International Review of Neurobiology Volume 29

INTERNATIONAL REVIEW

OF

Neutobiology VOLUME 29

Editorial Board

w . Ross ADEY JLJLIUS

AXELROD

SEYMOUR KETY KEITH KILLAM

Ross BALDESSARINI

CONANKORNETSKY

SIR ROGERBANNISTER

ABEL LAJTHA

FLOYDBLOOM

BORIS LEBEDEV

DANIELBOVET

PAULMANDELL

PHILLIP BRADLEY

HUMPHRY OSMOND

YURI BUROV

RODOLFOPAOLETTI

JOSE

DELGADO

SOLOMONSNYDER

SIR JOHN ECCLES

STEPHENSZARA

ELKES

SIR JOHN VANE

JOEL

H. J. EYSENCK

MARATVARTANIAN

KJELLFUXE

STEPHENWAXMAN

B o HOLMSTEDT

RICHARDWYATT

PAULJANSSEN

OLIVERZANGWILL

I NT ER NAT10NAL R EVIEW 0F

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

VOLUME 29

ACADEMIC PRESS, INC. Harcourt Brace Jovanovich, Publishen

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CONTENTS

Molecular Genetics of Duchenne and Becker Muscular Dystrophy

RONALD G . WORTON AND ARTHURH . M . BURGHES I. I1. I11. 1V. V. VI . V11.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Duchenne and Becker Muscular Dystrophy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Locating the DMD and BMD Genes at Band Xp21 . . . . . . . . . . . . . . . . . . . . . . . . . Approaches to Cloning the Responsible Gene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mutation at the DMD/BMD Locus.,. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Carrier Identification and Prenatal Diagnosis . . . . . .................... Prospects for the Future . . . . . . . . . . . . . . . . . . . . . . . .................... References . . . . . . . . . . . . . . . . . . . . .................................... Note Added in Proof . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2 2 18 32 46 56 61 64 75

Batrachotoxin: A Window on the Allosteric Nature of the Voltage-Sensitive Sodium Channel

GEORGEB. BROWN 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1. Electrophysiological Analysis of Batrachotoxin Effects . . . . . . . . . . . . . . . . . . . . . . . 111. Nature of the Binding Site . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Interactions with Other Sodium-Channel Neurotoxins and Ligands . . . . . . . . . . . . V. Role of Lipid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V I . Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...............

77 79 86 97 108

111 112

Neurotoxin-Binding Site on the Acetylcholine Receptor

THOMAS L . LENTZ AND I. I1. I11. IV. V.

PAUL

T.

WILSON

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Nicotinic AChR . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Structure and Function of Curaremimetic Neurotoxins . . . . . . . . . . . . . . . . . . . . . . Neurotoxin-Binding Site on the AChR ..................... .... Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V

117 118 122 131 151 154

CONTENTS

vi

Calcium and Sedative-Hypnotic Drug Actions

PETERL . CARLEN A N D PETERH . Wu I. I1. 111. IV. V.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Behavioral Effects . . . . . . . . . . . . . ................................... Electrophysiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . ................................. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

161 162 163 172 183 185

Pathobiology of Neuronal Storage Disease

STEVENU . WALKLEY I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1 . Experimental Animal Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I l l . Structural Changes in Neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Disordered Function of Neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. T h e Role of Gangliosides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI . Explaining Neuronal Dysfunction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII . Concluding Comments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ....... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

191 195 200 220 224 230 235 239

Thalamic Amnesia: Clinical and Experimental Aspects

STEPHEN G . WAXMAN I . Introduction .............. ........ I1 . Clinical Observations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I11. Experimental Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Theoretical Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Conclusions and Prospects: Future Questions. .

245 246 250 253

References . . . . . . . . . . . . . . . . . . . . . . . . . . Note Added in Proof . . . . . . . . . . . . . . . . .

Critical Notes on the Specificity of Drugs in the Study of Metabolism and Functions of Brain Monoamines

S. GARATTINI AND T. MENNINI I. I1. 111. IV.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Some Drugs Interfering with Monoamine Uptake and Release . . . . . . . . . . . . . . . . Drugs Acting at Monoarnine Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

259 260 268 276 277

CONTENTS

vii

Retinal Transplants and Optic Nerve Bridges: Possible Strategies for Visual Recovery as a Result of Trauma or Disease

E. TURNER, JERRY R . B L A I R , MAGDALENE SEILER, ROBERTARAMANT, THOMAS W. LAEDTKE, E. THOMAS CHAPPELL, AND LAURENCLARKSON

JAMES

I. 11.

111. I v. V. VI.

Introduction . . . . . . . . . . . . . . . . . . ......................... Intraocular Retinal Transplantatio ......................... Retinal Pigment Epithelium Grafted onto Bruchs Membrane . . . . . . . . . . . . . . . . Retinal Tissue Transplanted into the Central Nervous System . . . . . . . . . . . . . . . . . Peripheral Nerve Bridges and Optic Nerve Grafts: Enhancement of Retinal Ganglion Cell Axon Regeneration . ................. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . ..............................

281 283 296 297 302 305 306

Schizophrenia: Instability in Norepinephrine, Serotonin, and 7-Arninobutyric Acid Systems JOEL

I. 11. 111. IV. V.

GELERNTER AND DANIEL P. VAN

KAMMEN

........................................ Norepinephrine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Serotonin . . . . . . . . . . . . . . . . .

310

. . . . . . . . . . 311

. . . . . . . . . . . . . . . . . 323

Conclusions . . . . . . . . .

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

337

INDEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CONTENTS OF RECENTVOLUMES ..........................................

349 371

341

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MOLECULAR GENETICS OF DUCHENNE AND BECKER MUSCULAR DYSTROPHY

By Ronald G. Worton and Arthur H. M. Burghes Genetics Department and Research institute The Hospital for Sick Children Toronto, Ontario, Canada M5G 1x8, and Departments of Medical Genetics and Medical Biophysics University of Toronto Toronto, Ontario, Canada M5S 1A6

I. Introduction 11. Duchenne and Becker Muscular Dystrophy A. Clinical Features B. Genetic Considerations C. Manifesting Carriers D. Carrier Identification and Prenatal Diagnosis E. Nature of the Basic Defect 111. Locating the DMD and BMD Genes at Band Xp21 A. Females with X:Autosome Translocations B. Males with Deletions and Complex Phenotypes C. Linkage Analysis in DMD and BMD Families D. The Genetic Map of the X Chromosome Short Arm IV. Approaches to Cloning the Responsible Gene A. Cloning Sequences from the BB Deletion-The PERT 87 (DXS164) Region B. Cloning the t(X;21) Translocation Junction-The XJ (DXS206) Region C. The Physical and Molecular Map of the PERT 87-XJRegion D. Expressed Sequences-The DMD/BMD Gene Map V. Mutation at the DMDIBMD Locus A. Translocations in Affected Females B. Deletions in Males with Complex Phenotypes C. Deletions and Duplications in Males with DMDlBMD D. Recombination at the DMD/BMD Locus VI . Carrier Identification and Prenatal Diagnosis A. The Principles of Carrier Identification and Prenatal Diagnosis B. Practical Applications of Carrier Identification and Prenatal Diagnosis VII. Prospects for the Future References Note Added in Proof 1 INTERNATIONAL REVIEW OF NEUROBIOLOGY. VOL. 29

Copyright 0 1988 by Academic Press, Inc. AU rights of reproduction in any form reserved.

2

RONALD G . WORTON A N D ARTHUR H . M. BURGHES

1. introduction

Duchenne muscular dystrophy (DMD) is the most common and most devastating of the muscular dystrophies. Until recently most scientific studies of this genetic disease and its less severe counterpart, Becker muscular dystrophy (BMD), have focused on defining phenotypic markers. Recent advances in molecular biology have made it possible to search for the gene or genes responsible for these muscle-wasting disorders. During the past 5 years, the single gene responsible for both conditions has been localized to a small region in the middle of the short arm of the X chromosome, DNA probes have been isolated from this region for use in identification of carrier mothers and for prenatal diagnosis, and sequences from the disease locus itself have been isolated and characterized. These studies inspire hope that the gene responsible will soon be cloned in its entirety and the protein product will be isolated and its physiological function determined. Even at this point in time, many lines of investigation indicate that the genetic locus is very large and complex with a very broad spectrum of mutations leading to the disease. The purpose of this review is to summarize briefly some of the older, more classical studies on these diseases and then to focus on the molecular genetics of DMD and BMD as it has developed over the last 5 years. For a more in-depth treatment of the earlier work the reader is referred to reviews by Rowland (1980), Jones and Witkowski (1983a), and Moser (1984).

ii. Duchenne and Becker Muscular Dystrophy

A. CLINICAL FEATURES Duchenne muscular dystrophy was first described by Meryon in 1852 and subsequently by Little (1853) and Duchenne (1861, 1868). The disease is characterized by progressive muscular weakness and severe degeneration of skeletal muscle (Dubowitz, 1978; Gardner-Medwin et al. , 1978; GardnerMedwin, 1980). Clinical symptoms of the disease become apparent in the first 3 years of life. Most affected boys who are carefully examined show retarded motor development and about half of them are not walking before the age of 18 months (Gardner-Medwin d al., 1978). Somewhat later, patients develop a waddling gait, have difficulty climbing stairs, and are subject to frequent falls (Dubowitz, 1978). Cardiac involvement is common, and intellectual levels are lower in some patients (Dubowitz, 1965; Allen and Rodgin, 1960). One-third of DMD patients have an IQbelow 73 with the mean IQ between 70 and 85. A

DUCHENNE AND BECKER MUSCULAR DYSTROPHY

3

few patients who may be of special interest have an I Q below 50 (Emery, 1980). The skeletal muscle weakness is progressive and symmetrical, affecting first the proximal muscle of the lower extremities. The majority of D M D patients are chair bound by their early teens and thereafter contractures increase rapidly, leading to asymmetrical spinal deformities. Most patients die around the age of 20 of pneumonia related to chronic respiratory insufficiency. The treatment of D M D is purely symptomatic and is aimed at prevention of spinal deformities and muscle contraction by physiotherapeutic and orthopedic measures (Moser, 1984; Dubowitz, 1978). The disease affects mainly boys, a consequence of its X-linked mode of inheritance. A milder myopathic disorder which shows very similar clinical features along with X-linked inheritance was described by Becker and Keiner (1955). Becker muscular dystrophy is distinguished from Duchenne by its reduced rate of progression, resulting in a less severe phenotype. Onset is usually between 5 and 15 years and the disabling characteristics ensue much more slowly. The relatively mild course of Becker dystrophy allows some patients to continue a near normal routine up to 40 or 50 years of age. Cardiac complications do not seem to be a feature of the disease (Walton and Gardner-Medwin, 1981). Muscle tissue from dystrophic patients shows many different morphological changes even before clinical symptoms are apparent (Pearson, 1962; Hudgson et a l . , 1967). Typically, muscle biopsies from D M D patients exhibit variation in fiber size, focal areas of basophilic fibers, internal nuclei, and split fibers. A marked predoqinance of type I fibers is evident, along with infiltration of fat and connective tissue. Structural changes of fibers and whorled fibers are also characteristic (Dubowitz, 1985). A number of muscle enzymes and serum proteins have increased levels in the serum of affected boys. This increase is most dramatic for the muscle enzyme creatine kinase (CK) (Ebashi et al., 1959) in the serum of DMD and BMD patients, although it is somewhat elevated in other dystrophies as well. Other proteins, such as aldolase (Shapira et a l . , 1953), aminotransferase, hemopexin, and myoglobin (Adornato et al., 1978; Pennington, 1980), are also detected at high levels in DMD patients. Because there is considerable evidence to suggest that some of these proteins arise from the cytosol of damaged muscle fibers (Kobayashi et a!., 1979; Pennington, 1980; Rowland, 1980), the elevated levels are almost certainly secondary manifestations of the disease. Although unrelated to the basic defect these biochemical markers are still useful as diagnostic tests for neuromuscular disease. Originally, it was believed that BMD and DMD were closely related diseases. During the mid-1970s however, classical linkage analysis had

4

RONALD G . WORTON AND ARTHUR H. M. BURGHES

suggested that the gene responsible for Becker dystrophy might be close to the color-blind locus (Zatz et al., 1974; Skinner et a l . , 1974) on the long arm of the X chromosome and, as data accumulated to show that the Duchenne gene was on the short arm, it reduced the likelihood that the two diseases were related. However, as described in some detail below, more recent linkage analysis using DNA probes has shown that the BMD and D M D genetic loci are in a similar location on the X chromosome short arm, and deletion analysis has determined that the two disorders arise by mutation in the same gene. There are also the rare autosomally inherited dystrophies such as limb girdle and fascioscapulohumeral (FSH) types. These are distinguished by their mode of inheritance and clinical features, although limb girdle can be confused with Becker muscular dystrophy in isolated cases with no family history. Also, there is the rare X-linked Emery-Dreifuss dystrophy, which appears to be clinically different from BMD and DMD. There is now clear evidence that the Emery-Dreifuss gene is close to the hemophilia A (factor VIII) gene at the distal end of the long arm of the X chromosome (Boswinkel et al., 1985; Thomas et al., 1986a; Yates et al., 1986), as suggested by Thomas et al. (1972).

R. GENETIC CONSIDERATIONS Both D M D and BMD are X-linked genetic disorders, expressed almost exclusively in males. Table I summarizes data on the newborn frequency and the estimated mutation rates for D M D and BMD in comparison with other muscular dystrophies and with the X-linked mental retardation (fragile X) syndrome. Duchenne muscular dystrophy is the second most frequent X-linked

INCIDENCE AND

Disease Fascioscapulohumeral (FSH) Limb girdle (LGD)

TABLE I MLTATION R4TE OF THE DYSTROPHIES

Estimated incidence 4-9 x 4

10-5

Becker

2-3

Duchenne

1-3 x 10-

Fragilr X MR

10-5

5 x 10-4

Estimated mutation rate"

Reference

5 x 10-7

Morton and Chung (1959); Kloepfer and Emery (1974)

3

Morton and Chung (1959)

10-5

10-5 (male births)

Kloepfer and Emery (1974)

4-10 x lo-' (male births)

M . W. Thompson et al. (1981)

7 x 10-4 (male births)

Sherman et aE. (1984) (per sperm)

"Haidane formulae for DMD: V = % ( I - g I , for f = 0, V = % I , where Vis combined mutation rare. f is fertility rate for affected males (for DMD, f = O ) , and I is incidence of disease.

DUCHENNE AND BECKER MUSCULAR DYSTROPHY

5

disorder (second only to X-linked mental retardation), and is the most frequent of the common muscular dystrophies. Becker muscular dystrophy is considerably less frequent and the disease is not lethal. Duchenne muscular dystrophy is a lethal disease with zero fertility, so the mutations carried by male patients are not perpetuated in the family. This loss of mutant genes from the population is balanced by the generation of new mutations. According to Haldane (1956), if the mutation rate in male germ cells is the same as that in female germ cells, then one-third of all DMD cases are expected to be new mutants (Table I). A deviation from one-third would signify different mutation rates in the gametes of the two sexes. Although several studies have indicated that less than one-third of patients are new mutants (Roses et al., 1977; Pickard et al., 1978; Ionasescu et al., 1979), these studies all depended on somewhat unreliable means of carrier testing and have therefore been disputed (Moser, 1984). Other studies using C K testing as a means of carrier identification have upheld the value of one-third for new mutations (Davie and Emery, 1978; Gardner-Medwin, 1970; Williams et al., 1983; M . W. Thompson et al., 1981; Moser, 1984), as originally proposed by Chung and Morton (1959) from segregation analysis. Thus, it appears that the mutation rate for DMD is the same in male and female germ cells. In order to account for the relatively high mutation rate in DMD, suggestions of mutational hot spots at the DMD locus or merely a large gene which would allow a high probability of mutation have been advanced. Winter and Pembrey (1982) proposed that unequal crossing over might occur between the DMD gene and a nonfunctional pseudogene, leading to deletion of the DMD gene sequence on one X chromosome. This event could only occur at meiosis in the female. This model was proposed, in part, to explain why male and female mutation rates are equal at the DMD locus, but higher in sperm at the hemophilia A and Lesch-Nyhan loci. However, recent experiments using segregation analysis for hemophilia A and B show an equal mutation rate in both sperm and egg (Barrai et al., 1985), indicating that equal mutation rate in DMD is not unusual and therefore does not necessarily require female-specific mutational mechanisms to explain it. In addition, results of recent DNA studies make it unlikely that a pseudogene exists, but the idea of unequal crossing over between homologous sequences within the vicinity of the gene to cause deletions is still a viable one. The possibility that there may be more than one gene responsible for DMD arose in response to the observed phenotypic heterogeneity. It has been suggested, for example, that boys who are severely mentally retarded have a later age of onset and become confined to a wheelchair later; the fall in C K activity with age is less rapid and urinary excretion of certain amino acids is greater (Emery et al., 1979; Emery, 1980). Emery also finds that,

6

RONALD G . WORTON A N D ARTHUR H . M. BURGHES

in general, mentally retarded boys with D M D and boys of normal intelligence with DMD appear in different families. This has led to the suggestion of more than one gene for the disorder. While this seemed likely a few years ago, it seems less likely now, and one could explain the genetic heterogeneity in terms of different mutations in the same gene giving rise to slightly different phenotypes.

C. MANIFESTING CARRIERS Mothers of affected boys can be divided arbitrarily into three categories: definite carriers having an affected son and a previous affected male on the maternal side of the family, probable carriers with two or more affected sons but no other family history, and possible carriers who have a single affected child without family history (Smith et a l . , 1979; M . W . Thompson et al., 1967, 1981). Approximately 8 % of carriers have some clinical manifestations, ranging from pseudohypertrophy of the calves to marked proximal muscle wasting (Dubowitz, 1982). The wide variation in symptoms expressed by carriers is usually explained in terms of random X inactivation according to the Lyon hypothesis (Vogel and Motulsky, 1986). Since the choice of inactive X is random but at the time of inactivation is fixed for all progeny cells, the female develops as an X chromosomal mosaic in which a proportion of cells, about 50% on average, have an active wild-type gene at the D M D locus. In females with less than 50% active wild-type genes there is presumed to be a reduced amount of gene product, leading to mild manifestation of the disease. In very rare instances females have been observed who express D M D with Turner syndrome (XO) due to the rare occurrence of a D M D mutant allele on the single X chromosome of these girls (Walton, 1957; Ferrier et al., 1965; Jalbert et d.,1966). One such case has a chromosomal rearrangement as discussed below (Bjerglund-Nielsen and Nielsen, 1984). There have also been reports in the literature of fully manifesting females. Penn et al. (1970) reviewed 104 cases, considered them to be atypical of Duchenne, and suggested that most were autosomal dystrophies. Females with Duchenne-like dystrophy have been described with autosomal inheritance (Somer et al., 1985). In addition, a number of fully manifesting females have been described with D M D or BMD secondary to a translocation between an X chromosome and an autosome (reviewed by Boyd et al., 1986). These girls are described in some detail in Section II1,A because of their importance in defining the gene locus. There are also three interesting monozygous female twin pairs in which one has the full D M D phenotype and the other is phenotypically normal

DUCHENNE AND BECKER MUSCULAR DYSTROPHY

7

(Gomez et al., 1977; Meola et al., 1981; Olson and Fenichel, 1982; Burn et al., 1986). In the first two cases there was a family history of DMD; in fact, in one pair the unaffected twin had an affected child, confirming that she had the defective gene. It was assumed that in these girls there was nonrandom X inactivation such that in the unaffected girl the X carrying the normal gene remained active in most cells, whereas in the affected sister the X carrying the defective gene was the active one in most cells. In the latest case (Burn et al., 1986), this hypothesis was tested by fusing fibroblasts from each girl with mouse cells and growing the resultant hybrid cells in conditions that select for cells expressing the X-linked hprt (hypoxanthine phosphoribosyl transferase) gene on the active X chromosome. Hybrid cells quickly lost the inactive X chromosome. Using DNA markers (described in detail below) to determine which X chromosome was retained in the hybrid line it was found that all hybrids generated from the affected twin retained one X chromosome, whereas all hybrid lines generated from the normal twin retained the other X chromosome. This strongly suggests that the two girls have different active X chromosomes. Burn et al. (1986) go on to suggest that the first event might be an unusual X inactivation resulting in two distinct cell masses, each with a different active X, which then separate to initiate twin development. The result is monozygous twin girls discordant for the Duchenne phenotype.

D. CARRIER IDENTIFICATION AND PRENATAL DIAGNOSIS In reference to Duchenne muscular dystrophy, Harper has stated that “carrier detection in this condition is perhaps the single most important problem in the field of genetic counselling for mendelian disorders. . .” (Harper, 1982). The high incidence, the severe burden, and the high probability of transmission by an otherwise healthy carrier combine to make the identification of carriers of greater practical importance. Until the advent of DNA markers, described in Section III,C, all carrier testing was done by measuring secondary changes such as elevated creatine kinase. About 70% of D M D carriers have serum CK activity above the upper limit of normal. Although other enzymes are also elevated, CK is the most reliable indicator of carrier status (Dubowitz, 1982; Percy et al., 1979, 1981). Harper (1982) and Moser (1984) have reviewed the importance of carrier detection, along with the pitfalls of CK testing for carrier status. Pathological changes of muscle in carriers of DMD are minimal, but quantitative changes have been observed in some (Maunder-Sewry and Dubowitz, 1981). The advent of DNA markers at or near the site of the DMD/BMD gene locus has totally changed this picture and provided much more accurate methods for carrier identification as discussed in some detail below.

8

RONALD G. WORTON AND ARTHUR H. M. BURGHES

Based on the grossly elevated CK levels in the serum of affected boys, prenatal diagnosis has been attempted by fetal blood sampling and measurement of fetal C K levels (Mahoney el d . , 1977; Stengel-Rutkowski et d., 1977; Dubowitz et a l . , 1987). However, false negatives with C K testing have been reported (Ionasescu et a l . , 1978; Golbus et al., 1979), negating the value of the the CK test. As might be expected, DNA technology has had an enormous impact in this area and is discussed later.

E. NATUREOF T H E BASICDEFECT Despite more than 30 years of research into the cause of D M D and BMD the nature of the basic defect remains unknown. It is clear that the two diseases are genetic in origin and as such it is reasonable to conclude that the protein product of the D M D or BMD gene is missing or altered by mutation in affected individuals. Our review of this literature will be somewhat cursory in order that we might concentrate on the molecular genetics in later sections. For more extensive reviews of these concepts, the reader may refer to Rowland (1980), Lucy (1980), Jones and Witkowski (1983a), Moser (1984), and Witkowski (1986a,b). Research to date has focused mainly on D M D and has concentrated on the pathophysiology and biochemistry of patient tissue and cells. A number of possibilities have been thoroughly investigated and rejected. For example, a defect involving metabolic storage has been essentially ruled out by the lack of any compelling morphological evidence, and no abnormalities have been found in myosin or any of the other major contractile proteins of muscle (Rowland, 1980). An abnormality in circulation which could lead to ischemia and muscle degeneration (Demos et a l . , 1968; Engel, 1967) has been extensively studied in DMD, but no defect has been demonstrated and considerable evidence has been accumulated to refute the vascular hypothesis (Karpati et a l . , 1974; Bradley, 1977; Jerusalem et al., 1974a,b; Koehler, 1974; Paulson et al., 1974). A decrease in the number of functional motor units and in nerve impulse conductivity has led to the hypothesis of defective innervation of muscle (McComas et a l . , 1970, 1971a,b, 1974). The concept does not enjoy widespread support and the findings have been refuted (Ballantyne and Hansen, 1974; Panayiotopoulous, 1974). Nevertheless, type I fiber predominance (Dubowitz, 1985) and diffuse extrajunctional acetylcholine rcceptors can be seen in some D M D fibers, which may indicate some neuronal involvement (Engel, 1977). Vrbova (1983) has argued that incorrect fetal innervation could give rise to D M D and explain the selective fiber changes seen in dystrophy.

DUCHENNE AND BECKER MUSCULAR DYSTROPHY

9

A number of other active areas of investigation are summarized below, as some of these may give important clues as to the nature of the basic defect.

1 , Site of Expression

of the DMD Gene

Since the muscle is the prime target for destruction it is presumed that the normal gene is expressed in muscle cells. However, patient muscle is often not a suitable tissue in which to study the defective gene since secondary changes related to muscle necrosis and infiltration of muscle by fat cells and fibroblasts are expected to confound the picture by obscuring the basic defect. It, therefore, became relevant to ask whether the normal gene is likely to be expressed in other cell types or in muscle cells grown in culture. The question of muscle cell cultures expressing the basic defect has been addressed by comparison of cultured material from biopsies of normal and affected boys. For example, some investigators have observed abnormal morphology (Thompson et al., 1977), whereas others have reported no differences in morphology, histology, or growth of DMD muscle cultures or nerve muscle cocultures (Gallup et al., 1972; Witkowski, 1977). Muscle cultures from DMD have been shown to have decreased C K levels and also different forms of the enzyme (Ionasescu et al., 1981a; Franklin etal., 1981). In these studies and others, it is difficult to know whether the measured differences are intrinsic to the myoblast population and directly related to the disease or whether they simply reflect secondary pathologic changes including an increased proportion of adipocytes and fibroblasts present in the muscle cultures from patients. Cloned muscle cell lines have been established in an effort to overcome this problem (Yasin et al., 1980, 1982, 1983; Blau and Webster, 1981). Ionasescu et al. (1982) have studied DMD muscle clones and found that they also express less C K and, in particular, the CK-MM band is diminished. Whether this indicates subnormal differentiation is not known. Further comparison of muscle clones (Blau and Webster, 1981) from patients and controls revealed that DMD muscle cells are fully capable of initiating myogenesis in culture and do not differ from normal muscle in several important parameters of differentiation (Blau et al., 1983a). Studies of proliferative capacity, on the other hand, revealed a defect in a proportion of patient-derived clones with altered morphology, an extended generation time and cessation of growth at the 100-1000 cell stage (Blau et al., 1983b). In further studies with muscle cultures derived from carriers of DMD who were also heterozygous for glucose 6-phosphate dehydrogenase (GGPD) type (an X-linked gene), a mixture of defective and normal clones was found. This afforded an opportunity to determine if the defective clones always had the same GGPD type, as would be expected if the defective clones had always inactivated the same X chromosome, presumably the one carrying the normal DMD gene. Since the phenotype of the clones did not correlate with the

10

RONALD G . WORTON AND ARTHUR H . M. BURCHES

G6PD type, they concluded that the cellular growth defect must be a consequence of the disease process rather than a direct effect of the DMD gene (Hurko el a l . , 1986; Webster et a l . , 1986). Fibroblasts are easy to grow in large numbers and are available from a wide range of patients. While it could be argued that these cells are not likely to express the defect since D M D and BMD are myopathies, several studies using fibroblasts indicate that certain manifestations of the disease are expressed in these cells. For example, studies of fibroblasts from D M D patients have revealed abnormalities in protein synthesis (Rodemann and Bayreuther, 1986), phospholipid turnover (Rounds et al., 1980), and intercellular adhesiveness (‘Jones and Witkowski, 198313). As will be seen in the sections below, the evidence for perturbations in protein metabolism, for defects in the cell membrane, and for other specific cellular protein alterations comes from studies in whole muscle, muscle cell cultures, and cultured fibroblasts. Which of these perturbations, if any, is a primary manifestation of the gene defect and which are secondary to the disease process remains to be determined.

2. Studies

of Protein Metabolism

Duchenne dystrophy is a wasting disease, at least in the later stages of the disorder, suggesting the possibility of an imbalance between protein degradation and synthesis. Early studies of muscle biopsy tissue (Kar and Pearson, 1976, 1977) revealed higher levels of proteases, suggesting the possibility of increased protein degradation in D M D muscle. Elevated levels of 3-methyl histidine in urine of D M D boys has also been taken as an indication of an increase in muscle protein breakdown (McKeran et al., 1977; Ballard et al., 1979; Warnes et al., 1981). On the other hand, in vitro monitoring of DMD muscle synthesis and degradation revealed a reduced rate of protein synthesis but normal degradation (Rennie ef at., 1982), suggesting that the earlier results may have been secondary to the basic defect and related to tissue breakdown and the replacement of muscle with connective tissue. Studies in cell culture have not shown any consistent changes. Statham et al. (1980) in fibroblast culture and Neville and Harrold (1985) in muscle culture found no alteration in protein synthesis or degradation indicating either that these cultures do not express the defect or that the changes seen in uivo are secondary effects. In contrast to this, Ionasescu et al. (1976) reported a decrease in protein synthesis in muscle cultures perhaps related to the degree of fibroblast contamination and/or to the proportion of fused cells in the culture. Rodemann and Bayreuther (1986) found a decrease in protein synthesis and an increase in degradation in D M D fibroblast cultures. Also a defect in the ability of polysomes from D M D fibroblasts to support protein

DUCHENNE AND BECKER MUSCULAR DYSTROPHY

11

synthesis has been reported which would agree with altered protein synthesis (Boule et al., 1979). While these cell culture results appear to contrast markedly with a muscle biopsy study reporting an increase in the ability of polysomes to support protein synthesis (Ionasescu et al., 1977a), the studies are not strictly comparable, as biopsy material is heavily infiltrated with connective tissue. It is difficult to know the extent to which the findings in whole muscle tissue and in primary muscle culture are influenced by the connective tissue replacement of muscle. Thus, the results on protein synthesis and degradation are conflicting, but considering the apparently normal growth characteristics of DMD fibroblasts (R. G. Thompson et al., 1982) it appears that there is no reason to suspect a major alteration in protein metabolism. A massive increase in collagen in DMD muscle has led to the suggestion of a defect in collagen metabolism (Stephens et al., 1982; Duance et al., 1980). Studies of collagen synthesis in cultured fibroblasts have measured a decrease in intracellular collagen and an increase in extracellular collagen (Ionasescu et al., 1977b). R . G. Thompson et al. (1982) found a similar increase and suggested that it might be related to a very slight growth difference of the cells. In agreement, Dunn et al. (1984a) found a small increase in total collagen synthesis but an overlap between normal and DMD fibroblasts, while Rodemann and Bayreuther (1984) measured only intracellular collagen, finding increased degradation and a decreased synthesis. Studies with cloned muscle cells have also revealed an increase in total collagen synthesis (Ionasescu et al., 1982). In summary, cultured fibroblasts and myoblasts both appear to show a slight alteration in collagen synthesis, but the alteration is small and it would appear likely that this is a secondary response to the basic defect (R. G. Thompson et al., 1982).

3. Studies of Membranes A defect in the muscle cell membrane was first proposed when high levels of muscle enzymes were found in the serum of DMD patients (Schapira et al., 1953; Dreyfus et al., 1954; Dreyfus and Schapira, 1962). A simple model of leakage of enzymes through defective muscle membranes into serum is complicated, however, by the observation that the leakage appears to be selective and not related to protein size (Rowland, 1980). Since muscle tissue from patients is difficult to obtain in sufficient quanity for study, much of the evidence for a membrane defect comes from studies of other tissues such as erythrocytes and cultured fibroblasts. Any evidence in favor of a membrane defect is still indirect and much of it is controversial (Rowland, 1980). Furthermore, it is not clear whether any of the membrane abnormalities observed are a primary or secondary consequence of the genetic defect. The evidence for some alteration in the membrane comes from several sources.

12

RONALD G . U’ORTON AND ARTHUR H . M. BURGHES

a. Morphology. Focal areas of discontinuity, “delta lesions,” in the plasma membrane have been observed in electron microscope preparations of dystrophic muscle and are believed to be associated with breakdown of the plasma membrane (Mokri and Engel, 1975; Schmalbruch, 1975). While freeze-fracture studies carried out on D M D muscle have supported the concept of the membrane alteration (Schotland et al., 1977, 1980), cultured muscle cells and fibroblasts from D M D patients showed no changes in freezefracture pattern (Osame et al., 1981; Jones et al., 1983). b. Caz+ Znzux. A defect in the plasma membrane might result in elevated Ca2+within the muscle cell. This in turn might lead to excessive contraction of sarcomeres, excessive Ca2’ uptake into mitochrondria depleting cellular ATP, or stimulation of Ca2+ activated proteases, any one of which has been postulated to lead to the breakdown of muscle tissue (Cullen and Fulthrope, 1975; Cullen and Mastaglia, 1980; Wrogeman and Pena, 1976; Engel, 1977; Oberc and Engel, 1977; Duncan, 1978). Although there is little evidence to support these theories (Rowland, 1980), high calcium levels have been observed in muscle from D M D patients (Oberc and Engel, 1977; Bodensteiner and Engel, 1978; Capenter and Karpati, 1979; Maunder et al., 1977; Maunder-Sewry and Dubowitz, 1979). In studies of sarcoplasmic reticulum Samaha and Gergely (1969) found that DMD, BMD, and polymyositis patients showed a reduced rate of calcium uptake and an altered affinity for calcium. They subsequently showed alteration in some sarcoplasmic reticulum proteins (Samaha, 1977) and suggested that these changes and the apparent alteration in Cazt uptake were due to the type I fiber predominance and not therefore a direct consequence of the D M D lesion. In erythrocyte membranes Ca2+ATPase has been found to be more active (Hodson and Pleasure, 1977; Rutenbeek, 1979; Luthra et al., 1979; Dunn et al., 1982a) and Ca2+transport has been reported to be either enhanced (Mollman et al., 1980; Johnsson et a l . , 1983) or normal (Shoji, 1981; Pijst and Scholte, 1983). The difference in Ca2+ATPase is small (Dunn et al., 1982a; Luthra et al., 1979) and the assay depends on using the correct levels of Ca2+. Studies in cultured fibroblasts (Statham and Dubowitz, 1979) have shown no difference in Ca2+exchange, suggesting that these cells regulate their levels of Ca2+correctly. However, Fingermann et al. (1984) did find that patient fibroblasts are less sensitive to growth inhibition by high Caz+levels in the medium and in low Ca2+had alterations in protein synthesis and in their ability to replicate viruses. They suggested that Ca2+concentrations are slightly higher in D M D fibroblasts and that this fact might also account for the observed elevation of C1- flux (Pato et al., 1983). In summary, although calcium may be involved in the pathogenesis of the disease, it would appear that the elevated Ca2+level itself may be secondary to a membrane lesion.

DUCHENNE AND BECKER MUSCULAR DYSTROPHY

13

c. Membrane Enzymes. A generalized membrane defect might well have an effect on the activity of enzymes anchored in the membrane. The Na+, K+-ATPase is a widely studied marker of sarcolemma that, despite early reports of decreased activity, appears not to be altered in DMD (Rowland, 1980). Another major enzyme of the sarcolemma is adenylate cyclase. In D M D patient muscle (Mawatari et al., 1974) and erythrocytes (Mawatari et al., 1976) it has been reported to show reduced stimulation with epinephrine, although this has been refuted (Wacholtz et al., 1979; Fischer et al., 1978). More recently, Cerri et al. (1981) observed depressed adenylate cyclase activity and a reduced stimulation by epinephrine, concluding that the loss of basal activity was due to overall protein loss related to breakdown of the muscle itself, and hence the changes are most likely secondary to the disease. Gelman et al. (1980) assayed dipeptidyl aminopeptidase (DAP I or cathepsin C) and other lysosomal enzymes in normal and dystrophic fibroblasts and found a reduction in DAP I activity which they attributed to a possible alteration in the lysosomal membrane. d. Membrane Lipids. Abnormalities in the phospholipid profile of dystrophic muscle have been reported by many groups (Takagi et al., 1968; Kunze and Olthoff, 1970; Hughes, 1972; Kunze et al., 1973), but it has been considered likely that the changes observed may be accounted for by the replacement of muscle with fat and connective tissue (Pearce et al., 1981). This view would be consistent with the failure to detect changes in the lipids of cultured fibroblasts (Kohlschutter et al., 1976) and with studies of the enzymes involved in phospholipid metabolism which failed to demonstrate any differences between normal and DMD muscle (Kunze et al., 1980). Studies of turnover of phospholipids in Con A-challenged fibroblasts (Rounds et al., 1980) suggested a diminished rate of phosphatidylinositol resynthesis, but a subsequent study of the enzymes involved in the pathway failed to show any defect (Ziskind et al., 1981). A turnover study in muscle cell culture and muscle tissue (Ionasescu et al., 1981b) showing altered phospholipid synthesis is subject to the previously mentioned limitations of studies on such material. Whole-body 31P-NMR examinations of DMD muscle have revealed increased peaks of glycerol pGosphocholine (GPC) and serine ethanolamine phosphodiester (Newman et al., 1982). Barany et al. (1982), on the other hand, found GPC levels in extracts of normal muscle to be nonuniformly distributed (higher in arm than leg) but nearly absent from extracts of quadriceps from patients. The latter result and the discovery of an alternative pathway for the synthesis of phosphatidylcholine in voluntary muscle

14

RONALD G . WORTON AND ARTHUR H . M . BURGHES

(GPC is an intermediate) has led to the hypothesis that the alternative glycerol phosphodiester pathway in D M D muscle is defective (Infante, 1985a,b). Finally, an impaired @-oxidation of fatty acids in muscle has been suggested to explain the C K leakage from D M D muscle (Carrol et a l . , 1985). e. Cell Surfme Properties. Focal alterations in the binding of the lectin concanavalin A (Con A) at the cell surface of dystrophic muscle have been reported (Bonilla et a l . , 1978, 1980) but not confirmed in cultured muscle (Heiman-Patterson et a l . , 1981). Reduced binding of the lectin Ricinus communis I to D M D muscle plasma membranes has also been reported (Capaldi et a/. , 1984a,b) and attributed perhaps to secondary pathological changes. Appleyard el al. (1985) reported that HLA Class I antigens were not expressed on normal muscle but were expressed in polymyositis and the X-linked dystrophies, showing that some alteration must have occurred at the cell surface in DMD. Intercellular adhesiveness of skin fibroblasts is another indicator of cell surface properties. Cells from D M D patients have been found to be less adhesive and to show a different pattern of aggregation compared to normal controls (Jones and Witkowski, 1980, 1981; Pizzey and Jones, 1985). Cells from obligate D M D carriers, including presumably cells that had inactivated the normal D M D locus, showed normal aggregation (Jones and Witkowski, 1983b), suggesting that the aggregation pattern is not a direct cause of the gene defect. A potentially better approach was that of Hillier et al. (1985), who used cloned fibroblasts from a D M D carrier heterozygous for the G6PD marker and found that the cell aggregation phenotype correlated with one GGPD type. A major limitation of this study was the fact that only one aggregation-defective clone was examined. An expanded study is required to confirm these results. The glycoprotein fibronectin plays a major role in cell adhesion and it would be interesting to know if the altered adhesion properties might be a consequence of the reported increase in fibronectin on the surface of fibroblasts from D M D patients (McMurchie et a l. , 1979; Burghes et a l . , 1981). Unfortunately, studies of many celi lines failed to reveal consistent differences between normal and D M D samples (Burghes, 1984) and the hypothesis remains unconfirmed. The studies described above plus others reviewed by Witkowski (1986a,b) serve to illustrate the concept of a generalized membrane defect and argue for the expression of the defect in cultured cells. However, the studies fall short of determining the exact biochemical process or protein component that is affected.

DUCHENNE AND BECKER MUSCULAR DYSTROPHY

15

4. Protein Electrophoresis Electrophoretic techniques provide a way of searching for proteins altered by the disease process. One of the first successful applications of electrophoresis to genetic disease was the demonstration of an abnormal hemoglobin in patients suffering from sickle-cell anemia (Pauling et al., 1949). Since this time, there have been advances in the technique and two-dimensional gels can now separate 2000-3000 proteins (Fig. 1). When examining disease tissue by these techniques, some protein alterations are expected to be secondary to the pathology or to compensating metabolic changes, thereby complicating the search for the primary defect (Goldman et al., 1982; Merrill et al., 1983).

FIG. 1. Autoradiograph of a two-dimensional gel of [J5S]methionine-labeledprotein from skin fibroblasts. For isoelectric focusing,the cathode is on the right. Basic proteins are resolved on the right and acidic proteins on the left. High-molecular-weightproteins are resolved at the top of the gel. The arrows indicate the three protein spots that have recently been reported to be altered in DMD patients with deletions (Patel et al., 1986). Figure courtesy of Dr. M. J. Dunn, Hammersmith Hospital.

16

RONALD G . WORTON AND ARTHUR H . M . BURGHES

Erythrocyte membranes have been analyzed on one- and two-dimensional gels. No qualitative or quantitative differences were found in the onedimensional system (Roses et al., 1975; Dunn et al., 1982a) or the twodimensional system (Copeland et a l . , 1982) despite good resolution and detection sensitivity in the latter. a. Cultured Cells. Cultured muscle cells have the advantage that nonspecific effects of muscle breakdown are obviated. Two-dimensional gel analysis of primary muscle cultures and muscle cell clones has revealed no differences between D M D patients and normal individuals (Walsh, 1984), although the resolution did not extend into the high-molecular-weight region or into the basic protein region. Cultured fibroblasts are more easily grown and since there is evidence that they express the gene defect, extensive comparison of normal and DMD fibroblasts has been carried out. No qualitative differences have been found using SDS-polyacrylamide gel electrophoresis (PAGE), gradient gels, isoelectric focusing gels, and a series of detection systems (Pena et a l . , 1978; Burghes el al., 1981, 1982a-c, 1983). Minor quantitative variations have been attributed to variation between cell lines or secondary manifestations of the basic defect (Burghes et al., 1982b,c, 1984; Dunn et al., 1983, 1984b). The f35S]methionine-labeled proteins excreted into the medium by fibroblasts have also been subjected to one-dimensional analysis and no consistent changes were found (Dunn et al., 1983). Two-dimensional electrophoresis has also been used to compare skin fibroblast from normal and D M D individuals. No differences were detected by Coomassie blue staining (Willers et al., 1981) nor by [35S]methioninelabeling (Burghes et al., 1981). Rosenman et al. (1982) compared fibroblasts from DMD patients with normal controls using dual isotopic labeling and reported a 56,000-Da protein to be missing from D M D fibroblasts. This result was later negated when it was realized that the normal cell lines used in the experiments were all derived from human foreskin whereas the D M D biopsies all came from nongenital skin, and it was found that the 56,000-Da protein was specific to genital skin and not to D M D (R. G . Thompson et al., 1983). Further comparison by a 2D-PAGE system that resolves basic proteins also failed to detect any changes in DMD fibroblasts (Burghes et a l . , 1983; Dunn et a l . , 1984b). While no obvious changes in the 2D-PAGE profiles of normal and D M D fibroblasts have been observed, it is certainly possible that minor alterations have been overlooked. Even with dual-labeling techniques it is possible to miss differences in low-abundance proteins. Furthermore, the dual-labeling technique compares differences between individuals, whereas computer-assisted comparison between several patients and normals might prove more sensitive (Dunn et al., 1984a,b). Finally, a genetic disease such as D M D might often be

DUCHENNE AND BECKER MUSCULAR DYSTROPHY

17

the result of a mutation that alters the function of the protein without altering its abundance or its physical properties, as is the case for Lesch-Nyhan syndrome (Wilson et al., 1983, 1986; Merriel et a l . , 1983). For this reason patients with gene deletions or gross chromosomal alterations may have a better chance of showing a protein defect. It is of interest therefore that Pate1 et al. (1986) have described three protein spots that appear to be absent from fibroblast extracts of some known gene deletion patients. It is too soon to tell if the protein defect is primary or secondary to the basic defect in these patients. The affected proteins are shown in Fig. 1. 6. Muscle Tissue. Despite the difficulty in interpreting protein comparisons in muscle tissue several groups have examined the soluble proteins of muscle with isoelectric focusing (B. J. Thompson et al., 1981, 1982; Dunn et a l . , 1983), 2D-PAGE with Coomassie blue staining (Giometti et al., 1980, 1983), and with ['*C]iodoacetamide labeling (Giometti and Danon, 1985). In no case was any difference found between normal and DMD, forcing the conclusion that any alteration had to be in a low-abundance protein, below the detection limit of the technique, or involved no alteration in charge or molecular weight. With the recent molecular genetic studies revealing a very large gene as described in detail below, there is reason to focus attention on the large proteins of muscle that might be the product of such a gene. Highmolecular-weight proteins of muscle have been examined by SDS-PAGE. Capaldi et al. (1985) examined muscle on SDS gradient gels and found no change on standard protein staining. However, after blotting the gels and probing with the lectin Ricinus communis I, which had previously shown reduced binding to D M D muscle cells, a 370-kDa protein was found to be missing from DMD muscle. While it is possible that this is a result of secondary changes in the muscle, it is an interesting observation and merits further work. Recently, in response to the evidence for a large gene, Wood et al. (1987) examined the proteins nebulin (550 kDa) and titin (>lo00 kDa) by PAGE. They reported that nebulin, a structural protein that serves to align contractile fibers during contraction, was missing from D M D muscle but not from muscle of normal or other neuromuscular disease and suggested that it is a good candidate for the DMD gene. Without specific antibody staining of the gels it is difficult to determine whether the protein is absent or has undergone degradation. This is especially important for nebulin, as it is particularly susceptible to degradation by Ca2+-activated neutral proteases (Wang, 1984), and these are known to be elevated in dystrophic muscle (Kar and Pearson, 1976, 1977). (See Note Added in Proof regarding nebulin .)

18

RONALD G . WORTON AND ARTHUR H . M. BURGHES

A third component of muscle that could be a candidate for the D M D gene product is the sarcoplasmic reticulum protein that forms junctional feet linking the terminal cisternae to the transverse tubules (Cadwell and Caswell, 1982). This 300- to 325-kDa protein binds calmodulin and is rapidly degraded by Ca2+-activatedproteases (Seiler et al., 1984). Recently the Ca” gating channel of sarcoplasmic reticulum has been isolated and found to be composed of three proteins of 360, 330, and 175 kDa, one of which may be the feet protein (Inui et al., 1987). Combined with the fact that the Ca2+levels are elevated in DMD muscle, this protein would appear to be another good candidate for the product of the D M D gene. Thus, a great deal of effort has gone into the study of Duchenne muscular dystrophy. While many alterations have been observed in the tissues and cells of patients, most of these alterations are now felt to be secondary to the disease process. In the sections to follow, we describe work at the DNA level that has allowed a direct approach to the gene responsible for the disease, bypassing the need to know anything about the protein product. The direct approach is based on knowledge of the gene’s location on the X chromosome and on the availability of patients with structural rearrangements of the chromosome at the site of the gene. We review in some detail the studies that allowed the mapping of the gene and the approaches that led to its eventual isolation.

111. Locatlng the DMD and BMD Genes at Band Xp21

The genes responsible for Duchenne and Becker muscular dystrophy are located at band Xp21 in the middle of the short arm of the X chromosome. The human X chromosome is shown schematically in Fig. 2. The band Xp21 is in the short arm (p) about halfway between centromere and telomere. With high-resolution banding the dark band p21 is seen as two dark bands with a light space between, defining three high-resolution bands, p21.1, p21.2, and p2 1.3. The evidence for the D M D and BMD genes being located at p21 comes from three independent sources. The first is a series of patients with chromosomal translocations that involve breakage and reunion of the X chromosome at band p21, the second is a set of patients with visable chromosome deletions of the Xp21 region, and the third involves the mapping of the gene to Xp21 by genetic linkage analysis in families.

A. FEMALESWITH X:AUTOSOME TRANSLOCATIONS A female with Duchenne phenotype and a structural abnormality of the X chromosome was first reported by Berg and Conte (1974) in an abstract, but

DUCHENNE AND BECKER MUSCULAR DYSTROPHY

tel

-

19

22.3 -22.2 22.1 21.2

p ARM 11.3

cen

-

13 21.1

q ARM

tel

-

21.2

21.3 22 23 24 25 26 27 28

FIG. 2. The human X chromosome. The schematic on the left is the banding pattern of an X chromosome at metaphase. Bands are numbered according to international nomenclature. The schematic on the right is a high resolution banding pattern as seen in a prometaphase cell. The band Xp21 is seen as a single dark band on the left and as three bands, p21.1, p21.2, and p21.3 in the diagram on the right.

details of the abnormality were not available. In other abstracts (Verellen et al.) 1977, 1978; Greenstein et a l . , 1977) two additional cases were revealed, both reported to have X:autosome translocations and both with an exchange point at band p21 in the X chromosome. These initial observations were followed by a number of reports detailing further cases, and the involvement of a translocation exchange point at Xp21 was a consistent feature. A summary of the 14 published cases is presented in Table 11. Additional unpublished cases are listed below the table (reviewed by Boyd et al. 1986). The case described by Lindenbaum et al. (1979) was one of the first to be fully documented and showed a classic Duchenne phenotype and a complex chromosomal rearrangement with an inversion of the region Xp 11 to Xp2 1 as well as translocation of the terminal portion (from Xp21) to an autosome. In all the other cases, the translocation was simple and reciprocal; the distal end of the X chromosome had exchanged with the distal part of one of the autosomes. The autosomal exchange point was different in each case but the exchange point in the X was consistently within band Xp21. This led to the realization that the DMD gene might be located at band Xp21 and might be disrupted by the translocation, but it did not explain why these girls were affected with the disease since, in each case, there was a normal X chromosome )

Exchange point 'Translocation X:l

x:2 X:3 x:4 x:5 x:5 X:6 X:6 X:8 x:9 x:9 X:ll X:ll x:21

X chromosome

Autosome

Xp21.2< xp21.2 Xp21.2 or 21.3 xp21.1 xp21.1 xp21.2 xp21 xp21.2 xp21.1 xp21.2 xp21

lp34.1 o r 34.3 2q37.3 3q13.2 o r 13.3 4q26 5q35.3 5q31.1 6q16 6q2 1 8q24.3 9q22.3 9q2 1

xp21.1 xp21.2 xp21.1

1 lq13.5 11q23.3 21p12

Normal X inactivated (5%) 100 100 95 100 100 100 98

-

80 98 100

93

Clinical featuresb

Rcferenre

DMD DMD, mod. MR DMD, MR dysmorphir DMD DMD DMD, mod. MR DMD DMD Mild DMD (?BMD) DMD, mod. MR DMD, Turner's syndrome epilepsy, MR DMD Mild DMD (TBMD) Mild DMD (?BMD)

Lindenbauni et al. (1979) Holden el af. (1986) Canki et af. (1979) Saito et af. (1985) Jacobs et af. (1981) Nevin el af. (1986) Perez-Vidal d al. (1983) Zatz et af. (1981) Narazaki et af. (1986) Emanuel et al. (1983) Berjerglund-Nielsen and Nielsen (1984) Greenstein et af. (1978) Bjerglund-Nielsen ct af. (1983) Verellen-Dumoulin et al. (1984)

'All or most of these cases have had lymphoblastoid cell cultures grown up and frozen away by Dr. Y. Boyd and her colleagues at Oxford University. The lines are available for research purposes. In the review by Boyd el al. (1986) three additional unpublished cases are reported. One was a t(X;2)(p21;q14) from Dr. M. Zatz, the second was a t(X;3)(p21;q37)submitted to the cell bank by Professors P. Pearson and M. Ferguson-Smith, and the third was a case of t(X;15)(p21;q26) submitted by Dr. Ribiero. We are aware of two additional cases, a t(X;4) under investigation by Dr. U. Francke and another t(X;4) detected prenatally by Dr. D. van Dyke. This brings the total number of cases known by us to 19. *MR,mentalretardation. Mild cases of DMDmay be reinterpreted in some cases as BMD once the child has reached an age to make the distinction. 'This case had a complex rearrangement with an apparent inversion of the region Xpl 1 to Xp21 as well as translocation of the terminal portion (Xp21 to Xpter) to chromosome 1.

DUCHENNE AND BECKER MUSCULAR DYSTROPHY

21

present. The explanation comes from the observation that in nearly all cells examined from each of the translocation patients, the normal X chromosome (the one not involved in the translocation) was the one to replicate late in the cell cycle, a characteristic of the inactive X (Table 11). This nonrandom inactivation of the normal X chromosome is very different from the situation in most females, where X inactivation is random and results in approximately 50% of cells with inactivation of the maternally inherited X and 50% with inactivation of the paternally inherited X . The nonrandom pattern is, however, characteristic of girls with balanced X:autosome translocations including those whose translocations involve regions other than Xp21 and who do not have a dystrophic phenotype. A plausible explanation is that X inactivation in the embryo is random as for any female, but that cells with inactive translocation chromosomes may be at a selective disadvantage, especially if the attached autosomal sequences are also inactivated, leading to gene imbalance (Hagemeijer et al., 1977). In any event, the net result is an apparent nonrandom inactivation of the normal X. Whatever the mechanism, the consequence is clear; the normal DMD locus on the normal X chromosome is inactive, and girls who otherwise would be classified as heterozygous carriers manifest the disease. The corollary to this is that the single defective DMD gene in these girls must be located on the translocated X chromosome. Thus, the most striking feature that is common to all the cases in Table I1 is a break occurring in Xp21 with a balanced translocation rendering the normal X inactive. Furthermore, there is no family history of DMD in any of the girls’ families and the chromosomes, when examined in the parents, were normal. The inescapable conclusion is that the de nouo translocation is itself the cause of the DMD mutation and that it acts by disrupting the DMD genetic locus. This pinpoints the DMD locus to band p21 of the X chromosome. Recently, several of these translocations have been examined by highresolution chromosome banding to determine the precise breakpoints within the Xp21 band (Boyd and Buckle, 1986). This study revealed that in most cases the breaks in the translocations occurred in band Xp21.2 (Table 11). However, one case (Canki et a l . , 1979) appeared to have the break more toward the telomere in Xp21.2 or 21.3, and two others (Jacobs et al., 1981; Verellen-Dumoulin et al., 1984) appeared to break more toward the centromere in Xp21.1. Two additional cases (Saito et al., 1985; Narazaki et a l . , 1986) have also had their exchange points in X p 2 1 . 1 . Therefore, detailed cytogenetic analysis has indicated that the breakpoints are heterogeneous and may be located over a region of the X chromosome that, in molecular terms, amounts to 3000-4000 kilobases (kb) (3-4 million base pairs) of DNA.

22

RONALD G . WORTON AND ARTHUR H . M. BURGHES

It also appears that in some of the cases described in Table I1 the clinical course of the disease is milder, more resembling BMD than DMD. The exact differences in the clinical spectrum are difficult to determine as the translocation patients are all of different ages and some are too young to be precisely diagnosed (Dubowitz, 1986; Worton, 1986). Interestingly, there is no correlation between the apparent breakpoint position and the severity of the disease as might be expected if separate DMD and BMD genes were being disrupted (Boyd and Buckle, 1986). One explanation for the variable phenotype might relate to the variability of the autosomal exchange points, if the D M D gene should come under the influence of the transcriptional activity of the adjacent autosomal segment. An alternative explanation for the mild phenotype in some of the girls may be related to the fact that X inactivation is not completely nonrandom and the presence of a few cells (or a few nuclei in a muscle fiber) with an active normal X chromosome may be sufficient to alter the phenotype from the Duchenne to the Becker category. In this regard, two of the three patients said to have a mild dystrophy (Verellen-Dumoulin et al., 1984; Narazaki et al., 1986) had less than 100% (93 and 8 0 % , respectively) of the cells with an inactive normal X chromosome (Table 11), which is consistent with this hypothesis. In the third case of mild dystropy X inactivation was not examined. Although the case is strong that the translocation is the mutational event, it is possible that in some cases there was a preexisting mutation inherited on a rearranged X chromosome from a carrier mother. We have ruled out this possibility for the t(X;21) case (Fig. 3) by demonstrating that the translocation chromosomes were derived from the patient’s father. Since he carried no translocation in his somatic cells and since he did not have the disease, the translocation must have occurred in his germ cells and must have resulted in the mutation that caused the disease in his daughter (Kean et al., 1986). There are a number of ways a translocation could be envisioned to produce a mutation leading to DMD. One is to disrupt the gene itself, separating the sequences at one end from those at the other end, resulting in a truncated gene product. The second is to separate a regulatory sequence at one end of the gene from the gene itself, resulting in a reduction in gene expression. The third possibility is that what appears as a simple reciprocal translocation is really a more complex rearrangement with an associated deletion of a few million base pairs of DNA, resulting in deletion of all genes in the vicinity including the DMD or BMD genes. The fourth possibility is that the translocation causes an alteration of the chromatin structure surrounding the exchange point that might block the accessibility and therefore the expression of genes located at some distance along the chromosome. In the first two cases the D M D gene must be located at or very near the site of the translocation. In the latter two cases the gene could be located at some distance up to a few million base pairs from the

DUCHENNE AND BECKER MUSCULAR DYSTROPHY

23

FIG.3. Karyotype of a girl with DMD due to a reciprocal translocation between the short arm of the X chromosome and the short arm of chromosome 21. The top row shows pairs 1-6, the second row pairs 7-12, the third row pairs 13-18, and the fourth row pairs 19-22 and X. The arrows identify the two translocation chromosomes. The upper portion of the X, from band Xp21 to the short arm telomere, is translocated onto the upper end of chromosome 21. Although it is not visible in the microscope, molecular studies have revealed that a small amount of material from chromosome 21 has been translocated to the X chromosome. The translocation, therefore, is reciprocal. The chromosomes in the bottom row show slightly higher resolution banding. The X and the 21 are on the left and right, respectively. In the middle are the X-derived [der(X)] and the 21-derived [der(21)] chromosomes. The line runs through the exchange points in band Xp21 and band 2 1 ~ 1 2 The . band Zip12 is a light-staining region in the short arm of chromosome 21 that contains tandemly repeated copies of the genes that encode ribosomal RNA. A similar block of ribosomal genes is located on the short arms of each of the five pairs (13, 14, 15, 21, 22) of acrocentric chromosomes. A schematic diagram of the translocation is presented in Fig. 6.

site of the translocation. Detailed molecular studies of the translocations are just beginning as outlined below and will ultimately allow each of these models to be tested for several of the translocations.

24

RONALD G . WORTON AND ARTHUR H . M. BURGHES

B. MALESWITH DELETIONS AND

COMPLEX PHENOTYPES

The second category of patients to provide evidence that the D M D gene is at Xp21 is the boys with D M D and other X-linked disorders due to a cytologically visible deletion of band Xp21. A syndrome of Duchenne dystrophy or similar myopathy coupled with adrenal hypoplasia (AH), glyerol kinase (GK) deficiency, and mental retardation (MR) has been recognized for several years (Roig et al., 1977; McCabe et al., 1977a,b; Guggenheim et al., 1980; Toyofuku et al., 1981; Bartley et al., 1982; Renier et al., 1983). As indicated in Table 111, several of the cases occurred in families where there were two or more affected brothers or two affected males related through a female carrier. The pattern suggested X-linked inheritance of ail three conditions and suggested that the three genes might lie close together on the X chromosome. A somewhat different type of disease cluster was reported by Kouseff (1981) in a boy with chronic granulomatous disease (CGD) and Duchenne dystrophy and suggested that the genes for D M D and C G D may reside together on the short arm of the X chromosome. The phenotypes of this patient and the other patients described above are summarized in Table 111. The clustering of diseases in these patients suggested the possibility that the complex phenotype might be caused by deletion of a block of genes in close proximity to one another on the X chromosome. However, the chromosomes in most cases were initially reported to be normal by regular cytogenetic analysis. The first evidence for such a deletion came from two new patients also listed in Table 111. One of these was a young mentally retarded boy (initials BB) with DMD, CGD, retinitis pigmentosa (RP), and the McLeod red cell phenotype (Francke et al., 1985). Careful cytogenetic analysis by highresolution chromosome banding revealed a small but detectable deletion of part of band Xp21, Since a deletion of this size must remove a few thousand kilobases of DNA, it might well have deleted several genes including those responsible for DMD, CGD, and RP. In order to verify that the deleted segment was not reinserted into another part of the X chromosome or into another chromosome of the patient, it was necessary to study the DNA at the site of the deletion (Francke et al., 1985). At the time, a random DNA fragment from a human X chromosome had been cloned and had been shown to be derived from Xp21 near the D M D gene (Hoflcer et al., 1985). The DNA segment, called 754, is discussed in more detail in Section II1,C. The 754 DNA clone was radiolabeled and used as a probe to determine if the patient had sequences complementary to 754 DNA. The probe failed to hybridize with DNA from patient BB and confirmed that these sequences were indeed deleted from his X chromosome. The result also verified that these same sequences were not inserted elsewhere in his genome

TABLE I11 MALESWITH COMPLEX PHENOTYPES AND DELETIONS OF BANDxp21 Phenotype Patient and reference

GK

AH

DMD

MR

2 Brothers; Guggenheim et al. (1980) 2 Brothers; Toyofuku et al. (1981) Male; Kouseff (1981) Family 1 (2 aff.)n; Bartley et al. (1982); Patil et al. (1985) Family 2 (2 aff.)b; Bartley et al. (1982); Patil et al. (1985) Family 3; Bartley et al. (1986); Patil et al. (1985) Family 4: Patil et al. (1985) Male; Petzykowski ct al. (1982); Wieringa et al. (1985a) 3 Brothers; Renier et af. (1983); Wieringa et al. (1985a,b) BB; Francke et af. (1985) Male; Hammond et al. (1985); Old ct al. (1985) Male; Saito et al. (1986) Male 1; Dunger el al. (1986) Male 2; Dunger et al. (1986) SS AND JDd; Wilcox ef al. (1986) Male; Clarke et al. (1986) Male; Kenwrick ef al. (1987)

+

+ +

+

+

+

+ + + +

+

+

+ +

+ +

+

+

+

+

+

+

+ + +

+ + +

+

*

+ +

+

CGD

RP

OTC

Deletion Xp21

Hybridization to 754 ~

+

+

+ + +

+ + +

+ +

+ +

“Half cousins. *Second cousins. 3 i e d shortly after birth; DMD not demonstrated. dCousins; one retarded, the other near normal. Wow cytometry indicated a deletion of 9% of the X chromosome.

~~

+ + +

p21.2-21.3 p21.2-21.3

No Yes

+

+

+

+

p21.2-21.3 p21.2 Not done p21.2 p21.1-21.2

Yes Yes No No No

+ +

+

+ +

c

p11.2-Xp21

No

P21 None None p21.1-22.3c p21.1-22.1 None

Not done No Yes No No Yes

26

RONALD G. WORTON AND ARTHUR H. M. BURGHES

(Francke et al., 1985). We return to a discussion of patient BB in the section on cloning the D M D gene. The second patient confirmed to have a deletion at Xp2 1, also by highresolution chromosome banding, was an infant who died in the neonatal period with GK deficiency, AH, and ornithine transcarbamylase (OTC) deficiency (Hammond et al., 1985). DNA from this patient was tested for its potential to hybridize to probe 754 and to the O T C gene sequence. Both the 754 and the OTC probes failed to hybridize to the patient’s DNA, which confirmed, at the molecular level, the existence of the deletion that had been seen by cytogenetic analysis (Old et al., 1985). Following quickly upon these studies Patil et al. (1985) studied two families previously reported by Bartley et al. (1982), along with two other families. High-resolution banding showed a small deletion of Xp21.2-Xp21.3 in Families 1 and 2 and in the new Family 3 (Bartley et al., 1986). The son with a myopathy in Family 1 did not bind DNA probe 754. The son in Family 2 who has no myopathy and those in Families 3 and 4 with myopathy are all positive for 754 hybridization (Table 111). The investigators proposed that the GK and AH loci probably lie on the opposite side of the D M D locus from 754, although they could lie between D M D and 754 and still give the observed result. Since the 754 probe site had been mapped on the centromeric side of D M D (Hofker et a l . , 1975; Francke et al. , 1985), this suggested that the location for GK and AH was on the distal (telomeric) side of DMD. Similarly, Wieringa et al. (1985a,b) examined the patients described by Petzykowski et al. (1982) and Renier et al. (1983). They found that probe 754 failed to hybridize to DNA from each of these patients and that highresolution chromosome banding revealed a small deletion in Xp2 1.2. Since these reports, other patients have been described with cytologically visible deletions of Xp21. These include two unrelated retarded boys with GK, AH, and DMD, only one of whom is deleted for the probe 754 (Dunger et a ! . , 1986). and cousins with an inherited 6000-kb deletion (9% of the chromosome) that deletes the probe 754 (Wilcox et al., 1986). Despite apparently identical deletions, one of the two cousins has a profound mental handicap while the other is only mildly retarded (Wilcox et al., 1986). In summary, the data of Table I11 clearly indicate that deletions of the Xp21 region are found in boys with D M D and support the mapping of the DMD gene to that region. We return to discuss some of these patients at the molecular level after the sections on cloning of the D M D gene.

C. LINKAGE ANALYSIS IN DMD AND BMD FAMILIES The third line of evidence placing the DMD/BMD locus at Xp21 came from linkage analysis showing that the mutations in affected males segregated

DUCHENNE AND BECKER MUSCULAR DYSTROPHY

27

in families with DNA markers that mapped near the central part of the short arm of the X chromosome. DNA markers at the extreme ends of the short arm and markers on the long arm failed to segregate with the DMD or BMD mutant genes, indicating that these markers were sufficiently far from the DMD/BMD locus to appear unlinked, due to crossing over at meiosis. The ability to perform this type of analysis depended upon the discovery of DNA markers known as restriction fragment length polymorphisms, or RFLPs. Restriction fragments of DNA are generated by cleaving DNA with one of the numerous restriction enzymes that are available. Restriction enzymes cleave at specific recognition sites on the DNA molecule, and usually the recognition site is invariant when comparing DNA from different individuals. Some sites, however, are not invarient, and for sites on the X chromosomes, the DNA from any female may have a particular recognition site present on both X chromosomes, one of the two X chromosomes, or neither X chromosome. This natural variation is usually due to an innocuous base change in the noncoding portion of the chromosome that results in the creation or elimination of a specific recognition site, and the presence or absence of the site results in a corresponding variation (polymorphism) in the size of the restriction fragment released following digestion by the appropriate restiction enzyrne. For genetic analysis, the availability of these RFLPs is of great importance, because the presence or absence of a restriction site constitutes a genetic marker that is inherited in mendelian fashion, essentially the way a gene is inherited. Furthermore, RFLPs are known to occur at intervals of every few hundred base pairs along the length of each chromosome and therefore constitute an almost unlimited set of markers for genetic analysis (Botstein et al., 1980). The detection of an RFLP depends upon the availability of a cloned segment of DNA from the chromosome at or near the RFLP marker. This cloned segment is radiolabeled and used as a hybridization probe to detect the size of a restriction fragment released from an individual whose DNA has been digested with a restriction enzyme and fractionated according to size by electrophoresis in an agarose gel. T o detect the size of the fragment carrying sequences complementary to the probe, the size-fractionated DNA is first transferred by the Southern blot procedure (Southern, 1975) from the agarose gel to a solid support such as a nitrocellulose sheet. The radiolabeled probe is allowed to hybridize to the complementary sequences on the nitrocellulose sheet, and the exact position of the complementary sequences is determined by exposure of the sheet to an overlaid X-ray film. The size of the fragment detected determines whether the restriction site is

28

RONALD G . WORTON AND ARTHUR H. M. BURGHES

present or absent and hence constitutes the assay for the genetic marker. The concept of using RFLPs to study linkage in genetic disorders has been described in more detail by Housman and Gusella (1981) and by Housman et al. (1982). Thus, RFLPs provide DNA markers that can be followed in families, and cosegregation of a genetic defect with a marker implies close linkage between the gene and the marker. Since the cloned DNA segment that detects the RFLP marker can readily be mapped to a chromosome, or even to a specific region of a chromosome, mapping a closely linked marker effectively maps the nearby gene (Orkin, 1986). The first DNA probe to detect an RFLP linked to the DMD gene came from an X chromosome DNA library prepared by Davies et al. (1981). The Davies library consisted of human genomic DNA fragments inserted into the DNA molecule of bacteriophage X grown in Esherichia coli. T o enrich for fragments from the human X chromosome, Davies et al. (1981) prepared the library from the DNA of a female with four X chromosomes (karyotype 48, XXXX). Cells from this individual were blocked at mitosis to allow the highly condensed mitotic chromosomes to accumulate. The chromosomes were stained with a fluorescent dye and then fractionated according to size on a fluorescence-activated cell sorter (FACS). The peak of X chromosomes was collected and the DNA was digested with the restriction enzyme EcoRI and ligated to the bacteriophage DN.4 molecule that had also been digested with EcoRI. The recombinant DNA molecules were packaged into phage heads, and the intact phage particles were plated onto a lawn of E. coli. T o a first approximation, each of the 50,000 plaques was derived from a recombinant phage and each contained a different piece of human DNA. In such a phage library, therefore, most of the plaques contain a clonal isolate of a piece of the X chromosome. One of these clonal isolates, called XRC8, turned out to carry a 6.1-kb EcoRI fragment from the X chromosome short arm that showed linkage to the DMD gene (Murrey et al., 1982). The fragment was mapped to the X chromosome short arm using a panel of somatic cell hybrids. Such a panel consists of a set of human-mouse or human-hamster somatic cell hybrid lines, each carrying one or a few human chromosomes. The panel used by Murrey et al. (1982) included some hybrids made by fusing rodent cells to cells of patients with X chromosome structural rearrangements, so that some of the resulting cell hybrids contained only a portion of a human X chromosome. The DNA was extracted from each hybrid cell line, digested with EcoRI, and analyzed by the Southern blot procedure. The clone ARC8 hybridized with DNA from cell hybrids carrying an intact X chromosome but not with hybrids that were missing the distal third of the short arm. Thus, the XRC8 probe was localized to the distal third of the short arm of the X chromosome.

DUCHENNE AND BECKER MUSCULAR DYSTROPHY

29

Linkage of the ARC8 probe to the DMD gene was demonstrated by RFLP analysis, in this case involving a polymorphic site for the restriction enzyme TaqI.A Southern blot of a TaqI digest of human DNA was probed with ARC8 and a TqI fragment of either 3.2 kb (allele 1) or 5.3 kb (allele 2) or both was detected (Murray et a l . , 1982). Women who were heterozygotes with allele 1 on one X and allele 2 on the other X had both TuqI fragments. In families where D M D carrier mothers were heterozygous for the TaqI polymorphism, 80-90 % of the time the defective gene was found to be passed to their children with the polymorphic marker detected by XRC8. This level of cosegregation of the marker with the gene established that the two were linked. The lack of cosegregation in 10-20 % of children suggested a probability of recombination of 10-2076 in the chromosomal interval between the ARC8 polymorphism and the D M D gene. This provided the first linkage data assigning the D M D gene to the X chromosome short arm. In genetic terms 1 centiMorgan (1cM) is defined as a distance over which the recombination probability is 1%, and it corresponds very approximately to a physical distance of 1 million base pairs of DNA. Thus, the genetic distance between ARC8 and DMD is 10-20 cM and the estimated physical distance is 10-20 million base pairs, or 10,000-20,000 kb of DNA. The second probe for the D M D gene was the clonal isolate called L1.28 obtained from a plasmid library of total genomic DNA (Wieacker et al., 1984). The L1.28 marker was mapped by both somatic cell hybrid panels (Wieacker et al., 1984) and by direct in situ hybridization of the radioactive probe to human metaphase chromosomes (Hartley et al., 1984) and was found to localize over the proximal third of the X chromosome short arm. When used as a probe for Southern blot analysis, L1.28 detected a TqI polymorphism that also segregated with the DMD gene in 80-90% of children (Davies et a l . , 1983). This mapped L1.28 about 10-20 cM on the other side of the D M D gene from RC8, and the two results together indicated that the D M D gene must be located midway between the two probes, or at approximately the middle of the short arm at band Xp21.

D. THEGENETIC MAPOF THE X CHROMOSOME SHORTARM A number of additional probes and the O T C gene probe have now been mapped to the short arm of the X chromosome and have been found to be linked to the DMD and/or BMD locus. These are listed in Table IV and diagrammed in Fig. 4. Detailed linkage data is summarized by Goodfellow et al. (1985) in the report of the 8th International Conference on the Human Gene Map (published as a special issue of Cytogenetics and Cell Genetics 40,

