International Review of Neurobiology Volume 28

INTERNATIONAL REVIEW OF

Neurobiology VOLUME 28

Editorial Board W. Ross ADEY JULIUS

AXELROD

SEYMOUR KETY KEITH KILLAM

Ross BALDESSARINI SIR ROGERBANNISTER FLOYDBLOOM

CONANKORNETSKY

DANIELBOVET

PAULMANDELL

PHILLIPBRADLEY

OSMOND HUMPHRY

YURI BUROV

RODOLFO PAOLETTI

JOSE

DELGADO

SIRJOHN ECCLES JOEL

ELKES

ABELLAJTHA BORISLEBEDEV

SOLOMON SNYDER STEPHENSZARA SIRJOHN VANE

H. J. EYSENCK

MARATVARTANIAN

KJELL FUXE

STEPHEN

Bo HOLMSTEDT

RICHARD WYATT

PAULJANSSEN

OLIVER ZANGWILL

WAXMAN

INTERNATIONAL REVIEW OF

Neurobiology Edited by JOHN R. SMYTHIES RONALD J. BRADLEY Department of Psychiatry and The Neurosciences Program The Medical Center The University of Alabama at Birmingham Birmingham, Alabama

VOLUME 28

1986

ACADEMIC PRESS, INC. Harcourt Brace Jovanovich, Publishers

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COPYRIGHT .C 1986

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Xh 87 K K 89

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Z

I

CONTENTS Biology and Structure of Scrapie Prions

MICHAEL F! MCKINLEY AND STANLEY B. PRUSINER I. Introduction ........

.........................................

11. Biology of Scrapie ........................................................... 111. Structure of Scrapie Prions. ..........

2 16

IV. Chemical Characteristics of V. Polymorphic Forms of Prion VI. Conclusions ....................

46

...............................

.................. ..............................

51 53

Different Kinds of Acetylcholine Release from the Motor Nerve

S. THESLEFF I. Intermittent Secretions of ACh ......................................................

11. Continuous ACh Leakage (111) ...................................................... 111. Comments ...............................................................................

References ...............................................................................

59

73 78 80

Neuroendocrine-Ontogenetic Mechanism of Aging: Toward an IntegratedTheory of Aging

V. M. DILMAN, S. Y REVSKOY, AND A. G. GOLUBEV I. Introduction .............................................................................

89

11. The Main Postulate of the Neuroendocrine-Ontogenetic Theory:

T h e Law of the Deviation of Homeostasis ......................................... 111. T h e Age-Related Changes in the Reproductive Homeostate: T h e Unity of the Mechanisms of Development and Aging. Climacteric as a Normal Disease..................................................... IV. T h e Age-Related Changes in the Adaptive Homeostate. Hyperadaptosis as a Normal Disease .................................................................... V. The Age-Related Changes in the Energy Homeostate. Prediabetes and Obesity as Normal Diseases........................................................... VI. The Choice of Diseases in the Course of Aging. The Interrelations between Main (Noninfectional) Human Diseases ......... V11. Aging as a Disease and as a Stochastic Process ................................... VIII. The Influence of External Factors on the Mechanism of Aging and Diseases of Aging ...................................................................... V

90

93 95 100 109 120 129

vi

CONTENTS

1X. T h e Analysis of Geroprotectors From the Point of View of the Neuroendocrine-Ontogenetic Theory of Aging .................................. X. T h e Neuroendocrine-Ontogenetic Theory of Aging and the Evolution of Aging. ............................................ ng and the NeuroendrocrineXI. T h e Modern Theor Ontogenetic Theory.. ............................................ XII. Three Models of Medicine ............................................................. References .................................... .............................

132

143 150

The lnterpeduncular Nucleus

BARBARA J. MORLEY ............................................

I . Introduction ...................

............................. 111. Neurochemistry

V I. Behavior ....... VII. Summary and References ......

.................. .............. .............. ............................. ..................................................................

.................... ....................

157 158 164 171 173 175 176 179

Biological Aspects of Depression: A Review of the Etiology and Mechanisms of Action and Clinical Assessment of Antidepressants

S. I . ANKIER AND B. E. LEONARD I . Introduction

........................................................... .................................................

ssants ............................. IV. Clinical Assessment of New Antidepressants.. ..................................... V. Conclusions., .................................................. References .................................... .............................

183 185 2 12 231

Does Receptor-Linked Phosphoinositide Metabolism Provide Messengers Mobilizing Calcium in Nervous Tissue?

JOHNN. HAWTHORNE ............................................................ Polyphosphoinositides? ............................... 111. Inositol Trisphosphate and Diacylglycerol as Second Messengers .............. IV. Phosphoinositides of the Adrenal Medulla ......................................... I. Introduction.. .. I I . Phosphatidylino

V. Phosphoinositides and Receptors in Brain.. ........................................ VI. The Autonomic Nervous System and the Pituitary Gland ........................ VII. Lithium Chloride and Phosphoinositide Metabolism ....

241 242 245 250 254 259 262

CONTENTS VIII . IX . X. XI .

Phosphoinositidesand Diabetic Neuropathy ...................................... Polyphosphoinositides and the Retina .............................................. Inositol Tetraphosphate............................................................... Conclusions.............................................................................. References ...............................................................................

vii 263 266 267 268 269

Short-Term and Long-Term Plasticity and Physiological Differentiationof Crustacean Motor Synapses

H . L. ATWOOD AND J. M . WOJTOWICZ I . Introduction............................................................................. I1 . Release of Transmitters ............................................................... 111. Differential Synaptic Performance.............................. IV. Short-Term Facilitation ................................................................ V. Presynaptic Inhibition ............................................. VI . Long-Term Facilitation ................................................................ VII . Neurohormonal Modulation .........................................................

VIII . Activity-Dependent Long-Term Adaptation .. IX . Trophic Effects.......................................................................... X . Conclusion ............................................................................... References ...............................................................................

275 277 299 309 316 325 337 339 346 348 350

Immunology and Molecular Biology of the Cholinesterases: Current Results and Prospects

STEPHEN BRIMIJOIN AND ZOLTANRAKONCZAY I . Introduction............................................................................. I1 . Molecular Biology of the Cholinesterases.......................................... 111. Immunology of the Cholinesterases ................................................ IV. Conclusion ............................................................................... References ...............................................................................

363 367 372 405 406

INDEX...........................................................................................411

This page Intentionally Left Blank

BIOLOGY AND STRUCTURE OF SCRAPIE PRIONS By Michael P. McKinley and Stanley B. Prusiner Departments of Neurology and Biochemistry and Biophysics University of California, San Francisco San Francisco, California 94143

1. Introduction

Progress in purification of the infectious particles causing scrapie is beginning to lead to an understanding of their biology and structure. Numerous attempts have been made to purify the scrapie agent over the past three decades (Hunter, 1972; Millson et al., 1976; Siakotos et al., 1976; Diringer et al., 1983a; Mould et al., 1965; Marsh et al., 1984; Brown et al., 1978).Few advances in this area of investigationwere made until a relatively rapid and economical bioassay was developed (Prusiner et al., 1980a, 1982a). Over a period spanning nearly a decade, our investigationsof the molecular properties of the scrapie agent have been oriented toward developing effective procedures for purification. We began our studies by determining the sedimentation properties of the scrapie agent in fixed angle rotors and sucrose gradients (Prusiner et al., 1977, 1978a,b).Subsequent work extended those findings and demonstrated the efficacy of nuclease and protease digestions as well as sodium dodecyl sarcosinate gel electrophoresis in the development of purification protocols (Prusiner et al., 1980b,c). Once a 100-fold purification was achieved, convincing evidence demonstrating that a protein is required for infectivity was obtained (Prusiner et al., 1981; McKinley et al., 1981). Even before the scrapie protein was indentified, we began an intensive search for the putative nucleic acid genome within the scrapie agent. To date, we have failed to find this elusive nucleic acid (Prusiner, 1982, 1984a; Diener et al., 1982; McKinley et al., 1983a);indeed, our results are consistent with those reported by Alper and colleagues nearly two decades earlier (Alper et al., 1966, 1967, 1978). The requirement of a protein for infectivity and the extraordinary resistance of the scrapie agent to inactivation by procedures that modify or hydrolyze nucleic acids led to the introduction of the term “prion”to denote these infectious particles (Prusiner, 1982). 1 INTERNATIONAL REVIEW OF NEIJROBIOLOGY, VOL. 28

Copyright 8 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.

2

MICHAEL P. MCKINLEY AND STANLEY B. PRUSINER

II. Biology of Scmpie Prions

A. SCRAPIE PRIONS CONTAIN A SIALOGLYCOPROTEIN In our search for a scrapie-specific protein, a purification scheme was developed which led to the first identification of a macromolecule within the scrapie prion (Prusiner et al., 1982b, 1983, 1984; Bolton et al., 1982, 1984; McKinley et al., 1983b). This molecule is a sialoglycoprotein designated PrP 27-30 with an apparent molecular weight of 27,000-30,000 (Table I) (Bolton et al., 1985). Hydrolysis, denaturation, or selective chemical modification of PrP 27-30 resulted in a loss of scrapie infectivity. Considerable evidence indicates that the major protein found in purified prion preparations, PrP 27-30, is a component of the infectious particle (McKinley et al., 1983b; Prusiner et al., 1983; Bolton et al., 1984). The concentration of PrP 27-30 was found to be proportional to prion titer. Many attempts to dissociate native PrP 27-30 from scrapie prion infectivity were unsuccessful. Indeed, scrapie PrP 27-30 seems to be required for and inseparable from prion infectivity The development of a large-scale purification protocol has allowed us to raise antibodies against the protein (Prusiner et al., 1984; Bendheim et al., 1984, 1985). Other investigatorsusing purification steps similar to those developed by us seem to have demonstrated the presence of this protein in their preparations (Diringer et al., 1983b; Hilmert and Diringer, 1983).

FOR B. SEARCH

A

PRION GENOME

The size of the smallest infectious unit remains controversial, largely because of the extreme heterogeneity and apparent hydrophobicity of the TABLE I PROPERTIES OF HAMSTER SCRAPIE PrP 27-30 Composition Molecular weight

Properties Biological function Structure Occurrence

Sialoglycoprotein 27,000-30,000 sodium dodecyl sulfate-polyacrylamide gel electrophoresis 19,500 sodium dodecyl sulfate high-performance liquid chromatography (HPLC) Size and charge heterogeneity Protease resistant in native state Native conformation required for prion infectivity Polymerizes into amyloid rods Scrapie hamster brain Similar proteins in mouse scrapie as well as human, guinea pig, and mouse Creutzfeldt-Jakob disease (CJD)

BIOLOGY AND STRUCTURE OF SCRAPIE PRIONS

3

scrapie prion (Prusiner, 1982,1984b;Diringer and Kimberlin, 1983; Rohwer, 1984). Early studies suggested a molecular weight of 60,000- 150,000 (Alper et al., 1966).While an alternate interpretation of that data has been proposed (Rohwer, 1984), there is no firm evidence to suggest that these molecular weight calculationsare incorrect. In fact, sucrose gradient sedimentation, molecular sieve chromatography, and membrane filtration studies all suggest that a significant portion of the infectious particles may be considerably smaller than the smallest known viruses (Prusiner, 1982). However, the propensity of the scrapie agent to aggregate makes molecular-weight determinations by each of these methods subject to artifact. To date, no experimental data have been accumulated which indicate that scrapie infectivity depends upon a nucleic acid within the particle. Attempts to inactivate scrapie prions with nucleases, ultraviolet irradiation at 254 nm, Zn2+ catalyzed hydrolysis, psoralen photoinactivation and chemical modification by hydroxylamine have all been negative even using preparations which contain one major protein as determined by amino acid sequencing (Bellinger et al., 1986). While these negative results do not establish the absence of a nucleic acid genome within the prion, they make this possibility seem likely. Attempts to identify a nucleic acid in purified prion preparations by silver staining have been unsuccessful to date (Gilles et al., 1986).

C. CLONING OF PrP cDNA

We assumed that a RNA encoding PrP 27-30 protein would most likely be present in scrapie-infected brain during the exponential phase of prion formation and that it would polyadenylated. Scrapie infectivity increases logarithmically between the tenth and fortieth day after intracerebral inoculation with lo7 ID5,, units of scrapie agent and reaches a plateau after 50 days (Oesch et al., 1985). Between 60 and 65 days after inoculation, the animals developed signs of progressive neurological dysfunction leading to death about 2 weeks later (Prusiner et al., 1982a). Poly(A)+ RNA was isolated from scrapie-infected hamster brain 35 days after inoculation, and a cDNA library was prepared essentially by the procedure of Okayama and Berg (1982; Oesch et al., 1985). A set of 32 icosameric oligonucleotides, (5')GG(T/C)TT(A/G)TTCCA(T/C)TG(A/ G)TT(A/G)TG, was synthesized, based on the reverse translation of a seven amino acid segment of PrP 27-30, (N)His-Asn-Gln-Trp-Asn-LysPro(C) (Prusiner et al., 1984). Screening of 150,000 colonies with the 5'32P-labeledprobe mixture yielded a positive clone from which the recombinant plasmid pHaPrPcDNA-1 was isolated (Oesch et al., 1985).

4

MICHAEL P. MCKINLEY AND STANLEY B. PRIJSINER

D. THESTRUCTURE OF PrP cDNA

The plasmid pHaPrPcDNA-1 contained an insert of about 2 kb. A provisional restriction map was established and the insert was sequenced by the Maxam and Gilbert (1977) method. All regions were sequenced on both strands, except for two short stretches within the 3’-noncoding region, and across the restriction sites that served as origin for the sequencing (Oesch et al., 1985).The heteropolymeric sequence of that insert was comprised of 1918 nucleotides and was preceded by 33 G and followed by 56 A residues. The major N-terminal amino acid sequence of PrP 27-30 described earlier (Prusiner et al., 1984) corresponds to the sequence encoded from nucleotides 236 to 280 (Oesch et al., 1985).An open-reading frame extended from nucleotide 1 to 730; the first inframe ATG start signal was at nucleotide 11 and the TGA stop codon was at position 731 (Fig. 1). Subsequently, a larger PrP cDNA clone of 2096 nucleotides in hgtlO was obtained (Basler et al., 1986). Sequencing of this insert, as well as a genomic PrP DNA clone, suggests that an additional 14 amino acids at the N-terminus of the prion protein are probably translated from the PrP mRNA to form an N-terminal signal peptide. The ATG codon at the beginning of the signal peptide meets all the requirements for an initiation site for translation in eukaryotes (Kozak, 1983). The DNA sequence surrounding the ATG codon at nucleotide position 11 (Fig. 1) does not meet the requirements for an initiation site. This ATG codon encoded the first Met residue in our first cDNA clone (Oesch et al., 1985).

E. PrP 27-30 Is ENCODED IN THE HAMSTER GENOME Hamster chromosomal DNA was cleaved with individual restriction enzymes, electrophoresed through an agarose gel, and analyzed by Southern blotting (Southern, 1975), using the radiolabeled cDNA insert from pHaPrP as probe. As shown in Fig. 2, both normal and scrapie-infected animals (75 days after inoculation) gave the same restriction pattern, namely, a single band of 3.1, 6.5, and 7.2 kb after cleavage with EcoRI, BamHI, and Bglll, respectively. Two bands, a strong one at 7.2 kb and a weaker one at 4.8 kb were generated from digestion with HindIII, an enzyme that cleaves within the cDNA clone. Southern analysis of mouse and human DNA with the hamster PrP cDNA probe revealed single bands of 2.3 and 15 kb, respectively, after cleavage with EcoRI. No additional bands were seen in the hamster DNA under the less stringent hybridiza-

-

BIOLOGY AND STRUCTURE OF SCRAPIE PRIONS

5

tion conditions used in the latter analysis. These results are compatible with a single PrP gene in the hamster genome. The detection of related sequences in mouse (Chesebro et al., 1985), human (Oesch et al., 1985), rat, sheep, and goat, as well as yeast, Drosophilia, and nematode DNA (Westaway and Prusiner, 1986) suggests that this gene may have been relatively conserved during evolution. Genomic DNA from hamsters was isolated and restricted with either EcoRI or Hind111 (Basler et al., 1986). Genomic libraries were then constructed by cloning the fragments in Xgt 10. The PrP DNA clones were selected using the radiolabeled PrP cDNA. Four nucleotides upstream (5’) from the ATG initiation site is a splice site. Since the longer PrP cDNA of 2096 nucleotides spans this splice site, we were able to identify and locate the first exon (Basler et al., 1986).

F. DETECTION OF PrP mRNA TRANSCRIPTS Poly(A)+ RNA was prepared from brains of uninfected and scrapieinfected hamsters at different times after inoculation, electrophoresed through an agarose gel, and analyzed by Northern blotting (Thomas, 1980)using the cDNA insert of pHaPrP (Oesch et al., 1985).A major band of about 2.1 kb (marker not shown) was found in all samples; there was no significant quantitative difference between the samples obtained at different times after infection or between infected and control animals (Fig. 3). The lower molecular-weight RNA species are not a reproducible feature of these analyses and are attributed to mild sample degradation. RNAs from seven organs of uninfected animals were analyzed by a similar procedure. PrP-related transcripts were also detected in heart, lung, pancreas, spleen, testes, and kidney We failed to detect a reproducible hybridization signal in liver RNA. Using a mouse PrP cDNA, other investigators obtained similar results with Northern blots of mouse and hamster brain RNA (Chesebro et al., 1985). Although PrP mRNA was unchanged throughout the course of scrapie infection, we have found that the transcription of the PrP gene is developmentally regulated (McKinleyet al., 1986~). During the first 8 days after birth, no PrP poly(A)+ mRNA was found in the neonatal hamster brain by standard Northern blot analysis (Fig. 4).By 8 days of age, PrP mRNA levels are barely detectable, and by 20 days these levels have reached a maximum. Apparently the levels of PrP mRNA remain constant throughout the adult life of the hamster.

20

40

80

60

CCT

TCG

GGC

CAT

GGC

GTG cci A T C TGC A C T EAT GTT GGC cic TGC AAG ~ A C CGG CCA A A G GGA GGC A A C ACT GCA ACC CCA iAc CCT P h e Val A l a MET T r p T h r Asp Val G l y L e u C y s L y s LYS A r g Pro L y e P r o G 1 y G l y T r p Asn T h r G l y C l y Scr Arg T y r Pro 1 10 20

~~~c TTT

120

100

CAG

CCT

160

140

EGG

GGC

GGC AGC cci GGA GGC AAC &T TIC CCA CAC GGT GGC ACA TGG CIA ccc GCT GCT TGG GCA CAG Ccc CAT G l y C l n G l y Scr P r o G l y C1y Asn Arg T y r P r o P r o G l n G l y G l y G l y T h r T r p G l y G l n P r o H i s G l y C l y G l y T r p G l y C l n P r o H i . 30 40 50

ccc

180

200

220

GGT

240

260

CAA

CCC

GCC

GGC TGG G G A C A G ccc CAT GGT GGT GGC GGT C A G C A T GCT CGT Ccc TGG GGT GCA GCT ACC CAC AAT CAC TCG G 1 y C l y G l y T r p G1y C l n P r o His G l y G l y G l y T r p G l y C l n Pro His G 1 r C l y G l y v r p C l y C l n C l y G 1 y C1r T h r Ria A m Cln T r p 60 70 80 A

GGT

TCG

2 80

320

300

340

CAC

GCC

CCA A A A ACC AAC ATC A A G ATG GCC GCC GCT CCT ccc GCA GCG GCC cic CTG CCG CTT GCT ccc TAC ATC I a n L y a Pro Scr L y a J P r o L y s T h r Ann MET L y e Ilia M E T l A l a C l y A 1 a A l a Ale Ala C1y A 1 a Val Val r2ly Clr L c u 0 1 7 C l y T y r l M E T 90 100 0 110

AAC

ccc

ACT

ACT

ccc ATG

AAC A ~ C

360

cn

CTG

CGG

400

380 ACC ACG

Leu G l y S c r Ale MET Scr A r g 120

cic

TAT

TAC

CGC

CCA GTG GAC

CAC

150

ACC ACC

AAC GCG

GAG A A C

Thr Thr T h r Thr Lye G l y Clu Asn

520

ACA

600

5-30

TTC ACG G A G Phc Thr Glu

180

620

GAG

ACC

TAT

CAC ATC AAC ATA ATC GAG Ccc CTC CTG CAC ATC T G ACC ~ ACC CAC GAG Tlfr Aap I l c L j r I l c HET G l u Arg Val V a l G l u G l n MET C y e T h r T h r G l n Tyr C l a 190 200

660

640

GAG

140

500

TAC A A C A ~ CCAG A A C AAC TTT GTG CAC GAT TCT GTC AAC ATC ACC ATC AAC CAC CAC CTC T y r Ann Aan C l n I a n Ann P h c Val His A s p C y a Val Aan I l e T h r I l c L y a C l n H i m T h r Val 160 170

560

540

ACC

C

130

LBO

G l n V a l Tyr T y r A r g P r o Val A s p G l n

ACC

440

ATG

460 CAI

420

CAT TTT CGC A A T CAC TGC CAC CAC CCC TIC TAC CCT C A A AAC ATC A A C ccc TAC CCT AAC P r o MET M E T F I E P h c C 1 j A s n A s p T r p Clu Aap Ars T j r T j r Ars C l u AanjMET Awn Arg Tyr P r o A r n

ccc ATG

700

680

CTC

TCC

TAC TAC G A T CCA AGA AGC ACC GCG GTC CTG TTC TCC icc CCT CCT cic ATC CTC ATT TCC TTT CTC ATC L y e G l u Ser G l n Ala T y r T y r A s p G l y Arg A r g Scr Scr Ala Val L e u P h c Scr Ser P r o P r o Val I l c L e u L e u I l c Scr P h a L e u I l e 210 220 230 AAC

TCC CAG

CCC

720

TTC

CTG ATG

740

CCA

GTG TGA AGG A A C P h c L e u MET Val C 1 y 240

CCT ccc

780

760 TGC

TTG TAC TTC CTC

CTT CTT GTG

CTC

TAG CCT

CCG

BOO GGA CGG

CTT ATC

CAC CGT

Acc TCT

820

AAT

TTT

840

GGT GTC

TGA GGT

900 ACC

TCA TTC

CTG

TTT

GTC

TGA ATT

GAG

TCC ATC

TAT TCG ATC CAG AGC

CTT

TCG

CTA GTC AGG GCT TTG TTT

TGT T A A

TAA

ACC

ATC CAT

AAT AGG

TGG TCT

CGA AGG

AGC

CTT

CTA CAG

TCC TTC

GTG

A A A CCT

TTC ATT TCC

CTG T A A A A A TGT GGT TCA

CCT CCT

ATT AGG TCA

AEA TGA

AAT

AGC

CCC

TAG G A A GCA

CTC CAT TCA

GAC ATA

ACC ACT

CTG

CAT

CCG AAG

TAC

1800 TTC

CAT

GAA

TAG

TTA

TGA A A A

GAC

ATC

AGC

ATA TAC

TTC

ATG

GAC TTC

CAT

TGA CCA

TTT

cAc

Ccc

A A A AGT TTA

TAA ATG TTT

CCT AAC

TCG

TAC

ACA GAG

TTA

TCC CCT

GAA

GGC GCT

TAT TTG AAT

CGG

ACG ACA

cci ccc

TCA

TCT CTG

AAA

AT

ATT

AAT TAC

CCT ACT

inc

ATG

1340

CCA

TTT

GGA GAT

TEG CGT

GGC TCT C A A

EAG CCA TCA TAA

TTC

TTC

ATT TCT GTC

AAT

CTA

A A A ATT

1520

ACA GAC

CTC

CTA

GGT

TTA AGT TGC

Tcc

GTG

1600 TAA GGC

AAA

TCC

CTT TGT

1680

TAT

ATA

A A G TCT

AAC

1700

GAT GTT TTC

TGT

CTT

TAC A A G

1760 CCG GTA

TGG

ATT

1500

CAI

ACT

AGA CCT TCT

ETT

CTG

ATT

CGG

1780 GTG GCC

CCT

AGC TTT

CCT TCA

1860 TCT GCA TGT

EAC ACA

1620

CAC

GCT CTA

AAT

1240

TGG ATT

ATG

AGT

1160

CTC CCT A ~ GA A A CAG

ACA C A A ACT

GCG TTG CTT

GCA

GCA GTG ACA

1320

1840

C A A AGT GGA

cic

CTG ACT A A G ~ C A ACG GGA A ~ AA A A CIA

AGC CTT GCT

CAG TAG

980

AAT ATA ACA

TAT CTC

1580 AGT TTG

GGG AAT GTA

1140

1660 CAA CKG

CCC

1400

1820 ATA TGA

TTT

TGA TGT

1740

GTA ATG CAC

TCT

1480

1720 ATG

CTA

CAG

1220

TAE

ACT A ~ CA A A CTC

A A G GGA G ~ GATG GTT

TGC CCT

AGG

ccc

AAG AAG T C ~ CTG TTT GGC

1640 TGA CCA

ATG TGA

1560

1620 TTT

CCA

1460

ACA

A A A GTC

CTE

ccT

GGC

1060

1300

1540

TGT

TAA ATA

1380

1440 TTG

GAG

1280

1360 ACA

AGT

1200 AGG CAT TCC

1260 GAA

TAA CAC

CTA GTG

960

ACE GGC

1120

1180

ATC

CAG CAC

TTG GCA

1040

1100

AAC

Ccc

1020

1080 CCT

CTA ATG

940

1000 GGC

ccc CAT AGG

920

ACA TGC

880

860

CTT CTC

TCA CAT

TTT

ACA TCC

1880 CTA TAT

TTC

TAA CTT TGC ~ T G TCC

1900 TTG

TTT

TGT CAT AT;

FIG. 1. Nucleotide sequence of the pHaPrP cDNA insert and the amino acid sequence deduced for PrP. The numbering of the nucleotides (above sequence) and amino acids (below sequence) starts at the first nucleotide and amino acid, respectively, following the string of Gs. The underlined amino acids correspond to the peptides found by amino acid sequencing. Amino acid sequence A corresponds to the N-terminal sequence of PrP 27-30 (Prusiner et al., 1984).Sequences B and C correspond to CNBr peptides (Oesch et al., 1985).

8

MICHAEL P. MCKINLEY AND STANLEY B. PRUSINER

FIG. 2. Southern transfer analysis of genomic sequences related to pHaPrP. (A) Hamster DNA samples (10 Fg) were digested with restriction endonucleases, electrophoresed through a 0.9% agarose gel, and transferred to a nitrocellulose filter and hybridized with SSP-labeledcDNA insert of pHaPrP as described in the text. Lanes 1, 3, 5, and 7, Normal hamster brain DNA; lanes 2, 4, 6, and 8, brain DNA obtained 75 days after inoculation with scrapie agent. Lanes I and 2, BamHI; lanes 3 and 4, BglII; lanes 5 and 6, HindlII; lanes 7 and 8, EcoRI. (B) Detection of pHaPrP-related sequences in normal mouse and human DNA. DNA samples were digested with EcoRI and processed as described above. Lane 9, Hamster DNA; lane 10, mouse DNA; lane 11, human placental DNA. Sizes are indicated in kilobases. The filter was hybridized under conditions of reduced stringency. (From Oesch et al., 1985.)

G. PRION PROTEINS I N NORMALA N D SCRAPIE-INFECTED HAMSTER BRAINS Homogenates of normal and scrapie-infected (72 days postinoculation) harnser brains were electrophoresed in SDS-polyacrylamide gels with or without prior digestion with proteinase K. The proteins were electrophoretically transferred to nitrocellulose membranes (Towbin et uZ., 1979; Burnette, 1981) and visualized by Western immunobloting with antiserum raised against either PrP 27-30 (Bendheim et al., 1984) o r a synthetic peptide (Gly-Gln-Gly-Gly-Gly-Thr-His-Asn-Gln-Trp-Asn-LysPro-Gly-Gly-Cp) corresponding to the N-terminal 13 amino acids of PrP 27-30 (Barry et al., 1986). As shown in Fig. 5, lanes 1 and 3, diffuse bands of about 33-35 kDa were found in both infected and normal samples designated PrP 33-35% and PrP 33-35', respectively (Oesch et al., 1985;

BIOLOGY AND STRUCTURE OF SCRAPIE PRIONS

9

FIG.3. Detection of pHaPrP-related transcripts in RNA from normal and scrapieinfected hamsters. (A) Northern blot analysis of brain RNA. Samples of brain poly(A)+ RNA (10 pg) were electrophoresed through a 1% agarose gel, transferred to nitrocellulose, and hybridized with "P-labeled cDNA insert of pHaPrP. Film was exposed for 1 day at - 70°C with an intensifying screen. Lane 1 contains poly(A)+ RNA from uninoculated hamsters 40 days old. Lanes 2-5 contain poly(A)+ RNA extracted from scrapie-infected hamsters 24 hr, 20 days, 40 days, and 60 days after inoculation, respectively.(B) Northern blot analysis of RNA from brain (lane 6), heart (lane 7), and lung (lane 8). Each sample contains 10 pg of poly(A)+ RNA isolated from uninfected animals. Film was exposed for 3 days at - 70°C. (C) Slot blot of total RNA (25 pg) from various organs of 40-day-old uninoculated hamsters. Nitrocellulose filters were hybridized with the PrP cDNA. Film was exposed for 5 days. Slot 1, Brain; 2, heart; 3, lung; 4, pancreas; 5, liver; 6, spleen; 7, testes; and 8, kidney. (From Oesch et al., 1985.)

10

MICHAEL P. MCKINLEY AND STANLEY B. PRUSINER

FIG. 4. Northern analysis of PrP mRNA during development of the hamster brain. Samples of brain poly(A)+ RNA (5 pg) were electrophoresed through a 1% agarose gel, transferred to nitrocellulose, and hybridized with SPP-labeledORF of the cDNA insert of pHaPrP (Oesch et aL, 1985). Film was exposed for 3 days at - 70°C with an intensifying screen. Lane 1 contains poly(A)+ RNA from uninfected hamsters at 1 day before birth. Lanes 2-7 contain poly(A)' RNA from uninfected hamsters at 2, 4, 6, 8, 10, and 20 days following birth. (From McKinley et al., 1986c.)

Barry et al., 1986). After treatment of normal samples with proteinase K, the diffuse 33-35 kDa band (PrP 33-35') disappeared (lane 4). However, following proteinase K digestion of the scrapie-infected sample, a diffuse band (PrP 27-30) appeared at the position corresponding to 27-30 kDa (lane 2). Since identical results were obtained with antibodies raised against the synthetic peptide, it is highly likely that at least portions of PrP 3335%,PrP 27-30, and PrP 33-35' contain common amino acid sequences (Barry et al., 1986).

BIOLOGY AND STRUCTURE OF SCRAPIE PRIONS

11

FIG. 5. Immunoblots of Sarkosyl extracts from control and scrapie-infected hamster brains. Proteins from brain homogenates were extracted with a 0.1 % Sarkosyland aliquots were electrophoresed into SDS- 12% polyacrylamide gels, transferred to nitrocellulose paper, immunoblotted with rabbit antisera, and visualized by treatment with horseradish peroxidase-conjugated goat anti-rabbit IgC. Extracts from scrapie-infected brain (lanes 1 and 2) and normal brain (lanes 3 and 4) are shown. Proteinase K-digested samples are shown in lanes 2 and 4. Western blots were performed with PrP 27-30 antisera (lanes denoted a) and affinity-purified PrP 27-30 antibodies (lanes denoted b). Signal intensities do not reflect relative concentrations of PrP proteins. (From Oesch et al., 1985.)

H. FAILURE TO DETECT PrP cDNA-RELATED NUCLEIC ACIDS IN PURIFIED PRIONS The availability of cloned PrP cDNA allowed us to test one possible model of the scrapie prion, namely, that it contains a PrP-related nucleic acid. Purified infectious fractions were denatured by boiling in 1% SDS

12

MICHAEL P. MCKINLEY AND STANLEY B. PRUSINER

and digested with proteinase K. These conditions are known to severely reduce infectivity (Bolton et al., 1984)and disperse rod-shaped aggregates of prions (Prusiner el al., 1983). Under these conditions, we would expect virus-like packaged nucleic acid to be released. After phenol extraction, nucleic acids in the aqueous phase were collected by ethanol precipitation in the presence of carrier yeast tRNA and immobilized on nitrocellulose filters (Kafatos et al., 19'79; Thomas, 1980). The filters were hybridized with radiolabeled PrP cDNA probe, washed, and autoradiographed (Fig. 6). Double-stranded (ds) PrP cDNA or total brain RNA were added to some of the samples (subsequent to

A B C D E

t

1

FIG.6. Attempts to detect PrP-related sequences in preparations of infectious prions. Preparations of infectious scrapie prions were denatured by boiling, incubated with proteinase K, phenol extracted, precipitated, and immobilized on nitrocellulose (slots A, B, D, and E) and hybridized with "P-labeled EDNA insert of pHaPrP. pHaPrP cDNA insert, 10 pg, and brain RNA, 10 pg, were added to the samples in slots A and D, respectively, prior to proteinase treatment. Slot C contains 10 pg brain RNA dissolved directly into loading buffer. (From Oesch et al., 1985.)

BIOLOGY AND STRUCTURE OF SCRAPIE PRIONS

13

boiling) as internal controls to monitor recovery throughout the extraction process. PrP cDNA (10 pg) or brain total RNA (10 pg) mixed into the proteinase K digest of prions (slots A and D) gave distinct hybridization signals. In contrast, prions alone (slots B and E) gave no detectable signal. We then estimated the number of PrP-related nucleic acid molecules which are present per infectious unit. Each “slot”in Fig. 6 received samples derived from IDs0 units or 105 amol of infectious particles. We can detect 10 pg of the double-stranded 2.0 kb PrP cDNA insert, equivalent to 7.6 amol. If the signal arising from the prions alone were equivalent to that observed for the DNA control, then there would be 7.6 amol of ds nucleic acid per 105 amol of infectious particles, i.e., 0.072 (7.6 + 105) nucleic acid genomes per infectious unit. In fact, prions alone give a far weaker hybridization signal than the DNA control. To provide quantitation of the signals in Fig. 6, we scanned autoradiographs with an LKB 2202 ultrascan laser densitometer. Linearity of the autoradiographic signals was confirmed using standards. Signals in slots B and E were less intense by a factor of at least 30 than the DNA control (slot A). We conclude that any prion protein-related nucleic acid must be present at concentration of -0.002 PrP dsDNA or -0.004 PrP single-stranded (ss) DNA molecules per infectious unit. The number of RNA molecules was also assessed relative to the DNA internal standard. We assumed that DNA and RNA samples were bound to the nitrocellulose filters with the same efficiency and that the bound molecules are able to participate in hybridization reactions to similar extents. In 50% formamide, RNA-DNA heteroduplexes are more stable than DNA-DNA hybrids (Casey and Davidson, 1977). Under these assumptions, a hypothetical PrP RNA will give a signal at least equivalent to that of a ssDNA molecule. Hence, there are -0.004 PrP RNA molecules per infectious unit. We conclude that a PrP-related nucleic acid which encodes PrP 27-30 is not a component of the infectious particle.

I. THEPRIONPROTEIN

Our results show that the cDNA insert cloned in pHaPrPcDNA-1 encodes PrP 27-30, the polypeptide isolated from scrapie-infected hamster brains. A search of the GenBank and protein sequence data bases (Genetic Sequence Data Bank, 1985; Protein Sequence Data Base, 1985) has not revealed any meaningful homologies with any protein of known sequence. The deduced sequence has an extremely hydrophobic C-terminus, and a stretch of hydrophobic amino acids near the amino-terminus of PrP 27-30. These hydrophobic domains are probably buried within

14

MICHAEL P. MGKINLEY AND STANLEY B. PRUSINER

cellular membranes, since recent studies have shown that the prion protein is an integral membrane protein which spans the membrane bilayer at least twice (Hay et al., 1986; Bazan et al., 1986). Multiple forms of scrapie prions have been attributed to their hydrophobicity (Prusiner et al., 1978c), and numerous studies have documented the association of scrapie infectivity with membranes (Hunter, 1979). A striking feature of the predicted amino acid sequence of the PrP protein is the Occurrence of repeated sequences between codons 22 and 81, where two small repeats of GG(N/S)RYPare followed by a longer set of five repeats of P(H/Q)GGG(-/T)WGQ. Whether these repeated sequences might play a role in mediating cellular processing or polymerization of the PrP protein remains to be determined; however, these sequences do not seem to be required for infectivity as they are not present in PrP 27-30. At the nucleotide level, corresponding tandem repeats are also largely conserved (Table 11). Interestingly, a 50 kDa keratin protein which polymerizes into intermediate filaments has eight repeats of the tetrapeptide -GGGX- within its N-terminal segment where -X- is a hydrophobic amino acid (Marchuk et al., 1984). In the case of the PrP protein, the amino acid at position -X- is a tryptophan, which is also hydrophobic, in four out of five repeated tetrapeptides. The difference between predicted and observed molecular weights of PrP 27-30 (19K or less and 27-30K3, respectively) appears to be due to TABLE I1 REPEATED SEQUENCES I N PrP cDNA" 68 (23)

GCC GLY

GGa GLY

AgC Ser

CGa ARC

TAC TRY

CCT PRO

85 (28)

101 (34)

GGa GLY

GGc GLY

AaC Asn

CGt ARC

TAC TRY

CCa PRO

118 (39)

119 (40)

CtC PRO

GGT GLY

GGc GLY

GGC GLY

aca thr

146 (49)

ccc

CAg gln CAT HIS

GGT GLY

GGT GLY

170 (57)

ccc PRO

CAT HIS

GGT GLY

194 (65)

ccc

CAT

PRO

HIS

218

ccc

(73)

PRO

CAT HIS

PRO

TGG TRP

GGg GLY

CAa GLN

145 (48)

GGC GLY

TGG TRP

GGa GLY

CAG GLN

169 (56)

GGT GLY

GGC GLY

TGG TRP

GGa GLY

CAG GLN

193 (64)

GGT GLY

GGT GLY

GGC GLY

TGG TRP

GGt GLY

CAG GLN

217 (72)

GGT GLY

GGT GLY

GGC GLY

TGG TRP

GGt GLY

CAa GLN

241 (80)

"Theconsensus sequences are in uppercase letters and the deviant residues in lowercase letters. (From Oesch et al., 1985.)

BIOLOGY AND STRUCTURE OF SCRAPIE PRIONS

15

glycosylation (Bolton et al., 1985). Chemical deglycosylation of PrP 27-30 with hydrogen flouride or trifluoromethane sulfonic acid has yielded a protein of M , -20,000 by SDS-polyacrylamide gel electrophoresis (Aebersold et al., 1986). There are two potential N-glycosylation sites of the type Asn-X-Thr at codons 170-172 and 186-188, respectively.

J. PRIONPROTEINS IN UNINFECTED AND SCRAPIE-INFECTED BRAINS It appears that the PrP mRNA level is similar in both normal and diseased brain; however, there is a higher content of PrP protein in diseased brain. In scrapie-infected brain, PrP proteins are present in a different conformation and/or state of aggregation, as evidenced by their partial resistance to proteinase K and their polymerization into amyloid rods and filaments (Prusiner et al., 1983; McKinley et al., 1983b; Bendheim et al., 1984; DeArmond et al., 1985). Several explanations could account for the different properties of the PrP protein in normal and scrapie-infected brain. First, the PrP protein from scrapie-infected animals may have a different primary structure from the related normal protein due to point mutations or sequence rearrangements at the DNA or RNA level. In view of the Southern and Northern analyses, which showed no differences between PrP-related nucleic acids in normal and infected tissues, it is unlikely that any alteration would be substantial. Only the comparison of mRNA sequences at the single codon level from normal and scrapie-infected brain tissues will clarify this possibility; small changes in sequence can be of great significance (Ingram, 1957; Tabin et al., 1982; Capon et al., 1983; Varmus, 1984). Our finding that the entire open-reading frame of PrP is contained within a single exon reduces the likelihood that the mRNA encoding PrP 3335' is different from that encoding PrP 33-35% (Basler et al., 1986). Our cDNA clones have been constructed from mRNA isolated from scrapieinfected brains. The sequences from these clones are identical to the exons of our genomic clones constructed from the DNA of normal control hamsters. Indeed, it is possible that most of the PrP mRNA molecules encode PrP 33-35', while only a few encode PrP 33-35'". Whether or not sequencing a large number of PrP cDNA clones constructed from scrapie mRNA will elucidate differences between the scrapie and cellular forms of PrP remains to be established. A second explanation for the differences between the cellular and scrapie forms of the prion proteins is that they both have the same primary sequence but then undergo some posttranslational change which is different in scrapie-infected than in normal brain. Such posttranslational

16

MICHAEL P. MCKINLEY AND STANLEY B. PRUSINER

modifications could, for example, involve glycosylation, phosphorylation, acylation, or proteolytic cleavage. Third, the modified behavior of the PrP protein arises from some other component which interacts or fails to interact with it in infected cells.

111. Structure of Scmpie Prionr

Purification of scrapie prions to near homogeneity has shown that the infectious particles contain one major protein, PrP 27-30. Prion proteins polymerize into long filamentous structures in the brain and are found in rod-shaped structures in extracts from infected brain. These prion protein aggregates are ultrastructurally and histochemically identical to amyloid. Extensive purification of PrP 27-30 was required before convincing evidence linking the rods and prion infectivity could be obtained. Raising antibodies against PrP resulted in the demonstration that prion filaments in tissue and prion rods in extracts are composed of PrP. By negative staining, the individual rods measure 10-20 nm in diameter and 100-200 nm in length. In highly purified preparations, rods are usually found in large clusters or clumps, but individual rods can be dispersed by brief sonication. Prolonged sonication resulted in progressive fragmentation of the rods into a variety of small elongated particles and spheres; however, this profound alteration in ultrastructural morphology was not accompanied by a change in prion infectivity. Proteolytic enzyme digestion, alkali treatment, and detergent extraction did not yield an identifiable unit particle associated with infectivity. T h e lack of correlation between ultrastructural morphology of the rods and prion titers is consistent with the hypothesis that the rods are aggregates of prions. Clearly, the disruption of prion rods into amorphous globules, smaller rods, and spheres with no detectable loss of infectivity establishes that elongated structures, the size and shape of prion rods, or filaments are not required for infectivity. A. ULTRASTRUCTURAL STUDIES OF PRION-INFECTED TISSUES T h e complete molecular structure of the scrapie agent remains elusive, despite many intensive studies during the last two decades. Recent experimental results continue to support the hypothesis that the scrapie agent is different from both viruses and viroids (Alper et al., 1978; Diener et al., 1982; Prusiner, 1982). Numerous ultrastructural investigations have attempted to describe a unique particle either in situ or in fractions derived

BIOLOGY AND STRUCTURE OF SCRAPIE PRIONS

17

from scrapie-infected brains (Tables III-VII). Most of the early studies were focused on the description of structures found in thin-section preparations from scrapie-infected rodent and sheep brains (Table 111). A heterogeneous collection of elongated structures was described in these tissues, including tubules (David-Ferreira et al., 1968), rods (Field et al., 1967; David-Ferreira et al., 1968; Field and Narang, 1972; Narang, 1974b), fibrils (David-Ferreira et al., 1968), filaments (Field and Raine, 1966; DeArmond et al., 1985), and vacuoles-vesicles (Pattison and Smith, 1963; Field and Raine, 1964; Lampert et al., 1971; Bignami and Parry, 1972b). Concurrent investigations identified spherical and virus-like particles in both thin sections and extracts from infected brains (Table IV). Mouse brain preparations revealed enlarged synaptic terminals with spherical particles having a 32-36 nm diameter (David-Ferreiraet al., 1968).Smaller osmiophilic particles (23-nm diameter) were observed in arrays within postsynaptic processes in murine brain only (Baringer and Prusiner, 1978; Baringer et al., 1979). In the mid-l970s, many investigators attempted to correlate infectivity with a unique structure. These studies began to focus on samples derived from partially purified extracts of scrapie-infected tissue (Table V). The earliest studies on these preparations suggested an association of infectivity with fractions that lacked cellular membranes, although no specific structure could be identified (Prusiner et al., 1978c, 1979; Malone et al., 1979). A particle measuring 30-60 nm in diameter was identified in negatively stained preparations of murine brain and spleen fractions (Siakotos et al., 1979).Some investigators reported finding particles measuring 14 nm in diameter in preparations of scrapie mouse brains (Cho and Greig, 1975; Cho, 1976). Subsequent studies suggested that these particles were probably ferritin molecules (Cho et al., 1977).

B. PRIONRODSAND FILAMENTS In 1982, rod-shaped particles measuring 25 nm in diameter (rotary shadow) and 100-200 nm in length were reported in fractions which contained predominantly one protein (PrP 27-30) and which were partially purified from scrapie-infected hamster brains (Fig. 7) (Prusiner et al., 1982a) (Table VI). The rods were suggested to be either aggregates of the infectious prions or pathologic products of infection. The former consideration was shown to be correct when, in 1983, extensively purified fractions containing PrP 27-30, high levels of infectivity (ID50units/ ml), and rods were found (Prusiner et al., 1983).The morphologic analysis using uranyl formate to negatively stain the rods showed that their diameter varied between 10 and 20 nm with a mean of 15 nm. No unit structure could be discerned so that no direct correlation between the

TABLE 111 ELONGATEDSTRUCTURES I N SCRAPIE-INFECTEDTISSUES Structures reported

Size

Preparation Goat brain Mouse brain Mouse brain thin section Mouse brain thin section

Nerve cell vacuoles Small vesicles in nerve cells

z

Helical filaments with rod-like core in mitochondria Longitudinal tubules in nerve fibers of cerebellum

12-nm-wide filaments, 5-nm helical twists

Sheep brain thin section

30-75 nm diameter

Rat brain thin sections

Amyloid bodies and deposits

Rat brain thin sections

Ependymal cells with amorphous masses

Mouse and rat brain thin sections

Rod-like inclusions of parallel lamellae Enlarged cell processes with particles, rods, tubules, or fibrils

6 nm wide 32-36 nm diameter

Rat brain thin sections Mouse brain thin sections

Conclusions

Reference

Astroglial proliferation Hypertrophy of astroglial cells Vacuolation of “ground substance” Nerve cell vesicles, myelin degeneration, astroglial hypertrophy Nerve cell “spaces” and dense bodies in degenerating mitochondria Tubules common in older normal animals and in younger, scrapie-infected animals Congo red staining and concentric lamination indicate bodies are amyloid Reduction in ciliary movement of ependymal cells associated with degenerative changes Enlarged microglial cells have inclusions Biochemical and biophysical data suggest infectious scrapie particle is -2.5 nm

Hadlow (1961) Chandler (1961) Pattison and Smith (1963) Field and Raine (1964) Field and Raine (1966) Raine and Field (1967) Field and Narang (1972) Chandler (1967)

Field et al. (1967) David-Ferreira et al. (1968)

Vesicles and tubules

35 nm diameter

Elongated particles

60 by 20 nm

Dense bodies and granular vesicles

Vesicles = 60-150 nm diameter

Rod- or cucumbershaped bodies

20 X 60 nm

Rat b r d n thin section

Membrane-limited particles, tubules, and vesicles

Particles = 75 x 15-26 nm; tubules = 20 nm diameter; vesicles = 25-75 nm 35 nm

Sheep brain thin section

Elongated particles, vesicular bodies

60 X 20 nm, 100-110 nm diameter

Mouse and rat brain thin section

Rod-shaped particles

20 nm wide

Mouse brain thin section

Filaments

U p to 1500 nm long, 10-20 nm wide

Hamster brain thin sections

Vesiculotubular structures (rod or tubular)

Mouse brain thin sections Rat brain thin sections Sheep brain thin section

Mouse brain thin section

Arrays of vesicles and tubules in dendrites Granular inclusion bodies in neurons Degenerating nerve processes with a variety of structures, some seen in normal-aged animals Inclusion bodies in neurons; also tubules and nuclear rod-shaped inclusions Small particles enclosed in membrane in cell processes and nerve cell terminals

Lampert et al. (1971) Narang et al. (1972) Bignami and Parry (1972b)

Particles with electron-lucent center (associated with postsynaptic processes) not found in spleen or cell culture Stained with ruthenium red and lanthanum nitrate; may have nucleic acid core surrounded by polysaccharide coat Ruthenium red-lanthanum nitrate stain suggest polysaccharide coat aPrP 27-30 used with PAP immunocytochemistry to demonstrate reactive filaments in amyloid plaques in infected brains

Lamar et al. (1974)

Field and Narang (1972) Narang (1973)

Narang (1974~)

Narang (1974b)

DeArmond et al. (1985)

SPHERICAL

Structures reported

TABLE I V VIRWS-LIKE PARTICLES I N S C R A P I E - I N F E C T E D TISSUES

Size

Preparation

Conclusions

Reference

65- to 85-nm-diameter particles; 48-nm-diameter particles; and 15-nmdiameter filaments 35-nm particles

Human CJD brain thin sections

Filaments may be related to larger particles

Vernon et al. (1970)

Sheep brain thin sections

Neuronal vacuolation with cytoplasmic projections containing membranebound particle accumulations Neuronal vacuoles with distinctive appearances; some occasional viruslike particles Random and crystalline arrays in postsynaptic processes

Bignami and Parry (1971)

N ._

0

Virion-like particles and nucleoprotein-type filaments Round particles

Membrane-bound vacuoles and virus-like particles

Vesicles = 1-2 pm diameter; particles = 35-50 nm diameter

Sheep brain thin section

Osmiophilic particles

23 nm

Mouse brain thin section

Bignami and Parry (1972a) Baringer and Prusiner (1978)

Spherical osmiophilic particles

23 nm diameter

Mouse brain thin section

Spherical particles

30-60 nm

Mouse brain and spleen extracts

Two types of virus-like

75

particles (nonmembrane bound and membrane bound) Virus-like particles

14 nm

Mouse brain CsCl gradient

Virus-like particles

14 nm

Virus-like particles

14 nm

Hamster brain extract Mouse brain extract

X

15-26 nm

Sheep brain thin section

';?

Tubular and spherical particles

Mouse brain thin section

Particles in dilated postsynaptic process in murine, not hamster brain Singles and aggregates identified by ruthenium red staining Particles found in neurons enclosed in limiting membrane Particles in gradient fractions-infectivity not associated with particles Particles may be causative agent Particles in control and scrapie-affected brainpossibly ferritin Tubular appearance resolves into spherical particles by specimen tilting

Baringer et al. (1979) Siakotos et al. (1979) Narang (1974a)

Cho and Greig (1975)

Cho (1976) Cho et al. (1977) Baringer et al. (1981)

22

MICHAEL P. MCKINLEY AND STANLEY B. PRUSINER

TABLE V No DEFINABLE STRUCTURES IN SCRAPIE TISSUE EXTRACTS Structures reported

Size

Preparation Rat, mouse, and chimpanzee brain thin sections Subcellular fractions from hamster brain

Amorphous material

-

Mouse spleen extract

Nodefinable structure Nodefinable structure

-

Mouse spleen extract

-

Hamster brain extract-high-speed pellet Hamster brain extract

Nodefinable structure

-

Nodefinable structure No particles

-

-

Gel eluate of hamster brain homogenate Percoll density gradient

Conclusions

Reference

Status spongiosis results from fluid accumulation and vacuolated neurons Agent is component of endomembrane system Infectious particle not associated with organized membrane structure Agent not associated with membranes Agent not associated with membranes

Adornato and Lampert (1971)

Primarily amorphous structures; some fluffy 25- to 30-nm particles Particle has no definable unit structure N o association of particles with infectivity

Semancik et al. (1976) Prusiner et al. (1978~)

Prusiner et al. (1979) Malone et al. (1979) Prusiner et al. (1980b)

Prusiner et al. (1980d) Prusiner et al. (1980a)

rods and scrapie prion infectivity could be enumerated. This lack of a unit structure is another feature which distinguishes prions from viruses at the ultrastructural level. On the other hand, the heterogeneous appearance of the rods as well as their tendency to aggregate and cluster was thought to be reminiscent of amyloid. In histochemical studies, the prion rods were shown to be amyloid by their staining with Congo red dye and their green-gold birefringence under polarized light (Prusiner et al., 1983). Amino acid-sequencing studies on both electrophoretically purified PrP 27-30 and sucrose gradient-purified rods were completed in 1984 and revealed that the predominant protein in both samples had identical amino-termini (Prusiner et al., 1984).Subsequently,immunoelectron microscopy studies demonstrated that PrP 27-30 was a component of the rods (Barry et al., 1985).Recent investigations have shown that these elongated structures are artifacts of the preparative procedure and are not required for transmission of scrapie infectivity (McKinley et al., 1986a; Meyer et al., 1986).

BIOLOGY AND STRUCTURE OF SCRAPIE PRIONS

23

TABLE VI PROPERTIES OF PRION RODSAND FILAMENTS" ~~

Prion rods Dimensions Diameter Length Fine structure

10-20 nm 100-200 nm Variable structure due to extensive aggregation, indistinguishable from purified amyloids Purified fractions of prions from scrapie and CJD brain

Occurrence

PrP 33-35", PrP 27-30 Congophilic and show greengold birefringence Composed of PrP 27-30 which have been shown to correlate with titer; no unit structure for correlation with titer; can be sonically disrupted into infectious spheres Form upon detergent extraction of membranes containing PrP 33-35%

Composition Properties Relationship to infectivity

Origin

~~~~~

~

~

Prion filaments 10-20 nm 280-1500 nm Uniform diameter, indistinguishable from amyloid

Extracellular collections form amyloid plaques in scrapie, Creutzfeldt-Jakob disease (CJD), kuru, and GSS brain PrP 33-35"? Congophilic and show greengold birefringence Unknown

Form as PrP 33-35" released from scrapie-infected neurons?

~

"Properties of prion rods and filaments compiled from the following references: Prusiner et al. (1982a, 1983, 1984), McKinley et al. (1983c, 1986a).DeArmond et al. (1985), Meyer et al. (1986), Kitamoto et al. (1986).

C. SCRAPIE-ASSOCIATED FIBRILS In 1981, long, abnormal fibrils called "scrapie-associatedfibrils" ( S A F ) were identified in unpurified extracts of scrapie-infectedbrain. No mention of any possible relationship of SAF to the particle causing scrapie was made. SAF were distinguished from all other filamentous structures by their characteristic and well-defined morphology (Merz et al., 1981).Published electron micrographs of the scrapie-associated fibrils showed helically wound structures measuring 300-800 nm in length. While the chemical composition of these fibrils is unknown (Merz et al., 1981, 1983a),their ultrastructural morphology was well defined. Two types of scrapie-associated fibrils were observed: (1) those composed of two helically wound subfilaments measuring 12-16 nm in diameter and having a periodicity of either 40-60 nm or 80-110 nm and (2) those composed of four helically wound subfilaments measuring 27-34 nm in diameter and having a periodicity of 100-120 nm. At their points of greatest diameter, scrapie-

24

MICHAEL P. MCKINLEY AND STANLEY B. PRUSINER

FIG. 7. Ultrastructure of rotary-shadowed prion rods found in sucrose gradients. Large clusters of rods, which individually measure 100-200 by 25 nm, were detected in gradient fractions with a corresponding high scrapie prion titer. Bar, 100 nm. (From Prusiner, 1984a.)

associated fibrils were found to have spaces of either 2-4 nm or 3-4 nm between the subfilaments. On the basis of these ultrastructural features, the fibrils were stated to be different from intermediate filaments and amyloids (Merz et al., 1981, 1983a). In 1983, SAF were suggested to be either pathologic products of infection or the infectious particle (Merz et al., 1983a). These fibrils were said to correlate with infectivity, since they were found late during infection in both spleens and brains of scrapie-infected rodents. At this time, the brain titer was reaching a maximum, but the spleen titer was maximal long before SAF were found. No convincing data have been published which demonstrate a correlation between scrapie agent titers and brain SAF concentration. More important, there is clearly a lack of correlation between titers and spleen SAF concentration (Merz et al., 1984a). Scra-

BIOLOGY AND STRUCTURE OF SCRAPIE PRIONS

25

TABLE VII OF SCRAPIE-ASSOCIATED FIBRILS(SAF)" PROPERTIES Dimensions Diameter Length Fine structure

Occurrence Composition Properties Relationship to infectivity Origin

12-16 nm (Type I, two subfilaments) 27-34 nm (Type 11, four subfilaments) 100- 1000 nm Helically wound subfilaments with periodicities of 40-60 nm or 80110 nm (Type 1) or 100-120 nm (Type 11); subfilaments separated by spaces of 2-4 nm (Type I) or 3-4 nm (Type 11); readily distinguished from amyloid Extracts of synaptosomes from scrapie, CJD, kuru, and GSS brain; not seen in purified fractions or infected tissues Unknown Do not stain with Congo red dye Found in brain and spleen extracts late in disease; no correlation with spleen titer Unknown

Properties of scrapie-associated fibrils compiled from the following references: Merz et al. (1981, 1983a,b, 1984a,b).

pie-associated fibrils (Table VII) are distinct ultrastructurally from the prion rods (Table VI). Prion rods do not exhibit (1) regular substructure, (2)periodicity, or (3) spaces between adjacent filaments that are characteristic of scrapie-associatedfibrils. Equally important are the observations that the prion rods possess no regular or unit structure and that they are indistinguishable from many purified amyloids. In contrast, SAF have a regular identifiable structure, and they have been reported repeatedly to be different ultrastructurally from amyloids. Attempts to stain scrapieassociated fibrilswith Congo red dye have yielded negative results (Merz et al., 1981, 1983a,b);even a positive result would have been impossible to interpret because of impurities in the extracts. Some investigators (Diringer et al., 1983a, b) have used a purification protocol similar to that first developed by us (Prusiner et al., 1982a) to isolate prions. The rods in their purified fractions were identical to those described by us (Prusiner et al., 1982a; McKinley et al., 1983c),but they chose to call them scrapie-associated fibrils, in spite of the differences between SAF and prion rods described above. This is not only misleading, inappropriate, and incorrect, but it also causes confusion. Furthermore, this lack of precise terminology distorts the path of discovery. Recently, some investigators have claimed that scrapie-associatedfibrils as originally described are amyloid (Sommerville, 1985) and are found in purified fractions of prions (Gajdusek, 1985); neither assertion is true. A few of these investigatorssuggested that scrapie-associatedfibrils are filamentous animal viruses (Merz et al., 1984b), while others have claimed that they are pathologic products of infection (Multhaup et al., 1985). It will be

26

MICHAEL P. MCKINLEY A N D STANLEY B. PRUSINER

interesting to learn whether scrapie-associated fibrils are composed of PrP molecules or other proteins. Purification and characterization of the fibrils are needed before an understanding of their role, if any, in scrapie infection can be ascertained. To date, scrapie-associated fibrils as originally described have been seen neither in purified preparations of prions nor in prion amyloid plaques. In contrast, prion amyloid filaments, identical to the rods in morphology except for length, are found within the plaques (DeArmond et al., 1985); like the rods, these filaments are composed of prion proteins. Confusion about scrapie-associated fibrils has been compounded by the term SAF protein (Hilmert and Diringer, 1984; Multhaup et al., 1985). Using purification protocols similar to those developed by us (Prusiner et al., 1982a, 1983), some investigators have isolated a protease-resistant protein which they claim to have a nominal M , of 26,000 (Diringer et al., 1983b; Hilmert and Diringer, 1984).This appears to be the same protein which we first discovered and labeled PrP 27-30 (Bolton et al., 1982; Prusiner et al., 1982a, 1983; McKinley el al., 1983b,c). Like PrP 27-30 (Bolton et al., 1985), SAF protein is a glycoprotein (Multhaup et al., 1985) which exhibits niicroheterogeneity on SDS-polyacrylamide gels even though the M , designation of 26,000 does not reflect this apparent size heterogeneity. While the SAF protein and PrP 27-30 have the same properties and N-terminal amino acid sequence, they differ with respect to the molecular weights of their polypeptide chains. SAF protein was extracted with formic acid prior to hydrogen flouride deglycosylation; the resulting polypeptide had a M , of 7000 (Multhaup et al., 1985). Presumably, the formic acid extraction hydrolyzed the protein, since the cleavage of specific peptide bonds by formic acid is well documented (Schultz, 1967).Our studies show that the polypeptide backbone of PrP 27-30 has a M , of -20,000 by both cDNA sequencing (Oesch et al., 1985) and hydrogen flouride deglycosylation (Aebersold et al., 1986). SAF protein is considered to be a pathologic product of infection and unrelated to the "virus" causing scrapie (Multhaup et al., 1985). Many lines of evidence indicate that PrP 27-30 is the major macromoleculeof the purified scrapie agent, which is clearly not a virus. We believe that term scrapie-associated fibrils (SAF)should be reserved for fibrillar structures which fulfill the morphologic criteria that were orginally used to define SAF (Merz et al., 1981)and distinguish them from amyloids and other filaments (Table VII). It is of interest that scrapieassociated fibrils have been reported in extracts of brains from patients dying of Creutzfeldt-Jakob disease (CJD), kuru, and GSS (Merz et al., 1983b).

BIOLOGY AND STRUCTURE OF SCRAPIE PRIONS

27

D. PRIONRODSIN PURIFIED FRACTIONS Homogenates of scrapie-infected brains were extracted with detergents, sedimented, fractionated with ammonium sulfate, and subjected to electrophoresis through agarose gels containing sodium dodecyl sarThe prions were removed from cosinate (Sarkosyl)(Prusiner et al., 1980~). the gel by electroelution or by pulverizing the gel. Bioassays indicated that these eluates contained approximately lo7ID50 unitdm1 of scrapie infectivity. Generally, a 100-fold purification of prions with respect to cellular proteins was achieved by this procedure. The agarose gel eluates were evaluated by SDS-polyacrylamide gel electrophoresis and by electron microscopy. More than 50 protein bands were visualized by radioiodination and silver staining of the gels (Fig. 8). In contrast to the numerous protein species, ultrastructural exami-

FIG. 8. Detection of PrP 27-30 in partially purified fractions. Partially purified fraction was prepared from scrapieinfected hamster brain by Sarkosyl gel electrophoresis. Aliquots were concentrated 10-fold, radioiodinated with the Bolton-Hunter reagent, and electrophoresed into a 15%polyacrylarnide gel. The autoradiograph was exposed for 72 hr. PrP 27-30 is at the arrowhead.

28

MICHAEL P. MCKINLEY AND STANLEY B. PRUSINER

nation of concentrated gel eluates showed some globular and amorphous structures along with a few flattened rods (Fig. 9). The rotary-shadowed rods had a constant diameter of 25 nm and a variable length ranging between <50 and >300 nm (Table VIII). Most of the rods were 100-200 nm in length. By negative staining with uranyl formate, the diameter of the rods appeared to vary between 10 and 20 nm (Table IX). This variation in diameter may be due to a twisting of some rods as they dry onto the grid, since it is not a regular feature of all rods. Aggregates containing up to 15 rods were also found in these preparations. Rod-shaped particles were not observed in samples purified by gel electrophoresis from the brains of uninoculated controls or of hamsters inoculated with extracts of normal brain. The large number of protein bands seen in samples prepared by the agarose gel electrophoresis protocol demanded that we extend our purification scheme for prions. In order to accomplish this, it was first necessary to scale-up our protocol so that we would have sufficient material. Because the ultracentrifugation and electrophoretic steps were limited with respect to the volumes that could be processed, we sought procedures that could replace these steps. A protocol was developed which utilized

FIG. 9. Electron micrographs of fractions prepared by Sarkosyl gel electrophoresis. Scrapie agent titers -lo’* ID,, unitsiml. (A) Rotary-shadowed preparation of single, rodlike structures. (B) Negatively stained preparation of single, rod-like structures. (C) Rotary-shadowed preparation of aggregates of rod-like structures and contaminants. (D) Negatively stained preparation of aggregates of rod-like structures. Bar, 100 nni. (From McKinley et al., 1986a.)

29

BIOLOGY AND STRUCTURE OF SCRAPIE PRIONS

TABLE VIII DIMENSIONS OF ROTARY-SHADOWED PRIONRODS PREPARED BY SODIUM DODECYL SARCOSINATE ELECTROPHORESIS Dimensions Length (nm) 25-50 51-75 76- 100 101- 125 126-150 151-175 176-200 201-225 226-250 251-275 276-300 >301

Width (nrn) 24-26

Number of observations

Percentage of total

3 28 29 23 23 18 12 6 4 1 1 2 150

2 19 19 15 15 12 8 4 3 <1

150 150

<1 1 -

>99

100 100

TABLE IX DIMENSIONS OF NEGATIVELY STAINED PRIONRODS I N SUCROSE GRADIENT FRACTIONS Dimensions Length (nrn) 51-75 76- 100 101- 125 126- 150 151-1 75 176-200 201-225 226-250 25 1-275

Width (nm) >10 11-13 14-16 17-19 20-22 23-25

Number of observations

Percentage of total

8 23 48 52 33 26 6 3 1 200

11.5 24 26 16.5 13 3 1.5 0.5 100

20 24 60

75 20 1 200

4

10 12

30 37.5 10 0.5 100

30

MICHAEL P. MCKINLEY AND STANLEY B. PRUSINER

detergent extraction, polyethylene glycol (PEG) precipitation, ammonium sulfate fractionation, and sucrose gradient centrifugation in a reorienting vertical rotor (Prusiner et al., 1982a). Fractions from the bottom of the discontinuous sucrose gradients were found to be enriched for scrapie prions 1000-fold with respect to cellular proteins. SDS-polyacrylamide gel electrophoresis and electron microscopy were used to evaluate the purity of the fractions. A major protein, later designated PrP 27-30, was found by radioiodination (Fig. 10). Electron microscopy showed numerous rods (Fig. 11) identical to those observed in the less-purified fractions of scrapie prions isolated by agarose gel electrophoresis as described above. While the rod-shaped particles were found in all scrapie preparations, their uniqueness was questioned since some cylindrical structures of similar size and shape were observed in control fractions prepared in a vertical tube rotor (Fig. 12). Although similar to the prion rods, these cylindrical

-

FIG. 10. '251-Labeledsucrose gradient fractions analyzed by NaDodS0,-polyacrylamide gel electrophoresis. Aliquots were radiolabeled with '2'I-labeled Bolton-Hunter reagent. T h e samples were boiled for 2 rnin in 1.25% NaDodSO, and 1.25% p-mercaptoethanol prior to electrophoresis in 5-2076 linear gradient polyacrylamide gels. From the left: lanes 1 and 2, scrapie fraction 2; lane 3, scrapie fraction 3; lane 4, control fraction 2; lane 5, control fraction 3. Autoradiographic exposure was for 3 hr. The position of PrP 27-30 is denoted by an arrow. (From Prusiner el al., 1982a.)

FIG. 11. Ultrastructure of prion rods isolated from sucrose gradients. Electron micrographs of negatively stained scrapie prion-containing fractions from discontinuous sucrose gradients. Scrapie agent titer was > unitshl. (A) Aggregate of rods present in gradient fraction. (B ) Smaller aggregate of rods in same fraction. Bars, 100 nm.

32

MICHAEL P. MCKINLEY AND STANLEY B. PRUSINER

FIG. 12. Electron micrographs of rotary-shadowed normal brain fractions from discontinuous sucrose gradient fractions. (A) Arrays of cylindrical, rod-like structures. (B) Single, rod-like structure present in extensively purified normal brain extract. Bars, 100 nm. (From McKinley et al., 1986a.)

structures purified from control preparations displayed morphological characteristics that distinguished them from the prion rods (Prusiner et al., 1982a). As described below, control fractions prepared from zonal rotor sucrose gradients did not contain any cylindrical or rod-shaped structures.

E. COPURIFICATION OF RODS, PrP 27-30,

AND

INFECTIVITY

Examination of different fractions from vertical rotor sucrose gradients revealed several interesting relationships (Prusiner et d.,1982a). First, total protein had a bimodal distribution within the gradient. The majority of scrapie infectivity was associated with the small peak of protein near the bottom of the gradient. Numerous aggregates of amorphous material and flattened rod-like structures were detected in these highly purified fractions (Fig. 13). Second, protein and infectivity had minimum Ievels in the middle of the gradient. Ultrastructural examination of the middle of the gradients revealed a few rods but no aggregates (Fig. 13). Third, protein and infectivity reached a maximum at the top of the gradient, where most of the protein in the entire gradient was found. At the top of the gradient, numerous particles without any distinct shape or size were observed (Fig. 13). The significanceof the numerous rods found at the bottom of the sucrose gradients containing scrapie prions was unclear, since titers of top and bottom gradient fractions were similar. These observations raised the possibility that rods represented either an aggregated form of the infectious agent or a pathologic product of infection. While our conclusions about the significance of the rods remained

FIG. 13. Electron micrographs of selected fractions from a triton X-100/NaDodS04 discontinuous sucrose gradient. Samples were applied to grids coated with polylysine and then stained with uranyl formate. (A) Fraction 14, top of gradient. (B) Fraction 8, middle of gradient. (C) Fraction 2, bottom of gradient. Bars, 100 nm. Analysis of partially purified sucrose gradient fractions by radioiodination is described as follows. (D) Autoradiogram of Bolton-Hunter labeled fractions after gel electrophoresis. Position of PrP 27-30 is denoted by arrow.

34

MICHAEL

P. MCKINLEY AND STANLEY B.

PRUSINER

tentative, studies on the protein components of gradient fractions revealed that a unique protein (PrP 27-30) was present (Bolton et al., 1982; Prusiner et al., 1982a). The distribution of this protein paralleled that of scrapie infectivity with the bimodal distribution of each being coincident with the other. Many lines of evidence now indicate that this unique protein is a structural component of the scrapie prion. At that time, further studies were needed to establish a relationship among PrP 27-30, the rods, and scrapie infectivity.

F. RODSARE AGGREGATES OF PRIONS

A large-scale purification protocol employing zonal rotor centrifugation was developed to facilitate characterization of scrapie prions (Prusiner et al., 1983). Extensively purified fractions in these gradients had titers > IDs0 unitdm1 and specific infectivities between 3,000- and 10,000fold greater than homogenates. Greater than 90% of prion infectivity was found in a single peak near the bottom of the gradient, where one major protein (PrP 27-30) was also found (Prusiner et al., 1983). Other studies had convincingly demonstrated that PrP 27-30 was both a structural component of the prion and required for prion infectivity (McKinley et al., 1983b; Bolton et al., 1984). Electron microscopy of the gradient fractions from the infectivity peak showed numerous clusters of rod-shaped particles. Individual rods had dimensions similar to those described above (Fig. 14). Analogous fractions from uninoculated animals or animals inoculated with normal brain extracts failed to show any cylindrical structures. Virtually no contaminants could be detected in the scrapie fractions by either electron microscopy of SDS get electrophoresis (Prusiner et al., 1983). In fact, the presence of a single protein (PrP 27-30) in these fractions has been confirmed by Nterminal amino acid sequencing (Prusiner et al., 1984). Some rods in scrapie fractions appeared to be composed of globular subunits. Presumably, the subunits of the rods are PrP 27-30 oligomers, since PrP 27-30 is the only major protein in these fractions. These investigations demonstrated that rods are composed of PrP 27-30 and represent an aggregated form of the infectious scrapie prion. However, since the rods could not be readily dispersed into identifiable unit structures, no correlation between the number of rods and infectivity titers was possible (McKinley et al., 1986a). The arrays of rods of varying size and shape observed by electron microscopy provide ultrastructural evidence for the multiple molecular forms of prions previously reported in sucrose gradient sedimentation (Prusiner et al., 1978b) and purification studies (Prusiner et al., 1978c, 1980~).Additionally, size studies using ionizing radiation (Alper et al.,

FIG. 14. (A) Electron micrograph of extensively purified fraction of prions from discontinuous gradients. Preparation containing a large cluster of rods was negatively stained with uranyl formate. Bar, 100 nm. (B) SDS-polyacrylamide gel electrophoresis of extensively purified fraction containing prions from sucrose gradient. Position of PrP 27-30 is denoted by arrow. (From McKinley et al., 1986a.)

36

MICHAEL P. MCKINLEY AND STANLEY B. PRUSINER

1966), molecular sieve chromatography (Prusiner, 1982), and rate-zonal sedimentation (Prusiner, 1982) all indicate that the smallest or monomeric form of the prion may have a molecular weight as low as 50,000. Thus, the monomeric form of the infectious prion might contain as few as two PrP 27-30 molecules, while the rods may contain as many as lo" PrP 2730 molecules.

6. PRIONRODSFORMDURING DETERGENT OF MEMBRANES SOLUBIL~ZATION Homogenates of scrapie-infected brain (10% w/v) were prepared and processed as described by Prusiner et al. (1982). No elongated, rod-shaped structures could be found in these homogenates (Meyer et al., 1986). However, rods were seen in the homogenate after the addition of a partially purified sample of comparable infectivity in which rods had been previously observed (Meyer el al., 1986). This demonstrated that the elongated structures could be detected by our procedures if they were present in homogenates. Further support for the contention that elongated structures are not required for infectivity emanates from experiments in which scrapie-infected hamster brains were homogenized and then subjected to a subcellular fractionation to produce microsomes. This fraction had high infectivity but no rods or any other elongated structures (Fig. 15) (Meyer et al., 1986). When microsomes from scrapie-infected hamster brains were extracted with detergent, rods were formed (Fig. 15C and D). No rods were formed when control, uninfected brain microsomes were extracted (Fig. 15B). No difference in rod formation was detected when the anionic detergent, Sarkosyl, was compared to the nonionic detergent, octylglucoside. Under conditions of rod formation, PrP 33-35' was solubilized, while PrP 33-35& polymerized into the rods. These studies demonstrated the amphipathic characteristics of PrP 33-35%.

IV. Chemical Chamcteristics of Rods

A. ROD MORPHOLOGY AND PRION INFECTIVITY Do NOT CORRELATE The biochemical characteristics and ultrastructural morphology of a single scrapie p i o n or infectious particle are unkown. While the prion rods seem to be associated with infectivity, we attempted to identify an ultrastructural entity that is obligatory for infectivity. Our results are consistent with an earlier hypothesis, suggesting that the rods may

FIG. 15. Detergent solubilization of scrapie-infected brain microsomes produces prion rods. (A) Prion rods added to scrapie-infected microsomes. (B) Sarkosyl at a final concentration of 2% (w/v) added to normal microsomes; no rod-shaped structures were found. (C and D) Sarkosyl added to scrapie microsomes produced prion rods. All specimens were negatively stained with uranyl formate. Bars, 100 nm. (From Meyer et al., 1986.)

38

MICHAEL P. MCKINLEY AND STANLEY B. PRUSINER

represent an aggregated form of scrapie prions (Prusiner et al., 1982a, 1983). Earlier studies suggested that there is a rough correlation between the concentration of rods and prion titers. However, accurate particle counts were not possible because the unit structure of the prion is unknown, and conditions to disaggregate rod clusters into uniform particles had not been defined. Because of these problems, the relationship of the rods to scrapie prion infectivity was determined indirectly Having established the association among prions, PrP 27-30, and rods, we sought to identify the smallest infectious form of the prion by disrupting prion rods using various chemical treatments. Our attempts to disrupt clusters of rods and generate single structures using nondenaturing detergents such as Sarkosyl and Triton X-100 were unsuccessful (Table X). However, studies using high concentrations of the denaturing detergent SDS, which had previously been shown to diminish scrapie infectivity, revealed that, in combination with heat, the rods were destroyed. Aggregates of prion rods were heated between 25 and 100°C for 10-30 min alone or in the presence of SDS. No change in titer or rod structrure occurred upon exposure to heat. Similarly,at detergent concentrations of 2% SDS and temperatures of 65°C or less, no change in prion infectivity or rod morphology could be detected. Fractions exposed to 5% SDS at 37°C contained rods with smooth surfaces (Fig. 16A). Rods in samples heated to < 100°C in 10% SDS were disrupted into filamentous structures <5 nm in diameter (Fig. 16B). Exposure of purified preparations to SDS at 100°C resulted in the complete disruption of rods and reduction of titer by > lo2. A marked change in pH had contrasting effects on rod morphology TABLE X FURTHER ATTEMPTS 'ro DISAGCKEGATE PRIONSA N D PKESERVE INFECTIVITY' Treatment Sulfobetaine 3-1 4 Sodium dodecyl sarcosinate Triton X-100 Diethylpyrocarbonate Formamide Guanidinium hydrochloride Heat (100°C for 3 min) pH 3.5 pH 10 Proteinase K'

Concentration

Rod morphology

No change N o change No change 1% N o change 20 mM N o change 50% None present 6M Some unchanged No change I N HCI Some alteration 1 N NaOH 100-500 pg/ml No change 10% 10%

Prion infectivity No change N o change N o change Decreased by NDb Decreased by Decreased by No change Decreased by Decreased by

los

> lo3 10'

