International Review of Neurobiology Volume 32

International Review of

NEUROBIOLOGY VOLUME 32

Editorial Board W. Ross ADEY JULIUS

AXELROD

PAULJANSSEN SEYMOUR KETY

Ross BALDESSARINI

KEITH KILLAM

SIRROGERBANNISTER

CONANKORNETSKY

FLOYDBLOOM

ABELLAJTHA

DANIELBOVET

BORISLEBEDEV

PHILLIPBRADLEY

PAULMANDEL

YURI BLJROV

HUMPHRY OSMOND

Josh DELGADO

RODOLFO PAOLEFTI

SIRJ O H N ECCLES

SOLOMON SNYDER

JOEL

ELKES

STEPHEN SZARA

H. J . EYSENCK

MARATVARTANIAN

KJELL FUXE

STEPHEN WAXMAN

Bo HOLMSTEDT

RICHARD WYATT

International Review of

NEUROBIOLOGY Editedby

JOHN R. SMYTHIES D e p a h e n t of Neuropsychiatry Institute of Neurology National Hospital London England

RONALD J. BRADLEY Depahent of Psychiatry and The Neuropsychiatry Research Program The Medical Center The University of Alabama of Birmingham Birmingham, Alabama

V O L U M E 32

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CONTENTS O n the Contribution of Mathematical Models to the Understanding of Neurotransmitter Release

H. PARNAS,1. PARNAS,A N D L. A. SEGEL Introduction. ............................................. Fundamental Aspects of Synaptic Kelease . . . . . . . . . . . . . . . . . . . . . . . . . . The Relationships between Release and Ca'+. . . . . . . . . . . . . . . . . . . . . . . IV. Evaluating the Classical Calcitun .......... V. The Calcium-Voltage Hypothe V I . Conclusions.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . ...........

1. 11. 111.

1

3 10 28 35 45 46

Single-Channel Studies of Glutamate Receptors

M. S. P. SANSOM A N D P. N. K. USHERWOOD I.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

11. Channels Gated by Vertebralr (;lut;imate Receptors. . . . . . . . . . . . . . . . . 111. Channels Gated by 1nvertebr;ite Glutamate Receptors . . . . . . . . . . . . . . .

1V. Overview.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

51 57 74 100 101

Coinjection of Xenopus Oocytes with cDNA-Produced and Native mRNAs: A Molecular Biological Approach to the Tissue-Specific Processing of Human Cholinesterases

SHLOMO SEIDMAN A N D HERMONA SOREQ I.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

11. Cholinesterases: A Model Polymorphic Family of Enzynies. . . . . . . . . . . 111. Experimental Observations: A Biochemical Approach. . . . . . . . . . . . . . . I V . Xenopw Oocytes: Faithful b u t Complex Tools. . . . . . . . . . . . . . . . . . . . . . . V. Experimental Results: An Immunohistochemical Approach. . . . . . . . . . VI. Closing Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ....... References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

V

I07 111 118 123 126 1 so I35

vi

CONTENTS

Potential Neurotrophic Factors in the Mammalian Central Nervous System: Functional Significance in the Developing and Aging Brain

DALIAM. ARAUJO. JEAN-GUY CHABOT. AND RE'MI QUIRION ............... I . Introduction ............................. ............................... I1 . Nerve Growth Factor ...................... 111. Fibroblast Growth Fac

IV . V. VI . VII . VIII . IX . X. XI . XI1 .

...... Insulin and Insulinlike Growth Factors . . . . . . . . . . . . . Brain-Derived Neurotrophic Factor ......................... Ciliary Neurotrophic Factor ................... .......... Epidermal and Transforming Growth Factors .................. Platelet-Derived Growth Factor ................................... Interleukins and Other Lymphokines .............................. Hormones and Neurotransmitters as Neurotrophic Factors .......... Miscellaneous Factors with Potential Neurotrophic Activity .......... Concluding Remarks ...... ................................... References .......................... ....................

142 142 147 149 152 153 153 157 158 160 163 164 165

Myasthenia Gravis: Prototype of the Antireceptor Autoimmune Diseases

SIMONE SCHONBECK. SUSANNE CHRESTEL. AND REINHARD HOHLFELD I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175 I1 . Acetylcholine Receptor ........................................... 177 111. Anti-AChR Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181 IV . AChR-Specific T Lymphocytes.................................... 184 V . Role of the Thymus ...... ...... .... 190 VI . Treatment Strategies............................................ 193 References ...................................................... 195

Presynaptic Effects of Toxins

ALANL. HARVEY I . Introduction ...................... I1 . Toxins Affecting Neuronal Ion Channels .......................... 111. Toxins Affecting Release Mechanisms . . . . .

IV . Miscellaneous Toxins ............................................ V . Conclusions ....................... ............... References ......................................................

201 202 216 229 231 232

Mechanisms of Chemosensory Transduction in Taste Cells

MYLESH . AKABAS I . Introduction .................................................... I1 . Cell Biology of Taste Cells........................................

241 242

vii

CONTENTS

111. IV. V. VI. VII. VIII. IX.

.

Impediments to the Study of Taste Cells.. . . . . . . . . . . . . . . . . . . . . . . . . A Criterion for Taste Transduction Mechanisms. . . . . . . . . . . . . . . . . . . . Electrical Properties of the Lingual Epithelium . . . . . . . . . . . . . . . . . . . . . Electrophysiological Properties of Taste Cells. . . . . . . . . . . . . . . . . . . . . . . A Critique of Intracellular Recordings in Taste Cells.. . . . . . . . . . . . . . . Taste Transduction Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Summary. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

244 245 246 247 250 252 272 273

Quinoxalinediones as Excitatory Amino Acid Antagonists in the Vertebrate Central Nervous System

STEPHEN N. DAVIES AND GRAHAM L. COLLINGRIDGE .................. Introduction. . . . . . . . . . . . . . . . . .

I. 11. Binding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111.

IV. V. VI. VII.

. . .. .. . .. . .. . . . .. .. .. . .. . . . . . .. . . . .. .. . ., . . . . .. . .

Pharmacology. . . . . . . . . . . . . . . . . . . . . . Excitotoxicity .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Synaptic Physiology. . . ................ Conclusions. . . . . . .. . . . . . . . . . . . . . .. . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . .

28 1 283 284 284 29 1 292 30 1 30 1

Acquired Immune Deficiency Syndrome and the Developing Nervous System

DOUGLAS E. BRENNEMAN, SUSANK. MCCUNE,AND ILLANA GOZES I. Prologue ....................................................... 11. Clinical Features and Neurological Manifestations of Pediatric Acquired Immune Deficiency Syndrome. . .. . . . . . . . . . . .. . . . .. . . . . .. 111. Human Immunodeficiency Virus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. HIV External Envelope Glycoprotein: gp120. . . . . . . . . . . . . . . .. . . . . . V. Epilogue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

305

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

355 373

.

.

.

.

.

.

306 325 329 344 345

This Page Intentionally Left Blank

ON THE CONTRIBUTION OF MATHEMATICAL MODELS TO THE UNDERSTANDING OF NEUROTRANSMllTER RELEASE By H. Parnas and 1. Parnas Department of Neurobiology and Otto Loewi Center for Cellular and Molecular Neurobiology, The Hebrew University Jerusalem, Israel

and

L. A. Segel

Department of Applied Mathematics and Computer Science Weizmann Institute of Science Rehovot, Israel

I. Introduction A . Scope of This Review B. Remarks on the Role of hlotlcls 11. Fundamental Aspects of Syriaptit Release

111.

IV.

V.

VI.

A . T h e Calcium Hypothcsis B. Problems and Caveats C. ~ l ' w oExperimental hlethotls t o r Char-actcrizing Release: R.leasurerncnts o! Quanta1 Content (Amount of Release) and Kinetics D. Facilitation and Residual (hkiuiii T h e Relationships between Relc,~. A. Calcium Entry K. Dependence of Release 011 tlic Intracellulat- (:a'' (:onccntration C. Ca" Removal Mechanisnls Evaluating the Classical Calciiini I I! pothesis A . Major Features of Release and Facilitation B. Difficulties with the Classical C;alciunl IIypothesis C. Revisions to the Calcium I I!pothesis T h e <;alcium-Voltage Hypothesis A . Evidence T h a t the Othei- Factor Is Voltage-Dependent B. Formulation of the Calciun-Voltage Hypotheses C. Ways to Distinguish between the Calcium and the C.alcium-Voltage Hylmtheses Conclusions References

I. Introduction

A. SCOPE OF THIS REVIEW T h e present review is centered on neurotransmitter release in very fast systems. In particular, we discuss mechanisms of release after a rin-

gle pulse as well as twin pulse facilitation. Not examined in detail are events that take a longer time to develop, such as posttetanic potentiation (Magleby arid Zengel, 1975a,b, 1982; Zengel and Magleby, 1982), or long-term modulation (Atwood rt NI., 1973, 1983; Atwood and Wojtowicz, 1986), and that niay involve slower modulatory processes including second messengers (Dale ct nl., 1988; Yovell et nl., 1987; Klein e&nl., 1982) and phosphorylation or dephosphorylation of proteins (LlinAs el al., 1985; Greengard, 1978; Kravitz, 1988). Keviews by A t w o c d and Wojtowicz (1986) and Rugustine 01 ( I / . ( 1987) describe in detail many of the experimental results relating release to Ca“ , and there is no need for repetition. gather, we confront some of these experimental results with current views, particularly those embodied in mathematical models, on possible mechanisms involved in the release process. I n doing so, we point out some difficulties i n harmonizing accepted models with experimental results. Such a treatment may lead to reevaluation of accepted ideas related to neurotransmitter release.

Neurotransmitter release is a process of such preeminent importance that great experimental efforts have been made to understand it in considerable detail. As is often the case, when thorough understanding is sought, considerable complexity is revealed. In the face of-such coniplexity an experimenter must be guided by working hypotheses as to the nature of the mechanisms that are involved. Such hypotheses are often termed model.\. Models can be of considerable utility when they are essentially verbal and descriptive. Here, however, we are concerned with models that have been developed further into a formal niathernatical structure, a set of equations. We shall examine the contribution of‘such mathematical niodels to the elucidation of the mechanisms that generate and control neurotransmitter release. A t least three roles can be discerned for mathematical models of neurotransmitter release (and of other biological and nonbiological phenomena).

1. Prescriptz~~e role: Mathematical models provide a concise and precise statement of a verbal model. T h e construction of such a statement often reveals fuzziness in one’s initial thinking, or even holes in one’s initial logic. 2. Predictive role: From the equations of a mathematical model one

can generate testable predictions. These predictions can be rendered at least semiquantitative by nieasur-iiig the various parameters of the model. 3 . The volu of detevwiiiiiiig [hr ( w u i i t i u l fPature.s of (i r.o~nplox. s y f m : When the system u n d e r investigation is complex, models are invaluahle in the effort to ascertain which of the niany features of the system have a major influence o n a given phenomenon and which provide less important quantitative adjustments to the principal trends. (An example of this role would be a n effort to understancl the relative importance of' the many processes that might or do regulate intracellular C a 2 + .Elaboration of this crucial matter is given in this review.) It is worth making one general remark concerning the predictive function of models. Often the models contain several parameters. s o that it is fairly easy and not particulai-ly significant to obtain semiquantitative agreement with the results 0 1 a single experiment. Rut even if' a number of parameters a r e present, experience shows that with nonlinear models (which a r e characteristic of' complex phenomena) it is not a t all easy to obtain simultaneous semiquantitative predictions of'the ~-esults01' several experimen ts.

II. Fundamental Aspects of Synaptic Release

A.

-THE

CALCIUM HYPOTHESIS

Since t h e basic findings of Katz and his colleagues on synaptic transmission (Katz, 1969; for a review, see LlinAs, 1977), it has been accepted that Ca2+ ions are not only necessary but also sufficient for evoked release t o occur. These assumptions are the basis of the "calcium hypothesis" for neurotransmitter release. A direct consequence of the calcium hypothesis is the assumption that the temporal distribution of intracellular (:a2' controls the magnitude a nd tiap r.ou~:\eof release and, conversely, that the magnitude a n d time course of release can be taken (although there is no direct evidence for this) t o parallel the distribution of intracellular (:a" below the release site (for a review, see Augustine rt ( i l . , 1987). Analysis of' the validity of this assumption forms a major part of o u r stic-ceeding discussion. I n textbooks dealing with relcase, there appears the following minimal scheme to describe the (;a hvpot hesis: T h e action potential reaches the nerve terminal a nd depolarizes the membrane to open (:a" channels. (;a'+ ions then flow in and trigger the mechanisms leading to exocytosis.

4

H. PAKNAS rl u1

Release stops after the rapid removal o r binding of the elevated free Ca2+,at least of the free Ca'+ near the release site. This scheme has been so well accepted that for many years insufficient efforts have been made to inquire more deeply into the mechanism of release. At the same time, very elaborate work has been done to characterize Ca2+ channels (reviewed by Tsien, 1983; Tsien et al. 1986, 1988) and the behavior of Ca" currents under a variety of conditions (Llinas et al., 1981a,b; Augustine et al., 1985a,b), and an impressive body of findings has been accumulating on short-term and long-term modulation of transmitter release (Kandel and Schwartz, 1982; reviewed by Atwood and Wojtowicz, 1986).

B.

