Animal Models of Movement Disorders: Volume II (Neuromethods 62)

Neuromethods

Series Editor Wolfgang Walz University of Saskatchewan Saskatoon, SK, Canada



For further volumes: http://www.springer.com/series/7657

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Animal Models of Movement Disorders Volume II

Edited by

Emma L. Lane The Brain Repair Group, Welsh School of Pharmacy, Cardiff University, Cardiff, Wales, UK

Stephen B. Dunnett The Brain Repair Group, School of Biosciences, Cardiff University, Cardiff, Wales, UK

Editors Emma L. Lane The Brain Repair Group Welsh School of Pharmacy Cardiff University Cardiff, Wales, UK [email protected]

Stephen B. Dunnett The Brain Repair Group School of Biosciences Cardiff University Cardiff, Wales, UK [email protected]

ISSN 0893-2336 e-ISSN 1940-6045 ISBN 978-1-61779-300-4 e-ISBN 978-1-61779-301-1 DOI 10.1007/978-1-61779-301-1 Springer New York Dordrecht Heidelberg London Library of Congress Control Number: 2011936395 © Springer Science+Business Media, LLC 2011 All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Humana Press, c/o Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or d ­ issimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. Printed on acid-free paper Humana Press is part of Springer Science+Business Media (www.springer.com)

Preface to the Series Under the guidance of its founders Alan Boulton and Glen Baker, the Neuromethods series by Humana Press has been very successful since the first volume appeared in 1985. In about 17 years, 37 volumes have been published. In 2006, Springer Science + Business Media made a renewed commitment to this series. The new program will focus on methods that are either unique to the nervous system and excitable cells or which need special consideration to be applied to the neurosciences. The program will strike a balance between recent and exciting developments like those concerning new animal models of disease, imaging, in vivo methods, and more established techniques. These include immunocytochemistry and electrophysiological technologies. New trainees in neurosciences still need a sound footing in these older methods in order to apply a critical approach to their results. The careful application of methods is probably the most important step in the process of scientific inquiry. In the past, new methodologies led the way in developing new disciplines in the biological and medical sciences. For example, Physiology emerged out of Anatomy in the nineteenth century by harnessing new methods based on the newly discovered phenomenon of electricity. Nowadays, the relationships between disciplines and methods are more complex. Methods are now widely shared between disciplines and research areas. New developments in electronic publishing also make it possible for scientists to download chapters or protocols selectively within a very short time of encountering them. This new approach has been taken into account in the design of individual volumes and chapters in this series. Neuherberg, Germany

Wolfgang Walz

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Preface Movement is controlled by the interaction of many component parts of the central nervous system, from myelinated motor neurons projecting from the spinal cord to the higher neural processes in cerebellum and basal ganglia. This produces a highly controllable, functional system. However, this finely integrated network can be disrupted by injury and a range of disease processes that lead to significant motor dysfunction. Damage to single elements of this circuitry, which result from both sporadic and genetic conditions, can cause profound alterations in motor function. In order to understand these disorders and thereby facilitate recovery and repair, it is necessary to translate in vitro findings and hypotheses into animal representations of both functional and dysfunctional systems. These animal models range in species from lower orders, such as Drosophila and Caenorhabditis elegans, through vertebrate species including fish, to mammals, such as rodents and nonhuman primates. Each model has its own profile of face, construct, and predictive validities, all of which must be considered when selecting the most appropriate for the experiment in hand. Similarly, the assessment methods used will depend on the species and the outcome variables that need to be assessed and must be similarly scrutinized for validity to answer the postulated hypothesis. In the first volume (Neuromethods, vol. 61), we introduced the variety of tools used in the assessment of motor function, highlighting their advantages and limitations and noting important technical considerations. We first take a look through the clinician’s perspective on animal models of disease, before exploring both simple (e.g., Drosophila) and more complex (rodent and nonhuman primate) model systems and reviewing the use of genetic manipulations, behavioral assessments, and the increasing use of imaging techniques. We then take a journey, descending through the central nervous system, describing animal models of disorders that target different levels of motor control. One interesting development found through the process of formulating this volume was the overlap in rodent behavioral techniques that are used across a range of motor disorders. Importantly, despite their wide use, each laboratory has its own approach to each behavioral technique. Many of the standard tests appear simple on first inspection, but a critical eye is required, and seemingly insignificant manipulations can produce critical differences in the outcomes and interpretation of the data produced. The first volume went on to consider dopaminergic influences over motor control ­typified in the motor disorder, Parkinson’s disease. The interest in this disorder, aided by significant developments such as the accidental discovery of a toxin, MPTP, that produces pathology similar to the disease process, and identification of specific genes involved in the familial form of the disease have led to an extraordinary array of animal models. Furthermore, we not only need to model the disease, but also the consequences of pharmacological treatment, the development of another dopaminergic motor phenotype, l-dopa-induced dyskinesia. In the present volume (Neuromethods, vol. 62), we expand these themes to cover ­animal models of other basal ganglia disorders such as Huntington’s disease and multiple systems atrophy, then through neo- and allo-cortical systems to describe models of ischemia and eye movement control. Descending through the cerebellum, there is a description of

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the role of this complex nucleus in the control of fine motor function, disorders that affect this “little brain” and how they are represented in vivo. The last section considers the role of spinal cord systems, including the modeling of spinal cord injury, demyelinating disorders, and amyotrophic lateral sclerosis. We would like to take this opportunity to thank the teams that contributed chapters and in particular to acknowledge the more junior members who are often those actually at the coalface of these experiments. We also regret the loss of some chapters (thankfully, very few), a consequence of the ever-increasing demands on the time of researchers. We hope that this text will be a valuable reference for those studying motor disorders by covering methodologies in detail and providing the information necessary to consider both the appropriate models and assessment tools that can most informatively answer the key experimental issues in our field. Cardiff, UK

Emma L. Lane Stephen B. Dunnett

Contents Volume I Series Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Part I  Generic Methods of Assessment   1 Why Cannot a Rodent Be More like a Man? A Clinical Perspective . . . . . . . . . . . . . . 3 Anne E. Rosser   2 Zebrafish as a Vertebrate Model Organism for Studying Movement Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 Maria Sundvik and Pertti Panula   3 Methodological Strategies to Evaluate Functional Effectors Related to Parkinson’s Disease Through Application of Caenorhabditis elegans Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 Kim A. Caldwell and Guy A. Caldwell   4 Effects of Alpha-Synuclein Expression on Behavioral Activity in Drosophila : A Simple Model of Parkinson’s Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 Robert G. Pendleton, Xiaoyun C. Yang, Natalie Jerome, Ornela Dervisha, and Ralph Hillman   5 Neurological Evaluation of Movement Disorders in Mice . . . . . . . . . . . . . . . . . . . . . 65 Simon P. Brooks   6 Rodent Skilled Reaching for Modeling Pathological Conditions of the Human Motor System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87 Jenni M. Karl and Ian Q. Whishaw   7 High-Throughput Mouse Phenotyping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109 Sabine M. Hölter and Lisa Glasl   8 MRI of Neurological Damage in Rats and Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135 Mathias Hoehn   9 Quantification of Brain Function and Neurotransmission System In Vivo by Positron Emission Tomography: A Review of Technical Aspects and Practical Considerations in Preclinical Research . . . . . . . . . . . . . . . . . . . 151 Nadja Van Camp, Yann Bramoullé, and Philippe Hantraye

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10 Optical Approaches to Studying the Basal Ganglia . . . . . . . . . . . . . . . . . . . . . . . . . . 191 Joshua L. Plotkin, Jaime N. Guzman, Nicholas Schwarz, Geraldine Kress, David L. Wokosin, and D. James Surmeier 11 Electrophysiological Analysis of Movement Disorders in Mice . . . . . . . . . . . . . . . . . 221 Shilpa P. Rao, Véronique M. André, Carlos Cepeda, and Michael S. Levine

Part II Dopamine Systems 12 Genetic Models of Parkinson’s Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ralf Kühn, Daniela Vogt-Weisenhorn, and Wolfgang Wurst 13 6-OHDA Lesion Models of Parkinson’s Disease in the Rat . . . . . . . . . . . . . . . . . . . Eduardo M. Torres and Stephen B. Dunnett 14 6-OHDA Toxin Model in Mouse . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Gaynor A. Smith and Andreas Heuer 15 Rotation in the 6-OHDA-Lesioned Rat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Stephen B. Dunnett and Eduardo M. Torres 16 Of Rats and Patients: Some Thoughts About Why Rats Turn in Circles and Parkinson’s Disease Patients Cannot Move Normally . . . . . . . . . . . . . . . . . . . . . Gordon W. Arbuthnott 17 Comparing Behavioral Assessment of Sensorimotor Function in Rat and Mouse Models of Parkinson’s Disease and Stroke . . . . . . . . . . . . . . . . . . . . . . . Sheila M. Fleming and Timothy Schallert 18 Rodent Models of l-DOPA-Induced Dyskinesia . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hanna S. Lindgren and Emma L. Lane 19 Using the MPTP Mouse Model to Understand Neuroplasticity: A New Therapeutic Target for Parkinson’s Disease? . . . . . . . . . . . . . . . . . . . . . . . . . Giselle M. Petzinger, Beth E. Fisher, Garnik Akopian, Ruth Wood, John P. Walsh, and Michael W. Jakowec 20 The MPTP-Treated Primate, with Specific Reference to the Use of the Common Marmoset (Callithrix jacchus) . . . . . . . . . . . . . . . . . . . . . . . . . . . . Michael J. Jackson and Peter Jenner 21 Behavioral Assessment in the African Green Monkey After MPTP Administration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Eugene Redmond Jr.

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Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 437

Volume II Preface to the Series . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiii

Part I  Basal Ganglia   1 Behavioral Assessment of Genetic Mouse Models of Huntington’s Disease . . . . . . . Miriam A. Hickey and Marie-Françoise Chesselet   2 Excitotoxic Lesions of the Rodent Striatum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Máté D. Döbrössy, Fabian Büchele, and Guido Nikkhah   3 Combination Lesion Models of MSA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Daniela Kuzdas and Gregor K. Wenning   4 The Role of the Dorsal Striatum in Instrumental Conditioning . . . . . . . . . . . . . . . Mark A. Rossi and Henry H. Yin   5 3-Nitropropionic Acid and Other Metabolic Toxin Lesions of the Striatum . . . . . . Cesar V. Borlongan and Paul R. Sanberg   6 Functional Assessment of Subcortical Ischemia . . . . . . . . . . . . . . . . . . . . . . . . . . . Tracy D. Farr and Rebecca C. Trueman

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Part II Neo- and Allo-Cortical Systems   7 Functional Organization of Rat and Mouse Motor Cortex . . . . . . . . . . . . . . . . . . . G. Campbell Teskey and Bryan Kolb   8 Forebrain Circuits Controlling Whisker Movements . . . . . . . . . . . . . . . . . . . . . . . . Kevin D. Alloway and Jared B. Smith   9 An Approach to Understanding the Neural Circuitry of Saccade Control in the Cerebral Cortex Using Antidromic Identification in the Awake Behaving Macaque Monkey Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Kevin Johnston and Stefan Everling 10 Photothrombotic Infarction of Caudate Nucleus and Parietal Cortex . . . . . . . . . . . Toshihiko Kuroiwa and Richard F. Keep 11 Models of Rodent Cortical Traumatic Brain Injury . . . . . . . . . . . . . . . . . . . . . . . . Frances Corrigan, Jenna M. Ziebell, and Robert Vink 12 The Use of Commissurotomy in Studies of Interhemispheric Communication . . . . Ian Steele-Russell

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Part III Cerebellar and Brain Stem Systems 13 Genetic Models of Cerebellar Dysfunction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Robert Lalonde and Catherine Strazielle 14 Cerebellar Control of Fine Motor Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Rachel M. Sherrard 15 Cerebellum and Classical Conditioning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Richard F. Thompson 16 Assessments of Visual Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ma’ayan Semo, Carlos Gias, Anthony Vugler, and Peter John Coffey 17 The Role of the Pedunculopontine Tegmental Nucleus in Motor Disorders . . . . . . Nadine K. Gut and Philip Winn

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Part IV Spinal Cord Systems 18 Contusion Models of Spinal Cord Injury in Rats . . . . . . . . . . . . . . . . . . . . . . . . . . Kelly A. Dunham and Candace L. Floyd 19 Demyelination Models in the Spinal Cord . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Paul A. Felts, Damineh Morsali, Mona Sadeghian, Marija Sajic, and Kenneth J. Smith 20 Preparation of Spinal Cord Injured Tissue for Light and Electron Microscopy Including Preparation for Immunostaining . . . . . . . . . . . Margaret L. Bates, Raisa Puzis, and Mary Bartlett Bunge 21 Assessing Spinal Cord Injury . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Gillian D. Muir and Erin J. Prosser-Loose 22 Precise Finger Movements in Monkeys . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Roger Lemon

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Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 435

Contributors Kevin D. Alloway  •  Center for Neural Engineering, Pennsylvania State University, State College, University Park, PA, USA Margaret L. Bates  •  The Miami Project to Cure Paralysis, University of Miami Miller School of Medicine, Miami, FL, USA Cesar V. Borlongan  •  Department of Neurosurgery and Brain Repair, Center of Excellence for Aging and Brain Repair, University of South Florida College of Medicine, Tampa, FL, USA Fabian Büchele  •  Laboratory of Molecular Neurosurgery, Department of Stereotactic Neurosurgery, University Freiburg - Medical Center, Freiburg, Germany Mary Bartlett Bunge  •  The Miami Project to Cure Paralysis, University of Miami Miller School of Medicine, Miami, FL, USA; Department of Cell Biology & Anatomy, University of Miami Miller School of Medicine, Miami, FL, USA; Department of Neurological Surgery, University of Miami Miller School of Medicine, Miami, FL, USA Marie-Françoise Chesselet  •  Department of Neurology, David Geffen School of Medicine, UCLA, Los Angeles, CA, USA Peter John Coffey  •  Department of Ocular Biology and Therapeutics, Institute of Ophthalmology, University College, London, UK Frances Corrigan  •  Centre for Neuroscience Research, School of Medical Sciences, University of Adelaide, Adelaide, SA, Australia Máté D. Döbrössy  •  Laboratory of Molecular Neurosurgery, Department of Stereotactic Neurosurgery, University Freiburg - Medical Center, Freiburg, Germany Kelly A. Dunham  •  Department of Physical Medicine & Rehabilitation, University of Alabama at Birmingham, Birmingham, AL, USA Stefan Everling  •  Department of Physiology & Pharmacology, Robarts Research Institute, University of Western Ontario, London, ON, Canada Tracy D. Farr  •  Department for Experimental Neurology, Center for Stroke Research Berlin (CSB), Department for Experimental Neurology, Charité University Medicine, Berlin, Germany Paul A. Felts  •  Centre for Anatomy and Human Identification, University of Dundee, Dundee, UK Candace L. Floyd  •  Department of Physical Medicine & Rehabilitation, University of Alabama at Birmingham, Birmingham, AL, USA Carlos Gias  •  Department of Ocular Biology and Therapeutics, Institute of Ophthalmology, University College, London, UK Nadine K. Gut  •  Strathclyde Institute of Pharmacy & Biomedical Sciences, University of Strathclyde, Glasgow, Scotland, UK Miriam A. Hickey  •  Department of Neurology, David Geffen School of Medicine, UCLA, Los Angeles, CA, USA

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Kevin Johnston  •  Centre for Neuroscience Studies, Queen’s University Kingston, Kingston, ON, Canada Richard F. Keep  •  Department of Neurosurgery, University of Michigan, Ann Arbor, MI, USA Bryan Kolb  •  Canadian Centre for Behavioural Neuroscience, University of Lethbridge, Lethbridge, AB, Canada Toshihiko Kuroiwa  •  Department of Neurosurgery, University of Michigan, Ann Arbor, MI, USA; Department of Clinical Laboratory, Namegata District General Hospital, Namegata, Ibaraki, Japan Daniela Kuzdas  •  Division of Clinical Neurobiology, Department of Neurology, Innsbruck Medical University, Innsbruck, Austria Robert Lalonde  •  Départment de Psychologie, Université de Rouen, Mont-Saint-Aignan, France Roger Lemon  •  Institute of Neurology, University College London, London, UK Damineh Morsali  •  Department of Neuroinflammation, Institute of Neurology, University College London, London, UK Gillian D. Muir  •  Department of Biomedical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, SK, Canada Guido Nikkhah  •  Laboratory of Molecular Neurosurgery, Department of Stereotactic Neurosurgery, University Freiburg - Medical Center, Freiburg, Germany Erin J. Prosser-Loose  •  Department of Biomedical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, SK, Canada Raisa Puzis  •  The Miami Project to Cure Paralysis, University of Miami Miller School of Medicine, Miami, FL, USA Mark A. Rossi  •  Centre for Cognitive Neuroscience, Duke University, Durham, NC, USA Ian Steele-Russell  •  Departments of Psychiatry and Behavioral Science, Neuroscience and Experimental Therapeutics, and Veterinary Integrative and Biomedical Science, Texas A&M University System Health Science Center and Texas A&M University, TX, USA Mona Sadeghian  •  Department of Neuroinflammation, Institute of Neurology, University College London, London, UK Marija Sajic  •  Department of Neuroinflammation, Institute of Neurology, University College London, London, UK Paul R. Sanberg  •  Department of Neurosurgery and Brain Repair, Center of Excellence for Aging and Brain Repair, University of South Florida College of Medicine, Tampa, FL, USA Ma’ayan Semo  •  Department of Ocular Biology and Therapeutics, Institute of Ophthalmology, University College, London, UK Rachel M. Sherrard  •  UPMC-Univ Paris 6 and CNRS, UMR7102 Neurobiologie des Processus Adaptatifs, Paris, France Jared B. Smith  •  Center for Neural Engineering, Pennsylvania State University, State College, PA, USA Kenneth J. Smith  •  Institute of Neurology, University College London, London, UK Catherine Strazielle  •  INSERM U954 and Faculté de Médicine, Université Henri Poincaré, Vandœuvre-lès-Nancy, France

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G. Campbell Teskey  •  Department of Cell Biology and Anatomy, Hotchkiss Brain Institute, University of Calgary, Calgary, AB, Canada Richard F. Thompson  •  Program in Neuroscience, University of Southern California, Los Angeles, CA, USA Rebecca C. Trueman  •  The Brain Repair Group, School of Biosciences, Cardiff University, Cardiff, Wales, UK Robert Vink  •  Centre for Neuroscience Research, School of Medical Sciences, University of Adelaide, Adelaide, SA, Australia Anthony Vugler  •  Department of Ocular Biology and Therapeutics, Institute of Ophthalmology, University College, London, UK Gregor K. Wenning  •  Division of Clinical Neurobiology, Department of Neurology, Innsbruck Medical University, Innsbruck, Austria Philip Winn  •  Strathclyde Institute of Pharmacy & Biomedical Sciences, University of Strathclyde, Glasgow, Scotland, UK Henry H. Yin  •  Centre for Cognitive Neuroscience, Duke University, Durham, NC, USA Jenna M. Ziebell  •  Centre for Neuroscience Research, School of Medical Sciences, University of Adelaide, Adelaide, SA, Australia

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Part I Basal Ganglia

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Chapter 1 Behavioral Assessment of Genetic Mouse Models of Huntington’s Disease Miriam A. Hickey and Marie-Françoise Chesselet Abstract Huntington’s disease (HD) is a monogenetic, neurodegenerative disease. It is fatal, and although treatments are available for minor symptomatic relief, it remains incurable. Careful study of models of HD remains critical for elucidation of disease mechanisms and for the development of therapeutics. Models that rapidly develop to end stage are useful to assess efficacy of therapeutics for neuroprotection in relatively short experiments. However, the striatum, an area of brain traditionally associated with degeneration in HD already shows extensive atrophy by the time the symptoms manifest in patients, indicating that substantial degeneration occurs in the years preceding clinical onset. Thus, it is vital to also study models that allow analysis of the early pre-manifest disease stage. This requires the development of sensitive output measures to reveal deficits before the onset of obvious anomalies. Here, we briefly review the characteristics of ­several mouse models of HD and outline methods for analysis of behavioral deficits in both severe fast-­ progressing models and for early stages of disease in slowly progressive models. Key words: Knock-in, Transgenic, Viral vector, Behavior, Aggregate, Neuropathology, Therapeutic, Motor, Cognitive, Affective

1. Introduction Huntington’s disease (HD) is an autosomal dominant, genetic disorder that causes progressive neurodegeneration in the central nervous system (CNS). The disease, which affects up to 1 in 10,000 people worldwide, is caused by expansion of a trinucleotide repeat encoding glutamine (CAG) within exon 1 of the HD gene (1), leading to the expression of mutant huntingtin protein. The striatum undergoes extensive degeneration in HD, from an early stage in disease progression (2) although many other areas also degenerate. Traditionally, the triad of symptoms that are associated with

Emma L. Lane and Stephen B. Dunnett (eds.), Animal Models of Movement Disorders: Volume II, Neuromethods, vol. 62, DOI 10.1007/978-1-61779-301-1_1, © Springer Science+Business Media, LLC 2011

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HD include progressive decline in motor control and cognitive function, and psychiatric disturbances (3). Motor signs include chorea, dystonia, dysarthria, and dysphagia. Over time, the chorea gives way to bradykinesia (slowed movement) and rigidity (4). Cognitive dysfunction includes poor planning and impaired visuospatial skills (5–7). Modified rating scales have been developed to chart psychiatric symptoms, with much success (8). Apathy and irritability appear to be sensitive to disease progression, while depression is highly prevalent (8–10) and suicide is frequent (11). Once diagnosed, the disease progresses relentlessly over a period of 15–20 years. Although some symptoms may be improved with pharmacological treatments there are currently no effective neuroprotective or disease-modifying agents. Recent data show that many deficits and pathological changes are present in gene carriers that do not yet display sufficient symptoms for diagnosis of disease onset. These signs include changes in white matter and deficits in visual working memory, in odor recognition and identification, variability in tongue protrusion force and in timing of finger taps, in addition to increased incidence of apathy and irritability (2, 10, 12–16). Indeed several of these endpoints may be used for clinical trials over as little as 1 year (16). Other neuropathological changes also precede the onset of typical motor features and are present in gene carriers many years prior to the predicted age of onset. These changes include intranuclear inclusions (aggregates of insoluble mutant huntingtin), reduced striatal and whole brain volume, cortical thinning, and white matter loss, as well as hypothalamic changes (2,  12, 17–20). Intriguingly, cortical thinning has been shown to correlate with Total Functional Capacity, a measurement used in clinical trials for monitoring disease progression (20). In addition, the areas of cortical thinning, but not striatal volume, were different in patients with differing clinical phenotypes (20). The classical “selectivity” of neuronal degeneration in HD is relative rather than absolute, as outlined above. This has implications for the behavioral and pathological analysis of models of this disorder. Importantly, HD patients share symptoms with other neurodegenerative diseases, including Parkinson’s disease (PD) and Alzheimer’s disease (AD). Dystonia (co-contraction of antagonistic muscles) can complicate both PD and HD (21, 22); akinesia, a cardinal sign of PD, also occurs in patients with HD (23); as mentioned, cognitive disruption is present and may even be a presenting symptom in HD patients, whereas it dominates the symptoms in AD and can be present in PD (10, 16, 24, 25); both PD and HD patients show high prevalence of affective disorders and depression (9, 26); finally sleep disturbances are present in HD, AD, and PD (27). Thus, despite the presence of different pathologies, one should not be surprised to find similar behavioral deficits in models of these disorders, and indeed behavioral

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tests used for models of PD and AD can be utilized in the investigation of models of HD. An important consideration is that in patients with HD, clinical symptoms do not manifest until approximately 1⁄3 to 1⁄2 of striatum has degenerated (28). Thus, profound degeneration is necessary to cause clinical signs traditionally associated with the disease. However, in most models the neuronal cell loss that “defines” the disease in patients occurs late or is even absent in the lifetime of the animal. Nevertheless, profound behavioral anomalies can be detected with sensitive behavioral tests, indicating that the mutation induces neuronal dysfunction before causing cell death, mirroring observations in patients (29). An important caveat is that tests most commonly used to assess motor skills such as open field and rotarod are not always sensitive enough to detect the earliest deficits in models of basal ganglia dysfunction. Indeed, these tests are primarily sensitive to cerebellar or motor neuron deficits. In this chapter, we will present a brief overview of the most often studied mouse models of HD and will review the tests we found most useful in our own experience to assess early motor deficits in genetic mouse models of HD, with an emphasis on considerations to keep in mind for the choice of test and its interpretation.

2. Mouse Models of HD Prior to the discovery of genetic mutations, the only models available for HD were based on the use of neurotoxins that kill more or less selectively the neurons that are lost in patients. Toxin-based rodent models for HD were primarily based on local injections of quinolinic acid into the striatum or peripheral 3-nitroproprionic acid (3-NP; reviewed by (30)). However, since HD is caused by a single genetic mutation with high penetrance, expression of the mutated gene has high construct validity and there is little justification for the use of toxin models at this time. While no animal model is perfect, several genetic models of HD exist that replicate many features of the human disease. Genetic models of HD in rodents fall into two categories: mice or rats expressing a fragment of huntingtin as a transgene,1 or by viral vector-mediated gene transfer; and those expressing full length huntingtin, as a transgene, or as a knock-in2 mutation. In particular, mouse models based on expression of the full-length mutant

 Transgene: exogenous fragments of DNA. Generally inserted into the host DNA. May be introduced via a viral vector. If present in the germline, can be used to generate a transgenic line. 2  Knock-in: targeted insertion of cDNA into specific site in host organism’s genome. 1

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HD gene provide a long timeframe in which to examine the earliest behavioral and pathological manifestations of the mutation. Transgenes or viral vectors most often express the mutated protein in the context of a normal load of wildtype endogenous huntingtin, although a few models have been generated on a null background. In addition, the expression levels and site of expression can vary depending on the promoter used for the transgene, or the site of injection of the viral vector. Virus vector-mediated gene transfer has also facilitated the development of genetic models of HD in non-human primates (31, 32). The rapid progression of the neurodegenerative phenotype in these models is probably due to a high level of expression of mutant htt, as a result of a combination of strong expression from a heterologous promoter and transduction of infected neurons with multiple copies of the virus vector. In another rAAV-based model of HD in rats, quantitative RT-PCR revealed mutant htt expression to be over 100-fold that of the endogenous rat htt mRNA (33). Thus, cumulative expression of expanded polyQ proteins throughout the lifetime of experimental animals is not required to induce neuronal cell death; rather, acute overexpression of polyQ is toxic to adult neurons in vivo. There are several points to consider when comparing these models. Shorter fragments of huntingtin are more toxic than longer fragments (see for review (34)). Wildtype huntingtin is protective and important for development (35), and transgenic mice result in higher levels of expression of total (wildtype and mutated) huntingtin. Many kinds of peripheral cells become dysfunctional in HD (36, 37). Therefore viral vector-based models may not reflect the entire disease accurately. In contrast to transgenic models, knock-in models carry a mutation within their own endogenous huntingtin homologue (Hdh) gene. Thus they express a full length protein, theoretically in the proper genomic and protein context. However, the extent of human sequences inserted with the mutation determines the protein context surrounding the CAG repeat expansion and whether or not part of the human promoter is inserted with the mutation may influence the regional and temporal level of expression of the mutated protein. Typically, full-length models result in a more slowly progressing phenotype, which may aid in delineating the important early steps in disease progression. Despite radical differences in the timelines of disease progression between models, significant convergence in phenotypes and pathology is observed, as detailed below. It is important to consider animal size as a factor for eventual analysis of neurosurgical interventions for HD, or for analysis of pharmacokinetics and pharmacodynamics, either within the brain or periphery. Transgenic rats are more amenable than mice in a variety of behavioral tests and imaging studies, and successful rat models of HD have been extensively characterized (38, 39). In addition, several types of large animal models for HD now exist,

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including sheep (40), pigs (41), and non-human primates (32). However, the cost and facilities required will likely be limiting factors in their use. In addition, the paucity of behavioral research on sheep and pigs may impede therapeutics testing; however, steps are being made to address this issue (42). The vast majority of HD research utilizes mice, and here, we focus on behavioral testing of mice. 2.1. R6/2 HD Transgenic Mice

The R6 lines were the first successful transgenic mouse models of HD to be generated (43) and the R6/2 line remains the best characterized and most widely used of the fragment models. Indeed, this model develops several features observed in full-length htt models, albeit within a much faster timeframe (44–46). R6/2 mice express exon 1 of the human HD gene with approximately 150 CAG repeats (43). It is important to test for repeat length in each colony and in the animals actually used for the experiment because genetic drift occurs in these mice. This is particularly important in light of recent studies showing amelioration of disease in mice with “hyper-expanded” repeats (>300) (47, 48). Gender is also important to consider as male R6/2 mice tend to develop disease over a shorter time course than females (49). The original R6/2 mice have a rapid disease course. By approximately 10 weeks, their body weight plateaus, and thereafter declines, and the mice die prematurely at 13–16 weeks of age (43, 50). However, life span varies with source colony and husbandry techniques (51). These mice and other models expressing a fragment of the mutant protein can show glucose intolerance and diabetes, and this is also an important symptom to monitor (52). Furthermore, at late stages of disease, temperature regulation becomes dysfunctional, resulting in low body temperatures (53). R6/2 mice develop progressive motor deficits. By 6–8 weeks of age (depending on the colony) R6/2 mice begin to show overt deficits in gait, tremor, and poor coat condition (44). Cognitive deficits in aspects of visuospatial and procedural memory in R6/2 mice reminiscent of human HD are apparent in tests using the Morris water maze, T-maze, or Pavlovian fear conditioning, although age and disease progression can impact strategies used to complete cognitive tasks (54–57). Emotional responses are also dysfunctional at early (44) and late stages (58). Thus, these mice represent a severe and fast progressing model of HD, which can be used for in vivo high throughput behavioral testing at ages prior to overt phenotype development and severe pathology (44), although the timeframe to overt phenotype development is extremely short. The aggressive phenotype in R6/2 mice facilitates behaviorand, survival-based assays of therapeutic strategies in a relatively short time frame (59). However, institution-specific veterinary rules may dictate that the animal must be euthanized when a specific amount of weight is lost, and often prohibits the use of survival as an endpoint measure for neuroprotective studies (60).

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A limitation of the model is that early disease-related deficits may not be experimentally accessible in this model. It is also important to consider that R6/2 mice often exhibit phenotypes atypical of adult-onset HD, including epilepsy, cardiac dysfunction, and neuromuscular junction abnormalities (43, 61, 62). Nevertheless, this model clearly demonstrates in  vivo toxicity of exon 1 of mutant huntingtin and offers a wealth of behavioral, molecular, cellular and pathological endpoints for analysis. R6/1 mice, a sister line generated initially with a smaller repeat length, provide a longer timeframe to disease onset (43, 63). There may be some variability in onset of R6/1 phenotype, in different colonies, however a congenic line on the C57Bl/6 background has accelerated onset of behavioral dysfunction (63–66). Indeed with increasing C57Bl/6 background, we and others have also observed acceleration of phenotype and pathology in HD knock-in models (see below) (45, 67). In particular, rotarod impairments (see below) and learning deficits in the Morris water maze appear by 2m of age in the B6 R6/1 line, indicating substantial early dysfunction. 2.2. Full-Length Models of HD

Full-length models of HD include transgenic and knock in models. Full length Htt transgenic models include expression via a yeast artificial chromosome (68, 69), and more recently, via a bacterial artificial chromosome (70). Several lines of YAC HD transgenic mice have been generated that express a full-length human genomic mutant HD gene. YAC HD72 mice, with 72 CAG repeats, exhibit slower disease progression resulting from the lower levels of expression and perhaps the shorter repeat length. YAC128 mice express full-length HD containing 128 CAG repeats under the control of the native promoter (69). YAC128 mice have a more pronounced behavioral phenotype than YAC HD72 mice and exhibit a biphasic motor profile, with open field hyperactivity at 3 months followed by hypoactivity at 12 months. YAC128 mice have a normal gait but are impaired on the rotarod task by 6 months of age (69). Cognitive impairment precedes and then parallels progressive motor impairment (71). Both YAC lines show nuclear localization of htt fragments, which coincides with motor dysfunction and occurs first and most abundantly in the striatum, the region most vulnerable in HD (72). In addition, YAC128 mice show ~18% neuronal cell loss specifically in the striatum by 12 months of age (69). Carrying a stable poly CAA-CAG (n = 97) repeat to decrease somatic instability, the BAC HD mice develop progressive open field and rotarod deficits (49, 70). They show cortical and striatal atrophy and “dark neurons” without neuronal loss. Huntingtin aggregates and even diffuse huntingtin nuclear staining occur only in old age. These mice show consistent levels of both full length and N-terminal cleaved huntingtin between the ages of 2 and 12 months, while behavioral deficits and neurodegenerative changes progress.

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Both YAC and BAC transgenic full-length models show a gain in body weight over time (49), in contrast to the hallmark loss in body weight observed in HD patients despite increased caloric intake (73). Interestingly, overexpression of both wildtype and mutant full length huntingtin is associated with weight gain (74), suggesting that it is the overall greater level of the protein in these models that causes the weight gain. Theoretically, knock-in (KI) models should provide an optimal model of human HD as these mice carry the mutation in its appropriate genomic and protein context; however, this depends on the specific construct used (75). A number of KI mouse models have been generated to date. Of the various KI models, CAG140 (45, 76) and Hdh150 (46, 77, 78) are the best characterized. The Hdh150 mice harbor an expanded CAG repeat inserted into mouse Hdh, while CAG140 mice express a chimeric HD/Hdh protein, with human mutant exon 1 and part of the human promoter. Thus in contrast to the Hdh150, in these mice the protein context of the mutation includes both human and mouse sequences and the level and pattern of expression of the mutant Htt may be modified by the presence of human sequences in the promoter region. All KI mice eventually develop some evidence of neuronal degeneration, in the form of dark neurons in HdhQ111 mice and axonal degeneration in HdhQ72/80 mice in striatal projection areas. However, only CAG140 (45) and Hdh150 (77) mice show frank striatal ­neuronal loss, at approximately 2 years of age. Optical density for DARPP-32 is reduced by 1 year of age in CAG140 mice. In contrast to the YAC and BAC mice, both Hdh150 and CAG140 lines lose weight (45, 46, 77) at late disease stages, again a feature of the human disease. Most KI mice show overt, spontaneous gait deficits by 2 years of age (45, 77, 79). This late stage may recapitulate the overt phenotype that develops in R6/2 mice at approximately 8 weeks of age. Old Hdh150 mice show transcriptome anomalies similar to those of late stage R6/2 mice (46). Thus, these latter models (CAG150, Q111) require 2 years to progress to a point that is reached in 12 weeks in the R6/2 model, demonstrating the long timeframe that can be utilized for therapeutic and disease mechanism testing. In contrast, the CAG 140 mice have a much faster disease progression than these other KI mice; however, it is still much more protracted than in the R6/2 transgenics. We have recently found transcriptional deficits in CAG140 homozygous KI mice by 4m of age, many months prior to onset of overt phenotype (80), in agreement with the presence of behavioral and pathological deficits in these mice as early as 1 month of age (45, 76). Even on an identical genetic background, CAG140 mice develop aggregates much sooner than Hdh150 mice (81). Diffuse staining for aggregated huntingtin in striatal neurons is present from approximately 2m of age in CAG140 mice, with inclusions present from 4m (76, 81). In contrast, a diffuse nuclear localization is only

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observed at 7m in Hdh150 mice, and inclusions are evident by approximately 10m (77). Diffuse staining is observed in 6-weekold homozygous HdhQ111 knock-in mice, microaggregates by 5m and inclusions by 10m (79).

3. Methodological Considerations When Using Mice as Models of Neurodegenerative Diseases

As shown above, and in many reports that examine the effect of strain of mouse on behavioral tests, strain is extremely important to control, especially when lines are maintained on a mixed background (82–84). Non-sibling, distantly related pairs should be used for breeding for these colonies and this requires careful documentation of family lineages. The advent of congenic3 strains does not alleviate the effect of strain, since regular “refreshing” is necessary to prevent genetic drift. When mice are maintained on a hybrid background, careful consideration of generation number is also required when comparing results. Gender is an obvious factor to take into account when conducting any analyses, and in particular in behavioral research (45, 84–88). Although clear gender differences have not been documented clinically in HD patients, it is important to consider that males have been demonstrated to show more accelerated disease progression in R6/2 mice (49). Environmental enrichment for laboratory animals is now often required by animal research oversight committees, and this has profound effects on phenotype expression and disease course and can complicate comparison of results across laboratories (49). Indeed, prolonged environmental enrichment is beneficial in HD mouse models (89, 90) and the type of enrichment provided to mice should be detailed in methods sections. In addition, several laboratories now provide additional husbandry to advanced stage models of HD, including lowered water spouts and moistened chow (51, 90). These accommodations extend survival and should also be outlined in the methods. Additionally, prior to any behavioral testing, consideration should be given to whether the test subjects should be handled. We frequently do not handle our mice prior to testing the open field. However, mice are handled subsequently, to reduce stress in more involved tests of motor function, for example, rotarod (see below). Another important consideration is that, as in humans, the disease in these animals is progressive. Many models show hyperactivity followed by hypoactivity, and cellular

 Congenic: To generate a congenic strain mice from two genetically different strains are bred together. Resulting progeny are then bred again to the desired strain, up to 10 times (usually). Speed congenics may reduce the time taken, and involves analysis of the progeny for genetic markers of the desired strain, thus allowing only progeny with the most genetic markers of the desired strain to move forward to breeding.

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deficits change with age in R6/2 mice (29, 69, 76). Therefore, one needs to test at different times, carefully match groups for ages and take this possibility into account during statistical analysis of longitudinal data. Consideration of the effect of age on performance of wildtype mice is critical, since the window of genotype deficit may be reduced, due to declining performance of wildtype animals (45, 91). Finally, mice are nocturnal animals and the diurnal phase during which testing is conducted can profoundly influence the results. Testing in the dark phase, when mice are more active may provide more sensitivity, due to the higher normal level of activity (44, 45, 76). For these tests, it is useful to have access to a reversed phase animal room. Otherwise, one may choose tests that can be performed during the light phase. It is also important to schedule these tests after the animals have habituated to the dark or light, i.e., outside of the first/last hour of darkness or light.

4. Tests for Spontaneous Phenotype in Genetic Mouse Models of HD

5. Tests for Analysis of Early Motor Deficits in Mouse Models of HD

Most mouse models of HD do not develop obvious behavioral anomalies in their home cage until late in the disease. This obvious behavior is sometimes referred to as “symptomatic” or perhaps more accurately as “manifest”. When present, spontaneous home cage behavioral anomalies can be scored on a non-parametric scale for the presence or absence of tremor, and presence or absence of an unsteady or uncoordinated gait, in addition to turning ability and the width of the hindlimb base, the extent of piloerection (indicative of poor coat maintenance) and clasping. Interestingly, the CAG140 KI mice show these signs when approximately 40% of neurons and volume are lost (45), akin to the 1⁄3 to 1⁄2 loss observed in patients when obvious signs manifest (28).

As discussed earlier, one distinct advantage of mouse models of HD is the ability to analyze early stages of the disease, that are equivalent to the long pre-manifest phase of the disease that is now recognized in patients (16). This requires the use of specialized tests that challenge the motor system. Remarkably, many of the same tests used in R6/2 mice are able to detect deficits in the more progressive CAG140 mouse model at a very early age (44, 45). We will describe these tests starting with the most commonly used and then those we have found to offer superior sensitivity and/or ease of use.

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5.1. Open Field Test

One of the most well-known and well-used behavioral tests, the open field test can be conducted in the dark or light phase; however, it is important to maintain consistency within groups, and to disclose the period of the light phase used for testing. Behavior in the open field can be analyzed with manual methods, using video recording of activity for later analysis of rearing and locomotion, in addition to grooming or other behaviors, offering a low-cost alternative. When the required equipment is available, however, open field behavior can be analyzed automatically usually by recording infra-red beam breakages to identify the position of the mouse in X, Y and Z axes. With careful calibration to ensure accurate quantification of rearing and locomotion, automation can contribute information on speed, area occupied, number and length of individual movements, etc., more easily than video analysis. As stated above, hyperactivity is noted in several models at early stages of disease and is followed by hypoactivity (69, 76, 92, 93), thus this test can be used over time, or at specific ages. An issue associated with the open field is that it relies upon spontaneous locomotion. Factor analysis by various authors have found that exploration (94) and anxiety (95) can account for a large amount of variability in rodent open field activity. Thus, it is advantageous to use other tests in parallel.

5.2. Rotarod

Rotarod analysis can be performed either in the light or dark phase. It is easy to use, relatively automated, and relatively reproducible as long as the conditions of use are clearly defined. This allows for a comparison of mouse models between different laboratories. Paradigms include fixed speeds, or accelerating speeds, incorporating smooth or grooved axles, all of which greatly affect sensitivity (96). The standard apparatus is purchased from Ugo Basile, Varese, Italy, and it is used to measure the latency of mice to fall from the rotating rod. Usually, a few mice “cling” to the axle, and to prevent bias these are removed after three consecutive rotations and the latency at that time is used as the score for that animal. Sometimes the axle of the rotarod is covered with smooth rubber (“smooth axle”) to reduce clinging and to make the task more difficult than on the grooved axle. Duration and frequency of trials as well as speed can differ and it is important to document all parameters used in the published reports. Unfortunately, this test is particularly sensitive to motor neuron or cerebellar deficits, which are typically absent in HD; accordingly, HD mice tend to only show anomalies on the rotarod at advanced stages of the disease. In our experiments, mice were trained to run on the smooth axle that is accelerated from 4 to 40 rpm over 10 min (3 trials per day for 4 days) (44, 45, see also 90). Even with this protocol, however, deficits in rotarod ­performance of R6/2 mice were not detected until after initial climbing, and open field-rearing deficits and deficits in CAG140 KI mice are very subtle (44, 45). Many other models have now shown deficits in rotarod performance, including KI mice (97) and

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the BAC transgenic lines (49, 70), although there is some ­variability in rotarod impairments in YAC128 transgenic mice (49, 71). 5.3. Climbing

Climbing behavior can be measured without expensive equipment, either during the light or the dark phases of the day, and shows very early alterations in mouse models of HD. We found deficits as early as 4.5 weeks in R6/2 mice (44) and 1.5 months in the CAG140 KI mice (45). Mice are placed at the bottom of wire cylinder cages (diameter, 10.5 cm, height, 15.5 cm) and spontaneous activity is videotaped for 5 min to quantify the number of climbs (defined as all four paws on the side of the cage). The same tapes can be used to quantify the number of rears made by the mouse to monitor general activity. This test has now been used in several other transgenic mice and reveals deficits in BAC and YAC 128 transgenic mice (98, 99). A caveat is that when tested during the dark phase, gender effects are noted, and group sizes must be designed accordingly (Fig. 1).

5.4. Wheel Running

Running wheels are widely used in research on circadian rhythms and are fully automated. They represent a significant initial investment but are valuable for preclinical drug testing because of the high power to detect differences between groups, the minimal investigator time involvement, and the wealth of information obtained. We have successfully developed the use of running wheels

Fig. 1. Climbing activity in C57Bl/6J mice at 6.5 weeks of age. Mice were tested either during dark phase, or light phase (not both). Mice climb more during their active phase (dark phase). However, gender differences are observed in climbing during the dark phase, which would necessitate large mixed gender group sizes, in order to observe genotype differences, or treatment effects. Larger mixed gender group sizes (or single gender groups) are not necessary if mice are tested during the day phase. Data are shown as mean ± SEM. Data were analyzed using a completely randomized ANOVA, followed by Fisher’s PSD post hoc tests. Effect of phase F(1,30) = 16.5, p < 0.001. ^p < 0.05 compared to male, dark phase. *p < 0.05 compared to male, dark phase. **p < 0.01 compared to female, dark phase. N = 6–10.

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to assess motor deficits in both R6/2 and KI HD mice (44, 45, 100). Mice are placed in individual cages equipped with a running wheel (23  cm diameter, Mini Mitter Company Inc., Bend OR). Each rotation of the wheel is detected by a magnet and recorded by VitalView Data Acquisition Software V 4.0 (Mini Mitter Company Inc.), in 3 min bins. It is important to house running wheel cages in cabinets to minimize light and sound disturbance. Cabinets must be equipped with fans to allow air circulation, and light should follow the same pattern of light dark phases as in the vivarium. Running activity is recorded continuously and activity is recorded in light and dark phases. It is critical to only use mice of the same sex for the calculations as activity of females is different to that of males and also changes during the estrus cycle (45, 86, 87). Wheel running activity (speed of running) during dark and light phases can be calculated using ActiView V 1.2 (Mini Mitter Company Inc.). We have also analyzed patterns of activity over the dark (active) phase and found deficits in R6/2 and CAG140 KI mice (44, 45). In addition, by monitoring performance over time, it is possible to analyze motor learning using this test (45). Running wheel testing is highly amenable to therapeutic testing because of its high power to detect drug effects. For example, we have found that only 8 R6/2 transgenic mice are required to detect an improvement of 50% with 80% power (a = 0.05) at 4.5 weeks of age, while 5 male CAG140 homozygote KI mice are sufficient to detect a 50% improvement in performance at 6m of age (80% power, a = 0.05) (44, 45). Strain is also important to take into account with this test, as it can profoundly affect running performance. In our experience, B6129S4 mice are very poor runners (Fig.  2), while B6129/Sv (45) and B6CBA hybrids are extremely good runners (44).

Fig. 2. Illustration of the poor running activity in B6129S4 mice, compared with B6129Sv mice. B6129Sv mice were N3 on B6 background. Mice are wildtype. Data in gray (B6129Sv) are from Hickey et al. (45). Data are of mean ± SEM, and show mean running per 3 min bin, per night. Mice are housed individually in cages equipped with running wheels, as described in text. Data are collected automatically. These data demonstrate the importance of determining the suitability of each test for each strain as necessary.

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The pole task is commonly used in models of PD (101, 102) but also detects profound anomalies in HD mice. For this test, mice are placed on a vertical pole (1 cm in diameter, 60 cm high). The spontaneous behavior for mice is to turn downwards and descend the pole. After habituation to the task in 2 trials per day for 2 days, on the third day mice are given 5 trials. The time taken to turn to face downwards (t. turn) is measured, in addition to the total time to complete the task (t. total) and the time taken to descend (t. descend), following the turn. We found a marked increase in the total time to complete the task at 4 months in the CAG140 KI mice (45) and this has now also been observed in several lines of different repeat length R6/2 mice (Cummings et al. submitted).

6. Conclusion In conclusion, both simple but labor intensive (climbing cage, pole test) and almost or fully automated tests (open field, running wheels) can be used to detect early motor deficits in mouse models of HD. Using these tests does not require extensive prior training for the investigator but they require careful planning in term of phase of the day, age of the animals, and gender matching. We have preliminary data indicating that drug effects can be detected using these tests but a systematic evaluation of different lines of mice using the same tests in the same conditions is lacking. In the absence of this information, the use of a battery of tests is recommended with a selection of tests that have demonstrated the greatest sensitivity in detecting motor anomalies in HD models. References 1. The Huntington’s Disease Collaborative Research Group. (1993) A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington’s disease chromosomes. Cell 72: 971–983. 2. Paulsen JS, Langbehn DR, Stout JC et  al (2008) Detection of Huntington’s disease decades before diagnosis: the Predict-HD study. J Neurol Neurosurg Psychiatry 79: 874–880. 3. Vonsattel JP, DiFiglia M (1998) Huntington disease. J Neuropathol Exp Neurol 57: 369–384. 4. Harper PS (1996) Huntington’s Disease. London, Saunders. 5. Lemiere J, Decruyenaere M, Evers-Kiebooms G et al (2004) Cognitive changes in patients with Huntington’s disease (HD) and asymptomatic carriers of the HD mutation--a longitudinal follow-up study. J Neurol 251: 935–942.

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25. Watson GS, Leverenz JB (2010) Profile of cognitive impairment in Parkinson’s disease. Brain Pathol 20: 640–645. 26. Bassetti CL (2010) Nonmotor Disturbances in Parkinson’s Disease. Neurodegener Dis. [Epub ahead of print]. 27. Petit D, Gagnon JF, Fantini ML et al (2004) Sleep and quantitative EEG in neurodegenerative disorders. J Psychosom Res 56: 487–496. 28. Aylward EH (2007) Change in MRI striatal volumes as a biomarker in preclinical Huntington’s disease. Brain Res Bull 72: 152–158. 29. Levine MS, Cepeda C, Hickey MA et  al (2004) Genetic mouse models of Huntington’s and Parkinson’s diseases: illuminating but imperfect. Trends Neurosci 27: 691–697. 30. Schwarcz R, Guidetti P, Sathyasaikumar KV et al (2009) Of mice, rats and men: Revisiting the quinolinic acid hypothesis of Huntington’s disease. Prog Neurobiol. 90: 230–245 31. Palfi S, Brouillet E, Jarraya B et  al (2007) Expression of mutated huntingtin fragment in the putamen is sufficient to produce abnormal movement in non-human primates. Mol Ther 15: 1444–1451. 32. Yang SH, Cheng PH, Banta H et  al (2008) Towards a transgenic model of Huntington’s disease in a non-human primate. Nature 453: 921–924. 33. Franich NR, Fitzsimons HL, Fong DM et al (2008) AAV vector-mediated RNAi of mutant huntingtin expression is neuroprotective in a novel genetic rat model of Huntington’s disease. Mol Ther 16: 947–956. 34. Hickey MA, Chesselet MF (2003) Apoptosis in Huntington’s disease. Prog Neuropsy­ chopharmacol Biol Psychiatry 27: 255–265. 35. Reiner A, Dragatsis I, Zeitlin S et  al (2003) Wild-type huntingtin plays a role in brain development and neuronal survival. Mol Neurobiol 28: 259–276. 36. Phan J, Hickey MA, Zhang P et  al (2009) Adipose tissue dysfunction tracks disease progression in two Huntington’s disease mouse models. Hum Mol Genet 18: 1006–1016. 37. Sassone J, Colciago C, Cislaghi G et al (2009) Huntington’s disease: the current state of research with peripheral tissues. Exp Neurol 219: 385–397. 38. Kantor O, Temel Y, Holzmann C et al (2006) Selective striatal neuron loss and alterations in behavior correlate with impaired striatal function in Huntington’s disease transgenic rats. Neurobiol Dis 22: 538–547. 39. Nguyen HP, Kobbe P, Rahne H et al (2006) Behavioral abnormalities precede neuropathological markers in rats transgenic for

1  Behavioral Assessment of Genetic Mouse Models of Huntington’s Disease Huntington’s disease. Hum Mol Genet 15: 3177–3194. 40. Jacobsen JC, Bawden CS, Rudiger SR et  al (2010) An ovine transgenic Huntington’s ­disease model. Hum Mol Genet 19: 1873–1882. 41. Yang D, Wang CE, Zhao B et  al (2010) Expression of Huntington’s disease protein results in apoptotic neurons in the brains of cloned transgenic pigs. Hum Mol Genet 19: 3983–3994. 42. Morton AJ, Avanzo L (2011) Executive decision-making in the domestic sheep. PLoS One 6: e15752. 43. Mangiarini L, Sathasivam K, Seller M et  al (1996) Exon 1 of the HD gene with an expanded CAG repeat is sufficient to cause a progressive neurological phenotype in transgenic mice. Cell 87: 493–506. 44. Hickey MA, Gallant K, Gross GG et al (2005) Early behavioral deficits in R6/2 mice suitable for use in preclinical drug testing. Neurobiol Dis 20: 1–11. 45. Hickey MA, Kosmalska A, Enayati J et  al (2008) Extensive early motor and non-motor behavioral deficits are followed by striatal neuronal loss in knock-in Huntington’s disease mice. Neuroscience 157: 280–295. 46. Woodman B, Butler R, Landles C et al (2007) The Hdh(Q150/Q150) knock-in mouse model of HD and the R6/2 exon 1 model develop comparable and widespread molecular phenotypes. Brain Res Bull 72: 83–97. 47. Dragatsis I, Goldowitz D, Del Mar N et  al (2009) CAG repeat lengths > or =335 attenuate the phenotype in the R6/2 Huntington’s disease transgenic mouse. Neurobiol Dis 33: 315–330. 48. Morton AJ, Glynn D, Leavens W et al (2009) Paradoxical delay in the onset of disease caused by super-long CAG repeat expansions in R6/2 mice. Neurobiol Dis 33: 331–341. 49. Menalled L, El-Khodor BF, Patry M et  al (2009) Systematic behavioral evaluation of Huntington’s disease transgenic and knock-in mouse models. Neurobiol Dis 35: 319–336. 50. Stack EC, Kubilus JK, Smith K et  al (2005) Chronology of behavioral symptoms and neuropathological sequela in R6/2 Huntington’s disease transgenic mice. J Comp Neurol 490: 354–370. 51. Carter RJ, Hunt MJ, Morton AJ (2000) Environmental stimulation increases survival in mice transgenic for exon 1 of the Huntington’s disease gene. Mov Disord 15: 925–937. 52. van der Burg JM, Bacos K, Wood NI et  al (2008) Increased metabolism in the R6/2

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mouse model of Huntington’s disease. Neurobiol Dis 29: 41–51. 53. Weydt P, Pineda VV, Torrence AE et al (2006) Thermoregulatory and metabolic defects in Huntington’s disease transgenic mice implicate PGC-1alpha in Huntington’s disease neurodegeneration. Cell Metab 4: 349–362. 54. Bolivar VJ, Manley K, Messer A (2003) Exploratory activity and fear conditioning abnormalities develop early in R6/2 Huntington’s disease transgenic mice. Behav Neurosci 117: 1233–1242. 55. Lione LA, Carter RJ, Hunt MJ et  al (1999) Selective discrimination learning impairments in mice expressing the human Huntington’s disease mutation. J Neurosci 19: 10428–10437. 56. Murphy KPSJ, Carter RJ, Lione LA et  al (2000) Abnormal synaptic plasticity and impaired spatial cognition in mice transgenic for exon 1 of the Huntington’s disease mutation. J. Neurosci. 20: 5115–5123. 57. Ciamei A, Morton AJ (2009) Progressive imbalance in the interaction between spatial and procedural memory systems in the R6/2 mouse model of Huntington’s disease. Neurobiol Learn Mem 92: 417–428. 58. File SE, Mahal A, Mangiarini L et al (1998) Striking changes in anxiety in Huntington’s disease transgenic mice. Brain Research 805: 234–240. 59. Beal MF, Ferrante RJ (2004) Experimental therapeutics in transgenic mouse models of Huntington’s disease. Nat Rev Neurosci 5: 373–384. 60. Hockly E, Woodman B, Mahal A et al (2003) Standardization and statistical approaches to therapeutic trials in the R6/2 mouse. Brain Res Bull 61: 469–479. 61. Mihm MJ, Amann DM, Schanbacher BL et al (2007) Cardiac dysfunction in the R6/2 mouse model of Huntington’s disease. Neurobiol Dis 25: 297–308. 62. Ribchester RR, Thomson D, Wood NI et  al (2004) Progressive abnormalities in skeletal muscle and neuromuscular junctions of transgenic mice expressing the Huntington’s disease mutation. Eur J Neurosci 20: 3092–3114. 63. Brooks SP, Janghra N, Workman VL et  al (2011) Longitudinal analysis of the behavioural phenotype in R6/1 (C57BL/6J) Huntington’s disease transgenic mice. Brain Res Bull. 64. Naver B, Stub C, Moller M et  al (2003) Molecular and behavioral analysis of the R6/1 Huntington’s disease transgenic mouse. Neuroscience 122: 1049–1057.

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65. Pang TY, Du X, Zajac MS et al (2009) Altered serotonin receptor expression is associated with depression-related behavior in the R6/1 transgenic mouse model of Huntington’s disease. Hum Mol Genet 18: 753–766. 66. van Dellen A, Cordery PM, Spires TL et  al (2008) Wheel running from a juvenile age delays onset of specific motor deficits but does not alter protein aggregate density in a mouse model of Huntington’s disease. BMC Neurosci 9: 34. 67. Lloret A, Dragileva E, Teed A et  al (2006) Genetic background modifies nuclear mutant huntingtin accumulation and HD CAG repeat instability in Huntington’s disease knock-in mice. Hum Mol Genet 15: 2015–2024. 68. Hodgson JG, Agopyan N, Gutekunst CA et al (1999) A YAC mouse model for Huntington’s disease with full-length mutant huntingtin, cytoplasmic toxicity, and selective striatal neurodegeneration. Neuron 23: 181–192. 69. Slow EJ, van Raamsdonk J, Rogers D et  al (2003) Selective striatal neuronal loss in a YAC128 mouse model of Huntington disease. Hum Mol Genet 12: 1555–1567. 70. Gray M, Shirasaki DI, Cepeda C et al (2008) Full-length human mutant huntingtin with a stable polyglutamine repeat can elicit progressive and selective neuropathogenesis in BACHD mice. J Neurosci 28: 6182–6195. 71. Van Raamsdonk JM, Pearson J, Slow EJ et al (2005) Cognitive dysfunction precedes neuropathology and motor abnormalities in the YAC128 mouse model of Huntington’s disease. J Neurosci 25: 4169–4180. 72. Van Raamsdonk JM, Murphy Z, Slow EJ et al (2005) Selective degeneration and nuclear localization of mutant huntingtin in the YAC128 mouse model of Huntington disease. Hum Mol Genet 14: 3823–3835. 73. Stoy N, McKay E (2000) Weight loss in Huntington’s disease. Ann Neurol 48: 130–131. 74. Van Raamsdonk JM, Gibson WT, Pearson J et al (2006) Body weight is modulated by levels of full-length huntingtin. Hum Mol Genet 15: 1513–1523. 75. Menalled LB (2005) Knock-in mouse models of Huntington’s disease. NeuroRx 2: 465–470. 76. Menalled LB, Sison JD, Dragatsis I et  al (2003) Time course of early motor and neuropathological anomalies in a knock-in mouse model of Huntington’s disease with 140 CAG repeats. J Comp Neurol 465: 11–26. 77. Heng MY, Tallaksen-Greene SJ, Detloff PJ et  al (2007) Longitudinal evaluation of the Hdh(CAG)150 knock-in murine model of

Huntington’s disease. J Neurosci 27: 8989–8998. 78. Lin C-H, Tallaksen-Greene S, Chien W-M et al (2001) Neurological abnormalities in a knockin mouse model of Huntington’s disease. Human Molecular Genetics 10: 137–144. 79. Wheeler VC, Gutekunst CA, Vrbanac V et al (2002) Early phenotypes that presage lateonset neurodegenerative disease allow testing of modifiers in Hdh CAG knock-in mice. Hum Mol Genet 11: 633–640. 80. Hickey MA, Franich NR, Medvedeva V et al (in press) Mouse models of mental illness and neurological disease: Huntington’s disease. In: The Mouse Nervous System (Watson C, Paxinos G, Puelles L, eds). 81. Zhu C, Hickey MA, Medvedeva V et al (2009) Comparison of fully backcrossed CAG 140 and CAG 150 Knock-in mouse models of Huntington’s disease: Neuropathological, behavioral, and transcriptional analysis. In: Society for Neuroscience. Chicago: 240.16. 82. McKhann GM, 2nd, Wenzel HJ, Robbins CA et al (2003) Mouse strain differences in kainic acid sensitivity, seizure behavior, mortality, and hippocampal pathology. Neuroscience 122: 551–561. 83. Wahlsten D, Bachmanov A, Finn DA et  al (2006) Stability of inbred mouse strain differences in behavior and brain size between laboratories and across decades. Proc Natl Acad Sci U S A 103: 16364–16369. 84. Voikar V, Koks S, Vasar E et al (2001) Strain and gender differences in the behavior of mouse lines commonly used in transgenic studies. Physiol Behav 72: 271–281. 85. Cryan JF, Mombereau C, Vassout A (2005) The tail suspension test as a model for assessing antidepressant activity: review of pharmacological and genetic studies in mice. Neurosci Biobehav Rev 29: 571–625. 86. Kent S, Hurd M, Satinoff E (1991) Interactions between body temperature and wheel running over the estrous cycle in rats. Physiol Behav 49: 1079–1084. 87. Kopp C, Ressel V, Wigger E et  al (2006) Influence of estrus cycle and ageing on activity patterns in two inbred mouse strains. Behav Brain Res 167: 165–174. 88. Viberg H, Fredriksson A, Eriksson P (2004) Investigations of strain and/or gender differences in developmental neurotoxic effects of polybrominated diphenyl ethers in mice. Toxicol Sci 81: 344–353. 89. Benn CL, Luthi-Carter R, Kuhn A et al (2010) Environmental enrichment reduces neuronal intranuclear inclusion load but has no effect

1  Behavioral Assessment of Genetic Mouse Models of Huntington’s Disease on messenger RNA expression in a mouse model of Huntington disease. J Neuropathol Exp Neurol 69: 817–827. 90. Hockly E, Cordery PM, Woodman B et  al (2002) Environmental enrichment slows disease progression in R6/2 Huntington’s disease mice. Ann Neurol 51: 235–242. 91. Kurosaki R, Akasaka M, Michimata M et  al (2003) Effects of Ca2+ antagonists on motor activity and the dopaminergic system in aged mice. Neurobiol Aging 24: 315–319. 92. Luesse HG, Schiefer J, Spruenken A et  al (2001) Evaluation of R6/2 HD transgenic mice for therapeutic studies in Huntington’s disease: behavioral testing and impact of diabetes mellitus. Behav Brain Res 126: 185–195. 93. Menalled LB, Sison JD, Wu Y et  al (2002) Early motor dysfunction and striosomal distribution of huntingtin microaggregates in Huntington’s disease knock-in mice. J Neurosci 22: 8266–8276. 94. Jahkel M, Rilke O, Koch R et al (2000) Open field locomotion and neurotransmission in mice evaluated by principal component factor analysis-effects of housing condition, individual activity disposition and psychotropic drugs. Prog Neuropsychopharmacol Biol Psychiatry 24: 61–84. 95. Ramos A, Berton O, Mormede P et al (1997) A multiple-test study of anxiety-related behaviours in six inbred rat strains. Behav Brain Res 85: 57–69. 96. Monville C, Torres EM, Dunnett SB (2006) Comparison of incremental and accelerating

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protocols of the rotarod test for the assessment of motor deficits in the 6-OHDA model. J Neurosci Methods 158: 219–223. 97. Kennedy L, Evans E, Chen CM et al (2003) Dramatic tissue-specific mutation length increases are an early molecular event in Huntington disease pathogenesis. Hum Mol Genet 12: 3359–3367. 98. Menalled LB, Patry M, Ragland N et al (2010) Comprehensive behavioral testing in the R6/2 mouse model of Huntington’s disease shows no benefit from CoQ10 or minocycline. PLoS One 5: e9793. 99. Southwell AL, Ko J, Patterson PH (2009) Intrabody gene therapy ameliorates motor, cognitive, and neuropathological symptoms in multiple mouse models of Huntington’s disease. J Neurosci 29: 13589–13602. 100. Cepeda C, Cummings DM, Hickey MA et al (2010) Rescuing the Corticostriatal Synaptic Disconnection in the R6/2 Mouse Model of Huntington’s Disease: Exercise, Adenosine Receptors and Ampakines. PLoS Curr 2: RRN1182. 101. Fernagut PO, Hutson CB, Fleming SM et al (2007) Behavioral and histopathological consequences of paraquat intoxication in mice: effects of alpha-synuclein over-expression. Synapse 61: 991–1001. 102. Fleming SM, Salcedo J, Fernagut PO et  al (2004) Early and progressive sensorimotor anomalies in mice overexpressing wild-type human alpha-synuclein. J Neurosci 24: 9434–9440.

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Chapter 2 Excitotoxic Lesions of the Rodent Striatum Máté D. Döbrössy, Fabian Büchele, and Guido Nikkhah Abstract This chapter reviews the most common methods and protocols used to induce and assess excitotoxic lesions of the rodent striatum. Excitotoxic agents act through glutamate receptors to initiate intracellular cascades that can result in neuronal degeneration via both apoptotic and necrotic mechanisms. Whether it is glutamate, an endogenous excitatory amino acid, or an analogue, the neurotoxic potential of the excitotoxin is dose dependent. Excitotoxic lesions of the striatum, whose post-synaptic neurones are rich in glutamate receptors, make it possible to investigate structural and functional aspects and to establish animal models of certain basal ganglia diseases that can serve as a platform to investigate numerous therapeutic avenues. Although the rodent striatum is a relatively large target, the delivery of the excitotoxin requires precise spatial and temporal delivery that can be achieved only with stereotactic surgery. Key words: Animal models, Rodent, Basal ganglia, Excitotoxic lesion, Stereotactic surgery

1. Introduction Animal models of human brain diseases are essential tools to learn more about a specific or a family of diseases with shared pathology. Depending on the construct, the predictive and the face validity, the model can promote the understanding of the aetiology, the underlying neurobiological and behavioural consequences of the disorder, and offer a platform to develop therapeutic avenues that could be translated from the bench to the clinic. Excitotoxic lesions of the rodent CNS have become such a tool of choice for many investigators over the years whose research focuses on acute or chronic neuronal loss following accidents or progressive neurodegeneration. In particular, excitotoxic lesions

Emma L. Lane and Stephen B. Dunnett (eds.), Animal Models of Movement Disorders: Volume II, Neuromethods, vol. 62, DOI 10.1007/978-1-61779-301-1_2, © Springer Science+Business Media, LLC 2011

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of the striatum are used within the context of modelling basal ganglia diseases such as Multiple System Atrophy (MSA) and Huntington’s disease (HD), both predicaments that exhibit significant – but not exclusive – striatal neuropathology. Importantly, the development and availability of a wide range of transgenic animal models for HD and other diseases have not made excitotoxic lesion models superfluous: they have simply increased the model options for researchers to work with and ask different scientific questions. As with all scientific protocols, lesioning methods have evolved over a considerable time period in the past. Studies aiming at destroying the region of interest to induce a dysfunctional status are typically followed by the analysis of the behavioural and biochemical consequences of the lesion. These types of experiments using electrolytic lesions have been performed since the early part of the twentieth century (1). In this case, the lesion is induced by passing current through steel electrodes with the tip of one electrode lowered into the selected site while using an anal plug or an ear clip as the second electrode. Damage occurs as a consequence of the diffusion of metallic ions from the electrode tip into the tissue, and via the mechanical impact of bubble formation on the electrode tip. The size of the lesion is determined by the intensity and duration of the current, as well as by the length of the uninsulated electrode tip. From the 1950s, radio frequency lesioning became an alternative to electrolytic lesioning: this method causes tissue damage through generating heat in the tissue by passing high-frequency alternating current through the electrode tip. Neither of these two lesioning techniques is selective, and they can cause haemorrhages, and other unwanted side effects. Damage is produced in the tissue of choice, yes, but damage is done in all cell types and axons of passage, which compromises the validity of the model. The advent of excitotoxins in the 1970s allowed for the induction of more selective lesions through a physiologically relevant mechanism: excitotoxicity (2–5). Excitotoxins are glutamate analogues and constitute a class of neurotoxins. Their selective property regarding neurones arises because they can bind to different glutamate receptor subtypes, such as the N-methyl-d-aspartic acid (NMDA), the a-amino-3-hydroxy-5-methyl-4-isoxazole-proprionate (AMPA), or the kainate receptor, found principally on post-synaptic membranes of specific cells, thereby sparing axons of passage not carrying the respective receptors. Furthermore, by controlling various striatal lesion parameters such as the volume, dose, infusion rate, lesioning location, number of lesion sites and the excitotoxin used, the investigator, to a certain extent, can modulate the selectivity of the toxin and obtain a variety of cellular and behavioural outcomes.

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2. Excitotoxic Cell Death vs. Metabolic Toxins

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Two principal types of excitotoxic cell deaths have been described: indirect and direct. Indirect, or secondary, excitotoxicity describes a process which is initially dependent on intracellular defects and not on externally imposed factors. The cell is first made vulnerable, and then succumbs to excitotoxic neuronal death mediated by otherwise sub-toxic glutamate. The mechanism implicated in secondary excitotoxicity is referred to as metabolic or oxidative stress and is dependent on a progressive impairment of neuronal mitochondria-based energy metabolism (6). Examples of agents that have been used to model basal ganglia diseases by promoting secondary excitotoxicity are the metabolic toxins malonate and 3-nitropropionic acid (7–10), and these agents have been reviewed elsewhere (11, 12). Direct, or primary, excitotoxicity occurs when there is excessive or prolonged glutamate receptor activation and will be further reviewed in this chapter. In experimental animals, excitotoxic neuronal cell death is induced by the central injection of functional and structural analogues of glutamate such as kainic, ibotenic or quinolinic acids (13–19). The underlying mechanisms of glutamate receptor mediated toxicity are complex and context dependent, but consensus is that excessive calcium influx (20, 21) is a major factor, and that cell death can occur both via necrotic or apoptotic pathways (22). However, glutamate or glutamate analogue-­mediated excitotoxicity is not generating the pathology by itself, such as mutations or structural deficits in the ion channels might do; excitotoxicity is considered as a physiological response to an insult. The excitotoxin of choice at any one time depended on availability and the rationale for the use. During the early phase of the use of these agents, comparable behavioural deficits have been reported after kainic, ibotenic and quinolinic acids lesions of the striatum (13, 14, 16, 17, 19, 23, 24). However, kainic acid has been used more in the context of animal models of epilepsy due to its tendency to induce seizures: it is the most potent of the commonly used exogenous excitotoxins with a neurotoxic threshold of nearly two magnitudes lower than that of the other receptor-­ specific agonists (25–27). Although ibotenic acid is still in use, quinolinic acid has superseded all other agents and has proved to be the most relevant excitotoxin for the rodent striatum. Quinolinic acid is an endogenous tryptophan metabolite found in both the rat and human CNS (28). The function of the molecule is mediated by the NMDA receptor, onto which the quinolinic acid endogenous antagonist, kynurenate, can also bind (29). Similar to ibotenic acid, quinolinic acid is reported to have axonsparing neurotoxic properties following intrastriatal infusion.

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Dendritic swelling, tissue shrinkage, ventricle enlargements and absence of distant cell death have been observed. Animal models using quinolinic acid lesions have been shown to replicate the features of Huntington’s disease pathology well, including the differential vulnerability of striatal neurones. There are reports of depletion of GABA and substance P levels with quinolinic acid lesions, as well as relative sparing of somatostatin and neuropeptide Y, two neurotransmitters that are localised to aspiny neurons and co-localised with NADPH-diaphorase (30, 31). In experiments where histological examination was carried out 6–12 months after the lesion, somatostatin and neuropeptide Y immunoreactivity had almost doubled, and there were increases in concentrations of both 5-HT and its metabolite 5-hydroxyindoleacetic acid. Support for the use of chronic quinolinic acid lesions is further strengthened by observations of similarities between lesion cell death and the pattern of cell death seen in Huntington’s disease (19).

3. Methods and Protocols Methods and protocols concerning the preparation and application of excitotoxins can vary from lab to lab, often depending on the rationale behind the use. Pilot runs are highly recommended when the investigator uses the toxin for the first time, or even when the toxin comes from a newly prepared batch, as the potency of the preparations might differ. Furthermore, when the investigator deviates from previously used experimental parameters such as coordinates, dose, infusion speed, volume, or age of animal (e.g., switching from adult to young), testing protocol modifications with a half a dozen animals and approximately 2 weeks survival time is important. 3.1. Toxin Preparation and Storage

Different and equally valid protocols exist regarding the preparation of quinolinic acid. Preparing the toxin at the appropriate pH is essential, as this ensures the complete resolution of the toxin which is generally purchased in powder form. Measuring the pH is done using litmus paper, and if the agent is prepared in a too small volume, the paper itself could absorb a significant quantity of solution and change the concentration. This problem can be circumvented by dipping a needle tip into the toxin and spotting the needle onto the litmus paper, rather than trying to touch the paper to the toxin solution directly. The procedure to prepare a stock solution of 0.12 M quinolinic acid (QA; molecular weight = 167.12) is as follows. The aim is to prepare 6.25  ml of stock solution using 125  mg of research grade quinolinic acid.

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1. Dissolve 125 mg of QA in 750 ml PBS (pH = 7.4), add 50 ml of 10 M sodium hydroxide. 2. Sonicate the above solution for 15 min. 3. Add 3,200 ml PBS. The total volume at this stage will be 4 ml, and this is will permit the use of a pH meter. 4. Add 50 ml of 10 M sodium hydroxide to bring the solution to pH = 7.4; if pH needs to be adjusted use sodium hydroxide or concentrated hydrochloric acid. 5. Add 2,200 ml of PBS to obtain the required concentration of 0.12 M QA. 6. Check pH again, and if needed adjust to pH = 7.4. 7. Aliquot 50  ml into Eppendorfs, label and store in freezer at −20°C. If the required concentration is 0.09 M QA, then add 16.7 ml of PBS to a 50 ml aliquot of 0.12 M QA. The stock can be stored at −20°C safely for 12 months; beyond this time point a new batch should be made. The amount of QA injected is typically expressed either as “X” number of deposits of “Y” ml each of “Z” M (molarity): for example, four deposits of 0.2 ml of 0.12 M QA; or as “X” nmol (molality): for example, 96 nmol QA. Each deposit is infused with the micropump over 90 s, with 1 min between different vertical deposits, and a 3-min wait prior to removal of the cannula from the brain to eliminate/reduce lesion damage due to toxin re-flux. 3.2. Delivery Method and Lesion Set-Up

Fresh before use, a single 50 ml aliquot needs to be thawed out. During the surgery, the QA is kept on ice or in a fridge. As this volume is sufficient to lesion approximately 20–25 animals, it generally covers the day’s requirements even if two investigators are lesioning on two independent stations. At the end of day, the remaining QA needs to be disposed of, and each surgical session should start with a new aliquot. Excitotoxic lesions of the striatum require precision delivery of the toxin at the planned co-ordinates, of the required volume and at the required pace. These conditions can at best be fulfilled if using (a) an adapted stereotactic animal frame; (b) a thin steel cannula (recommended size: 30 gauge) that penetrates the brain with minimal co-lateral tissue damage, and which is fixed to the stereotactic arm and (c) a micropump capable of delivering sub-microliter volumes of toxin over minutes. The set-up is completed by polythene tubing with an inner diameter of approximately 0.28 mm, that connects up the steel cannula with a Hamilton syringe which itself is placed in the micropump. The Hamilton syringe-tubingcannula system needs to be filled with saline to make it completely airtight. Prior to lesioning, a tiny bit of air is taken up into the system by moving outwards the syringe’s plunger and this is followed by

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filling up the cannula and the tubing with QA. The small air bubble at the interface of the saline and the QA can be used as a visual cue confirming the movement of the solution. This is important particularly when the infusion starts, as it is a way to follow the steady expulsion of the agent, and allows for intervention if the cannula is blocked. 3.3. Post-operative Care

Performing excitotoxic lesions of the rodent striatum is a major intervention requiring anaesthesia and surgery, and as such, appropriate post-operative care must be given. Ensuring the welfare of the animal is required by law; equally importantly, it is also in the interest of the investigator who invests a lot of time and effort in training and testing the animals. There are two major concerns following surgery: short-term pain management, and short-term incapacity to eat. If gaseous anaesthesia is used, the animals will awaken relatively fast after the surgery, while following injectable anaesthesia the animals will remain unconscious for hours. This also means that the occasional and short-lasting motor side effects of the excitotoxin lesioning, e.g., barrel rolls or seizures, will become more apparent under gaseous anaesthesia and this needs to be managed. Below are guidelines for post-operative treatment of rats or mice following unilateral excitotoxic lesioning of the striatum. Always inform the animal care personnel in advance telling them which animals and when they will be operated on. 1. Starting on the day of surgery, and for the next 3 days, add 1 × 500  mg (250  mg for mice) paracetamol-soluble tablet in each water bottle. 2. At the end of surgery, while the animal is still in the frame inject 0.15  ml (0.03  ml for mice) diazepam intramuscularly into a hind leg muscle, and 5 ml (0.5 ml for mice) of saline/ glucose bolus subcutaneously in the scruff. 3. Following the operation, place the animal in a heated cage lined with paper, and ensure that it is not too hot and that animals already moving around will not disturb the fresh stitches on the animal’s head. Conscious animals need to be returned to their home cage. 4. Record relevant information on the cage cards, and in other documents as required by regulations. 5. Check the health status of the animals daily for the following 3 days. Animals not grooming or losing weight (all animals should be weighed at least once a week under normal circumstances) need to be put into a separate cage, fed with moist lab chow, and monitored regularly. Animals receiving bilateral striatal lesions will be more impaired following operation, and might need additional glucose saline injections over the days following surgery.

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3.4. Histological and Volumetric Assessment of the Excitotoxic Lesion

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Functional and behavioural assessment following excitotoxic lesions is an essential aspect in many studies using excitotoxic lesions of the striatum and this matter is dealt with by several chapters in this book. The survival time of the animal following the lesion will depend on the rationale for the study. If the investigator is particularly interested in lesion dynamics and factors affecting lesion development, the survival time will be measured in days or weeks; if the aim is to study functional deficits and repair strategies following striatal damage, the survival time will be measured in weeks or months. Assessing the selectivity of the neural toxin and measuring the extent of the excitotoxic damage, for example, requires classical immunohistochemical and image analysis methods which are described in detail elsewhere (32, 33). Briefly, the animals are terminally anaesthetized by intraperitoneal injection of an overdose of ketamine/rompun and transcardially perfused with 0.1 M PBS followed 4% paraformaldehyde in 0.1 M PBS. The removed brains are post-fixed in 4% paraformaldehyde for 1.5 h, then immersed in 30% sucrose until they sink. Tissue is serially sectioned commonly at 50 mm on a cryotome and collected as 4–6 series for standard immunohistochemistry or immunoflourescence, in case double labelling is needed. The most common markers used to assess the excitotoxic lesion are listed in Table 1, and typical stained coronal sections are depicted in Fig. 1. The stained, free-floating sections are mounted on slides, dehydrated, cover slipped and subjected to image analysis. Typically, sections stained with a marker that clearly delineates the extent of the striatal cell loss, such as DARPP-32 or NeuN, are used to quantify

Table 1 List of typical markers/staining methods used to visualize and quantify the lesion damage Target protein or molecule

Target cells

Reason

NeuN

Mature neurones

To visualise loss of striatal neurones

Cresyl violet or Nissl stain

All cells

To visualise the cell loss

DARPP-32

Striatal neurones

To visualise loss of medium spiny striatal projection neurones

Calbindin

Striatal neurones

To visualise loss of medium spiny striatal projection neurones

TH

Dopaminergic neurones

To visualise loss of nigrostriatal dopaminergic input

ChAT or AChE

Cholinergic interneurones

To visualise loss of the giant aspiny striatal cholinergic interneurons in the striatum

GFAP

Astrocytes

To identify astrocytic response to the lesion

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Fig. 1. Photomicrographs illustrating the effects of unilateral dorsal striatal injections of quinolinic acid. Sections from the control and the lesion groups are typically stained with a selection of markers specific for neurones (NeuN; a–b), dopaminergic afferents (TH, tyrosine-hydroxylase; c–d), medium spiny neurones (DARPP-32; e–f), general cell marker (Cresyl Violet; g–h), or cholinergic interneurons (Acetylcholinesterase, AChE, i–j). The control sections show regular staining and no anatomical deformation. However, lesioned sections have enlarged ventricles, collapsed axons of passage, shrunk striatal tissue, a necrotic core and exhibit a general reduction of staining in the striatum. Interestingly, the injection of 4 × 0.2 ml of 0.12 M QA produced apparently less damage then the injection of 2 × 0.5 ml of 0.09 M QA. This suggests that the excitotoxic lesion achieved is a function not only of the concentration of the QA used but also of the volume and the distribution of the toxin. For more details see (34, 35). Scale bar = 1 mm.

the striatal areas that survived or were affected by the lesion. Volumetric analysis is carried out by measuring the lost and remaining striatal tissue on the ipsilateral side, the unlesioned striatal tissue on the contralateral side, and the lateral ventricles; these values are quantified on each consecutive section throughout

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the anterior-posterior axis of the dorsal striatum. The final volume measurements for the various parameters are calculated by taking into account the sum of the areas and correcting for section thickness and sample frequency: Volume (mm3) = Sum of areas (mm2) × Section thickness (mm) × Sample frequency). 3.5. Factors Influencing Lesion Outcome

Excitotoxins act through glutamatergic receptors present on striatal projection neurones, and as such, factors that interact directly or indirectly with this transmission system can mitigate or accentuate the excitotoxic mechanism. Furthermore, co-ordinate selection, dose, and volume injected are important parameters of the experimental design that need to be considered within the overall scientific rationale. A selection of these issues is discussed below.

3.5.1. Anaesthesia

The choice of anaesthesia used during excitotoxic surgery of the striatum is an important factor to consider as it can influence the lesion outcome. For example, ketamine, a non-competitive NMDA receptor antagonist and widely used anaesthetic agent, shares its target with QA, and hence can mitigate the excitotoxic damage by exerting a neuroprotective effect. It is known for over two decades that ketamine can inhibit and modulate the neurotoxic effect of QA lesions in a dosage-dependent manner (36, 37). Therefore, it is remarkable that it is still used today as an anaesthetic for QA lesion experiments where neurotoxicity is the primary objective, for example, in rodent models of HD (see (38) for review). Early work that established the ketamine/QA combination in rodent models of HD did not recognise this as an issue when high dose of QA (e.g., 240  nmol) were being used (39). Due to the dose-dependent relationship between ketamine and QA, the neuroprotective interference of the two ligands can be overcome by either increasing the dose of QA or reducing the dose of ketamine. Controversially, the previously described profile of QA relating to its ability to achieve selective neuronal death akin to HD pathology, such as killing GABAergic medium spiny neurones while ­sparing somatostatin, neuropeptide Y, and NADPH-diaphorase immunoreactive striatal interneurons (30, 31) could be related to the lesion modifying aspect of ketamine and not to the inherent properties of the excitotoxin itself. Furthermore, ketamine is commonly used along with xylazine (Rompun), an alpha-2-adrenoreceptor agonist that works as a sedative and muscle relaxant (40), minimising side effects like tremor and muscle rigidity, while increasing the duration of anaesthesia by reducing its renal elimination (41, 42). Doing so, it may increase the bioavailability of ketamine thereby indirectly enhancing its neuroprotective effect, as has been suggested in a recent study where it was directly compared with isoflurane (38) (see Fig. 2). Isoflurane is a gaseous anaesthetic that provides for a more predictable alternative and has become the anaesthesia of choice in

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Fig. 2. Photomicrographs showing typical coronal sections of DARPP-32 stained QA lesioned rat striatum when using ketamine (a) or isoflurane (b) as anaesthetic during the surgery. The dotted lines delineate the lesion area. Analysis confirmed a significant role of the anaesthesia used during the surgery on the eventual striatal damage underlining the neuroprotective effect of ketamine in the context of QA excitotoxic lesions (c). For more details, see (38). CTX cortex, CC corpus callosum, V ventricle, L lesion, STR striatum, IC internal capsule. Scale bar = 1 mm. **, p < 0.001.

many labs working with rodent excitotoxic lesion paradigms (43,  44). Although isoflurane has been linked with neuropro­ tective effects as well, the only study – known to the authors of this chapter – that directly investigated this issue found no interaction between isoflurane and NMDA receptors (45). Isoflurane provides a more rapid induction, greater control and rapid recovery from the anaesthetised state, which means less trauma and danger for the experimental animals. Ketamine, on the other hand, is cheaper, easily applied and does not require additional equipment. The control

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over the depth of the anaesthesia, however, is far less, the risk of ­overdosing is higher, and of course, there is the issue of inappropriate use in the context of models of excitotoxic neuronal death. 3.5.2. Lesion Placement: Motor and/or Cognitive Effects

Most common excitotoxic lesions in the striatum are unilateral and this consistently produces impairments in contralateral paw use, or deficits in responding in the contralateral space. Bilateral lesions can be too debilitating, especially if behavioural testing of the animals is planned. The somatotopic organisation of the cortex, and the topographically organised afferent and efferent connections of the striatum mirror the diverse nature of deficits observed following striatal lesions in the rat (46). For example, regional striatal lesions have demonstrated the differential effects of the lateral and medial striatal contribution to motor functions (47). Dorsolateral striatal lesions selectively produce skilled forelimb deficits, while dorsoventral striatal lesions impair both skilled forelimb use and tongue reaching. Medial striatal lesions, receiving inputs mainly from the auditory and visual cortex, do not affect either measurement (48–51). In a choice reaction-time task, lateral and medial striatal lesions induce ipsilateral response bias and increase latency in response initiation, respectively (52). Non-motor, or cognitive behaviours can also be affected by appropriate targeting of the striatal excitotoxic lesion (53, 54). The striatum receives topographically organised projections from the whole of the neocortex, in particular the prefrontal cortex, providing a major system for the selection and initiation of cortically derived action plans. For example, habit, or procedural learning is characterised by gradual and stable acquisition of an association between a stimulus and a response, and is a form of memory believed to be mediated by the striatum where the sensory information is associated with a learned motor output (55– 58). Lesions of the anteromedial area of the striatum, a region that receives its afferents predominantly from associative, auditory and visual cortices, induces deficits in delayed alternation and spatial navigation tasks, for example, but leaves visual discrimination performance intact; however, lateral, particularly ventrolateral striatal lesions destroying cortical afferents from sensorimotor areas, disrupt performance on complex visual stimulus–response habit tasks (24, 59).

3.5.3. Striatal Afferents

The excitotoxic lesion of the rodent striatum relies on the excessive stimulation of the glutamate receptors found on the distal portions of the dendritic spines of the medium spiny striatal neurones. Studies have shown that the extent of the QA-induced lesion can be influenced not only by the availability of the glutamate receptors – e.g., in the presence of a ketamine, a non-competitive NMDA receptor ligand – but also by the state of other striatal afferents

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such as the corticostriatal glutamatergic or the nigrostriatal ­dopaminergic inputs that form synapses equally on the distal areas of the dendritic spines (60). The level of endogenous glutamate present in the striatum has a bearing on the extent of striatal lesion induced by excitotoxicity. For example, decortication, leading to the removal of the corticostriatal glutamatergic projection, protects the striatal cells from quinolinic acid-induced necrosis (14, 28). Conversely, glutamate levels within the striatum – and the sensorimotor cortex – have been shown to be higher on the side contralateral to an active, compared to an immobilised limb, and studies have indicated that the exclusive use of a limb can exaggerate the extent of tissue damage in rodent stroke model (61, 62), or following unilateral striatal lesion (44). Similarly, removal of the nigrostriatal dopaminergic input by 6-OHDA lesion has been shown to significantly reduce the vulnerability of the striatum to subsequent QA lesion (63), and this is particularly important to recognise when trying to establish the double lesion animal model for Multiple System Atrophy (see Kuzdas and Wenning, this volume, ch. 3). Dopamine acting on striatal postsynaptic D1 and D2 receptors can modulate ­glutamate signalling and excitatory currents by, respectively, increasing or decreasing NMDA and AMPA currents in the ­striatal neurons (64, 65), which will impact on the overall cell activity and subsequent excitotoxic damage.

4. Conclusions Despite the emerging transgenic animal models available to study basal ganglia diseases, producing striatal neuronal death using excitotoxic agents still enjoys strong scientific rationale. Excitotoxic lesions do not mimic the progressive degenerative processes that occur in Huntington’s disease or Multiple System Atrophy, for example; however, if the parameters are well chosen, they can recreate the cellular pathology at given stages of the disease with a high degree of authenticity. Once an excitotoxic striatal lesion model is established and characterised, it can open numerous research avenues looking into a multitude of scientific and clinical relevant issues such as neurodegeneration, neuroprotection, cell replacement therapy, circuit reconstruction and repair. The excitotoxic striatal lesions model has its limitations; but if the investigator makes use of it appropriately, it can serve as a highly powerful experimental tool to address scientific questions related to the structure and function of the basal ganglia.

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14. Schwarcz R, Whetsell WO, Mangano RM (1983) Quinolinic acid: an endogenous metabolite that produces axon-sparing lesions in rat brain. Science 219: 316–318 15. Schwarcz R, Foster AC, French ED, Whetsell WO, Köhler C (1984) Excitotoxic models for neurodegenerative disorders. Life Sci 35: 19–32 16. Schwarcz R, Fuxe K, Hökfelt T, Terenius L, Goldstein M (1980) Effects of chronic striatal kainate lesions on some dopaminergic parameters and enkephalin immunoreactive neurons in the basal ganglia. J Neurochem 34: 772–778 17. Schwarcz R, Hökfelt T, Fuxe K, Jonsson G, Goldstein M, Terenius L (1979) Ibotenic acidinduced neuronal degeneration: a morphological and neurochemical study. Exp Brain Res 37: 199–216 18. Beal MF (1992) Role of excitotoxicity in human neurological disease. Current Opinion in Neurobiology 2: 657–662 19. Beal MF, Ferrante RJ, Swartz KJ, Kowall NW (1991) Chronic quinolinic acid lesions in rats closely resemble Huntington’s disease. J Neurosci 11: 1649–1659 20. Choi DW (1987) Ionic dependence of glutamate neurotoxicity. J Neurosci 7: 369–379 21. Tymianski M, Charlton MP, Carlen PL, Tator CH (1993) Source specificity of early calcium neurotoxicity in cultured embryonic spinal neurons. J Neurosci 13: 2085–2104 22. Lau A, Tymianski M (2010) Glutamate receptors, neurotoxicity and neurodegeneration. Pflugers Arch 460: 525–542 23. Beal MF, Marshall PE, Burd GD, Landis DM, Martin JB (1985) Excitotoxin lesions do not mimic the alteration of somatostatin in Huntington’s disease. Brain Res 361: 135–145 24. Divac I, Markowitsch HJ, Pritzel M (1978) Behavioral and anatomical consequences of small intrastriatal injections of kainic acid in the rat. Brain Res 151: 523–532 25. Vincent P, Mulle C (2009) Kainate receptors in epilepsy and excitotoxicity. Neuroscience 158: 309–323 26. Ben-Ari Y, Cossart R (2000) Kainate, a double agent that generates seizures: two decades of progress. Trends Neurosci 23: 580–587 27. Coyle JT (1987) Kainic acid: insights into excitatory mechanisms causing selective neuronal degeneration. Ciba Found Symp 126: 186–203 28. Stone TW, Connick JH, Winn P, Hastings, MH, English M (1987) Endogenous excitotoxic agents. Ciba Found Symp 126: 204–220

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29. Perkins MN, Stone TW (1983) Quinolinic acid: regional variations in neuronal sensitivity. Brain Res 259: 172–176 30. Beal MF, Kowall NW, Ellison DW, Mazurek MF, Swartz KJ, Martin JB (1986) Replication of the neurochemical characteristics of Huntington’s disease by quinolinic acid. Nature 321: 168–171 31. Beal MF, Kowall NW, Swartz KJ, Ferrante RJ, Martin JB (1989) Differential sparing of somatostatin-neuropeptide Y and cholinergic neurons following striatal excitotoxin lesions. Synapse 3: 38–47 32. Döbrössy MD, Dunnett SB (2003) Motor training effects on recovery of function after striatal lesions and striatal grafts. Exp Neurol 184: 274–284 33. Döbrössy MD, Dunnett SB (2004) Environ­ mental enrichment affects striatal graft morphology and functional recovery. Eur J Neurosci 19: 159–168 34. Brasted PJ, Döbrössy MD, Robbins TW, Dunnett SB (1998) Striatal lesions produce distinctive impairments in reaction time performance in two different operant chambers. Brain Res Bull 46: 487–493 35. Döbrössy MD, Dunnett SB (2006) The effects of lateralized training on spontaneous forelimb preference, lesion deficits, and graft-mediated functional recovery after unilateral striatal lesions in rats. Exp Neurol 199: 373–383 36. Brady RJ, Swann JW (1986) Ketamine selectively suppresses synchronized afterdischarges in immature hippocampus. Neurosci Lett 69: 143–149 37. Henschke G, Wolf G, Keilhoff G (1993) Ketamine, but not glycine modulates quinolinate-induced neurodegeneration. Pol J Pharmacol 45, 339–347 38. Jiang W, Büchele F, Papazoglou A, Döbrössy M, Nikkhah G (2009) Ketamine anaesthesia interferes with the quinolinic acid-induced lesion in a rat model of Huntington’s disease. J Neurosci Methods 179: 219–223 39. Beal MF, Kowall NW, Swartz KJ, Ferrante RJ, Martin JB (1988) Systemic approaches to modifying quinolinic acid striatal lesions in rats. J Neurosci 8: 3901–3908 40. Lei H, Grinberg O, Nwaigwe CI, Hou HG, Williams H, Swartz HM, Dunn JF (2001) The effects of ketamine-xylazine anesthesia on cerebral blood flow and oxygenation observed using nuclear magnetic resonance perfusion imaging and electron paramagnetic resonance oximetry. Brain Res 913: 174–179 41. Gross M (2001) Tranquilizers, a2-adrenergic agonists and related agents. In: Adams HR (ed),

Veterinary Pharmacology and Therapeutics. Iowa State University Press, 299–342 42. Wright M (1982) Pharmacologic effects of ketamine and its use in veterinary medicine. J Am Vet Med Assoc 180: 1462–1471 43. Döbrössy MD, Dunnett SB (2007) The corridor task: striatal lesion effects and graft-mediated recovery in a model of Huntington’s disease. Behav Brain Res 179: 326–330 44. Döbrössy MD, Dunnett SB (2005) Training specificity, graft development and graft-mediated functional recovery in a rodent model of Huntington’s disease. Neuroscience 132: 543–552 45. Pearce RA, Stringer JL, Lothman EW (1989) Effect of volatile anesthetics on synaptic transmission in the rat hippocampus. Anesthesiology 71: 591–598 46. McGeorge AJ, Faull RL (1989) The organization of the projection from the cerebral cortex to the striatum in the rat Neuroscience 29: 503–537 47. Brasted PJ, Robbins TW, Dunnett SB (1999) Distinct roles for striatal subregions in mediating response processing revealed by focal excitotoxic lesions. Behav Neurosci 113: 253–264 48. Pisa M (1988) Motor functions of the striatum in the rat: critical role of the lateral region in tongue and forelimb reaching. Neuroscience 24: 453–463 49. Pisa M, Cyr J (1990) Regionally selective roles of the rat’s striatum in modality-specific discrimination learning and forelimb reaching. Behav Brain Res 37: 281–292 50. Whishaw IQ, Mittleman G, Bunch ST, Dunnett SB (1987) Impairments in the acquisition, retention and selection of spatial navigation strategies after medial caudate-putamen lesions in rats. Behav Brain Res 24: 125–138 51. Fricker RA, Annett LE, Torres EM, Dunnett SB (1996) The placement of a striatal ibotenic acid lesion affects skilled forelimb use and the direction of drug-induced rotation. Brain Res Bull 41: 409–416 52. Brown VJ, Robbins TW (1989) Elementary processes of response selection mediated by distinct regions of the striatum. J Neurosci 9: 3760–3765 53. Divac I (1984) The neostriatum viewed orthogonally. Ciba Found Symp 107: 201–215 54. Rogers RD, Baunez C, Everitt BJ, Robbins TW (2001) Lesions of the medial and lateral striatum in the rat produce differential deficits in attentional performance. Behav Neurosci 115: 799–811 55. Brooks SP, Trueman RC, Dunnett SB (2007) Striatal lesions in the mouse disrupt acquisition

2  Excitotoxic Lesions of the Rodent Striatum and retention, but not implicit learning, in the SILT procedural motor learning task. Brain Res 1185: 179–188 56. Brooks SP, Fielding SA, Döbrössy M, von Hörsten S, Dunnett SB (2009) Subtle but progressive cognitive deficits in the female tgHD hemizygote rat as demonstrated by operant SILT performance. Brain Res Bull 79: 310–315 57. Dunnett SB, Iversen SD (1981) Learning impairments following selective kainic acidinduced lesions within the neostriatum of rats. Behav Brain Res 2: 189–209 58. Reading PJ, Dunnett SB, Robbins TW (1991) Dissociable roles of the ventral, medial and lateral striatum on the acquisition and performance of a complex visual stimulus-response habit. Behav Brain Res 45: 147–161 59. Isacson O, Dunnett SB, Björklund A (1986) Graft-induced behavioral recovery in an animal model of Huntington disease. Proc Natl Acad Sci USA 83: 2728–2732 60. Parent A, Hazrati LN (1995) Functional anatomy of the basal ganglia. I. The cortico-basal ganglia-thalamo-cortical loop. Brain Res Rev 20: 91–127

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61. Bland ST, Gonzale RA, Schallert T (1999) Movement-related glutamate levels in rat hippocampus, striatum, and sensorimotor cortex. Neurosci Lett 277: 119–122 62. Bland ST, Schallert T, Strong R, Aronowski J, Grotta JC, Feeney DM (2000) Early exclusive use of the affected forelimb after moderate transient focal ischemia in rats : functional and anatomic outcome. Stroke 31: 1144–1152 63. Buisson A, Pateau V, Plotkine M, Boulu RG. (1991) Nigrostriatal pathway modulates striatum vulnerability to quinolinic acid. Neurosci Lett 131: 257–259 64. André VM, Cepeda C, Cummings DM, Jocoy EL, Fisher YE, William Yang X, Levine MS (2010) Dopamine modulation of excitatory currents in the striatum is dictated by the expression of D1 or D2 receptors and modified by endocannabinoids. Eur J Neurosci 31, 14–28 65. Surmeier DJ, Ding J, Day M, Wang Z, Shen W (2007) D1 and D2 dopamine-receptor modulation of striatal glutamatergic signaling in striatal medium spiny neurons. Trends Neurosci 30: 228–235

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Chapter 3 Combination Lesion Models of MSA Daniela Kuzdas and Gregor K. Wenning Abstract Multiple system atrophy (MSA) is a rapidly progressive neurodegenerative disorder presenting with parkinsonism, cerebellar ataxia, and autonomic failure in various combinations. The main pathological hallmark is the formation of (oligo-) glial cytoplasmic inclusions (GCIs) composed of alpha-synuclein (a-syn) aggregates (also called Papp–Lantos bodies). In the past decades, increasing insights into disease pathophysiology led to a variety of animal models as testbeds for interrentional therapies. Key words: Multiple system atrophy, Alpha-synuclein, Neurotoxin, Transgenic, Animal model, Parkinsonism, Ataxia, Autonomic failure, Glial cytoplasmic inclusion

1. Introduction Multiple system atrophy (MSA) is a fatal neurodegenerative disease with unclear etiology. The mean age of disease onset is 55  years and survival after disease onset lies between 6 and 9 years (1, 2). The prevalence is 1.9–4.9/100,000 and the incidence is 3/100,000/year in the population over 50 years (3, 4). Together with Parkinson’s disease (PD) and dementia with Lewy bodies (LBD), MSA is characterized as a primary synucleinopathy. However, in contrast to the former diseases where a-synuclein (a-syn) accumulates mostly in neurons to form Lewy bodies (LB) or Lewy neurites, in MSA a-syn accumulates mostly in oligodendrocytes to form eosinophilic, erythrophilic, sickle-shaped, oval or conical glial cytoplasmic inclusions (GCIs, also Papp– Lantos bodies) with fibrillar a-syn as their main component. The clinical symptoms range from parkinsonian L-DOPA refractory features resulting from striatonigral degeneration (SND) over cerebellar ataxia caused by olivopontocerebellar atrophy (OPCA)

Emma L. Lane and Stephen B. Dunnett (eds.), Animal Models of Movement Disorders: Volume II, Neuromethods, vol. 62, DOI 10.1007/978-1-61779-301-1_3, © Springer Science+Business Media, LLC 2011

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to the very characteristic autonomic failure, in different combinations. Neuropathological and functional imaging studies reveal that the unresponsiveness to L-DOPA results from a degeneration of substantia nigra pars compacta and its striatal target (5). In the Western world, the parkinsonian type of MSA (MSA-P) accounts for 80% of cases, whereas in the Asian area, MSA-C is present in the majority of patients (6, 7). Different animal models have been generated to reproduce the human disease pathology and research neurodegenerative mechanisms as well as establishing new therapeutical approaches. Like in animal models for PD, the application of neurotoxins is widely used. In rats, 6-hydroxydopamine (6-OHDA) in combination with quinolinic acid (QA) is administered via stereotaxic injection to induce SND. The most common approach in mice is the intraperitoneal application of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and/or 3-nitropropionic acid (3-NP), causing SND-Like neuronal loss in the substantia nigra and striatum. Since the a-syn involvement in MSA has been discovered, the development of animal models has shifted not only toward the generation of transgenic models, mostly overexpressing a-syn, but also other approaches such as overexpression of the a1B-adrenergic receptor (a1B-AR) (8–11).

2. Animal Models 2.1. Neurotoxins

Like in Parkinson’s disease (PD), many animal models are based on the administration of specific neurotoxins to cause the diseasecharacteristic neuronal loss in the relevant areas. Many protocols have been established, using one or more neurotoxins. We here want to give an overview on the most relevant approaches. The evaluation and analyses of these animal models have been done using different behavioral tests, and extensive pathological investigation (12). It should be kept in mind that there are not only species-­ dependent sensitivity differences to the different neurotoxins, but also big variations between different strains (13–19). The neuro­ toxins can be applied via stereotaxic surgery or via systemic intraperitoneal injections. This difference also has consequences for the lesion outcome; stereotaxic surgeries are usually done on one side only, whereas the systemic application leads to a bilateral lesion. A very useful tool in describing PD and MSA models is the rotation paradigm (see Dunnett and Torres, Volume I, chapter 15), where dopaminergic drugs like the dopamine-releasing substance amphetamine and the dopamine-receptor agonist apomorphine are used to induce lesion-specific rotations (20).

3  Combination Lesion Models of MSA

2.2. Neurotoxic Lesions in Rats

2.2.1. Single-Toxin, Double-Lesion Model

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One widely used neurotoxin to reproduce parkinsonian features in rats is 6-hydroxydopamine (6-OHDA). Chemically, it is an analogue to the neurotransmitter dopamine, but cannot pass the blood–brain barrier. Therefore, 6-OHDA cannot be applied systemically and has to be injected via stereotaxic surgery to cause the selective loss of dopaminergic neurons in the substantia nigra (21). In combination, the NMDA-agonist quinolinic acid (QA) is often applied to additionally lesion the striatal areas and complete the pathological picture of SND present in MSA-P. Also 3-nitropropionic acid (3-NP) has been used to cause striatonigral lesions in rats and mimic the human pathology of SND. 1. 1-Methyl-4-phenylpyridinium (MPP+): Unilateral striatal injections of 1-methyl-4-phenylpyridinium ion (MPP+), an active metabolite of MPTP, have been used in rats to replicate SND. Intrastriatal administration of MPP+ causes lesions in the striatum and leads to a retrograde damage of the dopaminergic system in the SN (17, 22, 23). Contraversive apomorphine-induced rotations are not observed in the MPP+ lesioned rats. The reason for this is the neurodegeneration in the striatum and SN. These lesions would lead to rotations in the opposite direction and therefore neutralize each other. This can also be regarded a rodent analogue of dopamine unresponsiveness as it is present in MSA patients. Also the asymmetry in thigmotactic scanning behavior toward the unlesioned side as known from 6-OHDA or QA lesioned models could not be detected (17). The stepping test to evaluate skilled motor behavior showed significant impairment when compared to sham-treated control groups, and in the paw reaching test, a significant contralateral deficit could be detected. Histopathological analysis of this animal model shows astrogliosis, metabolic failure, a discrete striatal lesion and striatal surface reduction, damage of the intrastriatal fibers and striatalcrossing fiber bundles and also a significant ipsilateral retrograde cell loss in the substantia nigra when compared to the sham-treated control group. Overall, the single-toxin rat model using unilateral striatal injections of MPP+, presenting with motor phenotype as well as some pathological parallels to the human SND, can be used to further research MSA. 2. 3-Nitropropionic acid (3-NP): Initially, injection of the mitochondrial inhibitor 3-NP has been used to reproduce the human pathology of Huntington’s disease (HD) in animal models, but the degeneration of intrinsic striatal neurons as well as the dopaminergic nigrostriatal systems suggested that it

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could be used as a valuable test bed for MSA research also (24–26). Unilateral stereotaxic injection of 3-NP in to the rat striatum causes significant neuronal loss in the striatum. Several studies have shown that the striatal surface area was significantly reduced and, in addition to the striatal degeneration, also retrograde loss of dopaminergic neurons in the SN as illustrated in Fig. 1b has been reported (26, 27). Significant dopamine depletion could be detected after this almost complete destruction of the striatum. Motor behavior tests of 3-NP versus sham lesioned control groups revealed impaired paw reaching in the animals.

Intact striatum and SN including projections

Rat models with stereotactic injections Single-toxin, doublelesion (3-NP) (26-28) Almost complete destruction of striatum, retrograde loss in SN

Sequential doubletoxin double-lesion (6-

OHDA+QA) (21, 29, 34, 38) Striatal outflow projections disrupted, global DA denervation

unilateral lesions

Simultaneous double-toxin double-lesion (6-OHDA+QA) (35) earlystage SND, lateral projection disrupted, medial projection preserved

striatum substantia nigra Mouse models with systemic administration Sequential double-toxin double-lesion (3-NP

bilateral lesions Simultaneous double-toxin double-lesion (MPTP+3-NP)

Single-toxin doublelesion (3NP) (51)

Sequential double-toxin double-lesion (MPTP 3-

Almost complete striatal damage, particularly lateral lesion

NP) (54)

MPTP) (54)

(57, 58)

Degeneration of SNc, less severe in the striatum

Degeneration of striatum, less severe degeneration in SNc

Neuronal loss in medial striatum, loss of DA neurons in mid and caudal levels of SNc

striatum substantia nigra Fig. 1. Lesion models. Illustration of the conditions of the rodent brains, in particular the striatum and substantia nigra, in healthy unlesioned animals (a) and the different lesion paradigms (b–h). The rat models of stereotaxic 3-NP (b), sequential administration of 6-OHDA + QA (c) as well as simultaneous stereotaxic application of 6-OHDA + QA (d) show unilateral lesions of the relevant areas striatum and substantia nigra to different degrees, whereas systemic application of 3NP (e), sequential injection of MPTP followed by 3-NP (f), sequential administration of 3-NP followed by MPTP (g) or simultaneous injection of MPTP and 3-NP (h) in mice lead to bilateral brain damage with different severity.

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Investigating the drug-induced rotation behavior, ipsiversive amphetamine induced rotations as well as ipsiversive rotations ­following apomorphine were reported. As will be explained later, the PD 6-OHDA lesion models lead to contraversive rotations after apomorphine injections. These differences can be explained by the striatal lesion and suggest that the partial reduction of dopaminergic neurons after 3-NP lesion does not significantly influence the rotation pattern. The behavioral impairment was directly correlated to the reduction of striatal surface area. In one study by Brundin and colleagues, they further analyzed the effects of alpha-phenyl-tert-butyl nitrone and found no improvement of the paw-reaching deficits after 3-NP lesion. Also neurorestorative approaches using embryonic neuronal grafts have been tried but a significant amelioration of behavioral deficits could not be achieved (27, 28). 2.2.2. Sequential Double-Toxin, Double-Lesion Model

A study by Wenning and colleagues (29) used 6-OHDA and QA in a sequential protocol (Fig. 1c). For this purpose, the two neurotoxins were stereotaxically injected unilaterally, but in one group 6-OHDA into the medial forebrain bundle (MFB) and 8  weeks later, QA into the striatum, and in the second group the protocol started with striatal injections of QA followed by secondary 6-OHDA injections into the MFB 8 weeks afterwards. Behavioral assessment tests, including the paw-reaching test or the side falling and stepping test, were performed before and after the administration of the second neurotoxin. Extensive histological analysis showed that in animals injected with QA first, the remaining striatal surface area was significantly reduced compared to animals with primary 6-OHDA nigral lesions. These findings correlate well with the relevant behavioral findings like the stepping test, where the contralateral backhand stepping pattern showed significantly higher impairment in the group with primary striatal QA lesion. Both experimental groups also presented severe motor impairment in the paw-reaching test. Loss of TH-positive neurons in the SN could be observed in both groups without significant differences between the different injection protocols. Microglial activation and astrogliosis are also present features of this lesion model. Ipsiversive rotation followed amphetamine injections have been observed; however, apomorphine induced contraversive rotations were absent. Investigators should keep in mind that potential differences in the response to apomorphine and amphetamine may be due to slight variations of the size and locus of the caused lesion (30). The ipsiversive rotation followed amphetamine administration is likely due to a stimulated dopamine release on the intact side (31–33). Kollensperger et al. also performed a cylinder test with animals treated after this sequential protocol (34). They found that after

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6-OHDA lesion a clear preference of wall contacts toward the ipsila­ teral paw representing the unlesioned side of the striatum and administration of dopaminergic agents could neutralize this asymmetry. The additional QA lesion created 8  weeks later, further shifted the paw use toward the ipsilateral side. The fact that the primary lesion decreases the effects of the secondary neurotoxin is suggested to be due to a release of neurotrophic factors such as BDNF or GDNF induced by the first lesion (29, 35–37). Transplantation of embryonic striatal cells could partly restore the dopamine response in regard to rotation behavior as well as complex motor behavior (21, 38). Furthermore, this double-toxin model has been used to test the effects of riluzole and minocycline. The glial activation inhibitor minocycline, which has been shown to have neuroprotective effects in an MPTP PD mouse model, did not lead to any beneficial effects in the sequential double-toxin, double-lesion model (39, 40). Riluloze is a therapeutic agent that is already FDA approved as treatment for amyotrophic lateral sclerosis (ALS) (41). The investigators report significant amelioration of motor impairment and a reduction in the striatal lesion size; however, they did not find significant differences in the number of dopaminergic neurons in the SNc between control and riluzole-treated animal groups. In summary, this model can be used to reproduce degeneration of the dopaminergic nigrostriatal and GABAergic striatopallidal pathways; however, it should be mentioned that the almost complete nigral and striatal degeneration does not reflect the early stages of human pathology with specific loss of ventrolateral nigral dopaminergic neurons and dorsolateral striatal target neurons (42–44). Therefore, the next model listed here might be more helpful to reproduce the early stage SND. 2.2.3. Simultaneous Double-Toxin, DoubleLesion Model

A method to generate a lesion model for MSA-P, more specifically mimicking the disease pattern of early-stage SND to closely reflect the human disease process, uses simultaneous striatal injections of 6-OHDA and QA (Fig. 1d) (35). The lateral striatum was chosen as target region for the surgery. This area receives projections from the motor cortex, afferent its dopaminergic innervation is restricted to the nigra and it has been reported previously that it is the functional homologue to the human sensorimotor putamen (45–48). The rotation behavior of this model was different compared to the sequential double-toxin double-lesion model. Due to the simultaneous 6-OHDA and QA injections, the characteristic 6-OHDA ipsiversive amphetamine and contraversive apomorphine induced rotation pattern was modified compared to single toxin control groups. This finding can be explained by the loss of dopaminergic responsiveness due to the destruction of striatal postsynaptic outflow (30, 49, 50). Also present in this model is astrogliosis and a

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significant striatal surface reduction, more restricted to the caudal than rostral striatum, and the loss of TH-immunopositive neurons in the SN was restricted to the medial part of the substantia nigra pars compacta after the stereotaxic administration of the two neurotoxins. Summing up, this model with its partial dopaminergic and striatal degeneration is a valuable model to investigate treatment options for early-stage SND. 2.3. Neurotoxic Lesions in Mice 2.3.1. Single-Toxin Double Lesion

2.3.2. Sequential Double-Toxin Double Lesion

Intraperitoneal injections of 3-NP lead to reversible striatonigral lesions (see Fig. 1e) (51). After the intoxication phase of 5 consecutive days, animals showed improving scores in evaluation of the motor behavior 3–5 days after the last 3-NP injections and a partial recovery after 7–10  days. The motor phenotype of this model ranges from early motor signs like slowing or transient hindlimb dystonia at low doses (two injections per day, doses increased over time from 10 to 50 mg/kg, total cumulated dose 340  mg/kg) to permanent hindlimb dystonia, hindlimb clasping, truncal dystonia or global postural abnormalities at higher doses (two injections per day, doses increased over time from 20 to 80 mg/kg, total cumulated dose 560 mg/kg). Even though low doses of 3-NP did not cause circumscribed lesions in the experiment performed by Fernagut et al., high dose administration led to lateral lesions or an almost complete destruction of the striatum with significant reduction of the striatal volume. That study also reported correlations between motor deficits and the remaining striatal volume, and also between the motor behavioral impairment and the estimated loss of cresyl violetnissl stained neurons in the lateral striatum post-intoxication. They further used MRI to detect the 3-NP-induced lesion in  vivo. The mentioned study described a useful SND model showing signs of age-dependent oxidative stress, apoptotic striatal neuronal cell death, and histopathologically circumscribed lesions. MPTP is a meperidine-derivate that leads to a selective degeneration of dopaminergic neurons in the SN and further degeneration of the striatal outflow pathways (52, 53). Stefanova et al. (54) tested the sequential, systemic application of MPTP and 3-NP. They analyzed two different procedures (a) a primary MPTP lesion, followed by 3-NP application compared to (b) a primary striatal 3-NP lesion followed by secondary administration of MPTP. In both approaches, the neurotoxins were injected twice a day intraperitoneally, MPTP for 2 consecutive days, and 3-NP over a period of 2  weeks. Primary MPTP lesions were shown to protect against the neurotoxic effects of 3-NP, which well correlates with results from rat models, where the primary striatonigral 6-OHDA injection had

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protective effects against QA lesions. Other previous studies also showed that dopamine depletion protects striatal neurons against various types of neurotoxins (55, 56). The behavioral analysis of these double-toxin models revealed no significant difference compared to the administration of 3-NP or MPTP alone regarding locomotor activity. On the other hand, when applying 3-NP prior to MPTP, Stefanova et  al. report reduced toxicity of MPTP to nigral dopaminergic neurons, which is similar to the findings in rat models where striatal QA lesions were protective against nigral 6-OHDA toxicity. In summary, these different administration patterns resulted in different lesion patterns and should be carefully chosen depending on the study design. The MPTP + 3-NP model pathology can be described as degeneration of SNc with a relative preservation of the striatum as represented in Fig.  3.1f, whereas the 3-NP-MPTP model had a more severe pathology including neuronal loss and glial pathology in the SNc as well as striatum, which can be seen in Fig. 1g. 2.3.3. Simultaneous Double-Toxin DoubleLesion

One approach described by Tison and coworkers (57, 58) is a mouse model with simultaneous systemic injections of the two neurotoxins MPTP and 3-NP (Fig. 1h). To assess the behavioral characteristics of this model, they used motor-tests such as the rotarod, beam-walking or the pole-test and were able to show significant motor impairment and altered gaitpatterns due to striatal dysfunction. Also an open-field test has been performed and detected a reduced amount of rearing in the MPTP + 3-NP treated group, as well as reduced mean and maximum velocity indicating lower activity levels. Histologically, they report bilateral striatal lesions with increased neuronal loss in the medial part of the striatum, significant reduction of striatal volume and significant loss of dopaminergic neurons in the mid and caudal levels of the substantia nigra pars compacta. Furthermore, when comparing MPTP + 3-NP groups to 3-NP or MPTP treated animals alone, they found a significantly increased number of astrocytes and also different distribution patterns showing that the simultaneous application of the two neurotoxins leads to a more severe pathology. This model can serve as a useful tool to mimic SND; however, after the 9-day intoxication phase, a recovery has been observed and after 3 weeks, the motor impairment was not detectable. The same group later tested whether the antiglutamateric drug riluzole can serve as a useful treatment option (58). They report dose-dependent neuronal rescue in histopathological analyses, reduced cell loss in the nigral and dorsolateral striatum, and less astroglial activation.

