/p75 (c) and trkA/TRPV1 (f). Cells that were trkAþ but p75 (green; c) will lose their trkA expression and develop c-ret/GFR-a expression as described in the text. TRPV1-expressing cells also express trkA (whitish cells; f) throughout development. Based on unpublished data from A Davenport, JC Petruska, and LM Mendell.
terminate. They also differ in that most trkAþ sensory neurons do not bind the lectin IB4. Developmental Effects of Other Neurotrophins
The neurotrophin NT-3 has been demonstrated to have effects on the development of large-diameter muscle afferent fibers known to express its high-affinity receptor, trkC. Depriving the developing rodent of a functional NT-3/trkC signaling system results in animals with an absence of sensory fiber projections into the ventral horn. This occurs primarily because the spindle afferent fibers responsible for these projections fail to survive. NT-3 also has been shown to be an important factor in promoting the final stages of muscle spindle axon elongation into the ventral horn to synapse with motor neurons. Furthermore, NT-3 is required for postnatal development of certain cutaneous receptors, specifically D-hairs and the Merkel cells associated with type I slowly adapting mechanoreceptors. Interestingly, BDNF appears to regulate the sensitivity of slowly adapting mechanoreceptors in the absence of any
effect on Merkel cells; that is, the effect may be on the properties of the mechanosensitive channels in the axon terminals. However, NT4/5, like BDNF, a ligand for the trkB receptor, is required for the survival of D-hair receptors (i.e., the two trkB ligands can elicit quite different actions).
Biological Effects of NGF In Vivo: Nociception It has become clear in the past few years that administration of NGF in the adult results in painful sequelae. It is now appreciated that this results from the binding of NGF to trkA receptors, although a possible independent role for the p75 receptor has also been demonstrated. The administration of NGF does not elicit spontaneous pain; rather, it sensitizes subsequent responses to nociceptive inputs (Figure 2). The duration of the sensitization in experimental models depends on the mode of administration of the NGF. Systemic administration of NGF results in sensitization lasting several days, whereas the effects
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Via trkA Although the rapid sensitizing effects elicited by local injections of NGF indicate a peripheral site of action, these studies cannot definitively prove that such a mechanism exists, nor do they indicate which cell types in the periphery are involved. Numerous cell types in the periphery are endowed with trkA receptors (e.g., mast cells and autonomic efferents). The effects of NGF can be reduced or even abolished by inactivation or elimination of sympathetic efferents and mast cells. Additionally, neutrophils have been shown to contribute to NGF-induced hyperalgesia. The relative contribution of these different cell types to NGF-elicited hyperalgesia is not known and their action may be coordinated. For example, mast cells contain NGF as well as expressing trkA receptors on their surface membrane, raising the possibility that NGF activation of mast cells causes an explosive release of NGF due to recurrent action of NGF (i.e., positive feedback). TrkA receptors are also well-known to be expressed on nociceptors themselves. These are also likely to contribute to the sensitization by NGF since numerous investigators have demonstrated that exposure of dissociated small-diameter sensory neurons to NGF acutely enhances their response to capsaicin or noxious heat (measured using Ca2þ imaging or patch clamp recording) (Figure 3(a)). The enhanced response of TRPV1 is likely due to its phosphorylation by various intracellular messengers activated by NGF. There is evidence that each of the intermediates mentioned previously in the general discussion of signaling pathways activated by NGF participates in sensitizing the response of TRPV1 to noxious heat or capsaicin. However, these studies, carried out under different conditions with different methods (patch clamping, Ca2þ imaging, and behavior), suggest that the signaling depends very much on the conditions of the experiment. Thus, the effectors activated by these signaling pathways appear to switch from axon growth and survival to sensitization after the animal is born, a proposition confirmed directly by the finding that NGF does not sensitize the response of TRPV1 to noxious heat in trkA-expressing cells on postnatal day 2, despite the fact that the two receptors are co-expressed.
Figure 2 Differing roles of NGF on nociceptors during development. Data obtained from rat.
of local administration last on the order of hours to 1 day. The onset of hyperalgesia occurs within minutes of administration, with thermal hyperalgesia occurring within 5 min. Mechanical hyperalgesia has been reported to begin several hours after administration, but in some behavioral studies it has been found to begin within 10 min (i.e., only slightly longer than the latency for thermal hyperalgesia). This is an important issue since the thermal hyperalgesia is generally ascribed to a direct peripheral action of NGF on sensory neurons. The locus of sensitization of mechanical stimulation is not well documented; a delayed response might be attributed to an indirect central action of NGF, whereas a rapid response might indicate a direct effect in the peripheral terminals. The nociceptive effects of NGF administration have been observed in animal models in which they are inferred from changes in the response to aversive stimuli; for example, the Hargreaves test, which measures putative nociceptive effects as a decrease in the latency of limb withdrawal from a standard noxious heat stimulus. However, more definitive evidence of the nociceptive effects of NGF administration has been obtained in humans as an unanticipated outcome of a phase I clinical trial for the use of NGF to counter the degenerative changes considered to contribute to the symptoms of Alzheimer’s disease. This trial had to be terminated early because of the pain experienced by the otherwise healthy subjects when NGF was administered systemically. In a subsequent study, NGF was administered locally to human volunteers and was also found to be associated with pain. These human studies provide important direct proof of the nociceptive effects of NGF.
Via p75 Evidence suggests that NGF can acutely influence the response of nociceptors by its action on the p75 receptor. As noted previously, NGF acting through the p75 receptor elicits enhanced levels of ceramide, which has been demonstrated to increase
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Figure 3 Examples of acute sensitization by neurotrophins: cell type, trk receptors, and receptors sensitized by neurotrophin action are displayed schematically (top). (a) The response (inward current, top traces) of a trkA-expressing cell (recorded in voltage clamp using dissociated DRG cell patch clamp) to noxious heat (lower traces) is acutely sensitized by NGF. (b) The response (inward current) of a lamina II cell (recorded in voltage clamp using spinal slice patch clamp) to exogenously applied NMDA is acutely sensitized by BDNF. (c) The response (voltage change in current clamp) of a spinal motor neuron (sharp electrode in neonatal hemisected spinal cord) to application of NMDA is sensitized by NT-3. (a) Adapted from Galoyan SM, Petruska JC, and Mendell LM (2003) Mechanisms of sensitization of the response of single dorsal root ganglion cells from adult rat to noxious heat. European Journal of Neuroscience 18: 535–541. (b) Adapted from Garraway SM, Petruska JC, and Mendell LM (2003) BDNF sensitizes the response of lamina II neurons to high threshold primary afferent inputs. European Journal of Neuroscience 18: 2467–2476. (c) Adapted from Arvanov VL, Seebach BS, and Mendell LM (2000) NT-3 evokes an LTP-like facilitation of AMPA/kainate receptor-mediated synaptic transmission in the neonatal rat spinal cord. Journal of Neurophysiology 84: 752–758.
the firing rate of dissociated nociceptors in response to a depolarizing ramp stimulus. This change in firing rate is associated with an increase in Naþ conductance and a decrease in Kþ conductance elicited by membrane depolarization. This action is in parallel with and independent of NGFs sensitization of the TRPV1 receptor via its effect on the trkA receptor. Inflammation-Induced Release of NGF into Peripheral Tissues
The role of NGF in hyperalgesia has been demonstrated by showing that the pain induced in various experimental models can be reduced or abolished by delivering an antibody to NGF in conjunction with the sensitizing agent. Another approach is to administer the immunoadhesin trkA-IgG, which binds with endogenous NGF. Both of these reduce or abolish inflammatory hyperalgesia. The implication of these experiments is that NGF is upregulated in inflamed skin, and this has been confirmed by direct measurement. Enhanced NGF levels have also been detected in the inflamed bladder and in the synovial fluid of inflamed joints, suggesting that NGF contributes generally to inflammatory hyperalgesia in peripheral tissues. The release of NGF is mediated by prior release of the cytokines tumor necrosis factor-a and
interleukin-1b; keratinocytes as well as mast cells are believed to be important sources of NGF (Figure 4). Long-Term Effects of NGF on Nociceptive Signaling
As indicated previously, regulation of gene activity is an important consequence of NGF binding to the trkA receptor. The expression of a number of genes has been shown to be upregulated by NGF administration in culture and in vivo, and these are considered to be important in mediating the long-term effects of NGF (see below). They include genes for ligand-gated ion channels involved in transducing nociceptive stimuli such as TRPV1 (gated by capsaicin/noxious heat), P2X3 (ATP), and ASIC3 (protons); genes for the neuropeptide transmitters SP and CGRP, important in nociception; and Naþ channel genes, both TTX sensitive (Nav1.3) and TTX insensitive (Nav1.8 and Nav1.9). Expression of the gene for another neurotrophin, BDNF, is also upregulated, and this is considered to be particularly important for the central effects induced by NGF. These and other similar changes are instrumental in mediating longer-lasting effects of NGF. The synthesis of additional receptors responsible for transducing nociceptive stimuli (TRPV1, P2X3, and ASIC3), enhances the sensitivity of nociceptors to noxious
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Figure 4 Some cellular mechanisms mediating NGF-induced sensitization of nociceptive afferents. NGF levels are upregulated in peripheral tissues after injury as a consequence of activation of inflammatory cells. NGF directly sensitizes nociceptor endings acutely and also contributes indirectly to sensitizing of these endings via the action of other agents released from immunocompetent cells. Direct administration of NGF activates many of these pathways and has virtually immediate sensitizing effects. After injury, the time course of sensitization will depend on the kinetics of NGF release, but it is generally several hours. NGF will also exert slower effects due to upregulation of other channels, such as TRPV1, bradykinin, and Nav1.8.
heat and other stimuli such as ATP and lowered pH. NGF also enhances expression of BK1 receptors, augmenting the effect of the sensitizing agent bradykinin. The elevated level of BDNF in the somata of trkAexpressing nociceptors after long-term exposure to NGF takes several hours to develop in vivo. This phenomenon is significant because BDNF has been shown to be released into the dorsal horn and to sensitize the glutamatergic synaptic response of cells in lamina II. This phenomenon is known as central sensitization (Figure 3(b)), to be distinguished from peripheral sensitization of nociceptors themselves (Figure 3(a)). Another long-term effect of NGF is the induction of terminal sprouting of nociceptors in the skin, which could also result in heightened sensitivity of these fibers.
The Effect of Other Neurotrophins and Glial-Derived Neurotrophic Factors on Peripheral Sensitization BDNF and NT-4, but not NT-3, have also been shown to sensitize the response to capsaicin in isolated DRG cells and noxious heat in the in vitro skin–nerve preparation. Interestingly, NT-3 reduces
the response of small DRG cells to NGF despite the fact that there are no trkC receptors on smalldiameter DRG cells. Members of the family of GDNFs, consisting of artemin, neurturin, and GDNF, have also been shown to sensitize the response of TRPV1 receptors in nociceptors. The sensitization is elicited at molar concentrations 10–100 times lower than that required for NGF-elicited sensitization of TRPV1mediated responses of nociceptors. Although all these substances elicit sensitization, artemin appears to be the most significant since it is the only one whose level in the skin is increased by the inflammatory mediator complete Freund’s adjuvant, as is its coreceptor GFR-a3. NGF and members of the GDNF family administered individually sensitize the behavioral response to noxious heat for several hours, but the combination of NGF and artemin sensitizes the response for up to 6 days.
Neurotrophins, Central Sensitization, and Neuropathic Pain Sensitization of AMPA/kainate synapses on lamina II cells by BDNF as described previously is distinct from the sensitization induced by neurotrophins at peripheral nerve endings. There are many examples of central and peripheral synapses whose efficacy is enhanced by neurotrophins. In the case of nociceptor synapses on cells of lamina II, this sensitization does not occur directly but, rather, as a consequence of sensitization of NMDA receptors located on the same neurons (Figure 3(b)), an effect also found in the NT-3/trkC system of spinal motor neurons (Figure 3(c)). PKC has been implicated in the coupling between sensitization of NMDA and AMPA/kainate receptors. As in the case of NGF-induced sensitization of the response of nociceptive neurons to capsaicin or noxious heat, sensitization of synaptic transmission persists for at least several minutes to an hour after removal of the neurotrophin, suggesting that the neurotrophin triggers long-lasting changes in the nociceptive pathway. BDNF has also been implicated in other mechanisms of central sensitization, specifically in eliciting neuropathic pain via microglia that are brought to an activated state by nerve injury. Nociceptor terminals co-release ATP and glutamate. ATP acts on activated spinal microglia via P2X4 and P2Y receptors to induce BDNF release. BDNF has been shown to inhibit the KCC2 Cl transporter in lamina I cells, shifting the Cl reversal potential to become positive with respect to the resting potential. This leads to a GABA-induced depolarization (rather than hyperpolarization) of lamina I cells, thereby enhancing activity in nociceptive pathways. The microglial and
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synaptic (trkA-expressing sensory neurons sensitized by NGF) sources of BDNF may act in synergy to produce postsynaptic sensitization throughout the superficial dorsal horn. NGF is an important intermediate in peripheral sensitization in inflammatory pain models (e.g., after complete Freund’s adjuvant or carrageenan). In neuropathic pain models (e.g., chronic nerve constriction injury), the evidence for direct NGF involvement is much less convincing. Nonetheless, neurotrophins may participate since there is evidence of increased BDNF expression in neighboring uninjured sensory neurons. Most of these models are essentially partial nerve injuries in that the system being examined involves plexus nerves. This is important because the spared neurons/axons project through a peripheral nerve containing fibers whose processes have been injured resulting in Wallerian degeneration. This degeneration is similar to peripheral tissue inflammation in many ways, not least of which is that denervated Schwann cells release neurotrophins, including NGF. Furthermore, since peripheral axonal injury induces influx of immune cells to the DRG, noninjured neurons resident in the same DRG as injured neurons are exposed to conditions known to induce sensitization (release of NGF by satellite cells, increased levels of immune mediators such as MCP-1, etc.).
Effects of Neurotrophins on Axonal Growth in Adults The ability of neurotrophins to promote neurite elongation during development as well as in culture has prompted efforts to determine whether they might enhance neurite growth in the adult animal in vivo. BDNF enhances regeneration of motor axons, and both NT-3 and BDNF improve elongation of spinal axons in conjunction with different permissive substrates. NGF has not been found to be as effective as BDNF and NT-3 in promoting regeneration of spinal axons, presumably since they express trkB and trkC but not trkA. On the other hand, regeneration of sensory neurons can be enhanced by NGF as well as by NT-3 and GDNF, but not BDNF. Regenerating dorsal root axons normally do not penetrate the spinal cord, but when exposed to neurotrophins, some growth into the spinal cord has been reported. Interestingly, the identity of the regenerating fibers is appropriate for the neurotrophin that was applied (i.e., nociceptors with NGF or GDNF, and spindle afferent fibers with NT-3). In addition to elongation, neurotrophins play a role in both adaptive and pathologic axonal branching plasticity in the adult. NGF is known to be a key player in the peripheral collateral sprouting of axons
from nociceptive DRG neurons and sympathetic neurons, a process by which noninjured neurons extend new axonal branches to innervate denervated skin. A pathologic version of this process may also occur after spinal cord injury where autonomic dysreflexia is associated with new central connections established by trkA-expressing DRG neurons into the sympathetic outflow circuitry. Remodeling of hippocampal neurons as part of memory processes involves BDNF, and BDNF, NGF, and NT-3 are likely involved to some degree in pathologic (e.g., epileptiform) processes. Evidence for significant NT-3-mediated adaptive plasticity is limited to conditions of nerve or spinal cord injury with exogenous NT-3.
Clinical Implications of Neurotrophin Biology A number of disorders of the peripheral nervous system have been attributed to alterations in NGF metabolism. The importance of neurotrophins as survival factors predicts that their absence during development should deplete specific populations of neurons according to their neurotrophin dependence. This has been advanced as the explanation of the rarely observed hereditary sensory and autonomic neuropathy type IV, in which the insensitivity to pain and reduced sweating are correlated with an absence of the NGF highaffinity receptor trkA. The infectious disease leprosy is characterized by hypoalgesia and reduced sweating from affected skin areas; this is accompanied by diminished NGF levels in skin biopsies from this areas. Diabetic neuropathy results in reduced nociceptive function; the magnitude of the capsaicin-evoked skin axon reflex (a measure of nociceptive function) is correlated with NGF levels in the skin. In adults in whom NGF acts to sensitize the response to certain nociceptive inputs, treatments that reduce NGF activity have been explored as possible treatment modalities in various pain states. In animal models, this approach has proven useful in models of experimental arthritis, visceral pain (pancreatitis and interstitial cystitis), or metastatic bone pain where more conventional treatments have not proven effective or have significant side effects. A humanized antibody to NGF (RN624) has been developed and is in clinical trials. See also: Neurotrophins: Physiology and Pharmacology.
Further Reading Anand P (2004) Neurotrophic factors and their receptors in human sensory neuropathies. Progress in Brain Research 146: 477–492. Arvanov VL, Seebach BS, and Mendell LM (2000) NT-3 evokes an LTP-like facilitation of AMPA/kainate receptor-mediated
Nerve Growth Factor 583 synaptic transmission in the neonatal rat spinal cord. Journal of Neurophysiology 84: 752–758. Chao MV and Hempstead BL (1995) p75 and Trk: A two-receptor system. Trends in Neurosciences 18: 321–326. Cowan WM (2001) Viktor Hamburger and Rita Levi-Montalcini: The path to the discovery of nerve growth factor. Annual Review of Neuroscience 24: 551–600. Galoyan SM, Petruska JC, and Mendell LM (2003) Mechanisms of sensitization of the response of single dorsal root ganglion cells from adult rat to noxious heat. European Journal of Neuroscience 18: 535–541. Garraway SM, Petruska JC, and Mendell LM (2003) BDNF sensitizes the response of lamina II neurons to high threshold primary afferent inputs. European Journal of Neuroscience 18: 2467– 2476. Hefti FF, Rosenthal A, Walicke PA, et al. (2006) Novel class of pain drugs based on antagonism of NGF. Trends in Pharmacological Sciences 27: 85–91. Huang EJ and Reichardt LF (2001) Neurotrophins: Roles in neuronal development and function. Annual Review of Neuroscience 24: 677–736. Levine JM and Mendell LM (2006) Trophic factors and their influence on regeneration. In: Selzer M, Clarke S, Cohen LG, Duncan PW, and Gage FH (eds.) Textbook of Neural Repair and Rehabilitation, vol. 1, pp. 405–420. Cambridge, UK: Cambridge University Press. Lewin GR and Barde YA (1996) Physiology of the neurotrophins. Annual Review of Neuroscience 19: 289–317. Lewin GR and Mendell LM (1993) Nerve growth factor and nociception. Trends in Neurosciences 16: 353–359.
McMahon SB, Mendell LM, Phillips HS, and Wall PD (1996) Neurotrophins and sensory neurons: Role in development, maintenance and injury. Philosophical Transactions of the Royal Society of London 351: 361–467. Mendell LM (1995) Neurotrophic factors and the specification of neural function. The Neuroscientist 1: 26–34. Mendell LM, Albers KM, and Davis BM (1999) Neurotrophins, nociceptors and pain. Microscopy Research and Technique 45: 252–261. Mendell LM and Arvanian VL (2002) Diversity of neurotrophin action in the postnatal spinal cord. Brain Research Reviews 40: 230–239. Petruska JC and Mendell LM (2004) The many functions of nerve growth factor: Multiple actions on nociceptors. Neuroscience Letters 361: 168–171. Pezet S and McMahon SB (2006) Neurotrophins: Mediators and modulators of pain. Annual Review of Neuroscience 29: 507–538. Shu X and Mendell LM (1999) Neurotrophins and hyperalgesia. Proceedings of the National Academy of Sciences of the United States of America 96: 7693–7696. Thoenen H and Sendtner M (2002) Neurotrophins: From enthusiastic expectations through sobering experiences to rational therapeutic approaches. Nature Neuroscience 5: 1046–1050. Zweifel LS, Kuruvilla R, and Ginty DD (2005) Functions and mechanisms of retrograde neurotrophin signalling. Nature Reviews Neuroscience 6: 615–625.
Retrograde Neurotrophic Signaling C Wu and W C Mobley, Neuroscience Institute, Stanford, CA, USA ã 2009 Elsevier Ltd. All rights reserved.
proximal axons or cell bodies. The use of this device and its variants has allowed study of the mechanisms for retrograde NGF and NT signaling. Microfluidic Chambers
Introduction The survival and differentiation of neurotrophin (NT)responsive neurons are critically regulated by the targets that these neurons innervate. However, the exact mechanism or mechanisms by which the target-derived NT signals produced at the axonal terminals are delivered to the cell body over a significant distance (>1 m in some neurons) remain unclear. These underlying mechanisms have been one of the most intriguing enigmas in modern neurobiology. The discovery of nerve growth factor (NGF) by Levi-Montalcini and Hamberger provided an important clue to the mechanism: A soluble NGF produced in the target dramatically influenced specific populations of neurons. It is now well established that NGF produced in and released from target tissues activates specific receptors at the distal axons of innervating neurons. The signals thus elicited are subsequently retrogradely transported to the cell bodies to regulate nuclear and cytosolic events that are known to be important for the survival and maintenance of healthy neurons. However, the generation, intracellular sorting, and maintenance of the signal remain the subjects of intensive investigation.
Compartmentalized Nerve Cell Chambers Are Used to Study Retrograde Axonal Traffic Campenot Chamber
In vitro experimental studies to support the need for retrograde NT signaling have benefited markedly from the use of compartmentalized neuronal cultures pioneered by Campenot and colleagues. In this experimental paradigm, the neuronal cell bodies and proximal axons are separated from distal axons by a Teflon divider sealed with silicone grease onto a collagencoated surface. Radiolabeled NGF (125I-NGF) can be added to the distal chamber and its retrograde transport from the axonal terminal to the proximal axon or the cell body can be quantitatively measured. Retrogradely transported signaling molecules can also be analyzed by comparing, by means of immunoblotting and immunostaining, the changes in distal axons to those in the
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A microfluidic device has been developed to serve as a platform for long-term cultures of primary neurons from the central nervous system in vitro to study axonal injury and regeneration. Though only recently introduced, it has already proven extremely useful for examining axonal transport. These miniature devices, fabricated with poly(dimethylsiloxane) (PDMS), afford the ability to precisely control, monitor, and manipulate cellular microenvironments. The microfluidic nerve cell chamber has been modified for study of the transport kinetics of retrograde axonal transport of NGF in rat embryonic dorsal root ganglion (DRG) neuronal cultures. The device is depicted in Figure 1. Dissociated DRG neurons are loaded into the left well, and both wells are supplied with standard growth medium (Figures 1(a) and 1(b)). Axons grow across the first microgroove and into the middle chamber in 2–3 days; many then traverse the second microgroove and emerge into the distal axon chamber in an additional 4–5 days. Among the many advantages of this device is that axons can be confined to the two straight microgrooves whose cross-sectional area (3 mm 3 mm) and length (in this case, 100 mm) are well defined. An example of rat embryonic DRG culture (7 days in vitro, i.e., DIV7) is shown in Figure 1(c). Furthermore, neurons can be loaded at a density at which only a single axon crosses the microgroove. This makes it possible, using microscopy, to more readily track labeled NGF and its signaling molecules. As demonstrated in Figure 2, distal axons from postnatal mouse DRG neurons that were cultured in the device for 7 days (Figure 1) were incubated with biotinylated NGF conjugated to streptavidin-Quantum dot 605, and the retrograde movement of labeled NGF was imaged in the proximal axons (Figure 2). Furthermore, the presence of a middle chamber makes it possible to modify the content of the environment (e.g., to administer various kinase inhibitors) through which the axon transmits neurotrophic signals.
Studying Retrograde NT Signaling In Vivo In vivo studies have most often used ligation to examine transport. In the ligature technique, after anesthesia has been administered, a nerve (in most cases the sciatic nerve) is surgically exposed, and one or
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c Figure 1 Schematic representation of a modified version of the microfluidic nerve cell chamber showing the top view (a) and side view (b). In (c), Rat E16 DRG neurons were cultured in the device for 7 days, and DRG axons crossed robustly the two microgrooves into the distal chamber. CB, cell body; PA, proximal axon; DA; distal axon; PDMS, poly(dimethylsiloxane).
more ligatures, separated by a desired distance, are placed. The animals are allowed to recover. Then, after a desired interval, they are sacrificed, and the nerve is taken for immunohistocytochemical, electron microscopic, and biochemical studies. The undamaged contralateral sciatic nerve may serve as control in some experiments. To define the extent of transport of a molecule or organelle, one typically measures the extent to which it accumulates on the most distal ligature (for retrograde transport) or the most proximal ligature (for anterograde transport). The use of a double ligature is useful in controlling for the effect of ligation-mediated injury on the possible local production of the marker of interest. In an alternative procedure, a segment of nerve can be surgically removed and placed on a slab of gelled agarose with both ends placed in a collecting chamber containing a physiological medium. The eluted axoplasm can be analyzed by one or more of the methods specified above. Another established technique that has been used to study retrograde NT signaling places the molecule of interest in the target area of afferent axons. For example, the hipposeptal cholinergic pathway has been used to examine NGF retrograde transport from the hippocampus to basal forebrain cholinergic neurons. In this experimental paradigm, either radiolabeled or fluorescently labeled NGF is stereotaxically injected into the frontal cortex or hippocampus of anesthetized animals. The animals are allowed to recover for a desired period before sacrifice and analysis. In these experiments, one can determine the extent of transport by measuring the accumulation of the labeled species in the septum using light microscopy for fluorescently tagged molecules or, in the case of radiolabeled molecules, in situ autoradiography or gamma counting in the septum.
Retrograde Neurotrophic Signaling: Proposed Mechanisms Signaling Endosome Hypothesis
Figure 2 Postnatal (P0) mouse DRG neurons were cultured in the microfluidic nerve cell chamber for 7 days. Biotinylated NGF that was conjugated to streptavidin-Quantum-dot 605 was added to the distal chamber. Movement of labeled NGF within the proximal axons was imaged in real time by an inverted microscope (100x oil). A series of time-sequenced frames shows the retrograde NGF transport.
This hypothesis posits that the NGF signal is transmitted via endocytosis of complexes containing NGF bound to its activated tyrosine receptor kinase A (TrkA) receptor (i.e., pTrkA), followed by retrograde transport of the ‘signaling endosome’ thus formed (Figure 3). The hypothesis points to signaling endosomes as an important source of retrogradely transmitted signals. It is consistent with the emerging view that cellular signaling pathways are highly organized and compartmentalized, features that are used to confer specificity and sustainability of signal transduction. The signaling endosome hypothesis is supported by an increasing body of experimental evidence from a number of laboratories. Studies in vitro have shown that (1) NGF at axon terminals must be internalized
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Figure 3 NGF–TrkA signaling pathways and retrograde axonal transport of signaling endosomes. (a) NGF activates its high-affinity tyrosine receptor kinase, TrkA, and elicits divergent signaling pathways. The four major pathways are (1) the transient SOS/Ras/C-Raf/ MEKK/MEK/Erk pathway; (2) the persistent C3G/Rap1/B-Raf/MEKK/MEK/Erk pathway; (3) the PI3K/Akt pathway; and (4) the PLCg pathway. Activation of these pathways effects neuronal functions of survival and differentiation. (b) Following endocytosis of NGF and activated TrkA signaling complex, the signaling endosome engages the dynein–dynactin complex to undergo retrograde axonal transport along the microtubule network (MT). NGF, nerve growth factor; TrkA, tyrosine receptor kinase A; FRS2, fibroblast growth factor receptor substrate-2; Shc, SH2-containing protein; SH2-B, SH2-containing protein B; Grb2, growth factor receptor bound protein 2; SOS, son of sevenless; Ras, a small GTPase; C-Raf, Raf kinase C; MEKK, MAPK/Erk kinase kinase; kinase; MEK, MAPK/Erk kinase kinase; Erk (or MAPK), extracellular signal-regulated kinase (or mitogen activated protein kinase); Gab2, Grb2-associated binding protein-2; GIPC, GAIP interacting protein; Shp2, SH2-containing protein phosphatase-2; CrkL, v-crk avian sarcoma virus CT10 oncogene homolog-like; C3G, Crk SH3 binding guaninenucleotide releasing factor; Rap1, Ras-related protein 1; B-Raf, Raf kinase B; PI3K, phosphatidylinoditiol-3-kinase; Akt, protein kinase B; PLCg, phospholipase-C-gamma; GAIP, Ga-interacting protein; Rab5, a member of the RAS oncogene family; P, phosphate.
and retrogradely transported to neuron cell bodies for the NGF signal to induce phosphorylation of cyclic adenosine monophosphate response elementbinding protein (CREB) and to increase survival of
immature neurons; (2) NGF-induced activation of TrkA in axon terminals is also required for survival; (3) NGF induces endocytosis of TrkA and is bound to pTrkA in endosomes; (4) retrograde NGF signals
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are decreased by inhibiting endocytosis; (5) NGF– pTrkA complexes are found in clathrin-coated vesicles together with activated components of the Ras/ extracellular signal-regulated kinase (Erk)1/2 pathway as well as phosphatidylinoditiol-3-kinase (PI3K) and phospholipase-C-g; (6) NGF signaling that is present in clathrin-coated vesicles is able in in vitro kinase assays to phosphorylate Elk-1, a downstream target of Erk1/2; (7) in what appears to be the next step in the maturation of the signaling endosome within the endosomal pathway, and a step that requires both dynein and intact microtubules, NGF also induces the formation of complexes containing pTrkA and activated components of the Ras-related protein 1 (Rap1)/Erk1/2 pathway in early endosomes; (8) NGF– pTrkA complexes are moved retrogradely in axons to cell bodies, as are the complexes of brain-derived neurotrophic factor (BDNF) and its phosphorylated tyrosine receptor kinase B (pTrkB); (9) retrograde transport of these complexes is blocked by disrupting microtubules; and (10) retrogradely transported pTrk is required for CREB phosphorylation, c-fos induction, protein kinase B (Akt) phosphorylation, and survival of immature neurons. In vivo and ex vivo observations are consistent in showing that (1) early endosomes containing NGF–pTrkA, together with activated components of the Rap1/Erk1/2 and PI3K pathways, are retrogradely transported in sciatic nerve; (2) increasing NGF levels in the target of innervation increases retrograde transport of these membranes and increases pErk1/2 in DRG neuron cell bodies; conversely; (3) decreasing NGF in the target of innervation by sequestering it with specific antibodies decreases the retrograde transport of endosomes containing pTrk and pErk1/2; and (4) it has been demonstrated recently in vivo that NGF was localized within early endosomes in cholinergic terminals, and after hippocampal injection, 125I-NGF was coimmunoprecipitated with antibodies against TrkA. NGF-Independent Retrograde Signaling
There is evidence that NGF retrograde signaling may not always be coupled to retrograde transport of NGF. Senger and Campenot found that while it took at least 30 min for 125I-NGF to arrive at the cell body/ proximal chamber at a detectable level following addition of 125I-NGF to the distal chamber in the compartmentalized culture of sympathetic neurons, TrkA and other signaling molecules were more rapidly activated in the cell body/proximal axons. Utilizing NGF that was covalently cross-linked to beads, thus rendering it incapable of internalization, Campenot’s group further demonstrated that this source of NGF
was able to trigger activation and axonal transport of TrkA and PI3K/Akt signaling proteins at levels adequate to support survival of sympathetic neurons for up to 30 h. Taken together, the findings suggest that signaling events initiated by NGF at the axonal terminals can be transported retrogradely without NGF. More recently, Mok and Campenot used Go6976 a very potent drug in inhibiting TrkA phosphorylation, to block TrkA phosphorylation in the cell body/ proximal axons in compartmentalized sympathetic neurons. Go6976 itself exerts no survival effects on sympathetic neurons deprived of NGF. After applying Go6976 to locally block TrkA phosphorylation in the cell body/proximal axons, NGF added to distal axons still resulted in activation of survival signaling pathways (e.g., pAkt, pCREB) in the cell body/proximal axons. These investigators concluded that retrograde transport of signaling molecules downstream of phosphorylated TrkA, but not phosphorylated TrkA itself, is necessary to support neuronal survival. Other Hypotheses
Other possible mechanisms for retrograde signaling can be envisioned. In a general sense, they conform either to the ‘wave model’ or to the ‘signaling effector model.’ The former suggests that NGF binds to and activates TrkA at the distal axonal terminals. Activated TrkA and/or signaling molecules downstream of activated TrkA are subsequently propagated and transported back to the cell body. The latter model postulates that second messengers such as ionic fluxes (e.g., cytosolic Ca2þ), instead of NGF–pTrkA, are retrogradely transported. There is little experimental data to support these alternatives, but given the paucity of knowledge for retrograde signaling mechanisms, continued attempts should be made to discover and characterize alternative models. The Nature of Intracellular Vesicles That Carry Retrograde NGF Signaling
There is no consensus about the type of endocytic vesicle or vesicles that transport retrograde NT signaling in axons. An early study suggested that lysomes/multivesicular bodies (MVBs) contained the majority of 125I-NGF that was retrogradely transported in cultured sympathetic neurons. Studies from Hendry laboratory showed that 125I-NGF transported from the eye to the sympathetic and sensory ganglia was concentrated in MVBs. These findings led to the proposal that MVBs are the major vesiclar carriers within axons for retrograde NT signaling. Depending on the experimental conditions, investigators from a numbers of laboratories discovered that
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NGF or its signaling components such as pTrk resided in pincher-positive complex structures, in ‘uncoated’ vesicles, in the trans-Golgi network, in late endosomes that are marked by Rab7, or in Rab5-positive early endosomes. It is possible that many different endocytic vesicle types are engaged in transporting NGF signals and that different neurons use a different complement of such organelles. Resolving this issue is an important next step in clarifying retrograde signaling pathways. Role of MT/Dynein in Mediating Retrograde Transport of NT Signaling
It is not surprising that retrograde transport of NT signaling endosomes in axons, like many other subcellular organelles (mitochondria, RNA particles, etc.), requires the microtubule network (MT) for movement. In support of this, infusion of nocodazole, a drug that causes the collapse of MT, into isolated sciatic nerves blocked retrograde NGF transport. Similarly, another MT collapse-causing drug, colchicine, suppressed Trk phosphorylation of cell bodies in response to neurite stimulation in DRG neurons. Colchicine pretreatment also blocked CREB phosphorylation in response to stimulation of the cell bodies, as well as the neurites. The dynein motor protein complex is primarily responsible for powering retrograde movement within a cell. It comprises many different subunits (the heavy chains, the intermediate chains, and the light chains). Evidence from Chao laboratory suggests that Trk associates with both the 14 kDa light and the 74 kDa intermediate chain of dynein. Furthermore, Segal and colleagues found that pTrk was found to co-localize with dynein, but not with the anterograde motor kinesin, in rat sciatic nerve axons. In addition, the 74 kDa subunit of dynein was found in retrograde transport vesicles that contain NGF–pTrkA isolated from rat sciatic nerve axons. More recently, disruption of the dynein function by introduction of the 50 kDa subunit (dynamitin) of dynactin, an established method to dissociate the dynein–dynactin complex, blocked retrograde transport of NGF and BDNF and led to atrophy and cell death in neurons. These results demonstrate that retrograde transport of NT signaling is driven by the dynein–dynactin complex.
Disrupted Retrograde Transport NT Signaling in Neurodegenerative Diseases Given the role that active retrograde NT signaling plays in the development of the nervous system, and evidence that continued production in targets of innervation are needed to maintain mature neurons, it is noteworthy that an increasing body of data points
to a link between defects in retrograde NT signaling and dysfunction and degeneration of neurons in neurodegenerative diseases. Salehi et al. recently demonstrated that in a mouse model of Down syndrome (Ts65Dn), the presence of one extra copy of the amyloid precursor protein selectively disrupted retrograde NGF transport and resulted in degeneration of basal forebrain cholinergic neurons (BFCNs), a pathological hallmark in Alzheimer’s disease. In an earlier study, Cooper et al. showed that NGF was retrogradely transported via the hipposeptal pathway. The age-related degeneration of BFCNs (decrease in cell numbers and profile area of cell bodies) in Ts65Dn mice could be reversed by direct intracerebroventricular NGF infusion. Similarly, a severe defect in retrograde NGF transport has also been reported by Yang and colleagues in cultured postnatal DRG neurons of a mouse model of giant axon neuropathy, a disorder that affects a number of neuronal populations in the peripheral and central nervous system. Defective retrograde NT transport has also been implicated in Huntington’s disease (HD), a condition that results from an expansion of polyglutamine tract (>37 glutamines) in huntingtin (htt). In a recent study, Rong et al. observed that htt-associated protein-1, a contributing factor to HD pathology, interacts with the dynein motor in sympathetic neurons. Mutant htt appears to influence this interaction and in so doing reduces the stability of intracellular TrkA. As a result, retrograde NGF signaling may well be compromised in the diseased neurons. Given that retrograde neurotrophic signaling may share mechanisms and protein machinery with other retrograde processes, it is conceivable that disruption or alteration in any components of the machinery will also inevitably affect retrograde NT signaling. Disruption of retrograde axonal transport has been suggested to contribute to the pathogenesis of a number of progressive disorders of motor neurons. In one study, transgenic overexpression of p50 was shown to result in inhibition of axonal transport and cause lateonset progressive degeneration in motor neurons. In addition, a family with chronic motor neuron disease has been shown to harbor a mutation in p150 glued, a protein that plays a critical role in the function of the dynein complex. Genetic mutations that affect the stability of the microtubules have been suggested to cause progressive motor neuronopathy in mice. A Trp524Gly substitution at the last residue of the tubulin-specific chaperone e protein (gene locus: chromosome 13) leads to a reduced number of microtubules in the sciatic and phrenic nerves. The affected mice develop progressive caudocranial degeneration of their motor
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axons from the age of 2 weeks and die 4–6 weeks after birth. Axonal degeneration apparently starts at the endplates and is prominent in the sciatic nerve and its branches and the phrenic nerve. Since intact microtubules are required for retrograde NGF transport, it is conceivable that retrograde NGF–pTrkA signaling is compromised in these mice as well, which may well in turn contribute to the disease. In another example, alsin, a putative guanine nucleotide exchange factor for Rab5, plays a critical role in regulating early endosomal fusion. Loss of alsin-2 function is believed to be the cause for a subset of amyotrophic lateral sclerosis (ALS) in humans, juvenileonset primary lateral sclerosis. In Als2(/) mice, endosomal transport of insulin-like growth factor 1 and BDNF receptors was selectively disturbed. In another mouse model of ALS, a missense mutation L967Q in Vps54 has been identified in the spontaneous autosomal recessive wobbler mutation. Axonal transport is impaired in wobbler mice, and Vps54 might be critical for retrograde vesicular transport and in particular for axonal transport in motor neurons. Since internalized NGF was found to localize in early endosomes in cholinergic neurons, retrograde NGF signaling is likely affected in these neurons as well. Recent studies have revealed that late endosomes that are marked by Rab7 may also play a role in modulating retrograde NGF–pTrkA signaling. In genetic studies, the locus for Rab7 has been implicated as the disease gene for Charcot-Marie-Tooth disease type 2B (CMT2B). CMT2B is a rare autosomal dominant genetic disorder that leads to peripheral sensory neuropathy with prominent axonal degeneration. Missense mutations in the highly conserved C-terminus of Rab7 (Leu129Phe, Val162Met, Lys157Asn) have been identified in CMT2B patients. It will be of great interest to examine whether retrograde NT trafficking or signaling is affected in neurons that harbor Rab7 mutations.
Summary Retrograde NT signaling plays a critical role in regulating survival and differentiation of specific populations of neurons in both the central nervous system and the peripheral nervous system. There is increasing evidence that NGF and other NTs bind to and activate their receptors at the axonal terminus to elicit the recruitment of an array of signaling molecules. Endocytosis of the NT–pTrk signaling complex is then sorted into a subpopulation of early endosomes or other cellular organelles to give rise to signaling endosomes. The signaling endosome driven by the dynein motor complex is retrogradely transported
via the MT to the cell body to effect gene expression and other somal events. Retrograde NT signaling thus appears to be a very complex process that involves numerous cellular components and events. Disruption of retrograde NGF and NT signaling may contribute or lead to many types of neurodegenerative diseases. See also: Neurotrophins: Physiology and Pharmacology.
Further Reading Campenot RB and MacInnis BL (2004) Retrograde transport of neurotrophins: Fact and function. Journal of Neurobiology 58: 217–229. Chao MV (2003) Neurotrophins and their receptors: A convergence point for many signalling pathways. Nature Reviews Neuroscience 4: 299–309. Cooper JD, Salehi A, Delcroix JD, et al. (2001) Failed retrograde transport of NGF in a mouse model of Down’s syndrome: Reversal of cholinergic neurodegenerative phenotypes following NGF infusion. Proceedings of the National Academy of Sciences of the United States of America 98: 10439–10444. Cui B, Wu C, Chen L, et al. (2007) One at a time, live tracking of NGF axonal transport using quantum dots. Proceedings of the National Academy of Sciences of the United States of America 104(34): 13666–13671. Deinhardt K, Salinas S, Verastegui C, et al. (2006) Rab5 and Rab7 control endocytic sorting along the axonal retrograde transport pathway. Neuron 52: 293–305. Delcroix JD, Valletta JS, Wu C, Hunt SJ, Kowal AS, and Mobley WC (2003) NGF signaling in sensory neurons: Evidence that early endosomes carry NGF retrograde signals. Neuron 39: 69–84. Heerssen HM, Pazyra MF, and Segal RA (2004) Dynein motors transport activated Trks to promote survival of targetdependent neurons. Nature Neuroscience 7: 596–604. Howe CL and Mobley WC (2005) Long-distance retrograde neurotrophic signaling. Current Opinion in Neurobiology 15: 40. Huang EJ and Reichardt LF (2001) Neurotrophins: Roles in neuronal development and function. Annual Review of Neuroscience 24: 677–736. LaMonte BH, Wallace KE, Holloway BA, et al. (2002) Disruption of dynein/dynactin inhibits axonal transport in motor neurons causing late-onset progressive degeneration. Neuron 34: 715–727. Levi-Montalcini R (1987) The nerve growth factor 35 years later. Science 237: 1154–1162. Salehi A, Delcroix JD, Belichenko PV, et al. (2006) Increased App expression in a mouse model of Down’s syndrome disrupts NGF transport and causes cholinergic neuron degeneration. Neuron 51: 29–42. Taylor AM, Blurton-Jones M, Rhee SW, Cribbs DH, Cotman CW, and Jeon NL (2005) A microfluidic culture platform for CNS axonal injury, regeneration and transport. Nature Methods 2: 599–605. Wu C, Lai CF, and Mobley WC (2001) Nerve growth factor activates persistent Rap1 signaling in endosomes. Journal of Neuroscience 21: 5406–5416. Zweifel LS, Kuruvilla R, and Ginty DD (2005) Functions and mechanisms of retrograde neurotrophin signalling. Nature Reviews Neuroscience 6: 615–625.
BDNF in Synaptic Plasticity and Memory N H Woo and B Lu, National Institutes of Health, Bethesda, MD, USA Published by Elsevier Ltd.
Introduction Among members of the neurotrophin family, brainderived neurotrophic factor (BDNF) stands out for its ability to regulate synaptic plasticity and various cognitive functions of the brain. A Medline search with the terms ‘BDNF’ and ‘synaptic’ yields more than 700 research articles, mostly published in the last 7 years. Given that neurotrophins were initially defined as secretory factors that promote neuronal survival and differentiation during development, the role of BDNF in synaptic modulation was not recognized until late 1990s. A number of observations have aided in the realization that the primary function of BDNF is to regulate synaptic transmission and plasticity, rather than neuronal survival. One is that BDNF is widely distributed in many regions of the adult brain, with levels much higher than any other neurotrophins. The other is that the expression of BDNF can be rapidly enhanced by neuronal activity under conditions relevant to synaptic plasticity. Because neuronal activity is known to be crucial for synaptic plasticity, it was hypothesized that activity-dependent synaptic modulation is mediated by BDNF. Indeed, early studies demonstrated that BDNF mimics neuronal activity in altering the number and/or strength of synaptic connections. Subsequent studies revealed a much more complex and interesting picture. On the one hand, BDNF regulates various forms of synaptic plasticity, leading to changes in neuronal circuitry subserving complex behaviors. On the other hand, many aspects of BDNF cell biology, such as transcription and secretion, are tightly controlled by neuronal activity. Complex interactions between BDNF and neuronal activity may offer a plethora of means to control sophisticated cognitive functions of the mammalian brain.
Cell Biology of BDNF All neurotrophins arise from their precursors as a result of proteolytic cleavage of the prodomain. Proneurotrophins have long been thought to be inactive precursors. However, this view was challenged a few years ago when proneurotrophins were shown to promote apoptosis via p75 neurotrophin receptor (p75NTR). This is opposite to the cell survival effect by mature neurotrophins, which act via their preferred
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tropomyocin-related receptor tyrosine kinase (Trk) receptors. Similarly, proBDNF and mature BDNF (mBDNF) have been shown to elicit opposite effects on synaptic plasticity (Figure 1). In recognition that proneurotrophins are biologically active, cleavage of proneurotrophins becomes an important regulatory mechanism that controls the direction of neurotrophin regulation. Signal Transduction
The biological functions of BDNF are mediated by two receptor systems: TrkB and p75NTR. It is well established that mBDNF binds TrkB with high affinity. Upon binding, BDNF triggers TrkB dimerization resulting in tyrosine phosphorylation in its cytoplasmic domain. These autophosphorylation events recruit a series of intracellular proteins that primes subsequent activation of several signaling pathways. Three classical signaling pathways have been identified: phosphatidylinositol-3-kinase (PI3K) pathway, phospholipase C-g (PLC-g) pathway, and Map-Erk Kinase (MEK)-MAPK pathway (Figure 1). The majority of BDNF actions described thus far are attributed to signaling cascades activated by TrkB. In addition to cell surface signaling, BDNF induces the endocytosis of TrkB. Rather than simply inactivating TrkB, the endocytosis of BDNF-TrkB complex results in the formation of BDNF-TrkB signaling endosomes, triggering signaling cascades different from those initiated from cell surface TrkB. This process is required for translation-dependent long-term functions, and is involved in retrograde propagation of BDNF signal from axonal terminals to cell body. All neurotrophins including proBDNF bind p75NTR, which triggers signaling events distinct from those by Trk receptors. The cytoplasmic domain of p75NTR lacks intrinsic catalytic activity. Upon ligand binding, several intracellular signal transduction cascades are activated, including nuclear factor kappa B (NFkB), Jun kinase, and sphingomyelin hydrolysis (Figure 1). Notably, p75NTR activation is associated with the initiation of apoptosis. For many years, p75NTR was considered a ‘low affinity’ neurotrophin receptor. Recent studies indicate that preferred ligands for p75NTR are proneurotrophins, with binding affinities just as high as that between mature neurotrophins and Trk receptors. Current data support a model that pro- and mature neurotrophins induce very different functions through two distinct receptor-signaling systems. An added complexity is the newly discovered coreceptor sortilin. The prodomain of proneurotrophins bind sortilin, whereas the mature domain binds p75NTR. The formation of
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Figure 1 Synthesis, trafficking, and receptor-signaling of BDNF. Initially synthesized in the endoplasmic reticulum (ER) as a precursor protein, proBDNF is properly folded in the ER and Golgi network and packaged into secretory vesicles. Subsequently, BDNF is sorted into either the constitutive or regulated secretory pathway, and transported to the appropriate site of release. The prodomain can be cleaved intracellularly by furin or protein convertases, resulting in the secretion of mature BDNF (mBDNF). Alternatively, proBDNF can be secreted and cleaved extracellularly by the tPA/plasmin cascade or metalloproteinases to yield mBDNF. Once secreted, proBDNF and mBDNF elicit diverse and often opposing biological actions via two distinct receptor-signaling systems. mBDNF binds TrkB, leading to the autophosphorylation of tyrosine residues in the tyrosine kinase domain. Consequently, three major signaling cascades can be activated by mBDNF-TrkB, including the PI3K pathway, ERK/MAPK pathway, and PLCg pathway. In contrast, proBDNF binds p75NTR, resulting in the activation of several signaling molecules, including NF-kB, JNK and RhoA.
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the sortilin–proneurotrophin–p75NTR triplex may be necessary for p75NTR signaling. Activity-Dependent Controls
A cardinal feature of BDNF is that its expression is regulated by neuronal activity. It is now recognized that a multitude of physiological stimuli can alter BDNF expression. For example, visual input and sensory stimulation of the whiskers control BDNF expression in the visual cortex and barrel cortex, respectively. In the superchiasmatic nucleus and amygdala, expression of BDNF is regulated by circadian rhythm and fear emotion. Remarkably, learning or exercise can also enhance BDNF expression in the hippocampus. Moreover, BDNF levels are also affected in a variety of pathological conditions associated with altered neuronal activity in the brain including seizure, Alzheimer’s, depression, and stress. In addition to regulation of BDNF gene expression, a new theme emerging from recent studies is that neuronal activity also controls many cellular processes of BDNF, including intracellular trafficking, secretion of BDNF, and perhaps cleavage of proBDNF. Transcription The genomic structure of BDNF is quite complex. In rats, there are at least four promoters controlling four short 50 exons. Each 50 exon is alternatively spliced onto a common 30 exon (exon V) encoding the pre-proBDNF protein. In humans, the latest study reported seven promoters and eight exons, with exon VIII being the common exon coding for preproBDNF. It has long been a puzzle why nature has designed multiple BDNF transcripts that encode exactly the same protein. Cumulative evidence now indicates that these transcripts are distributed in different brain regions, different cell types, and even different parts of the cells (soma vs. dendrites). Importantly, their expression can be altered in response to different physiological stimuli. For instance, exon III transcript is detected only in cell bodies, whereas exon IV transcript is present both in cell bodies and dendritic processes of neurons in the visual cortex. During cerebellar development, thyroid hormone treatment selectively primarily enhances the expression of exon II mRNA. Emerging evidence indicates that BDNF promoters are differentially involved in various neurological and psychiatric disorders. Promoter II-drive transcription can be suppressed by a neuronal silencer, and such suppression is removed by huntingtin, which binds and sequesters the silencer in the cytosol of cortical neurons. This is important for the survival of cortical neurons that projects to the striatum. In Huntington’s disease, the mutant huntingtin can no longer bind the
silencer, resulting in the translocation of the silencer into the nucleus and suppression of BDNF promoter II. Another striking example of BDNF promoter specific regulation involves MeCP2, which is a methyl-CpG-dependent transcriptional repressor that binds methylated DNA BDNF promoter III. Neuronal depolarization dissociates MeCP2 from promoter III, leading to the expression of exon III transcript in hippocampal neurons. Mutation in MeCP2, which occurs in 80% of Rett syndrome patients, abolishes this activity-dependent form of regulation. Promoter IV has been implicated in stress and is the major target of glucocortical and mineralocortical receptors. In clinical studies, a human single nucleotide polymorphism (SNP) in the exon IV has been associated with epilepsy and late-onset Alzheimer’s. Among all the promoters, promoter III has received much attention because it is by far the most effectively regulated by neuronal activity in the amygdala, hippocampus, and cortex. An increase in promoter III-driven transcription has been associated with long-term potentiation (LTP) and memory. Early work showed that BDNF gene expression was dependent on a rise in intracellular calcium and that application of high Kþ to cultured cortical neurons selectively enhanced exon III expression. Based on these observations, three elements in promoter III were characterized to be involved in Ca2þ-dependent expression of BDNF: the Ca2þ responsive sequence 1 (CaRE1) that binds Ca2þ responsive transcription factor (CaRF), the E-Box that binds upstream stimulatory factor (USF), and the classic cAMP responsive element (CRE) that binds cAMP responsive element binding (CREB). In addition, the transcription through promoter III is regulated by NF-kB and MeCP2. Taken together, transcription of BDNF exon III is tightly regulated by several mechanisms that couple neuronal activity with gene transcription. Processing and trafficking Like all neurotrophins, BDNF mRNA is translated into a precursor protein, pre-proBDNF, which enters into the endoplasmic reticulum (ER) lumen through its N-terminal ‘pre’ sequence (signal peptide). After the removal of the pre-sequence by signal peptidases in the rough ER, the protein is folded in the trans-Golgi network and then packaged into secretory vesicles. Once folded correctly, BDNF is sorted into one of two principal pathways, the constitutive (i.e., spontaneous release) or regulated (i.e., release in response to stimuli) secretory pathway (Figure 2). The BDNF-containing vesicles are trafficked to the appropriate subcellular compartment. In neuronal dendrites and spines, BDNF appears to be stored in a special type of
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Figure 2 Regulation of distinct forms of hippocampal synaptic plasticity by BDNF. (a) mBDNF facilitates E-LTP in neonatal hippocampus in which BDNF level is low. Application of tetanic stimulation to neonatal slices (p12-p13) induces only short-term potentiation (STP), which can be converted to E-LTP by exposure to exogenous BDNF. (b) mBDNF facilitates E-LTP by promoting vesicle docking. (c) mBDNF is also involved in L-LTP. Strong theta-burst stimulation (TBS) induces robust L-LTP in wild-type (WT), but not in BDNF þ/ mice hippocampal slices. (d) ProBDNF ! mBDNF conversion by tPA/plasmin is required for L-LTP. Strong TBS triggers the secretion of tPA, which cleaves plasminogen to form plasmin. Plasmin subsequently cleaves proBDNF to yield mBDNF, which binds TrkB and permits L-LTP expression. (e) proBDNF promotes hippocampal LTD. Slices from p75NTR/ mice fail to exhibit NMDA receptor-dependent LTD, whereas treatment with cleavage-resistant proBDNF enhances LTD in wild-type slice. (f) proBDNF binds to p75NTR to facilitate LTD, possibly through the regulation of NR2B, a distinct NMDA receptor subunit implicated in hippocampal LTD. EPSP, excitatory postsynaptic potential; fEPSP, field excitatory postsynaptic potential; LFS, low-frequency stimulation. (a) Reproduced from Figurov A, Pozzo-Miller L, Olafsson P, Wang T, and Lu B (1996) Regulation of synaptic responses to high-frequency stimulation and LTP by neurotrophins in the hippocampus. Nature 381: 706–709, with permission. (c) Reprinted with permission from Pang PT, Teng HK, Zaitsev E, et al. (2004) Cleavage of proBDNF by tPA/plasmin is essential for long-term hippocampal plasticity. Science 306: 487–491. Copyright 2004 AAAS. (e) Reproduced from NH Woo, Teng HK, Ciao C, et al. (2005) Activation of p75NTR by proBNF facilitates hippocampal long-term depression. Nature Neuroscience 8: 1069–1077, with permission.
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secretory granules that lack chromogranin A (CGA), a marker for large dense core vesicles (LDCV). In contrast, conventional BDNF-containing LDCVs have been found in axons and terminals, possibly through anterograde axonal transport. Nonneuronal cells such as fibroblasts and Schwann cell secrete neurotrophins constitutively, whereas principal neurons and neuroendocrine cells secrete neurotrophins in response to depolarization and a rise in intracellular calcium. The dendritic trafficking and synaptic localization of BDNF appear to be influenced by its own prodomain. This was implicated in a study that examined a SNP in the pro-region of the human BDNF gene. This SNP, located at nucleotide 196, produces a valine-to-methionine substitution at amino acid 66 (Val66Met). In cultured hippocampal neurons, fluorescence-tagged val-BDNF is distributed in cell body, as well as dendrites. A fraction of valBDNF is also located at synapses, as revealed by their co-localization with synaptic markers. In marked contrast, met-BDNF is largely located in cell body and proximal dendrites. Met-BDNF was rarely localized at distal dendrites and was absent at synapses. These results suggest that the prodomain, particularly the region containing Val66, is critical for dendritic trafficking and synaptic localization of BDNF. A long-held view is that proneurotrophins, particularly proNGF and proBDNF, are processed by intracellular proteases including the serine protease furin in the trans-Golgi network and the prohormone convertases (PC1/3) in the secretory granules. Recent studies have shown that a large fraction of BDNF in the brain is secreted in the proform, which is converted to mBDNF by extracellular proteases including plasmin or metalloproteinases (MMP3 or MMP7). Because proBDNF and mBDNF elicit distinct and often opposing biological actions through different receptors, proteolytic cleavage has now emerged as a new mechanism that determines the function of BDNF. Of particular interest is tissue plasminogen activator (tPA), an extracellular protease that converts the inactive zymogen plasminogen to plasmin. tPA is secreted from axonal terminals in response to neuronal activity. It is conceivable that neuronal activity could control proBDNF ! mBDNF conversion by triggering tPA secretion. Secretion BDNF is perhaps the only neurotrophin indisputably secreted in response to neuronal activity. In fact, majority of BDNF is sorted into the regulated, rather than the constitutive, secretory pathway. Experiments using green fluorescent protein (GFP)tagged BDNF revealed that BDNF can be secreted
from either pre- or postsynaptic sites. The amount of BDNF secretion depends on the pattern of neuronal activity. Generally, tetanus such as those used to induce LTP is more effective in inducing BDNF secretion than low-frequency stimulation. This has been demonstrated in cultured neurons as well as in slices that underwent different forms of plasticity. Studies of Val66Met SNP have drawn attention to the role of prodomain in activity-dependent BDNF secretion. In neurons transfected with met-BDNF, depolarization-induced secretion was selectively impaired while constitutive secretion remained normal. Subsequent studies demonstrate that proBDNF is co-localized with the neurotrophin coreceptor sortilin intracellularly in secretory granules, and that sortilin interacts specifically with the prodomain in a region encompassing Val66Met. Remarkably, inhibition of the interaction between the prodomain and intracellular sortilin attenuates secretion of BDNF induced by depolarization, suggesting that this interaction is critical for regulated secretion. However, sortilin could interact with the prodomain of other neurotrophins incapable of regulated secretion, making it less likely to be a specific mechanism for sorting. On the other hand, a sorting motif was recently identified in the mature domain of BDNF, but not nerve growth factor (NGF), that interacts with a well-known sorting receptor, carboxypeptidase E (CPE). Such an interaction was deemed essential for sorting proBDNF into regulated pathway vesicles for activity-dependent secretion. Given that the prodomain promotes proper folding of neurotrophins, it is conceivable that interaction between the prodomain and sortilin may hold proBDNF in a correct configuration, exposing the mature domain to the sorting receptor CPE, which sorts proBDNF into the regulated secretory pathway.
Roles of BDNF in Synaptic Plasticity The ability of the mammalian brain to adapt or modify itself in response to experience and/or environment depends on the plasticity of synaptic connections. Substantial evidence indicates that the number and strength of synapses is readily altered by neuronal activity. This process, known as synaptic plasticity, displays several physiological properties that substantiate its role as a cellular correlate for multiple cognitive processes, including learning and memory. These include the activity dependence and associative nature of induction as well as the input specificity of expression, all of which endow the vast storage and processing capacity of the mammalian brain. Remarkably, BDNF is involved in many of these features. An emerging theme is that BDNF plays a
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critical role in regulating several forms of synaptic plasticity in distinct regions of the brain, including the hippocampus and visual cortex. Input Specificity
Most activity-dependent forms of synaptic plasticity expressed in the brain are input specific, namely modifications at one synapse are not spread to synapses nearby. Experiments have shown that BDNF elicits its actions in a local and synapse-specific manner, with particular preference to active synapses. This unique property may be attributed to several key characteristics of BDNF signaling. First, secretion of BDNF is activity dependent and is likely to occur at synaptic sites. The control of BDNF secretion occurs relatively fast, usually in a timescale of seconds. Imaging studies demonstrate that BDNF is often co-localized with pre- and postsynaptic markers in hippocampal neurons, suggesting the synaptic localization of BDNF. Application of high-frequency stimulation (HFS) induces a rapid decay of GFP-tagged BDNF, indicating BDNF is secreted in an activity-dependent manner at synapses. Due to its negative charge, BDNF is thought to have a limited capacity for diffusion, and therefore constrains the actions of BDNF at or near its site of secretion. Second, there is good evidence that BDNF exon II and IV transcripts can be targeted into the dendrites of hippocampal neurons, and neuronal activity enhances such targeting. Recent studies have shown that dendritic BDNF mRNA can be translated locally into BDNF protein. Taken together, a possible scenario is that local synaptic activity triggers dendritic translation of BDNF and may serve as an alternative mechanism to ensure synapse-specific modulation by BDNF. Finally, local synaptic activity may ensure a better response of target synapses to BDNF by regulating TrkB trafficking. High-frequency neuronal activity has been shown to promote the insertion of TrkB into the surface membrane of hippocampal neurons. This process appears to be ligand independent and requires calcium influx and activation of Ca2þ/ calmodulin-dependent kinase II (CaMKII). BDNF secreted from active synapses/neurons recruits TrkB from extrasynaptic sites into lipid rafts, microdomains of membrane enriched at synapses. This lateral movement requires TrkB tyrosine kinase activity. Synaptic activity often induces a rise of postsynaptic cAMP. This local increase in cAMP concentration at active synapses facilitates translocation of TrkB into the postsynaptic density, and functions to gate synapse-specific effects of BDNF by controlling TrkB tyrosine phosphorylation locally. Finally, neuronal
activity promotes BDNF-induced TrkB endocytosis, a signaling event important for many long-term BDNF functions. All of these could contribute to a more efficient response to BDNF at active synapses. Early Phase Long-Term Potentiation
It is well established that BDNF plays a key role in LTP, a persistent enhancement of synaptic strength. LTP can be divided into an early phase (E-LTP) and a later phase (L-LTP). E-LTP is relatively short-lasting (1 h) and depends on protein phosphorylation, while L-LTP lasts many hours and requires new protein synthesis. Early work has focused on BDNF regulation of E-LTP in the hippocampus. In neonatal hippocampus in which BDNF levels are low, exogenous BDNF facilitates E-LTP induced by HFS (Figure 2(a)). In addition, exogenous BDNF facilitates LTP induced by subthreshold tetanus that normally induces weak potentiation. Conversely, in the adult hippocampus, a stage where endogenous levels of BDNF are relatively high, inhibition of BDNF activity either by functionblocking BDNF antibody or BDNF scavengers, TrkB immunoglobulin G (IgG), attenuates the expression of E-LTP. In agreement with pharmacological studies, genetically modified mice with mutation of either BDNF or TrkB gene exhibit severe impairments in E-LTP. Interestingly, heterozygous mice (BDNFþ/) with only half of the BDNF gene dosage show a similar degree of impairment as homozygous mice (BDNF/), arguing that a critical level of BDNF is important for hippocampal LTP. The impairment of LTP in BDNF-mutant mice is reversed by acute application of recombinant BDNF or by virus-mediated BDNF gene transfer. Deletion of BDNF gene selectively in the adult forebrain by inducible knockout approaches confirms that the effects of BDNF on E-LTP are not due to developmental abnormalities. The effects of BDNF on hippocampal E-LTP result primarily from alterations of presynaptic function (Figure 2(b)). Exogenous BDNF enhances synaptic response to HFS and paired pulse facilitation (PPF), two indicators of presynaptic function. In mice lacking BDNF, posttetanic potentiation (PTP) and PPF are significantly reduced. Electron microscopy reveals a reduction in the number of vesicles docked at presynaptic active zones in these mutant mice. Moreover, biochemical experiments using hippocampal synaptosomes indicate that BDNF modulates the levels or phosphorylation of synaptic proteins involved in vesicle docking and fusion, such as synapsin, synaptophysin, and synaptobrevin. Taken together, the presynaptic role of BDNF for the mobilization and/or docking of synaptic vesicles to presynaptic active
596 BDNF in Synaptic Plasticity and Memory
zones may allow hippocampal synapses to follow tetanic stimulation more effectively, resulting in the facilitation of hippocampal LTP. It is important to note that the biological effects of BDNF are not exclusively the result of its presynaptic actions. In the dentate gyrus, the induction of LTP requires postsynaptic BDNF signaling. A series of studies have demonstrated that BDNF can exert postsynaptic modulatory effects by modulating a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-type and N-methyl-D-aspartate (NMDA)-type glutamate receptors in neuronal cultures, and some potassium channels in hippocampal slices. In this respect, TrkB receptors have been observed to localize in the postsynaptic density of isolated synaptosomes prepared from cortical neurons. However, whether these postsynaptic modulatory effects of BDNF directly participate in LTP remains to be established. Late Phase Long-Term Potentiation
Several early studies suggest that BDNF may also play a role in late phase long-term potentiation (L-LTP). L-LTP-inducing tetanic stimulation selectively enhances the expression of BDNF and TrkB mRNAs in the hippocampus. BDNF transcription is regulated in part by CREB, a transcription factor required for L-LTP expression. The delayed and sustained enhancement of BDNF synthesis correlates well with the time course of L-LTP, which increases 2–4 h after L-LTP induction. More direct evidence comes from electrophysiology experiments showing a significant reduction in L-LTP recorded from slices treated with TrkBblocking antibody, or those from BDNF þ/ mice (Figure 2(c)). However, BDNF only regulates L-LTP induced by theta-burst stimulation (TBS) or application of the adenylate cyclase activator forskolin, but not by four spaced trains of HFS, a more standard protocol used to induce L-LTP. These results suggest that strong tetanic stimulation may induce signaling downstream of BDNF, bypassing the requirement of BDNF in L-LTP. Recent experiments have provided more in-depth insights as to the role of BDNF in L-LTP. In the presence of BDNF, weak tetanus that normally induces only E-LTP resulted in robust L-LTP. Moreover, perfusion of BDNF to hippocampal slices rescued L-LTP that is normally absent when protein synthesis is inhibited. It appears that BDNF is secreted largely in its precursor form (proBDNF), which is converted to mBDNF by extracellular proteases. Biochemical and genetic experiments indicate that this conversion is mediated by an enzymatic cascade that involves tPA and plasmin, two secreted
proteases found at hippocampal synapses. tPA has long been implicated in L-LTP, but the precise downstream effector(s) of tPA was not established. The current data support the notion that tPA cleaves the inactive zymogen plasminogen to form plasmin, which in turn cleaves proBDNF to generate mBDNF. Application of mBDNF, but not cleavageresistant proBDNF, completely reversed the L-LTP deficit observed in tPA and plasmin knockout mice. Thus, conversion of proBDNF to mBDNF by the tPA/ plasmin system is critical for L-LTP expression (Figure 2(d)). Two key cellular events associated with long-term synaptic plasticity are synaptic growth and de novo protein synthesis, both of which are regulated by BDNF. In addition to stimulating axonal growth, chronic application of BDNF to hippocampal slices increases the dendritic spine density of CA1 pyramidal neurons. This is particularly relevant since dendritic spines and protrusions are enhanced during L-LTP. In cell cultures, BDNF has been shown to increase mammalian target of rapamycin (mTOR)dependent translation of a panel of synaptically expressed transcripts including GluR1 and homer2 mRNAs in the dendrites of hippocampal neurons. However, exogenous BDNF applied immediately after strong TBS rescues L-LTP in hippocampal slices in which protein synthesis was blocked for the entire course of the experiments. This provocative result, together with the finding that gene expression of BDNF is stimulated by L-LTP-inducing tetanic stimulation, implies BDNF is a key protein synthesis product required for long-term modifications necessary for L-LTP expression. Long-Term Depression
In addition to its role in LTP, BDNF regulates longterm depression (LTD), a persistent reduction in synaptic strength induced by prolonged low-frequency stimulation. Expression of LTD is developmentally regulated and exists in several forms mediated by different glutamate receptors. The best-known form of LTD is the NMDA receptor (NMDAR)-dependent form, which is robustly expressed in young animals. mBDNF inhibits LTD in the visual cortex and hippocampus. Collectively, these observations point to a general theme that BDNF facilitates synaptic strengthening, but attenuates synaptic depression. Analogous to the survival and apoptosis effects of mBDNF and proBDNF, respectively, in the periphery, an important advance is that proBDNF, if uncleaved, enhances NMDAR-dependent LTD via p75NTR in the CNS. Compared to TrkB, the role of p75NTR in synaptic plasticity has not been studied until
BDNF in Synaptic Plasticity and Memory 597
recently. In p75NTR mutant mice (p75NTR/), NMDAR-dependent LTD was completely absent while other forms of plasticity including NMDARdependent LTP and NMDAR-independent LTD were intact. Biochemical experiments indicate that NR2B, a specific NMDAR subunit uniquely implicated in LTD, was significantly reduced in the mutant hippocampus. Whole-cell recordings revealed a severe reduction in NR2B-mediated synaptic currents in CA1 neurons of p75NTR/ mice. More importantly, application of cleavage-resistant proBDNF increased NR2B-mediated synaptic currents and enhanced LTD in hippocampal slices derived from wild-type mice but not in p75NTR/ mice (Figure 2(e)). These findings suggest that proBDNF is an endogenous ligand of p75NTR during development, which acts to enhance LTD via modulating NR2B function (Figure 2(f)). Learning, Memory, and Other Cognitive Functions
Given its central role in synaptic plasticity, numerous studies have examined how BDNF regulates the acquisition (learning) and retention (memory) of new information. Thus far, the strongest correlation is observed between BDNF and hippocampal-dependent forms of memory, which include declarative or episodic and spatial memory. During contextual learning, BDNF expression is rapidly and selectively upregulated in the hippocampus. When BDNF signaling is disrupted either by inhibitors or by genetic knockout, spatial learning is significantly impaired, as reflected by poor performance in the Morris water maze. In many cases
impairments in memory were also mirrored with LTP deficits. For instance, deletion of BDNF or TrkB gene in the adult forebrain results in a significant attenuation of contextual fear or spatial memories, as well as hippocampal LTP. A major advance came from a study on a SNP, which converts a valine (val) to a methionine (met) in the prodomain of the human BDNF gene. This SNP occurs with a frequency of approximately 19–25% in the Caucasian. Human subjects with the met allele exhibit lower hippocampal N-acetylaspartate (NAA), a putative measure of neuronal integrity and synaptic abundance. Functional imaging reveals an association of the met allele with abnormal hippocampal activation (Figure 3(a)). Most remarkably, subjects with the met-BDNF allele performed poorer in a hippocampal-dependent episodic memory task, but not in hippocampal-independent working memory and semantic memory tasks. In cultured neurons derived from rodent hippocampus, BDNF (valBDNF) is packaged in secretory granules that are distributed as puncta throughout cell body and dendrites, with some localized at synapses. In contrast, significantly less met-BDNF-containing granules are localized to dendrites and synapses. Moreover, regulated secretion of met-BDNF induced by neuronal depolarization, but not constitutive secretion, is significantly reduced. Thus, impairments in trafficking, synaptic targeting, and/or regulated secretion may explain the specific memory deficits seen in human subjects with the met allele (Figure 3(b)). These results represent the first demonstration of a role for
Val
R Pro Val-BDNF
R a
3.5 3.0 2.5 2.0 1.5 1.0 0.5 0
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R Impairments in:
b
− dendritic trafficking − synaptic targeting − regulated secretion
Figure 3 Impact of a SNP in the BDNF gene on cognitive brain function and intracellular trafficking of BDNF. The SNP converts a valine to a methionine in amino acid 66 located in the prodomain of BDNF (val66met). (a) Differences in fMRI responses between val/val and val/ met subjects during a memory task. Subjects with val/met genotype exhibit abnormal hippocampal activation (shown in red). The met/met subjects also exhibit deficits in hippocampus-dependent episodic memory. (b) Cellular phenotypes associated with the BDNF val66met SNP. Val-BDNF is distributed throughout a typical hippocampal neuron including distal dendrites and synapses. In contrast, met-BDNF is rarely localized at distal dendrites or synapses and fails to undergo depolarization-induced secretion. Failure of intracellular trafficking and activity-dependent secretion of BDNF may underlie the cognitive deficits observed in subjects with the met allele. (a) Reproduced from Egan MF, Kojima M, Callicott JH, et al. (2003) The BDNF val66met polymorphism affects activity-dependent secretion of BDNF and human memory and hippocampal function. Cell 112: 257–269, with permission from Elsevier.
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BDNF in human hippocampal function and of a single gene affecting human episodic memory. Genetic analyses in mice show that genes affecting memory performance often impact other cognitive functions. It is now recognized that deficits in BDNF may contribute to neurological and psychiatric disorders. In studies of drug addiction, LTP and LTD have emerged as candidate mechanisms for drug-induced alterations in the nucleus accumbens and ventral tegmental area. Several reports have demonstrated that BDNF modulates behavioral sensitization to cocaine. Substantial evidence also points to its role in depression. There is reduced BDNF expression in the hippocampus of animal models for depression; chronic treatment with antidepressants increases its levels. However, it is unclear whether antidepressants achieve their clinical effects on depression by upregulation of BDNF.
Conclusion Stemming from the multidisciplinary approaches used in present-day research ranging from cellular systems to behavior, BDNF is now recognized as a key regulator for synaptic circuits underlying many cognitive functions. New and unidentified role(s) of BDNF in plasticity and cognition will undoubtedly continue to surface and will provide abundant intellectual stimulation to drive future advances. Ultimately, understanding the actions of BDNF will aid in the development of therapeutic interventions that will alleviate a wide spectrum of neurological and psychiatric disorders derived from BDNF dysfunction. See also: Long-Term Depression (LTD): Metabotropic Glutamate Receptor (mGluR) and NMDAR-Dependent Forms; Long-Term Potentiation (LTP): NMDA Receptor Role.
Further Reading Barker PA (2004) p75NTR is positively promiscuous: Novel partners and new insights. Neuron 42: 529–533. Chao MV (2003) Neurotrophins and their receptors: A convergence point for many signaling pathways. Nature Reviews Neuroscience 4: 299–309. Chen ZY, Ieraci A, Teng H, et al. (2005) Sortilin controls intracellular sorting of brain-derived neurotrophic factor to the regulated secretory pathway. Journal of Neuroscience 25: 6156–6166. Egan MF, Kojima M, Callicott JH, et al. (2003) The BDNF val66met polymorphism affects activity-dependent secretion of BDNF and human memory and hippocampal function. Cell 112: 257–269. Figurov A, Pozzo-Miller L, Olafsson P, Wang T, and Lu B (1996) Regulation of synaptic responses to high-frequency stimulation and LTP by neurotrophins in the hippocampus. Nature 381: 706–709. Lee R, Kermani P, Teng KK, and Hempstead BL (2001) Regulation of cell survival by secreted proneurotrophins. Science 294: 1945–1948. Lou H, Kim SK, Zaitsev E, et al. (2005) Sorting and activitydependent secretion of BDNF require interaction of a specific motif with the sorting receptor carboxypeptidase E. Neuron 45: 245–255. Lu B (2003) BDNF and activity-dependent synaptic modulation. Learning and Memory 10: 86–98. Lu B, Pang PT, and Woo NH (2005) The yin and yang of neurotrophin action. Nature Reviews Neuroscience 6: 603–614. Nagappan G and Lu B (2005) Activity-dependent modulation of the BDNF receptor TrkB: Mechanisms and implications. Trends in Neuroscience 28: 464–471. Pang PT, Teng HK, Zaitsev E, et al. (2004) Cleavage of proBDNF by tPA/plasmin is essential for long-term hippocampal plasticity. Science 306: 487–491. Patterson SL, Abel T, Deuel TA, et al. (1996) Recombinant BDNF rescues deficits in basal synaptic transmission and hippocampal LTP in BDNF knockout mice. Neuron 16: 1137–1145. Poo MM (2001) Neurotrophins as synaptic modulators. Nature Reviews Neuroscience 2: 24–32. Sun YE and Wu H (2006) The ups and downs of BDNF in Rett syndrome. Neuron 49: 321–323. Woo NH, Teng HK, Siao CJ, et al. (2005) Activation of p75NTR by proBDNF facilitates hippocampal long-term depression. Nature Neuroscience 8: 1069–1077.
GFL Neurotrophic Factors: Physiology and Pharmacology M Saarma, University of Helsinki, Helsinki, Finland ã 2009 Elsevier Ltd. All rights reserved.
Introduction Growth factors are secretory proteins that bind to their cognate receptors at the plasma membrane of the cells and activate receptors that in turn trigger the activation of cellular biochemical signaling processes and lead to the cell proliferation, differentiation, migration, morphological changes, or induce the death of cells. Currently, about 100 growth factors that affect neurons have been described, but only those that mainly regulate the development and physiology of neurons are called neurotrophic factors. Neurotrophic factors are growth factors that regulate the number of neurons in a given population, neurite branching and synaptogenesis, adult synaptic plasticity, and maturation of neuronal phenotype. Neurotrophic factors include, for example, neurotrophins (NGF, BDNF, NT-3, NT-4), neurokines (CNTF, LIF, IL-6, CT-1, etc.), and glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs).
GFLs: Structure, Secretion, and Processing GDNF and the related factors artemin (ARTN), neurturin (NRTN), and persephin (PSPN) form the GDNF family of neurotrophic factors (Figure 1(a)). GFLs are synthesized by the cells as preproGFL proteins that are in the endoplasmic reticulum (ER) processed to proGFLs and secreted into the extracelluar space as mature GFLs or as proGFLs. The 211 amino-acidlong preproGDNF protein is finally processed into 134 amino acid mature GDNF monomer (Figure 1(b)) that is inactive and must homodimerize to generate a biologically active factor. All GFLs have seven conserved cysteines with similar relative spacing. Six of these cysteines are involved in the intramolecular S–S bonding whereas the seventh cysteine forms an intermolecular S–S bond stabilizing the homodimers of GFLs. Based on the conserved location of the seven cysteines, GFLs form a distant subfamily in the large transforming growth factor-beta (TGF-b) superfamily of growth factors. Orthologs of all four GFLs are found in all mammals, and also in teleost fishes. Interestingly, chicken genome lacks PSPN gene, but has a functional PSPN coreceptor GDNF family receptor a4 (GFRa4). Clawed frog Xenopus tropicalis genome surprisingly lacks the ortholog of NRTN.
Even more surprisingly insects have a receptor tyrosine kinase (RTK) rearranged during transfection (RET), a putative GFL coreceptor homolog but no obvious GFL homologs. The atomic structure of the major part of GDNF has been determined and GDNF together with all other TGF-b superfamily growth factors, including all GFLs, belong to the cystineknot family of proteins. The main structural elements of the GDNF molecule are helical regions and two fingers. Fingers 1 and 2 are involved in the binding of GDNF to its receptor GFRa1 (Figure 1(c)). In the GDNF structure the first 38 residues are disordered. Sequence analysis and experimental data indicate that this region binds heparin stabilizing the structure of GDNF and mediating GFL interactions with extracellular matrix (ECM). Recently, two laboratories have solved the crystal structure of ARTN. Although the overall fold of the ARTN covalent homodimer is similar to GDNF, the shape and the flexibility of elongated homodimer differs. ARTN also differs from GDNF in its overall charge and electrostatic distribution. Arginines in ARTN at positions 48, 49, and 51, as well as its amino terminus play a role in heparin binding. Although the three-dimensional (3-D) structures of NRTN and PSPN are not known, it is likely that their general spatial structure is very similar to GDNF and ARTN (Figure 1(c)). There are also heparin-binding regions in NRTN, but not in PSPN. The detailed mechanism of the secretion, processing, and activation of GFLs is poorly studied. Several cells and tissues can also secrete proGDNF in addition to mature GDNF (Figure 1(b)), suggesting that processing of proGDNF (and possibly all GFLs) can occur also in the ECM. Whether proGFLs can bind GFL receptors and trigger cellular signaling is currently not known.
GFL Receptors GFL receptor system is unique in several ways. First, GFLs can signal through three different receptor systems. Second, all three signaling receptors, RET, neural cell adhesion molecule (NCAM), and syndecans, are shared by four ligands, with the exception of PSPN that is not binding to syndecans. Third, the main GFL signaling receptor is the RTK RET, which is the only known RTK that does not bind the ligands directly but via coreceptors of GFRa family. Finally, RET activity is strictly regulated by Ca2þ ions, and in the intracellular part of RET in addition to tyrosine residues serines are also phosphorylated. The RET gene was identified in 1985 as a novel oncogene. Normal RET is the transmembrane RTK.
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Figure 1 (a) Schematic structure of GFL showing the relative lengths (and number of amino acids) of the mature GDNF (blue), NRTN (pink), ARTN (brown), and PSPN (yellow) molecules and their pre-(brown) and pro-(light green) domains, as well as relative positions of the seven conserved cysteine residues (black vertical lines). GDNF has two putative N-glycosylation sites (N). (b) The preregion of GFLs is cleaved off inside the cells during secretion. Cells can secrete mature GDNF and proGDNF, indicating that the processing of proGDNF can also occur in the ECM. (c) The 3-D structure of dimeric GDNF shows that it belongs to the cystine-knot family of proteins. The main structural elements of the GDNF molecule are helical regions and fingers 1 and 2 that are involved in its binding to GFRa1 coreceptor. The 3-D structure of ARTN is similar to that of GDNF. The 3-D structures of NRTN and PSPN are not known, but the modeling of NRTN shows that its spatial structure is very similar to GDNF. The binding of heparan sulfate to GDNF is shown (left). NRTN can also bind heparan sulfate. Putative binding site is shown (green).
In human cancer, especially in papillary thyroid carcinoma, the RET tyrosine kinase domain is fused with N-terminal regions of the variety of unrelated genes. In addition to these somatic rearrangements germ line mutations in RET are responsible for two inherited disorders: activating mutations cause multiple endocrine neoplasia 2 (MEN2) and inactivating mutations Hirschsprung’s disease (HSCR) or congenital central hypoventilation syndrome. The alternative splicing of the human RET gene gives rise to at least three splice isoforms. The larger of these encodes a protein of 1114 amino acids that contains 51 amino acids at the C-terminus (RET51) that are replaced by nine amino acids in the RET9 isoforms. RET51 has two additional tyrosines: Tyr1090 and 1096. Orthologs of RET have been identified in higher and lower vertebrates, as well as in Drosophila (D-RET).
The tyrosine kinase domain of D-RET is conserved as it is functional in vitro and activates similar intracellular signaling pathways as mammalian RET. The extracellular domain of D-RET is less conserved and fails to interact with mammalian GDNF/GFRa1 complex. Interestingly, the Drosophila genome does not encode putative GFLs, but has a GFRa-like gene, called munin. RET is the only RTK among the receptors of TGF-b superfamily factors, all others being the receptor serine–threonine kinases. Although RET is similar to other RTKs, it also has several special features. First, the extracellular domain of RET consists of four cadherin-like domains (CLD1–4) and a single cysteine-rich domain (CRD). RET binds one molecule of Ca2þ that is required for its correct folding and function as the GFL receptor. Second, RET is the only
GFL Neurotrophic Factors: Physiology and Pharmacology 601 RET CLD1 GDNF
NRTN
ARTN
PSPN
CLD2 Ca2+ CLD3 CYSrich domains GFRa1
CLD4
GFRa2
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Figure 2 GDNF-family ligands and receptor interactions. Homodimeric GFLs activate RET RTK by first binding to their cognate GFRa coreceptors. GDNF binds preferentially to GFRa1, NRTN to GFRa2, ARTN to GFRa3, and PSPN to GFRa4. Solid arrows indicate the preferred functional ligand–coreceptor interactions, whereas dotted arrows indicate putative cross-talk. GFRa proteins are attached to the plasma membrane through a GPI-anchor and consist of three (GFRa4 has only two) globular cysteine-rich domains joined together by adapter sequences. The extracellular domain of RET, that consists of CLD1–4 and one CRD interacts with all four GFL–GFRa complexes. Binding of Ca2þ ions to one CLD of RET is required for its activation by GFLs. RET intracellular part contains tyrosine kinase domain and tyrosine residues that become phosphorylated upon RET activation.
RTK that does not directly bind the ligands, that is, GFLs (Figure 2). Instead, RET is activated by binding to a complex formed by a GFL bound to its cognate glycosylphosphatidylinositol (GPI)-anchored coreceptor GFRa. Thus, GFLs interact with RET, but only in the presence of specific GFRa coreceptors. Four different GFL-GFRa pairs exist in mammals: GDNF-GFRa1, NRTN-GFRa2, ARTN-GFRa3, and PSPN-GFRa4 (Figure 2). With significantly lower-affinity GDNF can interact with GFRa2 and GFRa3, and NRTN and ARTN with GFRa1, but the physiological significance of these interactions is unclear. The GFL and GFRa homodimeric complex brings two RET molecules together, triggering transphosphorylation of specific tyrosine residues in their tyrosine kinase domains and activating intracellular signaling (Figure 3). GFRa Coreceptors
The GFL binding specificity to RET is determined by GFRa proteins that have unique binding affinities for each GFL. GFRas are cysteine-rich GPI-anchored proteins. Mammalian GFRa1–3 and most of the vertebrate GFRa consist of three CRDs, whereas
mammalian PSPN receptor GFRa4 lacks CRD1 and thus has only two CRDs (Figure 2). The structure of GFRa1 domain 3 revealed a novel protein fold: an all-a five-helix bundle stabilized by five disulfide bridges. The model for domain 2 was constructed, using its homology to domain 3. Recently, the crystal structure of ARTN in the complex with domains 2 and 3 of the GFRa3 was reported. These data reveal crucial amino acids in domain 2 of the GFRa3 receptor involved in ARTN binding and indicate that domains 2 and 3 define the potential RET binding site. Structural data on the ARTN-GFRa3 complex, on GFRa1 domain 3, and the conserved location of cysteine residues in GFRa1–4 CRDs predict a very similar general structural fold for all GFRas. Phylogenetic analysis indicates that all vertebrate classes from teleost fishes to mammals have orthologs for all four GFRa receptors. For several neuronal populations the full biological activity of GDNF is exerted only in the presence of TGF-b. It appears that TGF-b is required for the action of GDNF via GFRa1, but not for the action of NRTN, as TGF-b is increasing the surface location of GFRa1 but not of GFRa2.
602 GFL Neurotrophic Factors: Physiology and Pharmacology
a
b
c
d
Figure 3 GDNF interacts with coreceptor GFRa1 and activates RET. (a) Biologically active GDNF is a homodimer. In the absence of GDNF, GFRa1 and RET interaction is very weak. (b) A dimer of GDNF binds to the second CRD of GFRa1. (c) GDNF brings together two molecules of GFRa1. (d) GDNF-GFRa1 complex induces dimerization of two molecules of RET leading to transphosphorylation and activation of their tyrosine kinase domains.