PROBLEMS AND

CAVEATS

Little is known about the mechanisms linking the arrival of the action potential at the terminal and exocytosis. In addition to the fact that Ca'+ ions are involved (Katz, 1969) and that exocytosis occurs (Heuser and Reese, 1981; Heuser et al., 1979), very little is really established about intervening events. Although most researchers agree that the release of a single quantum occurs by exocytosis of a vesicle that contains the neurotransmitter (Katz, 1969, 1971; Heuser and Reese, 1981; Cohen and Van der Kloot, 1983; Atwood et al., 1987), there is some supporting evidence for the view that at least part of the release of quanta is via a nonvesicular process (Tauc, 1982; Dunant, 1986; Israel rt al., 1979). Moreover, very little is known of the mechanisms by which a vesicle fuses with the external membrane. Different models have been advanced in a variety of experimental or artificial systems (Duncan, 1983; Finkelstein et al., 1986; Cohen et al., 1980; Zimmerberg rt al., 1980a,b, 1985; Zimnierberg, 1987). At least one model, the chemosmotic model (Stanley and Ehrenstein, 1985), has been confronted by experimental results (Augustine et al., 1988; Zimmerberg et al., 1987)and is being reevaluated (E. F. Stanley and G. Ehrenstein, personal communication). In studying the process of release of neurotransmitters from nerve terminals, or the release of hormones and other conipounds from nonneuronal tissues, one should take into account that even though some common mechanisms may prevail [e.g., involvement of Cay+ ions, although Ca independent release is possible in some systems (Schwartz, 1986, 1987)], different mechanisms probably exist to account for the large differences seen in release properties. For example, in peripheral neuronal systems, such as the neuromuscular junctions of vertebrates and invertebrates or the giant synapse of the squid, at room temperature

MECHANIShlS O F NEL'KO7'KANShll'l . I X K KEI.EASk

5

the release process is very fast. LJnder propel- conditions. release starts l9 8 lb ) . This time course is after a delay of only 0.2 nisec ( l h 5 s et d., quite different from that seen in the sea urchin egg (Zininierherg. 1987) chromaffine cells (Douglas, 1968; Baker and Knight, 1984) or niast cells (Almers and Neher, 1987; Breckenridge and Almers, 1987), in which the release process is very slow, starting after long delays and lastiiig for many milliseconds if not seconcls. Even within the nervous system itself, we find responses that appear after long delays of several seconds (Dietzel et al., 1986; I. Parnas and Strumwasser, 1974). I n many of'these cases it is not clear what fraction of the niininial delay is produced by the release process itself and what fraction b y postsynaptic, second messenger niechanisms (Drummond et al., 1980; 1,einos and Levitan, 1984). Obviously, a mechanism that allows release to start after a delay of 0.2 msec m a y not be the same as one requiring much longer delays. As most nerve terminals are small and not amenable to certain experimental manipulations, there is a tendency to extrapolate to the nerve terminals results an d concepts obtained in experimental systems in which 1987). such experiments can be performed (reviewed by Augustine Pt d.. In view of the large difference in release properties seen in the dif-ferent experimental systems, such extrapolations should be made with great care.

c. T W O EXPERIMENTAL MErHO1)S FOR CHARACTERIZING KF:I.EAS~: OF RELEASE.) MEASUREMENTS OF Q U A N T A L CONTENT (AMOUNT KINETICS

AND

I n most electrophysiological experiments, the postsynaptic response serves as a measure of the ntnoitnt of transmitter released. Usually. the aniplitude of either the postsynaptic potential o r the postsynaptic current is used. O n e has then to assume proportionality between the amount of transmitter release and the postsynaptic response or to use corrections (Martin, 1965; but see McLxhlan and Martin, 1981). I n order to avoid such problems, the quantal content (Katz, 1971; Martin, 1965) should be determined if possible. In the squid giant synapse the quantum size is very small, in the range of 1 0 microvolts (Miledi, 1973; Charlton ot nl., 1982), and the synaptic responses are composed of a few thousand quanta (Katz and Miledi, 1970; Charlton et al., 1982). T h u s determination of quantal content is impossible and detection of very small levels of release is difficult. Consequently, the squid giant synapse, which is very convenient for the control of presynaptic membrane potential and for the measurement of presyriaptic calcium

6

I I . P \KNAS

rl n /

currents (Llinas el al., 1981a,b, 1982; Augustine (4 ul., 1985a,b), is less favorable for measurements of low levels of transmitter release. Neuromuscular junctions with their fine terniinals, however. are quite convenient foi- recording of.single quantal events at very low levels of release (Andreu arid 13arrett, 1980) but are inconvenient for the control of presynaptic 1iieniI)rane potential o r measuring presynaptic (;a‘+ currents. T h e crayfish opener neuromuscular system perniits some control of the presynaptic nienihrane potential by means o f a11 iritracellular microelectrode (Wqjtowicz and Atwood, 1983, 1984; Sivaramakrishnan et al., 1 988), a n d single qiianta can he detected when recordings ar e made from restricted rcgions with the macropatch electrode (C)udel, 1981), d u r i n g and after the depolarizing impulse (Atwoocl vt ( i l . , 1987; Wojtowicz et al., 1987). Nevertheless, the membrane potential at the release zone is not known, and the measurement o f (;a“ currents is not easy. I n the neuromuscular system, especially that of the craytish, it is possible to count the number of quanta released during each sweep, a n d statistical analyses (Poissoii o r binomial) can usefully be etnployed (Johnston an d Werning, 197 1). I3ased on these techniques, most of the w o r k o n release is concerned with the quantal content anti its dependence on C a y + ions or other variables. An important characteristic o f release, unfortunately not so frequently measured, is its k i w t i c s a s revelaed by the probability of a vesicle to be released (or of a unitary event to occur) as ;I function of‘the time after the pulse. We shall generally use the terms “kinetics” and “time course” interchangeably. For example, if a pulse releases 100 quanta, we may ask how many of these quanta were released in the first bin of time after the pulse, the second bin, etc’.Obviously, i f the 100 released quanta are nieasured as one event (synaptic potential o r current) it is difficult t o discern the kinetics of release. An attempt to discern the kinetics o f release from the time course of’ the average synaptic current has heen made hy V a n d e r Kloot ( 1988a,b) assuming the unitary synaptic current (mepc) to rise instantaneously and decay exponentially. At high temperatures these assumptions presumably hold. T h e n this new technique is very helpful and rapid. It is especialty useful for cases in which n i m y quanta ar e released following each pulse a n d single quanta cannot be discerned (e.g., the squid giant synapse). Van der Kloot (l9XRa,b) compared results of their n e w technique with those obtained with the “direct” technique of Kat7 and Miledi (l985a,b, a n d see below) a nd obtained good agreement. However, when the time course of release is studied under conditio~isthat may also alter the postsynaptic response, resulting in changes in rise tiirie or decay of the single current event, then this technique is less uset’ul. I n several

systems the shape o f t h e unitary syriaptic event

differeiit synapses o f t h e same axon (Atwootl, 1076) and incoi-rect coiiclusions ;is to the time course of release at such syiiiipses may be I-eactictl. Katz and Miledi (1965a,b)suggested a direct way t o ev;ilu;itc t h e tiiiie course of' release irrespective of the shape 01' the single events. At low quanta1 contents, the synaptic delay Iiistograrn represents t lie prolml~ility of release as a function of time, that is, the kinetics of I-elease(Fig. 1:l). Instead of giving one pulse that i ~ l c ~ man) c s quanta. the experiinetitei. gives many pulses, each releasiiig o i i average only one qir;iiitiriii. Exaniples of failures and single cltranta i-eleasecl af'tei. vai-iahle delays are tlepicted in Fig. 1B. I f tliesc tlclays are measui-ecl and a delay histograni constructed (Fig. 1A ) , the time coiii-se of release is obtained. In such A

(iiiepc) \ , ; i l k s i i i

C

8

Slope 4.2

8ol-F-

1-

I I

320-280-.

240-. v)

0

-.

v)

-0 Q)

200--

?!

'c

0 0

*

160-. _. .

120--

:"Id -

0

0

1

ll

2

msec FIG. I . Crayfish neuromusculai. I i i i i c t i o n opeiier I I I L I S C I ~ . ( A ) Kinetics of ~ . c l c a w,I\ depicted by a synaptic &lay hisrogi i i i i i . Nrrmhrr 01. pulses AV(pulac t l u i . , r t i o i i . 1 111. amplitude, -0.8 pamp) aiitl quarrral c o i i t t ' i i t t n arc given. 'I'eiiiper~~ruIc !I"(:, Y O I - I IKiiigu I~ solution. (1. Parnas. unpublished rxpci~iiiiciir.)(B) Saiiiplc rectri-(Is. Notice. t,iilurcs and single releases with variable de ((:) Img-log plot of (tic initi;rl ptraw ot r l ~ c s\lraptic tlcla) histogram in ( A ) . 'The slope pr e b a i r csrini;rte for. the r.clense coopel-arivit) 1. scc Scc-tioii 111,8,3.

8

H . PARNAS PI nl.

experiments, there is a minimal delay below which no quantum is released (irrespective of the number of pulses given). Obviously, this is the minimal time for the chemical reactions leading to release to take place. After the minimal delay, the probability of release increases to a peak and then decays to very low levels within a few milliseconds (in a temperaturedependent manner). Aumann and Parnas (1990) reconsidered the question of estimating the time course of release from measurements of the average synaptic current. The approach was similar to that o f Van der Kloot (1988a,b)but instead of approximating the unitary release by an instantaneous decay, they employed an average measured mepc. Good agreement with the considerably more difficult direct approach was obtained under a wide range of conditions. Unlike the direct approach, the Aumann and Parnas (1990) method can be employed at high quantal content. T h e quality of a delay histogram as a representation of the kinetics of release depends on the accuracy of the measurement of each quantum's delay and the size of the time bin taken for the histogram (H. Yarnas et al., 1986).With modern digital measuring devices high accuracy can easily be attained. It is also important to record a large number of single quanta events in order to obtain a complete histogram (H. Parnas and I. Parnas, 1987). I t should be pointed out that the area below the delay histogram is the total number of quanta released for a given set of pulses. Dividing this value by the number of pulses gives the quantal content m (see Fig. 1A). We now discuss various aspects of release that are associated with the dependence of the quantal content on Ca" ions.

D.

FACILITATION A N D

RESIDUAL CALCIUM

When twin pulses are given, the release elicited by the second pulse is generally higher than that elicited by the first. Such elevated release (or facilitation) was suggested by Katz and Miledi (1968) to be due to residual free Ca2+ remaining in the terminal after the first pulse. A major review of facilitation has been provided by Atwood and Wojtowicz (1986). Here w e restrict ourselves to a summary of the main experiments that provide evidence for residual free Ca" being the underlying cause of facilitation. 1. A close correlation has been shown to exist between the magnitude of influx of Ca2+and both the amplitude of twin pulse facilitation and its duration (Rahamimoff, 1968; H. Parnas el al., 1982a; I. Parnas et al., 1982a; Dude1 et al., 1982, 1983). Such correlation is difficult to envision if

MEC,HANISMSo r NECIRO I RANSMI I I L K K L L ~ A S ~

9

the entering Cay+ only starts a chain of events that are responsihle for facilitation. 2. Treatments that increase or decrease the free level of-intracellular Cay+ affect facilitation in the same manner (Kahaminioff rt al., 1978). Also, reducing [Na+Ioand hence inhibiting the extrusion of [Ca'+]i via the [Na+],-[Ca2+], exchanger prolongs facilitation ( I . Parnas rt al., 1982a; Meiri et al., 1986). 3. Equations for facilitation based on free residual Ca" describe both qualitatively and quantitatively various observed aspects of facilitation. In particular, the predicted time course of facilitation agrees with the experimental time course (Magleby and Zengel, 1982; Zengel and Magleby, 1982; I. Parnas and H. Parnas, 1986; Mallart and Martin, 1967, 1968). Also, the predicted magnitude of facilitation agrees with the observed magnitude. 4. An increase in the background level of intracellular Cay+reduces facilitation (Dudel, 1986). I'his result emerges naturally i f one assumes that the quanta1 content of release and facilitation are regulated by the same factor, the level of free Cay+,the key process being a saturating function of free Ca2+ concentration (H. Parnas and Segel. 1980; H. Parnas et al., 1982a). 5. Dyes that detect free Ca'+ show that the time course of' decay of the elevated Cay+ resembles the time course of facilitation (Connor et al., 1986). Similarly, Charlton et al. (1982) studied facilitation in the squid giant synapse after intracellular injection of Ca". Using ai-senazo I I1 [2,7-bis(2-arsenophenylazo)-I ,8-dihydroxynaphalene-3,ti-disulfonicacid] to monitor changes in intracellular free Cay+ concentration, they showed that in successive pulses the Ca" entry is the same and that the residual Cay+ lasts for severd seconds, again in good agreement tvith the time course of facilitation. T h e conclusion from these five findings is that twin-pulse facilitation (like the amount of evoked release) is regulated by the level of free Cay+ below the release sites. Other factors have been found to modulate release and that have been suggested to determine facilitation, for example synapsin I (Llinas et al., 1985; Nestler and Greengard, 1986; Greengard et al., 1987), may well be involved in twin pulse facilitation but cannot, we believe, be the key elements in determining it. Based on the adequacy of the residual Ca'+ theory to account for facilitation, H. Parnas and Segel(l980, 1988) and H. Parnas rf al. (l982a) suggested the use of facilitation to study the entry, release, and removal of Ca2+ in preparations (such as small nerve terminal) wherein these three processes are difficult to study directly. This matter will be discussed below.

10

l i . PAKNAS r/ ol

111. The Relationships between Release and C a Z i

Release increases with the amount of Cay+ions entering the terminal (Llinas P t al., 1981a.b; Augustine et al., 1985a,b) and levels of'f at high levels of extracellular Ca" . Saturation has been shown in all of the fast systems that have been examined: frog neuroinuscular-.junction (Dodge and Rahamimoff, 1967), mammalian neuromuscu1;irjunction (Choke et al., 1973), squid giant synapse (Ilin5s et ul., 1981a; Augustirie and Charlton, 1986), crustacean neuromuscular junctions (H. Parnas et al., 1982a), and leech neurons (Dietzel of ul., 1986). By now it is clear that the Ca'+ ions act from the inner side of the membrane (Katz and Miledi, 1977; Llinis, 1977; I.linas el al., 198la,b; Augustine et al., l985a,b). Consequently, we can divide our discussion of the involvement of C i 2 +in the release process into three main sections (H. Parnas and Segel, 198 1 ; H. Parnas et nl., 1982a): A, Entry of Cay+ as a function of membrane depolarization and extracellular C a y + concentration; €3, Release of neurotransmitter as a function of intracellular Ca2+ concentration; and C, Removal o f the excess intracellular (:a'+, restoring the normal level.

At small nerve terminals Cay+ currents cannot easily be measured directly. O u r present knowledge of the dependence of the total Cay+current on membrane potential and extracellular Ca"+ concentration conies from investigations of the squid giant synapse (Llinris rt al., l98la,b; Augustine rt ul., 1985a,b; Augustine and Charlton, 1986)o r from experiments on cultured neurons in which chemical synapses are established (Adams et al., 1989). I n these cases, the total Cay+ current is measured from a relatively large surface membrane, the soma, or the nerve terminal. 1. Dependence of Cu'+

Ciivwiits

on Extmcrllirluv Cn'

' Coucentratioii

It may be that because of "overlap" of calcium fluxes from nearby channels there is no simple relationship between the Ca'+ current per channel and the concentration ofCa2+ at a release site (Chad and Eckert, 1984; Simon and Llinas, 1985; Fogelson and Zucker, 1985; Zucker and Fogelson, 1986). Another possible complication stems from the fact that there a r e many different types of calcium channels. Perhaps because of their preferential proximity to release sites, only certain channels play a major role in the control of release. This is discussed by Smith and

MECHANISMS ( ) F N tl'KOTKANSX1 I T I E K REI.t:.\SE

11

Augustine (1988) and Hirning el (11. (1988). Nevertheless, there is good correlation between the total current and release. In systems wherein the total ~ a ' +current was measured, it was found to saturate a s a func-tion ot' extracellular Cay+ concentrntion at constant depolarization (Augustine and Charlton, 1986) (see Fig. 2A).

2 . Dvpendence of Ca2+ Currtwts 011 i2lrmDrme Potrntiul T h e different types of (;a'+ channels and their dependence o i i nienibrane potential are discussed in great detail by Tsien (1983, 198fi) and Tsien et al. (1988). T h e dependence of the Cay+currents on membrane potential is examined in the papers of LlinAs rt ul. (ISXla,b) and Augustine el ul. (1985a,b, 1987). Here we mentioned only some points that are relevant to the present discussion. 'The dependence of the Cay+current on rnernhrane potential is bellshaped (Fig. 2B). T h e peak of'the Cay+ current is at about 0 to - 10 niV. T h e channels seeni to be niaximally activated at above +20 m V with half maximal activation at - 15 niV. After a depolarizing pulse, t-di'I ciirrents decay over a period of a few microseconds (Augustine rt nl., 198'7). I t is important to emphasize that "with return to a negative resting potential, Cay+channels appear to close in a small fraction of a millisecond" (Augustine et al., 1987). This nieans that there is very little lingering entry of Ca2+ ions after a depolar-izing pulse or after an action potential in which most of the Cay+enters as tail currents. A *O01

zmm 200

/

a 4001 u

0

0

A 5 m v

- 30 rnV

-

I

a

-

-$ loo-

-

u 0

b+

0

20 Ce (mM)

OO

-

40 Membrane potential (p (mV)

FIG. 2. (A) Observed saturating dependence of Ca"+ current I ( .,. at tlit-ee Icvcls of depolarization, on the extracellular (:a'+ coticeriti-itlion C, (Augustiiic and <:ltarltoti. I %(i). (This and all succeeding figures itletititicd as d u e to other authors have been rrpt.odticed with permission.) (B) Observed bell-shaped dependence ol":a2 ' current I ( , , , on nicnihranc potential (From Llinas ~1 d.. 198I:i.)