lo2 lo3

~~~

"Prion rods were exposed to the individual treatments for 30 min at room temperature and then prepared for electron microscopy. 'ND, Not determined. 'Proteinase K digestions were performed for 30 h r at 37°C.

BIOLOGY AND STRUCTURE OF SCRAPIE PRIONS

39

FIG. 16. Disruption of prion rods by chemical treatments. (A) Effect of 5% SDS at 37°C for 30 min on extensively purified preparations containing prion rods. (B) Effect of 10% SDS at 85°C for 30 min on extensively purified preparations of prion rods. (C) Alteration of prion rod structure by exposure to pH 10, 30 min, 15°C.(D)Effect of proteinase K (100 pg/ml for 30 hr at 37°C) on structure of prion rods. Bars, 100 nm. (From McKinley et al., 1986a.)

40

MICHAEL P. MCKINLEY AND STANLEY B. PRUSINER

and prion titer. Reduction of sample pH to 3 by addition of 1 N HC1 did not alter the ultrastructure of the rods. Earlier studies had demonstrated that prion infectivity was unchanged at pH 3.5 (Prusiner, 1984a). However, the ultrastructure of prion rods was markedly altered at pH 10 (Fig. ISC) (Table X). While the general rod-like organization persisted, the structure of the individual rods was altered into a series of globular units. The globular appearance became quite pronounced when the pH was raised to 11. The relationship between infectivity and the prion rods was also investigated by subjecting purified fractions to protease digestion. Aliquots were held at 37°C in the presence of 100 pg/ml of proteinase K for 0-30 hr. Under these conditions, infectivity was decreased by a factor of > lo3 after 30 hr of digestion; however, no change in rod morphology or number could be discerned (Fig. 16D) (Table X). As observed in earlier studies (McKinley et al., 1983b), the change in concentrations of PrP 27-30 paralleled that of prion titers during proteolytic digestion. Why the rods do not fall apart under these conditions is unclear. Presumably, intermolecular forces favoring rod formation are sufficiently strong to maintain the structure of the rod even though the major protein component has been hydrolyzed to smaller polypeptides. One interpretation of these results is that they may reflect an internal molecule such as a nucleic acid which serves as skeleton for PrP 27-30 polymerization. Further studies are required to determine the mechanisms by which prion rods are formed and maintained. Similarly, exposure of the rods to 20 mM diethylpyrocarbonate did not alter the morphology, but reduced the titer by a factor of lo3(McKinley el al., 1981) (Table X).

B. ADDITIONALEVIDENCE THAT RODSARE AGGREGATES OF

PrP 27-30

Large clusters of rod-shaped particles are a constant feature of highly purified fractions prepared from scrapie-infected hamster brains (Prusiner el al., 1982a, 1983). Additionally, these fractions have a high ID50 units of priondml) and one major concentration of infectivity (protein (PrP 27-30) (Prusiner et al., 1983). Radiolabeling and SDS-polyacrylamide gel electrophoresis demonstrated that these fractions had a high degree of purity, and the rods were composed of PrP 27-30 molecules. The rods were apparently a form of the prion, since earlier studies had shown that PrP 27-30 was required for and inseparable from infectivity (McKinleyet al., 1983b).Previous studies using less purified fractions could not demonstrate whether the rods were a pathologic product of infection or an aggregate of the prion (Prusiner et al., 1982b).Subsequent to these studies,others faced the same dilemma because their preparations

BIOLOGY AND STRUCTURE OF SCRAPIE PRIONS

41

lacked sufficient purity due to protein contaminants (Diringer et al., 1983a). In fact, the numerous arrays of rod-shaped structures of varying size and shape identified by electron microscopy provided ultrastructural evidence for the multiple molecular forms of prions previously reported in sedimentation studies (Prusiner et al., 1977, 1978b,c). Antisera produced against gel-purified PrP 27-30 (Bendheim et al., 1984) provided us with an additional method to test whether the rodshaped particles found in prion preparations were composed of PrP 2730. Immunoelectron microscopy studies using colloidal gold demonstrated that gold particles were associated with prion rod aggregates (Fig. 17). These gold particles identified the locations of the binding of PrP antibodies to PrP 27-30. The number of gold particles decorating the prion rods was roughly proportional to the dilution of the antiserum. Due to grid incubations in blocking buffer proteins, the fine structural details of the prion rods were slightly masked (Barry et al., 1985; DeArmond et al., 1985). Binding of affinity-purified PrP antibodies to aggregates of prion rods was not a result of nonspecific association of immunoglobulin with large proteinaceous aggregates (Fig. 18). When tobacco mosaic virus (TMV), a heterologous structure similar in size and shape to prion rods, was mixed with prion preparations and analyzed using PrP 27-30 antisera, the colloidal gold was associated only with the rods. Conversely, the use of rabbit T M V antisera resulted in the association of colloidal gold with T M V only. Additionally, brief sonication of rod aggregates prior to immunoelectron microscopy produced small rod fragments and spherical particles, both of which associated with colloidal gold. Thus, the specificity of PrP 2730 antibodies for scrapie prion rods demonstrated that PrP 27-30 is a molecular component of these purified rods (Barry et al., 1985). Furthermore, the prion rods were also decorated with the colloidal gold when an antiserum raised against a synthetic peptide based on the amino acid sequence of PrP 27-30 was used as the primary antiserum (Barry et al., 1986). C. PURIFIED PRIONRODS The ultrastructure of prion rods is indistinguishable from many purified amyloids (Prusiner et al., 1983).Histochemical studies with Congo red dye have extended this analogy in purified preparations of prions as well as in scrapie-infected brain, where amyloid plaques have been shown to stain with antibodies to PrP 27-30 (Bendheim et al., 1984). Purified preparations of prions were stained with Congo red dye to further investigate observed ultrastructural similaritiesbetween prion rods and purified amyloid. By bright-field microscopy, numerous amorphous structures mea-

FIG. 17. Immunoelectron microscopy of aggregated prion rods. Prion rod aggregates are shown (A) before incubation, (B) after incubation in dilution buffers only, (C) after incubation in a 1/1000 dilution of immune serum from rabbit number 1, and (D) after incubation in a 1/ 5000 dilution of immune serum from rabbit number 2. Binding of rabbit Ig was determined after incubation with goat anti-rabbit IgGcolloidal gold diluted 1/60. Bars, 100 nm. (From Barry et al., 1985.)

FIG.18. Immunoelectron microscopy of sonicated prion rods and tobacco mosaic virus (TMV). Sonicated prion rods and TMV are shown (A) before incubation, (B) after incubation in dilution buffers only, (C) at 1/1000 dilution of immune serum, (D) at 1/5000 dilution of immune serum, and (E) at 1/1000 dilution of rabbit TMV antiserum. Binding of rabbit I g was determined after incubation with goat anti-rabbit IgG-colloidal gold diluted 1/60. Bars, 100 nm.

44

MICHAEL P. MCKINLEY AND STANLEY B. PRUSINER

suring 1-20 pm were observed (Prusiner et al., 1983). They exhibited a red color, indicating that they had bound the dye. Examination of these structures with crossed polarizers demonstrated a green color and birefringence (Fig. 19). A variety of green birefringent structures was observed. It is generally accepted that naturally occurring mammalian protein polymers exhibiting both a rod-like or fibrillary appearance by electron microscopy and green birefringence after Congo red staining should be classified as amyloid (Glenner, 1980). The green birefringence following Congo red dye binding to amyloid proteins has been attributed to domains within the protein having a high degree of beta structure. The predicted sequence for PrP 33-35 exhibits a few regions which have potential for beta structure (Bazan et al., 1986). Whether these domains within PrP 27-30 with potential for beta structure are sites for Congo red dye binding remains to be established.

D. PRIONFILAMENTS FORMPLAQUES Recent immunocytochernical studies using PrP 27-30 antisera have identified extracellular filaments composed of prion proteins within amyloid plaques of scrapie-infected hamster brain (DeArmond et al., 1985) (Fig. 20). These filaments measure approximately 16 nm in diameter and

FIG. 19. Light micrograph of extensively purified fractions of prions stained with Congo red dye and viewed under polarized light ( X 200).

BIOLOGY AND STRUCTURE OF SCRAPIE PRIONS

45

FIG. 20. Electron micrograph of a subependymal scrapie prion plaque. (A) The plaque consists of a loose aggregate of filaments in dilated extracellular space immediately beneath the ependyma (E). Astrocytes (Ac) form the other borders. Bar, 1 pm. (B) The filaments of the plaque are haphazardly organized and of variable length. Spherical electron-dense bodies and other irregular particles are interspersed among the filaments. Bar, 100 nrn. (From DeArmond el al., 1985.)

46

MICHAEL P. MCKINLEY AND STANLEY B. PRUSINER

up to 1500 nm in length. These findings raised the possibility that prionlike molecules might play a causative role in the pathogenesis of nontransmissible disorders such as Alzheimer’s disease (Prusiner, 198413). Amyloid proteins are prevalent in Alzheimer’s disease, but for many decades these proteins have been considered a consequence rather than a possible cause of the disease. Antibodies to PrP 27-30 did not react with normal filaments and tubules in brain tissue. The prion filaments have a relatively uniform diameter, rarely show narrowings, and possess all the morphologic features of amyloid. Except for their length, the prion filaments appear to be identical ultrastructurally with the rods which are found in purified fractions of prions (DeArmond et al., 1985; McKinley et al., 1986a).

V. Polymorphic Forms of Prions

Our studies suggested a general correlation between the concentration of rods and prion titers. However, we have been unable to make accurate particle counts because the unit structure of the prion is unknown. Both chemical and mechanical disruption of rod clusters failed to generate a population of uniform particles which could be quantitatively correlated with scrapie prion titers.

A. PRIONSBOUNDTO MEMBRANES Several experimental approaches indicate that the prion rods are not the smallest infectious unit. First, no elongated structures similar to prion rods could be identified in 10% homogenates of scrapie-infected hamster brains. Second, microsornal fractions contained no rods associated with prion infectivity. Third, detergent extraction of microsomal membranes isolated from scrapie-infected hamster brains was accompanied by the formation of prion rods. Thus, the prion rods in our purified fractions result from the detergent extractions used during purification.

B. SONICATION OF PRIONRODS Sonic disruption studies also indicate that the minimal infectious unit is smaller than a single rod. Homogenates and extensively purified gradient fractions disrupted by sonication for increasing periods of time

47

BIOLOGY AND STRUCTURE OF SCRAPIE PRIONS

TABLE XI DIMENSIONS OF SONICATED PRIONRODSAND SPHERES' Dimensions Spheres Diameter (nm) <10 11-13 14-16 17-19 20-22 23-25 26-28 >29 Rods Length (nm) <25 26-50 51-75 76- 100 101-125 >126 Width (nm) <10 11-13 14-16 17-19 20-22 >23

Number of observations

4 4 7 15 12 6 2 0 50

Percentage of total

8 8 14 30 24 12 4 0 100

-

1 23 45 19.5 9.5 2

2 46 90 39 19 4 200

100

22 44 56 40 28 10 200

22 28 20 14 5 100

11

Prion rods were ethanol precipitated from sucrose gradient fractions, resuspended in 20 mM Tris-OAc, pH 7.4, sonicated for 8, 12, or 16 min, and then negatively stained.

showed no change in infectivity (Table XI). Aliquots of the sonicated gradient fractions were examined by electron microscopy, and a marked change in the appearance of the prion rods was found (Fig. 21). Most of the rods were reduced to less than 70 nm in length (Table XI). Only 4% of the rods were less than 70 nm long prior to sonication, while nearly 70% were shorter than 70 nm after sonication. Since there was -20-fold decrease in structures greater than 70 nm as a result of sonic fragmentation, a change in titer should have been detected if the larger rod structure were required for infectivity. This was not observed; thus, we

FIG.21. Sonic fragmentation of prion rods in extensively purified gradient fractions. Electron micrographs of negatively stained samples which were sonicated for (A) 0.25 rnin, (B) 2 min, and (C) 4 min. Sonication for 0.25 min disrupts large rod aggregates into numerous smaller clusters. Extended sonication disrupted clusters and produced large numbers of fragments and spherical particles. Bars, 100 nm.

BIOLOGY AND STRUCTURE OF SCRAPIE PRIONS

49

conclude that the unit infectious particle is smaller than rods measuring 150 nm in length. Although other investigators claim a 10- to 20-fold increase in the titer of homogenates after sonication (Rohwer and Gajdusek, 1980), we observed no significant change in the titer of purified fractions after prolonged sonication.

C. GENERATION OF PRIONSPHERES In addition to short rods, many spherical particles measuring -20 nm in diameter were formed by the sonic disruption of the longer rods found in highly purified sucrose gradient fractions. These sonicated fractions were sedimented into continuous sucrose gradients resulting in a distribution of particles ranging from small spheres at the top of the gradient (-8 nm in diameter) to relatively undisrupted rods at the bottom (Table XI) (Fig. 22). Prion infectivity was found in each fraction. Analysis of fractions by 1251-labelingand Western blotting demonstrated the presence of PrP 27-30 as the primary protein component of the spheres. Additionally, the relationship between protein concentration and prion titer remained constant throughout the gradient, indicating that the specific infectivity associated with the spheres was comparable to that associated with the rods. These observations provide further evidence that the prion rods are aggregates of the infectious particle causing scrapie and that prions exist in a variety of forms (Table XII). The generation of polymorphic forms of prions appears to be dependent upon the method of sample preparation. Scrapie prion infectivity was unchanged following prolonged sonic disruption of the rods. If the rods contained a genomic nucleic acid molecule like the filamentous bacteriophage, then we would expect a reduction in titer upon fragmentation, since a single break in the genome renders the virion uninfectious. The structure and infectivity of bacteriophage MI3 were both markedly altered by sonic disruption. This filamentous virus was quickly broken into small fragments after exposure to sonic disruption for less than 15 sec (Fig. 23). In contrast to the lack of infectivity change seen in scrapie prions following extensive sonication, the infectivity of the M13 virus was reduced by a factor of >lo” upon brief sonication (data not shown). These studies provide further evidence that prion rods are not virions, since there is no recognizable correlation between ultrastructure and infectivity. Thus, we have not yet been able to describe the morphologic characteristics of the prion unit particle.

FIG. 22. Sucrose gradient separation of spheres from sonic disruption studies. Electron micrographs of negatively stained samples sonicated for 8 min, then sedimented into a 10-50s continuous sucrose gradient. (A) Fraction 6 containing predominately 8nm spheres. (B) Fraction 12 with pleornorphic-shaped particles 50 x 40 nm. (C) Fraction 20 consisted of rods with a slightly swollen appearance.

51

BIOLOGY AND STRUCTURE OF SCRAPIE PRIONS

TABLE XI1 POLYMORPHIC FORMSOF PRIONS~ Structure

Size

Preparation

No definable structure

100-200 nm length x 10-20nm diameter

Scrapie hamster and mouse brain homogenates Scrapie hamster brain purified fractions

50- 150 nm length X 8-16 nm diameter

CJD murine brain purified fractions Scrapie hamster brain thin sections

Spheres

Up to 1500 nm length by 10-20 nm diameter 9-20 nm diameter

Membrane vesicles

Micrometers in diameter

Rods

Filaments

Sonicated hamster brain extract purified on sucrose gradient Microsomal preparations from scrapieinfected hamster brain

Conclusion Particle has no unit structure Rods copurify with infectivity and PrP 27-30; IEM confirms PrP 27-30 is component of rods CJD prion proteins polymerize into rods PrP 27-30 antibodies identify filaments in amyloid plaques Elongated structures not required for infectivity Infectivity associated with membranes

Polymorphic forms of prions compiled from the following references: Semancik et al. (1976), Prusiner et al. (1978c, 1980a,b,d, 1982a,b, 1983), McKinley et al. (1983c, 1986a,b), Barry et al. (1985), Bendheim et al. (1985), Bockman et al. (1985), DeArmond et al. (1985), Meyer et al. (1986).

VI. Conclusions

While electron microscopic studies have not described the unit structure of the prion, they have been useful in demonstrating the amyloid nature of prion aggregates and the polymorphic forms of this unusual infectious particle. Once it was recognized that rod-shaped particles in purified preparations of prions are morphologically indistinguishable from many purified amyloids, subsequent studies showed that prion proteins aggregate to form filaments within amyloid plaques of scrapie-, CJD-, kuru-, and GSS-infected brains in animals and humans (Kitamoto et al., 1986). A small proportion of the PrP 33-35* protein in scrapie-infected hamster brains is found within prion amyloid plaques; the remaining PrP 33-35" is integrated into the cellular membranes, presumably neuronal.

FIG. 23. Sonic fragmentation of M 13 filamentous bacteriophage. (A) Negatively stained, unsonicated M 13 bacteriophage. (B) Negatively stained fragments of M13 after 30 sec sonication. Bars, 100 nm.

BIOLOGY AND STRUCTURE OF SCRAPIE PRIONS

53

Upon detergent extraction of the membranes, the PrP 33-35" is released and aggregates into prion amyloid rods. Besides the membrane and rod forms of the prion, ultrastructural studies have recently demonstrated a third form of the prion, spheres. The spheres were generated during sonication of the rods and separated from shortened rods by sucrose gradient centrifugation. We do not believe that the spheres are the smallest form of the prion. Thus, it is doubtful that the monomeric form of the prion can be defined by electron microscopy, unless it can be crystallized into a form where the infectious unit structure can be identified. Acknowledgments

The authors gratefully acknowledge the collaborative studies with Drs. L. E. Hood, C. Weissmann, R. Williams, T. 0. Diener, D. T. Kingsbury, and their colleagues. The contributions of Drs. David Westaway, Ronald Barry, and Michael Scott to these studies are acknowledged with pleasure. We also thank Ms. Darlene Groth and Ms. Monika Walchli, as well as Mr. Michael Braunfeld for expert technical assistance. The authors thank Ms. Lorraine Gallagher and Ms. Margaret Canevari for excellent editorial assistance. T h e studies described in this review were supported by research grants from the National Institutes of Health (NS22786, AG02132, and NS 14069), as well as by gifts from R. J. Reynolds Industries and The Sherman Fairchild Foundation.

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DIFFERENT KINDS OF ACETYLCHOLINE RELEASE FROM THE MOTOR NERVE By S. Thesleff Department of Pharmacology

University of Lund

5-22362 Lund, Sweden

Studies of transmitter release at the neuromuscular junction have revealed the presence of several distinct types of acetylcholine (ACh) secretion. The purpose of this presentation is to describe these processes and to discuss underlying release mechanisms and the possible physiological significance of the various kinds of transmitter action. It is not intended as a review coveringall aspects of the field. Excellent recent reviews already exist for most parts of the subject and reference will be made to them for further details. It is instead a rather personal presentation and interpretation of a complex area of research in which I have, to a small extent, been involved. Transmitter release from the motor nerve may be divided into those involving intermittent, quantal, or nonquantal release of ACh and those characterized by a continuous leakage of ACh. Figure 1 illustrates the three forms of ACh release (1-111) which will be considered. Intermittent secretion of ACh involves either a Ca2+-sensitive(I) or a Ca2+-insensitive(11) type of transmitter release process. The former characterizes phasic, nerve impulse evoked or spontaneous quantal ACh release and the latter the spontaneous intermittent, nonquantal secretion of ACh giving rise postsynaptically to so-called giant and slow-risingminiature end-plate potentials (Thesleff and Molgb, 1983).Molecular leakage of ACh is a continuous process originating not only from the presynaptic nerve (111) but also from the postsynaptic muscle cell.

I. Intermittent Secretions of ACh

A. CALCIUM-SENSITIVE QUANTAL RELEASE OF ACh (I) This is the mechanism responsible for neuromuscular transmission, i.e., the chemical transfer of a nerve impulse to the muscle fiber (Fig. 1, I). 59 INTERNATIONAL REVIEW OF NEUROBIOLOGY, VOL. 28

Copyright 6 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.

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Ca*+

FIG. 1. The diagram illustrates the three kinds of ACh secretion from the motor nerve (1-111) that will be considered in this presentation. I is a quantal, Ca2+-sensitive secretion of ACh, presumably originating from synaptic vesicles (SV) which discharge their content of ACh at active zones. I 1 is an intermittent, calcium-insensitive, but possibly cyclic AMP-sensitive, discharge of ACh from areas outside of the active zones, and 111 depicts a continuous, ATPase-dependent efflux of ACh from the terminal. The figure also illustrates the ionic mechanism responsible for nerve impulse evoked transmitter release, i.e., the presence of Na+ and K' channels in the axon and only Ca2+ and K + channels in the terminal. For further details, see text.

As elegantly shown 33 years ago, intracellular electrical recording at the synapse or end-plate region of a muscle fiber reveals the presence of spontaneous, small, intermittent electric potential changes of 0.5- 1 mV amplitude, rising rapidly within 1 msec and declining exponentially with a total duration of about 2 msec (Fig. 2B). These potentials are similar, but much smaller, than nerve impulse evoked end-plate potentials (epps) and were therefore called miniature end-plate potentials or mepps (Fatt and Katz, 1952; del Castillo and Katz, 1954). Subsequently Katz and coworkers in a series of classical papers that were also excellently reviewed (Katz, 1966, 1969)demonstrated the correspondence between mepps and the quantal components constituting the evoked epp. Furthermore, del Castillo and Katz (1955) postulated that each unit package of ACh, which when released produces a mepp, is preformed within a synaptic vesicle in the nerve terminal. The synaptic vesicle was supposed to accumulate ACh actively from its site of synthesis in the axoplasm. According to this socalled vesicular hypothesis for quantal ACh release, Ca2+inside the nerve

61

ACETYLCHOLINE RELEASE FROM THE MOTOR NERVE

terminal membrane is essential for the process which allows a transient fusion of terminal and synaptic vesicle membranes and thereby the release of a quantal package of ACh. Furthermore, it was suggested that this process was rapidly reversible so that a vesicle, having discharged its content, would quickly detach itself from the terminal membrane and start to reaccumulate the transmitter from the axoplasm (Katz, 1969, p. 15). Katz and Miledi (1965) demonstrated the Ca2+ dependency of this type of quantal ACh release. As amply supported by subsequent studies (see reviews by Llinas and Heuser, 1977; Silinsky, 1985) it is evident that the described spontaneous intermittent quantal secretion of ACh giving rise to the mepp is a Ca2+-sensitiveform of transmitter secretion. DepoIarization of the terminal part of the motor axon by the action potential increases the Ca2 conductance of the nerve terminal, allowing Ca2 ions to diffuse along their electrochemical gradient into the terminal and thereby activating ACh discharge. In the squid giant synapse, a linear proportionality is observed between Ca2+entry and the amount of transmitter released by this process (Llinh et al., 1981). Since the epp is the sum of a number of synchronous mepps evoked by the influx of Ca2+ during the nerve terminal action potential, one may call the process an electrically controlled Ca*+-sensitiveform of quantal ACh release. The vesicular hypothesis for ACh release has been critically examined in a number of electrophysiological,morphological, and biochemical studies of various types of cholinergic synapses, e.g., brain synaptosomes. electric organs of fish, and motor nerves from different species. Some of the results provide direct support for the hypothesis, such as the finding that synaptic vesicles actively accumulate and contain ACh (Whittaker et al., 1964; Israel et al., 1968; Whittaker et al., 1972) in amounts corresponding to that giving rise to a spontaneous mepp or a maximum of about 10,000 molecules of ACh (Kufller and Yoshikami, 1975; Fletcher and Forrester, 1975). Morphological studies have revealed depletion of synaptic vesicles followingstimulation of transmitter release to exhaustion and also evidence of vesicle fusion with axolemma (Peper et al., 1974: Heuser, 1977; Ceccarelli et al., 1979a,b; Heuser et al., 1974, 1979; PCcot-Dechavassine, 1982). By the use of extracellular marker molecules such as horseradish peroxidase, it has been possible to visualize what apparently constitutes the retrieval of vesicle membrane from the axolemma, i.e., vesicle reformation by an endocytic process (Heuser and Reese, 1973; Heuser, 1976; Ceccarelli and Hurlbut, 1980; Meldolesi and Ceccarelli, 1981). The membrane potential of the nerve terminal fails to influence the size of each quantum of ACh released (del Castillo and Katz, 1954), which fits with vesicular discharge, but argues against the possibility that quanta of ACh +

+

62

S.

THESLEFF

could be released from the cytoplasm along its electrochemical gradient (gated release of cytoplasmic ACh). Furthermore, no outward current which could correspond to ACh has been observed in mouse motor terminals. T h e expected ACh current would be 1.5 X lo-’‘ Nquantum for a 1-msec release period. This gives a current of about 1 nA in the presence of K +-channel blockers and is therefore larger than the currents carried by other ions (A. Mallart, personal communication). The shrinking or swelling of the nerve terminal should at least momentarily alter the ACh concentration in the cytoplasm and therefore also change the efflux through a gated channel. However, mepp amplitudes are not appreciably affected by massive alterations of the osmotic pressure of the extracellular solution that supports vesicular discharge of ACh (Van der Kloot, 1978). On the other hand, results have been published that at least superficially, are not readily reconciled with the vesicular hypothesis. For instance, it has repeatedly been demonstrated that newly synthesized ACh is released preferentially by nerve stimulation (Dunant et al., 1972; review by Israel et al., 1979). Since ACh is synthesized in the axoplasm and only subsequently accumulated in vesicles, this observation has been taken to indicate that stimulation preferentially releases ACh from the cytoplasm and that vesicular ACh is not primarily involved in the release process. Furthermore, it has been shown that neither the vesicular content of ACh nor the mean number of vesicles was modified by nerve stimulation at physiological frequencies (Dunant et al., 1972, 1974; Lynch, 1982). In contrast, the cytoplasmic-free ACh was depleted during such stimulation and renewed with precursor. Similarly, experiments using an inhibitor of precursor uptake (hemicholinium) or a “false”precursor have shown that the transmitter released by nerve stimulation is not stored in independent quanta but is continuously mixed with the cytoplasmic pool of transmitter (Elmqvist and Quastel, 1965; Large and Rang, 1978; Collier et al., 1979). Reports have also appeared questioning the validity of synaptic vesicle recycling on the ground that the labeling by extracellular horseradish peroxidase is too low to be consistent with synaptic vesicles undergoing continuous exo- and endocytosis along the presynaptic plasma membrane (Meshul and Pappas, 1984). On the basis of such conflicting evidence, there are proponents and opponents to the vesicular hypothesis for transmitter release. Their views have been excellently presented and summarized in several recent reviews (proponents: Zimmermann, 1979a; Meldolesi and Ceccarelli, 1981; Whittaker, 1984; opponents: Israel et al., 1979; Tauc, 1982). Opponents consider that the nerve terminal membrane contains a hypothetical structure, possibly located at “active”zones, that bind ACh to saturation and which, upon activation by Ca2+,releases ACh in a none-

ACETYLCHOLINE RELEASE FROM THE MOTOR NERVE

63

lectrogenic manner into the extracellular fluid. Isreal et al. (1979) have coined the term “operator” and Tauc (1982) the term “vesigate”for such a release mechanism. I believe that the contradiction between the vesicular and the cytoplasm-gate hypothesis for transmitter release, as discusssed by Zimmermann (1979b),Israel et al. (1979),and Whittaker (1984),could be overcome by certain assumptions. For instance, a portion of the synaptic vesicles have properties different from the rest, i.e., a functional heterogeneity exists among synaptic vesicles and certain vesicles are preferentially involved in transmitter release and these vesicles go through repeated cycles of exo- and endocytosis during transmitter release. Heterogeneity among cholinergic synaptic vesicles has been observed morphologically and biochemically (Zimmermann and Whittaker, 1977; Zimmermann, 1979a; Whittaker, 1984; and Agoston et al., 1985). Physiologically a functional heterogeneity is observed among quantal sizes (McLachlan, 1975;Doherty et al., 1984),and stimulation makes apparent metabolic and morphological heterogeneity of cholinergic synaptic vesicles (Zimmermann, 1979b). A motor nerve terminal contains 500-1000 “active”zones, and at each zone, a double row of 20-30 vesicles each are present (Couteaux and PCcot-Dechavassine, 1974). If vesicles in that position required the property to repeatedly discharge ACh, that population would constitute less than 10% of the total vesicular population in a terminal. Hence, changes in its content of transmitter would only marginally affect the total amount of vesicular ACh and thereby explain the failure to detect depletion during stimulation. The existence of a small population of synaptic vesicles, primarily involved in transmitter release and reaccumulation of ACh from the cytoplasm, would explain a preferential release of newly synthesized cytoplasmic transmitter and a lack of correspondence between the cytoplasmic and the total vesicular content of transmitter during stimulation. The idea that only a small portion of the total number of synaptic vesicles (operator vesicles; according to Isreal et al., 1979), presumably only those attached to the active zones of a nerve terminal under physiological conditions, participated in transmitter release as “shuttle gates” between cytoplasm and synaptic cleft is quite attractive. It would explain most, if not all, differences between the advocates of vesicluar and nonvesicular quantal transmitter release mechanisms (Israel and Dunant, 1979; Zimmermann, 1979b). The model would require that the vesicles discharge their content through a pore or a channel opening into the synaptic cleft. Upon discharge, the vesicles would quickly detach themselves and reaccumulate ACh from the cytoplasm. This would be the process of release during physiological stimulation,while during stimulation to transmitter exhaus-

64

S. THESLEFF

tion, a process involving the fusion and incorporation of synaptic vesicles with the axolemma would occur. The latter would involve the 90%or so “reserve”vesicles not readily available for release. In line with this view, Meldolesi and Ceccarelli (1981) propose that vesicular transmitter release and vesicle recycling is maintained by two different processes (Ceccarelli and Hurlbut, 1980).The first mechanism, as outlined above, would account for a fast recycling occurring at physiological frequency of stimulation, whereas a second, different mechanism would account for vesicles becoming completely incorporated into the axolemma and recycled as coated vesicles. The second mechanism might predominantly operate at higher frequencies of stimulation and during chemical stimulation by a Ca2+ionophore or black widow spider venom. A brief attachment of a synaptic vesicle to the axolemma and a discharge of ACh through a narrow membrane pore or channel, possibly by a cation exchange mechanism as suggested by Uvnas and Aborg (1984), is also attractive, because it offers an explanation for the release of ACh quanta of variable, particularly small, size, giving rise to small-amplitude mepps or sub-mepps (Uvnas and Aborg, 1984).It might therefore provide an explanation for the observation that various experimental procedures, which conceivably could affect the life-span of a secretory membrane pore, increase the number of sub-mepps present at the neuromuscular junction (see a review by Trernblay et al., 1983). Such a mechanism seems more plausible as an explanation for sub-mepps than the idea that one sub-mepp corresponds to the release of a single synaptic vesicle, whereas a mepp is caused by the simultaneous release of several vesicles (Wernig and Stirner, 1977). The mechanisms by which synaptic vesicles are attracted to the axolemma and made to discharge their content are so far unknown. It should, however, be mentioned that there is strong evidence that synaptic vesicles move along the surface of a specific set of presynaptic microtubules that direct the vesicles to dense bars at the presynaptic membrane. These dense bars constitute the active zone at which synaptic-vesicle discharge occurs. Freeze-fracture studies of motor end-plate active zones during or immediately after transmitter release reveal linear arrays of synaptopores perforating the presynaptic membrane (Dreyer et al., 1973; Heuser et al., 1974; Heuser, 1976; Akert and Tokunaga, 1980; Gray, 1983). Calcium ions, when entering the terminal along voltage-dependent channels, possibly exclusively located at active zones (Pumplin and Reese, 1978; Pumplin et al., 19Sl), may promote a vesicle movement, discharge, and detachment through a number of selective mechanisms, such as by activating actomyosin filaments, membrane phospholipase AP, adenylate cyclase, or cdlcium-calmodu~in and CAMP-dependent protein kinases.

ACETYLCHOLINE RELEASE FROM THE MOTOR NERVE

65

Screening of negative membrane charges by Ca" might also facilitate membrane apposition. The concerted action of many such events may explain how transmitter release is regulated (Greengard, 1978; Moskowitz and Puzskin, 1983; Cooper and Meyer, 19 4). However, as reviewed by Kelly et al. (1979), our knowledge about the iachemical steps involved in the release process is so far incomplete and marred by conflicting results caused by methodoIogica1 and experimental difficulties. In nerve impulse-evoked transmitter release not only intracellular Ca2+ but also the depolarization of the terminal directly controls the number of quanta released (Dudel, 1983; Dudel et ad., 1983). It seems that depolarization triggers a mechanism which acts as a gain control in the Ca2+-messengersystem. An example of such a mechanism would be inositol trisphosphate (Berridge and Irvine, 1984), but see also Rasmussen and Barrett (1984) and Kostyuk (1984) for other possible mechanisms. An interesting, but iittle-debated issue, is why a nerve impulse activates transmitter discharge only from a fraction of available active zones in the terminal. As previously mentioned, a frog motor-nerve terminal may contain about 1000 active zones. The quantum content of a normal epp is much smaller than that, about 200, and therefore only a part of the active zones is activated by a given impulse. Bennett and Lavidis (1982), Tremblay et al. (1984); and Dalonzo and Grinnell (1985) observed that the probability of quanta1 secretion from an active zone declined along the length of the terminal branch. Brigant and Mallart (1982) have shown that mammalian nerve terminals are practically devoid of Na+ channels while they are rich in K + and Ca2+ channels. Therefore, the terminal part of the axon cannot conduct an action potential and is depolarized passively by electrotonic spread. This initiates inward Ca2+and outward K + currents (Fig. 1). Normally the outward K + current overwhelms the inward Ca2+current and the terminal is repolarized and the Ca2+channels closed. Furthermore, the Ca"-dependent K + current is activated by Ca2+and therefore develops with a short delay after the start of the Ca2+ current (Mallart, 1984). Such ionic mechanisms are eminently suited as regulators of highfrequency phasic transmitter release but might also prevent the depolarization by an action potential from reaching the furthermost part of the nerve terminal network. These mechanisms provide an explanation of why drugs which block the outward K + current (see below) have such a dramatic potentiating effect on impulse-evoked ACh release. Another possibility is that heterogeneity exists in the probability of active zones to discharge transmitter. For instance, a proximodistal gradient might exist in the size of active zones as observed by Davey and Bennett (1982). A better knowledge of the role of aformentioned mech-

ti

66

S. THESLEFF

anisms in the regulation of transmitter discharge at individual active zones would undoubtedly be of great value for the understanding of phenomena such as activity-dependent facilitation and depression of transmitter release, as well as the mode of action of a number of drugs and toxins affecting the release process. 1. Effect of'Drugs and Toxins

Drugs or procedures which alter the free Ca2+concentration in the nerve terminal also alter the quantal release of ACh from the nerve, be it spontaneous, causing mepps, or impulse evoked, giving rise to epps. Table I enumerates a number of drugs and procedures which increase the level of free Ca2+ in the nerve cytoplasm, either by enhancing the influx of this ion from the extracellular medium or by releasing Ca2+from storage or binding sites within the teminal. This action greatly accelerates the frequency of the quantal discharge of ACh, giving rise to mepps. Several drugs selectively enhance nerve impulse-evoked Ca2+-semitive, quantal transmitter release without affecting spontaneous release. Such drugs are the aminopyridines (4-aminopyridine and SPdiaminopyridine) and tetraethylammonium, which all act by blocking the K + channel and thereby K currents in excitable tissues. These drugs enhance Ca2+influx into nerve terminals by prolonging the duration of depolarization due to the action potential by a blockade of the voltage-activated K + channels. For recent reviews of the pharmacology of these drugs see Thesleff (1980), Bowman and Savage (1981), and Glover (1982). Drugs which interfere with Ca2+entry into the terminal such as Mg2+ and the aminoglycoside antibiotics reduce evoked Ca2+-sensitive quantal +

TABLE I SUMMARY OF T H E EFFECTS OF VARIOUS PROCEDURES O N THE FREQUENCY OF CALCIUM-DEPENDENT FASTA N D CALCIUM-INDEPENDENT SLOWmepps AT NORMAL A N D BoTx POISONED ENDPLATES~ Agent or procedure

Normal muscle, fast mepps only

BoTx-poisoned, slow mepps only

Ca2+,8 mM K + , 20 mM K + , 0 mM Ouabain, 0.2 mM Ethanol, 0.5 M Mn2+, 10 mM Hypertonicity, 2~ Hypotonicity, 0.5X

Increase 4~ Increase 2 0 ~ Increase 15X Increase 5OX Increase IOX Increase 8~ Increase 8OX Decrease 0.3X

No change Increase 2X No change No change No change No change Decrease 0.3X Increase 3X

"From Thesleff el al. (1983). 'Approximate change times control.

ACETYLCHOLINE RELEASE FROM THE MOTOR NERVE

67

transmitter release (Elmqvist and Josefsson, 1962; Vital Brazil and PradoFranceschi, 1969; Molgo et al., 1979; Fiekers, 1983; review by Pittinger and Adamson, 1972), as expected from this mode of action. Of particular interest are the neurotoxins of Clostridium botulinum and Clostridium tetani which block Ca2+-sensitivequantal transmitter release from cholinergic nerve terminals. The block presumably results from an interference with the role of Ca2+ as a trigger of transmitter discharge, see reviews by Simpson (198l), Sellin (19Sl), and Mellanby (1984). Apparently, the toxins do not affect Ca2+entry into the nerve terminal (Gundersen et al., 1982; Dreyer et al., 1983) but reduce the efficacy of Ca2+ to release transmitter (Cull-Candy et al., 1976a). Molgo and Thesleff (1984) have suggested that botulinal toxins (BoTx), upon entering the nerve terminal by an endocytic mechanism, catalyze intracellular processes involved in the disposal of Ca2+from the cytoplasm. I n normal conditions, Ca2+ turnover in the nerve terminal is balanced so that the Ca2+ which enters the terminal reaches active zones in a sufficient amount to cause synaptic-vesicle discharge. An enhanced disposal of a Ca2+ would lower the concentration of this ion to a level in which the amount of Ca2+that entered the terminal would be insufficient to trigger this type of transmitter release. Mellanby (1984) has a somewhat similar suggestion. She proposes that the toxins inactivate a Ca2+-dependentenzyme within the nerve terminal which is involved in the release of transmitter, possibly by phosphorylating critical membrane proteins either on vesicles or on the presynaptic membrane. In that connection, it is interesting that Simpson (1984) has shown botulinum toxin type C2 to possess ADP-ribosylating activity.

2. Physiological Role The physiological importance of Ca2+-sensitive,quantal ACh release is to act as a chemical mechanism for propagating the phasic information contained in nerve impulses across the synaptic cleft. This type of ACh release mechanism is ideally suited to respond to high-frequency stimulation because of an efficient mechanism for phasic Ca2+ entry, which proportionately triggers transmitter release, and a subsequent rapid Ca2+ inactivation, which assures that each nerve impulse is faithfully transmitted. This mechanism is responsible for low- and high-frequency impulse propagation across the synaptic cleft and thereby for the neurogenic control of muscle tone and activity. Since the pattern of muscle activity has long-term consequences for the chemical and physiological properties of a muscle (Lomo and Westgaard, 1975; review by Lomo, 1976, one may consider that this type of transmitter release mechansim also is a part of the trophic influence exerted on muscle by the nerve.

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S. THESLXFF

B. CALCIUM-INSENSITIVE SECRETION OF ACh (11)

This type of ACh release is not involved in impulse propagation across the synaptic cleft, but represents a spontaneous intermittent form of ACh secretion which is particularly prominent when synaptic impulse transmission is blocked and during synaptic development (Fig. 1, 11). i t is characterized by spontaneous mepps with highly variable times-to-peak and amplitudes (see review by Thesleff and Molgo, 1983). Typically, such mepps have a prolonged time-to-peak, the mean exceeding more than twice that of Ca2+-sensitive quantal mepps or nerve impulse-evoked epps. In many instances, times-to-peak as long as 10 msec are recorded (Kim et al., 1984). The amplitude of such mepps is also highly variable, with generally much larger amplitudes than the Ca"-sensitive quantal mepp. Amplitudes as large as 15 mV are not uncommon and may therefore be of sufficient size to trigger an action potential in the muscle cell. Examples of Ca2+-insensitive and Ca2'-sensitive mepps are shown in Fig. 2A and B, respectively. Figure 2C illustrates Ca*+-sensitive multiquantal ePPs.

n

B

L FIG. 2. Examples of Ca2+-insensitive, intermittent secretion of ACh giving rise to slow-rising, large amplitude mepps in A. The record was obtained from a muscle poisoned by botulinum toxin. Record B illustrates intermittent, Cay+-sensitivequantal release of ACh giving rise to fast-rising, uniform amplitude mepps in a normal, untreated muscle. Record C is from the same fiber as A showing evoked epps. Record D is a recording from a normal, untreated muscle showing that slow-rising, giant mepps may also exist under normal conditions. Record E illustrates the effect of 4-aminoquinoline on a normal muscle. Note that the drug has induced a population of large-amplitude, slow-rising mepps of a type similar to that in A. Voltage calibrations are 1 mV for all tracings except C, for which it is 2 mV. Time calibrations are 2 msec for all tracings except B, D, and E for which it is 4 msec. Temperature, 30°C.

ACETYLCHOLINE RELEASE FROM THE MOTOR NERVE

69

Calcium-insensitive transmitter release is neither activated nor influenced by nerve impulses and resultant transmembrane Ca2+fluxes in the nerve terminal. Consequently, this type of transmitter release does not participate in impulse transmission. Spontaneous mepps resulting from the Ca2+-insensitivetype of transmitter release are uninfluenced in frequency by procedures which affect intra- or extracellular Ca2+concentrations of the nerve terminal (see Table I). Hypertonic media, which increase the frequency of Ca2+-sensitivemepps, decrease the frequency of Ca2 -insensitive mepps, while hypotonic solutions exert an opposite effect on both types of release (Table I). Furthermore, the temperature dependence of the two release processes is quite different; Ca*+sensitive, quantal ACh-release frequency is enhanced by temperatures with a Qlo of 2-3, while the Ca2+-insensitive,intermittent release rate is enhanced with a Qlo of about 12 and is virtually blocked at temperatures below 15°C (Thesleff et al., 1983). The first one to observe the Ca'+-independent type of intermittent ACh release was Liley, who in 195'7described unusually large mepps (giant mepps) occurring at a low rate in normal rat neuromuscuIar junctions. These potentials varied in frequency among fibers, but occasionally constituted up to 20% of all mepps recorded. Their frequency was unaltered by nerve stimulation, by nerve terminal depolarization,and by changes in the extracellular Ca2+ or Mg2+ concentration. They also differed, as reported by Liley, from nerve stimuIus-evokedquantal epps (Fig. 2C). Subsequently, Jansen and van Essen (1976) pointed out that the giant potentials had a slow and protracted shape which did not correspond to a simple summation of normal mepps, so that they could not be considered quantal in nature, as estimated from Poisson analysis of epp failure. According to Heinonen el al. (1982), ColmCus et al. (1982) and Kim et al. (1984)giant, slow-rising mepps constitute,on the average, 4% of all mepps recorded at rat neuromuscular junctions at 30°C with great variability in their frequency between fibers. Slow-rising, large-amplitude mepps of the Ca2 -insensitive type are present also at early stages of regenerating neuromuscularjunctions (Bennett et d.,19'73). These mepps constitute a majority of all mepps in this condition and are reduced in number when the synapse matures and normal fast-rising Ca2+-sensitive quantal mepps appear (Colm6us et al., 1982). Slow mepps are also present at neuromuscular junctions of dystrophic mice, strain 1291ReJ (Carbonetto, 1977), and in mice with a hereditary end-plate disease (Weinstein, 1980).They appear in skeletal muscle of chickens curarized during early development (Ding et al., 1983). Growth cones of embryonic cholinergic neurons in culture intermittently secrete large amounts of ACh by an apparently Ca'+-insensitive mechanism (Hume et al., 1983; Young and Poo, 1983). +

+

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S. THESLEFF

The Ca"4nsensitive type of transmitter release is particularly prominent in muscles totally or partly paralyzed by the use of botulinum neurotoxin t y p e A (Colmeus et al., 1982; Kim et al., 1984; Dolly et al., 1985).As previously mentioned, this neurotoxin blocks the Ca2+-sensitive, quantal release mechanism for ACh and thereby neuromuscular transmission. A few days after a neuromuscular block is produced slow-rising mepps of the Ca2+-insensitivetype increase in frequency so that, about 10- 14 days after poisoning, their occurrence reaches 0.3-1 Hz with large variations between fibers (see Fig. 2A). As the effects of the toxin subside and neuromuscular transmission is gradually restored, the frequency of these mepps is reduced, while the number of fast-rising Ca2 sensitive, quantal mepps increases. It is of interest that the occurrence of slowrising, Ca2+-insensitive mepps is more marked in muscles only partially paralyzed by botulinum toxin than in totally paralyzed ones (Kim et al., 1984). The difference between Ca2+-sensitive and Ca2+-insensitive ACh release is particularly evident in muscles poisoned with botulinum toxin since it is possible to observe both types of release simultaneously. At botulinum toxin type A poisoned junctions, it is possible to reintroduce the Ca2+-sensitivetype of quantal transmitter release by procedures which elevate the intracellular Ca2+ concentration. The administration of mitochondrial blocking agents such as dinitrophenol (Sellin et al., 1983) or of a Ca2 ionophore such as A23187 (Cull-Candy et al., 1976a)reintroduces fast-rising, Ca2+-sensitive quantal mepps which appear simultaneously with the slow-rising mepps. Similarly,it is possible to restore nerve impulseevoked quantal release of ACh by drugs such as the aminopyridines or tetraethylammonium (Lundh et al., 1977). Quanta1 epps evoked in such a manner are identical to fast-rising, Ca2+-sensitive mepps and without resemblance to the slow mepps representing Ca2+-insensitive release, which can be observed simultaneously at the same junction as in Fig. 2C (Sellin and Thesleff, 1981). +

+

1. Drugs Which Induce Calcium-Insensitive,

Intermittent Secretion of ACh

The calcium-insensitive secretion of ACh, as observed in BoTx-poisoned muscle, is stimulated in the presence of cyclic AMP or dibutyryl cyclic AMP and further enhanced in the presence of caffeine, a phosphodiesterase inhibitor. The effect of cyclic nucleotides is variable but generally characterized by an increase in the amplitude and frequency of the slow-rising mepps, the potentials frequently appearing as bursts of activity (Tabti et al., 1986). Thus, it seems possible that this type of spontaneous intermittent ACh secretion is somehow modulated by the intraterminal concentration of cyclic AMP.

ACETYLCHOLINE RELEASE FROM THE MOTOR NERVE

71

4-Aminoquinoline in a concentration of 100-250 p M induces, within minutes of its application to mammalian, but not to amphibian, skeletal muscle, the appearance of a population of mepps with a larger than normal amplitude and a prolonged time-to-peak (Fig. 2E). The slow and large mepps induced by 4-aminoquinoline have all the characteristics of a Ca2+-insensitive,intermittent, nonquantal secretion of ACh (Molgo and Thesleff, 1982; Thesleff and Molgo, 1983). They are unaffected in frequency by nerve stimulation, by nerve terminal depolarization, and by increases in intra- or extracellular Ca2+concentrations. Their frequency has a high positive temperature coefficient,Qlo of 12,and they are virtually absent at temperatures below 15°C (Thesleff et al., 1983). These effects of 4-aminoquinoline occur without observable changes in the number of fast-rising,Ca2+-sensitivequantal mepps or epps (Molgo and Thesleff, 1982). 4-Aminoquinoline exerts a similar effect on botulinum toxin-poisoned end plates in which it markedly enhances the frequency of slow mepps, but does not affect the number of fast mepps (Thesleff et at., 1983). At muscles with regenerating nerve terminals, the drug increases the frequency of slow mepps about three times without affecting the frequency of fast mepps (Molg6 et al., 1982). One the basis of those findings, Thesleff and Molgd (1983) have concluded that 4aminoquinoline,by a hitherto unknown mechanism, selectivelystimulates the Ca2+-insensitivetype of intermittent ACh secretion at mammalian neuromuscular junctions. Other drugs, ions, and procedures have been reported to induce the appearance of giant mepps. Thus, an acidic pH and hypertonic solutions, substitution of Na+ by Li+,and prolonged tetanic stimulation may induce giant mepps (PCcot-Dechavassine, 1970; Pkcot-Dechavassine and Couteaux, 1971, 1972; Benoit et al., 1973; Heuser, 1974). Drugs affecting microtubules such as vinblastine (Pkcot-Dechavassine, 1976) and phospholipase A2-containing elapid neurotoxins (e.g. taipoxin and notexin) (Cull-Candy et al., 1976b) have similar effects. In all these instances, the giant potentials have a fast rise time and the mepps are in some instances accompanied by the appearance of large or aggregated synaptic vesicles which has been interpreted to account for the discharge of larger than normal amounts of ACh. Thus, it seems possible that the giant mepps recorded in these conditions might reflect a modification of the Ca2+sensitive, quantal release system rather than a stimulation of the Ca2+insensitive type of intermittent ACh release.

2. Origin and Mechanism of Release The agent responsible for the slow type of mepps is presumably ACh, since tubocurarine blocks such potentials and cholinesterase inhibitors enhance and prolong their duration (Liley, 1957; Molg6 and Thesleff,

72

S. THESLEFF

1982). However, it cannot be excluded that other substances, possibly neuropeptides or ATP (Dowdall et al., 1974), are coreleased with ACh (Thesleff and Molg6, 1983). Presumably the ACh release originates from the nerve, since it is abolished by denervation, and only reappears following reinnervation of the end plate (Cull-Candy et al., 1976a; Molgo and Thesleff, 1982). Studies of the time course of the disappearance of fast and slow mepps, i.e., of Ca2+-sensitiveand -insensitive ACh release, respectively,followingdenervation by surgical sectioningof the motor nerve, showed that both kinds of secretion disappeared simultaneously. This is the opposite of that seen with rnepps which originate from Schwann cells in the amphibian. These potentials appear only after denervation with a few days delay following the cessation of neuromuscular transmission and the disappearance of fast mepps (Bevan et al., 1976). Kim et al. (1984) and Thesleff et al. (1983) have investigated the possibility that the Ca2+-insensitivetype of transmitter release might originate from nerve terminal sprouts, particularly since such sprouting is prominent in botulinum toxin-poisoned muscles in which this type of transmitter release dominates. However, when botulinum toxinpoisoned muscles were directly stimulated in viuo with a frequency pattern which inhibits nerve terminal sprouting no change was observed in the appearance and frequency of slow mepps. Neither did X-ray irradiation of poisoned muscles, a procedure which prevents sprouting, affect the frequency of such rnepps. Consequently there is, at present, no evidence that would indicate that the Ca"4nsensitive type of intermittent transmitter secretion might originate from structures other than the motor nerve terminal. Postsynaptic factors such as a heterogeneity among ACh receptor properties are unlikely as an explanation for the slow mepps, since normal, fast mepps and quanta1epps occur, or can be induced, concomitantly with the spontaneous appearance of slow mepps. Intra-end-plate differences in cholinesterase activity also seem to be excluded, since neostigmine only increases the amplitude and time course of slow mepps without affecting their frequency (Liley, 1957). Nerve terminals treated with 4-aminoquinoline have been examined for the presence of unusually large synaptic vesicles or subaxolemmal cisternae which might account for the release of the amounts of ACh responsible for the generation of slow, giant potentials. No ultrastructural alterations were, however, observed that could account for the release of such large amounts of ACh (PCcot-Dechavassine and Molg6, 1982).One must consider that the potentials might result from a protracted discharge of transmitter from a row or a cluster of normal-sized synaptic vesicles. Support for such a possibility is the observation that slow mepps sometimes have notches in their rising or falling phases indicating a composite nature. Sellin and Thesleff (1981) and Molg6 and Thesleff (1982) have

ACETYLCHOLINE RELEASE FROM THE MOTOR NERVE

73

discussed the possibility that the discharge of transmitter might occur from areas outside of the active zones, i.e., from sites more distant from the postsynaptic receptor which would tend to prolong the time-to-peak of the resulting mepps. Such a nonspecific release site in the axolemma might also explain the failure of Ca2+to influence the frequency of ACh discharges and why this type of ACh release dominates during development, i.e., before the presynaptic specializationof active zones. It cannot, however, be excluded that the discharge reflects a mechanism of protracted, gated release of ACh from the cytoplasmic pool of free transmitter. Calcium ions may be involved ip the discharge process but their presence seems less critical than with quantal release at active zones. Hence, I have used the terminology intermittent, Ca2+-' insensitive transmitter release for this type of ACh secretion.

3. Physiological Role Calcium-insensitive, intermittent secretion of ACh is particularly marked at junctions where neuromuscular transmission is blocked or impaired and during synapse development (botulinurn toxin poisoning, regenerating neuromuscular junctions, and growth cones). This suggests that the release process might constitute a chemical signal which assists the axon in finding and maintaining its proper synaptic connection. Intermittent release of large amount of ACh from the growth cone could be a chemical signal to responsive cells, which in turn could direct axonal growth by reply signals. Once an appropriate innervation was established, the release could be suppressed by (a) factor(s) coming from the target. In favor of the latter possibility, is the delay of several days in the onset of this type of secretion following paralysis by botulinum toxin and the gradual suppression of secretion, as neuromuscular transmission is reestablished (Kim et al., 1984). Presently, very little is known about the chemical signals which act as messengers between nerve growth cones and target cells. The described secretion of ACh might be such a messenger or one among a number of messengers in a complex system of signals operating between the two (see Schmitt, 1984).

11. Continuous ACh Leakage (111)

In addition to the intermittent quantal and nonquantal discharges of ACh previously described, there is biochemical (Mitchell and Silver, 1963; Fletcher and Forrester, 1975) and electrophysiological (Katz and Miledi, 1977; Vyskocil and IllCs, 1977) evidence that ACh may escape from nerve

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terminals by a continuous process (Fig. 1, 111). At frog and mammalian neuromuscularjunctions, a steady leakage builds u p an ACh concentration of the order of lo-* to lo-' M in the synaptic cleft (Katz and Miledi, 1977; Vyskocil et al., 1983). Since the leakage is continuous, the total amount of ACh secreted can be estimated to exceed the efflux due to spontaneous quanta1 discharge by two orders of magnitude, and therefore it accounts for the largest part of ACh released from resting muscle (Katz and Miledi, 1977; Vizi and Vyskocil, 1979). It should, however, be mentioned that not only the nerve but also the muscle synthesizes and releases ACh. The amount of ACh released under resting conditions from nonneural parts of normal rat diaphragms has been estimated to be 30-50% of that released from their neural parts (Dolezal and Tucek, 1983). Accordingly, denervation reduces resting release of ACh by 50-70% (Straughan, 1960;Mitchell and Silver, 1963; Card Linden et al., 1983). One way to detect spontaneous leakage of ACh from the nerve is to apply curare to the end-plate region of a muscle fiber. The resulting blockade of cholinergic postsynaptic receptors causes a local hyperpolarization amounting to 0.04-9 mV depending upon species and experimental conditions (Fig. 3). This hyperpolarization or H-response is enhanced in the presence of cholinesterase inhibitors indicating that its origin is a blockade of cholinergic receptors continuously stimulated by ACh. The ion selectivity of the receptor channel blocked by curare and giving rise to the H-response is similar to that of the channel giving the epp, i.e., presumably the channel of the nicotinic-cholinergic receptor (Vyskocil et al., 1983).The H-response is abolished by denervation in murine and rat muscle (Vyskocil et al., 1983; Dolezal et al., 1983) but not in frog muscle (Katz and Miledi, 1977), indicating that, at least in these mammals, nerve terminals are a main source of the ACh responsible for this effect. This also suggests that in frog muscle at least a part of the continuous leakage of ACh comes from the muscle or from the Schwann cell. T h e release of ACh which causes a persistent depolarization of the end plate is not affected by nerve stimulation (Vizi and Vyskocil, 1979; Katz and Miledi, 1981; Vyskocil et al., 1983). It is however, influenced by the Cay+ concentration of the medium. At physiological Ca2+ levels (2 mM), the release of ACh is maximal, while lowering or elevating the Ca2+ concentration reduces release (Vyskocil et al., 1983). Inhibition of the Na+*K+-activatedmembrane ATPase increases the continuous nonquantal leakage of ACh from nerve terminals, but activation of the enzyme has the opposite effect, (see Fig. 3A, C and D) (Paton et al., 1971; Vizi, 1973, 1977, 1978; Vizi and Vyskocil, 1979). Since Ca2+ ions have been shown to inhibit membrane ATPase (Skou, 1957; Somogyi, 1964), it has been suggested that Ca2+ entering the terminal during

ACETYLCHOLINE RELEASE FROM THE MOTOR NERVE a

b

TC -

TC

75

TC

TC

TC

4 TC 1

-

FIG. 3. Local curarization of the end-plate region of a mouse diaphragm causes, in the presence of cholinesterase inhibition by prostigmine 6 x M (a) or by pretreatment with soman (b), a hyperpolarizing response (H-response). Inhibition of Na+-K+activated ATPase (A) by ouabain 2 X M (B) or a K+-free solution (C) enhances the H-response, while activation of ATPase by readmission of K + (D) blocks the response. Horizontal bars indicate the time of tubocurarine (TC) diffusion from a pipette located in the end-plate area. From Vyskocil and 1116s (1978).

stimulation might affect the activity of the ATPase and thereby also control this type of ACh secretion (Vizi, 1978). However, as mentioned above, this has not been experimentally confirmed. Recently, Edward et al. (1985) suggested that nonquantal, continuous leakage of ACh from nerve terminals might be the result of synaptic

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vesicle membrane incorporation into the nerve terminal membrane. The synaptic vesicular membrane accumulates ACh synthesized in the cytoplasm by an active transport process which depends on a Ca2+-or a Mg2+ATPase system driving the uptake of ACh when linked to an internally acidic proton gradient (Anderson et al., 1982, 1983). If this ACh transport system maintains its orientation following the opening of the vesicle into the synaptic cleft or the incorporation of the vesicle membrane into the terminal membrane, it would move ACh from the axoplasm into the extracellular space and could thereby account for a continuous ATPasedependent secretion of ACh. Indeed, Edwards et al. (1985) observed that drugs which block this transport system (AH 5183 and quinacrine) also blocked the H-response and reduced the release of ACh. Similarly an alkaline pH (9.4) completely blocked the H-response, as might be expected if the ACh transport depended upon a pH gradient. Botulinum toxin type A which, as previously mentioned, blocks Ca2'sensitive, quantal discharge of ACh, also reduces, by about 40%, the continuous leakage of ACh from nerve terminals (Polak et al., 1981; Vyskocil et al., 1983; Dolezal et al., 1983). This effect occurs within minutes of toxin application to the nerve muscle preparation and therefore possibly reflects a mode of action different from its blocking effect on quantal transmitter discharge (Vyskocil el al., 1983). The results from several studies (Miledi et al., 1982a, b, 1983; Dolezal and Tucek, 1983) of ACh produced and released from muscle indicate that nonneural ACh is synthesized in the cytoplasm of the muscle, possibly by the enzyme, carnitine acetyltransferase (EC 2.3.1.7.) (Tucek, 1982), while the neural enzyme is choline acetyltransferase (EC 2.3.1.6.). The way the formed ACh leaves the muscle is not known. It might, however, be mentioned that endocytic and therefore presumably also exocytic activity is quite marked in muscle tissue (Thesleff et al., 1979; TPgerud and Libelius, 1985). Consequently, it seems possible that exocytic membrane vesicles might deliver ACh, located in the cytoplasm, to the extracellular fluid.

PHYSIOLOGICAL ROLE As already stated, the continuous secretion of ACh represents by far the largest portion of ACh released under resting conditions. Even if onethird to one-half of this leakage originates from the muscle cell, it is tempting to assume that the release from the nerve has a physiological function. In that context, it is interesting that Po0 (1984) and Sun and Po0 (1985) have demonstrated that growth cones of cholinergic neurons in

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culture have this mechanism of transmitter secretion. Furthermore Po0 et al. (1978, 1979), Po0 and Robinson (1977),and Lin-Liu et al. (1984) have demonstrated that a uniform electric field across the surface of an embryonic muscle cell results in the asymmetric accumulation of lectin and ACh-receptor proteins toward the cathodal pole of the cell. The accumulation of ACh receptors by the electric field causes the formation in the membrane of stable, localized receptor aggregates. The field strength required to induce a detectable receptor accumulation was between 1 and 1.5 Vlcm, corresponding to a voltage difference of 2-3 mV across the cell membrane. Thus, it is possible, as suggested by Po0 (1985), that the local continuous depolarization of the end-plate area in a muscle fiber by leakage of ACh generates an electric field of sufficient strength to cause the aggregation and immobilization of nicotinic-cholinergic receptors in the area of contact and that this mechanism might assist in the development of the postsynaptic receptor accumulation typical of an end plate. Ziskind-Conhaim el al. (1984) have demonstrated that ACh-receptor clusters at developing end plates arise from receptors that previously were uniformly distributed on the muscle surface. It might also be mentioned that the membrane proteins which constitute the Na' channel are present at a greater density at the end-plate region than in other parts of the muscle membrane (Thesleff et al., 1974; Betz et al., 1984). Other neurotrophic factors are, however, also likely to be involved (see a review by Fischbach et al., 1979). Botulinum toxin poisoning induces, in skeletal muscle, changes similar to those seen following denervation, i.e., chemical supersensitivity, fall of resting membrane potential, fibrillation potentials, and the development of tetrodotoxin-resistant action potentials (Thesleff, 1960;Josefsson and Thesleff, 1961; Mathers and Thesleff, 1978),but these changes are quantitatively less marked than those seen after surgical denervation. A subsequent blockade of cholinergic receptors by curare or a-bungarotoxin brings the denervation changes to the same level as those resulting from surgical denervation (Pestronk et al., 1976; Mathers and Thesleff, 1978; Drachman et al., 1982). On that basis these authors concluded that the motor nerve, even when transmission is paralyzed by botulinum toxin, exerts a trophic influence on the muscle and that this influence is mediated by ACh. In botulinum toxin-paralyzed muscles ACh is released intermittently in a Ca2+-insensitivemanner and also continuously a molecular leakage, as previously explained. It seems possible that these releases of ACh might have a trophic influence, not only on botulinum-poisoned muscles, but also on normal muscle (Thesleff and Sellin, 1980; Bray et al., 1982; Card Linden et al., 1983; McArdle, 1983). We have no knowledge of the functional role of nonneural ACh re-

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leased from the muscle. It has been suggested that it might participate in the control of the Na+ ,K+-ATPase of muscle fibers or play a role in the control of the metabolism of membrane phospholipids (for further details and references see Dolezal et aE., 1983; Dolezal and Tucek, 1983).

111. Comments

T h e aim of this presentation has been to show that a nerve may use the same chemical substance ACh as a transmitter of different kinds of messages. ACh is used not only as a transmitter of the nerve impulse across the synaptic cleft and thereby for motor control of the muscle, but presumably also as a messenger for other types of information. Release of ACh might serve as a signal which helps the axon to find its proper target, to establish the synapse, and to maintain its target cell (muscle) in an optimal-functional state (neurotrophism). ACh seems to be involved in all these functions, although the release mechanism for the substance may vary. A weakness of these speculations is that we do not understand the mechanisms by which ACh could exert such different actions on muscle. Are second messengers, such as Ca2+,calcium-mobilizing polyphosphoinositides, or cyclic nucleotides, involved in the transfer of information from the cholinergic membrane receptor to metabolic and catabolic regulating centers of the muscle cell? Perhaps the mechanisms are similar to those involved in the regulation of muscle metabolism by mechanical activity. Studies of acute denervation changes in skeletal muscle indicate that several mechanisms and factors participate and interact as neurotrophic influences (see reviews by Thesleff and Sellin, 1980; McArdle, 1983). The mechanisms of the different types of ACh release are also insufficiently understood. To what extent and how are synaptic vesicles involved, and is it possible that there are common steps in different release mechanisms? For instance could there be a relation between the described intermittent, Ca2+-insensitivesecretion of ACh and synaptic vesicles and possibly also with the spontaneous continuous efflux of ACh? Maybe it is the site of release, presynaptic active zones, or remaining axolemma, that determines the Ca2 sensitivity of the discharge process? Similarly, little is known about the biochemical steps which initiate ACh discharge. In fact, so little is known that it discourages speculations about the molecular mechanisms underlying different kinds of ACh secretion. Despite the above, it might be worthwhile to pursue certain ideas regarding possible mechanisms involved. Greengard and co-workers, ex+

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cellently reviewed by Nestler and Greengard (1984), consider phosphorylation of a specific synaptic vesicle protein (synapsin I) as a priming step in the vesicle fusion-ACh release process. Synapsin I phosphorylation is proposed to be regulated both by a calcium-calmodulin-dependent protein kinase and by a cyclic AMP-dependent kinase present in nerve terminals. Synapsin I is a protein closely associated with synaptic vesicles and, when phosphorylated, it is detached from the vesicle membrane. As hypothesized by Nestler and Greengard (1984), dephosphosynapsin I is the active form of the molecule and acts to hinder the association of neurotransmitter vesicles with the plasma membrane. Phosphorylation of synapsin I would therefore allow vesicle aggregation and fusion with the nerve terminal membrane. As previously explained (Fig. l), calcium-sensitive, intermittent secretion of ACh (I) is believed to originate from the active zones in nerve terminals while the calcium-insensitive type of intermittent secretion (11) might originate from areas outside of these specialized parts of the nerve terminal membrane. If voltage-dependent Ca2+channels are preferentially located at active zones, as believed, Ca'+ would primarily enter the terminal at such sites during stimulation and nerve terminal depolarization. Hence, synapsin I phosphorylation by a Ca2+-calmodulin-dependentkinase would be most prominent at those sites and thereby account for a preferential release of ACh from this part of the terminal. Changes in the level of cyclic AMP or another second messenger concentration in the terminal are presumably generalized and not located to specific areas. Therefore, protein kinases dependent on such messengers can be expected to phosphorylate synapsin I and thereby induce synaptic vesicle fusion and ACh release from all parts of the terminal. If accompanied by vesicle aggregation, giant, slow-risingmepps would result, as observed for the Ca2+-insensitive,but perhaps cyclic AMP-modulated, type of intermittent ACh release. Maybe a cyclic nucleotide-regulated secretion of ACh represents an embryonic, more primitive type of transmitter release which is followed by a Ca2+-regulated mechanism once active zones with functional calcium channels develop in the terminal. Skeletal muscle has been shown to contain a protein kinase inhibitor specific for cyclic AMP-dependent protein kinase (Walsh et d., 1971).The retrograde transfer of such an inhibitor from muscle to nerve as a result of neuromuscular transmission could explain the variability of this type of release between fibers and muscles and why cyclic AMP-dependent ACh release, to a large extent, is inhibited in synapses with intact transmission and its gradual reappearance and disappearance once transmission is respectively blocked and reestablished.

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One might also speculate that regulation of synapsin I phosphorylation by the cyclic AMP-dependent protein kinase present in nerve terminals is involved in an embryonic presynaptic chemoreceptor-regulated control of transmitter release. At present all these suggestions, however, are speculations, and this presentation raises far more questions than it answers. Acknowledgments 1 am grateful to Dr. E. Heilbronn, Stockholm, J. Molgo, Paris, and F. Vyskocil, Prague, for their valuable comments on the manuscript. References

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Heuser, J. E. (1974). A possible origin of the “giant” spontaneous potentials that occur after prolonged transmitter release at frog neuromuscular juncti0n.J. Physiol. (London) 234, 106P- 108P. Heuser, J. E. (1976). Morphology of synaptic vesicle discharge and reformation at the frog neuromuscular junction. I n “Motor Innervation of Muscle” (S. Thesleff, ed.). Academic Press, London. Heuser, J. E. (1977). Synaptic vesicle exocytosis revealed in quick-frozen frog neuromuscular junctions treated with 4-aminopyridine and given a single electrical shock. Neurosci. Symp. 2, 215-239. Heuser, J. E., and Reese, T. S. (1973).Evidence for recycling of synaptic vesicle membrane during transmitter release at the frog neuromuscu1arjunction.J. Cell B i d . 57, 315344. Heuser, J. E., and Reese, T S. (1981). Structural changes after transmitter release at the frog neuromuscular juncti0n.J. Cell Biol. 88, 564-580. Heuser, J. E., Reese, T. S., and Landis, D. M. D. (1974). Functional changes in frog neuromuscular junctions studied with freeze-fracture. J. Neurocytol. 3, 109- 131. Heuser, J. E., Reese, T. S., Dennis, M. J., Jan, Y., Jan, I., and Evans, L. (1979). Synaptic vesicle exocytosis captured by quick-freezing and correlated with quanta1 transmitter release J. Cell B i d . 81, 275-300. Hume, R. I., Role, L. W., and Fischbach, G. D. (1983). Acetylcholine release from growth cones detected with patches of acetylcholine receptor-rich membrane. Nature (London) 305,632-634. Israel, M., and Dunant, Y. (1979). On the mechanism of acetylcholine release. Prog. Bruin Res. 49, 125-139. Israel, M., Gautron, J., and Lesbats, B. (1968). Isolement des vksicules synaptiques de l’organe electrique de la Torpille et localisation de I’acetylcholine leur niveau. C . R. Hebd. Sances. Acad. Sci., Paris. 266, 273-275. Israel, M., Dunant, Y., and Manaranche, R. (1979). The present status of vesicular hypotheses. Prog. Neurobiol. 13, 237-275. Jansen, J. K. S., and van Essen, D. (1976).A population of miniature end-plate potentials not evoked by nerve stimulation.]. Physiol. (London) 258, 103-104P. Josefsson, J.-O., and Thesleff, S. (1961). Electromyographic findings in experimental botulinum intoxication. Acta Physiol. Scand. 51, 163- 168. Katz, B. (1966). “Nerve, Muscle and Synapse.” McGraw-Hill, New York. Katz, B. (1969). T h e release of neural transmitter substances. I n “The Sherrington Lectures,” Vol. 10. Liverpool Univ. Press, Liverpool. Katz, B., and Miledi, R. (1965). The effect of calcium on acetylcholine release from motor nerve terminals. Proc. R. SOC.London Sel: B 161,496-503. Katz, B., and Miledi, R. (1977). Transmitter leakage from motor nerve endings. Proc. R. SOC.London Sex B 196,59-72. Katz, B., and Miledi, R. (1981). Does the motor nerve impulse evoke “non-quantal” transmitter release? Proc. R . Soc. London Sex B 212, 131-137. Kelly, R. B., Deutsch, J. W., Carlson, S. S., and Wagner, J. A. (1979). Biochemistry of neurotransmitter release. Annu. Rev. Neurosci. 2, 399-446. Kim, Y.I., LGmo, T., Lupa, M. T., and Thesleff, S. (1984). Miniature end-plate potentials in rat skeletal muscle poisoned with botulinum toxin. J. Physiol. (London) 356, 587599. Kostyuk, P. G. (1984). Commentary. Metabolic control of ionic channels in the neuronal membrane. Neuroscience. 13, 983-989. Kuffler, S. W., and Yoshikami, D. (1975). The number of transmitter molecules in a

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NEUROENDOCRINE-ONTOGENETIC MECHANISM OF AGING: TOWARD AN INTEGRATED THEORY OF AGtNG By V. M. Dilman, S. Y. Revskoy, and A. G. Golubev N. N. Petrov Research Institute of Oncology of the USSR Ministry of Health Leningrad 188646, USSR

1. Introduction

The widespread feeling is that an integrated theory of aging is unattainable because of the multifactorial origins of aging. When the search for a basis of such a theory is nevertheless undertaken, it is sought as a rule at the cellular level, e.g., the Hayflick’s limit (Hayflick, 1976),or at the level of subcellular elements (e.g., DNA damage). It is believed that only a universal phenomenon with consequences at cellular, tissue, organ, and system levels may be suitable for the explanation of the universal character of the manifestations of aging. Given these general considerations, it seems unlikely that changes in the neuroendocrine system are appropriate candidates for the role of key factors in the mechanism of aging of higher vertebrates. However, the neuroendocrine theory of aging provides the basis for the description of such diverse phenomena as the mechanism of age-dependent on-and-off switching of the reproductive function, the mechanism of acceleration of development, and mechanism causing or promoting age-associated pathology: climacteric, obesity, prediabetes, psychic depression, age-linked immune disorders, atherosclerosis, and age-linked hypertension. This list should include several new syndromes, such as hyperadaptosis, metabolic immunodepression, and cancrophilia, and also classify climacteric as a disease. On the other hand, discrimination of the pathogenetic connections between the age-linked pathology and aging made it possible to regard many manifestations of the normal, physiological process of aging as a disease or a sum of diseases of homeostasis. In a final analysis, the neuroendocrine theory of aging and of age-linked diseases presented the opportunity to distinguish three models of the development of diseases, the ecological, genetic, and ontogenetic models, and, also, to reevaluate the role of stochastic processes in the development of certain diseases and, correspondingly, to propose the possibility of the key role of these processes in the development of diseases according to the involutionary INTERNATIONAL REVIEW OF NEUROBIOLOGY. VOL 28

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model. Finally, the neuroendocrine theory of aging is quite compatible with certain principles of the evolutionary approach to the analysis of the phenomenon of aging, in particular, with the dependence of the rate of aging upon the characteristics of reproductive function and with the postulate about the pleiotropic effects of some genes postponed to the late periods of ontogenesis. Thus, no other existing theory of aging gives such an opportunity to unite and explain so many fundamental phenomena inherent in development, aging, age-linked pathology, and natural death of mammals, including humans, by virtue of a single mechanism, the existence of which is not only postulated but, in many cases, is also demonstrated experimentally and clinically. Besides this, on the basis of the ontogenetic model of the development of diseases, the additional approaches toward the improvement of the metabolic state characteristic of an aged organism were elaborated. This permitted the reduction of the incidence of some chemically and virally induced tumors and to increase the mean life-span of the experimental animals by 20-25%, to pharmacologically restore the estrous cycle in old rats, to improve the course of postsurgery stress, to normalize the functions of the adaptive homeostate in patients with psychic depression, to diminish the metabolic shifts in 60-70% of cancer patients in clinical remission, and, in this way, to diminish or eliminate metabolic immunodepression, etc. (Dilman, 1981, 1983). In the present review, a brief discussion will be presented of these and other questions concerning the substantiation and documentation of the neuroendocrineontogenetic model of aging and the ways of its realization. Some considerations will also be presented and aimed to find the link between the stochastic processes of aging based on the accumulation of damage in cellular and subcellular structures and, conversely, the orderly processes connected with the mechanisms of the development of an organism.

II. The Main Postulate of the Neuroendocrine-Ontogenetic Theory: The Law of the Deviation of Homeostasis

The stability of the internal media of an organism, according to Claude Bernard (1878), is an inexpensible prerequisite of the “free existence of an organism.” In accordance with this, there functions in any living organism a number of complex homeostatic systems, keeping the internal media contents within certain relatively narrow limits. Glucose homeostate is a typical example of such a system. It follows, however, from general considerations that stability excludes the possibility of development, the

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programmed deviation from the stability being on the contrary an inexpensible prerequisite for implementation of a program of development. This notion is somewhat similar to the concept of homeorrhesis (Waddington, 1968). Therefore, a question naturally arises, “By what means may the deviation of homeostasis, necessary for development, be carried out?”In a developing fetomaternal system and in an organism after birth, this task is achieved by different means. In the course of pregnancy, there functions an additional endocrine gland, the fetoplacental system. The secretion of hormones by this system is not controlled by usual feedback loops, and therefore the production of the placental hormones increases in parallel with the mass of the placenta. The chorionic somatomammotropin, possessing the properties of the growth hormone, together with cortisol and, possibly, with other steroid hormones, decreases the utilization of glucose by muscles in the maternal organism. Correspondingly, during the second half of pregnancy, the postprandial blood glucose level substantially increases in the maternal organism, and, in turn, the blood insulin concentration increases. The combined influence of these two factors promotes increases in the body fat content by 5-6 kg. As a result of the increased fat mass, the spontaneous lipolysis increases, and the blood level of free fatty acids is elevated. This shift on the one hand causes additional inhibition of glucose utilization in the maternal organism with the channeling of glucose for supporting the demands of the fetus as a result. On the other hand, within the conditions of the elevated levels of free fatty acids (FFA), glucose and insulin, the synthesis of very low-density lipoproteins (VLDL) increases, which leads finally to elevated blood concentrations of VLDL, low-density lipoproteins (LDL), and correspondingly, of triglycerides and cholesterol (Felig, 1977). While the transfer of FFA across the placental barrier is limited (Dancis et al., 1973), a considerable amount of plasma cholesterol in a preterm fetus may be derived from maternal plasma cholesterol (Pitkin et al., 1972),supplying, as it is proposed (Dilman, 198l), for the buildup of plasma membranes of the rapidly proliferating fetal cells and for the synthesis of steroid hormones in the placenta (Hellig et al., 1970). Besides this, the described metabolic shifts cause metabolic immunodepression, i.e., suppress cellular (transplantational) immunity and serve as an additional factor preventing the rejection of the fetus as an allotransplantate. It may be concluded from what was stated above that the metabolic changes in a maternal organism (the elevated blood concentrations of glucose, insulin, free fatty acids, LDL, VLDL, etc.) serve in a final result for the implementationof the program of fetal development. In summary, it may be said that normal pregnancy is characterized by the same homeostatic changes as are the typical age-related diseases, such as

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obesity, the insulin-independent diabetes meltitus, atherosclerosis, and some others. The ways for the realization of the metabolic shifts in the fetomaternal system cannot be used, however, in a developing organism after birthanother mechanism must function. It does function and is based mainly, but not exclusively, upon the changes that occur at the hypothalamic level. Now we will consider this mechanism. In accordance with the existence of three main functions in any living system, i.e., the energy, reproductive, and adaptational functions, three corresponding homeostates may be distinguished. From the most general considerations, it is clear that it is stability in the functioning of these three systems that could render the realization of the developmental program of an organism impossible. Therefore, the changes must occur in these homeostatic systems that could ensure the elevation of what may be called the power or productivity of these systems and what serves for the demands of the growth of an organism and for the accomplishment of its development.' As long as stability ofthe three outlined systems is based upon negative feedback circuits that are interlocked at the hypothalamic level, only one way for the elevation of the power of these homeostatic systems can be envisioned, i.e., the gradual elevation of the threshold of the sensitivity of the corresponding hypothalamic centers to the peripheral regulatory signals, the hormones and the metabolites.' Consider an alternative situation that is the primary elevation of the activity of a peripheral component of a homeostatic system, e.g., the production of estrogens. In this case, the latter will inhibit by negative feedback the activity of the corresponding hypothalamic center, making the elevation of the power or the productivity of the whole system impossible. On the contrary, in the case of the primary

I It is known that in the course of maturation the production of sex hormones in females increases. Analogous processes can be observed in all three homeostatic systems (Dilman, 1981). These processes are designated here as indicators of the elevation of the power of the homeostatic systems. 'For the estimation of the state of any homeostatic system, two indices are of primary importance: the threshold of the sensitivity of a system and its sensitivity (reactivity) to a signal. T h e threshold of the sensitivity is the minimal strength of the signal that leads to the change in the registered state of the system, for instance, the minimal change in the concentration or the minimal dose of a hormone. The sensitivity (or reactivity) is the magnitude of a change under a challenge with a signal of a certain strength. Physiologically, the perception of a signal and the response to it always begins after it reaches a threshold value. After this, the magnitude of the response, i.e, the level of the sensitivity or of the resistance of the system, can be estimated. Methodologically, it is the threshold, rather than the sensitivity, that is estimated usually in cases of the homeostatic systems discussed (Dilman, 1981, 1983).

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elevation of the threshold of the sensitivity of the hypothalamic-pituitary complex to the influence of estrogens, this, occurring in parallel with increasing age, will ensure in a final analysis the elevation of the power of the corresponding homeostatic system. It is these sort of processes that occur, as will be shown below, in the course of postnatal ontogenesis in the reproductive homeostate, ensuring at first the switching-on of the reproductive function then the switching-off of it in the course of normal aging.

111. The Age-Related Changes in the Reproductive Homeostak: The Unity of the Mechanisms of Development and Aging. Climacteric as a Normal Disease

The hypothalamus of an immature organism possesses the maximal sensitivity to the inhibitory action of sex hormones. It was proposed (Dilman, 1981) that one of the consequences of this regulatory peculiarity is that the sexual maturation does not take place until a body reaches a certain size. Donovan and Van der Werff ten Bosch (1959)postulated that, before puberty, the sensitivity of the tonic sex center of the hypothalamus to the inhibition by sex hormones decreases with advancing age. Thus, it was shown, for instance, that in girls the blood level of estradiol increases in parallel with the levels of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) Uenner et al., 1972). Taking into consideration that estradiol inhibits tonic production of gonadotropins by negative feedback, it is possible to conclude that these changescharacterize the existence of the age-related elevation of the threshold of the sensitivity of the hypothalamus to the inhibitory action of estrogens. Therefore, the inhibitory effect of sex hormones on the hypothalamus decreases. Correspondingly, the secretion of gonadotropins increases which, in turn, leads to the elevation of the production of sex hormones, in particular, of estrogens by the ovaries. The power of the reproductive homeostate increases as a result, i.e., the sexual maturation occurs. Without treating the mechanism of switching-on the reproductive function in detail here (see Dilman, 1981), it is necessary to note that when the concentration of estrogens in the blood reaches a certain critical level they induce ovulation acting by positive feedback on the cyclic sex center of hypothalamus (Lu and Meites, 1977). Consequently, in this case, the mechanism of sexual maturation (i.e., one of the elements of the realization of the developmental program of an organism) acts at the expense of the deviation of homeostasis which, theoretically, may be referred to as a disease because any stable deviation of homeostasis should be regarded as a disease. However, it appears that the driving force of development, the deviation of homeostasis, continues

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to act after the age-related switching-on of the reproductive function. In

fact, during the reproductive period the secretion of gonadotropins continues to increase with age when the age-related decrease in the blood levels of classical estrogens is not yet pronounced (Dilman, 1981). This means that the elevation of the threshold of the sensitivity of the tonic center to the inhibitory action of sex hormones continues throughout the period of maturity This conclusion is supported by the data that, in old animals (Dilman and Anisimov, 1979)as well as in postmenopausal women (Mills and Mahesh, 1978; Isaacs and Havard, 1978),the dose of estrogens necessary for the inhibition of gonadotropic function is elevated. There is a number of reasons to believe that after sexual maturation the elevation of the threshold of the sensitivity of the cyclic sex center of the hypothalamus begins (Dilman, 1981, 1983).Therefore, in spite of the compensatory elevation of the activity of ovaries caused by their overstimulation, a period comes at a certain age when the level of sex hormones appears to be relatively low for the induction of ovulation (this is the reason why during the premenopausal period there often occurs follicular persistence which leads to the endometrial hyperplasis, hence creating the conditions for the arise of endometrial cancer). T h e age-linked switchingoff of the reproductive function occurs as a result. T h e experimental data confirm this conclusion (Lu et al., 1977).3 In summary, it should be stressed that the age-related switching-on of the reproductive function, i.e., one of the phenomena of development, and the age-related switching-off of the reproductive function, i.e., one of the phenomena of aging, are carried out by virtue of the same mechanism. 'Therefore, climacteric, i.e., a typical manifestation of physiological aging, is simultaneously a typical disease by definition, as far as any stable Some authors believe that the hypothalamic mechanism of the age-linked switchingoff of the reproductive function acts in certain species like the rat; but, in women, the primary changes occur in the ovaries (Meites et al., 1978) and are caused by the resistance of the ovaries to gonadotropic stimulation. But. it is quite possible that this resistance is caused by the very age-dependent elevation of the gonadotropin levels in women. while in rats this elevation is practically absent. T h e following may be proposed (Dilnian, 1981): under the influence of age-dependent elevation of gonadotropin levels, the impairment of the sensitivity of the ovaries to the action of gonadotropins may occur in a human female organism as a consequence of the reduction of the number of the receptors for estrogens or because of some more complex mechanism of desensitization (Freeman and Ascoli, 1981). T h e species-related differences in the age-dependent gonadotropin changes may be caused in turn by the stimulatory action of dopamine on the secretion of gonadotropins (Kamberi el al., 1970) in the rats, while in women dopamine acts as an inhibitor of the secretion of gonadotropins (Rakoff et al., 1978). At the same time, the content of dopamine in the hypothalamus decreases with age in both rats and humans (Finch. 1973; Robinson, 1975).

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deviation of the internal environment of an organism from the constancy may be qualified as a disease (the diseases that are analogous to climacteric in this sense may be mentioned for comparison: the steady elevation of blood-sugar concentration is classified as diabetes mellitus, and the steady elevation of blood pressure, as hypertensive disease). Climacteric, however, is a disease not only by definition. Age-linked hyperplasia of the theca tissue of ovaries leads to elevated production of androgens and, correspondingly,to their increased biotransformation into classical estrogens. Thus, it must be one of the causes of the age-related increase in the incidence of estrogen-dependent tumors, i.e., the mammary and endometrial carcinomas. The production of nonclassical estrogens (phenolsteroids) increases with age (Dilman, 1981). It is of interest, in this respect, that the elevated production of phenolsteroids was found in mammary cancer patients (Castagnetta et al., 1981). In the later age periods, the hormonal shifts characteristic of the climacteric cause osteoporosis, promote atherosclerosis, etc. Thus, along with the Law of Constancy of the Internal Environment, there coexists the Law of Deviation of Homeostasis, or more exactly, they both reflect the unity of the contradictions in the functioning of a living system which ensures the relative stability at any given moment on the one hand and the time-dependent changes in the functioning of certain systems on the other.

IV. The Age-Related Changes in the Adaptive Homeostate. Hyperadaptosis as a Normal Disease

The adaptation to different factors is carried out at all levels of an organism, but in vertebrates the central role in adaptation belongs to the hypothalamic-pituitary-adrenal system (Selye, 1950). Therefore, similar to the way in which the reproductive homeostate has been outlined, the adaptive system or homeostate may be distinguished. The regulation of adrenocorticotropic hormone (ACTH) secretion is carried out by different mechanisms which control: (1) the basal production of the hormone, (2) the circadian rhythm of its secretion, and (3) the stress-induced release' of ACTH. The basal secretion of ACTH is less sensitive to the inhibition by glucocorticoids than the circadian mechanism of ACTH secretion, which is under control of extrahypothalamic brain structures (Ceresa et ul., 1970). This is the reason why, in the case of dexamethasone administration at 11 PM for the short dexamethasone test, the necessary dose of the drug is two to three times lower than the dose

96

v. M. DILMAN et al.

that is necessary for the inhibition of the basal level of ACTH production. It is the latter mechanism that progressively changes in the course of aging (Dilman, 1981). It was experimentally shown that in this system the reduction of the sensitivity of the hypothalamic-pituitary complex to the inhibitory action of glucocorticoids occurs as the maturation of an organism takes place, similar to what happens in the reproductive system (Ostroumova and Dilman, 1972; Riegle, 1973, 1976). This phenomenon, probably, also occurs in man, although it is not documented with sufficient evidence (Dilman, 1981). For the purposes of estimation of this age-related process, the dexamethasone dose of 0.125 mg four times daily for 48 hr is routinely used in our laboratory (this dose is less than that needed for the discrimination between obesity and Gushing's disease). Table I demonstrates the age-related changes in the inhibitory action of dexamethasone as estimated in this test with the measurement of glucocorticoid excretion. Theoretically, the observed changes in the negative feedback must lead to an elevation of the blood level of glucocorticoids. Such an elevation was noted in rats by Barnett and Phillips (1976). However, in healthy humans the age-related increase in blood cortisol concentration was not observed, although in cases of age-specific pathology it is often encountered (Table 11). Perhaps the age-related changes in the adrenal tissue or its desensitization to the ACTH action (Malamed and Carsia, 1983) prevents the development of absolute hypercorticism. Relative hypercorticism, undoubtedly, arises in the course of aging as soon as the blood cortisol level does not decrease, or increases slightly while the levels of a series of corticosteroids with anabolic (cortisol-antagonizing) action decreases. The relative hypercorticism that develops as a result manifests itself by a number of signs, in particular by the age-related redistribution of body fat toward the Cushingoid type (excessive fat in the trunk and in the face with the diminished fat in the limbs) and by the corresponding metabolic shifts, in particular, revealed by the predinsolone-glucose test (Table 111). Of special interest is the fact that, although the short dexamethasone test of estimating the mechanism of circadian regulation of ACTH does not reveal age-related changes, this test does demonstrate the reduction of the sensitivity of this regulatory system in many patients with agespecific pathology, in particular, cases with atherosclerosis. At the same time, on the assumption that the mechanism of aging itself is the major cause of age-specific pathology, the exclusion of the individuals with such diseases from the general population must lead to the separation of the artifactual population with slowed down rate of age-related processes. Of interest in this respect are the results of the study on the course of stress reaction in two groups of cancer patients of the same age but with different

TABLE I THECOMPARISON OF THE RESULTS OF “SHORT” A N D “PROLONGED” DEXAMETHASONE TESTS (DMT) IN HEALTHY WOMEN AND IN MAMMARY CANCER PATIENTS OF DIFFERENT AGES Short DMT”

Prolonged DMTb

Mean age (years)

Number of observations

Initial

35.4 k 0.6 50.6 f 0.6

19 18

384 f 30.4 414 2 38.6

127 t 30.4 143 5 35.9

-67 -65

13.0 5 1.10 13.5 f 2.21

6.9 5 0.55 9.1 f 0.55‘

- 47

Mammary cancer patients I 36.5 f 0.7 I1 54.9 f 0.5

13 22

-

-

-

16.0 t 1.93 14.6 k 1.10

5.8 f 0.83 9.4 k 1.38‘

- 63

Study groups Healthy women I

I1

“Blood levels of 11-OHCS (nmolelliter). ’Excretion of 17-OHCS (nmole/24 hr). ‘ p = 0.05.

After Suppression dexamethasone (%)

-

-

Initial

After Suppression dexamethasone (%)

- 33

- 36

v.

98 THE

et al.

TABLE 11 RESULTS OF THE DEXAMETHASONE TEST I N PATIENTS W I T H CANCER, ENDOGENOUS DEPRESSION, A N D I N HEALTHY SUB~ECTS

Study groups Controls Ambulatory Hospital Endogenous depression Depression phase Remission phase Endometrial cancer Mammary cancer Stomach cancer Colon cancer Lung cancer Prostrate cancer a

M. DILMAN

Number of observations

Initial

After dexamethasone

sion (%)

Percentage of the DM-resistant subjects

85 24

397 2 13.8 339 f 24.8

138 t 11.0 221 t 24.8

65 35

41

52

568 t 22.1

455 t 24.8

19

69

29

450

f

27.6

163 2 19.3

64

10

57

430

2

30.4

273 2 16.6

37

67

13 36 55

378 ? 433 ? 436 ? 425 571 t

44.2 19.3 16.6 22.1 30.4

237 t 33.1 262 2 22.1 270 f 5.5 281 2 35.9 375 t 35.9

36 39 38 34 35

38 61 60 44 55

15

75

T h e blood levels of 1 I-OHCS" Suppres-

*

9

1 1-OHCS, 1 1-Oxycorticosteroids.

TABLE 111 THEINFLUENCE OF AGEON THE RESULTS OF PREDNISOLONE-GLUCOSE TEST IN MEN WITH ISCHEMIC HEARTDISEASE Age groups (years)

Number of observations

Before 50 50-59 After 60

25 31 17

Blood glucose level (mmole/liter) 1 hr

2 hr

10.9 f 0.40 12.2 -+ 0.45 13.5 2 0.42

8.0 f 0.39 9.4 f 0.35 11.3 t 0.32

sensitivities of the hypothalamic-pituitary complex to the inhibition by dexamethasone (Table IV). It may be seen that, during all periods of presurgery, surgery, and postsurgery stress, there was observed higher blood cortisol level in dexamethasone-resistantpatients than in dexamethasone-sensitive patients. The prolongation of high blood cortisol in this situation may be regarded as a direct consequence of the elevated threshold of the sensitivity of the control system to the cortisol action. These observations are in dissent with the conclusion of Blicher-Toft ( 1978)that

NEUROENDOCRINE-ONTOGENETIC MECHANISM OF AGING

99

TABLE IV THELEVELS OF 1 1-OXYCORTICOSTEROIDS IN RELATION TO THE SENSITIVITY TO DEXAMETHASONE IN STOMACH CANCER PATIENTS DURING RADICAL SURGICAL TREATMENT The period of blood sampling 1. Before operation 2. After premedication 3. The most traumatic period of the surgical interference 4. The end of the operation 5. After operation First day Third day Ninth day

Dexamethasone-sensitive mean age, 51.9 ? 3.8 years"

Dexarnethasone-resistant mean age, 51.5 f 2.9 yearsb

395 408 626

f f f

36.7 51.9 42.5

450 f 35.6 450 f 77.8 775 f 79.2

775

f

72.3

930

f

428 f 45.5 466 f 44.4 400 2 46.1

508 718 527

f 64.3 -C 119.5 f 42.5

41.7

" n = 14. bn = 8.

the more pronounced response to stress in middle-aged and elderly people, when compared with young people, is caused only by the faster cortisol clearance in the latter. In our studies, the excessive stress reaction occurred in subjects of the same age but with lower sensitivity of the hypothalamopituitary complex to the dexamethasone suppression. Thus, the age-related changes in the adaptive homeostate form one more disease of regulation of homeostasis which may be designated as hyperadaptosis. The excessive response of the adaptive system to stress is a specific marker of hyperadaptosis. With advancing age, a man begins to live as if in chronic stress even without really being stressed and, therefore, becomes more and more defenseless when a real stress challenges him. Besides existing opinions (Upton, 1977), the progressive vulnerability of an aging organism can be explained from this point of view of an excessive, damaging reaction to a supposed stress. It should be mentioned, however, that the state of hyperadaptosis makes poorer the prognosis for the clinical course of cancer (Saez, 1974),markedly increases the incidence of postsurgery complications (Ostroumovaet al., 1982), and, perhaps, may promote malignant transformation of cells. Hence, hyperadaptosis, as well as climacteric, is a disease not only by definition, but a disease proper, the appearance of which is connected with the action of factors ensuring the realization of the developmental program of an organism.

100

v. M. DILMAN et al. V. The Age-Related Changes in the Energy Homeostate. Prediabetes and Obesity as Normal Diseases

The energy homeostate may be divided into two sybsystems: one for the appetite regulation and one for the regulation of the fluxes of the energy substrates. We will see what changes must occur in these subsystems that lead to the regular, age-linked increase in body fat content. The constancy of body weight is a function of the state of homeostatic mechanisms. It is believed that in the hypothalamus, there are two interdependent centers of appetite regulation: the satiety center in the ventromedial hypothalamus and the appetite center in the lateral hypothalamus (Mayer and Arees, 1970). The neurons of the satiety center lead to the suppression of the appetite center. That is the reason why, when the bloodglucose concentration increases during food intake up to a certain level, the signals are generated for the termination of food intake. The hypothalamic system for the immediate control of food income into an organism may be designated as the "tactical appetite center." This center immediately reacts to the amount of energy substrates consumed by an organism, but it is not enabled for longtime regulation of the constancy of body weight on the basis of such a parameter as the postprandial hyperglycemia. As it was postulated by Woods and Porte (1978), the regulation of body weight is carried out by the hypothalamus on the basis of insulin, but not by glucose signal. The higher the body weight, or more exactly, the higher the body fat content, the higher is the concentration of insulin in the blood and cerebrospinal fluid. With an increasing level of insulin, the latter, acting on the ventromedial hypothalamus, stimulates reactions leading to a reduction of the amount of the food consumed. On the contrary, when the basal blood level of insulin decreases as a result of weight reduction, this leads to an increased appetite and to the elevation of body weight. The above hypothalamic system may be designated as the "strategical appetite center," as its activity serves for the constancy of fat content in an organism. However, the fact that these systems do not prevent the development of age-linked obesity in spite of double-regulation of appetite and body weight demands an explanation. To explain the mechanism of age-related impairment of appetite regulation, a proposal was put forward about the elevation of the threshold of the sensitivity of the hypothalamic centers of appetite regulation to regulatory signals (Dilman, 1958).4 The level of postprandial hypergly'This proposal was put forward in the period when the satiety center and the appetite center were not distinguished in the system of appetite regulation.

NEUROENDOCRINE-ONTOGENETIC MECHANISM OF AGING

101

cemia increases in the course of aging. This should lead to progressive appetite reduction with age and, correspondingly, to body weight reduction. If, however, this does not occur, it means that the sensitivity of the satiety center changes, i.e., the threshold of its sensitivity to the regulation by glucose elevates. As a result, in spite of an increasing level of postprandial hyperglycemia, the signals Tor the termination of Tood intake are generated later rather than in the earlier stage, i.e., when more food has been consumed than is necessary. In other words, the “weightingdevice” of the appetite center fails and a man involuntarily falls into error if he continues to follow the directions of his appetite as the measure of need for feeding. That is the reason why, in middle age, the appetite does not correspond to the energy expenditure, and a regulatory type of obesity develops. It is necessary to stress that 4 kg of excess weight, compared with an ideal norm, should be regarded as age-related obesity because this is enough for induction of blood lipid changes promoting atherosclerosis (Albrink et al., 1962). The considerations presented above permit one to hypothesize that the elevation of the threshold of sensitivity to the regulatory signals can be attributed not only to glucose but to insulin as well. Therefore, the strategical appetite center does not correct the deviations caused by the inadequate work of the tactical center. This follows from the absence of the normalization of body weight as a result of the increased insulin level caused by the obesity. It may also be hypothesized that the elevation of basal insulin level reduces the sensitivity of the hypothalamus to the regulatory action of insulin because of the down-regulation of the number of insulin receptors caused by hyperinsulinemia. In other words, in a certain sense it is not that a man grows fat because of overeating, but he eats too much because he is fat. On the whole, these changes may be summarized in the following model of the mechanism of the age-linked obesity: the age-linked elevation of the threshold of sensitivity in the tactical appetite center, the excessive food consumption, the accumulation of body fat, the elevation of basal insulin level, the elevation of the set point in the strategical appetite center, and the stabilizationof obesity. It is necessary to stress that, on the basis of availabledata, the elevation of the threshold of sensitivityof the appetite center to glucose may be referred to the reduction of the hypothalamic contents of noradrenaline (Saller and Stricker, 1976) and of serotonin (Breisch et al., 1976), although other neuromediators, in particular oligopeptides, also contribute to the appetite regulation (Morley and Levine, 1983). On the basis of these data, the increase in appetite as a consequence of a stress (Morley et al., 1983), which is often observed in middle-aged and elderly subjects (Johnson, 1947), may find an explanation. Within the conditions of stress, the age-linked reduction of biogenic amine levels adds

102

v. M. DILMAN et al.

to the stress-induced r e d ~ c t i o nThus, . ~ the age-related obesity is a disease of regulation. From this point of view, it is a normal disease of the same sort as are climacteric and hyperadaptosis because the basis of its pathogenesis is formed by the reduction of the sensitivity of the satiety center or, in other words, by the change of the set point for the perception of the glucose (or insulin) signal. The second subsystem of energy homeostate regulating the fluxes of energy substrates may be represented as a four-component system which regulates the relations between two main sources of energy, glucose and fatty acids, and two main hormones which control the utilization of these substrates, insulin and growth hormone (Dilman, 1981).6 Glucose and FFA are able to inhibit the secretion of growth hormone by acting on the corresponding hypothalamic areas (Glick et al., 1965). The intake of food, especially when it is enriched with carbohydrates, leads to the decrease in the secretion of growth hormone possessing insulin-antagonizing and lipolytic activity (Rabinowitz et al., 1965) and simultaneously stimulates the secretion of insulin, thus enhancing glucose utilization by tissue^.^ On starvation, in particular during sleep [sic nighttime], the secretion of growth hormone, on the contrary, increases. This leads to the intensification of lipolysis, with FFA becoming the main energy substrates as a result. The latter inhibits the glucose utilization by muscles in accordance with the Randle fat-carbohydrate cycle (Randle, 1965) which preserves glucose for the nervous tissues (Gomez et al., 1972). It is known that, in the course of aging, there occurs the reduction of the utilization glucose after its uptake by an organism, which is classified as the reduced glucose tolerance (O’Sullivan el al., 1971). Taking into account that this reduction goes with elevation of blood insulin level, the majority of authors came to the conclusion that the reduction of the tissue sensitivity to insulin action occurs in the course of aging (DeFronzo, 1979). The muscular tissue must be mainly responsible for this reduction, it T h e gut hormones cholecystokinin and bombesin inhibit stress-induced hunger by enhancing hyperglycemia (Levirie and Morley, 1981). is certainly taken into account, when the four-component energy homeostate is considered, that a whole number of hormones participate in the regulation of energy processes. However, they may be regarded as similar in the mode of action to one of the components of the outlined energy homeostate (the fat-mobilizing action of noradrenaline, prolactin, and ACTH may be taken as an example) or as acting by virtue of one of these components, for instance, the hyperglycemic action of glucagon (Dilman, 1981). ’It is necessary to take into account that the immediate reaction of insulin-secreting apparatus to the postprandial hyperglycemia decreases with age, whereas the late response increases. Correspondingly, in the first 5- 10 min after glucose injection, the decreased elevation in insulin concentration is observed in the elder subjects, whereas the overall insulin area increases with age.

NEUROENDOCRINE-ONTOGENETIC MECHANISM OF AGING

103

being the less sensitive to insulin action (Rabinowitz et al., 1965; Zierler and Rabinowitz, 1964). At the same time in the liver, the combined action of hyperglycemia and hyperinsulinemia must enhance VLDL secretion, leading to increased triglyceride transport and deposition into the adipose tissues, contributing to insulin resistance. This leads to the overall decrement in the sensitivity to insulin and the stimulation of insulin secretion. Age-linked obesity is usually connected with metabolic disturbances, manifested in increased blood triglycerides and cholesterol concentration (Table V). The mechanisms of this connection are still not clear. While taking into consideration that, in the Type I1 diabetes mellitus and obesity, the synthesis of VLDL is elevated (Angel, 1978; Greenfield et al., 1980; Reaven and Greenfield, 19Sl),it may be proposed that in the case of agerelated obesity, the same occurs (Dilman, 1981),albeit to a lesser extent. On the other hand, hypertriglyceridemia may also be caused by the agerelated reduction of the lipoprotein lipase activity in adipose tissue (Chen and Reaven, 1981)which is stimulated by insulin (Eckel et al., 1978).As to the age-linked elevation of the blood FFA levels (Davidson, 1979), it may be caused either by enhanced secretion of FFA by adipose tissue, being the consequence of its increased volume and of the insufficient inhibition of lipolysis by insulin, or by the reduced utilization of FFA caused by the decrease in the skeletal musculature and physical activity. The removal of the excessive blood FFA is carried out by virtue of their reesterification and transport into the adipose tissue as the fatty acids of the VLDL triglycerides (Have1et al., 1970). The relative roles of cholesterol synthesis and removal from the circulation in the origins of age-linked hypercholesteronemia in man is not established with sufficient evidence. Studies conducted on animals demonstrated the reduction of cholesterol synthesis in liver and of its turnover in the circulation (Hrfiza, 1971;Takeuchi et al., 1976).At the same time in man, the main cholesterol-containing fraction of lipoproteins, LDL, are derived in the circulation from VLDL (Eisenberg and Levi, 1975). That is why the question about the origins of age-related hypercholesteronemia is in a direct relation to the question about the origins of the age-related hypertriglyceridemia.8Hence, the central role in the origins of age-related HInestimation of the role of different factors in the origins of the age-linked hypercholesteronemia, the elevation of cholesterol synthesis in obesity (Nestel et al., 1973) should not be ignored, as this elevation can be observed within the middle-aged population (44-59 years). It is of importance in this connection that, within the age groups studied, the cholesterol production increases in parallel with the body weight (Nestel et al., 1968) and, correspondingly, with the body area and may not correlate with the level of triglycerides (Smith et al., 1976). The elevation in cholesterol synthesis is observed in diabetic disturbances also (Abrams et al., 1982), which are reminiscent with those characteristic of aging.