30

RONALD G. WORTON AND ARTHUR H . M. BURGHES TABLE IV DNA MARKERS FLANKING THE DMD

AND

BMD GENES

Genetic distance’ DNA probe __

Locus nameb

Chromosomal location

DXS9 DXS43 DXS41 DXS28 DMD/BMD DXS84 OTC DXS7

xp22 xp22 xp22 Xp21.3 xp21.1-21.2 xp21.1 xp21.1 Xpl 1.3

DMD

BMD

References’

~~~~

RC8 D2 99-6 C7 754 OTC L1.28

>15 cM 15 cM 15 cM 10 cM

10 cM 15 cM 20 cM

>15 CM 1, 3-6, 10-12, 14, 16, 20, 23, 26 15 cM 2, 7, 11, 14, 16, 20, 21, 23, 26 15 CM 2, 7, 11, 14, 16, 21-23, 26 10 cM 10-13, 19-21, 26

-

10 CM 9, 10, 13-16, 18, 19, 21-23, 26 15 CM 4, 8, 11, 16-18, 21, 23, 24, 26 20 CM 4-6, 10-12, 14-16, 21, 23, 26

“Genetic distance is estimated to the nearest 5 cM and is based on the tables provided by Goodfellow ef al. (1985) in the report of the eighth Human Gene Mapping Conference, plus more recent references. *he locus name is assigned by the Human Gene Mapping Conferences. The DXS designation means DNA segment, X chromosome, Sngle copy, and the number signifies the order of isolation of the segment. ‘References: 1, Murray d al. (1982); 2, Kunkel ct al. (1982); 3, Davies ef al. (1983); 4, Kingston et a / (1984); 5, Wieacker cf al. (1984); 6, Hartley et al. (1984); 7, Aldridge ef al. (1984); 8, Horwich el al. (1984); 9, Hofker ef al. (1985); 10, Bakker et al. (1985); 11, deMartinville ef al. (1985); 12, Ingle cf al. (1985); 13, Francke cf al. (1985); 14, Wilcox ef al. (1985); 15, Brown et al. (1985a); 16, Brown el al. (1985b); 17, Davies cf 01. (1985a); 19, Dorkins ef ai. (1985); 20, Fadda ef af. (1985); 21, Bertelsonef al. (1986); 22, Walkercf al. (1986); 23, Williamsetal. (1986); 24, Foxetal. (1986); 25, Lindgren ct al. (1984); 26, Bakker ef al. (1986).

1985). Probes D2 and 99-6 are cloned DNA fragments derived from a FACSsorted X chromosome library (Kunkel et al., 1982, 1985a) and map to the region of Xp22 (Aldridge et al., 1984). Several groups have shown linkage to the DMD gene by RFLP analysis (Table IV). Probe C7 was derived from a genomic library of J.-L. Mandel (unpublished) and also found to be linked to DMD (Dorkins et al., 1985) by RFLP analysis. Probe 754 was isolated from the library of Kunkel et al. (1982) and was found to map to Xp21 (Francke et al., 1985) and reveal RFLPs linked to the DMD locus (Hofker et al., 1985). Ornithine transcarbamylase is a urea cycle enzyme deficient in patients with an X-linked hyperammonemia. The OTC probe is a cDNA clone derived from the locus of this gene (Horwich et al., 1984; Davies et al., 1985a) and found to be localized at band Xp21 (deMartinville et al., 1985) and to reveal a set of RFLPs linked to DMD (Davies et al., 1985a,b). The closest probes to the DMD locus, both obtained by the random isolation approach, are probe 754 on the centromeric side (Hofker et al., 1985) and

DUCHENNE AND BECKER MUSCULAR DYSTROPHY

/ ]

RC8

] DMD ]

P21

21.3 21.2 21.1

L1.28

XP

\

8

31

RC8 D2 99-6 c7

DMD 754 OTC L1.28

FIG. 4. The physical map and linkage map of the short arm of the X chromosome. Schematic of the X chromosome short arm at metaphase (left) and at high resolution (center). The genetic map on the right shows the approximate distance of the short arm probes from the DMD gene in centimorgans (cM). One centimorgan is defined as the genetic distance over which the recombination frequency is 0.01. The correspondence between physical distance and genetic distance is approximately 1000 kb of DNA per centimorgan when averaged over the whole genome.

C7 on the telomeric side (Dorkins et al., 1985). The DXS84 locus (probe 754) was originally reported to show only 3-4% recombination with the DMD gene (Hofker et al., 1985; Wilcox et al. , 1985), but later studies gave recombination frequencies of 15-20% (Brown et al., 1985a). In three recent studies, values of 6 % (Bertelson et al., 1986), 10% (Walker et al., 1986) and 14% (M. W. Thompson et al., 1986) have been reported and the average recombination frequency for 754 has been estimated to be 10-1576 (Walker et a l . , 1986). This extensive variation is a simple reflection of the fact that, despite the large number of families analyzed, the number of recombination events is small and, therefore, the statistical error is great. A similar variation and uncertainty exists for the other closely linked probes. The closest probes have the fewest recombination events and therefore have the highest error. In the tabulation at the eighth gene mapping conference (Goodfellow et al., 1985), the linkage distances are presented as a probability table with the Lad score (log of the odds for linkage versus nonlinkage) representing the relative probability of linkage at genetic distances of 0, 5, 10, . . . 40 cM. The probability curves are not sharp peaks but are broad distributions that reflect the degree of uncertainty in the data. The linkage distances provided in Table IV are rounded to the nearest 5 cM but are still a fairly accurate representation of the best estimated values. Also provided in Table IV is linkage data for Becker muscular dystrophy. As previously mentioned in Section 11, Becker and Duchenne dystrophies were thought at one time to be the result of mutations in separate genes, with the

32

RONALD G . WORTON AND ARTHUR H. M. BURGHES

BMD gene located on the long arm and the DMD gene on the short arm of the X chromosome. The BMD mapping was based on investigations that suggested linkage of the BMD gene to the color blindness gene on the long arm (Skinner et al. , 1974; Zatz et al., 1974). Once it was established that the DMD gene was in the middle of the X chromosome short arm, Kingston et al. (1983, 1984) examined several BMD families for linkage to RC8 and L1.28 and found both markers segregating with the BMD gene with linkage at about 20 cM. As indicated in the table, subsequent analysis using newer probes confirmed linkage of the BMD and DMD genes to the same set of probes and, therefore, to each other (Dorkins et a l . , 1985; Wilcox et al., 1985; Roncuzzi et al. , 1985; Walker et al., 1986; Bertelson et a l . , 1986). Thus, linkage analysis has indicated that the two genes are very close together on the chromosome, or dternatively the two diseases are caused by mutations in the same gene. In summary, translocations in females, deletions in males, and linkage analysis in families all combined to map the DMD gene and the BMD gene to the band Xp21 on the X chromosome. While the existence of the newer probes is tremendously helpful in defining the gene location and in detecting RFLPs that are close enough to the gene to have some predictive value for carrier identification and prenatal diagnosis, the closest probes in Table IV are still 10 cM or more from the DMD/BMD gene. Since a genetic distance of 10 cM corresponds approximately to a physical distance of 10 million base pairs, the probes are still a long distance from the gene in molecular terms. The next section describes strategies that resulted in the isolation of closer probes from within the DMD/BMD locus itself.

IV. Approaches to Cloning the Responsible Gene

A large number of genes responsible for genetic diseases have now been cloned. The cloning strategy in most cases started from knowledge of the defective protein and involved one of three approaches. In the case of genes such as a- or P-globin, the messenger RNA (mRNA) is very abundant in specific cell types, thereby making it possible to purify the mRNA. A complementary DNA (cDNA) copy, prepared with the mRNA as template, was then prepared and inserted into a bacterial plasmid for replication in bacteria. In order to clone a gene whose message is not highly represented in the total mRNA of the cell, the total population of mRNA molecules is usually extracted from a tissue expressing the gene of interest, cDNA is prepared to the whole spectrum of mRNA, and the resulting cDNA molecules are inserted into bacterial plasmids or bacteriophage DNA for replication in bacteria. The

DUCHENNE AND BECKER MUSCULAR DYSTROPHY

33

collection of bacterial cells or phage particles each carrying a different cDNA derived from a different expressed gene, is a cDNA library. The bacterial cells are grown on agar plates where each cell grows into a colony, or the phage particles are spread on a confluent lawn of bacterial cells where each phage forms a zone of lysis, or a “plaque.” Each colony or plaque contains more than a million replicates of the gene sequence contained in the progenitor bacteria or phage. A cDNA library is different from a genomic library in that it contains only sequences from the expressed portion of the genome and no DNA fragments from the introns or from the regions between the genes. In a library of large enough size (about 100,000 independent colonies or plaques), the probability is high that sequences from the gene of interest will be represented in at least one colony or plaque in the library. In order to determine which one contains the gene of interest, the library is screened with radiolabeled probe, or antibody. In the former case, the probe is usually a short, synthetic nucleotide whose sequence is determined from the amino acid sequence of the protein. In the latter case, the probe is labeled antibody directed against the protein and, in that case, the library is prepared in a plasmid or phage system that allows the proper transcription and translation of the cloned sequence. In the case of the muscular dystrophies, the product of the defective gene is unknown. Any cloning approach based on knowledge of the gene product is therefore excluded, and new strategies based on the known location of the gene on the X chromosome had to be devised. What was clear was that cloning pieces of the X chromosome at random was unlikely to hit the gene simply because the human genome is so large. The genome (the total DNA in the human chromosome set) is about 3 x lo9 base pairs, or 3 million kb. The X chromosome contains about 5% of the genome, or about 150,000 kb. At the time the cloning work began in 1983 a large gene was considered to be about 50 kb in size, only 1 part in 3000 of the human X chromosome. It is therefore not surprising that of six random probes isolated from the X chromosome short arm, itself about 50,000 1:b in size, the closest were about 10,000 kb from the DMD/BMD locus. The new strategies discussed below were designed to improve the chances of finding a DNA clone from within the DMD/BMD gene locus. The approach of Davies and her colleagues was to search for musclespecific cDNA clones derived from the Xp21 region. Briefly the strategy was to test a set of DNA clones from the X chromosome library discussed above for their ability to hybridize with mRNA from muscle cells. One clone which was localized to the Xp21 region was found to share sequence homology with an mRNA expressed not only in muscle but in several cell types. The clone was found to have sequence homology to the gene encoding glyceraldehyde-3phosphate dehydrogenase (GAPDH) which is carried on another chromosome.

34

RONALD G . WORTON AND ARTHUR H . M. BURGHES

The cloned sequence from Xp21 was a nonfunctional copy of the GAPDH gene (a pseudogene) that is located near the DMD/BMD locus on the X chromosome (Benham et al., 1984). No other promising candidate for the DMD gene has been identified by this approach. Two strategies have led to the DMD/BMD locus. Kunkel and his colleagues at Boston Children’s Hospital took advantage of the BB deletion patient (Francke et al., 1985; Table 111) and devised a strategy to enrich for DNA fragments with no matching sequences in the patient. One of the clones obtained is within the DMD/BMD locus. Our own group focused on the patient with an X:21 translocation (Verellen et al., 1984; Table 11), utilizing sequences from chromosome 21 as a molecular probe to detect and clone a fragment spanning the X:21 junction. The X chromosomal portion of the junction fragment is within the DMD/BMD locus. Both approaches are described in detail below.

A. CLONING SEQUENCES FROM THE BB DELETION-THE PERT 87 (DXS164) REGION The strategy of Kunkel and his colleagues (Kunkel et al., 1985b) depended upon the availability of the deletion patient, BB (Francke et al., 1985, Table 111), and the use of the phenol-enhanced DNA reassociation technique (PERT) of Kohne et al. (1977). The strategy is described in Fig. 5. The source of the DNA to be cloned was a male with karyotype 49,XXXXY. This X chromosome-enriched DNA was digested to completion with the restriction enzyme MboI and was mixed with a 200-fold excess of DNA from the BB deletion patient that had been sheared to small size (about 1 kb) by sonication. The mixed DNA was denatured to single strands and allowed to reassociate for 5 days in the presence of 7 % phenol. The reassociated DNA was expected to be of three types (Fig. 5). Most molecules should involve association between two sheared pieces from BB, and the ends of these molecules are not easily ligated to plasmids for cloning. A much smaller fraction are hybrid molecules between a sheared fragment from BB and an M6oI cut fragment from the XXXXY male, and these also are not easily cloned. The rarest type is a perfect match between two MboI cut pieces from the XXXXY male, and the presence of an M6oI overhanging sequence at each end allowed preferential cloning of these molecules into BamHI cleaved (BamHI and MboI yield the same 4-bp overhang) plasmid DNA molecules. The latter type of molecule was expected to be greatly enriched for sequences from the region of the BB deletion since there were no sheared molecules from this region in the PERT reassociation mixture (Kunkel et a l . , 1985b).

DUCHENNE AND BECKER MUSCULAR DYSTROPHY

BBdeleted DNA

XXXXY DNA

1

1

Mbol

- - n r

Shear

rn

- - n r

35

s=s

rn

s=s

1 :200

1 8-8 -S

Denature & Re-anneal

s

S

S

rn

m-

m

-rn clone

FIG.5. The phenol-enhanced reassociation technique (PERT). DNA molecules are depicted with ends generated either by digestion with the restriction enzyme MboI (m) or by shearing (s) After mixing the DNA samples, with sheared molecules in ZOO-fold excess, the DNA is denatured and then reassociated in phenol (Kunkel et al., 1985b). The majority of reassociated molecules contain two sheared strands, others contain a sheared with an Mbd-digested strand, and the rarest contain two MboI-digested strands as shown. Only the latter have ends that are compatible with cloning and, as outlined in the text, these are enriched for sequences from within the BB deletion segment.

Of 125 initial clones from the PERT library, 18 were found to be derived from the X chromosome, and 4 of these failed to hybridize with DNA from the BB patient, indicating that their site of origin was from within the deleted region of the X chromosome (Kunkel et al., 1985b). Using hybrid cell lines carrying translocation-derived chromosomes from two female translocation patients (the X:21 patient of Verellen et al., 1984, and the X: 11 patient of Greenstein et al., 1980), three clones, PERT 55, 145, and 84, were found to hybridize to DNA sequences on the centromeric side of the translocation exchange points; one clone, PERT 87, matched sequences distal to the translocation exchange points (Kunkel et al., 1985b). These four cloned fragments were estimated to reside within a DNA segment of 1000-5000 kb in Xp21, a segment that must contain the DMDlBMD locus. Further studies of the PERT clones by Monaco et al. (1985) revealed rather spectacular results with PERT 87. Using a set of eight PERT clones, four described above plus four others, they labeled each clone and tested for

36

RONALD G. WORTON AND ARTHUR H . M. BURGHES

hybridization to Southern blots of DNA from a set of 57 male DMD patients. The PERT 87 clone was found not to hybridize with DNA from 5 of the 57 patients, suggesting that the PERT 87 sequence was deleted from their DNA. This suggested that deletions of the DMD gene caused the disease in these five boys and that PERT 87 DNA is within or near the DMD locus. The remaining seven PERT clones hybridized to all patient DNA samples. In order to isolate additional DNA from the PERT 87 region, the PERT 87 clone was used as a probe to screen genomic libraries constructed in X phage. Three phage clones identified by hybridization to PERT 87 were isolated and small subclones from the ends were used to identify clones containing fragments from further along the X chromosome. Chromosome walking along the X chromosome by the isolation of additional overlapping fragments has continued until a total of approximately 220 kb of genomic sequences have been isolated in 17 phage clones (for review see Monaco and Kunkel, 1987). After the first few walk steps, covering about 40 kb, the 57 male patients were again tested for hybridization with three subclones, PERT 87-1, 87-8, and 87-18, derived from the two ends and the middle of the 40-kb region. All three probes failed to hybridize with DNA from the same five patients, indicating that in all five the deletion is larger than 40 kb. An upper estimate to the size of the deletions was calculated as 250-600 kb based on the fact that seven of the eight original PERT clones did hybridize with the five deletion cases (Monaco ef a l . , 1985). A further indication that PERT 87 is close to the DMD gene came from linkage studies using RFLPs revealed by the subclone PERT 87-8. This probe revealed two RFLPs, one a variable BstXI site and one a variable TaqI site, and in a study of 34 offspring in 10 families no recombination was detected between the PERT 87 probe site and the DMD gene. The data demonstrated tight linkage between PERT 87 and the DMD locus. The Lod score was 7.6, meaning odds in favor of linkage of greater than 10’ to 1, but the confidence interval on the recombination fraction was 0-7%. In a subsequent study with additional PERT 87 probes, recombinants were found between PERT 87 and DMD at a frequency of about 5% (Kunkel et a l . , 1986; Bertelson d al., 1986), suggesting that some mutations that cause DMD are located a considerable distance from PERT 87 on the X chromosome. Subclones from the PERT 87 region have been sent to a large number of laboratories around the world for diagnostic and research purposes. The clones have been used to identify a large number of deletion patients (Kunkel ~t al., 1986) and these are discussed in more detail in Section V,C. We return to a more detailed discussion of the PERT 87 region and the expressed sequences that derive from the region after our description of the translocation cloning approach.

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37

B. CLONING THE t(X;21) TRANSLOCATION JUNCTION-THE XJ (DXS206) REGION The key to this approach (Worton et al., 1984; Ray et al., 1985) was the availability of the X:21 translocation patient (Verellen-Dumoulin et al., 1984; Table 11). The translocation in this patient is described as t(X;21) (p21;p12). The exchange point in the X chromosome is in band p21, as it is for all translocation cases. The exchange point in chromosome 21 is in band p12, a region of chromosome 2 1that stains poorly by conventionalstain and sometimes appears as a constriction separating the more distal “satellite” region from the short arm of the chromosome. Similar regions exist on all five pairs of acrocentric chromosomes (pair 13,14, 15,21and 22), and by in situ hybridization the region is known to carry multiple copies of the genes that encode 18 and 28 S ribosomal RNA (rRNA) (Henderson et al., 1973).Molecular studies of rRNA genes (rDNA) have shown the structure to be a tandem repeat of 44 kb in length. In each repeat unit the 18 and 28 S genes are part of a 14-kb transcribed region separated by a 30-kb nontranscribed “spacer” region (Wellauer and Dawid, 1979). The unique feature of this translocationpatient was the fact that the autosomal exchange point appeared to have occurred within one of the rDNA repeat units, and since the human rDNA complex had previously been isolated by molecular cloning it became feasible to use rDNA cloned fragments as probes to isolate a larger fragment spanning the X:21 junction. Assuming the translocation had disrupted the DMD/BMD gene, then one end of thejunction clone should contain sequences from the DMD/BMD locus. A schematic of the translocation chromosomes is shown in Fig. 6 . Our initial plan, therefore, was to prepare a genomic DNA library from the patient’s cells and identify a junction fragment from either translocation chromosome, the der(X) or the der(21) (Fig. 6).

RECIPROCAL

X

21

-

der (X)

der (21)

FIG.6. Schematic ofthe t(X;21) translocation. For simplicity, only the short arms are shown. The junction in the X is at the site of the DMD gene in Xp21. The junction in chromosome 21 is in a large block of tandemly repeated genes encoding 18 and 28 S ribosomal RNA. The two translocationderived chromosomes, der(X) and der(21), have ribosomal genes next to the DMD locus in Xp21.

38

RONALD G . WORTON AND ARTHUR H . M. BURGHES

One of the problems in this approach was the fact that the rDNA repeat units exist in 300-440 copies (Schmickel, 1973)distributed on the short arms of the 10 (5 pair) acrocentric chromosomes, and only one repeat unit should be disrupted by the translocation. In order to reduce the background of nonjunctional rDNA repeat units, it was essential, therefore, to separate the translocation-derived chromosomes from the acrocentric chromosomes by segregation in somatic cell hybrids, T o this end, patient fibroblasts were fused with a mouse cell line, and the hybrid cells were grown in a culture medium that selects for cells that carry an active human X-linked gene (Worton et ul., 1984). After several months, during which time most other human chromosomes were lost from the hybrid cells, one hybrid cell line was generated carrying the der(X) chromosome as the only human chromosome; another carried the der(2 1) chromosome. Southern blotting of DNA from these hybrids with a human rDNA probe provided by R . Schmickel and colleagues at the University of Pennsylvania revealed a hybridization signal equal to 3-5 copies of the rDNA repeat unit on the der(X) chromosome and 40-60 copies on the der(21) chromosome (Worton et ul., 1984). A more detailed Southern blot analysis with several rDNA probes revealed a set of restriction fragments in the der(X) chromosome that were different in size from the normal rDNA fragments and were thought to be segments spanning the translocation junction (Worton ef a l . , 1986). In particular, a 12-kb BarnHI fragment was visualized on a Southern blot of der(X) hybrid cell DNA probed with a human specific segment of the 28 S gene, and this was presumed to contain part of the 28 S gene at one end and part of the X chromosome at the other (Ray et a l . , 1985). To clone this fragment, DNA from the der(X) hybrid cell line was cleaved with BamHI, separated by electrophoresis on an agarose gel, and a region containing 11- to 13-kb fragments was isolated. These fragments (among them the putative X:21 junction) were ligated to BumHI-digested DNA from the X phage vector Charon 35. The recombinant phage was grown up as plaques on a lawn of E. coli and the plaques containing 28 S rDNA sequences were identified by their ability to hybridize the labeled 28 S gene probe. Several plaques were identified and in each one the human DNA insert was found to consist of a 12-kb BumHI fragment. One clone, XJ 1, was examined in detail and was found to contain 620 bp of rDNA attached to about 11 kb of X chromosomal DNA (Ray et a l . , 1985). The junction fragment, XJ 1, was subcloned in smaller pieces into a plasmid vector, and one of the subclones, XJ 1.1, was found to contain a 1-kb Nszl fragment of X chromosome that serves as a marker for the DMD/BMD locus. Evidence that the XJ region is within the DMD/BMD locus is similar to that for PERT 87. First, the XJ 1.1 probe detects no hybridization signal from DNA of a subset of D M D patients. With some exceptions, this subset includes the same patients who are deleted for the PERT 87 region (Monaco

DUCHENNE AND BECKER MUSCULAR DYSTROPHY

39

et al., 1985), indicating that many of the deletions that span PERT 87 also span the XJ region (Ray et al., 1985). More recently the XJ probe has been

found to detect deletions in boys who have their PERT 87 region intact (Hart et al., 1986; Thomas et al., 1986b), confirming that the XJ region contains

distinct sequences that appear to be part of the DMD/BMD locus. These deletions are discussed in more detail in Section V,C. The second type of evidence comes from linkage studies with RFLPs detected by the XJ probes. The subclone XJ 1.1 detects a TaqI variable site, and this polymorphism was found to segregate with the DMD gene in families. Initially, no recombinants were detected in 16 offspring of heterozygous carriers (Ray et at., 1985). O n subsequent testing with new probes XJ 1.2 and XJ 2.3, recombinants were found at a frequency of about 5% (M. W. Thompson et al., 1986). Thus, XJ behaves very much like PERT 87 in that it appears from deletion analysis to be part of the DMD/BMD locus, but some mutations still occur at a position some distance along the chromosome. The only human translocations that have been analyzed in molecular detail are tumor cell translocations that occur in the vicinity of oncogenes and are responsible for oncogene activation. In some cases, these translocations are found to have significant deletion of sequences at the translocation site (Varmus, 1984). It therefore seemed possible that the X:21 translocation might have an associated deletion and, if such a deletion was very large, then the myopathy in the patient might be the result of deleting a gene at some distance from the XJ site. In order to test this possibility it was necessary to examine the reciprocal junction on the der(21) chromosome to determine if all X chromosome sequences were present and accounted for between the two translocation chromosomes. The cloning of the junction in the der(21) chromosome by the use of rDNA probes was impractical because of the 40-60 copies of rDNA on this chromosome. Instead, our approach was to use the XJ 1.1 clone as a probe to screen a X library prepared from XXXXY male DNA and to isolate a clone, XJ 2, that extended toward the telomere of the X chromosome, crossing the site of the X:21 junction. A subclone from the telomeric side of the junction site was then used as a probe to identify and isolate a fragment from the der(21) chromosome that crossed back over the junction into the rDNA of the der(21). Sequencing through both junctions and comparison with the normal human X chromosome revealed a small deletion of 70 or 71 bp from the X chromosome and of 19 or 20 bp from chromosome 21 (Bodrug et al., 1987). Thus, although there is a deletion of a part of the X chromosome it is very small and the model of a large deletion removing a gene many kilobases from the XJ region is not tenable. O n this basis, it appeared that the translocation had occurred close enough to the DM D gene to have divided it into two parts, or to have separated it from a

40

RONALD G. WORTON AND ARTHUR H . M . BURGHES

nearby regulatory sequence. In either case the XJ region appeared to be part of the DMD/BMD locus. Chromosome walking from the original XJ clone has now been extended to cover 140 kb of the X chromosome contained in 10 phage clones derived from the XXXXY library. Subclones from the region have been sent to over 80 laboratories for diagnostic testing. Coupled with the P E R T 87 subclones they constitute a powerful set of probes to detect deletions within the DMD/BMD locus and to follow RFLPs for carrier identification and prenatal diagnosis.

C. THEPHYSICAL AND MOLECULAR MAP OF THE PERT 87-XJ REGION The physical and genetic map of the PERT 87-XJregion is shown in Fig. 7. From all the evidence discussed above, one would expect the XJ and PERT 87 sequences to be close together on the X chromosome and to lie within a region that is frequently deleted in affected males and disrupted by translocation in affected females. To determine the position of PERT 87 relative to XJ, the PERT 87 probes were tested for hybridization against DNA from the cell -300

C7

L1

J-66

-200

PERT

J-Bir

0

-100

87

230 kb

30 kb

10.1 3end

310

XJ

J-MD

5-47

140kb

5okb

50 kb

PERT

84

SOkb

#

send

II

\

I / 1 *

550

600

--

exons 8-20 s

I I

I t

105 kb

I

I

7 exons

intron

P .

I

350

754 cx5.4

Hip25

y / 11, / I 1, I A0 exons

680

,.

100

860

770

.

~

1500

FIG 7. The physical and genetic map of the DMDIBMD locus. The scale at the top is in kilobases. The blocks represent regions of cloned DNA and include PERT 87, XJ, and several others described in the text. The XJ block overlaps PERT 87 at one end and J-MD at the other. The various PERT 87 and XJ subclones are indicated by numbers below the blocks. Below the physical map is the genetic map indicating the position ofthe eight exons mapped in PERT 87 by Monaco ct al. (1986) and the two exons mapped in XJ by Burghes el al. (1987). The approximate position of seven other exom (Burghes cf af , 1987) is also indicated. The bottom line is the map of sf;I fragments as determined by pulsed field gel electrophoresis. The double-headed arrows indicate different sf;I fragments with unknown distances between (van Ommen ct a l . , 1986). The site (s) is an Sfl site in PERT 87 that separates a 350-kb and an 860-kb fragment. The site (p) is an Sfir site that fails to cut in some DNA molecules, perhaps because of methylation at the site.

.

DUCHENNE AND BECKER MUSCULAR DYSTROPHY

41

hybrids carrying the der(21) and the der(X) chromosome. The entire PERT 87 region was found to hybridize with DNA from the der(21) and not the der(X), demonstrating that PERT 87 lies distal to XJ on the X chromosome (Kunkel et al., 1985b; P. N. Ray and R. G. Worton, unpublished observations). The orientation of the XJ region of the chromosome was provided from the X:21 translocation since the XJ 1 clone contained sequences from the centromeric side of the translocation junction. The orientation of the PERT 87 region relative to the centromere and telomere was determined by deletion studies with PERT 87-15, 87-8, and 87-1 in which patients were identified who had deleted either 87-15 at one end or 87-1 at the other end, and only the latter had also deleted the XJ region (Kunkel et al., 1986). This provided orientation of PERT 87 with 87-1 on the XJ side toward the centromere as shown in Fig. 7. The distance between the PERT 87 and XJ regions was determined initially by pulsed field gel (PFG) electrophoresis. The pulsed field technique, when applied to DNA digested with an enzyme that has infrequent recognition sites, allows separation of DNA fragments in the size range from 20 to a few thousand kb (Schwartz and Cantor, 1984; van Ommen and Verkerk, 1986). Figure 7 shows the SjiI fragment sizes determined by van Ommen et al. (1986) with a series of probes from the DMD/BMD locus. Since one sf;I site was mapped in the PERT 87 region, this served to anchor the sf;I map relative to PERT 87 and XJ. One end of PERT 87 and the XJ 1.1 probe were found to be on the same Sf;I fragment of 860 kb (van Ommen et al., 1986) and on the same 400-kb SaCI fragment (Burmeister and Lehrach, 1986), demonstrating that the two regions were in close physical proximity. Definitive demonstration of the relationship between PERT 87 and XJ came through chromosome walking from XJ 1 in the telomeric direction tojoin with the PERT 87 region. The phage clone XJ 12 was found to have a restriction map that matched the centromeric side of the PERT 87 region and the sequences hybridized with the PERT 87-42 probe (P. N. Ray, C . Logan, H. Klamut, B. Belfall, C. Duff, A. H. M. Burghes, M. W. Thompson, I. Oss, andR. G . Worton, unpublished observations).The PERT 87 and XJ regions together contain over 350 kb of continuously cloned DNA sequence (Fig. 7). Four other sequence blocks are shown on the map in Fig. 7. One of these, PERT 84, came from the original PERT screen (Kunkel et al., 1985b) and mapped within the BB deletion. The three blocks of DNA 30-50 kb in length, called J-Bir, J-MD and 5-47, were isolated by Kunkel and his colleagues by deletion jumping (Monaco et al., 1987). For example, the patient M D had a deletion extending from within PERT 87 through XJ over to the J-MD region. In the patient’s DNA the PERT 87 region is connected directly to the J-MD region, so it was a simple matter to use a PERT 87 probe to identify a unique restriction fragment that spans the deletion junction and to identify a

42

RONALD G . WORTON AND ARTHUR H . M. BURGHES

clone containing this fragment from a library of patient DNA. One side of the clone was from PERT 87, the other side from the J-MD region. A subclone from the J-MD region formed the start point for a new 50-kb walk on the X chromosome (Monaco et af., 1987). The jump clones 5-47, J-Bir, and J-66 were obtained in similar manner, with 5-47 and J-Bir forming the focus for new chromosomal walks (Monaco et al., 1987). In our own walk from XJ we have crossed into the J-MD region on the centromere side as indicated in Fig. 7. The probe L1 is a random clone that came from one of the earliest X chromosome libraries (Kunkel et al., 1982). The Hip 25 clone was isolated in the laboratory of K. Davies by a reassociation technique in high inorganic phosphate (Hip) following essentially the PERT strategy (Smith et a l . , 1987). It maps within the XJ region (Kenwrick et al., 1987). The cX5.4 clone (DXS148) is from a random cosmid clone that also maps within the BB deletion (van Ommen et al., 1986). The C7 and 754 markers were described in Section II1,D of this review. The relative order of C7, L1, J-66, and J-Bir on the one side and 5-47 and PERT 84 on the other side came from deletion analysis as outlined in more detail below. The approximate distance between J-MD and 5-47 and between 5-47 and PERT 84 is known from PFG analysis of large DNA fragments (Fig. 7 ) . The C7, L1, J-66, and J-Bir probes all recognize different size fragments and the distance between them is not known. Burmeister and Lehrach (1986) also examined the region with enzymes that recognize a site containing a dinucleotide CG and found a cluster of recognition sites 200-300 kb from XJ 1.1 on the centromeric side. Since this type of CG-rich region is found commonly on the 5 ' end of several genes (Bird et al., 1985), it suggests the possibility that the 5 ' end (the front end) of the DMD/BMD gene might be in this region and, if so, the gene would be oriented in a centromere-to-telomere direction. This would agree with the expression studies described below. The pulsed field gel technology is now being applied to patient samples to map translocation exchange points and deletion end points (van Ommen et a l . , 1986; Kenwrick et al., 1987) as described below.

D .

EXPRESSED SEQUENCES-THE DMD/BMD GENEMAP

Over the past year or two the concept that has emerged is of an exceptionally large genetic locus responsible for DMD/BMD, perhaps 100 times larger than the average gene of 10-30 kb. There are three important lines of evidence leading to this picture. First, the deletions detected with the PERT 87 and XJ probes are large and varied, extending in both directions from the 350-kb PERT 87-XJ region (Kunkel et al., 1986; P. N. Ray, C . Logan,

DUCHENNE AND BECKER MUSCULAR DYSTROPHY

43

H. Klamut, B. Belfall, C. Duff, A. H. M. Burghes, M . W. Thompson, I. Oss, and R . G . Worton, unpublished observations). Second, high-resolution

chromosome banding has revealed that the translocation exchange points in female patients are heterogeneous within the band Xp21, suggesting a separation of 1000-3000 kb between the lowest exchange point in Xp21.1 and the highest in Xp21.2-21.3 (Boyd and Buckle, 1986). Third, 5% of families exhibit recombination between the site of mutation and the PERT 87-XJ region, indicating that at least some mutations occur at a considerable distance from PERT 87 and XJ (Kunkel et al., 1986; Fischbeck et al., 1986; M . W. Thompson et al., 1986). Whether this gigantic locus contained multiple genes responsible for muscular dystrophy or a single gene in the megabase size range remained an open question until the recent discovery of expressed sequences from the PERT 87-XJ region. The discovery of expressed sequences has several important implications. Not only will it allow the size and structure of the gene to be determined; it will also allow mutations of all types to be identified and characterized. Most importantly, it will ultimately allow the protein product of the gene to be identified and its function to be elucidated. As stated at the beginning of this section, most genes have been cloned from knowledge of the defective protein. In the case of DMD/BMD the process is the reverse, proceeding from knowledge of the chromosomal location to isolation of the chromosomal region and finally to identification of expressed sequences from the gene in this region. The problem of finding the expressed portions of the gene was essentially the problem of finding the exons of the gene within the PERT 87-XJ region when most of the region was expected to consist of noncoding intervening sequence (introns). The exons of the DMDIBMD gene were expected to hybridize to mRNA of muscle tissue, and some exons, at least, were expected to be conserved among mammalian species. On this basis, the approach taken to find expressed sequence was to use multiple DNA subclones from the PERT 87-XJ region, selecting those that are free of highly repeated sequence elements characteristic of noncoding DNA, and use each as a hybridization probe to determine if the sequence has homology to muscle mRNA, muscle cDNA, or the genomic DNA from other species. Using these strategies, two subclones, PERT 87-4 and PERT 87-25, were found to have strong homology to bovine, cebus, hamster, mouse, and chicken DNA (Monaco et al., 1986). The corresponding sequences from mouse were cloned by screening a mouse genomic library with PERT 87-4 and 87-25. The human and mouse clones were sequenced and compared and, in each case, a small region of high homology (putative exon) was found in a background of nonhomologous DNA (putative intron). The putative human and mouse exons contained “open reading frames” (no translational stop

44

RONALD G. WORTON AND ARTHUR H. M. BURGHES

signals) and were flanked by the expected splice junction signals that separate exons from introns. Since the splice junction signals are different on the 5 ' side and 3 side of an exon, the sequencing data allowed the direction of transcription (5 ' to 3 ') of each exon to be determined as centromere to telomere. Next, the conserved fragments were used as hybridization probes on Northern blots (RNA blots) of cytoplasmic mRNA from fetal muscle. One fragment, PERT 87-25, hybridized with a band on the Northern blot corresponding to an mRNA 16 kb in size. The P E R T 87-25 probe was then used to screen a cDNA library prepared from fetal muscle mRNA and five cDNA clones were identified. One clone, with a 1.0-kb insert, was used as a hybridization probe on a Southern blot of human DNA digested with HindIII, and eight distinct bands were visualized. The simplest interpretation is that the 1.0-kb cDNA clone contains sequences from eight different exons, and each exon in the genomic DNA is on a different sized Hind111 fragment. The cDNA clone failed to hybridize with DNA from the BB deletion patient, confirming that all eight exons mapped within the BB deletion (Monaco el al., 1986). In order to map the exons within the PERT 87 region the cDNA clone was hybridized to Southern blots of DNA from the 18 phage walk clones from the PERT 87 region. The 1.0-kb cDNA detected the original PERT 87-25 as expected, plus 6 other restriction fragments that carry putative exons. A different cDNA clone detected an additional hybridizing band in the PERT 87 region. These eight putative exons of the DMD/BMD gene were located over 130 kb of the 220-kb PERT 87 region and are shown in the exon map in Fig. 7. Finally, the 1.0-kb cDNA clone was found to hybridize with a 16-kb mRNA as did the original PERT 87-25 probe (Monaco et al., 1986). The size of the gene on the X chromosome can be estimated from these results. A 1.0-kb cDNA clone must contain only 1/16 of the total mRNA transcribed from the gene. Since this cDNA clone derives from eight exons located over a minimum of 130 kb of genomic DNA, the entire coding region might be expected to contain 8 x 16 = 128 exons distributed over 16 x 130 = 2000 kb of the X chromosome. While this estimate involves an extrapolation that may not be valid, the data are certainly consistent with a gene of these proportions. In our own laboratory, we have followed a similar path with the XJ subclones. The search for conserved sequences and for hybridization to muscle mRNA provided negative results with many subclones from the XJ region. However, our third approach, which was to screen a muscle cDNA library with multiple probes from the XJ region, yielded positive results. Two subclones, XJ 10.2 and 10.3 from phage walk clone XJ 10, detected positive signals on screening the adult muscle cDNA library of Lloyd et al. (1985) kindly provided by Dr. Y . Edwards. Two clones, one 1.4 kb and one 2.0 kb,

DUCHENNE AND BECKER MUSCULAR DYSTROPHY

45

have been examined in some detail (Burghes et al., 1987). On Northern blot analysis, these clones detect a 16-kb mRNA from fetal and adult muscle but not from skin fibroblasts. The cDNA clones also detect a series of bands on Southern blot analysis of human, mouse, and hamster DNA, demonstrating homology to multiple exons from each species. The 2.0-kb cDNA clone reveals 15 putative exons (13 bands) on a EcoRI or Hind111 digest of human DNA. Our own original hypothesis was that the (X;2 1) translocation had split the gene to cause the disease. If this is correct, then one might expect some of the exon-containing fragments to be derived from the telomeric side of the translocation exchange point and others to be derived from the centromeric side of the translocation exchange point. T o test this, the cDNA clones were used as hybridization probes on Southern blots of DNA from our hybrid cell lines carrying the der(X) and the der(21) chromosome. Eight exons (six bands) were derived from the der(21) chromosome and seven were derived from the der(X) chromosome. The simpliest interpretation is that the cDNA clone contains several exons from both sides of the junction, confirming that the translocation has indeed split the DMD/BMD gene to cause the disease. This is the first demonstration of a translocation splitting a gene to cause a genetic disease. To map exons within the XJ region, our cDNA clones have also been hybridized to Southern blots of DNA from the XJ phage walk clones. Only the phage clone XJ 10 gave a positive signal (two exons), indicating a lack of exons in the remainder of the XJ region. The XJ region, therefore, must contain an intron of greater than 100 kb upholding the picture of a megabase gene containing many small exons separated by large introns. The seven exons on the centromeric side of the junction are indicated in the exon map of Fig. 7. Six exons on the telomeric side have been localized to the PERT 87 region. This was determined by hybridization of our cDNA to Southern blots of DNA from patients with known deletions of the XJ and PERT 87 regions. Several of the hybridizing bands were absent from these DNA samples, and in some cases new bands were visualized, indicating that the cDNA detects an exon on a new “junction” fragment generated by deletion. These deletions are discussed in more detail in the next section, and some of them are shown schematically in Fig. 8. A Southern blot of patient DNA probed with a cDNA probe is shown in Fig. 9. This analysis showed that the six exons that map into the PERT 87 region overlap with the set of exons reported by Monaco et al. (1986). These exons are indicated in the exon map in Fig. 7. Overall, the evidence is now conclusive that the PERT 87 and XJ regions contain a total of 12 or more exons of a megabase gene that undoubtedly is the gene responsible for Duchenne and Becker muscular dystrophy. Studies are continuing to isolate the remainder of the gene and begin the process of determining the protein product (see Note Added in Proof).

46

RONALD C. WORTON A N D A R T H U R H. M. BURGHES

Tend I 1 1 1 I II I I I I1 / 1 1 I I I I I Send /

-

del 1 del 4 del 5 del 8

I

(

)

)

( (

del 10

( (

1

dup 2

i

del 9 del 11

-dup

dup3

k

FIG 8. T h e mutational map of the DMD/BMD locus. T h e schematic at the top is identical to that in Fig. 7. Immediately below this, arrows denote the positions of the known translocation exchange points, and below this is a schematic to show the extent of the deletions in five of the patients with multiple phenotypes. Their phenotypes are depicted in Table 111. As described in the text, the extent of the deletions determines the order of the probe sites and defines the site of the genes for G K , A H , D M D , C G D , and R P . T h e exon map in the center of the figure is the same as in Fig. 7. Below this are bars to indicate the regions duplicated in three duplication patients. Below this is indicated the extent of several deletions ascertained in our own laboratory. T h e deletion numbers correspond to the lane numbers in FiK. 9.

V. Mutation at the DMDlBMD Locus

Based on analysis of deletions, translocations, and recombination events the DMD/BMD locus seems to extend through J-Bir, PERT 87, XJ, and J-MD. This includes over 480 kb of cloned sequences with an additional unknown amount of material between J-Bir and PERT 87. Furthermore, there is a strong indication that the gene extends well beyond J-Bir in the telomeric direction. This section further documents some of the mutational events that are known to occur throughout this region.

A. TRANSLOCATIONS I N AFFECTED FEMALES As discussed earlier in this review, female patients with translocations were instrumental in defining the site of the D M D gene at band Xp2 1. While the

DUCHENNE AND BECKER MUSCULAR DYSTROPHY

47

FIG. 9. Southern blot of HindIII-digested DNA from 14 individuals probed with a 2-kb cDNA clone that indicates exons extending from the middle of PERT 87 to the centromeric side of XJ. The probes detects 13 bands corresponding to 15 exons of the DMD gene. All bands are present in patients 6 and 7, one of whom has no apparent deletion and the other of whom has a small deletion of the J-Bir region. Patient 2 has a major deletion of the entire PERT 87 and XJ region, removing all but two exons detected by this cDNA probe. Patient 1 has a deletion of only the two XJ 10 exons that lie on a single Hind111 fragment at the position of the arrow. The remaining patients have different deletions that remove one or more exons and a few of these deletions are depicted schematically in Fig. 8. Patient 5 has a new band (arrow) that represents a junction fragment at the edge of a deletion.

exchange points in the X chromosome are all in Xp21, high-resolution cytogenetic analysis of nine patients (Boyd and Buckle, 1986) provided evidence for an apparent heterogeneity in the point of exchange within band Xp21. Detailed analysis of other cases (Saito et a l . , 1985; Narazaki et al., 1985; Nevin et al., 1986) supported this heterogeneity. As indicated in Table 11, five cases have exchange points in Xp21.1, six cases in Xp21.2, and one at the upper edge of Xp21.2, possibly in Xp21.3. At the time when these

48

RONALD G . WORTON AND ARTHUR H . M. BURGHES

results were first reported their significance was striking because in molecular terms the distance between Xp2 1 . 1 and Xp21.3 is expected to be 3000-4000 kb. One had to question how translocations with exchange points spread over this distance could all cause the same disease, when the average human gene is perhaps one-hundredth that size. In order to examine this heterogeneity at the molecular level, Boyd and her colleagues have constructed a series of somatic hybrids containing either the X-derived translocation chromosome or the autosome-derived translocation chromosome from each of several translocation females. DNA from each hybrid cell line was examined by Southern blot analysis to test for hybridization with PERT 87 and XJ probes (Boyd et al., 1987). For translocations with an exchange point on the telomeric side of PERT 87 and XJ the probes were expected to bind DNA from hybrids carrying the X-derived chromosome, whereas those with an exchange point on the centromeric side of PERT 87 and XJ were expected to bind DNA from hybrids carrying the autosomal-derived chromosome. Both results were found (Boyd et al., 1987). The X:6 and the X:3 translocations (Canki et af., 1979; Zatz et al., 1981) mapped on the telomeric side of PERT 87 and XJ, whereas the X: 1 , X:5, and X: 1 1 translocations (Lindenbaum et al., 1979; Jacobs el al., 1981; Greenstein et al., 1980) mapped on the centromeric side. The correlation with cytogenetic data was excellent in that the X:3 and X:6 exchange points had been assigned to Xp21.2 or Xp21.3 and the X: 5 and X: 1 1 exchange points had been assigned to Xp2 1 . 1 . The exchange points are indicated in Fig. 8, and in fact further revision of the map has come from several sources. On the telomeric side of the t(X;3) and t(X;6) exchange points map between J-Bir and L1 (Monaco and Kunkel, 1987) and one of the t(X;9) translocations also maps distal to J-Bir (U. Francke, personal communication). O n the centromeric side the t(X;5) maps between J-MD and PERT 84 and the t(X; 1 1 ) between 5-47 and PERT 84 (Monaco and Kunkel, 1987). The t(X;l) exchange point has been examined by pulsed field gels and found to lie between Hip 25 and PERT 84, less than 220 kb from Hip 25 and less than 520 kb from PERT 84 (Kenwrick et al., 1987). The minimum distance between the most distal and the most proximal exchange points is 450 kb. The actual distance may be much greater as it includes unknown distances between J-Bir and PERT 87 on one end and between J-MD and PERT 84 on the other end. In addition, the distal exchange point may lie a considerable distance beyond the J-Bir region.

B . DELETIONS I N MALES WITH COMPLEX PHENOTYPES Deletions in boys with complex phenotypes were introduced in Section III,B, since they were instrumental in defining the location of the D M D gene.

DUCHENNE AND BECKER MUSCULAR DYSTROPHY

49

Now that there are a large number of probes from within and around the DMD locus it has been possible to reinvestigate these patients to determine the extent of the deletions. By locating the ends of the deleted regions and by comparing the extent of the deletions with the phenotype, the genes responsible for GK deficiency, adrenal hypoplasia, chronic granulomatous disease, and retinitis pigmentosa have been located relative to the available probes. The deletions in some of the boys from Table I11 are presented in Fig. 8. The Iowa families (Bartleyet al. ,1982,1986;Patil et al. ,1985) were further investigated by van Ommen d al. (1986). The deletion in Bartley’s family 1 extends from the centromeric side of 754 through XJ and PERT 87 but not through the L1 probe site. Furthermore, van Ommen indicates that in his laboratory only one of the DMD, GK, and AH patients examined was found to lack the L1 site. This places the DMD, GK, and AH genes all on the centromeric side of L l . The second Bartley family has two boys with GK deficiency and AH but not DMD. Their deletion includes C7 and L1 but not PERT 87-30 (van Ommen et al., 1986). Thus, the GK and AH genes must be between L1 and PERT 87-30 and the DMD gene cannot extend as far as L1 on telomeric side, However, the deletion in BB (Francke et al. , 1985) does not extend beyond J-Bir (Monaco and Kunkel, 1987) and since this patient does not have GK or AH these genes must lie between L1 and the BB deletion edge. Finally, the patient described by Kenwrick et al. (1987) is important because he has GK deficiency, AH, and DMD, yet his deletion does not extend to PERT 87-30 on the centromeric side. This demonstrates that at least a part of the DMD gene lies on the distal side of PERT 87-30. Together these data suggest the order C7-Ll-GK,AH-3 ’ end of DMD-PERT 87-30. At the other end of the complex, the deletion of Renier et al. (1983) (Wieringa 2 in Fig. 8) extends to the vicinity of cX5.4, as determined by the fact that the 1500-kb SfiI fragment normally detected by cX5.4 is reduced to 550 kb in this patient. Further studies mapped the deletion junction 120-190 kb distal of cX5.4 (van Ommen et al., 1986). Since the boys in this family do not have CGD or R P these genes must lie closer to the centromere than this deletion junction. On the other hand, the patient BB does have CGD and RP, and since his deletion includes 754 but not OTC, the genes must lie between 754 and OTC. Recently Orkin and his coleagues have determined that PERT 84 identifies an RFLP that segregates with the CGD gene in families with this X-linked disorder of phagocytic cells (Baehner et al., 1986). Sequences from phage clones related to several of Kunkel’s original PERT clones were tested for hybridization with mRNA enriched for sequences expressed in phagocytic cells. Phage clones derived from PERT 379 were found to hybridize the message and this clone was then used as a probe to isolate the CGD gene (Royer-Pokora et al. , 1986).

50

RONALD C . WORTON AND ARTHUR H. M. BURGHES

In summary, the deletion patients have allowed the order of the genetic markers and genes to be determnined as follows: telomere-C7-L1 -GK,AH-3 ’ end of DMD-PERT 87-XJ-5 ‘end of DMD-754-CGD, RP-OTC-centromere.

C. DELETIONS AND DUPLICATIONS I N MALES WITH DMD/BMD In the original studies with PERT 87, 5 of 57 boys with DMD were found to have a deletion of the PERT 87 probe. In order to obtain a better estimate of the deletion frequency, three PERT 87 probes (87-1, 87-8, and 87-15) were sent to laboratories around the world. Twenty-five laboratories contributed to the data on deletions and in a multiauthored paper (Kunkel et al., 1986) a total of 88 deletions were recorded in 1346 patients examined. The DNA from each of these 88 patients failed to hybridize with one or more of the PERT 87 probes. In over half of these, the deletion extended through the entire PERT 87 region, at that time 137 kb in size. In some patients, however, the deletion extended from within PERT 87 toward either the centromere or the telomere. Since these deletions did not define a minimum region of overlap, it suggested that there must be elements of the DMD gene on both sides of the PERT 87 region. Furthermore, one patient had a 45-kb deletion from entirely within the PERT 87 region, indicating that an important part of the gene must reside in this region, and the subsequent cDNA studies have confirmed that this region contains an exon of the DMD gene. A detailed map of the end points of many of these deletions is provided by Monaco el al. (1987). Of considerable interest was the fact that two of the deletion cases were classified as BMD, confirming that the two diseases are caused by mutations in the same gene (Kunkel et al. 1986; Hodgson et a [ . , 1986). What is now clear is that the three PERT 87 probes utilized in the collaborative study covered only a small portion of the gene (about 60 kb), suggesting that many deletions might have been missed. This became clear as we and others began to use the XJ probes for primary screening of male patients. Table V summarizes the deletions detected by three laboratories using a combination of PERT 87, XJ, and other probes. In Cardiff, a study of 165 unrelated patients (1 17 DMD, 48 BMD) revealed 9 deletion cases, all among the DMD patients (7.7 %) (Thomas d al., 1986). In London, a study of 140 independent DMD patients revealed 15 independent deletions (10.7 %) (Hart et a/. , 1986). Our own series of 120 independent DMD patients has revealed 9 deletions (7.5%) (Ray et al., 1987). In total, 32 deletions were found among 377 patients examined (8.5%), 7 of which (1.9%) were detected by XJ but not PERT 87 and 12 of which (3.2%) were detected by PERT but not XJ. Among our 120 patients, 1 was found to have a deletion of J-Bir but not PERT 87 or XJ, emphasizing the point that, as the number of probes grows, so does the

TABLE V DELETIONS ASCERTAINED WITH PERT 87

AND

XJ PROBES ~~

No. DMD Study

patients examined

Deletion of PERT 87 1, 8, 15

Deletion of XJ 1 . 1

Deletion of PERT 87 and XJ

Deletion total

Deletion frequency (76)

~~

Cardiff (Thomas et al., 1986a) London (Hart d a l . , 1986) Toronto (unpublished observations)b TOTAL % of TOTAL

117

4

4

1

9

7.7

140

6

2"

7

15

10.7

5 -

8 -

7.5 8.5

1 -

120 -

2 -

377

12

7

13

32

38

22

40

100

"One of these patients was ascertained with XJ 1 . 1 and later found to have a deletion extending into PERT 87-1 but not 87-8 or 87-15. 'A schematic showing the extent of some of these deletions is provided in Fig. 8.

52

RONALD G . WORTON AND ARTHUR H. M. BURGHES

number of detectable deletions. It would not be surprising if the frequency of cases with deletions exceeds 50% by the time the whole gene is cloned and characterized (see Note Added in Proof). While most of the deletions reported have been ascertained with genomic probes from the PERT 87 and XJ regions, the availability of cDNA clones dramatically increases the ability to detect and map the extent of deletions. In our own laboratory we have studied all patients with our 2.0-kb cDNA probe. This probe detects 13 bands on a Southern blot of EcoRI- or HindIII-digested genomic DNA, and these correspond to restriction fragments containing 15 exons of the gene. Figure 9 shows these bands from several deletion patients. The patient in lane 2 has a deletion that removes all fragments except for two weakly hybridizing bands that correspond to the first two exons of the gene. Another patient, lane 1 , has a deletion that removes only one hybridizing fragment; that fragment derives from XJ 10 and carries two exons. The patients in all the remaining lanes have deletions that remove a subset of the exons, leaving others intact. From knowledge of these deletions based on hybridization results with PERT 87 and XJ subclones it was possible to determine the relative map location of the exon containing fragment. The extent of some of these deletions is shown schematically in Fig. 8 and the numbers of the deletions correspond to the lane numbers in Fig. 9. Another type of mutation is duplication of part of the XJ and PERT 87 region. By carefully controlled hybridization reactions on Southern blots of patient DNA it has been possible to detect specific regions of XJ and/or PERT 87 that show an intensity of hybridization characteristic of female DNA, implying two copies of the region. By this means 4 of our 120 patients have been shown to have an increased hybridization signal for one or more probes. The extent of these duplications is shown in Fig. 8. In one of the three cases (dup 1, Fig. 8) only the two exons of XJ 10 are duplicated. The duplication appears to be tandem and the unique junction created by the tandem duplication has been cloned and the molecular details of the duplication event are being studied (Hu, Burghes, Ray, and Worton, unpublished observations). Figure 10 is a schematic of how a tandem duplication might arise by unequal crossing over. As can be seen in the figure, such an event gives rise to two recombinant chromosomes, one with a deletion and one with a duplication of the same region. In each chromosome there is a single unique recombination point. In the deletion chromosome it defines the point where the sequences flanking the deletion come together. In the duplication chromosome, is defines the ends of the duplicated segment. The second duplication (dup 2, Fig. 8) duplicates a larger region containing several exons in a boy with DMD. The third duplication (dup 3, Fig. 8) merits special attention because it is small, duplicates only a few exons, and occurs in a mildly affected boy classified as BMD.

53

DUCHENNE AND BECKER MUSCULAR DYSTROPHY 1

1

2

3

4

2

5

R1

5

3

4

R1

6

6

7

R 2 8 9 1 0

7

R 2 8 9 1 0

7

R2

1 1

1

2

3

4

R1

2

5

3

4 RllR2 8 9

6

7

R2lR15

10

6

8

9

10

FIG.10. Schematic of the process of exon duplication and deletion by unequal crossing-over within a gene. The exons in this hypothetical gene are shown as numbered black boxes. Homologous repeat sequence elements, R1 and R2, are located in two of the introns. Misalignment of the homologous chromosomes (or of the sister chromatids in a single chromosome after replication), followed by recombination between the R1 and R2 sequences results in two new chromosomes, one with a deletion and the other with a duplication of the sequences between the R1 and the R2 repeat units. In this example three exons are deleted or duplicated in the recombinant chromosomes.

The question often arises as to whether the site and size of the deletion (or duplication) correlates with the severity of the disease. To date no such correlation has become apparent. It is possible that certain exons will encode protein domains that have especially important functional roles, and duplication or deletion of these exons may result in a severe phenotype. There is another very important factor, however, and that is the number of nucleotides in the deleted or duplicated exons. For example, the duplication or deletion of an exon with 90 bp will add or eliminate 30 amino acids in the defective protein. This may have a relatively mild effect on the phenotype. The duplication or deletion of a 91-bp exon is another matter. Not only will it duplicate or delete 30 amino acids; it will also shift the reading frame for all remaining exons in the gene. This results in the translation of a new chain of amino acids after the duplicated or deleted segment until a stop codon is encountered, at which point the protein is truncated. The consequence of such a frame-shift duplication or deletion would be expected to be more severe than that of an in-frame duplication or deletion. A 14-kb duplication of seven exons in the LDL receptor gene, resulting in the addition of 373 amino acids to the LDL receptor protein, has recently been reported (Lehrman et al., 1987). This is the first report of a duplication causing a genetic disease. The DMD/BMD gene seems to be much more

54

RONALD G . WORTON AND ARTHUR H. M . BURGHES

prone to genetic rearrangement than smaller genes, and one would expect to encounter deletions and duplications of both the in-frame and the frame-shift type. In assessing the possible mechasnisms of duplication and deletion in the Duchenne gene, it would appear that unequal crossing over is not the sole mechanism. First, if it was the sole mechanism, then each crossover event should create both a deletion and a duplication and the frequency of duplications should equal the frequency of deletions. The frequency data do not support this. Second, unequal crossing over between X chromosomes at meiosis is a female-specific mechanism, as males have a single X chromosome. For deletions occurring with equal frequency per X chromosome in male and female germ cells the proportion of new mutants is expected to be one-third, whereas for deletions occurring only in female germ cells the frequency of new mutants is expected to be one-half (Haldane, 1956). In the latter case the relative proportion of familial and sporadic deletions should be 1: 1, in the former case it should be 2 : l . Kunkel et al. (1986) compared the incidence of deletion in familial and sporadic cases. The familial incidence was 8.3% and the sporadic incidence was 5.8%; the ratio of familial to sporadic was 1.4, suggesting that some but not all of the deletions might have occurred by a female-specific mechanism such as unequal crossing over. In at least one case (Bakker et a l . , 1986) RFLP analysis has allowed the detection of a recombination event in the vicinity of the D M D gene in a woman whose daughter carried a de novo deletion. This could be coincidence, but it is also possible that an unequal crossing over event resulted in both the deletion and the switch in polarity of the RFLP markers. In summary, duplication and deletion account for a high proportion of mutation at the D M D locus. The proportion will undoubtedly increase as new genomic and cDNA probes become available. The duplications and some of the deletions probably occur by unequal crossing over, but further study is required to determine the relative proportion that occur by this mechanism.

D. RECOMBINATION AT THE DMD/BMD Locus For an average-sized gene, one does not expect an appreciable frequency of recombination within the locus. Thus, any RFLP marker that fails to segregate with a disease gene 100% of the time is usually considered to be outside the gene locus. Conversely, an RFLP marker that maps inside the gene locus is expected to always segregate with the disease gene. In the X-linked muscular dystrophy families, this expectation is not upheld. Both the PERT 87 and the XJ probes map inside the DMD/BMD

DUCHENNE AND BECKER MUSCULAR DYSTROPHY

55

locus, yet they fail to segregate with the disease gene in approximately 5 % of meioses. This recombination frequency of 5% forced one to think of recombination events as occurring not in the interval between the RFLP and the gene, but rather in the interval between the RFLP and the site of the mutation in the gene. Furthermore, if mutations could occur on either side of the PERT 87-XJ region, then recombination events on either side of PERT 87-XJ might be found to reverse the phase between the disease gene and the marker. For an understanding of the DMD locus and for the practical application of PERT 87 and XJ probes, it became important to precisely determine the recombination frequency in families and to determine, whenever possible, on which side of the PERT 87-XJ region the recombination had taken place. Data has just begun to accumulate on these probes. For PERT 87, Fischbeck et al. (1986) studied 57 families and found 8 recombinations in 124 informative meioses. The best estimate of the recombination fraction (0) was 0.06 (6 cM) with a confidence interval of 0.02-0.12. Discarding the onegeneration families as possibly having an autosomal form of MD, the remaining 14 multigeneration families with X-linked patterns of inheritance gave 0 = 0.04 with confidence limits of 0.0-0.14. In a further series of 25 families (Bertelson et a l . , 1986), two PERT 87 recombinants were seen in 74 meioses (e = 0.03). We (M. W. Thompson et a l . , 1986) and others (Walker et al., 1986) have examined linkage for multiple probes, including those from the XJ and PERT 87 regions. In our study of 31 families (M. W. Thompson et al., 1986), crossing over was found between the mutation and PERT 87 in 5 of 64 meioses and between the mutation and XJ 1.1 in 2 of 45 meioses. One recombination event took place between XJ 1.1 and PERT 87; in fact, it took place in the interval between XJ 1.1 and XJ 2.3. In the study of Walker et al. (1986), the best estimate (maximum Lod . score) of recombination frequency was 0.05 for PERT 87 and 0.00 for XJ 1.1. In summary, a recombination fraction of 0.05 (linkage distance of 5 cM) is probably a reasonable value to use for both PERT 87 and XJ probes, but the confidence interval is still large and more data are required to establish better estimates. It is also important to know whether the recombinations occur on the centromeric side, the telomeric side, or both. There is insufficient data on this point at present, but in anecdotal information presented at meetings it appears that, in many PERT 87 or XJ recombinants, the disease gene segregates with the C7 or distal markers rather than with the 754 or proximal markers. This implies that the mutations in the recombinant chromosomes must have affected a part of the gene distal to PERT 87. In at least one case, the mutation appears to map distal to J-Bir (Monaco et a l . , 1987).

56

RONALD G . WORTON AND ARTHUR H. M. BURGHES

VI. Carder Mentificatlon and Prenatal Diagnosis

A. THEPRINCIPLES OF CARRIER IDENTIFICATION AND PRENATAL DIAGNOSIS

One of the most immediate benefits of the molecular genetic studies is the generation of genetic markers in and around the DMD gene that can be used for carrier identification and prenatal diagnosis. The genetic markers now available for diagnostic purposes are shown in Table VI. They include flanking markers D2, 99-6, and C7 on the telomeric side and 754 and OTC on the centromeric side. All the probes from J-Bir, PERT 87, and XJ are considered to be markers within the gene. Several of the probes reveal polymorphic sites with more than one enzyme. TABLE VI FOR CARRIER IDENTIFICATION AND PRENATAL DIAGNOSIS DNA MARKERS

Locus DXS43 DXS41 DXS28

DXS164

Probe name

Polymorphic enzyme

D2 99-6

PVUII PrfI EcoRV BumHI EgnI BamHI Tag1

c7 J Bir 87-30 87-15

XmnI

87-8 87-1

DXS206

DXS84

-

XJ 2.3 XJ 1.1 XJ 1.2 XJ 5.1 754 754-11

orc

BSfXI TaqI XmnI BsfNI MspI EcoRV

TaqI Tag1 BClI SphI PrtI EcoRI EumHI MspI

Constant bands

Polymorphic bands'

Heterozygote frequency'

6.0, 6.6 13.0, 22.0 8.0, 7.5 21, 1615 30.0, 8.0 9.4, 7.112.3 3.1, 3.3 2.8, 1.611.2 4.4, 2.2 3.8, 1.112.7 7.5, 8 . 7 3.1, 2.51.6 4.1, 1.8 12.0, 14.0 6.4, 7.8 3.1, 3.8 1.7, 2.0 17.0, 24.0 12.0, 16.0 2.4, 4.2 16.0, 5.4 6.6, 6.2 5.1, 4.4

.41 .41 .26 .40 .50 .21 .50 .29 .46 .38 .43 .46 .46 .50 .32 .32 .41 .47 .27 .38 .48 .39

"Alternate alleles are separated by commas; the slash separates two bands that occur as one allele. 'Heterozygote frequency is the proportion of females who have both alleles of the polymorphism.

DUCHENNE AND BECKER MUSCULAR DYSTROPHY

57

For most geneticdisorders, such a large set of probes would not be necessary to attempt carrier identification or prenatal diagnosis. One probe that revealed an RFLP at 1 cM distance from an X-linked gene would allow predictivetesting with 99% accuracy if the phase between marker and disease gene was known in the heterozygous mother. None of the probes listed in Table VI, not even those that reveal RFLPs within the introns ofthe gene, show such close linkage. The withingene probes stdl have a recombination frequency of about 0.05, so that one such probe, when used alone, has a diagnostic accuracy of about 95 % . For many genetic diseases, the diagnostic accuracy can be greatly improved by the use of flanking markers. An ideal and not atypical situation for a Duchenne family is illustrated in Fig. 11. The woman in generation 2 is an obligate carrier, since she has an affected brother and an affected son. She wishes to know if her daughter is also a carrier and she seeks prenatal diagnosis in her third pregnancy. The DMD gene (DMD = mutant allele; N = normal allele) is flanked by polymorphic markers A (alleles A l , A2) and B (alleles B1 and B2). The phase between DMD and the flanking markers on her X chromosome is known from the alleles in her father, since he must pass his chromosome carrying A1 N B2 directly to his daughter without recombination. Thus, one of her chromosomes is A1 N B2 and, since she is heterozygous for AUA2 and Bl/B2, her A2 and Bl alleles must be on the other chromosome with the DMD gene. Her affected son is A2 DMD B2 and therefore a recombination has occurred between the DMD gene and the B marker in the germ cells of his mother (Fig. 11). He is a recombinant and, in this instance, the DMD gene has segregated with the A marker and not the B marker. The fact that he is a recombinant does not alter the picture in his carrier mother.

FIG. 11. Pedigree to illustrate the use of flanking markers for carrier identification and prenatal diagnosis. Bars beside the family members represent a small portion of the X chromosome around the DMD gene. Flanking the DMD gene (normal allele = N) are RFLP markers A and B with alleles A1 and A2 and B1 and B2, respectively. The white chromosome has alleles A1 N B2, the shaded chromosome has alleles A2 N B2, and the black chromosome has alleles A2 DMD B1. The woman in generation 2 is an obligate carrier since she has an affected brother and an affected son. She wishes to know the carrier status of her normal daughter and seeks prenatal diagnosis for her unborn male child.

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RONALD G. WORTON AND ARTHUR H . M. BURGHES

To evaluate the carrier status of her daughter, a marker study reveals that she has A1 and A2 alleles and only the B2 allele at the B locus. Crucial to her study is her father, who carries alleles A2 and B2. One of her X chromosomes must have come from her father without recombination and is therefore A2 N B2. By deduction, her other chromosome carries A1 and B2. Since, in her mother, both the A1 and B2 markers are carried on the chromosome with the normal gene, the daughter’s maternally inherited chromosome is likely A1 N B2 and she is not likely to be a carrier. The only possibility that she is a carrier is if her maternally inherited chromosome was A1 DMD B2, and this would require a double crossover in her mother’s germ cells, once crossing over from A1 to DMD and then crossing back from DMD to B2. If the A marker is 10 cM from DMD and the B marker is 5 cM, then the probability of each crossover event is 0.1 and 0.05, respectively. The probability of both crossovers occurring in the same cell to give the A1 DMD B2 chromosome is 0.1 x 0.05 = 0.005. The chance that she is a carrier, therefore, is half of one percent. At prenatal diagnosis, the male fetus is found to have markers A2 and B1. Since these markers are in phase with the DMD mutant gene in the mother, the chance that the fetus is not affected is equal to the chance of a double recombination or 0.005. The chance that he will be affected is 99.5%. It is clear that flanking markers are very valuable, because the chance of an incorrect diagnosis depends on the probability of a double crossover and this is less than 1 % for flanking markers that are each less than 10 cM from the gene. Of course, if the fetus in Fig. 1 1 had received A1 and B1 (or A2 and B2, as did the affected son), then a recombination is evident in the interval between the A and B markers. Since it is not possible to tell whether the crossover took place between A1 and DMD or between N and B1, a prenatal diagnosis would not be feasible. This simple example contains the two key principles of carrier identification and prenatal diagnosis-the establishment of phase in a female by knowledge of her father’s markers, and the use of flanking markers to reduce the risk of misdiagnosis. An ironic twist in diagnostic testing for DMD is the fact that the closest genetic markers revealed by probes derived from within the gene cannot be used as flanking markers. In any family, one does not usually know whether the mutation in the gene has occurred on the telomeric side of J-Bir, on the centromeric side of XJ, or somewhere in between. Unless this is known, the J-Bir, PERT 87, and XJ probes have to be considered as single markers and any one is about as good as any other, with a recombination frequency of about 5 % . For more accurate testing, it is necessary to use the more distant C7 and 754 polymorphism as the closest known flanking markers.

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In terms of choosing which markers to use in diagnostic testing, the choice of available probes depends on the likelihood of being informative and on practical considerations such as cost and convenience. The most informative markers are those with the highest heterozygote frequency, since any marker is only of value in those woman who are heterozygotes. If allele 1 and allele 2 are equally prevalent in the population, then 25% of woman will be homozygous for allele 1, 25% will be homozygous for allele 2, and 50% will be heterozygotes. For a marker whose alleles are not equally prevalent in the population, the proportion of heterozygous women is less than 50% and the marker is less useful. Many of the markers listed in Table VI have heterozygote frequencies of 0.4-0.5 and therefore are good markers. In terms of cost and convenience, probes can be used in combination on the same Southern blot. For example, two of the markers are BumHI polymorphisms. One BumHI digest can be probed with J-Bir and PERT 87-15 and the two RFLPs read from the same blot. The O T C probe also has a BumHI polymorphism, but the band sizes are similar to J-Bir and, therefore, cannot be read from the same blot. Similar combinations apply to digests with PstI, T q I , XmnI, and MspI.

B. PRACTICAL APPLICATIONS OF CARRIER IDENTIFICATION AND PRENATAL DIAGNOSIS Before proceeding to the practical applications of this new technology, perhaps a word of caution is in order. In our own studies, we have encountered a family with two affected boys who have opposite alleles for all PERT 87 and XJ probes, but the same alleles for probes D2 and 754 (M. W. Thompson et al., 1986). A double recombination, though rare, is the obvious explanation. In this family, the use of PERT 87 and XJ probes alone would have resulted in misdiagnosis of the second son had it been attempted prenatally. As a second example, it has often been stated that when an affected boy has a deletion, and his mother has no deletion, she is to be considered a noncarrier. In one of our families, two affected brothers have the same deletion, but their mother has no deletion in her somatic cells. The most likely explanation is that she has a germ-line mosaicism, with a deletion present in one or more germ-line stem cells. Diagnostic testing after the birth of the first affected boy would have classified her as a noncarrier, and prenatal diagnosis would have been deemed unnecessary. Two other similar cases have come to our attention from other centers, suggesting that gonadal mosaicism may be a relatively frequent event.

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These rare but not altogether unexpected findings do not negate the value of predictive testing. They merely caution the investigator to realize that such testing in DMD and BMD families is in its infancy. All the answers are not yet available. For this reason, we have established our diagnostic laboratory as an experimental facility for 2 years and we inform the families that the test procedures are still in the experimental phase. Hopefully, the number of unexpected findings leading to misdiagnosis will be minimal. Based on the principles described above, a number of groups have reported prenatal testing for Duchenne and Becker muscular dystrophy. In the earlier studies, the distant probes RC8 and L1.28 were used as flanking markers and combined with Serum CK levels for carrier identification (Wieacker et al., 1983; Harper et al., 1983; Williams et al., 1986). Adding the closest probe 754 (Hofker et al., 1985), combined with probes on the other side, reduced the risk of error to below 1 % . Studies of Becker families, combining RFLP analysis with CK testing, allowed accurate carrier detection for this form of the disease as well (Kingston et al., 1985). Using the full spectrum of eleven probes available in 1985, Bakker et a/. (1985) reported the prenatal diagnosis of an affected fetus with 99% accuracy and, in another family, an affected boy was identified as a new mutant, allowing three females in the family to be classified as noncarriers. In a twin pregnancy of a potential carrier, eleven probes were used to reduce the carrier risk and to predict normal male fetuses (Lavergne et al., 1986). Two normal boys were delivered. In a recent issue of the Journal OfMedical Genetics devoted entirely to the molecular genetics of the X-linked muscular dystrophies, three groups report their experience with prenatal diagnosis and carrier detection. Old and Davies (1986) report on their first eight prenatal diagnoses. Six males were deemed at high risk and the pregnancies were terminated. One was deemed at low risk and one was female, and these pregnancies were continuing. In a Finnish study, Lindlof et al. (1986) used multiple probes, CK data, and the Baysian method of combining probabilities to identify 22 carriers and 51 noncarriers among 97 females at risk. Three prenatal diagnoses were attempted. In a similar study from the Netherlands, Bakker et al. (1986) examined 61 families and reported that carrier status was determined with 98% accuracy in 104 (36 carrier, 68 noncarrier) of 136 females. Markers were informative for prenatal diagnosis in 33 out of the 36 carriers and prenatal diagnosis was attempted in 23 male pregnancies. Thirteen had an assessed risk of less than 1% and the pregnancies continued; seven of these were born and confirmed to be normal at the time of the study. Three were given a high risk (>98 7%) and seven others were inconclusive due to a known crossover in the region. These ten pregnancies were terminated.

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In summary, carrier identification and prenatal diagnosis is now available. There are sufficient markers that 75 5% of females can benefit from carrier tests and prenatal diagnosis, with a test accuracy of better than 98% (Bakker el al. , 1986). Diagnostic service laboratories are being developed in many major centers. In our own center, we have recently established a diagnostic service facility to care for the needs of the 300 families on record at the Hospital for Sick Children. There is no question that the families are wanting such a service. For many women who are at risk because they have an affected male relative, the carrier test is of paramount importance. The fact that the carrier test is equally robust in determining noncarrier status means that many women (approximately two-thirds) can be given the good news and they can proceed with their families without worry. The others, who are determined to be carriers, have the option of prenatal diagnosis. It should be acknowledged that a 98% accuracy rate is not perfect. Furthermore, termination of pregnancy in cases of a positive diagnosis is unacceptable to some and, at best, a difficult choice for others. However, for most, it is a far better option than the previous choice of terminating all male pregnancies, a choice adopted reluctantly by many families who could not cope with the prospects of another affected child. Until such time as a cure or effective therapy become realities, prenatal diagnosis will continue to be a welcome option for many families seeking to have normal, healthy children.

VII. Prospects for the Future

Only 5 years ago it would not have been possible to predict the cloning of the Duchenne/Becker muscular dystrophy gene. The route to cloning the gene only became appparent because of the astute observations of the clinicians who recognized that girls with DMD and boys with a complex phenotype merited further investigation. This was followed by the painstaking work of the cytogeneticists who discovered the translocations in the affected girls and the deletions in the multiply affected boys. The molecular biologists who constructed the first X chromosome libraries and isolated the first flanking probes helped to further define the locus and verified that the mutant genes in boys with DMD and BMD mapped to the same place on the X chromosome as did the rare translocations and deletions. Once the flanking probes and then the PERT 87 and XJ probes became available, clinicians and scientists around the world became immediately involved, screening their patients for deletions and examining their families for recombination events. Overall, there has been a remarkable spirit of cooperation and collaboration such that the current picture of the DMD/BMD gene is

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truly the work of hundreds of individuals from many nations. The fact that the research has moved so far in such a short time is directly attributable to this healthy cooperation. The future will require a similar level of cooperation and collaboration. The major shift in emphasis will be away from the DNA toward the protein product of the gene. As the complete gene is isolated over the next several months it will be important to utilize cDNA subclones in expression vectors to synthesize peptides that can be used as immunogens to generate antibody directed to the D M D protein. Alternatively, cDNA sequence data can be used to determine amino acid sequence, and synthetic peptides can be prepared for use as immunogens. Once antibodies are available they can be used as probes to examine the protein in tissue sections, in cultured cells, and in cell extracts subjected to electrophoresis. Before long it should be possible to determine several properties of the protein including its size, it tissue distribution, and its subcellular localization. Determination of its function may be more difficult, but information on tissue distribution and subcellular location should provide helpful clues. The identification of the D M D protein can also be approached from the other direction. Certain large proteins of muscle such as the Ricintrs communicus binding protein, the membrane feet protein, or the structural protein, nebulin, have already been discussed in an earlier section. Studies are already underway, particularly with nebulin, to isolate the candidate protein, prepare monoclonal antibody, and use the antibody as probe to isolate the gene. Once the candidate gene is cloned its sequence can be compared to the PERT 87-and XJ-derived cDNA clones to test for identity. One way or another, one would anticipate that the protein whose malfunction is responsible for Duchenne muscular dystrophy will be identified and characterized in the nottoo-distant future (see Note Added in Proof). At that point it should be possible to begin the experiments that will lead to a complete understanding of the disease. Once again, the patients and their physicians will play an important role. It will become important to study a number of patients, each with a well-characterized genetic lesion (deletion, duplication, translocation, inversion, insertion, point mutation, regulatory mutation, etc.) in order to determine the effect of the lesion on the protein itself and on the function of the protein in the context of the cell. In this regard, perhaps the recent and elegant experiments with the membrane-bound LDL receptor will serve as a model. In patients with familial hypercholesterolemia due to defective LDL receptor, defects have been found in receptor synthesis, transport and movement in the membrane, as well as in ligand binding, ligand internalization, and receptor recycling (Brown and Goldstein, 1986). Clearly, the availability of a wide spectrum of mutations in the gene will be a major factor in experiments

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designed to lead to a complete understanding of the function of the normal protein in the cell. In some respects, naturally occurring mutations in Homo sapiens may not be sufficient for examining all aspects of the DMD gene and its protein product. There is tremendous advantage to genetic manipulation designed to perturb specific parts of the gene andlor its regulatory sequences. For this kind of analysis animal experiments will be of great value. There has not been a good animal model for X-linked dystrophy of the Duchenne type. The mdr mouse has been recently described as an X-linked form of dystrophy with similarities to DMD or BMD (Bulfield et al., 1984), but mapping data in the mouse has suggested that its location relative to other loci may be more similar to the map location of Emery-Dreifus muscular dystrophy on the human X chromosome (Avner et al., 1987). More recent unpublished observations have disputed this and suggest that the mdr locus is indeed the Duchenne equivalent in the mouse. One may not have to depend for very long on preexisting animal models, since the human DMDIBMD gene contains sequences that are homologous to sequences in mouse, hamster, and other species. It will certainly be possible to isolate and characterize the equivalent genes from these other species. It may also be possible to treat mice with X-rays or potent mutagens and then screen for new mutants with a dystrophic phenotype and with lesions in the DMD equivalent locus. It is curious that the mutation frequency in the human gene is so high, with a broad spectrum of spontaneous duplication and deletions, and yet mice with a similar phenotype are not routinely found. For this reason, one wonders if the mouse will be a good model even if the occasional mutation can be induced in the mouse DMD equivalent gene. Once the function of the DMD protein is clearly understood, the prospect of cure or therapy has to be addressed. The prospects will depend to a large extent on the nature of the protein itself. If the protein is a large structural protein of the muscle fiber, the probability of being able to modify the disease process through drug therapy may be slim. Unlike a humeral factor such as insulin, a defective structural protein will not be easily replaced or modified. Gene therapy is another prospect that is under intense investigation for certain diseases. The genetic diseases with the best prospects are those where a new gene can be introduced into bone marrow stem cells and the stem cells used to reseed the marrow. Even then, the gene product has to be an enzyme or humeral factor that can circulate in the body to correct the deficiency in all relevant tissues. Gene therapy for the correction of the somatic defect in muscular dystrophy would necessitate removal of muscle satellite cells, introduction of a normal DMD gene to provide the normal gene product, and then implantation

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of the corrected myogenic cells into all major muscles for subsequent inclusion into the growing muscle fiber. Even though some progress is being made in the area of muscle cell implantation in mice (Morgan et aE., 1986), the problems seem horrific in comparison with gene therapy using bone marrow stem cells. As an alternative, therefore, new genes might have to be introduced into the bloodstream via viral or other vehicles that will target the delivery to the muscles. While these approaches are not beyond the scope of one’s imagination, an assessment of their relative merits is beyond the scope of this review. Perhaps the biggest and certainly the most immediate benefit of the gene cloning will be in the area of carrier identification and prenatal diagnosis. As indicated above, many women can now be advised of their carrier status with a high degree of reliability. Those identified as noncarriers can proceed with their families without fear. Those identified as carriers can choose among the options, which include limitation of family size, adoption, or prenatal diagnosis. In the latter case the affected boys can be distinguished from the unaffected with a high level of reliability and the normal boys can be allowed to proceed to term with a minimum of risk. Even though the target of research must remain cure of the disease, prevention of second and third cases in families, through prenatal diagnosis, is a major step forward. If that is the only benefit of the gene cloning, then the effort will have been worthwhile. If an effective therapy or a cure comes from knowledge of the basic defect, that will be a bonus beyond our reasonable expectations.

Acknowledgments

The authors are grateful to many people for their help in putting together this review. In particular, Susan Coulson and Helen Worton deserve special credit for the hours they put into organization and entry of references, manuscript typing, and editing. Thanks are also in order for Olga Sitarz and Catherine Wallis for their effort in typing some of the numerous drafts. One of us, AHMB, is grateful to the Muscular Dystrophy Association (USA) for a postdoctoral fellowship, and RGW acknowledges the financial support of the Muscular Dystrophy Association of Canada, the Muscular Dystrophy Association (USA), and the Medical Research Council of Canada. We are grateful to our colleagues, especially Dr. M . W. Thompson, Dr. P. Ray, and Dr. X . Hu, and S. Bodrug, C. Duff, C. Logan, and B. Belfall for their many contributions to the work described herein from our laboratory.

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NOTEADDEDIN PROOF Since this review was written and submitted, many more publications have appeared. Three are important enough to mention here. The first is an article entitled “Complete cloning of the Duchenne muscular dystrophy (DMD) cDNA and preliminary genomic organization of the DMD gene in normal and affected individuals,” by M. Koenig, E. P. Hoffman, C. J. Bertelson, A. P. Monaco, C. Feener, and L. M. Kunkel in Cell 50, 509-517 (1987). It reports the isolation of the remainder of the cDNA from the DMD gene shows that the cDNA is 14 kb in size (rather than 16 kb as estimated earlier) and demonstrates that the transcript is formed from a minimum of 60 exons. The first 33 exons map over a distance of greater than 1000 kb with exons 32-34 roughly in the vicinity of J-Bir (see Fig. 7). The remaining exons are less well mapped but may spread over a similar distance. Exons 50-55 map in the region of 5-66 (Fig 7), and the remaining exons lie between 5-66 and L1 on the physical map. Most of the genomic region from PERT 87 to the 3’ end of the gene (about 1500 kb) remains to be mapped and cloned. The article also reports the use of cDNA to detect deletions throughout the gene. In addition to the many deletions that overlap the PERT 87-XJ region, there is a separate set of deletions that cluster around the middle of the cDNA at exons 37-45. Many of these start within a single intron (of unknown size) and extend for a variable distance toward the telomere. In total, the deletion frequency was over 50% in the 104 patients studied. The second article of importance is entitled “Is nebulin the product of the Duchenne muscular dystrophy gene?,” by H . Sugita, I. Nonaka, Y. Itok, A. Asakura, D. H. Hu, S. Kimura, and K. Maruyama in Proc. Jpn, Acad. Ser. B 63, 107-110 (1987). The answer to the question posed in the title is “no”! They used Western blot (immunoblot) analysis to show that protein recognized by antinebulin antibody was present as a 500-kDa band in muscle extracts from normal boys and from affected boys at a preclinical stage or symptomatic stage. They suggested that the earlier results of Wood et al. (see Section 11, E) was due to protein degradation, perhaps by calcium-activated neutral proteases that are activated in Duchenne dystrophy muscle. This result typifies the problems discussed in Section 11, E, that is, the difficulty in detecting the

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primary defect in tissue where secondary phenomenon predominate. Furthermore, it reopens the speculation as to the nature of the true product of the Duchenne muscular dystrophy gene. The third article adds to the speculation. In aletter to Ce//51 (October 9, 1987), R. G. Hammonds pointed out that the DNA sequence at the 5' end of the DMD gene reported by Koenig eta/. bore a strong similarity to the sequence at the 5' end of the a-actinin gene of chicken. The deduced amino acid sequences were most similar within 250 amino acids of the amino terminus of both proteins. a-Actinin is a normal component ofactin filaments, cross-linking them and connecting them to the cell membrane. The region of similarity with the DMD protein coincides with the actin-binding domain, suggesting that the DMD protein may also be an actin-binding protein. Although the identity and biological function are not revealed by the presumed homology, the clue is an important one that may help to narrow the search. With the stunning rate of progress to date, we would not be surprised if the true identity and function ofthe protein is known prior to final publication of this review. For the sake of thc boys with this dreadful disease, let us hope that this will be the case.

BATRACHOTOXIN: A WINDOW ON THE ALLOSTERIC NATURE OF THE VOLTAGE-SENSITIVE SODIUM CHANNEL By George B. Brown The Neuropsychiatry Research Program and Department of Psychiatry The University of Alabama at Birmingham Birmingham, Alabama 35294

I. Introduction 11. Electrophysiological Analysis of Batrachotoxin Effects A. General Effects B. Reversibility C. Effects of Stimulation D. Activation and Inactivation Parameters E. Selectivity Reduced F. Single-Channel Conductance Decreased 111. Nature of the Binding Site A. Structure-Activity Relationships B. The Protonated Form of BTX Is Active C. Microenvironment of the BTX Binding Site IV. Interactions with Other Sodium-Channel Neurotoxins and Ligands A. a-Scorpion Toxins and Sea Anemone Toxins B. Local Anesthetics, Anticonvulsants, and Related Compounds C. Pyrethroid Insecticides D. Tetrodotoxin and Saxitoxin E. Other Toxins V. Role of Lipid A. Solubilization of the Sodium Channel B. Photoaffnity Labeling C. Lipid Association with Purified Sodium Channel VI. Concluding Remarks References

1. introduction

The voltage-sensitive sodium-channel protein of excitable membranes mediates the fast rising phase of the action potential in a wide variety of tissues. Exactly how this is accomplished has been the subject of intensive investigation ever since Bernstein (1912) first advanced the hypothesis of rapid membrane conductance changes underlying electrical excitability.

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Electrophysiological studies, from the pioneering work of Hodgkin and Huxley (1952) onward, have now provided a precise phenomenological description of the sodium channel and the sequence of events occurring at that channel during an action potential. In brief, sodium channels in the resting state of an excitable cell exist in a closed, nonconducting conformation. In response to a local depolarization of the membrane the channef protein undergoes time- and voltage-dependent conformational changes, progressing through apparently multiple nonconducting states before reaching an open, conducting state permitting, selectively, the flow of sodium ions from extracellular to intracellular spaces down an electrochemical gradient. This process is termed activation. The open state is maintained only transiently before the channel closes, or inactivates, by adopting yet another conformation that is distinct from the closed, resting state. As the membrane potential recovers to resting values, the channel proteins returns to the closed, resting configuration without passing again through an open state. The whole cycle is complete within a few milliseconds. While these electrophysiological studies have provided a wealth of information, further progress in our understanding of the voltage-sensitive sodium channel has been greatly aided by the application of numerous specifically acting, naturally occurring neurotoxins and other pharmacological agents as molecular probes of sodium-channel structure and function. These compounds have been instrumental in such diverse tasks as assessing the density of sodium channels in various membranes, probing structure- function relationships, and ultimately the purification to homogeneity and molecular cloning of the channel protein. Three classes of specifically acting sodium channel neurotoxins in particular have been utilized extensively in these studies, including the heterocyclic guanidinium compounds tetrodotoxin and saxitoxin, the polypeptide neurotoxins from scorpion and sea anemone, and the so-called “lipid soluble” neurotoxins including batrachotoxin, veratridine, aconitine, and grayanotoxin. It is this latter class of neurotoxins, the structures of which are shown in Fig. 1, that forms the subject of this review. Experience with this group of sodium-channel neurotoxins over the last decade has led to the remarkable observation that essentially every functional characteristic or electrophysiological descriptor of the sodium channel is altered or modified upon binding of these compounds to a single, common site. Thus, binding of batrachotoxin, the prototypical and most potent member of the class, at once affects the voltage-dependent processes of sodium-channel activation and inactivation, changes the selectivity of the channel, and alters the single-channel conductance. In considering this broad spectrum of effects, one is led to invoke a model for sodium-chitnnc.1tiiiic.tion that incorporates not only extensive conformational r c ~ ~ I r ~ ~ i I i ~ c ‘ I i i c t)ut . i i t s idso . potential allosteric interactions among clilli.rc.ni p r i ) i c i n cloriiiiiiis. w \ . t , i . ; i l 1 1 1

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BATRACHOTOXIN

0

Batr achotoxin

OW3

Verotridine

OH

-

Aconitine

Grayonotoxin I

FIG.1. Structures of the lipid-soluble sodium-channel neurotoxins.

which may reflect upon a key structural element that includes the binding site for the lipid-soluble toxins. In this review I would like to focus on the body of work that underlies this construct, first from the electrophysiologicalviewpoint and then from the pharmacological and biochemicalapproaches. The experiments and the data to be described are of necessity selected, rather than all inclusive, with an emphasis placed on batrachotoxin along with veratridine, as these two compounds have been investigated more extensively to date. The interestedreader is encouragedto consult additional recent reviews of the voltage-sensitive sodium channel and sodiumchannel neurotoxins (Albuquerque and Daly, 1976;Narahashi, 1977,1982; Catterall, 1980, 1984; Daly, 1982a,b; Witkop and Gossinger, 1982; French and Horn, 1983; Khodorov, 1983a,b, 1985; Strichartz et al., 1987).

II. Electrophyslologlcal Analysls of Batrachotoxln Effects

A. GENERAL EFFECTS In the most general terms, the lipid-soluble sodium-channelneurotoxins are depolarizing agents, leading to a loss of membrane potential in virtually every

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sodium-dependent excitable membrane system in which they have been studied. Batrachotoxin (BTX) in particular is extremely potent in this regard, being capable of eliciting complete membrane depolarization at concentrations in the low nanomolar range. It is one of the most toxic nonprotein substances known. The toxin is a steroidal alkaloid isolated from the skin secretions of the frogs Phyllobates aurotaenia and Phyllobates terribilus (Daly , 1982b). Its use by the native Indians of Colombia, South America (the principal habitat of the frogs) for the preparation of poison blowgun darts and arrows has been reviewed (Myers et al., 1978). The LDso in mice is 2 pg/kg i.p., and it has been estimated that a lethal dose in humans would be less than 200pg (Albuquerque and Daly, 1976). Batrachotoxin is particularly active on the muscles of the heart, and death is principally due to paralysis of respiration. The pioneering work on the mechanism of action of BTX was carried out in the early 1970s by Albuquerque, Narahashi, and their colleagues (Hogan and Albuquerque, 1971; Albuquerque, 1972; Albuquerque et al., 1971, 1973a,b; Narahashi et al., 1971a,b). These early studies focused attention on a specific interaction of batrachotoxin with the voltage-sensitive sodium channel responsible for the rapid rising phase of the action potential. As a result of experiments in squid giant axon, rat phrenic nerve-diaphragm preparations, dog heart Purkinje fibers, and frog sartorius muscle, there was little doubt that the predominant effect of BTX was to maintain voltagesensitive sodium channels in an open configuration, leading to unabated sodium influx and membrane depolarization. Specificity for the sodium channel was indicated by the fact that, in each case, BTX-induced membrane depolarization was antagonized by absence of sodium or decreased sodium concentration in the medium, or by application of tetrodotoxin, a specific sodium-channel blocker. Further, muscle membranes from lobster and crayfish were unaffected by BTX (Albuquerque et al., 1972). In these species, Ca" ions rather than Na* ions carry the inward current during an action potential. Catterall has shown that BTX is without effect in a variant neuroblastoma cell line which lacks voltage-sensitive sodium channels (Catterall, 1977b). Of the many preparations tested which use sodium as the principal inward current-carrying species, only the frog which produces batrachotoxin (P.aumtaeniu) was found to be insensitive to the depolarizing action of BTX (Albuquerque et a l . , 1973). Thus, the animal which produces and stores this potent neurotoxin is essentially immune to its effects. Interestingly, veratridine retained some depolarizing activity in P. aurotaenia muscle, although the efficacy was greatly reduced compared to its effects on excitable membranes of the frog Runa pipiens. As will be discussed below, veratridine has been shown in other preparations to compete directly for the

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81

same binding site on the sodium channel as BTX. The fact that it retains activity, albeit reduced, suggests that the receptors for BTX and veratridine in P.aurotmiu are not identical but are probably made up of overlapping elements.

B. REVERSIBILITY The effect of BTX in intact preparations has generally been found to be irreversible. An example is found in the experiment described by Narahashi et al. (1971a) wherein a squid giant axon was initially depolarized by addition of 1 pikt BTX to the external bathing solution [artificial sea water (ASW)]. The preparation was then washed extensively with ASW containing, sequentially, tetrodotoxin (TTX), Tris-substituted 1 mM sodium ASW, and Trissubstituted 1 mM sodium ASW plus TTX. During this time, the membrane potential returned to a normal resting value. Upon finally changing the bathing solution back to ASW alone, an immediate depolarization ensued. Further washing with ASW for 90 min failed to repolarize the preparation. Similar results were obtained by Albuquerque et al. (1976) using the rat phrenic nerve-diaphragm preparation. This observed irreversibility has been difficult to reconcile with later studies which show binding or effects of batrachotoxin to be reversible in nonintact tissues. For example, Catterall (1975) has found that the activation of sodium channels in N18 mouse neuroblastoma cells is completely reversible with a half-time of 30 min at 36OC. Brown et al. (1981) showed that, following equilibration of a broken membrane preparation from mouse brain with a radiolabeled analog of BTX, BTX-B (see beIow), all radioactivity may be washed out by filtration on glass fiber filters within 1-2 min. In view of the lipid solubility of BTX, suggestions have been forwarded that the apparent irreversibility may simply be the result of BTX remaining trapped in the lipid phases during wash, coupled with the fact that diffusion barriers may be prominent in intact tissue and that only a small fraction of the sodium channels need to remain open in order to prevent repolarization. On the other hand, the effects of BTX are irreversible in the squid giant axon, which lacks significant diffusion barriers (Narahashi et al., 1971a), and the effects of the lipophilic depolarizing agents veratridine and grayanotoxin are readily reversible. The effects of aconitine are irreversible in squid giant axon. It must be admitted that at present there is not a completely satisfactory explanation for these observations. It would perhaps be interesting to measure the washout of radioactivity from a preparation which has been completely depolarized by radiolabeled BTX. In this way, the remote, but still untested, possibility that BTX-treated sodium channels in certain intact tissues remain irreversibly altered even in the absence of BTX could be directly examined.