+.

12

H . PARNAS el

a/.

3. Equation f o r Entry We now start to incorporate the salient accumulated information into a mathematical model. For this purpose, we shall use the notation of Table I in referring to the various calcium concentrations. The first to describe Ca2+entry as a saturating function of extracellular Ca2+concentration were Hagiwara and Takahashi (1967) followed by Akaike et al. (1978). A simple expression that implements the observed dependence of the Ca'+ current on the extracellular Ca" concentration C,, at a fixed depolarization 4, is the following equation for the total Cay+ entry Y:

Here F6 is the maximal possible Ca2+ entry (at high C,) for a given depolarization 4 and is the half-saturation coefficient (see Table 11). The maximal entry F6 is proportional to the number of open channels. One expects that F@will be an increasing, saturating function of the depolarization 4. The half-saturation coefficient A'$ reflects the dependence of the driving force on depolarization. The greater the voltage the lower the driving force, so K$' should increase with 4. The joint dependence of F@ and k$ on 4 assures the observed bell-shaped dependence of Yon 4 (Fig. 2B) while the saturation of Y as a function of C, follows from the observations of Fig. 2A. There seems no need for a power function in Eq. (1); that is, no cooperativity is assumed for Cay+entry (Augustine and Charlton, 1986). The above conclusions were reached employing direct measurements (in squid). Indirect techniques have also been used to monitor Ca2+ entry. Given that twin-pulse facilitation depends on residual Ca2+,a longer duration of facilitation is expected when more Cay+ions enter the terminal during the first pulse (H. Parnas and Segel, 1980). T h e longer duration is anticipated because it takes a longer time to remove the larger amounts of Ca2+.Thus the duration of facilitation reflects entry. Upon TABLE 1 NOTATION FOR V A R I O U S Ca"

Symbol C,

C

c*

<;ONCENTRATIONS

Meaning [Cay'],, the extracellular Ca2+ concentration [Ca'+Ii, the intracellular Ca'+ concentration Background value of [Ca'+], (or rest value after a long period of stimulation)

13

MECHANISMS O E N L L K O IKANShlITI'EK KE1.EASE

~I'ABLE[ I NOTATION FOR VARIOI:SPROC:E,SSES THAT GOVERNRELEASE Process Entry

Symbol

Y

V" Release

kf L L Ki

Removal

1 dCldt P

K,

Meaning

Cay+ entry (a tuiiction of depolar-ization 4 a n d [Ca' I,, I C,) Maximal entry (at large C?), at fixed depolarization 4 Half-saturation roefficient tor entry at fixed depolarizatioii 4 Am o u n t of i i ~ ~ i ~ - o t I a ~ i s ~release iiitter (as a function of [(;a'+], I c) Maximal relcase (at large C) Half-saturation coefficient 101-release (when 1 = 1 ; otherwise. a measure of saturation) Release cooperativity exponent Rate of removal Maximal removal rate Half-saturation coefficient for removal +

examining the duration of facilitation it was found (H. Parnas and Segel, 1980; H. Parnaset al., 1982a; Dudel etal., 1983, summarized by H. Parnas and Segel, 1988) that at constant depolarization, entry saturates and shows no cooperativity as a function of C,, and at a constant C, entry shows a bell-shaped dependence on depolarization. Because the same conclusions were obtained by monitoring facilitation and from direct measurements of Ca2+ currents, it is safer to project the results obtained in the large nerve terminals to small nerve terminals. These results also give further support to the residual theory of Katz and Miledi (1968)that residual free Ca2+ is the principal determinant of facilitation.

B. DEPENDENCE OF RELEASE ON CA*+

THE

INTRACELLULAR

CONCENTRATION

Three questions will be discussed in relation to the dependence of release on the intracellular (;a2+ concentration. ( 1 ) Does release following a pulse depend almost exclusively on the Cay+whose entry is induced by that pulse or is the relevant (;a'+ level obtained by a combination of entry and the background <:a2+ concentration? (2) Does the dependence of release on the intracellular C;a'+ concentration C exhibit saturation? (3) Does the dependence of release on C exhibit cooperativity? 1. Is the Ca2+ That Enters Follozvin,g th,e Pulse Almost Exclusiuely Responsible f o r Evoked Release?

T h e value of the background Ca" concentration at rest is about 100 nM (Baker, 1976, 1978; Baker and Umbach, 1987; (hnnoi- Pt d.,

14

M. I'AKNAS

Y/

nl.

1988). It is well established that (;a'+ enters the terminal via discrete channels. Based on this, some investigators conclude that the only Cay+ decisive for release is that which enters just prior to release and forms local domains of high Ca" concentration (Fogelson and Zucker, 1985; Simon and Llinas, 1985; Smith and Augustine, 1988). Nevertheless, there are many indications that there is no need for especially high Ca2+conceritrationsto induce release and hence that the calcium level that is formed by a combination of entry and the existing background level is decisive for release. Under conditions wherein voltage-dependent entry was absent, Dude1 (1989) obtained a close correlation between the level of evoked release and C,. From this he concluded that the change in C, caused changes in the background Cay+ level, which in turn affected release. 'This conclusion is supported by direct measurements, using Cay+electrodes, by Baker et al. (197 1) and Baker and Unibach (1987), that showed that the concentration of Cay+in the cytoplasm depends on C,. Indirect support is also furnished by observations that in axons poisoned with mitochondria1 inhibitors the intracelM a r Cay+increases about 50-fold (Baker and Umbach, 1987). Charlton et al. (1982) showed that release was increased following the injection of Ca2+ into the squid giant terminal. After repetitive firing, release increases (PTP), probably through the accumulation of Cay+ions (Magleby and Zengel, 1975a,b). Following application of Ca ionophore (H. L. Atwood, I . Parnas, H. Parnas, and J . M. Wojtowicz, unpublished results) or lipid vesicles filled with Ca" (Kahamimoff et al., 1978), release increases dramatically. Release can even be decreased by hjecting Ca2+ buffers (Adler et al., 1988). All the above treatments have a dispersed effect throughout the terminal and thus will not cause localized high Cay+concentrations near the release sites. It is thus difficult to believe that localized domains of high Cay+accumulation play the decisive role in controlling release and that the background Cay+ level should be ignored. On the contrary, these experiments lead to the conclusion that the appropriate description of the intracellular Ca2+ that is responsible for release is

c = Y + cs.

(2)

Here C denotes the intracellular Ca'+ concentration near the release sites, where C is obtained by adding the entry Y to the background level C, 'his equation was first suggested by H. Parnas and Segel(1980, 1981) and H. Parnas et al. (1982a) arid was later employed by Barton et al. (1983). From the above results it appears that in general Y and C,. should be regarded as functions of C,.

2. Suturation For the small nerve ter-iniitalsthere are no direct iiieaiis of'evaluating the dependence of' release oil 111eiiitracelldar Cay' concentration C. Information regarding the tlcpciitlcnce of. release o n the cxtrac-ellular [:a'+ concentration c,. caiiiiot autoriiaticaI1p tie applied t o the (Ieperidence on C. T h e C, depencleiicc o f release lumps both the entry :tiid the release processes. Entry depends OII (;,. while release clepentls o i i (,'. Nevertheless, the first clue to a possible relation between release antl C comes from the deper1dciic.e o f release on CC.Dodge and Kaliaiiiiinoff (1967) showed that release exhibits a siginoitl clependencc on (;<.. 'l'hat is, release saturates at higher Ievcls of C,. ;ind increases more tlian liiicarl\ C,, increases at relatively l o w \,slues ot. C,. 'These observations (xi1 lie represented in the k'ollowing IOi-niula for the release L,. as ;i function ot' the extracellular Ca' coiiceiiti-iitioii (;,.: +

-

Here the constants I, ,and K v , impectivelp, measure niaximal release antl provide an estimate of the level of saturation. (KJ'" is the halt-sat~ii-atioii concentration.) It has been observed ( f o i - example, by Rahaminioff, l!W3: H. l'itriias rt ul., lY8Pa; I. I'arrias PI (11.. I!)ti?b) that there is a tlecline in shoi-t-term facilitation (which is characteri/.etl b y a short intenal hetween t h e t \ v i n pulses) as C, is raised (Fig. 3 A ) . H. Parnas and Segel (1980) s h o w e d that this indicates that release s;itui-;ites as a function of the iritracrllulai. calcium concentration C (Fig. 3 H ) . 1,ater we shall incorporate t his finding in a counterpart of (3) that gives rele;ise as a t'ucntioii of' C.

3. Cooprrativity und Appcirrnt ( ~ o o / ) m i t t i ~ i t y T h e question of cooperat ivity in the process of release is of' iiiiportance as it bears on the mole(-iilar el'ents uiiderlying release. 'l'hc niaiii procedure that has been used t o evaluate the cooperative actioti ol' Cay' ions in the release process is measiireriieiit of the slope of the line relating log release to log C, (Dodge aiicl Kahamirnof'f, 1 9 6 i ) . Frecluciitly, i.elease is measured as a function ot' the (:a current, under the assumption that C, (Llirias rt cil., 19Xla.b: Augustine at ol., 19H5a.h; i2ugustine and Charlton 1986). 'l'his procedure is I ~ s c do n Eq. ( 3 ) , which relates release to C,. Accordiriglp, the value of the cooperativity I,. c-mi be estimated from the graph of log L , versus log C , at low C,,, since

-

L,

- C,'I

implies

log L,.

- I,.

log C,

16

H . PAKNAS

rf a/

A

B

C,(mM)

C

FIG.3. (A) Experiment showing dependence of short-term facilitation F , on the extracellular Ca2+concentration C., (Redrawn from I . Parnas et nl. 1982b).T h e box indicates the theoretically predicted relation between maximal facilitation F ” Y and the release cooperativity exponent 1. (B) T h e theoretical dependence of the total release L 1 on the intracellular Ca2+ concentration C as deduced from the esperinicntal result of. (A). ‘l‘he saturation follows from the observed decrease of’F,as a function of(,’,. ‘l‘hevalue ofthe cooperativity 1 is given by the boxed formula of (A).

Use of this procedure for estimating I, resulted in different values in different preparations. Even in the same preparation the variability was significant under various experimental conditions. In the frog neuromuscularjunction, Dodge and Kahamimoff( 1967) found a slope of four, but later Crawford (1974) found a slope smaller than one at very low quantal contents in the range 0.015-0.65. Andreu and Barrett (1980) reinvestigated this issue and found a power of four at very low levels of quantal content, as low as 0.002. In crayfish neuromuscular junction, Zucker (1974) and Staggs et d.(1 980) found a slope of one. 1. Parnas and Dudel (1982) and I. Parnas et al. (1982b) measured a slope of 1.6 in the proximal and 3 in the distal synapses of the axon innervating the opener muscle. For the crab, Linder (1973) found a slope of 3. Variability in measurements was also encountered in the giant synapse of the squid. Llinas et al. (1981a,b) describe a linear relationship between Ca‘+ currents and release. Later studies showed a sigmoid relation with saturation (Stanley, 1986; Augustine and Charlton, 1986), with a cooperativity exponent I, between three and four. In view of this variability, the question arises whether or not the slope of log L, versus log C, faithfully estimates the cooperative dependence of release on Cali concentration. If so, we are forced to assume that “this

MECHANISMS O b N E l ' K O rRAKSh1II"TF.K KELFASI.:

17

finding suggests that under some conditions a single ion" (or sometimes two, o r three), "may suffice to activate a release site" (Andreu and Barrett, 1980). A related interpretation has been suggested by Stanley (1986).He showed a slope of four for the graph relating log release to log C,, in the squid giant synapse, as long as low stimulus frequencies were employed. However, with higher frequencies of stimulation, the slope declined to 2.3 at 10 Hz and to 1.1 at 80 Hz. Stanley (11386) concluded that "the cooperative action of four C a y + ions is required to trigger the release mechanism and that the phenomenon of facilitation involves a reduction in Cay+cooperativity." I t may be, however, that the cooper-ativity in the release process is the same in all these preparations (probably four) and is the same for release elicited by a single pulse and that elicited by repetitive stimulation and that some other factor is responsible for the observed variations. This matter is discussed below. A priori, the observed cooperativity summed u p in Eq. (3) may reside in the entry process, in the release process, or both. We have already cited evidence that entry is noncooperative, so there must be a cooperative dependence of release L on the intracellular Ca'+ concentration C. H. Parnas et al. (1982a) showed that the cooperativity exponent 1 [see Eq. ( 4 ) ] can be estimated from observations of niaximal facilitation (at short intervals between pulses). T h e maximal facilitation, predicted to be 2', is found by extrapolating a graph of short-term facilitation Fs as a f'unction of C, to very small values of C , (see Fig. 3A). Facilitation values as high as 1 1 were obtained experimentally ( H . Parnas et al., 1982a; Dudel rt d , 1982; 1. Parnas et al., 1982a). There is thus no doubt that significant cooperativity is present. This result and the conclusion of H. Parnas and Segel (1980) cited above that release saturates are incorporated into the following equation for the total release L.r as a function of the intracelluIar Cay+concentration C:

I > ,(C) =

Lc' (K, + C)'

(4)

As stated in Table 11, L denotes maximal release and K , is a measure of saturation. Equations (1) and (2) should be used to evaluate C as the sun1 of entry and background Ca' ' . H . Parnas and Segel(1980),H. Parnas et al. (1982a),and Barton el al. (1983) showed that if the background Ca2+concentration is taken into account, then the apparent cooperativity 1, of Eq. ( 3 ) may be an underestimate of the true cooperativity 1 of Eq. (4). H. Parnas and Segel (1981) showed further that the key factor is the ratio between the concentration of the entering Ca2+and the background Cap+concentration.

18

t3. PAKNAS t’t ol

The maximum facilitation experiments Just cited lead to the conclusion that 1 > 3, but probably 1 = 4 (H. Parnas rt ul., 1982a; I. Parnas et al., 1982b). A more direct confirmation of’this analysis comes fi-om :he work of Augustine and Charlton ( 1986). These authors showed that while the Ca2+ current was linearly related to extracellular Ca2+ concentration, release was nonlinearly related t o the Ca’+ current, with an exponent of four. In order to circumvent the problems of measuring release at different extracellular Ca2+ concentrations where an “apparent cooperativity” is ) an alternative way to deterobtained, H. Parnas et NI. ( 1 9 8 6 ~developed mine the cooperativity from synaptic delay histograms. These authors demonstrated that the slope relating log release to log time at the beginning of the synaptic delay histogi-am is equal to the cooperativity (see Fig. 1C). A large number of’pulses should be used in one Ca2+concentrations under high depolarization and the bin size for the delay histogi-am should be carefully examined. Using this procedure, H. Parnas ef ul. ( 1 9 8 6 ~found ) slopes of approximately four for crayfish and lobster, and similar values were also found in the frog neiirornusculai-Junction (Matzner el ul., 1988). It seems then that the cooperativity in the squid (Augustine and Charlton, 1986), the frog (Dodge and Rahainirnoff, 1967; Matzner et ul., 1988), and crustaceans (Linder, 1973; I. Parnas et al., 1982b), is four. Higher values that are sometimes used (e.g., Fogelson and Zucker, 1985) do not seem justified. An important theoretical finding from the work of H. Parnas et ul., ( 1 9 8 6 ~ is ) associated with the step at which cooperativity exists. Such information cannot be extracted from nieasurenients of quanta1 content. However, measurements of the kinetics of release reveal that it is not the case that four Cap+ ions bind to a site (Dodge and Rahamimoff, 1967). Rather, four complexes of Ca2+witha key molecule (probably a protein) are involved in exocytosis o f a single vesicle. Our conclusion has provided a good example of the utility of theoretical models, for there seems no other way to deduce characteristics of an as-yet-unidentified molecule. The analysis concerning the value of cooperativity and the kinetic step at which cooperativity occurs may limit the candidates for the key molecule, which is probably a protein to which Ca‘+ ions bind (Duncan, 1983). In particular, calmodulin, which was suggested to be involved in the release process as a Cap+binding molecule (DeLorenzo, 1982; Duncan, 1983),is not likely to be the key molecule, as it binds four Cay+ ions. Smith and Augustine (1988) also discard calmodulin as the key molecule but for other reasons. Because of its assumed voltage sensitivity (see below) it has been suggested that the key molecule is membranous. In

addition, the finding that l'our (:a2' -key molecule complexes are involved in the exocytotic process leads to the suggestion that the key molecules are arranged in tlie menibi-ane in groups of four, locatecl in the release sites themselves, and fiicilitating cxocytosis by directly bincling the vesicle to the release site ( H . 1';irnas and I . Parnas, 1989).