Two proteins that are structurally similar to GFRa proteins – GAS1 and GFRAL – are described, but their relatedness to the GFL system is not yet clear. NCAM
NCAM plays an important role in neural development as the homophilic cell adhesion molecule. P140NCAM can also function as the alternative signaling receptor for all four GDNF family ligands. GFLs can directly bind to NCAM with low affinity, but do not trigger intracellular signaling. In the presence of cognate GFRa coreceptors GFLs can bind to p140NCAM with higher affinity and activate Src-like kinase Fyn and focal adhesion kinase FAK (Figure 4(a)). GFRa1 interaction with NCAM even in the absence of GDNF can inhibit NCAM-mediated cell adhesion. Thus GDNF–GFRa1– NCAM and GFRa1–NCAM interactions may have different biological readouts and physiological consequences. Current experiments indicate that GDNF by binding to GFRa1 and activating NCAM stimulates Schwann cell migration, hippocampal neurite outgrowth, and development of olfactory neurons in RET-independent manner. Further experiments are needed to unravel the in vivo roles of GDNF–GFRa1– NCAM and GFRa1–NCAM signaling and possible cross-talk of RET and NCAM in GFL signaling. Syndecans
Although the vast majority of GFL signaling utilizes GFRa/RET or GFRa/NCAM receptor systems, novel data indicate that additional GFL receptors exist. GDNF was originally purified by heparin-affinity chromatography, and later its interaction with heparan sulfates was documented. Recent experimental data indicate that heparan sulfates are required for GDNF signaling via GFRa1 and RET. GDNF,
ARTN, and NRTN, but not PSPN, can with high affinity bind to heparan sulfate proteoglycan (HSPG) syndecans (Figure 4(b)). Syndecans that bind GDNF can function as coreceptors and deliver GDNF to GFRa1/RET complex or activate directly Src signaling pathway triggering cell spreading and inducing neurite outgrowth of hippocampal neurons. GDNF interaction with syndecan also stimulates the migration of cortical neurons. Thus, GDNF can signal in two different ways – as a soluble protein and as a matrix-bound protein. In a soluble form it binds to GFRa1 and activates intracellular signaling via RET or NCAM. When bound to ECM (both transmembrane and soluble HSPGs), GDNF triggers qualitatively different signaling that is currently poorly defined.
Signaling Pathways The interactions of the intracellular part of RET with various signaling molecules are mainly triggered by the cognate ligands (GFLs), but in some cases also GFL-independently. GFL–GFRa homodimeric complex brings two molecules of RET together, triggering transphosphorylation of specific tyrosine residues of RET, followed by the activation of intracellular signaling cascades (Figures 3 and 5). Several cascades can be activated, which regulate cell survival, differentiation, proliferation, migration, chemotaxis, branching morphogenesis, neurite outgrowth, synaptic plasticity, etc. The mitogen activated protein (MAP) kinase pathway is involved in neurite outgrowth of the neurons. The phosphoinositide 3-kinase (PI3K)-Akt pathway is responsible for both neuronal survival and neurite outgrowth. GFL signaling also activates Src-family kinases, which elicit, for example, neurite outgrowth, neuronal survival, and kidney ureteric branching. In most cases tyrosine residues
GFL Neurotrophic Factors: Physiology and Pharmacology 603 NCAM
NCAM
IgG-like domain
GFRa1
Fibronectinlike domain
GDNF
FYN
FYN Signaling
a Syndecan
Signaling b Figure 4 RET-independent signaling by GFLs. (a) p140NCAM is an alternative signaling receptor for GFLs. It interacts with a GDNF– GFRa1 dimer leading to the activation of Fyn, a Src-like kinase. It is unclear, whether GDNF triggers NCAM dimerization. NCAM has five IgG-like domains and two fibronectin-like domains. (b) HSPG syndecans carry heparan sulfate side chains that bind with high affinity GDNF, ARTN, and NRTN. Unlike GFRa1, syndecans can bind many GFL molecules simultaneously. GDNF binding to syndecans can activate Src-family kinases or modulate signaling through GFRa-RET.
Tyr905, Tyr981, Tyr1015, Tyr1062, and Tyr1096 of RET are phosphorylated and docking proteins and intracellular proteins are bound and activated through interaction with these and possibly other RET phosphotyrosines (Figure 5). Interestingly, RET activation affects different downstream targets inside and outside lipid rafts that are the dynamic assemblies of cholesterol and sphingolipids scattered within the disordered phase of the lipid bilayer. Differences in GDNF
signaling through RET within and outside the rafts could lead to significantly different cellular responses. RET is also activated by several GFL-independent pathways. First, increase in the intracellular concentration of cyclic AMP level triggers protein kinase A (PKA)-dependent Ser696 phosphorylation of RET independently of GFL that regulates the migration of enteric neural crest cells in the developing gut. Second, in the mature sympathetic neurons, RET51
604 GFL Neurotrophic Factors: Physiology and Pharmacology
RET intracellular domain
S696 Y752
JNK
STAT3 Grb7/10
Y905
Src
Y981
Grb2
Y928 Y1015
PLCg
PKC
Y1062
Shc FRS2
Y1096 Y1096
AKT ERK
Rac
JNK
Grb2 Gab1
P13K
AKT
Grb2 SOS
RAS
ERK
Nck JNK RasGAP ERK Dok4/5/6 IRS1/2 Enigma p38MAPK ERK5
Dok1
Figure 5 GDNF-induced signaling pathways. The model shows the intracellular domain of RET and highlights the phosphorylated tyrosines and different intracellular RET-binding proteins and activated downstream signaling pathways. The phosphorylated tyrosine residues (Y752, Y905, Y928, Y981, Y1015, Y1062, Y1096) activate multiple signaling pathways. Serine 696 (orange) is phosphorylated GFL-independently by PKA, and Y1096 (orange) can be phosphorylated GFL-independently by NGF.
isoform becomes phosphorylated and functionally activated by nerve growth factor that signals via unrelated receptor TrkA. The physiological importance of this signaling is not yet known. Third, there is in vitro evidence that RET is also a dependence receptor that triggers apoptotic cell death when not bound by the ligands. However, the in vivo relevance of this RET-triggered apoptosis is completely unclear.
Biology and Physiology Mice that lack GDNF, GFRa1, or RET die soon after birth, whereas mice lacking other GFLs or GFRa coreceptors or alternative receptors NCAM or syndecans are viable and fertile. The neuronal phenotypes of the different GFLs and their receptor-knockout mice are summarized in Table 1. The strongly overlapping phenotypes of ligand and coreceptor knockouts demonstrate, under physiological conditions, a specific pairing of each GFL and corresponding GFRas. The vast majority of cells and tissues that are affected in GFL- or GFRa-knockout mice also express RET, indicating that in vivo this is the main signaling receptor for GFLs. GDNF-, GFRa1-,
or RET-deficient mice share a phenotype of kidney agenesis, and an absence of many parasympathetic and enteric neurons. Mice that lack NRTN or GFRa2 have similar deficits in enteric and parasympathetic innervation. Characterization of ARTN- and GFRa3-deficient mice revealed similar abnormalities in the migration and axonal projection pattern of the entire sympathetic nervous system. Ablation of GFRa4 or its ligand PSPN impairs thyroid calcitonin production in young mice. Both GDNF- and NCAM-deficient mice have impaired migration of the rostral migratory stream-derived neuronal precursors. Likewise, GDNF- and syndecan-deficient mice have an impaired migration of GABAergic cortical neurons. In some cases the ligand- and its receptor-deficient mice phenotypes differ. Mice lacking the long RET51 isoform seem to be normal, whereas mice lacking the short RET9 isoform were similar to mice lacking all RET isoforms. Only the short isoform can rescue the phenotype of the RET-null mutation in the kidney and enteric nervous system. In another study, however, homozygous RET9 and RET51 mice were viable and show normally developed kidneys. Knock-in mice with mutated Tyr1062 of RET had defects in the enteric nervous system similar to those
GFL Neurotrophic Factors: Physiology and Pharmacology 605 Table 1 Phenotypes of mice lacking GFLs and their receptors Gene knockout
RET
GDNF/GFRa1
NRTN/GFRa2
ARTN/GFRa3
PSPN/GFRa4
Gross phenotype
P0 lethal
P0 lethal
Viable, fertile. Pseudoptosis growth retardationb
Viable, fertile ptosis
Viable, fertile Gfra4/
Breathing defect
<40%, breathing defect Soma reduced
PNS Sensory: PG
ND
DRG
TG Autonomic Sympathetic
SMG
Enteric neurons
CNS Spinal motorneurons Brain
Other tissues
NS
NS
SCG and other ganglia; defect in migration and axon growth
Loss in whisker follicles SCG; migration defect
SCG; <35% in Gdnf/mice NS in Gfra1/ mice
Sympathetic cholinergic Parasympathetic Cranial: SPG OG
Soma size reduced, loss of heat sensitivity
Soma size, target innervation reduced Lack of ganglia Lack of ganglia
Lack of ganglia Lack of ganglia
Reduced number and soma size No neurons in bowel below stomach
Reduced number and soma size
Loss in various nuclei Substantia nigra dopamine neurons; NS No kidneys, moderate thyroid C-cell loss
No neurons in bowel below stomach
NS 40% reduced no. of neurons 42% reduced no. of neurons Moderate loss of fibers in small intestine
22–31% loss of neurons Impaired learning, reduced locomotor activitya No kidneys, testis degeneration in adult Gdnf+/ mice
No gross defects Impaired behavioral flexibility and memoryb ND
NS
NS
No gross defects
No gross defects
No gross defects
Pspn/: hypersensitive to cerebral ischemia
ND
Gfra4/ mice; calcitonin levels reduced in young mice
a
Gdfnþ/. Gfra2/ mice. NS, not significantly different from wild-type; ND, not determined; PG, petrosal ganglion; DRG, dorsal root ganglion; TG, trigeminal ganglion; SCG, superior cervical ganglion; SPG, sphenopalatine ganglion; OG, otic ganglion; SMG, submandibular ganglion. Adapted from Airaksinen MS and Saarma M (2002) The GDNF family: Signaling, biological functions and therapeutic value. Nature Reviews Neuroscience 3: 383–394.
b
observed in RET-deficient mice but with only slight histological changes in the kidneys. Dopamine neurons. GDNF and NRTN are potent survival factors for midbrain dopamine (DA) neurons and they signal through the GFRa1–RET receptor complex, which is expressed in developing and mature DA neurons. GDNF, but not NRTN, can also promote axon growth and sprouting of DA neurons. Analysis of GDNF-, NRTN-, GFRa1-, and RET-deficient mice reveals that GDNF signaling through GFRa1/RET is
not required for the embryonic development of DA neurons. However, DA neurons of the substantia nigra undergo programmed cell death (PCD) during postnatal development. This is a biphasic process with an initial major peak just after birth and a second minor peak 2 weeks after birth. Intrastriatal injection of GDNF diminishes the PCD event and GDNF-neutralizing antibodies augment it. Thus, GDNF seems to serve as a physiological targetderived neurotrophic factor for DA neurons during
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the first phase of PCD. This is also supported by the observation that GDNF is expressed prominently in the striatum during the first few postnatal weeks, when DA neuron target innervation is taking place. Results from mice, where RET or GFRa1 will be deleted from DA neurons, should demonstrate whether RET signaling is required for postnatal survival and maintenance of DA neurons. Motor neurons. In vitro, GDNF is the most potent survival factor for motor neurons (MNs). In GDNFand GFRa1-deficient mouse embryos, there is an increased (20–40%) loss of spinal and cranial MNs. RET-deficient mice also show significant losses in all motor neuron populations examined. Motor neuron survival is usually supported by synergistically acting trophic factors that are produced by muscle and glial cells. During development, GDNF is produced primarily by Schwann cells and by astrocytes throughout the nervous system. Muscle-specific expression of GDNF transgene or GDNF treatment in utero enhances survival of the MNs. In mice overexpressing GDNF under the control of an astrocytespecific GFAP promoter, the survival of MNs in brachial, lumbar, thoracic spinal cord, and the abducens nucleus was increased. GDNF also prevented MN PCD in several cranial motor nuclei. Furthermore, GDNF could support complete and relatively longterm survival of MNs following neonatal facial-nerve axotomy. Although exogenous NRTN supports motor neuron survival, this is mostly mediated by GFRa1, as no clear loss of MNs is found in NRTN- and GFRa2deficient mice. Continued subcutaneous injections of GDNF postnatally, or transgenic overexpression of GDNF in skeletal muscle, produce hyperinnervation of neuromuscular junctions. GDNF seems to induce this extra innervation by promoting terminal branching. Therefore, the main role of GDNF in postnatal MNs is to promote terminal axon branching and synapse formation. Parasympathetic neurons. In the cranial region, postganglionic parasympathetic neurons are located in small ganglia: ciliary, sphenopalatine, submandibular, and otic ganglia. In GDNF-, RET-, and GFRa1-knockout mice, the otic and sphenopalatine ganglia are absent, demonstrating that GDNF signaling is critically required for the development of these parasympathetic neurons. In addition, GDNF signaling through GFRa1/RET is required for the migration and proliferation of parasympathetic neuronal precursors during early embryonic development. Postnatally, NRTN is upregulated in target tissues and parasympathetic neurons continue to express RET and GFRa2. In line with that, NRTN- or GFRa2-deficient mice have deficits in parasympathetic target innervation in many regions. Taken together,
there is a shift from GDNF to NRTN signaling in developing parasympathetic neurons, where both growth factors have distinct roles. Enteric neurons. In mice that lack RET, GDNF, or GFRa1 enteric neurons and glial cells that are derived from the vagal and sacral neural crest are missing below the stomach. GDNF promotes the migration, proliferation, survival, and differentiation of multipotent enteric precursor cells that express cognate receptors. NRTN or GFRa2 mutant mice show a significant loss of the cholinergic, substance P-containing fine myenteric nerve plexus in the small intestine, and very little loss of enteric neurons. As the release of substance P in vitro from the NRTN-deficient mouse colon is reduced significantly, NRTN signaling could regulate neurotransmitter release. The function of GDNF and NRTN in the adult enteric nervous system is unknown. Sympathetic neuron development requires chemoattractant ARTN signaling through GFRa3/RET for migration and initial axon outgrowth. The role of GFLs in the postnatal sympathetic system is poorly studied. GDNF, NRTN, and ARTN in vitro support subpopulations of primary sensory neurons. Although GFLs can be survival-promoting factors for some sensory neurons, their specific roles for these neurons are poorly understood.
Kidney Development and Spermatogenesis Although GDNF is mainly known as the neurotrophic factor, it also plays a crucial role in kidney development and spermatogenesis. In mice in which GDNF, RET, or GFRa1 is knocked out, the kidneys show aplasia or severe hypodysplasia. GDNF is the major mesenchyme-derived factor that activates GFRa1/RET complex in the ureteric bud, thereby regulating ureteric budding and branching during nephrogenesis. Mutation of Tyr1015 in both RET51 and RET9 isoforms results in severe renal anomalies. In addition, loss of RET9 (Y1062)-mediated Akt/ MAPK activation resulted in renal agenesis or kidney rudiments, whereas mutation of this residue in RET51 had no obvious effect on AKT/MAPK activity and renal development. However, the precise role of GDNF/ RET signaling in renal branching morphogenesis and the specific responses of ureteric bud cells to GDNF remain unclear. The localized expression of GDNF in the metanephric mesenchyme, together with several types of negative regulation, is important to elicit and correctly position the initial budding event from the Wolffian duct. GDNF also promotes the continued branching of the ureteric
GFL Neurotrophic Factors: Physiology and Pharmacology 607
bud. However, it seems not to provide the positional information required to specify the pattern of ureteric bud growth and branching, as its site of synthesis can be drastically altered with minimal effects on kidney development. In the testis, GDNF is expressed by the Sertoli cells that regulate spermatogenesis in a paracrine manner, whereas RET/GFRa1 are found in the undifferentiated spermatogonia, including spermatogenic stem cells. Mice lacking one GDNF allele show partial depletion of spermatogenic stem cells, whereas mice overexpressing GDNF show clusters of undifferentiated spermatogonia that develop seminoma-like germ line tumors in adulthood. GDNF is thus a crucial paracrine regulator of the self-renewal and differentiation of spermatogonial stem cells. Actually, GDNF is the first factor known to determine the fate of spermatogenic stem cells. GDNF signaling is therefore an attractive target for the development of male contraceptives. However, such efforts are severely shadowed by the carcinogenic consequences of deregulated RET activation. NRTN, GFRa2, and NCAM are also expressed in the developing kidney and testis, but they have no renal or testis phenotype when knocked out. The function of GDNF and its receptors in kidney and testis should be very seriously considered when RET/GFRa1 agonists or antagonists to treat cancer or neurodegenerative diseases are developed.
Clinical Potential GFLs have received attention as potential therapeutic agents for the treatment of certain neurological diseases, in particular the Parkinson’s disease (PD). All current therapies for PD are symptomatic and do not slow down or reverse the underlying degeneration of midbrain DA neurons. GDNF has been an attractive treatment option since its discovery, because it is a naturally occurring protein that in vivo can protect and rescue nigrostriatal DA neurons that degenerate in PD. In various animal models of PD, GDNF can prevent the neurotoxin-induced death of DA neurons and can promote functional recovery. In the first clinical trial GDNF that was delivered into the lateral ventricles of patients with PD was without therapeutic efficacy and caused side effects, including weight loss. Later trials tested delivery of the GDNF into the site of the major DA loss in PD, namely in the caudate putamen. In a small open-label study with five patients clear positive clinical effects were found. In contrast, a more recent double-blind, placebo-controlled trial on 34 patients has shown no such effects. In this study patients with moderately advanced PD were randomly assigned intraputamenal GDNF or placebo. In addition, anti-GDNF antibodies were
reported in three patients out of 34. After several failures a question arises whether there is a future for GDNF in the treatment of PD. The ability to promote endogenous repair through the use of target-derived growth factor is still very attractive. This is further supported by recent improvements in GDNF delivery in animal models of PD that include: heparin coadministration, which increases GFL spread; novel viral vectors; and engrafted- or encapsulated-GDNF-producing neuronal cells, which maintain high levels of human GDNF production. Combined with safety measures, including means to control gene expression, these new vehicles for GDNF delivery look promising for PD therapy. GDNF is a survival factor for MNs and therefore it may have clinical importance for the treatment of amyotropic lateral sclerosis. In addition, GDNF can be useful in the treatment of various neurological traumas, as it supports the survival and axon regeneration of both MNs and sensory neurons. The combination of different GFLs might be the most efficient to promote functional regeneration of the sensory axons. Peripheral nerve injury often leads to chronic neuropathic pain. ARTN and GDNF have shown their effect in animal experiments and can be considered as drug targets for pain treatment. In experimental models of ischemia, exogenous GDNF administered before or just after anoxia can reduce ischemic brain injury. PSPN-deficient mice show normal development and behavior, but are hypersensitive to cerebral ischemia. More recent results highlight the importance of GDNF as a new target for drug addiction. In addition to GDNF, NRTN also has promise for PD therapy and for epilepsy, and GDNF and NRTN might modulate seizure susceptibility.
Small Molecules All known growth factors that activate RTKs bind directly to the receptor and trigger RTK dimerization. The only exception is GFLs that first bind to the ligandspecific GFRa-coreceptor that together with the ligand trigger RTK RET dimerization and activation. Unique mechanism of RET activation by GFLs also offers unique opportunities for rational drug design. Small number of GFRa receptors compared to large number of homologous RTKs predict less stringent specificity requirements for the designed drugs. Using highthroughput screening system XIB4035, a novel nonpeptidyl small molecule agonist for GFRa1 was described. This compound at 12 mM concentration induced RET phosphorylation and neurite outgrowth in Neuro 2A cells. Although the in vivo effects of XIB4035 are still unknown, the fact that a nonpeptidyl small molecule with high affinity binds to GFRa1 and
608 GFL Neurotrophic Factors: Physiology and Pharmacology
activates RET is remarkable and very encouraging. New information on the structure of GFL receptors and their complexes with GFLs is helpful to improve the specificity and efficacy of small molecules that will be beneficial for the treatment of PD.
Summary There are four members of the GDNF ligand family (GFLs): GDNF, NRTN, ARTN, and PSPN. These small secreted dimeric proteins, distantly related to TGF-b, promote the survival of central and peripheral neurons. GFL binds and activates the transmembrane RTK RET via specific GDNF family coreceptors a (GFRa). GDNF binds preferentially to GFRa1, NRTN to GFRa2, ARTN to GFRa3, and PSPN to GFRa4. Upon activation, RET dimerizes, transphosphorylates its tyrosine residues, and triggers activation of signaling cascades leading to survival, growth, or differentiation of cells. GDNF protects DA and MNs in vivo, making it a candidate for therapeutic use in several neurodegenerative diseases. Knockout mice revealed the function of GFLs and their receptors. GDNF / þ/ mice lack enteric neurons, as in human HSCR. GDNFand its receptor-deficient mice also lack kidneys, showing that GDNF is important in early kidney development. GDNF is also important for the regulation of stem cell differentiation in the testis. GDNF and NRTN regulate the development of enteric and parasympathetic neurons, ARTN controls the migration and initial growth of sympathetic neurons, and PSPN may protect the brain from ischemic insult and regulates calcitonin secretion. See also: Glial Growth Factors; Nerve Growth Factor.
Further Reading Airaksinen MS and Saarma M (2002) The GDNF family: Signalling, biological functions and therapeutic value. Nature Reviews Neuroscience 3: 383–394. Baloh RH, Enomoto H, Johnson J, and Milbrandt J (2000) The GDNF family ligands and receptors – Implications for neural development. Current Opinion in Neurobiology 10: 103–110. Cacalano G, Farinas I, Wang LC, et al. (1998) GFRalpha1 is an essential receptor component for GDNF in the developing nervous system and kidney. Neuron 21: 53–62.
Eigenbrot C and Gerber N (1997) X-ray structure of glial cell-derived neurotrophic factor at 1.9 A˚ resolution and implications for receptor binding. Nature Structural Biology 4: 435–438. Enomoto H, Araki T, Jackman A, et al. (1998) GFR alpha1-deficient mice have deficits in the enteric nervous system and kidneys. Neuron 21: 317–324. Gill SS, Patel NK, Hotton GR, et al. (2003) Direct brain infusion of glial cell line-derived neurotrophic factor in Parkinson disease. Nature Medicine 9: 589–595. Jain S, Encinas M, Johnson EM Jr., and Milbrandt J (2006) Critical and distinct roles for key RET tyrosine docking sites in renal development. Genes and Development 20: 321–333. Jing S, Wen D, Yu Y, et al. (1996) GDNF-induced activation of the RET protein tyrosine kinase is mediated by GDNFR-[alpha], a novel receptor for GDNF. Cell 85: 1113–1124. Kotzbauer PT, Lampe PA, Heuckeroth RO, et al. (1996) Neurturin, a relative of glial-cell-line-derived neurotrophic factor. Nature 384: 467–470. Leppa¨nen VM, Bespalov MM, Runeberg-Roos P, et al. (2004) The structure of GFRalpha1 domain 3 reveals new insights into GDNF binding and RET activation. EMBO Journal 23: 1452–1462. Lin LF, Doherty DH, Lile JD, Bektesh S, and Collins F (1993) GDNF: A glial cell line-derived neurotrophic factor for midbrain dopaminergic neurons. Science 260: 1130–1132. Manie S, Santoro M, Fusco A, and Billaud M (2001) The RET receptor: Function in development and dysfunction in congenital malformation. Trends in Genetics 17: 580–589. Meng X, Lindahl M, Hyvo¨nen ME, et al. (2000) Regulation of cell fate decision of undifferentiated spermatogonia by GDNF. Science 287: 1489–1493. Oppenheim RW, Houenou LJ, Johnson JE, et al. (1995) Developing motor neurons rescued from programmed and axotomy-induced cell death by GDNF. Nature 373: 344–346. Rossi J, Luukko K, Poteryaev D, et al. (1999) Retarded growth and deficits in the enteric and parasympathetic nervous system in mice lacking GFR alpha2, a functional neurturin receptor. Neuron 22: 243–252. Schuchardt A, D’Agati V, Larsson-Blomberg L, Costantini F, and Pachnis V (1994) Defects in the kidney and enteric nervous system of mice lacking the tyrosine kinase receptor RET. Nature 367: 380–383. Takahashi M, Ritz J, and Cooper GM (1985) Activation of a novel human transforming gene, RET, by DNA rearrangement. Cell 42: 581–588. Tomac A, Lindqvist E, Lin L-F, et al. (1995) Protection and repair of the nigrostriatal dopaminergic system by GDNF in vivo. Nature 373: 335–339. Trupp M, Arenas E, Fainzilber M, et al. (1996) Functional receptor for GDNF encoded by the c-RET proto-oncogene. Nature 381: 785–789. Wang X, Baloh RH, Milbrandt J, and Garcia KC (2006) Structure of artemin complexed with its receptor GFRa3: Convergent recognition of glial cell line-derived neurotrophic factors. Structure 14: 1083–1092.
Insulin-Like Growth Factor Signaling and Actions in Brain V C Russo and G A Werther, Royal Children’s Hospital and University of Melbourne, Parkville, VIC, Australia ã 2009 Elsevier Ltd. All rights reserved.
The Discovery of the Brain IGF System In 1957, Salmon and Daughaday reported a serum factor (or factors) that mediated the cartilage sulfation and longitudinal bone growth activity of the somatotrophic hormone (growth hormone, GH). This factor was termed ‘sulfation factor’ and was produced by hepatic cells after exposure to GH (see Figure 1). These circulating factors, which also showed insulin-like activity not suppressible by antiinsulin antibodies, were later found to have a biochemical structure similar to that of the b chain of insulin. Their structural homology with proinsulin led to their current designation as insulin-like growth factors 1 and 2 (IGF-1 and IGF-2). In the middle 1980s, a variant of IGF-1, then called des(1–3) or ‘truncated’ IGF-1, was identified in brain tissue. This truncated IGF-1 variant lacks the first three amino acids and is more potent than intact IGF-1 in various cell culture systems, probably due to its lower affinity for IGF-binding proteins (IGFBPs). This finding suggested brain synthesis of IGF-1 or its truncated variant. Following the isolation of IGF-1 messenger RNA (mRNA) from postnatal rat brain, a number of in situ hybridization studies demonstrated that IGF-1 and IGF-1 receptor (IGF-1R) mRNA is synthesized in the rat brain in specific regions, namely, olfactory bulb (Figure 2), hippocampus, and cerebellum. In addition, IGF-carrier proteins, later named as IGFBPs, were found to be expressed in similar brain regions. The finding of IGF-1 mRNA co-localization with IGF-1 receptors and the presence of IGFBPs suggested a paracrine or autocrine role for IGF-1, potentially modulated by IGFBP, in the developing brain (Figure 3).
IGF System Expression in the Nervous System IGF Ligands
IGF-1 plays a key role in the development of the nervous system, with demonstrated effects in many stages of brain development, including cell proliferation, cell differentiation, and cell survival. Although recent reports have demonstrated that postnatal
circulating IGF-1 might exert neurogenic and survival activity, systemic IGF-1 is not readily transported through the blood–brain barrier, and therefore local production of IGF-1 is considered the primary source of the ligand (autocrine and paracrine action) for brain cells. During embryogenesis, IGF-1 mRNA expression is detectable in many brain regions, with its expression being particularly high in neuron-rich regions such as the spinal cord, midbrain, cerebral cortex, hippocampus, and olfactory bulb (Figure 1). IGF-2 mRNA is abundantly expressed in the embryonic rat central nervous system (CNS); however, data are conflicting on IGF-2 expression in cells of neuroepithelial origin. IGF-2 is the most abundantly expressed IGF in the adult CNS, with the highest level of expression found in myelin sheaths, but it is also expressed in leptomeninges, microvasculature, and the choroid plexus, all nonneuronal structures that enable diffusion of growth factors to their sites of activity. IGFBPs
The mRNA expression profiles and location of the most abundant IGFBPs, 2, 4, and 5, in the normal developing and adult CNS are well defined, suggesting an important role for IGFBPs in the nervous system. IGFBP-1 is not expressed in the CNS, but its expression is induced under certain experimental conditions. IGFBP-2 is expressed early in embryogenesis and by embryonic day 10 is highly expressed in discrete neuroectoderm structures. Later in development, IGFBP-2 mRNA is highly abundant throughout the brain, particularly in brain regions undergoing continuous remodeling, as is the olfactory bulb (Figure 2), the cerebellum, and the hippocampus. IGFBP-2 expression correlates with and complements that of IGF-2, and both IGFBP-2 and IGF-2 protein are highly abundant in the cerebrospinal fluid and choroid plexus. IGFBP-2 associates to cell surface proteoglycans in rat brain tissue and neuronal cells, and IGF-1–IGFBP-2– proteoglycan complexes have been identified in rat brain tissue; their role is not yet well understood. Such findings suggest that IGFBP-2 may be involved in targeting IGFs to their cell surface receptors in the brain. The absence of a ‘brain phenotype’ in the IGFBP-2 / mouse suggests functional redundancy in the IGFBP family during development of the CNS. IGFBP-3 is normally expressed at a low level in the CNS, mainly in nonneuronal structures, including epithelial cells. The effects of IGFBP-3 gene deletion on CNS are either unknown or have not been reported. IGFBP-3 is found to be upregulated in rat
609
610 Insulin-Like Growth Factor Signaling and Actions in Brain
(Hypothalamus)
GHRH GH (Pituitary)
GH
Muscle cells (IGF-receptors)
Bone cells (IGF-receptors)
Liver
IGF
IGF
IGFBPs
IGFBPs
Figure 1 The somatomedin hypothesis. The circulating factor that was originally proposed to mediate the effects of growth hormone on longitudinal bone growth was subsequently found to be insulin-like growth factor 1 (IGF-1). However, the presence of IGF-1 and IGF-1 messenger RNAs in multiple tissues has necessitated the revision of the original ‘somatomedin hypothesis’ to include both autocrine and paracrine actions of IGF in addition to its classical endocrine aspects. The IGF-binding proteins (IGFBPs) present in serum and expressed in most tissues actively contribute to the endocrine, paracrine, and autocrine action of IGF-1. GH, growth hormone; GHRH, growth hormone-releasing hormone.
Section A-A A
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Figure 2 The insulin-like growth factor (IGF) system in the rat brain olfactory bulb. IGF-1 is expressed and synthesized in distinct areas of the rat brain, adjacent to regions rich in IGF-1 receptors (IGF-1Rs) and insulin-like growth factor binding proteins (IGFBPs; e.g., olfactory bulb, as shown). This provides strong evidence for an autocrine and paracrine action of the IGFs in the nervous system. IGF action is modulated by locally expressed IGFBPs. ONL, Olfactory nerve layer; GL, glomerular cell layer; EPL, external plexiform cell layer; MI, mitral cell layer; GRL, granular cell layer. Section A-A shows a hematoxylin–eosin staining of olfactory bulb. Area in the rectangle is enlarged and represented in the main drawing. Reproduced from Russo VC, Gluckman P, Feldman EL, and Werther GA (2005) The insulin-like growth factor system and its pleiotropic functions in brain. Endocrine Reviews 26(7): 916–943. Copyright 2005, The Endocrine Society.
Insulin-Like Growth Factor Signaling and Actions in Brain
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Figure 3 Circulating (endocrine) or locally expressed (i.e., in autocrine or paracrine system) insulin-like growth factor (IGF) complex. IGF-binding proteins (IGFBPs), in addition to regulating transport, diffusion, and half-life of IGFs, provide specific binding sites for the IGFs in the extracellular and pericellular space. This pericellular localization might contribute to the modulation of IGFs’ interactions with their receptors and downstream cellular signaling. The effects of the IGFBPs are also regulated by the presence of specific IGFBP proteases, which cleave the binding proteins, generating fragments with reduced or minimal binding affinity for the IGFs. ECM, extracellular matrix; IRS, insulin receptor substrate; rec, receptor.
cerebral cortical cells following GH stimulation. GH promotes proliferation of neural precursors, neurogenesis, and gliogenesis; these responses appear to be mediated in part by locally produced IGF-1 and its modulator IGFBP-3. No change in brain growth or phenotype has been reported in IGFBP-3 transgenic mice. IGFBP-4 is normally expressed at a very low level in the CNS, where its mRNA is found in a variety of brain cell types, including meningeal cells, astrocytes, and fetal neuronal cells. Whether ablation of the IGFBP-4 gene affects the CNS has not been reported. IGFBP-5 gene expression is highly abundant during early brain development. In rodents, IGFBP-5 appears to be co-expressed with IGF-1 in principal neurons of sensory relay systems, cerebellar cortex, hippocampal formation, and many other neuron-rich regions, including the olfactory bulb (Figure 2). Furthermore, in vivo, IGFBP-5 expression is specifically regulated by IGF-1. In Xenopus, IGFBP-5, together with three IGFs expressed in the early embryo, promotes anterior development by increasing growth of the head region. Thus, both IGF signal activation and IGFBP-5 appear to be required for anterior neural induction in Xenopus. Whether IGFBP-5 has similar functions in early mammalian neural development is not known. The effects of IGFBP-5 gene ablation in the brain have not been reported.
IGFBP-6 is poorly expressed in the nervous system, and information regarding its mRNA expression distribution, in both the developing and adult nervous system, is limited. IGFBP-6’s unique property of preferential binding to the IGF-2 ligand, coupled with the fact that this ligand is the most abundantly expressed IGF in the adult CNS, raises the possibility that the IGFBP-6–IGF-2 complex may have a unique role in modulating IGF-2 function in the adult brain. Preliminary analysis of the IGFBP-6 transgenic mouse shows reduced cerebellum size and weight, combined with altered differentiation of astrocytes. Abnormalities in the hypothalamus and pituitary were also reported. The effects on the brain of IGFBP-6 gene ablation remain unknown. IGF Receptors
Type 1 IGF receptors are highly expressed in many specific brain regions and cell types, particularly in plastic regions such as the developing cerebellum, midbrain, and olfactory bulb (Figure 2) and in the ventral floor plate of the hindbrain. Given that IGF receptors are expressed from early embryogenesis and throughout life and that their ligands also show a similar ‘temporal–spatial’ pattern of expression, it is evident that local brain IGF circuits are crucial modulators of the processes activated during brain
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development. The level of IGF-1R decreases to adult levels soon after birth but remains relatively high in the choroid plexus, meninges, and vascular sheaths. It is thus not surprising that knockout of the IGF-1R gene produced, in addition to in utero growth retardation, is a clear brain phenotype, namely, a small brain. The type 2 IGF receptor, which has low affinity for IGF-1, but a high affinity for IGF-2, and also binds mannose-6-phosphate, is selectively expressed in all major brain regions. It is therefore possible that the IGF-2–mannose-6-phosphate receptor, in addition to its role in transporting lysosomal enzymes, might also participate in control of postnatal brain development and possibly repair. Various atypical IGF-1R subtypes possessing distinct structures or binding properties have also been described (hybrid insulin–IGF-1Rs with the ability to bind insulin as well as IGFs). However, the physiological significance of hybrid and atypical IGF receptors is unclear. Early studies demonstrated that some brain regions (i.e., olfactory bulb and choroid plexus) expressing both the IGF-1 and IGF-2 receptors are also rich in
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insulin receptors. Insulin receptor density is elevated in the choroid plexus, olfactory bulb, limbic system, and hypothalamus, all regions concerned with olfaction, appetite, and autonomic functions. More recently, ablation of the insulin receptor in nestinpositive neurons has suggested a role for insulin in control of appetite suppression and reproduction.
IGF Signaling and Action in Brain Binding of IGF-1 (or IGF-2) to the a subunit of the type-IGF-1R (cysteine-rich domain) induces comformational changes of the intracellular b subunit with consequent induction of tyrosine kinase activity. Two major substrates, insulin receptor substrates 1 and 2 (IRS-1 and IRS-2), interact with the IGF-1R and become phosphorylated on multiple residues (Figure 4). Phosphorylated IRS-1 is then able to recognize and bind to the ‘Src homology polypeptide domain’ (SH domains) present in various signal transduction proteins (Figure 4). Molecules that bind IRS-1 include the growth factor receptor-bound (Grb-2), the adaptor oncogenic protein containing
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Figure 4 A model of key pathways and interactions involved between the insulin-like growth factor (IGF) and bcl-2 systems in glucosedeprived neuronal cells. Neuronal cell responses to glucose withdrawal (glucopenia) include activation of acute responses such as increased translocation of glucose transporter (GLUT) to cell surface and modulation of mitochondrial activities. These responses are followed by sustained activation of the extrinsic (death receptor cascade) and the intrinsic (mitochondrial cascade) proapototic pathways. These effects are potently regulated by IGF-1 via the phosphoinositide-3 kinase (PI3K) pathways, which lead to the upregulation of GLUT transcription and translocation, recovery of mitochondrial activities, and inhibition of intrinsic and extrinsic proapototic processes. A key element of IGF antiapoptotic action is Bcl-2, which promotes mitochondrial functions and potentiates glucose uptake. Akt, protein kinase B; IRS, insulin receptor substrate.
Insulin-Like Growth Factor Signaling and Actions in Brain
one SH2 and three SH3 domains, the protein tyrosine phosphatase Syp (SH-PTP2), and the p85 b-subunit domain of PI3K (see the section titled ‘The mitogenic pathway’). The Mitogenic Pathway
The IGF-1R mitogenic pathway begins with Grb-2, which is a small cytoplasmatic protein and acts as an ‘adapter molecule’ that links the guanidine nucleotide exchange factor for p21ras, termed mSOS (homologous to the Drosophila protein son of sevenless (SOS)), to IRS-1. The Grb-2–SOS complex can thus activate Rouss avian sarcoma (Ras), which recruits Raf (serin/threonin protein kinase), and they proceed to activate the mitogen-activated protein kinase (MAPK) pathway (MEK kinase, MEK, extracellular signal-regulated kinase (ERK) kinase, p90) and transcription factors and eventually induce activation of genes such as the early growth response genes c-jun and c-fos. Therefore, whether the IGFs are produced locally or reach brain cells systemically, binding to their receptor activates intracellular signaling, promoting neurotrophic, neurogenic, and neuroprotective/ antiapoptotic activities. A large number of in vitro studies have demonstrated that IGF-1 promotes mitogenesis and differentiation in glia, oligodendrocytes, neuronal cells, adult stem cells, and brain explants and regulates axon myelination via activation of MAPK, also known as ERK. There is also clear evidence that IGF-1 activation of the MAKP/ERK pathways enhances growth cone motility and promotes neurite outgrowth, suggesting that this pathway is likely to be involved in neurogenesis, migration, and repair. Supportive evidence for the importance of IGF and IGF receptor signaling and activation of downstream effectors has come from a number of studies in IGF null ( / ) mice. These studies have clearly demonstrated that most of the IGF functions determined in vitro also apply to the in vivo situation, affecting a wide range of brain cells. Thus IGF-1 null mice have reduced brain size and altered brain structures, including alteration of myelination processes. Furthermore, ablation of the Igf-1 gene has revealed deficits in the numbers of specific neurons and oligodendrocytes in the olfactory bulb, dentate gyrus, and striatum and in cochlear ganglion neurons. In addition to gross structural brain abnormalities, IGF null mice also show alteration of important brain metabolic functions such as reduced glucose uptake, the major source of energy for neuronal cells. These in vivo models also show that the IGF system affects several steps involved in development and organization of CNS architecture,
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including number of cells, connections, and regulation of the extracellular matrix. It is important to emphasize that the IGFs do not act in isolation or only discretely activate specific pathways. It is well recognized that the actions and effects of IGF signaling are influenced by or affect a large number of other growth factor/cytokine sytems and that IGF-induced IGF-1R phosphorylation activates complex and integrated intracellular signaling. The Survival and Metabolic Pathway
Binding of the IGFs to the IGF-1R may also activate signaling through the PI3K cascade (Figure 4). Protein kinase B (PKB), also known as serine/threonine kinase Akt, is one of the primary downstream targets of PI3K, and it is well characterized for its involvement in modulation of cell survival. Activated PKB/Akt in turn phosphorylates and inhibits several proapoptotic proteins, such as Bad, caspase-9, and the winged-helix family of transcription factors (FKHRL1, FKHR, and AFX), leading to cell survival in various systems, including hippocampal neurons. Activation of PKB/Akt appears to be also involved in the modulation of brain metabolic events such as glucose transport and utilization in neuronal cells. As mentioned earlier, IGF-1 is a major regulator of glucose uptake and utilization in brain. In an in vitro model of glucose stress (glucopenia), mimicking in vivo hypoglycemia, activation of PKB/Akt by IGF-1 was involved in both survival (inhibition of proapoptotic genes) and upregulation of glucose transporters at transcriptional and posttranslational levels (Figure 4). Another target of IGF-1, via the PKB/Akt pathway, is the glycogen synthase kinase 3b (GSK3b). IGF-1, similar to insulin, stimulates the phosphorylation of GSK3b, thus promoting glycogen and protein synthesis. Accumulation of glycogen, normally associated with astrocytes in mature brain, is abundant in IGF1-positive projecting neurons and postnatally in neurons in IGF-1-rich areas, suggesting that local autocrine and paracrine IGF-1 signaling via the PI3K/Akt pathway is involved in glucose metabolism and storage. The GSK3b substrate tau, involved in formation of the intracellular neurofibrillary tangles that contribute to the neuronal degeneration seen in Alzheimer’s disease, is hyperphosphorylated in IGF-1-null mice. Furthermore, GSK3b promotes apoptosis in neurons via mechanisms involving phosphorylation of tau and b-catenin. Thus IGF-1-induced inhibition of GSK3b, via the PI3K/Akt pathway, appears to be a critical mechanism of IGF-1 action in the brain.
614 Insulin-Like Growth Factor Signaling and Actions in Brain The IGFs Are Neurotrophic Factors
IGF-1 is involved in brain plasticity processes, and it specifically modulates synaptic efficacy by regulating synapse formation, neurotransmitter release, and neuronal excitability. IGF-1 provides constant trophic support to neuronal cells in the brain and in this way maintains appropriate neuronal function. Alteration of this trophic input may contribute to brain disease as seen in neurodegenerative disorders such as Alzheimer’s disease, ataxia telangiectasia, Huntington’s disease, and Parkinson’s disease, all variably responsive to IGF-1 treatment. Recent studies have shown that neurogenesis declines in brains of aged mice but is efficiently restored following IGF-1 administration via intracerebroventricular infusion.
The IGF System and Neuroendocrine Signaling Cross Talk in Brain In vivo, growth factors such as IGF-1 do not exist in isolation. Hence, the presence of other growth factors may further modulate IGF’s biological activity and cellular responses. This modulation can be seen in IGF’s neurogenic activity, which may be optimal only in the presence of such cytokines as fibroblast growth factor-2 (FGF-2) and a range of other local growth factors. In primary cultures of hypothalamic cells, FGF-2 combined with IGF-1 significantly increased the number of neurite-bearing cells above that seen when either one of the two growth factors was added in isolation. Their effects appear to be synergistic since blocking IGF-1 from binding with an antibody abrogates FGF2’s effects. In contrast, a recent study in neuroblastoma cells demonstrated that FGF-2 overrides IGF-1 mitogenic and survival activity via induction of neuronal differentiation and blockade of IGF-1’s antiapoptotic response. Interactions among local brain growth factors are thus complex and may vary under differing conditions. Recent reports have highlighted the importance of a number of serum (in contrast to brain-produced) growth factors, which included IGF-1, FGF-2, and other blood-borne neurotrophic factors (neurotrophins), in neuroprotective surveillance. All these factors exerted a tonic trophic input on brain cells, providing defense mechanisms ranging from blockade of neuronal death to upregulation of neurogenesis. Increased interest has thus recently been devoted to the interaction of IGF-1 with other ‘classic’ neurotrophic factors. Of a number of neuroprotective factors including IGF-1, glial cell-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), neurotrophin-4 and -3 (NT-4/5, NT-3), and ciliary neurotrophic factor, only IGF-1, GDNF, and
NT-4/5 were found to be potently neuroprotective of motor neurons. These findings point to the potential combined use of these neuroprotective factors in treatment of neurodegenerative diseases. Results from a number of phase III clinical trials with nerve growth factor, BDNF, and IGF-1 have indicated that these neurotrophic factors may find an application in degenerative disorders or injury of peripheral nerves and motor neurons. IGF-1 and GH
Although GH gene expression also occurs in the central and peripheral nervous systems, with brain GH imunoreactivity not affected by hypophysectomy, the potential cross talk between the IGF system and GH in the nervous system has not been fully investigated. Several experimental models, in vivo and in vitro, support a key role for GH in brain development. The Snell dwarf mouse (Pit1) and the GH-releasing hormone receptor-deficient little mouse exhibit microcephalic cerebra with hypomyelination and retarded neuronal growth with poor synaptogenesis. Postnatal GH administration normalizes neuronal growth in these mice. Recent in vivo studies have demonstrated that GH is involved in neuroprotection during hypoxic or ischemic brain injury. In these studies, GH-like immunoreactivity was demonstrated on injured brain cells, and it was shown that GH administered intracerebroventricularly is capable of preventing brain cell loss. Whether the neuroprotective effects of GH involve IGF-1 induction is not clear. However, some recent in vitro studies have demonstrated that GH promotes proliferation and differentiation of fetal cerebral cortical cells in primary culture and that these effects are mediated by IGF-1. There is some evidence that the GH-related hormone prolactin also possesses neurogenic properties. In female mice during pregnancy, prolactin stimulates production of neuronal progenitors in the forebrain subventricular zone. Whether prolactin’s neurogenic properties are a direct effect of its action or are mediated by other factors, including IGF-1, remains to be determined. IGF-1 and Erythropoietin
Erythropoietin (EPO) is traditionally known as a hematopoietic cytokine produced by the fetal liver and adult kidney in response to hypoxia. However, the expression of EPO and EPO receptors in the CNS and the upregulation of EPO by hypoxic–ischemic insult suggest that this cytokine is an important mediator of the brain’s response to injury. In fact, in vivo EPO administration protects hippocampal CA1 neurons and retinal neurons from ischemic damage and prevents brain injury
Insulin-Like Growth Factor Signaling and Actions in Brain
following a number of ‘insults’ by mechanisms involving both inhibitions of apoptosis and neurotrophic actions. In the brain, EPO expression, regulated by hypoxia-inducible factor-1a (HIF-1a), is mainly found in astrocytes. IGF-1 protection of primary neuronal cells exposed to low-oxygen concentration correlates with activation of HIF-1a expression. In the same studies using an in vivo model of hypoxic or ischemic brain injury, it was also shown that IGF-1 transcriptional activation correlates with that of HIF-1a, suggesting that HIF-1a might mediate some of the IGF-1 responses. Another study has shown that IGF-1 induces HIF-1a transcriptional activity in rat cerebral cortex and neuronal cells (PC12) and that this induction is abolished by a selective IGF-1R antagonist (JB-1). Furthermore, studies in hepatoma cells have shown not only that insulin, IGF-1, and IGF-2 induced HIF-1a expression but that HIF-1a is required for expression of IGF-2, IGFBP-2, and IGFBP-3. These findings suggest the presence of a potential complex synergistic cross talk between the IGF and the EPO systems, involving both activation of common intracellular signaling pathways and regulation of gene expression. The presence of synergistic cross talk between the IGF and the EPO systems in neuronal cells has recently been demonstrated by showing that EPO can exert a more immediate neuroprotective action when administered in concert with IGF-1. The neuroprotective mechanism, following coadministration of EPO and IGF-1, involved synergistic activation of the PI3K/Akt pathway. These findings further support the concept that the coadministration of synergistic neuroprotective agents, rather than a single agent, might provide improved therapeutic outcome. Thus, treatment with appropriate combinations of EPO and IGF-1 could be a future therapeutic strategy for a variety of acute neurological events. IGF-1 and Sex Steroids
Among the numerous endocrine systems regulating brain physiology, sex steroids play an important role. However, it appears that some of the effects of these hormones on the brain are mediated by trophic factors, including IGF-1. Estradiol and IGF-1 increase survival and differentiation of developing fetal rat hypothalamic neurons. In this case, estrogen-induced activation of the estrogen receptor requires the presence of IGF-1; both estradiol and IGF-1 use the estrogen receptor to mediate their trophic effects on hypothalamic cells. In vivo sex steroids affect IGF-1 levels in the endocrine hypothalamus, arcuate nucleus, and median eminence. Furthermore, increased clinical and basic evidence suggests that gonadal steroids affect the onset and
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progression of several neurodegenerative diseases and schizophrenia, as well as recovery from traumatic neurological injury such as stroke. Similarly, in the brain, both estrogen synthesis and estrogen receptor expression are upregulated at sites of injury. The neuroprotective effects of estrogen may be exerted independent of the classical nuclear estrogen receptors, involving modulation of IGF-1 signaling. This is supported by the fact that estrogen receptors and IGF-1R interact in the activation of PI3K and MAPK-signaling cascades and possibly in the promotion of neuroprotection. It is therefore possible that the decrease in estrogen and IGF-1 levels with aging may thus result in an increased risk for neuronal pathological alterations. These findings thus suggest the presence of potential complex synergistic cross talk between the IGFs and sex steroids involving activation of common intracellular signaling pathways; however, the precise mechanism remains unclear.
Future Directions Since 1957, a considerable body of investigation has been devoted to defining the role of the IGF system in many tissues, including the brain. There is no doubt that these pleiotropic factors, in concert with their receptors and binding proteins, are involved in controlling key processes in brain development and traumatic or degenerative disorders of the nervous system. Although the development of in vivo models such as the transgenic and knockout mice or disease and injury models have contributed to providing unique information about the growth-promoting, neurogenic, and neuroprotective actions of the IGF system, many questions remain. In particular, analysis of the signaling pathways activated by IGFs and their integration and cross talk with other brain growth factor systems is critical. Such insights will potentially lead to the development of improved approaches to prevention, management, and effective treatment of destructive, neurodegenerative, and malignant process of the nervous system. See also: Nerve Growth Factor.
Further Reading Beilharz EJ, Russo VC, Butler G, et al. (1998) Co-ordinated and cellular specific induction of the components of the IGF/IGFBP axis in the rat brain following hypoxic–ischemic injury. Brain Research 59: 119–134. Bondy CA and Cheng CM (2004) Signaling by insulin-like growth factor 1 in brain. European Journal of Pharmacology 490: 25–31. Bridges RS and Grattan DR (2003) Prolactin-induced neurogenesis in the maternal brain. Trends in Endocrinology and Metabolism 14: 199–201.
616 Insulin-Like Growth Factor Signaling and Actions in Brain Cardona-Gomez GP, Mendez P, DonCarlos LL, Azcoitia I, and Garcia-Segura LM (2001) Interactions of estrogens and insulin-like growth factor-I in the brain: Implications for neuroprotection. Brain Research. Brain Research Reviews 37: 320–334. Carro E, Trejo JL, Nunez A, and Torres-Aleman I (2003) Brain repair and neuroprotection by serum insulin-like growth factor I. Molecular Neurobiology 27: 153–162. Digicaylioglu M, Garden G, Timberlake S, Fletcher L, and Lipton SA (2004) Acute neuroprotective synergy of erythropoietin and insulin-like growth factor I. Proceedings of the National Academy of Sciences of the United States of America 101: 9855–9860. Drago J, Murphy M, Carroll SM, Harvey RP, and Bartlett PF (1991) Fibroblast growth factor-mediated proliferation of central nervous system precursors depends on endogenous production of insulin-like growth factor I. Proceedings of the National Academy of Sciences of the United States of America 88: 2199–2203. Edmondson SR, Werther GA, Russell A, Le Roith D, Roberts CT Jr., and Beck F (1995) Localization of growth hormone receptor/ binding protein messenger ribonucleic acid (mRNA) during rat fetal development: Relationship to insulin-like growth factor-I mRNA. Endocrinology 136: 4602–4609. Feldman EL, Sullivan KA, Kim B, and Russell JW (1997) Insulinlike growth factors regulate neuronal differentiation and survival. Neurobiology of Disease 4: 201–214. Giacobini MM, Olson L, Hoffer BJ, and Sara VR (1990) Truncated IGF-1 exerts trophic effects on fetal brain tissue grafts. Experimental Neurology 108: 33–37.
Jones JI and Clemmons DR (1995) Insulin-like growth factors and their binding proteins: Biological actions. Endocrine Reviews 16: 3–34. LeRoith D, Werner H, Faria TN, Kato H, Adamo M, and Roberts CT Jr. (1993) Insulin-like growth factor receptors: Implications for nervous system function. Annals of the New York Academy of Sciences 692: 22–32. Russo VC, Gluckman P, Feldman EL, and Werther GA (2005) The insulin-like growth factor system and its pleiotropic functions in brain. Endocrine Reviews 26(7): 916–943. Russo VC, Kobayashi K, Najdovska S, Baker NL, and Werther GA (2004) Neuronal protection from glucose deprivation via modulation of glucose transport and inhibition of apoptosis: A role for the insulin-like growth factor system. Brain Research 1009: 40–53. Scheepens A, Williams CE, Breier BH, Guan J, and Gluckman PD (2000) A role for the somatotropic axis in neural development, injury and disease. Journal of Pediatric Endocrinology & Metabolism 13(supplement 6): 1483–1491. Sepp-Lorenzino L (1998) Structure and function of the insulin-like growth factor I receptor. Breast Cancer Research and Treatment 47: 235–253. Torres-Aleman I, Naftolin F, and Robbins RJ (1990) Trophic effects of basic fibroblast growth factor on fetal rat hypothalamic cells: Interactions with insulin-like growth factor I. Brain Research. Developmental Brain Research 52: 253–257.
Glial Growth Factors B M Reuss, University of Go¨ttingen, Go¨ttingen, Germany ã 2009 Elsevier Ltd. All rights reserved.
Glial cells are a major source of growth factors in the healthy and injured central and peripheral nervous systems. Glia-derived growth factors regulate differentiation and function of glial cells in an autocrine fashion, and they modulate neuronal process formation, migration, and survival during development. An important function of glial growth factors is the regulation of cellular repair processes after injury, degeneration, and demyelination in the peripheral and central nervous systems. Changes in growth factor synthesis, release, and action are also associated with the formation of glia-derived brain tumors.
Expression of Growth Factors by Different Glial Subpopulations Satellite Cells
Satellite cells are specialized glial cells in the sensory ganglia ensheathing cell bodies of pseudounipolar sensory ganglion cells. During development, satellite cell-derived nerve growth factor (NGF) is an important determinant for neurite outgrowth from sensory neurons since, in the rat, neurite outgrowth from neonatal sensory neurons after removal of satellite cells is induced by NGF. After peripheral nerve injury in the rat, NGF and neurotrophin NT-3 induce sprouting of noradrenergic nerve terminals in the dorsal root ganglia. Satellite cell-derived NGF also affects expression of the nicotinic acetylcholine receptor in rat sensory neurons. NGF also seems to affect satellite cells since NGF depletion reduces reactivity of rat trigeminal satellite cells after inferior alveolar nerve injury. Other growth factors released from satellite cells are vascular endothelial growth factor (VEGF), the transforming growth factor (TGF)-bs, and TGF-a. Thus, VEGF plays an important role in the establishment and maintenance of blood vessels in the dorsal root ganglia during development, whereas TGF-b signaling is altered after peripheral nerve transection as indicated by changes in the distribution of TGF-b and the TGF-b type I and type II receptors in peripheral nerves and mechanoreceptors after traumatic injury. TGF-a and its receptor reveal strong upregulation in satellite cells in response to peripheral nerve lesioning.
Schwann Cells
Schwann cells are the myelinating cells of the peripheral nervous system and thus ensheathe with their processes axons in peripheral nerves. Perturbed Schwann cell functioning is a key feature of several demyelinating disorders, the most prominent of which is multiple sclerosis (MS). Schwann cells synthesize and release a wide variety of growth factors, such as neurotrophins, neuregulatory cytokines, TGF-bs, glial cell line-derived neurotrophic factor (GDNF), epidermal growth factors (EGFs), and platelet-derived growth factor (PDGF). High levels of NGF are found in Schwann cells in vivo and in primary cell cultures in vitro. NT-3 mRNA is expressed at low concentrations in cultured Schwann cells but is upregulated upon immortalization. In response to nerve injury, neurotrophins are differentially up- or downregulated in Schwann cells. Thus, low levels of NGF in normal Schwann cells are greatly increased after injury. Also, transection of the sciatic nerve leads to an increase in the mRNAs for brain-derived neurotrophic factor (BDNF) and NT-4 but to a decrease of that for NT-3. Schwann cells of the sciatic nerve also express ciliary neurotrophic factor (CNTF) beginning on postnatal day 8 and reaching adult levels on day 21. CNTF expression in Schwann cells is regulated by axonal factors since after axotomy, CNTF expression recovers only after regeneration of the axonal processes. Whereas in cultured Schwann cells mRNAs for CNTF and leukemia inhibiting factor (LIF), another neuregulatory cytokine, are high, myelinating Schwann cells in vivo show only CNTF expression and levels of LIF mRNA are low. However, retrograde transport of LIF and CNTF is increased after nerve lesioning. In Schwann cells and their precursors in vivo, both TGF-b2 and -b3 are expressed, whereas TGF-b1 is upregulated only after injury. Thus, in the transected sciatic nerve, TGF-b1 mRNA is induced in the distal nerve stump, whereas that for TGF-b3 is repressed. Also during axon regeneration, TGF-b1 mRNA is transiently increased. In contrast to the in vivo situation, in cultured Schwann cells expression of TGF-b1 mRNA is high but decreases when axonal contact is mimicked by the application of forskolin. A striking feature of the effects of TGF-b on Schwann cells is that application of TGF-b together with tumor necrosis factor-a (TNF-a) can induce Schwann cell death, whereas either cytokine alone is ineffective. Whereas in normal circumstances GDNF expression in Schwann cells and satellite cells is low, it shows transient upregulation after injury. Thus, GDNF is
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upregulated in the distal nerve stump of the transected sciatic nerve, as well as in the satellite cells of the trigeminal ganglion. Schwann cells are also known to express TGF-a and other neuregulins, as well as the neuregulin receptor ErbB1, on their surface. In accordance with this, neuregulins act as autocrine stimulators of Schwann cell proliferation during development. Similar observations have been made for PDGF, the expression of which in rat Schwann cells is high at birth, followed by a continuous postnatal decrease in nonmyelinating but persisting high levels in myelinating Schwann cells. Oligodendrocytes
Oligodendrocytes are the myelinating cells of the central nervous system (CNS) and thus, like Schwann cells in the peripheral nervous system (PNS), ensheathe axonal processes by their processes. Oligodendrocytes and their precursor cells also provide an important source of growth factors such as the neurotrophins and several TGF-b superfamily members. Thus, they express NGF, NT-3, and BDNF, probably mediating the autocrine control of proliferation and survival of immature oligodendrocytes and of O-2A precursor cells. In O-2A precursor cells, different TGF-b isoforms, such as TGF-b1 and -b3, are expressed, whereas mature oligodendrocytes express TGF-b2 and-b3. In cultured oligodendrocytes, all three TGF-b isoforms are present. Astrocytes
Astrocytes in the CNS mediate the transport of nutrients and waste products between blood vessels and neurons, and they also modulate neuronal activity and synaptic transmission. Due to their important role during gliosis and neuronal repair, growth factor synthesis by astrocytes has been intensely studied. Thus, astrocytes in the healthy CNS release NGF, NT-3, and NT-4, whereas reactive astrocytes after gliosis start to express BDNF together with NGF, a process which at least in vitro seems to depend on interleukin (IL)-1b and interferon-g (IFN-g). Astrocytes also release high amounts of fibroblast growth factor-2 (FGF-2), mRNA of which can be found in cortical, hippocampal, and spinal cord astrocytes with a strong postnatal increase. FGF-2 seems to act in an autocrine manner on astrocyte differentiation since in FGF-2/ mice astroglial gap junction coupling and neurotransmitter sensitivity are changed. Astroglial FGF-2 is also responsible for the induction of endothelial tight junctions and thus for the formation of the blood–brain barrier in the CNS. Astrocytes express neuregulatory cytokines such as CNTF, which can be found in type 1 astrocytes of the
optic nerve and the olfactory bulb. Also, primary cultures of astrocytes contain CNTF mRNA and protein. Astroglial CNTF expression is upregulated during brain lesions such as entorhinal cortex lesions, hippocampal deafferentiation, optic nerve transection, and ischemia. Also, LIF is expressed in cultured astrocytes of variable origins and is upregulated after trauma and during inflammation. However, astrocytes do not express EGF itself but synthesize other members of this growth factor family, such as TGF-a and the neuregulins. TGF-a has been found in astrocytes of the corpus callosum, striatum, and globus pallidum. Astrocytes also express ErbB1, the receptor for TGF-a, and therefore it is thought to act in an autocrine manner. In reactive astrocytes, TGF-a and its receptor are transiently reduced. Other neuregulins which can be found in cultured astrocytes are highly upregulated during reactive gliosis. Thus, GGF-2 and NDF have been detected in human white matter astrocytes of the spinal cord and in the cortex. Of the insulin-like growth factors (IGFs), only IGF-1 is expressed in astrocytes, which in vitro has been shown to promote astroglial proliferation in an autocrine manner. During remyelination, IGF-1 is upregulated in astrocytes, together with its receptor, and is also increased after ischemia, probably acting in a protective manner. With regard to the TGF-b superfamily members, in astrocytes a highly heterogeneous expression pattern can be found. Thus, astrocytes in the uninjured brain express only TGF-b2 and -b3, whereas after lesioning TGF-b1 is also upregulated. In contrast, GDNF is not expressed in astrocytes of the adult brain but can be found during early postnatal development. However, GDNF is released from cultured fetal human astrocytes and in astrocytes in vivo after brain damage. GDNF is also highly upregulated in gliomas in situ and in glioma cell lines. Ependymoglia
Ependymoglia line the ventricular wall and the choroid plexus and, thus, are important for substance transport and for establishing the blood–liquor barrier. FGF-2 is expressed in the choroid plexus epithelia of both lateral and third ventricles, in ependymal cells of the third ventricle, and along the lateral sides of the lateral ventricles. All positive cells reveal a similar distribution with apical labeling, as well as some cytoplasmic staining. This suggests a transepithelial transport of growth factors such as FGF-2 via ependymal cells into the underlying brain parenchyma. However, FGF-2 also seems to play a role in the autocrine regulation of ependymal cells since both EGF and FGF-2 have been shown to cause proliferation of
Glial Growth Factors
ependymal precursor cells in the adult rat spinal cord. Another growth factor which is expressed in ependymal cells, at least during development, is GDNF, which has been demonstrated in human prenatal cortical plate ependyme from the age of 10 weeks onward. Mu¨ller and Bergmann Glial Cells
Mu¨ller glial cells are persisting radial glial cells in the retina and express a large variety of cytokines and their receptors. Thus, expression of NGF, BDNF, and NT-3, along with neurotrophin receptors trkB and trkC, but not of trkA, has been reported. Also, glial maturation factor-b, another cytokine, is synthesized in these cells. Human Mu¨ller cells in vivo express high amounts of FGF-2. In addition, FGF-2 has been shown to downregulate IGF-1 in Mu¨ller cells. In vitro, Mu¨ller cells release TNF-a and nitric oxide upon stimulation with lipopolysaccharide and IFN-g. TGF-b2 and -b3 are also expressed in Mu¨ller cells. Glucose and pH influence expression of VEGF in Mu¨ller cells. VEGF183 has been reported as a Mu¨ller cell-specific splice variant of this growth factor. Mu¨ller cells also express CNTF and FGF-2. Bergmann glia, the persisting radial glia in the cerebellar cortex, express some growth factors and growth factor-related proteins. Thus, IGFBP2 is expressed in these cells during postnatal cerebellar maturation in parallel to the expression of IGF-1 in cerebellar Purkinje neurons. Bergmann glia cells also express IL-6.
Role of Glial Growth Factors in Glial Cell Differentiation and Function and the Formation of Glial Brain Tumors An important function of glial growth factors is paraand autocrine regulation of the differentiation and maturation of glial cells, a feature which is of utmost importance for pathological changes of the nervous system (Figure 1). Autocrine Regulation of Astrocyte Differentiation
With regard to autocrine regulation of astrocyte differentiation, studies from the 1980s reported that astrocyte-conditioned medium is able to inhibit astrocyte proliferation compared to fresh medium – a feature that has been attributed to the secretion of growth inhibitory factors by these cells. The complex network of para- and autocrine effects of glial cells during astrocyte development is highlighted by the fact that differentiation of O-2A progenitor cells into either oligodendrocytes or type 2 astrocytes seems to depend on growth
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factors released from type 1 astrocytes. Type 1 astrocytes have been shown to secrete PDGF, thus stimulating O-2A progenitor cell proliferation and oligodendrocyte differentiation. Later, type 1 astrocytes secrete CNTF, thus initiating differentiation of type 2 astrocytes. The LIF-receptor and its ligands play an important role in astrocyte differentiation. Thus, neural precursor cells isolated from LIFR/mice fail to generate glial fibrillary acidic protein (GFAP)-positive astrocytes, whereas precursors from heterozygous mice differentiate normally. Also, in vivo LIFR/ mice show low levels of GFAP. In contrast, animals deficient in LIF show reduced GFAP levels only in some brain regions, suggesting that LIF is not the predominant endogenous ligand for the LIF receptor. Region-specific effects of glia-derived factors on astrocyte differentiation can also be observed in the cerebellar cortex, in which granule cell precursors are differentiated into astroglial cells by sonic hedgehog and bone morphogenic peptides. Growth factor-stimulated cells initially express both GFAP and neuronal markers and later switch to S100-beta, a marker of differentiated astrocytes. Despite their important role in neuronal stem cell proliferation and differentiation, with regard to astrocytes, FGF-1 and FGF-2 are primarily involved in the regulation of astrocyte proliferation. Nevertheless, GFAP immunoreactivity is reduced in mice with a genetic defect in FGF-2. Also, Fgf-8b, a splice variant of this growth factor, has been shown to promote astroglial differentiation of a subpopulation of E15 cortical precursor cells in culture. Regulation of Oligodendrocyte and Schwann Cell Differentiation by Glial Growth Factors: Importance for Etiology and Treatment of MS
A central role in paracrine and autocrine regulation of oligodendrocyte differentiation is played by neuregulins, which in the normal human CNS are produced by astrocytes and neurons. Thus, NRG-1a and -1b and their receptors can be found in cultured oligodendrocytes from neonatal rat pups. Under these conditions, less differentiated oligodendrocytes contain both NRG isoforms in the cell bodies but not in the processes, whereas only NRG-1b was found in the nucleus. In contrast, differentiated oligodendrocytes contained both isoforms only in cytoplasm and cell processes. Similar effects could be observed for embryonic striatal oligodendrocyte precursor cells, which express Nrg-1 as well as its specific receptors, ErbB2 and ErbB4, but not ErbB3. Likewise, inhibition of Nrg-1 activity by the addition of soluble ErbB3 decreases the mitotic activity of Nrg-1 on oligodendrocyte precursor cells.
620 Glial Growth Factors CNS Ependymoglia: FGF-2 GDNF
Astrocytes: NGF, NT-3, NT-4 CNTF, LIF TGF-b2, TGF-b3 TGF-a FGF-2 Oligodendrocytes: NGF, BDNF, TGF-b2, TGF-b3
PNS
Development: Survival Differentiation Adulthood: Synaptic plasticity Aging
Degenerative diseases: Radial glia: NGF, BDNF, NT-3 Alzheimer’s disease Parkinsonism GMFb FGF-2 CNS lesions: IGF-I Inhibition of axon IFN-g, IL-6 regeneration TNF-a Glial brain tumors: TGF-b2 TGF-b3 Autocrine stimulation VEGF of tumor growth
Satellite cells: NGF, NT-3 VEGF TGF-bs TGF-a
PNS lesions: Stimulation of axon regeneration
Schwann cells: NGF, BDNF, NT-3, CNTF, LIF, TGF-b2, TGF-b3, GDNF EGF, TGF-a, Neuregulins PDGF Figure 1 Graphical summary of expression and effects of glial growth factors in the central and peripheral nervous systems. A sophisticated network of glia–neuronal, interglial, and autocrine actions of glial growth factors regulate normal development and function of the nervous system, degeneration and regeneration after nerve injury, as well as brain tumor growth.
Despite these in vitro findings, neuregulins seem to also be important for myelination in vivo since mice with a null mutation in the nrg-1 gene show defects in oligodendrocyte differentiation. This also seems to be important for the pathology of MS since in active and chronically active MS lesions, expression of astrocyte-derived neuregulin is reduced and thus may contribute to impaired remyelination in MS patients. Other glial growth factors influencing oligodendrocyte differentiation and myelination are PDGF and IGF-1. Thus, in spinal cord explants, NRG-1 and PDGF, but not LIF, enhance myelination. PDGF has also been shown to be a potent regulator of oligodendrocyte progenitor migration and proliferation in oligodendrocyte cultures. IGF-1 acts on myelin-forming cells to promote normal myelination and remyelination after injury. Thus, in experimental MS models, the neuregulin isoform GGF-2, IGF-1, and several neurotrophins promote remyelination following inflammatory demyelination. Differentiation-stimulating effects of IGF-1 on oligodendrocyte precursors have also been detected
in vitro, in which the addition of IGF-1 induced a high proportion of precursor cells to differentiate into galactocerebroside-positive oligodendrocytes, whereas the proportion of type 2 astrocytes was unaffected. In addition, IGF-1 promotes proliferation of O-2A precursor cells. Also in myelinating Schwann cells, growth factors released from glial cells are involved in remyelination after damage. Thus, in neuron–Schwann cell cocultures, GGF-1, an NRG-1 isoform, inhibits myelination by preventing axonal segregation and ensheathment. In addition, treatment of established myelinated cultures with GGF-1 results in demyelination that frequently begins at the paranodes and progresses to the internodes. In contrast, FGF-2 and TGF-b1 inhibited myelination but did not cause demyelination, suggesting that this effect is specific to the NRGs. The NRG receptor proteins erbB2 and erbB3 are expressed on ensheathing and myelinating Schwann cells and rapidly phosphorylate upon GGF-1 treatment. In Schwann cells, both NRG-1b and -1a are colocalized in cytoplasm and its processes. The Schwann
Glial Growth Factors
cell nucleus has weak immunoreactivity for both NRG-1 isoforms, although NRG-1b is predominant. ErbB2 and ErbB3 receptors, transducing the NRG-1 signal in Schwann cells, are found throughout cytoplasm and processes and are also localized in the nucleus. Stimulation of Schwann cells with mitotic agents induces nuclear translocation of NRG-1b. Regulation of Radial Glia Differentiation
Little information is available on the role of glial growth factors in the differentiation of radial glia. Thus, in the mammalian retina proliferation of Muller glia is stimulated by EGF in a dose-dependent manner, whereas astrocyte proliferation is stimulated by FGF-1 and FGF-2. In contrast, proliferation of glial precursor cells is stimulated by FGF-1, FGF-2, and PDGF, but not by EGF. The inhibitory role of FGF signaling for Muller cell differentiation is further supported by the fact that inhibition of the endogenous FGF receptor by cotransfecting a dominant-negative form results in an increased number of Muller cells, suggesting a balance between FGF signaling and other signaling cascades to modulate retinal precursor cell fate. Growth Factors in Glial Brain Tumors
Genetic changes in glial cells and their precursors lead to some of the most devastating human brain tumors, such as glioma, glioblastoma, and astrocytoma. During the process of tumor transformation, para- and autocrine stimulation of tumor cell proliferation is a key mechanism for tumor growth providing at the same time a starting point for pharmacological intervention of this process. Glioma cells are among the oldest known sources for a number of different growth factors, such as FGF-2 and GDNF, which have also been shown to affect glial cell proliferation in an autocrine manner. Thus, glioma cells express high levels of FGF-2 protein and high-affinity FGF receptors and therefore are mitogenically responsive to FGF-2. Likewise, a knockdown of GDNF or its receptor, GFR-a1, by antisense RNA results in reduced proliferation of rat C6 glioma cells, suggesting a role in autocrine growth stimulation. A growth factor that has been implicated in the regulation of glioblastoma growth is PDGF, which is coexpressed with its receptor in this tumor type and has been shown to regulate tumor cell proliferation. Because it is a secreted factor, PDGF not only has autocrine effects on producing cells but also paracrine actions on other tumor cells and on the tumor microenvironment. Thus, PDGF is involved in the regulation of tumor cell migration and tumor angiogenesis. In addition, human glioblastoma cells show a general
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increase in the expression of autocrine growth regulators such as TGF-a, TGF-b, and FGF-2 compared to normal human brain tissue. Also in astrocytomas, activity and regulation of a number of mitogenic signaling pathways is aberrant. Thus, upregulation of growth factor receptors such as EGFR, PDGFR, and c-Met, as well as signaling intermediates such as Ras, protein kinase C, and others which have been shown to positively regulate tumor proliferation and cell cycle progression, has been demonstrated.
Effects of Glial Growth Factors on Differentiation and Function of Neurons in Both Healthy and Injured Nervous Systems Glial growth factors are important regulators of normal neuronal differentiation and of neuron survival in the developing and adult brain. Disturbances of glial growth factor release are therefore supposed to play a role during CNS and PNS lesioning and in the etiology of various neurological disorders (Figure 1). Regulation of Neuronal Differentiation during Development
Glial growth factors are important for the migration of neuronal progenitors during development. Thus, intraventricular injection of NT-4 or overexpression of BDNF lead to defects in cortical layering. Cortical layering defects can also be observed in FGF-2 knockout mice. GGF-1 promotes the migration of neuronal progenitors along radial glial fibers. Glial growth factors can also influence axonal pathfinding, as shown for netrin-1 in the internal capsule. Netrin-1 is released from oligodendrocytes but not astrocytes. Together with netrin-1, engrailed-1 is directing axons of association neurons, projecting ipsilaterally to motor neurons in the spinal cord. Consistent with this, axonal pathways are altered in netrin-1-deficient mice. A number of glia-derived growth factors, such as TGF-a, have been shown to induce neuritogenesis. An important role for glial growth factors is the regulation of neuron survival during periods of ontogenetic neuron death. Thus, FGF-2 can prevent neuron loss in the chick ciliary ganglion. Ciliary neurotrophic factor is also able to rescue motor neurons in the embryonic chick lumbar spinal cord during ontogenetic cell death. Moreover, TGF-bs enhance survival of chick ciliary ganglionic neurons in vitro synergistically with different neurotrophins and CNTF. Surprisingly, immunoneutralization of TGF-bs during chick development in vivo enhances neuronal survival.
622 Glial Growth Factors Glia-Derived Growth Factors during Puberty and Aging
A late developmental event is puberty, during which gonadal steroids induce plastic changes in certain brain regions. In hypothalamic astrocytes, this is accompanied by the production of TGF-a and neuregulins, which elicit astroglial secretion of prostaglandin E2, stimulating the release of neuronal luteinizing hormone-releasing hormone. Hence, overexpression of TGF-a in the hypothalamus accelerates puberty, whereas blockade of TGF-a or neuregulin delays this process. Growth factor synthesis by glial cells is also changed during aging, in which, together with astrogliosis, a continuous upregulation of TGF-b1 can be observed. Also, a decrease in NGF has been observed in a senescence accelerated mouse strain. Likewise, in aging mice an increase in IL-6 and a decrease in IL-10 release from glial cells can be observed. Glia-Derived Growth Factors during Neurodegenerative Diseases
Neuronal degeneration during Parkinson’s disease (PD) and Alzheimer’s disease (AD) is accompanied by astrogliosis and thus upregulation of several gliaderived growth factors. On the other hand, some glial growth factors are downregulated in these diseases, such as FGF-2 expression, which is reduced in the substantia nigra of PD patients, and NGF expression, which is reduced in the hippocampus of both PD and AD patients. In vitro studies on growth factors such as FGF-2, GDNF, CNTF, and BDNF suggest that they
are important for survival of dopaminergic midbrain neurons. According to this, altered levels of glial growth factors are common in postmortem samples of the brains of AD patients. Thus, b-amyloid of senile plaques induces IL-1b and FGF-2, probably stabilizing the disease conditions. Whereas FGF-2 is expressed in astrocytes in the center of senile plaques, FGF-1 can be found in astrocytes surrounding the plaque. Also, other growth factors are upregulated in astrocytes of the brains of AD patients, such as endothelin-1, TGF-b2, IGF-1, and hepatocyte growth factor. In contrast, expression of BDNF is reduced in astrocytes surrounding senile plaques. Role of Glia-Derived Growth Factors in Neural Regeneration in the Central Nervous System
It has long been known that neuron regeneration after lesioning is enhanced by soluble proteins released from glial cells (Table 1). Thus, NGF prevents death and promotes fiber growth of transected cholinergic neurons. Similarly, the release of IGF-1 from astrocytes is involved in neuron regeneration after cuprizone-elicited demyelination of the CNS. Notably, IGFs are also potent regulators of remyelination following CNS damage. Glia-derived factors such as Nogo are also involved in the inhibition of axonal regeneration after spinal cord lesioning. Inhibition of the Nogo receptor could provide a means to overcome this obstacle to neuronal regeneration after spinal cord transection. However, Nogo is not the only axon outgrowth inhibitor
Table 1 Growth factor expression in different glial subpopulations SC
NGF BDNF NT-3 NT-4 CNTF LIF TGF-b1 TGF-b2 TGF-b3 GDNF TGF-a Neuregulins PDGF FGF-2 IGF-1
Oligodendrocytes
Astrocytes
Normal
Lesioned
Normal
Lesioned
Normal
Reactive
þþ þ þþ þ þþþ þ þþ þþþ þ þþ þþ þþ nd
þþþþ þþþ þþ þ þþ þþþ þ þ þþþ þþþþ nd nd nd nd
þþþ þþþ nd nd nd þ nd nd nd nd nd nd nd nd
nd nd nd nd nd þþþ nd nd nd nd nd nd nd nd
þþþ þþ þþ þ þ þþ þþ þ þþ nd þ nd
þþþþ þþþ nd nd þþþ þþþ þþþ nd nd þþþ nd nd nd þþþ nd
MG
BG
þþ þþ þþ nd þþ nd þþ þþ nd nd nd nd þþ þþ
nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd
BDNF, brain-derived neurotrophic factor; BG, Bergann glia; CNTF, ciliary neurotrophic factor; FGF-2, fibroblast growth factor-2; GDNF, glial cell line-derived neurotrophic factor; IGF-1, insulin like growth factor 1; LIF, leukemia inhibitory factor; MG, Mu¨ller glia; nd, not determined; NGF, nerve growth factor; NT, neurotrophin; PDGF, platelet-derived growth factor; SC, Schwann cells; TGF, transforming growth factor.
Glial Growth Factors
since other astroglial molecules, such as collagen IV, are similarly active. Also in brain regions in which axonal regeneration occurs throughout life, astrocyte-derived growth factors such as NGF, BDNF, GDNF, and neurturin seem to be involved since transplantation of astrocytes from the olfactory bulb to the spinal cord promotes axon regrowth after transection. PDGF upregulation in astrocytes of the facial nucleus after transection of the facial nerve is important for neuronal regeneration. Similarly, astroglial CNTF expression is increased in fields of axonal sprouting in the deafferented hippocampus after entorhinal cortex lesioning. Likewise, BDNF, NT-3, and GDNF are induced in astrocytes of the spinal cord adjacent to a lesion. Role of Glia-Derived Growth Factors in Regeneration of Peripheral Nerves
Transection of a peripheral nerve leads to apoptotic cell death of dorsal root ganglion neurons. This is probably due to a reduction in the expression of neurotrophins and neurotrophin receptors, and as a consequence, exogenously applied growth factors are able to counteract these effects. An important endogenous source for growth factor release in the distal nerve stump after peripheral nerve transection is Schwann cells, which release NGF, BDNF, NT-4/5, and p75. In addition to these factors, CNTF and LIF are needed for axonal regeneration, which act synergistically on nerve regeneration. Also, GDNF is upregulated in Schwann cells of the distal nerve stump
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after nerve injury and remains so for more than 5 months. See also: Gap Junctions and Hemichannels in Glia.
Further Reading Aloisi F (2003) Growth factors. Neurological Science 24 (Supplement 5): S291–S294. Bo¨ttner M, Krieglstein K, and Unsicker K (2000) The transforming growth factor-betas: Structure, signaling, and roles in nervous system development and functions. Journal of Neurochemistry 75: 2227–2240. Campbell K (2003) Signaling to and from radial glia. Glia 43: 44–46. Cui Q (2006) Actions of neurotrophic factors and their signaling pathways in neuronal survival and axonal regeneration. Molecular Neurobiology 33: 155–179. Decker L, Lachapelle F, Magy L, et al. (2005) Fibroblast growth factors in oligodendrocyte physiology and myelin repair. Ernst Schering Research Foundation Workshop 53: 39–59. English AW (2003) Cytokines, growth factors and sprouting at the neuromuscular junction. Journal of Neurocytology 32: 943–960. Miller RH (2002) Regulation of oligodendrocyte development in the vertebrate CNS. Progress in Neurobiology 67: 451–467. Panickar KS and Norenberg MD (2005) Astrocytes in cerebral ischemic injury: Morphological and general considerations. Glia 50: 287–298. Reuss B and von Bohlen und Halbach O (2003) Fibroblast growth factors and their receptors in the central nervous system. Cell and Tissue Research 313: 139–157. Schwab ME (2002) Repairing the injured spinal cord. Science 295: 1029–1031. Strelau J and Unsicker K (2003) Neuroregeneration. Advances in Neurology 91: 95–100. Teismann P, Tieu K, Cohen O, et al. (2003) Pathogenic role of glial cells in Parkinson’s disease. Movement Disorders 18: 121–129.
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ATYPICAL NEUROTRANSMITTERS A. Purines
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B. Endoannabinoids
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C. Gases
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Adenosine K A Jacobson and Z-G Gao, National Institutes of Health, Bethesda, MD, USA ã 2009 Elsevier Ltd. All rights reserved.
Introduction The behavioral stimulant effects of the alkylxanthine caffeine and related xanthines are well known. The mechanism of these effects at moderate xanthine doses is now known to be through antagonism of one or more subtypes of adenosine receptors (ARs), rather than through inhibition of phosphodiesterases or stimulation of calcium release, which occur only at higher concentrations of caffeine. At very high doses, caffeine depresses locomotor activity, which does not appear to be related to ARs. Adenosine, an endogenous agonist of the ARs, acts as a local modulator of the action of various neurotransmitters, including biogenic amines and excitatory amino acids. In fact, adenosine modulates the release of many, if not most, neurotransmitters, both in the brain and in the peripheral nervous system. The ARs activated by extracellular adenosine are classified as four subtypes: A1, A2A, A2B, and A3 (Figure 1). A2A and A2B ARs are relatively close in sequence identity (59%, for the human homologs), as are A1 and A3 ARs (49%, for the human homologs). Typically, adenosine has an affinity of 10–30 nM at the high-affinity binding sites of the A1 and A2A ARs. A2B AR has the lowest affinity (high micromolar) of all of the subtypes for adenosine, and its affinity at the A3 AR is intermediate. The classical second messengers associated with these subtypes are stimulation of adenylate cyclase through Gs proteins for the A2A and A2B subtypes and inhibition of adenylate cyclase through Gi proteins for the A1 and A3 subtypes. In recent years, it has become apparent that other effector mechanisms are important in the physiological actions of adenosine, such as phosphoinositide 3-kinase and mitogen-activated protein kinases (MAPKs), which may be activated through the b and g subunits of the G-proteins. In general, the role of adenosine in the brain and peripherally is protective: that is, when released or produced in response to organ stress or tissue damage, adenosine tends to increase the ratio of oxygen supply to oxygen demand. It also suppresses various cytotoxic processes, such as cytokine-induced apoptosis. In the brain, both neuronal and glial cell functions are regulated by adenosine. In the autonomic and enteric nervous systems, adenosine has been shown to modulate neurotransmitter release and
the interaction of neurotransmitters with receptors. ARs are critical in controlling bladder function via a neurological mechanism. In the intestine, adenosine also plays an essential role in modulating the function of neurons located in the myenteric plexuses between muscle coats and submucous plexus. In the peripheral immune system, adenosine has been shown to ‘put the brakes’ on excessive inflammation and to promote tissue protection against ischemic damage. In the cardiovascular system, adenosine promotes vasodilation, vascular integrity, and angiogenesis, and counteracts the lethal effects of prolonged ischemia on cardiac myocytes. Nearly every cell type in the body expresses one or more of the subtypes of ARs, indicating the fundamental nature of adenosine as a cytoprotective mediator in both the peripheral and the central nervous systems. Medicinal chemists have succeeded in designing and synthesizing for the ARs numerous selective and potent ligands that act competitively with adenosine at the orthosteric site of the receptors. Highly selective agonists and antagonists of the A1, A2A, and A3 ARs have been reported both as research tools and as experimental therapeutic agents, but for the A2B ARs only selective antagonists are currently known. Some of these selective agents are excluded from the central nervous system when administered peripherally, due to lack of penetration of the intact blood–brain barrier. Thus, these are important pharmacological probes for separating the central and peripheral actions of adenosine. It should be noted that nucleosides, such as adenosine and its analogs, by virtue of the hydrophilic 9-ribose moiety, tend to enter the brain only to a small degree, if at all. Most of the adenosine antagonists are nonnucleoside heterocyclic derivatives, which tend to be more hydrophobic than are the adenosine agonists. Therefore, a given adenosine agonist cannot be assumed to enter the brain in sufficient amounts to exogenously stimulate one of the ARs, unless there is supportive evidence for its action in the central nervous system. The half-life of adenosine in peripheral circulation can be as short as 1 s, and therefore its peripheral administration is unlikely to affect the concentration of extracellular adenosine in the brain. In contrast, a significant number of adenosine antagonists enter the brain when administered peripherally. The simple xanthines, caffeine and theophylline, readily permeate the blood– brain barrier. Envisioned therapeutic applications of AR agonists and antagonists include treatment of inflammatory bowel diseases, arrhythmia, bladder dysfunction, ischemia, neurodegenerative diseases (such as Parkinson’s
627
628 Adenosine ATP
ADP
AMP
Ado
ATP
ADP
AMP
Ado
A2A
A1 g
ai
b
(−)
g
Ino
b
PI3K
K+
(+)
PKB/AKT
cAMP
PKA
as
Nucleus
Gene expression
CREB
CREB
cAMP
PKA
MAPK NFk B Ca+ PKC
PKB/AKT
(+)
(−) PLC
ai
b g
A3
PI3K
aq
b
as
g A2B
Figure 1 Signaling pathways associated with the four subtypes (A1, A2A, A2B, and A3) of adenosine receptors. ATP, adenosine triphosphate; ADP, adenosine diphosphate; AMP, adenosine monophosphate; Adp, adenosine; Ino, inosine; CREB, cyclic AMP response element-binding protein; MAPK, mitogen-activated protein kinase; NFkB, nuclear factor-kappa B; PI3K, phosphatidylinositol 3-kinase; PKA, protein kinase A; PKB, protein kinase B; PKC, protein kinase C; PLC, phospholipase C.
disease and Alzheimer’s disease), pain, psychiatric disorders, and sleep disorders. The use of both genetic deletion of the receptor (now each of the subtypes has been deleted in mice strains) and selective agonists/ antagonists has contributed toward these concepts.