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GEORGE B. BROWN

c. EFFECTSOF STIMULATION In the absence of stimulation, the onset of BTX-induced membrane depolarization is relatively slow. Narahashi d al. (1971a) found that the rate of depolarization was slightly greater in squid giant axon when BTX was applied internally in the perfusion medium rather than externally. Rather than providing an indication of the relative location of the binding site, this observation addresses more the fact that, being lipophilic, BTX may access its binding site from either side of the membrane. The early studies of BTX-induced depolarization in a variety of tissues indicated that the rate of depolarization could be markedly enhanced if the preparation were stimulated. In fact, in the case of the electric organ from eel, BTX had no effect unless the preparation was stimulated (BartelsBernal et al., 1977). A similar result was obtained earlier for the effect of veratridine on eel electroplax (Bartels and Rosenberry, 1973). Veratridine alone at the concentration of 1 mMhad no effect on the resting membrane potential or action potential for periods up to 1 hr, but stimulation of the preparation at 2 Hz rapidly led to complete depolarization and loss of the action potential. This phenomenon of stimulus dependence was investigated in greater detail by Khodorov and his associates (Khodorov and Revenko, 1979; Revenko, 1977) in voltage-clamped frog node of Ranvier. Provided the stimulus pulse was of sufficient strength to activate normal sodium channels, repetitive pulsing dramatically increased the initial rate of appearance of BTX-modified channels (several hundred-fold) in this preparation. Similar results were obtained with neuroblastoma cells (Zubov et al., 1983). In this paradigm it was also observed that currents from modified sodium channels increased at the expense of those from normal, unmodified channels on a oneto-one basis. More recently, Rando et al. (1986) have shown that stimulation of frog sciatic nerve resulted in a biphasic depolarization in the presence of 100 nMBTX. The initial phase was extremely rapid and essentially complete after four stimuli at 1 Hz. The second phase was only slightly faster than that observed in the absence of stimulation, so that the overall t was increased only 15-fold. In the case of veratridine, depolarization was monophasic, complete after only one stimulus, and approximately 10,000 times faster than veratridine-induced depolarization in the absence of stimulation.

,,*

D. ACTIVATION AND INACTIVATION PARAMETERS From the foregoing results, it became clear that BTX exerts its depolarizing effects by binding readily only to the activated form of the sodium channel. The fact that BTX-modified channels then remain open at all but very hyperpolarized membrane potentials suggests that these channels fail to inactivate

BATRACHOTOXIN

83

and that the normal voltage dependence of the activation process is altered as well. A careful analysis of the effects of BTX on iodium-channel activation and inactivation parameters has been presented, primarily by Khodorov and his associates. Several lines of evidence demonstrate that BTX-modified channels fail to inactivate. 1 . Voltage-clamp records of frog myelinated nerve reveal that BTXmodified channels at the node of Ranvier continue to conduct even during sustained depolarizing command potentials at which unmodified channels fully inactivate (Khodorov et al., 1975). 2. In normal sodium channels, immobilization of a portion of the “on” gating current has been associated with the process of inactivation (Armstrong and Bezanilla, 1977; Nonner, 1980). In the presence of BTX, charge immobilization is abolished, and the “off” gating current is equal in magnitude to the “on” gating current (Dubois et al., 1983). 3. Quandt and Narahashi (1982) studied the activity of single sodium channels in patch-clamped N1E-115 neuroblastoma cells before and after modification with 10 BTX. In contrast to the unmodified channels, after BTX treatment the probability of channel opening remained constant over time during a step depolarization, showing lack of inactivation. Furthermore, the mean channel open time was found to be increased 25- to 50-fold in the presence of BTX.

Explanation for the fact that BTX-modified sodium channels remain open even at normal resting membrane potential has been found in the altered voltage dependence of channel activation. Measurements in BTXtreated, voltage-clamped frog node of Ranvier show that the steady-state sodium permeability (PNa)versus membrane potential curve is shifted fully 60-65 mV toward negative potentials relative to normal channels (Khodorov and Revenko, 1979). Likewise, the steady-state voltage dependence of the activation parameter (m,) is shifted 60 mV in the hyperpolarizing direction (Khodorov and Revenko, 1979). The kinetics of sodium-channel activation are also dramatically altered by BTX (Khodorov and Revenko, 1979; Huang et al., 1972). Calculations show that the rise in sodium current becomes exponential instead of sigmoidal, as in normal sodium channels, and the maximum time constant for activation ( T , ~ ) is greatly increased. In order to determine the origin of this effect, Khodorov and Revenko (1979) computed the voltage-dependent rate constants a, and 0, for channel opening and closing, respectively, as a function of voltage. The results of this analysis showed that in the presence of BTX the curve relating 0, to membrane potential was shifted by greater than 60 mV in the hyperpolarizing direction. Therefore 0, is decreased even at high negative

84

G E O R G E B. B R O W N

membrane potentials, thus accounting for the increase in T, and the shift in the voltage dependence of PNa.In BTX-modified sodium channels at normal resting membrane potentials the rate of channel opening far exceeds that of channel closing, leading to a steady-state abundance of open channels, whereas in unmodified channels the opposite occurs. A priori, these effects of BTX ought to be manifest as well in the gating currents (Q for channel activation, and such was found to be the case by Dubois et al. (1983). The steady-state Q dependence on membrane potential was virtually the same as that of PNa,being shifted approximately 50 mV in the hyperpolarizing direction. The total charge movement was not significantly different than for normal channels. Taken together with the kinetic analysis of channel activation, these results suggested that, in BTXmodified sodium channels, the three gating particles, whose sequential movement is normally considered to underlie the sigmoidal time course of sodium-channel activation, behaved as if they were aggregated and moving as a single unit. In this model, activation of BTX-modified sodium channels could be considered to follow a simple exponential transition from a single closed state to an open state. A subsequent study (Dubois and Schneider, 1985), however, revealed that the time course of channel activation was only fit by a single exponential if a delay was interposed between the onset of the potential change and the onset of sodium current. Further, it was found that the time constant for the gating current was not identical to the time constant for channel activation. These considerations require that activation of BTX-modified sodium channels be modeled by a multistep process involving at least two closed states, closed closed open. A similar model was also proposed for gating of BTX channels in neuroblastoma cells (Huang et al., 1984). The preceding discussion permits a first approximation of the possible physical correlates for BTX modification of sodium channels. Recall that the major effect of BTX can be seen to be on the voltage dependence of the closing rate constant, 0,. As discussed by Bezanilla (1985) for a simple two-state model, 0, for sodium-channel activation at any given potential is a function involving two variables: (1) the energy difference between the well for the active state and the peak of the barrier for transition to the closed state (the activation energy for closing), and (2) the fraction of the distance through the field where the peak of the barrier is located. With all else being equal, alteration of either one of these (or both) to account for the shift in voltage dependence of channel opening would seem to require a conformational change affecting the environment of the voltage sensor induced by the binding of BTX.

- -

BATRACHOTOXIN

85

E. SELECTIVITY REDUCED Batrachotoxin has been found to reduce the selectivity of the voltagesensitive sodium channel in a variety of preparations, including frog nerve node of Ranvier (Khodorov, 1978; Khodorov and Revenko, 1979), neuroblastoma cells (Zubov et al., 1983; Huang et a/. , 1979), and clonal muscle cells (Huang et al., 1979). Not only is the relative permeability for normally permeant species altered, but also the flux of relatively impermeant inorganic and organic cations such as calcium, cesium, and methylamine can be measured in modified channels. BTX-modified sodium channels remain impermeant to anions. Similar effects have been observed for purified channels which have been reconstituted in liposomes or planar lipid bilayers and treated with BTX, although the extent of selectivity reduction is generally not quite as great as that found in intact preparations (Barchi and Tanaka, 1984; Tamkun et al., 1984; Moczydlowski et al., 1984a; Hanke et al., 1984; Hartshorne et al., 1985). Veratridine (Frelin et al., 1981; Naumov et al. , 1979), aconitine (Campbell, 1982; Grishchenko et al., 1983), and grayanotoxin (Hironaka and Narahashi, 1977; Frelin et al., 1981; Seyama and Narahashi, 1981) similarly alter sodium-channel selectivity properties, although there are some quantitative differences compared to the effects of BTX. In general, veratridine and aconitine affect more pronounced changes than does BTX (Barchi and Tanaka, 1984; Tamkun et al., 1984). The permeability of BTX-modified sodium channels to methylamine is a particularly telling finding. This molecule cannot penetrate normal, unmodified channels, a fact which Hille (1971, 1972) used to help set a maximum physical dimension of 3 x 5 A for the selectivity filter of normal sodium channels. In order to accommodate the passage of methylamine, the size of the selectivity filter must be increased to a minimum size of 3.8 x 6 A (Huang et al., 1979), once again demonstrating a conformational change in yet another domain of the sodium channel induced by the binding of BTX.

F. SINGLE-CHANNEL CONDUCTANCE DECREASED As might be supposed from the BTX-induced alteration in ionic selectivity, the single-channel conductance is also modified by the toxin. In preparations where the two have been compared directly, single-channel conductance for BTX-modified channels is approximately half that of the native channels (Khodorov et al., 1981; Quandt and Narahashi, 1982; Huang et al., 1984). This is somewhat surprising in view of the evidence that BTX-modified

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GEORGE B. BROWN

channels have a selectivity filter with dimensions greater than normal. It is probable that the interactions of various cations with anionic sites within the selectivity filter are altered in the modified channels. In addition, the reduced single-channel conductance could possibly result from simultaneous movement through the channel of sodium and other ions that would otherwise be impermeant. Khodorov et al. (1981) have proposed a specific action of calcium ions on the sodium flux to account for the reduced single-channel conductance. In assessing the effects of BTX on voltage-sensitive sodium channels as revealed by the electrophysioiogical studies described above, one cannot help but be impressed by the fact that virtually every aspect of sodium-channel function is modified. Conformational changes in different domains of the channel subsequent to the binding of the toxin are also indicated. The central importance of BTX and the other lipid-soluble neurotoxins to the study of the sodium channel is therefore established, since no other toxin exerts such wideranging effects. Recent pharmacological and biochemical studies continue to support a model for BTX action in which binding at a single site results in conformational changes extending over multiple domains of the channel protein which may interact in an allosteric fashion.

111. Nature of the Binding Site

A. STRUCTURE-ACTIVITY RELATIONSHIPS The study of structure-activity relationships may often provide insights into the nature of a particular binding site. In the case of batrachotoxin, extensive studies of this type have been hampered by the relative paucity of starting materials for synthetic modification. A useful approach has been the modification of batrachotoxinin A (BTX-A), which lacks the pyrrole carboxylate group of batrachotoxin at the 20a-hydroxyl, but BTX-A is found as only a minor component of the venom from P. ausotaenia and P. tmibilus compared to the content of batrachotoxin. This venom remains the primary source of BTX-A. A complete synthesis of BTX-A has been accomplished by Wehrli and his collaborators in 36 steps (Imhof et al., 1972, 1973; most recently reviewed by Daly and Spande, 1986), but, the heroic and monumental nature of this accomplishment notwithstanding, the yields in such a lengthy synthesis were predictably and impractically low. Before examining some of the results of BTX modifications, it is helpful to review the general structural features of the molecule (see Fig. 1). The elegant X-ray crystallographic studies conducted by Karle and Karle (1969) revealed that the stereochemistry of the steroidal backbone corresponds to that of

a7

BATRACHOTOXIN

cholesterol in all points of comparison except at carbon 14. The cis AIB and C/D ring junctures along with the 3a-9a hemiketal form a rigid convex structure presenting the hydrophobic P face of the steroidal backbone. Several structural features of the molecule are unique to nature, including the 3a-9a hemiketal, which may be compared to the 4-9 hemiketal of veratridine, and the homomorpholino ring (with its weakly basic nitrogen atom) fused at the C/D ring juncture, resulting in a so-called “propellane” system. The 20a-2,4-dialkylpyrrole-3-carboxylate moiety is also unique to nature, and it is in this region where the majority of structure-activity relationships have been drawn. The toxicities of BTX and its homologs that co-occur in the venom along with a number of derivatives prepared by partial synthesis or selective modification are shown in Table I. Several points emerge from examination of this data, compiled from bioassays of toxicity in white mice, and similar conclusions result from investigations of the relative ability to depolarize rat phrenic nerve-diaphragm preparations (Warnick et nl., 1975). The steroidal backbone structure is apparently of major importance to activity, since reduction of the 3-9 hemiketal yields dihydrobatrachotoxin with greatly diminished TABLE I TOXICITY OF BATRACHOTOXIN AND ITS CONGENERS Compound Batrachotoxinin A 20-(2,4,5-trimethylpyrrole-3-carboxylate) Batrachotoxin Batrachotoxinin A 20-benzoate Batrachotoxinin A 20-(2,5-dimethylpyrrole-3-carboxylate) Homobatrachotoxin Batrachotoxinin A 20-(2,4-dimethyl-5-ethylpyrrole-3carboxylate) Batrachotoxinin A 20-(N-methylanthranilate) 3-0-methylbatrachotoxin 4-P-Hydroxybatrachotoxin Dihydrobatrachotoxin Batrachotoxinin A 20-(4,5-dimethylpyrrole-3-carboxylate) Batrachotoxinin A 20-(2,4-dimethyl-5-acetylpyrrole-3carboxylate) Batrachotoxin methiodide Batrachotoxinin A Batrachotoxinin A 20-(N,2,4,5-tetramethylpyrrole-3carboxylate) Batrachotoxinin A ZO-(pyrrole-Z-carboxylate) Batrachotoxinin A ZO-(p-bromobenzoate)

LD,, (pgikg)

1 2 2 2.5 3

Referencea

1 1 2 1 1

8 15 30 200 250 260

1 3

280 1000

1 1 1

> 1000 > 1000 > 1000

1 1 1

500

1 4 1 1

“References: 1, Tokuyama et al. (1969);2, Brown et al. (1981);3, Brown and Bradley (1985);4, Tokuyama and Daly (1983).

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GEORGE B. BROWN

potency. This is also the case with the naturally occurring 40-hydroxybatrachotoxin, perhaps suggesting the importance of a hydrophobic 0 face. Note as well that the permanently charged batrachotoxin methiodide is less potent but still retains some activity. At the moment it is not clear to what extent this reduced activity is due to impaired diffusion of this polar compound to the binding site. The striking sensitivity of potency to modifications in the 20a-ester region as indicated in the table was initially interpreted to mean that the pyrrole-3carboxylate moiety with its free and very weakly basic nitrogen was an essential and critically important component. This conclusion followed from the observed loss of activity resulting from removal of the ester altogether (BTXA), replacement with a p-bromobenzoate group, or methylation of the pyrrole nitrogen [batrachotoxinin A 20-(N,2,4,5-tetramethylpyrrole-3carboxylate)]. It was quite surprising, then, to discover that the simple benzoate derivative of BTX-A (batrachotoxinin A 20a-benzoate, BTX-B) was equipotent with BTX (Brown et al., 1981). Based on this finding, it now appears that the low potency of the p-bromobenzoate and pyrrole N-methylated derivatives could be primarily the result of steric constraints in this region of the binding site. Not only did the discovery of BTX-B reopen the question of structureactivity relationships in the ester region of BTX, but it also provided a longsought-after synthetic entry to a radiolabeled derivative of high specific activity and biological activity. Previous attempts to prepare such a derivative by nonspecific Wilzbach labeling or synthetic introduction of tritium into the pyrrole alkyl groups of BTX itself proved unsatisfactory due to low specific activity or instability of the product (G. B. Brown and J. W. Daly, unpublished observations). Selective esterification of BTX-A with p-[3H]benzoic acid prepared from catalytic tritiolysis of p-iodobenzoic acid led smoothly and in high yield to a remarkably stable [3H]BTX-B derivative with a specific activity limited only by the specific activity of the tritium used in the reduction (Brown et al., 1981). With this derivative in hand it became possible for the first time to evaluate directly the binding of batrachotoxin, its analogs, and other lipid-soluble toxins to the voltage-sensitive sodium channel. In general, these studies have shown an excellent correlation between activity and specific binding (Catterall et al., 1981; and see below). In the context of this discussion, however, it is important to note that in one of the first such applications of [3H]BTX-B (Brown et al., 1981), both BTX-A and dihydrobatrachotoxin were found to inhibit the binding of [3H]BTX-B with affinities only slightly less than that of BTX, even though these derivatives are poor depolarizing agents. In a separate study, BTX-A failed to antagonize responses to BTX in experiments in brain slices (Huang et al., 1972).

89

BATRACHOTOXIN

As mentioned previously, a large body of data indicates that the lipidsoluble neurotoxins all interact competitively at the same site on the voltagesensitive sodium channel (reviewed in Catterall, 1980). It is therefore instructive to compare structural features within the group in order to identify potentially important commonalities. Comparison of the crystal structures of aconitine and veratridine with that of BTX prompted Codding (1982, 1983) to extend the oxygen triad model first proposed by Masutani et al. (1981) to account for activity in a large series of grayanotoxins. This model proposes that a critical arrangement of three oxygen atoms is necessary for activity at the voltage-sensitive sodium channel. Figure 2 presents again the structures of BTX, veratridine, grayanotoxin, and aconitine with the three oxygens in each molecule indicated. Overlapping the crystal structures shows that a particular alignment can be found wherein each of the oxygen triads occupies a similar geometric arrangement. In each of the alkaloids, this triangle of oxygens is within 4-6 A of the nitrogen atom. Kosower (1983) has suggested that the function of the oxygen triad could be to complex a critical lysine ammonium moiety in the sodium-channel protein and that the neurotoxin amino groups could interact with a putative “exo-channel anion’’ normally involved in binding the lysine ammonium group.

Batrochotoxin

Verotridine

*OH

H . ,’u

YeOCOCH3 hru

Aconitine

HO r OH

Groyonotoxin I

FIG. 2. The oxygen triad model. The structures of the lipid-soluble toxins are shown with the three key oxygen atoms in each molecule indicated by a star.

90

GEORGE B. BROWN

The oxygen triad model has not been extensively tested, but some results, both pro and con, address its appropriateness. It does draw attention to a structural similarity among otherwise fairly diverse compounds with similar biological effects. Destruction of the oxygen triad in BTX by reduction of the 3-9 hemiketal results in marked loss of activity as predicted by the model. O n the other hand, veratroylzygadenine, which lacks the 12a and 17a hydroxyls of the veratridine oxygen triad, was found to be equipotent with veratridine in stimulating 22Na influx in neuroblastoma cells (Honerjager et al., 1982). As noted by Codding (1983), the model would suggest that the veratroyl moiety of veratridine is irrelevant to activity. Honerjager et al. (1982), however, found that veracevine, which lacks the veratroyl group of veratridine but maintains the oxygen triad, was ineffective in stimulating Na+ flux even at a concentration of 1 mM. Similarly, the importance of the 20~-esterregion of BTX is quite evident, but not part of the model which would predict, for example, that BTX-A would retain activity. BTX-A is a very poor depolarizing agent, but recall that it may still bind with relatively high affinity even though it fails to substantially activate the sodium channel. At least partial relief from these somewhat disparate observations may be found if one considers that the binding sites for this group of toxins are not identical, but only overlapping to the extent that they can adequately account for the competitive nature of their interactions and similar effects. Such a multipoint binding site model is reinforced by the observation mentioned earlier that veratridine will depolarize nerve and muscle preparations from the frog P. aurotmiu, albeit at reduced efficacy, even though BTX is completely inactive. Clearly, further work remains to be done in this important area. It is also clear that, in doing such work, a distinction must be drawn between binding affinity and efficacy, as several examples exist, some of which have been pointed out above, where these two do not vary in parallel.

B. THEPROTONATED FORMOF BTX Is ACTIVE Warnick et al. (1975) examined the effects of p H on the ability of batrachotoxin to depolarize rat diaphragm muscle and found that the potency increased on changing the pH of the medium from 6.0 to 7.2 to 9.0. Since BTX was thought to have a pK, of 7.5 (Marki and Witkop, 1963), it was concluded that the nonprotonated form of the toxin was the more active species. Bartels-Bernal et al. (1977) investigated the effects of p H on BTX-induced depolarization of electroplax from Electrophorus electricus. It was shown that, at pH 6.1, BTX had no effect on the action potential during repeated

BATRACHOTOXIN

91

stimulation. Changing the pH to 8.5 immediately resulted in a progressive prolongation of the action potential, which was reversed upon changing the p H of the bathing medium back to pH 6.1 as would be predicted if nonprotonated BTX were the more active species. However, this same phenomenon was observed when the quaternary methyl analog of BTX was substituted for BTX. Since the quaternary analog remains unchanged between these p H values, the results suggest instead that the macromolecular groups that interact with the toxin have pKa values in this range and that protonation of these groups leads to either reduced binding or reduced efficacy of BTX. To further investigate this relationship Brown and Daly (1981) measured the binding of [3H]BTX-B in broken membrane preparations of mouse cerebral cortex as a function of pH in the range 4-9. Binding measurements were carried out at 0-5OC in the absence of scorpion toxin. The resulting p H profile for specific binding is described by a bell-shaped curve wherein binding is negligible below pH 6 , maximal at pH 8.5, and approximately 66% of maximum at pH 9. Computer analysis of the data using a procedure developed by Cleland (1979) suggested that titration of two distinct ionizable groups with pKa values of 7.7 and 8.8 can account for this profile. Also in this study, the pK, of BTX was determined by titrimetric methods to be 8.2, which was considered to be in reasonable agreement with the pKa of 8.8 extracted from the pH profile, since the pK, by titrimetric methods was determined at higher temperature (25OC) and in a medium of lower dielectric constant than that of the binding assay. These considerations led to two possible interpretations of the data: (1) the protonated form of BTX-B is significantly more active than the nonprotonated form, and the protonation of a sodium-channel residue with pK, 7.7 produced a blockade of binding; or (2) both forms of BTX-B are equally active and there are two titratable residues of the sodium channel which account for the pH dependence of specific toxic binding. Continuing studies have lent support to the first interpretation, that the protonated form of BTX is the most active species ( G . B. Brown, unpublished data, 1987). The pH profile for specific [3H]BTX-B binding was determined again, only in this instance binding to synaptoneurosomes from mouse cerebral cortex was measured at 25 OC in the presence of a saturating concentration of scorpion toxin to increase the affinity of BTX-B binding. The resulting bell-shaped titration curve is shown in Fig. 3. The curve is shifted approximately 0.5 pH units to the left but is otherwise identical to the titration curve for toxin binding to mouse cerebral cortex at 0-5% in the absence of scorpion toxin (Brown and Daly, 1981). A shift along the pH axis is to be expected as a consequence of the temperature differential. The curve may be accounted for by two titratable residues with pK,s of 7.2 and

92

G E O R G E B. BROWN

7

6

5

8

10

9

PH

B "

2 I 5 0

c

.-c 2 2

-m m

(r

cn

c .--0

z c 1

-m 0 .-I 0 E LL

0 5

6

7

8

9

10

PH FIG 3 . T h e p H dependence of batrachotoxinin A benzoate binding. (A) Mouse brain synaptoneurosomes were equilibrated with 15 nM (3H]BTX-Bat the indicated pHs. Nonspecific hinding was determined from parallel incubations containing an excess of veratridine and has been subtracted from the data. T h e resulting specific binding at each pH is expressed as a fraction of that found at p H 7.5, where specific binding is maximal. (B) In this panel, the normal titration c u n e shown in (A) for specific BTX-B binding (filled squares) is compared to two separate superimposed conditions. T h e open squares show the results of holding the concentration of the

BATRACHOTOXIN

93

8.2, associated with the left and right arms ofthe curve, respectively. Since the pK, of BTX-B is known to be 8.2 at 25 OC, these data, as before, are consistent with the assignment of protonated BTX-B as the active species and the involvement of a sodium-channel residue with pKa 7.2 as a determinant of BTX-B binding. In order to test this possibility further, additional titrations were performed in which the concentration of either the protonated form or the nonprotonated form of BTX-B was held constant at all pHs (using a value of 8.2 for the pKa of BTX-B and calculating the appropriate total toxin concentration from the Henderson-Hasselbalch equation). As shown in Fig. 3, under conditions in which protonated BTX-B was held constant (open squares), the concentration of nonprotonated BTX-B increased by a factor of 30 between pH 7.5 and p H 9.0, yet specific binding increased by only a factor of 2. This result is consistent with the assignment of protonated BTX-B as the active species. In this case the slight increase in specific binding is a reflection of deprotonation of a sodiumchannel residue with pKa 7.2. This conclusion is reinforced by the experiment in which the concentration of nonprotonated BTX-B was held constant between pH 7.5 and p H 7 .O (open circle). If nonprotonated BTX-B were the active species and protonation of a sodium-channel residue with pKa 7.2 blocked binding, one would expect to see a decrease in specific [3H]BTX-Bbinding on going from pH 7.5 to pH 7.0 in this experiment. The finding that binding is essentially unchanged is consistent with the reciprocal effects of an increasing concentration of the active, protonated BTX-B species (as the nonprotonated form is held constant) concomitant with decreasing the binding affinity by protonation of a channel residue with pKa 7.2. Specific amino acid functional group modification experiments have focused attention on the possible involvement of a histidine or cysteine residue, the protonation of which results in inhibition of specific binding (Brown and Daly, 1981, and unpublished data.

C . MICROENVIRONMENT OF THE BTX BINDING SITE Additional information concerning the nature of the BTX binding site has been gleaned from studies using an active fluorescent analog of BTX,

protonated form of BTX-B constant at the pH 7.5 level as the pH is varied, and the open circle demonstrates the effect of maintaining the concentration of the nonprotonated form constant at the pH 7.5 level when the assay is carried out at pH 7.0. The total concentrations of ['HIBTX-B in each assay necessary to obtain these conditions were calculated from the Henderson-Hasselbalch equation using the experimentally determined value of 8.2 for the pKa of BTXB. As discussed in the text, these results are incompatible with preferential binding of the nonprotonated form of BTX-B.

94

GEORGE B. BROWN

batrachotoxinin A 20a-N-methylanthranilate(BTX-NMA). This compound, synthesized by reaction of batrachotoxinin A with N-methyl isatoic anhydride, retains biological activity and has been used successfully to visualize voltagesensitive sodium channels at mammalian nodes of Ranvier (Brown and Bradley, 1985). The biological properties are illustrated in Fig. 4. Free BTXNMA has an absorption maximum at 350 nm and an emission maximum at 425 nm. Angelides and Brown (1984) have evaluated the spectroscopic properties of BTX-NMA bound to the sodium channel. When BTX-NMA is added to rat brain synaptosomes alone, a 20-nm blue shift in the fluorescence emission maximum and a 4-fold enhancement are observed. The emission spectrum of the N-methylanthraniloyl moiety is highly dependent upon its microenvironment (Angelides, 1981), and this blue shift and spectral enhancement indicates that BTX-NMA under these conditions is located in a very hydrophobic environment. However, only a small fraction of the fluorescence emission was reduced when 1.8 pA4 BTX-B was included to displace specifically bound BTX-NMA, showing that the blue-shifted signal was due primarily to nonspecifically bound ligand. When BTX-NMA was added to synaptosomes which were pre-equilibrated with 1 scorpion toxin from Leiurus quinquestriatuc to enhance the specific binding affinity, an 8-fold increase in the fluorescence emission and a spectral shift of 10 nm to the red from the emission spectrum of the free BTX-NMA were observed. Of this signal, more than 70% could be displaced by 1.8 pA4 BTX-B, demonstrating that it arose from specific binding to the sodium-channel site. These findings are summarized graphically in Fig. 5. The dramatic 30-nm red shift to lower energy for the signal of specifically bound BTX-NMA from that of nonspecifically bound ligand is compatible with a model for toxin binding involving a redistribution from a hydrophobic environment, most probably represented by the membrane lipid, into a more hydrophilic region associated with the sodium-channel protein. It is also possible that BTX-NMA binds peripherally to protein domains with major exposure to lipid hydrocarbons. Conformational changes induced by scorpion toxin binding might then be reflected at this site, resulting in alteration of the microenvironment. Using the technique of fluorescence resonance energy transfer, Angelides and Brown (1984) find that BTX-NMA and the scorpion toxin are approximately 37 A apart when bound to their respective sites on the sodium channel, precluding a direct effect of scorpion toxin at the BTX site. Although it is not yet possible to define exactly the nature of the binding site for the lipid-soluble toxins, there do appear to be sufficient data to warrant a definition in general terms. Hydrophobic interactions are definitely an important factor, as the name of this class of toxins implies, yet there is now

1.0

-

-- z

0.8 0 8

-

0.6

-

0.4

-

0.2

-

\

0 -

FIG.4. Biological properties of batrachotoxinin A N-methylanthranilate. (A) The effects of 80 nMBTX-NMA on frog muscle action potentials are indicated. Experiments were carried out at 25 OC in Ringer’s solution. The toxin was diiuted into the buffer, and a random sampling of surface fibers was examined. (1) Control action potential recorded in normal Ringer’s solution before addition ofthe toxin. Stimulation at 1 Hz after 5 (2) and 15 (3) min ofexposure to 80 nMBTX-NMA. (4) Time course of 80 nMBTX-NMA (0) and 80 nMBTX-B ( 0 )induced depolarization at 25 OC of surface fibers of frog sartorius muscle. (B) The displacement of 7 nM [’HIBTX-B by BTX-B ( 0 )and BTX-NMA (m) on rat brain synaptosomesin the presence of 50 pg of crude Lcium venom is depicted. The inset shows a logit-log plot of the specific binding of BTX-NMA. (From Angelides and Brown, 1984.)

96

GEORGE B. BROWN

loo

I

10

385

425 465 505 W A V E L E N G T H (nm)

545

FIG 5. Fluorescence emission spectra of BTX-NMA. This figure depicts the corrected fluorescence emission spectra (excitation, 345 nm, 4-nm band passes) of 6 nM BTX-NMA free (-) and bound (- . -) to synaptosomal membranes (8.5 mg of proteinhl). The [SH]BTX-B binding capacity of these membranes was 1.9 pmollmg synaptosomal protein. Also shown are the fluorescence emission spectra of bound BTX-NMA in the presence of 1 pkftoxin V from L. p i n pltestriatus (---) and with 1.8 /.& BTX-B and 1pkf toxin V (. .) to displace specifically bound BTX-NMA. (From Angelides and Brown, 1984.)

..

ample evidence to point out the significance of more polar moieties. The oxygen triad model may indeed be relevant, indicating the importance of a particular arrangement of oxygens capable of hydrogen bonding interactions. Evidence that it is the protonated form of BTX that binds most avidly would seem to require a hydrophilic counterpart in the sodium-channel binding site for interaction, and this notion is supported by the observed red shift in the emission spectrum of BTX-NMA upon high-affinity binding. For BTX and its analogs, and perhaps for veratridine as well, other data draw attention to aromatic interactions with stringent steric constraints unlikely to be imparted by a hydrocarbon environment. Earlier electrophysiological studies (Albuquerque et ul., 1971) and more recent work with specific amino acid modifications (Brown and Daly, 1981)have raised the possibility of a role for cysteine or histidine residues, respectively. However, it should be pointed out that

BATRACHOTOXIN

97

these data do not require that the residue(s) be involved directly in the binding site of BTX. These residues could conceivably be located some distance from the binding site and still account for the observed effects by a mechanism involving conformational change in the protein consequent to modification. Taken together, these considerations, along with the additional information derived from studies with a photoactivatable BTX analog to be discussed below, prompt me to forward my own favorite hypothesis that the binding site for the lipid-soluble toxins lies at the interface of protein and lipid within the level of the hydrocarbon chains in a relatively inaccessible fold of the channel protein. In this view, both hydrophobic and hydrophilic interactions are required for binding and activity. Whatever the exact nature of this site is, the next section reinforces the notion derived from electrophysiological studies that this site is in a critical area relating to function, and shows that it is linked, directly or indirectly, to at least five other distinct binding domains of the voltage-sensitive sodium channel.

IV. Interactions wlth Other Sodium-Channel Neurotoxlns and Llgands

A. (Y-SCORPION TOXINS AND SEAANEMONE TOXINS The a-scorpion toxins and sea anemone toxins are basic polypeptides of approximate molecular weight 6000 to 7000 and 5000, respectively (Romey et at., 1976; Rochat et al., 1979; Beress, 1982). The effects of both of these groups of toxins on sodium-channel function are quite similar, although the details may vary somewhat depending on the particular toxin or the preparation in which it is tested (Koppenhoffer and Schmidt, 1968; Wang and Strichartz, 1983; Neumke et al., 1985; Nonner, 1979; Pelhate et al., 1984; Hu et at., 1983; Okamoto et al., 1977). In general, on binding to a site accessible from the extracellular side only, these toxins act to prolong the action potential by slowing down the process of inactivation. There is little effect on the process of activation, in contrast to the action of the P-scorpion toxins, a second class of scorpion toxins which may be found in the same venoms as the a-toxins (Cahalan, 1975; Wheeler et al., 1983; Couraud et at., 1982). In and of themselves, these polypeptide a-toxins are therefore poor depolarizing agents. Sea anemone toxins and a-scorpion toxins do not share significant sequence homologies, yet their similar electrophysiological effects and the

98

GEORGE B. BROWN

observation by direct binding measurements that they interact competitively in a variety of preparations (Ray et al., 1978; Jover et al., 1978; Catterall and Beress, 1978; Couraud et a l . , 1978; Catterall, 1979) suggest that they share a common binding site on the sodium channel. Of particular interest here with regard to function is the finding of Catterall and his collaborators that a-scorpion toxins and anemone toxins enhance the ability of the lipid-soluble neurotoxins to stimulate 22Na+flux (Catterall, 1975, 1976, 1977b; Tamkun and Catterall 1980). In the presence of toxin V from L. quinquestriatw, the affinity of the full activator BTX is increased 13-fold, from Kd = 700 nM to Kd = 50nM. For the partial activators veratridine and aconitine an increase in affinity is observed, but it is much less prominent. The predominant effect on these two toxins is revealed by an increase in the maximum stimulation of the rate of ion flux, i.e., their conversion to full activators. Catterall has proposed an allosteric model to account for this heterotropic positive cooperativity between the a-toxins and the lipid-soluble toxins (Catterall, 1977b). In this view, which is based on the classic theoretical description of Monod et al. (1965), the sodium channel may be assumed to exist in two states, active and inactive. The lipid-soluble toxins can bind to both states but with varying affinities. Thus, the full activator BTX binds with much greater affinity to the active state and pulls the equilibrium essentially completely in that direction, whereas the partial activators exhibit a much smaller ratio between the affinities for the active and inactive states. At equilibrium a significant fraction of the sodium channels then remain in the inactivated state with partial agonist bound. Phenomenologically, the effect of scorpion toxin is then qualitatively accounted for by an induced change in the equilibrium allosteric constant describing the transition between the two states. Since scorpion toxin acts to decrease the rate of inactivation, consideration has also been given to the possibility that the basis of this interaction may lie in the block of inactivation (Krueger and Blaustein, 1980). If this were the case, any manipulation which blocks inactivation should be accompanied by an increase in the potency of the lipid-soluble toxins. However, other agents which are known to block inactivation, such as chloramine T and NiZ+,have not been found to mimic the effects of the a-polypeptide toxins on the depolarization of frog sciatic nerve induced by BTX (Rando et al., 1986). The conformational changes that underlie the interaction between binding of these two classes of neurotoxins must therefore be more complex. Some indication of the extent of the conformational changes has been provided by the fluorescence resonance energy transfer studies by Angelides and Nutter (1983), who find that in rat brain synaptosomes the distance separating fluorescent derivatives of tetrodotoxin and L. guinquestriutus toxin V bound to their respective sites on the sodium channel increases from 35 to 42A upon binding of BTX. As

BATRACHOTOXIN

99

mentioned previously, the distance separating the BTX and scorpion toxin sites is approximately 37A, so that these conformational changes must be occurring through subunit-subunit interactions or by through-bond interactions in a single subunit over significant distances on the molecular scale (Brown and Angelides, 1984). Additional evidence for an allosteric interaction between the binding of a-scorpion toxin and BTX has been obtained by direct binding studies. As predicted by the allosteric model, BTX was found to increase the binding affinity of an 1251-labeledderivative of L. quinquestriatw toxin V to sodium channels in neuroblastoma cells (Catterall, 1977a). However, the observed 2-fold decrease in the K,, constitutes a fairly weak interaction, and this was only seen in depolarized preparations where the binding affinity of a-scorpion toxin is much reduced relative to that at normal resting membrane potentials. Once again, these findings suggest that the simple two-state model is less than adequate to fully account for the allosteric interaction. In examining the opposite side of this question, Catterall et al. (1981) found that, in agreement with the model, the affinity of [3H]BTX-Bbinding was increased 10-fold in the presence of the a-scorpion toxin. Not only did these results lend additional support to the conformational coupling between these two sites, but they provided the basis for a valuable and reliable filtration assay for direct binding studies of this site using [3H]BTX-Bas a probe. In the absence of scorpion toxin, the nonspecific binding component is too great, and the dissociation rate too fast, to permit convenient measurement of [3H]BTXB binding (Brown et al., 1981). In the presence of scorpion toxin the half-time for dissociation of specifically bound BTX-B from rat brain synaptosomes is increased to 60 min at 36 OC, and specific binding reflects the great majority of total bound label.

B , LOCALANESTHETICS, ANTICONVULSANTS, AND RELATED COMPOUNDS Many studies have revealed that local anesthetics antagonize the depolarizing effect of BTX and, conversely, that BTX can decrease the potency of local anesthetics, in an apparently competitive manner (Khodorov et al., 1975; Albuquerque et al., 1976; Revenko et a l . , 1982; Catterall, 1981; Cahalan, 1978; Krueger and Blaustein, 1980; Khodorov 1978; Huang and Ehrenstein, 1981). These studies, however, did not provide any specific indication of whether these effects were mediated by interactions at the same or different sites. With the availaWity of [3H]BTX-B and a reliable assay, it became possible to test these potential interactions more directly. Creveling et al. (1983) examined the ability of a series of local anesthetics of varying

100

GEORGE B. BROWN

potencies to displace specifically bound [3H]BTX-B from sodium channels in guinea pig cerebral cortex synaptoneurosomes. The results of these studies are shown in Fig. 6 . Analysis of the binding curves showed that the local anesthetics decreased the affinity of BTX-B binding without affecting the maximum number of binding sites, consistent with competitive inhibition. The apparent competitive nature of the inhibition allowed calculation of the inhibition constants K, from the IC50 values of the displacement. For a series of seven local anesthetics, these K, values were then compared to the Kis for inhibition of BTX-induced depolarization as shown in Table 11. Even though the K,s for blockade of BTX-induced depolarization were obtained under different conditions than those for inhibition of binding, the agreement between the two was good, and the rank order of potency for the two measures was the same with the exception of lidocaine ethiodide. This compound was found to be 18 times more potent in displacing BTX-B than in blocking depolarization. McNeal d al. (1985) have recently enlarged this data base by screening some 150 different compounds for their ability to displace specifically

I

1.o

1

I

1

, 6 1 1

1 1 I I I

10

LOCAL ANESTHETIC,

loo

I loo0

pM

FIG. 6. Inhibition of binding of [3H]BTX-B to a vesicular preparation from guinea pig cerebral cortex by local anesthetics. T h c binding of [3H]BTX-B(43 “M) in the presence of increasing concentrations of several local anesthetics is plotted as a percentage of [3H]BTX-Bbinding in the absence of local anesthetic versus the concentration of local anesthetic. Each data point represents the mean of three measurements. T h e Ki values are presented in Table 11, calculated from the concentration of local anesthetic yielding a 50% reduction in specific binding. From left to right the anesthetics tested were dibucaine (O-O), tetracaine ( 0 - O ) , QX-572 (A-A), hupivacaine methiodide (A---A), bupivacaine ( O--Q1), diphenhydramine (.--B), piperoO ) , lidocaine ethiodide (A-A), procaine (A-A), lidocaine (0-O), cocaine ( 0 caine ( 0-El),and benzocaine.).-.( (From Creveling el d.,1983.)

101

BATRACHOTOXIN TABLE I1 INHIBITION OF COMPARISON OF THE &S FOR LOCALANESTHETIC [3H]BTX-B BINDING AND BTX-INDUCED DEPOLARIZATIOV

Local anesthetic

Inhibition of BTX-B binding, K. ( p M )

Inhibition of BTX-induced depolarization, K , ( p M )

Dibucaine Tetracaine QX-572 Bupivacaine methiodide Bupivacaine Diphenhydramine Piperocaine Cocaine Diphenhydramine methiodide Lidocaine ethiodide Procaine Lidocaine Piperocaine methiodide Benzocaine

0.63 1.5 1.7 2.1 2.4 2.7 5.8 22 29 43 49 110 160 410

0.94 3.3 3.8 ND~ ND 10 19 26 ND 780 ND ND ND ND

'The K,s for inhibition of binding were determined from curves of the displacement of YHIBTX-B in the presence of scorpion venom (0.12 mg/ml) and 1.0 pM T T X in 30 min from guinea pig synaptoneurosomes. The values for nonspecific binding in the presence of 300 pM veratridine were subtracted from the total binding. The inhibition of BTX-induced depolarization of guinea pig synaptoneurosomes was determined by measuring the equilibrium distribution of [3H]TPMP' (86 p M ) at 30 min. The displacement to the right of dose-response curves for BTX at concentrations ranging from 1 to 100 nM were determined at three different concentrations of local anesthetics. The K,s were estimated by the method of least squares from replots of the apparent values and from Dixon plots. (From Creveling et al., 1983.) *Not determined.

bound BTX-B. The compounds included representatives of local anesthetics, catecholamine, histamine, serotonin, adenosine, y-aminobutyric acid (GABA), glycine, acetylcholine and calcium antagonists, tranquilizers, antidepressants, barbiturates, anticonvulsants, steroids, vasodilators, antiinflammatories, anticoagulants, and analgesics. The inhibition constants were in general agreement with the known local anesthetic properties of many of these drugs, leading the authors to suggest that this assay should be useful as a quantitative screening procedure for local anesthetic activity of new drugs and research agents. An excellent correlation between the ability of several local anesthetics to block sodium currents in electrophysiological experiments and the potency in inhibiting binding of [SH]BTX-B to rat brain synaptosomes was also reported by Postma and Catterall (1984). These workers extended the analysis of the mechanism of action by evaluating the effects of the local

102

GEORGE B . BROWN

anesthetics on the kinetics of BTX-B binding. Although there was no effect on the association rate, tetracaine, prilocaine, tocaimide, and lidocaine each increased the dissociation rate constant for BTX-B binding by amounts ranging from 2.3-fold for tetracaine to 8.5-fold for lidocaine. These results are incompatible with a model involving competition between local anesthetics and BTX-B for the same binding site on the sodium channel, but could be modeled by a competitive allosteric inhibition scheme as presented by Postma and Catterall (1984). Similar results have been reported for the anticonvulsant drugs diphenylhydantoin and carbamazepine. These two agents, used clinically in the treatment of grand mal and partial seizures, have been previously shown by electrophysiological methods to block sodium conductance in a variety of nonmammalian preparations at clinically relevant doses (Lipicky et al., 1972; Schwarz and Vogel, 1977; Suria and Killam, 1973; Schauf et al., 1974). Like the local anesthetics, their mechanism of action may be considered to involve rapid access to a specific binding site while the channel is in an open configuration followed by preferential binding to and resulting stabilization of an inactivated state of the sodium channel (Willow et al., 1985). Also like the local anesthetics, diphenylhydantoin and carbamazepine competitively inhibit the binding of [3H]BTX-B to sodium channels in rat brain synaptosomes with ICSOvalues in the mid-range of their respective therapeutic concentrations (Willow and Catterall, 1982). Continuing the analogy, these anticonvulsants were found to increase the dissociation rate constant for BTX-B binding without an effect on either the association rate or the maximum number of BTX-B binding sites, once again suggesting the existence of an indirect allosteric relationship between the anticonvulsant and the BTX-B binding sites (Willow and Catterall, 1982).

C. PYRETHROID INSECTICIDES Synthetic pyrethroids, structurally based upon the esters of the naturally occurring chrysanthemic acid [2.2 -dimethyl-3-(2-methylpropeny1)cyclopropane carboxylic acid], have found widespread use as potent insecticides (Elliott and Janes, 1978). These compounds are also known to have profound neurotoxic effects in mammals (Verschoyle and Barnes, 1972). The early synthetic pyrethroids, or type I pyrethroids, of which permethrin is prototypical (Fig. 7,11), and the more recent type I1 pyrethroids, represented by deltamethrin and cypermethrin (Fig. 7,1 and 111), elicit related but distinguishable symptoms upon intoxication. As shown in Fig. 7, the major difference in these two types of compounds rests in the presence or absence of the a-cyan0 group on the

103

BATRACHOTOXIN

-1. DELTAMETHRIN II. PERMETHRIN 111. CYPERMETHRIN

X

R

Br

CN H CN

CI CI

FIG. 7 . Structures of pyrethroid insecticides. The asterisk (*) represents asymmetric centers.

3-phenoxybenzyl alcohol portion. Structure-activity studies in a number of laboratories have also revealed strict stereochemical requirements for pyrethroid toxicity (Lawrence and Casida, 1982; Soderlund, 1979; Elliott et al., 1974). Within the cyclopropane carboxylate portion for all pyrethroids, only compounds having the cyclopropane C1 in the R configuration are active, whereas the a-cyano-bearing carbon S configuration yields a more toxic enantiomer than the R configuration for type I1 pyrethroids. Electrophysiological studies have provided strong evidence for an effect of both type I and type I1 pyrethroids at low micromolar concentrations on the voltage-sensitive sodium channel (Duclohier and Georgescauld, 1979; Vijverberg et al., 1982, 1983; Lund and Narahashi, 1983). Additional data supporting a sodium-channel site as a primary target for pyrethroids in mammalian neuronal tissue have been reported. Jacques et al. (1980) observed a synergistic enhancement by some pyrethroids of 22Na+uptake mediated by batrachotoxin, veratridine, and a-polypeptide neurotoxins in mouse neuroblastoma cells. Similar results were reported by Ghiasuddin and Soderlund (1985) using a rat brain synaptosomal preparation. Further, Bloomquist and Miller (1986) found that houseflies resistant to pyrethroids and DDT were also resistant to aconitine. Based upon these findings, Brown and his collaborators recently investigated the interaction of a series of pyrethroid insecticides with [3H]BTX-B binding in rat brain synaptoneurosome preparations (Brown and Olsen, 1984; Brown d al., 1987). Permethrin was found to have a weakly inhibitory effect on specific BTX-B binding, and deltamethrin, a potent member of the type I1 pyrethroids, increased the affinity of BTX-B binding %fold without altering the maximum number of binding sites. These findings were then extended to the complete set of eight stereoisomers of cypermethrin. Fig. 8 shows that an excellent correlation exists between the

104

GEORGE B. BROWN 400

RRS (100,000)

300

+ m

0

Piij

RRR (1400)

-!SRR(1100)

0

E

SRS(4300)

200

:

0-

-e

*

v) a-

5s

I

1

I 11111

10

I

I

10

I

I11111

I I I I I I

1

0

- 1

10

I

RSS (2)

+

SSS(1)

I I I

2 10

RSR(3)

-& 100

0

SSR (500)

3 10

Effector Concentratlon (Mlcromolar)

FIG.8. The effects of cypermethrin stereoisomers on BTX-B binding. Varying concentrations of the eight possible stereoisomers of cypermethrin ranging from 0.5 to 150 pi4 were included in a standard BTX-B binding assay and the resulting specific binding was recorded as a percentage of that in control assays which contained no pyrethroid. The right-hand margin indicates the stereochemistry for each compound (with reference to Fig. 7, asymmetric centers are listed left to right) and the corresponding felative insecticidal potency derived from assay of contact activity against fourth instar larvae of Hcliodis uirescms. (From Brown cf nl., 1987.)

ability of these stereoisomers to enhance binding of BTX-B and their insecticidal potency. Nontoxic isomers of this series were weakly inhibitory and, at high concentrations, even the active stereoisomers gave less than maximal enhancement. These results suggest that as a group the pyrethroids bind in a stereospecific manner to a distinct sodium-channel site resulting in a positively cooperative allosterism with BTX-B binding. However, they also share a nonspecific interaction, either with the sodium channel or with the membrane environment, leading to inhibition of binding. Whether or not enhancement

105

BATRACHOTOXIN

of binding is seen would then become a function of the relative difference between the specific and the nonspecific binding constants or the efficacy at these two sites.

D. TETRODOTOXIN AND SAXITOXIN For many years the blockade of voltage-sensitive sodium channels by the heterocyclic guanidinium compounds tetrodotoxin (TTX) and saxitoxin (STX) has been generally considered to be mechanistically uncomplicated with no significant interplay between their binding and channel-gating processes, conformations, or other sodium-channel neurotoxins (Ritchie and Rogart, 1977). Specifically, evidence that the binding site for the channel blockers and the BTX activator site were distinct and noninteracting has come from the inability of BTX to alter the binding of radiolabeled T T X or STX (Colquhoun et al., 1972; Catterall and Morrow, 1978). The complementary question of the effect of T T X or STX on the binding of BTX could not be adequately addressed by electrophysiological methods or **Na+flux experiments since the actions of the two are mutually occlusive. In order to further test these relationships, Brown (1986) examined the effects of T T X and STX on the specific binding of [3H]BTX-B to a vesicular preparation of mouse brain cerebral cortex. Both channel blockers markedly inhibit binding of [3H]BTX-B in a concentration-dependent and noncompetitive manner. As shown in Fig. 9, this inhibition is strongly temperature dependent, being negligible at 37 O C and maximum at 18OC, the

a

cn

30 25 20 TEMPERATURE (“C)

I5

FIG.9. Temperature dependence of T T X inhibition of [SH]BTX-Bbinding. Specific binding of [JHIBTX-Bin mouse brain cortical synaptoneurosomes in the presence ( 0 )or absence ( x ) of 1 phf TTX is shown as a function of incubation temperature. All incubationswere carried out for 60 min using the same tissue preparation and a constant concentration of [SH]BTX-B.Temperature was controlled by immersion of the assay tubes in thermostated water baths. (From Brown, 1986.)

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GEORGE 8. BROWN

lowest temperature investigated. At 25 OC, saturating concentrations of TTX reduce the binding affinity of [3H]BTX-B by a factor of 3. The concentration dependence for T T X inhibition of both specific [3H]STX and [3H]BTX-B binding is identical (Fig. lo), showing that inhibition of [3H]BTX-B binding is a result of TTX/STX binding at their specific sodium-channel site. Consideration of the temperature dependence and analysis of the underlying thermodynamic parameters suggest that binding of T T X or STX is attended by a significant conformational change that is reflected at the BTX-B binding site. Several other recent findings serve to strengthen the idea of conformational or allosteric interactions between the TTX/STX and BTX sites on the sodium channel. Sodium channels reconstituted in planar lipid bilayers and modified by BTX are blocked by T T X and STX in a voltage-dependent fashion not observed in normal channels (Krueger et al., 1983; Moczydlowski et al., 1984b). This same phenomenon has been observed in BTX-modified channels in frog node of Ranvier (Rando and Strichartz, 1986). In addition, in lobster walking leg axons BTX caused a significant but modest 10% reduction in the specific binding of [3H]STXdue to a decrease in STX binding affinity (Strichartz et a!. , 1986).

CONCENTRATION OF TETRODOTOXIN (MI FIG 10. Comparison of the concentration dependence of T T X inhibition of [SH]saxitoxin and ('HIBTX-B binding to mouse brain cortical synaptoneurosornes. Synaptoneurosomes from the same tissue preparation were incubated with a constant concentration of either ['Hlsaxitoxin (1 nM, e ) or ['HJBTX-B (20 nM, 0)and the indicated concentrations of unlabeled T T X for 1 hr at 25%. Specific binding was determined by filtration assays. The results are expressed as percentage of maximal inhibition, defined as the inhibition in the presence of 1 pA4 unlabeled T T X . The resulting displacement curves are superposable, giving a Ko.5 value of approximately 20 nM for T T X inhibition of both [3H]saxitoxin and [3H]BTXI3 binding.

107

BATRACHOTOXIN

E. OTHER TOXINS Even though the armamentarium of naturally occurring neurotoxins with specific actions on the voltage-sensitive sodium channel is already impressive and apparently without equal, new toxins continue to be discovered at a steady pace. Many of these, still in the early stages of investigation, have shown evidence of allosteric interactions with the BTX binding site. Examples include ciguatoxin from the dinoflagellate Gumbierdiscus toxicus (Bidard et al., 1984), a polypeptide neurotoxin from the coral Goniopora (Gonoi et al., 1986), and the polyether Pychodiscus brevis toxin from the Florida red tide dinoflagellate of that name (Catterall and Risk, 1981; Sharkey et a l . , 1987). Pychdiscus 6raris toxin 2 was found in direct binding studies to act allosterically at a new sodium channel site to increase the affinity of BTX-B binding in synergy with a-scorpion toxin and to increase the binding affinity of the @scorpion toxin I1 from Centruroides su&u su&us as well (Sharkey et al., 1987). To summarize the discussion of this section, the reader is referred to Table 111, where the interaction of these sodium-channel neurotoxins is delineated. In many of these interactions it becomes possible to discern a mechanistic pattern. Ligands that bind preferentially to an active state of the sodium channel stabilize that configuration and evoke an increase in the affinity of BTX binding. Conversely, agents that stabilize an inactive state produce inhibition of BTX-B binding. In general, these results can be modeled by a two-state allosteric system in which the effect of ligand binding can be interpreted as an alteration in the allosteric constant describing the transition between the states. However, we have also seen that the situation is apt to be more complex TABLE I11 ALLOSTERIC MODIFIERS OF BTX-B BINDING Compound

Effect

Reference

a-Scorpion toxin, sea anemone toxin

Increase affinity

Catterall ct al. (1981)

Local anesthetics

Decrease affinity

Creveling ct al. (1983); Postma and Catterall (1984)

Diphenylhydantoin and carbamazepine (anticonvulsants)

Decrease affinity

Willow and Catteral (1982)

a-Cyanopyrethroid insecticides

Increase affinity

Brown and Olsen (1984)

T T X and STX

Decrease affinity

Brown (1986)

Ciguatoxin Ptychodiscus brevis toxin

Increase affinity (?) Increase affinity

Gonoi ct al. (1986) Catterall and Risk (1981); Sharkey ct al. (1987)

108

GEORGE B. BROWN

than that. What is truly remarkable in all of this, however, is the great diversity of structures, each acting at a different site yet producing similar effects. The challenge remains to produce a model that can account for these findings. Since the lipid-soluble sodium-channel neurotoxins figure so prominently in these interactions, it will perhaps be instructive to consider the role of lipids in general in the next section.

V. Role of Lipid

A, SOLUBILIZATION OF THE SODIUM CHANNEL Some of the first observations directly implicating lipids in the normal physiology of the voltage-sensitive sodium channel arose from attempts to solubilize the protein with nonionic detergents as an initial step in its purification. Freshly solubilized material retains the ability to bind TTX and STX, but this property is rapidly lost with time unless the preparation is stabilized with a low concentration of phospholipid (Agnew and Raftery, 1979). Sodium channels also lose the ability to bind a-scorpion toxin and BTX, completely and irrevocably, as long as the channel proteins remain solubilized (Catterall et al., 1979). In a demonstration that a membrane environment is required for normal binding of BTX, reconstitution of purified sodium channels into artificial membranes is accompanied by recovery of lipid-soluble toxin binding. BTX and veratridine are now used rather routinely to activate purified sodium channels incorporated from the solubilized state into liposomes (Talvenheimo et al., 1982; Tamkun et a l . , 1984; Weigele and Barchi, 1982; Tanaka et al., 1983; Rosenberg et a l . , 1984; Barchi and Tanaka, 1984; Kraner et al., 1985). In this context it is also interesting to note that Hartshorne et al. (1986) report that under their conditions the presence of BTX or veratridine is required for successful fusion of sodium channel-containing liposomes with planar lipid bilayers. In contrast to the result with BTX, scorpion toxin does not bind to sodium channels reconstituted into phosphatidylcholine vesicles (Talvenheimo et al. , 1982). Addition of brain lipids to the reconstitution mixture results in recovery of scorpion toxin binding, but the affinity is lower than normal by an order of magnitude (Tamkun et al., 1984). Feller et al. (1985) find that addition of phosphatidylethanolamine or phosphatidylserine to the reconstitution mixtures in defined ratios restores high-affinity scorpion toxin binding and voltage dependence similar to that observed in native membranes. Thus, both the binding of BTX and of polypeptide toxins is dependent upon a membrane environment, with the latter exhibiting specific requirements at the level of the

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lipid head group. Interestingly, even in the presence of brain lipids or optimum ratio of phosphatidylserine and phosphatidylethanolamine, the marked allosteric interactions that normally characterize BTX and scorpion toxin binding are not in evidence (Feller et al., 1985). Apparently some functions of the voltage-sensitive sodium channel that are reflected in the binding of BTX and a-scorpion toxin have not yet been successfully reconstituted.

B. PHOTOAFFINITY LABELING Further indication of the involvement of lipids in the binding of BTX comes from work with photoactivatable derivatives of this toxin. Soldatov et al. (1983) used a [3H]o-azidobenzoate derivative of totally synthetic dihydrobatrachotoxinin A to investigate the composition of the BTX binding site in rat brain synaptosomes. Following ultraviolet (UV) irradiation, no reproducible incorporation of specifically bound label into protein components could be detected, but at least 70% of the specific binding was found to be covalently attached to membrane lipids. In additional experiments, pretreatment of synaptosomes with phospholipase A2 blocked the specific binding of [3H]dihydro-BTX, reminiscent of a similar effect on the binding of T T X reported previously by Reed (1981). Since analogous treatments with phospholipases C and D did not block specific binding, Soldatov and his colleagues suggested that the acyl chains of membrane lipids, not the head groups, could constitute a portion of the toxin binding site. Brown (1985) prepared the tritiated analog [3H]batrachotoxinin A 20a-oazidobenzoate (BTX-OAB) by partial synthesis and found this compound to retain binding affinity and specificity similar to that for BTX itself. Binding experiments show that BTX-OAB in the presence of a-scorpion toxin binds to the BTX site of rat brain sodium channels with a Kd of 45 nM. In initial experiments to covalently label the binding site with this ligand, rat brain synaptoneurosomes which had been pre-equilibrated with [3H]BTX-OAB under various conditions were irradiated and the resulting distribution of specifically bound label examined by SDS-polyacrylamide gel electrophoresis. Of the initial specific binding, 10% was irreversibly incorporated into the preparation following imadiation. Of this l o % , 90% was found to be associated with a component of apparent molecular weight less than 10,000 on SDS gels and could be extracted with chloroform/methanol, suggesting a lipid origin, As expected from the hydrophobic nature of BTX-OAB, radioactivity from nonspecifically incorporated material also contributed to the lowmolecular-weight peak on the gels. It must be emphasized, however, that a

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large fraction of this peak was lost under all conditions where specific binding was blocked, including excess unlabeled BTX-OAB or veratridine in the incubation mixture and the elimination of a-scorpion toxin to lower specific binding affinity. Two other small peaks of M, = 260,000 and 50,000 apparently associated with specifically incorporated label have been occasionally observed in these gels. The significance of these peaks is uncertain due to the paucity of counts, but it may be of some interest to note that Angelides et al. (1985) have identified target sizes of 280,000 and 48,000 Da for BTX-B binding by radiation inactivation analysis. The experiments with these photoactivatable derivatives of BTX, preliminary as they are, reinforce the hypothesis that lipid is intimately involved in the structure of the binding site and that this site may lie at the interface of protein and lipid.

c. LIPIDASSOCIATION WITH PURIFIED S O D I U M C H A N N E L Purified sodium-channel preparations from electric eel, mammalian brain, and skeletal muscle all have in common a large, 260,000-Da glycoprotein termed the a-subunit which runs as a diffuse band on SDS gel electrophoresis. In the case of the sodium channel from E. electricus, this anomalous behavior is correlated with an abnormal free electrophoretic mobility due to the association of high amounts of detergent as if the a-subunit were exceptionally hydrophobic (Thornhill and Levinson, 1986). Detailed compositional studies have shown that this underlying hydrophobicity is a consequence of the presence of large amounts of lipid which are tightly associated or covalently attached to the a-subunit. Levinson et a!. (1986) find that, relative to protein, fully 3 % of the a-subunit mass is accounted for by covalently bound fatty acids of which palmitate and stearate are the major species. In addition to this covalently attached lipid, an additional 3-5% is found in tight, noncovalent association with the a-subunit and cannot be extracted with detergent or chloroform/methanol. Although it is now clear that a variety of cellular proteins are posttranslationally modified by esterification with fatty acid or phospholipid (Schmidt, 1983; Magee and Schlesinger, 1982; Low and Kincade, 1985; Burn and Burger, 1987), the large amount found in the sodiumchannel a-subunit is without precedence. According to Levinson et al. (1986), the covalently attached lipid corresponds to 25 acyl chains per subunit. From the data currently available it is impossible to know exactly what the function of sodium channel-associated lipid is. An attractive possibility would include a role for anchoring or orienting the protein in the membrane in a manner that positions the voltage sensor(s) in the appropriate environment. If

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this were the case, then one might predict that any manipulation that perturbed these lipid-protein interactions would be reflected by changes in the voltage dependence of channel gating as is seen under the action of BTX and the other lipid-soluble neurotoxins. At the risk of stating the obvious, the sodium channel must function in a membrane environment and, if the results discussed above are any indication, further attention to the area of lipid-protein interactions in sodium-channel function would seem likely to yield new and valuable information. VI. Concluding Remarks

The perserverant reader will hopefully come away from the discussions of this review with a sense that the voltage-sensitive sodium channel is a remarkably dynamic structure whose normal, and abnormal, gating function may be accompanied by a spectrum of changes in configuration. Batrachotoxin and its analogs in particular, and the lipid-soluble sodium-channel neurotoxins in general, have been most useful tools in developing an appreciation of this phenomenon, providing, as it were, a window on the allosteric nature of the voltage-sensitive sodium channel. From the electrophysiological studies we have seen that no functional characteristic of the sodium channel escapes modification upon the binding of BTX. Does this mean that the control elements for each of these characteristics are in the immediate vicinity of the BTX binding site, or does the binding of BTX result in conformational changes which extend broadly and reflect upon control elements in different domains of the channel protein? Results from pharmacological and biochemical studies have come down solidly on the side of the latter possibility. Binding studies have demonstrated that the BTX site is allosterically coupled to neurotoxin binding sites in at least five other distinct channel domains. Some of these toxins bind only from the extracellular side, and some may access their binding sites more readily from the intracellular side, whereas the BTX site seems to lie in the hydrophobic interior of the membrane. Fluorescence resonance energy transfer measurements suggest that most of these different binding domains are separated from each other by tens of Angstroms. The emerging picture is therefore one in which the structural perturbations of specific, discrete events, such as the movements of gating charges or the binding of a neurotoxin, are distributed almost globally throughout the sodium-channel complex. The experimental work with BTX and its congeners in the last 15 years has established that its binding site is indeed special, and a better

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understanding of the nature of the site will almost certainly provide additional insights into the operation of the channel. The amino acid sequence(s) of the sodium channel from electric eel and rat brain is now known (Noda et al., 1984, 1986), and elegant models have already begun to appear to account for function within this framework (Numa and Noda, 1986; Greenblatt et al., 1985; Kosower, 1985). For the future, new studies using site specific mutagenesis, group-specific modification and affinity labeling, and further biochemical studies are to be encouraged to gain a better understanding of the BTX binding site and its relationship to the rest of the channel protein(s) and to the membrane environment. Along with our existing knowledge of this key site, this information will help provide the guidelines and much-needed constraints necessary for the development of models which will provide us, in time, with a molecular explanation of the voltage-sensitive sodium channel.

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Rosenberg, R . L., Torniko, S. A,, and hgnew, W . S. (1984). Proc. Natl. Acad. Sci. U . S . A . 81, 1329- 1343. Schauf, C . L . , Davis, F. A , , and Marder, J. (1974). J. P h a m c o l . Exp. Ther. 189, 538-543. Schmidt, M . F. G . (1983). Curr. Top. Microbiol. Immunol. 102, 101-129. Schwarz, J . R . , and Vogel, W . (1977). Eur. J . Pharmacol. 44, 241-249. Seyama, I . , and Narahashi, T . (1981). J . Pharmacol. Exp. Ther. 219, 614-624. Sharkey, R . G . , Jover, E., Couraud, F . , Baden, D. G., and Catterall, W . A. (1987). Mol. Pharmacol. 31, 273-278. Soderlund, D. M . (1979). Pestic. Biochem. Physiol. 12, 38-48. Soldatov, N . , Prasolava, T . , Kovalenko, V . , Petrenko, A , , Grishin, E., and Ovchinnikov, Yu. (1983). In “Toxins as Tools in Neurochemistry” (F. Hucho and Yu. Ovchinnikov, eds.), pp, 47-58. De Gruyter, Berlin and New York. Strichartz, G. R . , Rando, T., Hall, S . , Gitschier, J , , Hall, L . , Magnani, B., and Hansen Bay, C . (1986). In “Tetrodotoxin, Saxitoxin and the Molecular Biology of the Sodium Channel” (S. R . Levinson and C . Y. Kao, eds.), pp. 96-112. New York Academy of Sciences, New York. Strichartz, G . , Rando, T . , and Wang, G . K. (1987). Annu. Reu. Neurosci. 10, 237-67. Suria, A , , and Killam, E. K. (1973). Adu. Neurol. 27, 563-575. Talvenheimo, J. A , , Tamkun, M. M., and Catterall, W . A. (1982). J . Biol. Chem. 257, 1 1868-1 187 1 . Tamkun, M . M . , and Catterall, W. A. (1980). Mol. Pharmacol. 19, 78-86. Tamkun, M . M . , Talvenhemio, J . A , , and Catterall, W . A. (1984). J . Biol. Chem. 259, 1676- 1688. Tanaka, J . C., Eccelston, J. F . , and Barchi, R . L. (1983). J . Biol. Chem. 258, 7519-7526. Thornhill, W. B., and Levinson, S. R . (1986). In “Tetrodotoxin, Saxitoxin and the Molecular Biology of the Sodium Channel” (C. Y. Kao and S. R . Levinson, eds.), pp. 356-363. Annals of the New York Academy of Sciences, New York. Tokuyama, T . , and Daly, J . (1983). Tetrahedron 39, 41. Tokuyama, T . , Daly, J., and Witkop, B. (1969). J . Am. Ckem. Soc. 91, 3931-3938. Ulbricht, W. (1969). Ergeb. Physiol. Biol. C h m . Exp. P h a m k o l . 25, 379-383. Verschoyle, R . D., and Barnes, J. M. (1972). Pestic. Biochem. Physiol. 2, 308-311. Vi,jverberg, H. P. M., Ruigt, G. S. F . , and van den Berken, J . (1982). Pestic. Biochem. Physiol. 18, 315-324. Vijverberg, H . P. M . , van der Zalm, J . M . , van Kleef, R . G. D. M., and van den Berken, J. (1983). Biockim. Biopkys. Acta 728, 73-82. Wang, G . , and Strichartz, G. R . (1983). Mol. Pharmaco/. 23, 519-533. Warnick, J . E., Albuquerque, E. X . , and Sansone, F. M . (1971). J . Pharmacol. Exp. Ther. 176, 497-510. Warnick, J. E., Albuquerque, E. X . , Onur, R . , Jansson, S.-E., Daly, J . W., Tokuyama, T., and Witkop, B. (1975). J . Pharmacol. Exp. Ther. 193, 232-245. Weigele, J. B., and Barchi, R . L. (1982). Proc. Natl. Acad. Sci. U . S . A . 79, 3651-3655. Wheeler. K. P., Watt, D. D., and Lazdunski, M . (1983). pflugers Arch. 397, 164-165. Willow, M . , and Catterall, W. A. (1982). Mol. Pharmacol. 22, 627-635. Willow, M . , Gonoi, T . , and Catterall, W . A. (1985). Mol. Pharmacol. 27, 549-558. Witkop, B., and Gossinger, E. (1982). Alkaloids 21, 139. Zubov, A. N . , Naumov, A. P., and Khodorov, B. I. (1983). Gen. Physiol. Biopkys. 2, 75-77.

N EUROTOXIN-BI N DI NG SITE ON THE ACETYLCHOLINE RECEPTOR By Thomas L. Lentz and Paul T. Wilson Department of Cell Biology Yale University School of Medicine New Haven, Connecticut 08510

I. 11. 111. IV.

V.

Introduction The Nicotinic AChR Structure and Function of Curaremimetic Neurotoxins Neurotoxin-Binding Site on the AChR A. Specificity for ct Subunit B. Proximity to Reducible Disulfide C. Binding of Neurotoxins to Proteolytic Fragments of the (Y Subunit D. Comparison of Amino Acid Sequences of a Subunits, Other Subunits, and Other AChR E. Binding of Neurotoxins to Synthetic Receptor Peptides F. Monoclonal Antibodies as Probes of the Neurotoxin-Binding Site G. Site-Directed Mutagenesis H. Models of the Cholinergic Binding Site Conclusions References

1. lntroductlon

The nicotinic acetylcholine receptor (AChR) at the neuromuscular junction transduces a chemical signal, the neurotransmitter acetylcholine released in response to an action potential at the motor nerve terminal, into an electrical event in the muscle cell that eventually activates the contraction process. In response to acetylcholine, the AChR undergoes a conformational change in which a cation-selective channel is opened, allowing sodium ions to enter and depolarize the cell. Essential functions of the receptor during this process are the binding of acetylcholine to the AChR, the coupling of the binding event to elements of the receptor comprising the channel, the opening of the channel to allow inward passage of cations, and the mechanisms that modulate these steps. The current rapid accumulation of physiological, biochemical, and structural information on the AChR makes it likely that the molecular mechanisms underlying these events will be elucidated 117 INTERNATIONAL REVIEW OF NEUROBIOLOGY, VOL. 29

Copyright 0 1988 by Academic Press, Inc. AII rights of =production in any form reserved.

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over the next several years. Identification of the acetylcholine-binding site on the AChR would increase understanding of the mechanism by which binding of the ligand leads to the changes in the receptor that result in opening of the channel. Localization of this site is greatly facilitated by the use of snake venom curaremimetic neurotoxins, which bind to the receptor with considerably higher affinity than acetylcholine. As will be discussed later, there is evidence that the toxin-binding site includes the acetylcholine-binding site so that use of the toxins as biological probes should permit characterization of the site on the AChR involved in the binding of acetylcholine. The use of toxins as probes along with techniques employing other ligands, peptide fragments, monoclonal antibodies, synthetic receptor peptides, and genetic engineering are yielding considerable information on the toxin-binding site. Here, current information on the localization of the curaremimetic neurotoxin-binding site on the primary amino acid sequence of the nicotinic AChR is reviewed.

II. The Nicotinic AChR

The structure and biochemistry of the AChR are reviewed briefly in this section. Several thorough reviews of the physiological, structural, and biochemical properties of the AChR are available (Karlin, 1980; Changeux, 1981 ; Conti-Tronconi and Raftery, 1982; Barrantes, 1983; Popot and Changeux, 1984; Hucho, 1986; McCarthy et a!. , 1986). Three major classes of ligands interacting with the AChR are agonists, competitive antagonists, and noncompetitive inhibitors or antagonists. Ligands that activate opening of the ion channel are agonists, while agents like the curaremimetic neurotoxins that competitively inhibit agonist binding and action are competitive antagonists. Noncompetitive inhibitors block the response to agonists apparently by sterically blocking the closure and passage of ions through the channel. The agonist-binding sites are allosterically coupled to the binding sites of noncompetitive inhibitors, as binding of agents to either site changes the binding characteristics of the other site (Popot and Changeux, 1984). The AChR has at least three functional states. In the resting or closed state, the affinity for agonists is relatively low and the channel is closed. In the active or open state, the acetylcholine-binding sites are occupied by agonist and the channel is open. In the desensitized state, the affinity for agonists is high but binding of agonist does not lead to channel opening. Binding of two molecules of agonist per receptor monomer is followed by channel opening (Dionne et a l . , 1978; Neubig and Cohen, 1980; Sine and Taylor, 1980; Hess et

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al., 1982). The processes of channel opening and desensitization involve transitions in the conformation of the receptor (Nachmansohn, 1955; Karlin, 1969; Magleby and Stevens, 1972; Conti-Tronconi and Raftery, 1982; McCarthy et al., 1986; Mielke et a l . , 1986). Further resolution of the open state has been achieved by means of patch-clamp techniques, which have shown that during the open state of about 1 msec, the receptor can undergo rapid transitions on a microsecond time scale between the open state and the closed state (Colquhoun and Sakmann, 1981). At high concentrations of agonist or on prolonged exposure to agonist, the receptor undergoes a transition on a time scale of seconds to the desensitized state. Because acetylcholine concentration in the synaptic cleft is high during transmission (approaching millimolar), the high-affinity sites on the receptor may be associated with desensitization. The low-affinity site could be the same as the high-affinity site which changes affinity upon binding of agonist, or it could be a separate site located elsewhere on the receptor. Attempts to find separate low-affinity sites, however, reveal the presence of only the two high-affinity sites (Strnad and Cohen, 1985). The AChR was the first neurotransmitter receptor to be isolated and characterized biochemically. This was facilitated by the fact that the receptor occurs in high concentration in the electric organs of certain types of fishes such as rays (Torpedo) and electric eels (Electrophoncr). These organs are composed of electrocytes which are modified muscle cells with extensive nerve contacts (Heuser and Salpeter, 1979). The AChR is an integral membrane protein occurring in high density on the crests of the junctional folds at the neuromuscular junction (15,000-25,000 toxin-binding sites per pm2) (Salpeter and Loring, 1985). By electron microscopy, the AChR is a rosette-like structure, 80-90 in diameter, composed of subunits surrounding a central pit (Heuser and Salpeter, 1979; Kistler et al., 1982). Within the membrane, the receptor is 11 nm in length and extends 5.5 nm on the synaptic side of the membrane and 1.5 nm on the cytoplasmic side (Ross et a l . , 1977; Kistler etal., 1982; Sealock, 1982; McCarthy et al., 1986). A central channel traverses the entire length of the molecule beginning as a wide funnel at the synaptic end and narrowing abruptly at the level of the bilayer surface (Brisson and Unwin, 1985). The channel behaves as though it contains an aqueous environment (Lewis and Stevens, 1983). The AChR from the electric organ of Torpedo californica was observed by sedimentation analysis to have an apparent molecular weight of about 250,000 (Reynolds and Karlin, 1978). It is composed of four types of polypeptide chains with apparent molecular weights of 40,000 (a),50,000 (p), 60,000 (y), and 65,000 (6) as determined by gel electrophoresis in sodium dodecyl sulfate (Weill et a l . , 1974; Lindstrom et al., 1979; Raftery et al., 1980). The subunits form a pentameric complex in the stoichiometry of a&6. The receptor from

A

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Torpedo occurs as a dimer cross-linked by a disulfide between 6 chains (Hamilton et al., 1977). All of the subunits of the AChR are glycosylated (Anderson and Blobel, 1981). High-mannose-type oligosaccharide chains comprise over 70% of the carbohydrate in Torpedo AChR. Complex-type oligosaccharides exist mainly on the y and 6 subunits (Nomoto et al., 1986). Sites of phosphorylation are present on the cytoplasmic domains of each subunit (Huganir et al., 1984). It is generally accepted that the two a subunits are separated by another subunit. Two arrangements for the subunits around the channel have been proposed: ayapS (Holtzman et al., 1982; Karlin et al., 1983, 1986), and apcryS (Kistler d al., 1982; Hamilton et al., 1985). Channel opening and closing may be due to tilting of the subunits (Guy, 1984). The primary amino acid sequences of all of the subunits from several species have been elucidated from the nucleotide sequences of cDNA clones coding for the subunits (Noda d al., 1982, 1983a,b; Claudio et al., 1983; Devillers-Thiery et al., 1983, 1986; LaPolla et al., 1984; Nef et al., 1984; Boulter et al., 1985, 1986; Kubo et al., 1985). Based on the primary sequences, the molecular weights of the subunits are 50,000 (a),54,000 (p), 56,000 (y), and 58,000 (6). Thus, the molecular weight of the polypeptide is 268,000, which together with the carbohydrate yields a total actual molecular weight of about 290,000. Alignment of the sequences of the four subunits reveals a conspicuous homology among them and suggests the four chains are evolutionarily related (Noda et al., 1983a). After alignment, 19% of the positions are occupied by four identical residues, 20% by three identical residues, and, of the positions with three identical residues, 54% have a conservative substitution in the divergent chain (Noda et al., 1983a). The sequences of subunits are highly conserved across species. For example, mouse a subunit is 80% homologous with Torpedo, 86% homologous with chicken, 95% homologous with calf, and 96% homologous with human (Boulter et al., 1985). All a subunits thus far sequenced contain 437 amino acids. Numbering for the mature a subunit is employed in this review. In addition, in the calf, a subunit termed the E subunit, which shares highest homology with the y subunit, replaces the embryonic y subunit (Takai et al., 1985). The muscarink AChR is homologous with the 0-adrenergic receptor and rhodopsin but shows no amino acid sequence similarity with the nicotinic AChR (Kudo et al., 1986; Fukuda et al., 1987; Peralta et al., 1987). Analysis of the amino acid sequences shows four highly hydrophobic stretches of about 26 amino acids which were proposed to form membranespanning a-helices (Claudio et al., 1983; Devillers-Thiery et al., 1983; Noda et al., 1983a). Four such transmembrane segments would place both the amino (N) terminus and the carboxy (C) terminus on the extracellular surface. By means of Fourier analysis of hydrophobic periodicities, Finer-Moore and

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Stroud (1984) identified an amphipathic a-helix with a continuous hydrophobic face on one side and a hydrophilic face on the other. It was proposed that the amphipathic helix forms a fifth transmembrane segment which thus places the C-terminus in the cytoplasm (Fig. 1). It was also proposed that the amphipathic helices from each of the subunits form the wall of the water-filled, ionic channel, like the staves of a barrel. Kosower (1983a) proposed a model for the a subunit containing six transmembrane segments including two amphipathic helices between residues 334-357 and 368-391 forming part of the ionic channel. Finally, a model containing only three transmembrane helices has been proposed (Hawrot et a l . , 1987). Mielke et al. (1986), using circular dichroism spectroscopy, found only 15% a-helix for the receptor, which is sufficient helix to form three or possibly four transmembrane helices per subunit. Yager et al. (1984) employing Raman spectroscopy concluded 25 9% of the receptor is a-helix.

FIG. 1. Schematic diagram illustrating the secondary and transmembrane structure of the a subunit of the acetylcholine receptor (based on Finer-Moore and Stroud, 1984). Helices are indicated by coils, @-sheetsby straight lines, random coils as wavy lines, and &turns by turns of the chain. A site of N-linked glycosylation occurs at asparagine 141.A disulfide is formed by cysteine 128 and 142.Cysteine 192 and 193 form a disulfide (Kao and Karlin, 1986)and bind the affinity alkylating agent 4-(N-maleimido)benzyltrimethylammonium iodide (Kao et al., 1984). The model contains four hydrophobic transmembrane a-helices and a fifth amphipathic transmembrane helix. The amphipathic helices of all of the subunits may form the wall of the ionic channel. The acetylcholine-binding site has been proposed to occur in the vicinity of cysteine 192, 193.

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Antibodies raised against C-terminus peptides have been used to localize the C-termini of the subunits to the cytoplasmic side of the membrane, supporting a model with an odd number of transmembrane segments (Ratnam and Lindstrom, 1984; Young et al., 1985; Ratnam el al., 1986a). Similarly, using antibodies raised against synthetic peptides corresponding to different regions of the a subunit to determine extracellular or cytoplasmic locations, Ratnam d al. (1986b) proposed a model containing five transmembrane segments. However, unlike the other models in which residues 1-209 are considered to be extracellular, the model of Ratnam el a f . (1986b) proposed two transmembrane segments including an amphipathic helix in this portion of the molecule.

111. Structure and Function of Curaremimetic Neurotoxins

Venoms from snakes belonging to the families Elapidae (cobras, kraits, mambas, coral snakes, and others) and Hydrophidae (sea snakes) contain basic polypeptide neurotoxins that bind with high affinity (KD = 10-9-10-ii M ) to the nicotinic AChR and, like d-tubocurarine, competitively block the depolarizing action of acetylcholine (Lee, 1972; Karlsson, 1979). Examples of reported dissociation constants, determined from kinetic and equilibrium binding data, are for a-bungarotoxin (a-Btx) and Torpedo AChR 4.2 x M (Lukas et al., 1981) and for human receptor 5.0 x M (Stephenson et a l . , 1981). Dissociation constants for long and short neurotoxins are similar (5.7-8.2 x M), but the short neurotoxins associate with the receptor 6-7 times faster and dissociate 5-9 times faster than the long neurotoxins (Chicheportiche et al., 1975). The dissociation constant for a-cobratoxin has been reported to be 1.13 x loe9M (Klett et a l . , 1973) and 1.7 x lo-" M (Weiiand et a l . , 1976); for an a-neurotoxin from Naja nigricollis, 2.0 x lo-" M (Weber and Changeux, 1974); and for cobrotoxin, 3.3-4.9 x M(Endo et al., 1986). More than 80 of these curaremimetic neurotoxins have been sequenced (Karlsson, 1979; Dufton and Hider, 1983) and have been aligned and enumerated by Karlsson (1979) to characterize their maximal homology. The Karlsson alignment positions are employed here. The neurotoxins fall into two distinct size groups: short neurotoxins with molecular weights of about 7000 and composed of 60 to 62 amino acids, and long neurotoxins with molecular weights of about 8000 and containing 71 to 74 residues. The sequences of some of the commonly employed toxins are shown in the Karlsson consensus alignment positions in Fig. 2. wBtx from Bungarus multicinctus (Mebs et al., 1972) and a-cobratoxin from Naja naja siamensis (Karlsson, 1979) are long toxins and erabutoxin b from Laticauda sm$'imciata

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5

a-Bungarotoxin a-Cobratoxin Erabutoxin b Cobrotoxin dungarotoxin Conserved

10

15

20

25

35

30

40

45

50

55

60

65

70

75

IVCH:TTATiPSSA;TCPPGENLC;IRKMWCDAFCSSRGKYVELGCAATCPSKK-PYEEViCCST:DKCNHPPKR9PG

IRCF---ITPDITSKDCPNG-HVCYTKTWCDAFCSIRGKRVDLGC~TCPTVK-TGVDIQCCST-DNCNPFPTRKRP RICFNHQSSQPQTTKTCSPGESSCYHKQWSO-F---RGTIIERGC--GCPTVK-PGIKLSCCES-EVCNN LECHNQQSSQTPTTTGCSGGETNCYKKRWRD-H---RGYRTERGC--GCPSVK-NGIEINCCTT-DRCNN

RTCLISPSSTPQT---CPNGQDICFLKAQCDKFCSIRGPVIEQGCVATCPQFRSNYRSLLCCTT-DNCNH C CY W D E_ RG f G i CP CC CN H

U

U

ii FIG.2. Sequencesand alignment of amino acid sequencesof snake venom neurotoxins. Toxins are aligned in the Karlsson (1979) alignment positions which are employed throughout this review. Sequences are a-bungarotoxin from Bunprur multicinctur (Mebs et al., 1972),a-cobratoxin from Nuja nuja sianunrir (Karlsson, 1979), erabutoxin b from Laticaudu smz@rciata (Nishida et al., 1985), cobrotoxin from Nuja nuja aha (Yang ct al., 1969), and x-bungarotoxin from Bungoms mu~ticinclur(Grant and Chiappinelli, 1985). Residues highly conserved among both long and short curaremimetic neurotoxins and locationof disulfide bonds are shown. Amino acids are represented in the single-lettercode: A, Ala, alanine; C, Cys, cysteine;D, Asp, aspartic acid; E,Glu, glutamic acid; F, Phe, phenyldanine; G, Gly, glycine; H, His, histidine; I, Ile, isoleucine; K, Lys, lysine; L, Leu, leucine; M, Met, methionine; N, Asn, asparagine; P, Pro, proline; Q, Gln, glutamine; R, Arg, arginine; S, Ser, serine; T, Thr, threonine; V, Val, valine; W, Trp, tryptophan; Y, Tyr, tyrosine.

(Nishida et al. , 1985) and cobrotoxin from Nqu nqu atra (Yang et a l . , 1969) are short neurotoxins. Comparison of the primary structures shows that in long neurotoxins the N-terminus region is shorter, a fifth disulfide bond and other residues are inserted between positions 30 and 36, an Ma-Ma-Thr sequence is substituted for Gly 48, and the C-terminus tail is longer. The three-dimensional structures for a short toxin (Low et al., 1976; Tsernoglou and Petsko, 1976; Bourne e t a l . , 1984)and two long toxins (Walkinshaw et al., 1980; Agard and Stroud, 1982; Love and Stroud, 1986)have been determined and found to be similar. The toxins are saucer-shaped molecules with a convex and a concave face and overall dimensions of 40 8, x 30 A x 208,. The toxins consist of a long central loop flanked by two shorter loops projecting from a core region cross-linked by four invariant disulfide bridges (Fig. 3). The additional disulfide in the long neurotoxins is located at the tip of loop 2, making the end of the loop broader than in short neurotoxins. In a-cobratoxin, residues at positions 24-29 of loop 2, 40-44 of loop 2, and 58-62 of loop 3 form a triplestranded, antiparallel, &pleated sheet structure bonding loop 2 and the inner strand of loop 3. This triple-stranded @-sheetis a general structural property of all neurotoxins thus far examined, conferring, along with the four disulfide bonds in the core region, a degree of stability to the entire neurotoxin molecule. The tail of long toxins hangs down alongside the central loop. Nuclear magnetic resonance studies indicate that the long neurotoxins are more structurally rigid than the short neurotoxins, possibly as a result of stabilization of the triplestranded antiparallel @-sheetstructure through hydrophobic interactions with the long tail (Endo et al., 1981; Inagaki et al. , 1981).

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THOMAS I,. LENTZ AND PAUL T. WILSON 1

FIG 3 . Schematic diagram of a-bungarotoxin viewed from above. T h e Karlsson (1979) numbering scheme is used. T h e positions of loops 1 , 2 , and 3 are shown. Disulfide bonds are shown as heavy lines. Bold circles represent residues conserved among both long and short neurotoxins.

The neurotoxins show close amino acid homology with other nonneurotoxic peptides such as cardiotoxins, membrane toxins, cytotoxins, and other peptides present in snake venoms (Karlsson, 1979). Comparative sequence data have been used to deduce structure-function relationships (Karlsson, 1979; M&ez et a l . , 1984). Comparison of the sequences of nonneurotoxins reveals that the following residues conserved in neurotoxins are also present in the other toxins: Cys 3, 17, 24, 45, 49, 61, 62, 68; Thr/Phe 25; Gly 44; Pro 50; and Asn 69. These residues are, therefore, considered structurally invariant in neurotoxins and are most likely involved in disulfide pairing and conformation. Other residues are invariant in both long and short neurotoxins but not in nonneurotoxins and are considered functionally invariant residues. These residues are T r p 29; Asp 31; Phe, His, or Trp 33; Arg 37; and Gly 38. Position 42 is Asp or Glu and Lys 27 and 53 are highly conserved. Other residues are invariant in either long or short neurotoxins and, because they do not appear to be required for neurotoxicity, are classified as structurally invariant in long or short neurotoxins. Some of these, however, could account for functional differences between long and short neurotoxins.

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Another toxin isolated from the venom of B. multicinctus, unlike a-Btx, appears to bind to a neuronal AChR and block transmission at ganglia but binds to the neuromuscular AChR with much lower affinity (Chiappinelli, 1983, 1985). This toxin, which has been termed x-bungarotoxin (x-Btx) (Chiappinelli, 1983), appears identical to a toxin termed bungarotoxin 3.1 (Ravdin and Berg, 1979) and toxin F (Loring et a l . , 1986). x-Btx (Grant and Chiappinelli, 1985) shows considerable structural homology with the curaremimetic neurotoxins, especially the long neurotoxins, although it has a shorter tail (Fig. 2). x-Btx shows identity with all of the structurally invariant residues and most of the functionally invariant residues. An exception is the substitution of glutamine for invariant Trp 29, suggesting that Trp 29 is important in binding to the neuromuscular AChR. Other possibly significant differences are the presence of a positively charged lysine at position 32 and a proline at position 39 which could alter the orientation of invariant Arg 37 and Gly 38. A short neurotoxin from Nqa h z e annulqira which shows reduced neurotoxicity has a glycine substituted for the conserved Asp 31 (Endo et al., 1986). Numerous chemical modification studies have been performed in an effort to determine the contribution of individual residues to neurotoxin binding and toxicity (Karlsson, 1979; Dufton and Hider, 1983). These studies have shown that no single chemical modification completely abolishes toxicity or binding to AChR, although some modifications reduce toxicity. Modification of Arg 37 of a-cobratoxin with phenylglyoxal caused about a 75 % decrease in toxicity (Yang et al., 1974). Modification of all arginine residues by 1,2-cyclohexanedione in a-cobratoxin caused about an 8-fold decrease in toxicity and a 100-fold decrease in affinity for AChR (Martin et al., 1983). Modification of all the lysine residues by reductive methylation, in which the positive charge on the lysine residues is maintained, preserved 79% of the toxicity and 25% of the affinity of the unmodified toxin. Modification by acetylation with acetic anhydride, which abolishes the positive charge, preserved 32 % of the original toxicity but only 0.05% of the original affinity (Martin et al., 1983). Acetylation (Faure et al., 1983) or biotinylation (Lobe1 et al., 1985) of Lys 27 and Lys 53 caused a larger decrease in affinity than modification of other lysine residues. Acylation of Lys 52 in a-Btx was reported to decrease affinity 20-fold (Babbit and Huang, 1985). Nitration at Tyr 25 caused a 2- to 4-fold decrease in affinity (Martinet al., 1983), while nitrophenylsulfenylation of Trp 29 produced about a 10-fold decrease in affinity (Faure et al., 1983). Other studies have shown that chemical modification of T r p 29 causes a reduction in toxin binding or toxicity (Chang and Hayashi, 1969; Set0 et a l . , 1970; Allen and T u , 1985). The fact that toxins in most cases retain considerable affinity after modification of individual residues indicates that toxin binding to the receptor is most likely mediated by interactions at multiple sites.

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Reduction of the fifth disulfide at the end of loop 2 of long neurotoxins has little effect on toxicity (Chicheportiche et al. ., 1975; Martin et al., 1983). Even after complete reduction and carbamidomethylation of all the disulfides, a-cobratoxin retains an affinity of K D 3 X M (Martin et al., 1983). The latter finding indicates the native conformation of the polypeptide is not essential for toxicity. This conclusion is supported by the observation that a 33-residue peptide comprising residues 16-48 of the major toxin of Naja nqa philippinensis bound to receptor with an affinity of K D 2.2 x lo-’ M (Juillerat et al., 1982). In addition, Lentz et al. (1987) showed that short synthetic peptides corresponding to the tip of loop 2 of a-Btx competed binding of lZ5I-a-Btx binding to the AChR. An a-Btx 13-mer (residues 28-40) inhibited toxin binding with the same affinity as d-tubocurarine and an a-Btx 10-mer (residues 31-40) inhibited toxin binding with the same affinity as nicotine. These findings support the idea, discussed below, that the end of loop 2 interacts with the acetylcholinebinding site on the receptor. It can be expected that the specificity of neurotoxin binding to the receptor is mediated by interactions between sites on the receptor and the functionally invariant residues of the toxin. The concave surface of the toxin molecule comes into contact with the receptor. Most of the side chains of the functionally conserved residues occur on the concave surface (Low et al., 1976; Inagaki et al., 1981). Tsetlin et al. (1979, 1982) incorporated spin and fluorescent labels into various residues and observed changes in electron paramagnetic resonance and fluorescence spectroscopic signals after toxin binding. In addition, toxins derivatized at specific lysines with photoaffinity labels were cross-linked to the receptor. These studies indicated that a considerable portion of the concave surface of the neurotoxins including the region around Lys 27 and Lys 53 is involved in multipoint binding to the AChR (see Johnson and Yguerabide, 1985, for caution in interpretation of changes in fluorescence of labeled toxins upon binding to receptor). Similarly, using spin-labeled toxin derivatives and electron paramagnetic resonance spectroscopy, Rousselet et al. (1984) concluded that Lys 27 and Lys 53 are masked by the receptor upon toxin binding. Lys 27 and Lys 53 are less accessible to acetylation (Balasubramaniam et al., 1983) and the number of accessible amino groups on a-Btx is reduced when it is bound to AChR (Soler et al., 1983). Both acetylcholine (Beers and Reich, 1970) and d-tubocurarine (Sobell et al., 1972) are characterized by a positively char ed quaternary nitrogen atom away. It has been proposed and a hydrogen bond acceptor located about 6 that some of the functionally invariant residues of the neurotoxins mimic acetylcholine or d-tubocurarine. d-Tubocurarine is considered a competitive antagonist of acetylcholine, although it has been reported to act as a partial

x

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agonist on cultured embryonic rat myotubes (Trautmann, 1982). The guanidinium group of Arg 37 is the only cationic group common to all of the neurotoxins and may be the counterpart of the quaternary ammonium group of acetylcholine. A hydrogen-bonded ion pair between the guanidinium group of Arg 37 and the side chain carboxylate of Asp 31 has been proposed to stereochemically resemble acetylcholine (Tsernoglou et al. , 1978; Low, 1979). Inconsistent with this suggestion are nuclear magnetic resonance findings indicating that Asp 31 is hydrogen bonded to Trp 29 (Endo et al., 1981). Low and Corfield (1986) have pointed out that, although there is a watermediated hydrogen bond between Trp 29 and Asp 31 in erabutoxin b, there is also a hydrogen bond between Asp 31 carboxyl oxygen and Arg 37 amide nitrogen. Because the Arg 37 side chain is mobile, Asp 31 and Arg 37 could form an acetylcholinemimetic pair. Dufton and Hider (1977) proposed that Trp 29, Phe 33, Arg 37, and Lys 53 are organized into a pattern resembling that of d-tubocurarine, while Minez et al. (1984) proposed that Lys 27, Trp 29, Asp 31, Arg 37, and Glu 42, together with a variant residue at position 40, form the curare mimic. Martin et al. (1983) proposed that a concentration of positive charge in a-cobratoxin involving Ile 1, Arg 2, Arg 37, Gly 38, Lys 39, Arg 40, Arg 74, Lys 75, and Arg 76 may mediate initial attachment to the receptor. Endo et al. (1986) suggested that, for short neurotoxins, Lys 27 and LysIArg 30 are directly involved in binding. As noted above, Trp 29 is invariant among neurotoxins and chemical modification results in a decrease in toxicity, indicating this residue plays an important role in binding. Low and Corfield (1986) have proposed that Trp 29 lies within a hydrophobic “trp cleft” on the neurotoxin molecule. They suggest that, upon the initial interaction of the Asp 31-Arg 37 pair with the receptor, the water-mediated hydrogen bond between Trp 29 and Asp 31 is disrupted, leading to opening up of the cleft and permitting a hydrophobic interaction between cleft residues and a receptor tryptophan. A possible objection to a significant role for Trp 29 is that in a-Btx it projects from the opposite side of the /3 sheet than it does in a-cobratoxin and erabutoxin b (Dufton and Hider, 1983). However, in a-Btx bound to receptor, Trp 29 is not accessible to tryptophan-specific reagents, suggesting that in the bound state the Trp side chain lies on the concave surface (Fairclough et al., 1983). Recently it has been reported that Trp 29 is located on the convex side of a-Btx in the crystal but on the concave side in solution (Love and Stroud, 1986). The thermodynamics of neurotoxin binding to the AChR have also been studied and may provide useful information concerning the mechanisms of binding of neurotoxins to the AChR. Equilibrium values for the binding of N. naja siamensis a-cobratoxin were determined to be Gibbs free energy