4. Time Course ofKa1ra.w

So far we have discussed the deperidence of the quanta1 content on Ca2+, in particular on the iriti-acellular Ca2+ concentration C. Another aspect that needs consideration i n this context is the dependence of'the time course of release on C. Unfortunately there are its yet no direct techniques to resolve the time course of Ca'+ concentration ,just below the membrane. One widely accepted indirect inference is as follows: A t room temperat ure release induced by a pulse ceases af'ter about 2 insec (Katz and Miletli. 1968). Hence the intracellular Cay ' near tlie iiiembrane must be rapidly removed, in about 1 msec (Blaustein, 1988). T h e inference of the previous sentence rests o n the calciirni hypothesis that the presence of intrac.ellulai- Ca" is a necessary antl sufficient condition for release. However, ir may well be (as is stressed in this review) that the elevation of' intracellular Ca2+ is not sufficient to induce release. As long as the calcium hypothesis is not proven, it cannot be taken for granted that the rise and frill of release necessarily implies a parallel rise and fall of intrac.ellulai-(;a'+. More subtle arguments must be employed in any attempt to establish the connection between release and the intracellular Cay+concentration. T h e general features of the time course of evoked release after an impulse are seen in Fig. 1A. After the minimal latency the curve rises to a peak. giving a sigmoid shape 101. the initial part of the curve. The peak is significantly after the end of' the pulse. There is a niinimuni delay that usually outlasts the duration of the pulse (Katz and Miledi, 196.b; Datyner and Gage, 1980; Van der Kloot, 198%; H . Parnas ~t (11.. I
-

o

m

0

a

0

(9

0

=J

0

N

0

m

1"

0

DlUDnb 40 'ON

1"

W

D

ohuonb 40 O N

m

responded markedly to the clianges in calcium concentration (Fig. 4A). Datyner and Gage (1 980) also found that replacing <:a2+ by Bay+did not change the kinetics of release m t l that after a train of pulses the kinetics of release were still the same. Insensitivity of the time course of'release to conditions that alter the temporal distribution of intracellular (;a'+ concentration has been shown (Van der Klott, 19SSb;H. Parnes et ul., 1989). Andreu and Barrett (1980), working at low quanta1 contents of release at the neuromuscular junction, concluded that "at a given junction the duration of the evoked release was kept constant for all [Cay'I,,." Addition of Mg'+ ions, which compete with Ca" entry (Jenkinson, 1957; Dodge and Kahamimoff, 1967; H Parnas et al., 1982b), did not alter the delay histograms anti therefore the kinetics of'release (Matzner et al., 1988). Delay histograms were not changed by repetitive stimulation (Datyner and Gage, 1980; H. Parnas ~t ul., 1986a; Barrett and Stevens, 1972a,b) (Fig. 4B) or after addition of Cay+ ionophore ( H . L. Atwood, I . Parnas, H. Parnas, and J . M. Wojtowicz, unpublished).In particular, the minimal delay was not shorter. The time course of release was found to be insensitive to the level of the pulse depolarization (Dudel, 1984a; I . Parnas et al., 1984; H. Parnas et ( i l . , 1986a; 1989. See Fig. 4C). T h e finding that the time course of release is the same when Ba')+ replaces Ca'+ is of special interest. This is because Bay+ is less well buffered intracellularly than (;a" (Connor et ul., 1986; Tilloston and Gorman, 1983). Therefore if the only mechanism for termination of' release is the rapid removal of'certain ions from below the release sites, it is peculiar that release exhibits the same time course in (;a2+ or in Ra". In contrast to the insensitivity of the kinetic curve to conditions that alter the level and time course of intracellular calcium, the kinetics s h o w a strong dependence on temperature. The Q l o for minimal delay can tie as high as three or four (Katz and Miledi, 1965b; Dudel, 1984a; Barrett and Stevens, l972a,b; H. Parnas et al., 1989). The duration of the delay histogram is also sensitive to temperature. 'The Q I O is larger than 2 (H. Parnas et al., 1989; Dudel, l984a,b) (see Fig. 4D). Another parameter that strongly affects the time course and will be discussed later is post-

FIG.4. (A) Experimental dependcnre of a synaptic delay histogram on the extracellular Ca'+ concentration C,. (From Datyner and Gage, 1980.) Open dors, high C,; filled clots, low C,. Here and below, the right panel pi-esents the same data as the left panel except that each graph is normalized to its peak anrplitude. (B) Delay histograms of three successive pulses. (From Datyner and Gage, 1980.) ((:) Delay histograms at two levels of depolariLatiolr. Dashed line, high depolarization; solid line, low depolarization. (Redrawn from H. Parnas rl d.,1989.) (U) Delay histograms at two teniperatur-es. Open rectangles, high temperature; filled rectangles, low temperature. (Kedrawn from H. Parnas e t a / . , 1989.)

22

H. PARNAS el a1

pulse hyperpolarization (I. Parnas el al., 1986; Dudel, 1984b). (See Fig. 12 below.)

C . Ca2+REMOVAL MECHANISMS The third aspect that should be discussed in the context of the relation between release and Ca2+ is the removal of Ca2+ after it enters the terminal. This aspect is of special importance in the view of the assumed connection between fast termination of release and removal of Ca'+ (Blaustein, 1988; Fogelson and Zucker, 1985; Simon and Llinas, 1985). 1. The Nature of the Processes That Goziern Removal of CaZi As pointed out earlier, according to the calcium hypothesis the temporal behavior of Ca'+ controls the time course of release. If so, then following stimulation the intracellular Cay+ concentration C near the release sites should first increase and then decrease to a low level in less than 2 msec at room temperature. This is because at room temperature the duration of release is about 2 msec. The processes that could be responsible for this Cay+ removal can be constituted of one or more mechanisms from three groups: extrusion through the cell membrane, binding to internal organelles and/or buffers, and diffusion away from the release sites. Extrusion can involve Ca2+ pumps or [Na+Io++ [Ca"], exchangers. These processes have a time scale considerably longer than 1 msec (Blaustein, 1988). Therefore they cannot operate with the required speed to lower the intraceilular Ca'+ and terminate release. I t should be mentioned that the * [Cay'], exchange has been found to play an important role in regulating facilitation (Meiri et al., 1986; I. Parnas et al., 1982a; Arechiga et al., 1990). With respect to buffering, it is known that at rest 99.9% of the axoplasmic Ca" is in bound form. A work by Krinks et al. (1988) and a short review by Blaustein (1988) discuss the properties and binding capacities of several proteins from squid axoplasm and other neural elements. T h e sequestering mechanisms for Ca2+ can be divided into two groups, one that is energy-dependent, low-affinity, and high-capacity and one that is energy-independent, high-affinity, and low-capacity (Baker and Schlaepfer, 1975, 1978). If binding is to bring about the rapid termination of release, then the forward rate constant for binding must be large and the dissociation rate cannot be large. Hence the candidate mechanisms must involve a principal removal buffer with a low dissociation constant (i.e., with high ajjinity).

An attempt to estimate the dissociation constant and Ca" hitiding rate constant o f the principal buf1i.i- was made by Adler rt Ni. (I98X). -They injected several Ca' chelators into the crayfish nerve terminal antl tested for effects on release. BAP'I'A [ t , i s - ( o - ; i m i n o p h e t i o x ~ ) - e t h a i i e - ~ ~ r , ~ ~ , ~ V ' , ~ ~ ' tetraacetic acid] buffers with dissociation constants ratiging between 0.1-0.6 p M reduced release. Dinitro BAPTA, with a dissociation constant in the rnilliniolar range, \ v x It,ss effective. E(Y1-A [ethylene g l y d his (P-aminoethylether-N,N,h",N'-t~ti~aacetic acid], which has a similar dissociation constant to BAPTA but slower binding kinetics, WIS not effective. Thus it seems that the principal buffer may have fast bincling kinetics with a dissociation constant in the micromolar-teIis of micromolar range. (Further implications of these experiments will be discussed belolc.) Concerning the fast group of.buffers, however, Blaustein ( 1988) and Baker and Umbach (1987) point out that the ability to t,ut.fir (:a2' is limited in its capacity, and that the "cytosolic <:a'+ buffering, although rapid may be very limited in capacity, thus the Ca2+binding proteins may suffice to buffer the Ca" that enters mammalian nerve terminals during several 1-2 msec action potentials" (Blaustein, 1988). After repetitive firing, saturation of this compartment is rapidly achieved, followed by a rise in the free Ca2+ concentration (Baker and Umbach, 1987).Since the time course of termination of' release is not altered after repetitive stirnulation, even at very high rates (H. Parnas ut al., 1989), it appears that because of their rapid saturation high-affinity buffers cannot provide the dominant mechanism for terminating release. Diffusion, perhaps together with buffering, provides the remaining alternative for terminating release according to the calcium hypothesis. This is the prevailing view (Blaustein, 1988; Smith and Augustine, 1988; Augustine et al., 1987; Fogelson antl Zucker, 1985; Sinion and Ihnas, 1985). T h e possibility that such a mechanism can account for the time course of release is treated below.

2. The Use of Ca2 Indicators fo DrIermine Intrucellulur Spuliolrrnporal Changes in Ca2+ T h e use of Ca2+ indicators such as aequorin (Smith antl Zucker, methyl}1980), quin 2 [2-{[2-bis(carboxymethyl)-amino-5-methylphenoxy] 6-methoxy-8-bis(carboxymethyl)aminoquinoline] (Baker and Knight, 1984), arsenazo I11 (Miledi and Parker, 1981; Ross et al., 1986; Nich011s et al., 1986), an d fura 2 { 1 [2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-0xy]-2-(2'-amino-5-methylphenoxy)-ethane-~,N,~~'-tetraacetic acid} (Grynkiewicz et nl., 1985) may advance o u r understanding of the spatial a nd temporal changes in free Ca'+ concentration. O n the whole, arsenazo signals can now be obtained with good signal-to-noise

24

H . PAKNAS rl al.

ratio after single pulses (Ross and Werman, 1987; Nicholls et al., 1986; Ross et al., 1987). The arsenazo signals decay slowly in a few hundred milliseconds, especially after repetitive firing (see Fig. 5). The fastest decay of the arsenazo signal was seen by Ross and Werman (1987) in a distal dendritic region of the purkinje cell. T h e signal was more than halfway to its baseline in less than 50 msec. However, even this “f&” decline is slow compared to termination of release (1-2 msec). Using fura 2 and recording from myelinated axons, Lev-Ram and Grinvald (1987) also found relatively fast decays after single pulses in the range of 150200 msec. However, after a short train of 11 pulses (interpulse interval 15 msec), the decay of the fura 2 signals was much slower (V. Lev-Ram and A. Grinvald, personal communication). Sustained dendritic gradients of Ca2+were also seen by Connor et al. (1988). Smith and Augustine (1988) also stress that after a brief stimulus Ca2+ transients persist, with residual Ca2+often remaining a sizeable fraction of a micromolar several seconds after the stimulus. All these measurements confirm earlier indirect inferences (for example, from facilitation) that the intracellular Ca‘+ concentration remains sizeable long after the termination of release. If we accept these findings in their simplest form, namely that the time course of Ca2+ removal is slow, termination of evoked release cannot be due to removal of Ca2+ in less than 2 msec. By contrast, the conventional view, which we shall discuss below, points to the possibility that the increased level of intracellular Cay+concentration, “which persists for hundreds of milliseconds after a nerve pulse, is not very effective in eliciting transmitter release, but may be important in phenomena such as facilitation” (Miledi and Parker, 1981). One way to overcome this discrepancy between termination of release and the sustained Ca2+ signals is to assume that the Ca2+ concentration below the membrane, after the pulse, increases greatly to create localized domains of high Ca2+ concentration (perhaps in the millimolar range). It

RB

LIJ”

IIII-IIILIII I

‘

1

’ J10mV 10 sec

L10 Arsenazo absorbance

AA = 0.04

FIG. 5. (Top) Posttetanic potentiation. (Bottom) Corresponding arsenazo signal. (From Connor et al., 1986.)

MECHANISMS O F NI;LKO~IKANShlll"TEKRELEASE

25

is also necessary to assume that the removal of Cay+f'roni the neighborhood of the release sites is very rapid (Fogelson and Zucker, 1985; Simon and Llinas, 1985; Blaustein, l988), and that this rapid change is not picked up by the Cay+indicator, which detects the overall profile of the f- se Ca" throughout the terminal. There are several indications that this line of argument is untenable. One matter concerns the distribution of CayCwithin the terminal. T h e increase of Ca" concentration is not uniform below the membrane (Baker and Knight, 1984; Blaustein, 1988; Smith and Augustine, 1988). Treatments that increase Ca2+entry showed that the highest concentration of intracellular Ca2+ was always below the membrane even when Ca'+ reached the center of the axon (Requena and Mullins, 1979; Baker and Umbach, 1987). Therefore, i f the dyes indicate high levels of integrated Cay+long after the termination of release, then one would expect that the concentration just below the membrane (i.e., near the release sites) would be even higher. Baker and Knight (1984) and Krinks rt (11. (1988) put the transient peak concentration (near the membrane) in the micromolar, not millimolar range. This range is consistent with the buffer measurements, cited above, o f Adler et al. (1988) and also with recent experiments by Zucker and Haydon (1988), using the Cay+ cage nitr-5[{2-amino-5[l-hydroxy- 1-(2-nitro-4,5-methylenedioxyphenyl)methyl]-phenoxy)2-(2'-aminod'-methylphenoxy)ethane-N,N,N',N'-tetraacetic acid], which showed that t i pfvf (;a2-+is ef'fective for release but 1 0 pM Cay+ inhibits release. A more direct way to obtain the time course of Cay+ concentration below the membrane is to use a membrane parameter that is sensitive t o Cay+ .ions. An example is the C:a'+-dependent potassium conductance. T h e arsenazo signal and Ca2+-dependentpotassium conductance tlecav with similar time courses (Connor et ol., 1986). Also, the time course of facilitation resembles that of the dye signal. 'Fhis tinding supports the earlier conclusion that Cay+ remains high below the membrane for the length of time suggested by the indicators. T h e matter of localized domains was discussed earlier, with the conclusion that release depends on a combination of background Cay+ and newly entered Ca2+. The earlier discussion, together with the two preceding paragraphs, indicates that another explanation should be sought in order to explain the discrepancy between the sustained Cay' signal and the rapid termination of release after a pulse.