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This model has further been used in a study to investigate the potential treatment with human mesenchymal stem cells (59). For this purpose, mice received 9 days of double-toxin treatment before the bone-marrow-derived stem cells were administered via the tail vein on the following day. The behavioral as well as histopathological outcome of this study showed improvement in the stem-cell treated group compared to controls. Also upregulation of several cytokines including p-Akt, Bcl-2 and decreased levels of Bax and cytochrome C were detected. 2.4. Neurotoxic Lesions in Primates 2.4.1. Sequential Double-Toxin, DoubleLesion Model

A model described by Tison and colleagues uses a sequential, systemic intraperitoneal administration of MPTP followed by 3-NP injections in primates (60, 61). They report l-Dopa-responsive parkinsonian phenotype after MPTP injection, increased motor impairment and loss of l-Dopa response after secondary application of 3-NP. Histologically, they found severe dopaminergic cell loss in the SNc and bilateral subtotal striatal lesions. This model can serve as a useful tool to investigate the human pathology of l-Dopa unresponsive striatonigral degeneration in primates.

2.5. Transgenic Models in Mice

The neurotoxin models have been of great importance for researching MSA disease pathophysiology and they can still be applied depending on the experimental question. For therapeutic approaches such as neurorestorative cell transplantation trials, the lesion models can provide the necessary test bed. However, their utility is limited in neuroprotective studies that require insights into pathogenesis. The great advantage of transgenic MSA compared to neurotoxin models is, that transgenic models reproduce the overexpression, misfolding, and aggregation of human a-synuclein or a functional deficit of the degradation mechanisms at normal expression levels, resulting in the formation of GCIs and therefore leading to the characteristic neuronal loss (62). These transgenic MSA models can be used to identify intervention therapies by targeting different steps of the a-synuclein linked neurodegeneration. However, since it is still not fully understood how a-syn and GCIs cause neuronal loss and not all transgenic models reproduce striatonigral lesions or a motor phenotype, more research will be required in this field to generate research models even closer to the human disease that not only feature motor pathology but also autonomic nervous system degeneration. Since some transgenic MSA modes do not reproduce the characteristic neuronal loss, neurotoxins can be used to lesion the relevant areas and mimic the full-blown pathology of human MSA.

2.5.1. a-Syn Overexpressing Mouse Model Using PLP-Promoter

A well-established method has been developed by Stefanova et al. (63). They used the mitochondrial inhibitor 3-NP in an a-syn overexpressing transgenic mouse model to induce oxidative stress.

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The overexpression of a-syn in this model is linked to the proteolipid protein (PLP) promoter that is only active in oligodendrocytes and therefore leads to specific overexpression of human wild-type a-syn in oligodendrocytes (64). Histological analysis of this PLP-h-a-syn model showed halfmoon or sickle-shaped GCI-like insoluble a-syn positive inclusions in oligodendrocytes throughout the white matter. Further significant loss of dopaminergic neurons in the SN, microglial activation, and shortened stride length have been reported. Since this transgenic model did not reproduce the neuropathology of MSA, the effects of 3-NP injections were investigated. The findings reveal that already low doses of 3-NP, which did not have effects in the control group, led to motor impairment and loss of striatal neurons in PLP-h-a-syn mice. In addition, a significantly higher reduction of striatal volume has detected in transgenic animals with low dose 3-NP. In PLP-h-a-syn animals, a significant reduction of TH-immunoreactive neurons in the SNc was found at baseline levels, indicating that the a-syn pathology alone, is sufficient to cause this neuronal loss. Injections of 3-NP further increased this neuronal loss in PLP-h-a-syn animals. Significant neuronal loss after the injections of 3-NP was found in cerebellar purkinje cells, locus coeruleus, pontine nuclei, and inferior olivary nuclei in PLP-h-a-syn animals, but not in wt controls. Stefanova et al. further describe widespread astrogliosis throughout the striatum of 3-NP treated. Moreover, investigating the distribution and amount of activated microglia revealed significantly higher numbers in the PLP-h-a-syn group, further increased by 3-NP injections, but not or to a much lesser degree in control mice. This model was the first to represent the full-blown human MSA-pathology including SND, astrogliosis, microglial activation as well as the characteristic oligodendroglial a-syn inclusions. For this reason, the model has been used to test potential neuroprotective effects of a possible therapeutic agent. Rasagiline has been used in the transgenic model with additional 3-NP lesion and shown to have dose-dependent neuroprotective effects (65). 2.5.2. a-Syn Overexpressing Mouse Model Using MBPPromoter

The other transgenic MSA model that has been tested with additional 3-NP lesions, works under the control of the myelin basic protein (MBP) promoter (66). Since this promoter also has specific expression pattern in oligodendrocytes, this model too presents with overexpression of a-syn in oligodendrocytes (67). Motor impairment could be shown via rotarod and pole-test, and a-syn expression detected in the basal ganglia, brain stem, and cerebellum. To lead this model even closer to the human pathology, 3-NP has been administered twice daily over 6 days to cause the characteristic striatonigral lesions. They were able to show that the solubility pattern of a-syn changed after 3-NP injection. MBP-h-a-syn animals which received 3-NP treatment had significantly higher

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amounts of nitrated and oxidized a-syn in the insoluble urea fraction and decreased amounts of soluble nitrated, as well as oxidized a-syn in the TBS fraction compared to vehicle treated MBP-h-asyn mice, whereas the levels of total and phosphorylated a-syn were not affected by 3-NP. Histologically they found that the a-syn expression was significantly increased in the basal ganglia and frontal cortex of MBP-ha-syn animals with 3-NP treatment compared to the saline-treated group. Behavioral tests to investigate potential motor deficits have been performed. In the pole test, as well as the grip test, significant motor impairment could be detected; the MBP-h-a-syn + 3-NP group had the worst motor scores. The difference between nontransgenic mice receiving saline and MBP-h-a-syn + saline was not significant, illustrating that the severity of motor deficits strongly depends on the 3-NP administration. In summary, like the PLP-h-a-syn + 3-NP model, this model can serve as a useful tool for evaluating potential therapeutic agents, not only testing neuroprotective or neurogenic factors, but it can also find application in investigating the pathways of protein overexpression, misfolding, aggregation, oligodendrogliosis, and finally neurodegeneration (63, 66). Understanding these disease mechanisms will be of great importance for developing therapeutics interfering and inhibiting these mechanisms. 2.5.3. Other Transgenic Models

Currently there are 2 more transgenic mouse models of MSA, one using the phosphodiesterase (CNP) promoter to constitutively overexpress h-a-syn and the second model overexpresses a1Badrenergic receptor (a1B-AR), leading to an MSA-P-like phenotype (9, 11). However, these models have not yet been analyzed with regard to 3-NP sensitivity.

3. Methods The methods to evaluate and characterize the relevance of different animal models vary from histological quantifications using classical immunohistochemistry, molecular techniques such as western blots, and behavioral tests to investigate the motor impairment, which play a very important role in analyzing models for movement disorders (12). The major behavioral tests that have been used to evaluate MSA models are shown in Table 1, and the key features of each test are described in the following paragraphs. 3.1. General Neurological State 3.1.1. General Assessment

The motor behavior can be observed and the cardinal characteristics quantified, including slowing, transient or permanent hind- or forelimb dystonia, hindlimb clasping or truncal dystonia (23, 51, 54, 60, 61).

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Table 1 Behavioural tests

3.2. Locomotor Activity 3.2.1. Open-Field Test

Functional domain

Behavioral test

Key references

General neurological state

General assessment

(23, 51, 54, 60, 61)

Locomotor activity

Open-field test Rotation test

(35, 54, 57, 58) (21, 25, 26, 29, 35)

Balance and motor coordination

Cylinder test Rotarod Balance beams Gait analysis Climbing Grip strength Stepping test

(34) (57, 58) (57, 58) (51, 54, 57, 58) (23, 57, 58) (22, 23) (29, 34, 69)

Skilled limb use

Reaching test Staircase test

(22, 25, 26, 29, 35) (29, 35, 70)

Several parameters of motor activity can be obtained from openfield experiments, including global locomotion, maximum or mean velocity, or the number of rearings. MSA models often present with decreased activity and velocity and also a reduction of rearing events (35, 54, 57, 58).

3.2.2. Rotation Test

The rotation test is a very important tool for the characterization of animal models for movement disorders. It can be applied in unilateral lesion models to detect asymmetries. In the classical Ungerstedt PD model, ipsiversive rotations following amphetamine injection and apomorphine-induced contraversive rotations can be observed (68). Due to the additional striatal neuronal loss in MSA models, the contraversive rotations should not be present and therefore the rotation test can serve as a test to distinguish between unilateral PD and MSA lesion models (21, 25, 26, 29, 35).

3.3. Balance and Motor Coordination

The cylinder test can be used to detect motor asymmetries by placing the animal in a transparent cylinder and counting the wall touches for both fore paws (34). For example, animals with a unilateral 6-OHDA lesion would show a higher number of wall-touches with the ipsilateral (=unlesioned) than with the contralateral side.

3.3.1. Cylinder Test 3.3.2. Rotarod

Animal models for movement disorders like MSA or PD usually have impaired balance and coordination abilities and therefore do not manage to stay on the rotarod apparatus as long as healthy

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controls (57, 58). The test can be performed in different protocols, using a fixed speed or an accelerating mode. 3.3.3. Balance Beams

MSA animals need more time or have severe problems to traverse the beam and very often have an increased amount of slips (57, 58).

3.3.4. Gait Analysis

The gait analysis can be performed using special devices such as Digi-gait, where the animals are put on a treadmill and the paw movement is recorded with a camera, or more simple techniques analyzing the paw prints during spontaneous walking on paper. Parameters such as weight distribution or stride length can be obtained. Some MSA models show reduced stride length and an imbalance of weight distribution (in toxin models between lesioned and unlesioned side) (51, 54, 57, 58).

3.3.5. Climbing

One very common example of climbing tests is the pole test where mice are put head upwards on a vertical pole of approximately 1 cm thickness, and the needed time for a turn (when mouse has fully turned around, head downwards) and the total time needed to descend the pole are measured. MSA test animals often require more time and have more difficulties (often slips) due to their motor impairment (23, 57, 58).

3.3.6. Grip Strength

For the grip strength test, animals are put on top of a small-wired grid, before the grid is inverted and the time the mouse can remain clinging to the grid is measured (22, 23). This test has several pitfalls like the weight of the animals, which is not taken into consideration.

3.3.7. Stepping Test

The hind paws and the fore paw that is not being assessed have to be fixed by the experimenter before the animal can slowly be moved sideways (forehand as well as backhand direction) along a surface, the paw to be assessed touching the surface. The adjusting steps are counted and give information about motor-asymmetry. 6-OHDA lesions lead to a reduced number of adjusting steps on the contralateral paw (i.e. the paw under the control of the lesioned hemisphere) in particular when the animal moves in a forehand direction (29, 34, 69).

3.4. Skilled Limb Use

The reaching test can be used for the evaluation of more complex motor behaviors such as skilled forelimb use. For this test, animals have to reach through the bars of a wire mesh cage to retrieve food pellets. The success rate can be quantified and usually has lower scores in animals of movement disorders compared to wild-type animals (22, 25, 26, 29, 35).

3.4.1. Reaching Test

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3.4.2. Staircase Test

For the paw test, animals are put in Plexiglas boxes with a central platform between two staircases. Food pellets are put on each level of the staircase and numbers of eaten and taken ­pellets are quantified and a success rate calculated. This behavioral method not only tests skilled reaching, but also the animal’s ability to grasp the pellet. In mice it has been shown to be sensitive to cortical, basal ganglia, spinal, and ischemic lesions (70). MSA and PD animals have lower scores due to their motor impairment (29, 35, 70).

4. Conclusions In MSA, like in any other medical field, animal models which closely reproduce the human pathology are of great importance to uncover possible disease mechanisms and develop new treatment options. Environmental factors as well as genetic predisposition have been shown to be involved in the etiology of MSA; however, to what degree remains unclear today and requires further investigation (71–74). At this stage, the model organisms that replicate the human pathology the closest are transgenic approaches with additional application of neurotoxins. These mechanisms represent the combination of environmental and genetic factors, which is also considered to represent the underlying etiology of human MSA. However, the development of the “ideal” MSA model continues to be a still ongoing process. Even though scientists are able to mimic most of the human MSA features in the models available nowadays, and positive results from some ­animal studies have already led to several clinical trials in humans, not all of these clinical trials in humans have resulted in beneficial or disease-modifying effects (75, 76). Future MSA research must therefore be directed at optimizing the available models to improve preclinical screening programs of novel interventional strategies. References 1. Wenning G K, Tison F, Ben Shlomo Y, Daniel S E, Quinn N P (1997) Multiple system atrophy: a review of 203 pathologically proven cases. Mov Disord 12, 133–147 2. Wenning G K, Colosimo C, Geser F, Poewe W (2004) Multiple system atrophy. Lancet Neurol 3, 93–103 3. Bower J H, Maraganore D M, McDonnell S K, Rocca W A (1997) Incidence of progressive supranuclear palsy and multiple system atrophy in Olmsted County, Minnesota, 1976 to 1990. Neurology 49, 1284–1288

4. Wakabayashi K, Takahashi H (2006) Cellular pathology in multiple system atrophy. Neuropathology 26, 338–345 5. Gelb D J, Oliver E, Gilman S (1999) Diagnostic criteria for Parkinson disease. Arch Neurol 56, 33–39 6. Gilman S, Low P, Quinn N, Albanese A, BenShlomo Y, Fowler C, et al. (1998) Consensus statement on the diagnosis of multiple system atrophy. American Autonomic Society and American Academy of Neurology. Clin Auton Res 8, 359–362

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3  Combination Lesion Models of MSA 51. Fernagut P O, Diguet E, Stefanova N, Biran M, Wenning G K, Canioni P, Bioulac B, Tison. (2002) Subacute systemic 3-nitropropionic acid intoxication induces a distinct motor disorder in adult C57Bl/6 mice: behavioural and histopathological characterisation. Neuroscience 114, 1005–1017 52. Langston J W, Ballard P, Tetrud J W, Irwin I (1983) Chronic Parkinsonism in humans due to a product of meperidine-analog synthesis. Science 219, 979–980 53. Davis G C, Williams A C, Markey S P, Ebert M H, Caine E D, Reichert C M, Kopin I J (1979) Chronic Parkinsonism secondary to intravenous injection of meperidine analogues. Psychiatry Res 1, 249–254 54. Stefanova N, Puschban Z, Fernagut P O, Brouillet E, Tison F, Reindl M, Jellinger K A, Poewe W, Wenning G K (2003) Neuropatho­ logical and behavioral changes induced by various treatment paradigms with MPTP and 3-nitropropionic acid in mice: towards a model of striatonigral degeneration (multiple system atrophy). Acta Neuropathol 106, 157–166 55. Clemens J A, Phebus L A (1988) Dopamine depletion protects striatal neurons from ischemia-induced cell death. Life Sci 42, 707–713 56. Reynolds D S, Carter R J, Morton A J (1998) Dopamine modulates the susceptibility of striatal neurons to 3-nitropropionic acid in the rat model of Huntington’s disease. J Neurosci 18, 10116–10127 57. Fernagut P O, Diguet E, Bioulac B, Tison F (2004) MPTP potentiates 3-nitropropionic acid-induced striatal damage in mice: reference to striatonigral degeneration. Exp Neurol 185, 47–62 58. Diguet E, Fernagut P O, Scherfler C, Wenning G, Tison F (2005) Effects of riluzole on combined MPTP + 3-nitropropionic acid-induced mild to moderate striatonigral degeneration in mice. J Neural Transm 112, 613–631 59. Park H J, Bang G, Lee B R, Kim H O, Lee P H (2010) Neuroprotective effect of human mesenchymal stem cells in an animal model of double toxin-induced multiple system atrophyparkinsonism. Cell Transplant doi: 10.3727/096368910X540630 60. Ghorayeb I, Fernagut P O, Aubert I, Bezard E, Poewe W, Wenning G K, Tison F (2000) Toward a primate model of L-dopa-unresponsive parkinsonism mimicking striatonigral degeneration. Mov Disord 15, 531–536 61. Ghorayeb I, Fernagut P O, Stefanova N, Wenning G K, Bioulac B, Tison F (2002) Dystonia is predictive of subsequent altered

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Chapter 4 The Role of the Dorsal Striatum in Instrumental Conditioning Mark A. Rossi and Henry H. Yin Abstract This chapter is divided into three parts. In the first part, we introduce the theoretical foundation of instrumental conditioning and the commonly used methods to study it. In the second part, we review some recent work using these methods to investigate the role of the dorsal striatum in instrumental conditioning. In the third part, we describe in detail the methods and considerations in defining precise and informative experiments in operant analysis of striatal function. Key words: Instrumental conditioning, Operant, Learning, Action, Basal ganglia, Striatum, Reward

1. Introduction The term “instrumental” or “operant” conditioning is used to describe an experimental method for studying animal behavior in the laboratory as well as the behavioral phenomenon studied. As a method, instrumental conditioning is among the most widely used in psychology and neuroscience. As a phenomenon, there is still debate about what it is and how it can be explained. This chapter can be divided into three parts. In the first part, we describe the basic phenomenon of instrumental conditioning and the methods used to study it. In the second part, we review recent work using these techniques to study the role of the dorsal striatum in instrumental conditioning. In the third part, we describe in detail the methods and considerations in defining precise and informative experiments in operant analysis of striatal function.

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2. Instrumental Conditioning 2.1. The Phenomenon of Instrumental Conditioning

The phenomenon of instrumental conditioning was first studied by Thorndike and later investigated in detail by Skinner (1, 2). Thorndike’s method was simple. For example, he left a cat in a box and observed what it did to get out of the box. Skinner’s method was also simple. He left a hungry rat in a chamber, where it could press a lever to earn food pellets. The chief innovations introduced by Skinner were an automated experimental apparatus known as the operant chamber (or “Skinner box”) which permitted automatic and systematic manipulations of the relationship between the food and the behavior. The phenomenon of instrumental conditioning, as observed by Thorndike and Skinner, can be divided into three major phases. For the sake of simplicity, let us assume the standard scenario of a rat pressing a lever for food reward. 1. Exploration: The hungry animal generates a variety of exploratory behaviors in search of food. 2. Selection: the animal learns the relationship between the effective action (also called the operant) and the desired outcome (food). 3. Repetition: the animal repeats the successful action. To explain his observations, Thorndike proposed the “law of effect”: Of several responses made to the same situation, those which are accompanied or closely followed by satisfaction to the animal … will, other things being equal, be more firmly connected with the situation …; those which are accompanied or closely followed by discomfort…will have their connections with the situation weakened … The greater the satisfaction or discomfort, the greater the strengthening or weakening of the bond (2). The food reward is often called a reinforcer. A reinforcer, or reinforcement, is defined as some consequence of behavior that increases the future probability of that behavior. When food delivery follows lever pressing in a hungry rat, lever pressing will increase in frequency. According to Thorndike, the observation can be explained by the strengthening of some connection between the environmental stimuli (e.g., sight of the instrumental chamber) and the effective behavior (e.g., lever press). The behavior is not directed at all towards the food, but is simply generated randomly until it is reinforced or selected by the food that follows it. The assumption behind the law of effect is that behavior is always caused by some stimulus. If a particular stimulus–response relationship is not present, then it can be acquired using the reinforcement mechanism. The animal generates behavioral variants and the consequence of “satisfaction” will select the appropriate

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variant – the correct sensorimotor transformation. Instrumental conditioning is therefore analogous to evolution, beginning with the generation of variants and ending with the selection of the most “adaptive” variant and its differential amplification relative to other variants. The law of effect also assumes that consequences select behavior, that reinforcement is the independent variable and behavior is the dependent variable. But an independent variable is under the experimenter’s control, and this is not true of reinforcement, which depends on behavior. That is to say, in instrumental conditioning reinforcement is a dependent variable. If the food delivery depends on lever pressing, there is a feedback function from the action to the reward. A feedback function describes changes in a specific input variable (food) as a function of some behavioral output (lever press). It is the most fundamental feature of instrumental conditioning (3). If all behaviors produced by the rat will produce food, then it will not need to learn anything. The need for learning arises because there is only one feedback function, and the rat must discover it, i.e., it must learn the contingency between a specific action and the reward. Now note the term “action” used here. Pressing a lever is not a simple act. The rat can use its paws, its teeth, or its head. But even if it appears to use a highly stereotyped movement to press the lever every time, the underlying variability is not often noticed, as the repetition of the same neural output is not sufficient to reproduce the same action (4). To describe the entire class of behaviors recorded as “lever pressing,” Skinner coined the term “operant”. But if the operant is literally any behavior that leads to reinforcement, then how can reinforcement select behaviors the animal has never produced before? If what is repeated is literally a set of neural outputs in response to a set of inputs, then the rat would never have succeeded in pressing the lever. What is repeated in instrumental conditioning is not the action, but the reward. The “operant” cannot be defined independently of the reward. Rather than the selection of behavior by reinforcement, the so-called reinforcement is selected by the behavior. If the rate of lever pressing is a monotonically increasing function of the rate of reinforcement, then the animal would keep pressing the lever forever, so long as there is reinforcement. This is obviously not the case. If we leave the food in the instrumental chamber, the animal will reduce lever pressing and eat the food instead. But it is not correct to say that free reinforcement simply reinforces the eating behavior instead of the lever pressing, because at some point the animal stops eating as well. The concept of reinforcement inevitably leads to conceptual confusion. It is simpler to assume that the animal stops eating after a while because it has a set point for how much food it wants to eat, i.e., food intake is a controlled variable, just like body temperature.

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Thus whatever is called reinforcement is really a controlled variable. Control is defined as the ability of a system to vary its outputs in order to keep the value of the input at some desired, internally specified value. When a controlled variable is disturbed for whatever reason, the behavior of the animal will resist the disturbance (5). Thus, when the value of some essential variable, say food intake, departs from the desired range, then some internal error signal is produced that results in action. The action on the environment will lead to food (or input) through some feedback function (5). Of course, on this account, instrumental reinforcement is not restricted to food rewards. The animal can learn to perform actions to warm or cool itself, or to escape from shock. Food reward is usually chosen for the sake of convenience. We can therefore describe instrumental conditioning as follows: a disturbance (deprivation) is introduced to an essential variable being controlled by the animal (food intake), and a specific feedback function is set up by the experimenter so that the animal must perform a specific action (lever press) to defend the value of the controlled variable. The animal learns the function that transforms the error signal into the output, which can be any output that will change the value of the controlled variable (5, 6). The operant, then, is the action that serves as the output variable in the feedback function. It can be the rate of behavior, the duration of behavior, even the variability of behavior (1, 7). If the reward is contingent upon any of these parameters, then the animal will modify the behavior accordingly (8, 9). Returning to the example of a rat pressing a lever, if the rate of pellet delivery is the controlled variable, then the lever pressing is generated by some error signal – the difference between the desired value of the “pellet” variable and the actual rate of pellet discovery. When the rat is pressing the lever for pellets, it is largely acting as a control system, using the rate of pressing to control for the rate of pellet delivery. 2.2. Pavlovian Conditioning vs. Instrumental Conditioning

Now there are many behaviors associated with feeding. For example, salivation can be elicited not only by the food itself but also by predictors of food. The process by which a previously “neutral” stimulus, such as the sound of a metronome, comes to elicit the salivation is known as Pavlovian or classical conditioning (10). Salivation in response to dry foods in the mouth is an unconditional response. Through Pavlovian conditioning, animals can acquire “conditional” or “conditioned” reflexes. The distinction between Pavlovian conditioning and instrumental conditioning has remained controversial (11). Initially, it was thought to be based on a difference in the effector systems used – instrumental conditioning involves skeletomotor systems, whereas Pavlovian conditioning involves the autonomic system (12). But we now know that these two types of conditioning

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can engage the same effectors. It is also thought that Pavlovian conditioning involves the formation of stimulus–outcome associations, whereas instrumental conditioning involves the formation of action–outcome associations (13). Traditional terms such as stimulus, response, and outcome describe elements that can be discerned in any organism–environment relationship (14), and associations can be formed between any two terms. But experimentally it is difficult to show which of these associations are responsible for generating the behavior, despite repeated attempts (15). Moreover, associations, which can only vary in a single dimension of strength, do not explain the observed behavior. Clearly, in instrumental conditioning there is a feedback function from the behavior to the food delivery, and in Pavlovian conditioning there is not, i.e. the behavior does not cause the occurrence of the food (3, 16). But feedback is still present in Pavlovian conditioning. In fact, feedback can be found even in what are commonly known as reflexes. In the case of salivation in response to dry foods in the mouth, the observation of a reliable relationship between stimulus and response masks a feedback function. Salivation moistens the food, making it easier to swallow and digest (17, 18). But this particular feedback function can only change the value of a particular variable, i.e., dryness of the food; it does not cause food to appear. Reflexive behaviors, then, are generated to overcome particular types of disturbances to a set of controlled variables. Such disturbances are universal features of the organism’s environment, and control systems, with outputs which reliably defend the controlled variable against these disturbances, have been developed over millions of years. Such feedback functions are relatively fixed in two ways. First, the behavioral output can only change the value of a small set of pre-specified variables controlled by the organism. Being disturbance-specific, they cannot change the values of arbitrary variables. For example, salivation changes how dry food is, and the pupillary light reflex changes how much light reaches the retina. Secondly, the polarity of the feedback function is fixed. That is, salivation reduces dryness of food, and pupillary constriction reduces light on the retina; such behaviors cannot achieve the opposite results. As there are hundreds of such control systems already in place at birth, no learning is required for them to function. However, they can often be modified by experience, through a process like Pavlovian conditioning. Instead of responding to the unconditional stimulus, the animal responds to its predictor, the conditional stimulus, e.g., anticipatory salivation to the sound of the metronome predicting meat powder in Pavlov’s experiments. For this reason, Pavlov called the basic mechanism of association formation in Pavlovian conditioning “stimulus substitution.” Later investigators, however, were puzzled by instances in which the

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conditioned responses to predictors and the actual unconditional responses were clearly different (19, 20). For example, in fear conditioning, a number of anticipatory fear responses are generated in response to predictors of aversive events. Common conditional responses to a predictor of a painful stimulus such as electric shock could include freezing and conditioned analgesia with the anticipatory release of opioids, whereas the unconditional response is jumping (21). Thus conditional responses and unconditional responses do not have to be similar to each other, as in the case of salivation. A useful distinction is made between the preparatory and consummatory responses, both of which can be acquired through Pavlovian conditioning (19). What they have in common is the variable they control. In other words, there can be multiple outputs that affect the same variable. For example, to control the “heat” variable, there is a withdrawal reflex when the hand touches something hot such as a fire. It is engaged following abrupt disturbances to the controlled variable. A more gradual encroachment of the fire or the presence of predictors of the fire would allow the action of additional control systems producing behaviors such as moving away from the fire instead of having to respond to a direct contact with it. Responses that are appropriate in controlling for the perception of predictors (e.g., sight of smoke) cannot be unconditional because it is impossible to know a priori what all the predictors of danger are; they can only be learned through Pavlovian conditioning. Whether the conditional responses are similar to the unconditional responses or not, they share the same controlled variable, allowing the organism to prepare for the predicted disturbance to the controlled variable in advance. If the unconditional stimulus is a predator, then by freezing the animal can avoid contact with the predator altogether. If the unconditional stimulus is a food, then by approaching, the animal will move closer to the food. However, the chief limitation of such responses is the lack of flexibility following a reversal of feedback polarity, because for these control systems, the polarity of the feedback function between the behavior and the controlled variable is fixed. Approach always moves one closer to rewards. Escape always moves one away from danger. When the polarity of feedback is reversed, an animal ­cannot adjust its behavior accordingly. The animal equipped with only Pavlovian control systems cannot learn to move backwards or stand still in order to obtain rewards (22). It cannot discover new feedback functions in the environment. Since in nature approaching signs that predict reward (sign tracking) rarely reduces the probability of obtaining reward, the Pavlovian conditional responses are adaptive under most conditions, allowing the animal to use existing control systems to respond to arbitrary predictors of disturbances. But these behavioral outputs

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cannot adequately deal with arbitrary feedback functions which have not been incorporated into the genome of the animal in the course of evolution. The animal capable of only Pavlovian conditioning cannot learn to use the same behavioral output to control different types of variables, or to use different types of behavioral outputs to control the same variable. It cannot learn arbitrary relationships between behavior and the controlled variable (23). That is to say, it cannot learn feedback functions. As the relationship between the behavior of organisms and its consequences in the world is not always stable, the richer the ecological niche the greater uncertainty there will be in feedback functions. A definite movement cannot have a definite effect on the variable in question, because there are additional variables between this movement and the input variable (19). For example, if one wants to get apples from a tree, there is no specific output that is uniquely appropriate for controlling this input variable. Whether pulling or pushing or shaking is the suitable action will depend on the physical constraints that are part of the environment. The lack of feedback functions with a fixed polarity necessitates the development of mechanisms for instrumental conditioning. To exert control over desired commodities such as food it is necessary to discover arbitrary feedback functions through trial and error. This Pavlovian conditioning cannot accomplish. Hence the need for instrumental conditioning. 2.3. Experimental Manipulations of Feedback Functions

In instrumental conditioning, the relationship between the acquired instrumental action and the controlled variable is much more arbitrary. Learning involves the acquisition of novel and arbitrary action–outcome contingencies. The greater the capacity for instrumental conditioning, the more arbitrary this relationship can be. The experimenter designs and manipulates the feedback function. In this respect, it is different from other experimental methods used to study behavior in the laboratory. The experimenter defines what it takes to activate the food dispenser, but otherwise the animal controls whatever it wants to control. Note that the experimenter does not manipulate input (stimuli) while measuring output (behavior). Rather the input or food reward is controlled by the experimental subject. The experimenter only controls the feedback function available to the organism. This is commonly done using different so-called schedules of reinforcement (see Sect. 4 below). In addition to the feedback function, the experimenter can also manipulate motivational variables. For example, the extent of food deprivation will determine both how much food the animal wants at any given moment and its motivation to press the lever. Manipulating the degree of deprivation is a manipulation of the general motivational state. In addition, the experimenter can also use more specific motivational manipulations such as sensory specific satiety, e.g., pre-feeding with the same food reward that is

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earned by lever pressing can reduce desire for that reward relative to desire for other food rewards. These manipulations are all designed to affect the set points for the various control systems within the organism, consequently changing the behavioral output (24).

3. The Role of the Dorsal Striatum in Instrumental Conditioning

The scope of instrumental conditioning, then, is extremely broad, encompassing all behaviors normally referred to as “voluntary.” Its neural substrates include virtually all regions of the brain, depending on the specific nature of the task. It is beyond the scope of the present chapter to review all that is known about the neural substrates of instrumental conditioning (9, 25–29). We will restrict our discussion to recent findings on the role of the dorsal striatum in instrumental conditioning. The striatum is the input nucleus of the basal ganglia, a set of subcortical nuclei in the cerebrum. It can be divided into at least three major regions based on anatomical connectivity. The ventral striatum receives projections from the limbic cortical areas including the basolateral amygdala and the orbitofrontal and medial frontal cortical areas. The dorsomedial or associative striatum receives projections from association cortical areas. The dorsolateral or sensorimotor striatum receives projections from primary sensorimotor cortical areas (30, 31). These overlapping divisions constitute the striatal components of cortico-basal ganglia networks which together form the cerebrum (brain) division of the central nervous system. Massive lesions of the striatum, or removal of the dopaminergic afferents, can abolish voluntary behavior, resulting in conditions like akinesia and abulia. Such lesions can interfere with feeding and effectively disrupt instrumental conditioning. There is yet no work on the striatum on the exploratory process, though massive lesions or dopamine depletion in the striatum is known to reduce exploratory behaviors. Work on the neural substrates of song learning in song birds suggests that the basal ganglia are critical for the trial and error phase critical to the acquisition of bird song (32, 33). Work from our own lab (Yin, unpublished observations) also suggests a key role of the dorsal striatum in the initial exploratory behavior in instrumental conditioning. The ventral striatum, which includes the nucleus accumbens core and shell, plays a critical role in the motivational aspects of instrumental conditioning (34, 35). Lesions of the ventral striatum do not affect acquisition of lever pressing, but generally reduce the rate of pressing, especially under partial reinforcement schedules in which many presses are required to earn a reward. Depletion of

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dopamine in the ventral striatum, for example, is known to reduce lever pressing under fixed ratio schedules, especially when the ratio requirement is very high (36). Rats lacking dopamine in the ventral striatum do not persist in pressing the lever for food, suggesting a reduction in motivation. The nucleus accumbens core and shell are also critical for a phenomenon known as Pavlovianinstrumental transfer, in which Pavlovian conditional stimuli can increase the rate of instrumental lever pressing. This effect appears to require afferents to the accumbens from limbic cortical regions such as the basolateral amygdala (37–39). These afferents appear to send reward-specific or more general motivational signals to the ventral striatum. 3.1. Dorsomedial Striatum

The dorsomedial striatum or associative striatum has been shown to be critical for instrumental conditioning, in particular the acquisition of the action–outcome contingency (38, 40). Excitotoxic lesion or inactivation of the dorsomedial striatum results in lever pressing that is impervious to changes in outcome value or action– outcome contingency. For example, whereas rats receiving sham lesion of the dorsomedial striatum reduced lever pressing for a reward that has been devalued by selective satiety, those with dorsomedial striatal lesions did not reduce lever pressing for the devalued reward. Likewise, the lesioned rats also failed to show sensitivity to degradation of the action–-outcome contingency (40, 41). Similar effects were also found following lesions to other components of the associative thalmocortico–basal ganglia circuit (mediodorsal thalamus and medial prefrontal cortex) (38). Lesions of the dorsomedial striatum can also reduce the selectivity of the Pavlovian-instrumental transfer effect, suggesting that this area is critical for using some representation of the future outcome to guide performance (42). Studies using monkeys as subjects have shown similar results. The primate counterpart of the dorsomedial striatum, the caudate nucleus (43, 44), is known to show changes in anticipatory activity associated with the reversal of action–outcome contingency (45). The animal must make a saccade to a particular stimulus position, and both positive and negative expectation of reward modulates neural activity of caudate neurons engaged by the saccade task. Caudate neurons can fire in anticipation of the visual target, and the anticipatory activity changes according to changes in the action–outcome contingency. Thus the available evidence clearly suggests that dorsomedial striatum is critical for using the representation of the reward to guide actions. This flexible “goal-directed” system is able to track rapid changes in the feedback function for the reward, also known as changes in the action–outcome contingency. The controlled variable is some representation of the specific outcome (e.g., rate of reward).

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3.2. Dorsolateral Striatum

Whereas the dorsomedial striatum is critical for the use of reward representations to guide action selection, the dorsolateral or sensorimotor striatum appears to be critical for the automatization and recombination of action elements (26, 28, 46, 47). With extensive instrumental training or under feedback functions with high uncertainty in the time of reward availability, lever-pressing performance can become more automatic and habitual. Rather than using the incentive value of the outcome to guide output, the habit system, of which the dorsolateral striatum is a critical hub, appears to be guided by local feedback generated either by preceding actions or by Pavlovian predictors such as cues that are contiguous with the reward delivery. It is concerned with controlling the rate and sequence of behavior, as well as the local predictors of the outcome. The dorsolateral striatum appears to be critical for monitoring action-induced feedback and the sequencing of actions. When the reward is contingent upon the completion of a simple sequence consisting of two lever presses, mice with lesions of the dorsolateral striatum are significantly impaired at acquisition (28). When the dorsolateral striatum is lesioned or inactivated, instrumental lever pressing becomes more sensitive to the devaluation of the reward or to changes in action–outcome contingency even under conditions that promote habit formation (48–50). Likewise, human imaging work has shown activation of the posterior putamen which appears to be the primate counterpart of the dorsolateral striatum during habitual behavior (51). With habit formation, there appears to be a shift in the neural network controlling instrumental performance from the associative cortico–basal ganglia network to the sensorimotor cortico–basal ganglia network.

4. Materials and Methods Below we will outline the major steps of instrumental conditioning experiments. Rather than attempting to be comprehensive, we will focus on the rationale for the common methods used. Detailed explanations of the various schedules of reinforcement and the behavioral patterns typically generated are available in standard references (1, 5, 52–54). Standard instrumental chambers for mice and rats are commercially available (e.g., Med Associates, Vermont, USA). The behavioral data are typically stored and analyzed on a computer. 4.1. Training Protocols 4.1.1. Food Deprivation

Food deprivation to roughly 85% of ad libitum body weight is often used in instrumental conditioning experiments. It should be noted that such food deprivation is more similar to the conditions in the natural environment, where animals do not have access to

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unlimited food at all times. It is desirable to perform deprivation gradually to reduce the body weight to the desired level. However, even one day of deprivation can produce sufficient motivation for the acquisition of lever pressing. The animals should be well handled and habituated to the instrumental chamber. 4.1.2. Magazine Training

Since during lever press training food is delivered in the food magazine, animals are usually given at least one session during which they are exposed to food in the magazine. This session allows the animal to become acquainted with the chamber, to discover the location of food delivery, and to overcome any neophobia that makes it reluctant to consume a novel food in a novel environment.

4.1.3. Lever Press Training

Lever press training begins with a continuous reinforcement schedule (or FR1), in which each press is followed by reward delivery. Rats and mice will start to press the lever regularly after a few hours of training on this schedule. If an animal does not press at all after the first session, often it is helpful to leave some powder of the food pellet on the lever. The session ends after a pre-determined time (e.g., 1  h) or after a specified number of rewards have been earned.

4.1.4. Schedule of Reinforcement

A variety of schedules have been used to study instrumental behavior (52, 53). There are two major classes of instrumental schedules: Ratio schedules and interval schedules (53). In ratio schedules, the feedback function is a linear multiplier of the number of lever presses. For example, with a ratio of 1, each press results in a reward, and a ratio of 10 means 10 presses results in a reward. If the ratio is always the same, the schedule is called fixed ratio (FR). The ratio can also be varied to create variable ratio schedules, so that the feedback function will change after each reward. In interval schedules, the first lever press is rewarded only after some prespecified time interval has elapsed. If the time interval is always the same, then the schedule is called fixed interval (FI), but if it varies, then the scheduled is called variable interval (VI).

4.1.5. Post-training Tests for the Controlled Variable

Two types of manipulations can be done to assess the control system responsible for the lever-pressing behavior. In the first, the value of the outcome, typically a food reward, is manipulated after training. This approach indirectly changes the set points for the general motivational state and the specific incentive value. In the second, the feedback function is manipulated, typically by degrading or reversing some action–outcome contingency that has already been acquired.

4.2. Value Manipulations

The value of the outcome can be reduced by a devaluation procedure. There are two types of devaluation treatments: induction of taste aversion to the food reward with lithium chloride or selective

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satiety. General satiety devaluation and specific satiety devaluation are very different manipulations. Even when an animal is sated after consuming unlimited home chow, it may still be motivated to press the lever for a novel reward. Taste aversion induction is more similar to specific satiety devaluation in that its effects are limited to a specific food reward. Both procedures are performed after the completion of lever press training. Lithium chloride injections are given after consumption of the reward that is earned during training. The consumption of the same reward is measured on subsequent days. If the animal continues to consume the reward, another injection is given. This is repeated until a strong taste aversion to the food is developed, so that none or very little of it is consumed. Normally at least three injections are needed to produce sufficient aversion. Because the specific satiety devaluation procedure is reversible, it is more commonly used. In this test, the animal has free access to the reward for at least 1 h or until substantial amount (at least the amount earned in a whole training session) is consumed. Immediately following the free exposure, it is given a short probe test lasting 5 min. During this test, the levers are extended as during a normal training session, but no food is delivered. The purpose of such a probe test is to assess the memory of the action–outcome contingency. If the outcome value plays any role in the lever pressing, then the animal is expected to reduce the rate of pressing when the outcome has been devalued. But if performance is not sensitive to such a manipulation, then it can be said to be independent of outcome value. 4.3. Contingency Manipulations

What are traditionally called contingency manipulations include instrumental contingency degradation and omission. Instrumental performance is normally highly sensitive to such manipulations, but such sensitivity is significantly reduced following habit formation. Instrumental contingency degradation can be done in several ways. The easiest way is to introduce free reward independent of pressing. The rate of reward can be based on the average reward rate during the instrumental training session. Thus, suppose the animal earns 50 pellets per hour under a random ratio 20 schedule. Omission is a more radical change in the feedback function, effectively reversing the action–outcome contingency. Thus the reward previously earned by lever pressing is delivered non-­ contingently, and each press delays the delivery of the reward by some pre-specified time interval (50, 55, 56). For example, the reward is delivered once every 20  s, but any press delays the next reward delivery by 20 s. Omission normally results in the fastest reduction in the rate of lever pressing. But habit formation can reduce sensitivity to the imposition of the omission contingency.

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5. Summary The use of instrumental methods has considerably advanced our knowledge of the role of the dorsal striatum and other brain regions (8, 57–62), as well as the cellular and molecular mechanisms of behavior in mice (8, 28, 57, 63, 64). Studies using these methods have implicated the dorsal striatum and related cortical and thalamic structures in the acquisition and expression of instrumental conditioning. In particular, they have suggested that lever pressing can be mediated by multiple control systems sharing common effectors. The nature of the control can be revealed by specific manipulations of the outcome value and the action–outcome contingency. Damage to one control system can result in behavior that shows the characteristic signature of the alternative control system. Despite significant progress in this field, much remains unknown. In particular, it is not clear how these different types of control are implemented, e.g., how the brain represents the expected outcome or computes the difference between the desired value of a controlled variable and its current value. To answer these important questions, much more systematic investigations of the neural activity from different brain regions including the dorsal striatum during instrumental conditioning will be needed. The ability to use genetic tools to manipulate specific molecular mechanisms in the relevant neural circuits also appears to be a promising approach when combined with analytical behavioral assays. In short, the results that have been obtained in just a decade using instrumental conditioning demonstrate the tremendous power of this experimental tool. Rather than generating specific motor patterns, the cerebrum, which consists of cortico–basal ganglia networks, is largely responsible for the variation, selection, and goal-directed generation of specific action patterns. Instrumental conditioning may in fact be the most important higher function of the cerebral hemispheres. References 1. Skinner B (1938) The Behavior of Organisms. Appleton-Century-Crofts, New York 2. Thorndike EL (1911) Animal Intelligence: Experimental Studies. Macmillan, New York 3. Ashby WR (1960) Design for a Brain. Chapman & Hall 4. Bernstein N (1967) The Coordination and Regulation of Movements. Pergamon Press, Oxford 5. Staddon JER (1983) Adaptive Behavior and Learning. Cambridge University Press, Cambridge

6. Powers WT (1973) Feedback: beyond behaviorism. Science 179: 351–356 7. Neuringer A, Deiss C, Olson G (2000) Reinforced variability and operant learning. J Exp Psychol Anim Behav Process 26: 98–111 8. Yu C, Gupta J, Yin HH (2010) The role of mediodorsal thalamus in temporal differentiation of reward-guided actions. Front Integr Neurosci 4, pii: 14 9. Yin HH (2009) The role of the murine motor cortex in action duration and order. Front Integr Neurosci 3: 23

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10. Pavlov I (1927) Conditioned Reflexes. Oxford University Press, Oxford 11. Ellison GD, Konorski J (1964) Separation of the Salivary and Motor Responses in Instrumental Conditioning. Science 146: 1071–1072 12. Rescorla RA, Solomon RL (1967) Two-process learning theory: relationships between Pavlovian conditioning and instrumental learning. Psychol Rev 74: 151–182 13. Dickinson A (1989) Expectancy theory in animal conditioning. In: Klein SB, R. R. Mowrer (ed) Contemporary Learning Theories. Lawrence Erlbaum Associates, Hillsdale, NJ, pp 279–308 14. Bolles R (1972) Reinforcement, expectancy, and learning. Psychological Review 79: 394–409 15. Dickinson A (1997) Bolles’s psychological syllogism. In: Bouton ME, M. S. Fanselow, (ed) Learning, Motivation, and Cognition. APA, Washington D. C. 16. Kimble GA (1961) Hilgard and Marquis’ Conditioning and Learning (2nd edition). Appleton-Century-Crofts, New York 17. Sherrington CS (1906) The Integrative Action of the Nervous System. Yale University Press, New Haven 18. Hull C (1943) Principles of Behavior. AppletonCentury-Crofts, New York 19. Konorski J (1967) Integrative Activity of the Brain. University of Chicago Press, Chicago 20. Domjan M (2005) Pavlovian conditioning: a functional perspective. Annu Rev Psychol 56: 179–206 21. Fanselow MS (1998) Pavlovian conditioning, negative feedback, and blocking: mechanisms that regulate association formation. Neuron 20: 625–627 22. Hershberger WA (1986) An approach through the looking glass. Animal Learning & Behavior 14: 443–451 23. Timberlake W (1993) Behavior systems and reinforcement: an integrative approach. J Exp Anal Behav 60: 105–128 24. Dickinson A (1994) Instrumental Conditioning. In: Mackintosh NJ (ed) Animal Learning and Cognition. Academic, Orlando, pp 45–79 25. Balleine BW, Liljeholm M, Ostlund SB (2009) The integrative function of the basal ganglia in instrumental conditioning. Behav Brain Res 199: 43–52 26. Yin HH, Ostlund SB, Balleine BW (2008) Reward-guided learning beyond dopamine in the nucleus accumbens: the integrative functions of cortico-basal ganglia networks. Eur J Neurosci 28: 1437–1448

27. Yin HH, Knowlton BJ (2006) The role of the basal ganglia in habit formation. Nat Rev Neurosci 7: 464–476 28. Yin HH (2010) The sensorimotor striatum is necessary for serial order learning. J Neurosci 30: 14719–14723 29. Balleine BW, Dickinson A (1998) Goal-directed instrumental action: contingency and incentive learning and their cortical substrates. Neuropharmacology 37: 407–419. 30. Joel D, 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 31. McGeorge AJ, Faull RL (1989) The organization of the projection from the cerebral cortex to the striatum in the rat. Neuroscience 29: 503–537 32. Mooney R (2009) Neural mechanisms for learned birdsong. Learn Mem 16: 655–669 33. Brainard MS, Doupe AJ (2000) Interruption of a basal ganglia-forebrain circuit prevents plasticity of learned vocalizations. Nature 404: 762–766. 34. Cousins MS, Atherton A, Turner L, Salamone JD (1996) Nucleus accumbens dopamine depletions alter relative response allocation in a T-maze cost/benefit task. Behav Brain Res 74: 189–197 35. Corbit LH, Muir JL, Balleine BW (2001) The role of the nucleus accumbens in instrumental conditioning: Evidence of a functional dissociation between accumbens core and shell. J Neurosci 21: 3251–3260 36. Salamone JD, Correa M, Farrar AM, Nunes EJ, Pardo M (2009) Dopamine, behavioral economics, and effort. Front Behav Neurosci 3: 13 37. Corbit LH, Janak PH, Balleine BW (2007) General and outcome-specific forms of Pavlovian-instrumental transfer: the effect of shifts in motivational state and inactivation of the ventral tegmental area. Eur J Neurosci 26: 3141–3149 38. Balleine BW, O’Doherty JP (2010) Human and rodent homologies in action control: corticostriatal determinants of goal-directed and habitual action. Neuropsychopharmacology 35: 48–69 39. Shiflett MW, Balleine BW (2010) At the limbic­motor interface: disconnection of basolateral amygdala from nucleus accumbens core and shell reveals dissociable components of incentive motivation. Eur J Neurosci 32: 1735–1743 40. Yin HH, Ostlund SB, Knowlton BJ, Balleine BW (2005) The role of the dorsomedial striatum in instrumental conditioning. Eur J Neurosci 22: 513–523

4  The Role of the Dorsal Striatum in Instrumental Conditioning 41. Corbit LH, Janak PH (2010) Posterior dorsomedial striatum is critical for both selective instrumental and Pavlovian reward learning. Eur J Neurosci 31: 1312–1321 42. Corbit LH, Janak PH (2007) Inactivation of the lateral but not medial dorsal striatum eliminates the excitatory impact of Pavlovian stimuli on instrumental responding. J Neurosci 27: 13977–13981 43. Hikosaka O, Takikawa Y, Kawagoe R (2000) Role of the basal ganglia in the control of purposive saccadic eye movements. Physiol Rev 80: 953–978 44. Kawagoe R, Takikawa Y, Hikosaka O (1998) Expectation of reward modulates cognitive signals in the basal ganglia. Nat Neurosci 1: 411–416 45. Watanabe K, Hikosaka O (2005) Immediate changes in anticipatory activity of caudate neurons associated with reversal of position-reward contingency. J Neurophysiol 94: 1879–1887 46. Yin HH, Mulcare SP, Hilario MR, Clouse E, Holloway T, Davis MI, Hansson AC, Lovinger DM, Costa RM (2009) Dynamic reorganization of striatal circuits during the acquisition and consolidation of a skill. Nat Neurosci 12: 333–341 47. Miyachi S, Hikosaka O, Lu X (2002) Differential activation of monkey striatal neurons in the early and late stages of procedural learning. Exp Brain Res 146: 122–126 48. Faure A, Haberland U, Conde F, El Massioui N (2005) Lesion to the nigrostriatal dopamine system disrupts stimulus-response habit formation. J Neurosci 25: 2771–2780 49. Yin HH, Knowlton BJ, Balleine BW (2004) Lesions of dorsolateral striatum preserve outcome expectancy but disrupt habit formation in instrumental learning. Eur J Neurosci 19: 181–189 50. Yin HH, Knowlton BJ, Balleine BW (2006) Inactivation of dorsolateral striatum enhances sensitivity to changes in the action-outcome contingency in instrumental conditioning. Behav Brain Res 166: 189–196 51. Tricomi E, Balleine BW, O’Doherty JP (2009) A specific role for posterior dorsolateral striatum in human habit learning. Eur J Neurosci 29: 2225–2232

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52. Staddon JER (2001) Adaptive Dynamics: the Theoretical Analysis of Behavior. MIT Press, Cambridge 53. Ferster C, Skinner BF (1957) Schedules of Reinforcement. Appleton Century, New York 54. Catania AC, Reynolds GS (1968) A quantitative analysis of the responding maintained by interval schedules of reinforcement. J Exp Anal Behav 11: Suppl:327–383 55. Schwartz B, Gamzu E (1977) Pavlovian control of operant behavior. In: Honig W, Staddon JER (eds) Handbook of Operant Behavior. Prentice Hall, New Jersey, pp 53–97 56. Brown PL, Jenkins HM (1968) Auto-shaping the pigeon’s key peck. J Exp Anal Behav 11: 1–8 57. Yin HH, Zhuang X, Balleine BW (2006) Instrumental learning in hyperdopaminergic mice. Neurobiol Learn Mem 85: 283–288 58. Ostlund SB, Balleine BW (2005) Lesions of medial prefrontal cortex disrupt the acquisition but not the expression of goal-directed learning. J Neurosci 25: 7763–7770 59. Ostlund SB, Balleine BW (2007) Orbitofrontal cortex mediates outcome encoding in Pavlovian but not instrumental conditioning. J Neurosci 27: 4819–4825 60. Ostlund SB, Balleine BW (2008) Differential involvement of the basolateral amygdala and mediodorsal thalamus in instrumental action selection. J Neurosci 28: 4398–4405 61. Corbit LH, Balleine BW (2003) The role of prelimbic cortex in instrumental conditioning. Behav Brain Res 146: 145–157 62. Corbit LH, Muir JL, Balleine BW (2003) Lesions of mediodorsal thalamus and anterior thalamic nuclei produce dissociable effects on instrumental conditioning in rats. Eur J Neurosci 18: 1286–1294 63. Hilario MRF, Clouse E, Yin HH, Costa RM (2007) Endocannabinoid signaling is critical for habit formation. Front Int Neurosci 1:6 64. Yu C, Gupta J, Chen JF, Yin HH (2009) Genetic deletion of A2A adenosine receptors in the striatum selectively impairs habit formation. J Neurosci 29: 15100–15103

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Chapter 5 3-Nitropropionic Acid and Other Metabolic Toxin Lesions of the Striatum Cesar V. Borlongan and Paul R. Sanberg Abstract Huntington’s disease (HD) is a progressive neurodegenerative disorder associated with severe degeneration of basal ganglia neurons. Histologically, the striatum displays neurodegeneration of the intrinsic neurons of the striatum, whereas the behavioral symptoms are primarily characterized by progressive dementia and involuntary abnormal choreiform movements. This pathophysiology of HD has been linked to a genetic mutation in the huntingtin gene, which laid the foundation for generating transgenic and knock-in rodents that display a similar genetic defect. Despite this scientific advance in our knowledge of the disease pathophysiology over the last two decades, there is no cure for HD. Conventional animal models, via mitochondrial impairment and excitotoxicity-induced neurodegeneration, which have established cell death pathways in HD remain as mainstream platform for studying the disease. The focus of this book chapter is on the mitochondrial inhibitor 3-nitropropionic acid (3-NP). We previously demonstrated that systemic administration of 3-NP leads to a progressive locomotor deterioration accompanied by a very selective striatal degeneration resembling that of HD. Subsequent studies from our laboratory further showed that manipulating the time course of 3-NP injections leads to sustained hyperactivity (early HD) or hypoactivity (late HD). This mitochondrial impairment model differs mechanistically from excitotoxic lesions in that 3-NP irreversibly inhibits the mitochondrial citric acid cycle and leads to depressed ATP levels and elevated lactate concentrations. Neurochemical assays lend support to impaired oxidative energy metabolism as key neurodegenerative process in this 3-NP model. Because of the mechanistic and pathologic similarities between 3-NP lesions and HD, 3-NP is considered a suitable HD model. We review here the rodent and non-human primate 3-NP models and other mitochondrial inhibitors similarly employed to lesion the striatum. The goal is to provide a scientific rationale for the continued use of these mitochondrial impairment models which should complement the genetic models in further understanding the disease pathology and to serve as tools for evaluating experimental therapeutics for HD. Key words: Huntington’s disease, Mitochondria, Energy metabolism, Basal ganglia, Neurode­ generation, Experimental therapeutics, Animal model

Emma L. Lane and Stephen B. Dunnett (eds.), Animal Models of Movement Disorders: Volume II, Neuromethods, vol. 62, DOI 10.1007/978-1-61779-301-1_5, © Springer Science+Business Media, LLC 2011

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1. Introduction Huntington’s disease (HD) is characterized by a progressive neurodegeneration of the basal ganglia neurons, especially the intrinsic neurons of the striatum. The hallmark behavioral symptoms of HD consist of progressive dementia and involuntary abnormal choreiform movements (1, 2). Significant scientific milestones have been achieved since the publication of the seminal paper by George Huntington in 1872, foremost is the discovery of the defective HD gene and its localization to chromosome 4 (3, 4). The advent of HD transgenic mice harboring the aberrant huntingtin gene suggested the possibility of gene therapy for HD, but to date no such therapy for HD patients is available. Animal models which replicate the widely accepted cell death pathways of HD, namely excitotoxicity and mitochondrial impairment, may still offer an alternative approach for elucidating disease pathology and for optimizing of current treatments and/or development of new therapies for HD. In order to appreciate the mitochondrial impairment model, we provide a brief overview of the excitotoxin lesion models of HD.

2. Excitement in Excitotoxic HD Models Revisited

Traditionally, the bulk of investigations into the mechanisms of neuronal death in HD and other neurodegenerative diseases have focused on excitatory amino acids (EAAs, e.g., glutamate) as the primary cause of neuronal cell death (5, 6). The major precipitating factor implicated in glutamate’s neurotoxicity is the diminished mitochondrial energy production that could compromise cellular vulnerability (7, 8). Such alterations in oxidative phosphorylation may trigger a cascade of cell death events namely (a) ion-transporting enzymes being compromised, (b) neuronal depolarization, (c) alleviation of the Mg 2+ block of the NMDA receptor channel, and (d) a continual ion influx leading to persistent receptor activation and increased glutamatergic stimulation. Ultimately, the abnormal glutamate excitotoxicity predisposes neuronal degeneration (9). This excitotoxicity-mediated cell death has been well documented in in vitro studies using cultured neurons exposed to experimental oxidative stress paradigms, including increased toxicity to glutamate through glucose deprivation, disruption of glycolysis and blockade of the electron transport chain (10, 11). In parallel to these cell culture studies, Koroshetz et al. (12) provided the first in vivo evidence of impaired oxidative energy metabolism in HD by observing increased concentrations of lactate in the cerebral cortex as detected by nuclear magnetic resonance spectroscopy.

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That a dysfunctional oxidative energy metabolism is inherent in HD is further supported by biochemical analyses of platelets taken from HD patients demonstrating significant reductions in the activity of mitochondrial NADH:ubiquinone oxidoreductase (complex I) compared with age-matched controls (9). Interestingly, patients who were asymptomatic, but at risk for HD, displayed normal complex I activity (9). Characterization of neurochemical and histological alterations in the caudate-putamen of HD patients revealed the following pathological features (a) neurochemically, decreased levels of GABA, substance P, enkephalin, and choline acetyltransferase, and increased levels of somatostatin, neuropeptide Y, neurotensin, serotonin, and preserved dopamine, and; (b) histologically, sparing of NADPH-diaphorase neurons, parvalbumin neurons, acetylcholinesterase (large) neurons, and marked reductions of enkephalin and substance P neurons (9). Many of these histopathological characteristics of HD are observed following intrastriatal injections of excitotoxins, such as kainic acid (KA), ibotenic acid (IA), or quinolinic acid (QA), in both rodents and primates (13). For example, the degeneration of intrinsic striatal neurons along with irreversible reduction of the synthesizing enzymes glutamic acid decarboxylase (GAD) and choline acetyl transferase (ChAT) accompany KA and IA injections (14, 15). However, KA and IA lesions neither fully mimic the histopathology of HD nor are they present endogenously in the brain. On the other hand, QA injections into the rat striatum resulted in depletions of GABA and substance P levels with neuropeptide Y, somatostatin and NADPH-diaphorase levels being spared, thereby producing a closer resemblance to HD histopathology (16). These excitotoxin rodent models recapitulate some of the behavioral symptoms of HD, such as locomotor hyperactivity and poor performance in mnemonic and cognitive tests, but fail to include dyskinesias or chorea-like movements (17). Although the HD behavioral manifestations in rodent excitotoxin models may be limited, the primate excitotoxin models appear to exhibit a better repertoire of the behavioral sequelae of the disease (14).

3. 3-NP: A Mighty Mitochondrial Impairment Model

The routine delivery route of excitotoxins to produce striatal lesion is to directly inject the drug into the striatum. A systemic approach for producing striatal lesion was made possible by the discovery of the mitochondrial inhibitor 3-nitropropionic acid (3-NP), which has been shown to afford a selective striatal neurodegeneration in rodents provided that it is given in sufficiently low dose and over a period of weeks (16, 18–20). With fairly high doses of systemic 3-NP, extrastriatal cell death is observed in regions including the

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hippocampus and thalamus, as well as other brain regions (21). The selective effects of 3-NP to the striatum have been postulated to be due to this brain region displaying higher metabolic rates and being an NMDA receptor-enriched area. In addition, the selective striatal neurotoxicity of 3-NP has been ascribed to dysfunction of the blood–brain barrier and subsequent intracerebral hemorrhage (19, 20), in that a disturbance of the blood–brain barrier initiates deposits of serum/immune complement factors localizing around the vessel of the lateral striatum artery. Because the lateral striatal artery is a perforating artery, this vessel is often perturbed by the intracerebral artery during turbulent blood flow induced by the extravasation of serum proteins. 3.1. Striatal 3-NP Lesions

The resulting striatal lesions induced by 3-NP can be categorized into three distinct types based on histological appearance (22), namely (a) “Type I lesions” are described as small lesions with patchy neuronal loss in islands of the dorsal striatum with sparing of NADPH-diaphorase neurons. Slight axonal swelling is seen with AChE staining. (b) “Type II lesions” consist of larger scale neuronal loss of dorsal striatal cells than type I, with diaphorase neuronal sparing and shrunken islands of cells among packed traversing fiber bundles of the internal capsule similar to that seen in HD striatal pathology. (c) “Type III lesions” are characterized by neuronal loss throughout the dorsal striatum, with minimal involvement of the ventral striatum. Within the dorsal striatum, a distinct medial to lateral gradient of increased severity is also detected (23, 24). In addition, tyrosine hydoxylase fiber sparing accompanies types I and II and around the lesion perimeters in type III lesions (22). Neurochemical studies (16) reveal dose-dependent reductions in markers for both striatal intrinsic neurons such as GABA, substance P, somatostatin, and neuropeptide Y, as well as a reduction in markers for striatal afferents such as dopamine and its metabolites following intrastriatal or systemic administration of 3-NP. However, in contrast to intrastriatal injections, systemic administration of 3-NP displayed a sparing of dopamine concentrations with increased concentrations of dopamine 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) (16). The age vulnerability to 3-NP-induced pathology in animals is in agreement with the clinical adult age of onset of HD. Indeed, direct correlation exists between animal age and neuronal death (16, 22). In animals less than 6 weeks old, systemic 3-NP injection had no adverse effects while 25% of animals 7–14 weeks old demonstrated striatal damage and 80% of animals older than 16 weeks old showed striatal lesions or death (16, 22). Intrastriatal 3-NP injections also revealed a correlation between age and 3-NP-induced striatal damage (18). In support of this age-related effect of 3-NP, we noted a small but significant decrease of SCH23390 specific

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binding to D-1 dopamine binding receptors for 10-week-old rats injected with 3-NP (injected once every 4 days for 28 days) compared with 6-week-old 3-NP-treated rats or age-matched controls (24). These laboratory observations parallel the radioligand clinical studies indicating that the number of striatal dopaminergic receptors are actually reduced in HD (25). Altogether these findings highlight the clinical relevance of the age-dependent susceptibility of rodents to 3-NP-induced metabolic compromise. However, adult humans who accidentally ingested mildewed sugar cane contaminated with the 3-NP experienced gastrointestinal symptoms but rarely disorders of the central nervous system (17). In contrast, children who had eaten 3-NP-laced sugar cane experienced signs of severe striatal encephalopathy (17). Reconciling these discrepant results will require testing of varying 3-NP dosages in older rat subjects to further reveal in animals the age-related histopathological and behavioral deficits displayed by humans who ingested 3-NP. Although both excitotoxin and mitochondrial inhibitor models lead to striatal lesion (16, 22, 24), 3-NP mechanistically differs from excitotoxic lesions in that 3-NP irreversibly inhibits the mitochondrial citric acid cycle and leads to depressed ATP levels and elevated lactate concentrations (16, 22, 26). Because lowered glutamate levels and impaired oxidative energy metabolism precede neurodegeneration, including the cell death pattern seen in HD (21, 27), the mechanism of 3-NP-induced striatal atrophy may more closely resemble the pathological processes involved in HD than those produced by excitotoxins. 3.2. Behavioural Consequences of 3-NP Lesions

In agreement with solid clinical relevance of the neurodegeneration associated with 3-NP, the behavioral symptoms produced by this mitochondrial toxin are also reminiscent of HD, which can be categorized into three stages, namely (a) stage I, somnolence; (b) stage II, uncoordinated gait with stereotypical paddling and rolling movements; and (c) stage III, ventral and lateral recumbancy (23, 24). Single or repeated systemic 3-NP administration in mice resulted in lesions of the basal ganglia with an initial decrease in motor activity followed by occasional episodes of hyperactivity mad/or abnormal movements (e.g., tremors, head bobbing, head tilt, circling, tail rigidity and elevation) (17). In our studies with 3-NP (using the regimen of one injection every 4 days for 28 days) (5, 23), we observed that young (6- and 10-week-old) and mature (14- and 28-week-old) rats systemically injected with 3-NP displayed bradykinesia, and considerable paresis and paralysis, respectively. In concert with the observed age-dependent histopathological alterations, an age-dependent severity of behavioral deterioration also occurs in 3-NP-treated rodents, resembling that of HD. Both systemic and intrastriatal 3-NP treatments in rodents exhibited significant hypoactivity, with the latter drug regimen producing more profound hypoactivity (23, 24). This 3-NP-induced hypoactivity

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differs from the hyperactivity (considered to be a correlate of chorea) produced by excitotoxins (13). Our notion is that the hypoactivity, characterized by considerable stiffness and akinesia during periods of arousal may reflect certain behavioral aspects of both juvenile onset (Westphal variant) and advanced HD (23, 24, 28). The clinical progression of the disease entails deterioration of mental function over time (much shorter and faster rate in juvenile onset HD cases), in tandem with a decline in motor function from a choreic dyskinesia to a more disabling akinetic and Parkinsonian-like syndrome (2). With this in mind, the behavioral pathology produced by the excitotoxins seems to correspond to the early hyperkinetic stages of HD, while that induced by 3-NP appears to capture the later hypokinetic stages and, possibly, the juvenile onset (Westphal variant) of HD (29). The loss of overlying cortical and other extrastriatal neurons may explain the observed rigidity and dystonia with high dose systemic 3-NP (17, 21). However, the hypoactivity in younger animals lacks a corresponding histopathological manifestation in that no detectable gross striatal lesions accompany 3-NP treatment in 3- to 6-week-old animals (9, 26). It is possible that very mild neuronal, physiological, and neurochemical changes may proceed undetected at early stages of the disease coupled with the less vulnerable CNS at younger ages, but may still mediate the observed behavioral changes (23, 28). Notwithstanding these limitations, our results demonstrate that both young and mature animals display akinetic behavior after long-term and low-dose systemic administration of 3-NP with undetectable lesions and localized striatal lesions in young and old animals, respectively. Another advantage of 3-NP over the excitotoxin models is the demonstration of chorea in animals. Abnormal choreiform movements were observed in 3-NP-injected monkeys challenged with apomorphine (30). With chorea not observed in the rodent 3-NP paradigm, the discrepancy with the monkey data might simply reflect the absence of a convincing choreiform movement disorder in the rat, suggesting that chorea may be a higher primate’s feature. Recognizing that HD not only presents with motor but also learning and memory impairments, we explored the effects of 3-NP on a simple cognitive task (passive avoidance behavior) in rats (24). Using a step-down apparatus, systemically 3-NP injected rats did not differ significantly from vehicle-injected rats in acquiring the task (avoiding the electric shock when stepping down the platform), but were significantly deficient in retaining the task 24 h later. This impairment in memory retention was more pronounced in intrastiatally 3-NP-injected animals. A dose-dependent retention deficit was also noted, with animals receiving the largest dose (750 nmol) being the most cognitively impaired (31). These results show that in addition to motor abnormalities, 3-NP alters cognitive function in rodents, which may be the correlate of dementia seen in chronic stage of HD.

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The progressive nature of HD is another elusive clinical feature of the disease that remained a challenge until the introduction of 3-NP. As noted above, the deterioration of cognitive functions in HD (shorter period and more rapid rate in juvenile onset HD cases) is paralleled by the progression of motor effects from a choreic dyskinesia to a more disabling akinetic and Parkinsonian-like syndrome (29). Excitotoxic animal models have not been successful in replicating this progressive behavioral pathology of HD. Using our original systemic dosing regimen of 3-NP, we demonstrated the progressive locomotor alteration in rats (28). We recorded the locomotor activity over the course of 3-NP administration. Initially, systemically 3-NP-treated animals exhibited significant hyperactivity during the first two injections, reaching a plateau after the third injection (day 12), then showing hypoactivity from the fourth injection (day 16) onwards. Animals that received only two injections of 3-NP then thereafter injected with saline showed a significant hyperactivity that persisted for the duration of the experiment (28 days). This observation of progressive behavioral pathology is the first report of a selective striatal neurotoxic animal model that parallels the movement abnormality in HD. In addition, the adjunct co-treatment of dopamine agonist (i.e., methamphetamine), with chronic 3-NP injections led to hyperactivity at early 3-NP post-injection tests, but not at later post-lesion tests (26, 30). Moreover, the onset of hypokinesia coincides with the appearance of caudate nucleus lesions (32). Our histologic results also demonstrate that smaller lesions are visible at the onset of hypoactivity, but at the end of the long-term course of 3-NP administration, larger lesions were noted (28). However, the size of 3-NP striatal lesions are very unique considering that we used the systemic route of administration. Although we noted no visible striatal degeneration at periods of 3-NP-induced hyperactivity, this finding is not surprising since HD-related behavioral deficits can be observed with no apparent neuropathology. Indeed, early chorea in HD can occur with only very small lesions in the caudate-putamen (33). Our histological results also revealed that grossly visible striatal lesions correspond to the onset of hypoactivity. The small number of animals we studied for histology during the early period of 3-NP injections does not allow us to state conclusively that no neuropathological defects accompany the observed hyperactivity. The progressive locomotor deficits using 3-NP need to be extended to similarly reveal the progressive cognitive decline and striatal degeneration. First, while a cognitive dysfunction (passive avoidance) after a 28-day period of 3-NP administration has been reported, the onset of such a deficit remains unknown (28). Second, based on the overwhelming literature supporting the postulation that the pathology of HD and other neurodegenerative disorders involves an impairment in mitochondrial energy production which may lead secondarily to slow excitotoxic neuronal death (7, 16),

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a  similar progressive striatal degeneration likely accompanies the observed 3-NP-induced progressive behavioral pathology. These ongoing CNS alterations, to some extent, have been reflected in our observed progressive locomotor changes and worsening of striatal lesions from the early phases to latter hypoactive stages of 3-NP (28). Lastly, we emphasize that a major utility of this model is that it allows flexibility to investigate HD at different stages. An equally important scientific advance with the progressive behavioral pathology of 3-NP model is that experimental treatments can be evaluated at various times of disease progression. Along this line, our long-standing interest in stem cell therapy can be assessed at early HD when hyperactivity, mild cognitive dysfunction and minor, if any, striatal degeneration has started, or at advanced HD when hypoactivity, severe cognitive dysfunction, and more complete striatal damage have occurred. 3.3. Experimental Therapeutics in 3-NP Models

Based on the mitochondrial pathway of cell death mediating 3-NP, logical development of therapeutics primarily target protection of the mitochondria. Experimental compounds directed at maintenance of mitochondrial integrity include FK-506 (34, 35), cyclosporine A (36, 37), cystamine and cysteamine (38), and induction of cyclic AMP response element-binding protein or CREB (39). Still other treatments beyond mitochondrial protection have been assessed to combat 3-NP neurotoxicity including erythropoietin (40), NF-E2 related factor 2 activators (41, 42), cyclin-dependent kinase inhibitors (43), heat shock protein inducers (44), growth factors (e.g., BDNF (45), G-CSF (46), NGF (47), GDNF (48), VEGF (49)) coenzyme Q10 and/or creatine (50, 51) cannabinoids (52–54), NMDA receptor antagonist MK-801 (55, 56), anti-oxidants (57, 58), estrogen (59), melatonin (60), calpain inhibitors (61), adenosine protectors (62), and naturally occurring bile acid (63).

3.4. Cell Transplantation in 3-NP Models

A novel set of cell-based therapeutics has been explored in 3-NP models of HD. Our group was the first to demonstrate the potential of implanting allogeneic rat fetal cell transplants in the 3-NP-induced hypoactive model of HD to show whether fetal tissue transplantation can ameliorate behavioral deficits associated with a more advanced stage of HD (64). Following confirmation of significant hypoactivity, 3-NP-treated rats received bilateral intrastriatal solid grafts of fetal striatal (lateral ganglionic eminence, LGE) tissues from embryonic day 14 rat fetuses with each animal transplanted with approximately one third of each LGE into each lesioned host striatum. After a 3-month graft maturation period, animals that received fetal LGE grafts exhibited a significant increase in locomotor activity compared

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to their post-3-NP injection activity or to those 3-NP-treated rats that received vehicle alone. The observed functional recovery was accompanied by detection of surviving striatal grafts in those 3-NP-treated animals (64). A subsequent non-human primate study further provided evidence of efficacy of cell therapy in 3-NP model of HD (65). Monkeys that received 3-NP and received intrastriatal allografts displayed robust recovery in a frontal-type cognitive task 2–5 months following transplantation. In addition, reduced dystonia was also observed in the transplanted 3-NP-treated animals (65). Altogether, these results demonstrate that transplantation of fetal striatal cells stand as an efficacious treatment for HD. The mechanism of therapeutic benefits exerted by striatal grafts in 3-NP models may be due to prevention of the loss of striatal neurons and the cortical upregulation of BDNF (66). This protection of host neurons by striatal grafts is further elucidated in a proactive transplant paradigm whereby adult rats that received intrastriatal implantation of human neural stem cells (hNSCs) 1  week before 3-NP treatments displayed significantly improved motor performance and reduced loss of striatal neurons compared with control sham injections (67). Such pro-active neuroprotective effect by NSC grafts were also shown to confer an anti-oxidative effect by buffering and maintenance of homeostasis of the host tissue thereby reducing subsequent toxicity produced by oxidative stress (68, 69). Moreover, NSC derived from the MHP36 cell line also showed protective, rather than regenerative effects (70). Rats that received NSC grafts exhibited less impairments in the beam walk test and showed partial recovery of learning in the water maze, but did not display improvement in the staircase test. Serial magnetic resonance images (MRI) revealed that NSC grafts did not attenuate lesion volume but that they arrested the further deterioration of striatal damage over the course of the study. These data indicate that the preservation host tissue, instead of reducing lesion size, mediated maintenance of specific behavioral function (70). Using a modified MRI, focusing on blood oxygen level-dependent pharmacological parameters, revealed that the dopamine receptor 2-agonist bromocriptine acts detected attenuation of response of striatal projection cells following 3-NPA administration which was reversed by NSC grafts (71). Other types of stem cells, derived from bone marrow and adipose tissue, have also shown therapeutic benefits in 3-NP models (72, 73). Interestingly, the source of stem cells is also key to functional recovery because bone marrow harvested from transgenic mice harboring the human SOD1 G93A mutation, which induces amyotrophic lateral sclerosis through an unknown gain of toxicity and mitochondrial dysfunction, worsened the 3-NP pathologic manifestations (74).

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4. Other Metabolic Toxins Producing Striatal Lesions

4.1. Sodium Azide

The observation of 3-NP-induced progressive behavioral pathology is similarly achieved by other mitochondrial inhibitors. Here, we discuss two other mitochondrial inhibitors, namely sodium azide and malonic acid, which have been shown to produce some pathological symptoms resembling HD. Almost four decades ago, systemic injections of the mitochondrial complex IV, cytochrome c inhibitor sodium azide has been demonstrated to result in choreic-type movements in animals (75). Using a regimen of intramuscular injections (5 mg/kg of sodium azide, 7 days/week for 8 weeks or 5 days/week for 10 weeks), animals displayed choreic-like variable dyskinesia, the onset of which coincided with mitochondrial alterations in gray matters as revealed by electron microscopy. The dyskinesias were characterized as episodic lasting about 15 min and occurring one to three times a day, but disappeared in about 2 weeks. This timing of the disappearance of dyskinesia was critical to the progression of pathological changes, in that perivascular abnormalities were detected in the ventrolateral putamina, followed by neuronal cell loss, glial infiltration, and scars or necrotic lesions, whereas such morphological abnormalities ensue in the caudate nucleus and substantia nigra prior to and after the disappearance of dyskinesia, respectively. Such sodium azideinduced pathological changes appear reversible because withholding drug treatment halts the evolution of morphologic abnormalities, but continued drug regimen extended the pathological deterioration to the thalamus and mesencephalic tegmentum. These histologic and behavioral results indicate that the onset of dyskinesia highly correlated with striatal damage with consistent bilateral and symmetrical putaminal lesions. Moreover, the subsequent involvement of substantia nigra in the progression of this sodium azide-mediated pathological disruption signals the appearance of hypokinesia. The vulnerability of the striatum, as opposed to the substantia nigra, to the metabolic toxicity of sodium azide was thought to be due to the striatum being poorly supplied with oxygen (75). This initial study on sodium azide was replicated two decades later. Rhesus monkeys that received intrastriatal administration of sodium azide produced dose-dependent lesions characterized by neurochemical and histologic alterations in both spiny projection neurons (GABA, substance P) and aspiny interneurons (somatostatin, neuropeptide Y, NADPH-diaphorase). In parallel, animals that received systemically treatment with sodium azide (5 mg/kg/day for 20–100 weeks) displayed episodic variable dyskinesia, a choreoathetoid type, at 8–10  weeks post-injection, looked normal after 2 weeks, then developed a hypokinetic state, which is age dependent (76). Similar neurochemical profile was

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produced by the systemic sodium azide injection, but spared dopaminergic striatal afferents. Although striking similarities in the progression of behavioral impairments accompany sodium azide and 3-NP treatment, sodium azide produces a severe metabolic deficit that leads to a different pattern of striatal degeneration that involves secondary excitotoxic mechanism (76). That the chemical hypoxia induced by sodium azide produces a secondary excitotoxicity, leading to the activation of N-methyl-d-aspartate receptors (NMDA) is further demonstrated in another study demonstrating the effects of sodium azide on the immunohistochemical localization of kynurenine aminotransferase-I (77). Five days systemic treatment with sodium azide significantly reduced glial kynurenine aminotransferase-I immunoreactivity in the glial cells of the striatum, hippocampus, dentate gyrus, and temporal cortex. In addition, kynurenine aminotransferase-I reaction product accumulated around the ribosomes of neuronal endoplasmic reticulum suggesting de novo synthesis of kynurenine aminotransferase-I in the reactive nerve cells (77). With the observation of substantia nigral damage following chronic systemic injection of sodium azide, and the notion that mitochondrial electron transport chain function is selectively reduced in multiple tissues, including brain, from patients with Parkinson’s disease (PD), the use of this metabolic toxin has also been explored in investigations of pathological alterations associated with PD (78). In vivo striatal production of toxic hydroxyl radical in awake rats increased three- to fivefold after infusion of sodium azide. In addition to PD, chronic nonlethal sodium azide administration in rats has been proposed for the study of mitochondrial dysfunction and the role of environmental pollutants in brain and muscle tissues affected in certain neurodegenerative diseases (79). Because the resulting cell death in sodium azide has implicated the activation of NMDA receptors leading to excitotoxicity via the nitric oxide (NO) pathway, some experimental therapeutics, such as the anti-oxidant a-tocopherol (80), have been directed at inhibiting NMDA or NO to protect against sodium azide (e.g., MK-801 and manganese (81–83)). However, the sodium azide neurotoxicity was not blocked by either the nitric oxide synthase inhibitor nitro-l-arginine or by the NMDA channel blocker MK801 (78). An alternative non-NMDA cell death mechanism has been postulated for sodium azide toxicity in striatal neurons including ATP-sensitive potassium + channels which involves a tolbutamide-sensitive membrane hyperpolarization (84, 85), suggesting that targeting this non-NMDA pathway may arrest sodium azide neurotoxicity (86). Additionally, preservation of the mitochondrial ATP production through maintenance of key mitochondrial proteins, as accomplished by acetyl-l-carnitine may render protection of the mitochondria against sodium azide (87). Other neuroprotective

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compounds evaluated against sodium azide include delta opioid peptide receptor agonists (88), sigma-1 receptor agonists (89), and estrogens (90). 4.2. Malonate

Malonate is a reversible mitochondrial complex II, succinate dehydrogenase inhibitor. Following intrastriatal infusion, malonate produces both energy depletion and striatal lesions reminiscent of cerebral ischemia and HD (91, 92). The malonate-induced striatal lesions appear dose and age dependent (91, 93). Secondary excitotoxicity and the generation of reactive oxygen species mediate malonate-induced cell death. Because the striatal lesion produced by the low dose (1 mmol), malonic acid was completely blocked by the NMDA channel blocker MK801, this indicated the key contribution of “excitotoxicity” to malonate’s neurotoxicity (91). Furthermore, stimulation of both agonist- and voltage-dependent properties of NMDA exacerbated malonate’s neurotoxic effects lending support to the excitotoxicity cell death pathway of malonate (94). Aberrant-free radical formation and increased NOS activity were also observed following intrastriatal infusion of malonate in rats infused into the left striatum of rats (92, 95). That the increased production of hydroxyl radical contributes to malonate neurotoxicity is revealed by significantly increased conversion of salicylate to 2,3- and 2,5-dihydroxybenzoic acid, an index of hydroxyl radical generation, in knock-out mice with null mutation in glutathione peroxidase (GSHPx), a critical intracellular enzyme involved in detoxification of hydrogen peroxide to water, as compared with both heterozygote GSHPx knock-out and wild-type control mice (96). That malonate induced a severe glutathione (GSH)-mediated oxidative stress leading to the rapid development of the lesion is demonstrated in another study (97). The time course of reduction in GSH was shown to progressively decrease after malonate injection up to 40% of those of sham animals at 4 h, which was inversely correlated with increased concentrations of oxidized glutathione (GSSG) detected as early as 1 h after malonate treatment and sustained up to 4 h (97). These biochemical alterations were accompanied by similar time-dependent progression of lesion size which was maximal within 2 h of malonate injection, whereas edema continued to increase between 2 and 24 h (97). There is also laboratory evidence to suggest a key role of dopamine mediation in malonate neurotoxicity. Nigrostriatal dopaminedepleting agents such as 6-hydroxydopamine (6-OHDA) and reserpine combined with a-methyl-p-tyrosine significantly reduced malonate-induced striatal lesions concomitant with decreased generation of ROS (98). In contrast, treatment of l-DOPA or dopamine reinstated malonate toxicity and the generation of ROS in 6-OHDA-lesioned rats. These results suggest that malonate-induced dopamine toxicity in metabolically deficient neurons may involve dopa­mine transporter uptake-dependent,

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dopamine receptor-independent ­generation of ROS, and excessive stimulation of dopamine receptors (98). An earlier study showed similar results of dopamine depletion within the nigrostriatal pathway as altering the malonate neurotoxicity (92). This contribution of the nigrostriatal pathway to malonate neurotoxicity was further demonstrated in R6/1 transgenic HD mice expressing exon 1 of the HD gene with 115 CAG repeats (99). Despite maintenance of overall number of tyrosine hydroxylase-positive neurons and nigrostriatal connectivity, the size of tyrosine hydroxylase-positive neurons in the substantia nigra was reduced by 15%, and approximately 30% of these cells exhibited aggregated huntingtin in transgenic mice compared to wild-types controls. In vivo microdialysis further revealed that basal extracellular striatal dopamine levels were reduced by 70% in transgenic mice compared to their wild-type littermates. Local dopamine release following intrastriatal malonate perfusion in transgenic animals displayed a short-lasting, attenuated increase compared to wild-type mice. Finally, malonate-induced striatal lesion size was 80% smaller in transgenic animals compared to controls. Altogether, these results in conjunction with another study showing that mutant huntingtin causes a sublethal grade of metabolic stress and results in upregulation over time of cellular defense mechanisms against impaired energy metabolism and excitotoxicity (100), demonstrate that a dysfunctional nigrostriatal dopamine transmission mediated malonate-induced neurotoxicity (99). Experimental therapeutics that have been shown to ameliorate malonate neurotoxicity are the energy metabolism activators coenzyme Q10 and nicotinamide (101), NMDA receptor antagonist MK-801 (92), the free radical scavengers or antioxidants such as a-phenyl-tert-butyl-nitrone (95, 97), free radical spin trap n-tertbutyl-a-(2-sulfophenyl)-nitrone (102), and NF-E2-related factor 2 (103), the NOS inhibitors including aminoguanidine (95), the adenosine protectors (104), the dopamine receptor agonists (105), the monoamine oxidase acting drugs such as clorgyline (106), deprenyl (107) and transpeptidase called 1,2,3,4-tetrahydroisoquinoline (108), the glutamate uptake facilitator Valproate (109), the caspase-3 inhibitor (110), the anti-inflammatory agent minocycline (111), and the cannabinoid receptor agonists (112, 113).

5. Summary The brain region selectivity and relatively established time course of cell loss in mitochondrial toxin animal models make them appealing features for investigations into behavioral and histological components of neurodegeneration in HD, as well as development of novel neuroprotective and neurorestorative therapies for

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the disease. The progressive nature of the disease, although elusive initially in toxin models, has now been made available via the systemic administration of 3-NP. As noted above, several neuroprotective therapies have been tested in these metabolic striatal lesion models, and some have reached clinical trials for HD patients. A caveat of course with neuroprotective therapies in toxin models is the recognition that therapeutic effects may be limited to HD patients diagnosed, via genetic testing, early on prior to the onset of cell degeneration or behavioral symptoms. In contrast, when diagnosis occurs only after symptom manifestation, the use of neurorestorative therapies is indicated. To this end, stem cell therapy as a neurorestorative regimen has potential for HD, and such regenerative medicine approach has been tested in animal models of mitochondrial inhibitors. Despite these many advantages of mitochondrial toxin models, there are technical limitations that require careful consideration for data interpretation. These toxin models neither harbor the mutant huntingtin gene nor exhibit aberrant accumulation of misfolded proteins. That HD manifests behaviorally as both motor and cognitive, many of the toxin models have mainly characterized motor deficits, although there is a conscious effort among behavioral neuroscientists to investigate complex learning and memory functions especially in non-human primates. The advancement from the laboratory to the clinic of experimental therapeutics in HD, whether cell-, gene- or drug-based treatment, will eventually need to consider testing the efficacy and safety in both toxin and transgenic animal models not only because of creating a stringent criterion for successful translation of the therapeutic product, but that genetic defect and metabolic impairment are key factors mediating the pathological hallmarks of HD. References 1. Martin JB, Gusella JF (1986) Huntington’s disease: pathenogenesis and management. N Engl J Med 315:1267–1276 2. Shoulson I, Asbury A, McKhann GM, McDonald I (1986) Huntington’s Disease: The disease of the nervous system. Ardmore Medical Books (W. B. Saunders): Philadephia 3. Maragos WF, Young KL, Altman CS, Pocernich CB, Drake J, Butterfield DA, Seif I, Holschneider DP, Chen K, Shih JC (2004) Striatal damage and oxidative stress induced by the mitochondrial toxin malonate are reduced in clorgyline-treated rats and MAO-A deficient mice. Neurochem Res 29(4):741–746 4. The Huntington’s Collaborative Group (1993) A novel gene containing a trinucleotide repeat that is expanded and unstable on





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5  3-Nitropropionic Acid and Other Metabolic Toxin Lesions of the Striatum 53. de Lago E, Fernández-Ruiz J, OrtegaGutiérrez S, Cabranes A, Pryce G, Baker D, López-Rodríguez M, Ramos JA (2006) UCM707, an inhibitor of the anandamide uptake, behaves as a symptom control agent in models of Huntington’s disease and multiple sclerosis, but fails to delay/arrest the progression of different motor-related disorders. Eur Neuropsychopharmacol 16(1):7–18 54. Chin PC, Liu L, Morrison BE, Siddiq A, Ratan RR, Bottiglieri T, D’Mello SR (2004) The c-Raf inhibitor GW5074 provides neuroprotection in vitro and in an animal model of neurodegeneration through a MEK-ERK and Akt-independent mechanism. J Neurochem 90(3):595–608 55. Karanian DA, Baude AS, Brown QB, Parsons CG, Bahr BA (2006) 3-Nitropropionic acid toxicity in hippocampus: protection through N-methyl-D-aspartate receptor antagonism. Hippocampus 16(10):834–842 56. García O, Massieu L (2001) Strategies for neuroprotection against L-trans-2,4pyrrolidine dicarboxylate-induced neuronal damage during energy impairment in  vitro. J Neurosci Res 64(4):418–428 57. Binienda Z, Przybyla-Zawislak B, Virmani A, Schmued L (2005) L-carnitine and neuroprotection in the animal model of mitochondrial dysfunction. Ann N Y Acad Sci 1053: 174–182 58. Yang L, Calingasan NY, Chen J, Ley JJ, Becker DA, Beal MF (2005) A novel azulenyl nitrone antioxidant protects against MPTP and 3-nitropropionic acid neurotoxicities. Exp Neurol 191(1):86–93 59. Dykens JA, Simpkins JW, Wang J, Gordon K (2003) Polycyclic phenols, estrogens and neuroprotection: a proposed mitochondrial mechanism. Exp Gerontol 38(1–2):101–107 60. Borlongan CV, Yamamoto M, Takei N, Kumazaki M, Ungsuparkorn C, Hida H, Sanberg PR, Nishino H (2000) Glial cell survival is enhanced during melatonin-induced neuroprotection against cerebral ischemia. FASEB J 14(10):1307–1317 61. Bizat N, Hermel JM, Humbert S, Jacquard C, Créminon C, Escartin C, Saudou F, Krajewski S, Hantraye P, Brouillet E (2003) In vivo calpain/caspase cross-talk during 3-nitropropionic acid-induced striatal degeneration: implication of a calpain-mediated cleavage of active caspase-3. J Biol Chem 278(44): 43245–43253 62. Blum D, Gall D, Galas MC, d’Alcantara P, Bantubungi K, Schiffmann SN (2002) The adenosine A1 receptor agonist adenosine amine congener exerts a neuroprotective

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Chapter 6 Functional Assessment of Subcortical Ischemia Tracy D. Farr and Rebecca C. Trueman Abstract The middle cerebral artery occlusion (MCAO) is the most common rodent model to mimic large vessel occlusion in the human. The intraluminal filament MCAO technique should, theoretically, produce ischemia both in the cortex and striatum. However, collateral blood flow can contribute to maintenance of cerebral perfusion in the cortex at thresholds that sustain tissue viability. This means some animals may exhibit lesions only in the subcortical structures, particularly the posterior striatum, which may be desirable for certain experimental purposes. This chapter aims to describe, in detail, the steps and equipment required to perform the intraluminal filament model of MCAO in the rat. We also discuss relevant tips and tricks to identify rats with subcortical damage. In addition to a general discussion regarding basic requirements for behavioural experiments in rats with this type of injury, we provide a stepwise guide for using several sensitive behavioural tests. Key words: MCAO, Stroke, Behaviour, Subcortical ischemia

1. Introduction According to the World Health Organization, nearly 15 million people suffer an ischemic event each year. This makes stroke the third leading cause of death and the leading cause of disability in developed countries globally, and its incidence is predicted to increase in the coming years. The primary risk factor for stroke is atherosclerosis, which is a complex disease that begins with the accumulation of fatty materials, such as cholesterol, on the inside of arteries. The build up causes the vessels to lose elasticity and become hard (arteriosclerosis) and stenotic (narrow). The stenotic environment is also a haven for platelet accumulation, and clot thrombi can form around the atherosclerotic plaques. Clots and plaques can either break off and travel to the brain (embolic stroke) or eventually inhibit blood flow inside a brain artery (thrombotic stroke).

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The reduction in blood flow is the initiation point for the pathology associated with stroke, the complexity of which is beyond this chapter, but has been reviewed elsewhere (1, 2). A large vessel occlusion is the most common type of stroke (60%) and it is caused by either an embolic or thrombotic event in one of the major cerebral arteries: anterior, middle, or posterior. A lacunar infarction (26%) occurs when a thrombotic event develops in a small penetrating artery that supplies a subcortical structure; the most common subtype being pure motor hemiplegia syndrome in which thrombosis of the lenticulostriate branches produce ischemia in the internal capsule. By far the most common rodent model of focal stroke is the middle cerebral artery occlusion (MCAO), which is somewhat similar in etiology to large vessel occlusion in the human. As is the case with humans, the territory of the middle cerebral artery is the largest of all the cerebral arteries, and the proximal lenticulostriate branches supply the posterior striatum and internal capsule (3). MCAO should theoretically result in a cortical and subcortical ischemic lesion. However, Heubner pial anastomoses in the cortex have the ability to compensate for reductions in blood flow, which means the cortical component of this injury can be notoriously unpredictable (4). It has been suggested that animals exhibiting an insult confined to the subcortical structures (particularly the striatum) could be used to somewhat model pure motor hemiplegia syndrome in humans. This is not necessarily accurate, since the clinical pathology is typically associated with the internal capsule, and the damage produced in the striatum is generally considered to be selective neuronal death rather than true ischemia (5). Nevertheless, this type of insult is often preferred to a large vessel occlusion because animal welfare is generally better. Furthermore, studies which isolate these animals can be useful towards increasing our understanding of the function of the striatum, as well as a more targeted model for cell replacement therapies. The most common technique used to produce MCAO is the intraluminal filament model, which involves insertion of a blunted filament (or occluding device) into the internal carotid artery (ICA) with advancement until it reaches the origin of the MCA in the Circle of Willis. In this chapter, we explain the steps required to produce MCAO with this technique, as well as a detailed description regarding how to use behavioural tests in animals with subcortical only insults.

2. Materials and Methods The following sections will first describe the surgical procedures required for the intraluminal filament technique followed by a detailed discussion of different behavioural tests suitable for rats with striatal damage. With regards to the surgical procedure,

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Fig.  1. Photographs of hand made and commercially available filaments. From top to ­bottom, a filament made from fishing line, which has been blunted at the tip, a commercial filament from Doccol Company, USA, and a Xantopren coated Prolene filament.

the technique we will describe is modified from the original report that introduced the intraluminal filament technique in rats (6). Procedures for mice will not be described here, but can be found from other sources (7). 2.1. Filament Preparation

Many different types of filaments exist, including commercially available ones (Doccol Company, USA), but we will describe the most popular method by which to construct them (Fig. 1). 1. Wearing gloves, cut 3.0 surgical monofilament (Resorba, Germany), preferably nylon but Prolene or Mopylen have also been used, into 30-mm lengths. 2. Blunt one end with heat. The material determines how smooth the blunt end will be. 3. Mix Xantopren L silicon and Universal Activator (Heraeus Kulzer, Germany) and quickly dip the blunted end of the filament into the mixture before it hardens. 4. Measure the diameters of the silicon-coated tips under the microscope and select those with a range of 380–410  mm. Discard filaments that appear sharp. 5. Bend or mark the uncoated end approximately 20 mm away from the coated tip. 6. Store in a clean environment to avoid dust. 7. Ideally, filaments should be used only once. Alternatively, clean with 70% alcohol if re-use will be common.

2.2. Surgical Procedures

1. Ensure the surgical area and instruments are clean, preferably sterilized, between animals.

2.2.1. Animal Preparation and Care

2. Anaesthetize the animals with inhalable isoflurane in a 70:30 nitrous oxide:oxygen mixture.

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3. Core body temperature must be maintained at 36.7 ± 1°C throughout the duration of the procedure. The preferred method is automated heat blankets (Harvard, UK). 4. Shave and clean the incision sites with 70% alcohol or iodine scrub. 5. Provide adequate post-operative care. Animals that exhibit dehydration can be re-hydrated with daily subcutaneous injections of 2.5 mL of physiological saline and 5% glucose (Animal Care Limited, UK). Moistened diet should also be provided until weight stabilizes in a dish that is either secured to the cage or too heavy to allow disruption. 6. Assistance with pain relief can be provided according to the guidelines of your local veterinarian. We routinely mix 1 mg/mL of Paracetamol (Boots, UK) into the drinking water for 1 day before and up to 3 days after the surgery. 2.2.2. Laser Doppler Flowmetry

For an indirect measure of cerebral blood flow in the MCAO territory to confirm correct filament placement, Laser Doppler Flowmetry (LDF) can be used. Several different systems and probes exist (Moor Instruments, UK; PeriMed, Germany) but we will describe the method we find the most useful. 1. Place the animals on their side (the same side that the MCAO will take place). Shave and clean the area between the eye and ear taking care not to destroy the vibrissae. Make a small vertical incision (from top to bottom of head) using blunt dissection. 2. Place a calibrated 1-mm Laser probe against the skull through a small incision in the temporal muscle. Carefully rotate the rat into a supine position and secure the probe so that a stable baseline value can be observed. 3. It is advisable to record the measurements.

2.2.3. Middle Cerebral Artery Occlusion

The description that follows is to perform the MCAO on the right side of the animal, though there is no reason why it can not also be performed on the left side. 1. Make a 1- to 2-cm vertical incision along the midline in the prepared neck using blunt dissection. 2. Gently retract the right mandibular glands, pretrachial strap and sternomastoid muscles to expose the carotid artery (CA). The surgeon should be familiar with the anatomy of the CA (Fig. 2). 3. Gently dissect and retract the vagus nerve away from the CA. 4. Tie a silk suture around the CA as near to the chest as you can get. 5. Gently detach connective tissue from the CA using opening and closing movements of blunt forceps until the bifurcation

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Fig. 2. The vasculature supplying the rodent head. The CA branches into the ECA and ICA and both give off extra-cranial projections: pterygopalatine, occipital, superior thyroid, ascending pharyngeal, posterior auricular, external maxillary and lingual arteries.

of the external and internal carotid arteries (ECA and ICA, respectively) is visible. 6. Tie a silk suture on the ECA and another loose suture on the ICA taking care not to disrupt the small branches (Fig. 2). 7. Place a microclip above the loose suture on the ICA. 8. Once a closed system is ensured, use Vannas scissors to make a small hole in the CA below the bifurcation. 9. Insert the blunted end of the filament into the incision and advance it to the microclip. 10. Tighten the loose suture around the filament. 11. Release the microclip and advance the filament up the ICA. Take care to make sure the filament does not enter the pterygopalatine artery. Retract the ECA away (towards the left) to facilitate this. 12. The distance from the ECA and ICA bifurcation to the MCA is 17–22 mm in rats. Once the mark or bend of the filament nears the CA incision slow the advancement and watch the

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Fig. 3. Using Laser Doppler flowmetry to predict pathology. Laser Doppler recordings in the middle cerebral artery territory of an animal that exhibited a large cortical and subcortical stroke (a) and an animal with an insult confined to the striatum (b). The ischemic region is depicted in white on the 2,3,5-triphenyltetra-zolium chloride (TTC)-stained sections.

Laser Doppler carefully for decreases. Many labs use the Laser Doppler decrease as criteria for inclusion or exclusion. Generally, a stable decrease of approximately 80% of baseline predicts a large cortical and subcortical stroke (Fig. 3). 13. Once a decrease is observed, note the time. 14. During the desired occlusion time, typically 30, 60, 90, or 200 min, the animals can be left in place (for LDF) or recovered. It is also possible to produce a permanent occlusion by leaving the filament in place and recovering the animal. Generally, 30 min of MCAO produces striatal insults whereas longer occlusion times (60 min or more) produce larger strokes (typically cortical and subcortical). However, this must be tested and established in each laboratory with the desired animal strains and filaments. 15. Retract the filament and replace the microclip to allow release of the suture for filament withdrawal. Then tighten the suture again and remove the microclip. 16. Remove the ECA suture to re-establish perfusion in the extracranial territories. 17. Carefully replace the muscles and glands and suture the wounds.

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For complications and adverse effects of the intraluminal filament procedure, see Note 1, and for different modifications to the surgical procedure, see Note 2. Additionally, every laboratory should be equipped with basic histological techniques to evaluate the size of the insult, see Note 3. 2.3. Behavioural Tests

The most common symptom of human MCAO is contralateral hemiplegia and hemianesthesia, which is a combined sensorimotor deficit in the injured (contralateral) side of the body, face and extremities. As such, traditionally, the most common behavioural tests for rodents with stroke were designed to examine the difference in function between the intact (ipsilateral) and impaired (contralateral) side of the body (asymmetry tests). These tests include the bilateral asymmetry (adhesive removal test) (8, 9), spontaneous forelimb use asymmetry (cylinder test) (10, 11), paw reaching (staircase test) (12), stepping/paw placing tests (13, 14), druginduced rotation (15) and the disengage test (16). Many of these tests were originally developed for other unilateral models of basal ganglia disorders, such as Parkinson’s and Huntington’s disease. However, these tests also reveal deficits in animals with ischemia confined to subcortical areas, predominantly the striatum (17). The majority of these tests examine combined sensorimotor function; however, clever manipulations can be used to distinguish the sensory component from the motor aspect (see Note 7). It should be noted that animals with purely subcortical lesions following MCAO are prone to behavioural recovery. This is particularly evident if rigorous selection criteria are followed, such as excluding animals with any sign of haemorrhage on magnetic resonance imaging (MRI), and using a rigorous surgical method (see Note 1) to minimize the secondary effects of surgery. In our hands, tests of skilled motor function (paw reaching) and apomorphineinduced rotations are the most robust and least likely to recover (see Note 4).

2.3.1. General Considerations Prior to Behavioural Testing

One of the first considerations when designing a behavioural experiment involving MCAO rats should be appropriate control groups. When using the intraluminal filament model it is necessary to have a sham surgery group. These sham animals should undergo the same surgical preparation in the neck as the lesioned animals. This is necessary as the surgical procedure itself will have an effect on the behavioural responses of the animals, particularly if the rats are tested within the first week following surgery. The choice of behavioural tests should also be carefully considered, and a number of questions need to be addressed when ­making this decision. What is the purpose of the experiment? If you are assessing a potential therapy, what is the mechanism of the ­treatment? For example, unless you are examining a treatment that is potentially going to protect/replace a significant number of

Study Design

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medium spiny neurons within the striatum, you are unlikely to see a recovery in the number of apomorphine-induced rotations. Will there be sufficient time prior to surgery to train rats? Is food restriction a feasible option for tests such as paw reaching? What time points post-surgery are you testing? Testing in the first few days following surgery can result in spurious results because animals have not sufficiently recovered and can be lethargic. It is also important to consider how many times rats are going to be exposed to each test. Tasks such as the cylinder and adhesive removal can become ineffective if the rats are exposed to them too many times. Animals with lesions may also develop compensatory mechanisms on some tasks and/or recover function regardless of treatment. Rats should be tested long enough to see if there is recovery in non-treated animals. Many different factors govern whether functional recovery is evident on behavioural tests and learned compensation can contribute to recovery (for a review see (18)). For example, if you see improvement with a potential therapeutic, you must ask whether or not the therapeutic strategy truly caused “brain repair”; or merely sped up the compensation process via secondary mechanisms. One obvious secondary mechanism could be that the treatment merely increased the motivation of the animal, i.e. an injection of substance x could reduce lethargy by increasing hydration. Stringent controls and longitudinal testing with appropriate tasks should overcome such issues. Once the overall design of the experiment has been carefully planned it is then important to consider the smaller points that can have a large affect on behaviour. Housing, Handling and Test Environment

For behavioural testing, it is important that animals are well handled and unstressed. Rats are social animals and should ideally be housed in groups. We have found with the less severe subcortical lesions it is not necessary to house lesion and sham animals separately, provided moist diet is given regularly following surgery. However, occasionally one animal may need extra care and single housing following surgery. If possible, the rat should be reintroduced to the group once it has recovered sufficiently. However, this should be done with caution as adult male rats can become aggressive; although we have not found this to be an issue with male Wistar rats. If it is necessary to house sham and lesioned animals separately, ensure that the order of testing is arranged so that both groups are mixed, to prevent confounds in the data. The time of day rats are tested may affect performance and therefore it is also important to keep this constant. Rats should be habituated to both the test rooms and equipment before testing (except for the cylinder test). This will calm them and reduce the levels of anxiety. For the same reason, the test environment should be kept as uniform as possible, with unexpected noises and smells being avoided. A quiet radio in the background of the testing

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rooms will reduce startling of the animals when an unexpected noise happens. However, a radio should only be used when it will not affect the parameters of the task. For example, it is unwise to use a radio during spatial learning tasks, as the noise will act as an unintentional cue to the animals. Equally important as maintaining a constant test environment is having the same well-trained experimenter handling the animals. The rats may behave differently if being handled in an unfamiliar way, and small differences in technique on tests such as the lateralized stepping task can produce a large difference in the data obtained. It is also important that testing is carried out by somebody who is blinded to the condition of the animals, as some of the tests are easily unintentionally influenced. Food Restriction

Food restriction is required for a number of tests and there are a number of protocols that can be used. The method we use aims to maintain rats at 85–90% of their free feeding weight (the weight they are prior to food restriction). To achieve the desired weight range, the rats are given a set amount of food each day and weighed three times a week to ensure the quantity of food is correct. The level of food can be adjusted depending on the level of performance of the rats, provided that the weight remains above 85% of their free feeding weight. If the rats are not yet fully grown, the free feeding weight must be adjusted on a weekly basis in line with a growth curve, which can be obtained from the supplier. We do not food restrict animals until they have fully recovered from ­surgery and have regained lost weight. This therefore limits how close to surgery tests that require food restriction can be performed.

2.3.2. ApomorphineInduced Rotations

Apomorphine is an agonist to post-synaptic dopamine receptors, which are expressed on the medium spiny neurons of the striatum. In essence, when a rat with a unilateral lesion to the striatum is administered apomorphine, the drug will stimulate the intact ­striatum more that the lesioned striatum. This results in the rat ­circling ipsilateral to the side of injury, an action which can be quantified. However, the location of the striatal injury will influence the degree and direction of rotations (19). It should be noted that the mode of action of apomorphine when administered to rats with striatal MCAO lesions is similar to that of animals with excitotoxic lesions of the striatum, but different from that in rats with unilateral 6-OHDA lesions. In the latter instance, very low doses of apomorphine are administered to activate only the super-sensitive dopamine receptors of the dopamine-depleted striatum. This results in the rats circling contralateral to the side of injury (see Dunnett and Torres, volume 1, chapter 15). Within our laboratory we find that apomorphine-induced rotations provide a robust measure of striatal damage following MCAO. We describe here what we have found to be the best protocol to perform apomorphine-induced rotation in MCAO rats.

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Fig. 4. Rotometer. The model shown is from Med Associates, USA.

1. Inject the rat subcutaneously with 1  mg/kg of R-(−)Apomorphine hydrochloride hemihydrate (Sigma-Aldrich, UK), 0.2% ascorbate in physiological saline (see Note 5). 2. Place the rat into the bowl of an automated rotometer (Med Associates Inc, USA), with an elastic band or harness secured around the ribcage of the rat (Fig. 4). The band is connected to a wire, which is in turn attached via a pivot to the rotometer head. This head transforms the turns of the rat into electrical pulses that are interpreted by a computer interface. The rotometer bowl is located in a cylinder with removable lid to prevent rats climbing out. 3. Start program, and repeat above steps for each rat. 4. Record rotations for 60 min. 5. The software (MedPC Rotorat software, Med Associates Inc, USA) records the total number of rotations in both directions. 6. Net total number of rotations is determined (# clockwise – # counterclockwise turns). This can be expressed as rotations per minute or net total rotations. 7. Many companies sell automated rotometers, but the basic principles of all these setups are the same. If the equipment is not available, it is possible to score rotational behaviour manually. The injected rat is placed into a bowl either observed or filmed so that whole rotations clockwise and counterclockwise can be counted.