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AGfo2g8 = - 12.4 kcal/mol, enthalpy AHf0298 = + 17 kcal/mol, and entropy AS1 02g8 = + 99 e.u. (Maelicke et al., 1977), in substantial agreement with an earlier report (Klett et a/., 1973). Thus, from a thermodynamic standpoint, the high affinity ofequiiibrium binding between the neurotoxin and AChR molecules results from an extremely large and favorable entropy factor. As Maelicke et al. (1977) note, the magnitude of AS1 '298 is similar to AS$ "298 values for the heat denaturation of proteins. This suggests that the toxin-AChR complex undergoes significant conformational change. Changes in intrinsic fluorescence of the receptor (Endo et al., 1986) or in fluorescence of receptor with a covalently bound fluorophore (Gonzales-Ros et al., 1983) upon toxin binding, in fact, indicate that the toxin induces conformational changes in the receptor. In addition, changes in fluorescence upon binding of fluorescein-labeled a-cobratoxin (Kang and Maelicke, 1980;Johnson and Taylor, 1982) suggest that the conformation of the toxin is altered as well upon association with the receptor. However, in preformed toxin-receptor complexes, increase in affinity (decrease in dissociation rate) was not accompanied by changes in fluorescence of toxin and thus was not considered to result from conformational changes of the toxin portion of the complex (Kang and Maelicke, 1980). Perhaps more surprisingly, the equilibrium binding of the smaller cholinergic agonists and antagonists is also characterized by very large positive A S f o 2 9 8 values (Maelicke et al., 1977), again suggesting that the binding of these agents can also cause pronounced conformational changes in the AChR. Studies in which the fluorescent properties of the AChR upon binding of these ligands were monitored indicate that these ligands can also induce conformational changes in the AChR (Bonner et al., 1976; Barrantes, 1978; Kaneda et al., 1982). The fact that agonist binding results in activation of the ion channel whereas antagonist binding does not implies that the conformational changes induced in the AChR by these two classes of ligands differ significantly. The thermodynamic properties of the kinetics of binding of neurotoxins to the AChR have also been studied. Klett et al. (1973) reported activation parameters for association of the long neurotoxin, a-neurotoxin, to be AGf = + 10.4 kcal/mol, A H f = + 16.8 kcal/mol, and AS$ = +21.4 e.u. and activation parameters for dissociation to be AGI = +23.7 kcal/mol, AH1 = + 0 . 3 kcal/mol, and AS$ = - 78.5 e.u. The fact that, unlike association, dissociation is largely independent of temperature (i.e., AH$ nearly 0) implies that the mechanism of dissociation of the AChR-toxin complex is different from the mechanism of association and does not represent a simple reversal of the binding events that occur during association. The enthalpy and entropy of activation for association of short neurotoxins have also been determined. Endo et al. (1986) reported values of AH$ = + 4 . 0 kcal/mol and ASf = - 12 e.u./mol for cobrotoxin and AHf = +5.0 kcdlmol and ASf = - 14 e.u./mol for erabutoxin b. As expected for all three

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is positive. It is interesting, however, toxins, the enthalpy of activation (AH$) that the entropy of activation (ASS) is positive for the long neurotoxin and negative for the short neurotoxins. This implies that the mechanism of binding of the long toxin differs from that of the short toxins. It remains to be determined whether this difference reflects an underlying general difference between the binding mechanisms of the long and short toxins. It has already been mentioned that, in general, the short toxins associate faster than do the long toxins. That this may be the result of a different binding mechanism is supported by the observations of Endo et al. (1986) that long and short toxins had differing effects on the steady-state fluorescence of the AChR, suggesting to the authors slightly different binding modes. Although the exact mechanism of toxin binding to the receptor is unknown, the available knowledge of toxin structure-function relationships indicates two important features of binding: first, no one property of the toxin molecule has yet been identified that is absolutely essential for binding and, second, certain properties of the toxin molecule, as a whole, facilitate the binding event. As discussed above, chemical modification studies and studies involving labeled toxin derivatives indicate that the binding of toxin to the AChR is the result of contact between many points on the toxin molecule and the AChR. Thus the enzyme-substrate binding paradigm which is often used to describe the binding of ligand to receptor is not particularly suitable here. Multiple points of contact would account for, in part, the high affinity that characterizes the binding event and also for the resiliency of toxin binding in the face of chemical modification of individual amino acid residues. This resiliency is probably further aided by the relative rigidity of the toxin molecule itself. This rigidity is largely the result of the triple 0-sheet involving loops 2 and 3 and the presence of the four disulfide bonds in the core region and, as such, involves a sizeable portion of the toxin molecule. Disruption of the disulfide bonds, while not abolishing toxin binding, reduces affinity 10,000-fold (Martin et al., 1983), underscoring their importance in maintaining the overall conformation of the toxin molecule and the importance of the overall conformation to the binding event. This relative rigidity of conformation defines a template for toxin-AChR interaction and constrains the interactions of individual amino acid residues. At the same time, however, it also allows for a degree of variability in localized regions of the primary sequence since binding is more dependent on the overall fit of the toxin molecule with the AChR than on a small number of local interactions. This may, perhaps, explain how toxicity is preserved in toxin molecules where functionally invariant residues have been modified. Such modifications, whether the result of natural mutation or chemical reaction, are therefore unlikely to disturb greatly the overall conformation of the toxin molecule or the affinity.

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Toxin interaction with the receptor may involve local recognition involving shape and chemical complementarity between specific interacting residues followed by an induced fit accomplished by rearrangments of flexible regions in the toxin and/or receptor. Electrostatic interactions are known to play a role in the binding of neurotoxins to the AChR and may be particularly important in the initial binding event. In particular, binding seems to be facilitated by the presence of positive charges on the neurotoxin molecule. This conclusion is derived from chemical modification studies where positive charges have been neutralized with a resultant large decrease in affinity (Martin et al., 1983) and from binding studies in which the association rates were correlated with net positive charge (Endo et al., 1986). Charged groups on the toxin molecule may play a role first in orienting the toxin molecule in the most favorable position for interaction with the receptor surface (Low and Corfield, 1986). The facilitation of toxin binding by the presence of positive charges on the toxin molecule also implies that the corresponding areas of interaction on the AChR must possess negative charges that electrostatically interact with the positively charged toxin residues. It is attractive to postulate that the initial recognition event involves interaction between complementary structures on the toxin and receptor surfaces. Association of noncomplementary structures would be less likely as a result of large unfavorable enthalpies due to poor packing between residues and loss of hydrogen bonds made to water (Chothia and Janin, 1975). After the initial recognition event, a broad area of contact is formed between the receptor and the concave surface of the toxin. The toxin presents a relatively rigid surface across which charged and hydrophobic residues are arranged in alternating clusters or bands (Fig. 4). These regions can be expected to bind to complementary domains on the receptor surface with the interaction stabilized by multiple hydrophobic, ionic, and electrostatic interactions (Fairclough et a l . , 1983; Love and Stroud, 1986). Hydrophobicity (Chothia and Janin, 1975), aromatic-aromatic interactions (Burley and Petsko, 1985), and electrostatic forces (Getzoff et al., 1986) play important roles in stabilizing specific contacts. The ability of both the toxin and the AChR molecules to undergo conformational change may also facilitate the binding event. As an expression of the large favorable entropy of binding, this conformational change probably increases the number of contacts between the toxin and the AChR. This may occur by local side-chain readjustments or larger conformational changes in the receptor which improve stereochemical complementarity with toxin and allow interaction with previously buried residues. Induction of optimal complementarity between interacting surfaces is promoted by the entropically favored removal of bound water molecules that stabilize the positions of polar

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FIG.4. Mapping of hydrophobic (-) and charged domains (---)on a-bungarotoxin model. Hydrophobic residues (0) are those with a hydrophobicity value of -0.4 or less on the scale of Hopp and Woods (1981) (T, A, H , C, M, V, I, C, Y ,F, W). Positivelycharged residues ( +)are K and R and negatively charged residues ( - ) are D and E. Note that the mapping includes all toxin residues and is not restricted to those coming into contact with the receptor. The location of the domains is an approximation since the model is not identical to the three-dimensional structure of the toxin. See Fairclough d 01. (1983) for mapping of complementary domains on the acetylcholine surface relative to the a-carbon skeleton of a-bungarotoxin.

side chains and by increasing the contribution of hydrophobic and aromatic side chains to the interface (Getzoff et al., 1987). Such adjustments at the toxin-receptor interface would enhance the affinity of binding and produce a mutually locked complex.

IV. Neurotoxin-Bindlng Site on the AChR

A. SPECIFICITY FOR cr SUBUNIT The extracellular domain of the AChR molecule facing the synaptic cleft carries the neurotoxin-binding sites. Toxin binding to the surface was indirectly visualized with the electron microscope by Klymkowsky and Stroud (1979) by complexing antitoxin antibodies coupled with gold beads to toxin

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bound to receptor. a-Btx was observed to bind at two sites on the AChR by image averaging of AChR rosettes with bound a-Btx at 20 A resolution (Zingsheim et al., 1982; Bon et al., 1984). Similarly, two sites of toxin binding on the receptor monomer could be visualized after labeling the receptor with a complex of biotinylated a-cobratoxin and avidin, which is large enough to be visualized with the electron microscope (Holtzman et a l . , 1982). Toxin does not physically block the receptor channel (Zingsheim et al., 1982) but appears to bind on the top or outer perimeter of the receptor (Kistler et al., 1982; .Johnson et al., 1984). Affinity alkylating agents that act as either cholinergic agonists or competitive antagonists label only the a subunit (Karlin, 1969; Reiter et al., 1972; Karlin, 1980, 1983). Since these agents are competitive with the binding of neurotoxins, the latter are considered to bind at or very near the same site on the a subunit. a-Btx was observed to bind to the a subunit, but not other subunits, electroeluted from polyacrylamide gels (Haggerty and Froehner, 1981). Gershoni et al. (1983) and Oblas et al. (1983) observed binding of a-Btx to the a subunit after polyacrylamide gel electrophoresis and electrophoretic transfer onto immobilizing filters. a-Btx also binds to a subunit within the membranes of yeast cells transformed with a plasmid containing cDNA for the Torpedo a subunit (Fujita et al., 1986). In the studies in which toxin binding to isolated a subunit was observed, the affinity of binding was 0.1-0.2 p M , considerably less than the affinity for the intact receptor. A somewhat higher affinity was observed for toxin binding to a fusion protein containing residues 6-210 of the mouse a subunit (70-80 nM) (Barkas et al., 1987). It is not clear whether the reduced affinity of toxin binding to isolated a subunit is due to denaturation of the a subunit or to the absence of additional binding sites on other subunits. However, affinity of binding can be greatly increased by partial renaturation of the a subunit by 0.02% sodium dodecyl sulfate (KD3nlM) (Tzartos and Changeux, 1983) and by 0.1 % sodium cholate and 1 % lipids (asolectin) (KD 0.5 nM) (Tzartos and Changeux, 1984). The latter value approaches some reports of the affinity of a-Btx for native receptor, e.g., 4.2 x 10-''M (Lukas et al., 1981) and suggests the a subunit contains most or all of the toxin-binding site. In addition, newly synthesized a subunit in the mouse cell line BCJH-1 acquires the ability to bind a-Btx with high affinity (approximately the same as mature BC3H-1 AChR) prior to assembly with other subunits (Merlie and Lindstrom, 1983; Carlin et al., 1986; Merlie and Smith, 1986). Acquisition of toxin-binding activity may be due to formation of a disulfide necessary for formation of mature tertiary conformation (Merlie and Lindstrom, 1983). While the above evidence suggests that most or all of the determinants for toxin binding are on the a subunit, some studies have reported a possible

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interaction with other subunits. The diameter of the AChR is 85 A , while the x 30 A (Fairclough et al., 1983). Much of the toxin-binding surface is 20 latter could be accommodated by one subunit, but it is conceivable that portions of the toxin molecule contact other subunits. Covalent cross-linking of neurotoxins with the receptor have shown labeling of other subunits (Witzemann et al., 1979; Nathanson and Hall, 1980; Hamilton et a l . , 1985). Receptor was found to bind twice as much a-dendrotoxin as a-cobratoxin or a-Btx, suggesting that there may be other neurotoxin-binding sites on other subunits (Conti-Tronconi and Raftery, 1986).

A

B . PROXIMITY TO REDUCIBLE DISULFIDE Localization of the binding site for cholinergic ligands is greatly facilitated by the conclusive demonstration that a readily reducible disulfide lies close to the negatively charged subsite of the acetylcholine-binding site (Karlin, 1980; Karlin et a l . , 1986). In the resting conformation of the receptor, this disulfide can be reduced under very mild conditions (Karlin, 1980; Kao et a l . , 1984). Reduction of the disulfide by dithiothreitol causes a decrease in affinity of binding of cholinergic agonists (Walker et al., 1981, 1984) but does not significantly affect lZ5I-a-Btxbinding (Walker et a l . , 1981; Criado et a l . , 1986). Reduction was reported to slightly reduce the affinity of [3H]methyl-a-cobratoxin binding (Hamilton et al., 1985). Furthermore, the reduced disulfide can be affinity labeled by quaternary ammonium alkylating agents that are either agonists or competitive antagonists of acetylcholine (Karlin, 1969, 1980). These bifunctional reagents resemble cholinergic ligands at one end of the molecule and possess an alkylating group at the other end which covalently reacts with the reduced disulfide. One type of label, an example of which is 4-(N-maleimido)benzyltrimethylammonium iodide (MBTA), inhibits response of receptor to agonists. Another type, an example of which is bromoacetylcholine (BAC), results in covalent activation of the receptor (Silman and Karlin, 1969; Cox et a l . , 1979). The reactions of the two types of affinity labels with the reduced site are mutually exclusive (Damle et a l . , 1978). The reactions of both types are inhibited by agonists and competitive antagonists including the curaremimetic neurotoxins. Conversely, affinity labels inhibit the binding of agonists and competitive antagonists. The competition between affinity alkylating agents and the neurotoxins provides strong evidence that the neurotoxins bind at or very near the acetylcholine-binding site on the receptor. Based on the dimensions of the alkylating agents, it was determined that the acetylcholine-binding site is located about lOA from the reduced disulfide bond (Karlin, 1980; Karlin et a l . , 1986). This site on the receptor is

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considered to contain one or more residues with negatively charged side chains. A hydrophobic subsite may be located in the vicinity of the disulfide. It was hypothesized that receptor activation occurs when acetylcholine bridges the negative subsite and the hydrophobic subsite, causing an altered conformation around the negative subsite and a decrease of a few angstroms in the distance between the two subsites (Karlin, 1969). The two high-affinity acetylcholine-binding sites on the AChR monomer are not equivalent. The affinity alkylating agents react preferentially with one of the two sites on electric organ receptor because twice as much neurotoxin as affinity ligand is bound (Damle and Karlin, 1978; Damle et al., 1978). However, under conditions when labeling of affinity agent is saturated or maximal, both sites can be labeled (Wolosin et al., 1980; Dunn et al., 1983; Momoi and Lennon, 1986). d-Tubocurarine also interacts differently with the two sites (Neubig and Cohen, 1979; Sine and Taylor, 1981; Hamilton et al., 1985). Further evidence concerning the heterogeneity of sites is that some monoclonal antibodies raised against the receptor inhibit 50% of toxin binding, indicating that they may recognize only one of the two cholinergic-binding sites (James et al., 1983; Watters and Maelicke, 1983; Mihovilovic and Richman, 1984; Whiting et al., 1985). Neurotoxin binding at the two sites appears to be equivalent (Maelicke el a l . , 1977; Ellena and McNamee, 1980; Kang and Maelicke, 1980; Sine and Taylor, 1980). One explanation for the different binding characteristics of the two subunits is that they differ in extent of glycosylation (Lindstrom et al., 1983; Conti-Tronconi et al., 1984). Ratnam et al. (1986~)isolated two fragments of the a subunit generated by V8 protease treatment. Both fragments began at residue 46 of the CY subunit sequence. One fragment was more heavily glycosylated, while the other was less gylcosylated and was labeled by BAC after affinity labeling of one of the two sites on the intact receptor. Thus, it was suggested that one of the (II subunits is less glycosylated and reacts preferentially with affinity alkylating agents. More recent results indicate that the MBTAbinding fragment generated by V8 protease begins at residue 173 (Pedersen et al., 1986). The conflicting results were considered to be the result of comigration of the glycosylated fragment beginning at Val 46, but not affinity labeled, with the fragment beginning at Ser 173. Other evidence, furthermore, suggests that both subunits have high-mannose N-linked oligosaccharides and that there is little if any difference in the glycosylation of the two a subunits (Wilson et al., 1985; Nomoto et al., 1986). The most likely explanation for the differences in the binding characteristics of the two subunits is that each CY subunit has different adjoining subunits. Thus, there may be asymmetrical microenvironments in the binding sites caused by the interaction of each (II subunit with different subunits.

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According to current models of AChR secondary structure, four of the seven cysteine residues are located on the extracellular domain of the a! subunit. These residues are Cys 128,Cys 142,Cys 192,and Cys 193.Cys 128 and Cys 142 occur in all of the subunits, while Cys 192 and Cys 193 are restricted to the a subunit. Since it has been established that the cholinergicbinding site lies near a reducible disulfide, it becomes essential to determine which cysteines are labeled by the affinity ligands and which residues are disulfide cross-linked. Evidence presented by Kao et al. (1984)indicates that Cys 192 and possibly also Cys 193 are labeled by MBTA. Purified Torpedo AChR was affinity alkylated with [3H]MBTA and the isolated (Y subunit cleaved with cyanogen bromide. The fragments were separated by reversephase high-performance liquid chromatography. The labeled fragment as well as a labeled subfragment were partially sequenced and, based on this information, the MBTA-labeled fragment was identified as residues 179-209.This fragment contains the adjacent Cys 192 and Cys 193. These findings have been confirmed by Dennis et al. (1986)who showed that Cys 192 and 193 are labeled by 4-(N-maleimido)phenyltrimethylammonium iodide (MPTA). In contrast, Cahill and Schmidt (1984)proposed Cys 142 as the site labeled by MBTA based on the amino acid composition of a proteolytic fragment of the (II subunit labeled by MBTA. Initially it was proposed that a disulfide bond exists between Cys 128 and Cys 142 (Numa et al., 1983). Subsequently, it was suggested that a double disulfide bond exists between Cys 128 and Cys 193 and between Cys 142 and Cys 192 (Boulter et al., 1985, 1986b;Luyten, 1986). Kao and Karlin (1986) have presented evidence that a disulfide bond exists between Cys 192 and Cys 193.The receptor was reduced under mild conditions and alkylated with a mixture of [3H]MBTA,which under the conditions employed labels one of the two acetylcholine-binding sites, and N-[1 -"C]ethylmaleimide, which labels the remaining free sulhydryl group. Both labels were incorporated into a cyanogen bromide fragment containing Cys 192 and Cys 193, indicating that only these cysteine residues are reduced by mild reduction. In addition, two fragments, one containing Cys 128 and Cys 142 and the other containing Cys 192 and Cys 193,eluted as separate peaks and were not cross-linked to each other. This suggests that each fragment contains an internal disulfide bond. Criado et al. (1986), using monoclonal antibodies against a synthetic peptide comprising residues 127-143 of the (Y subunit, concluded that Cys 128 and Cys 142 form a disulfide bond. About 100 times as much dithiothreitol was required to inhibit binding of these antibodies to the AChR as was required to reduce the disulfide near the acetylcholine-binding site. These results suggest that the disulfide affecting the binding of the monclonal antibodies (i.e., between Cys 128 and Cys 142) is not located close to the cholinergic-binding site.

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The evidence discussed above indicates that the disulfide in proximity to the acetylcholine-binding site is located between Cys 192 and Cys 193. Another disulfide occurs between Cys 128 and Cys 142. Disulfide bonds rarely occur between adjacent cysteines, and it has been suggested that the unusual disulfide between Cys 192 and Cys 193 is involved in local conformational changes following agonist binding which lead to receptor activation (Kao and Karlin, 1986). The predicted cis peptide bond occurring between the adjacent cysteines can exist in two conformations. Agonist binding at the acetylcholinebinding site may induce a transition between these conformations leading to opening of the channel.

C. BINDINGOF NEUROTOXINS TO PROTEOLYTIC FRAGMENTS OF T H E (Y SUBUNIT Proteolytic digestion (Cleveland et al., 1977) of the a subunit produces patterns of cleavage fragments after gel electrophoresis characteristic for the protease used. If these fragments retain functions such as the ability to bind ligands and if the location of the peptides in the intact subunit can be determined, then such dissection of the receptor represents a useful tool in the localization of the ligand-binding site. Proteolysis of isolated subunits has also been utilized to compare the subunit structures of AChR from different sources (Froehner and Rafto, 1979; Nathanson and Hall, 1979) and to map the binding domains of monoclonal antibodies against the AChR (Gullick et al., 1981; Anderson et a l . , 1983; Ratnam et al., 1986a). Several investigators have observed binding of [3H]MBTA or lZ5I-Btxto proteolytic fragments of the a subunit. Gullick et al. (1981), after labeling a subunit with [3H]MBTA, observed a labeled 19-kDa fragment after V8 protease digestion and a labeled 27-kDa fragment after papain digestion. Tzartos and Changeux (1983), using a DEAE filter assay, detected binding of a-Btx to soluble 34-kDa and 27-kDa fragments generated by papain treatment. Wilson et al. (1984), after treating a subunit with V8 protease, papain, bromelain, or proteinase K , observed a-Btx binding to fragments immobilized on protein transfers and ranging in molecular mass from 7 to 34 kDa. The labeling of fragments with a-Btx was inhibited by prior affinity alkylation of receptor with MBTA. In no fragments were the MBTA and a-Btx sites separated, providing further evidence that the acetylcholine- and neurotoxin-binding sites are the same or very closely associated on the primary sequence. The apparent affinity of the fragments for a-Btx as determined from the ICSO value was 6.7 x 10-8M.This value agreed closely with the binding afinity of lO-’M observed with the immobilized, intact a subunit (Gershoni et al., 1983).

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Since the a subunit has only one site of N-linked glycosylation (Asn 141) and the presence of carbohydrate in fragments can be readily detected, it is possible to map fragments relative to this site. Wilson et al. (1985) mapped toxin-binding proteolytic fragments on the primary amino acid sequence in relation to Asn 141. Toxin-binding fragments were analyzed on the basis of the size of the fragments and the presence of Asn 141 as determined by susceptibility to digestion with endoglycosidase H. The toxin-binding site was found not to reside between amino acid residues 1 and 140 since toxin-binding fragments were detected that were considerably larger than 140 amino acids and lacked N-linked oligosaccharide chains. Thus, the toxin-binding site must be located on the C-terminal side of Asn 141. The size of the smallest toxinbinding fragment containing Asn 141 and the size of toxin-binding fragments generated by V8 protease indicated that the binding site lies within residues 153 to 241. Since the first membrane spanning region is considered to begin at residue 210, these results indicate that the binding site may be between residues 153 and 209. Considerable attention has been paid to proteolytic fragments generated by treatment of the a subunit with V8 protease. Among the fragments generated are at least two, but probably more (Wilson et al., 1985), fragments with molecular masses in the 17- to 20-kDa range. Most investigators have reported that the larger fragment (18-20 kDa) binds a-Btx or MBTA and is not glycosylated (Gullick et al., 1981; Wilson et al. , 1985; Neumann et a l . , 1985; Pedersen et al., 1986). Conti-Tronconi et al. (1984) reported that the larger V8 fragment is glycosylated but not nearly to the extent as the smaller fragment. An exception to these findings is a report by Oblas et al. (1986) that the 17-kDa glycosylated fragment bound a-Btx. Based on the observations that the larger toxin-binding fragment is not glycosylated, this peptide and the toxin-binding site have been suggested to be to the C-terminus side of Asp 152, the first V8 protease cleavage site beyond Asn 141 (Neumann et al., 1985; Wilson et al., 1985; Oblas et al., 1986). The larger V8 protease fragment, in addition to binding MBTA, a-Btx, and d-tubocurarine, contained a binding site for [3H]meproadifen mustard, an affinity label for the noncompetitive inhibitor binding site (Pedersen et al., 1986). Efforts to localize the V8 protease fragments on the primary sequence by N-terminal sequencing of the peptides have led to conflicting results. ContiTronconi et al. (1984) and Ratnam et al. (1986~)reported that both the larger and smaller fragments began at Val 46 and suggested that one a subunit is much more heavily glycosylated than the other. Oblas et al. (1986) and Pedersen et al. (1986) also reported that the smaller fragment began at Val 46. However, Pedersen et al. (1986) found that the larger fragment began at Ser 173, in agreement with the results of mapping studies which placed this

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fragment on the C-terminus side of Asp 152. Pedersen et al. (1986) also found that under certain conditions an incompletely cleaved form of the smaller fragment co-migrates with the larger fragment. Thus, sequencing of the contaminating fragment could account for the discrepancy in the identity of the N-terminus of the fragments as well as in the degree of glycosylation of the fragments. Finally, after covalent labeling of receptor with ligands, the a! subunit can be cleaved with cyanogen bromide and the fragments separated and identified by amino acid analysis and sequencing. In this manner, Kao et al. (1984) showed that 13H]MBTA was bound to a fragment comprising residues 179-207 of the a subunit sequence. Dennis et af. (1986) showed that cyanogen bromide fragment 179-207, in addition to being labeled with the affinity alkylating agent [3H]MPTA, was also the major fragment labeled by the cholinergic photoaffinity ligand p-(N,N-dimethy1amino)benzenediazonium fluoroborate (DDF). [SH]DDFwas incorporated to a lesser extent into two additional fragments, indicating other regions of the a! subunit chain may contribute to the cholinergic binding site. DiPaola et al. (1986) showed that, after photoaffinity labeling of the receptor with [3H]quinacrine azide, a noncompetitive inhibitor, a cyanogen bromide fragment composed of residues 208-243 which includes the first putative membrane-spanning region was labeled. These results suggest that the cholinergic-binding site and one of the noncompetitive inhibitor sites, which is associated with the receptor channel, are closely associated on the a! subunit. Both sites lie within residues 161 to 239, a sequence coded for by a single exon (Noda et al., 1983b), and it has been proposed that the two sites are part of one functional unit (DiPaola et al, , 1986).

D.

COMPARISON OF

AMINOACIDSEQUENCES OF (Y SUBUNITS,

OTHER SUBUNITS, AND OTHER AChR Evidence reviewed above indicates that the major cholinergic ligandbinding site is located on the a! subunit and includes a negatively charged subsite in proximity to a readily reducible disulfide. The similar pharmacological properties in a subunits from different species suggest that the acetylcholinebinding site has been highly conserved during evolution. A reasonable assumption, therefore, is that the residues comprising the binding site are located in proximity to a cysteine residue and that they are conserved in a subunits across species but possibly not in other subunits. The primary amino acid sequences of a! subunits from five species, a presumed neural AChR subunit, an AChR receptor protein from Drosophila, and Torpedo 0, y , and 6 subunits are shown in Fig. 5. Residues invariant in the (Y subunits and

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139

residues invariant or conserved (present in all but one sequence) in the AChR proteins are indicated. In this alignment, 77% of the residues are invariant among a subunits, while 25 % of a subunit residues are conserved in the other receptor proteins. Additional positions in the overall alignment are occupied by residues representing conservative substitutions. The sequences of the receptor proteins are compared to determine whether any information on the location of the binding site can be obtained. It should be kept in mind that deductions based on the linear sequence may not be representative of the situation in the folded structure of the protein where residues located apart on the primary sequence can be situated in close proximity in the native conformation. Cysteine residues are located on the ectodomain of the molecule at positions 128, 142, 192, and 193. One of these cysteines is about 10 A from the negative subsite in the folded structure of the a subunit (Karlin et al., 1986). Comparison of the regions containing these cysteines reveals that both are highly conserved among a subunits. However, the region surrounding Cys 128 and Cys 142 is conserved to a much greater degree in the other subunits than the region around Cys 192 and Cys 193. Cys 128 and Cys 142 are found in all of the AChR proteins, while Cys 192 and Cys 193 are present only in a subunits and the neuronal receptor. These comparisons suggest that Cys 192 and Cys 193 are involved in functions unique to a subunits, while Cys 128 and Cys 142 are related to functions common to all subunits. Based on the effects of mutation of these cysteines, Mishina et al. (1985) have proposed that the disulfide between Cys 128 and Cys 142 may be related to maintaining the conformation of subunits or the assembly of subunits. Two anionic subsites invariant among a subunits, Glu 129 and Asp 138, occur in proximity to Cys 128 and Cys 142. Asp 138 also occurs in the other receptor protejns and subunits. Around Cys 192 and Cys 193, negative subsites conserved in a subunits are Asp/Glu 180 and Asp 200. Asp 200 is also found in /3, y, and 6 and the other receptor proteins. Thus, Glu 129 and Asp/Glu 180 are the only anionic residues in proximity to a cysteine residue that are conserved in a across species and absent in 0, y, and 6. Based on this analysis, either Glu 129 or Asp/Glu 180 might seem to be likely candidates for the negative subsite within the acetylcholine-binding site. Arguments can also be made against Glu 129 or Asp/Glu 180 as possible negative subsites. First, if the neuronal protein is the same as the neuronal nicotinic AChR a subunit, it would be expected that this protein binds acetylcholine and should contain the negative subsite. However, in the neuronal protein a cationic lysine residue is substituted for these residues. The Drosophila protein has a negatively charged residue at position 180 but not at 129. Second, it has been assumed that the negative site is present in a but not in the other subunits. However, it has been suggested that since the AChR is a

140

THOMAS L. LENTZ AND PAUL T. WILSON

1 1. Torpedo a 2. Chicken a 3. Mouse a 4. Calf a 5. Human a 6. Rat nerve 7. Drosophila 8. Torpedo 8 9. Torpedo y 10. Torpedo 6 11. Conserved a 12. Conserved a l l

SEHETRLVANLL

25 EN YNKVIRPVEH~THFVDITVGLQL

50

IQL ISVDEVNQI~ETNVRLR

SEHETRLVAKLF ED YSSVVRPVEDHREIVQVTVGLQLIQLINVDEVNQIVTTNVRLK SEHETRLVAKLF ED YNSVVRPVEDHRQAVEVTVGLQLIQLINVDEVNQIVTTNVRLK SEHETRLVAKLF KO YSSVVRPVEDHRQVVEVTVGLQLIQLINVDEVNQIVTTNVRLK SEAEHRLFQYLF ED YNEIIRPVANVSHPVIIQFEVSMSQLVKVDEVNQIMETNLWLK SEDEERLVRDLF RG YNKLIRPVQNMTQKVGVRFGLAFVQLINVNEKNQVMKSNVWLR SVMEDTLLSVLF ET YNPKVRPAQTVGDKVTVRVGLTLTNLLILNEKIEEMTTNVFLN ENEEGRLIEKLL GO YDKRIIPAKTLDHIIDVTLKLTLTNLISLNEKEEALTTNVWIE VNEEERLINDLLIVNKYNKHVRPVKHNNEVVNIALSLTLSNLISLKETDETLTSNVWMD SEHETRLVA L Y V RPVE H V TVGLQLIQLI VDEVNQIV TNVRL E EL L Y EP V L L E !N! 75 100 125

1. QQWIDVRLRWNPADYGG~KKIRLPSDDVWLPDLVLYNNADGD~AIVH~KLLLDYTGKIMWTPPAIF~SYCEII 2. ADGDFAIVKYTKVLLEHTGKITWTPPAI FKSYCEI I 3. QQWVDYNLKWNPDDYGGVKKIHIPSEKIWRPDVVLYNNADGDFAIVK~KVLLDYTGHITWTPPAIFKSYCEII 4. QQWVDYNLKWNPDDYGGVKKIHIPSEKIWRPDLVLYNNADGDFAIVKFTKVLLDYTGHITWTPPAIFKSYCEII 5. QQWVDYNLKWNPDDYGGVKKIHIPSEKIWRPDLVLYNNADGDFAIVK~KVLLQYTGHITWTPPAIFKSYCEII 6. QIWNDYKLKWKPSDYQGVEFMRVPAEKIWKPDIVLYNNADGDFQVDDKTKALLKYTGEVTWIPPAIFKSSCKID 7 . LVWYDYQLQWDEADYGGIGVLRLPPDKVWKPDIVLFNNADGNYEVRYKSNVLIYPTGEVLWVPPAIYQSSCTID 8. LAWTDYRLQWDPAAYEGIKDLRIPSSDVWQPDIVLMNNNDGSFEITLHVNVLVQHTGAVSWQPSAIYRSSCTIK 9. IQWNDYRLSWNTSEYEGIDLVRIPSELLWLPDVVLENNVDGQFEVAYYANVLVY NDGSMYWLPPAIY RSTCP I A 10. HAWYDHRLTWNASEYSDISILRLPPELVWIPDIVL9NNNMQYHVAYFCNVLVRPNGYVTWLPPAIFRSSCPIN 11. QQW D L WNP DYGG K K I PS W PD VLYNNADGDFAIV TK LL TG I WTPPAIFKSYCEII 12. W D LW YG P W PD VL NN DG L G W PEA1 S C I

150

175

1. VTHFPFDQQNCTMKLGIWTYDGTKVSISPESDRP 2. VTYFPFDOONCSMKLGTWTYDGTMVVINPESDRP 3. VTHFPFDEQNCSMKLGTWTYDGSVVAINPESDQP 4.

5. 6. 7.

DLSTFMESGEWVMKDYRGWKHWVYYTCC DLSNFMESGEWVMKDYRGWKHWVYYACC DLSNFMESGEWVI KEARGWKHWVFYSCC VTHFPFDEQNCSMKLGTWTYDGSVVVINPESDQP DLSNFMESGEWVI KESRGWKHWVFYACC DLSNFMESGEWVIKESRGWKHSVTYSCC VTHFPFDEQNCSMKLGTWTVDGSVVAINPESDQP VTYFPFDYQNCTMKFGSWSYDKAKIDLVL IGSSM NLKDYWESGEWAII KAPGY KHE I KYNCC VTYFPFDQQTCIMKFGSWTFNGDQVSLALYNNKNFV DLSDYWKSGTWDIIEVPAYLNVYEGDSN VMYFPFDWQNCTMVFKSYTYDTSEVTLQHALDAKGERE VKEIVINKDAFTENGQWSIEHKPSRKNW RSD VTYFPFDWQNCSLVFRSQTYNAHEVNLQLSAEE GE AVEWIHIDPEDFTENGEWTIRHRPAKKNYNWQLTK

8. 9. 10. VLYFPFDWQNCSLKFTALNYDANEITMDLMTDTIDGKDYPIEWIIIDPEAFTENGEWEIIHKPAKKNIYPDKFP 11. VT FPFD QNC MKLG WTYDG V I PESD P DLS FMESGEWV K RGWKH V Y CC 12. V FPFD QKC Y -E G W -K

200 t - T r a n ~ m e m b r a n e ~1 ?- ~ 25?Transmembrane 21. PDTPYLO~TYHFIMDRIPLYFVVN~I IPCLLFSFLTGLVFYLPTDSG E~TLSISVLLSLTVFLLVIVELIPS 2. PDTPYLDITYHFLMQRLPLYFIVNVIIPCLLFSFLTGFVFYLPTDSG EKMTLSISVLLSLTVFLLVIVELIPS 3. PTTPYLDITVHFVMQRLPLYFIVNVIIPCLLFSFLTSLVFYLPTDSG EKMTLSISVLLSLTVFLLVIVELIPS 4. PSTPYLDITYHFVMQRLPLYFIVNVIIPCLLFSFLTGLVFYLPTDSG EKMTLSISVLLSLTVFLLVIVELIPS 5. PDTPYLDITYHFVMORLPLYFIVNVIIPCLLFSFLTGLVFYLPTDSG EKMTLSISVLLSLTVFLLVIVELIPS 6. EE IYaDITYSLY IRRLPLFYTINLIIPCLLISFLTVLVFYLPSDCG EKVTLCISVLLSLTVFLLVITETIPS 7. HPTE TDITFY I IIRRKTLFYTVNLILPTVLISFLCVLVFYLPAEAG EKVTLGISILLSLVVFLLLVSKILPP 8. DP SYEDVTFYLIIQRKPLFYIVYTIIPCILISILAILVFYLPPDAG EKMSLSISALLAVTVFLLLLADKVPE 9. DDTDFOEIIFFLIIORKPLFYIINIIAPCVLISSLVVLVYFLPAQAGGOKCTLSISVLLA9TIFLFLIAQKVPE 10. NGTNYQDVTFYLIIRRKPLFYVINFITPCVLISFLASLAFYLPAESG EKMSTAISVLLAQAVFLLLTSQRLPE 11. P TPYLDITYHF MQR PLYF VNVIIPCLLFSFLT VFYLPTDSG EKMTLSISVLLSLTVFLLVIVELIPS 12. DT R EL I PC L S L LvFyLP G EK != IS LL KFLC P

FIG.5. Sequences and alignment of amino acid sequences of acetylcholine receptor proteins. Sequences are ( 1 ) Torpedo califmica electric organ cx subunit (Noda et a l . , 1982); (2) partial sequence of chicken (Callus clonusticu) erythrocyte a subunit (Boulter ef a[., 1985); (3) mouse ( M u musculu) BCJH-1 cell (Y subunit (Boulter et a l . , 1985); (4) calf (Bos f a u w ) striated muscle (Y subunit (Noda et al., 1983b); (5) human (Y subunit (Noda et ul., 1983b); (6) rat (Rattu nomegicu) neuronal PC 12 cell protein (Boulter et al. , 1986b); (7) Drosophilu melunopastn neuronal protein (Hermans-Borgmeyer

141

NEUROTOXIN-BINDINGS I T E

+2?LTransmembrane

-I

1. 2. 3. 4. 5.

6. 7. 8. 9. 10. 11. 12. 1. 2. 3. 4. 5. 6. 7.

a.

9. 10. 11. 12.

3-

.

300

325

SKEKQEN SRDKPDK SROKQE SREKQDK SREKQDK TSLVIPLIGEYLLFTMIFVTLSIVITVFVLNVHYRTPTTHTMPTWVKAVFLNLLPRVMF MTRPTSGEGDTP TSLVLPL IAKY LLFTF IMNTVSILVTV IIINUNFRGPRTHRMPMYI RS IFLHYLPAFLF MKRP R TSLSVPIIIRYLMFIMILVAFSVILSVVVLNLHHRSPNTHTMPNWIRQIFIETLPPFLW IQRPVTTPSPDS TSLNVSL IGKYL IFVMFVSML IVMNCVI VLNVSLRTPNTHSLSEKIKHLFLGFLPKYLR MQLEPSEETPE TALAVPLIGKYLMFIMSLVTGVIVNCGIVLNFHFRTPSTHVLSTRVKQIFLEKLPRILH MSRADESEQPDW TSSAVPLIGKYMLFTM F V I S I I I T V VINTHHRSPSTH MP WVRK F I D T I P N MFFSTMKR S K -V V N RPTH F P MR T S &I Y F M 350 TSSAVPLIGKYMLFTMIFVISSIIITVVVINTHHRSPSTHTMPQWVRKIFIDTIPNVM~FSTMKRA

TSSAVPLIGKYMLFTMVFVIASIIITVIVINTHHRSPSTHTMPPWVRKIFIDTIPNIMFFSTMKRP TSSAVPLIGKYMLFTMVFVIASIIITVIVINTHHRSPSTHIMPEWVRKVFIDTIPNIMFFSTMKRP TSSAVPLIGKYMLFTMVFVIASIIITVIVINTHHRSPSTHVMPNWVRKVFIDTIPNIMFFSTMKRP TSSAVPLIGKYMLFTMVFVIASIIITVIVINTHHRSPSTHVMPNWVRKVFIDTIPNIMFFSTMKRP

K

IFAODIDISD

K

IFAEDIDISE

ISGKQVTGEVIFQ I SGKOGPVPVNFY K R~FTEDIDISD ISGKPGPPPMGFH K IFTEDIDISD ISGKPGPPPMGFH K IFTEDIDISD ISGKPGPPPMGFH KTRTFYGAELSNLNCFSRCRLQKLQGRLPLPR WDLWL LPHRRVKISNFSANLTRSSS KTRLPWMMEMPGMSMPAHPHPSYGSPAE LPKHISAIGGKQSKMEVMELSD LHHPNCKINRKVNSGGELGLGD KPTIISR ANDEYFIRKPA GDFVCPVDNARVAVOPERLFSE KPO PRRRSSFG IMIKAEEYILKKPR SELMFEEQKDRHGLKRVNKMTSDI D I QNDLKLRRSSSVG YISKAQEYFNIKSR SELMFEKQSERHGLVPRVTPRIGFGN K I F DIDIS ISGK F K -

c h ~ h i ~ a t h i Hc e?l i x~+ ~ 4?!-Transmembrane 4 -I 1. T P L ~ K N P DVKSAIEGVKYIAEHMKSDEESSNAAEEWKYVAMVIDHILLCVFMLICIIG SPLTKNP SVKNAIEGIKYIAETMKSDQESSN 2. 3. S P L I KHP EVKSAI EGVKYIAETMKSDOESNNAAEEWKYVAMVMDHILLGVFMLVCL I G SPL IKHP EVKSA IEG IKY IAETMKSDQESNNAAEEWKY VAMVMDH ILLAVFMLVCII G 4. SPLIKHP EVKSAIEGIKYIAETMKSDQESNNAAAEWKYVAMVMDHILLGVFMLVCIIG 5. SESVNAVLSLSALSP E IKEAI QSVKYIAENMKAQNVAKEIQDDWKYVAMVI D R I FLWVFILVCILG 6. LSP EASKATEAVEFIAEHLRNEDLYIOTREDWKYVAMVIDRLOLYIFFIVTTAG 7. GCRRESESESSDSIL MKWHLNGLTQPVTLPQDLKEAVEAIKY IAEQLESASEFDDLKKDWQYVAMVADRLFLYVFFVICSIG 8. GTTVDLY KDLANFAP E IKSCVEACNFI AKSTKEQNDSGSENENWVL I G K V I DKACFWI A L L L F S I G 9. NNENIAASDQLHDEIKSGI DSTNY IVKQ I KEKNAY DEEVGNWNLVGQTIDRLSMFI ITPVMVLG 10. PL K P VK AIEG KYIAE MKSD ES NAA EWKYVAMV D H I L L VFML C I G 11. IA WIv_ D G 12.

P K

425

437

1. TVSVFAGRLIELSQEG 2. 3. TLAVFAGRL IELHQQG 4. TLAVFAGRL IELNQQG 5. TLAVFAGRLIELNQQG 6. TAGLFLQPLMARDDT 7. TVGILMDAPH IFEY VDODRI I E I Y RGK a. TFSIFLDASHNVPPDNPFA 9. T L A I FLTGHFNQVPEFPFPGDPRKYVP 10. T I FIFVMGNFNHPPAKPFEGDPFDYSSDHPRCA 11. T VFAGRLIEL Q G 12. T F ef al., 1986); (8) Torpedo califomica electric organ p subunit (Noda el al., 1983a); (9) Torpcuh californica electric organ y subunit (Noda et al., 1983a); (10) Torpedo califomica electric organ 6 subunit (Noda ct at., 1983a); (11) residues present in all (x subunits (1-5); (12) residues present in all or nine (underlined) of the listed sequences (1-10). Numbering of amino acids is for mature a subunit. Sequences are aligned with a minimal number of gaps. Positions of putative transmembrane segments (Boulter et al., 1985) are indicated.

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THOMAS L. LENTZ AND PAUL T. WILSON

complex of highly homologous subunits derived from a common ancestor by means of gene duplications, then homologous binding domains might be present on some or all of the subunits (Conti-Tronconi and Raftery, 1986). In this case, Asp/Glu 200 or Asp 138 are possible candidates since they are present in all of the receptor proteins and subunits. Finally, the negatively charged binding site may not be a single anionic residue, but may be a domain containing several charged and/or polar residues. Such a domain need not contain identical residues in all AChR as long as its overall properties are preserved. Examination of other residues in the region of the extracellular a subunit cysteines reveals that both the region around Cys 192 and Cys 193 and that encompassing Cys 128 and Cys 142 contain a high proportion of hydrophobic residues. The region around Cys 192, 193 contains more aromatic residues (Tyr, Phe, Trp), which play an important role in protein-protein interactions (Burley and Petsko, 1985). Comparing two 30-residue segments encompassing these cysteines, nine aromatic amino acids occur between residues 176 and , five are present between residues 120 and 149. If aromatic 205 (Torpedo)while interactions make a major contribution to bindin*gof neurotoxins and AChR, the region in the vicinity of Cys 192 and Cys 193 appears to provide a more favorable environment for this type of interaction. The neuronal protein, which as noted above may not bind neurotoxins, contains six aromatic residues in the 120-to-149 region like the a subunit. In the 176-to-205 segment, it differs considerably from the a subunit and contains only five aromatic residues. O n the other hand, the Drosophila protein also contains only five aromatic residues between positions 176 and 205, although in Drosophila the protein with properties of the AChR binds a-Btx (Schmidt-Nielsen et al., 1977). The region between residues 176 and 205 in Torpedo also contains three negatively charged residues and eight residues with polar hydroxyl groups on their side chains. Thus, this region is also rich in residues that could potentially contribute to the anionic component of the toxin-binding site. In summary, comparsion of primary sequences does not provide conclusive evidence concerning the location of the neurotoxin-binding site. However, if the binding of toxin is mediated by multiple points of ionic and hydrophobic interaction, a region around Cys 192 and Cys 193 seems to represent the strongest candidate. This segment is highly conserved among a subunits and has multiple charged and hydrophobic residues. The neuronal protein which may represent the nontoxin binding neuronal nictonic AChR differs considerably from the musclelike a subunits in this region.

E. BINDINGOF NEUROTOXINS TO SYNTHETIC RECEPTOR PEPTIDES A potentially useful approach in identifying the neurotoxin-binding site is to determine the ability of synthetic peptides to bind neurotoxins. Binding

NEUROTOXIN-BINDINGSITE

143

characteristics of peptides corresponding to any portion of the subunit sequence can be compared. The role of individual residues in binding can be determined by chemical modification of peptides or by substitution of residues during synthesis. A disadvantage of this approach is that the peptides may not exist in the same conformation as in the intact receptor or that short peptides may not interact with toxin. However, the fact that the denatured, isolated a subunit (Haggerty and Froehner, 1981; Gershoni et al., 1983; Tzartos and Chargeux, 1983) and proteolytic fragments as small as 7 kDa (Wilson et al., 1984) retain the ability to bind neurotoxins indicates that this approach is feasible. McCormick and Atassi (1984) reported that a synthetic peptide corresponding to residues 125 to 147 of the Torpedo a subunit bound lZ5I-labeled a-Btx and [3H]acetylcholine.This peptide contains the region originally proposed by Noda et al. (1982) to represent the acetylcholine-binding site. The binding activity of the peptide was abolished by reduction with 2-mercaptoethanol. The validity of these results has been questioned based on the methodology that was employed to measure binding (Criado et al., 1986). In addition, Neumann et al. (1986b) reported that a peptide corresponding to residues 126-143 did not bind a-Btx. Subsequently, Wilson et al. (1985) synthesized a peptide comprising residues 173-204 of the Torpedo a subunit (32-mer). This sequence was chosen because it contains Cys 192 and Cys 193, shown by Kao et al. (1984) to be labeled by MBTA; it ends prior to the beginning of the presumed first membrane spanning region; and it contains four negatively charged residues (Glu 175, Asp 180, Asp 195, and Asp 200), one of which could represent the anionic subsite of the acetylcholine-binding site. The peptide was shown to bind lZ5I-a-Btxin a dot blot assay. a-Btx and d-tubocurarine inhibited lZ5I-Btxbinding to the peptide with Ic50values of 0.5 pA4 and 2 mM, respectively. More thorough studies indicate an affinity for a-Btx of 63 nM in the absence of sodium dodecyl sulfate and 7.2 nM in the presence of 0.01 % sodium dodecyl sulfate (Wilson et al., 1987). Thus, the affinity of toxin for the 32-mer is the same as for the isolated a subunit (Haggerty and Froehner, 1981; Gershoni et al., 1983). Furthermore, as is the case with the intact isolated a subunit (Tzartos and Changeux, 1983), partial renaturation of a-Btx binding to the 32-mer can occur in the presence of sodium dodecyl sulfate. These data suggest that the 32-mer possesses most or all of the determinants of the decreased affinity, a-Btx-binding site detected on the isolated a subunit. Mulac-Jericevic and Atassi (1986) then reported that a synthetic peptide corresponding to residues 182 to 198 of the Torpedo cr subunit bound lZ5I-a-Btx and lZ5I-labeleda-cobratoxin. Binding of toxins to this peptide was compared with binding to Torpedo and human peptides comprising residues 125 to 148. Assays were performed by measuring binding of labeled toxins to peptides coupled to Sepharose. It was found that peptide 182-198 bound 74% of

144

THOMAS L. LENTZ AND PAUL T. WILSON

the amount of labeled a-Btx as bound by intact AChR and that the human and Torpedo peptides 125-148 bound about half as much toxin as peptide 182-198. The affinities of a-Btx and a-cobratoxin for peptide 182-198 were 6.6 x lO-’M and 1 x lo-’ M, respectively, while for human peptide 125-148 the corresponding values were 1.5 x lO-’M and 2.2 x lo-’ M. It was concluded that residues 182-198 comprise a second toxin binding region in addition to residues 125-148 on the CK subunit. A 12-residue peptide comprising residues 185-196 of the Torpedo a subunit has been shown to bind 12s1-cx-Btxin a dot blot assay (Neumann et d.,1986a). Subsequently, the affinity of the 12-mer linked to Sepharose for a-Btx was reported to be 3.5 x M (Neumann et d.,1986b). Peptides corresponding to residues 1-20, 126-143, 143-158, 169-181, 193-210, and 394-409 of the a subunit did not bind toxin significantly. Reduction and carboxymethylation of the cysteine residues on peptide 185-196 inhibited its capacity to bind toxin. Interestingly, while the Torpedo 12-mer (185-196) bound a-Btx, the corresponding human peptide did not. Comparison of the Torpedo and human a subunit sequences between residues 185 and 196 shows that, in human, Ser is substituted for T r p 187 and Thr for Tyr 189. The lack of binding to the human 12-mer suggests that Trp 187 and Tyr 189 play an important role in neurotoxin binding. A receptor tryptophan residue has been reported to be present in the vicinity of the acetylcholine-binding site (Heidmann and Changeux, 1978; Tsetlin et d., 1982). Furthermore, chemical modification of the tryptophan in peptide 185- 196 by 0-nitrophenylsulfenylchloride and 2,4-dinitrophenylsulfenylchloridesignificantly reduced the ability of the peptide to bind toxin. Further evidence that the cholinergic-binding site of human and Torpedo AChR may differ comes from work by Momoi and Lennon (1986), who compared the binding of BAC to human muscle and Torpedo electric organ AChR. The human AChR had a higher binding affinity for BAC and an adjacent disulfide bond that was more readily accessible to reducing agents, suggesting a structural dissimilarity in the acetylcholine-binding region. Ralston et QL. (1987), investigated a-Btx binding to peptides corresponding to residues 127-143, 172-189, 194-212, 172-205, and 185-199 of Torpedo a-subunit coupled to Biodyne membranes. a-Btx was found to bind to 172-205 and 185-199 but not to the other peptides. Cold toxin blocked binding of labeled toxin to 172-205 with an ICSO value of 0.5 /.LM while a 100-fold higher concentration of cold toxin was required to block binding to 185-199. Because toxin bound to peptides 172-205 and 185-199 but not to peptides 172-189 and 194-212, residues 190-193 appear to be critical for binding. In addition, reduction and alkylation of 172-205 abolished toxin binding, indicating that Cys 192 and 193 must be disulfide cross-linked for toxin binding to occur.

NEUROTOXIN-BINDING SITE

145

The studies using synthetic receptor peptides most strongly point to the region around Cys 192 and Cys 193 as a major determinant in toxin binding. An exception was the report that a peptide including Cys 128 and Cys 142 bound toxin, but this same group later reported that a peptide containing Cys 192 and Cys 193 bound toxin with higher affinity. The affinities of toxin binding to the 32-mer (173-204), 34-mer (172-205), and the 17-mer (182-198) are comparable to the affinity of toxin binding to isolated a subunit (Gershoni et al., 1983), although this is less than to the intact receptor. The affinity of toxin for the 12-mer (185-196) and 15-mer (185-199) is less than for the 32-mer and the 17-mer. Recently, we have compared the binding of a-Btx to the 32-mer and the 12-mer under identical assay conditions. The affinity for the 12-mer under these conditions agrees almost exactly with the affinity reported by Neumann et al. (1986b) and is 500-fold less than the affinity of the 32-mer (Wilson et a l . , 1987). These results suggest that multiple determinants for toxin binding are distributed along the 32-mer sequence. It is not yet clear whether all these determinants directly interact with a-Btx or whether some are responsible for creating a conformation of the 32-mer more favorable for high-affinity binding.

F. MONOCLONAL ANTIBODIES AS PROBES OF THE NEUROTOXIN-BINDING SITE Monoclonal antibodies have proven to be extremely useful tools to study the synthesis, structure, and function of the AChR (Lindstrom, 1986). Antibodies are directed against relatively small regions of the antigen (5-7 amino acids). By mapping the epitopes of antibodies that affect function (e.g., ligand binding), these antibodies can be used to localize the cholinergic-binding site on the receptor. Some caution is necessary, however, because the antibodies are considerably larger than neurotoxins, and antibody binding to a site other than the cholinergic-binding site may interfere with toxin binding by steric hindrance. Most monoclonal antibodies raised against the receptor do not affect function (Lindstrom, 1986). However, several groups of investigators have shown that some monoclonal antibodies raised against electric organ AChR block the binding of a-Btx (Mochly-Rosen and Fuchs, 1981;James et al., 1983; Souroujon et al., 1983; Watters and Maelicke, 1983; Mihovilovic and Richman, 1984; Whiting et a l . , 1985; Lukas, 1986). Some of these antibodies inhibited only 50 % of toxin binding, suggesting they could distinguish between the two cholinergic-binding sites (James et al., 1983; Watters and Maelicke, 1983; Mihovilovic and Richman, 1984; Whiting et al., 1985). Some of the monoclonal antibodies cross-reacted with receptor from other species

146

THOMAS L. LENTZ A N D PAUL T. WILSON

(Mochly-Rosen and Fuchs, 1981; Souroujon et al., 1983; Watters and Maelicke, 19832; Lukas, 1986) while others did not (James et al., 1983; Whiting et a l . , 1985), suggesting that the toxin-binding region may contain both conserved and variable determinants. More recently, antibodies to synthetic peptides have been used to map the cholinergic-binding site. This approach has the advantage that, at least to the length of the peptide, the location of the antigenic site on the sequence of the receptor is known. Several groups have investigated the ability to inhibit toxin binding of monoclonal antibodies rasied against peptides located within residues 125 and 147. Monoclonal antibodies raised against peptides corresponding to residues 127-132 of the a subunit (Pliimer et a l . , 1984), a disulfide-looped peptide corresponding to residues 125-147 (Lennon et al. , 1985), and a disulfide-looped peptide corresponding to residues 127- 143 (Criado et al., 1986) failed to inhibit binding of a-Btx to the AChR. Similarly, cholinergic ligands did not interfere with antibody binding to the receptor (Plumer et al., 1984; Criado et a/. , 1986). In addition, Neumann et al. (1985) showed that antibodies raised against peptides comprising residues 1-20 and 126-143 bound to proteolytic fragments of the a subunit that did not bind a-Btx, while an a-Btx-binding fragment was not recognized by the antipeptide antibodies. In all cases, the antipeptide antibodies reacted with native receptor. Lennon et al. (1985) reported that immunization of rats with peptide 125-147 induced myasthenia gravis in rats and suggested that this region is a major antigenic determinant on the a subunit. Others did not report induction of myasthenia gravis after immunization with peptides within this region. Furthermore, some evidence suggests that this region is not fully exposed on the intact receptor. Ten times as much native receptor as peptide 126-143 was necessary to inhibit binding of '251-labeledAChR to the antibodies against the peptide (Neumann et al. , 1985). Similarly, receptor-rich membrane vesicles did not inhibit binding of serum antibodies raised against peptide 127-143 to the peptide, and monoclonal antibodies against the peptide did not bind as well to the membrane-bound receptors as to detergent-solubilized receptor (Criado et al., 1986). Although such differences in binding may reflect conformational differences between the peptide used as immunogen and the region in the intact receptor, the cyclized peptide used in the studies by Criado et al. (1986) may more accurately reflect the native conformation of this region. One of the monoclonal antibodies against peptide 127-143, in addition, crossreacted with other subunits. This antibody bound more effectively to membrane-bound receptors than two monoclonal antibodies that recognized only CY subunit. It was suggested that the part of the 127-143 sequence which is conserved between subunits (residues 135-143) is more exposed than the part (residues 127-134) that is unique to the a subunit (Criado et al. , 1986).

NEUROTOXIN-BINDING SITE

147

These studies employing antibodies are in general agreement with the results obtained with synthetic peptides that residues within the region 125-147 do not contribute to the cholinergic-binding site. Further evidence that this region is not a binding site is provided by the results indicating that this region may not be fully exposed on the surface of the intact receptor. Comparable studies utilizing other regions of the a subunit, especially in the vicinity of Cys 192 and Cys 193, would be helpful in determining the contribution of this or another region to the binding site. Ralston et al. (1987) reported that antibodies raised against a-subunit peptide 172-205 did not bind to the native receptor, indicating the conformation of this region in the native receptor is highly restrained.

G. SITE-DIRECTED MUTAGENESIS A potentially powerful tool in correlating receptor structure and function is site-directed mutagenesis (Stevens, 1984). With this technique, the cDNA encoding for the receptor is modified, e.g., by changing the codon for one amino acid to the codon for a different amino acid or by deleting portions of the DNA. The modified cDNA can be introduced directly into the nucleus or mRNA prepared from the cDNA is introduced into the cytoplasm of a cell lacking receptor. Upon synthesis of the protein in the cell, the functions of the receptors altered at specific sites can be compared. Mishina et al. (1985) have studied the function of AChR mutants synthesized in Xenopus oocytes after alteration of the a subunit by site directed mutagenesis of the cDNA. Some of these changes affected a-Btx binding as well as ion flux into the cell, None of a number of deletions in the a subunit on the C-terminus side of residue 249 had a significant effect on toxin binding. However, a mutant in which residues 224-237 were changed to Pro-Ser-Ser showed markedly decreased toxin binding. It was suggested that this mutation, which involved a deletion of part of the first transmembrane segment which follows the large extracellular portion of the a subunit, may have resulted in a conformational change in the region encompassing the toxin-binding site. Substitution of a Ser for either Cys 128 or Cys 142 virtually eliminated toxin binding as well as acetylcholine-induced ion flux. Substitution of Ser for Cys 192 and Cys 193 reduced toxin binding to 39 and 28%, respectively, relative to native receptor, while both changes eliminated ion flux. Because the mutation of either of the two cysteines contributing to a disulfide bond should have equivalent effects, the effects of these mutations were considered consistent with the presence of a disulfide bridge between Cys 128 and Cys 142 and between Cys 192 and Cys 193, in agreement with the conclusion of Kao and Karlin (1986). Conversion of Asn 141, the site of N-linked

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glycosylation, to Asp eliminated a-Btx binding and acetylcholine response, and it was suggested that N-glycosylation at Asn 141 is essential for maintaining a functional conformation of the a subunit. Caution is required in the interpretation of functional changes in the receptor resulting from the alteration of one or more residues, because such alterations can have multiple effects on the receptor which in turn indirectly affect the function being observed. If a deletion or alteration in a structural component has no effect, it seems reasonable to assume that the structure is not involved in that particular function. However, in cases where the function, e.g., toxin binding or ion flux, is lost, the mechanism by which function is altered must be determined. For example, loss of ability to bind toxin may be the result of alterations of the actual binding site, the conformation of the a subunit, or the assembly with other subunits. Indeed, mutants in which Cys 128 or Cys 142 was converted to serine contained substantially smaller amounts of a,y, and 6 subunits (Mishina et al., 1985). Thus, it was suggested that mutation of these cysteines affected the conformation of the a subunit or its assembly with other subunits. Since these cysteines occur in all four subunits, the proposed disulfide between them may be necessary for maintaining the conformation of the extracellular domain of the AChR molecule. In contrast, when Cys 192 or Cys 193 was converted into serine, the content of subunit polypeptides in the mutants was comparable with that of the wild-type receptor. Because these mutations also caused a reduction in binding affinity of agonist (ability of carbachol to inhibit a-Btx binding) and a loss of acetylcholine sensitivity, these residues were considered to be involved in the binding of acetylcholine and in the signal transduction that leads to opening of the channel (Mishina et al., 1985). Another approach to receptor function involving genetic engineering is to investigate the properties of fusion proteins expressed in bacteria. Barkas et al. (1987) subcloned specific restriction fragments of mouse a subunit cDNA into the 0-galactosidase gene fragment of Escherichia coli plasmid vector pUC8. The fusion proteins containing sections of the a subunit were tested for their ability to bind a-Btx and monoclonal antibodies against the main immunogenic region (MIR). a-Btx was found to bind to all fusion proteins containing residues 160-216 but not to proteins lacking this sequence. Gershoni (1987) showed that transformants expressing residues 166-200 of the a-subunit bound a-Btx, confirming the findings of Barkas et al. (1987). Mapping of the antibody-binding proteins showed that the MIR of the receptor is located between residues 6 and 85 and consists of at least two epitopes (Barkas et a l . , 1987). Peptide mapping techniques have localized the MIR between residues 46 and 127 (Ratnam et al., 1986a).

149

NEUROTOXIN-BINDING SITE

H. MODELS OF THE CHOLINERGIC BINDINGSITE Several models of the acetylcholine-bindingsite have been proposed based on deductions of the tertiary structure of the AChR from the primary structure. Shortly after the N-terminus sequence of the a subunit was determined by amino acid sequencing(Devillers-Thiery d ul., 1979; Hunkapiller etal., 1979), Smythies (1980) proposed a model for the acetylcholine-bindingsite in which residues 1-1 5 were predicted to form an a-helix and residues 17-22 a ,&sheet. Two of these segments in an antiparallel alignment would form four ionic pairs between Arg/Lys and Glu, each of which is complementary to acetylcholine. This model seems highly unlikely, since the two a subunits are not adjacent and antibodies raised against the N-terminus 10amino acids do not cross-react with the receptor, indicating that the N-terminus is inaccessible (Ratnam and Lindstrom, 1984). After the entire primary amino acid sequence of the a subunit became available (Noda et al., 1982), other structural models of the cholinergic-binding site were proposed. Noda d ul. (1982) first proposed that the acetylcholine-binding site is located in the region includingthe cysteine residues at positions 128and 142. Measurements on Corey-Pauling-Kolton space-filling models were consistent with the involvement of Asp 138(or Glu 129)as the negative subsite and His 134as a hydrophobic subsite in the binding of acetylcholine. An alternative region proposed was near Cys 192 and Cys 193 with Asp 195or Asp 200 as possible anionic subsites. Later, the same group, based on site-directed mutagenesis experiments, concluded that the Cys 128-142 disulfideis involved in maintaining the conformation of the subunit, while Cys 192 and Cys 193 are involved in the binding of acetylcholine (Mishina et al., 1985). Kosower (1983b) proposed a model in which acetylcholine binds to Lys 354 and Asp 371, which are considered to be in close proximity on adjacent amphipathic helices. Lys 354 is the fourth residue from the extracellular surface in the fourth transmembrane segment, and Asp 371 is the fourth residue in the fifth transmembrane segment in the a subunit secondary structure model proposed by Kosower (1983a). This model differs from others in that it places the acetylcholine-binding site within the channel itself. Smart et al. (1984) proposed that residues 135-142 of the a subunit form a cholinergic-bindingsite based on model building. A distance of 10 was proposed between Cys 142, the proposed alkylation site, and Asp 138, the proposed negative subsite. In addition, a model was presented for the binding of loop 2 of a-cobratoxin with this region of the a subunit. The following interactions between the toxin and the a subunit were proposed: stack between a subunit Phe 137 with toxin Trp 29, ionic interaction between Asp 138 and toxin Lys 27 and Lys 53, hydrogen bonding between Gln 139 and toxin Thr 51, and dipoleenhanced hydrogen bonding between Gln 140 and toxin Asp 42 and Lys 27.

A

150

THOMAS L. LENT2 AND PAUL T. WILSON

Luyten (1986) has proposed a model for the acetylcholine-binding site based on Corey-Pauling-Kolton models. Segments 125-145 and 185-200 were modeled. The model best fitting the structure-activity relationships of acetylcholine was considered to be segment 125-145. Acetylcholine was proposed to fit in a pocket on one face of an antiparallel /3-pleated sheet formed by residues 125-145. Acetylcholine fit into a cleft between the side chains of Ile 131, Thr 133, Asp 138, and Gln 140. A ring of negative charges is formed around the quanternary ammonium group of acetylcholine by Asp 138, T h r 133, and Gln 140. The P-pleated sheet is stabilized by a &turn at one end and a double cystine bridge at the other end involving Cys 142, 192, 193, and 128. Although it has now been determined that disulfides occur between Cys 192 and Cys 193 and between Cys 128 and Cys 142, it was felt that this arrangement would not alter the folding of the proposed site. Because deductions of three-dimensional structure from the primary sequence are unreliable, such detailed models of the binding site are highly speculative. However, the three-dimensional structure of the neurotoxins is known and Fairclough et al. (1983) have pointed out that the a-Btx region on the receptor surface will be complementary to the toxin’s concave surface, which comes into contact with the receptor. Thus, it is possible to map domains of the AChR surface within the binding interface as positively charged, negatively charged, hydrophobic, or hydrogen bonding based on the most attractive interaction with the side chains of the overlying toxin molecule. This approach is more likely to characterize accurately the overall characteristics of the receptor surface involved in the binding of neurotoxins. The size of the domains is determined by the maximum flexibility of toxin side chains on the fixed toxin backbone. A chargehydrophobic character map of the receptor was constructed which showed that the receptor-binding surface consists of rows of hydrophobic and ionic domains alternating every 5-8 A (see also Fig. 4). Beginning with the tip of loop 2 of a-Btx, a hydrophobic region lies under this tip which contains Phe 33. Next to this on the receptor surface is an ionic domain making contact with Asp 31, Arg 37, and Lys 39. The anionic subsite under Arg 37 may form part of the acetylcholine-binding site. Adjacent to this ionic domain on the receptor surface is a hydrophobic domain underlying Tyr 56 at the end of loop 3 , Trp 29, and Val 40. Next to this is another ionic domain with charged subsites underlying Lys 53, Lys 52, Lys 27, and Glu 42. It has been suggested that Lys 27 is near a sulfhydryl in bound toxin (Tsetlin et a[., 1982). The area under Lys 27 could be lOA from Arg 37, matching the distance between the MBTA-labeled disulfide and the anionic subsite. Adjacent to this anionic domain on the receptor surface is a large hydrophobic band underlying residues 46-50, Tyr 25, and most of loop 1 . Next to this, a cationic site underlies Glu 2 1 .

NEUROTOXIN-BINDING SITE

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V. Conclusions

Although precise localization and characterization of the neurotoxinbinding site must await X-ray crystallographic analysis of the toxin-receptor complex, certain conclusions can be drawn on the basis of present evidence. Neurotoxins bind to both a subunits on the AChR monomer. Most or all of the binding determinants are located on the a subunit, although some interaction with other subunits cannot be ruled out. Affinity alkylating agents that act as either agonists or antagonists bind to a cysteine residue that is located within 10 of the acetylcholine-binding site (Karlin, 1980). The affinity ligand MBTA labels primarily Cys 192 (Kao et al., 1984), which appears to be linked by a disulfide to Cys 193 (Kao and Karlin, 1986). O n the basis of peptide mapping, neurotoxins bind on the C-terminal side of Asn 141, the site of N-linked glycosylation. Comparative sequence data shows that most of the residues between positions 170 and 209 are highly conserved in a subunits of different species but not in the other subunits. This region contains an unusually high number of hydrophobic and aromatic residues as well as many negatively charged and polar residues (Fig. 6). Several groups have demonstrated binding of a-Btx to synthetic peptides matching in sequence portions of the segment 170-209. A 32-residue peptide (173-204) binds a-Btx with the same affinity as isolated a subunit (Wilson et a l . , 1985). Thus, present evidence strongly suggests that most of the determinants for neurotoxin binding are located between residues 170 and 209. The affinity of binding of a-Btx to isolated denatured a-subunit, peptide fragments, or synthetic peptides is considerably less than that to the intact receptor (Gershoni et al., 1983; Wilson et al., 1983, 1984). The reduced affinity could be the result of a loss of native conformation of the 170-209 region in the denatured protein. It is also possible that, in the native receptor, additional residues of the a-subunit or even of other subunits contribute to the neurotoxin-binding site. These residues could be widely separated on the linear sequence of the a-subunit but brought into proximity through folding of the native protein. 9

170

-

FMEs

G

- 9

+ - 9

E w VM KDY

8-8 9 9 9 + 9 CCTYYVWHKW P 9 0 - D T P Y L D I T Y H

R+ G

9 F I

+ MQR7-09

FIG. 6 . Torpedo culifornicu a subunit residues 170-209. This segment of the a subunit is located just prior to the first putative transmembrane segment. Turns in the sequence are located at regions predicted as 8-turns by secondary structure predictions. ( -) Negatively charged residues (Asp, Glu); ( + ) positively charged residues (Lys, Arg); (6)aromatic residues (Tyr, Phe, Trp).

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THOMAS L. LENTZ AND PAUL T. WILSON

Attention has also been directed toward the region around Cys 128 and Cys 142 as a possible binding site. This region also contains, in addition to the two cysteines, charged and hydrophobic residues and is highly conserved among a subunits, although it shows greater similarity with other subunits than does segment 170-209. The suggestion that this region is a binding site is based largely on theoretical considerations and model building but there is little direct experimental support. In fact, monoclonal antibodies directed against peptides within the region 125-147 do not block a-Btx (Plumer et al., 1984; Lennon et a l . , 1985; Criado et a l . , 1986). Peptide 127-143 does not bind a-Btx (Ralston et al., 1987). It seems likely that Cys 128 and Cys 142 are involved in maintaining the conformation of the a subunit or its assembly with other subunits (Mishina et al., 1985). The primary event in the binding of neurotoxins to the AChR is most likely interaction with the acetylcholine-binding site since toxin binding can be effectively competed by small cholinergic agonists in sufficient concentration. It cannot be ruled out, however, that toxins bind to an overlapping or even to a different site and that the apparent competition is due to another mechanism. For example, Kang and Maelicke (1980) reported that small cholinergic ligands accelerate the rate of dissociation of the toxin-receptor complex. One mechanism to explain this finding is that small ligands and toxin bind to separate sites on the receptor to form ternary complexes. Binding of one type of ligand to a preformed complex of another type of ligand causes structural rearrangements in the receptor whereby the affinity for the initially bound ligand is decreased. Thus, binding of neurotoxins and agonists could take place at separate sites and still be mutually exclusive if binding of one ligand causes a conformational change in the receptor that alters the binding site of the other ligand so that it cannot bind. Several observations indicate the neurotoxin- and acetylcholine-binding sites, if not the same, are very closely associated or overlapping. a-Btx binds to a synthetic peptide corresponding to residues 173-204 of the a-subunit with an affinity equal to that of the intact a-subunit, and this binding is competed by d-tubocurarine (Wilson et a l . . 1985). In addition, the sequence 179-207 is labeled by the affinity alkylating agents MBTA (Kao et al., 1984) and MPTA (Dennis et a l . , 1986) and the cholinergic photoaffinity ligand DDF (Dennis et al., 1986). Present evidence supports the hypothesis that toxin binding to the receptor involves a local recognition event between structurally and chemically complementary surfaces followed by an induced fit in which hydrophobic interactions are promoted by rearrangements and conformational changes in the toxin and receptor. The initial association between neurotoxin and the AChR may be due to complementarity in structure between residues near the end of loop 2 and the acetylcholine-binding site. Contact between complementary

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structures is thermodynamically most favorable and accounts for the specificityof the interaction (Chothia and Janin, 1975). The complementary surface on the a subunit is likely to include residues in the acetylcholine-bindingsite itself as well as neighboring residues, some of which may be involved in maintaining the correct conformation of the acetylcholine-binding site. This conclusion is supported by the observation that a-Btx binds to denatured a subunit under conditions where there is no specific ability ofthe agonist, carbamylcholine,to compete a-Btx binding (Tzartos and Changeux, 1983). This suggests that a portion of the complementary surface of the a subunit recognizedby a-Btx is independent of the conformation of the agonist-binding site. The affinity of a-Btx binding, however, increases under conditions that allow for the partial renaturation of the agonistbinding site (Tzartos and Changeux, 1984), suggestingthat residues important in the acetylcholine-binding site also contribute to the a-Btx-binding site as well. Acetylcholine is a hydrophobic cation whose binding to the receptor may be mediated by an ionic interaction involving the quaternary nitrogen, a hydrogen bond involving the carbonyl oxygen, and a hydrophobic interaction involving the methyl group (Beers and Reich, 1970). The complementaryreceptor surface can be expectedto include an anionic subsite, anucleophilicgroup, and a hydrophobic area near Cys 192. Models have been proposed in which neurotoxin residues form stereochemical analogs of acetylcholine or d-tubocurarine. Although further evidence is required to determine the validity of the models, it seems more likely that a group of charged residues including the cationic Arg 37 binds to a region of the receptor including the anionic subsite. In addition, it might be expected that the structural constraints of binding to the acetylcholine-bindingsite require similar geometries in all ligands which bind to this site and complementarity between the structures of these ligands and receptor surface. After the initial binding event, it is likely that the toxin-receptor interface is stabilized by an induced fit accompanied by local rearrangments in the flexible portions of the two molecules which facilitate the formation of multiple interactions between the concave surface of the toxin and the surface of the a subunit. The concave surface of the toxin contains a triplestranded 0-sheet structure'(Walkinshaw et al., 1980). The evidence reviewed here points most strongly to the conclusion that residues 170-209 of the a subunit form a neurotoxin-binding site and that most of the determinants of binding are located on this linear sequence. Because secondary structure predictions (Chou and Fasman, 1978; Gamier et al., 1978) indicate that this segment contains considerable /3-sheet structure and two &turns, it could form a surface structurally and chemically complementary to the alternating pattern of ionic and hydrophobic/hydrogen-bondingdomains on the concave surface of toxin (Fairclough et al., 1983; Fig. 4). The presence of invariant aromatic residues (Trp 29, Phe/His/Trp 33) and hydrophobic

154

THOMAS L. LENTZ AND PAUL T. WILSON

domains in neurotoxins and the high proportion of hydrophobic and aromatic residues on a subunit segment 170-209 indicate that, if binding occurs at this region, hydrophobic free energy (Chothia and Janin, 1975) would make a major contribution to the free energy required for association. Local side-chain displacements or larger conformational changes in the receptor and possibly also in the toxin may promote optimal stereochemical complementarity between toxin and receptor and increase the affinity of binding. Such a mechanism of toxin binding is consistent with other known protein interactions, including strong &domain interactions (Getzoff d al., 1986). In these interactions, binding energy depends on maximizing the hydrophobic surface area in the buried interface (Chothia and Janin, 1975; Getzoff et al., 1986), aromatic-aromatic interactions stabilize protein structure and ligand binding (Burley and Petsko, 1985), complementary hydrophobic and aromatic residues are prevalent in the binding sites of antibodies (Amit et al., 1986; Getzoff et al., 1987), and electrostatic forces stabilize specific contacts (Getzoff et al., 1986, 1987). Agonist binding is followed by conformational transitions in the AChR that result in channel opening and desensitization. Karlin (1969) proposed that the bridging of the negatively charged subsite and a hydrophobic subsite by acetylcholinecauses a local conformational change in the receptor. With the identification of the unusual disulfide between Cys 192 and Cys 193 near the acetylcholine-bindingsite, the possibility has been raised that a transition between conformations of the disulfide and peptide bonds between these residues occurs upon acetylcholine binding (Kao and Karlin, 1986). The initial conformational change occurring upon acetylcholine-binding leads to subsequent changes resulting in channel opening. An increase in &structure of the receptor upon binding of agonist has been observed (Mielke el al., 1986). It is of particular interest that binding sites for noncompetitive inhibitors, agents which block the ion channel are located on the first putative transmembrane segment which closely follows the putative acetylcholine-bindingregion (DiPaola et al., 1986). Thus, it seems possible that changes in conformation can progress sequentially along the polypeptide chain from the acetylcholine-bindingsite to elements of the channel.

Acknowledgments

The author’s research is supported by NIH grant NS 21896, NSF grant BNS 85-06404, and USAMRAA contract DAMDl7-86-6043.

References

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Amit, A. G., Mariuzza, R . A., Phillips, S. E. V., and Poljak, R . J. (1986).Scicnce 233, 747-753. Anderson, D. J., and Blobel, G. (1981).Roc. Natl. Acad. Sci. U . S . A . 78, 5598-5602. Anderson, D. J., Blobel, G., Tzartos, S . , Gullick, W., and Lindstrom, J. (1983).J.Neurosci. 3, 1773-1 784. Babbit, B., and Huang, L. (1985).Biochemistry 24, 15-21. Balasubramaniam, K., Eaker, D.;and Karlsson, E. (1983). Toxicon 21, 219-229. Barkas, T . , Mauron, A., Roth, B., Alliod, C . , Tzartos, S. J., and Ballivet, M. (1987).Science 235, 77-80. Barrantes, F. J. (1978).J. Mol. Bid. 124, 1-26. Barrantes, F. J. (1983).Int. Rev. Neurobiol. 24, 259-341. Beers, W.H., and Reich, E. (1970).Nature (London) 228, 917-922. Bon, F., Lebrun, E., Gomel, J., Van Rapenbusch, R., Cartaud, J., Popot, J.-L., and Changeux, J:P. (1984).J. Mol. B i d . 176,205-237. Bonner, R., Barrantes, F. J., and Javin, T. M . (1976).Nature (London) 263, 429-431. Boulter, J., Luyten, W., Evans, K., Mason, P., Ballivet, M., Goldman, D., Stengelin, S., Martin, G., Heinemann, s.,and Patrick, J. (1985).J. Neurosci. 5, 2545-2552. Boulter, J., Evans, K., Martin, G., Mason, P., Stengelin, S., Goldman, D., Heinemann, S., and Patrick, J. (1986a).J. Neurosci. Res. 16, 37-49. Boulter, J., Evans, K., Goldman, D., Martin, G., Treco, D., Heinemann, S., and Patrick, J. (1986b). Nature (London) 319, 368-374. Bourne, P. E., Sato, A., Corfieid, P. W. R., Rosen, L. S., Birken, S., and Low, B. W. (1984). Eur. J . Biochem. 153,521-527. Brisson, A., and Unwin, P. N. T . (1985).Nature (London) 315, 474-477. Burley, S. K., and Petsko, G. A. (1985).Science 229, 23-28. Cahill, S.,and Schmidt, J. (1984).Biochem. Biophys. Res. Commun. 122,602-608. Carbonetto, S. T . , Fambrough, D. M., and Muller, K. J. (1978).Roc. Natl. Acad. Sci. U . S . A . 75, 1016-1020. Carlin, B. E., Lawrence, J. C., Jr., Lindstrom, J. M., and Merlie, J. P. (1986).Roc. Natl. Acad. Sci. U . S . A . 83,498-502. Chang, C. C., and Hayashi, K. (1969).Biochem. Biophys. Rcs. Commun. 37,841-846. Changeux, J.-P. (1981).Harvey Lect. 75, 85-254. Chiappinelli, V. A. (1983).Brain Rcs. 277, 9-21. Chiappinelli, V. A. (1985).P h a m o l . Ther. 31, 1-32. Chicheportiche, R., Vincent, J.-P., Kopeyan, C., Schweitz, H., and Lazdunski, M . (1975). Biochemistry 14, 2081-2091. Chothia, C., and Janin, J. (1975).Nature (London) 256, 705-708. Chou, P.Y.,and Fasman, G. D. (1978).Adv. Emymol. 47, 45-148. Claudio, T.,Ballivet, M., Patrick, J., and Heinemann, S. (1983).Roc. Natl. Acad. sn'. U.S.A.80, 1 1 11-1 115. Cleveland, D. W., Fischer, S. G., Kirschner, M. W., and Laemmli, U. K. (1977).J.Biol. Chem. 252, 1102-1106. Colquhoun, D., and Sakmann, B. (1981).Nature (London) 294,464-466. Conti-Tronconi, B. M., and Raftery, M. A. (1982).Annu. Rev. Biochem. 51, 491-530. Conti-Tronconi, B. M., and Rafiery, M. A. (1986).Roc. Natl. Acad. Sci. U.S.A. 83,6646-6650. Conti-Tronconi, B. M., Hunkapiller, M. W., and Raftery, M. A. (1984). Roc. Natl. Acad. Sci. U.S.A. 81, 2631-2634. Cox, R . N., Karlin, A,, and Brandt, P. W. (1979).J.Membr. Biol. 51, 133-144. Criado, M., Sarin, V., Fox, J. L., and Lindstrom, J. (1986).Biochemistry 25, 2839-2846. Damle, V. N., and Karlin, A. (1978). Biochemistry 17, 2039-2045. Damle, V.N.,McLaughlin, M., andKarlin, A. (1978).Biochnn. Biophys. Res. Commun. 84,845-851. Dennis, M., Giraudat , J., Kotzyba-Hibert, F., Goeldner, M., Hirth, C., Chang, J.-Y., and Changeux, J.-P. (1986). FEBS Lett. 207, 243-249.

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CALCIUM AND S EDATIVE-HYPNOTIC DRUG ACTIONS By Peter L. Carlen Neurology Program Alcoholism and Drug Addiction Research Foundation Playfalr Neuroscience Unit Toronto Western Hospital, and Departments of Mediclne (Neurology) and Physiology and Institute of Medical Science University of Toronto Toronto, Ontarlo, Canada M5S 2S1

and Peter H. Wu Neurology Program Alcoholism and Drug Addiction Research Foundation Department of Pharmacology Unlversity of Toronto Toronto, Ontario, Canada M5S 2S1

I.

Introduction

11. Behavioral Effects 111. Electrophysiology

A.

Calcium Currents Calcium-Dependent Ionic Currents Calcium Currents and Second Messenger Systems Acute Sedative-Hypnotic Drug Actions in Relationship to Calcium and Calcium-Dependent Currents E. Tolerance, Dependence, and Chronic Neuronal Effects of SedativeHypnotic Drugs in Relationship to Calcium and Calcium-Dependent Currents F. Aging Interactions IV. Biochemistry A. Effects of Sedative-Hypnotic Drugs on Influx of Calcium B. Effects of Sedative-Hypnotic Drugs on Calcium Receptors C. Effects of Sedative-Hypnotic Drugs on Intracellular Regulation of Calcium Ion Concentration D. Calcium Second Messenger Systems and Drug Action V. Conclusions References

B. C. D.

1. introduction

Calcium participates in a large number of neuronal biological processes and its physiological role has been very extensively reviewed in recent years 161 INTERNATIONAL REVIEW OF NEUROBIOLOGY, VOL. 29

Copyright 0 1988 by Academic Press, Inc. AU rights of reproductionin any form reserved.

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(Hagiwara and Byerly, 1981; Tsien, 1983, 1987; Eckert and Chad, 1984; Rasmussen and Barrett, 1984; Rubin et al., 1985; Augustine et al., 1987; Miller, 1987; Ewald and Levitan, 1987). Our laboratory has conducted cellular electrophysiological experiments on the effects of commonly used and abused sedative-hypnotic drugs on central mammalian neurons and has reached the conclusion that many of their effects are calcium mediated. This review will focus on neurophysiological and biochemical data related to the interactions of sedative-hypnotic drug neuronal actions with calcium-mediated metabolic processes and calcium-dependent currents. For the sake of brevity, we have restricted the scope of the work cited herein. The second section will be a brief discussion of the behavioral effects of the three types of sedative-hypnotic drugs to be discussed in this article, i.e., alcohol, barbiturates, and benzodiazepines. These behavioral effects include acute sedative-hypnotic actions, tolerance, dependence phenomena (e.g., drug withdrawal hyperexcitability) and, at least in the case of chronic ethanol administration, brain damage. A description of the acute and chronic cellular neuronal electrophysiologicaleffects of these drugs will comprise the third section. We are stressing electrophysiological data initially because we feel that the expression of brain activity (and hence behavior) is via a summation of cellular and local circuit neuronal events, which, however, are ultimately based on biochemical and biophysical processes. The fourth section will entail a description of neuronal biochemical processes affected by these sedativehypnotic drugs.

II. Behavioral Effects

Many animal and human behavioral experiments have demonstrated the sedative actions of ethanol, barbiturates, and benzodiazepines (Boisse and Okamato, 1980; Harvey, 1985; Ritchie, 1985), although some barbiturates are better anticonvulsants than sedatives, others are convulsants, and some benzodiazepines have nonsedative or antagonistic properties (Henkeler et al. , 1981). Ethanol in low doses is stimulatory in certain species (Masur and Boerngen, 1980; Erickson and Kochbar, 1985) and it is a well known clinical observation that the elderly can show enhanced sensitivity or sometimes even a paradoxical excitatory response to sedative drugs such as benzodiazepines and barbiturates. This review will focus on those drugs that are considered to have primarily sedative properties. These sedative drugs all demonstrate a certain degree of cross-tolerance to each other (Kalant et ol., 1971; Okamoto, 1978; Boisse and Okamoto, 1980; Commissaris and Rech, 1983). Also, all three classes of drugs show the

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development of tolerance (Kalant et al., 1971; Boisse and Okamoto, 1980) to their sedative actions with repeated administration or even following a single dose (acute tolerance). After more prolonged administration, drug “dependence” can develop, which means that following drug withdrawal a central nervous system (CNS) hyperexcitable state results, often manifested by behavioral hyperactivity, shakes, tremors, and even seizures gaffe, 1985). Chronic administration of alcohol is well known to cause brain damage (Carlen et al., 1981). It is unclear whether chronic intake of benzodiazepines or barbiturates can cause any long-term brain dysfunction. Finally, extracellular brain calcium has been implicated in mediating the acute behavioral responses to ethanol (Erickson et al., 1980; Little et al., 1986; Morrow and Erwin, 1986).

111. Electrophyslology

A. CALCIUM CURRENTS Calcium currents and their electrophysiological actions are being extensively studied in many different types of neurons, and several recent reviews have been published (Hagiwara and Byerly, 1981; Tsien, 1983, 1987; Rubin et al., 1985; Augustine et al., 1987; Ewald and Levitan, 1987; Miller, 1987). Ca ions play an essential role in presynaptic transmitter release (Katz and Miledi, 1967), although presynaptic depolarization also plays a role (Dude1 et al., 1983; Parnas et al., 1986). When an action potential invades the nerve terminal, voltage-dependent Ca channels open, allowing Ca ions to flow from the M to the inextracellular fluid with a Ca concentration of approximately tracellular compartment with a much lower Ca concentration of lo-’ M or less. The rise in intracellular Ca in the presynaptic terminal somehow triggers the exocytotic release of quanta of neurotransmitter. Hence alterations in presynaptic Ca function can have profound effects on neurotransmitter release. T o study Ca currents directly by electrophysiological techniques, it is much easier to impale with a microelectrode the larger postsynaptic element (usually the soma) than the presynaptic terminal (the squid giant synapse being an exception, e.g., Augustine and Charlton, 1986). The advent of patch-clamp and single-electrode voltage-clamp techniques has permitted more accurate delineation of the various Ca currents of mammalian neurons. At present, it seems that there are probably three different types of neuronal Ca channels (“T,” “N,” and “L”) as measured by somatic recordings (Nowycky et al., 1985; Tsien, 1987). The T current is activated at relatively

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hyperpolarized potentials and is rapidly inactivating. The N current is activated at more depolarized voltages and is also rapidly inactivating. The L current is activated at relatively depolarized voltages and is slowly inactivating. All currents have different sensitivities to various Ca current-blocking agents. Yaari et al. (1987) showed, using whole-cell patch voltage-clamp recordings of acutely dissociated embryonic rat hippocampal neurons, low voltage-activated fully inactivating somatic Ca channels and high voltage-activated slowly inactivating Ca channels, predominantly located in the developing dendrites. Our laboratory, using the single-electrode voltage clamp in rat dentate granule neurons in in vitro slices, has demonstrated three types of Ca currents (Carlen et al., 1986; Nielsen et al. 1987), roughly corresponding to the descriptions of Nowycky et al. (1985) in chick dorsal root ganglion neurons. Brown and Griffith (1983) demonstrated a persistent slow inward Ca current in hippocampal CAI and CA3 neurons probably corresponding to the L current.

B. CALCIUM-DEPENDENT IONICCURRENTS Once intracellular Ca is raised, ion channel function can be affected. In 1972, Krnjevic and Lisiewicz showed in cat spinal motoneurons that inThis tracellular Ca ion injection activated a potassium conductance kK). powerful intrinsic inhibitory mechanism, i.e., Ca-dependent gK7 was also demonstrated in Aplysiu neurons by Meech (1972). During a train of Nadependent spikes in central neutrons, some inward Ca current is also generated, causing a postspike train afterhyperpolarization (AHP) which is due to Ca-dependent gK (Krnjevic el al., 1978; Hotson and Prince, 1980; Gustafsson and Wigstrom, 1981). Recently it has been shown in hippocampal neurons that the AHP is composed of two Ca-dependent gKs,an early (I,) and a late component (IAHP)(Lancaster and Adams, 1986). In addition, a Ca-activated gc, (Owen et ul., 1986) and a Ca-activated cation (Na and K) current (Kramer and Zucker, 1985) have been described. Several neurotransmitters activate Ca-dependent currents in neurons. For example, our laboratory has shown that the CAI neuronal dendritic hyperpolarizing response to focally applied y-aminobutyric acid (GABA) is a Cadependent gK (Blaxter et u l . , 1986). Finally, increased intracellular Ca concentration per se inactivates Ca currents in many neurons (Eckert and Chad, 1984), and this mechanism is key to the development of our working hypothesis as to how ethanol and barbiturates affect neurons and interact with Ca currents (discussed below). Recent observations suggest that Cadependent inactivation of the Ca current results from the activation of a Cadependent phosphatase during cellular entry and accumulation of Ca, and this inactivation is subsequently removed as the phosphatase activity

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declines and dephosphorylated sites are rephosphorylated through the action of an endogenous kinase (Chad and Eckert, 1986).

C. CALCIUM CURRENTS AND SECOND MESSENGER SYSTEMS There is now a growing literature concerning the interaction of cellular second messenger systems, neurotransmitters and neurohormones, and Ca currents (Kaczmarek and Levitan, 1987;Worley et al., 1987). There are two presently well studied receptor-regulated second messenger systems active in the CNS: (1) cyclic adenosine monophosphate (AMP), which activates specific phosphorylating enzymes called protein kinases, and (2) the phosphoinositide cycle whereby receptor activation causes the release of inositol-l,4,5-triphosphate(InsPs) and diacylglycerol (Rasmussen and Barrett, 1984). Guanosine triphophate-binding proteins (G proteins) operate in both of these systems. Tsien (1987) reviewed the modulation of voltagegated Ca channels in the surface membrane of excitable cells. These are complex interrelationships between the several ways of regulating intracellular Ca ion concentration and the effects of Ca ions on second messenger systems (Rasmussen and Barrett, 1984). Ca interacts with different types of protein kinases (Nestler et a f . , 1984; Nishizuka, 1986), with cyclic AMP, with guanine nucleotide-binding proteins (Dolphin, 1987), and with inositol phospholipids (Downes, 1983). Recent electrophysiological experiments have demonstrated interactions between second messenger systems, Ca currents, and Ca-dependent currents. Phorbol esters, which activate protein kinase C , blocked the ZAHp in hippocampal CAI neurons (Baraban et a l . , 1985; Malenka et al., 1986). Phorbol ester and protein kinase C enhanced a voltage-sensitive Ca current in Alplysia neurons (DeRiemer et al., 1985), whereas protein kinase C activation in embryonic chicken dorsal root ganglion neurons attenuated a voltage-dependent Ca current (Rane and Dunlap, 1986). Ca action potentials in Alplysia bag cell neurons were enhanced by intracellular microinjection of the catalytic subunit of cyclic AMP-dependent protein kinase C (Kaczmarek et al., 1980). Higashida and Brown (1986) gave evidence in voltage-clamped cultured neuroblastoma-glioma hybrid cells that extracellular application of bradykinin, which forms intracellularly InsPs, or intracellular injection of Ca or InsPs activated a Ca-dependent g ,. Paupardin-Tritsch et al. (1986)showed that intracellular injection of a cGMP-dependent protein kinase enhanced a Ca current and potentiated the serotonin-induced Ca current increase in snail neurons.

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D. ACUTESEDATIVE-HYPNOTIC DRUGACTIONS IN RELATIONSHIP TO CALCIUM AND CALCIUM-DEPENDENT CURRENTS Over the past several years, this laboratory has been examining the central actions of sedative-hypnotic drugs using intracellular electrophysiologicd recording techniques from mammalian neurons in in uitro brain slices. We have studied clinically relevant sedative doses of ethanol (Q20 mM), benzodiazepines (QlO-aM), and pentobarbital (QlO-*M). Most of this work has been presented elsewhere (Carlen et a l . , 1982a,b, 1983a,b; 1985a-c; Carlen, 1987; Durand and Carlen, 1984; O’Beirne et al., 1987). In hippocampal CAI neurons, all sedative drugs, when applied via the bath perfusate or by drop application, caused, within 1 to 2 min, a neuronal hyperpolarization of the neuron, usually, but not always, associated with decreased input resistance (or increased conductance). Spontaneously active neurons diminished or ceased firing. Intracellular injection of chloride had no effect, suggesting that this hyperpolarization was most likely due to an increased gK. Intracellular injection of cesium, which decreases several gKs, blocked the ethanol-mediated hyperpolarization (Carlen et al. , 1982b). Nicoll and Madison (1982) also showed that several general anesthetics hyperpolarized vertebrate neurons, probably by increasing gK. Extracellular perfusion of tetrodotoxin (TTX), which blocks Na channels and secondarily spike-evoked synaptic transmission, had no effect on these drugs’ actions. Zero Ca perfusate with added MnC12 also did not block the hyperpolarizing action of ethanol and pentobarbital, but did block the hyperpolarization caused by midazolam. We shall discuss this point later. To further examine sedative drug activation of the inhibitory gKs, other voltage responses dependent upon gK activation were examined. All three drug types enhanced the long-lasting orthodromic inhibitory postsynaptic potential (IPSP), which is mediated by a gK which is probably not C a dependent, although there is some debate on this issue. The postspike train long-lasting AHP is a Ca-activated gK (Ic and IAHP). In zero Ca perfusate, this AHP potential disappears. This conductance is ubiquitous throughout the cental nervous system and is a powerful intrinsic neuronal inhibitory mechanism. Enhancement of the inhibitory long-lasting AHP (IAHP) was the single most consistent response to ethanol (Carlen d al., 1982), pentobarbital (O’Beirne et al., 1987), midazolam (Carlen d al., 1983b), and clonazepam (Gurevich et al., 1984) in CAI neurons. The enhanced AHP was also seen with ethanol in dentate granule neurons (Niesen et al., 1986) and with pentobarbital in CA3 neurons (O’Beirne d al., 1987). These findings fed us to hypothesize that these drugs could act in large part by affecting C a currents or intracellular Ca metabolism and that the sedative drug-induced hyperpolarization and enhanced AHPs were due to increased Ca-dependent K currents.

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In TTX perfusate, higher threshold Ca spikes can be elicited in CAI neurons. Ethanol had no effect or slightly decreased the Ca spikes (Carlen et al., 1982a). Pentobarbital decreased the Ca spikes (O’Beirne d al., 1987). Since both these drugs tended to increase the membrane conductance, the decrease of the Ca spike could be in part due to a conductance increase to which these mainly dendritic Ca spikes could be very sensitive (MacDonald and Schneiderman, 1986). However, diminished Ca spikes were noted in cells with no apparent decrease in input resistance. This effect could be due to the inactivating effect of raised intracellular Ca on some inward Ca currents (Eckert and Chad, 1984). On the other hand, midazolam enhanced the Ca spikes, even though it also caused decreased input resistance (Carlen et al., 1983b). Zero Ca perfusate, with MnCh added, blocked the hyperpolarizing effect of midazolam, but not that of ethanol or pentobarbital. A specific benzodiazepine antagonist, Ro14-7437, caused a depolarization of hippocampal CAI neurons with increased input resistance, decreased AHPs, and, in the presence of TTX, diminished Ca spikes (Carlen et al., 1983a). Like midazolam, its effects were blocked by zero Ca perfusate. These data led us to the conclusion that the hyperpolarization caused by ethanol (Carlen et al., 1982a) and pentobarbital (O’Beirne et al., 1987) could be caused, at least in part, by an increased Ca-mediated gK which was triggered by raised [Ca], from intracellular stores, whereas the actions of the benzodiazepines were dependent upon extracellular Ca (Carlen et al. , 1983b). It is easy to explain the enhanced AHPs seen in the presence of midazolam as due to increased inward Ca current during the preceding train of spikes. However, based on the above data showing that ethanol and pentobarbital do not enhance Ca spikes, it seems that the enhanced AHPs in the presence of these two drugs are not due to enhanced inward Ca currents. There is more than one type of voltage-dependent Ca current in these neurons (Brown and Griffith, 1983): a spikelike current, which could be related to the N current, and a much more slowly inactivating current, which could be the L current as described by Tsien (1987). The interaction of sedative drugs with voltage-clamped Ca currents in central mammalian neurons has not been investigated to date except for one report by Llinas and Yarom (1986) showing that the low-threshold Ca current in inferior olivary neurons was blocked by low concentrations of monohydroxyl alcohols including ethanol (0.1 to 1 mM). If ethanol and pentobarbital do not enhance inward Ca currents, then their AHP-enhancing action could be related to some mechanism which enhances the gK sensitivity to the transiently increased [Ca],, resulting from a train of spikes. This could be related to second messenger activation.

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As mentioned above, ethanol and pentobarbital caused a tonic hyperpolarization, often with increased conductance, both in normal and in Cafree perfusate (Carlen d al., 1982a, 1985a; O’Beirne et ul., 1987). Also both drugs had no effect or decreased Ca spikes. Based on these data, we hypothesized that ethanol could act by raising intracellular free ionic Ca concentration from intracellular stores (Carlen et al., 1982a), which would tend to diminish inward Ca currents (Eckert and Chad, 1984). However, raised intracellular Ca concentration per se could hyperpolarize the neuron by augmenting g, (Krnjevic and Lisiewicz, 1972), even in the presence of zero Ca perfusate. Mention should be made here of probably the most favored explanation to date of sedative-hypnotic drug action, i.e., activation of GABA-mediated C1 conductance. In our hands, at pharmacologically relevant doses for sedation, no augmentation of GABA-mediated gc, was seen (Carlen et al., 1985b), although higher doses of pentobarbital (lom4M) (O’Beirne et al., 1987) and M) (Carlen et al., 1983b) did augment GABA actions. midazolam Nestros ( 1 980) reported that ethanol augmented GABA-mediated neurotransmission in cat cortex in uiuo using extracellular recordings. Shefner et al. (1982) showed that 30 mM ethanol enhanced the GABA action on locus coeruleus neurons using in uitro intracellular recordings. However, she demonstrated that the main mechanism of inhibition of spontaneous firing of these neurons was augmentation of the late postspike AHP (a Ca-mediated g,) and by decrease of the rate of depolarization preceding each spike (Shefner and Tabakoff, 1984). These data were interpreted as being due to ethanol enhancement of a long-lasting gK. Biochemical experiments, however, give much evidence for C1 channel and GABA involvement in ethanol actions ( M a n and Harris, 1987; Ticku and Burch, 1980). We shall now report the electrophysical work of other laboratories regarding the acute effects of sedative-hypnotic drugs with relation to Ca and Cadependent currents, starting with relevant experiments using alcohol. At the vertebrate neuromuscular junction in a perfusate without Ca and with added EGTA (a Ca chelator), ethanol increased spontaneous nonspike-induced neurotransmitter release (miniature endplate potentials), a finding compatible with increased presynaptic Ca concentration from intracellular sources (Quastel et al., 1971). Carlen and Corrigall (1980) also demonstrated raised spontaneous neurotransmitter release from rat neuromuscular junctions in the presence of ethanol. Bergman d al. (1974), using high doses of ethanol (>lo0 mM) in three different types of Aplysia neurons, showed blockade of inward Na and Ca currents. Schwartz (1983) showed in voltage-clamped Aplysiu neurons that the leakage and slow inward Ca currents were decreased, and outward K currents were increased by 4% ethanol. More recent experiments (Schwartz, 1985) demonstrated that the Ca-activated K current was increased and the

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Na- and Ca-dependent slow inward currents were reduced by ethanol. The reduction of the Ca-dependent slow inward current was not prevented by intracellular injection of EGTA, suggesting that ethanol-induced increased intracellular Ca concentration was not solely responsible for the reduced Ca current, although this author suggested that ethanol could act by increasing intracellular Ca concentration. Also in Aplysia neurons, Camacho-Nisi and Treistman (1985) showed that a relatively low dose of ethanol (50 mM) reversibly decreased the amplitude of the Ca current with no effects on Na currents. In single bullfrog atrial cells, ethanol, in anesthetic doses of 100 to 500 mM, significantly depressed the slow inward Ca current and the repolarizing K current (Takeda et al., 1984). In rat cultured dorsal root ganglion neurons, ethanol in pharmacologically relevant concentrations (0.05 to 0.30 g % ) decreased gK as a result of decreased inward Ca current (Oakes and Pozos, 1982a). C a spikes were also diminished (Oakes and Pozos, 1982b), although very low concentrations of ethanol, leaking from a nearby microejection pipette containing low concentrations of ethanol (0.05 to 0.10 g%), increased the Ca spike. This low-dose effect may be related to the excitatory behavioral effects of ethanol seen at lower doses in some species. Pozos and Oakes (1987) reported that other alcohols, containing one to five carbons, all produced a decrease in the Ca spike duration. They presented preliminary results in cultured neuroblastoma cells loaded with the intracellular fluorescent Ca dye indicator, fura 2, that ethanol doses of 0.15 g% and higher can increase intracellular free C a concentration. Vassort et al. (1986) demonstrated in squid axons injected with Arsenazo I11 that alcohols with a chain of 5 to 10 carbons increased intracellular free [Cali. Further biochemical evidence and discussion supporting the idea of ethanol and barbiturates raising intracellular free [Cali is presented in Section IV. In our experiments, pentobarbital acted similarly to ethanol postsynaptically on hippocampal pyramidal cells (Carlen et al., 1985a; O’Beirne et al., 1987). Ca spikes were diminished by M pentobarbital. Ca spikes were also decreased in cultured mouse spinal cord neurons by pentobarbital (25 to 600 p M ) , by phenobarbital (100 to 500 luM) (Heyer and MacDonald, 1982), and by convulsant barbiturates (Skerritt and MacDonald, 1984). In the leech Retzius neuron, several different barbiturates prolonged the action potential, compatible with the interpretation that barbiturates block a voltagedependent inward Ca current which activates a gK necessary for normal repolarization (Kleinhaus and Prichard, 1977). More recently it was demonstrated that Ca action potentials in leech nociceptive neurons were blocked by phenobarbital, pentobarbital, and methohexital (Johansen and Kleinhaus, 1986). Calcium spikes in Aplysia neurons were also blocked by pentobarbital (Goldring and Blaustein, 1982). Pentobarbital, on Helix

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neurons, increased the rate of voltage-dependent inactivation and decreased the maximal peak amplitude of the voltage-clamped Ca current (Nishi and Oyama, 1983a,b). Biochemical data also show that barbiturates and other sedative hynotics inhibit depolarization-induced Ca uptake into synaptosomes (Leslie, 1987). Benzodiazepines block Ca uptake in synaptosomes in micromolar concentrations (Leslie, 1987). Electrophysiological experiments in our laboratory on CAI neurons showed neuronal inhibition (i.e., membrane hyperpolarization and increased AHPs) by clonazepam (Gurevich et al. , 1984) and by midazolam (Carlen et al., 1983b) at low nanomolar concentrations. Midazolam, at 5 x M, consistently enhanced Ca spikes elicited in TTX. Skerritt et a!. (1984) showed that diazepam and its p-chloro derivative, Ro5-4864, inhibited intracellularly injected current-induced high-frequency repetitive firing in cultured mouse spinal cord neurons, with 50% inhibition by diazepam at 87.5 n M and by Ro5-4864 at and by diazepam (10 p M ) . C a action potentials were inhibited by 10 # diazepam and 10 pi4 Ro5-4864. The Kd of the high-affinity benzodiazepine sites in mammalian brain are in the lower nanomolar range. Cherubini and North (1985) observed that benzodiazepines, midazolam and diazepam in 100-300 pM concentrations, were able to decrease calcium action potentials in guinea pig myenteric neurons and the effects were reversibly blocked by Ro15-1788, a benzodiazepine antagonist.