3. Facilitation as a Tool to Study Remozial We have discussed relatively direct experiments concerning the processes that bring about the removal of the Cay+that enters following

26

H . PAKNAS pi a1

stimulation. Yet it is not possible to reach a clearcut conclusion regarding the nature of the most important processes that govern removal, in particular their relative importance in the spatiotemporal regulation of intracellular Ca2+.We now discuss an indirect approach that will bring us closer to a clear understanding of the nature of removal. In line with our discussion in Section II,D, we stress that facilitation is a potent indirect tool to follow the nature and time course of removal of Ca2+.As facilitation depends on the residual Ca" at the release sites, it is possible to test experimentally two principal models of Cay+ removal from these sites. In the first, we may assume that rapid diffusion is the dominant mechanism in removing the excess Ca'+. In this case the rate of removal of Ca'+ from near a release site will depend linearly on the concentration of Cay+.It must be emphasized that this relation still holds even if other processes such as buffers and pumps are included, providing that the dominant process is diffusion. A rapid diffusion model has been suggested to account for both release and facilitation (Stockbridge and Moore, 1984; Zucker and Stockbridge, 1983; Fogelson and Zucker, 1985) and for release alone (Simon and Llinas, 1985). This will be discussed below. The alternative assumption is that the dominant Ca2+removal mechanism from near the release site is satul-ative. Such mechanisms can be extrusion via the [Na+]o ++ [Ca'+]i exchanger, Ca" pumps, and binding to buffers (Blaustein, 1988). Figure 6A, modified from H. Parnas et ul. (1982a), shows predictions for experiments in which short- and long-term facilitation are measured as a function of the extracellular Ca'+ concentration C,. Also predicted (Fig. 6B) is the decay of facilitation with time at low and high C,. Figure 6C shows the duration of facilitation as a function of C,. For these calculations, the saturative equation (4) was taken for release. For the removal mechanisms, two different equations were studied. One equation (left column) assumes linear dependence on concentration as is characteristic of diffusion. The other equation (right column) assumes a saturative dependence on Ca'+ (which is the case if pumps, buffers, and/or exchangers are the dominant processes for removal. The predictions for the two cases are so different that they can be tested experimentally. T h e upshot of these experiments (Rahamimoff, 1968; H. Parnas et al., 1982a) is that the results of the right column and not the left column are affirmed, which shows that the removal mechanisms that govern the Ca2+ concentration below the release sites are saturative. (These experiments do not address the rapidity at which the Ca2+ is being removed.) This conclusion is in line with the finding of Baker and Umback (1987) of a high-affinity, low-capacity saturative compartment.

27

MECHANISM5 O b UE L K O 1 K.\N\hlI TTLK KFL E \5b

Nonsat urated removal F

Saturated removal

?,

‘.

L

t

t

F

F

\ , 0

0 0

Ce

0

Ce

“cI

FIG.6. Comparison o t theoretical 111-rdictionsconceiming the dependc1lc.eot f a c i l i t a t i o n F o n time (top row)and on extracellul;u- (;a’ conccnlration f;? (inidtile rot$). Solid (d,ishctl) line shows facilitation at short (long) intervals Iletwecn impulses. ~l’hchottom r o tlcpicts ~ the dependence of the duration of facilita~iori,TFon C,. (The arrow indicates a n inc~caac.) Left column: linear (nonsaturatetl) I-cinoval. Right column: satiu-atcd t - c n ~ ( ~ v a l . +

T h e essence of removal as a saturating process can be captured equation

in

the

Here p is the maximal removal rate, at saturation, and K p is the halfsaturation constant for removal. T h e finding of a saturative removal niechanisni is inconsistent with the hypothesis that diffusion is the dominant mechanism tor removal. This conclusion is supported tiy the high Q l o of the duration of‘release (mentioned above) a n d o f the time course of facilitation (Barrett and Stevens, l972b; Molgo and l’hesleff, 1984; Dudel, 1984a.b. 1986). T h e combination o f direct measurements (Cay+indicators) and indi-

28

H. PARNAS rl

a1

rect inferences (nature of removal in view of facilitation) allow the following conclusions. 1. If termination is due to removal of (;a2+, then only diffusion can supply the required rapid Cay+ removal. 2. Termination cannot in fact be due to the removal of Cay+since termination is not altered when the background level of Calf is modified. 3. Over the longer time scales that are characteristic of facilitation, the major removal processes are saturative in nature, which rules out diffusion.

Given that there is no requirement for a very rapid removal of Ca2+to bring about release termination, it is reasonable to assume that the same group of processes is responsible for removal of Ca2+over both short and longer time scales. It seems that the [NafIo [Ca'+]; exchange mechanism plays an important role in the regulation of the submembranal Ca concentration, at least for the residual Cay+that is important for facilitation. Blocking of the [Na+Io [Ca'']i exchange greatly prolongs facilitation in the crayfish neuromuscular system (I. Parnas et al., l982a; Arechiga et al., 1990) and in the frog neuromuscular system (Meiri et al., 1986).

-

-

IV. Evaluating the Classical Calcium Hypothesis

A. MAJORFEATURES OF KELEASEA N D FACILITATION We have discussed several features of release and facilitation. T o evaluate the various theories, it is helpful at this point to list a number of major features, all of which must be accounted for by a comprehensive theory. 1. Kinetics o j Release

1. T h e kinetics (i.e., the shape of the graph of release rate versus time) are to a great extent independent of the extracellular (;a2+ concentration, of the number of pluses given, and of other manipulations that might alter the intracellular Ca2+concentration prior to stimulation. 2. T h e kinetics are strongly dependent on temperature. 3. There is a minimal latent period wherein release is negligible. The duration of this period can outlast the duration of the pulse. For this to occur the pulse must be very short at high temperatures but can be as

long as 3 msec at low temperatures. At high depolarization, near the suppression potential for Cay+entry, release will start after the end of the pulse even if it is longer than 3 msec (Katz and Miledi, 1977; Idinas ~1 nl., 1981b). 4. Following the latent period the kinetic curve exhibits a steep rise. A log-log plot of this phase has a slope of approximately four, indicating cooperativity in release with an exponent of four. After attaining a maximum the curve drops steeply to low levels.

2. Quanta1 Content 1. T h e quantal content tti displays a signioid dependence 011 the extracellular calcium concentration C,. (For low C,, ni CL' where measurements of the "apparent cooperativity" I, are in range 1-4.) 2. T h e quantal content saturates as C, increases. 3. As we have discussed, it appears that the sigmoidicity and saturation effects (1) and (2) hold for the dependence of m on intracellular Ca2+. T h e sigmoidicity is generally interpreted as reflecting cooperativity. These considerations lead to Eq. (4) for release as a function of intracellular Cay+,with true cooperativity I = 4.

-

3 . Facilitution 1. We have summarized a body of evidence indicating that facilitation is due to the presence of residual intracellular <;a". 2. T h e duration of facilitation shows strong dependence on temperature. 3. At any temperature the duration of- facilitation is much longer (two orders of magnitude or more) than the duration of the evoked release.

B. DIFFICULTIES WITH

T HE c:I.ASSICAL C A L C I U M

HYPOTHESIS

In considering the above features in terms of classical calcium hypothesis, two major problems arise. It is difficult to explain the wide discrepancy in the durations of evoked release and facilitation. (This difficulty could be explained i f facilitation were not due to residual free Ca2+,but we have summarized what we regard as convincing evidence that residual free Ca2+is in fact the major determinant of facilitation.) It is also difficult to account for the insensitivity of release kinetics to manipulations that almost certainly alter the course of intracellular (;a'+ levels. This matter was discussed in detail by H. Parnas and Segel (1984),

30

H . PARNAS ul ul

H. Parnas et al. (1986b), and I. Parnas and H. Parnas (1988). There are also other problems, of which we shall now consider two examples. If one accepts an Ph power dependence of release as a function of intracellular Cay+, then the maximal possible facilitation should be 2 1 (Rahamimoff, 1968). T h e basic reason is that if all Ca2+ from the first pulse remains as residual <;a'+ during the second pulse then the concentration of Ca2+ is doubled. To derive an analytical formula for maximal facilitation let Li denote the release from the i(" pulse; i = 1,2. We define facilitation F by F

=

(6)

Ly/LI

Let 8t be the fraction of entry Y remaining after a time interval t. Neglecting residual calcium for simplicity we obtain from Eq. (4) =

Y(l + 8,) [ K , + Y ( l + 8,)

-1.

Kl+

Y

Y

(7)

For maximum facilitation Y should be small compared to K/. Also the time interval between pulses should be short, so that 8, -- 1 . This gives Fmax

=

2'.

(8)

Since we expect 1 = 4 (see above), maximum facilitation should be approximately 16. The assumption that all Ca" from the hrst pulse remains as free residual Ca", available for release during a second pulse, is in essence in contradiction to the <:a2+ hypothesis. This is because this hypothesis demands that most of the CaL+that enters during a pulse is removed rapidly to ensure termination of release (Smith and Augustine, 1988). Therefore, in practice the niaximal facilitation for twin pulses should be much smaller. Katz and Miledi ( 1968)measured facilitation in a TTX-treated preparation for closely spaced stimuli and found values of facilitation as large as 70. They remark that such levels of facilitation cannot be explained by the residual calcium hypothesis. Another difficulty arises from the fact that given the level of facilitation at a known extracellular Cay+ concentration C,, it is possible to estimate the residual intracellular Cay+concentration. T h e required calculations were provided by l . Parnas and H . Parnas (1986). They examined facilitation in crayfish with a 7-nisec interval between pulses. Entry Y was calculated from Eq. (4) using observed values of quanta1 content and the value KI = 1 p M , which was estimated from facilitation experiments. Given Y , KI, and I = 4, the facilitation measurements can be used to obtain Ot from Eq. ( 7 ) .T h e results are given in Table 111. Note that in the first

31 .I'AHLE 111

ESIIMATING

#o', ' I H F KtSIDL'Al. INTKA(:ELLl'I.AR

CONCENTRATION FROM E'A(.ILITA I I O N F

1

2

3.4 13 5

AND

2.7 2.0

(:a7

0.5.5 I:!

~1'0''

TRY 1'

0 28 0.6

experiment (C, = 13.5 nlizl) the iritracellular Ca2+concentration 7 msec after the first pulse (0.6p M ) is higher- than the initial level of inti-acellular Ca2+(0.53 p M ) . Yet release has long siiice termiiiated in the first case arid is .just beginning in the second. .l'hat is, in the first experiment release terminated at an intracellular (;a2+ level that is higher tfiari the level at which release commenced in the second experiment. This strong result will not be altered by a variety of modifications in details of the argument such as possible re-estimates 01' k', aiid 1. Given the various difficulties, there are two main approaches to follow. First, tlie classical calcium hypothesis can be refined, hoping thereby to diminish the existing problems. Such an approach has heen taken by several groups and will be discussed soon. T h e other approach, taken by the authors and J . Dudel, is t o exterid the calcium hypothesis to include another factor that together with Ca2+ is necessary to evoke release. According to this extension, the other factor determines tlie time course of release, and both the other factor and free (;a2+ determine the amount of release. Facilitation is due to residual free Ca2+.In this way there is 1 1 0 further conflict between the fast termination of release and the persistent facilitation. We now discuss the models that aim to refine the classical calcium hypothesis for release. 'Then the extended theory will be surveyed.

C. REVISIONS TO THE CALCIUM HYPOTHESIS 1. Ways to Terminate Releasf yet Mnintuin Facilitation

T h e question of the relative durations of evoked release and facilitation was already raised by Katz and Miledi ( I % % )who , suggested that the nonlinear dependence of release as a function ofCa'+ concentration can account for different time courses. These authors showed, however, that this explanation cannot account for the large differences observed. The ratio between the duration of facilitation arid that of evoked release should be (according to this explanation) at most the cooperativity 1, but

32

H. PARNAS el ul

the experiments show the duration of facilitation to be two orders of magnitude longer. A related approach coupled the assumption of nonlinear dependence of release on intracellular Ca2+with explicit calculation of the effects of Ca2+removal by diffusion (Zucker and Stockbridge, 1983; Stockbridge and Moore, 1984). Characterization of the conclusions from this approach will be given below in the context of our remarks on the similar but more comprehensive diffusion-domain models.

2. Diffusion-Domain Models f o r Release The essence of this group of models is that Ca'+ enters via patches of relatively densely packed Gag+ channels. The release sites are located in close proximity to one or several Cay+ channels. The spirit of this approach is best appreciated if channels and sites are both idealized as points in a planar cell membrane. The rate of release at a given site is assumed to be a function of the Ca" concentration at that point. Thus the time course of release is determined by the diffusion of Ca'+ toward and away from the release sites. Owing to the localized points of entrance of Ca'+, domains are formed below the Ca'+ channels wherein the Cay+ concentration is significantly higher than in more distant points (Chad and Eckert, 1984; Simon and Llinis, 1985; Fogelson and Zucker, 1985). For convenience, we thus term this group of models (which we have already briefly discussed) the diffusion-domain theories. Let us examine which of the basic observations listed in Section IV,A can be accounted for by the diffusion-domain theory. The various kinetic features can be reproduced with three exceptions. There is a dependence of shape on intracellular Cay+ prior to stimulation, albeit a weak one. There is a minimal latency but it is always much less than the pulse duration, and the major portion of the release occurs during the pulse (Fig. 7). With respect to quanta1 content, in their present form the diffusion-domain theories do not account for saturation. Finally, according to these theories facilitation only occurs for a few milliseconds. As stated by Simon and Llinas (1985), the diffusion-domain hypothesis contains some features and conclusions that are different from those of Fogelson and Zucker (1985). For example, the full kinetics of calciumbuffer interaction are employed. More important, from their supposition that release is determined by the Cay+ concentration at release sites located very close to the Ca'+ channels, Simon and Llinas (1985) deduce that release is best assumed to be a linear function of the intracellular Ca2+ concentration. This raises the problem of how to obtain the observed cooperativity of release measured as a function of the calcium current ZC, or of the extracellular Ca'+ concentration. Simon and Llinhs (1985) suggest possible ways to meet this difficulty.

33

"

0

2 4 t (msec)

6

0

'

I

t

(msec)

2

0

I

2

t ( rnsec)

FIG.7. (A) Numerical calculations ol'the release kinetics dL/dt. Redrawn ft-om H . Parnas rt al. (1989) using the calcium hypothesis as lormulated by Fogelson and Zucker (1985). Pulse duration is indicated by a heavy line on the time axis. Curves in (B) and ( C : ) arid the leftmost curves of (A) are norrnali/.etI to their peak antplitudes. (A) Low (--.--.) atitl high (-) depolarizations. Note that in conti-ast with experiment (H. Parnas rt a / . , 1989) facilitation is higher at the higher depolarization. (B) Normalized releases eliritetl by thc second (upper curve) and the first (lower curve) pulses at the low depolarization o f (A). ((1) As (B), except that the normalized high depolariration results of (A) are depicted.