TABLE V AGE-RELATED CHANGES IN THE METABOLIC PARAMETERS I N HEALTHY MEN Age groups (years) Parameters Blood glucose, fasting (millimolelliter) One hour after standard glucose load Two hours after standard glucose load Blood insulin, fasting (picomolelliter) One hour after standard glucose load Two hours after standard glucose load Deviation from the ideal body weight Cholesterol (millimolelliter) Triglycerides (millimole/liter)

4-19

20-29

30-39

40-49

50-59

4.2 f 0.14

4.4 f 0.14

4.7 f 0.14

4.7 t 0.23

4.7

4.6

5.5 t 0.24

6.5 5 0..55

7.3 f 0.51

7.8 f 0.44

4.7

0.27

.5.3 f 0.38

5.8 f 0.53

6.5 t 0.60

136 t 17.2

165 +- 26.5

136 2 20.8

179 f 43.0

265

265 f 20.8

416 t 110.5

452

703 5 143.5

631 f 114.8

308 f 92.5

459 t 121.9

847 t 57.4

631 t 81.4

+3.3 f 3.1 5.6 t 0.20 1.51 t 0.098

+2.3 f 2.4 5.9 t 0.32 1.71 t 0.104

rt

0.18

-

-

+ 3.4

f 6.3 4.9 f 0.31 1.06 rt 0.103

-8.3 4.4 1.14

rt

f f rt

2.5 0.19 0.074

2

78.9

+1.2 f 3.01 5.3 f 0.22 1.36 f 0.097

2

rt

0.19

93.3

NEUROENDOCRINE-ONTOGENETIC

MECHANISM OF AGING

105

hyperlipidemia is most possibly played by the reduction in the sensitivity of tissues to insulin which is manifested in the reduced glucose tolerance and the exaggerated postprandial hyperglucosemia and hypertriglyceridemia. Besides the possibility of primary postreceptor defects in the mechanism of insulin action (McConnell et al., 1982),the possibility of the inhibition of glucose utilization in muscles by FFA should be considered (Davidson, 1979). The blood FFA level is increased in middle age (Pickart, 1983),and it falls to a lesser extent than in earlier ages in response to the glucose load (Golay et al., 1982). This creates the conditions for the enhancement of the fat-carbohydrate cycle and reduces the use of glucose as an energy substrate (Muntoni el al., 1978). On the whole, the agerelated metabolic changes are reminiscent of those occurring in the insulin-independent diabetes mellitus (Type 11). In both cases, the simultaneous elevation of blood insulin and glucose, reflecting the reduction of glucose tolerance, creates the conditions for the shift of pathways of glucose metabolism from oxidative to lipogenetic (Dilman, 1981).’ Considering the causes of age-related reduction in the insulin sensitivity, the influence of hormonal background created by hyperadaptosis should be taken into account, and, also, the loss of adequacy in the response of the system controlling growth hormone secretion to food intake (Dilman, 1981). It is proposed that these age-related changes are caused by the prolongation of the program of the neuroendocrinological shifts, ensuring the realization of the developmental program of an organism through the mature period (Dilman, 1981).Thus, the age-related changes in the lipid and carbohydrate metabolism, predisposing to diabetes and atherosclerosis, may be caused to a significant degree by hypothalamic changes, ensuring the growth and development of a human organism in the earlier period of life. This is the reason for the main question in this case being “In what way and to what extent may the described metabolic changes be linked with the changes in the hypothalamic regulation of energy homeostate?” 91t has been found in recent years that in man, normally, a relatively small amount of glucose carbon may be converted into FFA carbon in adipose tissues (Bjorntorp and Sjostrom, 1978; Acheson et al., 1982). However, within the conditions of hyperglycemia and hyperinsulinemia or carbohydrate overeating, the incorporation of glucose into triglycerides as their glycermoiety is greatly enhanced in obese subjects (Bjorntorp and Sjostrom, 1978). It should be noted that, with obesity, the liver fatty-acid synthesis is markedly enhanced (Angel and Bray, 1979) with the product being able to be transported into adipose tissue as the fatty acids of VLDL triglycerides. At the same time, the role of adipose tissue in the production of the cholesterol precursor, squalene, increases in obesity. The lack of quantitative data do not permit precise estimation of the role of these processes in the age-related metabolic shifts.

v. M. DILMAN et al.

106

TABLE \'I AGE-RELATED DECLIKE I N THE REDUCTION OF THE BLOODGROWTH HORMONE LEVELB Y THE STANDARD GLUCOSE LOAD

Mean age (years)

Fasting blood growth hormone (nanograms/milliliter) One hour after glucose load Two hours after glucose load O n

2.4 2 0.5

1.0 k 0.1

1.2 2 0.2' 0.9 +. 0.2'

0.9 0.9

* 0.1d 5

0.1"

= 22.

'n = 27. ' - 50% = the degree of reduction compared to the fasting levels. - 10% = the degree of reduction compared to the fasting levels. ' -62% = the degree of reduction compared to the fasting levels.

Before maturity, the glucose load causes marked reduction of the blood level of growth hormone, acting via the hypothalamic glucoreceptors (Table VI). This creates the optimal conditions for glucose utilization. In basal condition, high blood concentrations of both FFA and growth hormone are observed in children (Heald et al., 1967).It may be proposed that the "fatty brake" of growth hormone secretion is inefficient in this period (Dilman, 1981). The data of Table VI demonstrate that, in middle age, the reduction of sensitivity of the hypothalamic system of growth hormone regulation is observed. This permits the assumption that, as a consequence, the precision of the energy homeostate is disturbed so that in postprandial conditions, hyperglycemia does not lead to sufficient inhibition of growth hormone secretion. In accordance with the model that is being described (Dilman, 1971, 1979, 1981),it is this shift that, together with the relative hypercorticism, reduces the sensitivity of muscle tissues to insulin and initiates the chain of metabolic changes leading to prediabetes and obesity.'' It should be stressed, however, that the reduction of the basal level of growth hormone is also observed in middle age. This reduction may be caused by the inhibition of the growth hormone secretion by FFA, since the administration of nicotinic acid, which inhibits lipolysis, causes an increase in the growth hormone level (Quabbe et al., 1977). Thus, as the age advances, two opposite processes occur in the energy homeostate: while the sensitivity of the hypothalamus to the glucose inhibition decreases, the sensitivity to the FFA inhibition, on the contrary, increases. It I " The paradoxically increased growth hormone secretions in subjects with the reduced carbohydrate tolerance (Grecu el al., 1983) is demonstrative in this connection.

NEUROENDOCRINE-ONTOGENETIC MECHANISM OF AGING

107

is not established, however, at what age the change in the domination of inhibitory actions occurs. It seems likely that this age period chronologically coincides with the period of the completion of sexual maturation and of the increase in body size." In any case, from 21-27 years of age, the existence of the fatty brake of the growth hormone secretion is already quite pronounced. At the same time, the presence of the fatty brake and the related age-linked decrease in the growth hormone level, i.e., the decrease in the action of the factor that, as it was assumed (Dilman, 198l), plays the leading role in the age-linked reduction of the carbohydrate tolerance and, correspondingly, in the development of obesity, do not already lead to the elimination of the age-related metabolic shifts. It is assumed that after obesity has been developed its presence alone is sufficient for the reproduction of obesity and of the related metabolic shifts because of the development of the metabolic "fatty shunt" (Dilman, 1981). In fact, obesity is linked with increased lipolysis and elevated blood FFA and triglyceride levels (Nestel and Whyte, 1968).This, in accordance with the fat-carbohydrate cycle (Randle, 1965; see also G6mez et al., 1972; Greenfield et al., 1980; ThiCbaud et al., 1982a,b),leads to hyperglycemia and hyperinsulinemia (Dilman, 198l), which enhances the synthesis of triglycerides in the liver and, as a result, ensures the restoration of the fat deposits in spite of the intensified lipolysis. The synthesis of triglycerides from glucose in the adipose tissue may contribute to this to some extent (Dilman, 1981)." The assumption of the existence of this fatty shunt, in fact, converts the four-component system of the energy homeostate into the free-component mechanism, lacking its central (hypothalamic) element.I3 At the same time, succeeding by means of a drug such as phenformin (which increases the sensitivity of muscle tissues to insulin) in the impairment of the functioning of the fatty shunt, it is possible to observe "The accumulation of body fat plays some role in the mechanism of age-linked switching-on of the reproductive function (Frisch and Revelle, 1970). It is not clear, however, if the fat accumulation stimulates the elevation of the threshold of the sensitivity of the sex center to estrogens or the transformation of androgens into estrogens, since certain levels of both processes are necessary for the achievement of sexual maturation. In spite of the important role that fatty acids play in the creation of the hormonalmetabolic changes in the course of aging, it is still not established if the increase of their role as the energy substrates occurs. The occurrence of this increase in reduced glucose tolerance (Felber et al., 1981) permits us to admit its occurrence in the course of aging (Dilman, 1981). "Perhaps this is due to the impairment of the temporal parameters of the response of the growth hormone secretion to food intake, i.e., inadequate functioning of energy homeostate, rather than the quantitative changes in the growth hormone secretion, is the most responsible for the central mechanisms in the age-related reduction of the glucose tolerance.

108

v. M.

DILMAN

et al.

the reduction of postprandial hyperglycemia of blood FFA level, of the levels of hyperlipoproteinemia, hypercholesteronemia, the weight of the body, etc. (Table VII). In summary, the three superhomeostates have much in common with respect to their changes, taking place with advancing age. These changes ensure the fulfillment of the demands of body growth, sexual maturation, and the increase in the adaptational capacities of an organism, i.e., the changes in fact accomplish the realization of the genetic program of the development of an organism. At the same time after the completion of the program, the driving force of these homeostatic changes does not disappear. Whatever ensured the deviation of homeostasis during postnatal growth continues to exist and to act after the completion of the growth, thus creating the situation of immediate transformation of the developmental program into a mechanism of development of the pathological processes coupled with this program. ‘I.ABLE V I I

INFLUENCE OF PHENFORMIN O N THE ENDOCRINE, METABOLIC, A N D IMMUNOLOGlCAI PARAMETERS I N MAMMARY CAXCER PATIEX33 I N REMISSION Parameter Body weight (kilograms) Body fat content (percentage) Serum contents of glucose in-2 hr after standard glucose load (millimolelliter) Insulin (picomole/liter) Growth hormone (microgramdliter) 1 1 -0xycorticosteroids (millimole/liter) Cholesterol (millimole/liter) Triglycerides (millimolelliter) Total-lipoproteins (units of extinction) Free fatty acids (millimolelliter) Somatomedin-like activity (unitslliter) Cholesterol in urine (millimole/literlmole of creatinin) Lymphocyte blast-transformation (cpm) Percentage of T lymphocytes Percentage of B lymphocytes Skin tests (diameter of papula, millimeters) DNCB PPD (1 : 1000) Candidin

Before treatment 80.4 f 2.7 44.0 f 0.8

After treatment 77.3

-C

2.4

41.6

k

0.6

8.2

rt

0.54

6.4

k

0.48

565 0.7

2 f

116.2 0.4

250 0.5

k k

63.8 0.2

425

5

13.8

309

rt

7.3 rt 2.3 2 677 2 811 f 1,800 rt 0.152 2

0.24 0.26 17 56 420 0.0127

14,521 f 3,820 55.6 % 2.4 31.8 2 2.7 3.5 5 2.0 3.2 2 3.3 3.2

2

2.0

6.3 2 2.1 f 568 2 639 2 800 2 0.096 ?

13.8

0.05 0.31 23 24 240 0.0204

38,221 2 5,130 56.0 k 2.3 37.6 f 3.2 8.5

2

1.2

15.1 2 5.0 12.4 2 1.8

NEUROENDOCRINE-ONTOGENETIC MECHANISM OF AGING

109

VI. The Choice of Diseases in the Course of Aging. The Interrelations between Main (Noninfectional) Human Diseases

Statistics show that, of the many hundreds of known diseases, only 56 pathological processes constitute the causes of deaths of 85 out of 100 subjects in middle age and elderly populations (WHO Yearbook, 1967). These diseases are atherosclerosis,cancer, hypertensive disease, the adultonset diabetes mellitus, and a group of respiratory diseases, including pneumonia and influenza, with the fatal outcome usually determined by the reduced resistance to infections. The increase with age in the incidence of each of these diseases may be connected with different factors, the damaging influences of the external environment being the first to come to mind. It was possible to see, however, in the example of climacteric, that there is a definite class of diseases that regularly develops on the basis of the processes that, in a theoretical, idealized consideration, may be represented as independent from the external environment and as a pure variant of pathological phenomena entirely dependent upon the internal factors of the development of an organism. In the immediate association with the age-connected regular hypothalamic shifts, there exists climacteric, prediabetes and obesity, hyperadaptosis, and, also, the age-linked psychic depression. The available data and the relevant proposals about the nature of the factors that cause these hypothalamic shifts will be considered below. Here it should be noted that these shifts are realized at least partly at the expense of the reduction of the biogenic amines in the hypothalamus, as initially proposed by Stoll (1972).In turn, this reduction causes the temper to fall and, in pronounced forms, may lead to typical psychic depression. Correspondingly, the resistance to the dexamethasone test is revealed in patients with the psychic depression (Carrol et al., 1976; Ostroumova et al., 1978), and, also, the excessive level of blood cortisol, indicating hyperadaptosis was found (see Table 11). Thus, the mood reduction occurring with age, is a by-product of the realization of the developmental program of an organism and may be regarded as a primary normal disease of the same sort as climacteric, age-related obesity, and hyperadaptosis. The primary normal diseases constitute the mechanism forming the secondary normal diseases. Metabolic immunodepression is a typical example of such secondary normal disease. After a number of years of studying the immunological phenomena, the factors for their regulation were sought within the immune system itself. However, the data on the immunosuppressive action of glucocorti-

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coids, followed by the discovery of the thymic endocrine function, radically broadened the sphere of the known interactions of the immune system. The data on the involvement of hypothalamic structures in the succession of the processes occurring in the course of the immune response (Besedovsky and Sorkin, 1977)were an important hallmark on this mechanism. Along with the elevation of the nerve impulse generation in certain liypothalamic structures and with hormonal changes such as the increase in the blood cortisol level, the development of certain metabolic shifts, in particular of hypertriglyceridemia and hypercholesteronemia, has been noted (Mathews and Feery, 1978; Kerttula et al., 1981). On the basis of these data, it may be assumed that the age-linked impairment of immunity (or perversion ot immunological reactions) may depend not only upon the decrease in the production of thymic factors but, also, upon the changes in the sensitivity of immunoreactive hypothalamic structures to humoral immune signals. (The age-related decrease in the sensitivity of an organism to pyrogens attracts attention in this respect, taking into consideration the possibility of the coupling of immune and temperaturecontrolling systems (Duff and Durum, 1983). However, in spite of the discrimination of the “network of immuneendocrine interaction” (Besedovsky and Sorkin, 1977) and the attention paid to the role of psychic factors, in particular of psychic depression in the decrease or perversion of immunological reactions (Solomon et al., 1974; Stein et al., 1976; Spector and Korneva, 19Sl), one more interaction remained underestimated, namely, the one between the immune system and the metabolic state. At the same time, the data were available on the one hand that the calorie or protein deficit can influence the state of the immune system (Chandra, 1980) and, on the other, that in such states as diabetes meliitus and obesity there often occurs the reduction of the resistance to infections and the impairment of immune reactions (Mahmoud et al., 1976; Delespesse et al., 1974). Starting from the year 1976, the concept of metabolic immunodepression emerged, i.e., of the inhibitory action of a certain combination of metabolic factors on the cellular immunity and on the phagocytic function of macrophages (Dilrnan, 1976,1979,1981;Golubev and Dilman, 1981). On the basis of this idea, the proposal was made to use the antidiabetic drug phenformin and the lipid-lowering drug ciofibrate for improvement of immunity. In 60-70% of patients with atherosclerosis and of cancer patients in clinical remission (respectively,Tables VIII and VII), the improvement of some parameters, characterizing the state of the cellular immunity, was achieved (Dilman et al., 1982; Dilman, 1981). The analysis of the data, obtained in the author’s laboratory, as well as of the data of the literature concerning the factors that induce metabolic

NEUROENDOCRINE-ONTOGENETIC MECHANISM OF AGING

111

TABLE VIII PATIENTS WITH ATHEROSCLEROSIS Parameter

Before treatment

After treatment

The influence of phenformin on the metabolic and immunological parameters 12.3 5 4.0 Excess of body weight (percentage) 9.3 ? 3.2 Serum cholesterol (millimole/liter) 7.87 ? 0.39 7.02 ? 0.28" 1.94 f 0.18 Serum triglycerides (millimolelliter) 1.54 f 0.1gb 659 f 47 576 2 32 Serum-free fatty acids (millimole) 28,047 ? 6,277 Lymphocyte blast-transformation (cpm) 39,731 & 9,466b 59.2 f 2.6 Percentage of T lymphocytes 63.4 ? 2.2b 15.0 f 2.1 Percentage of B lymphocytes 27.3 -c 2.6 44.4 ? 3.4 Percentage of monocytes, differentiated 57.0 -c 4.7" into macrophages in vitro Phagocytic index (number of iatex 6.92 ? 0.73 7.4 ? 0.56 particles per cell)

Lipid dynamics in female patients who have been treated with biguanides Scrum cholesterol 7.44 f 0.28 6.86 2 0.31" Serum triglycerides 1.54 ? 0.16 1.20 ? 0.15"

" p = 0.01, according to the Wilcoxon criteria. b p = 0.05, according to the Wilcoxon criteria. immunodepression, was made in other papers (Dilman, 1979, 1981; Golubev and Dilman, 1981; Golubev et al., 1983). In summary, these are the factors that act in the course of normal aging, namely, the increased blood concentrationsof VLDL, LDL, triglycerides, and fatty acids in conjunction with elevated blood insulin, the elevation of cholesterol content in the blood, and an elevation of cholesterol in lymphocytes that participate in metabolic irnrnunodepr~ssio~i.'~ Although metabolic irnmunodepression is a secondary disease, it is nevertheless a normal disease as soon as it regularly develops more or less rapidly in the course of normal aging. As Hallgren at al. (1973) noted, hyperglobulinemia, impaired lymphocyte blast transformation, and the elevated incidence of autoantibodies are typical signs of aging. There is no satisfactory explanation for this combination, but there are reasons to believe that certain relations between metabolic immunodepression and the autoimmune disorders exist. An explanation of these relations may be found in one of the mechanisms l4 By this combination of metabolic shifts (and by their origins), metabolic immunodepression differs from the irnmunosuppressivc action of polyunsaturated fatty acids (Meade and Mertin, 1978), the latter being the precursors of immunosuppressive pros taglandins.

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participating in the support of immunological tolerance, with are based on the clearance from the circulation of the antigens derived from an organism’s own tissues with the help of the cells belonging to the reticuloendothelial system, and, possibly, of the autoreactive T lymphocytes and autoantibodies. T h e depression of the functions of the reticuloendothelial tissue and of lymphocytes under the influence of the factors participating in metabolic immunodepression may create the conditions for the accumulation of an antigen u p to a level that makes the clonal expansion of the sensitized lymphocytes possible and, consequently, leads to the accumulation of autoantibodies and of autoaggressive cytotoxic lymphocytes. The existence of a mechanism of this sort is demonstrated in diabetes mellitus, accompanied by marked depression of the functions of the reticuloendothelial system (Iavicoli et al., 1982). These arguments permit the assumption that the increased incidence of autoimmune processes in an aging population may also be connected with metabolic immunodepression. Correspondingly, the inhibition of the development of autoimmune lesions in experimental animals by calorie restriction (Fernandez et nl., 1978) may be explained by the normalization of some age-related metabolic shifts. Metabolic immunodepression opens additional possibilities in the search for the explanation of some peculiarities of the immune changes in the second half of pregnancy, i.e., the reduction of the transplantational immunity with simultaneous satisfactory state of humoral immunity (Strelkauskas et al., 1978). Besides this, the last example illustrates the possibility that the phenomenon of metabolic immunodepression itself is a reflection of the processes that help to reduce the probability of the rejection of a fetus as an allotransplantate. Distinguishing the syndrome of metabolic immunodepression permitted us to find out the connections between obesity and Type I1 diabetes mellitus on the one hand and the age-related immune disturbances on the other and, also, between the peculiarities of the mechanism of development of this phenomenon and the conditions promoting atherosclerosis and cancer. For example, metabolic immunodepression may promote the damage of the capillary walls by the complexes of lipoproteins with autoantibodies by enhancing the autoimmune component of these processes [in this respect, the coupling of autoimmnune disorders with hyperlipidemias (Beaumont, 1980) is significant]. Besides this, the enrichment of macrophages with lipids suppresses the capability of the latter for the clearance of blood constituents (Chapman and Hibbs, 1977). As to the role of the described changes in the age-linked increase in the cancer incidence, the discussion of this major problem is beyond the scope of this review. It should be noted, however, that any steady disturb-

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ance of endocrine regulation, if it has cell proliferation-stimulating effect, must promote tumor development. For instance, the elevation of blood gonadotropins or of thyreotropin may cause the development of ovarial or thyroid cancers by stimulating cell proliferation in target tissues. Correspondingly, estrogens or thyroid hormones possessing the antistimulant action render an anticarcinogenic effect in both cases. In the two cases above, the decline in the level of the peripheral hormones, for instance, caused by subtotal castration or by dietary iodine deficit, is usually considered to initiate the disturbances in the hormonal homeostasis. Although such situationsare quite real in the course of aging, another variant of homeostatic disorders regularly occur, which is connected with the primary elevation of the sensitivity threshold of the hypothalamic-pituitary complex to the regulatory signals. Applying this principle to the reproductive system means that age-related elevation of the secretion of gonadotropins begins long before the menopausal decline in the secretion of classical estrogens. The analogous phenomenon occurs in the course of aging in the hypothalamic-pituitary-thyroid system, with the decline in the sensitivity to the inhibitory action of triiodothryronine being observed (Table IX) (Yevtushenko and Bobrov, 1978). Thus, the changes connected with the implementation of the Law of the Deviation of Homeostasis are able to promote cancer development. However, their influence is not limited by what was stated above. The analysis of experimental and clinical data reveals that no matter what is the cause of malignant transformation of a cell, there exist three conditions of general importance that promote cancer development. Namely, the probability of cancer development is higher with the higher number of proliferating cells, with the lower activity of the cell-mediated immunity and of macrophages, and with the lower activity of the DNA reparative system. At the same time, the hormonal-metabolic shifts inherent in aging promote the appearance of not only one of the above conditions, but, simultaneously, of two or three conditions facilitating the arisal of cancer. First, these age-related shifts induce metabolic immunodepression. Second, these shifts may stimulate the division of cells of certain types. The enhancement of cell division under the influence of hyperlipidemic system (Bierman and Albers, 1975) and of the serum of diabetic patients (Miller et al., 1977) may serve as examples. It was found that the somatomedin-like activity increases in the serum of patients with obesity, atherosclerosis, and adult onset diabetes mellitus (Vasilyeva et al., 1982) and that this activity decreases in these patients with phenformin treatment (Vasilyeva et al., 1980). It was experimentally shown that the cell proliferation in the wall of the aorta, induced by an inquiry to the wall, increases

TABLE IX

THE%ATE

OF T H E

THYROID HOMEOSTATE I N HEALTHY

W O M E N AND IN PATIENTS, AS ESTIMATEI] WITH T H E TRIIOUOTHYRONINE

TEST

Number Of

Study group Control

Fibroadenomatosis Mammary cancer

Endometrial cancer

“ p = 0.05. b p

= 0.01.

T4

Age observaCholesterol Reduction (years) tions (millirnole/liter) (percentage) 32 f 2.8

14

54 t 3.1

14

35

2.2

18

51 t 2.0

11

37

3.6

12

54 f 2.1

17

56 f 3.4

10

rt

rt

5.5 5.0 6.3 5.9 5.7 5.0 6.2 5.7 5.9 5.5 6.6 6.0 6.7 6.6

t 0.31

0.23 0.39 f 0.24 f 0.24 f 0.18 f 0.37 2 0.41 2 0.41 2 0.46 f f

2 0.44 f 0.40 If: 0.36 If: 0.42

-9 -6

- I4 -9

-7 -9

-

TSH (milliunits/liter) 7.5 5 5.2 2 11.3 2 9.2 2 6.9 +5.6 2 12.3 f 11.2 2 7.0 t 5.1 2 12.3 f 11.0 2 13.9 -c 13.0 t

1.16“ 0.87’ 1.00 1.15 0.83 0.74 1.21 2.00 1.15 0.96 1.31 1.42 0.88 1.31

Reduction (percentage)

(nanomolesl liter)

Reduction (percentage)

-31

117 t 1.3’ 84 f 14.1 93 f 7.7 80 f 5 . 1 86 If: 7.7 81 t 11.6 64 f 12.9 60 t 15.4 81 2 9.0 67 2 7.7 100 2 15.4 81 f 15.4 71 t 10.3 73 f 16.7

- 28

- 18 - 19 -9 - 27 -9 -6

- 14

-6 -6 - 17 - 19

+4

NEUROENDOCRINE-ONTOGENETIC

MECHANISM OF AGING

115

with age (Stemerman et al., 1982).15Finally, the age-related changes of homeostasis in the endocrine system may cause a number of proliferative effects by the elevated levels of hormones, the gonadotropins being an example. It is not yet clear if the metabolic shifts can influence the DNA reparative system, although a correlation was found between serum cholesterol and the reduction of the capacity of lymphocytes for UV-induced DNA repair (Dilman and Revskoy 1981). Thus, the same metabolic shifts promote the proliferation of nonlymphoid cells of certain types on the one hand and, on the other, they inhibit the cellular immunity, i.e., they induce the state of cancrophilia-a complex of metabolic conditions promoting cancer development. In favor of the role of cancrophilia in cancer development, the following experimental and clinical evidence may be cited as follows: (1) treatment of C3H mice with phenformin reduces the cumulative incidence of mammary carcinomas up to 20% in an experimental group, compared with 80% in the control group (Dilman and Anisimov, 1980); (2)phenformin reduces the DMBA-induced mammary cancer incidence in rats (Dilman et al., 1978);(3)calorie restriction improves the indices of cellular immunity in experimental animals and leads to prolongating the mean life-span (Fernandez et al., 1978; Weindruch et al., 1982); (4) numerous observations in the human populations show that excessive consumption of dietary fat and cholesterol and excessive calories in general correlate with the increased incidence of mammary, endometrial, prostate, colon cancers, and tumors of some other localizations. At the same time, if hypercholesteronemiapromotes cancer, a special explanation is necessary for the statisticaldata obtained in the longitudinal epidemiological studies, which demonstrated the subsequent lower cancer mortality among the subjects with the initial elevated serum cholesterol and the elevation of cancer mortality among those with the lower serum cholesterol (see Feinleib, 1981, for a review). As to the lower cholesterol level, several factors participating in the multifactorial origins of cancer may interfere in the estimation of the role of this physiological parameter. The low initial blood cholesterol level is most characteristic of men (not mentioning the cases in which clinically unmanifested cancer was present at the time of the examination of the blood parameters) in whom subsequent colon cancer develops. In these cases, lowered blood cholesterol may be caused by an elevated cholesterol l5

It should be noted that the relatively high cancer mortality rate, characteristic of 55-70 YedI S, Illdy bC LdUbed by the pIUbIlgCd ldleIlt pel iOd Of nldIly L d I l L t X b

LhC dlgC U f

initiated by the events that occur in the age period when the reduction of the cellular proliferative potential is insignificant.

116

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excretion within the feces, which is observed, in particular, in familial colon polyposis, which elevates the probability of colon cancer development (Broitman, 1981; Reddy et al., 1978). In accordance with this, data are available on the coincidence of gallstone disease with colon cancer (Weitz et al., 1983)and on the enhancement of chemical induction of colon cancer in animals by the cholesterol-loweringdrugs acting by the elevation of cholesterol excretion (Nigro et al., 1977). The range of the serum cholesterol level, in which the elevation of cancer incidence with lowering of serum cholesterol is observed, is substantially lower than the normal age-specific serum cholesterol values. This permits one to assume that the individuals with such a low cholesterol level constitute a separate subpopulation with some unidentified genetic defect leading, as it is proposed, to immune deficiency. In these cases, hypocholesteronemia is a marker of this defect. But, with the multiplicity of the factors that influence cancer development, the possibility of complications in interpreting the data concerning each separate factor, the blood cholesterol level in particular, should be kept in mind while analyzing the relation between cholesterol and cancer throughout the range of cholesterol concentrations.'" '"The high levels of hypercholesteronemia with the reduced cancer incidence in some populations cannot be a result of age-related processes that induce the syndrome of cancrophilia. Rather, they are a manifestation of familial hypercholesteronemias caused by the reduced levels of the receptors for LDL on the cells, in particular on the lymphocytes (Ho et al., 1977). T h e cholesterol content in the lymphocytes is normal in these cases in spite of the elevated blood cholesterol level. This permits one to assume that the rate of the development of metabolic immunodepression is lower in these cases, leading to the decrease in the cancer incidence. Thus, the individual with extremely high as well as with extremely low cholesterol levels differs from the main population in which hypercholesteronemia occurs as a consequence of definite metabolic shifts inherent in normal aging. It is this population in which the development of metabolic immunodepression and cancrophilia is characteristic. The following, however, should be taken into account. T h e atherosclerosis-related mortality increases with the elevation of the cholesterol level in the range of 220-270% mg (which corresponds to the age-related blood cholesterol elevation). I t is this range within which the cancer-related mortality is not dependent upon the blood cholesterol level in the age groups studied (Feinleib, 1981). For the explanation of the contradictions between these data and the data on the enhancement of cancer development by dietary hypercholesteronemia, it may be assumed that the influence of the age-related hypercholesteronemia on cancer development is not direct, but is mediated by metabolic immunodepression. T h e latter, as soon as it has developed, is not influenced by the elevation of hypercholesteronemia. It is possible to explain, from this point of view, the reason why the activity of the cellular immunity declines not more than 50% up to the age of fifty (Makinodan, 1978) and why the elimination of the corresponding metabolic shifts leads to the elimination of metabolic immunodepression. However, the longer is the time since metabolic immunodepression has developed; the higher is the probability of a tumor development as a result of the accumulation of stochastic damage at the cellular level (Dilman, 1983).

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117

The concept of cancrophilia provides the basis for the explanation of why a number of normal diseases are coupled with an increased cancer incidence and how they influence the course of cancer. Thus, for example, psychic depression and hypercorticism aggravates the clinical course of cancer (Saez, 1974; Bishop and Ross, 1970). These cancer patients have a tendency toward elevated hypertensive disease incidence (Dyer et al., 1975). A number of cancer localizations is promoted by obesity (Tannenbaum, 1959). The reduced carbohydrate tolerance is often observed in cancer patients with the normal or elevated insulin levels, which corresponds to Type I1 diabetes mellitus. On the whole, any normal disease pattern incorporates the elements of cancrophilia to a certain extent. What reflects the common etiology of these diseases is the absence of pronounced borderlines between them or the dependence of the diseases upon one another, their eminent connection being with the mechanism of aging. That is why at times all ten main diseases may be found in one patient. Such an age-related polypathology is exemplified by the data on the incidence of these diseases in women with endometrial cancer, presented in Table X (Dilman, 1981). It should be stressed that cancrophilia creates the conditions that only influence the probability of cancer development, but other factors determine whether it will occur in the first place. Such factors are connected with the state of oncogenes and transforming proteins (oncoproteins; Weinberg, 1983)and the transforming growth factors (Todaro et al., 1982) TABLE X ENDOMETRIAL CARCINOMA-ILLUSTRATION OF INTEGRATED DISEASE Parameter (or disturbance)

Frequency of disturbance ~

Arterial hypertension Obesity Hy percholesterolemia Age of menopause onset Climacteric bleeding Dexamethasone suppression test (psychic depression, hyperadaptosis, latent Cushingoid signs) Metabolic immunodepression Antibodies to thyroglobulin Birth of large baby (4 kg or more) Diabetes mellitus (chemical, latent, or overt) Suppression of blood growth hormone after glucose load

40% 21-72% 64.1%

51.9 years (49.5 years in control groups) 27.7-59% (14%in control) Resistance to inhibition in most patients (in control, 45.3%) In most patients 25% (in control 6.8%) 50% (in control 17.7%) 63-73% (in control, 20%) 0% (in age-matched control, 1 1 % ; in young control group, 50%)

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that constitute the system oncogene-oncoprotein-oncofactor regulating cell differentiation as is discussed elsewhere (Golubev and Dilman, 1984; Golubev, 1984). In this respect, it is of interest that cancrophilia can play a role in the initiation, promotion, and progression of tumor growth. It is believed now that the key role in initiation is played either by the increase in the dose of an oncogene as a result of the integration of a viral genome into a cell genome or by the activation of a cell oncogene activity caused by DNA or chromosome damage by chemical or radiation factors. The role of cancrophilia in the realization of these events cannot yet be determined with sufficient confidence. If the data i s confirmed that the activity of DNA reparative systems declines in parallel with the development of the metabolic shifts characteristic of aging, then the elimination of cancrophilia would facilitate the reduction of cancer incidence. It is also possible that certain hormonal shifts may play a direct role in the mechanism of initiation. For example, glucocorticoids may cause the activation of certain oncogenes (Govindan et al., 1982). Taking into consideration the regular development of hyperadaptosis in the course of aging, the carcinogenic significance of this factor may be considerable. As to the stages of promotion and progression, cancrophilia, by means of stimulating cell division, may promote tumor development, although the precise mechanisms remain obscure. It is of interest in this respect that the level of blood somatomedin-likeactivity increases in obesity, Type I1 diabetes, and in atherosclerosis,especially if the Type IIb hyperlipidemia is detected (Vasilyeva et al., 1982).Other relations of a similar sort are possible between the changes characteristic of aging and other growth factors. For example, the elevated blood lipid levels increase the tendency of platelet aggregation. This increases the possibility of the appearance of the metastases, which is known to be under the influence of the capability of the platelets to aggregate (Karpatkin el al., 1981). One of the consequences of platelet aggregation is the entrance into the circulation of the platelet-derived growth factor. Not only this, but other growth factors, in particular the transforming growth factors, are extracted from platelets (Childs et al., 1982). In summary, it is possible to assume that the metabolic shifts inherent in aging may facilitate the initiation as well as the promotion and progression of tumor growth. Thus, although the appearance o f a tumor is mainly dependent upon the result of the stochastic damaging effects on a cell, the presence of cancrophilia may increase the probability of a malignant process. Finally, for the last, but not the least of the ten normal diseases, the hypertensive disease, or more correctly, for the age-associated elevation of the blood pressure, its place in the pattern of the diseases produced by the action of the Law of the Deviation of Homeostasis has not been found.

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First, this situation is linked with the pathogenetic heterogeneity of the symptomatic complexes classified as the hypertensive disease, and, also, there is a lack of definite data on its pathogenesis. This makes it necessary to attempt distinguishing the hypothalamic forms of the hypertensive disease from the sum of the hypertensive syndromes. It seemed for a time that the reduction of the dopamine content in hypothalamus, indicated by the increase in the blood prolactin level in hypertensive patients, may be of importance in this respect (Stumpe et al., 1977). But, these data were not confirmed (Holland and Gomez-Sanchez, 1977), although the fact attracts the attention that, in the first study,young patients were examined, while, in the second, the subjects studied had a mean age of 51 years, when the metabolic shifts capable of inhibiting the prolactin secretion (obesity and hyperglycemia) may exist. The reduction of blood pressure in hypertensive patients under the influence of dopaminergic agonist bromocriptine was also observed (Sowers, 1981). The elevation of insulin level in hypertensive disease (Welborn et al., 1966),the often observed obesity and reduced tolerance to glucose (Lang, 1950),and the elevated cancer incidence all indicate the existence of some interconnection between the hypertensive disease and other normal diseases and compels the search for the basis of this interconnection. Hyperinsulinemia may constitute a part of this basis, since it elevates the renal sodium reabsorption (Bjorntorp, 1982; Drury, 1983). The development of the 10 main diseases may be considered within the framework of the common pathogenesis without referring to any other factors out of the sphere of the Law of the Deviation of Homeostasis. Moreover, the structure of this complex of diseases is predetermined by the structure of the three main homeostates of an organism, i.e., of the energy, adaptive, and reproductive homeostates, and by the way in which the changes in them are carried out that are necessary for the realization of the developmental program. These same factors also predetermine the choice of these 10 diseases out of the great number of others and their association with the process of normal aging. On the contrary, those homeostatic systems that do not demonstrate regular changes in the course of development do not participate in the molding of the coupled mechanism of aging and age-related pathology based on the implementation of the Law of the Deviation of Homeostasis. Thus, for instance, the ionic equilibrium in blood is supported at the same state throughout the period of development; hence the existence of the regular age-dependant pathological processes based upon the disturbances of this equilibrium are hardly possible (Dilman, 1981).This is the reason that the 10 normal diseases,which underlie the mortality in human population in middle and elderly ages, are significant in what may be called natural death increasing as the age increases. In many cases of death in old age, the causes of death

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are not determined precisely, and there arises the supposition of death because old age itself arises. It is more correct, from the point of view of the concept presented here, to classify these outcomes as death because of the diseases," since the old age in many respects is endowed with the properties of a disease, o r more correctly, of a sum of homeostatic diseases (Dilman, 1981).

VII. Aging as a Disease and as a Stochastic Process

In the course of normal aging, homeostasis deviates in the systems controlled by the reproductive, energy, and adaptive homeostates. The secretion of gonadotropins and nonclassical phenolsteroids increases, while that of the classical estrogens decreases. The production of androgen-like corticosteroids (ethiocholanolone, dehydroepiandrosterone, and other neutral 17-ketosteroids)decreases, creating the relative excess of the glucocorticoids action, especially in conjunction with the impairment of the rhythm of their spontaneous and stimulated secretion (hyperadaptosis). Sensitivity to insulin action decreases, as well as the tolerance to glucose; the blood concentrations of VLDL and LDL and, correspondingly, of triglycerides and cholesterol are increased. Blood pressure becomes elevated, the concentrations of the biogenic amines in the hypothalamus decline, etc. All the hormonal-metabolic shifts, considered above in more detail, form the basis for the development of normal diseases coupled with aging. At the same time, the regularity of all these shifts, inherent in normal aging, permits one to define the latter as a normal disease. In other words, in a certain sense, namely, in the sense of the homeostatic deviation, aging appears to be a disease or, more correctly, a complex of homeostatic diseases. From this point of view, it is impossible to demarcate clearly between aging as a disease and the normal diseases coupled with aging. Hence, it is of primary importance to find out w-hat the mechanisms are that participate in the age-related impairment of homeostasis. Of course, the chain of the phenomena between what is coded in the genome and what is realized at the level of the apparent processes that occur in aging cannot as yet be described with satisfactory precision. Only separate links of this chain are defined. However, what is known is of primary importance, since it must be taken into account in working out the measures directed against aging and the diseases that are "Especially, the possible role of hyperadaptosis, which cannot be estimated on the basis of postmortem investigation, should be taken into account.

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121

linked with it. In particular, the changes in the threshold of the sensitivity of the hypothalamus to the regulatory influences that lies at the basis of the age-linked deviation of the homeostasis may be connected with the following processes: 1. the age-linked decrease in the hypothalamic content of catecholamines and serotonin, as well as the changes in the balance between them (Robinson, 1975; Finch, 1976; Dilman et al., 1978); 2. the elevation of the activity of monoamine-oxidase in certain structures of the midencephalon (Robertson et al., 19'77); 3. the decrease in the number of the hormone receptors (Roth, 1979); 4. the decrease in the production of the hormones of the pineal gland, in particular of those of polypeptide nature (Ostroumova and DiIman, 1972; Dilman, 1981); 5. the metabolic shifts inherent in aging (Dilman, 1981). It should be noted that the relations between the hypothalamic activity and the energy metabolism are most distinctively revealed in the studies which demonstrated that an increase in caloric intake accelerates the agelinked switching-on of the reproductive function (Kennedy and Mitra, 1963; Frich, 1973), which in turn may be caused by the corresponding hypothalamic shifts. In general, the analysis of the changes related to the reproductive function, more than of those related to other systems, helps in understanding the relations between the development, aging, and the age-associated diseases, and, hence, in the consideration of the existing theoretical views on the mechanism of aging; 6. the secondary hormonal shifts, which are initiated by the primary hypothalamic changes and, in turn, affect the state of the hypothalamus. In this respect, the term which was introduced by C. Finch (1976), "the cascade mechanism of aging" is appropriate. In accordance with this mechanism, the decrease in the number of receptors for steroid hormones, for example, may lead to the decrease in the sensitivity of the hypothalamus to the regulatory signals and, in this way, to the cascade amplification of the deregulatory changes that have initially appeared in the hypothalamus. Aging is not an ordinary disease of homeostasis. Its manifestations also depend upon stochastic damage, progressing in quantity and in quality with advancing age, i.e., with the passage of time. This, for instance, has been known for almost 100 years that in the course of aging there has occurred in cells the progressive accumulation of lipofuscin (the age pigment) that has appeared to be the product of conjugations of lipid peroxides with the proteins. Such a ballast accumulation cannot but influence the cell functions, as well as cause other damage, in particular,

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damage caused by free radicals and occurring in all cells, especially in the postmitotic ones. In particular, the destruction of the genetic apparatus takes place, being reflected by the decrease in ribosomal RNA content, the derepression of the synthesis of certain proteins that are not characteristic of a given tissue, etc. (Cutler, 1978, 1980). The hypothalamus and other brain structures, connected with the implementation of the developmental program, must not radically differ in this respect from other tissues. Correspondingly,stochastic factors must contribute to the overall picture of aging, not only by creating the changes that occur in the peripheral tissues, but by creating the changes in the brain tissue as well. In particular, this process may be reflected in the agedependent neuron loss. However, this may be a secondary consequence of the atherosclerotic damage of the brain blood vessels. Consequently, the problem arises of the interrelations between the stochastic processes, considered to be the primary cause of aging by a number of authors (Cutler, 1978; Gensler and Bernstein, 1981),and the development of the regular, age-dependent symptoms of aging, reflecting the peculiarities of the developmental program of higher animals (Dilman, 1981). This problem will be treated in some detail below. Other aspects of this problem with respect to carcinogenesis, embryogenesis, and the Hayflick limit are discussed by Colubev (1984). Considering the role of the stochastic damage in aging, which may have other facets besides the mere imposition of additional features onto the picture of regular age-linked processes, it is necessary to keep in mind that the existing mechanisms of aging as neuroendocrine, among others, are a result of the evolutionary process. Hence the evolutionary aspects of the problem of aging constitute the natural basis for the general theory of aging, including and uniting the regular and the stochastic sides of this phenomenon. In the course of the biochemical evolution leading to the development of the present metabolic mechanisms,of primary importance was the fact that not a single compound participating in metabolic processes is monoreactive (Bartosz, 1981). However, of the enzymatically catalyzed reactions maintaining the life of an organism and being regulated in accordance with the demands of this maintainance, only those reactive potencies of these compounds that correspond to these demands are realized. All the other potencies constitute the basis of possible numerous nonenzymatic interactions as well as of the monomolecular reactions, such as racemization, functional group transformation, and splitting, etc., which lead to the impairment of the structure and function of macromolecules when the corresponding compounds are incorporated into their structure (McKerrow, 1979; Gensler and Bernstein, 1981).In recent times, a good deal of attention has been focused on the role in these processes

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of the reactions that involve oxygen and its free-radical forms (Fridovich, 1978; Soloway and LeQuesne, 1980; Halliwell, 1982). The development of the capacity to use oxygen for energy production was an extremely important evolutionary achievement, increasing the vitality of the organisms. However, it is this same achievement that appeared to be coupled with a multitudc of sidc reactions, since thc high-cncrgy outcome of the aerobic processes is ensured by the high activity of oxygen, easily participating not only in the enzymatically catalyzed interactions, but in many others, particularly, those associated with the reduced components of the mitochondrial and microsomal respiratory chains. The superoxide radicals generated by one-electron reduction of the oxygen molecule taking place in these interactions is one of the main initiators of free-radical chain processes, regarded as the main mechanisms of aging in the theories of Emmanuel (1977) and Harman (1981), which appeared in the mid-fifties. While the direct products of the reactions metabolize further, some of the side products accumulate. The accumulation of lipofuscin, for example, occurs in the most aerobic tissues such as those of the heart and brain (Strehler, 1962). In the course of the evolutionary elaboration of the metabolic pattern, the role of the damage caused by the accumulation of side reactions increased, and the pressure of natural selection toward increasing life-span was realized to an increasing extent via the development of the mechanisms preventing the self-destructive,endogenous processes. Thus, the evolution of longevity appears to be the evolution of the systems protecting the tissues from endogenous damage (Cutler, 1975, 1978). In particular, it was demonstrated that the life-span within the order of primates strictly correlates with the ratio of the activity in the liver and brain tissues of superoxide dismutase, an enzyme hindering the initiation of free-radical chains of superoxide radicals, to the consumption by these tissues of oxygen, upon which the production of these radicals is dependent (Tolmasoff et al., 1980). Thus, the existence of stochastic molecular processes in cellular selfdamage is an inexpensible consequence of biochemical evolution. Such processes take place in all tissues, the primary role belonging to different factors in different tissues and subcellular structures, which is reflected in the multiplicity of theories of aging based on one or several such factors and process. Here, the conclusion of Williams (1957) that the rates of the accumulation of the consequences of these processes with age in populations must be synchronized by the evolutionary process is of primary importance. This means that, there being several causes of death, the curves for dependencies of the mortalities upon age must be parallel. This is apparent in the mortality statistics in human populations (Upton, 1977). It follows from the above that the molding of the process of the

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individual postnatal development in evolution occurred with the necessity to accomodate the possibility of the participation of an organism in the self-reproduction of its population with the period of the existence of that organism. This period is determined by the relations between the processes of endogenous damage and the mechanisms that prevent this damage. T h e efficiency of the latter cannot increase indefinitely, since they compete for cell resources within the systems that provide the implementation by the cells of their functions in the organism. This efficiency appears to be fixed at a certain level corresponding to the rate of the succession of the generations that is optimal for the population in the given environment. Thus, between the rate of the implementation of a program of postnatal ontogenesis and the rate of accumulation of the consequences of stochastic damage, a correlation must exist. The notion of the neuroendocrine mechanisms of development and of their transformation into the mechanisms of aging, presented in this article and in more detail by Dilman (1981),allows us to propose a hypothesis concerning the basis of this correlation. The catecholaminergic systems appear to be an important part of the neuroendocrine mechanism of sexual maturation. At the same time, these mechanisms are the neural mechanisms which appear to be the most affected by the aging process (Robinson, 1975; Finch, 1976). In the hypothalamic nuclei, constituting the centers of the regulation of gonadotropin secretion, the most pronounced neuron loss is observed in rats (Hsu and Peng, 1978); although in other species, this process may not reach such an extremity and may be limited to the ultrastructural functional changes (Lamperti and Blaha, 1980) of the species of even to a racedependent degree of manifestation (Peng and Hsu, 1982). The measures compensating for the deficiency on the catecholaminergic mechanisms, in particular, DOPA administration, may lead to the restoration of the reproductive function. On the other hand, the pathways of catecholamine metabolism include the microsomal oxidase and hydroxylase systems, which belong to the sort of metabolic systems that are the most important sources of oxygen-free radicals (Goroshinskaya, 1979; Fucci et al., 1983). It should be noted here that the brain is the most aerobic organ, and its high lipid content provides the media for the spreading of the free-radical chain reactions. It should also be pointed out that it is the systems generating and metabolizing catecholamines that are the putative sources of the damage of the neural catecholaminergic mechanisms, but not the catecholamines themselves which can exhibit antioxidant properties in the appropriate conditions. Even without going into biochemical detail, the role of adrenaline metab-

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olism in the poststress complications, the prevention from which by the antioxidants is possible (Meerson et al., 1980), may be a confirmation of the possibility that the processes of catecholamine metabolism are coupled with the generation of damaging factors. Thus, it does not seem to be impossible that the neuroendocrine mechanism of the regulation of the rate of postnatal development, determining the temporary limits within which the switching-on or -off of the reproductive function is possible, has developed in the evolution on the basis of the self-damage of the neurons of the corresponding neural centers, with the central role in this damage belonging to the freeradical reactions as the product of the metabolism of catecholamines on which the functions of these neurons are based. If the functions of these neurons consist of the perception and transmission of the regulatory signals providing for the feedback of a regulator with the corresponding terminal target tissue, the damage of these neurons must lead to the enhancement of the functions of the target tissues. It is important that the changes in the peripheral part of the neuroendocrine system such as the rise of the blood level of estrogens may lead to the increased damage of the corresponding hypothalamic centers and the elevation of the secretion of some products. This mechanism possibly acts by the intensification of catecholamine metabolism. In the above case of prolactin, it has been shown that the hyperstimulation of prolactin secretion in old rats is caused by the loss of neurons in the tuberoinfundibular area, which is overstimulated by estrogens (Sarkar et al., 1983). It may be seen that the stochastic damage, accumulating in the central regulatory organs, spreads its consequences through definite channels, depending upon the organization of a system. The resulting damage acquires a certain degree of regularity. It is clear that the stochastic damage cannot be a mechanism providing for the order and the character of the events that, at the central, in particular, at the hypothalamic level, are characteristic of normal aging. It can only determine the rate of the succession of these events. As Gensler and Bernstein (1981) proposed, the damage of DNA, especially in the hypothalamic cells, may be the primary cause of agerelated changes in the functioning of the hypothalamus. The main role in this case, however, is believed to be played by the spontaneous processes such as the depurinization, which have little dependence upon the rate of the cell functioning. The distinctive feature of the hypothesis presented here is the importance of this very dependence. It is not excluded that the accumulation of the cell damage may be reproduced by the factors that are independent from the rate of cell functioning. It is known that the

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influences inducing the stochastic damage are able to accelerate the processes that are characteristic of normal aging. Thus, irradiation reproduces neuroendocrine manifestations of aging and accelerates the switching-off of the reproductive function in females (Prokudina, 1978). Certain chemical carcinogens may act in a similar way (Dilman, 1981). It is proposed that accelerated neurocndocrinc aging in subjects with Down’s syndrome is caused by a break in the balance in the system of protection from the oxygen free radicals and the enhancement of the damage of neurons by free radicals (Sinet, 1983). It remains unclear if these influences are able to accelerate not only aging but, also, development. Some observations support this possibility. Thus, for instance, the weak carcinogen DDT accelerates the elevation of body weight in animals (Tomatis et al., 1972). The prolongation of the light period, on the one hand, accelerates the switching-off of the reproductive function in old animals, and, on the other, it accelerates the sexual maturation, both effects being connected with the elevation of the threshold of sensitivity of the hypothalamus to estrogens (Hoffman, 1973). The acceleration of development in humans which regularly leads to the acceleration of the development of the age-related pathology (Dilman, 1981) is induced by a number of external influences (illumination, excessive nutrition, possibly stress, and toxic chemicals), although in most cases the mechanism of the effect is complex and cannot be unequivocally attributed only to the damaging influences of external factors. Thus, the hypothesis that the mechanism of aging and age-related pathology is a result of the canalization of the stochastic damage through the central regulatory systems cannot be properly estimated at present. In this respect, the analysis, in particular of the numerous data characterizing the complex cyclic pattern of the hormonal-metabolic shifts occurring during the development of an orgariisrri, is riecessary, especially of those coupled with a definite age period of intensified nocturnal secretion of gonadotropins (Beck and Wuttke, 1980) and of the diversed directions of the age-related changes in the energy homeostate, etc. In the search for an answer to the question about the mechanisms of changes of the threshold of the sensitivity of the hypothalamus to the regulatory signals, another hypothesis may be presented based on what is currently known about cell differentiation. At present, there are many arguments in favor of the role of cell disdifferentiation as one of the key phenomena in aging (Cutler, 1978, 1980). However, in certain systems, another point of view may be attributable, especially in the cases of the aging cell populations in which what is called aging appears to be the transition of the less-differentiated cells into the state of terminal differentiation (Kontermann, 1980). A number of authors believe that, in spite

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of the termination of morphogenetic processes in the hypothalamus in the prenatal ontogenesis, the functional development the hypothalamus proceeds through is during the period of postnatal ontogenesis (Mitzkevitch, 1978). For the earlier postnatal ontogenesis the most familiar is the example of the androgenization of hypothalamus, i.e., of the acquisition by the sex center of females of the type of functioning pertinent to a male organism under the influence of testosterone administration (Barraclough and Haller, 1970). The causes here may be peculiarities of the humoral and cellular microenvironment of the peptidergic neurosecretory cells (PNSC) producing the factors that control the activity of the anterior pituitary gland (liberines and statines) that do not provide for the complete maturation of these cells in the prenatal period (Polenov, 1968, 1979).18 Correspondingly, a portion of these processes takes place after birth. The changes in cell functions may be attributable to the changes in the state of cell differentiation, the PNSC included. The differentiation (specialization) may be reflected in the qualitative changes of the range of the secreted products as well as in the changes of the biological activities of the secreted products (Gyevai et al., 1982) as it occurs in the pituitary throughout ontogenesis (Chappel et al., 1983)and, also, in the quantity of the secreted products. The highly differentiated cells are characterized by the high sensitivity to the signals regulating the functions of these cells on the one hand and, on the other hand, by the reduction of their capacity to accept the regulatory signals that are not directly linked with the functions of differentiated cells. Acetylcholine, serotonin, melatonin, along with other neuromediators, belong to the range of the signals of the first type for the liberine-producing PNSC (Chumasov 1980; Richardson et al., 1982). The hormones of the peripheral endocrine organs, for which the PNSC have the receptors and which are able to suppress the production of liberines, may be referred to as to the signals of the second type. If so, the accumulation of the terminally differentiated cells could lead to the elevation of the threshold of sensitivity of the hypothalamus to these signals, leading to the enhancement of the secretion of liberines and, hence, of trophic hormones, i.e., could reproduce the characteristic features of the postnatal development of the neuroendocrine system as discussed above. The factors creating the conditions for the shift toward the accumulation of terminally differentiated cells may perhaps be linked with both the cellular and the humoral microenvironment of PNSC. In correspondence with the trend of the present article, much attention will

“In principle, the possibility of induction of neuronal differentiation in the hypothalamus of adult animals was demonstrated in birds (Goldman and Nottebohm, 1983).

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be given to the possible involvement in these phenomena of the internal media of an organism. The rate of hypothalamic development must depend upon the intensity of the signals that stimulate its function for the fulfillment of which the cells are to differentiate. Certain neuromediators and pineal factors may belong to the range of these signals. There exists the experimental evidence that permits one to believe that certain components of the energy metabolic system may modify the interaction of these signals with the PNSC. Thus, liposomes containing unsaturated fatty acids and phospholipids induce the neuroblastoma cells (representing the immature neural cells) to differentiate into neuron-like cells, fatty acids being the active component of the liposomes (Sandra et al., 1981). One mechanism of action of fatty acids may be the interference with the cell energy metabolism. In a similar manner, the elevation of the oxygen concentration may act. This may influence the red-ox state of the cells (Erkell, 1980).T h e maturation of the PNSC cells, expressed in the enhancement of functional activity of certain hypothalamic structures, triggers a cascade of changes in the target organs which in turn causes the hormonalmetabolic homeostasis to shift, leading to new changes in PNSC populations, and so on. The temporal characteristics of this process must depend upon the kinetics of PNSC differentiation, depending in turn upon not only internal, but, also, on external factors like diet and certain properties of the external media. If the exhaustion of stem cells occurs at a certain ontogenetic period, which must be different for the cells producing different neuropeptides, the accumulation of the damage in terminally differentiated cells becomes the principle factor that causes progressive changes in the activity of different hypothalamic structures and their eventual degeneration. It is also possible that the primary cause of the alterations in the functional activity of hypothalamus is not the changes in the structure of populations of PNSC, but the changes in the glial cells. The basis for considering such a possibility may be found in the data that demonstrate that certain steroid hormones (estrogens and testosterone) are able to stimulate neuroglia (Schipper at al., 1982; Goldman and Nottebohm, 1983), the latter being able to regulate the functions of neurosecretory cells (Allin, 1981). Thus, at present, several possibilities should be checked in order to find out what is the mechanism that causes the hypothalamic changes occurring during the postnatal ontogenesis. Additional information concerning this question may be found in the data on what may influence the process of aging and the age-associated diseases.

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VIII. The Influence of External Factors on the Mechanism of Aging and Diseases of Aging

A number of external influences are capable of accelerating the development of normal diseases and some of them of accelerating the process of aging. The latter effect is unequivocally proved in the case of irradiation (Sacher, 1977). The generation of secondary damaging products, the free radicals, is of primary importance in the mechanism of the acceleration of aging under this influence. Besides this, the irradiationinduced changes of the metabolic and the neuroendocrine homeostasis must be kept in Consideration, in particular, the elevation of the blood levels of cholesterol and FFA (Moroz and Kendysh, 1975), and, possibly, the impairment of feedback loops at the hypothalamic level (Prokudina, 1978). Thus, taking into consideration the multifactorial character of the irradiation effects and the existence of certain differences between radiational and natural aging, it is not possible to make the final choice between the roles of stochastic damage and of the action of the predetermined processes in natural aging on the basis of limited data on the influence of irradiation on the main homeostatic systems of an organism. Much more in this connection may be obtained from the data on the effects of stress on aging and age-associated diseases. Undoubtedly, many out of this group of diseases are exaggerated under the influence of stress. Such an effect may be clinically observed in the cases of climacteric, hyperadaptosis, psychic depression, hypertensive disease, atherosclerosis, Type I1 diabetes mellitus, cancer, immune deficiencies, autoimmune disorders, and obesity. In chronic stress, in particular, there are clinical observations that the appetite increases and the body weight grows in middleaged patients, which may be caused by the stress-induced decline in the biogenic amine contents in the hypothalamus. On the whole, the studies of the mechanism of the influence of stress on the process of aging may give much valuable information concerning the ways of realization of the neuroendocrine ontogenetic mechanism of aging. When referring to stress, of the two alternative energy substrates, glucose and fatty acids, the latter are preferable. Thus, the stress reaction involves not only the hypothalamic mechanisms that cause hyperglycemia but, also, increases 1ipoIysisby the elevation of blood levels of adrenaline, noradrenaline, corticotropin, somatotropin, prolactin,and by the suppression of insulin secretion. Usually, no attention is paid to the impossibility of the prolongation of the hypothalamic activity up to the time period necessary for the defense against the stressor without the elevation of the

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threshold of the sensitivity of the hypothalamus to regulatory signals including, first, the elevated blood cortisol, glucose, and FFA levels. Table XI demonstrates that dexamethasone,within the conditions of ether stress, does not inhibit hypothalamic-pituitary adrenal complex adequately in accordance with the above proposal. The mechanism of this elevation of the hypothalamic threshold is connected with the increased catabolism of hypothalamic catecholamines (Morgan et al., 19'75), which mediates the tonic inhibition of the ACTH secretion (Gruen, 1978) with the elevation of the blood ACTH level being the result. At the same time, taking into consideration the proposal that the intensified catecholamine metabolism should be coupled with intensified free-radical production, the increase in the overall cell damage in the hypothalamus may be expected as the result of repeated stress reactions. The serotonin metabolism also increases during stress (Morgan et al., 1975; Scapagnini et al., 1973), perhaps playing an additional role in the mechanism of the reduction of the hypothalamic sensitivity to dexamethTABLE XI THEINFLUENCE OF STRESS O N SENSITIVITY OF THE HYPOTHALAMIC-PITUITARY COMPLEX TO THE INHIBITORY ACTIONOF DEXAMETHASONE IN R A T S ~ Level of 1 1-OHCS6in blootl

Number Experimental

f P " P Control Stressed

P

After

Reduction

animals

Initial

dexamethasone

(percentage)

40 49

803 ? 45.3 1267 334 0.001

143 ? 18.5 1143 2 98.0 0.001

- 82 - 10 0.001

of

*

"Dexamethasone at a dose of 5 Wg/lOO g weight was administered at 10 AM, blood was sampled at 2 PM, and stressor treatment (periodic electrostimulation of tails, combined with immobilization) was performed from 10:30 AM up to 1:30 PM. 1 1-OHCS, 1 I-Oxycorticosteroid. TABLE XI1 THEINFLUENCE OF THE PINEAL POLYPEPTIDE EXTRACTON SENSITIVITY TO PREDNISOLONE IN OLDRATS" The preparation administered Prednisolone (0.05 mg/100 g body weight) Prednisolone + pineal extract (1 mg im, twice) From Ostroumova and Dilman (1972).

THE

Blood corticosterone levels (mcg %)

22.8 2 3.8 22.0 ? 3.6

22.2

?

11.1

?

2.6 2.8

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asone. It is of interest in this respect that the polypeptide pineal extract improves this sensitivity (Table XII). The increase in the role of fatty acids in energy metabolism, besides a wide range of influences on the development of age-related pathology, may intensify free-radical processes in the peripheral tissues. It is natural, if the hypothalamic threshold of the sensitivity in an adaptive homeostate increases with age, that a man becomes more subject to the deteriorating influences of chronic stress reactions, acquiring the features of hyperadaptosis as the age increases. Thus, the chronic stress must accelerate the process of normal aging, acting by enhancing both the hypothalamic and the metabolic components of aging. Overnutrition, causing the excessive body fat accumulation, as stress also does, must act on the two components of the mechanism of aging. In particular, overnutrition, causing the elevation of the blood concentrations of insulin and cortisol, may decrease the sensitivity of the hypothalamus to regulatory signals. This is due to the decrease in the number of the receptors for these hormones. By causing excessive weight or simply by increasing caloric intake, overnutrition intensifies energy metabolism. Both of these factors accelerate the development of the diseases of aging and, possibly, of aging itself. Perhaps the studies on the influence of hypercaloric diet on the acceleration of the switching-on the reproductive function will succeed in distinguishing these two influences. Of use in this respect would be the studies on the influence of diet enriched with fat or of obesity on switching-on the reproductive function within the conditions of the administration of the inhibitors of aromatases, catalyzing the biotransformation of androgens into estrogens. The influence of the increased illumination on the processes of maturation have already been mentioned. This influence is mediated mostly by the inhibition of the pineal activity and is of substantial theoretical importance. However, it is still not clear, how the pineal functions change in the course of aging, although it is undoubtedly certain that pineal polypeptides increase the sensitivity of the hypothalamic-pituitary complex to hormonal signals (Table XII). Unlike the factors listed above, that accelerate the development of the diseases coupled with the mechanism of aging, all those factors that either increase the sensitivity of the hypothalamic-pituitary complex to the regulatory signals or decrease the metabolism of fatty acids slow down the development of these diseases. First, this is the property of the dietary calorie restriction, of the decrease in the illumination, of the antidiabetic biguanides, pineal polypeptides (Dilman et al., 1979), L-DOPA, antioxidants, etc.

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v. M. DILMAN el al. IX. The Analysis of Geroprotectors from the Point of View of the Neuroendocrine-Ontogenetic Theory of Aging

The role of the dietary calorie restriction as a geroprotector is undoubted at present. As a matter of fact, this influence is regarded as the only one whose effect is reliable, with respect to the slowing down of the aging process, (Sacher, 1977).Although in experiment, the calorie restriction may decrease the incidence of certain age-specific pathological processes (autoimmune, malignant, and atherosclerotic) after having been initiated before maturity (Ross, 1972) as well as in the mature period of life, it is known for sure that only early onset calorie restriction can slow down the aging process. Such consequences of calorie restriction as the reduction of the blood levels of certain hormones, of cortisol and insulin in particular, the improvement of the carbohydrate tolerance, and slowing down of the fatty acid metabolism may be applicable to the ontogenetic mechanism of aging. All these effects are sufficient for the reduction of the rate of the development of age-associated diseases. Besides this, the inhibition of the accumulation of the endogenous cell damage may be suspected. It was shown that the rate of lipofuscin accumulation slows down under calorie restriction. The acceleration of sexual maturation under excessive nutrition provides the reason to believe that the slowing down of the elevation of the hypothalamic threshold may be responsible, at least partly, for the geroprotective effect of calorie restriction. Antidiabetic biguanides render their metabolic effects, as Muntoni (1974) proposed, via the inhibition of fatty acid oxidation. It was shown, also, that phenformin inhibits monoamine-oxidase (Feldman, 1975). This drug also elevates the sensitivity of tissues to insulin (Purello et al., 1982) and reduces the blood triglyceride and cholesterol concentrations (Tzagournis el al., 1968;Dilman, 1981). Phenformin also elevates the sensitivity of the hypothalamo-pituitary complex to the inhibitory action of estrogens, dexamethasone, and thyroxine (Dilman, 1981). The restoration of the estrous cycle under the influence of biguanides was observed in old rats (Anisimov, 1980). Phenformin also abolishes metabolic immunodepression in 60-7096 of patients (Dilman, 1981; Dilman et al., 1982). The data of Table XI11 demonstrate that phenformin increases the mean lifespan of mice and, also, reduces the incidence of spontaneous tumors (the latter effect is similar to that of the pineal polypeptide extract). Thus, it cannot be excluded that the proposal concerning the geroprotector possibilities of phenformin will be confirmed in further studies. As Cotzias et al. (1974, 1977) have shown, DOPA elevates the life-span of certain strains of mice. This observation is in correspondence with the

TABLE XI11 THEINFLUENCE OF PHENFORMINE AND DILANTIN ON THE LIFE-SPAN OF C3H FEMALE MICE AND ON THE INCIDENCE OF SPONTANEOUS TUMORS Mammary adenocarcinomas Experimental group Control Phenformine Diphenylhydantoin (Dilantin)

" p < 0.05.

Number of mice

Mean life-span (days)

Number of mice with tumors

Number of mice with tumors

30 25 23

450 f 19 555 ? 32" 558 ? 28"

24 (80%) 5" (20%) 8" (34.8%)

19 (63%) 4 (16%)" 7 (30.4%)"

Total number of tumors

Number of mammary tumors per mouse

30 4 7

1 .oo 1.oo

1.38

Leukemias 4 (13.3%) 1(4%) 2 (8.7%)

Other tumors 5

1 -

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data on the restoration of the estrous cycle in old rats on DOPA administration (Quadri et al., 1973) and the data on the influences of L-DOPA and L-9-tryptophan on the results of the dexamethasone test (Table XIV). With respect to antioxidants, these drugs prevent the development of a number of processes promoting age-related pathology and, also, increase the mean life-span without prolongation of the maximal life-span of experimental animals (for a review, see Harman, 1981). Therefore, one of the main tasks in the estimation of the antioxidant effect from the point of view of the concept presented above is the study on the influences of antioxidants on the threshold of the sensitivity of the hypothalamicpituitary complex to the inhibitory signals and on the temporal parameters of switching-on and -off the reproductive function. At the same time, the impression arises that the majority of influences, known to inhibit the rate of the development of age-specific diseases, are capable of inhibitory action on the pathogenetic mechanisms of normal diseases.

X. The Neuroendocrine-Ontogenetic Theory of Aging and the Evolution of Aging

The validity of any theory, including those of aging, should be estimated not only on the basis of its correspondence to facts within the limits of its applicability but, also, on the basis of its adjustability to the more general theories, in the present case, to the theories of not only of human TABLE XIV THEINFLUENCE OF L-DOPA, L-TRYPTOPHANE, DIAZEPAM, A N D PHENAZEPAM ON THE PARAMETERS OF THE DEXAMETHASONE TEST' Study groups (number of observations) L-DOPA (14) L-Tryptophane (20) Diazepam (6) Phenazeparn (27)

Levels of 11-OHCSb Treatment

Initial

Before After Before After Before After Before After

568 5 49.7 533 2 52.4 555 f 41.4 552 f 52.4 646 2 58.0 453 f 49.7' 574 f 38.6 411 35.9'

*

"From Nuller and Ostroumova (1980). '11-OHCS, 1 1-Oxycorticosteroid. ' p < 0.05.

After dexamethasone 317 ? 38.6 168 5 24.8' 331 f 41.4 226 f 38.6< 555 2 63.5 171 f 30.4c 397 f 30.4 187 k 24.8'

Reduction (percentage) -39 -52 -40 -59 -12 -63 -24 -55

f7 f 10 k

7

f 4" f 12 f 4' f f

8 5'

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or vertebrate aging, but of aging as a biological phenomenon of general importance and, first, to the evolutionary aspects of aging and longevity. Some general considerations concerning the inevitability of the appearance of biochemical processes underlying the manifestations of aging at the level of a whole organism are presented above. The general principles underlying the evolution of aging and longevity were analyzed by a IIUIIIber of authors (Medawar, 1952; Williams, 1957; Cutler, 1978, 1983; Kirkwood and Holliday, 1979; and others). Here, some particular examples of the realization of these principles in cases in which the neuroendocrineontogenetic model of aging can be applicable will be analyzed. The consideration of the problem of aging from an evolutionist’s point of view transfers the problem from the level of separate organisms to the population level. Here, the existence of aging is reflected in the elevation with age of the probability of death in each cohort, which shortens the period of existence of each such cohort and, correspondingly, the mean life-span of the organisms constituting this population. The most general question. which was resolved in the evolutionary analysis of aging, was why this phenomenon, being a factor that presumably reduces the reproductive potential of a population, was not eliminated by natural selection? Medawar (1952) was the first to show in general terms that in the cases of high mortality, because of random external causes, which is natural for wild populations, the potential immortality would be of no advantage compared with the population of organisms with limited life-spans. That is why the selection pressure is directed toward the optimization of the developmental and reproductive patterns even at the expenses of the lifespan. Medawar’s approach was further elaborated by Williams (1957),who proposed that the emergence of aging in such a case may be the consequence of the lasting appearance in a population of the genes with the favorable effects at the early stages of the life history of an organism in spite of their deleterious effects, manifested later in life. Aging must be the result of these delayed, pleiotropic effects. This idea is generally assumed because it gives an explanation for the appearance of aging in the course of evolution without referring to any adaptive value of aging and predetermined mortality per se. Nevertheless, some authors feel the argument in favor of the role in aging of the genes with delayed deleterious pleiotropic effects to be insufficient, since they consider it to be unclear what determines the switching of the action of these genes from beneficial to harmful (Sacher and Trucco, 1962; Kirkwood and Holliday, 1979; Gensler and Bernstein, 1981). As Cutler (1980) propnsed, it is neressary to distinguish the pleiotropic effects of the genes that underlie the basic functions and those effects of the genes that underlie the changes in these functions occurring during

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the development of an organism. The consequences of the polyreactivity of the compounds participating in metabolic, first, aerobic processes, may be the examples of functional pleiotropia. As an example of developmental pleiotropia, Cutler regards the neuroendocrine mechanism of aging, presented above: “If Dilman’s hypothesis is true, it is a perfect example of developmentally-linked pleiotropic biosenescent process” (Cutler, 1978, p. 343). It should be noted in this connection that the proposed mechanism makes the special mechanism for switching of the gene’s action from beneficial to deleterious unnecessary, since the action of the same genes controlling the deviation of homeostasis in the three main homeostates provides, in succession, for the action of the mechanism of growth and development and of the mechanism of aging. In the most demonstrative way, this pleiotropia is traced in the age-linked switching-on and -off of the reproductive function, both being accomplished at the expense of the elevation of the hypothalamic threshold. The ontogenetic theory of aging is in correspondence with a number of other phenomena, being the subjects of evolutionary considerations. In particular, the correlation exists between the length of the period of maturation and the length of the reproductive period when the species of organism with different life-spans are compared. This correlation may be based on the ontogenetic mechanism of aging. Consider the increase in the mortality in a population because of changes in the environment. The natural selection in this situation will act as a factor that increases the fraction of the organisms with the earlier appearance of the capacity for reproduction. In the species with the same principles of organizztion of ontogenesis as in mammals, this acceleration of sexual maturation must occur at the expense of the acceleration of the elevation of the “hypothalamic threshold.” At the same time, the steeper increase in the hypothalamic threshold must lead to the accelerated development of the ageassociated diseases and to the shortening of the life-span. The threshold or elevational mechanism of switching-off the reproductive function (Dilman, 1971,1981) also provides for the explanation of the gradual reduction with increasing age of the capacity for reproduction, which in humans is manifested in the follicular persistence and the anovulatory menstrual cycles. Of interest is the consideration of the supposition that the increase in longevity over the course of evolution, leading to the comparatively long human life-span, was caused by the elaboration of what was called the biological antiaging systems (Cutler, 1978). This proposal is supported by the data on the positive correlation between the species-specific life-span and the activities of superoxide dismutase (Tolmasoff et al., 1980) and the

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DNA reparative systems (Hart and Setlow, 1974; Francis et al., 1981). At the same time, in accordance with the principle of the transformation of the developmental program into the mechanism of aging, the latter must function with the rate determined by the rate of the realization of the developmental program. Taking these relations into account, the correlation of the activity of the biological antiaging mechanisms with the species-specificlife-span may be explained by the matching of the activity of the antiaging mechanisms to the existing life-span, if the rate of the action of the neuroendocrine mechanism of development and aging is what limits the longevity. In other words, the organisms with the short life-span, determined by the rate of the realization of the developmental program and, hence, by the hypothalamic component of the latter, do not need efficient antiaging mechanisms. It may be proposed, for example, that within the conditions of the restricted food resources, limiting the rate of growth and development, the organisms appear to be at an advantage with slowed down elevation of hypothalamic threshold. This leads to the selection of the organisms with increased life-spans and is often observed in desert animals like turtles and lizards. On the basis of the mechanisms of adaptation to the changes in the environment, the neuroendocrine-ontogenetic mechanism of aging may play a role in the mechanism of the elimination of the lesser fit organisms in the course of natural selection. The elder organisms are the most subject to all sorts of stress with accelerated aging as a consequence. The works of Christian (1968, 1976)provide an interesting example. When the density of a rodent population abnormally increases, the developing chronic stress leads to the elevated secretion of ACTH and cortisol and to the increased mortality of the aged animals because of infections, tumors, and glomerulonephritis, and, also, to the suppression of the reproductive function in young animals. It may be expected that animals with the more pronounced development of resistance in the central component of the adaptive homeostate would be more subject to development of hyperadaptosis and its associated diseases. Besides this, the younger animals with high sensitivity hypothalamus to feedback inhibition become less fertile, since under the influence of the stress-induced ACTH release, the inhibition of the gonadotropic function occurs (Christian, 1976).As a matter of fact, this is an example of the role of hypothalamic mechanisms in such a population phenomenon as the regulation of population density. The correlation of the rate of aging with the period necessary for the achievement of the final body size, besides other possible causes, may depend upon the connection between the time of the reproductive function switching-on and the achievement of a definite body size. The earlier

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is the time for the reproductive function switching-on, the smaller must be the body size, and the shorter must be the life-span when the physiologically similar biological species are compared. In limiting the body size, not only the mechanism of the inhibition of the basal secretion of growth hormone after the accumulation of a critical level of body fat, but, also, the changes in the sensitivity of the hypothalamic system of the growth hormone secretion to the fatty brake may play a role. At the same time, the accumulation of body fat participates in the switching-on of the reproductive function, that, in turn, leads to the increasing antisomatotropic actions of estrogens (Dilman, 1981). T h e correlation of brain weight with longevity may be linked, as Sacher noted (1970), with the homeostatic capacities. From the point of view of the ontogenetic model of aging, the improvement of this capacity means not only greater resistance of homeostatic systems to the deviating influences but, also, the reduction of the rate of the implementation of the program of the deviation of homeostasis. Thus, it cannot be excluded that the species-specific differences in longevity within certain taxonomic groups, including mammals, are determined to a significant extent by the differences in the rate of elevation of the hypothalamic threshold. However, the rate of the development of the age-linked pathological processes may depend not only upon the rate of the deviation of homeostasis but, also, upon the rate of accumulation of local damage. T h e case where the hypothalamic mechanisms definitely limit the life-span is the life cycle including a single period of reproduction, the so-called “big bang” reproduction. I n this case, abrupt hormonal changes occur that may be attributable to a program indispensible for reproduction or to environmental factors changing the hypothalamic threshold. T h e most familiar example is the case of the Pacific salmon (Wexler, 1976) in which death, after concluding the period of reproduction, is caused by the intensification of the neuroendocrine-ontogenetic mechanism of aging (Dilman, 1981). Recently, a similar case was found in mammals.’’ This supplies the reason to believe that the causes of natural death in such unrelated species as the Pacific salmon, the rat, and the human may be similar (Table XV); for details, see Dilman, 1981). Thus, definite mechanisms of the realization of a number of biological and evolutionary principles applied to the problem of aging may be proposed on the basis of the neuroendocrine-ontogenetic theory of aging.

Male, Australian, marsupial mice (Diamond, 1982).

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XI. The Modern Theories of Aging and the Neuroendocrine-Ontogenetic Theory

The fact that in the course of aging homeostatic disturbances take place has long ago been appreciated (Cannon, 1942; Shick, 1959; Verzar, 1957) and finds an increased support in more recent studies (Comfort, 1964; Timiras, 1975; Everitt and Burgess, 1976; Finch, 1976; Denckla, 1977; Samorajski, 1977). It is not enough to claim, however, that the homeostatic disturbances are characteristic of aging. The causes of these disturbances and their mechanism should be explored. Such an attempt is undertaken in a theory assuming that the impairment of homeostasis obeys the Law of the Deviation of Homeostasis, with the hypothalamic changes being of key importance for the implementation of this law (Dilman, 1978, 1981). The origins of the hypothalamic theory of aging are in the paper entitled “On the Age-Linked Elevation of the Activity of Certain Hypothalamic Centers” (Dilman, 1958). However, the initial ideas about the process of aging as the process of the elevation of hypothalamic activity gradually broadened. Such a mechanism is most fit for the description of the way in which climacteric occurs, where the elevation of FSH secretion is a consequence of the elevation of hypothalamic activity. Later on, this mechanism was associated with a more general phenomenon, the elevation of the threshold of the sensitivity of the hypothalamus to regulatory stimuli (Dilman, 1971, 1974). Later, Stoll(l972) proposed that the decline in the level of biogenic amines, in particular of dopamine, in the hypothalamus may be responsible for the decreased sensitivity of the hypothalamus. This hypothesis, in terms of the elevation of the threshold of hypothalamic sensitivity,was confirmed in our laboratory (Dilman, 1981). It was demonstrated, independently, that in the course of normal aging, there occurs in the brain and especially in the hypothalamus, a reduction of the level of biogenic amines (Robinson et al., 1972; Robinson, 1975); in particular, the metabolism of dopamine appears to be impaired (Finch, 1976). However, direct determination of biogenic amine concentrations in a human hypothalamus is impracticable. Therefore, the assumption of the link between the “neuromediator mechanism of aging” and the agerelated changes in the results of functional hypothalamic tests, e.g., the dexamethasone test, permitted the conclusion that in stress, hyperadaptosis, and psychic depression there must be a deficit of biogenic amines in the hypothalamus. In other words, the neuromediator mechanism of aging (which is correspondent to the concepts of Finch, 1973,1976;Cotzias

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~t al., 1974, 1977; and Samorajski, 1977) is a biochemical equivalent of the

functional hypothalamic changes. Further analysis of this problem revealed that, during the postnatal ontogenesis, the thresholds of the sensitivitiesof the hypothalamus shift into different directions. In the adaptive homeostate and, also, in the reproductive one, i.e., in the systems controlled by thc both positivc and negative fccdback, the threshold of the sensitivity of the hypothalamus increases with age. Changes in the energy homeostate are characterized by more complicated dynamics: the hypothalamic threshold to the inhibition by glucose elevates, while that by FFA declines. Finally, according to Ascheim (1976),the threshold of the sensitivity declines also in the complex hypothalamus-prolactin-estrogens. ‘Ihese data and, also, some biological considerations, permitted the description of the mechanism of the Law of the Deviation of Homeostasis in niore detail (Dilman, 1981, 1982) with no primary attention paid to the directions of the changes in the thresholds of the sensitivity of the hypothalamus in different systems, which may be the reduction in one system and the elevation in another, although the latter direction must be dominating, being necessary for the implementation of the developmental program of an organism. Moreover, the notion of the Law of the Deviation of Homeostasis does not necessarily imply that the hypothalamus is the only structure responsible for the implementation of the law. The extrahypothalamic changes, e.g., the alterations of the pineal activity, may cause the changes in hypothalamic sensitivity (Ostroumova and Dilman, 1972). Undoubtedly, other extrahypothalamic structures exist that are involved in the implementation of the Law of the Deviation of Homeostasis. But, of course, the interlocking of the feedback loops of the main homeostates at the hypothalamic level and the fact that the reprocessing of the influences from the rest of the CNS, as well as the external influences into the signals integrating the functions of an organism occurs mainly in the hypothalamus, make this structure the most responsible for implementing the Law of the Deviation of Homeostasis. It is necessary to stress that the role the Law of the Deviation of Homeostasis plays in the mechanism of development, aging, and ageassociated diseases by no means excludes the contribution of other factors, in particular of the exogenous and endogenous stochastic damage to the process of aging. On the whole, the picture of aging is created by the combination of all these factors. The following examples demonstrate this. The regular elevation of the rate of gonadotropin secretion, predetermined by the Law of Deviation of Homeostasis, may lead to the development of tumors in the ovaries as the result of the overstimulation. As far as carcinogenesis occurs as a result of mutations and other stochastic

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events at the level of the genetic apparatus of a cell, a correlation must exist between the phenomena covered by the Law of the Deviation of Homeostasis and the stochastic events occurring in cell transformation. It may be different from that discussed in Section VII of this article and be based, for example, in a particular case on the capability of the cellular cortisol-binding protein to bind when in complex with cortisol to the longterminal repeats of an oncogene (Govindan et al., 1982). This elevates the probability of derepression of an oncogene. The disturbed metabolic background may also influence the probability of events involved in the development of autoimmune disorders, thus contributing to the role of these disorders (Walford, 1969) in the mechanism of aging. Even such a seemingly local process as the cross-linking of collagen molecules may be slowed down by hypophysectomy and intensified by an excessive nutrition (Everitt and Burgess, 1976), i.e., it is interconnected with the processes controlled by the Law of Deviation of Homeostasis. The decline in the inductibility of enzymes, considered to be a key role in the mechanism of aging (Adelman, 1970), also appeared to be dependent upon the hormonal-metabolic background, but not only upon primary cellular changes (Finch, 1976). The mechanisms of aging, based upon the damage by free radicals, must also be subject to the influences of the metabolic processes, possibly being intensified by a shift toward the utilization of fatty acids as preferable energy substrates that occurs, for example, in stress. It cannot be excluded that even the mechanisms underlying Hayflick‘s limit within the conditions of an organism (not in vitro) are under influences that are of considerable importance in determining the rate of exhaustion of the limit of cell divisions. For example, the rate of cell divisions may be influenced by the blood somatomedin concentrations which, in turn, depend upon the metabolic state. Finally, the age-related changes occurring at the level of an organism, for instance, the elevation of blood cortisol and insulin levels, may exert secondary influences on the content of the corresponding hypothalamic receptors, thus establishingthe link with the decline of the hormone receptors, the importance of which in aging having been emphasized (Roth, 1979). As soon as the increased lipolysis intensifies the development of atherosclerosis, in particular in the blood vessels of the brain, the link may be established with the theories emphasizing the role of neuron loss in aging. All this permits one to state in an extreme, that “the metabolic disorders, induced by the hypothalamic regulatory shifts cause the changes in the somatic cells”(Dilman, 1981),on the one hand, while certain metabolic shifts, caused by overnutrition, for example, influence the sensitivity of hyperthalamus to regulatory signals.

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Thus, the ontogenetic model of aging describes the mechanisms of a number of phenomena, which in numerous other theories, are regarded as independent causes of aging; that is the reason why this model may be regarded as an integral theory of aging. It should be noted that the present state of the ontogenetic model is a result of gradual elaboration. In different periods, it was designated as hypothalamic and reflected the role of the hypothalamus in maintaining or deviating the constancy of the internal media, as elevational, reflecting the elevation of the power of the main homeostatic systems being a characteristic of many age-related changes, and, also, the immediate transformation of the developmental program into the mechanism of aging, etc. It is seen from the above, that this model in many respects corresponds to other existing models, developmental (Everitt, 1976), disregulatory (Lints, 1978), CNS-cybernetic (Meier-Ruge, 1975), and regulatory (Denckla, 1977). T h e ontogenetic model was formulated earlier than the aforementioned models and covers a wider range of phenomena. Finally, the consideration of the most important work of Cutler (1975, 1980, 1984), in comparison with the neuroendocrine-ontogenetic theory of aging (Dilman, 1971, 1981, 1983), is of special interest. Elaborating further the notion of aging as a by-product of a normal process of life, Cutler postulates the existence of two groups of phenomena constituting the basis of aging: (1) the by-product of metabolism; and (2) the by-product of development, “Aging is the result of by-product of the developmental and metabolic processes which have evolved to maintain the continuity of life” (Cutler, 1984).*’ Full correspondence to a postulate of the neuroendocrine theory of aging may be noticed in at least the first half of this statement. Considering aging to be too multifaceted, Cutler believes that it does not provide for the explanation of the dramatic differences between the longevity potentials of different species of mammals existing in spite of marked similarities in their structural and functional organizations (and the manifestations of aging). Cutler concludes that the evolution of longevity (species-specificlife-span) in mammals occurred by virtue of the elaboration of specific antiaging mechanisms. With respect to the developmentally linked antiaging process, it should be stressed that they are nothing else but the processes participating in the control of the rate of development and, so, belong to the sphere of the neuroendocrine-ontogenetic theory. In particular, the rate of the elevation of the hypothalamic threshold of sensitivity in the reproductive homeostate predetermines the time of the switching-on of the reproductive function, as well as the time of switching it off. Corre?“Theseprocesses are also designated by Cutler as continuously acting biosenescence processes (CABPs) and developmentally linked biosenescence processes (DLBPs).

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spondingly, the acceleration of development is at the same time the acceleration of the rate of the age-specific pathological processes (Dilman, 1971, 1981). This is in line with Cutler’s statement that “a decrease in the rate of development played a causative role in the evolution of longevity” If so, there is no need to distinguish between aging and antiaging mechanisms with respect to the developmentally linked processes. Cutler, however, does not make this discrimination.Further, if the age-related changes in the three main superhomeostates, i.e., in the energy, adaptive, and reproductional, occur at different rates in different individuals of the same species, leading to the existence of normal, accelerated, and delayed aging within this species, why is it not possible that this is what determines the interspeciesdifferences in longevity,i.e., the species-specificlife-spans? The correlation between the lengths of the prepubertate periods and the longevities in different species of mammals is in correspondencewith such a possibility and represents an argument in favor of the more important role of the DLBPs than that of CABPs in aging. If the DLBPs are what determines the life-span, then the interference with these processes after maturity would lead to the delay in the full manifestation of the factors that underlie natural death and so to the artificial prolongation of a lifespan, for instance, the slowing down of the changes in hypothalamic sensitivity.This is contrary to the conclusion (Cutler, 1984)that it is possible only by virtue of the interference with the processes of development. The peculiarities of the approaches to the treatment of the age-associated diseases, based on the neuroendocrine-ontogenetic theory, as well as the integrating property of the ontogenetic model, is most distinctively evidenced by the analysis of the origins of the diseases or, in a broader sense, by the analysis of the current models of medicine.

XII. Three Models of Medicine

The work on the theories of aging should ultimately be aimed at the elimination of the diseases coupled with the mechanism of aging, to slowing down of the aging process, and broadening of the human lifespan. The feasibility of this latter task is far from being obvious, since it may appear to be connected with the interference with the earliest periods of the growth and development of an organism. Therefore, consider in what way the ontogenetic theory of aging may influence the notion about the causes of the development of the main human diseases and, hence, about the means of preventing these diseases. At present, two tendencies in treating the causes of the development

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of diseases can be readily traced. In one of them, the main emphasis is made on the external factors such as trauma, overnutrition and undernutrition, pathogenic microorganisms, viruses, chemical and physical carcinogens, stress, etc. As Ludwig (1980) noted, the present model of medicine is essentially ecological. Correspondingly, the prevention from the action of the external, unfavorable conditions is considered to be the main preventive measure. T h e second model of medicine deals with inborn genetic causes of the development of the diseases or of the predisposition to their development. At the present time, about 2500 of the so-called hereditary diseases are known. Correspondingly, the counteraction against the development of such diseases or substitutive therapy until the correction of a genetic defect is possible are the preventive measures in this model of medicine. Certainly, in both models, not only the causes but, also, the conditions promoting or preventing the development of diseases are taken into consideration. At the same time, Sacher (1977) on the basis of the analysis of the experimental data and of the human life tables, has come to the conclusion that, at present, no method is available that could slow down the increase in mortality connected with aging. T h e analysis of Swedish life tables, performed by Gavrilov and Gavrilova (1979),have also shown that modern medication does not slow down the rate of age-dependent elevation of mortality. Ludwig (1980) emphasizes that the existing approaches do not ensure the counteraction against the main human diseases. He claims that, “To overcome this limitation is the true aim of gerontological research. I n initiating the revolutionary step from an environmentally oriented care to one centered on man himself, it becomes the very foundation of future scientific medicine.” The data arid arguments presented in this review and in earlier publications (Dilman, 1958, 1971, 1979, 1981) demonstrate, as H. Blumenthal states it (see epilogue in Dilman, 1981, pp. 327-329), the new medical model, in accordance to which not only the external damaging factors and the internal genetic defects are the causes of the main human diseases, but, also, the mechanism of development, i.e., the mechanism of ontogenesis. This thesis does not mean, however, that these diseases are programmed to the same extent, as in the postnatal ontogenesis. They arise with the regularity that is characteristic of ontogenesis because they are a by-product of the mechanisms of its realization. In other words, it is the normality of the deviation from the norm in the systems that obeys the Law of the Deviation of Homeostasis that leads to thc regular development of definite diseases in the process of natural aging. That is why it is not the violation o f the Law of the Constancy of the Internal Environment,

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but the strict implementation of the Law of the Deviation of Homeostasis which predetermines the development of a spectrum of definite diseases. It is when these diseases do not appear at the corresponding periods of ontogenesis that it is possible to suspect the violation of the physiological norm. This permits one to qualify the diseases, the development of which is defined by the Law of the Deviation of Homeostasis, as normal diseases (Dilman, 1981). Correspondingly along with the ecological and the genetic models of the diseases, the third model may be documented. It may be designated as the ontogenetic model of the development of diseases (Dilman, 1983). The emphasis, with respect to the preventive measures, is shifted in the ontogenetic model toward the slowing down of the development of disorders caused by the functioning of the mechanism of ontogenesis. The main consequences of the ontogenetic model are as follows: 1. In accordance with this model, the shifting or age-dependent norm assumed at present is misleading, because it is not the norm that it reflects, but the degree of the deviation from the norm of people of different ages. The norm should be constant for all age groups after cessation of growth. The physiological parameters (in particular blood triglyceride and cholesterol contents, blood glucose and insulin levels after the glucose load, the body weight, the blood pressure, and some others) estimated at the ages of 20-25, of course, in healthy subjects, constitute the ideal norm. Correspondingly, the estimation of biological or physiological age is the estimation of the degree of the developmentof the age-associateddiseases. 2. Stress, overnutrition, obesity excessive illumination, and a number of chemical carcinogens cause the decrease in the sensitivity of the hypothalamus to regulatory signals and/or the increase in the fatty acid metabolism. These properties of a number of external factors form the basis for the interrelation between the ecological and the ontogenetic models of the development of diseases. Hence, the influences that improve the sensitivity of hypothalamus to regulatory signals (L-DOPA and Ltryptophan) or diminish the utilization of energy substrates (food restriction), as well as the influences that protect against the damage caused by free radicals (antioxidants)are capable of slowing down the development of the age-associated diseases. 3. In accordance with the ontogenetic model of medicine, the appearance of any of the normal diseases must increase the probability of the appearance of other diseases of this group. Stoddard (1980) points out that “asbiotechnical data have multiplied, crossover similaritiesamong cases in different disease classes have become troublesomely frequent.” Therefore, the necessity exists for revision of the borders between diseases

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with more emphasis on the interrelations than on the differences between various categories of diseases. In fact, hyperadaptosis, metabolic immunodepression, and cancrophilia represent an attempt for the reconstruction of the generally assumed pattern of diseases. 4. The existence of common features in the pathogenesis of the main diseases and their connection with aging leads to the conclusion that the counteraction against these diseases may be achieved only by means of slowing down the aging process. In other words, the geriatrics proper must not be the subject of gerontological research, but must be the counteraction against the untimely development of the main human diseases. As Ludwig (1980) pointed out, “Medical care, one might say, remains in its infancy as long as it cannot forestall intrinsic pathogenesis as effectively as that originating in the environment.” 5. As it was noted earlier (Tsai et al., 1982),the effect of the elimination of two or more causes of death is r,umulative, but not additive, i.e., in the cases when the causes are eliminated separately, the sum of the additional years of being alive is less than in the cases when the same causes are eliminated as a group. It follows from the existence of a common mechanism of the main diseases that the pathogenetic treatment of one of these diseases must be efficient in preventing the development of other diseases. Food restriction may be one of the examples. 6. The mechanism of the diseases caused by internal factors in such unrelated species as the Pacific salmon, the rat, and the human are, in many respects, similar (Table XV). At the same time, beyond the regulatory mechanisms of diseases and death, there must exist more ancient mechanisms of aging and age-associated diseases. For example, the accumulation of lipofuscin in living cells, discovered more than a hundred years ago, cannot but influence their activity. The accumulation of lipofuscin that, in some cells, may occupy u p to 75% of the cytoplasm is a consequence of free-radical processes, the rate of which is only to a limited extent connected with external factors such as excessive dietary fat. At the same time, as it is stressed in the literature (Cutler, 1978),the generation of free radicals is an inevitable pleiotropic effect of the genes underlying the aerobic processes. Consequently, in this sense, the accumulation of lipofuscin can be neither referred to the genetic model nor to the ontogenetic one, the possible dependence of the predetermined processes of aging on the stochastic factors being excluded from consideration.“ The stochastic 21 in a certain sense, the generation of free radicals, coupled with the consumption of oxygen, may be regarded as an external damaging factor analogous to the harmful influence of inappropriate or excessive nutrition attributable to the ecological model of the development of diseases.