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Lateralized stepping is a method for assessing forelimb akinesia in the rat and was originally developed to evaluate unilateral lesion models of Parkinson’s disease (13, 14). The rat is restrained and moved in a fashion that should evoke adjusting movements of the free forelimb. The absence of these movements indicates deficits in initiation of motor responses of the forelimb. For each forelimb, the rat can be moved in two directions, towards the midline (forehand) or away from the midline (backhand). We have found that the forehand measure is more robust for assessing functional deficits in the MCAO model. This may be due to backhand measure invoking a vestibular response, due to a slight twisting in the rat’s trunk caused by the body being moved across the forelimb. Vibrissae-evoked placing requires the rat to be restrained in a similar way and examines the initiation of forelimb movement from the stimulation of the whiskers unilaterally. We have not found the vibrissae-evoked placing to be sensitive to the 30-min MCAO model, due to recovery. However, with larger lesions this test is effective. For issues with lateralized stepping and vibrissae invoked paw placing, see Note 7. Both tests are performed by restraining the rat in a similar manner and manipulating them. 1. Handle the rats prior to testing and introduce the restraining technique so that they are habituated to the procedure and are calm during data collection. 2. Restrain the rat in both hands so that all limbs are immobilized except for the forelimb that will be tested. 3. For lateralized stepping, tilt the animals at 45° with the snout downwards and the forelimb to be tested on the edge of a counter. Move the animals horizontally for 1 m in both directions. The speed of this movement should be kept constant. Normal animals should be capable of performing both “forehand” and “backhand” adjusting movements of their free fore paw. Record the number of placements for each forelimb in each direction. This should be repeated three times. 4. For vibrissae-evoked paw placing, turn the animals sideways and lift them upwards towards the edge of the bench until the whiskers contact the edge. This should elicit an ipsilateral forelimb placing response in normal animals (Fig. 5). Care should be taken to make the movement of the rat smooth, so a vestibular response is not induced. This procedure is performed ten times for each side and the number of correct placements is recorded for each forelimb. See Note 7 for alternative methods of vibrissae-evoked paw placing. 5. The data for both of these tests can be presented as total number of placements, or more commonly the contralateral score is expressed as a percentage of the total number of placements ((contralateral/(ipsilateral + contralateral)) × 100).

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Fig.  5. A depiction of the vibrissae-evoked forelimb placing test. The animal’s right forelimb is touching the edge of the table following vibrissae contact which was performed by the movements of the experimenter.

2.3.4. Disengage

The disengage test in its simplest form examines the response of a rat to stimulation of the whiskers on the ipsilateral and contralateral side of the face. However, the rat can also be tested while ­eating, which probes striatal function as the striatum has a crucial role in the ability of the animal to “disengage” from the eating behaviour (16, 20). Steps required to perform the disengage test are listed below. 1. Habituate the rats to chocolate for a number of days prior to testing. On day 1, give it in the home cage. For 2 days following this, it is advisable to give the chocolate to the rats in the test environment, so that they learn they only access the chocolate for a limited time. 2. Place the rats into an empty standard housing cage. To reduce exploration, use a cage to which the rat has been extensively habituated, or the rat’s own home cage. 3. From behind, without the rat seeing or hearing, stimulate the perioral region on either side with a 30-cm stick. Normal rats should quickly orient with the head to the stimulus. 4. Record the latency to respond (sec) for each side with a stopwatch. Setting a maximum time for responding is beneficial, for example, 10 or 60 s. 5. Provide the animals with a piece of chocolate (approximately 1 cm × 1 cm) and repeat. 6. Randomize the order of testing (with and without chocolate and side to side).

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7. This test can be repeated on consecutive days to reduce the variance in the data. See Note 8 for more information on this task. 2.3.5. Paw Reaching

Skilled motor function can be assessed using the staircase paw reaching task. A food restricted rat is placed in a staircase chamber, which contains sugar pellets. The box is designed in such a way that food can only be obtained from each staircase with the forelimb that is on that side of the staircase, and the rats must feel for the pellets with their paws (12) (Fig. 6). The task requires the rats to integrate motivational, sensory and skilled motor behaviour. We have found that this test is one of the least susceptible to functional recovery. The steps required to perform paw reaching are described below. 1. For a week prior to the commencement of paw reaching, food restrict the rats and maintain them between 85 and 90% of their free feeding body weight. 2. Give the rats sugar pellets in the home cage for 3–4 days prior to testing, to over come any neophobia of the new food source. 3. On day 1 of training in the chamber, provide excessive pellets in the steps to encourage the rats to reach. On following days, load each step with 2× 45 mg sucrose food pellets (Sandown Scientific, UK). 4. Place the rats inside the chambers for 15 min daily until performance has plateaued. 5. Record the total number of food pellets left on each step. Data can be examined as the total number of pellets eaten on each

Fig. 6. A depiction of a rat inside a staircase chamber reaching for food pellets placed into wells located on each step. Note the rat is only able to obtain food on each side of the middle partition with the forelimb that is on that side.

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side (pellets at start – pellets left) or the number of pellets displaced, on each side, can be worked out [(number of remaining pellets on each step − 2) + the number of pellets eaten]. See Note 9 for training and modifications to the paw-reaching protocol. 2.3.6. Bilateral Asymmetry (Adhesive Removal)

The bilateral asymmetry test examines sensorimotor asymmetries in rodents’ deficits by measuring the time taken to touch and remove sticky labels applied to the wrists (9). The time to touch is a measure of sensory function (time to notice); however, it should be noted that the rat has to initiate the movement to touch the marker, so it is not a pure sensory measure. The latency to remove assesses the fine coordination and motor skill required to remove the sticky label. The order of removal is also an important measure, as it can be used to examine contralateral sensory neglect and “tactile extinction”. Tactile extinction is the phenomena of sensory stimulation applied to the ipsilateral side masking detection of a stimulus simultaneously applied to the contralateral side. A more complex version of the task can be used to probe this phenomenon further (8) (see Note 10). As described before, in our experience a large amount of recovery is evident on this task when assessing animals with primarily subcortical infarcts. However, with larger cortical and subcortical lesions, this task is robust and regularly used. 1. Place square (7  mm × 7  mm) pieces of masking tape on the distal-anterior surface of the wrists of both forelimbs. 2. Place the rats into a Perspex cylinder (35 cm high, 32 cm diameter) to which they have been previously habituated and observe. Normal rats should remove both stimuli relatively quickly. 3. Use a stopwatch to record the time to touch and to remove each stimulus. If the rat does not finish removal, the trial is stopped after 5 min. 4. The order of removal, time to touch and time to remove the stimuli can be analyzed. 5. Repeat the procedure four times, with at least 10 min between each test session. 6. Data can be presented as limb bias, for example ((touch left/ (touch left + touch right)) × 100). See Note 10 for more information regarding this test.

2.3.7. Spontaneous Forelimb Use Asymmetry (Cylinder Test)

The cylinder test utilizes the natural behaviour of exploration and rearing that is exhibited by rats when placed into a novel environment. As the rat explores the cylinder, the number of weight bearing touches on the surface of the cylinder is counted with each paw (10, 11). In our hands we have found that this test is susceptible to recovery in rats that have undergone 30-min MCAO.

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1. Place the rats in a Perspex cylinder (37.5  cm high, 18.5  cm diameter) and observe. Normal rats should exhibit vertical exploratory movements with approximately equal use of both forelimbs. 2. Film the animals for the first 20 touches or 10 min with a digital camcorder (Panasonic, UK) positioning the camera in front of the cylinder at a downward angle. Place mirrors behind the cylinder to observe the movements made by the animals when their backs are towards the camera (Fig. 7). 3. Score the first 20 forelimb contacts with the cylinder wall from the video footage on a standard television screen. Contacts should be scored as an independent ipsilateral or contralateral placement, or a simultaneous placement (which occurs when the paws are simultaneously applied to the wall). 4. Express the number of each type of placement as a percentage of the total number of placements. For example, [(ipsilateral + ½ both) divided by (ipsilateral + contralateral + both)] × 100. See Note 11 for further discussion regarding problems and solutions for this task.

Fig. 7. A rat exhibiting vertical exploration of the cylinder during the spontaneous forelimb use asymmetry test.

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1. Adverse effects of the intraluminal filament procedure: There are three things, beyond the control of the surgeon, that produce unwanted side effects. (a) It is possible to produce ischemic lesions in the brain that are not in the territory of the MCA. This occurs because the filament blocks a few small ICA branches that arise before it joins the Circle of Willis. The anterior choroidal artery perfuses the internal capsule and thalamus and the hypothalamic artery supplies the hypothalamus. Therefore, often ischemic damage can be observed in the thalamus or hypothalamus, the latter of which can lead to postischemic hyperthermia (4). (b) In situations where a large MCAO has been produced, intracranial pressure becomes elevated, which can increase the chances of mortality. In very severe situations, animals can cease eating, drinking, grooming and mobility. This highlights the importance of proper post-operative care. Generally, edema begins to subside around 4 days after the MCAO and the difference in appearance of these severe cases between 3 and 4 days can be like night and day. (c) This model is well known to have low reproducibility (4, 21). Insufficient MCAO will produce animals with negligible striatal insults, which can appear perfectly normal after the procedure. This can be avoided to some degree using Laser Doppler. Generally, a successful MCAO can be predicted when a decrease in Laser Doppler signal of approximately 80% from baseline and remains during the entire procedure. If there is only a slight decrease in LDF, or an increase over time after filament insertion, the animals may not have sufficient MCAO. Additionally, there is also one severe adverse effect that can be produced by the surgeon. A common pitfall of this technique is the potential production of subarachnoid haemorrhage (SAH) by advancing the filament too far (22, 23). There are several ways to avoid this. The first is to use the mark on the filament only as a guideline. If resistance is felt, stop advancement. Additionally, observation of the Laser Doppler signal can greatly assist with this (24). If the signal drops to less than 10% of baseline, it can indicate SAH. Confirmation can be obtained by slightly withdrawing the filament. If there is no SAH, the signal should increase again, whereas the signal should remain negligible if an SAH is produced.

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2. Modification of the intraluminal filament procedure: The technique described in Sect. 2.2.3 is based on the initial description of the intraluminal filament procedure (6). However, it is also possible to choose a different site for insertion of the filament rather than the CA. Filament insertion into a transected ECA has been suggested in order to minimize collateral blood flow (25). It remains unclear whether this actually serves that purpose; nevertheless, this strategy is routinely employed in many laboratories (Fig. 8). The ECA gives off a number of extracranial branches that partially supply the head, mandible, tongue, pharynx, scalp and ear (3), if flow to these structures is lost, it could produce unwanted side effects for the animals. One group has reported that animals with ECA transection exhibited more severe weight loss and reduced performance in a neurological score when compared to animals without ECA

Fig. 8. A depiction of the surgical site during the intraluminal filament technique. (a) The filament is in place and protruding from the external carotid artery stump. The insert depicts a higher magnification of this. (b) The left carotid artery is reperfused at the conclusion of the procedure.

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transection with or without the presence of masticatory lesions (26). The same group subsequently performed filament insertion via the CA; only the corresponding incision was sealed in such a way (sutures and fibrin sealants) that permanent closure of the vessel was not required, thus somewhat restoring blood flow (27). We have directly compared the two methods and found that ECA transection has detrimental effects on body weight following surgery as well as some behavioural tests, which could mistakenly be attributed to the brain infarct (Data in preparation for publication). 3. Histological assessment of ischemic lesions: One of the fastest and most common methods to visualize an ischemic insult quickly and/or to estimate infarct volume without tissue fixation and histology is to stain large brain slices with 2,3,5-triphenyltetra-zolium chloride (TTC) (Fig. 3). In a normal brain, TTC is converted to formazan (a red product) by mitochondrial oxidative enzymes. In ischemic tissue, there will be no production of formazan, and provided the insult has evolved for at least 24  h, TTC staining can be used to visualize the infarct. Generally, fresh brains are extracted, placed into a chilled brain matrix (World Precision Instruments, USA), and immersed in cool tap water. The matrix allows for positioning of razor blades at equal intervals so that 1–2 mm slices can be obtained. Sections can be submerged in 1% TTC solution until the pink colour materializes. This process will be faster with higher temperatures and longer incubation times. Once the pink colour emerges the sections can be photographed. TTC is light sensitive so it should be stored in dark or foil-wrapped containers and keeps for quite some time. When standard histology is preferred, for example, to perform immunohistochemistry, a hematoxylin and eosin (H&E) stain can be easily performed on any type of tissue section (fresh frozen, fixed, or paraffin embedded) to differentiate ischemic tissue (Fig.  9). Hematoxylin stains the nucleus of all cells whereas eosin stains the cytoplasm a pale pink colour. Infarct volume estimations are often calculated from the region of tissue with a pale pallor of the eosin staining prior to cystic formation (Fig. 9a). After cystic formation (Fig.  8b), tissue loss is generally quantified. Acutely (<24 h) H&E does not reliably delineate the injured tissue unless you can examine the morphology of the nucleus by looking at the hematoxylin staining. However, cell morphology is quite poor in fresh frozen tissue (Fig. 9c, d) compared to paraffin-embedded tissue in which pyknotic nuclei can be easily visualized as early as 4 h post-MCAO, despite the lack of decrease in eosin pallor (Fig. 9e, f). 4. Behavioural tests for 30 min of the intraluminal filament procedure: Within our laboratory, a number of tests have been

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Fig. 9. Haematoxylin and eosin (H&E)-stained tissue sections from rat brains subjected to middle cerebral artery occlusion (MCAO). (a) Eight (20  mm) coronal sections from fresh frozen tissue at 24-h post-MCAO. The light pallor of the staining indicates the ischemic tissue. (b) Eight (6 mm) coronal sections from fixed, paraffin-embedded tissue at 28 days post-MCAO. Note the atrophy and the cyst where the dead tissue was phagocytosed and replaced by cerebral spinal fluid. Higher magnification images from the intact (c) and ischemic (d) striatum of fresh frozen H&E stained tissue at 24-h post-MCAO. Higher magnification images from the intact (e) and ischemic (f) striatum from paraffin-embedded tissue at 4-h post-MCAO.

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assessed for use with the 30  min MCAO model (producing predominantly subcortical damage). Several of these have been found to be insensitive and animals are prone to spontaneous recovery. These include: the adhesive removal test (28), the forelimb inhibition swim test (29), gait analysis (30), tapered balance beam (28), the cylinder test (28), grip strength (31), and the corridor test (32). We have included the adhesive removal and cylinder tests in the methods section of this chapter, as these tests are regularly used in the literature and with larger lesions, and are robust. 5. Preparation and administration of apomorphine: Apomor­phine is photosensitive and degrades quickly (oxidisation detected by the solution turning green); so care must be taken to store it in the dark and once in solution it must be used quickly. Ascorbate should be added to the solution to prevent oxidation; however, we have found if the solution is used immediately this may be omitted. Care should be taken as apomorphine does not dissolve easily and this can be a lengthy process. To speed up this step we make a solution that is half the concentration of that which we would normally use (1 mg of apomorphine in 2 ml of saline), and shake vigorously, keeping the vial covered. Apomorphine should be administered subcutaneously. There are two reasons for this. Firstly, apomorphine is more potent when administered subcutaneously rather than by an intraperitoneal injection (33, 34). Secondly, if apomorphine is administered via an intraperitoneal injection, the onset of drug action is faster, with maximum brain concentration achieved 10 min after injection. Whereas via the subcutaneous route, the maximum concentration is achieved by 17–20 min follow injection (34). This allows more time for getting the rat into the equipment before the onset of rotational behaviour. However, this time window is still short, therefore each rat should be injected and then placed in the rotometers, and data collection commenced before the next rat is injected. 6. Issues with stepping and vibrissae-evoked paw placing tests: These tests are susceptible to unintentional experimental bias, as small alterations in the technique, in particular the angle at which the rat is held, can alter the number of adjusting steps. Therefore, it is absolutely necessary that the experimenter is blinded to which animals are lesioned and/or treated. It is also essential that the same well-trained experimenter is used for all animals within a study. It is possible to distinguish the motor initiation deficit from the sensory deficit by using the crossover version of the task (35). This involves restraining the rat in such a way that it reaches for the bench with the paw opposite to stimulated whiskers. 7. Considerations for using the disengage task: One of the most important factors is that the rats are well habituated to the test

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environment. If the rat is engaged in exploratory behaviours they will not respond to the stimulus promptly. A cage with multiple holes drilled around the edge can be used for testing, if this is also the home cage it will reduce exploration (20). The size of the piece of chocolate given to the rat is also critical. It should not be so large that the rat cannot hold it between its forepaws. However, it should be large enough that the rat does not finish consuming the chocolate before the maximum time to respond is reached. We have found that using food-restricted rats is not advisable for the disengage task. The rats become very focused on the chocolate and even intact animals do not respond to the sensory stimulus. It is also advisable to keep the number of test sessions administered to a minimum, as the rats become used to the test and are less likely to respond when the stimulus is presented. 8. Training and modifications to the paw-reaching protocol: It is wise to train rats on the paw-reaching task prior to surgery. Training can take as long as 4  weeks and once animals have undergone MCAO surgery it can be hard to motivate them to learn to reach. It is not impossible to train animals with infarcts, but it will make the data more variable. Collecting baseline data prior to surgery also means that rats can be evenly grouped according to performance, as there will be variations even in the intact rats. Additionally, the number of sugar pellets used can be increased, and it has been argued that using a higher number of pellets per well is more sensitive (36). Other modifications of the task include colouring the pellets for each step, making it is possible to tell exactly how many pellets from each step have been dropped (37). 9. Issues and additions to the bilateral asymmetry test: The bilateral asymmetry test is highly susceptible to repeated testing issues. We have found that rats become unresponsive to the stimuli if the test is used over multiple time points. Additionally, there should be a number of weeks between each test point. For stimuli, we currently use masking tape cut into 7 mm × 7 mm squares; however, sticky dots can be used. Evaluation of the stickers should be carried out prior to testing, as we have found that some of these stickers are not adhesive enough. A more complex variation of the bilateral asymmetry task can be used to assess the magnitude of the sensory stimulus. To do this, once it has been established that the rat has a sensory asymmetry due to an injury, the size of the stimulus placed on the contralateral limb is increased and the size of the stimulus on the ipsilateral stimulus is decreased, until a sensory bias is no longer evident. The ratio of contralateral to ipsilateral limb gives a measure of the magnitude of the sensory asymmetry (8). 10. Spontaneous forelimb use asymmetry, problems and solutions: This is another test that can be detrimentally affected by repeated

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testing. The more the rat is exposed to the cylinder, the less it will explore. Also, on the day following surgery, many rats do not explore sufficiently to meet the 20 touch criteria. There are a number of things that can be tried to induce more exploratory behaviour. Testing the rats in a room with low or red light will improve the levels of exploration. Using a different test room for each time-point, therefore changing the environment outside the cylinder will encourage rearing. Also turning the lights on and off periodically during testing can encourage exploration. If you are testing over multiple time points, it is advisable to only leave the rat in the cylinder for the required 20 touches; therefore reducing the overall exposure to the cylinder. When scoring the cylinder, it is important that only decisive weight bearing touches on the side of the cylinder are scored. For example, it the rat performs a weight-bearing touch and then drags its paw down the edge of the cylinder touching it multiple times in a short period of time, without bearing weight on it, this should only count as one touch. It is important that the scorer is blinded and has a solid understanding of what constitutes a weight-bearing touch and what should be discounted.

Acknowledgements Our studies have been supported by grants from the European Union Framework 6 program (LSHB-CT-2006-037526, StemStroke), the Medical Research Council, the Alexander von Humboldt Foundation (TDF), and Cardiff University International Collaboration Award (RCT). References 1. Dirnagl U, Iadecola C, Moskowitz MA (1999) Pathobiology of ischaemic stroke: an integrated view. Trends Neurosci. 22: 391–397 2. Hossmann KA (2006) Pathophysiology and therapy of experimental stroke. Cell Mol. Neurobiol. 26: 1057–1083 3. Paxinos G (1995) The rat nervous system. Academic Press, San Diego 4. Carmichael ST (2005) Rodent models of focal stroke: size, mechanism, and purpose. NeuroRx. 2: 396–409 5. Hossmann KA (2011) Introduction to the pathophysiology of stroke. In vivo imaging of animal models. Springer Publishing, New York, in press 6. Koizumi J, Yoshida Y, Nakazawa T, Ohneda G (1986) Experimental studies of ischemic brain

edema, I: a new experimental model of cerebral embolism in rats which recirculation can be introduced in the ischemic area. Japanese Journal of Stroke 8: 1–8 7. Dirnagl U, and members of the MCAO-SOP Group (2009) Standard operating procedures (SOP) in experimental stroke research: SOP for middle cerebral artery occlusion in the mouse. Nature Proceedings hdl:10101/ npre.2009.3492.1 8. Schallert T, Whishaw IQ (1984) Bilateral cutaneous stimulation of the somatosensory system in hemidecorticate rats. Behav. Neurosci. 98: 518–540 9. Schallert T, Upchurch M, Lobaugh N, Farrar SB, Spirduso WW, Gilliam P, Vaughn D, Wilcox RE (1982) Tactile extinction: distinguishing

6  Functional Assessment of Subcortical Ischemia between sensorimotor and motor asymmetries in rats with unilateral nigrostriatal damage. Pharmacol. Biochem. Behav. 16: 455–462 10. Barth TM, Jones TA, Schallert T (1990) Functional subdivisions of the rat somatic sensorimotor cortex. Behav. Brain Res. 39: 73–95 11. Bland ST, Schallert T, Strong R, Aronowski J, Grotta JC, Feeney DM (2000) Early exclusive use of the affected forelimb after moderate transient focal ischemia in rats : functional and anatomic outcome. Stroke 31: 1144–1152 12. Montoya CP, Campbell-Hope LJ, Pemberton KD, Dunnett SB (1991) The “staircase test”: a measure of independent forelimb reaching and grasping abilities in rats. J. Neurosci. Methods 36: 219–228 13. Schallert T, Norton D, Jones TA (1992) A clinically relevant unilateral rat model of Parkinsonian akineasia. J Neural Transpl. Plast. 3: 332–333 14. Olsson M, Nikkhah G, Bentlage C, Bjorklund A (1995) Forelimb akinesia in the rat Parkinson model: differential effects of dopamine agonists and nigral transplants as assessed by a new stepping test. J. Neurosci. 15: 3863–3875 15. Ungerstedt U, Arbuthnott GW (1970) Quantitative recording of rotational behavior in rats after 6-hydroxy-dopamine lesions of the nigrostriatal dopamine system. Brain Res. 24: 485–493 16. Schallert T, Hall S (1988) ‘Disengage’ sensorimotor deficit following apparent recovery from unilateral dopamine depletion. Behav. Brain Res. 30: 15–24 17. Modo M, Stroemer RP, Tang E, Veizovic T, Sowniski P, Hodges H (2000) Neurological sequelae and long-term behavioural assessment of rats with transient middle cerebral artery occlusion. J. Neurosci. Methods 104: 99–109 18. Schallert T, Woodlee MT, Fleming J (2002) Disentangling multiple types of recovery from brain injury. In: Krieglstein J (ed) Pharmacology of cerebral ischemia. Medpharm Scientific Publishers, Stuttgart, pp 201–216 19. Fricker RA, Annett LE, Torres EM, Dunnett SB (1996) The placement of a striatal ibotenic acid lesion affects skilled forelimb use and the direction of drug-induced rotation. Brain Res. Bull. 41: 409–416 20. Hall S, Schallert T (1988) Striatal dopamine and the interface between orienting and ingestive functions. Physiol. Behav. 44: 469–471 21. Laing RJ, Jakubowski J, Laing RW (1993) Middle cerebral artery occlusion without craniectomy in rats. Which method works best? Stroke 24: 294–297 22. Bederson JB, Germano IM, Guarino L (1995) Cortical blood flow and cerebral perfusion

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pressure in a new noncraniotomy model of subarachnoid hemorrhage in the rat. Stroke 26: 1086–1091 23. Kuge Y, Minematsu K, Yamaguchi T, Miyake Y (1995) Nylon monofilament for intraluminal middle cerebral artery occlusion in rats. Stroke 26: 1655–1657 24. Schmid-Elsaesser R, Zausinger S, Hungerhuber E, Baethmann A, Reulen HJ (1998) A critical reevaluation of the intraluminal thread model of focal cerebral ischemia: evidence of inadvertent premature reperfusion and subarachnoid hemorrhage in rats by laser-Doppler flowmetry. Stroke 29: 2162–2170 25. Longa EZ, Weinstein PR, Carlson S, Cummins R (1989) Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke 20: 84–91 26. Dittmar M, Spruss T, Schuierer G, Horn M (2003) External carotid artery territory ischemia impairs outcome in the endovascular filament model of middle cerebral artery occlusion in rats. Stroke 34: 2252–2257 27. Dittmar MS, Vatankhah B, Fehm NP, Retzl G, Schuierer G, Bogdahn U, Schlachetzki F, Horn M (2005) The role of ECA transection in the development of masticatory lesions in the MCAO filament model. Exp. Neurol. 195: 372–378 28. Schallert T (2006) Behavioral tests for preclinical intervention assessment. NeuroRx. 3: 497–504 29. Stoltz S, Humm JL, Schallert T (1999) Cortical injury impairs contralateral forelimb immobility during swimming: a simple test for loss of inhibitory motor control. Behav. Brain Res. 106: 127–132 30. D’hooge R, Hartmann D, Manil J, Colin F, Gieselmann V, De Deyn PP (1999) Neuromotor alterations and cerebellar deficits in aged arylsulfatase A-deficient transgenic mice. Neurosci. Lett. 273: 93–96 31. Dunnett SB, Torres EM, Annett LE (1998) A lateralised grip strength test to evaluate unilateral nigrostriatal lesions in rats. Neurosci. Lett. 246: 1–4 32. Dowd E, Monville C, Torres EM, Dunnett SB (2005) The Corridor Task: A simple test of lateralised response selection sensitive to unilateral dopamine deafferentation and graft-derived dopamine replacement in the striatum. Brain Res. Bull. 68: 24–30 33. Barros HM, Braz S, Carlini EA (1989) Behavioural manifestations elicited by apomorphine, influence of the route of administration. Pharmacology 38: 335–340 34. Melzacka M, Wiszniowska G, Daniel W, Vetulani J (1979) Behavioral effects and cerebral pharmacokinetics of apomorphine in

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the  rat: dependence upon the route of administration. Pol. J. Pharmacol. Pharm. 31: 309–317 35. Woodlee MT, Asseo-Garcia AM, Zhao X, Liu SJ, Jones TA, Schallert T (2005) Testing forelimb placing “across the midline” reveals distinct, lesion-dependent patterns of recovery in rats. Exp. Neurol. 191: 310–317 36. Cordeiro KK, Jiang W, Papazoglou A, Tenorio SB, Döbrössy M, Nikkhah G (2010) Graft-

mediated functional recovery on a skilled forelimb use paradigm in a rodent model of Parkinson’s disease is dependent on reward contingency. Behav. Brain Res. 212: 187–195 37. Kloth V, Klein A, Loettrich D, Nikkhah G (2006) Colour-coded pellets increase the sensitivity of the staircase test to differentiate skilled forelimb performances of control and 6-hydroxydopamine lesioned rats. Brain Res. Bull. 70: 68–80

Part II Neo- and Allo-Cortical Systems

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Chapter 7 Functional Organization of Rat and Mouse Motor Cortex G. Campbell Teskey and Bryan Kolb Abstract Here we review both modern and emerging approaches that have lead researchers to make inferences about the functional organization of the rat and mouse motor cortex. We primarily focus on the use of cortical lesions and intracortical microstimulation (ICMS) in rats and mice because rats are the species that have undergone the most experimentation in recent decades and mice because they are highly amendable to a variety of genetic manipulations. We discuss the basic methodological approach and a series of electrical stimulation parameters that affect the derivation of the motor map. Key words: Rat, Mouse, Intracortical microstimulation, Movement, Motor map, Motor deficits

1. Introduction There is a long and rich history filled with both theory and experimentation asking the question, “What is the function of motor cortex?” Some have argued that the role of motor cortex is essentially to produce individual muscle movements whereas others have taken the position that it is to coordinate multiple muscle groups. From our perspective, the answer is that networks of interconnected neurons within the motor cortex in concert with subcortical structures produce the complex array of motor behaviours. We describe below how within motor cortex the role of interconnected neurons that generate behaviour is determined in large part by how the experimentation has been carried out, with short bursts of electrical stimulation producing simple movements and long trains of stimulation producing complex movements. Before we address the details of the methodological approaches, we should anatomically define motor cortex. Motor cortex is any and all areas of the six-layered neocortex that contain neurons

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which send their axons directly to the spinal cord. These corticospinal neurons can be found in traditionally named cortical areas such as cingulate cortex, granular (sensory) cortex and agranular (motor) cortex (1–3). The presence of a large layer IV, which has traditionally been named sensory cortex, does not preclude the presence of corticospinal cells and should also be defined as motor cortex when corticospinal neurons are present – in opposition to the common practice found in many stereotaxic atlases. In rats and mice, the term that is commonly used is sensorimotor cortex because of the mixed area that receives sensory input via the thalamus to layer IV and the presence of corticospinal projections with cell bodies in layer V. It is worth noting that this is also true in people. Forty percent of corticospinal neurons arise from the postcentral gyrus corresponding to Brodmann areas 1, 2, and 3, also known as sensory cortex (4). This is an important fact that should be carefully considered by those who hold chauvinistic views against rodents as useful models for understanding the organization of brain and behaviour. Historically, and still today, there have been two primary methods of studying motor cortex function (1) injuring the motor cortex; and (2) electrically stimulating the motor cortex. We consider the history of each before describing the methods currently used. 1.1. Motor Cortex Injury

The study of people with naturally occurring injuries to the cerebrum has a very long history but it was in the mid-1800s that clinical neurologists began to correlate functional outcomes with post mortem pathology. Perhaps the most famous example is Broca, who described severe aphasia in a patient who turned out to have a large injury to the left inferior frontal cortex (i.e., Broca’s area). The modern era of examining the effects of motor cortex injury on behaviour can be traced to the studies of Lawrence and Kuypers (5, 6). These authors cut the corticospinal tracts of monkeys at the level of pyramidal decussation in the medulla. Although there were many transient motor symptoms, there were two striking chronic deficits related to the use of the forelimb. First, the animals were unable to grasp food or objects with a pincer grasp of the thumb and index finger opposed. Second, once they had grasped the food with a whole-hand grip, they had difficulty releasing it. They would root for the food in their hands with their snouts. A later study by Passingham et al. (7) unilaterally removed the primary motor cortex and primary somatosensory cortex, from which a majority of corticospinal projections originate in monkeys. Although there were chronic deficits in speed and strength of movements, like Lawrence and Kuypers’s monkeys, the corticallyinjured monkeys could only make whole-hand grasping movements. The authors also showed that the monkeys were unable to make rotary movements of their wrist or elbow to grasp food if

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they were forced to reach through a hole in a clear plastic wall. Thus, taken together, the monkey studies show that the cortex is necessary for independent finger movements as well as for making skilled movements around the individual joints in the forelimb. 1.2. Motor Cortex Injury in Rats and Mice

The first study of forelimb use in rats or mice after motor cortex injury was done by Peterson and Francarol (8). These authors found that motor cortex injury shifted “handedness” in pretrained rats. Later, Castro (9) argued that motor cortex lesions disrupted reaching because the animals were impaired at using the digits, although he had no direct measure of this. Similarly, Kolb and Whishaw (10) reported that motor cortex lesions resulted in clumsy use of the forepaws as preoperatively trained rats had difficulty in opening latches on doors with their paws after the injury. They also had difficulty in manipulating large pieces of food and often resorted to standing on the food in order to gnaw it. Preinjury, they would sit on their hindpaws and hold the food in their forepaws to eat. An extensive series of studies by Whishaw and many colleagues (see reviews in (11–13)) shows that, like monkeys, rats with motor cortex lesions have many transient deficits but the most severe chronic difficulties are seen in grasping food. Specifically, rotary movements of the limb used in pronation and supination of the forepaw are supplanted by rotatory movements of the trunk. Trunk rotation is thus used as a crutch for the impaired rotatory movements of the limb. Although less well studied, it is clear that mice have similar forelimb and digit deficits after motor cortex injury when they are examined on similar tests to the Whishaw rat tests. There are three other striking chronic motor deficits after motor cortex injury in rats. First, there is an impairment in tongue and mouth control – the animals are unable to protrude their tongue past their teeth and they have difficulties in making fine mouth movements such as in manicuring the toe nails (14). Importantly, however, these deficits are highly specific to damage in the tongue or face area (15). Second, rats with motor cortex injuries have a change in forelimb use in swimming. Rats normally hold their forelimbs still under their chin when they swim (see Fig. 1) but after motor cortex injury there is a release of forelimb movements such that the animals “dog paddle” (10). Finally, injury to the hindlimb region leads to impairments in fine foot placing in uneven terrain or on narrow beams (10, 16). Motor cortex injury provides a useful technique not only for understanding the functions of the motor cortex but also for studying the effectiveness of rehabilitative treatments in stimulating functional recovery. We shall outline the current methods of injuring motor cortex and techniques of studying the evolution of functional outcomes after the injury.

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Fig. 1. Examples of behavioural measures used to identify motor dysfunctions after injury to the motor cortex. (a) Cylinder test. (b) Skilled reaching test. (c) Forepaw inhibition in swimming. (d) Sunflower seed opening. (e) Tongue extension. See text for details.

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The first documented use of a functional approach using electrical stimulation was carried out by Fritsch and Hitzig (17). They stimulated the cortical surface of dog brains with short DC discharges from a battery and reported five areas that gave rise to different types of movements. A few years later, Ferrier reported his experiments with dogs, cats, rabbits, guinea pigs and later with monkeys where he used seconds of continuous alternating current to induce movements (18, 19). Ferrier is credited as describing “purposive movements” and the first somatically arranged motor map. These two sets of researchers argued over the “correct” method of stimulation. Ferrier took the position that shorter trains of stimulation did not allow complex movements whereas Hitzig countered that longer stimulation trains evoked seizures (20). During the later part of the nineteenth century, Beevor and Horsley (21) working with monkeys used both short trains to elicit a muscle twitch and long trains to create a map of purposeful movements. They argued that both approaches were valuable ways to view cortical organization. While their later work was primarily based on short trains they also demonstrated that careful removal of the gray matter and stimulation of the remaining descending white matter tracts produced the same map of the body as did short train stimulation of the cortex itself. These influential researchers appear to have set the research field on a course typified by the view that the function of the motor cortex was defined by the simple activation of the descending connections to the spinal cord. Sherrington made many important insights into the function of motor cortex by stimulating the surface of the motor cortex with short bursts of electrical stimulation at the lowest intensity possible that allowed the observation of a movement. He examined the relation between cortical points by stimulating one site and observing the movement evoked by stimulation at another site. He noted that prior stimulation of one site could alter the movement at another site. Even though he was using short bursts of stimulation he reached the important conclusion that the map was not anatomically fixed but plastic and dependent on its activation history. That is to say the motor cortex is the repository of a large variety of sequences of movements (22). Several researchers still using cortical surface stimulation with short trains in primates, including humans, detailed multiple cortical maps (23–27). However, the “problem” with surface stimulation is that it requires relatively high intensity stimulation currents to elicit movements. Thus according to the view that larger currents lead to more current spread and deriving a map should not involve overlapping areas of excitation, the technique of surface stimulation leads to a relatively low resolution motor map. In order to stimulate with smaller current intensities and derive the highest resolution motor map possible, Asanuma pioneered the intracortical microstimulation technique for deriving movement representations.

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In the late 1960s and early 1970s, Hiroshi Asanuma and c­ olleagues worked in the motor cortex of cats (28, 29) and capuchin monkeys (30). They inserted fine insulated microelectrodes with an exposed tip into different layers of the motor neocortex and passed a minimal amount of cathodal (negative charge) current with short pulse-widths (0.2 ms) and short trains (40 ms trains of 13 pulses at 300 Hz) to elicit a muscle or joint movement. On average their current intensities were 1/100th the intensity of those used to evoke movement on the cortical surface. These “standard” short train methodologies have been successfully used for decades and have generated a substantial literature on the functioning of motor cortex. Hall and Lindholm (31) were the first to adopt the short train ICMS technique to work in rats. In the 2000s, Michael Graziano reinvigorated the use of long-train intracortical microstimulation methodologies. Using stimulation trains an order of magnitude longer than traditional ICMS (500  ms versus <50  ms), he evoked complex, coordinated movements involving multiple joints in monkeys (32, 33). The train duration used was similar to both the timescale of forelimb reaches and motor cortex neuron activity during movement in monkeys (34). It appears that train duration is a critical factor mediating evoked movements by ICMS. At behaviourally relevant time scales, complex movements similar to spontaneous behaviour are evoked. As train duration is reduced, the movements become truncated, eventually leading to individual joint flexions. Long-train ICMS has more recently been successfully applied in the rat (35). ICMS provides a valuable tool in the study of both the functional organization and sensitivity of cortical movement representations in addition to the nature and property of cortical-evoked movements (36). Below we provide a detailed description of the use of short-train stimulation techniques in both rats and mice and long-train stimulation techniques in rats.

2. Studying the Effects of Motor Cortex Injury 2.1. Inducing Injury

The motor cortex can be damaged selectively in a wide variety of ways, each of which requires slightly different equipment (Table 1). In most procedures a craniotomy is performed and the motor cortex is removed or damaged by gentle aspiration, electrocoagulation with a metal electrode, or stripping away of the fine surface blood vessels to produce ischemia (pial stripping). Other less focal procedures include either temporary or permanent occlusion of the middle cerebral artery. The cortex can also be damaged by injecting drugs that produce vasoconstriction such as endothelin-1 (ET-1). A final procedure is to give an i.v. injection of a photosensitive dye, Rose Bengal. When the skull of Rose-Bengal treated rats is illuminated with a

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Table 1 Equipment required for making selective motor cortex lesions Procedure

Specialized equipment

Suction

Aspiration pump, tubing and glass pipette

Electrocoagulation

Stainless steel jor platinum electrode, current generator

Pial stripping

Sterile cotton swab

MCA occlusion

Either vessel clamps (transient) or electrocautery machine (chronic)

Endothelin-1

Injection syringe and infusion pump

Rose Bengal

Fiber optic light system to deliver focused light

All procedures require a rodent stereotaxic device. All but Rose Bengal require fine surgical tools

focused beam of light for 10 min, cortical thrombosis is induced in the underlying tissue, at least partly due to the induction of apoptosis. The advantage of Rose Bengal is that no craniotomy is necessary. In those procedures using a craniotomy, the skull is exposed and the size of the desired motor injury is marked on the skull. The bone is thinned with a high speed drill and then the remaining bone is removed with fine rongeurs. The size of the opening will vary with the region of interest (see maps below). For aspiration, electrocoagulation, and pial stripping, the dura is then cut with a pointed (#11) scalpel blade and removed. We have found it best to also cut through the grey matter along the edges of the craniotomy in order to make it easier to delineate the size of the injury. The aspiration procedure involves using gentle suction and removing only the grey matter within the delineated region. For pial stripping, the cotton ball is dipped in sterile saline and then used to gently wipe off the surface blood vessels (e.g., (37)). Electrocoagulation is performed by delivering a 1-mA anodal current through an uninsulated stainless steel or platinum electrode during repeated (15 s each) traverses through the exposed cortex for a total of 2 min (e.g., (38)). There are several methods for the application of ET-1. These include the topical application across the surface of the cortex in the region of the craniotomy, multiple injections of the ET-1 into the grey and/or white matter, and injection of ET-1 adjacent to the middle cerebral artery. Dosages vary considerably across laboratories and require pilot work but, in general, the higher the dose, the larger the injury (see details in (39)). Occlusion of the middle cerebral artery (MCA) can be done at different points on the MCA beginning at the point it courses upward from the base of the brain, which produces significant cortical and

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subcortical injury, or at any point along the MCA as it spreads out over the cortex (e.g., (40)). The more the focal injuries of the cortex are seen, the more distal the occlusion is performed. A permanent occlusion is made by using electrocoagulation of the artery whereas transient occlusions are made by placing clips on the artery for 10–20 min and then removing the clips. The former procedure mimics a permanent blockade of the MCA, such as with a large emboli, whereas the latter procedure mimics transient attacks such as in vasospasm. For the Rose Bengal procedure, the photochemical Rose Bengal is infused into the femoral vein via a microinjection pump within 2 min (20 mg/kg). A light source is then positioned close to the skull, focussed on the desired area of cortex and turned on for 10 min. If the light is not cold (fiber optic), then the skull surface temperature must be monitored and the skull kept cool by cool air flow (e.g., (41)). 2.2. Measuring Behavioural Changes

There are many techniques to measure motor functions (see (12, 42) for extensive discussions). The choice of behavioural measures varies with the questions being addressed, however. In particular, studies in which the focus is on the details of behavioural changes in order to infer functions of the motor cortex typically use more sophisticated measures (see (12)) than studies looking for treatments to stimulate functional recovery. Most of our work has been in the latter category and we have developed a test battery to allow us to measure both forelimb and oral/facial functions (see Fig. 1). These measures are sometimes supplemented with measures of hindlimb coordination as rats traverse either a narrow beam (10) or a horizontal ladder (16). Similar measures can be used for mice, although the apparatus must be scaled down. We have been surprised to discover that all of the lesion techniques produce strikingly similar deficits with the severity varying mostly with the extent of injury rather than the etiology (37, 39). One important difference, however, is that the compensatory changes in the cortex vary depending upon the lesion etiology (37, 43). The simplest way to study this is to examine dendritic length and spine density using Golgi-type analyses of neuronal morphology (e.g., (37, 38)). For example, whereas the pial stripping stimulated dendritic growth in pyramidal cells in layer V of the forelimb region of the undamaged hemisphere, suction ablation did not. In contrast, suction increased spine density in the pyramidal neurons in the anterior cingulate cortex but pial stripping did not. Such differences could be important in designing rehabilitation procedures.

2.3. Cylinder Test (Forepaw Asymmetry)

Forelimb use for weight support during explorative activity is examined by placing individual rats in a transparent cylinder 20 cm in diameter and 30  cm high for 3  min (44). A mirror is placed underneath the cylinder at an angle to allow the observer to videotape the animal’s activity from a ventral point of view. Forelimb use

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is measured during vertical exploration. Each forepaw contact with cylinder wall is counted. An asymmetry score of forelimb use in wall exploration is calculated for each animal [(affected forelimb)/ (affected + unaffected)] to obtain a score where 0.5 represents perfect symmetry and any number closer to zero would suggest a decrease in the use of the affected limb. 2.4. Skilled Reaching

Training boxes for reaching are made of clear Plexiglas with the dimensions 26 cm high, 28 cm deep and 19 cm wide. The fronts of boxes are constructed of 2 mm bars separated from each other by a 9-mm gap. Animals are trained to reach for food fragments weighing ~30 mg each. Reaching success is calculated by dividing the number of successful reaches (food grasped and eaten) by the total number of reaches. It takes about 15 days for animals to reach a consistent baseline, which is about 60–70% for rats and 40–50% for mice.

2.5. Sunflower Seed Opening

The ability of animals to successfully open and consume seeds using their limb and digits is measured following procedures of Whishaw et al. (45). The animals are given experience with sunflower seeds for several days before testing. Five sunflower seeds are placed in a corner of a Plexiglas box (45 cm × 14 cm × 35 cm) that the animals have been accustomed to eating the seeds in. Their behaviour was video recorded on the fifth day. Two measures used to quantify the behaviour are videotaped and two measures recorded (1) time: the total amount of time spent on manipulating, opening the shell and consuming the seeds and (2) the number of pieces of shells after animals retrieve and eat the seeds. If the animal shredded the shell into pieces too small to count, a maximum of 30 pieces of shell is assigned.

2.6. Swimming Task

Rats are trained for several days to swim to a visible platform located at one end of a rectangular aquarium (120  cm × 43  cm × 50  cm) filled with warm water. During training, the animals are released from the opposite end of the pool and are given practice until they learn to swim directly to the platform without touching the pool walls. Normal rats and mice hold their forepaws immobile under their chins while swimming and use their hind limbs to propel through the water. Animals are videotaped on three swims and the number of forelimb strokes with either paw is counted. Normal rats typically make no strokes whereas normal mice are more variable and usually make some strokes.

2.7. Tongue Extension

This task requires animals to stick out their tongue to lick palatable food, which has been spread on a ruler (15). Animals are placed into the reaching boxes with the food tray removed. We have had good success with both peanut butter and a slurry of warm water and chocolate chip cookie. Normal rats can extend their tongues up to 12 mm whereas mice can reach about 3 mm.

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3. Standard “Short Train” Intracortical Microstimulation Methods

Table 2 lists the equipment necessary to perform ICMS in rats and mice.

3.1. Equipment 3.2. Electrodes

Insulated electrodes made from either glass or tungsten are typically used. The more traditional glass electrodes are created by heating and pulling borosilicate glass capillaries on a micropipette puller. The advantage of this system is that the electrodes can be custom made to have specific tip sizes, angles and impedances. The glass electrodes are filled with between 3.0 and 3.5 M NaCl and normally have impedance values in the 1–1.5 MW range. Tungsten electrodes are commercially available and can be reused for extended periods of time.

3.3. Anesthesia

Mapping at the correct anesthetic depth is probably the biggest “trick” to deriving an excellent map. This is because the anesthesia itself likely has the largest influence on the amount of current needed to induce a movement. For ethical reasons, the animal must be sufficiently anesthetized to prevent pain and yet if the anesthetic level is too deep then it may not respond to ICMS at all. It is critically important to revisit responsive points over the course of mapping the cortex and re-derive some of them to determine if the anesthesia level is fairly constant. A few physiological indices

Table 2 Equipment required for intracortical microstimulation experiments Rodent stereotaxic apparatus Microscope with camera connected to a computer Computer with imaging software Stimulator with monophasic and biphasic stimulation capability Microdrive Servo-controlled warming blanket Electrode puller and beveller if using glass electrodes Drill and fine surgical tools Disposables (liquid silicone to protect brain surface) Video camera and movement analysis software Amplifiers and filters for EEG and EMG analysis

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that can also be used as feedback indicating anesthesia level are breathing rate and vibrissae whisking as well as a foot reflex (in rats) or a tail reflex (in mice) in response to a gentle pinch (3, 46). Recording EEG and examining the amount of power at several frequencies in real time is also recommended to ensure that anesthesia levels are fairly constant (47). Deriving motor maps requires an anesthetic agent that has the following general characteristics (1) it maintains muscle tone so that stimulation-induced movements can be detected, (2) it has metabolic kinetics that give rise to a fairly constant anesthetic depth over several hours, (3) it allows a balance between excitation and inhibition so that reasonable levels of stimulation current intensities can be applied directly to layer V, and (4) it has a wide therapeutic window making it difficult to inadvertently kill the animal. For rats, researchers commonly use a combination of the NMDA receptor antagonist ketamine and the alpha-2 adrenergic agonist xylazine. Ketamine provides anesthesia and some analgesia while xylazine provides sedation, anesthesia and a little muscle relaxation. Rats initially receive an intraperitoneal (i.p.) injection of ketamine (100  mg/kg) and xylazine (5  mg/kg). Supplemental injections are required with either ketamine alone (25 mg/kg), or a cocktail of both ketamine (17 mg/kg) and xylazine (2 mg/kg) and are delivered i.p. as required throughout the surgical procedure to maintain a relatively constant level of anaesthesia. It is critically important to keep careful and detailed notes on the amount of drugs given to the animals and the length of the surgical procedure because the quantity of anesthetic needs to match between control and experimental conditions. Again, this is because the depth of anesthesia influences the amount of current used to determine a movement threshold. Mice will succumb if they are given the same dosage of ketamine and xylazine as rats. We have had success with an initial i.p. injection of ketamine (20  mg/kg) and xylazine (1  mg/kg). Supplemental injections of ketamine alone (5 mg/kg), or a cocktail of both ketamine (3.4  mg/kg) and xylazine (0.4  mg/kg) should be delivered i.p. as required throughout the surgical procedure to maintain a constant level of anaesthesia. 3.4. Craniotomy

Using a drill a craniotomy is performed by removing the skull and exposing the sensorimotor neocortex of one hemisphere. In rats this window extends approximately 4 mm anterior and 3 mm posterior from bregma and from midline to 5 mm lateral of midline. In mice, the window extends approximately 4  mm anterior and 3 mm posterior from bregma and from midline to 3 mm lateral of midline – yes, mice have proportionately bigger maps than rats. These windows allow for a complete mapping of the forelimb area and the window can be extended if the researchers are interested in probing for other body movements. The brain will often swell following the craniotomy. In order to reduce the pressure caused by

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edema, an 18-gauge needle is used to make a small puncture in the cisterna magna. Dura is then carefully removed from the cortical surface and a silicone fluid at body temperature is used to cover the neocortical surface to keep it from desiccating. 3.5. Stimulation Grid and Interpenetration Distance

A digital image of the exposed portion of the brain should be captured and displayed on a computer (Fig. 2). This image can then be overlaid with a grid. The grid directs the location of the electrode penetration points at the intersections of the grid lines and at a central point in the middle of each square except when a blood vessel is present at a penetration point, in which case, the point is not derived. The size of the grid should be a function of the current spread. Assuming a sphere of electrical activation, estimated effective current spread may be expressed by i = kr2; where i = applied current and r = distance from the electrode and k is a tissue-dependent current-­distance constant (48, 49). Since the vast majority of points have a movement threshold intensity of less than 40 mA we use that

Fig. 2. Photographic image of part of the surface of the rat left hemisphere with skull and dura removed. Pial vessels are clearly shown. A 500 by 500 mm grid (grey lines) is overlaid on the image. Coloured circles represent penetration points of the electrode (black = non-responsive, green = wrist, red = digit, dark blue = elbow, light blue = shoulder, pink = whisker, purple = jaw, yellow = neck, grey = trunk, white = hindlimb). The numbers represent the order in which the points were derived. The vertical yellow line indicates bregma. Left is anterior, right is posterior, top is medial, bottom is lateral.

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value for i. A k-value of 1,292 mA/mm2 has previously been used (49) but the reader should be aware that k-values can range (50). Given those values then the average radius of estimated effective current spread is 176 mm. In order to avoid overlapping penetration, that radius value needs to be doubled to yield 353 mm. Thus we employ a grid of 500 mm squares. This yields a minimum interpenetration distance of 353 mm (remember one point is derived in the centre of the grid squares). In mice a 250-mm grid has been employed (3). More recent evidence suggests that ICMS may elicit direct axonal activation leading to a much larger activation of sparse and distributed neuronal populations (51). Certainly more research into this question needs to be done. Nonetheless, interpenetration distance should be carefully considered to provide a balance between spatial resolution and the effects of current spread and repeated stimulation on acute changes in evoked responses (52). 3.6. Cortical Depth (Rats and Mice)

Movements can be driven from the cortical surface but as mentioned previously, the stimulation intensities are on the order of one hundred times larger than those applied directly to layer V. Anodal stimulation is generally more effective at a distance from the excitable site whereas cathodal stimulation is more effective close to the excitable site – an observation that we have confirmed in rats (53). Figure 3 shows that cathodal stimulation is generally more effective in both rats and mice as demonstrated by eliciting lower movement thresholds. In rats the most effective depth is between 1,400 and 1,700 mm from surface which corresponds to the cell bodies of layer V and their dendrites – the output layer of

Fig. 3. Mean movement threshold for neocortical depth versus current polarity in rats (left panel ) and mice (right panel ). Graphical representation of anodal (squares) and cathodal (circles) mean current intensities (±SEM) required to elicit forelimb movements at the indicated neocortical depth from surface. The graphs show that anodal current is more effective at eliciting forelimb movements at lower intensities at superficial neocortical depths. Cathodal current is more effective at eliciting forelimb movements at lower current intensities at deeper neocortical depths that correspond to the cell bodies and dendrites of layer 5.

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Fig. 4. Mean movement threshold by frequency of ICMS stimulation in rats (large circles ) and mice (small circles ). Mean movement thresholds (±SEM) were plotted according to the frequency of the ICMS stimulation, holding other stimulation parameters constant.

the motor neocortex. In mice, the most effective depth is between 600 and 1,000 mm (53). Stimulation sites can be marked by small electrolytic lesions or dye infusion through the micropipette. 3.7. Stimulation Parameters 3.7.1. Frequency

When the electrode tip is placed at 1,500 mm in rats and 800 mm in mice and the frequency of stimulation, within the 40 ms stimulation train, is varied, the following response curve displayed in Fig.  4 is generated. The optimal stimulation frequency is 250– 300  Hz in rats whereas in mice the curve is quite flat with little meaningful difference between 100 and 500 Hz. Mice appear to have slightly lower movement thresholds at the optimal frequencies compared to rats.

3.7.2. Number of Pulses (Train Duration)

When the frequency is held constant but the number of pulses in the train is varied, the optimal train duration is around 40 ms or 13 pulses. Mice, on the other hand, have a response profile that indicates that there is a range of train durations between 15 and 32 ms that are highly effective. Once again mice have lower movement thresholds at the optimal train durations compared to rats (Fig. 5).

3.8. Deriving a Movement Threshold for a Single Point

Using a pulse width of 200  ms, 13 cathodal pulses in a train at 300  Hz and having the train repeat once per second is a typical short train ICMS approach in rats. There are three general methods that can be used for deriving a point; overshoot, creeping-up and descending. The overshoot method starts with a current intensity at 0 mA and is rapidly increased until a movement is detected and then decreased until the movement is no longer present (54–57).

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Fig. 5. Mean movement threshold by number of pulses in a train in rats (large circles ) and mice (small circles). Mean movement thresholds (±SEM) were plotted according to the number of pulses in the stimulation train, holding other stimulation parameters constant.

In this way, no more than about ten trains of pulses (10  s) are delivered to a single site when determining a movement threshold which reduces the possibility that the stimulation itself influences the integrity of the map border (52). By rapidly increasing the current intensity, the investigator overshoots the movement threshold while decreasing the current more slowly allows the investigator to precisely determine when the movement disappears (the threshold). The creeping-up method also begins with a current intensity at 0 mA but the current is increased very slowly until a movement is detected. These two different methods are used because researchers have different concerns about the overall amount of current delivered and the influence of supra threshold current on the movement threshold. The creeping-up approach results in higher movement thresholds than the overshoot method because the generation of a large movement in the overshoot method and the sensory feedback that is also generated, lower the minimal amount of current to induce a movement. Note that both the overshoot and creeping-up methodologies give the movement threshold for one type of movement. However, the overshoot method can also give information about other ICMS-induced movements at the same penetration point. For instance, a single penetration point could yield an elbow threshold at 40  mA and a wrist threshold at 25  mA. The third methodological approach involves a descending stimulation series whereby the stimulation intensity starts at 60 mA and is then reduced until the last movement disappears. In this way, multiple movements that occur at higher intensities can also have their own threshold determined as the current is reduced without missing any movements which could occur with the overshoot method.

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A non-responsive point is designated when a maximum of 60 mA is delivered without generating a movement. The choice of 60 mA is somewhat arbitrary but is chosen in order to avoid both tissue damage and reaching the boiling point in the glass electrode tip. Other researchers have used currents in excess of 100  mA (58, 59). The use of biphasic stimulation has been touted as a solution to preventing tissue damage (see (50)). However, with short train stimulation we have found that the threshold for detecting a movement does not change over several hours when using cathodal stimulation, a result that appears to indicate healthy tissue. 3.9. Deriving a Motor Map

Landmark work by Nudo (52) showed that continuous ICMS could acutely change the boundaries of the motor map. For this reason, stimulation should be as brief as possible (as mentioned above) and the border of the map should be determined before internal map points are derived. Thus we recommend that the initial points be derived in the central area of the map and then points in a horizontal line are derived until a non-responsive point is observed. In a clockwise manner, generate the border with nonresponsive points or movements that are not of interest to the experimenter. Refer to Fig. 2 for the numbers that correspond to the order in which map points are derived. Note that for mice this simple clockwise method to establishing the border may not be the correct approach because responsive points are often found outside of borders established by this methodology. For mice many points outside of the “border” should be probed.

3.10. Motor Map Analysis

Imaging software can easily be used to visualize and analyze motor maps (Fig. 6). To begin an analysis of the total motor map area, the

Fig. 6. Colour-coded movement representation of the two forelimb areas from the same rat as depicted in Fig. 1; rostral forelimb area on the left and caudal forelimb area on the right (black = non-responsive, green = wrist, red = digit, dark blue = elbow, light blue = shoulder, pink = whisker, purple = jaw, yellow = neck, grey = trunk, white = hindlimb). The diamond shapes represent the forelimb area.

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grid size is halved, such that the distance between gridlines now becomes 250 mm. Lines are then drawn to surround each responsive site which will cross through or end at intersection points on the grid. This results in the ability to “block in” each penetration point within a 125-mm area in a diamond shape. If a point is not derived due to the presence of a blood vessel (for example, between points 16 and 17 in Fig. 2), then that area can be assumed to be either responsive or non-responsive depending on the movements evoked at neighbouring sites. In the case presented between points 16 and 17 in Fig. 2, the site below the blood vessel is assumed to be a whisker movement, and in this instance is used to provide a border for the forelimb movements below. Once all responsive movement areas are blocked in and a calculation of area is provided by the software, the researcher can perform several analyses related to motor map area, such as the size of the rostral forelimb area (RFA), the caudal forelimb area (CFA) and each movement category independently. Proportions of each movement type of the total map area, and the percent change in area of movements after experimental manipulations can easily be calculated. Other elements to analyze that may be of interest to the researcher are the presence of dual or triple movements as more than one movement type may be evoked at a single penetration site. Changes in movement threshold within each movement category can also be calculated.

4. Long-Train Intracortical Microstimulation Methods

Long-train microstimulation techniques are largely based on the methods previously discussed with some alterations. Foremost is the train duration of stimulation which is critical in shaping the evoked behavioural response. Train lengths on the order of 500 ms evoke complex, sequential, coordinated movements in rats (35). Train lengths that exceed the evoked movement result in a stable endpoint posture. Shorter train lengths result in movement truncation, resulting in individual joint flexions at values near conventional ICMS. Although stimulation intensity has been shown to influence the intensity and magnitude of the evoked response (35), we have found it to also influence the nature and quality of movements. For this reason, we do not perform threshold mapping when performing long-train ICMS; instead, maps are derived at a constant current intensity to allow for stability of evoked responses with repeated stimulation while maintaining an appropriate magnitude of postural changes for kinematic assessment. In addition to changes of train duration, train frequency is also decreased to 0.2 Hz, once every 5 s, to allow for a postural return to baseline and to minimize delivered current.

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Due to the nature of evoked movements, it is beneficial to video record mapping sessions for later movement assessment and classification. Electromyographic recordings would also be of aid in this endeavour. Finally, biphasic stimulation (cathodal lead) is used to avoid potential tissue damage. We have observed that evoked movements are maintained with repeated stimulation, suggesting that these stimulation protocols are not adversely affecting tissue integrity. This is also reflected by consistent movement sequences evoked with repeated stimulation at any given penetration site.

5. Notes 5.1. Anesthetics

Anesthetics interact with membranes, channels, and synaptic transmission thereby influencing the probability of action potential generation and transmission which in turn influences the cortical neuronal networks that drive movements (60). This means that the experimental manipulation under study could influence the efficacy of the anesthetic. For instance, since ketamine interacts with the NMDA receptor, experimental manipulations that also cause changes in NMDA receptor function could be influenced by this anesthetic during ICMS (61). One way to determine if your experimental manipulation is not influenced by a particular anesthetic is to perform ICMS under a different anesthetic that uses a different mechanism to achieve anesthesia. We have been collecting data with the anesthetic urethane. It does pose some challenges at inducing anesthetic depth in a reasonable time frame as well as maintaining a constant level of anesthesia but we believe it is a viable alternative to ketamine.

5.2. Polarity

Long-train stimulation paradigms typically use biphasic stimulation pulses (cathodal followed by anodal) to avoid possible tissue damage. We find no reason, beyond historical precedent, not to use biphasic stimulation even for short-train paradigms.

5.3. Map Size in Mice

One advantage to motor mapping in rats is that maps can appear to either enlarge or shrink in response to experimental manipulations when standard short-train stimulation paradigms are used (62–65). Thus motor maps’ size can be used as an indication of brain plasticity and a marker for the effectiveness of an experimental manipulation (66). We believe this is because only approximately half the map is typically observed when using conventional short train stimulation paradigms. The balance between excitation and inhibition may shift after experimental treatments exposing either more or less of the map (65, 67). However, it appears that in mice, at least using the typical rat stimulation paradigms, the whole map is

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derived and it cannot become significantly larger, although they could potentially become smaller. More work needs to be done to determine the stimulation parameters that would reveal half of the mouse motor map in a non-treated condition, allowing for treatment-induced change in both directions.

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Chapter 8 Forebrain Circuits Controlling Whisker Movements Kevin D. Alloway and Jared B. Smith Abstract The forebrain circuits involved in coordinating the bilateral movements of the whiskers have been elucidated by a series of neuronal tracing studies aimed at characterizing the interhemispheric connections of the primary motor (MI) cortex. In some experiments, different anterograde tracers were placed in the MI whisker region of each hemisphere. In other experiments, an anterograde tracer injection in one MI whisker region was paired with a retrograde tracer injection in the opposite hemisphere. In the last study, a retrograde tracer was placed in one of the forebrain targets of MI, thereby revealing the bilateral distribution of MI neurons that project to the injected target. The relative strengths of the interhemispheric connections of MI cortex were quantified by a variety of methods. In addition to measuring the density and spatial extent of terminal and somal labeling, we analyzed the amount of tracer overlap produced when different tracers are injected in the MI regions of both hemispheres. Key words: Bilateral coordination, Claustrum, Interhemispheric, Motor cortex, Neuronal tracing, Sensorimotor integration, Striatum, Thalamus, Whisking

1. Introduction 1.1. Methodologic Theory

In rats, a major component of behavioral exploration consists of repetitive (4–12  Hz) whisking movements in which whiskers on both sides of the head move synchronously at similar amplitudes and frequencies. Compared to whisker movements, the forelimbs are more likely to move independently. Given that bilateral coordination requires connections between both sides of the brain, the circuits controlling the forelimbs and whiskers should differ in the number and strength of their interhemispheric connections. In fact, our studies indicate that the whisker regions in MI have more interhemispheric connections than the MI forelimb regions (1–4).

Emma L. Lane and Stephen B. Dunnett (eds.), Animal Models of Movement Disorders: Volume II, Neuromethods, vol. 62, DOI 10.1007/978-1-61779-301-1_8, © Springer Science+Business Media, LLC 2011

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In general, the strength of connections between brain regions can be determined by injecting a neuronal tracer in one brain site and then quantifying the labeling patterns in other brain regions. If a retrograde tracer is injected, the number of retrogradely labeled neurons can be counted in each brain region that contains labeling. If an anterograde tracer is injected, the areal extent of the terminal labeling can be measured and the density of the labeled varicosities (terminal boutons and en passant synapses) in each target region can be estimated. Unfortunately, all of these parameters may vary substantially for different experiments because of variations in the amount of tracer that is injected and the spatial extent of its diffusion. Therefore, to determine the strength of the interhemispheric connections of specific regions in MI cortex, we normalized the data so that the relative strength of the interhemispheric connections with the MI whisker region can be compared with the corresponding connections of the MI forelimb region (5). After injecting a tracer into MI cortex, the number of labeled processes and the areal extent of the labeling are measured and compared across the two hemispheres. Despite variations in the size and spread of tracer injections across different experiments, the relative distribution of contralateral and ipsilateral labeling provides a convenient and normalized metric for assessing the bilateral connections of different parts of MI (see Fig. 1).

Fig. 1. Normalized distributions of MI projections to multiple forebrain targets following a unilateral tracer injection in the whisker (top) or forelimb (bottom) regions of MI. When each parameter (number of plotted varicosities or areal innervation) is summed across all targets, the total amount equals 100%. Compared to the MI forelimb region, injections in the whisker region produced more labeling in the contralateral hemisphere, especially in the thalamus and claustrum. Each bar represents mean labeling in seven rats; brackets represent SEM.

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When two anterograde tracers are injected into MI cortex, one in each hemisphere, the amount of tracer overlap throughout the brain indicates which regions might integrate motor information from both hemispheres. In other cases, in which an anterograde tracer is injected in MI on one side and a retrograde tracer is injected in the same MI region of the opposite hemisphere, the presence of dense overlap among anterogradely labeled terminals and retrogradely labeled neurons provides strong evidence for an interhemispheric circuit. Using these approaches, we found that the MI whisker region has many more interhemispheric connections than the MI forelimb region. This chapter will use results from the MI whisker experiments to illustrate the techniques in these studies. 1.2. Outline of the Procedures

Although neuronal tracing has a long history and represents a classic technique in systems neuroscience, the details of many circuits have not been fully elucidated. Neuronal tracing is a valuable tool for revealing new information about the functional connections in many systems, but its use requires skill with several techniques. Furthermore, knowledge of the pitfalls that can interfere with the acquisition and interpretation of tracing data is needed to develop an experimental design that puts the techniques to their optimal use. Briefly, the procedures that we use to elucidate the interhemispheric circuit connections of the MI whisker region are as follows.

1.2.1. Select the Tracer Injection Sites

For anterograde tracing studies, intracranial microstimulation (ICMS) is used to identify the MI cortical sites that evoke whisker movements. A stimulating electrode is moved from one site to another to evoke movements of the whiskers or other body parts. For retrograde tracing studies, ICMS is rarely used to identify an appropriate site for injecting the tracer into a MI forebrain target. Instead, results from the anterograde studies indicate the stereotaxic coordinates that should be used to locate an appropriate injection site.

1.2.2. Inject the Tracers

Neuronal tracers are injected by pressure or iontophoresis, and each method has strengths and weaknesses. Pressure enables injection of a specific volume of tracer, but the tracer may diffuse more widely than desired or might flow along the space that surrounds the injection needle. By comparison, if the tracer molecule is relatively small and has an ionic charge, the polarity and magnitude of an iontophoretic current can be controlled to produce a concentrated tracer deposit at the tip of the injection pipette.

1.2.3. Reconstruct the Labeled Connections

A microscopic reconstruction system is essential for plotting the locations of terminals and cell bodies that are labeled by anterograde and retrograde tracers, respectively. While viewing the labeling patterns at high magnification, the microscope stage is moved

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until a labeled structure appears under the cross hairs of the eyepiece. Optical transducers are mounted on the microscope stage so that each stage position is encoded by X–Y Cartesian coordinates. By plotting labeled structures and other anatomical landmarks, the system uses the coordinate data to reconstruct their relative locations. 1.2.4. Quantify the Labeled Connections

The labeling patterns in the plotted reconstructions are analyzed by several methods to determine the relative strength of the connections between MI cortex and its subcortical targets. For each anterograde tracer that is injected into an MI cortical site, the areal extent and relative density of terminal labeling are bilaterally measured in each subcortical target. At the most densely labeled sites in each target region, confocal microscopy is used to count the number of labeled varicosities in a standard area to determine the maximum density of MI projections to each brain region. If two anterograde tracers are injected into MI cortex, one tracer in each hemisphere, the amount of tracer overlap is measured throughout the forebrain to determine which targets might integrate information from both hemispheres. If an anterograde tracer injection in one hemisphere is paired with a retrograde tracer injection in the other hemisphere, the density and amount of terminal-soma overlap is measured to indicate the likelihood of an interhemispheric circuit connection. Finally, in separate experiments, a single retrograde tracer is injected into the forebrain regions that receive strong projections from MI. The labeled neurons appearing throughout MI cortex and the surrounding regions are plotted and counted. Adjacent sections labeled for Nissl material are used to determine the cytoarchitectonic borders that define each cortical area, and these boundaries are then projected onto the plotted reconstructions to enable quantitative comparisons of neuronal labeling in each MI region of the two hemispheres.

2. Materials 2.1. Tracers

1. Fluoro-Ruby (FR), Molecular Probes, Eugene, OR, D-1817, Prepare a 10% solution in 0.1 M sterile PBS for injection. Store solid and solution in light-tight container at 4°C. Shelf life exceeds 1 year when properly stored; solution stays stable for at least 6 months. 2. Alexa Fluoro (AF), Molecular Probes, Eugene, OR, D-2290. Prepare a 10% solution in 0.1 M sterile PBS for injection. Store solid and solution in light-tight container at 4°C. Shelf life exceeds 1 year when properly stored; solution stays stable for at least 6 months.

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3. Fluoro-Gold (FG), Fluoro-Chrome, LLC, Denver, CO, H-22845. Prepare a 2% solution in 0.9% sterile saline (physiological) for injection. Store solid and solution in light-tight container at 4°C. Shelf life exceeding 1  year when properly stored; solution stays stable for at least 6 months. 2.2. Filter Sets for Fluorescent Microscopy

1. Combined fluorescein isothiocyanate (FITC)/tetrarhodamine isothiocyanate (TRITC) filter for Fluoro-Ruby and Alexa Fluoro (Chroma Technologies, 51004v2). 2. UV filter for Fluoro-Gold (Chroma technologies, 11000v2).

2.3. Microstimulation and Tracer Injection Equipment

1. Hamilton microsyringe – Microliter#7002, 2 ml 2. Kopf model 5000 microsyringe holder 3. Ammeter 4. Constant current source (e.g., Bak Electronics Inc., Model BSI-2) 5. Electrode impedance tester (e.g., Bak Electronics, Model IMP-1) 6. Programmable digital timer, (e.g., Master 8 by A.M.P.I.)

2.4. Reconstruction and Analysis of Neuronal Labeling Patterns

1. AccuStage, Inc. Reconstruction System and MDPlot software, version 5.1 2. Microscope for light and fluorescent microscopy 3. Laser-based confocal microscope 4. Illustration and drawing software program (e.g., Canvas X, Deneba Systems)

3. Methods 3.1. Select the Injection Sites

Intracranial microstimulation (ICMS) is used to identify appropriate sites in the MI whisker region for injecting anterograde tracers. A pipette puller is used to produce a glass pipette that has a tip diameter of 1 mm and, after it is filled with hypertonic (3 M) saline, an impedance of 1 MW. The electrode’s impedance is always measured before placing it in the brain to insure its suitability for ICMS. At least three stimulating electrodes are made just prior to each experiment, and they are temporarily stored in a saline-filled electrode-holding jar to prevent the formation of salt crystals at the electrode tip. For ICMS, rats are anesthetized with intramuscular injections of ketamine HCl (20 mg/kg) and xylazine (6 mg/kg). After immobilizing the rat’s head in a stereotaxic instrument, a craniotomy is made over the MI cortical region and a small ground screw is placed

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Fig. 2. Schematic diagram of circuit connections for intracranial microstimulation in MI cortex and iontophoretic ejection of tracers such as Fluoro-Gold.

in the cranium overlying the cerebellum (see Note 1). As shown by Fig. 2, the constant current source must be connected with both the stimulating electrode and the ground screw to form a complete circuit for ICMS. Muscle twitches can be evoked from MI cortex only if the animal is in a shallow plane of anesthesia. If the animal is deeply anesthetized, ICMS will not evoke muscle twitches even if high stimulation currents are administered. Furthermore, repeated application of a large stimulation current may destroy the neuropil at the electrode tip. Within 60–90  min of the initial anesthetic injection, the rat usually enters a shallow anesthetic plane that is characterized by small, spontaneous whisker movements in which the peak-to-peak whisker motion is no more than 1 mm. This indicates the moment when ICMS is most likely to be successful in evoking a peripheral movement (see Note 2). Microstimulation mapping begins by lowering the stimulating electrode into the deep layers of MI cortex, which are located 1.2–1.7  mm below the pial surface. Initially, microstimulation consists of short trains (80 ms in length) of 0.7 ms current pulses that are separated by intervals of 3.3 ms. Hence, the experimenter activates the programmable timer so that each button press on the timer delivers a train of 20 pulses at a rate of 250 Hz (see Note 3). While observing the whiskers, the experimenter delivers a series of trains, usually at a rate of 1 Hz or less. Microstimulation should begin with suprathreshold positive currents of 100–150 mA. If the rat is lightly anesthetized and the tip of the stimulating electrode is in the deep layers of the MI whisker region, such currents will evoke robust movements of multiple whiskers, possibly on both sides of the face. The current level is gradually reduced to the lowest level that barely evokes twitches of a small number of whiskers on the contralateral face. The lowest level for evoking a single whisker twitch is usually about 10–20 mA. If ICMS does not evoke whisker twitches, the circuit connections and other factors must be carefully examined (see Note 4).

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Rat MI cortex contains two whisker regions that are distinguished by their responses to short and long pulse trains. Brief whisker retractions are evoked when short train pulses are administered to the medial agranular cortex, which is known as the retraction area in the MI whisker region (MI–Re). The MI–Re region is located about 1.7–2.5 mm rostral and 1.5–2.2 mm lateral to bregma in the adult rat. A second, more caudal whisker region evokes whisker protractions when it is stimulated by short (80 ms) pulse trains but evokes rhythmic (5–15  Hz) whisking movements when stimulated by long (1,400 ms) trains administered at 60 or 100 Hz (i.e., 0.7 ms pulses separated by 16 or 9.7 ms). This region, which is called the rhythmic whisking area (MI-RW), is located about 0.7–1.2  mm lateral and 0.6–2.4 mm rostral to bregma (6). To verify that a stimulation site is in one of these regions, we sequentially administer a series of short pulse trains and then a series of long pulse trains at each electrode penetration. The digital timer allows multiple programs to be stored in memory, and switching between short and long pulse trains is done quickly if both sequences of pulse trains have been programmed and stored in memory prior to the start of the experiment. Locating a suitable site for a tracer injection in MI cortex needs to be done rapidly because the animal’s plane of anesthesia is relatively shallow and reflexive movements could appear if the ICMS is not conducted efficiently. Furthermore, in cases where different anterograde tracers are injected into homotopical regions bilaterally, it is necessary to locate the same functional region in both hemispheres as quickly as possible. Another constraint is posed by the fact that even small tracer injections produce some diffusion, but the labeling data will not be interpretable if the tracer diffuses across the borders between the whisker and forelimb regions in MI. For these reasons, we use threshold ICMS currents to locate the B, C, or D whisker rows in MI because these representations are in the center of the MI whisker region. Small injections at these sites should reduce the likelihood that tracer will diffuse beyond the whisker representation. We usually stimulate 4–8 sites in each hemisphere. After locating the center part of the MI whisker region, ICMS is administered more laterally in 500 mm steps until we identify a site that elicits forelimb movements (see Note 5). After locating the forelimb-whisker border, the tracer is injected into a site located at least 500 mm away, thereby insuring that the tracer does not diffuse across this functional boundary. In rare cases, we have used ICMS to identify the whisker representations in subcortical brain regions that were injected with a retrograde tracer. For example, to determine whether the MI whisker region projects directly to the facial nucleus, which contains the cell bodies of the peripheral nerve fibers that innervate the whisker pad, we have injected a retrograde tracer into the lateral facial nucleus (see Fig. 3). In these cases, ICMS is used to locate sites in the facial nucleus that evoke whisker movements.

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Fig. 3. Examples of iontophoretic injections of the retrograde tracer Fluoro-Gold into the facial nucleus (a, a¢), dorsolateral striatum (b, b¢), and thalamus (c, c¢). AM anteromedial, AV anteroventral, DLS dorsolateral striatum, f fornix, LD laterodorsal, MD mediodorsal, mt mammillothalamic tract, sm stria medullaris, SpTT spinal trigeminal tract, VL ventrolateral, VM ventromedial, ZI zona incerta. Rectangles in (a) and (b) indicate regions in (a¢) and (b¢). Scale bars = 250 mm in (a, a¢), 200 mm in (b, b¢), 500 mm in (c, c¢).

For most forebrain targets of MI, ICMS is not effective for evoking whisker twitches. Tracer injections in these brain regions must rely on stereotaxic coordinates alone. Thus, after identifying regions in the thalamus, dorsolateral striatum, and claustrum that received projections from the MI whisker region, stereotaxic coordinates indicate where to place retrograde tracers in these regions (see Figs. 3 and 4). 3.2. Inject the Tracers

Two methods are used for injecting neuronal tracers into the brain: pressure or iontophoresis. Virtually all tracers can be injected with pressure, and this method enables control of tracer volume but not tracer diffusion. Pressure-injected tracers do not diffuse evenly in all directions, and a large portion of the injected tracer may flow backwards along the space surrounding the injection pipette. Furthermore, if the intended injection target is in a subcortical region, the tracer may leak from the injection pipette as it traverses the cortex and other structures along the route to the target site. This will interfere with the interpretation of the results. The iontophoretic technique is superior to pressure for injecting a tracer into a subcortical brain region. The main advantage is that iontophoresis relies on an electric potential to eject tracer molecules away from the pipette tip. This effectively restricts the tracer deposit

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Fig. 4. Bilateral labeling patterns of MI neurons produced by an injection of the retrograde tracer Fluoro-Gold into the left claustrum (a–c). Although neuronal labeling was present in multiple cortical areas of both hemispheres, labeling density was greatest in Agm of the right hemisphere (d–h). Scale bars = 1 mm in (a, h); 0.5 mm in (b); 250 mm in (c, e, f); 100 mm in (d, g).

to a small region. Furthermore, if the electrical polarity is reversed, the tracer molecules are retained in the pipette. This is advantageous because it prevents unwanted leakage of the tracer as the injection pipette is advanced to the injection site or when it is withdrawn from the brain after the injection is completed. As indicated by Figs.  3 and 4, we have used iontophoresis to deposit retrograde tracers into the facial nucleus, thalamus, striatum, and claustrum. For both pressure and iontophoretic tracer injections, a pipette puller must be used to produce a pipette that has specific tip dimensions. For pressure injections, we use thick capillary glass to produce pipettes with large tip diameters (i.e., 50–70 mm). Thick glass is used because a pressure-injection pipette must be robust enough

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for repeated usage. Furthermore, the tip of the pipette must be large enough to permit the flow of relatively viscous tracers such as Fluoro-Ruby. By comparison, pipettes used for iontophoresis are used only once and the tips are much smaller (20–30 mm) because tracers such as Fluoro-Gold are easily ejected at low current levels. 3.2.1. Pressure Injections

Pressure injections are performed with a Hamilton glass microsyringe in which a glass pipette has been cemented onto the end of the syringe needle. Although tracers can be injected directly into the brain from the metal needle of the Hamilton syringe, the metal needles are prone to becoming clogged when they are used repeatedly. Furthermore, inserting the metal needle into the brain produces more trauma than a glass pipette which has been pulled so that its tip is only 50–70 mm in diameter. Therefore, we cement a glass pipette onto the end of the syringe-needle and use the same injection assembly for several experiments. Because some tracer will adhere to the glass pipette, a separate injection assembly must be constructed for each tracer that will be injected by pressure. Before gluing the glass pipette to the Hamilton syringe needle with 5-min epoxy, the syringe is completely filled with mineral oil so that no bubbles are present in the glass syringe. The outer surface of the metal needle is wiped dry and epoxy is carefully placed around the end of the metal needle without entering the lumen of the needle. The Hamilton syringe is held by a holder so that the tip is facing upward when the epoxy is applied, and then the glass pipette is carefully lowered onto the metal needle so that the epoxy forms a visible seal that completely encircles the metal needle and the inner surface of the glass pipette (see Note 6). The epoxy must dry over 1–2 days, so the syringe-pipette assembly must be constructed well in advance of an experiment. To fill the pipette with tracer, the syringe assembly is placed in a syringe holder (Kopf Model 5001) that is specifically designed for Kopf stereotaxic instruments. A small amount of mineral oil is slowly expelled from the syringe-needle to insure that it completely fills the lumen of the pipette without the presence of any bubbles. After the tracer is subjected to agitation by a vortex, the tip of the glass pipette is lowered into the tracer reservoir and the syringe plunger is slowly retracted so that tracer flows upward into the lumen of the pipette. The interface between the tracer, which is colored, and the mineral oil, which is clear, is easily visualized through the glass pipette, and tracer is not allowed to flow into the metal needle. Once the pipette tip is filled with tracer, usually a volume of 200–250 nl, the syringe assembly and Kopf holder are placed on the rail of the stereotaxic instrument, and the tracer-filled pipette tip is lowered into the MI site identified by ICMS. The pipette is slowly lowered into MI cortex and a volume of 125–200 nl of Fluoro-Ruby or Alexa-Fluoro is injected between 1.2 and 1.7 mm below the cortical surface.

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Iontophoretic tracer injections require a complete electrical circuit in which a constant current source provides the electrical force that expels charged tracer molecules from the tracer-filled pipette. An ammeter must be present in the circuit to monitor current flow through the circuit, thereby insuring that the pipette tip does not become clogged. The programmable digital timer is used to activate the constant current source at precise intervals as shown by Fig. 2. As this diagram indicates, current flows from the constant current source to the ammeter and tracer pipette before returning from the ground screw back to the constant current source. With the exception of the connection between the ammeter and the tracer-filled pipette, all connections are made before the pipette is filled with tracer (see Note 7). When Fluoro-Gold is deposited iontophoretically into a subcortical structure, we fill the pipette by injecting the tracer into its back end. To do this, a small quantity of tracer is loaded into a plastic syringe equipped with a long (3 in.) Hamilton (90081) beveled needle. The needle is inserted into the back end of the glass pipette and is advanced to the tip of the pipette while visualizing the tip of the glass pipette with a dissecting scope. The tracer is slowly injected into the pipette so that 1–2  cm of the pipette is filled with tracer without any apparent bubbles. We never fill an iontophoretic pipette by dipping its tip directly into the tracer because this would coat the outer surface of the pipette with a layer of tracer, thereby permitting diffusion of the tracer into the brain tissue that surrounds the trajectory of the pipette’s penetration. The tracer-filled pipette is placed in a standard electrode holder seated on the stereotaxic instrument and is then lowered towards the craniotomy. To avoid the formation of salt crystals at the tip of the pipette, the craniotomy is filled with a small pool of saline, and the pipette is lowered into this pool. A silver wire is inserted into the pipette until it contacts the tracer inside the pipette, and a cable from the ammeter is connected to the silver wire to complete the iontophoretic circuit. While the pipette tip is in the saline pool over the cortical surface, the performance of the circuit is tested by activating the constant current source at a magnitude of 5  mA while watching the ammeter to verify the flow of current. If there is no current, the circuit connections must be checked. If current still does not flow, this suggests that the pipette is clogged and must be replaced. Before it enters the brain, the pipette is set to a negative polarity so that Fluoro-Gold is retained in the pipette while it is advanced to its subcortical target. Once there, the retention current is turned off, and the pipette is held in position without any current flow for 3–4 min to allow the brain tissue to rise with respect to the pipette. Subsequently, the ejection current is turned on (pipette is positive) and pulses are delivered in a duty cycle of 7 s on/off. The ammeter is continuously monitored to detect any interruption in current

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during the positive current pulse. If current magnitude drops, the pipette is becoming clogged. Sometimes this can be resolved by retracting the pipette a few hundred microns and then advancing it back to the target site. Otherwise, the pipette must be withdrawn and another inserted in its place (see Note 8). The size of the Fluoro-Gold injection depends on the magnitude of the injection current and the duration of the duty cycle. For large tracer deposits that extend 1 mm in diameter, a current magnitude of 5–6  mA for 15–25  min is used. To produce small tracer deposits with diameters of 0.5  mm of less, a current of 3–5 mA is applied, but the duration of the duty cycle is reduced to 10–15 min. The size of the tracer injection depends on many factors, however, and researchers must experiment with different current magnitudes and durations to produce the desired tracer deposit. After the duty cycle is completed, the current is turned off and the pipette stays in place for another 5 min. Subsequently, the retention current is turned on while the pipette is quickly withdrawn from the brain. 3.3. Reconstruct the Labeled Connections

Axonal transport of anterograde and retrograde tracers usually requires 10  days to produce maximal labeling of all connected regions throughout the brain. At the end of this period, the rat is deeply anesthetized with sodium pentobarbital and is transcardially perfused with 0.9% saline (300 ml), followed by 4% paraformaldehyde (300 ml), and then 4% paraformaldehyde with 10% sucrose. The brain is then stored in the fixative with 30% sucrose for at least 2 days, or until the brain sinks, before it is sectioned (see Note 9). Alternate serially ordered coronal sections of the brain (60–70 mm thick) are separated into two series, one for cytoarchitecture (thionin-stained Nissl labeling) and the other for viewing neurons and axon terminals labeled by the fluorescent tracers. Tissue sections for neuronal tracing are mounted on gelatin-coated glass slides, dried overnight, and are briefly dipped (5–10 s) in 100% ethanol and then xylene before being coverslipped with a mountant suitable for fluorescent microscopy (e.g., cytoseal).

3.3.1. Plot Anatomical Landmarks During Light Microscopy

Using conventional fluorescent microscopy, the labeling patterns are plotted with an AccuStage reconstruction system. By virtue of optical transducers mounted on the X–Y axes of the microscope stage, all potential stage positions are encoded by Cartesian coordinates. The researcher moves the stage while visualizing the labeling patterns at ×100 or ×200 (×10 eyepiece with a ×10 or ×20 objective), and when a labeled structure (i.e., neuron or terminal varicosity) appears under the crosshairs of the eyepiece, a foot pedal is pressed and the systems sends the Cartesian coordinates to the computer for storage and visual display on a monitor. Reconstructions begin by plotting the outline of the coronal section with respect to anatomical landmarks within the section.

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For this purpose, conventional light microscopy is used to visualize the unstained tissue. The AccuStage software program (MDPlot) allows the user to select different line elements and symbol types to represent plotted structures, and we generally select a solid white line (on a black background) to represent the external surface of the section. Subsequently, we plot the outline of the ventricles and any major fiber tracts (e.g., corpus callosum, external capsule, anterior commissure) that are easily visualized in unstained tissue sections. Sometimes it is possible to visualize the outlines of nuclei by adjusting the condenser on the microscope, and the outlines of these structures are also plotted if they are apparent. For all structures whose outlines are plotted, points are plotted at short intervals to produce a relatively smooth outline. 3.3.2. Plot-Labeled Terminals and Neurons During Fluorescent Microscopy

After the most salient structures are outlined, the tissue is viewed under fluorescent illumination to view the neural labeling patterns (see Note 10). After placing the appropriate filter set in the light path, anterogradely labeled axon terminals are plotted first because the larger amount of labeling in the neuronal soma is less likely to be quenched and, consequently, will persist during long exposure to intense lumination. As with most axonal projections, those from MI cortex form terminal arbors that contain beaded varicosities along the axon terminal. Ultrastructural examination indicates that these axonal enlargements contain vesicles and synaptic markers associated with en passant synapses (7, 8). Therefore, we plot the locations of labeled varicosities because these represent synaptic connections of the MI projection terminals. To plot labeled varicosities, a small dot size is selected from the software menu to represent the locations of these putative synapses. The color of the dot is determined by the color of the tracer, red for Fluoro-Ruby and green for Alexa-Fluoro. The density of labeled terminals may vary substantially from one location to another in each target structure. When the density of terminal labeling is sparse-to-moderate, it is possible to plot virtually all of the labeled varicosities. If the terminal labeling is dense, however, each labeled varicosity cannot be individually visualized and plotted. For this reason, labeled terminals are plotted so that the reconstructions represent proportional changes in labeling density. This is accomplished by repeatedly glancing back-andforth between the image appearing in the microscope and the reconstructed image displayed on the computer monitor as it progresses throughout the plotting process. For this purpose, the computer monitor is placed as close to the microscope as possible to afford rapid comparisons with what is seen in the microscope and what appears on the monitor. Although the number of plotted points may represent only a fraction of the actual number of labeled varicosities in the tissue, the reconstruction should accurately depict the locations where labeling is densest, and spatial changes

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in labeling density should be represented by proportional changes in the density of the plotted points. The neuronal cell bodies labeled by the tracers are plotted last. Because cell bodies are large, labeled soma are plotted as colored circles that are considerably larger than the colored dots that represent the plotted varicosities. Neurons labeled by Fluoro-Gold, for example, are represented as large gold circles. Although FluoroRuby and Alexa-Fluoro are used primarily to reveal the terminals of axonal projections, these tracers are also transported in the retrograde direction to a limited extent. Consequently, terminals and soma labeled by the same tracer injection may appear in several brain regions and should be plotted, as small dots and large circles, respectively, to represent both the efferent and afferent connections of the injected site (see Fig. 5). 3.4. Quantify the Labeled Connections

Several analytic approaches are used to assess the strength of the interhemispheric connections of MI cortex. Regardless of whether anterograde or retrograde tracers are injected, the amount of labeling in both hemispheres is quantified in a variety of ways to indicate the connection strength between MI cortex and other brain regions. When two different anterograde tracers are injected into MI cortex, one in each hemisphere, tracer overlap indicates the forebrain regions that might integrate MI inputs from both hemispheres. Alternatively, if an anterograde tracer in one MI region is paired with a retrograde tracer in the MI region of the other hemisphere, tracer overlap indicates an interhemispheric circuit that connects the MI regions in each hemisphere.

3.4.1. Bilateral Distributions of Anterogradely Labeled Terminals

After injecting an anterograde tracer into MI cortex, the labeled varicosities are bilaterally plotted in all forebrain regions that contain labeling. The total number of plotted varicosities throughout the whole forebrain is summed across all of the reconstructed sections and, as shown in Fig.  1, the amount of labeling plotted in each forebrain region can be expressed as a proportion of the total labeling in the forebrain. Hence, these normalized data indicate the relative strength of MI projections to different forebrain regions in both hemispheres. The spatial extent of terminal labeling in each forebrain target is also measured. For this analysis, each reconstructed section is subdivided into a grid of small bins (e.g., 50 mm2), and the number of square bins that contain one or more plotted varicosities is summed for each forebrain target that contains terminal labeling. The number of labeled bins in each forebrain region is then summed across sections, and the areal extent of labeling in each forebrain target is expressed as a fraction of the total area of innervation throughout the forebrain (see Fig. 1). In measuring the spatial extent of terminal labeling, the AccuStage software is flexible with respect to bin size and the

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Fig. 5. Bilateral labeling in the thalamus following injections of Alexa-Fluoro and Fluoro-Ruby into the MI whisker regions of the left and right hemispheres, respectively (a–c). Both tracers labeled corticothalamic terminals (small dots) on both sides of the thalamus, but neuronal labeling (large circles) were found only on the side of the tracer injection (d). Tracer overlap involving both labeled terminals and neuronal cell bodies was apparent in several parts of the thalamus (e, f ). Overlapping corticothalamic projections (g, h) from the two injection sites appeared more extensively throughout the thalamus than the terminal-soma overlap (i–k). Terminal–terminal overlap (h) was measured using 50 mm2 bins, while terminal-soma overlap ( j, k) was measured using 100 mm2 bins. CM centromedial, G gelatinosus, Rh rhomboid, PC paracentral, VPM ventroposteromedial. Scale bars = 1.0 mm for (d, g–k); 0.5 mm for (b); 250 mm for (a, c); 40 mm for (e); 20 mm for (f ).

threshold number of plotted varicosities that must reside in each bin in order to be counted. It is possible, for example, to use bins that are smaller than 50 mm2, to measure more precisely the spatial area that contains terminal labeling. Likewise, the threshold number of plotted varicosities required in each bin can be increased so that the measured area is based on regions that contain a minimum

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labeling density (e.g., two labeled varicosities per bin). We analyze the reconstructed data multiple times with different bin sizes and threshold counts so that our conclusions about the relative strength of the connections between MI and its forebrain targets do not depend on the specific parameters of the analysis. 3.4.2. Analysis of Tracer Overlap

Injection of two different anterograde tracers into MI, one tracer in each hemisphere, provides an opportunity for identifying forebrain regions that integrate information from the MI cortical areas in both hemispheres. As shown in Fig.  5, injections of FluoroRuby and Alexa-Fluoro into the right and left MI whisker regions, respectively, reveal labeled terminals on both sides of the thalamus for both tracers. To determine the amount of tracer overlap among the labeled terminals, the reconstructed sections were subdivided into grids of 50 mm2 bins. Bins in the thalamus that only contained varicosities labeled by Fluoro-Ruby were colored red, while those that only contained varicosities labeled by Alexa-Fluoro labeled bins were colored green, and bins that contained both tracers were colored white. As shown by Fig.  5 (panels g and h), analysis of tracer overlap indicated that as many as 20% of the labeled bins in a given section of the thalamus received projections from both MI injection sites. Although Fluoro-Ruby and Alexa-Fluor are transported most efficiently in the anterograde direction, these tracers are also transported in the retrograde direction. In contrast to anterograde labeling, in which projections from each MI whisker region terminated on both sides of the thalamus, retrogradely labeled cell bodies were located only on the side of the thalamus ipsilateral to the tracer injection. Nonetheless, as shown by the photomicrographs in Fig.  5 (panels e and f), corticothalamic axons labeled by one tracer often terminated in the vicinity of retrogradely labeled neurons labeled by the other tracer. Consequently, the presence of terminal-soma overlap suggests that the thalamus conveys information between the MI cortices of both hemispheres. Therefore, we measured the proportion of thalamic bins that contained terminals labeled by one tracer and neuronal soma labeled by the other tracer. As indicated by Fig.  5 (panels j and k), approximately 10–15% of the labeled bins in the thalamus, especially in the ventromedial, ventrolateral, and intralaminar nuclei, contained intermingled cell bodies and terminals that were labeled by different tracers. A more accurate approach for assessing the strength of interhemispheric circuit connections is to pair injections of two tracers that are optimally transported in opposite directions. For this purpose, we injected the anterograde tracer Fluoro-Ruby into the MI whisker region in one hemisphere and paired this with an injection of the retrograde tracer Fluoro-Gold in the MI whisker region of the opposite hemisphere. As shown by Fig. 6, injections of these

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Fig. 6. Bilateral labeling in the claustrum following injections of Fluoro-Ruby and Fluoro-Gold into the left and right MI whisker regions, respectively (a–c). Labeled terminals and cell bodies were densely intermingled in the right but not in the left claustrum (d–f ). The density of the labeled terminals was much greater in the right than in the left claustrum as determined from both the plotted reconstructions (h) and the varicosity counts obtained from images using confocal microscopy (g, i–m). Scale bars = 0.5 mm for (a–c); 50 mm for (d, e); 25 mm for (g, i–m).

tracers produced dense overlap in the claustrum located ipsilateral to the Fluoro-Gold injection, but very little overlap in the claustrum located ipsilateral to the Fluoro-Ruby injection. Thus, MI whisker projections to the contralateral claustrum are intermingled with claustral neurons that project to the MI whisker region of that hemisphere (see Note 11). These results indicate that interhemispheric cortico–claustro–cortical connections are structured for the transmission of information that could bilaterally coordinate the MI regions involved in regulating whisker movements (4).

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3.4.3. Density of Terminal Labeling

Following an anterograde tracer injection into MI cortex, estimating the density of labeled varicosities in different forebrain targets is a two-part process. Digital reconstruction systems (e.g., AccuStage system) can duplicate varicosity density if terminal labeling is relatively sparse. In densely labeled regions, however, the reconstructions can depict relative changes in labeling density, but the number of plotted varicosities is only a fraction of the actual number of labeled varicosities at each site. For this reason, reconstructions of varicosity labeling obtained by conventional fluorescent microscopy is useful for identifying where varicosity labeling is densest, but obtaining an accurate estimate of the maximum density of labeled varicosities requires confocal microscopy. To estimate the maximum density of labeled varicosities in a target brain region, we use a laser-based confocal microscope (TCS SP2 AOBS; Leica Microsystems) with a 63× oil-immersion objective to acquire high-resolution images of axonal terminals labeled by Fluoro-Ruby (543 nm excitation, 565–630 nm detection). The confocal microscope is used with an optical zoom of 120× to acquire images at sites where our reconstructions indicate that varicosity labeling is densest. Each image represents a small square region (e.g., 100 mm2) or counting box, in which all labeled varicosities can be visualized individually. Images are acquired at multiple depths of the section so that the final image represents the sum of luminance values for each pixel extending 15 mm through the Z-plane. The TIFF-based images are then imported into a drawing program (e.g., Canvas X, Deneba Systems), and persons who are blind to the experimental details of each image are given the task of counting the number of labeled varicosities. As indicated by Fig. 6 (panels j and m), this is done by placing a small green dot on each labeled varicosity so that the drawing program can be used to count the total number of dots placed in each image (see Fig. 6k, l). The green dots are placed on axonal enlargements that are at least twice the diameter of the axon (see Note 12). Given the size of the image, the density of the labeled varicosities is easily calculated from the total number of varicosities that were marked on the image. At least two individuals should tag the labeled varicosities on each image, and their scores are then averaged.

3.4.4. Analysis of Neuronal Labeling in MI

Retrograde tracers are placed in forebrain targets of MI cortex to characterize the bilateral distribution of the MI neurons that project to each forebrain target identified in the anterograde tracing studies. After plotting the bilateral locations of the retrogradely labeled neurons in MI, the adjacent thionin-stained sections are examined so that the cytoarchitectonic and laminar boundaries of Agm, Agl, and the cingulate cortex can be delineated. Once these boundaries have been determined, an outline of these boundaries is projected onto the plotted reconstructions so that the number and density of the retrogradely labeled neurons can be analyzed bilaterally with respect to each cytotectonic area in MI cortex.

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As seen in Fig. 4, a focal injection of Fluoro-Gold into the left claustrum revealed populations of retrogradely labeled neurons in the MI cortical areas of both hemispheres. The majority of the labeled neurons were located in the contralateral hemisphere, a finding that agrees with the anterograde tracing results showing that the MI whisker region projects more strongly to the contralateral than to the ipsilateral claustrum (see Fig. 6). Furthermore, the majority of the labeled neurons were in the medial and caudal part of the Agm region, which corresponds to the location of the MI-RW region, in which ICMS evokes rhythmic whisking behavior. Hence, the retrograde tracing studies are conducted to confirm and extend the findings produced by the anterograde tracing experiments.

4. Notes 1. The craniotomy must be done carefully so that the underlying cortex is not damaged. Bleeding from MI cortex reduces the likelihood that ICMS will evoke a muscle twitch. The placement of the ground screw may affect current flow and the gradient of the current density during ICMS. For experiments in which ICMS is administered in both hemispheres, midline placement of the ground screw is essential for bilateral mapping of MI. For this reason, the ground screw in these studies was placed over the cerebellum just behind the lambda suture lines. 2. Place a small piece of black plastic or black paper below the whiskers so that the smallest whisker movements evoked by ICMS are easily detected. 3. A programmable digital timer should be obtained that allows the storage of multiple programs because the same timer will be used to produce different pulse trains for ICMS and iontophoretic ejection of Fluoro-Gold or other neuronal tracers. This enables rapid switching from one program to another without wasting time during the experimental session. 4. If ICMS does not evoke a muscle twitch, then confirm that the ground screw is touching the dural surface and that all connections are correct (as shown in Fig. 2). Use an oscilloscope to confirm that the programmable timer sends a pulse train to the constant current source. If ICMS is still ineffective, then the electrode should be moved and tested at different sites in the cortex because many ICMS maps show a small number of unresponsive penetrations. When adjusting the electrode position, inspect its tip and make certain that it is not covered in  blood, which could block it. Finally, the batteries in the

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constant current source should be checked regularly to insure that they are not weak. 5. If the stimulating electrode is exactly at the border of the whisker and forelimb representations, ICMS will evoke movements of both the whiskers and the forelimb. 6. From experience we have learned that the tip of the metal needle must be flat, needles with beveled tips prevent the epoxy from forming a seal because the epoxy invariably flows into the lumen of the needle via capillary action. 7. The pipette should be filled with tracer just prior to being placed in the brain. A prolonged interval between filling the pipette and placing it in the brain increases the possibility that crystals will form at the tip of the pipette and prevent the ejection of tracer into the selected brain region. 8. The presence of a small amount of gold precipitate in the tip of the pipette is a good indication that the Fluoro-Gold was ejected. If too much precipitate is formed, however, this can clog the pipette and may cause a drop in current magnitude during the iontophoretic procedure. 9. Before sectioning, the brain must be stored for at least 2 days in paraformaldehyde with 30% sucrose. This is essential to avoid freeze-fracture tissue damage. Otherwise, the sectioned tissue will lack integrity and will be unusable for making accurate reconstructions of labeling patterns. 10. Some tracers, especially Fluoro-Ruby, produce labeling in pericytes that line the blood vessels. These perivascular cells are most prominent in the tissue near the injection sites and they are probably labeled because the tracer entered the vascular system during pressure injection. As such, labeled pericytes represent artifacts and should not be plotted. Fortunately, fluorescent pericytes are easily distinguished from labeled soma and axon terminals. 11. Analysis of terminal-soma overlap requires judgment with respect to setting the bin size and threshold number of labeled varicosities that constitute overlap. Bin sizes should not be too small when labeled cell bodies are analyzed because dendritic arbors may extend for hundreds of microns. Similarly, because postsynaptic neurons are more likely to be influenced by multiple presynaptic endings, the threshold count for labeled varicosities should be more than one (usually we require four or more) because multiple synaptic contacts need to be active to evoke a postsynaptic discharge. Having large bin sizes (e.g., 100 mm2) in which several labeled varicosities must be present with a labeled neuron produces a more realistic measure of potential synaptic connectivity between anterogradely labeled terminals and retrogradely labeled neurons.

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12. Persons responsible for plotting labeled varicosities must be carefully trained. For densely labeled images, untrained personnel have a tendency to plot brightly labeled varicosities while overlooking the labeled varicosities that are fainter in appearance. Also, if a brightly labeled axonal enlargement is apparent, but an axon does not emerge from two sides, the labeled structure should not be plotted because it may represent a labeled axon that was cut during histological sectioning. References 1. Alloway KD, Lou L, Nwabueze-Ogbo F, Chakrabarti S (2006) Topography of cortical projections to the dorsolateral neostriatum in rats: multiple overlapping sensorimotor pathways. J Comp Neurol, 499:33–48. 2. Alloway KD, Olson ML, Smith JB (2008) Contralateral corticothalamic projections from MI whisker cortex: Potential route for modulating hemispheric interactions. J Comp Neurol, 510:100–116. 3. Colechio EM, Alloway KD (2009) Bilateral topography of the cortical projections to the whisker and forepaw regions in rat motor cortex. Brain Struct Funct, 213: 423–439. 4. Smith JB, Alloway KD (2010) Functional specificity of claustrum connections in rats: An interhemispheric circuit for coordinating specific

types of bilateral movements. J Neurosci, 30: 16832–16844. 5. Alloway KD, Smith JB, Beauchemin KJ, Olson ML (2009) Bilateral projections from rat MI whisker cortex to the neostriatum, thalamus, and claustrum: Forebrain circuits for modulating whisking behavior. J Comp Neurol, 515:548–564. 6. Haiss F, Schwartz C (2005) Spatial segregation of different modes of movement control in the whisker representation of rat primary motor cortex. J Neurosci 25:1579–1587. 7. Kincaid AE, Wilson CJ (1996) Corticostriatal innervation of the patch and matrix in the rat neostriatum. J Comp Neurol 374:578–592. 8. Meng Z, Li Q, Martin JH (2004) The transition from development to motor control function in the corticospinal system. J Neurosci 24: 605–614.

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Chapter 9 An Approach to Understanding the Neural Circuitry of Saccade Control in the Cerebral Cortex Using Antidromic Identification in the Awake Behaving Macaque Monkey Model Kevin Johnston and Stefan Everling Abstract Saccadic eye movements are rapid conjugate movements of the eyes made to align the visual axis with objects of interest. Such movements are some of the most precise and well-controlled motor responses of which the nervous system is capable, and they display an enormous range of flexibility. Because saccades are controlled by a distributed network of brain areas including the cerebral cortex, basal ganglia, cerebellum, and brainstem, investigation of deficits in saccade control in movement disorders such as Huntington’s and Parkinson’s disease provide a potential window into their underlying neuropathology. A full understanding of pathological changes requires detailed investigations of the neural mechanisms of saccade control in the cerebral cortex. This requires invasive methods, which necessitate the use of an animal model. Here, we describe the rationale and use of the awake-behaving rhesus macaque model for the investigation of cortical control of eye movements. We focus on the technique of single neuron recording, particularly the use of antidromic identification of cortical projection neurons. Investigations of the circuit properties of the oculomotor system using this technique promise to provide unique insights into the neuropathology of movement disorders. Key words: Saccades, Eye movements, Microstimulation, Antidromic, Superior colliculus, Oculomotor, Electrophysiology

1. Introduction 1.1. Background and Historical Overview

Our ability to gather visual information from the surrounding environment is critically dependent upon the ability to move the eyes. Saccades are rapid eye movements made to align the visual axis with objects of interest. In humans and primates, such movements

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direct the fovea, the retinal region with the finest spatial resolution, to objects in the visual world and thus facilitate fine spatial analysis. Saccadic eye movements are arguably one of the best controlled motor behaviors of which we are capable. Within a total duration of as little as 300 ms, the oculomotor system is capable of determining the location of a suddenly appearing visual stimulus and sending control signals to the six extraocular muscles which will accelerate the eyes to velocities as great as 500 degree s−1 and then decelerate and land them accurately at the target location. This exquisite control system can be readily utilized to gain an understanding of the neural processes underlying the control of movement and the mechanisms by which disease processes adversely affect such control. Several aspects of the saccadic system render it amenable to such ends. First, because eye movements are restricted to rotations of the globes within the orbits, they are relatively simple to measure with a high degree of accuracy, and numerous eye tracking systems are commercially available which are capable of measuring eye movements with millisecond resolution. Second, many neurological conditions result in oculomotor deficits that can be readily observed and quantified. For example, patients suffering from disorders such as Huntington’s (1) and Parkinson’s disease (2) often exhibit difficulty initiating voluntary saccades, manifest in the form of increased saccade latencies. Third, because saccadic eye movements display an enormous range of flexibility, from short-latency reflexive saccades made to suddenly appearing stimuli, referred to as the “visual grasp reflex” (3), to purposive saccades directed to the mirror opposite location of a visual stimulus (4, 5), simple oculomotor tasks can be designed to evaluate different aspects of the control system, from initiation and suppression of movement to more cognitive functions such as working memory. Fourth, because rhesus monkeys can be trained to perform sophisticated eye movement tasks, in many cases identical to those carried out by human subjects, it is possible to perform detailed invasive physiological and anatomical investigations of the neural substrates of oculomotor control in a model system that in many ways approximates that of humans. In this sense, it is important to note the synergy of physiological and anatomical studies carried out in animals, and studies of changes in oculomotor control in disease states. Anatomical and physiological studies carried out in rhesus monkeys may provide the clinician with clues to the specific neural mechanisms disrupted in disease, while disruptive control changes due to disease states in humans may provide the basic scientist with insight regarding the normal operation of the system. As a result of this synergy, the system controlling saccadic eye movements is perhaps the most thoroughly understood sensory-motor system in the brain (6).

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Fig. 1. Cortical and subcortical areas of the saccadic eye movement system of the monkey. Top: medial view; bottom: lateral view. ACC anterior cingulate cortex, DLPFC dorsolateral prefrontal cortex, FEF frontal eye fields, LIP lateral intraparietal area, SC superior colliculus, SEF supplementary eye fields.

The saccade network is composed of a set of interconnected brain regions encompassing essentially the entire brain, including the cerebral cortex, thalamus, basal ganglia, cerebellum, and brainstem. At the cortical level, the saccade network includes the frontal (FEF) and supplementary (SEF) eye fields, dorsolateral prefrontal cortex (DLPFC) and anterior cingulate cortex (ACC), and the lateral intraparietal area (LIP) (Fig. 1). All of these areas send descending projections to the midbrain superior colliculus (SC), a region critical for saccade production that contains a topographic representation of saccade direction and amplitude. The SC, in turn, sends projections to structures in the brainstem directly responsible for saccade generation (7, 8). Much of our knowledge regarding the detailed neural mechanisms underlying the control of saccadic eye movements has been gained via recordings of the discharge characteristics of single neurons in rhesus monkeys trained to perform specific oculomotor tasks. Methods for extracellular recordings of single units in conscious animals were first developed by Hubel (9) and Jasper (10), and extended to include the trained, behaving, rhesus monkey by Edward Evarts (11), in his pioneering studies of the neural basis of movement control in the limb motor system. Single neuron recordings in the behaving primate are a powerful, and arguably the most direct approach to understanding the neural basis of behavior, since this method allows the observation of direct correlations between neuronal discharges and the specific components of behavior isolated by the experimental task being performed by the animal.

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The utility of this approach in oculomotor control in general, and the cortical control of saccades in particular, is well illustrated by investigations of the role of the FEF in oculomotor control. Early studies of electrical stimulation of the cortex by Ferrier (12) and others (13–16), revealed that stimulation of the frontal lobes of monkeys resulted in contralateral deviations of the eyes. Similar findings were subsequently obtained in humans (17, 18) and together, these results were generally accepted as evidence for a frontal eye field specialized for the initiation of eye movements. This “classical” view was subsequently challenged on the basis of single neuron recordings in the FEF of conscious monkeys, which demonstrated that neurons in this region increased their rate of discharge after rather than prior to the initiation of voluntary but untrained eye movements (19, 20). This discrepancy in findings was resolved by a series of investigations revealing a role of the FEF in control of goal-directed or purposive eye movements by combining recordings of single FEF neurons with strictly controlled oculomotor tasks. These studies demonstrated visual responses (21, 22) (and saccade-related discharges occurring prior to saccade initiation (23)) in FEF neurons. A direct role of FEF neurons in saccade initiation was subsequently demonstrated in single unit studies of monkeys performing the saccade countermanding task (24, 25), and it has been shown that this function is mediated by the connection between the FEF and SC (26). As the saga of the validation of the classical view of the FEF in saccade generation shows, dissecting the neural circuitry involved in the cortical control of saccadic eye movements requires the combination of strictly controlled behavioral tasks and physiological methods for recording the activity of single neurons. Further, from the anatomy of the saccade network, it is clear that saccade control depends not only upon local processes within the microcircuitry of a cortical area, but also the neural signals sent between nodes of the saccade circuit. This includes connections between not only cortical areas, but also those between cortical areas and subcortical structures with a direct role in saccade initiation, such as the SC. Indeed, as Evarts and colleagues (27) have stated: It should be appreciated that, insofar as the rest of the brain is concerned, the function of the cortex is mediated by its projection neurons. Only by influencing its subcortical targets…can the cortex affect behavior, either directly or indirectly via other cortical areas (p. 64).

A potentially fruitful approach to understanding the cortical control of saccadic eye movements is to employ a neurophysiological technique that permits investigation of the circuit properties of the saccade system. The technique of antidromic activation enables the identification of the cortical or subcortical projection targets of single neurons in the awake animal, and, when combined with behavioral tasks, a functional characterization of their response properties.

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Fig. 2. Schematic representation of experimental method for antidromic activation. (a) Waveform depicting activity showing artifact caused by microstimulation and stimulation-elicited action potential at the antidromic latency Ia. (b) Schematic representation of collision test. Spontaneous action potential triggers microstimulation pulse. If the stimulation latency Is is shorter than Ia plus the refractory period R, the two action potentials will collide and no action potential will be observed. (c) Same as (b), but the stimulation latency Is is longer than Ia plus the refractory period R. Therefore, microstimulation elicits an antidromic action potential.