E. TOLERANCE, DEPENDENCE, AND CHRONIC NEURONAL EFFECTS OF SEDATIVE-HYPNOTIC DRUGS IN RELATIONSHIP TO CALCIUM AND CALCIUM-DEPENDENT CURRENTS Unlike the biochemical literature, there is relatively little work done on the electrophysiologically measured effects of prolonged administration of sedative-hypnotic drugs in relationship to Ca or Ca-dependent currents. In the mammalian neuromuscular junction, ethanol increases spontaneous neurotransmitter release as measured by increased frequency of miniature endplate potentials (Quastel et al., 1971; Curran and Seeman, 1977; Carlen and Corrigall, 1980), which is a Ca-dependent process. Curran and Seeman (1977) showed tolerance to this action of ethanol in neuromuscular preparations removed from rats chronically exposed to ethanol, whereas Carlen and Corriglall (1980) did not. However, Carlen and Corrigall (1980) did show tolerqce to the in uitro ethanol-induced depression of the orthodromically evoked hippocampal CAI field potential at doses greater than 100 mM (supraanesthetic) and Durand el al. (1981) showed what appeared to be acute

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tolerance to perfused ethanol (100 mM) which initially depressed the CAI field potential, but within 30 min of perfusion, the field potential began to increase in size. This could be an in vitro correlate of acute tolerance. Rougier-Naquet et al. (1986) demonstrated hyperexcitability of CAI neurons in slices from rats undergoing ethanol withdrawal after 4 days of ethanol intubation (8- 12 g/kg/day) and in slices from ethanol-naive animals following 2 hr of slice perfusion with 100 mM ethanol. In both cases, the neuronal hyperexcitability associated with alcohol withdrawal showed, in intracellular recordings, diminished AHPs and decreased spike frequency adaptation to a 600-msec depolarizing current pulse. These results support the hypothesis that neuronal inhibition is depressed after ethanol withdrawal by means of decreased Ca-mediated gK. Whether this is related to alterations in Ca currents is not known. Hyperexcitability was also noted in in vitro hippocampal CAI neurons from drug-naive rats exposed to 20 n M clonazepam-containing perfusate for 2 or more hours and then withdrawn from drug (Davies et al., 1985, 1987) or in neurons from the rats fed clonazepam (10 to 50 mg/day in the rat chow) for 1 month following abrupt drug withdrawal (Davies and Carlen, 1984). No work to our knowledge has been done on the electrophysiology of neurons after subacute or chronic administration of barbiturates. Long-term administration of ethanol in the diet for 5 months is an adequate period for the development of behavioral and neuronal morphological deficits in rats. In hippocampal slices removed from such animals chronically exposed to ethanol via a liquid diet and then allowed to withdraw for 3 weeks, Durand and Carlen (1984) showed decreased AHPs and IPSPs in both dentate granule and CAI neurons. Certainly decreased &(s) helps to explain these findings. In the case of the decreased AHP, this ethanolinduced diminution implies a decreased Ca-mediated gK, which was hypothesized to be related to a state of chronically raised [Ca],, which is also toxic to cells and could be a cause of alcohol-induced brain damage (Durand and Carlen, 1984).

F . AGINGINTERACTIONS There are almost no data, to our knowledge, concerning the electrophysiology of sedative drug-neuronal interactions in aged compared to younger mature animals. Recent work from our laboratory (Niesen et al., 1986) in rat hippocampal dentate granule neurons showed that 20 m M ethanol inhibited neurons from young mature rats (6-8 months) by hyperpolarizing the membrane and augmenting the AHP. Presumably, these effects are similar to those noted in CAI neurons (Carlen et al., 1982a).

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However, in neurons from old rats (26-28 months), ethanol caused a slight depolarization, diminished spike-frequency adaptation to a 600 msec depolarizing current pulse, and diminished AHPs and IPSPs. The longlasting AHP is mediated totally, and the spike-frequency adaptation in part, by Ca-dependent g,. Hence, in old neurons, a moderately intoxicating dose of ethanol caused a neuronal disinhibition related in part to an altered effect on Ca-mediated g,.

IV. Biochemistry

In the preceding section, we presented evidence that the sedative-hypnotic drugs, i.e., ethanol, barbiturates, and benzodiazepines, may exert their pharmacological actions via Ca-dependent mechanisms. In this section we will present biochemical evidence that these drugs affect calcium homeostasis in neurons and might even alter second messenger function. The chemical gradient for C a across the neuronal membrane is approximately lO'-foId in favor of the extraneuronal calcium concentration. The influx of Ca into neurons is regulated by calcium channels. The intraneural Ca concentration is tightly regulated by the interplay of several factors, i.e., Ca channel influx, Na-Ca exchange, an ATP-dependent Ca pump, Ca-binding proteins, and intracellular C a uptake and release by the smooth endoplasmic reticulum and mitochondria (Rasmussen and Barrett, 1984). Sedativehypnotic drugs can affect intraneural C a by altering any or all of these factors. An alteration in intracellular C a concentration not only affects ionic conductances such as g,, but can also affect intraneuronal metabolic processes, since C a per se is a second messenger and affects other second messenger systems (Rasmussen and Barrett, 1984).

A. EFFECTS OF SEDATIVE-HYPNOTIC DRUGS ON INFLUX OF CALCIUM 1. Ethanol-Acute Eflects For the past 10 years, many groups have shown that ethanol, pentobarbital, and, to a lesser extent, benzodiazepines inhibit depolarization-evoked Ca influx into synaptosomes. It has been accepted that depolarizationinduced C a influx represents a mechanism for the mediation of presynaptic neurotransmitter release (Katz and Miledi, 1967). However, reviews of work done in this area indicate methodological problems (see Leslie, 1987). Earlier studies looking at the effects of ethanol on potassium-depolarized Ca uptake over a time course of minutes (slow-phase), have yielded conflicting

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results. Harris and Hood (1980) observed the inhibition of potassiumevoked Ca uptake by a concentration as low as 45 m M ethanol, whereas Blaustein and Ector (1975) did not observe any inhibition at an ethanol concentration of 100 mM. The reasons for these types of conflicting results could be that: 1. Ca influx through Ca channels and Ca channel opening time are known to occur in the millisecond range, whereas these studies observed Ca influx over a time course of minutes. These influx studies could therefore be looking at the summation of various cellular processes controlling [Cali. Ethanol may not affect all these processes equally. 2. Raised intracellular Ca plays a large role in the inactivation of many Ca channels (Eckert and Chad, 1984), and the inactivation time for the T and N types of C a channels is in the range of tens to hundreds of milliseconds. When Ca influx was examined over the time course of minutes, most Ca channels could have been largely inactivated. 3. The Ca uptake activity varies with different brain regions (Stokes and Harris, 1982). A synaptosomal preparation containing different amounts of tissue from the various brain regions may contribute to the differences seen in several laboratories.

Fast-phase Ca influx has been described by Nachshen and Blaustein (1980) and by Leslie et al. (1983a). This process probably correlates with the time course and pharmacology of L-type Ca channels (slowly inactivating Ca channels) (Leslie, 1987), possibly representing the neuronal mechanism of transmitter release (phasic release). Using a cortical synaptosomal preparation, Leslie et al. (1983a) and Daniel1 and Leslie (1986) showed that the fast-phase Ca influx was inhibited by low doses of ethanol, whereas the slow-phase Ca influx was insensitive to low concentrations of ethanol. In clonal neural cells, Messing et al. (1986) showed that acute exposure to ethanol produced a concentration-dependent decrease in depolarizationevoked 45Cauptake.

2 . Ethanol- Chronic Eflects Chronic administration of ethanol to rats results in a shift in the dose-response curve toward less ethanol inhibition of Ca influx. For example, Leslie et al. (1983b) demonstrated the development of tolerance in rats to the ability of ethanol in uitro to inhibit fast-phase Ca uptake in cerebral cortex. Wu et al. (1986) showed that the depolarization-induced Ca uptake measured at 20 sec following K depolarization was markedly inhibited in hippocampus and cerebral cortex but not in hypothalamus from rats made dependent on

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ethanol. In cultured clonal neural cells exposed to ethanol for 2-10 days, Messing et al. (1986) showed an increase in 45Cauptake to depolarization and an increase in the number of Ca channel-binding sites. The 45Cauptake was restored to normal following withdrawal of ethanol from the cultures. Very little has been done on human brain C a metabolism in alcoholism, Kapur et al. (1985) showed that cerebrospinal fluid (CSF) ionic Ca was lower in recently abstinent chronic alcoholic patients compared to neurological controls, whereas the total CSF Ca, serum ionic Ca, and total serum Ca were unchanged. How do biochemical studies explain electrophysiological data? Bear in mind that in most biochemical studies, the effects of ethanol on Ca entry are carried out in synaptosomal preparations (containing intact resealed presynaptic terminals), whereas the electrophysiological studies usually measure the postsynaptic neuronal somatic responses. T o date, we are not aware of any detailed studies comparing the properties of Ca channels in presynaptic and postsynaptic neuronal membranes and their sensitivities to the effects of sedative-hypnotic drugs.

3 . Barbiturates-Acute

Effects

Barbiturates inhibit synaptosomal calcium influx (Hood and Harris, 1980; Leslie et al., 1980b; Elrod and Leslie, 1980). It is interesting to note that pentobarbital reduced depolarization-induced Ca influx into synaptosomes and the Ca action potential in neurons in similar concentrations (Ondrusek et al., 1979; Heyer and MacDonald, 1982; Leslie et al., 1980b; Elrod and Leslie, 1980; Werz and MacDonald, 1985). Chandler et al. (1986) reported that 5-(2-cydohexylideneethyl)-5-ethylbarbituric acid (CHEB), a convulsant type of barbiturate, also inhibited the voltage-dependent Ca channels in brain synaptosomes. Since both convulsant and anticonvulsant barbiturates exert their effects through inhibition of voltage-dependent C a channels, it is difficult to explain the difference in these drugs’ actions based on Ca interactions alone.

4 . Barbiturates-Chronic

Effects

Chronic pentobarbital treatment results in an adaptive change of brain calcium channels similar to that observed with chronic ethanol treatment. The K-evoked Ca uptake was less sensitive to barbiturates in the chronically pentobarbital-treated animals (Leslie et a l . , 1980b).

5 . Benzodiazepines-Acute

Effects

There are high-affinity (Braestrups and Squires, 1977) and low-affinity (Shoemaker et a l . , 1981) benzodiazepine binding sites in brain. There is

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evidence that the peripheral type of benzodiazepines receptors is also present in brain (Shoemaker et al. , 1979). Electrophysiological and pharmacological studies show that peripheral-type benzodiazepine receptors are coupled to calcium channels in the heart (Mestre et al., 1985). Using the guinea pig papillary muscle preparation, Mestre et al. (1985) showed that Ro5-4864, an agonist of the peripheral type of benzodiazepine receptor, decreased the tension of the papillary muscle. This effect was reversed by increasing extracellular Ca concentration and also by PK 11195, an antagonist of the micromolar affhity peripheral-type benzodiazepine receptor. Biochemical studies on the effects of benzodiazepines on voltage-dependent Ca uptake have shown that benzodiazepines significantly inhibit "fast-phase' ' voltagedependent Ca uptake into mouse brain synaptosomes at concentrations which correlate with the KdS of low-affinity (micromolar) benzodiazepine receptors (Taft and Delorenzo, 1984). The ability of these benzodiazepines to inhibit Ca uptake is directly correlated to their hypnotic potency, suggesting that inhibition of presynaptic calcium entry may be linked with the hypnotic action of benzodiazepines (Leslie et al., 1986). However, Carlen et al. (1983b) demonstrated central mammalian neuronal inhibition by increased g, with concomitantly increased Ca spikes at nanomolar concentrations of midazolam. The Kdsof high-affinity benzodiazepine receptors are in the lower nanomolar range, as is the CSF concentration of these highly protein-bound drugs (Kanto et d.,1975).

6 . Benzodiazepines-Chronic Efects Chronic administration of chlordiazepoxide to animals resulted in decreased inhibitory effects of benzodiazepines on the K-evoked synaptosomal Ca uptake (Leslie et al.. 1980b), suggesting that voltage-dependent Ca channels underwent adaptive changes. It is important to note that, once again, the observation was made in presynaptic terminals. There are no data indicating whether the voltage-dependent Ca channels, which show adaptive changes to the effects of benzodiazepines, will also show adaptive changes to the effects of ethanol or pentobarbital, or vice versa, although it is assumed that all three drug classes show a certain degree of cross-tolerance following chronic treatment (Boisse and Okamoto, 1980). Finally, it should be mentioned that the data concerning sedative-hypnotic drug action on biochemically measured neurotransmitter release are quite confusing. This presumably reflects many methodological issues, in addition to the conceptual problem of possible sedative drug-induced increased presynaptic Ca concentration which would promote spontaneous transmitter release, but could have variable effects on action potential-evoked neurotransmitter release, depending on the degree of Camediated inactivation of the action potential-evoked nerve-terminal Ca current.

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B. EFFECTS OF SEDATIVE-HYPNOTIC DRUGS ON CALCIUM RECEPTORS 1. Ca Blockers Ca enters neurons via voltage-sensitive calcium channels. One way to investigate the functional roles of the Ca channel is to use drugs which are selective for blocking C a channels called Ca-channel blockers or antagonists. This area has been recently reviewed by Miller (1987) and Greenberg (1987). Cachannel antagonists include dihydropyridines (DHP), phenylalkamines, diltiazem, and bepridil. Although there is some controversy, reports indicate that there is single class of DHP-binding sites, since in either synaptosomal preparations or crude brain homogenates, binding studies show one class of binding site (Miller, 1987; Snyder, 1984). A recent study (Miller, 1987) using a brain synaptosomal preparation showed that Ca influx through voltagesensitive Ca channels was not blocked by DHPs when K depolarization was used as a stimulus, but the C a influx induced by Bay K 8644, a calciumchannel agonist, was completely blocked by DHPs in nanomolar concentrations, indicating that brain calcium channels appear to vary in sensitivity to the types of stimuli applied.

2. Ethanol-Acute

Effects

Hoffmeister et al. (1982) and Itil et al. (1984) observed that nimodipine enhanced sedative-hypnotic drug action. Isaacson et al. (1985) showed that nimodipine potentiated the sleep time and hypothermia induced by ethanol. These findings suggest that the sedative-hypnotic drugs, including ethanol, interact with DHP cerebral binding sites. In in vitro DHP binding studies, Greenberg and Cooper (1984) and Harris et al. (1985) observed that ethanol displaced [3H]DHP binding in brain. However, Harris et al. (1985) reported that I A4 ethanol was required to displace this binding by 30-40%. Greenberg and Cooper (1984) observed that ethanol inhibited specific [3H]nitrendipine binding with a K, value of 460 mM by decreasing binding affinity without altering the maximal number of binding sites (calcium channels), suggesting a competitive type of inhibition by ethanol. Furthermore, ethanol at a lower concentration did not modify the inhibitory actions of verapamil or diltiazepam on [3H]nitrendipine binding. These observations suggest that ethanol may not directly interfere with the operation of the calcium channel but may exert actions on the surrounding microenvironment of calcium channels which causes a change in the binding affinity of calcium-channel antagonists.

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We also observed that ethanol inhibited [SH]nitrendipinebinding to brain cortical synaptosomal membranes in vitro (P. H. Wu et al., unpublished data). The I C ~ O values for inhibition of [3H]nitrendipine binding by methanol, ethanol, propanol, butanol, and n-amylalcohol were 5400, 1075, 450, 170, and 75 mM, respectively. There is a linear relationship between these IC50 values and the carbon chain length, suggesting that the inhibitory effects on DHP binding may be related to the ability of alkanols to perturb brain synaptosomal membranes. Leslie (1987) concluded that, since very high ethanol concentrations are required to inhibit DHP binding, it is unlikely that ethanol exerts its pharmacological action by specifically and directly inhibiting voltage-dependent calcium entry into presynaptic nerve terminals.

3 . Ethanol- Chronic E’ects The effects of chronic ethanol administration on DHP binding have been shown by Lucchi et al. (1985). Ethanol was administered to rats through the drinking water. The very high-affinity and Ca-independent DHP binding (Kd = 0.05 “M) was slightly increased, whereas the Cadependent DHP binding was reduced to one-fifth of the controls in brain synaptosomal membrane preparations. Consistent with this binding study, Lucchi et al. (1985) also found that striatal slices from chronically ethanolfed rats showed greatly reduced K-stimulated Ca uptake. These results suggest that the effects of ethanol on calcium entry regulation may be a mechanism for the development of ethanol-induced tolerance, dependence, or even neurotoxicity. Wu et al. (1987) administered ethanol (10 g/kg/day) to groups of Wistar rats by intubation. Animals were sacrificed during the treatment period ranging from 1.5 to 9.5 days. The DHP-binding sites were labeled by [3H]nitrendipine. The results indicated that chronic ethanol treatment for 5.5 days resulted in an increase (approximately 40%) in the estimated B,, and Kd of the DHP-binding sites. It is interesting to note that the maximal increase in DHP binding occurred after 3.5 days of ethanol treatment. Wu et al. (1987a) also noted that the increase in DHP binding seemed to correlate with the time course for the development of behavioral tolerance to ethanol, suggesting that calcium channels may participate in the mechanism of ethanol tolerance. If “plasticity” of calcium channels is involved in the development of tolerance to ethanol, blockage of voltage-dependent calcium channels should be able to modify the development of ethanol tolerance. Wu et al. (1987b) showed that chronic nifedipine treatment of rats delayed the development of behavioral tolerance to ethanol in a moving-belt impairment test. The experiments showed that the time course for the development of tolerance to chronic ethanol

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administration (3 g/kg/day) was 21 days and 35 days, respectively, for the sham-treated and nifedipine-treated animals. In a recent report, Little et al. (1986) showed that calcium-channel blockers were able to abolish, prevent, or reduce seizures induced by audiogenic stimuli following ethanol withdrawal.

4. Barbiturates Pentobarbital anesthetic potency was significantly increased by verapamil (10 mg/kg), flunarizine (40 mg/kg), and nitrendipine (100 mg/kg) ( D o h and Little, 1986). Harris et al. (1985) showed that [3H]nitrendipine binding to rat cortical membranes was reduced by phenobarbital and pentobarbital at supraanesthetic ICSO values of 0.40 and 0.76 mM, respectively. These drugs reduced the Kd with little effect on the Bms.However, Harris et ~ l(1985) . found that the DHP-binding sites in their preparation did not seem to involve the voltage-dependent calcium channels since DHPs were not able to inhibit Ca influx. There are little data regarding the chronic effects of barbiturates.

5. Benzodiazepines Draski et al. (1985) showed that nimodopine (5 mg/kg) significantly potentiated the hypothermia and diminished motor activity induced by 2.5 or 5.O mg/kg diazepam, suggestingthat nimodipine may interact with diazepam receptor sites. On the other hand, nifedipine blocked the hypnotic effect of flurazepam. Also nifedipine (1 CUM) and nitrendipine (1 CUM) antagonized the effects of diazepam to increase Ca uptake (Mendelson et ul., 1984). These data suggest that calcium channels may be the sites where diazepam exerts its pharmacological action. This notion was further supported by Taft and Delorenzo (1984), who showed that micromolar concentrations of diazepam inhibited Ca uptake through voltagesensitive Ca channels and demonstrated that benzodiazepines function as Cachannel antagonists using [3H]nitrendipine as a Ca receptor probe. There is evidence suggesting a dissociation of calcium channels from peripheral-type benzodiazepines receptor sites (Bolger et al., 1986; Doble et al., 1985). However, the fact that calcium-channel blockers are able to block the behavioral effects of diazepam as well as the Ca-uptake mechanism regulated by benzodiazepines suggests a complex relationship between these two sites. We are not aware of data concerning the effects of chronic benzodiazepine treatment on DHP binding sites or benzodiazepine receptors.

C . EFFECTS OF SEDATIVE-HYPNOTIC DRUGS ON INTRACELLULAR REGULATION OF CALCIUM ION CONCENTRATION Intracellular calcium ions are subjected to many biochemical processes which serve to regulate their intracellular concentrations (Rasmussen and

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Barrett, 1984). These biochemical processes include calcium binding proteins, uptake by subcellularorganelles, extrusion of cytosolic Ca by an ATP-dependent Ca pump (Ca-ATPase), and the Na-Ca membrane exchange mechanism.

1. A TP-Dependent Calcium Pump The ATP-dependent Ca uptake into the smooth endoplasmic reticulum has been localized on the inner surface of synaptic plasma membrane fragments (McGraw et al., 1980) and has similar functions to the Ca pump of red cell membranes. Following acute administration of ethanol (4 glkg, i.p.) to mice, Garrett and Ross (1983) showed that, with the loss of the righting reflex [blood alcohol level (BAL) = 600 mg/%], Ca-ATPase and ATP-dependent C a uptake were inhibited. However, at the time of recovery of the righting reflex, the ATP-dependent Ca uptake remained inhibited while Ca-ATPase had returned to control levels, suggesting that ethanol may act to uncouple the enzyme and uptake process. T o elucidate the mechanism of the effects of ethanol on Ca-ATPase, Garrett and Ross (1983) showed that ethanol disrupted intracellular membranes such as smooth endoplasmic reticular membrane, resulting in fewer calcium ions being bound and hence increased free [Cali. Chronic ethanol administration results in the inhibition of ATPdependent Ca uptake in synaptic membranes (Ross, 1986). Membranes from ethanol-treated mice exhibited reduced capacity to take up Ca, and the addition of calmodulin to this preparation stimulated the chronic ethanolinhibited ATP-dependent Ca-uptake system. This suggests that chronic ethanol may cause a decrease in the calmodulin-regulated ATP-dependent Ca-uptake, thereby resulting in the diminution of the cytosolic buffering of intracellular Ca, causing a rise in cytosolic [Ca], in central neurons (Ross, 1986). Rudeen and Guerri (1985) observed that prenatal (in utero) and postnatal ethanol exposure decreased Ca-dependent ATPase activity in different brain regions. The longer the period of time that ethanol was consumed by the mother and prenatally and by the newborn postnatally, the greater the inhibitory effects of alcohol on this enzyme activity.

2 . Ca-Binding Protein The effects of ethanol treatment on calcium-binding activity in synaptosomal membranes prepared from hippocampus, cortex, and cerebellum were reported by Virmani et al. (1985). Using a chelator fluorescence probe (chlortetracycline) and 45Ca binding methods, Virmani et al. (1985) observed that, in acutely intoxicated and later dependent rats in withdrawal, the synaptosomal membranes from the hippocampus showed a more

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drastic increase in calcium-binding activity than did the synaptosomal membranes from the cortex and the cerebellum. However, the increase in Ca binding and Ca-binding sites seen by Virmani et al. (1985) cannot differentiate whether the binding occurs mainly on outer or inner synaptosomal membranes. Other lines of evidence indicate that ethanol does not inhibit calcium binding to Ca-binding protein in uztro. Ishii and Ohnishi (1985) employed a chelex resin method which is sensitive enough to measure free Ca concentration as low as low9M . They demonstrated that an ethanol concentration as high as 25% in nitro did not influence the Ca binding of troponin. Behavioral studies investigating the ethanol-induced sleeping time in mice correlated Ca prolongation of ethanol-induced sleeping time to activation of tyrosine and tryptophan hydroxylase through a calmodulin and calmodulin-dependent protein kinase mechanism (Sutoo ef a/., 1985). The inconsistent results in this area suggest that the effects of ethanol on intracellular protein C a binding probably do not account for the major effects of ethanol intoxication. We are unaware of data relating sedative-hypnotic drug action to intracellular Ca ion regulation by the smooth endoplasmic reticulum or mitochondria.

3. Nu-Ca Exchange Activating the Na-Ca exchange process reduces intracellular Ca which exchanges for extracellular Na (Michaelis and Michaelis, 1981). This has been shown to be a high-capacity intracellular C a regulating system with a K, of 40 ,uM (Gill et al., 1981). Michaelis et al. (1985) observed that the Na-Ca exchange activity in synaptic plasma membranes was inhibited even by low concentrations of ethanol (less than 25mM), while this concentration of ethanol did not have any effects on the ATPase-dependent Ca pump. Ethanol increases membrane fluidity (Chin and Goldstein, 1981). However, increasing membrane fluidity by inserting cis-vaccenic acid increased Na-Ca exchange activity, increasing the extrusion of intracellular C a (Michaelis et al., 1985). Whether the inhibitory effects of ethanol on the Na-Ca exchange antiporter system which thereby increases [Ca], can be extended to other sedative-hypnotic drugs is not yet known.

D. CALCIUM SECOND MESSENGER SYSTEMS AND DRUG ACTION Ca serves as an intracellular messenger (Williamson et a l . , 1981) for signal transduction when cell-surface receptors are activated. There are also other second messenger systems (Nairn et al., 1985; Worley et al., 1987) including cyclic AMP and cyclic GMP. It has been estimated that the cytosolic free C a is

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maintained in the range of 0.1-0.2 f l o r less (Rasmussen and Barrett, 1984). However, when neurons are stimulated by a-adrenergic or cholinergic agonists, the intracellular Ca concentration can rise up to 0.8 pA4 by mobilizing intracellular calcium stores (Reinhart et al., 1983; Berridge, 1984). The second messenger system involving Ca mobilization requires the breakdown of cellular membrane phosphatidylinositol by a phosphoinositide-specific phospholipase C in the plasma membrane, resulting in the intracellular release of inositol 1,4,5-triphosphate (Ins-l,4,5-P3) and diacylgylcerol (Nishizuka, 1984). Ins-1,4,5-P~causes the release of calcium from the endoplasmic reticulum (Streb et al., 1983; Gandhi and Ross, 1987). Diacylglycerol, in the presence of normal cytosolic levels of calcium, can increase the affinity of protein kinase C to calcium and can activate the enzyme to phosphorylate a variety of proteins and ion channels (see Section II1,C). Thus, receptor-mediated activation of phospholipase C leads to a complex network of intracellular signals resulting in increased phosphorylation of various soluble and membrane-bound proteins, thereby changing the state of neuronal function. Modification of the Ca second messenger system can therefore produce profound effects on neuronal activity. The precursor for the formation of inositol phosphates is the phosphatidylinositides in the cell membrane. The membrane phosphatidylinositides are in turn synthesized from myoinositol, which is formed de nouo from glucose-6-phosphate undergoing cyclization and dephosphorylation (Eisenberg, 1967), and can be obtained from the blood circulation by an active uptake system (Caspary and Crane, 1970; Hauser, 1969). Drugs that have effects on the synthesis or active uptake of myoinositol could therefore affect inositol phosphates and the Ca second messenger system. Allison and Cicero (1980) showed that acute ethanol administration (3 gkg) significantly depressed myoinositol-l-phosphate levels in the cortex by 60% within 30 min. In fact, a significant depression of myoinositol-l-phosphate was found 5 min after injection. The decreased myoinositol-1 -phosphate levels returned to normal 24 hr after the initial injection. The decreased myoinositol-lphosphate levels were correlated with blood alcohol levels and the time course of the change was correlated with the behavioral changes. It was suggested that myoinositol-1 -phosphate might participate in the acute pharmacological effects of ethanol. Allison et al. (1976) observed that lithium markedly increases myoinositoll-phosphate levels in brain cortex. Lithium has been shown to attenuate alcohol self-administration in both humans and animals, and to reverse some of the acute pharmacological effects of ethanol (Kline et al., 1974; Judd et al., 1977), suggesting that increased myoinositol-1 -phosphate can antagonize ethanol effects.

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i t has been shown (John et al., 1985) that synaptosomal phospholipase A2 and the phospholipid base-exchange enzymes are highly dependent on extracellular C a concentration and that these enzymes were significantly inhibited by the presence of 50 mM ethanol in vitro. When ethanol was administered to rats chronically, the activities of phospholiphase A2 and phospholipid base-exchange enzymes were increased according to the time course of treatment. The increase in the enzyme activities persisted through the period when the animals were undergoing a physical withdrawal from ethanol. The synaptic membranes obtained from chronically ethanol-treated animals showed less sensitivity to ethanol effects in uitro, suggesting that ethanol tolerance and dependence are associated with changes in membrane phospholipid metabolism and enzyme activities. Hudspith et al. (1985) showed that phospholipase C activity was inhibited in the presence of 50 m M ethanol in uitro but was slightly increased in the brains of animals treated chronically with ethanol. Also, the phosphatidylinositol turnover as the result of membrane depolarization was increased in the brains of the chronically ethanol-treated animals. These results suggest compensatory alterations in the activity of Ca-activated enzymes of phospholipid metabolism in brain tissue during the continued presence of ethanol in uiuo. Since polyphosphoinositides may occur in two forms in rat brain-metabolically inert and metabolically labile (Eichberg et al., 1971; Hauser et al., 1971; Urna and Ramakrishnan, 1983)-the increase or decrease in the metabolically active forms could therefore account for the observations of Hudspith et af. (1985). However, Shah et al. (1984) found that when female rats were allowed to consume ethanol during gestation and lactation, the turnover of the metabolically labile pools of phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-bisphosphate were impaired in the ethanol-exposed pups compared to the nonexposed pups. This result suggests that either the metabolically labile polyphosphoinositides easily accessible to hydrolyzing enzymes were absent in the brain tissue of ethanol-fed pups or the responsible degradative enzymes had become inactivated as the result of the chronic ethanol intake. If chronic ethanol administration indeed decreases the metabolically active polyphosphoinositides, the depolarization of brain synaptosomal membranes should not bring about an increase in the turnover of phosphatidylinositols, unlike what was shown by Hudspith et al. (1985). This apparent disagreement was resolved recently by a study reported by Hoek et al. (1987), who demonstrated the short-term effects of ethanol on C a homeostasis in isolated hepatocytes. Ethanol caused a rapid transient activation of the Ca-dependent phosphorylase a, not associated with changes in CAMPlevels. The activation peaked after 20-30 sec and declined slowly over a period of 5-10 min. Maximal activation was found with 200 mM ethanol,

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but a significant effect was observed with 25 m M ethanol. The effects of ethanol on Ca mobilization were investigated in the hepatocytes loaded with the intracellular calcium indicator, quin-2. The addition of ethanol caused a transient increase in cytosolic free Ca, with the same time course as noted for the effects of ethanol on the activation of the phosphorylase a activity. Once the hormone-sensitive Ca pools in the cell were depleted, the effects of ethanol on Ca mobilization were diminished, suggesting that the Ca mobilization affected by ethanol was activated by a second messenger system. To further elucidate the mechanism, Hoek et al. (1987) labeled hepatocytes with 32P,and the metabolism of inositol phosphates was investigated. Addition of ethanol to 32P-labeledhepatocytes caused a 5-7 % decrease in the level of [32P]phosphatidylinositol 4,5-bisphosphate and a 10-15 % increase in [32P]phosphatidylinositol-4-phosphateand [32P]phosphatidicacid. In the my~-[~H]-inositol-labeled hepatocytes, ethanol induced a 50-100% increase in the levels of inositol 1,4,5-triphosphate, inositol 1,3,4-triphosphate, and inositol biphosphate. More importantly, the changes in inositol 1,4,5-triphosphate levels due to ethanol (200 mM) paralleled the time course of the elevation of cytosolic free calcium levels and the activation of phosphorylase a. These results indicate that ethanol (acutely in vitro) increased the membrane phosphatidylinositide turnover via activation of hormone-sensitive phosphoinositide-specificphospholipase C activity. T h e activation of phospholipase C enhanced Ca mobilization through the inositol second messenger system. Whether ethanol affects CNS second messenger systems in a similai- manner is a matter for further research.

V. Conclusions

The relationship between calcium and sedative-hypnotic drug action is an emerging and fruitful area of research. We feel that the most productive approach is to combine, where possible, cellular electrophysiological studies with biochemical investigations. Electrophysiology, in part, suffers from being too specific (i.e., sampling of one neuronal type at a time), whereas biochemistry can be too nonspecific, since usually brain fractions involving many different types of neurons and other tissues are sampled. In the future, electrophysiological data should be obtained from several brain regions and more effort should be spent on examining the electrophysiological correlates of tolerance, dependence, drug-induced brain damage, and aging. Rather than examining C a spikes, more specific analyses of drug effects on whole-cell and membrane-patch voltage-clamped Ca currents should be undertaken. Future biochemical research could include more specific regional brain or the purer

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cell culture tissue measurements, better measures of acute drug actions, and further investigations into drug interactions with the [Ca], regulating mechanisms, including cytoplasmic organelles, and with the Ca second messenger system. More attention must be paid to lower and pharmacologically relevant drug concentrations, since many studies report drug effects using supra-anesthetic o r neurotoxicological concentrations. Technological advances will permit clearer answers to more precise questions. These include in vivo positron emission tomographic scanning, in vivo magnetic resonance imaging with spectroscopy, ion-selective microelectrodes, enzyme-specific microelectrodes, Ca-specific indicator dyes with neural imaging, and whole-cell and patch-clamp recording techniques. For example, patch-clamp recordings will permit direct measures of drug interaction with single ionic channel events. With regard to the data reviewed herein, all three drug types seem to block inward C a currents in neurons and depolarization-induced synaptosomal Ca influx, although micromolar concentrations of a benzodiazepine were required (Skerritt ef al., 1984; Taft and DeLorenzo, 1984). On the other hand, our laboratory showed enhanced Ca action potentials by nanomolar concentrations of midazolam, which may be pharmacologically more relevant. However, it must be mentioned that in guinea pig myenteric neurons, Cherubini and North (1985) showed decreased C a action potentials with picomolar concentrations. Our working hypothesis for acute neuronal sedative-hypnotic drug action is that ethanol and barbiturates, in pharmacological doses, increase intracellular Ca, which secondarily tends to inactivate inward Ca currents (Eckert and Chad, 1984), which would explain their inhibitory effects on depolarization-induced synaptosomal C a influx. Based on the hippocampal neuronal data wherein ethanol- and pentobarbital-induced hyperpolarizations were present even in zero C a perfusate and based on the synaptosomal Ca influx data, the source of the drug induced raised [Ca], was deduced to be intracellular. Published work to date on ethanol (none found for barbiturates or benzodiazepines) suggests that the Na-Ca pump or the CaATPase-dependent pump are inhibited, thereby raising [Ca],. The role of other [Cali homeostatic mechanisms remains to be explored for all three drug types. The data dealing with Ca-channel blockers and drug actions are incomplete and sometimes controversial. However, the study of Mendelson et al. (1984), showing that Ca-channel blockers antagonized the hypnotic effects of flurazepam and diminished the diazepam-induced increased synaptosomal Ca uptake, supports our hypothesis that phamacologicaal doses of benzodiazepines act by increasing [Ca], by increasing inward C a currents (Carlen et al., 1983b).

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This review has stressed the concept that acute sedative-hypnotic drug action is related to changes in intracellular Ca concentration. A transient rise in [Cali not only triggers ionic conductance changes (in this case increased gK which hyperpolarizes the neuron), but also interacts with the Ca second messenger system, thereby possibly activating several longer-term neuronal processes, depending on the cell type being examined. We hypothesize that the longer-term effects of sedative-hypnotic drugs such as tolerance and dependence phenomena will significantly involve Ca-second messenger interactions. Acknowledgments This work was supported by the MRC, OMH, and ABMRF. Secretarial assistance was provided by Yvonne Bedford.

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PATH0BI0LOGY OF NEURONAL STORAGE DISEASE By Steven U. Walkley Department of Neuroscience Rose F. Kennedy Center for Research in Mental Retardation and Human Development Albert Einstein College of Medicine Bronx, New York 10461

I. 11.

111.

IV. V.

VI.

VII.

Introduction Historical Aspects Experimental Animal Models A. Inherited Diseases B. Induced Diseases Structural Changes in Neurons A. The Storage Process B. Growth of Meganeurites and Neurites C . Alterations in Synaptic Connectivity D. Abnormalities of the Plasmalemma E. Spheroid Formation within Axons F. Changes in the Dendritic Domain Disordered Function of Neurons A. Clinical Manifestations of Disease B. Electrophysiological Studies The Role of Gangliosides A. Gangliosides and Neurite Growth B. Gangliosides and Synaptic Transmission Explaining Neuronal Dysfunction A. The Cytotoxicity Hypothesis B. Disordered Neuronal Geometry and Connectivity C. A Cascade of Events Concluding Comments A. The Promise of Therapy B. Storage Disorders and Neuroscience References

1. Introduction

The neuronal storage disorders are a complex family of diseases characterized by an abnormal accumulation of unmetabolized substances 191 INTERNATIONAL REVIEW OF NEUROBIOLOCY, VOL. 29

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within neuronal somata. A wide variety of enzymatic and molecular events underlies this storage process and these are most often known to be associated with the lysosomal system of affected cells. The storage process thus is seen primarily as a lysosomal event, although for some types of storage diseases, e.g., neuronal ceroid lipofuscinosis, this linkage is not firmly established. A complete discussion of the pathobiology of these diseases would necessarily encompass all pathogenetic events spanning from the molecular level to clinical manifestations of disease, clearly a much too ambitious task for a single review. Fortunately, many of the important enzymatic and molecular events known to be associated with different neuronal storage diseases have been discussed in earlier review articles (Adachi et al., 1978; Beaudet, 1983; Brady, 1983; Johnson, 1981; McKusick and Neufeld, 1983; O’Brien, 1983; Sandhcff and Christomanou, 1979; Sandhoff and Counzelmann, 1984), to which the reader is referred. The principle goal of this article is therefore to concentrate on the consequences of these primary metabolic disturbances at the level of individual neurons or of functional groups of neurons. In so doing an attempt will be made to develop a framework for understanding those factors making specific contributions to the malfunction of neurons and to the generation of those clinical manifestations which are characteristic of these diseases.

HISTORICAL ASPECTS Neuronal storage disorders first became recognized as distinct disease entities during the latter part of the last century, although limited clinical reports had appeared much earlier (see Rq4 et al., 1982). It was the initial clinical and ophthalmologic observations of Tay (1881) and the subsequent, independent clinical and pathological report by Sachs (1887) that can be cited as the first detailed descriptions of a neuronal storage disease. Later referred to by Sachs (1903) as amaurotic family idiocy to indicate the three hallmarks of the disease-blindness, familial taint, and mental retardation-this disorder in subsequent years became known as Tay-Sachs disease. Variations in age of disease onset, types of clinical signs, and severity of pathological features eventually led to recognition of other storage diseases and these too were identified at this time primarily by eponyms (e.g., Fabry’s disease and Niemann-Pick’s disease). Common to the many cases of Tay-Sachs disease described during this period were certain cytological features of brain cells. Sachs recognized that the “ganglion cells” (i.e., cortical pyramidal neurons) were severely affected in this disease and displayed marked swelling of their cell bodies and dendrites, but with axons appearing spared (Sachs, 1887, 1903; Sachs and Strauss, 1910). This swelling often pushed the nucleus to one side of the cell

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and ultimately appeared to cause the cells to degenerate. Particular emphasis was given to peculiar enlargements within the basilar dendrites of neurons, and these changes were often referred to as being pathognomonic for this disease (Sachs and Hausman, 1926). Advances in histological staining methods soon led to the view that neurons in Tay-Sachs disease contained a “lipoid” substance, and at least one investigator went so far as to suggest that a missing “ferment” or enzyme was responsible for this change (Bielschowsky, 1921). The actual biochemical nature of these materials accumulating within neurons in Tay-Sachs disease was not characterized until 1940, when Klenk recognized them as sialic acid-containing glycolipids and coined the term “ganglioside.” But it would still be almost three decades before it would be established that these diseases could be classified according to the primary storage of one particular ganglioside (Suzuki and Chen, 1967). In spite of the often remarkable observations and speculations of the early investigators of Tay-Sachs disease, a full understanding of the primary etiologic event underlying this disease process had to await major advances in cell biology and genetics. The rediscovery of Garrod’s concept of inborn errors of metabolism (1908), concomitant with the one gene-one enzyme concept of Beadle and Tatum (see Beadle, 1959), and the later discovery of lysosomes by de Duve and colleagues (1955) all were crucial in this process. In 1965 the concept of a heritable lysosomal enzyme deficiency underlying cell storage was first proposed by Hers, and in subsequent years a whole family of neuronal storage disorders growing out of select enzyme deficiencies was established (Suzuki, 1976). Perhaps not surprisingly, both the early and much of the modern work on storage diseases dealt primarily with those events underlying the initiation of the disease process, rather than with phenomena specifically responsible for altering the function of neurons. It was only after the establishment of storage diseases as lysosomal enzyme deficiency states that critical attention came to be focused on how the storage process might compromise cell function. The predominant view which emerged suggested that cell function was comprised simply through mechanical disruption of perikaryal cytoplasm and its organelles, with this process ultimately culminating in cell death (Desnick et al., 1976). This so-called “cytotoxicity hypothesis” was eminently logical but, as will be shown below, left many interesting secondary pathogenetic events unaddressed. The first truly modern hypothesis of cell dysfunction in neuronal storage disease came in 1976 when Purpura and his colleagues attempted to use established principles of neurobiology to address functional abnormalities at the cellular level in ganglioside storage disease (Purpura and Suzuki, 1976). Golgi studies of these disorders revealed with considerable clarity the remarkable changes occurring in neurons concomitant with the storage process (Fig. 1). These consisted of enlargements, not in the basilar dendrites as

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I

DW

f.

50um

FIG. 1. Camera lucida drawings of Golgi-impregnated layer 111pyramidal neurons from cerebral cortex of a 2.5-yr-old child with GMz gangliosidosis (AB variant). Asterisks identify cell bodies; M, meganeurites; and a , axons. Somata and meganeurites are separated by narrow necks (straight arrows). Secondary neurites are in abundance and occur primarily at the distal ends of meganeurites (curved arrows). Apical and basilar dendrites showed signs of degeneration and have been truncated for purposes of illustration. (Slightly modified fmm Purpura and Walkley, 1981,with permission.)

Sachs had believed (Sachs and Hausman, 1926) or in the apical dendrites as later erroneously conceived (Crome and Sterne, 1976), but interposed as a new cellular compartment, a “meganeurite, I’ occurring between cell soma and axon. The dendritic-like nature of meganeurite membrane and the formation of synapses on this new region of the cell led to an entirely new approach to understanding neuronal dysfunction in Tay-Sachs disease.

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II. Experimental Animal Models

Advances in understanding the pathogenesis of neuronal storage diseases has relied to a considerable degree on the availability of wellcharacterized animal analogs of known human disorders. Not only do these models allow for physiological studies directed at understanding neuronal dysfunction, but rapid tissue collection a n d o r fxation allows for a host of developmental, morphological, and neurochemical analyses which are generally impossible to accomplish with human postmortem tissue. Given this importance of experimental animal models in exploring the pathogenesis of neuronal storage disease, a brief overview of these diseases in animals is given below.

A. INHERITEDDISEASES Unlike human neuronal storage disorders, these conditions in animals were only rarely described as distinct disease entities prior to Hers’ (1965) development of the lysosomal storage disease concept (e.g., see Innes and Saunders, 1962). One of the earliest models was that of a degenerative neurological condition recognized in English setters at the Veterinary College of Norway (Hagen, 1953). A breeding colony of these animals was established and is still in existence. Originally referred to as “amaurotic idiocy,” detailed genetic and pathologic studies later established this disease to be a close analog of human juvenile ceroid lipofuscinosis (Koppang, 1982). Similar reports of unusual neuronal storage-like conditions also appeared in the U.S. and in Australia. A so-called lipid dystrophy was observed in a 2-year-old cocker spaniel and was described as being similar to late infantile or juvenile human idiocy (Ribelin and Kintner, 1956). The first cases of pseudolipidosis in Angus cattle, later discovered to be a-mannosidosis (see below), were reported in Australia at about the same time (Innes and Saunders, 1962). Although these diseases were recognized as being similar to certain human disorders and distinct from better-known encephalidites of animals, little else was known about them. Following Hers’ initial report and the subsequent classification of a variety of human storage disorders according to select enzyme deficiencies, a larger number of similar conditions in animals began to appear. Scattered reports in the late 1960s suggested that ganglioside storage diseases occurred in a variety of domestic species, including dogs, cats, swine, and cattle (for reviews, see Adachi, 1975; Baker et al., 1976, 1979). However it was not until Baker and colleagues published data on feline GMI gangliosidosis that the first animal

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model of this disease was fully documented by enzymatic, neurochemical, genetic, and pathologic criteria, and a research colony was established (Baker et al., 1971). This successful and early establishment of a breeding colony, the remarkable similarity to human juvenile GMI gangliosidosis, and the ready availability of mutants have made this particular model the cornerstone for studies directed at the pathogenesis of neuronal dysfunction in storage disorders. Kittens with this disease appear normal at birth and successfully achieve all developmental milestones. At about 4 months of age discrete head and hind limb tremors occur which slowly increase in intensity resulting in distinct gait abnormalities by 6 months of age. Spastic quadriplegia, impaired vision, and hyperacusis are all evident by 8 months of age. Seizure activity has been reported in those animals allowed to survive past 10 months. During advancing clinical signs at 7-9 months of age these animals remain alert and active, eat aggressively, and show little visceral involvement, thus making them excellent candidates for a variety of studies. In addition to feline GMI gangliosidosis, a variety of other ganglioside storage disorders have now been fully documented in animals. These include two canine models of GMI gangliosidosis, occurring in English springer spaniels (Alroy et al., 1985) and in a predominantly beagle phenotype (Read et al. , 1976). Both displayed 0-galactosidase deficiencies with neuronal and visceral storage resembling human GM1 gangliosidosis type 1, but differences in the oligosaccharide portion of storage materials suggested that the diseases in the two breeds may not be identical. Bovine GMX gangliosidosis also has been described and appears comparable to the type 2 form in humans based on clinical, morphological, and biochemical criteria (Donnelley et al., 1973). Storage of GM1 ganglioside also has recently been reported in sheep (AhernRindell et al., 1985, 1987) but appears to be associated with deficiencies of both P-galactosidase and a-neuraminidase. Available experimental evidence has suggested that this disorder may not be the same as combined P-galactosidase/a-neuraminidase deficiency in humans (galactosialidosis) where the 1 L protective protein” for this enzyme complex is apparently defective (D’Azzo et al., 1982). Both feline and canine models of GMz gangliosidosis also have been fully documented. The feline model appears to be a replica of Sandhoff’s disease, (GM:! gangliosidosis, type 2), as both major electrophoretic forms of 0-hexosaminidase (A and B) were deficient (Cork et al., 1977). Like feline GM1 gangliosidosis, this model also has been in colony production for a number of years and GMz mutants have been used in a variety of experimental studies. Canine GMZ gangliosidosis was only recently fully documented and this case appeared to be an authentic model of the AB variant form of the disease (Cummings et al., 1985; Ishikawa et al., 1987). Although not presently in colony production, its appearance in a number of

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animals of a single breed (Japanese spaniels) and geographic area make it likely that additional mutants and/or carrier animals could be located. Porcine GM2 gangliosidosisa also has been described but awaits complete documentation (Kosanke et al., 1978). Canine and feline models of mucopolysaccharide storage diseases with central nervous system (CNS) involvement (types I and VII) also have been documented and breeding colonies established (Spellacy et al., 1983; Haskins et al., 1983, 1984; Shull et al., 1982). Sphingomyelin lipidosis has been described in dogs and cats as well, but colonies are not presently available (Bundza et al., 1979; Wenger et al., 1980). Both a-and 0-mannosidosis have been documented in animals. Bovine a-mannosidosis was once a significant herd health problem in Angus cattle in New Zealand (Jolly, 1971), and experimental animals are still available from this source. Feline a-mannosidosis has been documented several times in different geographic areas (Burditt et al., 1980; J. F. Cummings et al., unpublished; Jezyk et al., 1986; Vandevelde et al., 1982) and may occur as both acute (3 months duration) and chronic (1 year duration) forms. Caprine P-mannosidosis has also been documented (Jones and Dawson, 1981) and represents the only incidence where the discovery of an animal model for a particular lysosomal hydrolase deficiency preceded the characterization of its human counterpart. Canine, feline, and ungulate models of fucosidosis, glycogenosis, and neuronal ceroid lipofuscinosis have also been described (Cho et a l . , 1986; Cook et al., 1982; Jolly et al., 1980; Kelly et al., 1983; Koppang, 1973/1974; Rafiquzzaman et al., 1976; Sandstrom et al., 1969). A complete listing of these inherited animal models of neuronal storage diseases is given in Table I.

B. INDUCEDDISEASES The rare and sporadic occurrence of neuronal storage diseases and the difficulties inherent in the successful development and long-term maintenance of breeding colonies have generated a sustained interest in the development of inducible models of these disorders. Given that inherited storage diseases are presently untreatable in any form, the likely prospect of being able to reverse an experimentally induced disease and to follow the fate of morphological and biochemical abnormalities in the CNS also has given impulse to these studies. Finally, agents which specifically interfere with normal lysosomal function have proved useful for studying normal physiological events related to these catabolic processes in cells. A variety of approaches to inducing lysosomal enzyme dysfunction in neurons has been

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STEVEN U. WALKLEY TABLE I ANIMAL MODELSOF INHERITED NEURONAL STORAGE DISEASE Disease

GM, gangliosidosis

GM2 gangliosidosis

Sphingomyelin lipidosis Mucopolysaccharidosis I Mucopolysaccharidosis VII Fucosidosis a-Mannosidosis 0-Mannosidosis Glycogenosis

Ceroid lipofuscinosis

Species

Selected references

Feline Canine Ovine Bovine Feline Canine Porcine Feline Canine Feline Canine Canine Canine Bovine Feline Caprine Bovine Canine Feline Canine Ovine

Baker ef d. (1971) Alroy cf 01. (1985); Read ef 01. (1976) Murnane cf d. (1986) Donnelly ef 01. (1973) Cork ct 01. (1977) Curnmings et 01. (1985) Kosanke ef 01. (1978) Wenger ef 01. (1980) Bundza d 01. (1979) Haskins ct al. (1983) Shull et 01. (1982) Haskins ef 01. (1984) Kelly ef 01. (1983) Jolly (1971) Burditt ct 01. (1980); Vandevelde cf 01. (1982) Jones and Dawson (1981) Cook ef al. (1982) Rafiquzzaman ct 01. (1976) Sanstrorn cf 01. (1969) Koppang (1973/1974); Cho et ~ l(1986) . Jolly cf 01. (1980)

tried, and some of the more successful agents and the types of storage induced are listed in Table 11. One of the most widely used of these approaches involves amphiphilic cationic drugs which are known to induce lysosomal storage following chronic administration (for reviews, see Blohm, 1979; Drenckhahn and LullmanRauch, 1979). Indeed, such deleterious side effects were first seen in humans following the use of some of these agents. Intracellular storage generally appears as membrane-bound, acid phosphatase-positive cytoplasmic inclusions with a crystalline or lamellated appearance. Biochemically, large quantities of polar lipids can be found in association with the administered drug and it has been suggested that many of these agents act through the formation of such drug-lipid complexes which are not readily catabolized by lysosomal hydrolases (Drenckhahn and Lullman-Rauch, 1979). Most of these agents apparently do not act through the inhibition of specific lysosomal hydrolases, although AY9944 has been suggested to cause a significant decrease in sphingomyelinase activity through impaired enzyme synthesis (Sakuragawa et al., 1977). Discontinuation of drug administration in all of these examples appears to lead to a gradual elimination of the stored material.

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TABLE I1

INDUCED MODELSOF NEURONALSTORAGE DISEASE Agent

Disease equivalent

Selected references

Chloroquine Chlorphentermine AY9944 Suramin Leupeptin AETT Conduritol-8-epoxide Swainsonine

Nonspecific storage Nonspecific storage Sphingomyelin lipidosis Mucopolysaccharidosis Ceroid lipofuscinosis Ceroid lipopigment storage Glucosylceramide storage a-Mannosidosis

Klinghardt (1977) Lullmann-Rauch (1974) Sakuragawa d al. (1977) Canstantopoulos ct al. (1980) Ivy et al. (1984) Spencer et al. (1979) Kanfer et al. (1975) Dorling ct al. (1978)

Experimental studies using amphiphilic bases have been limited primarily to induction of storage in nonneuronal tissues (Lullmann-Rauch, 1979) or in those areas of the CNS not protected by the blood-brain barrier (Frisch and Lullmann-Rauch, 1980). Some agents (e.g., AY9944) appear to be effective in inducing storage in rats only during the very early postnatal period (Suzuki and De Paul, 1971), whereas others (chloroquine, chlorphentermine) have proved effective in older animals (Adachi et al., 1976; Gleiser et al., 1968; Klinghardt, 1977). The nonspecific action of these agents on lysosomal hydrolases andlor the limited ability of some to penetrate the blood-brain barrier, however, have generally reduced their usefulness for producing experimental models of inherited neuronal storage diseases. This problem of drug access to the CNS has been circumvented in some studies through the use of injection or infusion directly into brain tissue. Chronic administration of chloroquine and of leupeptin, a thiol protease inhibitor (Toyo-oka et al., 1978), have resulted in significant intraneuronal storage in rats. The latter model closely resembled inherited neuronal ceroid lipofuscinosis and suggested that this disease may actually be due to a defect in this enzyme system (Ivy et al., 1984). A similar induction of storage of ceroid lipopigments has been reported following ingestion of acetyl ethyl tetramethyl tetralin (AETT) (Spencer et al., 1979). Finally, intracerebral injections into rats of the trypanocidal drug, suramin, has been reported to cause significant storage of glycosaminoglycans and gangliosides (GM2, GM3, GD3) and to replicate inherited mucopolysaccharidosis (Constantopoulos et al. , 1980). Reversible and irreversible (“suicide substrate”) inhibitors of lysosomal hydrolases have been used to induce specific forms of lysosomal storage, although in many cases such agents have been applicable only to tissue culture systems. For example, 0-GalMNT (0-D-galactopyranosylmethyl-p-nitrophenyltriazine)has been shown to be an effective suicide substrate for 0-galactosidase and P-glucosidase in human fibroblast cultures (Sinnott and Smith, 1978; Van Diggelen and Galjaard, 1980) but

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apparently has not proven effective in animal models. In contrast, conduritol-0epoxide has been found to irreversibly inhibit a-glucosidase and an experimental model of Gaucher’s disease has been produced in mice (Kanfer et al., 1975). A number of reversible inhibitors of lysosomal hydrolases also have been developed (e.g., D-galactal and galactolactone for /3-galactosidase), but as with the suicide substrates successful in vivo application has not been achieved (for review, see Lalegerie et al., 1982). The most successful of the reversible inhibitors of lysosomal hydrolases has been swainsonine, an indolizadine alkaloid found in certain plants (Dorling d al. , 1978; Molyneux and James, 1982) and in aplant fungus (Schneider d al., 1982). Chronic ingestion by livestock of plants of Swainsona and Astragalur spp. in Australia and the U.S., respectively, has long been known to result in a neurological condition which resembled inherited bovine a-mannosidosis (Hartley, 1971;James d al., 1970;Jolly, 1971; Van Kampen and James, 1969). But it was not until the seminal work of Dorling and his colleagues (1978) that the etiologic agent in Swainsonu (“darling pea”) plants was recognized. This alkaloid, named swainsonine, was characterized as an active site-directed and reversible inhibitor of lysosomal a-mannosidase. More recently, it has also been proven to inhibit Golgi membrane-associated a-mannosidase and thereby to interfere with glycoprotein processing (Tulsiani and Touster, 1983). Partially purified extracts from dried Astragalur (“locoweed”) hay also have been shown to contain a specific inhibitor of lysosomal a-mannosidase (Siegel et al., 1982), and this alkaloid was subsequentlyproven to be structurally equivalent to swainsonine(Molyneux and James, 1982). Swainsonine-induced a-mannosidosis has been shown to closely resemble inherited a-mannosidosis in essentially every aspect at the morphological level. Ultrastructural features of cytoplasmic storage vacuoles appear identical in the two diseases and are acid phosphatase positive (Novikoff et al., 1985; Walkley et al., 1986). As will be described in detail in a subsequent section (III,B), the phenomenon of ectopic dendritogenesis which is known to characterize most neuronal storage diseases is essentially identical in feline models of inherited and swainsonine-induced a-mannosidosis (Walkley and Siegel, 1985). Swainsonine-induced a-mannosidosis has also been successfully applied to the study of reversibility of storage-induced CNS lesions (Huxtable et al., 1982), including ectopic dendrite growth (Walkley et al., 1987).

Ill. Structural Changes in Neurons

A. THESTORAGE PROCESS The early studies on Tay-Sachs disease by Sachs (1887, 1903; Sachs and Strauss, 1910; Sachs and Hausman, 1926), Schaffer (1925), Bielschowsky

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(1921), Wolfsohn (1915), and others fully established that significant changes occurred within somata of neurons located throughout the CNS. The contour of nerve cells was found to be considerably distorted as somata were swollen and basilar dendrites appeared to develop proximal enlargements. Nissl substance was believed to disappear during the process of cell enlargement and was replaced by a fine, granular material. Normal intrasomatic fibrils, as could be seen with silver stains, were absent or were displaced to the cell periphery. Fibrils in axons and dendrites, in contrast, appeared normal. Sudan stains revealed that the bulk of the substance accumulating within swollen neuronal somata was lipid in nature. Cell death generally was not cited as a significant component of the disease process in the CNS, rather it was the existence of these striking changes in neuronal morphology which appeared most important to these early investigators. Apart from better defining the lipid materials accumulating within neurons in Tay-Sachs disease (Klenk, 1939-1940), the next major advance in understanding the cellular pathology of this and related storage disorders emerged with the advent of electron microscopy. Ultrastructural studies revealed all normal cellular constituents to be present within brain cells undergoing the storage process (Terry and Weiss, 1963) (Figs. 2 and 3). Although the Nissl substance appeared dispersed or diminished at the light microscopic level, electron microscopy (EM) revealed large numbers of ribosomes scattered throughout the cytoplasm. Other organelles such as mitochondria, the Golgi complex and endoplasmic reticulum, microtubules, and so forth could be found. But by far the most conspicuous feature of neurons at the EM level was the presence of characteristic cytoplasmic storage vacuoles or cytosomes. (Fig. 4). In Tay-Sachs disease, one of the first of the neuronal storage disorders to be studied ultrastructurally, cytosomes were found to consist largely of membranes and were referred to as membranous cytoplasmic bodies or mcb’s (Terry and Weiss, 1963). At the time of this initial ultrastructural characterization the nature of these storage vacuoles was not clearly understood. Lysosomes had recently been discovered (de Duve et al., 1955) and, indeed, acid phosphatase staining revealed that many of the mcb’s in Tay-Sachs disease were lysosomal in nature (Terry and Weiss, 1963). The latter finding was documented in greater detail the next year (Wallace et al., 1964), but the view that the production of such storage vacuoles was dikectly linked to absence of a particular lysosomal hydrolase was not proposed until later (Hers, 1965). These and subsequent ultrastructural studies have fully documented that storage diseases are characterized by the presence of specific types of cytoplasmic vacoules (Fig. 4). These cytosomes are most often found within neuronal perikarya, are generally less abundant in large dendritic processes, and are only rarely encountered in smaller dendrites or in synapses. They appear to be absent from axons. Although an explanation for this particular

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FIG. 2 . Electron micrograph of a typical neuron (n, nucleus) from the motor cortex of a 9-month-old cat with GMI gangliosidosis. 'The cell body is filled with characteristic membranous storage vacuoles. Also note that the neuropil immediately surrounding this neuron appears distorted (i.e., compressed) secondary to somatic expansion. Calibration bar equals 2.0 pm.

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FIG.3. Electron micrograph of a cortical neuron from a cat with GM? gangliosidosis at 6 months of age. Note the presence of a normal-appearing Golgi apparatus (G) with adjacent coated vesicles (curved arrow). Typical storage vacuoles (SV) are nearby. Other organelles (endoplasmic reticulum, ribosomes, mitochondria) appear normal, as does an adjacent axosomatic synapse (arrow). Calibration bar (lower left) equals 0.2 pm.

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FIG.4. Typical storage vacuoles from cortical neurons in a variety of feline models of neuronal storage diseases. (a) Membranous cytoplasmic body from GM,gangliosidosis, (b) membranous cytoplasmic body from GMz gangliosidosis, (c) zebra body-type inclusion as found in

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distribution has not been fully detailed in the literature, it is likely that it simply reflects the normal distribution of lysosomes within neurons rather than some specific characteristic tied to the degradative metabolic pathways of gangliosides or other cell constituents. The latter view is suggested by a similar distribution of cytosomes in a wide variety of neuronal storage diseases, from the gangliosidoses to a-mannosidosis. This predominance of storage vacuoles in cell somata leaves the surrounding neuropil appearing surprisingly normal at the EM level, apart from compression-related events seen late in the disease process. The normal ultrastructural appearance of synapses is particularly noteworthy in this regard (Fig. 3). The characteristic morphology of cytoplasmic storage vacuoles differs widely between different types of neuronal storage diseases and is directly related to the primary metabolic defect and to the nature of the material being stored. The mcb’s which characterize the gangliosidoses are 1.5-2.0 pm in diameter and are composed of closely packed electron-dense membranes often arranged in concentric fashion (Terry and Weiss, 1963) (Figs. 2-4a,b). In contrast , storage vacuoles in neurons in mucopolysaccharidosis are lamellated but the membranes tend to occur in stacks (“zebra bodies”) rather than swirls (McKusick a n d Neufeld, 1983) (Fig. 4c), whereas those in a-mannosidosis typically appear clear or contain flocculent or wispy material (Beaudet, 1983) (Fig. 4e). The early studies of Korey, Terry, and their colleagues (Samuels et al., 1963; Terry and Korey, 1963; Terry and Weiss, 1963) did much to elucidate the nature of the membranous inclusions in Tay-Sachs disease and their findings appear to have relevance to other storage diseases. The mcb was found to be composed of both lipid and protein in a ratio of 9: 1, with its characteristic lamellated structure being consistent with the hydrophobic and hydrophilic relationships between these molecules. Less than 50% of the dry weight of isolated mcb fractions was found to be ganglioside, and other lipid materials such as cholesterol and cerebrosides were also found in significant amounts. These and other studies (e.g., see Hers, 1973) suggest that the characteristic morphology of storage vacuoles (1) is due to the physicochemical properties of the stored material, (2) is a manifestation of the primary metabolic defect responsible for the disease, and (3) may reflect storage of heterogeneous materials as a consequence of the missing hydrolase activity or due to secondary events within the cell following this primary defect. Although a precise description of the origin of cytosomes in neuronal storage diseases is still

mucopolysaccharidosis type 1, (d) membranous inclusion from sphingomyelin lipidosis, (e) typical cytosomes found in a-mannosidosis (as induced with swainsonine), which can be seen to contain floccular and fibrillar materials as well as smaller vacuoles, (9 typical storage vacuoles of neurite-bearing pyramidal neurons in a-mannosidosis as visualized with a combined Golgi-EM procedure. The dark stippling seen in this electron micrograph is the result of the gold-toning process just prior to deimpregnation and allows for the visualization of Golgi-impregnated neurons at the EM level. Note the presence of membrane leaflets within the storage vacuoles. Calibration bar in each figure equals 0.2 pm.

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lacking, the most widely held view is that of fusion of autophagic vacuoles with primary lysosomes. The resulting digestive vacuole becomes residual within the cell due to the inherent lysosomal hydrolase defect (Hers, 1973). Although neuronal storage diseases are known to be characterized by specific kinds of storage vacuoles, some heterogeneity of the ultrastructural features of these cytosomes does occur within a given disease, indeed, even within a given neuron. For example, some storage vacuoles within neurons in ganglioside storage disease (e.g., in GM2 gangliosidosis) have a granular or vacuolar appearance which may or may not be accompanied by swirls of membrane leaflets (Fig. 3). Perhaps one of the most striking variations in storage vacuoles occurs in a-mannosidosis. Here occasional pyramidal neurons contain storage vacuoles with large numbers of membrane leaflets (sometimesresembling the zebra bodies of mucopolysaccharidosis), whereas neighboring cells contain vacuoles which are typical of the disease and do not possess membranous components (Fig. 4e,f). In considering the heterogeneity of storage vacuoles in ganglioside storage disease Sandhoffand Counzelmann (1984) suggestedthat these differenceswere the result of local variations in metabolic events in different kinds of neurons. Indeed, given the remarkable diversity of inputs to select neuron types and the unique roles that different neurons must play in the overall function of a local brain region, it would be surprising if these metabolic events were equivalent. As will be addressed in the next section, such variation in cytosomes in cortical pyramidal neurons in a-mannosidosis may hold important clues for understanding certain pathogenetic events in this disease. The relationship between known failures in specific degradative pathways and those related anabolic events in storage diseases are not clearly understood. Sandhoff and Counzelmann (1984) have suggested that in ganglioside storage disease the synthetic machinery for gangliosides may actually be increased, and in one study of GMI gangliosidosis preparations of synaptosomal membranes were found to have significantly elevated levels of GMI ganglioside (Wood et a l . , 1985). Thisjnding is important in that it suggests that the presence of a particular metabolic product in abundance in storage vacuoles reflects efevated leveh of the same material within its n o m l domain in the neuron. Gangliosides are integral membrane components, some forms of which (e.g., GM,) have been suggested to be concentrated in pre- and postsynaptic regions (Hansson et a l . , 1977). Their role in synaptic function, however, is not understood. As will be discussed in Section V,B, functional abnormalities in neurons secondary to the abnormal accumulation of specific gangliosides (e.g., at synapses) may be indicative of their role in normal neurons. Presently available data do suggest that the intrasomatic accumulation of gangliosides is not in itself cytotoxic, at least not in the initial stages of the disease. Neuron death in ganglioside storage disease has generally been reported to be significant only late in the disease process, long after the onset and progression of clinical

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signs. This is in marked contrast to certain other lysosomal storage diseases, e.g., Krabbe’s and Gaucher’s diseases, in which known cytotoxic agents (lysosphingolipids) are known to accumulate and are believed toxic to cells (Igisu and Suzuki, 1984). Very recently this proposal has been carried a step further by the demonstration that such lysosphingolipid derivatives may act through inhibition of protein kinase C (Hannun and Bell, 1987). As the latter enzyme is important in signal transduction across membranes and in cellular differentiation, loss of its activity could have profound effects on neurons. This could include not only cell death, but also alterations in a variety of neuronal operations such as signal transmission at synapses. Since the inhibition of protein kinase C conceivably could represent a common denominator for neuronal dysfunction in all of the sphingolipidoses, testing this new hypothesis will be very important in future studies.

B. GROWTH OF MEGANEURITES AND NEURITES Golgi staining of cerebral cortical neurons from children with ganglioside storage disease has clearly demonstrated the morphological changes induced in these cells as a consequence of this type of metabolic defect (Fig. 1). Cellular enlargements were found, not in the apical or basilar dendritic systems as previously believed, but interposed between cell somata and axons. Although careful review of the literature has revealed that at least one investigator accurately depicted these enlargements during the early studies of Tay-Sachs disease (Bielschowsky, 1921), their full significance clearly was not recognized until the Golgi and ultrastructural studies of Purpura and Suzuki (1976). In the latter, meganeurites were found to be composed of dendritic-like membrane, and ultrastructural studies revealed the presence of synapses on their surfaces. Some meganeurites possessed longer neuritic processes which in terminal disease displayed spines and resembled small dendrites. Two major hypotheses about gangliosides and storage diseases emerged from these studies. First, neuronal dysfunction in these diseases could result from a combination of distorted cell geometry (i.e., the insertion of a meganeurite just proximal to the axon) and the formation of anomalous synaptic connections on meganeurites. Second, the presence of abnormal quantities of gangliosides or closely related glycolipids was suggested to be associated with the recapitulation of dendritic growth features of these neurons. This latter hypothesis found support in then-current studies of ganglioside metabolism during early brain development (Suzuki, 1965) and, as discussed below (Section V,A) has since been further supported by a host of studies in which gangliosides have been applied to neurons in culture.

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A significant extension of these studies in humans has been possible through the use of well-characterized animal models of neuronal storage disorders. The availability of a large number of age-matched cats with GM1 gangliosidosis has allowed for systematic Golgi staining to be performed on the entire CNS in this disease and, as recently reviewed (Walkley, 1987), these studies have fully documented the distribution of meganeurites and neurites in this disease. Four major categories of Golgi-demonstrable morphological changes were described and these had limited distribution to select cell types in the CNS. Spiny meganeurites, i.e., those resembling the type described above in human ganglioside storage disease, were found on cortical pyramidal cells, multipolar cells of amygdala and claustrum, and some medium spiny cells of the neostriatum (Fig. 5a,b). Meganeurites without spines (aspiny m g a m r i t e s ) were occasionally seen on these same cell types as well as on certain neurons of subcortical and brain stem nuclei and on Golgi cells of the cerebellum. Some types of neurons lacked meganeurite formation but possessed tufts of short neuritic processes projecting from the axon hillock region of the cell (Fig. 5c). This secondary neurite growth occurred only on those types of neurons displaying spiny meganeurites. Finally, some types of neurons lacked meganeurites and neurite growth and showed either little or no change in morphology (Fig. 5d) or massive storage with a marked increase in somatic diameter. This latter change appeared mutually exclusiveof meganeurite formation and was believed to suggest important differences in these cell types, possibly at the level of the cytoskeleton. Comparative Golgi studies of a variety of animal models of neuronal storage diseases (GM1 and GM2 gangliosidosis, sphingomyelin lipidosis, mucopolysaccharidosis, a-mannosidosis) have revealed that as neurons become significantly involved in the storage process, t h same cell gpes appear to display neurite growth and meganeuritefonnation regardless qfthepn’mry metabolic defect. For example, axon hillock neurite growth has now been documented on cortical pyramidal neurons in GM1 and GM2 gangliosidosis (Purpura and Baker, 1978; S. U. Walkley and M. C. Rattazzi, unpublished), sphingomyelin lipidosis (Walkley and Baker, 1984), mucopolysaccharidosistype I (Walkley and Haskins, 1982), and a-mannosidosis (Walkley et al., 1981a); Walkley and Siegel, 1985). The only exception to this pattern in diseases studied to date is neuronal ceroid lipofuscinosis, where significant

FIG 5. Golgi-impregnated cortical neurons from a 9-month-old cat with GMI gangliosidosis. (a) Layer I11 pyramidal neuron with a meganeurite (large arrow) which is covered with spine and neurite growth (small arrows), (b) layer 111 pyramidal neuron with a meganeurite (arrow) which possesses only a few spines, (c) layer I11 pyramidal neuron with exuberant axon hillock neurite growth (large arrow) and one somatic neurite (upper arrows), (d) cortical intrinsic neuron with dorsally directed axon and no conspicuous changes in soma or dendrites. Arrowheads indicate axons; calibration bar in (a) equals lOpm and applies to all.

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storage in pyramidal neurons is accompanied only by aspiny meganeurite formation (Braak et al., 1984; S. U . Walkley, unpublished). Ultrastructural studies in human ceroid lipofuscinosis also have suggested that this disease is not characterized by ectopic synapse formation on meganeurites (Williams et al., 1977). For a-mannosidosis, inherited and swainsonine-induced models have been studied and have provided important insight into understanding the phenomenon of ectopic neurite growth. In both diseases only occasional layer 111 pyramidal neurons display axon hillock neurite growth, and ultrastructural studies in combination with Golgi staining have demonstrated the presence of unusual membrane-containing cytosomes in neurite-bearing neurons. Storage vacuoles of this type are in marked contrast to those seen with routine EM (e.g., compare Figs. 4e and 4f) and suggest that unique metabolic events are occurring in these cells relative to nonneurite-bearing neurons. The latter may include glycolipid, or importantly, ganglioside storage, although the one report of ganglioside analysis in a-mannosidosis (human) in the literature suggested no such abnormality (Ockerman, 1973). Given the observation the ectopic neurite growth occurs only on occasional layer I11 pyramidal neurons in a-mannosidosis, accompanying changes in gangliosides, if present, could be quite subtle. Alteration in ganglioside pattens either as a primary or secondary manifestation is known to occur in all other neuronal storage disorders with w r i t e growth. These include sphingomyelin lipidosis (Greenbaum et al., 1976; Wenger et al., 1980) and mucopolysaccharidosis (Gonatas and Gonatas, 1965; Ledeen et al., 1965; Shull et al., 1982), in addition to GMI and GM2 gangliosidosis. Indeed, alterations in gangliosides along with metabolic defects specifically involving lysosomal hydrolase activity, and similar ages of onset of disease, represent the known features in common among these diseases. As mentioned above, ceroid lipofuscinosis, which is not believed to be due to a lysosomal hydrolase deficiency, displays neither ectopic neurite growth nor new synapse formation. The comparative Golgi studies outlined above also suggest that rneganeurites and neurites are not different manifestations of the same phenomenon (Walkley, 1987). Meganeurites accommodate storage and inevitably are filled with characteristic storage vacuoles when examined ultrastructurally. They occur on certain kinds of neurons undergoing intracellular storage regardless of the storage material, whereas other types of neurons increase somatic volume rather than form meganeurites. Neurites, in contrast, do not accommodate storage, but do offer new surface area for ectopic synapse formation (see the next section). Ectopic neurite growth and new synapse formation are even more limited in distribution than meganeurites and appear to occur only on a subgroup of meganeurite-forming cell types. However, even in the latter, neurite growth can proceed in the absence of a

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meganeurite, presumably because intrasomatic storage has been insufficient to generate one. The question of whether a pyramidal neuron undergoing neurite growth is responding to a primary induction of these neurites (with new synaptic contacts forming secondarily) or vice versa is presently unanswered.

C.

ALTERATIONS IN SYNAPTIC CONNECTIVITY

Ultrastructural studies of human ganglioside storage disease by Purpura and Suzuki (1976) revealed that meganeurites were contacted by synapses and subsequently similar data were obtained for feline GM1 gangliosidosis (Purpura et a l . , 1978). Combined Golgi-EM studies using this same model revealed that axon hillock neurites also were contacted by synapses (Walkley et a l . , 1981b). As illustrated in Fig. 6, most of these synapses have prominent postsynaptic densities and can be classified as being asymmetrical. Given their apparent abundance on neurites, the signijicant degree of neurite growth on individual pyramidal cells, and the large number of neurite-bearing neurons in GA41 cat cortex, the amount of new synapse formation in this disease would appear to be substantial. Similar synapses also have been documented in feline GMz gangliosidosis (S. U. Walkley and M. C. Rattazzi, unpublished) and in swainsonine-induced a-mannosidosis (Walkley et a l . , 1987). In each case the majority of these synapses appeared asymmetrical, although occasional exceptions could be found. The source of presynaptic elements forming these synapses on ectopic neurites and on spiny meganeurites is unknown. However, their asymmetric morphology and the close proximity of neurites to the normal basilar dendritic system of pyramidal cells suggest that thalamocortical afferents be considered a likely candidate (see Section V1,C). More recently a second possible alteration in synaptic connectivity has been described in GM1 cat cerebral cortex (Walkley and Wurzelmann, 1986). Immunocytochemical studies using antibodies to glutamic acid decarboxylase (GAD) and directed at examining the GABAergic system in these animals revealed a similar density of innervation between neurons in diseased and normal cortex. However, as pyramidal neurons in the former were often considerably larger than those in normal cortex, the number of GADpositive terminals per cell was actually substantially increased in the GMI cats. Additionally, when meganeurites were clearly identified in these sections, they often appeared to be contacted by GAD-positive terminals. The asymmetrical terminals on secondary neurites, as described earlier, presumably are not GABAergic since the latter have been reported to be symmetrical in form (Houser et a l . , 1984). In recent years a variety of reports have appeared which indicate that the GABAergic system may be

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FIG 6. Golgi and ultrastructural features of a cortical pyramidal neuron with ectopic, axon hillock neurite growth, as derived from a 9-month-old cat with G M t gangiosidosis. Inset: Golglimpregnated neuron sitting adjacent to an impregnated blood vessel in glycerin mount prior to gold toning and deimpregnation. Numerous neurites (small arrows) can be seen projecting from the axon hillock region (compare to Fig. 5c). Electron micrograph: Axon hillock region of the same neuron seen in the inset, identifiable by the presence of gold stippling along inside of plasmalemma. Gold-labeled proftles surrounding the axon hillock represent sections through these neuritic processes and numerous synapses are evident on each (arrows). Calibration bar equals 1.0 p n . (Modified from Walkley ef a l . , 1981b, with permission.)

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capable of sprouting and forming new synapses in certain brain regions in response to local changes such as deafferentation (Goldowitz et al., 1982; Houser et al., 1983; Katsumaru et al., 1986; Nadler et al., 1974). The possibility of similar alterations in GABAergic connectivity in neuronal storage disease clearly deserves closer study.

D. ABNORMALITIES OF THE PLASMALEMMA Golgi studies of ganglioside storage diseases in man and animals have often revealed the presence of irregular surface features on neuronal somata (Purpura and Baker, 1978; Purpura and Suzuki, 1976). These have been studied in greatest detail using a combined Golgi-EM technique and cortical tissues from cats with GM1 gangliosidosis (Walkley et al., 1986b). A variety of surface membrane irregularities were identified over somatic and meganeurite regions of pyramidal neurons (Fig. 7). These ranged from small neuritic processes (microneurites) which did not appear to be associated with synapses to larger bleb-like protrusions. Microneurites projected into the neuropil surrounding the cell and often developed intricate relationships with structures therein (Fig. 7b,d). Surface blebs pushed the adjacent neuropil aside and possessed unusual membrane fusions (Fig. 7a). These alterations in cell surface features appeared distinct from the synapse-laden axon hillock neurites described earlier. However they did appear remarkably similar to the surface features of neuroblastoma cells following addition of bovine brain gangliosides to the culture medium (Roisen et al., 1981b; Spero and Roisen, 1984). Short “spikes” and blebs were reported with scanning EM and these also appeared distinct from the larger neuritic processes on these cells. Although the exact nature and significance of these abnormal surface features of neurons are unknown, they may be related to insertion of excess quantities of ganglioside into the plasma membrane of these cells. Recent studies have shown increased quantities of GMI ganglioside and cholesterol in synaptosomal preparations from GMI cats (Wood et al., 1985), and incorporation of gangliosides from culture medium into neuroblastoma and primary cell cultures has been documented (e.g., see Roisen et al., 1981b). Although the functional consequences of such increases in ganglioside in the plasmalemma are incompletely understood, Wood and colleagues (1985) have shown significantly reduced membrane fluidity in membrane samples derived from GM1 cat cortex.

E. SPHEROID FORMATION WITHIN AXONS Most of the early studies of Tay-Sachs disease suggested that this disorder was limited primarily to changes in neuronal somata and dendrites and that

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FIG.7. Abnormal features of the plasmalemma of cortical pyramidal neurons from a 9-monthold cat with GM1 gangliosidosis, as revealed by a combined Golgi-EM method. (a) Somatic surface bleb (arrow), (b-d) branched and unbranched microneurites (arrows). Processes of this type do not appear associated with new synapse formation. Calibration bar in each figure equals 0.2 pm. (Modified from Walkley ct a l . , 1981b, with permission.)

axis cylinders were spared (see Section 111,A). Later studies using newer histological stains, however, revealed large axonal dilations deep in white matter areas (Bielschowsky, 1921). Enlargements at the base of neuronal

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perikarya also were discovered with these silver staining methods and these came to be referred to as “axonal torpedoes,” presumably to differentiate them from dendritic enlargements which had been described previously (Crome and Stern, 1976). The discovery that enlargements adjacent to many neuronal somata were meganeurites and composed of dendritic-like membrane has in recent years tended to obscure the role of axonal pathology in storage diseases. These axonal, dendritic, and somatic changes occurring in neurons in storage diseases have now been fully documented through the use of a new histochemical technique as applied to feline GMI gangliosidosis (Walkley and Pierok, 1986). These studies used the ferric ion-ferrocyanide initial segmenthode of Ranvier stain of Waxman and Quick (1978) and safranin0 counterstaining. Resulting data clearly indicate that meganeurites form proximal to axonal initial segments and contain safranin-positive membranous cytosomes. Most likely these are equivalent to what have often been referred to as axonal torpedoes in earlier studies (e.g., see de Baecque et al., 1975). Other enlargements form distal to the initial segment, either within 100 Fm of the soma or deeper in white matter, and contain safraninnegative materials. Consequently, they do not resemble meganeurites. Ultrastructural studies have revealed that these axonal swellings contain large numbers of mitochondria, dense bodies, and tubulovesicular profiles (Fig. 8). Microtubules and neurofdaments are also found but generally are not in excess amounts. Typical lysosomal storage vacuoles (e.g., mcb’s) are limited to the soma, meganeurites, and proximal dendrites and are not seen in axonal spheroids. A summary diagram illustrating a cortical pyramidal neuron in ganglioside storage disease possessing both a meganeurite and an axonal spheroid and showing their relationship to one another is given in Fig. 9. h o n a l spheroids are commonly found in a wide variety of storage disorders and always appear similar at the ultrastructural level regardless of the primary lysosomal hydrolase defect (Fig. 8). They occur in both myelinated and unmyelinated portions of axons and are often large enough that interference with normal neuron function (i.e., action potential propagation) could be anticipated (see Section VI,C for discussion). The ultrastructurd appearance of these spheroids is remarkably similar to that described for certain types of neuroaxonal dystrophy (e.g., see Jellinger, 1973). The reason for their appearance in storage diseases is unknown but presumably they are secondary to a defect in the lysosomal system in cell somata. They may represent abnormalities in anterograde or retrograde transport in axons, but clear documentation of this is still lacking. Understanding the reasons for their formation in the neuronal storage disorders may shed light on pathogenetic mechanisms in the neuroaxonal dystrophies, where primarily metabolic abnormalities remain completely unknown.

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FIG.8. Electron micrographs of axonal spheroids as seen in cerebral cortex from a variety of feline models of neuronal storage disease. (a-c) Swainsonine-induceda-mannosidosis, (d) GMI gangliosidosis, (e) sphingomyelin lipidosis, (f)GMI gangliosidosis. Note that spheroids are both myelinated (a,c,e) and unmyelinated (b,d,f). Calibration bar in each panel equals 1.0 pm.

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I FIG. 9. Schematic illustration of a typical cortical pyramidal neuron in ganglioside storage disease showing location of axonal initial segment (is) relative to a meganeurite (mn), secondary neurites (sn), and axond spheroid (as). Other abbreviations: ah, axon hillock; ax, axon; and ac, axon collateral. (From Walkley and Pierok, 1986, with permission.)

F. CHANGES IN THE DENDRITIC DOMAIN The apical and basilar dendritic domains of neurons in ganglioside storage disease appear normal until late in the disease process. In cats with GMI

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gangliosidosis degenerative dendritic changes are not obvious in Golgi-stained neurons until 9 months of age, fully 5 months after onset of clinical symptoms. As indicated earlier, enlargements within apical or basilar dendritic domains are not representative of the cellular pathology of these diseases. The growth of meganeurites occurs at the base of the pyramidal cell in the axon hillock region and is proximal to the axonal initial segment. The formation of secondary neuritic processes appears to occur in the same area. Both are initiated uj?m the full maturation of the normal dendritic domain and appear separate from it (S. U. Walkley, unpublished). The appearance of meganeurites and secondary neurites does not coincide with generalized degenerative changes within the normal dendritic domain. Given the late appearance of the latter process, it can be suggested that it occurs secondary to the overall degeneration of nerve cells and/or with compression of the neuropil late in the disease. With minor exceptions, these events seem consistent across a variety of storage diseases which have been studied with the Golgi staining technique. In contrast to these findings in cortical pyramidal neurons and other cell types, changes in dendrites of Purkinje cells appear unique. These cells form conspicuous focal enlargements within their dendritic trees which may equal the volume of adjacent somata. Sometimes referred to as “megadendrites” (Goidman et al., 1981), these enlargements appear to represent an expansion of a normal dendritic compartment in the cell. They do not possess neurites or additional spine growth and therefore are not similar to meganeurites. Ultrastructurally some of these enlargements have been shown to contain storage vacuoles equivalent to those in cell somata. They have been reported to occur in a variety of neuronal storage disorders, and presumably their presence reflects characteristics of the distribution of the lysosomal system within this unusual type of neuron. The only neuronal storage disease where conspicuous dendritic changes have been reported in cortical neurons early in the disease course appears to be in inherited and swainsonine-induced a-mannosidosis (Walkley et al., 1981a, 1987). These changes appear as small, focal swellings within the apical or basilar dendrites (Fig. 10). They are often associated with dendritic spines and do not appear to be related to dendritic degeneration, at least not early in

FIG. 10. Ultrastructural and Golgi impregnation illustrations of dendritic spheroids as seen in inherited and swainsonine-induced a-mannosidosis. (a) Electron micrograph which demonstrates typical ultrastructural features of dentritic spheroids.Note that these swellings contain enlarged tubulovesicular profiles but not the typical storage vacuoles as are present in neuranal somata. (b) Golgi-stained dendrite illustrating typical appearance of dendritic spheroids (arrows), many of which bear normal-looking spines. (c) Higher magnification of normalappearing dendritic spine as seen in (a) (see arrows). Calibration bars in (a) and (c) equal 1.O pm; in (b), 5.0 prn.

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the disease. Ultrastructurally, these focal enlargements are found to be associated with tubulovesicular enlargements of unknown origin and not with accumulations of storage vacuoles. They may reflect abnormalities in dendritic transport secondary to a defect in a-mannosidase activity, but this possibility remains untested.

IV. Disordered Function of Neurons

A.

CLINICAL

MANIFESTATIONS OF DISEASE

Clinical symptoms in neuronal storage disorders vary widely depending on the nature of the primary metabolic defect. However, most of these diseases do have a number of characteristics in common. Affected individuals are most often normal appearing at birth with clinical onset occurring in infantile or juvenile stages of development, or least commonly during adulthood. Cessation of psychomotor development is followed by a gradual, but generally unrelenting, deterioration of CNS function. This progression of clinical symptoms varies in different disorders, but generally proceeds more rapidly in those with earlier onset. Deterioration of CNS function is most pronounced in ganglioside storage disorders, while clinical symptoms in mucopolysaccharidosis or a-mannosidosis often progress more slowly. Striking differences in clinical manifestations can be seen in storage diseases even when the same primary lysosomal hydrolase is involved. Although explanations for this remain incomplete, residual activity of the pivotal enzyme can be predictive in most cases. The less this activity, the more rapid the disease course. Critical to this evaluation, however, is the measurement of specific activity of the mutant enzyme against natural substrates and without the use of detergents in the assay procedure (Sandhoff and Christomanou, 1979). The involvement of nonneuronal tissues (visceral and skeletal) may also exert considerable influence over the overall course of the disease. Hexosaminidase deficiencies (the GM2 gangliosidoses) perhaps best illustrate the wide range of clinical symptoms and variation in disease onset and duration which can occur in storage diseases. Clinical conditions here include not only classical Tay-Sachs disease (hexosaminidase A deficiency), Sandhoff s disease (hexosaminidase A and B deficiency), and the so-called AB variant form (an activator protein deficiency), but also a growing number of intermediate forms. The latter have been seen as motor system disorders such as dystonia (Meek et al., 1984), spinocerebellar degeneration (Oonk et al., 1979), or adult-onset encephalopathies (Navon

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et al., 1981). These different disease forms appear to result from the variety of hexosaminidase isozymes with their separate gene loci and with the variety of natural substrates which are involved Uohnson, 1981; O’Brien, 1978). Multiple mutant alleles can give rise not only to a variety of homozygotes, but compound heterozygotes with defective hexosaminidase activity may also occur. The total number of mutations potentially affecting hexosaminidase enzyme activity has been suggested to be astronomical (Johnson, 1981). Animal models of neuronal storage disease often display strikingly similar clinical signs in comparison with their human disease counterparts. Mental retardation predominates in children with neuronal storage disease and is seen in conjunction with major motor system abnormalities. Since behavioral testing as a means to evaluate learning abilities in animals with storage diseases has not been exploited, these diseases are most often described in animal models by their impact on motor system function. The striking similarity between motor system dysfunction in children and animals with, for example, ganglioside storage disease, suggests that abnormalities in cerebral cortical functioning are likely to be similar. One interesting contrast between the clinical states seen in human and feline ganglioside storage disease involves the so-called “startle response.’’ This exaggerated response to loud sounds is characteristic of most cases of human GM1 gangliosidosis, particularly TaySachs disease. In feline models of ganglioside storage, however, it is characteristic of GMI storage and is generally not observed in the GM2 cats (H. J. Baker, M. C. Rattazzi, and S. U. Walkley, unpublished observations). It is also not present in other types of feline models of neuronal storage disease. The feline model of mucopolysaccharidosis (type 1) is also similar clinically to its human counterpart (Hurler’s disease). CNS changes secondaryto skeletal (vertebral) abnormalities are most common in the feline model, although some degree of intraneuronal storage and other changes is occurring within the CNS (Haskins et al., 1983). In contrast, feline a-mannosidosisdemonstrates prominent motor system abnormalities mostly associated with cerebellar dysfunction, including titubation and ataxia. In the latter stages of disease, opisthotonus is characteristic. Although cerebellardeficitsoccur in human a-mannosidosis,it is again the cerebralcorticaldysfunction(mentalretardation) which predominates (Beaudet, 1983). Swainsonine-induceda-mannosidosis in cats has been found to closely replicate the clinical syndrome observed in inherited feline a-mannosidosis (Walkley and Siegel, 1985).

B. ELECTROPHYSIOLOCICAL STUDIES Electroencephalographic(EEG) studies have been performed on children with a variety of neuronal storage disorders. In the gangliosidoses, EEGs were

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reported to be near normal in the early stages and as such to contrast sharply with other CNS disorders such as phenylketonuria and maple syrup urine disease (Pampiglione and Harden, 1984). Subsequent deterioration of EEG patterns varied according to the particular type of ganglioside storage disease. Rhythmic EEG activity was characteristically abnormal, and paroxysomal (spike) activity was uncommon even in those patients with a history of seizure activity. EEG changes also were not observed during prolonged startle responses in children with GM2 gangliosidosis. EEG patterns deteriorated with age and in Tay-Sachs patients often did so abruptly. This general deterioration of the EEG in ganglioside storage disease patients contrasted with those in mucopolysaccharidosis cases, in which only mild EEG alterations were the rule throughout the disease course (Hanefeld and Pampiglione, 1972). Similar studies in patients diagnosed as having neuronal ceroid lipofuscinosis have been performed and distinct differences were noted between different clinical forms of the disease (Harden and Pampiglione, 1982). Phasic EEG patterns were characteristically lost early in the infantile forms. In late infantile cases high-amplitude irregular slow-wave activity with multifocal spikes was seen, while in juvenile cases runs of slow spike wave discharges were more common. EEG patterns were suficiently distinct among the three groups so as to suggest major differences in pathological mechanisms underlying these types of neuronal ceroid lipofuscinosis. Electrophysiological studies in animal models of neuronal storage disease (feline GMI and GM2 gangliosidoses) have consisted of direct cortical surface recording and of single-unit intracellular recording with subsequent cell labeling using horseradish peroxidase or lucifer yellow (Purpura et al., 1980; Karabelas and Walkley, 1985). The latter technique has not only supplied information on the electrical activities of cortical neurons but also has given simultaneous data on cell morphology. Given the known changes in pyramidal neuron geometry and connectivity as revealed by Golgi staining and EM, this approach has been essential for a full assessment of functional changes in neurons. These studies were carried out in motor cortex of anesthetized cats with moderate to advanced GMI or GM2 gangliosidosis. Electrical stimulation was accomplished by placement of a stimulating electrode in the nucleus ventralis lateralis of thalamus. Input volleys to the ipsilaterd motor cortex were initially recorded with a ball electrode on the cortical surface. This evoked surface recording, as with spontaneous surface recordings, generally did not appear abnormal (Fig. 11). Intracellular recordings were performed using electrolyte-filled glass microelectrodes lowered into the motor cortex by a micrometer drive device. Synaptic activity was evoked in a variety of pyramidal and nonpyramidal cells and many of these were subsequently labeled with horseradish peroxidase or lucifer yellow. These studies clearly demonstrated that the presence even of large