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TABLE XV AGE-RELATED PATHOLOGY AND CAUSES OF NATURAL DEATHIN PACIFICSALMON, RAT,AND HUMAN Pacific salmon"

H y perglycemia H yperlipidemia H y percholesteronemia Adrenal cortex hyperplasia Thymic involution Obesity The infarction of myocardium, brain, kidneys, and other organs (atherosclerotic lesions)

Ratb H yperglycemia H yperinsulinemia H y pertriglyceridemia H ypercholesteronemia Adrenal cortex hyperplasia Thymic involution Excessive body weight Arterial hypertension nephrosclerosis, arteriosclerosis, constant estrous, myocardial infarction, pituitary adenomas, tumors

Human Hyperglycemia H yperinsulinemia H ypertriglyceridemia Hypercholesteronemia Relative excess of glucocorticoids Thymic involution Excessive body weight Obesity, climacteric, hyperadaptosis, diabetes mellitus of the obesed, metabolic immunodepression, autoimmune disorders, essential hypertension, psychic depression, cancrophilia (cancer)

"From Wexler (1971). bFrom Wexler (1976).

factors may play a considerable and sometimes crucial role in the development of diseases corresponding to any of the three models of their development. However, it cannot be excluded that there exist other diseases based entirely upon the stochastic mechanisms. As to the possible properties and manifestationsof these diseases, we are now quite ignorant because as it is assumed in the neuroendocrine-ontogenetic theory of aging the regulatory and ecological and genetic causes lead to death before any nosologically distinctive syndromes of this sort may appear. Nevertheless, the possibility of the existence of a fourth model of medicine, which may be called the involutionary model, cannot be excluded. Thus, we face different models of medicine, different approaches to understanding, prophylaxis, and treatment of diseases. For example, the development of atherosclerosis because of overnutrition, takes place in accordance with the ecologicalmodel. In cases of familial hyperlipidemias, the genetic model is applicable, while in the course of normal aging, atherosclerosis develops in accordance with the ontogenetic and the involutionary models. Correspondingly, the preventive measures are different: in the ecological model, it is the prevention from the action of harmful

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external influences; in the genetic model, it is the compensation for the defect; and in the ontogenetic model, it is the slowing down of the aging process (Table XVI). On the whole, a separate cause is considered to exist for each separate disease, e.g., for each infection of avitaminosis in the ecological model. On the contrary, in the ontogenetic model, there is one cause for many diseases. In the ecological model of diseases, those caused by external influences are strictly distinguished from the process of aging, which is considered to be a normal physiological phenomenon. On the contrary, in the ontogenetic model, aging is one of the principle causes of the main human diseases. In the ecological model, the discovery of a specific cure for each disease or a “magicbullet” using Paul Erlich’s expression, is the aim, while, in accordance with the ontogenetic model, there may in principle exist one cure for all of the main human diseases. This cure must possess one property; the ability to abrogate the action of the Law of the Deviation of Homeostasis after the completion of the development of an organism. Finally, the ontogenetic model creates the basis for elaboration of a unique complex of preventive measures when considering in unity the processes of pregnancy, development,acceleration of development,aging, and age-related pathology. This is, in particular, the reason to consider not the three models of development of diseases, but three models of medicine that imply not only differences in the etiology of diseases, but, also, the differences in the proposed measures for their prophylaxis and treatment. Each model exists independently, but, at the same time, each is interrelated with each other. Nevertheless, to distinguish between them is obligatory from the theoretical and practical points of view. The methods used for prophylaxis and treatment, according to the ecological model, are insufficient for prevention from the diseases that develop in accordance with the ontogenetic model, although the restriction of excessive calorie intake, phenformin, and antioxidants may have common pathogenetic pathways among those that they affect; the modulation of the sensitivity of the hypothalamus to the regulatory signals may appear to be at the crossroad. The utilization of the tests estimating this parameter permitted the explanation of the geroprotector effects of such preparations as L-DOPA,pineal polypeptides, and diphenylhydantoin (Dilantin). As to the fear that the prolongation of the human life-span will be connected with the wasteful predominance of elderly people in the society, the ontogenetic model renders it groundless, since the slowing down of the development of age-related pathology, in accordance with the ontogenetic model of its development, means the prolongation of the period of maturity in human life that is free of the age-related pathology characteristic of the present time.

TABLE XVI THREE MODELS OF MEDICINE Ecological

Genetic

Etiology: the external factors

Etiology: the genetic defects

Prophylaxis: the elimination of the external pathogenic factors Chemical or radiational cancerogenesis

Prophylaxis: compensation for the defect

Alimentary obesity Immunodepression, caused by overnutrition Alimentary atherosclerosis Maturity-onset diabetes mellitus (diabetes type 11) Exogenous psychic depression The enhancement of the autoimmune lesions by overnutrition Stress-induced hypertension

Stress-induced abnormalities in adaptation Psychogenic (hypothalamic) amenorrhea Carcinogenic, stress-induced radiational aging

Hereditary cancer or predisposition to cancer (retinoblastoma, familial poliposis, etc.) Familial obesity T h e DiGiorgio and Good’s syndromes, Burton’s diseases, etc. Familial hyperlipidemias Hereditary predisposition to diabetes (HLA-B8, HLA-BW15) Endogenous psychic depression Hereditary autoimmune disorders Hereditary hypertension, caused, for example, by the defect in the 11hydroxylation of steroids Types A and B psychological reactions The Stein-Leventhal syndrome Progeria, Werner’s syndrome

Ontogenetic Etiology: the mechanism of the development of an organism Prophylaxis: the slowing down of the rate of the development of a n organism Cancrophilia

Age-linked obesity Metabolic immunodepression Atherosclerosis Age-related decline of the carbohydrate tolerance (prediabetes) Age-linked psychic depression Age-related elevation of the titer of autoantibodies Age-related elevation of the arterial pressure H yperadaptosis Climacteric Normal aging

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v. M. DILMAN et al. Acknowledgments

The authors are indebted to Professor J. R. Smythes and Professor R. J. Bradley for the immense work they have done while preparing our manuscript for publication. For necessary information on further development of the authors’ ideas, as well as references on the most ’important recent works, one may refer to Dilman’s articles inJ. Theor Biol., 1986, 118, 73-81; in Med. Hypotksis, 1984, 15, 185-208; in Dilman’s monograph “The Four Models of Medicine” (in Russian), Medicina, Leningrad, 1986; and in Golubev’s article in Exp. Oncol., 1984, 6(5), 10-15.

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Tomatis, L., Turusov, V., Day, N., and Charles R. T. (1972). Int. J. Cancer 10,489-506. Tsai, S. P., Lee, E. S., and Kautz, J. A. (1982). Am. J. Epdemzol. 116, 376-384. Tzagournis, M., Seidenstricker, J. F., and Hamwi, G. J. (1968). Ann. N.Y. Acad. Sci. 148, 945-957. Upton, A. C . (1977). In “Handbook of the Biology of Aging” (C. E. Finch and L. Hayflick, eds.), pp. 513-535. Van Nostrand-Reinhold, London. Vasilyeva, 1. A,, Bershtein, L. M.,Ostroumova, M. N., and Dilman, V. M. (1980). Vopr Onkol. 9, 34-36. Vasilyeva, 1. A., Ostroumova, M. N.. and Dilman, V. M.(1982). Neoplasma 4,496-475. Verzar. F. (1957). Gerontology I, 363-370. Waddington, C. H., ed. (1968). “Towards a Theoretical Biology I . Prolegomena.” Aldine, Birmingham. Walford, R. L. (1969). “The Immunologic Theory of Aging.” Munskgaard, Copenhagen. Weinberg, R. A. (1983). Cancer 52, 1971-1975. Weindruch, R., Gottesman, S. R., and Walford, R. L. (1982). Proc. Natl. Acad. Sci. U.S.A. 79,898-902. Weitz, H., Mayring, K., Wiebecke, B., and Eder, M. (1983).Dtsch. Med. Wochenschl: 108,535I. Welborn, T. A,, Breckenridge, A., Rubinstein, A. H., Dollery, C. ’11, and Fraser, T. R. (1966). Lancet 1, 1336- 1337. Wexler, B. C. (1976). In “Hypothalamus, Pituitary, and Aging” (A. V. Everitt and 1. A. Burgess, eds.), pp. 333-361. Thomas, Springfield, Illinois. Williams, C. C . (1957). Evolulion 11, 398-411. Worid Health Organization. (1971). “World Health Statistics Annual, 1967,” Vol. 1. Woods, S. C., and Porte, D. (1978). Adrc Metab. Disor&rs 4,283-312. Yevtushenko, T. P., and Bobrov, Yu. F. (1978). Fiziol. Cheloveka (Hum.Physiol.) 4,560-563. Zierler, K. I,.. and Rabinowitz, D. (1964).J. Clin. Invesf.43,950-962.

THE INTERPEDUNCULAR NUCLEUS By Barbara J. Morley Research Division Boys Town National Institute for Communication Disorders in Children Omaha. Nebrasko 68131

1. introduction

The interpeduncular nucleus (IPN') is a midline structure located on the ventral surface of the midbrain. The IPN is bordered laterally by the cerebral peduncles, dorsally by the ventral tegmental nucleus, and rostrally by the mammillary bodies. The rostroventral portion of the IPN protrudes into the interpeduncular cistern (Berman, 1968). The IPN was first described by Fore1 (1872) and its morphology has since been studied in several species (i.e., Jansen, 1930; Cragie, 1930; Herrick, 1934, 1948).The IPN is an oval-shaped body, has little bilateral cellular differentiation, and consists primarily of small oval or fusiform neurons with round or oval nuclei and little perinuclear cytoplasm (Mizuno and Nakamura, 1974). The IPN has been considered to be an unspecialized nucleus, having a loose organization and no known function. More recently, the IPN has gained recognition because of the discovery that this nucleus contains several morphologically defined subnuclei and discrete subregional distributions of several putative neurotransmitters (i.e., Hamill et al., 1984; Kapadia and DeLanerolle, 1984; Morley et al.,

'

Neurochemical Abbreviations: ACh, acetylcholine; AChE, acetylcholinesterase; BuTx, a-bungarotoxin; CAT, choline acetyltransferase; CCK, cholecystokinin; DA, dopamine; DBH, doparnine-P-hydroxylase;2-DG, 2-deoxyglucose; GABA, y-aminobutyric acid; GAD, glutamic acid decarboxylase; leu-enk, leucine-enkephalin; LHRH, luteinizing hormone-releasing hormone; NE, norepinephrine; met-enk, methione-enkephalin; QNB, quinuclidinyl benzilate; SP, substance P; SS, somatostatin; T H , tyrosine hydroxylase; TRH, thyrotropin-releasing hormone; VIP, vasointestinal peptide. Anatomical Abbreviations: CS, Raphe, central superior nucleus; DTN, dorsal tegmental nucleus; FR, fasciculus retroflexus of Meynert; Hb, habenula; L-Hb, habenula, lateral nucleus; M-Hb, habenula, medial nucleus; IP, habenulointerpeduncular tract; IPC, interpeduncular nucleus, central nucleus; IPI, interpeduncular nucleus, posterior inner division; IPN, interpeduncular nucleus; IPP, interpeduncular nucleus, posterior outer division; NDB, nucleus of the diagonal band; SM, stria rnedullaris.

157 INTERNATIONAL REVIEW OF NEUROBIOLOGY. VOL. 28

Copyright 0 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.

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1985).T h e large number of neurochemicals present in the IPN has called attention to the fact that this nucleus is an integrative center and may have important functions that have been unrecognized previously.

II. Neumanatomical Considerations

A. CELLTYPES N o investigator has done an extensive study of cell types in the IPN of mammals or a comparative study of cell types between mammalian and nonmammalian species. In Golgi preparations of human tissue, Kemali and Casale (1982) identified four cell types: pyramidal-like, stellar, fusiform, and multipolar. Most of these cells were identified as “small” and were typically characterized by their close association with the vasculature. Unusual cellular characteristics have been described in cells of the IPN of the frog (Kemali, 1977) and human (Kemali and Casale, 1982). Kemali (1977) described a group of neurosecretory-like cells in the frog IPN which contain large dense granules and have beaded varicosities. The processes from these cells were found to be oriented in a dorsoventral direction, perpendicular to the axons from fasciculus retroflexus (FR) of Meynert. These processes each had two synapses, apparently originating from axons in the FR. The processes did not make synaptic contact with neurons, but gap junctions were observed all along their length. These processes apparently terminated in the subpial surface of the IPN, where they may make contact with the interpeduncular cistern. Similar cells with processes ending with foot-like expansions on blood vessels and the pial of the IPN have been described in human tissue (Kemali and Casale, 1982). Ciliated glial cells and neurons have also been identified in the frog IPN (Kemali, 1975). T h e morphological descriptions of the rat and cat IPN do not mention any neurosecretory-like cells.

B. CYTOARCHITECTURE AND NOMENCLATURE There are discrepancies in the literature with respect to the number of subnuclei within the IPN and the nomenclature used to identify these subdivisions. An early study by Edinger (1899) identified five subnuclei in the IPN of the dog, but most later studies have identified only two main subdivisions (Cajal, 1911; Castaldi, 1923; Calderon, 1927-1928; Brown, 1943; Taber, 1961).

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Berman’s (1968) classification of the IPN of the cat is similar to that of Edinger (1899) but includes only four major subdivisions. Ives (1971) subdivided the IPN of the rat into four subnuclei. Hemmendinger and Moore (1984) identified eight subnuclei and Hamill and Lenn (1984) identified seven subnuclei in the rat IPN. Recently, Lenn and Hamill (1984) have attempted to standardize the nomenclature used for describing subnuclei in the IPN. These authors make a good case for the use of standardized terminology in describing the results of studies of the IPN. Due to inconsistencies in nomenclature, it is not possible to adequately compare results obtained from different laboratories nor data generated by different techniques. The nomenclature used by various investigators is shown in Table I. This table should aid in understanding the anatomical and neurochemical descriptions that follow. The various subnuclei described by Lenn and Hamill (1984) are shown graphically in Fig. 1.

C. AFFERENT PATHWAYS A major input to the IPN originates in the habenula (Hb). The fibers descend in the habenulointerpeduncular tract (IP), which comprises the core of the FR of Meynert. When entering the IPN, the fibers decussate at least twice, forming a spiral or figure eight pattern (Cajal, 1911). T h e mammalian Hb is generally divided only into two distinct regions, the medial (M-Hb) and the lateral (L-Hb) subnuclei (Cajal, 1911). Based TABLE I NOMENCLATURE USEDTO DESCRIBE THE SUBNUCLEI IN THE IPN Lenn and Hamill (1984)

Hemmendinger and Moore (1984)

Hamill and Lenn (1984)

Berman (1968)

Rostral Apical

Rostral-dorsal Caudal-dorsal

Rostral Dorsal

Central Apical

Central

Rostral-ventral

Central

Intermediate

Caudal-intermediate

Intermediate

Lateral Rostral-lateral Dorsal-lateral

Caudal-lateral Rostral-lateral Dorsal-lateral

Lateral Interstitial Dorsal-lateral

Posterior (outer division) Posterior (inner division) Paramedian

Ives (1971) Pars dorsalis Pars dorsalis magnocellularis Pars medianus

Pars medianus

Pars lateralis

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BARBARA J. MORLEY

FIG. 1. The subdivisions of the IPN. This figure was reconstructed from Hamill and Lenn (1984) using the nomenclature proposed by Lenn and Hamill (1984).RI., Rostral-lateral; Lat, lateral; In t , intermediate.

on bilateral lesions to both the M-Hb and L-Hb and the presence of degenerating fibers in the IPN, the Hb has been determined to be the origin of the fibers terminating in the IPN (Mitchell, 1963; Way, 1975). In other studies, aimed at determining if both Hb nuclei project to the IPN, either the M-Hb or L-Hb was lesioned; the results of these studies suggested that the IPN receives afferents from both subdivisions of the Hb (Akagi and Powell, 1968; Smaha and Kaelber, 1973). Other studies using either the anterograde degeneration method and/or the transport of horseradish peroxidase (HRP) or tritiated amino acids, however, indicated that the M-Hb but not the L-Hb projects to the IPN (Herkenham and Nauta, 1979; Marchand et al., 1980; Distel and Ebbesson, 1981 ; Hayakawa and Zyo, 1982). In amphibians, the origin of fibers has been determined

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to be the dorsal habenular nucleus, a cell group analogous to the M-Hb of mammals (Kemali et al., 1980; Kemali and Guglielmotti, 1982). There is probably some topographical organization of the afferent projections to the IPN. The dorsal portion of the M-Hb is believed to project exclusively to the lateral zones of the IPN (Herkenham and Nauta, 1979). There is also evidence that the lateral part of the M-Hb projects to the dorsal region of the IPN. The IPN also receives significant input from the nucleus of the diagonal band (NDB) (Swanson and Cowan, 1979; Krayniak et al., 1980; Contestabile and Flumerfelt, 1981; Hayakawa and Zyo, 1982; Hamill and Fass, 1984), the dorsal tegmental nucleus (DTN) (Briggs and Kaelber, 1971; Marchand et al., 1980; Hayakawa and Zyo, 1982), mesencephalic raphe nuclei (Taber-Pierce et al., 1976; Azmita and Segal, 1978; Bobillier et al., 1979; Marchand et al., 1980; Hayakawa and Zyo, 1982), the central gray area and the locus ceruleus (Marchand et al., 1980). Most studies tracing projections to the IPN have concluded that the pathway from the raphe originates primarily from the raphe centralis superior (CS) (Bobillier et al., 1979), but some fibers may originate in the dorsal and medial raphe nuclei (Taber-Pierce et al., 1976; Azmita and Segal, 1978; Marchand et al., 1980; Hayakawa and Zyo, 1982). The input from the NDB to the IPN also has a specific organization. The origin of the cells is thought to be in the vertical limb of the diagonal band (Krayniak et al., 1980),and the greatest number of axons are believed to terminate in the dorsal region of the IPN (Hamill and Fass, 1984).

D. SYNAPTOLOCY The axons arising from the Hb travel in the IP and synapse in the IPN as they cross through it, forming synapses en passant with dendrites. Milhaud and Pappas ( 1966) described axodendritic and axosomatic synapses with round and dense core vesicles in the IPN of the cat. Both axodendritic and axosomatic synapses were associated with subjunctional bodies, unusual postsynaptic densities associated with synapticjunctions in the Hb and IPN. Mizuno and Nakamura (1974) described the ultrastructure of three types'of axonal endings in the rabbit IPN. Most endings contained round synaptic vesicles, while others contained round and flattened vesicles. A few endings contained only flattened vesicles. Following lesions of the Hb, they observed degeneration only in those endings with round vesicles. Leranth et al. (1975)have described two types of fibers and two types of axodendritic endings in the IPN of the rat; both types of fibers and synaptic endings were reported to originate in the FR.

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Murray et al. (1979) described two types of synapses in the rat IPN. In the rostra1 and caudal subdivisions, the axons from the FR were found to form a single axodendritic synaptic contact. In the caudal and parasagittal zones, they were also found to form crest synapses. Crest synapses were described as synapses in which two presynaptic terminals formed asymmetrical contacts with the parallel opposing sides of an attenuated dendritic appendage. Lenn (1976) has described four types of synapses in the rat IPN and suggested a more complicated synaptic arrangement than previous investigators. In Lenn's (1976) preparations, two major and two minor types of synapses were identified. T h e first major type of synapse identified was the S synapse. These synapses were characterized by spherical synaptic vesicles and asymmetrical contacts. The synaptic vesicle characteristic of the S synapse typically consisted of a population of agranular vesicles varying from 400 to 600 A in diameter with occasional large agranular vesicles with diameters of approximately 1400 A. Many S endings also contained some large granular vesicles with diameters approximately 1000 A. S endings were typically found to be asymmetric. Subjunctional bodies, when observed, were morphologically similar to those described by previous investigators (Milhaud and Pappas, 1966; Mizuno and Nakamura, 1974). However, subjunctional bodies were not observed frequently and were seen only when the tissue was rinsed with high molarity buffer. S synapses were found to be irregularly arranged along the lengths of axons as they passed through the IPN. Each axon made multiple synaptic contacts, always with dendrites. The most characteristic type of arrangement for these synapses was described as two axons approaching a single dendritic process with each forming a S synapse on opposite sides of the dendrite. These S (axodendritic) synapses were estimated to account for 90-95% of all synapses in the IPN of the rat (Lenn, 1976). The second major type of synapse described by Lenn (1976) was the crest synapse. T h e crest synapse was characterized by agranular synaptic vesicles and a few large granular vesicles and asymmetric synapses. These synapses could not be differentiated from S synapses on the basis of presynaptic vesicles. However, they could be distinguished on the basis of their contact with dendrites. Crest synapses made only paired synapses, i.e., axons made a parallel contact on opposite sides of a narrowed dendritic process. Following unilateral lesions, only one of the two axonal endings forming a crest synapse degenerated, indicating that crest synapses are typically formed by two different axons. Like S synapses, these synapses were also made en pussant. Also of interest was the observation that the

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same axon could make both an S synapse and a crest synapse (Lenn, 1976; Lenn et al., 1983). Minor synaptic types in the rat IPN were called F synapses and axosomatic synapses. F synapses were characterized by flattened vesicles and symmetrical contacts. These endings occurred throughout the IPN in an unpredictable arrangement, were not consistently related to either S or crest synapses, and were considered to be of unknown origin (Lenn, 1976). Axosomatic contacts were characterized by spherical synaptic vesicles and asymmetric contacts. Axosomatic contacts were reported to occur rarely, but others have observed them more frequently in other species (Mizuno and Nakamura, 1974). Following bilateral lesions to the Hb, ultrastructural studies revealed that both S and crest synapses degenerated (Lenn, 1976; Murray et al., 1979), while F and axosomatic synapses remained normal in appearance (Lenn, 1976). Lenn (1976) observed degeneration of nearly all synapses in the IPN following lesions to the Hb. In contrast, Leranth et al. (1975) reported that the majority of boutons did not show signs of degeneration following a lesion to the rabbit FR. Of particular interest are the observations that the synapses in the adult IPN retain plasticity. When a long survival time was allowed after a unilateral FR lesion, crest synapses were still present, indicating that remaining axons were capable of replacing the missing side of a crest synapse (Murray et al., 1979). The synaptology of the mammalian IPN is typical of chemical synapses, but Kemali (1974) has described both chemical and electrical synapses in the frog IPN.

E. EFFERENT PATHWAYS The major efferent pathway originating in the IPN projects to the DTN (Massopust and Thompson, 1962; Mitchell, 1963; Smaha and Kaelber, 1973; Hayakawa et al., 1981). Other significant projections include the mediodorsal nucleus of the thalamus (Hayakawa et al., 1981; Velayos and Reinoso-Suarez, 1982), the L-Hb (Massopust and Thompson, 1962), septum, anterior mammillary nucleus, NDB, and preoptic area (Hayakawa et al., 1981) and the ventral tegmental nucleus (Smaha and Kaelber, 1973; Hayakawa et al., 1981; Irle et al., 1984). Projections to the dorsal and/or lateral hypothalamus have also been demonstrated in some species (Massopust and Thompson, 1962; Smith et al., 1980; Kemali and Guglielmotti, 1982). There is also some electrophysiological evidence to indicate a projection from the IPN to the CS (Maciewicz et d., 1981).

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BARBARA J. MORLEY

E SPECIES DIFFERENCES The IPN of the rat, cat, monkey, and man may be successively larger in volume, but it is not clear what species differences would represent. They could simply reflect an increase in the size of existing cell types and pathways or the addition of new cell types and new pathways. There are little anatomical data bearing on this point, but species differences in the neurochemical composition of the IPN discussed below may shed some light on this issue.

111. Neurochemistry

A. AFFERENT NEUROTRANSMITTERS The vast majority of studies investigating the neurotransmitters present in terminals within the IPN have focused on the well-known cholinergic pathway thought to originate in the Hb. Biochemical studies have demonstrated extremely high concentrations of acetylcholine (ACh),choline acetyltransferase (CAT), and acetylcholinesterase (AChE) (Lewis et al., 1967; Kataoka et al., 1973,1977; Pdlkovits andJdcobowitz, 1974; Cheney et ul., 1975; Nakamura et al., 1976; Muth el ul., 1980; Flumerfelt and Contestabile, 1982; Rotter and Jacobowitz, 1984)and high-affinity choline uptake (Kuhar et al., 1975) in the IPN. The levels of these “cholinergic markers” are so high that this pathway may be regarded as one of the major cholinergic pathways in the brain. By biochemical assay, ACh has been found in both the M-Hb and L-Hb (Hoover et al., 1978).The level of CAT in the M-Hb is, however, considerably higher than that found in the L-Hb (Hoover et al., 1978). Both the M-Hb and L-Hb contain moderate levels of AChE (Hoover et al., 1978),but the AChE levels and turnover in the L-Hb were found to be higher than in the M-Hb (Lehmann and Fibiger, 1979; Flumerfelt and Contestabile, 1982). More recent immunocytochemical studies using monoclonal antibodies against purified CAT have confirmed the presence of cholinergic terminals throughout the IPN (Kimura et al., 1981; Houser et al., 1983). The CAT-immunoreactive boutons appear to contain round synaptic vesicles and form axodendritic synapses (Hattori et al., 1977). The presence of an intense cholinergic input to the IPN is supported by receptor binding studies (Yamamura et al., 1974; Kuhar et al., 1975; Hunt and Schmidt, 1978; Wamsley et al., 1981; Rotter and Jacobowitz,

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1984; Clarke et al., 1985). Most of the cholinergic receptors in the rat IPN are nicotinic and apparently have a ganglionic pharmacology (Clarke et al., 1985). There are also some muscarinic receptors (Kuhar and Yamamura, 1976; Rotter and Jacobowitz, 1984) and nicotinic receptors with a peripheral pharmacology (Hunt and Schmidt, 1978; Rotter and Jacobowitz, 1984), as determined by the binding of quinuclidinyl benzilate (QNB) and or-bungarotoxin (BuTx). The distributions of QNB and BuTx binding sites were compared with that of AChE in the rat IPN. Rotter and Jacobwitz (1984) reported an overlap among the distributions of labeling by QNB, BuTx, and AChE. Lesions to the Hb and FR have repeatedly been demonstrated to deplete CAT levels in the IPN (Kataoka et al., 1973; Kuhar et al., 1975; Mata et al., 1977; Contestabile and Fonnum, 1983). Based on biochemical assays and AChE histochemistry, several studies have concluded that the source of cholinergic IPN terminals is the Hb (Kataoka et al., 1973; Kuhar et al., 1975; Mata et al., 1977; Lehmann and Fibiger, 1979; Flumerfelt and Contestabile, 1982). The lesion studies have resulted in ambivalent results with respect to the origin of cholinergic neurons, with some researchers claiming that the cholinergic input to the IPN originates exclusively in the L-Hb. Based on electrolytic lesions and kainic acid lesions, Flumerfelt and Contestabile (1982) maintained that the source of AChE in the IPN originated primarily in the L-Hb. AChE is not, however, a specific marker for cholinergic neurons and is not necessarily correlated with cholinergic function. Intense AChE in the L-Hb might indicate a cholinoceptive function or a function unrelated to cholinergic mechanisms. Cuello et al. (1978) provided evidence that isolation of the M-Hb with a knife cut did not produce a depletion of CAT in the IPN, while lesions and knife cuts that severed connections of the L-Hb selectively depleted CAT activity in the IPN. Recent immunocytochemical studies, however, have demonstrated CAT-immunoreactivecells in the M-Hb but not L-Hb (Houser et al., 1983). Kimura et al. (1981) also reported intense fibers throughout the FR which could be traced from the M-Hb to the IPN in the cat. The hypothesis that at least some cholinergic neurons project from the M-Hb to the IPN is also supported by the observation that ['Hlcholine is retrogradely transported (in large quantity) to the M-Hb following an injection into the IPN (Villani et al., 1983). Other studies have suggested that the cholinergic input to the IPN originates exclusively from the septum (Contestabile and Fonnum, 1983). Lesions of the septum were found to deplete CAT activity in the Hb and IPN of the rat. Lesions to the septum produced decreased CAT activity

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BARBARA J. MORLEY

in the Hb and IPN, while lesions of the NDB made with kainic acid failed to deplete CAT in either the Hb or IPN. Other investigators have provided evidence that the cholinergic neurons projecting to the IPN originate in both the M-Hb and NDB (Gottesfeld and Jacobowitz, 1978; McGeer et al., 1979;Jacobwitz and Creed, 1983). Lesions of the Hb depleted CAT in the IPN by 95%. Lesions of the NDB or stria medullaris (SM) depleted CAT in the IPN by 45%, indicating that the M-Hb and NDB each contributed 50% of the cholinergic fibers in the IPN. Since fibers from the NDB travel in the SM and pass through the M-Hb, lesions to the M-Hb would sever connections from the NDB. Hence the contribution of cholinergic neurons originating in the NDB was not assessed in previous studies. CAT-positive neurons have been identified in the vertical limb of the diagonal band (Kimura el al., 1981; Houser el al., 1983). Most recently, Woolf and Butcher (1985) have completed an extensive study of the cholinergic projections to the IPN. Injections with the fluorescent tracers, propidium iodide or Evans blue, were made in the IPN, and tissue sections were processed for either CAT immunocytochemistry o r AChE histochemistry. The authors found a good correlation between intense AChE-containing neurons and CAT-positive neurons projecting to the IPN from several structures. T h e largest number of apparent cholinergic neurons projecting to the IPN were found in the basal forebrain (vertical and horizontal limbs of the diagonal band and the magnocellular preoptic area) and the dorsolateral tegmental nucleus. A few positive cells were also found in the medial septa1 nucleus, substantia innominata, nucleus basilis, and the pedunculopontine tegmental nucleus. A substantial number of cells in the M-Hb were labeled by the retrograde tracers, but the authors reported that these cells were only weakly CATimmunoreactive. Knife cuts that separated the Hb from the SM reduced immunoreactivity in the M-Hb, FR, and IPN. The authors concluded that the M-Hb contributes little to the cholinergic input to the IPN and that CAT immunoreactivity in the M-Hb may represent fibers originating from the basal forebrain either as collaterals en route to the IPN or as independent pathways. A second possible neurotransmitter in afferents to the IPN is y-aminobutryic acid (GABA). Contestabile and Fonnum (1983) measured the activity of glutamic acid decarboxylase (GAD),a somewhat specific enzyme in the synthesis of GABA, following lesions of the septum and NDB. GAD activity decreased in the Hb by 65% following lesion of the SM and 40% by kainic acid lesions of the NDB. These authors maintained that a GABAnergic pathway originates in the NDB and projects to both the M-Hb and JPN. This is a likely possibility since GAD-immunoreactive perikarya have been localized in the NDB (Kohler and Chan-Palay, 1983).

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167

However, Kataoka et al. (1973) failed to find a change in GAD activity in the IPN following extensive bilateral lesions to the Hb. Mata et al. (1977) found no change in GAD activity with short survival times (0-3 days) but a substantial linear increase in GAD after longer survival (8-30 days). Both peripheral-like and central-like benzodiazepine receptors have been localized in the IPN (Young and Kuhar, 1980; Benavides et al., 1984), suggesting a functional GABAnergic pathway to the IPN. A third possible pathway terminating in the IPN may contain substance P (SP). SP immunoreactivity has been reported to be present in a high concentration in the IPN (Brownstein et al., 1976),and SP-immunoreactive perikarya were localized in the M-Hb (Hokfelt et al., 1975; Cuello et al., 1978), suggesting that neurons in the M-Hb might project to the IPN. Hong et al. (1976)lesioned the M-Hb and found a depletion of 80% of the SP immunoreactivity in the IPN. Mroz et al. (1976)made lesions to the M-Hb and observed a depletion of SP in the IPN which paralleled the extent of the lesion. Maximum depletion was 70% and occurred with either bilateral, complete M-Hb lesions or bilateral lesions to both the MHb and L-Hb. Subsequently, Emson et al. (1977) and Cuello et al. (1978) provided evidence that isolation of the M-Hb with a knife cut produced a depletion of SP in the ventral tegmentum. These authors concluded that the M-Hb projects to the ventral tegmentum where the distribution of SP-immunoreactive terminals corresponds to the distribution of dopaminergic neurons. More recent immunocytochemical studies, however, have confirmed that SP-immunoreactivefibers are present throughout the IPN (Inagaki et al., 1981; Taban and Cathieni, 1983; Hamill et al., 1984; Kapadia and DeLanerolle, 1984). Other peptides present in fibers within the IPN include cholecystokinin (CCK) (Loren et al., 197913; Beinfeld and F’alkovits, 1982), leucineenkephalin (leu-enk)(RajNaik et al., 1981;Hamill et al., 1984),methionineenkephalin (met-enk) (Finley et al., 1981; Haber and Elde, 1982; Kapadia and DeLanerolle, 1984) vasointestinal peptide (VIP) (Loren et al., 1979a; Hamill et al., 1984), and somatostatin (SS) (Hamill et al., 1984; Kapadia and DeLanerolle, 1984; Morley et al., 1985). CCK is contained primarily in fibers located along the dorsal border in the rat (Hamill et al., 1984). VIP is also found in fibers in the dorsal region of the rat IPN (Hamill et al., 1984). A few SS-positive fibers have been localized in the rat IPN (Hamill et al., 1984)but in significantlylarger concentrations in several areas of the frog (Vandesande and Dierickx, 1980) and cat IPN (Morley et al., 1985). The heaviest concentrations of fibers immunoreactive for SP, CCK, and VIP are in the lateral zones. In support of the appearance of enk-immunoreactivefibers, radioreceptor assays indicate that there is a high density of opiate receptors throughout the IPN (Atweh and Kuhar, 1977; Geary and Wooten, 1983),

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but the densest areas are in the dorsal and central divisions (Atweh and Kuhar, 1977) in which enk-immunoreactive fibers are not densely concentrated. Fibers in the IPN also contain norepinephrine (NE) (Farley and Hornykiewicz, 1977; Gottesfeld, 1984), dopamine (DA) (Singhaniyom et al., 1982; Hamill et al., 1984; Gottesfeld, 1984), and serotonin (Nojyo and Sano, 1978). In immunoctyochemical preparations, antibodies to dopamine p-hydroxylase (DBH) produce a heavy punctate reaction product, suggesting punctate processes or fibers of passage. Following lesions to the Hb, Gottesfeld (1984) observed increased reactions for both N E and DA, suggesting the possibility of sprouting in synapses containing these neurotransmitters. Nojyo and Sano (1978) have investigated the ultrastructure of boutons in the IPN following treatment with dihydroxytryptamine, a chemical which produces degeneration in serotonergic neurons. They found that many of the degenerating serotonergic axons in IPN did not actually make synaptic contact, indicating that the presence of fibers positive for a neurochemical may not actually be making synaptic contact.

AND B. COMPARISON OF NEUROTRANSMITTER DISTRIBUTIONS AFFERENT ANATOMICAL PATHWAYS

The subnuclei containing terminals positive for these various neurochemicals are listed in Table 11. The dorsal pattern of the M-Hb is believed to contain the perikarya immunoreactive for SP (Cuello et al., 1978). The dorsal M-Hb is also believed to project exclusively to the lateral zones (Herkenham and Nauta, 19?9), which are also heavily labeled for SP (Hamill et al., 1984), AChE, and cholinergic receptor ligands (Rotter and Jacobowitz, 1984). The lateral part of the M-Hb projects to the dorsal region of the IPN where AChE and BuTx are heavily labeled, suggesting that a cholinergic projection from the lateral M-Hb, but CAT-positive cells have been localized exclusively in the ventral M-Hb (Houser et al., 1983). It has been suggested that the distributions of enk and CCK immunoreactivity are similar to the pattern of innervation of the NDB projection to the IPN (Hamill et al., 1984; Hamill and Fass, 1984). In addition, it should be pointed out that the heaviest projection from the NDB terminates in the dorsal region of the IPN in which AChE and BuTx binding is intensely labeled. There is an apparent overlap in the distributions of some peptides and cholinergic markers. The colocalization of ACh with several peptides

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TABLE I1 NEUROTRANSMITTER MARKERSLOCALIZED IN TERMINALS WITH SUBDIVISIONS OF THE IPN Rostral

Apical

SP VIP

SP

ss

SS' Leu-enk DBH 5-HT CAT

Leu-enk DBH CAT SP CCK SSb Leu-enk 5-HT' Met-enk CATb

Central

Intermediate

Lateral SP VIP

SS' Leu-enk DBH 5-HT CAT

CAT

SS' Met-enk CAT'

Dorsallateral

References'

SP

2 2 294 2 2 2 1

Leu-enk DBH

SPb SS' Leu-enk 5-HT Met-enk CATb

Rostrallateral

5-HT' Met-enk

5-HT

2,3 2 2,3,4 2 293 2 1

a 1, Kimura et al. (1981); 2, Hamill et al. (1984); 3, Kapadia and DeLanerolle (1984); 4, Morley et al. (1985). 'Significantly increased amounts were observed in the cat IPN in comparison with the rat IPN. Although these measurements are believed to be attributable to true species differences, some may reflect methodological differences between studies; for example, in some studies animals were injected with cholchicine prior to preparation for immunocytochemistry.

has been demonstrated in several parts of the nervous system. It would not be surprising to find that certain peptides and ACh are colocalized in some synapses in the IPN.

C. EFFERENT NEUROTRANSMITTERS Using immunoctyochemistry, the IPN has been found to contain cells immunoreactive for several peptides, including SF', met-enk, and SS (see Table 11). SP-positive cells are found in rostra1 areas of the rat (Hamill and Fass, 1984) and in the medial and dorsal nuclei of the cat (Kapadia and DeLanerolle, 1984). Met-enk positive cells are found in the rostrocaudal, median, and intermediate areas of the cat. These peptides are apparently not present in the same cells. SP-positive cells vary from 9 to 13 pm, while met-enk-positive cells are larger, averaging 12-18 pm (Kapadia and DeLanerolle, 1984).

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In the rat, a few SS-positivecells are seen along the ventral surface of the dorsal nucleus (Hamill et al., 1984). In the cat, there is a large number and more extensive distribution (see discussion below in Section 111,E). Of all of the putative neurotransmitters known to be in the IPN, SS is undoubtedly in the greatest concentration (in certain species).In addition, SS-postive fibers have been observed in apposition to the interpeduncular cistern, suggesting the possibility that these cells are identical to the neurosecretory cells described by Kemali (1977)and Kemali and Casale (1982) in the IPN of certain species. There is also evidence for the presence of serotonergic neurons in the IPN (Palkovits et al., 1977). Singhaniyom et al. (1982) and Hamill et al. (1984) have localized serotonergic neurons in the dorsal portion of the IPN. It has been approximated that 400 serotonergic neurons are present in the IPN and that these neurons are continuous with the B8 cell group of the median raphe (Singhaniyom et al., 1982). The subnuclei containing perikarya positive for these various neurochemicals are listed in Table 11.

D. HORMONES Receptors for thyrotropin-releasing hormone (TRH) are found throughout the limbic system of the monkey with a very high concentration present in the IPN (Ogawa et al., 1981). In addition, a measurable level of luteinizing hormone-releasing hormone (LHRH) was found in the IPN (Silverman at aE., 1979; Samson et al., 1980).

E. SPECIES DIFFERENCES There appear to be significant species differences in the number and distribution of fibers and cells containing certain peptides. In the rat, the number of cells containing most peptides is low. In comparison, the cat IPN contains large numbers of neurons immunoreactive for SP and SS (Kapadia and DeLanerolle, 1984; Morley et al., 1985). Although peptides may be contained within a few cells within the rat IPN, it appears that they may not represent a major pathway. These data suggest that the concentration of SP and cells in the cat IPC may represent new pathways. We have no evidence of the termination of either of these pathways, but SS-positive cells and fibers are present throughout the pedunculotegmental tract, and a distinct distribution of immunoreactive fibers is present in the TDP (Morley et al., 1985).

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We also cannot eliminate the possibility that some cells in the IPN of certain species are neurosecretory. For example, SS synthesized in the IPN may be released into the portal system and/or cerebral spinal fluid (CSF) and not be present in a neuronal pathway.

IV. Physiology-Pharmacology of the IPN

A. RECORDING AND IONTOPHORETIC STUDIES The first investigator to record electrophysiological activity from the IPN was Lake (1973). Lake recorded both excitatory and inhibitory responses in the cat IPN following stimulation of the Hb. The majority of the cells were found to be excited by ACh, including those that were inhibited by Hb stimulation. Atropine blocked the ACh-induced excitation, but was without effect on activity following Hb stimulation. Electrical activity of the rat IPN was studied in vitro in tissue slices. Following stimulation of the IP, excitatory postsynaptic potentials were recorded in the IPN (Ogata, 1979a).Spontaneousfiring could be increased by application of high concentrationsof L-glutamate (Glu).Twofold lower concentrations of ACh and SP also increased the firing rate. A low concentration of NE decreased responding. The iontophoresis of several substances on extracellular recordings in the IPN of the rat was investigated by Sastry (1978). Both ACh and SP produced excitation in the majority of cells, but the effects of ACh were more rapid than SP. Both ACh and SP enhanced the responding produced by electrical stimulation of the Hb. When ACh and SP were applied together, the stimulation of the Hb was significantly enhanced. A similar synergistic response could not be mimicked by the combined application of ACh and Glu or SP and Glu. The effect of atropine was to antagonize both Hb stimulation and the application of ACh, but the effect was slow (1-5 min for maximum effect). In a few cells, stimulation of the Hb resulted in short latency excitation followed by a weak, but prolonged, depression. Although ACh was found to excite these neurons, atropine did not block the response to ACh. SP enhanced responding in less than half of these cells. Other neurons responded in a more complicated fashion. In another study (Takagi, 1984),ACh was found to produce excitation in approximately 60% of the IPN cells recorded from guinea pig brain slices. Nicotine (nicotinic agonist) and carbamyl-f3-methylcholinechloride and muscarine (muscarinic agonists) also produced excitation. Excitation

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produced by ACh was blocked by both atropine (a muscarinic antagonist) and curare (a nicotinic antagonist). T h e authors concluded that the cholinergic receptors in the IPN have mixed nicotinic and muscarinic characteristics. A somewhat different view has been presented by Brown (Brown and Halliwell, 1981; Brown et al., 1983, 1984). In tissue slice preparations of rat IPN, these investigators observed excitation by nicotinic cholinergic agents applied to IPN neurons. In addition, however, they observed a depression of the presynaptic action potential recorded within the IPN. This depression in the peak height of the action potential was caused by nicotine and dimethylphenylpiperazinium (DMPP), but not nicotinic agonists, Glu and muscarine, suggesting that this effect is purely nicotinic. This is supported by the observation that the depressant effects of the cholinergic agonist, carbachol, were blocked by hexamethonium, curare, and mecamylamine (ganglionic blockers), but not BuTx (a peripheral nicotinic antagonist). These authors interpret their results as indicating that the depression of the amplitude of the compound action potential in the IPN is due to the activation of presynaptic nicotinic cholinergic receptors. The electrophysiological observation that nicotinic receptors in the IPN may be presynaptic is not surprising. Schwartz et al. (1984) have demonstrated that [’HIACh binding sites in the hypothalamus and the striatum with a ganglionic pharmacology are likely to be presynaptic. B. HORMONAL EFFECTS Physiological activity in the IPN of the rat has also been studied following the systemic administration of progesterone (Kawakami et al., 1979).Multiunit activity was found to be elevated in both the Hb and IPN, suggesting that these neuronal groups may be “sensitive”to progesterone and that hormone levels might regulate activity in this pathway.

C.

METABOLISM

Of particular interest is the observation that the IP is metabolically active during anesthesia (Duffy et al., 1981; Herkenham, 1981; McQueen et al., 1984).Following the administration of several anesthetics, it has been observed that 2-deoxyglucose (2-DG) uptake is enhanced in the M-Hb, IP, and the IPN. This is in contrast to most brain areas, where metabolism is lowered. This finding may relate to some unknown function.

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These data are also interesting because they can be used to substantiate a functional pathway between the septum, M-Hb, and IPN via the IP. Lesions of the septum (Herkenham, 1981) or SM (McQueen et al., 1984) prevent the enhanced metabolic activity In addition, increased 2-DG uptake is not observed in the L-Hb.

V. Development of the IPN

A. HISTOGENESIS The histogenesis of the IPN has been studied in the rat by Hanaway et al. (1971). Large IPN neurons were produced in the neuroepithelium of the aqueduct on embryonic days 11 through 15 (Ell-15), proliferation was maximum on days E14-15 and ceased after day E15. Proliferation of smaller neurons began on embryonic Ell and ceased after E22. Cells which form the IPN were found to be produced in the medial third of the basal plate. From this area, the neurons migrated ventrally in the raphe. Two columns of cells were formed by lateral movement to form an inverted fountain pattern. Cells from this same group migrated laterally from these columns to form the ventral tegmentum and substantia nigra (Hanaway et al., 1971). IPN neurons in monkey and human follow a similar developmental pattern (Lenn et al., 1978). Cells in the IPN of both monkey and man show differentiation of neuronal size and the development of Nissl substance only after they have reached their destination. Growth of neuronal perikarya and elongation of their dendrites proceed throughout the fetal period and into the postnatal period. Synaptogenesis and the formation of functional synapses is believed to begin late in primate prenatal development and parallel the deveiopment of dendritic growth.

B. MORPHOLOCICPOSTNATAL DEVELOPMENT

Although the rat IP was found to be distinct at birth, only occasional S synapses were reported by Lenn (1978a) to be present at this time. The number of S synapses increased until 28 days of age. Crest synapses developed during the period 8-14 days postnatal. The F synapses appeared sometime between 14 and 21 days of age (Lenn, 1978a). Following unilateral destruction to one M-Hb in neonatal rats, S synapses were reduced in number and delayed in their time of appearance

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(Lenn, 1978b). These results have been interpreted to suggest that the remaining FR axons form an increased number of synapses (Lenn, 1978b). Crest synapses were also present in the deafferentated IPN, apparently formed by remaining axons (Lenn, 197%; Hamill and Lenn, 1983). Following bilateral neonatal lesions of the M-Hb, S synapses never formed. Crest synapses, however, were present 30 days postnatal (Lenn, 1978b; Hamill and Lenn, 1983).Both F synapses and axosomatic synapses increased significantly (Lenn et at., 1979). These data have been interpreted to indicate that there are several presynaptic and postsynaptic mechanisms acting together during synaptogenesis (Hamill and Lenn, 1983). Two observations are of particular interest. First, that eliminating one-half of the M-Hb afferents to the IPN (ipsilaterai M-Hb lesion) produces only a 2% increase of non-M-Hb synapses, suggesting a preference for synapses formed by axons from the MHb (Lenn et al., 1983). Second, crest synapses persisted in the absence of M-Hb afferents. They could be formed by one normal and one heterologous fiber or two heterologous fibers, indicating the importance of postsynaptic factors in the formation of crest synapses, i.e., that certain IPN cells have a “signal” necessary for the formation of these synapses.

C. NEUROCHEMICAL POSTNATAL DEVELOPMENT

Several neurochemical measurements were made in the IPN and surrounding ventral tegmental area in rats from 3 to 30 days postnatal (McGeer et al., 1976). Levels of CAT were low at 3 days and increased linearly to 30 days of age. At 3 days, AChE and tyrosine hydroxylase (TH) levels are approximately 75% of their level at 30 days. T h e level of GAD was approximately 24% of its level at 30 days and increased linearly with age. These data suggest that cholinergic synapses in the IPN may not be functional until late in postnatal development. These data are not inconsistent with the morphological data reported by Lenn (1978a) which indicated that the number of S synapses, the major type of synapse in the IPN, increased in number and maturity until 28 days postnatal. Assuming the presence of GABAnergic synapses in the IPN, the data presented by McGeer et al. (1976) may indicate that these (presumed inhibitory) synapses develop more rapidly. T h e F synapses, which contain flattened synaptic vesicles, also show a more rapid morphological development (Lenn, 1978a). The postnatal development of SS immunoreactivity in the cat IPN has been studied (Morley el, al., 1985). In immunocytochemical preparations, SS was found to be present at birth and to increase rapidly in a linear fashion (Fig. 1). This increase was interpreted to reflect a slight increase

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in the number of immunoreactive cells, but primarily a substantialincrease in dendritic growth. The postnatal development of dendritic processes has been suggested in monkey and man (Lenn et al., 1978).The increase in SS could reflect the development of axodendritic synapses and/or the maturing of a certain population of dendrites.

VI. Behavior

A. AVOIDANCE BEHAVIOR Several studies investigating the effects of lesions on avoidance behavior have reported that lesions of the IPN produced significant deficits in the acquisition of several avoidance tasks (i.e., Wilson et al., 1972; Wirtshafter, 1981).Lesions to the IPN also affect avoidance retention (Thompson, 1960),but the effects on retention may be transient (Thompson and Rich, 1961). IPN lesions produce alterations in behavior similar to those produced by lesions to the dorsal tegmentum and septum (Wirtshafter, 198l),suggesting that a functional connection exists between the septum and dorsal tegmentum that is mediated by the IPN.

B.

EMOTIONAL BEHAVIOR

Cragg (1961) reported that electrical stimulation of the NDB, Hb, IPN, or the DTN produced panting and cutaneous vascular dilation in rabbits. In order to determine how this pathway produced panting and cutaneous dilation, they compared their results with the panting behavior known to result from restraint in rabbits. They found that bilateral lesions of the SM or the IP prevented panting during restraint. Cragg (1961)concluded that the Hb, IPN, and DTN formed a pathway that affects respiration by activating emotional factors. In other studies, using different measures of “emotionality”lesions to the IPN were without effect (Enloe, 1975). There seems to be considerable evidence for the modulation of behavior by “emotion” or limbic activity. The finding that SS-fibers were observed near the interpeduncular cistern is interesting in that it provides one possible way that the IPN could have a generalized effect on behavior. SS is found in the CSE The origin of the SS found in the CSF is unknown, but believed to be extrahypothalamic (Sorensen et al., 1981). A low level of SS in the human CSF is correlated with episodes of depression characterized by a “sad appearance,” tiredness, and insomnia (Agren and Lundquist, 1984).

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SS analogs have been found to reverse bombesin-induced hypothermia, presumably by increasing metabolism, and to increase oxygen consumption (Brown, 1982),also linking this neuron to functions associated with this peptide. C. SEXUAL BEHAVIOR Several studies have demonstrated the susceptibility of the M-Hb or IPN to hormones, including estrogen (Motta et al., 1968)and progesterone (Luttge and Hughes, 1976; Kawakami et al., 1979). The M-Hb has additionally been implicated in the control of feminine, but not masculine, behavior in rats (Modianos et al., 1974), and the IPN may be involved in lordosis control mechanisms (Luttge and Hughes, 1976). Most studies of hormonally mediated behavior have not investigated the possible role of the IP and IPN, and, therefore, it is not known if the hormones and/or hormonal receptors present in the IPN have any functional consequence. Considering that electrophysiological activity in the IPN is altered by the systemic administration of certain hormones (Kawakami et al., 1979),such studies appear justifiable.

D.

DEVELOPMENT

The IPN may have a special role during development. It has been hypothesized that the IPN mediates sucking, swallowing, and visceral functions necessary for digestion during the early neonatal period (Sarnat and Netsky, 1981), but that these functions are later suppressed by the activation of cortical systems. This hypothesis may be related to the developmental profile observable for SS (or some other peptide) and to the observation that the M-Hb, IP, and IPN are active during anesthesia.

VII. Summary and Future Directions

The IPN is a phylogenetically conserved area of the brain. Anatomically, the IPN is unique in that it is primarily an unpaired structure, but receives a significant input from a paired structure, the Hb. Solely on the basis of its anatomical connections it can be concluded that the IPN is an important integrative center for the limbic system. The IPN is highly vascularized and protrudes into the interpeduncular cistern. In some

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species IPN cells having morphologic similarities to neurosecretory cells have been identified. The synaptology of the IPN is reasonably clear. The major synapse, the S synapse, is asymmetric with two populations of vesicles. The origin of these synapses are axons of the IF'. A major portion of these axons originate in the M-Hb, while others originate in the NDB. Although the majority of cells in the IPN are small, there are populations of cells that vary slightly in size and in apparent neurochemical composition. Cytoarchitecturallp the IPN can be subdivided into at least eight distinguishableareas, but these subnuclei are not homogeneous with respect to their efferent projections nor their neurochemical composition. Cells in the IPN give rise to axons that project to several areas of the brain, with the efferents projecting to the tegmentum, septum, and hypothalamus. Neurochemically, the IPN is complex. It is clearly the recipient of one or more major cholinergic pathways. Axons originating from cholinergic neurons in the NDB apparently form axodendritic synapses containing round vesicles. This cholinergic input may be in part presynaptic and mediated largely by nicotinic receptors. In addition, GABA and SP are contained in pathways terminating in the IPN. Other neurochemicals, in less well-defined pathways, include VIP, the enkephalins, CCK, SS, NE, and DA. Within the IPN, there are cells which contain serotonin, SP, SS, and enkephalins. The distributions of these neurochemicallydefined cell populations apparently do not correlate with anatomically defined subnuclei, and the projections of these neuronal groups are not known. There are known species differences with respect to the quantity and distribution of the neurochemically defined cell groups. Despite the large amount of information available, much of the organization of the IPN remains unclear. Summarized below are some of the remaining questions and some possible future investigations that may eventually elucidate our understanding of the IPN. First, one major type of synapse exists in the IPN of the rat, accounting for 90-95% of the total number of synapses. Although other species may have a more complicated synaptology, no report to date has indicated that such is the case. These synapses apparently contain ACh since CAT immunoreactivity has been found in synapses of the cat IPN. Cat immunoreactivity has been observed in small cells within the M-Hb, but the number of cells showing immunoreactivityis lower than one might predict on the basis of the quantity of the cholinergic input to the IPN. This can be accounted for by assuming that many or all of these axons originate in the NDB and/or the dorsolateral tegmental nucleus, which have

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demonstrated projections to the IPN, and are known to contain CATimmunoreactive neurons. If the cholinergic input to the IPN originates from more than one source, then the terminals are apparently not differentiated by their morphological characteristics. The observation that the biochemical measurements indicate extremely high concentrations of ACh and CAT is in agreement with reports that the IPN contains a high concentration of the binding of [3H]ACh as well as other receptor ligands. Recent electrophysiological data suggests that nicotinic receptors in the IPN are presynaptic. This suggests that ACh regulates the release of another neurotransmitter in the IPN. Assuming that ACh is not the primary neurotransmitter in the IPN, then an important aspect of the neurochemistry of the input to the IPN is not known. A candidate for the primary neurotransmitter is SP. However, there is no evidence that ACh and SP coexist in the same neurons contributing to the IP or in the same synapses within the IYN. The distributions of other peptides and amines also seem not to be identical to that of CAT. One necessary line of research is the identification of the proposed unknown neurotransmitter(s). Second, the IPN has obvious neuroanatomical connections, but what remains unknown is which of the neurochemically and/or morphologically distinct subnuclei project to various areas of the brain. Describing the neurochemical and morphological characteristics of these proposed subsystems is also a needed line of research. The IPN undoubtedly has an important role anatomically as an integrative center of the limbic system. The evidence also indicates that cells within this nucleus are neurosecretory in at least some species. A third important line of research is determining if neurosecretory cells are present in the IPN of all species and what hormone@)are secreted. Fourth, an important aspect of both the neurochemical and neuroanatomical understanding of the IPN is to determine whether important species differences exist. There are obvious differences in the size of the IPN. Neurochemical data, however, suggest that certain species may have cells within the IPN containing neurochemicals that are not common to all species. These data imply the possibility that all efferent pathways from the IPN are not present in all species. Neuroanatomically, these pathways have not been defined. Fifth, many questions remain regarding development and plasticity. An important addition to this area will be the inclusion of neurochemical measures and the determination of the roles that certain neurochemicals play in synaptogenesis and plasticity. Last, but certainly not least, is the necessity of determining the functions of the IPN. Clearly, the IPN is involved in the acquisition of aversive

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tasks, and this finding is consistentwith its anatomical role as an integrative center for the limbic system. In addition, metabolic activity in the IPN is spared or increased during anesthesia, suggesting several possible behavioral functions related to sleep, to autonomic functions such as respiration, or to a generalized “emotional”state. Acknowledgments

This research was supported, in part, by grants BNS 8410198 from the National Science Foundation and Biomedical Research Support Grant 2 SO7 RR05834-05.

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Kimura, H., McGeer, P. L., Peng, J. H., and McGeer, E. G. (1981).J. Camp. Neurol. 200, 151-201. Kohler, C., and Chan-Palay, V. (1983).Neurosci. Lett. 34,259-264. Krayniak, P. E, Weiner, S., and Segel, A. (1980). Brain Res. 189, 15-29. Kuhar, M. J., and Yamamura, H. 1. (1976).Brain Res. 100,229-243. Kuhar, M. J., DeHaven, R. N., Yamamura, H. I., Rommelspacher, and Simon, J. R. (1975). Brain Res. 97, 265-275. Lake, N. (1973).Exp. Neural. 41, 113-132. Lehmann, J., and Fibiger, H. C. (1979).Life Sci. 25, 1939-1947. Lenn, N. J. (1976)J. Comp. Neural. 166,73-100. Lenn, N. J. (1978a).J. Comp. Neurol. 181,75-92. Lenn, N. J. (1978b).J. Comp. Neural. 181, 93-116. Lenn, N. J., and Hamill, G. S. (1984).Brain Res. Bull. 13,203-204. Lenn, N. J., Halfon, N., and Kakic, P. (1978).Anat. Embryol. 152,273-289. Lenn, N. J., Wong, V., and Hamill, G. S. (1979).Brain Res. Bull. 4, 343-348. Lenn, N. J., Wong, V., and Hamill, G. S. (1983).Neuroscience 9,383-389. Leranth, C. S., Brownstein, M., Zaborsky, L., Jaranyi, Z. S., and Palkovits, M. (1975).Brain Res. 99, 124-128. Lewis, P. R., Shute, C. C. D., and Silver, A. (1967).J. Physiol. (London) 191,215-224. Loren, I., Emson, P. C., Fahrengrug, J., Bjorklund, A., Alumets, J., Hakanson, R.,and Sundler, F. (1979a).Neuroscience 4, 1953- 1976. Loren, I., Alumets, J., Hakanson, R.,and Sundler, E (1979b).Histocbmistry 59,249-257. Luttge, W. G., and Hughes, J. R. (1976).Physiol. Behau. 17,771-775. McGeer, E. G., Parkinson, J., and McGeer, P. L. (1976).Exp. Neurol. 53, 109-114. McGeer, E. G., Scherer-Singler, U., and Singh, E. A. (1979).Brain Res. 168,375-376. Maciewicz, R., Foote, W. E., and Bry, J. (1981).Brain Res. 225, 179-183. McQueen, J. K., Martin, M. J.. and Harmar, A. J. (1984).Brain Res. 300, 19-26. Marchand, E. R., Riley, J. N., and Moore, R. Y. (1980).Brain Res. 193,339-352. Massopust, L. C., and Thompson, K. (1962).J. Comp. Neurol. 118,97-105. Mata, M. M., Schrier, B. K., and Moore, R. Y. (1977).E x f . Neural. 57,913-921. Milhaud, M., and Pappas, G. D. (1966).J. CellBiol. 30,437-441. Mitchell, R. (1963).J. Camp. Neurol. 121, 441-457. Mizuno, N., and Nakamura, Y. (1974).Brain Res. 65, 165-169. Modianos, D. T, Hitt, J. C., and Flexrnan, J. (1974). Beheu. Baal. 10, 75-87. Morley, B. J., Spangler, K., and Javel, E. (1985). Dev. Brain Res. 20,241-248. Motta, M., Fraschini, E, Giuliani, G., and Martini, L. (1968).Endocrinology 83, 1101-1107, Mroz, E. A., Brownstein, M. J., and Leeman, S. E. (1976).Brain Res. 113,597-599. Murray, M., Zimmer, J., and Raisman, G. (1979).J. Comp. Neural. 187,447-468. Muth, E. A., Crowley, W. R., and Jacobowitz, D, M. (1980). Neuroendocrinology 30, 329336. Nakamura, Y., Hassler, R., Kataoka, K., Bak, I. J., and Kim, J. S. (1976). Folia Psychiatl: Neural. Jpn. 30, 186. Nojyo, Y., and Sano, Y. (1978).Brain Res. 149,482-488. Ogata, N. (1979a).Experientia 35, 1202- 1203. Ogata, N. (1979b). Nature (London) 277,480-481. Ogawa, N., Yamawaki, Y., Kuroda, H., Ofuji, T., Itoga, E., and Kito, S. (1981).Brain Res. 205, 169-174. Palkovits, M., and Jacobowitz, D. M. (1974).J. Camp. Neural. 157,29-42. Palkovits, M., Saavedra, J. M., Jacobowitz, D. M., Kizer, J. S., Zaborsky, L., and Brownstein, M. J. (1977).Brain Res. 130, 121-134.

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BIOLOGICAL ASPECTS OF DEPRESSION: A REVIEW OF THE ETIOLOGY AND MECHANISMS OF ACTION AND CLINICAL ASSESSMENT OF ANTIDEPRESSANTS By S. 1. Ankier Charterhouse Clinical Research Unit Limited London EClM 6HR, England

and B. E. Leonard

Phormacology Department University College Galway, Republic of Ireland

1. Introduction

Depression is an illness which, in the United States alone, affects some 400,000 patients and has been rated as the tenth major cause of death (Hollister, 1978). For over two decades, it has been widely assumed that depressive illness is associated with an abnormalityin brain noradrenaline and/or serotonin metabolism (Schildkraut, 1965;Bunney and Davis, 1965). Since the initial hypothesis was proposed, numerous studies have been conducted on body fluids of depressed patients and on postmortem brain material from suicides, attempting to validate the hypothesis and determine the precise mechanism whereby the abnormality in central biogenic amine metabolism occurs. Studies of the changes in the major metabolites MHPG), serof brain noradrenaline (3-methoxy-4-hydroxyphenylg1yco1, otonin (5-hydroxyindole acetic acid, 5-HIAA), and dopamine (homovanillic acid, HVA) have helped only partially to validate the hypothesis. Thus it has been reported that a reduction in the cerebrospinal fluid (CSF) concentration of MHPG before treatment is associated with a favorable response to tricyclic antidepressants, whereas a slightly elevated CSF concentration of this noradrenaline metabolite is associated with a poor response to such drugs (Maas et al., 1972; Beckmann and Goodwin, 1975; Hollister et al., 1980). However, not all investigators could replicate these findings (Coppen et al., 1979; Maas et al., 1982). Despite the recent study by Maas et al. (1984) in which it was shown that a low urinary excretion of MHPG was associated with a greater response of patients 183 INTERNATIONAL REVIEW OF NEUROBIOLOGY, VOL. 28

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with bipolar affective disorder to tricyclic antidepressants, the consensus of opinion casts doubt on the usefulness of neurotransmitter metabolite studies in predicting the response of depressed patients to different types of antidepressants. This has been reviewed elsewhere (Leonard, 1982). A major change in research emphasis has occurred during the last decade following the detailed studies of Sulser and colleagues, who established that the delay in the onset of response to antidepressants, or electroconvulsive therapy (ECT), was correlated with a decrease in the functional responsiveness of the postsynaptic P-adrenoreceptors (Vetulani et al., 1976; Sulser, 1978). The discovery of P-adrenoreceptors on the human lymphocyte membrane led F’andey et al. (1979) to study changes in the activity of this receptor system in depressed patients; these investigators found that the responsiveness of the P-adrenoreceptors was decreased in the depressed patient. Other investigators have, however, shown that the numbers of f3-adrenoreceptors are increased in the untreated patient and return to control values following effective treatment (Healy et al., 1983). Clearly there is a significant divergence between these findings, and while the results of the study by Healy et al. (1983) would support the concept of P-adrenoreceptor desensitization (“down-regulation”) following effective treatment which has been shown to occur in the frontal cortex of the rat brain after chronic antidepressant administration (Sulser, 1978), the significanceof these findings to our understanding of the etiology of depression is still uncertain. A somewhat similar situation has arisen when attempts have been made to evaluate changes in ap-adrenoreceptors on the platelet membrane as a possible marker of noradrenaline autoreceptors. Thus various groups of investigators have shown that the density of these receptors is either reduced (Wood and Coppen, 1981), unchanged (Daiguji et al., 1981), or increased (GarciaSevilla et al., 1981; Healy et al., 1983) in the untreated-depressed patient. In contrast to these disparate results obtained from studies on depressed patients, the effects of chronic antidepressant treatments on the density of as-adrenoreceptors in rodent tissues have been more consistent and have shown that chronic drug treatment is associated with a decreased density of a*-adrenoreceptors in brain and other tissues innervated by the sympathetic system (Crews et al., 1978a,b, 1981). As it is uncertain whether the ap-adrenoreceptor on the platelet membrane is similar to the pre- or postsynaptic as-adrenoreceptor in the brain or even that the presynaptic a2-adrenoreceptorsfunction as autoreceptors on central noradrenergic terminals (Laduron, 1984), the relevance of the clinical findings to the etiology of depression remains an open question. Despite the well-established evidence that many clinically effective antidepressants reduce the reuptake of [’Hlserotonin into synaptosomes from rat cortex following acute administration and have a similar effect on the uptake of serotonin into platelets of nondepressed subjects, their

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effects on platelet serotonin uptake of depressed patients is qualitatively different. Thus several groups of investigators have shown that the platelet serotonin uptake in depressed patients is reduced and increases following effective treatment (Coppen et al., 1978; Tuomisto et al., 1979; Born et al., 1980; Healy et al., 1983). It is interesting that this increase in serotonin transport into the platelet occurs irrespective of the acute effect of the antidepressant on serotonin transport into rat cortical synaptosomes, which suggests that there may be a correlation between altered serotonin transport and the symptoms of depression. There is also evidence that the activity of the serotonin 2 (5-HT2) receptor on the platelet membrane is reduced in the untreated depressed patient, but returns to control values following effective treatment (Healy et al., 1983).This finding is supported by a number of experimental studies in which it has been shown that the postsynaptic response to ionophoreticallyapplied serotonin in most limbic areas of the rat brain was enhanced by chronically administered antidepressants (De Montigny and Aghajanian, 1978; De Montigny et al., 1981; Wang and Aghajanian, 1980). The acute and chronic effects of antidepressants on central serotoninergicfunction has been extensively reviewed by Willner (1985). The purpose of this review is to assess critically the evidence supporting the amine hypothesis of depression in the light of the recent studies on amine metabolites and receptor function and thereby to define more precisely the etiological basis of the illness. In addition, the possible role of other neurotransmitters and neuromodulators will be examined. However, such an approach would be incomplete unless an attempt were also made to define the mode of action of the various classes of antidepressants in current use and to examine the methods used to assess their efficacy in clinical trials. No attempt will be made to assess the limitations of the various animal models of depression, which are being used for the initial identification of putative antidepressants, as this has been the subject of several critical reviews elsewhere (Leonard and Tuite, 1981;Jancsar and Leonard, 1983; Cairncross et al., 1979; Vergnes and Karli, 1963; Porsolt et al., 1978).

II. Biochemical Changes in Depression

A. POSTMORTEM MATERIAL

While the precise relationship between suicide and depression is unclear (Goldberg and Huxley, 1980), it is widely accepted that primary or secondary depression is a major contributory cause for suicide. It is not unreasonable to predict, therefore, that direct evidence implicating the

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involvement of biogenic amines in the etiology of depression should come from postmortem studies. Results from studies conducted prior to 1976 showed that the concentration of serotonin andlor 5-HIAA in the brain stem or other regions innervated by the serotonergic system was reduced (Shaw et al., 1967; Bourne et al., 1968; Pare et al., 1969; Lloyd et al., 1974; Birkmayer and Riederer, 1975), whereas studies by Beskow et al. (1976) and Cochran et al. (1976), which had been carried out on more discrete regions, showed no change in either serotonin or its metabolite. There is no consistent evidence from postmortem studies that either noradrenaline or dopamine concentrations are affected. Of the more recent studies of postmortem brain, the study of Meyerson et al. (1982) is of particular interest. These investigators compared the brains of suicides with those obtained from victims of homicide in the same region of the United States. Using standard receptor ligand techniques to determine receptor numbers in different regions of the brain, they showed that the densities of muscarinic receptors and [3H]imipramine-bindingsites were increased in the cortex of the suicide victims. It was of interest that the density of the P-adrenoreceptors, which are thought to play a crucial role in depression, were unchanged. Interpretation of these results is complicated by the fact that a substantial minority of suicide victims are not endogenous depressives (Goldberg and Huxley, 1980) in addition to uncertainty of the relationship between receptor number determined by ligand-binding studies and the functional status of the various receptors. This is discussed elsewhere. The major difficulties arising from studies of the brains of suicides are due to ( 1 ) the difficulty in assessing the effect of postmortem change on the metabolism of the neurotransmitters, (2) the presence of drugs that may affect the metabolism of the neurotransmitters being investigated, and (3) the precise diagnostic classification of the patients at the time of death. It is difficult to see how such factors may be controlled satisfactorily. Until this is achieved, the relevance of results obtained from postmortem findings to the etiology of depression must be treated with caution. In a recent review, Rossor (1984) has critically examined the problems relating to postmortem brain studies.

B. AMINEMETABOLITESIN BODYFLUIDS Despite the inconclusive evidence from studies on brains from suicides, it might be anticipated that an analysis of neurotransmitters and their

metabolites in the CSF and urine of depressed patients would provide a direct assessment of the relationship between the symptoms and neurotransmitter status.

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Differences in the concentration of 5-HIAA in the CSF of depressed patients compared with nondepressed controls have been replicated by several groups of investigators. Thus Sjostrom and Roos (1972) found that both 5-HIAA and the major dopamine metabolite, HVA, were reduced in the CSF of depressed patients. Later Asberg et al. (1976) reported that not all depressed patients had a reduced CSF 5-HIAA concentration, and they postulated that a subgroup of patients exists whose symptoms are primarily attributable to a reduction in the concentration of brain serotonin. The introduction of probenecid to block the efflux of acid metabolites from the CSF enabled investigators to assess the turnover of at least some of the biogenic amines in the patients’brain. Using such a technique, several groups of investigators have shown that the rate of accumulation of 5-HIAA was significantly less in depressed patients than in the control group (Bowers, 19’72; Goodwin and Post, 1973; Van Praag et al., 1973). Most studies also reported that the accumulation of HVA was reduced, which suggested that the turnover of dopamine was diminished in some depressives. The probenecid technique cannot be used to assess central noradrenaline turnover, as the efflux of the principal CNS metabolite, MHPG, is not impeded by this drug. Studies of central noradrenergic function in depression have, therefore, been largely restricted to an analysis of MHPG in the urine. Maas et al. (1973) examined the urinary concentration of MHPG in a heterogeneous group of depressive patients and found that there was a subgroup of patients with subnormal excretion of the metabolite. These investigators later concluded that the reduced MHPG excretion occurs in patients with primary affective disorders (Maas et al., 1984). Several questions arise when one attempts to evaluate the MHPG studies. First, the lifestyle of the patient (e.g., exercise, changed diet, and circadian rhythm) may play a determining role in changing the CSF or urinary MHPG concentration. Second, it is assumed that MHPG is derived primarily from noradrenergic nerve terminals in the brain so that the concentration of this metabolite in the CSF reflects central noradrenergic activity In an attempt to answer the first question, Sweeney et al. (1978) showed that in female depressives there were no significant effects of physical activity on urinary MHPG levels. These investigators did show, however, that a relationship existed between changes in the concentration of MHPG in the urine and the degree of anxiety, which might suggest that those patients with lower baseline MHPG levels were those who were more prone to anxiety under stressful conditions. Unfortunately, no nondepressed controls were included in this study, and as the urinary excretion of this metabolite can vary fourfold in normals and is subject to a diurnal rhythm (Hollister et al., 1978), great caution must be exercised in extrapolating from MHPG excretion data.

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Furthermore, these investigators showed that in normal subjects the individual excretion pattern varied considerably and was uncorrelated with diet, physical activity,or the prevailing affective state (Hollister et al., 1978). As regards the source of CSF MHPG, there is evidence from animal studies that this metabolite arises, at least in part, from noradrenaline contained in nerve terminals adjacent to the cerebral ventricles and that a considerable portion of this metabolite is dependent on the activity of the neurons comprising the locus coeruleus (Ader et al., 1979). In man, however, while MHPG in the lumbar CSF is mainly of central origin, there is also a considerable contribution from the spinal cord (Chase et al., 1973; Ziegler et al., 1977),which further emphasizes the need to exercise caution in drawing conclusions regarding the pathogenesis of affective disorders from such studies. Several investigators, already referred to above, have shown that the concentration of CSF 5-HIAA in depressed patients shows considerable variation. There is also ample evidence to show that a wide variation occurs in the urinary excretion of MHPG. These variations in the concentrations of amine metabolites occur despite the apparent homogeneity of the patient population as assessed by standard-rating scales. Thus Sacchetti et a f .(1979)reported a wide variability in the concentration of this metabolite in the urine of 25 primary depressed patients, and they suggested that the age of onset of the disease may be one factor accounting for the variability; a positive correlation was found between motor retardation and low MHPG excretion. These investigators also reported that depressed patients with normal or elevated MHPG concentrations tended to respond to clomipramine and amitriptyline, which suggests that the ability of such drugs to reduce serotonin reuptake may be related to a primary defect occumng in serotonin rather than noradrenaline metabolism. However, it must be stressed that no control group was used in this study. In contrast, nondepressed controls were used in a study of depression in female patients by Maas and colleagues (De Leon-Jones et al., 1975; Maas et al., 1973), who reported that the MHPG excretion was reduced during depression. However, neither Goodwin and Post (1973) nor Schildkraut el al. (1978) found any change in the excretion of this metabolite in unipolar depressives. While such studies may be indicative of a tendency toward defective brain noradrenaline function in depression in a subgroup of patients, the results are by no means unequivocal. The range of values quoted [from 910 & 99 pg/24 hr by Sacchetti et al. (1979) to 1950 +- 177 fig/24 hr by Schildkraut et al. (1978)l are well within the limits found by Hollister et al. (1978) for normal subjects (900-3500 pg/24 hr), and it is noticeable that several of the clinical studies reporting changes in MHPG excretion do not contain adequate control data. Furthermore, there is evidence that prolonged stress can significantlyelevate the MHPG excre-

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tion and decrease P-adrenoreceptor sensitivity even in normal controls (Mackenzie et al., 1980). Maas et al. f1984), in a detailed study of 104 patients with affective disorder, determined the CSF concentrationsof MHPG, HIAA, and HVA before and following treatment with amitriptyline or imipramine in an attempt to determine whether changes in the pretreatment concentrations of any of these metabolites could predict subsequent therapeutic response. The results of this study showed that a reduction in the MHPG concentration was associated with a better response to drug therapy in the bipolar group of patients only, whereas a slightly raised concentration of this metabolite predicted a poor response. Studies on unipolar patients revealed that a reduction in the HIAA concentration was related to the subsequent response to drug treatment, which supports the hypothesis that there are at least two biochemical subtypes of affective disorders, one type being associated with a subnormal noradrenergic system, while the other reflects an abnormality in serotonergic transmission (Maas, 1975; Asberg et al., 1976). While such findings support the view that irregularities in amine function may underlie different types of affective disorders, other investigators have produced evidence which conflictswith this view. Thus Montgomery ( 1982) studied the antidepressant response of the “specific”noradrenaline and serotonin uptake inhibitors maprotiline and zimelidine, respectively, on subgroups of endogenously depressed patients who had reduced or normal basal CSF HIAA concentrations. No correlation could be found between the clinical response of the patients to either antidepressant and the pretreatment HIAA values. Thus patients with initially a low CSF HIAA (“serotonin deficient”) responded equally well to either drug as did those with initially normal CSF HIAA concentrations. Veith et al. (1983) have also failed to demonstrate differences in response to antidepressant treatments based on differences in pretreatment MHPG concentrations. These investigators showed that a reduction in the pretreatment urinary MHPG concentrations did not predict those patients who subsequentlyresponded to desipramine (which shows some selectivity in reducing noradrenaline reuptake) from those respoqding to amitriptyline, a drug which impedes the reuptake of both noradrenaline and serotonin. Mendlewicz and co-workers (1982) in their study of the comparison between mianserin and amitriptyline on monoamine metabolites in the CSF of depressed patients also failed to find a relationship between therapeutic response to treatment with either drug and the pretreatment CSF metabolite concentrations. These studies lead one to conclude that the concentrations of monoamine metabolites in body fluids is unlikely to be of value as markers of the depressed state or of response to drug treatment. Nevertheless, it is

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not without interest that changes in the plasma noradrenaline concentrations have been shown to reflect the mood of the patient (Wyatt et al., 1971; Shimizu and Fujita, 1981; Luck et al., 1983). If it is assumed that the concentration of plasma noradrenaline reflects the activity of both central and peripheral sympathetic systems, then the determination of changes in plasma noradrenaline concentrations, coupled with an assessment of peripheral receptor function, might provide a useful link between neurotransmitter status and the mood of the patient.

C. PERIPHERAL AMINE RECEPTORS The results of experimental studies on the chronic effects of antidepressant drugs on postsynaptic adrenoreceptors have led to a greater insight into the adaptive changes that occur following drug administration and have also enriched the methods that have been developed for the selection of new putative antidepressants (Olpe, 1982). However, has such an approach led to a greater understanding of the state-dependent biochemical changes that might occur in the depressed patient? If antidepressants decrease the functional activity of the adrenoreceptor-linked cyclase system, then it may be postulated that this receptor-enzyme complex is hyperactive in the untreated patient. Furthermore, as the activity of the postsynaptic receptor system reflects changes in the concentration of neurotransmitter in the synaptic cleft, it must also be assumed that the mechanisms governing the release of the neurotransmitter are abnormal in the depressed patient. As the release of noradrenaline from the nerve terminal is regulated, at least partially, by the presynaptic a-adrenoreceptors (Stiirke, 1977), it seems likely that the activity of this receptor system is also abnormal. While there is broad agreement that chronic antidepressant treatment attenuates postsynaptic adrenoreceptor activity in the limbic cortex of the rat brain, presumably as a consequence of a decrease in the activity of the inhibitory a*-synapticadrenoreceptors which are located presynaptically, studies on changes in monoamine receptor activities on the lymphocyte and platelet membrane are equivocal. Thus different groups of investigators have found that the density of a*-adrenoreceptors (which are presumed to be similar to the presynaptic a-adrenoreceptors on the neuronal membrane) is unchanged, decreased, or increased on the platelet membrane of untreated depressed patients (Healy et al., 1983, for review of literature). Garcia-Sevilla and colleagues (1981), who showed that the apadrenoreceptor density was increased in untreated depressives, also showed that the density of these receptors returned to control value following effective antidepressant treatment therapy, suggesting that the

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change in a2-adrenoreceptoractivity was a state-dependent process. These findings have been largely replicated in a more extensive study by Healy et al. (1983). Studies on the changes in P-adrenoreceptor activity on lymphocytes of depressives have also yielded equivocal results with F‘andey and colleagues (1979), showing a diminished response of the receptorlinked isoprenaline-stimulated cyclase in untreated patients, which presumably reflects a diminished P-adrenoreceptor activity, whereas Healy et al. (1983) have shown an increased (3-adrenoreceptor density, which may indicate an increased receptor activity. Undoubtedly, one of the main difficulties in drawing any firm conclusions from such conflicting findings lies in the heterogeneous population of patients used (some studying manic-depressives during the depressive phase of the illness, whereas other studied postpartum or endogenously depressed patients), the differences in the techniques used to assess the density of the adrenoreceptors, and the possible effects of circadian fluctuation on changes in the receptor sensitivity. Another possible explanation for the variation in the results obtained by different groups of investigators for the changes in the density of presynaptic a-adrenoreceptors in the depressed patient before and following therapy could be associated with a variation in the nutritional status during the different phases of the study. It is well known that anorexia and a loss in body weight are frequent symptoms of the disease; increased appetite is generally taken to be a sign of clinical improvement and many of the conventional (“tricyclic”)antidepressants increase body weight as a consequence of the changes in the intermediary metabolism of carbohydrates which they induce. There is clinical evidence that an inverse relationship exists between platelet a2-adrenoreceptor density and the plasma catecholamine concentrations (Davies et al., 1982); fasting has also been shown to induce a fall in the concentration of plasma noradrenaline (Jung et al., 1979). More recently, Luck et al. (1983) have shown that the plasma noradrenaline concentrations in a group of malnourished patients with anorexia nervosa were significantly lower than in age- and sex-matched controls; the decrease in plasma noradrenaline was associated with a rise in the platelet cy2-adrenoreceptordensity. From this study, it may be concluded that the nutritional status of the depressed patient must be critically assessed and taken into account when attempts are made to correlate changes in platelet a2-adrenoreceptor density with the psychiatric status of the depressed patient. In the studies of changes in platelet adrenoreceptor activity in the depressed patient already referred to, such factors as the loss in body weight and nutritional status of the patient before and following therapy have largely been ignored. One of the major problems which arises when attempts are made to interpret the changes in tritiated ligand binding to the platelet or lym-

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phocyte membrane concerns the relevance of such ligand-binding data to the functional role of the receptor. Apart from the study by Kafka et al. (1981), none of the platelet a2-adrenoreceptor-binding studies concurrently measured the response mediated by the receptor. 1x2-Adrenoreceptors, by modulating the release of noradrenaline, indirectly inhibit cyclic adenosine monophosphate (CAMP)synthesis at the postsynaptic receptor site, while prostaglandin El (PGEI) has the opposite effect. Thus the inhibition by noradrenaline of PGEl-stimulated cAMP synthesis can be used as an index of responsiveness of the ap-adrenoreceptor (Kafka et al., 1977). While two earlier studies reported no difference in the inhibition by noradrenaline of PGEl-stimulated cAMP synthesis, Murphy et al. ( 1974), Wang et al. (1974), and Sever et al. (1984) have recently shown that, in a group of 23 selected depressives, although the ol2-adrenoreceptor density (assessed by [SH]dihydroergocriptinebinding) was increased before treatment, the PGEl-stimulated cAMP response and its inhibition by noradrenaline were significantly reduced compared to the controls. Thus there was an apparent dissociation between the 1x2-adrenoreceptor-binding data (suggesting increased numbers of a*-adrenoreceptors) and the functional change which suggested hyposensitivity of the receptors. These changes were not correlated with the plasma noradrenaline concentrations which did not differ significantly from the controls. The results of such studies emphasize the need for caution when extrapolating from ligand-binding sites (which reflect binding sites which may not be associated with physiologically active receptor sites) to physiologically responsive receptor sites. Whether changes in platelet membrane receptors are a true reflection of those occurring in the brain remains an open question, but detailed studies by Checkley (1980) on the neuroendocrine responsiveness to different adrenoreceptor agonists in depressed patients suggest that central adrenoreceptors are functionally subnormal in the untreated patient. This implies that the changes in a2-responsivenessin the platelet may be a reflection of similar changes in the brain. Whether these changes reflect the density and activity of pre- or postsynaptic a2-adrenoreceptors is unknown.

D. ARE RECEPTORSON BLOOD CELLSUSEFULMARKERSOF CNS ADRENORECEPTORS? In addition to the difficulties of interpreting changes in adrenoreceptor density on platelets and lymphocytes, which have already been mentioned, recent experimental evidence has thrown doubt on the existence of autoreceptors in the mammalian central nervous system. This chal-

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lenges the theoretical basis of the receptor adaptation hypothesis of depression and the mode of action of antidepressants. Experimental evidence suggests that most presynaptic receptors are not autoreceptors (i.e., receptors which are sensitive to the transmitter originating from the nerve terminal on which it is located), but are receptors which respond to a different neurotransmitter released from an adjacent neuron. Evidence for the existence of a2-adrenergic autoreceptors is largely indirect and derived from experiments on stimulation-evoked release of exogenous neurotransmitter; for example, determining the tritium release following the electrical stimulation of brain tissue which had been preloaded with [’H]noradrenaline (Langer, 1981). However, as Laduron (1984) has concluded in his critical review of the evidence for the existence of adrenergic and dopaminergic autoreceptors, there is no evidence from such studies that the released tritium originates from the same intraneuronal compartment as the endogenous transmitter. Furthermore, it has been shown that tritiated ligands, most of which are basic compounds, are trapped via different intracellular compartments of intact cells which have a slightly acidic pH (Maloteaux et al., 1983). An additional complication arises with the possibility that a number of peptide cotransmitters coexist with the “classical”neurotransmitters, such as noradrenaline, and it is not unreasonable to assume that some of these cotransmitters may modulate noradrenaline release independently of any presumed action of noradrenaline on its autoreceptor. Furthermore, in vitro binding studies have failed to locate clonidine-binding sites at presynaptic sites (Tanaka and Starke, 1979), which suggests that the drug, which has been widely used as a marker for the cr2-adrenoreceptors, has failed to demonstrate the presence of such receptors on the presynaptic noradrenergic neuron. From such experimental studies, it may be concluded that changes in the density of a*-adrenoreceptors on the platelet membrane of the depressed patient before and during treatment do not necessarily represent presynaptic changes. It therefore seems more likely that change in a2adrenoreceptor activity is an epiphenomenon unrelated to the etiology of the illness. Despite well-established experimental evidence showing that all clinically effective antidepressants, following chronic administration, reduce the functional activity of postsynaptic P-adrenoreceptors in the frontal cortex of rat brain (Sulser, 1983),the search for suitable models of the padrenoreceptor complex in the peripheral tissues of depressed patients, which may be used as an indication of central receptor function, has met with limited success. It is well established that platelets (Steer and Atlas, 1982; Kerry and Scrutton, 1983) and lymphocytes (Williams et al., 1979) have type 2 P-adrenoreceptors on their membranes. So far, studies on changes in P-adrenoreceptor density in depression

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have been restricted to those receptors found on the lymphocyte membrane. However, such studies have not taken into account the nonuniformity of the lymphocyte population. Thus the numbers of B- and T-type lymphocytes vary independently of one another during the day (Richie et al., 1983). In addition, there is currently no agreement regarding the density of P-adrenoreceptors on the lymphocyte subtypes (Pochet and Delespesse, 1983), while evidence is now emerging that the receptors on lymphocytes undergo circadian changes which are not necessarily related to those governing the number of circulating cells (Titinchi et al., 1984). It may be concluded from such studies that extrapolation from changes in P-adrenoreceptors on lymphocytes to those in the brain of the depressed patient should be made with considerable reservation. It is possible that studies of the p-adrenoreceptor on the platelet membrane may provide more reliable information particularly as this type of membrane contains other types of amine receptors in addition to P- and ap-adrenoreceptors; this has led some investigators to conclude that the platelet is a useful model of the central neurons (Campbell, 1981).

E. PERIPHERAL MARKERSOF CENTRAL SEROTONERCIC FUNCTION It is well established that many effective antidepressants impede the reuptake of ['H]serotonin into cortical synaptosomes from rat brain and into platelets of nondepressed subjects who have received a single dose of antidepressants such as clomipramineor zimelidine. Such findings suggest that the transport system governing the uptake of serotonin into the synaptosome and platelet is similar, suggesting that the platelet may provide a useful model of the nerve terminal in the brain of the depressed patient. Several investigators have found that the transport of ['H]serotonin into the platelets of depressed patients is reduced before treatment, but returns to normal following effective therapy irrespective of the presumed mechanism of action of the antidepressant (i.e., whether a specific noradrenaline or serotonin uptake inhibitor); ECT has also been shown to be equally as effective as antidepressants in normalizing the serotonin uptake (Healy et al., 1983, 1984; Born et al., 1980; Tuomisto et al., 1979). The sensitivity of the serotonin receptor on the platelet membrane also appears to be subnormal in the untreated patient and returns to control values following effective drug treatment. There is experimental evidence to suggest that the serotonin receptor on the platelet membrane is of the 5-HTp-type(Lampugnani et al., 1982),which is widely distributed in limbic regions of the brain and whose activity is increased following

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chronic antidepressant treatment. These findings therefore suggest that the sensitivity of 5-HT2 receptors is decreased in the untreated patients and returns to normal values following effective treatment. Other investigatorshave failed to find changes in peripheral serotonin receptor sensitivity in depressed patients. Thus Wood et al. (1984b) reported no difference in the serotonin-induced aggregation response in the platelets obtained from drug-free depressed patients and their controls. The reason for the difference in the findings of Wood et al. (1984b) and Healy et al. (1983)is uncertain apart from the different methods used to prepare the platelet-rich plasma. The question also arises concerning the relevance of platelet serotonin receptors and those found in the brain. Thus although central serotonin 2-type receptors have a high affinity for [’Hlspiperone, Schacter and Grahame-Smith (1982) failed to show that this ligand could bind to the human platelet membrane. Leysen et al. ( 1984) have, however, convincingly argued that both the ligand-binding sites and the platelet aggregation responses are mediated by 5-HT2-type receptors. E ENDOCRINE MARKERSOF DEPRESSION Carroll et al. (1976a) and others (Meltzer and Fang, 1983; Asnis et al., 1981) have demonstrated clearly that hypersecretion of cortisol occurs in the depressed patient and that it is not readily suppressed by the administration of 1 mg of dexamethasone. Furthermore, the circadian rhythm which underlies the secretion of cortisol is blunted in the depressed patient. These observations have led to the development of the dexamethasone suppression test (DST) for the diagnosis of depression. The widespread application of the DST in recent years is confirmation of its usefulness and of the need to supplement standard clinical criteria of diagnosis with objective biochemical markers of the depressive state. The use of the DST has invariably shown that “false-positives”can occasionally arise in patients suffering from senile states, alcoholism, and anorexia nervosa (Ballin et al., 1983). Whether such abnormal endocrine profiles are attributable to secondary symptoms of depression or due to such factors as the nutritional status of the patients awaits elucidation. Carroll and colleagues, who have made the major contribution to the development and use of the DST, have recently received its application and reliability (Carroll et al., 1981). An interesting application of the DST has been its use in the identification of the subtypes of depressive disorder. Schlesser et al. (1980)have shown that nonsuppression of the cortisol level by dexamethasone can

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assist in separating those patients with primary unipolar depression (45% nonsuppression) from bipolar (85%) and controls (6%).These investigators further used the DST to differentiate familiar subtypes of unipolar depression; it was also found that nonsuppression, following dexamethasone administration, is correlated with a good response to antidepressant therapy, providing further evidence for a biological difference between these groups. Other investigators have studied the impaired growth hormone response to different types of physiological and pharmacological challenge and the hyporesponsiveness of the thyroid gland to thyroidstimulating hormone (TSH) stimulation. Such studies have also shown that bipolar (manic-depressive) patients differ from unipolar (endogenous) patients. This work has been reviewed by Brown et al. (1984) and substantiates the view that changes in neuroendocrine responsivenessmay be used to differentiate between the subtypes of depression. Despite the widespread interest in the use of endocrine markers in the study of affective disorder, little attention has been paid to the possible involvement of the nutritional status of the patient in affecting the endocrine response. This problem has been addressed by Fichter et al. (1984) in their study on the neuroendocrine disturbances that occur in depression, anorexia nervosa and starvation. These investigators found that weight loss, catabolic state and reduced caloric intake induced major changes in the response to dexamethasone suppression thyroid-releasing hormone (TRH) and growth hormone (GH) responsiveness to physiobgical challenge. This finding raises a serious question regarding the specificity of such tests as biological markers for depression. Is it possible to link the changes in glucocorticord secretion with changes in the adrenoreceptor status of depressed patients? So far studies have been limited to the interactions between circulating glucocorticoids and the adrenoreceptor-linked cyclase system in the rat brain. Thus Sulser and colleagues (1983) have shown that a decrease in circulating corticosteroids following adrenalectomy is associated with an increase in the responsiveness of the receptor-linked cyclase system to noradrenaline; this change was not associated with the density of P-adrenoreceptors as assessed by their affinity for [SH]dihydroalprenalol.By contrast, other investigators [e.g., Wagner et al. ( 1979)jhave shown that estradiol decreases the cortical adrenoreceptor density and reduces the sensitivity of the cyclase unit, suggesting that different steroid hormones have different modulating effects on different populations of adrenoceptors in different brain regions! These effects could possibly be affected by changes in the regulatory processes in the cell nucleus which are altered by the binding of the appropriate steroid to its nuclear receptor.

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In addition to changes in the cortisol response to various types of drug treatment, which have been well documented and have helped to validate the usefulness of the dexamethasone suppression test as a diagnostic marker of depression, other changes in pituitary-adrenal function have also been found in the depressed patient. 1. Growth Hormone (GH) Physiological stimuli such as insulin induced hypoglycemia and amino acids (e.g., dopa and 5-hydroxytrypotophan)have been shown to stimulate GH secretion in control subjects. Depressed patients exhibit a blunted GH response to both insulin and 5-hydroxytryptophan(Takahaski et al., 1974; Gruen et al., 1975). Other investigators have shown that an apparent abnormality in the GH response to dopa could not be substantiated when factors such as the age and sex of the patients were adequately controlled (Sacher et al., 1975). An abnormal GH response to TRH or luteinizing hormone (LH) challenge in depressed patients has been reported by Brambilla et al. (1978), which supports the hypothesis that the pituitaryadrenal axis is functioning subnormally in the depressed patient. Matussek et al. (1980) have shown that the elevation of the plasma GH concentration caused by infusion of clonidine is reduced in the depressed patient, an effect which may reflect decreased responsiveness in central ap-adrenoreceptors. Patients with obsessive-compulsive disorders also exhibit a similar response to clonidine (Siever et al., 1984), so that it still remains to be proved whether a blunted GH response is specifically related to depression.

2. Luteinazing Hormone (LH) Preliminary studies have shown that the concentration of plasma LH is reduced in depression (Altman et al., 1975; Rubin et al., 1981) and increased in mania (Benkert, 1975). Such changes may be a reflection of an alteration in the state of libido in patients with depression of mania (Winokur et al., 1969). While there is a paucity of detailed studies on changes in LH secretion in depression, Whalley et al. (1985)have recently studied changes in plasma LH, cortisol, and prolactin levels in young males with mania or schizophrenia. Their results showed that plasma LH concentrations were raised as were plasma prolactin and cortisol concentrations; no changes occurred in the plasma testosterone and sex hormone-binding globulin concentrations in either patient group as compared with the controls. Perhaps more detailed studies on changes in LH concentrations before and after treatment of depressed patients may identify a useful marker for the illness and of response to treatment.

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3. Prolactin Baseline plasma prolactin concentrations have been shown to be elevated in both bipolar and unipolar depressed patients (Cuenca et al., 1978), with an abnormal circadian rhythm in the secretion of prolactin reported in such patients (Halbreich et aE., 1979). Nielsen et al. (1980) have shown a correlation between changes in the plasma prolactin concentration and the response of the depressed patient to antidepressant treatment, suggesting that this hormone may provide a useful state-dependent marker.

G. NEUROTRANSMITTERS OTHER THAN BIOGENIC AMINES 1. Peptides and Depression Increasing evidence has accumulated in recent years to suggest that neuropeptides produced in the hypothalamus and in the extra hypothalamic regions of the mammalian brain may have a significant effect on brain function. Such "peptidergic" pathways probably play an important role in behavior (Guillemin, 1977). In recent years, interest in the possible role of vasopressin in central neurotransmitter processes has arisen largely as a consequence of the effects of this peptide on the restoration of memory in rats, following the extinction of a conditioned avoidance response (de Wied et al., 1977). Preliminary studies with a derivative of vasopressin (Gold et al., 1979) showed that three out of four depressed patients improved after such treatment. Until these studies are repeated using the double-blind procedure, the validity of such a finding is doubtful. The concentration of endorphins in the cerebrospinal fluids of depressed patients has also received considerable attention in recent years, and a hypothesis has been advanced that depression is associated with a hypoactive endorphinergic system, whereas mania is associated with a hyperactivity of this system (Emrich, 1982). The body of experimental evidence, so far, provides little support for this hypothesis despite the claim by Emrich (1984) that the activation of specific opiate receptors in the brain by, for example, P-endorphin, may have some beneficial effects in depression; the partial opiate receptor antagonist buprenorphine has been shown in an open trial to produce a 40% reduction in the depression score within 4 days of the start of treatment.

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2. GABA and Depression The y-aminobutyric acid (GABA) agonist progabide has been shown to be effective in the treatment of depression in two double-blind studies (see Bartholini et al., 1984), its therapeutic efficacy being similar to imipramine. The finding that the concentration of GABA in the CSF of depressed patients is lower than in controls (Gold et al., 1980) and that tricyclic antidepressants inhibit the reuptake of GABA thereby facilitating its effect (Harris et al., 1973)helps support the view that one of the actions of antidepressant drugs is to correct a deficit in the GABAergic system. If the therapeutic action of GABA-mimetic drugs can be fully substantiated, the conventional amine hypothesis of depression will have to be amended to account for the modulatory role which GABA and possibly other amino acids and peptides has on central noradrenergic and serotonergic processes. The possible mechanism whereby progabide produces its antidepressant effect has been investigated by Zivkovic et al. (1982),who found that the chronic administration of progabide reduces noradrenaline turnover in rat brain. This change is not associated with a decrease in the density of P-adrenoreceptors or in the activity of the postsynaptic adrenoreceptor cyclase. Thus progabide has a qualitatively different effect to conventional antidepressants on noradrenergic transmission. Bartholini and Morselli (1983) have speculated that GABA receptor agonists act by changing the firing rates of noradrenergic and serotonergic cells so normalizing central neurotransmission in the depressed patient; presumably this could be brought about by the drug-activatingGABA heteroreceptors located on such cell bodies. It is not without interest that conventional antidepressants such as amitriptyline and atypical antidepressants such as citalopram produce changes in GABA receptor density following their chronic, but not acute administration. Thus Pilc and Lloyd (1984) have shown that different types of antidepressant increase the GABA-B receptor density after chronic administration; the monoamine oxidase (MAO) inhibitor pargyline had a qualitatively similar effect to the other types of antidepressants tested. These studies suggest that antidepressants may owe at least part of their activity to a modulation of GABA-B receptors, thus providing a link between the GABAergic and monoaminergic system. a. Histamine and Depression. Many clinically effective antidepressants have potent antihistaminic properties which may contribute to their mode of action. Although it is generally considered that the antihistaminic effects of antidepressants contribute primarily to the sedative rather than the antidepressant effect of these drugs, Wood et al. (1983, 1984a) have

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shown that the accumulation of ['4C]histamine by platelets from female depressives was significantly decreased compared to controls. In a more extensive study, these investigators have shown that the platelet histamine accumulation rate was lowest in both depressed male and female patients and slightly higher in those being treated with lithium, compared to ageand sex-matched controls (Wood et al., 1984a). The meaning of these results is currently a matter of speculation. However, as the uptake of histamine in the human platelet is by passive diffusion, it is possible that these changes reflect a nonspecific abnormality in the transport of small molecules across active membranes. In support of this view, Pettergrew et al. (1982) have reported that a membrane abnormality occurs in the intact erythrocytes and lymphocytes of manicdepressives. 6. S-Adenosylmethionzne (SAM) and Depression. Studies undertaken since 1975have suggested that the methyl donor, SAM, has antidepressant properties. Initial studies were undertaken on schizophrenic patients as part of a program to evaluate the transmethylation hypothesis of schizophrenia. Two double-blind placebo controlled studies in depressed patients showed that SAM was as effective as clomipramine or amitritpyhe (Muscettola et al., 1982);Del Vecchio et al., 1978; Kufferle and Grunberger, 1982). Apart from a slight increase in the anxiety case, SAM appeared to be reasonably free from side effects. Preliminary studies on the antidepressant properties of SAM in the United Kingdom tended to confirm the Italian studies (Charney et al., 1981). The possible mechanism by which SAM brings about its effect is unclear, but Ordonez and Wurtman (1974) have examined the interrelationship between folate and the SAM metabolism in rat brain and suggested that the increase in the availability of folate as a cofactor for biogenic amine synthesis might contribute to the effect of SAM on central neurotransmission. In addition, experimental studies have shown that SAM increases the turnover of serotonin and noradrenaline in rat brain (Curcio et al., 1978) and also increases the concentration of HIAA in CSF of depressed patients (Agnoli et al., 1976). Whether these effects of SAM are secondary to the changes in membrane phospholipid methylation, which is thought to be the initial pathway for the transduction of receptormediated signals through the neuronal membrane (Hirata and Axelrod, 1980),is unproved. However, it seems possible that changes in neurotransmitter turnover, receptor sensitivity to biogenic amine neurotransmitters, or in endocrine function may be mediated by changes in membrane activity; such changes may be modulated by chronic SAM administration.

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Clearly, more double-blind studies are needed to confirm the efficacy of SAM as an antidepressant and also to elucidate its precise mechanism of action in modifying membrane processes.