When a neuron’s axon is electrically stimulated, an action potential is evoked which travels backward along the axon and invades the soma. This stimulation-evoked action potential can be observed in extracellular recordings (see Fig. 2). By recording the activity of single neurons in a given brain area, and applying electrical microstimulation to that area’s projection target, neurons sending a direct axonal projection to the target area can be identified. Because electrical stimulation can excite neurons either antidromically or transynaptically, and because no single criterion is diagnostic of antidromic activation, several criteria must be met by evoked action potentials to distinguish direct, antidromic responses from those evoked indirectly via intervening synapses (28–30). First, the latency between onset of stimulation and onset of the evoked action potential should be nearly constant at threshold stimulus intensity. Latencies of action potentials evoked transynaptically typically vary considerably due to variability in the duration of the synaptic processes intervening between stimulation and response, while those of antidromic action potentials are affected only by changes in the excitability of the membrane potential of the stimulated neuron (29, 31). Second, the threshold stimulus intensity required for evoking an action potential should remain stable, and increases in the intensity of stimulation should result in minimal changes in response latency. Synaptic activation typically results in variations in threshold intensity and large reductions in response latency as stimulus intensity is increased (28, 29). The third and most convincing criterion for antidromic activation is the collision test, which determines the presence of collision between spontaneously occurring and evoked action potentials (32–35). When a spontaneous action

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potential traveling along an axon in the forward, orthodromic direction encounters an antidromically evoked action potential traveling along the same axon in the opposite direction they will collide and both will be abolished (36). The collision test exploits this to establish antidromic activation. To apply the test, the occurrence of a spontaneous action potential in a neuron under study is used to trigger application of electrical stimulation of the target region of interest. If the spontaneous, orthodromic action potential, and the stimulation-evoked antidromic action potential are traveling along the same axon they will collide and no evoked action potential will be observed provided that both are traveling along the same axon at the same time. The time interval within which a collision is expected is referred to as the collision interval, I. With respect to the interval between spontaneous spike occurrence and delivery of stimulation, this interval may be defined as: I = L +R where L is the antidromic latency – the duration between stimulation onset and onset of the evoked antidromic action potential, and R is the refractory period of the axon. If the neuron under study is activated transynaptically, the orthodromic and antidromic action potentials will not collide, and a stimulation-evoked action potential will be observed. Neurons failing to meet any of the criteria noted above are disqualified from consideration as direct projection neurons. The technique of antidromic activation possesses characteristics advantageous to the study of the cortical control of eye movements. By identifying neurons projecting from one region of the oculomotor circuit to another, the technique enables an evaluation of the output signals of a given area. This approach has been employed to investigate signals sent between cortical areas and the superior colliculus; DLPFC to SC (37, 38), LIP to SC (39), FEF to SC (40–43), SC to FEF (44, 45). If the response properties of neurons in the target region are known, antidromic identification allows a direct comparison of output signals from one area to the target structure of interest, and the signals present within the target region. This approach has been applied to establish the progression of visual signals present in area LIP to more motor-related responses in the SC (46). Characterization of neural signals carried by parallel cortico-cortical and cortico-subcortical pathways may also be investigated. For example, Ferraina et  al. (47) identified LIP neurons projecting to the FEF or SC and established a visual bias of signals in cortico-cortical but not cortico-tectal pathways. Antidromic activation may also provide a means for investigating the local circuitry of cortical areas by providing supporting evidence for the classification and functional assessment of different neuronal types within a cortical region (48, 49). Because corticotectal neurons are located exclusively within layer V (50), identified

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neurons may also provide a landmark regarding the laminar location of a neuron under study. Studies aiming to derive a mechanistic understanding of the microcircuitry of the saccade system promise to provide further insight into disruptions in neural processing occurring in a variety of movement disorders, as many can be characterized as the result of disruptions in the function of neural circuits. Here, we aim to provide a basic guide to the study of neural circuits involved in the control of saccadic eye movements in the rhesus monkey model. We begin by briefly discussing the choice of the rhesus macaque as an animal model of the human oculomotor system, some typical behavioral paradigms, then proceed to application of experimental methods including training techniques and antidromic identification. 1.2. The Animal Model

The rhesus macaque has long been the animal model of choice for investigations of the neural basis of oculomotor control. The selection of this model has been based on several criteria. First, the overt oculomotor behavior of humans and rhesus macaques is strikingly similar. Both species produce saccadic and smooth pursuit eye movements with only minor quantitative differences (51). Further, rhesus monkeys can be trained on sophisticated behavioral paradigms, and their performance in tasks such as saccade countermanding (52) and the antisaccade task (53) is similar to that of human subjects. Second, there are substantial structural similarities between the oculomotor systems of humans and macaques. In humans and other primates, the SC contains a map of the contralateral visual field, while in other mammalian species the SC contains at least some representation of both ipsilateral and contralateral visual fields (54). There are also dense projections from frontal cortical areas to multiple layers of the SC in primates (55, 56), which are absent in non-primate species (57). Finally, putative homologies of many electrophysiologically defined areas of the saccade network in the rhesus monkey brain have been established in the human brain using comparative high-resolution fMRI (58–61) and cytoarchitectonic (62, 63) analyses. For example, a homologue of the monkey FEF has been localized to the junction of the superior precentral sulcus with the superior frontal sulcus (64–66). Detailed discussions of the primate model have been provided (67, 68).

1.3. Behavioral Tasks

A range of oculomotor tasks have been designed to investigate the control of saccades (Fig. 3). Such tasks range from simple visuallyguided saccade tasks, used to investigate basic processes of saccade control and the responses of single neurons to visual stimuli and saccades, to the antisaccade paradigm, which requires more sophisticated control mechanisms in the form of suppression of a response to a suddenly appearing visual stimulus and a voluntary saccade to the opposite location. Task performance is maintained by providing

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Fig. 3. Experiment saccade paradigms. The dashed circles indicate where the monkey has to fixate and the arrows indicate the direction of the saccade.

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fluid reinforcement for correct responses. Behavioral indices of performance in these tasks include saccadic reaction time (SRT), proportion of responses correctly directed to the target location, and accuracy of the saccade endpoint with respect to the target location. Accuracy may be expressed as either variability (referred to as endpoint scatter), or systematic shifts with respect to target location. Saccades falling short of the target location are termed hypometric, while those surpassing the target location are referred to as hypermetric. The relationship between performance of these tasks and neural activity is typically investigated by temporally aligning task events, such as visual stimulus onset, or behavioral events, such as saccade onset, with recorded neural activity and carrying out statistical analyses of changes in activity time-locked to those events. Some common paradigms are detailed below. 1.3.1. Visually Guided Saccades

The visually guided saccade task requires the animal to acquire fixation of a central visual stimulus which either (a) disappears a few hundred milliseconds before the appearance of a peripheral visual stimulus (gap task), (b) disappears simultaneously with appearance of the peripheral stimulus (step task), or (c) remains visible while the ­peripheral stimulus is presented (overlap task) (see Fig.  3a). The duration of presentation of the central fixation stimulus is typically several ­hundred milliseconds, and is varied in order to avoid anticipatory responses and thus ensure that the saccades are truly visually guided. A well-described observation is the reduction of saccadic reaction times in the gap task compared to the overlap task in healthy human subjects (69) and also in non-human primates (70). In addition to this so-called gap-effect, many subjects also generate saccades with extremely short reaction times in the gap condition. These ­saccades are termed express saccades and have reaction times between 90 and 120 ms in humans (71, 72), and 70 and 90 ms in monkeys (73, 74). Neural correlates for the gap effect have been found in the FEF (41, 75), DLPFC (76), LIP (77), and the SC (78, 79).

1.3.2. Delayed Saccade Task

This class of tasks inserts a delay between visual stimulus presentation and saccade onset in order to dissociate neural responses related to visual stimulus onset from those associated with saccade initiation (see Fig. 3b). In the visually guided delayed saccade task, animals are required to fixate a centrally presented visual stimulus for a variable period of a few hundred milliseconds, following which a peripheral stimulus is presented. The fixation and peripheral stimuli remain present concurrently, typically for a delay of several hundred milliseconds. Offset of the fixation stimulus serves as a “go” signal instructing the animal to make a saccade to the location of the peripheral stimulus. A variation of this task is the memory-­guided delayed saccade task (80), which differs in that the peripheral visual stimulus is presented briefly rather than remaining

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present for the duration of the delay period. Following the delay, the animal is required to generate a saccade to the remembered location of the visual stimulus. Because it requires a saccade to a remembered location, this version of the task has been used extensively to investigate the neural basis of working memory. In most cortical nodes of the saccade circuit, single neurons exhibit changes in activity time-locked to visual stimulus onset, saccade onset, and sustained delay period activity during this task: LIP (81), FEF (23, 82), and DLPFC (82–84). 1.3.3. Saccade Countermanding Task

This task has been used extensively to investigate the neural basis of saccade initiation in a range of cortical areas including FEF (24, 25), SEF (85), ACC (86), and the SC (87). The saccade countermanding task probes the ability to control initiation of voluntary saccades by infrequently presenting a stop-signal in the context of a standard visually-guided saccade task, and requiring animals to withhold a saccade in response to the stop-signal (see Fig. 3c). Two types of trials are typically presented. On no-stop signal trials, animals are required to acquire and maintain fixation on a central fixation stimulus for a period of several hundred milliseconds. A peripheral visual stimulus is presented concurrent with fixation offset and the animal is required to saccade to the stimulus location. On stop-signal trials, reappearance of the fixation stimulus serves as a stop signal. At variable durations following its initial disappearance, the fixation stimulus reappears and the animal is required to withhold a saccade. Behavioral performance on stop trials task can be modeled as a race between go and stop processes that accumulate toward a fixed threshold (88), with the process reaching threshold first being the behavior that is expressed. Using this model, it is possible to compute the time required to cancel a planned movement; the stop signal reaction time (SSRT). Neural activity recorded concomitantly with performance of this task must meet two criteria to be considered sufficient for saccade initiation. First, discharge must differ between trials on which a saccade is produced and those on which a saccade is cancelled. Second, such changes in activity must occur before the SSRT. Recordings of ­single neurons in behaving monkeys have shown that the only cortical area with signals sufficient to control initiation of saccades is the FEF (24, 25). A similar finding has been obtained in the SC (87).

1.3.4. Antisaccade Task

The antisaccade task (4) probes the ability to suppress an automatic saccade toward a flashed peripheral stimulus in favor of a voluntary saccade to its mirror location and has been used extensively to investigate inhibitory control of saccades and the neural correlates of response preparation or preparatory set (89). In a typical antisaccade experiment, animals are required to fixate on a centrally presented fixation stimulus. Upon presentation of a peripheral visual stimulus, they are required to either generate a saccade

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toward the peripheral stimulus (prosaccade trial) or generate a ­saccade of equal amplitude away from the peripheral stimulus in the opposite direction (antisaccade trial) (see Fig.  3d). Pro and antisaccade trials may be performed either in blocks of trials or randomly interleaved, and task instructions are typically provided to monkeys via the color of the central fixation point. Monkeys can be trained to perform the antisaccade task (53, 90), and numerous single neuron studies have investigated the roles of cortical areas and the SC in this task (see (89) review).

2. Methods 2.1. Behavioral Training

Rhesus macaques can be readily trained to perform oculomotor paradigms using the method of successive approximations (91). This method involves successively rewarding oculomotor responses that are progressively more similar to the desired response. Fluids such as small amounts of water or fruit juice are typically used as rewards. Training in oculomotor paradigms is undoubtedly facilitated by the fact that saccades naturally occur with high frequency, the result being that animals are able to quickly learn contingencies between oculomotor responses and reward. This technique was first applied to training of oculomotor responses in monkeys by Fuchs and Robinson (51, 92). Oculomotor training requires monitoring of eye movements and precise timing of reward delivery in relation to task events. This can be accomplished using commercially available technologies for eye tracking and computerized behavioral control systems. Successive steps in the training progression are outlined below.

2.1.1. Fixation Training

Since most saccade tasks require the monkey to actively fixate prior to or during the presentation of a peripheral saccade target, this is a necessary first step in training on all oculomotor paradigms. A ­further benefit of this training is that monkeys readily learn that moving the eyes to and fixating visual stimuli generally results in reward, which facilitates subsequent saccade training. The simplest method for training animals to fixate is to present a small picture of an interesting object on an otherwise blank ­display. The natural curiosity of the animals will typically result in their looking at this stimulus, and this behavior can then be reinforced by providing reward when the animal’s eyes are on target. Initially, rewards can be delivered when the eyes are on target for only a short time. Over successive responses, the required fixation duration may be gradually increased. Following this, the size of the stimulus and area within which eye position must be maintained may be reduced. Finally, the picture may be replaced with a small fixation spot or cross.

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2.1.2. Saccade Training

Once monkeys have learned to fixate a small visual stimulus for a sufficient period of time, they may progress to saccade training. As with fixation training, this stage is facilitated by the natural curiosity of the animals; they will generally saccade toward a suddenly appearing visual stimulus without any specific training. The ­simplest training method is thus to simply add a peripheral visual stimulus to the fixation training protocol and modify the reward contingency such that the reward is delivered when the animal actively fixates and subsequently makes a saccade toward the peripheral visual stimulus upon its appearance. Initially, the animal may be rewarded for ­saccades directed to a large area surrounding the visual stimulus. The spatial requirements of this response can be made successively more precise by gradually shifting reward to only those saccades made within a restricted area. In addition, the ­number of directions and amplitudes at which the peripheral ­stimulus is presented may be restricted to simply left and rightward directions in the early stages of training, and subsequently progressed.

2.1.3. Specialized Training

Once the monkey is able to reliably make visually guided saccades, training in more specialized tasks may commence. Here, we ­provide as an example the general approach we have employed to train monkeys to perform the antisaccade task. In this task, the color of the central fixation stimulus serves as a behavioral instruction to perform either a pro- or antisaccade. A first step in training on this task is thus to establish a contingency between fixation color and the required saccade. This can be accomplished by training the animal to follow a simple matching rule. On some trials, a colored fixation stimulus may be presented, followed by two simultaneously appearing peripheral stimuli, one of which matches the color of the fixation stimulus and another that does not. To train the animal to follow a matching rule, saccades made to the peripheral stimulus matching the color of the fixation stimulus are rewarded, while those to the peripheral stimulus of non-matching color are not. For example, a red fixation stimulus may be presented, followed by red and green stimuli at opposite directions and equal distances from fixation. The animal would then be rewarded for saccades to the location of the red peripheral stimulus. On other trials, a green fixation stimulus would be presented, and saccades to the green peripheral stimulus rewarded. It is generally easiest for monkeys to learn this color-matching rule if trials with a given color are presented in blocks of several trials in a row, followed by several trials with the other fixation color. Following this, fixation colors may be presented in a randomly interleaved fashion. Once monkeys are able to perform this color matching task, antisaccade training may commence. In this phase of training, the luminance of one of the colored peripheral stimuli is gradually reduced until eventually the animal is generating a saccade to a blank location. For example, over a number of days, the luminance of the

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green stimulus may be reduced but saccades to its location will ­continue to be rewarded. Because the animal has previously learned a color-matching rule, it will initially make saccades to the red peripheral stimulus when presented with a red fixation ­stimulus, and the green peripheral stimulus when presented with a green fixation stimulus. As the luminance of the green peripheral ­stimulus is reduced and it becomes progressively less visible, the animal will gradually learn to make saccades away from the location of the red peripheral stimulus. Once the green peripheral stimulus is no longer visible and the animal is able to reliably make saccades toward the red peripheral stimulus when presented with a red fixation ­stimulus, and away from the red peripheral stimulus when presented with a green fixation stimulus, the color of the red peripheral stimulus may be gradually desaturated. An animal whose performance has been shaped in this manner will make saccades toward the peripheral visual stimulus (prosaccades) when presented with a red fixation stimulus, and away from the peripheral stimulus when presented with a green fixation stimulus. As with training of color matching, this training may progress more readily when colored fixation ­stimuli are initially presented in blocks of trials, and randomly interleaved only when animals have obtained a high level of performance. 2.2. Antidromic Identification 2.2.1. Experimental Setup

2.2.2. Place-Stimulating Electrodes in the Target Region: SC

Neurophysiological experiments using antidromic identification do not require a great deal of specialized equipment that is not generally available in a standard laboratory set up for single neuron recordings in awake-behaving macaques. Commercially available microstimulators and photoelectric stimulus isolation units typically used for microstimulation of the brain should suffice. An oscilloscope that can be triggered by microstimulation onset, and from which voltage traces can be saved is required. No specialized procedures are required to prepare animals for such experiments. Standard recording microelectrodes and microelectrode drives are sufficient. Because most cortical areas involved in the control of saccadic eye movements project to the superior colliculus, we detail below the steps involved in antidromic identification in the context of an experiment seeking to identify cortical neurons sending a direct projection to the SC – cortico-tectal projection neurons. Because the technique of antidromic identification depends on activation of axons within the target region (30), stimulating electrodes must be placed with care. In this regard, the known anatomy of projections to the SC can be exploited. The majority of axons entering the SC do so via the rostral pole (93), and the probability of antidromically stimulating a cortico-tectal axon is concomitantly greater when stimulation is applied at this collicular site. A similar logic has been employed with regard to antidromic identification of corticospinal neurons, in which stimulation is typically applied at the medullary pyramids, through which all corticospinal axons pass (35).

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For this reason, it is useful to map the topography of the SC by determining the response fields of single SC neurons and performing microstimulation. Because the intermediate layers of the SC contain a topographic map of saccade direction and amplitude, with smallest amplitudes represented at the rostral pole (94), this region of the SC can be readily identified by observing the direction and amplitudes of the response fields of SC neurons, and the direction and amplitudes of saccades evoked by microstimulation. As the influence of cortical outputs on saccade generation are typically under study, stimulation electrodes should be located in the intermediate layers of the SC. Practically speaking, electrodes should be located at a depth at which saccades can be evoked at the lowest possible stimulation intensity. Stimulation electrodes may be advanced daily, or fixed in place within the recording chamber using epoxy (see Note 1). The latter method has the advantage that multiple stimulation electrodes may be fixed at multiple ­locations across the SC intermediate layers, thus maximizing the likelihood of antidromically activating an incoming axon. 2.2.3. Isolate the Activity of Single Neurons in the Cortical Area of Interest

Before a candidate neuron can be tested for antidromic activation, it must be well isolated and stable. It is obviously futile to attempt to identify an individual projection neuron using the criteria for antidromic identification if the action potentials under study are being elicited by multiple units. As is always the case with recordings of single neurons, a bias toward isolation of larger neurons exists (see Note 2). The use of modern microdrives, which allow multiple recording electrodes to be advanced simultaneously, and multiple channel recording systems, which allow simultaneous isolation of multiple single neurons, can greatly increase the yield of antidromically identified neurons in a study, by virtue of the simple fact that multiple single neurons can be isolated and tested for antidromic ­identification simultaneously. In addition, because cortico-tectal projection neurons often have a “patchy” distribution across layer V of the cortex (50, 95) that results in small, localized regions containing larger numbers of cortico-tectal neurons, distributing electrodes across the cortical surface of an area of interest allows for a parallel, spatially broad search for such patches, which may subsequently be restricted when regions of the cortex with particularly high yields of projection neurons are identified. The researcher may then focus their efforts on these areas to maximize the yield of identified neurons (see Note 3).

2.2.4. Apply Micro­ stimulation to the Projection Target: SC and Determine Whether an Action Potential Is Evoked in the Neuron Under Study

Once the action potentials of a single unit have been reliably isolated, the neuron under study can be tested for antidromic activation. Single stimulation pulses may be applied to one or more electrodes within the SC (see Note 4). Single oscilloscope traces of the recording record should be triggered by the stimulation pulse. A typical record will show a stimulation artifact caused by application

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of the stimulus pulse. If stimulation evokes action potentials in the axon of the neuron under study an action potential will be observed in the oscilloscope trace following the stimulation artifact (Fig. 2a). If an action potential is reliably evoked by application of stimulation, the experimenter may proceed to apply the previously noted criteria for distinguishing antidromic from transynaptic activation. Application of these criteria is described below. 2.2.5. Apply Criteria for Antidromic Activation

Once it has been established that stimulation of the SC evokes an action potential, the criteria of constant latency, constant threshold, and the collision test may be applied. To determine constant latency, the current threshold for ­evoking an action potential in the neuron under study must first be determined. This may be accomplished by systematically varying the amount of current applied and determining the level at which an action potential is evoked on 50% of stimulus applications (29). Once threshold has been determined, stimulation pulses at this threshold level should be repeatedly applied, and the latency of the evoked action potential with respect to the onset of the stimulation noted. If the neuron under study is antidromically rather than ­transynaptically activated, the latency of the evoked action potential with respect to the onset of stimulation will remain relatively constant. This may be defined as the antidromic latency (Ia) (see Fig. 2). Typically, latency variability will be quite low (29), usually within 20% of Ia (28). The criterion of constant threshold should also be applied by periodically repeating the procedure described above for determining threshold and noting the threshold value. If the criteria of constant latency and threshold are met, the collision test should be applied. This test is the most definitive method for differentiating antidromic from transynaptic activation (30, 35) (see Note 5). To apply the test, the experimental setup must allow for triggering of the stimulation pulse and oscilloscope sweep by a spontaneous action potential emitted by the neuron under study. Stimulation pulses should be triggered by the spontaneous action potential, and the latency between onset of the ­spontaneous action potential and stimulation onset (Is) should be varied. If Is is less than the antidromic latency, Ia, plus the refractory period (R) of the axon of the neuron under study (the ­collision interval), the orthodromically traveling spontaneous spike and the stimulation evoked antidromic spike will collide, and no action potential should be observed in the record following the stimulation artifact (Fig. 2b). Conversely, if Is is greater than the collision interval, Ia + R, the orthodromic action potential will pass the stimulated region of the axon before the antidromic spike is evoked, and no collision will result. In this case, an action potential will be observed in the recording record following the stimulation artifact (Fig. 2c). The test should be repeatedly applied and confirmed at a range of values of Is both greater and less than the collision

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interval. If collision is reliably observed, and the criteria of constant latency and threshold are met, the neuron under study may be considered a cortico-tectal projection neuron. Behavioral paradigms and data collection should then commence (see Note 5). The collision test may be applied throughout the recording session in order to confirm isolation of the unit under study.

3. Notes 1. The use of fixed versus movable stimulation electrodes has been discussed (30). Both methods have some advantages. Use of movable electrodes allows determination of the threshold intensity for antidromic stimulation as a function of electrode depth, and thus may be useful for determining the optimal location for placement of stimulating electrodes. Pare and Wurtz (39) used this method to investigate threshold for antidromic stimulation of cortico-tectal LIP neurons as a function of the depth of the stimulating electrode within the SC, and found lowest thresholds for depths at which SC neurons showed saccade-related activity. Conversely, the use of multiple fixed electrodes may increase the yield of identified neurons, since multiple SC sites can be simulated within each session, and the duration of each session is reduced as stimulation electrodes need not be advanced each day. An optimal approach may be to begin experiments using antidromic identification using a movable stimulation electrode to determine optimal depths for evoking antidromic responses at sites across the collicular surface, and subsequently fixing electrodes in place at these depths and locations. 2. In any extracellular recording experiment, a substantial bias exists toward isolation of larger neurons. This is due to the fact that action potential discharges of larger neurons involve a greater flow of membrane current. The amplitude of extracellularly recorded action potentials is therefore greater for larger neurons, and may be detected at greater distances from the recording electrode than action potentials of smaller neurons. This bias toward larger neurons may be especially pronounced in the case of experiments employing antidromic identification, since action potentials can be evoked with lower stimulation intensities in axons of large diameter (96), which are generally associated with larger soma (97). The combination of these effects induces a “double bias” toward recordings of larger neurons, and may produce misleading results when the response properties of neurons in a given behavioral task are pooled to determine population activity (28, 98). This is particularly

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problematic given the large range of sizes of pyramidal cortical neurons (56, 99, 100). The issue of sampling bias may be addressed in two ways. First, random rather than selective sampling procedures may be employed. This simply involves careful attempts to isolate and test each neuron encountered. This process may be facilitated by current multi­channel acquisition processors that allow offline sorting of spikes, though it is impossible to remediate truly poor isolation. Second, a procedure correcting for sampling bias may be applied. This procedure involves estimating the effective volume of neural tissue recorded from, which may be determined from the conduction velocity of neurons in the neural sample. Conduction velocity can be readily computed based on antidromic latency, and the known distance of anatomical projections between cortical areas and the SC. The relationship between effective volume, veff and conduction velocity has been determined to be:

veff = kv 3/ 2 where k is a constant and v is the conduction velocity of the neuron (98). This relationship holds because of the relationship between conduction velocity, axon size, and soma size (97). This correction procedure has been described in detail (28). Examples of its ­application may be found in Humphrey and Corrie (98) who applied it in the limb motor system, and Sommer and Wurtz (42) who applied it in investigation of cortico-tectal FEF neurons. 3. In general, it is best to apply the minimal intensity of stimulation necessary to elicit antidromic identification. Use of excessive currents or durations may have deleterious effects on both the neuron under study and connected neurons. This is a further reason to determine optimal stimulation depths, as described above. In addition, current spread is increased as stimulation intensity is increased (96), and may in some cases activate tissue outside the target area. An additional negative effect is evocation of a larger stimulation artifact in the recording record that may obscure evoked antidromic action potentials occurring at short latencies. This may result in selectively decreased yield of projection neurons activated at short latencies, and introduce bias into experimental results. Experiments employing antidromic identification of cortico-tectal neurons typically employ biphasic square-wave pulses of 0.15–0.3 ms in duration (37, 39, 41, 42). 4. Under some conditions, errors may occur in the estimation of the collision interval, which may lead to inconsistent results when the collision test is applied. Such results may result in the

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rejection of candidate neurons. This issue has been discussed in detail (35). 5. Because experimental setup, isolation of single neurons, and application of stimulation and the tests for antidromic activation can be time-consuming, awake-behaving animal subjects may exhibit some restlessness during each recording session. This can be manifested as movement, which can adversely affect the stability of recordings and thus the process of antidromic identification. A ­simple method that may alleviate this is to display movies or other videos on the stimulus display monitor while the experimenter goes through these procedures. Typically, this will alleviate boredom on the part of the animal subject, and reduce restlessness and movements. Once a neuron has been isolated and identified, behavioral tasks and data collection may commence as normal.

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Chapter 10 Photothrombotic Infarction of Caudate Nucleus and Parietal Cortex Toshihiko Kuroiwa and Richard F. Keep Abstract Basal ganglia infarction is typically caused by the occlusion of deep arteries and the formation of relatively small lesions called lacunes. In this report, we describe a method to induce basal ganglia infarction by photothrombosis and show some of the characteristics of the method in comparison to the more standard cortical photothrombotic model. A polymethylmethacrylate optic fiber with 50-cm length and 0.5-mm diameter is connected to a light source. The edge of optic fiber is beveled with 30 degree of angle, and the light intensity at the tip kept at 1,700 lux. Male Sprague–Dawley rats or Mongolian gerbils are anesthetized, and the optic fiber was stereotaxically placed into left caudate nucleus or parietal cortex. Rose Bengal dye (20 mg/kg) is intravenously injected, and the brain exposed to cold white light for 10 min via the optic fiber. In our experiments, morphological examination revealed an infarct with thrombosed parenchymal vessels surrounded by a layer of selective neuronal death in both the caudate nucleus and cortex. The pattern and distribution of open field locomotion after caudate infarction in gerbil was significantly different from those of cortical infarction. Photothrombotic infarction is inducible in the basal ganglia by the stereotaxic implantation of thin optic fiber. This method is useful for the study of neurological changes after basal ganglia infarction. Key words: Polymethylmethacrylate optic fiber, Photothrombosis, Histology, Infarction, Neurological deficits

1. Introduction Neurological deficits differ between basal ganglia and cortical infarcts. Basal ganglia infarction is typically caused by the occlusion of deep small parenchymal arteries. While numerous animal models of cerebral ischemia induced by large intracranial artery occlusion have been developed (1), few models of basal ganglia infarction have been reported. We recently developed a new method to induce caudate infarction by photothrombosis via a stereotaxically

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implanted plastic optic fiber (2). In that study, we observed changes typical of ischemic tissue in the caudate, i.e., decreased local cerebral blood flow, ischemic neuronal and parenchymal changes, blood–brain barrier disruption, edema, and neurological deficits. Here, we describe the method to induce basal ganglia infarction by using plastic optic fiber. We also discuss the characteristics and limitations of the new method.

2. Materials and Methods 2.1. Animal Preparation

Experimental protocols were approved by the University of Michigan Committee on the Use and Care of Animals. Male Sprague–Dawley rats weighing 270–320 g or male Mongolian gerbils weighing 50–70  g (both from Charles River Laboratories; Portage, MI) were used. Animals had free access to food and water before surgery. Anesthesia was induced by inhalation of 4% isoflurane in a mixture of nitrous oxide/oxygen mixture (70%/30%) and maintained by 2% isoflurane administered through a facemask. Rectal temperature was maintained at 37.5°C with the use of a feedback-controlled heating pad (Model 74; Yellow Springs Instrument Co). Left femoral vein and artery were cannulated for drug injection and for monitoring blood gases and blood pressure.

2.2. Photothrombosis Procedure

Polymethylmethacrylate optic fibers coated with polyethylene with 0.5 mm in diameter and 50 cm in length (Eska CK-20, Mitsubishi Rayon) were used for cold lighting. The optic fiber with an attachment handmade of plastic syringes and pipettes was connected to a light source with dichroic halogen bulb and parabolic reflector (Model LG-PS2; Olympus) (Fig. 1a, b). The edge of the fiber was beveled with 30 degree of angle (Fig.  1c). Illuminated region was spherical (Fig. 1d), and the light intensity (luminous emittance) at the tip of optic fiber was monitored and kept constant at 1,700 lux. The anesthetized animals were placed in a stereotaxic holder (Fig. 1e) and the optic fiber was placed to the left caudate nucleus (stereotaxic coordinates of rat: posterior to bregma = 0  mm, left = 3  mm, depth = 4.5  mm, gerbil: posterior to bregma = 0  mm, left = 2  mm, depth = 3 mm) (Fig. 1f) or left parietal cortex (stereotaxic coordinates of rat: posterior to bregma = 4 mm, left = 4 mm, depth = 0 mm, gerbil: posterior to bregma = 3 mm, left = 3 mm, depth = 0 mm) via a small burr hole (3, 4). Rose Bengal dye (20 mg/mL saline, 1 mL/kg, disodium 4,5,6,7-tetraiodofluorescein; Sigma) was infused via the femoral vein and light exposure for 10 min followed immediately. After light exposure, the optic fiber and catheters were removed, and the incisions were closed. Control animals underwent caudate or cortex light exposure without Rose Bengal dye infusion.

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Fig. 1. (a) Light source (Olympus, Tokyo, Japan) with an attachment of optic fiber. (b) The attachment of optic fiber ­handmade of plastic syringes and pipettes. (c) Schematic drawing of the tip of the optic fiber. The diameter is 0.5 mm. (d) Spherical lighting from the optic fiber. (e) Position of the optic fiber and stereotaxic frame. (f) An optic fiber stereotaxically implanted to rat.

2.3. Histopathology and Blood–Brain Barrier Changes

For light microscopy, the animals were reanesthetized with 4% isoflurane and transcardially perfused with 4% paraformaldehyde in 0.1 mol/L phosphate-buffered saline (pH 7.4). The fixed brains were removed and kept in 4% paraformaldehyde for 6 h. A coronal slab of brain tissue containing the center of the lesion was cut, embedded in paraffin, sectioned, and prepared for hematoxylin and eosin staining. Electron microscopy was also performed on caudate tissue from the rats at 4 h after photothrombosis. The animals underwent transcardiac perfusion with 4% glutaraldehyde. Specimens containing the photothrombosis-induced lesion were excised, embedded in epon, and prepared for ultrastructural examination. For the assessment of blood–brain barrier change, the animals were anesthetized and injected with Evans Blue solution (2 mL/kg, 2% in saline). After 1 h, brains were removed and cut coronally (1-mm-thick) on a brain-slicing matrix at the level of optic fiber implantation. The coronal section was stained with 2,3,5-triphenyl tetrazolium chloride to visualize the area of mitochondrial dysfunction and Evans blue leakage.

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2.4. Neurological Deficits

Neurological deficits were examined in gerbils by the recording of open field locomotion. The animals were placed in an open field 90 cm × 90 cm in size and their spontaneous locomotion was traced with a video-tracking system (PanLab, Barcelona, Spain) for 5 min, and length of stay in the center and the periphery, both equal to 50% of total area, of open field was calculated.

3. Model Characteristics In our hands, the mortality rate was zero in these stroke models. The blood gases and blood pressure were stable and within the normal range. At 24  h after light exposure in the basal ganglia model, an oval, lightly stained, ischemic lesion was present in the caudate around the tip of the optic fiber (Fig. 2a, b). Histologically, the lesion was an ischemic infarction (Fig.  2c). Four hours after

Fig. 2. (a) An example of 2,3,5-triphenyl tetrazolium chloride staining 1 day after photothrombosis (bar = 1.0 mm). A round lesion indicative of infarction is seen in the center of the caudate nucleus. Evans blue was injected intravenously as a tracer of blood–brain barrier opening before sacrifice and an area of blue staining is seen within the lesion. (b) Schematic drawing of the stereotaxic placement of the optic fiber in the caudate nucleus. (c) Typical lesion 1 day after photothrombosis (hematoxylin and eosin staining; scale bar = 1.0 mm). (d) A small thrombosed blood vessel (about 10 mm diameter) 4 h after light exposure (bar = 5 mm). (e) Typical electron micrograph of a large microvessel within the periphery at 4 h after ictus filled with platelets and are surrounded by swollen perivascular end-feet of astrocytes (bar = 50 mm). (f) Histology 4 h after light exposure demonstrating neuronal pyknosis and microvacuolation (bar = 5 mm).

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photothrombosis, many thrombosed microvessels with pyknotic and eosinophilic neurons and numerous microvacuoles were seen in the lesion (Fig.  2d, f). Electron microscopy revealed platelet thrombus formation within parenchymal small vessels (Fig.  2e). The lesion became infarcted at 24 h after photothrombosis. Histological changes after cortical photothrombosis were identical to caudate photothrombosis (Fig. 3). Control animals developed no tissue injury except for that caused by the needle track.

Fig. 3. (a) Typical lesion 4 days after cortical photothrombosis. Coronal section of the lesion showing central infarction and lesion periphery (hematoxylin and eosin staining; scale bar = 0.2 mm). (b). Higher magnification of the infarction periphery (square in Fig. 3a) demonstrating a layer of pyknotic neurons and microvacuolation (bar = 50 mm). These changes are identical to those seen after caudate infarction.

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Fig. 4. (a) Typical trace of locomotion 24 h after caudate photothrombosis in Mongolian gerbil. The animal developed circulation. The locomotion was evenly distributed both in the center and periphery. (b) Typical trace of locomotion after cortical photothrombosis. Circulation was not observed, the locomotion was distributed mostly in the periphery of the open field.

Recording of locomotion in gerbil after photothrombosis revealed different pattern and distribution between caudate and cortical lesioning. The trace showed circulation after caudate photothrombosis (Fig. 4a). The animal moved evenly both in the center and periphery of the open field. After cortical photothrombosis, the animal developed linear locomotion mainly in the periphery of the open field (Fig. 4b).

4. Discussion of Model Watson et al. originally developed an animal model of cortical photothrombotic infarction (5). The method has high reproducibility in the size and location of ischemic lesion. We have reported a model of photothrombotic infarction in the deep brain structures using an optic fiber stereotaxically implanted to deep brain structure (2). We found polymethylmethacrylate optic fibers were suitable for cold lightning because of low transmission loss (about 0.25 dB/m or 3% loss/m) at the absorption wave length of Rose Bengal (520–550 nm) and negligible transmission of the infrared light (CK-20 Specification sheet DPF0811–15, May 2001; Mitsubishi Rayon) (6). A preliminary study revealed no temperature increase of 1 mL water in a chamber 10 min after light exposure. In fact, tissue injury and neurological deficits were mild in

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control rats that underwent optic fiber implantation and light exposure without Rose Bengal dye infusion. Histological examination early after light exposure with Rose Bengal showed an almost spherical infarct around the tip of the optic fiber surrounded by a peripheral area of selective neuronal death and ischemic edema. Thrombotic occlusion of small parenchymal vessels was found in the center and periphery of the lesion, and local cerebral blood flow (CBF) was decreased by 85% in the lesion center. We observed that the infarcted area turned to a small cyst 6 weeks after photothrombosis (2). These histological changes are very similar to cortical photothrombosis (5). This is, thus, a model of deep localized brain ischemia that induces subsequent infarction. Significant neurological deficits after a caudate hematoma have been noted (7). We have reported that neurological changes after caudate photothrombosis were similar to those after caudate hematoma (2). Thus, caudate photothrombosis causes significant reductions in vibrissae-elicited forelimb placing, greater forelimb use asymmetry and more bias in a corner turn test (2). These neurological deficits generally were maximal at day 1 after lesioning and then decreased over the period of 4–6 weeks (2). This suggests that this model may be useful in studying interventions designed to improve recovery after a stroke. The results presented here also revealed that the pattern and distribution of open field locomotion after caudate infarction in gerbil was significantly different from those of cortical infarction. Thus, photothrombotic caudate infarction is a useful model for future studies on motor disturbances after basal ganglia lesion. The current study used a 0.5-mm-diameter plastic optic fiber. The needle tract lesion was significantly smaller than the photothrombotic lesion. We have reported that there were no behavioral deficits in the animals that just had the optic fiber insertion plus light without Rose Bengal infusion, and the local CBF decrease did not reach ischemic level except for the area adjacent to the optic fiber (2). However, many mechanisms have been shown to be involved in the tissue injury by brain penetration (8). Therefore, a thinner optic fiber that could transmit enough cold light for lesioning and still retaining enough stiffness for accurate stereotaxic implantation might prove to be advantageous. Although our studies have focused on caudate and cortex lesions, it should be possible to use this technique to cause ischemic lesions in a variety of other brain areas provided that it is possible to insert the optic fiber to the correct location and that the injury caused by fiber insertion (for deep brain lesions) is minimal compared to that induced by photothrombosis. The technique should also be adaptable to species other than the rats and gerbils used here. Photothrombosis has been used to generate cortical lesions in mice (8). It may also be useful in causing

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deep brain lesions in mice with the caveat, again, that the lesion must be significantly larger than that caused by insertion of the fiber alone. The technique may also be useful for large animal models of stroke. Different-sized lesions can be generated by varying the illumination intensity and the diameter of the fiber optic (2). Thus, we have generated caudate lesions from 1 to 7  mm3 in rats (2) but larger deep brain lesions may be possible. Apart from the histological and behavioral endpoints described above, the caudate photothrombosis model can be used to examine other stroke-related parameters. For example, we have used [14C]-N-isopropyl-p-iodoamphetamine to measure CBF in the rat model using either tissue sampling or autoradiography (2). The model would also be amenable to measurements using positron emission tomography (PET). The lesion also results in significant brain edema which can be measured by wet weight/dry weight or specific gravity measurements (2). Perilesional edema might also be examined using magnetic resonance imaging, allowing sequential examination. The caudate photothrombotic lesion also causes significant blood–brain barrier disruption. Our initial studies employed Evans blue albumin to assess that disruption (2), but a wide range of markers and techniques can be used to assess such disruption. A concern over any animal model of stroke is whether it will be amenable to therapeutic modulation. This has not been extensively studied in the caudate photothrombosis model, although we have found that tissue plasminogen activator (tPA) can reduce infarct size and neurological deficits in the model (2). A number of groups have shown that injury in the cortical photothrombosis models is susceptible to therapeutic modulation (e.g. (8)).

5. Summary Cortical photothrombotic models of stroke have gained in popularity, in part because of the ease of surgery. In this chapter, we have described that photothrombosis can also be used to reproducible ischemic lesions in the caudate nucleus of rat and gerbil. It likely can also be used to produce ischemic lesions in other brain regions and other species. The lesion size can be varied by changing light intensity and the diameter of the optic fiber. There are changes in cerebral blood flow, tissue morphology, blood–brain barrier integrity, brain edema, and neurological deficits expected from ischemic lesions. There are differences between human cortical and basal ganglia infarcts and this new animal model should allow investigation of those differences and how basal ganglia infarcts might be treated.

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References 1. Hossmann K-A, Pathophysiology of focal brain ischemia. Pathology & Genetics Cerebrovascular Diseases. International Society of Neuropathology Vol. 5, pp 201–214, 2005 2. Kuroiwa T, Xi G, Hua Y, Nagaraja TN, Fenstermacher JD, Keep RF. Development of a rat model of photothrombotic ischemia and infarction within the caudoputamen. Stroke 40(1): 248–53, 2009 3. Paxinos G, Watson C. The Rat Brain in Stereotaxic Coordinates, 6th ed. San Diego: Elsevier Academic Press, 2005 4. Loskota WJ, Lomax P, Verity MA. A stereotaxic atlas of the Mongolian gerbil brain (Meriones unguiculatus). Ann Arbor, MI: Ann Arbor Science Publishers, 1974 5. Watson BD, Dietrich WD, Busto R, Wachtel MS, Ginsberg MD. Induction of reproducible brain infarction by photochemically ini­

tiated thrombosis. Ann Neurol. 17: 497–504, 1985 6. Boquillon M, Boquillon JP, Bralet J. Photo­ chemically induced, graded cerebral infarction in the mouse by laser irradiation evolution of brain edema. J Pharmacol Toxicol Methods. 27: 1– 6, 1992 7. Hua Y, Schallert T, Keep RF, Hoff JT, Xi G. Behavioral tests after intracerebral hemorrhage in rat. Stroke 33: 2478 –2484, 20028. Collias JC, Manuelidis EE. Histopathological changes produced by implanted electrodes in cat brains; comparison with histopathological changes in human and experimental puncture wounds. J Neurosurg. 14(3): 302–28, 1957 8. Kim GW, Sugawara T, Chan PH. Involvement of oxidative stress and caspase-3 in cortical infarction after photothrombotic ischemia in mice. J Cereb Blood Flow Metab. 20(12): 1690–701, 2000

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Chapter 11 Models of Rodent Cortical Traumatic Brain Injury Frances Corrigan, Jenna M. Ziebell, and Robert Vink Abstract The absence of a pharmacological therapy that is effective in the treatment of traumatic brain injury (TBI) highlights the need for further research into the secondary mechanisms that are initiated by the traumatic event, and which determine eventual neurological outcome. Various animal models of TBI exist, with each attempting to replicate different aspects of human brain injury. If an effective pharmacological therapy is to be developed and accepted by the neurotrauma community, it is critical that there is consistency across various laboratories in how these models are applied. The present review critically analyses the benefits and pitfalls of the common rodent models of TBI that are widely used today. Key words: Neurotrauma, Injury models, Rats, Closed head injury, Anaesthesia, Gender

1. Introduction Traumatic brain injury (TBI) remains a significant health problem, with an estimated ten million people affected annually by a TBI serious enough to result in death or hospitalisation, and an overall mortality rate of 15–30 persons per 100,000 in industrialised countries (1, 2). Survivors are often left with long-term or lifelong disability, and in the USA alone, this cohort of disabled survivors currently totals 2% of the population (3). Moreover, the cost globally for rehabilitation of individuals following head injury is estimated to be around US $500 billion a year (4). The resultant injury is divided into two phases, the primary phase encompassing the cellular damage occurring at the moment of injury, and the secondary phase of ongoing neurodegeneration that is the result of activation of a number of injury cascades including excitotoxicity, oxidative stress and inflammation (5, 6). Contemporary medical management focuses on surgical and critical care since the interrelationship between the secondary injury cascades and eventual Emma L. Lane and Stephen B. Dunnett (eds.), Animal Models of Movement Disorders: Volume II, Neuromethods, vol. 62, DOI 10.1007/978-1-61779-301-1_11, © Springer Science+Business Media, LLC 2011

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neurological outcome are unknown. Accordingly, no effective treatments are available to reduce the secondary damage. Nonetheless, the prospect for success is promising, emphasising the necessity for further research into secondary injury. In particular, the development and use of appropriate animal models of TBI is needed to increase our understanding of the complex pathophysiological events triggered by the traumatic event, allowing the subsequent development of novel treatments that may reduce the resultant brain injury.

2. Traumatic Brain Injury It is widely accepted that TBI is the result of the head impacting with an object, from acceleration/deceleration forces that produce vigorous movement of the brain within the skull, or from varying combinations of these mechanical forces (7). Mechanical forces (rotation, acceleration/deceleration and direct force applied to the head) generated at the moment of injury damage the blood vessels, axons, nerve cells and glia of the brain in a focal, multifocal or diffuse pattern of involvement. The type and severity of the resulting injury depend on the nature of the initiating force, as well as its site, direction and magnitude (8). Contact forces generated when the head strikes or is struck by an object generally produce focal injuries which are characterised by surface contusions that may be accompanied by skull fracture or intracranial haematoma formation (9). Surface contusions occur where the brain encounters the irregular surface of the skull directly beneath the site of impact (coup) or opposite to the site of impact (contre coup) and involve the underlying cortical, and with more severe injuries, subcortical structures (10). In contrast acceleration/deceleration forces that result from violent unrestrained head movement are associated with diffuse axonal injury (DAI), the scattered destruction of white matter tracts throughout the brain (11, 12). This axonal injury not only occurs at the time of impact, but is a progressive event evolving from an initial focal perturbation of the axon to ultimate axonal disconnection (secondary axotomy) (13, 14). However, both focal and diffuse injuries can co-exist, with, for example, direct impacts to the head seen in falls, motor vehicle accidents and assaults capable of producing significant acceleration/ deceleration forces leading to DAI, as well as the more obvious contact forces causing focal injuries (9).

3. Animal Models TBI encompasses a range of different events from purely focal injuries to purely diffuse injuries with varying combinations of the two in between. Due to the heterogeneous nature of human TBI, it is not

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possible for one model to replicate all the events that occur. Thus a number of different models have been developed to replicate different aspects of TBI, including the simple cortical lesion models produced through focal freezing (15), stab injury (16), cortical aspiration (17), electrolytic lesioning (18) and chemical (excitotoxic) lesioning (19), as well as the more complex models that are widely used today. These more complex animal models are classically divided into those that produce a predominantly focal injury, such as in the controlled cortical impact (CCI) and lateral fluid percussion (FP) injury models, and those that produce a diffuse injury, such as in the impact-acceleration models and midline FP. However, like human TBI, there is some degree of overlap between the two divisions. With any model of TBI, it is important to ensure that the severity of histological and behavioural changes increase with the amount of mechanical force applied, thus mimicking the range of injury severity seen in human TBI (20). Furthermore, the response to injury should be reproducible when different investigators perform the same method. Although large, gyrencephalic animal models are necessary to investigate some specific aspects of TBI, rodents are the most commonly used species today, largely because they are easily available, require less specialized equipment and can be assessed for motor and cognitive outcome on a variety of well-characterised functional tests (20). Moreover, the ability to genetically manipulate rodents means that the specific neurologic, histopathologic and behavioural sequelae of TBI in the absence or overexpression of certain gene products can be studied (21). This review will focus on the techniques needed to produce each of the three most common rodent TBI models used today in such a way as to ensure that injury severity is comparable across laboratories. 3.1. Controlled Cortical Impact Model

The controlled cortical impact injury is the most commonly used model to produce a non-penetrating focal injury, characterised by the presence of a contusion with intraparenchymal petechial haemorrhages, extensive tissue loss and cell death. The model was first characterised in the ferret (22), before being adapted for use in the rat (23), mouse (24) and pig (25), with rodents currently the animals of choice. In the original model, a midline location was used (22), but like FP, a lateral location is now preferred (23), allowing the area of damage to be concentrated in one hemisphere. Indeed, this injury model causes significant cell loss within the cortex, hippocampus, dentate gyrus and thalamus of the damaged hemisphere, with smaller degrees of contralateral damage observed with higher degrees of injury (26). Although it is thought of as a predominantly focal model, studies over the last 5 years have demonstrated extensive axonal degeneration following CCI involving the ipsilateral cortex, including the frontal, somatosensory, parietal and occipital cortices, the hippocampus, dorsolateral thalamus, parts of the caudate nucleus, hypothalamus and the optic tectum.

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Additionally, areas of the contralateral cortex and hippocampus are affected, although to a lesser extent, due to anterograde degeneration of fibres arising from the ipsilateral cortical and hippocampal neurons that project via the corpus callosum or hippocampal commissure (27). This axonal degeneration has been associated with an increase in cytoskeletal degradation peaking at 24 h (28), suggesting that much of the axonal injury following this model is the result of activation of the secondary injury cascade, and not simply mechanical damage. Apart from axonal injury and cell death, the CCI model is also associated with significant oedema formation, with increases in brain water content seen as early as 2 h post-injury and peaking at 24  h (29). Along with the intraparenchymal haemorrhage, this oedema formation causes a rise in ICP and reduction in cerebral perfusion pressure (30). Motor and cognitive deficits have been detected using a variety of different assessment tasks including the rotarod (31–33), grid walk (34), Morris Water Maze (24, 35, 36) and Barnes Maze (37), with these deficits still reportedly present up to 1 year after the injury (31, 38). 3.1.1. Methodology

CCI injury is produced by using a piston with a specific diameter tip to strike the exposed dura at a precise velocity, thus causing rapid cortical deformation with resultant formation of a contusion (23). To expose the dura, a craniotomy is first performed of sufficient size to comfortably accommodate the tip, with the size of the craniotomy generally 1 mm in diameter greater than the tip, and most commonly located in the centre of the parietal bone midway between the lambda and bregma. The animal is placed in a stereotaxic frame and the craniotomy performed by carefully thinning a circular trench with a hand-held electric drill or a trephine until the bone within the craniotomy moves independently in all directions. While drilling, repeatedly test the craniotomy site to gauge progress and thus prevent damaging the dura. Also flush repeatedly with saline to remove any bone debris. Under magnification, the resultant bone flap can be removed with fine forceps or a scalpel, carefully working around the circumference without injuring the underlying dura. If intending to replace the bone flap, store in sterile saline while CCI is induced. The tip of the CCI device is angled perpendicularly to the dura and lowered so that it is just touching the dura; this needs to be confirmed either by visual inspection, force sensors, or by using a continuity circuit. The tip is then driven into the brain under constant force (hydraulic, pneumatic or electromagnetic) at the desired speed and to the predetermined depth. In the rat, the most commonly used parameters are an impact between 2- and 3-mm depth with a 5- or 6-mm impactor tip (39) and in mice a 2.5–3 mm tip with depths typically between 0.5 and 1.5 mm (24, 40, 41). To ensure reliability, the velocity of the impactor should be measured with an oscilloscope. Following injury the skull plate is replaced and sealed with dental

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cement or a similar substance to prevent infection and to maintain pressure within the skull. It should be noted that some laboratories prefer not to replace the bone flap as this acts as a decompressive craniectomy to reduce secondary injury. The skin incision can then be sutured and the animal placed in a cage on a heating pad to recover from the anaesthesia. As the CCI injury causes minimal damage to the brainstem, there should be minimal mortality. 3.1.2. Notes

The obvious advantage of this model is that it is highly reproducible and that the severity of the injury is easy to manipulate with the extent of damage increasing as greater mechanical forces are applied. Studies in rats and mice have shown that both behavioural and histological responses can be graded with changes in either depth or velocity of the impact. Amounts of acute haemorrhage, cortical cavitation and hippocampal cell death correlated with either increasing depth or velocity of impact (26). Furthermore, both velocity (37) and depth of impact (33, 35, 38) have been found to affect the degree of motor and cognitive deficits seen post-injury, which relate to progressive increases in cortical and hippocampal cell damage. However, despite the findings that both impactor depth and velocity affect the severity of the injury, recent computer modelling data have suggested that impact depth and impactor shape, rather than impact velocity are most important (39). Indeed a flat tip was predicted to result in a contusion volume almost double that of a spherical tip, while changing velocity had little effect (39). A recent study which used an electromagnetic actuator to drive the impactor tip rather than the traditional pneumatic device, found that the degree of overshoot was greater in the pneumatic device as it is unable to stop the high speed impactor the instant it reached its preset depth (33). Thus studies showing a graded response to changes in velocity may relate to changes in impact depth as overshoot increases with speed. Interestingly, the impact depths needed to produce histologically similar injuries in this electromagnetic study were much greater, with a 2-mm depth of impact found to most closely represent the histological damage seen with a 1-mm depth in other studies (40). This is unlikely to be solely due to differences in overshoot and may also relate to differences in technique with respect to setting the zero point, where the tip touches the dura before impact. This highlights the necessity of calibrating the parameters of each individual device to produce the desired level of injury. Indeed, it has previously been noted that that injuries produced in different laboratories with different parameters can produce histologically similar injuries (42), while the same parameters in different laboratories can produce dissimilar injuries. For example, an injury produced using a pneumatic device set to deliver a 1-mm deformation at 6.0 m/s caused greater cortical ablation and hippocampal cell loss in one study (24) than another (43).

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3.2. Fluid Percussion Model

The FP model was originally developed for use in the cat and rabbit (44), and later adapted for use in the rat (45, 46) and mouse (47). While originally described in a midline position, the lateral version has become more commonly used given the ease of detecting unilateral motor deficits. Today, the lateral FP model is the most extensively used and characterized model of experimental TBI (10). It is known to cause a focal contusion with petechial, intraparenchymal and subarachnoid haemorrhages and tissue tears (46, 48). At a moderate level of injury, 20–25% of animals die within the acute post-traumatic period, generally from respiratory failure and pulmonary oedema (49). In surviving animals, cell death is prominent within the ipsilateral cortex, particularly the motor and sensory cortex, hippocampus, thalamus and cerebellum (50). The contralateral cortex and hippocampus are usually spared (51, 52), although a slight change in injury parameters can cause damage to these areas (53–55); this contralateral damage may represent reversible rather than irreversible cell injury (56). Significant axonal injury is also evident, with a progressive loss of myelinated axons up to a year following TBI, particularly in the fimbria and external capsule (57). The combination of the cortical contusion and extensive axonal injury makes this a suitable model to look at the effects of combined focal and diffuse injury. In contrast to lateral FP injury, mild to moderate midline FP injury is not associated with an overt cortical contusion, but only scattered petechial haemorrhages throughout the corpus callosum and brainstem (58, 59). Extensive axonal injury is seen in the subcortical white matter, corpus callosum, lateral neocortex, thalamus, internal capsule and fimbria of the hippocampus (27, 59). Neuronal cell death is evident within the hilus, but not the CA2/3 regions of the hippocampus, which is unlike in the lateral FP model where cell loss is significant in both these regions (54). More severe levels of injury are not employed due to the high mortality rate associated with brainstem involvement (60). Regardless of a midline or lateral injury site, the FP model causes disruption of the BBB, oedema formation and the induction of an inflammatory response (50, 60, 61). Furthermore, motor and cognitive deficits are still present up to a year following injury (62–64) and have been detected on numerous tests including the beam walk (64), rotarod (65), MWM (52, 66) and radial arm maze (64, 67, 68).

3.2.1. Methodology

In the FP model, injury results from the application of a rapid fluid pressure pulse onto the intact dura to cause a brief displacement and deformation of the brain tissue. The injury device consists of an assembly with a pendulum, a cylindrical reservoir filled with sterile isotonic saline (37°C), a pressure transducer with a male Luer lock and a stage for positioning the animal. To induce injury, the animal is attached to the device by connecting the male Luer

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lock on the device to a corresponding female Luer lock positioned on the animal’s skull within a craniotomy. The pendulum is released from a pre-determined height to impact a foam-covered piston on the saline-filled reservoir, thus creating a fluid pressure pulse which is delivered to the intact dura via the saline-filled Luer lock connection. The pressure pulse is measured by the pressure transducer positioned at the end of the saline reservoir and immediately before the Luer lock connection to the animal, and is recorded on a storage oscilloscope. In order to prepare the animal for an FP injury, the craniotomy is first performed to allow the female Luer lock to be attached. For lateral FP injury in rats, a 4.8-mm craniectomy is performed centrally over the left parietal bone, centred 4.0  mm lateral to the sagittal suture, while in the midline version the craniectomy is centred over the sagittal suture (45). To induce a lateral FP injury in mice, a 2-mm craniectomy is created 0.5 mm lateral to the sagittal suture (47), although the size of the craniotomy does vary between different groups (50). It is important to ensure that the dura is not torn while the craniectomy is being performed as this will alter the characteristics of the injury. A rigid Luer lock needle hub is then secured to the skull with cynoacrylate adhesive and dental acrylic. In the rat, the hub fits inside the craniotomy site, while for mice the hub fits outside the craniotomy (49). Some laboratories induce the injury immediately after surgery at a point when the cynoacrylate adhesive and dental acrylics are dry and firm, whereas others wait until the next day. If not performed immediately after surgery, the hub is capped, the incision site sutured and the animal allowed to recover. To perform the injury the hub is filled with 37°C saline and connected to the injury device via the male Luer lock. In mice it is common to use a piece of extension tubing, while rats can be directly connected. The addition of extension tubing will decrease the pressure arriving at the dural surface depending on the length and flexibility of the tubing. Prior to connecting the animal, it is important to check that there is no trapped air within the fluid percussion device, pressure transducer or saline connection as air bubbles will decrease the pressure pulse intensity. Air bubbles can be removed by tilting and tapping the affected component such that bubbles are expelled through the reservoir opening. It is also important to ensure that the foam covered piston on the end of the saline-filled reservoir moves freely as any resistance will reduce the intensity of the pressure pulse; vaseline gel can be used to lubricate the rubber o-ring around the piston. After injury, the oscilloscope trace should be smooth with rise, plateau and fall components; sharp changes indicate air bubbles (49). 3.2.2. Notes

Similar to the CCI model, FP is highly reproducible, with the level of injury easily altered by changing the height from which the pendulum is released, thereby increasing the intensity of the fluid

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pressure pulse delivered to the dura. Nonetheless, the technique does have a steep injury curve where higher levels of injury severity (as set by the increased angle of the pendulum before release) result in an exponential increase in mortality. In both the lateral and midline versions, disruption of the BBB and post-injury haemorrhage increase with higher magnitudes of injury (46, 69), corresponding with worsening of motor (46) and cognitive performance (70). One study, however, found that although hippocampal cell death increased between a mild to moderate lateral FP injury, there was no further increase in damage following a severe injury, suggesting a threshold of maximal cell damage had been reached (71). Atmospheric pressure is commonly used as an indication of injury severity, although this only indicates the pressure produced at the end of the apparatus and not the amount of pressure delivered to the animal. This may vary between laboratories due to slight differences in the injury device, the construction of the injury hub, connection of the animal to the device and the size and site of the craniotomy. Thus an injury caused by generation of atmospheric pressures averaging 2.7 in a LFP model has been reported as severe (72), moderate (49) and mild (73). A better way of determining injury severity is through objective measures such as return of reflexes, levels of mortality, performance on standardized motor and cognitive tests or the degree of histological damage. Indeed the length of unconsciousness time has been used instead of atmospheric pressure in several papers as a measure of severity (74–76). The position of the craniotomy site should also be carefully monitored, particularly with lateral FP, as only small changes in position can alter the pattern of injury observed. A more lateral craniotomy will create a lesion in the ipsilateral cortex, hippocampus and other subcortical structures, while a more medial position, no more than 3.5 mm from the midline in the rat will result in bilateral damage to the hippocampus with a reduction in the amount of damage in the ipsilateral cortex (55). Furthermore, a more caudal or medial craniotomy position will worsen cognitive deficits as observed on the MWM, without affecting righting reflex time or motor outcome as determined by the beam walk, indicating that this is not the result of an increase in severity of the injury (77). 3.3. ImpactAcceleration Model

The impact-acceleration model was first characterised in Sprague– Dawley rats and has yet to be adapted for use in other species, although a modification of the technique has been adapted to rat pups (78). In contrast to the LFP, CCI and other closed head injury models, impact-acceleration produces a largely diffuse injury with widespread axonal injury particularly in the long tracts of the brain stem as well as within the corpus callosum and internal capsule (79). This axonal injury is not only caused by the primary

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event but is part of the secondary injury process, evolving from local cytoskeletal disruption and impaired axonal transport, progressing to axonal swelling and if severe, detachment and retraction bulb formation (13). Subarachnoid haemorrhage is also present following injury and worsens with increasing injury severity (79, 80). Although cell death is less prominent than in focal injury models, significant damage is evident within the hippocampus and in the cortex directly under the impact site (81–83). Furthermore, this model is associated with the induction of an inflammatory response, with breakdown of the BBB and significant increases in brain water content (84). Animals have been shown to be impaired on a wide variety of motor and cognitive tasks including the rotarod (85), beam balance (86), Barnes Maze (87) and MWM (86). 3.3.1. Methodology

The trauma device consists of a brass weight within a Plexiglas or plastic tube, which is allowed to freely fall from a designated height onto a stainless steel disc that is fixed to the animal’s skull. As the animals are placed on a foam cushion, the force of the weight accelerates the animal’s head into the foam, which causes rapid deceleration. The metal helmet acts to protect the skull from fracture and to disperse the energy of the impact over the surface of the brain. To perform the injury, a midline incision is made on the scalp and the fascia cleared away to expose the skull. A 9 mm in diameter, 3-mm thick stainless steel disc is then attached to the skull centrally between lambda and bregma using a polyacrylamide adhesive. It is important to ensure the disc is placed centrally to prevent lateralization of the injury and that it is also firmly bonded to the skull to ensure full translation of the force of the falling weight to the animal’s head. The animal is then strapped onto a foam pad and positioned directly under the tube such that the lowered weight can be positioned centrally over the helmet. The density of the foam is crucial in producing a consistent injury, so it is important to replace it frequently as continued use will alter its characteristics and thus the type of injury delivered (88, 89). To deliver the injury, the weight is raised to its predetermined height (usually 1.0–2.1 m) from where it is released to impact the steel disc on the animal’s skull. The animal must be removed immediately after impact to prevent rebound injury where the weight bounces up and returns to again impact the skull. The injury device should be maintained regularly to ensure that the brass weight is falling freely without impediment from the inside of the tube. Notching at the upper end of the tubing where the line attached to the weight rubs as the weight is pulled up is also a common problem, with the addition of a metal ring inset at the top of the tubing as a useful addition to reduce friction (88).

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After injury, check the animal for skull fracture, a complication of the procedure (90). To minimize the number of animals experiencing this complication, animals need to be in the correct weight range, typically between 350 and 400  g (89). However, slight genetic differences in skull development in different animal colonies may necessitate the use of heavier animals (85, 91). Regardless, animals toward the lower end of a set weight range are prone to skull fractures due to thinner skulls, while those over the upper end may be less injured as increased skull thickness absorbs some of the impact force. 3.3.2. Notes

This model exhibits a high mortality rate if animals are not ventilated or resuscitated after injury, given that apnea invariably occurs as a result of brainstem injury (79). Manual resuscitation throughout the apneic period (5–10 min) is usually adequate to improve survival, although frequent stimulation of the diaphragm is required to facilitate independent respiration. In ventilated animals, the effects of apnea can be replicated by introducing a secondary hypoxic episode through transient reduction of inspired oxygen to 12% (89, 92). This will increase axonal injury and exacerbate neuroinflammation (92). Although secondary insults have also been employed following the CCI (93) and FP (94, 95) models, this is primarily to replicate a clinical situation involving multiple other injuries that can compromise the respiratory or circulatory systems. In contrast, hypoxia is an integral component of the impact-­ acceleration model as it is a caused by the brain injury itself. By ventilating and giving each animal a consistent period of hypoxia post-injury, mortality can be decreased and the consistency of injury among animals improved, as without ventilation animals will demonstrate variable periods of apnea (79). The advantages of this model are that it is inexpensive, easy to perform and produces graded axonal injury (79, 96). Motor and cognitive deficits worsen with increasing injury severity (86). A mild injury is produced by releasing a 450-g weight from 1 m, a moderate injury by releasing the 450  g weight from 2  m and a severe injury by releasing a 500-g weight from 2.1 m (89). The impact-acceleration model is one of the most consistent across laboratories, in contrast to the CCI (39) and FP (50) models where injury parameters can vary. This may be due to the fact that only one strain of rat has been used to date, removing the need to alter injury parameters to accommodate for variation among different strains. Despite this, the severity of the injury may differ with the use of different apparatus. For example, while some groups have detected cognitive deficits in MWM acquisition following a moderate injury (86, 97) others have not (68, 98). This may relate to slight differences in density of the foam as this is a critical parameter. It determines the rate of deceleration of the head after impact, the distance the head has travelled, the degree of flexion of the

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neck, and the rebound of the animal from the foam block. Indeed, in some cases, the head may rebound onto the base of the tube, an event that can only be detected using high-speed video cameras. Another source of variability can be caused by the weight skewing slightly as it falls, impacting the sides of the tube and changing the dynamics of the impact. Interestingly, in one study of animals injured with the same parameters (500 g weight from 2.1 m), only those that took longer than 12 min to regain their righting reflex exhibited cognitive impairment (99). Thus, it is important to use these independent measures, in righting time and performance on standardized functional tasks, to ensure consistency of the injury delivered.

4. Experimental Design As discussed, each of the four models replicate different aspects of human TBI and have their own advantages and disadvantages. The CCI and FP models employ computer-based measurements of the applied load (impactor velocity, pressure pulse), allowing precise adjustments to be made to the device and achieving a controlled range of injury severity within a particular study (20). Furthermore, this technology allows the assessment of each individual injury that is delivered, reducing the variability between animals. However, these models require trephination, a feature that does not represent a clinically encountered injury situation. In contrast, the impactacceleration model imparts force to the intact skull as would occur in human TBI. However, the forces applied are less controlled, with the potential of lateralization of the hit, thus increasing the variability among animals. While this may more accurately mimic the heterogeneous clinical situation, it may also require an increase in the number of animals required to achieve statistical significance in a study. Ultimately the choice of model depends on the information being sought. As well as choice of model, there are other important variables that will influence outcome after TBI. In any experiment it is important to use sham (uninjured) animals that undergo identical surgical treatment without receiving TBI, thus controlling for systemic variables such as the influence of anaesthesia, operative procedure, head restraint and physiological parameters (20). Choice of anaesthesia is important and depends on the type and duration of the surgery being performed. Inhalation anaesthetics are useful for short procedures as their effects wear off rapidly allowing for an accurate assessment of righting time post-injury, a measure of injury severity. However, it has been well documented that the choice of anaesthetic influences outcome (87), with isoflurane being neuroprotective following TBI, improving both functional

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outcome and reducing hippocampal cell death (100, 101). In addition, different strains of rats (102, 103) and mice (104) respond differently to TBI. For example, Fisher 344 rats exhibit a 45% mortality rate compared to a 0% mortality in Sprague–Dawley rats following a mild LFP injury (103). The strain of the animal also influences performance on motor and cognitive tests, so it is essential to ensure the animals being used are able to successfully complete a given task before beginning an experiment (105, 106). Finally, gender has been shown to influence outcome, with the genderrelated hormones shown to be protective after TBI (107). Even time within the oestrus cycle is an important factor. Female rats also perform differently on a number of functional tests (87), particularly with respect to baseline performance. Accordingly, males have been the historical preference in experimental TBI studies.

5. Conclusion Extensive research has created a better understanding of the complex secondary injury processes that are set in motion after TBI, and paved the way for the development of potential therapeutic interventions. In order to conduct this research, different animal models are required to replicate the highly heterogeneous human TBI, with the CCI, lateral FP, midline FP and impact-acceleration models all examining different aspects of head injury encompassing both focal and diffuse injuries. It is important to ensure that these models are used in a consistent manner, with an increase in reporting of objective measures such as righting time and standardized neurological scores allowing others to interpret the severity of the injury delivered.

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els of mild neurotrauma. J Neurotrauma 24: 1147–1160 74. Giza CC, Prins ML, Hovda DA, Herschman HR, Feldman JD (2002) Genes preferentially induced by depolarization after concussive brain injury: Effects of age and injury severity. J Neurotrauma 19: 387–402 75. Hoane MR, Tan AA, Pierce JL, Anderson GD, Smith DC (2006) Nicotinamide treatment reduces behavioral impairments and provides cortical protection after fluid percussion injury in the rat. J Neurotrauma 23: 1535–1548 76. Li HH, Lee SM, Cai Y, Sutton RL, Hovda DA (2004) Differential gene expression in hippocampus following experimental brain trauma reveals distinct features of moderate and severe injuries. J Neurotrauma 21: 1141–1153 77. Floyd CL, Golden KM, Black RT, Hamm RJ, Lyeth BG (2002) Craniectomy position affects morris water maze performance and hippocampal cell loss after parasagittal fluid percussion. J Neurotrauma 19: 303–316 78. Cernak I, Vink R, Zapple DN, Cruz MI, Ahmed F, Chang T, Fricke ST, Faden AI (2004) The pathobiology of moderate diffuse traumatic brain injury as identified using a new experimental model of injury in rats. Neurobiol Dis 17: 29–43 79. Foda MA, Marmarou A (1994) A new model of diffuse brain injury in rats. Part ii: Morphological characterization. J Neurosurg 80: 301–313 80. Heath DL, Vink R (1995) Impact acceleration-induced severe diffuse axonal injury in rats: Characterization of phosphate metabolism and neurologic outcome. J Neurotrauma 12: 1027–1034 81. Yamamoto M, Marmarou CR, Stiefel MF, Beaumont A, Marmarou A (1999) Neuroprotective effect of hypothermia on neuronal injury in diffuse traumatic brain injury coupled with hypoxia and hypotension. J Neurotrauma 16: 487–500 82. Rafols JA, Morgan R, Kallakuri S, Kreipke CW (2007) Extent of nerve cell injury in marmarou’s model compared to other brain trauma models. Neurol Res 29: 348–355 83. Thornton E, Vink R, Blumbergs PC, Van Den Heuvel C (2006) Soluble amyloid precursor protein alpha reduces neuronal injury and improves functional outcome following diffuse traumatic brain injury in rats. Brain Res 1094: 38–46 84. Donkin JJ, Nimmo AJ, Cernak I, Blumbergs PC, Vink R (2009) Substance p is associated with the development of brain edema and

functional deficits after traumatic brain injury. J Cereb Blood Flow Metab 29: 1388–1398 85. Harford-Wright E, Thornton E, Vink R (2010) Angiotensin-converting enzyme (ACE) inhibitors exacerbate histological damage and motor deficits after experimental traumatic brain injury. Neurosci Lett 481: 26–29 86. Beaumont A, Marmarou A, Czigner A, Yamamoto M, Demetriadou K, Shirotani T, Marmarou C, Dunbar J (1999) The impactacceleration model of head injury: Injury severity predicts motor and cognitive performance after trauma. Neurol Res 21: 742–754 87. O’Connor CA, Cernak I, Vink R (2003) Interaction between anesthesia, gender, and functional outcome task following diffuse traumatic brain injury in rats. J Neurotrauma 20: 533–541 88. Piper IR, Thomson D, Miller JD (1996) Monitoring weight drop velocity and foam stiffness as an aid to quality control of a rodent model of impact acceleration neurotrauma. J Neurosci Methods 69: 171–174 89. Marmarou CR, Prieto R, Taya K, Young HF, Marmarou A (2009) Marmarou weight drop injury model. In: Chen J, Xu ZC, Xu XM, Zhang JH (eds) Animal models of acute neurological injuries, Humana Press, New York, pp 393–407 90. Marmarou A, Foda MA, van den Brink W, Campbell J, Kita H, Demetriadou K (1994) A  new model of diffuse brain injury in rats. Part  i: Pathophysiology and biomechanics. J Neurosurg 80: 291–300 91. Cook NL, Vink R, Donkin JJ, van den Heuvel C (2009) Validation of reference genes for normalization of real-time quantitative rtpcr data in traumatic brain injury. J Neurosci Res 87: 34–41 92. Hellewell SC, Yan EB, Agyapomaa DA, Bye N, Morganti-Kossmann MC (2010) Posttraumatic hypoxia exacerbates brain tissue damage: Analysis of axonal injury and glial responses. J Neurotrauma 27: 1997–2010 93. Clark RS, Kochanek PM, Dixon CE, Chen M, Marion DW, Heineman S, DeKosky ST, Graham SH (1997) Early neuropathologic effects of mild or moderate hypoxemia after controlled cortical impact injury in rats. J Neurotrauma 14: 179–189 94. Bramlett HM, Dietrich WD, Green EJ (1999) Secondary hypoxia following moderate fluid percussion brain injury in rats exacerbates sensorimotor and cognitive deficits. J Neurotrauma 16: 1035–1047 95. Matsushita Y, Bramlett HM, Alonso O, Dietrich WD (2001) Posttraumatic hypothermia is neuroprotective in a model of

11  Models of Rodent Cortical Traumatic Brain Injury traumatic brain injury complicated by a ­secondary hypoxic insult. Crit Care Med 29: 2060–2066 96. Kallakuri S, Cavanaugh JM, Ozaktay AC, Takebayashi T (2003) The effect of varying impact energy on diffuse axonal injury in the rat brain: A preliminary study. Exp Brain Res 148: 419–424 97. Berman RF, Verweij BH, Muizelaar JP (2000) Neurobehavioral protection by the neuronal calcium channel blocker ziconotide in a model of traumatic diffuse brain injury in rats. J Neurosurg 93: 821–828 98. Maughan PH, Scholten KJ, Schmidt RH (2000) Recovery of water maze performance in aged versus young rats after brain injury with the impact acceleration model. J Neurotrauma 17: 1141–1153 99. Schmidt RH, Scholten KJ, Maughan PH (2000) Cognitive impairment and synaptosomal choline uptake in rats following impact acceleration injury. J Neurotrauma 17: 1129–1139 100. Statler KD, Kochanek PM, Dixon CE, Alexander HL, Warner DS, Clark RS, Wisniewski SR, Graham SH, Jenkins LW, Marion DW, Safar PJ (2000) Isoflurane improves long-term neurologic outcome versus fentanyl after traumatic brain injury in rats. J Neurotrauma 17: 1179–1189 101. Statler KD, Alexander H, Vagni V, Holubkov R, Dixon CE, Clark RS, Jenkins L, Kochanek PM

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Chapter 12 The Use of Commissurotomy in Studies of Interhemispheric Communication Ian Steele-Russell Abstract An overview is given of the structure and function of the mammalian cerebral commissures, with an emphasis on their role in interhemispheric communication. A major focus is placed on the use of commissurotomy as a method of selective disconnection of interhemispheric sensory-motor integration. In order for commissure section to be effective it is crucial that the sensory input is under total experimenter control. For example, in the split-brain preparation, the optic chiasma must also be sectioned as well as the corpus callosum in order to restrict the monocular visual input to a single hemisphere. Special consideration is given to differences in the commissural organisation in different species. These differences can mean that the same lesion can have different effects in various animal species. Finally, detailed surgical protocols for callosal and chiasma section are given separately for monkey, cat, rabbit, and rat. Each such surgical protocol is specifically tailored to each species’ neurosurgical vulnerabilities. Key words: Interhemispheric communication, Cerebral commissures, Corpus callosum, Anterior commissure, Optic chiasma, Monkey, Cat, Rabbit, Rat

1. Introduction A conspicuous feature of the mammalian brain is its duplex ­anatomy. It consists of two separate hemispheres that are solely united by the various intercerebral commissures or decussations. The principal function of these commissures is to unify the separate hemispheres into a single functional identity. It would therefore follow that commissurotomy would be a vital experimental approach to elucidate the anatomical substrate responsible for the unity of conscious awareness. Early work on the forebrain commissures failed to show any significant functional deficits following their transection in monkey Emma L. Lane and Stephen B. Dunnett (eds.), Animal Models of Movement Disorders: Volume II, Neuromethods, vol. 62, DOI 10.1007/978-1-61779-301-1_12, © Springer Science+Business Media, LLC 2011

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or man (1–5). In the minority of papers reporting deficits ­following commissurotomy, these findings were confounded by the presence of extensive collateral brain damage in addition to commissure transection. Before commissure section can be useful in split-brain research, the sensory input pathways must be both identified and lateralised. With mammalian vision the retinal projections from each eye bifurcate in the optic chiasma such that both lateral hemiretinae project ipsilaterally via the optic chiasma and the lateral geniculate nucleus (LGN) to primary visual or striate cortex (V1); whereas the two nasal hemiretinae decussate in the chiasma to project contralaterally via the LGN to V1. This means the each eye innervates (equally in humans and primates) both visual cortexes. Thus interocular information transfer is mediated both by the optic chiasma and the corpus callosum. Midline transection of the optic chiasma will ­disrupt visual integration via the chiasma, leaving the callosal pathways responsible for visual integration. It is only in the split-brain animal, where both the chiasma and the corpus callosum are sectioned, that there is no interocular transfer of information following monocular training. Although both the haptic and visual modalities provide a ­spatial map, the central integration of tactile or somatosensory information is very different from the visual system. It is polysensory, ­consisting of touch, temperature, proprioception, pain, and other components. Traditionally these different senses are transported by two separate ascending pathways in the spinal cord (a) the dorsal column lemniscal system (DCLS), and (b) the spinothalamic ­system (STS). The DCLS was considered to be the pathway for discrete touch in terms of stereognosis, feature identification, ­tactile localisation, etc. In contrast, the STS was believed to convey less precise forms of tactile sensitivity, as well as temperature and pain (6). The contemporary view concerning the pathways involved in touch has modified this traditional scheme (7–9). The DCLS is seen to be more concerned with touch involving tactile search (10). Complete DCLS transection eliminates the ability to learn tactile discriminations, involving active search of the tactile stimuli’s differential features, e.g., reading Braille. In contrast, tactile problems that are independent of feature extraction, such as texture, roughness, smoothness, localisation, pain, and temperature survive DCLS transection. In addition to the functional differences between the two somatosensory systems, there are additional critical anatomical ­differences between these systems. The DCLS is completely crossed in the cord via the dorsal column nucleus. This connects the ­various somatic points of the body surface with the contralateral ­hemisphere. The thalamic termination of this crossed pathway is the ventral posterolateral thalamic nucleus (VPL), which contains only ­contralateral cells (11). In contrast, the STS terminates in both the

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ipsilateral and contralateral hemispheres. Ipsilaterally, it targets the VPL and the VPM (ventroposteromedial thalamic nuclei); whereas the contralateral projections are exclusively targeted to the VPM nucleus (11). It should be remembered that the ipsilateral pathway of the STS is idiosyncratic. It is derived from fibres that originate from doubly crossing fibres (12). Tactile information enters the STS, using the right dorsal horn and then crosses to the left dorsal horn before entering the STT. The second decussation is at the level of the PO thalamic nucleus, where the STS fibres cross back to the right hemisphere. This double decussation of the STS fibres provides the anatomy for a “functional ipsilateral” relay of tactile information in the brain stem. From this brief description of the somatosensory system (SSS), it is clear that the SSS is very different from the visual pathways, which are unimodal and dedicated exclusively to visual information processing. In contrast, the somatosensory pathways are ­polymodal. They further present considerable difficulties in isolating a discrete information pathway by commissurotomy to a single cortical hemisphere. The auditory system, with its multiple decussations across the midline, also presents a major challenge to surgically isolating auditory input to a single cortical hemisphere. For these reasons, I shall restrict the present coverage, exclusively on the use of disconnection techniques such as commissurectomy or chiasma section, to the visual system. The cerebral commissures, or cerebral decussations, consist of the corpus callosum, the hippocampal commissure, the anterior commissure, the posterior commissure, the habenula, and the optic chiasma. The present overview will focus solely on the corpus ­callosum, the optic chiasma, and the anterior commissure, as there is little or no literature on either the habenula or posterior commissures. Reference to the hippocampal commissure per se and memory is available but sparse. Furthermore, although it is possible to transect the corpus callosum leaving the hippocampus intact, the converse has never, to my knowledge, been reported. It should be noted that the monkey, cat, rabbit, and rat section of the corpus callosum alone usually includes a section of the hippocampal commissure.

2. Functional Anatomy 2.1. Anterior Commissure

The anterior commissure (AC) in humans contains a majority of very fine fibres (13). For the rat, rabbit, guinea pig, and mouse, 80% of the fibres are less than 1 mm in diameter (14). The same appears true of the AC in cat as determined by electron microscopy. In most mammals, the major component of the AC is that the ­rostral portion has major projections to the olfactory system. In the cat, the evidence for neocortical projection of the AC to the

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neocortex is moot (15). For the rabbit, van Alphen (16) claims that all areas of neocortex send axons through the AC, but none of these projections were found to terminate in the contralateral ­neocortex. They all projected to the basal telencephalon. In humans and primates only, there is a very marginal projection of the AC with connections to the olfactory tubercle and other olfactory areas. The vast majority of projections go to the inferior temporal area and to the hippocampus. There are none to the superior ­temporal gyrus or the amygdala (15). Different techniques have questioned these findings. Lesions in the superior temporal gyrus by pial asphyxiation (which do not produce damage to the underlying white matter (17)) have resulted in Marchi degeneration in the rostral half of the AC (18). Pandya et al. (19) have both confirmed and extended these earlier findings. They confirmed that the anterior part of the superior temporal gyrus projected through the AC. However, comparable lesions to the caudal part did not project to the AC. Using small discrete lesions, Whitlock and Nauta (20) found no AC projection from the superior temporal gyrus. However, they found a massive input from the middle temporal gyrus to the AC, with far less input from lesions in inferior temporal cortex. Finally Akert et al. (21) reported nearly total degeneration of the AC following lesions to the cortex of the temporal pole or superior, middle, and inferior temporal gyri in macaques. 2.2. Corpus Callosum

The splenium of the corpus callosum has two principal functions in interhemispheric integration of vision: firstly, depth discrimination deriving from both unit recording and behavioural experiments (22–25), and secondly, the callosum has control of ocular vergence movements (26). In addition, the corpus callosum, in conjunction with the optic chiasma, plays a major role in the integration of the two halves of the visual field in most mammals including even the rabbit with its laterally sited eyes and minimal binocular field ­(27–29). The callosal commissure also conveys information between limbic and visual systems involved in the recognition of the significance/ meaning of visual signals which are aversive or attractive (30–32). The corpus callosum has also been used as a major tool in recovery of function research. This was first demonstrated in dog by Imamura (33) working in Vienna with Exner and von Economo. Imamura first induced contralateral visual neglect, by a unilateral lesion of the frontal eye field (FEF), which spontaneously ­recovered within 4–6 weeks. Section of the callosum resulted in the immediate and permanent return of neglect, indicating that the previous recovery was due to the intact FEF acquiring control of eye movements formerly governed by the damaged side. This important finding was later repeated using dogs by Yoshimura (34). The callosal role in the mediation of recovery from contralateral neglect was tested in macaque monkeys with somewhat different results (35). A unilateral FEF lesion readily produced contralateral neglect,

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whereas a lesion of sulcus principalis did not. The neglect was ­transient, showing full recovery within a month. Contrary to these original findings on dog, we found the section of the commissures reinstated neglect for only a few days, after which visual attention showed no lateral asymmetry. More importantly, it was also noted that the recovery in contralateral attention involved saccadic head scanning. In a later report using eye movement recording, we established that the recovery did not involve the return of the original visual attention mechanisms via ocular scanning. There was no functional recovery per se, the monkey’s visual attention was mediated by saccadic head movements, which substituted for the original oculomotor control programme (36). Finally, in addition to the sharing of sensory input to each hemisphere, the corpus callosum also plays a major role in memory formation such that the information is stored in both hemispheres. For further information on the vast literature on the mnemonic role of the corpus callosum see: Mountcastle (37), Steele-Russell et al. (38), Leporé et al. (39) and Zaidel and Jacobini (40). 2.3. Anatomy of the Corpus Callosum

The human corpus callosum has been estimated to contain approximately 200 million fibres (13), which is in agreement with the later estimates given by Blinkov and Glezer (41). They (Blinkov and Gleser) give fibre population estimates for other mammalian species as follows: dog 22 million, cat five million, rabbit six ­million. The rat has been found to have 1.1 million fibres by Cumming (42), and the mouse was determined by Tomasch and Macmillan (13) to have 300,000 fibres, which is five times greater than the fibres in the mouse pyramidal tract. In humans, the ratio of callosal to pyramidal tract fibres is much greater at 100:1. SzentágothaiSchimert (43) early reported that the corpus callosum in most mammals has a large proportion of either small (£1 mm) or nonmyelinated fibres.

2.3.1. Callosal Connections

The forebrain commissures consist of axons of cortical neurons that cross from one hemisphere to the other. Accordingly, commissural fibres are one of the major sets of extrinsic connections of the cerebral cortex. A conspicuous feature of the interhemispheric connections by the corpus callosum is the heterogeneity of the density of the commissural connections. Some regions of the ­hemisphere have dense contralateral connections, while others have virtually none (44–49). Furthermore, there are also considerable interspecies differences. Finally there are differences between neonates and adults in the density of interhemispheric fibres (50). Two general statements can be made (a) the higher the species on the phylogenic scale the more restricted or discrete are the ­commissural connections, (b) with increase in maturity there is a corresponding development of discrete areal innervation of callosal cross-connections.

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Fig. 1. Distribution of degenerating terminals in the neocortex of macaca mulatta ­following hemispherectomy (from Myers (45)).

In adult rhesus monkeys, some cortical areas such as parts of the pre- and postcentral gyri, the inferior parietal lobe, and the caudal superior temporal lobe, the caudal superior temporal gyrus have dense interhemispheric connections. In contrast, such areas as the pre- and postcentral gyri, some areas of the prelunate gyrus and V1 have negligible callosal innervation (see Fig. 1). Areas of the sensorimotor cortex that control somatic sensory or motor representation of axial, or midline parts of the body tend to have heavy commissural connections. Other areas that are concerned with ­sensory motor control of distal parts of the extremities are sparsely innervated (46, 47, 51). Higher order association areas, in the frontal and the parieto-temporo-occipital region, have variable densities of callosal innervation. 2.3.2. Type of Callosal Connections

The corpus callosum fibre organisation is heterogeneous, ­containing fibres that project to either homotopic or heterotopic sites in the opposite hemisphere. All cortical areas that have contralateral connections send some fibres to homotopic areas. Projections to heterotopic sites such as the primary somatic sensory cortex ­project not only to the contralateral S1, but also to the neighbouring S2, as well as to the supplementary (SSA) areas (52). Further, the inferior temporal lobule projects not only to the contralateral inferior temporal lobule, but also to the opposite superior temporal sulcus, cingulate gyrus, and parahippocampal area (53). Since these areas also receive ipsilateral projections from the inferior parietal lobule (46, 54–57), it is evident that the contralateral projections parallel ipsilateral cortical projections (53). The contralateral connections are less dense than the ipsilateral ones, where as a rule termination zones in the opposite hemisphere are more restricted than in the ipsilateral hemisphere.

12  The Use of Commissurotomy in Studies of Interhemispheric Communication 2.3.3. Topological Organisation of the Corpus Callosum

3. Methods of Transection of the Commissures and Optic Chiasma in Split Brain Animals

3.1. General Considerations 3.1.1. The Newton–von Gudden Principle

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Clinical and experimental evidence indicates that discrete lesions of the forebrain commissures differentially affect cortical functions. These observations are in keeping with the topological organisation of the commissures. For example, in humans tumours of the anterior part of corpus callosum result in a behavioural syndrome similar to schizophrenia, while invading other parts of the callosum does not (58). Similarly, anterior lesions result in certain forms of apraxia, while posterior lesions can result in deficits of reading and writing (59). Further selective lesions of posterior parts of the corpus callosum result in impairments of auditory function in humans (60). In the primate, lesions of both the splenium of the corpus callosum and the anterior commissure selectively disrupt visual functions. However, the visual deficits of the two lesions are qualitatively different (61).

Techniques of callosal section differ profoundly across different species due to major differences in anatomy and physiology between simple and complex species. In particular, there are profound differences between rodent vision and other mammals. Its nocturnal specialisation obviously make the rat unsuitable for split-brain brain research, involving lateralisation of visual information (see Table 1). The first important anatomical variable concerns the Newton–von Gudden principle. This describes the effect of phylogenic changes in the positioning of the eyes in the head on binocularity, and the proportion of contralateral versus ipsilateral fibres in the optic chiasma (see Table 1). Animals with laterally sited eyes have a reduced binocular field, as well as a corresponding increase in the number of crossed fibre projections in the optic chiasma. This is seen very clearly in the rabbit (which has maximal laterally sited eyes in the mammalian order) and has only 22° of binocularity combined with 90% crossed fibres in the optic chiasma. This contrasts with the monkey’s frontally sited eyes, and 130° of binocularity accompanied by 50% of crossing of fibres in the optic chiasma. This means that transection of the optic chiasma in different split-brain species will have dramatically different effects on the size of the total visual field. In the split-brain monkey, the monocular visual field will be only 50% following section of both the chiasma and callosum as compared with the split-brain rabbit (including chiasma section) with a monocular visual field of 5–6%. The split-brain monkey will have little or no visual learning impairment, following visual field reduction; whereas the rabbit will be forced to acquire and adopt entirely novel visual scanning strategy, without which visual ­learning is impossible (62).

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Table 1 Interspecies differences in visual function and anatomical structure Monkey

Cat

Rabbit

Rat

Vision

Diurnal

Crepuscular

Crepuscular

Nocturnal

Retina

Foveate

Area centralis Area centralis Concentric

Lens

Biconvex

Biconvex

Biconvex

Spherical

Binocular field

130°

100°

22°

50°

Convergence

35°

20°

12°

Not known

Pursuit movements Present

Present

Present

None

Binocular vision

Present

Present

Present

Not known

Optic nerve fibres

1,210,000 154,000

265,000

75,000

Ipsilateral fibres

50%

40%

10%

10%

Acuity (cycles/°)

64 Hz

6 Hz

4 Hz

0.3 Hz

Extrastriate areas

28

10

6

None

3.1.2. Presence of a Falx

The presence of a falx in the supracallosal space between the cerebral hemispheres is a critical factor which facilitates callosal section. This is an extension of the dura mater which extends into the space above the corpus callosum between the cerebral hemispheres in certain mammals. It is present in primates, dogs, and cats. It is not present in rabbits, rats or mice. The falx facilitates retraction of the hemisphere without any direct contact with the pia mater and other vasculature of the medial neocortex or cingular cortex. Accordingly in primates, cats, and dogs it is relatively safe to avoid such midline damage with section of the corpus ­callosum. In animals lacking a falx, such damage is virtually unavoidable – where the major endeavour is to minimise it to acceptable levels. Also in split-brain rabbits, rats, and mice, it is essential to use a surgical control group where the callosum had been exposed but not sectioned.

3.1.3. Chiasma Bisection and the Lateral Visual Fields

With the possible exception of cetaceans, there is a remarkably consistent organisation in the decussation of the retinal ganglion cell projections in the chiasma. The lateral hemiretinae are the source of the ipsilateral retinal projections, which consist predominantly of cortical binocular cells. The nasal hemiretinae give rise to the contralateral projections, which are exclusively cortical monocular cells. The ipsilateral projections process pattern information from the binocular part of the visual field; whereas the contralateral fibres respond to movement signals from the lateral monocular

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crescent of the visual field (see a discussion of this issue by Russell et al. (63)). The contralateral system also provides a critical ­pathway via the nucleus of the optic tract to the cerebellar oculomotor ­control centres. This retino-cerebellar system controls optokinetic nystagmus and the vestibulo-ocular reflex, as well as conditioned movements of the nictitating membrane response in Pavlovian conditioning (62). 3.1.4. Brain Rigidity

4. Surgical Techniques of Midline Sensory Disconnection

4.1. Animals Possessing a Falx 4.1.1. Section of the Corpus Callosum in Monkey and Cat

A fourth consideration is the major change that occurs in the ­rigidity of the brain tissue as a function of phylogeny. The brains of animals, such as the rat or mouse, have very little structural rigidity or firmness. Hence direct mechanical manipulation of the brain using retractors is hazardous. The retractor blades can penetrate into the wall of the midline cortex. In addition, the blood vessels are very fragile, such that moving them to access a commissure can be difficult. In the rabbit, the brain tissue is more resilient than in mouse or rat. It can, however, be “hardened” by the use of i.v. injection of mannitol or albumen. Unfortunately, this technique is not effective in either rats or mice.

In this section, a detailed description is given of the optimal methods for section of the corpus callosum, the anterior and posterior ­commissures, as well as for chiasma section. In each case, the methodology described is one which we have either modified or developed over the last 30 years to produce consistent results and minimal collateral damage (Fig. 2). Separate and complete descriptions of these procedures will be given for the monkey, the rabbit, and the rat. This is to enable the reader to focus on the procedural details for the species relevant to their research. Nonetheless, it should always be remembered that great differences exist between ­organisation of the mammalian visual pathways (see Table  1). Unless otherwise stated, it should be assumed that these procedures should be carried out using a full sterilisation procedure for the animal as well as the surgeon. A bone flap is created in the left hemisphere to enable the entire left cortical hemisphere to be retracted from the confines of the cranium in order to permit lateral retraction of the falx to access the corpus callosum without compressing the surface vasculature of the retracted hemisphere. As the procedure is lengthy, the bone flap approach is essential to avoid ischæmic cortical damage. It also prevents the boney flap from necrosis. In both the monkey and the cat, a short-lasting anæsthetic is used to secure the animal in an appropriate head holder, with the

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Fig. 2. Specialised surgical instruments for commissurotomy procedures. (a) A microknife curved to form a linear extension of the hand. It has a cutting blade on the lower edge with a bevelled back on the obverse. side (b) A large speculum/hemisphere retractor suitable for monkey and cat procedures. (c) A microspeculum/hemisphere retractor suitable for rabbit procedures. The blades of the retractors are counter sprung in the open position. During surgery they are manually held together during insertion between hemispheres and then gently released to separate the medial walls between hemispheres.

head positioned horizontally. Using a set of pædiatric jaw ­retractors, the trachea is intubated with a small calibre tube. The animal can then be transferred to gaseous anæsthesia for the remainder of the procedure. It is essential to observe a stringent sterile procedure throughout the callosal section procedure to avoid any infection in the deep regions of midline structures of the brain. The eyes are covered with vasoline to protect the cornea from either dehydration or the effects of sterilisation of the scalp. The entire dorsal surface of the head is first closely shaved with a razor, washed with surgical soap followed by a Zephiran sterilisation of the dorsal ­surface of the head for 15 min. The head is then draped with a sterile covering creating a ­window that exposes the operative area only throughout the ­operation. An incision is made in the right side of the skull approximately 1  cm over the central suture parallel to the midline, extending from the anterior to the posterior pole of the cranium. From either end of this longitudinal incision, two lateral incisions are added extending down the lateral aspect of the dorsotemporal aspect of the left side of skull to expose the temporalis muscle, which is left attached to the skull. The periosteum is blunt dissected back to each side of the temporalis muscle. In order to shrink the

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Fig. 3. Bone flap schematic. Showing a circular array of trephine holes that are connected by use of a Gigli saw to complete the bone flap which remains attached to the temporalis muscle (see text for further details).

brain and reduce the tension on the blood vessels, 50 ml of 20% mannitol, using a microdrive, are injected i.v. at a rate of 10 ml per min. Throughout this transfusion period, care is taken to maintain the temperature of the mannitol blood temperature, using a water bath with a temperature feedback control from the mannitol bag. A circular shaped bone segment, starting 0.5 cm in the right hemisphere crossing over into the left hemisphere, is then freed but it remains attached to the temporalis muscle. The bone flap is made by first creating a perimeter of six 5 mm diameter trephine holes as illustrated in Fig. 3. A Gigli saw was threaded via a surgical guide between the trephine holes and the inter-trephine hole. The bone bridges are cut to free the bone flap, with the exception of the part underlying the temporalis muscle. The bone bridges between trephine holes in the right hemisphere are ­chamfer cut at 45° to provide a support ledge when the flap is replaced. Following this, a 3-mm wide flat spatula is inserted between the trephine holes over right hemisphere to drill 0.5 mm diameter holes on either side the chamfered cut. This enables the bone flap to be secured in place, using silver sutures at the end of the procedure. The flap is cautiously elevated at the midline crest, using a dental elevator and small curette to separate the flap from any dural adhesions. Typically these are greatest at the midline region above the central

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sinus. The bridge between trephine holes beneath the ­temporalis muscle is separated by elevating the flap until it breaks from the ­calvarium remaining attached to the temporalis muscle. The bone flap is then wrapped in gauze soaked in Ringer’s solution, and draped laterally away from the hemisphere. To retract the dura mater, a dural hook is used to snare the dura of the left hemisphere, 1 cm lateral to the central sinus. The dura is then elevated and opened by a fine pair of scissors, and a slit parallel to the central sinus is extended from the frontal to the occipital pole of the brain. The dural incision is then extended towards the central sinus at both ends to allow the dural flap to be reflected over the midline as a covering to retract the central sinus against the medial wall of the non-retracted hemisphere. Care is taken to avoid extending the dural section into the sagittal sinus. On occasion there are adhesions between the dura and communicating veins attached to the sinus. No attempt is made to ­separate or ligate the vessels. A slit is made in the dura on each side of the vein, which enables the dura to be turned back as a discontinuous fold. Cottonoid strips, soaked in Ringer’s solution, are placed over both the exposed dura and cortex. The number of communicating veins from the cortex into the sagittal sinus are sparse, being rarely more that one or two. Accordingly, it is possible to preserve them by working around them. Ligation of large veins, which on occasion is done, can result in œdema and damage to the exposed hemisphere. Using a ­speculum and a small gauge aspirator (with a finger controlled pressure release valve) are cautiously inserted between hemispheres. In either the monkey or the cat, there are rarely adhesions between the medial wall and the retracted hemisphere, or the falx in the posterior callosal region. Frequently, there is a substantial arterial plexus over both the splenium and the body of the callosum dorsal to the anterior massa intermedia. Care is essential to avoid damage to these arteries. With the surgical microscope, a dental elevator is used to manipulate cottonoid balls soaked in sterile Ringers which in turn manipulate these vessels and to enable access to the callosum. This is sectioned using the speculum with one hand and alternating the sucker and angled knife with the other hand. With a mixture of care and caution, the section can be accomplished with little or no damage to the medial walls of the hemispheres. Starting the transection at the splenial end of the callosum, csf from the underlying third ventricle will flow into the section area, which is easily controlled by low pressure suction. As the section continues rostrally, the massa intermedia can be seen as a distinct grey structure in contrast to the bright white of the corpus callosum. The arterial plexus at the anterior rim of the mass intermedia is a critical landmark that indicates the sectioning procedure is on the midline. Following the completion of the transection and staunching any blood seepage from fine vessels, the cavity is irrigated with Ringers at blood temperature to see if there is any further ­bleeding.

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Only when it is certain that there is no vascular leakage, are the hemispheres re-opposed. Before replacing the bone flap, the entire exposed surface of the exposed hemispheres is carefully covered with a pre-shaped gel film cover, which is tucked under the margins of the bone defect. Then the bone flap is secured in place by silver sutures, holding the chamfered edges of the bone flap together. The margins of the periosteum are sutured across the bone defect, as are the margins of the scalp, which are closed with discontinuous mattress sutures, to prevent any damage due to any scratching of the incision by the animal after recovering consciousness. Before the anæsthesia is turned off, 30 cc of body temperature sterile isotonic saline is given i.v. to protect against any dehydration. Cortisone treatment is also given to prevent post operative brain œdema in combination with appropriate pain medication. Both treatments are continued for the next 5 days. On the first post-operative day, the animal should be alert and without any motor deficits. 4.1.2. Section of the Optic Chiasma in Monkey and Cat

Section of the optic chiasma and the anterior commissure can be achieved via an extension of the dorsal approach to the corpus ­callosum first pioneered by Dandy (2). The disadvantage of this procedure in neither monkey or cat it is neither a necessary nor an optimal approach in these animals. Another disadvantage of the approach is that access to the optic chiasma is of necessity by threading through the circle of Willis (which is both hazardous and extremely difficult). Downer (31), adapted the transbuccal procedure of Cushing for accessing dissecting tumours of the chiasma, to the macaque and the cat, to section the optic chiasma. This approach has advantages over the dorsal route, in that the entire chiasma is visible via the surgical microscope. It avoids the circle of Willis, and does not involve the heroic passage between hemispheres from cortex to the ventral surface of the brain. Using a short-lasting anæsthetic, the animal is intubated with a small calibre tube, and then transferred to gaseous anæsthesia. The monkey is placed in a supine position with the head dorsiflexed and secured in a head holder. The eyes are covered with vaseline, and then covered with surgical tape to prevent any corneal dehydration. The mouth is held open with a pædiatric jaw retractor. Using full aseptic procedure, the field around the mouth is covered with sterile drapes. The mouth is then thoroughly washed with a solution of isotonic saline followed by Zephiran for 15  min. A “U”- shaped incision is made through the soft palate through the nasopharynx. The incision begins laterally and caudally just anterior to the isthmus of the fauces, and is extended rostrally to approximately 2 mm over the caudal margin of the bony palate, and then is curved around to the other side and returned to the same point where it was begun. The underlying membraneous ­septum is severed, and the flap thus formed is reflected from the field. The spinous process

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of the vomer is removed with a small pair of rongeurs. With a small curette the mucosa overlying the vomer and sphenoid bones is also removed. The entire vomer and buccal cavity are filled with Zephiran (1:5,000) for a further 15 min. From this stage onwards, all instruments used in the surgical field are placed in 70% alcohol on being removed from the field. They are only returned to the incision via a sterile saline rinse in an attempt to keep the field sterile. A dental burr (with a long shank and large spherical cutter) is centred on the suture separating the sphenoid bone from the vomer, and a hole drilled through into the cranial cavity. The hole is then enlarged anteriorly in the midline until the optic nerves can be seen joining to form the optic chiasma, and posteriorly until the anterior hypophysis is clearly visible. Throughout this approach, sterile bone wax is applied into bone trabiculae of the cavity to control bone bleeding. Using a Zeiss long focus surgical microscope, the optic chiasma is cut longitudinally in the midline (Fig. 4). A preshaped rectangle of gel film is placed over the chiasma, carefully tucking the sides of the film beneath each side of the rectangular boney defect. A fast curing liquid bone cement is then applied with a dental spatula to seal the cavity. This will set within a few minutes to form a hard, bone-like plug, sealing the cavity. The edges of the palatal flap are then closed

Fig. 4. Section through the CC and HC in monkey.

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with interrupted ophthalmic silk sutures, as well as a continuous plain cat-gut suture. Post operatively the monkey is given antibiotics and analgesics daily for 10 days. The day after recovery from anæsthesia, all animals are examined for asymmetries in the diameter of their Behr’s pupillary reflexes (64). Any asymmetry would be a strong indicator that the chiasma section had deviated from the midline. During the post-operative recovery period, all animals are fed a liquid diet via oesophageal intubation for 2 weeks, following which they are gradually adjusted to solid food. This is a necessary precaution to avoid any fistulation of the palate incision. 4.2. Animals Without a Falx 4.2.1. Section of the Corpus Callosum in Rabbit

The procedure in the rabbit differs from that in higher mammals. The lack of a falx and also the lack of the brain’s structural rigidity together make it impossible to manipulate the brain without ­causing major damage to brain tissue. Accordingly, intravenous mannitol is used to both harden and shrink the brain. This makes it possible to use retractors to separate the hemispheres to access the corpus callosum. All rabbits are given a short lasting anæsthetic by an injection in the ear vein. They are placed in a restraining stock, and then intubated using a small calibre tracheal tube and transferred to gaseous anæsthesia. Following this, they are placed in a head holder with the skull aligned horizontally. Constant core body temperature is maintained using a thermal blanket with a rectal temperature feedback probe throughout the procedure, and also for 1 h postoperatively. At the same time, such vital signs as heart rate and respiration are monitored. In order to both harden and shrink the brain, 50 ml of 20% mannitol is injected via an ear vein at a rate of 10 ml per min, using a micro drive. Throughout this transfusion period, care is taken to maintain the temperature of the mannitol at blood temperature using a water bath with temperature feedback control from the mannitol bag. Both eyes are covered with vasoline, held in place by surgical tape, to prevent corneal dehydration. The dorsal surface of the head is then shaved using electric clippers with a vacuum collection of fur. The residual stubble is removed, using a standard depilatory cream to ensure a smooth surface. After removal of the cream, the area was cleansed with 70% alcohol and sterilised with Zephiran for 15 min. After this preparation phase, full sterile precautions are observed throughout the surgical procedure. An incision is made in the right hemisphere 5 mm parallel to the central suture, extending rostrally from the frontonasal suture to bregma. The head is then draped with a sterile covering creating a window that exposes only the operative area throughout the surgical procedure. An incision is made on the right side of the skull approximately 5 mm over the central suture parallel to the midline extending from the anterior to the posterior pole of the cranium. From either end of this,

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two lateral incisions are added extending down the lateral aspect of the dorsotemporal aspect of the left side of skull to expose the border of the temporalis muscle which is attached to the dorsolateral attachment ridge of the skull. Using a small curette, the entire attachment of the temporalis muscle is separated from the skull, and the muscle is blunt dissected from the vertical lateral aspect of the skull. A 5-mm diameter trephine hole is made in the skull, posterior to bregma and across the sagittal suture. A small dental elevator is used to detach the disc of bone as well as to free any dural adhesions to the ventral surface of the margins of the bone defect. Using rongeurs in combination with a dental elevator, the bone defect is then extended towards the intersection of the frontonasal and ­sagittal sutures at the extremity of the frontal poles and then subsequently caudally to expose the occipital pole. Throughout this part of the procedure, care is required to avoid damage to the ­central sinus, and also to ensure that the walls of the extended bony defect are devoid of any projecting bone spicules. The bone defect is then extended laterally over the dorsal and temporal regions of the skull, over the left hemisphere to permit retraction of this hemisphere with minimal risk of vascular compression. Using a long focus Zeiss surgical microscope, a longitudinal incision is made in the dura mater, of the left hemisphere, 2–3 mm to the side of the sagittal sinus and extending along the length of the hemisphere. The incision is then cautiously box cut to the ­central sinus and the entire dural flap is draped over the sinus, and the margin of the right hemisphere. Using a miniature pair of retractors (see Fig.  5), the two hemispheres are cautiously separated at the frontal pole where access between them is facilitated by the relative scarcity of surface vessels, extending from the central sinus to the left hemisphere. Where possible, each such vessel is preserved by using the retractors discontinuously to retract the hemispheres between the crossing vessels to minimise any tension on them. In this way, it is possible to preserve the integrity of the vascular bed of the majority of the medial wall of both hemispheres. At all times the retractors are used with gel foam wadding, soaked in warm (core temperature) sterile Ringer’s solution to gently ­separate the walls of the hemispheres. This is mainly achieved by displacement of the left hemisphere through the bone defect, which minimised cortical vascular damage due to compression. The space between the hemispheres is filled with Ringers for 5 min, to observe if there is any blood seepage. Following this, the cavity is emptied using low pressure suction and the CS and hippocampal commissure are cut using a scleral microknife, from the genu to the splenium. The cavity is again filled with warm Ringer’s solution to test for vascular seepage. In the event of no seepage, all gel foam waddings are removed, and the left hemisphere is gently returned in place using a 5-mm spatula. A panel of gel film, contoured to the

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Fig. 5. Section of the OC in monkey.

shape of the bone defect is then inserted below the margins of the defect, to protect the brain in the absence of a true bone flap. The margins of the incision are then closed, using discontinuous mattress sutures. To compensate for the fluid loss, due to the copious diuresis induced by the mannitol injection, a 50 cc infusion of isotonic Ringer’s solution is given at the rate of 10 ml per min using the ear vein. Cortisone treatment is given to prevent post-operative brain œdema, in combination with appropriate pain medication. Both treatments are continued for the next 5 days. On the first post-operative day, the animal should be alert and without any motor deficits. 4.2.2. Section of the Optic Chiasma in Rabbit

The usual transbuccal approach to the chiasma that is used in primates or cat, is not feasible in the rabbit, due to its small mouth which denies surgical access to the hard palate. Accordingly, a dorsal approach to access the optic chiasma medially between the frontal lobes is the only alternative approach. All rabbits are first given a short lasting anæsthetic by an injection in the ear vein. They are then intubated using a small calibre tracheal tube, and transferred to gaseous anæsthesia. Following this, they are then placed in a head holder with the skull aligned horizontally. Constant core body temperature is maintained,

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using a thermal blanket with a rectal temperature feedback probe throughout the procedure, and also for 1 h postoperatively. At the same time, such vital signs as heart rate and respiration are monitored continuously. In order to both harden and shrink the brain, 50 ml of 20% mannitol are injected via an ear vein at a rate of 10 ml per min, using a microdrive. Throughout this transfusion period, care is taken to maintain the temperature of the mannitol at blood temperature using a water bath with temperature feedback control from the mannitol bag. To prevent corneal dehydration, both eyes are covered with vasoline, which is then covered by surgical tape. The dorsal surface of the head is then shaved using electric clippers with vacuum ­collection of fur. The residual stubble is removed using a standard depilatory cream to ensure a smooth surface. After removal of the cream, the area is cleansed with 70% alcohol, and sterilised with Zephiran for 15 min. After this initial preparation phase, full sterile precautions are observed throughout the surgical procedure. An incision is made in the right side of the skull approximately 5 mm over the central suture parallel to the midline extending from the anterior to the posterior pole of the cranium. From either end of this, two lateral incisions are added extending down the dorsotemporal aspect of the left side of skull to expose the border of the temporalis muscle which is attached to the dorsolateral attachment ridge of the ­cranium. The head is then draped with a sterile towel creating a window that exposes only the operative area throughout the surgical procedure. Using a small curette, the entire attachment of the temporalis muscle is separated from the skull, and the muscle is blunt dissected from the vertical lateral aspect. The skull is then exposed and a 5-mm diameter trephine hole is made, posterior to bregma and over the sagittal suture. A small dental elevator is used to detach the dural adhesion to the ventral surface of the skull. The bone defect is then extended towards the frontonasal suture to expose the medial aspect of both hemispheres as far as the ­extremity of the frontal poles, and subsequently laterally over the dorsal ­surface of the left hemisphere to permit retraction of this hemisphere with minimal risk of compression. Using a long focus Zeiss surgical microscope, a longitudinal incision is made in the dura mater 2–3  mm to the side of the sagittal sinus, extending from bregma to the frontal pole. The dura mater is carefully draped over the margin of the right hemisphere to expose the sagittal sinus. A miniature pair of flat-bladed falx retractors (see Fig. 2) are used to separate the two hemispheres at the frontal pole, where access between them is facilitated by the bifurcation of the sagittal sinus. In this way, it is possible to preserve the integrity of the vascular bed of the majority of the medial wall of both hemispheres. Using the Trendelburg of the operating table, the rabbit’s head and body are aligned upward by 30° in order to approach the chiasma

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Fig. 6. View of the OC section in rabbit through the surgical microscope. The intact vessels of the circle of Willis are shown over the complete chiasma section.

without compromising either the genu of the callosum or the ­anterior ­commissure. Micro-retractors are used with gel foam wadding, soaked in warm sterile Ringer’s solution, to gently open a pathway to the anterior dorsal surface of the circle of Willis and the optic chiasma (Fig. 6). This is achieved by displacement of the left hemisphere. Extreme care is required to avoid damaging either the ethmoidal anastomatic vessels or the anterior cerebral artery when displacing them to access the anterior arch of the chiasma. The entire optic chiasma is then sectioned midsagittally with a scleral microknife, taking pains to avoid contact with either the anterior hypothalamic regions overlying the posterior chiasma or the anterior pituitary gland. Great care is necessary to avoid vascular damage when retracting the midline vessels in the circle of Willis because such damage will invariably vitiate the procedure and the animal’s viability. The completeness of the chiasma section is visible directly from the anterior to posterior arches of the chiasma using the long focus surgical microscope (see Fig. 7). The chiasma region is then repeatedly irrigated with warm sterile Ringers at blood temperature to check for any vascular seepage. Between such lavages, the various gel foam packs are removed. After which, the retracted frontal lobes are gently returned to their midline position, using a 5-mm wide spatula. The dura mater is replaced and covered with a sheet of gel film contoured to fit

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Fig. 7. (a) Midline section of the OC in rabbit. The absence of midline damage, due to the mannitol pre-treatment can be seen. (b) dorsal aspect of the brain following OC section. (c) ventral view of the chiasma OC section.

within the confines of the craniotomy. The scalp is then closed with ­discontinuous mattress sutures. To maintain hydration, 70 ml of warm Ringer’s solution is injected subcutaneously after the procedure and before the animal regains consciousness. Cortisone treatment is given to prevent post-operative brain œdema in combination with appropriate pain medication. Both treatments are continued for the next 5 days. The day after recovery from anaesthesia, all rabbits are examined for asymmetries in their Behr’s pupillary reflex to check on possible asymmetries in chiasma section. During the post-operative recovery period, the animals are fed a liquid diet via oesophageal tube for at least 10 days, following which they are gradually adjusted to solid food. Some animals during this 14-day period can either fail to regain normal eating or show symptoms of progressive pulmonary œdema (65–67). Such animals should be euthanised if no improvements are observed during this period.