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250msec' 12-

A

B2 I

I

-1

FIG.1 1. Electrophysiological recordings from 8- to 9-month-old cats with GM, gangliosidosis. (A) Recruiting response recorded over surface of motor cortex following repetitive thalamic (nucleus ventralis lateralis) stimulation (at arrows). (B1 -B3) Examples of intracellularly recorded spontaneous spike discharges in one pyramidal neuron. (B4) Spontaneous spike discharge from another cell [note time-base change for (B4)].

quantities of gangliosides within neurons in the form of membranous cytosomes did not lead to immediate signs of cell dysfunction. Such neurons most commonly displayed resting electrical potentials and spontaneous spike discharges similar to normal cells (Fig. 11). These studies also demonstrated a predominance of inhibitory postsynaptic potentials (ipsp's) in cortical neurons following thalamic stimulation, with excitatory postsynapic potentials (epsp's) being only rarely encountered (Fig. 12). Normally, neurons of motor cortex in cats display epsp's or epsp-ipsp sequences following thalamic activation (Noda and Yamamoto, 1984). Subsequent intracellular labeling of neurons in diseased cats revealed that the ipsp activity was occurring in a variety of cells, both pyramidal and nonpyramidal, and was not limited to pyramidal neurons with meganeurites or secondary neurites. Subsequent immunocytochemical studies, as discussed earlier (Section III,C), indicated that the GABAergic system is fully intact in the cerebral cortex of GMI cats. Indeed, the number of GAD-positive terminals per neuron appeared increased (Walkley and Wurzelmann, 1986).

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A3

A2

12. Intracellular electrophysiological recordings from neurons of motor cortex in 8- to 9-month-old cats with GM, gangliosidosis. (Al-A4) Recordings of ipsp activity elicited by electrical stimulation of thalamus (nucleus ventralis lateralis). (B) Thalamic stimulation evoked epsp activity in a cortical neuron. (C) Interneuron burst discharge.

Intracellular electrophysiological studies also have been reported for Neuro-2a cells and primary cultures of rat brain following chronic administration of gangliosides (Dimfel et al., 1981). Acute (24-hr) changes were not seen in membrane potential, but whole cell input resistance did increase slightly. Similarly, in chronic studies (5 weeks) using primary cell cultures, membrane resistance was not significantly altered, and the only remarkable finding was a higher incidence of inhibitory potentials. This was considered unusual for these culture conditions, as ipsp's were only rarely encountered in control cultures (Dimfel et al., 1981).

V. The Role of Gangliosides

As discussed earlier, many neuronal storage disorders are known to be characterized by the abnormal accumulation of gangliosides andfor by alterations in patterns of specific types of gangliosides. In the gangliosidoses these changes are of major proportion and logically follow from specific defects in ganglioside degradative pathways. Although massive accumulation of a single ganglioside (e.g., GMI or GM2) will occur depending on the enzyme defect,

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minor alterations in other ganglioside species are also routinely encountered. As described earlier (Section III,B), the mucopolysaccharidoses and sphingomyelin lipidosis, although not ganglioside storage disorders per se also are well documented as having significant increases in certain gangliosides (particularly GM2 and GM3). Even a-mannosidosis, previously reported to be free of ganglioside alterations (Ockerman, 1973), has recently been implicated as well. Ultrastructural studies have revealed membranous inclusions in a select population of cortical neurons in the feline model of this disease, and this may suggest storage of gangliosides or other glycolipids is occurring in at least this one small population of cells (Walkley et al., 1987). Although the function of gangliosides in the mammalian CNS is not understood, a large body of data has developed over the years suggesting numerous roles in a variety of cell processes. These include an association with dendritogenesis and myelination during early brain development, as receptors for growth factors, neurotransmitters, and hormones, as regulators of synaptic events or in cell-cell contacts, and so forth (e.g., see Fishman and Brady, 1976). Being integral membrane molecules, gangliosides are well suited to play some of the above roles. They are found with their hydrophobic, ceramide portion embedded in the neuronal membrane and with their carbohydrate portion extending over the external surface of the cell. This carbohydrate moeity consists of sialic acid, hexoses, and N-acetylated hexosamines, and as such represents that portion of the molecule which provides structural diversity. Gangliosides are negatively charged and are known to bind calcium ions avidly (Ledeen and Yu, 1973). Given the discovery that significant alterations occur in ganglioside patterns and concentrations in a variety of storage diseases, it is possible that these changes represent a common denominator for initiation of some of the abnormal structural and functional events which occur in these diseases. Such a linkage, if firmly established, could reveal important insights into the role of gangliosides in normal nervous systems. Two proposed actions of gangliosides are appropriately reviewed here, first, that they may play a pivotal role in the induction of neurite growth (i.e., dendritogenesis), and second, that they are critically involved in functional events at chemical synapses.

A. GANGLIOSIDES AND NEURITE GROWTH The demonstration by Purpura and Suzuki (1 976) that ganglioside storage diseases were characterized by ectopic neurite growth led to the hypothesis that the presence of an altered pattern of gangliosides, or accumulation of a specific ganglioside or closely related glycolipid, was responsible for this

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recapitulation of embryonic growth features of neurons (see Section 111,B). The greater abundance of neurite growth in ganglioside storage disease relative to other human storage disorders was a critical observation in the development of this hypothesis, and this view has been fully confirmed by more detailed studies on a wide variety of storage diseases in animals (Walkley, 1987; Walkley and Baker, 1984; Walkley et al., 1981a; Walkley and Haskins, 1982). In the years immediately following Purpura’s observation a large number of studies appeared which demonstrated that chronic ganglioside administration to neurons in culture would cause neuritic sprouting and elongation of neurites (for a recent review, see Ledeen, 1984). In one of the first such reports (Roisen at al., 1981a), a mixture of bovine brain gangliosides was applied to primary neurons in culture (chick embryo dorsal root ganglia) and to an established neuronal culture line (Neuro-2a cells). Results showed that the degree of axonal elongation exhibited by sensory ganglia cells was increased by the addition of gangliosides, as were the length and number of neuroblastoma cell processes. The suggestion was made that gangliosides were incorporated into the cell membrane and acted as acceptor molecules for growth-promoting substances present in the media. Later studies indicated that this induced neuritogenesis was not due to protein contaminants in the mixed bovine brain gangliosides and also that many types of individually purified gangliosides could induce the neuritogenic response (Byrne et al., 1983). This latter study also showed that neural glycosphingolipids had littie or no capacity to induce neurite growth, thus suggesting the importance of sialic acid in this process. Sialic acid alone, however, also was not neuritogenic. Ultrastructural studies of neuroblastoma cells induced to sprout neurites by ganglioside administration appeared to indicate the importance of microfilaments in this process (Spero and Roisen, 1984). In contrast, microtubules and neurofilaments appeared unchanged in ganglioside-treated cultures. However, in cultures with colcemid-disrupted microtubules, gangliosides failed to induce the usual level of neuritic sprouting. The mechanisms by which gangliosides act to induce neurite growth in neuronal cultures are not understood. The influence of gangliosides on membrane fluidity has already been described (Section II1,D). Gangliosides are also known to be potent stimulators of adenylate cyclase (Partington and Daly, 1979) and phosphodiesterase activity (Davis and Daly, 1981) and may therefore critically influenceCAMPlevels. Indeed, neuroblastoma cultures treated with gangliosidesare known to have elevated intracellular CAMP(Dimpfelet al. , 1981). Gangliosides have also been shown to be involved in the regulation of mRNA for tubulin sequences (Rybak d al., 1983). A 9-fold increase in the latter occurred following 2 weeks of chronic ganglioside treatment in a somatic neurohybrid clonal cell line (SBPlBl), whereas action mRNA remained unchanged.

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In addition to studies showing ganglioside-stimulated neuritogenesis in cultured neurons, a variety of reports also have appeared which seem to indicate that a similar process may occur in the intact nervous system following ganglioside administration (also reviewed by Ledeen, 1984). These reports include both the peripheral nervous system, where nerve-muscle synapses have been shown to be responsive to ganglioside treatment in a number of experimental conditions (Gorio et al., 1980), and in the CNS as well. For the latter, parented injections of GMi ganglioside appear to result in reduced dopaminergic cell loss and more rapid recovery following unilateral hemitransection of the nigrostriatal pathway (Agnati et al., 1983; Toffano et al., 1983). Following septal lesions in the hippocampus, postlesion recovery of cholinergic function also was shorter in ganglioside-treated animals (Wojcik et al., 1982). These studies suggest a role for gangliosides not only in neuritepromoting actions but also as neurotrophic factors (Ledeen, 1984). Mechanisms of action of gangliosides in these in vivo models are poorly understood, and their relationship to the tissue culture data and to events documented in ganglioside storage diseases is unclear.

B . GANGLIOSIDES AND SYNAPTIC TRANSMISSION Although controversy has surrounded the issue of localization of gangliosides in plasmalemmal domains (e. g., see Ledeen, 1978),'considerable evidence exists which suggests that they are located primarily at synaptic regions of neurons (De Robertis et al., 1976; Hansson et al., 1977). Consequently, speculation on the possible role(s) that gangliosides might play in synaptic transmission has been widespread. For example, Morgan and colleagues (1976) have suggested that gangliosides may regulate the synaptic events by binding cations or, alternatively, by acting as receptors for neurotransmitters. It is well known that gangliosides can act as receptors for bioactive factors, e.g., cholera toxin, but there is little evidence that they act as receptors in general (Hakomori, 1984). An alternative view is that gangliosides act as secondary, auxiliary receptors, cofactors, or modulators of protein receptors (Bremer and Hakomori, 1984; Dawson and Berry-Kravis, 1984; Lancetti et al., 1984). In relation to neurotransmitters and their receptors, a variety of indirect evidence also suggests a link between GMI ganglioside and the GABAergic system. For example, early studies indicated a correlation between tissue concentrations of gangliosides and of GABA (Lowden and Wolfe, 1964), and antibodies to GMI ganglioside have been reported to enhance GABA release in rat brain slices (Frieder and Rapport, 1981). Anti-GM1 antibody injections into rat cerebral cortex also have been shown to induce EEG spiking and seizure activity (Karpiak et al., 1976, 1981). Cholera toxin binding to GMI ganglioside results in similar epileptiform

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activity (Karpiak et al., 1981), and numerous studies exist which indicate a linkage between perturbation in GABAergic function and seizure activity (e.g., see Roberts, 1986). Finally, in relation to the neurotransmitter, acetylcholine, synaptosomal preparations high in choline acetyltransferase activity have been reported to be richer in gangliosides than noncholinergic synaptosomal preparations (De Robertis et al., 1976). The feline model of GM1 gangliosidosis has been used in a number of studies directed at assessing alterations in events at the synapse secondary to this defect in ganglioside metabolism. As described earlier, synaptosomes prepared from cerebral cortex of these animals contained increased amounts of GM1 ganglioside, as well as phospholipid and cholesterol, and showed reduced membrane fluidity (Wood et al., 1985). In earlier studies, Singer and colleagues (1981) reported a reduction in reuptake of a number of neurotransmitters (GABA, glutamate, norepinephrine) in a variety of brain regions in GMI cats. This reduction appeared unassociated with comparable alterations in neurotransmitter levels or neurotransmitter-synthesizing enzyme activity. More recently the metabolism of another cortical neurotransmitter, acetylcholine, has been closely examined in this same model and marked increases in acetylcholine synthesis and K+-stimulated release were detected (Baker and Jope, 1985; Jope et al., 1986). Additionally, high-affinity choline transport was increased in cerebral cortex in G M I mutant cats. As discussed by Baker and Jope, these data suggest two possibilities. First, they may indicate that cholinergic nerve terminals have proliferated within cerebral cortex of GM1 cats. These conceivably would be associated with the new synapses forming on ectopic neurites and meganeurites of pyramidal neurons. However, an argument against this correlation is that ultrastructural characteristics of most neuritic synapses do not resemble cortical cholinergic synapses (e.g., see Wainer et al., 1984). Immunocytochemical studies involving choline acetyltransferase staining in GM1 cats have revealed an abundant distribution of cholinergic fibers within the cerebral cortex of these animals, and comparative studies with agematched normal cats are in progress (S. U. Walkley, unpublished). A second way to explain these data is that there has been an upward regulation of cholinergic metabolism within existing cholinergic fibers in G M I cat cortex. This latter process could occur as a result of changes in synaptic membrane composition which interfere with the regulation of synthesis and release of acetylcholine (Jope et al., 1986). Possible interactions between GMI ganglioside and cholinergic systems also have been suggested by other recent studies. Hefti and colleagues (1985) have shown that ganglioside administration to septal neurons in dissociated cell cultures stimulates the activity of choline acetyltransferase. Intramuscular injections of GM1 ganglioside following deafferentation of rat hippocampus

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have been reported to enhance postlesion responses of ChAT and acetylcholinesterase activity, suggesting that GMI ganglioside facilitates regrowth of new cholinergic nerve terminals (Oderfeld-Nowak et al., 1984). Similarly, animals receiving unilateral lesions of the nucleus basalis magnocellularis and repeated administration of GM1 ganglioside revealed marked attenuation of the loss of high-affinity choline uptake observed in control animals. However, the short time duration between lesioning and the effect on choline uptake observed in controls was suggested to argue against a role for neuritic sprouting. Rather a direct action on choline uptake or increased activity of residual cholinergic cells was considered responsible (Pedata et al., 1984). Considerable speculation also surrounds the cation-binding capacity of gangliosides relative to synaptic transmission. The importance of calcium in synaptic events is well established (Katz and Miledi, 1967). Calcium ions enter the presynaptic terminal as a result of invasion of the action potential and local membrane depolarization. Once internalized, Ca2 is believed responsible for fusion of synaptic vesicles and subsequent neurotransmitter release. Since gangliosides are known to bind calcium avidly (Quarles and Folch-Pi, 1965), their role in this aspect of synaptic transmission could be considerable. Rahmann et al. (1982), like Morgan and colleagues (1976), has stressed the importance of such a relationship by proposing that formation of ganglioside-Ca2 complexes is associated with stability (closure) of the presynaptic membrane, whereas their dissociation may be linked with opening of the membrane. Svennerholm (1980) has proposed a similar hypothesis. He suggests that calcium is originally bound to the negatively charged hydrophilic portion of the ganglioside in the presynaptic membrane. Its removal by local impulse invasion and depolarization leads to instability of this membrane and to fusion of presynaptic vesicles. He also suggests that gangliosides may play a role in reuptake of positively charged neurotransmitter molecules, thus implying importance of gangliosides in the full cycle of local events in the synaptic junction. Recent experimental studies using brain slices with ganglioside patterns altered by neuraminidase treatment support such a role for gangliosides in neurotransmission (Wieraszko and Seifert, 1984). In a related study Jope and colleagues (Koenig et al., 1987) examined calcium fluxes within the synaptosomes of cats with GMI gangliosidosis and calcium influx was found to be significantly reduced in the diseased animals. Since neurotransmitter release is a calcium-dependent process, and as gangliosides have a pronounced ability to complex with calcium, disruption of intranerve terminal calcium homeostasis may be a critical factor underlying neuronal dysfunction. Although these data may offer an explanation for the decreased epsp activity recordable in GM1 cat cortex following thalamic stimulation (Karabelas and Walkley, 1985) (Section IV,B), the persistent and +

+

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possibly increased amount of ipsp activity is not readily explained. The increased acetylcholine release data described above is also not consistent with these findings on calcium flux, although Jope and colleagues suggest that increased calcium within presynaptic terminals may be responsible for this. Recent studies in which normal rat synaptosomes were treated with GM1 ganglioside offer additional support for these studies of feline GMI gangliosidosis. Such exogenous ganglioside application was found to significantly influence the time course of Ca2+ channel activity during the depolarization-dependent Ca2 uptake process and suggested that gangliosides may act as potent modulators of synaptosomal calcium fluxes (Domanska-Janik et al., 1986). +

VI. Explaining Neuronal Dysfunction

A. THECYTOTOXICITY HYPOTHESIS As described earlier (Section I,A), this hypothesis is a logical outgrowth from the initial studies on storage diseases. In its simplest form it suggests that at some point during the storage disease process a threshold is reached after which further lysosomal accumulation becomes toxic, and the cell is no longer able to perform its normal physiological operations (Desnick et a l . , 1976). Inherent in this view is a mechanical disruption of the cell cytoplasm with subsequent defects in organellar and molecular trafficking and recycling and, ultimately, cell death secondary to these events. Ultrastructural examination of a storage disease like GMl gangliosidosis at end stage (1 1- 12 months in the GMI cats) reveals evidence in support of this hypothesis. Neurons are found to be severely impacted with mcb's with regions of normal cytoplasm and normal organelles being difficult to locate. Meganeurites of massive size are interposed between neuronal somata and axons and, although microtubules and neurofilaments may be seen passing through, they are tightly surrounded by mcb's which fill this cell compartment. These ultrastructural studies also reveal occasional neurons with dark cytoplasm and other features suggestive of dying neurons, whereas the neuropil contains profiles of degenerating processes, many of which appear to be small axons. Golgi studies carried out at end stage GMI gangliosidosis demonstrate significant degeneration of normal dendrites in addition to the presence of very large meganeurites and of secondary neurites. Many dendrites appear severely compromised by storage-induced compression of the neuropil, and loss of dendritic spines is obvious in many areas. Routine light microscopy reveals variable amounts of gliosis as well as some loss of myelin at this late

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stage in the storage process. Some overall loss of neurons also can be detected. Although these findings give support to the idea of a storage-induced cytotoxic effect, examination of the CNS early in the disease process gives a very different view. For example, again drawing on the detailed studies of GM, cats, morphological evaluation of the cortex in 6-month-old animals reveals widespread but only moderate intraneuronal storage. Furthermore, neither Golgi staining nor routine light and electron microscopic studies reveals signs of neuronal degeneration. Indeed, apart from the presence of mcb’s and occasional axonal spheroids, the ultrastructural features of cerebral cortex, including synapses, appears quite unremarkable. Golgi studies reveal normal-appearing apical and basilar dendrites on pyramidal cells, and dendritic spines are abundant and appear normal. Meganeurites are present and are of moderate size and many axon hillocks display prolific growth of neurites. Thus many features of neuronal storage are present in GMI cats at 6 months of age, but degenerative changes and cell loss are not apparent. Zmportantb, however, the clinical manifestations of GM1 storage are conspicuous at this stage of disease and have been since 3 months of age. The studies summarized above appear to indicate that the cytotoxic effects of storage in most of the neuronal storage disorders discussed here are limited to the end stage of the disease process and do not contribute in significant fashion to the onset and progression of clinical symptoms. Exceptions would necessarily include those disorders in which known cytotoxic agents accumulate, e.g., Krabbe’s disease (Igisu and Suzuki, 1984), but these conditions most often fall into the class of diseases known as leukodystrophies rather than neuronal storage disorders per se. A second exception may be the very selective cell loss reported to occur in some types of neuronal storage disease (e.g., sphingomyelin lipidosis) (Braak et al., 1983), but even here developmental studies linking cell loss with early manifestations of disease are lacking. Overall, presently available studies of the cellular pathology of neuronal storage disease suggest that cytotoxic events associated with cell death predominate only in the latter stages of the disease process. Thus other events at the cellular and molecular levels must be intimately involved in this disease process during the earlier stages, and some of these are explored below.

B . DISORDERED NEURONAL GEOMETRY AND CONNECTIVITY The Golgi studies carried out by Purpura and colleagues a decade ago clearly demonstrated the striking morphological changes induced in neurons as a result of the lysosomal hydrolase activity defect and subsequent storage process. Since meganeurites appeared to occur between cell soma and axonal

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initial segment, it was hypothesized that postsynaptic potentials would be attenuated before reaching this sodium channel-rich region and neuronal output would be altered as a result. Additionally, as meganeurites and associated secondary neurites were found to be contacted by new synapses, and since these connections were located just proximal to the axonal initial segment, their presence was believed to offer another factor which could lead to altered neuronal output. In spite of the compelling nature of this hypothesis, data to fully support or refute it are still not available. Electrophysiological studies did not reveal significant differences in evoked synaptic activity in meganeurite or neuritebearing neurons versus other nearby cells displaying storage but no incorporation of new dendritic-like membrane (Karabelas and Walkley, 1985). But combined Golgi-EM studies have clearly documented the significant amount of new synapse formation of these axon hillock neurites and meganeurites, and some influence of this new synaptic input over neuronal output is highly probable. One likely view is that this ectopic dendrite growth has been induced to form by the intraneuronal accumulation of a specific neurite growth-promoting factor (possibly a ganglioside) as a direct result of the primary metabolic defect. If so, this resulting axon hillockassociated synaptic input presumably would be aberrant and would cause neuronal output to be abnormal. However, if ectopic neurite growth occurs later in a chain of events initiated by the storage of abnormal amounts or patterns of gangliosides, it does not necessarily follow that its presence, along with new synaptic input, will cause neurons to malfunction. Indeed, under these conditions it is conceivable that this new synaptic input might play a compensatory role. Whether the synaptic input to axon hillock neurites and meganeurites is contributory to neuronal dysfunction, or to normal function under other adverse disease conditions, remains one of the most important unanswered questions on the pathogenesis of neuronal storage disease. Not only is the effect of synaptic input at axon hillock neurites unknown, but also the source of this input remains to be established. As illustrated in Fig. 13 and as discussed earlier (Section III,C), a variety of possibilities exist. GABAergic connections may be enhanced over neuronal somata in cortex of GM1 cats, but the ultrastructural features of neuritic synapses (being primarily asymmetrical, see Fig. 6) make it unlikely that they form the input to these process. From this ultrastructural view, a most likely explanation would be that they are derived from thalamocortical afferents, since these form asymmetrical contacts (White, 1981). As described earlier (Section V,B), some features of cholinergic metabolism are enhanced in the cerebral cortex of GMI cats and this may reflect the presence of increased numbers of cholinergic afferents in the cortex

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I

IV

TC

ACH

?

FIG. 13. Schematic illustration depicting some of the many possible (but unproven) synaptic relationships of extrinsic and intrinsic connections relative to meganeurite and secondary neuritebearing pyramidal neurons in laminae 11-111 of neocortex in neuronal storage disease. GABAergic neurons may synapse on neuronal somata in excess (1) and also could form meganeurite (2) and secondary neurite (3) synapses. Cholinergic afferents (ACH) may proliferate and form connections on meganeurites (4) and on secondary neurites (5). Thalamocortical (TC) afferents may synapse with meganeurites (6) and with secondary neurites (7) in addition to normal placement adjacent basilar dendrites (8). Other afferents (?)or intrinsic neurons (?)may also supply synaptic input to secondary neurites (9) or to meganeurites (10). See text for details.

of these animals. Again, existing ultrastructural data on the cholinergic synapses in normal cortex (Wainer et al., 1984) suggest that neuritic synapses may be unrelated to this input. However, as with the GABAergic system, altered patterns of cholinergic connectivity may still be occurring as a result of the disease process. Finally, ectopic synapses on axon hillock neurites and meganeurites may be derived from other intrinsic or extrinsic sources or, indeed, from a variety of sources. Understanding the origin and functional consequences of these connections is central to a full description of the

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pathogenesis of neuronal storage disease and may have relevance to modifiability of connections in normal nervous systems.

C. A CASCADE OF EVENTS As reviewed previously (Sections 111-V), there is now a large body of evidence indicating that a variety of changes occur in neurons coincident with the primary metabolic defect in storage diseases, and clearly any number of these could have profound influence over the normal operations of neurons. These data are persuasive of another approach to understanding the pathogenesis of neuronal storage diseases. The suggestion here is that following the defect in activity of one or more lysosomal hydrolases (due to any one of an array of potential molecular faults), an expanding cuscude of events is set in motion which ultimately culminates in neuronal dysfunction and subsequent clinical manifestations (Fig. 14). Conceivably, certain of these events, e.g., massive synaptic connectivity changes in select brain regions (Sections II1,B and C), abnormal Ca2 homeostasis in presynaptic terminals (Section V,B), or inhibition of protein kinase C (Section III,A) may emerge as a principal investigator of cell dysfunction in certain storage diseases. But for the +

Led 1

PRIMARY GENE DEFECT

1

Level 2

LYSOSOMAL HYDROLASE DEFECT

/

LBYd 3

I

\

PRIMARY METABOLIC ABNORMALITIES

SECONDARY

I

\

Level 4

-

I

I

NEURON TYPE-SPECIFIC CHANGES IN CELL STRUCTURE,

-

FUNCTION. CONNECTIVITY. AND VIABILITY

i i 1 i i i i i I 1 1 i 1 i 1 1 FLINICAL MAN~FESTAT~ONS OF NEUR~NALSTORAGE

1 I

DISEASE^

FIG 14. The pathogenetic mechanisms involved in neurond storage disorders are suggested to be most appropriately viewed as an expanding cascade of events. See text for details.

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sake of hypothesis development and testing, and particularly in order to sort out these multiple events relative to one another, the concept of an expanding cascade to explain neuronal dysfunction would seem to be the most productive approach. This cascade is viewed as expanding rather than as a simple series of events because available data clearly indicate that many complex phenomena are involved. However, the relationship of these events to one another is largely unknown. For example, two areas of investigation reviewed here in some detail, (1) the role of abnormal Ca2 homeostasis in synaptic terminals, and (2) the presence of ectopic neuritogenesis and new synapse formation in select neuronal populations, both may be related to the presence of abnormal quantities of gangliosides. Each possibility has considerable experimental support from in vitro and in uiuo studies, yet the explanation for how such diverse actions result from a single event, i.e., abnormal ganglioside metabolism, remains unknown. However, a common feature of each event is the synapse, at least if ectopic neuritogenesis is considered to be simply added surface area for new synapse formation. Although changes such as those described above may emerge as being chiefly involved in the onset and progression of clinical symptoms in neuronal storage diseases, numerous other phenomena should not be overlooked. Axonal spheroid formation, for example, is found in abundance in many areas of the CNS in most types of neuronal storage diseases. Based on theoretical assumptions regarding action potential propagation relative to step increases in axonal diameter (as would occur at spheroids), it is conceivable that impulse blockade could occur (Goldstein and Rall, 1974). Clearly this deserves closer study, as does the role of intrasomatic lysosomal pathology in the generation of these focal abnormalities in distinct regions of the axon. Two other events reviewed here which could have important influence over normal neuronal operations include (1) displacement of the axonal initial segment by the meganeurite and/or the abnormal trafficking to, or recycling of, normal glycoprotein components of this sodium channel-rich region of the neuron, and (2) abnormal membrane fluidity which conceivably could alter a variety of events occurring at the plasmalemma. +

VII. Concluding Comments

A. THEPROMISE OF THERAPY Speculation as to not only the cause, but also the potential cure for the neuronal storage disorders far predated the actual elucidation of the lysosomal enzyme abnormalities underlying these diseases. It was Bielschowsky, for

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example, who suggested in 1921 not only that diseases like Tay-Sachs could be due to a missing “ferment,” but also that treatment for such conditions might be possible through the injection of extracts taken from normal brains. This remarkable suggestion, though too far ahead of its time to be taken seriously, has been echoed anew in more recent times. For the discovery that specific lysosomal hydrolase deficiencies are responsible for these diseases has led inevitably to consideration of the logical cure-enzyme replacement therapy (ERT) . As reviewed elsewhere (Desnick et al., 1976; Hers, 1973; Kolodny, 1976; Lloyd and Griffiths, 1979; Rattazzi et al., 1979; von Specht et al., 1979), the E R T approach has been only partially successful and to date appears limited to amelioration of storage in nonneuronal tissues. But now with the availability of bone marrow transplant techniques (Parkman, 1986), populations of normal lysosomal hydrolase-producing cells can permanently replace defective ones, either through compatible donor grafts or genetic engineering (Anderson, 1984). The hope remains that chronic release of normal enzyme into the circulation will find its way past the blood-brain barrier and reverse neuronal storage. To support this notion there is evidence for protein uptake from blood by at least certain types of CNS neurons, including motoneurons of the brain stem and spinal cord (Broadwell and Brightman, 1976). Whether this would occur for lysosomal hydrolases is unknown, as is the possible transneuronal transport of such proteins. Other means by which circulating enzyme may ameliorate CNS changes would be through normalization of endothelial and perithelial cells and by contact with cells of the choroid plexus. Elevation of normal enzyme levels in cerebrospinal fluid (CSF) may in time lead to reduced storage in some neuronal populations, as there is again evidence for direct uptake of proteins from the CSF by at least certain types of neurons (see Borges et al., 1985). Thus through one or more of these mechanisms, peripheral E R T may reduce intraneuronal storage. The use of animal models of neuron storage diseases to test such procedures offers hope for answers to these questions on therapy, and preliminary findings are encouraging (Shull et al., 1987). But will successful correction of the primary metabolic abnormality in storage disorders lead to normalization of other cascade-related pathogenetic events? In terms of cytoplasmic storage vacuoles and of axonal and dendritic spheroids, the answer is clearly affirmative (Huxtable et al., 1978; Walkley et al., 1987). But recent studies on ectopic dendrite growth and new synapse formation suggest that reversal of these phenomena may not occur (Walkley et al., 1987). Using swainsonine to induce a-mannosidosis in cats, ectopic axon hillock-associated neurite growth was initiated on susceptible cell populations (e.g., pyramidal neurons of cerebral cortex). Withdrawal of the a-mannosidase inhibitor clearly led to the disease reversal, but despite

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normalization of the CNS in most respects, ectopic neurites and their synapses remained intact. Although further studies will be necessary to fully elucidate the significance of this finding (relative to neuron function), the suggestion is that ERT following significant disease development may not lead to the normalization of all aspects of brain morphology. From a neurobiological perspective, these studies also suggest the likelihood that those mechanisms responsible for the initiation of pyramidal neuron dendrites are not the same as those required for their maintenance.

B. STORAGE DISORDERS AND NEUROSCIENCE As indicated by studies reviewed here, the neuronal storage disorders are rich in questions relating to cell biology of the neuron. An understanding of the pathobiology of these disorders will likely carry with it new insights into events at all levels of nervous system operation, from molecular phenomena to interaction of neuronal ensembles in the generation of specific behaviors. The highly discrete character of the primary metabolic lesions, and the now considerable data detailing deficient activity of specific lysosomal hydrolases, recommends these “experiments of Nature” as remarkable tools for further study. Indeed, few experimental probes are presently available which can surpass the exquisite metabolic scalpel inherent in these disorders. Swainsonine, that remarkable indolizadine alkaloid now known to selectively and reversibly inhibit the a-mannosidase enzyme, represents one of the few of these experimental probes active in vivo and capable of crossing the blood-brain barrier. The availability of animal models of neuronal storage diseases thus represents an important resource through which to study these primary metabolic defects and the cascade of events which ensue secondarily. As detailed here, some of the phenomena which characterize the neuronal storage disorders are unique, indeed, as in the case of ectopic dendritogenesis and synaptogenesis, they are unprecedented. In no other disease condition or stage of development of the nervous system can this same event be found. Certainly there is support for the view that this ectopic dendritogenesis is not so much abnormal as it is displaced in time, i.e., a recapitulation of embryonic features of developing neurons (Purpura, 1979). But this change in cell geometry and connectivity is at its greatest extent a massive alteration in what otherwise is a highly ordered operation. The amount of new synaptic surface area on neurons in, e.g., the gangliosidoses, is considerable, and its placement is such that significant influence over neuronal output is likely. Whether this process is eventually found to be a key component of neuronal dysfunction or is in some way

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compensatory and beneficial to neuron function relative to other storage disease-induced phenomena, an understanding of it can be anticipated to lend considerable insight into those factors regulating dendritogenesis and synapse formation in normal nervous systems. The storage disorders may also play an important part in further elucidating the function(s) of gangliosides in the mammalian CNS. Implicated by currently available data are pivotal roles in neuritogenesis and in neurotransmitter release from synaptic terminals. Examining these events in ganglioside storage disease, e.g., by using the feline model of GM1 gangliosidosis, may eventually offer a common explanation for the role of GM1 ganglioside in such diverse phenomena. A third conspicuous abnormality occurring in major proportion in the storage disorders is that of the axonal spheroids. Remarkable for their morphological similarity across a variety of lysosomal enzyme deficiencies, little is presently known as to the events immediately responsible for their formation. Their close similarity to those axonal changes present in another, much less well characterized family of diseases-the neuroaxonal dystrophies-make their study all the more important. A somewhat similar, but more limited, phenomena in terms of distribution among storage diseases is that of the dendritic spheroids. Found only in a-mannosidosis, their presence appears to suggest that there is a link between dendritic integrity and the normal function of the lysosomal a-mannosidase enzyme. As with axonal spheroids, however, any such linkage most likely is indirect since the dendritic spheroids are not characterized simply by the accumulation of cytosomes. In these and related ways do the neuronal storage disorders offer themselves as windows on complex events in the nervous system. For clearly what begins as a highly discrete molecular defect ends as a host of cellular malfunctions. The challenge then is to follow the threads linking those events, and in so doing to understand not only the pathogenesis of these diseases, but also to gain insight into normal cellular operations. In this the centenary year of Barnard Sachs’ original observations, such a quest has never seemed more possible, or indeed, more potentially rewarding.

Acknowledgments

The author wishes to thank Drs. Dominick Purpura and Henry Baker for their collaboration during the course of these studies, as well as M . Buschke, M . Huang, and S . Wurzelmann for excellent technical assistance, and V. Ahrcns for manuscript preparation. Particular thanks go to Mrs. Buschke for her transtation of the Bielschowsky article. Much of the work reported in this review has been supported by grants from the National institute of Neurological, communicative Disorders and Stroke, and the National Tay-Sachs and Allied Diseases Foundation, Inc.

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THALAMIC AMNESIA: CLINICAL AND EXPERIMENTAL ASPECTS By Stephen G.Waxman Department of Neurology Yale University School of Medicine New Haven, Connecticut 06510, and Center for Neurological Diseases and Regeneration Research Veterans Administration Medical Center West Haven, Connecticut 06510

I. 11. 111. IV. V.

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Introduction Clinical Observations Experimental Studies Theoretical Considerations Conclusions and Prospects: Future Questions References Note Added in Proof

1. lntroductlon

One of the most interesting and important aspects of human behavior involves learning and memory. There is no question that clinical disorders of memory not only provide syndromes that are of significant theoretical interest, but also constitute, in some patients, a source of marked disability. The alcoholic Korsakoff syndrome, for example, constitutes a clinical problem of significant magnitude. In this regard, information about the neural basis for memory, derived from clinical or experimental observations, is of considerable importance. It is the purpose of this article to summarize the available evidence concerning one particular type of memory disorder that has a discrete neuroanatomical substrate, i.e., thalamic amnesia. Recognition of this syndrome is of obvious clinical importance, since it can occur as a result of disorders (e.g., neoplasm, infarction, hemorrage) that require prompt and accurate diagnosis and treatment. This disorder also, however, has theoretical importance in pointing toward some of the central nervous system (CNS) structures that are required for intact memory function in mammals. Previous studies on the effects of focal lesions on memory have suggested the involvement of a number of structures. These include the hippocampus 245 INTERNATIONAL REVIEW OF NEUROBIOLOGY, VOL. 29 '

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AU rights ofrepduction in any form reruved.

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(Scoville and Milner, 1957; Penfield and Milner, 1958; Victor et al., 1971; DeJong et al., 1969; Olds, 1970; Mohr et a l . , 1971; Iverson, 1976), temporal lobe (Serafetidines and Falconer, 1962; Dimsdale et al., 1964; Geschwind and Fusillo, 1966; Van Buren and Borke, 1972; Benson et al., 1974), temporal stem (Horel et al., 1975; Horel, 1978), fornix (Nathan and Smith, 1950; Hassler and Reichert, 1957), and cerebellar nuclei (Thompson et al., 1984). There is evidence implicating each of these areas in memory function within the mammalian brain. The purpose of this article is not to negate the importance of these areas in memory function, but rather to call attention to another nuclear area, the mediodorsal thalamus, which may play a role in memory, and to describe the amnestic deficits which may occur as a result of relatively discrete lesions of this diencephalic region.

II. Clinical Observations

Even the early clinicopathological studies suggested that thalamic lesions could result in disorders of memory. For example, in one early study, Smyth and Stern (1938) concluded that, in tumors originating in lateral thalamic areas, sensory signs predominated at early stages. In contrast, in those cases where thalamic tumors originated medially, behavioral disturbances constituted the earliest clinical abnormalities. The behavorial changes included marked abnormalities of memory. Considerable information concerning the role of the thalamus in memory disorders was provided by the classical clinicopathological study of the alcoholic Wernicke-Korsakoff syndrome carried out by Victor et al. (1971). These authors studied 245 patients with this syndrome and carried out 82 postmortem examinations. The thalamus was commonly involved, with the mediodorsal nucleus being most frequently affected. The Victor et al. (1971) study included an attempt to relate the memory disorder to the site(s) of pathology in the diencephalon and mesencephalon. The results showed a high degree of correlation between memory deficit and pathology of the mamillary bodies, mediodorsal nuclei of the thalamus, and medial part of the pulvinar. Victor et al. noted that it was not possible, on the basis of their results, to determine with certainty which one of these structures is crucial for memory, but emphasized the following observations. Within the series of patients studied, there were five patients who displayed the signs of acute Wernicke’s disease (ataxia, ophthalmoparesis, confusional state) but did not show evidence for memory defect; notably, in each of these cases, the medial dorsal nucleus of the thalamus was found to

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be normal on histological examination. Of the 43 cases in which the medial dorsal nucleus was available for study, these five patients without amnesia were the only cases in which the mediodorsal nucleus was spared. The mamillary bodies, on the other hand, showed significant pathological changes in all five of the above patients, leading Victor et al. to conclude that pathological changes could occur in the mamillary bodies in the absence of memory defect. Additionally, this study revealed five other cases in which the medial dorsal nucleus was the only thalamic nucleus involved; each of these cases was characterized by a severe defect of memory. While the pulvinar was not available for study in some of these latter cases so that its role in memory loss could not be fully assessed, on the basis of their extensive study, Victor et al. were led to suggest that, in terms of memory function in the Wernicke-Korsakoff syndrome, the most crucial lesions were located in the medial dorsal nucleus of the thalamus. Studies of patients with neoplastic, vascular, or traumatic lesions have provided additional evidence for a role of the thalamus in memory function. Data from patients with tumors can present difficulty with interpretation because of the possibility of tumor spread or mass effect, with compression and/or invasion of neighboring structures. Nevertheless, analysis of data from patients with diencephalic tumors reveals that memory loss is often a prominent part of the clinical picture. In an early analysis of 26 patients with brain tumors and prominent memory loss (Williams and Pennypacker, 1954), deep midline supratentorial tumors were found in 15. Four patients displayed a classical confabulatory amnesic syndrome, and in all four there were lesions involving the floor and/or walls of the third ventricle. McEntee et al. (1976) described a patient who abruptly developed a memory deficit as the presenting feature of tumor (metastatic from a small cell carcinoma of the lung) invading the region surrounding the posterior portion of the third ventricle and upper brain stem; notably, the mamillary bodies, mamillothalamic tracts, and anterior thalamus were not involved. The authors suggested that this case provides support for the hypothesis that pathology of the medial dorsal thalamic nuclei, without concurrent pathology of the mamillary bodies, can result in an amnestic syndrome. Ziegler et al. (1977) described a patient in which abrupt memory loss was associated with a glioblastoma infiltrating the thalamus. Extension of the tumor in this patient to the medial aspect of both temporal lobes makes interpretation of this case difficult, although the brunt of pathology was localized to the thalamus. Vascular lesions of the thalamus associated with amnesia have been described by a number of authors. Several case reports of medial thalamic infarction (Mills and Swanson, 1978; Guberman and Stuss, 1983) and unilateral thalamic hemorrhage (Watson and Heilman, 1979), while not focusing explicitly on memory loss, describe the abrupt onset of severe memory

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deficit in the context of these thalamic lesions. A number of other studies have described memory disorder in patients with unilateral or bilateral thalamic infarctions or hemorrhages. Schott et al. (1980) described a 46-year-old, previously healthy male who developed “thalamic amnesia” in the context of a bilateral symmetrical infarction of the medial part of the thalamus. Winocur et al. (1984) published a detailed report describing the acute onset of anterograde amnesia in a 38-year-old man whoisuffered a bilateral infarction of the medial thalamus, presumably due to infarction in the territories of the paramedian thalamic arteries. The patient appeared to have no general intellectual loss and he performed well on standard memory tasks testing immediate recall. He displayed, however, significant impairment in terms of verbal and nonverbal material over delays of as little as several seconds. There was also impairment of recognition memory. Interestingly, remote memory was relatively spared; this patient displayed primarily an anterograde memory disorder, with little impairment of retrograde memory function. Winocur et al. (1984) concluded that this patient’s disorder involved a deficit in acquiring andlor encoding information, and not in the retrieval stage of memory. They further suggested that amnesia resulting from diencephalic lesions is most readily interpreted as a deficit of learning, a view supported by studies on patients with alcoholic Korsakoffs syndrome (Squire, 1981). An anterograde amnestic syndrome in a patient with a circumscribed infarction of the mediodorsal thalamic nucleus on the left side was reported by Speedie and Heilman (1982). Notably, in this case the deficit was most marked for verbal material; retention of visual material was relatively preserved in this patient. In discussing this patient, the authors noted that, as a result of its interconnections with frontal cortex, the mediodorsal thalamus might facilitate memory function by participating in the “chunking” or sequencing of information according to specific categories or semantic structures; according to this hypothesis, mediodorsal lesions would interfere with memory by impairing these frontal executive functions. Speedie and Heilman (1983) subsequently described another patient with a discrete lesion of the right rnediodorsal thalamic nucleus who displayed an anterograde memory deficit for visuospatial material. The authors emphasized, on the basis of their studies on these patients, that interconnections between mediodorsal thalamic nuclei and frontal, temporal, or both lobes are involved in thalamic amnestic disorders. Amnesia can also occur as a result of thalamic hemorrhage. Choi et al. (1983) described three patients with unilateral thalamic hemorrhage (leftsided in two, right-sided in one) and memory deficit. Although previous contralateral brain disease in the patient with right-sided hemorrhage made it

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difficult to examine, in this series, the relationship between unilateral rightsided thalamic lesions and amnesia, the authors did conclude that bilateral thalamic lesions are not always required to produce verbal amnesia. They also noted that behavioral changes can occur as a result of thalamic hemorrhage in patients with an otherwise relatively benign clinical picture and emphasized the importance of early recognition of thalamic hemorrhage when it presents as an amnestic syndrome. A patient with right-sided thalamic hemorrhage and the abrupt onset of amnesia has been described by Waxman et al. (1986). This patient developed signs of thalamic hemorrhage while in the hospital and, as a result of this, memory function had been tested immediately prior to, as well as following, damage to the thalamus. Figure 1 illustrates the C T scan showing this patient’s lesion. In addition to a severe amnestic syndrome, this patient developed, concomitant with hemorrhage into the thalamus, left-sided hemiinattention and motor neglect, as well as profound indifference and hypokinesia; each of the above deficits has been described in patients with thalamic lesions (Heilman et al., 1978; Watson et al., 1979; Cambier et al.,

FIG. 1 . CT scan showing right-sided thalamic hemorrhage in patient with severe memory disorder. Modified from Waxman cl al. (1 986).

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1980; Schott et a l . , 1981). This patient’s amnestic syndrome was characterized by severe anterograde and retrograde memory deficits and included confabulatory responses. As shown in Fig. 1, this patient’s C T scan revealed cerebral atrophy, and there was a history of carotid artery disease with a prior left carotid endarterectomy. However, in this patient memory was known to be intact prior to acute deterioration from the thalamic hemorrhage, and memory loss developed with a time course matching that of the thalamic bleeding. In view of this, it appears likely that amnesia in this patient was related to the thalamic hemorrhage. This case also illustrates the point that amnesia need not occur as an isolated finding, but can also occur (even though it is not necessarily part of a global dementia or diffuse encephalopathy) together with other focal behavioral changes. Finally, a number of cases of traumatic lesions of the thalamus have been described and are highly instructive. Teuber et af. (1968) described a patient who developed a profound amnestic syndrome as a result of a stab wound to the basal brain; the lesion was the result of a fencing accident in which a foil entered the right nostril, penetrated the base of the brain and coursed upward in the diencephalon. This patient displayed a marked anterograde amnesia, which was more severe for verbal than for nonverbal material. The patient was grossly deficient in terms of paired associative learning. He exhibited a profound amnesia for events which occurred since the injury, which occurred in 1960, although memory for events in the premorbid period prior to 1960 was relatively preserved (Squire and Slater, 1978). In this patient, C T scans localized the lesion to the left mediodorsal thalamus (Squire and Moore, 1979). Since the lesion in this patient was produced by a focal traumatic injury, and since the C T scan revealed no evidence of CNS damage outside of the thalamus on the left, this case provides strong clinical evidence for the role of the thalamus in memory.

111. Experimental Studies

Recent experimental studies in animals provide additional evidence for a role of the thalamus in memory and point toward involvement of the mediodorsal thalamic nucleus. Early studies by Walker (1940), Chow (1954), and Peters et al. (1956) failed to demonstrate a deficit in learning after medial thalamic lesions in mammals. In these early studies, however, the experimental lesions were quite small and did not necessarily include the magnocellular medialis dorsalis or included only part of this nucleus.

THALAMIC AMNESIA

25 1

In 1964, Schulman provided evidence, based on studies of monkeys with large lesions of the dorsomedial thalamic nucleus produced by radiation, that performance in delayed-reaction tests and conditioning could be impaired as a result of medial thalamic lesions. Briese and Olds (1964) studied the effects of electrical stimulation of the mediodorsal thalamus during periods of retention in monkeys and suggested that this nucleus might play a role in memory processing, although they were careful to note that they could not exclude a more nonspecific effect, e.g., in terms of modivation or arousal. Subsequent electrophysiological studies (Chandler and Liles, 1977) examined subcorticalevoked potentials during conditioning of the eye-blink response; interestingly, these studies showed an increase in amplitude of the evoked activity in mediodorsal nucleus throughout the period of conditioning, suggesting a possible role of the nucleus in conditioning. These early studies were less than definitive because they required total destruction of the mediodorsal thalamus in order to produce reproducible behavioral abnormalities and since the lesioning technique employed also caused damage to neighboring thalamic nuclei. Nevertheless, these early experimental studies provided some of the early evidence based on experimental lesions that suggested a role of the mediodorsal thalamus in memory. More recent studies have provided much firmer evidence that lesions of the mediodorsal thalamus can produce memory impairment in primates. Isseroff et al. (1982) produced bilateral radiofrequency lesions in the mediodorsal nucleus of juvenile (2- to 3-year-old) rhesus monkeys and subsequently studied the behavioral results of these lesions in terms of delayed spatial responses, visual pattern discrimination, and delayed alternation responses (Goldman et al., 1971). The delayed spatial response and delayed alternation tasks were used in this study because they are sensitive to spatial memory impairments of the type observed after damage to the prefrontal cortex, which has extensive connections with mediodorsal thalamus (Nauta, 1964; Johnson el al., 1968). Isseroff et ul. (1982) found that performance on spatial delayed alternation tasks was significantly impaired in monkeys with mediodorsal thalamic lesions and that there was a significant correlation between the degree of behavioral impairment and the degree of damage to the posterior half of the nucleus. There was also a significant correlation between damage to posterior parts of the nucleus and impairment in terms of delayed alternation responses, which are also a measure of spatial memory. The authors concluded that experimental lesions of the mediodorsal thalamic nucleus can produce a specific syndrome, characterized by spatial memory loss, that is similar to that observed after lesions of prefrontal cortex.

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Aggleton and Mishkin (1983a,b) have carried out several instructive ablation studies which demonstrate memory loss following restricted medial thalamic lesions in monkeys. In a preliminary study, these workers examined the question of whether medial thalamic lesions could produce amnesia. In animals with diencephalic lesions involving the medial aspects of the anterior and medial thalamic nuclei, there was significant impairment in terms of relearning of a one-trial object recognition test, nonmatching to sample. These lesions did not result in abnormalities in the learning of pattern discrimination or spatial delayed responses (Aggleton and Mishkin, 1983a). In a subsequent study, these workers subdivided the original medial thalamic lesion into two components: an anterior component, centered on medial portions of the anterior thalamic nuclei, and a posterior component, centered on the magnocellular portion of the medial dorsal thalamic nuclei. Lesions were made surgically by suction and were verified histologically. Both lesions resulted in impairment on tests designed to assess both recognition and associative memory (Aggleton and Mishkin, 1983b). Taken in the context of the earlier finding that medial thalamic lesions do not impair performance in terms of pattern recognition or delayed-response tasks (Aggleton and Mishkin, 1983a), these results were interpreted as providing evidence for a similarity between diencephalic behavorial changes in monkeys and amnesia of diencephalic origin in man. In discussing the question of which thalamic nuclei were responsible for memory deficits, Aggleton and Mishkin (1983b) provided a number of arguments suggesting that the magnocellular division of the medial dorsal nucleus is critically involved. They noted that this nucleus is a major thalamic target of the amygdala, which has also been implicated in memory. They further suggested that there might be a specific link between lesions in this thalamic nucleus and associative memory. Another ablation study (Zola-Morgan and Squire, 1985) examined the effects of mediodorsal thalamic lesions in monkeys using a recently developed test of memory (Squire and Zola-Morgan, 1983) that is sensitive to human amnesia. In this study, bilateral electrolytic lesion were made in the mediodorsal nuclei and monkeys were then examined via a delayed nonmatching-tosample test. Notably, monkeys with mediodorsal lesions were impaired in terms of learning the task when delays of 8, 15, and 60 sec and 10 min were used; as the delay was increased from 15 sec to 10 min, the performance of the lesion group deteriorated sharply. In contrast, the lesioned monkeys performed at normal levels on a second test designed to examine skill-based tasks in amnesic individuals. As in the above study, the results of this experiment show that circumscribed lesions of the mediodorsal thalamus can produce a significant amnestic syndrome in experimental animals.

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253

Thus, recent studies (Isseroff et al., 1982; Zola-Morgan and Squire, 1985) provide experimental support for the idea that lesions within mediodorsal thalamus can produce an amnestic syndrome in monkeys. In these studies, it was pointed out that these results do not imply that damage to other brain areas does not produce memory deficit. Moreover, as pointed out by Markowitsch (1982), the possibility remains that the mediodorsal nucleus itself might not constitute a crucial site of information processing or storage, but that fibers of passage, coursing through the nucleus, might be damaged by lesions of the nucleus. In this respect, it should be recalled, however, that the number of axons passing through the mediodorsal nucleus is small, although the fasciculus retroflexus (habenulo-interpedular tract) is located in contact with cells of the nucleus (Tombol, 1968); whether damage to this tract contributes to the amnestic syndrome remains unclear at this time. Fuster and Alexander (1973) studied changes in unit activity in mediodorsal thalamus of the monkey and attempted to correlate these with the learning of delayed response tasks. They observed alterations in firing patterns which led them to suggest that a significant subclass of neurons in the parvocellularis portion of the mediodorsal nucleus is related to short-term processes. This observation is an important one, since it rests on the recording of ongoing neuronal activity rather than on the production of lesions. The important conclusion is that the mediodorsal thalamic nucleus may play a role in memory, and that discrete lesions within mediodorsal thalamus, whether occurring in isolation or in the context of more widespread disease, can lead to an amnestic syndrome. Production of this syndrome in lesioned animals, moreover, provides a new experimental model which may bear similarity to amnestic disorders seen in the clinical domain and may permit the experimental dissection of the contribution, from lesions within various regions of the central nervous system to clinical disorders of memory.

IV. Theoretical Conrideratlons

Given that lesions of the mediodorsal thalamus do produce an amnestic syndrome, the question arises as to the significance of this finding in terms of understanding the neural basis for memory. Since there have been only a few studies on the mediodorsal nucleus (see Fuster and Alexander, 1973) which focused on synaptic or neuronal plasticity (see, e.g., Kandel and Schwartz, 1982) as a result of learning, little can be said about possible cellular or molecular changes in the thalamus that accompany learning.

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Moreover, the available data are certainly not sufficient to purpose the localization of engrams per se within thalamic nuclei. On the other hand, a consideration of thalamic projections may provide some information which is relevant to the role of the thalamus in memory. In this regard, Isseroff et al. (1982) have commented on the possible role of medial thalamic nuclei in a system including dorsolateral prefrontal cortex and its subcortical projections, including caudate nucleus and mediodorsal and other thalamic nuclei, that may be involved in memory function. The available evidence suggests, in this regard, that lesions of mediodorsal thalamic nuclei can produce a variety of behavioral deficits that are similar to those observed following damage to the prefrontal projection fields of these nuclei (Peters et al., 1956; Schulman, 1964). Moreover, the existence of connections between mediodorsal thalamus and prefrontal cortex has been established anatomically (Akert, 1964; Nauta, 1964; Kievit and Kuypers, 1977) and is further supported by the observation of cells with similar physiological responses (in terms of delayed task performance) in both areas (Fuster and Alexander, 1973), so that thalamic nuclei might be expected to play a role in the function of this system. There is also evidence for projections between mediodorsal thalamic nuclei and the amygdala (Nauta, 1962) and hippocampus (Valenstein and Nauta, 1962). Thus, appropriately localized mediodorsal thalamic lesions may interfere with synaptic input or output from these areas; in view of their proposed role in memory, this would provide another mechanism for the production of amnestic deficits as a result of thalamic lesions. Markowitsch (1982) suggests that the mediodorsal nucleus can be considered as a nodal point in the pathway bringing limbic system-processed information (e.g., from the fornix fiber system and/or reticular formation) to the prefrontal cortex. It has also been suggested (Kovner et al., 1981) that parallel circuits, involving the pulvinar and its cortical target (temporoparietal association cortex) and the mediodorsal nucleus and its prefrontal cortical target, are necessary for normal memory function; according to this hypothesis, encoding involves the pulvinar pathway, and further processing depends on mediodorsal nucleus. As pointed out by Markowitsch, the possibility of two or more parallel pathways involed in memory is consistent with observations (Victor et al., 1971) that there are often multiple lesions in patients with the Korsakoff syndrome.

V. Conclusions and Prospects: Future Questions

On the basis of the data reviewed above, it seems clear that a syndrome of thalamic amnesia can be produced by relatively focal lesions of the mediodorsal

THALAMIC AMNESIA

255

thalamus. The evidence for this syndrome derives from both clinical observations and experimental studies involving discrete lesions of thalamic nuclei. One important question concerns the degree to which unilateral, as opposed to bilateral, thalamic lesions can produce memory deficits. In many of the human cases cited there was evidence of contralateral brain disease. Even in those cases where there is no structural evidence for contralateral disease, the possibility of “diaschesis” (Kushner et al., 1984) could always be raised. In the experimental studies cited, behavioral abnormalities were noted after the production of bilateral lesions. Thus, the question of whether unilateral thalamic lesions can lead to amnestic abnormalities will require further study. It should be noted, in this respect, that the clinical reports include a number of cases (Speedie and Heilman, 1983; Choi et al., 1983; Waxman et al., 1986) in which amnestic syndromes developed with a time course suggesting that memory was impaired as a result of a unilateral thalamic lesion. While it might be argued that the thalamic lesion was superimposed on previously existing contralateral disease, the important point that emerges is that discrete thalamic lesions can produce acute and profound deterioration of memory. If unilateral thalamic lesions can produce memory disorder, an important question will concern the degree to which handedness affects outcome in patients with unilateral diencephalic disorders. In this context, the present author has seen a left-handed patient who developed, on the basis of an infarction in the territory of the left posterior cerebral artery, a syndrome of alexia without agraphia together with a dense retrograde and anterograde amnesia. While the memory disorder may well have reflected pathology of other structures in the territory of the posterior cerebral artery (e.g., temporal lobe or hippocampus), this case illustrates that, in left-handers as well as right-handers, unilateral cerebral disease can produce memory deficit. Observations on thalamic amnesia have important theoretical implications because, while they do not localize the engram to the thalamus per se, they demonstrate a role of thalamic nuclei in memory processes-in this sense, it is becoming increasingly clear that the mediodorsal thalamus must be viewed as part of a mnemonic system and that damage to thalamic components of the system may interface with memory function. It is interesting, in this regard, that a number of authors have commented on the fact that multiple lesions are most likely to produce memory deficits-this observation may reflect a degree of redundancy in memory systems. From a clinical point of view, thalamic amnesia is important since it provides, for the clinician, significant localizing information in addition to the traditional signs of thalamic damage (Walshe et al., 1977) which may point toward pathology. Moreover, memory dysfunction can occur early in the

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progression of thalamic disease, before other localing signs become apparent. Since early recognition of thalamic lesions such as tumors and hemorrhages can be lifesaving, this is a very important point. Finally, the syndrome of thalamic amnesia may provide an interesting experimental model in which memory is impaired on the basis of a wellcircumscribed and focal lesion. Noninvasive modes of imaging (e. g. , MRI scanning) promise to make these lesions more easily studied in humans. Moreover, the availability of a primate model promises to be of considerable value. As further patients with this syndrome are studied and further experimental studies are carried out, it may be possible to dissect out the role of thalamic nuclei in memory systems. Acknowledgments

This work was supported in part by the Medical Research Service, Veterans Administration.

References

Aggleton, J. P., and Mishkin, M. (1983a). Neuropsycholosia 21, 189-197. Aggleton, J . P., and Mishkin, M. (1983b). Exp. Brain Res. 52, 199-209. Akert, K. (1964). In “The Frontal Granular Cortex and Behavior” M. Warren and K. Akert, eds.), pp. 372-396. McGraw Hill, New York. Benson, D. F., Marsden, C. D., and Meadows, J . C. (1974). Acta Neurol. Scad. 5 0 , 133-145. Briese, E . , and Olds, J . (1964). Exp. Neurol. 10, 493-508. Cambier, J., Elghozi, D., and Strube, E. (1980). Reu. Neurol. (Pan$) 136, 105-116. Chandler, S. D., and LiIes, S. L. (1977). Exp. Neurol. 55, 368-380. Choi, D., Sudarsky, L., Schachter, S., Biber, M., and Burke, P. (1983). Arch. Neurol. 40, 611-613. Chow, K. L. (1954). Arch. Neurol. Psychiatr. 71, 762-771. DeJong, R . N., Itabashi, H.H . , and Olson, J. R. (1969). Arch. Neurol. 20, 339-348. Dimsdale, H . , L o p e , V., and Piercy, M. (1964). Neuropsycho~o~a 1 , 287-298. Fuster, J . M . , and Alexander, G. E. (1973). Brain Res. 61, 79-91. Geschwind, N . , and Fusillo, M. (1966). Arch. Neurol. 15, 137-146. Goldman, P. S . , Rosvold, H., Vest, B., and Galkin, T. (1971).J. Compar. Physiol. Psychol. 7 7 , 212-220. Guberman, A , , and Stuss, D. (1983). Neurology, 33, 540-546. Hassler, R., and Riechert, T . (1957). Arch. Psychiutr. Nnucn Kr. 200, 93-122. Heilman, K . M . , Schwartz, H.D., and Watson, R. T . (1978). Neurolosy 28, 229-232. Horel, J. A. (1975). Bruin 101,403-445. Iverson, S. (1976). Inl. Rev. Ncurobtol. 19, 1-49. Isseroff, A , , Rosvold, H. E . , Galkin, T.W., and Goldman-Rakic, Y. S. (1982). Brain Res. 232, 97-114. Johnson, T . N., Rosvold, H . E., and Mishkin, M. (1968). Exp. Neurol. 21, 20-34. Kandel, E. R . , and Schwartz, J . (1982). Scicnce 218, 433-442. Kievit, K., and Kuypers, H. G. J . M. (1977). Exp. Brain Res. 29, 299-322. Kovner, R . S., Mattis, S., Goldmeier, E., and Davis, L. (1981). Brain L Q ~ s 12, . 23-32.

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Kushner, M., Alavi, A,, Reivich, M., Dann, R., Burke, A,, and Robinson, G. (1984). Ann. Neurol. 15, 425-434. McEntee, W. J., Biber, M. P., Perle, D. P., and Benson, D. F. (1976). J . Neurol. Neurosurg. Psychiutr. 39, 436-441. Markowitsch, H . J. (1982). Neurosci. Biobehav. Rev. 6 , 351-380. Mills, R. P., and Swanson, P. D. (1978). Ann. Neurof. 4, 149-155. Mohr, J., Leicester, J., Stoddard, L., and Sidman, R . (1971). Neurology 21, 1104-1113. Nathan, N., and Smith, 0. A. (1971). J . Comp. Physiol. Psychol. 74, 68-73. Nauta, W. J. H . (1964). In “The Frontal Granular Cortex and Behavior” M. Warren and K. Akert, eds.), pp. 397-409. McGraw-Hill, New York. Olds, J. (1970). In “The Neural Control of Behavior” (R. E. Whalen, R. F. Thompson, M. Verzeano, and N. Weinberger, eds.), pp. 257-293. Academic Press, New York. Penfield, W., and Milner, B. (1958). Arch. Neurol. Psychiatr. 79, 475-497. Peters, R . H., Rosvold, H. E., and Mirsky, A. F. (1956).J. Comp. Physiof. Psychof. 49, 111-116. Schott, B., Mauruiere, F., Laurent, B., Serclerat, O., and Fischer, C. (1980). RCZJ. Neurol. (Paric) 136, 117-130. Schott, B., Laurent, B., Mauruiere, F., and Chauzot, G. (1981). Reu. Neurol. ( P ~ n i137,447-455. ) Schulman, S. (1964). Arch. Neurol. 11, 477-499. Scoville, W. B., and Milner, B. (1957). J . Neurof. Neurosurg. Psychiutr. 20, 11-21, Serafetidines, E. A,, and Falconer, M . A. (1962). J. Neurof. Neurosurg. Psychiutr. 25, 251-256. Smythe, G. E., and Stern, K. (1938). Bruin 61, 339-374. Speedie, L. J . , and Heilman, K. M. (1982). Neuropsychologia, 20, 597-604. Speedie, L . J., and Heilman, K. M . (1983). Arch. Neurol. 40, 183-186. Squire, L. R . (1981). Trends Neurosci. 3, 52-54. Squire, L. R., and Moore, R . Y. (1979). Ann. Neurof. 6 , 5003-506. Squire, L. R., and Slater, P. C . (1978). Neuropsychologia 16, 313-322. Squire, L. R., and Zola-Morgan, S. (1983). In “The Physiological Basis for Memory” (J. A. Deutsch, ed.), 2nd Ed., pp. 200-268. Academic Press, New York. Teuber, H.-L., Milner, B., and Vaughn, H . G., Jr. (1968). Neuropsychologiu 6, 267-282. Thompson, R . , Clark, G. A., Donegan, N. H., Lavond, D. G., Madden, J., Mamounas, L. A., Mauk, M. D., and McCormick, D. A. (1984). In “Neuropsychology of Memory” (L. R . Squire and N. Butters, eds.), pp. 424-442. Guilford, New York. Q Hung. 16, 183-203. Tombol, T . (1968). A C ~ Morphof. Valenstein, E. S., and Nauta, W. J. H. (1959). J . Comp. Neurol. 113, 337-362. Van Buren, J . M., and Borke, R . C. (1972). Bruin 95, 599-632. Victor, M., Adams, R . D., and Collins, G. H . (1971). “The Wernicke-Korsakoff Syndrome.” Davis, Philadelphia. Walker, E. A. (1957). Arch. Neurol. Psychiatr. 78, 543-552. Walshe, T. M., Davis, K. R., and Fisher, C. M . (1977). Neurology 27, 217-222. Watson, R. T., and Heilman, K. M . (1979). Neurofogy 29, 690-694. Watson, R. T., Heilman, K. M., Cauthen, J. C., and King, P. A. (1973). Neurology23, 1003-1007. Waxman, S. G., Ricaurte, G. A., and Tucker, S. B. (1986). J. Neurol. Sci. 75, 105-112. Williams, M., and Pennypacker, J. (1954). J . Neurol. Neurosurg. Psychiutr. 17, 115-123. Winocur, G. M., Oxbury, S., and Roberts, R . (1984). Neuropsychologiu 22, 123-144. Ziegler, D. K., Kaufman, A., and Marshall, H. E. (1977). Arch. Ncurof. 34, 545-548. Zola-Morgan, S . , and Squire, L. R . (1985). Ann. Neurol. 17, 558-564.

a.

NOTEADDEDI N PROOF Since this article was written, another report of thalamic amnesia appeared (Mori, E., et d. (1986). Ann. Neurol. 20, 671-676.1. This report describes a patient with impairment of verbal memory after infarction of the left anterior thalamus.

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CRITICAL NOTES ON THE SPECIFICITY OF DRUGS IN THE STUDY OF METABOLISM AND FUNCTIONS OF BRAIN MONOAMINES By S. Garattini and T. Mennini lstituto di Ricerche Farmacologiche “Mario Negri” 20157 Milan, Italy

I. Introduction 11. Some Drugs Interfering with Monoamine Uptake and Release A. &Amphetamine and the Noradrenergic System B. Amineptine and Dopamine Uptake C . Fenfluramine and Serotonergic Neurons 111. Drugs Acting at Monoamine Receptors A. Importance of the Choice of Ligand B. Interaction with Receptors by Drugs Which Are Highly Metabolized C . Brain Regional Differences D . Species Differences in Brain Receptor Subtypes IV. Concluding Remarks References

I. lntroductlon

In psychopharmacology it is common to employ drugs known to interact with a neurotransmitter system as a tool for studying the mechanism of action of psychotropic drugs. This approach has led to considerable progress in understanding the mechanism of action of drugs, but it must be applied with caution. The selectivity of drugs exerting a given biochemical effect must always be established under the same precise experimental conditions since it is very difficult to extrapolate across doses, routes of administration, times after dosing, animal species, brain regions, etc. In a drug interaction study, comparisons must be made at equal brain-or brain area-concentrations to make sure no “metabolic” interaction has occurred. In fact, one drug may change the effect of another by affecting its disposition as regards organ distribution, protein binding, and formation of active metabolites. Moreover, the specificity frequently lies within a narrow dose range, outside which interactions may occur with other chemical mediators. Interference with a chemical mediator in a certain area is not necessarily the same in all brain areas. The links between various chemical mediators may 259 INTERNATIONAL. REVIEW OF NEUROBIOLOGY, VOL. 29

Copyright @ 1988 by Academic PRSS,Inc. AU rights of repduction in m y form reserved.

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differ widely depending on the brain structure considered. For instance the dopaminergic and cholinergic systems are linked in the striatum (Consolo et al., 1974; Ladinsky et al., 1978) but not in other brain areas (Consolo et al., 1978). Similar considerations apply when measuring interactions with neurochemical receptors because different subtypes exist in various brain areas and in different animal species. This review will give some examples about the use of drugs in studying monoamine functions, with the aim of cautioning against oversimplified conclusions from pharmacological experiments. Particular attention will be paid to drugs acting on uptake and release of monoamines and to drugs acting on monoamine receptors. The chemical structures of some drugs mentioned in the text are given in Fig. 1.

11. Some Drugs interfering with Monoamlne Uptake and Release

A , &HETAMINE AND THE NORADRENERGIC SYSTEM d-Amphetamine is a drug frequently used as a tool to study catecholaminergic effects elicited by release of noradrenaline (NA) and dopamine (DA). In addition this drug inhibits catecholamine uptake and, in uifro, is a specific inhibitor of noradrenaline uptake (Garattini et al., 1978). Its IC~O is 80 nM for inhibition of noradrenaline uptake by rat brain synaptosomes, while 500 nM are required to inhibit dopamine uptake by 50% and 12 f l is the concentration giving half-maximal inhibition of serotonin (5HT) uptake (Garattini et al., 1978). Different doses of amphetamine result in brain levels in the range of those inhibiting monoamine uptake in vitro (Table I). In order to establish which effect of &hetamine is likely to occur in vivo, Table I sets out the ratios between brain levels (2 hr after i.p. injection of different amphetamine doses) and the concentrations giving half-maximal inhibition of monoamine uptake. The probability of uptake inhibition occurring in vivo rises as the ration exceeds 1. The results suggest that brain amphetamine concentrations 2 hr after i.p. injection of 0.7 m g k g (Cho et al., 1973) are enough to give considerable inhibition of NA uptake without affecting DA and 5HT uptake; at a higher dose (1.8 mgkg) DA uptake is affected to a considerably greater degree Uori et al.., 1978), and at the dose of 15 m g k g (Belvedere et al., 1973) the brain levels can inhibit the uptake of all three m i n e s . This may be important in explaining the different effects obtained with amphetamine in rats: 1.25 mg/kg i.p. can reduce food intake, an effect related to the activation of the noradrenergic

SPECIFICITY OF DRUGS IN STUDY OF MONOAMINES

bcr3

261

+

CHZ-CH -N-Hz I C 1 2H5 FENFLURAMINE

( P m P ) PVRIMIDINVLPIPERAZINE

HN

N

W

N

N

I

t

- ( CH2) 3- N -C

TRAZODONE

0”

(mCPP1m-CHLOROPHENYLPIPERAZINE

AMINEPT IN€

NH

I

( c H2)6- Cook! FIG. 1 . Chemical structures of some drugs mentioned in this review.

system (Quattrone et al., 1977); 10 mg/kg induce stereotyped behavior typical of dopaminergic stimulation (Quattrone et al., 1977). An increase in the turnover of 5HT in the brain has also been reported (Reid, 1970) after high doses of amphetamine. The fact that different doses of d-amphetamine interact with different monoaminergic systems is also reflected in the biochemical and behavioral effects induced by repeated treatment with this drug. The effects of long term treatment with different doses of d-amphetamine indicate that monoamine

262

S. GARATTINI AND T . MENNINI TABLE I RATIOSBETWEEN AMPHETAMINE BRAINLEVELS AND in VItrO CONCENTRATIONS FOR INHIBITION OF MONOAMINE UPTAKECARRIER^ Amphetamine dose (mg/k!3)

Noradrenaline

Dopamine

Serotonin

0.7 1.8 15.0

4 20 625.000

<1 3

<1 <1 4

100

'The rations were calculated by dividing amphetamine brain concentrations (luM) 2 hr after i . p . injection of different doses by the in uitro IC,O(luM) for inhibition of monoamine uptake.

pathways are differently affected: two daily i.p. injections of 1.25 m g k g of d-amphetamine for 28 days did not alter the number of binding sites for 5HT and catecholamines and no tolerance arose to the anorectic activity (Bendotti et al., 1982). A similar schedule but with 10 m g k g significantly reduced the number of 8-adrenergic receptors in hippocampus and dopamine receptors in striatum and nucleus accumbens (Bendotti et al., 1982). This treatment did not modify the anorectic effect of 1.25 mg/kg d-amphetamine, but it markedly reduced the amphetamine motor hyperactivity and completely blocked the stereotyped licking and biting induced by apornorphine (Bendotti et al., 1982). These effects cannot be easily extrapolated across animal species or even across different strains of the same species, as it is well established, for instance, that certain strains of mice, such as C3H, are resistant to some effects of d-amphetamine (Weawer and Kerley, 1962; Caccia et al., 1973; Dolfini et a / . , 1969a,b). Potentiation by tricyclic antidepressant drugs (TCAs) of amphetamineinduced locomotor hyperactivity or hypertermia has been suggested as a test to differentiate this class of psychotropic drugs (Morpurgo and Theobald, 1965) and neuroleptics, which are characterized by the fact that they block several actions of amphetamine (Bradley and Hance, 1957; Janssen et al., 1967). The hypothesis explaining the in viuo interaction between TCAs and amphetamine was based on the fact that TCAs, by blocking NA uptake at nerve terminals (Carlsson et a l . , 1966, 1969), would increase the concentrations of NA released by amphetamine at the receptors. This attractive hypothesis is no longer tenable, at least in a simple way, because TCAs interact with amphetamine by raising its concentration in the brain (Valzelli et a l . , 1967). A series of studies has been conducted in this respect on the interaction between desipramine and amphetamine (Garattini et al., 1976). It was shown

SPECIFICITY OF DRUGS IN STUDY OF MONOAMINES

263

that the prolongation of amphetamine hypertermia was temporally related to an increased brain level of amphetamine in rats (Valzelli et al., 1967). This increase is due to the fact that desipramine blocks amphetamine hydroxylation (Dingell and Bass, 1969; Consolo et al., 1967) by liver microsomal enzymes (Axelrod, 1954), thus reducing its major metabolic pathway in rats. Since hypertermia is directly related to brain amphetamine concentrations, it appears that when equal concentrations of amphetamine in brain are considered, animals pretreated with desipramine are actually less responsive than controls (Garattini, 1969). That the potentiating effect of desipramine is almost entirely related to an inhibition of amphetamine hydroxylation is indirectly confirmed by the fact that desipramine does not potentiate amphetamine and does not raise brain amphetamine concentrations in mice (Dolfini et al., 1969c), a species in which amphetamine is only poorly hydroxylated (Dring et al., 1966). The interaction between TCAs and amphetamine in rats can be minimized when amphetamine is given orally, because TCAs block gastrointestinal motility and reduce the oral absorption of several drugs in relation to their anticholinergic activity (Consolo and Garattini, 1969). At the same time, therefore, there is less amphetamine being absorbed but also less drug being metabolized after TCA pretreatment. However, a recent paper reports potentiation of amphetamine’s effects after chronic TCA treatment (Spyraki and Fibiger, 1981). In these conditions, no differences were found in brain concentrations of amphetamine, so the effect of TCAs may not be simply related to metabolic interactions (Spyraki and Fibiger, 1981). Probably a supersensitivity of postsynaptic dopamine receptors is responsible for this potentiation of amphetamine’s effects (Spyraki and Fibiger, 1981).

B. AMINEPTINE AND DOPAMINE UPTAKE Amineptine is a new antidepressant agent (Roster, 1979; Samanin et al., 1977; Poignant, 1979; Borsini et al., 1981) which differs from other antidepressants in its selectivity on the dopaminergic system (Garattini et al. , 1986). It has been reported to inhibit dopamine uptake in vitro (Ceci et al., 1986) and in vivo (Garattini et al., 1986) without affecting noradrenaline neurons (Garattini et al., 1986). However the ability of amineptine to inhibit dopamine uptake is reduced after long term administration (Ceci et at., 1986), as is the basal accumulation of dopamine in synaptosomes from rats chronically treated with the drug (Ceci et al., 1986). It seems therefore that the presynaptic carrier for dopamine is modified by long-term amineptine, resulting in an apparent reduction of the maximum number of uptake sites and of its sensitivity to the drug’s inhibitory effect (Ceci et al., 1986). It is of

264

S. GARATTINI AND T. MENNINI

interest to note that in vivo, too, amineptine lost its effect of brain dopamine metabolism after 3 weeks of daily dosing, as indicated by the fact that a challenge with 40 m g k g did not elicit the usual rise in striatal homovanillic acid (Garattini et a l . , 1986). As to its acute effect on 3,4-dihydroxyphenylaceticacid (DOPAC), i.p. injection of 40 m g k g to rats resulted in a persistent, clear-cut decrease of DOPAC levels in the nucleus accumbens (Fig. 2a), as revealed by in vivo voltammetry recordings (De Simoni et al., 1986), while in the striatum only a slight and transient decrease of DOPAC can be documented after in vivo treatment (DeSimoni et al., 1986) (Fig. 2b). Thus for amineptine, as discussed later for buspirone, it appears that the interaction with a particular neuronal system in the brain occurs specifically in

T .

.

. . .2L

.

-12

.

.

.

0

. . 12

. 24

.

. . 36

. 48

.

. . . 60 72

.

. Q4

.

.

I

.

. 108

min

.

120

FIG 2. W P A C levels in the nucleus accumbens (a) and in the caudate (b) of unanesthetized, freely moving rats: effect of amineptine, 40 m g k g i.p. (-, n = 6) and saline (-----, n = 5). The arrow indicates the time of injection. W P A C was recorded every 2 min, but the levels are shown here every 6 min.

SPECIFICITY OF DRUGS IN STUDY OF MONOAMINES

265

a selected brain region responsible for the drug's pharmacological activity. Amineptine may therefore be considered a unique tool drug for studying the dopaminergic system but only under well-defined conditions. It should be stressed that the selective effect of amineptine on dopamine uptake can be demonstrated in vivo by determining the destruction of dopaminergic or noradrenergic neurons after intracerebral administration of the neurotoxin 6-hydroxydopamine(6 OHDA). Table II shows the specific effects of amineptine or dopamine in relation to other agents (Garattini et d.,1975, 1986; Samanin et al., 1975) which nonselectively inhibit 6 OH-DA uptake in noradrenergic neurons. A selective effect of 6 OH-DA uptake in noradrenergic neurons can be obtained using desipramine (Garattini d al., 1975).

C . FENFLURAMINE AND SEROTONERGIC NEURONS Fenfluramine is an anorectic agent (Garattini et al., 1979) known to interfere with the serotonergic system and at the same time is an inhibitor of the uptake and a releaser of serotonin (5HT). Being a racemate, it comprises both d and 1optical isomers which in viuo give rise to, respectively, the d and 1 forms of an N-deethylated metabolite, norfenfluramine. Therefore four compounds are found in the brain after administration of fenfluramine (d- and I-fenfluramine and d- and 1-norfenfluramine), as shown in Table 111. These four compounds, however, have different effects on 5HT, as shown in Table IV; d-fenfluramine (dF)for instance is 3, 18, and 28 times more active on 5HT uptake than d-norfenfluramine (dNF), I-fenfluramine (IF), and Cnorfenfluramine(fNF), respectively (Mennini et al., 1985). dNF is over 5 times more active than dF on noradrenaline uptake and on 5HT spontaneous release. Furthermore, dNF appears to be much more effective than dF in releasing TABLE I1 EFFECT OF VARIOUS DRUGS ON THE DESTRUCTION OF DOPAMINERGIC OR NORADRENERGIC TERMINALS INDUCED BY 6-HYROXYDOPAMINE(6 OH-DA)

Drug and dose (mgk) None Amineptine (20) Nomifensine (10) Desipramine (10) Desipramine (30) &Amphetamine (5)

Dopamine

('3% of control) 40 7 5" 100"

Noradrenaline (% of control) 15 24 48O

60"

32 774

'Significantly different from 6 OH-DA alone.

9ff 63'

266

S. GARATTINI AND T. MENNINI TABLE 111 IN RATS" DOSE-DEPENDENT KINETICSOF d, ~-FENFLURAMINE Brain stem levels (pglg i S.E.)

Dose of fenfluramine ( m g k i.P.1

dFb

3

1.0

15 25

* 0.05

12.7 f 1.1 1 7 . 1 f 0.4 ~

dNFb

KFb

lNFb

1.4 i 0.1 1.8 i 0.1 2.7 + 0.2

0.4 i 0.01 7.8 1.0 9.4 f 0.6

2.5 0.2 5.5 f 0.3 10.5 i 0.5

~~

*

*

~

T h e optical isomers of fenfluramine and norfenfluramme were determined in the brain stem 2 hr after 1 p injection of different doses of fenfluramine bAbbreviations d F , d-fenfluramine, dNF, d-norfenfluramine, lF, I-fenfluramine, INF, [-no rfenfluramine

TABLE IV EFFECTSOF THE STEREOISOMERS OF FENFLURAMINE (F) AND NORFENFLURAMINE (NF) ON MONOAMINE MECHANISMSO Amine mechanisms

dF

Binding (Go pM) 5HTI 5HTi Dz (3-NA LYI-NA (YI-NA

7.0 >30.0 >30.0 >30.0 >3 0 .0

25.0

Uptake (ICSOluM) NA DA 5HT

10.0 20.0

Release SC25 5 H T (normal) 5 H T (reserpine)'

0.5 5.0

> 100.0

dNF

4.2 2.2

lF 2.7

INF

4.5

2.2 3.0

>30.0 10.0

> 30.0

> 30.0

>30.0

6.0

>30.0

15.0

7 .O

5.4 >30.0

1.8 7.0 f .4

13.0 38.0 8.9

n.d.' n.d.

1 .O 0.2

3.0

2.0

1.0

2.0

20.0

14.0

'['HISHT uptake, release, and binding were studied by the method described by Mennini et al. (unpublished data). Data are means of four replications varying less than 10%. ICsois the drug concentrations producing 50% inhibition of uptake or binding and is calculated from the log dose-effect plot of the data, using three to four drug concentrations. SD25 is the drug concentrations stimulating [3H]5HT release by 25%, calculated from the log dose-effect plot of the data using three drug concentrations. % release stimulation = (% release with drugs/% release of controls x 100) - 100. bn.d., Not determined. T h e animals received 10 m g k g reserpine intraperitoneally 18 hr before death. Data for C A binding are ICso obtained from three to four replications varying less than 5%. 5HT2 and CA-receptor binding was determined as described by Mennini ct al. (1985) using the following ligands: [3H]piperone (5HTa and Dz), [3HJdihydroalprenolo1 (BNA), [3H]WB4101 (a,NA), and ['H]clonidine (al-NA).

SPECIFICITY OF DRUGS IN STUDY OF MONOAMINES

267

5HT from the extragranular pool, as shown by experiments in synaptosomes obtained from reserpinized animals (Mennini et al., 1981; Borroni et al., 1983) (see Table IV). These in vitro results could hardly anticipate the in vivo findings reported in Table V. In vivo the administration of dF or IF results in a complete dissociation of effects on brain monoamine metabolites (Invernizzi et al., 1986); at doses of 2.5-5 mgkg orally dF only lowers 5-hydroxyindoleacetic acid (5HIAA) leaving intact the metabolites of dopamine and noradrenaline, while IF increases the levels of MHPG- so4 (metabolite of noradrenaline) and DOPAC (metabolite of dopamine) leaving intact the 5HT metabolite. At the same doses (data not shown) dF,differently from IF, also lowers 5HT. On increasing the doses of dF up to 20 mgkg orally it is possible to raise MHPG.SO4 by about 30% without any change in the levels of DOPAC. Therefore resolution of fenfluramine into the two optical isomers has resulted in compounds which in vivo are relatively selective for 5HT (dF) or for catecholamines (IF). These data oblige us to look critically at several papers which draw conclusions on the role of 5HT from findings with racemic fenfluramine at relatively high doses. Furthermore (see Table III), fenfluramine undergoes dosedependent kinetics, i.e., at higher doses there is a disproportionate increase of brain levels particularly for I- and d-fenfluramine (Caccia et al., 1981a); this makes high doses of fenfluramine even less suitable as a tool for studying the serotoninergic system. Another important aspect of fenfluramine is the time between drug administration and the observation of its effect. If dF is utilized, at an early time there is more dF than dNF in brain, but at a longer time the opposite is true (Caccia et al., 1981a). As already mentioned, when dF predominates quantitatively there is mostly an inhibition of 5HT uptake, while the presence of TABLE V EFFECTS OF d AND A-ISOMERS OF FENFLURAMINE (F) ON MONOAMINE METABOLISM I N THE RATBRAIN" Percentage of controls Dose ( m g k P.0.)

dF 2.5 dF 5.0 IF 2.5 IF 5.0

5HIAA 74 70 93 86

f 4'

f 7' f 14 f 7

MHPG-SO4

DOPAC

96 f 4 95 f 5 137 + 14' 140 f 5'

85 f 4 89 4 126 f 9' 136 f lob

*

'5HIAA was measured 4 hr after injection and MHPG-SO4 and DOPAC, were measured 1 hr after injection of d and 1 isomers of fenfluramine. % < 0.01 vs. control (Dunnett's test).

< 0.05.

268

S. GARATTINI AND T. MENNINI

dNF adds an important component of 5HT release particularly from the extragranular 5HT pool (see Table IV). Therefore the indirect serotonergic agonist effect of dF may be sustained by different mechanisms in relation to time. 111. Drugs Acting at Monoamine Receptors

A. IMPORTANCE OF THE CHOICE OF LIGAND The advent of receptor binding assays has heralded a new approach in psychopharmacology: the labeling of receptors for neurotransmitters with radiochemical compounds at high specific activity (Snyder, 1984). However, with a few exceptons such as [3H]-5HT the molecules available for receptor binding are principally drugs believed to act selectively at a neurotransmitter receptor. Examples are antagonists for ( ~ 1 , (YZ, and P-adrenoceptors, and neuroleptics for dopamine-2 receptors. In such cases, care must be taken to establish that the binding assay does represent the receptor for the neurotransmitter and not a drug or a chemical binding site unrelated to the neurotransmitter receptor. Moreover, since the specific binding of a ligand is indirectly determined by evaluation of nonspecific binding measured in the presence of a high concentration of an unlabeled compound, the choice of this compound becomes decisive in defining the binding under study. Figure 3 presents an example of evaluation of the binding of [SH]spiperone, a spirodecanone derivative to dopamine receptors in the striatum (Seeman, 1981). If unlabeled spiperone is used the specific binding amounts to more than 90% of total binding. However, only a portion (80%) of this is shared by another neuroleptic of a different chemical class, e.g., ( + )butaclamol. This indicates that in the first case the spirodecanone binding sites are also evaluated. Moreover, spiperone and certain other neuroleptics interact with high affinity at both dopamine and 5HT receptors (Leysen et al., 1978), so stereospecific binding as defined by the difference between ( + ) and ( - )butaclamol to define “specific” binding of [3H]spiperone exclusively to dopamine receptors would clearly yield misleading results (Quick et al., 1978); more selective drugs (Quick d al., 1978;Leysen et al., 1981 ; List and Seeman, 1981) must be used to define [3H]spiperone binding to dopamine or 5HT receptors, like 2-amin0-6,7-dihydroxy-l,2,3,4-tetrahydronapthalene (ADTN) and sulpiride or ketanserine, respectively. Another example is [3H]imipramine, used to label 5HT presynaptic uptake carrier (Langer et al., 1980). 5HT inhibits [SH]imipraminebinding only by 5076, while desmethylimipramine (DMI) completely inhibits it. This

SPECIFICITY OF DRUGS IN STUDY OF MONOAMINES

269

SPECIFIC AND STEREOSELECTIVE

TOTAL BINDING

I

NONSPECIFIC, NONSATURABLE

.- - ---- --

----BINDING TO

FlLTER NONE

(-I

I+)

SPIPERONE

BUTACLAMOL IN

VITRO

ADDITION

FIG.3. Binding of [SH]spiperone to dopamine receptors in the striatum.

means that only a portion of total imipramine binding is related to the 5HT uptake site, the other being a drug binding site not located on 5HT nerve terminals. In fact, if [3H]imipiraminebinding is determined in the presence of an excess of DMI, lesion of serotonergic neurons results only in about a 40% decrease of specific imipramine binding (Mennini et al., 1986a; Severson and Wilcox, 1986). If an excess of unlabeled 5HT is used in place of DMI, about a 60 % decrease of imipramine binding is obtained by serotonergic lesions (Severson and Wilcox, 1986). Since [3H]imipramine binding has been studied in different physiological and pathological conditions, such as depression, chronic treatment with antidepressants, and aging, the choice of the drug used to define “specific” imipramine binding becomes particularly crucial in correlations with 5HT uptake. For instance, a decrease of the maximal velocity of the 5HT uptake system was reported in 24-month-old rats (Brunello et al., 1985) together with an increase of [3H]imipraminebinding sites (Brunello et a l . , 1985). The effect of aging on total [3H]imipramine binding was also reported by others and related to an increase in the myelin content of aged brain (Severson, 1986). However, when only the portion of [3H]imipramine binding related to 5HT uptake is considered, an age-related decrease in the density of the physiologically significant component of [3H]imipramine binding has been

270

S. GARATTINI A N D T. MENNINI

documented (Severson, 1986), in agreement with the age-related decrease in serotonin uptake (Brunei10 et a l . , 1985).

B. INTERACTION WITH RECEPTORS BY DRUGS WHICHAREHIGHLY METABOLIZED Most of the studies concerning interactions between drugs and receptors have been done in vitro; however, their conclusions call for a critical approach since it must be established at least whether the drug under study crosses the blood-brain barrier and whether metabolites are formed since they may have their own interactions with brain receptors or may modify the parent drug’s effect. Some examples taken from work by this laboratory help clarify these points. Buspirone and its congener gepirone are new nonbenzodiazepine anticonflict agents (Weissman et al., 1984) with reported antianxiety activity (Seidel et al., 1985). Unlike benzodiazepine (BDZ), buspirone does not interact with the GABA-CI-BDZ receptor complex (Riblet et al., 1980; Garattini et al., 1982). This fact prompted several investigators to characterize the possible “target” of buspirone activity using in uitio receptor-binding techniques (Glaser and Traber, 1983; Wood et a l . , 1983). However, the in viuo relevance of these data, summarized in Table VI, can be questioned considering the particular pharmacokinetics of the drug. After i.v. injection of 10 mgkg of buspirone to rats, the drug is rapidly cleared from blood with a t1,2 of 30 min (Caccia et a l . , 1982, 1983). After the same dose given orally, the drug is not detectable in blood or brain within limits of sensitivity of the method (Caccia et a l . , 1983). As indicated in Table VII, the route of administration of buspirone or gepirone is critical in determining the presence of the parent drug in the brain, while in any case a common metabolite 1-pyrimidylpiperazine (PmP) is present at relatively high concentrations independently of the route of administration (Caccia et al., 1982, 1983, 1985). As shown in Table VI, the profile of the interactions of PmP with brain monoamine receptors is quite different from the parent compounds buspirone or gepirone. Thus in order to extrapolate the possible mechanism of action from in vitra data, it is important to consider the effect of the metabolite on receptors and to compare these data with the brain concentrations of parent compounds after in vivo administration. Since buspirone is active in the rat conflict test at doses as low as 1 m g k g orally (Riblet et a l . , 1982), brain concentrations of the parent compound are probably sufficient to act at 5HTIA receptors but are far from being effective on the other receptor sites although drug accumulation at given subcellular sites cannot be excluded. The accumulation of PmP in the brain

271

SPECIFICITY OF DRUGS IN STUDY OF MONOAMINES TABLE VI EFFECTOF BUSPIRONE AND THEIR COMMON METABOLITE 1-PYRIMIDYLPIPERAZINE (PmP) O N DIFFERENT BINDINGSITES IN THE RAT BRAIN" Receptor 5HT1 5HTIA 5HTiB 5HTz alNA azNA

[ 'HILigand ['H]5HT ['HIPAT [125I]CYP ['HISPI ['HIKET ['H ]WB4104 ['HH]CLO ['HIYOH ['HIDHA ['HIDA [3H]SPI ['HIADTN

Buspirone 1.4 3.2 1.5 9.0 2.2 1.0 1.3 4.6 2.4 7.1 6.0

Gepirone

PrnP

x x lo-' x 10-4

4.3 x 10-6 5.2 10-7

x 10-7 x 10-6

8.0 x 6.4 x

2.7 x 3.0 x 10+ 1.1 10-5 > 10-5

x 10-6 10-5

8.0 x

x

8.0 x

x 10+ x 10-4 10-7 8.0 x

-

8.8 x

> lo-' 7.4 x 10-4 4.2 x 1.7 x

> 10-4 1.0 2.5 2.6 2.0 5.0

x x x x x

10-5

10-7 10-6 10.' > lo-' > 10-5

'Standard deviations were always lower than 15%. IC5, (M) was calculated by nonlinear fitting of curves for inhibition of specific [3H]ligand binding, using six to eight drug concentrations. The following conditions were used for receptor-binding assays: ['H]5HT 2 nMcortex 100 vol., 10 iuU 5HT for nonspecific binding; ['HIPAT 2 nM, cortex 100 vol., 10 pi4 5HT: [1z51]i~d~cyanopindolol 0.15 nM in presence of 30 @4, ( -)isoprenoline, cortex 500 vol., 10 f l 5HT; ['Hlspiperone 0.7 nM, cortex 100 vol., 1 a k e t a n s e r i n ; ['Hlketanserin 0.6 nM, cortex 200 vol., 1 ,LLV methysergide; ['H]WB4101 0.22 nM, cortex 100 vol., 100 ( -)NA: [3H]clonidine 0.4 nM, cortex 100 vol., 100 f l (-)NA: ['Hlyohirnbine 2.2 nM, cortex 200 vol., loo&( -)NA: ['H]dihydroalprenolol 1 nM, cortex 100 vol., 1 pi4 ( f)propranolol; ['Hldopamine 1 nM striaturn 400 vol., 10 (+)butaclamol; [3H]spiperone 0.22 nM, striaturn 400 vol., 1 pM ( +)butaclamol; ['HIADTN 8nM, striaturn 120 vol., 1 @4 (+)butaclarnol. Final incubation volume was 1 ml. All radiochemicals were purchased from N.E.N. Incubations were carried out for 15 min at 37% (5HT1, 5HT,A, 5HT2, DA,, and DA,), for 20 min at 25% (alNA, a2NA, and PNA), for 30 min at 23% (DA,), or for 90 rnin at 37% (5HT,B).

presumably results in concentrations more than sufficient to act at 5HTIA and particularly a2-noradrenergic receptors. The pharmacological activity may be the result of the complex interaction between parent drugs and metabolite. In fact, although drug interactions with particular subtypes of 5HT receptors have been described as producing anxiolytic activity (Glaser and Traber, 1985), an activation of 5HT receptors per se does not seem responsible for this pharmacological activity. In fact, 5HT antagonists are reportedly active in the rat conflict test (Stein et al., 1973; Geller et al., 1974; Robichaud and Sledge, 1969), while agonist result in suppression of this behavior (Stein et al., 1973) and BDZ anxiolytics do not affect serotonin metabolism (Rastogi et a/. , 1977). The same applies for the activity of PmP as an a2-noradrenoceptor antagonist (Sanghera and German, 1983), since there is a decrease, not an increase, in the output of the noradrenergic

272

S. GARATTINI A N D T. MENNINI

TABLE VII AREAL'NDER THE C U R V EOF PLASMAANII BRAINAFTER ADMINISTRATION O F RUSPIRONE O R GEPIRONF O R T H E I R COMMON METAROLITE l-~RIMlDl't.PtPERAZINE(PmP)" Plasma AUC (nmol/mllmin) D r uy and routc

Brain A U C (nmollglmin)

Parent dru!:

PmP

Parent drug

Pm P

433 532

a9 1 a3 1

1613

-

349 318 1350

-

7484

n.d.6

350

n.d.

356

n.d. n.d.

1530 1571

Intravenous Buspirone Gepirone PmP Oral Buspironr Gepirone PmP

1382

"25 pmollkg i . v . or oral 'n.d , Not detectable.