111. Mechanisms of Action of Antidepressants

A. STRUCTURE-ACTIVITY STUDIES ON NORADRENALINE-UPTAKE INHIBITORS Because of their long clinical availability, it is not surprising to find that the most extensive studies on structure-activity relationships have been made on the tricyclic antidepressants. Thus Salama et al. (1971) studied the effects of several different tricyclic antidepressants on ['Hlnoradrenaline uptake into rat cortical slices and showed that some of the most potent noradrenaline-uptake inhibitors contained a dihydrodibenzazepine ring (e.g., desipramine with an IC50 of 7.1 X lo-' M). The inhibition of noradrenaline uptake was found to be decreased if the number of carbon units in the side chain was increased or decreased from the optimal value of three: branching of the side chain also reduced the potency With regard to substitutions on the side chain N , Salama et al. (1971) found that compounds in the dibenzocycloheptatriene (protriptyline) series which contained-NH2, NHCH3, and N(CH& were equipotent; N-ethyl,N-isopropyl, and N-butyl derivativeswere only weakly active. Horn et al. (1971) studied the amine uptake inhibitory properties of the amitriptyline series and showed that the secondary amine (nortriptyline) had only one-tenth of the potency of the tertiary amine (amitriptyline)in inhibiting noradrenaline uptake into hypothalamic synaptosomes; others have shown that the difference between these drugs w a s only fourfold (Salama et al., 1971; Maxwell et al., 1969). In addition to the effects of changes in side-chain substitution on noradrenaline uptake in vitro, Maxwell et al. (1969)noted that if the rings of a tricyclic antidepressant were coplanar, then the compound was only a weak inhibitor of amine reuptake, whereas uptake inhibition was dramatically increased if the phenyl rings were held at dihedral angles >90" and <180".These investigators suggested that the low potency of coplanar tricyclic compounds could be attributed to the projection of the ring system into the binding site for the nitrogen atom. Later studies showed that several bicyclic antihistamines also had weak noradrenaline-uptake

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inhibitory properties in vitro, showing that the tricyclic ring was not essential for uptake inhibition (Maxwell et al., 1974).This has been verified more recently with the establishment of the bicyclic compound viloxazine as an effective antidepressant. Studies on the optical isomers of desipramine and other noradrenaline uptake inhibitors (Maxwell et al., 1970a) suggest that the phenylethylamines and the amine-uptake inhibitors bind by a single phenyl ring to a hydrophobic surface, while the N binds to a negatively charged area within the same plane as the phenyl ring. A hydrophobic area is believed to be adjacent to the negatively charged area in the receptor site; this hydrophobic area receives any methyl substitution on the N-side chain. A detailed discussion on the structure activity relationship for amine reuptake in the tricyclic antidepressant series has been published by Maxwell and White (1978). For optimal interaction with the noradrenaline uptake system, derivatives of phenylethylamine must occupy the fully extended (antiperiplanar) conformation (Maxwell et al., 1970b; Bartholow et al., 1977). As most derivatives of phenylethylamine are conformationally mobile, a variety of conformations can be assumed by the drug in the region of the transport site for noradrenaline. Determination of the nature of the amine-uptake site may help in evaluating the relationship between the structure and activity of specific noradrenaline-uptake inhibitors. The most widely accepted model of the transport site is based upon that described by Bogdanski and Brodie ( 1969),which suggests that a carrier noradrenaline-Na+ complex is transported to the inner surface of the neuronal membrane, where the Na+ and noradrenaline dissociated form the carrier, an effect facilitated by the low amine and Na' concentrations. The low amine concentration is maintained by monoamine oxidase, while the sodium ions are removed by the Na' ,K+-ATPasepump. The mechanism may be represented diagrammatically as follows: +

IInner membrane1

NA

-MA0 Astorage +met abol i t e s

+ Nd

K+ NA, noradrenaline; Na. sodium; K + , potassium ions; 0 ,carrier.

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Despite the attraction of the hypothesis linking catecholamine uptake to Na+,K+-ATPaseactivity, studies attempting to validate this hypothesis (following the administration of antidepressant and other types of psychotropic drugs to rats) have proved inconclusive. Thus Leonard and McNulty (1977) found that, although amitriptyline inhibited the activity of this enzyme in vitru, when the drug was administered acutely or chronically to rats, the enzyme activity was increased in several brain regions. If inhibition of amine reuptake by amitriptyline is linked to membranebound ATPase, one would anticipate that the enzyme activity would be decreased, not increased, following administration. It would appear that changes in Na+,K+-ATPasefollowing antidepressant treatment are coincidentally, rather than causally, linked to the amine transport site. It is not the purpose of this article to review extensively the relationships between chemical structure and pharmacological activity of the various classes of antidepressants, but to give an overview of the less controversial aspects of the subject. Detailed aspects of the structureactivity requirements for noradrenaline-uptake inhibitors have been published by Patil et al. (1975) and Ross (1984), while the conformational preferences of drugs, specifically inhibiting noradrenaline uptake, have been assessed by Rutledge et al. (1984). In addition to the conformational structure, chirality may also play an important role in determining the M A 0 or catecholamine-uptakeinhibitory properties of an antidepressant. For example, the cis-isomer of tranylcypromine has only one-third the potency of the trans-isomer in inhibiting M A 0 in vivo, even though it appears to be equipotent with the trans-isomer as a monoamine oxidase inhibitor MA01 in vitro (Zirkle et at., 1962;Zeller and Sarkar, 1962).Among the tricyclic antidepressants, the bridged derivative maprotiline and its side-chain-hydroxylated derivative oxaprotiline are potent and selective inhibitors of noradrenaline uptake into the rat heart and brain in vitro (Pinder et al., 1977; Waldmeier et al., 1977). Detailed studies of the (R)( -) and (S)-( +) enantiomers of oxaprotiline (Waldmeier et al., 1982)show that the pharmacologically activity form of the drug resides in the (S)-( ) configuration. This corresponds to the (S)-configurationof noradrenaline and presumably inhibits the transport site for the amine. Further studies by Delini-Stula et al. (1983) have shown that the stereospecific action of oxaprotiline is maintained after subchronic administration as only the (S)(+) form antagonizes the central depressant effects of clonidine. These effects are paralleled by a reduction in P-adrenoreceptor sensitivity, as indicated by a decrease in the P-adrenoreceptor density in rat cortical membrane coupled with a decrease in functional activity of the adenylate cyclase system. Despite the well-established pharmacological differences

+

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between the oxaprotiline enantiomers, preliminary clinical studies suggest that both enantiomers are equally effective as antidepressants (DeliniStula et al., 1983). If confirmed by more extensive studies, this finding emphasizes that caution must be taken in extrapolating from experimental studies to the therapeutic activity in depressed patients. A detailed review of the differences between the pharmacological properties of chiral stereoisomers of various classes of antidepressants has been made by Nickolson and Pinder (1984) and by Ross (1984). Caution must be exercised in extrapolating from in vitro data to the chronic administration of amine uptake inhibitors. For example, compared with some of the newer nontricyclic antidepressants, drugs such as imipramine and amitriptyline show only slight selectivity in inhibiting serotonin compared to noradrenaline uptake after acute administration. Following chronic administration, both drugs produce secondary amine metabolites (desipramine and nortriptyline, respectively), which show some selectivity for inhibiting noradrenaline reuptake. Similarly,the serotonin uptake inhibitor clomipramine is metabolized to its desmethylated derivative during chronic administration. Therefore, both noradrenaline and serotonin reuptake will be impeded by the parent compound and its metabolite in this situation. Even with the newer nontricyclic antidepressants such as nomifensin, which show considerable specificity in inhibiting noradrenaline uptake in vitro, there is evidence from in vivo studies that one of its principal metabolites, 4-hydroxynomifensin, selectively impedes the reuptake of serotonin (Leonard et al., 1984). Thus it seems unlikely that the specific uptake inhibitory properties of many antidepressants have any relevance to their activity on amine transport following their chronic administration.

B. STRUCTURE-ACTIVITY STUDIES ON SEROTONIN-UPTAKE INHIBITORS It is well established that the tertiary amine tricyclic antidepressants, such as imipramine and amitriptyline, are 5-10 times more potent in inhibiting serotonin than noradrenaline reuptake. Horn and Trace (1974), in their study of the effects of a series of tricyclic compounds on the uptake of 5-r3H]HT into rat hypothalamic homogenates, found that altering the length of the side chain by one or more carbon units (from its optimal length of three units) decreased the uptake inhibitory effect 7- to 8-foId, while OL or @-methylsubstitution in the side chain reduced the potency 17- to 33-fold, respectively These investigators also showed that

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chlorine substitution into position three on the tricyclic ring increased the potency !&fold,while dimethylamino-substitution in this position did not affect the potency. Substitution of a sulfur atom for the 2C bridge in imipramine and in clomipramine, which give promazine and chlorpromazine, respectively,reduced serotonin-uptake inhibition by 30- to 50-fold, respectively.

ON MONOAMINE C. STRUCTURE-ACTIVITY STUDIES OXIDASE INHIBITORS

The general structure, which describes most of the known monoamine inhibitors (MAOIs), can be described by the formula Aryl-X-N-R2

I

RI

where aryl is a phenyl, an indole, or a substituted analog; X is an aliphatic group containing one or more C or 0 groups; N is an amino, amide, or hydrazine N; R1 is -OH or -CHs; and R2 is hydrazine, propargyl, or cyclopropyl. In the most potent irreversible MAOIs, the R2 group binds irreversibly to the enzyme. In some MAOIs, a cyclopropyl group may be introduced into the X position (e.g., tranylcypromine), thereby markedly increasing the inhibitory potency. P-Carbolines may be considered as cyclized indolealkylamines and conform to the structural features of this general formula. The requirement for the aromatic ring in all MAOIs, together with an N-group, suggests that the inhibitor must bear some resemblance to the substrate if it is to bind to the active center of the enzyme. The most potent MAOIs contain an aromatic group separated from the amine-N group by at least one carbon unit. It is thought that the initial enzymeinhibitor interaction is initially weak and reversible; the attachment to the enzyme surface becomes irreversible if other features of the MAOIs molecule provide favorable binding properties. Fujita ( 1973) has suggested that the aromatic moiety is bound to an electron-rich noncatalytic site of the enzyme and that changes in M A 0 inhibitory potency, as a result of substitution on the aromatic ring, is usually dependent on the steric effects of these substitutents. A detailed review of the structure-activity relationships of different types of MAOIs has been written by Maxwell and White

(1978).

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D. CHRONIC EFFECTS OF ANTIDEPRESSANTS ON NEUROTRANSMI-M-ER FUNCTION Antidepressants are often classified in terms of their effects on the presynaptic sites on the neurons of biogenic amines. Thus until the discovery of such atypical antidepressants as iprindole, mianserin, and trazodone, antidepressants were classified as monoamine oxidase inhibitors or amine reuptake inhibitors. Such a classification was undoubtedly helpful in the development of a plethora of drugs based on chemical modifications of imipramine or phenelzine. This was perpetuated by the use of animal models which could only select molecules with MA01 or reuptake inhibitory properties, but which could not identify antidepressants such as mianserin or trazodone that do not act in this way in vim. It may be argued that the very success of MAOIs and amine reuptake inhibitors helped to provide the basis for the amine hypothesis of depression. Recently, a number of serious deficits have been found in the amine hypothesis, some of which have already been referred to. Thus a delay occurs between the initial administration of the antidepressant and the time of onset of the therapeutic effect. This delay in onset occurs irrespective of the chemical nature or acute pharmacological profile of the drug. As a delay also occurs with nonpharmacological treatments such as electroconvulsive shock therapy or REM sleep deprivation, it may be presumed that all effective antidepressant treatments bring about adaptational changes in one or more central neurotransmitter systems which take several weeks to become established. The lack of correlation between the acute effects of antidepressants on the amine reuptake processes with the antidepressant response is further compounded by the lack of correlation between the potencies of the drugs in inhibiting amine reuptake and their therapeutic potencies. Furthermore, several drugs were developed on the basis of their amine uptake inhibitory properties in animal models of depression only to be found ineffective when administered to depressed patients. This can be exemplified by the phenyl propylamine derivative gamfexine which was shown to be 30 times more potent than imipramine in reversing reserpine-induced ptosis in mice (Gershon et al., 1967). Similarly, the benzylammonium compound BL-KR 140 was shown to reverse reserpine-induced hypothermia in mice, a routine method used in screening compounds for their potential antidepressant activity, but failed to show any antidepressant activity in initial clinical studies (Hekimiran et al., 1968). At least ten compounds of varying structure and potency in animal models of depression have been reported to be therapeutically ineffective (Kelwala et al. 1983), thereby serving as a warning regarding the extrapolation of the acute effects of psychotropic drugs on

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neurotransmitter systems in animals to their therapeutic effects following chronic administration. A further limitation of the amine hypothesis of depression arises when one considers the effect of amine precursors on depressive symptoms. Thus if the depression arises as a consequence of a deficit in brain biogenic amines, one would anticipate that the administration of the amine precursors of the catecholamines and indole alkylamines (L-dopa and L-tryptophan, respectively) would lead to a rapid reversal of the symptoms. Clinically, the response of depressed patients to either treatment has been found to be equivocal. It must, therefore, be concluded that antidepressant treatments bring about their therapeutic effects indirectly and not directly by modifying the reuptake or metabolism of biogenic amine neurotransmitters. This subject has been reviewed critically by Sulser (1982). In the last decade, there has been a major switch in research emphasis from the pre- to the postsynaptic neurons regarding the mode of action of antidepressant drugs. The initial studies of Vetulani et al. (1976) and Banerjee et al. (1977) indicated that various chronic antidepressant treatments changed the number and responsiveness of postsynaptic P-adrenoreceptors, while acute treatments were without effect. These findings were of considerable conceptual value because they established for the first time a single biochemical event which could be specifically induced by both pharmacological and nonpharmacologicaltreatments, irrespective of the acute effects of the treatments. Subsequent studies have shown that, in addition to tricyclic antidepressants, MAOIs and ECT, “atypical”antidepressants such as iprindole and mianserin also reduce the functional activity of the P-adrenoreceptor. On the other hand, barbiturates, anticonvulsants, benzodiazepines, antihistamines, and butyrophenones have no effect. This has been reviewed by Sulser (1983). It is interesting to note that chlorpromazine has an “antidepressant”profile in this biochemical model. While this may be an example of a false-positive, it should be remembered that this drug has been shown to have antidepressant properties (Overall et al., 1966). Although the effects of antidepressant treatments on the functional activity of cortical P-adrenoreceptors has proved to be of value in studying known antidepressants, it remains to be established whether this will provide a completely reliable model for the selection of novel antidepressants. In this respect, it has been shown that the specific serotonin-uptake inhibitor, fluoxetine, which has been shown to be an antidepressant in several clinical studies, does not change the density or functional activity of cortical P-adrenoreceptors (Mishra et al., 1979). Relatively high doses of trazodone (Clements-Jewery, 1978) and buproprion (Ferris et al., 1983) have however been shown to decrease 13-adrenoreceptor density.

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Moreover, studies on the effect of the triazolobenzodiazepine alprazolam on the functional activity of cortical adrenoreceptors suggests that this atypical antidepressant may have a similar neurochemical profile to those regarded as tricyclic antidepressants. The relevance of the desensitization of cortical P-adrenoreceptors for the selection of antidepressants is supported by electrophysiologicalstudies in which the subsensitivity of postsynaptic P-adrenoreceptors has been established. Thus subsensitivity of postcerebellar Purkinje cells (Siggins and Schultz, 1979) and cortical pyramidal cells (Olpe and Schellenberg, 1980) has been reported following the microiontophoretic administration of noradrenaline to rats which had been treated with chronic, but not acute, antidepressants; MAOIs, "typical," and "atypical" antidepressants were found to be effective. Other studies in which the rate of firing of noradrenergic neurons in the rat hippocampus were studied showed that only chronic treatment with desipramine resulted in a suppression of the firing rate (Huang, 1979). Thus electrophysiologicaland biochemical studies largely support the hypothesis that chronic antidepressant treatment reduces the sensitivity of postsynaptic P-adrenoreceptors. Furthermore, the time necessary for the reduction in p-adrenoreceptor sensitivity to occur (about 14 days) approximates to that needed for the antidepressant effect to become apparent in patients. In addition to their effects on P-adrenoreceptors, most antidepressants have also been studied for their effects on other biogenic amine receptor densities. Consistent changes in the receptor densities only become apparent following the chronic ( 14-21 days) administration of antidepressant drugs. The results of studies on a selected group of typical and atypical antidepressants on serotonergic, adrenergic, muscarinic, and dopaminergic receptor sites are summarized in Table I. The effects of the antidepressants are compared with those of methysergide and chlorpromazine, psychotropic drugs with well-established actions on central neurotransmission but which do not exhibit antidepressant properties. In addition to their ability to reduce the density of postsynaptic padrenoreceptors, the typical and atypical antidepressants listed in Table I have been shown to cause a subsensitivity in the responsiveness of the Padrenoreceptors to noradrenaline; mianserin and zimelidine, which do not affect the P-adrenoreceptor density, reduce the functional sensitivity of these receptors, whereas fluoxetine is without effect (Sulser and Mobley, 1981). This lack of effect of the therapeutically active antidepressants on P-adrenoreceptor density suggest that a change in the P-adrenoreceptor density is not a prerequisite for the functional desensitization of these receptors. It may be speculated that these antidepressants act on the coupling mechanism between the receptor site and the adenylate cyclase subunit. Because of the well-established finding that all clinically effective anti-

TABLE I EFFECTS OF CHRONIC ADMINISTRATION OF ANTIDEPRESSANTS ON THE DENSITY OF BIOCENIC AMINERECEPTORS IN RAT BRAIN"^^ Receptor type ~

Tricyclic antidepressants Amitriptyline Imipramine Desipramine Atypical antidepressants Iprindole Fluoxetine' MA01 Pargyline Serotonin antagonist Methysergide Neuroleptic Chlorpromazine

~~

~

Serotonin 1 5-[$H]HT

Serotonin 2 [ 'HISpiperone

a-Adrenergic ['H]WB4 101

P-Adrenergic ['HIDHA

Muscarinic [$H]QNB

Dopamine ['HISpiperone

NC'

p < O.Old

NC

p < 0.01

NC NC NC

p < 0.85d p < 0.05 p < 0.05

NC NC NC

NC NC NC

NC

p < 0.01 NC

NC NC

p < 0.05

p < 0.01

NC

NC NC

NC NC

p < 0.05

p < 0.01

NC

p < 0.05

NC

NC

NC

NC

NC

NC

NC

NC

NC

NC

NC

NC

NC

p < 0.01f

"From Green and Nutt (1983). [3H]WB4101, a,-adrenoreceptor antagonist, dimethoxyphenoxyethylamino methyl benzodioxan; ['HIDHA, the p-adrenoreceptor antagonist, dihydroalprenolol; ['HIQNB, the muscarinic receptor antagonist, quinuclidinyl benzilate. "C, No change relative to controls. dDecrease in receptor density relative to controls. 'After Wong and Bymaster (1981). /Relative to controls.

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depressants reduce the functional responsivenessof the P-adrenoreceptors in rat brain, it has been suggested that changes in the postsynaptic receptors must reflect an increase in neurotransmitter release, presumably as a consequence of a decrease in the sensitivity of the noradrenergic autoreceptors. This hypothesis is supported by the finding that the atypical antidepressant mianserin (Fludder and Leonard, 1979) and tricyclic antidepressants (Crews and Smith, 1978b) decrease the sensitivity of the noradrenergic autoreceptors following chronic treatment. Since the autoreceptors inhibit the release of noradrenaline, the blockade or desensitization of these receptors by chronic drug treatment would facilitate transmission and so decrease the sensitivity of the postsynaptic receptor sites. This simple hypothesis has been challenged by the observation that not all antidepressants which decrease the functional activity of postsynaptic P-adrenoreceptors decrease the density of the presynaptic autoreceptors (Sugrue, 1981). Furthermore the results of experiments in which the density of a*-adrenoreceptors, presumed to be similar to the noradrenergic autoreceptors, were determined using ['Hlclonidine are conflicting with both a decrease (Smith et al., 1981) and an increase (Tsukamoto et al., 1982) in a2-adrenoreceptor numbers being reported following the chronic administration of antidepressants. With the plethora of changes in the densities of various neurotransmitter receptors following antidepressant administration, involving histamine 1- and 2-type receptors (Green and Maayani, 1977; Kanof and Greengard, 1978) and dopamine autoreceptors (Chiodo and Antelman, 1980) in addition to serotonergic and noradrenergic receptors (Wang and Aghajanian, 1980; Garcia-SeviIIa et al., 1981) it is difficult to draw any conclusions regarding the primary site of action of antidepressants on neurotransmission in the rat brain. In addition to the uncertainty regarding the existence of autoreceptors, few investigators appear to distinguish between ligand-binding sites and receptor sites, which has undoubtedly added to the confusion when attempting to interpret the mechanism of action of antidepressants on rat brain. As Sulser (1982) has emphasized in his review, to qualify as a receptor, the dual function of "recognition and response" must be demonstrated. Thus irrespective of its affinity and number of specific sites, a binding site for a ligand cannot be designated a receptor unless a close relationship between occupancy and biological response can be demonstrated; this has been considered in detail by Hollenburg and Cuatrecasas (1978). Furthermore, it must be emphasized that changes in sensitivity reflect the functional responsiveness of the cell to a neurotransmitter, and the number of receptors as determined by ligand-binding studies need not nec-

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essarily parallel the changes in cellular responsiveness. This is not surprising since not all receptors need be occupied for an optimal response to be elicited. Presumably all available receptors would be occupied by a ligand and, therefore, incorrect conclusions could be drawn regarding the relationship between receptor number and the subsequent physiological response of the cell. So far only the studies in which the changes in receptor-linked adenylate cyclase activity have been monitored, following chronic antidepressant treatment, appear to have addressed this question rigorously. Whether all antidepressant treatments produce their effect through a final common pathway which involves the functional desensitization of postsynaptic P-adrenoreceptors is still a matter of conjecture. Several studies have shown that chronic antidepressant treatment also results in the increased responsiveness of serotonin receptors in rat brain; such an effect occurs following the chronic administration of tricyclic antidepressants, mianserin, adinazoiam or ECT, MAOIs (particularly the MAO-A inhibitor, clorgyline, but not the MAO-B inhibitor, deprenyl), and of such specific serotonin-uptake inhibitors as zimelidine and indalpine (De Montigny et al., 1984). These changes in rat brain appear to simulate the functional change in serotonin receptor responsiveness in platelets from depressed patients in which the aggregation response to serotonin is reduced to approximately 50% of the control value before the start of treatment, but returned to control values following effective drug treatment (Healy et al., 1983, 1985). How the changes in noradrenergic and serotonergic receptor function, following chronic antidepressant treatment, can be linked is a matter of conjecture but studies by Brunello et al. (1982) have shown that desipramine cannot reduce the density of f3adrenoreceptors in the rat cortex and hippocampus following specific lesions of the serotonergic system with 5,7-dihydroxytryptamine.This was confirmed by Sulser ( 1983), which suggests that both the serotonergic and noradrenergic systems are involved in the regulation of the functional activity of noradrenaline-coupled adenylate cyclase in the cortex of the rat brain. Presumably a serotonin heteroreceptor located on the noradrenergic terminals could play a key role in linking these systems. Whether the other classical neurotransmitters (such as acetylcholine, dopamine, and GABA), the putative transmitters (phenylethylamine,histamine, and adrenaline), and the peptide cotransmitters (as exemplified by the enkephalins, endorphins, and vasopressin) play any major role in modifying the responsiveness of the noradrenergic pathway to the chronic effects of antidepressant treatments is unknown.

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IV. Clinical Assessment of New Antidepressants

There is increasing need for more well-designed and carefully controlled clinical studies to assess the ever-growing list of putative antidepressants (Ankier, 1986). Each time a new clinical trial is being planned, it is crucial to consider the specific aim of the study, the study design, and also to anticipate and resolve logistic problems. These considerations lead to the preparation of a protocol which should provide a complete specification for the study management and be accompanied by examples of record forms, rating scales, grading cards, and any other relevant information or documentation. As such, the protocol is a formal document agreed between the study sponsor, the clinical investigator, the patient, and the scientific community and assists in accurate communication between all those involved. Protocols are constructed under specific headings, and those items to be considered for antidepressant studies are basically the same as those used for studies on any other therapeutic class of drug. The preparation of a protocol is helped by considering headings in published checklists (Lionel and Herxheimer, 1970; Chaput de Saintonge, 1977;Warren, 1978; Peterson and Fisher, 1980; Friedman et al., 1981). Reference to the guidelines for the adequate reporting of some methodological and patient variables (Kupfer and Rush, 1983a,b) will further help to ensure that essential considerations for the conduct of antidepressant trials are not overlooked. It is also useful to develop a standard “in-house”version as a “master” reference which should be updated in the light of one’s and others’ experiences. Having drafted an early outline of the protocol, discussions with all members of the clinical trial “team,” to include a medical biostatistician, are necessary with the trial coordinator being responsible for distilling advice and information given both on theoretical and practical aspects. The protocol will require frequent redrafting until agreement is achieved. individual and collective commitment to the success of the study must also be established at this stage. This important process is normally very demanding, and it is essential to allocate sufficient time to this activity when scheduling the timing of a trial. Important factors requiring careful consideration when preparing a protocol for an antidepressant study, as well as some more general points of particular importance, are now discussed.

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A. THEINVESTIGATOR The design and performance of any scientifically valid clinical trial is the joint responsibility of the sponsor and the clinical investigator. The sponsor must choose the best available study coordinator (also known as the monitor) as their representative. The coordinator should be suitably qualified, have administrative ability, and possesses personal qualities of tact, enthusiasm, and leadership. The study coordinator’s choice of an appropriate investigator is most important, and this may be approached in different ways. For example, the investigator may already be known through publications, a previously successful cooperation, or by a good reputation. The trial coordinator should make every reasonable effort to ensure that the investigator chosen has appropriate facilities, is both interested in and motivated by the project, has the time to be involved personally, will cooperate, keep information confidential and that the investigator has access to an adequate number of suitable patients to meet the study objective. The investigator should be trained adequately, experienced, competent, and reliable. The “professional investigator,”who is observed to be overcommitted to several concurrent trials and who, by his/ her attitude, reveals commercial rather than scientific motivation, should normally be avoided. Such judgments are very difficult to make but become easier with experience. The study coordinator should remember that their own good reputation also depends on the successful conclusion of valid clinical trials. Lastly, the personal relationship between the investigator and the study coordinator cannot be overestimated. The successful completion of a clinical trial is not simply a mechanical process, but depends on the harmonious interaction of those people involved.

B. PATIENT POPULATION TO BE STUDIED Ordinary sadness is a normal disturbance of mood caused by a stress or loss. Sometimes this mood disturbance is outside the limits accepted as “normal” and leads to a syndrome involving depressive symptomatology, which for research purposes may be classified as “reactive”or “endogenous’’ depression using the Newcastle rating scale (Kiloh et al., 1972). Reactive (anxious, neurotic, or situational) depression appears suddenly and is precipitated by acute and stressful events, for example, a bereavement. Endogenous depression (sometimestermed “psychotic”depression

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when more severe symptoms are present) is thought to be associated with biochemical abnormalities which may be genetically determined. Most endogenously depressed patients suffer from depression alone (unipolar), but some may experience depression with intermittent episodes of mania (bipolar manic-depression). Moreover, patients with endogenous depression may experience a life-disturbing event compficating their illness with neurotic symptoms (called “mixed” depression) (Klerman, 1975; Hamilton, 1979; Kiloh, 1980; Klein et al., 1981). These features should be recorded at trial entry.

Diagnosis of Depression No one classification of depressive illness is ideal, although several useful sets of criteria have been evolved. A study organized by the Medical Research Council (1965) used a carefully defined set of criteria which has subsequently proved a useful research tool. The primary manifestation and major symptom of the depressive illness was specified as being a persistent alteration of mood (with or without diurnal variation)exceeding customary sadness, being evident to the examiner, and which is accompanied by one or more of the following symptoms: self-depreciation and a morbid sense (or delusional ideas) of guilt, sleep disturbance, hypochondriasis, retardation of thought and action, and agitated behavior. Other classifications of primary depression based on the collective opinions of leading researchers have been developed, for example, the St. Louis or the Feighner criteria (Feighner et al., 1972). Subsequently, the Research Diagnostic Criteria (RDC) were developed (Spitzer et al., 1978). It is derived from the St. Louis criteria, but considers both inclusion and exclusion criteria as well as the symptoms, signs, duration or course of illness, and the severity of impairment. In some cases, certain symptoms or symptom clusters are of diagnostic significance only if they persist beyond a certain stated duration. Diagnostic terms are frequently defined in the criteria themselves to avoid possible ambiguity. The schedule for Affective Disorders and Schizophrenia (sADS) is a structured interviewing procedure with rating scales designed to elicit information so an RDC diagnosis may be made (Endicott and Spitzer, 1978). Thus a probable case of “major depressive disorder” is defined when a patient has been persistently depressed or sad for at least a week and also has four of the following symptoms: (1) loss of appetite or body weight; (2) difficulties in sleeping; (3) complaints of tiredness or lack of energy; (4) agitation or retardation; ( 5 ) loss of interest or pleasure in activities that had been pleasurable; (6) feelings and thoughts of self-reproach or guilt; (7) complaints of indecision, slowed thinking, or difficulty with concentration; or

BIOLOGICAL ASPECTS OF DEPRESSION

2 15

(8) suicide preparations or attempts or recurrent thoughts of death or suicide. For a “definite”case of depression the depressed mood must be associated with five of the symptoms lasting at least 2 weeks. Recently the Diagnostic and Statistical Manual (DSM 111)of the American Psychiatric Association has become established as an FDA requirement. Affective Disorders are divided into “Major Affective Disorders” (bipolar and major depression) to include a full affective syndrome and “Other Specific Affective Disorders,”which include a partial affective syndrome of at least 2 years duration. This latter class includes cyclothymic and dysthymic disorders. A third class, “AtypicalDisorder,”has been added to define depressions that do not fulfill the criteria for the duration or severity of the two specific subclasses.The DSM 111 has the merit of taking into account the personality,precipitatingstress,and the physical condition of the patient. It provides clear-cut categories defining all types of affective disorders without splitting them into psychoses, neuroses, and personality disorders. There are other diagnostic systems currently accepted as useful for defining depression. For example, the Depression Component of the Minnesota Multiphasic Personality Inventory (Graham, 1977), the Present State Examination (Wing et al., 1974)or the W.H.O. International Classification of Diseases, 9th edition, or the so-called ICD-9 (197’7).It is suggested that the DSM 111 and one other currently recognized diagnostic criterion be used in an antidepressant study. Since anxiety states and depressive syndromes are represented by overlapping clusters of symptoms (Foa and Foa, 1982), the degree of anxiety should be assessed. Meanwhile, it is hoped that the search to identify specific physiological, biochemical, or pharmacological markers will succeed in providing an objective screening procedure for the diagnosis of depressive subtypes.

C. INITIAL SEVERITY The minimum acceptable severity of the patients’ depression should be assessed at trial entry using at least one clinician-ratedmethod. Criteria chosen tend to be pragmatic, which ensures an adequate number of patients are entered into the study, but they should remain relevant. Kupfer and Rush (1983a,b) recently reported on the views of a group of clinical investigators attending an international conference on the origins of depression. They could not arrive at a consensus about the use of any particular rating scale to assess the severity of depression. It was noted that many available scales are alleged to measure the severity of depressive

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symptomatology but that a cross-scale comparison study was needed to confirm if each was measuring the same phenomena. Although relatively time-consuming to administer correctly and consistently, the (HAM-D) (Hamilton, 1967) is widely used for this particular purpose with a total score of 17 often being used as a suitable but arbitrary minimum measure of severity at entry. The severity of the patients’ illness may also be described using a single global scale with say a three, five, or even a seven point scale and/or by the use of a visual analog scale either by the investigator or completed by the patient. Whatever techniques are used to assess the initial severity of illness, they should also be used to measure drug response during the study for comparative purposes. While the debate about the relative merits of various scales continues, the use of several approaches is advocated.

D. WHERETO TEST AN ANTIDEPRESSANT Antidepressants may be prescribed for hospital in- or outpatients or for general practice patients, hence the need to conduct valid clinical studies in both settings. However, most patients suffering from depression are managed in general practice (Wheatley, 1973) with only 2% being referred to a psychiatrist (Watson and Barber, 1981).Recruitment in general practice studies, therefore, has the possibility of achieving large numbers of patients, and since they are unlikely to be receiving concomitant medication, the possibility of complications arising from drug-drug interactions is minimized. Unlike the hospital psychiatrist, the type of depression most often seen by the general practitioner tends to be reactive. Although the general practitioner may not normally separate depression from anxiety states perhaps due to the enormous pressure on their time, clear operational definitions must be adhered to. It is often the policy in general practice for ambulant working patients to be maintained on an antidepressant drug at a dose below those at which side effects occur; however, it is important that antidepressants are tested in this setting using adequate doses as defined in the protocol. From an ethical point of view, efficacy must be assessed properly, utilizing the smallest number of carefully selected patients (Wheatley and Little, 1982). The hospital psychiatrist is trained extensively in the accurate diagnosis and in carefully rating depression and also has more time than the general practitioner to investigate and monitor patients intensively. If side effects occur during a hospital inpatient study, compliance is less likely to be compromised since reassurance can be given by staff who are more readily accessible. There are, however, problems with recruiting suitable

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cases for antidepressant studies in the hospital setting. Thus most patients referred to hospital by the general practitioner will already be on medication (Bouras et al., 1983) and probably be drug treatment failures. They may even be suffering from social, physical, or personality problems. It may be more sensible and easier for depressed patients to be recruited for a clinical study as soon as their illness is diagnosed and hence much future psychopharmacological research might well be conducted by psychiatrists attached to general practice (Little et al., 1978). The alternative approach is for collaborative multicenter hospital studies, and this possibility is considered below. E. INFORMED CONSENT

A clinical study on an antidepressant must be a compromise between an ideal scientific experiment and an investigational procedure in which both moral and legal principles are observed. Thus although the random selection of patients is a scientific ideal, the individual should be given the right not to provide informed consent. Although to obtain “informed consent” is considered necessary by ethics committees and regulatory authorities, Hamilton (1983) has described it as a “lawyers’ myth.” To inform a patient fully about a study, there will be a considerable amount of detail, some of it of a highly technical nature which needs to be communicated. A severely depressed patient will not be able or competent to make a decision about risks versus benefits before entering a clinical trial, since their ability to comprehend information may be poor and/or their judgment impaired. If the information given to the patient is condensed so as to enhance comprehension, then paradoxically they cannot be said to be in a position to give fully informed consent. There may be doubt about the validity and also the duration of consent given by a depressed patient. Then again perhaps a patient appears to consent but, subsequently, they may develop feelings of guilt and doubts. It may be prudent, therefore, for a responsible relative or friend of the patient to be present when informed consent is being sought and to obtain their signature as a witness. Moreover, the investigator has the obligation to evaluate the original consent of the patient throughout the trial and to permit a patient to withdraw from a study, if the patient wishes to at any time. Regardless, it remains the professional responsibility of the trialist to be reasonably convinced that the possible benefits of a new treatment are likely to outweigh its disadvantages, as compared with other treatments or even none at all (Hamilton, 1983). In any event, if valid clinical data is to be obtained, the cooperation of the patient is essential. Such cooperation also enhances

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B. E. LEONARD

compliance by what has been described as an “attention-placebo”effect (Myers and Calvert, 1984).

E MULTICENTERED STUDIES The difficulty of recruiting sufficient patient numbers for antidepressant studies in a reasonable time is not new (Little et al., 1978). One unsuccessful approach was to set up a special hospital clinic to which patients were specificially referred (Bouras et al., 1983). Thus the collaboration of several centers in a national or international multicenter study, using an identical core protocol, has remained necessary for statistical purposes. Such studies have the merit that a wide cross section of depressed patients can be studied relatively quickly. To mitigate against a local bias and to ensure an homogeneous recruitment of patients, some stratification may be necessary. Perhaps when improved methods of quantifying antidepressant efficacy emerge, the number of patients required for assessment will be reduced. At the outset, decisions about timing, details of the protocol, the data analysis required, and publications must be established between study sponsor and the collaborating investigators. In the case of a long-term study, the selection, commitment, and training of suitable personnel may need to be repeated due to a “turnover” of staff. Copeland and Gourlay (1973) have reported on the differences in the use of terms and definitions occurring even within one culture. Thus the understanding by each investigator on any special procedures and in the proper use of the chosen rating scales must be checked, unified, and cross-validated before patient recruitment commences. These measures will help reduce the variability between centers which could mask subtle differences between treatments. There is a need for strong central coordination particularly to ensure adequate communication between all members of “the team.” The central coordinator must visit each center regularly to monitor performance (Ferris and Ederer, 1979; Mowery and Williams, 1979). It is also sensible to have a coordinator at each center with responsibility for monitoring adherence to the protocol, avoiding a “drift”in performance, supervising the quality of the data being collected, and maintaining and promoting continued enthusiasm.

G. COMPLIANCE It may be incorrect to assume that patients have taken their medication as planned. Moreover, patients may not attend when required, record forms may not be completed adequately, or laboratory procedures may

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not be followed correctly. The reasons for such noncompliance may be related to many factors, such as insufficient attention to details in the planning and preparation of a study, transportation difficulties making patient attendance a problem, complex dosing regimens, inadequate labeling of the medication, or by inaccurate, complex, or incomplete instructions given to the patient by the investigator. The noncompliance might also be due to a clinically significant reason such as an unpleasant or unacceptable characteristicof the medication or even to the patient feeling better and incorrectly believing that medication may be discontinued. Even though potentially useful pharmacological effects may be obscured, the data on all patients randomized to the study should normally enter the statistical analysis for efficacy. This “pragmatic approach” or “analysis by intention to treat” is preferable, since noncompliance itself may be an integral feature of the treatment being studied and this would be relevant to its clinical usage (Schwartz and Lellouch, 1967; Schwartz et al., 1980). Provision should be made to monitor and enhance compliance. It may often be helpful to involve a member of the patient’s family A patient record card could be kept or the investigator could make a “pill”count on returned and unused medication. Unfortunately, these approaches are open to criticism, since the patient could claim an accurate drug compliance while actually having discarded unused medication before visiting the investigator. Another approach is to perform a plasma, urine, or saliva assay, although detection of low concentrations of drugs may present technical problems, and venepuncture may be ethically unacceptable if performed solely to assess compliance. Moreover, assays are open to misleading conclusions, since patients might only take medication prior to an interview in the knowledge that a check on compliance was to be performed. Assay results may also lead to false conclusions about drug compliance due to large differences reported between different subjects receiving the same dose of an antidepressant (Coppen and Perris, 1976). One strategy reported to improve compliance is to provide comprehensible information to the depressed patient (Leyet d., 1976),although Myers and Calvert (1984) showed that the cognitive or educational content is less important than an attention-placebo effect. Reasonable procedures to monitor compliance should be established, and the interpretation of study results should take into account the degree to which the study was adequately performed.

H. USEOF PLACEBO Patients referred to a psychiatrist by their general practitioner frequently enter the hospital while on medication. This may mask a valid diagnosis of the patients’ condition and hence lead to inception of an

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incorrect treatment after inclusion in a clinical study. A drug-free period is thus justified during which time a placebo might be given. This out” procedure, usually for about 7 days long, allows the initial severity of the patients’ depression to be assessed and also helps avoid possible drug interactions with the study medication. It identifies “placebo-responders: that is those patients who dramatically improve on placebo, who should not enter the active medication phase of a clinical trial. The possibility exists that a depressed patients’ clinical condition could deteriorate during the placebo “screeninglwash-out”period with the risk of suicide being a major consideration. Therefore, during the placebo period the patients’ well-being must be monitored carefully, and an “escape” to active medication must be permitted in the protocol on ethical grounds. It is possible that an apparent deterioration of a patient’s symptoms during the placebo period may not be due to the depression getting worse, but rather to symptoms, such as increased anxiety and sleep disturbance, caused by an acute benzodiazepine withdrawal phenomenon (Hallstrom and Lader, 1981; Petursson and Lader, 1981). A careful note of medication taken by the patient before consideration for the trial is therefore necessary. The use of placebo (negative control) as a parallel treatment to an established medication (positivecontrol) and a test medication is a further problem (Joyce, 1982). It can be argued that it is unethical to perform an antidepressant study without a placebo control group, since such a design cannot adequately assess the pharmacological efficacy of a test medication, particularly as a placebo response has been reported in about 30% of depressed patients (Medical Research Council, 1965). However, it may be improper to deny an established medication to patients with severe depression or who are potentially suicidal. Where doubt exists about the efficacy of the established medication, then the use of placebo would appear justified, particularly if an advantage in terms of side effects were also anticipated for the test medication. One possible compromise is in the use of an “active”placebo. This approach has been used (Russell et al., 1978; Richards et al., 1982; Ather et al., 1985)on the basis that a benzodiazepine, such as diazepam, is known to be effective in reducing the symptoms of anxiety associated with depression, but with only limited activity on the core symptoms of depression. Dose-response studies (Wittenborn, 1977), which compare fixed “high” and “low” dosages of the test medication, avoid using a placebo group altogether. However, a valid inference may only be possible if the chosen high dose is significantly more effective than the chosen low dose. I. ASSESSING SEVERITY OF DEPRESSION

To compare the efficacy of two or more treatments across different patients, clinicians, studies, and settings requires reliable means to assess

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the change in severity of the patients’ depression. Since suitable objective laboratory-based data are impossible to collect in the depressed patient, rating scales have been developed in an attempt to quantify such subjective information. The main symptoms are affective (e.g., sadness and anxiety) and biological (e.g., insomnia, lack of energy, and loss of appetite), and interview techniques are highly effective when combined with self-rating assessments. Rating scales may be completed by an observer, who will make an evaluation based on clinical judgment, or can be completed by the patient. These rating scales exist in an attempt to categorize the depressive syndrome into items to which numbers may be attributed so that a total score can be derived. Reliable assessment of the behavior of patients is easiest for the observer, who will have standards against which to evaluate the intensity of a symptom, but most difficult for the patient, who may lose insight into their own condition. Thus some symptoms, such as agitation, hypochondriasis, or depersonalization, are likely to be rated inaccurately by the patient. Therefore, self-rating scales are likely to be most useful in assessing patients with mild to moderate depression, in which full insight is retained, although they make the assumption that definitions of terms, such as “pessimism,”“mood,”or “guilt,”are stable across different cultures or across patients from different socioeconomic backgrounds. The choice of a rating scale, although often done in an arbitrary and ad hoc fashion, is critically important. It should reflect the degree of severity of the depression and be reliable both between raters and between test and retest. If a rating scale is too short, it will tend to have a low reliability, while if it is too long, it may become very difficult to use. However, for a study where the number of patients available is limited, then the scale chosen should have the highest validity and reliability, regardless of the difficulties involved, although it should not be used in isolation from other means of assessing the patient’s clinical condition. The patient should be carefully supervised initially by the investigator in the use of self-rating scales,and it is important for investigators themselves to be trained in the proper use of any scaIe chosen for a clinical study. The apparent simplicity of some rating scales makes them vulnerable to error, as they appear easy to administer and usually yield quantifiable data. Visual analog scales have been developed to circumvent the limitations in the use of language for measurement of a feeling (Clarke and Spear, 1964; Aitken, 1969). They have been reported to be particularly suitable for the measurement of change (Zealley and Aitken, 1969) and have been further validated (Luria, 1975). Basically, the visual analog scale is a continuous line 100 mm long with both ends labeled on which the rater (usually the patient) makes a mark bisecting the line to define their feelings within a chosen time frame, such as ‘‘normall$ “over the last week,”or “at

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that particular moment." A score is derived from the distance of the mark (to the nearest millimeter) from one end of the line or the other. The exact wording of the questions asked and the labels used are important, since patients and psychiatrists often mean different things by the use of certain words. The patient has only their own experience by which to judge the severity of the symptom or syndrome being rated and might overreact once they appreciate what they are expected to do. Patients may not be able to project onto the scale what they are asked to rate, and for the elderly patient, conceptualization of the scale dimensions may be impossible. It is also possible that the medication under investigation could impair the patients' ability to assess an effect. The observer scales may suffer from observer bias, particularly in the general assumption that patients are more ill before they commence a clinical trial than at the end of the study (Seldrup, 1977).Other factors, such as whether the line should be horizontal or vertical or whether the subject should have knowledge of their previous recording and use that to represent the midpoint for the current assessment (Seldrup and Beaumont, 1975), may influence the results obtained, Often such scales are developed ad hoc, so proper validation is essential. Scales assessing the overall current global severity, based on discrete categories to grade continuous phenomena, are used to represent the investigator's general impression of the severity of the patient's illness. Assessment of therapeutic or global improvement compared to the start of active treatment, also using categories, is used to monitor the progress of a patient in a clinical study. Most rating scales are prepared in a written form and should be used only if they are of a length that will engage the concentration and secure the cooperation of the depressed patient being assessed. They may be inappropriate for the elderly, those of low intelligence, or the illiterate. They cannot be used in very severe degrees of depressive illness, since patients may not be accessible to questioning or may be incapable of giving meaningful answers. ECT may be most suitable for such patients who should really be excluded from a clinical study comparing the efficacyof chemical antidepressants. The HAM-D, originally described in 1960, was revised subsequently (Hamilton, 1967) and is now widely used in clinical research. There are 21 items used to assess responses either on a three-point (e.g., middle or delayed insomnia, general or gastrointestinal somatic symptoms, genital symptoms, insight, and loss of weight) or five-point (e.g., depressed, guilt, suicide, initial insomnia, work and interests, retardation, agitation, psychic or somatic anxiety, and hypochondriasis) basis, depending on the feasibility of grading the responses coarsely or finely. The depressive symptoms are presumed to be part of a depressive syndrome and not secondary to

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some other condition such as schizophrenia. A total score is calculated based on the patient’s condition, which should not be assessed more than once a week to avoid minor fluctuations; items such as loss of weight and loss of libido are unlikely to alter in shorter periods. The total score does not always reflect the quantity or even the quality of the mood disturbance. Thus, although a total score may have decreased sufficiently to indicate an apparent improvement, highly significant symptoms of depression, such as the possibility of suicide, may actually have become more of a risk. Many items are concerned with psychomotor symptoms and on the somatization of mood, making the HAM-D more appropriate for rating endogenous depression. Cognitive disturbances are represented, but less so than are affective changes. Rarer events (or diurnal mood variation, which reflects the type of depression), such as depersonalization and derealization, obsessive-compulsive symptoms, and paranoid symptoms, are excluded, although other workers have reported these items to be useful (Guy, 1976; Lader, 1981).Several other modifications of the HAMD have appeared in the literature (Hedlund and Vieweg, 1979),so that it is important to specify which version has been used in any study. Clinical improvement has been defined by some workers as a reduction of initial score by 50% or a reduction of the total score to less than 10. In some studies, individual items have been analyzed or symptoms have been grouped into rational clusters for the purposes of defining changes in the patients’ condition (Hedlund and Vieweg, 1979).The approach to be used should have some consistency with real practice and be decided upon before the study commences. The HAM-D was designed to be administered independently by two trained raters with adequate clinical experience in assessing the severity of depression in patients already having been diagnosed as suffering from depression. When used in this way, the interrater reliability of the total score has been found to be high. It is not a diagnostic tool and was never designed to measure change, and yet the latter purpose is exactly that for which it has become a standard instrument. Bech et al. (1981) examined the consistency of the HAM-D and concluded that the original 17-item scale was inadequate. They developed a melancholia subscale (itemsof depressed mood, guilt, work and interests, retardation, psychic anxiety, and general somatic symptoms) claimed to be capable of comparing patients on a one-dimensional depression continuum, which would save the time of investigators. Other scales have been used to rate the severity of depression, with the HAM-D being the principal validating instrument, and some of the more widely used ones are considered below. A subset of items from the regular sADS make up a version designed

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to measure change. It is known as the sADS-C and is used at follow-up evaluations when sADS had been used initially for diagnostic purposes (Endicott and Spitzer, 1978). The Beck Depression Inventory (Beck et aZ., 196l), which contains items referring to symptoms and attitudes, is now administered as a self-rating scale with the subject reading the questions to themselves (Bech et al., 1975). This scale places more emphasis on cognitive or psychodynamically orientated aspects than the HAM-D, which itself emphasizes physiological correlates, with less than a third of the Beck items referring to somatic or behavioral features of depression. Since cognitive disturbances are prominent in “mild”or “neurotic”depressions and psychomotor and somatic disturbances are prominent in “severe” or “psychotic” disturbances, a self-administered scale such as the Beck may be a more accurate measure in the neurotically depressed, while the HAM-D may be more appropriate in the psychotically depressed (Prusoff et al., 1972). Bech et al. (1975) found that the total scores of the HAM-D and Beck scales were comparable both for baseline ratings and for change ratings, although both scales failed to differentiate adequately between moderate and severe depression measured by a global clinical assessment. Bailey and Coppen (1976) reported that HAM-D and Beck scales compared well in about two-thirds of the depressed patients studied, but that the Beck showed less percentage change over time than did the HAM-D. The Wakefield self-assessmentdepression inventory (Snaith et al., 1971) was derived from the Zung self-rating depression scale (Zung, 1965),which lacks items on guilt, insight, and retardation, and has been reported to correlate well with the HAM-D. The Wakefield has the merit of brevity and simplicity with 12 questions each being scored 0, 1, 2, or 3, according to the response obtained. However, Kearns et al. (1982) have suggested that the Wakefield inventory should be discarded as a research instrument, since its ability to discriminate between different grades of severity was found to be low when compared with several available depression rating scales. In fact the Wakefield self-assessmentdepression inventory has been superseded. An item analysis and the addition of 10 further items has resulted in the establishment of the Leeds scales for measurement of depression and of anxiety (Snaith et al., 1976).Zigmond and Snaith (1983) recently developed a self-assessment mood scale specifically for detecting and assessing the severity of depression and of anxiety in the setting of a hospital medical outpatient clinic. The concepts in emotional and somatic illness were carefully separated, and the scale is thus unaffected by the presence of bodily illness. The modified Zung (1974)self-rating depression scale contains 20 items, has good concurrent validity with the HAM-D for mildly to moderately depressed patients, but not so for the more severely depressed patient (Carroll et al., 1973). This may be attributable to the

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lack of such items as retardation, guilt feelings, and hypochondriasis in the Zung scale, although it is doubtful whether patients with marked features of this kind are capable of rating themselves accurately (Lader, 1981). A rating instrument developed recently to assess the patients’response to treatment and concerned exclusively with the psychic symptoms of depressive illness (i.e., the patient’s report of mood) is the MontgomeryAsberg scale (Montgomery and Asberg, 1979). It is derived from the comprehensive psychopathological rating scale (Asberg et al., 1978) and comprises the 10 items found to show the largest change with treatment and the highest correlations to overall change. The items chosen for the rating scale are (1) apparent sadness, (2) reported sadness, (3) inner tension, (4)reduced sleep, (5) reduced appetite, (6) concentration difficulties, (7) lassitude, (8) inability to feel, (9) pessimistic thoughts, and (10) suicidal thoughts. The rating itself is based on a clinical interview, moving from broadly phrased questions about symptoms to more detailed ones which aIlow a recise rating of severity. For each of the 10 items on the Montgomery- sberg scale, the rater must decide whether the rating lies on the defined scale steps (0,2,4, or 6) or between them (1,3, or 5). There are clear definitionsguiding the rater as to the definition of each symptom and examples of the value to be ascribed to a particular rating. It is claimed that the Montgomery-Asberg scale is a more precise measure of change than the HAM-D so that significant differences between treatments might be revealed with smaller numbers of patients. This is an important ethical point, since it would mean that fewer patients will need to be exposed to possibly inferior treatment. In a comparative study, this rating scale performed about equally as well as the established HAM-D (Kearns et al., 1982). The two scales, however, have different applications in clinical trial work with, for example, the use of the HAMD being more appropriate when a broad assessment of the psychic, behavioral, and somatic features of depression is important. On the other hand, the Montgomery-Asberg scale, which is exclusively concerned with the psychic symptoms of depressive illness, is particularly suited for investigations on patients suffering from concurrent physical illness or who are likely to experience marked somatic side effects from the antidepressant being tested or from a concurrent medication. Unlike the HAM-D, the Montgomery-Asberg scale is suitable for rating the severity of depression at time intervals of less than 1 week. Hence it is a useful instrument for assessing the onset of action of an antidepressant in which relatively frequent measurement of the patients’ condition is indicated early on during active treatment. Computers are now in regular use for assessment of psychiatric pa-

x

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tients. An automated system modified for the self-rating of depression which utilizes a microcomputer has been developed (Carr et al., 1981). It is based on the HAM-D, since existing scales tended to concentrate on mood disturbance. The computer displays the questions asked by the investigator with three to five possible answers. Such computerized techniques enable the collection of information from patients to include “delicate” questions, such as about suicide or sex, and encourages patients to admit to symptoms and feelings that would perhaps otherwise be denied. Also it enhances the speed of data collection and processing, enables an investigator to monitor relatively large numbers of patients reliably and accurately, and establishes a standardized format between centers. The system is simple enough for relatively untrained staff to operate and supervise. Since data are stored on small disks with distant computers not being involved, the information collected remains confidential. Cost is still a limiting factor to routine use in large multicenter studies, and the production of “numbersby a computer” may appear to be more convincing than a simple rating scale. The value of an experienced clinicaljudgment is still a powerful instrument for measuring changes in the severity of depression. Although there may be professional antipathy from experiences with mainframe computers (Carr and Ancill, 1983), this new technical development is likely to be useful for taking the patients’ history, assessing the severity of depression, and evaluating new rating scales. Work on psychological, physiological, biochemical, and/or pharmacological changes during depression may help to identify a test, or group of tests, which will provide specific and objective measurements of the patients’ condition. Meanwhile, there being no ideal scale for antidepressive research which would be suitable for all kinds of patients and situations, well-validated and familiar scales known to be reliable should be used with new ones (for example the Hospital Anxiety and Depression Scale; Zigmond and Snaith, 1983, for patients being assessed in a general medical clinic) also being tried out. There is a real problem in choosing any particular rating instrument for a trial to quantify depression in view of the large number available with each having their own individual sources of error. In general, it is sensible for both investigator and patient rated scales to be used for the assessment of the severity of depression in a clinical trial. At least two appropriate investigator rated scales should be used and complemented by a patient rated scale, a global scale, andlor a visual analog scale. The choice of investigator scales depends on factors such as the severity of the depression as well as whether assessment of psychomotor symptoms and somatic aspects of mood and/or cognitive aspects is important to achieve the study aim. Thus, whereas the HAM-D is particularly useful for the former, other scales such as the Beck may be

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more useful for the latter purpose. Finally, since the accurate completion of rating scales is essential for the success of antidepressant studies, particular attention should be paid to the careful design of clear and simple record forms and with the provision of grading cards to assist in their correct completion. Factors, such as the print chosen, the layout adopted, and the phrasing of questions, particularly for visual analog scales designed on an ad hoc basis, are all relevant factors (Wright and Haybittle, 1979).

J. ASSESSING ADVERSEEVENTS Although antidepressants are well tolerated by most patients, they can produce undesirable effects. This may lead to poor patient compliance resulting in an apparent treatment failure. The severity of side effects usually diminish after the first few days of treatment, and the outcome of a study could be affected if this possibility is explained to the patient. Assessing side effects is notoriously difficult, since many preexist a study as symptoms of the depression itself (e.g., fatigue, dry mouth, dizziness, nausea, and drowsiness)or emerge during treatment without being drug related (Klein at al., 1981). The use of a placebo-treated control group is, therefore, important, although symptomatic complaints may be reported even in healthy volunteers taking placebo (Green, 1964; Reidenberg and Lowenthal, 1968). Nevertheless, the use of a placebo allows assessment of the extent to which possible side effects are drug induced. However, although comparisons with a placebo remain the most useful approach to assess the incidence, severity, and frequency of side effects, this technique provokes both ethical and legal problems (Lasagna, 1979). It should be remembered that, although drug-drug interactions may be minimized by the use of exclusion criteria, when a patient enters a clinical study, the recent (prestudy) withdrawal of a medication might itself precipitate an adverse reaction. Moreover, for hospital inpatients, there is always the possibility that communication between patients may produce a “halo” effect with an “epidemic-like spread of reports of an adverse effect (Lasagna, 1981). These considerations emphasize the importance of carefully noting recent medication and preexisting complaints and of using controls to evaluate adverse reactions which occur during a clinical study. There are two fundamental approaches to eliciting adverse subjective feelings in a clinical study (Lasagna, 1981).First, an open-ended question such as, “Did you experience any problem with the medication you took?”, which will evoke a volunteered response. This approach tends to elicit the

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more important side effects. Second, a checklist with specifically anticipated events (and also a few not to be anticipated, as a control), plus an open-ended unspecified category, may be administered. This latter approach has the disadvantage of collecting some relatively trivial data, probably of no clinical significance, although one cannot be certain about this in the absence of a placebo control. It has been suggested that both types of approaches should be used with side effects being rated on severity (1, mild; 2, moderate; 3, severe; and 4,very severe), frequency (1, once; 2, infrequently; 3, often; and 4, continually), and also on the investigator’s opinion regarding the relationship of the event to medication (0, not related; 1, possibly related; and 2, definitely related). Since all these factors have mutual effects, a total side-effects score may be derived by multiplying together the individual scores obtained (Priest, 1976; Pugh et al., 1982) to represent the overall intensity, persistence, and relevance of any given side effect.

K. STATISTICAL CONSIDERATIONS There are two statistically valid options in designing a clinical study, namely, to perform a sequential analysis or to use a fixed sample size determined in advance. A sequential analysis is not generally suitable for antidepressant studies, since there is not usually one major criterion on which to choose a treatment success or a failure. Consequently, a fixed sample size is used which, for ethical and economic reasons, should be the minimum number of cases required to achieve a valid conclusion. Determination of sample size involves the statistical concepts of Type I and Type I1 errors, the probability of the statistical errors (aand f3, respectively), and the size of the therapeutic effect regarded as clinically important. The Type I error arises when it is incorrectly claimed that the treatments differ (i.e., the null hypothesis is true and the investigator rejects it incorrectly). The Type I1 error occurs when the treatments actually do differ, but the clinical study failed to demonstrate this difference, (i.e., the null hypothesis is false, and the investigator fails to reject it). Unlike the a-probability, which defines the probability of incorrectly rejecting the null hypothesis and which can be specified exactly for each experiment, the @-probabilitycan only be specified by making assumptions about the size of the experimental effect. It is the “power” (defined as 1 - p) or sensitivity of an experiment which expresses the probability that a study will find a treatment difference, if one exists. The precision of a clinical trial can be enhanced by increasing the aprobability level, thus changing the magnitude of the effect under study,

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or by increasing the group size. The investigator is normally unwilling to increase the a-probability level beyond the conventional maximum and accepted value of 5% although it may sometimes be possible to enhance the apparent magnitude of an effect under study by reducing uncontrolled sources of variability, such as the biological heterogenicity in diagnostic groups (Buchsbaum and Rieder, 1979). It is the manipulation of sample size which is the usual way of increasing the power of a clinical study, having first made a value judgment on the required difference to be regarded as clinically relevant. In general, the smaller the difference judged to be clinically significant, the greater the number of patients required. However, in practice, the size of a clinical trial is a compromise between statisticalconsiderations, particularly the required power (usually 80% is suitable) to find the difference decided upon as being clinically significant, and practical considerations, such as the budget, manpower, time constraints, and the anticipated recruitment rate (Altman, 1980, Gore, 1981). It seems to be common experience that the latter is likely to be an overestimation of the truth, and yet, inappropriate enthusiasm is not unknown to result in a study being initiated at a center where the required number of patients cannot actually be recruited in a reasonable time period. Hence a more realistic recruitment rate should be established by monitoring the situation during the period that the study is being setup. If it is discovered that sufficient patient numbers are not available, then clearly the study should not be performed or a multicenter approach be employed. Although the actual power chosen for a particular study is arbitrary rather than optimal (Rothpearl et al., 1981),the various considerations involved in deciding its value help avoid serious problems of interpretation between statistical significance and clinical importance when the study results become available. Since antidepressant studies involve evaluations at regular intervals over several weeks, much data are generated. Separate significance tests should not be performed at each time point, since such multiple testing will lead to confusion and probably spurious conclusions. Pocock (1983) illustrated this point by citing a published example involving 45 hospitalized patients with primary depression in which over 100 significance tests had been used to describe the results. It is more appropriate to use an analysis of variance technique with appropriate significance tests being applied only to identify sources of statistical difference if an overall difference is found. Alternatively,a two sample t-test for treatment difference between scores at baseline value and the final assessment or baselins value and the last two assessments divided by two could be used. The latter option avoids placing too much emphasis on the score obtained on the final day. A further possibility, which gives equal "weight" to each time

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point, is to use the difference between baseline score and the mean of all scores on treatment. Whichever approach is to be used, the decision should be taken before the study commences, and this should be specified clearly in the protocol.

V.

Conclusions

Ten years ago, Leonard (1975) reviewed the possible neurochemical basis of depression and the neuropharmacologicalaspects of its treatment. In the past decade, several conceptual leaps have been made with respect to our understanding the possible etiology of the disease. In experimental studies, this is reflected in the change of emphasis from behavioral and neurochemical studies following the acute administration of antidepressant drugs to chronic studies in which subtle changes in neurotransmitter function have been assessed in discrete brain regions. The major advantage of this approach lies in the correlation between the changes in neurotransmitter receptor function and the onset of the response to treatment. Whether these associations are causally or coincidentally related still remains to be proved. The second major conceptual advance has involved studies of receptor function in depressed patients both preceding and following antidepressant treatment. Despite the previous enthusiasm for studies in which amine metabolites were determined in the cerebrospinal fluid, details of which were reviewed by Leonard (1975), the conclusions based on such studies have been at best equivocal. Recent studies on postmortem brains from suicide victims have also failed to provide convincing evidence in favor of specific and discrete changes in neurotransmitter functions. Such studies are likely to be of only limited value until a mechanism is developed for the rapid analysis of receptor activity and neurotransmitter concentrations shortly after the death of adequately diagnosed patients. The neuropharmacologist is, therefore, left with studying peripheral markers of neurotransmitter function (as exemplified by changes in the endocrine responsiveness,pharmacological responses to the administration of indirectly acting sympathomimetic arnines, changes in receptor activity on platelets and lymphocytes, and in neurotransmitter uptake into platelets) in the hope that the results reflect neurotransmitter function in the brain of the patient. The results from the changes in ['H]serotonin uptake into platelets and in the amine receptor responsiveness on the platelet membrane (as assessed by determining the aggregatory rate to serotonin and noradrenaline) appear to provide the experimenter with useful state-dependent markers of depression.

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The fact that the reduction in the serotonin-uptake rate into platelets of depressed patients has been reported by investigators in different countries may lead to a useful biochemical test for depression. While more sophisticated experimental and clinical studies have been undertaken in the last decade, uncertainty surrounding the precise mode of action of antidepressant drugs has increased largely due to the realization that any change in one pathway will initiate a change in all other pathways with which it is in contact. These interrelationships in the neurotransmitter systems subserving the limbic regions, cortex, nucleus accumbens, and the basal ganglia of the rat brain have been reviewed by Leonard (1984). The recent establishment of internationally approved criteria for the diagnosis of depression has undoubtedly been of major technical importance in a field in which objective data are virtually impossible to collect. The widespread use of effective antidepressant drugs has helped to restrict the number of patients being hospitalized to those who are more severely ill and/or who are nonresponsive to usual drug treatment. Therefore, most clinical trials of antidepressants are, of necessity conducted initially on hospitalized depressed patients. This trend could lead to an atypical subpopulation of depressed patients being selected for study. Perhaps it is more relevant to study depressed nonhospitalized patients, since this is where most patients with depression are found initially. Since there is no ideal rating scale suitable to quantify depression in all types of patient and different situations, it is important to include well-validated and familiar scales. In the future, the use of computers to assess depression is likely to become more commonplace. Despite all the difficulties that have arisen in proposing a comprehensive hypothesis of depression, a disorder in neurotransmitter function in which the biogenic amines are the prime candidates would still seem to be the most reasonable hypothesis available. Perhaps the next decade will provide new insights into the neurobiology of depression that will enable a more valid conclusion to be reached. References

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Sethy, V. H., and Hodges, D. H. (1982). Res. Commun. Chem. Pathol. Pharmacol. 36, 329332. Shaw, D. M., Camps, F. E., and Eccleston, E. G. (1967).Be J. Psychiatry 113, 1407-1411. Shimizu, J.. and Fu.jita, M. (1981). In “New Vistas in Depression” (S. Z. Lange, ed.). Pergamon, Oxford. Sever, L. T., Kafka, M. S., Targurn, S., and Kake, C. R. (1984). Psychzatc Res. 11,287-302. Siggins, G . R., and Schultz, J. E. (1979). Proc. N.Y. Acad. Sci. 76,5987-5991. Sjostrorn, R.,and Roos, B. E. (1972). Eu,: J. Clin.Pharmacol. 4, 170-176. Smith, C. B., Garcia-Sevilla, J. A., and Hollingsworth, R. J. (1981). Brain Res. 210,413-418. Snaith, R. P., Ahmed, S. N., Mehta, S., and Hamilton, M. (1971).Psychol. Med. I, 143-149. Snaith, R. P., Bridge, G. W. K., and Hamilton, M. (1976). B c J . Psychiatry 128, 156-165. Spitzer, R. L., Endicott, J.. and Robins, E. (1978). Arch. Gen. Psychiatry 35, 773-782. Starke, K. (1977). Rev. Physiol. Biochem. 77, 1- 124. Steer, M. L., and Atlas, D. (1982). Biochim. Biophys. Acta 686, 240-244. Sugrue, M. F. (1981). Lije Sci. 28,377-384. Sulser, F. (1978). Pharnacopsychiatry lI,43-52. Sulser, E (1982). In “Typical and Atypical Antidepressants” (E. Costa and G. Racagni, eds.), Vol. 1. Raven, New York. Sulser. F. (1983).J. Clin. Psychiatry 44, 14-20. Sulser, F., and Mobley P. L. (1981). In “Neuroregulators: Bare and Clinical Aspects” (E. Usdin, J. M. Davis, and W. E. Bunnep eds.). Wiley, New York. Sulser, F., Janowsky, A. K., Okada, E, Manier, D. H., and Mobley, P. L. (1983). Neurophurmacology 22, 425-431. Sweeney, D. R., Maas, J. W., and Henniger, G. R. (1978). Arch. Gen. Psychiatry 35, 14181423. Takahashi, S.. Kondo, H., Yoshirnura, M., and Ochi, Y. (1974). In “Psychoneuroendocrinology Proc.” (N. Hatotani, ed.), p. 3238. Karger, Basel. Tanaka, T., and Starke, K. (1979). I% S. Arch: Pharmacol. 309,207-215. Titinchi, Al Sharora, M., Patel, K., Kerr, S., and Clark, B. (1984). Clin. Sci. 66,323-328. Tsukamoto, T., Asakura, M., and Hasegawa, K. (1982). In “New Vistas in Depression” (S. Z. Langer and M.Briley, eds.). Pergamon, New York. Tuomisto, J., Tukiainen, E., and Ahlfors, U. G. (1979). Psychopharmacology 65, 141-147. Van Praag, H. M., Korf, J., and Schut, J. (1973). Arch. Gen. Psychiatry 28,827-832. Veith, R. C., Bielski, R. J., Bloom, V., Fawcett, J. A., Narasi Mhachari, N., and Friedel, R. 0. (1983).J. Clin. Psychopharmacol. 3, 18-27. Vergnes, M., and Karli, P. (1963). C.R.S. Soc. Biol. 157, 1061-1063. Vetulani, J., Stawarz, R. J.. Digell, J. V., and Sulser, E (1976). Arch. Pharmacol. 293, 109115. Wagner, H. R., Crutcher, K. A., and Davis,J. N. (1979). Brain Res. 171, 147-151. Waldrneier, €? C., Baurnann, P. A., Wilhelim, M.,Bernasconi, R., and Maitre, L. (1977). Euc J. Phurmacol. 46,387-392. Waldmeier, P. C., Maitre, L., Storm, A., and Baumann, P. A. (1982). Biochem. Pharmacol. 31,2169-2176. Wang, R.Y., and Aghajanian, G. K. (1980). Commun. Psychophurmacol. 4, 83-90. Wang, Y. C., Pandey, G. N., Mendels, J., and Frazer, A. (1974).Psychopharmacology 36,291300. Warren, M. D. (1978). Be Med. J. 1, 1195-1196. Watson, J. M., and Barber, J. H. (1981). Health Bull. Soc. Home Health Dept. 39, 112-116. Whalley, L. J., Christie,J. E., Bennie, J., Dick, H., Blackburn, I. M., Blackwood, D., Sanchez Watts, G., and Finch, G. (1985). BI:J. Med. 290,99-102. Wheatley, D. (1973). “Psychopharmacology in Family Practice:’ Appleton, New York.

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DOES RECEPTOR-LINKED PHOSPHOINOSITIDE METABOLISM PROVIDE MESSENGERS MOBILIZING CALCIUM IN NERVOUS TISSUE? By John N. Hawthorne Departmentof Biochemistry University Hospital & Medical School

The University of Nottingham Medical School Nottingham NG7 2UH, England

I. Introduction

Three inositol lipids are found in the membranes of nerve tissue, phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), and phosphatidylinositol4,5-bisphosphate (PIPz).The structure of PIPz,which may be the most important of the three, is shown in Fig. 1. As a group, the lipids are known as phosphoinositides,while PIP and PIP:! are referred to as the polyphosphoinositides.Myelin is the richest source of these, which is a pointer to their significance as plasma membrane constituents. They were first studied by Folch (1949)and are dealt with in a number of reviews (Downes and Michell, 1982;Hawthorne, 1983;Hokin, 1985). The most exciting recent development in the phosphoinositide field comes from Berridge (1983)who, with a number of collaborators, particularly Irvine (see review by Berridge and Irvine, 1984),has provided evidence that inositol1,4,5-trisphosphate(IPS),released on receptor-linked hydrolysis of PIPZ, is a second messenger mobilizing calcium from intracellular stores. The other hydrolysis product, diacylglycerol, is also considered to be involved in signal transduction by the activation of protein kinase C (Nishizuka, 1984).This dual messenger theory has been hailed with some enthusiasm as the long-awaited explanation of the receptorlinked phosphoinositide changes first shown by Hokin and Hokin (1953). Paradoxically, though the poly phosphoinositidesare found in higher concentrations in nervous tissue, most of the evidence for the dual messenger theory comes from other cell types. In this review, I shall attempt a critical assessment of the theory with particular emphasis on brain and nerve, though it will be necessary to discuss some work with adrenal medulla and retina. Research in this field is moving so quickly that I cannot hope to be comprehensive. Some reviews have already been mentianed and for INTERNATIONAL REVIEW OF NEUROBIOLOGY, VOL 28

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I

CH20COR’ FIG. 1. PhosphatidylinositoI4,5-bisphosphate.

suitably dedicated readers there are several more (Michell, 1975; Hawthorne and Pickard, 1979; Fain, 1982, 1984; Farese, 1983; Abdel-Latif, 1983; Berridge, 1984; Hirasawa and Nishizuka, 1985; Majerus et al., 1985; Downes and Michell, 1985), as well as a whole book devoted to inositol and phosphoinositides (Bleasdale et al., 1985). The Michell (1975) review has been particularly influential in directing research on phosphoinositides toward the control of intracellular calcium concentration.

11. Phosphatidylinositol or the Polyphosphoinositides?

When the link between receptors and the inositol lipids was first demonstrated by Hokin and Hokin (1953), their chemical structures were still unknown. Most workers then considered that mammalian tissues contained two such lipids, phosphatidylinositol,probably analagous in structure to the long-known phosphatidylcholine, and the brain diphosphoinositide of Folch (1949), now known to be a mixture of PIP and PIP:! along with some PI and phosphatidylserine. An early review (Hawthorne, 1960) gives some idea of the relatively uncultivated field of those days and Lowell Hokin’s review (Hokin, 1985) gives a first-hand account of the pioneer work on muscarinic receptors and the phosphoinositides. A major difficulty, and one still with us, is that the phosphoinositide response cannot easily be demonstrated in cell-free systems. Fain (1984) reviews progress from his and other laboratories with plasma membrane fractions, but this is very recent. With regard to nervous tissue, the best that the early workers could do was to demonstrate phosphoinositide effects in synaptosomes, methods for the preparation of which became available in 1960. Durell et al. (1968) showed that inositol monophosphate and inositol bisphosphate were released when [SH]inositol-labeledsynaptosomes were incubated with acetylcholine.They suggested that PI and

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PIP were hydrolyzed by the phospholipase C route to diacylglycerol and the inositol phosphates. The synaptosomes had been labeled in viuo, a method more suitable for phosphoinositide studies than labeling in vitro with ['H]inositol or [32P]orthophosphate,since phospholipid synthesis requires enzymes within the nerve ending. Only the phosphoinositides of presynaptic and intraterminal membranes will be labeled in vitro, leaving those of the attached postsynaptic membranes unlabeled. In preparations which consist entirely of nerve-ending particles then, labeling in vitro will only detect phosphoinositide responses to presynaptic receptors. It may not be possible to prepare such synaptosomes however. Fisher et al. (1980) have provided evidence that synaptosomal fractions of guinea pig hippocampus contain particles exhibiting a postsynaptic muscarinic phosphoinositide response ([32P]orthophosphatelabeling in vitro). It is suggested that these may be dopaminergic or other cholinoceptive nerve-endingsor dendrite-derived particles (dendrosomes). Pre- and postsynaptic phosphoinositide responses become matters of terminology (see also Agranoff and Fisher, 1982). Returning to the main theme after this synaptosomal digression, though Durell et al. (1968) provided evidence for a polyphosphoinositide, or at least a PIP response to cholinergic activation and also emphasized the importance of brief incubations (3 min or less), these observations were neglected for a long time. One of the problems was that labeling experiments were measuring phosphoinositide synthesis while the receptor-linked responses involved phosphoinositide hydrolysis. Increased incorporation of [32P]orthophosphateinto PI, one of the commonest measures of the phosphoinositide response, was in reality several steps removed from the initial response. Schacht and Agranoff (1972) showed that synaptosomal labeling of polyphosphoinositides with [32P]orthophosphate was inhibited by acetylcholine, but the effect was not sensitive to atropine. Yagihara and Hawthorne (1972) saw no changes in polyphosphoinositide labeling in similar experiments. For some time, the emphasis then shifted to PI and phosphatidate, and some evidence was provided that their metabolism might be important in synaptic vesicle membranes (reviewed by Abdel-Latif, 1983).Emphasis on PI as the lipid responding to activation of receptors was reinforced by two studies, though neither involved nerve tissue. Hokin-Neaverson (1974) showed that acetylcholine decreased the concentration of PI in pancreas, and Jones and Michell(l974)showed the same for rat parotid. Loss of PI and formation of diacylglycerolcould be measured chemically. This seemed to be good evidence for the hydrolysis of PI to diacylglycerol and inositol phosphate. A number of other publications supported the concept that PI hydrolysis was the receptor-linked event, and Michell(l975) suggested that

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this was the key event in calcium gating, since the receptors involved were all considered to raise the cytosol Ca2+concentration. His theory is outlined in Fig. 2, and it received support from various laboratories including that of Berridge (Berridge and Fain, 1979). I have never been happy with it, partly because a number of phosphoinositide-linked receptors do not increase intracellular Ca2+and partly because PI seemed an unsuitable lipid for a plasma membrane function (Hawthorne and Pickard, 1979; Hawthorne, 1982). It occurs in most cell membranes and has no specific connection with the plasmalemma. As Fig. 2 shows, its hydrolysis at the cell surface and its resynthesis in the endoplasmic reticulum provide severe transport problems. When we described the kinase which synthesizes PIP:, in rat brain (Kai et al., 1968),its activity was compared with the monoesterase and diesterase which hydrolyze PIP:!. The hydrolytic enzymes are about 100 times as active as the kinase. The rates were of the same order as those for acetylcholine synthesis and destruction. For phosphatidic acid on the other hand, the rate of hydrolysis was not as high in comparison to its synthesis. We made the following comment: "Comparison of these rates suggested that phosphatidic acid is involved in the intermediary metabolism of brain lipids but that triphosphoinositide (PIP2) has a different function. It resembles a transmitter substance in that the capacity for its destruction is very great." The importance of the polyphosphoinositides was emphasized in a review t w o years later (Hawthorne and Kai, 1970),which pointed out their plasma membrane localization and the great affinity of PIP:! for calcium

ca2+gate

agonist

plasma membrane

FIG. 2. The Michell PI theory of calcium gating

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(Dawson, 1965).Though we had seen stimulation of PIP kinase by acetylcholine in crude brain homogenates (Kai et al., 1966),we suggested in the review that polyphosphoinositideswere more important in the axolemma, but not particularly at synapses. The metabolism of PI seemed more important in connection with synaptic transmission. The first clear evidence that polyphosphoinositides responded to receptor activation was provided by Abdel-Latif while on sabbatical leave in Nottingham (Abdel-Latif et al., 1977). This marks the shift in interest toward PIP and PIP:, though the Michell PI and calcium-gating theory persisted for a few years. Abdel-Latif et al. worked with iris smooth muscle and showed a muscarinic loss of labeled PIP2 which was attributed to phosphomonoesterase activity the PIP2 being converted to PIP. Further work showed that the cholinergic stimulus caused breakdown by the phosphodiesterase route, giving inositol trisphosphate and diacylglycerol (Akhtar and Abdel-Latif, 1980). As in synaptosomes (Griffin and Hawthorne, 1978), the effect seemed to be secondary to influx of Ca2+. It seemed then, that polyphosphoinositide hydrolysis by phospholipase C was the receptor-linked event, but there was no reason to think that this caused influx or mobilization of Ca2+. Studies with hepatocytes and secretory tissues restored the link with calcium, though it is not possible to give a full account here. The key paper is that of Kirk et al. (1981), showing that vasopressin, a Ca2+mobilizing hormone, induces loss of PIP2 from hepatocytes, but that hepatocyte PIP2 is not sensitive to ionophore-mediated Ca2+influx. The Michell group therefore suggested that polyphosphoinositidebreakdown leads to mobilization of Ca2+ (Creba et al., 1983). Abdel-Latif and coworkers had found that in iris muscle PIP2 hydrolysiswas caused by calcium ionophores, but more detailed study showed that the ionophore was releasing noradrenaline and that this in turn stimulated PIP2 breakdown (Akhtar and Abdel-Latif, 1984). The iris muscle work thus supported the concept that polyphosphoinositide metabolism controlled Ca2+mobilization. Work from other laboratories on this subject is reviewed by Hokin (1985).

111. lnositol Trisphosphate and Diacylglyced as Second Messengers

A. THEDUALMESSENGER THEORY Study of PIP2 hydrolysis stimulated by 5-hydroxytryptamine in blowfly salivary glands showed release of inositol bisphosphate and trisphosphate within 5 sec, leading Berridge (1983) to suggest that one or another of

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agonist

plasma membrane \

\ \

\ \

\

RESPWSES

endoplasmic reticulum

FIG. 3. The dual messenger theory of PIPp breakdown

these compounds might be a second messenger mobilizing Ca2+from an intracellular store. Further work by Berridge, Irvine, and others has shown that inositol trisphosphate is the active compound, and the evidence is outlined in reviews by Berridge (1984), Berridge and Irvine (1984), and Hokin (1985). Nishizuka ( 1984)and colleagues have shown that many tissues contain a protein kinase requiring phosphatidylserine and Ca2 for activation. Full activation of this protein kinase C can be obtained with low concentrations (1- 10 p M ) of Ca2+ if diacylglycerol is provided. Further information is given in reviews by Nishizuka et al. (1984) and Kikkawa and Nishizuka ( 1985). Since receptor-linked hydrolysis of phosphoinositides by the phospholipase C (diesterase) route yields diacylglycerol, both Nishizuka (1984) and Berridge (1984) see this hydrolysis as a dual messenger system. T h e concept (Fig. 3) is that inositol trisphosphate acts as a calciummobilizing agent, while diacylglycerol released at the same time and reinforced by the increased cytoplasmic Ca2+,activates kinase C. A number of substrates for kinase C are known (Kikkawa and Nishizuka, 1985),but the precise effect in any particular cell is not well understood at present. Though it is beyond the scope of this review, activation of kinase C by the tumor-promoting phorbol esters is of considerable interest. +

B. EVIDENCE FOR THE THEORY Resting concentrations of free cytosolic Ca2+ are usually 100 nM or thereabouts. T h e increased concentration necessary for physiological

24'1

RECEPTOR-LINKED PHOSPHOINOSITIDE METABOLISM

events such as secretion may come from extracellular sources by the opening of membrane channels or from an intracellular store associated with the endoplasmic reticulum and not the mitochondria. The Berridge theory that inositol trisphosphate is a calcium-mobilizing messenger applies of course only to the release of intracellular Ca2+.Since the trisphosphate is highly polar and water-soluble, it does not penetrate cell membranes. The early evidence for its role in releasing Ca2+ had to be provided by somewhat artificial systems. Hepatocytes or pancreatic acinar cells were treated with saponin or digitonin, or washed in Ca2 -free solutions so that they became permeable to inositol phosphates. As indicated by the loss of half the cytosolic lactate dehydrogenase (Streb and Schulz, 1983),this process severely damages the cells. The cells are then incubated with ATP and an ATP-generating system in a medium resembling cytosol with Ca-EGTA buffers to set free Ca2 concentrations at suitably low levels (see for example Burgess et al., 1984). Under these conditions, Ca2+is taken up into membrane-bound organelles from which it can be released by inositol 174,5-trisphosphate,but not by other inositol phosphates such as the 1-phosphate or 1,4- bisphosphate. Experiments with mitochondria1poisons indicate that release is not from mitochondria, and work with microsomal fractions from pancreas (reviewed by Berridge and Irvine, 1984)suggests that the inositol trisphosphate-sensitive pool of Ca2 is associated with endoplasmic reticulum or vesicles. Do we know whether this pool of Ca2+is the one from which calcium is released when cell-surface receptors are activated? This is a key question, and the only direct answer so far comes from the original study of pancreatic acinar cells by Streb et al. (1983). This showed that leaky cells lost Ca2+when treated with carbachol, the cholinergic drug long known to stimulate secretion of amylase. If inositol trisphosphate was applied first, the carbachol response was abolished, suggesting that both agents act on the same pool of Ca2+.In this work, the cells were made permeable to inositol phosphates by incubation in a Ca2+-freemedium, while for other cells types, detergents have been used. It may be that the link between the agonist receptor and the Ca2+store is destroyed by detergent. For example, platelet-derived growth factor but not inositol trisphosphate causes Ca2+efflux from intact Swiss 3T3 cells, while cells made leaky with saponin were sensitive to inositol trisphosphate but not to the growth factor (Berridge et al., 1984). +

+

+

C. UNRESOLVED PROBLEMS 1. Which Inositol Phosphates Are Released? The Berridge theory proposes that PIP2 hydrolysis in response to receptor activation releases inositol 1,4,5-trisphosphate as the calcium-

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mobilizing messenger. This is then inactivated by a specific phosphatase (Downes et al., 1982; Seyfred et al., 1984) which converts the 1,4,5-phosphate to the inactive 1,4-phosphate. Supporters of the Berridge theory assume that only PIP2 breaks down in response to receptor activation. In the majority of cases this is not true, however. The effects are usually measured as loss of labeled lipid or production of labeled inositol phosphates, [32P]orthophosphateor [3H]inositol being the precursors used. Equally rapid loss of labeled PIP and PIP2 is usually seen, and quite often there is loss of PI as well. That PIP as well as PIP2 may be hydrolyzed is admitted by Berridge (1984), but he assumes “for the sake of simplicity” that PIP2 is the primary substrate linked to the receptors. Since nature is not always simple, the point requires some attention. The phosphoinositide-specific phospholipase C has been purified from sheep seminal vesicles by Wilson et al. (1984) and shown to attack all three phosphoinositidesin the presence of Ca2+,a point we made long ago with a crude enzyme preparation from intestinal mucosa (Atherton and Hawthorne, 1968).Of more interest, however, is the finding of Wilson et al. that when Ca2+ was removed with EGTA, only the polyphosphoinositides were hydrolyzed, though Ca2+ activated the hydrolysis of all three lipids. I am not aware of an enzyme which hydrolyzes PIP2 and not PIP, though vasopressin (200 nM) in a medium with high KCl and 240 nM Ca2+ decreased rat liver plasma membrane [32P]PIP2by 20% in 1 min, without affecting PIP or PI (Seyfred and Wells, 1984). The plasma membrane enzyme was not specific for PIP2, however. It has been argued that the receptor-linked loss of PIP is due to its conversion to PIP2 by the action of PIP kinase. As pointed out in the original description of this kinase (Kai et al., 1968), it is much less active than the phospholipase C, so this explanation seems unlikely. It is not easy to design suitable kinetic experiments to decide between loss of labeled PIP by conversion to PIP2 or by hydrolysis to diacylglycerol and inositol 1,4-bisphosphate. An attempt, using nonequilibrium labeling of platelet phosphoinositides with 32P,has been made by Wilson et al. (1985). These workers conclude that most of the loss of PI on stimulation with thrombin is due to direct phospholipase C hydrolysis rather than conversion to PIP and PIPp. Chemical estimation of lipid phosphorus showed a thrombininduced loss of 7 nmol PI/lOg platelets in 30 sec. Over the same period there was a loss of about 1 nmol PIP2and no change in PIP concentration. The experiments provide no evidence of a direct phospholipase C hydrolysis of PIP, but do not exclude the possibility. At present, therefore, the evidence does not justify the widespread assumption that only PIP2 is hydrolyzed in response to activation of receptors. If PIP is also hydrolyzed with the release of inositol 1,4-bisphosphate we should have the distinctly odd situation in which a receptor was

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simultaneously releasing the second messenger in both its active (inositol 1,4,5-trisphosphate)and inactivated (inositol 1Abisphosphate) form. Furthermore, if PI is hydrolyzed, as it appears to be in the platelet, should we be looking again at the suggestion of Michell and Lapetina (1972) that the initial product of this hydrolysis, inositol 1,2 cyclic phosphate (Dawson et al., 1971) is some sort of second messenger? 2. Does Receptor-Linked Polyphosphoinositide Hydrolysis Always Lead to Mobilization of Calcium? Much of the current interest in this field goes back to Michell’s (1975) review which pointed out that most receptor systems involving phosphoinositide turnover also raised the concentration of intracellular calcium. As pointed out already, myelin is the richest source of the polyphosphoinositides, yet it is in the nervous system that evidence linking their metabolism with calcium mobilization is weakest. Michell’s PI theory of calcium gating has been described already, but it now seems clear that entry of calcium through plasma membrane channels, either voltage-controlledor receptor-controlled, does not usually involve the phosphoinositides. Such gating of Ca2+ is well-known in nervous tissue. Current theories place PIPz in control of mobilization of this ion from intracellular stores and avoid gating altogether. Polyphosphoinositidebreakdown in several well-defined systems does not cause mobilization of Ca2+,however. Examples are the muscarinic receptors of adrenal medulla, heart, and probably brain. It may be that there are no stores of Caz+ in the endoplasmic reticulum of the cells concerned, so that inositol trisphosphate is released but is ineffective. Even so, there should be some function for the phosphoinositide response in these cases. They will be considered in more detail in later sections. 3. Is Activation of Kinase C Linked to Phosphoinositzde Hydrolysis?

The dual-messenger theory assumes that receptor-linked hydrolysis of PIPz releases diacylglycerol which activates protein kinase C, leading to various effects within the cell. Direct evidence for this is lacking, however. Support for the proposed link between PIP2 and kinase C activation comes from the following indirect evidence. Phosphorylation of proteins in vitro by kinase C requires Ca2+and phosphatidylserine but the Caz+ requirement is greatly reduced in the presence of diacylglycerol. The enzyme then becomes active at Caz+concentrations (lo-’ M) equivalent to those within cells. On application of a synthetic diacylglycerolwith one short chain (l-oleoyl2-acetylglycerol) to intact platelets, a 40K protein is phosphorylated by kinase C. Thrombin activation leads to phosphorylation of this protein also, together with one of 20K molecular weight. Calcium ionophores alone cause the 20K protein to be phosphorylated in

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platelets. It is therefore suggested (Hirasawa and Nishizuka, 1985) that Caz and diacylglycerol act synergistically upon kinase C. Since thrombin also causes hydrolysis of platelet phosphoinositides to release diacylglycerol, it is reasonable to consider that this is the physiological source of the diacylglycerol needed for kinase C to act. It will be apparent that this evidence is indirect and two other points should be considered. First, the diacylglycerol released from phosphoinositides will be predominantly the 1- stearoyl2-arachidonoylcompound, yet kinase C shows no specificity for this. It is activated by a range of diacylglycerols, even those with two short-chain saturated acids such as dioctanoyl glycerol (Kikkawa and Nishizuka, 1985). Second, in cells such as hepatocytes, many diacylglycerols will be present as intermediates in triacylglycerol and phospholipid synthesis. Unless there is some special compartmentation-and kinase C is a soluble enzyme-it is difficult to see why it should not be fully supplied with diacylglycerol without any phosphoinositide hydrolysis. There is no doubt that the kinase C system is important in many receptor mechanisms, but the evidence for its link with the breakdown of PIPzseems less than overwhelming. It would certainly be more economical in terms of cellular ATP to provide the diacylglycerol by PI hydrolysis, as is perhaps the case in platelets (Wilson et al., 1985). +

IV. Phosphoinositidesof the Adrenal Medulla

A. A MUSCARINICPHOSPHOINOSITIDE RESPONSE We have been interested in this topic since finding that PI kinase was present in the bovine chromaffin granule membrane (Buckley et al., 1971). It had been known for some time that cholinergic stimulation increased labeling of PI and phosphatidic acid by 32Pin slices of the adrenal medulla (Hokin et al., 1958;Trifaro, 1969).Nicotinic cholinergic receptor activation in the bovine gland causes release of catecholamine, but the phosphoinositide response is muscarinic (Mohd. Adnan and Hawthorne, 1981; Fisher et al., 1981b). Nicotinic stimuli cause the influx of Caz+ required for secretion, but muscarinic drugs do not significantly affect accumulation of 45Ca(Fisher et al., 1981b). Experiments with the perfused gland indicate that activation of the muscarinic receptors reduces the nicotinic secretion of catecholamine (Swilem et al., 1983).Acetylcholine also reduces nicotinic secretion in isolated bovine adrenal chromaffin cells, and the effect is blocked by atropine (Derome et al., 1981).Experiments from our

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laboratory with slices of the bovine gland (Mohd. Adnan and Hawthorne, 1981) appeared to show that muscarinic receptors can cause secretion, since atropine partly blocked the secretory action of acetylcholine or carbachol. The experiments were indirect, however, and it seems more likely that the muscarinic receptor is inhibitory (Livett, 1984). Cheek and Burgoyne (1985) found no secretory effect on isolated chromaffin cells when they used the muscarinic drug methacholine. However, inhibition of secretion due to nicotine was only seen with high concentrations of methacholine. The conclusion that the bovine chromaffin cell has an inhibitory muscarinic receptor is therefore somewhat controversial. Muscarinic drugs produce a typical phosphoinositide effect in bovine chromaffin cells, as shown by the rapid loss of labeled PIP and PIP2 and the production of inositol tris-, bis-, and monophosphates (A. F. Swilem, H. Yagisawa, and J. N. Hawthorne, unpublished). The phosphoinositide changes were not seen if a calcium-free medium was used. Quin 2 studies showed no change in intracellular free Ca2+in response to the muscarinic stimulus. Nicotine on the other hand, raised the cytosolic Ca2+ concentration. Cheek and Burgoyne (1985)and Kao and Schneider (1985)found that muscarinic agonists caused a small rise in Ca2+ as measured by quin 2, though this change was much less than that caused by nicotine. In the unpublished work mentioned above we found no such rise. If the muscarinic receptor is inhibitory, it is difficult to see how a small increase in Ca2+could explain this action, when much more Ca2+is provided from extracellular sources by the nicotinic opening of plasma membrane channels. On the other hand, Kao and Schneider (1985) suggest that the Berridge mechanism may explain the small muscarinic Ca2+ increase, inositol trisphosphate releasing this ion from intracellular stores. I am not aware of any evidence for an endoplasmic reticulum store of this type, though the chromaffin granules themselves store Ca2+.As mentioned already, the granules have a PI kinase and both PIP and PIP2 can be formed on the cytoplasmic face of the granule (Hawthorne et al., 1980). Attempts to localize the muscarinic loss of [32P]PIand phosphatidate in perfused bovine adrenal medulla were not successful (Azila and Hawthorne, 1982), but there was a significant loss of PI from the chromaffin granules. Such experiments are not easy to extend to the polyphosphoinositides because these lipids are so readily hydrolyzed during the subcellular fractionation procedure, so we do not know at present whether the muscarinic stimulus causes loss of polyphosphoinositides from the chromaffin cell plasma membrane, from the chromaffin granules, or from both.

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B. KINASEC POTENTXATES CATECHOLAMZNE RELEASE BY Ca2+ Using bovine chromaffin cells made leaky by brief exposure to intense electric fields, Knight and Baker (1983) showed that 12-O-tetradecanoylphorbol-13-acetate (TPA) made secretion of catecholamines more sensitive to calcium in a medium containing MgATP. They concluded that kinase C, which is known to be activated by TPA, promotes secretion. Similar conclusions were reached by Brocklehurst and Pollard (1985) who used digitonin-permeabilized cells. The evidence is of course indirect, and though TPA increases catecholamine release from normal chromaffin cells in culture (Brocklehurst et al., 1985), the involvement of the nicotinic receptor, which provides the physiological rise in Ca'', has not been shown in any of these experiments. Nevertheless, protein kinase C is well documented in the bovine chromaffin cell (Brocklehurst et al., 1985). T h e Berridge dual messenger theory (Fig. 3) suggests that diacylglycerol from PIP2 hydrolysis is the physiological activator of kinase C. This cannot be true of the adrenal medulla since nicotinic receptors are not linked to phosphoinositide hydrolysis. Kinase C seems to potentiate the secretory effects of the Ca2' influx caused by these receptors. Phosphoinositide hydrolysis on the other hand is a muscarinic effect associated with modulation of secretion. Livett (1984) suggests that under basal conditions the bovine chromaffin cell is subjected to inhibitory control, possibly through cyclic GMP, and that when acetylcholine concentration exceeds M the nicotinic activation overcomes this. At present it seems that kinase C activation is secondary to the nicotinic influx of calcium.

C. POSSIBLE MECHANISMS OF SECRETION It is generally accepted that catecholamines are released from the chromaffin cell by Ca2+-dependent exocytosis but the molecular mechanism is not well defined. It has been suggested that a spectrin-like protein on the chromaffin granule membrane interacts with F-actin (Aunis and Perrin, 1984). In his recent review, Burgoyne (1984) discusses other work on actin and suggests a model in which Ca2' influx releases chromaffin granules from the cytoplasmic actin network as a preliminary to exocytosis. There is evidence that several cytosolic proteins interact with the granule membrane. Calmodulin is one of these and there is a specific calmodulin-binding protein in the membrane (Bader et al., 1985). There is evidence (reviewed by Burgoyne, 1984) that calmodulin is involved in the