Sources of Adenosine, Its Transport Mechanisms, and Metabolism Adenosine production in the synapse is not through vesicular release in response to nerve firing, as is the case for classical neurotransmitters. Rather, adenosine acts as a local autacoid, the release of which increases upon stress to an organ or tissue. Most cells in culture and in situ produce adenosine and release it extracellularly, which tends to influence the outcome of pharmacological studies if not properly controlled. The levels of extracellular adenosine in a given tissue or organ may vary widely, depending on stress factors present, leading to highly variable basal levels of stimulation of the ARs by endogenous adenosine. The source of extracellular adenosine may be both from inside the cell, where it is present in millimolar
concentrations, and from the breakdown of extracellular adenine nucleotides, which activate signaling pathways through their own superfamily of receptors (P2Y metabotropic and P2X inotropic receptors). Several nucleotide precursors of adenosine, such as ATP and adenosine diphosphate (ADP), act at P2 receptors, but the monophosphate, AMP, which does not act at any of the P2 receptors, is weakly active as an agonist of ARs. The level of extracellular adenosine may be as low as 20 nM in the resting brain and as high as 100 mM in severe ischemic conditions in the brain or elsewhere in the body. As a hydrophilic small molecule, adenosine does not diffuse across the plasma membrane freely; rather, it may pass through an equilibrative transporter such as the ENT1 nucleoside transporter. Levels of extracellular adenosine may also rise as a result of the enzymatic hydrolysis of extracellular adenine nucleotides by widely occurring ectonucleotidases or as a result of cell lysis. Ectonucleotidases, which are also ubiquitously expressed on the cell surface but with characteristic distribution patterns, cleave the phosphate moieties of adenine nucleoside 50 -phosphate derivatives, to culminate in the formation of adenosine. There are many classes
Adenosine 629
of ectonucleotidases; however, the most relevant species in the family of ectonucleoside triphosphate dihydrolases (E-NTPDases) are apyrase (NTPDase1, which converts ATP and ADP to AMP) and NTPDase2 (which converts ATP to ADP). A separate enzyme ecto50 -nucleotidase (CD73) converts AMP to adenosine. CD73 is characteristically found on the surface of astrocytes but not of neurons. Unlike classical neurotransmitters, adenosine does not have a rapid synaptic uptake system (as for the biogenic amines), or a rapid chemical inactivation system (as for acetylcholine). Rather, adenosine is metabolized extracellularly by widespread enzymes adenosine kinase (AK; to produce AMP) and adenosine deaminase (to produce inosine) and therefore is inactivated with respect to the ARs. The metabolite inosine may have its own biological actions. For example, there is evidence that inosine activates the A3 AR. Inhibition of the metabolism of extracellular adenosine or its uptake proteins is being explored for therapeutic purposes. AK inhibitors have been proposed for the treatment of pain and seizure; however, the promising clinical development of these efficacious compounds has been discontinued due to their toxicity.
Receptor Structure, Signaling Pathways, and AR Regulation Receptor Structure
All of the ARs are G-protein-coupled receptors (GPCRs), which structurally consist of seven transmembrane helices connected by extracellular and cytoplasmic loops. There is a strong sequence homology between A1 and A3 ARs and between A2A and A2B ARs. The neurotransmitter receptors that are closest in primary sequence to the ARs are the biogenic amine receptors. Each of the ARs has been modeled both in overall three-dimensional structure and in the nature of molecular recognition in the putative ligand binding site, based on homology to bovine rhodopsin or by other methods, such as the recently developed threading assembly refinement method. Essential residues for the binding of agonists and antagonists and for the activation of the receptors have been defined through modeling and mutagenesis. Use of the molecular models to design de novo new ligands has not yet met with extensive success. However, confidence in the receptor models and in their predictive abilities has increased gradually since the first models were reported in the early 1990s. The neoceptor approach to reengineering GPCRs in general, and ARs specifically, has validated binding site hypotheses for adenosine at its receptors.
The binding and activation steps of receptor action have been dissected computationally, although not yet in a global fashion, largely due to the lack of a suitable template for the activated conformation. The conformational dynamics of the activation of the A3 AR have been approximated with respect to isolated portions of the receptor. Signaling Pathways
Two AR subtypes, A1 and A3, couple through Gi to the inhibition of adenylate cyclase, while the other two subtypes, A2A and A2B, stimulate adenylate cyclase through Gs or Golf (for A2A). The A2B AR is also coupled to activation of phospholipase C through Gq. Furthermore, each of these receptors may couple through the b and g subunits of the G-proteins to other effector systems, including ion channels and phospholipases. ARs have been found to couple to MAPKs in a variety of circumstances, leading to effects on differentiation, proliferation, and cell death. The A3 AR can reduce apoptosis through the activation of Akt. ARs can couple to ion channels; for example, activation of the A1 AR can induce the influx of calcium ions or the efflux of potassium ions. Cross-talk occurs between ARs and other receptors. For example, an otherwise subthreshold concentration of acetylcholine (ACh), as might be present in the Alzheimer’s brain, still produces a strong calcium signal when the A1 AR is costimulated. Cross-talk occurs with the striatal dopamine receptor system, in which a direct physical association (dimerization) occurs between A2A and D2 receptors, and between other subtypes. A2A AR stimulation also transactivates the neuroprotective Trk neurotrophin receptor pathway, even in the absence of neurotrophic growth factor. Recently, heterodimers of adenosine A1 receptors and either P2Y1 or P2Y2 nucleotide receptors have been characterized pharmacologically. Regulation
Similar to the function and regulation of other GPCRs, both activation and desensitization of the ARs occur after agonist binding. Interaction of the activated ARs with the G-proteins leads to second-messenger generation and classical physiological responses. Interaction of the activated ARs with G-protein receptor-coupled kinases (GRKs) leads to their phosphorylation. Downregulation of ARs should be considered in both the basic pharmacological studies and with respect to the possible therapeutic application of agonists. AR responses desensitize rapidly, and this phenomenon is associated with receptor downregulation, internalization, and degradation. Mutagenesis has been applied to analyze the molecular basis for the differences in the kinetics
630 Adenosine
of the desensitization response displayed by various AR subtypes. The most rapid downregulation among the AR subtypes is generally seen with the A3 AR, due to phosphorylation by GRKs.
Novel and Definitive Ligands Potent and selective AR antagonists have been prepared for all four AR subtypes, and selective agonists are known for three subtypes. Thus, numerous pharmacological tools are available for in vitro and in vivo use. Potent and selective A2B AR agonists are yet to be reported, although several research groups have identified lead compounds. Molecular modeling of the ARs and ligand docking have provided insights into the putative binding sites of all of the subtypes, which has aided in ligand design. Adenosine Agonists
Until recently, nearly all AR agonists have been purine nucleoside derivatives. With the synthesis of pyridine3,5-dicarbonitrile derivatives, there is now an example of a nonnucleoside chemical class that fully activates ARs. Medicinal chemists have extensively explored the structure–activity relationships of adenosine derivatives as agonists of the ARs (Figure 2). In general, for the adenine moiety of adenosine, modifications at the N6 position have led to selectivity for the A1 AR and the A3 AR, and modifications at the 2 position, especially with ethers, secondary amines, and alkynes, have led to selectivity for the A2A AR. Commonly used A1 AR agonists that are N6-cycloalkyl derivatives are cyclopentyladenosine (CPA), its 2-chloro analog CCPA, and endo-norbornyl adenosine (S()-ENBA) (Figure 2(a)). At the human ARs, S()-ENBA is more highly A1 AR-selective than are CPA and CCPA. Adenosine amine congener (ADAC) is an amine-functionalized congener designed for covalent coupling to carriers with the retention of A1 AR activity. The A2A AR-enhancing effects of substitution at the 2 position are further boosted with a uronamido substitution at the 50 position of adenosine (Figure 2(b)). For example, the 50 -N-ethyl derivatives CGS21680 and ATL146e are both selective in binding to the rat A2A AR and less selective at the human subtypes. When the 50 -N-alkyluronamide group alone is present, high affinity at the A2A AR, but not selectivity, is typically observed, as with the nonselective agonist 5’-N-ethylcarboxamide adenosine (NECA). The N6-substituted A1 AR agonists NNC-21-0136 and GR79236 and the doubly substituted selodenoson have been clinical candidates. A1 AR agonists are of interest for use in treating cardiac arrhythmias (for which adenosine itself, under the name Adenocard, is in widespread use). AMP579 is a carbocyclic nucleoside that activates both A2A and A1
ARs. A2A AR agonists such as ATL-146e, binodeson, CVT-3146, and MRE0094 have been clinical candidates. Such agonists are of interest for use as vasodilatory agents in cardiac imaging and in suppressing inflammation. Numerous exceptions to the preceding generalizations concerning selectivity of specific substitution sites on adenosine have been found. For example, the mono-N6-substituted derivative of adenosine, N6-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)-ethyl] adenosine (DPMA), is potent and selective at the rat A2A AR. At the human ARs, the A2A AR and A1 AR affinities of DPMA are roughly equal. N6-Arylmethyl derivatives of adenosine, such as N6-benzyl derivatives, tend toward selectivity for the A3 AR. MRS3997, an adenosine derivative that is monosubstituted at the 2 position with a (6-bromotryptophol)ether moiety, is a promising lead for the A2B AR. It is to be noted that MRS3997 fully activates human A2A and A2B ARs equipotently. The A3 agonists (Figure 2(c)) IB-MECA and ClIB-MECA are widely used as selective agonists of the A3 AR, although yet more selective agents are now known (e.g., the conformationally locked (N) methanocarba derivative MRS3558 and the 40 -thio nucleoside LJ568). CP-608039 is a selective A3 agonist that was developed for cardioprotection. Adenosine Antagonists
The structures of A3 antagonists are typically more chemically diverse than are the classical xanthine antagonists of the A1 and A2 receptors (Figure 3). The dihydropyridine derivatives MRS1191 and MRS1334 and the pyridylquinazoline derivative VUF5574 are potent, selective A3 AR antagonists in the human, but are weak or inactive at the rat A3 AR. Nevertheless, MRS1191 has been used successfully in murine species. MRS1220 is very potent at the human but not at the rat or the mouse receptor. The pyridine derivative MRS1523 and the nucleosides MRS1292, however, are selective A3 AR antagonists in both rats and humans. Selective A3 AR antagonists, such as the aforementioned compounds and the heterocyclic derivatives OT-7999 and MRE-3008-F20 and the adenine derivative MRS3777, are in general of interest for the treatment of cancer, stroke, inflammation, and glaucoma. Variations in the relative efficacy of nucleosides, depending on structure, have been noted. This phenomenon is especially pronounced for the A3 AR, at which changes on the adenine moiety (N6 and 2 positions) and ribose moiety can either reduce efficacy to the point of pure antagonism (i.e., combination of 2-Cl and N6-(3-iodobenzyl)) or can guarantee
Adenosine 631
a robust, nearly full activation of the A3 AR (i.e., 50 -uronamide). Although the classical AR antagonists are xanthines derivatives such as caffeine and theophylline, today
an enormous diversity of heterocyclic structures have been reported as AR antagonists. The micromolar affinity of the naturally occurring antagonists has been greatly exceeded with the introduction of selective
Nonselective agonists NH2 N
N
N
N
N HO
S
NH2
Cl
N
N
CH2CH3
HN
O
C2H5NHCO
HO
O
N
OH
Adenosine, Adenocard
N
N
HO
OH C2H5NHCO
NECA
HO
OH
AMP579 A1 agonists
H NH
N
N
N HO
N
N HO
O
N
N N
N
N
HO
NH
NH N
N R
NHCOCH2
O
H2N(CH2)2NHCOCH2 O HO
HO
OH
HO
R = H CPA 2.3
OH
S(-)-ENBA 0.38
R = Cl CCPA 0.82
ADAC
CH3
0.85 (r)
OH S
N
N Cl
S
NH
N
N
HO
HN
N
NH
N
N
HO HO
HO
OH
NNC-21-0136 10 (r) a
N
N C2H5NHCO
O
O
HO
Figure 2 Continued
N
N
O
N
N
OH
OH
GR79236 3.1 (r)
RG 14202, selodenoson 1.1 (r)
OH
632 Adenosine A2A agonists
NH2
CH3O
N
N RCO(CH2)2
(CH2)2NH
N CH3NHCO
N
N
N
N
N HO
O
O
CH3
HN
HO
N
N
HO
OH
OH
CVT-3146 290
R = OH, CGS21680 15 (r)
N
N HO
N
N
CH3CH2NHCO
CH3O
NH2
R = H2N(CH2)2NH, APEC 5.7 (r)
O DPMA 153 HO
OH
NH2 NH2 N
N N
CH2C
C
N O
CH3CH2NH
N
N
N Cl
(CH2)2O
HO
N
N
HO O
O
O
HO
NH2 N
N
CH3OC
N
N
NH HO
O
HO
OH
OH
OH
MRE0094 59
ATL-146e 0.5 Binodenoson 270 A2B agonists O HN CO HN N
N NC
N O
CN
H2N
N
NH2
S
CH3CH2NH
HN
(CH2)2O
N
N
HO
N
O
O
HO
N
N
Br
O Bay 60-6583 ∼10
NH2
O
HO
OH
OH
Compound 12b 82
MRS3997 128
b Figure 2 Continued
antagonists of subnanomolar affinity. High selectivity of xanthines at the A1 (e.g., the epoxide derivative BG 9719 and the more selective BG9928; KW3902), A2A (e.g., KW6002), and A2B (e.g., MRS1706 and MRS1754) ARs has been achieved (Figure 3). Although xanthines that are selective for the A3 AR are not available, certain cyclized derivatives of xanthines, such as PSB-11, are A3 AR selective. In general, modifications of the xanthine scaffold at the 8 position
with aryl or cycloalkyl groups has led to selectivity for the A1 AR, and modifications at the same position with alkenes (specifically, styryl groups) has led to selectivity for the A2A AR. 8-Cyclopentyl-1, 3-dipropylxanthine (DPCPX) is highly A1 AR selective in the rat but less A1 AR selective among the human AR subtypes. The 8-styrylxanthine derivatives KW6002, CSC, and MSX are moderately potent A2A AR antagonists. Some 8-styrylxanthine derivatives,
Adenosine 633 A3 agonists I
I NHCH2
NHCH2 N
N
Cl
N
N
N
N
CH3NHCO
CH3NHCO
O
HO
N
N X
HO
OH
OH
X = O, Cl-IB-MECA (CF102) 1.4 IB-MECA (CF101) 1.8 X = S, LJ568 0.38 CH3
Cl
Cl N O
O
NHCH2
N
N R
Cl
N
N
CH3NHCO
N
N
HN
N
N
O CH3NH
O
HO H2N
HO
OH
CP-608039 5.8
MRS3558 0.29
c Figure 2 Structures of (a) nonselective and A1, (b) A2A and A2B, and (c) A3 adenosine receptor agonist probes used as pharmacological tools, and in some cases as clinical candidates. The Ki values (nM) in binding to the appropriate human adenosine receptor are indicated after the name (all are human adenosine receptors, unless indicated ‘R’ for rat). Values indicated for the A2B adenosine receptor are functional EC50 values. Bay 60-6583 is a nonnucleoside that potently and selectively activates the A2B adenosine receptor. MRS3997 is a mixed agonist for the A2A and A2B adenosine receptors.
especially CSC, have been discovered to inhibit monoamine oxidase-B as well. A persistent problem in the use of xanthine derivatives as AR antagonists of the A1 AR is their interaction at the A2B AR. Use of adenine derivatives, such as the inverse agonist WRC0571, provides A1 AR-selective antagonists having low affinity at the A2B AR. The triazolotriazine ZM241,385 and the pyrazolotriazolopyrimidines SCH-58261 and SCH442,416 are highly potent and selective A2A AR antagonists. SCH442,416 displays >23 000-fold selectivity for the human A2A AR (Ki, 0.048 nM), in comparison to human A1 AR, and an IC50 > 10 mM at the A2B and A3 ARs. A2A AR antagonists, such as the xanthine KW6002 and the nonxanthines SCH58261, SCH442416, VER6947, and VER7835, are of interest for use in treating Parkinson’s disease.
There is a marked species dependence of antagonist affinity at the A3 AR. Therefore, commonly used antagonists must be treated with caution in species other than humans. In general, one must be cognizant of potential species differences for both AR agonists and antagonists. Radioligands
Radioligands commonly used for the ARs are A1 agonist [3H]CCPA, antagonist [3H]DPCPX, A2A agonist [3H]CGS21680, antagonist [3H]ZM241,385 or [3H]SCH58261, A3 agonist [125I]I-AB-MECA, and antagonist [3H]PSB-11. Ligands for in vivo positron emission tomographic (PET) imaging of A1 and A2A ARs have been developed. For example, the xanthine [18F]CPFPX and the nonxanthine [11C]FR194921 have been developed as centrally active PET tracers
634 Adenosine
for imaging the A1 AR in the brain. Potent fluorescent ligands have been reported for A1, A2A, and A3 ARs.
under consideration for disease treatment. Such modulators, either positive enhancers or negative allosteric inhibitors, might have advantages over the directly acting (orthosteric) receptor ligands. The action of the allosteric compounds would depend on the presence of a high local concentration of
Allosteric Modulation
In addition to directly acting AR agonists and antagonists, allosteric modulators of agonist action are
A1 antagonists
O
H
CH3(CH2)2 N
N
R=
HO
R N
N
O
HN
HN N
N
N
(CH2)2CH3 N
N
DPCPX 3.9
N
N
N-0861 700
BG 9719 (CVT-124) 0.43
KW3902 0.72
(CH2)2
CO2H
CH(CH3)2
CH3
CH3 O
CH3
N
N
WRC-0571 1.7
O
O
H
CH3(CH2)2 N
CH3
N
N
N N
O
BG 9928(BIO-9002) 29
N
N (CH2)3F
N
CPFPX 4.4
FR194921 2.9
A2A antagonists O
NH2
CH3
CH3(CH2)n
N
N
HO
(CH2)2NH
N
N
N
N X
O
N
CH3(CH2)n
N
O
N
N
ZM241, 385 1.6
X = 3,4-(OMe)2, n = 1, KW6002 39 X = 3-Cl, n = 2, CSC 54 (r)
RO
NH2 N
O
N
N H2N
N
N
R = phenyl, VER 6947 1.1 R = 2-thienyl, VER 7835 1.7
C NHCH2-R O a Figure 3 Continued
N
N
O
N
(CH2)n N N
R = H, n = 2, SCH 58261 5.0 R = CH3, n = 3, SCH 442416 0.048
Adenosine 635 A2B antagonists O CH3(CH2)2 N
N
H3CCONH
NH
N O N
N
O
(CH2)3
H
CONH
N
N
R
N H
N
(CH2)2CH3
O
R = CN, MRS1754 2.0 OSI P 339391 0.5 R = COCH3, MRS1706 1.4 O
N
N
CF3
N-CH2
H3C
N
N
N
N
N
O
N
N
H
CH3(CH2)2 N
H
CH3(CH2)2 N O
O
O
CO
NH
(CH2)2CH3
(CH2)2CH3
O
CVT-6883 8.3
N SO3H N
N
O A3 antagonists
O MRE 2029-F20 3.0
H
CH3(CH2)2 N
H
PSB-1115 53
I
N N
NHCH2 N
N
C O
N
N
CH3CH2
O
N O
C
O
HN O
HO
O
H
O
CH3
H
R
N
H R = H, MRS1191 31
OH
MRS1292 29
VUF5574 4.0 CH3CH2
F3C
N
H
HN
O
N
N
N O
N
NH
N
N
CH3(CH2)3
N
N
N CH3
N
N MRS3777 47
N
CH3O
R = NO2,MRS1334 2.7
N
O
N
I
PSB-11 3.5
N H
NHCONH N
N
NHCH2
CH3(CH2)2 O
O CH3CH2S
N
N
O O(CH2)2CH3
N
N S
N
N CH3(CH2)2
N
OCH3
OT-7999 0.61
CH3CH2
N
N
HO MRE 3008-F20 0.82
MRS 1523 19
OH
LJ1251 4.2
b
Figure 3 Structures of selected (a) A1 and A2A and (b) A2B and A3 adenosine receptor antagonist probes used as pharmacological tools, and in some cases as clinical candidates. The Ki values (nM) in binding to the appropriate human adenosine receptor are indicated after the name (all are human adenosine receptors, unless indicated ‘R’ for rat).
adenosine, which often occurs in response to a pathological condition. In some cases (dependent on tissue, receptor subtype, and other conditions), one would wish to boost the adenosine effect, and therefore an allosteric enhancer would be useful; in other
cases, the elevated adenosine may be detrimental, in which case one would want to apply a negative modulator. Positive allosteric modulators have been explored for the A1 (benzoylthiophene derivatives) and A3 (imidazoquinoline derivatives) AR subtypes.
636 Adenosine
Role of ARs in Autonomic Nervous System Disorders Distribution
ARs are widely distributed in the autonomic and enteric nervous systems. Distribution of neural ARs in the human intestine has been investigated. Messenger RNAs (mRNAs) of subtypes of AR are differentially expressed in neural and nonneural layers of the jejunum, ileum, colon, and cecum. The A1 AR is expressed in jejunal myenteric neurons and colonic submucosal neurons. The A2A AR is also found in other neurons, but A2B AR immunoreactivity is more prominent than that of the A2A AR in myenteric neurons, nerve fibers, and glia. The A3 AR largely occurs in substance P-positive jejunal submucosal neurons and less in vasoactive intestinal peptide (VIP) neurons. The AR that mediates the relaxation of bladder strips induced by AR agonists such as CGS21680 and NECA has been classified as A2A; however, the highest mRNA levels are found for A2B transcripts in the bladder. However, reports of tissue distribution based on mRNA levels might not correspond to protein levels. Thus, quantitative determination with a potent and selective A2B AR radioligand may be necessary. Western blot analysis shows that all four ARs are expressed in the uroepithelium. A1 ARs are prominently localized to the apical membrane of the umbrella cell layer, whereas A2A, A2B, and A3 ARs are localized intracellularly or on the basolateral membrane of umbrella cells and the plasma membrane of the underlying cell layers. Various subtypes of ARs have also been detected in the heart, blood vessels, kidney, and other organs throughout the body. A1, A2A, and A2B ARs are all expressed on normal human airway smooth muscle cells, and both A1 and A2A ARs are expressed in vagal pulmonary C fibers. A1 and A2A ARs are highly expressed in gastric mucosa. In the brain, the A1 AR is widely expressed in almost all areas. Pre- and postsynaptic activation of the A1 AR inhibits synaptic transmission, in part by suppressing the release of excitatory transmitters. The A2A AR is less widely expressed, with greatest density in the striatum, nucleus accumbens, and olfactory tubercle. The A2B and A3 ARs are expressed at low density in most brain regions, and are implicated in purinergic signaling in neuronal–glial interactions.
Functions of ARs in the Autonomic and Enteric Systems The enteric nervous system contains several hundred million neurons located in the myenteric plexuses
between muscle coats and submucous plexus. ARs in the enteric nervous system are critical for the control of motor and secretomotor functions. Adenosine is known to suppress intestinal motility by activating putative neural A1 ARs in the small intestine. A2A and A2B ARs in the myenteric neurons were also suggested to contribute to effects of adenosine on motility. ARs in circular muscle may contribute to the postjunctional actions of adenosine on motility. Adenosine directly modulates intestinal tone in the rat by causing relaxation via A2B ARs or contraction via A1 ARs in longitudinal muscle cells. Electrophysiological studies in rodents provided evidence for pre- and postsynaptic A1 AR-mediated inhibition of slow synaptic transmission and presynaptic inhibition of fast synaptic transmission. A2B AR gene expression products are widely expressed in the mucosa of the human intestinal tract, where they are postulated to be involved in the pathophysiology of diarrheal diseases. Both A2A and A3 ARs have been found in mucosal tissues, suggesting that they influence secretion and/or absorption in the human intestine. The ARs are involved in neuroplastic changes occurring in inflamed gut. The A2A AR modulates the activity of colonic excitatory cholinergic nerves via facilitatory control on inhibitory nitrergic pathways, and such a regulatory function is enhanced in the presence of bowel inflammation. Activation of the A2A AR inhibits stress-induced gastric inflammation and damage. Thus, selective A2A AR agonists may be useful for preventing ulcers and gastric inflammation. Both A1 and A2A ARs are involved in gastrin release. The modification of AR expression by changes in intraluminal acidity may represent a novel regulatory feedback mechanism to control gastric acid secretion. The discharge of urine from bladder is controlled by the nervous system. The causes of bladder dysfunction related to the nervous system include multiple sclerosis, traumatic or developmental brain or spinal cord injury, or Parkinson’s disease. Although it is generally agreed that ACh acting on smooth muscle muscarinic receptors is the primary neurologic mechanism controlling bladder emptying, neural stimulation of the bladder is only partially inhibited in many cases by the muscarinic receptor antagonist atropine. The atropine-resistant component of parasympathetic contraction was later found to be ATP sensitive. There is plenty of evidence to suggest that ARs also play an important role in bladder function. It has been shown that adenosine-evoked membrane hyperpolarization and relaxation of bladder smooth muscle is mediated by A2A AR-mediated activation of KATP channels via adenylate cyclase and elevation
Adenosine 637
of cAMP. High mRNA levels of A2B transcript are also found in the bladder. Adenosine was found to be released from the uroepithelium, which was potentiated tenfold by stretching the tissue. It is generally accepted that the sensation of bladder fullness is relayed through the mucosal layer by afferent nerves, which are activated by the release of neurotransmitters such as ATP from the urothelium when it is stretched as the bladder fills. It is suggested that adenosine reduces the force of nerve-mediated contractions by acting predominantly at the presynaptic sites at the nerve muscle junction via the A1 AR. The autonomic nervous system, a complex and self-organized entity, plays a key role in regulating cardiovascular function. In the heart, a number of intrinsic nerves in the atrial and intra-atrial septum have been shown to release ATP, ACh, 5-hydroxytryptamine, and other neurotransmitters. Adenosine may regulate both the release and the interaction with their receptors of these neurotransmitters. Adenosinemediated myocardial protection has been suggested to be through a neurogenic pathway. The infarctreducing effect of intravenous adenosine in intact rats was blocked with both the ganglionic blocker
hexamethonium and the nitric oxide synthase inhibitor No-nitro-L-arginine. The A1 AR was determined to be the primary subtype involved in the modulation of norepinephrine release from cardiac nerve terminals using isolated rat hearts. Vascular tone in blood vessels is controlled by perivascular nerves and the endothelial cells. Adenosine acts directly on smooth muscles as well as modulates the release of neurotransmitters, such as ACh and ATP. A study of neurotransmitter release following electrical depolarization of nerve endings from the rat mesenteric artery suggests that activation of the presynaptic A2A AR and A3 AR modulates neurotransmission by inhibiting the release of norepinephrine but not neuropeptide Y. The A1 ARs, not P2 receptors, inhibit prejunctionally sympathetic neurotransmission in the hamster mesenteric arterial bed. Activation of both A1 AR and A2A AR is required to attenuate neurogenic coronary constriction due to sympathetic stimulation. In the respiratory system, adenosine has been shown to stimulate vagal pulmonary C fiber terminals through activation of the A1 AR, and subsequently cause bronchoconstriction, which was significantly attenuated by A1 AR antagonists. Recent evidence suggests
Table 1 Exploration of the role of adenosine receptors in disorders of the nervous system Condition/system
Model a
Subtypes implicated
Relationship
Aggression
A1 KO A2A KO b-Amyloid A2A KO Various Swim A3 KO Various 3-NP 3-NP A1 KO Ischemia Ischemia Ischemia A1 KO A2A KO A3 KO Constriction injury Formalin A1 KO A2A KO 6-OHDA MPTP Haloperidol ADA KO mice A2A KO
A1 A2A A2A A2A A1 A2A A3 A1 A1 A2A A1 A1 A2A A3 A1 A2A A3 A1 A2A A1 A2A A2A A2A A2A A2B A2A
Increased aggressiveness Increased aggressiveness Reduced neurotoxicity by antagonists Increased anxiety Agonist protects Antagonist improves Increased despair-like behavior Agonist protects in some models Agonist protects striatal damage Antagonist protects striatal damage No change Agonist protects Antagonist protects Chronic agonist protects No effect in adults, benefits newborns Detrimental in newborns, benefits adults Detrimental in adults Agonist protects Agonist protects Increased thermal nociception Lowers thermal nociception Antagonist protects Antagonist protects Decreased catalepsy by antagonists Antagonist protects Agonist lost effect
Alzheimers’s disease Anxiety Cardiac arrhythmias Depression Epilepsy Huntington’s disease Memory Neurodegeneration
Pain
Parkinson’s disease
Pulmonary inflammation Sleep disorders a
ADA, adenosine deaminase; KO, knockout; 6-OHDA, 6-hydroxydopamine; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; 3-NP, 3-nitropropionic acid.
638 Adenosine
that both A1 AR and A2A AR are involved in the activation of vagal sensory C fibers in the lung. The A1 AR has also been suggested to be directly involved in the mobilization of calcium in human bronchial smooth muscle cells. The A2B AR is known to mediate mast cell degranulation in large animals and humans, and A2B AR antagonists are of potential clinical applications for asthma. A1 AR agonists reduce pain signaling in the spinal cord, where the receptors are highly expressed. In humans, infusion of adenosine in the spinal cord is effective in decreasing postoperative pain. Recent studies suggest that A1 ARs might be more important in chronic pain than in acute pain. The A1 AR agonists are being evaluated in phase II clinical trials for the treatment of pain and migraine. An A1 ARselective allosteric enhancer is also used in clinical trials as a treatment for neuropathic pain. Peripherally administered A2A AR agonists have an antinociceptive effect. In summary, in the peripheral nervous systems, ARs are related to gut inflammation, rheumatoid arthritis, ischemia, Crohn’s disease, constipation, bladder disorders, and a variety of other conditions. In the brain, ARs appear to regulate important functions in cerebral ischemia, dementia, Parkinson’s disease and other neurodegenerative diseases, pain, sleep disorders, anxiety, and schizophrenia (Table 1). Genetic Deletion of ARs
Deletion of each of the four subtypes has been carried out, and the resulting single-AR knockout (KO) mice are viable and not highly impaired in function. The pharmacological profile indicates that the analgesic effect of adenosine is mediated by the A1 AR, and analgesia is lost in mice in which the A1 AR has been genetically eliminated. Genetic KO of the A1 AR in mice removes the discriminative-stimulus effects, but not the arousal effect, of caffeine, and increases anxiety and hyperalgesia. Study of A2A AR KO mice reveals functional interaction between the spinal opioid receptors and peripheral ARs. A1 AR KO mice demonstrate a decreased thermal pain threshold, whereas A2A AR null mice demonstrate an increased threshold to noxious heat stimulation, supporting an A1 AR-mediated inhibitory and an A2A AR-mediated excitatory effect on pain transduction pathways. KO of the A2A AR eliminates the arousal effect of caffeine. Genetic KO of the A2A AR also suggests a link to increased anxiety and protects against damaging effects of ischemia and the striatal toxin 3nitropropionic acid. Genetic KO of the A3 AR leads to increased neuronal damage in a model of carbon monoxide-induced brain injury. Neutrophils lacking A3 ARs show correct directionality but diminished
speed of chemotaxis. Although studies on A2B ARs KO mice have been reported, the importance of A2B ARs in the brain still awaits future investigation. In conclusion, adenosine is a ubiquitous neuromodulator of many functions, by activating one or more of the widely distributed AR subtypes. Selective AR agonists and antagonists have been developed, some of which are in advance stages of clinical trials for therapeutic applications. The use of both knockout animals and selective drugs has contributed toward elucidation of the physiological role of adenosine and the signaling pathways involved.
Further Reading Burnstock G (2007) Physiology and pathophysiology of purinergic neurotransmission. Physiological Reviews 87: 659–797. Christofi FL, Zhang H, Yu JG, et al. (2001) Differential gene expression of adenosine A1, A2a, A2b, and A3 receptors in the human enteric nervous system. Journal of Comparative Neurology 439: 46–64. Costanzi S, Ivanov AA, Tikhonova IG, et al. (2007) Structure and function of G protein-coupled receptors studied using sequence analysis, molecular modelling, and receptor engineering: Adenosine receptors. In: Frontiers in Drug Design and Discovery, vol. 3. Hilversum, The Netherlands: Bentham Science Publishers, Inc. Fields RD and Burnstock G (2006) Purinergic signalling in neuronglia interactions. Nature Reviews Neuroscience 7: 423–436. Franco R, Casado V, Mallol J, et al. (2006) The two-state dimer receptor model: A general model for receptor dimers. Molecular Pharmacology 69: 1905–1912. Fredholm BB, IJzerman AP, Jacobson KA, et al. (2001) International Union of Pharmacology. XXV. Nomenclature and classification of adenosine receptors. Pharmacological Reviews 53: 527–552. Fredholm BB, Chen JF, Masino SA, et al. (2005) Actions of adenosine at its receptors in the CNS: Insights from knockouts and drugs. Annual Review of Pharmacology and Toxicology 45: 385–412. Jacobson KA and Gao ZG (2006) Adenosine receptors as therapeutic targets. Nature Reviews Drug Discovery 5: 247–264. Linden J (2005) Adenosine in tissue protection and tissue regeneration. Molecular Pharmacology 67: 1385–1387. McGaraughty S, Cowart M, Jarvis MF, et al. (2005) Anticonvulsant and antinociceptive actions of novel adenosine kinase inhibitors. Current Topics in Medicinal Chemistry 5: 43–58. Moro S, Gao ZG, Jacobson KA, et al. (2006) Progress in pursuit of therapeutic adenosine receptor antagonists. Medicinal Research Reviews 26: 131–159. Ruggieri MR Sr. (2006) Mechanisms of disease: role of purinergic signaling in the pathophysiology of bladder dysfunction. Nature Clinical Practice Urology 3: 206–215. Schulte G and Fredholm BB (2003) Signalling from adenosine receptors to mitogen-activated protein kinases. Cell Signaling 15: 813–827. Yan L, Burbiel JC, Maass A, et al. (2003) Adenosine receptor agonists: From basic medicinal chemistry to clinical development. Expert Opinion in Emerging Drugs 8: 537–576. Yu L, Huang ZL, Mariani J, et al. (2004) Selective inactivation or reconstitution of adenosine A2A receptors in bone marrow cells reveals their significant contribution to the development of ischemic brain injury. Nature Medicine 10: 1081–1087.
Adenosine Triphosphate (ATP) G Burnstock, Royal Free and University College School of Medicine, London, UK ã 2009 Elsevier Ltd. All rights reserved.
Early History Drury and Szent-Gyo¨rgyi, in 1929, were the first to demonstrate the potent extracellular actions of adenosine 50 -triphosphate (ATP) and adenosine on the heart and coronary blood vessels. In 1948, Emmelin and Feldberg demonstrated that intravenous injection of ATP into cats caused complex effects that affected both peripheral and central mechanisms. Injection of ATP into the lateral ventricle produced muscular weakness, ataxia, and a tendency of the cat to sleep. Application of ATP to various regions of the brain produced biochemical or electrophysiological changes. Holton presented in 1959 the first hint of a transmitter role for ATP in the nervous system by demonstrating the release of ATP during antidromic stimulation of sensory nerves supplying the ear artery. Buchthal and Folkow recognized a physiological role for ATP at the neuromuscular junction in 1948, finding that acetylcholine (ACh)-evoked contraction of skeletal muscle fibers was potentiated by exposure to ATP. Presynaptic modulation of ACh release from the neuromuscular junction by purines in the rat was also reported. The existence of nonadrenergic, noncholinergic (NANC) neurotransmission in the gut and bladder was established in the mid-1960s. Several years later, after many experiments, Burnstock and his colleagues published a study that suggested that the NANC transmitter in the guinea pig taenia coli and stomach, rabbit ileum, frog stomach, and turkey gizzard was ATP. The experimental evidence included mimicry of the NANC nerve-mediated response by ATP; measurement of release of ATP during stimulation of NANC nerves with luciferin-luciferase luminometry; histochemical labeling of subpopulations of neurons in the gut with quinacrine, a fluorescent dye known to selectively label high levels of ATP bound to peptides; the later demonstration that the slowly degradable analogue of ATP, a,b-methylene ATP (a,b-meATP), which produces selective desensitization of the ATP receptor, blocked the responses to NANC nerve stimulation. Soon after, evidence was presented for ATP as the neurotransmitter for NANC excitatory nerves in the urinary bladder. The term ‘purinergic’ was proposed in a short letter to Nature in 1971, and the evidence for purinergic transmission in a wide variety of systems was presented in Pharmacological Reviews in 1972 (Figure 1). This
concept met with considerable resistance for many years. This was partly because it was felt that ATP was established as an intracellular energy source involved in various metabolic cycles and that such a ubiquitous molecule was unlikely to be involved in extracellular signaling. However, ATP was one of the biological molecules to first appear, and therefore it is not really surprising that it should have been utilized for extracellular, as well as intracellular, purposes early in evolution. The fact that potent ectoATPases were described in most tissues in the early literature was also a strong indication for the extracellular actions of ATP. Purinergic neurotransmission is now generally accepted, and a volume of Seminars in Neuroscience was devoted to purinergic neurotransmission in 1996.
Purinergic Cotransmission Another concept that has had a significant influence on our understanding of purinergic transmission was that of cotransmission. Burnstock wrote a commentary in Neuroscience in 1976 titled, ‘‘Do some nerves release more than one transmitter?’’ This position challenged the single-neurotransmitter concept, which became known as ‘Dale’s Principle,’ even though Dale himself never defined it as such. The commentary was based on hints about cotransmission in the early literature describing both vertebrate and invertebrate neurotransmission and more specifically, with respect to purinergic cotransmission, on the surprising discovery in 1971 that ATP was released from sympathetic nerves supplying the taenia coli as well as from NANC inhibitory nerves. The excitatory junction potentials (EJPs) recorded in the vas deferens were blocked by a,b-meATP, a selective desensitizer of P2X receptors (Figures 2(a) and 2(b)). This finding clearly supported the earlier demonstration of sympathetic cotransmission in the vas deferens in the laboratory of Dave Westfall, following an earlier report of sympathetic cotransmission in the cat nictitating membrane. Purinergic cotransmission was later described in the rat tail artery and in the rabbit saphenous artery. Noradrenaline (NA) and ATP are now well established as cotransmitters in sympathetic nerves (see Figure 3 (a)), although the proportions vary in different tissues and species, during development and aging, and in different pathophysiological conditions. ACh and ATP are cotransmitters in parasympathetic nerves supplying the urinary bladder. Subpopulations of sensory nerves have been shown to utilize ATP in addition to substance P and calcitonin generelated peptide; it seems likely that ATP cooperates
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Figure 1 Purinergic neuromuscular transmission depicting the synthesis, storage, release, and inactivation of adenosine 50 -triphosphate (ATP). ATP, stored in vesicles in nerve varicosities, is released by exocytosis to act on postjunctional P2 purinoceptors on smooth muscle. ATP is broken down extracellularly by ATPases and 50 -nucleotidase to adenosine, which is taken up by varicosities to be resynthesized and restored in vesicles. Adenosine acts prejunctionally on P1 purinoceptors to modulate transmitter release. If adenosine is broken down further by adenosine deaminase to inosine, it is removed by the circulation. Adapted from Burnstock G (1972) Purinergic nerves. Pharmacological Reviews 24: 509–581, with permission from the American Society for Pharmacology and Experimental Therapeutics.
with these peptides in axon reflex activity. ATP, vasoactive intestinal polypeptide and nitric oxide (NO) are cotransmitters in NANC inhibitory nerves, but that they vary considerably in proportion in different regions of the gut. More recently, ATP has been shown to be a cotransmitter with NA, 5-hydroxytryptamine, glutamate, dopamine and g-aminobutyric acid (GABA) in the central nervous system (CNS) (see Figure 3(b)). ATP and NA act synergistically to release vasopressin and oxytocin, which is consistent with ATP cotransmission in the hypothalamus. ATP, in addition to glutamate, is involved in long-term potentiation in hippocampal CA1 neurons associated with learning and memory.
Receptors for Purines and Pyrimidines Implicit in the purinergic neurotransmission hypothesis was the presence of purinoceptors. A basis for distinguishing two types of purinoceptor, identified as P1 and P2 for adenosine and ATP/adenosine diphosphate (ADP), respectively, was recognized in 1978. This helped resolve some of the ambiguities in earlier reports, which were complicated by the breakdown
of ATP to adenosine by ectoenzymes so that some of the actions of ATP were directly on P2 receptors, whereas others were due to indirect action via P1 receptors. At about the same time, two subtypes of P1 (adenosine) receptor were recognized, but it was not until 1985 that a pharmacological basis for distinguishing two types of P2 receptors (P2X and P2Y) was proposed. A year later, two further P2 receptor subtypes were named, a P2T receptor selective for ADP on platelets and a P2Z receptor on macrophages. Further subtypes followed, perhaps the most important being the P2U receptor, which could recognize pyrimidines such as uridine 50 triphosphate (UTP) in addition to ATP. However, to provide a more manageable framework for newly identified nucleotide receptors, Abbracchio and Burnstock proposed in 1994 that purinoceptors should belong to two major families: a P2X family of ligand-gated ion channel receptors and a P2Y family of G-protein-coupled receptors. This was based on studies of transduction mechanisms and the cloning of nucleotide receptors: P2Y receptors were cloned first, in 1993, and a year later P2X receptors were cloned. This nomenclature has been widely adopted, and currently seven P2X subtypes and eight P2Y
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b Figure 2 (a) EJPs in response to repetitive stimulation of adrenergic nerves (white dots) in the guinea pig vas deferens. The upper trace records the tension, the lower trace the electrical activity of the muscle recorded extracellularly by the sucrose gap method. Note both summation and facilitation of successive junction potentials. At a critical depolarization threshold, an action potential is initiated which results in contraction. (b) The effect of various concentrations of a,b-methylene ATP (a,b-meATP) on EJPs recorded from guinea pig vas deferens (intracellular recordings). The control responses to stimulation of the motor nerves at 0.5 Hz are shown on the left. After at least 10 min in the continuous presence of the indicated concentration of a,b-meATP, EJPs were recorded using the same stimulation parameters. (a) Reproduced from Burnstock G and Costa M (eds.) (1975) Adrenergic neuroeffector transmission. In Adrenergic Neurones: Their Organisation, Function and Development in the Peripheral Nervous System, pp. 51–106. London: Chapman and Hall, with permission from Springer Science and Business Media. (b) Reproduced from Sneddon P and Burnstock G (1984) Inhibition of excitatory junction potentials in guinea-pig vas deferens by a,bmethylene-ATP: Further evidence for ATP and noradrenaline as cotransmitters. European Journal of Pharmacology 100: 85–90, with permission from Elsevier.
receptor subtypes are recognized. Four subtypes of P1 receptor have been cloned and characterized. P2X receptors in general mediate fast neurotransmission but are sometimes located prejunctionally to mediate increase in release of cotransmitters, for example, glutamate in terminals of primary afferent neurons in the spinal cord. P2X3 and P2X2/3 receptors are prominent in sensory neurons and are involved in nociception. P2X7 receptors are involved in cell death. P2Y receptors are particularly involved in prejunctional inhibitory modulation of transmitter release, as well as long-term (trophic) events such as cell pro liferation. P2Y1 receptors are widespread in many regions of the brain, while the P2Y2 receptors have been
localized on pyramidal neurons in the hippocampus and prefrontal cortex, on supraoptic magnocellular neurosecretory neurons in the hypothalamus, and on neurons in the dorsal horn of the spinal cord. In addition, mRNA but not protein has been reported for P2Y4 and P2Y6 receptor subtypes in the cerebellum and hippocampus, while P2Y12 receptor mRNA has also been described in the cerebellum and P2Y13 in the cortex. In the periphery, P2Y1,2,4,6 receptors have been described on subpopulations of sympathetic neurons, P2Y2 and P2Y4 receptors in intracardiac ganglia, P2Y1 and P2Y2 receptors on sensory neurons (although P2Y4 and P2Y6 mRNA have also been reported) while P2Y1 receptors appear to be the dominant subtype on enteric neurons. P2Y1,2,4,6 functional receptors have been described on astrocytes in the CNS and also on microglia, where functional P2Y12 receptors have also been identified. P2Y1 and P2Y2 receptors have been located in Schwann cells and oligodendrocytes, where functional P2Y12 receptors also appear to be present. P2Y2 (and/or P2Y4) receptors are expressed on enteric glial cells. There is also emerging evidence for P2Y receptors on stem cells.
ATP Release and Degradation There is clear evidence for exocytotic vesicular release of ATP from nerves, and the concentration of nucleotides in vesicles is claimed to be up to 1000 mmol l1. It was generally assumed that the main source of ATP acting on purinoceptors was damaged or dying cells. However, it is now recognized that ATP release from many cells is a physiological or pathophysiological response to mechanical stress, hypoxia, inflammation, and some agonists. There is debate, however, about the ATP transport mechanisms involved. There is compelling evidence for exocytotic release from endothelial and urothelial cells, osteoblasts, astrocytes, mast, and chromaffin cells, but other transport mechanisms have also been proposed, including ATP binding cassette transporters, connexin hemichannels, and plasmalemmal voltage-dependent anion channels. Much is now known about the ectonucleotidases that break down ATP released from neurons and nonneuronal cells. Several enzyme families are involved: ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases), of which NTPDase1, 2, 3, and 8 are extracellular; ectonucleotide pyrophosphatase of 3 subtypes; alkaline phosphatases; ecto-50 -nucleotidase; and ecto-nucleoside diphosphokinase. NTPDase1 hydrolyzes ATP directly to adenosine monophosphate (AMP) and UTP to uridine diphosphate (UDP), while NTPDase2 hydrolyzes ATP to ADP and 50 -nucleotidase AMP to adenosine.
642 Adenosine Triphosphate (ATP) Cotransmission − sympathetic nerves
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b Figure 3 (a) Cotransmission in sympathetic nerves. Adenosine 50 -triphosphate (ATP) and noradrenaline (NA) from terminal varicosities of sympathetic nerves can be released together. With NA acting via the postjunctional a1-adrenoceptor to release cytosolic Ca2+, and with ATP acting via P2X1-gated ion channels to elicit Ca2þ influx, both contribute to the subsequent response (contraction). IP3 is inositol triphosphate, EJP is excitatory junction potential. (b) Schematic diagram of the principal cotransmitters with ATP in the nervous system. Nerve terminal varicosities of (i) sympathetic, (ii) parasympathetic, (iii) enteric (NANC inhibitory), (iv) sensory-motor neurons, and (v) central nervous system (CNS). (a) Adapted from Kennedy C, McLaren GJ, Westfall TD, and Sneddon P (1996) ATP as a cotransmitter with noradrenaline in sympathetic nerves – Function and fate. In: Chadwick DJ and Goode J (eds.) P2 Purinoceptors: Localization, Function and Transduction Mechanisms, pp. 223–235. Chichester: John Wiley and Sons, with permission from John Wiley & Sons. (b) Reproduced from Burnstock G (2007) Physiology and pathophysiology of purinergic neurotransmission. Physiological Reviews 87: 659–797, with permission from The American Physiological Society.
Physiology of Purinergic Neurotransmission Purinergic signaling appears to be a primitive system that is involved in many nonneuronal and neuronal mechanisms, in both short-term and long-term (trophic) events, including exocrine and endocrine secretion, immune responses, inflammation, mechanosensory transduction, platelet aggregation, endothelial-mediated vasodilatation and in cell proliferation,
differentiation, migration, and death in development and regeneration. The first clear evidence for nerve–nerve purinergic synaptic transmission was published in 1992. Synaptic potentials in the coeliac ganglion and in the medial habenula in the brain were reversibly antagonized by the antitrypanosomal agent suramin. Since then, many articles have described either the distribution of various P2 receptor subtypes in the brain and
Adenosine Triphosphate (ATP) 643
spinal cord or electrophysiological studies of the effects of purines in brain slices, isolated neurons, and glial cells. Synaptic transmission has also been demonstrated in the myenteric plexus and in various sensory, sympathetic, parasympathetic, and pelvic ganglia. Adenosine produced by the ectoenzymatic breakdown of ATP acts through presynaptic P1 receptors to inhibit the release of excitatory neurotransmitters in both the peripheral and the central nervous systems. Purinergic signaling is also implicated in higher order cognitive functions, including learning and memory in the prefrontal cortex.
CNS Control of Autonomic Function Functional interactions seem likely to occur between purinergic and nitrergic neurotransmitter systems; these interactions might be important for the regulation of hormone secretion and body temperature at the hypothalamic level and for cardiovascular and respiratory control at the level of the brain stem. The nucleus tractus solitarius (NTS) is a major integrative center of the brain stem involved in reflex control of the cardiovascular system; stimulation of P2X receptors in the NTS evokes hypotension. P2X receptors expressed in neurons in the trigeminal mesencephalic nucleus might be involved in the processing of proprioceptive information.
Neuron–Glia Interactions ATP is an extracellular signaling molecule between neurons and glial cells. ATP released from astrocytes might be important in triggering cellular responses to trauma and ischemia by initiating and maintaining reactive astrogliosis, which involves striking changes in the proliferation and morphology of astrocytes and microglia. Some of the responses to ATP released during brain injury are neuroprotective, but at higher concentrations, ATP contributes to the pathophysiology initiated after trauma. Multiple P2X and P2Y receptor subtypes are expressed by astrocytes, oligodendrocytes, and microglia. ATP and basic fibroblast growth factor (bFGF) signals merge at the mitogenactivated protein kinase cascade, which underlies the synergistic interactions of ATP and bFGF in astrocytes. ATP can activate P2X7 receptors in astrocytes to release glutamate, GABA, and ATP, which regulate the excitability of neurons. Microglia, immune cells of the CNS, are also activated by purines and pyrimidines to release inflammatory cytokines such as interleukins 1b (IL-1b) and IL-6 and tumor necrosis factor a. Thus, although microglia
might play an important role against infection in the CNS, overstimulation of this immune reaction might accelerate the neuronal damage caused by ischemia, trauma, or neurodegenerative diseases. P2X4 receptors induced in spinal microglia gate tactile allodynia after nerve injury. P2X7 receptors mediate superoxide production in primary microglia and are upregulated in a transgenic mouse model of Alzheimer’s disease, particularly around b-amyloid plaques.
Purine Transmitter and Receptor Plasticity The purinergic neurotransmission field is expanding rapidly; there is increasing interest in the physiology and pathophysiology of this neurosignaling system, and therapeutic interventions are being explored. The autonomic nervous system shows marked plasticity: that is, the expression of cotransmitters and receptors shows dramatic changes during development and aging, in nerves that remain after trauma or surgery, and in disease conditions. There are several examples where the purinergic component of cotransmission is increased in pathological conditions. The parasympathetic purinergic nerve-mediated component of contraction of the human bladder is increased to 40% in pathophysiological conditions such as interstitial cystitis, outflow obstruction, idiopathic instability, and also some types of neurogenic bladder. ATP also has a significantly greater cotransmitter role in sympathetic nerves supplying hypertensive compared to normotensive blood vessels. Upregulation of P2X1 and P2Y2 receptor mRNA in hearts of rats with congestive heart failure has been reported, and there is a dramatic increase in expression of P2X7 receptors in the kidney glomerulus in diabetes and hypertension.
Neuroprotection In the brain, purinergic signaling is involved in nervous tissue remodeling following trauma, stroke, ischemia, or neurodegenerative disorders. The hippocampus of chronic epileptic rats shows abnormal responses to ATP associated with increased expression of P2X7 receptors. Neuronal injury releases fibroblast growth factor, epidermal growth factor, and platelet-derived growth factor. In combination with these growth factors, ATP can stimulate astrocyte proliferation, contributing to the process of reactive astrogliosis and to hypertrophic and hyperplasic responses. P2Y receptor antagonists have been proposed as potential neuroprotective agents in the cortex, hippocampus, and cerebellum. Blockade of
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A2A (P1) receptors antagonizes tremor in Parkinson’s disease. ATP–MgCl2 is being explored for the treatment of spinal cord injuries.
Dual Purinergic Neural and Endothelial Control of Vascular Tone and Angiogenesis ATP and adenosine are much involved in the mechanisms underlying local control of vessel tone in addition to cell migration, proliferation, and death during angiogenesis, atherosclerosis, and restenosis following angioplasty. ATP, released as a cotransmitter from sympathetic nerves, constricts vascular smooth muscle via P2X receptors, whereas ATP released from sensory-motor nerves during ‘axon reflex’ activity dilates vessels via P2Y receptors. Furthermore, ATP released from endothelial cells during changes in flow (shear stress) or hypoxia acts on P2Y receptors in endothelial cells to release NO, resulting in relaxation (Figure 4). Adenosine, following breakdown of extracellular ATP, produces vasodilatation via smooth muscle P1 receptors.
Pain and Purinergic Mechanosensory Transduction The involvement of ATP in the initiation of pain was recognized first in 1966 and later in 1977 using human skin blisters. A major advance was made when the P2X3 ionotropic receptor was cloned in 1995 and shown later to be predominantly localized in the subpopulation of small nociceptive sensory nerves that label with isolectin B4 in dorsal root ganglia whose central projections terminate in inner lamina II of the dorsal horn. A unifying purinergic hypothesis for the initiation of pain was proposed in 1996 with ATP acting via P2X3 and P2X2/3 receptors associated with causalgia, reflex sympathetic dystrophy, angina, migraine, and pelvic and cancer pain. This has been followed by an increasing number of published reports expanding on this concept for acute, inflammatory, neuropathic, and visceral pain. P2Y1 receptors have also been demonstrated in a subpopulation of sensory neurons that colocalized with P2X3 receptors. A hypothesis was proposed that purinergic mechanosensory transduction occurred in visceral tubes and sacs, including ureter, bladder, and gut, where ATP, released from epithelial cells during distension, acted on P2X3 homomultimeric and P2X2/3 heteromultimeric receptors on subepithelial sensory nerves, initiating impulses in sensory pathways to pain centers in the CNS (Figure 5(a)). Subsequent studies of bladder, ureter, gut, tongue, and tooth pulp have produced
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Platelets Figure 4 A schematic representation of the interactions of adenosine 50 -triphosphate (ATP) released from perivascular nerves and from the endothelium (Endoth.). ATP is released from endothelial cells during hypoxia to act on endothelial P2Y receptors, leading to the production of endothelium-derived relaxing factor (EDRF), nitric oxide (NO), and subsequent vasodilation (–). In contrast, ATP released as a cotransmitter with noradrenaline (NA) from perivascular sympathetic nerves at the adventitia (Advent.)–muscle border produces vasoconstriction (+) via P2X receptors on the muscle cells. Adenosine (ADO), resulting from rapid breakdown of ATP by ectoenzymes, produces vasodilation by direct action on the muscle via P1 receptors and acts on the perivascular nerve terminal varicosities to inhibit transmitter release. Reproduced from Burnstock G (1987) Local control of blood pressure by purines. Blood Vessels 24: 156–160, with permission from S. Karger AG, Basel.
evidence in support of this hypothesis. P2X3 knockout mice were used to show that ATP released from urothelial cells during distension of the bladder act on P2X3 receptors on subepithelial sensory nerves to initiate both nociceptive and bladder voiding reflex activities. In the distal colon, ATP released during moderate distension acts on P2X3 receptors on lowthreshold intrinsic subepithelial sensory neurons to influence peristalsis, whereas high-threshold extrinsic subepithelial sensory fibers respond to severe distension to initiate pain (see Figure 5(b)). ATP is also a neurotransmitter released from the spinal cord terminals of primary afferent sensory nerves to act at synapses in the central pain pathway. Using transverse spinal cord slices from postnatal rats, excitatory postsynaptic currents have been shown to be
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b Figure 5 (a) Schematic representation of the hypothesis for purinergic mechanosensory transduction in tubes (e.g., ureter, vagina, salivary, and bile ducts and gut) and sacs (e.g., urinary and gall bladders and lung). It is proposed that distension leads to the release of adenosine 50 triphosphate (ATP) from the epithelium lining the tube or sac, which then acts on P2X2/3 receptors on subepithelial sensory nerves to convey sensory (nociceptive) information to the central nervous system (CNS). (b) Schematic of a novel hypothesis about purinergic mechanosensory transduction in the gut. It is proposed that ATP released from mucosal epithelial cells during moderate distension acts preferentially on P2X3 receptors on low-threshold subepithelial intrinsic sensory nerve fibers (labeled with calbindin), contributing to peristaltic reflexes. ATP released during extreme distension also acts on P2X3 receptors on high-threshold extrinsic sensory nerve fibers (labeled with isolectin B4 (IB4)) that send messages via the dorsal root ganglia (DRG) to pain centers in the CNS. (a) Adapted from Burnstock G (1999) Release of vasoactive substances from endothelial cells by shear stress and purinergic mechanosensory transduction. Journal of Anatomy 194: 335–342, with permission from Blackwell Publishing. (b) Adapted from Burnstock G (2001) Expanding field of purinergic signaling. Drug Development Research 52: 1–10, with permission of Wiley-Liss, Inc.
mediated by P2X receptors activated by synaptically released ATP, in a subpopulation of less than 5% of the neurons in lamina II, a region known to receive major input from nociceptive primary afferents. There is an urgent need for selective P2X3 and P2X2/3 receptor antagonists that do not degrade
in vivo. Pyridoxal-phosphate-6-azophenyl-20 , 40 -disulphonic acid is a nonselective P2 receptor antagonist but has the advantage that it dissociates about 100 to 10 000 times more slowly than other known antagonists. The trinitrophenyl-substituted nucleotide TNPATP is a selective and very potent antagonist at both
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P2X3 and P2X2/3 receptors. 5-((3-Phenoxybenzyl) [(1S)-1,2,3,4-tetrahydro-1-naphthalenyl]amino carbonyl)-1,2,4-benzenetricarboxylic acid (A-317491) is a potent and selective nonnucleotide antagonist of P2X3 and P2X2/3 receptors, and it reduces chronic inflammatory and neuropathic pain in the rat. Antisense oligonucleotides have been used to downregulate the P2X3 receptor, and in models of neuropathic (partial sciatic nerve ligation) and inflammatory (complete Freund’s adjuvant) pain, inhibition of the development of mechanical hyperalgesia was observed within 2 days of treatment. P2X3 double-stranded-short interfering RNA also relieves chronic neuropathic pain and opens up new avenues for therapeutic pain strategies in humans. Tetramethylpyrazine, a traditional Chinese medicine used as an analgesic for dysmenorrhoea, is claimed to be a P2X receptor antagonist, and it inhibited significantly the first phase of nociceptive behavior induced by 5% formalin and attenuated slightly the second phase in the rat hindpaw pain model. Antagonists to P2 receptors are also beginning to be explored in relation to cancer pain.
development and thus might be useful in the treatment of Me´nie`res disease, tinnitus, and sensorineural deafness. ATP, acting via P2Y receptors, depresses sound-evoked gross compound action potentials in the auditory nerve and the distortion product otoacoustic emission, the latter being a measure of the active process of the outer hair cells. P2X splice variants are found on the endolymphatic surface of the cochlear endothelium, an area associated with sound transduction. Sustained loud noise produces an upregulation of P2X2 receptors in the cochlear, particularly at the site of outer hair cell sound transduction. P2X2 receptor expression is also increased in spiral ganglion neurons, indicating that extracellular ATP acts as a modulator of auditory neurotransmission that is adaptive and dependent on the noise level. Excessive noise can irreversibly damage hair cell stereocilia, leading to deafness. Data have been presented that release of ATP from damaged hair cells is required for Ca2þ wave propagation through the support cells of organ of Corti, involving P2Y receptors, and this might constitute the fundamental mechanism to signal the occurrence of hair cell damage.
Special Senses Eye
P2X2 and P2X3 receptor mRNA is present in the retina and receptor protein expressed in retinal ganglion cells. P2X3 receptors are also present on Mu¨ller cells, which release ATP during Ca2þ wave propagation. ATP, acting via both P2X and P2Y receptors, modulates retinal neurotransmission, affecting retinal blood flow and intraocular pressure. Topical application of diadenosine tetraphosphate has been proposed for the lowering of intraocular pressure in glaucoma. The formation of P2X7 receptor pores and apoptosis is enhanced in retinal microvessels early in the course of experimental diabetes, suggesting that purinergic vasotoxicity might have a role in microvascular cell death, a feature of diabetic retinopathy. The possibility has been raised that alterations in sympathetic nerves might underlie some of the complications observed in diabetic retinopathy; ATP is well established as a cotransmitter in sympathetic nerves, raising the potential for P2 receptor antagonists in glaucoma. P2Y2 receptor activation increases salt, water, and mucus secretion and thus represents a potential treatment for dry eye conditions. Ear
Both P2X and P2Y receptors have been identified in the vestibular system. ATP regulates fluid homeostasis, cochlear blood flow, hearing sensitivity, and
Nasal Organs
The olfactory epithelium and vomeronasal organs contain olfactory receptor neurons that express P2X2, P2X3, and P2X2/3 receptors. It is suggested that the neighboring epithelial supporting cells or the olfactory neurons themselves can release ATP in response to noxious stimuli, acting on P2X receptors as an endogenous modulator of odor sensitivity. Enhanced sensitivity to odors was observed in the presence of P2 antagonists, suggesting that low-level endogenous ATP normally reduces odor responsiveness. It has been suggested that the predominantly suppressive effect of ATP on odor sensitivity could be involved in reduced odor sensitivity that occurs during acute exposure to noxious fumes and might be a novel neuroprotective mechanism. See also: Adenosine; Purines and Purinoceptors: Molecular Biology Overview.
Further Reading Abbracchio MP and Burnstock G (1994) Purinoceptors: Are there families of P2X and P2Y purinoceptors? Pharmacology and Therapeutics 64: 445–475. Abbracchio MP and Burnstock G (1998) Purinergic signalling: Pathophysiological roles. Japanese Journal of Pharmacology 78: 113–145. Bodin P and Burnstock G (2001) Purinergic signalling: ATP release. Neurochemical Research 26: 959–969.
Adenosine Triphosphate (ATP) 647 Burnstock G (1972) Purinergic nerves. Pharmacological Reviews 24: 509–581. Burnstock G (1987) Local control of blood pressure by purines. Blood Vessels 24: 156–160. Burnstock G (1999) Release of vasoactive substances from endothelial cells by shear stress and purinergic mechanosensory transduction. Journal of Anatomy 194: 335–342. Burnstock G (2001) Expanding field of purinergic signaling. Drug Development Research 52: 1–10. Burnstock G (2001) Purine-mediated signalling in pain and visceral perception. Trends in Pharmacological Sciences 22: 182–188. Burnstock G (2002) Purinergic signalling and vascular cell proliferation and death. Arteriosclerosis, Thrombosis and Vascular Biology 22: 364–373. Burnstock G (2001) Purinergic signalling in gut. In: Abbracchio MP and Williams M (eds.) Handbook of Experimental Pharmacology, Vol. 151: Purinergic and Pyrimidinergic Signalling II: Cardiovascular, Respiratory, Immune, Metabolic and Gastrointestinal Tract Function, pp. 141–238. Berlin: Springer. Burnstock G (2001) Purinergic signalling in lower urinary tract. In: Abbracchio MP and Williams M (eds.) Handbook of Experimental Pharmacology, Vol. 151: Purinergic and Pyrimidinergic Signalling I: Molecular, Nervous and Urinogenitary System Function, pp. 423–515. Berlin: Springer. Burnstock G (2003) Purinergic receptors in the nervous system. In: Schwiebert EM (ed.) Current Topics in Membranes, Vol. 54: Purinergic Receptors and Signalling, pp. 307–368. San Diego, CA: Academic Press. Burnstock G (2004) Cotransmission. Current Opinion in Pharmacology 4: 47–52.
Burnstock G (2006) Pathophysiology and therapeutic potential of purinergic signalling. Pharmacological Reviews 58: 58–86. Burnstock G (2007) Physiology and pathophysiology of purinergic neurotransmission. Physiological Reviews 87: 659–797. Burnstock G and Costa M (eds.) (1975) Adrenergic neuroeffector transmission. In Adrenergic Neurones: Their Organisation, Function and Development in the Peripheral Nervous System, pp. 51–106. London: Chapman and Hall. Burnstock G and Knight G (2004) Cellular distribution and functions of P2 receptor subtypes in different systems. International Review of Cytology 240: 31–304. Dunn PM, Zhong Y, and Burnstock G (2001) P2X receptors in peripheral neurones. Progress in Neurobiology 65: 107–134. James G and Butt AM (2002) P2Y and P2X purinoceptor mediated Ca2þ signalling in glial cell pathology in the central nervous system. European Journal of Pharmacology 447: 247–260. Kennedy C, McLaren GJ, Westfall TD, and Sneddon P (1996) ATP as a co-transmitter with noradrenaline in sympathetic nerves – Function and fate. In: Chadwick DJ and Goode J (eds.) P2 Purinoceptors: Localization, Function and Transduction Mechanisms, pp. 223–235. Sneddon P and Burnstock G (1984) Inhibition of excitatory junction potentials in guinea-pig vas deferens by a, b-methylene-ATP: Further evidence for ATP and noradrenaline as cotransmitters. European Journal of Pharmacology 100: 85–90. Zimmermann H (2001) Ectonucleotidases: Some recent developments and a note on nomenclature. Drug Development Research 52: 44–56.
Purines and Purinoceptors: Molecular Biology Overview G Burnstock, Royal Free and University College School of Medicine, London, UK
death that occur in development and regeneration are also mediated by purinergic receptors.
ã 2009 Elsevier Ltd. All rights reserved.
P1 Receptors Early Studies A seminal paper describing the potent actions of adenine compounds was published by Drury and Szent-Gyo¨rgyi in 1929. Many years later, ATP was proposed as the transmitter responsible for nonadrenergic, noncholinergic transmission in the gut and bladder, and the term ‘purinergic’ was introduced by Burnstock in 1972. Early resistance to this concept appeared to stem from the fact that ATP was recognized first for its important intracellular roles in many biochemical processes, and the intuitive feeling was that such a ubiquitous and simple compound was unlikely to be utilized as an extracellular messenger, although powerful extracellular enzymes involved in its breakdown were known to be present. Implicit in the concept of purinergic neurotransmission was the existence of postjunctional purinergic receptors, and the potent actions of extracellular ATP on many different cell types also implicated membrane receptors. Purinergic receptors were first defined in 1976, and 2 years later a basis for distinguishing two types of purinoceptor, identified as P1 and P2 (for adenosine and ATP/ADP, respectively), was proposed. At about the same time, two subtypes of the P1 (adenosine) receptor were recognized, but it was not until 1985 that a pharmacological basis for distinguishing two types of P2 receptor (P2X and P2Y) was made. In 1993, the first G-protein-coupled P2 receptors were cloned and a year later two iongated receptors were cloned, and in 1994 Abbracchio and Burnstock, on the basis of molecular structure and transduction mechanisms, proposed that purinoceptors should belong to two major families: a P2X family of ligand-gated ion channel receptors and a P2Y family of G-protein-coupled purinoceptors. This nomenclature has been widely adopted and currently seven P2X subtypes and eight P2Y receptor subtypes are recognized, including receptors that are sensitive to pyrimidines as well as purines. It is widely recognized that purinergic signaling is a primitive system involved in many nonneuronal as well as neuronal mechanisms, including exocrine and endocrine secretion, immune responses, inflammation, pain, platelet aggregation, and endothelial-mediated vasodilatation. Cell proliferation, differentiation, and
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Four different P1 receptor subtypes, A1, A2A, A2B, and A3, have been cloned. All are G-protein-coupled receptors (GPCRs). At 318 amino acids in length, the A3 subtype is the shortest, while A2A is the longest (412 residues). Their N-termini are relatively short (7–13 residues in length), as are their C-termini (32–120 residues). In the transmembrane domains (TMI–TMVII), human adenosine receptors share 39–61% sequence identity with each other and 11–18% identity with P2Y receptors. Each of the four human P1 receptor genes contains an intron within the coding region, located immediately after the end of the third transmembrane domain (see Figure 1(a)). P1 receptors couple principally to adenylate cyclase. A1 and A3 are negatively coupled to adenylate cyclase through the Gi/o protein a subunits, whereas A2A and A2B are positively coupled to adenylate cyclase through Gs. The human A2B receptor has also been observed to couple through Gq/11 to regulate phospholipase C activity, and the A3 receptor may interact directly with Gs. A number of P1 subtype-selective agonists and antagonists have been identified (see Table 1). Alteration or opening of the ribose ring drastically reduces affinity. The hydroxyl group at the 20 position is needed for both affinity and activity. The most selective agonist for the A1 subtype is 2-chloro-N6cyclopentyladenosine (CCPA). CGS 21680 is the most selective A2A agonist; NECA is the most potent A2B receptor agonist. 2-Cl-IB-MECA is 11-fold selective for the human A3 receptor and about 1400-fold selective for the rat A3 receptor. In general, methylxanthines such as caffeine and theophylline are weak P1 receptor antagonists. DPCPX (8-cyclopentyl-1,3dipropylxanthine) is an A1 receptor antagonist with subnanomolar affinity. The most selective A2B receptor antagonist is MRS1754. MRE3008-F20 is the most selective human A3 receptor antagonist. The diverse physiological effects mediated by the different P1 receptor subtypes, particularly modulation of the cardiovascular, immune, and central nervous systems, have been confirmed by transgenic knockout mice for A1, A2A, and A3 receptors. In contrast to knockout studies, overexpression of either A1 or A3 subtypes in transgenic mice has a cardioprotective effect.
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Agonist & antagonist recognition site S
S HN2
Binding of 5⬘-substituted agonists
S V
VI S
VII
H
Extracellular
H I
IV II
III
Intracellular
a
COOH
S-S Extracellular surface
S-S H5
M1
NH2
Plasma membrane
M2
COOH b
s s
NH2
c
Intracellular surface COOH
Figure 1 Membrane receptors for extracellular ATP and adenosine. The P1 family of receptors for extracellular adenosine comprises G-protein-coupled receptors signaling by inhibiting or activating adenylate cyclase (a). The P2 family of receptors binds extracellular ATP or ADP, and comprises two types of receptors (P2X and P2Y). The P2X family receptors are ligand-gated ion channels (b), and the P2Y family members are GPCRs (c). (a) Reproduced from Ralevic V and Burnstock G (1998) Receptors for purines and pyrimidines. Pharmacological Reviews 50: 413–492, with permission from the American Society for Pharmacology and Experimental Therapeutics. (b) Reprinted by permission from Macmillan Publishers Ltd: [Nature] (Brake AJ, Wagenbach MJ, and Julius D (1994) New structural motif for ligand-gated ion channels defined by an ionotropic ATP receptor. Nature 371: 519–523), copyright (1994). (c) Adapted from Barnard EA, Burnstock G, and Webb TE (1994) G protein-coupled receptors for ATP and other nucleotides: A new receptor family. Trends in Pharmacological Sciences 15: 67–70, with permission from Elsevier.
P2X Receptors Molecular Structure
The first cDNAs encoding P2X receptor subunits were isolated in 1994. Members of the family of ionotropic P2X1–7 receptors show a subunit topology of intracellular N- and C-termini possessing consensus binding motifs for protein kinases; two
transmembrane-spanning regions (TM1 and TM2), the first involved with channel gating and the second lining the ion pore; a large extracellular loop, with ten conserved cysteine residues forming a series of disulfide bridges; a hydrophobic H5 region close to the pore vestibule, for possible receptor/channel modulation by cations; and an ATP-binding site, which may involve regions of the extracellular loop adjacent
Receptor
Main distribution
Agonists
Antagonists
Transduction mechanisms
CCPA, CPA, S-ENBA
DPCPX, N-0840, MRS1754
Gi/o #cAMP
A2A A2B
Brain, spinal cord, testis, heart, autonomic nerve terminals Brain, heart, lungs, spleen Large intestine, bladder
CGS 21680, HENECA NECA (nonselective)
GS "cAMP GS "cAMP
A3
Lung, liver, brain, testis, heart
IB-MECA, 2-Cl-IB-MECA, DBXRM, VT160
KF17837, SCH58261, ZM241385 Enprofylline, MRE2029-F20, MRS1754, MRS1706 MRS1220, L-268605, MRS1191, MRS1523, VUF8504
Smooth muscle, platelets, cerebellum, dorsal horn spinal neurons
a,b-meATP ¼ATP ¼ 2-MeSATP (rapid desensitization), L-b,g-meATP ATP ATPgS 2-MeSATP a,b-meATP (pH þ zinc sensitive) 2-MeSATP ATP a,b-meATP Ap4A (rapid desensitization) ATP a,b-meATP, CTP, ivermectin ATP a,b-meATP, ATPgS
TNP-ATP, IP5I, NF023, NF449
Intrinsic cation channel (Ca2þ and Naþ)
Suramin, isoPPADS, RB2, NF770 TNP-ATP, PPADS, A317491, NF110
Intrinsic ion channel (particularly Ca2þ) Intrinsic cation channel
TNP-ATP (weak), BBG (weak) Suramin, PPADS, BBG
Intrinsic ion channel (especially Ca2þ) Intrinsic ion channel
P1 (adenosine) A1
P2X P2X1
P2X2 P2X3 P2X4 P2X5 P2X6 P2X7 P2Y P2Y1
P2Y2 P2Y4 P2Y6
Smooth muscle, CNS, retina, chromaffin cells, autonomic and sensory ganglia Sensory neurons, NTS, some sympathetic neurons CNS, testis, colon Proliferating cells in skin, gut, bladder, thymus, spinal cord CNS, motor neurons in spinal cord Apoptotic cells in, for example, immune cells, pancreas, skin Epithelial and endothelial cells, platelets, immune cells, osteoclasts Immune cells, epithelial and endothelial cells, kidney tubules, osteoblasts Endothelial cells
P2Y11
Some epithelial cells, placenta, T cells, thymus Spleen, intestine, granulocytes
P2Y12
Platelets, glial cells
P2Y13
Spleen, brain, lymph nodes, bone marrow
P2Y14
Placenta, adipose tissue, stomach, intestine, discrete brain regions
(Does not function as homomultimer) BzATP > ATP 2-MeSATP a,bmeATP
Gi/o Gq/11 #cAMP "IP3
Intrinsic ion channel KN62, KN04, MRS2427, Coomassie brilliant blue G
Intrinsic cation channel and a large pore with prolonged activation
2-MeSADP ¼ADPbS > 2MeSATP ¼ADP > ATP, MRS2365 UTP ¼ATP, UTPgS, INS37217
MRS2179, MRS2500, MRS2279, PIT
Gq/G11; PLC-b activation
Suramin > RB2, AR-C126313
UTP ATP, UTPgS
RB2 > suramin
UDP > UTP ATP, UDPbS
MRS2578
Gq/G11 and possibly Gi; PLC-b activation Gq/G11 and possibly Gi; PLC-b activation Gq/G11; PLC-b activation
AR-C67085MX > BzATP ATPgS > ATP 2-MeSADP ADP ATP
Suramin > RB2, NF157, 50 -AMPS
Gq/G11 and GS; PLC-b activation
CT50547, AR-C69931MX, INS49266, AZD6140, PSB0413, ARL66096, 2-MeSAMP MRS2211, 2-MeSAMP
Gi/o; inhibition of adenylate cyclase
ADP ¼ 2-MeSADP ATP and 2-MeSATP UDP glucose ¼ UDP-galactose
Gi/o Gq/G11
BBG, brilliant blue green; BzATP, 20 - and 30 -O-(4-benzoyl-benzoyl)-ATP; cAMP, cyclic AMP; CCPA, chlorocyclopentyl adenosine; CPA, cyclopentyl adenosine; CTP, cytosine triphosphate; IP3, inosine triphosphate; IP5I, diinosine pentaphosphate; 2-MeSADP, 2-methylthio-ADP; 2-MeSATP, 2-methylthio-ATP; NECA, 5’-N-ethylcarboxamido adenosine; NTS, nucleus tractus solitarius; PLC, phospholipase C; RB2, reactive blue 2. Adapted and reproduced from Burnstock G (2003) Introduction: ATP and its metabolites as potent extracellular agonists. Current Topics in Membranes 54: 1–27, with permission from Elsevier.
650 Purines and Purinoceptors: Molecular Biology Overview
Table 1 Characteristics of purine-mediated receptors
Purines and Purinoceptors: Molecular Biology Overview Table 2 Potential coassembly of P2X receptor subunitsa
P2X1 P2X2 P2X3 P2X4 P2X5 P2X6 P2X7
P2X1
P2X2
P2X3
þ
þ þ
þ þ þ
P2X4
þ
P2X5
P2X6
þ þ þ þ þ
þ þ
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Table 3 Chromosomal localization of human P2X receptorsa P2X7
Subunit
Chromosome
Accession number
17p13.2
þ
P2X1 P2X2 P2X3 P2X4 P2X5 P2X6 P2X7
X83688 AF190826 Y07683 Y07684 AF016709 AB002059 Y09561
þ þ
a
11q12 12q24.31 17p13.3 22q11 12q24.31
a
P2X receptor subunits carrying either one of two epitope tag units were expressed in pairs of HEK293 cells. þ, Subunits immunoprecipitated with antibody to one epitope could be detected with an antibody to the second epitope. Reproduced from Torres GE, Egan TM, and Voigt MM (1999) Hetero-oligomeric assembly of P2X receptor subunits. Specificities exist with regard to possible partners. Journal of Biological Chemistry 274: 6653–6659, with permission from the American Society for Biochemistry and Molecular Biology.
Accession numbers are those for the original submission of cDNA sequences. Chromosomal localizations are from human genome databases. P2X2 chromosomal location is not yet determined. The mouse gene is located on chromosome 5, in a region that is syntenic with the extreme end of the long arm of human chromosome 12 (some 6 MB from the P2X4 and P2X7 genes). Reproduced from North RA (2002) Molecular physiology of P2X receptors. Physiological Reviews 82: 1013–1067, with permission from the American Physiological Society.
to TM1 and TM2 (see Figure 1(b)). The P2X1–7 receptors show 30–50% sequence identity at the peptide level. The stoichiometry of P2X1–7 receptors is thought to involve three subunits, which form a stretched trimer or a hexamer of conjoined trimers. All of the P2X receptor subunits have consensus sequences for N-linked glycosylation. The pharmacology of the recombinant P2X receptor subtypes expressed in oocytes or other cell types is often different from the pharmacology of P2Xmediated responses in naturally occurring sites. Several contributing factors may account for these differences. First, heteromultimers as well as homomultimers are involved in forming the trimer ion pore. P2X2/3, P2X1/2, P2X1/5, P2X2/6, P2X4/6, and P2X1/4 receptor heteromultimers have been identified (Table 2). P2X7 does not form heteromultimers, and P2X6 will not form a functional homomultimer. Second, spliced variants of P2X receptor subtypes might play a part. There are seven genes for P2X receptor subunits. P2X4 and P2X7 subunit genes are located close to the tip of the long arm of chromosome 12. P2X4 and P2X7 subunits are among the most closely related pairs in amino acid sequences. P2X1 and P2X5 genes are also very close together on the short arm of chromosome 13. The remaining genes are on different chromosomes (Table 3). The genes vary considerably in size (e.g., mP2X3 ¼ 40 kb; hP2X6 ¼ 12 kb). The fulllength forms have 11–13 exons, and all share a common structure, with well-conserved intron/exon boundaries. Many spliced forms of the receptor subunits (or fragments) have been described. Several fulllength nonmammalian vertebrate sequences have been identified. There are no reports of homologous sequences from invertebrate species, although there is considerable functional evidence that extracellular
ATP and other nucleotides can directly gate ion channels in invertebrates. Recent advances have been made by the preparation of knockout mice for P2X1, P2X2, P2X3, P2X4, and P2X7 receptors, and transgenic mice that overexpress the P2X1 receptor. P2X Receptor Subtypes
P2X1 receptors A cDNA encoding the P2X1 receptor was isolated by direct expression in Xenopus oocytes, beginning with a cDNA library made from rat vas deferens. Human and mouse cDNAs have also been cloned and expressed. The homomeric P2X1 receptor is a cation-selective channel that shows little selectivity for sodium over potassium. It has a relatively high permeability to calcium. A major property of the P2X1 receptor is the mimicry of the agonist actions of ATP by a,b-methylene ATP (a,b-meATP), which distinguishes P2X1 and P2X3 receptors from the other homomeric forms. 20 ,30 -O-(benzoyl-4-benzoyl)-ATP (BzATP) is also an effective agonist. P2X1 receptors are blocked by suramin and pyridoxal-phosphate-6-azophenyl-20 ,40 disulfonic acid (PPADS), but there are newer antagonists that are more P2X1-selective (see Table 1). A valuable antagonist at P2X1 receptors is 20 ,30 -O(2,4,6-trinitrophenyl)-ATP (TNP-ATP), which has an IC50 of about 1 nM. Desensitization means the decline in the current elicited by ATP during the continued presence of ATP. In some P2X receptors this decline occurs in milliseconds (fast desensitization: P2X1, P2X3), and in others it occurs 100–1000 times more slowly (slow desensitization: P2X2, P2X4). Recovery from desensitization is extremely slow. Treatment with
652 Purines and Purinoceptors: Molecular Biology Overview
apyrase allows P2X1 receptors to recover from desensitization. Adenoviral expression of a P2X1 receptor– green fluorescent protein construct in vas deferens shows the receptor to be localized in clusters, with larger ones apposing nerve varicosities. P2X2 receptors The rat P2X2 receptor cDNA was isolated from a library constructed from nerve growth factor (NGF)-differentiated PC12 cells by testing pools for functional expression in Xenopus oocytes. The human receptor cDNA was amplified from pituitary gland. There are no agonists currently known that are selective for P2X2 receptors. However, P2X2 receptors are potentiated by protons and by low concentrations of zinc and copper. There are no selective antagonists for P2X2 receptors. The P2X2 receptor is generally described as nondesensitizing, compared with the P2X1 and P2X3 receptors. When oocytes are injected with RNAs encoding P2X2 receptors, and also the a3 and b4 subunits of nicotinic receptors, they show responses to both ATP and acetylcholine; these can be selectively antagonized with appropriate receptor blockers. However, with concomitant application of both agonists, the resultant current is less than the expected sum of the two independent currents, indicating an interaction between the two receptors. Heteromeric P2X1/2 receptors P2X1 and P2X2 receptor subunits have been co-expressed in defolliculated Xenopus oocytes and the resultant receptors were studied under voltage clamp conditions. Coexpression yielded a mixed population of homomeric and heteromeric receptors, with a subpopulation of novel pH-sensitive P2X receptors showing identifiably unique properties that indicate the formation of heteromeric P2X1/2 ion channels. It has been claimed that trimeric P2X1/2 receptors incorporate one P2X1 and two P2X2 subunits. P2X3 receptors P2X3 receptor subunit cDNAs were isolated from rat dorsal root ganglion cDNA libraries, from a human heart cDNA library, and from a zebra fish library. The mimicry of ATP by a,b-meATP makes these receptors similar to P2X1 and distinct from the other homomeric forms. 2-MethylthioATP is as potent as or more potent than ATP at P2X3 receptors. The antagonists suramin, PPADS, and TNP-ATP do not readily distinguish between P2X1 and P2X3 receptors, but NF023 is about 20 times less effective at P2X3 than at P2X1 receptors. Similar to P2X1 receptors, desensitization is fast and recovery is very slow. P2X3 receptors are prominently expressed on nociceptive sensory neurons.
Heteromeric P2X2/3 receptors Direct association between P2X2 and P2X3 receptor subunits has been shown by co-immunoprecipitation. P2X2/3 heteromeric channels can be defined on the basis of a sustained current elicited by a,b-meATP. P2X2/3 receptor channels and, like homomeric P2X2 receptors, are potentiated by low pH, and do not desensitize rapidly. The P2X2/3 heteromer, like the homomeric P2X3 receptor, is blocked by TNP-ATP, as well as PPADS and suramin. IP5I is much more potent for blocking P2X1 and P2X3 homomers than for blocking the P2X2/3 heteromers and is therefore useful to distinguish between P2X3 and P2X2/3 receptors. P2X2/3 receptors have been identified in subpopulations of sensory neurons, sympathetic ganglion cells, and brain neurons. P2X4 receptors cDNAs for the rat P2X4 receptor were isolated independently from superior cervical ganglion, brain, hippocampus, and pancreatic islet cells. Human, mouse, chick, and Xenopus cDNAs have also been isolated. Homomeric P2X4 receptors are activated by ATP, but not by a,b-meATP. The most useful distinguishing feature of ATP-evoked currents at P2X4 receptors is their potentiation by ivermectin. When the application of ATP is of short duration, P2X4 receptors operate as cation-selective channels; the calcium permeability is relatively high. When the application of ATP is continued for several seconds, the P2X4 receptor channel becomes increasingly permeable to larger organic cations such as N-methyl-D-glucamine (NMDG). Desensitization at P2X4 receptors is intermediate between that observed at P2X1 and P2X2. The rat P2X4 receptor is unusual among the P2X receptors in its relative insensitivity to blockade by the conventional antagonists suramin and PPADS. Currents evoked by ATP at the mouse P2X4 receptor are actually increased by PPADS and suramin, probably because of their ectonucleotidase inhibitory activity. Heteromeric P2X1/4 receptors Co-injection of P2X1 and P2X4 subunits into Xenopus oocytes showed that both subunits were present in trimeric complexes of the same size. Voltage clamp experiments revealed functional P2X receptors with kinetic properties resembling those of homomeric P2X4 receptors and a pharmacological profile similar to that of homomeric P2X1 receptors. Preliminary results show that the P2X1 receptor from the vas deferens and the P2X4 receptor from salivary gland form complexes of the same size as the recombinant trimeric complexes expressed in oocytes.