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The effectiveness of the mannitol pre-treatment can be seen ­post-operatively in the lack of any collateral brain damage subsequent to the radical brain manipulation (see Fig. 7). 4.2.3. Section of the Corpus Callosum in Rats

Only pigmented rats are suitable for split-brain research as they possess a better visual system than the “standard” albino animal. Furthermore, it is well documented that albino rats lose all of their retinal photoreceptors by 30 days of age when housed in modern brightly lit housing conditions (68). For these reasons, albino rats are a totally inappropriate choice for visual split-brain research. The rat is similar to the rabbit in some respects. It is without a falx and has a low gel factor of the brain tissue, which precludes any direct manipulation such as hemisphere retraction without massive brain damage. The use of high osmotic concentration of mannitol to both shrink and harden the brain substance is not an option that can be used in the rat, as it does not respond to such a procedure. This is similar to the chinchilla strain of rabbits, which also are nonresponsive to either mannitol or albumin. Chinchilla rabbits, similar to rats, have maximally concentrated urine along with permanently high circulating levels of ADH. To my knowledge, the chinchilla strain of rabbits is unique within the lagomorph species, which is usually attributed to their ancestral desert adaptation. All attempts at dorsal transection of the corpus callosum in the rat (69, 70) have, without exception, caused major and extensive bilateral damage to the medial walls of the cortex, as well as the cingulate gyri and the sagittal sinus, accompanied by major secondary damage to the cortex as well as dorsal limbic structures. Moreover, on many occasions the damage is excessively asymmetrical. Our ability to modify and to improve the dorsal approach to callosum section in rat, despite numerous variations, has without exception, been completely unsuccessful. Post-mortem examination of a rat brain with the left ­hemisphere removed, indicated that a posterior stereotaxic approach to the corpus callosum could be feasible via the midline aspect of the vertical lobe of the cerebellum, using a hypodermic needle enclosing a retractable knife (see Figs.  8 and 9a). For further details, see Goodman and Steele-Russell (71). For section of the corpus callosum rats, they are first anaesthetised with sodium pentobarbital (50  mg/kg) via i.m. injection. A  small calibre tracheal tube is then inserted to ensure that the ­airways remained patent during the procedure. They are then placed in the head holder of the stereotaxic device. Both eyes are covered with vasoline and surgical tape to prevent corneal dehydration. Constant core body temperature is maintained using a thermal blanket with a rectal temperature feedback probe throughout the procedure, and postoperatively until recovery. The dorsal surface of the head is then shaved using electrical clippers with vacuum

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Fig. 8. (a) Knife schematic for CC transection in rat. (b) Serial reconstruction of the CC transection in rat. Abbreviations: AC anterior commissure, CCG genu of the corpus callosum, CCSP splenium of the corpus callosum, CCT trunk of the corpus callosum, CIC inferior collicular commissure, CSC superior collicular commissure, F fornix, HbC habenular commissure, HC hippocampal commissure, OC optic chiasma, PC posterior commissure.

collection of fur. The area is cleansed with 70% alcohol and ­sterilised with Zephiran for 15 min. The head is levelled such that the corpus ­callosum is parallel to the horizontal plane. A midline incision is made in the scalp, using the central suture as a guide. Right and left skin flaps are created by lateral extensions to the midline incision, which are reflected laterally by mosquito haemostats. The head is then draped with a sterile towel to create a window that exposes only the operative area throughout the surgical procedure. After this initial preparation phase, sterile precautions are observed throughout the surgical procedure, using the alcohol-sterile saline method. A 3-mm trephine opening is made in the skull at the intersection of the most posterior coronal suture and the sagittal suture. This also demarcates the confluence of the transverse and superior sagittal sinuses. Using a small curette, the cranial levator auris longus muscle is then separated from the occipital crest. A small pair of rongeurs are used to create an opening 1.5 mm wide from the posterior edge of the trephine defect to occipital crest and continuing over the crest for 3 mm down the caudal surface.

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Fig. 9. (a) OC knife. (b) Serial sections of the transected optic chiasma (see text for further details).

The carrier needle, with the knife retracted, is then positioned in the midline, and lowered until the dorsal surface of the needle is in level with the dorsal surface of the transverse sinus where the needle tip is 1 mm posterior to the sinus. This measurement determines the vertical zero point coordinate. The needle is then ­elevated to clear

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the cerebellum, moved posterior over the exposed cerebellum and lowered 1.75 mm below the vertical zero point. It is then advanced until the tip of the needle slightly engages the dural covering of the cerebellum. This point then serves as the anterior – posterior zero point. The needle is then inserted 8 mm through the vertical lobe of the cerebellum until it penetrates the tentorium cerebellar membrane, then raised 0.5 mm, and the tungsten knife deployed from the carrier needle. The knife is then advanced 7  mm further to transect the splenium and trunk of the callosum. The needle is then tilted down from horizontal by 4°, and advanced a further 5 mm at this orientation. The needle is withdrawn 5 mm, the orientation is then returned to horizontal and the knife is withdrawn a further 7 mm. The knife is then retracted into the needle carrier, and the assembly withdrawn from the brain. The bone defect in the skull is washed with warm saline until there is no blood seepage, which almost never occurs. A panel of gel film, contoured to the shape of the bone defect is inserted below the margins of the defect to protect the brain in the absence of any bone flap. The incision is then closed using interrupted mattress sutures. Cortisone treatment is given to prevent post-operative brain œdema in combination with appropriate pain medication. Both treatments are continued for the next 5 days. On the first post-operative day, the animal should be alert and without any motor deficits. The results of using this procedure clearly show it to be remarkably successful. We have used this method of callosal transection over the years on a large number of rats with little or no midline damage (72). The only failing is that on minority of occasions there is some sparing of the callosal genu (see Fig. 9b). For visual experiments, this is not a disadvantage. All visual information transfer by the callosum uses splenial fibres in the rat. Additionally there is no anterior commissure involvement in the rat, which is mainly involved with integrating the olfactory lobes and the paired amygdalae. As against these advantages, it must be emphasised that the proper control procedure would be a group of rats that have undergone the complete surgical procedure without the knife being extruded from the needle. Normal animals do not provide adequate control comparisons. To my knowledge, this control ­surgery has never been used. The measurements cited in the above stereotaxic procedure are purely heuristic. As the rat brain increases in size throughout the first year of its life, it is crucial to use a post-mortem rat where one hemisphere has been removed to permit accurate measurements. This should be done for each cohort of rats used in each experiment. 4.2.4. Section of the Optic Chiasma in Rats

The same approach to section of the chiasma that is used in the rabbit is unsuitable in the rat. The rat does not respond to mannitolinduced hardening of the brain which makes it impossible to retract

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the hemispheres to section the chiasma using visual guidance. An inspired solution to the problem was provided by Crowne et al. (73) who devised a special cutting tool that would section the chiasma stereotaxically. It is possible to use this instrument because the chiasma in the rat is slack unlike the other mammals considered, and also the vasculature in the circle of Willis is sparse in comparison to them. All rats are preoperatively treated with dexamethasone (0.1 mg, i.m.) to reduce intra-cranial pressure and anæsthetised an hour later with sodium pentobarbital (50 mg/kg, i.p.). Ketamine and xylazine are avoided because ketamine raises cerebral blood flow and intracranial pressure, and the surgery entails risks of bleeding and increased intracranial pressure from puncture of the third ventricle. Animals are placed in a Kopf DKI 900 stereotaxic instrument in the “fiat skull” orientation, with the incisor bar set at −3.9 mm. To ensure the fiat skull, the heights at Lambda and Bregma are routinely checked. The micromanipulator is tilted posteriorly 10° and the anterior–posterior, medial–lateral, and height coordinates are measured from bregma using the caudal tip of the knife blade. A skull opening 3 mm wide and 5–6 mm long is made over the sagittal sinus, and the dura is carefully incised just lateral to the sinus. Since cortex adjacent to the medial wall (area PCm) is invariably damaged, we balanced the side of dural incision and retraction in the chiasma-sectioned and split-brain groups. The knife is positioned midsagittally and advanced +0.8 mm. Using a small spatula, the sagittal sinus is retracted and the knife entered between the hemispheres. The knife is lowered approximately 9.75–10.00  mm from the surface of the brain at bregma. Transection of the chiasma is accomplished by stubbing the blade gently but firmly against the sphenoidal bone by delicate rotation of the micro-manipulator. To ensure complete section, the knife is slightly raised and advanced in three 0.5-mm steps, lowering and cutting at each step. Withdrawal of the knife and routine closure completed the procedure. Postoperative nursing is frequently required that included fluid replacement with lactated Ringer’s solution and feeding with wet mash and pieces of apple. Within a week, however, animals typically began to resume normal eating and drinking. A few animals lose no more than 15 g of body weight and do not require special care. Three weeks are allowed for recovery. One major caveat should be made concerning the consequences of this procedure on residual vision. Unlike the rabbit, little or no rigorous visual ­psychophysical test procedures have either been developed or used in chiasma-sectioned rodents. In the light of the considerable ­literature showing rats can solve visual tasks without any photoreceptors, any conclusion concerning visual capability in these ­animals (chiasma sectioned rats) must remain uncertain (68).

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5. Concluding Remarks The present overview of the use of commissurotomy techniques focussed on the role of disconnection studies of the neural mechanisms underlying the functional unity of the brain. In anatomical terms, the main focus has been on the information flow between the cerebral hemispheres at a cortical level. A survey is given of the anatomy and function of the ­mammalian commissural system with a special emphasis on the changes that exist between different species. Such differences can be profoundly dependent on the sensory modality involved, e.g., the visual comparison between monkey and rat determines that commissurotomy will be radically different between the two species. The same can also be said for both tactile and motor systems in these species. The motor system in primates is both behaviourally and anatomically highly subserved to detailed visual guidance. In rodents, lacking detailed vision and being nocturnally specialised, this is not the case. As has been earlier mentioned (68), albino rats, which are frequently used for behavioural experiments, lose their photoreceptors in standard laboratory housing conditions. This could be, and frequently is, a major limitation on visual research. The remainder of the chapter gives details of commissurotomy procedures for surgical transection of the corpus callosum, anterior commissure, and the optical chiasma in the four principal laboratory species of monkey, cat, rabbit, and rat. Detailed procedural information is provided for each species, as the same surgical procedure in one species must be done differently in another. References 1. Trendelenburg W, Hartman F (1927). Zur Frage der Bewegungsstörungen nach Balkendurchtrennung an der Katze und am Affen. Zschr ges exp Med. 54: 578–533. 2. Dandy WE (1930) Changes in our conceptions of localization of certain functions in the human brain Am J Physiol 93: 643–692 3. Akeleitis AJ (1944) A study of gnosis, praxis and language following section of the corpus callosum and the anterior commissure J Neurosurg 1: 94–102 4. Bridgman CS, Smith KU (1945) Bilateral neural integration in visual perception after section of the corpus callosum. J Comp Neurol 93:57–66 5. Kirschbaum WR (1947) Agenesis of the corpus callosum and associated malformations J Neuropath & exp Neurol 6: 78–94 6. Mountcastle VB, Darien-Smith I (1968) Neural mechanisms of in somesthesia In : VB Mountcastle (Ed.) Medical Physiology, St Louis, Mosby Press

7. Semmes J, Mishkin M (1965) Somatosensory loss in monkeys after ipsilateral cortical ablation J Neurophysiol 28: 473–486 8. Azulay A, Schwartz AS (1975) The role of the dorsal funiculus of the primate in tactile discrimination Exp Neurol 46: 315–332 9. Wall PD (1970) The sensory and motor role of impulses travelling in the dorsal columns toward the cerebral cortex Brain 93: 505–524 10. Gibson JJ (1962) Observations on active touch. J Psychol 69: 477–491 11. Poggio GF, Mountcastle VB (1960) A study of the functional contribution of the lemniscal and spinothalamic systems to somatic ­sensibility Bull Johns Hopkins Hosp 106: 266–316 12. Kerr FWL (1975) The ventral spinothalamic tract and other ascending systems of the funiculus of the spinal cord. J Comp Neurol 159: 335–356

12  The Use of Commissurotomy in Studies of Interhemispheric Communication 13. Tomasch J, Macmillan (1957) A quantitative analysis of the human anterior commissure Acta anat 30: 902–906 14. Nakamura K, Omuri S, Omuri Singo (1960) Measurement of the nerve fibers in the anterior commissure of the guinea pig and mouse Bull Kobe Med Coll 19: 660–664 15. Fox C J, Schmitz JT (1943) A Marchi study of the distribution of the anterior commissure in the cat J comp Neurol 79: 297–314 16. van Alphen HAM (1969) The anterior commissure of the rabbit. Acta Anat 74 suppl 57: 111pp 17. Steele-Russell, I., J.F. Hobbelen, M.W. van Hof, and S.C. Pereira (1984) The effect of devascularization of the visual cortex on visual function in the rabbit Behav. Brain Res. 14:1, 69–80 18. Sunderland S (1940) The distribution of commissural fibres in the corpus callosum in the macaque monkey J neurol Psychiat 3: 9–18 19. Pandya DN, Hallet M, Mukerjee SK (1969) Intra and interhemispheric connections of the neocorticalauditory system in the rhesus monkey Brain Res 14: 49–65 20. Whitlock DG, Nauta WJH (1956) Subcortical projections from the temporal neocortex in macaca mulatta J comp Neurol 106: 183–212 21. Akert K, Gruesen RA, Woolsey CN, Meyer DR (1961) Klüver-Bucy syndrom in monkeys with neocortical ablations of the temporal lobe Brain 84: 480–498 22. Mitchell DE, Blakemore C (1970) Binocular depth perception and the corpus callosum Vision Res 10: 49–54 23. Steele-Russell I, van Hof MW, Pereira SC (1983) Angular acuity in normal and commissure-sectioned rabbits Behav Brain Res 8: 3, 167–176 24. Elberger AJ (1982a) The corpus callosum is a critical factor for developing maximum visual acuity Dev Brain Res 5: 350–353 25. Elberger AJ (1982b) The functional role of the corpus callosum in the developing visual system; A Review Prog Neurobiol 18: 15–79 26. Westheimer G, Mitchell DE (1969) The sensory stimulus for disjunctive eye movements. Vision Res 9: 749–755 27. van Hof MW Steele-Russell I (1977) Binocular vision in the rabbit. Physiol Behav 18: 121–128 28. Steele-Russell I, van Hof MW, van der Steen J, Collwijn, H. (1987) Visual and oculomotor function in chiasma-cut rabbits Exp Brain Res 66: 61–73 29. Russell MI, Castiglioni JA, Setlow B, SteeleRussell I (2010) Differential retinal localisation of pathways for pattern and movement vision Advances in Biomed Res 10: 76–81

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30. Downer JL de C (1961) Changes in visual gnostic functions and emotional behavior following unilateral temporal pole damage in the split-brain monkey Nature (Lond) 191: 50–51 31. Downer JL de C (1962) Interhemispheric integration in the visual system. In: Mountcastle VB (Ed): Interhemispheric relations and cerebral dominance, pp 87–100. Baltimore: John Hopkins Press 32. Horel JA, Keating EG (1969) Partial KlüverBucy syndrome produced by cortical discontinuity. Brain Res 16: 281–284 33. Imamura A (1903) Ueber die kortikalen Störungen des Sehaktes und die bedeutung des Balkens Pflügers Arch 100: 495–531 34. Yoshimura K (1909) Über die Beziehungen des Balkens zum Sehakt Pfügers Arch ges Physiol 129: 425–460 3. 35. Crowne DP, Yeo CH, I Steele-Russell (1981) The effects of unilateral frontal eye fields in the monkey: visual-motor guidance and avoidance behaviour Behav Brain Res 2: 165–187 36. van de Steen H, I Steele-Russell, James GO (1986) Effects of unilateral frontal eye-field lesions on eye-head coordination in monkey J Neurophys 55 4: 696–714 37. Mountcastle VB (ed) (1962) Interhemispheric relations and cerebral dominance. Baltimore: John Hopkins University Press. 38. Steele-Russell I, van Hof MW, Berlucchi G (eds) (1979) Structure and function of the cerebral Commissures, Baltimore: University Park 39. Leporé F, Pitto M, Jasper HH (eds) (1986). Two hemispheres - one brain: functions of the corpus callosum. In: Neurology and neurobiology, vol 17, New York: Alan R Liss 40. Zaidel E, Jacobini M (eds) (2003). The parallel brain: the cognitive neuroscience of the corpus callosum. The MIT press, Cambridge, Massachusetts, London, England 41. Blinkov SM, Glezer II (1968) The human brain figures and tables 482 pp. Haigh (Transl) New York Plenum Press 42. Cumming WJK (1969) The fibre-content of the corpus callosum of the albino rat J Anat (Lond) 104: 189–201 43. Szentagothai-Schimert J (1942) Die Bedeutung des Faserkalibers und der Markesheidendicke im Zentralnervesystem Z Anat Entwickl Gesch 111: 201–223 44. Ebner FF, Myers RE (1962) Commissural connections in the neocortex of monkey Anat Rec 142: 22 45. Myers RE (1965) The neocortical commissures and interhemispheric transmission of information. In Ettlinger EG (Ed) Functions of the corpus

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callosum. Ciba Foundation Study Group No 20, pp 133–143 Boston: Little Brown 46. Jones EG, Powell TPS (1968) The commissural connections of the somatic sensory cortex in cat J Anat (Lond) 103: 433–455 47. Karol EA, Pandya DN (1971) The distribution of the corpus callosum in the rhesus monkey Brain 94: 471–486 48. Pandya DN, Gold D, Berger T (1967a) Interhemispheric connections of the precentral motor cortex in the rhesus monkey Brain Res 15: 594–596 49. Van Essen DC, Newsome WT, Bixby JL (1982) The pattern of interhemispheric connections and its relationship to extrastriate visual areas in the macaqe monkey J Neurosci 2; 265–283 50. Innocenti GM (1986) What is so special about callosal connections? In: Leporé F, Ptito M, Jasper HH (Edits) Two hemisphere-one brain: functions of the corpus callosum Alan R Liss Inc, New York 51. Killacky HP, Gould JH III, Cusik CG, Pons TP, Kaas JH (1983) The relationof corpus callosum connections to architectonic fields and body surface maps in sensorimotor cortex of new and old world monkeys J comp Neurol 219: 384–419 52. Pandya DN, Vignolo LA (1969) Interhemis­ pheric projections of the parietal lobe in the rhesus monkey Brain Res 15: 49–65 53. Hendreen JC, Yin TCT (1981) Homotopic and heterotopic callosal afferents of caudal inferior parietal lobule in in macaca mulatta J comp Neurol 197: 605–62 54. Pandya DN, Kuypers HGJM (1969) Corticocortical connections in the rhesus monkey Brain Res 13: 13–36 55. Seltzer B, Pandya DN (1976) Some cortical projections to the parahippocampal area in the rhesus monkey Exp Neurol 50: 146–160 56. Seltzer B, Pandya DN (1978) Afferent cortical connections and architectonics of the superior temporal sulcus and surrounding cortex in the rhesus monkey 57. Seltzer B, Pandya DN (1978) The distribution of posterior parietal fibers in the corpus callosum of the rhesus monkey Exp Brain Res 55: 147–150 58. Nasrallah HA, McChesney CM (1981). Psychopathology of the corpus callosum tumours Biol Psychiat 16: 663–669. 59. Geschwind N (1967) Brain mechanisms ­suggested by the study of interhemispheric connections In Millikan CH, Darley FI (Eds) Brain mechanisms underlying speech and

language New York: Grune & Stratton pp 103–108 60. Musiek FE, Reeves A (2008) Effects of partial and complete corpus callosotomy on central auditory function In: Leporé F, Ptito M, Jasper HH (Edits) Two hemisphere-one brain: functions of the corpus callosum Alan R Liss Inc, New York 61. Doty RW & Negrão N (1973) Forebrain commissures and vision. In: Jung R (ed) Central processing of visual information, Part B. Handbook of sensory physiology, Vol VII/3B. Springer-Verlag Berlin Heidelberg New York 62. Steele-Russell I, Russell MI, Castiglioni JA, Werka T, Stetlow B (2008). The role of sensory pathways in Pavlovian conditioning in rabbit Exp Brain Res 185: 199–213 63. Russell MI, Castiglioni JA, Setlow B, SteeleRussell I (2010) Differential retinal localisation of pathways for pattern and movement vision Advances in Biomed Res 10: 76–81 64. Behr C (1925) Die Lehre von den Pupillenbewegungen Berlin. 65. Gamble JE, Patton HD (1953) Pulmonary edema and hemorrhage from preoptic lesions in rats Am J Physiol 172: 623–631 66. Maier FW, Patton HD (1956a) Neural substrate change involving in the genesis of “preoptic pulmonary edema’ Am J Physiol 184: 345–350 67. Maier FW, Patton HD (1956b) Role of the splanchnic nerve and the adrenal medulla in the genesis of ‘preoptic pulmonary edema’ Am J Physiol 184: 351–355 68. Anderson KV, O’Steen WK (1971) Photically evoked responses in rats exposed to continuous light. Exp Neurol 30:555–564 69. Bureš J, Burešova O, Fifkova E (1964) Interhemispheric transfer of a passive avoidance reaction J comp physiol Psychol 57: 326–330 70. Cydell A (1970) The use of hypothermia in split-brain surgery on the rat Behav Res meth Instrum 2: 65–66 71. Goodman ED, Steele-Russell I (1974) Splitbrain rat: a new surgical approach. Physiol Behav 13: 327–330 72. Steele-Russell I, Safferstone JF (1973). Lateralisation of brightness and pattern discrimation learning in the callosum-sectioned rat Brain Res 49: 497–498 73. Crowne DP, Monica F. Novotny, SteeleRussell  I (1991) Completing the split in the split-brain rat: transection of the optic chiasm Behav Brain Res 43: 185–19

Part III Cerebellar and Brain Stem Systems

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Chapter 13 Genetic Models of Cerebellar Dysfunction Robert Lalonde and Catherine Strazielle Abstract Mice with spontaneous mutations or with genetic modifications serve as models of spinocerebellar atrophy (SCA) and Friedreich’s ataxia. ATXN1, ATXN2, ATXN3, and ATXN7 transgenic mice mimic SCA1, SCA2, SCA3, and SCA7, respectively, while Spnb3 and Atxn8os null mutants mimic SCA5 and SCA8, respectively, with age-related onset of cerebellar pathology and motor-coordination deficits. The onset in spontaneous mutations generally occurs prior to or during the weaning period, providing information on the consequences of cerebellar lesions throughout the animal’s lifetime. Prevalent tests to screen cerebellar dysfunction include the stationary beam, suspended wire, inclined grid, rotorod, exploratory activity, and spatial orientation. Cartographies of regional metabolism and neurotransmitter uptake sites and receptors provide valuable insight into the functional impact of cerebellar lesions throughout the brain and their relation with behavioral deficits, to facilitate pharmacotherapies aimed at mitigating symptoms of cerebellar­related degenerative diseases. Key words: Cerebellum, Spinocerebellar atrophy, Genetics, Motor coordination, Rotorod, Spatial orientation, Regional brain metabolism, Biogenic amines

1. Introduction The behavioral effects of cerebellar degeneration are studied with spontaneous murine mutations, with onset prior to or during weaning, namely Grid2Lc (Lurcher), Grid2ho (hot-foot), Rorasg (staggerer), Agtpbp1pcd (Purkinje cell degeneration), Cacna1aln (leaner), Cacna1arol (rolling mouse Nagoya), Relnrl (reeler), nervous, and pogo, as well as the Dstdt (dystonia musculorum) spinocerebellar mutant, the latter with neuropathological and behavioral features resembling Friedreich’s ataxia. All these mutants show ataxia, as defined by a wide-spread gait to help maintain posture, as well as motor-coordination deficits.

Emma L. Lane and Stephen B. Dunnett (eds.), Animal Models of Movement Disorders: Volume II, Neuromethods, vol. 62, DOI 10.1007/978-1-61779-301-1_13, © Springer Science+Business Media, LLC 2011

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Genetically modified mice serve as models of spinocerebellar atrophy (SCA) and Friedreich’s ataxia (1, 2), generally with onset during the adult period. Human SCA1, SCA2, SCA3/MachadoJoseph, SCA5, and SCA7 diseases are caused by CAG polyglutamine triplet expansions, leading to cerebellar and brainstem damage with ataxia and action tremor. In SCA3/Machado-Joseph disease, there is prominent damage to substantia nigra pars compacta, causing parkinsonian symptoms. In patients with Friedreich’s ataxia, spinocerebellar tracts degenerate, as well as cerebellar neurons, peripheral sensory nerves, dorsal root ganglia, and cranial nerve ganglia. Friedreich’s ataxia is caused by a GAA polyglutamate triplet expansion deleting FRDA, which encodes frataxin, involved in iron transport in mitochondria (3, 4). FRDA KO mice mimic Friedreich’s ataxia. Experimental models of SCA include ATXN1, ATXN2, ATXN3, and ATXN7 transgenic mice, as well as Spnb3 and Atxn8os knockout (KO) mice as models of SCA5 and SCA8, respectively.

2. Neuropathology of Cerebellar Mutants 2.1. Grid2  (Lurcher) Lc

The semi-dominant Lurcher mutation causes a gain-in-malfunction of the Grid2 gene, encoding the GluRd2 ionotropic glutamate receptor (5), functionally related with the amino-methyl-isoxazolepropionate (AMPA) receptor (6). GluRd2 mRNA is predominantly expressed in Purkinje cells (7). Depolarization of their membrane potential (5) is probably responsible for their nearly complete disappearance from postnatal weeks 2–4 (8). The massive degeneration of granule cells seen in Grid2Lc mutants is secondary to the absence of Purkinje cellinduced trophic influence (9, 10). Likewise, the 60–75% loss in inferior olive neurons (8, 11) and 30% loss in deep cerebellar nuclei (12) are considered to be secondary consequences of Purkinje cell atrophy.

2.2. Grid2 ho (Hot-Foot)

Two autosomal recessive hot-foot alleles (4J and Nancy) cause different deletions in the coding sequences of Grid2 (13, 14). For the ho-4J allele, the truncated GluRd2 protein is expressed in Purkinje cell soma without being transported to the surface (15). The main neuropathologic finding in the mutants is defective parallel fiberPurkinje cell innervation and mildly depleted cerebellar granule cells, resulting in cerebellar ataxia and a hopping gait reminiscent of mice walking on a hot plate (16).

2.3. Rora sg (Staggerer)

The autosomal recessive staggerer mutation deletes Rora, encoding a retinoid-like nuclear receptor involved in neuronal differentiation and maturation, highly expressed in Purkinje cells (17). Rora null mutants have almost the same phenotype (18, 19).

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Rorasg Purkinje cells decline in number before postnatal day 5, with 25% of them remaining at the end of 1 month (20). The granule cell loss is secondary to Purkinje cell degeneration (21) and by the end of 1  month is nearly complete (22). Inferior olive neurons decrease by 60% as early as postnatal day 24 (23), remaining low in adults (24), but the number of deep nuclei appears intact (25), indicating retrograde not anterograde degeneration as a consequence of Purkinje cell loss. 2.4. Purkinje Cell Degeneration

The autosomal recessive Purkinje cell degeneration mutation affects the Agtpbp gene, encoding ATP/GTP binding protein 1 (26). In normal mouse brain, Agtpbp1 mRNA is prominent in Purkinje cells, olfactory bulb mitral cells, thalamic neurons, and retinal photoreceptors, structures prone to degenerate in the mutant. Several alleles of the affected gene have been discovered, including 1J, 2J, and 3J. The Purkinje cell population of Agtpbp1pcd-1J mutants declines almost completely from postnatal weeks 3–4 (27, 28). The late onset degeneration of granule cells (29) and deep nuclei (30) appears to be secondary consequences of Purkinje cell loss, together with retrograde degeneration of the inferior olive, reaching 20% on postnatal week 3 (31) and 50% at 10 months of age (32).

2.5. Nervous (nr)

The gene of the nervous (nr) mutation has yet to be discovered. The autosomal recessive nervous mutation causes a major loss in Purkinje cells in the hemispheres but less so in the vermis, granule cell degeneration being less severe and of later onset (33–35). As with other mutants with Purkinje cell atrophy, there is retrograde degeneration of the inferior olive (36).

2.6. Pogo Mutants

The pogo mutation of a yet-to-be-discovered gene causes focal swellings of vermal Purkinje axons and cell loss with vacuolated granule cells (37, 38). The ataxic mutants have a tendency of falling backwards as a result of exaggerated hindlimb extension while rearing.

2.7. Relnrl (Reeler)

The autosomal recessive reeler mutation affects the Reln gene (39). Two alleles have been discovered: Jackson (J) and Orleans (Orl), the former with a deletion of the entire gene and the latter with a 220-bp deletion, causing a frame shift (40). The gene encodes reelin, an extracellular matrix protein involved in neural adhesion and migration at critical stages of development (39). The mutants display architectonic disorganization in several brain regions including the cerebellum and inferior olive (41–43). Reln mRNA is highly expressed in granule cells of normal mouse brain (44), the main type depleted in the mutant (45). Presumably as a consequence of a 50% loss in Purkinje cells (42), inferior olive neurons diminish by 20% (46, 47).

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2.8. Cacna1a ln (Leaner) and Cacna1a rol (Rolling Mouse Nagoya)

Autosomal recessive leaner and rolling mouse Nagoya mutations concern Cacna1a, encoding the voltage-dependent P/Q type alpha-1A subunit calcium channel (48–50). In Cacna1arol, the synaptic morphology between parallel fibers and Purkinje cell dendrites is abnormal (50). Both mutants are characterized by ataxia and seizures.

2.9. Dst dt (Dystonia Musculorum)

The autosomal recessive dystonia musculorum mutation deletes Dst, encoding dystonin, a cytoskeletal organizing protein associated with neurofilaments and microtubules (51). Dst expression is especially marked in dorsal root and cranial nerve ganglia, main sites of neurodegeneration in the mutant (52). Several alleles have been discovered, including Jackson (j), Albany (Alb), and Orleans (Orl). As in patients with Friedreich’s ataxia (2), spinocerebellar and spinothalamic tracts degenerate, as well as peripheral sensory nerves, dorsal root ganglia, and cranial nerve ganglia 5, 7, 9, and 10 (53, 54). Like the mutants cited above, they display ataxia, but unlike them, most of them locomote by crawling.

2.10. FRDA Null Mutants

Like patients with Friedreich’s ataxia, FRDA null mutants show anomalies in spinal cord and dorsal root ganglia combined with cerebellar damage (55). In particular, somatosensory conduction was slowed down after stimulation of the sciatic nerve. Cerebellar granule cells appear depleted and Purkinje cell arborization reduced, leading to ataxia by 10 months of age.

2.11. SCA Transgenic Mice

ATXN1/Q82 transgenic mice containing the entire human ataxin-1 protein with 82 CAG trinucleotide repeats have been generated, the transgene being driven by the Purkinje-cell specific mouse Pcp2 promoter (56). At 1 year of age, the molecular layer of ATXN1/Q82 transgenic mice is thinner and contains ectopic Purkinje cells, though cell loss appears minor (57). Unlike the natural mutations cited above, whose ataxia appears during postnatal development, the onset of ataxia in ATXN1/Q82 transgenic mice occurs at 3 months of age (57). Ataxia was delayed in a conditional ATXN1/Q82 model with delayed transgene expression (58). On the contrary, a second model of SCA1, Atxn1/Q78 knock-in (KI) mice do not display ataxia (59). As a model of SCA2, ATXN2/Q75 transgenic mice with the mutated gene containing 75 CAG repeats of ataxin-2 and driven by the self Atxn2 promoter lose calbindin-28K expression in Purkinje cells, with shrinkage of their dendritic arborization and soma, though with no apparent ataxia (60). Similarly, ATXN2/ Q58 transgenic mice encoding ataxin-2 with 58 CAG repeats regulated by the Purkinje-cell specific Pcp2 promoter lose calbindin28K expression in Purkinje cells but do not have ataxia (61). Several models of SCA3 have been behaviorally characterized. ATXN3/Q79 transgenic mice were generated with 79 CAG

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repeats with or without the full length ataxin-3 protein, controlled by the Purkinje cell-specific L7 promoter, with only the latter showing ataxia by 1  month of age, associated with lost calbindin-28K expression in Purkinje cells (62) ATXN3/Q84 transgenic mice were generated with the mutated gene containing 84 CAG repeats on a yeast artificial chromosome construct with enhancers and regulatory elements in physiological ranges (63). The mutants display Purkinje cell loss with ataxia and a flattened body posture. ATXN3/ Q71 transgenic mice with the mutated gene containing 71 CAG repeats and driven by the mouse Prp (prion protein) promoter have intranuclear inclusions in deep cerebellar nuclei and reduced cell numbers in substantia nigra pars compacta (64). The mutants display ataxia, tremor, and weak grip strength. ATXN3/Q148 transgenic mice with the mutated gene containing 148 CAG repeats and driven by the rat Htt (huntingtin) promoter have intranuclear inclusions in darkly stained Purkinje cells, though no ataxia or tremor was reported (65). The Spnb3 KO mouse serves as a model of SCA5 (66). The gene encodes spectrin-b3, highly expressed in cerebellum, particularly Purkinje cell soma and dendrites. Loss of spectrin-b3 may cause excitotoxicity, as the protein interacts with the glutamate transporter in Purkinje cells (67). Spnb3 null mutants show Purkinje cell loss, leading to ataxia and tremor (66). As a model of SCA7, ATXN7/Q92 transgenic mice contain 92 CAG trinucleotide repeats of ataxin-7 driven by the mouse Prp promoter, causing ataxia, falls, and whole-body tremor by 2 months of age (68). A second transgenic model of SCA7, ATXN7/Q90, has 90 CAG repeats of the mutated gene driven by the murine Pcp2 promoter expressed in Purkinje cells. Nuclear inclusions were found in these cells, but no ataxia was present up to 1 year of age (69). A third transgenic model, ATXN7/Q52, has 52 CAG repeats of the mutated gene driven by the PDGFb promoter. Purkinje cells contain nuclear inclusions and soma and dendritic arbor appear shrunken, leading to ataxia and wobbly locomotion (70). The Atxn8os KO mouse serves as a model of SCA8 (71). The gene is normally expressed in Purkinje cell dendrites and soma. Atxn8os KO mice have a thinner molecular layer in the cerebellum, leading to locomotion abnormalities in the form of longer stance and stride times but with no visible ataxia. 2.12. Unc13c Null Mutants

Unc13c mRNA is almost exclusively found in Purkinje and granule cells of cerebellum (72). Targeted deletion of Unc13c caused abnormal synaptic transmission at the parallel fiber-Purkinje cell synapse, but no overt neuropathology (73).

2.13. Calb1 Null Mutants

Calb1 encodes calbindin D28K, a cytoplasmic Ca2+-binding protein particularly enriched in Purkinje cells (74). Dendritic calcium transients associated with complex spikes were affected in Purkinje cells of Calb1 null mutants, causing ataxia and tremor (75).

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2.14. Hzf Null Mutants

Hzf null mutants are deficient in a RNA-binding protein highly expressed in Purkinje cells (76). These mutants also display ataxia and tremor.

2.15. Plcb4 Null Mutants

Plcb4 null mutants are deficient in phospholipase C b4 subunit, highly expressed in Purkinje and cerebellar granule cells (77). Consequently, the mutants are deficient in parallel fiber-Purkinje cell synapses and display ataxia.

3. Motor Coordination Tests The principal methods used for evaluating motor coordination in rodents are stationary beam, suspended wire, inclined or vertical grid, and rotorod tests (Table 1). For each test, a 15-min intertrial interval is often used as a rest period. The primary measure is the latency before falling, whose validity depends on whether the animal exerts an effort to stay on the apparatus. An unmotivated animal usually leaps forward instead of dropping straight down, which sometimes happens when the characteristics of the apparatus are far beyond their capabilities, as when the initial speed of a rotorod is too fast. This maladaptive response should not be considered equivalent to a fall. Instead, the trial should not count, the subject being placed back at once until this misbehavior stops. After a few unsuccessful attempts, the animal can be re-assessed 15 min later. If this behavior persists, the subject should not be included in the analysis. 3.1. Stationary Beam

In the stationary beam test, the animal either stays put or locomotes along the beam, usually closed at either end to prevent escapes. Distance traveled and number of slips are used as auxiliary measures to distinguish immobile from moving subjects. The beam should be placed at least at a height of 30 cm from a padded surface, to prevent temptations of jumping off deliberately and any type of injury. For optimal performances, the beam should be covered with masking tape or some other material to increase traction. The beam is usually circular, since square-shaped ones facilitate clinging to the side. The stationary beam test is sensitive to natural mutations with ataxia, as seen in Grid2Lc (78, 79), Grid2ho-Nancy (14, 79), Grid2ho-4J (14), Rorasg (79–81), nervous (82), Relnrl-Orl (83), pogo (84), and Dstdt-J (85, 86), as well as non-ataxic Cacna1arol heterozygotes (87). When a thick square-shaped beam was substituted for a round one to facilitate clinging against the side, Rorasg mutants were still impaired (86), but not Grid2Lc (78) or Agtpbp1pcd-1J (87). Thus, a partial loss of Purkinje cells in Rorasg caused a worse outcome than their nearly total disappearance in the other two mutants, presumably

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Table 1 Motor performance by cerebellar or spinocerebellar mutants vs. controls Mutants

Stationary Ataxic or not beam

Suspended string

Inclined grid

Rotorod References

Grid2Lc

Ataxic

↓

↓

↓

↓

(78, 79)

Grid2ho-Nancy

Ataxic

↓

↓

?

↓

(14, 79, 95)

Grid2ho-4J

Ataxic

↓

Normal

?

↓

(14)

Rora

Ataxic

↓

↓

?

↓

(79, 95)

Agtpbp1pcd

Ataxic

Normal

↓

Normal

↓

(87)

Nervous

Ataxic

↓

↓

↓

↓

(82)

Pogo

Ataxic

↓

?

?

?

(84)

Ataxic

↓

↓

?

↓

(83)

Cacna1ala het

Non-ataxic

?

↓

?

↓

(92)

Cacna1arol het

Non-ataxic

↓

↓

?

↓

(90)

Dst

Ataxic

↓

↓

↓

?

(85)

FRDA KO

Ataxic

?

?

?

↓

(55)

Calb1 KO

Non-ataxic

↓

?

?

?

(75, 89)

Unc13c KO

Non-ataxic

?

?

?

↓

(73)

Hfz KO

Ataxic

?

↓

?

↓

(76)

Plcb4 KO

Ataxic

?

?

?

↓

(77)

ATXN1/Q82 Tg

Ataxic

↓

?

?

↓

(57)

Atxn1/Q78 KI

Non-ataxic

?

?

?

↓

(59)

ATXN2/Q75 Tg

Non-ataxic

?

?

?

↓

(60)

ATXN3/Q84 Tg

Ataxic

↓

?

?

?

(88)

ATXN3/Q71 Tg

Ataxic

?

?

?

↓

(64)

ATXN3/Q148 Tg

Non-ataxic

?

?

?

↓

(65)

Spnb3 KO

Ataxic

↓

Normal

?

↓

(66)

ATXN7/Q92 Tg

Ataxic

?

?

?

↓

(68)

ATXN7/Q90 Tg

Non-ataxic

?

?

?

↓

(69)

ATXN7/Q52 Tg

Ataxic

?

?

?

↓

(70)

Atxn8os KO

Non-ataxic

?

?

?

↓

(71)

Reln

sg

rl-Orl

dt

↓ Lower, normal, ? untested vs. non-ataxic controls, KO = knockout, Tg = transgenic, het = heterozygotes

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because the presence of the remaining cells causes worse dysfunction than their absence. The stationary beam test is also sensitive to cerebellar anomalies in genetically modified mutants, such as ATXN3/Q84 ataxic transgenics (88), as well as Spnb3 (66) and Calb1 (75, 89) KOs, as well as nonataxic Cacna1a heterozygotes (90), in addition to ATXN1/Q82 transgenics prior to ataxia (57). The bar deficit in the ATXN3/Q84 transgenic was alleviated with dantrolene, a calcium stabilizer (88), indicating its sensitivity to pharmacologic intervention. 3.2. Suspended Wire or Coat-Hanger

In the wire suspension test, the animal either stays put or locomotes upside down while clinging to a thin metal or plastic wire closed at either end. Because the animal must carry its own body weight, this test is more dependent on muscle strength than the previous one. To obtain measurements of movement time and climbing abilities, the animal may reach and then climb at either end on a diagonal wire of a coat-hanger. As with beam tests, the horizontal wire should be placed at least at a height of 30 cm from a padded surface. Despite cerebellar damage, the animal may land on four feet, or on its side, as the air-righting reflex is still active. Some cerebellar mutants are susceptible to convulsions when placed upside down. In such cases, the animal’s fall should not count. Instead, the animal should rest awhile and the trial repeated after recovery. Latencies before falling from the coat-hanger were lower than normal in natural mutations with ataxia, such as Grid2Lc (78, 79), Grid2ho-Nancy (79), Rorasg (79–81), Agtpbp1pcd-1J (87), Relnrl-Orl (83), and Dstdt-J (85, 86). Latencies before falling were also lower in Grid2Lc mutants placed on the suspended wire (91). On the contrary, ataxic nervous mutants did not fall sooner than controls, probably helped by relatively preserved Purkinje cells at the level of the vermis, crucial in the control of posture and equilibrium, though their movement times were longer (82). However, nonataxic Cacna1arol (90) and Cacna1aln (92) heterozygotes were deficient in the wire suspension test, though ataxic Spnb3 (66) and Hfz (76) KOs were not. Thus, on one hand, ataxia does not necessarily lead to deficits in hanging from a wire. On the other, some non-ataxic mutants are deficient, indicating no obligatory relation between the two signs.

3.3. Inclined or Vertical Grid

In the inclined or vertical grid test, the animal is placed on a metal surface oriented from 0 to 180°. As with the previous test, the grid should be placed at least at a height of 30 cm from a padded surface. In comparison to non-ataxic controls, the number of falls from a vertical grid increased in Grid2Lc (78), Rorasg (81), and Dst dt-J (85, 86) mutants, but not in Agtpbp1pcd-1J (87). As noted above, the superior performance of Agtpbp1pcd-1J relative to Rorasg on the same grid may be explained by nearly total as opposed to

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partial Purkinje cell depletion, the former being more favorable to behavioral performances. Their superior performance relative to Grid2Lc may be ascribed to later onset of Purkinje cell depletion or to relative preservation of cerebellar granule cells. Their superior performance relative to Dstdt-J is attributed to the latter’s spinocerebellar and lemniscal atrophy leading to crawling in addition to ataxia. 3.4. Rotorod

Unlike the above-named tests, animals placed on the rotorod move in synchrony with a moving surface. An animal with postural deficits but with preserved muscle strength may cling to the rotating beam without moving, though cerebellar lesions usually cause hypotonia. To distinguish moving from immobile animals, the latter may be removed from the beam after turning twice around, as if it fell, since passively rotated animals tend to repeat this strategy until the end of testing. A more time-consuming approach is to videotape and measure time spent in passive rotation. In most models, the rod is placed lower than 30  cm above an unpadded surface, since animals are less inclined to jump away from a moving beam. However, it is judicious to start the test at the slow pace of 4 revolutions per minute (rpm), accelerating up to 40 rpm, because at faster initial speeds animals tend to jump off deliberately. If the beam is not rotated at the start, animals sometimes fall off immediately when surprised by the first jerk of beam movement. The rotorod test is sensitive to natural mutations with ataxia, as seen in Grid2Lc (91, 93–95), Grid2ho-Nancy (14, 95), Grid2ho-4J (14), Rorasg (80, 95), Agtpbp1pcd-1J (87), nervous (82), and Relnrl-Orl (83), as well as non-ataxic Cacna1ala (92) and Cacna1arol (90) heterozygotes. The rotorod test is also sensitive to cerebellar anomalies in genetically modified mice. ATXN1/Q82 transgenic mice were impaired on the rotorod as early as 5 weeks of age, prior to signs of ataxia, when the main histopathology is the accumulation of cytoplasmic vacuoles inside Purkinje cells (57). Moreover, non-ataxic Atxn1/Q78 KI mice were impaired in the same test (59). Rotorod performance improved in the conditional ATXN1/Q82 model with delayed transgene expression relative to the original transgenic (58). After stopping transgene expression in the conditional model, rotorod performance was also ameliorated (96). Nonconditional ATXN1/Q82 transgenics crossbred with mice overexpressing the inducible form of rat HSP70, encoding a molecular chaperone involved in protein folding, had Purkinje cells with thicker and more arborized dendritic branches than the single transgenic and better rotorod performance (97). Rotorod performance also improved after RNA interference (98), intranasal insulin-growth factor (99) or injections of taurine-conjugated ursodeoxycholic acid (100). As with the SCA1 model, FRDA null mutants were impaired on the rotorod prior to ataxia (55).

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Other ataxic mutants showing impairments on the rotorod include ATXN3/Q71 (64), ATXN7/Q92 (68), and ATXN7/ Q52 (70) transgenics, as well as Spnb3 (66), Atxn8os (71), Hfz (76), and Plcb4 (101) KOs. Moreover, non-ataxic ATXN2/Q75 (60), ATXN3/Q148 (65), and ATXN7/Q90 (69) transgenic mice, as well as Unc13c (73) KOs mice were deficient on this test. Rotorod impairments are also found after drug injections directly into the cerebellum. Indeed, intracerebellar injections of CB1 receptor agonists reduced rotorod performances in rats (102, 103).

4. Exploratory Activity Motor activity and spontaneous alternation tests (Table 2) are relevant to cerebellar pathology. Motor activity in the open-field and T- or Y-mazes provides an estimate of motor capabilities as well as general arousal levels. Spontaneous alternation testing provides an estimate of the motivation to explore unfamiliar environments. 4.1. Open-Field and T-Maze Activity

When evaluated for distance travelled in a T-maze, Rorasg mutants (104) were hypoactive relative to non-ataxic controls, due at least in part to ataxia and frequent falls, especially after attempted rears

Table 2 Exploratory activity by cerebellar or spinocerebellar mutants vs. controls Mutants

Ambulatory activity

Spontaneous alternation

References

Grid2Lc

↑

↓

(105)

Grid2ho-Nancy

↓

↓

(109)

Grid2

Normal

?

(108)

Rora

↓

↓

(104)

Agtpbp1pcd

↑

↓

(106)

Nervous

↑

↓

(107)

Reln

?

↓

(114)

Reln

Normal

↓

(83)

Dstdt

↓

?

(85)

ATXN1/Q82 Tg

↑

?

(57)

ATXN3/Q71 Tg

↓

?

(64)

ho-4J

sg

rl-J rl-Orl

↑ Higher, ↓ lower, normal, ? untested vs. non-ataxic controls

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251

against the walls. But despite ataxia and stumbling, Grid2Lc (105), Agtpbp1pcd-1J (106), and nervous (107) mutants were hyperactive in the same maze relative to their respective controls. Non-ataxic ATXN3/Q148 mutants were likewise hyperactive in the openfield (65), as well as ATXN1/Q82 mutants prior to ataxia (57). The lower activity of Rorasg relative to Grid2Lc and Agtpbp1pcd-1J reproduces their profile on the square-shaped stationary beam, only Rorasg being deficient (78, 81, 97). Thus, activity levels in cerebellar mutants gauge the extent of motor deficits in a complementary manner to tests measuring balance and equilibrium. Exploratory activity was normal in ataxic Relnrl-Orl (83) and Grid2ho-4J (108) mutants, though the Grid2ho-Nancy mutants were hypoactive (109), perhaps indicating an allele-specific deficit. ATXN3/Q71 transgenic mice with cerebellar and basal ganglia dysfunction were also hypoactive (64), as well as the Hfz KO (76) and Dstdt-J mutants (85), the latter result being expected due to their crawling phenotype. 4.2. Spontaneous Alternation

5. Spatial Orientation in Water Mazes

After entering one of two maze arms in a T- or Y-maze, rodents have a strong tendency to enter the opposite side (110). The spontaneous alternation test is sensitive to natural mutations with cerebellar atrophy, as shown in Grid2Lc (105, 111, 112), Grid2hoNancy (109), Rorasg (104, 113), Agtpbp1pcd-1J (106), nervous (82), Relnrl-Orl (83), and Relnrl-J (114). However, these results did not extend to the ataxic Hfz KO (76). In general, the results are concordant with a role for the cerebellum in disinhibitory tendencies, as seen in animals with damage to the vestibular system (115), the limbic system (116, 117), and the prefrontal cortex (118), attributable to two-way vestibulo–cerebello–cortical connections. The gross motor control implied in turning right or left precludes involuntary entries into a maze arm as the main reason for their perseveration.

The lurching gait so characteristic of Grid2Lc mutants on dry ground is no longer evident in the water, as these show good swimming abilities (119). Nevertheless, Grid2Lc mutants were deficient in spatial orientation toward a hidden platform as well as visuomotor guidance toward a visible one, distance swum and escape latencies being increased (91, 112, 120–122) (Table 3). These results confirm a role for the cerebellum in the visual guidance of movement (123). The same pattern was evident in Relnrl-Orl mutants with ectopias in cerebellum and hippocampus (83) as well as Cacna1ala heterozygotes (92). In contrast, Rorasg (113), Agtpbp1pcd-1J (124), and nervous (82) mutants were deficient

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Table 3 Water maze performance by cerebellar or spinocerebellar mutants vs. controls Mutants

Hidden platform

Visible platform

References

Grid2Lc

↓

↓

(112, 120–122)

↓

Normal

(113)

Agtpbp1

↓

Normal

(124)

Nervous

↓

Normal

(82)

Relnrl-Orl

↓

↓

(83)

Dst

?

↓

(129)

Cacna1ala heterozygotes

↓

↓

(92)

Rora

sg pcd

dt

↓ Lower, normal, ? untested vs. non-ataxic controls

only in the hidden platform subtest, concordant with a role for cerebello-limbic or cerebello-neocortical connections in spatial orientation, further supported by findings of the same dissociation in rats with immunotoxin-induced degeneration of Purkinje cells (125) as well as electrolytic lesions in lateral cerebellar cortex or dentate nucleus (126–128). Due to their susceptibility to drowning, Dstdt-J mutants cannot be tested in the hidden platform version (129). In the visible platform subtest, escape latencies were higher than controls but path lengths were lower. Unlike the cerebellar mutants described above, the Dstdt-J line is characterized by slower swim speed and intact directional guidance, presumably due to spinocerebellar as opposed to cerebellar atrophy.

6. Regional Brain Metabolism The effects of cerebellar damage on brain metabolism have been examined by high-resolution cytochrome oxidase (COX) histochemistry. COX is the fourth enzyme of the mitochondrial electron transfer chain of oxidative phosphorylation, immediately prior to ATPase. COX labeling is a specific marker of neuronal activity, since the contribution of glial cells on oxidative metabolism is minimal (130). Because COX histochemistry is expressed as a function of tissue weight, a loss in neuron numbers does not necessarily lead to reduced enzymatic activity. Studies in brain metabolism offer insights into the functional impact of cerebellar dysfunction on remaining cells of this structure as well as cerebellarrelated pathways.

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Table 4 Variations of cytochrome oxidase (COX) activity in cerebellar cortex and deep nuclei of cerebellar mutants Mutants

Molecular layer

Purkinje cell layer

Granule cell layer

Deep nuclei

References

Grid2Lc

Normal

Absent

Normal

↑

(132)

Grid2

↑

Normal

Normal

Normal

(133)

Normal

↑

Normal

↑

(80)

Normal

↑

Normal

↓

(136)

Rora

ho-Nancy

sg

Relnrl-Orl

↑ Higher, ↓ lower, normal, absent vs. non-ataxic controls

In cerebellar cortex of 15- and 25-day-old Grid2Lc mutants, Purkinje cells could be observed prior to their degeneration. COX activity in these cells was higher than that of wild-type, but unchanged in the molecular layer (131). Hypermetabolism in Purkinje cells probably reflects chronic depolarization of their membrane potential (5). In adult mutants where these cells have disappeared, COX activity of shrunken molecular and granule layers did not differ from that of wild-type, indicating normal metabolic activity relative to tissue weight but higher metabolic activity relative to absolute levels (132). COX activity relative to tissue weight was higher than controls in deep cerebellar and lateral vestibular nuclei (Table 4), attributable to lost gamma-amino butyric acid (GABA)-ergic inhibitory input from Purkinje cells, since excitatory synapses have higher energy demands than inhibitory ones (130). COX activity of Grid2Lc mutants was also elevated in several regions directly connected with the cerebellum, probably as a result of the hypermetabolic deep nuclei, the only pathway out of the cerebellum. In an opposite fashion, COX activity decreased in inferior olive, precursor to retrograde degeneration as a consequence of Purkinje cell atrophy. Regional brain metabolism of Grid2ho-Nancy mutants differed substantially from Grid2Lc in view of their milder cerebellar atrophy (133). COX activity in the molecular layer of Grid2ho-Nancy mutants was higher than that of non-ataxic controls, probably due to increased neural activity of cerebellar afferents in response to defective dendritic organization of Purkinje cells. Unlike Grid2Lc, COX activity in deep cerebellar nuclei and inferior olive was unchanged in Grid2ho-Nancy probably because of the preservation of Purkinje cells. Rorasg mutants share many neuropathological characteristics with Grid2Lc, in particular massive degeneration in cerebellar cortex. Like Grid2Lc, COX activity of Rorasg mutants was higher than that of non-ataxic controls in deep cerebellar nuclei and cerebellar

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target structures such as red and interpeduncular nuclei, but unchanged in granular and molecular cerebellar cortex, though elevated in Purkinje cells (80). Once again, hypermetabolism in deep nuclei is probably due to decreased GABAergic input from depleted Purkinje cells. Unlike Grid2Lc, COX staining of Rorasg mutants was normal in inferior olive, presumably because of a stabilization in retrograde degeneration. In contrast to the previous two mutants, COX activity decreased in thalamic nuclei, probably as a direct consequence of Rora deletion, since this gene is highly expressed there in normal mouse brain (134, 135). Like Rorasg and Grid2Lc, COX activity in unfoliated cerebellar cortex of Relnrl-Orl mutants was unchanged (136). The normal enzymatic value in the granule cell layer is concordant with unchanged values of deoxyglucose uptake existing in this mutant (137), implying normal synaptic activity despite granule and Purkinje cell loss. Like Rorasg, COX activity increased in correctly positioned Purkinje cells, but, unlike the previous three mutants, COX activity diminished in roof nuclei (interpositus and dentate combined, no longer identifiable separately). Because optical density readings of sections stained with methylene blue showed no change of coloration per surface area, the hypometabolism in roof nuclei may be due to increased GABAergic influence from hypermetabolic Purkinje cells prior to their disappearance.

7. Biogenic Amines Regional analyses of dopamine, noradrenaline, and serotonin (5-hydroxytryptamine, 5HT) uptake sites and receptors are relevant in view of understanding lesion effects on local and distant anatomical pathways, as the cerebellum receives and sends information from and to areas rich in biogenic amines (138–145). Biogenic amines modulate synaptic activity at the dendritic arborization of Purkinje cells. A cartography of dopamine and 5HT uptake sites as a function of protein weight and for atrophied regions as total area binding (multiplying specific binding with tissue volume) was determined by quantitative autoradiography in Grid2Lc mutants (146, 147) (Table 5). Relative to wild-type, 5HT uptake increased in Grid2Lc cerebellar cortex and deep nuclei, while 5HT total binding increased only in the latter. These results indicate that elevated 5HT uptake in deep nuclei serves as a compensatory response to cerebellar atrophy. On the contrary, dopamine uptake was normal in the striatum of Grid2Lc mutants, one possible explanation as to why their activity levels are not decreased in spite of ataxia (105). Like Grid2Lc, 5HT uptake increased in cerebellar cortex and deep nuclei of Agtpbp1pcd-1J mutants, with total binding increasing

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Table 5 Variations of cerebellar noradrenaline and 5HT transporters in cerebellar cortex (ctx), deep nuclei (dcn) or total (tot) cerebellum and of dopamine uptake in striatum of cerebellar or spinocerebellar mutants Mutants

Dopamine

Noradrenaline

5HT

References

Grid2Lc

Normal

?

↑ ctx ↑ dcn

(146, 147)

Agtpbp1pcd

?

↑ ctx ↑ dcn

↑ ctx ↑ dcn

(148–150)

Relnrl-Orl

Normal

↑ tot

↑ tot

(151–154)

Dst

?

↑ ctx ↑ dcn

↑ dcn

(155, 156)

dt

↑ Higher, normal, ? untested vs. non-ataxic controls

in the latter (148). These results indicate that Purkinje cell atrophy may be responsible for changes in the 5HT system, since granule cells degenerate minimally in this mutant. Likewise, noradrenaline uptake increased in cerebellar cortex and deep nuclei, though no change occurred for total area binding, indicating normal innervation of a shrunken structure (149). However, total area binding in cerebellar cortex diminished for a1 and b receptors and augmented for a2 receptors, indicating changes in noradrenaline transmission as a result of cerebellar atrophy. Regionally selective actions were also reported for dopamine uptake in the same mutant, increasing in deep nuclei but not in cerebellar cortex (150). When areaadjusted for atrophy, dopamine uptake decreased in cerebellar cortex. Unlike Grid2Lc, dopamine uptake increased and D2/D3 receptor binding decreased in dorsal striatum of this mutant. An increase in monoamine uptake inside an atrophied cerebellum is also a feature of Relnrl-J mutants, a result found for dopamine (151–153), noradrenaline (154), and 5HT (154) (Table 5). Like Grid2Lc, dopamine uptake was normal in the striatum of Relnrl-J mutants (152). Increased 5HT uptake in deep nuclei though not in cerebellar cortex was also seen in spinocerebellar Dstdt-J mutants (155) (Table  5). Moreover, their noradrenergic transporter binding rose in both regions (156). These monoaminergic changes may be due to functional compensation for missing mossy fiber input from spinal cord. Changes in monoaminergic systems have given rise to clinical and experimental pharmacotherapy. The dopamine uptake inhibitor, amantadine, improved reaction times, and movement times in patients with SCA and Friedreich’s ataxia (157), as well as movement

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times in the Grid2Lc mouse model (158). Likewise, facilitation of 5HT neurotransmission by high-dose buspirone or 5-hydroxytryptophan aids some human ataxic symptoms (159–161). By choosing appropriate behavioral tests in ataxic mutants, there is a potential in uncovering better molecules to mitigate motor coordination defects in these incurable diseases. References 1. Klockgether T, Evert B (1998) Genes involved in hereditary ataxias. Trends Neurosci 21:413–418 2. Koeppen AH (1998) The hereditary ataxias. J Neuropathol Exp Neurol 57:531–543 3. Campuzano V, Montermini L, Moltò MD, Pianese L, Cossée M, Cavalcanti F, Monros E, Rodius F, Duclos F, Monticelli A et al (1996) Friedreich’s ataxia: autosomal recessive disease caused by an intronic GAA triplet repeat expansion. Science 271:1423–1427 4. Delatycki MB, Camakaris J, Brooks H, EvansWhipp T, Thorburn DR, Williamson R, Forrest SM (1999) Direct evidence that mitochondrial iron accumulation occurs in Friedreich ataxia. Ann Neurol 45:673–675 5. Zuo J, De Jager PI, Takahashi KA, Jiang W, Linden DJ Heintz N (1997) Neurode­ generation in Lurcher mutant mice caused by mutation in the delta 2 glutamate receptor gene. Nature 388:769–773 6. Landsend AS, Amiry-Moghaddam M, Matsubara A, Bergersen L, Usami S, Wenthold RJ, Ottersen OP (1997) Differential localization of delta glutamate receptors in the rat cerebellum: coexpression with AMPA receptors in parallel fiber-spine synapses and absence from climbing fiber-spine synapses. J Neurosci 17:834–842 7. Takayama B, Nakagawa S, Watanabe M, Mishina M, Inoue Y (1996) Developmental changes in expression and distribution of the glutamate receptor channel delta 2 subunit according to the Purkinje cell maturation. Dev Brain Res 92:147–155 8. Caddy KWT, Biscoe TJ (1979) Structural and quantitative studies on the normal C3H and Lurcher mutant mouse. Philos Trans Roy Soc Lond (Biol) 287:167–201 9. Vogel MW, McInnes M, Zanjani HS, Herrup  K (1991) Cerebellar Purkinje cells provide target support over a limited spatial range: evidence from Lurcher chimeric mice. Dev Brain Res 64:87–94 10. Wetts R, Herrup K (1982) Interaction of granule, Purkinje and inferior olivary neurons

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47. Shojaeian H, Delhaye-Bouchaud N, Mariani J (1985) Neuronal death and synapse elimination in the olivo-cerebellar system. II. Cell counts in the inferior olive of adult X-irradiated rats and weaver and reeler mice. J Comp Neurol 232:309–318 48. Doyle J, Ren X, Lennon G, Stubbs L (1997) Mutations in the Cacnl1a4 calcium channel gene are associated with seizures, cerebellar degeneration, and ataxia in tottering and leaner mutant mice. Mamm Genome 8:113–120 49. Mori J, (2000) Reduced volatge sensitivity of the activation of P/Q-type Ca2+ channels is associated with the ataxic mouse mutation rolling Nagoya (tg(rol)). J Neurosci 20: 5654–5662 50. Plomp JJ, van den Maagdenberg AMJM, Kaja S (2009) The ataxic Cacna1a-mutant mouse Rolling Nagoya: an overview of neuromorphological and electrophysiological findings. Cerebellum 8:222–230 51. Brown A, Bernier G, Mathieu M, Rossant J, Kothary R (1995) The mouse dystonia musculorum gene is a neural isoform of bullous pemphigoid antigen 1. Nature Genet 10: 301–306 52. Bernier G, Brown A, Dalpé G, De Repentigny Y, Mathieu M, Kothary R (1995) Dystonin expression in the developing nervous system predominates in the neurons that degenerate in dystonia musculorum mutant mice. Mol Cell Neurosci 6:509–520 53. Duchen LW, Strich SJ (1964) Clinical and pathological studies of an hereditary neuropathy in mice (Dystonia musculorum). Brain 87:367–378 54. Janota I (1972) Ultrastructural studies of an hereditary sensory neuropathy in mice (dystonia musculorum), Brain 95:529–536 55. Simon D, Seznec H, Gansmuller A, Carelle N, Weber P, Metzger D, Rustin P, Koenig M, Puccio H (2004) Friedreich ataxia mouse models with progressive cerebellar and sensory ataxia reveal autophagic neurodegeneration in dorsal root ganglia. J Neurosci 24:1987–1995 56. Burright EN, Clark BH, Servadio A, Matilla T, Feddersen RM, Yunis WS, Duvick LA, HZoghbi HY, Orr HT (1995) SCA1 transgenic mice: a model for neurodegeneration caused by an expanded CAG trinucleotide repeat. Cell 82:937–948 57. Clark HB, Burright EN, Yunis WS, Larson S, Wilcox C, Hartman B, Matilla A, Zoghbi HY, Orr HT (1997) Purkinje cell expression of a mutant allele of SCA1 in transgenic mice leads to disparate effects on motor behaviors,

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Chapter 14 Cerebellar Control of Fine Motor Function Rachel M. Sherrard Abstract The cerebellum is a brain region that is fundamental in controlling movement. However, because ­dysfunction anywhere within the motor system, from the cortex to the muscle, will result in abnormal movement, examining cerebellar motor control requires a combination of tests which evaluate different aspects of this structure. First we describe tests of dynamic postural reflexes for the vestibulo-cerebellum – righting reflex and vestibular drop. These are followed by tests of quadruped locomotion – from simple foot print ­analysis, through crossing a narrow bridge, to complex coordination for manoeuvring along a wire. More detailed analysis of the cortico-nuclear circuit is made using gait synchronisation on the rotarod and finally motor learning. Key words: Cerebellum, Equilibrium, Balance, Gait, Motor synchronisation, Rotarod, Motor coordination, Motor learning, Ataxia ­

1. Introduction The cerebellum is a key region involved in balance and motor ­control, in particular regulating the coordination and timing of fine movements. Because the cerebellum receives ascending sensory and descending motor information and in turn projects to many key motor centres (e.g., the red nucleus, motor thalamus and striatum), disorders affecting this structure greatly influence motor control. Extra-cerebellar afferents converge upon cerebellar ­cortical Purkinje neurons, indirectly via granule neurons and their parallel fibre axons to convey information about the context of movement, but also directly through inferior olivary climbing fibres to indicate errors in performance (1–3). The correct function of these cortical circuits is required for motor learning. In addition to genetic mouse models that affect the cerebellum (see Chap. 13), there are various lesion studies (4–9) that aim to

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increase understanding of cerebellar motor function, which is important for the development of potential treatments for human cerebellar dysfunction, e.g., spinocerebellar ataxias, idiopathic cerebellar atrophy or olivopontocerebellar atrophy. Many of the methods used to evaluate cerebellar function are essentially the same or similar to those used for general sensorimotor testing. Therefore it is their application and interpretation that must be carefully modified to ensure that it is cerebellar (dys)function that is being assessed. This chapter includes methods used for testing rodent cerebellar motor control. It is divided into three ­sections each of which assesses different aspects (1) general motor function being fundamental tests of postural reflexes (righting reflex, vestibular drop), gait (foot prints, crossing a narrow bridge) and more complex tasks such as manoeuvring along a wire and climbing a ladder; (2) gait synchronisation which describes more refined testing using the rotarod; and (3) motor learning tests to evaluate the function of different cerebellar regions and cerebellar cortical integrity using the rotarod and swimming tests.

2. Materials and Methods As tests of cerebellar motor control involve several observations (e.g., ability, time taken, distance progressed, response direction bias, foot slips, etc) it is advisable to create specific grids containing each parameter to be tested, on which the outcome can be easily and quickly written. An example template is given in Fig.  1. All testing should be video recorded for subsequent off-line analysis and/or confirmation of written observations. 2.1. General Motor Function

General tests of cerebellar motor control are performed three times a day for 5 days. We find that if they are continued for longer, the animals clearly become bored and performance decreases. For each test, there is an upper time limit of 3 min. This value was calculated from the performance of normal animals, which is then multiplied threefold to allow adequate time for an ataxic animal to attempt the test without confounding performance through becoming fatigued (10); see Note 1. For each animal on each day, the data for the three tests are averaged. This involves giving a numeral to ­qualitative data such as sidedness (Fig. 1).

2.1.1. Dynamic Postural Reflexes

The cerebellum and its vestibular circuitry play key roles in dynamic postural adjustment. Postural reflexes are quickly and easily tested but form only a relatively crude index of cerebellar dysfunction and thus are not always discriminative for minor lesions. They are particularly useful in unilateral cerebellar lesions and those undertaken during development. The two main tests are the righting reflex and

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BRIDGE Date: Animal ID 1.

Crossed Time taken bridge (Y/N) (sec)

Distance crossed

Delay start Paw slips time (number/side)

Freezing ( / )

Gait (crawl/walk)

Falls/time

T1 T2 T3

2.

T1 T2 T3

3.

T1 T2 T3

4.

T1 T2 T3

5.

T1 T2 T3 Side: left = 1, right = 2

Gait: crawl = 1, walk or run = 2

Fig. 1. An example template for scoring motor behaviour. Although this describes the bridge test, similar parameters apply to all the fundamental tests and the rotarod. T1–T3 indicate the trial number on each testing day. Qualitative data such as sidedness of movement or type of gait will need to be given a numerical score for subsequent inter-group comparisons (5, 10). These can be identified on the templates.

vestibular drop. Neither of these tests requires special equipment although, to reduce stress levels, it is better not to have the room lighting too bright. 1. Righting reflex. Animals are momentarily held supine by the shoulders and hip-girdle on a flat surface and released. Normal animals will immediately turn over to recover their normal prone quadruped stance. The presence/absence of the reflex, time taken and direction of response are noted (see Note 2). 2. Vestibular drop. When an animal is suspended by the tail it arches its body to bring its head up to the level of its tail. This procedure also tests truncal muscular strength as well as vestibular function. The presence/absence of the reflex, denoted success/failure, within 60  s (the short upper time limit is to minimize distress), time and direction taken are observed (see Note 3a–c). 2.1.2. Quadruped Locomotion

The patterns of simple gait can be tested through a bridge-crossing task and footprint analysis. 1. Foot print analysis. This test is very simple and it often used as a general indicator of motor function, but it also provides useful information about cerebellar ataxia.

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Equipment. While this test can be done on an open surface without specialist equipment, it is easier to create a narrow ­passage which is well lit at the start and has a dark escape box at the other end. We used an alley 19 cm wide and 80 cm long for adult rats, which is also suitable for mice (see Note 4a). Method. Footprints are made using waterproof ink to mark the paws and are revealed on absorbent white paper lining the passage. 1. Either front and hind paws are marked in different colours (11, 12), or just the hind paws are coloured (10, 13) using washable ink (see Note 4b). 2. Animals are placed in the corridor facing away from the tester toward the dark escape box. 3. The animals are enticed to walk forward simply by ­turning on a light at the “start” end of the corridor (see Note 4c). Analysis. Regularity of footprints, their overlap in normal ­animals, stride length (left–left and right–right), stride width (left–right) and the angle of external rotation of the hind foot are measured (10, 13). Rotation angle is measured from the back of the heel to the centre of the third toe and it is increased in ataxic animals (Fig. 2; see Note 4d). 2. Crossing a narrow bridge. Crossing a narrow bridge is another general motor test which can provide information about cerebellar motor control. Equipment. The bridge needs to be at least 60 cm above a foam padded base (to prevent injury in the event of a fall) and 60 cm long (Fig. 3). The addition of distance markers along the side facilitates data acquisition. We use a wooden bridge with a square profile, which makes the bridge width important; if it is too narrow, ataxic animals cannot cross it without falling and therefore tend to freeze soon after they start (see Note 5a). For adult rats with unilateral cerebellar lesions, 3  cm width is a ­difficult and discriminatory task (5, 10), but mice require <2 cm width. Method. Animals are placed on one end of a bridge and have to progress to a box on the other end (see Note 5b). Analysis. The ability to traverse the bridge, distance covered if the bridge was not crossed, time taken, foot-slips and type of gait (crawl or walk) are recorded (Fig. 1 gives a template of data for crossing a bridge; see Note 5c, d). 2.1.3. Complex Locomotor Skill

The complex coordination and muscular strength of the animals also provide useful insights into cerebellar function. This can be tested by climbing a ladder and the ability to progress along a wire.

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Fig.  2. An example of rat hindpaw prints showing the measures that can be taken. Although stride length can be measured as shown from one footprint (left) to the next (right); it is more useful for animals with unilateral cerebellar lesions to measure the length between foot-strikes of the same side, as these can be asymmetric. q = angle of rotation between the axis of the foot and the direction of walking.

Fig. 3. Schema showing a narrow bridge to test locomotor function. It is 60 cm long and 60 cm above the base but width will vary depending on species.

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1. Ascending a ladder. Climbing a ladder can also be used to assess coordinate gait as it demands repeated even stepping to place each foot on the subsequent rung. While it provides only a relatively crude screen of cerebellar ability, it is useful to assess maturation of motor function in animals lesioned during development. It is not sufficiently discriminatory in animals with unilateral cerebellar lesions Equipment. A steel ladder 8 cm wide comprises 18 rungs, each 5 mm in diameter and 2 cm apart rising at an angle of 25° to the vertical to a dark covered platform (Fig.  4). The whole ladder is placed on the edge of a bench/table so that from its base there is a drop to the floor. This is necessary to induce the animal to climb. Method. Animals are placed on the bottom rung so that they climb to the top (see Note 6). Analysis. Successful arrivals at the top platform, time on ladder and number of rungs climbed, plus number of hindfoot slips are recorded. Time spent in pauses or freezing is subtracted from the total time taken. 2. Progression along a wire. Although hanging from a wire (or coat hanger) is generally used as a test of muscular strength, a horizontal wire provides

Fig. 4. Schema showing a ladder for assessing locomotor function. It has 18 rungs, each 5 mm in diameter and 2 cm apart. It rises at an angle of 25° to the vertical to a dark covered platform.

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a test of trunk and limb muscle coordination. Animals will use their tail and hind limbs to progress along a wire to a box at one end to escape from falling (10, 14). Equipment. The apparatus consists of a horizontal wire ­suspended between upright supports on which there are escape platforms. The wire is 3  mm in diameter, 70  cm long and 60 cm above a foam base. Method. Animals are suspended by their front paws from the middle of the wire (see Note 7a) and are required to climb along the wire to a box at either end. Analysis. The ability of the animals to reach and enter the box, the distance traversed, the direction taken, time taken, falls and the use of supporting hind limbs are recorded (see Note 7b). 2.2. Motor Synchronisation Task

A key cerebellar function, which directly correlates to human ­cerebellar dysfunction, is the synchronisation of repetitive movements (e.g., walking or running 15). The rotarod is a sensitive measure of this task as it is able to detect even minor abnormalities of gait synchronisation (13, 15–17). Moreover, it is also simple enough for animals with highly disordered cerebellar cortex or unilateral cerebellar lesions to still perform the task at slow speeds (10, 18, 19) and therefore allow lesion/treatment comparisons (see Note 8). Equipment. The rotarod is a horizontal cylinder, which can either be made “in house” or is commercially available for mice, rats or combined (e.g., Panlab/Harvard Apparatus Rota Rod LE8500). Commercially available apparatuses have automatic timers to measure when the animal falls; whereas this has to be manually timed for “in house” machines (see Note 8a). The rod is mounted 50 cm above a foam base. It is 30–50 cm long, covered in adhesive plaster to allow grip and divided into lanes for each animal. Cardboard/ plastic circles make a useful option for adjusting the number and size of lanes. The cylinder rotates about its long axis at either constant or accelerating speeds. Method. The machine is set to rotate at either constant or ­accelerating speed. 1. The constant speed chosen varies with the experimental paradigm/degree of ataxia, but increments of 10 rpm starting from 10 rpm provide a discriminatory range for cerebellar testing. 2. Accelerating tests start at 4 rpm and increase by 4 rpm every 30 s over 5 min to a maximum of 40 rpm (see Note 8b). 3. Irrespective of the test, the animal is placed on the rotating cylinder perpendicular to the direction of rotation facing away from the tester. This is best achieved by allowing the animals to walk off the open palm onto the rotating rod. In order to maintain position on top of the rod and not fall off, it has to

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walk forwards synchronising stepping frequency and stride length to the speed of rotation. For each trial, the parameters recorded are: (a) Total time on the rod. (b) Time walking and time spent in error (clinging or walking backwards). (c) Time to first error (fall or cling). The trial ends either when the animal falls or 180  s is reached (but see Note 8c). 4. There are between three and ten trials per day with an intertrial interval of at least 3 min (10, 20); see Note 8d). Analysis. For each animal on each day the mean walking latencyto-error (fall or cling) and/or total walking time (total time – time spent clinging/reverse walking) is calculated. The mean time on the final day at each speed is used to compare the performance of the experimental groups (see Note 8e). 2.3. Motor Learning

In addition to coordination and synchronisation of repetitive movements, cerebellar control of fine movement also involves motor learning (10, 15, 20, 21). The type of motor learning described here is not classical conditioning reflexes such as the ­eyeblink reflex (e.g., review 22), but rather the learning of motor patterns required for complex movements. The capacity to evaluate this more cognitive aspect of cerebellar motor control becomes crucial to detect differences between groups which may be missed by less discerning tests and to differentiate the function of different regions of the cerebellum, specifically its cortical circuit.

2.3.1. Learning Motor Synchronisation

As the cerebellar cortex regulates gait synchronisation and motor learning, differences between experimental groups may result from motor skills deficit due to the lesion/pathology, an inability to learn the task or a combination of both. These different (dys)functions can be evaluated using data from the rotarod. Equipment and method. See constant speed testing of gait ­synchronisation above (Sect. 2.2). Analysis. Discrimination between deficits in motor performance and learning can be assessed by comparing the differences in errorlatency (relative motor deficit) in untrained animals (first day of testing), when any between-group differences will be due only to motor impairment, with trained animals whose performances will reflect both motor and learning ability (10, 15); Fig. 5). The relative motor deficit is calculated as (control group time − ­experimental group time)/control time; 10, 15). The relative motor deficit in animals that can learn a task will remain similar or decrease (a to b in Fig. 5), whereas in animals which are unable to learn the task,

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Fig. 5. Learning impairment calculation from data obtained from a 10-rpm constant speed rotarod. Control animals learn to walk for the maximal time without error within the first training day. Although young lesioned animals (Yx) animals learn more slowly over 3 days, they improve their relative deficit to controls from the start of training (“a”) to the end of training (“b”), which demonstrates learning. In contrast, mature lesioned animals (Mx) do not improve error-latency or relative impairment (compare “c” to “d”) with training, i.e. they did not learn. Consequently, the increased relative deficit between Mx and Yx animals (“a”–“c” vs. “b”–“d”) reveals an inability to learn gait synchronisation in the Mx group. Significant differences between Yx and control; #p < 0.05: significant differences between Mx and control; *p < 0.05, **p < 0.01. Reprinted from (10), Copyright (2005), with permission from Elsevier.

the relative motor deficit to the control group remains almost unchanged or increases (Fig. 5 points “c” to “d”; see Note 9). 2.3.2. Learning Motor Patterns

In addition, the cerebellum is also involved in spatial navigation (21, 23–26), specifically its role to learn the motor patterns required to make a direct path to a specific target and link them to inputs from the environment (21, 27). Since cerebellar lesions have less effect on swimming than on terrestrial locomotion (9), comparing swimming ability to visible vs. hidden platforms in a Morris water maze (28) adds further evaluation of cerebellar motor learning (but see Note 10). Equipment. The water maze is a circular pool (120 cm in ­diameter for rats, 90 cm for mice) filled with water (21°C; see Note 10a) and a clear 15-cm diameter Plexiglas escape platform is positioned in one quadrant (see Note 10b). The maze is located in a room with numerous extra-maze cues (24, 29–32), white noise and a videotracking camera above (Fig. 6; see Note 10b). Method. Swimming is evaluated both when the platform is ­hidden (learning motor patterns) and visible (capacity to orientate to a target). 1. The first tests are made with the platform submerged 2  cm below the water surface in a hidden platform test. The animal’s starting position is randomly selected from one of four entry points (N, S, E and W) and the animal is released facing the

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Fig. 6. Water maze. Learning of motor patterns can be tested in a Morris water maze (28). The circular pool is placed in a room with several extra-maze cues (to aid learning) and an overhead camera for recording swimming trajectory. To accommodate animal strains with poor visual acuity, the cues should be large shapes strongly contrasting to their background. The platform (grey dotted outline to indicate that it is transparent and therefore invisible to the animal) is initially hidden under the water. The animal is released from one of four starting points (N, S, E, and W) and uses the cues to find the platform.

pool wall and given 120 s to find the escape platform (33). The measured variables in each trial are (1) escape latency, (2) total quadrants crossed, (3) percentage of direct swims, defined as a swim path that did not deviate outside a 20-cm wide corridor from the rat’s entry to the platform (33, 34), and (4) success to locate the goal (see Note 10d). 2. Tests are repeated 5× per day for 6  days with at least 5  min between tests (see Note 10e). 3. Given that inability to find a hidden platform may be due to inability to swim to it, a second test is made, preferably using a set of animals from each group that have not learned the task, in which the platform is raised above the water level and a coloured flag attached to ensure visibility. Animals are released into the pool (as above) and undergo four training sessions (five trials/ session) to evaluate motor dysfunction in orientating and swimming to the platform. Measured variables are escape latency, total quadrants crossed and search score (see Note 10f).

3. Notes 1. Habituation

Although it is usual to allow a learning and habituation phase, it is important to know that doing this before any lesion/treat-

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ment will significantly affect the outcome (17, 35) as a key cerebellar function of motor learning will have already been activated. In our experience a calm, experienced tester can begin these simple tests with lesioned/treated animals, which are naïve to the motor tests. 2. Righting reflex

In the righting reflex, normal animals will turn in either direction with equal frequency, but often they will turn away from the tester or a bright light source. In order to avoid a false directionality of response, it is necessary to randomly change the orientation in which the animals are held, i.e. head to the left for one test and head to the right for another. 3. Vestibular drop

We have found that the vestibular drop test requires a few specific details: (a) It is important not to use tail suspension test equipment that is created for antidepressant function. In this commercially available apparatus the distal tail is held, from which position it is impossible for the animal to stabilise its base in order to turn and re-orientate itself. Also there is no “escape” onto which the animal can climb, so truncal coordination and strength cannot be tested. It is best to hold the tail about ⅓–½ its length; holding the base of the tail makes the task easier for the animal. (b) When testing adult rats it is advisable to wear gloves as the animals have to grip hard to climb onto the testers hand, this can scratch badly. (c) Although the upper time limit is set at 60  s, some really ataxic animals become quickly distressed and make violent circular movements in an attempt to right themselves. In our experience, animals that do this never succeed in the task and we stop it early and exclude these animals from the testing. 4. Footprints

(a) A newer version of this test can be made using a Catwalk XT (Noldus Information Technology, The Netherlands) which films the animal’s gait from below as it walks on a glass-floored corridor (36, 37). The advantage is that this provides dynamic investigation of gait beyond that available from foot-prints. However, it requires significant financial outlay for infrastructure (approximately €25 000). (b) We recommend placing the animals’ feet onto ink pads that are made for printing stamps as this limits the amount of ink on the feet – too much can be easily smudged by the tail, thereby distorting the data.

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(c) It is important that the animals are not frightened into running along the alley as this makes them rise onto their toes so the information about heel-strike and foot rotation is harder to see. (d) The acquisition of footprints is very quick, only needing a single test for each animal. However, the manual analysis is time-consuming and requires several consecutive nonsmudged footprints. 5. Crossing a bridge

There are several confounders to the bridge test: (a) Freezing. Ataxic animals which have fallen from the bridge tend to freeze soon after they start in order to avoid falling. The time spent stationary needs to be subtracted from the overall time to cross. (b) We find the animals need to be trained that there is a safe box across the bridge. This involves initially placing the animal on the bridge close to the box and showing them their safe goal. For young animals, placement of a littermate in the safe box speeds this learning. (c) Interest. Like many tasks, performance crossing the bridge depends on the animals’ motivation. Mice in particular start to cross the bridge, then stop and turn around. In these cases, the animal has strictly “failed” the test (crossing the bridge) so data should be excluded from the results; however data about the distance travelled in the upper time limit give an indication of ability and speed. (d) Sensitivity. In the analysis of data from this test, ability (% successful crossing) and time taken are not particularly discriminatory between groups as any animal whose foot-base approaches the width of the bridge is equally disabled (5, 10, 20). On the other hand, the number of hind foot slips and strategy used (crawling or walking) clearly differentiates groups (10). 6. Climbing a ladder

In practise, this test is not particularly discriminatory and will be affected in other non-cerebellar motor dysfunction (12). Moreover, the test is not suitable for mice which are much more likely to explore climbing around the sides and back of the ladder rather than just climb it! Such behaviour cannot be included in the data and wastes time. 7. Progression along a wire

(a) When presenting the animal to the wire it is important to release their body weight slowly so the animal takes its own weight. Letting go too quickly can cause the animal to fall

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straight away. This has two adverse effects; it confounds the data giving a falsely poor performance and teaches the animal that the consequence of a fall is not very serious, so those that find the task difficult tend to elect to drop off rather than struggle to complete the task. This is most noticeable with adult rats as they are relatively heavy for their fore limbs. (b) This variation of hanging from a wire is particularly discriminatory of ataxia as the ability to progress along the wire strongly correlates with the attachment of at least one hind limb (10), which in turn needs coordination of trunk and hind limb muscles. 8. Rotarod This apparatus is widely used for general sensorimotor testing, and will show abnormal results in many motor dysfunctions which have their origin outside the cerebellum. To test cerebellar function, there are specific parameters to note. (a) Although automatic time-to-fall is a useful measure for general motor ability, it is not a sensitive indictor of cerebellar function, which requires that the gait is synchronised to the speed of rotation. As rodents hate to fall, they will undertake whatever alternative strategies they can to avoid it; e.g., clinging to the rod and being passively rotated (very common) or turning around, lying with their abdomen in contact with the rod and shuffling backwards. Neither of these indicates correct cerebellar function yet will not be detected as “error” by automatic time-to-fall devices. (b) Although the accelerating rotarod is quick and simple, it takes only three trials per day for 5 days, it is a less sensitive assessor of cerebellar dysfunction than is the constant speed protocol. (c) Constant speed rotarod testing is given an upper time limit of 180 s because animals that can walk on the rod for this period are able to maintain their equilibrium for a much longer time (15–20  min; 16). However, when making between-group comparisons, setting an upper time limit can give a false indication of ability to do the task if one group reaches the limit and another has not (16). In this circumstance, the less able group will have reached their maximum performance, whereas the “good” group has been stopped prematurely. This will decrease the “real” intergroup differences and may mask statistically significant differences. (d) The number of trials varies with the experimental paradigm and information required. The accelerating protocol is performed three times per day. It is best to test all the animals

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for trial 1 before progressing to trial 2 as this increases the inter-trial interval to allow for recovery. This is important as the 5-min trial and fast speeds at the end can induce fatigue and falsely impair performance in later trials (11, 12). In the constant speed protocol, animals undergo 5–10 trials a day for 7 days, with at least a 3-min interval between each trial. This long time-consuming protocol allows performance to reach asymptote and the animal to learn the task (see Sect. 2.3). (e) The validity of the data is highly sensitive to animal motivation. There needs to be a clear balance between giving the animal enough trials to optimize their performance, without over-testing so that animals which can do the task get bored (with associated worsening performance) and those which cannot become frustrated. The latter is often demonstrated either as animals that jump off the rotarod immediately without trying to walk on it or through increasingly aggressive behaviour when the animals are brought to the testing area. If non-cooperation continues, testing should stop. 9. Motor learning

An example calculation for motor learning. In Fig. 5, control animals easily learn to walk on a rotarod turning at 10  rpm. Also young lesioned animals (Yx) can also improve their error latency from 116 to 170 s, but this takes 3 days of training. To assess whether these animals really learn the task, relative motor deficits ({control time − Yx time}/control time) are compared in untrained and trained animals. Comparison of error latency with controls reveals a relative motor deficit of 30% on day 1 (untrained, {166 − 116}/166 = 0.30; “a” in Fig. 5) in comparison with only a 6% deficit after 9 days training ({180 − 170}/ 180 = 0.057; “b” in Fig.  5), an improvement that indicates learning. In the mature lesioned animals (Mx), there is no evidence of learning with both error latency and the motor deficit relative to controls remaining similar before and after training (“c” vs. “d” in Fig. 5). Comparison of the two lesion groups emphasises the learning impairment of Mx animals. Compared to Yx, Mx animals relative performance deficit increased from 4% ({116 − 111}/116 = 0.043: “a” vs. “c” in Fig.  5) in untrained animals to 22% after training ({170 − 132}/170 = 0.22: “b” vs. “d” in Fig. 5) confirming that Yx animals learned but Mx did not. 10. Morris water maze

This apparatus is widely used for testing cognitive function, and mnemonic difficulties of any origin will affect this task.

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However, in an otherwise normal animal with only a cerebellar abnormality, it does give useful discriminative test of cerebellar cortical function in motor learning and allows discrimination between different regions of the cerebellum. Spatial learning (hidden platform protocol) involves the cerebellar hemispheres whereas visuomotor guidance to a visible target requires midline vermal function (38). (a) The relative water temperature can affect the results; it needs to be sufficiently cold to provide incentive to escape (39). (b) It is important that the escape platform is not visible to the animal as it swims. Also the animal must be visible to the camera/tester. To avoid putting things into the water (non-toxic paint, polystyrene beads, etc.), either the platform and pool must be painted the same colour (both black or both white) or the platform should be transparent. To accommodate both pigmented and albino animals, we use a pool made of grey polypropylene (standard in institution workshops or commercially available, e.g., Panlab/Harvard Apparatus) and a platform made of clear Plexiglas. (c) To avoid biasing the swim direction, the experimenter must either not be visible or always remain in the same fixed spatial relation throughout all testing (27, 40). (d) Cerebellar motor learning can be evaluated with the hidden platform and visible platform paradigms (39), although testing navigation skills usually includes a probe test (two trials without the escape platform) and a retrieval test 7 days later (four trials with the hidden platform in its original position). These are not described in this chapter on motor control. (e) On each day, between trials the animals are dried and placed in warm boxes. These need to be to one side so that the animals cannot watch the others perform. Spatial learning tasks can be learned by observation in both animals (41, 42) and humans (43), which may confound the results, wherein improvement of the task (apparent “motor learning”) may be completely unrelated to cerebellar motor control. (f ) Increased time taken or quadrants crossed indicates motor dysfunction. However, even very ataxic animals, which are  unable to learn a motor pattern to solve the hidden maze, can usually orientate and swim to a visible platform (20, 21, 24, 33).

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36. Cendelin J, Voller J, Vozeh F (2010) Ataxic gait analysis in a mouse model of the olivocerebellar degeneration. Behav Brain Res 210:8–15 37. Chen X, Kovalchuk Y, Adelsberger H, Henning HA, Sausbier M, Wietzorrek G, Ruth P, Yarom Y, Konnerth A (2010) Disruption of the olivo-cerebellar circuit by Purkinje neuron-specific ablation of BK channels. Proc Natl Acad Sci USA 107:12323–12328 38. Joyal CC, Meyer C, Jacquart G, Mahler P, Caston J, Lalonde R (1996) Effects of midline and lateral cerebellar lesions on motor coordination and spatial orientation. Brain Res 739:1–11 39. Martin LA, Goldowitz D, Mittleman G (2003) The cerebellum and spatial ability: dissection of motor and cognitive components with a mouse model system. Eur J Neurosci 18:2002–2010 40. Le Marec N, Dahhaoui M, Stelz T, Bakalian A, Delhaye-Bouchaud N, Caston J, Mariani J (1997) Effect of cerebellar granule cell depletion on spatial learning and memory and in an avoidance conditioning task - studies in postnatally x-irradiated rats. Dev Brain Res 99:20–28 41. Graziano A, Leggio MG, Mandolesi L, Neri P, Molinari M, Petrosini L (2002) Learning power of single behavioral units in acquisition of a complex spatial behavior: An observational learning study in cerebellar-lesioned rats. Behav Neurosci 116:116–125 42. Leggio MG, Molinari M, Neri P, Graziano A, Mandolesi L, Petrosini L (2000) Representation of actions in rats: The role of cerebellum in learning spatial performances by observation. Proc Natl Acad Sci USA 97:2320–2325 43. Torriero S, Oliveri M, Koch G, Lo Gerfo E, Salerno S, Ferlazzo F, Caltagirone C, Petrosini L (2011) Changes in Cerebello-motor Connectivity during Procedural Learning by Actual Execution and Observation. J Cog Neurosci 23:338–348

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Chapter 15 Cerebellum and Classical Conditioning Richard F. Thompson Abstract More is known about the neuronal basis of classical conditioning of eyeblink and other discrete responses than for any other aspect of learning. The cerebellum and its associated afferent and efferent circuits are the essential neuronal substrates for this basic form of associative learning and memory. Key methods, procedures, and issues for classical conditioning of the eyeblink response in rabbits, humans, mice, and rats are reviewed. Key words: Cerebellum, Interpositus nucleus, Eyeblink conditioning, Nictitating membrane, Conditioned response, Unconditioned response, Alpha response

1. Introduction Classical conditioning of the eyeblink response is quite possibly the best understood form of basic associative learning and memory in terms of neuronal substrates. For standard conditions, where the conditioned stimulus (CS) precedes and overlaps with the unconditioned stimulus (US), the entire essential circuit has been identified, including the CS pathway, the US pathway, and the conditioned response (CR) pathway. Neuronal plasticity occurs in the cerebellar cortex and the cerebellar interpositus nucleus and evidence strongly supports the view that the essential memory trace is formed and stored in the interpositus nucleus (1). These same results apply to the conditioning of any discrete response learned with an aversive US, e.g., limb flexion, head turn, etc. and apply to all mammalian species studied, to the extent tested, including humans. Here we focus on procedures and methods of measurement and on the nature of the behavioral responses.

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2. A Bit of History The first eyeblink conditioning study was by Cason in 1922 (2). He arranged a mechanical coupling attached to the subject’s eyelid to break a contact that controlled a clock, providing a response latency measure, but no measurement of the actual eyeblink response. He used an auditory CS paired with an orbital shock US in humans. In early studies, the US was sometimes a slap in the face, using a little padded hammer (3). Precise measurement of the behavioral response was first achieved by Hilgard in 1931 (4) using a photochronograph. An artificial eyelash of stiff paper was attached to the eyelid and cast a shadow on a free falling photographic plate. Spence and Taylor (5) in 1951 first used a lightweight rotary potentiometer connected to the eyelid with a thread, an amplifier and an inkwriter to obtain detailed records of eyelid movements. This became a standard method in the field for both human and animal studies. The first infrahuman animal studies were by Hilgard and Marquis in 1935 and 1936 (6, 7), where they conditioned four dogs and four monkeys with a light CS and a corneal airpuff US. In the early 1960s, Isadore Gormezano, then at the University of Indiana, introduced the rabbit as an ideal preparation, focusing on extension of the nictitating membrane response, as did Alan Wagner, at Yale, using external eyelid closure. Like humans and dogs but unlike mice and rats, rabbits tolerate restraint well.

3. The Reflex Eyeblink Response (UR)

Since so much work on the neural substrates has been done on the rabbit, we focus on this preparation. First a few words about the reflex response. It is a coordinated response involving simultaneous and perfectly correlated external eyelid closure and eyeball retraction controlled by the retractor bulbus muscle with resulting passive extension of the nictitating membrane (NM) response (e.g., (8, 9)). With a corneal airpuff US, the onset latency of the NM extension is a minimum of about 25 ms (with a 3-psi air pressure). In a very careful study by Evinger and associates (10), using guinea pig with electrical stimulation of the supraorbital branch of the trigeminal nerve as a US and recording the EMG activity of the orbicularis oculi (eyelid muscle), there were two response components: the first having a latency of 7–9 ms. and the second from 17 to 19 ms. With mechanical recording of the NM extension resulting in a slight lag, and the use of an airpuff with a several microseconds rise time (which smears the response in to one component), these data are consistent. In terms of reflex paths, there are direct

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projections from neurons in regions of the trigeminal nucleus to the accessory abducens and abducens nuclei (innervating the retractor bulbus muscle) and the facial nucleus, controlling the external eyelid (11, 12), as well as indirect projections relaying via the brainstem reticular formation, at least to the facial nucleus (13). In humans and other primates, the NM is vestigial; the eyeblink response is generated strictly by external eyelid closure controlled by the orbicularis oculi muscle innervated by the facial nerve. Hence, all methods of recording involve measurement of the eyelid response and/or EMG activity of the orbicularis oculi muscle. Methods and procedures for eyeblink conditioning, with an emphasis on rabbit, are given in great detail and in elegant fashion in the Handbook of Classical Conditioning, written by Lavond and Steinmetz (14), see Chaps. 2–5, covering stimulus presentations, measurement of behavioral responses, recording neuronal data and computer analysis of data. Here I will simply note a few aspects of our procedures and results that I feel are particularly relevant. In rabbit we have typically used measurement of extension of the NM, by suturing a small loop of thread to the outer edge of the NM and connecting it to a rotary minipotentiometer, delivering a DC voltage to our computer system. The rabbit is held in a standard restraint box and the US is typically a 3-psi air puff delivered from a nozzle a few cm from the eye. The external eyelids are often held open so extension of the NM does not alter the properties of the US.

4. The Conditioned Eyeblink Response (CR)

Gormezano et al. (8) showed in separate studies in the rabbit that learning of eyeball retraction, NM extension, and external eyelid closure all had essentially identical acquisition functions. Simultaneous recording of NM extension and external eyelid closure during acquisition and extinction showed that they were, in essence, perfectly correlated – both within trials and over training (9, 15). They are all components of the same global CR. It has been suggested that different motor nuclei might somehow exhibit different CRs in eyeblink conditioning (16). This possibility is not supported at all by the evidence. The CR and the UR are similar in the sense that to a large degree the same muscles and motor nuclei are engaged in both. However, they differ fundamentally in a number of respects (17). The minimum onset latency of the CR to a tone in well-trained rabbits, measured as NM extension, is about 90–100 ms; the minimum onset latency of the NM extension UR to a 3-psi corneal airpuff is about 25 ms. Perhaps most important, the variables that determine the topographies of the UR and the CR are quite different.

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The topography of the UR is under tight control of the properties of the unconditioned stimulus, e.g., stimulus intensity, rise-time, and duration. In marked contrast, the topography of the CR is largely independent of the properties of the US and is determined primarily by the CS–US onset interval – the CR peaking at about the onset of the UR over a wide range of CS–US onset intervals. The degree and speed of learning is also under control of this CS–US onset interval. For rabbits, best learning occurs with an interval of about 200–500  ms between CS onset and US onset. The best interval range for humans is somewhat longer. This key property of the CR cannot be derived from the properties of the US or the UR.

5. Alpha/Startle Responses These are responses that are elicited by the onset of the CS without training and are unlearned. The alpha response was first noted in humans by Hilgard in 1931 (4). It has a much shorter onset latency than the learned CR and can so be distinguished. A bright light CS elicits an alpha response in most mammals, including rabbits. However, a tone with a several microseconds rise time does not elicit such responses in rabbits (it is necessary to turn on a tone CS with an electronic switch to avoid the onset click that can elicit an alpha response). Use of a tone CS can be a problem in mice. A tone CS at the best frequency range for mice, around 8 kHz, will elicit a startle response – a “jump” – that includes an unlearned eyeblink response (an alpha response) even with a several microseconds rise time. Better is to use a lower frequency tone of 1–2  kHz which mice can hear but which does not elicit startle/alpha responses. A  most unfortunate example of this was published by Koekkoek et al. (18). Using mice, following a cerebellar lesion (Interpositus nucleus) that we and all others found to abolish the eyeblink CR, they reported the occurrence of CRs. However, the CRs they reported had onset latencies of about 20 ms and were clearly unlearned alpha/startle responses and not CRs. When using mice, one must always test the CS to be used in advance to avoid this problem. Use of lower intensities, lower frequencies, and a several microseconds rise time are all helpful.

6. Use of Mouse and Rat Unlike rabbits and humans, mice and rats do not tolerate restraint well at all. Indeed, a standard method to induce substantial stress in these animals is simply immobilization. Consequently, methods have been developed to train these animals while they are freely

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moving. Skelton (19) first developed the method in rats, Stanton et al. (20) adapted it to infant rats and then to mice (21). In brief, four insulated electrode wires are implanted in the orbicularis oculi muscle (controlling the eyelid response) dorsal to the eye and connected to a small head stage fixed to the skull. Two electrodes record the EMG and two are used to deliver the shock US. It is necessary to blank the amplifier during the actual delivery of the brief shock train but the remainder of the UR after the shock has ended can be recorded (see (22) for details). We have found that the US intensity should be adjusted so that it elicits a brief head-turn response to obtain best learning. Because of the extraordinary advances in genetics, the mouse has become a favorite preparation in biology. In the case of basic associative learning, particularly eyeblink conditioning, mutant and transgenic mice have added greatly to our knowledge (see e.g., (23)). Unfortunately, confusion has been introduced in the field in a paper by Koekkoek et al. (24) claiming that the EMG measure of the eyeblink response in mouse had problems. They reported that their EMG measure (taken from the orbicularis oculi muscle ventral to the eye) was contaminated by other muscle actions, e.g., those controlling the upper lip and vibrissa. Skelton ((19); personal communication) examined various recording loci from the orbicularis oculi muscle and found that recordings from regions other than dorsal to the orbit (e.g., ventral to the orbit) were contaminated by adjacent muscle actions due to sniffing and jaw movements. Freeman (personal communication) found that when stimulating the cerebellar nuclei, EMG responses dorsal to the orbit occurred only when lid movements were elicited and not during nose twitches, vibrissae, jaw movements, head turns, etc. In short, dorsal recordings are not contaminated at all by other muscle actions but ventral recordings are. The Koekkoek et al. (24) objection to EMG recordings applies only to their ventral recordings, not to dorsal recordings.

7. Non-invasive Measures of Eyelid Closure

In the past few years, several methods have been developed to measure closure of the external eyelids non-invasively. This is particularly useful not only for humans but also for other primates. In my view the best of these was developed by Disterhoft and associates at Northwestern University (25). In brief, an infrared beam is shined on the eye by an LED and lenses. The amount of reflected light is converted to a DC voltage signal by a phototransistor, thus providing a measure of eyelid closure (the translucent surface of the cornea reflects less light than the opaque eyelid or, in rabbit, the NM). This method has been used with great success in humans, rabbits and rats. Indeed, this and similar methods are now standard for use in humans.

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References 1. Thompson RF, Steinmetz JE (2009) The role of the cerebellum in classical conditioning of discrete behavioral responses. Neuroscience 162:732–755 2. Cason G, (1922)The conditioned eyelid reaction. J Exp Psychol 21:153–195. 3. Switzer SA (1930) Backward conditioning of the lid reflex. J Exp Psychol 13:76–97 4. Hilgard ER (1931) Conditioned eyelid reactions to a light stimulus based upon the reflex wink to a sound. Psychol Monogr 41: No 184. 5. Spence JT, Taylor JA (1951) Anxiety and strength of the UCS as determiners of the amount of eyelid conditioning. J Exp Psychol 42: 183–188. 6. Hilgard ER, Marquis DG (1935) Acquisition, extinction, and retention of conditioned lid responses to light in dogs. J Comp Psychol 19:29–58. 7. Hilgard ER, Marquis DG (1936) Conditioned eyelid responses in monkeys with a comparison of dog, monkey and man. Psychol Monogr 47:186–198 8. Gormezano I, Schneiderman N, Deaux E, Fuentes I (1962) Nictitating membrane: classical conditioning and extinction in the albino rabbit. Science 136:33–34. 9. McCormick DA, Lavond DG, Thompson RF (1982) Concomitant classical conditioning of the rabbit nictitating membrane and eyelid responses: correlations and implications. Physiol Behav 18:769–775 10. Pellegrini JJ, Anja K, Horn E, Evinger C (1995) The trigeminally evoked blink reflex. I. Neuronal circuits. Exp Brain Res 107:166–180 11. Cegavske CF Harrison TA, Torigoe Y, (1987) Identification of the substrates of the unconditioned response in the classically conditioned rabbit nictitating membrane response pre­ paration. In: I Gormezano, WF Prokasy, RF Thompson (eds), Classical Conditioning. Lawrence Erlbaum, Hillsdale NJ 12. Berthier NE, Desmond JE, Moore JW (1987) Brainstem control of the nictitating membrane response . In: I Gormezano, WF Prokasy, RF Thompson (eds), Classical Conditioning. Lawrence Erlbaum, Hillsdale NJ 13. Tamai Y, Iwamoto M, Tsujimoto T (1986) Pathway of the blink reflex in the brainstem of the cat: Interneurons between the trigeminal nucleus and the facial nucleus. Brain Res 380:19–25 14. Lavond DG, Steinmetz JE (2010) Handbook of Classical Conditioning. Kluwer Boston

15. Lavond DG, Logan CG, Sohn JH, Garner WD, Kanzawa SA (1990) Lesions of the cerebellar interpositus nucleus abolish both nictitating membrane and eyelid EMG conditioned responses. Brain Res 514:238–248 16. Delgado-Garcia JM, Evinger C, Escudero M, Baker R (1990) Behavior of accessory abducens and abducens motoneurons during eye retraction and rotation in the alert cat. J Neurophysiol 64:413–422 17. Gormezano I, Kehoe EJ, Marshall-Goodell BS (1983) Twenty years of classical conditioning research with the rabbit. In: JM Sprague, AN Epstein (eds) Progress in Physiological Psychology. Academic Press, New York. 18. Koekkoek SKE, Hulscher HC, Dortland BR, Hensbroek RA Elgersma Y, Ruigrok TJH De Zeeuw CL (2003) Cerebellar LTD and learning-dependent timing of conditioned eyelid responses. Science 301:1736–1739 19. RW (1988) Bilateral cerebellar lesions disrupt conditioned eyelid responses in unrestrained rats. Behav Neurosci 102:586–590 20. Stanton ME, Freeman JH Jr, Skelton RW (1992) Eyeblink in the developing rat. Behav Neurosci106:657–665 21. Aiba A, Kano M, Chen C, Stanton ME, Fox GD, Herrup K, Zwingman TA, Tonegawa S (1994) Deficient cerebellar long-term depression and impaired motor learning in mGluR1 mutant mice. Cell 79:377–388 22. Chen LBao S, Lockard JM, Kim JK, Thompson RF (1996) Impaired classical eyeblink conditioning in cerebellar lesioned and Purkinje celldegeneration mutant mice. J Neurosci 16:2829–2838 23. Kim JJ, Chen L, Bao S, Sun W, Thompson RF (1996) Genetic sissections of the cerebelar circuitry involved in classical eyeblink conditioning. In: S Nakanishi, AJ Sil va, S Aizawa, M Katsuki (eds). Gene Targeting and New Developments in Neurobiology, Japan Scientific Societies, Tokyo 24. Koekkoek SKE, Den Ouden WL, Preey G, Highstein SM, De Zeeuw CL (2002) Monitoring kinetic and frequency-domain properties of eyelid responses in mice with magnetic distance measurement techniques. J Neurophysiol. 88:2124–2133 25. Thompson LT, Moyer JR Jr, Akase E, Disterhoft JF (1994) A system for quantitative analysis of associative learning Part I. Hardware interfaces with cross-species applications. J Neurosci Meth 54:109–117

Chapter 16 Assessments of Visual Function Ma’ayan Semo, Carlos Gias, Anthony Vugler, and Peter John Coffey Abstract The visual system is the part of the central nervous system that detects light. It is sub-served by the ­photoreceptor detectors within the eye, and this information allows creatures to build a representation of the visual world as well as regulating a whole range of subconscious physiological processes. Here we describe seven techniques to probe the functionality of the visual system in rodents. They encompass direct electrical recordings and functional anatomy, as well as reflexes and behavioural response outputs of light detection. Used in concert, these techniques can enable the assessment of which photoreceptor classes are functioning as well as to indicate whether light information is processed at a sub-cortical and/or cortical level. Key words: Retinal degeneration, ERG, Optokinetic, Circadian, Optical imaging, Visual cortex, Visual function

1. Introduction The visual system is the part of the central nervous system that detects light. It is sub-served by the photoreceptor detectors within the eye, and derives information that allows creatures to build a representation of the visual world as well as to regulate a range of subconscious physiological processes. The eye has direct connections to many brain nuclei/regions; in the rodent these include: the hypothalamus [suprachiasmatic nucleus (SCN)], the mid-brain [superior colliculus (SC)] and the thalamus (lateral geniculate nucleus (LGN) and pretectum). The geniculate nucleus is then connected to the visual cortex (VC). The visual system is often divided into two pathways, image forming and non-image forming. Image-forming pathways enable organisms to build a visual

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representation of the world and are concerned with the spatial location of light or radiance. Non-image forming pathways are those that are concerned with measuring the overall levels of light or irradiance, regulating functions such as the pupillary light reflex and circadian photoentrainment. There are three classes of photoreceptors within the mammalian retina, rods, cones and intrinsically photosensitive melanopsin ganglion cells. Rods have the highest sensitivity, operating in the lowest light levels (scotopic). Cones are less sensitive but due to light adaptation are able to operate over a broad range of lighting conditions to support image-forming vision (mesopic to photopic). The melanopsin cells are the least sensitive, encoding high irradiance signals in order to support non-image-forming responses (Fig. 1). The photoreceptor classes also vary in the wavelengths of light to which they are most sensitive based on the opsin photopigment that they express. In rodents, rods and melanopsin cells express one type of opsin, whereas there are two types of opsin that can be expressed by cones, a short wavelength and a long wavelength sensitive one. The peak sensitivities (lmax) for opsins in rats and mice are summarized in Table 1. In this chapter, we describe seven methods to probe the functionality of the visual systems in rodents. Together, these approaches can allow one to elucidate the photoreceptive systems that are functioning, and also gain insight into whether light is perceived at both cortical and sub-cortical levels (Table 2).

Fig. 1. Approximate operating ranges for rods, cones and melanopsin ganglion cells (GCs). The high sensitivity rod pathway saturates at relatively low light levels; however, recent work suggests that in light-adapted conditions new undefined rod pathways can play a role in non-image forming responses at high irradiances (indicated by the dashed green line) (1).