locus coeruleus with anxiolytic agents like diazepam (Sanghera and German, 1983). It is therefore possible that the combined activity of the two compounds results in the final pharmacological effect. That the effect of PmP on az-adrenoceptors is of the antagonistic type is shown by the fact that this compound antagonizes the inhibitory effect of clonidine on gastrointestinal motility and the potentiating effect of clonidine on hexobarbital sleeping time. These two effects of PmP are shared by buspirone, although this compound has no affinity for a?-adrenoceptors (see Table VI). Furthermore, PmP and buspirone counteract the lowering of brain M H P G - S O , induced by clonidine (Bianchi and Garattini, 1986) (see Table VIII), an effect completely unexpected for buspirone on the basis of its effect on monoamine receptors in vitro. Another example of differences in action of the parent drug and its metabolite is offered by the antidepressant agent trazodone and its analog etoperidone. These two drugs are metabolized with the formation of m-chlorophenylpiperazine (mCPP), a compound which accumulates in the brain (about 25 times the plasma level (Caccia et a l . , 1981b) and has 5 H T agonistic properties (Samanin d a / . , 1979). Here, therefore, is an example where the parent compound (trazodone) is considered an antiserotonin agent (Mai et al., 1979) and a metabolite showing serotoninlike activity. This discrepancy between effects can also be observed by studying the i r i teraction of the parent compounds and their common metabolite on 5 H T receptors. As indicated in Table IX, trazodone and etoperidone show higher affinity for 5HT2 than for 5 H T I receptors, while the opposite is true for mCPP (Samanin ct af., 1980). Thus when trazodone or etoperidone are administered in viuo their effect of 5 H T receptor subtype changes with the dose

SPECIFICITY OF DRUGS IN STUDY OF MONOAMINES

273

TABLE VIII EFFECT OF BUSPIRONE AND PmP ON CLONIDINE-INDUCED DECREASE OF CORTICAL LEVELSOF TOTAL MHPG IN RATSa

Treatment

MHPG (nmol/g)

Saline PmP Clonidine PmP and clonidine

0.49 0.67 0.31

Saline Buspirone Clonidine Buspirone and clonidine

0.56 f 0.04 0.73 f O . O l b 0.37 f 0.05b 0.59 f 0.04

f

0.04

* 0.04' * 0.02' 0.46 * 0.02

'TmP (2 mgkg i.p.) was given 10 min and buspirone (15 mgkg i.p.) 30 min before clonidine (0.1 mgkg s.c). MHPG levels were measured 3 hr after clonidine. Results are the means S.E. of four to six animals. 'p < 0.05 vs. saline by Dunnett's test.

*

and with the time after their administration in relation to the formation of their metabolite mCPP. This discussion could be extended to other drugs as shown in Table X, which reports that a number of drugs form an arylpiperazine metabolite which accumulates in brain, where it may interact with monoamine receptors in a different way from the parent compound (Caccia et al., 1984).

C. BRAINREGIONAL DIFFERENCES Often an interaction observed with a given receptor in the brain is extrapolated to all the brain areas or the effect observed in a given brain area is TABLE IX EFFECTS OF TRAZOWNE, ETOPERIDONE, AND mCPP ON 5HT RECEFTORSUBTYPES" Nanomolar ICSOon Compound

5HTI

5HTa

Ratio of 5HTJ5HTi

Trazodone Etoperidone mCPP

500 3000 200

30 120 400

0.06 0.04 2.00

"5HTI and 5HTa receptor binding was determined in rat cortex using, respectively, [3H]5HT and ['Hlspiperone.

274

S. GARATTINI AND T. MENNINI TABLE X SOMEDRUGSWHICHAREMETABOLIZED I N RATSWITH OF AN ARVLPIPERAZINE

THE

FORMATION

Drug

Metabolite present in brain“

Interaction of the metabolite with monoamine receptors

Tr az odon e Etoperidone Mepiprazole Buspirone Gepirone Piribedil Methopertone Urapidil Emiprazine Niaprazine Eupiprazole Antraferine Oxypertine Azaperone

mCPP mCPP mCPP PmP PmP PmP oMPP oMPP oMPP pFPP OCPP mTPP PP PYP

5HT1 5HT1 5HTI cr2-Adrenoceptors az-Adrenoceptors cr1-Adrenoceptors 5HT1 5HT1 5HTI 5HT1 5HT1 5HT, a*-Adrenoceptors al-Adrenoceptors

“mCPP, I-(m-chloropheny1)piperazine;PmP, 1-(2-pyrimidyl)piperazine; oMPP, 1-(o-methoxypheny1)piperazine;pFPP, 1-(p-5uomphenyl)piperazine; oCPP, 1-(o-chloropheny1)piperazine;mTPP, 1-(m-trifluoromethylphenyllpiperazine; PP, 1-phenylpiperazine; Py P, 1-(2-pyridy1)piperazine.

“generalized” to other brain regions. This is not always correct. Studies of the mechanism of action of buspirone and gepirone have in fact shown regional differences. The affinity of the two drugs for 5HT1 receptors markedly differs if one considersjust three brain regions (Mennini d af., 1986b), being higher in the hippocampus, intermediate in the cortex, andlowest in the striatum (Table XI). This may be due to the regional heterogeneity of 5HT receptor subtypes or their environment to which the drugs may bind allosterically. Considering the in vivo concentrations of buspirone and gepirone after oral administration, the drugs seem to act on 5HT receptors in hippocampus but not in striatum. This is confirmed in vivo, since a dose-dependent decrease of 5HIAA can be obtained in hippocampus (Mennini et al., 1986b) but not in striatum (Cimino et al., 1983) of buspironetreated rats. In the case of the common metabolite PmP the interaction with 5HT1 receptors shows much less affinity and is fairly homogeneous in the three brain areas considered.

D. SPECIES DIFFERENCES IN BRAINRECEPTOR SUBTYPES The existence of multiple serotonin receptors is postulated principally on the basis of radioligand-binding experiments. Alternative interpretation, like the

TABLE XI INHIBITION OF [3H]5HT BINDING I N THREE RATREGIONS“ Hippocampus Drug Buspirone Gepirone PmP

Ki

(4

3.4 f 0.7 x lo-’ 6.1 f 1.1 x lo-’ 1.1 f 0.1 x 10+

Cortex

K . (W

Slope

0.6 f O.lb 0.8 + 0.1 0.9 f 0.1

1.0 3.1 1.9

f f

f

0.3 x lo-‘ 0.6 x 0.7 x

Striatum Slope

0.5 0.5 0.9

O.lb 0.lb f 0.6 f f

Kc

(4

2.0 f 1.1 x lo-’ 1.7 i 0.3 x 1.1 f 0.2 x

Slope

0.5 0.8 0.8

O.lb 0.1 f 0.1 f f

“[3H]5HTconcentration was 2 nM; nonspecific binding was determined in the presence of 10 pA4 unlabeled 5HT, 8-10 drug concentrations were used for each curve The values were obtained as parameters ( f SD) analyzing the displacing curves with the logistic function. bp < 0.01 different from 5HT slope in the same area (Student’s t test)

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existance of allosteric sites near the 5HT receptors, could equally fit such complex inhibition curves of the labeled ligand (Gobbi et al., 1986; Rothman, 1986; Rothman et af., 1983). However, these reports clearly indicate the possibility that drugs (and their metabolites) interact with 5 H T receptors in a heterogeneous way, having different affinities in different brain regions and distinguishing between pre- and postsynaptic serotonin receptors (Verge et al. , 1985). Peroutka and Snyder (1979) described the existence of 5HT1 and 5HT2 receptors in rat brain, having preferential affinities for serotonin agonists and antagonists, respectively. In 1981 Pedigo et ai. introduced the concept of 5HT1A and 5HTlB receptors, discriminated in rat cortex by spiperone and other neuroleptics. Recently the presence of 5HT1C subtypes in the pig choroid plexus was described (Pazos et al., 1984a). The drug R U 24969 displays nanomolar affinity for 5HTIA and even higher affinity at non5HT1A sites present only in rats and mouse brain, but is weakest at a second component of non-5HT1A sites which can be identified in all regions and species analyzed (Heuring et af., 1986). Thus, the non-5HT1A sites displaying high affinity for R U 24969 appear to label a molecular form of 5HT receptors (5HTIB) that is unique to rats and mouse brain. Behavioral metabolic and physiological effects of R U 24969 in the rat and mouse may be irrelevant to its effects in other species. Thus it becomes particularly important to characterize human brain receptors before extrapolation can be made from laboratory animal to human pharmacology. The pharmacological characterization of 5HT2 receptors in human brain clearly indicates that these sites are different from rat brain 5HT2 receptors and closer to cat and pig brain receptors (Pazos et al., 1984b). Some drugs, particularly spiperone, piremperone, ketanserin, mianserin, and ( + )butaclamol showed similar affinities in the four species considered. In contrast, drugs such as mesulergine, methysergide, cinanserin, and dLSD behaved differently in displacing [3H]ketanserin from 5HT2 binding sites in rat, pig, and human cortices. The differences between rat and pig were particularly marked when [3H]mesulergine was used as ligand. A much lower density of sites was found in both pig and human brain, although the affinity of the sites labeled in the three species was similar. 5HT, which had a low affinity for 5HTz sites, showed good affinity for [3H]mesulergine binding sites, suggesting that the sites labeled in pig and human brain by mesulergine are not the 5HTz receptors, but have the characteristics of the 5HT1C receptors (Pazos et al., 198413). IV. Concluding Remarks

It is customary to characterize drugs acting on the central nervous system on the basis of their interaction with monoamines, particularly in relation to

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uptake, release, and receptors. These studies are mostly done in vitro and there is the tendency to extrapolate the findings to their effects in vivo. This frequently leads to a somewhat oversimplified classification of drugs. In some cases, when drugs show a selective effect in vitro toward a given monoaminergic system, they are utilized as tool drugs in pharmacological studies with the aim of detecting whether that monoaminergic system is important in explaining the action of a new drug. This review has given examples to show how cautious one must be in the use of this approach. In order to utilize correctly a drug as an investigative tool it is important to detect whether the drug passes the blood-brain barrier. In addition brain regional distribution should be studied in order to obtain quantitative data relating the dose(s) employed to given brain concentration(s). Knowledge of these concentrations is essential to establish whether an effect observed in vitro is likely to occur in vivo. Furthermore, since several psychotropic drugs are highly metabolized, the chemical nature of the major metabolites must be established; as shown in this review some metabolites may not be detectable in blood or urine, while they nevertheless accumulate in the brain. These metabolites must therefore be studied for their effects on brain monoaminergic systems; some may be inactive but some may show activity similar to the parent compound and in some cases may be different or even antagonistic in relation to the administered drug. Thus the effect in vivo may become very complex because of these different effects, which may be further complicated by differences arising in relation to the animal species and the route of administration. Regional differences present in the various brain areas must also be taken into consideration. In conclusion, there is a need for a more critical analysis of the activity of psychotropic drugs where kinetic and metabolic studies prerformed in brain are integrated with the central biochemical and pharmacological effects.

References

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Caccia, S., Cecchetti, G., Garattini, S., and Mennini, T. (1973). Br. J . Pharmacol. 4 9 , 400-406. Caccia, S., Dagnino, G., Garattini, S., Guiso, G., Madonna, R., and Zanini, M. G. (1981a). Eur. J . Drug Mctab. Pharmacokinct. 6 , 297-301. Caccia, S., Ballabio, M., Samanin, R., Zanini, M . G., and Garattini, S. (1981b). J. Phann. Pharmacol. 33, 477-478. Caccia, S., Garattini, S., Mancinelli, A., and Muglia, M . (1982).J. Chromatogr. 252, 310-314. Caccia, S., Muglia, M., Mancinelli, A,, and Garattini, S. (1983). Xenobiotica 13, 147-153. Caccia, S., Notarnicola, A,, Fong, M . H., and Benfenati, E. (1984). J . Chromatogr. 283, 2 1 1-221. Caccia, S., Fong, M. H., and Guiso, G. (1985). Xenobiotica 15, 835-844. Carlsson, A., Fuxe, K . , Hamberger, B., and Lindqvist, M . (1966). Acta Physiol. Scand. 67, 48 1-497. Carlsson, A , , Corrodi, H., Fuxe, K., and Hokfelt, T . (1969). Eur. J . Pharmacol. 5 , 367-373. Ceci, A., Garattini, S., Gobbi, M., Mennini, T . (1986). Br. J . Phannacol. 88, 269-275. Cho, A. K., Hodshon, B. J., Lindeke, B., and Miwa, G . T . (1973). J . Phann. Sci. 62, 1491-1494. Cimino, M . , Ponzio, F., Achilli, G . , Vantini, G., Perego, C., Algeri, S., and Garattini, S. (1983). Biochem. Phannacol. 32, 1069-1074. Consolo, S., and Garattini. S. (1969). Eur. J . Pharmacol. 6 , 322-326. Consolo, S., Dolfini, E., Garattini, S., and Valzelli, L. (1967).J. Pharm. Pharmacol. 19, 253-256. Consolo, S., Ladinsky, H., and Garattini, S. (1974). J . Phann. Pharmacol. 26, 275-277. Consolo, S . , Ladinsky, H., Bianchi, S., and Ghezzi, D. (1978). Brain Res. 135, 255-263. De Simoni, M . G., Dal Toso, G., Algeri, S., and Ponzio, F. (1986). Eur. J . Phannacol. 123, 433-439. Dingell, J. V . , and Bass, A. D. (1969). Biochem Phamurcol. 8, 1535-1538. Dolfini, E., Garattini, S., and Valzetti, L. (1969a). Eur. J . Phamtocol. 7, 220-223. Dolfini, E., Garattini, S., and Valzelli, L. (196913). J . Phannacol. 21, 871-872. Dolfini, E . , Tansella, M., Valzelli, L., and Garattini, S. (1969~).Eur. J . Pharmacol. 5 , 15-190. Dring, L. G., Smith, R. L., and Williams, R. T. (1966).J. Phannacol. 18, 402-404. Garattini, S. (1969). In “Importance of Fundamental Principles in Drug Evaluation” (D. H . Tedeschi and R. E. Tedeschi, eds.), pp. 129-139. Raven, New York. Garattini, S., Borroni, E., Mennini, T., and Samanin, R . (1978). In “Central Mechanisms of Anorectic Drugs” (S. Garattini and R. Samanin, eds.), pp. 127-143. Raven, New York. Garattini, S., Jori, A., Manara, L., and Samanin, R . (1975). In “Chemical Tools in Catecholamine Research” (G. Jonsson, T. Malforms, and Ch. Sachs, eds.), Vol. 1, pp. 303-309. North-Holland Publ., Amsterdam. Garattini, A., Jori, A , , and Samanin, R . (1976). Ann. N . Y. Acad. Sci. 281, 409-425. Garattini, S . , Caccia, S., Mennini, T., Samanin, R., Consolo, S., and Ladinsky, H . (1979). Curr. Med. Res. Opin. 6 (Suppl. I ) , 15-27. Garattini, S., Caccia, S., and Mennini, T. (1982).J. Clin. Psychiatr. 43, 19-22. Garattini, S., Mennini, T . , and Ponzio, F. (1986). Meet. DopamineDepressione, Oct. 1984, Rome, in press. Geller, I., Hartman, R. J., and Croy, D. J . (1974). Res. Commun. Chem. Pathol. Phannacol. 7, 165-174. Glaser, T., and Traber, J. (1983). Eur. J. Pharmacol. 88, 137-138. Glaser, T., and Traber, J. (1985). Naunyn-Schmicdebng’s Arch. Pharmacol. 329, 211-215. Gobbi, M . , Barone, D., Dagnino, G., Verotta, D., and Mennini, T. (1986).J. Recept. Res. 6, 27-46. Heuring, R . E., Schlegel, J . R., and Peroutka, S. J. (1986). Eur. J . Phannacol. 122, 279-282. Invernizzi, R . , Berettera, C., Garattini, S., and Samanin, R . (1986). Eur. J . Pharmacol. 120, 9-15.

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RETINAL TRANSPLANTS AND OPTIC NERVE BRIDGES: POSSIBLE STRATEGIES FOR VISUAL RECOVERY AS A RESULT OF TRAUMA OR DISEASE By James E. Turner, Jerry R. Blair, Magdalene Seiler, Robert Aramant, Thomas W. Laedtke, E. Thomas Chappell, and Lauren Clarkson Department of Anatomy Bowman Gray School of Medicine Wake Forest University Winston-Salem, North Carolina 27103

I . Introduction 11. Intraocular Retinal Transplantation A. Retinal Transplantation into the Anterior Chamber B. Vitreal Chamber Grafts: Transplantation into an Adult Retinal Lesion Site 111. Retinal Pigment Epithelium Grafted onto Bruch’s Membrane IV. Retinal Tissue Transplanted into the Central Nervous System A. Introduction B. Methods C. Graft Differentiation D. Connections to Host Brain E. Functional Significance V. Peripheral Nerve Bridges and Optic Nerve Grafts: Enhancement of Retinal Ganglion Cell Axon Regeneration A. Introduction B. Peripheral Nerve Bridges C. Target Tissue Connections VI. Summary References

1. lntroductlon

Intracerebral, brain stem, and spinal cord grafting of embryonic neural tissue in mammals has recently become a valuable tool for the analysis of neuronal growth and differentiation, as well as for studies on neuronal regeneration and the formation of new connections in the central nervous system (Bjorklund and Stenevi, 1984). In particular this approach may have considerable potential for reconstructive replacement of damaged neuronal tissue within the brain, spinal cord, and visual system. Central nervous system (CNS) grafting has been successful because the brain, spinal cord, eye, and 281 INTERNATIONAL REVIEW OF NEUROBIOLOGY, VOL. 29

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optic nerve have no lymphatic systems. In addition, the blood-brain barrier keeps immune cells out of the CNS, thus making these areas immunologically privileged sites. Of all the areas in the body, the CNS is least likely to reject a graft of foreign tissue. Embryonic grafts placed into the adult CNS have not only survived, matured, and established new connections, but have been shown to produce neurotransmitters such as dopamine, acetylcholine, norepinephrine, and serotonin and the hormones vasopressin and gonatotropin-releasing hormone (Kolota, 1983; Roberts, 1983). It is not too surprising that brain grafts have corrected experimentally induced motor deficits, memory losses, and hormone deficiencies by either secretion and diffusion of substances from the graft or by reinnervation of denervated areas by the graft (Arendash and Gorski, 1982; Froy et al., 1983; Labbe et a l . , 1983; Roberts, 1983). Grafting studies of a different nature involving the implantation of peripheral nerve segments into the CNS have refuted the dogma that these neurons are incapable of axonal regeneration. Through an elegant series of experiments it was demonstrated that lesioned adult mammalian CNS neurons from many diverse areas could be induced to regenerate their axons considerable distances in uivo through peripheral nerve bridges inserted into these regions (Aguayo et a l . , 1981; Benfry and Aguayo, 1982; David and Aguayo, 1981). Therefore, the peripheral nerve appears to offer the appropriate environment for the stimulation of CNS regeneration and may serve to reroute lesioned axons from terrain normally found inhospitable to such growth. In vertebrates such as bony fishes, amphibians, birds, and mammals, the neural retina can regenerate from pigmented epithelium early in embryonic life (Columbre and Columbre, 1970). This capacity persists throughout life in some amphibians and is maintained until metamorphosis in some tailless varieties (Columbre and Columbre, 1970). However, adult mammalian vertebrates are not endowed with the capacity to regenerate the retina at the histological level or organization. The only exception is the regeneration of photoreceptor outer segments under certain lesion conditions (Blight and Hart, 1977). In other words, the adult mammalian retina, like many other areas of the CNS, will not by itself regenerate in response to a lesion or diseased state (Foulds, 1979). Under these conditions and the realization of today’s modern science, there appear on the horizon several means by which the damaged retina and optic nerve may be repaired to initiate return of visual function: (1) by replacing lost retinal tissue or several of its individual cell populations through a grafting technique, (2) by rescuing retinal cells and eliciting a would-healing response through the application of a neurotrophic stimulus, and (3) by stimulating the regeneration of damaged retinal ganglion cell axons through a supportive environment to their appropriate visual

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centers, where they make meaningful synapses. This article attempts to review some of the historical as well as the current literature just emerging which begins to offer possibilities for development of future strategies for visual recovery as a result of trauma or disease.

II. lntraocular Retlnal Transplantation

A. RETINAL TRANSPLANTATION INTO THE ANTERIOR CHAMBER The mammalian eye, principally the anterior chamber, has been a favorite site for the placement of a vast array of tissues for over 100 years. Possibly the earliest recorded attempt of an in oculo graft was that of Van Dooremal in 1873. However, there was no attempts made to transplant mammalian retina to the eye until 1959, when Roy0 and Quay placed embryonic rat retina into the anterior chamber of the maternal rat’s eye. After this single set of experiments by Roy0 and Quay, a quarter of a century would pass before their work would be repeated and extended by del Cerro et al. (1985a). Roy0 and Quay’s initial studies established that the immature retina would indeed survive and continue to develop when placed in the anterior chamber of the maternal rat’s eye. Additionally, they reported that the grafted cells often migrated from the anterior chamber to the posterior chamber. This was followed by migration of certain elements, considered by Roy0 and Quay to be retinal ganglion cells, posterior to the lens and through the empty hyaloid canal to contact the regenerating hyaloid artery at the optic disk. The initial observations made by Roy0 and Quay in reference to survival and continued differentiation of immature retinal grafts to the anterior chamber of the eye were supported by the recent study by del Cerro et al. (1985a). However, they did not report the same migratory phenomenon observed by Roy0 and Quay. This difference in results may have arisen from differences in experimental design and methods utilized in the two grafting studies. Roy0 and Quay most likely included connective tissue elements in their grafts which may have contributed to the greater migration of graft elements. Unfortunately, the identity of the migrating elements was not indisputably established as being either of neuronal or of connective tissue origin. In addition the study by del Cerro et al. (1985a) extended the earlier anterior chamber grafting paradigm to include hosts from different strains of rats (Lewis and Fischer) than the source of the donor tissue (Long Evans). The

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study also provided transmission electron micrographs (TEMs) of select structures in the retinal grafts which were comparable to those observed in the normal adult retina. These electron micrographs revealed the presence of cell types found in the normal adult retina as well as synapses typical of contacts between neural elements of the retina including characteristic ribbon synapses. They also reported the close apposition of the graft tissue to elements of the host cornea and iris. Consequently, there exist a paucity of investigations dealing with retinal grafts to the anterior chamber of the eye, which is surprising when one considers the great number of studies that have dealt with the placement of more ectopic tissues to this same site (Greene, 1967). However, even more surprising is the lack of a body of literature dealing with retinal transplantation to the vitreal chamber of the mammalian eye, the normal in uiuo environment for retinal tissue.

B. VITREAL CHAMBER GRAFTS: TRANSPLANTATION INTO AN ADULTRETINAL LESION SITE Neuronal transplantation work is directed toward the study of the development of the tissue in question or its possible use in alleviation of clinical deficits or both. In such cases investigation should move toward study of tissues in the environment in which they have the maximum opportunity for good survival, normal development, and reestablishment of lost function. For the retina this points to the placement of retinal grafts in the normal retinal environment, which is within the vitreal chamber of the eye. There are studies that suggest that the vitreal environment may be unique even from that of the anterior chamber and that the retina itself itself may elucidate substances which are supportive of neuronal survival and growth (Politis, 1986). A few studies have been published dealing with the placement of ectopic tissues into the vitreal chamber to examine the trophic influences of these tissues on the damaged retina (Stenevi and Bjorklund, 1978; Turner et al., 1986). A recently developed model has been described which begins to deal with retinal grafts as a means for repair of the damaged adult mammalian retina (Turner and Blair, 1986). In addition, there are several published abstracts that allude to this possibility (del Cerro et a/. , 1985b, 1986). The results of this previous study, taken in combination with those reported in anterior chamber studies, suggest that the immature retina is a very plastic tissue capable of surviving the stresses of the transplantation procedure over a wide range of conditions. This plasticity, together with the observations that grafts develop rather normal laminar and synaptic architecture, while readily integrating with the damaged adult retina, supports the

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plausibility of using such a paradigm for the repair of diseased or damaged retina in humans. This recent study (Turner and Blair, 1986) showed that very good survival could be achieved with retinal grafts, > 90 5% , and that they would continue to develop in an essentially normal manner both at the histological and ultrastructural levels (Figs. 1 and 2). However, this study did not rely upon embryonic donor tissue, which was required for the anterior chamber work as well as for retinal grafting to more ectopic sites (see Sections II,A and IV,B). Rather, this study was able to achieve the reported results using postnatal tissues, 1- and 2-day neonatal retina. This also suggests that the host vitrealretinal environment may present a uniquely supportive milieu for retinal grafts. These grafts readily filled the lesion gap (Fig. 3A-F) bridging the cut edges of the lesioned host retina (Figs. 1C and E and 4A-F). At points of contact between host and graft retinal tissues the plexiform and, at times, cellular layers of the two readily integrated without any obvious barriers separating them (Fig. 4B-F). Transmission electron micrographs revealed all the normal cellular constituents to be present in the graft along with normal synaptic configurations (Fig. 2A-H). Scanning electron microscopy (SEM) of the grafts in situ revealed the dense population of neuronallike cells occupying the ganglion cell layer (GCL) of the graft and extending processes which collected on the vitreal surface of the graft (Fig. 5A-C). These fibers left the graft as fascicles or sheets to contact and course along the vitreal surface of the host retina for up to 2 mm before penetrating into the optic fiber layer (OFL) of the host as revealed by light microscopy and SEM (Figs. 5D and 6A-E). Studies recently conducted in this laboratory (Blair and Turner, 1986) have begun to further establish the parameters within which retinal grafting to the lesioned adult retina can occur. These studies have examined the role of the lesion environment as well as providing a more detailed analysis of donor age influence on the ultimate success of the grafting paradigm. It was observed that neonatal graft tissue survived ( > 93 5%) and fused ( > 81%) in the host lesion site regardless of the length of the lesion conditioning period prior to graft delivery of from 0 to 8 weeks (Table I). There were no significant differences observed among the different lesion groups utilizing statistical analysis of an evaluation index based on select histological characteristics. The only minor differences observed were related to reduced barrier formation between graft and host tissue in the more stable 4- and 8-week groups (Fig. 7). These results revealed the receptive nature of the damaged adult retina over an extended posttraumatic period. Even more flexibility was added to the model through the donor age studies. While the majority of our work has dealt with neonatal retinal grafts, there existed a real need for contrasting studies utilizing donor tissue of varying degrees of maturity. As a result studies were conducted in which retinal

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af

FIG. 1. Photomicrographs showing newborn retinal graft survival and continual cell differentiation for up to 4 weeks in the host lesion sites. (A) Graft (G) 1 week after implantation into the vicinity of the host lesion site (L) ( x 74). (B) Higher magnification of (A) showing that the graft at 1 week after implantation comprises mostly differentiating cells in what was the neuroepithelial layer. Developing areas of the outer plexiform layer (OPL) and cell layers have not yet emerged except for an apparent retinal ganglion cell layer (GCL) and a small inner plexiform layer (IPL) ( x 186). (C) Graft (G) 2 weeks after implantation showing increased appearance of plexiform layers and a healthy population of differentiating cells ( x 74). (D) Higher magnification of (C) showing that the neuroepithelial layer is beginning to be segregated into ONL and INL ( x 186). (E) Graft (G) 4 weeks after implantation showing signs of histotypic differentiation into adult characteristics. Compare general area 1 of the graft with area 2 of the intact host retina for similarities ( x74). (F) High magnification of (E) showing the full extent of the graft differentiation process into outer nuclear layer (ONL), OPL, inner nuclear layer (INL), IPL, and GCL. Also note indications of an outer limiting membrane (OLM) ( x 186).

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tissue from E14, E16, E20, PN1, and PNlO donors was transplanted to a fresh lesion site in adult hosts. Again, amazing flexibility was revealed to be a characteristic of the immature retina in this model. Retinal grafts from all groups exhibited excellent survival ( > 97 %) and fusion ( >95 %) in the lesion site (Table I). These results are to some degree in contrast to those reported by other investigators dealing with other portions of the CNS (Bjorklund and Stenevi, 1984) or with transplantation of the retina to other sites (McLoon and McLoon, 1984). Statistical analysis of the data collected using an evaluation index revealed significant differences between PNlO grafts and all other groups in several histological categories, even though no contrasts were revealed by the survival and fusion data (Fig. 8). The most critical points of consideration are the lack of GCL components and the preponderance of outer nuclear layer (ONL) elements in the PNlO grafts in contrast to the other groups. This difference very likely arises from the formation and consolidation of synaptic connections by ganglion cells and INL neurons of the PNlO grafts prior to their being collected for grafting. This might result in making these populations less able to withstand the rigors of grafting. In the same light, the photoreceptors are among the last neurons to be born and mature in the retina, thus conserving their ability to survive the procedure when harvested from donors older than PN 1 or PN2. The recent studies by del Cerro d al. (1985a) as well as our own studies have firmly established the capacity of immature mammalian retina to survive and continue to develop when utilized for in oculo grafting. However, if these grafts are to have any significance in a clinical setting, certain critical cell populations must survive and proper relationships between those populations must develop. This holds for populations both in the graft itself and in the host retina. Among the most functionally significant of these cell populations are the retinal ganglion cells. Without the ganglion cells, which are the only output cells from the retina to higher visual centers, there would be a little hope of reestablishing lost visual function due to trauma, disease, or inherited disorders except through lateral graftlhost interactions within the retina. Although light and electron microscopic evidence supports the presence of these cells in the grafts, no studies have been performed so far which utilize markers specific for retinal ganglion cells. This situation has recently been addressed through preliminary studies in our laboratory utilizing an antibody to a cell-surface glycoprotein restricted in the mammalian retina to only ganglion cells (Beale and Osborne, 1982; Barnstable and Drager, 1984). This monoclonal antibody (OX-7, Sera Labs) is specific for the Thy-1.1 allele which is found in all rat strains tested to date (Morris, 1985). The antibody was used in conjunction with the ABC method to localize the antigen in both control and grafted retinal tissue. The result was very specific labeling of OFL, GCL, and inner plexiform layer (IPL) of the host retina as previously reported by other investigators. A similar pattern of labeling was seen utilizing tissue from 12-week-old grafts. In addition, clear

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FIG. 2. Transmission electron micrographs of portions of all the layers of a retinal graft 4 weeks after transplantation. (A) The outermost surface of the retinoid showing healthy, intact ganglion layer cells (GLC). As indicated by scanning electron micrographs, there is an absence of a continuous optic fiber layer (OFL) and outer limiting membrane. The darker appearing cells (N) may be neuroepithelial cells or developing glia. Bar, 2 pm. (B) A high-magnification micrograph of the inner plexiform layer (IPL) showing a typical ribbon (arrowhead) synapse of a developing dyad apparently consisting of a bipolar cell segment (B) contacting two poafsynaptic elements (P) which may be amacrine and ganglion cell dendrites. Bar, 1 pm. (C) Higher magnification of the IPL showing a conventional synapse with distinguishable synaptic vesicles (arrowheads) in the presynaptic element. Bar, 1 pm. (D) A higher magnification electron micrograph of the outer plexiform layer (OPL) showing a portion of a typical ribbon (arrowhead) synapse of a developing triad consisting of photoreceptor (P), horizontal (H), and bipolar (B) cell processes. Bar, 1 pm. (E) A micrograph demonstrating a portion of the inner nuclear layer (INL) with its varied cell types. Bar, 2 pm. (F) A section of the graft showing differentiating photoreceptor cell nuclei (P) as they aggregate about a luminal surface filled with microvilli (M), to form a

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FIG. 3. Photomicrographs depicting the temporal condition of the retinal lesion site without a gr& for up to 3 months after initial trauma. V, Vitreous body; Sc, sdera; L, lesion site; S, suture. (A) Retinal lesion site 1 week after transection. Note that between the cut retinal tips (arrows) there is an intense RPE phagocytic response (P) which essentially fills the die-back zone (between arrows). (B) Retinal lesion site 2 weeks after transection. Note the greatly diminished cellular activity in the die-back zone (between arrows). (C) The lesioned retina at 3 weeks after transection showing the die-back zone clear of cellular debris and activity (between arrows). (D-F) The dieback zone depicted at 5 (D), 8 (E), and 16 (F) weeks after transection showing no migration of the host retinal cells into the lesion site from the cut edges. All plates x 74. rosette. Note the presence of an outer limiting-like membrane (arrowheads). Bar, 2 pm. (G) Higher magnification of (F) showing the junctional complexes of the outer limiting-like membrane (arrowheads) and microvilli (M) protruding into the lumen of a rosette. Bar, 1 pm. (H) A micrograph of a portion of the distal end of an inner segment (P) of a developing photoreceptor cell with its cilium (C) protruding into the luminal space of a rosette. Bar, 0.5 pm.

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FIG 4.A series of photomicrographs taken at 4 weeks after newborn grafts were implanted into a 5-week-old retinal lesion site. This series shows that the newborn retinas can be grafted to the lesion site. In many instances the graft will bridge the cut edges of the retina by a merging of plexiform and possibly cellular layers. (A) The leading edge ofa retinal graft (G) placed within the lesion site between the cut edges of the retina (arrows). Note that a clear distinction can be made between host and graft tissue ( x 73). (B) A greater portion of the graft (G) seen 150 pm from (A), further into the lesion area. Note that as in (A), a clear delineation between host retinal tips (arrows) and graft tissue is visible ( x 9 3 ) . (C) Higher magnification of (B), showing the retinal bridging phenomena more clearly. Note that host and graft plexiform layers have merged (arrow) ( x 182). (D) A photomicrograph of another host-graft integration phenomenon at 1 week after implantation into the old lesion. Note areas of obvious merging between host and graft plexiform

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FIG 5. Scanning electron micrographs showing cellular features on the retinal graft surface as well as details of host-graft fiber integration on the host vitreal surface 4 weeks after implantation. (A) Micrograph of a retinal graft (G) showing the absence of both an inner limiting membrane and a continuous optic fiber layer which allows for direct observation of the ganglion layer cells (GLC) and the inner boundary of the inner plexiform layer (IPL). Bar, 3 0 pm. (B) Higher magnification of graft in (A). Note the cablelike bundle of fibers (arrowheads) coursing across the graft surface. Bar, 8 pm. (C) Higher magnification of the graft seen in (A) and (B) above. Note the shapes of distinct GLC soma, the fibrous nature of the IPL, and the bulbous enlargements possibly corresponding to synaptic terminals (ST)both within the IPL and on the surface of cells in the GLC (arrowheads). Bar, 1.5 pm. (D) Micrograph showing an area of host-graft optic fibers integration on the host vitreal surface. The fascicles of graft fibers (GF) course diagonally and those of the host OFL (HF) course predominately in the horizontal direction. Note the branching of GFs which join with the HFs to run with them in the horizontal direction (arrowheads). Bar, 1.5 pm. regions at the cut retinal tips (arrows) ( x 73). (E) Higher magnification of (D) showing merging of plexiform layers in greater detail (arrow) ( x 290). (F) A photomicrograph showing that retinal grafts (G) are capable of merging with intact host retina (H) outside of the lesion area. At 2 weeks after implantation graft and host are seen to merge sharing the same IPLs (P) ( x 73).

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FIG.6. Photomicrographs of fiber bundles extending from the newborn retinal grafts and their association with the optic fiber layer (OF) of the host retina 4 weeks after implantation.(A-D) Light micrographsof three different retinal grafts (G) showing the fasciculation ofprocesses (arrowheads)

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' + SEM. dAnalysiz,by chi-square test for several proportions: no significant differences found among the groups in any column.

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FIG. 8. Graph comparing the evaluation index ( E L ) scores of E14, E16, E20, PN1, and PNlO retinal grafts placed in fresh (unconditioned) lesion sites. All tissues were collected at a time equivalent to 4 weeks postnatal in order to match the developmental ages of the grafts. The grafts were analyzed for differences in group mean E.I. score (A), graft survival (B), graft lamination (C), overall graft organization (D), presence of nonneuronal barriers (E), and lesion repair by the graft (F). Analysis of the group mean E.I. scores revealed a significant reduction in the E.I. score of only the PNlO grafts (p < .001).In addition, evaluation of the individual criterion scores (B-F) showed significant differences for the PNlO grafts (p < .03) for all criteria except that of nonneuronal barriers. Vertical lines represent the SEM. Numbers in parentheses indicate number of grafts analyzed for each group.

staining of ganglion cells was seen in host retina as well as in graft tissue. These preliminary results strongly indicate the presence of ganglion cells within retinal graft tissue, thereby opening the door for further investigation

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01.

dealing with communication between graft and host tissues and the functional state of these ganglion cells.

ill. Retlnal Pigment Eplthellum Grafted onto Bruch’s Membrane

Transplantation of cultured retinal pigment epithelium (RPE) to a denuded area of Bruch’s membrane is a relatively new investigative paradigm. To date all of the literature in this area has been derived from the work of Peter Gouras and his colleagues. This group first reported in 1983 that RPE cultured from postmortem human donors could be pipetted directly over an area of Bruch’s membrane in a monkey host (Gouras et al., 1983). The procedure involved an anterior “Open Sky” approach described in detail by Gouras et al. (1984). Briefly, the cornea of an anesthetized owl monkey is incised along 250 degrees of its perimeter and folded over saline gauze. The lens and liquid vitreous are then removed, providing an adequate view of the retina. A 0.5-cm2 area of the retina is then cauterized, cut on three sides, and folded back until the experiment is complete. At this point several drops of CaZ+-Mg2+-free Hank’s solution with EDTA and trypsin can be pipetted onto the RPE for approximately half an hour. Afterward the RPE can be gently aspirated off of Bruch’s membrane. The experimental group of animals then receive cultured RPE over the denuded Bruch’s membrane. In one control group the host epithelial layer is left intact and in another control group it is removed, but neither receive transplanted tissue. The results demonstrated that the human RPE cells attach to the denuded surface within 2 hr of transplantation and forn an epithelial-like monolayer that adheres closely to Bruch’s membrane. Since the donor cells had been subcultured in tritiated thymidine, autoradiography left no doubt that the cells under observation were those cultured from human donors. Furthermore, the transplanted cells have the same characteristics in the graft site as they do on the culture plate with the exception that they adhere more tightly to the former. Specifically, they maintain their apical-basal polarity, exhibit close contact with neighboring cells, and have the same cytoplasmic structure as in vitro. If the cells are allowed to incubate for several days, they eventually begin to behave like host RPE cells beneath an area of detached retina. These changes have been described in monkey (Machemer, 1968; Kroll and Machemer, 1968, 1969). Namely, the apical surface begins to bulge into the subretinal space and other cells from the same area migrate away from Bruch’s membrane and extended processes over the apical surfaces of adjacent cells. Although macrophages accumulate in and around the transplant with time, the authors did not feel that grdft rejection would be a major hurdle for

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this research. Instead, they are more concerned with whether the neural retina can be reapposed on the graft site and whether this will prevent the cellular proliferation seen both in the transplant situation and in the case of simple retinal detachment. In an effort to more easily and effectively accomplish reapposition of the detached retina, Gouras and his co-workers developed a closed-eye technique which they reported recently in a rabbit model (Lopez tt al., 1986). Using a 30-gauge cannula inserted into the vitreous through a pars plana incision, the authors are able to create a bleb detachment by injecting 0.1 cc of balanced salt solution. The host RPE can then be removed using the diamond-dusted undersurface and rabbit RPE cultured in tritiated thymidine can then be injected onto the denuded Bruch’s membrane. Within 24 hr the retina has become reapposed and intact outer segments can be seen within 0.1 mm of transplanted RPE cells that have attached to Bruch’s membrane. Based on the findings of Marmor tt al. (1980) and Negi and Marmor (1984), Gouras and his colleagues concluded that the transplanted cells homed in on the patch of denuded Bruch’s membrane because of the flow of subretinal fluid out of the subretinal space is greatest in areas of damaged retinal epithelium. In short, the most recent results of Gouras and his co-workers show that human or rabbit W E cells can be maintained in tissue culture and transplanted onto a denuded area of Bruch’s membrane (Gouras et al., 1986). This can be accomplished beneath a small area of detached retina so that within several hours of graft tissue has attached to Bruch’s membrane of the host and to overlying host neural retina and survives in that fashion for at least several days. From here these investigators plan to go on to determine the fate of outer segments adjacent to transplant cells identified by autoradiography over extended periods of time.

IV. Retinal Tissue Transplanted into the Central Nervous System

A. INTRODUCTION It was not until 1980 that the first studies concerning transplantation of the retina to CNS were published. By placing the retina into a foreign environment the relationship between intrinsic and extrinsic factors determining retinal development could be studied. The extent to which the graft can differentiate toward its normal organization and make specific connections with the host brain can be addressed in this model. In addition, how donor and host ages influence the development of the graft are two important areas which can be addressed by such studies.

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B. METHODS Only a few research groups have transplanted retina to the CNS. Freed and Wyatt (1980) transplanted by stereotaxical injection whole dissociated eyes of 14- to 15-day-old rat fetuses into the lateral geniculate nucleus or as a control into the frontal cortex of adult blind rats. The host eyes were removed 3 weeks after grafting. Light-evoked responses were recorded from the grafts, which were examined after a survival time of 1-4 months. The research group of McLoon and Lund has developed a routine and easily reproducible transplantation method. Specifically, E l 4 retinas, dissected free of RPE and vascular elements, are transplanted by means of a glass pipet onto the the left superior colliculus of neonatal rats (McLoon and Lund, 1980a). Contralateral eye enucleation was done initially in half and later in all of the host animals. The authors have also transplanted cultured (McLoon et al., 1981) or dissociated and reaggregated retinas (McLoon et al., 1982) and used adult hosts instead of newborn rats (McLoon and Lund, 1983). The grafts and the adjoining host tissues were examined after a survival time of 1-4 months. Graft projections to the host brain were traced by either graft lesion and subsequent examination for degenerating pathways (Fink-Heimer method) or by injection of H R P into the graft or into the superior colliculus of the host. Host eyes were injected with [3H]proline in order to compare the projections of the host and the transplanted retina. Embryonic retinas have also been transplanted to other areas of the CNS, e.g., occipital cortex (Matthews et ul., 1982; Matthews and West, 1982; McLoon and Lund, 1984), inferior colliculus (McLoon and Lund, 1982), and spinal cord (McLoon et d.,1983). Matthews and West (1982) have transplanted [3H]thymidine-labeled El5 retina either alone or together with a second graft from superior colliculus or thalamus into the occipital cortex.

C . GRAFTDIFFERENTIATION Both McLoon and Lund as well as Matthews reported 70-80 % survival of grafts independent of graft localization and survival time (for review see McLoon and McLoon, 1984; Matthews, 1986). Graft survival seemed to be dependent on its revascularization by the host (McLoon and McLoon, 1984; McLoon and Lund, 1983; Matthews et al., 1982). Retinal grafts did not develop when taken after E l 6 (McLoon et al., 1981). Freed and Wyatt (1980) had only observed surviving retinal cells when placing the grafts into the dorsal lateral geniculate nucleus. The grafts that consisted of neuroepithelium at the time of transplantation developed a laminar pattern resembling mature retina. Laminae were

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organized in folded sheets or in rosettes (ganglion cells outside, receptor cells inside). The best condition for graft development existed when retina taken from an E14-15-day donor was placed near the superior colliculus of a neonatal host with an enucleation of the contralateral eye (McLoon and McLoon, 1984). Laminar organization was reported to be affected by the following: donor age (McLoon and Lund, 1980b; McLoon et al., 1983), host age (McLoon and Lund, 1983), dissociation of the graft prior to transplantation (McLoon et al., 1982), long-term explant culture (McLoon et al., 198l), and graft placement (McLoon and McLoon, 1984; Matthews, 1986). If the graft is placed far away from its target area (e.g., occipital cortex, spinal cord) the majority of the ganglion cells (GCs) will generally not develop and no axons will extend from the graft (Matthews et al., 1982; McLoon et al., 1983; McLoon and Lund, 1984). However, if retina is grafted together with superior colliculus (SC), more ganglion cells will survive and axons will extend from the retina toward the SC graft (Matthews and West, 1982). In other words, GCs survived in the graft only after contact with target tissue. By injecting horseradish peroxidase (HRP) into the superior colliculus of the host McLoon and Lund were able to identify several types of GCs within the graft (e.g., McLoon et al., 1982; Perry et al., 1985). However, no immunohistochemical staining of GCs within the graft has been published. A GC layer can be identified mostly on the outer surface of the graft. Most GC dendrites are restricted to the IPL of the graft; however, their axons may course through the whole graft (Perry et al., 1985). Large GCs are only present when neurite outgrowth from the graft takes place (Matthews and West, 1982; McLoon et al., 1983; McLoon and Lund, 1984). Matthews has regarded cells located in the presumptive ganglion cell layer as GCs and counted them. He determined the ratio of GCs to cells in the inner and outer nuclear layer in the graft. This ratio was very low in areas without neurite outgrowth i.e., without contact with the SC co-graft (Matthews and West, 1982). Ganglion cells did not survive when the graft was placed in nontarget areas of the CNS (Matthews et al., 1982; McLoon and Lund, 1984). All cell types normally found in the inner nuclear layer could be identified by morphological criteria (Lund and McLoon, 1980; Matthews et al., 1982; McLoon and McLoon, 1984). In addition, specific classes of amacrine cells could be identified immunohistochemically (McLoon and McLoon, 1984). Photoreceptor cells differentiated from the graft 10-15 days after the transplantation (Matthews, 1982). They developed inner segments and contorted outer segments (Matthews d aL, 1982). The development of outer segments seemed to be dependent on the presence of RPE cells inside the rosettes (McLoon d al., 1982). Glial cells with radially oriented processes showed immunoreactivity for the Miiller cell specific antibody G3 (Barnstable, 1980) and for an antibody against

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glial fibrillary acidic protein (McLoon and Karten, 1983), which is characteristic for Miiller cells in injured retina (Bignami and Dahl, 1979). The Muller cells formed an external limiting membrane (McLoon et al., 1982; Matthews et af., 1982) around the inner segments of the receptor cells. However, the presence of a inner limiting membrane was not mentioned in any publication and was not discerned in any of their micrographs. Within the graft, all types of synapses normally occurring in the retina can be found after 1 month of survival (Lund et al., 1983), i.e., dyad ribbon synapses and conventional synapses in the IPL as well as triad ribbon synapses and a few conventional synapses in the outer plexiform layer (OPL), which is relatively thin (Lund and McLoon, 1980). Synapses develop within the graft 20-30 days after transplantation (Matthews et al. , 1982). Amacrine-amacrine synapses (dyad ribbon synapses) have been reported to predominate in the graft IPL, probably because of the altered input (Matthews, 1986).

D. CONNECTIONS TO HOSTBRAIN If placed sufficiently close to the SC of the host, fibers extend from the graft (presumably from ganglion cells) and terminate in the superficial layers of the SC of the host (McLoon and Lund, 1980b). Since this projection is specific, no axonal outgrowth or termination is seen to project toward nonretinorecipient nuclei. When the retina is grafted on top of the inferior colliculus (IC), axons grow through the IC and terminate directly into a neonatal host, the graft also projects to other visual nuclei such as the dorsal terminal nucleus, posterior pretectal nucleus, olivary pretectal nucleus, and dorsal laterd geniculate nucleus (Lund and McLoon, 1980; McLoon and Lund, 1980b). However, this will not happen if the retina is kept in uitro prior to grafting (McLoon et af., 1981). If embryonic retinas are grafted to an adult host, mainly unmyelinated fibers extend from the graft and penetrate the SC of the host only up to 2 mm (McLoon and Lund, 1983). In the neonatal host, most retinal ganglion cell graft fibers are myelinated. No neurite outgrowth occurs when the graft is placed too far away from the target area (Matthews et al., 1982; McLoon et al., 1983; Matthews and West, 1982; McLoon and Lund, 1984). The graft projections are specific but no evidence is found for topographiocally ordered projections (McLoon and Lund, 1980b). The extent of neurite outgrowth and synapse formation with the SC of the host is dependent on the available synaptic space (McLoon and Lund, 1480b). If the contralateral eye is enucleated in a neonatal host at the time of grafting when the retinocollicular projection has not yet developed, the projection from the graft toward the host SC seems to be much denser than the projection from the normal control eye to the contralateral tectum (“overshooting”). Without enucleation, only

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a few fibers extend from the graft to the SC of the host (McLoon and Lund, 1980a). Thus, the graft competes directly with the host retina for the available synaptic space. Therefore, it is not surprising that axons of a graft in an adult host terminate 2 mm from the point of entry, since the retinocollicular projection is fully established (McLoon and Lund, 1983). In a neonatal host, axons from the graft terminate over the whole extent of the SC (McLoon and Lund, 1980a). Less neurite outgrowth is seen with older grafts, probably because the more mature ganglion cells are damaged by axotomy through the grafting procedure (McLoon and Lund, 1980a). The graft projection to the SC of the host does not depend on whether the graft is well fused with the SC of the host (McLoon and Lund, 1980a). However, if the graft is placed in a remote area such as the occipital cortex, fibers extend from the graft only in the direct contact zone between the grafted retina and a co-grafted SC (Matthews and West, 1980). The extent of the projection of the graft is not affected by dissociation and reaggregation prior to grafting (McLoon et al., 1982) but is affected by explant culture (McLoon et al., 1981). In the last case only connections to the SC of the host and not to other visual nuclei can be found. No evidence was obtained for projections from the host brain to the graft (McLoon and Lund, 1980a; McLoon and McLoon, 1984).

E. FUNCTIONAL SIGNIFICANCE Can the retinal graft respond to light stimuli and transfer this information to the SC of the host? So far, only two articles have been published using electrophysiological techniques (Freed and Wyatt, 1980; Simons and Lund, 1985). Freed and Wyatt have transplanted whole dissociated eyes into the forebrain (near the dorsal lateral geniculate nucleus) of “blind” adult rats. After light-flash stimulation of the graft in the forebrain, “short-latency negative waves” (NI) sometimes followed by excitatory positive responses were recorded. In three grafts only NIs were found. The authors suggested a defect in the coupling of the receptor to the bipolar cells as the cause for the predominance of the NI response. Some long-latency responses gave indirect hints for a connection of the graft to the host brain, but no other evidence could be shown. Simons and Lund (1985), upon grafting E l 4 retinas to unilaterally enucleated neonatal hosts, could record light-evoked responses from the graft which were similar to a normal electroretinogram (ERG). Cells with “on” responses as well as cells with “on-off” responses could be found. It could also be shown that cells in the tectum of the host could be activated by electrical stimulation of the graft. Thus, according to the authors retinal

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grafts could respond to light and make functional connections to the SC of the host.

V. Peripheral Nerve Bridges and Optic Nerve Grafts: Enhancement of Retlnal Ganglion Cell Axon Regeneration

A. INTRODUCTION It is widely known that the mammalian visual system does not regenerate but can undergo abortive sprouting following injury to the optic nerve or retina (Ramon y Cajal, 1928; Goldberg and Frank, 1980; Richardson et a l . , 1982; McConnell and Berry, 1982; AUcutt et al., 1984). Ramon y Cajal was among the first investigators to describe the degenerative events which occur following optic nerve transection in the rabbit model (1928). He described an initial traumatic degeneration characterized by the formation of a necrotic zone near the wound, followed by a secondary degeneration phase occurring later and confined to the proximal stump. Both degenerative and regenerative aspects of retinal GC projections have been investigated more recently in rat and mouse models, primarily through surgical damage to the (adult) optic nerve. Following optic nerve crush surgery, GC axons undergo a slow retrograde degeneration, characterized by astroglial scar formation at the lesion site, early Wallerian degeneration of all axons in the distal stump (in relation to cell bodies), and the distal hypertrophy and progressive degeneration of axons in the proximal stump (Misantone et al., 1984; Allcutt at a l . , 1984). Optic nerve transection, on the other hand, apparently accentuates this degenerative response proximal to the lesion site, as a central core of necrosis develops in the proximal stump (Richardson et al., 1982; Grafstein and Ingoglia, 1982). Since these surgical procedures result in the eventual degeneration of all optic nerve axons, it is surprising to find that the loss of retinal GCs is incomplete. Indeed, as reviewed by Misantone et al. (1984), previous optic nerve lesion (transection and crush) studies have reported the survival of approximately 40-60’36 of the neurons in the GCL. Although this cell loss is often accomplished by the shrinkage of remaining GCs, cell size distribution tends to recover (at later time intervals) to near-normal values (Grafstein and Ingoglia, 1982; Misantone et al., 1984). This survival of cells in the GCL remains a puzzling issue but may be explained by the presence of ectopic amacrine cells in the GCL which may account for as much as 50% of the total cells in some species.

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Abortive sprouting of CNS neurons following transection presents an interesting model for regeneration studies. McConnell and Berry (1982) observed randomly regenerating fibers as early as 14 hr postlesion (PL) following optic fiber layer lesion in the mouse retina. Optic nerve damage has also been shown to promote a few nonfasciculated fibers growing for short distances from the ends of surviving axons (Ramon y Cajal, 1928; Grafstein and Ingoglia, 1982; Allcutt et al., 1984). These GC fibers in the optic nerve do not undergo a sustained regeneration but have been shown to grow until at least 100 days PL following intraretinal transection (McConnell and Berry, 1982). In addition, peripheral nerve implant experiments by Turner et al. (1986) suggest possible trophic interactions which may enhance GC survival and axonal fasciculation. Further studies are therefore necessary to better understand the regenerative potential of GC axons.

B.

PERIPHERAL NERVE BRIDGES

1 . Peripheral Nerve Grajs into the Retina Peripheral nerve bridges have been frequently used in recent years to analyze the regenerative potential of CNS tissue into a PNS environment. These bridges offer an alternative pathway for regenerating CNS axons and are thought to mediate this growth by trophic andfor substrate influences. Based on earlier work done in the spinal cord (David and Aguayo, 1981), similar grafts have been used in the adult rat retina (So and Aguayo, 1985; Berry et al., 1986). The first retinal “bridgework” published was done by So and Aguayo (1985). In their study, a short segment of autologous sciaticnerve was used as a “bridge” for GC axon growth out of the retina. Although the distal portion of this graft was left as a blind end underneath the scalp, the graft did not provide a regenerative pathway for those axons severed during its insertion into the retina. After 4-12 weeks, the peripheral nerve graft was transected and analyzed. Sciaticnerve grafts contained fibers which grew to distances equivalent or greater to those of normal retinal GC axons. H R P staining and fluorescent dye double labeling confirmed the presence of regenerating axons in the graft, originating from GCs that had lost their normal projections through the optic tract. Labeled somas in the retina indicated that all normal GC sizes were represented. In addition, physiological experiments (Keirstead et al., 1985)showed that some GCs regenerating axons into these grafts had light-inducedresponsessimilarto those of normal GCs. This work by So and Aguayo therefore supports findings by previous investigators (McConnell and Berry, 1982) which showed that retinal GCs have the

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JAMES E. TURNER cf ~ l .

inherent capacity to regenerate, if transected intraretinally . A similar study described in Berry et al. (1986) supported this work, showing that severed GC axons can regrow directly into the peripheral nerve graft.

2 . Peripheral Nerve Grafts into the Optic Nerve Similar studies have also been done to examine the regenerative potential of GC axons extraretinally. In a majority of these experiments, segments of peripheral nerve are inserted into the proximal region of the optic nerve and the distal end is sutured ectopically. This methodology is not new, however. A series of optic nerve graft experiments are reviewed in an early article by Ramon y Cajal (1928) and indicates the regenerative potential of optic nerve axons into these grafts in the rabbit model. Two recent grafting methods have been used successfully in the rat to elicit the regenerating GC axons. Politis and Spencer (1986) established a model to examine the regenerative response from traumatized optic nerve axons. They exposed a portion of intradural tissue, crushed one-third of the cross-sectional area of the optic nerve, and then sutured in a segment of peroneal nerve. Berry et al. (1986), on the other hand, transected the optic nerve and anastamosed a segment of sciatic nerve to the stump. Retrograde staining through the grafted nerve identified labeled GCs in both models and regenerating axons were found within the grafts. These HRP-filled GCs represented the normal size distribution in cotrol retinas (Berry et al., 1986). Axonal regeneration has also been shown by Stevenson (1985) in the hamster model, after peripheral nerve segments were inserted into the optic tract. Since different nerve implantation methods have been used in these peripheral nerve bridge studies, it is important to compare the results from a technical standpoint. Complete nerve transection offers the additional advantage of ruling out collateral sprouting of intact and crushed optic nerve fibers. Although these peripheral nerve segments transplanted to the host optic nerve can therefore support regenerating GC axons, this response only involved a small number of axons relative to the retina-peripheral nerve implants. These optic nerve graft results contradict work done by Richardson et al. (1982), which failed to show optic axon regeneration into the peripheral nerve graft.

C . TARGET TISSUE CONNECTIONS Peripheral nerve grafts to the retina or optic nerve provide an attractive model for studing CNS axon regeneration into the permissive PNS substrate. Although these grafts can support regenerating axons for distances equivalent and greater than those of normal projections in situ (So and

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Aguayo, 1985), a few studies have investigated the regenerative capability to reinnervate their native target sites. Previous studies using peripheral nerve grafts between the proximal and distal stumps of transected CNS tracts have shown that regenerating axons can penetrate through these bridges, yet their reentry into the CNS is limited to only a few millimeters (David and Aguayo, 1981; Kao et al., 1977). Ross and Das (1986) have established a model to investigate the regenerative growth of CNS neurons to their target sites. Based on earlier studies (Ross and Das, 1981, 1982a,b, 1983) which showed that rat retinal GCs are capable of sustained axonal regeneration, these investigators analyzed the effects of neocortical transplants on this regenerative response. Their experimental design (Ross and Das, 1986) involved partially transecting the brachium of the SC or optic tract in adult rats and transplanting El7 neocortex to the lesion site. Anterograde H R P labeling (from the intravitreal injection site) was used to visualize the regenerative growth of these GC axons. Regenerating fibers penetrated these transplants in approximately 60% of the cases examined. In general, these afferents entered the transplants across well-integrated interface regions, adjacent to hypertrophied proximal axonal segments from the retinofugal protection. Furthermore, 25% of animals with transplants contained HRP-labeled axons which passed through the transplant, reinnervated the distal stump, and projected to deafferented optic layers of the SC. Afferent fibers in the transplant were first detected 14 days after surgery, while fascicles of axons entering the distal stump of the optic tract were seen after 30 days. Although synaptogenesis at the target site was not examined (ultrastructurally), these experiments did report mammalian CNS axonal regeneration to the target site. In a similar fashion, peripheral nerve bridgework provides a convenient model for studying the potential target tissue connectivity of regenerating GC axons. Based on the model established by So and Aguayo (1984), Vidal-Sanz and his colleaguesexamined this regrowth through a peroneal nerve bridge from the retina to the region of the SC (1986). Anterograde tracing revealed many axons which appeared to terminate at the graft-host (midbrain) interface but also a few labeled axons which penetrated the host tissue for up to 1 mm. Furthermore, some of these projections terminated in growth conelike dilations and electron microscopy suggested synaptogenesis taking place. Although these findings (Vidal-Sanz et al., 1986) are preliminary they indicate the potential for peripheral nerve bridge-mediated CNS regeneration to the target site.

VI. Summary

From the review of the current literature it is quite evident that some exciting prospects are on the horizon which will help to better explain the development

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and functioning of the visual system. In addition, the new technology of CNS tissue grafting coupled to other newly emerging technologies (i.e. , microsurgical, microinjection, and micromanipulative techniques coupled with our knowledge of immunosuppressive methods) will allow for a realistic approach in exploring possible strategies for visual recovery as a result of trauma or disease within the near future. One specific area of research that hopefully will emerge from this new body of knowledge comes from the realization that at the present time there is no effective therapy for practically all types of hereditary retinal degenerative disorders in man. It would seem most appropriate to take advantage of the new neuronal transplantation technology mentioned in this article and the availability of hereditary retinal degeneration models in the hope of developing new methods for a therapeutic approach to this problem. Such an approach could involve replacing the abnormal, absent, and/or lost host retinal cells with tissue from healthy donors by means of a grafting technique with the goal of arresting and/or reversing the disease process. Of course, this is but one example of the many challenges in this area of research which increasingly appear to be within our grasp.

References

Aguayo, A. J . , David, S., and Bray, G . M. (1981).J. Exp. Biol. 95, 231-240. Allcutt, D., Berry, M . , and Sievers, J. (1984). Deu. Brain Res. 16, 231-240. Arendash, G. W . , and Gorski, R . A. (1982). Science 217, 1272-1278. Barnstable, C . J . (1980). Nature (London) 286, 231-235. Barnstable, C . J . , and DGger, U. C. (1984). Neuroscience 11, 847-855. Beale, R., and Osborne, N. N . (1982). Ncurochm. I n f . 4, 587-595. Benfey, M . , and Aguayo, A. J . (1982). Nature (London) 296, 150-152. Berry, M . , Rees, L., and Sievers, J. (1986). I n “Neuronal Transplants and Regeneration, Proceedings in Life Sciences” (G. D. Das and R . B. Wallace, eds.), pp. 181-228. SpringerVerlag, Berlin. Bignami, A,, and Dahl, D. (1979). Exp. Eye Res. 28, 63-69. Bjorklund, A . , and Stenevi, V. (1984). Annu. Reu. Neurosci. 7, 279-308. Blair, J . R . , and Turner, J . E. (1986). SOC. Neurosci. Abstr. 12, 561. Blight, R., and Hart, J . C . D. (1977). Br. J. Opthalmol. 61, 573-587. Columbre, J. L . , and Columbre, A. J. (1970). Nature (London) 228, 559-560. David, S., and Aguayo, A. J. (1981). Science 214, 931-933. del Cerro, M . , Gash, D. M., Rao, G. N., Notter, M . F., Wiegand, S. J., and Gupta, M. (1985a). Inuest. Opthalrnol. Vis. Sci. 26, 1182-1 185. del Cerro, M., Gash, D. M., Rao, G. N . , Notter, M. F., Wiegand, S . J., and del Cerro, C. (1985b). SOC.Neurosci. Abstr. 11, 15. del Cerro, M . , Gash, D. M., Notter, M . R., Wiegand, S.J., Jiang, L. Q., and del Cerro, C. (1986). SOC.Neurosci. Absfr. 12, 561. Foulds, W . S. (1979). Br. J . Opthalmol. 6 3 , 71-84. Freed, W. J . , and Wyatt, R. J . (1980). Lif Sci. 27, 503-510.

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Froy, P. J., Dunnett, S. B., Iversen, S. K., Bjorklund, A., and Stenevi, V. (1983). Science 219, 4 16-41 9. Goldberg, S., and Frank, B. (1980). Exp. Ncurol. 70, 675-689. Gouras, P., Flood, M. T., Eggers, H., and Kjeldbye, H. (1983). Inuest. Opthulmol. Vis. Sci. 24, 142A. Gouras, P., Flood, M. T., and Kjeldbye, H . (9184). An. Acad. Bras. Cienc. 56, 431-443. Gouras, P., Flood, M. T., Kjeldbye, H., Bilek, M. K., and Eggers, H . (1985). Curr. Eye Res. 4, 253-265. Gouras, P., Lopez, R., Brittis, M., Kjeldbye, H. M., and Fasano, M. K. (1986). In “Retinal Signal Systems, degenerations and Transplants” (E. Agardh and B. Ehinger, eds.), pp. 271-286. Elsevier, Amsterdam. Grafstein, B., and Ingoglia, N. A. (1982). Exp. Ncurol. 76, 318-330. Greene, H. S. N. (1967). In “In Vivo Techniques in Histology” (G. H. Bouine, ed.), pp. 80-1 12. William & Wilkins, Baltimore. Kao, L. C., Chang, L. W., and Bloodworth, J . M. B. (1977). J. Neurosurg. 46, 757-766. Keistead, S. A., Vidal-Sanz, M., Rasminsky, M., Aguayo, A. J. M., and So, K.-F. (1985). Bruin Res. 359, 402-406. Kolota, G. (1983). Science 221, 1277. Kroll, A. J., and Machemer, R. (1968). Am. J. Opthulmol. 66, 410-427. Kroll, A. J., and Machemer, R. (1969). Am. J. Opthulmol. 68, 58-77. Labbe, R., Firl, A,, Jr., Mufson, E. J., and Stein, D. (1983). Science 221, 470-472. Lopez, R., Brittis, M., Gouras, P., Fasano, M. K., and Kjeldbye, H. (1986). Znuest. Opthulmol. Vis. Sci. 27, 318A. Lund, R . D., and McLoon, S. C. (1980). Soc. Ncurosci. Abstr. 6 , 825. Lund, R. D., McLoon, L. K., McLoon, S. C., Harvey, A. R., and Jaeger, C. B. (1983). In “Nerve, Organ and Tissue Regeneration” (F. J. Seil, ed.), pp. 303-323. Academic Press, New York. McConnell, P., and Berry, M. (1982). Bruin Res. 241, 362-365. Machemer, R. (1968). Am. J . Opthulmol. 66, 396-410. McLoon, L. K., and Lund, R. D. (1982). Soc. Ncurosci. Abstr. 8 , 452. McLoon, L. K., McLoon, S. C., and Lund, R . D. (1981). Bruin Rcs. 226, 15-31. McLoon, L. K., Lund, R . D., and McLoon, S. C. (1982).J. Comp. Ncurol. 205, 179-189. McLoon, L. K., Sharkley, M. A., and Lund, R. D. (1983). SOC. Ncurosci. Abstr. 9, 373. McLoon, S. C . , and Karten, H. J. (1983). Soc. Ncurosci. Abstr. 9, 854. McLoon, S. C., and Lund, R. D. (1980a). Bruin Res. 197, 431-495. McLoon, S. C., and Lund, R. D. (1980b). Exp. Bruin Res. 40, 273-282. McLoon, S. C., and Lund, R . D. (1983).J. Comp. Ncurol. 217, 376-386. McLoon, S. C., and Lund, R. D. (1984). Dcu. Bruin Res. 12, 131-135. McLoon, S. C., and McLoon, L. K. (1984). In “Neural Transplants: Development and Function” (J. R. Sladek, Jr. and D. M. Gash, eds.), pp. 99-124. Plenum, New York. Marmor, M. F., Martin, L. J., and Tharpe, S. (1980). Invest. Opthulmol. Vis. Sci. 19, 1016-1029. Matthews, M. A. (1986). In “Neuronal Transplantation and Regeneration” (G. A. Das and R. B. Wallace, eds.), pp. 126-147. Springer-Verlag, New York. Matthews, M. A,, and West, L. C. (1982). Anut. Embryol. 163, 417-433. Matthews, M. A., West, L. C., and Riccio, R. V. (1982).J. Neurocytol. 11, 533-557. Misantone, L. J., Gershenbaum, M., and Murray, M. (1984).J. Ncurocytol. 13, 449-465. Morris, R . (1985). Deu. Ncurosci. 7, 133-160. Negi, A., and Marmor, M. F. (1984). Arch. Opthulmol. 102, 445-449. Perry, V. H., Lund, R. D., and McLoon, S. C. (1985).J. Comp. Ncurol. 231, 353-363. Politis, M. J. (1986). Bruin Res. 364, 369-371. Politis, M. J . , and Spencer, P. S. (1986). Exp. Neurol. 91, 52-59.

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Ramon y Cajal, S. (1928). In “Degeneration and Regeneration ofthe Nervous System” (R. M. May, ed.), pp. 583-596. Hafner, New York. Richardson, P. M . , Issa, V. M. K., and Shemie, S. (1982). J . Neurocytol. 11, 949-966. Ross, D. T., and Das, G. D. (1981). Anat. Rec. 199, 217A. Ross, D. T., and Das, G. D. (1982a). Anal. Rec. 202, 161A. Ross, D. T., and Das, C. D. (1982b). SOL.Ncurosci. Absfr. 8 , 758. Ross, D. T., and Das, G. D. (1983). Anat. Rec. 205, 167A. Ross, D. T . , and Das, G. D. (1986). In “Neural Transplantation and Regeneration” (C. D. Das and R . B. Wallace, eds.), pp. 181-228. Springer-Verlag, Berlin. Royo, P. E., and Quay, W. B. (1959). Growth 23, 313-336. Simons, D. J . , and Lund, R . D. (1985). Deu. Brain Rcs. 21, 156-159. So, K. F . , and Aguayo, A. J . (1985). Brain Res. 328, 349-354. Stenevi, U., and Bjorklund, A. (1978). Pros. Brain Rcs. 48, 101-110. Stevenson, J . A . (1985). Exp. Neurol. 87, 446-457. Turner, J . E., and Blair, J. R. (1986). Dcu. Brain Res. 26, 91-104. Turner, J. E., Blair, J . R., and Chappell, E. T. (1986). Brain Res. 376, 246-254. Van Doorernall, J . C. (1873). Ondnr. Physiol. Lab. Ufrccht Hooscsch. 3.R.11, 277-290. Vidal-Sanz, M . , Bray, G . M . , and Aguayo, A. J . (1986). SOL.Ncurosci. Abstr. 12, 700.

SCHIZOPHRENIA: INSTABlLlTY IN NOREPINEPHRlNE, SEROTONIN, AND .y-AMINOBUTYRIC ACID SYSTEMS By Joel Gelernter National institute of Mental Health Clinical Neurogenetlcs Branch Bethesda, Maryland 20892

and Daniel P. van Kammen Veterans Administration Medical Center Pittsburgh, Pennsylvania 15208, and Western Psychiatric Institute and Clinic University of Pittsburgh School of Medicine Plttsburgh, Pennsylvania 15213

I.

Introduction

11. Norepinephrine A. B. C. D. E. F. G. H. I. J. K.

Brain Norepinephrine CSF Norepinephrine CSF Norepinephrine Metabolites CSF and Brain DBH Plasma Norepinephrine Plasma DBH CAMP Stimulation with PGEl in Platelets a*-ReceptorBinding in Platelets az-Receptor Function as Assessed by Clonidine Challenge Plasma MHPG Urinary Catecholamines L. Adrenergic Drugs M. Norepinephrine in Schizophrenia: Discussion 111. Serotonin A. Brain 5HT and 5HIAA Studies B. CSF 5HIAA Studies C. Platelet 5HT Uptate and Related Studies D. Serotonergic Drugs E. Serotonin in Schizophrenia: Discussion IV. y-Aminobutyric Acid A. CSFGABA 8. Plasma GABA C. GABAergic Drugs D. GABAergic Effects of Classical Antipsychotics E. GABA in Schizophrenia: Discussion V. Conclusions References

309 INTERNATIONAL REVIEW OF NEUROBIOLOGY, VOL. 29

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1. Introduction

Although dopamine (DA) has long been implicated in the pathophysiology of schizophrenia (Carlsson and Lindqvist, 1963; Sedvall et a l . , 1981; Randrup and Munkvad, 1972; Snyder, 1977; van Kammen, 1979), it has been clear for some time that malfunction in this neurotransmitter system is not sufficient of itself to account for all the clinical pathology. The initially lustrous concept of DA overactivity as the etiology of schizophrenia now appears to be too simplistic, and some of the data are more supportive of DA hypoactivity (Chouinard and Jones, 1979; van Kammen et al., 1986). While no other single neurotransmitter system is likely to be implicated to replace DA as the major source of schizophrenic psychopathology, several other transmitter systems that interact with DA have yielded exciting results. Presently, the most enduring monoaminergic findings in schizophrenia are noradrenergic (Hornykiewicz, 1982; van Kammen and Antelman, 1984) and serotonergic (Stahl et a l . , 1985a,b). The y-aminobutyric acid (GABA) system is less well studied. Are these findings epiphenomena or of etiologic or illness modifying significance? One of the major problems in schizophrenia research is the episodic nature of the illness. One must distiriguish between findings present at all times in the schizophrenic individual (trait dependent) and those that are characteristic only of more symptomatic periods and may disappear interepisodically (state dependent). Most evidence supports a state-dependent disorder of DA dysregulation (Bowers et al., 1980a,b; Cleghorn et al., 1983; Post et al., 1975; van Kammen et a l . , 1985b). There is also evidence for norepinephrine (NE)-related state-dependent changes (van Kammen et al., 1985a,b). State dependency is not established as clearly for the serotonin (5HT) and GABA system but could be present; both of these interact with the DA and NE systems. We now have the neuropeptide neuromodulators to contend with as well. Neuropeptides are often coreleased with the classical neurotransmitters (Lundberg and Hokfelt, 1983); for example, neuropeptide Y, which is the most abundant of the neuropeptides in the central nervous system (CNS) (Adrian et al., 1983), can be coreleased with NE. Much less is known about the specific behavioral effects of the neuropeptides than about the classical transmitters. It appears that while the classical transmitters act over a relatively brief time course, the neuropeptides can act over seconds and perhaps even longer. Supposing that they may act to potentiate the effects of classical neurotransmitters, it is easy to see that blocking a particular monoamine may not be a sufficient step to block its effects, if some of these effects are shared with a peptide which can be coreleased with it anyway and go unblocked. It

SCHIZOPHRENIA

31 1

is clear that we will not be able to complete a biochemical understanding of schizophrenia until more is known about the role of peptidergic transmission. In this article we will examine selectively some of the evidence linking the nondopaminergic NE, 5HT, and GABA systems with schizophrenia and emphasize those findings that have been replicated most frequently. We will indicate relationships with other systems, especially DA, and with clinical syndromes whenever possible. We will also advance some concepts that may facilitate the understanding of these diverse and conflicting data, such as their relation to state dependency, clinical instability, stress sensitivity, relapse, and genetic vulnerability. We will try to demonstrate that the concept of a one-neurotransmitter system disorder in schizophrenia can no longer be sustained. With all of this in mind we now proceed to the norepinephrine system.

Ii. Norepinephrine

In 1971, Stein and Wise proposed that anhedonia in schizophrenia was caused by a brain dopamine b-hydroxylase (DBH) deficiency. The NE deficiency that would result from impaired conversion of DA to NE would lead to aberrant DA metabolites with cytotoxic properties, explaining the progressive deteriorating course of the disease. Hornykiewicz (1982) suggested that NE regulates the sensitivity of the DA system. Other authors have also suggested that schizophrenia is a disorder of both the DA and the NE systems (van Kammen and Antelman, 1984). Van Kamrnen and Antelman (1984) stressed that most of the schizophrenic behaviors thought to be dopamine mediated can also be mediated or modified by norepinephrine (‘Joseph et al., 1979b). Antelman and co-workers (Antelman and Caggiula, 1977; Antelman and Chiodo, 1983) reviewed the evidence for an NE and DA interaction in the brain and concluded that during minimal stress it facilitates them. When, under conditions of high stress or disturbed NE regulation, NE is unable to sustain its modulatory influence on DA, those behaviors can become deregulated. Presumably, behaviors that involve NE are stimulus bound, while DA behaviors are motorically based (Kokkinidis and Anisman, 1980). Both of these kinds of behaviors are disturbed in schizophrenia.

A. BRAINNOREPINEPHRINE Post-mortem studies of brains of schizophrenics have provided evidence for NE involvement in schizophrenia. Farley et al. (1978) provided the first

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evidence for elevated NE in limbic areas of brains from paranoid schizophrenic patients. Carlsson (1980) reported increased NE levels in the midbrains of paranoid schizophrenic patients. Bird d d.(1979a,b) and Crow d al. (1979) also reported increased NE levels in schizophrenic brains. Kleinman d d. (1979, 1981) studied NE and some of its metabolites in nucleus accumbens of paranoid schizophrenic patients; no difference was found in NE, but an increase in 3-methoxy-4-hydroxyphenylglycol (MHPG), the primary CNS NE metabolite, was demonstrated in hypothalamus of psychotic patients. Farley et al. (1980) found elevated NE in limbic areas of brains of schizophrenics. Bridge d a!. (1987) studied brain concentration of catecholamines in schizophrenics 45 yr old and older for whom medical records were available for symptom assessment; the areas studied were hypothalamus and nucleus accumbens. Patients had significantly higher MHPG than controls in both areas. Hypothalamic MHPG and homovandic acid (HVA) also correlated with chart observations of depressed mood in the patients, and there was an inverse correlation between nucleus accumbens MHPG and DA and cognitive function.

B. CSF NOREPINEPHRINE Lake d nl. (1980) reported elevated cerebrospinal fluid (CSF) NE levels in 35 drug-free schizophrenics compared to 29 control subjects. The difference in CSF NE could be accounted for when considering the paranoid subgroup alone. All three subgroups considered (paranoid, undifferentiated, and schizoaffective mainly schizophrenic) had signs suggestive of generally increased noradrenergic outflow, including increased heart rate and diastolic blood pressure. Kemali et nf. (1982) reported increased CSF and plasma NE levels in 46 drug-free schizophrenic patients. While the latter finding was noted to be consistent with an increased level of arousal in the schizophrenic patients, the former was felt to be indicative of a globally increased level of central noradrenergic activity. This finding was repeated by Kemali et al. (1985a), who compared 20 pateints with Research Diagnostic Criteria (RDC) acute schizophrenia with 20 controls who had no psychiatric illness. CSF NE was significantly increased in the schizophrenic patients. CSF NE was also found to be significantly correlated with arousal as measured by computerized EEG. There was no relationship between CSF NE and psychosis ratings. Gomes et al. (1980) found additional evidence for increased NE levels in schizophrenic CSF; they found that chronic schizophrenics had significantly higher NE levels than acute patients and a control group. Unfortunately the 11 chronic schizophrenic patients considered here were on antipsychotic drugs at the time, as were 5 of the 10 acute patients. Antipsychotic drug treatment can affect CSF NE levels (see below).

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Thus, schizophrenia has been associated fairly consistently with increased brain and CSF NE levels, particularly in paranoid patients, i.e., these levels appear at first to be trait markers only. However, the studies quoted above all relied on single CSF catecholamine measurements. Linnoila et al. (1983) investigated the reliability of this method by making repeated determinations of CSF monoamine and metabolite levels in 15 drug-free schizophrenic patients, up to four measurements per patient. They found that levels of NE varied from lumbar puncture (LP) to LP essentially unpredictably, although some of the intraindividual changes in NE values could be related to changes in psychosis ratings (Y = 0.62, N = 9 women; p < 0.05). This suggests that CSF NE levels may be state dependent as well. Van Kammen et al. (1985b) also supported NE state dependency in a preliminary study which showed that CSF NE was higher in patients who slept less the night before the LP (Y = 0.44, N = 53, fi = 0.0008) (Fig. i), as do Sternberg et al. (1981), who demonstrated that pimozide treatment caused a significant decrease in CSF NE relative to improvement in

CSF NE AND SLEEP

I

I

0.2

0.4

0.6

0.8

I

1

1

1.2

1.4

1.6

I

1.8

NE FIG. 1. CSF NE, expressed in pmol/ml correlated significantly with the hours of sleep the night before the LP; this may explain the lack of correlation between drug-free LP to drug-free LP within patients. Whether relationships between psychosis and CSF NE are observed depends on whether patients are in a stable or in a decompensated, relapsed state. I = -0.44,

fi

=

0.0008,N

=

53.

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JOEL GELERNTER AND DANIEL P. VAN KAMMEN

psychotic symptoms (p < 0.02). Decreased sleep is frequently observed in the prepsychotic phase and early in psychotic episodes, and its restoration is often one of the first signs that the patient is responding to antipsychotic treatment. Gattaz et al. (1983) reported that patients who were receiving antipsychotics had elevated CSF NE, whereas drug-free patients could not be distinguished from controls by this measure. Bagdy et al. (1985) reported a slight decrease in CSF NE at 2 weeks following withdrawal from long-term antipsychotic treatment. One possible explanation of the decreased levels following antipsychotic withdrawal in some studies is that they may be related to clinical state, as some patients improve temporarily after withdrawal (Marder et ul., 1979); another is that some antipsychotics block a-receptors, leading to an additional increase in NE levels, particularly at high doses (Rice et al., 1984). If the proposed interaction of NE and DA activity actually exists, then CSF values might well relate to each other; indeed, CSF NE correlated with CSF DA (Y = 0.64, N = 34, p C 0.001) and DASOi (Y = 0.35, N = 35, p < 0.05) (D. P. van Kammen, unpublished observations). On the other hand, MHPG and NE levels do not correlate with each other in schizophrenia as they do in affective disorder (Berrettini et al., 1985). Preliminary data from our group indicate that the elevated NE spinal fluid levels after probenecid in drug-free patients are significantly associated with decreased platelet monoamine oxidase (MAO) activity (7 = 0.64,p < 0.0003) (van Kammen d al., 1981). Furthermore, low platelet M A 0 activity has been associated with stimulus-seeking behavior, paranoid schizophrenia, and high risk for psychiatric disorders. Most groups report that platelet M A 0 is decreased because of prolonged antipsychotic treatment (DeLisi et al., 1981a). We found that psychosis ratings and platelet M A 0 activity correlated significantly 3 to 6 months after antipsychotic withdrawal (Y = 0.53, N = 21, p = 0.05). This interaction suggests that M A 0 activity is partially under state-dependent influences as well, because there is not evidence of an acute antipsychotic effect on M A 0 activity and after 3 to 6 months any indirect drug effects on platelets should have been washed out.

C . CSF NOREPINEPHRINE METABOLITES Shopsin et al. (1973) reported that schizophrenic patients had normal CSF MHPG levels, although some schizophrenic (and manic) subjects had levels outside the range of control subjects. Similarly, other groups did not find significantly altered CSF MHPG in acutely ill schizophrenic patients or in normal control subjects either (Berger et al., 1980; Faull d al., 1984; Gershon

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et al., 1976; Post et al., 1975; Sedvall et a l . , 1981). Post et al. (1975) observed that MHPG levels were not different between psychotic and remitted patients, in contrast to HVA levels, which declined. Van Kammen et al. (1982a) noted that CSF MHPG obtained prior to an amphetamine challenge test predicted the direction of change in psychosis following the d-amphetamine infusion; those patients with the lowest levels of MHPG tended to improve with d-amphetamine. This was consistent with Post et al. (1975), who had reported previously that schizophrenic patients who rated themselves as experiencing high subjective stress showed significantly lower levels of MHPG, while patients with higher MHPG levels reported more hallucinations. Sedvall et al. (1981) found that schizophrenic patients with psychotic relatives tended to have either higher or lower CSF MHPG levels than those without. Kopin et al. (1983) reported that plasma-free MHPG was highly correlated with CSF MHPG and suggested that free MHPG is able to cross the blood-brain barrier with relative facility. Consequently, a significant proportion of MHPG found in the CSF is thought to be of peripheral origin. In patients with cortical atrophy, CSF MHPG was decreased compared to those with normal C T scans (40 13.8 versus 34 5.7 ng/ml, p = 0.055) and correlated significantly with degree of atrophy (Y = -0.37, N = 22, p = 0.04) (D. P. van Kammen, unpublished data, 1982a). This is a much smaller difference than for CSF HVA or 5HIAA, suggesting that plasma MHPG may have contributed to the levels. One of us found CSF MHPG, sleep, and psychosis to correlate with each other (36 days drug free). CSF MHPG decreases significantly following antipsychotic (chlorpromazine) treatment in some studies (Ackenheil et al., 1974; Alfredsson et al., 1983; Sedvall et al., 1976), which is similar to the pimozide-related decrease in CSF NE levels (Sternberg et al., 1981). However, not all CSF studies have shown such a decrease in MHPG with antipsychotic treatment (Gattaz at al., 1982), nor has the decrease been found with all antipsychotics (WodeHelgodt et al., 1977). Finally, Jimerson et al. (1975) did not find differences in CSF vanillylmandelic acid (VMA) between drug-free schizophrenics and normal subjects. There are no reports of CSF normetanephrine in schizophrenia.

*

D. CSF

AND

*

BRAINDBH

As DBH catalyzes the conversion of DA to NE, its level in the CSF may reflect the general level of noradrenergic function; many variables, such as changes in posture, can affect plasma DBH and possibly CSF DBH as well. Wise and Stein (1973) reported decreased DBH activity in 1973 in the brains of nine schizophrenic patients, with particularly low values obtained

316

JOEL GELERNTER AND DANIEL P. VAN KAMMEN

in the hippocampus and diencephalon. Wyatt et al. (1975) reported nonsignificantly lower brain DBH levels in 10 schizophrenic patients. Both groups have been criticized for their assay methods. Cross et al. (1978) did not observe significant differences in brain DBH activity between normals ( N = 12) and schizophrenic patients ( N = 12). Brain atrophy on C T scans is associated with decreased CSF levels of NE and DBH (van Kammen et al., 1983, 1984, 1985a) (for 5HIAA, see below). Decreased CSF DBH (and HVA) levels were found in CSF of 11 schizophrenics with brain atrophy [defined by ventricle brain ratio (VBR) greater than two standard deviations above that of normal population, or cortical atrophy] compared to 22 schizophrenics with normal head C T scans (van Kammen et al., 1983). Major problems in the assessment of DBH in schizophrenia autopsy studies are the lack of information on brain atrophy, the small number of brains studied, and the time lag between death and autopsy. Sternberg et al. (1981) studied CSF DBH longitudinally in 30 schizophrenic and schizoaffective-schizophrenicpatients, compared with 27 normal controls. The mean CSF and DBH levels were not significantly different for the two groups, nor was there any relationship of DBH to age or sex. In the patient group, DBH was not related to clinical subtype. In the drug-free condition, CSF DBH activity was not related to severity of psychosis. Both patient and control groups showed a wide range of DBH values. Six patients were psychotic at admission and nonpsychotic at discharge; DBH activity did not change. Eleven patients had paired LPs during the drug-free condition and while receiving pimozide; no pimozide effect on DBH activity was found. In toto, CSF DBH, when followed longitudinally, was remarkably constant. However, premorbid and present level of social and sexual function correlated with CSF DBH; better-functioning patients had lower levels. The low-DBH group had more “reactive”-type schizophrenic patients (by Phillips scale), compared to more “process” schizophrenic patients in the high-DBH group (Sternberg et al., 198213). The antipsychotic drug responders in the group had significantly lower mean DBH activity (Sternberg et al., 1983). The report of decreased CSF DBH in schizophrenic patients with brain atrophy appears at first to be nonconsistent with this, unless one posits that low DBH has several possible etiologies (e.g., genetic or environmental).

E. PLASMA NOREPINEPHRINE Zander et al. (1981) reported that during antipsychotic treatment in chronic, hospitalized schizophrenic patients, plasma NE levels were significantly elevated. After a 30-day drug-free period, NE levels decreased

SCHIZOPHRENIA

317

but were still elevated above values of normal subjects. Similarly, MuellerSpahn et al. (1986) found that serum NE was elevated in two different groups of chronic schizophrenic patients while they were treated with antipsychotics. One group was withdrawn and measured again after 5 days; the other group was withdrawn and remeasured after 12 days. The first group showed a significant decrease in plasma NE after drug withdrawal. Both groups still had significantly elevated plasma NE compared to controls after antipsychotic withdrawal. Ackenheil et al. (1985) reported, besides evidence of elevated plasma levels, that plasma NE response to “stress” in chronic schizophrenic patients was less than in normal subjects. Gjerris et al. (1981) reported that hypoglycemia induced significantly smaller increases in plasma NE in acute schizophrenic patients than in normal controls. Castellani et al. (1982) noted that a group of young drug-free schizophrenic patients had supine plasma NE levels similar to control subjects, but standing levels were significantly more elevated. They reported increased heart rate in schizophrenics, which is also consistent with increased circulating NE. This increase was felt to be consistent with decreased peripheral a2-receptor sensitivity (see clonidine challenge, below). The evaluation of antipsychotic treatment effects on NE levels showed that haloperidol did not increase NE levels, but chlorpromazine, which is a much better az-blocker, did. Similarly, Rice et al. (1984) found that plasma NE levels were normal in drug-free schizophrenic patients but were increased after 2 weeks of chlorpromazine treatment. When NE levels are measured in schizophrenic patients treated with a variety of antipsychotics, the a2-receptor effects of these drugs need to be taken into account. Kemali et al. (1982) reported that plasma NE levels did not correlate with central NE levels or psychopathology, which contrasts with the report by Ziegler et al. (1976), who observed a significant correlation between NE levels in plasma and CSF in normal controls (Y = 0.78, p < 0.0001).

F. PLASMADBH Plasma DBH studies have not yielded consistent results. Plasma and CSF DBH levels correlate significantly, but brain atrophy may affect this relationship in schizophrenic patients (Meltzer et al., 1984b). There is a negative correlation between serum DBH and VBR in affective disorder patients, and possibly for schizophrenics as well (Meltzer et a l . , 1984b) as for CSF DBH and VBR. Plasma DBH levels were increased (Markianos et al., 1976) or decreased (Fujita et al., 1979) in schizophrenic patients, but most groups reported no differences from controls. Markianos and Tripodianakis (1985) reported low plasma DBH in demented schizophrenics. Of interest is the

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JOEL GELERNTER AND DANIEL P. VAN KAMMEN

biological evaluation of the schizophrenic Genain quadruplets; plasma DBH was decreased in all four women (DeLisi et al., 1981a).

G. c A M P STIMULATION WITH PGEl

IN

PLATELETS

Rotrosen et al. (1978, 1980), Kaflca et al. (1979, 1980b, 1985), and Kafka and van Kammen (1983) observed that the accumulation of [3H]cAMP production following P G E l stimulation in drug-free schizophrenic patients was lower than in normal subjects. This decrease is also seen following inhibition with NE. Decreased NE inhibition of PGEIstimulated cAMP production is also seen in depression, essential hypertension, and idiopathic orthostatic hypotension. However, that relatively decreased inhibition by NE of PGEI-stimulated cAMP production in depressed but not in schizophrenic patients is observed suggests that the mechanisms involved in schizophrenia and depression are not identical. The schizophrenic Genain quadruplets also showed decreased PGEl-stimulated cAMP production (DeLisi d al., 1981a). Further studies showed that adenyl cyclase activity was also decreased in schizophrenic patients (Kafka et al., 1985). The data suggest an alteration in the adenyl cyclase catalytic unit or a dissociation between receptor, regulator unit, and adenyl cyclase which leads to the decreased [3H]cAMP production following PGEl stimulation (i.e., a?-receptor function) in schizophrenia. The CNS implications of subsensitive PGEl receptors are presently unclear.

H. CY~-RECEPTOR BINDING IN PLATELETS Kafka et al. (1985) reported increased [3H]dihydroergocryptine (13H]DHE) binding on platelets in 10% of a group of schizophrenic patients ( N = 83), but significantly in depression ( N = 23), essential hypertension ( N = 22), and some forms of orthostatic hypotension. Because this increased binding is also observed with [3H]clonidine, but not with [3H]yohimbine or [3Hjrauwolscine, the binding site is presumably a high-affinity a2 site. It is not clear if the increased receptor sites in schizophrenia are functionally intact, because of the decreased cAMP production (see above); they could be desensitized. Corresponding brain studies have not been reported yet but are underway ( G . N. KO d al., personal communication). Rice et al. (1984) did not find elevated [ 3H]clonidine but found decreased [3H]yohimbine (a2-receptor antagonist) binding in platelets from drug-free schizophrenic patients. Binding measured with both [3H]yohimbine and [3H]clonidine was decreased after 2 weeks of treatment with chlorpormazine.

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319

Rotman d al. (1980) reported no difference in NE uptake by platelets between groups of 22 acute schizophrenics and 15 normal controls.

FUNCTION AS ASSESSED BY I. CX~-RECEPTOR

CLONIDINE CHALLENGE

Unlike binding studies, which provide information about as-receptors that is strictly anatomical, clonidine challenge can assess their function indirectly through physiological and pharmacological measures. A blunted growth hormone (GH) response to clonidine has been a consistent finding in affective disorder patients and may in fact represent an affective disorder trait marker; this test has also been used to assess schizophrenic patients. As opposed to some of the studies with affective disorder patients, which have often used variable clonidine dosage based on the subject’s weight, all of the studies quoted below relied on a standard i.v. dosage of 0.15 mg. Matussek et al. (1980) studied G H response to clonidine in 32 controls, 8 schizoaffective patients, 10 schizophrenics, and depressed patients. The endogenous depressed patients and the schizoaffectives showed a reduced G H response, but the schizophrenics did not differ from the controls. The schizophrenics had been free of antipsychotics for at least 4 weeks. Lal et al. (1983) studied 13 male chronic schizophrenic patients who had been withdrawn from antipsychotics for at least 2 weeks and 18 normal male controls; they found no difference in G H response to clonidine (peak response) between the two groups. MuellerSpahn et al. (1986) studied two groups of male schizophrenic patients, described above in Section II,E with paired clonidine infusion tests before and after either 5 or 12 days of antipsychotic drug withdrawal. G H response to clonidine was significantly lower than for the age- and sex-matched controls after 12 days drug free but not under the other conditions. These results are difficult to interpret because of the variable doses of clonidine on a m g k g basis, but evidence for altered an-receptor function in schizophrenia based on this test is scanty at this point. One of us found that G H response to oral clonidine in 12 chronic schizophrenics (van Kammen et al., 1987) does appear to be blunted compared to a sex- and age-matched control group and that normal G H response to clonidine predicted a good antipsychotic response to it as well. Kemali et al. (1985b), studying diurnal rhythms of hormones in schizophrenics and controls, found that the expected G H surge during sleep was not seen in 10 of 23 patients. This finding may be related to the blunted G H response to clonidine seen in some schizophrenics, because normal G H release appears to be under as-receptor control.

320

JOEL GELERNTER AND DANIEL P. VAN KAMMEN

J. PLASMA MHPG Plasma MHPG levels have been determined in schizophrenia only recently. KO et ~ l(1985) . did not find differences in plasma MHPG between a small group of schizophrenic and normal subjects. According to Sternberg et QZ. (1982a), acute clonidine administration, but not placebo, suppresses plasma MHPG in normal subjects, but not in schizophrenic patients. We discussed the relationship between CSF and plasma MHPG levels above.

IS. URINARY CATECHOLAMINES Gershon et al. (1976) observed that 24-hr urinary MHPG was elevated in the schizophrenic population, although CSF MHPG was not different between drug-free schizophrenic and depressed patients. Joseph et al. (1979a) studied urinary MHPG excretion in acute schizophrenic patients before and during a trial of two isomers of flupenthixol and placebo. Higher pretrial urinary MHPG excretion was associated with better social functioning at 1 yr after the trial in male subjects, but not in the females. 6-Flupenthixol treatment decreased urinary MHPG excretion, but not following the active isomer a-flupenthixol and placebo. Higher pretrial urinary MHPG excretion was associated with better social functioning at 1 yr after the trial in male subjects but not in the females. @-Flupenthixol decreased urinary MHPG excretion, but the active isomer a-flupenthixol and chlorpromazine did not. They concluded that lower urinary MHPG may be a predictor of poor outcome in schizophrenia, but MHPG excretion may also change as a result of clinical state. These results have to be . studied 13 never-mediated schizophrenic pareplicated. Deakin ct ~ l (1979) tients. Five of these, the most severely ill, excreted less MHPG and failed to habituate to loud tones. The practical value of urinary MHPG levels is limited because of the difficulty in obtaining reliable urinary collections from schizophrenic patients the large variations from day to day in MHPG excretion, and the variable CNS contribution to urinary MHPG. Nevertheless, low MHPG seems to be associated with a more severe (less drug-responsive) illness across studies. Barbeito et al. (1984) found elevated urinary NE levels in a sample of 2 1 schizophrenic patients, some medicated and some unmedicated, compared to 12 controls. They also found, in a subsample of the schizophrenics, that urinary NE correlated with plasma NE levels.

SCHIZOPHRENIA

32 1

L. ADRENERGIC DRUGS 1 . Clonidine Clonidine is a specific az-receptor agonist which has the effect of decreasing central adrenergic outflow. In recent years, several small open studies have suggested that clonidine may have an antipsychoptic effect in schizophrenia (Elizur et al., 1980; Freedman et al., 1982; Jouvent et al., 1980; Lechin et al., 1980; Sedvall et al., 1976). They only double-blind placebo controlled study in what may be regarded as an adequate dosage range, by Freedman et al. (1982), reported that in eight schizophrenic patients with tardive dyskinesia, clonidine had an antipsychotic effect equal to haloperidol. Martin et al. (1984) reported that clonidine increased CSF HVA and decreased MHPG but did not change 5HIAA in patients with an alcohol-related amnestic syndrome. Memory did not improve in these subjects. In a doubleblind study of eight drug-free schizophrenic patients, memory improved significantly with 5 weeks of clonidine treatment, independent of improvement in psychosis, and CSF MHPG declined significantly (p < 0.01) (Fields et al., 1987). Clonidine may also be effective in akathisia (Zubenko et al., 198413) and tardive dyskinesia.

2 . Prazosin Hommer et al. (1984) reported that 6 weeks of the at-antagonist prazosin had no antipsychotic effect in schizophrenic patients, but it did increase autonomic arousal.

3 . Propranolol Since Atsmon et al. (1972) reported improvement with “high doses” of the @-antagonist propranolol, other studies have appeared with mixed results (Bigelow et al., 1979; Gardos et al., 1973; Hansen et al., 1980; King et al., 1983; Pugh et al., 1983; Yorkston et al., 1977, 1981). Most negative studies used only 4 weeks of treatment with 1000 mg of the drug or less, or used propranolol alone (Belmaker et al., 1979; Peet et al., 1981). CSF studies indicated increases in HVA and 5HIAA but no changes in MHPG at 21 days (King et al., 1983), but more sustained treatment led to decreases in 5HIAA. We (D. P. van Kammen, S . C . Schulz, and D. E. Sternberg, unpublished data, 1981) noted an increase in CSF MHPG but no change in CSF HVA. Baseline CSF monoamine metabolites did not identify responders, but lymphocyte @-receptoractivity ([3H]isoproterenol binding) was lower in the patients who improved

322

JOEL GELERNTER AND DANIEL P . VAN KAMMEN

(D. P. van Kammen el al., unpublished data, 1981). This decreased &receptor activity may reflect increased circulating NE levels. The length of treatment and the lack of clearly positive studies have greatly decreased interest in further studies with propranolol. Unfortunately, studies identifying biochemical conditions associated with subsequent behavioral improvement with propranolol have not been reported. Propranolol is helpful in patients with akathisia (Adler et al., 1985; Zubenko et al., 1984a) and may be of use in tardive dyskinesia (Bacher and Lewis, 1980), and it may affect habituation (Gruzelier et al., 1981).