Purines and Purinoceptors: Molecular Biology Overview
P2X5 receptors The P2X5 receptor cDNA was first isolated from cDNA libraries constructed from rat celiac ganglion and heart. A P2X receptor was also cloned from embryonic chick skeletal muscle. The only human cDNAs reported are missing exon 10 (hP2X5a) or exons 3 and 10 (hP2X5b). A feature of the currents elicited by ATP in cells expressing the rat P2X5 receptor is their small amplitude, compared with the currents observed with P2X1, P2X2, P2X3, or P2X4 receptors expressed under similar conditions. The currents otherwise resemble those seen at P2X2 receptors: they show little desensitization, are not activated by a,b-meATP, and are blocked by suramin and PPADS. P2X5 mRNA is highly expressed in developing skeletal muscle. Heteromeric P2X1/5 receptors P2X1 and P2X5 subunits can be co-immunoprecipitated and the defining phenotype of this heteromer is a sustained current evoked by a,b-meATP, which is not seen for either of the homomers when expressed separately. Cells expressing the heteromeric receptor are very sensitive to ATP, concentrations as low as 3 or 10 nM evoking measurable currents. The sensitivity to the antagonist TNPATP is intermediate between the sensitive homomeric P2X1 receptor and the insensitive homomeric P2X5 receptor. P2X6 receptors The rat P2X6 receptor was cloned from superior cervical ganglion cDNA and from rat brain. The human equivalent was isolated from peripheral lymphocytes as a p53-inducible gene. This was originally designated P2XM to reflect its abundance in human and mouse skeletal muscle. The P2X6 receptor appears to be a ‘silent’ subunit, in the sense that no currents are evoked by ATP when it is expressed as a homomultimer in oocytes or HEK293 cells. It appears that the P2X6 subunit is only functionally expressed as a heteromultimer. Heteromeric P2X2/6 receptors P2X2 and P2X6 receptors have been found to co-immunoprecipitate after expression in HEK293 cells. Oocytes expressing this combination have subtly different responses to ATP as compared to oocytes expressing only P2X2 receptors. The most convincing of these differences is the fact that at pH 6.5 the inhibition of the current by suramin is clearly biphasic; one component has the high sensitivity of homomeric P2X2 receptors, whereas the other component is less sensitive. P2X2/6 receptors are prominently expressed by respiratory neurons in the brain stem. Heteromeric P2X4/6 receptors P2X4 and P2X6 receptors form a heteromeric channel when co-expressed
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in oocytes. The subunits can be co-immunoprecipitated from oocytes and HEK293 cells. The principal functional evidence for co-expression is that currents elicited by ATP are larger in oocytes 5 days after injection of mRNAs for P2X4 and P2X6 than after injection of P2X4 alone. However, the phenotype of the heteromer differs only in minor respects from that of P2X4 homomers. P2X4/6 receptors are prominent in adult trigeminal mesencephalic nucleus and in hippocampal CA1 neurons. P2X7 receptors A chimeric cDNA encoding the rat P2X7 receptor was first constructed from overlapping fragments isolated from superior cervical ganglion and medial habenula; full-length cDNAs were subsequently isolated from a rat brain cDNA library. Human and mouse cDNAs were cloned from monocyte and microglial cells, respectively. The main feature of the P2X7 receptor is that in addition to the usual rapid opening of the cation-selective ion channel, with prolonged exposure to high concentrations of ATP it becomes permeable to larger cations (e.g., NMDG) and later undergoes a channel-to-pore conversion to allow the passage of large dye molecules such as ethidium and YO-PRO-1, and this usually leads to cell death. Evidence for P2X7 receptor activation includes inward currents and increase in [Ca2þ]i; other end points involve uptake of YO-PRO-1 or similar fluorescent dyes which bind to nucleic acid, and structural changes in the cell, such as membrane blebbing. BzATP is a potent agonist at the P2X7 receptor. There are five main types of blockers (see Table 1): ions (calcium, magnesium, zinc, copper, and protons), the suramin analog NF279, Coomassie brilliant blue G (which is most effective at rat P2X7 receptors), oxidized ATP, and KN62, which is selective for the human P2X7 receptor. ATP or BzATP induces remarkable changes in the appearance of HEK293 cells transfected with the rat P2X7 receptor. After continuous application of BzATP (30 mM) for about 30 s, the plasma membrane begins to develop large blebs, and after 1 or 2 min, these become multiple and sometimes coalesce. Blebs are usually preceded by the appearance of smaller vesicles (<1 mm in diameter), which are shed from the cell and appear to release inflammatory cytokines.
P2Y Receptors Molecular Structure
The first P2Y receptors were cloned in 1993. Since then several other subtypes have been isolated by homology cloning and are assigned a subscript on the basis of cloning chronology (P2Y4, P2Y6,
654 Purines and Purinoceptors: Molecular Biology Overview
P2Y11). The long-awaited Gi-coupled ADP receptor (P2Y12) of platelets was finally isolated by expression cloning in 2001, while P2Y13 and P2Y14 receptors were characterized later during a systematic study of orphan receptors. At present, there are eight accepted human P2Y receptors: P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, and P2Y14 (see Table 1). The missing numbers represent either nonmammalian orthologs, or receptors having some sequence homology to P2Y receptors, but for which there is no functional evidence of responsiveness to nucleotides. p2y3 may be a chicken ortholog of P2Y6, while p2y8 and tp2y could be the Xenopus and turkey orthologs of P2Y4, respectively. p2y7 is a leukotriene B4 receptor. p2y5 and p2y10 are considered as orphan receptors. A p2y8 receptor cloned from the frog embryo appears to be involved in the development of the neural plate. p2y9 was reported to be a novel receptor for lysophosphatidic acid, distant from the Edg family. P2Y15 was recently introduced to designate the orphan receptor GPR80/GPR99 on the basis that it would be a receptor for adenosine 50 -monophospahte (AMP), but it is now firmly established that it is not a P2Y receptor, but rather a receptor for aketoglutarate, as recently underlined in a report by the International Union of Basic and Clinical Pharmacology (IUPHAR) P2Y Subcommittee. In contrast to P2X receptors, P2Y receptor genes do not contain introns in the coding sequence, except for the P2Y11 receptor. Site-directed mutagenesis of the P2Y1 and P2Y2 receptors has shown that some positively charged residues in TM3, TM6, and TM7 (see Figure 1(c)) are crucial for receptor activation by nucleotides. From a phylogenetic and structural (i.e., protein sequence) point of view, two distinct P2Y receptor subgroups characterized by a relatively high level of sequence divergence have been identified. The first subgroup includes P2Y1,2,4,6,11 subtypes and the second subgroup encompasses the P2Y12,13,14 subtypes (see dendrogram in Figure 2). Most of the P2Y receptor subtypes are still lacking potent and selective synthetic agonists and antagonists. However, ADPbS is a potent agonist of P2Y1, P2Y12, and P2Y13 receptors. 2-Methylthio-ADP (2MeSADP) is a potent agonist (EC50, in nM) at P2Y1, P2Y12, and P2Y13 receptors. Selective antagonists have been identified for some P2Y receptor subtypes (see Table 1). P2Y receptor-mediated responses occur in nonneuronal and nonmuscular cell types, as well as in the nervous system, involved in both short- and long-term signaling. Second Messenger Systems and Ion Channels
P2Y1, P2Y2, P2Y4, and P2Y6 receptors couple to Gproteins to increase inositol trisphosphate (IP3) and
cytosolic calcium. Activation of the P2Y11 receptor by ATP leads to a rise in both cAMP and in IP3, whereas activation by uridine 50 -triphosphate (UTP) produces calcium mobilization without IP3 or cAMP increase. The P2Y13 receptor can simultaneously couple to G16, Gi, and, at high concentrations of ADP, Gs. The activation of several P2Y receptors is commonly associated with the stimulation of several mitogen-activated protein (MAP) kinases, in particular extracellular signal-regulated protein kinase-1/2. In recent years, GPCRs in neurons and other excitable cells have been found to modulate the activity of voltage-gated ion channels in the cell membrane through certain actions of activated G-proteins. Such actions are now well established in closing (or, in certain cases, in opening or potentiating) various classes of Kþ channels and voltage-gated Ca2þ channels. ATP (or UTP, or their products ADP or UDP) present at synapses, plus ATP diffusing from astrocytes, activates P2Y receptors on distinct subsets of brain neurons, regulating their activities by the coupling of those receptors to specific ion channels. While ion channel couplings of P2Y receptors are primarily of importance in neurons, they have in a few cases been detected also in various other tissues (e.g., in cardiac muscle cells). Among the channels with which the superior cervical ganglion cell membrane is well endowed are two types of voltage-gated channels, which are important in receptor-based regulation of neuronal activity, the Ca2þ channel of the N-type and the M-current Kþ channel. P2Y Receptor Subtypes
P2Y1 receptors Human, rat, mouse, cow, chick, turkey, and Xenopus P2Y1 receptors have been cloned and characterized. In most species, ADP is a more potent agonist than ATP is and their 2-methylthio derivatives are even more potent. UTP, UDP, CTP, and GTP are inactive. At present, the most potent and selective agonist known is the N-methanocarba analog of 2-MeSADP, MRS2365 (EC50 of 0.4 nM). The most effective antagonists to display selectivity for the P2Y1 receptor are MRS2179, MRS2279, and MRS2500 (see Table 1). Site-directed mutagenesis studies on the human P2Y1 receptor have shown that amino acid residues in TM3, TM6, and TM7 are critical determinants in the binding of ATP. Four cysteine residues in the extracellular loops, which are conserved in P2Y receptors, are essential for proper trafficking of the human P2Y1 receptor to the cell surface. P2Y1 mRNA expression is highest in various regions of the brain, prostate gland, and placenta, and has also been detected at varying levels in other organs.
Purines and Purinoceptors: Molecular Biology Overview
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Figure 2 (a) Dendrogram to show relatedness of 29 P2X receptor subunits. Full-length amino acid sequences were aligned with Clustal W software using default parameters. The dendrogram was constructed with Tree View. h, human (Homo sapiens); r, rat (Rattus norvegicus); m, mouse (Mus musculus); gp, guinea pig (Cavia porcellus); c, chicken (Gallus gallus); zf, zebra fish (Danio rerio); f, fugu (Takifugu rubripes). The ovals indicate the apparent clustering by relatedness into subfamilies. (b) A phylogenetic tree (dendrogram) showing the relationships among the current members of the P2Y receptor family (human P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, and P2Y13 receptors) and the human UDP-glucose receptor (here indicated as the P2Y14 receptor). The P2Y receptors can be divided into two subgroups, shown with blue and purple backgrounds. Sequences were aligned using Clustal X and the tree was built using the Tree View software. (a) Reproduced from North RA (2002) Molecular physiology of P2X receptors. Physiological Reviews 82: 1013–1067, with permission from the American Physiological Society. (b) Reproduced from Abbracchio MP, Boeynaems J-M, Barnard EA, et al. (2003) Characterization of the UDP-glucose receptor (re-named here the P2Y14 receptor) adds diversity to the P2Y receptor family. Trends in Pharmacological Sciences 24: 52–55, with permission from Elsevier.
P2Y1 receptor knockout mice have been generated. These mice are viable with no apparent abnormalities affecting their development, survival, and reproduction. Platelet counts are normal, but shape change is abolished. Transgenic mice overexpressing the P2Y1 receptor specifically in the megakaryocytic/platelet lineage have also been generated.
P2Y2 receptors P2Y2 receptors have been cloned and pharmacologically characterized from human, rat, mouse, canine, and porcine cells or tissues. P2Y2 receptors are fully activated by ATP and UTP, whereas ADP and UDP are much less effective agonists. The g-thiophosphate, UTPgS, has been shown to be a potent hydrolysis-resistant agonist of P2Y2
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receptors, as is the recently developed P2Y2 receptor agonist, INS37217 (Up4dC). Suramin acts as a weak competitive antagonist of human and rat P2Y2 receptors. AR-C126313 and the related aminotetrazole derivative AR-C118925, flavanoids, and tangeretin have been claimed recently to be effective antagonists. P2Y2 receptors can directly couple to PLCb1 via Gaq/11 protein to mediate the production of IP3 and diacylglycerol, which are second messengers for calcium release from intracellular stores and protein kinase C (PKC) activation, respectively. Expression of P2Y2 receptor mRNA and protein has been detected in many tissues. P2Y2 receptor activation increases the synthesis and/or release of arachidonic acid, prostaglandins, and nitric oxide. P2Y2 receptor expression in smooth muscle cells is upregulated by agents that mediate inflammation. P2Y2 receptors have been shown to play a role in the wound healing process. P2Y2 receptor activation increases Cl secretion and inhibits Naþ absorption in epithelial cells. A P2Y2 receptor knockout mouse has been produced that is defective in nucleotide-stimulated ion secretion in airway epithelial cells. P2Y2 receptors have been shown to inhibit bone formation by osteoblasts, and N-type calcium currents in neurons. P2Y4 receptors Human, rat, and mouse P2Y4 receptors have been cloned and characterized. UTP is the most potent activator of the recombinant human P2Y4 receptor. In contrast, the recombinant rat and mouse P2Y4 receptors are activated equipotently by ATP and UTP. Up4U (INS365) and dCp4U (INS37217) are agonists of the human P2Y4 receptor. Reactive blue 2 effectively blocks rat P2Y4 receptors, but only partially blocks human P2Y4 receptors. Suramin is a weak antagonist at the P2Y4 receptor. The structural determinants of agonism versus antagonism by ATP are located in the N-terminal domain and the second extracellular loop. In the human and the mouse, P2Y4 mRNA and protein was most abundant in the intestine, but was also detected in other organs. P2Y4-null mice have apparently normal behavior, growth, and reproduction, but the chloride secretory response of the jejunal epithelium to apical UTP and ATP is abolished. P2Y6 receptors The mouse, rat, and human P2Y6 receptors are UDP receptors. The uridine b-thiodiphosphate, UDPbS, and Up3U are selective agonists of the P2Y6 receptor and are more stable to degradation. INS48823 is also a potent P2Y6 agonist. A 1,4di-(phenylthioureido)butane derivative (MRS2578) has recently been shown to selectively inhibit UDPinduced PLC activity. A unique feature of the P2Y6 receptor is its slow desensitization and internalization.
A wide tissue distribution of P2Y6 mRNA and protein has been demonstrated, with the highest expression in spleen, intestine, liver, brain, and pituitary. P2Y11 receptors Among P2Y receptors, the human P2Y11 receptor has a unique profile. Its gene is the only P2Y receptor gene that contains an intron in the coding sequence. The potency of its natural agonist ATP is relatively low and it is dually coupled to PLC and adenylyl cyclase upon stimulation. ATPgS is a more potent agonist than ATP is. The P2Y12 antagonist AR-C67085MX acts as a potent agonist at the P2Y11 receptor. Suramin behaves as a competitive antagonist of the hP2Y11 receptor. P2Y12 receptors The human, rat, and mouse P2Y12 receptors have been identified and characterized. ADP is the natural agonist of this receptor. The P2Y12 receptor is heavily expressed in platelets, where it is the molecular target of the active metabolite of the antiplatelet drug clopidogrel. Potent direct competitive P2Y12 antagonists are also available, including the 50 triphosphate derivative AR-C69931MX compound, named cangrelor. The P2Y12 receptor has also been shown to be expressed in subregions of the brain, glial cells, brain capillary endothelial cells, smooth muscle cells, and chromaffin cells. The P2Y12 knockout mice that have been generated display the phenotype of clopidogrel-treated animals. P2Y13 receptors The human, mouse, and rat P2Y13 receptors have been identified and characterized. ADP and Ap3A are naturally occurring agonists of the P2Y13 receptor. The P2Y13 receptor is primarily coupled to a Gi/o protein. However, cangrelor, which is an antagonist of the hP2Y12 receptor, is also an antagonist of the human and rat P2Y13 receptors. Recently MRS2211, a derivative of PPADS, was shown to selectively antagonize the human P2Y13 receptor. The P2Y13 receptor is strongly expressed in the spleen, followed by placenta, liver, heart, bone marrow, monocytes, T cells, lung, and various brain regions. P2Y13-null mice have been generated recently, but no phenotype has been characterized to date. P2Y14 receptors From a phylogenetic and structural point of view, the P2Y14 receptor (previously known as GPR105 or UDP-glucose receptor) is 47% identical to the P2Y12 and P2Y13 receptors. The gene for this receptor has been found in human chromosome 3q24-3q25, where a cluster of other related GPCRs, consisting of P2Y1, P2Y12, and P2Y13 receptors and the orphan receptors GPR87, GPR91, and H963, have been found. The P2Y14 receptor couples to the Gi/o family of G-proteins and is activated by
Purines and Purinoceptors: Molecular Biology Overview
UDP-glucose as well as UDP-galactose, UDP-glucuronic acid, and UDP-N-acetylglucosamine. At present, no selective antagonists are available. P2Y14 mRNA is widely distributed in the human body. Both chemoattractant and neuroimmune functions have been claimed for the P2Y14 receptor. Receptor Dimerization and Cross-Talk
It is now recognized that interactions between GPCRs can take place through the formation of oligomers, or downstream of the receptor through the action of second messengers. The former process is commonly referred to as receptor dimerization. The latter process is known as receptor cross-talk. There is evidence that the human P2Y2 receptor forms homodimers. An example of dimerization involving P2Y receptors with non-P2Y receptors is rat P2Y1 and adenosine A1 receptors co-expressed in HEK293 cells. It has also been shown that the P2Y1 and A1 receptors are co-localized in neurons of the rat cortex, hippocampus, and cerebellum. The formation of oligomers by P2Y receptors is likely to be widespread and to greatly increase the diversity of purinergic signaling. P2X receptors and P2Y receptors are often expressed in the same cells. Thus, there is the possibility of bidirectional cross-talk between these two families of nucleotide-sensitive receptors. For example, the P2X1 receptor may have a priming role in activation of P2Y1 receptors during platelet stimulation. See also: Adenosine; Adenosine Triphosphate (ATP); P2X
Receptors.
Further Reading Abbracchio MP, Boeynaems J-M, Barnard EA, et al. (2003) Characterization of the UDP-glucose receptor (re-named here the P2Y14 receptor) adds diversity to the P2Y receptor family. Trends in Pharmacological Sciences 24: 52–55. Abbracchio MP, Burnstock G, Boeynaems J-M, et al. (2006) International Union of Pharmacology. Update and subclassification of the P2Y G protein-coupled nucleotide receptors: From
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molecular mechanisms and pathophysiology to therapy. Pharmacological Reviews 58: 281–341. Brake AJ, Wagenbach MJ, and Julius D (1994) New structural motif for ligand-gated ion channels defined by an ionotropic ATP receptor. Nature 371: 519–523. Barnard EA, Burnstock G, and Webb TE (1994) G protein-coupled receptors for ATP and other nucleotides: A new receptor family. Trends in Pharmacological Sciences 15: 67–70. Boeynaems J-M, Communi D, Gonzalez NS, et al. (2005) Overview of the P2 receptors. Seminars in Thrombosis and Hemostasis 31: 139–149. Burnstock G (1978) A basis for distinguishing two types of purinergic receptor. In: Straub RW and Bolis L (eds.) Cell Membrane Receptors for Drugs and Hormones: A Multidisciplinary Approach, pp. 107–118. New York: Raven Press. Burnstock G and Knight GE (2004) Cellular distribution and functions of P2 receptor subtypes in different systems. International Review of Cytology 240: 31–304. Fredholm BB, Ijzerman AP, Jacobson KA, et al. (2001) International Union of Pharmacology. XXV: Nomenclature and classification of adenosine receptors. Pharmacological Reviews 53: 527–552. Hollopeter G, Jantzen H-M, Vincent D, et al. (2001) Identification of the platelet ADP receptor targeted by antithrombotic drugs. Nature 409: 202–207. Jacobson KA, Jarvis MF, and Williams M (2002) Purine and pyrimidine (P2) receptors as drug targets. Journal of Medicinal Chemistry 45: 4057–4093. King BF and Burnstock G (2002) Purinergic receptors. In: Pangalos M and Davies C (eds.) Understanding G Protein-Coupled Receptors and Their Role in the CNS, pp. 422–438. Oxford: Oxford University Press. Neary JT, Rathbone MP, Cattabeni F, et al. (1996) Trophic actions of extracellular nucleotides and nucleosides on glial and neuronal cells. Trends in Neurosciences 19: 13–18. Nicke A, Baumert HG, Rettinger J, et al. (1998) P2X1 and P2X3 receptors form stable trimers: A novel structural motif of ligandgated ion channels. EMBO Journal 17: 3016–3028. North RA (2002) Molecular physiology of P2X receptors. Physiological Reviews 82: 1013–1067. Ralevic V and Burnstock G (1998) Receptors for purines and pyrimidines. Pharmacological Reviews 50: 413–492. Surprenant A, Rassendren F, Kawashima E, et al. (1996) The cytolytic P2Z receptor for extracellular ATP identified as a P2X receptor (P2X7). Science 272: 735–738. Torres GE, Egan TM, and Voigt MM (1999) Hetero-oligomeric assembly of P2X receptor subunits. Specificities exist with regard to possible partners. Journal of Biological Chemistry 274: 6653–6659. Webb TE, Simon J, Krishek BJ, et al. (1993) Cloning and functional expression of a brain G-protein-coupled ATP receptor. FEBS Letters 324: 219–225.
P2X Receptors Z Li, S Harris, and T M Egan, Saint Louis University School of Medicine, St. Louis, MO, USA ã 2009 Elsevier Ltd. All rights reserved.
Introduction P2X receptors are ligand-gated ion channels that open in the presence of extracellular adenosine triphosphate (ATP). They are found in both simple parasitic organisms such as Schistosoma mansoni and more complex animals, such as humans. Activation usually results in a rapid depolarization of the target cell. This occurs as ATP binds to an extracellular site and opens an integral transmembrane pore that allows small monovalent and divalent cations to flow down their electrochemical gradients. At physiological membrane potentials, the overriding effect of activation is an influx of sodium and calcium (Figure 1(a)). The resulting rise in intracellular sodium depolarizes the membrane and renders the cell hyperexcitable. The influx of calcium activates a number of intracellular calcium-dependent processes such as muscle contraction and synaptic vesicle fusion. Some P2X receptors, particularly those expressed in muscle, also have a demonstrable permeability to chloride, although the concurrent influx of sodium still causes depolarization. At times, P2X receptors initiate the movement of larger molecules across the membrane. For example, the release of proinflammatory interleukin-1b from macrophages is stimulated by activation of native P2X7 receptors, and activation of some recombinant receptors (e.g., rat P2X2 P2X4, and P2X7 receptors and human P2X5 receptors) expressed in model cells leads to the opening of membrane pores permeable to the large nucleic acid stains, ethidium and YO-PRO-1 (Figure 1(b)). The original hypothesis that dye uptake reflects a time-dependent dilation of the intrinsic ion channel (also known as the ‘pore-dilation’ hypothesis) has come under attack. Specifically, recent data suggest that separate pathways mediate the ATP-gated movement of small cations and large dyes; the evidence that supports this claim is discussed in more detail below.
Genes and Gene Products The first two P2X receptor genes (P2X1 and P2X2) were identified in 1996 by expression cloning from tissues derived from rat. These two then served as templates for the subsequent discovery of five additional rat genes (P2X3–P2X7) by polymerase
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chain reaction. Orthologous sequences are found in a number of organisms, including trematodes, fish, frogs, chickens, rodents, cows, and humans. Paralogs and splice-variants have also been described. The seven full-length rat genes encode proteins that vary in length from 384 to 595 amino acids and are 40–50% identical in sequence. Functional receptors are oligomeric complexes of three subunits that selectively combine in both homomeric and heteromeric fashions. Each subtype has a phenotype that is defined by (1) the amplitude of the ATP-gated current; (2) the relative permeability of cations and anions; (3) the percentage of the current carried by calcium; (4) the ability to initiate uptake of nucleic acid stains; (5) the rate and extent of desensitization and resensitization; (6) the ability to form heteromeric complexes with other family members; and (7) sensitivity to a range or agonists, antagonists, and allosteric modulators. Six of the seven genes (P2X1–5, P2X7) are capable of encoding functional homomeric receptors that mediate ATP-gated transmembrane currents in heterologous expression systems (Figure 1(c) and (d)). The P2X6 receptor is the exception. Biochemical analyses suggest that monomers of this subtype are partially processed and retained by the endoplasmic reticulum. Despite this fact, there are occasional reports of ATP-gated ionic currents generated in cells expressing only the P2X6 gene. These findings are somewhat controversial and may reflect the unintended formation of heteromeric receptors containing recombinant P2X6 and native (but apparently silent) non-P2X6 subunits. This hypothesis is plausible because several laboratories have reported that the P2X6 subtype forms functional heteromeric complexes in a subunit-specific manner when co-expressed with other family members. Indeed, most P2X receptors are capable of forming functional heteromers that display unique phenotypes when expressed in heterologous systems. Although there are numerous examples of functional heteromeric receptors made of two different subtypes, there is no report of a P2X receptor containing three different family members, even though the existence of such a receptor is possible. Structure and Function
Because the crystal structure of the receptor is unsolved, a range of less direct biochemical and pharmacological approaches was used to develop a preliminary map of the protein as it sits in the membrane. All functional P2X receptors have the following topology: intracellular N-terminus/first transmembrane segment (TM1)/extracellular segment/second transmembrane segment (TM2)/intracellular C-terminus (Figure 1(e)).
P2X Receptors 659 Ca2+-permeable P2X channels
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Figure 1 (a) P2X receptors are cation channels permeable to sodium and calcium. The traces show total membrane current (black traces) and fura-2 fluorescence recorded from a HEK-293 cell expressing homomeric rat P2X2 receptors. In this experiment, a decrease in fluorescence (excitation 380 nm; emission 510 nm) indicates an increase in intracellular free calcium. An application of 10 mmol l1 ATP (solid black bar) causes an inward current and a decrease in fura-2 fluorescence. The fluorescence was calibrated, filtered, and differentiated to give the ATP-gated calcium current (blue trace). About 7% of the total current is carried by calcium (compare the amplitudes of the blue and black traces). In the inset, the peak currents of the total current and calcium current are normalized to show that their time courses are identical, as expected if all the rise in intracellular calcium comes from the inward flow of calcium current through the P2X channel. (b)YO-PRO-1 uptake. The traces show the averaged data from 45 test (filled black circles) and 12 control (empty red squares) experiments. In the test experiments, ATP (600 mmol l1) was applied to six to ten HEK-293 cells expressing the P2X2 receptor and a reporter protein (AsRed). The drug was applied 2 min after the start of the recording. YO-PRO-1 fluorescence (491/509 nm) was collected by a photomultiplier. Transfected cells showed little background fluorescence in the absence of ATP but fluoresced brightly during agonist application (solid bar). Control cells expressed only the reporter protein. (c) The traces show ATP-gated currents recorded from seven different HEK-293 cells transiently expressing one subtype of P2X receptor. All the cells except those expressing the P2X6 receptor show inward current in response to ATP. In some cases (P2X1 and P2X3 receptors), the receptors show rapid desensitization during the ATP application. In another case (P2X5 receptor), the size of the current is relatively small compared with the other subtypes. The concentrations of ATP are as follows: P2X1 and P2X3, 1 mmol l1; P2X2 and P2X4, 30 mmol l1; P2X5, 100 mmol l1; and P2X7, 1 mmol l1. (d) The current traces of (c) are shown at an expanded timescale to show the rate of onset and desensitization of the six functional homomeric receptors. (e) A representation of a P2X subunit. Functional receptors are formed of three subunits; the diagram shows one subunit sitting in the membrane (gray shaded area). Both the N- and C-termini are intracellular. Two transmembrane domains (1,2) span the lipid bilayer, and these are connected by a large extracellular loop (E) that makes up the bulk of the receptor protein. (f) Oligomeric receptors comprise three subunits that form a cation-permeable pore. Although the pore contains parts of both transmembrane domains, TM2 seems to contribute more than TM1 does to the permeation pathway.
Individual subunits are arranged in a head-to-tail fashion so that the TM1 of each subunit abuts the TM2 of its neighbor (Figure 1(f)). The presence of only two transmembrane segments distinguishes P2X receptors
from other ligand-gated cation channels, which typically possess three or more. The N-tail is relatively short (about 25 amino acids) and contains a consensus site for protein kinase C-mediated phosphorylation;
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removal of this site leads to an accelerated fade in the amplitude of the ATP-gated current of the P2X2 receptor. The C-tail is longer (about 25–250 amino acids) and more variable and contains multiple sites that mediate subtype-specific effects. These effects include receptor desensitization; membrane trafficking; altered permeation; and interactions with proteins, phospholipids, and lipid polysaccharides. P2X7 receptors with their C-tails altered by truncation or site-directed mutagenesis have normal sodium and calcium currents but lose the ability to show a timedependent increase in the permeability of larger cations like N-methyl-D-glucamine (NMDGþ). It is interesting that the time-dependent increase in permeability to ethidium and YO-PRO-1 is unaffected by a truncation that obliterates NMDGþ permeability, suggesting that separate pathways underlie pore dilation (i.e., the gradual increase in permeability to NMDGþ) and dye uptake (i.e., the gradual increase in permeability to nucleic acid stains). Both transmembrane segments line the pore and may contain sites affecting channel function. An explicit role for the TM1 has not been forthcoming, although data suggest that it is involved in transducing agonist binding into channel gating. The functional domains of the TM2 are better described. Within the TM2 are specific amino acids that affect the two properties that P2X receptors share with all ion channels, permeation and gating. Further, the TM2 helps direct the assembly of subunits into oligomeric complexes and is partly responsible for determining the rate and amount of desensitization experienced by different family members. The two transmembrane segments are connected by a long string of amino acids that form the extracellular extent of the receptor. This string contains ten conserved cysteines, some of which participate in disulfide bonds that help define the tertiary structure of the protein. In contrast; disulfide bonds do not form between adjoining subunits; so they are not involved in stabilizing the quaternary structure. The extracellular loop also contains consensus sequences for the N-linked glycosylation involved in targeting the receptor to the cell surface membrane, for agonist and antagonist binding sites, and for intersubunit binding sites responsible for allosteric modulation of the ATP response by hydrogen ions and zinc. The ATP Binding Site
The ATP binding site resides at the interface of neighboring subunits of the multimeric receptor complex. The negative charge of the phosphate chain of ATP is attracted to the formal positive charge of conserved lysines and arginines located near the TM1 and TM2,
and the adenine ring is coordinated by aromatic amino acids. Different family members respond to different agonists. For example, ab-methylene ATP is a potent agonist at P2X1 and P2X3 receptors but has little or no effect on P2X2, P2X4, P2X5, and P2X7 receptors. Further, the median effective concentration (EC50) of ATP on the P2X7 is much higher than that of all other family members. The differences in agonist (and antagonist) profiles suggest that the structure of the agonist and antagonist binding sites varies in a subunit-specific manner.
Ion Permeation and Gating P2X receptors are generally classified as ligand-gated cation channels because most of their physiological effects result from the inward flow of cations across the cell surface membrane. However, some subtypes initiate the movement of both cations and anions across cell surface membranes, making the cation channel classification somewhat inaccurate. Cation Permeability
All P2X receptors are freely permeable to the three most important physiological cations: sodium, potassium, and calcium. Sodium and potassium permeate equally well. Ding and Sachs have suggested that cations interact with an anionic binding site of low to moderate field strength located at about 20% of the electrical distance from the extracellular surface of the channel. If P2X receptors have only two transmembrane segments, then this binding site must be made of parts of TM1 and/or TM2. The only conserved formal negative charge in these domains is an aspartate in TM2 (e.g., Asp349 of the P2X2 receptor). Neutralizing Asp349 has no effect on cation permeability, making it unlikely that it contributes to regulation of current flow through the channel. Rather, the site may be formed of polar amino acids that reside in a narrow constriction of the pore, although such a model is not applicable to all P2X subtypes (see below). An alternative hypothesis is that other parts of the protein form a reentrant pore loop that forms a selectivity filter in the pore, analogous to the P loop of the potassium-selective KcsA channel or the M2 loop of the glutamate receptor. However, no such reentrant loop has been identified for any P2X receptor to date. The relative permeability to calcium is higher than that to sodium or potassium and varies in a subunitspecific manner. For example, about 6% of the current through the P2X2 receptor is carried by calcium, and this is large enough to facilitate excitatory neurotransmission onto stratum radiatum interneurons of the
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hippocampus. By contrast, the calcium currents of P2X1 and P2X4 receptors are twice as large as those of the P2X2 receptor. The molecular mechanism(s) responsible for the subunit-specific variability are unknown. Two complementary methods have been proposed. First, site-directed mutagenesis of hydroxylbearing amino acids within the TM2 of the P2X2 receptor decreases calcium flux, perhaps by interfering with a binding site involved in partially dehydrating permeating ions as they pass through a narrow constriction in the pore. However, the polar residues are missing in some of the subtypes, and therefore hydroxyls are not fully required for calcium flux throughout the family. Second, the COO side chains of acidic amino acids found at the extracellular ends of the transmembrane domains may provide an electrostatic force that interacts with calcium in the mouth of the channel pore. Indeed, subtypes that possess these formal charges (i.e., P2X1 and P2X4 receptors) transduce significantly higher calcium fluxes than those that do not (i.e., P2X2 receptors). Anion Permeability
Hume and Thomas were the first to describe an anion-permeable, ATP-gated ion channel. They measured a shift in the reversal potential of the ATP-gated current of chick skeletal muscle when extracellular chloride was replaced by larger anions. Such a shift suggests that the outward movement of chloride contributes to the ATP-evoked depolarization. The responsible gene was cloned by Soto and others and found to encode a chick P2X5 receptor capable of transducing significant chloride flux in heterologous expression systems. Orthologs have been described in zebra fish, bullfrog, mouse, rat, and humans. Most of these are poorly characterized because their ATPgated currents are small (e.g., less than 100 pA even at the extremes of physiological membrane potentials), and therefore their anion permeability is unknown. North et al. described a full-length human P2X5 receptor that is different from other P2X5 receptors in that it transduces large (nA) currents in response to ATP. Like the smaller currents of the chick P2X5 receptor, the human ortholog displays a significant permeability to chloride. Pore Dilation versus Dye Uptake
Sodium, potassium, calcium, and chloride carry most of the ATP-gated membrane current during the first few seconds of application of ATP. Longer applications evoke an additional conductance as the membrane permeability to large organic cations (like NMDGþ) gradually increases. The increase in permeability suggests that the narrowest portion of the
pore dilates by at least 3 A˚ from an initial diameter of 9–11 A˚. At the same time that the permeability to NMDGþ begins to increase, cells expressing P2X2, P2X4, P2X5, or P2X7 receptors also gain the ability to take up large nucleic acid dyes (i.e., ethidium bromide or YO-PRO-1). A simple hypothesis that could explain both effects is that the P2X pore moves through three states. The first is the nonconducting, closed state seen in the absence of ATP. The second is open in the presence of ATP and permeable to small cations with geometric diameters of <11 A˚. The third opens in the continued presence of ATP and with a delay; it occurs when the initial open state widens to a new diameter that is big enough to allow large molecules to pass through the channel. The three-state model is sufficient to explain much of the biophysical data currently available. However, at least in the case of the P2X7 receptor, recent data suggest that the uptake of dye does not occur through the pore of the P2X7 channel per se. Surprenant, North, and others have shown that deleting a short stretch of 18 amino acids just intracellular to the end of the TM2 (called the Cys-rich region because it contains many cysteines) prevents the change in NMDGþ permeability but has no effect on the uptake of ethidium. Such an effect is difficult to explain if both molecules use the same pathway to enter the cell. Further, both the Surprenant and Dahl laboratories have shown that RNA interference directed against recently discovered pannexin-1 hemi-channels significantly reduces dye uptake without effecting cation flux through the pore of the P2X7 channel. Thus, it seems that the P2X7 channel uses two different methods to move large ions across the cell surface membrane. The first is a gradual dilation of the integral P2X pore that ultimately results in an increase in permeability to NMDGþ. The second is an indirect activation of ethidium-permeable pannexin-1 channels that may result from a protein–protein interaction with the P2X7 receptor. These findings are exciting and novel, but additional experiments are needed to prove the hypothesis they seem to support.
Pharmacology Although the field has advanced in the past few years, it remains difficult to discriminate among P2X receptors on the basis of pharmacology alone. Gever et al. have provided an excellent review of the recent advances in purinergic pharmacology. Not only do many of the currently available reagents discriminate poorly among the P2X receptors themselves; these same drugs also act on ATP-gated metabotropic P2Y receptors. However, a few pharmacological trends do hold true. P2X receptors can
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be divided into three general classes on the basis of the phenotype of the ATP-gated current and the pharmacology of the receptor. The first class includes the P2X1 and P2X3 receptors. These receptors display agonistgated currents that desensitize rapidly (<1 s), have a high affinity for both ATP and its analog a,b-methylene ATP (the EC50 for both agonists is about 1 mmol l1), and are blocked by nanomolar concentrations of the purinergic antagonist TNP-ATP. The second class includes P2X2, P2X4, and P2X5. These receptors show a much slower rate of desensitization (tens of seconds), are less sensitive to ATP (EC50 about 6– 30 mmol l1), are relatively insensitive to a,b-methylene ATP, and are blocked by micromolar concentrations of TNP-ATP. Heteromeric receptors that contain members of both the first and the second classes generally adopt the agonist and antagonist sensitivities of the first class; that is, they show robust responses to low concentrations of both ATP and a,b-methylene ATP and are readily antagonized by TNP-ATP. The third class is made of the P2X7 receptor alone. This receptor shows the lowest ATP potency (EC50 > 100 mmol l1) and is the only member of the family in which prolonged activation is reported to result in cell vesiculation and death.
Distribution and Physiology P2X receptors are expressed throughout the body. They are found in a range of tissues, including nerve, muscle, glia, epithelium, bone, and hemopoietic. Most tissues express more than one subtype, and current dogma suggests that the majority of native receptors are heteromeric complexes of two or more subtypes. The P2X1 receptor is predominately expressed in smooth muscle. Genetically altered mice that lack this receptor are infertile because the ATP-mediated contraction of the vas deferens is missing. Further, P2X1 receptors are also widely expressed in the cardiovascular system, where they are found in heart, blood vessels, and platelets. However, no obvious effect on cardiovascular function is found in P2X1knockout mice, and the role of ATP in these tissues is still unknown. P2X2 and P2X4 receptors are found in neurons and glia throughout the central and peripheral nervous systems, where they facilitate neurotransmission by both presynaptic and postsynaptic actions. In contrast, the distribution of the P2X3 receptor is more restricted. It is predominately found on the sensory afferent neurons of the peripheral nervous system, where it mediates gustation; mechanosensory regulation of urinary bladder function; and transmission of persistent, chronic, neuropathic and inflammatory pain. P2X5 receptors are found in skeletal muscle and may play a role in regulating prolifer-
ation and differentiation of tumors. P2X7 receptors are found in mast cells, macrophages, and lymphocytes, where they participate in the immune response.
Presynaptic and Postsynaptic Effects In general, two separate but related functions are described for these receptors in neurons. First, P2X receptors allow cations to enter the cell, leading to membrane depolarization. Direct somatic depolarization by release of endogenous ATP has been difficult to demonstrate, perhaps because of the rapid degradation of the nucleotide by endonucleotidases. However, good examples exist including depolarization of smooth muscle in the vas deferens and neurons of the celiac ganglia, spinal cord, myenteric plexus, medial habenula, hippocampus, and somatosensory cortex. The outcome of depolarization is the propagation of excitation from one cell to the next. The second effect of ATP is presynaptic. P2X receptors increase intracellular calcium in nerve terminals, both directly, by conducting calcium, and indirectly, by activating voltage-gated calcium channels. In both cases, the rise in intraterminal calcium can lead to an increased probability of neurotransmitter release.
Conclusion P2X receptors are some of the newest members of the ligand-gated ion channel superfamily. Their unique topology, ubiquitous distribution, high calcium flux, and ability to induce the movement of very large (up to about 900 kDa) molecules across the cell-surface membrane suggest that they play important roles in regulating the homeostasis of a range of organisms. In upcoming years, a high-resolution map of a P2X receptor structure may well be forthcoming; this map, in combination with functional studies, will greatly improve our understanding of the molecular physiology and pharmacology of the receptor. Further, recent studies that demonstrate direct protein– protein interactions of P2X receptors and hemichannels, other ligand-gated channels, and Fe65 suggest that the influence of ATP in the physiology and pathophysiology of humans is underappreciated. See also: Adenosine; Adenosine Triphosphate (ATP); Purines and Purinoceptors: Molecular Biology Overview.
Further Reading Gever JR, Cockayne DA, Dillon MP, Burnstock G, and Ford AP (2006) Pharmacology of P2X channels. Pflu¨gers Archiv: European Journal of Physiology 452: 513–537. Khakh BS and North RA (2006) P2X receptors as cell-surface ATP sensors in health and disease. Nature 442: 527–532.
P2X Receptors 663 North RA (2002) Molecular physiology of P2X receptors. Physiological Reviews 82: 1013–1067. Roberts JA, Vial C, Digby HR, et al. (2006) Molecular properties of P2X receptors. Pflu¨gers Archiv: European Journal of Physiology 452: 486–500.
Samways DS, Li Z, and Egan TM (2006) Binding, gating, and conduction of ATP-gated ion channels. In: Arias H (ed.) Biophysical Aspects of Ligand-Gated Ion Channel Receptor Superfamilies, pp. 419–443. New York: Research Signpost.
Endocannabinoid Role in Synaptic Plasticity and Learning B Lutz, Johannes Gutenberg University, Mainz, Germany G Marsicano, Johannes Gutenberg University, Mainz, Germany; and U862 Centre de Recherche INSERM Franc¸ois Magendie Universite´ Bordeaux 2, Bordeaux, France ã 2009 Elsevier Ltd. All rights reserved.
Introduction The records of cannabinoids date back more than 5000 years, to when the therapeutic and psychotropic actions of extracts of the hemp plant Cannabis sativa were first documented. The long history of this plant has been very vivid and has not ceased to elicit a lot of attention in our society. As with other natural products with strong pharmacological effects in humans, there has been great interest in understanding what the active components of extracts from C. sativa are and how they act on the human body, but success was hampered for a long time because of technical reasons. Only in 1964 was the group of Raphael Mechoulam able to chemically identify D9-tetrahydrocannabinol (D9-THC) as the major psychoactive constituent of C. sativa (Figure 1(a)). However, it took another 25 years to identify the mechanisms underlying these effects. Specific binding proteins for D9-THC, called cannabinoid receptors, were discovered. Potent derivatives of D9-THC such as CP55940 and HU-210 (Figures 1(c) and 1(d)) were instrumental in the pharmacological and molecular identification of these novel receptors, which belong to the G-protein-coupled receptor family. It is worth mentioning that in addition to D9-THC, C. sativa extracts contain many other nonpsychoactive cannabinoids, such as cannabidiol (Figure 1(b)), which do not bind to cannabinoid receptors but which have interesting pharmacological effects with therapeutic potential. The molecular mechanisms underlying these effects have not yet been elucidated, but further investigation may lead to interesting insights, possibly even novel endogenous physiological mechanisms. Finally, the cloning of the cannabinoid receptor enabled the identification of the body’s own molecules that can elicit effects similar to those of D9-THC and that bind to and activate cannabinoid receptors. These endogenous ligands represent a novel class of lipid signaling molecules, called endocannabinoids (Figure 2). Endocannabinoids are derivatives of the polyunsaturated eicosanoid arachidonic acid and can act as versatile neuromodulators at the synaptic level in both the central and peripheral nervous systems. Multiple roles
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in nonneuronal cells (such as the regulation of lipid metabolism, bone mass, and immune responses) have also been reported. Discovered in 1992 and 1995, respectively, the two major endocannabinoids are N-arachidonoyl ethanolamine (AEA), named anandamide, and 2-arachidonoyl glycerol (2-AG) (Figures 2(a) and 2(b)). Both of them bind to and activate cannabinoid receptors. Recently, other endocannabinoids (such as 2-arachidonoyl glycerol ether and Narachidonoyl dopamine (Figures 2(c) and 2(d))) have been isolated. To date, two receptors have been characterized, both belonging to the class of seventransmembrane receptors, coupled to the pertussis toxin-sensitive G-protein, Gi/o. Cannabinoid type 1 (CB1) receptors are predominantly expressed in neurons, but important sites of expression have also been found in peripheral nonneuronal cells, such as adipocytes and hepatocytes. Cannabinoid type 2 (CB2) receptors are present mainly in immune cells, but recent investigations were able to show functional receptors in central nervous system (CNS) neurons, too. This article focuses mainly on the CB1 receptor and its role in synaptic plasticity and memory processing as the present knowledge of CB2 receptors in the CNS is sparse. However, it would not come as a surprise if in the near future, the relevance of CB2 receptors in the CNS is better understood and receives more attention than is the case to date. Endocannabinoids and cannabinoid receptors, together with the enzymes of endocannabinoid biosynthesis and degradation, constitute the endocannabinoid system. This system has recently emerged as an important regulator of a plethora of physiological processes in the CNS, including pain, appetite, reward, memory processing, and neuronal excitability. These functions appear to involve the fine-tuned regulation of synaptic transmission, which ultimately ensures homeostatic conditions of the nervous system. During pathological processes in the nervous system, dysregulations of the endocannabinoid system may occur. The endocannabinoid system in this state may be a therapeutic target for the treatment of particular nervous system diseases by agents that either stimulate or inhibit the activity of the endocannabinoid system, depending on the pathological conditions. During the past few years, this concept has drawn a lot of attention to the endocannabinoid system.
Cannabinoid Receptor CB1 and the Modulation of Intracellular Signaling The prototypical signaling after activation of CB1 receptor by both plant-derived cannabinoids and
Endocannabinoid Role in Synaptic Plasticity and Learning
endocannabinoids is the inhibition of adenylate cyclase via Gi/o protein, resulting in a decrease of intracellular cyclic adenosine monophosphate (cAMP) production (Figure 3), although coupling to Gs was also reported
OH
HO Δ9-THC
a
under some experimental conditions. CB1 receptordependent inhibition of the cAMP signaling pathway can stimulate A-type potassium channels, which are negatively regulated by phosphorylation provided by the cAMP-dependent protein kinase A (PKA). A direct coupling of the Gi/o protein to inwardly rectifying potassium channels (GIRK1 and GIRK2) and the Gi/o-dependent inhibition of N-type and P/Q-type voltage-gated Ca2þ channels lead to an enhanced Kþ efflux and to an inhibition of Ca2þ influx, respectively. Thus, CB1 receptor activation leads to a general decrease of neuronal excitability. In addition, a multitude of other intracellular signaling systems are activated, of which many have been implicated in synaptic plasticity and memory processing. Finally, immediate early genes, including brain-derived neurotrophic factor and the transcription factors zif268 and c-fos, are switched on after CB1 receptor activation.
OH
O
Cannabidiol
b HO
OH OH
OH
O HO c
CP55940
d
HU-210
Biochemistry of Endocannabinoids
Figure 1 Chemical structures of plant-derived and synthetic cannabinoids. (a) D9-Tetrahydrocannabinol (D9-THC), identified in 1964 by Raphi Mechoulam, is the major psychoactive component in extracts from the hemp plant C. sativa. It binds to and activates the cannabinoid receptors. (b) Cannabidiol, a major constituent of C. sativa, is not psychoactive, as it does not bind to cannabinoid receptors. However, it is able to elicit distinct pharmacological effects by mechanisms that have not been elucidated in detail. (c) CP55940, a synthetic derivative of D9-THC developed by Pfizer in 1974, is 40 times more potent than D9-THC on cannabinoid receptors. The radiolabeled CP55940 was pivotal in the discovery and molecular characterization of the long-sought endogenous D9-THC receptor, named cannabinoid receptor type 1 (CB1 receptor), as published in 1990. (d) HU-210, developed by the Hebrew University, is the most potent activator of cannabinoid receptors known to date (100 times more potent than D9-THC).
Endocannabinoids are remarkable signaling molecules with multiple faces. As they are biosynthetic derivatives from arachidonic acid, they belong to the class of bioactive eicosanoids, such as prostaglandins, thromboxanes, and leukotrienes. These three wellknown classes of lipophilic signaling molecules have been shown to act locally, in either an autocrine or a paracrine manner. For endocannabinoids, similar modes of actions also occur both in the CNS and in peripheral tissues. Different from ‘classical’ neurotransmitters, endocannabinoids cannot be stored in vesicles. Therefore,
O
O OH
OH
O
N H
a
665
OH
b 2-Arachidonoyl glycerol
Anandamide
O OH O
N H
OH
c
2-Arachidonoyl glycerol ether
d
OH OH
N-Arachidonoyl dopamine
Figure 2 Structure of endocannabinoids. (a) Anandamide (N-arachidonoyl ethanolamine, AEA), the first endocannabinoid, discovered in 1992 and named from the Sanskrit word ananda, meaning ‘internal bliss.’ (b) 2-Arachidonoyl glycerol (2-AG). (c) 2-Arachidonoyl glycerol ether (noladin ether). (d) N-Arachidonoyl dopamine (NADA). All these four compounds bind to and activate cannabinoid receptors, but with different efficacies. AEA and 2-AG are the major endocannabinoids; the relevance of noladin ether in physiological processes has still to be elucidated. NADA represents a novel category of compounds as it is a condensation product of arachidonic acid with an amino acid derivative. Other lipids with similarity to AEA and 2-AG, such as N-arachidonoyl serine, dihomo g-linolenoyl ethanolamide, docosatetraenyl ethanolamide, and oleamide, have been characterized, but these compounds do not bind or bind only with low affinity to the cannabinoid receptors CB1 and CB2.
666 Endocannabinoid Role in Synaptic Plasticity and Learning EC
K +ir
+
Ca2+
KA
CB1 Gi/o +
–
P –
Adenylate cyclase
P
–
PKC ATP cAMP + PI3-K
PKA +
+ FAK
+ JNK
+ + PKB/AKT Raf-1
+ ERK
+ Calcineurin
+ p38
+ IEG c-fos c-jun zif268 BDNF Figure 3 Schematic representation of the possible cannabinoid type 1 (CB1) receptor-mediated signaling pathways. Activation of CB1 receptors by both D9-THC and endocannabinoids (EC) leads to the stimulation of Gi/o proteins that, in turn, inhibit the adenylate cyclase-mediated conversion of adenosine triphosphate (ATP) to cyclic adenosine monophosphate (cAMP). Then cAMP molecules can bind the regulatory subunits of protein kinase A (PKA) and induce the liberation of the catalytic subunits. Activated PKA can phosphorylate A-type potassium (KþA) channels, causing a decrease of the Kþ efflux. Given the negative effect of CB1 receptor activation on adenylate 2þ cyclase, the final result is an activation of Kþ A channels. Activated Gi/o can also directly inhibit voltage-gated N- and P/Q-type Ca þ channels and activate inwardly rectifying potassium (K ir) channels. The latter two effects are controlled by protein kinase C (PKC), which, after activation, can phosphorylate CB1 receptors in the third cytoplasmatic loop and uncouple the receptor from the ion channels. CB1 receptor activation can also stimulate several intracellular kinases, such as focal adhesion kinase (FAK), phosphatidyl inositol-3-kinase (PI3-K) and its downstream effectors protein kinase B (PKB) (also known as AKT) and Raf-1, extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated kinase. CB1 receptor activation can also lead to increased expression levels of calcineurin, also called protein phosphatase 2B. Stimulation of cytoplasmic kinases can also mediate CB1 receptor-induced expression of immediate early genes (IEG), such as the transcription factors c-fos, c-jun, and zif268 and brain-derived neurotrophic factor (BDNF). It is important to note that several of these CB1 receptor-regulated signaling pathways have been shown to be involved in the modulation of synaptic plasticity and memory processing. P, phosphorylation.
biosynthesis (Figures 4 and 5) and degradation pathways of these compounds are the central regulatory steps in the activation and termination of endocannabinoid signaling. These pathways are currently being characterized, but some features are known: (1) Biosynthetic precursor molecules are phospholipids stored in the membrane. These phospholipids constitute a large pool and are not only specific for endocannabinoid synthesis but are also used for other
metabolic processes in the cell. (2) The ondemand biosynthesis of endocannabinoids can be regulated by several intracellular pathways, including Ca2þphospholipase C (PLC)- and/or cAMP-dependent mechanisms. (3) Biosynthesis of the two major endocannabinoids, anandamide and 2-AG, can occur independently of each other in different pathways, and regulation of the on-demand synthesis of anandamide and 2-AG can also differ. (4) Biosynthetic pathways
Endocannabinoid Role in Synaptic Plasticity and Learning Acyl Acyl
O O P O O–
+
Arachidonoyl Acyl
NH2
667
O O P O R O–
Phosphatidyl ethanolamine (PE) N-Acyltransferase (Ca2+, cAMP) (NAT) Acyl Acyl
O O P O O–
O +
N H
HO Acyl
O O P O R O–
N -Arachidonoyl-PE (NArPE)
PLA2 (Ca2+)
Acyl HO
O O P O– O– N-Arachidonoyl 1-acyl-lyso-PE
NAPE-PLD (Ca2+)
Lyso-PLD
O HO
N H
+ Phosphatidic acid
Anandamide (N-Arachidonoyl ethanolamine) Figure 4 Schematic representation of the biosynthetic pathways leading to anandamide (N-arachidonoyl ethanolamine, AEA). As a first Ca2þ-dependent (and possibly also cyclic adenosine monophosphate (cAMP)-regulated) reaction catalyzed by N-acyltransferase (NAT), N-arachidonoyl-PE (NArPE) is generated from two membranous precursor molecules, phosphatidyl ethanolamine (PE) and an arachidonoyl phosphoglyceride. Then, anandamide can be synthesized by two different Ca2þ-dependent pathways, either by N-acyl-PE-phospholipase D (NAPE-PLD) or by phospholipase A2 (PLA2) and lyso-PLD. Note the redundancy of the pathways.
appear to be redundant, as gene inactivation experiments have not yet been able to pinpoint conclusively the crucial enzymes. Pharmacological experiments using inhibitors have provided good evidence for particular pathways, but such inhibitors may not be completely specific for one particular enzyme. (5) Degradation enzymes (FAAH and MAGL for anandamide and 2-AG, respectively), although clearly able to degrade the respective compounds, appear not to be completely specific. In particular, 2-AG and its precursors are at the crossroads of several metabolic pathways and are important precursors or/and degradation
products of phospho-, di- and triglycerides. FAAH is also able to degrade many other acyl ethanolamides, which are not considered endocannabinoids. (6) After action, endocannabinoids can be taken up into the cells by facilitated transport mechanisms, although the endocannabinoid membrane transporter has not been cloned yet. (7) The differential physiological functions of anandamide and 2-AG are not completely understood, although many electrophysiological studies have identified 2-AG as the most likely to act as a retrograde neurotransmitter. (8) Anandamide is promiscuous and able to modulate the activity of
668 Endocannabinoid Role in Synaptic Plasticity and Learning Acyl Arachidonoyl
Acyl Arachidonoyl O O P O⫺ O⫺
O O P O Inositol O⫺
Phosphatidyl inositol Phosphatidic acid phosphohydrolase
PLC
PLA1
HO Arachidonoyl
Acyl Arachidonoyl OH
sn -1-Acyl-2arachidonoyl glycerol
O O P O Inositol O⫺
sn -1 lyso phosphatidyl inositol
DAGL (Ca2+)
Lyso-PLC HO Arachidonoyl OH
2-Arachidonoyl glycerol (2-AG) Figure 5 Schematic representation of the biosynthetic pathways leading to 2-arachidonoyl glycerol (2-AG). Membranous arachidonoyl phosphoglycerides serve as precursors to react with the enzymes phosphatidic acid phophohydrolase, phospholipase C (PLC, which can be regulated by G-protein-coupled receptors), and/or phospholipase A1. 2-AG is then formed by the enzymes diacylglycerol lipase (DAGL) and lyso-PLC. The pathways are redundant, as are the pathways for anandamide synthesis. PLA1, phospholipase A1.
several other receptors, in particular vanilloid TRPV1 receptor (anandamide stimulates Ca2þ influx, cation current, and depolarization), TASK-1 (anandamide inhibits outward Kþ current), low-voltage-activated T-type Ca2þ channels (anandamide blocks Ca2þ current), 5-hydroxytryptamine type 3 serotonin receptor (anandamide acts as a negative allosteric modulator), and glycine receptor (anandamide and 2-AG act as inhibitors). In conclusion, the current level of knowledge about endocannabinoids is not yet comparable with what is known about ‘classical’ neurotransmitters, and further intensive investigations are necessary to fully illuminate the on-demand biosynthesis of endocannabinoids. This is exemplified by the observation that genetic deletion of the anandamide-synthesizing enzyme NAPE-PLD does not lead to a deficit in either basal or stimulated anandamide levels. Thus, either NAPE-PLD is not the important enzyme, or redundancy and/or efficient compensatory pathways exist to synthesize anandamide. During past years, several other bioactive lipid mediators have been described which have structural similarities with endocannabinoids and which act, at least in some cases, via cannabinoid receptors CB1 and/or CB2. These compounds include dihomo g-linolenoyl ethanolamide, docosatetraenoyl ethanolamide, N-arachidonoyl serine, and oleamide.
Specific pharmacological effects in vivo have been described for some of them, but for many of these compounds, endogenous functions remain to be deciphered, and possible corresponding high-affinity receptors have yet to be identified. Therefore, the term endocannabinoid may not be correct in a strict sense, but it is important to note that the prototypical structures of anandamide and 2-AG have initiated the discovery of novel endogenous compounds that may be components of yet-to-be-discovered signaling systems in the body. This is exemplified by recent observations that GPR55, a G-protein-coupled receptor, may be considered a novel ‘cannabinoid’ receptor. Recent data indicate that endogenous peptides could also modulate the activity of cannabinoid receptors, thus opening new perspectives in the field of the endocannabinoid system.
Distribution of the Endocannabinoid System in the Nervous System In the adult nervous system of rodents and humans, CB1 receptors are abundantly and widely expressed in neurons of numerous brain areas, in the spinal cord, and in peripheral neurons such as dorsal root ganglia and enteric nervous system. The various expression sites in the brain correspond very well with
Endocannabinoid Role in Synaptic Plasticity and Learning
the different physiological functions of the endocannabinoid system, including, to mention only a few, in hippocampus, cerebral cortex, and amygdala for memory processing; in striatum for motor activity; in the nucleus accumbens, cerebral cortex, and ventral tegmental area for reward processing; and in hypothalamus for the regulation of the neuroendocrine axis, feeding behavior, and energy balance. CB1 receptors are also expressed in glial cells, such as astrocytes and microglial cells. The latter expression sites have not been implicated to date in the regulation of synaptic transmission, but they are likely involved in CB1 receptor-mediated neuroprotection and repair mechanisms. CB1 receptors are also highly expressed in the developing nervous system of the embryo, where they are particularly highly present in proliferating neuronal precursor cells. Matching this embryonic expression, CB1 receptors are also described in neuronal precursors in the dentate gyrus and subventricular zone of the adult brain, promote the proliferation of these neuronal precursor cells, and endorse the switch of these newly born cells toward a glial fate, as shown in the dentate gyrus. In neurons of the adult brain, functional CB1 receptors are shown to be localized predominantly at presynaptic sites and, as recently shown for particular cortical g-aminobutyricacidergic (GABAergic) neurons, on the soma. Some CB1 receptor protein can also be detected intracellularly in the cytoplasma of the soma and in axonal projection, but the functional significance of these expression sites is still a matter of investigation, and the possibility exists that these CB1 receptors are merely proteins that have been newly synthesized and transported to their final sites of action. CB1 receptors present at the presynapse mediate the endocannabinoid-mediated retrograde suppression of neurotransmitter release, while their expression at the soma is likely responsible for an autocrine endocannabinoid-mediated self-inhibition of neuronal activity. These features will be discussed later as they represent remarkable mechanisms for the short- and long-term modulation of excitatory and inhibitory transmission in the nervous system and in synaptic plasticity, with profound implications for the quest to understand how phenomena such as memory processing function. The complexity of CB1 receptor expression is further enhanced by the fact that CB1 receptors and other components of the endocannabinoid system are found in diverse neuronal subpopulations. CB1 receptor messenger RNA (mRNA) and protein are highly abundant in GABAergic interneurons (in particular, in cholecystokinin-containing GABAergic interneurons). The expression levels in GABAergic neurons may be counted at the highest levels known
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for any receptor in the brain. This feature contrasts very much with the expression in cortical glutamatergic neurons. Here, the expression levels of both mRNA and protein may be estimated to be as much as 50–100 times lower than in GABAergic neurons (Figure 6). It is, however, remarkable that CB1 receptor expression at these low levels in glutamatergic neurons is necessary and sufficient to mediate several functions, such as protection from abnormal neuronal activities and some of the central effects of D9-THC. All other components of the endocannabinoid system also show a differential distribution. For the two major endocannabinoids, anandamide and 2-AG, levels differ depending on brain area and the diurnal cycle, but they can also grossly change in response to physiological stimuli or during pathological conditions of the brain. To terminate the signaling, the enzymes for the degradation of endocannabinoids, fatty acid amide hydrolase (FAAH, degrading anandamide) and monoacylglycerol lipase (MAGL, degrading 2-AG), are also present in the brain. It is interesting that both enzymes exhibit differential expression patterns in cortical areas, in particular in the hippocampal formation. FAAH is present in pyramidal neurons of CA1 and CA3 and in granule cells of the dentate gyrus, but not in GABAergic interneurons. MAGL is expressed similarly to CB1 receptor, present both in GABAergic interneurons and pyramidal neurons. Remarkably, FAAH is expressed specifically at the postsynapse, while MAGL is present in the presynaptic terminal. The differential distribution of FAAH and MAGL might underlie the different functions of the two major endocannabinoids, anandamide and 2-AG, in neurons. The regional distribution of the putative enzymes for the biosynthesis of endocannabinoids has not been studied in detail yet.
Cannabinoids and the Suppression of Neurotransmitter Release Exogenously applied cannabinoids were shown to suppress GABAergic transmission in several brain regions, including hippocampus, amygdala, striatum, nucleus accumbens, and substantia nigra. These effects were inhibited by specific CB1 receptor antagonists (e.g., SR141716, also called rimonabant) and were absent in slices derived from CB1 receptor-deficient mice. Similar results were obtained for glutamate transmission. Furthermore, cannabinoids were also shown to inhibit the evoked release of other neurotransmitters, such as noradrenalin and acetylcholine in the hippocampus, dopamine in the striatum, and serotonin in the cerebral cortex. An understanding of the underlying mechanisms has been advanced by insights into the biochemistry and neuroanatomy of
670 Endocannabinoid Role in Synaptic Plasticity and Learning
a GAD/CB1
b
VGLUT1/CB1
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Figure 6 Distribution of messenger RNA (mRNA) encoding CB1 receptor visualized by in situ hybridization. (a) View of the hippocampal formation of an adult mouse brain. Brown-colored precipitation, CB1 receptor mRNA; blue staining, cell nuclei. Note the very high expression in scattered neurons (GABAergic interneurons, black arrow) and the low but distinct expression in pyramidal cells (glutamatergic neurons, white arrow). (b) Co-expression (black arrow) of CB1 receptor mRNA (red staining) with the GABAergic marker g-aminobutyric acid decarboxylase (GAD; silver grains). (c) Co-expression (white arrow) of CB1 receptor mRNA (red staining) with the glutamatergic marker vesicular glutamate transporter type 1 (VGLUT1; silver grains).
the endocannabinoid system. The principal mechanisms by which the endocannabinoid system can modulate synaptic transmission are depicted in Figure 7. Several specific modulatory mechanisms of synaptic transmission and the involvement of the endocannabinoid system in short- and long-term synaptic plasticity are discussed briefly in the following sections. This field of study is expanding rapidly.
Endocannabinoids and Short-Term Synaptic Plasticity When a neuron is activated postsynaptically, it produces endocannabinoids, which travel in a retrograde direction to the presynaptic site and transiently (<1 min) suppress presynaptic neurotransmitter release. The phenomenon of short-term depression (STD) was first observed about 15 years ago, when a brief depolarization of hippocampal principal neurons was shown to trigger a transient suppression of
GABAergic synaptic input. This was named depolarization-induced suppression of inhibition (DSI). Critical features of DSI were defined as follows: (1) DSI induction requires an increase in postsynaptic Ca2þ. (2) DSI has a presynaptic location of expression. (3) Therefore, a retrograde messenger must exist mediating the inhibition of neurotransmitter release. The molecular nature of such a retrograde messenger has remained elusive for a long time. However, in 2001, endocannabinoids, which are stimulated by Ca2þ and act at presynaptic CB1 receptors, were discovered to be the postulated retrograde transmitter (Figure 8). Several areas besides the hippocampus were found to express DSI, including substantia nigra, dentate gyrus, and cerebellum. In addition, this phenomenon has been observed for glutamatergic transmission, is called depolarization-induced suppression of excitation (DSE), and is found in areas, including hippocampus, cerebral cortex, hypothalamus, ventral tegmental area, and cerebellum.
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Figure 7 Representation of the synthesis, signaling, and degradation of endocannabinoids at the synaptic site. Key steps and mechanisms are depicted. In the postsynapse, Ca2þ and G-protein-coupled receptor-induced activation of Gq protein can initiate the synthesis of endocannabinoid (EC), which reaches the synaptic cleft by facilitated diffusion through the EC membrane transporter (EMT). Cannabinoid type 1 receptor (CB1) is localized at the presynaptic site, where it is activated by EC and then reduces Ca2þ-induced release of neurotransmitter. AEA, anandamide; 2-AG, 2-arachidonoyl glycerol; DAGL, diacylglycerol lipase; FAAH, fatty acid amide hydrolase; MAGL, monoacylglycerol lipase; mGluR, metabotropic glutamate receptor; M1/M3, muscarinic acetylcholine receptor types M1 and M3; NAPE-PLD, N-acyl phosphatidyl ethanolamine phospholipase D; NArPE, N-arachidonoyl phosphatidyl ethanolamine; PLCb, phospholipase Cb.
Clearly, the number of different brain areas with proven CB1 receptor-dependent DSI/DSE expression will grow even further in the near future. Synthesis and release of endocannabinoids is a complex process. For the initiation of endocannabinoid production, two pathways have been described in the hippocampus. (1) Postsynaptic increase of Ca2þ is necessary and sufficient to trigger endocannabinoid production. (2) Activation of postsynaptic metabotropic glutamtate receptors (mGluRs) or muscarinic acetylcholine receptors with exogenous agonists is also sufficient to trigger endocannabinoid release. It is interesting that metabotropic induction of DSI does not
require a large increase in Ca2þ. It is suggested that the metabotropic pathway triggers endocannabinoid production by PLCb and that PLC activity is facilitated by increases in Ca2þ. In this model, PLC acts as a coincidence detector, integrating two different postsynaptic processes: metabotropic receptor activation and Ca2þ increase. Two contributions for the intracellular Ca2þ increase are described: Influx through voltage-gated Ca2þ channels, and release from intracellular Ca2þ stores via ryanodine-sensitive receptors. After their synthesis, endocannabinoids travel to the presynapse. This process is not understood at all. It is not clear how highly lipophilic molecules such as
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Postsynapse Figure 8 Schematic summary of endocannabinoid-mediated short-term plasticity, called depolarization-induced suppression of excitation (DSE; depicted on the left side) and depolarization-induced suppression of inhibition (DSI; depicted on the right side). Two events can induce endocannabinoid production. (1) Postsynaptic step depolarization (experimentally by a shift from 60 mV to 0 mV) or action potential at the postsynapse induces Ca2þ influx via voltage-gated calcium channels. Ca2þ influx may be amplified by enhancement of Ca2þ release from intracellular stores via ryanodine receptors. (2) Brief tetanic stimulation of excitatory afferents or group I metabotropic glutamate receptor (mGluR) activation by agonists can initiate endocannabinoid synthesis. Alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) activation may also contribute to this induction process. Phospholipase C (PLC) and diacylglycerol lipase (DAGL) are required for mGluR-mediated processes, indicating 2-arachidonoyl glycerol (2-AG) as an important candidate for this pathway; 2-AG will activate presynaptic cannabinoid type 1 (CB1) receptors, leading to a short depression of neurotransmitter release (<1 min). Depending on the synaptic input that is measured, either DSI or DSE will be observed. DAG, diacyl glycerol; GABA, g-aminobutyric acid.
endocannabinoids are able to diffuse so rapidly across the synaptic cleft. Binding proteins may help to increase the solubility in the hydrophilic environment of the synaptic cleft. In the cholecystokinin-expressing GABAergic interneurons of the hippocampus, the CB1 receptordependent effects at the presynaptic site are mediated mainly by an inhibition of N-type Ca2þ channels by a direct interaction with the CB1 receptor-induced liberation of Gi/o bg subunits, but a direct effect on the vesicle release machinery , and/or Kþ channel activation also have to be considered. In synapses of hippocampal GABAergic interneurons onto principal neurons, the time-to-onset of inhibitory postsynaptic current in the voltage step protocol (60 mV to 0 mV in a particular frequency) to induce DSI was determined to be about 410 ms. In experiments using caged anandamide that
can be photoactivated to exert its action, the times for three steps were determined: Time for the Ca2þ induction was 60 ms, time for endocannabinoid synthesis, 190 ms, and time to activate CB1 receptors, 160 ms. When mGluRs were activated to induce DSI, time for endocannabinoid synthesis was reduced to 75 ms. These observations suggest that different pathways can lead to the on-demand synthesis of endocannabinoids and that the time to activate CB1 receptors is an important rate-limiting step. The detailed mechanisms for post- and presynaptic events can vary in different brain regions, as shown for the cerebellum in particular. There are also indications that, depending on the stimulus, variations between one synapse and another within a distinct brain region exist. These features thus provide a high level of complexity to endocannabinoid-mediated modulation of synaptic plasticity.
Endocannabinoid Role in Synaptic Plasticity and Learning
Endocannabinoids and Long-Term Plasticity CB1 receptor-mediated processes also have a long-term impact on synaptic transmission. Longterm depression (LTD) of synaptic transmission can occur via a presynaptic CB1-dependent mechanism. Once established, LTD does not require continuous CB1 activation. The postsynaptic trigger is similar to the one inducing short-term depression, usually Ca2þ increase and/or activation of mGluRs. As compared with the ‘common’ LTD forms on excitatory synapses, endocannabinoid-mediated LTD is independent of postsynaptic glutamate receptors of the N-methyl-D-aspartate (NMDA) type. The wide occurrence of endocannabinoid-mediated LTD in the brain indicates that the endocannabinoid system is able to make long-term modifications to neural circuits and behaviors such as learning and memory. Homosynaptic LTD on Excitatory Synaptic Input
Homosynaptic LTD on excitatory synaptic input was first discovered in corticostriatal and corticoaccumbal projections. For example, at glutamatergic cortical inputs of dorsal striatal GABAergic medium spiny neurons, high-frequency stimulation of this synapse (100-Hz trains) induces a form of LTD shown to depend on the activation of CB1 receptors. This process follows a scheme similar to the one described for DSI (Figure 8) and presents the following hallmarks: (1) requirement of postsynaptic intracellular Ca2þ increase; (2) postsynaptic loading with anandamidesuppressed synaptic transmission via a presynaptic mechanism, which is CB1 receptor dependent and which suggests that anandamide can travel from the post- to the presynaptic site; and (3) dependence on a convergence of several input signals, including group I mGluRs, D2 dopamine receptors, and L-type Ca2þ channels. The dorsal striatum and nucleus accumbens are relevant for a variety of goal-oriented behaviors, dorsal striatum for motor planning and reward-driven motor learning and nucleus accumbens in processing of rewarding environmental stimuli and in drug addiction. Therefore, the involvement of endocannabinoids on this LTD is very relevant to understanding several motor- and reward-related behaviors. Heterosynaptic LTD on Inhibitory Synaptic Input
This form of CB1-dependent synaptic plasticity was first discovered in amygdala (called LTDi) and hippocampus (called I-LTD), two brain areas critically involved in emotional and declarative memory. They are heterosynaptic forms of plasticity, induced by repetitive activity of excitatory synaptic
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inputs onto glutamatergic neurons, resulting in LTD of nearby inhibitory synapses of CB1 receptor- and cholecystokinin-expressing GABAergic interneurons and thus generating a long-term decrease in GABA release (Figure 9). As GABAergic inputs regulate the balance between excitatory and inhibitory drive in the neural network, this durable disinhibition may have long-term effects on excitability and on the synaptic plasticity of excitatory synapses, finally facilitating the formation of new memories by use-dependent processes. However, similar to the continuing discussion of whether and in which manner long-term potentiation (LTP) on excitatory synapses is an electrophysiological correlate of memory processing, it is likely to be very difficult to establish a functional link between heterosynaptic endocannabinoid-dependent LTD and memory processing and behavior. In contrast to DSI and homosynaptic LTD, heterosynaptic LTD does not require postsynaptic Ca2þ increase, but activation of group I mGluRs is both necessary and sufficient for the induction of LTDi/I-LTD. However, there are distinct differences between LTDi and I-LTD. In the hippocampus, I-LTD is induced by moderate- to high-frequency stimulation (10–100 Hz) and by theta-burst stimulation. PLC and diacylglycerol lipase are involved in generating 2-AG, as postsynaptic inhibition of these enzymes inhibited I-LTD. In the amygdala, LTDi is induced by low-frequency stimulation (1 Hz, 100 stimuli), and inhibitors of adenylate cyclase and PKA, but not of PLC and diacylglycerol lipase, did affect the induction of LTDi. It is suggested that a particular isoform of mGluRs may be linked to the adenylate cyclase-PKA pathway, which then activates N-acyltransferase, the first step in the synthesis of anandamide. Genetic inactivation of the anandamide-degrading enzyme FAAH facilitates the formation of LTDi, further suggesting a role of anandamide but not 2-AG in this process. However, more experiments will be necessary to fully reveal this cAMP-regulated pathway of endocannabinoid production. Time-Dependent LTD
This form of plasticity of glutamatergic transmission has been observed in pyramidal neurons in layer V of the visual cortex. Here, LTD on glutamatergic synapses is triggered by pairing of presynaptic stimulations with postsynaptic depolarizations or action potentials. The time schedule of the pairing of the stimuli is important for LTD formation; thus, it is also called timing-dependent LTD. Increases of postsynaptic Ca2þ lead to the production of endocannabinoids, which then inhibit glutamate release from the presynapse. Presynaptic, but not postsynaptic,
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While in DSI/DSE and endocannabinoid-dependent LTD, retrograde signaling from the post- to the presynapse occurs, an autocrine mechanism has recently been described in low-threshold-spiking (LTS) neocortical GABAergic interneurons (Figure 10). This function occurs after sustained firing of the interneuron. Intracellular Ca2þ rises after opening of voltage-gated Ca2þ channels in the somatodendritic compartment, and 2-AG is synthesized from membranous precursors. Then 2-AG most likely travels laterally within the membrane and activates CB1 receptors, leading to a Gi/o-dependent inhibition of Kþ channels and a subsequent hyperpolarization of LTS GABAergic interneurons, which lasts at least 30 min. As GABAergic interneurons are centrally involved in oscillatory processes in neural networks, this endocannabinoid-mediated modulation of synaptic transmission may also be relevant for memory processing.
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2-AG Postsynapse b Figure 9 Heterosynaptic endocannabinoid-mediated long-term depression (LTD) of inhibitory current. (a) Gamma-aminobutyricacidergic (GABAergic) input onto a principal glutamatergic neuron is activated and g-aminobutyric acid (GABA) is released. (b) In heterosynaptic LTD of GABAergic transmission, a coincident stimulation of an excitatory input onto the same principal glutamatergic neuron provokes the synthesis of endocannabinoids at the postsynaptic site. Cannabinoid type 1 (CB1) receptor-expressing GABAergic terminals receive the endocannabinoid signaling and reduce GABA transmission. This effect persists as long as 30 min. Metabotropic glutamate receptor (mGluR) activation is necessary and sufficient to induce LTD. The induction of endocannabinoid synthesis may differ in hippocampus and amygdala. First, while medium- to high-frequency stimulation is required for hippocampal I-LTD, only low-frequency stimulation induces LTDi in amygdala. Second, hippocampal I-LTD requires phospholipase C (PLC) and diacylglycerol lipase (DAGL) activity and involves 2-arachidonoyl glycerol (2-AG), while LTDi involves cyclic adenosine monophosphate (cAMP) pathway to generate presumably anandamide (AEA). AC, adenylate cyclase; DAG, diacylglycerol; Glu, glutamate; NAT, N-acyltransferase; PKA, protein kinase A.
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Figure 10 Mechanism of endocannabinoid-mediated slow self-inhibition. Depolarization of distinct neocortical g-aminobutyricacidergic interneurons (so-called low-threshold spiking interneurons, which also express cholecystokinin) opens somatodendritic Ca2þ channels and induces the synthesis of 2-arachidonoyl glycerol (2-AG), which binds to cannabinoid type 1 (CB1) receptors present on the same neurons. CB1 receptor activation inhibits inwardly rectifying Kþ channels, finally leading to a hyperpolarization of the neuron.
Endocannabinoid Role in Synaptic Plasticity and Learning
Endocannabinoids and Memory Processing The quest to understand the roles of the endocannabinoid system is impelled by the well-known observation that exogenous cannabinoids (e.g., from smoking marijuana) impair certain forms of memory. However, application of exogenous cannabinoids to animal models and subsequent behavioral analysis have not been particularly successful in elucidating this process of impairment. An important reason is that the endocannabinoid system is activated on demand, whereas exogenous application of agonists lacks temporal and spatial selectivity. As discussed earlier, the endocannabinoid system is involved in many different forms of modulation of synaptic transmission. These modulations can be either of short duration or longlasting and represent important mechanisms of synaptic plasticity. Short-term and long-term suppression of both glutamate and GABA transmission is a major effect, but the endocannabinoid system is also able to influence LTP at excitatory synapses, an electrophysiological phenomenon hotly discussed in memory research. In hippocampal slices, an induction of DSI with subsequent subthreshold high-frequency stimulation (which would normally not induce LTP) enables the formation of LTP. Thus, DSI transiently reduces the inhibitory drive in the system and makes synapses more excitable and can thereby strengthen synaptic connections. Thus, the endocannabinoid system provides multiple possibilities for influencing memory processing. To produce results that enable one to draw solid conclusions about the role of the endocannabinoid system in memory processing, the most powerful approach has been the application of specific CB1 receptor antagonists to animals, in particular to rodents, and the use of mice deficient in CB1 receptor. In several behavioral paradigms, distinct phenotypes have been observed. Fear Conditioning
In this behavioral test, the mouse hears a loud but per se harmless tone. Then, the tone co-terminates with a mild electric foot shock. The animal will form an association of the tone and the context (the conditioning chamber) with the electric shock. In subsequent exposures to the tone or the context, the mouse will show a fear reaction (e.g., freezing, an innate defense response in rodents) as a harmful shock is expected. If the shock does not occur anymore, the mouse will gradually start to decrease the freezing reaction, a
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phenomenon called extinction. It has been shown that the endocannabinoid system is required for the appropriate extinction of both tone freezing and contextual freezing. It is noteworthy that acquisition and consolidation are the same in wild-type and CB1 receptor-deficient mice. As fear conditioning depends on the amygdala, electrophysiological properties have been characterized. CB1 receptor-deficient mice show increased LTP, normal LTD, and abolished LTDi. How these changes can be understood in relation to impaired extinction remains to be elucidated. Morris Water Maze
This assay investigates learning abilities in a spatial memory task. Mice have to learn the location of an invisible escape platform in a small water tank by using contextual cues to orientate themselves in the space. Efficiency and accuracy in learning this location do not depend on the endocannabinoid system, but in the reversal test, when the position of the platform is transferred into the opposite quadrant of the tank, CB1 receptor-deficient mice have shown impaired ability to find the new position and keep on searching in the original position much longer than control mice do. This behavior can be interpreted as impaired extinction of the previously acquired memory trace and impaired flexibility toward a new environment. Thus, the phenotype is similar to the one observed for fear conditioning. A possible interpretation is that the Morris water maze represents a strong aversive event for mice, and thus systems similar to those for fear conditioning may be required in a CB1 receptor-dependent manner. Object Recognition and Social Recognition
In this assay, rodents have to learn whether a new object or another rodent can be classified as familiar or unfamiliar. Normal animals spend more time with unfamiliar than with familiar objects or animals. CB1 receptor antagonist-treated rodents and CB1 receptor-deficient mice show an enhanced ability to prefer novel objects and animals, at least when mice are young and middle-aged. This means that the endocannabinoid system acts to inhibit the formation and stability of memory traces in this period of age. In old animals, however, CB1 receptor-deficient mice are inferior to wild-type mice and show neurodegeneration in the hippocampus. This may be due to the observation that the endocannabinoid system has important functions in the regulation of stress responses, mediated by the hypothalamic–pituitary– adrenal axis. Lifelong absence of CB1 receptors may
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cause sustained increases of corticosterone, leading finally to neurodegenerative effects in the hippocampus. Regarding electrophysiological properties, it is interesting that LTP is enhanced in CB1 receptordeficient mice compared with wild-type controls, as analyzed in young and middle-aged animals. Appetitively Motivated Learning Task
In this task, in order to obtain a food reward, micehave to learn to poke a hole at the location where a light appears. After learning, mice are tested after 1 week to evaluate memory consolidation. After this, extinction is induced by omitting the food reward when they poke the correct hole. Neither the learning phase nor consolidation nor extinction depends on the endocannabinoid system in this task. Thus, if there is no aversive component, the endocannabinoid does not seem to be involved in extinction processes.
Conclusion During the past few years, the endocannabinoid system has emerged as an important modulator of synaptic plasticity. Both short-term and long-term effects on synaptic strength and efficacy have been reported at GABAergic and glutamatergic synapses. Numerous brain circuits are modulated by endocannabinoids. The effects are mediated either by presynaptically expressed CB1 receptors and retrograde endocannabinoid transmission or by autocrine mechanisms. The endocannabinoid system is involved in several memory systems, particularly in the extinction of emotional memories and the acquisition of object and social recognition memory. However, further investigation is needed to bridge the gap between the functions of this neuromodulatory system in electrophysiological processes in vitro and the behavior of the intact organism. See also: Long-Term Depression (LTD): Endocannabinoids and Cerebellar LTD; Neuromuscular Junction (NMJ): Presynaptic Short-Term Plasticity of Neuromuscular Transmission; Synaptic Plasticity: ShortTerm Mechanisms.
Further Reading Alger BE (2002) Retrograde signaling in the regulation of synaptic transmission: Focus on endocannabinoids. Progress in Neurobiology 68: 247–286. Bilkei-Gorzo A, Racz I, Valverde O, et al. (2005) Early age-related cognitive impairment in mice lacking cannabinoid CB1 receptors. Proceedings of the National Academy of Sciences of the United States of America 102: 15670–15675. Chevaleyre V, Takahashi KA, and Castillo PE (2006) Endocannabinoid-mediated synaptic plasticity in the CNS. Annual Review of Neuroscience 29: 37–76. Di Marzo V, Bifulco M, and De Petrocellis L (2004) The endocannabinoid system and its therapeutic exploitation. Nature Reviews. Drug Discovery 3: 771–784. Gerdeman GL, Ronesi J, and Lovinger DM (2002) Postsynaptic endocannabinoid release is critical to long-term depression in the striatum. Nature Neuroscience 5: 446–451. Heinbockel T, Brager DH, Reich CG, et al. (2005) Endocannabinoid signaling dynamics probed with optical tools. Journal of Neuroscience 25: 9449–9459. Howlett AC, Barth F, Bonner TI, et al. (2002) International Union of Pharmacology. XXVII. Classification of cannabinoid receptors. Pharmacological Reviews 54: 161–202. Mackie K (2005) Distribution of cannabinoid receptors in the central and peripheral nervous system. Handbook of Experimental Pharmacology 168: 299–325. Marsicano G, Wotjak CT, Azad SC, et al. (2002) The endogenous cannabinoid system controls extinction of aversive memories. Nature 418: 530–534. Monory K, Massa F, Egertova M, et al. (2006) The endocannabinoid system controls key epileptogenic circuits in the hyppocampus. Neuron 51: 455–466. Nicoll RA and Alger BE (2004) The brain’s own marijuana. Scientific American 291: 68–75. Pagotto U, Marsicano G, Cota D, Lutz B, and Pasquali R (2006) The emerging role of the endocannabinoid system in endocrine regulation and energy balance. Endocrine Reviews 27: 73–100. Piomelli D (2003) The molecular logic of endocannabinoid signalling. Nature Reviews Neuroscience 4: 873–884. Varvel SA, Anum EA, and Lichtman AH (2005) Disruption of CB (1) receptor signaling impairs extinction of spatial memory in mice. Psychopharmacology (Berl) 179: 863–872. Wilson RI and Nicoll RA (2001) Endogenous cannabinoids mediate retrograde signalling at hippocampal synapses. Nature 410: 588–592. Wotjak CT (2005) Role of endogenous cannabinoids in cognition and emotionality. Mini Reviews in Medicinal Chemistry 5: 659–670.
Long-Term Depression (LTD): Endocannabinoids and Cerebellar LTD D M Lovinger, National Institutes of Health, Rockville, MD, USA Published by Elsevier Ltd.
Endocannabinoid-Dependent Long-Term Depression Is a Form of Synaptic Plasticity Glutamatergic synapses are capable of long-lasting changes in their ability to communicate. These enduring changes in efficacy are often referred to as long-term plasticity. Long-term depression (LTD) a long-lasting decrease in synaptic efficacy, occurs at glutamatergic synapses throughout the brain. Subtypes of LTD with different mechanisms have been identified. One recently discovered form of LTD involves endogenous fatty acid derivates that are agonists for cannabinoid receptors (called endocannabinoids). Endocannabinoid-dependent LTD (eLTD) is widespread in the forebrain. This form of plasticity has been observed at the following glutamatergic forebrain synapses: . Glutamatergic synapses between deep-layer cortical projection neurons and striatal medium spiny projection neurons. . Glutamatergic synapses on medium spiny projection neurons in the nucleus accumbens. . Glutamatergic synapses between pairs of layer 5 thick-tufted neurons in the primary visual cortex. . Glutamatergic synapses between layer 4 neurons and layer 2/3 neurons in the whisker barrel field sensory cortex. A form of eLTD induced by the application of the addictive drug amphetamine has been observed at glutamatergic inputs to neurons in the lateral amygdala. The synapses that exhibit LTD in this case arise from extrinsic cortical inputs to the amygdala. In addition, eLTD has been described at g-aminobutyric acid (GABA)ergic inhibitory synapses in other brain regions, including the basolateral amygdala and hippocampus. It is likely that this form of plasticity will be found elsewhere in the nervous system in the coming years. A form of LTD found within the cerebellum also has mechanistic similarities to eLTD (see the section titled ‘Cerebellar long-term depression’).
Endocannabinoids Endocannabinoids are fatty acid analogs that are metabolites of membrane lipids. These compounds are formed inside of neurons as a result of lipid breakdown catalyzed by phospholipases. There are
two major endocannabinoids in the body: arachindonylethanolamide (AEA, also known as anandamide) and 2-arachindonyl glycerol (2-AG). Increases in intracellular calcium play a prominent role in endocannabinoid formation because they stimulate the enzymes necessary for biosynthesis of the compounds. The biological actions of endocannabinoids are produced via interactions with several target proteins, the most prominent of which are the cannabinoid receptors. Cannabinoid receptors were originally discovered as targets for D9-tetrahydrocannabinol (D9-THC), the psychoactive ingredient in marijuana and other drugs derived from the Cannabis sativa plant. These receptors couple to G-proteins and activate biochemical signals inside cells. In the brain, the CB1 type of cannabinoid receptor (CB1R) is the major endocannabinoid target. AEA and 2-AG act as agonists at the CB1R. Most CB1Rs in the brain are located on presynaptic terminals of GABAergic and glutamatergic synapses. The CB1Rs generally couple to Gi/o-type G-proteins. They usually inhibit neurotransmitter release.