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secretory process. Creutz et al. (1985) describe the binding to chromaffin granules of phospholipase C capable of hydrolyzing PI. Since 2 mM Ca2+ was required for binding, this is unlikely to be physiologically significant in relation to exocytosis. Protein phosphorylation may be involved in the secretory process and its control, as the kinase C work suggests. There are also calmodulin and cyclic AMP-dependent protein kinases in the chromaffin granule membrane (Burgoyne, 1984).These and the kinase C of cytosol and membrane fractions from bovine adrenal medulla are compared by Wise and Costa (1985). In comparison with Ca2+ or cyclic AMP, cyclic GMP had little effect on protein phosphorylation. Boonyaviroj and Gutman (1977, 1979) showed that PGE2 inhibited catecholamine release from rat, human, and bovine adrenals, and micromolecular concentrations of Ca2+can release arachidonic acid from digitonin-treated chromaffin cells (Frye and Holz, 1985). The phorbol ester TPA enhanced release, suggesting that kinase C modifies phospholipase A2 activity, though the evidence would be more indirect than usual, even in this field.

D. MECHANISM OF A MUSCARINIC INHIBITION There are two problems here. First, it is not absolutely certain that the muscarinic receptor is inhibitory. Second, since we know so little of the molecular mechanism underlying secretion in response to nicotinic drugs, it is not likely that muscarinic inhibition of secretion can be better defined. Several points are clear, however, and these are made below. Muscarinic and not nicotinic stimuli lead to phosphoinositide breakdown, most rapidly of PIP and PIP2. The diacylglycerol released does not appear to activate kinase C, since the effects attributed to this enzyme relate to secretion of catecholamines rather than its inhibition. If the phosphoinositide changes are related to protein phosphorylation or dephosphorylation, these protein changes should reduce secretion in some way. Muscarinic stimulation, as in other cells, increases cyclic GMP production (Derome et al., 1981), and this in turn could affect protein phosphorylation, as well as antagonizing the effects of cyclic AMP. Finally, muscarinic agonists do not act by raising cytosolic Ca2+concentration. Whatever the function of the muscarinic phosphoinositide changes turns out to be, neither of Berridge’s two messengers seem to be involved (Fig. 3).

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E. EXCITATORY MUSCARINIC RECEPTORS I N ADRENAL MEDULLA

Not all chromaffin cell muscarinic receptors inhibit secretion. There is variation between species, and though the bovine cell muscarinic receptors are not able to increase secretion, those of the cat, chick, and guinea pig do. The PC 12 cell line from a rat pheochromocytoma has muscarinic receptors which do not provoke secretion and nicotinic receptors causing catecholamine release. Nerve growth factor induces differentiation of PC12 cells so that they resemble neurons and at the same time develop many more muscarinic receptors. The release of inositol phosphates by carbachol in such differentiated cells has been studied by Vincentini et al. (1985). Quin 2 showed a rise in intracellular Ca2+ even in calcium-free media containing 1 mM EGTA, suggesting that the muscarinic stimulus released the cation from an internal store. If this store was depleted by prior treatment with ionomycin, there was no Ca2+ rise with carbachol. Ionomycin caused no release of inositol phosphates, but carbachol produced all three, the trisphosphate in largest quantity as judged by [3H]inositol radioactivity. It was released more rapidly than the bis- or monophosphates. This work therefore provides good evidence that inositol trisphosphate can mobilize Ca2+from internal stores though it should be remembered that these are abnormal cells. In addition, the phosphoinositide response is muscarinic, while secretion is caused by nicotinic activation, so there is a problem in linking the two events, exactly as in the bovine adrenal medulla. No similar information is available at present for the normal rat chromaffin cell, as far as I am aware.

V. Phosphoinoritides and Receptors in Bmin

A. INTRODUCTION The adrenal chromaffin cell provides a relatively simple model of an adrenergic neuron and so has been studied in greater detail than cells obtained from the mammalian brain. Phosphoinositide studies in brain slicesor synaptosomal fractions from brain regions such as cerebral cortex are difficult to interpret. This is because of the variety of cell types represented. A number of receptors are known to be linked to phosphoinositide metabolism and these may be pre- or postsynaptic. Interpretation of results from brain tissue is therefore more difficult.

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B. CALCIUM AND THE PHOSPHOINOSITIDE RESPONSE Early work made use of labeling with [32P]orthophosphateand gave a measure of resynthesis rather than receptor-mediated breakdown. Some of it has been mentioned in Section I1 and it will not be discussed further here. It has been well reviewed by Abdel-Latif (1983). As in many of the cell types, muscarinic receptors of brain are linked to phosphoinositide metabolism. Pharmacological studies indicate that there are different classes of muscarinic receptor, but both MI and M2 types seem to have this link (Lazareno et al., 1985). While excitatory responses could involve a rise in cytosol Ca2+,it is unlikely that inhibitory muscarinic receptors increase the concentration of this ion (Brown and Masters, 1984). Possible links with calcium will be emphasized in this section therefore. Using only '*P-labeling, Griffin et al. (1979)showed that the muscarinic PI response of synaptosomes from guinea pig forebrain was abolished by EGTA. From studies with calcium-EDTA buffers it was concluded that a concentration greater than M was required in the incubation medium for the phospholipid effect. It was concluded that presynaptic receptors were involved and that they differed from the muscarinic receptors in, for instance, pancreas or parotid gland, which mobilize Ca2+.More recent work with synaptosomes showed that muscarinic stimuli decreased the pools of [32P]PIPand [32P]PIP2(Fisher and Agranoff, 1981). Studies with the calcium ionophore A23187 and with calcium chelators showed that the concentrations of these phosphoinositidesbut not of PI were lowered when Ca2+ entered the nerve ending. At the same time, there was increased labeling of phosphatidate by 32P.The experiments suggest that PIP2 and PIP were hydrolyzed by the phospholipase C route though relatively little inositol trisphosphate was detected in comparison with the bisphosphate (Griffin and Hawthorne, 1978). Fisher and Agranoff (1981) showed that A23187 enhanced the muscarinic breakdown of the polyphosphoinositides. The phosphoinositide response to several different muscarinic agonists, measured as release of inositol monophosphate from brain slices in the presence of Li+ (see Section VI), has been studied by Jacobson et al. (1985). This and the studies of Fisher et al. (1983, 1984) are consistent with the coupling of the low-affinity form of the muscarinic receptor to phosphoinositide hydrolysis. Regional differences in the coupling system have been described for guinea pig brain by Fisher and Bartus (1985). The full agonist oxotremorine-M was a more powerful stimulator of phos-

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phoinositide breakdown in neostriatum than in cerebral cortex, for instance. Such regional differences are not easily detected by radioligandbinding methods. An enzyme which may be involved in these muscarinic responses has been detected in synaptosomal membrane preparations by Van Rooijen et al. (1983). The enzyme is a phospholipase C acting on the polyphosphoinositides rather than PI, but it required unphysiologically high concentrations of Ca2+for activity. As the authors suggest, loss of a modulator such as calmodulin could account for this. E. Brown et a/. (1984) have studied the release of [3H]inositolphosphates from rat cerebral cortex slices in response to various agonists. They concluded that cholinergic muscarinic, a-adrenergic, and histamine H receptors were all linked to phosphoinositide metabolism. The histamine H I response has been investigated more fully by Daum et al. (1984) and Kendall and Nahorski (1985a). A further study of inositol phosphate release from rat cerebral cortex slices showed a response with phenylephrine but not with clonidine, suggesting that the a1 receptor rather than the a:!is linked to the phosphoinositides (Schoepp et al., 1984). The problem of the calcium requirement for receptor-linked [3H]inositol phosphate release has been studied in rat cerebral cortex slices by Kendall and Nahorski (1984). They confirm earlier observations that phosphoinositide effects are abolished completely by 0.5 mii4 EGTA and demonstrate that phosphoinositide synthesis is inhibited by Ca2 . This point is often overlooked, though it was demonstrated in the original enzyme studies (see review of Hawthorne and Kai, 1970).At 10 pM Ca2+, however, the carbachol (presumably muscarinic) response was seen in full, while the histamine response was abolished. Depolarizing concentrations of K also stimulated phosphoinositide breakdown whether the medium contained 1.3 mM CaC12or no added calcium, but this K + effect was not seen in the presence of 0.5 mM EGTA. The calcium ionophore A23187 also stimulated phosphoinositide breakdown. There is a markid contrast between these brain responses and the muscarinic and cx-adrenergic responses in rat parotid (Jones and Michell, 1975) as was emphasized by Griffin et al. (1979). In parotid the phosphoinositide response is unaffected by 0.2 mM EGTA, while in brain it is completeiy abolished. This is hard to understand when the phospholipase C responsible for the receptor-linked hydrolysis requires Ca2+ in both tissues, as far as we know. Nor can we exclude the possibility that polyphosphoinositide loss is secondary to the influx of Ca2+in brain. Kendall and Nahorski (1985b) depolarized brain slices in a high K + medium and assayed release of ['H]inositol phosphates. There was an increased release in the presence of the dihydropyridine calcium channel +

+

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activator BAY-K-8644, and this was markedly less if the medium contained no added Ca2+.The effect was stereospecifically suppressed by dihydropyridine antagonists. Phosphoinositide changes in these experiments are probably secondary to the release of various neurotransmitters by the depolarizing conditions. There is evidence of nonmitochondrial stores of calcium in brain, associated with special vesicles or endoplasmic reticulum, and so the Berridge scheme in which inositol trisphosphate releases intracellular Ca2+ could apply. At present, however, the phosphoinositide results provide no real support for this concept. Moreover, there are inhibitory receptors similar to the muscarinic receptors of adrenal medulla which trigger release of inositol trisphosphate, but do not mobilize Ca2+. Masters et al. (1984) have studied the muscarinic phosphoinositide response in human astrocytoma cells. They loaded the cells with 45Cafor 18-20 hr so that equilibrium with exchangeable calcium was achieved. Muscarinic but not nicotinic stimuli then caused efflux of 45Ca.Pilocarpine and oxotremorine caused submaximal calcium effluxes, while carbachol and acetylcholine were much more effective. The same applied to the ability of these compounds to release inositol 1-phosphatefrom cell phosphoinositides. The phosphoinositide effect paralleled the efflux of calcium, and it was suggested therefore that calcium mobilization resulted from inositol trisphosphate production, in accord with the Berridge theory. Muscarinic agonists lower cyclic AMP concentration in these cells, possibly as a consequence of Ca2+mobilization and resulting activation of cyclic AMP diesterase. In a further study of these cells, Masters et al. (1985) showed that carbachol treatment for 90 min desensitized the calcium efflux response, but not the release of inositol phosphate, suggesting that the site at which the calcium effect desensitized is distal to the receptor-linked phosphoinositide hydrolysis. In mouse NIE-115 neuroblastoma cells there is a muscarinic phosphoinositide response which resembles that in parotid gland in not being inhibited by EGTA (Cohen et al., 1983). Muscarinic agonists raise cyclic GMP concentration in these cells, and it was suggested that phosphoinositide hydrolysis mobilized Ca2+to activate guanylate cyclase. Studies with aequorin, the calcium-sensitive photoprotein, show no rise in cytoplasmic Ca2+when the neuroblastoma cells are stimulated with carbachol, however (review of McKinney and Richelson, 1984). Cohen et al. (1983) measured the muscarinic phosphoinositide response only by the increased labeling of phosphatidylinositol. The NIE-115 neuroblastoma muscarinic receptor hyperpolarizes the cells while the NG 108-15 neuroblastoma has a depolarizing muscarinic receptor,yet the phosphoinositide response is the same in each cell line.

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The neuroblastoma-glioma NG108-15 and 1321 NI human astrocytoma cells have been compared by Hughes et al. (1984), who showed that pertussis toxin blocked the muscarinic decrease in cyclic AMP only in the former. Since pertussis toxin ADP-ribosylates the guanine nucleotide regulatory protein N1,which inhibits adenylate cyclase, the authors conclude that there is a muscarinic action via this cyclase in the neuroblastoma cells, but that in the astrocytoma cells cyclic AMP diesterase is activated by Ca2+ mobilization. Perhaps the muscarinic receptor is linked to phosphoinositide metabolism in the astrocytes but not in the NG108-15 neuroblasts. To summarize a confusing section, it is certainly not possible to draw the general conclusion for brain tissue that muscarinic receptors mobilize calcium by phosphoinositide breakdown and release of inositol trisphosphate. The situation appears much more complex, as the review of McKinney and Richelson (1984) shows. As already mentioned, the phosphoinositides mostly contain the 1stearoyl P-arachidonoyl glycerol moiety. Van Rooijen et al. (1985) have shown that muscarinic stimulation of nerve endings from guinea pig cerebral cortex conserves these species. In other words, the diacylglycerol released by muscarinic drugs is converted back to phosphoinositides via diacylglycerol kinase and conversion of the resulting phosphatidate to CDP-diacylgl ycerol.

C. PRESYNAPTIC AND POSTSYNAPTIC MUSCARINIC RECEPTORS It seems clear that both these types of receptors exist in brain, and the muscarinic effects of phosphoinositide metabolism seen in synaptosomes have been assumed to relate to presynaptic receptors. Fisher et al. (1980)have shown that the muscarinic phosphoinositide response in synaptosomes prepared from hippocampus was not affected by a fornix lesion which removed the cholinergic input. Direct lesion of hippocampal neurons with the neurotoxin ibotenicacid reduces the phosphoinositideeffect, however (Fisher et al., 1981a). The authors conclude that the effect is postsynaptic in the hippocampus and that it is seen in synaptosomal fractions because these contain dendrite-derived particles or nerve endings which have postsynaptic surface-receivinginput from a third neuron. Nevertheless, it seems likely that there are brain presynaptic receptors which are linked to the phosphoinositides. What is described as presynaptic may be a matter of terminology when several neurons interact.

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D. PROTEIN B50 Protein B50 is characteristic of brain and appears to be associated showed that the peptide with presynaptic membranes. Zwiers et al. (197’6) ACTH 1-24 inhibited the phosphorylation of this protein. Linked with this effect is the increased formation of PIP:! by PIP kinase (Jolles et al., 1980), suggesting that phosphorylated B50 inhibits the kinase. It seems likely that the B50 kinase is identical in brain with kinase C (Aloyo et al., 1983). Studies in vitro with partly purified PIP kinase from rat brain cytosol showed that addition of the phosphorylated B50 inhibited the kinase more effectively than the dephosphorylated form of the protein (Van Dongen et al., 1985).Jork et al. (1984)found that dopamine decreased the phosphorylation of protein B50 in a synaptosomal membrane fraction from rat hippocampus incubated with labeled ATP. When slices of hippocampus were incubated with dopamine, however, there was an increased phosphorylation of B50 in plasma membranes prepared from the slices and incubated with ATP. Studies of PIP phosphorylation in the two systems showed increases when the synaptosomal plasma membranes were incubated in the presence of dopamine but decreases when the membranes were prepared from slices previously incubated with dopamine. This latter effect was blocked by haloperidol. No conclusions were drawn about the mechanism of dopamine action on phosphorylation of B50,but it is interesting that Simmonds and Strange (1985)noted inhibition of thyrotropin-releasing hormone (TRH)-induced phosphoinositide breakdown by dopamine in dissociated bovine anterior pituitary cells. Gispen et al. (1985)suggest that the B50 phosphorylation might regulate the receptor-mediated breakdown of PIP2. This breakdown would release diacylglycerol to stimulate kinase C phosphorylation of B50,and this in turn would decrease the formation of PIP:! from PIP. My own view is that kinase C will be shown to have a more fundamental role in signaling, but the link with PIP2 formation deserves further study.

VI. The Autonomic Nervous System and the Pituitary Gland

A. THEAUTONOMIC SYSTEM The classical study showing that electrical stimulation of sympathetic ganglia increased labeling of PI is that of Larrabee and Leicht (1965).

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Since then, such ganglia have been investigated in more detail and the phosphoinositide response has been shown to be muscarinic (reviewed by Abdel-Latif, 1983). Bone et al. (1984) have studied this muscarinic effect in isolated rat superior cervical sympathetic ganglia and have shown that it involves release of inositol trisphosphate and inositol bisphosphate. Vasopressin gave a much greater phosphoinositide hydrolysis than the muscarinic drug bethanechol. The vasopressin-induced release of inositol phosphates was also seen in calcium-free media. A vasopressin-like peptide has been identified in noradrenergic neurons of sympathetic ganglia in several species, including the rat (Hanley et al., 1984). Further work by Bone and Michell(l985) showed that depolarization of the ganglia in high K + media also caused release of inositol phosphates, provided that the media contained Ca2+.This suggested that the phosphoinositide breakdown was activated by endogenously released transmitter. Evidence was provided that an unidentified peptide neurotransmitter might contribute to this breakdown. The nodose ganglion of the rat responded to electrical stimulation through the vagus nerve by release of inositol phosphates (Briggs et al., 1985).This release was not affected by nicotinic or muscarinic antagonists. The nodose ganglion is usually considered to be without synapses or to have very few, yet the phosphoinositide response to electrical stimulation was as great as in the superior cervical ganglion. Briggs et al. suggest that peptides may be responsible for the nodose ganglion phosphoinositide changes and that these peptides are released nonsynaptically. The work of Bone and Michell (1985) is clearly relevant. Briggs et al. (1985) also studied release of inositol phosphates by electrical stimulation of superior cervical ganglia. As found by Bone and Michell(1985),the release was not seen in Ca2'- free media, suggesting that it depended on transmitters released by electrical stimulation. Neither atropine nor hexamethonium abolished the release of inositol phosphates caused by this stimulation, and it was argued that nicotinic receptors were partly responsible for phosphoinositide hydrolysis. This is in conflict with many other studies and since electrical release of vasopressin-like peptides would give a much larger phosphoinositide response than the cholinergic transmitters (Bone et al., 1984), the interpretation of Briggs et al. (1985) may be incorrect.

GLANDPHOSPHOINOSITIDE METABOLISM B. PITUITARY The rat pituitary tumor cells known as GH3 have been studied by several workers, since they release prolactin in a calcium-dependent manner. Thyrotropin-releasing hormone (TRH or thyroliberin) promotes this

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release, and at the same time there is polyphosphoinositide hydrolysis (Rebecchi and Gershengorn, 1983; Martin, 1983; Macphee and Drummond, 1984). Experiments with quin 2 show that cytoplasmic-free Ca2+ concentration is increased by TRH, and inositol trisphosphate can mimic this release in GH3 cells permeabilized by saponin (Gershengorn et al., 1984).

An interesting paper by Drummond and Raeburn (1984) describes a prolonged reduction of PI concentration in the GHS cells treated with TRH, without loss of PIP or PIP:,. The lipids were measured as [3H]inositol radioactivity after labeling to equilibrium. The authors suggested that the cells could preserve the level of PIP:, in the face of a considerable reduction of cellular PI. More recently Drummond (1985) has provided evidence for the mobilization of Ca2+at a low level of TRH-receptor occupancy caused by inositol trisphosphate and a second inhibitory response at higher receptor occupancy. The inhibitory component is considered to be related to kinase C, which may have a negative feedback role. Ronning and Martin ( 1985), however, find that phorbol esters which activate kinase C stimulate prolactin release. This would not be associated with the fall in cytosol Ca2+claimed by Drummond to be providing feedback inhibition. A more detailed study by Albert and Tashjian (1985) indicates a complex Ca2+ response to TRH and to phorbol esters. The latter produced a rapid increase in cytoplasmic Ca2+concentration, followed by a decrease to below resting level and then a slow increase to a plateau above the initial level. Ionomycin and phorbol ester together produced effects similar to TRH. The authors suggest that the phorbol ester-induced Ca2+ rise is due to influx through voltage-dependent channels, but that the TRH-induced Ca2+ spike may be mediated by inositol trisphosphate action on intracellular stores. Their interpretation of kinase C action is therefore quite different from that of Drummond (1985). It is not difficult to propose schemes involving Berridge’s two putative messengers (Fig. 3), but whether they relate to conditions within the cell is another matter. A further complication is provided by the effect of a muscarinic receptor in GH3cells which inhibits secretion of prolactin. Activation of this receptor lowers cyclic AMP levels, but also reduces cytosolic Ca2+(Schlegel et al., 1985). Muscarinic receptors are usually linked with phosphoinositide metabolism, and so it is not easy to envisage inositol trisphosphate not only mobilizing Ca2+in response to TRH as outlined above, but also decreasing Ca2+ in response to muscarinic stimuli. However, the GH3 muscarinic receptor has no phosphoinositide response (S. Simmonds, H. Yagisawa and J. N. Hawthorne, unpublished) and recent studies suggest that there may be two types of muscarinic receptors, or one receptor which may link to different G-proteins.

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The ability of dopamine to decrease the TRH-induced breakdown of phosphoinositides in bovine pituitary cells has been mentioned in Section V,D (Simmonds and Strange, 1985).

VII. lithium Chloride and Phosphoinositide Metabolism

Allison and Stewart ( 1971) found that free myo-inositol concentration was reduced in the brains of lithium chloride-treated rats, There was a simultaneous increase in the brain inositol l-phosphate level (Allison et al., 1976; Sherman et al., 198l), since lithium inhibits the phosphatase converting this substance to inositol (Hallcher and Sherman, 1980).Allison (1978) also showed that the effects of lithium chloride in vivo were inhibited by atropine and that cholinergic drugs raised the concentration of inositol phosphate in brain without affecting that of free inositol. Berridge et al. (1982)introduced the use of lithium chloride to amplify agonist-dependent phosphoinositide responses measured in vitro and this practice is now widespread. Inhibition of the inositol 1-phosphatase increases the production of inositol phosphate in such experiments with relatively little effect on inositol bisphosphate and inositol trisphosphate. This could be interpreted as evidence for direct phospholipase C action on PI, rather than a specific action on the polyphosphoinositides, but it is likely that the inositol l-phosphate comes from the higher phosphates. Sherman et al. (1985) discuss this question in relation to the effects of lithium chloride injections on the concentrations of the various inositol phosphates in rat cerebral cortex. The Berridge scheme envisages selective agonist stimulation of PIP2 with release of inositol 1,4,5-trisphosphate.As outlined in Section III,C, this trisphosphate is inactivated by conversion to the 1,4-bisphosphate and then to the inositol l-phosphate. It is not possible at present to decide whether all the inositol 1-phosphate arises from PIP2 in this way. The initial product of PI hydrolysis is the inositol 1,2 cyclic phosphate (Section III,C), and though there was some technical difficulty, Sherman et al. (1985) consider that the increase in this cyclic phosphate caused by lithium is not sensitive to atropine and so may not be related to muscarinic receptor activation and subsequent PI hydrolysis. Nevertheless, lithium can increase the concentration of inositol 1,2 cyclicphosphate, a point which deserves further study. Sherman et al. (1985) calculate that phosphoinositide metabolism produces D-inositol l-phosphate about seven times more rapidly than the brain can synthesize inositol from glucose-6-phosphate. Interestingly, L-inositol l-phosphate, rather than the D-compound, is an intermediate in this synthesis. Batty and Nahorski (1985) have shown differential effects of lithium

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on muscarinic inositol phosphate release from slices of rat cerebral cortex. While lithium potentiated the carbachol- induced accumulation of the mono- and bisphosphates, it inhibited inositol trisphosphate production. The authors comment that brain slices are very different from hepatocytes in their response to lithium, and they also conclude that receptor-mediated hydrolysis of PI and PIP in brain tissue should not be disregarded. Minneman and Johnson (1984),for instance, found that norepinephrine had no effect on accumulation of inositol trisphosphate in rat cerebral cortex slices, but that it increased the bisphosphate concentration. There was apparently a reduction in inositol monophosphate. The incubation medium contained 10 mM lithium chloride, but it should be noted that the incubations were for 2 hr. Effects were associated with the &-receptor. Lithium chloride is effective in the control of manic-depressive illness, and Sherman et al. (1981) suggested that its effect on phosphoinositide metabolism may contribute to its therapeutic action. Physostigmine has a similar effect in raising brain inositol phosphate concentration and can cause temporary remission of mania. Berridge et al. (1982) pointed out that the lowered concentration of inositol due to lithium may reduce phosphoinositide synthesis,since there is a limit to the transport of inositol from blood to brain, as there is to endogenous synthesis from glucose. This is discussed in the review of Berridge (1984).The therapeutic action may be effective because lithium increases inositol phosphate at the expense of inositol in stimulated cells. As a result, these cells have a reduced capacity for PI synthesis so that the phosphoinositide-signaling system will be damped down in the most active cells. There has been a tendency to oversimplify by concentrating on muscarinic actions, however, when adrenergic and other receptors may also be involved. It is also possible that lithium, through its resemblance to sodium, may have quite separate effects on nerve excitation. This could involve sodium and potassium fluxes and the transport of sodium by the Na+, K+-ATPasesystem. The transport of inositol into peripheral nerve is an energy-dependent process linked to Na+ transport (Greene and Lattimer, 1982; Gillon and Hawthorne, 1983). If the same is true of brain, lithium could also affect this system, apart from any actions on membrane potential.

VIII. Phosphoinositides and Diabetic Neuropathy

It has been known for some time that the concentration of free inositol is reduced in sciatic nerve of streptozotocin-diabeticrats (Greene et al., 1975;Palmano et al., 1977)and in postmortem nerve samples from diabetic patients (Mayhew et al., 1983).Though there is also an increased sorbitol

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level in such nerves, the lack of inositol may be more important in producing the nerve conduction defects which are the first indications of peripheral neuropathy in diabetic patients (see review of Winegrad et al., 1983). It is possible that the reduced nerve inositol concentration in diabetes is due to competition from glucose for the carrier responsible for the Na+-linked active transport of inositol (see Section VII). There is also evidence that axonal transport is defective in diabetic animals (Mayer and Tomlinson, 1983), one consequence of which could be reduced enzyme activity. Enzymes of polyphosphoinositide synthesis, particularly PIP kinase and CDP-diacylglycerol inositol phosphatidyltransferase (for PI synthesis), are less active in nerves from diabetic rats (Whiting et al., 1979). Drugs such as sorbinil, which inhibit aldose reductase, can both reduce the excess nerve sorbitol in diabetic animals and restore the inositol concentration to normal. At the same time, there is correction of the slowed motor nerve conduction (Gillon et at., 1983). Das et al. (1976) reported a reduction of the sodium-pump ATPase activity in sciatic nerve of streptozotocin-diabeticrats. This has been confirmed by Greene and Lattimer (1983), who showed that activity could be restored through feeding by a diet containing 1% inositol. A more direct measurement of the pump can be obtained by measuring ouabain-sensitive “Rb+ uptake, and this has also been shown to be reduced in rats 6 weeks after streptozotocin injection, though not at 4 weeks (Simpson and Hawthorne, 1986).Greene and Lattimer (1984) report that sorbinil, which corrects the nerve inositol deficiency in diabetic animals, also prevented the fall in ouabain-sensitive ATPase. They have suggested that a defect in PI synthesis as a result of the low inositol concentration is a major factor in diabetic neuropathy. This PI defect would in turn lower the activity of the sodium pump, though at present we do not understand in biochemical terms how this regulation might come about. Charalampous (1971) showed that KB cells had defective Na+ and K + transport when cultured in a medium containing insufficient inositol. If the phosphoinositides are linked to protein kinase C, as suggested by Berridge and Nishizuka, it is possible that sodium transport might be regulated by a phosphorylation mechanism involving these lipids. There is evidence that the Na+,K+ATPase in Ehrlich ascites tumor cells can be phosphorylated (Spector et al., 1983). If inositol is required in some way for effective Na+ transport and its own transport into nerve is Na+ linked, there is the possibility of selfreinforcing damage to the nerve conduction system,as Greene et al. (1985) suggest in a recent review which discusses several aspects of the biochemical changes related to diabetic neuropathy.

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There are several studies of phosphoinositide metabolism in peripheral nerves of diabetic animals. In sciatic nerves from diabetic patients, there is a reduced total lipid inositol (Mayhew et al., 1983), and in diabetic rats, there is a decline in PI synthesis (Hothersall and McLean, 1979; Bell and Eichberg, 1985). Segments of sciatic nerve or endoneurial preparations from this nerve have been used in vitro to study phosphoinositide synthesis in diabetic and control animals. Bell and Eichberg (1985) found lower incorporation of ['H]inositol into PI of diabetic rats, but no significant change in PIP or PIP:! labeling. Using endoneurial preparations, Hawthorne et al. (1985) found less labeling of PI by 32Pin the diabetics, but greater labeling of polyphosphoinositides.Though the results for the polyphosphoinositidesdiffered in detail, some increased labeling was also reported by Natarajan et al. (1981) and Bell et al. (1982). Berti-Mattere et al. (1985) have made an important advance by showing that in diabetic rats the increased [32P]PIP2and [32P]PIPcan be reversed by insulin treatment. Other phospholipids in epineurium-free nerves from diabetic animals showed increased labeling, but insulin did not prevent the rise. Thus, abnormal polyphosphoinositide metabolism is at least in part a consequence of hyperglycemia. The function of phosphoinositides in peripheral nerve is not understood, though electrical stimulation has long been known to affect these lipids (White et al., 1974) and a theory linking the polyphosphoinositides with ion fluxes through their ability to bind calcium was put forward by Hawthorne and Kai (1970). The early work involved long periods of stimulation and a more recent study was better designed (Goswamiand Gould, 1985). Rat sciatic nerves were prelabeled by intraneurial injection under anesthetic and later stimulated electrically in situ, the rats being reanesthetized. Changes due to 100 Hz stimulation for 5 min were small and only appeared in PI (loss of [32P]-and ['H]inositol label) and PIP (corresponding increases). Statistical information was not provided, and to judge from SD, the changes may not be highly significant. Changes were not seen in the other phospholipids studied, nor did a 20-min period of stimulation change the labeling of inositol phosphates significantly Current thinking about phosphoinositide function relates it to receptors, yet polyphosphoinositidesare particularly associated with myelin and the axolemma, where such receptors are as yet undefined. The nodose ganglion releases inositol phosphates on stimulation, yet is traditionally considered to be without synapses (Section VI,A). Further study of polyphosphoinositide metabolism in nerve and its relation to conduction processes seems particularly important. The results could radically change present views about the function of these lipids.

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IX. Polyphosphoinoritider and the Retina

It has been known for some time that light provokes changes in the labeling of retinal phosphoinositides, but this seemed to be associated with nerve cells in the synaptic layer next to to the photoreceptor cells, rather than with the rod and cone cells themselves (Anderson and Hollyfield, 1981; Anderson et al., 1983). More recently, however, a polyphosphoinositide loss in response to illumination has been localized in the photoreceptor cells (Ghalayini and Anderson, 1984; Millar and Hawthorne, 1985). This effect occurs in rod outer segments and so could be related directly to some aspect of transduction. J. E. Brown et al. (1984) used the rudimentary ventral eyes of the crab Limulus polyphemus to show that light increased the concentration of inositol trisphosphate. At the same time, there was a reduction in ['H]inositol labeling of PIP2, but no change in '*P labeling. Intracellular injection of inositolI,4,5-trisphosphatecaused depolarization resembling the response to light. A transient effect was obtained with inositol bisphosphate. The authors considered that inositol trisphosphate might be an authentic excitatory messenger in response to light. In a similar study with inositol 1,4,5-trisphosphate, Fein et al. (1984)point out that a single photon opens about 1000 ionic channels in the Limulus photoreceptor, suggesting the need for amplification by a biochemical messenger. They also consider that the trisphosphate excites and adapts the ventral photoreceptors in a manner similar to light. In general, the photoreceptor results do not support the theory that inositol trisphosphate acts to mobilize calcium as part of the response to light. Breakdown of PIP and PIP2 is a general response to illumination, but in vertebrate photoreceptors, light causes hyperpolarization, while in Limulus it causes depolarization. The simple theory that mobilization of Ca2+is essential for transduction is not valid, and at least in the vertebrate rod cells, many workers now consider that the key event on illumination is hydrolysis of cyclic GMP which causes closing of plasma membrane channels (Haynes and Yau, 1985; Cobbs et al., 1985). Perhaps the phosphoinositides are more important in adaptation to light. Waloga et al. (1985) in a brief abstract describe experiments in which inositol 1,4,5- trisphosphate was injected into the outer segment of salamander rods. They concluded that injection of the trisphosphate can decrease the receptor potential induced by dim light and that light can decrease the response of the rod to this compound. In another abstract from the same meeting, Rubin and Brown (1985) injected various substances into the photoreceptor cells of Limulus. After EGTA injection, the

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depolarization due to inositol trisphosphate was reduced, as would be expected if it mobilizes internal Ca2+,but the sensitivity to light was increased. It was concluded that a light-induced increase in the trisphosphate is unlikely to be a step in the single file unique chain of reactions mediating excitation. Irvine et al. (1985) at the same time reported that inositol 1,3,4-trisphosphateis the isomer predominantly released by light in Limulus, rather than the 1,4,5-compound.As a final contribution to this complicated picture, Yau and Nakatani (1985) find that, contrary to previous reports, light reduces the concentration of cytoplasmic-freeCa2+in retinal rod outer segments of the’toad eye. Yoshioka et al. (1983a) studied the effect of light on incorporation of [Y-’~P]ATPinto PIP, PIP2, and phosphatidate of microvilli membranes from the octopus eye. There was a loss of radioactivity with all three lipids. Isobutylmethylxanthine,the inhibitor of cyclic GMP diesterase, also lowered the incorporation of 32Pinto PIP and PIP2 by about 75% in a 30 min incubation, but increased the labeling of phosphatidate. These effects did not seem to be mediated by changes in cyclic GMP concentration. Yoshioka et al. (1983b) homogenized the heads of fruit fly mutants with visual defects and incubated with [y-’*P]ATP for 2 min. The mutants labeled phosphatidate less effectively,and in one case, there was increased PIP labeling. In a more recent study, Yoshioka et al. (1985)show that these norp A mutants of Drosophila lack the phospholipase C hydrolyzing PI. The work probably provides the first example of a mutation in a higher organism which affects this enzyme. In summary,the photoreceptor studies provide no convincingevidence that inositol trisphosphate acts as a second messenger in the light-transduction process. Nor can it be claimed that hydrolysis of PIP or PIP2 is consistently linked with mobilization of calcium. Nevertheless, these lipids respond rapidly to light, and the phosphoinositides may play some part in light-adaptation. It will be important to investigate kinase C effects and the possible relation between phosphoinositide metabolism and the phosphorylation of rhodopsin.

X. lnoritol Tetmphotphate

As this review was in its final stages, an interesting new development was reported at a meeting in the University of Birmingham, England, and is now in press (Batty et al., 1985).Carbachol stimulation of muscarinic receptors in slices of rat cerebral cortex caused the rapid formation of a new inositol phosphate, probably the inositol 1,3,4,5-tetraphosphate.It is

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suggested that this may be the precursor of the inositol1,3,4-trisphosphate and possibly the 1,4,5-compound as well. Santiago-Calvo et al. (1963)reported a phosphoinositide with four phosphates per inositol by a combination of 32P-labelingand large-scale extraction techniques, but Seiffert and Agranoff (1965) failed to confirm this. It seems likely that if such a PIP3 (phosphatidylinositol-3,4,5-trisphosphate) exists in brain, its concentration must be considerably smaller than that of PIPp and PIP. Nevertheless, the results of Batty et al. may force a reconsideration of existing theories about the functions of inositol 1,4,5-trisphosphate.

XI. Conclusions

The phosphoinositide field has seen explosive growth over the past few years, and it is certain that our present ideas about the function of these lipids in relation to receptor activation will be out of date in a few more years. Though the polyphosphoinositides were discovered in brain, their function in neurons and nerve fibers remains obscure. The work suggesting that inositol 1,4,5-trisphosphatemobilizes calcium from intracellular stores is best documented for pancreas. At present, there is no convincing evidence for this concept in nervous tissue and a number of examples provide difficulties. These include the inhibitory muscarinic receptors of adrenal medulla and probably those of brain and GH3 cells and the phosphoinositide responses in photoreceptor cells. The links between phosphoinositide metabolism and protein kinase C are probably important and need to be defined more clearly. In peripheral nerve and some ganglia, phosphoinositide turnover is linked in some way to excitation, but the receptor aspect is missing so far. There may be a connection with both the Na+,K+-ATPaseand the Ca*+-ATPasesystems. The detection of inositol tetraphosphate will open up new possibilities, especially if the presence of the corresponding lipid is confirmed. It may be that the phosphoinositides provide a very complex signaling system which has functions other than calcium mobilization, at least in the nervous system. If the pun is not too awful, the phosphoinositides will continue to be exciting lipids. Acknowledgments

My thanks are due to jenny Paxton for her skill and patience in preparing the manuscript. I am grateful to J. Eichberg, R. H. Michell, S . R. Nahorski, S. H. Simmonds, and P. G. Strange for making available to me their papers now in press.

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SHORT-TERM AND LONG-TERM PLASTICITY AND PHYSIOLOGICAL DIFFERENTIATION OF CRUSTACEAN MOTOR SYNAPSES By H. L. Atwood ond J. M. Wojtowicz Department of Physiology Universityof Toronto Toronto, Ontario, Canada M5S 1A8

I. Introduction

Synaptic plasticity has been much studied recently. The early studies of alterations in synaptic transmission resulting from previous impulse activity in the transmitting neuron were carried out at vertebrate (particularly frog) neuromuscularjunctions and also at neuromuscularjunctions of crustaceans (review Atwood, 1976). More recently work on well-defined invertebrate central synapses has led to cellular hypotheses for synaptic plasticity (Klein et al., 1980). The advent of studies on mammalian brain slices (in which parts of brain circuits are isolated in simplified form) has led to a large volume of work on long-term potentiation in the hippocampus (review Erulkar, 1983). The complexity of the central nervous system (even in invertebrates) is a disadvantage for attempts to define cellular mechanisms of synaptic plasticity. In addition, inaccessibility of individual synapses to refined recording techniques may limit the level of analysis, although, recently, indirect methods have been developed to address the problem (e.g., Redman and Walmsley, 1983). Crustacean neuromuscular junctions provide accessible synapses which show both short-term and long-term plasticity and have features in common with many central synapses. They have long been recognized as useful models for the study of synaptic plasticity, and recent work on them has continued to provide insights into basic mechanisms. The aim of the present review is to summarize and evaluate recent work on plasticity in crustacean motor synapses, with a view to extracting general features that may be more widely applicable. Many phenomena can be included under the general label of "plasticity.'' We will not consider all of them, but will restrict our attention to a subset that can be observed particularly well at crustacean neuromuscular junctions. In brief, these include the following: 275 INTERNATIONAL REVIEW OF NEUROBIOLOGY, VOL. 28

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1. Short-term facititation. Following one impulse or a brief train the probability of release of transmitter by a subsequent impulse is enhanced for one to several seconds (review Zucker, 1982). This phenomenon is particularly striking at many crustacean neuromuscular junctions and is important in grading muscular contractions. 2. Low-frequency and high-frequency depression. At certain specialized synapses associated with phasic motor neurons, substantial depression of transmitter release occurs after a single impulse. This effect cannot be explained on the basis of transmitter depletion (Zucker and Bruner, 1977). At higher frequencies of stimulation, these same synapses show transient short-term facilitation followed by depression. The depression observed at higher frequencies of stimulation is different from that seen at very low frequencies and may be attributable to depletion of transmitter. 3. Long-term facilitation. Prolonged trains of impulses lead to a gradual enhancement of transmission that persists for minutes to hours after termination of the inducing activity. The effect is presynaptic in origin; its role in the normal control of muscular activity is not certain. 4 . Long-term adaptation. When a neuron experiences repeated bouts of supernormal impulse activity or supernormal subthreshold activation, synaptic physiology and structure shift in an adaptive fashion (Lnenicka and Atwood, 1985a,b). The reverse change occurs when impulse activity is reduced below normal values (Pahapill et al., 1985).This semipermanent modification of synaptic transmission very likely occurs normally, since seasonal shifts in synaptic physiology corresponding with periods of altered activity have been noted in crayfish. 5. Presynaptic inhibition. In crustacean limb muscles, heterosynaptic effects occur in which an inhibitory neuron’s activity depresses release of transmitter at excitatory neuromuscular junctions. This phenomenon, which is now known to be widespread in central nervous systems, was first analyzed definitively at crustacean neuromuscular junctions (Dude1 and Kuffler, 1961b). 6. Neurohormonal modulation. Circulating neurohormones (in particular, serotonin and octopamine) accentuate synaptictransmission through presynaptic and postsynaptic actions. Central as well as peripheral effects are known in crustaceans (review Kravitz et al., 1985). The advantages of crustacean motor neurons for studying these phenomena lie first of all in the large size and identifiability of the individual neurons. Individual peripheral synapses, the large conducting axons, and the neuronal cell body can be studied with refined analytical techniques. Participation of any of these structures in phenomena such as long-term facilitation and long-term adaptation can be assessed experimentally.

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Coupled with such advantages for experimentation, crustacean motor neurons offer central-type synaptic physiology not available in more specialized synapses such as the squid ganglionic giant synapse. However, not all of the analytical techniques recently applied to the squid synapse have been adapted to the much smaller crustacean synapse, and this, as we shall see, presents problems for definition of ultimate mechanisms. It should be noted at the outset that considerable controversy has been generated by different studies of the phenomena exhibited at crustacean motor synapses. This controversy continues. In what follows, we shall attempt to present both sides of controversial issues along with our own current interpretations, which may point the way to further work.

II. Release of Transmitters

Consideration of short-term and long-term plasticity at crustacean neuromuscular junctions requires assessment of the general mechanism of synaptic transmission. In particular, the factors governing release of transmitter by nerve impulse are important for our discussion, since at present the phenomena of synaptic plasticity at crustacean neuromuscular junctions are thought to be entirely presynaptic in origin. Most of the detailed knowledge of transmitter release has been derived from work in frog neuromuscular junctions, squid giant synapse, and Torpedo electroplaque. A smaller body of work on the crustacean neuromuscularjunction has accrued. We shall examine possible applications of recent work on other systems to the crustacean neuromuscular junction. It is assumed that some, but not necessarily all, of the principles underlying transmission at the more intensively studied synapses will be valid at crustacean neuromuscular junctions. At the outset, however, it must be conceded that there remain major controversies about the mechanism of transmitter release even in the beststudied synapses. Two major unresolved issues are at follows: 1. Is nerve-evoked release carried out by synaptic vesicles or is it nonvesicular? 2. Is calcium entry into the nerve terminal the necessary and sufficient condition for nerve-evoked transmitter release or is a membrane potentialdependent activation of a calcium-binding protein or “active site” also necessary? We will deal with these issues briefly in turn.

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A. VESICULAR AND NONVESICULAR RELEASE Nerve-evoked release of transmitter is quantal in nature. ‘The exact size of the quantal unit, and whether it is formed of subunits, has been much discussed in studies of the vertebrate neuromuscularjunction without definitive resolution (review Tremblay et al., 1983). To date, there is no evidence for subunits at crustacean neuromuscularjunctions. In addition to quantal release by nerve impulses, nonquantal (molecular) release of acetylcholine is prominent at the frog neuromuscular junction (Katz and Miledi, 1977).This release appears to take place steadily at a low level, but the total amount of acetylcholine involved exceeds considerably that released as quantal units. The issue of whether such release takes place in the intact animal or only in isolated preparations has not been dealt with. In crustaceans, tonic release of the inhibitory transmitter y-aminobutyric acid (GABA) and its action on presynaptic receptors of the excitatory nerve terminal have been proposed to account for changes of transmission induced by picrotoxin in crab muscles (Parnas et al., 1975). In addition, it is known that glutamate, the putative excitatory transmitter of many crustacean muscles, is released in the absence of nerve impulse activity, though part of this background release may be from muscle fibers. It seems likely that nonquantal (tonic) release of transmitter occurs at crustacean neuromuscular junctions, but its magnitude and physiological significance are not known. Given that synaptic potentials can be attributed to quantal release, there remains the question of whether quantal release is vesicular. When synaptic vesicles were discovered by electron microscopy and observed to be of uniform size at the neuromuscular junction, the proposal that they constitute the morphological counterpart of the quantal unit became popular. Much evidence supports this view which has been upheld by several reviews within the past decade (e.g., Tremblay et al., 1983; Whittaker, 1984). The most refined attempt to demonstrate the morphological features of vesicular release has utilized rapid freezing techniques in which frog neuromuscular junctions are frozen at precise times during the transmission cycle and subsequently studied by freeze fracture and replication in the electron microscope (Heuser and Reese, 1973, 1981).Surface features of the nerve terminal membrane can then be visualized. At the time of release, openings of synaptic vesicles are apparent along the rows of membrane particles associated with “active zones” of the neuromuscular junction. The active zone is characterized by an electron-dense structural component associated with the presynaptic membrane, by large mem-

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brane particles on the external surface of the membrane, and frequently by evidence of vesicular fusion (Couteaux and Pecot-Dechavassine, 1970; Heuser and Reese, 1981). The prominent membrane particles along the active zone are putative calcium channels (Pumplin et al., 1981). The number of vesicle openings is large when transmitter release is enhanced by an agent (4-aminopyridine, 4-AP) which prolongs the action potential; but when a normal nerve impulse is carried, the “images of release” are reduced to very small numbers (Heuser, 1977). The initial stages of exocytosis are not clearly visualized without 4-AP, which slows the membrane changes. Nevertheless, the correlation between the number of images of release and quantal content of transmission suggests that exocytosis of transmitter from synaptic vesicles could form the basis for quantal release. The number of vesicle openings correlates well with the estimated quantal content of transmission over a wide range as would be expected if each vesicle represented one quantal unit of release (Heuser et al., 1979). Heuser and Reese (1973, 1981)have proposed a cycle of vesicle fusion at the active zone which starts with formation of small pores at the point of vesicular contact, progresses rapidly to a large opening, and ends with a flattening of the vesicular structure into the presynaptic membrane. Prominent membrane particles presumably derived from synaptic vesicles appear in clusters at the point of coalescence; subsequently, they appear to spread away from the active zone. Synaptic vesicle antigens can be detected on the presynaptic membrane of the electroplaque junctional membrane, suggesting incorporation of vesicle membrane after exocytosis (Buckley et al., 1983). A recent study by Torri-Tarelli et al. (1985) confirmed the general features of vesicular fusion during transmitter secretion of the frog neuromuscular junction, and in addition improved the temporal resolution so that fusion events could be detected at the time that secretion of transmitter started. These authors propose that fused vesicles do not flatten into the presynaptic membrane, but instead are rapidly retrieved by the nerve terminal. Incorporation of vesicular membrane into the presynaptic terminal apparently is responsible for expansion of the latter’s membrane surface during sustained, vigorous transmission. Retrieval and recycling of terminal membrane occurs less rapidly at sites away from the active zone. Extracellular markers such as ferritin and horseradish peroxidase (HRP) have been used to demonstrate the formation of coated vesicles and larger membranous cisternae responsible for membrane recycling (review Heuser, 1977). Although the morphological evidence at the neuromuscular junction favors the view that the synaptic vesicle is the morphological counterpart

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of the quantal unit, this view has been challenged by a number of contrary observations, drawn mostly from other synapses (review Israel et al., 1979; Tauc, 1982). Perhaps the most cogent of these observations comes from work on the Torpedo electroplaque (which is in fact a modified muscle). The transmitter, acetylcholine, is found in vesicles and also in the nerve terminal cytoplasm. The vesicutar compartment appears to be more stable and exchanges less readily with the extracellular environment during transmission than the cytoplasmic compartment. The extravesicular pool of transmitter declines first during intense neurosecretory activity. However, a subfraction of synaptic vesicles has been described ("small, dense vesicles") which takes up acetylcholine more rapidly than the rest of the vesicle population (Zimmerman and Denston, 1977; Zimmerman and Whittaker, 1977) and could function as the "vehicle of release" at the electroplaque synapse. Similar "recycling" and "reserve" vesicles have been described in other preparations (Agoston et al., 1985). However, in the electroplaque this population is a small fraction of the total, and the evidence to date supports the idea that many synaptic vesicles are relatively inert and do not cycle rapidly. Evidence at other synapses also suggests that recently formed quantal units are released first during stimulation (Collier, 1969; Molenaar et al., 1973). In Aplysia neurons, injection of acetylcholinesterase gradually abolishes transmission in a manner that suggests preferential contribution of the cytoplasmic pool of acetylcholine to active transmission (Tauc et al., 1974; Tauc, 1982). Vesicular acetylcholine is postulated to be less affected by the hydrolyzing enzyme and to exchange very slowly with the cytoplasmic pool. A number of other observations that raise problems for the original form of the vesicular hypothesis are discussed extensively in recent reviews (Tauc, 1982). Since the size of the quantal unit is not affected by presynaptic membrane potential changes (Cohen et al., 1983), it seems unlikely that cytoplasmic acetylcholine (which is positively charged) is being liberated through a conventional membrane channel. There must exist a mechanism for prepackaging available transmitter for release at the active zone. Alternatives for the vehicle of release are either a saturable carrier, perhaps formed by coalescence of membrane proteins, some of which bind acetylcholine, or an "operative vesicle," postulated to be associated with the presynaptic membrane and in rapid equilibrium with the cytoplasmic transmitter pool (Israel et al., 1979).The latter could be the subpopulation of metabolically active, rapidly cycling vesicles described by Zimmerman

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and Denston (1977). Rapidly cycling vesicles could account for many observations, including the morphological ones of Heuser, Reese, and coworkers at the neuromuscular junction. Thus exocytosis is likely an indication of transmitter release, but only a small part of the vesicular population seen in electron micrographs may be able to participate. In a recent review, Dunant (1986) has restated the nonvesicular hypothesis, which is bolstered by new observationson release of acetylcholine from reconstituted membrane vesicles. A membrane-bound structure (”mediatophore”)is proposed as the key element of the secretory mechanism. A group of these structures, charged with acetylcholine, generates a quantal unit when synchronous discharge is triggered by an associated calcium-binding protein. Subminiature potentials would result from calcium-independent release of acetylcholine by a single mediatophore. Functionally, there is no way to decide between this hypothesis and the others mentioned above. Eventually, more refined biochemical and ultrastructural evidence will help in this regard. At crustacean neuromuscular junctions, vesicle fusion with the presynaptic membrane and subsequent membrane recycling appear to occur as at the frog neuromuscular junction (Thompson and Atwood, 1984). Labeling of vesicles with HRP is more extensive at synapses which release more transmitter. There is evidence for exocytosis selectively at putative active zones at lobster neuromuscular junctions and axodendritic synapse of the abdominal stretch receptor organ (Govind and Chiang, 1979; Schaeffer, 1984; Pearce et al., 1986). So far, the crustacean studies that have been conducted have not employed quick freezing methods, but have been made on aldehyde-fixed material in which exocytosis and other membrane-dependent processes are considerably slowed (Smith and Reese, 1980). We will assume that the general mechanism of release is similar in crustacean and frog neuromuscular junctions, although overall synaptic organization and morphology are quite different.

B. Is THE CRUSTACEAN QUANTAL UNITATTRIBUTABLE TO A SINGLE VESICLE? Quanta1nature of transmission at crustacean neuromuscular synapses is well documented. It is possible to record individual quantal units (miniature excitatory junction potentials, or min.ejps) intracellularly without a significant spatial decay, especially in small muscle fibers (Baxter et al., 1985; Wojtowicz and Atwood, 1986). Alternatively, one can record extracellularly by means of the “loose patch” electrode (Dudel, 1981; Lnenicka

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and Mellon, 1983a; Atwood et al., 1986).In all reported cases, considerable variation in size of individual quanta has been observed. Typically, amplitudes span about fivefold in range and are distributed in approximately normal manner regardless of the method of recording. It is not known why quantal amplitudes vary. The discovery of subminiature end-plate potentials (sub-mepps) at the frog neuromuscular junction revealed the possibility that the majority of mepps are composed of several sub-mepps, each corresponding to release of one vesicle (Kriebel and Gross, 1974; see Tremblay et al., 1983, for review). Thus the release of a variable number of vesicles at a single release zone could account for variability of quanta. T h e above multivesicular hypothesis does not seem likely in the case of crustacean neuromuscular synapses. First, recent experiments by Atwood et al. (1986) showed that quantal amplitudes remain constant when the rate of release is altered u p to 25-fold by direct depolarization of the nerve terminal with an intracelluiar microelectrode. If vesicles were released freely at each release zone, one would expect a greater number participating in each quantum as the rate of release was enhanced. This, however, was not the observed result: quantal size remained constant. Second, simple calculations show that an average vesicle is likely to contain a sufficient amount of transmitter to activate all receptors in a single synaptic cleft ('Fable I). T h e table demonstrates that a concentration of the order of 0.5 mM of glutamate is attained during a release of the contents of a single vesicle if it is assumed that the vesicle contains isosmotic glutamate. This concentration is higher than the 0.2 mM required to saturate the postsynaptic response of the muscle membrane to glutamate (Dudel, 1977). An estimated 6000 elementary events (postsynaptic channels), each contributing a postsynaptic current of about 0.5-1.0 PA, were thought to TABLE I ESTIMATE OF AMOUNT OF TRANSMXITER RELEASEDB Y EACHVESICLE A T A TYPICAL SYNAPSE I N THE CRAYFISH OPENER MUSCLE Amount of transmitter Vesicle diameter Vesicle volume Vesicle content Cleft volume Cleft concentration

500 A 6.6 X 10-201iter 9.9 x lo-'' mole" (6000 molecules) 2 x lo-'' literb 0.5 mM'

References Atwood and Morin (1970)

Jahromi and Atwood (1974)

"Assuming isosmotic concentration (150 mM) of glutamate salt present in vesicle. 'Assuming surface area of I Fm2 and 200 A width. 'Assuming uniform mixing of the content of one vesicle within synaptic cleft.

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participate in one min.ejp in crab neuromuscular synapses (Crawford and McBurney, 1976). This estimate appears to be grossly different from that derived from a direct recording of channel fluctuations at crayfish synapses obtained by Franke and Dudel (1985; see also Stettmeier et al., 1983). Here, the single channel conductance was approximately 100 pS, contributing a current of 8 PA, and the number of channels in a min.ejp was estimated to be 100-200. The results of the latter study are more likely to be accurate at least in the case of crayfish synapses, since they are consistent with the magnitude of min.ejps in this species. One- to twonanoamp currents corresponding to min.ejps have been recorded by Lnenicka and Mellon (1983a),Franke and Dudel (1985),Finger (1985),and Wojtowicz and Atwood (unpublished voltage clamp data). Therefore, assuming that four molecules of glutamate are necessary to open one channel (Dudel, 1977), only 400-800 molecules are required to produce one min.ejp. The calculation in Table I indicates that up to 6000 molecules could be released from one vesicle; this would provide complete saturation of postsynaptic receptors. If the estimate given above is correct, each synaptic cleft together with an associated release zone could act as a unit of transmission. It follows that the number of quanta released simultaneously at a given recording site is ultimately limited by the number of synaptic contact regions with an associated release zone and not by the number of vesicles. Furthermore, the number of quanta appearing synchronously can be taken as the number of active synaptic contact regions. Such an interpretation of quantal units was presented in a recent study by Wojtowicz and Atwood (1986) in which an estimate of the binomial parameter n was taken as the number of active release zones, following earlier suggestions by Zucker (1973)and work on inhibitory inputs to the Mauthner cell of fish by Korn et al. (1982). This result will be considered in more detail later in this article. However, if a synaptic contact and its active zone act as a unit, how can one explain quantal variability?Electron microscopy reveals considerable variation in the surface contact areas of individual synapses (Atwood and Marin, 1983). This may be one source of variability of quantal size since the number of postsynaptic glutamate receptors may vary with synaptic contact area. Larger synapses may possess more receptors and thus generate a larger postsynaptic current in response to a saturating dose of glutamate. However, other factors such as possible differences in receptor sensitivity and density or different amounts of glutamate released as a quantal unit should also be examined. Some central inhibitory interneurons in the fish (Mauthnercell system) possess synapses which are thought to act as units of transmission. Combined binomial and morphological analysis demonstrated that the binomial parameter n is equal to the number of synaptic boutons (Korn et al.,

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1982). Furthermore, electron microscopic observations showed only the zone (presumed functional synapse) per bouton (Triller and Korn, 1982). Calculations by Faber et al. (1985) indicated that the contents of one vesicle were more than adequate to saturate a postsynaptic receptor zone; this accords with our own predictions for the crayfish synapse. It is puzzling, however, that no account of quantal variability was taken by Korn et at. (1982) in their statistical analysis and interpretation of results. To our knowledge, such variability is a prominent and integral characteristic of every synapse where quantal units have been observed.

C. CALCIUM CHANNELS AT THE SYNAPSE The experiments of Katz and Miledi (1967, 1969) on squid giant synapse demonstrated clearly that membrane calcium channels are located in higher density at synaptic terminals than elsewhere on the axon. Furthermore, they showed that when calcium entry was prevented, by setting the membrane potential approximately to the calcium “equilibrium potential” (Hagiwara et d., 1969; Lee and Tsien, 1984), no transmitter was released until the membrane potential was restored to a more negative value, at which point calcium ions entered the terminal for a short time through the opened voltage-sensitive calcium channels. Thus, the general role of calcium channels in phasic release of transmitter and their relative abundance at the point of transmitter release were established. Further work in the squid giant synapse using intracellular calcium indicators has confirmed the entry of calcium through voltage-sensitive calcium channels during membrane depolarization (Llinas et al., 1976; Charlton et al., 1982) and has provided precise measurement of calcium current as a function of membrane potential. The calcium channels of the squid giant synapse differ from many others that have been studied elsewhere [e.g. molluscan neurons (Brown et al., 1981; Eckert and Tilletson, 1981);Paramecium (Hennesseyand Kung, 1985)], in showing little or no inactivation during prolonged depolarization (review Eckert and Chad, 1984). Their kinetics of activation are much slower than for voltage-sensitive Na channels, and this difference is greater at low temperatures: less calcium current and less transmitter release occur at temperatures below 12°C (Charlton and Atwood, 1979) in spite of a larger and longer action potential. Recently, attempts have been made to link the physiological calcium channel to a morphological feature of the presynaptic membrane. Large membrane particles are seen with freeze-fracture techniques along the active zones of the frog neuromuscular junction (Heuser, 1977) and at

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other vertebrate neuromuscular junctions (Walrond and Reese, 1985). Similar presynaptic membrane particles are seen at neuromuscularjunctions of insects (Rheuben, 1985) and crustaceans (Schaeffer, 1984; Pearce et al., 1986), along with vesicle openings in glutaraldehyde-fixed material (Fig. 1). At the giant synapse of the squid, large particles of the same type are found at the numerous presynaptic active zones at which transmitter release must occur. Since the time available during synaptic transmission for movement of calcium from its point of entry to the site of transmitter release is short (perhaps 200-500 ysec) (Llinas, 1977), calcium channels must occur very close to the points of release (Parsegian, 1977). The location of large membrane particlesjust where one would expect to find the voltage-sensitive synaptic calcium channels is strong circumstantial evidence that these particles are the morphological equivalent of the calcium channel. On this basis, calculations have been made at the squid giant synapse to estimate whether the number of particles can account for the observed calcium current seen under voltage clamp conditions (Pumplin et al., 1981).

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_,/*--*.-

0.125 gm

FIG. 1. Arrangement of prominent membrane particles (putative calcium channels) a t active zones of lizard tonic (A) and phasic (B), frog phasic (C), and lobster (D) neuromuscularjunctions. [(A-C) After Walrond and Reese, 1985; (D) After Pearce et al., 1986.1 In A-C, active zone particles (small solid circles) and associated synaptic vesicles (large hatched circles) are drawn to scale. T h e double array of particles at lizard phasic (B) neuromuscularjunctions could provide more intense calcium entry at the site of a synaptic vesicle, and consequently a greater probability of release of the vesicle. The lobster neuromuscular synapse (D) (dashed outline) has two active zones on the face of the presynaptic membrane, each with an irregular array of large membrane particles (small solid circles) and a surrounding array of vesicle openings captured during glutaraldehyde fixation (larger solid circles).

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The total number of large membrane particles (92 A) at the active zones is about 1 . 1 x lo7for a typical squid giant synapse. With a total membrane calcium current of 200-300 nA under voltage clamp, an estimate for single-channel conductance of 0.2 pS results; this is not greatly different from values of 0.1-10 pS for single calcium channels of other cells obtained with more direct measurements (Hille, 1984). Because of time- and voltage-dependence of these channels, only about 10% of them would be opened by a standard action potential of 70 mV amplitude and 1 msec duration. If one accepts the view that the large membrane particles at active zones are calcium channels, some observed differences in synaptic transmission can be explained. For example, phasic and tonic vertebrate neuromuscular junctions have different patterns of distribution of these particles (Fig. 1). The output of transmitter at a vertebrate phasic junction is greater for a single nerve impulse than that at a tonicjunction. The pattern of distribution of the putative calcium channels would subject synaptic vesicles at the active zone to more active calcium at the phasic junction (Fig. 1); thus, the probability of transmitter release would be enhanced. In general, then, it seems quite possible that certain aspects of synaptic performance could be linked to the distribution of active calcium channels. In all of the above work, there is an inherent assumption that the observed membrane particles are all equivalent in their properties. It is possible, however, that some of them are latent or inactive calcium channels which cannot be recruited to the active state by a single nerve impulse. Furthermore, the metabolic condition of the nerve terminal may lead to altered phosphorylation of some of the calcium (or other) channels, thus affecting their voltage sensitivity or responsiveness to gating ions (Ewald et al., 1985). Possible changes in responsiveness of membrane calcium channels could be a basis for longer term synaptic modification. At crustacean neuromuscular junctions, morphological measurements of synapse size and active zone size were not able to predict transmitter release at physiologically different synapses (Atwood and Marin, 1983). Active zone particles have now been seen in freeze-fracture preparations (Schaeffer, 1984), but a complete quantitative correlation of transmitter release with the occurrence of these particles has not yet been attempted in crustaceans. D. DEPOLARIZATION, CALCIUM, AND TRANSMITTER RELEASE

The nerve impulse, on its arrival at or near the synaptic terminal, opens voltage-dependent calcium channels located near the sites of transmitter release. Calcium entry, measured at the squid giant synapse as total

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presynaptic calcium current (Zca), is thought to flow mainly after the peak of the nerve impulse, when calcium channels have been opened by depolarization and the electrical driving force on calcium (Em- Ec,) is increased due to membrane repolarization. Thus, most of the calcium entry associated with a single nerve impulse is in the form of a “tail current” which occurs during the repolarizing phase of the impulse (Llinas, 1977). The nerve impulse contributes an increment of free calcium ion (Cai) to the internal environment (Fig. 2). The reaction leading to transmitter secretion is dependent upon this increment. When extra free calcium is not available, the reaction proceeds at a very slow rate, and quanta1 secretion of transmitter is in the form of randomly occurring spontaneous miniature potentials. The normally low rate of spontaneous transmitter release reflects the low level of intracellular Ca2+,according to present orthodox views. Several processes act to remove free Ca2+once it has entered (Fig. 2). These include uptake by intracellular organelles and calcium-binding proteins and extrusion through the membrane surface by an ATP-utilizing calcium pump and by Na-Ca exchange. Removal processes may govern the time course of short-term facilitation (Parnas et al., 1982a,b). When the action potential is prolonged or enlarged (for example, by blocking repolarizing potassium channels), transmitter release is greatly augmented. The general view is that increased Zca leads to a greater increment of Cai. Changes in amplitude of the presynaptic action potential can also affect transmitter release, though less strikingly. In general, there Internal compartments

Membrane extrusion

Ca0

FIG.2. The major processes governing the fate of intracellular calcium (Cai)entering through membrane calcium channels from the extracellular environment (Ca,). In the model of F’arnas et aZ. (1982a), saturation kinetics describe the processes of calcium entry and removal and transmitter release.

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is a steep relationship between membrane potential and recruitment of calcium channels. This has been modeled as a fifth power relationship in the squid giant synapse (Llinas et al., 1976, 19Sl), implying voltage-dependent cooperative action of five channel subunits to open a calcium channel. The role of membrane depolarization in controlling Zca has been well established, and there is substantial evidence also for the role of Cai in neurosecretion (Charlton et al., 1982). However, a number of recent observations have been adduced in support of a second role of membrane depolarization-recruitment of a calcium-binding protein or active site. According to this view, membrane depolarization does two things: (1) it opens calcium channels and (2) it activates a voltage-sensitive protein or active site, which serves as the substrate for the calcium-activatedreaction. Thus at present, there are two hypotheses to account for the role of the nerve impulse in releasing transmitter: the “calcium hypothesis” in which the extent and time course of transmitter release are governed only by intracellular calcium and the “voltage hypothesis”, or more correctly the “calcium-voltage hypothesis” in which an additional voltage-activated step is postulated to affect both the time course of release and the amount of secretion (Dudel et al., 1983; Parnas and Segel, 1984; Parnas et al., 1986a,b; see Fig. 3). The evidence for a second effect of presynaptic membrane depolarization is based mainly on experiments on crayfish and frog neuromuscular junctions in which an extracellular “macropatch” electrode was used to polarize or depolarize the nerve terminal (Dudel, 1983b, 1984a,b; Dudel et al., 1983). The transmembrane potential of the nerve terminal is not known in such experiments, but is assumed to reflect the applied extracellular current. Thus, it must be stressed that several uncertainties surround the interpretation of these experiments, and it is not surprising that they are currently under dispute (Zucker and Lando, 1986). Since the outcome of the debate on these alternative hypotheses will have important implications for future work on transmitter release and plasticity at crustacean neuromuscular junctions, we shall briefly review here some of the available evidence and the inferences that are being drawn from it. The contentious observations are several. First, experimental work on crayfish terminals by Dudel et al. (1983) indicated dissociation between available intracellular calcium (measured by the extent of short-term facilitation)and phasic transmitter release. It was observed that a brief train of nerve impulses was followed by a period during which a weak extracellular pulse to the nerve ending, ineffective by itself in releasing transmitter, caused a large increase in transmission. Nerve impulses admit

289

PLASTICITY AND DIFFERENTIATION OF SYNAPSES T h e calcium hypothesI8

Depolarlzatlon

The voltage hypothesis

-7

U Dopolarization

Elevated

Activated

Release s i t e s or receptor

Transmltter

or r e c e p t o r

1

Impulse

A:

Actlvation by deP01arizstion klD

(Inactive form) T

B:

k-,"

SActlve form)

Reacilon with calcium

s + Ca

c: n(CaS) ReIeese

+V

Release of transmitter k3

X L (Postsynsptlc response)

FIG. 3. The calcium hypothesis and the voltage hypothesis of transmitter release (top, from Zucker and Lando, 1986) and a further explanation of the voltage hypothesis (bottom, after Parnas and Parnas, 1986). In the kinetic scheme for the voltage hypothesis, T represents a n unreactive protein or "release site," S represents an active form, V represents the vehicle of transmitter release, and L represents the postsynaptic response (current flow due to a secreted quantal unit). Rate constants k l D and k k l H are associated with depolarization and hyperpolarization, respectively (step A in the sequence); kp and L2are associated with the calcium reaction (step B in the sequence); and ks is associated with secretion of transmitter (step C). Cooperativity of calcium action is assumed to occur in step C with A the number of units of the active complex required for release of a single quantal unit.

calcium, but a weak pulse that does not release much transmitter presumably admits very little calcium. If its inability to admit calcium persists after the train of impulses, then the release of transmitter must be attributed to a different depolarization-mediated event interacting with the residual calcium remaining from the nerve impulse train. A second observation stems from experiments in which two pulses were applied to the nerve ending, and quantal release measured for both.

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Quanta1 content for the first pulse increased progressively as its amplitude was made larger. However, facilitation of quanta1 release by a constantamplitude second pulse, which is thought to indicate the level of Cai established by the first pulse, first increased and then declined as the amplitude of the first pulse was increased. The calcium entry expected for particular membrane depolarization can be estimated from the bellshaped curve of Fig. 4, which was obtained from the measurements on the squid giant synapse. Thus, facilitation was used as an indirect measure of calcium entry. T h e experiment was taken to indicate that the first pulse admitted less calcium when its amplitude was increased (due to approach of the membrane potential to &,), but that transmitter release for this pulse nevertheless became larger because a second depolarization-activated process was augmented. Essentially the same results have been obtained at the frog neuromuscular junction treated with tetrodotoxin (TTX) to block nerve terminal action potentials and stimulated by macropatch electrodes (Dudel, 1984a,b). Here, too, there is an apparent dissociation between calcium entry and transmitter release, suggestive of an additional fast voltagedependent reaction. A third argument is based upon the observation that the time course of transmitter release remained rather constant when depolarizing pulses of different amplitude were applied to the nerve terminal (Dudel, 1984a). T h e invariance of the time course of phasic transmitter release under 1 .o

1.0

*

(0

e

a 0

c

0.1

u m n 0.0 1 Prosynaptic pulse p o t e n t i a l I m V )

0.5 1.0 0.1 Ica normalized t o p e a k

1.0

FIG. 4. The relationship between calcium entry (lcs) and postsynaptic current (PSC) at the squid giant synapse as determined by voltage clamp measurements (after Augustine el al., 1985a). In B, data from the ascending (I) and descending (111) limbs of the calcium voltage-current curves are plotted on iinear and logarithmic coordinates, revealing a third power relationship which suggests cooperation of three calcium ions for each unit of transmitter secretion.

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conditions in which entry of calcium is presumed to vary greatly has been used as evidence that a fast voltage-dependent process additional to calcium entry serves as a limiting factor in the process of transmitter secretion (Parnas et al., 1986a). This rate-limiting step is postulated as a potentialdependent component of release, which acts as the limiting factor even though Cai may vary in response to different levels of depolarization. The voltage hypothesis elaborated by Dudel and co-workers (Dudel, 1983b, 1984a,b; Dudel et al., 1983) postulates that the kinetics of transmitter release are governed by levels of potential-dependent “activators” and “repressors.”Modulating effects of both depolarization and hyperpolarization have been described, and a complex kinetic scheme has been elaborated (Dudel, 1984b). In recent work, the modulating effects of hyperpolarizing and depolarizing pulses applied to the crayfish nerve terminal by a macropatch electrode were investigated (Parnas et al., 1986b).The transmitter-releasing effectiveness of test pulses applied to the terminal was dramatically reduced by hyperpolarizing pulses applied immediately before or after the test pulse. The results can be described by a model in which the reactive state of a potential-dependent membrane component is governed by membrane potential of the nerve terminal. The powerful modulating effects of hyperpolarization are suggestive of a potential-dependent effect on membrane properties. Recent experiments by Zucker and Lando (1986),designed to test the voltage hypothesis, have led these authors to conclude that it cannot be supported. Their counterarguments are based upon additional experiments and upon current models of the distribution of Cai in the squid giant synapse. They attribute the supernormal effect of a weak extracellular pulse following a train of nerve impulses to a period of supernormal excitability set up in the nerve terminal by depolarizing afterpotentials (Zucker, 1974b).The weak extracellular pulse may elicit an action potential during this period, thereby producing substantial release of transmitter. The result of the two-pulse experiment is attributed to the different voltages experienced by synaptic active zones under the thick rim of the extracellular loose-patch electrode in comparison to its central region. A small pulse causes active zones under the lumen of the electrode to release transmitter, while a large pulse could suppress release from these release points (due to approach of the membrane potential to Eta) while recruiting additional release sites under the edge of the electrode. According to this explanation, decline of facilitation measured by the second pulse is due to less calcium entry in nerve endings within the lumen of the macropatch electrode, while the increased release for the first pulse reflects

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recruitment of increased numbers of release sites away from the lumen. Such an effect would obviate the need to postulate a second voltagedependent step for release. In recent experiments on crayfish nerve terminals (Wojtowicz et al., 1986),intracelluiar electrodes were used to deliver controlled stimulating pulses to the nerve terminal, thus avoiding the “rim effect” analyzed by Zucker and Lando ( 1986) for extracellular stimulating electrodes. The two-pulse experiment gave results of the same general form as seen previously for the extracellular experiment (Dudel et al., 1983). However, the potential changes in the transmitter-releasing nerve terminals are not accurately known, so it is not certain that a large pulse delivered intracelluiarly brings the membrane potential close to EG. This is inferred indirectly from the magnitude of short-term facilitation following the first pulse. In addition, tail current at the end of the pulse would not be eliminated when the membrane potential during the pulse approaches E G , so there will always be at least some entry of calcium. We must conclude that terminal membrane potential and calcium entry are both unknown entities in these experiments and that the debate will only by finally resolved when methods are found that will permit the relevant measurements. The recent experiments reported by Dudel ( 1984a,b) on frog neuromuscularjunctions and by Parnas et al. (1986b) on crayfish neuromuscular junctions add additional evidence for modulation of release by hyperpolarizing and depolarizing pulses applied just before or just after a stimulating pulse. The observations were interpreted as evidence for control of rapid voltage-dependent activator and repressor steps by the membrane potential. Zucker and Lando (1986) concede that membrane potential could play a role in modulating release. But once again we are faced with uncertainties about membrane potential changes and calcium entry. Katz and Miledi (1967) showed effects of pre- and poststimulus hyperpolarization in the squid giant synapse; poststimulus hyperpolarization was very effective in reducing transmitter release, but mainly through reduction in duration of the preceding depolarization. Their results and discussion (pp. 424-425) illustrate the difficulty of interpreting such experiments. At present, the experiments on crayfish and frog nerve terminal are suggestive, but do not lead to clear support for the voltage hypothesis of transmitter release. More experimental evidence is required to determine whether there is a second potential-dependent process. In the giant synapse of the squid, evidence from voltage-clamped presynaptic nerve terminals in which 1, was measured indicated until recently that more transmitter was released by large depolarizations approaching Eca than by small depolarizations giving an equivalent Zca

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(Llinas et al., 1976). More recent experiments in which transmitter release was restricted to a small isopotential segment of the presynaptic terminal have shown that this effect is minimal (Smith et al., 1985; Augustine et al., 1985a). In addition, new theoretical models of entry and distribution of intraterminal Ca2+(Simon and Llinas, 1985; Zucker and Fogelson, 1986) can account for apparent voltage dependence without the need for an additional voltage-dependent process. The work on the squid giant synapse is more advanced than that on other synapses and represents a “gold standard,” because presynaptic membrane potential and calcium current can be measured and controlled more accurately. This does not mean that the squid synapse is a good model for the other synapses. The large size of the presynaptic terminal and the predominance of synaptic depression at the squid synapse have implicationsfor intraterminal calcium distribution and transmitter release that serve to differentiate this synapse from those in crustacean muscles. Short-term and long-term plasticity (apart from synaptic depression) are poorly shown at the squid synapse. Even if it should prove finally that there is no second voltage-dependent step in transmitter release at the squid synapse, such a step would not be ruled out at other synapses. But in this case, the need for unambiguous experimental evidence to support the claim at the other synapses would be even stronger. In conclusion, much recent work on the squid giant synapse and on frog and crustacean neuromuscularjunctions supports the central role of calcium in transmitter release. The existence of a second voltage-dependent process is suggested by a number of observations, but not yet confirmed in an unambiguous manner. Such a mechanism, if it exists, could explain several unresolved phenomena of short-term facilitation and presynaptic inhibition at crustacean neuromuscular junctions (Sections IV and V). E. DYNAMICS OF CALCIUM AT

THE

SYNAPSE

1. Squid Giant Synapse Calcium entry at the squid giant synapse (and presumably at other synapses) is voltage- and time-dependent. As the membrane potential is made more positive, calcium entry (ZcJ at first increases and then decreases (Fig. 4). The ascending limb of the bell-shaped curve reflects increasing gG, while the descending limb reflects decreased driving force for Ca with little further increase in gc,. Transmitter release (measured in Fig. 4 as postsynaptic current under voltage clamp) follows Icain a nonlinear manner. For both ascending and

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descending limbs of the Zca curve, transmitter release increases as a third power function of Zca (Fig. 4). The third power relationship between transmitter release and Zca may reflect the requirement for several calcium ions to act cooperatively within the axon terminal for secretion of a single transmitter quantum, as originally proposed by Dodge and Rahamimoff (1967). However, this issue is currently not resolved. Since calcium enters through discrete channels, and, once inside, rapidly diffuses away from the entry point, the calcium concentration at locations where the transmitter is released will be determined by time-dependent "calcium domains" around individual calcium channels (Chad and Ecker, 1984)and possibly also by the degree of overlap with adjacent domains (Zucker and Fogelson, 1986). Two recent models have been developed to explain the influence of calcium on transmitter release at the squid giant synapse. Both postulate entry of calcium and creation of a local calcium domain at the calcium channel. However, in one model (Simon and Llinas, 1985),it is postulated that transmitter release is linearly related to the calcium concentration within the local domain and that apparent nonlinearities are explained by the requirement for more than one calcium channel to open for release of one quanta1 unit. Overlap of calcium domains is not considered to be significant in this model. In the second model (Zucker and Fogelson, 1986), it is postulated that transmitter release is actually dependent upon the fifth power of calcium concentration at the local release site and that variations in spacing of calcium channels, and hence in overlap of adjacent calcium domains, modify the apparent calcium cooperativity of release (Figs. 5 and 6). This model emphasizes the importance of overlap of adjacent calcium domains. The two models make different assumptions about the diffusion constant for calcium within the terminal and about the spatial relationship between the calcium channel and the site of liberation of the transmitter. It is not possible at present to decide which of the assumptions used in these models are valid. Both models explain the fact that persistence of intracellular calcium, measured directly (Charlton el al., 1982),long outlasts the acute phase of transmitter release. Calcium concentration at an active zone for release will decline rapidly, even though total intraterminal calcium declines slowly (Zucker and Stockbridge, 1973). Both models also emphasize the importance of the microscopic localization of calcium for transmitter release at the active zone. Thus, the number and spacing of calcium channels at the active zone could be an important determinant of synaptic performance. In the model of Zucker and Fogelson (1986), the spacing of calcium channels around the point of transmitter release changes with membrane potential, and overlap between adjacent channel domains is an important

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2 0I

10

0 -300

Distance (nm)

0

3 0

Distance (nm)

FIG. 5. One version of the calcium domain hypothesis as applied to secretion of transmitter at individual release sites in the squid giant synapse (after Zucker and Fogelson, 1986). Open calcium channels (open circles) are separated by 218 nm when the membrane voltage is set to - 20 mV (A) or by 101 nm when the membrane voltage is set to 0 mV (B). In each case, curves 1,2, and 3 represent spatial distribution of submembrane free calcium concentration near open calcium channels at 1.0, 2.4, and 3.4 msec after the beginning of the voltage pulse. The active calcium concentration at release sites (solid circles) increases as the open calcium channels become more closely spaced. The curves lie in the plane perpendicular to the presynaptic membrane and through two open channels.

factor in determining the calcium concentration at the release point (Fig. 5). An apparent voltage dependence of transmitter release (Llinas et al., 1981),which according to recent experiments may be very minimal at the squid synapse (Augustine et al., 1985b), could arise from changes in the spacing of open channels with membrane potential (Zucker and Fogelson, 1986). Similarly, changes in channel spacing could give rise to a calcium cooperativity for release that is lower than the true value (Fig. 6). The model of Zucker and Fogelson (1986) provides an attractive way to visualize the limits and variation of apparent cooperativity of transmitter release. If submembrane calcium concentration were in the form of a uniformly distributed layer, the apparent cooperativity measured from the relationship between postsynaptic response and presynaptic calcium current would approximate the true biochemical cooperativity. If, on the other hand, calcium were to enter at widely separated channels with no overlap of calcium domains, available active calcium at each nearby transmitter-releasing zone would decline as the membrane potential approaches Eta, even though Ica for the whole terminal increases due to recruitment of more calcium channels. This would lead to a less than linear dependence of release on calcium current. Hence, values of calcium

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I 5

I 10

I 15

Assumed calcium cooperativity

FIG. 6. Relationship between observed and assumed actual calcium cooperativity, predicted by the model of Zucker and Fogelson (1986). The degree of calcium cooperativity was varied from 1 to 20, and the simulated peak rate of transmitter release was plotted against presynaptic calcium current at the end of a 2.5-msec pulse, using logarithmic coordinates. T h e midrange slope of this curve is plotted as a function of the assumed calcium cooperativity. The obsemed value is always less than the assumed actual value.

cooperativity between the extremes can be generated by varying the spacing of the calcium channels, with the true value always greater than that observed experimentally (Fig. 6). The models developed for the squid giant synapse provide useful insights into the factors that may be important for performance of other synapses. However, crustacean nerve terminals are physiologically and morphologically quite different from those of the squid, and it cannot be assumed that features characteristic of the squid synapse will be faithfully replicated elsewhere.

2. CrustaceanSynapses Models of calcium cooperativity and kinetics at crustacean synapses have not yet evolved to the single-channel level. However, the dependence of transmitter release and short-term facilitation upon calcium can be gained from available models of crustacean terminals. The possibility of

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a voltage-dependent step additional to calcium entry (Fig. 3) must be considered. The most comprehensive model for crustacean nerve terminals has been put forward by F’arnas et al. (1982a),on the basis of theoretical work by Parnas and Segel(l980, 1981).The model does not make any assumptions about the morphology of the nerve terminals or the distribution of calcium channels. The approach employed was to study transmitter release as a function of external calcium (Ca,) and to examine the time course and amplitude of short-term facilitation (for twin pulses of variable separation) also as a function of Ca,. Sets of equations derived from saturable and nonsaturable kinetic processes were then matched with the experimental results, and conclusions were drawn about the true nature of calcium entry into the terminal, calcium removal from the terminal, and calcium dependence of release. The original model (F’arnas et al., 1982a) was subsequently modified (Parnas and Segel, 1984) on the basis of further experimental and theoretical work to include an additional voltage-dependent step for transmitter release, as outlined above (Fig. 3). For nerve terminals of the crayfish opener muscle, experiments on the effect of calcium on transmitter release had earlier suggested a linear relationship (Bracho and Orkand, 1970). In crabs, a more-than-linear relationship was reported (Linder, 1973). More recent measurements on crayfish indicate values for apparent calcium cooperativity ranging from 1.6 to 4 (Dudel, 1981; E’arnas et al., 1982a).The apparent cooperativity is thought to be an underestimate of the true value (Parnas et al., 1982a; Zucker and Lara-Estrella, 1983; see also Fig. 6). In the case of the crayfish terminal, limitations on entry of calcium (which was postulated to be saturable) could mask the true cooperativity for release (Parnas and Segel, 1981). We can add now the possible effects of calcium channel spacing (Fig. 6). On balance, it seems likely that there is a nonlinear reiationship between active calcium at crustacean active zones and transmitter release and that the experimentally observed value is an underestimate of the real value. Parnas et al. (1982a) deduced that entry of calcium saturates as a function of Ca,,. Saturation of entry through calcium channels has been observed and studied in other systems (e.g., Akaike et al., 1978). In the crayfish, the time course and amplitude of short-term facilitation do not change much, as Ca,, is increased above 9 mM, which suggests that calcium entry is limited. At the normal Ca,, of 13.5 mM, saturation of calcium entry would be predicted, and the amount entering for a single impulse would be far below that needed to activate fully the release process.

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Another way of looking at this would be to postulate that only a small proportion of the available calcium channels are activated by one impulse. When the impulse is broadened, for example by cesium ion (Atwood and Lang, 1973; Zucker and Lara-Estrella, 1979), release for a single impulse is enormously increased, suggesting a much greater entry of calcium, possibly through a larger number of opened channels. Removal of calcium from active zones was postulated to be a saturable process, in agreement with the known characteristics of calcium removal processes studied elsewhere. For example, calcium extrusion at the surface of the squid axon (Dipolo, 1973),calcium uptake by mitochondria (Carafoli and Crompton, 1975), and calcium sequestration by elements of the endoplasmic reticulum in nerve terminals (Blaustein and Ector, 1976; Blaustein et al., 1978) all show saturation kinetics. Two mechanisms involved in removal have been postulated in order to account for changes in the duration of facilitation under different external Ca and Na concentrations (Parnas et al., 1982b). Membrane NaCa exchange was implicated by the lengthening of short-term facilitation in low Na,. Although the time course of calcium buildup and decline following an impulse has not yet been measured directly in crayfish terminals, the general notion of saturable calcium removal from the terminal as a whole accords well with the rather slow time course of intracellular calcium decline at the squid giant synapse (Charlton et al., 1982).However, as already described for the squid synapse (Fig. 5), removal or extrusion of the calcium that has entered the terminal may not be the dominant factor in determining the concentration of calcium at an active zone. The kinetics of calcium diffusion away from the surface into the terminal and the spacing of calcium channels may be of considerable importance in this regard. Saturation of the transmitter release process is postulated also in the model of Parnas et al. (1982a). In part, this conclusion is based upon the observation that short-term facilitation measured by closely spaced twin pulses is reduced in elevated Ca, (i.e., release cannot increase past a saturated level even when conditions favorable to an increase in intracellular Ca exist). Experimental support for the idea of saturable release is found in studies which show maximal values for the excitatory postsynaptic potential (EPSP) under conditions of high-frequency stimulation (Bittner, 1968) or broadened nerve impulses (Atwood and Lang, 1973). However, once release has been jacked up to maximal levels, an unknown amount of synaptic depression may become superimposed upon the release process. The fact that only one or a few quanta can be "seen" at once for a given synapse (Section II,B) may also give apparent saturation below the true level. Morphological work suggests that release is ultimately limited by the

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available active zones, which, though numerous on crayfish opener nerve terminals, are nevertheless finite in number. Termination of evoked release in the presence of persistent intracellular Ca is postulated to be due to the rapid temporal subsidence of a depolarization-dependent process or material (Parnas and Segel, 1984). This hypothesis reconciles the short time course of evoked release with the more prolonged time course of the short-term facilitation (thought to depend upon elevated intracellular Ca). As noted above (Fig. 5), an alternative is the rapid diffusive collapse of the calcium domains near the active zones for transmitter release. Unfortunately, present experimental evidence is not refined enough to decide this issue definitively at the crayfish terminal. The general picture that presently emerges for the crayfish nerve terminal is that calcium entry during a single nerve impulse is limited, thus recruiting only a small fraction of the immediately available transmitter store. This fraction was estimated at about 5-15% in typical nerve endings by Parnas et al. (1982a). Transmitter release probably proceeds through a step involving cooperative action of two to five calcium ions. Release apparently is saturable, but in the crayfish nerve terminal the saturation level is far above the level of release attained by a single nerve impulse. Release may be terminated either by diffusion of calcium from active release zones or by rapid disappearance of a voltage-activated cofactor. Clearly the crayfish opener nerve terminal performs well below capacity for a single nerve impulse, and thus there is much opportunity for synaptic plasticity at this type of terminal.

111. Differential Synaptic Performance

A. TYPES OF SYNAPSE In order to discuss the phenomena of synaptic plasticity it is first necessary to review briefly the different types of crustacean neuron and their associated synapses. More extensive reviews have appeared previously (Atwood, 1976, 1982). Crustacean motor neurons can be broadly classified as phasic or tonic according to the patterns of impulse activity they normally exhibit. Phasic motor neurons are typically silent most of the time and are recruited for production of rapid muscular responses. Examples include the motor neurons supplying the fast abdominal flexor and extensor muscles in crayfish and lobsters (Kennedy and Takeda, 1965a).Tonic motor neurons, in contrast, maintain a low level of intermittent activity much of the time,

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which speeds up when more powerful muscular responses are required. The neurons supplying the slow (or tonic) abdominal flexor and extensor muscles of crayfish and lobsters are of this type (Kennedy and Takeda, 196513). So, too, are many of the motor neurons supplying decapod limb muscles. However, some of the limb muscles receive both phasic and tonic motor neurons (the classical “fast” and “slow” axons of the claw closer muscles of crayfish and lobsters being the best known). The phasic (fast) neuron evokes rapid muscular responses which soon fatigue, while the tonic (slow) neuron evokes slower muscular responses which are very resistant to fatigue. The differences in muscular response are due in part to the connectivity patterns of the motor axons within the muscle. Limb muscles often contain fast-acting and slow-acting muscle fibers with characteristic membrane properties, myofibrillar proteins, and intrinsic contraction rates (Atwood, 1972). Phasic axons tend to innervate the fast-acting muscle fibers more heavily, while tonic axons preferentially supply the slow-acting muscle fibers. Properties of synapses associated with phasic and tonic axons are also very important for muscular performance. When the excitatory postsynaptic potentials (EPSPs)set up by a phasic motor neuron are measured by intracellular electrode in a responding muscle fiber, one usually sees a substantial electrical event (often several millivolts in amplitude) during the initial stages of stimulation. The EPSP is large enough in many fibers to trigger an action potential (with a resulting twitch contraction). However, the response fades rapidly with maintained stimulation, even at rather low frequencies (1-5 Hz). Thus, the diagnostic hallmarks of phasic synapses are relatively high initial output of transmitter and rapid depression of transmission with repeated activity. EPSPs set up by a tonic motor neuron are often very small in amplitude (less than 1 mV) for the first few impulses of a train. However, this is not invariably the case: some fibers within a muscle may show quite large EPSPs for the same axon. Several investigations have shown that the difference is linked to low-frequency output of transmitter, and it is possible to distinguish, for tonic motor neurons, “high-output’’ and “lowoutput” synapses supplying different muscle fibers (review Atwood, 1982)’ High-output synapses appear to release more transmitter per

‘

In a few recent papers, high-output and low-output synapses of the tonic crayfish opener-stretcher motor neuron have been referred to as fast and slow synapses, respectively (Parnas et al., 1982d). This terminology seems confusing, since none of the synapses of the opener-stretcher motor neuron are like those of the phasic (fast) motor neuron of the claw closer muscle.

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impulse at low impulse frequencies and show less short-term facilitation and more rapid depression than low-output synapses. But neither type shows the very rapid depression characteristic of phasic synapses. Comparative morphological studies have been carried out on synaptic terminals of phasic and tonic motor neurons (Atwood and Jahromi, 1977; Hill and Govind, 1981). Such studies may help ultimately to explain some features of synaptic performance. A diagrammatic overview of the main features is given in Fig. 7' . Terminals of phasic axons are usually filiform without pronounced varicosities. Mitochondria1 content of the terminals is rather low, as is the relative number of synaptic vesicles. Numerous well-defined and typically uniform synaptic contacts appear on the terminal (Atwood and Jahromi,

1977). Terminals of tonic axons are typically varicose with pronounced enlargements and bottlenecks. Mitochondria are larger and more numerous than in phasic axon terminals, and synaptic vesicle populations are larger. Low-output terminals have more individual synaptic contacts per unit length of terminal than do their high-output counterparts. Individual synaptic contacts average larger in high-output terminals (Atwood and A

y-

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FIG.7. Three types of synaptic terminal described for phasic (A) and tonic (B) motor axons. High-output and low-outputterminals are often associated with different branches of the same tonic motor axon.

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Marin, 1983). The presynaptic dense bodies (thought to be counterparts of the vertebrate active zone) are larger and more complex for some highoutput synapses. B. PHYSIOLOGICAL DIFFERENTIATION It is important to note at the outset that the mechanisms responsible for the various forms of synaptic plasticity are very likely present to at least some degree at all of the different crustacean synapses. Thus, they all probably have the potential to exhibit short- and long-term facilitation, depression, etc. The net effect actually observed for a given stimulus presumably depends upon the membrane channels, morphology, and metabolic properties of the synaptic terminal. From this general postulate, we can make some inferences about the important factors that determine performance of the rather different types of crustacean synapse. The classical picture of the transmitter economy of the nerve terminal (Fig. 8B) emphasizes the existence of a readily releasable pool of transmitter, which can be identified as a subfraction of the synaptic vesicles at synapses (Fig. 8A). Calcium channels are postulated to be associated closely with the active zones of the synapses. Adjacent synapses on a nerve terminal are usually separated from each other by glial cell processes (Fig. 8A). 1. Phasic and Tonic Synapses The morphological features of phasic nerve terminals and the observed rapid depression of transmitter output with repetitive stimulation suggest substantial depletion of the readily available transmitter pool with each impulse and relatively slow replenishment. Depression at frequencies of 4 Hz and above probably involves depletion of transmitter (Bryan and Atwood, 1981).Lower supplies of mitochondria and perhaps of metabolic enzymes in phasic terminals will decrease the availability of transmitter in the metabolic pool (Fig. 8B) and lead to more rapid depression. In such synapses, depression will mask short-term facilitation, even though the mechanism for the latter is available. The initial high output of transmitter at phasic nerve terminals can be partly accounted for by the observation that they appear to be capable of conducting an action potential to the synaptic release zones (Dudel et al., 1984). In contrast, there is now good evidence that many of the terminals of the more tonic crayfish opener motor axon are inexcitable (Dudel, 1982,1983a).In phasic axons, a full-fledged action potential reach-

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A

B ~

Metabolic Pool of Transmitter I

L

:tion and removal

FIG. 8. T h e morphology (A) and transmitter economy (B) of a crustacean nerve terminal. (A) A large synaptic terminal of the excitatory motor axon in the stretcher muscle of the lobster (Hornarm) possesses numerous discrete synapses (S), some of which are supplied with clusters of synaptic vesicles (V) while others are not. Synapses are generally separated by glial cell processes (G). The terminal contains numerous mitochondria (M). (After Fritz et al., 1980.) (B) The classical view of transmitter mobilization from a metabolic pool and its release by entry of calcium (C&) through specific channels in the synaptic membrane of the nerve terminal.

ing the synapsewill maximize calcium entry and hence transmitter release. Although the amount of transmitter released by a single impulse may be close to the saturation level, there is nevertheless some ability to augment release by posttetanic potentiation (Parnas and Atwood, 1966;Lnenicka and Atwood, 1985b; Fig. 20). Short-term facilitation is present in

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phasic terminals, but it decays more rapidly than in tonic terminals of the same muscle (Linder, 1973). No good explanation of this observation is presently available. T h e phenomenon of low-frequency depression associated with phasic motor axons (Zucker and Bruner, 1977; Bryan and Atwood, 1981; Lnenicka and Atwood, 1985b) remains an enigma. Depression occurs after a single impulse and levels off rapidly with additional widely spaced impulses (see Fig. 20). It cannot be explained by a depletion process (Zucker and Bruner, 1977). A tentative hypothesis is that some of the available synapses become inactivated after one or a few impulses and are effectively removed from the responding pool of synapses. This implies inequality of the individual synapses along the nerve terminal. An hypothesis similar to this was proposed to explain features of depression at the frog neuromuscular junction (Betz, 1970). However, there is presently no concrete evidence for such a mechanism. Calcium-dependent inactivation of calcium channels (Eckert and Chad, 1985) at some synapses but not others would explain the result, but two types of calcium channel seem necessary for this. 2. High- and Low-Output Synapses of Tonic Axons Tonic motor axons such as that of the crayfish opener muscle show transmitter release for a single nerve impulse that is much larger for some terminals than for others (Atwood, 1982). With repetitive stimulation, transmitter release reaches a maximal value at much lower frequencies for the high-output terminals (Bittner, 1968; Fig. 9). However, when the frequency of stimulation is increased sufficiently, the transmitter output for a muscle fiber innervated by low-output terminals can be driven u p to a level comparable to, or even exceeding, that seen for a fiber innervated by high-output terminals. Thus, the maximal rate of transmitter release may be comparable for the two types of terminal, but the rate of transmitter released by a single nerve impulse or by a short train is greater for the high-output terminal, release is closer to saturation, and a larger fraction of the immediately available store of transmitter is utilized. Why does a single impulse release more transmitter at the high-output terminal? T h e possibilities include (1)differences in excitabilityor of spike invasion in the terminals, (2) differences in synaptic morphology and active zones, (3) differences in resting calcium concentration or cooperativity of calcium in the release process. a . Ex~ztabilityor Spike Invasion. T h e hypothesis of nonexcitable synaptic terminals in the crayfish opener excitor axon was developed originally by Dudel (1965a) on the basis of observations of the extracellularly recorded nerve terminal potential and failure to excite action potentials