M. NOREPINEPHRINE I N SCHIZOPHRENIA: DISCUSSION Depending on state and perhaps also on the type of symptoms an individual can express, it thus appears that the NE abnormality in schizophrenia can be in either direction, with overactivity in some circumstances and underactivity in others (Hornykiewicz, 1982; van Kammen and Antelman, 1984). (This is also the case with the proposed DA disturbance.) This disturbance could either involve levels of NE, also reflected in its metabolites, in brain, CSF, plasma, or urine, or in receptors, as there are reports of abnormal receptor function at least peripherally, or both. Related findings are summarized in Table I. Increased NE levels in brain, CSF, and plasma appear to be associated with paranoid schizophrenia. It is conceivable that this association is specific; increased NE levels could be associated with this subtype, which is partially homologous with the “type I” syndrome (see below). This is the syndrome proposed to be associated with the hyperdopaminergic state (Crow, 1980). Type I symptomatology, which is episodic and state dependent, resembles paranoid schizophrenia as defined by Tsuang and Winokur (1974). Chronic, TABLE I NOREPINEPHRINE ABNORMALITIES IN SCHIZOPHRENIA Brain NE and MHPG levels are increased in some areas CSF NE is episodically elevated in drug-free patients CSF NE and MHPG decrease with antipsychotic treatment relative to clincial improvement Plasma NE is elevated in chronic, drug-free, and medicated patients There is decreased PGE,-stimulated CAMPproduction in platelets a2-Receptor abnormalities: increased [’HIDHE binding; possibly altered G H response to clonidine CSF DBH decreased in patients with brain atrophy, good premorbid function, and low discharge psychosis ratings Certain adrenergic drugs can have antipsychotic effects

SCHIZOPHRENIA

323

clinically stable paranoid schizophrenicpatients, who have developedtype I1 (i.e., negative) symptoms, do not show these elevated levels (Gattaz d al., 1983). We propose that the variable CSF NE levels in drug-free patients are state dependent. With the chronicity of the illness, state-dependent fluctuations may decrease or disappear. We suggest that a dysregulation of the NE systems leads to the prepsychotic phase and can then expose the underlying DA disturbance, when NE is activated beyond the system’s capacity with or without environmental stimulation. Such stimulation does not have to be extreme if the NE system is regulated abnormally; this could occur, for example, in the presence of subsensitive as-receptor function or increased receptor binding (Kafka et al., 1980a, 1985; Kafka and van Kammen, 1983; Sternberg et al., 1982a) or decreased DBH activity (Sternberg et al., 1982b, 1983). Low plasma and CSF DBH activity have not consistently been reported in schizophrenia, and the early findings of decreased brain DBH activity could not be replicated. However, detailed clinical descriptors such as premorbid social function and C T scan readings were not provided (van Kammen et al., 1982d). The patients with low CSF DBH activity in the Sternberg et al. study (1982b, 1983) were patients with good premorbid function who had good antipsychotic drug response. Reanalysis of the available data showed that only two of the patients with CT scan abnormalities (van Kammen et al., 1983) were included in the CSF DBH study (Sternberg et al., 1983), suggesting that the illness of most of the patients in these studies was the type I syndrome. Presumably, in good premorbid patients with hyperactive DA systems, low DBH may lead to isolated psychotic episodes in some patients (and to the full deteriorating schizophrenic syndrome in others). Indeed, alcoholic subjects with low CSF DBH activity were more likely t o experience psychotic episodes with disulfiram (which is a DBH inhibitor) than other alcoholic subjects (Major et al., 1979). On the other hand, low DBH in the presence of decreased DA function could lead to increasingly prolonged episodes and negative symptoms (van Kammen and Gelernter, 1987). At this time, interpretation of these findings of low DBH in seemingly inconsistent groups of patients has to wait. Antihypertensive drugs have a long history in the pharmacotherapy of schizophrenia, starting with reserpine. Propranolol has found a role in the treatment of akathisia; propranolol and clonidine may eventually be useful as antipsychotics for some patients, either alone or in combination with other agents.

111. Serotonin

Over a quarter of a century ago, serotonin (5-hydroxytryptamine, 5HT) was hypothesized to be involved in psychotic disorders (Woolley and Shaw,

324

JOEL GELERNTER AND DANIEL P. VAN KAMMEN

1954). In one of the earliest 5HT-related CSF studies, Bowers (1975) suggested that the probable serotonergic mechanism of action of LSD was a reason to study 5HT metabolism specifically in psychosis. Studies of serotonergic systems in schizophrenia have proven fruitful since. We will discuss brain and CSF studies, which focus on 5HIAA (the major CNS metabolite of 5HT), as they pertain to schizophrenia and briefly to aggressive behavior and violent suicide; whole-blood and platelet 5 H T studies, which led to the proposal of serotonergic abnormalities as a schizophrenia-associated trait marker; and some of the recent studies of serotonergic drugs in schizophrenia.

A. BRAIN5HT AND 5HIAA STUDIES Korpi et al. (1986) found increased serotonin in the basil ganglia of chronic schizophrenics (and suicide victims); they also found increased 5HIAA in occipital cortex in schizophrenics. Most brain areas assessed did not show any significant difference in these measures. Fourteen of thirty chronic schizophrenics were considered drug free; 5HT and 5HIAA did not differ between the medicated and drug-free groups. Crow et al. (1979) also found elevated serotonin in schizophrenic autopsy brains, in putamen in this case (the only area assessed for serotonin). Five of their nine patients had been receiving antipsychotic medication. Autopsy studies of 5HIAA in the temporal cortex, hippocampus, and putamen revealed no deficit or differences in 5HIAA (Joseph et al., 1979a;Jus et al., 1958). However, Farley et al. (1980), who analyzed several more forebrain areas, did find increased 5HIAA in the stria terminalis region of chronic schizophrenic patients. Bennett et al. (1979) reported a 40-50% decrease in [SH]LSDbinding in the cortex of schizophrenic autopsy brains. In spite of several replications by the same group, Crow et al. (1979) did not find such a difference in their patients.

B.

CSF 5HIAA STUDIES

CSF 5HIAA in schizophrenia has consistently been negatively associated with states of motor activity, agitation, and arousal (Bowers, 1975, 1978a,b; Kirstein et al., 1976), and this has led to interest in its potential use as a subtype marker. Bowers (1975) reported that patients with acute schizophrenia had lower CSF 5HIAA than chronic patients, and Gattaz et al. (1982) found lower CSF 5HIAA in paranoid schizophrenic patients than in controls. CSF 5HIAA was also correlated positively with grandiosity, hallucinations, and

SCHIZOPHRENIA

325

thought disturbance but correlated negatively with motor retardation. Bowers (1973) reported significant relationships between 5HIAA and unusual thought content, anxiety, perceptual disorganization, suspiciousness, and total BPRS scores. King et al. (1985) measured CSF and platelet 5HIAA, which were both positively correlated with mannerisms and posturing (BPRS). CSF 5HIAA did not correlate with other BPRS symptoms. These authors speculated that the increased serotonergic activity seen in this type of patient could explain such motor defects as eye-tracking abnormalities. Thus, it appears that decreased serotonergic activity is partially associated with agitation or increased arousal in schizophrenic patients, whereas increased serotonergic activity is associated with mannerisms and posturing or other positive symptom items. Bowers (1978a) was the first author to point out possible state-dependent influences on CSF 5HIAA. Increased CSF 5HIAA could be a marker associated with a family history of schizophrenia, that is, a trait marker. Sedvall et al. (1980; Sedvall and Wode-Helgodt, 1980) evaluated CSF monoamine levels in 60 normal subjects, 28 with a family history of schizophrenia and 32 without. They demonstrated that subjects with a family history of schizophrenia had significantly higher or lower CSF 5HIAA levels than subjects with a family history of depression; the subjects themselves were not regarded as abnormal behaviorally. Lindstrom (1985) also found increased CSF 5HIAA in drug-free schizophrenic patients with a family history of schizophrenia. He found that CSF 5HIAA levels in patients were otherwise not statistically different from levels in controls, although low levels were correlated with delusions and sadness. Gattaz et al. (1982) found decreased 5HIAA levels in schizophrenic patients regardless of medication status. Bowers (1978a,b) reported lower CSF 5HIAA in “better-prognosis” patients. Gerner et al. (1984) showed a trend for increased CSF 5HIAA in chronic schizophrenics. Finally, in a study comparing acutely ill schizophrenic patients with controls, Post et al. (1975) found no statistical difference in CSF 5HIAA (after probenecid). Low CSF 5HIAA is reportedly associated with increased risk for violent suicide (van Praaz, 1986) and poor impulse control. This has been independently demonstrated in several schizophrenic populations with violent attempts or completed suicides, controlled for height and sex (Banki et al., 1984; Ninan et al., 1984); these authors suggested that the low CSF 5HIAA relates to a vulnerability to commit violence against self or others, independent of diagnosis. In one study decreased hypothalamic 5 H T was found in suicide victims (Korpi et al., 1986). The association with increased arousal in schizophrenics and the increased suicide risk and loss of impulse control in general make CSF 5HIAA worthwhile to pursue in schizophrenia.

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JOEL GELERNTER AND DANIEL P. VAN KAMMEN

C. PLATELET 5HT UPTAKEAND RELATED STUDIES Several groups have focused on platelet 5HT uptake and blood as possible markers of biological abnormalities in schizophrenia. The most consistent finding in this area has been for decreased platelet 5HT uptake schizophrenic patients (see Table 11). While it has been shown that at least chlorpromazine has an effect on platelet 5HT uptake (Arora and Meltzer, 1983), some groups have found this abnormality in drug-free patients and others in medicated patients. A possible explanation for this discrepancy is suggested by Humphries et al. (1985), who found that the value obtained for platelet 5HT uptake can depend on when during the day the sample is drawn. Some groups (Meltzer et al., 1981b; Arora and Meltzer, 1982) did not find abnormal 5HT uptake in platelets from schizophrenics at all. TABLE I1 A N D WHOLE-BLOOD SEROTONIN FINDINGS’ PLATELET References

Number of patients (N)

Results

Feldstein ef d.(1959)

22 sz (chronic) 15 “acute psychotic’’ 17 c (med status not given)

“Acute psychotic” patients had lower blood 5HT than chronic sz or controls

Todrick et af. (1960)

29 sz (med status not given) 20 c

No significant difference in schizophrenics for platelet 5HT

Garelis et d.(1975)

16 sz (chronic): 12 m, 7 df (3 in both groups) 20 c

Higher blood 5HT in chronic sz df patients than in either m patients or controls

Joseph et d.(1977)

10 df sz (chronic) 6 m sz (chronic) 14 c

Unmed: no elevated platelet 5HT. Med: elevated platelet 5HT. 5HT and M A 0 levels not related

DeLisi d d.(1981a)

33 m sz (6 of these also studied df) 23 c

Increased whole-blood 5HT in sz patients with abnormal C T scans; no difference in 5 H T concentration between medicated and df conditions

Freedman d al. (1981)

33 chronic sz; 32/33 20 undiff. 13 paranoid 11 c

Elevated mean platelet 5HT in patients, more marked in the CU group: lower platelet M A 0 activity in patients

Stahl ct al. (1983)

19 df sz and sa 17 m sz and sa 25 c (56 other patients)

Increased platelet 5HT in chronic sz patients; antipsychotic medication did not alter platelet 5HT concentration. 5HT uptake did not differentiate between patient and control groups (continued)

327

SCHIZOPHRENIA TABLE I1 (continued) References Jackman et d. (1983)

Number of patients (N) 41 df sz and sa

34 c

Results Increased platelet 5HT found only in black patients. Platelet 5HT neg. correlated with lack of insight and conceptual disorganization. Subsample of 21 patients: no correlation between 5HT and C T scan findings

. Modai et ~ l (1979)

10 sz (acute)(all tested both m and df) 10 c

Platelet 5HT uptake in patients lower than in controls in both df and m conditions

Rotrnan Et ~ l (1980) .

22 df sz (acute) some of whom were also studied m 15 c

Platelet 5HT uptake lower in patient group; no correlation with BPRS scores during treatment

Meltzer et ~ l (1981b) .

12 sz (chronic) 7sz (acute) (plus other psychiatric patients) 20 c

No difference in platelet 5HT uptake or K, found in schizophrenics

Rotman ut ~ l(1982b) .

20 sz (10 df, 10 rn) and their families

Medicated patients had decreased platelet uptake at high 5HT concentration. 18/20 sz patients had lower 5HT uptake than their families at 5HT concentration
Arora and Meltzer (1982)

1 1 df sz (acute) , 32 df sz (chronic) 42c

No difference in platelet 5HT uptake between groups

. Rotrnan ut ~ l (1982a)

12 m sz 12 c

Platelet 5HT uptake decreased in , of 5HT patient group; V uptake was correlated with IMI binding

Lingjaerde (1983)

23 df sz 19 m sz 70 c

Decreased platelet 5HT uptake in df patients; increased K, in rn patients

Arora and Meltzer (1983)

19 sz, before and during CPZ treatment

No patientkontrol 5HT uptake difference, unrnedicated; platelet 5HT uptake decreased in CPZ-treated patients

6OC

Humphries et d. (1985)

23 psychiatric (various diagnoses) 22 c

Platelet 5HT uptake varies by time of day

“Abbreviations: c, control; df, drug free; m, medicated; sa, schizoaffective; sz, schizophrenic.

328

JOEL GELERNTER AND DANIEL P. VAN KAMMEN

Despite the decreased platelet uptake of 5HT in schizophrenics, net 5HT in their platelets has generally been found to be increased, as is whole-blood 5HT (Garelis et al., 1975;Joseph et al., 1977; DeLisi et al., 1981b; Freedman et al., 1981; Stahl et al., 1983;Jackman et al., 1983). This apparent inconsistency has been explained by the presence of an abnormal K , (e.g., Lingjaerde, 1983) as well as an altered V,,,. DeLisi et al. (1981) found that increased whole-blood 5HT was present in patients with abnormal C T scans, but this was not confirmed by Jackman et al. (1983). These abnormalities in platelet and blood serotonin are not unique to schizophrenia and have been found in other disorders, medical and psychiatric, as well.

D. SEROTONERGIC DRUGS Since the observation that serotonergic drugs can have psychotomimetic effects, it has been speculated that antiserotonergic drugs could have antipsychotic effects. There gave been two recent small studies investigating the effect of fenfluramine in schizophrenia: one on chronic patients (Shore et al., 1985) and one specifically investigating its effects on negative symptoms (Stahl et al., 1985b). Fenfluramine decreases CSF 5HIAA in man, and in animals it causes a decrease in whole brain 5HT. In the study by Shore et al. (1985a), fenfluramine did not significantly affect BPRS subscales, but there was a trend for improvement on the global BPRS. Stahl et al. (1985a) found trend-level improvement in negative symptoms with fenfluramine. S . R . Marder and D. P. van Kammen (unpublished data, 1978) observed improvement in two patients’ psychotic symptoms only after withdrawal from fenfluramine. A pilot study with setoperone showed promising results (Ceulemans et al., 1985). Setoperone is an antipsychotic drug with more pronounced 5HTblocking than DA-blocking properties. Thirty-four type I1 schizophrenic patients completed this study; mean BPRS improvement was 50%, compared with previous treatment with conventional antipsychotics. The authors felt that setoperone may have an “antiautistic” effect. An investigation of the “atypical” antipsychotics clozapine and loxapine showed that, after chronic or acute administration, these drugs caused downregulation of 5 H T ( S z ) receptors in rat brain, but no upregulation of D:! receptors (Lee and Tuang, 1984). These authors hypothesized that these drugs would be less likely to cause tardive dyskinesia than the standard antipsychotics. Thus, even though specifically serotonergic agents have not yet been demonstrated to be useful for the treatment of schizophrenia in most controlled studies, some antipsychotic drugs have significant effects on serotonergic systems that may be related to their therapeutic usefulness.

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There is a report using L-tryptophan (LTP) as an agent for serotonergic challenge in schizophrenic patients (Cowen et al., 1985) which demonstrated decreased G H response to LTP in schizophrenics compared to normal controls; the schizophrenics were receiving antipsychotics at the time of the investigation, so the results are difficult to interpret. Given the relationship between adrenergic function and G H release and the fact that most antipsychotics are potent a-receptor blockers (and, as the authors note, can have 5HT blocking properties as well), it seems more plausible that this finding is a result of the use of medication than a marker for schizophrenia.

E. SEROTONIN IN SCHIZOPHRENIA: DISCUSSION Serotonergic dysregulation is a factor in some schizophrenic patients (Table 111), whether it be-of primary or secondary importance; presumably, 5HT plays a permissive role in the regulation of DA activity. Increased CSF 5HIAA is associated with a family history of schizophrenia, chronic illness, and motor abnormalities such as mannerisms and posturing. Low CSF 5HIAA is associated with arousal and increased likelihood of violence to self or others, or with a more acute illness and better premorbid function. Low CSF 5HIAA is also found in patients with large lateral ventricles and cortical atrophy by C T scan. This seems inconsistent and suggests differences in patient samples. The meaning of increased whole-blood or platelet 5HT (Table 11) remains obscure, particularly in those with C T scan brain atrophy. One explanation may be that increased 5HT uptqke indicates less 5HT at the receptor sites, if platelet dynamics reflect neuronal mechanisms.

TABLE I11 SEROTONIN ABNORMALITIES IN SCHIZOPHRENIA ~

Brain serotonin and 5HIAA are increased in some areas CSF 5HIAA is lower in acute and paranoid patients and higher in chronic patients CSF 5HIAA is negatively correlated with agitation and arousal CSF 5HIAA shows increased variance in normals with schizophrenic relatives Fenfluramine may decrease negative symtoms Antipsychotic drugs can affect 5HT receptor populations Platelet serotonin is increased in some schizophrenic patients; platelet serotonin uptake can be decreased

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JOEL GELERNTER A N D DANIEL P. VAN KAMMEN

IV. Y -Amlnobutyrlc Acid

y-Aminobutyric acid (GABA) is a major inhibitory neurotransmitter in the CNS. Drugs that potentiate GABAergic transmission tend to have anxiolytic, anticonvulsant, and muscle relaxant properties, whereas drugs that inhibit GABA activity are known as convulsants and anxiogenics. Distributed very widely in the CNS, GABA inhibits dopaminergic transmission, among other functions. GABA receptors have so far been divided into two main groups, GABA-A and GABA-B. GABA-A receptors are sometimes associated with benzodiazepine (BDZ) receptors which, when bound with BDZ, increase the GABA-A receptor’s affinity for substrate (Enna, 1985). GABA-B receptors are associated with adenyl cyclase and may inhibit NE, DA, and 5HT release, whereas GABA-A receptors increase NE release (Suzdak and Gianutsos, 1985). GABA-A and GABA-B receptors have different distributions in the CNS (Enna, 1985). Development of GABAergic drugs with specificity for either receptor may help solve the confusing behavioral reports. Roberts (1972) hypothesized that decreased GABA could be involved in schizophrenia; strategies to study GABA function in schizophrenia were outlined by van Kammen (1977). It has been hypothesized that the relative DA excess proposed in schizophrenia stems from a GABA deficiency (van Kammen, 1977). If this were the case, CSF GABA levels could be altered; enzymes responsible for GABA metabolism could have abnormal activity in brain or CSF; and finally, GABAergic drugs should have an effect on schizophrenic symptoms. In this section, we review GABA-related studies in schizophrenia. However, the most robust findings linking altered CSF GABA with psychiatric illness to date are those of low GABA in depression (Gerner et a l . , 1984; Gerner and Hare, 1981; Gold et a l . , 1980). The importance of the GABA system in schizophrenia has been reviewed extensively (Enna, 1985; Garbutt and van Kammen, 1983; Meldrum, 1982).

A. CSFGABA Several groups have looked to the CSF as a direct reflection of GABAergic activity in the brain, with varying results. The first of these groups (Lichtshtein et al., 1978) examined CSF GABA in 17 schizophrenic patients compared to 9 normals. Although there were no significant differences between the schizophrenic and control subjects, it was noted that six of the seven lowest GABA levels were found among the schizophrenics. An enzymatic fluorometric assay was used for this study, and its methodology has been questioned. McCarthy et al. (1981) found increased CSF GABA by radioimmunoassay in chronic schizophrenics compared to all other groups; however, there were

33 1

SCHIZOPHRENIA

only 7 patients in that group (compared to 9 acute schizophrenics and 75 others). In a study where there was no significant difference in CSF GABA between drug-free and medicated psychotic patients and controls. Bowers et al. (1980a) reported a significant negative correlation between GABA and anxiety, agitation, and conceptual disorganization. Gattaz et al. (1986) studied CSF from 19 schizophrenic patients before and after 3 weeks of antipsychotic treatment; the medication did not have a significant effect on CSF GABA. They also reported a lack of correlation between GABA levels and changes in behavioral ratings. However, their standard for classifying a patient as drug free was 3 weeks without medication, which may not have been sufficient. Another study, with only 3 schizophrenic patients (out of 84 with various neurological and psychiatric disorders), found a trend for decreased GABA in the schizophrenic patients' CSF (Kuroda et at., 1982). Gold et al. (1980), in a larger study (comparing 14 psychotic patients with 17 who were depressed and 20 controls), found a trend for lower levels in depressed patients. In a comparison of 30 drug-free schizophrenic patients and 39 controls (van Kammen et al., 1982c), there was no significant difference in CSF GABA, but this was felt to be because the control subjects were older (see below). They found that recently ill schizophrenics had significantly lower GABA. GABA levels increased with duration of illness (Fig. 2). Gerner and Hare (1981) and Gerner et al. (1984) also found a negative correlation with age in normal and depressed subjects but not in schizophrenic patients. Gold et al. (1980) did not find any age-GABA relationship. Gerner et al. (1984) did not report altered CSF GABA in their schizophrenic group either. There was a trend for increased GABA with short-term pimozide treatment and a trend for positive correlation of GABA levels with psychosis. Blunted affect correlated with CSF GABA

4oor

r = .56

100

"=a p = I

0

I

I

5 10 15 YEARS DURATION OF ILLNESS

< ,001 I

20

FIG. 2. CSF GABA levels increased with duration of illness, which is defined by first appearance of psychotic symptoms.

332

JOEL GELERNTER AND DANIEL P . VAN KAMMEN

levels (van Kammen et al., 1982~).Bowers et al. (1980b) did not find a significant relationship with duration of illness, blunted affect, or premorbid functioning, but found agitation and conceptual disorganization (BPRS) to correlate with CSF GABA and to correlate inversely with plasma and CSF HVA. They obtained probenecid LPs which were done in the afternoon, when CSF GABA levels are somewhat higher than in the morning.

B . PLASMAGABA Plasma GABA may also reflect central function; this is supported by animal data. A study considering GABA levels in control subjects and patients with various psychiatric disorders (Petty and Sherman, 1984) found no statistical difference in mean levels of schizophrenics, but an increased scatter was seen. The validity of this method is supported by the finding of decreased plasma GABA levels in depressed patients also, in whom CSF GABA levels are known to be low, However, GABA has a short half-life, so single blood samples may not reflect actual or mean levels. In addition, there is a report measuring plasma glutamic acid decarboxylase (GAD, which catalyzes the synthesis of GABA from glutamic acid) in schizophrenia and other psychiatric illnesses. Kaiya et al. (1982) found that, while GAD levels were not statistically different between schizophrenic and control subjects, plasma GAD levels increased when the patients were given antipsychotic drugs with anticholinergics. In contrast, GAD levels were lower in depressed patients.

C . GABAERGICDRLJGS 1. Benzodiazepines Benzodiazepines have shown the most promise as adjunctive agents in the treatment of schizophrenia. Jimerson et al. (1982) found some improvement in two of five schizophrenic patients placed on high doses of diazepam. Lerner et al. (1979) compared haloperidol and diazepam in acutely psychotic schizophrenics and found them about equally effective. Nestoros (1980) added diazepam in high doses to antipsychotic drugs in their 10-patient sample of chronic schizophrenic patients. Seven of these experienced marked or moderate improvement, and none got worse. The improvement was seen in paranoid schizophrenic patients. Wolkowitz et al. ( 1986) found alprazolam added to an antipsychotic to be beneficial for both positive and negative symptoms in the two chronic schizophrenics they studied. In open studies,

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333

Beckmann and Haas (1980) found an antipsychotic effect for high doses of diazepam alone, especially in paranoid patients; Csernansky et al. (1984) added alprazolam to patients’ other medications and reported reduction of negative symptoms. Thus, there is some indication that patients who have not fully responded to antipsychotics do well with high doses of BDZs; BDZs in more usual doses may potentiate the effects of antipsychotics in some of these patients as well.

2 . Baclofen Baclofen is a GABA-B receptor agonist, and it is used as a muscle relaxant. Since an early positive study in 1975 (Fredericksen, 1975), results with this agent for schizophrenia have been negative. Drugs that enhance GABA activity in the brain have sometimes been associated with inducing either schizophreniform or toxic psychosis in schizophrenic patients, and baclofen is in this category. Simpson d al. (1976) studied 12 “treatment-resistant” schizophrenic patients treated with baclofen; 9 of these worsened, 2 showed no change, and 1 reportedly improved slightly. Davis et al. (1976) also reported worsening of schizophrenic symptoms with baclofen. D. P. van Kammen et al. (unpublished data, 1980) observed that a chronic paranoid patient who for years reported only an occasional residual auditory hallucination decompenstated severely with baclofen (Fig. 3). This agent, then, can provoke an exacerbation in schizophrenic symptoms.

3 . Other Agents Valproic acid (VPA) inhibits GABA transaminase, which is involved in the breakdown of GABA; thus it causes increased brain GABA levels. In a small double-blind trial of its effects in schizophrenia, VPA did not have an effect on BPRS ratings. This study used patients refractory to neuroleptic treatment (KO et al., 1985). Very few patients have been studied with muscimol. Tamminga et al. (1979, 1983) noted a worsening in psychosis with it. D. P. van Kammen gave it to two patients in a double-blind 4-week trial; one worsened as if intoxicated, and one was unchanged (unpublished data, 1979) (Fig. 4). Also of interest is that Hanada et al. (1984) found increased [SH]muscimolbinding in schizophrenic brains. y-Hydroxybutyrate, which, like y-butyrolactone, turns off DA neurons, showed variable antipsychotic and worsening responses, which sometimes resembled amphetamine effects (Schulz et al., 1981). It induced akathisia and other extrapyramidal side effects. The largely negative results with baclofen and VPA contrast with the studies with BDZs, which have been quite promising. Benzodiazepines are

Placebo 13

2mg

Pimozide

14 mg

.6 mg

I

98

105

112 119 126

I

133 140

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147

154 161

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175

DAYS

FIG 3. A 32-year-old white, single, male patient with chronic paranoid schizophrenia who reported mainly occasional voices was treated with baclofen (Lioresal), which led to a severe worsening of symptoms. H e heard the voice of the unit psychiatrist, believed others to be communists, and that God told him to leave. After discontinuation, an initial pimozide treatment was ineffective. A second attempt with pimozide decreased his psychotic behavior below baseline levels. O n day 119, he became acutely suicidal and eloped in spite of steady improvement. It was assumed that his dose of pirnozide (14 mg) was too high. Pimozide withdrawal was associated with drug withdrawal improvement.

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SCHIZOPHRENIA

w

z

9-

4 v,

$ I

Y P

7-

654-

a

u

5

35 DAYS

FIG. 4. A 21-year-old white, single, woman with paranoid schizophrenia who experienced only minimal psychotic symptoms during a drug-free evaluation responded to muscimol treatment with an acute psychotic worsening. Subsequently, tolerance developed until the next dose increase. The patient complained that it felt as if she was "walking under water." Besides increases in idiosyncratic psychotic symptoms, the patient also felt as if she was intoxicated.

anxiolytics, and anxiety is often a prominent feature in psychotic or prepsychotic conditions; it is unclear to what extent this explains the results. Added interest has come to GABA research by the recent discovery of an endogenous diazepam-binding inhibitor (Alho et al., 1985). So far there are no reports of this substance in man. Finally, GABA agonists have been used in tardive dyskinesia with mixed results (Casey et al., 1980; Gerlach et al., 1975; Stahl et ul., 1985a; Tamminga et al., 1979).

D. GABAERGICEFFECTS O F CLASSICAL ANTIPSYCHOTICS There are some reports of GABAergic effects of haloperidol in animals. Kornhuber et al. (1984) investigated the effects of chronic (39 days) haloperidol at two dosage levels (3 m g k g and 0.8 mgkg) in rats and discovered an increase in striatal GABA at the higher dosage level but not at the lower dosage. The GABA increase was felt to be indicative of a release of an inhibitory effect of DA neurons on GABA neurons. Rastogi et al. (1982) also studied the effects of fairly long-term (5 or 30 days) haloperidol treatment on brain GABA levels in rats; they used dosages of 1 m g k g and 10 mgkg.

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JOEL GELERNTER AND DANIEL P. VAN KAMMEN

With the lower dosage, given for 30 days, striatal GABA was increased after treatment. In rats treated with 10 m g k g of haloperidol for 5 days, GABA was increased in striatum, frontal cortex, and midbrain. An earlier report (Kim and Hassler, 1975) had demonstrated decreased GABA in striatum and substantia nigra in rats 1 hr after a single very large (10 mgkg) dose of haloperidol. The time course demonstrated for development of increased GABA is consistent with that of the onset of antipsychotic effects with this drug. Mao et al. (1977) studied the effects of haloperidol on GABA turnover and found that haloperidol treatment, whether acute (single dose) or chronic (1 week), shortens the turnover time in nucleus accumbens. Prolonged treatment increased turnover time in both substantia nigra and globus pallidus.

E. GABA IN SCHIZOPHRENIA: DISCUSSION van Kammen et al. (1982~)have presented evidence of decreased CSF GABA in young, recently ill, and good premorbid schizophrenic patients, whereas GABA levels were increased with duration of illness and chronic neuroleptic treatment. With aging, CSF GABA levels decrease in normals but increase in schizophrenic patients, perhaps as the patients progress from type I to type 11 disease. Bowers (1978a) reported that increased agitation was associated with lower GABA but higher CSF HVA levels. Decreased CSF GABA levels in both schizophrenic and affective disorder patients suggest that these two diagnoses share some disturbances, although the similarities change with the progression of illness. Certain neurological diseases are associated with decreased CSF GABA as well, e.g., Huntington’s disease, suggesting the nonspecificity of decreased GABA levels. Table IV summarizes GABAergic abnormalities in schizophrenia. State-dependent changes in GABA activity cannot be excluded. The inverse relationship of GABA activity compared to decreased DA activity with increasing duration of illness and the increased variance in plasma GABA are TABLE IV 7-AMINOBUTYRIC ACID ABNORMALITIES IN SCHIZOPHRENIA ~~~

~~

CSF GABA is lower in some young drug-free patients CSF GABA increases with duration of illness CSF GGBA is higher in patients with negative symptoms Plasma GABA levels fluctuate more in schizophrenics than in other groups GABA-B agonists (e.g., baclofen) can worsen schizophrenic patients GABA-A agonists (BDZs) can be effective in schizophrenia when added to classical antipsychotics Classical antipsychotics have GABAergic effects

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intriguing. GABAergic agents, with the exception of the BDZs, have largely failed to fulfill their promise of therapeutic effects in schizophrenia. The worsening with baclofen, a GABA-B agonist , could be a diagnostic challenge test similar to that with amphetamine (Angrist and van Kammen, 1984) but without its controlled-drug stigma. Whether GABA-B antagonists can have antipsychotic effects remains untested. For the most part, studies relating GABA to schizophrenia have not yielded consistent results so far. Methodological differences abound in CSF GABA studies. In evaluation studies of plasma, CSF, or brain GABA levels, it is important to realize that GABA levels increase as the samples stay at room temperature for longer than 1 hr (Hare d al., 1979). Animal studies suggest that after a single dose haloperidol can decrease brain GABA levels, but with longer-term use GABA levels increase in some areas. This is consistent with GABA having a role in response to classical antipsychotic medications. However, these effects are seen most clearly in animals when they are given much higher doses on a m g k g basis than are therapeutic in humans, so whether or not they really apply in the treatment of schizophrenia is open to conjecture. Presumably this result could apply at least to those individuals proven to respond only to unconventionally high doses of antipsychotics, doses that far exceed those necessary to saturate the brain’s DA receptors. An important factor inhibiting our understanding of GABAergic mechanisms in schizophrenia is that while GABA serves as an inhibitory neurotransmitter on a single synapse basis in some DA systems, it is also capable of having the opposite effect of potentiating DA transmission (for review, see Garbutt and van Kammen, 1983). We are left with an unclear picture of GABA’s role in schizophrenia (van Kammen d al., 1982~).In any event, the evidence does not support a primary GABA disorder in schizophrenia.

V. Conclusions

Monoamines regulate or modify many behaviors, so pharmacological interventions that affect monoamine activity can change them. These relationships have spawned various hypotheses which emerging biological findings in schizophrenia have unseated sequentially. Even though we might try to blame our inability to nail down a comprehensive simple neurotransmitter hypothesis on previous chronic antipsychotic treatment, the evidence that neuroleptics may be culpable, rather than state of illness, length of treatment, or the duration of the illness itself is not very strong. Besides the multiple

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interactions between the different systems, peripheral and central monoaminergic mechanisms may influence each other, but they are not necessarily regulated the same way. Conceivably, NE, 5HT, and GABA fulfill permissive roles, regulate the impact of the environment on the organism, and may affect the sensitivity of the DA system. Considering that as we learn more the interrelationships of neurotransmitter systems that figure in schizophrenia seem to get more and more complicated, one can hardly be blamed for pessimism for the prospects for a unified field theory of schizophrenia. Many neurotransmitter systems proposed initially to be responsible for schizophrenic symptomatology have found more important places in the study of affective disorders, with abnormalities in both disorders: an NErelated hypothesis for schizophrenia was soon abandoned (at first) for a decreased NE hypothesis in depression (Bunney and Davis, 1965; Schildkraut et al., 1965); low CSF GABA and 5HIAA levels have been found most consistently in affective disorder patients. The reviewed data of NE, 5HT, and GABA activity suggest that unstable (acute) schizophrenic patients may biologically resemble affective disorder patients early in the illness. Meltzer et al. (1981a,b) pointed out that there are some biological similarities (in, for example, CPK increases and M A 0 activity) between the two major “functional psychoses.” Further support for this relationship appears in the study by Kendler et a/. (1985), who found that schizophrenic patients have an increased incidence in their relatives of schizoaffective disorder, as well as schizophrenia and paranoid and atypical psychoses, although there was no increase in affective disorders. Worse yet, some of these findings are not only present in schizophrenia and affective disorders (PGEI-induced stimulation of cAMP production, elevated CNS NE, decreased urinary MHPG, decreased CSF GABA) but also in hypertension (PGEl-induced stimulation of cAMP production, increased ISHIDHE binding), ‘‘type A” personality (increased whole-blood 5HT), brain atrophy (low CSF SHIAA, NE, MHPG, and DBH), or suicide (low CSF 5HIAA). None of these findings are specific for schizophrenia; for that matter, it is possible that none of the DA system-related findings are either (Table V). Certain of these findings can be sorted out by recognizing subgroups of schizophrenic patients which are evident clinically. Groups of patients at opposite ends of the behavioral spectrum might be expected to differ biologically as well. For example, the reports of increased or decreased NE, 5HT, and GABA activity tend to be consistent with a division of schizophrenics into subgroups connoting hyper- or hypoarousal (Kornetsky and Mirsky , 1966; Zahn d al., 1981). Other clinical dichotomies include acute versus chronic, good antipsychotic responders versus poor responders, patients with good

339

SCHIZOPHRENIA TABLE V VARIABLES IN SCHIZOPHRENIA' STATE-AND TRAIT-DEPENDENT

Variable NE (brain, CSF, plasma) MHPG (brain, CSF, plasma, urine) DBH (brain, CSF, plasma) a2Receptor binding (platelets) PGE,-stimulated CAMPproduction 5HT (blood, platelet) 5HIAA (CSF) GABA (CSF, plasma)

State dependent

Trait dependent

Yes Partially No Possibly Possibly Yes Possibly Possibly

No

Yes Probably Probably Yes Possibly

-

"All of these have been found to be different in schizophrenic patients compared to normals. None of the findings are specific for schizophrenia.

premorbid function versus those with impaired function, those early in the illness versus those in its later stages, and patients without brain atrophy versus those with it. A less clearly dichotomous grouping opposes patients with predominantly positive symptoms versus those with predominantly negative symptoms; the grouping is less clearly dichotomous because positive and negative symptoms can coexist. Certain of these groups of symptoms have been roughly cordoned into the type I and type I1 syndromes as proposed by Crow (1980); although this is a valuable distinction to make conceptually, it is also important to recognize when it does not fully apply clinically. Negative symptoms may co-vary with psychotic symptoms in paranoid and undifferentiated schizophrenic patients, for example (Rosen et al., 1984). We propose yet another dichotomy: stable versus unstable. We suggest that the stable and unstable conditions can be defined biochemically and by drug response (van Kammen et al., 1985b). In general, physiological variables change with psychological fluctuation, but in stable psychological states-even when they are abnormal-physiological measures remain normal. If this holds true for the neurochemistry as well, it may explain why so many chronic schizophrenic and remitted acute patients who are clinically stable can show no difference compared to normals in biochemical findings (Castellani et al., 1982) or in response to pharmacological challenge tests (Angrist et al., 1980; Janowsky and Davis, 1976; van Kammen et al., 1982a,b), thus the difficulty in identifying useful trait markers. The systems reviewed here regulate the interaction between the organism and its environment. If these systems are unstable, the organism cannot deal

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effectively with the demands put upon it. The variable CSF NE levels in a given patient or the increased variance of plasma GABA levels are consistent with biochemical instability too. With increasing chronicity, many patients become more stable-less responsive to antipsychotics, but also less psychotic-and may develop more negative symptoms. Antipsychotic drugs can induce stability and negative symptoms as well. GABA-A receptors may be involved in these negative symptoms (Csernansky et al., 1984). Chronically hospitalized patients frequently display the type I1 syndrome, which can become rather stable in the natural history of the disease. We expect to find clinical instability to be associated with state-dependent markers. In stable patients normal or low values are found, while in unstable patients increased values or normal values with increased variance are seen. Therefore, the discrepant biological findings in schizophrenia related to the NE, 5HT, and GABA (or DA) systems may be explained by an episodic instability in neurochemical and clinical states, particularly in young, good premorbid, medication-responsive schizophrenic patients. We have stressed elsewhere the need for longitudinal assessment in schizophrenia to rule out state-dependent effects, even though in normal subjects the variables under study are stable. Also, a diverse mix of drug-free patients should be included to identify the clinical conditions associated with each variable. King et al. (1984) developed a mathematical model for DA which could explain the chaotic and unstable phenomena. This model may be relevant for the systems reviewed here as well. Because several different biochemical parameters are disturbed, presumably for different endogenous reasons, it is possible that some of these effects could be caused or aggravated by a variety of factors such as a membrane disorder (Stevens, 1972), cell loss due to some sort of CNS viral infection (Torrey et af., 1982), prenatal or perinatal trauma (Reveley et al., 1984), or an autoimmune disorder (Rudin, 1979). Some of the biochemical findings might be consequences of environmental deprivation or overstimulation or of the patients’ place in their social network. These disturbances, and others as well yet unidentified, may contribute to or result from stress sensitivity, i.e., neurochemical instability, leading to the syndrome of schizophrenia. The last decade has seen a cascade of new approaches and new technologies applied in the psychobiological evaluation of schizophrenia, and the enthusiasm over single neurotransmitter system hypotheses has abated. Understanding how these biochemical findings relate to course , relapse, prognosis, drug response, and side effects in schizophrenia is the issue now. None of these findings appears to be specific for schizophrenia when taken in isolation, and none begins to explain schizophrenic symptomatology in full. The implied dysregulation or instability of the systems may take us closer. It appears that schizophrenia is a multineurotransmitter system disease. It could be caused by a factor that interferes with or interacts with any of these systems.

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A Acetyl ethyl tetramethyl tetralin, neuronal storage disease and, 199 Acetylation, neurotoxin-binding site and, 125 Acetylcholine muscular dystrophy and, 8 neuronal storage disease and, 228, 230 retinal transplants and, 282 Acetylcholine receptor, neurotoxin-binding site and, see Neurotoxin-bindingsite Acetyltransferase, neuronal storage disease and, 228 Acid phosphatase, neuronal storage disease and, 198, 200, 201 Aconitine, batrachotoxin and, 78 binding site, 89 electrophysiological analysis, 81, 85 interactions, 98, 103 Activation batrachotoxin and, 78, 84 calcium and, 165, 173, 181, 182 neurotoxin-binding site and, 128, 129, 133, 136 Adenyl cyclase, schizophrenia and, 318, 329 Adenylate cyclase muscular dystrophy and, 12 neuronal storage disease and, 226 Adhesion muscular dystrophy and, 10, 14 retinal pigment epithelium and, 296 Adipocytes, muscular dystrophy and, 9 Adolase, muscular dystrophy and, 3 Adrenal hypoplasia, muscular dytrophy and, 25, 26, 49 a-Adrenergic agonists, calcium and, 181 Adrenergic drugs, schizophrenia and, 321, 322, 329 fl-Adrenergic receptor monoamines and, 262 neurotoxin-binding site and, 120 After hyperpolarization (AHP), calcium and, 164, 166-168, 170-172 349

Aging calcium and, 171, 172, 183 monoamines and, 269 Agitation, schizophrenia and, 325, 331, 336 Agraphia, thalamic amnesia and, 255 Akathisia, schizophrenia and, 321-323, 333 Alcohol calcium and, 162 behavioral effects, 163 biochemistry, 174, 179, 181 electrophysiology, 169, 171 schizophrenia and, 321, 323 thalamic amnesia and, 245, 246, 248 Alexia, thalamic amnesia and, 255 Alkanols, calcium and, 177 Alkylation, neurotoxin-binding site and, 151, 152 models, 149 proteolytic fragments, 136 reducible disulfide, 132, 133, 135 synthetic peptides, 144 Alleles muscular dystrophy and, 57-59 neuronal storage disease and, 221 retinal transplants and, 287 Allosterism batrachotoxin and, 78, 111 electrophysiological analysis, 86 interactions, 98, 99, 102, 106, 107 lipids, 109 neurotoxin-binding site and, 118 Alprazolam, schizophrenia and, 332, 333 Amacrine cells peripheral nerve bridges and, 302 retinal transplants and, 299, 300 Amineptine, monoamines and, 263-265 Amino acids batrachotoxin and, 93, 112 muscular dystrophy and, 5, 33, 53, 62 neurotoxin-binding site and, 118 comparison, 138-142 curaremimetic neurotoxins, 122, 124, 129

350 models, 149 mutagenesis. 147 nicotinic acetylcholine receptor, 120 proteolytic fragments, 137, 138 reducible disulfide, 135 Amino groups, batrachotoxin and, 89 y-Aminobutyric acid calcium and, 164, 168 schizophrenia and, 310, 311, 329, 330, 336-340 antipsychotics, 335, 336 CSF, 330-332 drugs, 332-335 plasma, 332 Aminorransferase. muscular dystrophy and, 3 Ammonium, neurotoxin-binding site and, 133, 150 Amnesia schizophrenia and, 321 thalamic, see Thalamic amnesia Amphetamine monoamines and, 260-263 schizophrenia and, 315, 333, 337 Amygdala neuronal storage disease and, 208 thalamic amnesia and, 252, 254 Anesthetics batrachotoxin and, 99-102 calcium and, 169, 184 Anorectic activity. monoamines and, 262, 265 Anterior chamber, retinal transplants and, 283, 284 Antibodies muscular dystrophy and, 17, 33, 62 neuronal storage disease and, 211. 227 neurotoxin-binding site and, 154 models, 149 nicotinic acetylcholine receptor, 122 probing, 145-147 reducible disulfide, 135 subunit, 131 retinal transplants and, 287, 299 Anticholinergics, schizophrenia and, 332 Anticonvulsanu batrachotoxin and, 101, 102 calcium and, 162, 174 Antidepressants, monoamines and, 269 Antigens muscular dystrophy and, 14

INDEX neurotoxin-binding site and, 145, 146 retinal transplants and, 287 Antipeptide antibodies, neurotoxin-binding site and, 146 Antipsychotic drugs, schizophrenia and, 338, 340 y-aminobutyric acid, 331-333, 335-337 norepinephrine, 312, 314-317, 319, 321, 323 serotonin, 324, 328, 329 Anxiety, schizophrenia and, 325, 331 Anxiogenics, schizophrenia and, 329 Anxiolytic activity, monoamines and, 271, 272 Anxiolytics, schizophrenia and, 329, 335 Aplysia, calcium and. 164, 165, 168, 169 Apomorphine. monoamines and, 262 Arginine, neurotoxin-binding site and, 125 Artificial sea water, batrachotoxin and, 81 Arylpiperazine, monoamines and, 274 Ataxia, neuronal storage disease and, 221 ATP calcium and, 172, 189 muscular dystrophy and. 12 ATPase calcium and, 180, 184 muscular dystrophy and, 12 Atrophy, schizophrenia and, 338, 339 norepinephrine, 315-317 serotonin, 329 Autopsy, schizophrenia and, 316, 324 Autoradiography, retinal pigment epithelium and, 296, 297 Autosomes, muscular dystrophy and, 6 gene location, 18-24 mutation, 48, 55 Axonal torpedoes, neuronal storage disease and, 215 Axons neuronal storage disease and, 194 dysfunction, 231-233, 235 neuroscience, 238 spheroid formation, 213-217 structural changes, 201, 207, 211. 213, 218 therapy, 236 peripheral nerve bridges and, 302-305 retinal transplants and, 282, 299-301 thalamic amnesia and, 253

351

INDEX

B Baclofen, schizophrenia and, 333. 337 Bacteria muscular dystrophy and, 32, 33 neurotoxin-binding site and, 148 Barbiturates, calcium and, 162, 184 behavioral effects, 162, 163 biochemistry, 172, 174, 178 electrophysiology, 164, 169, 171 Batrachotoxin, 11, 77-79, 112 binding site microenvironment. 93-97 protonation, 90-93 structure-activity relationships, 86-90 electrophysiological analysis activation, 82-84 general effects, 79-81 reversibility, 82 selectivity reduction, 85 single-channel conductance, 85, 86 stimulation, 82 interactions anesthetics, 99-102 pyrethroid insecticides, 102-105 saxitoxin, 105, 106 tetrodotoxin, 105, 106 toxins, 97-99, 107, 108 lipids photoaffinity, 109, 110 purified sodium channel, 110, 111 solubilization. 108, 109 Batrachotoxin A (BTX-A), 86, 88, 94 Batrachotoxin A benzoate (BTX-B) binding site, 91-93 electrophysiological analysis. 81 interactions, 99-102, 104, 106, 107 lipids, 110 Batrachotoxin A 20a-N-methylanthranilate (BTX-NMA), 94, 95 Batrachotoxin A 20a-o-azidobenzoate, 109, 110

Batrachotoxin methiodide, 88 Becker muscular dystrophy, see Duchenne muscular dystrophy Benzodiazepine calcium and, 162, 184 behavioral effects, 162, 163 biochemistry, 172, 174, 175, 178 electrophysiology, 166, 167, 170 monoamines and, 270, 271

schizophrenia and, 329. 332, 333, 337 Bepridil, calcium and, 176 Blindness, neuronal storage disease and, 192 Bone marrow muscular dystrophy and, 63, 64 neuronal storage disease and, 236 Brachium, peripheral nerve bridges and, 305 Brain damage, calcium and, 162, 163, 171, 183

Brain stem neuronal storage disease and, 208, 236 retinal transplants and, 281 thalamic amnesia and, 247 Bromelain, neurotoxin-binding site and, 136 Bruch’s membrane, retinal pigment epithelium and, 296, 297 a-Bungarotoxin, neurotoxin-binding site and, 132, 133 curaremimetic neurotoxins, 122 models, 150 monoclonal antibodies, 145, 146 mutagenesis, 147, 148 proteolytic fragments, 136, 137 synthetic peptides, 143-145 Buspirone, monoamines and, 264, 270, 272-274

Butaclamol, monoamines and, 268, 276 Butanol, calcium and, 177 7-Butyrolactone, schizophrenia and, 333

C Calcium batrachotoxin and, 80, 85, 86, 101 muscular dystrophy and, 12, 18 neuronal storage disease and, 225, 229, 230, 234, 235

Calcium. sedative-hypnotic drugs and, 161, 162, 183-185 behavioral effects, 162, 163 biochemistry, 172 influx, 172-175 intracellular regulation, 178-180 receptors, 176-178 second messengers, 180-183 electrophysiology, 163, 166-170 aging, 171, 172 currents, 163-165 effects, 170, 171 second messengers, 165

352

INDEX

Calmodulin calcium and, 179, 180 muscular dystrophy and, 18 Carbamazepine, batrachotoxin and, 102 Carbamidomethylation, neurotoxin-binding site a n d , 126 Carbamylcholine, neurotoxin-binding site and, 153 Carbohydrates neuronal storage disease a n d , 225 neurotoxin-binding site a n d , 120, 127, 137 Carboxymethylation, neurotoxin-binding site and, 144 Cardiac complications, muscular dystrophy and, 2, 3 Cardiotoxins. neurotoxin-binding site and, 124 Carrier identification, muscular dystrophy and, 6 , 7 applications, 59-61 future prospects, 64 principles, 56-59 Catalysis. schizophrenia and, 315, 318 Catecholamine batrachotoxin and, 101 monoamines and, 260, 262, 267 schizophrenia and, 313, 320 Cauterization, retinal pigment epithelium and, 296 cDNA muscular dystrophy and cloning. 32, 33, 43-45 future prospects, 62 gene location, 30 mutation, 50, 52 neurotoxin-binding site and, 120, 132, 147, 148 Central nervous system calcium and, 163, 165, 166, 183 monoamines and, 276 neuronal storage disease and animal models, 197, 199, 200 disordered function, 220-222 dysfunction, 231, 235 gangliosides, 225. 227 neuroscience, 238 structural changes, 201, 208 therapy, 236, 237 peripheral nerve bridges and, 303-305 retinal transplants and, 281. 282, 297, 306 connection to host brain, 300, 301

functional significance, 301, 302 graft differentiation, 298-300 methods, 298 vitreal chamber, 287 schizophrenia a n d , 310, 340 y-aminobutyric acid, 329 norepinephrine, 312, 320 thalamic amnesia and, 245, 250, 253 Cerebellum calcium and, 179, 180 neuronal storage disease and, 208, 221 thalamic amnesia and, 246 Cerebral cortex batrachotoxin and, 89, 100 calcium and, 166, 173 neuronal storage disease and disordered function, 221, 223 dysfunction, 231, 232 gangliosides, 227, 228 structural changes, 207, 211 Cerebrosides, neuronal storage disease and, 205 Cerebrospinal fluid calcium a n d , 174 neuronal storage disease and, 236 schizophrenia and, 338-340 yaminobutyric acid, 330-332, 336, 337 norepinephrine, 312-316, 321-323 serotonin, 324, 325, 329 Ceroid lipofuscinosis, see Neuronal ceroid lipofuscinosis Cesium, batrachotoxin and, 85 Chlordiazepoxide. calcium and, 175 m-Chlorophenylpiperazine, monoamines and, 272, 273 Chloroquine, neuronal storage disease and, 199 Chlorpromazine, schizophrenia and, 317, 318, 320, 326 Cholesterol batrachotoxin and, 87 neuronal storage disease a n d , 205, 213, 228 Choline, neuronal storage disease and, 228, 230 Cholinergic agonists calcium and, 181 neurotoxin-binding site a n d , 128, 152 Cholinergic binding sites models, 149, 150 monoclonal antibodies, 145-147

INDEX mutagenesis, 148 reducible disulfide, 134, 135 synthetic peptides, 144 Cholinergic function, neuronal storage disease and, 227-229, 232, 233 Cholinergic ligands, neurotoxin-binding site and, 133, 138, 152 Cholinergic systems, monoamines and, 260 Chromatin, muscular dystrophy and, 22 Chromosomes, muscular dystrophy and basic defect, 17, 18 cloning, 33, 37-41, 43, 45 future prospects, 57, 58 gene location, 18, 19, 21, 22, 25-29 mutation, 48, 52 Chronic granulomatous disease, muscular dystrophy and, 25, 49, 50 Chronic neuronal effects, calcium and, 170, 171 Chunking, thalamic amnesia and, 248 Cinanserin, monoamines and, 276 Circular dichroism spectroscopy, neurotoxin. binding site and, 121 Claustrum, neuronal storage disease and, 208 Clonazepam, calcium and, 166, 170, 171 Clonidine monoamines and, 272, 273 schizophrenia and, 318-321, 323 Cloning batrachotoxin and, 78, 85 muscular dystrophy and, 2, 32-34 basic defect, 9, 11, 14, 16 BB deletion, 34-36 expressed sequences, 42-45 future prospects, 61, 64 gene location, 25-30 mutation, 49, 51, 52 PERT, 40-42 translocation, 57-40 neurotoxin-binding site and, 120 Clozapine, schizophrenia and, 328 a-Cobratoxin, neurotoxin-binding site and, 153 curaremimetic neurotoxins, 122, 123, 125-128 models, 149 subunit, 132, 133 synthetic peptides, 143, 144 Cobrotoxin, neurotoxin-binding site and, 122, 128

353

Colcemid, neuronal storage disease and, 226 Collagen, muscular dystrophy and, 11 Color-blind gene, muscular dystrophy and, 4, 32 Concanavalin A, muscular dystrophy and, 13, 14 Conditioning, thalamic amnesia and, 251 Connective tissue muscular dystrophy and, 3, 10, 13 retinal transplants and, 283 Contralateral brain disease, thalamic amnesia and, 255 Contralateral tectum, retinal transplants and, 300 Convulsants calcium and, 169, 174 schizophrenia and, 329 Cornea, retinal transplants and, 284, 296 Cortex, calcium and, 179, 180 Cortical atrophy, schizophrenia and, 315, 329 Creatine kinase, muscular dystrophy and, 3, 5, 7, 8 basic defect, 9, 14 carrier identification, 60 Cross-linkage, neurotoxin-binding site and curaremimetic neurotoxins, 126 nicotinic acetylcholine receptor, 120 reducible disulfide, 135 subunit, 133 synthetic peptides, 144 Crossover, muscular dystrophy and, 54, 58, 60 Cross-tolerance, calcium and, 162, 175 CT scans, schizophrenia and norepinephrine, 315, 316, 323 serotonin, 328, 329 Curaremimetic neurotoxins, 118, 122-131, 133 Cyanogen bromide, neurotoxin-binding site and, 135, 138 Cyclic adenosine monophosphate calcium and, 165, 180, 182 neuronal storage disease and, 226 schizophrenia and, 318 Cypermethrin, batrachotoxin and, 103, 104 Cysteine batrachotoxin and, 93, 96 neurotoxin-binding site and, 151, 152 amino acid sequences, 138, 139, 142 models, 149, 150 mutagenesis, 147, 148 reducible disulfide, 135, 136 synthetic peptides, 144

354

INDEX

Cytoplasm calcium and, 184 muscular dystrophy and, 44 neuronal storage disease and animal models, 198, 200 dysfunction, 230 neuroscience. 193, 236 structural changes, 201, 205 neurotoxin-binding site a n d , 119-122. 147 retinal pigment epithelium and, 296, 297 Cytosol calcium and, 179-181. 183 muscular dystrophy and, 3 Cytosomes, neuronal storage disease and disordered function, 223 neuroscience, 238 structural changes, 201. 205, 206, 210, 215 Cytotoxicity, neuronal storage disease and, 193 dysfunction, 230, 231 structural changes, 206, 207 Cyrotoxins, neurotoxin-binding site and, 124

D Deafferentation, neuronal storage disease and, 213, 228 Degeneration peripheral nerve bridges and, 302 retinal transplants and, 298, 306 Degradation calcium and, 182 muscular dystrophy and, 10, 11, 17 neuronal storage disease and, 205, 224 Deletion muscular dystrophy and, 4, 5 basic defect, 17 carrier identification. 59 cloning, 39-42, 44, 45 future prospects, 61, 63 gene location, 18, 22, 23, 25, 26 mutation, 46, 48-54 neurotoxin-binding site and, 147 Deltamethrin, batrachotoxin a n d , 103 Delusion, schizophrenia and, 325 Dendrites calcium and, 164 neuronal storage disease a n d , 192, 194 animal models, 200 dysfunction, 230-232

neuroscience, 238 structural changes, 201, 207, 213, 215, 217-220 therapy, 236 retinal transplants and, 299 Dendritogenesis, neuronal storage disease and, 200, 225, 237, 238 a-Dendrotoxin. neurotoxin-binding site and, 133 Dependence, calcium and, 162, 183, 185 behavioral effects, 163 electrophysiology, 170, 171 Dephosphorylation, calcium and, 165, 182 Depolarization batrachotoxin and binding site, 88 electrophysiological analysis, 80-83 interactions, 97-101 calcium a n d , 184 biochemistry, 172-174, 176, 182 electrophysiology, 163, 164, 167, 170-172 neuronal storage disease a n d , 229, 230 neurotoxin-binding site and, 117, 122 Depression monoamines and, 269 schizophrenia and y-aminobutyrk acid, 330-332 norepinephrine, 318-320 serotonin, 325 Deprotonation. batrachotoxin and, 93 Desensitization, neurotoxin-binding site and, 118, 119. 154 Desipramine, monoamines a n d , 262, 263, 265 Desmethylimipramine, monoamines and, 268, 269 Diacylglycerol, calcium and, 165, 181 Diaschesis, thalamic amnesia and, 255 Diazepam calcium and, 170, 178, 184 monoamines and, 272 schizophrenia a n d , 332, 333, 335 Diencephalon schizophrenia and, 316 thalamic amnesia and, 246, 255 clinical observations, 246, 247, 250 experimental studies, 252 Differentiation muscular dystrophy and, 9 retinal transplants a n d , 281. 297-300 Dihydrobatrachotoxin, 87, 88, 109

355

INDEX Dihydropyridines(DHP), calcium and, 176-178 Diltiazem, calcium and, 176 Diltiazepam, calcium and, 176 Dipeptidyl aminopeptidase, muscular dystrophy and, 13 Diphenylhydantoin, batrachotoxin and, 102 Disinhibition, calcium and, 172 Distal hypertrophy, peripheral nerve bridges and, 302 Disulfides, neurotoxin-binding site and, 151, 154 amino acid sequences, 138 curaremimetic neurotoxins, 126, 129 models, 150 monoclonal antibodies, 146 mutagenesis, 147 reducible, 133-136 synthetic peptides, 144 Dithiothreitol, neurotoxin-binding site and, 133, 135 DNA muscular dystrophy and, 2, 4, 7, 8 basic defect, 18 carrier identification, 56 cloning, 32-36, 38, 40-45 future prospects, 62 gene location, 21, 22, 25-30 mutation, 48, 52 neurotoxin-binding site and, 147 DOPAC, monoamines and, 264, 267 Dopamine monoamines and, 260 receptors, 268, 269 uptake, 262-265, 267 retinal transplants and, 282 schizophrenia and, 310, 311, 338 y-aminobutyric acid, 330, 333, 335, 337 norepinephrine, 311, 314-316, 323 serotonin, 328, 329 Dopamine P-hydroxylase (DBH), schizophrenia and. 311, 515-318, 323 Dopaminergic cells, neuronal storage disease and, 227 Dopaminergic systems, monoamines and, 260, 261, 265 Dorsal lateral geniculate nucleus, peripheral nerve bridges and, 298, 300 Dorsal terminal nucleus, retinal transplants and, 300 Dorsomedial thalamic nucleus, thalamic amnesia and, 251

Drosophilu, neurotoxin-binding site and, 138, 139, 142 Drugs, monoamines and, see Monoamines, drugs and Duchenne muscular dystrophy, 2 basic defect, 8, 9 gene expression, 9, 10 membranes, 11-14 protein electrophoresis, 15-18 protein metabolism, 10, 11 carrier identification applications, 59-61 principles, 56-59 carriers, 6-8 clinical features, 2-4 cloning, 32-34 BB deletion, 34-36 expressed sequences. 42-45 PERT 87-XJ region, 40-42 t(X;21) translocation junction, 37-40 future prospects, 61-64 gene location, 18 female translocations, 18-24 linkage analysis, 26-29 male deletions, 25, 26 X chromosome, 29-32 genetics, 4-6 mutation, 46 female translocation, 46-48 male deletions, 48-54 recombination, 54, 55 prenatal diagnosis applications, 59-61 principles, 56-59 Duplications, muscular dystrophy and future prospects, 63 mutation. 50-54 Dystonia, neuronal storage disease and, 220

E Ectopic tissues peripheral nerve bridges and, 302 retinal transplants and, 284, 285 EDTA, retinal pigment epithelium and, 296 EGTA, calcium and, 168, 169 Electroencephalographic studies, neuronal storage disease and, 221, 222, 227 Electron microscopy muscular dystrophy and, 12

356 neuronal storage disease and disordered function, 222 dysfunction, 231, 232 structural changes, 201-203. 205, 210, 211, 213, 216, 219 neurotoxin-binding site and, 131, 132 retinal transplants and, 284, 287, 305 Electrophoresis batrachotoxin and, 109, 110 muscular dystrophy and cloning, 38, 41 cultured cells, 16, 17 future prospects, 62 gene location, 27 muscle tissue, 17, 18 protein, 15, 16 neuronal storage disease and, 196 neurotoxin-binding site and, 119, 132, 136 Electrophysiology calcium and, 162, 163, 166-170, 183 aging, 171, 172 biochemistry, 175 currents, 163-165 effects, 170, 171 second messengers, 165 neuronal storage disease and, 221-224, 232 retinal transplants and, 301 thalamic amnesia and, 251 Electroretinogram, retinal transplants and, 301 Embryonic tissue, retinal transplants and, 281-283 central nervous system, 297, 298, 300 vitreal chamber, 285 Emery-Dreyfuss dystrophy, 4, 63 Encephalopathies, neuronal storage disease and, 220 Endoglycosidase, neurotoxin-binding site and, 137 Endoplasmic reticulum calcium and, 172, 179-181 neuronal storage disease and, 201 Endothelial cells, neuronal storage disease and, 236 Enucleation, retinal transplants and, 300 Enzyme replacement therapy, neuronal storage disease and, 236, 237 Enzymes calcium and, 165, 181, 182

INDEX monoamines and, 263 muscular dystrophy and, 3, 7 basic defect, 9, 11, 13, 14 carrier identification, 56 cloning, 34, 41 future prospects, 63 gene location, 27-29 neuronal storage disease and, 192, 193 animal models, 196-199 disordered function, 220, 221 gangliosides, 224 neuroscience, 237, 238 structural changes, 207 therapy, 235 neurotoxin-binding site and, 129 Epinephrine, muscular dystrophy and, 13 Epithelium, retinal transplants and, 282, 296, 297 Epitopes. neurotoxin-binding site and, 145, 148 (3-Epoxide, neuronal storage disease and, 200 Equilibrium batrachotoxin and, 98 neurotoxin-binding site and, 127, 128 Erabutoxin. neurotoxin-binding site and, 122, 127, 128 Erythrocytes, muscular dystrophy and, 11-13, 16 Escherichia coli muscular dystrophy and, 28, 38 neurotoxin-binding site and, 148 Fsterification, batrachotoxin and, 88, 110 Ethanol, calcium and, 162, 184 behavioral effects, 162, 163 biochemistry, 172-183 electrophysiology, 164, 166-172 Etoperidone, monoamines and, 272, 273 Excitatory postsynaptic potentials, neuronal storage disease and, 223, 229 Exocytosis, calcium and, 163 Eye, retinal transplants and, 281, 283, 284

F Fasciculus retroflexus, thalamic amnesia and, 253 Fascioscapulohumeral dystrophy, 4 Fat, muscular dystrophy and, 3, 13 Fatty acids batrachotoxin and, 110

INDEX muscular dystrophy and, 14 Fenfluramine monoamines and, 265-268 schizophrenia and, 328 Fibrils, neuronal storage disease and, 201 Fibroblasts muscular dystrophy and, 7 basic defect, 9-14. 16, 17 cloning, 38, 45 neuronal storage disease and, 199 Fibronectin, muscular dystrophy and, 14 Flunarizine, calcium and, 178 Fluorescence batrachotoxin and binding site, 93, 94, 96 interactions, 98 lipids, 111 calcium and, 169, 179 neurotoxin-binding site and, 126, 128, 129 peripheral nerve bridges and, 303 Fluorescence-activated cell sorter, muscular dystrophy and, 28, 30 Flupenthixol, schizophrenia and, 320 Flurazepam, calcium and, 178, 184 Fractionation, muscular dystrophy and, 28 Frontal cortex retinal transplants and, 297 schizophrenia and, 336 Fucosidosis, neuronal storage disease and, 197 Fusion batrachotoxin and, 87 muscular dystrophy and, 7, 10 neuronal storage disease and, 229 neurotoxin-binding site and, 132, 148 retinal transplants and, 294, 301

G 0-Galactosidase neuronal storage disease and, 196, 199 neurotoxin-binding site and, 148 Gambaerdiscw toxicw, batrachotoxin and, 107 Ganglion cell layer peripheral nerve bridges and, 302 retinal transplants and, 285, 287, 299 Ganglion cells peripheral nerve bridges and, 302-305 retinal transplants and, 282, 283

357

central nervous system, 299-301 vitreal chamber, 287, 295, 296 Gangliosides, neuronal storage disease and, 193 animal models, 195, 196, 199 disordered function, 220-224 dysfunction, 235 neurite growth, 225-227 neuroscience, 238 role, 224, 225 structural changes, 205-207, 210, 211, 213, 215, 217 synapses, 227-230 Gangliosidoses, neuronal storage disease and, 205, 221 Gaucherh disease, neuronal storage disease and, 200, 207 Gene expression, muscular dystrophy and, 9, 10 Gene therapy, muscular dystrophy and, 63, 64 Genetic counselling, muscular dystrophy and, 7 Gepirone, monoamines and, 270, 272, 274 Glial cells, retinal transplants and, 299 Glioblastoma, thalamic amnesia and, 247 Glioma, calcium and, 165 Glucose 6-phosphate, calcium and, 181 Glucose 6-phosphate dehydrogenase, muscular dystrophy and, 9, 10, 14 or-Glucosidase,neuronal storage disease and, 200 6-Glucosidase, neuronal storage disease and, 199 Glutamic acid decarboxylase neuronal storage disease and, 211, 223 schizophrenia and, 332 Glutamine, neurotoxin-binding site and, 125 Glyceraldehyde-%phosphate dehydrogenase, muscular dystrophy and, 33, 34 Glycerol kinase deficiency, muscular dystrophy and, 25, 26, 49 Glycerol phosphocholine, muscular dystrophy and, 13, 14 Glycerol phosphodiestercholine, muscular dystrophy and, 14 Glycine, neurotoxin-binding site and, 125 Glycogenesis, neuronal storage disease and, 197 Glycolipids, neuronal storage disease and, 193, 207, 210, 225

358

INDEX

Glycopmtein batrachotoxin and, 110 muscular dystrophy and, 14 neuronal storage disease and, 200, 235 retinal transplants and, 287 Glycosaminoglycans, neuronal storage disease and, 199 Glycosphingolipids, neuronal storage disease and, 226 Glycosylation,neurotoxin-binding site and, 151 mutagenesis. 148 nicotinic acetylcholine receptor, 120 proteolytic fragments, 137, 138 reducible disulfide, 134 GM, gangliosidosis, neuronal storage disease and animal models, 195, 196 disordered function, 222 dysfunction, 230 neuroscience, 238 structural changes, 206, 208, 210, 211, 213, 215, 217, 218 synapses, 227-230 GM, gangliosidosis, neuronal storage disease and animal models, 197 disordered function, 221, 222 structural changes, 208. 210, 211 Golgi complex. neuronal storage disease and disordered function, 22 dysfunction, 231, 232 structural changes, 201, 207, 208, 210, 211, 213, 218 Golgi membrane, neuronal storage disease and, 200 Gonadotropin-releasing hormone, retinal transplants and, 282 G o n i o ~ m batrachotoxin , and, 107 Grafting peripheral nerve bridges and. 303-305 retinal transplants and, 281-284, 306 B N C ~membrane, S 296, 297 central nervous system. 298-302 vitreal chamber, 284-296 Grayanotoxin, batrachotoxin and, 79, 81. 85. 89 Growth hormone, schizophrenia and. 319, 329 Guanidium, batrachotoxin and, 78 Guanine nucleotide-binding proteins, calcium and, 165 Guanosine triphosphate-binding proteins, calcium and, 165

H Hallucinations, schizophrenia and, 315, 324, 333 Haloperidol, schizophrenia and. 317, 321, 332, 335-337 Handedness, thalamic amnesia and, 255 Heart batrachotoxin and, 80 calcium and, 175 Hemoglobin, muscular dystrophy and, 15 Hemophilia A. muscular dystrophy and, 4, 5 Hemopoxin, muscular dystrophy and, 3 Hemorrhage, thalamic amnesia and, 247-250, 256 Hepatocytes, calcium and, 182, 183 Heterogeneity monoamines and, 274, 276 muscular dystrophy and, 5, 6 cloning, 43 gene location, 21 mutation, 47, 48 neuronal storage disease and, 205, 206 neurotoxin-binding site and, 134 Hexobarbital, monoamines and, 272 Hexosamines, neuronal storage disease and, 225 Hexosaminidase, neuronal storage disease and, 220, 221 6-Hexosaminidase, neuronal storage disease and, 196 Hexoses, neuronal storage disease and, 225 Hippocampus calcium and, 184 biochemistry, 173, 179 electrophysiology, 164-167, 169-171 monoamines and, 262, 274 neuronal storage disease and, 227, 228 schizophrenia and, 316, 324 thalamic amnesia and, 245, 254 Histamine, batrachotoxin and, 101 Histidine, batrachotoxin and, 93, 96 Histology, muscular dystrophy and, 9 Homeostasis calcium and, 172, 182, 184 neuronal storage disease and, 229, 234, 235 Homogeneity batrachotoxin and, 78 monoamines and, 274 Homology batrachotoxin and, 87, 97

359

INDEX muscular dystrophy and, 5 cloning, 33, 43, 45 future prospects, 63 neurotoxin-binding site and amino acid sequences, 142 curaremimetic neurotoxins, 122, 124, 125 nicotinic acetylcholine receptor, 120 Hormones calcium and, 183 neuronal storage disease and, 225 retinal transplants and, 282 schizophrenia and, 319 Horseradish peroxidase peripheral nerve bridges and, 303-305 retinal transplants and, 298. 299 Hyaloid artery, retinal transplants and, 282 Hybridization calcium and, 165 muscular dystrophy and, 7 cloning, 33-38, 40, 41, 43-45 gene location, 25-29 mutation, 48-50, 52 neuronal storage disease and, 198, 201, 205 Hydrogen batrachotoxin and, 96 neurotoxin-binding site and, 153 curaremimetic neurotoxins, 126, 127, 129 models, 149, 150 Hydrolysis, calcium and, 182 Hydrophobicity, neuronal storage disease and, 205, 225 6-Hydroxydopamine, monoamines and, 265 5-Hydroxyindoleacetic acid, monoamines and, 267 Hydroxyl groups, neurotoxin-binding site and, 142 Hydroxylation, monoamines and, 263 Hyperactivity calcium and. 163 monoamines and, 262 Hyperacusis, neuronal storage disease and, 196 Hyperammonemia, muscular dystrophy and, 30 Hypercholesterolemia, muscular dystrophy and, 62 Hyperpolarization batrachotoxin and, 82, 83

calcium and, 184. 185 electrophysiology, 164, 166-168, 170, 171 Hypertension, schizophrenia and, 318, 338 Hypertermia. monoamines and, 262, 263 Hypoglycemia, schizophrenia and, 317 Hypokinesia, thalamic amnesia and, 249 Hypothalamus calcium and, 173 schizophrenia and, 312, 325 Hypothermia, calcium and, 176, 178

I Idiocy, neuronal storage disease and, 195 Imipramine, monoamines and, 268, 269 Immunogens muscular dystrophy and, 62 neurotoxin-binding site and, 146 Immunohistochemical staining, retinal transplants and, 299 Immunoreactivity, retinal transplants and, 299 Inactivation batrachotoxin and, 82, 98, 110 calcium and, 175, 182 Infarction, thalamic amnesia and, 248, 255 Inferior colliculus, retinal transplants and, 298, 300 Inhibitory postsynaptic potential calcium and, 166, 171, 172 neuronal storage disease and, 223, 224, 230 Inner nuclear layer, retinal transplants and, 287 Inner plexiform layer, retinal transplants and, 287, 299, 300 Inositol phosphates, calcium and, 165, 181, 183 Inositol phospholipids, calcium and, 165 Insulin, muscular dystrophy and, 63 I Q muscular dystrophy and, 2, 3 Iris, retinal transplants and, 284 Irradiation, batrachotoxin and, 109 Ischemia, muscular dystrophy and, 8

K Ketanserin, monoamines and, 268, 276 Korsakoff syndrome, thalamic amnesia and, 245-248, 254

360

lNDEX

Krabbe's disease, neuronal storage disease and, 207, 231

L Lactation, calcium and, 182 Laminae, retinal transplants and, 298 Later geniculate nucleus, retinal transplants and, 298 LDL receptor, muscular dystrophy and, 53, 62 I~zeiurusquinquestnhtuls. batrachotoxin and, 94, 98, 99 Lens, retinal transplants and, 283, 296 Lesch-Nyhan syndrome, muscular dystrophy and, 17 Leukodystrophies. neuronal storage disease and. 231 Leupeptin, neuronal storage disease and, 199 Ligands batrachotoxin and, 107, 109 monoamines and, 268-270, 276 muscular dystrophy and, 62 neurotoxin-binding site and, 118, 151-154 curaremimetic neurotoxins, 128 nicotinic acetylcholine receptor, 118 proteolytic fragments, 136, 137 reducible disulfide, 134 Light microscopy neuronal storage disease and, 201, 230 retinal transplants and, 285 Linkage, muscular dystrophy and, 3, 4 carrier identification, 57 gene location, 26-29, 31 Lipid dystrophy, neuronal storage disease and, 195 Lipids batrachotoxin and, 78, 79, 111 binding site, 88, 89, 94, 97 electrophysiological analysis, 79, 81, 85, 86 interactions, 98, 106, 108 photoaffinity labeling, 109, 110 purified sodium channel, 110, 111 solubilization, 108, 109 muscular dystrophy and, 13, 14 neurotoxin-binding site and, 132 Liposomes, batrachotoxin and, 85, 108 Lithium, calcium and, 181 Liver, monoamines and, 263

Localization neuronal storage disease and, 227 neurotoxin-binding site and, 136, 151 retinal transplants and, 298 Locus coeruleus, monoamines and, 272 Loxapine, schizophrenia and, 328 LSD. schizophrenia a n d , 324 Lysine batrachotoxin and, 89 neurotoxin-binding site and, 125, 126 Lysis, muscular dystrophy and, 33 Lysosomal hydrolase, neuronal storage disease and animal models, 197-200 disordered function, 220 dysfunction, 231, 234 neuroscience, 237 structural changes, 201, 206, 210, 215 therapy, 236 Lysosomes muscular dystrophy and, 13 neuronal storage disease and, 192, 193 animal models, 195. 197, 198 dysfunction, 230, 235 neuroscience, 238 structural changes, 201, 205-207, 215, 218 therapy, 235 Lysosphingolipids, neuronal storage disease and, 207

M Macrophages. retinal transplants and, 296 Main immunogenic region, neurotoxinbinding site and, 148 4-(N-Maleimido)benzyltrimethylammonium iodide, neurotoxin-binding site and, 133, 135-138, 143, 150-152 Mannose. neurotoxin-binding site and, 120, 134 a-Mannosidase, neuronal storage disease and animal models, 200 neuroscience, 237, 238 structural changes, 220 therapy, 236 a-Mannosidosis, neuronal storage disease and animal models, 195, 197, 200 disordered function, 220, 221 gangliosides, 225

361

INDEX neuroscience, 238 structural changes, 205,206,208,210,

211,218 therapy, 236 0-Mannosidosis, neuronal storage disease and, 197 Medial dorsal nucleus, thalamic amnesia and, 246,247,252,253 Mediodorsal nucleus, thalamic amnesia and clinical observations, 246,247 experimental studies, 250-253 theoretical considerations, 253,254 Mediodorsal thalamus, thalamic amnesia and, 246,255 clinical observations, 248,250 experimental studies, 251,253 theoretical considerations, 253,254 Megadendrites, neuronal storage disease and,

218 Meganeurites, neuronal storage disease and,

194

muscular dystrophy and, 4-6,25,26 neuronal storage disease and, 192,221 2-Mercaptoethanol, neurotoxin-binding site and, 143 Mesencephalon, thalamic amnesia and, 246 Mesulergine, monoamines and, 276 Methanol, calcium and, 177 Methohexital, calcium and, 169 3-Methoxy-4-hydroxyphenylglycol (MHPG), schizophrenia and, 312,314,315,320,

321 Methyl groups, neurotoxin-binding site and,

153 Methylamine, batrachotoxin and, 85 Methylation, batrachotoxin and, 88 Methysergide, monoamines and, 276 Mianserin, monoamines and, 276 Microfilaments, neuronal storage disease and, 226 Microneurites, neuronal storage disease and,

213

disordered function, 223 dysfunction, 230-233,235 gangliosides, 228 structural changes, 207-211,213,215,218 Meiosis, muscular dystrophy and, 5,27,54,

55 Membranes batrachotoxin and, 11, 77, 112 electrophysiological analysis, 79,80,

82-84 interactions, 104 lipids, 108,111 calcium and, 177,179-183 muscular dystrophy and, 11-14,16 neuronal storage disease and, 198,201,

206 neurotoxin-binding site and curaremimetic neurotoxins, 132 monoclonal antibodies, 146 nicotinic acetylcholine receptor, 120 proteolytic fragments, 137 synthetic peptides, 143,144 Memory retinal transplants and, 282 schizophrenia and, 321 thalamic amnesia and, 245,246,255,256 clinical observations, 246-248,250 experimental studies, 250-253 theoretical considerations, 253,254 Mental retardation

Microtubules, neuronal storage disease and,

201,215,226,230 Midazolam, calcium and, 166-168,170,175,

184 Midbrain, schizophrenia and, 336 Mitochondria calcium and, 172,180 muscular dystrophy and, 12 neuronal storage disease and, 201,215 Mitosis, muscular dystrophy and, 28 Monoamines drugs and, 259,260,276,277 amineptine, 263-265 d-amphetamine, 260-263 fenfluramine, 265-268 interaction, 270-273 ligand, 268-270 regional differences, 273,274 species differences, 274-276 schizophrenia and, 313. 319,321,325,337 Monoclonal antibodies muscular dystrophy and, 62 neurotoxin-binding site and, 118,152 probes, 145-147 proteolytic fragments, 136 reducible disulfide, 134,135 retinal transplants and, 287 Morphology calcium and. 171

362

INDEX

muscular dystrophy and. 3, 8, 9, 12 neuronal storage disease and animal models, 195-197, 200 disordered function, 222 dysfunction, 231 neuroscience, 238 structural changes, 201, 205, 207, 208 therapy, 237 retinal transplants and, 299 mRNA muscular dystrophy and cloning, 32, 33, 43-45 mutation, 49 neuronal storage disease and, 226 neurotoxin-binding site and, 147 Mucopolysaccharidosi, neuronal storage disease and animal models, 197, 199 disordered function, 220-222 gangliosides, 225 structural changes, 205, 208, 210 Muller cells, retinal transplants and, 299, 300 Mutation batrachotoxin and, 112 muscular dystrophy and, 2, 4-6, 46 basic defect, 17 carrier identification, 58. 60 cloning, 36, 39, 43 female translocation, 46- 48 future prospects. 61-63 gene location, 21, 22, 26, 27, 31, 32 male deletions, 48-54 recombination, 54, 55 neuronal storage disease and, 196, 197, 221 neurotoxin-binding site and, 139, 145-149 Myelin monoamines and, 269 neuronal storage disease and, 230 retinal transplants and, 300 Myelination. neuronal storage disease and, 225 Myoblasts, muscular dystrophy and, 9 , 11 Myogenesis, muscular dystrophy and, 9 Myoglobin. muscular dystrophy and, 3, 8 Myoinositol, calcium and, 181 Myoinositol-1-phosphate, calcium and, 181

N Nebulin, muscular dystrophy and, 17 Necrosis muscular dystrophy and, 9

peripheral nerve bridges and, 302 Neocortex, peripheral nerve bridges and, 305 Neostriatum, neuronal storage disease and, 208 Neuraminidase, neuronal storage disease and, 229 wNeuraminidase, neuronal storage disease and, 196 Neurites neuronal storage disease and disordered function, 223 dysfunction, 231-233 gangliosides, 225-229 structural changes, 207-211, 213, 218 therapy, 236, 237 retinal transplants and, 299-301 Neuritogenesis, neuronal storage disease and, 226, 227, 235, 238 Neuroaxonal dystrophy, neuronal storage disease and, 215 Neuroblastomas batrachotoxin and electrophysiological analysis, 80-85 interactions, 99, 103 calcium and, 165 neuronal storage disease and, 213, 226 Neuroepithelium, retinal transplants and, 298 Neurofilaments, neuronal storage disease and, 215, 226, 230 Neuroleptics monoamines and, 262, 268, 276 schizophrenia and, 336, 337 Neuronal ceroid lipofuscinosis, neuronal storage disease and, 192 animal models, 195, 197, 199 disordered function, 222 structural changes, 208, 210 Neuronal storage disease, 191, 192 animal models, 195 induced diseases. 197-200 inherited diseases, 195-197 disordered function clinical manifestations, 220, 221 electrophysiological studies, 221-224 dysfunction cascade of events, 234, 235 connectivity, 231-234 cytotoxicity, 230, 231 gangliosides, 224, 225 neurite growth, 225-227

363

INDEX synaptic transmission, 227-230 history, 192-194 neuroscience, 237, 238 structural changes dendritic domain, 217-220 growth, 207-211 plasmalemma, 213 spheroid formation, 213-217 storage process, 200-207 synaptic connectivity, 211-213 therapy, 235-237 Neurons peripheral nerve bridges and, 302 retinal transplants and, 281 schizophrenia and, 335 Neuropil. neuronal storage disease and, 213, 218, 230 Neuroscience, neuronal storage disease and, 237, 238 Neurotoxin-binding site, 117, 118, 151-154 amino acid sequences, 138-142 curaremimetic neurotoxins, 122-131 models, 149, 150 monoclonal antibodies, 145-147 mutagenesis, 147, 148 nicotinic acetylcholine receptor. 118-122 proteolytic fragments, 136-138 reducible disulfide, 133-136 subunit, 131-133 synthetic peptides, 142-145 Neurotoxins batrachotoxin and, 78, 79 binding site, 89 electrophysiological analysis, 79, 80, 86 interactions, 97-108 lipids, 111 monoamines and, 263 Neurotransmitters monoamines and, 259, 268 neuronal storage disease and, 225, 227-229, 238 retinal transplants and, 282 schizophrenia and, 310, 337. 338, 340 Nicotine, neurotoxin-binding site and, 126 Nicotinic acetylcholine receptor, neurotoxinbinding site and, 117-122, 139 Nifedipine, calcium and, 177, 178 Nimodipine, calcium and, 176, 178 Nitrendipine, calcium and. 176-178 Nitrogen batrachotoxin and, 88 neurotoxin-binding site and, 126, 153

Noradrenaline, monoamines and, 260, 262, 265, 267 a,-Noradrenergic receptors, monoamines and, 271 Noradrenergic system monoamines and, 260-263, 265, 272 schizophrenia and, 310, 312, 315 Norepinephrine retinal transplants and, 282 schizophrenia and, 310, 311, 322, 323, 338-340 adrenergic drugs, 321, 322 brain, 311, 312 CAMP, 318 CSF, 312-314 CSF metabolites, 314, 315 DBH, 315, 316 plasma, 316, 317 plasma DBH, 317, 318 plasma MHPG, 320 receptor, 318, 319 urinary catecholamines, 320 Norfenfluramine, monoamines and, 265, 266 Northern blot, muscular dystrophy and, 44,45 Nucleotides muscular dystrophy and, 33, 53 neurotoxin-binding site and, 120 Nucleus accumbens monoamines and, 262 schizophrenia and, 312, 336

0 Occipital cortex retinal transplants and, 298, 301 schizophrenia and, 324 Oligosaccharides neuronal storage disease and, 196 neurotoxin-binding site and, 120, 134, 137 Olivary pretectal nucleus, retinal transplants and, 300 Oncogenes, muscular dystrophy and, 39 Opisthotonus, neuronal storage disease and, 221 Optic nerve bridges, retinal transplants and, 281-283, 302-306 Ornithine transcarbamylase, muscular dystrophy and carrier identification, 56, 59 gene location, 26, 29, 30 mutation, 49, 50

364

INDEX

Outer nuclear layer, retinal transplants and, 287 Outei plexiform layer, retinal transplants and, 299, 300 Oxygen batrachotoxin a n d , 89. 96 neurotoxin-binding site and. 127

P Papain, neurotoxin.binding site and, 136 Paranoid schizophrenia, 338, 339 y-aminobutyric acid, 332, 333 norepinephrine, 312, 314, 322, 323 serotonin, 324 Paroxysornal activity, neuronal storage disease a n d , 222 Pentobarbital, calcium and, 184 biochemistry, 172, 174, 175. 178 electrophysiology, 166-169 Peptides muscular dystrophy and, 62 neurotoxin-binding site and, 118, 151, 152. 154 curaremimetic neurotoxins, 122, 124, 126 monoclonal antibodies, 146, 147 mutagenesis. 148 nicotinic acetylcholine receptor, 122 proteolytic fragments, 136, 137 reducible disulfide, 136 synthetic peptides. 142-145 Perikarya. neuronal storage disease and, 193, 201, 215 Peripheral nerve bridges, 281-283. 302, 303, 305, 306 optic nerve, 304 retina, 303, 304 target tissue connections, 304, 305 Peripheral nervous system neuronal storage disease and, 227 prrlpheral nerve bridges and. 305, 304 Peroneal nerve, peripheral nerve bridges and, 304 pH, batrachotoxin and, 91, 92 Phagocytes. muscular dystrophy a n d , 49 Phenobarbital, calcium and, 169, 178 Phenol-enhanced reassociation technique, muscular dystrophy and carrier identification. 56, 59 cloning. 34, 35, 38-45

future prospects, 61 mutation, 46, 48, 50-52. 54, 55 Phenotype muscular dystrophy and, 2, 3, 5-7 basic defect, 9, 14 future prospects, 61, 63 gene location, 18, 19, 21-23, 25, 26 mutation, 48-50. 53 neuronal storage disease and, 196 Phenylalkamines, calcium and, 176 Phenylglyoxal, neurotoxin-binding site and, 125 Phorbol esters, calcium and, 165 Phosphatases, calcium and, 164 Phosphatidylcholine batrachotoxin and, 108 muscular dystrophy a n d , 13 Phosphatidylethanolamine. batrachotoxin and, 108, 109 Phosphatidylinositides, calcium and, 181, 183 Phosphatidylinositol calcium and, 181, 182 muscular dystrophy and, 13 Phosphatidylserine, batrachotoxin and, 108, 109 Phosphoinositide, calcium and, 165, 181 Phospholipase C, calcium and, 181-183 Phospholipids calcium and, 165, 182 muscular dystrophy and, 10 neuronal storage disease and, 228 Phosphorylation calcium and, 165, 181 neurotoxin-binding site a n d , 120 Phyllobales aurotaenia, batrachotoxin and, 80, 81, 86 Phyllobates tern'bilus, batrachotoxin and, 80, 86 Pimozide, schizophrenia and, 313, 315, 316, 331 Piremperone, monoamines and, 276 Placebos, schizophrenia and. 320, 321 Plasma, schizophrenia a n d , 340 y-aminobutyric acid, 332, 336 norepinephrine, 312, 315-318, 320, 322, 323 Plasma membrane calcium and, 179, 181 muscular dystrophy and, 12, 14 neuronal storage disease a n d , 213 Plasmalemma, neuronal storage disease and, 213, 227, 235

365

INDEX Plasmids muscular dystrophy and, 32-34 neurotoxin-binding site and, 132, 148 Platelets, schizophrenia and, 314, 318, 319, 324-328 Pneumonia, muscular dystrophy and, 3 Polarity, retinal transplants and, 296 Polyacrylamide gel electrophoresis, muscular dystrophy and, 16, 17 Polymyositis, muscular dystrophy and, 12, 14 Polypeptides batrachotoxin and, 78 interactions, 97, 98, 103, 107 lipids, 108 neurotoxin-binding site and, 154 curaremimetic neurotoxins, 126 mutagenesis, 148 nicotinic acetylcholine receptor, 119, 120 Polyphosphoinositides, calcium and, 182 Polysomes, muscular dystrophy and, 10, 11 Posterior pretectal nucleus, retinal transplants and, 300 Potassium calcium and, 164, 172, 173 neuronal storage disease and, 228 Prazosin, schizophrenia and, 321 Prefrontal cortex, thalamic amnesia and, 251, 254 Prenatal diagnosis, muscular dystrophy and, 8 applications, 59-61 future prospects, 64 principles, 56-59 Proliferation, retinal transplants and, 297 Proline neurotoxin-binding site and, 125 retinal transplants and, 298 Propanol, calcium and, 177 Propranolol, schizophrenia and, 321-323 Proteases muscular dystrophy and, 10, 12, 17 neurotoxin-binding site and, 134, 136, 137 Protein batrachotoxin and, 77, 78, 111 binding site, 89, 94, 97 electrophysiological analysis, 86 lipids, 108-111 calcium and, 175, 179-181 monoamines and, 259 muscular dystrophy and, 2, 3 basic defect, 8, 10-14 cloning, 32, 33, 43, 45

electrophoresis, 15-18 future prospects, 62, 63 mutation, 46, 53 neuronal storage disease and, 196, 205, 226, 236 neurotoxin-binding site and, 151, 154 amino acid sequences, 138, 139, 142 mutagenesis, 148 nicotinic acetylcholine receptor, 119 subunit, 132 Protein kinase C calcium and, 165, 181 neuronal storage disease and, 207, 234 Protein kinases, calcium and, 165, 180 Proteolysis, neurotoxin-binding site and fragments, 136-138 monoclonal antibodies, 146 reducible disulfide, 135 synthetic peptides, 143 Protonation, batrachotoxin and, 91, 93, 96 Pseudogenes, muscular dystrophy and, 5 Pseudohypertrophy, muscular dystrophy and, 6 Pseudolipidosis, neuronal storage disease and, 195 Psychosis, 338, 339 y-aminobutyric acid, 331-333, 335 norepinephrine, 312-316, 321, 323 serotonin, 324, 328 Psychotropic drugs, monoamines and, 259, 277 Pulsed field gel technology, muscular dystrophy and, 42 Pulvinar, thalamic amnesia and, 246, 247, 254 Purification batrachotoxin and, 78 electrophysiological analysis, 85 lipids, 108, 110, 111 neurotoxin-binding site and, 135 Purkinje cells, neuronal storage disease and, 218 Putamen, schizophrenia and, 324 1-Pyrimidylpiperazine, monoamines and, 270-274 Pyrrole-3-carboxylate, batrachotoxin and, 88

R Radiation, thalamic amnesia and, 251 Radioactivity, batrachotoxin and, 81, 109 Rum pipiens, batrachotoxin and, 80 rDNA, muscular dystrophy and, 37-39 Receptors, schizophrenia and, 314, 317-323, 329

366

INDEX

Recombination, muscular dystrophy and carrier identification, 57, 58 future prospects, 61 mutation, 54. 55 Redundancy, thalamic amnesia and, 255 Regeneration peripheral nerve bridges and, 302-305 retinal transplants a n d , 281, 282 Replication, muscular dystrophy and, 32 Repolarization, batrachotoxin and, 81 Restriction fragment length polymorphisms, muscular dystrophy and carrier identification, 57, 59, 60 cloning, 36, 39, 40 gene location, 27-30 mutation, 49, 54, 55 Retention, thalamic amnesia and, 251 Retinal pigment epithelium, Bruchs membrane and, 296-299 Retinal transplants, 281-283, 305, 306 anterior chamber, 283, 284 Bruchs membrane, 296, 297 central nervous system, 297 connection to host brain, 300, 301 functional significance, 301, 302 graft differentiation, 298-300 methods, 298 vitreal chamber, 284-296 Retinitis pigrnentosa, muscular dystrophy and, 25, 49, 50 Retrieval, thalamic amnesia and, 248 Revascularization, retinal transplants and. 298 Rhodopsin, neurotoxin-binding site and, 120 Ribosomes, neuronal storage disease and, 200 Ricinur communts. muscular dystrophy and, 14, 17, 62 rRNA, muscular dystrophy and, 37

s Sandhoffs disease, neuronal storage disease and, 196, 220 Sarcolemma, muscular dystrophy and, 13 Sarcomeres, muscular dystrophy and, 12 Sarcoplasmic reticulum, muscular dystrophy and, 12, 18 Saxitoxin, 78, 105. 106 Scanning electron micrographs, retinal transplants and, 285. 291

Scar formation, peripheral nerve bridges and, 302 Schizophrenia, 310, 311, 337-340 y-aminobutyric acid, 329, 330, 336, 337 antipsychotics, 335, 336 CSF, 330-332 drugs, 332-335 plasma, 332 norepinephrine, 311, 322, 323 adrenergic drugs, 321, 322 brain, 311, 312 CAMP, 318 CSF, 312-314 CSF metabolites, 314, 315 DBH, 315, 316 plasma, 316, 317 plasma DBH, 317, 318 plasma MHPG, 320 receptor, 318, 319 urinary catecholamines, 320 serotonin, 323, 324, 329, 330 brain, 324 CSF, 324, 325 drugs, 328, 329 platelet, 326-328 Schizophreniform, schizophrenia and, 333 Sciatic nerve batrachotoxin and, 82 peripheral nerve bridges and, 303, 304 a-Scorpion toxins, batrachotoxin and interactions, 97-99, 107 lipids, 108-110 Sea anemone toxins, batrachotoxin and, 97-99 Second messengers. calcium a n d , 184, 185 biochemistry, 172, 180-183 electrophysiology, 165 Sedimentation, neurotoxin-binding site and, 119 Seizures, neuronal storage disease and, 196, 222, 227, 228 Sepharose, neurotoxin-binding site and, 143, 144 Serine, neurotoxin-binding site and, 148 Serotonin batrachotoxin and, 101 calcium and, 165 monoamines and receptors, 268-276 uptake, 260-262, 265-268 retinal transplants and, 282

INDEX schizophrenia and, 310, 311, 323, 324, 329, 330, 338-340 brain, 324 CSF, 324, 325 drugs, 328, 329 platelets, 326-328 Short-latency negative waves, retinal transplants and, 301 Sialic acid, neuronal storage disease and, 193, 225, 226 Sickle-cell anemia, muscular dystrophy and, 15 Sodium, calcium and, 166, 169, 180. 184 Sodium channel batrachotoxin and, see Batrachotoxin neuronal storage disease and, 232, 235 Sodium dodecyl sulfate, neurotoxin-binding site and, 143 Southern blot, muscular dystrophy and carrier identification, 59 cloning, 38, 44, 45 mutation, 52 Spectroscopy calcium and, 184 neurotoxin-binding site and, 121, 126 Spheroid formation, neuronal storage disease and, 213-217, 231, 238 Sphingolipidoses, neuronal storage disease and, 207 Sphingomyelin lipidosis, neuronal storage disease and, 197, 208, 210, 225 Sphingomyelinase activity, neuronal storage disease and, 198 Spinal cord neuronal storage disease and, 236 peripheral nerve bridges and, 303 retinal transplants and, 281, 298 Spinal cord neurons, calcium and, 169, 170 Spine, neuronal storage disease and, 207, 218 Spinocerebellar degeneration, neuronal storage disease and, 220 Spiperone, monoamines and, 268, 269, 276 Spirodecanone, monoamines and, 268 Startle response, neuronal storage disease and, 221, 222 Stem cells, muscular dystrophy and, 59, 63, 64 Stereotaxical injection, retinal transplants and, 298 Steroids, batrachotoxin and, 80, 86, 87, 107 Stress, schizophrenia and, 311, 340

367

Striatum monoamines and, 262, 264, 269, 274 schizophrenia and, 336 Subclones muscular dystrophy and cloning, 36, 38-40, 42-44 future prospects, 62 mutation, 52 neurotoxin-binding site and, 148 Substantia nigra, schizophrenia and, 336 Suicide, schizophrenia and, 324, 325, 338 Sulfhydryl groups, neurotoxin-binding site and, 135 Sulpiride, monoamines and, 268 Superior colliculus peripheral nerve bridges and, 305 retinal transplants and, 298-301 Suramin, neuronal storage disease and, 199 Swainsonine, neuronal storage disease and animal models, 200 disordered function, 221 neuroscience, 237 structural changes, 210, 211, 218 therapy, 236 Swelling, neuronal storage disease and, 192, 201, 215 Synapses neuronal storage disease and, 194 connectivity, 211-213 disordered function, 222 dysfunction, 231-235 gangliosides, 225, 227-230 neuroscience, 237, 238 structural changes, 201, 205, 207, 210 therapy, 236, 237 retinal transplants and, 283, 284 central nervous system, 300, 301 vitreal chamber, 284, 285, 287 schizophrenia and, 337 thalamic amnesia and, 254 Synaptogenesis, peripheral nerve bridges and, 305 Synaptoneurosomes, batrachotoxin and, 100, 102, 109 Synaptosomes batrachotoxin and interactions, 98, 99, 101, 103 lipids, 109 calcium and, 184 biochemistry, 172-177, 179, 180, 182 electrophysiology, 170

368

INDEX

monoamines and, 260, 263, 267 neuronal storage disease a n d , 206, 213, 228, 230

T Tardine dyskinesia, schizophrenia and, 321, 322, 328, 335 Target tissue, peripheral nerve bridges and, 304, 305 Tay-Sachs disease, 192-194 disordered function, 220-222 structural changes, 200, 201, 205, 207, 213 therapy, 236 Trctum, retinal transplants and, 300, 301 Temperature batrachotoxin and, 91 neurotoxin-binding site and, 128 Temporal cortex, schizophrenia and, 324 Xetrodotoxin , 78 calcium and, 166, 167 electrophysiological analysis, 80, 81 interactions, 98, 105, 106 lipids, 109 Thalamic amnesia, 245, 246, 254-256 clinical observations, 246-250 experimental studies, 250-253 theoretical considerations, 253, 254 Thalamus neuronal storage disease and, 222, 223, 229 retinal transplants and, 298 Thiol protease, neuronal storage disease and, 199 Thymidine, retinal transplants and, 296-298 Titin, muscular dystrophy and, 17 Titubation, neuronal storage disease and, 221 Tolerance, calcium and, 162, 183, 185 behavioral effects, 163 electrophysiology, 170, 177 Torpedo, neurotoxin-binding site and amino acid sequences, 138, 142 curaremimetic neurotoxins, 122 nicotinic acetylcholine receptor, 120 subunit, 132 synthetic peptides, 143, 144 Torpedo californica, neurotoxin-binding site and, 119 Transcription, muscular dystrophy and. 22. 33, 44

Translation, muscular dystrophy and, 33 Translocation, muscular dystrophy and, 6 cloning, 34-43, 45 future prospects, 61 gene location, 18-24 mutation, 46-48 Transmission electron micrographs, retinal transplants and, 284, 285, 288 Trauma peripheral nerve bridges and, 302, 304 retinal transplants and, 283, 287, 306 schizophrenia and, 340 Trazodone, monoamines and, 272, 273 Tricyclic antidepressant drugs, monoamines and, 262 Tritium, batrachotoxin and, 88 Troponin, calcium and, 180 Trypsin, retinal transplants a n d , 296 Tryptophan, neurotoxin-binding site and, 127, 144 L-Tryptophan, schizophrenia and, 329 Tryptophan hydroxylase, calcium and, 180 d-Tubocurarine, neurotoxin-binding site and, 126, 127, 137. 151, 153 Tubulin, neuronal storage disease and, 226 Tumon muscular dystrophy and, 39 thalamic amnesia and, 246, 247, 256 'liurner syndrome, muscular dystrophy and, 6 Tyrosine, calcium and, 180

U Urinary catecholamines, schizophrenia and, 320

v Valproic acid, schizophrenia and, 333 Vasopressin, retinal transplants and, 282 Ventricle brain ratio, schizophrenia and, 316, 317 \'erapamil, calcium and, 176, 178 Veratridine, 78, 79 binding site, 87, 89, 96 electrophysiological analysis, 80-82, 85 interactions, 97, 103 Iipids, 108, 110

INDEX Viruses muscular dystrophy and, 64 schizophrenia and, 341 Vitreal chamber, retinal transplants and, 284-296 Vitreous, retinal transplants and, 296, 297

W Wallerian degeneration, peripheral nerve bridges and, 302 Wernicke’s disease, thalamic amnesia and, 246, 247 Withdrawal calcium and behavioral effects, 163 biochemistry, 174, 182 electrophysiology, 171 schizophrenia and, 314, 317, 319, 328 Wound healing. retinal transplants and, 282

X X chromosomes, muscular dystrophy and, 2, 4, 6, 7 basic defect, 9, 18 carrier identification, 57, 58 cloning, 33-37, 39-42, 44 future prospects, 61 gene location, 18, 19, 21, 27-32 mutation, 54 Xenopus, neurotoxin-binding site and, 147 X-linked inheritance, muscular dystrophy and, 3 carrier identification, 57, 60 cloning, 38 future prospects, 63 mutation, 49, 54 X-rays muscular dystrophy and, 63 neurotoxin-binding site and, 151

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CONTENTS OF RECENT VOLUMES

Volume 19

Biochemical Aspects of Newtransmission in the Developing Brain Joseph T Coyle

Do Hippocampal Lesions Produce Amnesia in Animals? Swan D. Iuersen

The Formation, Degradation, and Function of Cyclic Nucleotides in the Nervous System John W. Daly

Synaptosomal Transport Processes Giulio Levi and Maunzio Raiten'

Fluctuation Analysis in Neurobiology Louis J. DeFelice

Glutathione Metabolism and Some Possible Functions of Glutathione in the Nervous System Marian Orlowski and Abraham Karkowsky

Peptides and Behavior Georges Ungar

Neurochemical Consequencesof Ethanol on the Nervous System A m n K. Rawat

Biochemical Transfer of Acquired Information S R.Mitchel1,J. M. Beaton, and R.J. Bradley

Octopamine and Some Related Noncatecholic Amines in Invertebrate Nervous Systems H. A . Robertson and A . Y: Juo7io Apormorphine: Chemistry, Pharmacology, Biochemistry E C. Colpaert, W . E M. Van Bever, and J. E. M. E Leysen

Aminotransferase Activity in Brain M . Benuck and A. Lajtha The Molecular Structure of Acetylcholine and Adrenergic Receptors: An All-Protein Model J. R. Smythies Structural Integration of Neuroprotease Activity Elena Gabrielescu Lipotropin and the Central Nervous System W . H . Gis@en,J. M. van Ree, and D. de Wied

Thymoleptic and Neuroleptic Drug Plasma Levels in Psychiatry: Current Status Thomas B. Cooper, George M.Sim@son,and J. Hillay Lee

Tissue Fractionation in Neurobiochemistry: An Analytical Tool or a Source of Artifacts Pierre Laduron

SUBJECT INDEX

Choline Acetyltransferase: A Review with Special Reference to Its Cellular and Subcellular Localization Jean Rossier

Volume 20

SUBJECT INDEX

Functional Metabolism of Brain Phospholipids G. Bnan Ansell and Sheila Spanner Isolation and Purification of the Nicotine Acetylcholine Receptor and Its Functional Reconstitution into a Membrane Environment Michael S. Briley and Jean-Piewe Changeux

371

Volume 21 Relationshipof the Actions of Neuroleptic Drugs to the Pathophysiology of Tardive Dyskinesia Ross J. Baldessanni and Daniel Zany

372

CONTENTS OF RECENT VOLUMES

Soviet Literature on the Nervous System and Psychobiology of Cetacea Theodore H. Bullock and Vladimir S. Gurevich Binding and Iontophoretic Studies on Centrally Active Amino Acids-A Search for Physiological Receptors E V DeFeudis Presynaptic Inhibition: Transmitter and Ionic Mechanisms R . A . Ndcoll and B. E. Alger

Mechanisms of Synaptic Modulation William Shain and David 0. Carpenter Anatomical. Physiological, and Behavioral Aspects of Olfactory Bulbectomy in the Rat B. E. Leonard and M . Tuite The Deoxyglucose Method for the Measurement of Local Glucose Utilization and the Mapping of Local Functional Activity in the Central Nervous System Louis Sohaloff INDEX

Microquantitation of Neurotransmitters in Specific Areas of the Central Nervous System Juan M. Saavedra Physiology and Glia: Glial-Neuronal Interactions R . Malcolm Stewart and Roger N. Rosaberg Molecular Perspectives of Monovalent Cation Selective Transmembrane Channels Dan W Urry Neuroleptics and Brain Self-Stimulation Behavior Albert Wauquier

Volume 22

Volume 23 Chemically Induced Ion Channels in Nerve Cell Membranes David A . Mathers and Jeffery L. Barker Fluctuation of Na and K Currents in Excitable Membranes Berthold Neumcke Biochemical Studies of the Excitable Membrane Sodium Channel Robert L. Barchi Benzodiazepine Receptors in the Central Nervous System Phil Skolnick and Steven M. Paul

Transport and Metabolism of Glutamate and GABA in Neurons and Glial Cells Arne Schousboe

Rapid Changes in Phospholipid Metabolism during Secretion and Receptor Activation E I: Crews

Brain Intermediary Metabolism in Vdvo: Changes with Carbon Dioxide, Development, and Seizures Alexander L. Miller

Glucocorticoid Effects on Central Nervous Excitability and Synaptic Transmission Edward D. Hall

N,N-Dimethyltryptamine: An Endogenous Hallucinogen Steven A . Barker,John A . Monti, and Samuel ?: Christian Neurotransmitter Receptors: Neuroanatomical Localization through Autoradiography L. Charles Murrin Neurotoxins as Tools in Neurobiology E. G. McCeer and l? L. McCeer

Assessing the Functional Significance of LesionInduced Neuronal Plasticity Oswald Steward Dopamine Receptors in the Central Nervous System Ian Cresse, A . Leslie Morrow, Stuart E. LefJ David R. Sibley, and Mark W Hamblin Functional Studies of the Central Catecholamines I: W Robbim and B. J. Even'tt

CONTENTS OF RECENT VOLUMES Studies of Human Growth Hormone Secretion in Sleep and Waking Wallace B. Mendelson Sleep Mechanisms: Biology and Control of REM Sleep DennisJ. McGinty and Rent! R. Drucker-Colin INDEX

Volume 24

373

Volume 25 Guanethidine-Induced Destruction Sympathetic Neurons Eugene M. Johnson, J r and Pamela Toy Manning

of

Dental Sensory Receptors Margaret R. Byers Cerebrospinal Fluid Proteins in Neurology A. Lowenthal, R. Crols, E. De Schutter, J. Gheuens, D. Karcher, M . Noppe, and A . Tasnier

Antiacetylcholine Receptor Antibodies and Myasthenia Gravis Bernard W Fulpiw

Muscarinic Receptors in the Central Nervous System Mordechai Sohlovsky

Pharmacology of Barbiturates: Electrophysiological and Neurochemical Studies Max Willow and Graham A . R. Johnston

Peptides and Nociception Daniel Luttinger, Daniel E. Hernandez, Charles B. Nemeroff, and ArthurJ. Prange, Jr

Immunodetection of Endorphins and Enkephalins: A Search for Reliability Alejandro Bayon, William J. Shoemaker, Jacqueline E McGinty, and Floyd Bloom

O n the Sacred Disease: The Neurochemistry of Epilepsy 0. Carter Snead III Biochemical and Electrophysiological Characteristics of Mammalian GABA Receptors Salvatore J. Enna and Joel I! Gallagher Synaptic Mechanisms and Circuitry Involved in Motorneuron Control during Sleep Michael H. Chase Recent Developments in the Structure and Function of the Acetylcholine Receptor E J. Barrantes

Opioid Actions on Mammalian Spinal Neurons W . Zieglgansberger Psychobiology of Opioids Albert0 Oliverio, Claudio Castellano, and Stefan0 Publisi-Allegra Hippocampal Damage: Effects on Dopaminergic Systems of the Basal Ganglia Robert L. Isaacson Neurochemical Genetics V: Cscinyi The Neurobiology of Some Dimensions of Personality Marvin Zuckerman, James C. Ballenger, and Robert M. Post INDEX

Characterization of a,- and a2-Adrenergic Receptors David B. Bylund and David C. U’Prichard Ontogenesis of the Axolemma and Axoglial Relationships in Myelinated Fibers: Electrophysiological and Freeze-Fracture Correlates of Membrane Plasticity Stephen G. Waxman, Joel A . B h c k , and Robert E. Foster INDEX

Volume 26 The Endocrinology of the Opioids MarkJ. Millan and Albert H e n Multiple Synaptic Receptors for Neumactive Amino Acid Transmitters- New Vistas Najam A . Shanif

374

CONTENTS OF RECENT VOLUMES

Muscarinic Receptor Subtypes in the Central Nervous System Wayne Hoss and John Ellu

Excitatory Transmitters and Epilepsy-Related Brain Damage John W Olney

Neural Plasticity and Recovery of Function after Brain Injury John k: Marshall

Potassium Current in the Squid Giant Axon J o h n R . Clay

From Immunoneurologyto Immunopsychiatry: Neuromodulating Activity of Anti-Brain Antibodies Bmnislav D. JankoviE Effect of Tremorigenic Agents on the Cerebellum: A Review of Biochemical and ElectrophysiolagicalData V: G. Long0 and M . Massotti INDEX

INDEX

Volume 28 Biology and Structure of Scrapie Prions Michael €! McKinley and Stanley B. h i n e r Different Kinds of Acetylcholine Release from the Motor Nerve S. Thesleff

Volume 27

Neuroendocnne-Ontogenetic Mechanism of Aging: Toward an Integrated Theory of Aging V: M . Dalmun, S. Y. Reuskoy, and A . G. Golubev

The Nature of the Site of General Anesthesia Keith W Miller

The Interpeduncular Nucleus Barbam J. Morley

The Physiological Role of Adenosine in the Central Nervous System Thomar V: Dunwiddie

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

Somatostatin, Substance,'F Vasoactive Intestinal Polypeptide, and Neuropeptide Y Receptors: Critical knresrment of Biochemical Methodology and Results Anden U n d h , Lou-LouPeterson, and Tamas Bartfai Eye Movement Dysfunctions and Psychosis Philip S. Holzman Peptidergic Regulation of Feeding J . E. Morley, T J. Bartness, B. A . Gomell, and A . S. Levine Calcium and Transmitter Release I m Cohen and Willaam Van der Kloot

Does Receptor-Linked Phosphoinositide Metabolism Provide Messengers Mobilizing Calcium in Nervous Tissue? John N. Hawthorne Short-Term and LongTerm Plasticity and Physiological Differentiation of Crustacean Motor Synapses H. L. Atwood and J. M . Wojtowicr Immunology and Molecular Biology of the Cholinesterases: Current Results and Prospects Stephen Bn'mijoin and Zoltan Rakoncray INDEX