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Table 1 Peak sensitivity of rodent photoreceptor opsins Opsin

Peak sensitivity (nm)

Reference

Rod

498 (rats, mice)

(2)

Long wavelength cone

508 (mice), 509 (rats)

(3, 4)

Short wavelength cone

360 (mice), 358 (rats)

(5, 6)

Melanopsin

479 (mice), 484 (rats)

(7, 8)

Table 2 Summary of the visual system brain regions that are involved in, or that can be recorded by, each technique Visual system region Retina Midbrain

Hypothalamus/ Visual thalamus cortex

Electrical recordings

+

+

Optical imaging

+

Functional anatomy

+

Wheel running

+

Pupillometry

+

+

+

Optokinetic head-tracking

+

+

+

+a

Behavioural light aversion

+

+

+

+

+

+ +

+

+

+

+

 This response can be enhanced by involvement of the visual cortex (9, 10)

a

2. Electrical Recordings of the Visual System

2.1. Electro­ retinography

Sensory information in the nervous system is processed and transmitted through electrical signals. Electrophysiological techniques aim to measure these signals using different visual stimulation protocols at various locations and spatial scales to understand the function of the visual system. Most components required to record electrical signals are the same across various electrical visual function modalities. Electroretinography (ERG) is a well-established non-invasive technique used to diagnose patients with retinal degeneration or in animal studies. When a light stimulus impinges on the retina, a flow

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of extracellular current is initially generated at the photoreceptors and subsequently in other retinal cell types. Given the parallel arrangement of the retinal layers, extracellular currents will only be aggregated in the radial direction while lateral currents will be cancelled out. This current can be measured using extraocular electrodes in the form of a small potential and it is commonly known as the electroretinogram (ERG). Therefore, while the ERG originates from many different cells types and layers within the retina it is represented by a single electrical trace. The ERG trace is made of a combination of signals giving rise to several characteristic peaks and troughs within the initial hundreds of milliseconds after stimulus onset (Fig. 2). These characteristic features of the ERG response are used as indicators to estimate the functional condition of the retinal cells that give rise to them. We only describe here those components that are most commonly used in rodent studies. 1. The a-wave Following light absorption by the photoreceptor outer segments a chain reaction is initiated leading to the neurotransmitter release in their synaptic terminals. Blocking synaptic transmission using l-glutamate suppresses subsequent neural activity while leaving intact the photoreceptor response. Under this condition, a negative potential is generated in the initial tens of milliseconds following stimulus onset, while the subsequent

Fig. 2. ERG components: a-wave, b-wave, and oscillatory potentials (OPs). Top trace shows an ERG recording from a mouse following a single flash of broad spectrum light in a dark adapted condition. Medium trace shows the same ERG response as in the top picture after filtering out the OPs. Bottom trace shows the OPs obtained from the top trace after filtering out the a-wave and b-wave components.

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positive-going signal is abolished. The a-wave component corresponds to this initial negative component of the ERG trace and is therefore considered to be a direct reflection of photoreceptor activity. 2. The b-wave Following the a-wave, a positive signal appears in the ERG trace reaching a maximum before it declines. This component of the ERG is called the b-wave. The b-wave is abolished in mGluR6 knockout mice (11) or by pharmacologically blocking (12) the mGluR6 receptor. Given that mGluR6 receptors are specific to ON-centre bipolar cells, the b-wave is considered to be generated by this type of cell. Additional studies have also shown that the amplitude and latency of the b-wave is modulated by third order neurons (13). 3. Oscillatory potentials At bright stimulus intensities, the ERG trace shows high frequency wavelet components in the 100  Hz range on the ascending part of the b-wave known as the oscillatory potentials (OPs). The OPs can be separated from the a-wave and b-wave by using a band-pass filter between ~80 and 150 Hz. Dopaminergic amacrine cells appear to be involved in generating this component of the ERG and it appears to be affected in retinal degenerations both in human and rat models of retinal disease. 2.2. Brain Electrophysiology

1. Visual Evoked Potential (VEP) Visual-evoked potentials are a gross measure of neural activity. Although borders of the active region cannot be clearly defined using this technique, the amplitude and latency of the response have been used clinically to assess visual function. Since the visual cortex lies at the surface of the brain, an evoked electrical response can be detected non-invasively simply by placing a low impedance electrode on the surface of the skull or epidurally. The ability of the visual system to resolve a certain type of visual stimulus at the level of the visual cortex can then be assessed using VEPs (14). As in the ERG, a judicious choice of visual parameters can be used to determine the cortical response of the different visual pathways. 2. Extracellular electrophysiological recordings Electrophysiological responses at the cellular level can be examined by inserting a small electrode made of metal (tungsten or platinum-iridum) insulated with thin glass into the brain. The tubing is filled with an electrolytic solution to allow electrical contact between the neural tissue and the electrode. There are also metal electrodes with a small conductive area that can be directly inserted in the brain without the glass tubing. There are commercial electrodes that allow multiple recordings across

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its length (channels) with the additional option of arranging several of them in a spatial array (Neuronexus, Ann Arbor, MI, USA). Extracellular recordings give an indication of the electrical activity of surrounding neural tissue. 3. Local field potential As a result of an action potential, the presynaptic neuron releases glutamate at a synapse onto the postsynaptic neuron. This leads to the opening of glutamate receptor channels resulting in a net flow of current inside the neuron. An extracellular electrode detects this as a negative potential. A nearby electrode of approximately 1 MW impedance is able to measure this potential known as the local field potential (LFP). It is generally assumed that the LFP reflects the incoming electrical signal and local neural processing of tissue up to a couple of millimetres surrounding the electrode (15). In order to extract this signal, a band-pass filter between ~1-300 Hz is applied to the electrical recording. Figure 3 shows an example of the cortical recordings taken from an electrode with 16 different channels. 4. Single unit and multiunit activity Multiunit activity (MUA) is believed to reflect the aggregate spiking activity of neurons within approximately a few hundred micrometers surrounding the electrode (15). The same electrophysiological recording taken to measure the LFP

Fig. 3. Local field potential (LFP) recordings from the rat visual cortex using a 16-channel electrode following a single flash of broad spectrum light.

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can be used to study MUA by simply using a band-pass of ~300–3,000 Hz. Furthermore, the spiking activity of individual neurons can be estimated by assigning them the detected spikes based on the spike characteristics (spike sorting). 2.3. Materials

1. Fixed frame A stable mechanical support is required to avoid vibrations from surrounding equipment or natural movements such as respiration that could introduce noise in the recordings or even break the electrode. A nose clamp and/or ear bars are ideal to fix the head of the animal. Specifically, brain electrical recordings require the accurate location of visual structures with respect to some visual landmarks and a stereotaxic frame is used for this purpose (Harvard Apparatus, Kent, UK). 2. Electrode An electrode is an electrical conductor that provides a path for electricity to enter or leave a medium. The shape of the electrode depends on the visual structure that is to be measured. The electrical connection between the electrode and the amplifier should be kept at a minimum distance and away from potential sources of interference, since any noise introduced at this point will be subsequently amplified. In order to reduce interference with the mains, the body of the animal (e.g., the base of the tail) should be connected to the ground of the amplifier. The use of a Faraday cage should be considered as an option to block environmental electromagnetic noise. Custom-made electroretinogram (ERG) electrodes can be manufactured using simple components. Stainless steel or gold wire electrodes with a round shape or saline-soaked cotton-wick electrodes can be used to measure the ERG. Alternatively, ERG electrodes are also commercially available (LKC Technologies, Gaithersburg, MD, USA). It is important to maintain a good electrical connection at all times by applying a transparent electrolytic solution such as methylcellulose between the cornea and the electrode. 3. Voltage amplifier The analogue electrical signal measured is generally very small and needs to be amplified (103–104) with voltage amplifiers in the order of volts before it can be digitized (Digitimer, Letchworth, UK). 4. Signal filtering The aim of filtering is to remove the unwanted noise from the signal. Very low frequencies (drift) are usually removed before digital conversion to prevent the signal drifting out of the desired voltage range. Unnecessary high frequencies can also be removed at this point by low pass filtering (Digitimer, Letchworth, UK).

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5. Analogue-to-digital converter (ADC) The electrical signals need to be converted from an analogue to a digital signal so that the voltage can be interpreted by a computer. It is important to make sure that the sampling rate is at least twice the size of the maximum frequency of the signal of interest to avoid aliasing. Current commercial ADCs also have a resolution of at least 16-bits which allows for a wide range of voltage levels to be measured (CED, Cambridge, UK). 6. Digital signal processing In order to increase the signal to noise ratio, the same visual stimulus is generally presented several times and averaged at corresponding time steps. Further processing steps such as discrete Fourier analysis might also be carried out to highlight the frequency components of interest. 2.4. Methods 2.4.1. Electroretinography

Given that different cells respond preferentially to a set of stimulus conditions, visual parameters and presentation protocols should be chosen according to the retinal cell type and visual pathway that we want to examine. Table  3 shows a range of stimulus options grouped under different criteria. While there are a large number of stimulus combinations that can be used, we only present here an overview of the most common in rodent studies. 1. Flash ERG The use of a single full-field flash of broad spectrum (white) light at various intensity levels and pre-adaptation to light (light adaptation level) is commonly used both in human and rodent ERG. The strengths of this method lie in its simplicity and ability to give an indication of visual function from photoreceptors and various cell types of the neural retina. While there are international guidelines for human ERG recordings to this type of stimulation, no such standard exist in animal

Table 3 Visual stimulation ERG modalities Light intensity

Spectral properties

Spatial luminance pattern

Scotopic range

Broad spectrum

Flash ERG

Photopic range

Narrow spectrum

Pattern ERG

Number of flashes

Adaptation to light

Single flash

Dark adaptation

Double flash

Light adaptation

Flicker

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studies including the rodent, and recordings are taken with a much wider range of stimulus conditions. The flash ERG stimulus is ideally spanning the entire visual field of the animal by means of a Ganzfeld although the use of a diffuser or a fibre optic near the eye is also common. It is important to control the light level over a wide range of intensities and limit the duration of the stimulus to the shortest time possible, and no longer than the integration time limit of the photoreceptors (approximately 15 ms for cones and 100 ms for rods). In order to test rod function at low intensity levels, the rodent needs to be dark adapted for a prolonged period of several hours and the flash of light needs to be presented at increasing intensity levels. At the higher intensities, ERGs will be generated from a combination of rod and cone function. In order to selectively test cone function, visual stimuli can be delivered after a period of light exposure at a rod-saturating level (approximately 5–30 cd/m2) of at least 10 min which is deemed to be sufficient in order to render the retina into a light-adapted state. Using this type of stimulus the a-wave, b-wave and oscillatory potentials can be extracted from the ERG. 2. Flicker ERG Given that cones are capable of regenerating at a much faster rate than rods, a stimulus presented at a high rate will be mainly a reflection of cone activity. The response has the resemblance of a sinusoidal signal at rates higher than 3 Hz and individual components of the ERG can no longer be identified. Instead, the amplitude and phase of the response at the stimulus frequency is useful in determining cone physiology. The amplitude of the response is reduced at high flicker frequencies until it is no longer distinguishable from noise (critical flicker frequency). 2.4.2. Brain Electrophysiology

Visual stimulation described in Table  3 for ERG recordings are also applicable to any of the electrophysiological recording modalities in the brain. Extracellular recordings of the superior colliculus (SC), lateral geniculate nucleus (LGN) or visual cortex (VC) are taken using stereotaxic coordinates as the reference followed by post-mortem confirmation of the electrode position. By sampling the region of interest at regular intervals using various visual stimulus parameters and, by combining these responses, one can examine the properties of individual neurons or small clusters of neurons (16). The superior colliculus is a particularly important visual structure in the rodent given that over 70% of retinal ganglion cells (RGC) project to the superficial layers of the superior colliculus and that it contains a retinotopic map of visual space. Therefore, electrophysiological recordings of the SC give an accurate description of RGC output. The VC in the rodent also contains a topographic representation of visual space and is crucial for high visual acuity given its ability to resolve high spatial frequencies.

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3. Optical Imaging of Intrinsic Signals There is a close link between the electrical activity of a population of neurons and the underlying metabolic activity. Local changes in electrical activity result in local changes in blood volume, blood oxygenation, and light-scattering properties of tissue. These phenomena can be exploited to determine the location of cortical activity by measuring the stimulus-evoked changes in tissue reflectance (Fig.  4). The technique used to measure these intrinsic changes is known as optical imaging of intrinsic signals (17). It offers the possibility of simultaneously sampling a substantial area of the rodent cortex (several millimetres) with good spatial resolution (mm range) and temporal resolution (peak changes within several seconds of stimulus onset). 3.1. Materials

1. Camera A camera with a good signal to noise ratio is required to measure changes in the signal in the order of 0.1–1% with respect to baseline. A high quality CCD camera (DALSA, Billerica, MA, USA) with an analogue-to-digital converter of no less than 10-bits resolution per pixel will achieve this. The camera should be able to capture images synchronized with the visual stimulus (NI LabView, Newbury, UK) at a constant rate of at least 2 Hz for an extended period of time in order to capture the temporal profile of the response. The optical imaging response returns slowly to baseline and consequently it is also important to avoid inter-stimulus intervals shorter than 15 s.

Fig. 4. A schematic of the optical imaging set up.

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2. Lens system It is important to focus below the cortical surface (~500– 700 mm) and collect images at a shallow depth of field in order to reduce blood vessel artefacts in the functional maps. It is also necessary to collect images with a field of view large enough to include the brain structures of interest (several millimetres) at good spatial resolution (~10–50  mm/pixel) and with a long enough working distance to allow proper alignment of light illumination (more than 3 cm). The use of a macroscope made of a tandem-lens combination of two 50-mm camera lenses or a 50  mm with a 135-mm lens will fulfil all these requirements. 3. Cortical illumination The light source to illuminate the cortex has to provide the intensity, stability and wavelength required. Tungsten halogen lamps (Newport, Irvine, CA, USA) powered by a stabilized power supply are suitable for this purpose. A light guide is sufficient to achieve certain uniformity in the illumination over the cortex. Interference filters (Edmund Optics, York, UK) are interposed between the light source and the light guide to deliver narrow band in the 570–730 nm range. 3.2. Methods

Urethane on its own or in combination with other drugs (e.g., ketamine) is generally administered to the rodent in optical imaging experiments since it provides a stable level of anesthesia for a prolonged period of time. Barbiturates and gas anesthetics (e.g., isoflurane) have also been used in optical imaging although it is important to bear in mind that the effects of anesthetics on neurovascular coupling may be different between species and strains. Mucus secretions can make breathing difficult and a subcutaneous injection of atropine has been routinely carried out in the past. However, atropine can affect pupil dilation and recent studies have shown that it can also affect retinal activity (18), so it is advisable to avoid its use in the future. Instead, performing a tracheotomy which is a relatively fast and simple procedure can facilitate breathing and provide a route for artificial ventilation. The physiological condition of the animal should be monitored and controlled whenever possible. Maintaining core body temperature constant for the duration of the experiment at approximately 37°C is critical and a regulated heating pad using a rectal thermoprobe should always be used. A pulse oxymeter for rats and mice has been recently made commercially available (Starr Life Sciences, Oakmont, PA, USA) and monitors relevant physiological parameters such as heart rate, breathing rate and blood oxygenation non-invasively. This device is highly recommended since it avoids difficult artery cannulation in such small species. While large species require the opening of the skull, this is not required in rodents. Optical imaging can be performed in rats by thinning the skull and in the case of mice by leaving

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the skull intact. In order to increase the translucency of the skull, silicon oil or saline should be applied and maintained for the duration of the experiment. Optical imaging measurements are generally taken at a single wavelength in order to emphasize a specific component of the reflected light changes: blood volume (570 nm), blood oxygenation (605  nm) or scattering (>650  nm). Optical imaging applications to study visual function in rodents have been comparatively less used than other electrophysiological techniques. High contrast gratings presented at various locations of the visual field have been used to study the retinotopic organization of the visual cortex (19). A high contrast thin bar moving at constant speed across the visual field in different directions has also proved to be efficient for this purpose (20) as well as for the study of the plastic properties of the visual cortex (21). Prolonged periods of spatially uniform stimulation have also been shown to elicit strong cortical responses (22). In order to identify the region of activity, an initial ratio analysis is first carried out by removing and dividing a reference image collected previous to stimulus onset, from the images acquired after stimulation to normalize for uneven illumination. A general linear model has also been used to fit the measured signal to a temporal model of the response and noise, and subsequently calculate the map of activity in terms of z scores (Fig.  5). Alternative methods attempt to separate the cortical response from background noise without assuming a shape of the temporal response by making use of their underlying differences in variance (principal component analysis) or other measures of independence (23, 24).

Fig. 5. Cortical map of activity following high contrast gratings stimulation. Left picture shows the z scores map of activity. a anterior, p posterior, l lateral, m medial. The right trace shows the average temporal response of the most active pixels in percentage changes from baseline.

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4. Functional Anatomy The activation of retinal circuitry by visual stimulation can be assessed by several functional anatomical methods which include the visualisation of neurotransmitter signalling using both indirect (25, 26) and direct methods (27). By far the most commonly applied methodology involves the use of immunohistochemistry for the immediate early gene c-fos. The transcription of c-fos is associated with neural activity, as it is activated following membrane depolarization (28). Light stimulation induces Fos expression in retinal neurons and has been demonstrated in both sub-cortical and cortical regions in the brain. Comparative anatomical techniques can be employed to assess qualitative and quantitative differences in light-induced Fos in these regions to assess visual function. In the retina, immunohistochemistry for Fos was first used to measure the activation of retinal dopamine neurons in response to stroboscopic illumination in the chick (29) but is equally effective in revealing the rod/cone-driven activation of amacrine cell interneurons of the rodent retina (30, 31). Immunohistochemistry for Fos can be used in combination with a second, cell-type specific marker to identify light-driven neural activation of amacrine cells (Fig. 6) (30–33) or intrinsically photosensitive, melanopsin-expressing retinal ganglion cells (34– 36). Importantly, in the wildtype rodent retina, a linear relationship exists between light intensity and the detection of Fos expression in amacrine cells of the inner retina (31). The Fos induction in this retinal cell type has been shown to decline with advancing retinal degeneration in dystrophic mice (37) and as such can provide a sensitive measure of regional losses in rod/cone function. Below we describe a relatively simple functional anatomical method, which employs immunohistochemistry for Fos to visualise lightdriven activation in specific inner retinal circuits. We have recently used this technique to measure the spatial decline and preservation of retinal function following stem-cell grafting into the sub-retinal space of dystrophic RCS rats (38). Light-induced Fos has been described in a growing list of retinorecipient brain regions and nuclei. Given that different regions of the visual system are specialized in responding to different types of stimuli, the optimal light stimulation parameters for Fos induction will vary and would need to be defined on a region-by-region basis. Such in-depth analysis is yet to be performed for many visual system targets. One region that has been extensively studied is the suprachiasmatic nucleus (SCN) within the hypothalamus. In mammals, the SCN is the master circadian rhythm generator (39, 40). The cells of the SCN respond to increasing levels of

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Fig. 6. Light-driven Fos activation in dopaminergic amacrine cells of the rat retina. Confocal images from flat-mounted retina showing Fos labelled in green and tyrosine hydroxylase in red. (a) Following 90 min of light. (b) Following 90 min of darkness. Arrows point to nuclear Fos in (A) verses cytosolic Fos in (B), scale bar 100 mm.

irradiance and appear to function as irradiance detectors (41). Fos expression in the SCN can be light induced but it is expressed in an endogenously rhythmic manner in constant conditions. Under a 12:12 light dark (LD) cycle Fos levels peak 2 h after dawn and are low during the night (42, 43). In constant darkness (DD) there is a peak of Fos expression during the mid-subjective day (44–46). Fos expression in the subjective night is low, and light stimulation up-regulating it during this period can be informative to assess visual function (Fig. 7). Light-induced Fos in the SCN is phase dependent and only occurs at times when exposure to light will cause phase shifts of the endogenous clock (43, 45, 47–50). The levels of light-induced Fos correlate to both the intensity and duration of the light exposure and show a linear–temporal relationship to the number of photons applied over durations of 5–47.5  min (51). Melanopsin-positive ganglion cells provide the majority of the projection to the SCN (52–57). As such light-induced Fos in the SCN survives the degeneration of the outer retinal photoreceptors (47, 58–60). Mice with only melanopsin ganglion cells as functional photoreceptors retain

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Fig. 7. Light-induced Fos in the suprachiasmatic nuclei of mice. (a) The mouse remained in darkness and was perfused at the same circadian time (CT) as (B). (b) The mouse received a 15 min light pulse at CT16 and was perfused 90 min later. Fos has been visualized using DAB-nickel; relative quantification of Fos can be made by densitometry analysis. Scale bar 100 mm.

light-induced Fos in this structure (61, 62). Indeed the level of induced Fos is unattenuated between wildtype animals and those with only melanopsin cells at monochromatic wavelengths ~505 nm (~1013 photons/cm2/s) (62). Melanopsin knockout mice still display photically induced c-fos to broad-spectrum white light (70– 280 lux) in the SCN but the level is attenuated compared to wildtypes (63). Other sub-cortical brain regions that show light-induced Fos in rodents include the vLGN (62, 64), IGL (48, 62, 64–67), ventrolateral preoptic nuclei (VLPO) (68), and OPN-pretectal regions (64, 69, 70). These structures (with the exception of the VLPO) show impairment in Fos induction following loss of rod and cone photoreceptors (vLGN (62, 64), IGL (61, 62, 64), OPN-pretectum (61)). These regions receive input not only from melanopsin, but also non-melanopsin positive ganglion cells (56, 71); as such, rod and cone activity may be responsible for most light activated Fos within them. Light-induced Fos in the superior colliculus of the midbrain seems to be dependent on the functioning of rod photoreceptors as it is not detectable in a rat with degenerated rods and cones (64). Indeed the degeneration of the retina causes a confounding nonlight-dependent Fos activity in this region. The visual cortex also responds to light activation with Fos induction (72), and this still occurs in mice with only melanopsin photoreceptors (18). To what extent different types of light stimuli and different photoreceptor classes lead to Fos induction in the visual cortex still needs further investigation.

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4.1. Methods

The specific parameters of light stimulation required to induce Fos will depend on the system under investigation; optimal light stimulation may need to be verified in advance. Continuous pulses of light of 15 min or longer are typically used to assess Fos induction in regions associated with irradiance detection such as the SCN (59, 62), while discontinuous stimuli may be optimal for others such as the SC (64). Dark adaptation may need to be considered as well as circadian time at the point of sampling. Induction of c-fos transcription occurs rapidly and the protein has a half-life of about 2 h (73); to assess acute Fos induction animals are generally killed between 90 min and 2 h from the beginning of light stimulation. Animals are then deeply anaesthetised with sodium pentobarbital (60 mg/kg) and perfused with 0.1 M PBS (pH 7.2) followed by 4% paraformaldehyde (in 0.1 M PBS). The brains and eye are removed and post-fixed overnight at 4°C. Tissues to be cryostat sectioned need to be cryoprotected overnight at 4°C in 30% sucrose solution (in 0.1  M PBS), and then frozen with a dry ice/acetone slurry. Brains should be cryosectioned (~30–40  mm thick) while retinae can be sectioned (~10– 15 mm) thick or removed and processed free floating as flat-mounts. Immunostaining for Fos can be carried out using standard protocols, two examples are described below, the first for double immunofluorescent labeling in the retina and the second for densitometry analysis of Fos label in brain regions: 1. Immunofluorescent double labeling for Fos and tyrosine hydroxylase in the retina Tissue should first be blocked in 5% normal donkey serum (NDS) 1 h (in 0.3% triton X-100 (BDH) in PBS (0.1 M pH 7.2) for sections or flat-mount retina. Then incubated concurrently with sheep anti-tyrosine hydroxylase (Chemicon AB1542, 1:1,000 dilution, a pan-TH antibody recognizing all phosphorylation states) and rabbit anti-c-fos (Calbiochem PC38, at 1: 5,000 dilution) antibodies in 1% NDS in 0.3% triton/PBS at room temperature overnight. Then washed in a couple of changes of PBS before incubation in secondaries: FITC anti-rabbit and TRITC anti-sheep antibodies (preabsorbed to multiple species and especially designed for multiple labelling; Jackson Immuno Research; catalogue no: 713-095147 (FITC), 711-025-152 (TRITC)) diluted 1:200 in 2% NDS (0.3% triton/PBS). Nuclei can be stained with DAPI (1 min incubation in DAPI diluted 1:5000 in PBS) and then tissue should be washed extensively in PBS and Tris buffer (0.05  M, pH7.5). Flat-mounts can now be mounted onto slides (ganglion cell layer up) and all slides can be coverslipped using Vectashield (Vector Laboratories, Burlingame, CA). Co-localisation can be viewed on a microscope using the appropriate epifluorescent filter cubes, but confocal microscopy is preferable as it enables accurate determination of co-localization

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of Fos-positive label in the nuclei of the tyrosine hydroxylase positive cells (30). 2. Densitometry analysis of Fos induction in brain sections In brain sections, this can be done reliably using diaminobenzidine (DAB) chromogen development which produces a dark brown/black reaction product. The following is a detailed protocol, described from (74). Free floating sections should first be incubated in 50% ethanol, 0.9% NaCl, and 0.05% H2O2 to block endogenous peroxidase at 4°C for 1 h. Then washed in PBS (0.01 M, pH7.4), 0.3% triton, and 0.1% sodium azide (PBSTA) and then incubated in blocking serum containing PBSTA with 1.5% normal goat serum for 60 min at 4°C. The primary antibody should then be applied to the sections; primary antibody rabbit anti-Fos (Ab-5 Oncogene Research Products Calbiochem) at a final dilution of 1:20,000 in blocking serum at 4°C for 72 h. The sections are then rinsed twice in PBSTA and incubated in the secondary biotinylated antibody for 2 h at room temperature (BA-1000 goat antiserum, Vector Laboratories; final dilution 1:200) followed by two further rinses in PBSTA. Avidin biotin amplification is then carried out by incubation in the avidin-biotin-peroxidase complex for 2 h at room temperature (Vectastain ABC Elite Kit, PK-6100, Vector Laboratories). Brain sections are then washed in Tris buffer 0.05 M, pH 7.4 twice for 10 min and then transferred to chilled 4°C Tris buffer containing 0.02% 3,-3¢diaminobenzidine, 0.5% nickel ammonium sulphate and 0.001% H2O2. The development of the DAB nickel chromogen can be visually monitored. After which sections should be washed in Tris buffer before mounting them onto gelatinized slides, air-dried, dehydrated in a series of alcohols, cleared in xylene, and coverslipped with DePeX. Images can be captured from a microscope using a digital camera (e.g., Spot RT colour digital camera (Diagnostic Instruments Inc., Sterling Heights, USA)). Relative amounts of Fos can be quantified by calculating the integral optical density of the chromogenic label (74). This analysis can be done using image analysis software such as Image Pro Plus (Media Cybernetics UK, Finchampstead, UK). 4.2. Notes

1. Fos can be induced as part of many different cellular pathways, including apoptosis, cellular death (75–77) acute stress (78), and in response to injury (79). As such appropriate controls (usually animals maintained in darkness) should always be used to identify light-induced Fos specifically. 2. If freezing artefact (tissue shattering) is observed, then this is likely to be due to insufficient time in sucrose solution. We recommend overnight exposure of fixed eyes to 30% sucrose

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solution. Removal of lens is important when processing sectioned eye material. 3. It is important to use 3% triton with the blocking/primary antibody incubation stages because, without this, penetration of antibodies will be insufficient to visualise amacrine cells in the inner nuclear layer. 4. When flat-mounting retinae, it is important to post-fix the eyes for at least 2 h prior to attempting removal of the retina. If the retinae are not sufficiently fixed, they will disintegrate upon immuno-processing with the 3% triton solution. 5. Try to perfuse animals rather than just remove the eyes, as it is more difficult to obtain flat-mounts from fresh tissue. 6. Always use an anti-fading mounting medium such as Vectashield as, otherwise, fading of fluorescent signal will make it difficult to quantify the extent of fos activation.

5. Measurement of Wheel Running Behaviour

Wheel-running behaviour of mice can be used to assess visual functional at the sub-cortical level. Its use has been well studied in mice and to a lesser extent in rats. The circadian clock of mammals, the SCN within the hypothalamus is photo-entrained by light signals that are mediated by the retinohypothalmic tract projection from the eye (80). Locomotor behaviour is a clock-controlled output, and is demonstrated by wheel running in rodents. Wheel-running behaviour is generally analysed using an actogram (Fig. 8). As nocturnal animals, when mice are photo-entrained they will restrict wheel running to the dark phase under light dark (LD) conditions. When animals are released into constant darkness then their activity will no longer be entrained, their periodicity (t) will be controlled by the endogenous clock and t will probably not equal external time (24  h). Their activity rhythms are said to be free running (through time). The t of mice (in constant darkness) is usually <24 h, as such with each successive day they will commence wheel running slightly earlier. The full cycle of activity (between activity onsets on each successive day) is considered to last 24 circadian hours, 1 h lasts t/24 h. The start of subjective night, circadian time 12 (CT12) is defined as the time at which mice start wheel running. Light has a distinct effect on the circadian system at different circadian times. Pulses of light during the subjective night (CT12-24) can cause phase shifts in the subsequent onsets of activity. Conversely pulses of light during the subjective day (CT0-12) do not. Pulses of light as short as 15 min in duration, in the early part of the subjective night (CT1218) cause phase delays, and those in the late subjective night

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Fig. 8. An actogram showing wheel-running behaviour of a mouse. The black blocks represent bouts of wheel-running activity; each day is plotted on a successive line. For the first 4 days, the animal is maintained in an LD cycle with lights on from 5:00 to 17:00; it restricts its activity to the dark period. On the 5th day, the lights remain switched off and the animal’s wheel running activity free runs through time. On the 14th day, the animals receives a 15-min pulse of light at CT16; this causes a phase delay in the onset of activity in subsequent days.

(CT18-24) phase advance the activity rhythm. In mice, the largest phase shifts are elicited to pulses of light that phase delay the activity during the early part of the subjective night (81). There is a positive correlation between the magnitude of the phase shift and the intensity of light (over about 2 log units) given for the same duration (82). Melanopsin-positive ganglion cells provide the majority of the projection to the SCN (52–57). If they are specifically ablated then mice are unable to photoentrain their locomotor activity (83) or phase shift following a light pulse (84). Although the melanopsin photopigment alone is sufficient to phase shift and entrain the clock in mice (61, 85–87), it is not absolutely required (63, 88, 89). Rods are able to drive photoentrainment and phase shifting from scotopic to surprisingly photopic levels of light (1, 88). Indeed in normal mice the circadian clock has a peak spectral sensitivity matching that of rod opsin (90) and rods contribute to entrainment to dim levels of light (91). Cones do appear to signal to the circadian system (92, 93); however, this appears to be attenuated by light adaptation, but can be revealed by providing a discontinuous light stimulus (1). The magnitudes of phase shifts to monochromatic pulses of light (505–515  nm) are surprisingly unattenuated in retinally degenerate mice (lacking rods and most cones) (59, 85, 94, 95) (over an irradiance range of 109–1013 photons/cm2/s) when compared

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to congenic wildtypes. Melanopsin knockout animals do show deficits in phase shifting (to monochromatic light of 480  nm) across a range of light intensities (1010–1013 photons/cm2/s) (89), indicating that a phase-shifting pulse of light in this spectral range would be informative to probe the visual system of a rodent. Wheel running can also be used to assess the acute effects of light on locomotor behaviour (masking). Pulses of bright light (>10  lux) during a rodent’s active phase suppresses locomotor behaviour (negative masking); by contrast, low illumination pulses (~0.014–4 lux) enhance locomotor behaviour, (positive masking) (96, 97). The precise brain regions that are involved with negative masking are currently unknown (98), but positive masking is abolished by lesions to the dorsal lateral geniculate nucleus (99). Negative masking is absent in mice in which melanopsin ganglion cells have been ablated indicating that as for circadian responses these cells provide the conduit to brain regions controlling the response (83, 84, 100). Negative-masking responses survive outer retinal degeneration while positive masking requires the presence of rods and cones (96, 101). Recent action spectra analyses indicate that positive masking may largely be rod driven, while cones as well as the melanopsin system are implicated in negative masking in wildtype animals (97). Masking responses are preserved in melanopsin knockout animals, although they are impaired (89, 102). Negative masking is reduced to irradiances above 100  lux and is not sustained during a 3-h light pulse (98). Mice lacking the melanopsin photopigment and properly functioning rods and cones are unable to photoentrain, phase shift their locomotor rhythms or display negative masking (86, 102). 5.1. Methods

Locomotor behaviour is assessed using singly housed animals in cages with running wheels installed. The cages must be situated in a light-tight box in which the lighting conditions can be regulated and the temperature and humidity controlled. The revolutions of the wheel can be recorded and analysed with software such as Chronobiology Kit (Stanford Software Systems, Santa Cruz, CA) or Clocklab (Actimetrics, Wilmette, IL). For phase-shifting experiments, animals are removed from the wheel-running cages and placed into a specialized chamber that is coated inside to ensure that there is full internal reflection of the light. The light source for this needs to be relatively bright so a xenon-arc lamp (Lambda DG-4, Linton Instrumentation) connected via a light guide into the pulsing chamber or LED light sources of particular wavelengths can be employed. The precise parameters used can be varied as to the needs of the experimenter, what follows is a generalized methodology that is often used to assess the phase-shifting capability in mice. Mice are initially entrained to a 12  h light (L): 12  h dark (D) cycle. Normal mice can entrain to a broad range of light intensities

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(e.g., (88)). The definition of the threshold for entrainment for a particular subject in itself can be informative as to the functionality of the visual system. However, for phase-shifting experiments, the entrainment light is often ~500 lux. Entrainment is generally continued for 7–14 days. The entraining light is then discontinued and the free-running activity monitored in constant darkness (DD) for 7 days. Entrainment is confirmed if the phase of locomotor activity on initial transfer to DD is coincident to the dark phase of the previous LD cycle. On the 8th day at CT16, the animal is removed and placed into the pulsing chamber. Light stimulation (of defined intensity and bandwidth) is applied for 15 min and then the animal returned to its running wheel cage for a further 10 days in constant darkness. Any handling or husbandry of mice during this period should be done under infrared light with the aid of night vision equipment. The magnitude of the phase shift can be calculated using the monitoring software and is defined as the difference (in circadian time) between the regression line through steady state activity onsets prior to and post-pulse (94). Methods to assess masking require the animal to remain in the running wheel cage throughout the duration of the light pulse. As such the simulating light needs to be controlled remotely. Masking is generally assessed by applying the stimulating light (1–3  h in duration) 1–2  h after lights are switched off during a period of photoentrainment. 5.2. Notes

1. The majority of photoentrainment and phase shifting assays have been carried out using male animals, so for comparative studies it may be advisable to use males. 2. When studying mutant animals, age-matched congenic or littermate controls should be used for comparison as there is much inter-strain variability in light-induced responses (103). 3. Other environmental factors apart from light can influence an animal’s circadian rhythm. As such, it may be advisable to ensure that other factors (e.g., temperature) are controlled and that sham “light” pulses are carried out in which animals are handled in a similar manner but no light pulse is administered.

6. Pupillometry The pupillary light reflex (PLR) provides a fast and relatively simple method to probe the function of the visual system. As this technique does not require anesthesia, pupillometry can be carried out repeatedly on the same animal, providing a swift way to probe functioning photoreceptors. In rodent models of retinal degeneration, one can use the PLR to document the changes in photoreceptive inputs

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that occur over time (95, 104) and to monitor functional changes following interventions (105). Multiple photoreceptors contribute to provide the full dynamic range of the PLR response (1, 7, 86, 106). Animals with all three photoreceptive systems intact show a pupil constriction to a broad range of irradiances (from approx 108 to 1015 photons/cm2/s to 500 nm light). Animals with only melanopsin photoreceptors demonstrate a PLR. However, the dynamic range is reduced (1012–1015 photons/cm2/s to 500 nm) and the peak spectral sensitivity is well matched by that for melanopsin (479 nm) (8). At the lower intensities, a (108–1011 photons/cm2/s) cone, rather than rod, photoreceptors appear to mediate pupil constriction (1). Melanopsin is required, however, for the pupil to attain a full pupil constriction at high irradiances (~1014 photons/cm2/s). The steady state pupil area attained by a melanopsin knockout mouse to a bright light stimulus is three times that of a wildtype animal. At lower irradiances (~1011 photons/cm2/s), the pupil constriction of the melanopsin knockout appears similar to that of the wildtype both in terms of speed and amplitude of the response (106). The PLR in mammals can be mediated by central pathways, the retina sends afferent projections (via melanopsin and some non-melanopsin positive ganglion cells (52, 56, 84)) to the olivary pretectal nuclei (OPN) in the thalamus which controls the PLR, this in turn innervates the Edinger Westphal (EW) nucleus in the midbrain which via the parasympathetic nervous system results in activation of muscarinic acetylcholine receptors on the sphincter pupillae muscle causing it to contract (107–109). In addition to this central mechanism, the irises of some mammals (at least rats and hamsters) are intrinsically photosensitive (110, 111). Thus it is advisable to record the consensual pupil response; this also provides a quick measure to assess whether the optic nerve of the stimulated eye is intact. 6.1. Methods

A significant diurnal rhythm in the murine PLR has been noted previously (7). Consequently, the measurements should be taken at a restricted time during the L:D cycle, e.g., between 6 and 2 h before lights are switched off (when mice are maintained on a 12L:12D cycle). Since this technique can be used on unanaesthetized mice and rats, held steady under mild restraint, a period of habituation and handling prior to recording the pupil constrictions is advisable. Rodents should be dark adapted (1–1.5 h) prior to recording. The following provides a description of the type of equipment that could be used to stimulate and record the PLR from a rodent (Fig. 9). An infrared light source (LEDs >850 nm are often used) can be used to illuminate the eye for recording as rodents are not sensitive to this wavelength and their pupils will remain dark adapted until stimulated. Any computer monitors or other stray sources of light should be covered with a long pass filter, for example,

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Fig. 9. Schematic of the pupillometry set up.

medium red #27 Lee Filters, Andover, UK (passing wavelengths >600 nm), although ideally any stray light should be baffled from the recording arena. The pupil can be recorded using a digital camera such as a CCD fitted with a macro lens situated in a parallel plane to the cornea. A long pass filter interposed between the camera lens and the subject’s eye to block any light of <665 nm prevents any stimulation light from overexposing the recorded image. The animal can be positioned in front of the camera using an infrared image converter such as night vision goggles. Light stimulation to the contralateral eye to the one being recorded can be provided by a broad-spectrum light source such as a xenon-arc lamp (Lambda DG-4, Linton Instrumentation) with a quartz fibre optic light guide. An adirectional light stimulus can be generated using a diffusing sphere attached to the end of the light guide; this can be constructed from a ping pong ball (86). The light stimulation should ideally be controlled with an electronic shutter (MellesGriot) and synchronized with the image capture from the recorded eye using software such as LabVIEW (National Instruments). The pupil area from captured images can be analysed using software such as TINA 2.10 g image analysis software (Isotopenmeßegeräte GmbH, Germany) or MATLAB. The PLR is a relatively fast response and although the speed of constriction does change with the intensity of irradiance, the maximum constriction is reached within the first 10–15 s (when irradiance is in the range 1011–1014 photons/cm2/s). As such recordings and stimulations tend to be about 1 min in duration. This is also a practical duration for the restraint of an unanesthetized rodent. 6.2. Notes

1. To control for any intrinsic defect in the iris sphincter pupillae muscle of the animals being studied, a few drops of 1 M carbachol administered to the eye in darkness, should result in pupil constriction within about 5 min. 2. When studying the consensual PLR, application of a mydriatic eye drop, e.g., 1% atropine sulphate into the stimulated eye allows to both maximise the retinal area illuminated as well as normalizing the pupil size between different subjects.

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7. Assessment of Optokinetic Head-Tracking

Spatial vision in rodents can be measured using the optomotor response. A grating of vertical black and white stripes moving across the visual field of an animal can elicit a head-tracking response, the animal moving its head in synchrony with the gratings. Acuity can be measured by increasing the spatial frequency (cycles/degree) of the stripes until the head tracking response is no longer detectable. Contrast sensitivity can also be measured using this technique by altering the contrast of the stripes (112, 113). This response in rodents is largely sub-served by sub-cortical structures such as the thalamic accessory optic system and the superior colliculus in the midbrain (114). Unilateral or bilateral lesions of the visual cortex do not substantially reduce the visual acuities in either rats or mice when they are measured using the optomotor response (114, 115). The visual cortex can, however, modulate the activity of these sub-cortical pathways to enhance the acuity measured by head-tracking. This cortical involvement is induced following monocular deprivation in adult mice (9) and also following experience of optomotor testing during early postnatal (from 15 to 30 days of age) life in rats (10). Indeed other behavioural methods testing visual acuity in rodents (e.g., the visual water maze) do result in consistently higher acuity levels than those obtained from head-tracking and these responses are impaired by lesions of the visual cortex (116, 117). The optomotor response is controlled primarily by rod and cone photoreceptors. It can be used as a reliable indicator of animals with outer retinal degeneration as they are unable to head track to grating stimuli (118). Both rod and cone photoreceptors can apparently mediate the head-tracking response independently, as it is elicited in normal mice at both photopic (30  cd/m2 and higher) and also scotopic conditions (<0.01  cd/m2) luminances. Mice with dysfunctional rods or those actually lacking the rhodopsin photopigment display optomotor responses but only at higher photopic luminance levels (113, 119). When cone function is knocked out, mice can still head track. Deficits in tracking to photopic levels of light have been reported in one cone mutant (GNAT2cpfl3) (113), while not in another (CNGA3−/−) (119), the latter displayed similar tracking at both scotopic and photopic light levels. This discrepancy may reflect differences in the experimental protocols or differences in the cone dysfunctions caused by the two different mutations. Mice lacking functional rod and cone photoreceptors are unable to head track to grating stimuli using solely melanopsin photoreceptors, demonstrating rod/cone importance in eliciting this response (53, 119). Head-tracking can be used to assess visual acuity of the two eyes independently in rats and mice. Stripes moving in the clockwise

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direction (movement from left to right) test the left eye while counter clockwise movement (from right to left) tests the right eye (115). This allows one to test treatments to one eye in comparison to the other in a within animal-controlled experiment. For example, it has been used to test the efficacy of transplants into the left eye in retinally degenerate rats (38). Recently, spatial acuity as measured by optokinetic tracking has been restored to mice and rats with outer retinal dystrophy by optogenetic gene therapy, by insertion of microbial opsins into remaining inner retinal neurons (120, 121). 7.1. Methods

Optomotor responses have been measured effectively using two alternative techniques, using a rotating drum or using monitors simulating a virtual rotating drum (Fig. 10). Rotating drum method: A clear plastic cylinder is situated in the middle of a motorized rotating drum which has a vertical grating pattern displayed on the inside wall. Varying the widths of the gratings subtends to different spatial frequencies at the centre of the drum. (Dimensions for a system used for mice: perspex cylinder 15 cm diameter and 18 cm high and the drum 63 cm diameter and 35 cm high). The drift speed of the drum to elicit head-­tracking responses is generally set at around 12°/s for both rats (115) and mice (118, 122). The illumination can be provided from above by light bulbs or diffused LEDs. The animal’s movements can be recorded using a digital camera or an infrared sensitive camera (in which case infrared illumination needs to be provided). Headtracking responses can then be analysed from the videos. The response is generally quite clear to an observer; an animal while watching the gratings will move its head in an angular motion at a similar speed as the gratings in order to maintain fixation on the pattern.

Fig. 10. Optomotor responses. (a) Measurement using a rotating drum with a stripe pattern displayed for the rodent to view from a transparent container in the centre of the drum. (b) Measurement using a virtual drum created by four inward facing computer monitors displaying a grating pattern with the rodent placed on a platform in the centre (112).

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However, programs have been written to enable statistical analysis of their movements with respect to the grating pattern and have been used to automate the detection of head-tracking behaviour (119). Thresholds of acuity in mice measured using a rotating drum are 0.3–0.4 cycles/degree, and they are most sensitive to spatial frequencies of 0.1–0.2 cycles/degree (119). Virtual method: A platform is placed in the centre of an arena comprising four inward facing computer monitors (17 in. monitors have been used for mice and 19 in. for rats (115)). The monitors display a vertical grating pattern that creates a virtual cylinder, and the gratings are extended with floor and ceiling mirrors. Again the movement of the animal can be monitored using a digital camera. A commercially available virtual system for assessing the optomotor response in both mice and rats, OptoMotry is available from CerebralMechanics (Lethbridge, Alberta, Canada) (112). The OptoMotry software allows the rapid assessment of visual acuity by an observer, using a staircase procedure to obtain a threshold for visual acuity, and can be completed within about 40 min per subject (115). The threshold optomotor acuity measured in this way for rats is approximately 0.54 cycles/degree while for mice it is slightly lower at approximately 0.39 cycles/degree (115). Cortex-dependent enhancement of this response increases acuity to 0.8 cycles/degree in rats (10) and 0.55 cycles/degree in mice (9). The spatial frequencies used to determine acuity thresholds tend to be between 0.03 and 1 cycles/degree in rats, and 0.03–0.6 cycles/degree in mice. 7.2. Notes

1. As with other behavioural tests, age and sex (123) should be controlled for. 2. It should also be noted that head-tracking is much less pronounced in albino rats (115) while it has been reported as undetectable in one strain of albino mice (124). 3. Retinal physiology changes throughout the light/dark cycle, which may well impact on measurements of acuity (125).

8. Assessment of Behavioural Light Aversion

The ability to measure light intensity and move relative to a gradient in this stimulus is known as phototaxis (126). Adult rats and mice show behavioural light aversion (BLA) to acute (10–30 min) light exposure in the open field (127–130). The “light/dark box test” has been used extensively in drug development (131) and to investigate human photophobia in mouse models of migraine (132, 133). However, the neural circuitry mediating BLA in rodents is not well understood. One study has implicated both subcortical

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(superior colliculus, lateral geniculate) and cortical (visual cortex) processing in rats (134). The photoreceptive inputs to the BLA in rodents have not been thoroughly dissected. It seems that both rod/cone and melanopsin systems contribute. Neonatal rodents display negative phototaxis during a developmental period when only the melanopsin cells are photosensitive (135–137), and indeed in melanopsin knockout mice, this behaviour is abolished (136). In adults, though the melanopsin system does not appear to be as important, the targeted destruction of melanopsin cells has no impact on the light:dark preference of mice (100). Rats with retinal degeneration which primarily affects the rods/cones show a progressive loss of BLA over time, with no response detectable by 7 months to a 5 min light exposure (138), However, over a longer time period of 22  h, mice lacking rods and most cones display a preference for dark nesting areas (139). In fact, mice lacking rods and cones can show behavioural light aversion to broad spectrum white light stimuli (~1,300  lux), but this is only more clearly revealed after 20 min during a 30-min trial. Melanopsin knockout animals also show BLA (18), implying that rods/cones and melanopsin photopigments are required for the normal response. Pre-treatment of the eyes with atropine greatly enhances the BLA response and increases the sensitivity of the technique (the response of mice with only melanopsin cells becomes almost equivalent to wildtypes). The precise mechanisms by which this works is unclear, but may be by both increasing the illumination on the retina by dilation of the pupil and possibly by directly acting pharmacologically to enhance retinal function (18). BLA has also been restored in animals with outer retinal degeneration and those lacking melanopsin and functional rods and cones by transducing remaining retinal cells (cones (140) and retinal ganglion cells (18)) to express microbial opsins. 8.1. Methods

An open field arena used to test behavioural light aversion essentially comprises an open brightly lit area and a dark compartment. An example of an arena for testing mice is shown in (Fig. 11). The arena is square (26 × 26 cm) and is divided into an open front-half and an enclosed back-half with a small door through which the mouse can enter the enclosed area. The insert in the back-half of the arena is impervious to visible light but not to infrared beams. The location of the animal during the trial can thus be monitored using a grid of infrared beams produced by a monitoring system such as TRUSCAN (Coulburn Instruments, Inc Allentown, PA). A video tracking system (e.g., View Point Life Sciences, Montreal, Canada) could also be employed to monitor the location of the animal. The illumination of the lit side of the arena can be made with any suitable light source, for example, an LED array, ideally diffused to provide homogenous illumination across the arena floor.

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Fig. 11. An open field arena comprising a brightly lit open and dark enclosed area to test the light aversion behaviour of mice.

Care should be taken to avoid inadvertently heating the open side of the arena, of which case a heat filter could be used. The duration of the BLA trial would depend on the specific photoreceptor system being tested, but at least 20–30 min is required to detect the normal melanopsin contribution to this response. 8.2. Notes

1. The use of naïve mice in protocols designed to test visual function may be advantageous. Although habituation is used in some light/dark choice protocols (141), this can reduce the amount of time spent in the dark (142) which may mask subtle light responses. 2. Other environmental factors could influence the behaviour and should be controlled (e.g., temperature, loud noises, odours etc). 3. If atropine pre-treatment is required then a drop of 1% atropine sulphate (Minims, preservative free) should be applied bilaterally. This is long-acting, so the animal can recover for at least 30 min in the home cage prior to behavioural testing.

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Chapter 17 The Role of the Pedunculopontine Tegmental Nucleus in Motor Disorders Nadine K. Gut and Philip Winn Abstract In this chapter, we discuss why the pedunculopontine is an appropriate target in studying movement disorders, explaining its association with both the pathology and treatment of Parkinsonism. We discuss how various laboratories, including our own, have approached experimental examination of the pedunculopontine and some of the findings that have emerged. These lead us finally to reflect on the nature of movement disorders and how they necessarily involve not just the control of musculature but additionally the complex cognitive processes of decision making – the processes that underlie not just “doing” but also “choosing” and how these are embedded deeper in brain than is often appreciated. Key words: Action selections, Basal ganglia, Decision making, Deep brain stimulation, Mesencephalic locomotor region, Parkinsonism

1. Why Study the Pedunculo­ pontine? 1.1. A Note on Nomenclature

In our laboratory we have followed the convention of abbreviating pedunculopontine tegmental nucleus to “PPTg”. This is the abbreviation used in Paxinos and Watson’s stereotaxic atlases of the rat and mouse brain, the most widely used of their kind. Others talk of the pedunculopontine nucleus (PPN or PPT) or the tegmental pedunculopontine nucleus (TPP), and an older literature uses the Latin form nucleus tegmenti pedunculopontinus (NTPP), though this is rarely used now. Does the choice of term matter? Probably not, if the community in general recognizes that all of these terms and abbreviations refer to the one thing. Internally, the PPTg was originally described as containing a pars compactus and a pars dissipatus. These terms are being used again, though not with particular clarity. It was argued in the late

Emma L. Lane and Stephen B. Dunnett (eds.), Animal Models of Movement Disorders: Volume II, Neuromethods, vol. 62, DOI 10.1007/978-1-61779-301-1_17, © Springer Science+Business Media, LLC 2011

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1980s that the PPTg consisted solely of cholinergic neurons – in Mesulam’s classification (1), the Ch5 group – but this is not the case. Interdigitated and adjacent to the Ch5 neurons are very many glutamate- and GABA-containing neurons (and possibly others not yet classified) (2), and it has become apparent that the PPTg is not a homogeneous structure, in terms of either neuronal neurochemical identity or the connectivity of its different parts. As such, terms such as pars compactus and pars dissipatus that identify subregions are needed. In our lab we have simply split the PPTg in two – posterior and anterior PPTg. The posterior PPTg contains the bulk of what was thought of as the pars compactus, anterior PPTg the dissipatus. However, while understood by readers of the specialized PPTg literature, our terms are not widely adopted. The development of a clear description of the internal organization of the PPTg, and the naming of parts, is a challenge to be met. 1.2. PPTg, Movement, and Movement Disorders

In this chapter, we first consider why the PPTg is thought of as a motor structure, one that might then naturally be involved with movement disorders. Classic accounts of PPTg function emphasize sleep state switching (3, 4) and the control of locomotion and there is indeed an abundance of evidence showing that the PPTg has a role in these. It is our contention, however, that these are narrow views of what the PPTg is about and that in order to grasp its role in movement disorders, one needs a wider perspective (for reviews, see (5, 6)). So to start, in this first section we briefly consider evidence suggesting that the PPTg is involved in the pathophysiology of Parkinsonism; that it forms part of the “mesencephalic locomotor region” (MLR); and finally, that it might be considered part of the basal ganglia family of structures. (Note that we have not attempted a comprehensive review of the literature here, citing selected references only.).

1.3. Parkinsonism and Related Disorders

Progressive cell loss of dopaminergic neurons in the substantia nigra (SN) is not the only pathophysiological characteristic of Parkinson’s disease (PD). Neuronal loss is present in various brainstem nuclei including subnuclei of the reticular formation, the raphe system and the coeruleus–subcoeruleus complex (7). In the PPTg, significant overall cell loss of 40% was identified, averaged across various levels along the rostrocaudal axis of the PPTg (8). Later, differential loss of cholinergic and non-cholinergic neurons was noted (9). In Rinne’s study, the average loss of cholinergic neurons was similar to that reported by Zweig et al. (36%), but non-cholinergic neurons were also reduced by 23% and the overall reduction of all neurons in the PPTg across the rostrocaudal axis was reported as 27%. Cell degeneration and atrophy was found in surviving neurons showing a 14% (all neurons) to 26% (cholinergic neurons only) decrease in cell size. The loss of cholinergic cells correlated with disease severity according to the Hoehn and Yahr stages (10).

1.3.1. Loss of Neurons

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1.3.2. Altered Input from, and Input to, the Basal Ganglia

In the 6-OHDA rodent model of PD, changes in PPTg neuronal activity have been observed. Increased activity observed in the subthalamic nucleus (STN) appears to be related to increased activity in the PPTg, the former possibly caused by the latter (11). This significant increase in activity in the PPTg was previously described by Mitchell et al. (12) in MPTP lesioned primates and confirmed in rodents by Carlson et  al. (13) – it is thought to represent an increased level of GABA-mediated inhibition of the PPTg due to changes in pallidal output activity.

1.3.3. Relationship to Loss of Dopamine Neurons in Substantia Nigra

The simultaneous appearance of cholinergic and catecholaminergic neuron degeneration in PD suggests the possibility of common etiologic factors causing degeneration or cell loss in the SN and PPTg, which directly projects to the substantia nigra pars compacta (SNc) and receives input from the substantia nigra pars reticulata (SNr) (14). According to Braak et  al. (7), both nuclei, SN and PPTg, initially show degeneration through development of Lewy neurites (LN) and inclusion of Lewy bodies (LB) in stage 3, leading to substantial damage and cell loss in the SN and PPTg in stage 4. The susceptibility to develop LNs and LBs lies in the nature of the neurons affected. Neurons with long thin axons, either unmyelinated or sparingly myelinated, are more likely to form LNs and LBs (15). Both SN and PPTg contain neurons with long, thinly myelinated axons, developing long LNs followed by LBs. However, in early Parkinsonism, PPTg neurons are intact, which could either mean that they might contribute to the maintenance of DA functioning or that their increased activity leads to the degeneration of DA neurons. The nature of this relationship – protective, destructive or indifferent? – remains unknown.

1.3.4. DBS in Humans

Recently, the PPTg has received attention because of demonstrations that low frequency deep brain stimulation (DBS) here might have therapeutic benefit for patients. The involvement of the PPTg in PD, its relationship with the basal ganglia and the presumed PPTg motor function drew attention to the possibility that this structure could be targeted for DBS (16). The results of the studies carried out are exciting, but variable and inconclusive. The first results on PPTg DBS in PD patients were published in 2005. Preliminary results were encouraging: two independent centres claimed improvements of gait dysfunction and postural instability with low frequency DBS (17, 18). These raised the hope of being able to address by PPTg DBS motor dysfunctions that were not especially well treated by other therapeutic regimes. But the use of the UPDRS in these studies is problematic: many items rely on subjective accounts and often complete subscales were analysed as a whole, combining different deficits. Furthermore, because only very few patients were tested, questions remained unanswered regarding, for example, long-term effects, cost-benefit, and the

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actual degree and nature of the effect. It remained unclear whether PPTg stimulation could be seen as an alternative or an addition to DBS in the STN, or whether it would make medication unnecessary. Finally and most importantly which stimulation parameters were most beneficial and which part of the PPTg should be targeted? Subsequent studies that included more patients with bilateral STN and PPTg stimulators produced rather inconclusive results. STN stimulation was clearly better than PPTg stimulation, which on its own only led to motor amelioration over the first day of stimulation (which could conceivably be due to a placebo effect). Stimulating PPTg and STN together was only beneficial in combination with l-DOPA (19). A closer look at specific features of gait, by limiting the study to patients with severe freezing of gait (FOG) and postural instability, also led only to an increase in stride length when combining low STN with low PPTg stimulation. The beneficial effect of unilateral PPTg stimulation on gait, contralateral tremor and bradykinesia (20) and an increased REM sleep threshold resulting in more REM periods (21) are also only based on few case studies. Furthermore, these authors acknowledge that they cannot rule out the possibility of the current spreading from the electrode into adjacent structures (20). Indeed, the studies to date have generated discussion about the actual target site of the stimulators between different laboratories – target site turns out to be the biggest uncertainty (22–24). The most recent work on PPTg DBS has used double blind studies and PD patients either in an advanced stage or with severe FOG unresponsive to l-DOPA or STN stimulation (25). However, results are still not convincing: no significant changes in motor outcome could be shown and the results were variable, reaching from fair improvement to worsening of FOG in the 1-year follow-up assessment. It is of interest to note in the present context that, just as with rodent models of PPTg function, DBS produced changes in working memory and in addition (surprisingly) language abilities – grammar, speech complexity, speech fluency and semantics were tested (26) as well as delayed recall, executive functions and phonetic verbal fluency (27). A reduction in reaction time in the N-back task might be taken to show facilitation of information processing in the content of working memory, but small group sizes and ceiling effects in some of the patients only allowed the authors to discuss trends in the improvement of the other functions. Overall, the results of low frequency DBS in PPTg on motor performance are (at best) inconclusive. However, this in itself might be important. Hypotheses about the effect of PPTg DBS on Parkinsonian symptoms – and in particular gait and postural difficulties – are based on the assumption that the PPTg is a part of the MLR. Is it really so? A brief discussion of this proposition is required.

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1.4. The Mesencephalic Locomotor Region

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The MLR is defined functionally (rather than anatomically) as an area of the mesencephalon from which it is possible to elicit locomotor activity on a treadmill in decorticate animals by means of electrical stimulation (which modulates spinal locomotion oscillators). Locomotion-inducing sites that project to the spinal cord include cholinergic PPTg neurons (28, 29). The role of the PPTg in locomotion and postural control is additionally defined by connections with motor cortex and basal ganglia (30). Locomotion can be induced by electrical or chemical stimulation within the PPTg pars compacta in cat and rat (28), while electrical stimulation of the lateral part of the PPTg suppresses muscle tone in decerebrate cats (31). Stimulation-induced locomotion can be blocked by injections of the muscarinic receptor antagonist atropine sulphate into the medioventral medulla, where efferent cholinergic fibres from PPTg terminate (28). Furthermore, when the primate PPTg was inhibited by local injection of the GABA agonist muscimol, a significant decrease of motor activity was observed (32). Conversely, stimulating the PPTg with the GABA antagonist bicuculline, after the same primate had been made Parkinsonian by MPTP, has been reported to alleviate akinesia (32). Different types of lesions of the PPTg in primates have led to apparent Parkinsonianlike motor symptoms. Hypokinesia and rigidity have been reported on the contralateral side after an injection of kainic acid unilaterally into the PPTg (33). Thermal (34) as well as kainic acid (35) lesions of PPTg in primates produced transient hypokinesia when only one hemisphere was lesioned, which developed to include profound paucity and slowness of movement after a subsequent lesion in the contralateral hemisphere. There are, however, reasons for caution in interpreting these deficits – the use of kainic acid is problematic, and the recovery times in these experiments was short (see (5) for further discussion of this issue). In contrast to this association of PPTg with the MLR, Takakusaki’s group reported in 2003 that the locomotor region described as the MLR was mainly located within and around the cuneiform nucleus (CNF) though including the dorsal part of the PPTg, with the ventrolateral PPTg as a part of an inhibitory system – this was after observing muscle tone suppression after chemical and electrical stimulation of the ventrolateral PPTg in decerebrate rats (36). Changing the focus from PPTg to CNF as the main representative of the MLR is supported by Fos expression following treadmill locomotion in rats, detected in CNF but not in PPTg (37). However, bilateral excitotoxic lesions of the CNF showed no effect on spontaneous or amphetamine-induced locomotion (38) though such lesions produce anxiety-like behaviours in rats (39). Is the PPTg critical for the production of locomotion? Lesions of the whole PPTg do not reduce spontaneous locomotor activity (40) or have an effect on amphetamine-stimulated locomotion (41). Moreover, decorticate animals appear able to make appropriate

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selections of simple movements, actions and goals (6). Rather than simply being a “movement centre”, many studies show that the functions of the PPTg are quite diverse. Ros et  al. describe the large variability of projections from PPTg neurons, which gives an idea of how the PPTg could be involved not only in diverse behavioural processes including the control of movement, but also sensorimotor coordination, sleep–wake mechanisms, attention and autonomic functions (42) as well as reinforcement processes and learning (43). Evidence that, while involved, the PPTg is not necessary for locomotion challenges the notion of the PPTg being synonymous with the MLR (and indeed it remains a question as to whether the notion of a MLR is needed at all (38)). Ultimately this leads to a question as to whether or not the PPTg is an adequate target for PPTg DBS in PD for the alleviation of gait disturbances. The supposed association of the PPTg with the MLR is a doubtful basis for PPTg DBS. 1.5. The Relationship Between PPTg and Basal Ganglia

Situated in the caudal mesopontine tegmentum, the PPTg is a phylogenetically ancient structure with many ascending and descending connections. It reaches rostrally from the substantia nigra, just below the red nucleus, to the lateral tip of the superior cerebellar peduncle caudally. From this position, PPTg has extensive connections with a variety of structures above and below. It has connections to the musculature via the spinal cord; connections with deep cerebellar nuclei, the trigeminal complex, and the mesencephalic and medullary reticular formations; and connections with the locus coeruleus, raphe nuclei, laterodorsal tegmental nucleus (LDTg, with which it is intimately interconnected; see (6) for review). However, most prominent is its influence on the basal ganglia and the input it receives from here, supporting the proposal that the PPTg should be understood as a part of the basal ganglia family (44). The PPTg has direct and reciprocal projections to nuclei such as the globus pallidus (GP) (notably the internal segment [Gpi]) and the STN: afferent projections to here from PPTg are cholinergic, glutamatergic and GABAergic and efferent connections to PPTg are glutamatergic. Connections with the striatum are made indirectly via cholinergic and glutamatergic projections from PPTg to the SNc and ventral tegmental area (VTA). Additionally the PPTg receives GABAergic projections from the basal ganglia output nucleus, SNr. There is some evidence for direct connections to the PPTg from dorsal striatum (ventrolateral caudate-putamen) in rodents but no substantial evidence for a connection with the ventral striatum, though there is input to PPTg from the extended amygdala (45). Connections with the striatum are also made via thalamic nuclei which of course also gives PPTg profound influence over cortical activity: virtually every thalamic nucleus appears to receive cholinergic input from the PPTg (Fig. 1) (for reviews of PPTg connections, see (5, 46)).

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Fig. 1. Illustration of the location, connections and composition of the PPTg. (a) Sagittal view on a rat brain section (taken from the atlas of Paxinos and Watson (94)). Schematic representation of the principal afferent and efferent connections of the PPTg with forebrain and brainstem nuclei. Black arrows represent cholinergic connections, dotted arrows GABAergic and dashed arrows glutamatergic connections. Grey arrows represent unclassified connections. (b) Schematic graded distributions of glutamatergic, cholinergic and GABAergic neurons within the PPTg. The gradient represents the rostro-caudal distribution of each neuron type based on the cell identification of Wang and Morales (2) and Mena-Segovia et al. (95). GPe globus pallidus externa, GPi globus pallidus interna, LC locus coeruleus, LH lateral hypothalamus, LDTg laterodorsal tegmental nucleus, SC superior colliculus, SN substantia nigra, STn subthalamic nucleus, VTA ventral tegmental area.

2. How to Study the Pedunculo­ pontine

In order to study the behavioural (and inferentially, psychological) functions of the PPTg, scientists have typically applied the classic methods of behavioural neuroscience to problems of movement disorder. For the most part, data have generalized across species, but this is not always the case. It is widely accepted that the structure and connectivity of the PPTg is similar across species, up to and including humans. Imaging of human brain has not often touched on the brainstem, but at least some studies have produced data that are entirely compatible with conclusions based on rodent experiments (47). What has been harder to accommodate with the rodent literature are studies of movement in primates.

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Perhaps the most difficult aspect of dealing with the PPTg – alluded to already in the section on DBS – is direct intervention through stereotaxic surgery. In humans there is still uncertainty about the precise location of PPTg. In experimental animals, stereotaxic surgery presents a number of problems (1) if one wishes to lesion the whole structure, multiple small infusions of neurotoxin are required because PPTg is a relatively long thin structure; (2) exposure of the surface of the brain prior to penetration within a device exposes a large superficial blood vessel – the superior sagittal sinus – that can be damaged while drilling skull, or which can require moving aside in order to access brain below; (3) we have found that the introduction of excitotoxic agents into the PPTg in both hemispheres simultaneously is often fatal to rats and as such we do not use this approach. Rather, we make lesions in one hemisphere then return 7  days later to make lesions in the contralateral one. Targeting the PPTg by stereotaxic surgery is a challenge. In rats, when the skull is in a level position during surgery, the PPTg sits right underneath the superior sagittal sinus. By elevating the incisor bar on the stereotaxic frame, problems with the sinus can be avoided, and the vessel not touched by inserted pipettes, cannulae or electrodes. The angle to move the incisor bar to is calculated by multiplying the distance between the interaural line and the back of the incisor bar by the sine of 8°29¢ (0.147), as described by Whishaw et al. (48). The cholinergic neurons of the PPTg are intimately associated with those of the adjacent LDTg (much as the DA-containing neurons of SNc are continuous with those of the VTA). Stereotaxic access to the LDTg is even more difficult because the bulk of it is embedded within the central gray, close to the cerebral aqueduct and access to it is also obstructed by superficial blood vessels. By raising the incisor bar as described above helps avoid the vessels; refining the stereotaxic coordinates by means of pilot studies, using fine tipped glass pipettes, as well as measuring the dorsoventral distance to the LDTg from dura rather than skull has helped our lab improve the accuracy and reliability of infusions into the LDTg. Other labs have used angled approaches to the LDTg, though this can be difficult because it adds an extra potential for stereotaxic error. Stereotaxic surgery of human PPTg has proven to be even more difficult. One major point of discussion, as mentioned above, is the correct localization of the nucleus. Neurosurgeons describe the PPTg as “unfamiliar territory” pointing out the difficulty of defining the borders of the PPTg correctly because of its small size and reticular nature. Individual differences between subjects are also more of an issue than one faces working with laboratory animals of a standard age and size. Preoperative neuroimaging should be of benefit here, but the fact that the PPTg is not clearly visible in T1-weighted MR images makes this difficult (49, 50).

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Common approaches to refining localization during functional neurosurgery are microelectrode recording and measuring local field potentials, which remain only an approximation because the specific physiological characteristics of human PPTg neurons are as yet undefined (49). However, by recording cell activity and plotting the corresponding neurons in the coordinate system of a human brainstem atlas, these approaches can provide useful information for electrode implantation (51). Post-operative structural magnetic resonance imaging should be obtained, both in order to match these with intra-operational microelectrode recording and to create evidence for functional characteristics of PPTg neurons of PD patients (23). Localization by direct proton density MR imaging is also a technique described as being useful during surgery in order to address these problems (49). 2.1. Lesions 2.1.1. Excitotoxic Lesions

Discrete lesions of the whole PPTg in our lab are successfully made by the neurotoxin ibotenic acid. Infusions of ibotenate are made up as 0.12 M solution in phosphate buffer with a final pH adjusted to pH 7.0 using 2 M NaOH. This technique spares fibres of passage but it damages all neuronal types (52). In order to avoid creating lesions of adjacent structures but still targeting the complete PPTg, multiple placements of low volumes are necessary. When delivered in a volume of 200  nl in two injection sites per hemisphere the whole structure can be affected. The toxin is infused using a 0.5–1 ml syringe or by pressure ejection from a drawn glass micropipette (tip diameter, 30–40 mm) for better control and left in situ for 5 min after infusion. Bilateral lesions are performed in two separate unilateral surgeries separated by 7 days, as experience immediately proved that bilateral ibotenate infusions made in this region in only one surgery resulted in high mortality rates. During the immediate post-operative recovery period convulsive activity and barrel-rolling are to be expected with any excitotoxin. Rats need to be monitored closely during this period until this activity disappears. It is rarely acknowledged, but important to note that different excitotoxins have different diffusion characteristics which need to be examined in pilot experiments; see (39) for an elaboration of this point. What are the behavioural consequences of such lesions? As outlined above, excitotoxic lesions of the rodent PPTg do not have an effect on either spontaneous or amphetamine-induced locomotion (induced by either systemic administration or microinjection directly into the nucleus accumbens) (40). Other basic behavioural patterns such as feeding, drinking or grooming are not affected either (53). It can, however, be shown that the PPTg is involved in complex associative processing. Lesions of the PPTg affect responding for conditioned reinforcement. PPTg lesioned rats show an increased rate of response on non-reinforced levers, as opposed to control rats that learn to discriminate between the levers and focus

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almost exclusively on the reinforced lever. This indiscriminate lever pressing reflects a disruption in the process of reward-related behaviour but not motor deficit per se (40). The same inability to learn the association between a conditioned stimulus and a reward has been shown in experiments using different experimental paradigms (54) after bilateral excitotoxic lesions of the whole PPTg. Complex cognitive processing as required to complete the delayed spatial win-shift radial maze task has also shown to be affected by whole bilateral PPTg lesions in the absence of any locomotor deficits (55), an effect that cannot be attributed to disrupted motivation (55, 56). Here it made no difference whether or not the rats received the lesions before or after being trained on the task. They made significantly more errors than sham-operated rats and showed therefore impairment in making the appropriate choice of the arm to enter in the maze (55). Given the strong connections to midbrain DA neurons and their importance for establishing associations between action and reinforcement (57), an involvement of the PPTg in drug reward was expected. Responding for intravenous administration of amphetamine (58) and heroin (59) is affected by lesions of the whole PPTg; self-administration of nicotine was investigated with separate lesions of the anterior PPTg (aPPTg) and posterior PPTg (pPPTg) because of the different relationships these have with midbrain DA neurons – pPPTg preferentially innervates the VTA and aPPTg the SNc. Posterior but not anterior lesions affected nicotine administration. On the other hand, lesions of aPPTg cause significantly reduced baseline levels of locomotor activity which is not observed after pPPTg lesions (60). In respect to drug selfadministration studies, PPTg lesions appear to abolish the ability to learn a specific contingency between an action and its outcome, unless the association between action (lever pressing) and reward (outcome) has been made prior to the lesion. However, when the requirements for obtaining a reward change rats with PPTg lesions that previously performed well show impairments (58, 61). These data strongly support the contention that the PPTg is involved in learning about the selection of appropriate actions rather than movement per se. 2.1.2. Transient Lesions

Another method of studying the behavioural functions of the PPTg is inactivation of the structure by direct pharmacological manipulations rather than by permanently lesioning it. This can be achieved, for example, by infusion of the GABA agonist muscimol into the PPTg (62). For this purpose, guide cannulae are placed above the PPTg and by means of injectors, muscimol can be administered prior to the testing session. In our lab, a dose of 50 ng/hemisphere has shown to produce an effect in the PPTg, however other researchers observed effects only after higher doses (62). A much smaller amount is necessary to inactivate, for example, the VTA (63).

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Fig. 2. Mean (±SE) consumption of 20% sucrose solution (in grams solution) over 90 min following posterior PPTg inactivation by muscimol microinjection or sham inactivation by saline microinjection. (Muscimol: 0.05 mg/0.3 ml per hemisphere infused at 0.3 ml/min via 30-gauge stainless steel cannulae inserted via permanent indwelling guides. Sham inactivation involved an identical procedure but infusion of the vehicle solution [0.9% saline] only).

The use of muscimol is especially interesting in the study of the PPTg’s role in learning because it allows re-tests of rats after inactivation, and the use of within subject controls. Furthermore, long interruptions between training and testing due to surgeries can be avoided. In line with data obtained from ibotenic acid-lesioned rats, inactivation of the pPPTg by means of muscimol in our own lab (D. Wilson and D. MacLaren, unpublished data) caused rats to significantly overconsume 20% sucrose solution compared to rats with sham inactivation (Fig. 2). This validates that the technique has similar effects to ibotenic acid lesions and allows for further studies in behavioural paradigms suited to inactivations rather than lesions. A change in firing pattern from tonic to burst firing in midbrain DA neurons is implicated in reward-related learning (64). Maskos (46) explains how the cholinergic neurons of the mesopontine tegmentum, including the PPTg, may be essential for this change: restoration of the high-affinity nicotinic–cholinergic receptor subunit b2 in the VTA restores burst firing, which is completely absent in the mouse knockout of the same receptor subunit (65). An inactivation study offers support for this hypothesis: inactivation of the PPTg by means of 1% lidocaine hydrochloride silences conditioned

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responses of midbrain DA neurons which were present before the inactivation (66). Using muscimol for temporary inactivations Corrigall et al. have shown that the inactivation of the whole PPTg lead to reduced nicotine self administration in rats which had learnt to lever press for nicotine prior to inactivation (67). 2.1.3. Lesions Selective for Cholinergic Neurons

Because the PPTg is a heterogeneous structure with different interdigitated neurons, isolating the functions of the different neuron types is of considerable interest. This cannot be achieved with neurotoxins such as ibotenic acid. The first neurotoxin that was thought to be selective for cholinergic neurons was ethylcholine mustard aziridinium ion (AF64A) which operates through choline transport mechanisms and causes a decrease in cellular choline acetyltransferase activity (68, 69). It has been used in the PPTg (70) but it has been shown that even low doses of AF64A produce a local non-specific lesion in the injection site (71). AF64A has not been a success in PPTg or elsewhere in brain. The best-known fusion toxin selective for cholinergic neurons is 192 IgG saporin, which has been invaluable in study of basal forebrain cholinergic neurons. It is ineffective on PPTg neurons because they lack the necessary 192 IgG nerve growth factor receptor (72). However, the fact that only cholinergic neurons in the mesopontine – the Ch5 group in the PPTg and the Ch6 group in the LDTg – express the urotensin II receptor has allowed for the development of a selective fusion toxin (DTx/UII). The conjugation of the diphtheria toxin (DTx) catalytic portion and an active UII-targeting domain created a toxin that selectively binds to cholinergic PPTg neurons causing cell death over a period of 21 days (73). The only published report of behavioural effects of this toxin has to be interpreted with great caution as the behavioural effects in terms of changes in gait and posture are the result of the bilateral injection of DTx/UII in a concentration (20–30%) that causes non-selective lesions rather than selective cholinergic lesions, as the authors report themselves (74). In recent work in this lab (75), selective lesions of cholinergic neurons in the posterior PPTg made with DTx/UII that produced significant loss of cholinergic neurons (measured by choline acetyltransferase immunohistochemistry) but not non-cholinergic neurons (measured by NeuN immunohistochemistry) caused only minor behavioural effects. This raises intriguing questions about the substrate for the robust behavioural effects seen after excitotoxic lesions.

2.2. Stimulation

In the past, the stimulation of the PPTg has been achieved by electrical stimulation or neurochemical stimulation using the muscarinic cholinergic antagonist scopolamine or agonist carbachol, the GABA receptor antagonist bicuculline, N-methyl-d-aspartate (NMDA) or l-trans-pyrrolidine-2,4-dicarboxylic acid (PDC), which blocks l-glutamate uptake and presumably prolongs the

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activity of endogenous glutamate. Electrical and neurochemical stimulation of the PPTg has been shown to activate DA neurons in SNc and VTA (76, 77) and to increase burst firing without affecting population activity (78). Furthermore, PPTg stimulation causes changes to phasic DA release in the nucleus accumbens (79). In a more detailed analysis, stimulation of the LDTg showed that its excitatory input causes DA midbrain neurons to release DA in the ipsilateral striatum with a triphasic pattern of an initial rapid release, a short reduction in release and a sustained increase (80); it is thought likely that the same pattern follows PPTg stimulation. In order to examine the influence of the PPTg on neuronal activity of the thalamus, Fos expression was measured after PDC infusion into the PPTg – increased expression of Fos was seen in the centrolateral, ventrolateral and reticular nuclei (81). This stimulation caused no change in locomotion of the rats, so the increase in thalamic activity can be attributed to the PPTg stimulation itself rather than being an artefact of the injection procedures; care needs always to be taken in measuring Fos activity in freely moving animals. The use of a glutamate reuptake blocker is particularly helpful: the PPTg has substantial glutamatergic inputs. Direct injection of exogenous glutamate has little effects because it is rapidly removed by high affinity uptake processes. Structural analogues of glutamate can be problematic also, because of their capacity to induce excitotoxic lesions; this indeed is what excitotoxins are. Blockade of reuptake effectively prolongs the activity of endogenous glutamate – levels of glutamate might therefore achieve levels that are at the maximum of what might normally be physiologically present, but there is the virtue of achieving stimulation targeted through normal synaptic processes. 2.3. Electro­ physiological Recordings

Electrophysiological recordings of PPTg neuronal activity in experimental animals are not numerous – the difficulties of access to the PPTg are in part the cause of this – but they nevertheless allow examination of its physiological characteristics and are particularly interesting because they show a range of different activities in response to locomotion and voluntary movement. Garcia-Rill identified three types of neurons in the area of the PPTg that display rhythmic activity in decerebrate rats in response to locomotion (82). Non-bursting, possibly cholinergic Type II neurons relay feedback of sensory information from the spinal cord and provide the main inputs back into the thalamus and the SNc. They can be divided into ON and OFF cells. The ON cells show a tonic firing pattern during locomotion which ceases or decreases when locomotion stops. The OFF cells also exhibit a tonic firing pattern which increases before the cessation of locomotion and decreases when the locomotion frequency increases. Those neurons might be important for the maintenance of gait. Bursting non-cholinergic type I neurons show a bursting pattern of firing during locomotion

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which are primarily innervated by GABAergic GPi and possibly SNr which provide the main PPTg outputs to the spinal cord. These neurons might be important for the initiation of programmed movements, determining therefore the initiation and frequency of stepping (for review see (15)). Electrophysiological recording studies show that the PPTg receives polymodal sensory information and is involved in rewardrelated learning. In a conditioned lever-pressing task, cats were trained to hold down a lever until an auditory signal indicated that releasing the lever would lead to a food reward. PPTg neurons responded very early to the stimulus when the conditioned movement had to be executed as quickly as possible, which could be interpreted as a signal triggering the previously learned action. PPTg neurons furthermore reacted with a sustained response when the cat was expecting the reward and when the food reward was given (83). This resembles DA activity in SNc and ventral striatum in relation to reward and expectation of reward (57). To investigate further the involvement of PPTg neurons in reward-related responding PPTg neuron activity was recorded while monkeys were executing a visual saccade task. First of all PPTg neurons showed burst firing not only during saccades but also during target fixation, suggesting a role in relaying the signal indicating the necessary performance of goal-directed behaviour (84). The existence of abundant excitatory projections to midbrain DA neurons from the PPTg is consistent with Kobayashi’s hypothesis that the PPTg is involved in reward prediction error computing (85). Recording studies showed that during rewarded trials of a visual saccade task, the PPTg activity increased gradually after the onset of the fixation target and remained elevated until reward delivery, whereas the activity decreased during trials when no reward was delivered. According to the authors, this shows that a high level of prediction of an upcoming reward was associated with enhanced activity of PPTg neurons. This, together with an abrupt increase in firing in response to completely unpredicted rewards supports the claim of an implication of the PPTg in prediction error signalling (85). Subsequent studies identified neurons in PPTg that responded in a graded manner to stimuli predicting reward and a separate population of neurons that responded to the reward itself (86). In addition to these studies, the PPTg has been investigated for its role in sleep states, and in particular, transitions between REM sleep, slow wave sleep and the waking state. There is no doubt that PPTg neurons are active during the waking state and REM sleep, but quiet during slow wave sleep. As noted above, the PPTg has a profound control over thalamic activity – and hence cortical activation and information processing – that has been the object of study of sleep labs worldwide (see for example (4)). As is the case with locomotion, however, while the PPTg is most clearly

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involved with sleep regulation, this is only one among many functions and does not identify a unique function or the nucleus. 2.4. Imaging

Other methods of measuring neuronal activity are quantification of Fos-immunoreactivity, 2-deoxyglucose autoradiography, and in situ hybridization for the first subunit of cytochrome oxidase messenger RNA. Increased activity in the PPTg in 6-hydroxydopamine lesioned animal models for PD is shown by 2-deoxyglucose autoradiography (12) and measurement of cytochrome oxidase (10) provides support for a role of the PPTg in the physiopathology of PD. Given its proposed role in the regulation of nicotine reinforcement (87) Lanca et al. investigated the extent of nicotinic activation in the LTDg and PPTg (70) by measuring Fos expression after systemic administration of nicotine. Nicotine-induced Fos activity was found to be above baseline levels in both the PPTg and LDTg, and double labelling of Fos and nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d – this is a nitric oxide synthase that is, in this region of brain, selectively expressed by cholinergic neurons) showed that it is mainly non-cholinergic neurons that show nicotine-induced activity. These results are interesting especially in the light of later findings that show involvement of the PPTg in nicotine self-administration (88). Moreover, these animal data are consistent with human imaging studies that show elevated activity in the PPTg in abstinent nicotine addicts exposed to stimuli associated with the drug (47).

2.5. New Horizons: Optogenetics

Optogenetics is a new technique that allows for reversible activation of specific neuronal populations (see Plotkin et al., Volume I, chapter 10). Optogenetic techniques for activating or disabling neurons (depending on the opsin used) transiently have enormous potential for functionally dissecting the properties for targeted groups of neurons from within a heterogeneous population (89). Though not done yet, it is undoubtedly feasible to produce transgenic rat or mouse lines in which cholinergic neurons express Cre. Cre (Cre recombinase) is an enzyme that can be used to delete a segment of DNA flanked by LoxP sites (that is, “floxed”). The deletion effectively produces a mutation linked to specific cells. Deploying a CRE-inducible adeno-associated virus vector carrying the gene encoding the light-activated cation channel channelrhodopsin-2 (fused with yellow fluorescent protein; ChR2-EYFP) could allow optogenetic modulation of cholinergic neurons in the PPTg in a temporally accurate and reversible manner. Mouse models are available for this (90); for our purposes, the production of a rat model would have considerably more power given the more extensive behavioural repertoire of the lab rat compared to mouse.

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3. What Have We Learned? In this review, we have outlined a number of the ways in which it is currently possible – and might in the immediate future be possible – to manipulate or monitor the PPTg. Numerous labs around the world have engaged with experimental studies of PPTg functions using a raft of different techniques – lesioning it in whole or part to determine what the consequence of its absence are; stimulating it, recording from it or imaging it. What is clear from the literature is that, while once there might have been reluctance to engage experimentally with such a deep brain structure, the work of many labs – and using a range of lab animal species – has shown that it is (with care) perfectly possible to engage with the PPTg just as it is with forebrain structures where such techniques are used routinely. What is also of interest is that, en masse, the studies point not just to involvement of the PPTg with locomotion and sleep – the traditional functions – but to a much wider engagement with processes that might be called “cognitive”. These include processing relating to reward and reinforcement, learning, attention and the selection of appropriate actions. Anyone about to start examining the PPTg needs to take cognizance of this breadth of functional properties. Interest in the PPTg in regard to movement disorders has been greatly increased by the possibility that low frequency DBS here might improve gait and locomotion in Parkinsonian patients. There were two reasons for looking at the PPTg in regard to Parkinsonism (1) it is clearly altered in the disease, both through neuronal loss and through changed activity, most likely the result of changed input from the basal ganglia; and (2) it remains the case that the PPTg is considered still as a “motor structure” whose primary function is the production of coordinated movement. In this review, we have tried to shift the focus from the PPTg as a brainstem nucleus responsible for locomotion towards the idea that it has an even more complex function. This discussion resembles the debate about the “mysterious motor functions” of the basal ganglia in the 1980s (91). Since the late seventeenth century, the basal ganglia were thought to be the “seat of motor power”, a concept that held well into the twentieth century (92). The basal ganglia are undoubtedly involved in movement disorders but new knowledge about the anatomical and physiological properties of the basal ganglia made it impossible to maintain this idea of the basal ganglia as a structure of “pure” motor control. Therefore, the understanding of what a movement disorder actually is and the concept of motor control had to be reassessed. In the 1982, Robert Wartenberg Lecture David Marsden analyzed in detail those “mysterious motor functions” (91).

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Electrophysiological studies had revealed that the basal ganglia nuclei did not show activity in relation to specific movements but rather to more complex stimuli. Further, discrete lesions of the basal ganglia caused deficits similar of frontal lobe syndrome (a comparison we have also suggested for deficits observed after PPTg lesions (53)). The basal ganglia were seen to be involved in what could be thought of in some sense as non-motor functions. Marsden summarized: “the striatum takes in information from virtually all areas of the nervous system to undertake some unknown transformation into instructions to the output zones of the GPi and SNr whose own output is coded in relation to motor behavior” (p. 518) and that the basal ganglia were responsible for the “automatic execution of learned motor plans” (p. 534). This shifted basal ganglia function towards a function that critically involved learning. Disorders such as PD are very much understood in these terms – not as muscular disorders per se but involving problems with the selection and execution of actions. Similarly, we believe that the PPTg needs to be thought of as being involved not simply in the control of movement per se – the production of coordinated locomotion, gait and posture. The PPTg is evidently part of the basal ganglia family of structures and needs to be understood in “basal ganglia” terms as a structure concerned with learning about which actions should be expressed in response to particular stimuli. What makes it special in this context is its location: it functions as a basal ganglia output station but also regulates the basal ganglia through connections with the thalamus and midbrain DA neurons. In the past, we have compared the structure and function of the PPTg with substantia nigra, but two things in particular discriminate it from this (1) the extent of its descending connections that have control over motor (and associated autonomic) systems in the brainstem and spinal cord; and (2) the very fast activation by sensory stimuli. These could be taken to suggest that the PPTg contributes to forebrain activity, sending information upwards; that it has a part in mediating outflow from the forebrain; and that it has the capacity to operate independently, generating fast motor output in response to a first pass analysis of incoming sensory data (6). Its involvement in movement disorders will only be fully understood if we are able to contextualize them in terms of what the functions of the PPTg are.

4. Summary 1. There are good reasons for understanding the PPTg not as a “locomotor centre” responsible for the production of locomotion (in the sense of muscular activity), but as a nucleus strongly

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connected anatomically and functionally to the basal ganglia, and therefore involved in higher order functions important in processes of action selection. Motor control needs rather to be understood as a process that implicates the cognitive process of action selection. By attributing an integrative role to the PPTg, as the above outlined findings suggest, the PPTg should be understood as an important structure involved in this process of action selection. 2. Defining the PPTg as a part of the MLR carries a problem: Laboratories have based their studies and hypotheses regarding PPTg DBS in PD patients on this idea, but examination of the PPTg DBS literature shows that this is not the case and the stimulation of this area so far could not be confirmed to be significantly beneficial for gait and posture deficits in PD patients. 3. One of the major problems of working in the PPTg – in animal models and human functional neurosurgery – is stereotaxic access. This is possible, but requires careful attention. Outside of this, however, interventions in PPTg can be followed by all of the normal methods that one would expect to see applied in behavioural and cognitive neuroscience. 4. The idea of the PPTg as a target site for DBS remains exciting. Further and more detailed studies need to be carried out taking in consideration different factors such as the PPTg’s involvement in cognitive processes and its heterogeneous structural identity. What is the most beneficial target site? What should the stimulation parameters be in terms of stimulation frequency, pulse width and intensity to get an optimal effect? Which symptoms can actually be targeted with PPTg DBS? Controlled animal studies are necessary to address these questions. Combining those results with the ambitious and interesting work done in human PPTg DBS so far will hopefully lead to real therapeutic benefit for PD patients.

Acknowledgements The authors acknowledge the assistance of Duncan MacLaren in preparing this chapter. Work in our lab is supported by grants from the Wellcome Trust (081128/Z/06/Z) and the Medical Research Council (G0901332).

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26. Ferraye MU, Debu B, Fraix V, et  al. (2010) Effects of pedunculopontine nucleus area stimulation on gait disorders in Parkinson’s disease. Brain 133:205–214 27. Stefani A, Galati S, Pierantozzi M, et al. (2009) Motor and Non-motor Effects of PPN-DBS in PD Patients: Insights from Intra-operative Electrophysiology. In: Groenewegen HJ, Berendse HW, Cools AR, Voorn P, Mulder AB (eds) Basal Ganglia Ix. Springer, New York, pp 573–587 28. Alessandro S, Ceravolo R, Brusa L, et al. (2010) Non-motor functions in parkinsonian patients implanted in the pedunculopontine nucleus: Focus on sleep and cognitive domains. Journal of the Neurological Sciences 289:44–48 29. Garcia-Rill E, Houser CR, Skinner RD, Smith W, Woodward DJ (1987) Locomotion-inducing sites in the vicinity of the pedunculopontine nucleus. Brain Research Bulletin 18:731–738 30. Skinner RD, Kinjo N, Henderson V, GarciaRill E (1990) Locomotor projections from the pedunculopontine nucleus to the spinal cord. Neuroreport 1:183–186 31. Garcia-Rill E (1986) The basal ganglia and the locomotor regions. Brain Res 396:47–63 32. Lai YY, Siegel JM (1990) Muscle tone suppression and stepping produced by stimulation of midbrain rostral pontine reticular-formation. Journal of Neuroscience 10:2727–2734 33. Nandi D, Aziz TZ, Giladi N, Winter J, Stein JF (2002) Reversal of akinesia in experimental parkinsonism by GABA antagonist microinjections in the pedunculopontine nucleus. Brain 125:2418–2430 34. Kojima J, Yamaji Y, Matsumura M, et al. (1997) Excitotoxic lesions of the pedunculopontine tegmental nucleus produce contralateral hemiparkinsonism in the monkey. Neuroscience Letters 226:111–114 35. Aziz TZ, Davies L, Stein J, France S (1998) The role of descending basal ganglia connections to the brain stem in Parkinsonian akinesia. British Journal of Neurosurgery 12:245–249 36. Munro-Davies LE, Winter J, Aziz TZ, Stein JF (1999) The role of the pedunculopontine region in basal-ganglia mechanisms of akinesia. Experimental Brain Research 129:511–517 37. Takakusaki K, Habaguchi T, OhtinataSugimoto J, Saitoh K, Sakamoto T (2003) Basal ganglia efferents to the brainstem centers controlling postural muscle tone and locomotion: A new concept for understanding motor disorders in basal ganglia dysfunction. Neuroscience 119:293–308 38. Shojania K, Livingston CA, Pylypas S, Jordan LM, Nance DM (1992) Descending projections

of the mesencephalic locomotor region (MLR) based upon treadmill induced c-fos protein and anterograde tract tracing. Society for Neuroscience Abstracts 18:1410 39. Allen LF, Inglis WL, Winn P (1996) Is the cuneiform nucleus a critical component of the mesencephalic locomotor region? An examination of the effects of excitotoxic lesions of the cuneiform nucleus on spontaneous and nucleus accumbens induced locomotion. Brain Research Bulletin 41:201–210 40. Walker SC, Winn P (2007) An assessment of the contributions of the pedunculopontine tegmental and cuneiform nuclei to anxiety and neophobia. Neuroscience 150:273–290 41. Inglis WL, Dunbar JS, Winn P (1994) Outflow from the nucleus accumbens to the pedunculopontine tegmental nucleus: a dissociation between locomotor activity and the acquisition of responding for conditioned reinforcement stimulated by d-amphetamine. Neuroscience 62:51–64 42. Olmstead MC, Franklin KBJ (1994) Lesions of the pedunculopontine tegmental nucleus block drug-induced reinforcement but not amphetamine-induced locomotion. Brain Research 638:29–35 43. Ros H, Magill PJ, Moss J, Bolam JP, MenaSegovia J (2010) Distinct types of non-cholinergic pedunculopontine neurons are differentially modulated during global brain states. Neuroscience 170:78–91 44. Alderson HL, Winn P (2005) The pedunculopontine and reinforcement. Basal Ganglia VIII 56:523–532 45. Mena-Segovia J, Bolam JP, Magill PJ (2004) Pedunculopontine nucleus and basal ganglia: distant relatives or part of the same family? Trends in Neurosciences 27:585–588 46. Zahm DS, Williams EA, Latimer MP, Winn P (2001) Ventral mesopontine projections of the caudomedial shell of the nucleus accumbens and extended amygdala in the rat: Double dissociation by organization and development. Journal of Comparative Neurology 436:111–125 47. Maskos U (2008) The cholinergic mesopontine tegmentum is a relatively neglected nicotinic master modulator of the dopaminergic system: relevance to drugs of abuse and pathology. Br J Pharmacol 153 Suppl 1:S438-445 48. Smolka MN, Buhler M, Klein S, Zimmermann U, Mann K, Heinz A, Braus DF (2006) Severity of nicotine dependence modulates cue-induced brain activity in regions involved in motor preparation and imagery. Psychopharmacology 184:577–588

17  The Role of the Pedunculopontine Tegmental Nucleus in Motor Disorders 49. Whishaw IQ, Cioe JDD, Previsich N, Kolb B (1977) Variability of interaural line vs stability of bregma in rat stereotaxic surgery. Physiology & Behavior 19:719–722 50. Zrinzo L, Zrinzo LV, Tisch S, Limousin PD, Yousry TA, Afshar F, Hariz MI (2008) Stereotactic localization of the human pedunculopontine nucleus: atlas-based coordinates and validation of a magnetic resonance imaging protocol for direct localization. Brain 131:1588–1598 51. Fu YL, Gao WP, Zhu MW, Chen XG, Lin ZG, Wang SG (2009) Computer-assisted automatic localization of the human pedunculopontine nucleus in T1-weighted MR images: a preliminary study. International Journal of Medical Robotics and Computer Assisted Surgery 5:309–318 52. Shimamoto SA, Larson PS, Ostrem JL, Glass GA, Turner RS, Starr PA (2010) Physiological identification of the human pedunculopontine nucleus. Journal of Neurology Neurosurgery and Psychiatry 81:80–86 53. Rugg EL, Dunbar JS, Latimer M, Winn P (1992) Excitotoxic lesions of the pedunculopontine tegmental nucleus of the rat. 1. Comparison of the effects of various excitotoxins, with particular reference to the loss of immunohistochemically identified cholinergic neurons. Brain Research 589:181–193 54. Winn P (1998) Frontal syndrome as a consequence of lesions in the pedunculopontine tegmental nucleus: A short theoretical review. Brain Research Bulletin 47:551–563 55. Inglis WL, Olmstead MC, Robbins TW (2000) Pedunculopontine tegmental nucleus lesions impair stimulus-reward learning in autoshaping and conditioned reinforcement paradigms. Behavioral Neuroscience 114:285–294 56. Keating GL, Winn P (2002) Examination of the role of the pedunculopontine tegmental nucleus in radial maze tasks with or without a delay. Neuroscience 112:687–696 57. Keating GL, Walker SC, Winn P (2002) An examination of the effects of bilateral excitotoxic lesions of the pedunculopontine tegmental nucleus on responding to sucrose reward. Behavioural Brain Research 134:217–228 58. Schultz W (2002) Getting formal with dopamine and reward. Neuron 36:241–263 59. Alderson HL, Latimer MP, Blaha CD, Phillips AG, Winn P (2004) An examination of d-amphetamine self-administration in pedunculopontine tegmental nucleus-lesioned rats. Neuroscience 125:349–358 60. Olmstead MC, Munn EM, Franklin KBJ, Wise RA (1998) Effects of pedunculopontine tegmental nucleus lesions on responding for intravenous

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heroin under different schedules of reinforcement. Journal of Neuroscience 18:5035–5044 61. Alderson HL, Latimer MP, Winn P (2008) A functional dissociation of the anterior and posterior pedunculopontine tegmental nucleus: excitotoxic lesions have differential effects on locomotion and the response to nicotine. Brain Structure & Function 213:247–253 62. Alderson HL, Brown VJ, Latimer MP, Brasted PJ, Robertson AH, Winn P (2002) The effect of excitotoxic lesions of the pedunculopontine tegmental nucleus on performance of a progressive ratio schedule of reinforcement. Neuroscience 112:417–425 63. Samson HH, Chappell A (2001) Injected muscimol in pedunculopontine tegmental nucleus alters ethanol self-administration. Alcohol 23:41–48 64. Hodge CW, Haraguchi M, Chappelle AM, Samson HH (1996) Effects of ventral tegmental microinjections of the GABA(A) agonist muscimol on self-administration of ethanol and sucrose. Pharmacology Biochemistry and Behavior 53:971–977 65. Mena-Segovia J, Winn P, Bolam JP (2008) Cholinergic modulation of midbrain dopaminergic systems. Brain Research Reviews 58:265–271 66. Maskos U (2007) Emerging concepts: novel integration of in vivo approaches to localize the function of nicotinic receptors. Journal of Neurochemistry 100:596–602 67. Pan WX, Hyland BI (2005) Pedunculopontine tegmental nucleus controls conditioned responses of midbrain dopamine neurons in behaving rats. Journal of Neuroscience 25:4725–4732 68. Corrigall WA, Coen KM, Zhang JH, Adamson KL (2001) GABA mechanisms in the pedunculopontine tegmental nucleus influence particular aspects of nicotine self-administration selectively in the rat. Psychopharmacology 158:190–197 69. Sandberg K, Schnaar RL, McKinney M, Hanin I, Fisher A, Coyle JT (1985) AF64A: An Active Site Directed Irreversible Inhibitor of Choline Acetyltransferase. Journal of Neurochemistry 44:439–445 70. Hanin I (1996) The AF64A model of cholinergic hypofunction: An update. Life Sciences 58:1955–1964 71. Lanca AJ, Adamson KL, Coen KM, Chow BLC, Corrigall WA (2000) The pedunculopontine tegmental nucleus and the role of cholinergic neurons in nicotine self-administration in the rat: A correlative neuroanatomical and behavioral study. Neuroscience 96:735–742 72. Rodriguez M, MantolanSarmiento B, GonzalezHernandez T (1998) Effects of ethylcholine mustard azirinium ion (AF64A)

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on the choline acetyltransferase and nitric oxide synthase activities in mesopontine cholinergic neurons of the rat. Neuroscience 82:853–866 73. Book AA, Wiley RG, Schweitzer JB (1994) 192 IgG-Saporin .1. Specific lethality for cholinergic neurons in the basal ganglia forebrain of the rat. Journal of Neuropathology and Experimental Neurology 53:95–102 74. Clark SD, Alderson HL, Winn P, Latimer MP, Nothacker HP, Civelli O (2007) Fusion of diphtheria toxin and urotensin II produces a neurotoxin selective for cholinergic neurons in the rat mesopontine tegmentum. Journal of Neurochemistry 102:112–120 75. Karachi C, Grabli D, Bernard FA, et al. (2010) Cholinergic mesencephalic neurons are involved in gait and postural disorders in Parkinson disease. Journal of Clinical Investigation 120:2745–2754 76. MacLaren DAA, Wilson, D.I.G., Scott, N.W., Winn, P. Investigating the effects of lesions in the cholinergic mesopontine tegmentum on the locomotor response to nicotine. 491.13/ CCC14. 2010 Neuroscience Meeting Planner. San Diego, CA: Society for Neuroscience, 2010. Online. 77. Kelland MD, Freeman AS, Rubin J, Chiodo LA (1993) Ascending afferent regulation of rat midbrain dopamine neurons. Brain Research Bulletin 31:539–546 78. Chapman CA, Yeomans JS, Blaha CD, Blackburn JR (1997) Increased striatal dopamine efflux follows scopolamine administered systemically or to the tegmental pedunculopontine nucleus. Neuroscience 76:177–186 79. Lodge DJ, Grace AA (2006) The hippocampus modulates dopamine neuron responsivity by regulating the intensity of Phasic neuron activation. Neuropsychopharmacology 31:1356–1361 80. Floresco SB, West AR, Ash B, Moore H, Grace AA (2003) Afferent modulation of dopamine neuron firing differentially regulates tonic and phasic dopamine transmission. Nature Neuroscience 6:968–973 81. Forster GL, Blaha CD (2003) Pedunculopontine tegmental stimulation evokes striatal dopamine efflux by activation of acetylcholine and glutamate receptors in the midbrain and pons of the rat. Eur J Neurosci 17:751–762 82. Ainge JA, Jenkins TA, Winn P (2004) Induction of c-fos in specific thalamic nuclei following stimulation of the pedunculopontine tegmental nucleus. European Journal of Neuroscience 20:1827–1837 83. Garcia-Rill E, Skinner RD (1988) Modulation of the rhythmic function in the posterior midbrain. Neuroscience 27:639–654

84. Dormont JF, Conde H, Farin D (1998) The role of the pedunculopontine tegmental nucleus in relation to conditioned motor performance in the cat I. Context-dependent and reinforcement-related single unit activity. Experimental Brain Research 121:401–410 85. Kobayashi Y, Inoue Y, Yamamoto M, Isa T, Aizawa H (2002) Contribution of pedunculopontine tegmental nucleus neurons to performance of visually guided saccade tasks in monkeys. Journal of Neurophysiology 88:715–731 86. Kobayashi Y, Okada KC (2007) Reward prediction error computation in the pedunculopontine tegmental nucleus neurons. In: Balleine BW, Doya K, Odoherty J, Sakagami M (eds) Reward and Decision Making in Corticobasal Ganglia Networks. Blackwell Publishing, Oxford, pp 310–323 87. Okada K, Toyama K, Inoue Y, Isa T, Kobayashi Y (2009) Different Pedunculopontine Tegmental Neurons Signal Predicted and Actual Task Rewards. Journal of Neuroscience 29:4858–4870 88. Corrigall WA, Coen KM, Adamson KL (1994) Self-administered nicotine activates the mesolimbic dopamine system through the ventral tegmental area. Brain Research 653:278–284 89. Alderson HL, Latimer MP, Winn P (2006) Intravenous self-administration of nicotine is altered by lesions of the posterior, but not anterior, pedunculopontine tegmental nucleus. European Journal of Neuroscience 23:2169–2175 90. Deisseroth K, Feng GP, Majewska AK, Miesenbock G, Ting A, Schnitzer MJ (2006) Next-generation optical technologies for illuminating genetically targeted brain circuits. Journal of Neuroscience 26:10380–10386 91. Kravitz AV, Freeze BS, Parker PRL, Kay K, Thwin MT, Deisseroth K, Kreitzer AC (2010) Regulation of parkinsonian motor behaviours by optogenetic control of basal ganglia circuitry. Nature 466:622–626 92. Marsden CD (1982) The mysterious motor function of the basal ganglia – The Robert Wartenberg Lecture. Neurology 32:514–539 93. Lanska DJ (2010) Chapter 33: the history of movement disorders. Handb Clin Neurol 95:501–546 94. Paxinos G, Watson C (2005) The rat brain in stereotaxic coordinates. Elsevier Academic Press, San Diego. 95. Mena-Segovia J, Micklem BR, Nair-Roberts RG, Ungless MA, Bolam JP (2009) GABAergic neuron distribution in the pedunculopontine nucleus defines functional subterritories. Journal of Comparative Neurology 515:397–408.

Part IV Spinal Cord Systems

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Chapter 18 Contusion Models of Spinal Cord Injury in Rats Kelly A. Dunham and Candace L. Floyd Abstract Spinal cord injury (SCI) is a devastating condition affecting approximately 262,000 Americans with 12,000 new cases each year. In addition to the obvious motor and sensory deficits, SCI decreases life expectancy, independence and overall quality of life while increasing patient health care costs dramatically. Much has already been elucidated about SCI mechanisms and injury progression using rodent models of thoracic SCI; however, no pharmacological interventions have proven to be overwhelmingly effective in the human SCI population. Given the dire outcomes following SCI, discovery of novel pharmacological interventions for the treatment of SCI remains a top priority. The characterization and use of highly clinically relevant models of SCI are therefore of utmost importance in the quest to find pharmacological interventions as well as to further our understanding of SCI mechanisms and injury progression. The method outlined here describes a rat cervical hemicontusion SCI model which closely resembles the etiology and progression of human SCI. Application of more clinically relevant models of SCI, including this cervical hemicontusion model, will allow the field to move forward toward novel drug discovery and treatment of human SCI. Key words: Spinal cord injury, Translational studies, Cervical injury, Contusion, Rat model, Forelimb

1. Introduction 1.1. Classification of Spinal Cord Injury

Spinal cord injury (SCI) is a devastating and debilitating condition that affects an estimated 262,000 persons in the United States with approximately 12,000 new cases occurring each year (1). Motor vehicle accidents, falls, violence, and sporting accidents are the most common causes of SCI (2), affecting primarily young adults between the ages of 16 and 30 (1). Although there are various causes of SCI, the majority of cases can be classified into four injury categories based on gross morphology as well as imaging and histological assessments (3, 4), as summarized in Table 1. The first of these, solid cord injury, is characterized by a cord that grossly appears normal without discoloration, softening or

Emma L. Lane and Stephen B. Dunnett (eds.), Animal Models of Movement Disorders: Volume II, Neuromethods, vol. 62, DOI 10.1007/978-1-61779-301-1_18, © Springer Science+Business Media, LLC 2011

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Normal

Meningeal disruption, connective tissue scar

Loss of cord topography

Fluid-filled cyst, rim of spared tissue surrounding epicenter, no connective tissue scar

Solid Cord Injury

Laceration

Maceration (compression)

Contusion

20 50

Hemorrhage, necrosis, glial scar

20

Fiber disruption across the lesion, systemic cell infiltration Fiber disruption across the lesion

10

Clinical frequency (%)

Demyelination, motor neuron loss

Histological features

Methods this chapter

Overview this chapter

Bates et al., ch. 20

Felts et al., ch. 19

Chapter

Full or unilateral contusion, MASCIS, OSU, IH or other modified weightdrop devices

Aneurysm clip, epi- or sub-dural balloon

Full or hemi-transection, selective tract suction

None

Animal models

a Summary of features of main types of SCIs and the applicable animal model. The four most common types of SCI observed clinically are listed in the leftmost column. The main clinical features of each are highlighted. The animal model that best mimics each type of SCI is listed and the chapter in this volume that discussed that model is referenced, when applicable

Gross features

Injury type

Clinical features

Table 1 Features of spinal cord injury typesa

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cyst formation. However, upon histological analysis dorsal column demyelination and motor neuron loss can be observed (3). Injuries belonging to this category are rarely observed clinically (10% of patients) and currently there are no animal models in use to study this type of injury. The second category is that of laceration, which is most commonly observed clinically due to penetrating objects or sharp bone fragments (3). Injuries of this type are characterized by disruption of the meninges resulting in widespread systemic cell infiltration and a connective tissue scar formed at the lesion epicenter that often adheres to the overlying dura (2, 3). Animal models of lacerating injury include bilateral or hemitransection of the cord in which the cord is exposed and tissue severed with a sharp object. Alternatively, suction may be applied to selectively ablate motor or sensory tracts (5). Benefits of laceration models include the ease with which one can target lesion site and size as well as the potential ability with which this model can be used to quantify regeneration techniques following injury (5). This injury type, however, is observed relatively rarely clinically (21% of cases (6)), and as such, laceration models do not reflect the majority of human SCIs. The third ­category of injury is that of cord maceration or compression. This injury results from massive compression of the cord resulting in large disruption of the spinal cord anatomy such that cord topography is lost (2, 3). Very few fibers are observed crossing the length of the lesion, distinguishing this injury type from that of contusion. Compression injury is modeled in animals using aneurysm clips (7) and epidural (8) or subdural (9) balloons resulting in prolonged disruption of blood flow to cord tissue (5). About 20% of clinical cases fall into the compression category. The fourth injury category is that of contusion, characterized by contusive injury to the spinal cord resulting in areas of hemorrhage and necrosis that ultimately give rise to a fluid-filled cyst at the lesion epicenter with a rim of spared (although generally dysmyelinated) tissue at the pial surface of the cord (3, 10). The meninges remain intact resulting in less systemic cell infiltrate and a scar surrounding the lesion consisting of virtually no connective tissue components, distinguishing this injury type from laceration injury. The majority of clinical cases of SCI (70%) are contusivetype injuries with some degree of compression, acute or chronic, resulting from fracture/dislocation of the spinal column and ­subsequent compression of the cord (4, 5, 10). As such, there are many animal models of this type of SCI that have been shown to have pathophysiological elements comparable to those observed in human cases of SCI (5). 1.2. Animal Models of Contusive SCI

Various mechanisms to induce contusion injuries to the spinal cord have been employed for almost a century and have evolved from simple weight-drop techniques to sophisticated computer-controlled

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devices capable of controlling precise injury parameters. These advances have resulted in animal injury models that have been well characterized, are reproducible, and produce models with a high degree of relevance to human SCI pathophysiology (4). As contusion and compression often both contribute to observed cases of SCI (5), both types of injury models will be described here. The earliest contusion model was that described by Allen in 1911 in which weights were dropped onto canine spinal cords exposed by laminectomy (11). By dropping weights from different distances gravity is employed to apply differing forces and result in differing compression of the spinal cord. Others later applied this model to make the first assessments of histological changes (12), vasculature impairments (13), myelin breakdown and edema (14) following SCI. In 1975, force transducers were added to the weight-drop model to quantify the force of impact in primate models (15). Subsequently, canine, feline, and primate models were used to describe progressive tissue damage processes in finer detail as well as the effects of putative therapeutics in SCI (16, 17). Metabolic dysfunction, myelin breakdown, free radical production, respiratory function, ionic shifts, blood–brain-barrier breakdown, cellular immunity, glutamate release and the roles of necrotic and apoptotic cell death were described as reviewed by Young (10). This gave rise to the current understanding of a biphasic injury process in which primary mechanical tissue trauma causes complex, interacting biochemical processes resulting in ­secondary progressive tissue damage (2, 18). The elucidation of these delayed secondary injury mechanisms formed the rationale for using therapeutics targeting these processes to limit motor deficit. In 1985, a weight-drop contusion device similar to those described previously was used in a rat model (19, 20). Use of contusion SCI models in the rat now dominates the field of SCI research and has led to quantitative assessments of injury-induced alterations in ascending and descending fiber tracts, neurons and neuronal networks, gliosis, and motor and sensory functional deficits/recovery (10). Indeed the effects of methylprednisolone, the only clinically accepted acute pharmacological treatment of SCI, were evaluated pre-clinically using the contusion model in the rat (21). 1.3. Physical Characteristics of Contusive SCI

All models of contusive SCI essentially involve the application of a force to the spinal cord, leaving the meningeal layers intact (3). The spinal cord is tolerant of acute compression or stretching by 1/3 its normal length with no significant damage if the deforming force is applied slowly. However, prolonged deformations (longer than 20  min) or rapid deformations (past the critical velocity of 0.5–1.0 m/s) result in cell damage and areas of infarct (10, 22). Contusive forces cause tissue to move along the rostral-caudal axis of the spinal cord with the movement being greatest in the

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middle of the cord and decreasing toward the pial surface and with greater distance from the site of contusion (10). These differential movements account in part for the distribution of tissue damage typically observed following contusive SCI. Longitudinal movement of tissue shears cells in the gray matter and stretches axons in the white matter (10). Myelinated axons stretch and ultimately break at the nodes of Ranvier when stretching velocities approach 0.5–1.0 m/s, however lower stretching velocities can also result in axonal damage by interfering with membrane channel function, leading to alterations in intracellular ionic concentrations. Large myelinated axons are most vulnerable to contusion, with those at the pial surface most likely to survive (23). This results in a spared rim of tissue surrounding the lesion epicenter. Smaller axons are more likely to be damaged in response to ischemic insults as a result of their smaller surface area to volume ratios (23). However, most cases of SCI include both contusive and compressive components and as such both injury processes are likely to be at work (4, 5). In contusive SCI models, it has been shown that the rate of cord compression correlates linearly with tissue damage and behavioral recovery (10). 1.4. Contusion Impactors and Devices

The weight-drop device employed by Allen in 1911 was composed of weights dropped from differing heights onto the exposed spinal cord, and as such, employed gravity to apply differing forces onto the spinal cord (11). Limitations of early impactor devices included the lack of precise force measurements being applied to the cord, inability to account for displacement of the vertebral column and movement of the animal during impact, and limited range of forces available to the experimenter. Injury parameters have since evolved from gram-centimeter used to describe the height of a weight dropped onto exposed spinal cord to more precise parameters describing the physical nature of the injury such as force applied, tissue compression, impact velocity, and duration (24). The New York University (NYU) impactor is a weight-drop device designed in 1989 to circumvent some of the limitations of earlier impactor systems (25). Briefly, a 10-g rounded steel rod with a diameter slightly smaller than the vertebral column is threaded onto a rod and placed into a tube positioned above the dorsal aspect of the spinal cord exposed by laminectomy. The weight is then dropped from various vertical heights through the tube directly onto the spinal cord at the mid-thoracic level. The spinal column is supported and suspended to prevent impact energy absorption by the animal’s body mass and to minimize column displacement. Digital optical potentiometers assess the location and timing of the dropped weight providing information about the velocity of the weight at maximum impact as well as biomechanical parameters upon impact (26). These sensors also serve to measure displacement of the spinal column whereupon this extraneous

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movement is subtracted to give accurate measurements of spinal cord tissue displacement. These features increase the reliability and reproducibility of the device and allow for exclusion of impact severities which fall outside accepted values. This system, however, does not account for rebound of the weight and a second impact from about 10% of the original height occurs, possibly adding ­significant injury effects (27, 28). The Ohio State University electromagnetic spinal cord injury device (ESCID) was designed in 1992 and is distinguished from other injury devices in that it utilizes an impactor driven by a solenoidcontrolled device as opposed to weights dropped from various heights to initiate injury (24). This device measures cord displacement as well as the force imparted to the tissue and a range of injury severities can be produced by the researcher by specifying tissue displacement values. Rapid contusive injuries (<25 ms) are produced that eliminate rebound to generate a single impact. The ESCID is adaptable to both mice and rats. The Infinite Horizon (IH) impactor (Precision Systems and Instrumentation, Lexington, KY) is a commercially available impactor system (see Fig. 1). This device differs from the ESCID in that the parameter used to define injury severity is the force applied to the tissue as opposed to tissue displacement (4, 29). The device makes use of a stepping motor that moves the impactor tip vertically with a precision of 1/1,200th of an inch and superfast microcontrollers that sample the force applied at the impactor tip to allow a high precision feedback. This results in a highly accurate injury device and reproducible injury effects. Measurements of applied force and tissue displacement over the course of the impact sequence as well as impactor tip velocity at the time of maximal impact are immediately displayed, thus decreasing error introduced by displacement of the spinal column and allowing the experimenter to assess injury accuracy. Additionally, various compression durations can be implemented in which the impactor tip is held at the preselected force on the surface of the spinal cord for a predetermined time period to model a contusion/compression type injury. As with the ESCID, the IH Impactor eliminates complications caused by rebound of the impactor tip onto the tissue and a wide range of tip diameters allows experimenters to create full contusions in rats and mice as well as rat hemicontusions. As cord diameter changes with animal age and this parameter can change the effects of impact onto the tissue (10), multiple impactor tip sizes allow a wide age range of animals to be utilized with this device. This model has been characterized in both thoracic and cervical SCI and behavioral as well as recovery data are in agreement with those described using the NYU impactor (1, 29, 30). The main advantages of the IH impactor include the increased range of injury severities produced, the ability to produce contusion/

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Fig. 1. Picture of the Infinite Horizons (IH) impactor device. The IH impactor device is used in the protocol described in this chapter. The device comprises an impactor tip which is driven by a stepping motor and controlled by feedback from force and position sensors. The animal is stabilized by clamping Adson micro-forceps onto the spinal column. The spinal cord contusion is induced by impacting the spinal cord with the impact tip. The spinal cord was exposed by a laminectomy at the vertebral segment(s) of interest.

compression-type injuries, the flexibility with which the experimenter can change impactor tip sizes to suit their needs, and the fact that it is commercially available. 1.5. Cervical SCI

The early models of contusive SCI were performed in the midthoracic region of the spinal cord resulting in hindlimb deficits while allowing the animal to ambulate to some extent via preserved function of the forelimbs (10). The mid-thoracic injury model has been used throughout the last century and has increased our understanding of SCI pathophysiology and progression substantially and led to characterization of widely used quantitative analyses of hindlimb motor and sensory function. However, over 50% of clinical cases of SCI occur in the cervical region (31). Indeed, clinical cases of SCI rarely occur at the

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mid-thoracic region for several reasons (3, 32). First, the cervical spinal column is highly flexible and as such the vertebrae are relatively unstable and at greater risk for fracture than at the midthoracic region where the vertebrae are stabilized by the ribcage (22). Second, a smaller epidural space in the cervical region increases the likelihood that fracture of the vertebrae will result in impingement upon the spinal cord (22, 24). Although over 50% of clinical cases of SCI occur in the midcervical region, most research is performed using a mid-thoracic injury model (31). Several key anatomical differences between these two regions, however, question the application of results obtained from mid-thoracic models to a predominantly cervical SCI clinical population (31). The first of these is the relative gray–white matter distribution. The thoracic cord is composed almost entirely of white matter, with the gray matter innervating the small intercostal musculature (31). Alternatively, the cervical cord has the largest gray matter area by volume of all spinal cord regions, as lower motor neuron pools innervating the upper extremity are housed in this region (33). As such, deficits evaluated in animal models of midthoracic SCI are primarily attributable to white matter damage, whereas deficits following cervical injury include both white and gray matter pathology (30, 31). As the physical properties, metabolic demands, and cell types of white and gray matter differ, it stands to reason that the pathophysiology following SCI may also differ between these two cord regions (24). Secondly, target innervations of these regions differ. As mentioned previously, functional deficits resulting from mid-thoracic injury are typically quantified through hindlimb motor function and hind-fore limb coordination which is based on hindlimb ­locomotor abilities that are attributable to white matter sparing. In contrast, the cervical region controls the upper extremities in both rats and humans. For example, carbanocyanine tracing studies have demonstrated that cervical motor neuron topography is conserved between rats and humans and that rats are capable of complex prehensile movements of the forepaw comparable to those seen in the human hand (33, 34). Consequently, quantitative assessments of forelimb function have recently been characterized in rodent ­models of cervical SCI (30–32, 35). These assessments appear to be capable of demonstrating more subtle changes in motor function than the classical hindlimb function assessments, which may reveal subtle effects of potential therapeutics that may be missed if employed in a mid-thoracic injury model. As cervical spinal cord lesions result in fore- and hindlimb ­dysfunction, hemicontusion models have been developed to ensure mobility and health of the animal (36). The motor and sensory deficits observed following this type of injury reflect those observed in the clinical example of Brown-Sequard syndrome, a unilateral injury of the spinal cord (37). The corticospinal tract (CST) is the

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main descending motor tract. Since the majority of CST fibers decussate in the medulla (pyramidal decussation) and therefore above the level of the lesion, the C5 hemicontusion injury results in impairments in motor function in the ipsilateral forelimb and less pronounced deficits in the ipsilateral hindlimb. No motor deficits are observed contralateral to the injury (30, 32, 34). The spinothalamic tract, the main ascending tract responsible for pain sensation, decussates at the level it enters the spinal cord. Therefore, a C5 hemicontusion injury results in contralateral ­neuropathic pain-like behaviors in dermatomes below the level of the lesion and ipsilateral in the dermatome corresponding to the lesion site. Taken together, these studies indicate that a cervical hemicontusion injury model in rats appears to correlate well with motor and sensory deficits observed in human cases and can be a powerful tool in the quest to discover novel interventions for the treatment of SCI.