An important problem to be faced by the diffusion-domain hypotheses is the necessity to account for the observed marked temperature dependence of release kinetics. This feature cannot be provided by a theory in which diffusion is the major regulator of the intracellular Ca'+ concentration and wherein release is a direct reflection of this concentration. T h e reason is of course that the diffusivity L) is weakly independent of temperature. A priori there are several ways to revise the diffusion-domain hypotheses in order to obtain suitable temperature dependence. One such way is to use the frequently employed notion of an effective diffusivity, which is the product of the actual diffusivity and a factor that depends on the rate constants for the binding and dissociation of Cay+to intracellular sites (see Fogelson and Zuckei-, 1985, for example). As emphasized in a detailed critique of such theoretical treatments of Cay+binding by Simon and Llinas (1985), an effective diffusivity can be used only if there is negligible saturation in binding and if the binding and dissociation reactions are sufficiently fast. To show that the diffusion-domain model, as supplemented by an effective Cay+ diffusivity, could account for the observed temperature dependence, it would have to be demonstrated that changes could occur in the binding rate constants that sirnultaneously give the observed temperature dependence and also permit the rate constants and amount of binding to remain within ranges permitting the use of an effective diffusivity. In our discussion of buffering, evidence was given that the high-affinity buffers are relevant for rapid (:a'+ removal. We pointed out that these high-affinity buff-ers appear to saturate rapidly and to have a dissociation constant in the micromolar range,

34

H. PAKNAS

Y/

d.

which casts considerable doubt on the neglect of saturation and hence on the use of an effective diffusivity. In any case, there remains the problem of the temperature dependence of the minimal latency. This feature is due primarily to diffusion just under the membrane from the channels to the release sites. Here Ca2+binding should not be a major factor. Temperature dependence can be obtained while retaining the major tenet of the calcium theory that Ca" is a necessary and sufficient factor for release by discarding the implicit assumption that the rate-limiting step in release is Ca'+-dependent. If a succeeding step is relatively slow, and if that step is temperature-dependent, then there is a chance to explain the observed temperature dependence. The trouble now is that the theory runs afoul of the fact that the kinetics of neurotransmitter release have a shape that is virtually independent of the intracellular calcium concentration C prior to stimulation (see Fig. 4B). With the addition of a "succeeding step" the diffusion-domain theories seem to provide time courses whose shapes are very sensitive to C. (See Fig. 8, top row.) This matter and the consequences of other possible ways to obtain

lk;y;l lwr

0

0

2

4

6

.-.' ; 0

I

0

2

2

I

t (msec)

I (rnsec)

I ( rnsecl

t (msec

I

FIG. 8. Panels (A, B, and C) parallel (A, B. and C) of Fig. 7 except for the inclusion of an additional slow step linking Ca" to exocytosis. Now the peak of the release can occur after the pulse (as in experinients) but there is more marked dependence ofdlldt on Ca". Panels (D, E, and F) correspond to ( A , B, and C) using the calcium-voltage hypothesis. (Redrawn from H. Parnas and I. Parnas, 1989.)

temperature dependence with a modified diffusion-domain hypothesis are discussed by H. Parnas et a/.( 1989). We have shown that rehiement of the classical calcium hypothesis to include localized accumulation of Ca2+ and its diffusion to anti away from the nearby release sites does not seem to abolish the difficulties of' the classical theory. Moreover, we have examined experimental data that cast doubt on the existence of' the postulated refinements. Particularly in view of the strong temperature dependence of release termination aiid facilitation, and the saturation of longer-term removal, diffusion cannot be the dominant process that regulates iiitracellular <;a'+. Localized Ca2+ domains cannot be essential for evoked release in view of the evidence that the background Ca'+ level also can play a major role in determining release. It is possible that there are other means, as yet unknown, that will allow retention of the franiework of the calcium hypothesis and yet will account for all of the exper-ilnental results. Be that as it may, there are strong reasons to explore an alternative approach. Thus, in the following section, w e discuss extension of the calcium hypothesis to include another factor that together with Cay+controls release. We remark that faced with Occam's razor one might feel obliged to make further vigorous efforts to avoid the introduction of another factor. But the pristine simplicity of the original calcium hypothesis seems irretrievably lost. The present versions of the refined calcium hypothesis are no sinipler than the alternative that we have proposed.

V. The Calcium-Voltage Hypothesis

A. EVIDENCE THAT THE O T H E R

k-ACTOR I S

VOLTAGE-DEPENDENT

1. Unexpected Additional Releasv hy Depolarizataon

Several lines of experinieiit point to voltage being the activator of the other factor. Llinas et al. ( 1 98 1a,b), studying the relation of release to Ca2+ currents in the squid, observed that at high depolarization the (;a"+ current produced more release than the same Ca'+ current obtained at a low depolarization. (Such an experiment is possible because of the bellshaped dependence of Cay+current on membrane potential.) In a later study, Augustine et al. ( 1985a.h) claimed this hysteresis to be an artifact of' improper clamping. With a three-electrode voltage clamp, they found less hysteresis. Llinas et al. (1981b) explained the hysteresis as resulting from a direct

36

H. PARNAS

P!

a/.

effect of membrane depolarization on the release mechanism. Others (Simon and Llinas, 1985; Fogelson and Zucker, 1985) reasoned as follows. At low depolarization only a few channels open. As a result there is no overlap of Ca“ from the various channels. At high depolarization, many channels open. While the current via a single channel diminishes (owing to the lower driving force), there is overlap of Ca‘+ from the various channels and a higher concentration at the release sites might thereby be obtained. However, upon further examining the Fogelson and Zucker (1985) model, Hovav et al. (1990) showed that for the same total Cay+ current, irrespective of whether it comes from one or several channels, the resulting release is the same. Hence, the last explanation of the observed hysteresis is less convincing than a direct voltage effect. In a less direct way hysteresis was also obtained in the crayfish and frog neuromuscular junctions (Dudel, 1984a; Dudel et al., 1983). Since it is impossible to perform direct measurements of Ca2+ currents in small nerve terminals, Dudel et al. (1983) used an indirect method, measuring constant test pulse facilitation (amplitude and duration). The facilitation obtained following a constant test pulse given after variable prepulses will depend on the residual Ca2+ stemming from that prepulse. The larger the entry of Ca2+ during that prepulse, the larger the facilitation. Dudel et al. (1983) found that, in such experiments, there was a region in which increasing membrane depolarization produced more release at the prepulse, but a smaller facilitation of the constant test pulse. The dependence of the test pulse facilitation on membrane potential was bellshaped and thus was similar to the dependence on membrane potential of the Ca2+ currents. Hence similar hysteresis is seen as for the squid. Judged by facilitation, at the higher depolarization, more release is obtained for the same Cay+ entry compared to the lower depolarization. Alternative explanations to the results of Dudel et al. (1983) were given by Zucker and Land6 (1986) and were in turn challenged by I. Parnas and Parnas ( 1986).

2 . Effect of Depolarization on Minimal and Maximal Release Another support for the role of voltage comes from the work of Cooke et al. (1973) and Dudel (1989) who found that at very low C,, when almost no Ca2+enters, the level of release (“minimal release”) increases as depolarization increases. In a theoretical analysis H. Parnas and Segel (1981) showed that this finding (together with others, for example the depolarization dependence of the level of the maximal release) is best explained if membrane depolarization affects the release machinery itself. They suggested that the maximal level of release (at high C,) increases as depolarization increases.

MECHANISMS 01'NEC' K O ~ I ' K A N S h I I ' l ~ ~ I ERELEASE R

37

3. Release with Very Little Entry oJCu2+ T h e most direct indication of the role of voltage should come from experiments in which the terminals are loaded with Ca2+ and synchronized release is obtained upon depolarization without further entry of Ca'+. Under such conditions the other factor is formed during the depolarization and can act with the Ca2+whose level was elevated prior to depolarization. Such an attempt was made by Zucker and Land6 (1986) and Zucker et al. (1986), who tried to increase the intracellular Ca2+ level by the use of metabolic inhibitors and to obtain release in zero Cay+solutions. As they could not detect any extra release by depolarization, they concluded that there is no need to postulate an additional role to membrane depolarization in the release process. Unfortunately, there are at least two problems with these experiments. In the crayfish neurorn~~scular junction, the opening of postsvnaptic glutamate channels requires Ca2+ions in the bathing solution (Hatt et al., 1988).Therefore, in zero Ca'+ solution, it is more difficult to detect release because of the postsynaptic suppression. Another problem stems from the direct effects of prolonged elevated intracellular Cay+concentration on the release process. Adams et al. (1985) showed that when metabolic inhibitors such as NaCN, FCCP, or ruthenium red were applied to the bathing solution, the evoked release was depressed even though the mepp frequency increased. At the same time, the postsynaptic response to ionophoresis of glutamate was not altered. Release was temporarily restored by injection of EGTA into a terminal poisoned by a metabolic inhibitor, while for the control injection of EGTA had no effect. Adams et al. (1985) concluded that an increase in the intracellular level of Ca'+ can also block synchronized release. Prolonged injection of Cay+ ions into the presynaptic terminal has been reported to reduce transmitter release (Miledi and Slater, 1966; Kusano, 1970). These findings indicate that it is difficult to detect release in zero Ca'+ solutions, at least in the crayfish neuromuscular preparation, and that prolonged exposure to elevated intracellular Ca2+concentration has its difficulties. It would therefore appear that at this stage the experiments of Zucker and Land6 ( 1 986) or Zucker et al. ( 1 986) cannot be used to refute the possible role of voltage in inducing release. Indeed, Hochner et al. ( 1 989) succeeded in demonstrating that when the intracellular level of Ca2+ was elevated by photolysis of nitr-5 (see Section V,C,3 below), depolarization evoked release without a concomitant entry of Ca2+.These findings substantiate results obtained earlier employing a somewhat different approach toward the same goal (Dude1 et al., 1983). They reasoned that if release stops after a train of pulses, even with high residual Ca2+,it should be possible to evoke release by a

38

11. PAKNAS el

depolarizing pulse, which by itself lets in a very small amount of Ca'+. This is because the depolarizing pulse will produce an additional amount of the other factor, which will act in tandem with the elevated intracellular Ca'+ to evoke release. Dudel et al. (1983) employed a 1-msec pulse, which when given alone released only 0.006 quanta per pulse on the average. T h e nerve was then stimulated by a train of pulses in order to produce facilitation, presumably by increasing the intracellular Ca'+ concentration. When release after the train had subsided, the same test pulse released nine quanta on average, showing facilitation of more than 1500. A possible role of a change in excitability due to the repetitive stimulation (Zucker and Lando, 1986) is overruled in view of the achievement of similar results in the presence of T T X (I. Parnas and H. Parnas, 1986).

B. FORMULATION OF T H E CALCIUM-VOLTAGE HYPOTHESIS

By indicating that the second factor is depolarization-dependent, the experiments listed above provide support for the role of membrane potential in release from synapses. The authors and J. Dudel therefore extended the calcium hypothesis to the calciutn-voltage hypothesis for neurotransmitter release. T h e essence of this hypothesis is that both Ca2+ and another factor, which is voltage-dependent, are necessary to evoke release. Both factors are limiting prior to stimulation and become available as a consequence of depolarization. T h e amount of release (quanta1 content), is determined by both factors. However, the time course of release is mainly determined by the other factor. We now briefly summarize the main steps that led to the formulation of the calcium-voltage hypothesis in its present form. Evidence given in previous sections has established the likelihood of a depolarizationdependent step, other than the entry of Cap+, in the release process. Perhaps the most natural way to incorporate such a step in a model is to render depolarization-dependent one of the rate constants that links the accumulated calcium to release (Llinas et al., 1981a,b) (Fig. 9A). This way turns out not to be suitable, however, as it always provides maximum release at the end of the depolarizing pulse (Fig. 9B). One is thus led to the idea that the depolarization-dependent step must occur in parallel to calcium accumulation (H. Parnas et nl., 1986a). To be more precise, it is reasonable to assume the existence of an entity T whose shift to a conformation S is induced by depolarization. Convergence of the two parallel steps is assumed by assuming that T does not bind Ca2+but S does. It is expected that T will be found in the membrane, in order for it to sense the change in membrane potential.

39

M E C H A N I S M S O F N E l ~ R C ~ I ' K A N S h l I T I ' I . : KK E L E A S E

t (msec)

C

*p,

1-S S+nCo--(SC")

t imsec )

D

1-S

s+ co

-

(SCI

dt

2 4 6 8 1 0 t (msec)

2 4 6 8 1 0 t (msec)

FIG. 9. (A) Illustration of the hypothesis that the Ca" complex formation parameter k:! of' Eq. (9) is relatively high during thc period of. depolarixation. ( K ) C:alculatiotr, using the hypothesis of (A), of the synaptic tlrlay histograin. (Redrawn I.roin H. Patnas and I . Parnas. 1989). (C) Theoretical delay histogram w l i e n cooperativity resides in the hitiding of rl (:a'+ ions to S. (D) Similar to (C), except that cooperativity resides in the binding of rt Ca"-S complexes to the vesicle V Parameters for (C) and (D) as in H . Parnas et (11. (lW6a).

An additional important question is the precise location of the cooperative step. This question is best evaluated from measurements of'kinetics. Iffour Ca2+molecules bind to one S then the histogram rises without any noticeable delay (Fig. 9C). If, however, nS-Ca'+ complexes ot S with Ca'+ (i.e., SC complexes) bind to the vesicle, then the histogram rises iii a sigmoid manner after a delay (Fig. 9D), as observed experimentally. ) that the cooperativity exponent 1 Indeed, H. Parnas et al. ( 1 9 8 6 ~showed of Eq. (4) can be obtained from nieasurements of the initial rise o f the delay histogram (see Fig. 1C). 'I'he nieasurements show that 1 = 4, as mentioned earlier. Furthermore, the steep rise in the delay histogram (which yields 1 = 4) indicates that four complexes of (SC) [see Eq. (Y)] bind a vesicle to permit its secretion. This rules out the possibility that the key step in regulating the time course of release is the binding to some molecule (such as calmodulin) of four Ca'+ ions. T h e steep rise in the time course of- release and the cooperative dependence of the quanta1 content (both with the same exponent) can, however, both be explained by the assumption that four S-C:a'+ complexes (SC) are responsible for the release of a single vesicle.

40

H . PARNAS eta!.

The considerations thus far can be summed u p in the following basic kinetic scheme.

Here, as earlier, the superscript d, indicates voltage dependence. To insure the appropriate T ++ S transition it is assumed that kf increases with depolarization while k? 1 increases with hyperpolarization. T h e concentration of vesicles is given by V. The process of release after a single pulse is represented schematically in Fig. 10. At rest most Ca2+ channels are closed, and most of the Ca2+ binding sites are in the T form. Some particles are in the S form. Intracellular Ca'+ concentration is low. When the membrane is depolarized by the action potential, Ca2+channels open, and the T molecules transform to the S conformation. S molecules then bind Cap+ ions to form a complex CS. Four such complexes are required for the release of

Ca+

Membrane at rest

+

W Membrane depolarized

'Release configuration

FIG. 10. Diagram of the Ca'+-voltage hypothesis.