Induction and Expression Mechanisms of eLTD eLTD is initiated by the coactivation of presynaptic glutamatergic afferents and the postsynaptic neuron. Experiments designed to elicit eLTD usually involve electrical stimulation of afferent fibers and electrophysiological recording from inside the postsynaptic neuron in brain-slice preparations. However, eLTD can also be induced in dual recordings from a single presynaptic neuron that forms a glutamatergic synaptic connection with a single postsynaptic neuron. The patterns of activation necessary for eLTD induction differ at different synapses. Induction protocols range from one-to-one pairing of pre- and postsynaptic action potentials (called spike timing-dependent synaptic plasticity (STDP)) at low frequencies of activation, to high-frequency afferent activation paired with postsynaptic depolarization. There are several general features of eLTD at different synapses and variants that are specific to a given synapse. The induction of eLTD involves a sequence of events that starts with presynaptic activation. Repetitive activation of excitatory glutamatergic synapses stimulates postsynaptic signals that are involved in the induction of most forms of eLTD. The sequence of events involved in eLTD induction is: 1. Presynaptic glutamate release. 2. Depolarization leading to the activation of voltage-gated calcium channels and a rise in 677
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postsynaptic calcium concentration (these steps are not required for eLTD of inhibitory synapses in the basolateral amygdala and hippocampus). 3. Activation of metabotropic glutamate receptors (mGluRs), leading to phospholipase activation. 4. Formation and release of endocannabinoids from the postsynaptic neuron. 5. Activation of presynaptic CB1Rs. The initial postsynaptic depolarization necessary for most forms of eLTD arises from the activation of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-type glutamate receptors subsequent to the presynaptic release of glutamate. The rise in postsynaptic calcium can come from several sources. In most forms of eLTD a voltage-gated calcium channel is activated by depolarization, and this allows calcium influx to the postsynaptic neuron. In the case of eLTD in striatum, an L-type calcium channel plays this role. A T-type channel appears to be the predominant calcium channel involved in eLTD induction in the whisker barrel cortex, with L-type channels playing a lesser role at this synapse. Activation of mGluRs can also contribute to the rise in postsynaptic calcium. These receptors activate the enzyme phospholipase C, which catalyzes the breakdown of phosphatidylinositol into diacylglycerol (DAG) and inositol trisphosphate (IP3). The IP3 generated by this reaction acts on receptors that release calcium from intracellular stores, and this is another source of calcium that can contribute to endocannabinoid production and eLTD induction. The DAG formed by mGluR activation of phospholipase C can also contribute to endocannabinoid formation. The enzyme known as DAG lipase catalyzes the formation of 2-AG. Inhibitors of DAG lipase prevent eLTD at several synapses, suggesting that 2-AG is a retrograde signal in this form of plasticity. The key role of mGluRs in eLTD is underscored by the fact that the application of mGluR agonists can produce such plasticity at several synapses. In contrast to several other forms of long-term synaptic plasticity, eLTD usually does not require activation of N-methyl-D-aspartate (NMDA)-type glutamate receptors. The exception is eLTD in the cortex (Figure 1). Endocannabinoids formed in the postsynaptic neuron traverse the synapse in a retrograde direction (post-to-presynaptic) and activate CB1Rs. The retrograde signal appears to be restricted to presynaptic terminals impinging on the postsynaptic neurons that produce the endocannabinoids. This finding is consistent with the idea that endocannabinoids do not diffuse more than 10–20 mm from their site of release. The CB1Rs activated by endocannabinoids then initiate molecular events leading to a long-lasting decrease in
the probability of neurotransmitter release. Synergistic effects of CB1Rs and other receptors may be required for eLTD induction at many synapses. For example, eLTD at synapses in visual cortex requires the activation of NMDA-type glutamate receptors, most likely presynaptic receptors that contain the NR2B subunit. Activation of a presynaptic NMDA receptor has also been implicated in eLTD at synapses in the whisker barrel cortex. Expression of eLTD becomes independent of CB1R activation within minutes after the induction paradigm (Figure 2). Thus, the expression mechanism is set into motion by CB1R activation, but eLTD persists well beyond the brief initial period of receptor activation. It is not yet clear which endocannabinoid acts as the retrograde signal, but studies in the hippocampus and sensory cortex suggest a role for 2-AG based on the fact that an inhibitor of the DAG lipase enzyme prevents eLTD induction. The molecular mechanisms underlying sustained eLTD expression are poorly understood at present. The induction of eLTD can also be modulated by neurotransmitters other than those directly involved in induction or expression. For example, in dorsal striatum dopamine acts on D2-type receptors to increase the likelihood that LTD will be initiated. However, recent evidence indicates that the D2 receptor is not strictly necessary for eLTD induction or expression because the receptor can be bypassed, for example, by combined depolarization and activation of postsynaptic mGluRs. A similar role for dopamine is probably involved in amphetamine-induced eLTD in the amygdala. As mentioned previously, the expression of eLTD has been observed at both excitatory glutamatergic and inhibitory GABAergic synapses in different brain regions. However, even in cases in which the depression is expressed as a decrease in GABAergic transmission, the initiation process usually involves glutamatergic transmission that activates mGluRs.
Cerebellar LTD The LTD observed at parallel fiber (PF) inputs to cerebellar Purkinje cells (PCs) was the first form of LTD characterized in the brain. Theoretical models of cerebellar function predicted that LTD at this synapse might have a crucial role in motor learning and that the coactivation of PFs and climbing fibers (CFs) would activate this type of plasticity. Consistent with these predictions, it has been found that paired PF and CF activation at low frequencies does indeed produce a long-lasting decrease in the efficacy of synaptic transmission at the PF–PC synapse. This form of LTD has been shown to occur in vivo and has also been found in brain slices and in single Purkinje
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Figure 1 Induction mechanisms of four types of eLTD: (a) homosynaptic LTD; (b) heterosynaptic LTD; (c) spike timing LTD; (d) cerebellar LTD. The diagrams show the molecular mechanisms proposed to participate in initiation of eLTD at the striatal neocortical and cerebellar glutamatergic synapses (a, c), and amygdala and hippocampal GABAergic synapses (b). Note that eLTD expression is probably presynaptic in the amygdala, hippocampus, neocortex, and striatum but postsynaptic in the cerebellum. eLTD in the neocortex and striatum is homosynaptic, whereas in the amygdala and hippocampus it is heterosynaptic. Cerebellar LTD requires pairing of afferent inputs. 2-AG, 2-arachindonyl glycerol; AC, amygdala complex; AEA, arachindonylethanolamide; AMPAR, alpha-amino3-hydroxy-5-methyl-4-isoxazole propionic acid receptor; AP, action potential; cAMP, cyclic adenosine-30 ,50 -monophosphate; CB1R, CB1 receptor; CF, climbing fiber; DAG, diacylglycerol; DGL, lower layer of the dentate gyrus; eCB, endocannabinoid; eLTD, endocannabinoiddependent long-term depression; GABA, g-aminobutyric acid; Glu, glutamate; LTD, long-term depression; mGluR, metabotropic glutamate receptor; NAT, N-acetyltransferase; NMDAR, N-methyl-D-aspartate receptor; NOS, nitric oxide synthase; PF, parallel fiber; PKA, protein kinase A; PLC, phospholipase C; TBF, theta-burst frequency. Reproduced, with permission, from the Annual Review of Neuroscience, Volume 29, ã2006 by Annual Reviews. Chevaleyre V, Takahashi KA, and Castillo PE (2006) Endocannabinoid-mediated synaptic plasticity in the CNS. Annual Review of Neuroscience 29: 37–76.
neurons grown in cell culture. In the cell culture preparation, the activation of afferent inputs can be replaced by the application of receptor antagonists to induce LTD and examine mechanisms of expression. A large array of molecules has been implicated in cerebellar LTD, including many that also participate in eLTD. There is consensus about the involvement of the following mechanisms: . Presynaptic release of amino acid transmitters from CFs and PFs. . Postsynaptic depolarization and activation of voltage-gated channels, including calcium channels.
. Activation of AMPA-type glutamate receptors and receptors containing the glutamate d2 subunit. . Activation of type I mGluRs. . Increased postsynaptic intracellular calcium and sodium. . Activation of G-proteins and phospholipases. . Protein phosphorylation/dephoshorylation, involving the activation of enzymes such as protein kinase C (PKC), cyclic guanosine monophosphate (cGMP)dependent protein kinase and protein phosphatases PP1 and PP2A. The AMPA receptor itself is an important target for phosphorylation.
680 Long-Term Depression (LTD): Endocannabinoids and Cerebellar LTD 3 µM SR141716A (3 min)
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Figure 2 Time course of endocannabinoid release and CB1 activation during striatal LTD. Expression of LTD is prevented when CB1 antagonist is applied 1 min after high-frequency stimulation (HFS) but not when application begins 10 min after induction. Results with application at 3 min are intermediate. Calibration ¼ 100 pA, 25 ms. EPSC, excitatory postsynaptic current; LTD, long-term depression. Reproduced from Ronesi J, Gerdeman GL, and Lovinger DM (2004) Disruption of endocannabinoid release and striatal long-term depression by postsynaptic blockade of endocannabinoid membrane transport. Journal of Neuroscience 24: 1673–1679, copyright 2004 by the Society for Neuroscience, with permission.
. Internalization of GluR2-containing AMPA receptors. . Transcription and translation (in late-phase LTD). Other molecular mechanisms have been implicated in the induction of cerebellar LTD, but they may play modulatory roles or may represent alternative pathways for activating the molecular mechanisms involved in LTD induction. For example, activation of nitric oxide synthase, leading to formation of the gaseous messenger nitric oxide (NO), has been implicated in cerebellar LTD induction. However, NO is not necessary for LTD in the cell culture preparation. This is most likely due to the fact that LTD in cell culture results from the strong activation of phosphorylation by the PKC pathway. In the slice preparation, NO released from stellate-type interneurons in response to glutamate activation of NMDA receptors appears to participate in LTD induction by the activation of guanylate cyclase, leading to the formation of cGMP. This intracellular second messenger, in turn, stimulates cGMP-dependent PKC to phosphorylate a substrate that inhibits the activity of the protein phosphatases PP1 and PP2A. Phosphatase inhibition presumably stabilizes GluR2 in the phosphorylated state and promotes AMPA receptor internalization.
Expression of cerebellar LTD almost undoubtedly involves postsynaptic mechanisms. Decreased AMPA receptor-mediated responses are observed that are tightly coupled to LTD expression. No reliable evidence of a presynaptic change associated with cerebellar LTD has yet been observed. The predominant cellular mechanism believed to be important for the expression of this form of LTD is the internalization of AMPA receptors at PF–PC synapses. Phosphorylation of the AMPA receptor itself, most likely via PKC-mediated transfer of phosphate to serine 880 on the GluR2 subunit, is a key step in this process. Phosphorylation at this residue enhances protein interacting with C kinase 1 (PICK1) binding to GluR2, and this protein–protein interaction results in the subsequent internalization of GluR2-containing AMPA receptors by a process of endocytosis involving the coating of the vesicles by the molecule clathrin. Reduction in the number of synaptic AMPA receptors leads to the reduction in the amplitude of the PF synaptic responses. It has also been suggested that the activation of CB1Rs plays a role in cerebellar LTD. Postsynaptic mGluR activation and increases in intracellular calcium stimulate endocannabinoid production in Purkinje neurons and the subsequent activation of
Long-Term Depression (LTD): Endocannabinoids and Cerebellar LTD
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Figure 3 Extinction of aversive memories is impaired in the CB1 receptor knockout mouse: (a) after 1 day of conditioned freezing; (b) after 6 days of conditioned freezing; (c) pain thresholds; (d) unconditioned freezing; (e) open arm entries in the elevated plus maze; (f) horizontal open-field locomotion; (g) stronger conditioning regimen. Conditioned freezing extinguishes over several trials in CB1þ/þ but not in CB1/ mice after 1 day (a) or 6 days (b) of conditioning. Pain thresholds, unconditioned freezing, open arm entries in the elevated plus maze, and horizontal open-field locomotion do not differ across the different mouse lines. In (g), some extinction is observed in CB1/ mice with a stronger conditioning regimen, but extinction was still slower and less complete in comparison to CB1þ/þ mice. Black symbols indicate CB1/ mice; white symbols indicate CB1þ/þ mice. CS, conditioned stimulus. Reprinted by permission from Macmillan Publishers Ltd: Nature, Marsicano G, Wotjak CT, Azad SC, et al. (2002) The endogenous cannabinoid system controls extinction of aversive memories. Nature 418(6897): 530–534, copyright 2002.
presynaptic CB1Rs. A recent study suggested that these molecular events take place during the induction of cerebellar LTD. The presynaptic CB1 activation leads to the release of another factor, possibly NO, that then acts on the postsynaptic neuron to induce LTD. Other forms of synaptic plasticity occur in the cerebellum that can interact with PF LTD. The CF synapses
are capable of expressing LTD when activated by sustained 5 Hz electrical stimulation in cerebellar slices. CF LTD may also involve a postsynaptic expression mechanism and may be involved in pruning back excessive innervation by this powerful synaptic input. Recent studies have characterized a form of long-term potentiation (LTP) that reverses PF LTD. This form of LTP is activated by sustained low-frequency PF input in
682 Long-Term Depression (LTD): Endocannabinoids and Cerebellar LTD
the absence of CF coactivation. It is expressed postsynaptically and involves phosphatase activation.
Functional Roles of eLTD The roles played by eLTD in the function of neural circuits and in behavior are not yet fully understood. The suppression of inhibitory transmission produced by eLTD at GABAergic synapses produces a longlasting increase in the potential for excitation of the postsynaptic neuron by glutamatergic synaptic inputs. In the hippocampus, this can lead to the priming of LTP at glutamatergic synapses to CA1 pyramidal neurons. The suppression of inhibition allows for greater excitation of the postsynaptic neuron in response to the activation of glutamatergic inputs. This increases the likelihood of NMDA receptor activation and thus enhances the probability of LTP induction. Roles for eLTD in learning and memory have been postulated, as is the case for other forms of persistent synaptic plasticity. The only example to date in which eLTD has been linked to a specific form of learning is in the extinction of fear conditioning. The basolateral amygdala (BLA) is one site of information storage important for forming conditioned fear responses to previously neutral stimuli by pairing them with aversive stimuli. Endocannabinoids in the BLA appear to play a role in this extinction, and eLTD at GABAergic synapses within this brain region is a candidate mechanism for the decrease in efficacy of inhibitory transmission thought to underlie extinction (Figure 3). The discovery that eLTD accounts for spike timingdependent, long-lasting depression of excitatory transmission in the visual cortex suggests a role for this plasticity in cortical development and information storage. Plasticity at cortical glutamatergic synapses is thought to shape patterns of information flow through cortical circuitry during development by setting relative efficacies of thalamocortical and cortico-cortical synapses. These efficacies are shaped by sensory experience. For example, ocular dominance plasticity is a well-characterized change in cortical neuron responses to visual input. Visual experience, including deprivation, alters the strength of neuronal responses from the different eyes, and these experience-dependent changes may involve eLTD and other forms of synaptic plasticity. Within the rodent whisker barrel field sensory cortex, eLTD may also play roles in information storage related to tactile stimuli, as well as in synaptic development. A form of LTD that shares expression mechanisms with eLTD occurs during deprivation of sensory input from facial whiskers, although it is still to be determined if this LTD requires endocannabinoids or the CB1R.
Endocannabinoids and cannabinoid receptors are implicated in neural responses to drugs of abuse, including not only cannabis-based drugs but other addictive substances. Reducing CB1R activity with either antagonists or gene-targeted knockout reduces the self-administration of a variety of drugs of abuse. It has been suggested that eLTD might play a role in neuroadaptations to drugs and drug-related conditioning and that these processes might contribute to addiction. In the nucleus accumbens, eLTD is lost following chronic exposure to D9-THC. The loss of plasticity in this case is most likely a consequence of a drug-induced decrease in the number of CB1Rs (receptor downregulation). The loss of eLTD may contribute to behavioral tolerance to cannabinoid drugs. The eLTD induced by amphetamine in the lateral amygdala could play a role in drug-related conditioning, such as conditioned place preference. Cerebellar LTD is thought to be part of an erroradaptation system involved in motor learning in which PF synapses activated during incorrect performance are depressed based on feedback from the CF inputs. This can come into play during fine-tuning of the vestibulo-ocular reflex. Cerebellar LTD may also play roles in Pavlovian eyeblink conditioning and locomotor adaptation. See also: Endocannabinoid Role in Synaptic Plasticity and Learning; Long-Term Depression (LTD): Metabotropic Glutamate Receptor (mGluR) and NMDAR-Dependent Forms; Long-Term Potentiation (LTP): NMDA Receptor Role; Metabotropic Glutamate Receptors (mGluRs): Functions.
Further Reading Alger BE (2002) Retrograde signaling in the regulation of synaptic transmission: Focus on endocannabinoids. Progress in Neurobiology 68: 247–286. Belmeguenai A and Hansel C (2005) A role for protein phosphatases 1, 2A, and 2B in cerebellar long-term potentiation. Journal of Neuroscience 25(46): 10768–10772. Bender VA, Bender KJ, Brasier DJ, and Feldman DE (2006) Two coincidence detectors for spike timing-dependent plasticity in somatosensory cortex. Journal of Neuroscience 26: 4166–4177. Chevaleyre V and Castillo PE (2003) Heterosynaptic LTD of hippocampal GABAergic synapses: A novel role of endocannabinoids in regulating excitability. Neuron 38(3): 461–472. Chevaleyre V, Takahashi KA, and Castillo PE (2006) Endocannabinoid-mediated synaptic plasticity in the CNS. Annual Review of Neuroscience 29: 37–76. DeZeeuw CI, Hansel C, Bian F, et al. (1998) Expression of a protein kinase C inhibitor in Purkinje cells blocks cerebellar LTD and adaptation of the vestibulo-ocular reflex. Neuron 20: 495–508. Gerdeman GL, Ronesi J, and Lovinger DM (2002) Postsynaptic endocannabinoid release is necessary for long-term depression in the striatum. Nature Neuroscience 5(5): 446–451.
Long-Term Depression (LTD): Endocannabinoids and Cerebellar LTD Gerdeman GL and Lovinger DM (2003) Emerging roles for endocannabinoids in long-term synaptic plasticity. British Journal of Pharmacology 140: 781–789. Hansel C and Linden DJ (2000) Long-term depression of the cerebellar climbing fiber-Purkinje neuron synapse. Neuron 26: 473–482. Hansel C, Linden DJ, and D’Angelo E (2001) Beyond parallel fiber LTD: The diversity of synaptic and non-synaptic plasticity in the cerebellum. Nature Neuroscience 4(5): 467–475. Hoffman AF, Oz M, Caulder T, and Lupica CR (2003) Functional tolerance and blockade of long-term depression at synapses in the nucleus accumbens after chronic cannabinoid exposure. Journal of Neuroscience 23(12): 4815–4820. Huang YC, Wang SJ, Chiou LC, and Gean PW (2003) Mediation of amphetamine-induced long-term depression of synaptic transmission by CB1 cannabinoid receptors in the rat amygdala. Journal of Neuroscience 23(32): 10311–10320. Ito M (2001) Cerebellar long-term depression: Characterization, signal transduction, and functional roles. Physiological Reviews 81(3): 1143–1195. Ito M and Kano M (1982) Long-lasting depression of parallel fiberPurkinje cell transmission induced by conjunctive stimulation of parallel fibers and climbing fibers in the cerebellar cortex. Neuroscience Letters 33: 253–258.
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Kreitzer AC and Malenka RC (2005) Dopamine modulation of state-dependent endocannabinoid release and long-term depression in the striatum. Journal of Neuroscience 25(45): 10537–10545. Maldonado R, Valverde O, and Berrendero F (2006) Involvement of the endocannabinoid system in drug addiction. Trends in Neurosciences 29: 225–232. Marsicano G, Wotjak CT, Azad SC, et al. (2002) The endogenous cannabinoid system controls extinction of aversive memories. Nature 418(6897): 530–534. Robbe D, Kopf M, Remaury A, Bockaert J, and Manzoni OJ (2002) Endogenous cannabinoids mediate long-term synaptic depression in the nucleus accumbens. Proceedings of the National Academy of Sciences of the United States of America 99(12): 8384–8388. Ronesi J, Gerdeman GL, and Lovinger DM (2004) Disruption of endocannabinoid release and striatal long-term depression by postsynaptic blockade of endocannabinoid membrane transport. Journal of Neuroscience 24: 1673–1679. Safo PK and Regehr WG (2005) Endocannabinoids control the induction of cerebellar LTD. Neuron 48: 647–659. Sjostrom PJ, Turrigiano GG, and Nelson SB (2003) Neocortical LTD via coincident activation of presynaptic NMDA and cannabinoid receptors. Neuron 39(4): 641–654.
Nitric Oxide G Wilson and J Garthwaite, University College London, London, UK ã 2009 Elsevier Ltd. All rights reserved.
Introduction Nitric oxide (NO) is a unique type of cell-to-cell signaling molecule found in almost all tissues of the body and throughout the central nervous system. NO rose to biological fame in 1987 when it was identified as the factor released from endothelial cells lining blood vessels to cause relaxation of the underlying smooth muscle, thereby influencing blood flow and blood pressure. The following year, it was found to be the mysterious substance known to be produced following activation of the N-methyl-D-aspartate (NMDA) class of glutamate receptors on brain neurons to relay signals to neighboring cells, and also a molecule generated by activated macrophages as part of their repertoire of defense against foreign organisms. These three diverse functions – smooth muscle relaxation, neural communication, and immune defense – remain at the core of NO biology and are subserved largely by specialized NO-generating proteins known, respectively, as endothelial, neuronal, and inducible NO synthases (eNOS, nNOS, and iNOS, respectively). As well as being a key physiological signaling molecule, NO has been invoked as an important player in many different disorders affecting humans, including neurodegenerative diseases, pain, atherosclerosis, and septic shock. The physicochemical properties of NO set it apart from conventional signaling molecules, such as neurotransmitters. First, like oxygen and carbon dioxide, NO lacks chemical specialization. It does, however, possess an extra (unpaired) electron, making it a radical. Whereas some other radicals are chemically reactive and can cause damage to cells, NO is stable in physiological concentrations, which are thought to be approximately 1 nM or less. Second, also like oxygen and carbon dioxide, NO diffuses very quickly through membranes, obviating the need for a specialized release mechanism and giving it the ability to act on neighboring cells within microseconds of its manufacture. This property gives the recipient cell temporal information about the events causing NO to be formed. Finally, NO binds very avidly to heme groups possessing vacant coordination sites. Indeed, the capacity of hemoglobin to bind and inactivate NO was well known long before NO was recognized as a biological messenger, and this property proved to be
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an important one in developing the hypothesis that the biological messenger was NO and also in testing that hypothesis. Although many cellular heme groups do not possess the necessary vacant binding site, this property has been exploited to provide highly sensitive NO detectors within cells, initiating physiological NO signal transduction.
How Is NO Made? The NO synthase family of enzymes catalyzes the stereoselective conversion of the amino acid L-arginine to NO. The reaction requires molecular oxygen and L-citrulline is formed as a by-product. The enzymatic conversion of L-arginine into NO was first shown in 1989 and, later, the three different isoforms were identified in molecular terms (chronologically, nNOS, eNOS, and then iNOS). The enzymes exist as homodimers, with each constituent subunit containing an N-terminal oxygenase domain and a C-terminal reductase domain. The former contains binding motifs for heme, tetrahydrobiopterin (BH4, a cofactor), and the substrate, L-arginine. The reductase domain possesses binding sites for NADPH, FAD, and FMN. Functionally, electrons are donated by NADPH to the reductase domain and proceed via the FAD and FMN redox carriers to the oxygenase domain, whereupon interaction with the heme iron and BH4 at the active site catalyzes the generation of NO. The two domains are linked by a binding site for calmodulin and the binding here of calmodulin complexed with calcium facilitates electron flow through the enzyme (Figure 1). The calcium dependence of NO synthesis varies with the NOS isoforms, with nNOS and eNOS having a much higher requirement than iNOS. The calcium insensitivity of iNOS is the result of the lack of an autoinhibitory loop insert within its FMN-binding subdomain that, in the other two isoforms, acts by destabilizing calmodulin binding at low calcium concentrations. Loss of eNOS activity at low calcium concentrations is circumvented when the protein is phosphorylated, typically by the kinase known as Akt, or protein kinase B. Physiologically, eNOS phosphorylation may be the most important device for maintaining NO output in blood vessels. Knowledge of the enzymatic mechanism for NO synthesis led to the identification of inhibitors, which have become invaluable tools for investigating the biological roles of NO. First-generation inhibitors were L-arginine derivatives with substituents on the guanidino nitrogen normally used for NO synthesis,
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Figure 1 NO biosynthesis. The NO synthases are homodimers, with each subunit containing a reductase and oxygenase domain. Electrons donated by NADPH proceed through the reductase domain via FAD and FMN redox carriers. On reaching the oxygenase domain, electrons interact with the heme group and BH4 cofactor at the active site to catalyze the reaction of L-arginine with molecular oxygen, yielding NO. CaM, calmodulin.
compounds such as NG-monomethyl-L-arginine and NG-nitro-L-arginine. These inhibitors are able to inhibit all NOS isoforms, but compounds showing good selectivity for nNOS or iNOS over the other isoforms are now available.
NO Generation in the Nervous System The nNOS isoform is found predominantly in neurons and has a distribution in the brain and spinal cord as wide as that of the major neurotransmitters glutamate and g-aminobutyric acid, although the amounts vary from region to region (Figure 2(a)). In some brain areas, such as the cerebellum, nNOS is found in virtually all cells, whereas in others, such as the striatum and cerebral cortex, it is found in a subpopulation of interneurons comprising only a few percentage of the total neuronal number. Nevertheless, even in those regions that are relatively sparsely endowed with nNOS-containing neurons, dense nNOS-containing fiber networks are to be seen ramifying throughout the neuropil, suggesting that the majority of neurons receive NO signals. In many of these brain regions, NO formation is coupled to the stimulation of the NMDA class of glutamate receptor, which is found in almost all excitatory synapses. This special relationship is the result of the NMDA-receptor-associated ion channels having a high permeability to calcium ions and of the compartmentalization of the nNOS and NMDA receptor proteins into a complex at synaptic
sites. The scaffold for this complex is postsynaptic density-95 (PSD-95) protein, which contains several PDZ domains – the modular protein–protein interaction motifs. The N-terminus of nNOS contains a PDZ domain which interacts with the second PDZ domain on PSD-95. PSD-95 also binds the C-terminal PDZ domain of NMDA receptors, leading to the colocalization of the two proteins. In general, NMDA receptors play only a minor role in fast synaptic transmission but are brought into action when there is a concomitant depolarization of the postsynaptic membrane which, by removing a block of the NMDA receptor channel by magnesium ions, allows calcium ions to pass into the cell. One result of NMDA receptor activation in many synapses is the induction of long-term increases or decreases in synaptic strength, phenomena considered relevant to learning and memory formation and ones in which NO is also commonly involved. In addition to the coupling with NMDA receptors postsynaptically, there are other instances in which NO is formed presynaptically. The best examples of this neurotransmitter-like mode of action are to be found in the peripheral nervous system, where NO is released from nonadrenergic, noncholinergic nerves to act on smooth muscle (e.g., in the intestine) and relax it. The trigger for NO release here is the activation of voltage-dependent calcium channels by invading action potentials. Also, in parts of the brain, nNOS is concentrated strongly in axons (e.g., in the parallel fibers in the cerebellum) and it may perform
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Figure 2 The NO–cGMP signaling pathway in rat brain. Shown are the distributions of (a) NO synthase as indicated by NADPH diaphorase histochemical staining and (b) NO-stimulated cGMP accumulation after perfusion in vivo of the NO donor, sodium nitroprusside. The images are of adjacent parasagittal sections and are displayed as heat maps, with red indicating strong staining intensity and blue indicating weak staining intensity. Generally, there is a good match between the presence of NO synthase and cGMP production. Pictures of unmodified sections and experimental details can be found in Southam E and Garthwaite J (1993) The nitric oxide–cyclic GMP signalling pathway in rat brain. Neuropharmacology 32: 1267–1277.
an analogous ‘orthograde’ transmitter function at these locations. Because it is such an unconventional transmitter, developing a conceptual framework for synaptic NO signaling has been problematic, not least because NO is very difficult to measure at physiological concentrations and in discrete synaptic locations. By analogy with the numbers of NMDA receptors, there may be only 10–100 nNOS molecules per synapse. Assuming that the nNOS has a similar activity to that exhibited by the purified enzyme and that, once made, it is dispersed by diffusion, active NO concentrations (in the 0.1–1 nM range) would only be found very locally, within a submicrometer radius. Hence, NO from nNOS may only be able to function in subsynaptic dimensions, implying that it signals between closely juxtaposed elements, such as the pre- and postsynaptic specializations. Although nNOS is the main isoform in the nervous system, emerging evidence suggests that NO from
eNOS in the microvasculature can also influence neurons. This represents a conceptual departure from the traditional view of brain function, in which signal processing is perceived as being performed by neurons with the support of nearby glial cells. On the other hand, any point in the brain is maximally only approximately a cell diameter (approximately 25 mm) away from a capillary and the geometry of the capillary network is ideal for distributing a molecule such as NO within a tissue volume, just as it is for oxygen. At least in vitro, the global NO signal deriving from the endothelium can influence the membrane potential of axons in the optic nerve and the capacity for synaptic plasticity in the hippocampus.
How Does NO Act? Curiously, the answer to this question preceded the identification of NO as a biological messenger. In the 1970s, it was found that NO was a powerful
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activator of guanylyl cyclase (GC) enzymes, causing the synthesis of cyclic GMP (cGMP). This observation provided crucial evidence for the hypothesis that endothelium-derived relaxing factor was NO because the factor was found to cause relaxation through cGMP generation. Because of its location in the soluble fractions in tissue homogenates, the enzyme activity became known as ‘soluble’ GC, but it is now clear that the proteins constitute major physiological receptors for NO and we refer to them here as NO(GC) receptors. In the rodent brain, NO-evoked cGMP accumulation has a similarly broad distribution to that of the NO-forming enzyme (Figures 2(a) and 2(b)). The NO(GC) receptors are designed to capture and transduce low-level NO signals, even very transient ones. The ligand-binding site is a specialized heme group that excludes oxygen, allowing NO to bind without becoming oxidized, in contrast to its reaction with oxyhemoglobin when nitrate ions are formed. The proteins to which the heme is attached are heterodimers of a- and b-subunits and exist in two main isoforms. The b1 subunit appears to be the common subunit and it is partnered by either a1 or a2. Both isoforms are found abundantly in brain tissue but have an uneven distribution, with some areas or cells showing prominently a1b1 and others showing a2b1. A special feature of the a2b1 isoform is that it binds to pre- or postsynaptic scaffold proteins, which would be convenient for transducing localized NO signals from nNOS, whereas the a1b1 may have a less localized distribution in neurons and can also be found in some glial cells. A b2 subunit is present in low levels at the mRNA level, but there is no evidence that a b2 protein is expressed. The binding of NO to the vacant coordination site on the heme group of the NO(GC) receptor is extremely rapid, being nearly diffusion limited. Initially, a six-coordinate nitrosyl–heme complex is formed, but the subsequent snapping of a bond between the heme and a nearby histidine residue results in a five-coordinate species. This bond breakage causes a conformational change that propagates to the catalytic domain, accelerating conversion of GTP into cGMP by 1000-fold or more. Although both subunits contribute to the catalytic domain, there is only a single active site. The second ‘pseudosymmetric’ site, which is thought to be analogous to the forskolin binding site in adenylyl cyclase, may have a regulatory function. Binding of NO to other heme groups is so tight that it often takes hours or days for it to unbind, a property that would preclude a dynamic signaling role for NO. Although of high affinity (dissociation constant of approximately 1 nM), binding of NO to the NO(GC) receptor heme is readily reversible so
that, on removal of NO, the associated GC activity switches off in the 100 ms timescale. Overall, the properties of the receptors mean that they can sense NO concentrations of approximately 0.1 nM or more, convert them into approximately 1000-fold higher concentration of cGMP within 1 s, and then cease activity when NO production stops, again within 1 s. Thus, although many details of their functioning remain to be explained, the NO(GC) receptors are highly effective proteins for transducing NO signals into greatly amplified levels of cGMP with good temporal fidelity. Whether or not other NO receptors exist remains to be determined.
Inactivation of NO Brain tissue avidly consumes NO such that its halflife at physiological concentrations is estimated to be in the 10 ms timescale. Although rapid, this process is unlikely to contribute much to localized NO signaling because diffusion alone ensures rapid dissipation of the molecules from small volumes. Active NO inactivation is more likely to be of importance in shaping the NO concentrations when there are many nearby sites of simultaneous NO synthesis. Despite much research, the means by which the NO signal is actively quenched remains unclear. Reaction with oxyhemoglobin in red blood cells in the vasculature probably plays some role. Chemically, NO can also react with oxygen, but the rate is too slow to be of physiological importance. NO can, however, react very rapidly with lipid peroxyl radicals, although this process is likely to be significant mainly in pathological conditions associated with oxidative stress. The major mechanism of NO consumption in normal brain tissue remains to be identified.
Downstream Targets On being generated in response to NO, cGMP may act on protein kinases to initiate phosphorylation cascades, on phosphodiesterases responsible for the hydrolysis of cGMP and/or cAMP, or on cyclic nucleotide-gated/modulated ion channels located in cell membranes. cGMP-dependent protein kinases (cGKs) are activated by submicromolar cGMP concentrations and then phosphorylate serine or threonine residues. There are three types: cGKIa and -1b (splice variants) and cGKII. Activation appears to involve a conformation change that removes the autoinhibitory domain from the catalytic site. Autophosphorylation can also occur, causing a more persistent kinase activity that could outlast the original cGMP signal. In brain, cGKIa is concentrated in Purkinje cells in the cerebellum,
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neurons that also express a 23 kDa target protein known as G-substrate which functions as a protein phosphatase inhibitor. By engaging this cascade in Purkinje cells, NO contributes to synaptic plasticity and motor learning in the cerebellum. The hippocampus is enriched in the cGK1b isoform, which is important in the NO-dependent initiation of synaptic plasticity in this region, whereas cGKII is more widespread, being found particularly abundantly in the cerebral cortex, thalamus, olfactory bulb, and superior colliculi. One role of cGKII is in the regulation of circadian rhythms. Phosphodiesterases (PDEs) catalyze the hydrolysis of cyclic nucleotides to the corresponding noncyclized monophosphates. In mammals, there are 11 PDE families encoded by genes with several splice variants that may exhibit different subcellular locations or tissue distributions. Most of the PDEs can hydrolyze cGMP; those with the greatest affinity are PDE1–3, -5, -6, and -9 through -11. The first family, PDE 1, is a widely expressed, calcium/calmodulindependent isoform that hydrolyzes both cGMP and cAMP. Competition between the two cyclic nucleotides could occur, potentially allowing a rise in one to inhibit breakdown of the other. This scenario is more probable with PDE3, however, because this isoform binds both cGMP and cAMP with similar affinities but hydrolyzes cGMP at a much lower maximal rate. In effect, therefore, cGMP can act as a PDE3 inhibitor and raise the levels of cAMP. Although there is little information available on PDE3 in brain, there is evidence that cGMP signals are transduced through this mechanism in terminals of the vagus nerve in the heart, with the increased level of cAMP ultimately causing increased release of acetylcholine. Another cGMP-regulated PDE, PDE2, is a dual-substrate enzyme equipped with a noncatalytic cGMP binding site (called a GAF domain) whose occupation stimulates enzyme activity. PDE2 is found widely in the brain and plays a major role in hydrolyzing NO-evoked cGMP responses in regions such as the cortex, hippocampus, and striatum. In some peripheral tissues, a rise in cGMP causes a lowering of cAMP levels through PDE2 activation. In the adrenal gland, for example, engagement of this mechanism results in a decrease in aldosterone secretion. However, there is no clear evidence for a similar cross-talk mediated by PDE2 in the brain. The final category of known receptors for cGMP are members of the family of cyclic nucleotide-gated/ modulated ion channels. Cyclic nucleotide-gated (CNG) channels are heteromeric proteins that are directly opened in response to the binding of cAMP or cGMP. They were first discovered in retinal rod photoreceptors and then in olfactory receptor cells and retinal cone cells, but there is now evidence for
a more widespread distribution, both within the brain and outside. Molecular cloning has identified two subfamilies of subunits, with one (CNGA1–4) being the core subunits (with all except CNGA4 being channel-forming subunits) and the other (CNGB1 and -B3) having a modulatory effect on ion selectivity, ligand sensitivity, and gating properties. cGMP is the preferred agonist for rod- and cone-type channels, whereas olfactory-type channels are relatively nonselective. Under physiological conditions, CNG channels carry inward currents of sodium and calcium ions and are relatively voltage independent. Unfortunately, the pharmacology of CNG channels is poorly developed, making studies of their participation in NO signal transduction difficult. Nevertheless, there is evidence that in response to activation of the NO–cGMP pathway, the channels can promote neurotransmitter release from presynaptic terminals and increase neuronal firing postsynaptically. Related structurally to CNG channels are the hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels. As the name suggests, HCN channels are activated at more hyperpolarized membrane potentials (typically negative to approximately 60 mV) and convey a depolarizing, cationic current, known as Ih, largely carried by sodium ions. The channels have an important role in cells that fire rhythmically, such as thalamocortical neurons. Here, the hyperpolarization that follows action potential firing activates HCN channels, which provides a depolarizing drive leading to the activation of other voltage-dependent conductances and further action potential generation. This hyperpolarization-dampening role is also important in setting the resting membrane potential of neurons. Two of the four known HCN channel isoforms (HCN2 and HCN4) are particularly sensitive to regulation by cyclic nucleotides, which shift activation to more depolarized membrane potentials, speeding up rhythmic firing or making the resting membrane potential more depolarized. Being tenfold more potent, cAMP is generally regarded as the natural ligand, but there are several examples in which the NO–cGMP pathway brings about changes in neuronal excitability using HCN channels.
What Does NO Do in the Central Nervous System? NO has been implicated in numerous behaviors in mammals, including learning and memory formation, feeding, sleeping, reproductive behavior, aggression, and anxiety. Interestingly, some of these roles appear conserved across many millions of years of evolution. For example, the jellyfish Aglantha digitale (phylum Cnidaria) first appeared approximately half a billion
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years ago and contains a very primitive nervous system consisting of a nerve net, photoreceptors, and mechanosensitive nerve cells. NOS-containing neurons are found in the tentacles and NO plays a key role in swimming behavior associated with feeding, with cGMP being the effector molecule. The NO– cGMP pathway is also important in the feeding behavior of mollusks and honeybees, in which it contributes to the formation of long-lasting associations between scent and food reward. In mammals, NO regulates both food intake and the passage of food through the intestine. At a more cellular level, NO and cGMP have frequently been found to participate in synaptic plasticity, which refers to the capability of synapses to adjust their strength enduringly upward or downward in response to brief periods of altered input, and which is commonly regarded as a cellular correlate of learning and memory formation. Long-term potentiation in the hippocampus, cerebral cortex, cerebellum, amygdala, and spinal cord has been reported to involve the NO–cGMP pathway, as has long-term depression (LTD) in the cerebellum and striatum. The precise roles of NO remain to be elucidated. In some cases, it appears to act as a retrograde transsynaptic messenger, conveying information about postsynaptic NMDA receptor activity to the presynaptic terminal and influencing neurotransmitter release. In others (e.g., in cerebellar LTD), it appears to be formed presynaptically and act on the postsynaptic neuron. Later phases of the plasticity may require cGMPdependent alterations in gene expression. In addition to its abundant physiological roles, NO is important in brain pathology. The emphasis of much research has been on the damaging effect of NO, which is usually considered to be caused by the reaction between NO and superoxide ions, forming peroxynitrite. Peroxynitrite can react with proteins, lipids, or DNA and also break down to yield the noxious hydroxyl and nitrogen dioxide radicals. An alternative mechanism contributing to damage by NO is inhibition of mitochondrial respiration. This occurs in competition with oxygen at the level of cytochrome c oxidase, the terminal complex of the respiratory chain. There has been a shift toward the appreciation of the important protective roles of NO. Protection by NO may be multifaceted. First, NO is a very potent inhibitor of lipid peroxidation which causes membrane damage in many neuropathological conditions. It does so by reacting directly with lipid peroxyl radicals, preventing propagation
of the oxidative damage. Second, NO release from endothelial cells is important in maintaining blood flow and preventing adhesion of platelets and leukocytes to vessel walls. Third, NO exerts antiapoptotic effects on many different cell types through multiple mechanisms, including the inhibition of caspases, the release of neurotrophic factors, and the stimulation of the expression of cytoprotective genes. Finally, brain repair may be augmented by the NO–cGMP pathway enhancing the generation of new neurons (neurogenesis) and the formation of new blood vessels (angiogenesis). See also: Long-Term Depression (LTD): Metabotropic Glutamate Receptor (mGluR) and NMDAR-Dependent Forms; Long-Term Potentiation (LTP): NMDA Receptor Role.
Further Reading Alderton WK, Cooper CE, and Knowles RG (2001) Nitric oxide synthases: Structure, function and inhibition. Biochemical Journal 357: 593–615. Bender AT and Beavo JA (2006) Cyclic nucleotide phosphodiesterases: Molecular regulation to clinical use. Pharmacological Reviews 58: 488–520. Bicker G (2005) STOP and GO with NO: Nitric oxide as a regulator of cell motility in simple brains. Bioessays 27: 495–505. Craven KB and Zagotta WN (2006) CNG and HCN channels: Two peas, one pod. Annual Review of Physiology 68: 375–401. Duncan AJ and Heales SJ (2005) Nitric oxide and neurological disorders. Molecular Aspects of Medicine 26: 67–96. Garthwaite G, Bartus K, Malcolm D, et al. (2006) Signaling from blood vessels to CNS axons through nitric oxide. Journal of Neuroscience 26: 7730–7740. Garthwaite J (2005) Dynamics of cellular NO-cGMP signaling. Frontiers in Bioscience 10: 1868–1880. Gibbs SM (2003) Regulation of neuronal proliferation and differentiation by nitric oxide. Molecular Neurobiology 27: 107–120. Hofmann F, Feil R, Kleppisch T, and Schlossmann J (2006) Function of cGMP-dependent protein kinases as revealed by gene deletion. Physiological Reviews 86: 1–23. Hopper RA and Garthwaite J (2006) Tonic and phasic nitric oxide signals in hippocampal long-term potentiation. Journal of Neuroscience 26: 11513–11521. Keynes RG and Garthwaite J (2004) Nitric oxide and its role in ischaemic brain injury. Current Molecular Medicine 4: 179–191. Pilz RB and Broderick KE (2005) Role of cyclic GMP in gene regulation. Frontiers in Bioscience 10: 1239–1268. Prast H and Philippu A (2001) Nitric oxide as modulator of neuronal function. Progress in Neurobiology 64: 51–68. Susswein AJ, Katzoff A, Miller N, and Hurwitz I (2004) Nitric oxide and memory. Neuroscientist 10: 153–162.
Role of NO in Neurodegeneration Y-I Lee, T M Dawson, and V L Dawson, The Johns Hopkins University School of Medicine, Baltimore, MD, USA ã 2009 Elsevier Ltd. All rights reserved.
Introduction Nitric oxide (NO) was first identified as endotheliumderived relaxing factor. NO is produced by many different cells in multicellular organisms, where it acts as an unprecedented biological messenger molecule. NO is synthesized by the enzymes NO synthases (NOSs), which catalyze the essential amino acid L-arginine into NO and L-citrulline. In the central nervous system (CNS), NO is an unusual neuronal messenger molecule in that conventional neurotransmitters are released by exocytosis from the nerve terminal, but because of its small size and ability to move through lipids, NO reaches its targets through diffusion. NO forms covalent and noncovalent linkages with protein and nonprotein targets. Conventional neurotransmitters undergo reversible interactions with cell surface receptors. NO is inactivated by diffusion away from its targets, by forming covalent linkages to the superoxide anion, or by scavenger proteins. In contrast, conventional neurotransmitters are terminated by presynaptic reuptake or enzymatic degradation. Because NO does not use conventional means for control of its biological actions, NO depends upon its small size, reactivity, and diffusibility more than any other biological molecule to exert its biological effects. NO is involved in a wide range of physiological processes, and under conditions of excess formation in the presence of superoxide anion it can mediate a series of pathophysiological actions. The multifaceted actions of NO in brain physiology include mechanisms related to development, synaptic function and neural plasticity, and neuronal survival and neuronal cell death.
NO in the Nervous System NO is produced by a group of enzymes designated NOS. There are three members of the NOS family: neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS). A fourth family member was proposed, mitochondrial NOS (mtNOS), but studies indicate that mtNOS is nNOSa – it is coded by the nNOS gene, and nNOS knockout mice do not have mtNOS. All the NOSs share between 50% and 60% sequence homology. In the nervous system, nNOS is expressed in neurons, eNOS is predominantly
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expressed in the endothelium of blood vessels, and iNOS is expressed in glial cells. nNOS and eNOS are Ca2þ-calmodulin-dependent enzymes that are constitutively expressed, although both isoforms can be induced following a variety of physiological and pathological stimuli. In contrast, iNOS does not require Ca2þ-calmodulin for activation and its regulation depends on de novo synthesis. Despite its highly diffusible nature, NO can exert very specific effects within the CNS and peripheral nervous system (PNS). Under physiological conditions, NO facilitates neurotransmitter release and uptake via neuron-glial communication, modulating the release of neurotransmitters, including glutamate, g-aminobutyric acid (GABA), substance P, and acetylcholine. nNOS and eNOS are regulated via posttranslational modification, including phosphorylation, and nNOS levels are dynamically regulated through transcription. Cerebral blood flow is regulated through NO released from endothelial cells as well as autonomic nerves within the adventitia. NO may also regulate cerebral blood flow through activitydependent activation of NOS in neurons that influence small cerebral arterioles. In the brain, nNOS is linked to the postsynaptic membrane near N-methyl-D-aspartate (NMDA) receptors via the postsynaptic density protein95, and nNOS function is regulated through its interactions with the protein inhibitor of NOS (PIN) and carboxyl-terminal PDZ ligand of NOS (CAPON) (Figure 1). In the gut, NO functions as the nonadrenergic, noncholinergic (NANC) neurotransmitter where it mediates relaxation of smooth muscles associated with peristalsis. Penile erections are mediated, in part, through the neurotransmitter action of NO. The diffusion properties and the short half-life of NO make it an ideal candidate for playing a role in nervous system morphogenesis and synaptic plasticity. In the nervous system, NO was first recognized as a messenger molecule that mediates increases in cyclic GMP (cGMP) levels that occur after activation of glutamate receptors, particularly those of the NMDA subtype. In this capacity, NO modulates long-term potentiation (LTP), which is essential for learning and memory. LTP is inhibited by administration of NOS blockers or genetic deletion of NOS isoforms, confirming the role of NO in synaptic plasticity. Nerve growth factor (NGF)-induced differentiation of pheochromacytoma cells (PC12) has been shown to be NO dependent with NO acting as a cytostasis factor. NO may play a role in the developmental processes of the nervous system, in that it is transiently expressed in the cerebral cortical plate during critical periods of neuronal development. NO is also critical for the neurite outgrowth and differentiation in murine cortical
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Neuronal activity
Glu
Glu Glu
Adjacent cells
GLUT
Fe-Heme proteins ~ Guanyly cyclase
Ca2+ NMDA-R PSD-95
Fe-S proteins ~ Aconitase
Astrocyte
CAPON nNOS L-Arg
NO L-Cit
NO
R-S-NO ~ Ras, parkin
Postsynaptic nNOS synapses
Figure 1 NO signaling in the CNS. Glutamate activation of NMDA receptor results in stimulation of neuronal NO synthase (nNOS) which is functionally coupled to the NMDA receptor by PSD-95, a scaffolding protein. The tethering of nNOS near the NMDA receptor facilitates its activation by Ca2þ resulting in the generation of NO. CAPON binds nNOS and regulates its interaction with PSD-95 and thus regulates the generation of NO. NO diffuses between cells and can activate a number of cell signaling pathways in adjacent cells. NO can react with the heme center of soluble guanylyl cyclase (GC) activating GC to produce cyclic GMP (cGMP). NO can react with iron–sulfur (Fe–S)-containing proteins such as aconitase to regulate their activity. NO may S-nitrosylate (R-S-NO) proteins such as Ras or parkin, thus regulating complex cell signal cascades.
neurons. NO may play a role in the molecular maturation of adult spinal cord motor neurons and also in the initial pattern of connections in ocular dominance columns in the developing visual system. It has also been suggested to play a prominent role in the activity-dependent establishment of connections in both the developing and regenerating olfactory neuronal epithelium. Thus, NO may play an important role in synaptic refinement in the developing nervous system. In the immune system, NO generated by iNOS is released in bursts of high concentrations and is a key cytotoxic weapon for the destruction of bacteria. The CNS immune cell counterparts, microglia and astrocytes, also have iNOS, which generates a burst of NO in response to injury and can contribute to neural injury. The expression of iNOS occurs following trauma, following stroke, or during neurodegenerative diseases and is thought to contribute to neural injury. Although NOS is the main NO source, in some special situations this molecule can be synthesized by other mechanisms. NO can be produced by the xanthine oxidase pathway or by H2O2 and L-arginine in a nonenzymatic reaction or by the reduction of nitrites in acid and reducing conditions, such as occurs in ischemic tissue.
NO
ONOO-Mitochondrial dysfunction -PARP activation -Tyrosine nitration -Lipid peroxidation S-NO -Parkin -GAPDH/Siah
Pathophysiologic
Fe-Heme -Guanyl cyclase -Cyclooxygenase Fe-Sulfur centers -Aconitase -NADH succinate oxidoreductase -NADH ubiquitin oxidoreductase S-NO -Ras -Erk -JNK Physiologic
Figure 2 Physiological and pathophysiological NO signaling events.
NO Molecular Targets There are several molecular mechanisms by which NO elicits cellular signaling (Figure 2). The unique chemical properties of NO facilitate its role as an important signaling molecule in the biological system by reacting directly with biological molecules or indirectly
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following reaction with other reactive oxygen species. NO reacts with the ferrous ion in the heme complex in enzymes such as guanylate cyclase (GC), P450, and hemoglobin to form an iron nitrosyl complex. The binding of NO induces a conformational change in the protein structure activating these proteins. The prototype reaction is that of NO on GC leading to the production of cyclic guanosine-30 ,50 -monophosphate (cGMP). cGMP is a second messenger that activates protein kinases. In the nervous system, NO was first recognized as a messenger molecule that regulates cGMP accumulation through activation of glutamate receptors, particularly those of the NMDA subtype. cGMP, in turn, binds to and activates a cGMP-dependent protein kinase (G kinase), which phosphorylates specific proteins on serine or threonine residues and also activates phosphodiesterase and ion channels. NO increases prostaglandin production by activation of cyclo-oxygenase, another hemecontaining enzyme. Numerous enzymes (including cis-aconitase, NADH succinate oxidoreductase, and NADH ubiquinone oxidoreductase) that contain nonheme iron in iron sulfur clusters are also regulated by NO. NO also stimulates the RNA-binding function of the iron-responsive element binding protein in brain slices while diminishing its aconitase activity. In recent years, the importance of protein S-nitrosylation of cysteine thiols by NO in physiological and pathophysiological actions has been appreciated. S-nitrosylation is a posttranslational protein modification that increases or decreases the activity of the target protein. Some known biological targets for S-nitrosylation include glyceraldehyde-3-phosphate dehydrogenase (GAPDH), caspases, transglutaminase, aromatases, mitochondrial aldehyde dehydrogenase, cyclooxygenase-2, G-proteins, p21-Ras, RAC1, cdc42, Dexras1, ERK, JNK, p38, and PKBa/AKT1. S-nitrosylation of the monomeric GTPase Ras activates this enzyme, triggering the MAPK and PI3K pathways in an NO-dependent and cGMP-independent manner. A recent study reports that NO donors promote HIF-1a accumulation via an increase in transcription and translation. It is not yet known how this occurs, but it is possible that these effects are due to activation of Ras and PI3K through NO-mediated nitrosylation of Ras. NO has inhibitory effects on cell cycle progression and promotes cell differentiation, in part due to activation of the retinoblastoma gene product (pRb) by S-nitrosylation. Nuclear factor kappa beta (NF-kb), a transcription factor that induces iNOS expression, is inhibited by S-nitrosylation. Thus, NO inhibition of NF-kb could be an autoregulatory feedback mechanism to regulate NO production. Another autoregulatory mechanism is the prevention of eNOS
dimerization by S-nitrosylation, resulting in decreased eNOS activity. S-nitrosylation is also involved in the pathological activation of matrix metalloproteases in stroke and neurodegenerative diseases. NO regulates NMDA receptor activity through nitrosylation, thus reducing calcium permeability. One of the important features of S-nitrosylation is that the modification is reversible, and a specific protein sequence of acidic– basic motifs seems to be necessary. As usual, the modification is also selective, since only a specific cysteine is generally nitrosylated in a protein. Oxygen and other free radicals can also react with NO to form reactive nitrogen oxide species and exertits effects on different biological molecules. Superoxide anion can react with NO to form peroxynitrite, a potent oxidant with many cellular targets. Peroxynitrite damages proteins by reacting with the tyrosine residues to form 3-nitrotyrosine. It can also induce lipid peroxidation and cause DNA damage. It is thought that many of the acute toxic effects of NO are mediated by peroxynitrite.
Neurotoxic Effects of NO In the CNS, glutamate excitotoxicity due to activation of the NMDA receptor is mediated in large part by NO. Under physiological conditions, the binding of glutamate activates NMDA receptors in the postsynaptic neuron that result in Ca2þ influx, which in turn activates nNOS. Then NO either via cGMP or by S-nitrosylation suppresses NMDA receptor activity, thereby limiting further Ca2þ influx and thus protecting neurons. However, under pathophysiological conditions such as occur during stroke, trauma, or neurodegenerative stress, NO participates in neuronal damage through disruption of this system and generation of peroxynitrite that activates subsequent cell death cascades. NO regulation of the release of glutamate contributes to the initial toxic events. When NO concentrations are low, there is a decrease in glutamate release despite elevated cGMP levels. However, under increased NO production and excessive increases in cGMP levels, the inhibitory effect on glutamate release is reversed, and glutamate release is increased. Under the pathological conditions of excess glutamate release and NMDA receptor activation, activation of NOS leads to cell death. Excitotoxicity can be blocked by several classes of NOS inhibitors or by nNOS gene deletion. nNOS knockout mice are resistant to focal and transient global ischemia, suggesting a role for nNOS in CNS injury. The neuronal toxicity of NO is mediated by peroxynitrite, which inhibits mitochondrial respiration and manganese superoxide dismutage (MnSOD) activity,
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thus establishing a feed-forward loop for peroxynitrite generation. Peroxynitrite damages DNA, which activates the nuclear enzyme poly(ADP-ribose) polymerase (PARP) that leads to the release of the cell death effector apoptosis-inducing factor (AIF) from mitochondria. AIF can induce phosphatidyl serine exposure on the plasma membrane, and in the nucleus it facilitates DNA cleavage and nuclear condensation (Figure 3). In addition, glutamate receptor-mediated excitotoxicity activates parallel cellular responses, resulting in the activation of proteolytic enzymes, lipid peroxidation, ROS and RNS formation, and activation of programmed cell death cascades. Under some conditions, NO can activate the ceramide–Jun kinase pathway and the NF-kb–Jun kinase pathway, which regulate the expression of a large number of genes involved in both neuroprotection and neurotoxicity. The biological significance of NO stimulation of these pathways depends on the cell type and the environment. NO can also play a protective role via the blocking of caspases (cysteine–aspartate proteases), which form a complex family of proteolytic enzymes. It is not yet clear whether NO acts as a master regulator, determining which cell death pathway will be activated in neuronal injury, but these data are suggestive that NO production could direct neurons away from a classic apoptotic type of cell death and toward an excitotoxic form of cell death acutely and perhaps activate other apoptotic cascades in the late remodeling phase of neuronal injury and recovery.
Oxidative and nitrosative stress plays a pivotal role in ischemic-reperfusion injury during stroke. The increase of NO production in the presence of increased superoxide anion leads to brain damage. Brain ischemia triggers a cascade of events, including membrane depolarization, increase in intracellular Ca2þ concentrations via increase of extracellular glutamate concentration, and overstimulation of NMDA receptors, leading to activation of calcium-dependent nNOS. Released NO rapidly reacts with superoxide produced in excess during reperfusion resulting in peroxynitrite formation, and nitrosylation or nitration of proteins. Furthermore, ischemia or reperfusion eventually induces the expression of iNOS. This isoform is not normally present in the CNS, but it can be detected after inflammatory, infectious, or ischemic damage, as well as in the normal aging brain. Activation of nNOS or induction of iNOS mediates ischemic brain damage through mechanisms previously discussed. On the other hand, eNOS is thought to act as a neuroprotective agent by enhancing blood supply to the injured tissue. Therefore, NO generated by nNOS or iNOS contributes to injury following stroke, whereas NO generated by eNOS is protective by maintaining blood flow (Figure 4).
NO and Neurodegenerative Diseases Abnormal activation of glutamate neurotransmission may contribute to neurodegenerative processes and
Glutamate 2+
Ca
NMDA-R Mitochondria ROS Ca2+ NOS
Respiration NO
O2.−
Energy production
ONOOAIF ONOO-
DNA damage
DNA fragmentation Nuclear Condensation
PARP activation Poly-ADP ribosylation Nicotinamide
NAD Energy depletion + 4ATP
Figure 3 Mechanism of NO-mediated neurotoxicity. NMDA receptor (NMDA-R) activation causes an increase in intracellular calcium levels, which activates NOS, as well as other potential free radical-generating enzymes. These free radicals then damage DNA and also inhibit mitochondrial function, leading to more free radical formation. The damaged DNA activates PARP-1, which transfers ADP-ribose groups to nuclear proteins consuming NAD. NAD is resynthesized from nicotinamide (NAm), a reaction that consumes four high-energy equivalents of ATP. PARP activation also elicits the release and translocation of AIF from the mitochondria into the nucleus, initiating a caspase-independent cascade of cell death.
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Ischemia Neurons nNOS
Glial cells Macrophages
Endothelium & smooth muscle eNOS
NO
nNOS iNOS
NO
NO
Cell damage
Protection
Figure 4 Dual roles for NO in brain ischemia. NO is involved in the mechanisms of neurotoxicity after cerebral ischemia. Following a stroke, the generation of NO can be either beneficial or detrimental to the brain depending on the which NOS isoform is activated and in which cellular compartment. NO generated from eNOS in endothelial cells provides protection by inducing vasodilation. Inhibition of eNOS can drastically increase stroke injury. NO produced by nNOS in neurons or iNOS in glia results in the formation of toxic peroxynitrite and cellular damage. Inhibition of nNOS or iNOS greatly reduces brain injury following stroke.
diseases such as Parkinson’s disease (PD), Alzheimer’s disease (AD), amyotrophic lateral sclerosis (ALS), and multiple sclerosis (MS). Neurodegenerative disorders are usually marked by a progressive loss of a selective group of neurons and are associated with aging. One of the common features of neurodegeneration is the presence of protein aggregates and increased indices of oxidative stress. PD is a common neurodegenerative disorder characterized by impairment in motor function. It is another common age-related neurodegenerative condition associated with selective loss of dopaminergic neurons in the substantia nigra of the midbrain region. In PD, postmortem pathological studies have revealed mitochondria complex I dysfunction, reduced glutathione and ferritin levels, increased lipid peroxidation, nitrotyrosine immunoreactivity, and increased levels of iron. Genetically engineered animals reveal that NO generated by both iNOS and nNOS contributes to pathology. In the experimental 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intoxication model of PD, NOS inhibition in wildtype mice or gene deletion of the iNOS or nNOS gene protects dopaminergic neurons. In PD patients, polymorphonuclear cells have increased expression of nNOS and increased NO production and accumulation of nitrotyrosine-containing proteins, reflecting an increase in nitrosative stress in these patients. Glial-derived neuronal factor (GDNF) is protective in animal models of PD, although its effect on patients remains controversial. GDNF action leads
to the inhibition of nNOS activity and neurotoxicity. R-(–)-deprenyl (deprenyl) slows the progression of disability in early PD. Deprenyl prevents S-nitrosylation of GAPDH, which blocks the binding of GAPDH to Siah, preventing the GADH/Siah death cascade. In MPTP-treated mice, low doses of deprenyl prevent binding of GAPDH and Siah1 in the dopamineenriched corpus striatum and it is neuroprotective. PD is mostly sporadic, but rare familial cases are also found. Mutations in a-synuclein, parkin, DJ-1, PINK1, and LRRK2 have been linked to rare familial forms of PD. Point mutations or replication of a-synuclein results in autosomal dominant PD, which prompted intense investigation into the actions and pathobiology of this protein. The hallmark lesions of PD, the Lewy body, contain a-synuclein that is modified by nitration of tyrosine residues. It is possible that dityrosine cross-linking can generate stable oligomers that lead to aggregates. Both wild-type and disease-causing a-synuclein mutants can be nitrated, and the nitration induces the formation of a-synuclein inclusions with similar biochemical characteristics to protein extracted from human PD tissue. In addition to nitration, nitrosylation may also play a key role in both sporadic and familial PD, as there is a greater than twofold increase in S-nitrosylated proteins in PD. Parkin is an E3 ligase that adds ubiquitin on specific protein substrates by both K48 and K63 linkages. Disease-causing mutations and loss-of-function mutants lead to the accumulation of toxic parkin substrates in both mouse and humans. Parkin can be S-nitrosylated. This modification results in loss of function as an E3 ligase and inhibition of the protective function of parkin. In sporadic cases of PD, parkin is S-nitrosylated, suggesting that in the more common form of PD, parkin function may be compromised. While parkin and DJ-1 do not interact physiologically, under conditions of oxidative and nitrosative stress there is a gain of function and these two proteins interact. It is possible that this interaction results in the sequestering of DJ-1 into insoluble aggregates terminating its normal biological actions. The pathophysiological significance of this interaction between DJ-1 and parkin is not yet known but is an area of active scientific investigation. Actions of oxidative and nitrosative stress on PINK1 or LRRK2 await investigation. AD is the most common chronic progressive neurodegenerative condition; it was originally described by Alzheimer about a century ago. Brains from AD patients are characterized by extracellular aggregates of amyloid b-peptide (Ab) that form the neuritic plaques and intracellular neurofibrillary tangles due to the hyperphosphorylation of tau protein. NO can contribute to AD neurodegeneration in several
Role of NO in Neurodegeneration 695
ways. Ab fibrils are toxic, in part, by inducing ROS formation, which can produce peroxynitrite by reacting with NO. Increased expression of iNOS and eNOS in astrocytes has been associated with the presence of neuritic plaques indicating increased nitrosative stress in AD. Additional studies indicate that Ab is critically involved in the induction of an inflammatory response through activation of astrocytes and microglia, eliciting subsequent production of inflammatory cytokines and neurotoxic factors, including NO, superoxide, and prostaglandins (via COX-2). Ab deposition in brain vessels correlates with a decrease in the expression of eNOS in endothelial cells that can lead to decreased regulation of cerebral blood flow. In cell culture and experimental animal models, Ab elicits NO-mediated excitotoxicity, neuroinflammation, and oxidative stress. Similar stress events are observed in patient tissue, implicating a role for NO in the progression of AD. ALS is a neurodegenerative disease characterized by selective death of motor neurons in the cerebral cortex, brain stem, and spinal cord. The role of NO in ALS is controversial. There is evidence that nitrotyrosination induces motor neuron death and that there is irreversible inhibition of the mitochondrial respiratory chain in these cells, suggesting a role for NO. However, mice transgenic for the human disease-causing mutation in SOD-1 when crossed with the nNOS knockout mice did not derive any benefit and progressed to disease and death. On the other hand, treatment with a nonselective NOS inhibitor slightly decreased motor neuron degeneration in an ALS mouse model. It is possible that NO from different isoforms could contribute to progressive neurodegeneration in ALS and exacerbate disease progression. In postmortem tissue, there are reactive astrocytes that contain protein inclusions, express iNOS and COX-2, display nitrotyrosine immunoreactivity, and have a loss of the glutamate transporter, EAAT2. The combination of excitotoxicity and nitrosative stress could contribute to the development and acceleration of the disease.
Conclusion NO is a multifunctional molecule that is needed for physiological functions, especially in the brain. NO
has clearly revolutionized our thought about aspects of neuronal transmission. It is capable of regulating various body functions directly, via a cGMP-mediated mechanism or a cGMP-independent mechanism. NO is an important mediator of a variety of physiological effects in the nervous system; however, excessive NO production under pathological conditions could be destructive to the brain. Understanding the role of NO in these processes as well as the disposition and activation of NO will lead to a better understanding of the basic processes underlying normal and pathological neuronal functions, and hopefully to the development of selective protective therapeutic agents against neurodegenerative diseases. See also: Long-Term Depression (LTD): Metabotropic Glutamate Receptor (mGluR) and NMDAR-Dependent Forms; Long-Term Potentiation (LTP): NMDA Receptor Role; Nitric Oxide.
Further Reading Ahern GP, Klyachko VA, and Jackson MB (2002) cGMP and S-nitrosylation: Two routes for modulation of neuronal excitability by NO. Trends Neuroscience 25: 510–517. Chung KK, Dawson TM, and Dawson VL (2005) Nitric oxide, S-nitrosylation and neurodegeneration. Cellular and Molecular Biology 51: 247–254. Contestabile A and Ciani E (2004) Role of nitric oxide in the regulation of neuronal proliferation, survival and differentiation. Neurochemistry International 45: 903–914. Dawson VL and Dawson TM (2004) Deadly conversations: Nuclearmitochondrial cross-talk. Journal of Bioenergetics and Biomembranes 36: 287–294. Emerit J, Edeas M, and Bricaire F (2004) Neurodegenerative diseases and oxidative stress. Biomedicine and Pharmacotherapy 58: 39–46. Esplugues JV (2002) NO as a signaling molecule in the nervous system. British Journal of Pharmacology 135: 1079–1095. Guix FX, Uribesalgo I, Coma M, and Munoz FJ (2005) The physiology and pathophysiology of nitric oxide in the brain. Progress in Neurobiology 76: 126–152. Moro MA, Cardenas A, Hurtado O, Leza JC, and Lizasoain I (2004) Role of nitric oxide after brain ischemia. Cell Calcium 36: 265–275. Thippeswamy T, Mckay JS, Quinn JP, and Morris R (2006) Nitric oxide, biological double-faced janus – Is this good or bad? Histology and Histopathology 21: 445–458.