2. Materials 2.1. Tools for Pre-surgical Preparation of Rats (See Note 1)

Electric fur trimmer or other method for removal of fur

2.2. Surgical Tools

Ear Punch (Fine Science Tools 24210-02) (see Note 2)

Petroleum ophthalmic ointment (e.g., PURALUBE ointment) Povidone Iodine 5% solution (e.g., BETADINE solution) Antimicrobial Skin Cleanser (e.g., 2% chlorhexidine scrub, CHLORHEXIDERM solution)

Scalpel handle (Fine Science Tools 10003-12) and blade(s) (Henry Schein 100-8976) Heiss tissue retractor (Fine Science Tools 17011-10) Tissue forceps (Fine Science Tools 11023-10) Vannas spring scissors (Fine Science Tools 91500-09) Friedman-Pearson rongeur (Fine Science Tools 16021-14) Olsen-Hegar needle holder (Fine Science Tools 12102-12) 2.3. Disposable Supplies for Surgery

Sterilization wrap (Henry Schein 100-6950) (see Note 3) Cotton balls size zero (Richmond Dental, 100505) Non-woven gauze sponges 2″ × 2″ (Butler Animal Health Supply, 019923) Cotton-tipped applicator sticks (Fisher 23-400-101)

2.4. Anesthesia and SCI Surgery

Ketamine KETAVED 100 mg/ml (Vedco) and xylazine ANASED 100 mg/ml (Lloyd Laboratories) (see Note 4)

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T/Pump heating pad (Gaymar) Fiber-Lite MI-150 illuminator (Dolan-Jenner 660000391010) Suprasorb absorbable 3/0 suture (Vedco) Non-absorbable monofilament 3/0 suture (Monomid) Sterile latex gloves (Cardinal Health 2D7251) 2.5. Recovery and Post-operative Care

Ringers solution (Hospira 0409-7953-03) Carprofen, injectable (RIMADYL) 50 mg/ml (Pfizer): dilute 1:10 with sterile water Enrofloxacin, injectable (BAYTRIL) (Bayer) Vetwrap bandaging tape (3M, 1404) Nutri-cal dietary supplement Chew Guard Spray (or other topical bite deterrent) Antiseptic first-aid spray (e.g., Solarcaine first aid medicated spray) Topical lidocaine gel (e.g., TOPICANE topical gel)

3. Methods for Cervical Hemicontusion (See Note 5) 3.1. Surgical Preparation (See Note 6)

1. At the surgical preparation station, administer anesthesia to rat as approved by Institutional Animal Care and Use Committee (IACUC). For example, administer ketamine/xylazine cocktail (100/10 mg/kg) via intraperitoneal injection. 2. Shave and aseptically prepare the neck region with repeating betadine and/or chlorhexidine scrubs. 3. Apply sterile ophthalmic ointment to the cornea, carefully without touching or damaging the fragile tissue. 4. Once the animal has reached a depth of anesthesia appropriate for surgery, move to the surgical procedure station (see Fig. 2).

3.2. Surgical Procedure (See Note 7)

1. Place the animal on a warming pad to maintain body temperature throughout surgery. Cover animal with sterile drape, exposing only surgical site as appropriate. 2. Palpate the prominent spinous processes of the second cervical vertebrae (C2) and second thoracic vertebrae (T2) and make a midline incision through the skin between these two bony landmarks. 3. Make a midline incision completely through the superficial musculature from C2 to T2. The musculature will separate revealing the deeper cervical musculature. A fissure between the right and left sides will be apparent. Separate the deep musculature down the midline with a blunt probe, no cutting is necessary (see Note 8).

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Fig. 2. Pictures of rodent surgery station where SCI surgery (laminectomy) is conducted. Pictured in panel (a) is a typical organization of the rodent surgery station. The rat is placed on the heat source (labeled as heating pad) to maintain body temperature. Inhalant anesthesia (e.g., isoflurane) can be administered through a nose cone and regulated via an attached anesthesia vaporizer and flow regulator. A light source with fiber optic light guides improves the visualization of the anatomical landmarks during surgery without producing heat on the tissue. A spine elevator can be positioned under the animal to improve the orientation of the spinal column. Both sterile suture and sterile gloves are used to maintain the sterility of the surgery. Adjacent to the heating pad as pictured in panel (b), the surgical instruments (labeled as sterile tools ) and surgical supplies are sterile and maintained in a sterile field. A waste bin for non-hazardous waste is positioned near the sterile field for disposal of soiled surgical materials. All sharps are disposed of in an appropriate bin (sharps disposal ). A chemical sterilant is on hand to wipe surfaces of the spinal impactor, as appropriate.

4. Using tissue retractors, separate the left and right sides of both muscle groups, revealing the deep paravertebral musculature directly overlying the spinal column. Make bilateral longitudinal incisions in the musculature along either side of the spinal column from C2 to T2. 5. Using forceps grasp the deepest musculature and remove completely with spring scissors.

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If necessary, reposition retractors so the cervical vertebral column can be visualized. Using the scalpel, remove any remaining muscle tissue from the dorsal spinal column. Use cotton-tipped swabs to clear and dry the dorsal aspects of the vertebrae. 6. Using a blunt probe, identify the prominent vertebral arch of C2 and use this landmark to identify C5. Alternatively, identify the vertebral arch of T2 to identify C5 (see Note 9). Firmly grasp the vertebral body of C2 with tissue forceps and slightly lift the entire vertebral column upward (see Note 10). This minimizes the vertebral curvature to perform the laminectomy. Using rongeurs gently remove the intervertebral tissue rostral and caudal to the C5 vertebral body. The spinal cord will be visible. 7. Without touching the spinal cord, conduct a bilateral laminectomy by carefully removing the dorsal aspect of the lamina of C5 using rongeurs (see Fig. 3a and Note 11). Ensure that there are no sharp edges or fragments of bone protruding. A surgical microscope or surgical loops may be used, as appropriate. 8. Place animal on spine stabilization platform (fixation plate) provided for use with the IH Impactor (see Fig. 3b). Stabilize the spinal column using toothed Adson forceps connected to supporting arms on the platform. Clamp the spinous process of T2 and the vertebral body of C2 (see Fig. 3b). Tighten the clamps only enough to stabilize animal as over-tightening can fracture C2 and/or damage the spinal cord. Ensure the dorsal aspect of the spinal cord is perpendicular to the impactor tip. Check that the body of the rat is slightly elevated from the surface of the stabilization platform. 9. Align the impactor tip over the area to be contused (see Fig. 3b and Note 12). Lower the tip to the point that it makes contact with the cord but does not result in movement of the animal. Move the tip upward vertically by turning the z-axis manual position controller by one full turn. 10. Using the IH controller software, impact cord at the desired pre-selected force (see Note 13). Remove the animal from the stabilization platform. Replace animal on heating pad at the surgery station and replace the sterile drape. 11. Suture the deep and superficial musculature in layers using Vicryl absorbable suture. Make two sutures for each layer. Avoid placing a suture directly over the laminectomy/contusion site. Using non-absorbable suture, bring the skin together and suture tightly (see Note 14). 12. Record and/or save impact parameters for future reference.

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Fig. 3. Illustration of the C5 laminectomy and set-up for the hemicontusion SCI. Panel (a) illustrates the positioning of the forceps, tissue retractor, and rongeurs for conducting a laminectomy at C5. Panel (b) illustrates the stabilization of the spinal column and alignment of the impactor tip prior to induction of the C5 hemicontusion injury. Note that the Adson micro-forceps of the fixation plate are clamped on C2 and T2. The impactor tip is centered (along the rostral/caudal axis) in the C5 segment and located laterally as to impact the dorsal surface of ½ of the exposed spinal cord. Note that the surgical drape is omitted from both the illustrations so the anatomy of the rat and resultant positioning of the instrumentation can be better appreciated.

3.3. Post-surgical Recovery

1. Create the surgical recovery station by warming clean cage(s) from underneath with a heating pad(s) (lowest setting). Either remove or cover bedding to prevent accidental aspiration by recovering animals. Ensure that food and water are accessible to injured animals (see Note 5). 2. Fashion a compression bandage by cutting two holes in a strip of Vetwrap through which the forelimbs can be inserted.

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Place 1–2 pieces of non-woven gauze over the incision site and secure with Vetwrap. As the Vetwrap will compress the wound, ensure that the animal can move and breathe unhindered by the bandage. 3. Administer 0.1  mg/kg Rimadyl/Baytril and 3  ml sterile Ringer’s solution subcutaneously. 4. Place animal in a clean, warm cage at the recovery station. Animals may be housed with cage mates (i.e., multiple animal housing) after awaking from anesthesia and return of ambulation (as appropriate for the level and severity of SCI). 3.4. Daily Care of SCI Animals

Animal care should be performed at least twice daily (a.m. and p.m.) for the duration of the experiment. A list of responsibilities and frequency for care of SCI animals follows: 1. Evacuation of the urinary bladder: Depending on the location and severity of the SCI, many animals will experience spastic bladders with concomitant urinary retention. In these cases, the urinary bladder must be manually evacuated multiple times per day (at least 2× day and up to 4× per day) at regular time intervals (see Note 15). This is done by the Crede method such that the abdomen of the rat is compressed by applying a continuous pressure, usually with the experimenter’s forefinger and/or thumb. The pressure should be enough to expel the urine but not too much as to damage the bladder. Male animals require greater abdominal pressure for the Crede method than female animals. 2. Measurement of body weight: Once per day, preferably each morning, weigh the animals and track body weight. If animal weight is 10 g less than pre-operative weight or 5 g less than the previous day’s value, give 1 cc Nutri-cal dietary supplement orally at morning and evening care sessions. 3. Administration of fluids, antibiotics, and pain medication: Every day for the first 5 days post-SCI, administer 3 cc sterile Ringer’s twice per day (a.m. and p.m.). Also administer systemic antibiotics and pain medication (e.g., enrofloxacin and carprofen) according to IACUC protocol. For example, we administer Baytril once per day in the a.m. and carprofen in the a.m. and p.m. for 5 days post-SCI. 4. Survey animals for signs of discomfort: At least once per day, evaluate the animals for overt signs of distress/discomfort. The two most common post-SCI complications we have observed with the C5 hemicontusion SCI are contralateral biting of the hind paw and ipsilateral over-grooming of the face and back. We apply topical lidocane gel and bite deterrent spray on the affected area (a.m. and p.m.) to alleviate discomfort and reduce the behavior until the behavior resolves.

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4. Notes 1. The authors have no affiliation with any company or brand. However, when possible, we have supplied specific manufacturers and catalogue numbers as a guide. Several manufacturers and distributors for surgical instruments and supplies are available and comparable products from other companies could readily be substituted for those listed. 2. In our experience, housing the animals in groups (3–5/cage) reduces stress and improves the health of post-surgical animals. However, this renders animal identification a critical issue. We use a combination of ear punches and tail markings to identify animals. If another identification method is used, the ear punch is unnecessary. 3. To limit post-operative complications, it is important that sterile surgical tools and surgical field be maintained throughout surgery. Surgical packs are made prior to surgery by cutting sterilization wrap into 18″ × 11″ rectangles. A second rectangle 5″ × 3″ is cut and a diamond shape is cut out of the middle to make a sterile drape. This surgical drape, a small handful of cotton balls, gauze sponges, and cotton-tipped applicators are added to the middle of the larger rectangle and it is closed up and sealed with autoclave tape. We autoclave all surgical tools and surgical packs prior to surgery as a more cost-effective alternative than purchasing sterile materials. 4. The anesthesia regimen should be in accordance with the Guide for the Care and Use of Laboratory Animals and only with approval of the Institutional Animal Care and Use Committee, or equivalent. We use a cocktail of ketamine/ xylazine (100 mg/kg and 10 mg/kg) administered by intraperitoneal injection. 5. The methods described here detail the procedure for inducing a hemicontusion injury at the vertebral level C5. These same techniques can be applied with slight modification to other levels of the rat spinal cord including thoracic and lumbar regions. Also, the same techniques can be applied to a bilateral contusion using the 2.5 mm impactor tip with the IH Impactor as described by Scheff et al. (29). 6. To maintain sterility of the surgical field, three separate stations are set up: a surgical preparation station, a sterile surgical station, and a post-surgery recovery station. These stations can be in the same room but in a separate area. 7. All surgical and post-surgical care protocols should be conducted in accordance with the Guide for the Care and Use of

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Laboratory Animals and only with approval of the Institutional Animal Care and Use Committee, or equivalent. 8. For the recovery of the animals, it is important to make ­controlled midline incision with as minimal cuts as possible. Careful surgical technique will minimize bleeding. This is of particular concern in the cervical region as there is extensive musculature covering the spine but also is important in the thoracic and lumbar regions. 9. Proper identification of vertebral segments is crucial to ensure induction of the injury at the proper spinal level. It is useful to have available anatomical diagrams such as those included in A Colour Atlas of Anatomy of Small Laboratory Animals, Volume 2 (38). As an additional reference tool, we use an anatomically mounted rat skeleton that was purchased from a distributor of scientific education materials such as http://www.naturewatch.com, item #805p. 10. We place an elevating pad (spinal elevator) under the animal to position the spinal column in a straighter orientation, facilitating the laminectomy. 11. If any muscle movement or twitch is observed during this ­procedure, the spinal cord has been mechanically stimulated and potentially damaged by the surgical instruments. Care should be taken to avoid touching the spinal cord. It is generally helpful to lift the spinal column higher (i.e., step 9) to open more space for the rongeur tips. Also, only insert the tips of the rongeur and break small portions of the lamina to ­complete the laminectomy. 12. For the C5 cervical hemicontusion, ensure that the position of the impactor tip is correct. The tip should be positioned in the middle of the C5 segment along the rostral-caudal axis so that motor neuron pools in the distal portions of C4 or C6 are not directly contused. Additionally, the tip should be just lateral to the dorsal sulcus to ensure a unilateral contusion; however, the tip should not be positioned too far lateral as to “slide” off the side of the spinal cord during impact. 13. The IH Impactor functions as follows: A pre-selected force value is entered into the controller software. The stepping motor applies a force to move the impactor tip downward one step whereupon the force applied to the impactor tip is sampled. If the force at the tip is less than the desired force, the motor drives the impactor tip downward again, at which time force is sampled again. The device continues this sequence of downward movements until the force at the impactor tip is equal to or slightly exceeds the preselected force. At this point, the downward movement of the tip is reversed and the tip is withdrawn back to the start position. Since the steps of the motor are very small

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(1/1,200th of an inch) and the sampling time rapid, the entire process appears to occur as one movement. 14. If  inducing a cervical SCI, the sutures should be spaced 2–3 mm apart. However, if inducing a thoracic or lumbar SCI, the sutures can be placed 5–6 mm apart. This is due to the posture of the animal and that there is a greater range of motion associated with the neck than the trunk. 15. In all cases, the bladder should be palpated daily to check for distension associated with urine retention. In our experience, animals with a C5 hemicontusion will have very limited retention of urine and will resume voluntary micturition within 1 week post-SCI. However, rats with SCIs induced in the thoracic and lumbar regions typically will have prolonged deficits in micturition and will require Crede method management for weeks to months post-SCI. Return of micturition is dependent on injury severity and tissue sparing, and as such should be carefully monitored and documented. References 1. National Spinal Cord Injury Statistical Center (2010) Spinal Cord Injury Facts and Figures at a Glance. www.nscisc.uab.edu. Accessed 6 May 2010 2. Norenberg MD, Smith J, Marcillo A (2004) The pathology of human spinal cord injury: Defining the problems. J Neurotrauma 21: 429–440 3. Bunge RP, Puckett WR, Becerra JL et al (1993) Observations on the pathology of human spinal cord injury. A review and classification of 22 new cases with details from a case of chronic cord compression with extensive focal demyelination. In: FJ Seil (ed) Advances in Neurology. Raven Press, Ltd., New York 4. Grill RJ (2005) User-defined variables that affect outcome in spinal cord contusion/compression models. Exp Neurol 196:1–5 5. Rosenzweig ES, McDonald JW (2004) Rodent models for treatment of spinal cord injury: research trends and progress toward repair. Curr Opin Neurol 17:121–131 6. Stokes BT, Jakeman LB (2002) Experimental modelling of human spinal cord injury: a model that crosses the species barrier and mimics the spectrum of human cytopathology. Spinal Cord 40:101–109 7. Rivlin AS, Tator CH (1978) Effect of duration of acute spinal cord compression in a new acute cord injury model in the rat. Surg Neurol 10:38–43 8. Vanicky I, Urdzikova L, Saganova K et al (2001) A Simple and Reproducible Model of Spinal

Cord Injury Induced by Epidural Balloon Inflation in the Rat. J Neurotrauma 18:1399–1407 9. Martin D, Schoenen J, Delree P et  al (1992) Experimental acute traumatic injury of the adult rat spinal cord by a subdural inflatable balloon: methodology, behavioral analysis, and histopathology. J Neurosci Res 32:539–550 10. Young W (2002) Spinal cord contusion models. In: L McKerracher, G Doucet, S Rossignol (eds) Progress in Brain Research. Elsevier Science B.V. 11. Allen AR (1911) Surgery of experimental lesion of spinal cord equivalent to crush injury of fracture dislocation of spinal column. A preliminary report. JAMA 57:878–880 12. Guth L, Richardson KC, Baker CA et al (1977) Neurohistological and enzyme histochemical staining of adjacent sections in series cut from normal and traumatized spinal cords. Exp Neurol 57:179–191 13. Ducker TB, Assenmacher DR (1969) Microvascular response to experimental spinal cord trauma. Surg Forum 20:428–430 14. Wullenweber R, Ebhardt G, Collmann H, Duisberg R (1978) Spinal cord blood flow after experimental trauma in the dog. I. Morphological findings after standardized trauma. Adv Neurol 20:407–414 15. Daniell HB, Francis WW, Lee WA et al (1975) A method of quantitating injury inflicted in acute spinal cord studies. Paraplegia 13: 137–142

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16. Parker AJ, Smith CW (1976) Functional recovery from spinal cord trauma following dexamethasone and chlorpromazine therapy in dogs. Res Vet Sci 21:246–247 17. Goodkin R, Campbell JB (1969) Sequential pathological changes in spinal cord injury: a preliminary report. Surg Forum 20:430–432 18. Rowland JW, Hawryluk GWJ, Kwon B et  al (2008) Current status of acute spinal cord injury pathophysiology and emerging therapies: promise of the horizon. Neurosurg Focus 25:1–17 19. Wrathall JR, Pettegrew RK, Harvey F (1985) Spinal cord contusion in the rat: production of graded, reproducible injury groups. Exp Neurol 88:108–122 20. Noble LJ, Wrathall JR (1987) An inexpensive apparatus for producing graded spinal cord contusive injury in the rat. Exp Neurol 95:530–533 21. Behrmann DL, Bresnahan JC, Beattie MS (1994) Modeling of acute spinal cord injury in the rat: neuroprotection and enhanced recovery with methylprednisolone, U-74006F and YM-14673. Exp Neurol 126:61–75 22. Panjabi MM, White AA (1980) Basic biomechanics of the spine. Neurosurgery 7:76–93 23. Blight AR, Decrescito V (1986) Morphometric analysis of experimental spinal cord injury in the cat: the relation of injury intensity to survival of unmyelinated axons. Neuroscience 19:321–341 24. Stokes BT, Noyes DH, Behrmann DL (1992) An electromechanical spinal injury technique with dynamic sensitivity. J Neurotrauma 9:187–195 25. Kwo S, Young W, Decrescito V (1989) Spinal cord sodium, potassium, calcium and water concentration changes in rats after graded contusion injury. J Neurotrauma 6:13–24 26. Gruner JA (1992) A monitored contusion model of spinal cord injury in the rat. J Neurotrauma 9:123–126 27. Koozekanani SH, Vise WM, Hashemi RM et al (1976) Possible mechanisms for observed pathophysiological variability in experimental spinal cord injury by the method of Allen. J Neurosurg 44:429–434

28. Ford RW (1983) A reproducible spinal cord injury model in the cat. J Neurosurg 59: 268–275 29. Scheff SW, Rabchevsky AG, Fugaccia I et  al (2003) Experimental modeling of spinal cord injury: Characterization of a force-defined injury device. J Neurotrauma 20:179–193 30. Dunham KD, Siriphorn A, Chompoopong S et al (2010) Characterization of a graded cervical hemicontusion spinal cord injury model in adult male rats. J Neurotrauma 27: 2091–2106 31. Pearse DD, Lo Jr TP, Cho KS et  al (2005) Histopathological and behavioral characterization of a novel cervical spinal cord displacement contusion injury in the rat. J Neurotrauma 22:680–702 32. Gensel JC, Tovar CA, Hamers FPT et al (2006) Behavioral and histological characterization of unilateral cervical spinal cord contusion injury in rats. J Neurotrauma 23:36–54 33. McKenna JE, Prusky GT, Whishaw IQ (2000) Cervical motoneuron topography reflects the proximodistal organization of muscles and movements of the rat forelimb: A retrograde carbocyanine dye analysis. J Comp Neurol 419:286–296 34. Soblosky JS, Song JH, Dinh DH (2001) Graded unilateral cervical spinal cord injury in the rat: evaluation of forelimb recovery and histological effects. Behav Brain Res 119:1–13 35. Anderson KD, Sharp KG, Hofstadter M et  al (2009) Forelimb Locomotor Assessment Scale (FLAS): Novel Assessment of Forelimb Dysfunction After Cervical Spinal Cord Injury. Exp Neurol 220:23–33 36. Choi H, Liao WL, Newton KM et  al (2005) Respiratory abnormalities resulting from midcervical spinal cord injury and their reversal by serotonin 1A agonists in conscious rats. J Neuroscience 25:4550–4559 37. Rumana CS, Baskin DS (1996) Brown-sequard syndrome caused by cervical disc herniation: case report and literature review. Surg Neurol 45:359–361 38. Popesko P, Rajtova V, Horak J (1992) Anatomy of small laboratory animals. Elsevier, Amsterdam

Chapter 19 Demyelination Models in the Spinal Cord Paul A. Felts, Damineh Morsali, Mona Sadeghian, Marija Sajic, and Kenneth J. Smith Abstract Disruption of axonal conduction within the central nervous system has obvious, negative consequences on numerous functions, including the ability to execute movement successfully. One important cause of axonal conduction deficits is primary demyelination, that is, the loss of the myelin sheaths but sparing of the axons which they surround. Such demyelination leads to conduction deficits ranging from action potential slowing and loss of transmission fidelity, to conduction block, and this latter, most severe consequence is almost inevitably the first consequence of the loss of a whole internode(s) of myelin. Several methods have been developed to induce primary demyelination in the spinal cord and some of the more common of these will be discussed in this chapter. Key words: Demyelination, Myelin, Multiple sclerosis, Experimental autoimmune encephalomyelitis, Ethidium bromide, Lysophosphatidylcholine

1. Introduction Demyelination is a characteristic feature of a number of disorders of the central nervous system (CNS) that can disturb normal function, and the most obvious deficit is motor weakness or paralysis. For example, acute disseminated encephalomyelitis is a monophasic demyelinating disease which can produce persistent severe motor disability (1), and subacute sclerosing panencephalitis, which results from persistent measles virus infection of the CNS, can ­produce a variety of motor signs and deficits (2). However, the most common central demyelinating disorder is multiple sclerosis (MS). MS is characterized by multiple plaques of inflammatory demyelination in the white matter of the CNS which result in a wide variety of motor and sensory deficits. Indeed the breadth of symptomatology is

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unsurprising because the location of the inflammation and demyelination dictates which functional pathways will be disrupted, and the lesions occur semi-randomly throughout the CNS. There is no known disorder directly analogous to MS in ­animals other than humans, but several animal models of the disease have been developed. None of the models so far recapitulate all the facets of MS, but the different models are useful in mimicking particular aspects of the disease. Animal models of MS can, in general, be subdivided into three groups (1) those in which autoimmunity is provoked to induce inflammatory demyelinating lesions throughout the CNS, although particularly within the caudal spinal cord, (2) those in which focal lesions are induced at predictable sites, and (3) those utilizing virus infection to induce demyelination. For the most part, the first group is used to examine the interplay between the immune and nervous systems, in light of the widely supported belief that inflammation and autoimmunity play a significant role in lesion initiation and progression in MS. In this regard, a wide variety of models have been developed based on different types of immunization. Such models come under the umbrella term experimental autoimmune encephalomyelitis (EAE), and have been used extensively to dissect the possible role of various antigens, immune cells, cytokines etc., in the disease. The second group of animal-based MS models is based on ­producing focal demyelinating lesions within the CNS. These lesions are often produced by injecting one or more factors directly into a region of CNS white matter. Generally the experiments are designed either to assess the effect of the injectate itself in causing demyelination, or to examine the repair processes that subsequently occur (see Note 4.1). The fact that the lesions can be induced at precise locations means that they are ideal for study by techniques such as electrophysiology (e.g., (3, 4)). Relatively focal demyelinating lesions can also be produced by feeding rodents with the copperchelating agent cuprizone (bis-cyclohexanone-oxaldihydrazone), although these lesions are predominately not in the spinal cord, but in discrete regions of the white matter of the brain including the corpus callosum and the superior cerebellar peduncle (5). Finally, the third group of animal-based MS models utilize viral infection to produce demyelination. Viruses which have been shown to produce primary demyelination in animals include Theiler’s murine encephalitis virus, the JHM strain of murine hepatitis virus, and Semliki Forest virus. Given that several human demyelinating conditions result from viral activity, this group of demyelinating models are important tools in understanding human demyelinating disease. There are numerous excellent reviews of viral models of central demyelination (e.g., (6, 7)) and because the expertise of the authors lies in autoimmune and focal lesions, the remainder of this chapter will concentrate on these models of spinal cord demyelination.

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One of the main applications of this model is to investigate the mechanisms underlying autoimmune-mediated inflammation, demyelination and axonal damage in CNS, which are events believed to be important in MS. Its other applications include the discovery and testing of therapeutic targets and novel strategies for treating MS. There are several models of EAE induced using different immunogens and in different species, and the subtle differences in outcome can provide models that resemble the various forms and stages of MS rather closely. EAE is arguably the best model of MS there is, but whether it is a good model is open to debate (8–10). EAE is induced by stimulating an immune response directed against CNS antigens. EAE can be induced via either active or passive methods. In active EAE, animals are immunized with a range of different immunogens including purified (typically myelin) proteins, recombinant proteins, synthesized peptides, or even whole spinal cord and brain homogenates (Fig. 1). Those most commonly used today are: myelin basic protein (MBP) (11), proteolipid protein (PLP) (12), myelin-associated glycoprotein (MAG) (13), and myelin oligodendrocyte glycoprotein (MOG). Passive EAE is more complicated to induce and it involves the administration of encephalitogenic T cells directed against CNS antigens. Splenocytes and lymph nodes are removed from donor animals that have been immunized with the antigen of interest a few days earlier (the precise time is dependent upon the antigen and animal strain) and the cells are treated with the appropriate antigen in vitro to produce encephalitogenic T cells. The T cells are then transferred to naive

Fig. 1. Light micrographs of toluidine blue-stained sections showing regions of the spinal cord of a female DA rat following induction of active EAE using whole spinal cord homogenate as the encephalitogen. (a) A region containing several demyelinated axons (e.g., arrows ) is shown. Arrowheads indicate examples of axons which are still surrounded by myelin sheaths. (b) A small blood vessel (asterisk ) exhibiting a perivascular cuff of inflammatory cells.

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animals where disease is induced (14, 15). Most active and passive protocols function by activating the myelin-specific CD4+ T cells that have escaped immune tolerance and circulate in the periphery of naïve animals (16). Activation of CD4+ T cells enables them to cross the blood–brain barrier more efficiently than naïve T cells (17, 18). Recently, models of passive transfer EAE induced with encephalitogenic CD8+ T cells have been developed (19, 20). 2.1. Choice of Animal Species

A number of different animal species including mice, rats, guinea pigs, rabbits, macaques, rhesus monkeys and marmosets have been used as suitable candidates for EAE (21–23). Marmoset models seem to resemble human disease, both clinically and pathologically (24–26); however, due to various reasons such as the number of immunological tools, the availability, lifespan, and fecundity of the animals and the reasonable resemblance of the induced disease to MS, mice and rats have become the most commonly used species today (21–23, 27, 28). We will therefore only focus on the EAE models induced in these two species. It is important to note that not all strains of mice and rats are susceptible to EAE. The decision as to which species and strain to use for EAE depends on the research question. Different species and different breeds will develop different forms of EAE, each representing a good model for the different stages of MS (27, 28). A large amount of research has concluded that certain antigens work best in certain species, strains and genders which further reflects the heterogeneity of EAE (21, 23, 27, 28). Table 1 summarises the optimal and most well-established EAE models.

2.2. Inducing Active EAE in Rat

This is the most important step in inducing EAE as the severity of the induced disease to a great extent depends on a thoroughly homogenised emulsion. The emulsion is typically a 1:1 ratio by volume of a solution containing the antigen of interest (in our laboratory this is typically recombinant MOG (rMOG) in female DA rats) and an emulsifier (usually incomplete Freund’s adjuvant (IFA) or complete Freund’s adjuvant (CFA)); each animal typically receives a 200 ml portion of the total emulsion (0.1 mg of antigen per animal). When calculating the amount of antigen needed, always allow for at least an extra volume of up to 200 ml as losing some of the emulsion while preparing for injection is inevitable. To make the emulsion, two glass syringes (Hamilton, Bonaduz, Switzerland) are connected together using a metal connector. The plunger on one syringe is taken out before connecting to avoid vacuum build up. The connected syringes are then placed vertically in a glass container such as a beaker with the top syringe being the one with no plunger. The empty barrel is then filled by slowly adding the adjuvant and then the antigen using a pipette. The plunger is then put slowly back in. The two connected syringes are then

2.2.1. Preparing the Emulsion

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Table 1 EAE models Strain

Preferred gender

Immunogen

References

Mouse SJL/J B10.S B10.PL PL/Jb ASWb NZW C57BL/6 NOD

Male Female Female Female Male Male No difference No difference

PLP139–151a, MBP PLP139–151 MBP MBP MOG92–106 MBP MOG35–55 MOG35–55

(28, 44–46) (45) (28, 45, 47) (45, 47) (47) (47) (45, 47) (47)

Rats DA

Female

(9, 48, 49)

LEW.AVI

Male Female

Recombinant MOG, MBP Spinal cord homogenate Recombinant MOG, MBP Recombinant MOG Spinal cord homogenate

Lewis

Female Male

(48, 49) (49, 50)

held horizontally and the plunger is pushed firmly but slowly to facilitate mixing of the antigen with the adjuvant as the mixture passes through to the second syringe. The mixture is passed between the two syringes for at least 30–40 times or until the emulsion is thick and homogenous in texture. At this stage, the plunger of the syringe containing the emulsion is drawn back slightly to produce a gentle vacuum, and the empty syringe and connector are disconnected and removed to be replaced with a needle. To eliminate air bubbles remaining in the emulsion, which would result in under-dosing, a 23–25G needle is placed on the syringe and air bubbles are removed by holding the syringe vertically and tapping and expelling the air through the needle. If air bubbles have inadvertently been included, they can be difficult to dislodge if the emulsion is appropriately thick. 2.2.2. Preparing Animals for Injection

Under light anaesthesia (e.g., 2% isoflurane) the area of the back immediately adjacent to the base of the tail is shaved, and the skin scrubbed with iodine-based disinfectant.

2.2.3. Injecting the Emulsion

Gently pull the skin from the shaved area to create a small “tent,” insert the tip of the sterile needle into the pulled tent making sure that the needle is in a subcutaneous space, and gently manipulate

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the needle laterally to create a small pocket for injection. Inject 200 ml of the emulsion, avoiding injecting intradermally. Allow the needle to remain in place for a few seconds and then gently remove it, closing the puncture wound between the fingertips while doing so. Clean the injection site using iodine and gently push the bolus of injected fluid rostrally to avoid the emulsion leaking out of the injection site. Transfer the animal to a recovery chamber. 2.3. Preparing Spinal Cord Homogenate

Another form of active EAE utilizes whole spinal cord homogenate (SCH) rather than a specific myelin protein or peptide. Immunising with SCH is similar to the method already described in Sect. 2.2., but here we explain the steps to create the SCH.

2.3.1. Extracting the Spinal Cord

In our experience, this EAE model works best in male DA rats, but it can also be employed in Lewis rats. The whole spinal cord of one adult male rat is sufficient for immunizing three adult male rats. Once the animal is sacrificed, the spinal cord is dissected free and the dura removed. Sterile saline should be applied as required to prevent the tissue from drying out. Each cord is weighed and the weight noted to ensure that sufficient cord tissue is obtained for the proposed usage, and the cord is then wrapped in a small piece of aluminium foil and left to freeze on dry ice. The frozen cords are stored at −80°C.

2.3.2. Preparing the Homogenate

On the day of immunization, the required weight of spinal cord tissue is defrosted on ice. Using a sharp blade the cord is minced into smaller pieces to facilitate making the homogenate with adjuvant. In this model of EAE, we find it is best to use CFA to achieve effective disease. As explained in Sect. 2.2.1., the pieces of spinal cord will be added to CFA in the glass syringe and carefully emulsified as explained. The injection method is the same as described in Sect. 2.2.3. In our experience, the first symptoms of EAE (rMOG and SCH rat models) occur between 9 and 11 days post immunization (DPI). Depending on the quality of the injected emulsion, a slightly earlier or later onset can occur, typically preceded by weight loss in the animals.

2.4. Induction of Passive EAE

Passive transfer EAE is a useful model particularly as the inflammatory demyelination induced in animals in this way is almost entirely spared of degeneration. Thus the effects of primary demyelination can be studied without the confounding factor of significant axonal loss. In addition, this form of EAE is highly reproducible among an immunised cohort of animals, enabling reduction of animal numbers and more stringent experimental controls. The first step in inducing passive EAE is preparing the encephalitogenic cell line. The second step is transfer of these cells into naive recipients. We have typically used rMOG as the encephalitogenic antigen in the DA strain of rat.

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Eight or ten days post-immunization with rMOG (100  mg per a­ nimal in IFA), using techniques similar to those described above for induction of active EAE, animals are sacrificed by an overdose of carbon dioxide and the draining (inguinal) lymph nodes and spleens are harvested into sterile Hank’s buffered salt solution (HBSS). Spleens are used as a source of antigen-presenting cells (APCs) and do not have to be harvested from immunised animals. For efficiency, we harvest the tissue from 5 to 6 actively immunised animals. The lymph nodes and the spleens are dissociated ­separately using the plunger of a 2-ml syringe to pass the tissue through a 70-mm mesh cell strainer, and washed three times in sterile HBSS. The number of cells in the single cell suspension is then estimated using trypan blue staining and a haemocytometer. Harvested lymphocytes are subjected to a two-phase in vitro protocol in order to produce a high number of activated, antigenspecific T cells. In the first phase, the lymph node cells are co-­ cultured with APCs pre-incubated (pulsed) with the antigen (rMOG). During this process, any lymph node cells which were in  vivo primed against rMOG will receive survival stimuli from APCs and change their morphology to that of activated (blast) cells. The blast cells are larger than non-activated lymphocytes and of lower density than non-activated lymphocytes, and thus they can be separated by density gradient centrifugation. To produce blasts, washed and counted APCs are first ­irradiated with 30 Gy (Cobalt 60 source), centrifuged for 8 min at 680 × g at room temperature, and the pellet re-suspended in RPMI 1640 medium. The irradiated cells are incubated (pulsed) with antigen (20 mg/ml of rMOG) for 90 min at 37°C, in a 5% carbon dioxide atmosphere. After 90 min, the lymph node cells are added to antigenpulsed APCs at a ratio of 1:6–1:10 (lymph node cells : APCs), to give a final concentration of cells in the co-culture of 3 × 106/ml of RPMI 1640 medium supplemented with 10% foetal calf serum (FCS). After 48–72 h, depending upon the specific response of the cells to culture conditions (see Note 4.2), cells from the culture are washed, re-suspended in 6 ml of HBSS, very carefully layered on 3 ml of Lymphoprep solution (Frenius Kabi Norge, Oslo, Norway) and then centrifuged for 30 min at 1,462 × g. At this stage, it is very important to not allow mixing of the two phases and to disable the centrifuge brake. After centrifugation, the blasts are carefully ­collected from the interface with a pipette, washed in HBSS and counted in a haemocytometer. The second phase is the expansion of the blasts in a medium enriched with IL-2. The blasts are resuspended in RPMI 1640 medium supplemented with 20 U/ml of IL-2 and cultured for a further 48–72 h. The exact duration of culture with IL-2 depends on blast activation state and number (see Note 4.2). In general,

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blasts proliferate well at higher densities (70% covering of flask ­surface or more). When cells reach over 100% covering density in the culture flask, they are washed again and the whole cycle of ­re-stimulation with APCs is repeated. Several cycles of re-stimulation may be necessary to achieve the adequate numbers of cells needed for passive transfer. The vast majority of activated blasts produced in this way are CD4+ T cells. For cell transfer, naive DA rats are anaesthetised (2% isoflurane in oxygen) and a small area of the tail is cleaned with iodine ­solution. A small incision is made on the lateral side of the tail base and a short length of the tail vein exposed and cleared of the ­surrounding connective tissue. The caudal end of the exposed vein is ligated using a 4.0 suture. A loop of suture is tied around the rostral end of the exposed vein, but not tightened. Using iris scissors a small cut is made in the vein, cutting approximately half of the vein circumference. This incision is used to insert a cannula (Fine Science Tools, Canada) attached to a 1-ml syringe containing 500–700 ml of a cell suspension in PBS. The cannula is then secured by tightening the knot at the rostral end of the exposed tail vein, and the cell suspension is slowly injected. After the cells are injected, the ­cannula is carefully removed, the rostral end of the exposed part of the vein ligated and the wound closed using a 4.0 suture. Sterile technique is applied throughout the procedure. Cells may also be injected via the intraperitoneal route. We find that passive transfer of ten million encephalitogenic T cells generated using the described protocol induces very reproducible, monophasic EAE in DA rats. The disease onset is at day 4 or day 5 post-transfer. All animals reach the peak of the disease on day 6 post-transfer, characterised by a paralysed tail, inability to spread toes and ataxic gait. These signs of motor deficit last for 3 days. All animals completely recover by day 10 post-transfer. Pathological characteristics at the peak of disease are inflammatory infiltrates and demyelination, with only small numbers of degenerated axons present. 2.4.2. Passive EAE in C57BL6 Mice

In C57BL6 mice, the donor group is immunized with MOG35–55/ CFA. After 11 days, spleens are isolated from the immunized mice and splenocytes are restimulated in  vitro with MOG35–55, IL-12 and anti-IFNg antibodies for 3 days to generate encephalotigenic T cells. These are then injected to the recipient mice as before.

2.5. Induction of Active EAE in C57/BL6 and B6/CBA F1 Mice

In this model, EAE is induced by immunizing the mice with MOG35–55 emulsified in CFA. The presence of Mycobacterium tuberculosis in the emulsion is a very potent stimulus for priming the immune system in mice (29). In our laboratory M. tuberculosis is used at a concentration of 5 mg/ml in IFA.

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2.5.1. Preparation of MOG35–55 /CFA Emulsion

Each mouse is immunized with a total amount of 200  mg of MOG35–55 in 200 ml of the emulsion, resulting in a final antigen concentration of 1 mg/ml. The emulsion contains a 1:1 ratio (by volume) of MOG35–55 solution: CFA and is administered to each mouse by subcutaneous injection. In practice, the MOG35–55 peptide is dissolved in sterile saline at a concentration that is twice the final concentration, to allow for the dilution. Due to loss of some of the emulsion in the dead space of the hub of the syringe, excess emulsion should initially be prepared. We calculate the total volume of emulsion needed by multiplying the number of mice to be immunized by 1.5, and multiply that number by 200 ml, then this number is divided by two which gives the ­volume of both the MOG35–55 peptide and the CFA needed. The ­emulsion is prepared using two glass syringes following the procedure ­outlined in 2.2.1.

2.5.2. Preparation of Pertussis Toxin

Pertussis toxin from Bordetella pertussis (Calbiochem, Nottingham, UK) is re-suspended in sterile saline to a concentration of 50 mg/ml stock solution. Dilutions are then made so that a final concentration of 30 (ng/ml) in sterile saline is achieved. Each mouse will receive 0.1 ml of pertussis toxin in saline i.p. on the day of immunization followed by a second dose 48 h later.

2.5.3. Preparation of the Animals for the Injections

Day 0: Mice with mean age 14 weeks (range 10 weeks to 16 weeks, depending on colony supply) are chosen and weighed. We have observed that mice lose weight 1 or 2 days before showing symptoms of EAE so it is important and helpful to keep a daily record of their weight. Day 1: Mice are anaesthetised with isoflurane (2% in air) and a standard method is used to assess the level of anaesthesia ­(e.g., pinch of the hind limb toes). Once the mouse is fully anaesthetised, all the injection sites on the skin are cleaned with ­antiseptic solution such as povidone-iodine prior to injection. 2 × 100 ml subcutaneous injections of emulsion (intradermal injections are to be avoided) are made into the sacral haunches (5–10  mm rostral to the base of the tail). The needle is left inserted into the subcutaneous space for 5–10 s after injection, to avoid leakage of the emulsion. After the injection, a bolus mass should appear under the skin that persists for at least 2 weeks. This step is then followed, under the same anaesthesia, by intraperitoneal injection of 100 ml of the 30 ng/ml pertussis toxin, Day 3: The second intraperitoneal injection of pertussis toxin is made, as described for day 1. Day 7: The second round of immunization is carried out by repeating the MOG35–55/CFA emulsion injection as on day 1, but this time the emulsion is injected ~1 cm rostral to the previous location around the lumbar flanks.

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Table 2 Grade scale for clinical signs Clinical sign

Score

Slight floppy tail (1/2 paralysis)

0.5

Slight tail spasticity

0.5

Floppy tail

1

Tail spasticity

1

Slightly impaired righting reflex

1.5

Impaired righting reflex

2

Slight Hind Limb weakness

2.5

Hind limb weakness in one leg

2.5

Slight hind limb spasticity

2.5

Hind limb spasticity in one leg

2.5

Hind limb weakness

3

Hind limb spasticity

3

Hind limb weakness in one, paralysis in other

3.5

Very hind limb weakness in both legs

3.5

Hind limb spasticity causing immobility one leg

3.5

Very hind limb spasticity

3.5

Hind limb paralysis

4

Hind limb spasticity (paralysis both limbs)

4

Slight forelimb weakness

4.5

Fore limb weakness one leg

4.5

Slight fore limb spasticity

4.5

Fore limb weakness

5

Fore limb spasticity

5

Fore limb weakness one, paralysis one

5.5

Very fore limb weakness

5.5

Fore limb spasticity, immobility in one limb

5.5

Very fore limb spasticity

5.5

Fore limb paralysis

6

Fore limb spasticity, immobility of both

6

Moribund

7

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Mice are monitored daily to assess the magnitude of any neurological deficit and to detect weight gain or, more importantly, weight loss. In our laboratory, the clinical severity of EAE in mice is scored using a grading scale (0–7) that is outlined in Table 2. The symptoms of EAE tend to appear 9–14 days after immunization, but this timing can vary depending on the strain of mice used.

3. Focal Demyelinating Lesions

3.1. Preparation and Filling of Injection Pipettes

A variety of agents have been used to induce primary demyelination in the mammalian CNS following direct injection into white ­matter tracts, including diphtheria toxin (30), 6-aminonicotinamide (31), lysophosphatidyl choline (32), galactocerebroside anti-serum (33), ethidium bromide (34), ionomycin (35) and lipopolysaccharide (36). The use of direct injection into central white matter to produce demyelination circumvents the potential problem of the blood– brain barrier preventing systemically administered agents gaining access to the CNS. In addition, many of the demyelinating agents that have been utilized (e.g., ethidium bromide) are toxic and so cannot be administered systemically. Finally, focal lesions allow the deleterious effects resulting from the conduction block following demyelination to be confined to particular pathways, such as the dorsal columns, that produce minimal neurological deficits. Indeed, in our experience even large lesions of the dorsal columns do not produce any obvious deficits, although careful locomotor analysis may reveal more subtle deficits (37). The injection of demyelinating agents into the dorsal columns has been historically achieved using small gauge needles (30), but better (less traumatic) results can be achieved using drawn glass micropipettes (3). Glass capillaries of 1.0 or 1.2 mm outside diameter are pulled on a pipette puller, using a setting to provide a relatively rapid taper. We use a Sutter Instruments Flaming/Brown style programmable puller, although a less sophisticated instrument will certainly suffice. The pipette tips as initially pulled are too flexible to allow penetration of the pial surface, and so are carefully broken, while being viewed under a dissecting microscope, to an internal diameter of approximately 15 mm using no. 5 watchmaker’s forceps. Pipette tips can then be inspected with a standard light microscope and tip diameter measured with an ocular graticule calibrated with a stage micrometer (see Note 4.3), although the precise measurements are not very important. The glass micropipette is then attached to a length of microbore plastic ­tubing of appropriate inside diameter, the other end of which is attached to a hypodermic needle (18 or 19G depending upon the outside diameter of the glass micropipette, and hence the diameter

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of the tubing). The hypodermic needle is then attached to a 5–10 ml hypodermic syringe via an intervening three-way stopcock. In order to deliver an accurate volume of injectate, we use an analytical balance to weigh a number of pipettes from the same batch of pipette glass. We then fill the pipettes with distilled water and re-weigh them, and by measuring the length of fluid in each pipette, we determine the average length necessary to contain a volume of 0.5 ml. We then use a drawing program to construct a scale with appropriate graduations which, when attached to the pipette, show through the pipette and fluid, providing a scale with minimal parallax error. The pipette is then mounted in a pipette holder (e.g., World Precision Instruments no. M3301EH) which is placed in a single axis manipulator via a small clamp which allows it to be immersed into a fluid for filling. Very small volumes can be used, and we routinely fill pipettes from 100 ml of solution in an Eppendorf tube. Proper positioning of the fine tip of the pipette in a small volume of fluid can be aided by placing the pipette under a small amount of pressure using the syringe, as this will produce visible bubbles once the tip reaches the solution (this technique cannot be used with low surface tension fluids, where large long-lasting bubbles form). The pipette is then filled by suction using the syringe; however, it should be noted that, particularly with very fine tips, excessive vacuum can cause dissolved gases to come out of solution and form bubbles. Such bubbles are to be avoided as they can compress during the subsequent delivery of fluid and result in the delivery volume being less than that indicated by the scale. 3.2. Surgical Procedure

Surgeries are carried out on a steel table top or plate to allow ­magnetic bases to be used. The anaesthetised animal is placed on an electrically heated blanket with rectal probe feedback to maintain body temperature (e.g., FHC no. 40-90-8D). Choice of anaesthetic is generally not critical; we routinely use isoflurane as it allows the depth of anaesthesia easily to be adjusted during the procedure. The animal should be shaved at the surgical site and an antiseptic such as povidone–iodine solution applied to the skin. Sterile technique should be used throughout the surgical procedure (see Note 4.4). For an injection into the dorsal columns, a skin incision is made above the appropriate vertebrae, and we typically use vertebrae in the lower thoracic region, where the vertebrae are close to the surface. Once the correct vertebral level is identified, an incision is made either side of the spinous processes of the target vertebrae and extended one vertebra caudally. The remainder of the surgery is carried out using a stereomicroscope, preferably an operating microscope equipped with through-the-lens illumination (which avoids shadows) and a working distance of at least 150 mm. A small amount of paraspinal muscle is removed from either side of the

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spinous process to allow access and, using very fine rongeurs, the posterior-lateral quadrant of the lamina is removed from the target vertebra (we typically use T11 or 12), exposing the spinal cord. Care must be taken not to allow the jaw of the rongeurs to extend too far under the lamina during this procedure to avoid damage to the spinal cord. A small eyelid retractor can be used to hold the field open. With approximately 1–2 mm of dura mater covering the spinal cord exposed, a small hole is made in the dura just lateral to the dorsal vein using the point of a small sapphire knife (e.g., Duckworth and Kent, cat. no. 4-4-100s) to cut the dura open. At this point, the vertebral column is stabilized by attaching a clamp (which is fixed to the post of a magnetic base) to the spinous process of the target vertebra. Following the injection (see below), the muscle and fascia are sutured and the skin incision closed with wound clips, which are removed 7–10 days after the surgery. 3.3. Injection

For injection, the pipette holder with attached injection pipette is mounted on a three axis hydraulic micromanipulator (we use a Narashige, model MO-103), which in turn is mounted on a standard three axis mechanical micromanipulator via a small bar. The mechanical manipulator is attached to the post of a magnetic base so that it can easily be moved into or out of the surgical field. The mechanical micromanipulator provides coarse positioning, and the hydraulic manipulator provides fine positioning. For dorsal column injections, the pipette tip is positioned, through the hole in the dura, just lateral to the dorsal vein and just touching the spinal cord. The hydraulic manipulator is mounted such that the final “angle of attack” of the pipette is 16° from the vertical, and this angle positions the pipette tip, when inserted, within this roughly triangular-shaped tract. After noting the depth on the hydraulic micromanipulator, the tip is then advanced into the spinal cord. Slight dimpling of the cord is typical as the pipette is advanced, with the tissue rebounding as the pipette tip penetrates the pial surface. The tip is then inserted to the desired injection depths for injection. In our experience, two injections of 0.5 ml, at depths of 700 and 400 mm, give good lesion placement. Too rapid an injection may cause reflux of injectate up the needle track; however, we do not see such reflux when injecting at a rate of approximately 0.5 ml over a period of approximately 30 s. We will typically leave the pipette in place after the final injection for a period of perhaps 30  s, although with these small volumes, we have not observed reflux even when the pipette was withdrawn immediately, including when using ethidium bromide solutions which are dark red in colour. An example of an injection using the above procedure (but substituting a black tracer to visualise the location of injection), is shown in Fig. 2, along with a representative lysophosphatidylcholine lesion in the dorsal column.

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Fig. 2. Light micrographs of rat spinal cord sections. (a) The cord has been injected with a dark tracer using the method described in Sect. 4.3. (although, in this case, with three injections along a single track). Arrows indicate the location of the injectate within the dorsal columns. (b) The clear area located unilaterally within the dorsal columns represents a large region of demyelination induced by the injection of lysophosphatidyl choline.

3.4. Use of Dorsal Column Lesions for In Vivo Electrophysiology

The difficulty in isolating white matter tracts in the CNS complicates experiments to assess the electrophysiological effects of a demyelinating lesion in vivo. Although the optic nerve represents one such isolated conduction pathway, it is not easily accessed over a significant length. In several experiments (e.g., (3, 38–40)), we have made use of the dorsal columns, usually in the rat, to examine the consequences of demyelination and remyelination in vivo. These tracts are composed chiefly of topographically ordered, ascending primary afferent axons (41), as well as a discrete descending motor ­pathway, the corticospinal tract, which is located in the ventral-most position within the dorsal columns. The majority of ascending sensory fibres leave the dorsal columns within 2–3 segments of entering the spinal cord; however, a substantial number extend to the ­dorsal column nuclei in the brainstem. Thus if a lesion of proper position and size is induced in the dorsal columns, it is possible to ensure that all directly conducted (i.e., non-synaptic) impulses pass through the lesion by stimulating the dorsum of the spinal cord rostral to the lesion (in paralysed, ventilated, anaesthetised animals) and recording the antidromically conducted compound action potentials from dorsal roots (or their associated peripheral nerves) that enter the spinal cord caudal to the lesion. Such techniques can be used to determine the conduction properties of the lesion at a single time point, or serial recordings can be made by utilizing implanted, or intermittently placed percutaneous, electrodes. Single recordings in terminal experiments have the advantage that the histology of the lesion can be determined and correlated with the physiological responses.

4. Notes 4.1. Repair of Focally Induced Lesions

Following induction of experimental focal demyelinating lesions in the CNS, repair by remyelination is typically extensive, particularly in rodents. This property has proven useful for the study of repair;

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however, it has been problematic when the experimenter wishes to study demyelinated lesions of relatively long standing. One way around the problem has been locally to irradiate the injection site and adjacent spinal cord. Both X-rays (42) and beta irradiation (43) have been used for this purpose. The former method seems to be more effective but can produce radiation damage which can limit the survival period. In our experience remyelination typically restores secure conduction (3). Interestingly, the remyelination, particularly in the rodent spinal white matter, is often dominated by Schwann cells rather than by oligodendrocytes, and such “peripheral” repair is also effective at restoring conduction (38). 4.2. Cell Culture Considerations

The exact time point at which blast cells should be separated from APCs cannot be decided beforehand, as it depends on the interaction between APCs and lymphocytes and subsequent lymphocyte proliferation. At the start of the procedure (first 2–3 cycles), lymphocytes usually respond slowly to re-stimulation with APCs, as there are few antigen-specific cells in the culture. Furthermore, the number of antigen-specific cells generated in lymph nodes in vivo depends on the success of active EAE induction and varies between animals. In our hands, at the beginning of the in vitro stimulation protocol, the cells should be kept in co-culture with APCs for about 3 days. As the number of antigen-specific T cells present during re-stimulation cycles increases and as these cells are acquiring activated phenotype, they require less time in the co-culture with APCs. However, it is important to pay close attention to acidification of culture media as the cells are using up nutrients even though few are proliferating. Therefore, if the media is showing signs of acidification (e.g., turning yellow), it might be necessary to separate cells and change the media. Similar criteria apply to culture in media enriched with IL-2.

4.3. Pipette Tips

Breaking pipette tips generally produces a sharp edge which penetrates the pial surface with only minor dimpling (i.e., <0.4 mm). With experience, it is possible to assess the “sharpness” of the tip under the compound microscope and discard unsuitable pipettes which have broken with a blunt surface. We have used a pipette tip beveller equipped with a diamond grinding disk to produce what appears to be a sharp point on the ends of the pipettes; however, in general we did not find that this produced easier penetration than an unaltered broken tip with a sharp exposed edge.

4.4. Sterile Technique and Surgical Complications

In our experience, with proper sterile technique, infections following surgery are extremely rare, and prophylactic antibiotic is not necessary. Sterile surgical gloves are used, and a surgical field is established by placing a sterile cloth with an opening in the centre over the incision site. The surgical kit is autoclaved and includes aluminium foil which is used to cover any surfaces that must be touched (e.g., ­controls of the stereomicroscope, micromanipulators etc).

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Acknowledgements Research in the authors’ laboratories is supported by grants from the Medical Research Council UK, the European Union (NeuroproMiSe), the Multiple Sclerosis Society of GB and NI, and the Brain Research Trust. References 1. Kawashimal S, Matsukawa N, Ueki Y et  al (2009) Predicting the outcome of acute disseminated encephalomyelitis by apparent diffusion coefficient imaging: Two case reports. J Neurological Sciences 280:123–126 2. Garg RK (2008) Subacute sclerosing panencephalitis. J Neurology 255:1861–1871 3. Smith KJ, Blakemore WF, McDonald WI (1981) The restoration of conduction by central remyelination. Brain 104:383–404 4. Honmou O, Felts PA, Waxman SG, Kocsis JD (1996) Restoration of normal conduction properties in demyelinated spinal cord axons in the adult rat by transplantation of exo­ genous Schwann cells. J Neuroscience 16: 3199–3208 5. Blakemore WF (1973) Demyelination of the superior cerebellar peduncle in the mouse induced by cuprizone. J Neurological Sciences 20:63–72 6. Drescher KM, Sosnowska D (2008) Being a mouse in a man’s world: what TMEV has taught us about human disease. Frontiers Bioscience 13:3775–3785 7. Lane TE, Hosking MP (2010) The pathogenesis of murine coronavirus infection of the central nervous system. Critical Reviews Immunology 30:119–130 8. Nelson AL, Bieber AJ, Rodriguez M (2004) Contrasting murine models of MS. International Multiple Sclerosis J 11:95–99 9. Sriram S, Steiner I (2005) Experimental allergic encephalomyelitis: a misleading model of multiple sclerosis. Annals Neurol 58:939–945 10. Steinman L, Zamvil SS (2006) How to successfully apply animal studies in experimental allergic encephalomyelitis to research on multiple sclerosis. Annals Neurol 60:12–21 11. Martenson RE, Deibler GE, Kies MW (1969) Microheterogeneity of guinea pig myelin basic protein. J Biol Chem 244:4261–4267 12. Olitsky PK, Tal C (1952) Acute disseminated encephalomyelitis produced in mice by brain proteolipid (Folch–Lees). Proc Soc Exp Biol Med 79:50–53

13. Poduslo SE (1983) Proteins and glycoproteins in plasma membranes and in the membrane lamellae produced by purified oligodendroglia in culture. Biochimica Biophysica Acta 728:59–65 14. Ben-Nun A, Wekerle H, Cohen IR (1981) The rapid isolation of clonable antigen- specific T lymphocyte lines capable of mediating autoimmune encephalomyelitis. Eur J Immunol 11:195–199 15. McDevitt HO, Perry R, Steinman LA (1987) Monoclonal anti-Ia antibody therapy in animal models of autoimmune disease. Ciba Found. Symp. 129:184–193 16. Seamons A, Perchellet A, Goverman J (2003) Immune tolerance to myelin proteins. Immunol Res 28:201–221 17. Hickey WF (1991) Migration of hematogenous cells through the blood–brain barrier and the initiation of CNS inflammation. Brain Pathol. 1:97–105 18. Brabb T, von Dassow P, Ordonez N et al (2000) In situ tolerance within the central nervous system as a mechanism for preventing autoimmunity. J Exp Med 192:871–880 19. Huseby ES, Liggitt D, Brabb T, Schnabel B, Ohlen C, Goverman J (2001) A pathogenic role for myelin-specific CD8(+) T cells in a model for multiple sclerosis. J Exp Med 194:669–676 20. Sun D, Whitaker JN, Huang Z et  al (2001) Myelin antigen-specific CD8+ T cells are encephalitogenic and produce severe disease in C57BL/6 mice. J Immunol 166:7579–7587 21. Steinman L (1999) Assessment of animal models for MS and demyelinating disease in the design of rational therapy. Neuron 24:511–514 22. Gold R, Hartung HP, Toyka KV (2000) Animal models for autoimmune demyelinating disorders of the nervous system. Molecular Med Today 6:88–91 23. Baxter AG (2007) The origin and application of experimental autoimmune encephalomyelitis. Nature Rev Immunol 7:904–912 24. Pomeroy IM, Matthews PM, Frank JA, Jordan EK, Esiri MM (2005) Demyelinated neocortical

19  Demyelination Models in the Spinal Cord lesions in marmoset autoimmune encephalomyelitis mimic those in multiple sclerosis. Brain 128:2713–2721 25. Pomeroy IM, Jordan EK, Frank JA, Matthews PM, Esiri MM (2008) Diffuse cortical atrophy in a marmoset model of multiple sclerosis. Neurosci Letters 437:121–124 26. Pomeroy IM, Jordan EK, Frank JA, Matthews PM, Esiri MM (2010) Focal and diffuse cortical degenerative changes in a marmoset model of multiple sclerosis. Multiple Sclerosis 16:537–548 27. Mix E, Meyer-Rienecker H, Zettl UK (2008) Animal models of multiple sclerosis for the development and validation of novel therapies - potential and limitations. J Neurology 255 Suppl 6:7–14 28. Krishnamoorthy G, Wekerle H (2009) EAE: an immunologist’s magic eye. Eur J Immunol 39:2031–2035 29. Fillmore PD, Brace M, Troutman SA et  al (2003) Genetic analysis of the influence of neuroantigen-complete Freund’s adjuvant emulsion structures on the sexual dimorphism and susceptibility to experimental allergic encephalomyelitis. Am J Pathol 163:1623–1632 30. McDonald WI, Sears TA (1970) Focal experimental demyelination in the central nervous system. Brain 93:575–582 31. Blakemore WF (1975) Remyelination by Schwann cells of axons demyelinated by intraspinal injection of 6-aminonicotinamide in the rat. J Neurocytology 4:745–757 32. Blakemore WF, Eames RA, Smith KJ et  al (1977) Remyelination in the spinal cord of the cat following intraspinal injections of lysolecithin. J Neurological Sciences 33:31–43 33. Kaji R, Suzumura A, Sumner AJ (1988) Physiological consequences of antiserum-mediated experimental demyelination in CNS. Brain 111:675–694 34. Blakemore WF (1982) Ethidium bromide induced demyelination in the spinal cord of the cat. Neuropathology Appl Neurobiology 8:365–375 35. Smith KJ, Hall SM (1994) Central demyelination induced in vivo by the calcium ionophore ionomycin. Brain 117:1351–1356 36. Felts PA, Woolston A-M, Fernando H et  al (2005) Inflammation and primary demyelination induced by the intraspinal injection of lipopolysaccharide. Brain 128:1649–1666 37. Jefferies NA, Blakemore WF (1997) Locomotor deficits induced by experimental spinal cord demyelination are abolished by spontaneous remyelination. Brain 120:27–37

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38. Felts PA, Smith KJ (1992) Conduction properties of central nerve fibers remyelinated by Schwann cells. Brain Res 574:178–192 39. Felts PA, Kapoor R, Smith KJ (1995) A mechanism for ectopic firing in central demyelinated axons. Brain 118:1225–1231 40. Smith KJ, Felts PA, John GR (2000) Effects of 4-aminopyridine on demyelinated axons, synapses and muscle tension. Brain 123:171–184 41. Smith KJ, Bennett BJ (1987) Topographic and quantitative description of rat dorsal column fibres arising from the lumbar dorsal roots. J Anat 153:203–215 42. Blakemore WF, Patterson RC (1978) Suppression of remyelination in the CNS by X-irradiation. Acta Neuropathologica 42:105–113 43. Felts PA, Smith KJ (1996) Blood-brain barrier permeability in astrocyte-free regions of the central nervous system remyelinated by Schwann cells. Neuroscience 75:643–655 44. Bernarnd CC, Carnegie PR (1975) Experimental autoimmune encephalomyelitis in mice: immunologic response to mouse spinal cord and myelin basic proteins. J Immunol 114:1537–1540 45. Scheikl T, Pignolet B, Mars LT, Liblau RS (2010) Transgenic mouse models of multiple sclerosis. Cellular Molecular Life Sci 67:4011–4034 46. Waldner H, Whitters MJ, Sobel RA, Collins M, Kuchroo VK (2000) Fulminant spontaneous autoimmunity of the central nervous system in mice transgenic for the myelin proteolipid proteinspecific T cell receptor. Proc National Academy Sci USA 97:3412–3417 47. Stromnes IM, Goverman JM (2006) Active induction of experimental allergic encephalomyelitis. Nature Protocols 1:1810–1819 48. Brehm U, Piddlesden SJ, Gardinier MV, Linington C (1999) Epitope specificity of demyelinating monoclonal autoantibodies directed against the human myelin oligodendrocyte glycoprotein (MOG). J Neuroimmunology 97:9–15 49. Gold R, Linington C, Lassmann H (2006) Understanding pathogenesis and therapy of multiple sclerosis via animal models: 70 years of merits and culprits in experimental autoimmune encephalomyelitis research. Brain 129: 1953–1971 50. Gold R, Giegerich G, Hartung HP, Toyka KV (1995) T-cell receptor (TCR) usage in Lewis rat experimental autoimmune encephalomyelitis: TCR beta-chain-variable-region V beta 8.2-positive T cells are not essential for induction and course of disease. Proc National Academy Sci USA 92:5850–5854

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Chapter 20 Preparation of Spinal Cord Injured Tissue for Light and Electron Microscopy Including Preparation for Immunostaining Margaret L. Bates, Raisa Puzis, and Mary Bartlett Bunge Abstract An important outcome measure of effects of treatment for experimental spinal cord injury is excellent histology. One way to achieve this is to prepare the tissue for electron microscopy, which ensures the best preservation and requires resin embedding. “Semi-thin” sections of resin-embedded tissue (0.6–1.5 mm in thickness) may be stained and viewed in the light microscope to detect morphological features of the lesion such as various cell types, myelin and blood vessels. Specific areas of interest then may be chosen for thin sectioning preparatory to examination in the electron microscope when better resolution is required, such as searching for non-myelinated nerve fibers. The steps required to prepare the tissue for both light and electron microscopy are detailed in this chapter. A method for assessing the total numbers of axons, myelinated and non-myelinated, is included. Immunostaining for both light and electron microscopy also is described. Key words: Tissue preservation, Resin embedding, Green fluorescent protein label, Myelinated axons, Schwann cells, Non-myelinated axons, Spinal cord injury

1. Introduction Testing more and more potential treatments to reverse the outcome of spinal cord injury in recent years has given much hope to those who have been so injured. Determining the efficacy of all these therapeutic strategies to repair the injured spinal cord requires a combination of outcome measures. A variety of behavioral tests are used to evaluate both sensory and motor outcome; they are described elsewhere in this volume. After preservation by full body perfusion, frozen or paraffin sections are stained with standard dyes such as hematoxylin and eosin or luxol fast blue to assess overall histology, including tissue sparing, lesion volume, and neuronal loss. Emma L. Lane and Stephen B. Dunnett (eds.), Animal Models of Movement Disorders: Volume II, Neuromethods, vol. 62, DOI 10.1007/978-1-61779-301-1_20, © Springer Science+Business Media, LLC 2011

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Besides performing neuroanatomical tracing to determine the parent somata of damaged axons and the resultant response of the axons, immunostaining contributes by allowing the identification of some of the types of responding axons. Immunocytochemistry also enables detection of the macrophage/microglia response as well as additional aspects of the reaction of the immune system. The responses of glia, the progression of cell death/apoptosis, and vasculature changes can be documented. The emphasis in this chapter will be on the preparation of spinal cord tissue embedded in resin for electron microscopy with its ensuing examination in the light microscope (LM) and, when appropriate, in the electron microscope (EM). The preparation of 1  mm “semi-thin,” toluidine blue-stained sections of tissue prepared for electron microscopy but examined in the LM provides for superior histological examination. Whereas scrutiny in the EM may not be necessary, appropriate areas in the semi-thin sections can be chosen for the requisite thin sectioning preparatory to study in the EM. Myelinated axons are easily detected in the LM and, furthermore, myelination by oligodendrocytes or Schwann cells can be distinguished in the semi-thin sections. Macrophages and blood vessels are well seen. EM examination is necessary to detect individual, small diameter, non-myelinated axons. Also, intracellular detail and formed extracellular matrix content are resolved in the EM in contrast to the LM. We describe here the procedures that have worked well in our laboratory for many years.

2. Materials 2.1. Tissue Processing and Embedding

For perfusion, heparin (100 ml) is injected into the rodent heart before initiating perfusion with 300–400 ml of 0.9% saline. Then the rat is perfused with 300–400 ml of 4% paraformaldehyde in 0.1 M phosphate buffer or phosphate-buffered saline (PBS, 900  ml distilled water, 100 ml 10 M NaOH, 40 g paraformaldehyde powder, stirred until clear, 30–60 min; 100 ml × 10 PBS). This is kept in the cold room until used. If pH is not 7.4, HCl or NaOH can be added in small amounts (5–10 ml at a time) for adjustment. For processing tissue, phosphate buffer (PO4) stocks, doubledistilled water (DDW), 0.2 M monobasic PO4 (Solution A), and 0.2 M dibasic PO4 (Solution B) are combined at a ratio of 28 (A):72 (B) to prepare Sorensen’s PO4 buffer at 0.2 M and pH 7.2. From this 0.2 M stock buffer, dilutions can be made to yield 0.15 M, 0.1 M and 0.05 M PO4 buffer. All buffers are kept at 4°C, except Solution B which may be stored at room temperature (RT). The glutaraldehyde fixative for electron microscopic examination (EM fixative) is prepared as follows: 0.05 M Sorensen’s PO4 buffer, 2% glutaraldehyde [25% in vials, Electron Microscopy

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Sciences (EMS), Fort Washington, PA], and 100  mM (3.7%) sucrose. It is stored at 4°C. Osmium tetroxide (OsO4) is a fixative used after glutaraldehyde. OsO4 crystals (1 g vials, EMS) are dissolved in DDW to make a 4% stock solution. As OsO4 is highly toxic and volatile, it should be handled only in a fume hood while wearing latex gloves. The stock solution of aqueous 4% OsO4 should be stored in a sealed dark glass jar within a larger sealed dark glass jar at 4°C in a refrigerator dedicated to toxic materials. From the stock, a 1:1 dilution of the OsO4 can be made with 0.2 M PO4 buffer for 2% OsO4 in 0.1 M PO4 which can be diluted further 1:1 with 0.1 M PO4 for 1% OsO4 in 0.1 M PO4 buffer and stored as above. The stock Epon/Araldite (E/A) embedding medium is mixed and stored at 4°C in a capped bottle and allowed to come to RT before opening in the fume hood to avoid condensation of water in the stock mixture that will cause the embedded blocks to be soft and, perhaps, unusable. In addition, resin components once opened are stored in a vacuum desiccator at RT. The stock resin is made by combining Epon (Embed, 50 ml), Araldite 502 (50 ml), and dodecenyl succinic anhydride (DDSA, 120 ml, all from EMS). Thereafter, for infiltration and embedding, the hardener 2, 4, 6-Tri (dimethylaminomethyl) phenol (DMP-30) is added at 0.1  ml per 5  ml resin using a disposable 1  ml tuberculin syringe. For overnight infiltration, the resin is mixed 1:1 with propylene oxide (PO, EMS) in a disposable tripour beaker. For embedding, fresh resin is mixed prior to infiltration in the vacuum desiccator. Remaining resin is stored, well wrapped, in the refrigerator to be used a few hours later (after coming to RT) for embedding tissue in molds. 2.2. Staining Resin Sections for the LM

Semi-thin, 1-mm sections are stained on a mild to moderate hot plate with Richardson’s stain for 1–2  min (Figs.  1 and 2). Richardson’s stain contains 1% toluidine blue, 1% methylene blue, and 1% borax dissolved in DDW. It is passed through a 0.22 mm acetate syringe filter before staining. Slides are rinsed well with DDW and replaced on the hot plate until dry. Often cutting semithin sections at <1 mm (as thin as 600 nm) may improve the clarity of cellular detail and enable high quality imaging. One percent p-phenylene diamine (PPD), a good stain for myelin (Fig. 2), is prepared in 1:1 methanol/isopropanol. It should be stored in an amber bottle and allowed to rest at least 5 days before using. As PPD is a toxic and permanent stain, gloves should be worn and work should be done in the fume hood. It is filtered with Whatman #1 filter paper before each use into a staining jar and slides are submerged for 20–30 min and rinsed ×2 in isopropanol until the rinses are clear. PPD can be stored for reuse. Because of rinsing after staining with 2–3 changes of isopropanol, a pencil, not a Sharpie, should be used to label the slides. A final rinse in DDW prepares the slide for the application of Richardson’s stain

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Fig. 1. This toluidine blue-stained, semi-thin resin section shows areas of the spinal cord on the right and a Schwann cell implant on the left. The oligodendrocyte-myelinated axons appear close together due to the lack of intervening extracellular matrix. The Schwann cell-myelinated axons are more separated and occasionally show an adjacent nucleus (see arrows). This is one of the main clues for identifying Schwann cell-myelinated axons; oligodendrocyte nuclei in relation to the myelin are not evident because their nuclei are distant from the myelin sheaths that they formed. Scale bar, 20 mm.

Fig. 2. Transverse, semi-thin sections of the sciatic nerve from a normal mouse (40 days old). Sections were stained with toluidine blue (a) or both p-phenylene diamine and toluidine blue (b, c) to highlight myelin. (c) Myelin is further emphasized following bright field background correction. Scale bar, 10  mm. (Courtesy of Paul Morton, Miami Project to Cure Paralysis).

next to reveal more cellular detail in the section. Ethanol may be substituted for isopropanol in this procedure. 2.3. Staining Sections for the EM

For typical electron microscopy, 150 mesh, formvar-coated grids are used for viewing sections in the transmission EM. Formvarcoated grids may be purchased (EMS) or grids can be coated in the laboratory by using a 0.5% formvar solution in dichloroethane.

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Because success in coating grids may be inversely related to the humidity in the atmosphere, in Miami we buy the formvar-coated grids. Formvar-coated slot grids (2 × 1 mm, EMS) also may be used to be able to view the entire section without interfering grid bars. These may be purchased coated, or grids can be coated in the laboratory by using a 1.0% formvar solution in dichloroethane. Sections on grids are stained in two steps, first with uranyl acetate and then with lead citrate. Uranyl acetate is prepared at least 1 day prior to staining and must be crystal clear before using. Uranyl acetate (4% in methanol) is passed through a 0.22 mm acetate syringe filter. The grids are stained in uranyl acetate for 20–22  min before rinsing in 50% methanol, then DDW. Uranyl acetate is stored, undisturbed, in the dark at RT. Lead citrate (0.25%) is filtered as well. The grids are stained in lead citrate for 3–5 min, followed by rinsing in DDW. Lead citrate is stored in a polypropylene-capped tube at 4°C. Allow it to come to RT before staining grids. Place 25 mg of lead citrate in 9 ml DDW, add 2–4 drops of fresh 10 N NaOH and shake until dissolved. Add DDW for a final volume of 10 ml. An example of a thin section so stained appears in Fig. 3.

Fig. 3. This electron micrograph illustrates sheaths of myelin formed around larger diameter axons (a) by Schwann cells. Smaller diameter axons (u) are not myelinated but ensheathed by Schwann cell cytoplasm. At the a* is an axon around which the Schwann cell is in the process of forming myelin. Extracellular matrix, including collagen fibrils, fills the space in between the Schwann cells. A basal lamina is seen to coat the Schwann cell exterior. Scale bar, 1 mm.

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2.4. Anti-GFP (aGFP) Immuno­cytochemistry for the LM and EM

Vibratome sections or 250–300 mm thick slices are dehydrated and rehydrated for better penetration. A steady and experienced hand is able to cut slices with a double-edged razor blade at approximately 200–300 mm thickness. These sections can be floated in the reagents in a multi-well dish to enable immunostaining on both sides of the slice. The critical blocking of endogenous peroxidases step includes 1% H2O2 in methanol before absolute ethanol and rapid rehydration. The blocking solution used is tris-buffered saline (TBS), and blocking agents are 5% normal goat serum (NGS) and 0.5% bovine serum albumin (BSA). Throughout the antibody incubations, 3% NGS in TBS is used. After antibody incubation for development of staining, TBS alone is used, and final rinses of the immunostained tissue are in 0.15 M PO4. All steps of the protocol are performed at RT on a rotator. The primary antibody is polyclonal chicken aGFP (1:500, Millipore, Temecula, CA) and the secondary antibody is biotinylated goat anti-chicken IgG (1:1,000, Santa Cruz Biotechnology, Santa Cruz, CA), both diluted in 3% NGS/TBS. For chromagen precipitate development, tissue slices are first incubated in horseradish peroxidase streptavidin (Vector, Burlingame, CA) in TBS, followed by rinses in TBS, then staining monitored with a Vector VIP substrate kit for peroxidase (1:100 in TBS) for optimal staining intensity. Tissue slices are rinsed in 0.15 M PO4 for viewing in the LM. Positive staining will be purple. Slices should not be allowed to dry. Embedding the reacted tissue sections for the EM entails a 2-h fixation in EM fixative followed by an overnight fixation in 1% OsO4 in 0.1 M Sorensen’s PO4. This could be shortened to 2 h on a rotator in the fume hood. The embedding procedure proceeds as stated in processing tissue for the transmission EM. Penetration of the reagents is limited to the surface of the tissue, perhaps 50 mm deep, so care should be taken not to section through the VIP stain when cutting 1 mm sections for viewing in the LM. Examine the sections either unstained or after a short time (10 s) in Richardson’s stain, to optimize visualization of the aGFP chromagen. When the stained area is located, it can be thin sectioned and placed on grids to be examined in the EM. First examine and photograph unstained grids for best visibility of the reaction product. A 5–10 min exposure to 4% uranyl acetate after the aGFP staining enables a better view of the morphology. When immunostaining semi-thin resin sections not destined for examination in the EM, they require an incubation with sodium methoxide to remove OsO4 bonds and to unmask the antigens. Sodium methoxide, a strong oxidizing agent, is a saturated solution of sodium hydroxide in 100% methanol. It should not be used until it has matured in the dark for 4 weeks.

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3. Methods for Tissue Preparation 3.1. Preservation by Perfusion

3.2. Dissection of the Tissue

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Whole body perfusion by injection of fixative into the heart is best for adequate tissue preservation. Typically animals are perfused with ice cold 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, following an initial injection into the left ventricle of the terminally anesthetized rat of 100 ml of heparin followed by perfusion of cold physiological saline. In an adult rat weighing 200  g, for example, 300–400 ml of saline and 300–400 ml of cold fixative are introduced sequentially into the animal. The spinal cord including the spinal column is carefully extirpated and placed in the same fixative overnight at 4°C. Usually the cord is not removed from the bony encasement until after the overnight fixation. Glutaraldehyde may be added to paraformaldehyde but the concentration of the former must not exceed 0.1% if the tissue is to be immunostained. Because some antigens are hardier than others, a higher percent of glutaraldehyde may be used to improve ultrastructure. It is best to determine the allowable amount of glutaraldehyde for satisfactory immunostaining when starting a new experiment. The spinal cord is removed extremely carefully from the bony encasement. Great care should be taken not to touch the spinal cord. The tissue then may be cut into variously sized segments for immersion in 30% sucrose in PBS or 0.1 M phosphate buffer (+0.01% sodium azide) if only general light microscopy and immunostaining are to be performed on frozen cryostat (10–20  mm thick) or paraffin sections. Tissue should be kept in the sucrose solution until it sinks to the bottom of the vial, 1–2 days. Tissue destined for the EM must not be exposed to this concentration of sucrose or frozen. Thus, separate animals need to be prepared, or the tissue can be subdivided after the post-perfusion preservation so that some slices of the spinal cord are set aside for electron microscopic preparation. A technique used in our laboratory for the complete transection/Schwann cell injury model is to take a 0.5–1 mm transverse slice from the middle of the Schwann cell bridge for electron microscopy and then subject the remaining neighboring bridge/spinal cord segments to sucrose treatment, freezing, and immunostaining. When preparing tissue for frozen sectioning, it may be surrounded by gelatin for stability. Twelve grams of gelatin are added to warmed 100 ml PBS +0.01% sodium azide. The gelatin is then layered on the bottom of an embedding chamber and gelled in the cold room. The piece of tissue with excess sucrose solution removed is placed and oriented on the gelatin. Careful rostral and caudal labeling, etc., should be done at this time. Fresh gelatin is added slowly to cover the tissue and then placed in the cold room. The tissue block is now ready for sectioning longitudinally or transversely.

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3.3. Processing Tissue for the Transmission EM

Preparing spinal cord for EM assessment requires considerable time due to four overnight steps. It involves fixation in glutaraldehyde (overnight), followed by secondary fixation in OsO4 (overnight), then dehydration and penetration of the embedding resin (overnight) and ending with polymerization in a mixture of E/A (overnight). The tissue must be trimmed to a size appropriate for penetration of fixatives and resins, having at least one dimension no greater than 1 mm in thickness. If orientation is important, i.e., rostral vs. caudal, it may be necessary to mark the tissue in some way, such as making a notch or adding a suture, when embedding. Once trimmed, the specimen is placed in the EM fixative and left overnight or up to 1 week. Specifically, the method is as follows: 1. Place trimmed cord slices in EM fixative in labeled 3-ml capped glass shell vials at 4°C and, after 1 rinse of this fixative, leave overnight or up to 1 week. A variation is to use 2.5% glutaraldehyde in 0.1 M cacodylate buffer plus 5% sucrose (1). 2. While working in the fume hood and wearing latex gloves, and after rinsing ×3 in 0.15 M PO4 buffer, 5–10 min each, replace buffer with 1% OsO4 in 0.1 M Sorensen’s P04 [or cacodylate (1)] buffer. Still in the fume hood, place vials on a rotator at a slow setting, covered, for 30 min to 1 h before storing the vials overnight at 4°C. Because it is a heavy metal, the penetration of OsO4 is limited, particularly in the lipid-rich milieu of the spinal cord that is densely packed with myelin membranes. Penetration of the fixative is approximately 0.5  mm, making the 1 mm or less dimension of the trimmed tissue essential in an intact spinal cord. For injured spinal cords, penetration may be much better because of the loss of integrity of the tissue and cavitation. If the osmium does not reach the center of the specimen, the central tissue will remain white and the inadequately fixed membranes will be too poor to assess. Therefore, 1% OsO4 and overnight fixation is employed rather than 2% OsO4 and 1 h fixation that is generally used. Do not leave in OsO4 longer than overnight. If necessary, the tissue can be rinsed in 0.1 M PO4 buffer and stored in the buffer at 4°C until ready to proceed. The use of OsO4 serves a dual purpose because it is both an excellent cross-linking agent and provides contrast to the tissue for imaging. Because the tissue will become black on contact with OsO4, most distinctive physical markings will be lost, necessitating the orienting maneuver recommended earlier. 3. The next day, place the stock E/A resin in the fume hood and allow to come to RT for later use. Uncap the specimens in the fume hood and remove OsO4 by pipette. Discard into a labeled waste container for pick-up by Environmental Health and Safety. Do not discard into the sink. Rinse ×3 in 0.15 M PO4, 5–10 min each, while on the rotator in the hood. Place rinses in the same waste container.

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4. Dehydrate tissue in a series of chilled graded ethanols, 25, 50, 70 and 95% at ×2 for 5–7 min each, continuing to use the rotator in the hood. Further dehydrate in 100% ethanol ×5 at RT for 10 min each. Use a different pipette for each change to ensure that all water is removed. If necessary to halt at this stage, hold in 70 or 95% ethanol. Once the dehydration sequence is started, it should continue to embedding if possible. 5. Before exposing the specimen to the embedding resin mixture, perform two changes in PO at 5  min each. The resin is not compatible with ethanol but because PO is miscible with both ethanol and resin, it is used to facilitate the penetration of resin. PO can be used only in glass or polypropylene vessels, not polystyrene or tissue culture plastic that will soften. PO is highly volatile and should be used in the fume hood while wearing gloves. PO is added very quickly to the vials after the absolute ethanol is removed to avoid drying of the tissue. Extreme care also must be taken while rinsing in PO to avoid evaporation from the surface of the tissue. 6. Replace PO with a mixture of 1:1 PO and E/A with DMP-30 (see Materials) to aid resin penetration into the tissue. Because of viscosity of the resin, polyethylene disposable pipettes are used to transfer resin. Cap vials and keep in the hood overnight but no longer. 7. The next morning, allow the stock E/A once again to come to RT in the hood. We prepare enough embedding resin to not only penetrate the specimens but also for the actual embedding of the tissue in molds. Add DMP-30 (see Materials) to the E/A in a tripour beaker and thoroughly but carefully stir to mix evenly. Bubbles are unavoidable, but should be minimized as much as possible. When ready, remove the PO/resin mixture and add E/A with hardener to the tissue. Place the vials of tissue in a vacuum desiccator for 2–4 h. The remaining embedding medium is capped and covered with parafilm and stored at 4°C. Leaving the tissue in fluid resin for more than 5  h can result in partial polymerization within the vial and make it difficult to remove tissue from the vials for embedding. Thirty minutes prior to embedding, allow the remaining chilled E/A with DMP-30 to come to RT. Small identifying labels can be embedded with the tissue in flat embedding molds. Once the resin is ready, it is added to these molds and the tissue is then added and oriented. Try to avoid introducing bubbles at this time. It is important to think how the tissue will be cut on the microtome when orienting the tissue. If it needs to be initially embedded and then remounted for the proper angle of the cutting face, the excess embedding medium is saved, capped, and wrapped as before and stored at 4°C for glue. Once embedded, place the tissue blocks in an oven set at 64°C and allow to polymerize overnight or over the weekend.

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3.4. Sectioning and Staining Spinal Cord for Light and Electron Microscopy

Traditionally, embedded tissue to be cut for the EM should be trimmed to 1 mm2 and sectioned on an ultramicrotome for silver/ gold-reflecting sections that are placed on a grid for viewing in the EM. For initial perusal of the tissue, however, a larger block may be trimmed and semi-thin sections cut, mounted on a glass slide, stained with toluidine blue, and viewed in the LM. For some studies, the semi-thin section may be all that is needed to assess the spinal cord tissue. Myelinated axons can be clearly seen and identified as myelinated by oligodendrocytes or Schwann cells. To resolve unmyelinated fibers, basal lamina, collagen matrix, and cell–cell relationships, it is necessary to utilize the EM. For examination in the LM, the cured resin blocks are mounted in an ultramicrotome chuck and trimmed using a doublesided razor blade to include all the tissue within a trapezoidalshaped area. Although the size limit depends solely on the width of the knife used to section the block, the width should be <6 mm. The actual face of the block can be greater than 6 × 6 mm (36 mm2); the base (width) of the trapezoid should be no more than 6 mm but the height can depend upon the sweep of the microtome chuck. Therefore, large blocks can be cut for viewing in the LM. Glass knives or Histotech diamond knives (Diatome, Hatfield, PA) can be used to prepare semi-thin sections. The semi-thin sections (0.6–1  mm) are collected from the knife boat (filled with DDW) and transferred to a drop of DDW on a cleaned glass slide. The slide is placed on a hot plate to evaporate the water, thus causing the sections to adhere to the slide. A drop of Richardson’s stain (see Materials) is placed on the sections and the slide is returned to the hot plate for about a minute. The stain is rinsed off the slide with DDW and returned to the hot plate for drying. The slide is now ready to be viewed in the LM. In our laboratory, we avoid coverslipping these stained slides until photographing or counting in order to minimize possible fading of the stain. A toluidine blue stain, like Richardson’s, is the traditional staining method for viewing sections in the LM. The EM resins are resistant to most histological staining techniques. Toluidine blue is a metachromatic stain that reveals good membrane and nuclear details, neuronal Nissl bodies, and is excellent for visualizing myelin (Figs. 1 and 2). PPD also can be used for resin-embedded tissue to assess spinal cord myelin. Because it stains myelin brown (Fig. 2) while other cellular details remain essentially unstained, it is helpful when counting myelin sheaths. The thickness of the sections for PPD staining should be 1–1.2 mm to emphasize the myelin. A brief counterstain with toluidine blue can be used to restore the cellular topography of the spinal cord tissue (Fig. 2). The slides are dried on a hot plate, counterstained with Richardson’s stain for 30  s, rinsed with water, and the slides returned to the hot plate to dry.

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After semi-thin sections have been collected and assessed, the spinal cord tissue embedded in resin blocks can now be sectioned for the EM. Again, the size of the block to be sectioned is dependent upon the width of the knife or size of the grid. This runs counter to traditional electron microscopy where a block trimmed to 1 mm2 is the rule. In our laboratory, we routinely cut a complete transverse section of rat spinal cord to fit entirely on a 150mesh copper grid. Formvar-coated grids are used if the mesh is 150 or less, or the grids contain only one slot. Grids are stained with 4% uranyl acetate in methanol for 20 min carefully covered, followed by thorough rinsing in 50% methanol, then DDW, both delivered from a squirt bottle. These grids are stained in 12 multiwell glass dishes. Another possibility is to use 4% uranyl acetate in 50% ethanol on which the grids may be floated, section side down. Rinsing is as above. Grids, tissue side up, are then placed within a drop of 0.25% lead citrate for 4  min, and then rinsed in DDW. Because the staining solution is placed on dental wax, parafilm, or Teflon sheets, the fluid forms a drop into which the grid may be submerged (or floated). Both stains should be filtered before use (see Materials). Staining must be performed very carefully to avoid dirt or precipitate settling on the sections which would make the sections unusable.

4. Light and Electron Microscopic Analysis of Injured Spinal Cord

Toluidine blue-stained, semi-thin sections of spinal cord are assessed in the LM for intact myelin formed by oligodendrocytes or Schwann cells (SCs), neuronal loss, macrophages, hemorrhage, and vascularization. The general appearance of the tissue is noted. How much spinal cord tissue is spared during the injury? Is there cavitation? Are there meningeal cells invading the spinal cord? If there is a graft, how has it survived and has it integrated with the host tissue? These and other questions can be answered at the LM level. In our laboratory, SCs have often been transplanted, either into a previously contused rat spinal cord or as a bridge of SCs placed between two completely transected stumps of spinal cord (2). One method of assessing the success of the transplantation model is to count the number of SC myelinated fibers in the implant and, in the case of the SC bridge, to count the number of blood vessels that have formed inside. Depending on the number of SC myelinated fibers, counts can be done by a direct method, i.e., counting every peripheral myelin profile using a 10 × 10 eyepiece graticule and a 63× oil immersion objective lens, or by counting representative myelin profiles using the Stereoinvestigator (MicroBrightField, Williston, VT) program. If the counts appear to be <1,000, the direct method

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is the easiest and most accurate method. If >2,000, the use of Stereoinvestigator is more efficient. For both methods, counting the blood vessels in each field helps to assess the health of the graft. If the PPD staining method is used for counting myelinated axons, it may be difficult to see the blood vessels without a counterstain of toluidine blue. It is important to note that all the myelin, whether central or peripheral, is stained but can be distinguished. In general, SC myelin is much better preserved than oligodendrocyte myelin. It stains deep blue (or dark brown in the case of PPD staining) compared to less intense staining of central myelin. There will be numerous SC nuclei associated with peripheral myelin, reminiscent of a signet-ring appearance (Fig. 1); very few nuclei appear associated with central myelin. Although there is a 1:1 relationship between the SC and the myelinated axon, the nucleus will not always be present in the section. But the ubiquitous rim of cytoplasm, while not visible, is covered by basal lamina and associated collagen, leading to more space between SC myelinated axons than between central myelinated axons which appear often to touch each other. SCs myelinate single axons, peripheral and central alike, when invading, transplanted into, or bridging injured spinal cord. A SC can ensheathe but does not myelinate multiple axons. There can be 7–20 times more unmyelinated fibers related to SCs than myelinated ones. The LM does not provide the resolution necessary to adequately assess the number of unmyelinated fibers present in the spinal cord. In order to count unmyelinated fibers, it is necessary to employ the EM (Fig. 3). If there are ten times more unmyelinated fibers in an injured spinal cord and there are 1,000 myelinated fibers, there could easily be 11,000 fibers associated with SCs. Direct counting of that many fibers in the EM is not feasible. In order to get a valid assessment of the number of unmyelinated axons, a ratio of myelinated to unmyelinated axons can be determined. It is important not to bias the ratio by preferentially choosing to photograph SC unmyelinated axons next to SC myelin profiles. The magnification of the photographs must be high enough to be able to identify and count unmyelinated axons, but low enough to see an adequate area of spinal cord tissue. The number of photographs should be adequate for statistical quantification, but low enough to make the project reasonable in scope. The entire cross-section of the spinal cord is scanned in the EM to map the total tissue area on the grid. Then the grid is scrutinized at points in the corners (A,B,C,D) and center (E) of each grid square to mark on the map those areas containing SC-associated axons whether they are myelinated or unmyelinated. These are termed “hits.” After scanning, the number of hits in each area (A,B,C,D,E) is counted. The goal is to randomly select 20 areas containing SC-associated axons per grid. The preference is to

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choose the central area of each grid square, far from the grid bar. Sometimes additional areas need to be included to have 20 areas of SC-associated axons. If there are 20–25 hits in the E regions, electron micrographs are taken at 6,600 magnification of those areas. If there are fewer than 20, then A, B, C, and D areas will be used to choose 20 areas for photography, i.e., B may have a total of nine hits and D may have a total of 12, enabling 21 areas to photograph. After the photographs are taken, the myelinated and unmyelinated axons are counted. Care must be taken to accurately count the unmyelinated fibers; it is easy to mistake a vacuole, an inclusion or, particularly, swollen rough endoplasmic reticulum, for an unmyelinated axon. After counting is completed, an unbiased ratio of myelinated/unmyelinated axons within the injured/transplanted spinal cord is obtained. This ratio can be used, knowing the count of myelinated axons from the LM analysis, to determine the total number of SC-associated axons (3).

5. Immunostaining of ResinEmbedded Sections

5.1. Immunostaining of Semi-Thin Sections for the LM

Immunocytochemical analysis is an important outcome measure. Whereas viewing 10–20 mm sections in the LM is valuable, questions that require superior histology or better resolution may arise. For example, what cell type has been transduced by a virus carrying genes for enhanced green fluorescent protein (EGFP)? Is it distributed throughout axons if neurons have been infected? Immunostaining may be performed on 1-mm resin sections or tissue prepared in such a way as to view antibody staining in thin sections for the EM. Antigenicity in tissue embedded in resin may be recovered to enable immunostaining of the same block that has been processed for LM and EM viewing. The tissue must have been fixed in glutaraldehyde for not more than 24 h, and the technique involves neutralizing the OsO4 fixative, unmasking the antigens by etching the resin to expose the antigenic sites. Sections 1-mm thick are placed on an array of drops of water on a Surgipath slide and heated on a hot plate to adhere to the slide. Each section is circled with a diamond tipped pen to identify placement. The slides are then placed in a 64°C oven overnight to ensure adherence to the slide during immunostaining. The next day, the slides are immersed in sodium methoxide/methanol at a 1:1 dilution for 45 min in the dark. The following procedure is recommended: 1. Rinse the slides in methanol ×4 at 5 min each. 2. Immerse the slides in 3% hydrogen peroxide (H2O2) in methanol for 20 min in the dark. 3. Wash the slides in distilled water ×3 at 5 min each.

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4. Check the slides to make sure the OsO4 has been removed before immersing them in 8% formic acid for 10 min. 5. Wash in water for 10 min, then place in 0.1% Triton in PBS for 10 min. The slides are now ready to be stained. Circle the sections with a PAP pen and stain every other section. The alternate sections may be stained later with toluidine blue for tissue morphology. 6. Place the slides in a blocking solution of 20% normal goat serum (NGS) in PBS for 30 min. 7. Blot and apply the antibody at a dilution ×2–5 more concentrated than normal in 2.5% NGS in PBS with 0.1% Triton. For instance, if the antibody is usually diluted 1:1,000, dilute at 1:200 for this staining procedure. Incubate the sections at RT for 1 h or overnight at 4°C. 8. Wash the slides in PBS with 0.1% Triton ×3 for 5 min each. 9. Apply biotinylated goat-anti-rabbit or goat-anti-mouse at a 1:200 dilution in PBS or ×2–5 the concentration used normally. 10. Incubate the slides with secondary antibodies at RT for 1 h. 11. Rinse slides ×3 for 10 min each with TBS. Prepare the horseradish peroxidase streptavidin (Vector) 1:250 in TBS and incubate for 1 h at RT. 12. Rinse the slides with TBS ×3 for 10 min at RT. 13. Prepare the diaminobenzidine (DAB) solution using the ImmPACT DAB peroxidase substrate kit (Vector). 14. View the slides in the DAB solution in the LM using 4× and 10× magnification to check the reaction. Stop the reaction by placing slides in water for 5 min at RT immediately after the desired stain intensity develops. 15. Dry the slides and coverslip with Vectamount permanent mounting medium (Vector). If using the Vector-VIP Kit instead of DAB, prepare the VIP according to the manufacturer’s instruction. Stop the reaction by placing slides in water for 5 min at RT immediately after the desired stain intensity develops. Dry the slides and coverslip with Vectamount medium. When using DAB, a counterstain of toluidine blue (10  s) on a warm hot plate may be employed prior to coverslipping. When using VIP, do not use a counterstain. 5.2. Immunostained Thin Sections for the EM

The gene for green fluorescent protein (EGFP), introduced into cells by way of virus, is a useful compound to follow cells after transplantation. The technique to detect it in thin sections is as follows. 1. Fix the tissue in 4% buffered paraformaldehyde up to 10 days.

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2. Slice the rat spinal cord transversely rostral to caudal (including the transplantation site) in a Vibratome or with a double-edged stainless steel razor blade at a thickness of 250–300 mm. 3. Label each slice according to its distance from the lesion/ implant to be embedded separately. 4. Wash the tissue slices in 0.15 M PO4 (pH 7.5) and rapidly dehydrate in ethanol (70 and 95% at 5 min each). Followed by 1% H2O2 in 100% methanol for 10 min to block endogenous peroxidase, then in 100% ethanol for 5 min and rapidly rehydrate. 5. Rinse slices in TBS and block with 5% NGS and 0.5% BSA in TBS for 1 h before incubation for 18 h in chicken anti-GFP polyclonal antibody, diluted 1:500 in the blocking solution. Negative control slices should remain in blocking solution only. 6. After incubation wash the slices in 3% NGS/TBS ×3 for 20 min each, and then incubate for 2  h in biotinylated goat antichicken IgG at a dilution of 1:1,000 in 3% NGS/TBS. 7. Wash slices in TBS before incubation in horseradish peroxidase streptavidin for 2 h. 8. After washing in TBS ×3 for 10 min each, the Vector VIP substrate kit for peroxidase is used with a modified reagent concentration of 1:100 and 10  min incubation in substrate to produce good staining intensity. 9. Perform all steps of the protocol at room temperature on a rotator. 10. The presence of immunolabeling is detectable in the LM. 11. Rinse the slices in 0.15 M PO4 ×3 at 5 min each. 12. Post-fix the tissue slices in EM fixative (see Materials) at RT for 2 h. 13. While in glutaraldehyde, examine every sample in the LM to detect the label and to map its location to guide the trimming of the sample for thin sectioning. 14. Further fix samples in 1% OsO4 in 0.1 M PO4 (pH 7.4) overnight. 15. Rinse in 0.15 M buffer and rapidly dehydrate in cold graded ethanols. 16. Follow the procedure leading to embedding as detailed above. 17. For less sectioning and thus faster analysis of the tissue, glue some consecutive blocks together with E/A, cut in half and remount to provide transverse or longitudinal sections.

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Fig. 4. This figure illustrates the EGFP reaction product as seen in the EM. Arrows point to Schwann cells that contain the product, having been transduced by way of a virus to label the cells. At least four non-myelinated axons (u) can be seen. (Courtesy of Dr. Damien Pearse and Raisa Puzis, Miami Project to Cure Paralysis). Scale bar, 1 mm.

18. Stain semi-thin sections in these samples with toluidine blue or leave unstained to detect the presence of immunolabel in the LM. 19. After trimming to be able to thin section only the immunolabeled area, collect silver/gold sections on 150 mesh, formvar-coated copper grids. 20. Examine both negative control and EGFP labeled samples and photograph in the EM without uranyl acetate or lead citrate staining. The EGFP label is apparent as a dense precipitate (Fig. 4). 21. Prepare additional sections for uranyl acetate staining to better appreciate the tissue ultrastructure.

6. Technical Remarks This chapter attempts to stress the value of examining semi-thin resin sections of tissue for superior histology. These sections provide a valuable addition to the array of outcome measures employed to assess potential treatments following injury. Preservation is outstanding because the tissue is prepared as if it were to be studied in the EM but it is not necessary to proceed to use of the EM if not warranted.

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Also, having the 1 mm sections can provide the possibility of indentifying desired regions to be thin sectioned for EM analysis. An ultramicrotome is required for sectioning the resin blocks at 1 mm thickness and for ultrathin sectioning. Either glass knives prepared in the laboratory or Histotech diamond knives, less expensive than those required for thin sectioning, can be used. A glass knife is used to obtain a smooth and complete block face for sectioning and can be used to cut semi-thin (and even thin) sections but it quickly becomes dull whereas a Histotech knife, although more costly than glass, can last for several years before resharpening is necessary. An ultradiamond knife to acquire thin sections lasts for several years and can be resharpened when dull or adds too many knife marks to the sections. Another point to be stressed is the necessity for cleanliness during preparation for EM analysis. Dirt or stain precipitate is magnified as well as the image and can interfere with study of tissue ultrastructure. Finally, if digital imaging rather than exposure of film is used for capturing the tissue images in the EM, staining with uranyl acetate and lead citrate may not be necessary to provide good contrast. Described here is a protocol for whole mount tissue EGFP immunolabeling as an example of antibody staining for EM evaluation. This protocol, compatible with the standard immunoperoxidase protocol and processing of tissue for EM in our laboratory, is based on results of a pilot protocol optimization study. We use this protocol to detect EGFP-labeled axons after stereotaxic injection of adeno-associated viral vectors carrying the gene for EGFP into the brainstem (4). We decided to use pre-embedding antibody staining to accomplish both successful immunolabeling (especially for low levels of EGFP) and tissue ultrastructure preservation. An EGFP method of detection using an electron dense chromogen for peroxidase can provide improved visualization of EGFP at both the LM and EM levels. Good preservation of antigenicity and ultrastructure and the possibility of high labeling resolution with low background contamination are tightly connected to the fixation procedure. In this protocol, we use 4% buffered paraformaldehyde as the primary fixative for regular immunocytochemistry and electron microscopic preparation. Comparison of tissue stored at 4°C in this fixative for 1 through 4 weeks did not show a difference in antigenicity or tissue morphology. With prolonged fixation time, however, paraformaldehyde forms reversible peptide bonds and irreversible methylene bridges that eventually reduce immunoreactivity. A next step in this protocol after antibody staining is postfixation in EM fixative and then 1% OsO4. This preserves the ultrastructure of the cells, as well the color (in the LM) and texture of the chromogen precipitate that labels the EGFP, and it also reduces background interference. Whereas the conditions of fixation are critical in retaining EGFP in the cells and maintaining antigenicity, the penetration of

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the reagents into the tissue is also important. For LM studies, the detergents TritonX-100 and Tween-20 are routinely used to disrupt membranes to allow increased penetration of the immunological reagents. To protect cell integrity for the EM, detergents and the antigen-unmasking technique are eliminated from the protocol. Instead we use rapid dehydration–rehydration that aids in penetration of large molecules throughout the tissue. Thickness of the tissue slice also should be considered; we use 250–300  mm slices to prevent damage to the fragile graft area in the injured spinal cord and possible mechanical loss of the EGFP. Immunolabeling also can be limited by the density of the tissue itself. Slices through the intact region of the spinal cord and control uninjured areas show penetration of staining in the 30–50 mm range, compared to the more disrupted injured graft area where the reagents may have penetrated throughout the slice. To find the best substrate for immunoperoxidase labeling in the EM, we compared DAB and Vector-VIP reaction products. For the LM, the difference between the two chromogens is based on color as DAB is brown and Vector-VIP is dark purple. Because we prefer VIP for the LM, as it provides more intense and crisp staining, we tested it in the immunoEM protocol. Whereas both chromogens are suitable for electron microscopy, we found that DAB presents as a granular precipitate, strongly enhanced by OsO4. Vector-VIP yields larger electron dense granules that are easily and clearly visible in the EM. Ultrathin sections, unstained with uranyl acetate and lead citrate, easily reveal the presence of the electrondense VIP particles at the EM level and show good contrast. The level of background contamination is lower in the Vector-VIP procedure than in the DAB reaction. The DAB reaction product, however, is insoluble in ethanol compared to the Vector-VIP which requires rapid dehydration in ethanol before embedding in E/A. This combination of immunostaining and electron microscopy provides consistent and repeatable results with intense immuno­ staining and acceptable ultrastructure preservation. It can be used for tissue explants and cell cultures as well as whole mounts. This protocol is not limited to aGFP labeling but can be used for different antibodies with adjusted concentrations [see a previous report using this pre-embedding antibody staining technique in (5)].