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FIG. 9. Frequency-dependent postsynaptic depolarization produced by high-output and low-output terminals of the motor axon to the crayfish opener muscle. (From Bittner, 1968.)The maximal effect is reached at lower frequencies by high-output terminals, but the maximal attainable effect is greater (at high frequencies) for the fibers supplied by low-output terminals.

by focal stimulation. Subsequent work by Zucker (1974a)provided evidence for focal elicitation of action potentials by stimulation applied through microelectrodes at transmitter-releasing terminals. In addition, some of the original interpretations of nerve terminal potentials are probably erroneous (Ortiz, 1972).More recently, however, applicationof loose-patch microelectrodes for recording and stimulating has provided consistent evidence for nonexcitability of most of the terminals in the crayfish opener muscle (Dudel, 1982, 1983a). Proximal (high-output) terminals of the crayfish opener muscle apparently experience a larger potential change during an action potential than distal (low-output) terminals of the same axon (Dudel, 1983a), even though both types are usually inexcitable when tested by pulses applied through a loose-patch electrode. In response to a single impulse, proximal terminals release transmitter that is equivalent in amount to that produced by a large pulse applied directly to the terminal. In contrast, distal synapses, for a single impulse, release 5-10% of the transmitter that can be generated by a large direct pulse. From this, it appears that the proximal synapses are operatingcloser to the saturation level for transmitter release for a single impulse. This could occur if they experience a larger potential change during a single impulse (Dudel, 1983a),as originally proposed by Atwood and Bittner (1971) and by Sherman and Atwood (1972). If a larger potential change is experienced by high-output synapses, the entry of calcium for a set of equivalent calcium channels should be

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greater. If cooperativity of calcium in transmitter release is the same, the terminals gaining more calcium after an impulse would be expected to release more transmitter and to attain a release rate closer to the saturation level (Fig. 10). Results of experiments in which nerve terminal potentials were broadened by treatment with Cs+ lend qualitative support to this hypothesis. High-output terminals and low-output terminals both increase transmitter output for a single impulse, but the relative increase at low-output terminals is far greater (Atwood and Lang, 1973). The low-output terminals thus appear to operate normally far below their maximal level of transmitter release, while high-output terminals are closer to the saturation point at low frequencies of stimulation (Fig. 10). b. Synaptic Morphology and Active Zones. The observation that individual synapses of high-output terminals are, on average, larger than those of low-output terminals of the same axon (Sherman and Atwood, 1972) stimulated a number of studies in which synaptic morphology was compared. T h e relatively small active zones of crayfish opener muscle excitatory synapses Uahromi and Atwood, 1974)were found to occur, on average, one per synapse, with some synapses having none and others (relatively few) having two or three. Clusters of synaptic vesicles were usually found at active zones (Fig. 8), but were absent at synapses that did not possess

Saturation W v)

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INTRACELLULAR Ca2+ (Relative units) FIG. 10. A simplified possible interpretation of transmitter release by a nerve impulse at low-output and high-output terminals. Both are assumed to operate with a fifth power relationship between intracellular calcium and transmitter release. Entry of calcium is postulated to be greater at high-output synapses with consequent closer approach of transmitter release to the saturation level.

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an active zone. Inhibitory terminals, known to have a higher quanta1 output of transmitter than excitatory synapses on a given muscle fiber (Atwood and Bittner, 198l), had more active zones per synapse, (Jahromi and Atwood, 1974) and often, individual active zones were of larger size (Atwood and Kwan, 1976). In high-output and low-output terminals of the accessory flexor muscle in the American lobster, individual active zones were found to be larger in the former type (Govind and Chiang, 1979; Govind et al., 1982).While this was also true in comparable terminals of a crab muscle, there was no simple correlation between the number and size of active zones and transmitter output at low frequencies of stimulation (Atwood and Marin, 1983). The general picture emerging from ultrastructural comparison of low- and high-output terminals is that low-output terminals have more synapses per unit length of terminal than high-output synapses,but fewer and smaller active zones per synapse (Atwood and Marin, 1983).Presently, it is not known whether the larger active zones have more calcium channels, or whether the calcium channels are differently spaced. Observations on putative calcium channels at active zones of arthropod synapses are just beginning to appear (Rheuben, 1985). In crab muscles, a morphological difference at the macroscopic level has been recently discovered by Tse (1986). High-output terminals often have a palmate configuration with terminal boutons radiating out from the point of contact of the main axon branch with the muscle fiber to supply a relatively dense array of boutons in a restricted area of innervation. Low-output terminals of the same axon have the better known linear and more diffuse configuration (cf. Florey and Cahill, 1982),which provides a less concentrated array of boutons over a larger area of the muscle fiber’s surface. The more concentrated configuration of the highoutput terminal could serve to promote the electrical effect of an action potential in the boutons, which are possibly inexcitable. c. Resting Calcium Concentration or Cooperativity. Quanta1 release of transmitter and short-term facilitation were compared in high-output and low-output terminals of the crayfish opener muscle by Parnas et al. (19826). The magnitude and kinetics of short-term facilitation provided data which could be used in the model of transmitter release (Parnas et al., 1982a) to predict which parameters differ in the two types of terminal. Using focal stimulation of terminals with a loose-patch electrode, Parnas et al. (1982d) found a steeper dependence of transmitter release on extracellular calcium in low-output terminals and a higher maximal output in the highoutput terminals for a single pulse. Short-term facilitation was much more sensitive to external calcium concentration in low-output terminals. When parameters in the model of F’arnas et al. (1982a) were adjusted to match

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the experimental results, it was found that a higher maximal entry of calcium did not provide a good explanation of the results. Higher resting level of intracellular calcium in the high-output terminals or a higher cooperativity of calcium action in low-output synapse was chosen as more likely alternatives. Higher entry of calcium in high-output terminals was rejected because it predicts an increased slope of the relationship between extracellular calcium and release for those terminals, contrary to the experimental results. To account for the higher transmitter output, an increase in the level of release at saturation (with high external calcium) was proposed for high-output synapses. To account for the results of focal stimulation, Parnas et al. (1982d) suggested that cooperativity for calcium action in the terminal would have to be twice as great for low-output terminals (i.e., six) as for high-output terminals (i.e., three). Alternatively, resting calcium would have to be about 20 times higher in the high-output terminals. Differences in apparent cooperativity of calcium action could arise through variation in distribution of calcium channels at the active zones, as discussed above (Section 11). However, there is no concrete evidence for this as yet in morphologica1studies. The larger active zones of individual high-output crustacean synapses imply more calcium channels near the site of transmitter release. This could increase the probability of releasing a quantum of transmitter as at vertebrate phasic neuromuscular junctions (Walrond and Reese, 1985; Fig. 1) and also, according to the model of Zucker and Fogelson (1986), increase the apparent cooperativity of release by increasing the degree of overlap of calcium domains. The first effect is consistent with the physiological observations; the significance of the second effect cannot be judged on present evidence, but if present it would be contrary to the predictions of the model of Parnas et al. (1982d). A 20-fold difference in resting calcium seems unlikely. With a fourth power relationship between active calcium and transmitter release, one would see a 160,000-fold difference in spontaneous release of transmitter. In fact, the spontaneous release of transmitter in pairs of proximal and distal muscle fibers is not greatly different (Bittner, 1968),and there is no direct evidence that resting calcium concentrations vary greatly in different terminals of the same axon. d. Conclusions. The differences in terminal morphology, active zone size, and possible size of electrical event in the terminal bouton, all suggest that a single impulse causes more release of transmitter at terminals of the high-output type by causing a greater influx of calcium. This would cause the high-output terminal to perform closer to its saturation level for release (Fig. 10) and would explain such features as the smaller extent of short-term facilitation. The slower decay of short-term facilitation in

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high-output terminals (Linder, 1974)could be accounted for by less rapid kinetics of calcium removal. Results for “strong”and “weak”frog neuromuscularjunctions (Pawson and Grinnell, 1984, 1985) are in accord with this view. Strong junctions exhibit a higher rate of spontaneous quantal transmitter release and a higher quantal content of impulse-evoked release. The difference in spontaneous release disappears in low-calcium solution. Leakage of calcium through calcium channels is a possible cause for the difference in spontaneous release, and a larger impulse-linked calcium entry can account for the difference in evoked transmitter release. Recent morphological evidence corroborated the view that there are more calcium channels at the strong junction (Propst and KO, 1985). We propose that the larger number of individual synapses on lowoutput crustacean terminals (Govind et al., 1982;Atwood and Marin, 1983) provides a situation in which more short-term facilitation can arise through recruitment of additional synapses during repetitive activity (Fig. 9). Thus, a single impulse recruits a greater percentage of available synapses on high-output terminals, through combined electrical and morphological properties of the terminal, and places transmitter output closer to saturation. This leaves less room for plastic changes such as short-term facilitation in the high-output terminals.

IV. Short-Term Facilitation

Crustacean neuromuscular synapses of tonic motor axons present an ideal system for study of short-term facilitation. At moderate frequencies of impulse production, there is little competition from depression to obscure facilitation. Additional processes, such as augmentation and potentiation (characteristic of frog neuromuscular junction: Magleby, 1973a,b) or long-term facilitation (Section VI), do not become prominent during short trains of impulses. One way in which short-term facilitation can be studied is to present two impulses with variable spacing and to observe the time course of decline in amplitude of the second response in comparison with the first (“twin-pulse facilitation”). A second method is to study the growth in amplitude of the EPSP during short trains of impulses at various frequencies (“short-train facilitation”).Still a third method which has been recently employed is to apply local stimulation to the nerve terminal with a macropatch electrode (F’arnas et al., 1982a-d). This method allows adjustment of pulse width and amplitude in twin-pulse experiments; but, as

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noted in Section XI, difficulties of interpretation can arise due to lack of information about the values of membrane potential in the stimulated terminals. An advantage of using macropatch electrodes to measure transmitter release is that counts can be made of the number of quanta1 units liberated by each impulse. This provides a more exact picture of transmitter release than obtained by intracellular measurements of the EPSP. Facilitation measured by any of these methods is usually taken as the ratio of the amplitude of the EPSP or number of quanta for the second or subsequent impulse or pulse in relation to the first. A variant approach is to take the proportional change for the second or subsequent event in relation to that of the first impulse or pulse. Kinetic properties of facilitation that can be derived from these measurements include ( I ) the magnitude of facilitation following one impulse, (2) the rate of decay following stimulation, and (3) the manner in which facilitation accumulates with successive impulses. Synapses of the crayfish opener muscle (particularly those of the lowoutput type) exhibit very pronounced facilitation in response to a train of stimuli. Growth of the EPSP is frequency- and time-dependent with marked differences among synapses (Fig. 9). There have been several types of explanation advanced over the years to account qualitatively and quantitatively for the growth and decay of short-term facilitation (Zucker, 1982). These fall into three main categories. 1. Changes in electrical events in the nerve terminal. 2. Residual calcium in the nerve terminal. 3. Progressive recruitment of calcium channels. We shall consider each of these briefly in turn in light of recent work.

A. POSSIBLE MECHANISMSOF FACILITATION 1. Changes in Electrical Events

The idea that spike amplitude or afterpotentials might change with repetitive activity to effect facilitation was proposed for crayfish neuromuscular junctions by Dudel (1965a). Extracellular records at synaptic foci suggested that nerve terminal potentials increased in amplitude during facilitation. Subsequent work has shown that such recordings contain an artifact (Ortiz, 1972). Occurrence of a depolarizing afterpotential following the impulse was established by Zucker (1974a), but shown not to be responsible for facilitation.

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lntracellular recordings from synaptic terminals of both squid (Charlton and Bittner, 1978) and crayfish (Wojtowicz and Atwood, 1984) have shown clearly that small spike amplitude changes do occur, but that they do not follow a pattern that can be used to explain facilitation. In crayfish, the peak voltage attained by the action potential is actually reduced after a short train (Fig. 13 and 16), largely due to the emergence of a hyperpolarizing change in the membrane potential (activationof an electrogenic sodium pump). The depolarizing afterpotential associated with a single impulse becomes a hyperpolarization after a few closely spaced impulses (Baxter and Bittner, 1984), but facilitation proceeds regardless of these changes. It must be concluded that voltage changes per se have, if anything, a small effect which is submerged by more significant events. In passing, it can be noted that both the hyperpolarization and the reduced peak voltage attained by the action potential after a few impulses would be predicted to reduce transmitter release, according to the voltage hypothesis (Section 11; Dudel, 1984b). A voltage effect, if present, is not dominant.

2. Residual Calcium The proposed nonlinear relationship between active intracellular calcium and transmitter release (Section 11) provides the basis for residual calcium models of facilitation (Katz and Miledi, 1968).A very small residuum of active calcium could provide enhanced release of transmitter with a subsequent impulse. In the squid giant synapse, direct measurements have shown that calcium ion entering the terminal persists long enough to account for facilitation (Charlton et al., 1982). This is a necessary condition for the residual calcium hypothesis. Recent measurements (Dudel, 1981)have shown that the relationship between external calcium and transmitter release is nonlinear for crayfish terminals, rather than linear as proposed previously (Bracho and Orkand, 1970; Zucker, 1974b).Since cooperativity of calcium action within the terminal is probably greater than can be observed from dependence of transmitter release on external calcium (Fig. 6), a residual calcium mechanism could very well describe facilitation. Indeed, the most recent models operate under this assumption (Parnas et al., 1982a; Zucker and Lara-Estrella, 1983). The most straightforward version of the residual calcium model, developed for frog neuromuscularjunctions (Zengeland Magelby, 1981)and then modified and adapted for crayfish motor terminals, is illustrated in Fig. 11, from the work of Zucker and Lara-Estrella (1983). Each increment of active calcium generated by an impulse decays exponentially with time, but can contribute to total available intraterminal calcium; closely spaced impulses can build up the intraterminal calcium well above resting values.

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FIG. 11. The residual calcium hypothesis, as applied to the crayfish opener nerve terminal by Zucker and Lara-Estrella (1983). Each impulse (arrow below abscissa) contributes a set amount of "entry calcium" (Ca,) to the normal resting level (Ca.). When impulses are closely spaced, residual calcium (Ca,) builds up in the terminal.

Calcium entering at a time when residual calcium has been built up by preceding impulses would evoke an enhanced phasic release of transmitter. Magnitude of transmitter release would be predicted from a power function of active calcium concentration. So also would increased spontaneous release of transmitter, if it is influenced by active calcium. Zucker and Lara-Estrella (1983) found that both facilitation of the EPSP and enhanced frequency of spontaneous miniature potentials seen after a short train of impulses decayed with two time constants. The magnitude and time course of the facilitatory effects on these two manifestations of transmitter release differed, but when a value for calcium cooperativity of five was adopted, along with time constants of decay estimated from the data on spontaneous miniature potential frequency, a good (but not perfect) fit was obtained for magnitude and time course of EPSP facilitation. Exact values for some of the parameters (such as resting and entry calcium) are not known, and this, together with other assumptions, could easily distort the exactitude of the modeling. Entry of calcium, cooperativity of calcium action inside the terminal, and processes involved in removal of active calcium will all contribute to the magnitude and duration of short-term facilitation. A comprehensive attempt to model all of these processes with assigned parameters was developed by Parnas and Segel (1981) and Parnas et al. (1982a), as de-

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scribed previously (Section 11).Experimentally,Parnas et al. (1982a)found that the time course and magnitude of facilitation are sensitive to external calcium especially at concentrations below that at which entry of calcium is thought to saturate. The effects of altered external calcium on facilitation were modeled by equations incorporating principles of saturation kinetics, since best fits to experimental data were produced when entry, removal, and transmitter release were all assumed to be saturable. The importance of removal processes in controlling the duration of facilitation was emphasized in studies of facilitation in which external sodium (Parnas et al., 1982b) or magnesium (Dude1 et al., 1982) were varied. Reduction of Na prolonged facilitation, suggesting that Na-Ca exchange acts as one removal process. Prolongation of facilitation by elevated Mg was also thought to reflect perturbation of Na-Ca exchange (Parnas et al., 1982b,c). The biphasic time course of decay of facilitation immediately following an impulse can be explained (at least in the squid giant synapse) by diffusion of entry calcium away from release zones (Zucker and Stockbridge, 1983). In this system, there is no need to postulate an additional process in order to get the observed early decay of facilitation. However, the squid terminal may not be a perfect model for that of the crayfish or crab. It has been found, for example, that the two phases of decay of short-term facilitation are differentially affected by changing the temperature (Stephens and Atwood, 1983). The more restricted volume of the crustacean bouton could well necessitate additional removal processes to maintain calcium homeostasis, although diffusion from the release zones could turn out to account for at least the initial phase of decay On the other hand, Linder (1974) postulated from his kinetic studies that the same process was involved in both early and late phases of decay in the crayfish opener muscle. Invariance of time course of decay over a wide amplitude range for facilitation favors diffusion as a possible mechanism. Further study of possible mechanisms responsible for decay of facilitation in crustacean terminals is warranted to sort out the unsolved problems. Problems for the simple residual calcium model are posed by studies in which attempts were made to model the growth of facilitation during a train of impulses (Bittner and Schatz, 1981). Models based upon linear summation of increments of facilitation remaining after each impulse in a train, or upon a power law, cannot account for the observed growth of synaptic potentials during repetitive stimulation (Bittner and Sewell, 1976). The assumption of a constant amount of calcium entering with each impulse could not readily account for the observed growth of facilitation with long trains of impulses. However, modifying the model to incorporate a progressive increase in calcium influx produced a satisfactory fit to the data (Bittner and Schatz, 1981).

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3. Progressive Recruitment of Calcium Channels Zucker (1974b)suggested that facilitation in the crayfish opener muscle could be explained by progressive increase in calcium entry for successive spikes. Although direct measurements of calcium entry at the squid giant synapse showed that this mechanism does not occur there (Charlton et al., 1982), it has recently been found in bovine adrenal chromaffin cells (Hoshi et al., 1984). Since the squid giant synapse is physiologically quite different from the crayfish neuromuscular synapse and since various calcium channels can be quite diverse in their gating properties (Tsien, 1983),we cannot rule out the possibility that calcium channels are progressively recruited or that more than one type of calcium channel may exist at the crustacean synapse. A recent report on mouse neuromuscular junction (Mallart, 1985)has raised the possibility that the prominent calcium-activated potassium current may modulate facilitation. The repolarizing potassium current is depressed by successivestimuli, perhaps by channel inactivation. Enhancement of calcium current results. Such an effect could account for the observed growth of facilitation in crayfish terminals if the same channels were present.

B. STATISTICAL PARAMETERS OF FACILITATION Following the discovery that release of transmitter at the crayfish neuromuscular junction could be best described as a binomial statistical process at low frequencies of stimulation (Johnson and Wernig, 1971), several studies were conducted to examine changes in the statistics of transmitter release with short-term facilitation. The binomial distribution incorporates a mean probability of response, p, for a fixed number of responding units, n . The physical meaning to be attached to these parameters has been discussed in several papers on crustacean neuromuscular junction (Zucker, 1973; Wojtowicz and Atwood, 1986) and other systems. Zucker’s (1973) suggestion, that n might represent the number of responding release sites at the crustacean neuromuscular junction has been, since strongly supported by combined morphological and physiological studies of another system, the inhibitory input to the Mauthner cell of fish (Korn et al., 1982). There, the number of synaptic boutons of individual inhibitory neurons matches n of the computed binomial distribution. Further, each bouton can be assumed to behave in all-or-nothing fashion for each impulse (releasing either one quanta1 unit, or none).

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We have already suggested (Section 11) that for a nerve terminal of the crayfish opener muscle, each of the numerous small synapses could behave as a unit generating one quantal unit of release. This view would bring the crayfish nerve terminal into line with the findings in the Mauthner cell. The probability p is assumed to be uniform across the responding population in the simple binomial model. Zucker (1973)suggested that p could be a compound probability, representing a product of the probabilities of two processes, tentatively identified with the rate of vesicular reoccupation of release sites following an impulse, and the probability that a primed site will be activated by an impulse. The latter event was thought to be responsible for observed alterations in the compound p, since the former event was believed to occur with relatively constant high probability Recent work on vertebrate central synapses (Redman and Walmsley, 1983)and those of the fish Mauthner cell (Korn et al., 1986) suggests that response probability of individual synapses or boutons is very likely not uniform in most real situations. Instead, individual responding units may vary in their response probabilities with at least some being normally completely unresponsive ( p = 0). This view accords well with the morphological picture of the crayfish nerve terminal: active and inactive synapses are inferred from their morphological appearance (Jahromi and Atwood, 1974; Atwood et al., 1978; Fig. 8). Determinations of n and p during short-term facilitation have not given uniform results. Wernig (1972a) found that at frequencies up to 5 Hz, n increased, while above 5 Hz, p increased. Zucker (1973) found a facilitatory increase only in p. More recently, Hatt and Smith (1976) and Smith (1983) have established that nonuniform probabilities of release may occur during growth of facilitation produced by a train of impulses. Later in the train a more stationary situation exists. Facilitation was associated with increase in n for frequencies of stimulation up to 10 Hz and with increase mainly in p at higher frequencies. Both Wernig (1972b) and Dude1 (1981) have reported that the higher quantal contents obtained by raising external calcium are attributable to an increase in p. The general picture that emerges from this work can be summarized by stating that the crustacean nerve ending possesses a number of potential release sites (synapses) of unequal potency, that facilitation (due to elevated intraterminal calcium) acts to recruit release sites (increase in n), and that, additionally,average probability of release at individual synapses rises with higher intraterminal calcium concentrations. The residual calcium mechanism may account for many aspects of the time course of

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facilitation, but in addition, growth of facilitation probably involves recruitment of synapses (“responding units”). Whether the differences between easily recruited and poorly recruited synapses reside in their complement of calcium channels or even in different types of calcium channel has yet to be determined.

V. Presynaptic Inhibition

A. INTRODUCTION Modification of excitatory transmission by inhibitory neurons is an important form of short-term plasticity in crustacean neuromuscular systems. Functionally, presynaptic inhibition can be thought of as the inverse of short-term facilitation; but unlike the latter, it requires a second participating neuron. Most crustacean muscles receive innervation from peripheral inhibitory neurons, all of which inhibit muscle fibers directly. The inhibitory transmitter substance (y-aminobutyric acid or GABA) acts on receptors at the inhibitory neuromuscular junction (Fig. 12), and the resulting increase in chloride conductance (gcJ directs the membrane potential of the muscle fiber toward the chloride equilibrium potential (Fatt and Katz, 1953). Increased chloride conductance creates a countercurrent which opposes depolarizing electrogenesis of action potentials in normally spiking muscle fibers (Parnas and Atwood, 1966). In addition, the reduced membrane time constant renders summation of excitatory postsynaptic potentials less effective. All of these effects inhibit contraction of muscle fibers. Some of the limb muscles of decapod crustacean are known to undergo presynaptic inhibition when impulses in the inhibitory axon closely precede those in an excitatory axon (Dudel and Kuffler, 1961b). Presynaptic inhibitory influences may be exerted both in the central nervous system (where they inhibit impulse production by an excitatory axon; Wiens, 1982) and at the periphery ihrough axo-axonal synapses (Atwood and Morin, 1970). The peripheral effect is known to involve reduction of the quantal output of excitatory transmitter substance; that is, each excitatory impulse releases fewer quantal units of transmitter when the inhibitory axon is firing appropriately and the quantal units do not change in size (Dudel and Kuffler, 1961). Effects of the inhibitory transmitter on excita-

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i Muscle FIG. 12. Receptor complement of excitatory (EX) and inhibitory (IN) nerve terminals of crayfish opener muscle. (After Atwood, 1982.) Pre- and postsynaptic receptors are shown for the inhibitory neurotransmitter GABA and for circulating neurohormones octopamine (Oct) and serotonin (5-HT). Postsynaptic receptors are indicated for the excitatory neurotransmitter (glutamate) and for the putative neurohormone doparnine (DA).

tory postsynaptic receptors have been ruled out by these observations. . ~ ~ . ~ _ Usually the presynaptic effects of a single inhibitory nerve impulse are maximal 1-3 msec after its arrival at the axon terminal and wear off completely in another 5-10 msec (e.g., Wiens and Atwood, 1975). This indicates that inhibitory synaptic transmission and subsequent removal of the inhibitory transmitter from the axo-axonal synapse are the processes that limit the time course of the presynaptic effect. Following a train of inhibitory impulses, a longer lasting aftereffect is sometimes observed (Rathmayer and Florey, 1974).The mechanism is not known, but saturation of removal processes is a possibility. An additional tonic effect, possibly due to nonquantal or molecular release of inhibitory transmitter, was described at crab neuromuscular junctions by Parnas et al. (1975) and was thought to alter the dependence of excitatory transmitter release on external calcium. These observations were not replicated in crayfish (Staggs et ad., 1980); however, the experiments are disputed (Parnas and Dudel, 1982).The functional significance of tonic inhibition remains uncertain. ~

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B. MECHANISMSOF PRESYNAPTIC INHIBITION The primary event in presynaptic inhibition appears to be action of GABA on receptors of the excitatory terminals. Presynaptic GABA receptors differ pharmacologically from postsynaptic receptors of the same muscle (Dudel and Hatt, 1976); but the main functional event, opening of membrane chloride channels, is apparently similar (Takeuchi, 1976). Such an action could reduce the release of transmitter substance by one or more of the following mechanisms: 1. Hyperpolarization or depolarization of the excitatory nerve terminal. 2. Blockage of impulse conduction in branches of the excitatory axon. 3. Reduction in amplitude of the excitatory action potential. 4. Electrical isolation of terminal boutons from the influence of the action potential. In addition, another possibility not related to chloride channel activation, namely, inactivation of synaptic calcium channels has been suggested in other systems (Klein and Kandel, 1980) and must be considered. We shall examine each possibility in turn. 1. Membrane Potential Change

Presynaptic inhibition of central processes in both vertebrate (review Nicoll and Alger, 1979) and invertebrate nervous systems (Kirk and Wine, 1984) is accompanied by depolarization of the inhibited terminals (often referred to as primary afferent depolarization, or PAD, when it occurs in terminals of sensory axons). Such depolarization could reduce transmitter release by inactivating axonal sodium channels and reducing the action potential. In fact, the action potential does get smaller under such circumstances. Such a mechanism is unlikely to be of any significanceat the crustacean neuromuscular junction. The effects of depolarizing or hyperpolarizing the nerve terminal during passage of an action potential have been studied directly (Wojtowicz and Atwood, 1983, 1984). Depolarizing the nerve terminal reduces the action potential, as expected, but there is a large increase, rather than a decrease, in the output of transmitter (Fig. 13). Conversely, hyperpolarizing the terminal increases the action potential, but reduces transmitter release. These findings are paralleled by observations on the modulating effect of depolarizing and hyperpolarizing pulses at the frog neuromuscular junction (Dudel, 1984b). Intracellular recordings from the excitatory nerve terminal in the crayfish opener muscle have shown that either depolarizing or hyperpo-

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.ii-2 0 msec

r B

L

a

1

FIG. 13. Effects of hyperpolarizing or depolarizing a nerve terminal of the crayfish opener motor axon. (From Wojtowicz and Atwood, 1984.)The presynaptic action potential (a) is increased in amplitude by hyperpolarization (B), but the EPSP recorded in the muscle fiber (in) is reduced. Conversely, depolarization reduces the action potential but increases the EPSP substantially (C).

larizing shifts in membrane potential can occur during presynaptic inhibition or GABA application (Baxter and Bittner, 1981). In either case, reduction of transmitter output occurs. Thus, membrane potential changes induced by inhibitory transmitter action are not in themselves an important feature of the mechanism.

2. Blockage of Impulse Conduction There have been a few direct demonstrations of intermittent failure of impulse conduction in branches of an excitatory motor axon during presynaptic inhibition (e.g., Dudel, 1965~).These events probably occur in subterminal branches rather than in terminal boutons. However, extracellular recordings along nerve terminals of crabs in which presynaptic inhibition is intense (Atwood and Bittner, 1971) have not shown many examples of such blockage; usually, the nerve terminal potentials do not change much in shape or amplitude (Tse, 1986). Increased chloride conductance, especially at or near bottlenecks and branch points, could create

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conditions that enhance the probability of impulse conduction block (Spira et al., 1969, 1976). Although some axo-axonal synapses occur at bottlenecks and branch points of the excitatory axon Uahromi and Atwood, 1974), the majority occur on terminals (Smith, 1978; Atwood et al., 1984). Impulse blockade could account for some cases of presynaptic inhibition in which transmitter release is almost eliminated and excitatory facilitation disappears (Wiens and Atwood, 1975). In many cases, however, excitatory facilitation persists during inhibition in spite of a substantial reduction of transmitter output (Atwood and Bittner, 1971), implying continued presence in the terminal of the action potential or its electrotonic residuum. Besides, definitive proof for elimination of the action potential has not been obtained even for the cases in which excitatory facilitation is eliminated.

3. Reduction of the Excitatoy Action Potential impalement of preterminal axon processes in the crayfish opener muscle has permitted direct demonstration of a reduction in the amplitude of the excitatory action potential (Baxter and Bittner, 1981). This corroborates earlier studies with focal extracellular electrodes (Dudel, 1965c; Takeuchi, 1976).The reduction in amplitude is attributed to increased gcl, as in the muscle fiber, and not to a change in membrane potential per se. Reduction of transmitter output at synapses could thus be due in part to the reduced amplitude of the excitatory action potential, given the voltage sensitivity of the release process. However, the reduction in amplitude of the action potential in the crayfish axon and in central interneurons (Glantz et al., 1985)is only about 576, yet the accompanying reduction of transmitter output is 50-60% in the published records. There are two reasons to suspect that the recorded reduction of the action potential does not entirely explain presynaptic inhibition. First, in cases in which a depolarized membrane potential occurs during inhibition, an increase in transmitter release would be expected even though the action potential is reduced (Wojtowicz and Atwood, 1983, 1984; Fig. 13). Secondly, since calcium entry leading to evoked transmitter release is actually a “tail current” following the action potential, there may not be a large reduction in calcium entry with a 5% reduction in the peak of the action potential, since in this voltage range gc, would not be much altered (see Fig. 4). It seems likely that an additional mechanism is involved. 4. Electrical Isolation of Terminal Boutons

The electrical changes recorded in the axon may be much less than those occurring in terminal boutons, where many of the inhibitory axoaxonal synapes occur. In tonic motor axons, the terminal structure is

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highly varicose and terminal boutons are joined to the preterminal axon branch by narrow bottlenecks of variable diameter (Florey and Cahill, 1982). This varicose structure is particularly evident in terminals subjected to intense presynaptic inhibition (Tse, 1986). Although membrane potential changes cannot yet be recorded from terminal boutons, an electrical model can be formulated, using membrane electrical parameters measured in axons. If the terminals of tonic motor axons are electrically inexcitable (Dudel, 1982, 1983), the boutons probably do not experience a regenerative action potential, but rather a passively propagated waveform slightly smaller than the action potential. When an inhibitory axo-axonal synapse is active on a terminal bouton, the increase in gClattributed to one quanta1 unit of inhibitory transmission (Finger and Stettmeier, 1981) is sufficient to cause a large decrease in the voltage of the bouton (Atwood et al., 1984; Fig. 14). The attenuation increases when the diameter of the connecting bottleneck is smaller. The model predicts that the extent of presynaptic inhibition is dependent on terminal morphology and on number and distribution of

5

40-

2 0-

flection caused by an action potential in the main axon branch (M) is attenuated in the nonexcitable bouton when inhibitory synapses 2 or 3 are activated (curves c and d), but not when a synapse (1) in the main axon is activated (curve b). Without inhibition, the bouton is isopotential with the main

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axo-axonal synapses. The variable strength of inhibition along individual axon terminals could be explained by these features. In addition, strategic placement of inhibitory axo-axonal synapses could reduce transmitter outputof-seme excitatory synapses (those on inhibited terminal boutons) almost to zero, while allowing transmission at other synapses (those on preterminal axon branches) to proceed almost normally. The result would be fewer active synapses participating in transmission during inhibition, and this would be seen physiologically as a reduction in quantal content. The reduction in probability of release should be unequally distributed among the participating synapses. Evidence for this situation has been obtained in strongly inhibited crab terminals (Tse and Atwood, 1985; Tse, 1986). Recordings were obtained with a macropatch electrode of 10-20-pm tip opening from active terminal locations, and synaptic currents were recorded with and without inhibition (Fig. 15). Binomial statistical analysis provided a good description of transmission in terms of the parameters and p . Parameter n was

Synaptic current of excitor axon during preaynaptic inhibition

n = 1 p.0.16

Synaptic current of exeitor axon n.16

p=O.99

I

1

0

5

10

1

1

1

15

1

1

1

20

Number of quantal units FIG. 15. Binomial statistics of transmitter release determined by focal recording from a high-output terminal with a macropatch electrode before and during presynaptic inhibition (Tse and Atwood, 1985). T h e opener-stretcher motor axon of the shore crab fPuchygrapsw) was stimulated at 1 Hz throughout with optimally timed inhibitory stimulation added to cause presynaptic inhibition. With no inhibition, 16 putative release sites were involved, each with very high probability of release. During inhibition, the number of release sites was drastically reduced (to one), along with probability of release.

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interpreted as the number of active release sites; typically, this was found to have a value between 8 and 20 before inhibition and a lower value after inhibition. Parameter p (probability of release) decreased on average during inhibition, but it is clear that this value is not the same for all units of release (active zones) during inhibition. The analysis suggests that some of the participating synapses have their probability of release reduced almost to zero during inhibition, while others are able to transmit with reduced probability. This result is in agreement with the predictions of the electrical model (Atwood et al., 1984).

5. Reduction of Calcium Current A mechanism involving reduction of presynaptic calcium current by inhibitory transmitter action, as suggested for other systems (Klein and Kandel, 1980), seems unlikely at the crustacean neuromuscularjunction. As discussed above, vehicles of release at the excitatory synapse must be very close to the point of calcium entry, and the significant calcium channels are probably located at synaptic active zones (Fig. 12). Inhibitory axoaxonal synapse are generally located several micro-meters away from the excitatory active zones, and frequently the two synpases are separated by intervening glial cell processes which presumably take up transmitter. Yet, inhibitory synaptic action occurs with a typical synaptic delay of 1-3 sec. There is not enough time for GABA to act at a more distant synapse in time available, and one must conclude that the action of GABA following a single nerve impulse is confined to the inhibitory axo-axonal synapse.

c. PRESYNAPTIC INHIBITION AND EXCITATORY FACILITATION An apparent paradox occurs in many excitatory nerve terminals: short-term facilitation can take place during presynaptic inhibition even though transmitter output is severely depressed (Atwood and Bittner, 1971). Transmitter release often rebounds to preinhibitory levels after inhibition is relieved. Reduced transmitter release implies reduced calcium entry into synaptic boutons, according to the prevailing view; yet short-term facilitation requires residual calcium. How can full-fledged facilitation be expressed with reduced calcium entry? In squid giant synapses, it has been shown that facilitation of transmitter release can occur with rather small depolarizations of the presynaptic terminal (Charlton and Bittner, 1978). However, the absolute amount of transmitter release does not approach that evoked by a normal presynaptic spike. In the crayfish terminal, spike amplitude and terminal depolarization are undoubtedly reduced during inhibition (Baxter and Bittner,

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1981), and antifacilitation of transmitter release often occurs during a train of inhibitory stimuli (Atwood and Bittner, 1971), yet fully facilitated transmitter release can appear with the first noninhibited impulse. In such cases, terminal boutons must be depolarized sufficiently during inhibition for intracellular calcium to remain at an elevated level; this would allow for expression of facilitation after inhibition. However, with a highly nonlinear dependence of transmitter release on intracellular calcium, it would still be difficult to achieve the rebound effect if available calcium in boutons were reduced in comparison with the noninhibited situation. Extension of present calcium models (Simon and Llinas, 1985; Zucker and Fogelson, 1986) may have to be undertaken to test whether the calcium hypothesis (Fig. 3) can account for this effect. If, however, a voltage-dependent step in transmitter release actually occurs (Parnas and Segel, 1984), the effect could be explained. Submaximal depolarization during inhibition may be sufficient to allow the normal calcium tail current to enter the bouton following the impulses, but not sufficient to activate the voltage-dependentstep fully. Thus, active calcium would be present, but transmitter release would not be fully expressed until the first full-sized action potential occurs. This possibility must be kept in mind, in view of the fact that the presence or absence of a voltage-dependent step in transmitter release is still an open question for the crayfish terminal.

CONSEQUENCES OF PRESYNAPTIC INHIBITION D. FUNCTIONAL In the decapod crustacean limb, independent use of two of the distal muscles (stretcher and opener) can be achieved only when their respective inhibitory neurons are recruited. However, postsynaptic inhibition could serve this role, so there must be another advantage to having the additional mechanism of presynaptic inhibition. One advantage which has been demonstrated more clearly in.crustacean sensory systems (Bryan and Krasne, 1977) is conservation of transmitter supply in excitatory terminals. This would not only conserve energy in the excitatory neuron, but would also ensure a strong synaptic response when the inhibition is removed. Furthermore, short-term facilitation can occur in many of the inhibited excitatory terminals (Dude1 and Kuffler, 1961). The combination of these effects would increase the rate of muscular contraction just after a period of inhibition, since facilitated release of transmitter, drawing upon a maximal immediately available store, could take place. Such an effect was apparent in records obtained from muscles of freely moving crabs (Atwood and Walcott, 1965).

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The timing of arrival of inhibitory impulses at the neuromuscular junction must be carefully controlled to achieve maximal presynaptic inhibition. Central control of timing apparently occurs in fiddler crabs, which use complex claw movements in courtship display (Spirito, 1970). However, there is little evidence for such control in the less-sophisticated freshwater crayfish (Wilson and Davis, 1965). From the limited sample of species studied to date, the tentative conclusion can be drawn that the presynaptic mechanism is more heavily utilized in advanced species with complex behavior. In crab limb stretcher muscles, the most intense presynaptic inhibition occurs in muscle fibers of the slow-acting, nonspiking type which are recruited for slow movement or postural activity (Sherman, 1977). Selective elimination of these fibers from the pool of muscle fibers recruited for rapid movement, by both pre- and postsynaptic inhibition, permits more rapid movement (Bush, 1962; Atwood, 1973; Ballantyne and Rathmayer, 1981).Presynaptic inhibition is thus an important mechanism contributing to the frequency response of the muscle.

VI. Long-Term Facilitation

A. INTRODUCTION In the last review dealing with the subject of long-term facilitation (LTF) in crustaceans (Atwood, 1976),the following picture of the phenomenon emerged. Repetitive stimulation of a tonic synaptic connection progressively enhances the amplitude of evoked synaptic potentials. The rise in EPSP amplitude is most rapid in the first few seconds, but continues for 10-30 min at which time a plateau is attained with the synaptic responses exceeding the control values by up to 50-fold. Upon the cessation of the stimulation, the responses recover partially within 5-20 min. The remaining phase of recovery is a much slower process which can take hours. Accumulation of Ca in the synaptic terminals was considered to be the underlying cause of LTE Summation of accumulated Ca with the incoming flux accompanying each impulse would be expected to enhance synaptic release. The following recovery would represent the dissipation of Ca from the terminals. However, experiments by Atwood and co-workers provided evidence for significant accumulation of sodium ions (Na) in the terminals during LTE Moreover, conditions which favored the accumulation of Na such as a blockade of Na-K pump by ouabain tended to

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enhance LTE Thus, although the contribution of Ca to LTF could not be denied, Na was thought to play a significant or perhaps even a dominant role in the process. Several aspects of this scheme, however, remained uncertain. First, the evidence for accumulation of Na was based on extracellular measurements of nerve terminal potentials and prone to errors such as movement of the recording electrode with respect to the nerve terminal or the accumulation of extracellular K which would be expected to alter the terminal spike. Second, there was no direct evidence for augmentation of release by intracellular Na. Third, no compelling evidence was available to rule out a possible change in the sensitivity of postsynaptic receptors during LTE Fourth, relative magnitudes of contributions of Ca and Na to LTF were not known. Fifth, the exact nature of a process through which Ca andlor Na could enhance synaptic transmission was not known. Sixth, it had not been established if LTF could occur in a living, intact animal. Seventh, contribution of neurohormones to LTF had not been investigated. Some of these questions have been answered or at least addressed in more recent studies. It is the object of the present review to summarize and analyze these later studies.

B. CONDITIONS NECESSARYFOR ESTABLISHMENT OF LTF LTF appears to be a common phenomenon in crustaceans. It was first described and named in classical tonic neuromuscular junctions (NMJ) (Sherman and Atwood, 1971) such as the opener muscle of a crayfish (Procambarus clarkiz) and the stretcher muscle of a crab (Grapsus grapsus). Later the phenomenon was observed in the stretcher muscle of a spider crab (Libinia emarginata) (Atwood et al., 1983), the Blue Crab (Callinectes sapidus), and a shore crab (Pachygrapsw crassipes) (Atwood et al., 1975). An analogous phenomenon can also be observed in a phasic NMJ such as the fast axon in the claw closer muscle of the crayfish, although in this case, LTF appears to be partially masked by depression (Lnenicka and Atwood, 1985b). In fact, a depression can sometimes intervene in LTF even in tonic NMJs. It appears that the ambient temperature plays a significant role in the relative magnitudes of LTF and depression, the latter being more dominant at higher temperatures. LTF on the other hand is most prominent when measured at or near the temperature to which animals are acclimated (Jacobsand Atwood, 1981a). In most studies, LTF was produced by stimulation at 5-30 Hz for 1030 min. When stimulation in excess of these ranges wasattempted, depression obscured the results (Swenarchuk, 1975).The question could arise as

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to how many action potentials are necessary to produce LTF? The answer has to be qualified by the fact that LTF is not an all-or-none event, but rather a graded phenomenon; nevertheless, it appears that in order to obtain a clear LTF with all its characteristic features one needs to apply approximately 12,000 action potentials (Wojtowicz and Atwood, 1985a). This corresponds to stimulation at 20 Hz for 10 min. LTF is typically composed of tetanic phase (i.e., gradual growth of response during stimulation) and at least three distinct phases of decay Phase I lasts seconds, phase I1 minutes, and phase I11 (sometimescalled the aftereffect) hours and perhaps days (Wojtowicz and Atwood, 1985a). The three phases do not always occur together. In particular, phase I11 was not observed in studies of Acosta-Urquidi (1980) and Atwood et al. (1983). It appears that phase I11 requires particularly intense stimulation for its induction and is the most labile of all LTF components. On the other hand, a strong phase I11 can be produced even when the initial components are altered by ionic substitutions (see below) or by concomitant depression (e.g., Lnenicka and Atwood, 198513). Recently, Baxter et al. (1985)discovered the phenomenon of long-term potentiation (LTP) at the crayfish NMJ. This type of enhancement of synaptic transmission can be induced with very brief (1-20 sec)but intense trains of stimuli (15-100 Hz) applied at room temperature. The events accompanying such stimulation are difficult to analyze due to the contravening muscle contraction. However, the magnitude and the time course of the decay of LTP resemble those of LTE Clearly in the case of LTP, only hundreds, not thousands, of stimuli were needed. Very little information is available so far about the mechanism of LTP in crustacea, although it seems certain that only presynaptic factors are involved (see below). C. POSSIBLE MECHANISMS OF LTF 1. Accumulation of Ions It has been proposed that LTF results from the accumulation and subsequent dissipation of Na and Ca in the synaptic terminals (Atwood, 1976). Several studies have appeared which substantiate this view. A tandem of papers by Atwood et al. (1975) and Swenarchuk and Atwood (1975) provided evidence for changes in the externally recorded nerve terminal potential (NTP) during LTF. It was found that the amplitude of NTP decreases, while its latency increases during repetitive stimulation. Both effects were to be expected if Na accumulated in the terminals. These findings were recently confirmed by direct intracellular recordings (Fig. 16).

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A

B

FIG. 16. Correlation of presynaptic (A) and postsynaptic (B) potentials during LTE 1, Control records (stippled) are superimposed on data recorded 30 sec following the beginning of 20-Hz tetanus. Hyperpolarization and increase in amplitude of action potential are visible on the presynaptic record. Postsynaptic potential was increased about 12-fold. 2, Ten minutes after onset of tetanus, there was reduction of amplitude of action potential and increase in postsynaptic potential. 3, Thirty minutes after the end of tetanus, postsynaptic poteotials were enhanced about 2-fold with no corresponding presynaptic change. All records are averages of 100 responses; arrows indicate stimulus artifacts. Postsynaptic records at 30 sec and 10 min are shown at reduced gain (one-half of control).

Detailed data presented in a paper by Wojtowicz and Atwood (1985a) and outlined in Fig. 16 revealed that action potentials and membrane potential of the presynaptic terminal undergo characteristic changes during LTE Initially during the first 30 sec of tetanic stimulation, amplitudes of action potentials are slightly enhanced, while membrane potential becomes hyperpolarized. The hyperpolarization appears to be the result of activation of an electrogenic sodium pump presumably due to a sudden influx of a large amount of sodium ions and possibly due to a voltagedependent mechanism (Holloway and Poppele, 1984). The initial increase

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in the amplitude of action potentials may be caused by the hyperpolarization (see for example, Wojtowicz and Atwood, 1984). During sustained stimulation, action potentials become progressively smaller and their latency longer, indicating that the Na pump is not capable to keep pace with Na influx. Thirty minutes after the tetanus, the action potential amplitude and the membrane potential return to their control values. Yet, a significant enhancement of postsynaptic potentials persists at that time (Figs. 16 and 17).The small enhancement of the action potential seen in Fig. 1, was not a consistent finding (see Wojtowicz and Atwood, 1986, for composite data from a series of experiments). Thus, it is clear, from the evidence presented, that neither the accumulation of sodium nor alteration of presynaptic action potential contribute directly to the enhancement of synaptic transmission lasting longer than 30 min.

20

80

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Tlme (min)

FIG. 17. Time course and ionic dependence of LTE Outer envelope outlines time course of LTF in normal conditions with sodium and calcium conductances intact. Stippled area illustrates components of LTF (intraterminal stimulation) which remain when influx of sodium is blocked by tetrodotoxin. Hatched area depicts a sodium-dependent component of LTF. Sample records corresponding to points 1, 2, and 3 are given in Fig. 16 for normal LTE

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It has been found that conditions which favor accumulation of sodium ions in the terminals, such as application of a sodium pump blocker ouabain, removal of potassium ions from the extracellular medium (Atwood el al., 1975; Swenarchuk and Atwood, 1975), or loading of the terminals via a sodium ionophore monensin (Atwood et al., 1983),enhance synaptic transmission at the crustacean NMJs. These experiments suggested that Na played a role in LTF; however, the relative contributions of Na and Ca in LTF in normal conditions (not favoring a large accumulation of Na) remained uncertain. An attempt to settle this question was made by Swenarchuk and Atwood (1975) who blocked the entry of Ca during LTF by the application of 6 mM manganese (Mn) and measured the decay phase of LTE The results indicated that the blockade of calcium entry by Mn during tetanic stimulation did not prevent establishment of the aftereffect of LTF, suggesting a requirement for sodium in this phase of LTF. However, similar experiments utilizing 30 mM magnesium to substitute for calcium in the perfusate led to variable results, i.e., in about half of the experiments the aftereffect was blocked, while in others it was still present. An alternative approach to this problem has been undertaken recently by Wojtowicz and Atwood (1985b), who developed a technique of stimulating the synaptic release by direct activation of synapses with intraterminal current injections. Since the method can be applied in the presence of T T X , i.e., when the influx of Na into the terminals is blocked, any influence of Na on LTF should be absent. It has been found that upon stimulation with direct depolarizing pulses, a modified LTF develops which is deprived of its progressive growth during prolonged stimulation. A part of its decay phase is also absent under these conditions (Fig. 17). It can be concluded therefore that the chief role of Na in LTF is to sustain a high level of release during prolonged activity and to add a component to the decay phase. However, the experiments suggest that the rapidly rising phase of LTF as well as the prolonged aftereffect (Phase 111) can develop in the absence of the influx of Na into the terminals. Direct injections of Na into presynaptic terminals of the crayfish (Wojtowicz and Atwood, 1985a) as well as of the squid (Charlton and Atwood, 1977)have demonstrated that Na is capable of enhancing transmitter release. It is not known however what is the exact nature of this process. The most plausible schemes are those which involve sodiumcalcium exchange systems, since these have been demonstrated in certain preparations such as synaptosomes (Blaustein and Oborn, 1975)or squid axons (Baker et al., 1969). In both cases the increase in the intracellular content of sodium is thought to lead to an increase in the internal calcium concentration which in turn would be expected to enhance the synaptic

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release. It is not known, however, if such mechanisms operate in crustacean nerve terminals. Clearly more work is needed in this area. Yet another mechanism which may account for the role of Na in LTF derives from the work of Birks, who found increased acetylcholine stores in sympathetic ganglia of the cat upon stimulation of the preganglionic nerve (Birks, 1977)and after ionic manipulationswhich were likely to inhibit the sodium pump (Birks, 1983).Both procedures were thought to increase net Na influx into the terminals, therefore a role for Na in stimulation of acetylcholine synthesis was proposed. It is not yet known if Na can increase the amount of neurotransmitter in crustacean axons. A possibility that sodium promotes transmitter release directly (without mediation of calcium) also needs to be kept in contention. Recently, patch-clamp data have been obtained from isolated nerve terminals of crab's sinus gland, and a population of channels whose duration of opening can be prolonged by sodium has been observed (Lemos et al., 1986). The significance of this particular finding for transmitter release is still obscure, but such direct measurements of ionic currents from nerve terminals constitute a promising line of investigation of processes underlying LTF. In summary, both sodium and calcium are necessary for the full expression of LTF. While both ionic species participate in the process during and shortly after the tetanic stimulation, the aftereffect does not appear to depend directly on accumulation of sodium. According to the most recent evidence, the late phase of LTF (Phase 111) can be largely expressed in the absence of sodium influx into the terminals, but is dependent on calcium (Wojtowicz and Atwood, 1985b).

2. Presynaptic versus Postsynaptic Effects during LTF In the early study by Sherman and Atwood (1971), the authors noted that there was no change in the input resistance of the postsynaptic muscle fibers during LTF and concluded that the locus of the phenomenon is primarily presynaptic. This notion is supported by recent findings of Wojtowicz and Atwood (1986),who found no change in the amplitude of miniature EPSPs during long-lasting decay phase of LTF and thereby ruled out an increase in the sensitivity of postsynaptic receptors. Baxter et al. (1985) have also reported that LTP at the crayfish NMJ does not involve an increase in the miniature EPSPs and therefore is not likely to involve postsynaptic events. However, a possible change in the receptors during early phases of LTF cannot be ruled out without further experimentation. Presynaptic locus for LTF is difficult to ascertain directly. Even though no lasting changes in presynaptic action potential or resting membrane

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potential have been found at times longer than 30 min following tetanus (Fig. 16), a localized alteration of presynaptic potential in the vicinity of release sites remains a possibility. Direct measurements in this region are not feasible at present.

3. Intracellular Mechanisms Associated with LTF Quanta1analysis has been applied frequently to quantify and elucidate the mechanisms of synaptic facilitation at crustacean NMJs (Atwood, 1976). Its application to LTE however, was difficult due to a number of technical reasons. First, long-term focal recording from synaptic release sites is prone to errors due to inevitable movement of muscle fibers during prolonged trains of stimuli. Second, the amplitude of quantal units is difficult to measure because of the relatively infrequent spontaneous release at crustacean NMJs. Third, in most phases of LTF, the EPSPs are not stationary i.e., their average amplitude varies in time. By utilizing small muscle fibers in the opener muscle of juvenile crayfish, Wojtowicz and Atwood (1986) were able to circumvent the first two problems. In the small fibers EPSPs can be recorded intracellularly without a significant spatial decay and consequently the quantal analysis can be performed without the need for focal extracellular recording and its pitfalls. The chance of recording spontaneous miniature EPSPs is greatly improved, since all release sites can be sampled simultaneously. Moreover, one can release quanta on command by applying depolarizing current pulses intracellularly to the terminals. The problem of nonstationarity limited the application of the technique to the relatively stable long-lasting phase of LTF. A binomial model was used to interpret statistical, quantal fluctuations of EPSPs. The data provide good fit to the model in the majority of cases and indicate that the enhancement of synaptic transmission during LTF can be fully accounted for by an increase in the number of active release sites (binomial parameter n ) and a small reduction of the average probability of release (binomial parameter p) without a change in the quantal size. Baxter et al. (1985) used a different approach to analyze quantal fluctuations during LTI? They found that the distributions of amplitudes of evoked EPSPs in certain preparations can be reasonably described by the Poisson function. The Poisson model assumes a large number of releasable quanta each with a low probability of release; thus, it represents a limiting case of the binomial model. The analysis indicated an increase of quantal content without a change in quantal size, suggesting a presyn-

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aptic locus of change during LTI? It should be pointed out that Baxter et al. (1985) used low frequency stimulation (0.3 Hz) to collect their data for analysis. Under these conditions, quantal output tends to be very low at crayfish synapses. Perhaps probability of release was sufficiently low to produce a good fit of fluctuations with the Poisson distribution. Both the binomial and the Poisson distributions have been applied to the crustacean NMJs in the past (Atwood, 1976) with the binomial approach being more generally accepted. Recent application of the binomial distribution to other preparations made this method particularly attractive, since it has been found that the binomial parameter n can be equated with the number of releasing elements such as synaptic boutons (Korn et al., 1982).In crustacean NMJs, the parameter n may represent the number of active release zones (synapses) which can be recruited under a variety of conditions-one being frequency facilitation (Smith, 1983)and another LTF (Wojtowicz and Atwood, 1986) (see Section 11,B). A possible morphological correlate of the increase in the number of active release zones has been found by Chiang and Govind (1986). Using electron microscopy, they observed an increase in the number of synaptic junctions associated with dense bars upon stimulation of the lobster NMJ in the presence of ouabain. Since in the lobster the dense bars appear to be more numerous and larger at synapses which have relatively large quantal output (Meiss and Govind, 1980),the dense bar may represent a structural or an ionic feature in the vicinity of an active synapse (Govind and Chiang, 1979; Govind and Meiss, 1979). A direct comparison of the binomial parameter n and of the number of dense bars is needed to settle this question. The meaning of the binomial parameter p is less well understood, but it may represent the availability of transmitter or of Ca in the vicinity of the release zones. To this effect Acosta-Urquidi (1980)has proposed that there is an increase in the mobilization of transmitter during the first 510 min of the recovery phase of LTE Thus, an increase in the parameter p would be expected at that time. Unfortunately, due to technical limitations mentioned previously, no such estimates have been obtained so far. In summary, the available evidence suggests that LTF is caused by an increase in transmitter release. Although statistical analysis cannot be definite without more direct evidence, it appears that LTF may be the result of an increase in the number of active, transmitter-releasing units (possibly release zones). The exact mechanism which could account for such increase is not known. However, recent findings showed that longterm changes in synaptic terminals are dependent on calcium ions (see above), thus a gate has been opened to a new field of investigation.

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D. ARE NEUROHORMONES INVOLVED? Two circulating neurohormones, octopamine and serotonin, have been found to cause a long-lasting enhancement of synaptic transmission not unlike that in LTF (Breen and Atwood, 1983; Dixon and Atwood, 1985; Section VII). Furthermore, LTF was potentiated by these two hormones (‘Jacobs and Atwood, 1981b). One may ask therefore if a neurohormone or some other substance is secreted from an axon or the surrounding tissue during repetitive stimulation, leading to LTF and to the prolonged enhancement of synaptic transmission. It would appear that LTF does not require such a mechanism, since its rising phase and the initial phase of decay can be satisfactorilyexplained by accumulation of Ca and Na ions in the terminals (Fig. 17). The longlasting aftereffect (Phase 111) does not appear to involve any neurosecretory process either, since it can be produced by the local intracellular stimulation of a single axonal terminal branch (Wojtowicz and Atwood, 1985b). It is possible however that neurohormones play a role in modulating active NMJs in the normal in vivo conditions.

E. POSSIBLE PHYSIOLOGICAL SIGNIFICANCE It has been established that LTF can be induced in intact animals and that it leads to an enhancement of muscle tension (Jacobs and Atwood, 1981a).The frequencies of stimulation which lead to the induction of LTF in the opener muscle of the crayfish are well within the frequency range observed in freely moving animals. Wilson and Davis (1965) observed barrages of muscle potentials in the crayfish claw with mean frequencies up to 33 Hz upon stimulating freely behaving animals by “touching, pinching or scratching various parts of the body.” Figure 18 demonstrates that significant muscle tension (corresponding to 50% opening of the claw) develops when the excitor axon is stimulated with bursts of action potentials at 30 Hz lasting 1 sec (control). Prolonged 20-Hz tetani at first produced minimal muscle tension. After 10 min of continuous stimulation, however, a significant tension developed. Following the tetanus, the individual contractions were enhanced (LTF). It is clear, therefore, that LTF can lead to markedly improved performance of the whole muscle. Generally, however, the activity in motor axons is irregular as opposed to the steady trains of impulses used in most studies. Jacobs and Atwood (1981a) have found that LTF can be produced when stimuli are applied in a bursting pattern. More studies utilizing irregular patterns of stimulation seem warranted. Moreover, it has been

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Tension transducer

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FIG.18. LTF in crayfish opener muscle: functional consequences. Muscle contraction (B) corresponding to bursts of stimuli at 30 Hz are shown before (control) and after a bout of sustained activity. (A) Illustration indicates the movement (opening) of the claw produced by the stimulation. Twenty-hertz tetanus for 10 min resulted in long-term facilitation of the mechanical responses.

found that in the living animals the activity of the excitatory axon in the crayfish claw is usually accompanied by activity in the inhibitory one (Wilson and Davis, 1965). It is not known at present if the activity of the inhibitor has influence on LTE Finally, one may wonder about physiological advantage that LTF may bring to behaving animals. As pointed out by Bittner (1968), the highly facilitating synapses in the crayfish opener muscle may serve the purpose of extending the useful range of tensions developed by the muscle. Thus, enhancement of synaptic responses lasting hours or even days may in fact shift the muscle into the higher tension level within this range and result in a more active muscle (or more vigorous animal) without any modification in the central nervous system. In this light, the suggestion of Wilson and Davis (1965) that “a crayfish thinks with his claws” seems particularly intriguing.

E LTF AND POSTTETANIC POTENTIATION LTF has been compared to posttetanic potentiation (PTP), as both phenomena can have similar time courses under certain conditions (Atwood, 19’76). E’TP, together with its preceeding tetanic phase, have been

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studied primarily at the amphibian neuromuscular junction. In normal physiological conditions, PTP is obscured by a dominant phenomenon of synaptic depression; however, the depression can be minimized by a prior reduction of the synaptic output. A series of articles by Magleby and Zengel(1975a,b, 1982) describes and quantifies PTP under the condition of reduced synaptic release. The phenomenon can be distinguished from the accompanying processes of synaptic facilitation and augmentation by its characteristically long duration. PTP can be explained by a residual calcium hypothesis, but considerable stretching of this concept is required to allow for its relatively long time constant which can reach minutes. In contrast to the short-term synaptic facilitation which can be well described by a residual calcium hypothesis (Zucker, 1982), the mechanism of PTP remains uncertain. A new light on the subject was shed by Erulkar and Rahamimoff (1978), Misler and Hurlbut (1983), and others (see review by Erulkar, 1983), who found that PTP can develop even when tetanic stimulation is applied in the absence of extracellular Ca. It was therefore proposed that the accumulation of Na is the primary factor in PTP and the enhancement of synaptic release is due to the increase of Ca uptake in exchange for Na extrusion from the synaptic terminals. These studies apparently contradicted an earlier report of Weinreich (1971), who observed PTP during the blockade of Na channels with tetrodotoxin and thus concluded that Na is not involved in the phenomenon. The discrepancy might be explained by differences in the procedures used to induce PTP. Misler and Hurlbut, for example, used relatively long trains of stimuli (5-10 min), whereas Weinreich utilized short bursts. Perhaps the long tetani produce more net Na influx than the short ones. In view of the uncertainties about the mechanism of PTP at the frog NMJ, it is difficult to compare it with LTF at the crustacean NMJ. A tentative analogy can be drawn between one phase of LTF (phase 11 in Wjtowicz and Atwood, 1985a) and PTP at the amphibian NMJ on the basis of their similar time courses and a probable dependence on the influx of Na. In conclusion, LTF comprises the tetanic phase and three phases of decay which may be compared with other forms of synaptic plasticity. The phenomenon is certainly more complex than PTP, but it would appear that LTF incorporates PTP as one of its components. Finally, it should be stressed that LTF at the crustacean NMJ occurs under normal conditions, whereas the physiological significance of PTP at the amphibian NMJ is doubtful in view of the dominant synaptic depression.

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VII. Neurohormonal Modulation

Although long-term facilitation and long-term adaptation have been shown to be produced by altered activity of a single excitatory neuron, neuromuscular function can be affected by the influence of other neurons, often in a long-lasting fashion, by virtue of the presence of presynaptic and postsynaptic receptors for neurotransmitters and neurohormones (Fig. 12). Presynaptic receptors for GABA are responsible for the short-term modulation achieved by presynaptic inhibition, while presynaptic receptors for serotonin (5-HT) and octopamine can set up long-lasting potentiation of transmission. We will deal here with the latter effects. Both 5-HT and octopamine appear to function as neurohormones in the intact animal. Their localization and general actions are reviewed by Kravitz et al. (1985).

A. SEROTONIN (5-HT) Enhancement of excitatory synaptic transmission by 5-HT in the opener muscle of the American lobster and crayfish involves a presynaptic effect leading to increased quanta1 content of impulse-evoked transmission (Dudel, 1965b)and elevated spontaneous release (Glusman and Kravitz, 1982). Brief exposure of nerve terminals to 5-HT leads to enhancement lasting 30 min to 1 hr, which decays in two phases from an initial peak (Glusman and Kravitz, 1982). Receptors for 5-HT may be of two types, accounting for the two phases of potentiation, and they appear to be more heavily concentrated on terminal boutons than elsewhere on the axon (Dixon and Atwood, 1985). Preliminary evidence suggests that exposure to 5-HT leads to phosphorylation of specific proteins in the neuromuscular preparation (Goy et al., 1984), but the location and function of these is not presently known. Involvement of the cyclic AMP system of the presynaptic terminal is a possibility (Kravitz et al., 1985). In fact, treatments that elevate cyclic AMP levels duplicate some of the effects of 5-HT exposure (Enyeart, 1978). Development of enhanced transmission does not require presence of extracellular calcium during exposure of the preparation to 5-HT in either lobster or crayfish (Glusmanand Kravitz, 1982; Dixon and Atwood, 1985).In the lobster, it was reported that enhancement is also independent

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of extracellular sodium but in the crayfish opener muscle, reduction of extracellular sodium during 5-HT treatment eliminates the development of long-lasting potentiation almost completely (Dixon and Atwood, 1985). Apparently, an important initial event in the action of 5-HT is an increased sodium conductance of the nerve terminal, leading to a characteristic Nadependent depolarization. Intracellular recording from presynaptic nerve terminals has revealed that 5-HT induces sodium-dependent depolarization, with concomitant reduction in amplitude of the action potential (Dixon and Atwood, 1985). The depolarization subsides before transmission returns to normal and is abolished in low-Na solution. Although depolarization of the nerve terminal would be expected to enhance transmission (Fig. 13),the increase in EPSP amplitude produced by exposure to 5-HT is greater than that attributed to depolarization per se, and since the enhancement outlasts the depolarization, it is clear that another factor is involved. The intracellular recordings strongly suggest that broadening of the action potential as seen in the molluscan sensory neurons (Klein et al., 1982) is not an important feature of the 5-HT effect in crustaceans. Inactivation of repolarizing K channels can probably be ruled out in the crayfish terminal. What are the other possibilities? One is an increase in intracellular calcium, generated from interal sources (Glusman and Kravitz, 1982). Such an effect is thought to be produced by an increase in intracellular Na (Rahamimoff et al., 1978). This would explain the increased spontaneous release and also the development of enhanced transmission in the absence of extracellular calcium. An increase in the resting level of intracellular calcium would lead to enhanced transmitter release by nerve impulses, given a nonlinear relationship between active intracellular calcium and release (Dudel, 1981;Parnas et al., 1982a).In molluscan neurons, a similar effect of 5-HT (in addition to spike broadening) has now been reported (Boyle et al., 1984). However, experiments in which short-term facilitation was measured by twin pulses before and after application of serotonin have recently provided another possibility (Grayson, 1985). Short-term facilitation and initial EPSP amplitide are both enhanced by 5-HT, but the facilitation measured at short time intervalsbetween impulses is increased much more than that measured at longer time intervals. This cannot readily be modeled by increasing the resting intracellular calcium concentration. However, a small change in calcium cooperativity (from five to six in the model of Zucker and Lara-Estrella, 1983) provides an excellent fit to the data. Such a change in apparent cooperativity could take place if, for example, the spacing of active calcium channels in the active zone were to decrease,

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providing more overlap of calcium domains (Zucker and Fogelson, 1986). Seen in this light, a possible effect of 5-HT would be to recruit calcium channels at the synapse from a high-threshold to a low-threshold form. In turn, this could increase the number of active synapseson the terminal, since one determinant of the potency of a synapse is the number of calcium channels available during depolarization by the impulse.

B. OCTOPAMINE In the American lobster, octopamine’s effect on the opener muscle is almost entirely postsynaptic (Battelle and Kravitz, 1978; Kravitz et al., 1980). However, in the crayfish, enhancement of EPSPs by a presynaptic mechanism has repeatedly been reported (Florey and Rathmayer, 1978; Fischer and Florey, 1983). Confirmation was made by recording quanta1 content at individual nerve terminals before and after treatment with octopamine (Breen and Atwood, 1983). The effect is more subtle and more variable than that of 5-HT. An interesting feature of presynaptic modulation by octopamine in the crayfish is its interaction with ongoing low-level impulse activity. At very low frequencies, well below the point at which LTF normally appears, exposure of the preparation to octopamine gives rise to a long-lasting LTF-like enhancement of EPSP amplitude, which is much greater than can be gained by octopamine alone (Breen and Atwood, 1983).The mechanism has not been studied further in the crayfish. In Aplysiu, very similar phenomena produced by 5-HT have been investigated; they are attributed to calcium-dependent enhancement of cyclic AMP-mediated protein phosphorylation (Carew et al., 1983; Walters and Byrne, 1985). Activity-dependent enhancement of synaptic transmission by a neurohormone provides a mechanism for neurohormonally controlled selective strengthening of active pathways in the nervous system.

VIII. Activity-Dependent Long-Term Adaptation

The motor neurons of crustaceans, like those of vertebrates, are differentially active under normal conditions. Neurons of the phasic type are silent most of the time, and fire in high-frequency bursts during periods of intense activity, while neurons of the tonic type are active during normal posture and locomotion, typically firing much of the time at a moderate frequency (Section 111).

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Synaptic physiology and morphology are correlated with the normal role of the motor neuron. Phasic neurons typically produce large synaptic potentials with rapid depression, while tonic neurons produce small to intermediate synaptic potentials which show facilitation rather than depression. Phasic neurons have thinner, more filiform terminals with fewer mitochondria and synaptic vesicles than tonic motor neurons. A. LONG-TERM ADAPTATION IN A PHASIC MOTORNEURON In recent work, it has been shown that both morphology and physiol-

ogy of motor neurons can be altered by changes in their ongoing patterns of impulse activity. Such changes are most easily observed in a phasic neuron in which impulse activity is normally very low (typically only 3-4 impulses/hr in the fast neuron of the claw closer muscle of isolated, unrestrained crayfish (Pahapill el al., 1985). Activity of the fast motor neuron of the closer muscle in the crayfish claw can be manipulated either by directly stimulating the neuron with implanted electrodes (Lnenicka and Atwood, 1985a) or by altering sensory input to the claw motor neurons, through immobilization of the claw (Pahapill et al., 1985)or through artificial stimulation of claw sensory receptors (Lnenicka and Atwood, 1985~; Atwood and Lnenicka, 1986). The major physiological effects of altering the impulse activity in the fast closer axon are shown in Fig. 19. After 3 days or more of exposure to periods of extra impulse activity, the pattern of transmitter release at individual synapses changes considerably. Initial amplitude of the EPSP is reduced substantially, but the ability to sustain release is enhanced: the synapses are less fatigable (Lnenicka and Atwood, 1985a). The two effects appear to represent separate processes within the neuron, since they appear at different times after activity has been altered (Lnenicka and Atwood, 1985b; Pahapill et al., 1985). Furthermore, most of the effect can be attributed to activation of the central processes of the neuron. Sectioning the axon and stimulating its distal end does not produce physiological adaptation (Lnenicka and Atwood, 1985b). On the other hand, activation by artificial stimulation of sensory inputs which impinge directly on the motor neuron (Wiens, 1982) leads to synaptic adaptation, even though the impulse activity of the motor neuron is not much enhanced (Lnenicka and Atwood, 1985~).When the motor axon is reversibly blocked midway along its length by microapplication of tetrodotoxin, stimulation of the central end of the neuron, without activation of its terminals, leads rapidly (within 3 days) to marked reduction of initial EPSP amplitude and to reduced synaptic depression. In contrast, stimu-

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30 min

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FIG. 19. mg-term adaptation of EPSPs resulting from repeated stimuAon in vivo of the fast axon of the crayfish closer muscle. The fast axon in the experimental claw was stimulated for 2 hdday at 5 Hz for 14 days. EPSP values represent mean values from 10 fibers in 5 experimental claws (A)and 10 fibers from the 5 contralateralcontrol claws (0). Bars show standard error of the mean. Inset shows representative records of EPSPs. Calibration, 20 msec, 4 mV. (From Lnenicka and Atwood, 1985a.)

lation of the distal end of a blocked axon may produce enhancement of EPSP amplitude lasting for up to 1 day (a long-term facilitation effect; Lnenicka and Atwood, 1985b), but not the characteristic reduction of initial EPSP amplitudes (Fig. 19). In some preparations, however, reduced depression is observed in this experiment. Immobilization of the claw, which reduces impulse activity and sensory drive of the fast neuron, leads to enhancement of initial EPSP amplitude and increased depression with repetitive stimulation (Pahapill et al., 1985). Thus, peripheral synaptic adaptation can be modulated in either direction by altering the “experience”of the neuron’s central processes. Adaptation occurs even though the total impulse production by the fast motor neuron does not change greatly in response to altered sensory drive. The ability of the neuron to express posttetanic potentiation and longterm facilitation is modified by establishment of long-term adaptation. The naive fast axon generates a supernormal EPSP following a period of maintained stimulation,even though depression occurs during the period of inducing stimulation (Lnenicka and Atwood, 1985b). Once long-term adaptation has been established by conditioning stimulation, the magnitude of the posttetanic increase in EPSP amplitude is much reduced (Fig. 20). Immobilization of the claw leads to the opposite effect. In summary, when the phasic neuron has experienced increased synaptic drive or increased impulse activity, all of the physiological changes

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FIG. 20. Low-frequency and high-frequency depression and long-term facilitation (LTF) in the phasic motor axon of the crayfish closer muscle and effects of age and previous conditioning. (A) Depression and LTF in large (a) and small ( 0 )crayfish. (From Lnenicka and Atwood, 1985b.) Stimulation at low frequency (0.1 Hz) leads to depression of the EPSP (low-frequency depression). During a 30-min period of conditioning stimulation at 5 Hz, further depression occurs (high-frequency depression). Resumption of stimulation at 0.1 Hz leads to an enhanced EPSP which does not show depression. Enhancement is more pronounced in younger animals. EPSP amplitudes are expressed as a percentage of the initial amplitude. Each point represents the mean value for six muscle fibers. (B) Long-term adaptation modifies the expression of LTE (After Pahapill, 1985.) LTF, induced as illustrated in A, is shown in muscles of thrcc groups of animals: untreated controls (Il), animals previously subjected to conditioning stimulation to induce longterm adaptation (111). and animals in which the claw had been immobilized for 15-20 days (I). LTF is reduced by long-term adaptation and enhanced slightly by immobilization, which reduces activity.

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equip it for a more tonic role. Conversely, reduced experience causes the neuron to acquire more phasic characteristics. The physiological changes are accompanied by morphological ones. In activity-conditioned neurons, the terminals acquire a varicose structure in place of the more filiform morphology seen in naive neurons (Atwood et al., 1985; Lnenicka et al., 1986). The varicosities of conditioned neurons contain most of the synapses and possess enlarged, multibranched mitochondria. Accumulation of mitochondria and other intracellular organelles could be responsible for the varicosities (Sasaki-Sherrington et al., 1984). The varicose structure is similar to that found in tonic motor neurons, though not as pronounced (Jahromi and Atwood, 1974; Florey and Cahill, 1982). Increased resistance to depression is very likely linked to the increase in terminal mitochondrial volume. No marked changes in size or number of synapes were observed in electron micrographs of the nerve terminals. Thus, the lower initial output of transmitter from adapted terminals may be linked to changes in terminal excitability, synaptic calcium channels, or altered intracellular sequestration of calcium. Another case of morphological adaptation in identified neurons has been described in Aplysia (Bailey and Chen, 1983). Synapses of sensory neurons on follower cells mediating the gill-withdrawal reflex were analyzed in marked cells. Habituation of the reflex was accompanied by reduction of active zones and of synaptic vesicles closely associated with active zones; sensitization of the reflex was accompanied by morphological changes in the reverse direction. The results imply a correlation between altered synaptic efficacy and structural (cytoskeletal) changes. Known alterations in calcium influx could lead to long-term synaptic alteration. As in the crustacean terminal, the number of functional synapses appears to be an important variable related to (and perhaps determining) the strength of response. A general hypothesis for these changes must involve regulation of protein synthesis and/or axoplasmic transport by the neuronal soma (Fig. 21). One possibility is that parts of the nuclear genome are responsive to ion fluxes resulting from depolarization of the central processes of the fast motor neuron. At least two genetic events could be initiated: one linked to mitochondrial protein synthesis (Schatz and Mason, 1974) and one leading to a “down-regulation”of transmitter output at some of the peripheral synapses. It is conceivable that in a naive terminal, most of the synapses are able to release transmitter for the first impulse, while in a conditioned terminal, a proportion of the synapses acquire a high threshold for transmitter release, possibly due to modification or removal of synaptic calcium channels (Fig. 21). Indeed, the removal or inactivation of calcium channels at some of the synapses could be an adaptation to the increased entry of calcium entailed by a more persistent mode of impulse transmission. Increased mitochondrial volume in the terminals could also

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A

Axonal transport

l r of synaptic terminals

B Naive terminal

c Conditioned terminal

FIG. 21. Hypothetical scheme for long-term adaptation. (A) Long-term neuronal adaptation is modulated by synaptic activity at the central processes of a neuron and leads to axonal transport of “transforming” message or material to the periphery. Morphological and physiological changes of the terminals result. (B) Some synapses are removed from the responsive “pool” by a factor (X) that is increased by long-term adaptation.

act to prevent calcium buildup in the terminals by sequestering excess calcium during maintained impulse activity. Improved calcium homeostasis could explain the curtailment of LTF after a burst of activity (Fig. 20). Another feature of adaptation, seen to good advantage in neurons stimulated centrally during reversible block of transmission to the periphery with tetrodotoxin, is the removal of low-frequency depression. This phenomenon cannot be satisfactorily accounted for by transmitter

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depletion (Zucker and Bruner, 1977). The presence of synapses that can be readily inactivated (perhaps by local calcium entry) could account for the phenomenon of low-frequency depression in naive terminals, while the inactivation of such synapses by a factor making its appearance during conditioning could lead to diminution or disappearance of low-frequency depression, along with lower initial transmitter release. Physiological differentiation of synapses on a single terminal must be postulated in this model. Another form of long-term adaptation, possibly related either to activity or to nutrition, has been described in tonic motor axons of crayfish by Bittner (1968) and in crabs by Acosta-Urquidi (1978). Synaptic terminals of animals maintained in captivity for a long time produce smaller than normal EPSPs, and in some cases, may only release significant amounts of transmitter after a period of stimulation (“dormant”synapses). Here, LTF may be necessary to enable the synapses to regain function. This observation strengthens the view that many synapses may not release transmitter unless recruited by either short-term or long-term facilitation. B. AGEDEPENDENCE OF LONG-TERM ADAPTATION

The fast closer motor neuron of the crayfish claw changes its physiological properties with age. In small crayfish, the pattern of transmitter release does not show as much depression as in older crayfish. Possibly, changing levels of impulse activity in the fast motor neuron contribute to this difference, since small crayfish are more active than larger ones. In very large animals, transmission of the fast axon rapidly depresses with sequential impulses. Paradoxically,the muscle fibers in these older animals acquire the histochemical properties of slow fibers, from an earlier condition in which many of them had fast histochemical properties (Govind and Pearce, 1985). Synaptic properties and muscle fiber properties drift physiologically in opposite directions with age. Presumably, the slow axon is responsible for determining the properties of the muscle fibers in the older animals, while the fast axon gradually becomes trophically and physiologically impotent. Long-term adaptation can be produced most readily in animals of intermediate age and size (Lnenickaand Atwood, 1985a).In older animals, a rather small increase in resistance to the depression induced by maintained stimulation occurs, but the initial EPSP amplitude remains large and low-frequency depression still occurs after 7- 14 days of conditioning. Clearly, the neuron loses its ability to adapt to altered patterns of activity with age. This could indicate loss of genomic responsiveness.

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In other crustaceans, age-dependent alteration of neuronal responsiveness to activity may also occur. Asymmetry of neuromuscular performance in claws of the American lobster develops with age, eventually leading to appearance of the slow-acting crusher and fast-acting cutter claws (Govind, 1984). Determination of claw properties is established at a critical period early in development. There is evidence that the more active claw eventually becomes the crusher. Animals raised in an environment in which sensory stimulation of both claws is minimal often fail to develop a crusher claw. Possibly, central comparison of sensory input from the two claws leads to determination of neuromuscular properties (and muscle fiber type) during an early stage of development. Thereafter, in lobsters, the pattern is fixed, although claw reversal occurs in other species, particularly in alpheid shrimps (Mellon, 1981).

IX. frophic Effects

A. RESPONSES OF MOTOR AXONS TO DECENTRALIZATION Some motor axons of decapod limb muscles have the remarkable ability of retaining physiological function and morphological integrity of severed peripheral axonal processes for months (Hoy et al., 1967; Atwood et al., 1973). The decentralized axon of the crayfish opener muscle can still release quanta of transmitter substance, but the amplitude of the EPSP declines at first rapidly, then gradually (Velez et al., 1981). The decline of EPSP amplitude is more dramatic for terminals that produce a relatively large EPSP initially. Hypertrophy of glial cells around the severed distal axon and evidence for transfer of large molecules from glial cells to the axon indicate that synthetic activity of the glial cell maintains the axon. Eventually, ephaptic condition of impulses between the outgrowing central process of the neuron and its distal end lead to functional restoration of transmission (Bouton and Bittner, 1981). Still later, the outgrowing central axon displaces the remaining distal processes, and new synapses are presumably formed. Electron microscopic examination of the terminals in a decentralized lobster motor axon has provided evidence that axonal branches retract after decentralization (Chiang and Govind, 1984). In addition, the number of presynaptic dense bodies (indicative of active zones) decreases. Thus, the decrease in transmitter output evident in decentralized axons may reflect synaptic turnover, as well as loss of ability to maintain the normal

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supply of transmitter. Individual synapses may be decommissioned fairly rapidly (within 24 hr); this could account for the rapid initial loss of EPSP amplitude after decentralization (Velez et al., 1981).

B. NERVE-MUSCLEINTERACTION

Changes in neuronal activity apparently have little immediate effect on crustacean muscle fibers, since denervation (decentralization)produces little change in ultrastructure or electrical properties over a period of weeks or months (Atwood et al., 1973; Boone and Bittner, 1974). However, there is evidence for neuronal effects on muscle development at critical periods (Govind, 1984) and during regeneration (Quigley et al., 1985). In addition, some evidence for retrograde influence of muscle fibers on synaptic properties has been advanced. In the accessory flexor muscle of the American lobster, different branches of the motor axon form synapses of the same physiological type when contacting a particular muscle fiber (Frank, 1973).In crayfish, experimental alteration of muscle fiber growth produces an appropriate adaptive adjustment of synaptic strength (Lnenicka and Mellon, 198313).Adjustment of transmitter output to match the size of the muscle fiber is also seen during normal growth in both lobster (De Rosa and Govind, 1978;Govind et al., 1982)and crayfish (Lnenicka and Mellon, 1983a). The ultimate solution to the problem of synaptic differentiation must be sought through further studies on synaptic components during development and aging.

C. AXONAL INTERACTIONS Long-term changes in synaptic plasticity have been described in abdominal muscles of the rock lobster (Panulirus pennicillatus) following selective elimination of either an inhibitory or an excitatory axon from the total complement of axons supplying a particular muscle. When an inhibitory axon is eliminated by injection of pronase, EPSPs become larger and more prolonged (Dude1et al., 1981;Parnas et al., 1982~). This effect appears to be mainly postsynaptic in origin and is attributed to prolonged open time of excitatory (glutamate-operated) postsynaptic channels. Selective elimination of a common excitatory axon leads to enhanced transmission for the remaining excitatory innervation (Parnas et al., 1984). The effect requires central continuity of the remaining excitatory axons

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and is presynaptic in origin. Presynaptic mechanisms are involved: early strengthening is attributed to increased release at existing synapses, while with time, increased sprouting of axonal terminals and formation of new synapses apparently occurs. Both nerve-muscle and axo-axonal trophic interactions may be involved in reinnervation of crayfish slow abdominal flexor muscles by the pool of six motor axons (Ely and Velez, 1982; Hunt and Velez, 1982; Clement et al., 1983). Different members of the slow flexor motor pool differ in their connectivity patterns and synaptic properties when presented during regeneration with a similar population of target muscle fibers. Intrinsic axonal growth programs, nerve-muscle interaction, and axonal competition may all come into play in this complex situation. The role of trophic interactions in determining synaptic properties is still very much an undeveloped area of investigation. Clearly, long-term plastic changes are prominent in crustacean neuromuscular systems and play an important role in maintenance of normal synaptic function.

X. Conclusion

In this survey, we have emphasized some of the recent physiological and morphological work and modeling that is of most immediate relevance to an overview of synaptic properties and plasticity in crustacean neuromuscular junctions. Clearly, there are a number of unresolved issues that have a bearing on our final view of the determinants of performance. The best available data on the acute control of synaptic transmission comes from the squid giant synapse. All important features of transmission and short-term facilitation can reasonably be accounted for on the basis of calcium entry and subsequent distribution within the terminal; there seems to be no need to propose a voltage-dependent second step for that synapse. The most recent work (Augustine et al., 1985a,b) has removed basis for this proposal in the squid synapse to a large extent; and new models of calcium domains (Zucker and Fogelson, 1986) can accommodate a small degree of "hysteresis" in the voltage-release curves, purely on the basis of the calcium hypothesis. However, as good as the models are for the squid giant synapse, it is not the physiological equivalent of the crustacean or vertebrate synapse. Processes such as long-term facilitation cannot be observed in the squid synapse. Other processes may also exist at the crustacean synapse and not at the squid synapse. Therefore, we must still keep an open mind on the question of an additional voltage-dependent step in secretion at the cray-

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fish synapse. Neither the present experimental evidence nor its interpretation are conclusive at this stage. If a second voltage-dependent step exists, it could account for some presently mysterious phenomena, such as persistence of short-term facilitation during presynaptic inhibition. Models of calcium entry and removal provide a useful framework for general consideration of transmitter release and short-term facilitation, but in some instances they have pointed to conclusions that seem unlikely (Section V). The most realistic models will be those that consider both morphological and physiological data, rather than one aspect in isolation. The concept of the synapse as a functional unit, behaving like the allor-nothing boutons of Mauthner cell innervation (Korn et al., 1982), is one that we have used to explain several of the phenomena of plasticity at the crustacean neuromuscular junction. It is reasonable to suppose that small synapses such as those of low-release terminals in the crayfish opener muscle behave as an all-or-nothing unit; larger synapses may depart from this model. An exact test of this proposal has not been devised. Morphological evidence suggest that not all synapses on a single terminal are functionally equivalent. Thus, plasticity may in some instances be brought about by adding or subtracting synapses from the functional pool. Presynapticinhibitionand low-frequencydepressioncould represent short-term subtraction of functional synapses; facilitation probably involves short-term addition to the pool. Long-term facilitation and longterm adaptation are viewed as semipermanent alterations of the functional pool. The molecular mechanisms responsible for long-term plasticity can be approached through analysis of single identifiable crustacean neurons that express these phenomena. Acknowledgments

Research by the authors was supported by grants from the Medical Research Council of Canada and NSERC, Canada. Ms. M. Hegstrom-Wojtowicz and Ms. J. Bilyk helped to prepare the manuscript. Dr. Robert Zucker, Dr. C. K. Govind, Dr. I. Parnas, and Dr. H. Parnas kindly supplied previews of manuscripts presently in press and gave us permission to incorporate several items into our figures. We acknowledge the following authors and/or publishers for granting us copyright permission; Oxford University Press for material in Fig. 1 (after Walrond and Reese, 1985) and in Fig. 19 (after Lnenicka and Atwood, 1985a); Dr. M. P. Charlton for material in Fig. 4 (after Smith et al., 1985); Dr. G. D. Bittner and the Rockefeller University Press for material in Fig. 9 (after Bittner, 1968); Dr. R. S. Zucker and the Rockefeller University Press for material in Fig. 11 (after Zucker and Lara-Estrella, 1983); The American Physiological Society for material in Fig. 13 (after Wojtowicz and Atwood, 1984); Alan R. Liss, Inc. for material in Fig. 14 (after Atwood et al., 1984); and Dr. R. S. Zucker and the American Association for the Advancement of Science for material in Fig. 3 (after Zucker and Lando, 1986).

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IMMUNOLOGY AND MOLECULAR BIOLOGY OF THE CHOLINESTERASES: CURRENT RESULTS AND PROSPECTS By Stephen Brimijoin Department of Pharmacology Mayo Clinic Rochester, Minnesota 55905

and Zoltan Rakonczay Central Research Laboratory Medical University Sreged 6720, Hungary

1. Introduction

A. GENERAL The cholinesterases, a group of enzymes with near-universal distribution across the animal kingdom, have long been recognized for their medical, military, agricultural, and neurobiologic importance. These enzymes are usually divided into two families: “true”or acetylcholinesterase (AChE, officially designated acetylcholine acetylhydrolase, EC 3.1.1.7) and “pseudo-” or butyrylcholine esterase (BuChE, officially designated acylcholine acylhydrolase, EC 3.1.1.8). The two enzyme families can be distinguished on the basis of substrate preferences and kinetics, sensitivity to selective inhibitors, and tissue localization, but they show many points of resemblance, including mechanism of action. For a general survey of the cholinesterase literature, the reader is referred to Silver’s monograph (1974) and to recent symposium proceedings (Brzin et al., 1984). Interest in the cholinesterases has risen during the last few years as a result of several developments. One stimulus to research was the recognition that both AChE and BuChE occur in a variety of molecular forms defined in terms of the nature and number of their subunits. As reviewed by MassouliC and Bon (1982) and Brimijoin (1983), evidence is accumulating that each of these forms has a characteristic location inside or outside the cell, a characteristic fate, and perhaps a characteristic functional role. In consequence, there have been many studies on the dynamics of the cholinesterases and their molecular forms. To list just a few of the INTERNATIONAL REVIEW OF NEUROBIOLOGY, VOL. 28

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Copyright 0 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.

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resulting insights, one might cite evidence that AChE forms are differentially distributed at the synapse (Hall, 1973), synthesized and turned over at different rates (Brimijoin and Carter, 1982), transported along nerves in different proportions and at different velocities (DiGiamberardino and Couraud, 1978; Brimijoin, 1979), and differentially subjected to exocytotic release (Gisiger and Vigny, 1977; Skau and Brimijoin, 1978; Kimhi et al., 1980; Lazar and Vigny, 1980). Another major development has been the increasing success in using affinity chromatography to obtain highly purified cholinesterases for structural study (Rosenberry et al., 1974;Taylor et al., 1974;Ott et al., 1975; Rakonczay et al., 1981; Mintz and Brimijoin, 1985a). Information on the purification, properties, and distribution of molecular forms of AChE in mammalian brain was recently reviewed by Rakonczay ( 1986). The latest development has been the application of modern immunology and molecular biology to the study of cholinesterases. Below we deal in some detail with the goals and results of these two approaches. To lay the groundwork, a brief sketch of cholinesterase biochemistry is given first.

B. BASICSTRUCTURE AND MOLECULAR HETEROGENEITY OF CHOLINESTERASES The building blocks of AChE and BuChE are globular catalytic subunits with apparent molecular masses of 70,000-85,000 Da in their native form. Each subunit possesses a single active site, containing a reactive serine residue. The subunits tend to be highly glycosylated. For example, carbohydrate comprises 24% of the mass of human serum BuChE (Haupt et al., 1966; Muensch et al., 1976) and 15%of the mass of human erythrocyte AChE (Niday et al., 1977). Deglycosylated ChE subunits display molecular masses close to 65,000 Da. Thanks to elegant experiments by Massoulib and co-workers (Massou1% and Rieger, 1969; Bon et al., 1973, 1976; Vigny et al., 1978b)and other groups (Rosenberry and Richardson, 1977; Dudai et al., 1973; Dudai and Silman, 1974),we can now explain many of the molecular forms of AChE and BuChE as combinations of catalytically indistinguishable globular subunits, with or without a "collagen-like"tail unit (Fig. 1). Only a limited range of combinations are found in tissue extracts from sources as diverse as insect and mammal. The purely globular tail-free forms consist of monomers, dimers, or tetramers, if detergents are used to stop insoluble aggregates from forming. Globular cholinesterases are stable, noninterconverting forms that are soluble in media of low OT high ionic strength.

CHOLINESTERASES

365

A8

A4 FIG. 1. Organization of subunits in the molecular forms of the cholinesterases. Each circle represents a catalytic subunit. Globular forms are designated by the symbol G. The linear elements are the collagen-like tails, found only in the asymmetric forms, designated by the symbol A. T h e locations of disulfide bridges were deduced from studies of electroplax AChE (Rosenberry and Richardson, 1977; Anglister and Silman, 1978), but a similar pattern is believed to occur in AChEs and BuChEs from many sources.

The tailed or asymmetric forms contain one, two, or three globular tetramers as their catalytic component (Fig. 1). Asymmetric cholinesterases are solubilized only at high ionic strength (e.g., 1.0 M NaCI). Both the globular and asymmetric forms are reproducibly identified by their sedimentation properties during ultracentrifugation on continuous sucrose density gradients. The basic arrangement of AChE and BuChE forms is neatly described by the classification scheme of Bon at al. (1979) in which globular and asymmetric forms are designated G or A, respectively with subscripts for the number of subunits. According to this scheme, the major AChE form of brain is G4 (globular tetramer), while a characteristic form at peripheral cholinergic synapses is A12 (tailed triple tetramer). When isolated from mammalian tissues, G4 AChE sediments at 10-11 S, while AI2 AChE sediments at 16 S.

366

STEPHEN BRIMIJOIN AND ZOLTAN RAKONCZAY

The catalytic subunits of oligomeric forms are held together by disulfide bonds and by noncovalent-hydrophobic interactions. Disulfide bonds contribute to thermostability and are essential for the existence of free dimers (Vigny et al., 1978b; Edwards and Brimijoin, 1983),but not for the structural integrity of tetramers or for the catalytic activity of any form. Lockridge and La Du (1982) showed that the native tetramer of human serum BuChE dissociates into monomers, dimers, and trimers after mild tryptic proteolysis. Such treatment cleaves a peptide of less than 5000 Da from one end of the catalytic subunit, probably the C-terminus. This region therefore contains interchain disulfide bonds as well as the residues responsible for hydrophobic interactions between adjacent subunits. Disulfide bonds are also involved in the association between globular and tail units of asymmetric forms (Rosenberry and Richardson, 1977; Anglister and Silman, 1978). Apart from the number of subunits and the presence or absence of a tail unit, globular cholinesterases can be differentiated by their interactions with detergents. Some AChE or BuChE can be solubilized from any given tissue with aqueous media alone, while the remaining extractable enzyme requires detergent for solubilization. This behavior is presumed to reflect the existence of a hydrophilic, releasable form and a hydrophobic, menibrane-associated form, respectively (asymmetric AChE is uniformly hydrophilic). Although detergent-free and detergent-containing extracts do not yield pure preparations of either species, progress has been made in characterizing the structural correlates of the two solubility forms. For example, it has been shown that phosphatidylinositol-specific phospholipase C selectively solubilizes membrane-bound AChE from several sources including Torpedo electroplax synaptosomes and bovine or rat erythrocytes (Futerman et al., 1985a). Direct biochemical investigation confirms the inference that covalently bound inositoi is a component of the membrane-anchoring domain of hydrophobic Torpedo AChE (Futerman et al., 1985b,c). Work with human erythrocyte AChE has generated a similar picture, although the role of inositol is less clear, since phospholipase C is not an effective solubilizer. Chromatographic analyses of papain digests show that the membrane-binding domain of erythrocyte AChE is a small glycolipid linked to the C-terminus of the main polypeptide via ethanolamine (Dutta-Choudhury and Rosenberry, 1984; Rosenberry et al., 1986). This glycolipid has not been fully characterized, but its lectinbinding properties are consistent with the presence of inositol. It should be emphasized that the proposed linkage of membraneassociated AChEs to the plasmalemma is highly unusual, though studies of trypanosomal surface antigens have established a precedent (Holder, 1983; Ferguson et al., 1985). The implication is that AChEs are not integrally incorporated in the lipid bilayer, but are more loosely attached, extrinsic proteins. This implication has not yet been proven for all types