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Subject Index Notes Cross-reference terms in italics are general cross-references, or refer to subentry terms within the main entry (the main entry is not repeated to save space). Readers are also advised to refer to the end of each article for additional cross-references – not all of these cross-references have been included in the index cross-references. The index is arranged in set-out style with a maximum of three levels of heading. Major discussion of a subject is indicated by bold page numbers. Page numbers suffixed by T and F refer to Tables and Figures respectively. vs. indicates a comparison. This index is in letter-by-letter order, whereby hyphens and spaces within index headings are ignored in the alphabetization. Prefixes and terms in parentheses are excluded from the initial alphabetization. A A1 receptors see P1 receptor(s) A2 receptors see P1 receptor(s) a-Amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors see AMPA receptor(s) Ab peptide see Amyloid beta (Ab) Abp1, endocytosis and SV cycle 85t Absence (petite mal) seizure(s) GABAB receptors 365 somatosensory cortex 365–366 Acetylcholine (ACh) 478, 479f anatomy see Cholinergic neurons/systems cotransmission see Cholinergic neurons/systems degradation see Acetylcholinesterase (AChE) hydrolysis see Acetylcholinesterase (AChE) inactivation see Acetylcholinesterase (AChE) at motor nerve terminals see Neuromuscular transmission at neuromuscular junction see Neuromuscular transmission neurotransmission see Cholinergic neurotransmission receptors see Acetylcholine receptors (AChRs) storage 480 synthesis 478 ChAT see Choline acetyltransferase (ChAT) choline transport 478 see also Cholinergic neurons/systems Acetylcholine binding protein (AChBP) astrocyte-mediated neuromodulation 117–118 role 504 structure 374, 504, 505f Acetylcholine receptor-aggregating factor (AGRIN) 506 Acetylcholine receptor-inducing activity (ARIA), nicotinic acetylcholine receptors 506 Acetylcholine receptors (AChRs) 481 location 480 muscarinic see Muscarinic acetylcholine receptors (mAChRs) neuromuscular junction see Neuromuscular junction, acetylcholine receptors nicotinic see Nicotinic acetylcholine receptors (nAChRs) peripheral 482t responses 482t subunits 180, 182f Acetylcholinesterase (AChE) 245, 246f, 481 acetylcholine binding sites 481 acetylcholine hydrolysis 478, 481 stages 481 acetylcholine inactivation 481 ColQ see Collagen-tailed acetylcholinesterase (ColQ-AChE) distribution 481 inactivation see Acetylcholinesterase (AChE) inhibitors inhibitors see Acetylcholinesterase (AChE) inhibitors neuromuscular junction 186 basal lamina component 177 collagen-tailed (ColQ) 177 see also Neuromuscular transmission pharmaco-histochemical technique 487–488 Acetylcholinesterase (AChE) inhibitors Alzheimer’s management 492–493 Acetyl CoA, synthesis 479
N-Acetyltransferase (NAT) neuropeptide synthesis 513, 513f ACTH see Adrenocorticotropic hormone (ACTH) Actin/actin filaments b-actin filopodial motility regulation 6–7 cadherin interaction 38–40 clathrin-coated pit dynamics 90 integrin-mediated signaling 178 motor proteins see Myosin(s) postsynaptic development 24 dendritic spines dynamic interaction 24–25 motility regulation 7–9 striated muscle 141 Actin-associated proteins clathrin-coated pit dynamics 90 Action potential(s) conduction/propagation gap junctions 197 filopodial motility regulation 7 muscle(s) 180, 184 activation threshold 180, 183f, 184 Nav1 channels and 184 safety factor 186 species differences 186, 187f see also Neuromuscular transmission Activator protein-1 (AP-1) neuropeptide granule biogenesis 516 Active protein kinase B see Akt/PKB protein kinase Active zone (AZ) 11, 52–58, 53f, 84 components 11, 16, 54 cytoskeleton/cytomatrix see Cytoskeleton of the active zone (CAZ) non-specific 54–55 proteins 56t AZ specific 55, 55f RIMS see RIMs scaffolding 11, 84, 90 regulatory functions 16 specific 54–55 structural 16 core active zone 90 definition 52 morphology 52 dense projections 53–54 mammalian synapses 53–54, 53f neuromuscular junction of frog 53f, 54 neuromuscular junction 159, 160f, 161f as anatomical correlate of binomial n 163 muscle fiber type differences 159–160 slam freezing studies 160 species differences 159–160 peri-active zone 90 synaptic vesicle release 11, 16, 52 see also Neurotransmitter release; Synaptic vesicle(s) Activity-dependent plasticity 31 mGluR-dependent LTD 327 NMDA receptor dependent LTD 327
697
698 Subject Index Activity-dependent plasticity (continued) at PSD 101 short-term see Short-term synaptic plasticity synaptic competition and 35 see also Synaptic competition see also Hebbian plasticity Adaptor proteins clathrin-mediated endocytosis see Clathrin-mediated endocytosis (CME) mGluR regulation 294 postsynaptic density see Postsynaptic density (PSD) see also Scaffold proteins Addiction alcoholism see Alcoholism molecular mechanisms see Addiction, molecular mechanisms treatment 372 see also Substance abuse Addiction, molecular mechanisms dopamine role see Dopamine GABAB receptor ligands 365 glutamate role 238, 598 mGluR2/3 agonists 299 mGluR5 298 melanin-concentrating hormone 555 neuropeptide release 523–524 orexins (hypocretins) and 555 synaptic plasticity glutamate receptors as targets long-term depression 598 long-term potentiation 598 Adenosine 627–638 connexin permeability differences 213 ectonucleotidases 628–629 functional roles 627 glial cells and astrocytes ATP release and neuromodulation 116–117 see also Astrocytic calcium waves metabolism 628 neuromuscular transmission astrocytes and 49 nucleotide precursors 628–629 receptors see P1 receptor(s) sources 628 transport mechanisms 628 Adenosine A1 receptors see P1 receptor(s) Adenosine A2 receptors see P1 receptor(s) Adenosine A3 receptors see P1 receptor(s) Adenosine amine congener (ADAC) 630 Adenosine monophosphate kinase (AMPK) striatal dopamine D2 receptors and 411 Adenosine receptor(s) see P1 receptor(s) Adenosine triphosphate (ATP) 648 degradation to adenosine, neuromodulation and 116–117 gliotransmission and see Adenosine triphosphate, transmitter role neurotransmission and see Adenosine triphosphate, transmitter role production 243 transmitter function see Adenosine triphosphate, transmitter role vesicular neurotransmitter transporters 258–259 Adenosine triphosphate, transmitter role 639–647 central nervous system autonomic function control 643 cotransmission norepinephrine 420–421 purinergic 639 degradation 641 ectonucleotidases 641 ear 646 eye 646 gliotransmission astrocytic release 49, 115, 116, 118f glial–neuronal signaling 643 historical aspects 639 nasal organs 646 neuromuscular transmission astrocytes and 49 neuroprotection 643 peripheral nervous system mechanosensory transduction 644 pain transduction 644 smooth muscle contraction 644, 644f receptors 640 plasticity 643
purine 640 pyrimidine 640 release mechanisms 641 exocytotic 641 Adenylate cyclase see Adenylyl cyclase(s) Adenylyl cyclase(s) dopamine receptor transduction 410 GABAB receptor transduction 362 inhibition via CB1 receptors 664–665 Ad fibers NGF role in postnatal development 577 Adhesion molecules see Cell adhesion molecules (CAMs) Adrenaline see Epinephrine Adrenergic (PNMT-containing) neurons neuropeptide coexistence galanin 528 neuropeptide Y 528 neuropeptide Y (NPY) 538, 539 substance P 528 Adrenergic receptors 416t, 424–429 antagonists intermittence mechanisms 419 see also Beta-blockers astrocytes see Astrocyte(s) catecholamine stimulation effects 424 classification 425f history 424 polymorphisms 428 regulation 427 signal transduction 427–428 structure 424 a-Adrenergic receptors 416t a1 receptors 416t, 426 a1A receptors 426 a1B receptors 426 a1D receptors 426 a1L receptors 426 activation of 426 cloning of subtypes 426 distribution 424 molecular characteristics 426 pharmacological characteristics 426 signal transduction pathways 428 structure 426 a2 receptors 416t, 426 a2A receptors 426–427 a2B receptors 426–427 a2C receptors 426–427 a2C(del) receptors 428 a2D receptors 426–427 activation of 427 cloning of subtypes 426–427 location 424 molecular characteristics 427 pharmacological characteristics 427 polymorphisms 428 signal transduction pathways 428 structure 427 b-Adrenergic receptors 416t, 427–428 asthma 427 b1 receptors catecholamine sensitivity effects 427 polymorphisms 428 b2 receptors 416t catecholamine sensitivity effects 427 polymorphisms 428 b3 receptors 416t tryptophan-64 to arginine polymorphism effects 428 downregulation 427–428 drugs acting at antagonists see Beta-blockers nonselective antagonists 427 location 424 molecular characteristics 427 pharmacological characteristics 427 structure 427 Adrenergic system neurons see Adrenergic (PNMT-containing) neurons organization 431t non-locus coeruleus 435 see also Epinephrine Adrenoceptors see Adrenergic receptors
Subject Index a-adrenoceptors see a-Adrenergic receptors b-adrenoceptors see b-Adrenergic receptors Adrenocorticotropic hormone (ACTH) pituitary secretion CRH role 544 synthesis precursor peptides 511–512 Afadin, nectin binding 43 Afferent neurons see Sensory afferent(s) Afterhyperpolarization (AHP) 5HT4 receptor 476 Age/aging autonomic nervous system see Autonomic nervous system (ANS) glial cell growth factors 622 neuroendocrine see Neuroendocrine aging Aggression/aggressive behavior molecular/neurochemical mechanisms adenosine receptors 637t Agnatha neuromuscular transmission efficacy 148–149 skeletal muscle innervation 142 central fibers 142–143 ‘muscle units’ 142 Agomelatine, depression 465–466 Agrin, neuromuscular junction role 44, 177 binding partners 177 functional importance 177 interactions 177 NMJ development and AChR aggregation 126, 177 Akt/PKB protein kinase cell survival signaling IGF signaling and 613 Alcohol addiction/dependence see Alcoholism consumption/intake baclofen effects 365 neuropeptide Y and 539 GABAA receptors and 357 Alcohol dependence see Alcoholism Alcoholism neural substrates dopamine D2 receptor 400 see also Addiction Allopregnanolone GABAA receptors 353 Allosteric modulation GABAB receptors 365 mGluRs 293–294 Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors see AMPA receptor(s) ALS see Amyotrophic lateral sclerosis (ALS) Alsin gene/protein retrograde neurotrophic signaling defects 589 Alteplase see Tissue plasminogen activator (TPA) Alternative splicing NMDA receptors 277 Trk receptors 570 Alzheimer’s disease, neurotransmitter systems adenosine receptors 637t cholinergic deficits 492 M1 receptor activation 492–493 M3 receptor activation 492–493 therapeutic targets 492–493 dopamine D2 receptors 400, 402f glutamate mGluRs 307 nitric oxide 694–695 serotonergic system cloned mouse receptor human receptor differences 468 rat receptor differences 468 5-HT6 receptor and 468 Alzheimer’s disease, pathology/pathogenesis ‘cholinergic hypothesis’ see Alzheimer’s disease, neurotransmitter systems glia role growth factors 622 endothelin-1 622 hepatocyte growth factor 622 TGF-b1 622
699
neurotransmitter dysfunction see Alzheimer’s disease, neurotransmitter systems retrograde neurotrophic transport signaling defects 588 Alzheimer’s disease, therapy 573–574 cholinergic drugs AChE inhibitors see Acetylcholinesterase (AChE) inhibitors neurotrophins 573–574 nerve growth factor 573–574 Amacrine cells connections bipolar cells see Bipolar cells (retina) neuropeptides coexistence 528 Amine(s) catecholamines see Catecholamine(s) a-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors see AMPA receptor(s) Amino acid(s) L-amino acids vs. D-amino acids 333 neurotransmitters neuropeptide coexistence 528 D-Amino acid oxidase (DAAO) control 334 D-serine degradation 333, 334, 337 Aminopeptidase(s), neuropeptide synthesis 513, 513f Aminopeptidase P (APP), neuropeptide Y synthesis 538 2-Aminopropyl-methylphosphinic acid (3-APMPA), GABAB receptor agonists 363, 363f 2-Aminopropyl-phosphinic acid (3-APPA), GABAB receptor agonists 363, 363f Ammonia GABA metabolism 343–344 Ammon’s horn see Hippocampus AMPA receptor(s) 96–97, 230, 233f, 268–275, 260–267 calcium permeability 263 definition 268 desensitization 262–263 synaptic depression and cerebellar LTD 680 endocytosis and see AMPA receptor trafficking excitatory postsynaptic currents 250–251, 323 expression/distribution 260 synaptic localization 23, 99 functions 261 engineered agonist-binding domain 261–262 glutamate concentrations 250 group I mGluRs and 305–306 kainate effects 233 metabolism 239–241 neuropeptide synaptic potentials 568 pharmacology 264 agonists 264, 265f partial 263 antagonists competitive 264 noncompetitive 265 kainate receptor agonist/antagonist interaction 313 pore-blocking molecules 266 positive allosteric modulators 265, 266f posttranscriptional modifications 263 RNA editing 263 posttranslational modifications 264 phosphorylation 264 protein interactions 325 SALMs binding 42 PSD complexes 96–97, 99 adaptor/scaffolding proteins 98f, 99 development 100 expression and ‘desilencing’ silent synapses 16–17 plasticity and 101 proteomic approaches 99 PSD-95 and 271 rapid cycling 99 Ras GTPases and 100 regulation phosphorylation by CaMKII 270 subunits/structure 230–232, 260, 261f, 268 assembly 260 chromosomal locations 260 cytoplasmic C-terminal domains 260–261 flip/flop splice variants 263 GluR1 268, 273–274
700 Subject Index AMPA receptor(s) (continued) GluR1long 270 GluR2 268, 273–274 agonist binding domain 261, 261f conformational changes 262, 262f efficiency reduction effects 263 expression 260 RNA editing 263 structure 261–262 GluR2short 270 GluR3 268, 273–274 GluR4 268 expression 260 GluR5-deficient animal models 318 long subunit role 270 N-terminal domains 260–261 functions 261 pore 260–261, 262 rotational symmetry 262–263 short subunit role 270 topology 260, 261f synaptic efficacy 99 synaptic plasticity 99 activity-dependent maturation 27 activity-dependent translation in dendrites 101 astrocytic TNFa role 113–114 long-term depression see Long-term depression, glutamate role long-term potentiation see Long-term potentiation, glutamate role synaptic transmission 321 trafficking see AMPA receptor trafficking AMPA receptor binding protein (ABP) 271 mGluR2-dependent long-term depression 329 AMPA receptor trafficking 27–28, 268 activity-dependent membrane insertion 270 GluR1long 270 PDZ protein 270 receptor phosphorylation 270 endocytosis and 272 clathrin-mediated 272 fate of 273 GluR1 fate 273–274 GluR2 fate 273–274 GluR3 fate 273–274 NSF 273–274 small GTPase signaling 273 endoplasmic reticulum to synapse 268, 269f anterograde trafficking 268 dendritic spines 268–270 exocyst 268–270 GluR1 268 GluR2 268 glutamate receptor interacting protein 1 (GRIP1) 268 microtubules 268 Rab GTPases 268–270 Rho GTPases 268–270 trans-Golgi network (TGN) 268 glutamate receptor interacting protein (GRIP1) 271 lateral diffusion 272 membrane insertion 325 NSF-dependent cycling 270 GluR1long 270 GluR2short 270 pentaxin-induced clustering 271 protein interacting with C kinase 1 (PICK1) release 272 scaffold proteins 271 degradation 272 PSD-93 272 SAP-97 270 selective delivery 268 slot proteins 270 transmembrane interacting proteins (TARPs) 271 Amphetamine(s) actions endocannabinoid LTD and 677 mechanisms of action dopaminergic system and dopamine D2 receptor and 406 vesicular monoamine transporter inhibition 256 Amphibian(s) neuromuscular system neuromuscular junction 146 skeletal muscle innervation 143
Amphiphysin endocytosis and SV cycle 85t, 95 Amygdala synaptic plasticity LTD and endocannabinoids and 677 Amygdaloid complex see Amygdala Amyloid beta (Ab) Alzheimer’s disease 694–695 FGF-2 induction 622 interleukin-1b induction 622 endothelial (eNOS) effects 694–695 Amyotrophic lateral sclerosis (ALS) animal models SOD1 knockout mice synapse elimination and 153, 156 etiology/pathogenesis genetic factors SOD1 mutations 155–156 see also Familial amyotrophic lateral sclerosis (ALS) nitric oxide and 695 superoxide dismutase 1 (SOD1) 695 ubiquitin–proteasome system 155 familial see Familial amyotrophic lateral sclerosis (ALS) management/therapeutic strategies neurotrophin therapy 574 GDNF family ligands (GFLs) 607 Anacusis see Hearing loss Analgesia kainate receptor antagonists 319 mGluR2 299 mGluR3 299 see also Anesthesia Anaplerosis 243, 343–344 Andriezen, William Lloyd, glial metabolism 239 Anesthesia mechanisms of action GABAA receptors 353 Angiotensin II (Ang II) parasympathetic neuron modulation 560 receptors see Angiotensin receptor(s) Angiotensin receptor(s) AT1 internalization 558 sympathoeffector junction modulation 561 Aniracetam 266 Annelids gap junctions 208 Anterior latissimus dorsi, chicken 145 Anterograde axonal transport AMPA receptor 268 Antianxiety agents see Anxiolytic agents Antiapoptotic agents nitric oxide 689 Anticholinesterases see Acetylcholinesterase (AChE) inhibitors Anticonvulsants see Antiepileptic drugs (AEDs) Antidiuretic hormone (ADH) see Vasopressin Antiepileptic drugs (AEDs) peptidergic interneurons and 568 Anti-opiate activity opioid receptors and 536 Antipsychotic drugs (neuroleptics) 392–409 dopamine receptor occupancy/binding antagonism studies 392 historical aspects 392 mGluR2/3 agonists 299 in vivo experiments 392 Antisense RNA neuropeptide storage 517–518 neuropeptide Y (NPY) 539 Anuran(s) skeletal muscle innervation 143, 144f see also Frog(s); Toad(s) Anxiety adenosine receptors 637t glutamate 237 pathological see Anxiety disorders as stress response CRH and 547 CRH receptors and 547 see also Fear; see also Stress
Subject Index Anxiety disorders neurochemistry GABAergic mechanisms 365 glutamatergic mechanisms 297–298, 300 treatment 352 pharmacotherapy see Anxiolytic agents see also Fear Anxiolytic agents LY35470 299 mGluR2/3 agonists 299 AP-1 see Activator protein-1 (AP-1) AP2 adaptor complex 89–90 AMPA receptor cycling 99 AMPA receptor-dependent LTP 329 endocytosis and SV cycle 85t synaptic vesicle biogenesis 77–79 mGluR2-dependent long-term depression 330 recruitment to CCVs 89 AP180, endocytosis and SV cycle 85t Apo-E–cholesterol, glial-mediated synaptogenesis 13, 47 Apomorphine-R-(-), dopamine receptor kinetics 394t Apoptosis neurotrophin effects 572 p75NT receptor 570–572 Apoptosis-inducing factor (AIF) nitric oxide, neurotoxic effects 692–693, 693f Appetitive behavior motivational learning/performance endocannabinoids and 676 see also Addiction Arachidonic acid (AA) endocannabinoid derivatives 664, 665f, 677 N-Arachidonoyl dopamine 664, 665f 2-Arachidonoylethanolamine (2-AG) 664, 677 degradation 665–668 long-lasting self inhibition 674, 674f receptor see Cannabinoid receptor(s) structure 665f synthesis 665–668, 668f N-Arachidonoylethanolamine (AEA) 664, 677 degradation 665–668 receptor see Cannabinoid receptor(s) structure 665f synthesis 665–668, 667f 2-Arachidonoyl glycerol ether 664, 665f ARC-239, adrenergic receptors 427 Arecoline, muscarinic receptors 500–501 Arginine vasopressin (AVP) see Vasopressin Aripiprazole 409 Arousal 555 CRH receptors and 547 melanin-concentrating hormone and 555 rodent models 555 orexins (hypocretins) and 555 rodent models 555 rodent models melanin-concentrating hormone 555 orexins (hypocretins) 555 see also Waking state Arrestin(s) b-arrestins mGluR signaling 302 NPY receptor internalization 542 signal transduction adrenergic receptors 427–428 Artemin (ARTN) 599 GFRa coreceptors, structure 601 knockout mice 605–606, 605t structure 599, 600f sympathetic neurons 606 Arterial baroreceptor reflex see Baroreflex ASC-1 (amino acid transporter), D-serine clearance 336, 337 Ascending reticular activating system (ARAS) ‘cholinergic hypothesis of geriatric memory dysfunction’ 490–491 Ascidians connexins 211 Aspartate neuropeptide coexistence 528 Association neurons see Interneuron(s) Associativity definition 323 NMDA receptor-dependent LTP 323
Asthma b-adrenergic receptor development 427 Astrocyte(s) 239 calcium waves see Astrocytic calcium signaling communication see Astrocytes, communication and signaling development differentiation CNTF 619 FGF-1 619 FGF-2 619 glial fibrillary acidic protein (GFAP) 619 growth factors 619 leukemia inhibitory factor receptor 619 PDGF 619 region-specific 619 ischemic stroke see Ischemic stroke (cerebral ischemia) excitation 120 calcium oscillations 120 responses 120, 121f spontaneous 120 see also Astrocytes, communication and signaling functional roles 618 gap junctions see Astrocytic gap junctions GFAP see Glial fibrillary acidic protein (GFAP) glycine concentrations 334 growth factors 618, 622t axonal regeneration 623 CNTF 618 fibroblast growth factor-2 618 insulin-like growth factor 1 618 leukemia inhibitory factor 618 NGF 618 NT-3 618 NT-4 618 TGF-a 618 TGF-b 618 metabolism see Astrocytes, metabolism/metabolic functions morphology/structure microdomains 336 neuromodulation and 112, 114 number of synaptic contacts/cell 112–113 see also Glial cells, neurotransmission modulation neurotransmitter regulation SNAP-23 121–123 synaptotagmin IV 121–123 see also Glial cells, synaptic functions proliferation 643–644 see also Glioma(s) receptor expression see Astrocytes, communication and signaling signaling see Astrocytes, communication and signaling synaptic functions of see Glial cells, synaptic functions transmitter release see Astrocytes, communication and signaling Astrocyte–neuron metabolic unit 243 Astrocytes, communication and signaling calcium signaling see Astrocytic calcium signaling cannabinoid receptor expression 668–669 gap junctions see Astrocytic gap junctions gliotransmitters/neuromodulators 112, 120–125 ATP release 49, 115, 116 D-serine release 49, 115, 116 exocytotic regulation 120, 123, 335 bafilomycin A1 120–121 clostridial toxins 120–121 dense core secretory granules 123 kiss-and-run 121–123 a-latrotoxin 120–121 properties 121–123 synaptic-like microvesicle compartments 121 glutamate 49, 112, 115, 116f nonexocytotic release 123 gap-junction hemichannels 123–124 plasma membrane transporters 124–125 purinergic P2X7 receptors 123–124 volume-regulated anion channels 123–124 D-serine, NMDA receptor activation 335 transmitters associated 120 see also Astrocytic calcium signaling receptor expression glutamate receptors mGluRs 299 see also Glial cells, neurotransmission modulation
701
702 Subject Index Astrocytes, metabolism/metabolic functions aerobic glycolysis 239 GABA metabolism 340 glutamine transporter expression 342 transporter inhibition 342 gap junctions and see Astrocytic gap junctions glutamate and 242 recycling 239 glycogen storage 241 Astrocytic calcium signaling 112 calcium waves see Astrocytic calcium waves GABAB-receptor mediated 116–117 gap junctions and 116–117 intercellular signaling see Astrocytic calcium waves see also Astrocytic gap junctions Astrocytic calcium waves 112–113 gap junctions 116–117, 191 see also Astrocytic gap junctions Astrocytic gap junctions 192–192, 213, 215–216, 215f calcium signaling and 116–117 Ca2þ waves (intercellular) 216–217 see also Astrocytic calcium waves see also Astrocytic calcium signaling connexins 215–216, 216t connexons (hemichannels) 215f coupling 216 functional roles 216, 217f metabolic trafficking 191–192 organization 189 permeability 191, 192 connexin composition and 189–190 Astrocytoma(s) 621 growth factor receptors 621 Astroglia see Astrocyte(s) Asynchronous neurotransmitter release 164 AT1 see Angiotensin receptor(s) AT2 see Angiotensin receptor(s) ATP see Adenosine triphosphate (ATP) ATP-binding cassettes, nonexocytotic gliotransmitter release 124–125 ATP-dependent vesicular membrane transporter 2 (VMAT-2) 80 Atrial natriuretic factor (peptide) see Atrial natriuretic peptide (ANP) Atrial natriuretic peptide (ANP) receptors 561 A-type Kþ currents (IA) striatum 411 Auditory cortex anatomy/organization tonotopic activity-dependent synaptic competition and 33 see also Tonotopic organization Auditory system(s) central pathways see Central auditory pathways development activity-dependent synaptic competition 32 central pathways see Central auditory pathways dendritic arbor formation 29 peripheral anatomy/physiology 32–33 tonotopic organization 32–33 synaptic competition/pruning and 32–33 Auerbach’s plexus see Myenteric plexus Autapses 13–14 Autism/autistic spectrum disorder (ASD) genetics PSD mutations and 101–102 neural substrates/neurobiology kainate receptors 320 Autoinhibition neuropeptide release 519–521 serotonergic neuronal pathways 472 Autonomic ganglia activity-dependent synaptic competition 34 parasympathetic see Parasympathetic nervous system preganglionic innervation 34 sympathetic see Sympathetic nervous system Autonomic nervous system (ANS) components/organization 415 ganglia see Autonomic ganglia ganglia see Autonomic ganglia neurochemistry see Autonomic nervous system, neurochemistry Autonomic nervous system, neurochemistry acetylcholine see Cholinergic neurons/systems GABAB receptors 361–362
NANC system see Non-adrenergic noncholinergic (NANC) neurons neuropeptides see Neuropeptide(s) norepinephrine see Norepinephrine/noradrenergic transmission peptidergic receptors 559 serotonin 450 Autoreceptors definition 388–389 dopamine 388 GABAB receptors neurotransmitter release regulation 368 paired-pulse depression 368–369, 370f NMDA receptor-dependent LTP 369 Autoregulation oxytocin 524 vasopressin 524 Auxilin, endocytosis and SV cycle 85t Avian pancreatic polypeptide (APP), retina 528 AVP see Vasopressin Axo-axonal synapses pyramidal cell output control 355 Axonal arbor(s) definition 27 filopodia, synaptic precursor function 5 Axonal guidance cues axonal regeneration see Axonal regeneration netrin-1 621 Axonal injury/degeneration regeneration see Axonal regeneration retrograde neurotrophic signaling defects 588–589 see also Neurodegeneration/neurodegenerative disease Axonal regeneration glial cell-derived growth factors 622–623 astrocytic 623 Axon branching motor neurons 150 Axosomes, NMJ synapse elimination 153 Axosome shedding 48–49
B B1-B9 midline nuclei, serotonergic neuronal pathways 470 Baclofen 371–372 alcohol consumption 365 cocaine addiction 365 GABAB receptor agonists 363, 363f Bafilomycin astrocyte neurotransmitter release 120–121 vesicular neurotransmitter transporter inhibition 253–254 Bam 22, opioid peptides 532 b-amyloid see Amyloid beta (Ab) Baroreflex 5HT-7 receptors 452 Barrel(s) see Barrel cortex Barrel cortex ablation, effects 32 development ablation effects 32 activity-dependent plasticity synaptic competition 32 dendritic spine formation 3–4 signals 32 Basal forebrain 490 anatomy/connectivity 490 cholinergic neurons see Basal forebrain cholinergic neurons (BFCNs) nucleus basalis see Nucleus basalis magnocellularis (of Meynert) (NBM) functions 490 memory role 490–491 trophic factor dependency 491 Basal forebrain cholinergic neurons (BFCNs) retrograde neurotrophic transport signaling defects 588 Basal ganglia, motor functions mGluR5 and 298 Basal lamina components 174 NMJ postsynaptic basal lamina see Neuromuscular junction, basal lamina ‘Basalocortical cholinergic pathway’ 489, 490f Basal optic nucleus of Meynert see Nucleus basalis magnocellularis (of Meynert) (NBM) Basket cells GABAA receptor subtypes 351, 351f, 355–356
Subject Index Bassoon 16 CAZ role 56t, 57, 92t, 94 see also Piccolo BDNF see Brain-derived neurotrophic factor (BDNF) Behavioral flexibility, norepinephrine effects 440 Bendorphin, opioid peptides 532 Benzamides disadvantages 447–448 dopamine D2 receptor neuroimaging 446–447 Benzodiazepines effects 352 mechanism of action GABAA receptor binding 352, 356 sensitivity 352 Bergmann glia glial–synapse relationship 50 growth factors 619, 622t IGF-1 619 Beta-amyloid see Amyloid beta (Ab) Beta-blockers norepinephrine effects 429, 429t Biarylpropylsulfonamides 266 Bicarbonate GABAA receptors and 347 depolarization 357–358 Binomial statistics, transmitter release 163, 164f factors underlying probability (p) 164 number of release sites (n) and countable AZs 163 Biogenic amines dopamine see Dopamine neuropeptide coexistence 528 norepinephrine see Norepinephrine/noradrenergic transmission serotonin see Serotonin uptake 420 Biologically active peptides, neuropeptides see Neuropeptide(s) Bipolar cells (local circuits) see Interneuron(s) Bipolar cells (retina) ON cells mGluR6 300–301 retinal ganglion cell connections see Retinal ganglion cells (RGCs) Bird(s) motor systems neuromuscular junction 146 skeletal muscle innervation 145 Bladder contractility human control 643 BMPs see Bone morphogenetic proteins (BMPs) Boltzmann relation, gap junctions, voltage gating 200 Bone morphogenetic proteins (BMPs) retrograde homeostatic modulation and 131 Bony fish(es), skeletal muscle 143 innervation 143, 144f primary motor neurons 143 secondary motor neurons 143 slow fibers 143 Boutons see Synaptic bouton(s) Bradykinin receptors B2 receptors, sympathoeffector junction modulation 561 Brain(s) development see Brain development electroencephalography see Electroencephalography (EEG) fast excitatory signaling 245 functional reorganization see Brain plasticity/reorganization gonadal steroids and see Gonadal hormone(s) plasticity see Brain plasticity/reorganization receptors see Receptor(s) white matter see White matter (WM) Brain-derived nerve growth factor see Brain-derived neurotrophic factor (BDNF) Brain-derived neurotrophic factor (BDNF) 590 activity-dependent controls 592 processing 592 secretion 594, 595 trafficking 592 transcription 592 developmental role cell differentiation/morphology 573 synapse elimination at NMJ 154–155 synapse formation role 13 disease/dysfunction and 622 depression 598
drug addiction 598 electrophysiology 568–569 filopodial motility regulation 7 functional roles 590, 595–596 gene expression 590, 592 calcium dependence 592 dendritic translation 595 genetically modified mice studies 595 sorting motif 594 gene structure 592 exons 595 promoter III 592 glial cell production 622t Mu¨ller glial cells 619 oligodendrocytes 618 historical aspects 570, 590 neural regeneration and peripheral nerve regeneration 623 neuropeptide coexistence 526f, 528 polymorphism SNPs 594, 597–598, 597f proBDNF vs. mBDNF 590, 591f receptor binding p75 receptors see p75 receptor(s) secretory pathway 592–594, 593f signal transduction 590, 591f p75NTR binding 590–592, 596–597 sortilin 590–592, 594 synaptic function 573 synaptic plasticity role 594 cognitive function 597 input specificity 595 learning 597 LTD 596 LTP and early-phase LTP 593f, 595 late-phase LTP 593f, 596 memory 597 TrkB and 590, 595 therapeutic use 574t Brain development cerebral cortex see Cortical development forebrain see Forebrain GABA role 340 midbrain see Midbrain myelination and see Myelination NMDA receptors see NMDA receptors, developmental role Brain stem anatomy/physiology cholinergic system 490 midbrain see Midbrain serotonergic neurons 450 Branched-chain amino acids (BCAA), GABA metabolism 343–344 Branched-chain aminotransferase (BCAT), GABA metabolism 343–344 Brimonidine 427 BRL44408, adrenergic receptors 427 Bruchpilot see ELKS/CAST/ERC proteins Bulk membrane invagination, synaptic vesicles at NMJ 165 Burst firing neuropeptide release 519–521, 520f oxytocin neurons 524 Butyrylcholinesterase (BChE) 481
C C1 neurons, innervation targets 435 CA1 region see Hippocampus Ca2þ ions see Calcium ions (Ca2þ)/calcium signaling CA2 region see Hippocampus CA3 region see Hippocampus Cable properties/cable theory muscle fiber properties 182, 183f membrane capacitance 184 spatial factors 183 M-Cadherin(s), NMJ basal lamina 178 N-Cadherin(s) 38, 40t axon growth/guidance filopodia stabilization 9 NMJ basal lamina 178 synaptic stabilization 16
703
704 Subject Index Cadherin(s) 38 actin cytoskeleton interaction 38–40 biological functions 38–40 catenin interactions 40 classification 38 intracellular signaling pathways 38–40 long-term potentiation and 38 NMJ basal lamina 178 catenin interactions 178 presynaptic development 16 structure 38–40, 39f catenin-binding 38–40 see also Protocadherin(s) Cadherin-11 40t Cadherin-like domains, RET 600–601 Caenorhabditis elegans development presynaptic development 127–128 Cajal see Ramo´n y Cajal, Santiago Calbindin, dopamine 389 Calcineurin plasticity role long-term depression NMDA receptor-dependent 328 Calcitonin gene-related peptide (CGRP) autonomic nervous system parasympathetic neuron modulation 560 neurotransmitter coexistence acetylcholine 528 GABA 528 storage 525–526 Calcium (Ca2þ) see Calcium ions (Ca2þ)/calcium signaling Calcium-activated Kþ channels (KCa) norepinephrine-induced blockade 439 Calcium and neurotransmitter release, SNAREs sensitivity 70f, 73 Calcium/calmodulin-dependent kinase II (CaMKII) AMPA receptor activation 264 CaMKII-b isoform filopodial motility 7–9 functional roles filopodial motility regulation 7–9 PSD protein 96–97, 99–100 NMDA receptor interactions 96–97, 281 serotonergic neuronal pathways 472 Calcium/calmodulin-dependent serine protein kinase (CASK) neurexin binding 14 presynaptic development role 14–15 synaptic vesicle tethering and 15 RIM1 interactions 16 Calcium channel(s) GABAB receptor, conductance 360 synaptic vesicles 79 voltage-gated see Voltage-gated calcium channels (VGCCs) see also Calcium currents Calcium currents R-type 61–62 see also Voltage-gated calcium channels (VGCCs) Calcium-dependent activator protein for secretion (CAPS), neuropeptide release 520f, 521 Calcium-dependent neuropeptide release 519 Calcium-dependent NMDA receptors 280, 280f Calcium-dependent synaptic transmission see Calcium ions and neurotransmitter release Calcium/diacylglycerol-dependent protein kinase C, group I mGluRs 305 Calcium ions (Ca2þ)/calcium signaling axonal growth/guidance filopodial motility regulation 7 Ca2þ-activated Kþ channels and see Calcium-activated Kþ channels (KCa) cytosolic levels 59 GABAB receptors and 362 gap junctional communication astrocytic calcium waves 116–117, 191 junction permeability 191 glial cells astrocyte excitation 120, 335 astrocyte signaling see Astrocytic calcium signaling intracellular mGluR5 signaling 295f, 297 neuronal death and see Neurodegeneration/neurodegenerative disease noradrenergic intermittence mechanisms 419, 420f neurotransmission see Calcium ions and neurotransmitter release
nitric oxide (NO) and 684 NMDA receptors 277–278, 279–280 long-term depression and 328, 328f SNAREs sensitivity 70f, 73 synaptic plasticity role see Calcium ions and synaptic plasticity synaptic vesicle cycle see Calcium ions and neurotransmitter release see also Astrocytic calcium signaling Calcium ions and neurotransmitter release 162, 170 classical (Katz) theory and calcium hypothesis 162, 162f asynchronous release and 164 binomial statistics (normal extracellular Ca2þ) 163, 164f factors underlying probability (p) 164 number of release sites (n) and countable AZs 163 Poisson statistics (low extracellular Ca2þ) 162 neuropeptide release 516–517, 519, 565–567 oxytocin 520f, 522–523 synaptic plasticity and see Calcium ions and synaptic plasticity voltage-gated Ca2þ channels and 59–66, 62 calcium entry 62 calcium sensor 63, 63f Power–Law function 62 excitation–response coupling 59, 60f Heuser–Reese cycle 59, 60f Hodgkin cycle 59, 60f steps 59 ‘microdomains’ 62 synaptotagmin role 62 Calcium ions and synaptic plasticity LTD 321, 323–324, 328, 328f NMDA receptor dependent 328, 328f short-term synaptic depression 172 short-term synaptic enhancement 170 voltage component 171 Calcium sensor, synaptotagmin-1 70f, 73 Calcium signaling see Calcium ions (Ca2þ)/calcium signaling Calcium waves (astrocytes) see Astrocytic calcium waves Calretinin 389 Calyx of Held synaptic vesicles vesicle priming 371 vesicle recruitment depression 371 Campenot, Robert, retrograde neurotrophic signaling 587 Campenot chambers retrograde neurotrophic signaling 584 cAMP signaling see Cyclic AMP Cannabidiol 664, 665f Cannabinoid(s) cannabidiol 664, 665f endogenous see Endocannabinoid system group I mGluR retrograde signaling 304 history 664 memory effects 675 neurotransmission suppression 669 nonpsychoactive 664 receptors see Cannabinoid receptor(s) tetrahydrocannabinol 664 derivatives 664 see also Cannabis Cannabinoid receptor(s) 664, 677 CB1 receptors 664 cerebellar LTD and 680–681 distribution 668–669, 677 brain 669 CCK-containing GABAergic interneurons 669, 670f glia 668–669 knockout mice age-related hippocampal degeneration 675–676 fear extinction 675 object/social recognition 675–676 spatial memory 675 signal transduction 664, 666f cAMP pathway 664–665 sympathoeffector junction modulation 561 distribution 668 GPR55 as 668 Cannabis 664 see also Cannabinoid(s); Cannabinoid receptor(s); Endocannabinoid system Capacitance, cell membrane see Membrane capacitance Carbenoxolone gap junction blockade 205 Carbon fibers, voltammetry electrode design 442, 443f
Subject Index Carbonyl cyanide m-chlorophenylhydrazone (CCCP), vesicular neurotransmitter transporter inhibition 253–254 Carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), vesicular neurotransmitter transporter inhibition 253–254 Carboxypeptidase (CPE) BDNF sorting 594 metal ion requirement 513 missense mutations 514 type 2 diabetes mellitus 514 neuropeptide synthesis 513 Cardiac arrhythmias adenosine receptors 637t Cardiac muscle 141 Cardiovascular control central, serotonin role 451, 452f Cardiovascular disease neuropeptide Y 539 Cardiovascular reflexes see Cardiovascular control Cardiovascular system control see Cardiovascular control CRH receptors 548 disease see Cardiovascular disease CART see Cocaine- and amphetamine-regulated transcript peptide (CART) Cartilaginous fish(es) skeletal muscle aberrant fibers 143 innervation 143, 144f red fibers 143 white fibers 143 CASK see Calcium/calmodulin-dependent serine protein kinase (CASK) CAST (cytomatrix of the active zone-related structural protein) see ELKS/CAST/ERC proteins Cat(s) ATP mode of action studies 639 Cataplexy 555 with narcolepsy see Narcolepsy Catecholamine(s) metabolism chlorpromazine metabolite 392–393 Catechol-O-methyltransferase (COMT) catecholamine metabolism 421–422 dopamine 386 isoforms 386–387 a-Catenin filopodial stabilization 9 Catenin(s) cadherin activation 40 NMJ 178 Caudoputamen complex (CPu) dopamine D2 receptor 400, 400f CaVs see Voltage-gated calcium channels (VGCCs) CAZ see Cytoskeleton of the active zone (CAZ) Cdc42 activation dendritic filopodial motility 6–7 PSD proteins 100 ‘Ceiling effect’, dopaminergic neuroimaging 447 Cell adhesion molecules (CAMs) filopodial stabilization 9 NMDA receptor trafficking 287 synapses 11, 12f, 38–45, 39f ECM-mediated interactions 38, 39f, 40t, 44 CNS 44 integrins 44–45 neuromuscular junction 44 immunoglobulin superfamily 38, 42 PSD 99, 100 synaptogenesis 13, 15f postsynaptic development 100 Cell–cell junctions nonsynaptic 11 synapses see Synapse(s) Cell contact, glial-mediated synaptogenesis 47 Cell culture synapse formation in 13–14 Cell membrane capacitance see Membrane capacitance lipids synaptic vesicle cycle regulation 94 membrane deformation and 94
Central auditory pathways 32–33 calyx of Held see Calyx of Held cortex see Auditory cortex thalamus 32–33 Central nervous system (CNS) brain see Brain(s) ECM-mediated interactions 44 gap junctions 213 neurotransmission calcium channel types 62 see also Neurotransmission neurotrophins and 573 see also Neurotrophin(s) nitric oxide role 688 see also Nitric oxide (NO) receptors/ion channels calcium channel types 62 regeneration/repair glial cell growth factors 622 see also Neural regeneration/repair synapses see Synapse(s) tachykinin receptors see Neurokinin (NK) receptors tachykinins see Tachykinin(s) Cerebellar ataxia glutamate 238 Cerebellar plasticity long-term depression (LTD) 678 LTP and 681–682 mechanisms 679–680 AMPA receptor-mediated 680 endocannabinoids and 679f, 680–681 NO and 680 postsynaptic 680 see also Endocannabinoid system and synaptic plasticity occurrence 678–679 theoretical models of cerebellar function 678–679 long-term potentiation (LTP) LTD reversal 681–682 Cerebellar radial fibers see Bergmann glia Cerebellum anatomy/physiology afferent systems 33–34 activity-dependent synaptic competition 33 Bergmann glia and 50 climbing fibers 33–34, 50 mossy fibers see Mossy fiber(s) topographic maps 33–34 astrocyte–synapse relationship and 50 cortex GABAB receptors 371 Purkinje cells see Purkinje cell(s) development activity-dependent synaptic competition 33 norepinephrine distribution 415 plasticity see Cerebellar plasticity Cerebral blood flow (CBF) hypoperfusion see Ischemic stroke (cerebral ischemia) nitric oxide regulation 690 Cerebral cortex, anatomy/organization neurons see Cortical neuron(s) norepinephrine distribution 415 pyramidal cells (projection neurons) see Pyramidal neuron(s) Cerebral infarction see Stroke Cerebral ischemia see Ischemic stroke (cerebral ischemia) Cerebrovascular accident (CVA) see Stroke cGMP see Cyclic GMP (cGMP) cGMP-dependent protein kinases (cGKs) 687–688 CGP 35348, GABAB receptor antagonists 363, 363f CGP 36742, GABAB receptor antagonists 363, 363f CGP 44532, GABAB receptor agonists 363, 363f CGP 52432, GABAB receptor antagonists 363f CGP 55845, GABAB receptor antagonists 363f CGRP see Calcitonin gene-related peptide (CGRP) Charcot–Marie–Tooth disease type 2 (normal nerve conduction velocity) type 2B, retrograde neurotrophic signaling 589 Chemical induction mGluR-dependent LTD 327 NMDA receptor-dependent LTD 327 Chemical synapse(s) 11 evolution 158 see also Neurotransmitter(s)
705
706 Subject Index Chemoattractants (axon pathfinding) see Axonal guidance cues Chemorepellant signaling (axon pathfinding) see Axonal guidance cues Chicken(s) neuromuscular junction 146 skeletal muscle innervation 145 Chirality D-serine 333, 334f CHL-1 41–42 Chloride channel(s) hyperpolarization 347, 348f synaptic vesicles 80–82 Chloride ions (Cl-) excitatory amino acid transporters 247 GABAA receptor depolarization 357–358 m-Chlorophenylpiperazine (mCPP) 464, 465 Chlorpromazine catecholamine metabolites 392–393 historical aspects 392 mechanism of action 392 mitochondrial enzymes 392 sodium–potassium ATPase 392 Cholecystokinin (CCK) 561, 562f animal knockouts, processing peptidases 514 biological actions 561–562, 562–563 coexistence CRH 517, 528 oxytocin 521–522, 528 in GABAergic interneurons, cannabinoid receptor expression and 669, 670f short-term depression 672 receptors see Cholecystokinin receptors sources 561–562 synthesis posttranslational processing 512 precursor peptides 511 targets 562 Cholecystokinin-8 GABA coexistence 528 Cholecystokinin receptors 562 agonists, disease treatment 562 antagonists disease treatment 562 gastrointestinal expression 562 therapeutic significance 562 localization 562–563 Cholesterol glial-mediated synaptogenesis 13, 47 Choline 478, 486 recycling 478 transport 478 see also Choline transporter (CHT1) uptake 478 Choline acetyltransferase (ChAT) 479 localization 486 refilling synaptic vesicles 165 synthesis 479 Cholinergic basal forebrain see Basal forebrain cholinergic neurons (BFCNs) Cholinergic hypothesis Alzheimer‘s disease 492–493 see also Basal forebrain ‘Cholinergic hypothesis of geriatry memory dysfunction’ 490–491 Cholinergic neurons/systems 486, 490 astrocytic modulation via binding proteins 117–118 basal forebrain see Basal forebrain cholinergic neurons (BFCNs) brain stem 490 cell groups 487–488, 489f cerebral cortex human vs. rodents 488–489 synapses 486 CNS pathways 486–493 endocannabinoid effects suppression and 669–670 historical aspects 486 interneurons location 501 muscarinic receptors 501 learning and memory role 490–491 see also Alzheimer’s disease, neurotransmitter systems; Cognition see also Acetylcholine at motor nerve terminals see Neuromuscular transmission nomenclature 487–488 regulation 480
synapses cerebral cortex 486 CNS 486 presynaptic boutons 486–487 vesicles 486–487, 487f thalamus 490 Cholinergic neurotransmission 478–485, 486 acetylcholine release 480 cotransmission/coexistence 528 CGRP 528 galanin 528 somatostatin 528 extrasynaptic transmission 486–487 historical aspects 486 neuromuscular junction see Neuromuscular transmission physiological responses 484t, 485 receptors see Acetylcholine receptors (AChRs) regulation 480 5-HT6 receptor 468 Cholinergic receptors see Acetylcholine receptors (AChRs) Cholinesterase inhibitors see Acetylcholinesterase (AChE) inhibitors Choline transporter (CHT1) 478, 486 localization 478, 486 synaptic vesicle co-localization 478 Chondrichthyes see Cartilaginous fish(es) CHPG, mGluR5 agonists 297–298 Chromaffin cells large dense-core vesicles (LDCVs) 521 Chromogranin(s) neuropeptide storage 517–518 CIC3 254t Ciliary neurotrophic factor (CNTF) glial cells 622t astrocytes 618 differentiation 619 neurodegenerative disease and 622 neuronal regeneration peripheral nerve 623 Schwann cells 617 Circadian clock(s) see Circadian rhythm(s) Circadian rhythm(s) neural mechanisms BDNF role 592 norepinephrine regulation 440 Circular dichroism, SNAREs 67 Citric acid cycle see Krebs cycle Clathrin endocytosis and SV cycle 85t initial purification 89 neuronal content 89 neuropeptide granule biogenesis 515 Clathrin adaptors 89–90 classic AP-2 89–90 stonin 2 90 Clathrin-coated pits (CCPs) dynamics 90 formation 89 accessory proteins 89–90 adaptor proteins 89–90 maturation 90 stabilization 89–90 Clathrin-coated vesicles (CCVs) AMPA receptor cycling 99 coat formation 89 synaptic vesicle protein content 89 Clathrin-mediated endocytosis (CME) accessory proteins 89–90 adaptor proteins see Clathrin adaptors AMPA receptors 272 long-term depression and 328 see also AMPA receptor trafficking clathrin-coated pit formation 89 clathrin-coated pit maturation 90 NMDA receptors synaptic trafficking 288f PIP2 and phospholipid regulation 89–90, 94 sorting motifs 89–90 synaptic vesicles 89 CAZ role 94 cycling pathway and 84 endocytic zones 84, 90
Subject Index identification of role 89 at the NMJ 165, 166f vesicle-specific adaptors 90 Claustrum, 5HT-2 serotonin receptor 474 Climbing fibers 33–34 activity-dependent synaptic competition 33–34 Clonidine adrenergic receptor activation 427 Clostridial neurotoxins (CNTs) astrocytes, neurotransmitter release 120–121 SNARE protein interactions 82 SNAREs 69–70 see also Tetanus toxin (TeNT) Clozapine D4 receptor affinity 401 CNG knockout models see Cyclic nucleotide-gated (CNG) channels Cnidaria connexins 211 CNQX, kainate receptor antagonists 314, 314t CNS see Central nervous system (CNS) Cocaine addiction baclofen and 365 hypocretin actions 555 Cocaine- and amphetamine-regulated transcript peptide (CART) 553 oxytocin coexistence 521–522 Cochlea ATP neurotransmission 646 Cognition GABAB receptor ligands and 365 gap junctions and neuronal oscillations 223 nicotine effects see Nicotine Cognitive performance locus coeruleus impulse activity 433 Colchicine, retrograde neurotrophic signaling 588 Collagen(s) type IV binding partners 175 a chains 174–175 NC1 domain 174–175 trimer formation 175 genes 174–175 meshwork formation 175, 175f MMP cleavage 175, 179 NMJ basal lamina 174 type XVIII, NMJ basal lamina 177 Collagen-tailed acetylcholinesterase (ColQ-AChE) 177 neuromuscular junction 177 Collateral branching see Axon branching Colon ATP release 644 ColQ acetylcholinesterase see Collagen-tailed acetylcholinesterase (ColQ-AChE) Compensatory endocytosis 165 synaptic vesicles at the NMJ 165, 166f, 167f methods for study 165 types 165 Competency model, retinal development see Retinal development Complexin(s) SNARE interaction 70f, 73–74, 73f COMT gene/protein see Catechol-O-methyltransferase (COMT) Confocal microscopy noradrenergic intermittence mechanisms 419 Congenital central hypoventilation syndrome, RET mutations 599–600 Congenital megacolon see Hirschsprung disease Congenital myasthenic syndrome (CMS) 506 Connexin(s) 193, 219 communication role 189 functional consequences 191 between glial cells 189 between neurons 189 structure and 193 compatibility 194 connexon (hemichannel) role 191, 194, 196f, 211 assembly 203 clustering 204 docking 198f, 203 function 189 transport 203, 204f expression/distribution 189, 194, 195 gap junctional communication pannexin convergence 211
707
gap junction formation 213 genetic manipulation research 218 knockout mice studies 210–211 molecular mass 213 nomenclature 193 permeability 190 protein interactions 189, 190 selectivity 190 gating 190 ionic 190 size-based 190 structure 189 communication and 193 subtypes 213 vertebrate 211 mammalian 195 Connexin 26 (Cx26) mutational effects 210 Connexin 30 (Cx30) 189–190 mutational effects 210 Connexin 31 (Cx31) mutational effects 210 Connexin 32 (Cx32) 190 adenosine permeability 213 mutational effects 210 Connexin 36 (Cx36) 189, 193 blockers 220–221 conductance 201 epilepsy 224 expression 222 knockout mice 223, 224 Connexin 37 (Cx37), conductance 201 Connexin 43 (Cx43) 189–190 adenosine permeability 213 astrocytes 213 gliotransmitter release 123–124 oculodentodigital dysplasia 218 patch clamp recordings 191 Connexin 45 (Cx45) 190 Connexin 46 (Cx46) expression 211 Schwann cells 216 Connexin 47 (Cx47), oligodendrocytes 213 Connexin-mimetic peptides, gap junction blockade 205 Connexon (hemichannel) 191, 194, 213, 215 assembly 203 clustering 204 composition 219 proteins see Connexin(s) docking 198f, 203, 213 factors influencing gating 215 formation 194f, 196, 198f, 203, 205f function 189, 216, 217f, 218 glia nonexcytotic gliotransmitter release 123–124 opening probability 204f, 205f, 211 structure 213, 214f transport 203, 204f voltage sensitivity 202 Constant potential amperometry see Voltammetry Cooperativity definition 323 NMDA receptor-dependent LTP 323 Coronary vessels, 5HT-3 serotonin receptors 451–452 Cortical barrels see Barrel cortex Cortical chandelier, pyramidal cell output control 355 Cortical cup perfusion, microdialysis 445 Cortical development endocannabinoid LTD and 682 Cortical neuron(s) cholinergic neurons see Cholinergic neurons/systems coupling 209 postsynaptic differentiation, receptor transport 20–21 pyramidal cells (projection neurons) see Pyramidal neuron(s) Corticotropin see Adrenocorticotropic hormone (ACTH) Corticotropin-releasing factor (CRF) see Corticotropin-releasing hormone (CRH) Corticotropin-releasing hormone (CRH) 544–550 coexisting peptides/transmitters cholecystokinin 517, 528 oxytocin 528 vasopressin 521–522, 527
708 Subject Index Corticotropin-releasing hormone (CRH) (continued) depression and 547 digestion/gastrointestinal tract 547 gastrointestinal motor function 548 direct injection studies 547–548 cardiovascular effects 548 discovery 544 energy balance and feeding regulation 547 fear/anxiety neurochemistry 547 metabolism 547 pregnancy and 549 Fas ligand expression 549 immunosuppressive effects 549 placental expression 549 receptors see Corticotropin-releasing hormone receptors (CRHRs) stress response network 544 anxiety 547 arousal 547 rodent models 547 see also Hypothalamic–pituitary–adrenal (HPA) axis; Stress structure 544, 545f synthesis/release 547 by PVN 544 Corticotropin-releasing hormone binding protein (CRH-BP) 546 affinity 547 gene 546–547 structure 546–547 Corticotropin-releasing hormone receptors (CRHRs) 544 multiple transcripts 545 signaling 546 structure 544–545, 546f two-step binding model 546 type 1 544–545 agonists 547–548 anxiogenic effects 547 depression 547 feeding 547–548 structure 546f type 2 544–545 cardiovascular effects 548 CRHR2a 545–546 CRHR2b 545–546 CRHR2g 545–546 distribution 545–546 feeding 547–548 knockout mice 548 soluble 546 structure 546f Cortistatin somatostatin receptor-binding 557–558 Cotransmission purinergic 639 Coupling coefficients, gap junctions 198–199 CP55940 664, 665f CPG15 40t Cross-talk 114–115 excitatory amino acid transporters 248, 249f, 250 limitation 250 spillover and synaptic cross-talk 114–115 see also Synaptic spillover Cx32 gene, Schwann cells 216 Cxs see Connexin(s) Cyclic adenosine monophosphate (cAMP) see Cyclic AMP (cAMP) Cyclic AMP (cAMP) CB1 receptor signaling 664–665 mGluR signaling group I mGluRs 304 group III mGluRs 306 mGluR1 292 mGluR5 292 Cyclic AMP-dependent protein kinase A see Protein kinase A (PKA) Cyclic AMP-GEFII/Epac2 CAZ role in clathrin-mediated endocytosis and 94 Cyclic GMP (cGMP) synaptic plasticity role 689 Cyclic nucleotide-gated (CNG) channels nitric oxide 688 Cyclothiazide 266 Cysteine–glutamate exchanger mGluRs 303 nonexocytotic gliotransmitter release 124–125
Cytokine(s) glial cells neurotransmission 49, 113 Schwann cells 617 receptors NMDA receptor effects 282–283 Cytomatrix assembled at the active zone (CAZ) see Cytoskeleton of the active zone (CAZ) Cytomatrix of the active zone-related structural protein (CAST) see ELKS/CAST/ERC proteins Cytoplasmic macromolecules neuropeptide granule biogenesis 516 Cytoskeleton actin see Actin/actin filaments microtubules see Microtubule(s) PSD and 100 Cytoskeleton of the active zone (CAZ) 90 endocytosis role 94 molecular organization 91, 91f, 92t Bassooon/Piccolo 94 ELKS/CAST/ERC proteins 91, 94 Muncs 91–94 RIMs see RIMs neurotransmitter release and 90–91 protein transport 77
D DAG see Diacylglycerol (DAG) Dagga see Cannabis Dale‘s Principle 639 serotonin 450 Damage minimization, microdialysis 445–446 Dasm 40t Dasm1 42 DAT see Dopamine transporter (DAT) Deafness see Hearing loss Degenerative diseases see Neurodegeneration/neurodegenerative disease Delta opioid receptor see Opioid receptor(s) Dendrites, translation in 101 Dendrites/dendritic arbor definition 27 development 27–30 branching capacity 28–29, 29f lifespan 28, 28f synaptic input function 29, 30f see also Dendritic spines; Synaptogenesis filopodia see Filopodia ion channels glutamate receptor blockade effects 29 Dendritic shafts, peptidergic interneurons 567–568 Dendritic spines development/morphogenesis origins 3 see also Rho GTPase(s); Synaptogenesis filopodia see Filopodia glutamate regulation see Dendritic spines, glutamate regulation history see Dendritic spines, history morphology/structure 3 Eph receptors see also Ephrins/Eph receptors filopodia see Filopodia peptidergic interneurons 567–568 remodeling/plasticity AMPA receptor translation and plasticity 101 synapses formation filopodia 11–12 synaptic plasticity and AMPA receptors and 101 Dendritic spines, glutamate regulation glutamate receptor expression AMPA receptors 268–270 LTP and 101 Dendritic spines, history 3 Dendrodendritic gap junctions 207 Denervation neuromuscular junction and partial denervation at birth and synapse elimination 151
Subject Index Dense core secretory granules (DSGs) 80 astrocytic exocytosis 123 Dense projections, species variations 54 Depolarization-induced suppression of excitation (DSE), endocannabinoids and 670–671, 672f Depolarization-induced suppression of inhibition (DSI), endocannabinoids and 670–671, 672f Depolarizing bipolar cells (DBCs) see Bipolar cells (retina) Depotentiation (DP) metaplasticity 325–326 NMDA receptor activation 322 see also Long-term depression (LTD) Deprenyl, GADH/Siah death cascade prevention 694 Depression see Depressive illness Depressive illness BDNF regulation 598 management agomelatine 465–466 neural basis see Depressive illness, neural basis pathogenesis/pathophysiology adenosine receptors 637t GABAB receptors 365 glutamate 237 5-HT1A receptors PRX-00023 457–459 VPI-013 457–459 5-HT2C receptor 465–466 5-HT7 receptor 468–469 see also Depressive illness, neural basis Depressive illness, neural basis neuropeptides 523–524 Desensitization kainate receptors see Kainate receptor(s) neuropeptide receptors 567 NMDA receptors 280 Diabetes mellitus neuropathy see Diabetic neuropathy type 2 (T2DM) b3-adrenergic receptor mutation 428 carboxypeptidase missense mutations 514 Diabetic neuropathy neurotrophins 582 Diabetic retinopathy 646 Diacylglycerol (DAG) synaptic plasticity and behavior role presynaptic Ga signaling 136 Dialysis membrane, microdialysis 444 Dialytrode, microdialysis 445 Diazepam GABAA receptors 352 Diencephalon dopamine 383, 385 hypothalamus see Hypothalamus thalamus see Thalamus Differential splicing see Alternative splicing 4,40 -Diisothiocyanostilbene-2,20 -disulfonic acid (DIDS), vesicular glutamate transporter inhibition 258 Dileucine motif, synaptic vesicle biogenesis 77–79 Dimerization mGluR activation 293–294 neuropeptide Y receptors 542 N,N-Dimethyltryptamine (DMT) structure 471f serotonin vs. 470 DIMM gene/protein neuropeptide storage 518 Dipeptidyl-peptidase IV neuropeptide Y synthesis 538 (+)-7-OH-Dipropylaminotetralin, dopamine receptor kinetics 394t Discharge frequencies, neuropeptides 567 Diurnal rhythm(s) see Circadian rhythm(s) Dizocilpine, NMDA receptors 237 DJ-1 (PARK7) parkin interaction 694 Dliprin-a see Liprin-a DNQX, kainate receptor antagonists 314, 314t Dogfish skeletal muscle innervation 143, 144f Domestic cat (Felis catus) see Cat(s) Dopamine (DA) 383–391, 410–413 anatomy see Dopaminergic neurons/systems dysregulation
neuroimaging see Monoamine neuroimaging parkinsonism see Parkinsonian syndromes/parkinsonism schizophrenia 384, 391 functional significance 383 gap junction regulation 209 life cycle 385, 386f M5 receptor relationship 501 metabolism 386 catechol O-methyltransferase 386 monoamine oxidase 386 motivational system 410 see also Addiction; Dopaminergic neurons, reward role neuromodulatory functions 390 neurotransmission see Dopaminergic neurons/systems pleasure/reward and see Dopaminergic neurons, reward role postsynaptic signaling 389 long-term depression 390 long-term potentiation 390 pyramidal cell dendritic spines 390 somatodendritic release 390 volume transmission 390 receptors see Dopamine receptor(s) regulation 388 autoreceptors 388 cp-localized peptides/proteins 389 extrinsic 389 firing pattern 388 reward and see Dopaminergic neurons, reward role striatal 411 glutamatergic afferents 411 inhibitory role 411 receptor coactivation 411 spiny striatal cells 411–412 see also Nigrostriatal dopaminergic pathway; Striatum synthesis 385, 410 Dopamine b-hydroxylase (DBH) norepinephrine synthesis 414 Dopamine receptor(s) 392–409 agonists see Dopaminergic agonists classes of 395 current research 408 Parkinson’s disease 408 D1 receptors agonists kinetics 393, 394t antagonists, kinetics 393, 395t antipsychotic dose correlation 396f discovery 393 drug abuse 410–411 EPSC 410–411 EPSP 410–411 frontal cortex 410–411 historical aspects 393, 396f medium spiny neurons 411–412 nomenclature 395–398 Parkinson’s disease 408 receptor interactions D2 receptors 405 signal transduction adenylate cyclase activation 410 G-protein-linked 410 structure 396f amino acid sequence 396f D2 receptors addiction role alcoholism 400 agonists/partial agonists kinetics 394t Alzheimer’s disease 400, 402f amino acid sequence 397f antagonists/inverse agonists kinetics 395t see also Antipsychotic drugs (neuroleptics) antipsychotic occupation, parkinsonism and 409 D2High 403 negative cooperativity model 405, 407f, 408 in psychosis 406 psychosis/schizophrenia theory 406, 408f in schizophrenia 406 supersensitivity 405 ternary complex 403–405 D2High to D2Lowregulation 403, 405f, 406f
709
710 Subject Index Dopamine receptor(s) (continued) D2Low 403 discovery 393 antagonist/agonist properties 393–395 function/distribution 399 caudate-putamen 400, 400f gene expression 400, 401f presynaptic terminals 399 historical aspects 393 antipsychotic drug dose correlation 397f butaclamol stereoselection 393 IPSPs 399 neuroimaging benzamide tracers 446–447 low sensitivity 447–448 PET 446–447, 447f radiotracer effects 447–448 SPECT 446–447 nomenclature 395–398 Parkinson’s disease/parkinsonism antipsychotic occupation 409 polymorphisms 397f, 399 psychomotor functions 399 psychosis/schizophrenia 395, 398f, 400, 405 amphetamine sensitivity 406 D2High 406, 408f hippocampal lesions vs. 406–408, 408f methylphenidate sensitivity 406 molecular basis 406 receptor interactions 405 adenosine A2 receptor 405 dopamine D1 receptor 405 signal transduction 410 striatum 411 AMPA-mediated currents 411–412 AMP/protein kinase A cascade 411 A-type Kþ currents 411 GABA-mediated currents 411–412 glutamate-mediated currents 411–412 structure 397f amino acid sequence 397f variants 397f, 399, 399f D3 receptors 400 agonists 400 in development/testing 400 kinetics 394t schizophrenia therapy 400 amino acid sequence 402f antagonists kinetics 395t signal transduction 410 striatum 411 structure 400 D4 receptors 400 agonists kinetics 394t antagonists, kinetics 395t clozapine affinity 401 gene expression 401 polymorphisms 400, 404f dopamine sensitivity 400–401 schizophrenia 401 signal transduction 410 structure 400, 403f D5 receptors agonists kinetics 394t amino acid sequence 404f signal transduction 410 D6 receptors 402 forms 389–390 in health and disease 392–409 historical aspects 392 antipsychotic drug antagonism studies 392 kinetics 394t nomenclature 398, 398f regulation 402 dopamine affinity states 402–403, 405f signal transduction 402–403 G-proteins 410 Dopaminergic agonists dopamine receptor kinetics 394t
Dopaminergic neurons, reward role 410 see also Addiction Dopaminergic neurons/systems 383 A11 neurons 385 A12 neurons 385 A13 neurons 385 A14 neurons 385 diencephalon 383, 385 endocannabioids and, suppression of neurotransmission by 669–670 frontal 410 drug abuse 410–411 electrophysiological data 410–411 long-term depression (LTD) 411 long-term potentiation (LTP) 411 pyramidal cell/GABAergic interneuron connections 411 G-activated potassium current (IRK) 412 GDNF and 605–606 GDNF family ligands (GFLs) and 605–606 main pathways 384f mesencephalon 383 neuroimaging 446 ‘ceiling effect’ 447 compound numbers 447 limitations 447 radioligand effects 447 see also Monoamine neuroimaging neurturin and 605–606 pathways mesocortical 384 mesolimbic system 384 nigrostriatal system 383 pleasure and reward see Dopaminergic neurons, reward role puberty and see Puberty striatal see Striatum ventral mesencephalon 412 Dopamine transporter (DAT) 388 voltammetry 443–444 Dorsal raphe nuclei micturition 454 serotonergic neuronal pathways 470 serotonin 454 Dorsal root ganglia (DRG) neurons NGF retrograde axonal transport kinetics 584 nociceptors peptidergic 525–526 Down’s syndrome (DS) retrograde neurotrophic transport defects 588 Downstream processing, long-term depression (LTD) 328 DPDZ proteins see PDZ protein(s) DRG see Dorsal root ganglia (DRG) Drosophila development dendrite formation 5 synapse elimination, glial cells and 48 synaptogenesis presynaptic development 127 Drosophila melanogaster Bruchpilot see ELKS/CAST/ERC proteins neuropeptides storage 518 synaptic transmission SNAREs 69–70 Drosophila neuromuscular junction neuromuscular receptors postsynaptic ionotropic glutamate retrograde signaling and 127 retrograde signaling 129–130 homeostatic signaling 129–130 synapse development 127 vesicle recycling 165–166 Drosophila neuromuscular junction, larval 127 Drug(s) abuse see Substance abuse addiction see Addiction design/development neuropeptide Y receptors 542 discovery peptidergic receptors 558 serotonergic pathway studies 476 Drugs of addiction/abuse see Substance abuse Duchenne muscular dystrophy (DMD) dystroglycan complex 177–178
Subject Index Dye(s) gap junction coupling studies 220, 221 asymmetry 202 scrape loading 202–203 Dynamin(s) endocytosis and SV cycle 85t clathrin-coated pit dynamics 90 Dynein(s) gephyrin binding 376–377 retrograde neurotrophic signaling 588 Dynorphin animal knockouts 514 coexistence oxytocin 521–522 vasopressin 521–522, 528 synthesis precursor peptides 511 a-Dystrobrevin dystroglycan complex components 177 a-Dystroglycan 177 b-Dystroglycan 177 Dystroglycan complex components 177 Duchenne muscular dystrophy and 177–178 dystrophin/utrophin binding 177–178 NMJ basal lamina 177 Dystrophin dystroglycan complex binding 177–178
E EAATs see Excitatory amino acid transporters (EAATs) Ear(s) P2 receptors 646 Eating disorders Ghrelin 523–524 a-melanocyte-stimulating hormone (a-MSH) 523–524 neuropeptide release 523–524 neuropeptide Y (NPY) 523–524 orexins (hypocretins) 523–524 Ecstasy see MDMA (methylenedioxymethamphetamine: ecstasy) Ecto-nucleoside triphosphate diphosphohydrolase(s) (E0NTPDases), ATP degradation 641 Ectonucleotidase(s) adenosine triphosphate degradation 641 classes 628–629 EEG see Electroencephalography (EEG) Efferent neurons see Motor neuron(s) (MNs) EGF see Epidermal growth factor (EGF) Eicosanoids endocannabinoids as 665 Elderly people see Age/aging Electrical coupling gap junctions 198 cytoplasmic continuity 198 fast network oscillations 223 neurons 191 Electrical signaling action potential see Action potential(s) pyramidal neurons 224 Electrical synapses Inhibitory 193, 209 extrinsic hyperpolarizing potential 209 Electrochemical gradients glutamate transport 246 Electroencephalography (EEG) applications/studies gap junctions epilepsy 224 neuronal oscillations 219 Electron microscopy (EM) active zone 159–160, 160f, 161f peptidergic interneurons 567–568 postsynaptic density analysis 96, 97f Electron tomography, active zone 159–160, 161f Electrophysiology dopaminergic systems 410–411 neuropeptides see Neuropeptides, electrophysiology serotonin receptors 472 5HT-1 472 5HT-2 474
711
5HT-3 475 5HT-4 476 5HT-6 475 5HT-7 476 ELKS active zone 56t, 57 see also ELKS/CAST/ERC proteins ELKS/CAST/ERC proteins CAZ role 91, 92t, 94 Endocannabinoid system biochemistry 665, 671f degradation enzymes 665–668 independent pathways 665–668, 667f, 668f induction of synthesis/release 671 precursors 665–668 redundancy 665–668 biological actions/roles 664, 677 differential effects of individual cannabinoids 665–668 plasticity and 682 retrograde signaling 128, 669, 678 classical neurotransmitters vs. 665–668 developmental role 682 distribution adult brain 669 CCK-containing GABAergic interneurons 669, 670f, 672 differential distribution of components 669 glial cells 668–669 neurons 668–669 endocannabinoids 664, 665f, 677 2-arachidonoylethanolamine see 2-Arachidonoylethanolamine (2-AG) learning and memory 664–676, 682 appetitive learning 676 fear conditioning 675, 681f, 682 Morris water maze, and 675 object /social recognition 675 see also Endocannabinoid system and synaptic plasticity neuromodulatory functions neurotransmission suppression 669, 671f see also Endocannabinoid system and synaptic plasticity reward/addiction role 682 signal transduction 666f, 664, 671f ‘on demand’ synthesis/release 665–668, 672 receptors see Cannabinoid receptor(s) retrograde signaling 669, 678 TRPV1 activation and 665–668 synaptic plasticity and see Endocannabinoid system and synaptic plasticity transporters 665–668 Endocannabinoid system and synaptic plasticity 664–676, 677–683 glutamatergic (depolarization-induced suppression of excitation) 670–671 long-term depression (eLTD) 670, 677 amphetamine-induced 677 cerebellar 678, 679f functional roles 682 cortical development 682 drug abuse and 682 error adaptation system 682 learning/memory role 675 LTP priming 682 general features 677–678 heterosynaptic LTD on inhibitory input 673, 674f, 679f homosynaptic LTD on excitatory input 672f, 673, 679f induction/expression mechanisms 677 AMPA receptors and 678 cerebellar 679f Heterosynaptic 673, 679f homosynaptic 679f mGluRs and 673, 678, 680–681 neuromodulation of 678 sequence of events 677–678 timing-dependent 679f VGCCs and Ca2þ increases 673–674, 678 long-lasting self-inhibition 674, 674f memory role 675 NMDA-dependent (cortex) 678, 679f, 680f NMDA independent mechanism 673, 677–678 occurrence 677 time-dependent LTD 673, 679f short-term depression 670, 672f GABAergic (depolarization-induced suppression of inhibition) 670–671, 672f
712 Subject Index Endocannabinoid system and synaptic plasticity (continued) glutamatergic (depolarization-induced suppression of excitation) 670–671, 672f initiation of synthesis/release 671 memory role 675 transport to presynapse 671–672 Endocrine system hormones 193 PACAP see Pituitary adenylate cyclase activating polypeptide (PACAP) VIP see Vasoactive intestinal peptide (VIP) Endocytosis AMPA receptors see AMPA receptor trafficking clathrin-mediated see Clathrin-mediated endocytosis (CME) dynamins see Dynamin(s) endosomes synaptic vesicles at the NMJ 165 kiss and run see Kiss-and-run fusion membrane deformation and 95 synaptic vesicles see Synaptic vesicle endocytosis Endophilin endocytosis and SV cycle 85t, 95 Endoplasmic reticulum (ER) neuropeptide synthesis 511, 525–526 receptor trafficking kainate receptors 310–311 Endoproteolytic cleavage, neuropeptide synthesis 511 b-Endorphin synthesis precursor peptides 511–512 Endosome(s) endocytosis synaptic vesicles at the NMJ 165 transport 589 Endothelial NOS (eNOS) see Nitric oxide synthase(s) (NOS) Endothelin-1 Alzheimer’s disease 622 Endothelin receptors sympathoeffector junctions, presynaptic modulation 561 Endothelium-derived relaxing factor see Nitric oxide (NO) Endplate current (EPC) AChR role 180 from EPC to EPP 182 Endplate potentials (EPPs) 180, 181f, 182 miniature see Miniature endplate potentials (mEPPs) muscle fiber passive cable properties and 182, 183f Energy expenditure neuropeptide Y and 539 Enhancers mGluR activation 294 Enkephalin(s) animal knockouts 514 coexistence GABA 528 galanin 527 glutamate 528 oxytocin 521–522, 528 retina 528 serotonin 528 eNOS see Nitric oxide synthase(s) (NOS) Entactins (nidogens), basal lamina 176 Enteric nervous system, development neurotrophic factors prenatal development role GDNF 606 GDNF family ligands (GFLs) 606 neurturin 606 Enterochromaffin cells (ECs) serotonin 454 Ependymal cells growth factors 618 Ependymal glia see Ependymal cells Ependymoglia see Ependymal cells Ependymoglial cells see Ependymal cells Ephaptic transmission, definition 193 EphB2 gene/protein NMDA receptor trafficking 287 Ephrins see Ephrins/Eph receptors Ephrins/Eph receptors glial-mediated synaptic stability and 49–50 postsynaptic development role 22
Epidermal growth factor (EGF) Schwann cells 617 Epilepsy EEGs 224 pathophysiology see Epilepsy, pathophysiology treatment group III mGluRs 300 pharmacotherapy see Antiepileptic drugs (AEDs) Epilepsy, pathophysiology adenosine receptors 637t connexins in connexin 36 224 galanin and 523–524 gap junctions 224 glutamate excitotoxicity and 237 ion channels kainate receptors and 319–320 neuropeptide Y 523–524 see also Epilepsy, neurodegeneration Epinephrine autonomic nervous system 425–426 cell groups associated 434 dopamine receptor kinetics and 394t non-locus coeruleus impulse activity 435 non-locus coeruleus projections 435 receptors 427 see also Adrenergic receptors see also Adrenergic (PNMT-containing) neurons Epinephrine-releasing cells, vesicular monoamine transporters (VMATs) 255–256 Eps15 endocytosis and SV cycle 85t Eps15R, endocytosis and SV cycle 85t Epsin(s) endocytosis and SV cycle 85t Equilibrative nucleoside transporters (ENTs) nonexocytotic gliotransmitter release 124–125 Erythrokeratodermia, connexin 31 210 Erythropoietin (Epo) injury response role 614–615 Escape responses gap junctions 208 crustaceans 208 synaptic delays 208–209 teleost fish 208 Estradiol IGF-1 and 615 neuroprotection 615 Ethanol see Alcohol Ethyl alcohol see Alcohol N-Ethylmaleimide-sensitive fusion proteins see NSFs (N-ethylmaleimide-sensitive fusion proteins) Etomidate, GABAA receptors and 353 Evans blue, vesicular glutamate transporter inhibition 258 Evolution neuromuscular junction (NMJ) 158 Evolutionary conservation neuropeptide precursor peptides 511 Excitation 347 Excitatory amino acid transporters (EAATs) 246f, 248f cellular localization 247 EAAT1 (GLAST) distribution/localization 247, 248f EAAT2 (GLT1) distribution/localization 247, 248f EAAT3 (EAAC) 342 distribution/localization 247 EAAT4 distribution/localization 247 EAAT5, distribution/localization 248 energetics 246 genes 245 glutamate clearance 245 molecular structure 245, 246f Pyrococcus horikoshi 245–246 synaptic functions 248 cross-talk limitation 248, 249f, 250 glutamate concentration maintenance 250 glutamate recycling 248 spillover 248, 249f, 250, 251 synapse specificity 250 uncoupled ionic conductances 247
Subject Index Excitatory junctional potential (EJP) definition 418 high-resolution studies 418, 418f P2X receptor blocking 639, 641f Excitatory junction current (EJC), noradrenergic intermittence mechanisms 419, 419f Excitatory postsynaptic current (EPSC) 250–251 AMPA receptors 323 dopamine D1 receptor 410–411 peptidergic interneurons 568 postsynaptic kainate receptors 317 Excitatory postsynaptic potentials, (EPSPs) 5HT-1A serotonin receptor 473–474 dopamine D1 receptor 410–411 NMDA receptors 276, 277f Excitatory synapses 19 glutamatergic see Glutamate/glutamatergic transmission neurotransmitter release 96 presynaptic side 96 signal reception/processing 96 Excitatory transporters see Excitatory amino acid transporters (EAATs) Exocyst complexes AMPA receptor 268–270 NMDA receptor 286 Exocytic fusion pore, vesicular neurotransmitter transporters 254 Exocytosis astrocytes 121–123 calcium role 82 see also Calcium ions and neurotransmitter release gliotransmitters 120, 123 evidence 120–121 kiss-and-run fusion see Kiss-and-run fusion microdialysis 446 neuropeptide release 529–530 neurotransmitter release see Neurotransmitter release synaptic vesicles see Neurotransmitter release Extinction endocannabinoids and 675, 681f, 682 Extracellular domain, mGluRs 302 Extracellular matrix (ECM) functional roles 174 synapses 174 CAMs and see Cell adhesion molecules (CAMs), synapses NMJ see Neuromuscular junction, basal lamina Extracellular peptidases, neuropeptide release 521–522 Extraocular muscles mammals 144f, 145 slow (nonexcitable multiply innervated) fibers 188 Extrasynaptic receptor(s) glutamate spillover 251 muscle nAChRs 505–506 NMDA receptor trafficking 289 subunit localization 289 see also Volume transmission (VT) Extrasynaptic transmission see Volume transmission (VT) Extrinsic apoptotic pathway see Apoptosis Extrinsic hyperpolarizing potential 209 Eye(s) purinergic receptors P2X receptors 646
F Falck–Hillarp method, serotonin detection 470 Familial amyotrophic lateral sclerosis (ALS) gene mutations SOD1 155–156 kainate receptors 320 Fananserin, serotonin receptor research 463 Fas see Fas/Fas ligand Fasciclin II (FasII) 41 Fas/Fas ligand CRH and pregnancy 549 Fast network oscillations, gap junctions and electrical coupling 223 Fast prepotentials (FPPs) 221 Fast-scan cycle voltammetry see Voltammetry Fear conditioning/conditioned learning endocannabinoids and 675 Fear extinction, pharmacology endocannabinoids 675, 681f, 682
Feeding/feeding behavior hormones melanin-concentrating hormone 553 intake regulation see Food intake regulation orexins (hypocretins) 553 stress and CRH 547 CRH receptors 547–548 Fenfluramine neuroimaging 448 Fibroblast growth factor(s) (FGFs) developmental role synapse formation/maturation 13 Fibroblast growth factor 1 (FGF1) astrocyte differentiation 619 radial glia differentiation 621 Fibroblast growth factor 2 (FGF2) b-amyloid induction 622 glial cells 622t astrocytes 618 differentiation 619 gliomas 621 Mu¨ller glial cells 619 radial glia differentiation 621 Schwann cell differentiation 620 IGF-1 and 614 neurodegenerative diseases 622 neuronal survival 621 Fibronectin binding partners 176 NMJ basal lamina 176 AChR aggregation and 176 receptors see Integrins Field effects, definition 193 Fight-or-flight response norepinephrine 414, 424 Filopodia adhesion molecule-mediated stabilization 9 axonal dendritic vs. 6 growth cones actin cytoskeleton 6–7 synaptic precursor function 5 arbor growth 5 motility 5–6 role 5–6 synaptogenesis 5 dendritic spines axonal vs. 6 extension 11–12 synaptic precursor function 3 axonal contacts 3 branch formation 4, 5f models 3 synaptotropic growth 4 extension dendritic spines 11–12 function 6 motility regulation 6 b-actin 6–7 brain-derived neurotrophic factor 7 calcium signaling 7 intrinsic 6 neuronal activity 7 neurotransmitters 7 signaling proteins 6–7 trial synapse formation 6 synaptic precursor function 3–10 synaptogenesis 4–5, 6 Firing patterns serotonergic neuronal pathways 470–471 Fish(es) bony see Bony fish(es), skeletal muscle neuromuscular system neuromuscular junction 146 skeletal muscle innervation, motor unit use patterns 146 teleost see Teleost fish Flamingo gene/protein 40t Flight or fight response see Fight-or-flight response Fluphenazine, dopamine antagonism 392 FMRFamide (Phe-Met-Arg-Phe-NH2) receptors 557 FMRP see Fragile X mental retardation protein, (FMRP)
713
714 Subject Index Focal extracellular measurement, noradrenergic intermittence mechanisms 419 Food intake regulation endocannabinoids and see Endocannabinoid system neuropeptides neuropeptide Y (NPY) 539 neuropeptide Y1 receptor 540, 542 neuropeptide Y5 receptor 542 ghrelin see Ghrelin taste role reward value see Food reward value Forebrain basal see Basal forebrain cholinergic input 490–491 diencephalon see Diencephalon non-locus coeruleus cell clusters 435 telencephalon see Telencephalon Fractional release, norepinephrine 416–417 Fragile X mental retardation protein (FMRP) LTD role mGluR-dependent 331 Fragile X syndrome (FXS) pathology/pathogenesis mGluRs and 307 mGluR5 297 mGluR-dependent LTD 331 Freeze-fracture electron microscopy, active zone 159–160, 161f Frequency facilitation see Presynaptic facilitation Frog(s) active zone 53f, 54 neuromuscular system neuromuscular junctions 146 skeletal muscle 143 innervation 143, 144f Frontal cortex (lobe) dopamine 410 Frontal lobe see Frontal cortex (lobe) Furin animal knockouts 514 neuropeptide granule biogenesis 516 FXS see Fragile X syndrome (FXS) FXYD proteins see Sodium pump (Naþ/Kþ-ATPase) Fyn tyrosine kinase NMDA receptor internalization 289 NMDA receptor phosphorylation 281–282 postsynaptic development pathways 24
G GABA (g-aminobutyric acid) see GABA/GABAergic transmission GABA, development role activity-dependent synaptic competition 35 somatosensory cortex development 32 GABAA receptor(s) 347–354 activation 355 phasic inhibition 356, 358–358 tonic inhibition 351, 357, 358–358 agonists 351 drug development 351–352 negative 351–352 positive 351–352 allosteric modulators 351 benzodiazepines 352 intravenous anesthetics 353 neurosteroids 353 subtype-selective drugs 352 anesthesia and 353 antagonists 351 picrotoxin 351–352 bicarbonate permeability 347 desensitization 356–357 distribution specificity 355–356 synaptic 355 drug-binding ethanol 357 subtype-selective drugs 352 historical aspects 347 potassium/chloride cotransporter, 2 358 reversal potential 357–358
subtypes a2b2g1 356 a4b6 351 a4bd, sleep-promoting ligands 353 a6bd 351 abg2 352 enrichment 350 subunits/structure 347, 355–356 a1 351 a2 351 a5 357 assembly 348, 349, 349f, 350f abg subunit combinations 349 ion permeable pore formation 355 kinetics 356–357 loss-of-function mutations 355 properties 355–356 d 357 extracellular N-terminal domain 348, 349 g2 anchoring role 349, 356 cysteine residues 349–350 g2 subunit 373 gene family 347 clusters 347 selectivity filter 349 transmembrane domains 349 synaptic functions 355–359 depolarization bicarbonate ions 357–358 chloride ions 357–358 distribution 355 excitation 355, 357 gephyrin and 349, 356 hyperpolarization 358 inhibitory 357 inhibitory postsynaptic currents 350–351 kinetics 355–356 kinetics 356–357 neurotransmission 347 occupancy 350, 356–357 presynaptic inhibition and 357 synchrony 357 GABAB receptor(s) 360–367, 368–372 absence epilepsy and 365–366 activation 363, 364t, 368 agonists 371 allosteric modulation 365 anatomy/physiology 362 hippocampus 362 lateral geniculate nucleus 362 neuronal hyperpolarization 362 antagonists 371 Ca2þ conductance 360 distribution 361 autonomic nerve terminals 361–362 brain 362 heart atrium 361–362 drug binding see GABAB receptor ligands effector mechanisms 362 adenylate cyclase 362 Ca2þ reduction 362 Kþ increase 362 historical aspects 360 inhibitory postsynaptic potentials 368, 369f interactions 360 Kir3.2 subunit co-localization 368 ligands see GABAB receptor ligands long-term potentiation 371 antagonists 369–371 NMDA receptor-dependent and 323 mGluR coupling 371 potassium conductance 360, 368 function 368 presynaptic regulation 368 autoreceptors 368 reduction 371–372 structure 360 C-terminus 360 GABAB1 subunit 360, 361f GABAB2 subunit 360, 361f splice variants 360
Subject Index transmembrane region 360 subtypes 361 vesicle recruitment depression 371 GABAB receptor ligands 363 Agonists 363, 363f absence epilepsy 365–366 2-aminopropyl-methylphosphinic acid (3-APMPA) 363, 363f 2-aminopropyl-phosphinic acid (3-APPA) 363, 363f baclofen 363, 363f CGP44532 363, 363f Antagonists 363, 363f absence epilepsy 365–366 CGP 35348 363, 363f CGP 36742 363, 363f CGP 52432 363f CGP 55845 363f 2-hydroxy saclofen 363, 363f phaclofen 363, 363f saclofen 363 SCH50911 363, 363f therapeutic uses 364, 371 anxiety 365 cognitive function 365 depression 365 drug addiction 365 mesolimbic reward center effects 365 nociception 364 acute pain models 364 chronic inflammation 364 hyperalgesia 364 neuropathic pain 364–365 skeletal muscle relaxation 364 spasticity therapy 364 GABA/GABAergic transmission 340 anatomy/physiology brain GABA concentrations 341 cerebral cortex neocortex 340 development 340 astrocytes, metabolism 239–241 development role see GABA, development role plasticity CB1 expression on CCK-containing neurons 669, 670f short-term depression and 672 suppression of neurotransmission by 669–670 long-term 677 short-term 670–671, 672f glutamine transporter expression and 342 interneurons micturation 454 presynaptic kainate receptors 317 serotonin and 454 metabolism 340–346 catabolism 340, 342 clearance pathways glial uptake 341 isotopic studies 341–342 neuronal reuptake 341 inherited disorders 345 nitrogen homeostasis 343 carrier cycles 343–344 neuropeptide coexistence CGRP 528 cholecystokinin-8 528 enkephalin 528 neuropeptide Y 528 neuropeptide Y (NPY) 521–522, 538 vasoactive intestinal polypeptide (VIP) 528 nonprincipal cells see Interneuron(s) presynaptic kainate receptors and 316–317, 316f receptors see GABA receptor(s) release cytoplasmic pathways 344 mGluR1 inhibition 294–295 vesicular pathways 344 striatal dopamine D2 receptors and 411–412 synapses/synaptic functions 342 activity-dependent competition 35 somatosensory cortex development 32 hippocampal pyramidal cells 350–351, 351f synaptic vesicles 76
synthesis 340–346 cytoplasmic 344 precursors 341, 342 extracellular glutamate 342 glutamate/GABA–glutamine cycle 342, 343f polyamines 343 vesicular 344 transporters see GABA transporters (GATs) see also Inhibitory synapses GABA receptor(s) GABAA see GABAA receptor(s) GABAB see GABAB receptor(s) regulation 342 GABA shunt 340 metabolic compartmentation 340 GABA-transaminase (GABA-T) 340, 342 GABA transporters (GATs) 342 expression 341 Gaboxadol GABAA receptors 350f, 353 benzodiazepines vs. 353 GAD see Glutamic acid decarboxylase (GAD) Gaddum, serotonin 469 GADH/Siah death cascade, prevention 694 Galanin coexistence/cotransmitters acetylcholine 528 aspartate 528 enkephalin 527 luteinizing hormone-releasing hormone 528 neuropeptide Y 527 nitric oxide 528 norepinephrine 528 serotonin 528 substance P 527 vasopressin 521–522 epilepsy and 523–524 Galen noradrenaline and autonomic nerves 414 Gallamine, muscarinic receptors 498 g-aminobutyric acid see GABA/GABAergic transmission g-vinyl GABA, vesicular GABA transporter inhibition 257 Gammahydroxybutyrate (GHB) GABA synthesis 341 Gamma oscillations gap junctions 223 abolishment 223 studies 223 Gap junction(s) 193–212, 194f action potential propagation 197 biophysical properties 213 coupled cells 213 drugs 215 electrophysiological analysis of single channels 214–215 open junctions 213 pore issues 213 permeability influences 214–215 pH and 214–215 blockade 223 pharmacological 205 calcium sensitivity 205 central nervous system 213 circuit parameters 198, 199f communication via 189–192 brain connexins 189 intercellular 190 calcium waves 191 between glial cells 189 ionic permeability 191 junction locations 191 regulation 190 research directions 192 see also Connexin(s); electrical coupling (below) conductance 198–199, 199f definition 193 degradation 204 dendrodendritic 207 disease associations epilepsy 224 genetic disease and 210 distribution 197
715
716 Subject Index Gap junction(s) (continued) electrical coupling 198, 199f, 221f Cajal’s neuron 207 cytoplasmic continuity 198 evidence 220 fast network oscillations 223 features 222 junctional membrane capacitance 198–199 regulation 204 steady state potentials 198–199 evolution 211 connexin–pannexin convergence 211 formation 203 functions 216, 217f glial cells 213–218 astrocytes see Astrocytic gap junctions communication via 189 intercellular calcium waves 116–117, 191 metabolic exchanges via see Astrocytic gap junctions oligodendrocytes see Oligodendrocyte(s) Schwann cells 216, 216t growth 203–204 hemichannels see Connexon (hemichannel) heterocellular 194 heterotypic channels 200 gating asymmetry 202 rectification 202 historical aspects 219 homocellular 194 homotypic channels 200 internalization 204 interneurons 221 isolation techniques 195–196 kinases 204 modulation 219–220 neuromuscular junction see Neuromuscular junction (NMJ) neuronal oscillations and 219–225 cognitive relevance 223 electroencephalograms 219 types 223 permeability 202 phylogeny of proteins invertebrates 214f vertebrates 214f principle neurons 221 properties 193, 219 regulation 190, 209 phosphorylation 205 pH sensitivity 205 single channels conductance measurement 201 gating 201, 201f symmetry 201 spikelets 223 structure 194f, 195, 197f, 219, 220f connexins 193, 213 see also Connexin(s); Connexon (hemichannel) connexon see Connexon (hemichannel) innexins 213 junctional particles 195–196 pannexins 193, 213 symmetry 195–196, 196f study methods 220 conduction increase procedures 221 dye-coupling 192–192, 202, 203f, 220, 221 asymmetry 202 scrape loading 202–203 infrared differential interface contrast microscopy 220 whole-cell patch recording 220 transmission between neurons 189, 199f, 205 escape responses 208 plasticity 209 postsynaptic potential 205–206 spatial summation 209 spikelets 206–207, 221, 222, 223 synaptic delay 199f, 205–206 synchronization 207 temporal summation 209 turnover 203 uncoupling agents 220–221 voltage gating 200, 200f Boltzmann relation 200
Fast 201 Sensitivity 201–202 Slow 201 steady state currents 200 Garter snake, skeletal muscle innervation 143 Gastrointestinal dysfunction 562–563 Gastrointestinal motility cholecystokinin receptors 562 disease treatment 562 CRH receptors 548 peptidergic receptors 561 Gastrointestinal tract dysfunction see Gastrointestinal dysfunction food intake regulation see also Feeding/feeding behavior; Food intake regulation motility see Gastrointestinal motility serotonin 454 enterochromaffin cells 454 Gating gap junctions 190 asymmetry, heterotypic channels 202 ligand-mediated see Ligand-gated ion channels GDNF see Glial cell line-derived neurotrophic factor (GDNF) GDNF family ligands (GFLs) 599–608 biology 604 dopamine neurons 605–606 enteric neurons 606 motor neurons 606 parasympathetic neurons 606 sympathetic neurons 606 definition 599 GFRa coreceptors see GFRa kidney development 606 knockin mice 604–605 knockout mice 605–606, 605t physiology 604 processing 599 receptors 599 GFRa coreceptors see GFRa heparan sulfate proteoglycan (HSPG) 602, 603f neural cell adhesion molecules (NCAMs) 599, 602, 603f syndecans 599, 602, 603f secretion 599 signaling pathways 602, 602f, 604f MAP kinase pathway 602–603 phosphoinositide 3-kinase (PI3K)-Akt pathway 602–603 protein kinase A 604–605 RET 602–603, 604f species 599 spermatogenesis 606 structure 599, 600f synthesis 599 as therapeutic targets 607 amyotropic lateral sclerosis 607 ischemia 607 Parkinson’s disease 607 GDNF family receptor a4 (GFRa4) 599 GFL binding 599, 600f Gene expression CNS development mGluR5 297 dopamine D2 receptor 400, 401f Genetic disorders gap junctions and 210 Genomic disorders see Genetic disorders Gephyrin 373, 375 sggregation 377 binding proteins 376, 376f dynein binding 376–377 evolutionary origin 375–376 expression 375 GABAA receptors and 349, 356 glycine receptor interactions microtubule linking 378 trafficking role 377 structure 375 oligomerization patterns 375–376 variants 375 WW domain 376–377 GFRa 601 knockout mice 605–606, 605t phylogenetic analysis 601