41

MECHANISM\ O E N C I ' R 0 1 RANSMI 1 I L K KLLE 1st

one quantum of transmitter. When the action potential is over and the membrane repolarizes, Cay+ channels close rapidly, and S molecules transform back to the T state. The S -+T transformation causes termination of release. This occurs even though Ca'+ concentration may remain higher than normal below the release sites. When a second action potential reaches the terminal, the depolarized membrane lets in fresh C;a" to add to the residual (:a'+. Again, 'r molecules transform to S . The amount of S is the same, because the depolarization produced by the action potential is the same, and there is no residual S at intervals longer than 5-7 msec (I. Parnas and Parnas, 1986). T h e higher Ca2+concentration will act with the same amount of S , resulting in facilitation. Thus, at constant depolarizing pulses, facilitation for (temperature-dependent) times longer than a few milliseconds reflects only the residual Ca2+.Kelease after the second pulse will end with the same time course because the S + T transformation occurs at the same rate, as long as the shape of the action potential is not altered. After several or many pulses, intracellular Ca" concentration increases, whether by accumulation of residual Ca'+ or by recruitment from internal stores (Blaustein, 1988). T h e amount of release will be greatly facilitated and the duration of facilitation will increase and last as long as the Ca'+ concentration is elevated. Yet the time course of release, after each of the pulses, will be the same, since it is determined by the S -+ T transformation. In summary, the amount of' release, or the quanta1 content, is determined by both the intracellular (:a2+ concentration and the amount of S . T h e time course of release is virtually independent of Ca2+and is determined mainly by the T * S transformation.

c. W A Y S T O DISTINGUISH HETWEEN T H E C A L C I U M A N D T H E c ; A L C l U M VOLTAGE HYPOTHESES 1 . Initial Time Course of Release

T h e theoretical result that the slope of log release versus log time, as release builds up, indicates the cooperativity for both the calcium and the calcium voltage hypotheses was used to differentiate between the two If changes in Ca2+concentration in hypotheses (H. Parnas et al., 1986~). time determine the sigmoidicity, then this sigmoidicity should be affected by the resting Ca2+concentration and, more significantly, by the ratio of the entering Ca'+ to the resting Cay+ concentration. T h e higher this ratio, the higher the measured slope, reaching a maximum of four. On S transforthe other hand, if the sigmoidicity is determined by the T

-

42

H. PARNAS p t d.

mation, then the slope should not depend on the mode of variability of either the intracellular Ca" concentration or the ratio of Ca2+ entry to the resting level of Cay+. The predictions for the behavior of the slope in three different experimental conditions is given in Fig. 11. According to the Ca2+ hypothesis, the slope should increase with the level of depolarization (up to a peak), increase with the duration of the depolarizing pulse (as long as the suppression potential is not reached), and decrease when the intracellular Ca2+concentration is increased. For the calcium-voltage hypothesis, however, the slope should decrease with the level of depolarization (starting from values higher than four, and reaching a minimum of four), it should be independent of pulse duration, and it should be independent of the intracellular Cay+concentration. When these predictions were tested by experiment, all of the results for the three predictions were qualitatively in accord with the calciumvoltage hypothesis (H. Parnas et al., 1 9 8 6 ~ ) .

1 - 1

Pulse duration

Ca"hypothesis

g -0ul

:I

Pulse amplitude

Ca"- rest

-

t

2

1

2

time (msec)

L

M H depolarization

hypothesis m-.-\

1 2 3 . 4

time (msec)

L

M

H

depolarizat ion

0,Ol

0.05

0.l

JJM

FIG. 1 1. Schematic representation of' theoretical comparison between the Ca2' and Ca'+-voltage hypotheses. Plotted are the initial slopes o f log-log delay histograms as a function respectively of the pulse duration, pulse amplitude (low, medium, and high depolarization) and the resting level of Ca" . (Redrawn from I . Parnas and H. Parnas, 1988.)

43 2. Efject of Hjperpolurazatiori In the calcium voltage hypothesis as summarized in Eq. (9) there is an increase with hyperpolarization of the rate constant A!! 1 that is responsible for the S to T transition. I t is therefore expected that postpulse hyperpolarization will affect the time course of release. In particular, it will reduce the peak release amplitude and also will make more rapid the decline of release from its peak value. Together, these effects should cause a reduction in the total amount of release. Dudel (1984b) and I. Parnas rt ul. (1986) checked whether a hyperpolarizing post pulse would af'fect release. Indeed, when different brief l-msec hyperpolarizing pulses were given with zero delay after a depolarizing test pulse, release was reduced. Stronger hyperpolarizing pulses produced stronger block, with maximal inhibition of release of' about 65%. In recent experiments an 80%'block was also seen (Arechiga rt al., 1990). An example (with a 50%.block) is seen in Fig. 12. The hyperpolarizing pulses were effective not only when given with a zero delay after the depolarizing test pulse, but also when given with some delay. T h e per-

Time (msec)

FIG. 12. T h e effect of postpulse 1iypei-polari;lation on delay histogl-ams. Fittten hundred pairs of pulses were given. alternating between dcpolai-ization alone ( 1 nisec. -0.4 pamp) and the same depolarization followed by a p o ~ t p ~ l shyperl'c'lai-ization e (0.5 msec. + 0.5 pamp). T h e histogram with the additional hyperpolarization is ,tippled. Note that the beginning of'the histogwins is the same but with the postpulse hyperpolari~ation, there is a shift of. the peak to the left and a faster decay fi-om the peak. ( I . l'arnas, unpublished experiment. Conditions as in Fig. 1.)

44

H. PARNAS eta/

centage of inhibition was smaller when the delay was increased, showing no reduction in release with 5 msec delay (at approximately 5°C). Even though Ca2+channels close with repolarization in a fraction of a millisecond, (Augustine et al., 1987), the possibility exists that the hyperpolarizing post pulses increased the rate of channel closing and by doing so decreased the Ca‘+ entry of the test pulse, although in order to block 80% of the release the reduction in Ca2+entry must be substantial. This possibility was also tested by I. Parnas et al. (1986). They showed that a hyperpolarizing post pulse affected the release of a test pulse, but not the facilitation of a second test pulse given 10 msec later. Since facilitation was the same, they argued that the residual Ca2+ after the hyperpolarizing test pulse was the same and therefore that the Ca2+entry during the first pulse was not affected by the hyperpolarizing post pulse. This point was reinvestigated and confirmed by Arechiga et al. (1990) employing a large number of pulses (also see Dudel, 1984b). Especially in view of the findings that the time course of release is independent of changes in Ca2+concentration (Datyner and Gage, 1980; Barrett and Stevens, 1972a,b; Andreu and Barrett, 1980; H. Parnas et al., 1986a; H. Parnas and I. Parnas, 1987; Matzner et al., 1988), it is important to see whether changes in membrane potential will modulate the time course of release. It turns out that a short hyperpolarizing pulse not only affects the quanta1 content but also alters the time course of release. It shifts the peak toward shorter delays and shortens the duration of the release (see Fig. 12). Such results were obtained using both crayfish (I. Parnas et al., 1986) and frogs (Dudel, 1984b; Arechiga et al., 1990). 3. The Use of Caged Ca2+

A group of photolabile Ca‘+ chelators has been developed by Tsien (1988) and his colleagues (Tsien et al., 1988). These compounds have different dissociation constants for Ca2+ binding in the dark and after a flash of light. Therefore elevation of free Ca2+in the cytoplasm is possible by intracellular injection of such a compound (nitr 5, nitr 7) (Tsien and Zucker, 1986) followed by a flash of light. This treatment can also be given in zero Ca‘+ solutions, thus eliminating voltage-dependent entry of Ca2+.Such a trial was done by Zucker and Haydon (1988), usingcultured neurons of the snail Helisoma. Release was evoked by a flash of light after nitr 5 injection into a presynaptic cell, but the authors could not modulate the release by a slow ramp depolarizing pulse. They concluded that voltage has no “direct” effect on the release process. Unfortunately, this work cannot be employed as evidence for or against any postulated process of evoked release. Only miniature potentials were recorded, the depolarization was by a slow ramp and not an

MECHANISMS O F NELTKO I’RANSMI 1”lk:R KbX.EASt

45

action potential, and synchronized evoked release was not shown even in the presence of Cap+in the bathing solution. There is no controversy as to the basic finding of an “additional role” of membrane depolarization in the release process (Zucker and Land6, 1986), but there are differences of opinion as to the interpretations of these results. Thus the effects of nitr 5 or other “Ca” cages” should be tested in preparations wherein the voltage effect has been demonstrated, employing a fast synchronized synapse. This program has been carried out by Hochner et al. (1989),who injected nitr 5 into nerve terminals in the crayfish neuromusculai- junction. By means of synaptic delay histograms they observed elevation in spontaneous release following a flash of light. However, under conditions wherein no entry of Ca2+ took place, evoked release occurred only after depolarization.

VI. Conclusions

T h e classical calcium hypothesis for neurotransmitter release is that elevation of intracellular Cay+ is necessary and sufficient to induce release. For decades, convincing evidence has existed that accumulation of intracellular Ca is indeed necessary for release. However, some findings cast doubt on the hypothesis that the initiation, time course, and termination of release are determined solely by the temporal variation of intracellular Ca‘+ at the release sites. These findings include persistent levels of intracellular Ca2+(as indicated by calcium dyes) in contrast with the rapid termination of release, insensitivity of the tiiiie course of release to teniporal changes in the intracellular (;a2+ concentration, and anomalous effects of depolarization and hyperpolarization. There are two major options in the face of these findings. One option is to ramify the classical calciuni hypothesis by placing emphasis on details such as the local distribution of calcium in the neighborhood of the release sites. At present this approach has not succeeded in providing a comprehensive extended calcium hypothesis that can overcome all the problems mentioned above. Moreover, a new problem arises with the emphasis on diffusion as the major controlling tactor in regulating the spatiotemporal distribution of calcium, whose precise course is at the heart of the domain hypotheses. Observations of strong temperature dependence cannot be accounted for by theories that are centered on the role of diffusion. T h e other option, a natural one, is to assume that release is also strongly controlled by a second factor, in addition to Ca“. Experiments

46

H PAKNAS rf al.

in neuromuscular junctions indicate that this second factor is depolarization-dependent. The calcium-voltage hypothesis has been developed to the point that it harmonizes all the ma-jor experimental findings. Why has it taken so many years to perceive difficulties in the classical calcium hypothesis? A major reason resides in the fact that the dominant experiments to date have initiated release by action potentials while varying the extracellular calcium concentration. This has the effect of keeping one factor (depolarization) constant while varying the other. Voltage clamp experiments have the same feature. Another major point is that most experiments deal with the total neurotransmitter release (quanta1 content measurements) and this property indeed is dominantly affected by calcium. Years ago, Katz and Miledi (1965a) showed how to measure the time course of release, but these experiments are to this day technically much more difficult and painstaking, and naturally were not carried out in the absence of strong motivation. Given the suspicion that two factors play essential roles in controlling release, the obvious strategy is to attempt to hold each separate factor constant while varying the other. With modern experimental techniques (caged calcium compounds, injectable intracellular calcium buffers) it is indeed now becoming possible to “clamp” the intracellular calcium concentration while varying depolarization. This line must be vigorously pursued in the future. In addition, considerable theoretical and experimental effort must be concentrated on elucidating the biophysical processes and molecular steps that are involved in the control of neurotransmitter release. Acknowledgments

The authors are grateful to J . Dude1 for reading the nianuscript and making valuable comments, t o S. Fliegelmann and C. Weintraub for expert typing, and to Y . Barbut for skilled preparation of figures. I . Parnas is the (;reenfield Professor of Neurobiology. L. Segel is the Benson Professor of Mathematics. This work was partially supported by a grant from the DFG and the Goldie Anne trust to 1. Parnas.

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48

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Dudel, J. (1990). I n “Neuromuscular Junction” (L. C. Sellin, R. Sibelius, and S. Thesleff, eds.) Elsevier (Biomed. Div.) Amsterdam (in press). Dudel, J., Parnas, I., and Parnas, H. (1982).Pfliiegers Arch. 393,237-242. Dudel, J., Parnas, I., and Parnas, H. (1983). Pfliiegers Arrh. 399, 1-10. Dunant, Y . (1986)Prog. Neurobiol. 26, 55-92. Duncan, C. J. (1983). Cell Calczzrm 4, 171-193. Finkelstein, A., Zimmerberg, J., and Cohen, F. S . (1986).Annu.lRev. Physiol. 48, 163-174. Fogelson, A. L., and Zucker, R. S. (1985) L3iophys.j. 48, 1003-1017. Greengard, P. (1978). Science 199, 146-152. Greengard, P., Browning, M. D., McGuiness, T. L., and Llinas, R. (1987). 1u “Molecular Mechanism of Neuronal Responsiveness” ( Y . H. Ehrlich, R. H. Lenox, E. Kornecki, and W. D. Berry, eds.), pp. 135-153. Plenum, New York. Grynkiewicz, G., Poenie, M., and Tsien, R . Y . (1986).j.Biol. Chem. 260,3440-3450. Hagiwara, S., and Takahashi, K. (1967).J.Gen. Physiol. 50, 583-601. Hatt, H., Frdnke, C . , and Dudel, J. (1988).Pfliiegtr.7 Arch. 411, 17-26. Heuser, J. E., and Reese. T . S. (1981).J. Cell Biol. 88,564-580. Heuser, J. E., Reese, T. S.,Dennis, M. J., Jan, Y . ,Jan, L., and Evans, I.. (lY79).J.Cell Bzol. 81,275-300. Hirning, L. D., Fox,A. P., McCleskey, E. W., Olivera, B. M . Thayer, S. A,, Miller, R. J.. and Tsien, R. W. (1988)Scirnce 239,56-61. Hochner, B., Parnas, H., and Parnas, 1. (1989). Nature 342,433-435. Hovav, G., Parnas, H., and Parnas, I. (1990). L3iophy.J. (submitted). Israel, M., Dunant, Y., and Manaranche, R. (1979). Prog. Neurohiol. 13,237-275. Jenkinson, D. H. (1957).J.Physiol. ( L ( J ~ V138,434-444. L) Johnson, E. W., and Werning, A. (1971).J.Physiol. ( I ~ ~ n d o218, n ) 757-767. Kandel, E. R., and Schwartz,J. H. (1982). Srience 218,433-443. Katz, B. (1969). “The Release of Neural Transmitter Substances.” Liverpool Univ. Press, Liver pool. Katz, B. (1971). Science 173, 123-126. Katz, B., and Miledi, R. (1965a). Pror. R . Soc. London, Ser. B 161,483-495. Katz, B., and Miledi, R. (1965b).J. Physzol. (London) 181,656-670. Katz, B., and Miledi, R. (1968).J. Physzol. ( L o d o n ) 195,481-492. Katz, B., and Miledi, R. (1970).]. Physzol. (London) 207, 789-801. Katz, B., and Miledi, R. (1977). P m . R . Soc. London, Ser. B 196,465-469. Klein, M., Camarado, J., and Kandel, E. R. (3982). I ’ m . Nutl. Acad. Sci. U.S.A. 79,57135717. Krdvitz, E. A . (1588). Science 241, 1775-1781. Krinks, M. H., Klee, C. B., Pant, H. C., and Gainer, H. (l988).J.Neuroscz. 8,2172-2182. Kusano, K. (1970).J. Neurohiol. 1,435-457. Lemos, J. R., and Levitan, I . B. (1984).J.G m . P h p o l . 83, 269-285. Lev-Ram. V . , and Grinvald, A. (1987).Biophy.\.J. 52, 571-576. Linder, -1‘. M. (1973).J. Gen. Physiol. 61, 56-73. Llinas, R. R. (1977). Soc. Neuroscz. Symp. 2 109-160. Llinas, R. R., Steinberg, 1. Z.,and Walton, K. (1981a).Biophys.J. 33, 289-322. Llinas, R. R., Steinberg, 1. Z., and Walton, K. (l98lb). Bzophys.]. 33, 323-352. Llinls, R. R., Sugimori, M., and Simon, S. M. (1982). Proc. Natl. Acad. S c i . , U.S.A. 79, 24 15-24 19. Llinas, R. R., McGuinness, T. L., Leonard, C. S., Sugimori, M., and Greengard, P. (1985). Pror. Natl. Acad. Sci. U.S.A. 82, 5035-3039. Magleby, K. I.., and Zengel, J . E. (1975a).J. Physiol. (London)245, 163-182.