CHOLINESTERASES

367

of AChE-bearing cells. Since about 90% of brain AChE is membraneassociated (Rakonczay, 1986), it will be particularly important to understand the mode of enzyme anchoring in neurons. At present there are indications that brain AChE is anchored somewhat differently from the red cell enzyme (Rosenberry et al., 1986), but the basic principle of the membrane linkages may still be the same. The asymmetric forms of AChE, and probably of BuChE as well, are subject to a different kind of anchoring. Minor amounts of these forms can be detected intracellularly, where they are probably assembled and glycosylated (Younkin et al., 1982; Rotundo, 1984a). The bulk of the asymmetric AChE, however, is located specifically in the synaptic cleft, attached to the basal lamina. Abundant evidence supports the concept that the tail unit is the component responsible for the interaction with glycosaminoglycans and other constituents of the basal lamina (Hall and Kelly, 1971; Bon et al., 1978; Brandan and Inestrosa, 1984). The basis for the interaction is probably ionic, but the chemical nature of the tail unit itself is still debatable. In summary, the molecular forms of AChE and BuChE have been relatively well characterized in recent years, though we can hardly claim a complete understanding. Recognizing that the globular forms occur both in hydrophilic and hydrophobic variants, whereas the asymmetric forms are exclusively hydrophilic, a total of nine possible entities can be identified. These entities bear no simple relation to the isoenzymes identified by their mobility during gel electrophoresis under nondenaturing conditions (Gisiger et al., 1978; Kasi and Rakonczay, 1982). Most of the latter are probably charge isomers resulting from differing types or degrees of carbohydrate substitution. Conceivablythe cholinesterasesharbor further types of microheterogeneity that are still unrecognized. In any case, investigating the biochemical and physiological significance of the known forms is a major challenge before us. Meeting this challenge will require the applicationof an array of sophisticated modern methods along with the classical techniques. A primary purpose of the present review is to consider the promise and accomplishments of modern immunological and molecular approaches to the biology of cholinesterases.

II. Molecular Biology of the Cholinestemser

A. In Vitro TRANSLATION The methods of molecular biology are rapidly transforming our knowledge of the cholinesterases. Studies on the molecular biology of the vertebrate cholinesterases may be said to open with Soreq's ex-

368

STEPHEN BRIMIJOIN AND ZOLTAN RAKONCZAY

periments demonstrating the in uitro translation of AChE in Xenopus oocytes injected with mRNA from various sources (Soreq et al., 1982). Soreq’s work revealed the remarkable ability of the Xenopus cells, not only to translate an exogenous AChE message into functional enzyme, but also to process the foreign AChE for secretion into the bathing media. Since the secreted enzyme can be quantitated by sensitive assays for catalytic activity (Johnson and Russell, 1975; Parvan et al., 1983), this system forms the basis for a practical in uitro translation assay. Such assays may be useful in exploring the regulation of cholinesterases during development, in altered physiological states (e.g., denervation), and in different tissues. In one study, Soreq et al. (1984) used the oocyte translation assay to test the possibility that transcriptional events contribute to cholinesterase heterogeneity. They were able to detect cholinesterase-inducing mRNAs sedimenting at 32, 20, and 9 S. The relative amounts of these three different size classes of mRNA were different in preparations from primary glioma, meningioma, and embryonic brain. Furthermore, brain mRNAs inducing AChE and BuChE synthesis had a different size distribution. In vitro translation experiments also identified different translation products and multiple mRNAs encoding for AChE in Torpedo (Sikorav et al., 1984; Maulet et al., 1985). Taken together, these findings make it clear that the heterogeneity of brain cholinesterases is not limited to the posttranslational level. The obvious question still to be answered is whether the mRNA heterogeneity reflects differential processing of a single transcript or the operation of diverse structural genes. Another interesting application of En uitro translation has been to investigate the relation between human AChE and that of Drosophila. The latter enzyme is under the control of Ace, a genetic locus mapped to region 87BE on chromosome 3 (Hail and Kankel, 1976) and recently cloned (Bender et al., 1983; Hall et al., 1983). Soreq et al. (1985) found a DNA fragment of Ace which hybridized with Drosophila mRNA that induced AChE synthesis in the oocyte system. They used this fragment to identify homologous sequences in a human genomic DNA library, one of which hybridized with a 7 kb human mRNA that (1) was capable of inducing AChE synthesis by Xenopus oocytes and (2) was notably scarce in acetylcholinesterase-deficienttumor cells. Because the DNA sequences have not been compared, we are not in a position to analyze the homology between the insect and human genes. It is tempting to infer that Ace and its human counterpart represent structural genes for AChE. However, as pointed out by Soreq et al. (1985),it is too early to exclude the alternative possibility that both genes code for a protein that indirectly regulates the expression of endogenous Xenopus AChE. This issue could be resolved by gene sequencing or by unequivocally establishing the species designation of the translation products.

369

CHOLINESTERASES

AND SEQUENCING B. GENECLONING

Major efforts have recently been devoted to obtaining full sequence cDNA probes to determine primary protein structure. Taylor’s group focused on 11 S Torpedo AChE, using oligonucleotide probes synthesized to correspond with two nonoverlapping sequences within a 26-residue CNBr fragment and a third sequence from the N-terminal region. A clone that hybridizes all three probes hasjust been isolated and sequenced (Schumacher et al., 1986a,b).By comparing the inferred amino acids with the sequences of tryptic peptides corresponding to a large portion of the native protein, it was possible to confirm the correct reading frame and demonstrate the likelihood that the clone does code for the Torpedo enzyme. A similar analysis of human serum BuChE is nearing completion in another laboratory (La Du and Lockridge, 1986). The information obtained in this way is still limited and it is difficult to predict its full impact, but it is already providing insights of great importance. The new cDNA sequence data (Fig. 2) show that Torpedo AChE is a 5’75 amino acid residue protein, calculated mass 65,612 Da (Schumacher et al., 1986a,b). The 5’ end of the cloned DNA encodes for a 13-residue DDHSELLVNTKSGKVMGTRVPVLSS

HISAFLGIPFAEPPVGNMRFRRPEP KKPWSGVWNASTYPNNCQQYVDEQF PGFSGSEMWNPNREMSEDCLYLNIW

VPSPRPKSTTVMVWIYGGGFYSGSS TLDVYNGKYLAYTEEVVLVSLSYRV GAFGFLALHGSQEAPGNVGLLDQRM ALOWVHDNIQFFGGDPKTVTIFGES’; AGGASVGMHILSPGSRDLFRRAILQ SGSPNCPWASVSVAEGRRRAVELGR NLNCNLNSDEELIHCLREKKPQELI DVEWNVLPFDSIFRFSFVPVIDGEF FPTSLESMLNSGNFKKTQILLGVNK OEGSFFLLYGAPGFSKDSESKISRE DFMSGVK L S V P H A N D L G L D A V T L O Y TDWMDDNNGIKNRDGLDDIVGDHNV ICPLMHFVNKYTKFGNGTYLYFFNH RASNLVWPEWMGVIHGYEIEFVFGL PLVKELNYTAEEEALSRRIMHYWAT FAKTGNPNEPHSOESKWPLFTTKEQ

KFIDLNTEPMKVHQRLRVQMCVFWN QFLPKLLNATETIDEAERQWKTEFH

RWSSYMMHWKNOFDHYSRHESCAEL

25 50 75 100 125 150 175 200 225 250 275 300 325 350 375 400 425 450 475 500 525 550 575

FIG.2. Amino acid sequence of Torpedo AChE. The sequence was deduced from fulllength cDNA coding for hydrophilic 11 S enzyme (Schumacher ct al., 1986a).The active site serine, position 200, is marked by an asterisk.

370

STEPHEN BRIMIJOIN AND ZOLTAN RAKONCZAY

peptide rich in hydrophobic amino acids and not found in the mature electropiax AChE. This region probably represents a leader peptide. The active site serine sits at position 200. Since histidine is thought to participate in ACh hydrolysis via a charge relay system (Cunningham, 1957; Froede and Wilson, 1971), it is interesting to note that the nearest such residues are located at positions 181 and 209. Schumacher et al. (1986b) suggest that the most likely candidate for an active site histidine within an appropriate cystine loop is located at position 264. Significantly,AChE lacks the histidine near position 57 that would have been predicted by analogy with chymotrypsin’s active site (Davie et al., 1979).The sequences Asn-X-Ser or Asn-X-Thr occur in four places (positions 59,416,457, and 533), marking potential sites of N-linked glycosylation. The derived sequence also contains eight cysteine residues, at positions 67, 94, 231, 254, 265, 402, 521, and 572. At least one of these must be involved in disulfide bonding of adjacent subunits in dimers and larger forms, but the location of the bonds remains to be determined. Schumacher et al. (1986b) emphasize the relative absence of hydrophobic residues outside the leader peptide and consider the hydropathy profile to be consistent with that of a secreted, globular protein. In fact, there seem to be no good candidates for membrane-spanning regions. This finding is not surprising since the 11 S AChE of Torpedo is a hydrophilic form. If membrane-associated forms of the enzyme (i.e., 5.7 S dimers) prove to be similar in structure, we will understand why they must be peripherally attached to cell membranes by glycophospholipid linkages (Futerman et al., 1985a,b). Although the information on BuChE is less complete, it is already possible to make some comparisons with AChE. Human serum BuChE consists of 580 amino acid residues per subunit (Lockridge, 1984),just 5 more than in Torpedo AChE. As Fig. 3 shows, the active centers of these proteins are highly homologous, with identity in 20 of 29 residues and conservative replacement in 5 others (Lockridge, 1984; La Du and Lockridge, 1986; MacPhee-Quigley et al., 1985).There is also striking homology at the C-terminus, with identity in four of six residues and conservative replacement in one more, corresponding to a point mutation. On the other hand, homologies at the amino-terminus are scarce. Comparisons between Torpedo AChE and the mechanistically similar serine hydrolases show little homology except with aliesterases and carboxylesterases (Schumacher et d.,1986b).The evidence thus suggests that AChE did not arise from serine proteases by the simple mechanism of gene duplication and genetic drift. It is also worth noting the absence of significant homologies between Torpedo AChE and any of the subunits of the nicotinic acetylcholine receptor, despite the parallel localization of these molecules in the electroplax. One surprising finding emerging from the

371

CHOLINESTERASES

Active Site Peptides:

C-Terminal Peptides AChE: m A E [ 5 BuChE: E S C V G L 580 N-Terminal Peptides

10

10

or

10

FIG.3. Homology between Torpedo AChE and human serum BuChE. Data were taken from Lockridge (1984), Lockridge and La Du (1982), and Schumacher et al. (1986a,b). T h e sequences of peptides from three regions of the molecules are compared. Identical sequences are enclosed in boxes. Note that the difference between alanine (A) and valine (V) in the C-terminal peptides corresponds to a point mutation (single base substitution). The N-terminal peptides are compared in two alignments: (1) starting from the initial residues of both enzymes and (2) starting with the initial residue of AChE, but the sixth residue of BuChE (allowing for the greater length of the latter). Neither alignment reveals many points of identity.

cDNA analysis is a striking global homology between Torpedo AChE and the C-terminal portion of bovine thyroglobulin. Although the active site serine at position 200 is not conserved in thyroglobulin, 60% of the AChE residues between positions 144 and 195 are identical with those in the larger protein (Schumacher et al., 1986a,b).This structural homology is not accompanied by any known functional resemblance. Additional studies on the genomic organization of Torpedo AChE will shed light on the mechanism responsible for the structural diversity of AChE forms. It does appear that most of these forms are explainable as different assemblies of the same basic units, but there is evidence that some of them represent distinct gene products. We have already noted observations of heterogeneity at the mRNA level. Also, in studies of the Torpedo enzyme, Taylor and co-workers have shown that the catalytic subunits of hydrophobic 5.7 S AChE differ from those of hydrophilic 11 S AChE in their peptide maps (Lee et al., 1982) and in their reactivity with monoclonal antibodies (Doctor et al., 1983). A search for separate cDNA clones representing soluble and membrane-bound AChE is therefore in order. Further progress in the molecular biology of the cholinesterases will

372

STEPHEN BRIMIJOIN AND ZOLTAN RAKONCZAY

undoubtedly be rapid. Obviously, the more proteins in this family that are sequenced, the better we will understand their structural interrelations. Site-directed mutagenesis will help to define the catalytic and structural roles of individual amino acid residues. Of special interest is the possibility of engineering molecules with altered anionic sites, which might clarify the mechanism of substrate-induced AChE inhibition. Judicious modifications of cysteine residues or hydrophobic amino acids might give rise to unusual oligomeric enzyme assemblies for studies of the physiological role of AChE forms. Much more sophisticated studies of enzyme regulation will certainly be feasible. Finally, expression vectors could be put to use in obtaining large quantities of pure enzyme for basic studies or possibly even for therapeutic use. Of course, not all of the problems of cholinesterase biochemistry and physiology are approachable through molecular biology. In the next section, we take up immunological methods, which have great potential for measuring and localizing particular types of enzyme and for a variety of other interesting purposes.

111. Immunology of the Cholinestemses

A. OVERVIEW AND GUIDE TO TABLES Immunological reagents have been used to study the cholinesterases for over 15 years. Work started with poorly delineated polyclonal antisera and progressed to today's elaborately characterized monoclonal antibodies. Investigations have tended to focus on four general areas: (1) structure and function-including the comparison of equivalent enzymes from different tissues or animal species, (2) localization-including the microscopic visualization of special enzyme forms, (3) quantitation-including the immunoassay of cholinesterase protein, and (4) synthesis and metabolism-including the examination of individual translation products in vitro. Progress in all these areas is considered in the following sections. For the reader's convenience, a good deal of the information has been summarized in tabular form, antibody by antibody. To provide a condensed summary of the uses of antibodies in the study of cholinesterases,we have tried to list every cholinesterase-directed monoclonal antibody or polyclonal antiserum recorded in the literature. In Tables I-VII, the antibodies are grouped according to the animal species

CHOLINESTERASES

373

from which the immunizing antigen was obtained. Polyclonal antisera are denoted by the symbol P; monoclonal antibodiesare individually identified by the code names assigned in the initial publications. For the benefit of those wishing to use antibodies that may still be available, the species crossreactivities are noted where known, along with certain other noteworthy properties. Besides the original description of the antibody, references are made to as many specific applications as possible. Most of these applications are dealt with in the text below.

B. IMMUNOCHEMICAL APPROACHES TO ENZYME STRUCTURE AND FUNCTION Detailed knowledge of antibody-binding sites, or epitopes, on the surface of the AChE molecule would aid in several undertakings. A partial list would include studies of (1) theter-d quaternary structure of the enzyme, (2)the relation between enzyme structure and function, (3) the orientation of the enzyme in the cell membrane and basal lamina, and (4) the interrelationships among the solubility classes and size isomers of AChE. Polyclonal antisera have been helpful in dealing with these issues, but monoclonal antibodies are better suited to experiments on the mapping and biochemical characterization of enzyme epitopes. Work with these reagents is getting underway, and the results are mounting rapidly. 1. Number and Location of Epitopes Most monoclonal antibodies would be expected to recognize nonrepeating epitopes, one per catalytic subunit of AChE. This type of behavior has seldom been demonstrated. One exception is a series of antibodies known to be directed toward nonoverlapping epitopes on human erythrocyte AChE (Brimgoin et al., 1983).Analysis of the sedimentation velocity and gel filtration of immune complexes showed the expected stoichiometry with both monomeric and dimeric AChE (Brimijoin and Mintz, 1985). Larger enzyme forms could not be characterized in the same way, but computer modeling of the AChE-binding curves suggested that steric factors prevent a given antibody from occupying all its epitopes at the same time. Indirect immunolabeling of intact erythrocytes showed that most of the antibodies were directed toward exposed regions of the membrane-bound AChE. One antibody that did not bind to intact erythrocytes, despite good affinity for detergent-solubilized AChE, probably recognized enzyme regions buried in the membrane (Brimijoin and Mintz, 1985).

TABLE I ANTIBODIES To BuChE Antigen source (antibody code) Crude human plasma BuChE (P)“

$2

Partially purified human plasma BuChE (P)“

Partially purified human plasma BuChE (P)”

Highly purified human plasma BuChE (P)“

Species crossreactivity

-

Special properties

Applications

Reference

Ouchterlony immunodiffusion: cross-reacting material absent from sera of homozygotes for “silent” allele Ouchterlony immunodiffusion: cross-reacting material absent from sera of homozygotes for “silent” allele Ouchterlony immunodiffusion: approximately normal content of immunoreactive BuChE in sera of homozygotes for “atypical” allele Radial immunodiffusion, immunotitration: similar Vmaxof “usual” and “atypical” BuChEs, but 30% less immunoreactive protein in “atypical” phenotype

Hodgkin et al. (1965)

Rubinstein el al. (1970)

Rubinstein et a1 (1976, 1978)

Eckerson et al. (1983)

Purified rat brain BuChE (P)” Purified human plasma BuChE (monoclonals B,12-1 and Bz18-5) Purified human plasma BuChE (monoclonals B212-1 and B218-5)

2

Purified human plasma BuChE (monoclonals E34E10-2, E3-9B11-2, and five additional antibodies) “P, polyclonal.

I

Man and higher primates only (Bz12-1); most mammals except guinea pig and rat (B218-5)

-

Cow, Torpedo (E3-4E10-2); Torpedo (E3-9B11-2)

No cross-reactivity with AChE

No cross-reactivity with AChE

Reacts with deglycosylated BuChE (E3-4E10-2); reacts with reduced, denatured BuChE but not deglycosylated BuChE

Immunocytochemistry : localization of cerebellar BuChE to nonneuronal cells (especially astrocytes) Immunochemical comparison by antibody affinity: no difference detected between “usual” and “atypical” BuChE Immunochemical comparison by antibody affinity: progressive modification of BuChE during evolution of higher primates

Barth and Ghandour (1983) Brimijoin et al. (1983)

Mintz et al. (1984)

B. P. Doctor (personal communication)

TABLE 11 ANTIBODIES TO HUMAN AChE Antigen source (antibody code)

t3

Species crossreactivity

Special properties

Erythrocyte: purified AChE (P)"

Weak competitive inhibitor

Erythrocyte: purified AChE (P)"

Reaction with detergent-soluble brain AChE, not with BuChE Reaction with detergent-soluble brain AChE, not with BuChE Preference for GS versus GI AChE

Brain: purified AChE (P)"

$

Primarily man, but also dog, pig, cow, and horse

Erthrocyte: wholemembrane and purified AChE (P)" Brain: detergentsoluble forms (P)"

Erythrocyte (P)"

Primarily man, but also dog, pig, cow, and horse

Moderate preference for detergent- versus salt-soluble AChE, no reaction with BuChE

Applications Agglutination reaction localizes AChE to external surface of red cell

Reference Niday et al. (1977)

-

Sptrensen et al. (1982)

-

S6renson et al. (1982)

Immunological evidence that dimeric AChE is native form in human erythrocyte Tandem crossed immunoelectrophoresis: demonstration of immunochemical identity between detergent-soluble AChE of brain and erythrocyte Solid-phase assay of AChE activity: detection of AChE in amniotic fluid, screening for neural tube closure defects

Ott et al. (1983)

Gennari and Brodbeck (1985); Sptrensen et al. (1985)

Norgaard-Pedersen et al. (1983); Hangaard et al. (1984)

Erythrocyte (monoclonal AEl-AE5)

Man, monkey, rabbit, guinea pig, dog, and cow. Not rat, chicken, or frog

Cross-react with muscle and brain AChEs

(AEl and AE3)

Lu -3

-3

(AE1-AE4)

Erythrocyte (monoclonais A163, A123, and An45)

Man only (A163); man and monkey only (A123); monkey, rabbit, guinea pig, but not dog, burro, sheep, goat, pig, cow, or rat (A245)

No reaction with BuChE

Immunocytochemistry : immunoperoxidase and immunofluorescence staining of neuromuscular junction (light microscope) Immunocytochemical localization of AChE in monkey visual cortex Demonstration of immunochemical equivalence of Gl and G, forms from human brain Solid-phase assay of AChE activity: detection of AChE in amniotic fluid; screening for neural tube closure defects Immunoprecipitation of AChE from radiolabeled erythrocytes. Demonstration of AChE deficit in paroxysmal nocturnal hemoglobinuria Quantitative determination of antibody affinity shows immunochemical identity of brain and erythrocyte AChE

Fambrough et al. (1982)

Hedreen et al. (1984)

Marsh et al. (1984)

Brock et al. (1985)

Chow et al. (1985)

Brimijoin et al. (1983)

(continued)

TABLE I1 (continued) _

Antigen source (antibody code)

Species crossreactivity

Special properties

“P, Polyclonal.

~

~~

Applications

~

A,63, A123, and A245

_

_

~

~

~

_____

Rapid purification of rabbit brain AChE by immunoaffinity chromatography Study on localization, number, and affinity of antibody-binding sites on different AChE molecular forms Quantitative imrnunodisplacement assay of tissue extracts; irnmunofluorescence cell sorting; characterization of AChE deficit in paroxysmal nocturnal hemoglobinuria

Reference ~~

~

Mintz and Brimijoin (1985a) Brimijoin and Mintz (1985)

Brimijoin el al. (1986)

CHOLINESTERASES

379

2. Comparison of Hydrophilic and Hydrophobic AChEs Immunochemical methods are suitable for determining whether the different molecular forms of the cholinesterases are composed of structurally distinct subunits. Attempts to answer this question have revealed immunochemical differences between the hydrophobic forms of AChE (membrane-associatedenzymes) and the hydrophilic forms (water-soluble enzymes). The first such evidence was obtained by Zanetta et al. (1981), who raised a rabbit antiserum against Triton-solublized AChE from rat brain membranes. This antiserum had low cross-reactivity for the watersoluble forms of AChE from the same tissue. A similar result was later reported by Sorensen et al. (1982), although Marsh et al. (1984) were unable to repeat it. Taking the findings at face value, it seems that at least some antisera can recognize immunologic differences between membrane-bound and soluble AChE of mammalian brain. Certain monoclonal antibodies also distinguish among the solubility classes of brain AChE. For example, one of our antibodies to rabbit AChE binds hydrophilic brain enzyme much less readily than its hydrophobic counterpart (Mintz and Brimijoin, 198513; Rakonczay and Brimijoin, 1985). This antibody, however, is not directed toward the hydrophobic linkage per se, since it binds the purely hydrophilic serum AChE just as avidly as any of the molecular forms from brain. In addition, the antibody shows a consistently higher affinity for Gq AChE than for GI forms. The basis for these preferences remains unknown. Further studies of brain AChE might exploit the fact that the hydrophobic linkage of membrane-bound cholinesterases is quite sensitive to proteases (Lockridge and La Du, 1982; Dutta-Choudhury and Rosenberry, 1984; Weitz et al., 1984). It would be worth determining whether partial proteolysis simultaneouslyimpairs the ability to combine with nonionic detergent and with form-selective antibody. This is one way to map epitopes with respect to specialized portions of the polypeptide chain. Immunochemical differences between hydrophilic and hydrophobic AChEs have also been observed in a study of monoclonal antibodies to the Torpedo electroplax enzyme (Doctor et al., 1983). One antibody was found to bind the hydrophobic 5.6 S dimer 100 times better than the hydrophilic tetramers from proteolysed asymmetric forms. Perhaps there is a common explanation for these results and the similar findings with brain enzymes. Reasonable candidates are (1) specific targeting of the hydrophobic elements responsible for linking AChE to cell membranes or (2)targeting of other structural features that happen to differ among the solubility classes of AChE. In either case, posttranslational mechanisms are a conceivable source of the immunochemical heterogeneity. However, Doctor et al. (1983) found at least two distinct peptides in the

TABLE 111 ANTIBQDIES TO RODENT AChE ~

~~

~

~

~~

Antigen source (antibody code) W

W 0

Mouse brain: partially purified soluble AChE (P)”

Rat brain: membranebound G, AChE (P)”

Rat brain: membranebound G4 AChE (P)”

~

Species crossreactivity Rat

Special properties Precipitating antiserum

I

Rat = mouse > man > cow; no reactivity for chicken, eel, or ray

No precipitation of “soluble” AChE; modest enzyme inhibition Up to 70% inhibition of AChE from rat or man, but not cow

Applications

Reference

To show immunological identity among muscle, brain, and erythrocyte AChE. To show immunological differences between AChE of rat superior cervical ganglion (strong reaction) and BuChE (no reaction) To demonstrate imrnunochemical dqference between soluble and membranebound brain AChE To demonstrate irnmunochemical similarities between soluble and membrane-bound brain AChE

Adamson (1977); Vigny el al. (1978b); Koelle et al. (1979)

Zanetta et al. (1981)

Marsh

et

al. (1984)

Rabbit brain: membrane-bound C, AChE (monoclonals F543, F,49 and nine additional antibodies)

Rabbit only (Fs43); rabbit and guinea pig (F443); various mammals but not nonmamrnalian enzymes (other antibodies)

Rat brain: membranebound C, AChE (seven monoclonals ZR1-ZR7)

Mouse, man, rabbit, guinea pig, cow, and cat, but not chicken, frog, or eel (ZR1); various mammals but not mouse or nonmammalian enzymes (other antibodies)

~~

~~

"P,Polyclonal.

>90% inhibition of AChE activity, but n o effect on BuChE (Fs43); partial discrimination between size isomers and solubility forms (F.43); no affinity for BuChE Bind Gr in preference to GI; does not bind erythrocyte AChE or BuChE (ZR1); no affinity for BuChE (ZR1ZR7)

Analysis of relation between binding sites for antibody, curare, propidium iodide, decamethoniurn, Nmethylacridiniurn, and diisopropyl fluorophosphate

Mintz and Brimijoin (1985b); Brirnijoin et al. (1985); Rakonczay and Brimijoin (1985)

Immunofluorescence cytochemistry of rat spinal cord and striatum (ZR1ZR7 pool)

Rakonczay and Brimijoin (1985, 1986)

TABLE I V ANTIBODIESTO BOVI~SE AChE Antigen source (antibody code)

Species crossreactivity

Special properties

Bovine erythrocyte: commercial preparation (P)"

-

Bovine erythrocyte commercial preparation (P)" Bovine caudate nucleus (PI"

-

Stabilizes enzyme against heat and partially reactivates denatured AChE; similar effect with Fab and F(ab')n Weakly inhibitory (2550%), but stabilizing

Not with Electrophorw

Bovine caudate nucleus (P)"

Man, dog, pig, and horse

Fetal bovine serum (monoclonals 10H 10, 10C05, and five additional antibodies

Eel, Torpedo, man (10H 10); Torpedo (10C05)

P, Polyclonal.

Inhibitory (50%)

electricw

Weak preference for membrane-bound versus soluble form Cross-reactivity with human BuChE (10H 10)

Applications

Reference

-

Michaeli et al. (1969a,b); Holmes et al. (1973a)

-

Holmes et 41. (1973b)

Analysis of cross-reactivities by immunodiffusion, immunoelectrophoresis, and microcomplement fixation

Greenberg et al. (1977)

Spjrensen et al. (1985) -

B. P. Doctor (personal communication)

TABLE V ANTIBODIES TO AChE OF Electrophorus electricw Antigen source (antibody code) Electric organ: commercial preparation (P)" Electric organ: purified, globular (P)"

Species crossreactivity

Not cow (erythrocyte) -

Special properties Inhibitory

u l

Electric organ: purified, globular, and asymmetric forms (P)" Electric organ: purified, globular, and asymmetric forms (P)" Electric organ: purified, globular, and asymmetric forms (P)" Electric organ: purified, globular, and asymmetric forms (P)" Same antibody

"P, Polyclonal.

Not cow (brain)

Williams (1969) Fluorescence, immunocytochemistry: electroplax, brain, and spinal cord Fluorescence, immunocytochemistry: electroplax, brain, spinal cord, and muscle Analysis of immunochemical effects of neuraminidase

Torpedo

Not cow (erythrocyte)

Reference

(70-80%)

Electric organ: commercial preparation (P)"

$

Applications

Equivalent reactivity for all molecular forms; weakly inhibitory Inhibitory (50%)

Studies on inhibition indicated antibodies not active site directed

Benda et al. (1970)

Tsuji et al. (1972)

Rieger et al. (1973, 1976)

Gurari et al. (1974)

Greenberg et al. (1977)

Schastosoma mansoni

Analysis of relation between asymmetric AChE and collagen

Anglister et al.

Evaluation of cytotoxicity toward parasites

Tarrab-Hazdai et al.

(1979)

(1984)

TABLE V I ANTIBODIES TO Torpedo AChE Antigen source (antibody code) Torpedo mamorata (P)”

w

g

Torpedo califomica globular and asymmetric forms (monoclonals 4E3, 4E7, and seven others) Torpedo c a l f m i c a globular forms (monoclonals TclO1104; Tc201-204)

Species crossreactivity E lectropkorus electricus

-

Narhe japonica (in varying degrees)

Nark japonica nerve terminal membrane (Nj501)

-

Narke japonica nerve terminal membrane (Nj601)

-

“P, Polyclonal.

Special properties

100-fold preference for 5.6 S globular dimer (4E7); selectivity for asymmetric forms (4E3) Marked enzyme inhibition (T,102, 104,202, and 204); 100-fold preference for detergent-soluble AChE (Tc201) Markedly inhibitory Reacts with all molecular forms

Applications Demonstration of cross-species homologies Analysis of structural differences among catalytic subunits

Reference Rieger et al. (1976) Doctor et al. (1983)

Sakai et al. (1985)

Western blot analysis of AChE subunits: fluorescence immunocytochemistry Immunoaffinity chromatography

Abe et al. (1983) Sakai et al. (1985)

TABLE VII

MISCELLANEOUS ANTIBODIES TO AChE ~

Antigen source (antibody code) Nematode worm (Na#mstrongylur brmiliensis)

Species crossreactivity

-

Special properties

-

Chicken brain (P)"

Only chicken and quail

No reaction with BuChE; equivalent affinity for all moIecular forms

Chicken brain (monoclonal 1A2)

Only chicken and quail

No reaction with BuChE; equivalent affinity for all molecular forms

"P, Polyclonal.

Applications

Reference

Active (natural) and passive immunization of rats affected survival of infesting worms and AChE production by the parasites Immunoprecipitation of radiolabeled AChE synthesized in organ culture. Immunoblotting of AChE after SDS gel electrophoresis; immunocytochemistry of neuromuscular junction (light microscope) Immunoprecipitation of radiolabeled AChE synthesized in organ culture. Immunoblotting of AChE after SDS gel electrophoresis; immunocytochemistry of neuromuscular junction (light microscope)

Jones and Ogilvie (1972)

Rotundo (1984a,b)

Rotundo (1984a,b)

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STEPHEN BRIMIJOIN AND ZOLTAN RAKONCZAY

tryptic digest of each AChE class from Torpedo. As discussed in Section 11, the immunologic and biochemical evidence of diversity in catalytic subunits is thus consistent with the results of in vitro translation experiments pointing toward genetic or transcriptional heterogeneity. A search for multiple AChE genes is certainly warranted. 3. Comparison of AChEs from Different Tissues Definite but mysterious immunologic differences are also being observed among the AChEs of different tissues, particularly brain and erythrocyte. Most of the reported AChE antisera and antibodies show equivalent affinity for the enzymes from these two sources (Fambrough et al., 1982; Brimijoin et al., 1983), but there are exceptions. Some antisera against human erythrocyte AChE are markedly poorer than antisera against human brain AChE in binding AChE from horse brain (S6rensen et al., 1985). More striking is the behavior of ZR1, a monoclonal antibody to rat brain AChE. This antibody is virtually incapable of binding the AChE of rat erythrocytes, though it does bind brain AChE forms of the same size and solubility class (Rakonczay and Brimijoin, 1985, 1986).The immunochemical results are consistent with lectin-binding data, suggesting biochemical differences between neural and erythrocyte AChE (Meflah et al., 1984). At present, tissue-specific properties of AChE are most likely to reflect differences in posttranslational processing, such as the nature or extent of glycolysis. However, studies of AChE binding by the form-specific antibody ZR1 have failed to uncover interactions with any of several lectins including concanavalin A, Ricinzls communis, Lotus tetragonolobus, wheat germ agglutinin, or soybean lectin (2. Rakonczay and S. Brimijoin, unpublished results). 4. Immunologic Studies of AChE Tail Components

The tail component of asymmetric AChE forms that predominate in synaptic basal lamina is often considered collagen-like.This view is based on the high content of hydroxyproline and hydroxylysine, the tendency to associate with polyanionic compounds such as chondroitin sulfate, and the sensitivity to collagenase (Bon et al., 1978; Silberstein et al., 1982; Anglister et al., 1979). The tail component also has a collagen-like triplehelical structure (Mays and Rosenberry, 1981).Despite these properties, it is controversial whether the tail component is closely related to collagen. The relevant immunochemical data are conflicting. Anglister et al. (1979) reported that asymmetric AChE from the electric eel reacted with an antiserum to collagen from rat tail tendon. On the other hand, Grassi et al. (1983) were unable to demonstrate specific interaction between asymmetric AChE of eel or cow and any of a large series of antibodies, including

CHOLINESTERASES

387

antisera against type I collagen from rat skin, type 111collagen from cow, and type IV collagen from mouse, chicken, or cow. Weak cross-reactivity was observed with antisera to type I collagen from rat tendon and type IV collagen from man, but the specificity of the cross-reactivitywas doubtful. Thus, antibody binding of iodinated AChE was inhibited by excess globular AChE (i.e., a tail-less form), but not by excess collagen (Grassi et al., 1983). The results of this competition experiment were attributed to the presence of serum components that bind AChE and other proteins without any clear antigen specificity To clarify the issue, the immunologic relation between AChE and collagen should be reexamined with the aid of monoclonal antibodies raised against various forms of these proteins from a single species. 5. Immunologic Analysis of BuChE

It is well recognized that there are multiple alleles coding for BuChE. These alleles were identified and defined many years ago on the basis of their substrate kinetics and sensitivity to inhibitors (for review see Harris, 1980). In man, there are at least three such alleles, commonly designated EP (coding for the “usual type” serum BuChE), Ef (coding for “atypical” serum BuChE), and Ei (associated with a “silent phenotype” with greatly deficient amounts of serum BuChE activity and immunoreactivity). There is clinical interest in these enzyme variants, since they correspond to individual differences in susceptibility to muscle relaxant drugs such as succinylcholine, used as adjuvants in general anesthesia (Kalow and Genest, 1957; Kalow and Davies, 1958). The detailed genetic information makes immunochemical methods superfluous for defining gene products, but an antibody that distinguished among the different types of enzyme would certainly have clinical value. Unfortunately, no such antibodies are known. The rabbit antisera obtained by Eckerson et aE. (1983) and the monoclonal antibodies obtained in this laboratory (Brimijoin et al., 1983) all have identical affinity for human serum BuChE of the normal and atypical varieties. This finding is not surprising since the difference between the two proteins is beiieved to be minor. Recent analysis of peptide fragments containing the active site serine, for example, showed identical amino acid sequences (Lockridge et al., 1985). It is nonetheless worth attempting to generate active-site directed monoclonal antibodies to BuChE that might recognize the features responsible for the altered catalytic efficiency of the atypical enzyme. 6. Effects of Antibodies on Esterase Activity A surprising number of antibodies against AChE are able to interfere with the cataIytic function of this enzyme (see Tables I-VII). In fact, the

388

STEPHEN BRIMlJOIN AND ZOLTAN RAKONCZAY

very first reported AChE antiserum showed this property (Williams, 1969), though only 1 out of 23 in our recent series of monoclonal antibodies was inhibitory. Do the inhibitory antibodies bind to a component of the catalytic site? If so, they could be used profitably for studies of the chemistry of this region. If not, they might help us understand how allosteric and conformational effects can influence cholinesterase activity. It seems improbable that many of the inhibitory antibodies are directed toward the active site of AChE, but there have been few attempts to settle the question experimentally. Gurari et al. (1974) found that their antiserum no longer inhibited electric eel AChE after conversion to monovalent Fab fragments. They also noted that inhibition was only evident when assays were performed with “good” (i.e., rapidly hydrolyzed) substrates. It was therefore suggested that the antiserum might act by restricting the diffusion of substrate into the active site. Very few antibodies are capable of inhibiting AChE by more than 70%. This is a reason for suspecting targets other than the active site. With plyclonal antisera, such as the ones described by Williams (1969),Gurari et al. (1974),and Marsh et al. (1984),limited inhibition might be explained by a specialized subfraction of immunoglobulin in very low titer. This explanation does not apply to monoclonal antibodies such as the one obtained by Abe et al. (1983) against Torpedo AChE or the one obtained in our laboratory against rabbit brain AChE (Mintz and Brimijoin, 1985b). The latter antibody is of particular interest for its considerable power M) and potency (50%blockade at lo-’ M). (>90%blockade at Detailed study of the inhibitory antibody to rabbit brain AChE (Brimijoin et al., 1985) revealed several interesting findings, which may be summarized as follows: (1) the effect on enzyme kinetics was complex with a decrease in V,,, and a large increase in optimal substrate concentration, (2) similar effects were produced by Fab fragments, (3) enzyme labeling by f’H]diisopropyl fiuorophosphate (DFP)was blocked, (4)antibody affinity for DFP-labeled enzyme was moderately reduced, (5) antibody affinity was also reduced in the presence of reversible inhibitors such as curare and decamethonium, and (6) the antibody interfered with the inhibition of AChE by propidium iodide, a fluorescent probe for the secondary anionic site. Most of the observations could be explained by hypothesizing that the antibody recognizes an epitope in or near the secondary anionic site of AChE. However, fluorescence analysis of propidium binding in the presence and absence of antibody showed only weak, nonstoichiometric competition. We have therefore concluded that the antibody-induced inhibition of AChE results from conformational changes that interfere with efficient catalysis. A recent report by Sakai et al. (1985) described four monoclonal

CHOLINESTERASES

389

antibodies that inhibit AChE from Torpedo californica. The effect was always greater with asymmetric AChE than with detergent-soluble,membrane-associated forms. Two of the antibodies actually caused total inhibition of the hydrophilic, asymmetric AChE. The differential effect on the two types of AChE may well be related to the bulky detergent micelles needed to keep the hydrophobic forms in solution. A likely result is masking of certain epitopes or steric hindrance to antibody binding. In view of Saki’s findings, future experiments on the mechanism of antibody-induced inhibition should focus on hydrophilic AChE.

C. INTERENZYME AND INTERSPECIES COMPARISONS OF PROTEIN STRUCTURE 1. Comparisons between AChE, BuChE, and Related Enzymes From studies on substrate kinetics, mechanism of action, and activesite chemistry, the cholinesterases are classed with other aliphatic and aromatic esterases. Furthermore, all of the esterases show numerous points of resemblance to the serine proteases: trypsin, chymotrypsin, and thrombin (Augustinsson, 1948, 1963). The biochemical similarities might imply a common evolutionary origin. However, as mentioned earlier, the amino acid sequence information derived from cDNA clones offers little support for this view. If these great enzyme families did stem from a single ancestor, the divergence must have occurred early on, while the divergence of AChE and BuChE came much later (Augustinsson, 1968). Thus, as already discussed, the amino acid sequencesof AChE and BuChE show substantial homology, but comparisons of the cholinesterases with any of the serine protease enzymes reveal very few points of resemblance (Schumacher et al., 1986a,b). It is therefore appropriate to suggest that the biochemical similarities reflect convergent evolution instead of ancestral relationships. When cDNA clones have been obtained for a large number of cholinesterases, the exact relationships among the primary structures of these enzymes will be determined. Meanwhile, comparative analyses of antibody reactivities allow us to begin cataloging similaritiesand differences among many enzymes, even though it is difficult to determinejust what structural features account for the observations. Immunochemical information bearing on the phylogeny of the cholinesterases is still limited. Most of the antibodies and antisera obtained to date have rather narrow specificities. For example, the vast majority of the polyclonal or monoclonal antibodies raised to AChE have no measurable affinity for BuChE (Vigny et al., 1978b;Koelle et al., 1979;Brimijoin

390

STEPHEN BRIMIJOIN AND ZOLTAN RAKONCZAY

et al., 1983; Mintz and Brimijoin, 1985b; Rakonczay and Brimijoin, 1986; Rotundo, 1984b). Likewise, none of the antibodies or antisera raised against BuChE react with AChE. This lack of interenzyme cross-reactivity provided early evidence against the notion (Koelleet al., 1977) that BuChE constitutes a precursor of AChE. Of course the idea has now been ruled out by gene-sequencing data. It has been proposed that cholinesterases are related to, or even identical with, serum aryl acylamidases (George and Balasubramanian, 1980, 1981). Choiinesterase and aryl acylamidase activities copurify by several chromatographic and electrophoretic methods, starting from tissues such as eel electroplax and mammalian brain, red cell, and sera (Tsujita and Okuda, 1983; George and Balasubramanian, 1981). Putatively homogeneous preparations of AChE and BuChE exhibit aryl acylamidase activity (George and Balasubramanian, 1981; Majumdar and Balasubramanian, 1984).With one or two exceptions, purified aryl acylamidases have cholinesterase activity (George and Balasubramanian, 1981). Immunological evidence supports the proposed association between cholinesterases and arylamidases. Antisera raised against eel electroplax AChE precipitate aryl acylamidase activity from the same tissue (Majumdar and Balasubramanian, 1984). Also, antisera against human serum aryl acylamidase precipitate this enzyme activityand cholinesteraseactivity to equivalent degrees in all cross-reacting species (George and Balasubramanian, 1981, 1983).Such results do not absolutely rule out cross-contamination as an explanation for the associated enzyme activities. Nevertheless, at least in the case of human serum BuChE, the argument that cholinesterase and aryl acylamidase activities do represent partially overlapping active centers on the same molecule is certainly defensible. It would be worthwhile to consider the question further by quantitative affinity determinations with a large series of monoclonal antibodies. Active site titrations should also be carried out to establish whether the correspondence between esterase and acylamidase loci is one-to-one. The relation between cholinesterasesand peptidases is another subject that calls for immunological investigation. Recent work has established that purified, electrophoretically homogeneous preparations of BuChE and AChE are capable of hydrolyzing neuropeptides such as substance P (Lockridge, 1982; Chubb et al., 1980). It remains unclear whether the two enzymatic functions are located on the same catalytic subunits. Chatonnet and Masson (1985) have shown that the peptidasic site associated with human plasma BuChE shares many properties with the esteratic site, including the presence of essential serine and histidine residues. These results might suggest overlapping sites on the same molecule. However, the same authors cited preliminary results indicating that the number of

CHOLINESTERASES

391

titratable peptidasic sites was a small fraction of the number of esteratic sites. The most plausible location of the peptidase activity is therefore (1) on an isozyme devoid of cholinesterase activity or (2)on a rare variant with a mutated active site possessing both cholinesterase and aryl amidase activity. The alternative possibilities might be distinguished with the aid of suitably selective monoclonal antibodies to human BuChE.

2. Comparisons of Cholinesterases from Dqferent Animal Species Immunochemical comparisons of the cholinesterases from different animal species have been somewhat more fruitful than comparisons across enzyme classes, perhaps because one can deal with smaller evolutionary gaps. Even in this case, narrow specificities are the rule. It is well-known that polyclonal antisera to eel electroplax AChE tend not to cross-react with the AChE from bovine caudate nucleus and vice versa (Greenberg et al., 1977).Rotundo’s polyclonal and monoclonal antibodiesto chicken brain AChE fail to recognize the cholinesterase of any nonavian species (Rotundo, 1984b). Most monoclonal antibodies to mammalian AChEs react only with the comparable enzyme from other mammals (Brimijoin et al., 1983; Mintz and Brimijoin, 1985b; Rakonczay and Brimijoin, 1986). This sort of narrow specificity was used advantageouslyin one instance to investigate the phylogenetic relationshipsamong primate BuChEs. The study of BuChE phylogeny focused on a monoclonal antibody to the enzyme from human plasma BuChE. This antibody (B212-1)had little or no affinity for the cholinesterases of subprimate species or even for the BuChE of squirrel monkey (Brimijoin et al., 1983). When plasma samples from a series of higher primates were tested, on the other hand, there was excellent cross-reactivity (Fig. 4).Quantitative measurements established the following order of affinities for the BuChEs of different species: man > chimpanzee = pigmy chimpanzee > gorilla S orangutan > gibbon S monkey (Mintz et al., 1984). It is interesting to note the correspondence between these results and the phylogenetic order deduced from global complementarity of total cellular DNA (Tobias, 1975).Since the nature of the epitope recognized by Bz12-1 is unknown, we cannot say whether the progressive alteration in immunochemistry reflects changes in the primary structure of BuChE. Quite possibly the reactivity is altered because of modifications of protein conformation during the later stages of primate evolution. Some observations suggest that monoclonal antibodies to mammalian cholinesterases are often directed toward epitopes that are sensitive to protein conformation or to posttranslational processing. For example, the majority of our own anti-AChE antibodies exhibit reduced affinity for enzyme that is sodium dodecyl sulfate (SDS)-treated,radioiodinated, or

392

,-lOOr

i05 W

a

40

20

STEPHEN BRIMIJOIN AND ZOLTAN RAKONCZAY

B

FIG. 4. Affinities of monoclonal antibodies for serum BuChE from higher primates. Serum samples, with estimated BuChE concentration
heat-denatured (S. Brimijoin, K. P. Mintz, and Z. Rakonczay, unpublished results). As mentioned earlier, Soreq et al. (1982) has demonstrated that Xenopus oocytes injected with human brain mRNA synthesizeand secrete what is presumed to be human brain AChE. In an attempt to characterize this translation product, Soreq tested it with several monoclonal antibodies known to have high affinity for human brain AChE (Brimijoin et al., 1983). Since none of the antibodies specifically bound the secreted enzyme (H. Soreq, personal communication), it clearly lacked the appropriate epitopes. A possible explanation is that posttranslational processing of human AChE in amphibian oocytes fails to establish the true native conformation of the enzyme, even though it supports activation and secretion. 3. Future Applications of Species-Spec$c Antibodies Regardless of whether conformational or sequential determinants are involved, antibodies with sharply defined species specificity may be helpful in determining the source of cholinesterases in mixed cell systems. For

CHOLINESTERASES

393

example, antibodies with exclusive preference for mammalian and avian AChEs, respectively, can clarify the cellular origin of synaptic enzyme in mixed cultures of neurons and myocytes. De La Porte et al. (1986) have just used such antibodies to prove that the AChE patches on innervated myotubes arise solely from the muscle cells. Species-selective antibodies could also reveal the genomic origin of the enzyme in chimeric cells (e.g., rat-mouse hybridomas). Suitable immunochemical tools are available for genetic analysis of AChE (see Tables 1-VII), but the experiments remain to be tried.

D. IMMUNOCYTOCHEMISTRY 1. Rationale and Applications

A principal use for antibodies in neurobiology is the microscopic localization of important proteins, i.e., immunocytochemistry. The principles of immunocytochemistry are well described in standard works (Sternberger, 1979). It can be mentioned in passing that most methods depend on the binding of an unlabeled primary antibody which is detected by means of a labeled conjugate (e.g., fluorescein- or peroxidase-tagged anti-IgC from another animal species). If the primary antibody binds tightly enough to survive rinsing and washing steps, spectacular sensitivity can be obtained with the right conjugate, especially avidin-biotin systems that increase the density of labeling (Hsu et al., 1981). The specificity of the immunocytochemical reaction is also potentially great, but relies critically upon the binding properties of the primary antibody. Elaborate controls are often necessary. With polyclonal antisera, absorption experiments are commonly performed to confirm loss of reactivity after removal of components reacting with authentic antigen. With monoclonal antibodies, it is more usual to use only “irrelevant” IgG from nonimmunized animals or from a random hybridoma. This sort of control is sometimes inadequate, as we shall see. Immunocytochemistry is crucial for studies of peptides and nonenzymatic proteins, whose cellular and subcellular distribution cannot easily be revealed in other ways. The cholinesterases, on the other hand, have been approachable through a variety of histochemical procedures based on enzyme activity. Thus, thiocholine methods (Koelle and Friedenwald, 1949; Karnovsky and Roots, 1964) have been used for over 35 years, with various modifications, to identify cholinesterase-containing cells and tissues at the light and electron microscope level. Although the methods are not intrinsically specific for acetylcholinesterase or butyrylcholinesterase, the two enzyme families can be delineated by the use of appropriate enzyme inhibitors. What can immunocytochemistry tell us about cholinesterases that we

394

STEPHEN BRIMIJOIN AND ZOLTAN RAKONCZAY

could not discover with classical histochemistry? T h e potential answers are (1) the localization of enzyme subtypes that do not differ in terms of catalytic activity (i.e., the size or solubility isoforms of AChE or BuChE, (2) the localization of inactive enzyme, (3) the identity of living cells bearing the enzyme on their external surfaces, and (4) the changes in localization of enzyme molecules over time. Under favorable circumstances, immunocytochemical methods may in addition yield superior definition of fine neuronal processes.

2. Technical Obstacles At present, the potential advantages of immunocytochemistry are incompletely achieved, in part because of inherent obstacles. Immunocytochemical results are particularly sensitive to the changes that take place in tissue antigens during preparation for microscopy. Comparatively mild fixation (2% formalin for 2 hr) sometimes eliminates antigen-reactivity with certain antibodies. T h e glutaraldehyde fixation procedures favored for electron microscopy are even more damaging. Nevertheless, to preserve basic tissue structure and to facilitate penetration of antibodies into the cell interior, some fixation is usually needed. The difficulty is to balance structural preservation and antibody access on the one hand, with antigenic reactivity on the other. This balance can frequently be struck with antisera, heterogeneous mixtures of many different antibodies, some of which may behave suitably under any given set of conditions. The all-ornone nature of monoclonal antibodies, although desirable for other applications, poses special problems. It is usually necessary to compare several fixation regimes, differing in composition, concentration, and time of exposure to fixative (acetone or acid ethanol are sometimes superior to formalin; occasionally one can avoid fixation altogether). Permeabilizing agents like Triton X-100 need to be evaluated. In addition, multiple dilutions of primary antibody must be tried. Finally, if indirect methods are used, different secondary antibodies must be investigated. Since each antibody is a special case, establishing the right conditions for monoclonal immunocytochemistry is labor intensive and not always successful. An even greater obstacle to successful immunocytochemistry is the task of obtaining antibodies that have a desired specificity along with high affinity for their antigenic targets. Antigen-bound antibody with low affinity is lost while nonspecifically bound antibody is rinsed away from tissue sections. Low affinity is commonly encountered with monoclonal antibodies unless hybridomas are screened for high affinity (antigendissociation constants of lov6 to lo-' M are typical in our experience), Recently an anti-cholinesterase antibody with Kd = 1W9M was shown to dissociate from AChE with a half-time of 13 min (Brimijoin et al., 1985). This probably represents the lower limit of cytochemically useful affinity.

CHOLINESTERASES

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3. Immunocytochemistry of Nonmammalian Chohesterases Despite the obstacles, polyclonal and monoclonal antibodies have been successfully used to localize cholinesterases at the light microscope level. Benda et al. (1970) reported the first immunocytochemical study of AChE in the electric eel. They examined the enzyme in brain, spinal cord, and electroplax by means of indirect immunofluorescence with a polyclonal rabbit antiserum. In the electroplax, there was bright fluorescence staining along the external membrane foldings of the innervated face, while the noninnervated face stained faintly but distinctly. The authors were impressed by the absence of diffusion artifacts. A direct comparison of classical histochemistry and irnmunofluorescence in the same tissue revealed strikingly similar results (Tsuji et al., 1972). There have been few recent immunocytochemical studies of AChE in electroplax. Immunofluorescence staining patterns suggested to Abe et al. (1983) that a monoclonal antibody to Nurkejaponica synaptosomes was actually an anti-acetylcholinesterase.Kushner (1984)investigated another monoclonal antibody (TOR-23) raised to synaptosomes from the electric organ of a related fish, 'T: californica. Like antibody 4E7 of Doctor et al. (1983),TOR-23 preferentially binds the hydrophobic G2 AChE of synaptosomes, but reacts poorly with the hydrophilic enzyme forms of the postsynaptic electroplax (F? Kushner, personal communication). Not surprisingly, immunoperoxidasestaining showsthat TOR-23's target is largely confined to presynaptic tissue (Buckley et al., 1983). This finding is significant because the gross preponderance of postsynaptic enzyme mak& it difficult to demonstrate presynaptic AChE by other methods. It is worth mentioning that TOR-23 stains a rare antigen of rat brain. The antigen, however, is not AChE since, according to F! Kushner (personal communication),(1) it has a molecular weight of 175,000 under reducing conditions and (2) the staining is primarily in nonesterase positive regions, although there is some overlap with known cholinergic systems. Also tests in our laboratory show that the antibody fails to react with any molecular form of solubilized rat brain AChE (S. Brimijoin and F? Hammond, unpublished results). Although they are of considerable interest in their own right, these findings underscore the dangers of uncritically interpreting immunohistochemical data obtained with heterologous antibodies. Even homologous antibodies can give misleading results. For example, working with a monoclonal antibody to choline acetyltransferase of Drosophilu, P. Salvaterra (personal communication) noted strong cross-reactivity with the product of another gene that was 10-fold more abundant in the neural DNA. The immunochemical-staining pattern of the antibody, therefore, probably represented the combined localization of two structurally dissimilar proteins. One must always bear in mind the possibility that unrelated antigens bear equivalent epitopes, especially when the specificity of the

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antibody probes has not been directly demonstrated. Neither an absorption control nor an irrelevant IgG control will ensure against misleading results in such cases.

4. Cholinesterase Immunocytochemistry in Mammals Some cholinesterase immunocytochemistry has been carried out in mammals. Barth and Chandour (1983) used a specific polyclonal rabbit antiserum to characterize cellular localization of BuChE in rat brain. They showed bright immunofluorescence staining of astrocytic cell bodies and processes, but no staining of neurons, observations nicely complementing those obtained with classical methods and specific enzyme inhibitors (Brown and Palay, 1972; Hosli and Hosli, 1970; Kasa et al., 1965; Kasa and Csillik, 1966). One difference was a lack of immunostaining in capillary endothelia, which are typically positive by the activity-based methods. As Barth and Ghandour (1983) suggest, the BuChE of these structures may be immunochemically distinct from the predominant form in brain. Most immunocytochemical studies of mammalian AChE have been performed with monoclonal antibodies. Such a study was described by Fambrough et al. (1982)in their original report of monoclonal antibodies to human erythrocyte AChE. There was good staining in neuromuscular junctions from several animals, including monkey, guinea pig, rabbit, dog, and cow, but not rat, chicken, or frog. Two of the antibodies were subsequently used by Hedreen et al. (1984)for a thorough irnmunocytochemical examination of the monkey visual cortex. According to these authors, the antibody method, utilizing a peroxidase-labeled second antibody, improved the visualization of fine neural processes (Fig. 5). High definition enabled the demonstration of several distinct plexuses of AChE-containing axons, which were especially dense in layers I, IIB, IV, and VIB of Hassler and Wagner (Hedreen et al., 1984). Immunocytochemistry of rodent AChE became possible recently, when monoclonal antibodies with reasonable affinity for the rat brain enzyme were finally obtained (Rakonczay and Brimiljoin, 1986). Indirect immunofluorescence experiments with these antibodies revealed specific staining of large motor neurons in the ventral horn of the rat spinal cord, as well as densely packed smaller neurons in the caudate nucleus (Rakonczay and Brimijoin, 1986). Axonal networks in the rat brain have not yet been demonstrated by this approach. Some of our anti-rat AChE antibodies have an interesting selectivity for particular molecular forms of AChE (poor binding of G1enzyme from erythrocytes and brain, in comparison with larger forms). Unfortunately, the most selective antibodies have the lowest affinity overall (Rakonczay and Brimijoin, 1986).This fact has so far precluded form-specific immunocytochemistry.

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FIG. 5. Immunocytochemistry of AChE in monkey visual cortex. Fine axonal processes with dark-staining enlargements (arrows) are seen in sections from layer IIIC (A) and layer I (B). Magnification approximately X 450. (Taken from Hedreen et al., 1984; reprinted with permission from the Journal of Comparative Neurology, Alan R. Liss, Inc.).

5. Vital Immunocytochemistry As mentioned earlier, immunostaining of live cells is a potentially powerful application of specific anticholinesterase antibodies. AChE-containing cells tend to expose the enzyme on their external surface (McIsaac and Koelle, 1959; Tennyson et al., 1968; Brimijoin et al., 1978; Younkin et al., 1982; Fernandez and Stiles, 1984; Rotundo and Fambrough, 1980a; Goudou et al., 1985).Therefore vital immunostainingmay identify cholinesterase-bearing neurons in tissue culture. Combined studies of choline

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acetyltransferase and AChE show that not all esterase-positive neurons are cholinergic, but the exceptions are largely confined to specific brain regions, like cerebellum or substantia nigra (Eckenstein and Sofroniew, 1983; Levey et al., 1983; Satoh et al., 1983; see also review by Greenfield, 1985). AChE therefore continues to be a useful marker for presumptively cholinergic cells, and it is the only cholinergic marker known to be accessible in intact cells. Human erythrocytes were the first live cells studied with AChE antibodies. Indirect radiolabeling showed measurable and selective binding to the cell surface (Brimijoin and Mintz, 1985). The binding could be visualized in the fluorescence microscope with the aid of fluorescein isothiocyanate (F1TC)-labeled second antibodies (S. Brimijoin, unpublished results). It was also possible to count and select cells according to the intensity of the fluorescence labeling, by means of a commercial fluorescence-activated cell sorter. We used this method to characterize the distribution of AChE among separate populations of red blood cells in patients with paroxysmal nocturnal hemoglobinuria (Brimijoin et al., 1986).The results revealed a separate class of enzyme-negative red cells, which are absent from normal blood. Other evidence suggests that these cells are responsible for the characteristic hemolytic anemia (Rotoli et al., 1984). In principle, the techniques of vital immunostaining are directly transferable to neuronal cell culture. One could certainly identify AChE-positive neurons in an established culture. Theoretically one should also be able to enrich cultures in cholinergic cells, e.g., by imrnunofluorescence cell sorting of dissociated spinal cord. It is not yet known, however, if surface AChE is abundant enough for reliable detection in the embryonic or fetal cells that must be used for this purpose.

E. IMMUNOAFFINITY PURIFICATION Until the advent of affinity chromatography based on ligands for the active centers of AChE or BuChE, pure cholinesterasescould be obtained only from the most highly enriched sources (Leuzinger and Baker, 1967). Affinity methods developed during the past decade to surmount this difficulty made use of resins containing N-methylacridinium (Dudai et al., 1972), phenyltrimethylammonium (Berman and Young, 1971), or procainamide (Lockridge and L a Du, 1978; Ralson et al., 1985) to mention just a few. Judicious application of lectin-affinity chromatography in tandem with ion-exchange chromatography also proved effective, especially in purifying AChE from the rat brain (Rakonczay el al., 1981). Many ligand

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affinity methods suffer from low yields, however, and require several weeks to isolate microgram quantities of pure AChE from sources like mammalian brain. For this reason, there has been justifiable interest in the prospects of immunoaffinity chromatography. Early attempts to use polyclonal antisera for immunoaffinity purification of AChE gave little enrichment and low yield (K. P. Mintz and S. Brimijoin, unpublished results). This is a characteristic difficulty with polyclonal antibody-affinity columns, which contain an array of antibodies with different binding properties. Since much of the immunoglobulin is nonspecific, the enzyme binding capacity of a polyclonal affinity column is low. Also, because conditions optimal for desorption from one antibody are unsuitable for the next, elution requires rather harsh conditions (e.g., very low pH or strong chaotropic agents) which easily lead to enzyme inactivation. The irreproducible nature of polyclonal antisera and their inherent lack of expandability further hinder general use of this method. With monoclonal antibodies, immunoaffinity purification becomes practical. All that is required is a single antibody whose affinity for the enzyme is normally high ( K d C lo-’ M), but can be sharply reduced (Kd > M )under conditions tolerated by the enzyme. In order to achieve good recovery during the elution step, it may be necessary to avoid antibodies with especially high affinity. Chaotropic agents such as isothiocyanate will elute many proteins from antibody columns, but we have found that they inactivate AChE rapidly. Antibody binding is poor below pH 5, but mammalian AChEs are not acid stable. In our hands, the gentlest and most effective way to elute AChE from antibody columns is by raising the pH. Brain AChEs show remarkably little loss of activity after 1 hr in glycine buffer at pH 11, conditions under which many immune complexes dissociate. We used immunoaffinity chromatography with alkaline elution to purify AChE from rabbit brain (Mintz and Brimijoin, 1985a).The increase in specific activity was 2000-fold in one step with 50-8076 yield. Another step (i.e., ligand-affinity chromatography) was required to obtain electrophoretically homogeneous enzyme. Even so, fewer than 10 days were required to generate 150 pg of electrophoretically homogeneous AChE from 100 g of whole brain. One of our antibodies is sufficiently salt-sensitive to elute at neutral pH, but high ionic strength (2 M NaC1). This antibody (F461)was originally raised to the AChE of rabbit brain (Mintz and Brimijoin, 1985b)but crossreacts with human AChE. The readily eluted F461 immunoaffinity col~umnshave proved useful in purifying AChE from the human erythrocyte (Brimijoin et al., 1986) and human brain (Z. Rakonczay and S. Brimijoin, unpublished results). Again, final purification on affinity columns of

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phenyitrimethylammonium Sepharose yielded homogeneous enzyme preparations. Starting with richer sources, the electroplax of ?: californica or N. juponica, Sakai et al. (1985) produced electrophoretically homogeneous AChE by immunoaffinity chromatography on monoclonal antibody Nj 601 (originally raised to the Narke enzyme). Acid tolerance of the fish enzyme allowed elution at pH 2.8 with excellent recovery. Given the increasing availability of monoclonal antibodies in virtually unlimited quantity, we expect similar methods to find wide application. This will certainly be true if conditions can be found that give one-step purification in good yield from material of low enrichment.

E IMMUNOASSAYS OF CHOLINESTERASES 1. Rationale In attempts to measure the local concentration of cholinesterases in various tissues, biochemists and physiologists are blessed with choices among assays of great sensitivity, specificity, and convenience (Ellman et al., 1961; Potter, 1967;Johnson and Russell, 1975; Parvari et al., 1983). The remarkable turnover number of the cholinesterases favors their detection in minute quantities. For example, AChE activity can be routinely measured by radioenzymatic methods in submilligram samples of peripheral nerve (Brimgoin and Dyck, 1979), which we estimate to contain no more than 30 pg of the enzyme. It is also fortunate that there are no known endogenous inhibitors of AChE or BuChE. This fact supports the presumption of proportionality between enzyme activity and enzyme protein content. However, the presumption could be challenged whenever there is a physiologically or pathologically meaningful change in cholinesterase activity such as those taking place in denervated muscle (Guth, 1968; Hall, 1973; Linkhart and Wilson, 1975; Carter and Brimijoin, 1981; Collins and Younkin, 1982). Active-site titration and other special methods could be used to resolve some questions. Unfortunately, even titration methods cannot distinguish between irreversible changes at the active center and outright disappearance of the enzyme. Considerations of this sort have fueled controversy over the interpretation of deficits of AChE activity in the red blood cells of patients with the hemolytic disorder known as paroxysmal nocturnal hemoglobinuria (Rosse and Dacie, 1966). Is the enzyme missing or just enzymatically silent? The possibility of proteolytic enzyme inactivation. makes conventional methods useless for comparing the intraneural fluxes of AChE by rapid anterograde and retrograde axonal transport. Finally,

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of course, activity-based methods do not measure enzyme that has been inactivated, deliberately or accidentally, by exposure to irreversible antagonists. These and other considerations provoke interest in antibody methods for quantitation of cholinesterase protein.

2. Precipitin-Based Assays for BuChE The earliest “immunoassays”for cholinesteraseswere made with Ouchterlony immunodiffusion in which the intensity and location of precipitin bands in agar gel are roughly related to the concentration of immunoreactive protein. Immunodiffusion enabled Hodgkin et al. (1965) and Rubinstein et al. (1970) to demonstrate the absence of BuChE from sera of patients who were homozygous for the silent allele and totally lacked circulating BuChE activity. The same method was employed by Rubinstein et al. (1976, 1978) in a study of homozygotes for the atypical form of BuChE (slow hydrolyzers of the muscle relaxant, succinylcholine).In this case, the results showed approximately normal amounts of circulating immunoreactivity. The Ouchterlony method does not lend itself to precise quantitation. Eckerson et al. (1983) recently reevaluated the amount of immunoreactive BuChE in homozygotes for the atypical allele. Using radial immunodiffusion and immunotitration, they recorded a 30% deficit of immunoreactivity, exactly parallel to the deficit in total enzyme activity when measured at V , , , . This result is consistent with other findings indicating that the atypical enzyme differs from the usual form in affinity for substrate, but not in maximal velocity or turnover number (Lockridge and La Du, 1982). 3. Immunoassaysfor AChE Immunoassays for AChE have been slow to appear. One obstacle has been the extreme scarcity of the enzyme in samples of biological interest. The trace quantities of AChE in typical extracts of mammalian muscle or nerve, for example, fall below the detection limits of methods that depend on precipitin bands. Some investigators (N@rgaard-Pedersenet al., 1983; Hangaard et al., 1984; Brock et al., 1985) have used “solid-phase immunoassays” to screen amniotic fluids for AChE, an index of developmental defects in neural tube closure. Because immobilized antibodies concentrate AChE from dilute samples while rejecting related esterases and reversible inhibitors, sensitivity, specificity,and accuracy are all increased. What is measured, nonetheless, is the intrinsic activity of sampled AChE as determined in a conventional thiocholine-based enzyme assay. The methods are therefore sensitive to pharmacologic or genetic effects on enzyme activity, and they cannot detect inactive enzyme.

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Assays to quantitate AChE protein require a different approach. One stimulus €or the development of true AChE irnmunoassays has been the need to characterize the deficit of AChE activity in red cells of patients with paroxysmal nocturnal hemoglobinuria. Chow et al. (1985) measured the binding of ‘251-labeledanti-mouse IgC to erythrocytes treated with saturating concentrations of specific monoclonal antibody against human AChE. A striking deficit was noted in the abnormal samples, which correlated with the percentage of complement-sensitive (i.e., defective) cells. In addition, it was shown that antibody-induced precipitation of radioactivity from extracts of radioiodinated red cells was reduced or absent in the case of patient samples. Both of these methods may be viewed as primitive AChE immunoassays, and they clearly demonstrate the loss of immunoreactive protein in the abnormal samples. a. Immunodisplacement Assay for AChE. We have developed a general method, which, like radioimmunoassay, is based on the principle of immunodisplacement (Brimijoin et al., 1986). The immunodisplacement assay measures the competition between sample AChE and purified AChE for binding to a specific monoclonal antibody on a solid-phase support. In a conventional assay of this type, the pure standard is radioiodinated for sensitive detection and also for distinction from the sample protein (Berson and Yalow, 1968). Our assay instead uses the native AChE activity of the standard as an “intrinsic label.”To distinguish it from the standard, sample AChE is irreversibly inactivated by an organophosphate anticholinesterase (added while the sample is binding to the solid-phase antibody). The anticholinesterase is then rinsed away, and the residual binding capacity for the AChE standard is determined. The immunodisplacement assay detects and measures any material, enzymatically active or not, which reacts with the antigen-binding site of the solid-phase antibody. The specificity of the assay has been demonstrated by comparing the immunoreactivity of purified, electrophoretically homogeneous red cell AChE with that of crude red cell extracts. The results show that crude and purified samples have identical immunodisplacement ability, when expressed in terms of the amount of AChE activity added (Brimijoin et al., 1986). Because the present antibodies do not bind all AChEs with the same efficiency, assay standards must be of the same molecular form as the sample AChE. Using Gr AChE standards from human blood, we have found that normal erythrocytes each contain about 300 copies of the membrane-associated enzyme dimer. We have used the same method to confirm the conclusion that the red cells of patients with paroxysmal nocturnal hemoglobinuria are deficient in immunoreactive AChE (Brimijoin et al., 1986). These results are especially interesting in view of the immunofluorescence cell sorting data, which show that the

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AChE deficit is confined to a specific subpopulation of erythrocytes that totally lack the enzyme (Brimijoin et al., 1986). Like a radioimmunoassay,the immunodisplacement assay yields precise data only over a narrow range of antigen concentrations. Maximal sensitivity is reached when a small amount of antigen occupies an appreciable fraction of the antibody present. The sensitivity of the immunodisplacement assay therefore depends critically on the affinity of the solidphase antibody. Using our highest affinity monoclonal antibody (Kd between lo-'' and lo-' M), half-maximal displacement occurs with 100 ng of AChE, and the threshold for detection is about 10 ng. These figures compare favorably with enzyme-linked immunosorbent assay (ELISA) methods in which AChE is bound nonspecifically to a solid phase (e.g., polystyrene wells) and detected with unlabeled anti-AChE followed by phosphatase-conjugated second antibody. With the antibodies available to us, a reliable ELISA signal requires several hundred nanograms of AChE per sample (S. Brimijoin, unpublished results). b. Two-Sate Zmmunoassay for AChE. Two-site methods promise to dramatically increase the sensitivity of AChE immunoassay (S. Brimijoin and Z. Rakonczay, unpublished results). The principle is straightforward. A specific monoclonal antibody is first bound to polystyrene at alkaline pH , followed by rinsing and exposure to AChE-containing samples. Bound AChE is quantitated with a radioiodinated second antibody that recognizes an unrelated epitope (sufficiently distant from the first antibody to avoid binding interference). Our preliminary results show a strictly linear relationship between the amount of radioactivity bound and the amount of AChE added over a wide range. The detection threshold is only 100 pg (i.e., 100 times lower than that of the displacement assay). Since performance depends less critically upon antibody affinity than is usual with other methods, the two-site assay will be useful for biologically interesting samples that have little AChE, such as mammalian nerve and muscle. In addition, the assay has inherent advantages of specificity. Extraneous antigens can interfere only if they are recognized by each of two antibodies, directed toward separate epitopes and thereby possessing entirely different specificities. This feature tends to limit artifacts owing to crossreactive materials in crude tissue extracts. G. IMMUNOPRECIPITATION AND STUDIES OF CHOLINESTERASE METABOLISM

As mentioned in Section I, there has been an explosive increase in investigations of the cellular dynamics of the cholinesterases, that is, the

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events surrounding synthesis, assembly, packaging, intracellular transport, metabolism, and release of enzyme. Full treatment of these subjects is beyond the scope of the present review, but we will end by considering examples of how immunological methods have been and may be applied. Rotundo has used immunoprecipitation to characterize the synthesis of AChE in cultured avian muscle. The key reagents were a polyclonal antiserum and a monoclonal antibody, both raised against the AChE of chicken brain, and cross-reacting with denatured enzyme (Rotundo, 1984b; see Table VII). With these antibodies, Rotundo was able to selectively immunoprecipitate AChE that had been radiolabeled intracellularly with [35S]methioninein primary cultures of chicken embryo muscle (Rotundo, 1984b). The precipitated radioactivity migrated as a broad band during SDS gel electrophoresis. When the experiment was repeated with material that had been treated with endoglycosidase H to remove carbohydrate residues, the broad band was resolved into two narrow bands corresponding to polypeptides about 10,000 Da smaller than before. A similar result was obtained by treating the muscle cultures with tunicamycin to block glycosylation in the Golgi apparatus, although the amount of immunoprecipitable radioactivity was greatly reduced (Rotundo, 1984b). The separate bands of deglycosylated AChE, termed alpha (a) and beta (p), were initially thought to represent distinct polypeptide chains arising within the same cell and incorporated in a constant ratio in the multisubunit oligomeric AChE forms. It has since been established that the a-and f3-chains are products of different alleles, only one of which is present in any pure cell line (R. L. Rotundo, personal communication). Another striking observation from the same experimental system concerns the degradation of AChE. The work has yet to be reported in detail, but the immunoprecipitation results strongly suggest that a large portion of the newly synthesized monomeric enzyme is rapidly degraded before being activated. The evidence for this view is as follows. Over 80% of the immunoprecipitable translation product that is radiolabeled in avian muscle cultures by ["S]methionine incorporation subsequently disappears with a half-time of less than 1 hr (Rotundo, 1984~).For the most part, this pool of enzyme consists of monomeric forms that lack catalytic activity, though some dinieric AChE behaves similarly. By contrast, the larger forms are much more stable. From previous results, the major fate of C4 AChE appears to be transference into long-lived membrane-associated pools or outright release from the cells (Rotundo and Fambrough, 1980a,b). Further work is necessary to characterize the metabolism of AChE forms and to test the generality of the conclusions reached so far. It is already time to raise the possibility, however, that AChE regulation is

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primarily posttranslational, at the level of the assembly into oligomeric forms. Attempts are currently underway in several laboratories to characterize the level of AChE-coding mRNA in various cells and tissues by in vitro translation assays. Naively, one would look for elevated mRNA levels when cellular AChE activity is rising (e.g., at certain stages of development) and for depressed mRNA levels when enzyme activity is falling (e.g., in denervated muscle). The immunoprecipitation data, on the other hand, point toward a different and potentially more complex mechanism.

IV. Conclusion

The techniques surveyed here represent just a few examples of an array of new methods being applied to the study of cholinesterases. The work accomplished in this way is far from exhausting the possibilities offered. In the next few years, the combined approaches of immunology and molecular biology may resolve many problems concerning the structure, function, localization, and dynamics of these enzymes. UTe look forward to a clearer picture of the homologies between BuChE and AChE and the precursor-product relations among the molecular forms of these enzymes. Rapid and convenient purification procedures, perhaps starting with genetically engineered organisms, will make it easy to obtain enzyme preparations suitable for large-scale structural studies. It will become feasible to observe particular enzyme forms specifically and directly or even to induce their synthesis in certain types of cells, aiding attempts to clarify their physiological significance. Sensitive immunoassays will be available for experiments in which measurements of enzyme activity are an unreliable guide. It will be possible to track enzyme molecules as they make their way through the cell and to gain increased understanding of the regulation of their synthesis, assembly into complex forms, delivery to destinations, and metabolism. Unanticipated developments, however, may prove the most exciting. Acknowledgments

We thank Luanne Wussow for typing the manuscript and tables and for preparing the figures, Pamela Harnrnond for proofreading and editorial assistance, and Dr. John Hedreen for permission to reproduce his micrographs. Some of the work described in this article was supported by grant NS 11855 from the National Institute of Cornmunicative Disorders and Stroke. At the time of writing, Dr. Rakonczay was a Senior Research Fellow of the Mayo Foundation.

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INDEX

A

localization in subnuclei, 168 amino acid sequence, Torpedo, 369-371 future studies, 404-405 immunoaffinity chromatography, 399400 from Torpedo electropax, 400 immunochemistr y aryl acylamidase activity, 390 brain and erythrocyte forms, 386 distinction from BuChE, 389-390 epitopes, number and location, 373 hydrophilic and hydrophobic forms, 379,386 inhibition by antibodies, 387-389 tail components, collagen role, 386387 immunocytochemistry in human erythrocytes, live, 3 9 b - . : . , monkey visual cortex, 396-397 rat CNS, 396 Torpedo electropax, 395 immunoprecipitation, in cultured avian muscle, 404 quantitative immunoassys in human erythrocytes, 402-403 immunodisplacement, 402-403 two-site methods, 403 structure, 364-367; see also Cholinesterases translation in Xenopus oocytes injected with AChE mRNA, 368,392 ACh, see Acetylcholine AChE, see Acetylcholinesterase ACTH (adrenocortocotrophic hormone), see Adrenocorticotropin Action potentials, in crustacean synapses long-term facilitation and, 328-329, 331-332 presynaptic inhibition and, 318-319, 320 S-Adenosylmethionine (SAM) antidepressant properties, 200-201 central neurotransmission and, 200

Acetylcholine (ACh) continuous leakage from motor nerve and muscle, 73-78 ACh receptors and, 77 botulinum toxin and, 76 curare-induced H-response and, 7475 extracellular Ca2+and, 74 mechanism of, 75-76 Na+, K+-ATPaseand, 74-75 neurotrophic effect on muscle and, 77 physiological role, 76-78 intermittent release from motor nerve nonquantal, Ca2+-insensitive 4-aminoquinoline and, 71, 72 botulinum toxin and, 68,70,73 caffeine and, 70 CAMPand, 70,79-80 giant mepp-inducing treatments and, 71 mechanism of, 72-73 physiological role, 73 slow spontaneous mepps, 68-72 temperature and, 69 quantal, Ca'+ -sensitive, 59-68 botulinum toxin and, 67-68 cytoplasm-gate hypothesis, 63 drugs affecting Ca2+entry and, 66-67 fast evoked mepps and, 60-62,64, 66, 68, 70 physiological role, 67 temperature and, 69 vesicular hypothesis, 60-63 in IPN afferents, 164 nonquantal release in frog, 278 mechanism of, 280-281 Acetylcholinesterase (AChE) in afferents to IPN, 164-165 411

412

INDEX

Adrenal medulla catecholamine secretion, Cas+-induced, 252-253 protein kinase C and, 252-253 phosphoinositides, muscarinic response, 250-251 carbachol effect, 254 mechanism of. 253-254 as-Adrenoreceptors, during depression CAMPsynthesis and, 192 in CNS, indirect evidence, 192-193 on lymphocytes and platelets. 190-192 nutritional status and, 191 presynaptic, antidepressants and, 190192 P-Adrenorereptors, during depression in platelets and lymphocytes, 193- 194 as model for CNS receptors, 193194 postsynaptic antidepressant effects, 207-208 Ad renocort icoi ropin (ACTH) inhibition by glucocorticoids, 95-96 age-related changes, 96 dexaniethasone test, see Dexamethasone suppression test Aging adaptation and, 95-99 hyperadaptosis, 99 relative hypercorticism, 96 cancer and, 97-99, 112-118 external factor effects illumination increase and, 131 irradiation and, 129 overnutrition and, 131 stress and, 129- 131 as homeostatic disease complex, 120 hypothalamic activity and, 121, 122, 125-128 hypertension and, 118-119 I.ow of Deviation of Homeostasis and, 91, 95, 118-119, 140-141, 144-145 metabolic i mmu node pression and, 109112 models, 143-149 ecological, 144 prophylaxis, 147- 149 genetic, 144 prophylaxis, 147- 149 ontogenetic, 145- 147

prophylaxis, 147- 149 neuroendocrine-ontogenetic theory evolution and, 134-138 geroprotectors and, 132-134 modern theories of aging and, 139143 obesity and, 100- 108 appetite regulation, 100- 102 energy substrate fluxes. 102-108 psychic depression and, 98, 109 reproduction and, 93-95 climacteric as normal disease, 9495 stochastic damage and, 121-126 y-Aminobutyric acid (GABA) in afferents to IPN, 166 in crustaceans nonquantal release, 278 presynaptic inhibition by, 316-319 in depressed patients in CSF, low level, 199 therapeutic action, 199 Aminopyridines Ca2+-sensitiveACh release stimulation, 66 K' channel blocking, 66 4-Aminoquinoline Ca?+-insensitive intermittent ACh release stimulation, 71 nerve terminal ultrastructure unaffected by, 72 Amitriptyline, inhibitory effects on MA0 activity, in nitro, 203 noradrenaline uptake, 201 serotonin uptake, 204 Anesthetics, 2-deaxyglucose uptake stimulation in IPN, 172-173 in medial habenula, 172-173 Antibodies, in cholinesterase studies to bovine AChE, 382-383 (table) to human AChE, 376-378 (table) to human BuChE, 374-375 (table) reaction with BuChE from higher primates, 391-392 to rodent AChE, 380-381 (table) distinction between rat and mouse AChE, 393 to Torpedo AChE, 384-385 (table) inhibitory action, 388-389

INDEX

Antidepressants chronic effects on neurotransmitters, 206-212 amine hypothesis limitations, 206207 biogenic amine receptor density and, 208-209 noradrenergic autoreceptor sensitivity and, 210-211 postsynaptic P-adrenoreceptors and, 207-208, 211 putative, clinical assessments compliance monitoring, 218-219 depression diagnosis, 213-215 informed consent, 217-218 initial depression severity, 215-216 investigator, choice of, 213 multicentered studies, 218 placebo use, 219-220 rating scales, choice of, 220-227, see also Depression side effects, rating of, 227-228 statistical considerations, 228-230 test conduction by general practitioners, 216 hospital psychiatrists, 216-217 structure-activity studies noradrenaline uptake innhibitors acute and chronic administration, 204 mechanism of action, 202-204 phenylethylamine derivatives, 202 tricyclic, 201-202 serotonin uptake inhibitors, 204205 MA0 inhibitors, 205 Appetite, regulation by hypothalamic satiety center age-related impairment, 100- 102 insulin signals and, 100- 101 Aryl acylamidase, cholinesterases and, 390 Atherosclerosis patients, phenformin effects, 110- 111 Atropine, IPN excitation inhibition, 171 Axons, crustacean excitatory, elimination, presynaptic effects of, 347-348 inhibitory, elimination, postsynaptic

413 effects of, 347 motor, decentralization glial cell hypertrophy, 346 neurotransmitter release and, 346347

B Bacteriophage M 13, sonication-reduced infectivity, 49-50 Behavior, IPN-affected avoidance, 175 emotional, 175-176 sexual, 176 Botulinum toxin effects on intermittent ACh release Ca'+-insensitive, 68, 70, 73 Ca'+-sensitive, 67-68 Brain in depression, postmortem studies, 185-186 phosphoinositide metabolism lithium chloride effect, 262-263 muscarinic responses, Ca2+role in astrocytoma cells, human, 257, 258 in neuroblastoma cells, mouse, 257-258 in slices, regional differences, 255256 in synaptosomes, 242-243,255 postsynaptic, 258 presynaptic, 255,258 phospholipase C, 255, 256 protein B50,259 BuChE, see Butyrylcholine esterase Butyrylcholine esterase (BuChE), from human serum amino acid sequence, 370-371 immunochemistry aryl acylamidase activity, 390 atypical enzyme forms, 387 distinction from AChE, 389-390 higher primate BuChE, 391-392 peptidase association with, 390-391 immunodiffusion, 401 tetramer structure, 366 Butyrylcholine esterase, in rat brain, immunocytochemistry, 396

414

INDEX

C Caffeine, Ca''-insensitive intermittent ACh release and, 70 Calcium channels, in synapses crustacean, 284-286, 314 frog and other species synapses, 284285 squid giant, 284-286 Calcium ion in crustacean motor synapses long-term adaptation and, 343-344 long-term facilitation and, 325-327, 329-331.334 neurotransmitter release and, 293, 296-299,306-309 presynaptic inhibition and, 323 serotonin-induced transmission enhancement and, 338-339 short-term facilitation and, 311-313 mobilization, PIP2 and, 244-245, 249 nonmitochondrial stores in brain, 257 nonquantal ACh release independence from, 68-73 phosphoinositide muscarinic responses and in adrenal medulla, 250-251 in brain, 255-258 in sympathetic ganglia, 260 in pituitary gland phorbol ester effect, 261 polyphosphoinositide hydrolysis and, 261 TRH-induced mobilization, 261 quanta1 ACh release dependence from, 59-68 secretion in adrenal medulla and, 252253 Calmodulin, secretion in adrenal medulla and, 252-253 CAMP,see Cyclic AMP Cancer, age-related cancrophilia concept, 115-118 cholesterol blood level and, 115-116 dexamethasone suppression test, 9699 endometrial carcinoma, polypathology, 117 metabolic immunodepression and, 113 obesity and, 117 phenformin effects during remission, 107- 108

proliferation of nonlymphoid cells and, 113-115 Carbachol IP4 induction in cerebral cortex slices, 267-268 phosphoinositide release from adrenal medulla and, 254 CAT, see Choline acetyltransferase Catecholaminergic system, age-related changes, 124-125 Cerebrospinal Huid (CSF) in depression, decrease of 5-HIAA, 187, 189 HVA, 187, 189 MHPG, 183, 188, 189 SS, 175 Cholesterol, blood level age-related increase, 103- 104, 113- 114 T I effect, 113-114 in atherosclerosis patients, phenoformin and, 110-111 cancerogenesis and, 115-116 in cancer patients phenformin effects, 107- 108 TSeffects, 113- 114 Choline acetyltransferase (CAT), in IPN afferent fibers, 164-166 localization in subnuclei, 168-169 during postnatal development, 174 Cholinesterases, see also Acetylocholinesterase, Butyrylcholine esterase amino acid sequences, 369-372 antibodies to, see Antibodies future studies, 405 immunoaffinity purification, 398-400 imniunochemistry, 373-393 immunocytochemistry, 395-398 immunoprecipitation, 403-405 quantitative immunoassays, 400-403 structure asymmetric (tailed), 365, 367 attachment to basal lamina, 367 globular, 364-367 membrane-associated, 366-367 reaction with detergents, 366 Collagen, in asymmetric AChE, 364-365, 386-387 Corticosterone, blood level prednisolone treatment and, 130-131 pineal polypeptide extract effect, 130- 131

INDEX

Crustacean motor synapses long-term adaptation, phasic neurons age dependence, 342,345-346 CaZ+role, 343-344 EPSP amplitude and, 340-342,345 long-term facilitation and, 342, 344345 low-frequency depression removal and, 340-342,344-345 morphological changes, 343 neurotransmitter release and, 340, 343,345 scheme of, 343-344 long-term facilitation Ca2+accumulation and, 325-327, 329-331,334 comparison with posttetanic potentiation, 335-336 EPSP amplitude rise and, 325,332 induction of, 326-327 long-term adaptation and, 342, 344345 muscle tension enhancement by, 334335 Na+ accumulation and, 325-331, 334 neurohormones and, 334 neurotransmitter release and, 333 phases of, 327 postsynaptic potentials and, 328-329, 331 presynaptic potentials and, 328-329, 331-332 quanta1 analysis, 332-333 binomial distribution, 333 neurotransmitter release, 278, 281-284, 302-303 Ca2+role in, 293, 296-299, 306-309 phasic, 300-304 EPSP and, 300-301 low-frequency depression and, 304 presynaptic inhibition action potential and, 318-319, 320 advantages of, 324-325 Caz+current reduction and, 323 excitatory facilitation during, 323324 GABA receptor in muscles and, 316318 impulse conduction blockage and, 319-320 terminal bouton changes

415

electrical isolation, 319-322 neurotransmitter release reduction, 322-323 short-term facilitation, tonic neurons Ca2+channel progressive recruitment and, 314 electrical events and, 310-311 EPSP and, 309-310,312 residual Ca2+and, 311-313 statistical parameters, 314-316 tonic cooperativity for Ca*+action, 307309 excitability or spike invasion, 304306 high-output, 301-302 low-output, 301 morphology, active zones and, 306307 neurotransmitter release, 306-309 postsynaptic depolarization, 304-305 transmission enhancement by octopamine, 339 serotonin, 337-339 trophic effects; see also Axons, crustacean excitatory axon elimination and, 347-348 inhibitory axon elimination and, 347 motor axon decentralization and, 346-347 muscle fiber growth and, 347 CSF, see Cerebrospinal fluid Cyclic AMP (CAMP) Ca2+-insensitiveintermittent ACh release stimulation, 70 synapsin I phosphorylation by, 79-80

Depression adrenoceptors in CNS, 192-194 peripheral, 190-192 on platelets and lymphocytes, 190194 age-linked as primary normal disease, 109 resistance to dexamethasone test, 98, 109 diagnosis systems, 214-215 endocrine markers, 195- 198

416

INDEX

G 4 K A and, 199

histamine and, 199-200 hypothalamus role, 109 lithium chloride treatment, 263 neuropeptides and. 198 neurotransmitter metabolites in CSF and urine, 186- 190 postmortem brain studies, 185- 186 SAM and, 200-201 srrotonin receptors. 194-195 severity rating scales, 215-216, 220-227 Beck Depression Inventory, 224, 226 computerized technique, 225-226 HAM-D, 216,222-226 Hospital Anxiety and Depression Scale. 226 Montgomery-Asberg scale, 225 visual analog scales, 221 -222 Wakefield self-assessment depression inventory, 224-225 SS low level in CSF, human, 175 subtypes, DST and, 195-196 types of, 213-214 Dexamethasone suppression test (DST) agedependent in women, 96-97 in cancer patients, 96-99 during surgery, 96,98-99 depression assay, 98, 195- 196 diazepam and, 134 DOPA and. 134 phenazepam and, 134 in stressed animals, 130 tryptophan and, 134 Diabetes, streptozotocin-induced, rat phosphoinositide metabolism in sciatic nerve, see Sciatic nerve Diaglycerol as PIP, hydrolysis product, 245-246 protein kinase C activation, 246 Ca'+ synergism, 249-250 DNA age-related damage in hypothalamus, 125 complementary (cDNA), for PrP 27-30 protein library preparation, 3 nucleotide sequence, 4,6-7 DOPA dexamethasone test and, 134 effect on GH during depression, 197 life-span increase, mouse, 132

Dopamine hypothalamic, age-related changes, 139 in IPN afferents, 168 protein B50 phosphorylation in brain and, 259 Dorsal tegmental nucleus (DTN) efferent pathways from IPN to, 163 DST, see Dexamethasone suppression test DTN, see Dorsal tegmental nucleus

E ECT, see Electroconvulsive therapy EGTA, phosphoinositide breakdown in brain and, 255-257 Electroconvulsive therapy (ECT), serotonin uptake in depression, and, 194 Endorphins, in CSF during depression, 198 Enkephalins, in IPN afferent fibers, 167-168 efferent fibers, 169 localization in subnuclei, 169 EPSP, see Excitatory postsynaptic potentials Erythrocytes, human AChE in normal subjects distinction from brain form, 386 localization in subpopulations, 398 AChE in paroxysmal nocturnal hemoglobulinuria activity deficit, 400 immunoassays, 402-403 immunocytochemistrp vital, 398 Estrogen, IPN susceptibility to, 176 Evolution, aging and, 134-138 biological antiaging systems and, 136137 body size limiting and, 138 developmental pleiotropia, genes and, 135-136 limited life-span advantage for population, 135 natural selection and, 137 species-specific differences, 138 Excitatory postsynaptic potentials (EPSP), crustacean inhibitory axon elimination and, 347 long-term adaptation and, 340-342, 345

417

INDEX

long-term facilitation and, 325, 332 motor axon decentralization and, 347 in phasic motor neurons, 300-301 presynaptic inhibition and, 319 short-term facilitation and, 309-310, 312

F Fatty acids, in aging blood level increase, 107 reduction by phenformin, 107-108, 110-111 growth hormone reduction by, 106-107 Fetoplacental system functions during pregnancy, 91-92 metabolic changes and, 91

G GABA, see y-Aminobutyric acid GABA receptors in crustacean synapses, 316-318,337 during depression, antidepressant effects, 199 GAD, see Glutamate decarboxylase Genes developmental pleiotropia during aging and, 135-136 for PrP 27-30 protein detection in scrapie-infected hamster, 4-5,8 related sequences in various mammals, yest, insect, and nematode, 5 Geroprotectors dietary calorie restriction, 132 DOPA, 132, 134 phenformin, 132-133 GH, see Growth hormone Glial cells crustacean, motor axon decentralization and, 346 hypothalamic, age-related changes, 128 Glucose, in aging blood level increase, 103-105, 107 phenformin effect, 107-108 prednisolone effect, 96, 98 effect on growth hormone secretion, 106- 107 utilization reduction, 102- 105 Glutamate decarboxylase (GAD),in IPN

afferent fibers, 166-167 during postnatal development, 174 Growth hormone (GH) age-related decline fatty acids and, 106-107 hypothalamic glucoreceptors and, 106 during depression clondine stimulation reduction, 197 responses to DOPA, insulin, TRH, and LH, 197

H Habenula 2-deoxyglucoseuptake, anesthetics and, 172-173 IPN afferent pathways from, 159-161 lesions, IPN synapses and, 163 multi-unit activity, progesterone and, 172 stimulation, IPN neuron responses to, 171 Hepatocytes, PIP2,vasopressin effect, 245 5-HIAA, see 5-Hydroxyindoleacetic acid Histamine, during depression antidepressant effects, 199-200 in platelets, low level, 200 Homeostasis adaptive, aging and, 95-99 energy, aging and, 100-108 Law of Deviation of, 91, 95, 118-119, 140-141, 144-145 reproductive, aging and, 93-95 Homovanillic acid (HVA), in CSF during depression, 187, 189 5-HT (hydroxytryptamine),see Serotonin HVA, see Homovanillic acid 5-Hydroxyindole acetic acid (5-HIAA), in CSF during depression, 187-189 Hyperadaptosis, as aging sign, 99 Hypercorticoidism, as aging sign, 96 Hypertension, in aging connection with other normal diseases, 119 hypothalamic forms of, 119 Hypothalamo-pituitary complex, sensitivity to glucocorticoids age-related changes, 96-99 effect of stress during surgery, 98-99

418

INDEX

Hypothalamus in age-linked diseases metabolic immunodepression and,

110 psychic depression and, 109 age-related changes DNA damage and, 125 dopamine decrease and, 139 inhibition by sex hormones, 93-94 neuron loss and, 122,124 overnutrition and, 131 satiety center sensitivity and, 100-

102,121 stress and, 129-131 postnatal ontogenesis glial cell changes, 128 PNSC differentiation, 127-128

I illumination, aging and, 131 Imipramine, serotonin uptake inhibition,

204 structure changes and, 204-205 Immunoaffinity chromatography, AChE purification, 399-400 Immunocytochemistry application, 393-394 cholinesterase studies, 395-398 technical obstacles, 394 Immunodiffusion, BuChE from human serum, 401 Inositol, in diabetic sciatic nerve, 263-264 sorbinil effect, 264 Inositol I-phosphatase, in brain lithium chloride effect, 263 Inositol phosphate (IP), in brain lithium chloride effect, 262-263 Inositol tetraphosphate (IP4),carbacholinduced in cerebral cortex slices,

267-268 Inositol 1,4,5-trisphosphate (IP,) in brain, lithium chloride effect, 263 Ca2+mobilization and, 246-247 release by PIP, hydrolysis, 241,243,

245-246 in retina, light-induced increase, 266-

267 as second messenger in Ca2+mobilization, 241,246-247

Insulin in aging appetite regulation by hypothalamus and, 100-101 blood level increase, 102- 105 phenformin effect, 107-108,110-

111 effect on GH during depression, 197 phosphoinositides in diabetic sciatic nerve and, 265 fnterpeduncular nucleus (IPN) action potential depression, mechanism of, 172 anatomy afferent pathways from lateral and medial habenula,

159- 161 from NDB, 161 cell types, 158 efferent pathways, mainly to DTN,

163 species differences, 164 subnuclei, 158-159, 160 synapses, 161-163 behavioral effects lesion-induced avoidance, 175 sex hormone treatment and, 176 stimulation-induced emotion activation, 175- 176 2-deoxyglucose uptake, anesthetics and,

172-173 development postnatal neurochemistry, 174-175 synaptogenesis, 173- 174 prenatal, histogenesis, 173 excitatory responses to ACh, 171-172 habenula stimulation, 171 nicotinic cholinergic agents, 172 SP, 171 functions during development, 176 future directions, 176- 179 hormonal level, 170 multiunit activity, progesterone and,

172 neurotransmitters afferent, 164-168 efferent, 169- 170 localization in subnuclei, 168- 169

INDEX

species differences, 170- 171 Ionophore A23187, phosphoinositide breakdown in brain and, 255,256 IP, see Inositol phosphate IPS,see Inositol 1,4,5-trisphosphate IP4, see Inositol tetraphosphate IPN, see Interpeduncular nucleus Irradiation, aging acceleration, 129

L LH, see Luteinizing hormone Lipofuscin, accumulation in aging, 121, 123 Lithium chloride effects on phosphoinositide metabolism in brain inositol 1,2-cyclicphosphate increase, 262 inositol 1-phosphatase inhibition, 263 IP increase, 262-263 IPSdecrease, 263 manic-depressive illness control, 263 Luteinizing hormone (LH), during depression G H abnormal response to, 197 plasma level reduction, 197 Luteinizing hormone-releasing hormone, in IPN, 170 Lymphocytes, during depression a2-adrenoreceptors, 190- 192 P-adrenoreceptors, 193- 194

MAOI, see Monoamine oxidase inhibitors mepps, see Miniature end-plate potentials Metabolic immunodepression, age-linked autoimmune disorders and, 111-112 hypothalamus role, 110 phenformin effects in atherosclerosis patients, 110-111 in cancer patients, 107- 108 as secondary normal disease, 109, 111 3-Methox yindole-4-hydrox yphenyglycol (MHPG), during depression CSF level decrease, 183, 188, 189 urinary excretion decrease, 187-189

419

Miniature end-plate potentials (mepps), in intermittent ACh release evoked, fast, Ca2+-sensitive,60-62, 64, 66, 68, 70 spontaneous, slow Ca2+-insensitive,6872 Monoamine oxidase inhibitors (MAOI), antidepressant activities, 202-203, 205 Motor nerve terminal, ACh release nonquantal, Ca*+-insensitive, intermittent, 68-73 slow mepps and, 68-72 quantal, Ca2+-sensitive, intermittent, 59-68 active zones in membrane and, 62, 63-66 fast mepps and, 60-62,64,66, 68, 70 inward Ca2+and outward K + currents, 60, 65 synaptic vesicle attraction to axolemma and, 64 vesicular hypothesis, 60-63 Muscarinic receptors in adrenal medulla phosphoinositide responses and, 250-251 secretion inhibition and, 253 secretion stimulation and, 254 in brain Ca2+mobilization and, 255-258 in depressives, postmortem, 186 phosphoinositide responses, 255-258 Muscles ACh continuous leakage, 73-78 ACh receptors and, 77 curare effect, 74-75 Na+,K+-ATPaseand, 78 pathway to extracellular fluid, 76 avian, cultured, AChE metabolism, 404 CAMP-dependent protein kinase inhibitor, 79-90 crustacean interaction with nerves, 347 tension, long-term facilitation and, 334-335 iris, phosphoinositide muscarinic responses, 245 motor nerve trophic influence, ACh role, 77

420

INDEX

N Na ,K -ATPase antidepressant actions and, 202-203 in diabetic sciatic nerve, 264 sorbinil effect, 264 NDB, see Nucleus of diagonal band Neuromuscular junctions amphihian, posttetanic potentiation, 335-336 Ca" and Na+ role, 336 crustacean, see Crustacean motor synapses Neurons age-dependent loss, 122, 124, 141 crustacean, see also Crustacean motor synapses phasic, 299-300 long-term adaptation, 339-346 tonic, 299-300 short-term facilitation, 309-316 Neurotransmitter release calcium channels at synapse and, 284286 calcium and voltage hypotheses of, 288-293 in crustacean, see Crustacean motor synapses depolarization and, 286-292 nonquantal (tonic) acetylcholine (ACh), mechanism of, 278,280-281 GABA in crustacean, 278 quantal, by vesicular fusion crustacean, mechanism of, 281284 frog, 279, 281 Nicotinic cholinergic receptors, in IPN, 172 Noradrenaline, see Norepinephrine Norepinephrine in IPN fibers afferent, 168 efferent, 170 localization in subnuclei, 169 in plasma, depressed patient mood and, 190 turnover in brain, progabide effect, rat, 199 +

+

uptake in vitro, antidepressants and, 201 -204 Nucleus of diagonal band (NDB), IPN afferent pathways from, 161

0 Obesity, in aging hypercholesterolemia and, 103- 104 hyperglycemia and, 102-105, 107 hyperinsulinemia and, 102-105, 107 hypothalamic satiety center sensitivity and, 101-102 Octopamine, in crustacean motor synapses long-term facilitation and, 334 transmission enhancement by, 339 Octopamine receptors, in crustacean nerve terminals, 317 Overnutrition, aging acceleration, 131 11-Oxycortocosteroids, dexamethasone effect on blood level, see Dexamethasone suppression test Oxygen, free radicals, age-related accumulation, 123, 141

P Paroxysmal nocturnal hemoglobinuria AChE in erythrocytes, 398,400,402403 Peptidases, association with BuChE, 390391 Peptidergic neurosecretory cells (PNSC) damage accumulation with aging, 128 differentiation during postnatal ontogenesis, 127-128 liberine production, 127 Phenformin effects on C3H female mouse life-span increase, 132-133 spontaneous tumor reduction, 132133 on metabolic and immunobiological parameters in atherosclerosis patients, 110- 111 in mammary cancer patients, 107108, 110

INDEX

Phorbol ester, effects in pituitary gland Ca2+increase, 261 prolactin release and, 261 protein kinase C activation, 261 Phosphatidylinositol (PI) receptor-linked hydrolysis in synaptosomes, 243 Ca2+gating and, 244 Phosphatidylinositol4,5-bisphosphate WP2) affinity for Ca2+,244 breakdown, IP2 and IPS release, 245246 dual messenger theory, 245-247 receptor-linked mechanism of, 247249 Ca2+mobilization and, 244-245, 249 in diabetic sciatic nerve, 265 insulin effect, 265 in nerve tissue membrane, 241 as PIP hydrolysis product, 248-249 structure, 242 synthesis and hydrolysis in brain, 214 vasopressin-induced loss from hepatocytes, 245 Phosphatidylinositol4-phosphate(PIP) in diabetic sciatic nerve, 265 insulin effect, 265 receptor-linked conversion to PIP,, 248-249 Phosphoinositides catecholamine secretion in adrenal medulla and, 252-254 hydrolysis in pituitary gland, 261-262 muscarinic responses in adrenal medulla, 250-251 in brain, 255-258 in cervical sympathetic ganglion, 260 in iris smooth muscle, 245 in synaptosomes, 242-243,255 in retinal photoreceptors adaptation to light and, 266-267 responses to light, 266-267 Phospholipase C, in brain, hydrolysis of PIP and PIPz, 255,256 PI, see Phosphatidylinositol Pineal polypeptides, sensitivity to prednisolone and, 130-131

421

PIP, see Phosphatidylinositol4-phosphate PIP,, see Phosphatidylinositol4,5bisphosphate Pituitary gland, GH, tumor cells phosphoinositide metabolism, 261-262 protein kinase C, 161 TRH stimulation of Ca2+spike, 261 prolactin release, 260 Platelets, during depression a*-adrenoreceptors, 190-192 CAMPsynthesis and, 192 as model for CNS receptors, 192,194 nutritional status and, 191 P-adrenoreceptors, 193- 194 histamine low level, 200 serotonin receptors, 194-195 PNSC, see Peptidergic neurosecretory cells Polyphosphoinositides in retina, light effects and, 266-267 in synaptosomes, muscarinic responses, 243-244 Potassium ion, in motor nerve terminals ACh release and, 60,65,66 Prednisolone age-dependent effect on blood glucose, 96,98 corticosterone blood level and, 130-131 pineal polypeptide extract effect, 130- 131 Pregnancy fetoplacental system functions, 91-92 metabolic changes during, 91 Progabide, antidepressant effect, 199 Progesterone, effects on IPN multiunit activity increase, 172 sexual behavior and, 176 Prolactin, during depression, 198 Protein B50 phosphorylation, dopamine effect, 259 PIP* formation and, 259 in presynaptic membranes, 259 Protein kinase C activation by diaglycerol, 246 Cazf synergism, 249-250 in adrenal medulla CaZ+influx and, 252 secretion promotion, 252,253

422

INDEX

in pituitary gland phorbol ester-activated, 261 phosphoinositide metabolism and, 261 prolactin release and, 261 Protein kinase inhibitor, in muscles CAMP-dependent ACh release and, 7980 PrP 27-30, scrapie-specific aggregation in rods, 17, 22, 30, 34,4041 microelectron microscopy, 42-43 amino acid sequence, 13- 15 comparison with protein from SAF, 26 copurification with infectivity, 32-33 hydrophobicity, 13-14 identification as syaloglycoprotein, 2 Psychic depression, see Depression

R Reproduction, age-related changes climacteric as normal disease, 94-95 hypothalamus inhibition by sex hormones, 93-94 sexual maturation, 93-94 switching-on, switching-off mechanism, 94 Retina, phosphoinositide responses to light in Drosophila phospholipase C-lacking mutant, 267 in Limullcs polyphumus, 266-267 in octopus, 267 RNA, messenger (mRNA) AChE, Drosophila, translation in Xenopw oocytes, 368 AChE, human translation in Xenopus oocytes, 368 immunologic analysis, 392 PrP, in hamster developmental regulation, 5, 10 isolation from scrapie-infected brain, 3, 5 transcripts in infected and uninfected animals, 5, 9

S SAM. see S-Adenosylmethionine

Sciatic nerve, in diabetic rat inositol decrease, 263-264 restoration by sorbinil, 264 Na ',K -ATPase inhibition, 264 reversal by sorbinil, 264 PIP and PIP, increase, 265 reversal by insulin, 265 Scrapie infectivity-associated particles, see Scrapie prions tissue extracts, lack of definable structures, 17, 22 Scrapie-associated fibrils (SAF) distinction from rods, 25-26 protein, isolation and properties, 26 ultrastructure, 23-25 Scrapie prion rods formation during microsome extraction with detergent, 36-37 immunoelectron microscopy, 41-43 morphology, infectivity and, 36-40 polymorphic forms, 51 (table) as prion aggregates, 17, 22, 34,40-42 in purified fractions dimensions, 28-29 distinction from rods in normal brain, 30,32 PrP 27-30 detection, 17, 22, 30, 34, 40-41 electron microscopy, 42-43 ultrastructure, 28, 30-31, 34-35 similarity to amyloids, 41, 44 sonication, 46-51 infectivity unaffected by, 49 short rods generated by, 47-49 spheres generated by, 47-51 Scrapie prions elongated structures, 18- 19 (table) fibrils, 17, 23-26, see also Scrapie-associated fibrils filaments, 17, 23 (table), 26 from amyloid plaques, 44-45 comparison with rods, 23, 46 identification as sialoglycoprotein PrP 27-30,2 multiple molecular forms, 34 in normal and infected hamster brain, 8, 10-11 explanations of differences, 15- 16 properties, 2 purification, 1-2, 27-34 +

423

INDEX

rods, see Scrapie prion rods Serotonin in crustacean motor synapses long-term facilitation and, 334 transmission enhancement by, 337339 Ca2+role, 338-339 Na+-dependent depolarization and, 338 in depressed brain, postmortem, 186 uptake during depression antidepressant effects, 194, 204-205 ECT effect, 194 in platelets, 194 in synaptosomes, 194 Serotonin receptors, in crustacean nerve terminals, 317 Sex hormones hypothalamus inhibiton by, 93 age-related changes, 93-94 Sodium ion, in crustacean motor synapses long-term facilitation and, 325-331, 334 serotonin-induced depolarization and, 338 Somatostatin (SS) in CSF, low level during depression, 175 in IPN afferent fibers, 167 efferent fibers, 169-170 localization in subnuclei, 169 during postnatal development, 174175 species differences, 170 Sonication bacteriophage M 13, infectivity reduction by, 49-50 scrapie prions infectivity unaffected by, 49 polymorphic form generation, 46-51 Sorbinil, effects on diabetic sciatic nerve inositol level and, 264 Na+,K+-ATPaseactivity and, 264 SP, see Substance P SS, see Somatostatin Squid, giant synapse Ca2+channels, 284-286 depolarization and, 286-292 neurotransmitter release, 284-293 calcium and voltage hypotheses of, 288-293

dynamics, Ca" entry and, 293-296 Stress, age-related diseases and, 129 hypothalamic mechanism, 129-131 Substance P (SP), in IPN afferent fibers, 167 efferent fibers, 169 localization in subnuclei, 168-169 species differences, 170 Superoxide dismutase, in liver and brain primate life-span and, 123 Sympathetic ganglia, electrical stimulation phosphoinositide responses to, 259260 Synapses crustacean, see Crustacean motor synapses giant of squid, see Squid, giant synapse of IPN axons axodendric and axosomatic, 161 major and minor types, 162-163 postnatal development, 173-174 Synapsin I, phosphorylation ACh release and, 79-80 Synaptosomes receptor-linked responses by phosphoinositides, 242-243,255 polyphosphoinositides, 243-244 serotonin receptors, depression and, 194

T Ts, see Triiodothyronine T1, see Thyroxine Thyroid-stimulating hormone (TSH) in depression, thyroid gland hyporesponsiveness to, 196 Thyrotropin-releasing hormone (TRH) Caz+ mobilization and, 261 in depression, GH abnormal response to, 197 in IPN, 170 phosphoinositide hydrolysis and, 261262 prolactin release and, 260 Thyroxine (T4), sensitivity to Ts in aging, 113- 114 in cancer patients, 113-114 Torpedo californica, electropax acetylcholine (ACh) release by synapses, 280

424

INDEX

acetylcholinesterase (AChE) amino acid sequence, 369-371 antibodies to, 384-385 (table) inhibitory action, 388-389 hydrophobic, structure, 366 immunoaffinity chromatography, 399-400 imniunocytochemistry, 395 T R H , see Thyrotropin-releasing hormone Triglycerides, blood level age-dependent increase, 103- 105. 107 reduction by phenformin in atherosclerosis patients, 110-111 in cancer patients, 107-108 Triiodothyronine ( T S ) , effects on lipid metabolism and Tt in aging, 113-114

in cancer patients, 113- 114 TSH, see Thyroid-stimulating hormone

V Vasointestinal peptide (VIP), in afferents to IPN, 167 Vasopressin depression improvement by, 198 PIPp loss from hepatocytes and, 235

X Xenopus oocytes, AChE synthesis, induction by Drosophila AChE mRNA,368 human AChE mRNA, 368,392