Subject Index structure 601 transforming growth factor-b effects 601 GGF-1, Schwann cell differentiation 620 Ghrelin biological activity eating disorders 523–524 neuropeptide Y regulation 538 GIRKs (G-protein-activated Kþ channels) see G-protein-activated Kþ channels (GIRKs) GIT see G-protein-coupled receptor kinase-interacting protein (GIT) GLAST (EAAT1) see Excitatory amino acid transporters (EAATs) Glia see Glial cells (glia) Glial cell line-derived neurotrophic factor (GDNF) glial cell production 622t gliomas 621 Schwann cells 617–618 knockout mice, motor neurons in 606 neurodegenerative disease and 622 peripheral nerve regeneration 623 peripheral sensitization 581 Sertoli cells and spermatogenesis 607 as survival/neuroprotective factor dopamine neurons 605–606 synapse elimination at the NMJ 154–155 therapeutic use/gene therapy Parkinson’s disease 694 see also GDNF family ligands (GFLs) Glial cell line-derived neurotrophic factor, ENS role development and 606 Glial cell line-derived neurotrophic factor-related ligands (GFLs) see GDNF family ligands (GFLs) Glial cells (glia) astrocytes see Astrocyte(s) cannabinoid receptors 668–669 cell classes 189 communication role see Glial cells, communication and signaling growth factors see Glial growth factors (GGFs) metabolism 239–244 astrocytes see Astrocyte(s) glutamate astrocytic 242 glycogen see Glial glycogen metabolism metabolic exchanges via gap junctions see Astrocytic gap junctions microglia see Microglia neuromodulatory functions see Glial cells, neurotransmission modulation neuropeptides see Neuropeptides oligodendrocytes see Oligodendrocyte(s) phagocytosis, synapse elimination and 48 plasma membrane transporters 245–252 in retina see Retinal glial cells signaling see Glial cells, communication and signaling synaptic functions see Glial cells, synaptic functions tumors growth factors and 621 Glial cells, communication and signaling astrocytes see Astrocytes, communication and signaling gap junctions 190 astrocytes see Astrocytic gap junctions connexin expression 189 metabolic exchanges via see Astrocytic gap junctions receptor/channel expression 5HT-3 serotonin receptors 452 group I mGluRs 304 neuropeptides see Neuropeptide(s) potassium channels see Potassium channel(s) transmitters see Gliotransmitters see also Glial cells, neurotransmission modulation Glial cells, neurotransmission modulation 49, 112–119 astrocyte morphology/structure and 112, 114 number of synaptic contacts/cell 112–113 SON of lactating rats and glutamatergic transmission 114–115 astrocytes as mediators of 112 Ca2þ-role 112, 115–116 see also Astrocytic calcium waves cytokines and 49, 113 TNFa role 113–114 heterosynaptic depression and 49, 114–115 purines and 116–117 integrative functions 112–113, 114–115 retina as model 112, 114 transmitter binding protein release 117 transmitter/neuromodulator release 49, 112, 115
717
ATP/adenosine release 49, 116, 118f D-serine release 116, 117f astrocyte-specific transmitter 115 NMDAR glycine site binding 115, 116 NMDAR-mediated LTP 49, 116 glutamate release 49, 115 action on NR2B-containing NMDA receptors 115–116, 116f Ca2þ-dependence 112 mechanism of action 115–116 modes of action 115 ‘tripartite synapse’ concept and 112, 113f types of modulation 114 Glial cells, synaptic functions 46–51 Astrocytes 46, 47f ablation effects 50 synapse formation/maturation 13, 46, 48f synaptic stability and 49, 50 synaptic transmission and see Glial cells, neurotransmission modulation cytokines and microglial 49 microglia 112 cytokines and plasticity 49 neurotransmission modulation see Glial cells, neurotransmission modulation at the NMJ see Perisynaptic Schwann cells (PSCs) oligodendrocytes 112 Schwann cells see Perisynaptic Schwann cells (PSCs) synapse formation/maturation 13, 46 contact role 47 GABAergic hippocampal neurons 47 PSCs and NMJ formation 46–47 RGC synaptogenesis 13, 46, 48f secreted factors 47 Apo-E–cholesterol 13, 47 Thrombospondins 13, 47 synapse induction in multiple neuronal classes 46 synapse location 49 synapse pruning/elimination 48 Drosophila development 48 PSCs and NMJ development 48–49, 155 synapse structure/stability 49 Eph receptors/ephrins and 49–50 glial ablation effects 50 hormonal responses and 50 synaptic activation 333 second messenger pathways 333 synaptic strength/plasticity see Glial cells, neurotransmission modulation ‘tripartite synapse’ 46, 47f, 112, 113f Glial fibrillary acidic protein (GFAP) astrocyte differentiation 619 Glial glycogen metabolism glycogen mobilization 239 Glial growth factors (GGFs) 617–623 Aging 622 Alzheimer’s disease 622 axonal regeneration 622–623 biological roles 619, 620f brain tumors and 621 CNS neural regeneration 622 definition 617 in development 621 neurodegenerative disease 622 nicotinic receptors and 506 peripheral nerve regeneration 623 puberty 622 radial glia differentiation 621 Glioblastoma multiforme (GBM) 621 PDGF 621 Glioblastomas see Glioblastoma multiforme (GBM) Glioma(s) 621 FGF-2 621 GDNF 621 Gliotransmitters 115, 333 astrocytes see Astrocytes, communication and signaling criteria 120, 122t definition 120 exocytotic regulation 120, 123 AMPA receptor activation 123 bafilomycin A1 120–121 clostridial toxins 120–121 dense-core secretory granules 123 evidence 120–121
718 Subject Index Gliotransmitters (continued) a-latrotoxin 120–121 non-vesicular pathways 335, 336f SNAP-25 120–121 SNARE proteins 120–121 synaptic-like microvesicle compartments 121 TNF-a signaling 123 neuromodulation by see Glial cells, neurotransmission modulation neurotransmitters vs. 121–123 nonexocytotic release 123 gap-junction hemichannels 123–124 plasma membrane transporters 124–125 purinergic P2X7 receptors 123–124 volume-regulated anion channels 123–124 D-serine see D-Serine Global cerebral blood flow (CBF) see Cerebral blood flow (CBF) GLUA1 230–232 GLUA2 230–232 GLUA3 230–232 GLUA4 230–232 Glucagon animal knockouts, processing peptidases 514 Glucocorticoid(s) cortisol see Cortisol neuropeptide Y regulation 538 see also Hypothalamic–pituitary–adrenal (HPA) axis Glucose degradation see Glycolysis MCH neuron excitability 553–555 metabolism glutamate/GABA–glutamine cycle 342 GLUK1 232 GLUK2 232 GLUN1 230 GLUN2 230, 232–233 Glutamate see Glutamate/glutamatergic transmission Glutamate/aspartate transporter (GLAST) see Excitatory amino acid transporters (EAATs) Glutamate decarboxylase see Glutamic acid decarboxylase (GAD) Glutamate dehydrogenase (GDH) Expression 343–344 Glutamate/glutamatergic transmission 229–238, 260, 264, 340 clearance, EAATs 245 concentration maintenance, EAATs 250 cytoplasmic concentrations 246 dendritic spine regulation see Dendritic spines, glutamate regulation endocannabinoids and long-term depression 677 short-term depression 670–671, 672f see also Endocannabinoid system and synaptic plasticity exocytosis regulation 121 filopodial growth role 7, 8f glial cells astrocytes release and neuromodulation 49, 112, 115, 116f homeostasis and brain nitrogen 343 metabolism 229, 243 glial see Glial cells (glia) metabolic fate 243 see also GABA/GABAergic transmission neuropeptide coexistence enkephalin 528 pituitary adenylate cyclase activating peptide (PACAP) 528 substance P 528 neurotransmitter role 192 astrocyte gap junctions 192 synaptically released 260 receptors see Glutamate receptor(s) recycling astrocytes 239 excitatory amino acid transporters 248 release 229 detection 229 peptidergic interneurons inhibition 567–568 D-serine co-storage 335 striatum dopamine D2 receptors and 411–412 synapses formation 12–13
Synthesis 243 transport see Glutamate transport/uptake uptake see Glutamate transport/uptake Glutamate receptor(s) 230 activity-dependent maturation ‘silent’ synapses 23, 27–28, 28f agonists 233f antagonists 234f clustering at the PSD 96 AMPA receptors 96–97, 99, 100 Development 100 mGluRs receptors 96–97, 98–99 NMDA receptors 96–97 scaffolding/adaptor proteins 96–97, 98f AMPA receptor-linked 98f, 99 developmental expression 100 mGluR-linked 98–99 multiple interfaces 99 NMDA receptor-linked 97–98 posttranslational modification and plasticity 101 Shanks 97–98, 98–99, 98f see also Stargazin; Postsynaptic density (PSD); Narp ionotropic see Ionotropic glutamate receptor(s) (iGluRs) metabolism role 239–241 metabotropic see Metabotropic glutamate receptors (mGluRs) phylogram 232f signaling 230 G-protein-linked see Metabotropic glutamate receptors (mGluRs) synaptic transmission 321 Glutamate receptor-interacting protein (GRIP) 233–234 AMPA receptors and Localization 99 mGluR2-dependent long-term depression 329 signaling 311 Glutamate receptor interacting protein 1 (GRIP1), AMPA receptor 268, 271 Glutamatergic neurons see Glutamate/glutamatergic transmission Glutamatergic synapses see Glutamate/glutamatergic transmission Glutamatergic transmission see Glutamate/glutamatergic transmission Glutamate synthase 240f, 243 Glutamate transport/uptake 229, 239, 250–251 electrochemical gradients 246 inhibition 248f, 250 plasma membrane 229 Glutamic acid decarboxylase (GAD) isoforms 344 regulation 345 Glutaminase, metabolism 239 Glutamine metabolism 239 Glutamine synthase see Glutamine synthetase Glutamine synthetase glutamate metabolism 340 Glutamine transporter 342 Glutaminyl cyclase (QC) metal ion requirement 513 neuropeptide synthesis 513 Glutathione (GSH) metabolic role 242 reducing potential 242 synthesis 242 Glutathione peroxidase (GPx) reactive oxygen species scavenging 242 Glycine see Glycine/glycinergic transmission Glycine/glycinergic transmission glial cells and astrocytic concentrations 334 NMDA receptor coagonist 115, 116, 276, 280 receptors see Glycine receptor(s) see also Inhibitory synapses Glycine-insensitive NMDA receptors 280 Glycine receptor(s) 373–379 assembly 374 channel opening 373 chloride conductance 373 developmental switching 375 diffusion properties 378 single-particle tracking 378 dynamics 377 stability 377 synaptic activity 377 ubiquitination 377 gephyrin and 373, 375
Subject Index aggregation 377 binding proteins 376, 376f evolutionary origin 375–376 expression 375 microtubule linking 378 structure 373, 375 oligomerization patterns 375–376 variants 375 WW domain 376–377 trafficking role 377 historical aspects 373 ligand-gated ionotropic receptor family 373 molecular diversity 375 postsynaptic anchoring 377 purification 373 redistribution 377 spastic syndromes associated 378 human hyperexplexia 378 mouse startle syndromes 378 strychnine sensitivity 375, 377 subunits/structure 373, 374f expression sites 375 hydrophobic segments 373–374 isoform expression 375 N-terminal domains 373–374 organization 373–374 Stoichiometry 374–375 topology 373, 374, 374f trafficking 377 Glycine-sensitive NMDA receptors 280 Glycogen 241 metabolism see Glycogen metabolism Glycogen metabolism glia mobilization 239 lysis/breakdown see Glycogenolysis Glycogenolysis lactate production 241 Glycogen synthase kinase-3 (GSK-3) GSK-3b signaling IGF signaling 613 substrate see Tau gene/protein Glycolysis aerobic, astrocytes 239 brain metabolism astrocytes 239 norepinephrine and 239 Glycosylphosphatidylinositol-linked cell surface receptor a (GFRa) see GFRa Glycyrrhetinic acid, gap junction blockade 205 Golgi apparatus neuropeptide granule biogenesis 514 neuropeptide synthesis 511, 525–526 Golgi-associated g-ear-containing adenovirus death protein (ADP)ribosylation-factor-binding-protein (GGA) neuropeptide granule biogenesis 515 Golgi epithelial cells see Bergmann glia Gonadal hormone(s) IGF-1 and 615 neuroprotection and 615 G-protein(s) dopamine D1 receptor signal transduction 410 dopamine receptors signal transduction 410 Gaq/11 short-term synaptic plasticity 135 Gi/o, short-term synaptic plasticity 134 Gs short-term synaptic plasticity 135 kainate receptor signaling 318f, 319 receptor-linked see G-protein-coupled receptor(s) (GPCRs) short-term synaptic plasticity 135 G-protein-activated Kþ channels (GIRKs) ventral mesencephalon, dopamine 412 G-protein-coupled receptor(s) (GPCRs) 302, 557 adrenergic receptors and 428 ‘constitutive activity’ 558 family B 557 FMRFamide family 557 muscarinic receptors 494 neurotensin receptors 557 noradrenergic neurotransmission 416t, 421–422 opioid receptors 534–535
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orphan receptors 557–558 promiscuity 557–558 signal transduction 302 structure 302, 629 synaptic plasticity role short-term synaptic plasticity 134 G-protein-coupled receptor kinase(s) group I metabotropic glutamate receptor desensitization 292 mGluR5 desensitization 292 G-protein-coupled receptor kinase-interacting protein (GIT) CAZ role in clathrin-mediated endocytosis and 94 G-protein-dependent, inwardly rectifying potassium current (GIRK) see G-protein-activated Kþ channels (GIRKs) Gradient guidance see Axonal guidance cues Granin protein family neuropeptide granule biogenesis 515–516 Grb-2, IGF-1 mitogenic pathway 613 GRIP see Glutamate receptor-interacting protein (GRIP) Growth factor(s) 568 glial see Glial growth factors (GGFs) retrograde signaling 128 see also Neurotrophin(s) Growth hormone (GH) neuropeptide Y regulation 538 neuroprotective effects 614 regulation insulin-like growth factor 1 (IGF-1) 614 Growth hormone-releasing hormone (GHRH) animal knockouts 514 GSK-3 see Glycogen synthase kinase-3 (GSK-3) GTPase(s) hypocretin receptors 552 GTP-binding proteins (GTPases) see GTPase(s) Guanosine triphosphate (GTP)-binding proteins (Gq) see GTPase(s) Guanylate kinase-associated proteins (GKAPs) NMDA receptor clustering at the PSD 97–98, 98f functional role 98 PSD-95 interactions 22 Guidance cues (axonal growth) see Axonal guidance cues Guide to Receptors and Channels (GRAC), serotonin receptors 456, 457f, 458t, 459t, 460t Gut see Gastrointestinal tract
H Habc domain SNAREs 67–68, 68f syntaxin 1 68, 69f Hair cell(s) cochlear 32–33 activity-dependent synaptic competition 32–33 Haloperidol catecholamine metabolites 392–393 dopamine antagonism 392 Hamburger, Victor, target-derived molecules and retrograde signaling 126 Hargreaves test, nerve growth factor 579 Hashish see Cannabis HCN channels see Hyperpolarization-activated cyclic nucleotide gated (HCN) channels Hearing loss connexin mutations connexin 26 and 210 Heart 5HT-3 serotonin receptors 451–452 autonomic innervation sympathetic postganglionic input 557 GABAB receptors 361–362 Hebbian plasticity activity-dependent synaptic competition and 35 visual system 35 see also Long-term depression (LTD); Long-term potentiation (LTP) Hebb’s postulate activity-dependent synaptic competition and 35 evidence for validity 35 see also Hebbian plasticity Hebb’s rule see Hebb’s postulate Hemichannel see Connexon (hemichannel) Hemoglobin, nitric oxide 684 Heparan sulfate proteoglycans (HSPGs) GFL receptors 602, 603f NMJ basal lamina 176
720 Subject Index Heparan sulfate proteoglycans (HSPGs) (continued) agrin 177 collagen XVIII 177 perlecan 177 unique features 176–177 Hepatocyte growth factor/scatter factor (HGF/SF) Alzheimer’s disease 622 Heptanol, gap junction blockade 205 Hereditary diabetes insipidus 512 Heroin 532 Heterosynaptic long-term depression (LTD) activity-dependent synaptic competition 35–36 astrocytes and 49, 114–115, 116–117 endocannabinoids and 673, 679f Heuser–Reese cycle 59 High-affinity plasma membrane glutamate carriers, nonexocytotic gliotransmitter release 124–125 High-frequency oscillatory activity 224 High-frequency stimulation (HFS) long-term potentiation induction 321 neuropeptide release 516–517, 519–521 High-pressure liquid chromatography (HPLC), microdialysis 444 High-voltage activated (HVA) calcium channels see Voltage-gated calcium channels (VGCCs) Hip1 gene/protein endocytosis and SV cycle 85t HIP1R, endocytosis and SV cycle 85t Hippocampal formation see Hippocampus Hippocampal long-term potentiation (LTP) CA1 region 369, 370f mechanisms glial cells and 49 NMDA receptors and D-serine and 49 Hippocampal plasticity aging and CB1 receptors 675–676 long-term depression (LTD) endocannabinoid-dependent 673, 674f long-term potentiation (LTP) see Hippocampal long-term potentiation (LTP) short-term depression 670–671 Hippocampus anatomy/physiology cholinergic pathways retrograde neurotrophic signaling 585 interneurons subtypes 221 neurons postsynaptic differentiation 20 postsynaptic differentiation, proteins 20–21 pyramidal cells see Pyramidal neuron(s) CA1 region long-term potentiation 369, 370f see also Hippocampal long-term potentiation pyramidal cells see Pyramidal neuron(s) tonic inhibition elimination 357 glutamate spillover 251 glutamate uptake inhibition 248f, 250 neurotransmitters/receptors GABAA receptors 357 GABAB receptors 362, 368 norepinephrine distribution 415 serotonin 5HT-1A receptor 473–474 5HT-1 receptor 473 5HT-2A receptor 474 5HT-3 receptor 475 5HT-4 receptor 476 plasticity see Hippocampal plasticity Hirschsprung disease (HSCR) gene defects c-ret mutation 599–600 Histochemical techniques serotonin localization 470 Hodgkin cycle, Ca2þ-mediated neurotransmitter release 59, 60f Homeostasis definition 129 presynaptic modulation, retrograde signaling 129, 130f Homer proteins mGluR complexes 98–99 activation 294
signaling 302–303 PSD-95 interactions 22 Shanks binding 99 Homocarnisine, GABA synthesis 341 Homocarnisinosis, GABA metabolism 345–346 Homologous desensitization 292 Homosynaptic long-term depression (LTD) endocannabinoids and 672f, 673, 679f Homosynaptic long-term potentiation (LTP) activity-dependent synaptic competition 35–36 Hormone(s) astrocyte–synapse relationship and 50 see also Endocrine system HPA axis see Hypothalamic–pituitary–adrenal (HPA) axis 5-HT see Serotonin (5-HT)/serotonergic neurons 5-HT receptors see Serotonin (5-HT) receptor(s) HU-210 664, 665f Hubel, David H ocular dominance columns (ODCs) 31 see also Visual deprivation Human hyperexplexia, glycine receptor defects 378 Huntington’s disease (HD) pathology/pathogenesis adenosine receptors 637t BDNF reduction 592 kainate receptors 320 retrograde neurotrophic signaling defects 588 therapy neurotrophins 574 Hydrogen carbonate (HCO3) see Bicarbonate 2-Hydroxy saclofen, GABAB receptor antagonists 363, 363f 5-Hydroxytryptamine (5-HT) see Serotonin (5-HT)/serotonergic neurons Hyperalgesia GABAB receptor ligands 364 neural basis sympathetic nervous system NGF role 579, 580 Hyperexplexia, glycine receptor defects 378 Hyperpolarization chloride channels 347, 348f cyclic nucleotide-gated channels and see Hyperpolarization-activated cyclic nucleotide gated (HCN) channels GABAA receptors 358 GABAB receptors 358 Mauthner cells 209 see also Afterhyperpolarization (AHP) Hyperpolarization-activated cyclic nucleotide gated (HCN) channels nitric oxide 688 structure subunits 688 Hypersecretion, growth hormone (GH) see Growth hormone (GH) Hypocretin 1 (Hcrt1) structure 551 Hypocretin 2 (Hcrt2) structure 551 Hypocretins see Orexins (hypocretins) Hypothalamic–pituitary–adrenal (HPA) axis CRH see Corticotropin-releasing hormone (CRH) glucocorticoids see Glucocorticoid(s) neurochemistry group III mGluRs 306–307 PVN see Paraventricular nucleus, HPA axis role SCN see Suprachiasmatic nucleus (SCN) Hypothalamo-neurophysial system see Neurohypophysis Hypothalamus hormones see Hypothalamus, neuroendocrine role magnocellular neurons see Magnocellular hypothalamo–neurohypophysial system (mHNS) neuroendocrine functions see Hypothalamus, neuroendocrine role neuropeptides 527 rhythmic activity see Hypothalamus, rhythmicity temporal organization of behavior see Hypothalamus, rhythmicity Hypoxia-inducible factor-1a (HIF-1a), neuroprotection 615 Hypoxia–ischemia (HI) IGFs and 615
I Ibotenic acid 264 IGFs see Insulin-like growth factor (IGF) system Imaging studies see Neuroimaging
Subject Index Immune system nitric oxide 691 Immunoglobulin superfamily synapses 38, 42 Immunosuppression pregnancy, CRH and 549 Impulse-to-impulse basis, noradrenergic neurotransmission 421, 421f Inferior olive (IO) synchronously active cell coupling 207 Inflammation chronic GABAB receptor ligands 364 Infrared differential interface contrast (IR-DIC) microscopy, gap junctions 220 Ingestive behavior see Feeding/feeding behavior Inhibition striatal dopamine 411 Inhibitory postsynaptic currents (IPSCs), 5HT-3 serotonin receptor 475 Inhibitory postsynaptic potentials (IPSPs) 5HT-1 serotonin receptor 473 dopamine D2 receptor 399 GABAB receptors 323, 368, 369f Inhibitory synapses 19 GABAergic transmission see GABA/GABAergic transmission see also GABA/GABAergic transmission; Glycine/glycinergic transmission Innexins 189, 195 iNOS see Nitric oxide synthase(s) (NOS) Inositol 1,4,5-triphosphate (IP3) signaling gap junctional communication 190, 191 mGluRs 98, 294, 295f Input resistance, muscle fibers 182–183 Input specificity definition 322 NMDA receptor-dependent LTP 322 Insomnia treatment 353 Insulin NMDA receptor effects 282 Insulin-like growth factor (IGF) system 609–616 binding proteins see Insulin-like growth factor binding proteins (IGFBPs) discovery of brain IGF system 609 somatomedin hypothesis 609, 610f truncated IGF-1 609 future research directions 615 ligands 609 IGF-1 see Insulin-like growth factor-1 (IGF-1) IGF-2 see Insulin-like growth factor-2 (IGF-2) nervous tissue expression 609 IGF binding proteins 609, 611f IGF ligands 609 IGF receptors 611 rat olfactory bulb 610f neuroendocrine cross-talk 614 IGF-1 and erythropoietin 614 IGF-1 and GH 614 IGF-1 and gonadal steroids 615 neuroprotective surveillance and 614 as neurotrophic factors 614 receptors see Insulin-like growth factor receptor(s) signaling and brain actions 612, 612f intracellular substrates 612–613, 612f mitogenic pathway 613 survival and metabolic pathway 613 Insulin-like growth factor-1 (IGF-1) CNS neuron regeneration 622 discovery 609 disease associations 614 glial cells 622t astrocytes 618 Bergmann glial cells 619 oligodendrocytes differentiation 620 knockout mice 613 nervous system development and 609 nervous system expression 609, 610f neuroendocrine cross-talk 614 erythropoietin and 614 FGF-2 and 614 gonadal steroids and 615 growth hormone regulation 614 neuroprotective functions 614 erythropoietin/HIF-1a and 615
estradiol and 615 growth hormone and 614 receptors see Insulin-like growth factor receptor(s) signaling 612, 612f mitogenic pathway 613 survival and metabolic pathway 613 ‘truncated’ (des(1-3)) 609 Insulin-like growth factor-2, (IGF-2) discovery 609 nervous system expression 609 receptors see Insulin-like growth factor receptor(s) Insulin-like growth factor binding proteins (IGFBPs) nervous system expression 609, 611f IGFBP-1 609 IGFBP-2 609, 610f IGFBP-3 609–611 IGFBP-4 610f, 611 IGFBP-5 610f, 611 IGFBP-6 611 truncated IGF-1 affinity 609 Insulin-like growth factor receptor(s) downstream signaling 612, 612f IGF-1R 610f, 611–612 atypical subtypes 612 mitogenic pathway 613 survival and metabolic pathway 613 IGF-2R 612 insulin receptor co-expression 612 nervous system expression 611 Insulin receptors IGF receptor co-expression 612 Insulin receptor substrate 1 (IRS-1) 612–613, 612f Insulin receptor substrate 2 (IRS-2) 612–613, 612f Integrin(s) 40t actin cytoskeleton and 178 ECM-mediated interactions 44–45 kinase regulation by 178 L1-family cell adhesion molecule interaction 41–42 netrin binding 43 subunits/heterodimer formation 178 synapses see Integrins, synaptic functions Integrins, synaptic functions glial-mediated synaptogenesis 47 neuromuscular junction postsynaptic basal lamina 178 a7 subunit and 178 intracellular signaling 178 presynaptic role 178 vertebrate NMJ development 127 Intercellular communication peptides 557 volume transmission see Volume transmission (VT) wiring transmission see Wiring transmission (WT) Interleukin-1b (IL-1b) b-amyloid induction 622 ‘Intermittent release model’, norepinephrine measurement 417–418 International Union of Basic and Clinical Pharmacology (IUPHAR) opioid receptors 532–533 serotonin receptors 456 Interneuron(s) 220 electrically coupled 222 localization 220–221 gap junctions 221 Interneuronal network gamma (ING) 223 Intersectin endocytosis and SV cycle 85t Interstitial collateral axon branching see Axon branching Intracellular calcium see Calcium ions (Ca2þ)/calcium signaling Intracellular signaling cadherins 38–40 see also Signaling pathways Intracellular sorting neurotrophins 570 Intracellular vesicle(s) see Vesicle(s) Intracerebroventricular injection, pancreatic polypeptide (PP) 540 Intracranial self-stimulation (ICSS) 555 Intrafusal muscle fibers 188 mammals 144f, 145 Intravenous anesthetics, GABAA receptors 353 Invertebrate(s) GPCRs see G-protein-coupled receptor(s) (GPCRs) innexins 213
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722 Subject Index Invertebrate(s) (continued) neuromuscular junctions see Neuromuscular junction, invertebrate in vivo push–pull cannulae, neuropeptide metabolism 567 in vivo recordings neuropeptide synaptic potentials 568 Inwardly rectifying potassium channels (Kir) 202 GABAB receptor IPSPs 368 5HT-1 serotonin receptor and 472–473 Ionic selectivity, gap junctions 190 Ionotropic glutamate receptor(s) (iGluRs) 230, 232f astrocytes see Astrocyte(s) classes 260, 308 AMPA receptors see AMPA receptor(s) kainate receptors see Kainate receptor(s) NMDA receptors see NMDA receptor(s) nomenclature 230 pharmacology 230t, 232 protein partners 233 schematic subunit 232f signaling 232 structure 230, 230t Irritable bowel syndrome (IBS) 5-HT3 receptor studies 466 5-HT4 receptor studies 466 Ischemia/ischemic-like insults cerebral see Ischemic stroke (cerebral ischemia) GDNF family ligands (GFLs) 607 glutamate 237 see also Ischemic stroke (cerebral ischemia) Ischemic stroke (cerebral ischemia) nitric oxide role 694f Islet beta cell neuropeptides see Neuropeptides
J Jugular vein, 5-HT2B receptor 465
K Kþ see Potassium ions (Kþ) Kainate AMPA receptor activation 264 Kainate receptor(s) 230, 232, 233f, 308–312, 313–320 agonists 313, 314, 314t AMPA receptor interaction 313 SYM2081 314 AMPA effects 233 antagonists 313, 314, 314t AMPA receptor interaction 313 CNQX 314, 314t DNQX 314, 314t NBQX 314, 314t pain 319 pilocarpine antagonism 316–317 as cation channels 313 desensitization 313 recovery 313–314 speed 313–314 distribution 310 diversity 308 dual signaling 319 G-proteins 318f, 319 protein kinase C activation 319 voltage-dependent Ca2þ channels 319 expression regulation 310 glutamate binding 311–312 metabolism role 239 neuropathology/disease 319 autism 320 epilepsy 319–320 familial amyotropic lateral sclerosis 320 Huntington’s disease 320 obsessive–compulsive disorder 320 pain 319 schizophrenia 320 pharmacological aspects 314 postsynaptic responses 317
demonstration 317 excitatory postsynaptic currents 317 functions 317 GluR5-deficient animal models 318 kinetics 317 long-term depression (LTD) 318 long-term synaptic plasticity 318 LY382884 318 retina 317–318, 318f properties 313 rectification properties 310 subunits/structure alternative splicing 308, 309f binding domain 308, 311, 311f desensitization 308 GluR5 308, 311–312 variants 308–309 GluR6 308, 309f, 310, 311–312 agonist binding 311–312 GluR7 308, 310 variants 308–309 KA1 310 KA2 310 mRNA editing 310 synaptic neurotransmitter release 314, 315f GABA 316–317, 316f GABA interneurons 317 long–term potentiation 314–316 mechanisms 316f, 317 mossy fiber–CA3 synapses 314–316 postsynaptic 314–316 presynaptic 314–316 signaling 317 Kappa opioid receptor see Opioid receptor(s) Katz, Sir Bernard basis of synaptic function at the NMJ 159, 160 see also Neuromuscular transmission, presynaptic events neurotransmitter release 160 Katz theory of neurotransmission 160 Kþ channels see Potassium channel(s) a-Ketoglutarate (a-KG) 243 glutamate metabolism 340, 341f regeneration 340–341 Kidney(s) development, GDNF family ligands (GFLs) 606 KIF1A, synaptic vesicle transport 76–77 KIF1Bb, synaptic vesicle transport 76–77 Kinase(s) see Protein kinase(s) Kinesin(s) axonal transport 25 postsynaptic development, KIFs 25 synaptic vesicle transport 76–77 Kinetics NMDA receptors 277–278, 279f Kirs see Inwardly rectifying potassium channels (Kir) Kiss-and-run fusion endocytosis 79 exocytosis astrocytes 121–123 synaptic vesicles 84, 87f, 165, 166f active zone location 90 Knockin animal(s) GDNF family ligands (GFLs) 604–605 Knockout animal(s) developmental studies NMJ agrin/MuSK 178 furin 514 glucagon processing peptidases 514 G-protein-coupled P2X purinoceptors 422 insulin-like growth factor-1 (IGF-1) 613 insulin-like growth factor-1 (IGF-1), roles 613 laminin b2 knockout mice 176 mGluRs group II mGluRs 306 group III mGluRs 306–307 mGluR2 306 mGluR2-dependent long-term depression 330 mGluR3 306 mGluR4 306–307 mGluR6 300–301 muscarinic receptors 501
Subject Index neuropeptides neuropeptide Y 523–524 synthesis 514 neurotransmission Munc-13 16 neurotrophic factors artemin 605–606, 605t GDNF family ligands 605–606, 605t GFRa coreceptors 605–606, 605t neurturin 605–606, 605t persephin 605t RET 605–606, 605t opioid receptors 533 P1 receptor(s) 638 P2Y1 receptors 655 PACE4 514 proinsulin 514 serotonin transporter see Serotonin transporter (5-HTT/SERT) synaptic function SNAP-25 70 SNAREs 70 synaptobrevin 2 70 synaptic plasticity and mGluR2-dependent long-term depression 330 synaptogenesis MuSK and agrin knockouts 126 TSP and glial-mediated 47 Wnt signaling pathway mutants 128 type 2 CRH receptors 548 Krebs cycle 243 GABA metabolism 340
L L1-family cell adhesion molecules (L-CAMs) 38, 41–42 binding moieties 41–42 integrin interaction 41–42 neurocan interaction 41–42 structure 39f, 41–42 L-655708, GABAergic action 351–352 Lactate glycogen metabolism 241 Lactate dehydrogenase (LDH) pyruvate 239 Lactation neurohypophysis and D-serine 337–338 Lamina I cells, BDNF inhibiting effects 581–582 Laminin(s) NMJ 175 extrajunctional vs. synaptic cleft 176 retrograde signaling in formation 127 receptors see Integrin(s) subunit structure 175–176 Laminin a4 neuromuscular junctions 44 presynaptic development, retrograde signaling 127 Laminin b2 40t knockout mice 176 neuromuscular junctions 44 N-type calcium channels 44 postsynaptic NMJ basal lamina 176 presynaptic development retrograde signaling 127 Laminin b4 40t Lamprey(s) skeletal muscle innervation 142–143 Large conductance calcium-activated potassium channels see Calciumactivated Kþ channels (KCa) Large dense-core vesicles (LDCVs) 516, 517f, 519, 525–526 amine-containing 529–530 chromaffin cells 521 distribution 529 exocytosis 521 morphology 522 pancreatic b cells 521 peptide-containing 529–530 Large granular vesicles, neuropeptide storage 525, 526f Large GTPases see Dynamin(s) LAR-interacting protein 1 see Liprin-a LAR receptor protein kinases 43
LAR-RPTP 40t late bloomer 127 Late endosome(s) retrograde neurotrophic signaling 587–588 Lateral diffusion, AMPA receptor 272 Lateral dorsotegmental (LDT) nuclei cholinergic input 490 Lateral geniculate nucleus (LGN) GABAB receptors 362 inputs development, activity-dependent synaptic competition 31 Lateral giant axons, tail flip response 208 a-Latrotoxin 21 neurexin interactions 43–44 LDCVs see Large dense-core vesicles (LDCVs) Lens (crystalline) gap junction turnover 204, 206f Leprosy nerve growth factor 582 Leptin feeding regulation/energy balance MCH relationship 553–555 Leucine, as nitrogen source 343–344 Leukemia inhibitory factor (LIF) glial cell production 622t astrocytes 618 Schwann cells 617 peripheral nerve regeneration 623 receptor, astrocyte differentiation 619 LGN see Lateral geniculate nucleus (LGN) LIF see Leukemia inhibitory factor (LIF) Ligand-gated ion channels 5HT-3 serotonin receptor 475 glial cells see Glial cells, communication and signaling glycine receptors 373 see also Ionotropic receptors Ligature technique, retrograde neurotrophic signaling studies 584–585 Lipid peroxidation nitric oxide and 689 Liprin(s) functions active zone 57 Liprin-a function active zones 56t, 57 presynaptic development 127–128 Liprin-b active zone 57 Lizard(s) skeletal muscle innervation 143 Local circuitry neurons see Interneuron(s) neuropeptide receptors 567 Local perfusion, microdialysis 445, 445f Locus coeruleus (LC) 430 anatomy/physiology afferent projections 430, 433f efferent projections 430, 431t neurotransmitter inputs 430 norepinephrine system see Locus coeruleus norepinephrine system (LC-NE) impulse activity 433 cognitive performance 433 modes 433 decision completion 433–434, 434f sleep 433 stress 433 waking 433 neural circuit response properties 439 output effects 439 Locus coeruleus norepinephrine system (LC-NE) 415, 426 impulse activity 435 Long-term depression (LTD) activity-dependent synaptic competition 35 AMPA receptors and see Long-term depression, glutamate role amygdala endocannabinoid-dependent 673 dopamine role 390 frontal dopamine and 411 endocannabinoids and see Endocannabinoid system and synaptic plasticity experience-dependent learning and
723
724 Subject Index Long-term depression (LTD) (continued) glutamate receptors see Long-term depression, glutamate role heterosynaptic astrocytes and 49 endocannabinoids and 674f homosynaptic endocannabinoids and 672f induction 327 activity-dependent 327 chemical induction 327 maintenance, AMPA phosphorylation 331 mGluRs and see Long-term depression, glutamate role molecular mechanisms intracellular events 328 calcium influx 328f, 328 downstream processing 328 nitric oxide 680 see also Synaptic plasticity, molecular mechanisms NMDA receptor-dependent see Long-term depression, glutamate role NMDA receptor-independent, endocannabinoid LTP 673, 677–678 postsynaptic receptors, knockout mice 23–24 D-serine and 337 time-dependent, endocannabinoids and 673 Long-term depression, glutamate role AMPA receptors and 268 cerebellar LTD 680 clathrin-mediated endocytosis 328 desensitization and 680 endocannabinoid-mediated LTD 678, 680 maintenance 331 phosphorylation 331 subunits 329 trafficking 329 AP-2 complex 329 internalization 329 mGluRs and 304, 327–332 activity-dependent induction 327 chemical induction 327 endocannabinoid-mediated LTD 673, 678, 680–681 expression mechanisms 328 group II mGluRs 306 intracellular events 328, 328f maintenance antagonist studies 331 fragile X mental retardation protein (FMRP) 331 fragile X syndrome 331 protein synthesis 331 mGluR,1 294–295, 330 mGluR2 329 AMPA receptor binding protein (ABP) 329 AP-2 330 cerebellar 329–330 glutamate receptor interacting protein (GRIP) 329 knockout mice 330 NSF interactions 330 phosphorylation 330 protein interacting with C kinase 1 (PICK1) 329 protein kinase C 329 presynaptic neurotransmitter release 329 receptor endocytosis 329 subunit preparation dependency 327–328 NMDA receptors and 278–279, 327–332, 596–597, 678, 679f activity-dependent induction 327 calcineurin 328 calcium influx 328, 328f chemical induction 327 developmental role 278–279 endocannabinoid-mediated LTD 678, 679f, 680f expression mechanisms 328 maintenance 331 subunit composition 327 postsynaptic kainate receptors and 318 Long-term potentiation (LTP) activity-dependent synaptic competition 35 AMPA receptors and see Long-term potentiation, glutamate role dopamine 390 frontal dopamine and 411 GABA inhibition role 323 GABA receptors 323 glutamate receptors and see Long-term potentiation, glutamate role hippocampal see Hippocampal long-term potentiation (LTP) homosynaptic
activity-dependent synaptic competition 35–36 induction 321 high-frequency stimulation 321 pairing 321 molecular mechanisms 324, 325 cadherins and 38 glutamate receptors and see Long-term potentiation, glutamate role nitric oxide and 690–691 see also Synaptic plasticity, molecular mechanisms neurotrophins 573 NMDA receptor-dependent see Long-term potentiation, glutamate role phases 324 early (E-LTP) 324–325 late (L-LTP) 324–325 postsynaptic density and 101 priming by LTD 682 D-serine and 337 Long-term potentiation, glutamate role AMPA receptors and 268, 325 PSD and 100 PSD distribution and 101 PSD role 101 see also AMPA receptor(s) hippocampal see Hippocampal long-term potentiation (LTP) mGluRs group I mGluRs and 304 mGluR5 297 NMDA receptors and 321–326 associativity 323 cooperativity 323 GABAB receptor role 369 autoreceptors 323, 369, 370f induction protocol 369–371 glial release of D-serine and 49, 116 group I mGluRs and 304 input specificity 322 metaplasticity 325 PSD role 101 signaling mechanisms 323 see also NMDA receptor(s) presynaptic kainate receptors 314–316 Long-term synaptic plasticity depression see Long-term depression (LTD) mechanisms glutamate and see Long-term depression, glutamate role; Long-term potentiation, glutamate role postsynaptic kainate receptors 318 see also Synaptic plasticity, molecular mechanisms potentiation see Long-term potentiation (LTP) Lou Gehrig’s disease see Amyotrophic lateral sclerosis (ALS) Low-frequency stimulation NMDA receptors 321 Low-voltage activated (LVA) calcium channels see Voltage-gated calcium channels (VGCCs) LSD (lysergic acid diethylamide) structure 471f serotonin vs. 470 LTD see Long-term depression (LTD) LTP see Long-term potentiation (LTP) Luteinizing hormone-releasing hormone (LHRH) biological effects 524 galanin coexistence 528 LY35470, anxiety disorders 299 LY334740 298–299 LY341495 234f, 298–299 LY354740 235, 237 LY379268 298–299 LY382884, postsynaptic kainate receptors 318 Lysergic acid diethylamide (LSD) see LSD (lysergic acid diethylamide)
M Macroendocytosis (ME), synaptic vesicles at the NMJ 165–166, 166f Magnesium ions (Mg2þ) NMDA receptor blockade 276–277, 278f, 321 Magnetic resonance spectroscopy (MRS) applications/studies SNAREs 67 Magnocellular hypothalamo–neurohypophysial system (mHNS) neuropeptide coexistence 526, 527 oxytocin synthesis/secretion 522
Subject Index see also Oxytocin vasopressin synthesis/secretion 522 see also Vasopressin see also Neurohypophysis MAGUKs (membrane-associated guanylate kinases) long-term potentiation 325 NMDA receptors 286 PSD-95 see PSD-95 Mammal(s) neuromuscular system intrafusal muscle fibers 144f, 145 neuromuscular junction 145, 146 see also Neuromuscular junction (NMJ) skeletal muscle innervation 144f, 145 afterhyperpolarization 147 axon diameter:cell body size ratio 145 en grappe terminals 145 en plaque terminals 145 graded voluntary contraction 148 motor unit:muscle fiber ratio 145 motor unit recruitment 148 motor unit use patterns 147 ‘size principle’ 148 twitch fibers 145 pannexins 213 MAP kinase signaling pathway GDNF family ligands (GFLs) 602–603 group I mGluRs 305 IGF-1 mitogenic pathway 613 MAPK signaling see MAP kinase signaling pathway Maps/representations see Neural maps/representations Marijuana see Cannabis Martin–Bell syndrome see Fragile X syndrome (FXS) Mass spectroscopy postsynaptic density analysis 96 Maternal bonding, oxytocin see Oxytocin Matrix metalloproteinase(s) (MMPs) NMJ basal lamina 179 collagen IV cleavage 175 Mauthner cells hyperpolarization 209 interneuron axon convergence 209 action potential generation 206f, 209, 210f teleost fish escape response 208 inhibitory neuron excitation 208 MaxiK (BK) channels see Calcium-activated Kþ channels (KCa) MDMA (methylenedioxymethamphetamine: ecstasy) vesicular monoamine transporter inhibition 256 a,b-meATP mimicry, P2X1 receptors 651 MeCP2 BDNF expression 592 Medial pontine reticular formation, 5HT-2 serotonin receptor 474 Medial raphe nuclei serotonergic neuronal pathways 470 Medium-spiny neurons striatal dopamine 411–412 Mefloquine, gap junction blockade 205 Melanin-concentrating hormone (MCH) 551–556 brain actions 553 addiction 555 arousal 555 feeding 553 metabolism 553 motivation 555 discovery 551 distribution 551, 552f energy balance and feeding regulation 553 inputs 553, 554f localization 551 neuroanatomy 551, 552f neuronal membrane protein expression 553 outputs 553, 554f overexpression 553–555 receptors 552 binding proteins 553 distribution 552 MCHR1 552 MCHR2 552 signaling cascades 552 GPCR coupling 552 structure 551
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a-Melanocyte-stimulating hormone (aMSH) eating disorders and 523–524 oxytocin release 522–523 Melanoma mGluR1 294–295 Melanotropes precursor peptides 511–512 Melanotropin, posttranslational processing 512 Memantine NMDA receptors 237 Membrane(s) capacitance see Membrane capacitance cell (plasma) see Cell membrane endocytosis see Endocytosis exocytosis see Exocytosis fission, dynamins see Dynamin(s) permeability, neuropeptide synaptic potentials 568 Membrane-associated guanylate kinases see MAGUKs (membraneassociated guanylate kinases) Membrane capacitance muscle fiber passive cable properties and 183f, 184 Membrane trafficking proteins synaptic vesicles and 82 see also Endocytosis; Exocytosis; Synaptic vesicle cycle Memory cellular mechanism adenosine receptors 637t BDNF 597 synaptic plasticity see Synaptic plasticity consolidation see Memory consolidation disorders see Memory disorders drug effects cannabinoid effects 675 endocannabinoids and 675 appetitive learning 676 fear conditioning 675 Morris water maze 675 object/social recognition 675 neural basis amygdala see Amygdala basal forebrain 490–491 cholinergic neurons basal forebrain 490–491 frontal lobe see Frontal cortex (lobe) spatial see Spatial memory synaptic plasticity see Long-term depression (LTD); Long-term potentiation (LTP); Synaptic plasticity Memory consolidation synaptic-level consolidation endocannabinoids and 676 see also Synaptic plasticity Memory disorders neurobiology cannabinoids and 675 cholinergic hypothesis see also Basal forebrain Mental conditions see Psychiatric disorders Mental disorders see Psychiatric disorders Mental illness see Psychiatric disorders Mescaline structure 471f Mesencephalon see Midbrain Mesocortical dopaminergic system 384 Mesolimbic dopaminergic system 384 reward role GABAB receptor ligands 365 ventral striatum see Striatum Metabolism glucose see Glucose melanin-concentrating hormone 553 metabolic exchanges via gap junctions see Astrocytic gap junctions orexins 553 see also Orexins (hypocretins) Metabotropic GABA receptors see GABAB receptor(s) Metabotropic glutamate receptors (mGluRs) 234, 235f, 292–301, 302–307 activation 249f, 251, 292 dimerization 293–294 D-serine efflux 335, 335f Homer proteins 294 IP3 receptor 294, 295f ligand-binding 293–294 PI3K enhancer (PIKE) 294
726 Subject Index Metabotropic glutamate receptors (mGluRs) (continued) scaffold proteins 294 agonists 296f clinical applications 297t allosteric modulators 293–294 antagonists 296f clinical applications 297t biochemistry 231t cell biology 294 classification 292 diseases/disorders 307 Alzheimer’s disease 307 fragile X syndrome 307 Parkinson’s disease 307 schizophrenia 307 expression 303 functional roles 303 LTD see Long-term depression, glutamate role neuromodulation 303 synaptic plasticity 304 GABAB receptor coupling 371 glutamate sources 303 afferent repeated stimulation 303 cysteine–glutamate exchanger 303 group I 292, 303t, 304 homologous desensitization 292 kinases 305 Ca2þ/diacylglycerol-dependent protein kinase C 305 MAPK/ERK 305 protein kinase A 305 protein kinase C 305 NMDA receptor-dependent long-term potentiation 304 pharmacology 305 agonists 305 antagonists 305 plasticity 305 AMPA receptors 305–306 NMDA receptors 305–306 retrograde signaling 304 cannabinoids 304 scaffolding proteins 302–303 signaling 292, 302–303 cyclic AMP 304 phospholipase C 304 structure 293f synaptic locus 304 glia 304 Kþ channel modulation 304 voltage-gated calcium channels 304 group II 292, 303t, 306 agonists 306 distribution 306 knockout mice 306 long-term depression 306 structure 293f synaptic adaptation 299 group III 292, 303t, 306 agonists 300, 306 antagonists 300 cyclic AMP production 306 distribution 300, 306–307 epilepsy therapy 300 HPA axis 306–307 knockout mice 306–307 structure 293f historical aspects 292, 302 human disease 294 localization 231t, 235 mGluR1 292, 294, 303t agonists 295–297 antagonists 294–295 chronic pain 298 competitive antagonists 295–297, 296f cross-desensitization, mGluR5 298 distribution 294–295 GABAB receptor coupling 371 GABA release inhibition 294–295 knockout animals 294–295 long-term depression 294–295, 330 melanoma 294–295 postsynaptic 294–295 Purkinje cells 294–295
signaling 292 cyclic AMP 292 phosphatidylinositol 3-kinase pathway 292 PI hydrolysis 292 structure 294 mGluR2 298, 303t agonists 298–299, 306 therapeutic uses 299 analgesia 299 antagonists 298–299 distribution 298, 306 knockout mice 306 long-term depression see Long-term depression, glutamate role signaling 298 structure 298 synaptic adaptation 299 mGluR3 298, 303t agonists 298–299, 306 therapeutic uses 299 analgesia 299 antagonists 298–299 distribution 298, 306 astrocytes 299 microglia 299 knockout mice 306 signaling 298 structure 298 mGluR4 299, 303t activation 300 agonists 300, 306 Parkinson’s disease 300 epilepsy therapy 300 knockout mice 306–307 structure 299–300 mGluR5 235–236, 292, 297, 303t agonists 295–297, 297–298 CHPG 297–298 MPEP 297–298 therapeutic uses 297–298 chronic pain 298 desensitization 292 GPCR kinases 292 mGluR1 cross-desensitization 298 developmental expression patterns 297 distribution 297 basal ganglia motor circuit 298 drug addiction 298 fragile X syndrome 297 genetics 297 long-term potentiation 297 NMDA receptors and 297 signaling 292, 297 cyclic AMP 292 intracellular Ca2þ 295f, 297 phosphatidylinositol 3-kinase pathway 292 PI hydrolysis 292 structure 297 mGluR6 236, 299, 303t ON bipolar cells 300–301 distribution 307 knockout animals 300–301 structure 300 mGluR7 299, 303t anxiety 300 distribution 306–307 epilepsy therapy 300 functions 300 structure 300 mGluR8 299, 303t anxiety 300 epilepsy therapy 300 functions 300 structure 300 pharmacology 231t, 235, 294, 307 physiological functions 236 protein partners 236 PSD complexes 96–97 adaptor proteins/scaffolding molecules 98 regulation 294 adaptor proteins 294 signaling 235 b-arrestin 302
Subject Index Homer proteins 302–303 IP3 signaling 98 phosphorylation 302 regulation 302 structure 234, 292, 302 extracellular domain 302 intracellular C-terminus 292–293 N-terminus extracellular domain 292–293 seven transmembrane region 292–293, 302 ‘venus fly-trap’ region 293–294 subunits 235 synaptic plasticity astrocytes and heterosynaptic depression 114–115 endocannabinoid LTD and 673, 678, 680–681 LTD role see Long-term depression, glutamate role ‘venus fly-trap’ 234 Metaplasticity definition 325–326 long-term potentiation NMDA receptor-dependent LTP 325 Met-enkephalin (met-ENK) precursor peptides 511 Methyl-CpG-binding protein 2 see MeCP2 3,4-Methylenedioxymethamphetamine see MDMA (methylenedioxymethamphetamine: ecstasy) Methylphenidate (MPH) dopamine D2 receptor sensitivity 406 MGS0039 298–299 Microdialysis 444 advantages 444, 445 compounds 445–446 damage minimization 445–446 damage response 446 exocytosis 446 identifications 444 sample purification 445–446 detectors 444 dialysis membrane 444 disadvantages 445 sensitivity 446 spatial resolution 446 temporal resolution 446 historical aspects 444 cortical cup perfusion 445 dialytrode 445 local perfusion 445, 445f push–pull cannula perfusion 445 HPLC 444 method 444, 445f neuropeptide metabolism 567 theory of 444 timescales 444 Microdisk electrodes, voltammetry electrode design 442 Microelectrode recordings noradrenergic neurotransmission 418 Microfilament-regulating proteins, gephyrin role 356 Microfluidic chambers, retrograde neurotrophic signaling 584, 585f technique advantages 584 Microglia activation 643 cannabinoid receptors 668–669 cytokine release synaptic modulation and 49 mGluR3 receptors 299 Microiontophoresis 436 Microtubule(s) AMPA receptors and 268 functional roles retrograde neurotrophic signaling 588 colchicine 588 nocodazole 588 synaptic vesicle transport 76–77 transport 25 Midbrain dopamine 383 non-mammalian vertebrates see Optic tectum Migraine management serotonin receptor-targeted drugs 461, 462 Miniature endplate potentials (mEPPs) 160–161, 181, 181f, 418 asynchronous release and 164 Ca2þ concentration and 162
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Miniature excitatory postsynaptic currents (mEPSCs) synaptic transmission models 106, 109 Minimal membrane-fusion machinery, SNAREs 70 Mitochondrial enzymes, chlorpromazine mechanism of action 392 Mitogen-activated protein kinase (MAPK) signaling see MAP kinase signaling pathway MMPs see Matrix metalloproteinase(s) (MMPs) mocha mouse 80 Modulatory signaling receptors, NMDA receptors see NMDA receptor(s) Monoamine(s) neuroimaging see Monoamine neuroimaging release studies 442–449 microdialysis see Microdialysis voltammetry see Voltammetry Monoamine neuroimaging 448 dopaminergic system see Dopaminergic neurons/systems serotonergic disorders see Serotonin (5-HT)/serotonergic neurons, neuroimaging see also Positron emission tomography (PET); Single photon emission computed tomography (SPECT) Monoamine oxidase(s) (MOAs) catecholamine metabolism 421–422 dopamine catabolism 386 Monocular deprivation see Visual deprivation Monte Carlo (MC) methods synaptic transmission models 105 diffusion equation 105 Morphine 532 addiction MCH neurons 555 5-HT3 receptor 466 Mossy fiber(s) 33–34 CA3 synapses, presynaptic kainate receptors 314–316 Mossy fiber–CA3 synapses, presynaptic kainate receptors 314–316 Motivation melanin-concentrating hormone 555 orexins (hypocretins) 555 Motor endplate see Neuromuscular junction (NMJ) Motor neuron(s) (MNs) 150 endplate see Neuromuscular junction (NMJ) morphology axon branching 150 motor unit component 150 see also Motor unit(s); Neuromuscular junction (NMJ) neuromodulation serotonergic 474 neurotrophic factors GDNF family ligands (GFLs) 606 RET 606 size principle 148 vertebrate spinal 142 Motor neuron development axon branching 150 NMJ formation and target innervation precision 174 see also Neuromuscular junction development, mammalian Motor neuron disease (MND) see Amyotrophic lateral sclerosis (ALS) Motor unit(s) definition 150 force production factors regulating 150 maturation (mammalian) precision 174 size 150 reduction by synapse elimination 150 evidence 151 vertebrate 142 see also Motor neuron(s) (MNs); Muscle(s)/muscle fiber(s); Neuromuscular junction (NMJ) MPEP, mGluR5 agonists 297–298 MPins/LGN protein, NMDA receptor trafficking 286 Mu¨ller cells (glia) D-serine role 337 growth factors 619, 622t BDNF 619 FGF-2 619 NGF 619 NT-3 619 TNF-a 619 VEGF 619 Multiple endocrine neoplasia 2 (MEN2), RET mutations 599–600 Multiple production, neuropeptide precursor peptides 511–512
728 Subject Index Multiple sclerosis (MS) Schwann cells 617 Multiple transcripts, CRH receptors 545 Multivesicular bodies (MVBs) retrograde neurotrophic signaling 587–588 Munc13 genes/proteins active zone 56t CAZ role 91–94, 92t knockout mice 16 neuropeptide release 521 Munc18 gene/protein 16 SNARE interactions 70f, 72 syntaxin 1 binding 68–69, 68f, 72 Mu-opioid receptor see Opioid receptor(s) Muscarinic acetylcholine receptors (mAChRs) 494–502, 495f acetylcholine binding 494, 495f, 498 activation 501–502 agonists 482t amino acid sequence alignment 494, 499f antagonists 482t, 500–501 arecoline 500–501 coding regions 494–495 distribution 485 gallamine 498 gene structure 498 groups 494 history 494 interneurons 501 kinase activity effects 502 knockout mice studies 501 ligands 495f, 498 M1 receptors activation 485 gene structure 498–500 role indications 501 signal transduction 501 M2 receptors 494, 495f activation 485 gene structure 498–500 role indications 501 signal transduction 501 M3 receptors 498 activation 485 gene structure 498–500 role indications 501 signal transduction 501 M4 receptors, signal transduction 501 M5 receptors dopamine relationship 501 role indications 501 signal transduction 501 phosphate activity effects 502 physiological functions 500 signal transduction 500f, 501 structure 494 subtypes 482t, 494 evolutionary relationship proposal 500f properties 496t transmembrane domains 498f Muscle(s)/muscle fiber(s) action potential generation 180, 184 activation threshold 180, 183f, 184 Nav1 channels and 184 safety factor 186 species differences 186, 187f see also Neuromuscular transmission classification/types NMJ efficacy and 159 nonEx-MIF see NonEx-MIF (electrically, inexcitable, multiply innervated muscle fibers) slow see Slow muscle fiber(s) contraction neural control (innervation) 141 synaptic delays 208–209 development differences between muscle types 150 NMJs (mammalian) see Neuromuscular junction development, mammalian force/effort factors regulating 150 see also Motor neuron(s) (MNs) motor units see Motor unit
neural control (innervation) 141 autonomic 141 contraction 141 see also Neuromuscular junction (NMJ) neuromuscular junction see Neuromuscular junction (NMJ) passive cable properties and EPP generation 182, 183f input resistance 182–183 membrane capacitance and 184 muscle diameter and 182 spatial factors 183 polyaxonal innervation see Polyaxonal (polyneuronal) muscle innervation size passive cable properties and diameter 182 Muscle-specific kinase (MuSK) 44 neuromuscular junction development 126, 178 knockout mice and 178 Muscle spindles slow (nonexcitable multiply innervated) fibers 188 MuSK see Muscle-specific kinase (MuSK) Mute synapses see Silent synapses Myasthenia gravis (MG) 506 congenital see Congenital myasthenic syndrome (CMS) Myelination regulation/control neuregulins 620 see also Oligodendrocyte(s); Schwann cell(s) Myenteric plexus CCK receptors 562 Myosin(s) striated muscle 141 transport using see Myosin-mediated transport Myosin-mediated transport NMDA receptor trafficking 286–287
N Naþ see Sodium ions (Naþ) NADPH metabolic role 240f, 241–242 Naþ/Kþ-ATPase see Sodium pump (Naþ/Kþ-ATPase) Naþ pump see Sodium pump (Naþ/Kþ-ATPase) Narcolepsy pathology/pathogenesis melanin concentrating hormone 555 orexin system/orexin receptor 555 Narp postsynaptic development role 22 Nasal organs, P2 receptors 646 Natriuretic peptide receptors sympathoeffector junctions, presynaptic modulation 561 Nav see Voltage-gated sodium channels (VGSCs) NBQX, kainate receptor antagonists 314, 314t NC1 domain, type IV collagen a chains 174–175 NCAMs see Neural cell adhesion molecules (NCAMs) Ncs-1 gene/protein short-term synaptic plasticity 133 Negative cooperativity model, dopamine D2 receptor 405, 407f, 408 Neocortex 5HT-2 serotonin receptor 474 anatomy/organization interneurons subtypes 221, 222 Neocortical areas (fields) primary sensory areas auditory see Auditory cortex NEPH family adhesion molecules 42 Nephrin 42 Nerve growth factor (NGF) 576–583 cell differentiation/morphology 573 cholinergic basal forebrain neurons 491, 492f clinical applications 573–574, 574t Alzheimer’s disease 492–493, 573–574 peripheral neuropathy 574–575 developmental role 577 axon growth 577 cell survival 577 postnatal 577 Ad high-threshold mechanonocieptors 577 glial cells 622t astrocytes 618 Mu¨ller glial cells 619
Subject Index oligodendrocytes 618 Schwann cells 617 historical aspects 570, 576 neural injury and regeneration peripheral nerves 623 see also clinical applications (above) NGF 2.5S 576 NGF 7S 576 nociception 578, 579f Hargreaves test 579 hyperalgesia 578–579 long-term effects 580 nociceptor (peripheral) sensitization 579, 580f, 581 via p75 579 via trkA 579 retrograde signaling 584 Campenot chamber studies 584 independent 587 microfluidic chamber studies 584 satellite cells 617 targeted deletion 572 Nerve terminal impulse (NTI), noradrenergic intermittence mechanisms 419 Netrin(s) 40t, 43 afadin binding 43 integrin binding 43 structure 39f Netrin-1 axonal growth/guidance axonal pathfinding 621 Neurabin, postsynaptic development 24–25 Neurabin II (spinophilin) postsynaptic development 24–25 Neural cell adhesion molecules (NCAMs) 38, 40t, 41 genes 41 GFL receptors 599, 602, 603f neuromuscular junction 41 polysialic acid binding 41 structure 39f, 41 Neural maps/representations development/maturation activity-dependent synaptic competition barrel cortex/somatosensory maps 32 retinotopic maps 31–32 tonotopic maps 32–33 sensory see Topographic maps/representations see also Topographic maps/representations Neural regeneration/repair axonal see Axonal regeneration ischemic stroke see Ischemic stroke peripheral nervous system see Peripheral nerve regeneration/repair similarity to developmental NMJ synapse elimination 152 Neuregulin(s) (NRGs) glial cells 622t oligodendrocyte differentiation 620 myelination 620 Neuregulin-1 (NRG-1) NRG-1a oligodendrocyte differentiation 619 Schwann cell differentiation 620–621 NRG-1b oligodendrocyte differentiation 619 Schwann cell differentiation 620–621 Neurexin(s) 21, 38, 43 CASK interactions 14–15 extracellular interactions neuroligins see Neurexin–neuroligin complex a-latrotoxin binding 43–44 neuroligin recognition see Neurexin–neuroligin complex presynaptic development 14 synapse stabilization 15–16 structure 39f, 43–44 synaptic differentiation 44 Neurexin–neuroligin complex 14 b-Neurexins 40t Neuritis see Neuropathy Neuroactive steroids see Neurosteroids Neurocan L1-family cell adhesion molecule interaction 41–42 Neurodegeneration/neurodegenerative disease adenosine receptors 637t in epilepsy see Epilepsy glutamate role 237
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gonadal hormones and 615 growth factors/neurotrophic factors BDNF 622 CNTF 622 fibroblast growth factor-2 (FGF-2) 622 GDNF 622 glial cell growth factors 622 retrograde signaling disruption 588 see also Growth factor(s); Neurotrophin(s) nitric oxide role 690–695 prevention see Neuroprotection/neuroprotective agents synapse elimination vs. 152 Wallerian see Wallerian degeneration see also Apoptosis; Neurotoxicity Neurofascin 40t, 41–42 localization 42 Neurohypophysis anatomy/organization hypothalamic connections magnocellular system see Magnocellular hypothalamo–neurohypophysial system (mHNS) see also Hypothalamic–pituitary–adrenal (HPA) axis remodeling 337–338 D-serine levels 338 Neuroimaging dopamine see Dopaminergic neurons/systems monoamines see Monoamine neuroimaging SNAREs 69–70 Neuroinflammation see Inflammation Neurokinin (NK) receptors 559–560 NK-1 structure 525 Neurokinin A (NKA) 559–560 Neuroleptics see Antipsychotic drugs (neuroleptics) Neuroligin(s) 38, 40t, 43 neurexin recognition 14 postsynaptic density and differentiation 21 aggregation 21 clustering effects 22 isoforms 21 knockout mice 21 NMDA receptor recruitment 23, 287 presynaptic development 14, 128 synapse stabilization 15–16 structure 39f Neuroligin-1, postsynaptic differentiation 20 scaffold role 22 Neuroligin-2 postsynaptic differentiation 21, 22 Neuromodulation acetylcholine see Acetylcholine (ACh) endocannabinoid system endocannabinoid LTD 678 see also Endocannabinoid system mechanisms GPCR-mediated mGluRs 303 Neuromuscular junction (NMJ) 150, 158, 160f, 175f acetylcholine receptors see Neuromuscular junction, acetylcholine receptors acetylcholine release see Neuromuscular transmission acetylcholinesterase role see Acetylcholinesterase (AChE) agrin see Agrin, neuromuscular junction role basal lamina see Neuromuscular junction, basal lamina cell types 155, 174 motor neurons see Motor neuron(s) (MNs) muscle fibers see Muscle(s)/muscle fiber(s) Schwann cells see Perisynaptic Schwann cells (PSCs) see also Motor unit(s) definition 141 development see Neuromuscular junction, development evolution 158 invertebrates see Neuromuscular junction, invertebrate as model synapse 158 norepinephrine measurement 416 plasticity see Neuromuscular junction, plasticity postsynaptic terminals see Neuromuscular junction, postsynaptic presynaptic terminals see Neuromuscular junction, presynaptic (motor nerve terminal) synaptic efficacy 159 specializations and 158
730 Subject Index Neuromuscular junction (NMJ) (continued) transmission across see Neuromuscular transmission vertebrate 147f boutons 145 frog short-term plasticity studies 168 neurotransmitters 146 postsynaptic membrane 146, 147f presynaptic, species differences 159 ‘quantal content’ of release 146 transmission efficacy 145–146 variations in 145, 146f voltage-gated sodium channels 146 voltage-gated ion channels 146 sodium channels see Neuromuscular junction, postsynaptic Neuromuscular junction, acetylcholine receptors 160f, 180, 182f acetylcholine binding 180–181 aggregation/clustering fibronectin role 176 functional fold location 174, 184 open channel conductance 180–181 subunit composition 180, 182f Neuromuscular junction, basal lamina 174–179 AChR expression/aggregation and see Neuromuscular junction, acetylcholine receptors cell surface and membrane receptors 177 cadherins 178 dystroglycan complex 177 integrins 178 intracellular signaling 178 presynaptic role 127, 178 MuSK see Muscle-specific kinase (MuSK) components 174 acetylcholinesterase 177 collagen IV 174, 175f fibronectin 176 HSPGs 176 laminins 175 laminin a4 44 laminin b2 44 nidogens (entactins) 176 ECM-mediated interactions 44 functional roles 174 neural cell adhesion molecules (NCAMs) 41 protease regulation 179 matrix metalloproteinases (MMPs) 179 collagen IV cleavage 175 tissue inhibitors of MMPs (TIMPs) 179 Neuromuscular junction, development 127f invertebrates 150 C. elegans 127–128 mammalian see Neuromuscular junction development, mammalian Schwann cells and 46–47 Neuromuscular junction, invertebrate development 150 C. elegans 127–128 Neuromuscular junction, plasticity short-term presynaptic 168–173 enhancement (increased transmitter release) 168 augmentation 169 comparison between species 170 component definitions 168 facilitation 169 kinetic properties 170 mechanism 170 see also Calcium ions (Ca2þ)/calcium signaling potentiation and PTP 169 relationship between components 170 time constants 169–170 model development 173 short-term depression 168, 171, 172f mechanisms 172 short-term synaptic memory 173 techniques used to study 168 component analysis using media modification 168, 169f endplate potentials and 168 frog NMJ preparations 168 see also Short-term synaptic plasticity Neuromuscular junction, postsynaptic 184 folds 174, 184, 185f acetylcholine receptor location 174, 184 Nav1 channel location 184–185
Nav1 channels 184 location 184–185 neurotransmission and see Neuromuscular transmission, postsynaptic events structure–function relationship 185–186, 185f Neuromuscular junction, presynaptic (motor nerve terminal) neuroplasticity and see Neuromuscular junction, plasticity neurotransmission see Neuromuscular transmission, presynaptic events structural properties 158 active zone 159, 160f boutons 159, 159f functional independence 158–159 vertebrate species differences 159 Neuromuscular junction, synapse elimination 150–157 consequences 150 reduction in motor unit size 150 molecular mechanisms 154 neurotrophic factors 154 proteases and ubiquitin–proteasome system 155 muscle fiber type specificity and selective elimination 154 neurodegenerative disease and 155 differences 156 similarities 153, 156 SOD mutant ALS mice and 153, 156 nonneuronal cell types and 155 polyaxonal to monoaxonal (p to m) transition 150 axonal regeneration similarity 152 elimination vs. degeneration 152 evidence for electrophysiological 150–151 histological 151, 151f Jansen, Brown and van Essen‘s study 151 partial denervation at birth and 151 real-time in vivo imaging 152 axosomes 153 fate of losers 153 local competition 152 retraction bulbs 153 transgenic XFP mice 152 synaptic competition and 156–157 activity-dependent 34, 154, 156–157 conclusions about 154 intrinsic MN hierarchies 153 local 151–152 asynchrony 152–153 axonal transport and 153 endplate takeover 152–153 flip–flop behavior 153 in vivo imaging 152 see also Synaptic competition timing/rates 151 Neuromuscular junction development, mammalian agrin role 126 central synapses vs. 11–12 early development AChR accumulation agrin-induced 126 see also Neuromuscular junction, acetylcholine receptors polyaxonal innervation 150 change to monoaxonal see Neuromuscular junction, synapse elimination evidence for 150 Schwann cells 155 timing of muscle innervation 150 motor neuron development 150 ACh release from growth cones 150 precision 174 see also Motor neuron development motor unit maturation muscle fiber homogeneity development 154 perisynaptic Schwann cells and early development 155 synapse elimination 48–49, 155 synaptogenesis 46–47 postsynaptic maturation 126 presynaptic maturation 126 synapse elimination see Neuromuscular junction, synapse elimination retrograde signaling and 126, 127f skeletal muscle fiber development other muscle types vs. 150 Neuromuscular synapse see Neuromuscular junction (NMJ) Neuromuscular transmission 180
Subject Index acetylcholine role 145–146, 158, 180 quanta 161–162, 181 sites of release 182 termination see Acetylcholinesterase (AChE) see also Neuromuscular junction, acetylcholine receptors adenosine triphosphate (ATP) and see Adenosine triphosphate, transmitter role digital nature 158 efficacy vesicle processing and 166 endplate current (EPC) 161–162 endplate potentials (EPPs) 160–161 miniature endplate potentials see Miniature endplate potentials (mEPPs) neurotoxins and see Neurotoxin(s) NMJ plasticity and see Neuromuscular junction, plasticity postsynaptic events see Neuromuscular transmission, postsynaptic events presynaptic events see Neuromuscular transmission, presynaptic events reliability 186 retrograde signaling and development and 126, 127f homeostatic signaling and 129–130 safety factor 186 postsynaptic specialization and 186 presynaptic specialization and 186 species differences 186, 187f toxin effects see Neurotoxin(s) Neuromuscular transmission, postsynaptic events 180–188 acetylcholinesterase and 186 endplate current (EPC) 161–162 AChR role 180 acetylcholine activation 180, 182f single quantum-induced current see Miniature endplate potentials (mEPPs) sites of ACh release and 182 termination of action 186 from EPC to EPP 182 endplate potentials (EPPs) 160–161, 180, 181f, 182 miniature see Miniature endplate potentials (mEPPs) muscle fiber passive cable properties and 182, 183f input resistance 182–183 membrane capacitance 184 muscle diameter and 182 spatial factors 183 muscle action potential generation 180, 184 activation threshold 180, 183f, 184 Nav1 channels and 184 safety factor 186 species differences 186, 187f slow (nonexcitable multiply innervated fibers: nonEx-MIF) fibers 186 higher vertebrates intrafusal fibers 188 mammalian extraocular fibers 188 lower vertebrates (frog) 187 structure–function relationship and 185–186, 185f Neuromuscular transmission, presynaptic events 158–167 anatomical considerations 158 active zones 159 species differences 159 synaptic strength and 159 classical (Katz) transmitter release 160 asynchronous release 164 binomial statistics 163, 164f difficulties/problems 163 expected fluctuations 163 factors underlying probability (p) 164 general features 163 number of release sites (n) and countable AZs 163 calcium hypothesis see Calcium ions and neurotransmitter release experimental events seen 160–161 Poisson statistics 162 probability of release (p) 163 binomial statistics and 164 Ca2þ concentration and 162, 164 quantal hypothesis 161 motor neuron action potential 180 vesicle processing 164 endocytosis (compensatory) 165, 166f bulk membrane invagination 165 clathrin-mediated 165 macroendocytosis 165–166 models 165, 166f study methods 165, 167f
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evolutionary conservation 165–166 mobilization of reformed SVs to AZ 165 proportion of vesicles recycled 164–165 refilling 165 strategies for low vs. high-level use 165–166 synaptic strength and 166 Neuron(s) action potentials see Action potential(s) brain 28–29 development neuropeptide Y (NPY) 539 excitability see Neuronal excitability gap junctional communication 190 connexin expression 189 electrical coupling 191 see also Gap junction(s) hyperpolarization, GABAB receptors 362 morphology/structure brain function 27 cytoskeletal filaments see Cytoskeleton dendrites see Dendrites/dendritic arbor secretory pathway see Secretory pathway sodium pump (Naþ/Kþ-ATPase) see Sodium pump (Naþ/Kþ-ATPase) survival FGF-2 621 TGF-b 621 see also Neuroprotection/neuroprotective agents; Neurotrophin(s) Neuronal activity developmental filopodia motility regulation 7 oscillatory see Oscillatory neuronal activity see also Neuronal excitability Neuronal atrophy see Neurodegeneration/neurodegenerative disease Neuronal degeneration see Neurodegeneration/neurodegenerative disease Neuronal excitability glucose effects on MCH neurons 553–555 see also Neuronal activity; Neuroplasticity Neuronal firing rates 5HT-1 receptor 450–451 5HT-2 receptor 450–451 5HT-3 serotonin receptors 450–451 5HT-4 serotonin receptors 450–451 Neuronal nitric oxide synthase (nNOS) 684 dystroglycan complex component 177 expression 690 location 685, 686f regulation 690 Neuronal plasticity see Neuroplasticity Neuronal regeneration see Neural regeneration/repair Neuronal spikes see Action potential(s) Neuronal transport postsynaptic scaffolding proteins 25 Neuropathic pain GABAB receptor ligands 364–365 glutamate receptors and mGluR5 agonists 297–298 Neuropathy diabetes mellitus and see Diabetic neuropathy pain see Neuropathic pain therapy nerve growth factor (NGF) 574–575 Neuropeptide(s) actions 557 clinical potential 558 coexistence/cotransmission 525–531, 564, 565f acetylcholine 528 amino acids 528 biogenic amines 528 brain-derived neurotrophic factor 526f, 528 co-storage 525, 557 definition 564 distinct receptor complexes 530 fast vs. slow cotransmitters 530 functional implications 530 hypothalamus 527 neurotransmitters 516, 521–522 and neurotransmitters 528 nitric oxide 528 permutations and chemical coding 557 postsynaptic regulation 530 presynaptic regulation 530 retina 528
732 Subject Index Neuropeptide(s) (continued) spinal cord 526f, 527 target cells 530 conservation 564 definition 525 differential distribution 516–517 electrophysiology see Neuropeptides, electrophysiology functional roles 557, 517, 525, 565–567 metabolism see Neuropeptide metabolism neurotransmission 528 slow-acting 529 intracellular Ca2þ 529 neurotransmitters vs. 519, 525, 564 antagonism 522 storage 519 synergy 522 synthesis 519 receptors see Peptidergic receptors release see Neuropeptides, release sensory systems see Neuropeptides, sensory systems storage see Neuropeptides, storage synthesis see Neuropeptides, synthesis Neuropeptide metabolism 564 microdialysis 567 peptidases 521–522, 567 in vivo push–pull cannulae 567 Neuropeptides, electrophysiology 564–569 peptidergic interneurons 567 antiepileptic agents 568 dendritic shafts 567–568 dendritic spines 567–568 electron microscopy 567–568 excitatory postsynaptic current (EPSC) 568 glutamate release inhibition 567–568 Kþ currents 567 use-dependent plasticity 568 synaptic potentials 568 AMPA receptors 568 membrane permeability 568 NMDA receptors 568 selective agonists 568 selective antagonists 568 transgenic mice 568 in vivo recordings 568 volume transmission 564, 565f concentrations 564 definition 564 peptide-induced receptor trafficking 564, 566f Neuropeptides, release 519–524, 525, 529, 557, 564 Ca2þ-mediated release 516–517, 565–567 calcium-dependency 519 coexistence, combinations 529–530 discharge frequencies 567 exocytosis 529–530 exocytosis machinery 565–567, 566f high-frequency stimulation 516–517 molecular release mechanisms 521 Ca2þ-dependent activator protein for secretion (CAPS) 520f, 521 large dense-core vesicle exocytosis 521 Munc13 521 vesicle/membrane fusion 521 nonuniformity 522 axon terminal vs. somatodendritic release 522 between neurons 522 within neurons 522 temporal changes 522 physiological effects 523 addiction 523–524 depression 523–524 eating disorders 523–524 plasma membrane fusion 564–565 signal termination 521 extracellular peptidases 521–522 SNARE machinery 521 Stimulus 519 time-scale 519 autoinhibition 519–521 burst-pattern firing 519–521, 520f high-frequency firing 519–521 latencies 519–521 transient pores 529–530
Neuropeptides, sensory systems 525–526 Neuropeptides, storage 511–518, 525 cell localization 519, 520f coexistence/co-storage 525, 557 genetics 517 antisense RNA studies 517–518 chromogranin 517–518 DIMM gene/protein 518 Drosophila 518 granule biogenesis 514 activator protein (AP-)1 516 clathrin 515 cytosolic proteins 516 furin 516 golgi-associated g-ear-containing adenovirus death protein (ADP)-ribosylation-factor-binding-protein (GGA) 515 golgi complex 514 granins 515–516 membrane remodeling 515 phosphofurin acidic cluster-sorting protein (PACS-)1 516 selective aggregation 515–516, 515f sorting by retention model 515 sorting for entry model 514–515 synaptotagmin IV 516 trans-Golgi network 514 transmembrane domain proteins 515 vesicle-associated membrane protein-4 (VAMP-4) 516 large dense-core vesicles see Large dense-core vesicles (LDCVs) large granular vesicles 525, 526f Neuropeptides, synthesis 525, 511–518, 557, 564 endoplasmic reticulum 511, 525–526 endoproteolytic cleavage 511 genes 564 Golgi complex 511, 525–526 posttranslational modifications 512, 564 precursor peptides 511, 512f conservation 511 multiple production 511–512 NH2-terminal signal sequence 511 N-linked oligosaccharides 511, 512f protein folding disorders 512 regulation 512 precursor-processing enzymes 512 N-acetyltransferase 513, 513f aminopeptidase 513, 513f carboxypeptidase, (CPE) 513 glutaminyl cyclase, (QC) 513 knockout animals 514 peptidylglycine a-amidating monooxygenase (PAM) 513 propeptide/prohormone convertases (PCs) 512–513 regulation 513–514, 513f transgenic animal models 564 trans-Golgi network (TGN) 511, 512f, 526, 527f Neuropeptide Y (NPY) 538–543 antisense RNA 539 biological activity 539 alcohol consumption 539 eating disorders 523–524 energy expenditure 539 epilepsy 523–524 food intake 539 neuronal development 539 reproductive regulation 539 seizures 539 coexistence/cotransmission 538 aspartate 528 co-storage 560 GABA 521–522, 528, 538 galanin 527 norepinephrine 528, 538, 539 retina 528 somatostatin 538 disease/dysfunction cardiovascular dysfunction 539 distribution 538 expression 538 genetics 538 knockout animals 523–524 seizures 539 NPY2-36 538 NPY3-36 538 receptors see Neuropeptide Y receptor(s)
Subject Index regulation 538 ghrelin 538 glucocorticoids 538 growth hormones 538 protein kinase A 538 protein kinase C 538 structure three-dimensional 538, 539f synthesis 538 aminopeptidase P 538 dipeptidyl peptidase IV 538 peptidylglycine a-amidating monooxygenase 538 Neuropeptide Y receptor(s) 540 dimerization 542 drug development 542 internalization 542 b-arrestin 2 542 pancreatic polypeptide (PP) binding 540 peptide YY (PYY) binding 540 regulation 542 Y1 receptor 540, 540t agonists 540 antagonists 542 drug development 542 feeding behavior 540, 542 localization 540 selective antagonists 540 Y2 receptor antagonism 540–541 Y2 receptor 540, 540t agonists 542–543 biological activity 540–541 drug development 542–543 localization 540 as presynaptic autoreceptor 540–541 structure 540 sympathoeffector junction, presynaptic modulation 560 Y1 receptor antagonism 540–541 Y4 receptor 540, 540t, 541 agonists 542–543 distribution 541–542 drug development 542–543 rat vs. human 541–542 structure 541–542 Y5 receptor 540, 540t, 542 antagonists 542 drug development 542 feeding behavior 542 Neurophysiology norepinephrine 430–441 Neuroplasticity activity-dependent see Activity-dependent plasticity ATP neurotransmission 643 gap junctions and transmission between neurons 209 neuromuscular junctions see Neuromuscular junction, plasticity pain and see Pain synaptic see Synaptic plasticity visual development, neural activity in see Visual development, neural activity role Neuroprotection/neuroprotective agents ATP neurotransmission and 643 IGFs and neuroprotective surveillance 614 Neuroproteomics postsynaptic density analysis 96, 99 AMPA receptor composition 99 cytoskeletal proteins 100 Neuropsychiatric disorders see Psychiatric disorders Neuroradiology see Neuroimaging Neuroregeneration see Neural regeneration/repair Neurosteroids GABAA receptors and 353 Neurotensin (NT) coexistence, retina 528 receptors see Neurotensin receptor(s) Neurotensin receptor(s) 557 Neurotransmission 84 action potentials see Action potential(s) classical (Katz) theory 160 cross-talk 114–115 at the NMJ see Neuromuscular transmission synaptic vesicle cycling see Synaptic vesicle cycle transmitter release see Neurotransmitter release see also Neuromodulation
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Neurotransmitter(s) 84 autonomic see Autonomic nervous system (ANS) endocannabinoid suppression 669–670, 671f filopodial motility regulation 7 gliotransmitters vs. 121–123 see also Gliotransmitters mechanisms of action 84 neuromodulators see Neuromodulation neuropeptide coexistence 516, 521–522 postsynaptic differentiation induction 21 presynaptic regulation, GABAB receptors 368 release see Neurotransmitter release small synaptic vesicles (SSVs) 519, 525, 526f, 564–565 distribution 529 spillover and synaptic cross-talk 114–115 see also Synaptic spillover synthesis 79 transporters/uptake excitatory amino acids see Excitatory amino acid transporters (EAATs) proton electrochemical gradients 80 Neurotransmitter release astrocytes and gap junctions and glutamatergic transmission 192 neurotoxins and 120–121 SNAP-23 121–123 synaptotagmin IV 121–123 see also Astrocytes, communication and signaling asynchronous release 164 calcium role see Calcium ions and neurotransmitter release classical (Katz) theory 160 calcium hypothesis see Calcium ions and neurotransmitter release quantal hypothesis 161 quantal nature 161 readily releasable vesicles and 11 spillover limitation 248, 249f, 250, 251 target-dependent control 128, 128f homeostasis and 130 see also Retrograde signaling vesicular see Synaptic vesicle(s) see also Neuromuscular transmission Neurotrophic factors see Neurotrophin(s) Neurotrophic–pTrk signaling complex 589 Neurotrophic signaling, retrograde see Retrograde neurotrophic signaling Neurotrophin(s) 570–575 axonal growth 582 cell survival 572 apoptosis 572 CNS 573 peripheral nervous system 573 central sensitization 581 clinical implications 582 developmental roles 578 cell differentiation/morphology 573 BDNF 573 NGF 573 NT3 573 dysregulation/dysfunction 614 family members 576 historical aspects 570 neuropathic pain 581 nicotinic acetylcholine receptors and 505 pharmacology 573, 574t Alzheimer’s disease 573–574 amyotropic lateral sclerosis 574 availability 574 delivery methods 574 Huntington’s disease 574 Parkinson’s disease 573–574 spinal cord injury 574 stroke 574 production 570 receptors 570, 576 p75 see p75 receptor(s) specificity 570 Trks see Trk receptor(s) release 570 intracellular sorting 570 retrograde signaling see Retrograde neurotrophic signaling Schwann cells 617 signaling mechanism 576 p75 577 trkA 576
734 Subject Index Neurotrophin(s) (continued) synaptic competition role activity-dependent 36 NMJ synapse elimination and 154–155 synaptic function 573 BDNF 573 long-term potentiation 573 synaptic plasticity 573 synthesis 570 therapeutic uses BDNF see Brain-derived neurotrophic factor (BDNF) clinical trials 574 CNTF see Ciliary neurotrophic factor (CNTF) GDNF see Glial cell line-derived neurotrophic factor (GDNF) neurotrophin-3 see Neurotrophin-3 (NT-3) NGF see Nerve growth factor (NGF) see also Growth factor(s) Neurotrophin-3 (NT-3) clinical applications 574t developmental role cell differentiation/morphology 573 glial cells 622t astrocytes 618 Mu¨ller glial cells 619 oligodendrocytes 618 historical aspects 570 regeneration/repair role peripheral nerve regeneration 623 satellite cells 617 signaling 576 synapse formation/maturation 13 Neurotrophin-4 (NT-4) astrocytes 618 glial cell production 622t historical aspects 570 peripheral nerve regeneration 623 Neurturin (NTN) 599 dopamine neurons 605–606 enteric nervous system 606 knockouts 605–606, 605t structure 599, 600f Neutrin-G ligands (NGLs) 43 Newt(s), skeletal muscle innervation 143 NGF see Nerve growth factor (NGF) NGL (adhesion molecule) function 21–22 interactions 21–22 NH2-terminal signal sequence, neuropeptide precursor peptides 511 Nicotinamide adenine dinucleotide phosphate (reduced) see NADPH Nicotine addiction 506 responses to see Nicotine response Nicotinic acetylcholine receptors (nAChRs) 481, 503–507 agonists 482t allosteric states 505 antagonists 482t diversity 503 gene expression 506 historical aspects 503 homology 503 intrinsic cationic channel 503, 504f, 505 permeability 503 muscle compartmentalized expression acetylcholine receptor-inducing activity 506 during development 505–506 electrical activity 505 extrasynaptic distribution 505–506 neurotrophic factors 505 postsynaptic distribution 505–506 pathological roles 506 nicotine addiction and see also Nicotine physiological roles 506 posttranslational regulation 505–506 subtypes 482t subunits/structure 481–484, 503 activation gate 504–505 assembly 503, 504f structure 503–504
transmembrane topology 503 ACh binding domain 503–504, 504f cytoplasmic segments 503–504 transcription 505–506 Nicotinic receptors see Nicotinic acetylcholine receptors (nAChRs) Nidogen-2, basal lamina 176 Nidogens (entactins), basal lamina 176 collagen IV binding 175 Nigrostriatal dopaminergic pathway 383 Nitric oxide (NO) 684–689 antiapoptotic effects 689 autonomic transmission see Nitric oxide, transmitter role biosynthesis 684, 685f calcium dependence 684 central nervous system 688, 690, 691f role indications 690–691 cerebral blood flow regulation 690 D-amino acid oxidase regulation 334 development 684 diffusion role 690 disease role 689 neurodegeneration 690–695 Alzheimer’s disease 694–695 amyotrophic lateral sclerosis 695 Parkinson’s disease 694 downstream targets 687 cGMP-dependent protein kinases 687–688 cyclic nucleotide-gated channels 688 HCN channels 688 phosphodiesterases 688 EDRF hypothesis 686–687 free radical effects 692 functions 684, 686 nervous system role 685 gut effects 690 historical aspects 684 immune system 691 inactivation of 687 inhibitors 684–685 lipid peroxidation 689 molecular targets 691 cGMP accumulation regulation 691–692 nervous system generation 685 neurotoxic effects 692 apoptosis-inducing factor 692–693, 693f brain ischemia effects 693, 694f neurotransmitter function see Nitric oxide, transmitter role NO (GC) receptors 687 binding effects 687 oxygenase domain 684, 685f oxygen effects 692 pathophysiological signaling events 691f peroxynitrite 689, 692 physicochemical properties 684 physiological signaling events 691f PSD-95 685 reductase domain 684, 685f serine racemase regulation 334 S-nitrosylation 692 autoregulatory mechanisms 692 structure 684 synaptic plasticity 689 long-term depression and cerebellar LTD 680 long-term potentiation and 690–691 Nitric oxide (NO)-GMP signaling pathway 689 rat brain 686–687, 686f Nitric oxide, transmitter role coexistence/cotransmission galanin 528 somatostatin 528 Nitric oxide synthase(s) (NOS) 690 endothelial (eNOS) 684, 690 amyloid b effects 694–695 expression 690 phosphorylation 684 inducible (iNOS) 684, 690 expression 690 mitochondrial implications 690 neuronal (nNOS) see Neuronal nitric oxide synthase (nNOS) Nitrogen homeostasis, GABA metabolism 343
Subject Index S-Nitrosylation biological targets 692 nitric oxide 692 NK receptors see Neurokinin (NK) receptors N-linked oligosaccharides, neuropeptide precursor peptides 511, 512f NMDA receptor(s) 96–97, 230, 233f, 276–283 activation 123, 284, 322, 371 co-agonists 333 glutamate concentrations needed 250 glycine binding site 336–337 structure–function relationships 336–337 without LTP 322 agonists 336–337 GABA metabolism 344 biological properties 276 calcium-dependence 280, 280f degradation 290 ubiquitination 290 desensitization 280 developmental see NMDA receptors, developmental role disease and dysfunction neurodegenerative disease 338 functional properties 277 Ca2þ permeability 96–97, 277–278, 279–280 co-agonists 115 glycine as 276, 280, 336–337 D-serine as 280 as coincidence detector 279 endogenous allosteric modulators 280, 281f oxidizing agents 280–281 polyamines 280–281 zinc 280–281 glutamate binding 276 ion channel opening 276 Mg2þ blockade 96–97, 276–277, 278f D-serine as coagonist 115 gene(s) alternative splicing 277 glycine-insensitive 280 glycine-sensitive 280 high single channel conductance 276 kinetics 277–278, 279f learning and memory role 278–279 see also synaptic plasticity (below) neuropeptide synaptic potentials 568 nitric oxide and 684 activation effects 685 neuronal NOS 690 NR1 subunits 284–285 C2’ cassette 284–285 PDZ binding motif 284–285 splice variants 284–285 NR2 subunits 23 NR2A 285–286, 287–289 NR2B 23 receptor stability 287–289 YEKL motif 289 NR3 subunits 284 phosphorylation 281 calcium/calmodulin-dependent protein kinase II (CaMKII) 281 effects 281 Fyn 281–282 H-Ras 282 protein kinase A 281 protein kinase C 281 serine/threonine phosphatase reversal 282 Src 281–282, 282f physiological function 277 physiological modulation 280 presynaptic 279 PSD complexes 96–97 development 100 recruitment, neuroligins 23 scaffolding/adaptor proteins 97–98 PSD-95 see PSD-95 receptor interactions CaMKII interactions 96–97 GABA receptors potassium conductance 368 group I mGluRs and 305–306 mGluR5 and 297 modulatory signaling 282
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cytokine receptors 282–283 insulin 282 PACAP 282 protein kinase C-Src signaling 282 receptor for activated C kinase 1 (RACK1) 282 receptor protein tyrosine kinases 282 types of 282 SALMs binding 42 signal transduction calcium entry 96–97 see also Calcium ions (Ca2þ)/calcium signaling protein kinase C 282 ‘silent’ synapses 16–17 spillover 322 subunits/structure 28, 230, 283, 284 developmental changes PSD development 100 subunit switching 289 receptor complex 283 structure–function relationship 284, 285t topology 284 synaptic plasticity 278, 289 cortical endocannabinoid-mediated LTD 678, 679f, 680f LTD role see Long-term depression, glutamate role LTP role see Long-term potentiation, glutamate role spike-timing dependent 278–279, 279f synaptic transmission EPSPs 250–251, 276, 277f properties 321 trafficking see NMDA receptor trafficking NMDA receptors, developmental role long-term depression 278–279 synaptic development/synaptogenesis ‘silent’ synapses 23, 27–28, 28f NMDA receptor trafficking 284–291 dendritic 286, 286f KIF17 286–287 membrane-associated guanylate kinases 286 mPins/LGN 286 myosin motors 286–287 vesicles 286–287 developmental switching 289 endoplasmic reticulum, role 284 extrasynaptic 289 subunit localization 289 phosphorylation 290 kinases associated 290 subunit effects 290 regulation 287 synaptic 287 clathrin-mediated internalization 287, 288f NR2/PSD-95 interactions 287 PDZ proteins 287 N-methyl-D-aspartate receptors see NMDA receptor(s) NMJ see Neuromuscular junction (NMJ) NMR see Magnetic resonance spectroscopy (MRS) No, Lorente de, Golgi impregnation studies 355 Nociceptin (orphanin FQ) knockouts 514 Nocodazole, retrograde neurotrophic signaling and 588 Non-adrenergic noncholinergic (NANC) neurons 639 cotransmission 639–640 Non-ergoline derivatives, dopaminergic agonists see Dopaminergic agonists Nonexcitable multiply innervated fibers (nonEx-MIF) see Slow muscle fiber(s) NonEx-MIF (electrically, inexcitable, multiply innervated muscle fibers) 141 anuran 143 mammals 145 Nonpeptidergic nociceptors, dorsal root ganglia (DRG) see Dorsal root ganglia (DRG) Noradrenaline see Norepinephrine/noradrenergic transmission Noradrenergic neurotransmission see Norepinephrine/noradrenergic transmission Norepinephrine (noradrenaline) see Norepinephrine/noradrenergic transmission Norepinephrine/noradrenergic transmission 394t, 414–423, 424–429 acetylcholine release inhibition 480–481 anatomy/physiology 415, 416t, 430–441 cell groups 430, 434, 435f cerebellum 415 cortex 415 hippocampus 415 locus coeruleus see Locus coeruleus norepinephrine system (LC-NE)