Acknowledgment Funding in the authors’ laboratory is from NIH NS 09923 and 38665, the Miami Project to Cure Paralysis, the Buoniconti Fund, and the Christopher and Dana Reeve Foundation. MBB is the Christine E. Lynn Distinguished Professor of Neuroscience.

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References 1. Zhang L, Ma Z, Smith GM, Wen X, Pressman Y, Wood PM, Xu X-M (2009) GDNF-enhanced axonal regeneration and myelination following spinal cord injury is mediated by primary effects on neurons. Glia 57(11):1178–1191. 2. Bunge MB (2008) Novel combination strategies to repair the injured mammalian spinal cord. J Spinal Cord Med. 31:262–269. 3. Xu XM, Guénard V, Kleitman N, Bunge MB (1995) Axonal regeneration into Schwann cell-seeded guidance channels grafted into transected adult rat spinal cord. J Comp Neurol 351:145–160.

4. Shihabuddin LS, Brunschwig J-P, Holets VR, Bunge MB, Whittemore SR (1996) Induction of mature neuronal properties in immortalized neuronal precursor cells following grafting into the neonatal CNS. J Neurocytol 25:101–111. 5. RR Williams, DD Pearse, MB Bunge (2010) Expression of the developmental transcription factor MASH-1 in the brainstem enhances the regeneration of noradrenergic axons across a Schwann cell bridge. The 40th Annual Meeting for the Society of Neuroscience.

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Chapter 21 Assessing Spinal Cord Injury Gillian D. Muir and Erin J. Prosser-Loose Abstract Functional recovery is the ultimate goal of research into experimental therapy for spinal cord injury (SCI). The effective use of animal models of SCI requires functional assessment methods that can be reliably repeated in different laboratories. The aim of this chapter is to describe some key features of behavioural methodology which inform our laboratory’s decisions regarding appropriate assessments in rat models of SCI. These include recognition of the type of data being measured, assurance of appropriate sampling methods to improve reliability, and considerations of the animals’ motivation during completion of the behavioural tasks. We then illustrate these principles with methods used in our laboratory, a major emphasis of which has been biomechanical analysis of limb action during overground locomotion. We also describe analysis of skilled limb movements during more challenging tasks such as ladder locomotion and forelimb pellet retrieval. Our focus throughout is on objective quantitative assessment of movement that can be reliably used to assess functional capabilities in rat SCI models under different lesion or treatment conditions. Key words: Rat, Behaviour, Spinal injury, Locomotion, Ground reaction force, Skilled movement, Velocity, Forelimb, Reaching

1. Introduction Functional recovery is the ultimate goal of research into experimental therapy for spinal cord injury (SCI). Thus, the approaches and methods used to assess recovery in animal models of SCI are crucial elements in the search for successful therapies. Over the past decades, a wide variety of tests and measurements have been developed and used to assess recovery after spinal injury and these have been thoroughly summarized in several comprehensive reviews (1–6). The effective use of animal models requires methods that can be reliably used in different laboratories. Thus, behavioural assessment methods need to be chosen with care, and particular attention paid to the robust nature of the methods (7). This chapter

Emma L. Lane and Stephen B. Dunnett (eds.), Animal Models of Movement Disorders: Volume II, Neuromethods, vol. 62, DOI 10.1007/978-1-61779-301-1_21, © Springer Science+Business Media, LLC 2011

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will describe some basic principles underlying the choice of appropriate methods of behavioural analysis and will then apply these principles to methods used in our laboratory, focusing on robust, quantitative measurement used to assess behaviour in spinal-injured rats.

2. Where to Start? Some Initial Considerations

2.1. Which Behaviour Should Be Examined?

There are two main considerations to be taken when approaching behavioural assessment. First, which behaviour should be examined, and second, what specifically should be measured from that behaviour ? Like most SCI studies, this chapter will focus on the rat as an animal model. Rats are the most commonly used species for experimental SCI research, in part due to their convenient size and their wide behavioural repertoire. Nevertheless, the basic principles of assessment can be applied to behavioural measurement in all species. The answer to this question starts with an assessment of the functional capabilities of the animal after the spinal lesion, which in turn is dependent upon the type of lesion. One of the biggest contributing factors to the methodological diversity in SCI studies is the range of different lesion types produced in rat models. Spinal lesions can be inflicted by transection or contusion, and can be located in the cervical or thoracic regions of the spinal cord (8–12). Lesions may completely sever all spinal projections or may lesion only a subset. Partial injuries can be of varying severity but generally involve at least the dorsal portion of the spinal cord, largely due to ease of surgical access. So, how does one determine the capabilities of the rat following SCI? An obvious place to start is with overground locomotion. Careful observation of the animals as they move around in their cages and in an open field will quickly reveal how well they are able to use their limbs and, in turn, which behavioural tests might provide appropriate assessment. Experimentally injured animals can be generally classified into three main groups (1) those that are unable to move overground, (2) those that can move overground but with clearly abnormal limb movements and paw positions, and (3) those that locomote well or with only mild deficits (2). More detailed classification of overground locomotor abilities in rats with thoracic spinal cord lesions can be obtained using the Basso, Beattie and Bresnahan locomotor rating scale (BBB) (10). This scale categorizes the functional capacity of the hindlimbs from complete paralysis (score of 0) to normal movement (score of 21). The three main groups described above correspond to BBB scores of 0–9 (unable to locomote), 10–15 (locomotion with abnormal limb movements) and 16–21 (locomotion with good limb placement).

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Similar rating scales have been developed to rate forelimb function after cervical lesions (13–15). If an animal is classified as being unable to locomote, further locomotor analysis is obviously limited. However, some analysis of limb function can be performed by providing weight support while the animal steps on a moving treadmill (16, 17). Swimming is another behaviour that can be analyzed in some animals unable to support their weight overground (18–20). Several reviews provide comprehensive lists of tests which can be applied to animals with different functional capabilities (1, 2). In our laboratory, we focus on the contribution of different spinal pathways to motor behaviour. Therefore the spinal lesions we inflict involve only a small proportion of spinal white matter so as to avoid the confounding effect of simultaneous damage to multiple pathways (21–24). Consequently, the animals locomote well after surgery, allowing us to assess subtle changes in overground locomotion using sensitive kinetic and kinematic measurements (see Sect.  3.1 below). We are also able to examine the animals’ performance on more challenging tasks, such as ladder crossing (Sect. 3.2) and forelimb pellet retrieval (Sect. 4.1) with the aim of distinguishing the contributions of specific spinal pathways to each behavioural task. 2.2. What to Measure from Behaviour? The Nature of Behavioural Data

Once a decision has been made about the set of behavioural tasks that spinal-injured rats are capable of, the next step is to decide what aspect of each task to measure. There are many aspects of any particular behaviour that can be measured in some form (1, 2). The possibilities are numerous and can initially be overwhelming. In our lab, there are several general considerations that guide our decision making with respect to the behavioural measures we use. These can be classified into three basic “principles”: 1. Employ quantitative measures over ranked or categorical measures whenever possible. 2. Ensure methods provide representative sampling so that the data is reliable. 3. Consider the rat’s point of view with respect to motivation and compensation.

2.2.1. Levels of Measurement: Quantitative vs. Ranked Measures

Measurement is the process of assigning numbers to observations according to specified rules (25). There are several levels of behavioural measurement well described in textbooks on analysis of behaviour in humans and in animals (25, 26). For the measures that have been used to assess behaviour in rat models of SCI, the most important distinction is between quantitative measurements and ranked measurements. Quantitative measurements are those that are made on a true numerical scale (also known as interval or ratio scales), such as length of stride during overground locomotion, or numbers of foot slips made while crossing a ladder. Ranked

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measures are those in which the investigator has assigned numbers to particular sets of characteristics on an ordinal scale (e.g., locomotor rating scores, rung walking scores) (10, 14, 15, 27). Quantitative measurements hold several advantages over ranked measures. While the scores in ranked measures can be assigned in a meaningful way, the differences between scores on these derived scales are not meaningful. For example, on the BBB scoring system, a score of 18 represents an animal with better functional capabilities than a score of 15, but a change from a score of 15 to 18 does not represent the same functional improvement as from 12 to 15. This contrasts with quantitative measures where differences between data are meaningful and hold value, for example, a difference between 15 vs. 18 cm in stride length compared to 12 vs. 15 cm represents an improvement of 3 cm in both cases. The use of parametric statistical analysis with quantitative measures represents another advantage over ranked measures. Ranked measures can be derived for most behavioural tasks and are frequently employed in SCI studies because of their ease of use, their applicability to specific behavioural conditions and, in some cases, their ability to represent a wide range of functional capabilities (10, 28, 29). Nevertheless, because the ranking of the categories relative to each other is the only consistent characteristic of score measurements, categorical data should be analyzed using nonparametric statistics (26). Alternately, statistical analysis can be performed on quantitative measures that are associated with the scores, such as time to reach a particular functional category, for example, the number of days to reach a score of 15. In this way, the benefits of the categorical measures (ease of use, wide range of applicability) can be employed while still using more powerful parametric statistical analysis on appropriate data. The other main advantage of using quantitative data over ranked data is the robustness with which the former measures can be applied to animals with different lesions and treatment conditions. Ranked measures might be reliable and valid for the lesion model for which they were derived, but they can lack robustness. This is because the derived categories will not necessarily account for the characteristics displayed by rats with different lesions or during recovery from different therapy. When thus applied, they can become inaccurate measures of the behavioural capabilities. This is familiar to anyone who has tried to “fit” an animal into a descriptive category for which they do not possess all the characteristics. Thus, use of ranked measures should be restricted to the conditions for which they were derived and shown to be reliable and valid (15, 29). 2.2.2. Sampling Considerations: Reliability and Representativeness

For any behavioural measurement, it is necessary to ensure that the sampling methods produce data that reliably represent the abilities of the animal. An essential factor to consider here is the inherent

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variability in the performance of an individual animal. The more performance varies, the higher the number of samples that will need to be acquired. Assuming that variability is due to random error ((25) and see below), the mean of the samples should provide the desired representative measure. Importantly, performance can be variable within the same sampling session, and/or between sampling sessions on different days. For example, in our laboratory, we have found that reaching success during forelimb pellet retrieval (see Sect. 4.1, below) varies noticeably from day to day, even in rats well trained to perform the task. We have determined that sampling reaching success over three consecutive days and averaging the result provides a representative measure of each animals’ performance. Therefore it is important as an investigator to be aware of the variability that may occur in any given task and sample accordingly. Even for the same task, there are differences in the amount of variation which occur in different measures. We assess overground locomotion in rats using a variety of quantitative measures as rats repeatedly traverse a runway (see Sect. 3.1, below). We have found that, for an individual rat moving at the same speed overground, there is very little variation in stride length between repeated passes, but other measures, such as the vertical force exerted by each limb, can vary considerably more. By collecting data from a number of different passes for each trial, we have determined that data collected from ten passes per rat will produce a representative measure of peak vertical force for each limb. Thus sampling methods need to take into consideration the inherent variability in the measured values for each task. Taking the mean of data values sampled from the same animal is only valid, if, as stated previously, the variation between those values is due to random error. For this assumption to be correct, the values must be sampled independently of one other. If this is not the case, that is, if what occurs during one sample is partly dependent upon another, then systematic error is introduced. An example of the latter situation occurs when samples are taken from successive strides during locomotion, either overground or over a horizontal ladder. A stride is one complete locomotor cycle, e.g., from the start of ground contact of a limb to the contact of the same limb in the subsequent cycle. Any single stride is clearly not independent of the stride prior to it, because many variables (e.g., the positions of the paws, the length or timing of each step) depend, in part, upon what was occurring in the previous stride. A shorter step or a slip on a ladder rung will alter how the rat makes subsequent steps. Therefore, samples should not be taken from successive strides. We accomplish this by sampling only one stride per pass (for each limb) as the rat repeatedly traverses the runway or the ladder. We also allow a correction stride, which is not analyzed, following any errors.

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Another important consideration which affects behavioural data sampling is the speed with which an animal carries out a particular task. This is particularly important not only for locomotion, as many locomotor measures vary with speed (see Sect. 3.1), but also applies to other behavioural tasks. After injury, animals often perform a task more slowly than prior to injury. Some measures address this aspect directly, e.g., the number of food pellets retrieved in a fixed period of time. Nevertheless, attention should be paid to the speed with which an animal accomplishes a task and consideration given to how this might affect the measured values. For example, one could question the validity of comparisons made between the numbers of foot slip errors made by a spinal cord-injured animal slowly and carefully stepping across a horizontal ladder and those made by a sham-operated animal stepping quickly across the same ladder. 2.2.3. The Rat’s Point of View: Motivation and Compensation

The analysis of behaviour after SCI necessarily requires that the rat “do something” or perform a task so that we can measure it. From the point of view of the animal, there needs to be some motivation to partake in any particular behaviour, whether it be unconditioned, e.g., spontaneous exploration, or conditioned by the experimenter. During spontaneous exploration, the animal is motivated by natural curiosity to freely explore a novel environment, within which the animal can be observed for particular movements. This applies to the rank scoring of limb movements during exploration in an open field (10, 14), for example, or measurement of forepaw contacts with the walls of a cylinder in the paw preference test (30). Conditioned behaviour, such as reaching for a food pellet through a small opening, requires that the animals be motivated to achieve a particular goal, in this case to obtain a food reward (31). Conditioning an animal to perform a task such as pellet retrieval is valuable in rat SCI models for several reasons. First, conditioning is a requirement for some tasks, such as pellet retrieval, where rats do not initially perform well but will show improvements after repeated practice. Second, when animals are well versed in a particular task, such as pellet retrieval or even ladder crossing, the experimenter can examine the effect of a particular manipulation or treatment on performance of a well-known task. This is in contrast to measurement of performance during the acquisition of a new task. An example of the latter situation is examination of the ability of a rat to cross a horizontal ladder in which the rung positions can be varied and are altered for each session (27, 28). In these cases, the task is assessing the ability of the animal to cross a new rung pattern each day (i.e. learning in addition to motor performance), rather than the ability of the animal to perform a well-known pattern (strictly motor performance). While both situations are valid, the investigator should be aware of what is being asked of the animal in any particular experiment and in turn what exactly is being measured. A third advantage of conditioning is that it tends to

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reduce differences in motivation that affect performance on a particular task. These differences can occur between individuals as well as within the same individual at different times during an experiment, especially before and after SCI. Finally, awareness of compensatory behaviour following SCI is an important consideration, especially when taken from the point of view of the rat. When animals perform a task after experimental SCI, their external motivation remains the same, e.g., the acquisition of the food reward, but in order to attain the reward, they may alter the way they accomplish the task (32). These compensatory actions are undoubtedly many faceted but can involve moving more slowly, shifting weight to less affected limbs, or using the less affected forepaw to retrieve a food pellet. These compensations represent many of the challenges of interpreting sensorimotor behaviour after experimental manipulation and of distinguishing recovery of movement from replacement by new movements (12, 24). In our laboratory, we see compensatory behaviour by rats in the ladder task following SCI, for example, when animals use the wrist rather than the palm to contact the ladder rung. It is difficult to deal with these behaviours upon analysis since they are not as severe as actual errors (for example, a foot slip) and do occasionally occur in unlesioned animals. How these abnormal behaviours are assessed in the context of the spinal injury and subsequent treatment should be decided ahead of time by the investigator.

3. Assessing Locomotion Locomotor tasks assess the coordinated functioning of all four limbs, although it is clear from research in rats as well as other quadrupeds that the contributions of the hindlimbs and forelimbs during locomotion are not equivalent. The neural control of locomotion is arranged hierarchically in the central nervous system (33). Many of the salient features of stepping (alternating flexion and extension of each limb, right-left alternation, transitions from swing to stance) are controlled at the level of the spinal cord working in concert with sensory feedback from the limbs (34). Input from higher centres, namely the brainstem and cerebrum, are required for balance, speed control, locomotion over uneven or challenging surfaces and of course voluntary adjustments to locomotion (35). We will discuss the methods we use to examine locomotion in spinal-injured rats, both overground and under more challenging conditions. 3.1. Overground Locomotion

Research in our laboratory has focused on the quantitative assessment of overground locomotion in rats (12, 21, 23, 24, 36–39). We use kinetic (relating motion with mass and force), and kinematic (analysis of motion apart from mass and force) techniques which

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are standard methods to quantify movement performance in many animals, including humans (40). These methods provide highly robust and objective assessments that are applicable to many functional conditions. We have developed many of these techniques in our lab in order to determine the functional contributions of specific spinal pathways to overground locomotion in the rat. 3.1.1. Ground Reaction Force Measurement

We are one of the few laboratories which have used biomechanical assessments of ground reaction forces (GRF) during locomotion in rats. Ground reaction forces are the forces which act through each limb to support the animal’s weight, and provide propulsion and lateral stability as the animal moves overground. Subtle differences in the use of individual limbs during locomotion, such as changes in weight support or propulsive effort, can be detected by GRF measurement. These measurements require specialized equipment, namely force platforms or plates, which are capable of detecting forces in three orthogonal directions (Fig.  1a). Force plates are readily available for human locomotion research, as GRF measurement is a standard technique in biomechanical assessments of gait. There are also force platforms sized appropriately for smaller animals, including rats (AMTI, Inc, Watertown, MA). Our laboratory uses custom-built force platforms (41). Three platforms, each measuring approximately 11 cm × 11 cm, are positioned level with the surface of the runway (180  cm × 15  cm) and arranged in series. The size of the plate surface is important. If the plates are too small, the rat will miss contact with a plate as it passes over. If the plates are too large, more than one limb will contact the plate at one time, which does not allow the investigator to distinguish which limb is responsible for the forces measured. Each platform measures force in three orthogonal directions (vertical, fore-aft and medio-lateral). The position of the limb on each platform can be calculated from the ratio of the input from vertical force sensors located at each corner of the platform (41). Outputs from the platforms are amplified, digitally sampled (1,000  Hz) and acquired simultaneously with digital video recordings (Midas, Xcitex Inc, Boston, MA). A digital camera (125 Hz, Motionscope 1050; Redlake MASD Inc.) is positioned perpendicular to the runway. An infrared beam positioned across the runway ahead of the series of three force platforms triggers simultaneous GRF and digital video recordings each time the rat approaches the platform. A second beam positioned on far end of the platforms stops data recording and an LED display indicates the time taken for the rat to traverse between the beams, providing a measure of average velocity over the platforms. Data acquired from our custom-built force platforms is analyzed using custom-written software, although commercially available force platforms will have accompanying software. Data for each pass is digitally filtered using a fourth order Butterworth Filter (cut-off 40 Hz). For each pass, the force data from each plate are

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Fig. 1. Ground reaction forces produced by rats moving along a runway. (a) Rats are conditioned to move repeatedly along a runway in which three force platforms (plates 1–3) are embedded in the ground surface. Ground reaction forces (forces exerted through the limbs onto the ground) are recorded in three orthogonal directions: vertical (red arrow), fore-aft (blue arrows) and lateral (green arrows). The rat contacts each platform first with a forelimb followed by the ipsilateral hindlimb, so that forces from the right and left limbs can be recorded separately (right and left in (b–d)). (b) Peak vertical forces are approximately equal for fore- and hindlimbs, although impulse (area under the curve, indicated in light blue) differs between fore- and hindlimbs. (c) Forelimbs generate most of the braking forces while the hindlimbs generate more propulsion. (d) Lateral forces are small for both fore- and hindlimbs. For each graph, solid lines represent mean data from unlesioned animals (n = 12), thin lines are ±standard error.

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matched with the identity of the limb that has contacted the plate, using time-synchronized digital video recording (Fig.  1a and Sect. 3.1.3). Data for each limb from different passes (ten passes minimum) across the plates are averaged to obtain a representative force for each limb. In order to compare performance between individuals and between groups of animals, we examine key features of the GRF profile for each limb such as peak force (e.g., maximum vertical, maximum braking and maximum propulsive forces) and impulse (e.g., area under the force vs. time graph for vertical, braking and propulsive forces) (Fig. 1b, c). The value of these variables are determined for each pass and averaged for each limb of an individual animal. 3.1.2. The Importance of Locomotor Velocity

In order to increase the chances of acquiring data when the rats are moving at a steady speed across all three platforms, the rats are con­ ditioned to repeatedly traverse back and forth along the runway for a food reward. A steady speed is necessary because ground reaction forces are only consistent when net forward acceleration is 0, since animals use their limbs differently if they are accelerating or decelerating. Rats must be motivated to shuttle to and fro but at the same time must be relaxed enough so that they are not dashing from one end of the runway to the other. We achieve this by conditioning the animals over several weeks to expect a food reward (a Cheerio) as they reach either end of the runway, so that they shuttle calmly back and forth across the platforms. In addition to ground reaction forces, many other locomotor parameters can be rapidly and accurately obtained from the force plates. The plates record the position and time of contact of each paw as the limb is placed on the platform, so that spatial and temporal variables can be obtained for individual limbs (stride lengths, stance time) and as well as for interlimb coordination (timing overlap) (22). An important consideration for measurement of all locomotor parameters, including ground reaction forces, is the speed with which the animal is travelling. Stance time, stride lengths, peak forces are just a few variables which differ significantly at different speeds. It is important to note that, after injury, animals will often move more slowly than prior to injury and many locomotor parameters change simply because of the animal’s reduced velocity. For valid comparisons of locomotor parameters, it is essential that pre- and post-surgical data be collected at the same speed. Because we cannot control the speed with which animals move overground unrestrained (unlike with treadmill locomotion, for example), we record many passes during pre-surgical data collection. We record the speed with which the animal moves for each pass and then, during analysis, we group the data for each individual according to speed. This ensures that there will be pre-surgical data sampled at a number of different velocities,

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allowing us to match pre-surgical data to post-surgical data at comparable speeds. 3.1.3. Kinematic Data Collection

Digital video recording of rats as they move over the force platforms allows the identification of the limbs which contact each platform (e.g., right or left, forelimb or hindlimb), information which is necessary for the correct interpretation of GRF data. Using custom-written software, the data from force plates will also provide limb timing, step distances and limb overlap measurements using the onset and off-set timing of individual limbs on all three force platforms (22). This information provides very precise timing and distance measurements because sampling rate of the GRF data (1,000  Hz) is much higher than that of most digital video data (e.g., 25–60 Hz), the more common method with which to obtain limb timing and step distance information. We also use digital video to record the position and orientation of the limb segments in each video frame as the animal moves through the stride cycle. To identify the position of limb segments in each video frame, reflective markers are placed at the ends of each segment prior to recording. Markers (2-mm diameter) can be fashioned from reflective tape (3M, St. Paul, MN, USA) shaped into a shallow cone and glued to the ends of each limb segment or over joints which have been clipped free of fur. During analysis, the markers are identified in each video-frame, allowing computerassisted digitization of segment positions throughout the stride (ProAnalyst; Xcitex Inc., Boston, MA, USA). This information is then used to construct a model of each limb as a series of linked segments (Fig. 2). The kinematic data obtained from this analysis (e.g., limb segment positions and joint angle changes throughout the stride) are used to quantitatively describe the action of each limb during locomotion (42–45).

3.1.4. Mechanical Analysis of Limb Movement

The simultaneous collection of GRF data and kinematic data allows incorporation of kinetic information into the linked segment model of the limb described above, thus providing information about the mechanical action of the limbs (40). Joint torques and joint power are two commonly determined measures, which can be obtained from kinetic and kinematic data using a method called inverse dynamic analysis (40). This method is commonly used for human gait analysis, and has proven valuable for the diagnosis of pathological gait. Biomechanical analyses have been investigated only recently in laboratory rodents (46, 47). In our laboratory, we have used the inverse dynamic method to examine joint torques and joint power for the joints of all four limbs after peripheral nerve injury (Bennett et al., unpublished data). After loss of ankle extensor innervation, we found that rats alter the power at the joints of the lesioned hindlimb in complex ways, and additionally increase the net mechanical power produced by the unlesioned limbs to compensate for the reduced power of the lesioned limb. Thus,

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Fig. 2. Video image of rat trotting over force platforms as it moves past the camera. Computer-assisted digitization of reflective markers placed on the limb segments allows the estimation of joint positions (hip, knee, ankle) and segment locations in each video frame. Reconstruction of the limb as a series of linked segments allows calculation of joint angles (red shading) throughout the stride cycle. Simultaneous collection of video data and ground reaction forces allows the calculation of joint torques and powers by synchronizing limb segment positions in each video frame with the magnitude and position of the ground reaction force vector (dashed line) obtained from force plate measurements.

inverse dynamic analysis can provide quantitative biomechanical information on how rats use their limbs before and after injury. 3.2. Skilled Locomotion

Skilled locomotion refers to locomotor tasks that require the rat to move over challenging terrain. Rats are naturally nimble and are able to negotiate many obstacles and structures. In our laboratory, we examine their ability to move across a horizontal ladder for a food reward (11, 22–24). Other researchers have similarly examined locomotion over ladders, grids or along beams of different or tapering widths (1, 13, 48–50). We prefer to examine skilled locomotion over a horizontal ladder rather than a grid because the wire ladder rungs present a consistent surface for each paw. A grid, with its support bars running in two dimensions, can present a slightly different surface for each paw, depending on the position of the paw relatively to the grid bars. The beam is similar to the ladder in that it presents a consistent surface for each paw placement, although the texture of the beam surface will have an effect on the amount of friction afforded to each paw. Similar to conditioning for the collection of kinetic and kinematic data (Sect. 2.1 above), we condition rats to shuttle repeated back and forth across a horizontal ladder for a food reward as they reach either end (Fig. 3a). The ladder apparatus is a 124-cm long runway, in which the central 80 cm portion of the floor of the runway consists of a horizontal ladder (2  mm diameter wire rungs placed at 2-cm intervals (24)). Rats are considered sufficiently ­conditioned when they cross the ladder willingly up to ten times in succession. Consistent with our measurement of overground locomotion (Sect. 3.1 above) and skilled forelimb use (Sect. 4.1 below),

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Fig. 3. (a) Video images of rats crossing a horizontal ladder. A mirror placed below the ladder and positioned at 45° to the horizontal allows lateral and ventral views to be recorded simultaneously. Rats are conditioned to repeatedly shuttle back and forth across the ladder so that multiple independent strides can be recorded so as to obtain a representative sample of ladder performance. (b) Video images of rats reaching for a food pellet from a shelf located outside the reaching box. Rats are conditioned to retrieve a food reward at rear of the reaching box after each reaching attempt, to ensure that each reach is independent of the previous attempt.

we therefore examine the effect of our experimental manipulations on the performance of well-known ladder task (see Sect.  2.2.3). Rats are digitally videotaped from a lateral perspective as they repeatedly cross the ladder. A mirror angled at 45° beneath the ladder allows both the lateral and ventral views to be recorded in each video frame (Fig. 3a). During analysis of digital video, data is collected from only one stride per pass to ensure independence of different samples from the same animal (Sect. 2.2.2). We collect ten samples from each animal to obtain a representative measure of each animal’s performance. In keeping with the use of quantitative measures, we assess whether each paw made a correct step (full palmar or plantar contact with the rung maintained throughout weight support), an abnormal step (e.g., wrist only) or an error (e.g., paw contact

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with a rung was missed or lost, resulting in loss of equilibrium). For each paw, these three events are counted over the ten strides, providing quantitative measurement along with an indication regarding the quality of paw placement.

4. Assessing Forelimb Use Rats have a natural ability to use their forelimbs for many behaviours in addition to locomotion, including exploration and manipulation of objects, particularly food. While there are many tests available to assess forelimb function (1, 2), our laboratory has focused on the pellet retrieval task (see Karl and Whishaw, Volume I, ch. 6, and below), and the paw preference task. Originally developed to assess forelimb function in experimental brain lesions (51), these tests are thought to be analogous to assessments of arm and hand function in humans (52). Tests of forelimb function have also proven useful for assessing the effects of cervical SCI in rat models (23, 24, 53–55). 4.1. Skilled Reaching

Although rats are skilled at using their paws to handle food, they nevertheless need to be conditioned to use their forepaws to prehend food, a task they normally accomplish with their mouths. The Whishaw skilled pellet retrieval task (Karl and Whishaw, Volume I, ch. 6) is a well-described task which can be used to assess forelimb function under many experimental conditions. Our use of the task varies little from the original descriptions of this test (56, 57). Rats are placed in a rectangular box (45 cm × 12 cm) with a ledge attached to the outside of the box. A flavoured sugar pellet (4.0 mm × 3.3 mm, 45 g, BioServe, Inc) is placed into a shallow depression on the ledge. The rat can access the pellet through a vertical opening in the wall of the box which is wide enough for a paw to fit through but not the rat’s snout. With repeated attempts, rats learn to use their forepaw to reach the pellet and with further exposure will learn to grasp the pellet with the digits to successfully retrieve it (Fig. 3b). As for many behavioural tasks, there are a variety of methods with which to score performance. In our laboratory, we count the number of successful attempts out of 20 possible reaches. A successful reach is one in which the pellet is grasped with the digits, brought to the mouth and eaten in one attempt. If the rat misses the pellet on the first attempt, or drops it before it reaches its mouth, this is not considered a successful reach. Other studies have used different measures to assess reaching success, including counts of the number of pellets successfully retrieved in a fixed amount of time, or the number of attempts used to retrieve a single pellet (2, 58). These endpoint measures are useful because they provide a quantitative score against which performance can be

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compared before and after experimental SCI or therapy. Nevertheless, endpoint measures do not provide any information regarding how the animal is performing the task (2). After injury, rats frequently use a variety of “abnormal” movements to reach and retrieve pellets and similar endpoint scores can be achieved using different movement strategies (32). To address this, a more detailed analysis of reaching behaviour is required (57, 59). The reaching action of the forelimb can be divided into ten sequential components, and frame-by-frame analysis of videotape recordings allows the scoring of the presence or absence of each component (57, 59). We have used this method to examine the reaching movements used by rats after combined pyramidal tract and spinal cord injury (23, 24). Importantly, reaching performance can be affected not only by injury to the cervical spinal cord affecting the reaching forelimb but also by lesions which affect other limbs or parts of the body (60, 61). Rats need to maintain a stable stance in order to successfully complete the reaching task, so that lesions that affect hindlimb control, for example, can also alter the animal’s ability to successfully retrieve sugar pellets (61). In addition, appropriate orientation of the rat’s body in front of the vertical opening will favour successful reaches (62). This is an important confounding factor in this task – if a rat adopts a less favourable position, all the reaches performed from that particular stance will be affected. In order to avoid this confound and ensure independence of each reaching attempt, we, like others, allow only one reaching attempt each time the animal approaches the vertical opening. We do this by conditioning the rats to return to the back of the reaching box to obtain a food reward after each reach. 4.2. Paw Preference

The other forelimb task that we assess in our laboratory is the paw preference task. This is an assessment of forelimb use during exploratory behaviour. No conditioning is required and so this is an assessment of spontaneous and relatively unskilled use of forelimbs. We use methods previously well described (30, 50). Rats are placed in a cylindrical chamber (20-cm diameter) and digitally videotaped for 10 min as they explore the chamber. The number of times each forepaw touches the cylinder wall is counted. The number of contacts for each limb is analyzed as a percentage of total contacts, thus providing a measure of limb use symmetry which can be compared before and after injury or experimental treatment.

5. Summary The effective use of animal models of SCI requires reliable and valid methods that provide relevant assessments of the animal’s functional capabilities. This chapter described some of the basic principles on which our choices of appropriate methods of

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behavioural analysis are based and has attempted to demonstrate how we apply these principles in our laboratory. Of course, there are a variety of excellent tasks and measurements that have been employed in the literature, and careful application of these methods to functional assessments in animal models will be essential in the ongoing investigation for successful SCI treatments. References 1. Sedy J, Urdzikova L, Jendelova P, Sykova E (2008) Methods for behavioral testing of spinal cord injured rats. Neurosci Biobehav Rev 32:550–580 2. Muir GD, Webb AA (2000) Mini-review: assessment of behavioural recovery following spinal cord injury in rats. Eur J Neurosci 12:3079–3086 3. Goldberger ME, Bregman BS, Vierck-CJ J, Brown M (1990) Criteria for assessing recovery of function after spinal cord injury: behavioral methods. Exp Neurol 107:113–117 4. Kunkel BE, Dai HN, Bregman BS (1993) Methods to assess the development and recovery of locomotor function after spinal cord injury in rats. Exp Neurol 119:153–164 5. Wrathall JR (1992) Behavioral endpoint measures for preclinical trials using experimental models of spinal cord injury. J Neurotrauma 9:165–167 6. Basso DM (2004) Behavioral testing after spinal cord injury: congruities, complexities, and controversies. J Neurotrauma 21:395–404 7. Sharp KG, Flanagan L, Yee KM, Steward O (2011) A re-assessment of a combinatorial treatment involving Schwann cell transplants and elevation of cyclic AMP on recovery of motor function following thoracic spinal cord injury in rats . Exp Neurol in press 8. Webb AA, Muir GD (2005) Sensorimotor behaviour following incomplete cervical spinal cord injury in the rat. Behav Brain Res 165:147–159 9. Cheng H, Almstrom S, Gimenez LL, Chang R, Ove OS, Hoffer B, Olson L (1997) Gait analysis of adult paraplegic rats after spinal cord repair. Exp Neurol 148:544–557 10. Basso DM, Beattie MS, Bresnahan JC (1995) A sensitive and reliable locomotor rating scale for open field testing in rats. J Neurotrauma 12:1–21 11. Kanagal SG, Muir GD (2008) The differential effects of cervical and thoracic dorsal funiculus lesions in rats. Behav Brain Res 187:379–386 12. Webb AA, Muir GD (2002) Compensatory locomotor adjustments of rats with cervical or thoracic spinal cord hemisections. J Neurotrauma 19:239–256

13. Sandrow-Feinberg HR, Zhukareva V, Santi L, Miller K, Shumsky JS, Baker DP, Houle JD (2010) PEGylated interferon-beta modulates the acute inflammatory response and recovery when combined with forced exercise following cervical spinal contusion injury. Exp Neurol 223:439–451 14. Martinez M, Brezun JM, Bonnier L, Xerri C (2009) A new rating scale for open-field evaluation of behavioral recovery after cervical spinal cord injury in rats. J Neurotrauma 26:1043–1053 15. Anderson KD, Sharp KG, Hofstadter M, Irvine KA, Murray M, Steward O (2009) Forelimb locomotor assessment scale (FLAS): novel assessment of forelimb dysfunction after cervical spinal cord injury. Exp Neurol 220:23–33 16. Timoszyk WK, Nessler JA, Acosta C, Roy RR, Edgerton VR, Reinkensmeyer DJ, de LR (2005) Hindlimb loading determines stepping quantity and quality following spinal cord transection. Brain Res 1050:180–189 17. Cha J, Heng C, Reinkensmeyer DJ, Roy RR, Edgerton VR, de Leon RD (2007) Locomotor ability in spinal rats is dependent on the amount of activity imposed on the hindlimbs during treadmill training. J Neurotrauma 24:1000–1012 18. Magnuson DS, Smith RR, Brown EH, Enzmann G, Angeli C, Quesada PM, Burke D (2009) Swimming as a model of task-specific locomotor retraining after spinal cord injury in the rat. Neurorehabil Neural Repair 23:535–545 19. Smith RR, Brown EH, Shum-Siu A, Whelan A, Burke DA, Benton RL, Magnuson DS (2009) Swim training initiated acutely after spinal cord injury is ineffective and induces extravasation in and around the epicenter. J Neurotrauma 26:1017–1027 20. Smith RR, Shum-Siu A, Baltzley R, Bunger M, Baldini A, Burke DA, Magnuson DS (2006) Effects of swimming on functional recovery after incomplete spinal cord injury in rats. J Neurotrauma 23:908–919 21. Webb AA, Muir GD (2003) Unilateral dorsal column and rubrospinal tract injuries affect overground locomotion in the unrestrained rat. Eur J Neurosci 18:412–422

21  Assessing Spinal Cord Injury 22. Muir GD, Webb AA, Kanagal S, Taylor L (2007) Dorsolateral cervical spinal injury differentially affects forelimb and hindlimb action in rats. Eur J Neurosci 25:1501–1510 23. Kanagal SG, Muir GD (2008) Effects of combined dorsolateral and dorsal funicular lesions on sensorimotor behaviour in rats. Exp Neurol 214:229–239 24. Kanagal SG, Muir GD (2009) Task-dependent compensation after pyramidal tract and dorsolateral spinal lesions in rats. Exp Neurol 216:193–206 25. Martin P, Bateson P (2007) Measuring Behaviour, an introductory guide. Cambridge University Press, Cambridge, UK 26. Kerlinger FN (1986) Foundations of Behavioral Research. Holt, Rinehart and Winston, Inc., New York 27. Metz GA, Whishaw IQ (2002) Cortical and subcortical lesions impair skilled walking in the ladder rung walking test: a new task to evaluate fore- and hindlimb stepping, placing, and coordination. J Neurosci Methods 115:169–179 28. Metz GA, Whishaw IQ (2009) The ladder rung walking task: a scoring system and its practical application. J Vis Exp 29. Basso DM, Beattie MS, Bresnahan JC, Anderson DK, Faden AI, Gruner JA, Holford TR, Hsu CY, Noble LJ, Nockels R, Perot PL, Salzman SK, Young W (1996) MASCIS evaluation of open field locomotor scores: effects of experience and teamwork on reliability. Multicenter Animal Spinal Cord Injury Study. J Neurotrauma 13:343–359 30. Schallert T, Fleming SM, Leasure JL, Tillerson JL, Bland ST (2000) CNS plasticity and assessment of forelimb sensorimotor outcome in unilateral rat models of stroke, cortical ablation, parkinsonism and spinal cord injury. Neuropharmacology 39:777–787 31. Whishaw IQ, Pellis SM (1990) The structure of skilled forelimb reaching in the rat: a proximally driven movement with a single distal rotatory component. Behav Brain Res 41:49–59 32. McKenna JE, Whishaw IQ (1999) Complete compensation in skilled reaching success with associated impairments in limb synergies, after dorsal column lesion in the rat. J Neurosci 19:1885–1894 33. Grillner S (1975) Locomotion in vertebrates: central mechanisms and reflex interaction. Physiol Rev 55:247–304 34. Grillner S, Wallen P (1985) Central pattern generators for locomotion, with special reference to vertebrates. Annu Rev Neurosci 8:233–261 35. Armstrong DM (1986) Supraspinal contributions to the initiation and control of locomotion in the cat. Prog Neurobiol 26:273–361

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36. Muir GD, Whishaw IQ (2000) Red nucleus lesions impair overground locomotion in rats: a kinetic analysis. Eur J Neurosci 12:1113–1122 37. Muir GD, Whishaw IQ (1999) Ground reaction forces in locomoting hemi-parkinsonian rats: a definitive test for impairments and compensations. Exp Brain Res 126:307–314 38. Kanagal SG, Muir GD (2007) Bilateral dorsal funicular lesions alter sensorimotor behaviour in rats. Exp Neurol 205:513–524 39. Webb AA, Muir GD (2004) Course of motor recovery following ventrolateral spinal cord injury in the rat. Behav Brain Res 155:55–65 40. Winter DA (1990) Biomechanics and Motor Control of Human Movement. John Wiley and Sons, Inc, New York 41. Biewener AA, Full RJ (1992) Force platform and kinematic analysis. In: Biewener AA (ed) Biomechanics: Structures and Systems. Oxford University Press, Oxford, pp 45–73 42. Couto PA, Filipe VM, Magalhaes LG, Pereira JE, Costa LM, Melo-Pinto P, Bulas-Cruz J, Mauricio AC, Geuna S, Varejao AS (2008) A comparison of two-dimensional and threedimensional techniques for the determination of hindlimb kinematics during treadmill locomotion in rats following spinal cord injury. J Neurosci Methods 173:193–200 43. Fischer MS, Schilling N, Schmidt M, Haarhaus D, Witte H (2002) Basic limb kinematics of small therian mammals. J Exp Biol 205:1315–1338 44. Pereira JE, Cabrita AM, Filipe VM, Bulas-Cruz J, Couto PA, Melo-Pinto P, Costa LM, Geuna S, Mauricio AC, Varejao AS (2006) A comparison analysis of hindlimb kinematics during overground and treadmill locomotion in rats. Behav Brain Res 172:212–218 45. Thota AK, Watson SC, Knapp E, Thompson B, Jung R (2005) Neuromechanical control of locomotion in the rat. J Neurotrauma 22: 442–465 46. Johnson WL, Jindrich DL, Roy RR, Reggie E, V (2008) A three-dimensional model of the rat hindlimb: musculoskeletal geometry and muscle moment arms. J Biomech 41:610–619 47. Wehner T, Wolfram U, Henzler T, Niemeyer F, Claes L, Simon U (2010) Internal forces and moments in the femur of the rat during gait. J Biomech 43:2473–2479 48. Soblosky JS, Colgin LL, Chorney-Lane D, Davidson JF, Carey ME (1997) Ladder beam and camera video recording system for evaluating forelimb and hindlimb deficits after sensorimotor cortex injury in rats. J Neurosci Methods 78:75–83

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49. Jeffery ND, Blakemore WF (1997) Locomotor deficits induced by experimental spinal cord demyelination are abolished by spontaneous remyelination. Brain 120 (Pt 1):27–37 50. Schallert T, Woodlee MT (2005) Orienting and Placing. In: Whishaw IQ, Kolb B (eds) The behavior of the laboratory rat: a handbook with tests. Oxford University Press, Oxford, pp 129–140 51. IQ, O’Connor WT, Dunnett SB (1986) The contributions of motor cortex, nigrostriatal dopamine and caudate-putamen to skilled forelimb use in the rat. Brain 109 (Pt 5):805–843 52. Whishaw IQ, Pellis SM, Gorny BP (1992) Skilled reaching in rats and humans: evidence for parallel development or homology. Behav Brain Res 47:59–70 53. Soblosky JS, Song JH, Dinh DH (2001) Graded unilateral cervical spinal cord injury in the rat: evaluation of forelimb recovery and histological effects. Behav Brain Res 119:1–13 54. Schrimsher GW, Reier PJ (1992) Forelimb motor performance following cervical spinal cord contusion injury in the rat. Exp Neurol 117:287–298 55. Schrimsher GW, Reier PJ (1993) Forelimb motor performance following dorsal column, dorsolateral funiculi, or ventrolateral funiculi lesions of the cervical spinal cord in the rat. Exp Neurol 120:264–276 56. Whishaw IQ, Gorny B, Sarna J (1998) Paw and limb use in skilled and spontaneous reaching

after pyramidal tract, red nucleus and combined lesions in the rat: behavioral and anatomical dissociations. Behav Brain Res 93:167–183 57. Whishaw IQ, Whishaw P, Gorny B (2008) The structure of skilled forelimb reaching in the rat: a movement rating scale. J Vis Exp 58. Diener PS, Bregman BS (1998) Fetal spinal cord transplants support the development of target reaching and coordinated postural adjustments after neonatal cervical spinal cord injury. J Neurosci 18:763–778 59. Metz GA, Whishaw IQ (2000) Skilled reaching an action pattern: stability in rat (Rattus norvegicus) grasping movements as a function of changing food pellet size. Behav Brain Res 116:111–122 60. Miklyaeva EI, Whishaw IQ (1996) HemiParkinson analogue rats display active support in good limbs versus passive support in bad limbs on a skilled reaching task of variable height. Behav Neurosci 110:117–125 61. Miklyaeva EI, Castaneda E, Whishaw IQ (1994) Skilled reaching deficits in unilateral dopaminedepleted rats: impairments in movement and posture and compensatory adjustments. J Neurosci 14:7148–7158 62. Whishaw IQ (2005) Prehension. In: Whishaw IQ, Kolb B (eds) The behavior of the laboratory rat; a handbook with tests. Oxford University Press, Oxford, pp 162–170

Chapter 22 Precise Finger Movements in Monkeys Roger Lemon Abstract Many movement disorders interfere with skilled movements of the hand and digits, and patients give a high priority to restoration of skilled hand function. Because of both neuroanatomical and behavioural adaptations, the best available model for the study of skilled hand movements is the non-human primate, and this model may be required to study the effects of therapeutic approaches on hand function. A wide variety of different methods are available for qualitative and quantitative assessment of hand and digit function in non-human primates. These methods can document the kinematics, dynamics and functional organisation of hand and digit movements, as well as providing more direct measures of the efficiency of the hand in, for example, efficient retrieval of food rewards and the use of tools. They include the use of high speed digital video and motion analysis techniques. The techniques available are able to provide a quantitative documentation of the characteristic deficits resulting from movement disorders and other neurological diseases, and are sensitive enough to detect significant improvements in hand function due to therapy. Key words: Non-human primate, Macaque, Hand, Digits, Prehension, Precision grip

1. Why Is It Important to Study Skilled Digit Movements?

The hand is the principal organ through which we interact with our environment. Skilled hand function contributes to many different aspects of our daily life and is crucial for our technology, communication, culture and social interaction (1). The loss of hand function is devastating. In a survey of quadriplegic patients, the regaining of arm and hand function was ranked as most important (2), above bowel and bladder control, sexual function, and standing/walking. Movement disorders almost invariably disrupt the normal function of the hand. The early neurologists recognised that the poverty and weakness of hand movements were negative features of movement disorders. Positive signs in the hands, such as tremor and dyskinesia, are further characteristic features of these disorders.

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2. Animal Models In selecting an animal model appropriate for study of movement disorders, it is important not to ignore the devastating effects of lost or deteriorated hand function in human patients (3, 4). There are key differences in the functional capacity of the hand and forelimb between rodent and non-human primate models, and these reflect both neuroanatomical and behavioural features. Although the motor systems of vertebrates are highly conserved, some key features have undergone pronounced evolutionary change. The most telling examples are the motor cortex and corticospinal tract (CST). There is marked variation between rodents and primates in the pattern of CST terminations within the spinal cord (Fig. 1). In rodents, the CST projects mainly to the dorsal horn and intermediate zone, whereas in many primates the CST additionally terminates in the ventral horn and can influence motoneuron activity both directly and indirectly. The direct cortico-motoneuronal projection, unique to primates, is particularly significant for the control of motoneurons innervating hand muscles. There is evidence that these neuroanatomical changes are associated with important behavioural adaptations in the control of the hand.

Fig. 1. Relationship between the development of the CST and the emergence of fine motor control abilities. In rodents, there are no direct connections between CS neurons and the cervical motoneurons which innervate forelimb muscles–brainstem pathways and spinal interneurons relay cortical input to motor neurons. Most of the CST fibres in rodents travel in the dorsal columns. In non-human primates and humans, direct CS connections with motoneurons have evolved, together with an increase in the size and number of the CS fibres. This is reflected in an increase in the size of the excitatory postsynaptic potential (EPSP) elicited by cortical neurons in hand motoneurons. The primate CST is located mostly in the lateral columns, and a significant proportion of CS fibres (~10%) descend ipsilaterally (but actually many of these cross and still terminate contralaterally; see (44)). Development of the CST correlates with the improvement in the index of dexterity, particularly in the ability to perform finger-thumb precision grip (with permission from Courtine et al. (4)).

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Fig. 2. (a) Sequence of photographs showing macaque monkey using thumb and index finger to retrieve food from a small well on a Klüver board (with permission from (9)). (b) Brinkman board that requires macaque monkeys to use eye–hand control to retrieve food pellets (black) from the board. Access to pellet is via angled slots in the board. Protruding pegs prevent use of purely tactile cues to locate pellet (with permission from (19)). (c) Rosette device designed to test visuo– motor coordination of precision grip in macaque monkeys. The central food well is accessed by slots on either side, which are highlighted by a white outline. Blind slots located at other orientations do not give access to the reward. The rosette can be rotated on each trial. The monkey has to orient its hands and fingers correctly to gain access to the reward (see (28)).

The “index of dexterity” is a measure of the degree of functional adaptation of the hand that allows relatively independent use of the digits and the employment of different types of grasp (5, 6). In the dexterity index, a true precision grip involving pad-to-pad opposition between index finger and thumb is the highest form of dextrous capacity (Figs. 2a and 3a). It is found in humans and in great apes, and is given an index of 7. An opposable thumb, allowing a somewhat more limited form of precision grip is found in dextrous monkeys such as the Old World macaque (Rhesus monkey) and the New World capuchin (Cebus monkey), and has an index of 6. Monkeys with prehensile digits but only capable of a side grip between thumb and index finger (pseudo-opposition) (index 5) include the New World squirrel monkey, while the marmoset, the most widely used non-human primate in modern research, has

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Fig. 3. (a–c) Sequences showing a macaque performing a trained precision grip movement which involved displacing two spring-loaded levers, one with the index finger and one with the thumb. A plastic cover with two slots above the lever (not shown) restricted access to these two digits and prevented digits 3–5 from contributing; these digits were flexed out of the way. (d) Modified Brinkman board used to assess skilled hand function in monkeys subjected to incomplete spinal lesions and treatment with anti-NoGo antibody (21). Food pellets were placed in wells accessed via slots cut in the board, some horizontal, some vertical. Monkeys used a precision grip (opposition of thumb and index finger) to retrieve food pellets from the board, and monkeys were scored for the number of pellets retrieved in 30 s. (e) Manipulandum designed to test “individuation” of digit movements in the macaque. A pistol-grip device is shown with a monkey’s hand in place, with each digit positioned between two microswitches. An overhead view of a monkey’s finger between two microswitches is shown in detail below. The LED display used to cue different movements (flexion or extension) is shown on the right. 1 = thumb 2–5 = index, middle, ring and little finger, respectively, W = wrist (with permission from (46)).

prehensile digits but a non-opposable thumb, and has a dexterity index of 4. There is a correlation between the number of direct connections between cortex and motoneurons and the level of manual dexterity of different non-human primate species (Fig. 1; (6–8)). Accordingly, interruption of the cortical projections to the spinal cord in humans and non-human primates causes a major impairment in fine motor function of the hands and feet (9–12) and this is most severe in humans (13). Corticospinal lesions in rats do not affect overground locomotion and induce sensorimotor rather

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than motor impairments in forelimb function (3, 14), in keeping with the pattern of corticospinal termination in this species. So we can conclude that if we want to measure the effect of treatments or therapies that might help to recover loss of hand function, a non-human primate model will be needed. The macaque monkey is much more dextrous than the marmoset, and of course its ability to perform dextrous movements, including the use of tools, is supported by an enlarged frontal lobe and a highly developed cognitive system.

3. Biomechanical Considerations Most of the measures to be described in this article are in some way designed to test and challenge the capacity for non-human primates to perform skilled manipulation of a device or challenging retrieval of food rewards. These functional tests are to some extent all dependent on the capacity to make what Lawrence and Hopkins (15) called “relatively independent finger movements” (RIFM). That is, the capacity to execute “fractionated” digit movements, best exemplified by the control of index finger and thumb. These authors were careful to use the term “relative”: in fact, even in humans, there are limits to the extent to which the different digits can be moved in a completely independent manner (16). The thumb is the exception to this rule. Studies have shown that there are both biomechanical and neural constraints on independent finger movements (17, 18). The biomechanical constraints arise because of the complex interactions between the many different muscles acting on the digits. Some muscles (such as the extensor digitorum communis and the flexor digitorum profundus, FDP) act on more than one digit. Their motor units are partitioned in such a way that when one digit is moved, it allows associated movements of neighbouring digits. Compared with humans, macaque monkeys have a somewhat restricted repertoire of grasps, partly due to the fact that the monkey has a relatively shorter thumb length compared with finger length (18). Macaques also have more interconnections between the tendons of the FDP and the origins of the lumbrical muscles (18), which increases the mechanical constraints on the independence of individual digits. Although a small number of muscles actually control the forces exerted at the fingertip (such as FDP), many others are needed to stabilise the articulated bony chain that extends from the shoulder through to the elbow, wrist, hand and phalanges. Only with such stability can accurate tip-to-tip forces be developed. The hand has a very large number of degrees of freedom, and it is very difficult to monitor movements and forces at every single joint, let alone the activity in all the different muscles that underpins this movement.

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4. Measuring RIFM The early studies of Lawrence and Kuypers (9) on the effects of pyramidotomy on fine finger movements in the macaque monkey adapted the original Klüver board (Fig. 2a). This board contains food wells of varying diameter and depth; the smallest wells required precise co-ordination of thumb and index finger to “winkle out” the reward. After section of the pyramidal tract, monkeys were unable to retrieve food, especially from the smallest wells. Very young monkeys, who lack the capacity for RIFM and in whom the CST is immature, also fail on these tests (15). Monkeys use visual cues to direct their reach to the appropriate well and to orientate their hand and digits to grasp the reward; once contact with the well and reward is made, additional tactile/ haptic cues help the monkey to retrieve the reward. Brinkman and Kuypers (19, 20) developed a board which forced monkeys to use visual rather than tactile cues to retrieve food pellets (Fig. 2b). In this board, protruding pegs, of the same diameter and shape as the food pellets made it difficult to use touch alone to find the real pellets; in addition, slots which gave access to the real pellets were cut into the board at a variety of different angles, again forcing the monkey to use vision to guide its approach to the reward. Only monkeys with intact eye–hand control could retrieve food pellets with normal speed and accuracy (Fig. 2b). Monkeys show hand preference for different tasks, so it may be important to determine this for the particular task that is adopted.

5. Timed Tests of RIFM A modified Brinkman board was used by Freund et al. (21) to test macaque monkeys with incomplete spinal lesions (Fig.  3d), in which some subjects were treated with the neurite growth inhibitor, anti-Nogo A, to promote recovery. In studies of this kind, there are inevitably large variations in the size and type of lesion, so the methods used have to be very sensitive to detect any consistent changes in control vs. treated animals. The board used by Freund et al. (21) had both vertical and horizontal slots (Fig. 3d). Intact, control monkeys find it more difficult to retrieve pellets from the horizontal slots compared with the vertical slots, probably because they have to adopt a rather unusual hand orientation to do so. Lesioned monkeys often showed much bigger impairments on the horizontal slots (as measured by the number of pellets retrieved in a fixed test time of 30  s) than on the vertical slots. In a further examination of these data, Freund et al. (22) measured the “contact time.” This was defined as the time interval between the first

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contact of the index finger with the food pellet and the final ­successful grasp of the pellet utilising pad-to-pad opposition of the index finger and thumb. This contact time is a useful additional measure because it focuses on the dextrous grasp and excludes other components of the retrieval score in the timed period, such as the reaching time and any delay in the monkey eating or chewing the reward before starting a new retrieval. Nudo et al. (23) used a modified Klüver board to examine the prehensile performance of squirrel monkeys. These authors counted the number of attempts (“flexions per retrieval”) made by the monkey to retrieve the reward (a banana flavoured pellet) from the board. This approach was important in demonstrating the plasticity of the motor system after an experimental cortical lesion, and in demonstrating the power of intensive rehabilitation in promoting plasticity and functional recovery (24). Moore et  al. (25) used timed tests on a Klüver-type board to assess changes in dexterity in aged macaques. Owl and squirrel presented with a board of varying well size rapidly adapt a particular two or three digit strategy to optimise rapid food retrieval, and Xerri et al (26) showed that this caused long-term plastic expansion of the representations of these digits in the somatosensory cortex. Timed tests on a Klüver board were used by Belhaj-Saif and Cheney (27) to assess hand function after unilateral pyramidal tract lesions in macaques, and to show the compensatory function of the rubrospinal tract.

6. Visual Guidance of Food Retrieval from the Brinkman Board

In a further development of the Brinkman board, Haaxma and Kuypers (28) designed a “rosette” device that presented the food in a narrow slot, allowing the monkey to insert its index and thumb on either side of the food pellet (Fig. 2c). The slot was highlighted by a white line which cued the monkey to orientate its hand and digits in such a way as to retrieve the reward. Additional “blind” slots were present: these did not give access to the pellet, and were not highlighted. The device could be rotated between trials, so that the highlighted slot was presented at different orientations on every trial. Monkeys with normal eye–hand coordination and able to correctly use the visual cue to appropriately plan its grasp rarely make errors on this task; those rendered apraxic by lesions to the occipito-frontal white matter lesions were at chance as to which orientation they chose, often inserting their digits into the blind slots (28). Rotating the Klüver board containing the rewards makes additional demands on the monkey’s ability to plan its reach-to-grasp movement precisely so that it is timed to coincide with a moving target. This test was first introduced by Asanuma and Arissian (29)

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and subsequently used by Kaeser et  al. (30), who used a board rotating at 10 revolutions per minutes, and varying the direction of rotation from clockwise to anticlockwise. They used this test to look for a second phase of recovery in fine hand function after monkeys were treated with autologous stem cells derived from the prefrontal cortex. Examples of both the static and rotating Brinkman board can be found at: http://www.unifr/neuro/rouiller/motorcontcadre.htm.

7. Other Tests of Precision Grip A potential drawback of the Klüver board is that it does not allow easy visualisation of individual digit movement, nor of the dynamics and kinematics involved. A pioneering study by Hepp-Reymond and Wiesendanger (31) used a small hand-held device that monitored the precision grip forces exerted by the monkey, and this was combined with simultaneous EMG recordings from hand muscles (32). The force required was indicated to the monkey by a visual display. This approach demonstrated a significant deterioration in the level of precision grip force that follows damage to the pyramidal tract, and also showed that the rate at which grip force increased was also much slower, reflecting the slowness and weakness of movement mentioned in the Introduction. Another approach was used by Muir and Lemon (33): this required the monkey to exert independent control of the index finger and of the thumb to move two spring-loaded levers into a required target zone, and hold them both there for 1 s in order to earn a food reward (Fig. 3a–c; (34, 35)). The displacement of each digit was in the order of a few mm, and forces required were <1.0 N. A cover mounted above the manipulandum levers restricted access to the thumb and index and prevented other digits from contributing, thereby highlighting the fractionation of digit movement and hand muscle activity (36, 37). Using a robotic device it is possible to simulate spring loads with different properties and these can be changed in either a blocked manner or trial-by-trial. Other investigators have used a small reward mounted in a device that constrains the monkey to use a precision grip to retrieve it. Darian-Smith (38) reviewed the use of high speed digital video to capture macaques grasping a reward held in a device that required the monkey to develop different precision grip force levels to release the reward; these ranged from 0.5 to 2.0  N (see Fig. 4). This approach allows the precise kinematics of the grasp to be documented in great detail. The time taken to collect a glucose tablet held in a spring clip was used by Galea and Darian-Smith (39) to document the maturation of precision grip in neonatal macaques; while such movements are absent at birth, the speed

Fig. 4. Frame sequences from digital video illustrating the recovery of voluntary hand movements and sensorimotor function following a dorsal root lesion that removed all detectable input from digits one to three and the thenar palm. This monkey was unable to perform the reach-retrieval task at all for the first 2 months and then gradually recovered the ability to oppose the digits and to efficiently grasp and remove the target object. Note the failed attempt to retrieve the target 1 week following the lesion and the altered but successful retrieval at 20 postlesion weeks(with permission from (38)).

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and fluency of the grip develop rapidly over the first 5–6 months of life, and then show a slow but prolonged improvement into the third year. Fine movements of the digits are essential for social grooming, and young macaques are reported to begin grooming at around 5–6 months old (40). Digital video was also used by Sasaki et al. (41) to demonstrate that unilateral lesions to the lateral corticospinal tract at the C5 level, which would have interrupted descending corticospinal control of the spinal segment in the cervical enlargement supplying hand and digit muscles, caused an immediate impairment of precision grip. Over the following weeks, the slowness and clumsiness of the grip recovered significantly, and this was again documented by digital video. Recovery may have involved a number of different mechanisms (42), including ipsilateral propriospinal systems and uninjured but slowly conducting corticospinal fibres (43), and possibly a contribution due to collateral sprouting from intact corticospinal fibres descending on the unlesioned side of the cord (44).

8. Slow Finger Movements Williams et al. (45) investigated the brain mechanisms of control of slow flexion-extension movements made with a single digit. In this task, the monkey inserted its index finger into a narrow tube. The tube restricted movement to flexion/extension around the metacarpophalangeal (MCP) joint and a motor generated a torque opposing the flexion movement. During each trial, the palm and digits 1, 3, 4, and 5 lay horizontally against a flat surface and the elbow and upper arm were held in a sleeve. The monkey was given visual feedback of finger position and had to capture targets which required a movement at the MCP of either 24° or 12°; the target then moved slowly (a linear ramp lasting 1 s) to a new position (12° or 24°, respectively), and the monkey had to track the cursor.

9. A Task Involving Individual Movements of All Five Digits

Schieber (46) introduced a task in which the monkey placed its hand in a device such that each digit was in contact with small switches which could be activated by extension (dorsal switch), or flexion (ventral switch) movements (Fig. 3e). The switches for digits 2–5 were mounted in the horizontal plane, while that for the thumb was mounted in the vertical plane. By extending or flexing a digit a few mm, the monkey could close either the dorsal or ­ventral switch, respectively. A further switch could be activated by wrist

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movements. A display of five LEDs indicated to the monkey which switch was to be closed (“instructed” digit and movement). The monkey was rewarded for closing one switch alone and holding it closed for 500 ms. The forces exerted by each digit were monitored by strain gauges mounted on the lever arm of each switch. Interestingly, in view of the biomechanical constraints on individual digit movement (see above, Sect. 3), this task showed that while monkeys could learn to close a single switch, they almost always exerted some force with the neighbouring digit (e.g. middle finger when index finger was the “instructed digit”). In contrast, thumb flexion acted independently and had a high “individuation index”.

10. Investigation of Different Classes of Grasp in Non-human Primates

Many studies have investigated how non-human primates adapt the grasp to suit the object or device they are required to control. Early studies led by Sakata’s lab used a wide variety of different objects to evoke different grasp configurations to explore grasprelated activity in the posterior parietal cortex (47) and has been widely used since (e.g. (48, 49)). Objects such as a ring could involve just a single digit, a small cube evoked a side grip between two digits (thumb and index), while larger objects, such as a sphere, involved a whole hand grasp. Each object was mounted on a horizontal, spring-mounted shuttle, and a number of these shuttles were placed together on a carousel device. In a delayed response paradigm, monkeys first observed the object and then, after a variable delay, were required to grasp the object and displace it by a controlled amount and hold it there. The amount of force required to pull the object was determined by the strength of the shuttle spring. The grip force exerted on the object is known to be linearly correlated with the load or pull force (50). Rotation of the carousel between trials allowed the experimenter to present the objects to the monkey in a pseudorandom order. Grasp could be carried out either in the dark or the light. In this type of study, it is important to make sure that as wide a range of objects as possible is used to exploit the full-grasping repertoire of the monkey’s hand, and prevent the monkey using a strategy which allows it to grasp all the objects in the same way! If the monkey is adopting a specific set of grasps, then detailed EMG analysis of hand and digit muscles reveal specific temporal and spatial patterns of muscle activity to grasp the different objects (51). It should be stressed that monitoring of EMG activity from multiple muscles always complements kinematic and dynamic analyses and provides direct evidence for the CNS control of grasp-specific activity.

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11. Motion Analysis of Prehension in Non-human Primates

12. Tool Use by Non-human Primates

Motion capture approaches have been important for trying to classify some of the basic synergies of human hand movements and this is an important step in reducing the scale of the problem for Brain Machine Interface control of basic grasp functions. Detailed trial-by-trial analysis using high-speed digital video of finger kinematics and simultaneously recorded neuronal activity was introduced by Ro et  al. (52). Their imaging methods allow direct correlation of full-frame, full-field video images with the actual spike trains. The implementation of digital video provides high-resolution snapshots of the hand motor behaviour every 33.3 ms, and a precise calibration and display of the synchronously recorded electrophysiological activity digitized at rates up to 44.5 kHz on the same platform. Analysis of these data allows construction of raster plots and histograms of repeated behavioural trials using the timing of the corresponding video frame for alignment. These analyses reveal functional classes of cortical neurons signalling specific stages of prehension (53). Reach and grasp in the monkey were studied using motion analysis by Roy et  al. (54) who used the Optotrak method. Macaque monkeys were trained to reach out, grasp and lift one of two different objects in a pseudorandom order. Markers were taped to the wrist, and to the nails of the thumb, index and middle fingers to monitor wrist motion and grasp aperture. These authors found that that object size and its location affected both reaching and grasping. The maximum grip aperture to retrieve objects of different size depended strongly not only on object size but also grip selection. Their findings argue against a strict postulate of independence between the visuo-motor channels, favouring instead the idea of variable degrees of coordination between the reach and grasp components depending on the task demands. There were many similarities between the reach-and-grasp kinematics of humans and monkeys. Active sensors, such as those used in the Optotrak technology, and gloves, such as Cyberglove, which have both been widely used in human studies (e.g. (55, 56)) are not easy to attach to the monkey’s hand without the danger that the monkey may interfere with or remove the marker or sensor and, perhaps more importantly, the sensor itself may prevent the monkey from using natural, unencumbered grasping movements.

Many species of non-human primates use tools in the wild, and some can be trained to use them in the laboratory. Macaques can learn to manipulate a rake and employ it as a tool, using it to

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retrieve food rewards placed beyond their peri-personal space. This was first shown by Warden et al. (57), and developed by Iriki et al. (58). This task has now allowed a number of the sensorimotor mechanisms involved in tool use to be explored for the first time (59, 60) including the grey and white matter changes that occur when the monkey learns the task for the first time (61). Macaques learn to use the rake as a tool remarkably quickly, usually within 2–3 weeks.

13. Conclusions A wide variety of different methods are available for qualitative and quantitative assessment of hand and digit function in non-human primates. These methods allow some insights into the kinematics, dynamics and functional organisation of hand and digit movements, as well as providing more direct measures of the efficiency of the hand in, for example, retrieving food rewards. The method of choice will depend upon the particular parameter (e.g. speed of movement, force control, fluency of grasp, etc.) most affected in the disease model being studied. The techniques available are able to provide a quantitative documentation of the characteristic deficits resulting from movement disorders and other neurological diseases, and are sensitive enough to detect significant improvements in hand function due to therapy. References 1. Napier JR. Hands. George Allen and Unwin (London) 1980. 2. Anderson KD. Targeting recovery: priorities of the spinal cord-injured population. J Neurotrauma 2004; 21: 1371–1383. 3. Lemon RN, Griffiths J. Comparing the function of the corticospinal system in different species: Organizational differences for motor specialization? Muscle Nerve 2005; 261–279. 4. Courtine G, Bunge MB, Fawcett JW et al. Can experiments in nonhuman primates expedite the translation of treatments for spinal cord injury in humans? Nat Med 2007; 13: 561–566. 5. Napier HR. Prehensibility and opposability in the hands of primates. Symp Zool Soc Lond 1961; 5: 115–132. 6. Heffner RS, Masterton RB. Variation in form of the pyramidal tract and its relationship to digital dexterity. Brain Behav Evol 1975; 12: 161–200. 7. Heffner RS, Masterton RB. The role of the ­corticospinal tract in the evolution of human digital dexterity. Brain Behav Evol 1983; 23: 165–183.

8. GA, Strick PL. Corticospinal terminations in two New-World primates: Further evidence that corticomotoneuronal connections provide part of the neural substrate for manual dexterity. J Neurosci 1993; 13: 5105–5118. 9. Lawrence DG, Kuypers HGJM. The functional organization of the motor system in the monkey. I. The effects of bilateral pyramidal lesions. Brain 1968; 91: 1–14. 10. Porter R, Lemon RN. Corticospinal function and voluntary movement. Physiological Society Monograph. Oxford: Oxford University Press; 1993. 11. Lang CE, Schieber MH. Differential impairment of individuated finger movements in humans after damage to the motor cortex or the corticospinal tract. J Neurophysiol 2003; 90: 1160–1170. 12. Courtine G, Roy RR, Raven J et al. Performance of locomotion and foot grasping following a unilateral thoracic corticospinal tract lesion in monkeys (Macaca mulatta). Brain 2005; 128: 2338–2358.

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27. Belhaj-Saif A, Cheney PD. Plasticity in the Distribution of the Red Nucleus Output to Forearm Muscles After Unilateral Lesions of the Pyramidal Tract. J Neurophysiol 2000; 83: 3147–3153. 28. Haaxma R, Kuypers H. Role of occipito-frontal cortico-cortical connections in visual guidance of relatively independent hand and finger movements in rhesus monkeys. Brain Res 1974; 71: 361–366. 29. Asanuma H, Arissian K. Experiments on functional role of peripheral input to motor cortex during voluntary movements in the monkey. J Neurophysiol 1984; 52: 212–227. 30. Kaeser M, Brunet JF, Wyss A et al. Autologous adult cortical cell transplantation enhances functional recovery following unilateral lesion of motor cortex in primates: a pilot study. Neurosurgery 2011. 31. Hepp-Reymond MC, Wiesendanger M. Unilateral pyramidotomy in monkeys: effects on force and speed of a conditioned precision grip. Brain 1972; 36: 117–131. 32. Smith AM, Hepp-Reymond MC, Wyss UR. Relation of activity in precentral cortical neurons to force and rate of force change during isometric contractions of finger muscles. Exp Brain Res 1975; 23: 315–332. 33. Muir RB, Lemon RN. Corticospinal neurons with a special role in precision grip. Brain Res 1983; 261: 312–316. 34. Baker SN, Philbin N, Spinks R et al. Multiple single unit recording in the cortex of monkeys using independently moveable microelectrodes. J Neurosci Methods 1999; 94: 5–17. 35. Baker SN, Jackson A, Spinks R, Lemon RN. Synchronization in monkey motor cortex during a precision grip task I. Task -dependent modulation in single-unit synchrony. J Neurophysiol 2001; 85: 869–885. 36. Lemon RN. Cortical control of the primate hand. Exp Physiol 1993; 78: 263–301. 37. Bennett KMB, Lemon RN. Corticomotoneuronal contribution to the fractionation of muscle activity during precision grip in the monkey. J Neurophysiol 1996; 75: 1826–1842. 38. Darian-Smith C. Monkey models of recovery of voluntary hand movement after spinal cord and dorsal root injury. ILAR J 2007; 48: 396–410. 39. Galea MP, Darian-Smith I. Postnatal maturation of the direct corticospinal projections in the macaque monkey. Cereb Cortex 1995; 5: 518–540. 40. Hinde RA, Rowell TE, Spencer-Booth Y. Behaviour of socially living rhesus monkeys in their first six months. Proc Zool Soc Lond 1964; 143: 609–649.

22  Precise Finger Movements in Monkeys 41. Sasaki S, Isa T, Pettersson LG et al. Dexterous finger movements in primate without monosynaptic corticomotoneuronal excitation. J Neurophysiol 2004; 92: 3142–3147. 42. Nishimura Y, Onoe H, Morichika Y, Perfiliev S, Tsukada H, Isa T. Time-dependent central compensatory mechanisms of finger dexterity after spinal cord injury. Science 2007; 318: 1150–1155. 43. RN. Cortico-motoneuronal system and dexterous finger movements. J Neurophysiol 2004; 92: 3601–3603. 44. Rosenzweig ES, Courtine G, Jindrich DL et al. Extensive spontaneous plasticity of corticospinal projections after primate spinal cord injury. Nat Neurosci 2010; 13: 1505–1510. 45. Williams ER, Soteropoulos DS, Baker SN. Coherence between motor cortical activity and peripheral discontinuities during slow finger movements. J Neurophysiol 2009; 102: 1296–1309. 46. Schieber MH. Individuated finger movements of Rhesus monkeys: A means of quantifying the independence of the digits. J Neurophysiol 1991; 65: 1381–1391. 47. Taira M, Mine S, Georgopoulos AP, Murata A, Sakata H. Parietal cortex neurons of the monkey related to the visual guidance of hand movement. Exp Brain Res 1990; 83: 29–36. 48. Murata A, Fadiga L, Fogassi L, Gallese V, Raos V, Rizzolatti G. Object representation in the ventral premotor cortex (Area F5) of the monkey. J Neurophysiol 1997; 78: 2226–2230. 49. Umilta MA, Brochier TG, Spinks RL, Lemon RN. Simultaneous recording of macaque premotor and primary motor cortex neuronal populations reveals different functional contributions to visuomotor grasp. J Neurophysiol 2007; 98: 488–501. 50. Johansson RS, Westling G. Coordinated ­isometric muscle commands adequately and erroneously programmed for the weight during lifting task with precision grip. Experimental Brain Research 1988; 71: 59–71.

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Index A Abducens nucleus.............................................................283 Accessory abducens nucleus.............................................283 Acetylcholine (Ach)........................27–28, 73, 308, 322–335 Acetylcholine receptors....................................................308 Acetylcholinesterase (AChE).................................27–28, 73 Action control systems.......................................................60 Active EAE.............................................. 365–368, 370–373 Adrenergic receptors.........................................................255 Aggregates huntingtin................................................... 3, 4, 8–10, 83 a-synuclein.......................................................37, 45–47 Agtpbp1pcd–1J mutant mice.......................... 241, 243, 247–255 Alexa-fluoro..................................... 142–143, 148, 151–154 Alpha/startle responses.....................................................284 Alpha-synuclein (a-syn).................................... 8, 37, 45–47 Alzheimer’s disease...........................................................4–5 Amacrine cells.......................................... 291, 299–300, 304 a-Amino–3-hydroxy–5-methyl–4-isoxazole-proprionate (AMPA)............................................. 22, 32, 242 6-Aminonicotinamide......................................................373 AMPA receptors................................................................22 AMPA. See a-Amino–3-hydroxy–5-methyl–4-isoxazoleproprionate Amphetamine........................ 17, 38, 41–42, 48, 77, 190, 330 Analgesia.......................................................60, 127, 225, 358 Analogue-to-digital converter (ADC).............................294 Anesthesia................................................... 29–31, 126–127, 134, 203–204, 223–224, 227–228, 353–354 Anetromedial thalamic nucleus (AMN)...........................146 Animal husbandry.....................................7, 10, 26, 307, 358 Animal identification.......................................................359 Animal models basal ganglia diseases................................................3–12 epilepsy.........................................................................23 EAE..../........................................................363–378 HD..........................3–15, 22–24, 32, 71–72, 77, 83–84 MS......................................................................363–365 MSA.................................................................32, 37–50 PD.................................................................... 335, 338 saccade control....................................................161–178 spinal cord injury.................345–347, 352, 401–416, 420

stroke....................................................91–112, 183–190 traumatic brain injury.........................................193–204 Anterior commissure (AC)............................... 151, 211–236 Anterograde tracing..................................................139–157 Anteroventral thalamic nucleus (AVN)............................146 Antidromic activation....................................... 161–178, 376 Antigen-presenting cells (APCs)..................... 369–370, 377 Antisaccade task.......................................................167–172 Apomorphine....................................38–43, 76, 97–100, 110 Aspiration lesion............................................... 122–123, 195 Ataxia cerebellar................................. 37–38, 241–256, 263–277 discriminatory.............................................................269 experimental models...........................................275–276 Friedrich’s...........................................................241–242 spinocerebellar............................................ 241–246, 255 spinal..........................................................................370 Atropine............................................297, 309, 313–314, 325 ATXN3/Q79 mice...................................................244–251 Autoimmune lesions.................................................363–378 Autonomic failure...................................................37–38, 45 Autonomic nervous system........................... 45, 58, 326, 337 a-wave............................................................... 290–291, 295 Axon pathways. See Anterograde tracing; Retrograde tracing Axon tracing.............................................................139–157 Axonal transport.......................................................150, 201

B BAC HD transgenic mice..........................................8–9, 13 Balance and motor coordination. See Raised bridge; Rotarod tests Barnes maze.............................................................196, 201 Basal ganglia........................................................ 3–112, 161, 163, 183–190, 251, 321–328, 336–337 Beam balance. See Raised bridge test Behavioral light aversion (BLA) test........................312–314 Behr’s pupillary reflex...............................................225, 230 Bicuculline................................................................325, 332 Bilateral asymmetry (adhesive removal) test......................97, 104, 110–111 Bilateral coordination.......................................................139

Emma L. Lane and Stephen B. Dunnett (eds.), Animal Models of Movement Disorders: Volume II, Neuromethods, vol. 62, DOI 10.1007/978-1-61779-301-1, © Springer Science+Business Media, LLC 2011

435

Animal Models of Movement Disorders 436  Index

  

Biogenic amines. See Dopamine (DA); Noradrenaline (NA); Serotonin (5-HT) Blood flow.................................................79, 91–92, 94, 106, 108, 184, 189–190, 222–223, 236, 296–298, 347 Blood vessels............................................122–123, 128, 133, 158, 186, 194, 219, 221, 222, 297, 328, 365, 381–382, 391–392 Blood-brain barrier.......................39, 74, 184–185, 190, 198, 348, 366, 373 Body weight.................................................7, 9, 64–65, 103, 108, 235, 248, 274, 358 Bone flap approach...........................................................219 Brain rigidity....................................................................219 Bridge crossing. See Raised bridge test Brinkman board.......................................................422–426 b-wave......................................................................... 290–291

C Cacna1aln and Cacna1arol mutant mice.............................244 CAG repeats....................................3, 6–9, 83, 242, 244–245 CAG140 HD knock in mice....................................9, 11–15 Calb1 mutant mice...........................................................245 Calbindin............................................................ 27, 244, 246 Callosal transection..................................................211–236 Camera................49, 105, 126, 203, 271, 272, 296–297, 303, 309–312, 408, 412 Carbachol.................................................................309, 332 Cat............................. 121–122, 211–223, 227, 236, 325, 334 Catwalk XT appratus.......................................................273 Caudate nucleus................63, 73, 77, 80, 183–190, 195, 326 Centromedial thalamic nucleus (CMN)...........................153 Cerebellar ataxia animal models..................37–38, 241–255, 265, 269, 275 human disease.......................................37, 254–256, 263 Cerebellum cortex...................252–255, 263–264, 269–270, 277, 281 deep nuclei...........................242–245, 253–255, 285, 326 lesions..........................241, 249, 252, 264–273, 276, 284 mutations............................................................241–256 Cerebral asymmetries...............................................215, 225 Cerebral commissures...............................................211–236 Cervical hemicontusion model......................... 345, 350–361 CFA. See Complete Freund’s adjuvant Channelrhodopsin....................................................310, 335 Chiasma bisection.................................... 217–219, 223–236 Cholesterol.........................................................................91 Choline acetyl transferase (ChAT)....................... 27, 73, 332 Chorea................................................................ 4, 73, 76–77 Chromosome..................................................................4, 72 Circadian rhythms.................................... 287–189, 299–307 Circle of Willis.........................................................106, 223 Classical conditioning. See Pavlovian conditioning Claustrum..................................139–140, 146–147, 155, 157 Climbing tests...................12–13, 48–49, 248, 264, 269, 274

Closed head injury............................................................200 Cognitive tests.............4–8, 31, 73, 76–79, 84, 162, 195–204 Commissurotomy.....................................................211–236 Complete Freund’s adjuvant (CFA).........................366–371 Complex motor skills. See Motor tests Compression-type injuries........................................350–351 Conditioned analgesia........................................................60 Conditioned eyeblink response (CR).......................281–284 Coenzyme Q10............................................................78, 83 Cones.........................................288, 295, 301, 305–306, 313 Controlled cortical impact (CCI) model..................195–204 Contusion model......................................................345–351 Contusion, impactors and devices............................349–351 Corneal dehydration.........................................................228 Corpus callosum.................. 30, 151, 196, 198, 200, 211–236 Corridor test.....................................................................110 Cortical depth..........................................................129–130 Cortical lesions......................................... 194–195, 198–200 Corticospinal tract................................... 118, 173, 352–353, 422, 424–425, 428 Countermanding task...............................................168, 170 Craniotomy......................................................127–128, 143, 149, 157, 196, 199–200, 230 Cresyl violet stain...................................................27–28, 43 Cuneiform nucleus (CNF)...............................................325 Cylinder test...........................................41, 48, 97, 104–105, 110, 120, 124–125 Cytochrome oxidase (COX) activity.........................252–254

D DARPP–32........................................................9, 27–28, 30 Deep brain stimulation (DBS)................................323–324, 326, 328, 336, 338 Deep cerebellar nuclei......................................................255 Delayed response tasks................31, 167–170, 324, 330, 429 Demyelination...................................viii, 346–347, 363–378 Dexamethasone................................................................235 Digital signal processing...................................................294 Digits.................................................119, 125, 414, 419–429 Diptheria toxin.................................................................373 Disengage test.....................................97, 102–103, 110–111 Dog....................................................121, 214–215, 218, 282 Dopamine (DA)...............................73, 82–83, 99, 254–256, 328, 331–332, 337 Dopamine receptors..........................................32, 75, 83, 99 Dorsal column lemniscal system (DCLS)........................212 Dorsal striatum............................28–29, 55–67, 74, 255, 326 Dorsolateral striatum....................................................62, 64 Dorsomedial striatum...................................................62, 63 Dose-dependency.................................21–22, 24, 29, 43–44, 46, 73–74, 76, 80, 82, 99, 124, 330, 332, 371 Double lesion models, MSA...................... 37–38, 41–43, 45 Dstdt mutant mice.............................................................244 Dynamic postural reflexes................................4–5, 264–267

Animal Models of Movement Disorders 437 Index     

Dyskinesia.......................................... vii, 73, 76–77, 80, 419 Dystonia.......................................4, 43, 47, 76, 79, 241, 244 Dystrophic mice and rats..........................................299, 311

E Electrodes...........................................................22, 122–123, 126–132, 141–145, 149, 157–158, 173, 176, 285, 290–295, 324, 328–329, 376 Electrolytic lesions...................................... 22, 130, 196, 252 Electrolytic solutions................................................291, 293 Electromyography (EMG)..............................126, 282–283, 285, 426, 429 Electron microscopy (EM)................................ 80, 185–187, 213, 381–398 Electrophysiology.........................................v, 163–167, 289, 291–292, 295, 298, 331, 333–335, 337, 364, 376, 430. See also Volume I, ch. 11 Electroretinography (ERG)......................................289–295 EM. See Electron microscopy EMG. See Electromyography Endothelin–1 lesions................................................122–123 Energy metabolism (in HD)..............................................72 Enkephalin.........................................................................73 Environmental enrichment.................................................10 Environmental influences...................................... 10, 50, 56, 58, 60–61, 64–65, 81, 91, 98–99, 104, 112, 161, 293, 307, 314, 406 Epilepsy..........................................................................8, 23 Equilibrium......................................................................275 ERG. See Electroretinography Ethidium bromide....................................................373, 375 Ethylcholine mustard aziridinium ion (AF64A)..............332 Evoked movements..........................................................134 Excitotoxic lesions cerebellum..................................................................245 cortex...................................................................... 93, 195 striatum.........................21–32, 63, 71–73, 77, 81–83, 99 striatal afferents......................................................31–32 PPTg............................................................ 325, 328–330 toxin preparation....................................................24–25 Experimental auto-immune encephalomyelitis (EAE) models.....................................................364–378 Experimental design..................................... 97–98, 403–406 Exploration.......................................................................105 Extracellular recording.....................................................295 Eye movements........................................................161–178 Eyeblink conditioning..............................................281–285

F Falx............................................. 218–219, 222, 225, 228, 231 Ferret............................................................................. 195 Fixation (surgical).....................................................356–357 Fixation (tissue)........................................ 108, 386–388, 397 Fixation (visual)........................................ 169–173, 311, 334

Flash ERG...............................................................294–295 Flicker ERG.............................................................294–295 Fluid percussion (FP) model.................... 195, 198–200, 202 Fluoro-gold..............................................................142–158 Fluoro-ruby...................................... 142–143, 148, 151–155 Focal demyelination..................................................372–376 Foetal calf serum (FCS)....................................................369 Food manipulation...................................... 49–50, 103–104, 118–119, 125, 406–407, 413–415, 421–426 Food restriction.................................64–65, 98–99, 103, 111 Food reward................................................. 56–66, 103–104, 184, 334, 406–407, 410, 412–414, 419–426 Foot-print stepping test. See Gait analysis Forebrain commissures. See Anterior commissure (AC); Corpus callosum, Optic chiasm Forelimb use. See Skilled reaching tests Fos expression....................................299–304, 325, 333, 335 FRDA mutant mice.................................. 242, 244, 247, 249 Freezing (behaviour)............................60, 265, 268, 274, 324 Freezing (lesions)..............................................................195 Freezing (tissue preparation)....................................303, 387 Friedreich’s ataxia............................................. 241–242, 244 Frontal eye field.........................163–164, 166, 169, 177, 214

G GABA...................24, 29, 42, 73–74, 80, 253–254, 322–334 GABA receptors.......................................................332, 325 Gait analysis.............................................7–9, 11, 44, 48–49, 75, 110, 241–242, 252, 263–269, 273–274, 323–324, 326, 332–333, 336–338, 370, 408, 411 Galactocerebroside anti-serum.........................................373 Gender......................... 7, 10, 13–15, 193, 204, 312, 366–367 Genetic models. See Mutant mice; Transgenic mice Gerbil......................................................................183–190 GFP. See Green fluorescent protein Glial cytoplasmic inclusions...................................37, 45, 47 Glial fibrillary acidic protein (GFAP)................................27 Globus pallidus (GP)................................. 42, 323, 326–327 Glutamate receptors..................................21, 22, 29–31, 291 Glutamic acid decarboxylase (GAD)..................................73 Glutaraldehyde..................186, 382–383, 387–388, 393, 395 Glutathione (GSH)............................................................82 Goal-directed behaviour..................................... 63, 164, 334 Golgi stain........................................................................124 Granule cells (cerebellum)................ 242–246, 249, 253–255 Green fluorescent protein (GFP)..................... 386, 393–398 Grid walk/climb tests...................................... 196, 241, 246, 248–249, 412–414 Grid2ho (hot-foot) mutant mice..................................241, 253 Grid2Lc (Lurcher) mutant mice..................................242, 254 Grip strength................................................ 48–49, 110, 245 Growth factors............................................. 42, 78, 249, 332 Ground reaction force (GRF)...................................408–411 Guinea pig................................................ 121, 213, 282, 366

Animal Models of Movement Disorders 438  Index

  

H Habituation........................................ 15, 272–273, 308, 314 Haemorrhage................................................. 22, 74, 97, 106, 195–201, 346–347, 391 Hand use (primates).................................................419–431 Hank’s buffered salt solution (HBSS)..............................369 HD. See Huntington’s disease (HD) Hematoxylin and eosin (H&E) stain......................108–109, 186–187, 381 Hemisphere displacement........................................229–231 Heroin..............................................................................330 Heterogeneous population................................................335 Hindlimb clasping.................................................. 11, 43, 47 Hindlimb deficits.....................................................351–352 Hippocampus...................................................... 74, 81, 195, 198–201, 204, 206, 213–216, 232, 251 Histological assessment....................................... 27–28, 108, 143, 185–187, 189, 299–304, 383–385, 397–398, 401–416 Hot-foot mouse mutation........................ 241, 242, 245–253 Huntingtin........................................3–6, 8–9, 71–72, 83–84 Huntington’s disease (HD) animal models and behavior..............3–15, 21–32, 71–84 human pathology and symptoms......................... vii, 3–5, 22, 24, 32, 39, 71–76, 97 6-Hydroxydopamine (6-OHDA).......................... 32, 38–44, 48–49, 82, 99, 323. See also Volume I, ch. 13, 14 5-Hydroxyindoleacetic acid (5-HIAA)..............................24 5-Hydroxytryptamine (5-HT). See Serotonin (5-HT) Hypothalamus..................................................... 4, 106, 195, 229, 287, 289, 299, 304 Hzf null mutation.............................................................246

I Ibotenic acid......................................... 23, 73, 329, 331–332 ICMS. See Intracortical microstimulation 192 IgG saporin lesions....................................................332 Imaging (histology)...........................126, 132, 383, 388, 397 Imaging (in vivo)..........................................6, 38, 64, 79, 97, 190, 287, 289, 296–299, 327–329, 335. See also Volume I, ch. 8–10 Immunization....................................364, 368–369, 371, 373 Impact energy absorption.................................................349 Impact-acceleration model.......................................200–203 Impactor devices cortex..................................................................195–203 spinal cord..........................................................349–351 Inclined grid test.............................................. 241, 246–248 Incomplete Freund’s adjuvant (IFA).................................366 Instrumental conditioning............................................55–67 Intracortical microstimulation (ICMS)...........................122, 126–134, 143–145, 173 Intraluminal filament procedure......................... 93, 106–108

Iontophoresis.................................... 141, 144–150, 157–158 Ischaemia. See Stroke Isoflurane........................................29–30, 93, 184–185, 203, 297, 355, 367, 370–371, 374

K Kainic acid...................................................... 22–23, 73, 325 Ketamine....................................................... 27, 29–31, 127, 134, 143, 235, 297, 353–354, 359 Kinematic analysis................................................... 133, 403, 407–408, 411–412, 419, 426, 429–431 Kinetic analysis................................. 403, 407–408, 411–412 Klüver board.............................................................425–426 Knock-in mice cerebellar mutations.................................... 244, 247, 249 mutant huntingtin..............................................6–10, 71 Knock-out mice cerebellar models........................................ 242, 245, 247 metabolic lesions...........................................................82 nicotinic receptors.......................................................331 visual system models..................................................291, 301, 306, 308, 313 Kynurenate.........................................................................23 Kynurenine aminotransferase-I..........................................81

L Ladder climbing task.................264–268, 401–407, 412–413 Laser Doppler flowmetry..................................... 94, 96, 106 Lateral dorsal thalamic nucleus (LDN)....................146, 153 Lateral geniculate nucleus (LGN)............................212, 287 Law of effect.................................................................56–57 L-dopa.................................................vii, 37, 38, 45, 82, 324 Learning circuits cerebellum..........................................................281–285 striatum...................................................... 57–59, 62–67 Learning..........................55–67, 76, 271–272, 276, 281–286 LED light sources............................................................306 Lever pressing..............................................................57–67 Lewy bodies................................................................37, 323 Light aversion test............................................ 304, 312–314 Light microscopy (LM)............................................381–398 Light stimulation..............................................................307 Limb function..........................................................407–416 Lipopolysaccharide...........................................................373 Lithium chloride..........................................................65–66 Local field potentials........................................................292 Locomotor tests.......................................................266–268, 304–306, 407–414 Locomotor velocity...................................................410–411 Lurcher mouse mutation.................................................241, 242, 245–253 Lymph nodes.................................................... 365, 369, 377 Lymphocytes....................................................................377 Lysophosphatidylcholine.......................... 363, 373, 375–376



M Macaque. See Non-human primates Macrophages............................................................382, 391 Magnetic resonance imaging (MRI)........................... 79, 97, 190, 328–329 Maladaptive response.......................................................246 Malonate...........................................................23, 80, 82–83 Mammillothalamic tract...................................................146 Mannitol.................................................................. 219, 221, 227–231, 234–235 Manual dexterity (primates).............................................422 Marmoset........................................................... 366, 421, 423 Masking.................................................... 104, 111, 306–307 Maze learning tasks Barnes maze.......................................................196, 201 Radial arm maze.................................................198, 330 T-maze...........................................................7, 250–251 Water maze.................................................. 7, 8, 79, 196, 198, 200–203, 251–252, 271–272, 276–277, 310 Y-maze...............................................................250–251 MBP. See Myelin basic protein MBP-h-a-syn animals.................................................46–47 MBP-promoter............................................................46–47 MCA. See Middle cerebral artery MCAO. See Middle cerebral artery occlusion Mediodorsal thalamic nucleus (MDN).............................63, 67, 146, 153 Melanopsin........................................288, 301, 306, 308, 313 Melanopsin knock-out mice..................... 301, 306, 308, 313 Memory..................................................................4, 7, 31, 66, 76, 84, 145, 162, 169–170, 213, 215, 281, 324 Mesencephalic locomotor region (MLR)........................322, 324–326, 338 Metabolic toxins.....................................................23, 80–83 Metacarpophalangeal (MCP)...........................................428 1-Methyl–4-phenylpyridinium (MPP+)............................39 MI. See Motor cortex Microglia................................................................41, 46, 382 Microstimulation. See Intracortical microstimulation Middle cerebral artery (MCA)........................... 92, 122, 123 Middle cerebral artery occlusion (MCAO)...............91–112, 123–124 Midline disconnection cat and monkey...................................................219–225 rabbit.................................................................... 225–231 rat.......................................................................... 231–235 Mid-thoracic region.................................................351–352 Mitochondria.......................................23, 39, 45, 53, 71–84, 108, 185, 242, 252 Mitochondrial impairment...................................73–79, 185 MK-801................................................................................ 83 Mongolian gerbil. See Gerbil Monkey. See Non-human primates Monochromatic pulses.............................................304–305

Animal Models of Movement Disorders 439 Index      Monocular deprivation.....................................................310 Morris water maze. See Water maze Motor cortex (MI) forelimb region...................................................140–141 lesions...................................118–120, 122–124, 192, 425 organization.........................117–118, 140, 145–147, 152 stimulation.................................. 118, 121–122, 126–135 whisker regions............................127, 132–135, 139, 154 Motor learning....................14, 263–264, 270–273, 276, 277 Motor synchronisation.............................................269–271 Motor tests. See also Volume I, ch. 2–7 cylinder test......................................41, 48, 97, 104–105, 110, 120, 124–125 open-field tests.................................................. 8, 10, 12, 15, 44, 48, 183, 186, 188–189, 250–251, 312–314, 402, 406 pole test...................................................... 15, 44, 46–47 raised bridge test.............................................. 44, 48–49, 79, 110, 119, 124, 198, 200, 201, 241, 246–248, 251, 266, 274, 412 rotarod.........................................................12–13, 48–49, 201, 241, 246–250, 275–276 rotameter.....................................38–42, 48, 97–100, 110 single pellet reaching test....................................412–414 skilled reaching tests......................49, 119, 125, 352, 412 wheel running tests....................................... 13, 304–307 whisker test.........................................................101–102 Mouse.................................................3–15, 43–45, 241–256, 117–135, 196–197, 199, 204, 241–256, 266, 269–271, 274, 282–285, 287–314, 350, 366, 370–373 Movement disorders. See Huntington’s disease (HD); Multiple sclerosis (MS); Multiple system atrophy (MSA); Parkinson’s disease (PD); Spinal cord injury (SCI); Spinocerebellar ataxia (SCA) MPP+. See 1-Methyl–4-phenylpyridinium MPTP. See N-methyl–4-phenyl-tetrahydropyridine MS. See Multiple sclerosis MSA. See Multiple system atrophy Multiple sclerosis (MS) animal models.....................................................364–378 human disease.....................................................363–365 Multiple system atrophy (MSA) human disease...................................................22, 37–38 lesion models....................................................32, 38–45 transgenic models...................................................45–47 Multiunit activity......................................................292–293 Muscimol.......................................................... 325, 330–332 Mutant mice behavioural assessment.......................................245–253 cerebellar mutants...............................................241–256 neuropathology...................................................241–144 visual impairments......................................................310 Myelin basic protein (MBP)......................... 46–47, 365, 367

Animal Models of Movement Disorders 440  Index

  

Myelin oligodendrocyte glycoprotein (MOG).........373–381 Myelin-associated glycoprotein (MAG)...........................365 Myelinated axons...................................... 382, 384, 390, 392

N NADPH-diaphorase...........................24, 29, 73–74, 80, 335 Neonatal animals......................................................313, 426 Nervous (nr) mutant mice........................ 241, 243, 245–253 NeuN.................................................................... 27, 28, 332 Neurodegeneration.................................................. 3–4, 6, 8, 10, 21, 32, 37–39, 45, 47, 71–85, 193, 244 Neuronal labeling analysis................................ 147, 155–157 Neuropathic pain..............................................................353 Neuropetide Y............................................ 24, 29, 73–74, 80 Neuroplasticity......................................... 134, 281, 298, 425 Neuroprotection................ 3–4, 7, 29–32, 45–47, 79–84, 203 Neurotensin........................................................................73 Neurotrophic factors..................................... 42, 78, 249, 332 Neurovascular coupling....................................................297 Newton–von Gudden principle................................217–218 Nictitating membrane...................................... 219, 281–282 Nigral lesions.....................................37–46, 80–81, 242, 322 3-Nitropropionic acid (3-NP)................................. 5, 38–41, 43–47, 71, 73–81, 84 NMDA receptors............................................ 22–23, 29–32, 39, 74, 78, 81, 127, 134 NMDA. See N-methyl-D-aspartate N-methyl–4-phenyl-tetrahydropyridine (MPTP)..................... vii, 38–40, 42, 45, 323, 325 N-methyl-D-aspartate (NMDA)............. 22, 32, 81–82, 332 Nomenclature...........................................................321–322 Non-human primates.....................................vii, 6–7, 45, 63, 71, 73, 76, 79–80, 84, 118–122, 161–178, 211–227, 236, 282–285, 323, 325, 327, 334, 348, 366, 419–431 Non-motor functions.......................................................337 Noradrenaline (NA).................................................254–256 Norepinephrine (NE). See Noradrenaline Normal goat serum (NGS)............................... 386, 394, 395 Novel environment..................................... 65, 104, 250, 406 3-NP. See 3-Nitropropionic acid

O Occipital lobe........................................... 195, 216, 222, 226 Oculomotor tasks............................................... 62, 164, 171 6-OHDA. See 6-Hydroxydopamine Olfactory bulb..................................................................243 Olfactory tubercle.............................................................214 Open-field tests.............................. 8, 10, 12, 15, 44, 48, 183, 186, 188–189, 250–251, 312–314, 402, 406 Operant chamber..........................................................56, 64 Operant conditioning. See Instrumental conditioning Optic chiasm............................................................211–236 Optic nerve............................................... 218, 224, 308, 376

Optical imaging....................................................... 287, 289, 296–299, 335. See also Volume I, ch. 10 Optogenetics............................................................311, 335 Optokinetic head-tracking....................... 287, 289, 310–312 Optokinetic nystagmus.....................................................219 OptoMotry software................................................310–312 Orbicularis oculi muscle...................................................285 Oscillatory potentials........................................ 290–291, 295

P Papp-Lantos bodies. See Glial cytoplasmic inclusions Paraformaldehyde...............................27, 150, 158, 185, 302, 382, 387, 394, 397 Parietal lobe...............................163, 183–189, 195, 216, 429 Parkinson’s disease (PD). See also Volume I, ch. 12–21 animal models................................. 38, 81, 101, 322–324 human disease..................................4, 7, 37, 81, 161, 322 Parvalbumin.......................................................................73 Passive avoidance test...................................................76–77 Passive EAE.............................................................368–370 Pavlovian conditioning................................. 58–60, 281–285 Paw reaching. See Skilled paw reaching PCD mouse mutation.....................................................241, 243, 245–253 Pcp2 promoter...................................................................244 PD. See Parkinson’s disease (PD) Pedunculopontine tegmental nucleus (PPTg)..........321–338 Pertussis toxin...................................................................371 Phase shifting...........................................................304–306 Photocell beams.......................................... 12, 285, 313, 408 Photoreceptors................................................................231, 235–236, 243, 287–291, 294–295, 300–301, 305–307, 310, 313–314 Photothrombotic infarction......................................183–190 Phylogeny....................................................215, 217, 219, 326 Pig................................................................................7, 195 Pilot tests............................................ 24, 123, 328–329, 397 Pipette puller.....................................126, 143, 147–148, 373 Plcb4 mutant mice............................................................246 PLP. See Proteolipid protein PLP-h-a-syn animals........................................................46 PLP-promoter..............................................................45–46 Pogo mouse mutation................................ 241, 243, 245–253 Pole test............................................................ 15, 44, 46–47 Polymethylmethacrylate optic fiber..................................184 Polymodal sensory information........................................334 Post-operative care............................. 26, 235, 354, 357–360 p-Phenylene Damien (PPD)............................ 383, 390, 392 Precision grip (in primates)......................................419–431 Prefrontal cortex....................................31, 63, 163, 251, 426 Prehension (in primates)..................................................430 Primate. See Non-human primates Progressive lesions............................................................313 Protein aggregation. See Aggregates

Animal Models of Movement Disorders 441 Index     

Proteolipid protein (PLP) EAE models...............................................................367 promoter.................................................................45–46 Pupillary light reflex................................. 230, 288, 307–309 Pupillometry..................................................... 289, 307–309 Purkinje cell degeneration (PCD) mutant mice.......241–255 Purkinje cells.............................................. 46, 241–255, 263 Putamen..............................................42, 64, 73, 77, 80, 326

Q Quadruped locomotion............................................265–267 Quantitative anatomy.................................... 27–28, 47, 108, 152, 254, 299, 303, 335, 348, 391–393 Quantitative measurement of behaviour.............. 12–13, 125, 401–408, 412–415, 419 Quinolinic acid (QA)............................. 5, 23–32, 38–44, 73

R R6/1 and R6/2 HD transgenic strains......................7–15, 83 Rabbit................................121, 198, 211–236, 281–286, 366 Radial arm maze.......................................................198, 330 Raised bridge test..................... 44, 48–49, 79, 110, 119, 124, 198, 200, 201, 241, 246–248, 251, 266, 274, 412 Rat.......................... 21–32, 37–43, 71–84, 91–112, 117–135, 139–159, 231–234, 183–190, 193–204, 218, 231–236, 263–277, 282–285, 287–314, 321–328, 345–361, 363–378, 381, 398, 401–416 Reaching. See Skilled reaching tests Recombinant MOG (rMOG)..................................367–369 Reconstruction and neuronal labeling pattern analysis.....143 Reeler mouse mutation............................. 241, 243, 245–253 Reflex eyeblink response (UR).................................282–283 Reflexive behaviours.............................59, 60, 127, 162, 200, 203, 219, 225, 230, 248, 263–265, 270, 282, 287–288, 307, 372 Reflexive eye movement........................... 162, 219, 270, 282 Regional brain metabolism.......................................252–254 Regional brain metabolism............................... 252–254, 296 Relatively independent finger movements (RIFM)...................................................423–425 Reliability...........................................196, 328, 350, 401, 404 Reln rl (reeler) mutant mice................................................243 Reserpine............................................................................82 Resin embedding......................................................393–396 Retina.......................... 59, 162, 212, 218, 231, 243, 287–314 Retinal ganglion cells................................ 218, 300, 305, 313 Retinally degenerate rats..................................................311 Retractor bulbus muscle...................................................282 Retrograde tracing....................................................139–157 Reward..............................................56–57, 61, 63, 330–331 Reward-related learning...................................................331 Rhesus macaque. See Non-human primates Righting reflex................... 200, 203, 248, 263–265, 273, 372 Righting reflex..................................................................273

Ringer’s solution............................................... 226–227, 354 RNA interference...............................................................49 Rods..................................................288, 295, 301, 305–306, 310, 313 Roof nuclei.......................................................................254 Rorasg (staggerer) mutant mice...................................242–243 Rose Bengal...............................122–124, 183–184, 188–189 Rotarod........................................................................12–13, 48–49, 201, 241, 246–250, 269–271, 275–276 Rotating drum test...................................................311–312 Rotation................................................................. 38–42, 48, 97–100, 110 Rotometer tests................................................... 48, 99–100, 110. See also Volume I, ch. 15 Royal College of Surgeons (RCS) dystrophic rat.............299

S Saccadic eye movement............................................161–178 Salivation......................................................................58–60 Sampling bias...................................................................177 Satiety..................................................................... 61, 63, 66 Schedules of reinforcement..........................................61–65 Schwann cells (SCs)........................................................377, 381, 384–393 SCI. See Spinal cord injury Semi-thin resin sections........................... 382–384, 396–397 Sensorimotor pathways................................... 31–32, 42, 57, 62, 64, 118, 127, 216 Sensorimotor tests............................................. 97, 104, 264, 275, 326, 407, 422, 427, 431 Serotonin (5-HT)......................................... 24, 73, 254–256 Sex. See Gender Sexual function.................................................................419 Signal filtering..................................................................293 Single pellet reaching test........................................412–415. See also Volume I, ch. 6 Single unit activity....................................................292–293 Skilled hand use....................................... 412–415, 419–431 Skilled reaching test.............................................. 31, 41, 49, 103–104, 119–120, 125, 352, 412–415, 419 Sleep-wake mechanisms............................... 4, 322, 324, 326 Slow finger movements....................................................428 Sodium azide lesions....................................................80–82 Solenoid-controlled devices..............................................350 Somatostatin........................................................... 24, 29, 73 Somatotopic map....................................... 31, 128, 132–134 Spatial orientation Dst dt-J line...................................................................252 Grid2 Lc mutants..........................................................251 Reln rl-Orl mutants.........................................................251 Specialization...........................................................172–173 Spectral sensitivity............................................................308 Spectrin-b3......................................................................245 Spinal cord homogenate (SCH).......................................368

Animal Models of Movement Disorders 442  Index

  

Spinal cord injury (SCI) animal models............................. 345–361, 401–416, 420 behavioural analysis.................................... 381, 401–416 human injury.............................................. 345–346, 352 human pathology........................................................348 Spinocerebellar ataxia (SCA)............................ 241–256, 264 Spinocerebellar atrophy. See Spinocerebellar ataxia Spinothalamic tract.................................. 212–213, 244, 353 Split-brain research..........................................................231 Spnb3 knockout mouse.....................................................245 Spontaneous alternation...........................................250–251 Spontaneous forelimb use................................. 111–112, 325 Stab injury.........................................................................195 Staggerer mouse mutation........................ 241, 242, 245–253 Staircase test..................................................... 48, 50, 79, 97 Startle response................................................................284 Stepping test......................................39, 41, 48–49, 101–102 Stereotactic surgery....................................39, 118, 123, 126, 141, 143, 146, 148–149, 183–189, 196, 231, 234–235, 293, 295, 321, 328, 338, 397 Stimulation grid............................................... 128–129, 132 Stimulation-induced movements............. 118–121, 130–131, 143–145, 325 Stria medullaris................................................................146 Striatonigral lesions......................................................38–39 Striatum......................................3–15, 21–32, 37–50, 55–67, 71–84, 91–112, 139, 146–147, 254–255, 263, 326, 333, 334, 337. See also Caudate nucleus; Putamen Stroke/ischemia.................................... 32, 91–112, 183–190 Substance P..................................................................24, 73 Substantia nigra..................................37–46, 80–81, 83, 242, 245, 322–323, 326–327, 337 Subthalamic nucleus (STN).....................................323, 327 Sucrose consumption........................................................331 Sunflower seed opening............................................120, 125 Superior colliculus....................................161, 163, 166, 173, 232, 287, 295, 301, 310, 313, 327 Suprachiasmatic nucleus (SCN)....................... 287, 299–305 Surface technique......................121, 219–235, 354, 374–375 Surgical instruments........................................ 122–123, 220, 224, 353, 355, 359–360 Surgical methods............................................. 25–26, 93–97, 219–235, 353–358, 370–371, 374–375 Suspended wire. See Wire suspension test Swimming test...........................120, 125, 251, 264, 271, 403 a-syn. See Alpha-synuclein Synchronisation task.................................................269–271

T Temporal lobe..................................................... 81, 214, 216 Thalamus........................ 67, 74, 80, 106, 118, 139, 146–147, 153–154, 163, 196, 198, 212–213, 243, 254, 263, 287, 289, 308, 310, 326–327, 334, 337

Thalamus, individual nuclei anteromedial (AMN).................................................146 anteroventral (AVN)...................................................146 centromedial (CMN)..................................................153 lateral dorsal (LDN)...........................................146, 153 lateral geniculate (LGN)....................................212, 287 mediodorsal (MDN).............................. 63, 67, 146, 153 ventrolateral (VLN)............................................146, 153 ventromedial (VMN).................................................146 ventroposterior (VPN)............................... 153, 212–213 Therapeutic testing....................... 7, 9, 14, 21, 38, 45, 47, 71, 78, 83–84, 204, 323, 348, 352, 365, 381, 419 Thin section immunostaining..................................394–396 Thoracic cord...................................................................351 Threshold determination.......................... 130–133, 145, 165 Tissue dissection...............................................................387 Tissue perfusion............................................... 185, 382, 387 Tissue plasminogen activator (tPA)..................................190 T-maze.................................................................7, 250–251 Tongue extension................................................ 31, 120, 125 Tool use (primates)...................................................430–431 Topological organisation..................................................217 Tract tracing. See Anterograde tracing; Retrograde tracing Transection procedure..............................................211–236 Transgenic mice cerebellar mutants...............................................241–256 HD models...............................................................3–15 MSA models..........................................................45–50 SCA models.......................................................244, 245 Transmission electron microscopy............................388–389 Transplantation retina...........................................................................311 spinal Cord.........................................................391–395 striatal......................................................... 42, 45, 78–79 Traumatic brain injury (TBI) models.......................193–204 Trephine hole...........................................................221, 226 Trigeminal nerve...............................146, 282–283, 286, 326 2,3,5-Triphenyltetra-zolium chloride (TTC).............96, 108 TRUSCAN......................................................................313 Tyrosine hydroxylase.................27–28, 74, 83, 300, 302–303

U Unc13c mutant mice.........................................................245 Unconditioned response (UR)..................................282–283 Unilateral lesion impairments...................................... 39, 42, 49, 97, 104–105, 111, 124, 125, 189, 230–231 Untrained animals............................................................270 Urethane...................................................................134, 297 Urinary bladder evacuation...............................................358 Ursodeoxycholic acid........................................................249

V V1. See Visual cortex Varicosities................................................ 140, 142, 150–159

Animal Models of Movement Disorders 443 Index     

Vasculature supplying the head.....................................94, 95 Velocity, ground reaction forces................................410–411 Ventral posterolateral thalamic nucleus (VPL).........212–213 Ventral striatum............................................................62–63 Ventrolateral preoptic nuclei (VLPO)..............................301 Ventrolateral thalamic nucleus (VLN).....................146, 153 Ventroposterior thalamic nucleus (VPN)........................153, 212–213 Vertical grid..............................................................248–249 Vestibular drop test........................................... 263–265, 273 Vibrissae-evoked forelimb placing test. See Whisker test Video tracking..................................... 12–13, 105, 124–126, 134, 178, 186, 203, 249, 264, 271, 311, 313, 408–415, 419, 426–430 Viral vectors.................................................. 3, 5–6, 364, 397 Virtual rotating drum.......................................................311 Visual acuity............................................. 272, 295, 310, 312 Visual cortex (V1)............................................. 31, 212, 216, 287, 289, 291–292, 295, 298, 301, 310, 313, Visual evoked potentials (VEPs)......................................291 Visual function.........................................................287–314 Visual stimulation............................................................289, 294–295, 311–312 Visually-delayed saccade task...................................168–170 Visually-guided saccade tasks........................... 168–173, 334 Voltage amplifier..............................................................293 Volumetric analysis.............................................................28

W Water maze................................ 7, 8, 79, 196, 198, 200–203, 251–252, 271–272, 276–277, 310 Weight-drop techniques...........................................347–349 Wheel running tests............................. 13–14, 289, 304–307 Whisker test.............................................................101–102 Whisking movements...............................................139–140 White matter tracts isolation............................................376 Wire suspension test..................241, 246–248, 268–269, 369 Withdrawal reflex.......................................................60, 127 Working memory......................................... 4, 162, 170, 324 World Health Organisation...............................................91

X X ray..................................................................................377 Xylazine............................... 29, 127, 143, 235, 353–354, 359

Y YAC128 HD transgenic mice.....................................8–9, 13 Yeast artificial chromosome (YAC)..............................8, 284 Yellow fluorescent protein (YFP).....................................335 Y-maze.....................................................................250–251

Z Zona incerta.....................................................................146