736 Subject Index Norepinephrine/noradrenergic transmission (continued) non-locus coeruleus projections 431t, 435 physiological function 436 postganglionic sympathetic neurons 415 serotonergic pathways and 471–472, 471f, 476 sympathetic neurons 425–426 sympathetic nervous system (SNS) vas deferens 415–416, 417f biogenic amine uptake 420 biosynthesis 414, 424 dopamine b-hydroxylase 414 tyrosine hydrolase 414 cellular mechanisms associated 439 chemical structure 424, 425f clinical implications 440 cotransmission acetylcholine 639–640 ATP 420–421, 639, 642f effects mediated by 425 autonomic 425 CNS 426 electrophysiology 418 microelectrodes 418 endocannabinoids and, suppression of neurotransmission by 669–670 ‘fight-or-flight’ response 414, 424 functional implications 440 glycolysis 239–241 GPCRs and 416t, 421–422 P2X purinoceptors 421 knockout mice 422 P2Y purinoceptors 421 historical aspects 414, 424 impulse-to-impulse basis 421, 421f intermittence mechanisms 419 adrenoceptor blockers 419 confocal microscopy 419 excitatory junction current (EJC) 419, 419f focal extracellular measurement 419 intracellular calcium 419, 420f nerve terminal impulse (NTI) 419 optical resolution 420 measurement 416 ‘fractional release model’ 417 fractional release studies 416–417 ‘intermittent release model’ 417–418 neuromuscular junctions 416 metabolism 421–422, 424, 425 neurochemistry 424 ontogeny, in brain 425 phosphorylation cascade 439 physiological function 436 postsynaptic actions 436 modulatory effects 436 receptors 421–422 b-blocker effects 429, 429t a-blockers and 429, 429t see also Adrenergic receptors release 424, 432 reuptake 421–422, 424 storage 424 structure 414, 415f sympathomimetic drug effects 428, 429t transport proteins see Norepinephrine transporter (NET) Norepinephrine reuptake inhibitors (NRIs) 421–422 Norepinephrine transporter (NET) 425 NP2 (Narp) see Narp NPY see Neuropeptide Y (NPY) NPY2-36, neuropeptide Y (NPY) 538 NPY3-36, neuropeptide Y (NPY) 538 NR1 subunits see NMDA receptor(s) Nr-CAM 41–42 NRGs see Neuregulin(s) NSFs (N-ethylmaleimide-sensitive fusion proteins) postsynaptic receptor maintenance 23–24 receptor interactions AMPA receptor trafficking 270, 273–274 GluR1long 270 GluR2short 270 mGluR2-dependent long-term depression 330 synaptic vesicle recycling 79–80 NST (nucleus of the solitary tract) see Nucleus tractus solitarius (NTS) NT-3 see Neurotrophin-3 (NT-3)
N-terminal region mGluRs 292–293 SNAREs 67–68 NTS see Nucleus tractus solitarius (NTS) NTs see Neurotrophin(s) Nuclear magnetic resonance (NMR) spectroscopy see Magnetic resonance spectroscopy (MRS) Nucleus accumbens (NAc) anatomy/physiology dopamine 383 learning and memory endocannabinoids and homosynaptic LTP 672f, 673 5HT-2 serotonin receptor 474 Nucleus basalis magnocellularis (of Meynert) (NBM) cholinergic neurons 488–489 Nucleus laminaris development, activity-dependent synaptic competition 33 Nucleus magnocellularis (NM), synapse elimination 32–33 Nucleus of solitary tract (NTS) see Nucleus tractus solitarius (NTS) Nucleus of the solitary tract (NST) see Nucleus tractus solitarius (NTS) Nucleus paragigantocellularis lateralis (PGi), locus coeruleus excitation 430 Nucleus tractus solitarius (NTS) P2X receptor stimulation 643 Numb (and homologs) endocytosis and SV cycle 85t Numb-like, endocytosis and SV cycle 85t Nutrition homeostasis melanin-concentrating hormone 553 orexins (hypocretins) 553
O Object recognition endocannabinoid role 675 Obsessive–compulsive disorder (OCD) etiology glutamate role kainate receptors 320 OCD see Obsessive–compulsive disorder (OCD) Octanol gap junction blockade 205 Ocular dominance columns (ODCs) activity-dependent plasticity and endocannabinoid LTD and 682 synaptic competition 31 Hebb’s rule and 35 temporal patterns of 35 visual deprivation experiments 31, 35 Hubel and Wiesel’s, work 31 see also Visual deprivation see also Visual development, neural activity role Oculodentodigital dysplasia (ODDD) connexin, 43 218 Oculomotor muscles see Extraocular muscles Odorant receptors (ORs) see Olfactory (odorant) receptor(s) 8-OH-DPAT 5HT-1A serotonin receptor 473–474 5HT-1 serotonin receptor 473 heart vagal tone effects 451–452 Older people see Age/aging Oleamide 5-HT7 receptor 468–469 Olfactory (odorant) receptor(s) development activity-dependent synaptic competition 33 Olfactory bulb development activity-dependent synaptic competition 33 synchronously active coupling 207 Olfactory cortex (lobe) see Piriform cortex Olfactory system(s) development activity-dependent synaptic competition 33 topographic mapping activity-dependent synaptic competition 33 olfactory bulb see Olfactory bulb Olfactory tubercles 5HT-2 serotonin receptor 474 5HT-4 serotonin receptor 476
Subject Index Oligodendrocyte(s) connexin expression 190 development differentiation growth factors 619 IGF-1 620 neuregulins 620 NRG-1a 619 NRG-1b 619 PDGF 620 gap junctional communication 190, 213, 215–216 connexins 216, 216t coupling 216 growth factors 618, 622t BDNF 618 differentiation and 619 nerve growth factor 618 NT-3 618 transforming growth factor-b 618 synaptogenesis and 46 Oligodendroglia see Oligodendrocyte(s) ON–OFF RGCs see Retinal ganglion cells (RGCs) Onuf’s nucleus, 5HT-5A serotonin receptors 454 Opiate(s)/opioid(s) 532–537, 533f autonomic nervous system parasympathetic modulation 559 bam 22 532 history 532 endogenous ligands 532 molecular components 532 physiology 533, 534f genetics approach 533 pharmacology 533 precursor peptides 511 preprodynorphin gene 532 preproenkephalin gene 532 preproopiomelanocortin gene 532 receptors see Opioid receptor(s) Opioid receptor(s) 532–537, 535f, 559 delta (d) 532–533 genetics approach 533 genes/genetics 532–533 heterogeneity 536 mutagenesis data 536 pharmacological subtype existence 536 kappa (k) 532–533 mu (m) 532–533 genetics approach 533 physiological role 533, 534f genetics approach 533 gene knockout mice studies 533 pharmacology 533 signaling 534 desensitization mechanisms 535–536 G protein-coupled receptors 534–535 intracellular loops 534–535 regulation 535 structure 534 binding 534 extracellular domains 534, 535f chronic opiates 536 anti-opioid systems 536 sympathoeffector junctions, presynaptic modulation 561 Opium 532 Optical resolution, noradrenergic intermittence mechanisms 420 Optic pathway BDNF role 592 development activity role see Visual development, neural activity role NMDA receptors see NMDA receptors, developmental role retina see Retina Optic tectum development dendritic arbor formation 29 Orexins (hypocretins) 551–556 brain actions 553 addiction 555 arousal 555 metabolism 553 motivation 555 narcolepsy 555 discovery 551
disease associations eating disorders and 523–524 distribution 551, 552f energy balance and feeding regulation 553 gene knockout mice 555 mutational effects 556 Hcrt ataxin 3 mice 555 inputs 553, 554f neuroanatomy 552f outputs 553, 554f receptors 552 affinity 552 distribution 552 Hcrt ataxin 3 mice 553 Hcrtr1 552 Hcrtr2 552 serotonergic pathways 471–472, 471f signaling cascades 552 structure 551 Oriens-lacunosum moleculare (OLM) (hippocampal), GABAA receptor selective control 355 activation 355 ORL-1 (orphanin FQ/nociceptin) receptor 532–533 Orphanin FQ see Nociceptin (orphanin FQ) Orphan receptors 557–558 5-HT2C receptor 465 Oscillatory neuronal activity fast, gap junctions and electrical coupling 223 gap junctions and see Gap junction(s) Osteichthyes see Bony fish(es), skeletal muscle Oxidizing agents, NMDA receptors and 280–281 Oxymetazoline 427 Oxytocin astrocyte–synapse relationship and 50 biological activity autoregulation 524 burst activity 524 lactation see Lactation copeptides cholecystokinin 521–522, 528 cocaine- and amphetamine regulated transcript 521–522 corticotropin-releasing hormone (CRH) 528 dynorphin 521–522 enkephalin 521–522, 528 magnocellular neurons 526, 527 parvocellular neurons 527 extracellular half-life 521–522 lactation see Lactation secretion 522–523 Ca2þ mobilization 520f, 522–523 a-melanocyte-stimulating hormone 522–523 priming 520f, 523 synthesis/storage hypothalamus magnocellular neurons 522 supraoptic nucleus 517 precursor peptides 511
P P1 receptor(s) 640, 648 A1 receptor 627, 628f, 629 agonists structure 633f antagonists structure 635f A2 receptors dopamine D2 receptor interactions 405 A2A receptor 627, 628f agonists structure 633f antagonists structure 635f autonomic nervous system 636 A2B receptor 627, 628f agonists structure 633f antagonists, structure 635f A3 receptor 627, 628f, 629 agonists
737
738 Subject Index P1 receptor(s) (continued) structure 633f antagonists structure 635f structure 633f adenylate cyclase coupling 648 autonomic nervous system 636 disorders 636 distribution 636 role indications 637t enteric nervous system 636 genes 648, 649f deletion effects 638 knockout mice models 638 ligands agonists 630 nonselective 633f allosteric modulation 634 antagonists 630, 635f definitive 630 novel 630 radio 633 regulation 629 selective agonists 648, 650t signaling pathways 628f structure 629, 648 subtypes 648 physiological effects 648 P2 neuron development, activity-dependent synaptic competition 33 P2X receptors 649, 658–663, 659f activation 658 effects 641 agonists 650t antagonists 645–646, 650t ATP binding site 660 ATP neurotransmission role 640–641 glial cells 123–124, 643 distribution 662 excitatory junction potentials 639, 641f expression/distribution 643 function 658 genes 658 products 658 subtype phenotype 658 ion permeation and gating 660 anion permeability 661 cation permeability 660 ASP349 neutralizing effects 660 calcium 660 potassium 660 sodium 660 pore dilation vs. dye uptake 661 noradrenergic transmission and 421, 422 P2X1 651, 658 antagonists 651 a,b-meATP mimicry 651 desensitization 651–652 distribution 662 P2X2 652, 658 ATP neurotransmission role 646 distribution 662 P2X3 652 antagonists 652 ATP neurotransmission role 646 a,b-meATP mimicry 652 distribution 662 nociception 644 P2X4 652 activation 652 antagonists 652 ATP neurotransmission role 643, 652 desensitization 652 distribution 652, 662 P2X5 653 ATP neurotransmission role 653 distribution 662 P2X6 653, 658 P2X7 653, 658 activation 653 agonists 653 C-tail 658–660
distribution 662 expression 646 nonexcytotic gliotransmitter release 123–124 permeability 653 pore dilation vs. dye uptake 661 splice variants 646 pharmacology 651, 661 classes 661–662 physiology 662 postsynaptic effects 662 presynaptic effects 662 structure 658 C-tail 658–660 N-tail 658–660 TM1 658, 659f TM2 658, 659f functional domains 660 subtypes 651 heteromeric a,b-meATP-mediated currents 652 P2X1/2 652 P2X1/4 652 P2X1/5 653 P2X2/3 652 P2X2/6 653 P2X4/6 653 suramin effects 653 voltage clamp experiments 652 subunits/structure 649, 649f coassembly 651t genes 651 chromosomal locations 651t spliced forms 651 P2Y receptors 650t, 653 activation 654 effects 641 agonists 650t, 654 antagonists 643–644, 650t, 654 cross-talk 657 genes 654 glial cells ATP neurotransmission role 643 ion channels and 654 noradrenergic neurotransmission 421 orthologs 653–654 P2Y1 654 agonists 654 antagonists 654 knockout mice 655 structure 654 P2Y2 655 activation 655–656 agonists 655–656 expression 656 PLCb1 coupling 656 P2Y4 656 activation 656 antagonists 656 P2Y6 656 agonists 656 P2Y11 656 activation 654 agonists 656 antagonists 656 P2Y12 656 agonists 656 antagonists 656 expression 656 P2Y13 656 activation 654 P2Y14 656 activation 656–657 gene 656–657 signaling second messenger systems 654 see also G-protein(s) subtypes 654 subunits/structure 653 dimerization 657 p75 receptor(s) 570, 576 apoptosis role 570–572 BDNF signal transduction 590–592
Subject Index genetically modified mice studies 596–597 neurotrophin binding 576 physiological role 570–572 signaling 570–572, 571f, 577 signaling mechanism 577 Trk co-expression 570–572 see also Trk receptor(s) PACAP see Pituitary adenylate cyclase activating polypeptide (PACAP) PACE4 convertase animal knockouts 514 Pain adenosine receptors 637t clinical (chronic pathological) mGluR1 298 mGluR5 298 neuropathic see Neuropathic pain glutamate role 237 kainate receptors and 319 models see Pain models transduction ATP neurotransmission 644 P2X3 receptors 644, 645f Pain models GABAB receptor ligands 364 Paired pulse depression (PPD) GABAB receptors and neurotransmitter release 368–369, 370f Paired pulse facilitation see Presynaptic facilitation Pairing, long-term potentiation induction 321 Palmitoylation postsynaptic assembly regulation 25 Pancreatic b cells large dense-core vesicles (LDCVs) 521 Pancreatic peptide see Pancreatic polypeptide (PP) Pancreatic polypeptide (PP) 540 discovery 538 gene 538 intracerebroventricular injection 540 NPY receptor binding 540 retina 528 Pannexin-mimetic peptides, gap junction blockade 205 Pannexins (Pxs) 219, 189, 193 evolution 195 connexin convergence 211 hemichannel formation 212 mammals 213 Parallel fibers 33–34 activity-dependent synaptic competition 34 plasticity at Purkinje cell synapses see Cerebellar plasticity Parasympathetic nervous system GDNF family ligands (GFLs) 606 peptidergic receptors and modulation 559 angiotensin II 560 CGRP 560 opioid peptides 559 PACAP 560 tachykinin peptides 559 VIP 560 see also Neuropeptide(s) Paraventricular nucleus (PVN) HPA axis role see Paraventricular nucleus, HPA axis role neuroendocrine functions CRH synthesis 544 see also Corticotropin-releasing hormone (CRH) see also Hypothalamic–pituitary–adrenal (HPA) axis; Paraventricular nucleus, HPA axis role Paraventricular nucleus, HPA axis role CRH secretion 544 see also Corticotropin-releasing hormone (CRH) PARK2 gene/protein see Parkin Parkin DJ-1 interaction 694 Parkinson’s disease etiology 694 Parkinsonian syndromes/parkinsonism dopamine D2 receptor and 409 mGluR5 agonists 297–298 Parkinson’s disease see Parkinson’s disease (PD) Parkinson’s disease (PD) clinical features 694 etiology/pathogenesis see Parkinson’s disease, pathology/pathogenesis management see Parkinson’s disease, therapy neural basis see Parkinson’s disease, pathology/pathogenesis pathology see Parkinson’s disease, pathology/pathogenesis
pathophysiology see Parkinson’s disease, pathology/pathogenesis treatment see Parkinson’s disease, therapy Parkinson’s disease, pathology/pathogenesis 392 adenosine receptors 637t dopamine dysfunction 383–384 dopamine receptors and 408 D1 receptor 408 see also Dopamine (DA); Substantia nigra GDNF family ligands (GFLs) 607 genetic factors parkin (PARK2) mutations 694 glial growth factors 622 glutamate role mGluRs 307 Lewy bodies 694 nitric oxide 694 nitrosylation role 694 postmortem findings 694 see also Dopaminergic neurons/systems Parkinson’s disease, therapy 573–574 growth factor/neurotrophin therapy GDNF 694 pharmacological dopaminergic agonists see Dopaminergic agonists mGluR4 agonists 300 Parvocellular (parvicellular) system (neurosecretion) neuropeptide coexistence oxytocin 527 vasopressin 527 PVN see Paraventricular nucleus (PVN) PDZ domain(s) 310–311 postsynaptic density proteins 100 AMPA receptor adaptor proteins 99, 270 Shanks 97–98 PDZ protein(s) 310–311 Pedunculopontine tegmental (PPT) nucleus cholinergic input 490 Pentaxin, AMPA receptor clustering 271 Pentose phosphate pathway (PPP) 241 PEPA 266 Peptide(s) intercellular communication 557 neuroactive see Neuropeptide(s) receptors see Peptidergic receptors Peptide-induced receptor trafficking, volume transmission 564, 566f Peptidergic interneurons see Neuropeptides, electrophysiology Peptidergic receptors 525, 557–563, 567 autonomic nervous system 559, 559t enteric neurons gastrointestinal motility and 561 parasympathetic modulation 559 angiotensin II 560 CGRP 560 functional implications 560 opioid peptides 559 PACAP 560 tachykinin peptides 559 VIP 560 sympathoeffector junction modulation 560 angiotensin II AT1 receptors 561 bradykinin B2 receptors 561 cannabinoid CB1 receptors 561 endothelin receptors 561 natriuretic receptors 561 NPY Y2 receptors 560 opioid receptors 561 PACAP receptors 561 somatostatin receptors 561 VIP receptors 561 desensitization 567 endocytosis 558 ligands, clinical potential 558 local circuitry 567 peripheral organs 559, 559t recycling 558 structure 567 G-proteins 525 transduction pathways 558 Peptide tyrosine tyrosine (PYY) 539 discovery 538 distribution 538 genetics 538
739
740 Subject Index Peptide tyrosine tyrosine (PYY) (continued) neuropeptide Y receptor binding 540 PYY3-36 539 Peptide tyrosine tyrosine 3-36 (PYY3-36) 539 Peptide YY see Peptide tyrosine tyrosine (PYY) Peptidylglycine a-amidating monooxygenase (PAM) metal ion requirement 513 neuropeptide synthesis 513 neuropeptide Y synthesis 538 Pergolide dopamine receptor affinity/kinetics 394t Peripheral nerve regeneration/repair BDNF 623 CNTF 623 GDNF 623 glial cell growth factors 623 Schwann cell responses see Schwann cell(s) Peripheral nervous system (PNS) neuromuscular junctions see Neuromuscular junction (NMJ) neuropeptides see Neuropeptides, sensory systems neurotrophins 573 tachykinins see Tachykinin(s) Peripheral neuropathy see Neuropathy Peripheral tissues peptidergic receptors 559 Perisynaptic fibroblasts, NMJ synapse elimination and 155 Perisynaptic Schwann cells (PSCs) ablation effects 50 development 46–47 NMJ plasticity and see Neuromuscular junction, plasticity synapse elimination and 48–49, 155 synaptogenesis 46–47 terminal 174 Perlecan NMJ basal lamina 177 Peroxynitrite (ONOO-) nitric oxide 689, 692 Persephin (PSPN) 599 knockout mice 605t species 599 structure 599, 600f PET see Positron emission tomography (PET) Phaclofen, GABAB receptor antagonists 363, 363f Phagocytosis glial cells synapse elimination and 48 Pharmacokinetics, monoamine neuroimaging see Monoamine neuroimaging Pharmacology AMPA receptors see AMPA receptor(s) metabotropic glutamate receptors (mGluRs) 294, 307 group I mGluRs 305 neurotrophins see Neurotrophin(s) P2X receptors 651 Phasic inhibition, GABAA receptor activation 356 Phencyclidine (PCP) NMDA receptors 237–238 4-[2-Phenylsulfonylaminoethylthio]-2,6-difluorophenoxyacetamide (PEPA) 266 Phosphate-activated glutaminase (PAG), GABA metabolism 343–344 Phosphatidylinositol (PI), hydrolysis mGluR1 signaling 292 mGluR5 signaling 292 Phosphatidylinositol 3-kinase (PI3K) IGF survival and metabolic pathway 612f, 613 mGluR signaling 292 synaptic vesicle cycle and 94–95 Phosphatidylinositol 3-kinase (PI3K)–Akt pathway GDNF family ligands (GFLs) 602–603 NGF role in transport 587 see also Akt/PKB protein kinase Phosphatidylinositol 3-kinase enhancer (PIKE) mGluR activation 294 Phosphatidylinositol-4,5-biphosphate (PIP2) clathrin-mediated endocytosis 89–90, 94 Phosphatidylinositol phosphate kinase (PIPK), type 1g, endocytosis and SV cycle 85t, 94–95 Phosphodiesterase(s) (PDEs) family members 688 nitric oxide 688
Phosphodiesterase 1 (PDE 1) 688 Phosphodiesterase 2 (PDE 2) 688 Phosphodiesterase 3 (PDE 3) 688 Phosphofurin acidic cluster-sorting protein (PACS-)1, neuropeptide granule biogenesis 516 Phosphoinositide-3 kinase (PI3K) see Phosphatidylinositol 3-kinase (PI3K) Phospholipase C (PLC) dopamine receptor transduction 410 group I mGluR signaling 304 Phospholipid(s) synaptic vesicle cycle and 89–90, 94 Phosphorylation gap junctions 205 mGluR2-dependent long-term depression 330 mGluR signaling 302 receptor modulation see Receptor(s) vesicular monoamine transporters (VMATs) 256 see also Protein kinase(s) pHuorin imaging, synaptic vesicle cycle 84–87, 88f Phylogeny/phylogenetics GFRa coreceptors 601 PI3K see Phosphatidylinositol 3-kinase (PI3K) Piboserod, 5-HT4 receptor studies 467 Piccolo 16 active zone 56t, 57 CAZ role 92t, 94 clathrin-mediated endocytosis and 94 see also Bassoon PICK1 (protein interacting with kinase C) AMPA receptor localization 99 Picrotoxin GABA receptors blockade 351–352 PIKE see Phosphatidylinositol 3-kinase enhancer (PIKE) Pilocarpine kainate receptor antagonists and 316–317 Pimavanersin, serotonin receptor research 463 PIP2 see Phosphatidylinositol-4,5-biphosphate (PIP2) Piriform cortex serotonin 5HT-2 receptor 474 Pituitary adenylate cyclase activating polypeptide (PACAP) autonomic neurons parasympathetic modulation 560 coexistence glutamate 528 retina 528 NMDA receptor effects 282 receptors PAC1 see Pituitary adenylate cyclase activating polypeptide receptor (PAC1) sympathoeffector junctions, presynaptic modulation 561 see also Vasoactive intestinal peptide receptors PKA see Protein kinase A (PKA) PKB see Akt/PKB protein kinase PKC see Protein kinase C (PKC) Placenta CRH expression 549 Plasmalemma see Cell membrane Plasma membrane see Cell membrane Plasticity see Neuroplasticity Platelet-derived growth factor (PDGF) astrocyte differentiation 619 glial cell production 622t glioblastoma 621 oligodendrocyte differentiation 620 radial glia differentiation 621 Schwann cells 617 PLC see Phospholipase C (PLC) Poisson statistics transmitter release 162 Polyamine(s) AMPA receptor blocking 266 Kir channel blockade see Inwardly rectifying potassium channels (Kir) NMDA receptors 280–281 Polyaxonal (polyneuronal) muscle innervation developmental 150 bioassays and 154 change to monoaxonal see Neuromuscular junction, synapse elimination
Subject Index evidence for 150 electrophysiological 151 histological 151, 151f Polysialylation, neural cell adhesion molecules 41 Positron emission tomography (PET) applications/studies dopaminergic transmission D2 receptors 446–447, 447f Posterior pituitary gland see Neurohypophysis Postmitotic cell migration, retinal development see Retinal development Postsynaptic density (PSD) 11, 19, 52, 84, 96–102, 126 assembly/development 100 neurological disease and 101–102 physiological role 101 importance 96 signal transduction 101 structural changes and plasticity 101 protein composition 96 cytoskeletal proteins 100 spinophilin (neurabin II) 24–25 integral membrane proteins 99 regulatory/signaling proteins 99 CaMKII 96–97, 99–100 plasticity and 101 Ras GTPases 100 Rho GTPases 100 scaffolding/adaptor proteins 96–97, 98f AMPA-receptor-linked 98f, 99 developmental expression 100 functional role 98 Homer 98–99 mGluR-linked 98–99 multiple receptor interfaces 99 neurological disease and 101–102 NMDA-receptor-linked 97–98 PDZ domains 100 PICK1 99 posttranslational modification and plasticity 101 PSD-95 see PSD-95 Shank see Shank see also PDZ domain(s) rearrangements 100 activity-dependent changes 101 dendritic AMPA receptor expression 101 LTP 101 posttranslational modification and 101 see also Synaptic plasticity developmental assembly 100 cell adhesion molecules 100 size/structure 96, 97f, 97t microdomains 101 study methods 96 differential centrifugation 96 electron microscopy 96, 97f molecular/biochemical approaches 96 proteomics 96, 99 AMPA receptor composition 99 cytoskeletal proteins 100 Postsynaptic density 95 protein see PSD-95 Postsynaptic development 19–26 adhesion molecules 100 assembly regulation 25 lipid modifications 25 PSD-95 phosphorylation 25 ubiquitination 25 differentiation governing principles 20 induction 21 neurotransmitters 21 neuroligin-1 20 excitatory synapses 19 presynaptic development and 11–12 PSD see Postsynaptic density (PSD) scaffolding proteins 19, 20f definition 19 F-actin 24 localization 22 neuronal transport 25 organization 22 proteomic approaches 19
741
synaptic adhesion molecules 22 NMDA receptor clustering 23 signaling pathways 24 Postsynaptic membrane 11 assembly/development see Postsynaptic development receptors see Postsynaptic receptors see also Postsynaptic density (PSD) Postsynaptic potential (PSP) 5HT-2 serotonin receptor 475 excitatory see Excitatory postsynaptic potentials (EPSPs) inhibitory see Inhibitory postsynaptic potentials (IPSPs) Postsynaptic receptors 23 sensitivity 250 see also Receptor(s) Post-tetanic potentiation (PTP) 132 calcium role 170–171 residual free Ca2þ hypothesis 170–171 definition 169–170 neuromuscular junction 169 study methods 168 time constant of decay 169–170 Posttranslational modifications (PTMs) cholecystokinin synthesis 512 neuropeptide synthesis 512, 564 vesicular monoamine transporters (VMATs) 256 Potassium (Kþ) conductances 5HT-1 serotonin receptor 473 GABAB receptors 360 peptidergic interneurons 567 Potassium channel(s) calcium-activated see Calcium-activated Kþ channels (KCa) G-protein activated see G-protein-activated Kþ channels (GIRKs) inward rectifier (Kir) channels see Inwardly rectifying potassium channels (Kir) modulation, group I mGluRs 304 Potassium/chloride cotransporter 2 (KCC2) 358 Potassium ions (Kþ) GABAB receptors 362 gap junction permeability 191 see also Potassium (Kþ) conductances; Potassium channel(s) Pramipexole monohydrate dopamine receptor kinetics 394t Prazosin 422 adrenergic receptors 426, 427 Precursor-processing enzymes, neuropeptide synthesis see Neuropeptides, synthesis Prepiriform cortex see Piriform cortex Preprodynorphin 532 Preproenkephalin 532 Preprohypocretin 551 addiction 555 Prepromelanin concentrating hormone (ppMCH) 553–555 Preproopiomelanocortin gene 532 Prepyriform cortex see Piriform cortex Presynapse 11 assembly see Presynaptic development development see Presynaptic development different target cell synapses 128, 128f glutamate spillover 251 quanta 76, 77f receptors see Presynaptic receptors structure 11, 158 active zone see Active zone (AZ) boutons see Synaptic bouton(s) synaptic efficacy/strength and 159 Presynaptic bouton see Synaptic bouton(s) Presynaptic density 126 Presynaptic development 11–18 assembly vesicular intermediates 20–21 components active zone and cytomatrix 16 differentiation mechanisms cellular 16 mobile precursors and axonal transport 11–12 molecular 13 agrin 126 cadherins 16 CASK role 14–15 g-protocadherins 16 integrin-mediated signaling 127
742 Subject Index Presynaptic development (continued) laminins 127 mechanisms of action 14 neurexins 14, 15–16 neuroligin 14, 15–16, 128 retrograde signals 126 scaffolding/cytoskeletal molecules 127–128 SynCAM 14–15, 15f, 128 Wnt signaling 128 glial role see Glial cells, synaptic functions initial events axonal growth cones and 11–12 en passant synapses (synaptic boutons) 11–12 secreted molecules and FGFs 13 glutamate 12–13 neurotrophins 13 Wnts 13 maturation of nascent sites 11–12 activity role 12, 16 background activity and 17 ‘desilencing’ and 16–17 short-term plasticity and 17 functional changes 16 short-term plasticity and 17 silent synapses 16–17 stages 12, 13f synapse stabilization 15 synaptic vesicles CASK role 15 excitosis and 12–13 recycling changes 12, 15 model systems/experimental analysis imaging studies 12 invertebrate systems C. elegans 127–128 Drosophila 127 vertebrate NMJ 126, 127f multiple stages 11–18 postsynaptic development and 11–12 prepackaged molecules 12, 14 Presynaptic facilitation 132 calcium role residual free Ca2þ hypothesis 170–171 components 169 definition/derivation 168 neuromuscular junction 169 study methods 168 Presynaptic homeostasis, retrograde signaling and 129, 130f Presynaptic receptors 5HT-1A 472 D2 receptors 399 kainate receptors 317 NMDA receptors 279 serotonin receptors, 5HT-1A 472 Primary afferent neurons see Sensory afferent(s) Primary auditory cortex see Auditory cortex Primary olfactory cortex see Piriform cortex Principle neurons, gap junctions 221 Profilin CAZ role in clathrin-mediated endocytosis and 94 Progressive motor neuronopathy, retrograde neurotrophic signaling defects 588–589 Proinsulin non-cleavage, animal knockouts 514 Prolactin neurogenic properties 614 Proline-rich synapse-associated protein (ProSAP) see Shank Proneurotrophins 590, 594 Proopiomelanocortin (POMC) synthesis granule biogenesis/storage 514 precursor peptides 511–512 Propeptide/prohormone convertases (PCs) metal ion requirements 513 neuropeptide synthesis 512–513 Propofol, GABAA receptors 353 Propranolol adrenergic receptors 427 N-Propyl-norapomorphine-R-(-), dopamine receptor kinetics 394t
Prosencephalon see Forebrain Prostaglandin E2 (PGE2) puberty 622 Proteasome(s) synaptic elimination at the NMJ 155 Protein folding disorders/misfolding neuropeptide precursor peptides 512 see also Neurodegeneration/neurodegenerative disease Protein interacting with C kinase 1 (PICK1) 233–234 AMPA receptor 272 mGluR2-dependent long-term depression 329 Protein kinase(s) gap junction activation 204 integrin-mediated regulation 178 long-term potentiation 323–324 Protein kinase A (PKA) GDNF family ligands (GFLs) 604–605 glutamate receptors and group I mGluRs 305 NMDA receptor phosphorylation 281 neuropeptide Y regulation 538 phosphorylation reaction 264 serotonergic neuronal pathways 472 Protein kinase B (PKB) see Akt/PKB protein kinase Protein kinase C (PKC) 264 behavior role see Protein kinase C (PKC), synaptic plasticity and behavior glutamate receptors and group I mGluRs 292, 305 kainate receptor signaling 319 mGluR2-dependent LTD 329 NMDA receptors 281, 282 neuropeptide Y regulation 538 PKCa, postsynaptic receptor localization 23–24 synaptic plasticity and see Protein kinase C (PKC), synaptic plasticity and behavior Protein kinase C (PKC), synaptic plasticity and behavior long-term potentiation mGluR2-dependent 329 Protein kinase C-Src signaling, NMDA receptor effects 282 Protein–lipid interactions, membrane deformation and synaptic vesicle cycle 95 Protein motifs endocytic sorting motifs 89–90 Protein targeting to glycogen 241, 242f Proteolysis synaptic elimination at the NMJ 155 Proteomics see Neuroproteomics a-Protocadherin 40–41 d-Protocadherin 40–41 Protocadherin(s) 40, 40t genes 40–41 structure 39f, 40–41 g-Protocadherins, presynaptic development 16 Proton–acetylcholine exchanger, refilling vesicles at the NMJ 165 Proton electrochemical gradient neurotransmitter uptake 80 vesicular neurotransmitter transporters see Vesicular neurotransmitter transporters Proton pump(s), neurotransmitter uptake 80 PRX-00023, depression studies 457–459 PSD-93, AMPA receptor 272 PSD-95 functional roles 98 AMPA receptors and 271 NMDA receptors and clustering 97–98, 98f trafficking 287 plasticity 101 neurological disease and 101–102 nitric oxide 685 postsynaptic development and 19, 22, 100 function 24 knockout mice 19–20 localization 22, 23 regulation 23–24 signaling pathways 24–25 overexpression 19–20
Subject Index phosphorylation 25 proteomic studies 19 scaffold role 21–22 signaling pathways 24 splice variants 19 protein interactions NR2 subunits 287 stargazin binding 99 structure posttranslational modification 101 Psychiatric disorders genetic factors PSD mutations and 101–102 neuroimaging 448 see also Psychopathology Psychogenic networks, HPA axis see Hypothalamic–pituitary–adrenal (HPA) axis Psychological diseases/disorders see Psychiatric disorders Psychomotor functions, dopamine D2 receptor 399 Psychosis dopamine D2 receptor 406 Puberty glial cell growth factors 622 Pulmonary inflammation, adenosine receptors 637t Purine(s) 648–657 astrocytic Ca2þ wave regulation see Astrocytic calcium waves neurotransmission see Purinergic transmission receptors see Purinoceptor(s) Purinergic receptors see Purinoceptor(s) Purinergic transmission 639, 640f, 642 ATP see Adenosine triphosphate (ATP) cotransmission 639 mechanisms associated 648 mechanosensory transduction 644 physiology 642 receptors see Purinoceptor(s) Purinoceptor(s) 648–657 ATP-mediated activation 640 historical aspects 648 P1 (adenosine) receptors see P1 receptor(s) Purkinje cell(s) 33–34 dendrites weaver mutant mice 3–4 GABAB receptor expression 371 mGluR1 294–295 PAG expression 343–344 plasticity at parallel fiber synapses see Cerebellar plasticity Push–pull cannula perfusion, microdialysis 445 Pyramidal neuron(s) cortical GABAergic interneuron connections 411 dendritic spine synapses, dopamine and 390 electrical signaling 224 hippocampal GABAergic terminals 350–351, 351f Pyridoxine (vitamin B6) epilepsy 345–346 Pyriform cortex see Piriform cortex Pyrimidine receptors 640 Pyrococcus horikoshi excitatory amino acid transporters 245–246, 246f Pyroxidal-phosphate-6-azophenyl-20 ,40 -disulphonic acid, P2 receptors 645–646 Pyrrolidones 266 Pyruvate, lactate dehydrogenase 239 Pyruvate carboxylase (PC) glutamate metabolism 340 metabolism 243 Pyruvate dehydrogenase, astrocytes 239, 240f PYY see Peptide tyrosine tyrosine (PYY)
Q Quantal content neuromuscular junction (ACh) 161–162, 181 presynaptic terminals 76, 77f Quantal hypothesis 161 Quinagolide, dopamine receptor kinetics 394t (-)Quinpirole, dopamine receptor kinetics 394t Quisqualate, metabotropic receptors 235
743
R Rab3-interacting molecules see RIMs Rab GTPases AMPA receptor 268–270 membrane trafficking 82 SNARE interactions 72 Rab-interacting molecule (RIM) see RIMs Rac GTPases PSD proteins 100 Radial fibers of Mu¨ller see Mu¨ller cells Ramo´n y Cajal, Santiago dendritic spines 3 Golgi method 355 Rap GTPase(s), PSD proteins 100 Raphe nuclei (RN) dorsal see Dorsal raphe nuclei serotonin receptors 5HT-1 473 5HT-1A 473 Rapid eye movement (REM) sleep see REM sleep RAS (reticular activating system) see Ascending reticular activating system (ARAS) Ras GTPase(s) PSD proteins 100 H-Ras, NMDA receptor phosphorylation 282 Rat(s) jugular vein, 5-HT2B receptor 465 kainate receptor distribution 310 stomach fundus receptor 463 vibrissae see Vibrissae Reactive oxygen species (ROS) NADP 242 Readily releasable vesicle pools (RRPs) 11, 79–80 probability of release and 11 short-term synaptic plasticity 132 Receptor(s) coactivation, striatal dopamine receptors 411 desensitization synaptic depression and 680 extrasynaptic see Extrasynaptic receptor(s) neuropeptide coexistence 530 orphan receptors 557–558 phosphorylation AMPA receptor 270 mGluRs 302 NMDA receptor see NMDA receptor(s) presynaptic see Presynapse Receptor for activated C kinase 1 (RACK1), NMDA receptor effects 282 Receptor tyrosine kinase(s) (RTKs) NMDA receptor effects 282 short-term synaptic plasticity 134 Reconstitution experiments, SNAREs, membrane fusion 70–71, 70f Recreational drugs see Substance abuse Red fibers, muscle 141–142 Reelin(s) 40t Regeneration see Neural regeneration/repair Remote memories see Memory consolidation REM sleep molecular mechanism serotonin receptors 5HT-1 473 Repair see Neural regeneration/repair Representations (neural) see Neural maps/representations Reproduction neuropeptides and neuropeptide Y (NPY) 539 Reptile(s) skeletal muscle innervation 143 Repulsive guidance molecules (RGMs) see Axonal guidance cues Reserpine vesicular monoamine transporter inhibition 256 Reserve vesicle pool (RP) 79–80 synapsin-mediated regulation see Synapsin(s) Residual bound Ca2þ hypothesis 170–171 problems with 171 Respiratory system adenosine effects 637–638 RET gene/protein GDNF family ligands (GFLs) 599 cadherin-like domains 600–601
744 Subject Index RET gene/protein (continued) congenital central hypoventilation syndrome 599–600 identification 599–600 interaction 600–601, 601f knockout mice 605–606, 605t motor neurons 606 mutations 599–600 signaling 600–601 signaling pathways 602–603, 604f structure 600–601 as therapeutic target 607–608 Reticular activating system (RAS) see Ascending reticular activating system (ARAS) Reticulospinal neurons lordosis behavior 435f Retina anatomy/physiology amacrine cells see Amacrine cells bipolar cells see Bipolar cells (retina) ganglion cells see Retinal ganglion cells (RGCs) glial cells see Retinal glial cells neuronal cell types see Retinal neurons postsynaptic kainate receptors 317–318, 318f development see Retinal development neurons see Retinal neurons neuropeptide coexistence avian pancreatic polypeptide (APP) 528 enkephalin 528 neuropeptide Y 528 pancreatic polypeptide (PP) 528 pituitary adenylate cyclase activating peptide (PACAP) 528 somatostatin 528 substance P 528 D-serine role 337 topographic maps and see Retinotopic maps Retinal axons, filopodia 5–6 Retinal bipolar cells see Bipolar cells (retina) Retinal development activity-dependent synaptic competition 31–32 Retinal ganglion cells (RGCs) dendrites 4–5 calcium imaging 7–9 development astrocytes and synapse formation/maturation 13, 46, 48f see also Retinal development neuropeptide coexistence 528 retinotopic maps see Retinotopic maps Retinal gap junctions 191 Retinal glial cells Mu¨ller cells see Mu¨ller cells neurotransmission modulation and 112, 114 Retinal neurons axonal filopodia 5–6 bipolar cells see Bipolar cells (retina) ganglion cells see Retinal ganglion cells (RGCs) specification see Retinal development Retinotopic maps development activity-dependent synaptic competition and 31–32, 35 ocular dominance see Ocular dominance columns (ODCs) retinotopic see Retinotopic maps Retraction bulbs, NMJ synapse elimination 153 Retrograde neurotrophic signaling 128, 584–589 compartmentalized nerve cell chamber studies 584 Campenot chamber 584 microfluidic chambers 584, 585f technique advantages 584 neurodegenerative diseases 588 proposed mechanisms 585 intracellular vesicles 587 microtubule/dynein role 588 NGF-independent retrograde signaling 587 ‘signaling effector model’ 587 signaling endosome hypothesis 585, 586f evidence 585–587 ‘wave model’ 587 in vivo studies 584 hippocampal cholinergic pathway 585 ligature technique 584–585
Retrograde signaling endocannabinoids 128, 669, 678 group I mGluRs see Metabotropic glutamate receptors (mGluRs) growth factors 128 historical aspects 126 neuromuscular transmission development and 126, 127f homeostatic signaling and 129–130 neurotrophins see Retrograde neurotrophic signaling serotonin 450 transynaptic influences 126–131 homeostatic signaling 129 BMPs and 131 calcium channels and 130 chronic blockade of central synapses 129, 130f definition 129 experimental evidence 129 presynaptic transmitter release and 130 protein synthesis-independent process 130 putative molecular mechanisms 130 time frame 130 presynaptic release probability and 128, 128f behavioral effects 128–129 different targets/different properties 128, 128f invertebrates 128–129 mammals 129 synapse formation 126 C. elegans model 127–128 central synapses 128 Drosophila model 127 laminins and 127 neuroligin and 128 neuromuscular junction 126, 127f scaffolding proteins 127–128 SynCam and 128 Wnt signaling 128 see also Presynaptic development RG12, short-term synaptic plasticity 136–137 RGS-2, short-term synaptic plasticity 136–137 Rho GTPase(s) AMPA receptor 268–270 PSD proteins 100 Rhythmic neuronal activity see Oscillatory neuronal activity RIBEYE CAZ role 94 RIM1a CAZ role 91 presynaptic membrane role 16 RIM-binding proteins active zone 56–57, 56t CAZ role 92t RIMs active zone 56, 56t protein interactions RIM-binding proteins 92t synaptic transmission role 16 CAZ and 91, 91f, 92t ‘Ripple’ activity, electrical signaling 224 Ritalin see Methylphenidate (MPH) Ritanserin, sleep studies 465 Rodent(s) barrel cortex see Barrel cortex stress response models, CRH 547 Rose Bengal, vesicular glutamate transporter inhibition 258
S Saclofen, GABAB receptor antagonists 363 Safety factor neuromuscular transmission 186 postsynaptic specialization and 186 presynaptic specialization and 186 species differences 186, 187f Salamander(s) skeletal muscle innervation 143 SALM1 protein, NMDA receptor trafficking 287 SALMs 42 AMPA receptor binding 42 NMDA receptor binding 42 NMDA receptor trafficking 287 SALP 40t
Subject Index Sample purification, microdialysis 445–446 SAP-97 AMPA receptor 270 Sarcoglycan(s) dystroglycan complex components 177 Satellite cells (glial) growth factors 617 nerve growth factor 617 neurotrophin-3 617 transforming growth factor-a 617 transforming growth factor-b 617 vascular endothelial growth factor 617 SB, 242084 454 Scaffold proteins glutamate receptors and AMPA receptors 98f, 99 mGluRs activation 294 group I 302–303 NMDA receptors 97–98 PSD-95 see PSD-95 PSD clustering 23 postsynaptic density see Postsynaptic density (PSD) postsynaptic development 19, 100 definition 19 F-actin 24 localization 22 neuronal transport 25 organization 22 proteomic approaches 19 synaptic adhesion molecules 22 NMDA receptor clustering 23 presynaptic development 127–128 RIMs see RIMs synaptic vesicle endocytosis and 84–95 as spatial regulators 90 see also Synaptic vesicle cycle; Synaptic vesicle endocytosis SCH50911, GABAB receptor antagonist 363, 363f Schizophrenia clinical features negative symptoms DSM-IV criteria 232–233 positive (psychotic) symptoms DSM-IV criteria 232–233 diagnosis/diagnostic criteria DSM-IV criteria 232–233 management pharmacological antipsychotics see Antipsychotic drugs (neuroleptics) D3 receptor agonists 400 mGluR2/3 agonists 299 mGluR5 agonists 297–298 neural basis see Schizophrenia, neural basis Schizophrenia, neural basis dopamine role 384, 391 D2 receptor and 400, 406 D4 receptor and 401 glutamatergic dysfunction 234, 237 kainate receptors and 320 mGluRs and 297–298, 299, 307 NMDA receptors 234 serotonin receptor research 463 Schwann cell(s) development see Schwann cell development gap junctions 216 growth factors/receptors 617, 622t ciliary neurotrophic factor 617 cytokines 617 epidermal growth factors 617 glial cell line-derived growth factor 617–618 leukemia inhibiting factor 617 nerve growth factor 617 neurotrophins 617 platelet-derived growth factor 617 transforming growth factor-a 618 transforming growth factor-b 617 tumor necrosis factor-a 617 multiple sclerosis 617 myelination and multiple sclerosis 617 neuromuscular junction (NMJ) see Perisynaptic Schwann cells (PSCs) synaptogenesis and 46
Schwann cell development growth factors and differentiation 619 FGF-2 620 GGF-1 620 NRG-1a 620–621 NRG-1b 620–621 TGF-b1 620 Scopolamine memory 490–491 Scrape-loading dye transfer technique gap junctions 202–203 Sdk-1 42 Sdk-2 42 Sea urchin, connexins 211 Sec1p, SNARE interactions 72 Second messenger systems DAG see Diacylglycerol (DAG) glial cells synaptic activation 333 P2Y receptors and 654 Secretory pathway kiss-and-run exocytosis 84–87 synaptic location 90 Seizure(s) neuropeptide Y and 539 see also Epilepsy Seizure disorder see Epilepsy Selective aggregation, neuropeptide granule biogenesis 515–516, 515f Selective serotonin reuptake inhibitors (SSRIs) 457, 464 Senescence synaptic plasticity 337 see also Age/aging Sensitivity microdialysis 446 Sensory afferent(s) repeated stimulation, mGluRs 303 Sensory neuron(s) see Sensory afferent(s) ‘Septal–hippocampal cholinergic pathway’ 489–490 D-Serine 333–339 as astrocytic gliotransmitter 115, 116, 117f NMDA-mediated LTP and 49, 116 chirality 333, 334f clearance 336 degradation 334 expression 337–338 glutamate co-storage 335 localization 334 NMDA receptor coagonist 115, 116, 280 plasticity 336 release 335 sites 336 synaptic transmission role 336 NMDA receptors 336–337 synthesis 334 Serine racemase (SR) 333 distribution 334 expression 337–338 regulation 334 nitric oxide 334 Serine/threonine phosphatase, NMDA receptor phosphorylation reversal 282 Serotonergic neuronal pathways 470 behavioral correlations 476 drug studies 476 clinical correlations 476 drug studies 476 firing patterns 470–471 functions of 470–471 input density 470 localization 470 B1-B9 midline nuclei 470 dorsal raphe nuclei 470 median raphe nuclei 470 noradrenergic activation 476 regulation 471f, 471 autoinhibition 472 calcium/calmodulin protein kinase II (CaMKII) 472 hypocretin system 471–472, 471f intracellular recordings 471–472 noradrenergic inputs 471–472, 471f protein kinase A 472 short-wave sleep 471–472
745
746 Subject Index Serotonergic neuronal pathways (continued) slow depolarizations 471–472, 472f tonic firing patterns 471–472 tryptophan hydrolase 472 waking states 471–472 whole cell recordings 471–472 ‘wake-on’ systems 470–471, 471–472 Serotonergic receptor(s) see Serotonin (5-HT) receptor(s) Serotonin see Serotonin (5-HT)/serotonergic neurons Serotonin (5-HT) receptor(s) 450, 456–469 5-HT1 cerebral cortex 474 electrophysiology 472 hippocampus 473 inhibitory postsynaptic potentials (IPSPs) 473 inwardly rectifying potassium channels 472–473 neuronal firing rates 450–451 8-OH-DPAT 473 potassium conductance 473 raphe nuclei 473 REM sleep 473 serotonin affinity 450–451 structure 472 subcortical regions 473 5-HT1A 456 agonists 451–452, 457, 458t partial 457 silent 457 airways 453 antagonists 458t autoreceptors 456–457 cell body (somatodendritic) 456–457 terminal 456–457 bladder 453, 454 cardiovascular 451–452 depression and PRX-00023 457–459 VPI-013 457–459 EPSPs 473–474 features 458t hippocampus 473–474 in vivo studies 456–457 knockouts embryonic/early postnatal 462 location 461–462 8-OH-DPAT 473–474 presynaptic activation effects 457 raphe nuclei 473 SSRIs and 457 5-HT1B 460, 473 agonists 458t, 460 antagonists 458t, 460 chromosomal location 460 features 458t human vs. rodent 460 knockout mice 461 locations 460 terminal autoreceptor 460 5-HT1D 461 agonists 458t, 461 antagonists 458t, 461 chromosomal location 461 features 458t 5-HT1E 461 chromosomal location 461–462 features 458t human brain binding studies 461–462 5-HT1F 462 agonists 459t, 462 antagonists 459t, 462 antimigraine proposals 462 chromosomal location 462 features 459t 5-HT2 462 binding residues 462 cerebral cortex 475 claustrum 474 electrophysiology 474 medial pontine reticular formation 474 motoneurons 474 neocortex 474 neuronal firing rates 450–451
nucleus accumbens 474 olfactory tubercle 474 piriform cortex 474 postsynaptic potentials 475 signal transduction 474 subcortical regions 474 5-HT2A 462 actions 462–463 agonists 459t, 462 antagonists 451, 459t, 462, 463 bladder 453 cardiovascular regulation 451 cerebral cortex 475, 475f classical ‘D’ receptor 462 features 459t hippocampus 474 5-HT2C comparisons 463, 464 regulatory effects 472 UP states 476 5-HT2B 463 agonists 459t antagonists 459t, 463–464 chromosomal location 463 features 459t ligand clinical trials migraine 464 pulmonary hypertension 464 rat stomach fundus receptor 463 5-HT2C 464 agonists 459t, 464 antagonists 454, 459t, 464 bladder 453 cerebral cortex 475 characteristics 464, 465 m-chlorophenylpiperazine (mCPP) 464, 465 features 459t gene 464 5-HT2A comparisons 463, 464 INI isoform 464 orphan receptors 465 rat vs. human sequence comparisons 464 VSV isoform 464 5-HT3 459t, 466 antagonists 466 bladder 453 cardiovascular 451–452 electrophysiology 475 endocannabinoids and 665–668 features 466 glial cells 452 gut 454 hippocampus 475 IPSCs 475 irritable bowel syndrome studies 466 ligand-gated ion channels 475 morphine and 466 neuronal firing rates 450–451 pioneering animal studies 466 serotonin affinity 450–451 structure 475 vagal sensory afferents 452, 453f 5-HT4 466 afterhyperpolarization 476 agonists 459t, 467 partial 467 antagonists 459t, 467 bladder 453 electrophysiology 476 features 459t gut 454 hippocampus 476 irritable bowel syndrome studies 466, 467 locations 467 neuronal firing rates 450–451 olfactory tubercle 476 striatum 476 substantia nigra 476 5-HT5 467 gene location 467 pharmacological properties 467 5-HT5A antagonist 459t, 467
Subject Index bladder 453 features 459t localization study findings 467 Onuf’s nucleus 454 5-HT5B, features 459t, 467 5HT-6 467 Alzheimer’s disease 468 cholinergic neurotransmission regulation 468 electrophysiology 475 pharmacophores 468 5-HT7 468 airways 453 antagonists 460t, 468 baroreceptor reflex 452 bladder 453, 454 central cardiovascular regulation 451, 452 depression 468–469 electrophysiology 476 features 460t oleamide 468–469 splice variants 468 5-HT8 antagonists 459t features 459t affinity 450–451 cerebral cortex 5-HT1 474 5-HT2 475 5-HT2A 475, 475f 5-HT2C 475 classification 456 subtypes 457f electrophysiology 472 future research 469 genetics multiple genes 456 G-protein-coupled family members 456 subtypes 450 transduction pathways 473f Serotonin (5-HT)/serotonergic neurons 450–455, 470–477 anatomical locations airways 453 receptors 453 autonomic nervous system enteric nervous system role 454 cardiovascular regulation 451, 452f receptor agonists/antagonist effects 451 receptor subtypes 451 cotransmission/coexistence neuropeptides enkephalin 528 galanin 528 substance P 528 definition 470 dopamine receptor kinetics and 394t endocannabinoids and suppression of neurotransmission by 669–670 gastrointestinal 454 enterochromaffin cells 454 historical aspects 470 Falck–Hillarp method 470 histochemical localization 470 ligand-gated receptors see Serotonin (5-HT) receptor(s) micturation role 453 dorsal raphe nucleus 454 GABAergic interneurons 454 receptors 453 SB 242084 effects 454 neuroimaging 448 psychiatric disorders 448 see also Monoamine neuroimaging neuronal pathways see Serotonergic neuronal pathways neurotransmission 450 autonomic functions 450 brain stem neurons 450 Dales principle 450 evidence for 451 retrograde neural transmission 450 volume transmission (VT) 450 wiring transmission (WT) 450 see also cotransmission/coexistence (above) receptors see Serotonin (5-HT) receptor(s)
structure 457f, 471f d-lysergic acid diethylamide (LSD) vs. 470 N,N-dimethyltryptamine (DMT) vs. 470 synthesis 456 transporter see Serotonin transporter (5-HTT/SERT) Serotonin transporter (5-HTT/SERT) 456 SERT see Serotonin transporter (5-HTT/SERT) Sertoli cells, GDNF 607 Sessile tunicates see Ascidians Seven-pass (Flamingo-like) transmembrane cadherins see Cadherin(s) Seven transmembrane region, mGluRs 292–293, 302 Sex hormone(s) see Gonadal hormone(s) Sex steroid(s) see Gonadal hormone(s) Shank 98f developmental expression 100 functional role 98 neurological disease and 101–102 protein interactions glutamate receptor binding mGluR association 98–99 NMDA receptor association 97–98 Homer binding 99 protein-binding domains 97–98 PSD-95 interactions 22, 24–25 structure PDZ domains 97–98 Short-term depression (STD) 132, 670–671 components 172 definition 171 endocannabinoids and 670, 672f experimental analysis 168 masking of synaptic enhancement 171–172 mechanisms 172 Ca2þ channel inactivation 172 depletion (of neurotransmitter) 172 incomplete vesicle refilling 172 vesicle depletion 172 vesicle mobilization and 172–173 vesicle recycling and 172 neuromuscular junction 168, 171, 172f see also Neuromuscular junction, plasticity synaptic characteristics and 172 Short-term enhancement (STE) see Presynaptic facilitation Short-term facilitation (STF) see Presynaptic facilitation Short-term potentiation 324–325 Short-term synaptic memory 173 Short-term synaptic plasticity 132–137 changes in transmitter release 168 component relationships 170 interspecies comparisons 170 depression see Short-term depression (STD) enhancement 168 augmentation see Synaptic augmentation calcium role 170 residual free Ca2þ hypothesis 170–171 voltage component 171 facilitation see Presynaptic facilitation masking by synaptic depression 171–172 potentiation see Post-tetanic potentiation (PTP) synaptic vesicles and the presynaptic membrane 171 kinetic components 168 model development 173 neuromuscular junction 168–173 see also Neuromuscular junction, plasticity neurotransmitter release activity-dependent regulation 132 active zone proteins 132 kinase pathways 134 NCS-1 133 SNARE regulation 133 G-protein-mediated regulation 134 Gi/o-coupled receptors 134 Gq/11-coupled receptors 135 Gs-coupled receptors 135 presynaptic G-protein signaling effectors 135 Ga 135 Gb/g. 136 G-protein regulation 136 targets 135 presynaptic maturation and 17 presynaptic mechanisms 171
747
748 Subject Index Short-term synaptic plasticity (continued) short-term synaptic memory and 173 Short-wave sleep, serotonergic neuronal pathways 471–472 Sidekick cell adhesion molecules 40t, 42 Signaling endosome hypothesis 585, 586f evidence 585–587 Signaling pathways apoptosis see Apoptosis dopamine receptors 402–403 GDNF family ligands (GFLs) see GDNF family ligands (GFLs) GPCRs see G-protein-coupled receptor(s) (GPCRs) NMDA receptors see NMDA receptor(s) serotonin receptors 473f 5HT-2 474 Silent synapses 23 activity-dependent maturation 27–28, 28f ‘desilencing’ 16–17 see also AMPA receptor(s) glial cell thrombospondin-induced synapses 47 NMDA receptor expression 16–17 postsynaptic development and 100 presynaptic development and 16–17 Single nucleotide polymorphisms (SNPs) brain-derived neurotrophic factor 594 Single-particle tracking, glycine receptor diffusion properties 378 Single photon emission computed tomography (SPECT) applications/studies dopamine D2 receptors 446–447 Size-based selectivity, gap junctions 190 Skeletal muscle 148 cadherin expression 178 contraction force 142 definition 141 development cadherin expression 178 NMJ formation see Neuromuscular junction development, mammalian origin of fiber types 154 diversity of 141 electrically excitable 141–142 function 174 inexcitable fibers 141 innervation motor 142 phylogenetic variation 142 intermediate forms 142 motor unit use patterns 146 peak force 142 relaxation, GABAB receptor ligands 364 spasticity see Spasticity twitch 141–142 SKF 38393 394t Slam freezing electron microscopy, active zone (AZ) 160 Sleep see Sleep/sleep states Sleep disorders adenosine receptors 637t insomnia see Insomnia Slot proteins, AMPA receptor 270 Slow depolarizations, serotonergic neuronal pathways 471–472, 472f Slow muscle fiber(s) 141, 186 higher vertebrates intrafusal fibers 188 mammalian extraocular, fibers 188 lower vertebrates (frog) 187 ‘Small brain’ see Cerebellum Small GTPases PSD 100 Small synaptic vesicles (SSVs), neurotransmitters 519, 564–565 Smooth muscle structure 141 SM proteins, SNARE interactions 72 SNAP-23, astrocytic exocytosis 121–123 SNAP-25 acetylcholine release 480 gliotransmitter exocytotic regulation 120–121 identification 69 knockout mice 70 membrane trafficking 82 neurotransmitter release 67 short-term synaptic plasticity 134 SNARE interactions 72 SNARE specificity effects 74 tomosyn interaction 72–73
SNAP-29, short-term synaptic plasticity 134 SNAPs synaptic vesicles 79–80 SNARE complex 67–75 acetylcholine release 480 calcium channel signaling 335 functions 69 clostridial neurotoxins 69–70 Drosophila studies 69–70 historical aspects 69 imaging studies 69–70 knockout animal models 70 minimal membrane-fusion machinery 70 sequence analysis 69 future work 74 gliotransmitters, exocytotic regulation 120–121 interactions 71 active zone proteins 16 exocytosis components 72, 73f Ca2þ sensitivity 70f, 73 complexins 70f, 73–74, 73f Munc 18s 70f, 72 Rab proteins 72 Sec1p 72 SM proteins 72 SNAPs 72 irrelevant 71–72 membrane fusion 70, 335 component interactions 71 objections to 71 reconstitution experiments 70–71, 70f structural data 70–71 zippering model 69–70, 70f membrane trafficking 82 neuropeptide release 521 neurotransmitter release 67 regulation 72 short-term synaptic plasticity 133 specificity 74 structure 67, 68f circular dichroism 67 classification 67 Habc domain 67–68, 68f NMR 67 N-terminal region 67–68 synaptic vesicle recycling 79–80 Vps33p 72 v-SNAREs 121 Social bonding, oxytocin see Oxytocin Social recognition, endocannabinoid role 675 Sodium see Sodium ions (Naþ) Sodium/chloride sensitive transporter, D-serine clearance 336 Sodium-dependent transporter(s) D-serine clearance 336 Sodium ions (Naþ) gap junction permeability 191 glutamate transport 239, 240f Sodium/potassium ATPase see Sodium pump (Naþ/Kþ-ATPase) Naþ/Kþ-ATPase see Sodium pump (Naþ/Kþ-ATPase) Sodium–potassium pump see Sodium pump (Naþ/Kþ-ATPase) Sodium pump (Naþ/Kþ-ATPase) chlorpromazine mechanism of action 392 Sodium waves, astrocytes 241 Soluble N-ethylmaleimide-sensitive factor (NSF) attachment proteins see SNAPs Somatosensory cortex absence epilepsy 365–366 barrel cortex see Barrel cortex GABAB receptors 365–366 Somatosensory system development activity-dependent synaptic competition 32 maps/representations see Somatotopy/somatotopic organization somatotopic representation see Somatotopy/somatotopic organization Somatostatin clinical potential 558 coexistence acetylcholine 528 neuropeptide Y (NPY) 538 nitric oxide 528 retina 528 receptors see Somatostatin receptor(s)
Subject Index Somatostatin 2 see Cortistatin Somatostatin receptor(s) cortistatin binding 557–558 sympathoeffector junctions, presynaptic modulation 561 Somatotopy/somatotopic organization barrel cortex see Barrel cortex development/maturation activity-dependent synaptic competition 32 SON see Supraoptic nucleus (SON) Sortilin BDNF signaling 590–592 Sorting by retention model, neuropeptide granule biogenesis 515 Sorting for entry model, neuropeptide granule biogenesis 514–515 Spasticity pathophysiology glycine receptor defects 378 human hyperexplexia 378 startle syndromes 378 treatment GABAB receptor ligands 364 Spatial memory assessment in animals water maze 675 neurobiological substrates endocannabinoid system 675 Spatial resolution microdialysis 446 SPECT see Single photon emission computed tomography (SPECT) Spermatogenesis GDNF family ligands (GFLs) 606 Spermine AMPA channel-blocking 266 Spike(s) see Action potential(s) Spikelets, interneuronal gap junctions 206–207, 221, 222, 223 kinetic properties 223 Spike timing-dependent plasticity (STDP) NMDA receptors 278–279, 279f Spillover see Synaptic spillover Spinal cord injury (SCI) management 574 Spinal cord pain systems adenosine effects 638 Spinal muscular atrophy (SMA) animal models NMJ synapse elimination and 156 gene mutations 155–156 type 1 (Werdnig–Hoffman disease) gene mutations 155–156 Spindle fibers see Microtubule(s) Spinophilin (neurabin II) postsynaptic development 24–25 Spiny projection neurons, medium see Medium-spiny neurons Sprouting, synaptic plasticity see Synaptic plasticity Src homology domains IGF signaling peptides 612–613, 612f Src kinase(s) integrin-mediated regulation 178 NMDA receptor phosphorylation 281–282, 282f postsynaptic development 24 S-SCAM/MAGI-2 (PDZ protein), postsynaptic development role 24 stargazin (Stg) 99 function 23 PSD interactions AMPA receptor binding 98f, 99 PSD-95 binding 99 Startle hyperexplexia (STHE) 378 Startle response pathology glycine receptor defects 378 disease characterization 378 oscillator phenotype 378 spasmodic phenotype 378 spastic mutation 378–379 Stellate ganglion neuropeptide Y 557 projections 557 Steroid hormone(s) corticosteroids see Corticosteroid(s) sex steroids see Gonadal hormone(s) Stonin 2, endocytosis and SV cycle 85t, 90
749
Striatum anatomy see Striatum, anatomy/physiology learning and memory role endocannabinoids and 673 goal-directed behavior 673 physiology see Striatum, anatomy/physiology Striatum, anatomy/physiology dopamine system 383 receptors D1 receptors 411–412 D2 receptors 411–412 D3 receptor 411 see also Dopamine receptor(s) matrix 383 medium spiny cells see Medium-spiny neurons nucleus accumbens see Nucleus accumbens patch (striosome) 383 serotonin 5HT-4 expression 476 see also Dopaminergic neurons/systems Stroke cerebral ischemia see Ischemic stroke ischemic see Ischemic stroke management 574 pathogenesis/pathophysiology glutamate 237 Subcortical pathways/systems serotonin receptors 5HT-1 473 5HT-2 474 see also Substantia nigra (SN) Submandibular ganglion (SMG) 559 VIP action 560 Submaxillary muscles, frogs 143 Substance abuse addiction see Addiction dopaminergic system and D1 receptors 410–411 see also Dopaminergic neurons, reward role endocannabinoid LTD and 682 frontal cortex, dopamine 410–411 management alcoholism see Alcoholism noradrenergic neurons, effects 436 Substance P (SP) 559–560 coexistence aspartate 528 galanin 527 glutamate 528 norepinephrine 528 retina 528 serotonin 528 receptor see Neurokinin (NK) receptors, NK-1 synthesis precursor peptides 511 Substantia nigra (SN) anatomy/physiology 5HT-4 receptors 476 Subtilisins, synthesis 512–513, 513f Succinic semialdehyde dehydrogenase (SSADH), GABA metabolism disorders 345–346 Succinyl-CoA synthetase 341f Sulfation factor see Insulin-like growth factor (IGF) system Sumatriptan, serotonin receptor 461 Superior cervical ganglion (SCG) membrane channels 654 Superior olivary complex (SOC) development, activity-dependent synaptic competition 33 Superior olivary nucleus see Superior olivary complex (SOC) Superior olive see Superior olivary complex (SOC) Superoxide dismutase 1 (SOD1) mutants/mutations familial ALS and 155–156 see also Amyotrophic lateral sclerosis (ALS) reactive oxygen species scavenging 242 Suprachiasmatic nucleus (SCN) anatomy/physiology locus coeruleus innervation 430 Supraoptic nucleus (SON) lactating rats, astrocytic plasticity and synaptic transmission 114–115 oxytocin 517 Suramin P2X2/6 effects 653
750 Subject Index SV2 254t, 259 Syd1, presynaptic development 127–128 Syd-2 see Liprin-a Syg1 42 presynaptic development 127–128 structure 42 Syg2, presynaptic development 127–128 SYM2081, kainate receptor agonists 314 Sympathetic nervous system (SNS) historical aspects 414 neurotrophic factors artemin (ARTN) 606 GDNF family ligands (GFLs) 606 Sympathetic noradrenergic system see Sympathetic postganglionic neurons Sympathetic postganglionic neurons norepinephrine distribution 415 Sympathoeffector junctions, presynaptic modulation, peptidergic receptors 560 Synaphin(s) see Complexin(s) Synapse(s) 76, 333 cadherins see Cadherin(s) central nervous system (CNS), retrograde signaling homeostatic signaling and 129, 130f synaptogenesis and 128 chemical signaling 11 evolution 158 ‘cross-talk’ 114–115 definition 52, 193 development see Synaptogenesis elimination see Synaptic pruning/elimination excitatory see Excitatory synapses features mature 27 nascent 27 formation see Synaptogenesis GABAergic neurons 342 glial cells and see Glial cells, synaptic functions inhibitory see Inhibitory synapses maturation 27 steps 27, 28 ultrastructural studies 28 neural cell adhesion molecules see Neural cell adhesion molecules (NCAMs) neuromuscular see Neuromuscular junction (NMJ) other cell–cell junction vs. 11 plasticity see Synaptic plasticity pruning see Synaptic pruning/elimination retrograde signaling see Retrograde signaling silent (mute) see Silent synapses specificity, excitatory amino acid transporters 250 cross-talk limitation 250 ‘spillover’ see Synaptic spillover stabilization 15 glial cells and see Glial cells, synaptic functions LTP/LTD role 35–36 structure/morphology 12f adhesion molecules and 11, 13 asymmetry 11 central synapses 11 postsynaptic see Postsynaptic membrane postsynaptic structures see Postsynaptic membrane presynaptic see Presynapse presynaptic structures see Presynapse vesicles see Synaptic vesicle(s) transmission across see Synaptic transmission Synapsin(s) membrane trafficking 82–83 Synaptic adaptation, mGluRs 299 Synaptic adhesion molecules postsynaptic development 22 Synaptic augmentation calcium role 170–171 neuromuscular junction 169 study methods 168 see also Neuromuscular junction, plasticity Synaptic bouton(s) development en passant boutons 11–12 NMJ 145, 159, 159f Synaptic cell adhesion-like molecule (SALM), postsynaptic receptors 23 Synaptic cell adhesion molecules (SynCAM) see SynCAM
Synaptic cleft laminin 176 Synaptic competition activity-dependent 31–37 auditory system 32 autonomic ganglia 34 cerebellum 33 mechanisms 35 GABAergic inhibition 32, 35 Hebb’s rule 35 LTD/LTP link 35 postsynaptic factors 34 proximity effects 34 temporal activity patterns 35 trophic factor hypothesis 36, 154–155 neuromuscular junction 34 olfactory system 33 sensory map maturation barrel cortex/somatosensory maps 32 cerebellar maps 33–34 olfactory maps 33 retinotopic maps 31–32 tonotopic maps 32–33 somatosensory system 32 visual system 31 see also Synaptic pruning/elimination definition 31 neuromuscular junction see Neuromuscular junction, synapse elimination see also Synaptic pruning/elimination Synaptic depression AMPA receptor desensitization and cerebellar LTD 680 endocannabinoids and 664–676 long-term see Long-term depression (LTD) short-term see Short-term depression (STD) Synaptic differentiation, neurexins 44 Synaptic efficacy (strength) AMPA receptor numbers and 99 GABAA receptor numbers 350 glial cell contribution see Glial cells, neurotransmission modulation NMJ anatomical correlates 159 synaptic vesicle processing and 166 Ramon y Cajal and 31 see also Synaptic competition; Synaptic plasticity; Synaptic pruning/ elimination Synaptic elimination see Synaptic pruning/elimination Synaptic facilitation see Presynaptic facilitation Synaptic formation see Synaptogenesis Synaptic fragmentation, NMJ synapse elimination 153 Synaptic-like microvesicle compartments (SLMVs) gliotransmitter exocytotic regulation 121 morphology 121 protein expression 121 Synaptic plasticity 689 activity-dependent see Activity-dependent plasticity aging and senescence 337 depression see Synaptic depression endocannabinoids and see Endocannabinoid system and synaptic plasticity glial cell contribution see Glial cells, neurotransmission modulation long-term see Long-term synaptic plasticity metaplasticity see Metaplasticity molecular mechanisms see Synaptic plasticity, molecular mechanisms pain role see Pain properties 594–595 short-term see Short-term synaptic plasticity spike-timing dependent plasticity see Spike-timing dependent plasticity (STDP) synapse-specific alterations postsynaptic density and 101 trophic factors 590–598 Synaptic plasticity, molecular mechanisms endocannabinoids and see Endocannabinoid system and synaptic plasticity neurotrophins and 573 signaling pathways BNDF/TrkB receptors 594 see also Brain-derived neurotrophic factor (BDNF) glutamate receptor-mediated LTP role see Long-term potentiation (LTP)
Subject Index mGluRs 304 NMDA receptor role see NMDA receptor(s), synaptic plasticity see also Glutamate receptor(s) Synaptic pruning/elimination activity-dependent auditory system development 33 autonomic ganglia development 34 barrel cortex development 32 NMJ development 34 visual system development 31 see also Synaptic competition activity-independent, auditory system 32–33 definition 31 glial cells 48, 155 see also Glial cells, synaptic functions NMJ development see Neuromuscular junction, synapse elimination synaptogenesis balance 11–12 Synaptic remodeling 31 see also Synaptic competition; Synaptic efficacy (strength); Synaptic pruning/elimination Synaptic spillover synaptic cross-talk 114–115 Synaptic strength see Synaptic efficacy (strength) Synaptic transmission 60f glutamate receptors 321 models see Synaptic transmission models retrograde signaling see Retrograde signaling synaptic vesicles see Synaptic vesicle(s) transmitter release see Neurotransmitter release see also Neurotransmission; Neuromuscular transmission Synaptic transmission models 103–111 cholinergic transmission at neuromuscular junction 106 insights from modeling 106 further directions 110 hippocampal synapses 107 insights from modeling 109 glutamate diffusion in extracellular space 110 mEPSC amplitude 109 mEPSC time course 109 quantal variability origins 109 kinetic constraints 108 morphological constraints 107 modeling approaches 103 continuum methods 103 finite difference methods 104 finite element methods 104 acetylcholine diffusion 104–105 information requirements 106 Monte Carlo 105 synaptic response measurement, quantal analysis 106 Synaptic vesicle(s) 11, 76–83 biogenesis 76, 78f AP2-dependent fusion 77–79 dileucine motif 77–79 precursors 76–77, 77–79 clustering 79–80 composition 80, 81f chloride channels and 80–82 membrane trafficking proteins 82 neurotransmitter uptake proteins 80 protein stoichiometry 80 SNAP 79–80 synapsins see Synapsin(s) cycling see Synaptic vesicle cycle Drosophila mutants 84, 85t endocytosis see Synaptic vesicle endocytosis features 76 GABAergic 76 heterogeneity 76 morphology 76 lipid bilayer 76 surface 76 neurotransmitter release activity-dependent regulation 132 active zone proteins 132 NCS-1 133 SNARE regulation 133 ‘pools’ see Synaptic vesicle pools presynaptic development and active zone proteins 16 CASK role 15
recycling changes 12, 15 SV exocytosis 12–13 protein folding/chaperone proteins and 11 quantal content NMJ vesicles 161–162, 181 synaptic plasticity short-term plasticity and 171 synaptic depression 172 as transmitter quantum 161–162 transport/trafficking 76–77 see also Synaptic vesicle cycle Synaptic vesicle cycle 78f, 79, 84, 87f endocytosis see Synaptic vesicle endocytosis endosomes 87 membrane fusion/release (exocytosis) active zone proteins and 16 kiss-and-run mode 84, 87f, 165, 166f location 90 see also Kiss-and-run fusion SNAREs and see SNARE complex stimulation frequency and mode 84–87 see also Neurotransmitter release pHuorin imaging 84–87, 88f refiling/recycling 79, 164 budding 80 incomplete, synaptic depression and 172 intrasynaptic Ca2þ and 11 NMJ see Neuromuscular transmission, presynaptic events synaptic response rate and 11 regulation membrane lipids 94 membrane deformation and 95 phosphoinositides 94 protein scaffolds as spatial regulators 16, 90 CAZ and 94 see also Cytoskeleton of the active zone (CAZ) vacuolar bulk retrieval 87 Synaptic vesicle endocytosis (SVE) 84–95 clathrin-mediated 87f, 89 CAZ role 94 clathrin-coated pit formation 89 clathrin-coated pit maturation 90 at the NMJ 165 proteins involved 84, 85t specialized areas for 84, 90 SV cycle and 84 vesicle fission 90 at the NMJ 165, 166f, 167f clathrin-mediated 165 macroendocytosis 165–166 models 165, 166f study methods 165, 167f types 165 vacuolar bulk retrieval 87–89 protein scaffolds and 90 CAZ and 94 see also Cytoskeleton of the active zone (CAZ) vacuolar bulk retrieval 87, 87f Synaptic vesicle exocytosis see Synaptic vesicle cycle Synaptic vesicle pools readily releasable see Readily releasable vesicle pools (RRPs) reserve pool 11 see also Endocytosis; Exocytosis; Neurotransmitter release Synaptobrevin membrane trafficking 82 Synaptobrevin 2 gliotransmitters, exocytotic regulation 120–121 identification 69 knockout mice 70 neurotransmitter release 67 SNARE specificity effects 74 Synaptogenesis 11–18, 27, 29–30 activity-dependent 31–37 presynaptic maturation and 16 see also Synaptic pruning/elimination; Synaptic competition adhesion molecules and 13, 15f postsynaptic development 100 variety 13–14 autapses 13–14 dendritic spine filopodia and 11–12 differentiation, neurexins 44 error prone nature 15–16
751
752 Subject Index Synaptogenesis (continued) filopodia see Filopodia glial role see Glial cells, synaptic functions inherent neuronal ability 13–14 injury-induced 13–14 multiple stages 11 neurexin–neuroligin complex see Neurexin–neuroligin complex neurexins see Neurexins NMJ central synapses vs. 11–12 developmental see Neuromuscular junction, development postsynaptic specializations PSD development 100 see also Postsynaptic membrane presynaptic development see Presynaptic development promiscuity 13–14 retrograde signaling 126 central synapses 128 neuromuscular junction 126 stabilization and 15 steps associated 27 synapse elimination relationship 11–12 see also Synaptic pruning/elimination timing 11–12 Synaptojanin(s) endocytosis and SV cycle 85t Synaptotagmin(s) calcium sensitivity binding sites 62–63 endocytosis and SV cycle 85t N-glycosylation 77–79 Synaptotagmin-1 C2 domains 82 as Ca2þ sensor 70f, 73 functions fast vesicle exocytosis 82 neurotransmitter release 67 short-term synaptic plasticity 133 Synaptotagmin-4 astrocytic exocytosis regulation 121–123 neuropeptide granule biogenesis 516 short-term synaptic plasticity 133 Synaptotropic hypothesis 4–5 SynCAM 38, 40t, 43 filopodia stabilization 9 postsynaptic differentiation 21 synapse formation and 14 CASK as downstream target 14–15 presynaptic development 14–15, 15f, 128 Syndapin(s), endocytosis and SV cycle 85t Syndecan(s) GFL receptors 599, 602, 603f Syndecan-2 40t SynGAP postsynaptic development 24 Syntaxin-1 Habc domain 68, 69f identification 69 Munc18 binding 68–69, 68f, 72 neurotransmitter release 67 SNAP-25 interactions 72 tomosyn interactions 72–73 Syntrophin(s) dystroglycan complex components 177 Szenta´gothai, Ja´nos, Golgi impregnation studies 355
T Tachykinin(s) CGRP coexistence 517 PNS 559 receptors see Neurokinin (NK) receptors storage 525–526 substance P see Substance P (SP) synthesis precursor peptides 511 Tachykinin receptors see Neurokinin (NK) receptors Tail flip response (crustaceans), lateral giant axons 208 Tamsulosin, adrenergic receptors 426 Target-derived neurotropins see Neuropeptides, sensory systems TASK-1 receptor, endocannabinoids and 665–668
Tau gene/protein phosphorylation IGF-1 null mice 613 TCA cycle see Krebs cycle Tectal neurons, dendritic branch formation 4 Telencephalon olfactory bulb see Olfactory bulb ventral, cholinergic cell bodies 488–489 Teleost fish escape response 208 Temporal activity patterns, activity-dependent synaptic competition 35 Temporal lobe epilepsy see Epilepsy Temporal resolution microdialysis 446 Tench, skeletal muscle innervation 143, 144f Tetanus toxin (TeNT) NMDA receptor-dependent LTP 321 cooperativity 323 Tetrabenazine, vesicular monoamine transporter inhibition 256 Tetrahydrocannabinol (THC) 664 structure 665f Tetramethylpyrazine 645–646 Thalamus anatomy/physiology cholinergic innervation 490 Therapeutic drugs see Drug(s) Theta burst stimulation (TBS) GABAB autoreceptors and NMDA receptor-dependent LTP 369–371 THIP see Gaboxadol Thrombospondin(s) glial-mediated synaptogenesis 13, 47 knockouts 47 receptors see Integrins Thyrotropin-releasing hormone (TRH) PVN secretion 517 Time-dependent long-term depression, endocannabinoids and 673, 679f TIMP2, NMJ basal lamina 179 Tissue inhibitors of metalloproteinases (TIMPs) NMJ basal lamina 179 Tissue plasminogen activator (tPA) 594 long-term potentiation 596 TNF-a see Tumor necrosis factor a (TNFa) Toad(s) skeletal muscle innervation 143 Tomosyn short-term synaptic plasticity 133–134 SNAP-25 interaction 72–73 syntaxin 1 interactions 72–73 Tonic firing patterns serotonergic neuronal pathways 471–472 Tonic inhibition, GABAA receptors 351 Tonic muscle fibers see Slow muscle fiber(s) Tonotopic organization activity-dependent synaptic competition and 32–33 auditory cortex activity-dependent synaptic competition 33 thalamus 32–33 activity-dependent synaptic competition 32–33 Topographic maps/representations auditory see Tonotopic organization development/maturation activity-dependent synaptic competition auditory system 32–33 olfactory system 33 retinotopic maps 31–32 somatosensory maps 32 somatosensory system 32 tonotopic maps 32–33 visual system 31–32 retinotopic maps see Retinotopic maps olfactory activity-dependent synaptic competition 33 see also Olfactory bulb somatosensory see Somatotopy/somatotopic organization visual system see Retinotopic maps Torpedo nicotinic acetylcholine receptor 347–348 Transbilayer cholesterol distribution (TCB) see Cholesterol Transforming growth factor a (TGFa) astrocytes 618 glial cell production 622t
Subject Index puberty 622 satellite cells 617 Schwann cells 618 Transforming growth factor b (TGFb) superfamily age-related changes 622 astrocytes 618 GFRa coreceptor effects 601 neuronal survival 621 oligodendrocytes 618 satellite cells 617 Schwann cells 617 Transforming growth factor b1 (TGFb1) Alzheimer’s disease 622 glial cell production 622t Schwann cell differentiation 620 Transforming growth factor b2 (TGFb2) glial cell production 622t Transforming growth factor b3 (TGFb3), glial cell production 622t Transgenic animals mice neuropeptide synaptic potentials 568 XFP mice 152 neuropeptide synthesis 564 oligodendrocytes see Oligodendrocyte(s) SOD1 mice (ALS model) see Amyotrophic lateral sclerosis (ALS) synapse elimination at the NMJ 152, 153 see also Knockout animal(s) Transgenic labeling astrocytes see Astrocyte(s) trans-Golgi network (TGN) AMPA receptor 268 neuropeptide granule biogenesis 514 neuropeptide synthesis 511, 512f, 526, 527f Transient pores, neuropeptide release 529–530 Transmembrane domains GABAB receptors 360 neuropeptide granule biogenesis 515 Transmembrane interacting proteins (TARPs), AMPA receptor 271 Transporter proteins 245–252 endocannabinoids 665–668 kinetics, constant potential amperometry 443 vesicular transmitter transporters see Vesicular neurotransmitter transporters Transport vesicles, NMDA receptor trafficking 286–287 Transsynaptic adhesion 21 Transsynaptic influences, retrograde signaling see Retrograde signaling Trial synapse formation filopodial motility regulation 6 Tricarboxylic acid (TCA) cycle see Krebs cycle Trimeric GTP-binding proteins (G proteins) see G-protein(s) 3,4,5-Trimethoxyphenethylamine see Mescaline ‘Tripartite synapse’ concept 46, 47f, 112, 113f Trisomy 21 see Down’s syndrome (DS) Trk receptor(s) 570, 571f p75NT receptor co-expression 570–572 signaling 572, 572f TrkA receptor(s) 576 NGF binding 587 signaling mechanism 576 transport, NGF role 587 see also p75 receptor(s) TrkC receptor(s) differential splicing 570 Trophic factor(s) activity-dependent synaptic competition 36 see also Growth factor(s); Neurotrophin(s) Tropomyosin-related kinase (Trk) receptors see Trk receptor(s) TRPV1 (VR1) receptor(s) activation cannabinoids and 665–668 Tryptophan hydroxylase (TH) serotonergic pathways 472 serotonin 456 T-type Ca2þ channels see Voltage-gated calcium channels (VGCCs) Tumor necrosis factor a (TNFa) gliotransmitter exocytotic regulation 123 homeostatic retrograde signaling 129 Mu¨ller glial cells 619 production astrocytes 113–114
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Schwann cells 617 synaptic modulation and glial release 49 synaptic plasticity and, astrocytic production and 113–114 Turtle(s) skeletal muscle innervation 143 Twitch fibers 141–142 anuran 143 mammals 145 reptiles 143–145 urodeles 143 Two-step binding model, CRH receptors 546 Tyrosine dopamine 385–386 norepinephrine synthesis 424 Tyrosine hydrolase norepinephrine synthesis 414 Tyrosine kinase A (trkA) receptors, neurotrophins/neurotrophic factors 576 signaling 576 phosphorylation at Tyr490 576–577 phosphorylation at Tyr751 576–577 phosphorylation at Tyr785 576–577 Tyrosine sulfotransferase, cofactors 513
U Ubiquitination glycine receptor dynamics 377 NMDA receptor degradation 290 postsynaptic assembly regulation 25 Ubiquitin–proteasome system neurodegenerative disease and ALS 155 synaptic elimination at the NMJ 155 Ultrafast oscillations, gap junctions 223 generation 224 Unc-13 genes/proteins CAZ role 92t Unc-17 gene/protein, vesicular acetylcholine transporter (VAChT) 256 Uncoupled ionic conductances, excitatory amino acid transporters 247 Uncoupling agents, gap junctions 220–221 Unwin, Nigel, Torpedo nicotinic acetylcholine receptor 347–348 UP states, 5HT-2A serotonin receptor 476 Urinary bladder see Bladder Urocortin(s) cardiovascular effects 548 Urocortin-I discovery 544 Urodeles skeletal muscle innervation 143 see also Salamander(s) Uroepithelium, adenosine 637 Urotensin-I (UI), discovery 544 Use-dependent plasticity, peptidergic interneurons 568 Utrophin(s) dystroglycan complex binding 177–178
V Vacuolar bulk retrieval, synaptic vesicles 87, 87f Vacuolar-type Hþ-ATPase (V-ATPase) 254t vesicular neurotransmitter transporters 253 Vagal afferents sensory, 5HT-3 serotonin receptors 452, 453f Valinomycin, vesicular neurotransmitter transporter inhibition 253–254 Vannilloid receptor 1 see TRPV1 (VR1) receptor(s) Vascular endothelial growth factor (VEGF) Mu¨ller glial cells 619 satellite cells 617 Vascular tone adenosine effects 637 Vas deferens norepinephrine distribution 415–416, 417f Vasoactive intestinal peptide (VIP) autonomic nervous system parasympathetic nerves modulation 560 coexistence/cotransmission GABA 528 receptors see Vasoactive intestinal peptide receptors
754 Subject Index Vasoactive intestinal peptide receptors sympathoeffector junctions, presynaptic modulation 561 see also Pituitary adenylate cyclase activating polypeptide receptor (PAC1) Vasopressin biological activity autoregulation 524 coexistence corticotropin-releasing hormone (CRH) 527 CRH 521–522 dynorphin 521–522, 528 galanin 521–522 magnocellular neurons 526, 527 parvocellular neurons 527 extracellular half-life 521–522 hypothalamic synthesis magnocellular neurons 522 precursor peptides 511 peripheral action 522–523 pituitary storage/release priming 520f, 523 Ventral forebrain see Basal forebrain Ventral telencephalon, cholinergic cell bodies 488–489 Ventrotemporal (VT) retina development see Retinal development Venus fly trap domain 293–294 Versican 40t Vertebrate(s) neuromuscular connection patterns 141–149 see also Neuromuscular junction (NMJ); Skeletal muscle Very fast oscillations (VFOs), gap junctions 223 Vesamicol 480 vesicular acetylcholine transporter inhibition 257 Vesicle(s) clathrin adaptors see Clathrin adaptors docking 480 membrane fusion see Vesicle fusion retrograde neurotrophic signaling 587 late endosomes 587–588 multivesicular bodies 587–588 secretory pathway see Secretory pathway synaptic see Synaptic vesicle(s) Vesicle-associated membrane protein-2 (VAMP-2) see Synaptobrevin 2 Vesicle-associated membrane protein-4 (VAMP-4), neuropeptide granule biogenesis 516 Vesicle fusion neurotransmitter release theory SNAP-25 67 SNAREs see SNARE complex synaptobrevin 2 67 synaptotagmin-1 67 syntaxin 1 67 see also Synaptic vesicle(s) synaptic vesicles see Synaptic vesicle cycle Vesicle pools see Synaptic vesicle pools Vesicular acetylcholine transporter (VAChT) 77–79, 80, 253, 254t, 256, 486 affinity 256 expression 256 inhibitors 257 vesamicol 257 localization 486 stoichiometry 256 structure 480 unc17 gene 256 Vesicular GABA transporter (VGAT) 253, 254t, 257 discovery 257 expression 257 inhibitors 257 g-vinyl GABA 257 sequence 257 Vesicular glutamate transporter(s) (VGLUTs) 121, 229, 253, 254t, 257 expression 257–258 heterogeneity 123 inhibitors 258 4,4’-diisothiocyanostilbene-2,2’-disulfonic acid (DIDS) 258 Evans blue 258 rose Bengal 258 mechanism of action 258 structure 257
Vesicular glutamate transporter 1 (VGLUT1) 254t expression 257–258 structure 254–255 Vesicular glutamate transporter 2 (VGLUT2) 80, 254t expression 257–258 structure 254–255 Vesicular glutamate transporter 3 (VGLUT3) 254t expression 258 Vesicular inhibitory amino acid transporter (VIAAT) 80 Vesicular monoamine transporter(s) (VMATs) 253, 254, 254t epinephrine-releasing cells 255–256 granule sorting 516 inhibitors 256 amphetamines 256 MDMA 256 reserpine 256 tetrabenazine 256 mechanism of action 255 phosphorylation 256 posttranslational modification 256 regulation 256 specificity 255–256 structure 254–255 Vesicular monoamine transporter 1 (VMAT1) 254t Vesicular monoamine transporter 2 (VMAT2) 254t Vesicular neurotransmitter transporters 253–259, 254t, 255f advantages 253 historical aspects 253 neuromodulators 258 ATP 258–259 zinc 258 proton electrochemical gradient 253, 255f bafilomycin 253–254 carbonyl cyanide m-chlorophenylhydrazone (CCCP) 253–254 carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) 253–254 exocytic fusion pore 254 inhibitors 253–254 vaculolar-type Hþ-ATPase 253 valinomycin 253–254 Vesicular trafficking acetylcholine 480 Vibrissae somatosensory cortex connections see Barrel cortex trimming, filopodial motility 7 Visual cortex brain damage, functional recovery see Visual cortex, reorganization/plasticity development endocannabinoid LTD and 682 functional reorganization see Visual cortex, reorganization/plasticity norepinephrine effects 436–437, 437f, 438f plasticity see Visual cortex, reorganization/plasticity Visual cortex, reorganization/plasticity endocannabinoid-induced LTP and 678, 680f, 682 use-dependent plasticity Hebbian learning rule 35 Visual deprivation activity-dependent plasticity and endocannabinoid LTD and 682 synaptic competition 31 Hebb’s rule and 35 Visual development, neural activity role synapse pruning/elimination 31 visual deprivation effects see Visual deprivation see also Ocular dominance columns (ODCs); Visual system development Visual field maps see Retinotopic maps Visual pathway see Optic pathway Visual system development activity-dependent synaptic competition 31 Hebb’s rule and 35 endocannabinoid LTD and 682 ocular dominance see Ocular dominance columns (ODCs) VMAT-2 see ATP-dependent vesicular membrane transporter 2 (VMAT-2) Voltage-gated calcium channels (VGCCs) biochemical requirements 59 features 65 functional roles 59 synaptic plasticity and
Subject Index endocannabinoid LTD 678 synaptic transmission and 170 neurotransmitter release see Calcium ions and neurotransmitter release retrograde homeostatic signaling and 130 group I mGluRs 304 high-voltage-activated 60 kainate receptors 319 L-type channels (Cav1) 61, 64–65 Cav1.3 subunit (CACNA1D gene) 64–65 synaptic plasticity and endocannabinoid LTD and 678 molecular pharmacology neurotoxin binding 61–62 neuromodulation see Voltage-gated Ca2þ channel neuromodulation N-type channels (Cav2.2) 60–61, 61f, 64f, 65t permeation 65 P/Q-type channels (Cav2.1) 60–61, 64f, 65t R-type channels (Cav2.3) 60–61 selectivity 65 SNARE interactions see SNARE complex structural requirements 59 subfamilies/types 64 Cav2 family 62 development 65 discovery 60 subunits/structures 60–61, 61f T-type (Cav3, low-voltage-activated) 60, 61 endocannabinoids and 665–668 LTD role 678 Voltage-gated sodium channels (VGSCs) Nav1 subfamily neuromuscular transmission 184 neuromuscular junction 184 Voltage gating, gap junctions 200 Boltzmann relation 200 fast 201 sensitivity 201–202 slow 201 steady state currents 200 Voltage-operated calcium channels (VOCCs) see Voltage-gated calcium channels (VGCCs) Voltammetry 442 constant potential amperometry 443 transporter kinetics 443 definition 442 dopamine release autoreceptor control 444 kinetics 444 electrode design 442 advantages 442 carbon fibers 442, 443f microdisk electrodes 442 fast-scan cycle voltammetry 443 advantages 443 disadvantages 443 mechanism of action 443 voltammogram 443 monoamine transporter kinetics 443 dopamine transporter 443–444 exogenously applied substrate 444 Voltammogram, fast-scan cycle voltammetry 443 Volume-regulated chloride/anion channels (VRACs) antagonists 123–124 gliotransmitters, nonexocytotic release 123–124 Volume transmission (VT) definition 442 peptide-induced receptor trafficking 564, 566f serotonin 450 see also Neuropeptides, electrophysiology VPAC1 see Vasoactive intestinal peptide receptors VPAC2 see Vasoactive intestinal peptide receptors VPI-013, 5-HT1A receptor depression studies 457–459 Vps33p, SNARE interactions 72 V-SNAREs 121
W ‘Wake-on’ systems, serotonergic pathways 470–471, 471–472 Waking, locus coeruleus impulse activity 433 Waking state serotonergic pathways 471–472 see also Arousal; Sleep/sleep states Wallerian degeneration synapse elimination vs. 152 Water maze endocannabinoid system role 675 WAY-100635, cardiopulmonary reflex effects 451–452 Weaver mutant mice, Purkinje cell dendrites 3–4 Werdnig-Hoffman disease (SMA type 1), gene mutations 155–156 Whisker(s) see Vibrissae White fibers, muscle 141–142 Whole cell recording patch clamp methods gap junctions 220 serotonergic neuronal pathways 471–472 Wide-field ganglion cell (alpha cells) see Retinal ganglion cells (RGCs) Wiesel, Torsten ocular dominance columns (ODCs) 31 see also Visual deprivation Willardiine 264 5-substituted Willardiines, AMPA receptor function 263 wingless gene/protein synapse formation/maturation in 13 see also Wnt signaling Wiring transmission (WT) serotonin and 450 see also Gap junction(s); Neurotransmission; Synapse(s) Wnt3 synapse formation/maturation 13 Wnt7a synapse formation/maturation 13 Wnt signaling retrograde signaling in central synapse formation 128 synapse formation/maturation 13
X Xanthine 627 Xenopus laevis development synaptogenesis BDNF role 7 XIB4035 607–608 X-linked Charcot–Marie–Tooth disease (CMTX) connexin 32 210
Y Yohimbine adrenergic receptors 427 Yxx motifs 89–90
Z Zebrafish retina retinal ganglion cells dendrites 4–5 skeletal muscle innervation 143 tectal dendritic branch formation 4 Zero flux boundaries, synaptic transmission models 103–104 Zinc NMDA receptors 280–281 vesicular neurotransmitter transporters 258 Zinc metallopeptidases, neuropeptide metabolism see Neuropeptide metabolism Zippering model membrane fusion, SNAREs 69–70, 70f ZnT3 254t, 258
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