MECHANISMS O F NI<,UKO’IKANSML’I‘TEK KEIXASE

49

Magleby, K. L., and Zengel, J. E. (l975b).,/.Ph;.\ro/. ( k J d 0 f C ) 245, 183-208. Magleby, K. L., and Zengel, J. E. (IW2).,/. C r n . P h y o L 80, 613-ti38. Mallart, A., and Martin. A. R. (1967). /. Phy.tid. (Lond(~?r) 193, 679-6!M. Mallart, A., and Martin, A. R. (l!Ki8).,/.I’//~.WJ/. (Londwr) 196, 593-604. Martin, A. R. (1965). Physid. Reil. 46, 6 - 6 6 . Matzner, H., Parnas, H., and Parnas, 1. ( 1 9 8 8 ) . J . Phyiol. ( I m z d o n ) 398, 109- 12 1. McLachlan, E. M . , and Martin, A. K. ( l 9 8 I ) . j .f’/tyio/, (Lorrdo~t)311, 307-324. Meiri, H., Zelingher, 1.. and Kahaniirnoff, R. (1986).In “Calcium Neuronal Function and ‘Transmitter Release” (R. R;ilraminioff and B. Katz. eds.), pp, 2:lY-236. hlartiniis Nijhoff, Boston, Massachusetts. Miledi, R. (1973). Proc. R. SOC.London. S e t . H 183, 421-425. Miledi, R., and Parker, I . (1981). Pror. R. Sor. L o d o n Ser. H 212, 197-21 1. Miledi, R., and Slater, C . R. (l966).,/.Ph;sro/. ( L ( ~ d o n814, ) 473-498. Molgo, J., and Thesleff, S. (1984). Binin R r \ . 297, 309-3I6. Nestler, E. J., and Greengard. P. ( I W i ) . P r ~ gHI-nrn . Re.\. 69, 323-339. Nicholls, J . G.. Ross, W. N., and Arcchiga, 11. (1986). Nrtrrosri. Absrt. 12, :170. Parnas, H., and Parnas, 1. (1987).,/.f’hytol. ( b f / d f J ? / ) 388, 487-493. Parnas, H. and Parnas, I . (1989).I n ‘ X k 4 t o Cell Signalling: From Experinirnt to 1 heorctical Models” (A. Goldbeter, ed.) pp. 15-59 Academic Press, 1.oncion. Parnas, H., and Segel, L. (198O).J. Thwt. W d . 84, 5-29. Parnas, H., and Segel, L. (lUSl).,/. T ~ N JH,i.d . 91, 125-169. Pal-nas, H.. and Segel, L. (1984).J. Thror..H i d . 107, 345-365. Parnas. H., and Segel, I.. (1988). !’rug. Nrtrrobzol. 32, 1-9. Parnas, H., Dudel,]., and Parnas, 1. (1!)82a) Pf(iirgrn Arch. 393, 1-14. Parnas, H., Dudel,]., and Parnas, I . (19821)). f’fliirgrrc Awh. 395, 1-5. Parnas, H., Dudel, J., and Parnas. I. ( I W j ; i ) . PfiIirgrn Arch. 406, 121-130. Parnas, H., Parnas, I . . and Dudel. J. ( 1986h).f r i “Calcium. Neuronal Function and ‘Transmitter Release” (R. Rahaminiofl ;ind 13. Kati. eds.), pp. 215-238. Rlartinus Nijhoff, Boston, Massachusetts. Parnas, H., Parnas, I . , and Segel, 1.. ( I!)X6c).,/. Th(,or.H i o l . 119, 4XI-.ZY!). Parnas, H., Hovav, G., and Parnas, I. (l98!1). L l t ~ p h ; s . J .55, 859-874. Parnas, I . , and Dudel, J. (1!)82).J. ! V r P / t I ’ ~ J / J / d . 13, 75-77. Parnas, I., and Parnas, H. (198S).J. ! ’ h y . ~ r o / . ( P a m )81, 289-:105. Parnas. I., and Parnas, H. (1988).H ~ ~ p l i (:hrm. y . ~ 29, 85-113. Parnas, I . and Strumwasser, F. ( 1‘374).,/. h’rirrophysro/. 34, 609-620. Parnas, I., Parnas, €I.,and Dudel,J. (10X2a). Pfiflii+yrs Atch, 393, 2:12-256 Parnas, I., Parnas, H., and Dudel,]. (I!)X‘Lh). PJliirgrr.cA,ch. 395, 261-270. Parnas, 1. Dudel, J., and Parnas, € I . (I!)84) Nrzrto.\cr. Lrtl.50, 1.57- 162. Parnas, I., Parnas, H.. and Dudel,,]. ( I 9 X t i ) P j / i i r g m Atch. 406, 131-I:~7. Rahamimoff. K. (1968).J. I’hyszd. ( h r d o t r ) 195, 47 1-480. Raharnimoff, K., Meiri, H . , Erulkar I).. a l l d Bal-enhOk. y. (1978). Pf-OC.,vU/[.i q ( ( / d . LS
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Smith, S.J.. arid Augustine, G. J. (1988). Ttrnd~Nrto-mci. 11, 458-464. Smith. S . J . . and Zucker, R. S. (1980).J . Physiol. (London)300, 167-196. Staggs, D. R., Pofcher, E., L’Heureux, K., Ortiz. C. L.. and Orkand, R. K. (1980). J . Neurohiol. 11, 629-632. Stanley, E. F. (1986).J. Nunrosci. 6, 782-789. Stanley, E. F., and Ehrenstein, G. (1985). Lifr Sci. 37, 1985-1995. Stockbridge, N., and Moore.1. W. ( l 9 8 4 ) . j .Nrurosri. 4, 803-81 1. T a w , L. (1982). Physiol. Re?!.62, 857-893. Tillotson, 1). L., and Gornian, A. I,. F. (1983).Cell. Mol. Neurohiol. 3, 297-310. Tsien, R. W. (1983).Annu. Rrib. I’hysiol. 45, 34 1-58. Tsien, R. W .(1986). I n “l\ieuromodulation” ( I . B. Levitan and L. K. Kacrmarek, eds.). Oxford Univ. Press, London and New York. Tsien, R. W. (1988). Tt-rndsNrurosci. 2, 419-424. Tsien, R. W., and Zucker, K. S. (1986).Biophy.,]. 50, 843-853. Tsien, R. W., Lipscombe. I).. Madison, D. V., Rley. I(. R., and Fox, A. P. (1988). 7rrnd.y Neut-om. 11, 431-438. Van der Kloot, W. (l988a).J . P h ~ s i o l (London) . 402, 595-603. Van der Klott, W. (l988b).J . Physiol. ( I m d o n )402, 605-626. Wojtowicz,.]. M., and Atwood, 13. L. (1983).,/. A’eurobzul. 14, 385-390. U‘qjtowicz. J . M., and Atwood, H. 1.. (1984).,/. Ncuropliyiol. 52, 99-1 13. Wqjtowicz,]. hi., Parnas, I., I’ai-nas, I]., and Atwood, H. L.(1987).Can.J . P h y i o l . Pharmacol. 65, 105-108. Yovell, Y., Kandel, E.R., Dudai. Y.. arid Abranis, T. W.(1987). P w r . A’citl. Acad. Sri. U.S.A. 84,9285-9289. Zengel, J. E.. and Magleby, li. I>. (l9X2).J. Grn. I’hyswl. 80, j83-6 1 I . Zimmerherg, J. (1987). H i o m . Rcj. 7, 251-2(’,8. Zimrncrberg, J.. Cohen, F. S..arid Finkelstein, A. (198Oa).,/. G e n . Plyzol. 75, 24 1-250. Zimmerberg, J . , Cohen, F. S..and Finkelstein. A. (1980b). Scirucr 210,906-908. Zirnrnerberg, 1.. Sardet, C., and EpeI, D. (1985).J. Cull Hid. 101, 2398-24 10. Zimrnerberg,]., Curran, M., Cohen, F. S., and Brodwick, M. (1987). Proc. Natl. Acad. Sci. U.S.A. 84, 1585-1589. Zucker, R. S.(1974).,1. IJ/iwd.(London) 241, 69-89, Zucker, K. S., and Fogelson, A. L,. (1986).Proc. h’c~tl. Arcid. Sci. U . S . A . 83, 3032-3036. Zucker, R. S., and Haydon, P. G. (1988). Nalurr (London) 355, 360-362. Zucker, R. S.,and Lando, I.. (1986) Scienrr 231, 574-579. Zucker, R. S., and Stockbridge, N . (1983).]. ,Yrurosci. 3,1263-1269. Zucker, R. S., Land6, L., and Fogelson, A. (1986).,/.Physiol. (Paris)81, 237-245.

SINGLE-CHANNEL STUDIES OF GLUTAMATE RECEPTORS By M. S.

P. Sansom and P. N.R. Usherwood Department of Zoology University of Nottingham University Park Nottinghom NG7 2RD, U.K.

I. Inti-oduction 11. Channels Gated by Vertebrarc A . Noise Studies

(;lutaiii;ite

Kcceptoi-s

B. Single-Channel Studies C . Full Conductances, Subcoirtlut t.iriccs. a i d Llnityirig Hypotheses f o i Glutamate Receptors 1). Noncompetitive AiitdgOiiiSlll of \'crtcl)I;uc (;lutainate Rcceptoi-s E. Glycine Modulation of NMI):2-K IZctivity F. Desensitization of Maiiiiii;iliitii (;lut;imatc Receptors 111. (:hannels Gated by Invertelware ( ~ I i i r a i i i a t cKcreptors A . D-GluR B. H-GluR IV. Overview Kcterences

1. Introduction

From inauspicious beginnings in the middle of'this century studies o f glutamate receptors and their role in excitable cell function have reached their early adulthood. T h e time is past when proposals that L-glutaniic acid might be a neurotransmitter were countered by arguments about the ubiquity of this amino acid in animals and, therefore, its unsuitability as a signal niolecule in nervous systems. -1here is now general acc-eptance that this amino acid is the transmitter. at arthropod excitatory nerve-muscle junctions and strong support for its role as a neurotransmitter in central nervous systems (CNS) of vertebrate and invertebrate animals. Over the past decade o r so single-channel studies of glutamate receptor (GluK) function undertaken using patch clamp techniques have contributed significantly to o u r understanding of glutamatergic transmission. T h e purpose of this chapter is t o review this held and, from o u r untlcrstancling of the properties of G~LIK, to develop a comparative view of' these

52

M. S. P. SANSOM A N D P. N. R. USHERWOOD

important macromolecules, and to comment on possible future developments. McLennan (1983) and Mayer and Westbrook (1987) have extensively reviewed the physiology and pharmacology of GluR in mammalian CNS, and a recent edition of Trend5 in Neuroscience (volume 10, 1988) was devoted to a subtype of mammalian GluR, the N-methyl-D-aspartate (NMDA) receptor. In addition, Shinozaki (1988) has discussed the comparative pharmacology of GluR. Little purpose would be served in covering this ground again, yet any discussion of the channels gated by GluR must inevitably be related to the pharmacological properties of their gating membrane proteins. For this reason we shall present a brief summary of GluR pharmacology, if only to identify the complexity of the subject and to provide a further opportunity briefly to advertise the fact that the blanket terms “excitatory amino acids” and “excitatory amino acid receptors” are, at best, misleading and in truth erroneous (Usherwood, 1978). The ubiquitous distribution of GluK (coupled either directly or indirectly to ion channels, but excluding those involved with transmembrane transport of L-glutamic acid) throughout the animal kingdom is illustrated in Table I. Single-cell organisms such as the protozoan Paramecium respond to L-glutamate, so one may assume that there are GluR on the surface membranes of organisms without nervous systems. There appears to be a division between the vertebrates and those invertebrates with nervous systems in terms of the bodily dispositions of postjunctival GluR. They are found in the CNS of both groups of animals, but in the former they are found peripherally in the afferent nervous system, whereas in invertebrates any peripheral location appears to be restricted to motor systems (Usherwood, 1978; Nistri and Constanti, 1979; Duce, 1988). The use of ligand binding techniques to investigate GluR of vertebrate CNS has been a major contributory factor in the identification of different classes or subtypes of receptor (see review by Foster and Fagg, 1984), although the original classification of GluR subtypes was based initially upon electrophysiological data (e.g., Watkins and Evans, 198 1). According to pharmacological criteria, three GluR subtypes have been clearly identified so far, in both biochemical and electrophysiological studies: the N-methyl-D-aspartate-sensitive receptor (NMDA-R), the quisqualate-sensitive receptor (the Quis-R) and the kainate-sensitive receptor (the Kain-R). A further subtype, the L-2amino-4-phosphonobutyrate-sensitivereceptor has been identified in biochemical studies, but as yet has no electrophysiological correlate. When considering any classification of vertebrate CNS GluR based upon pharmacological differences it is important to bear in mind that, appar-

53

SINGLE-CIlANNt 1 GLU 1 AMATE KECEPIOKS

TABLE 1 PHYLOGENIC D ~ S T R I R I I TOF~ GLUTAMATE ON RECEPTORS

Phylum Protozoa '4nnelida Mollusca

Location

Physiological response Pharmacological at resting potential tY pe

Peripheral Hyperpolarization Central Depolarizatiori Central Depolariration Hyperpolarization Hyperpolari7ation

Arthropoda Insecta

Peripheral Depolarization

H yperpolariration Depolarization H yperpolarimt ioii Crustacea Peripheral Depolarization Hyperpolarization Central Depolarization H yperpolariration Protochordata Central Depolarization Pisces Central Depolarization Central

Mamrnalia

Central

Depolarization

-

Ibotenate Kainatel quisqualate I tmtenate Quisqualate (ibotenate & aspartate)* I hotenate -

Ibotenate Quisqualate ?

Quisqualatei kainate Kainate I botenate Quisqualate h'-rnethyl-oaspartate

Ion channel

Keference5"

Na', K + Na', K', Ca"

I. 2 3 4. 5

(:I-

6 7. 8

K'

Na'. K + ,Ca"

9. 10, I I

(;ICINa', K', Cay' CI-

12, I S 14 14. 15 1 6 , 17, 18 10

20 20 21 22

23

24, 25

Key to references: 1. Preston and llshei-wood (1988a); 2. Preston and Ushei-wood (1988h); 3 . Sargeant et al. (1977); 4. Adams and Gillespie (1988): 5. Walker (1976); 6 . Piggott P/ ul. (1975); 7. Oomura el a/. (1974): 8. Yokoi et ul. (1977); 9. <;ration et al. (1979); 10. Bodrn rt crl. (19x6); 11. Kits and Usherwood (1988): 12. Lea and Usherwood (1973a,b): 13. Dudel rt ul. (19891)): 14. Giles and Usherwood (1985); 15. Wafford and Sattelle (1986): 16. Onodera and Takeuthi (1Y75); 17. OnoderaandTakeuchi(l976): 18. Deakin (1983); 19. FrankePtuL (I986a);20. Kober-ts and Walker (1982); 21. Matthews arid Wickelgren (1979); 22. Ishida P/, a/. (1984): 23. Tachibana (1983); 24. Watkins and Evans (1981); 25. Foster ant1 Fagg (1984). * Mixed pos'junctional receptor population ($1).

ently, only the Kain-K has a restricted structure-activity profile; the other two receptor subtypes are sensitive to a wide variety of glutamate agonists, including those that identify the other GluK subtypes. This comment is relevant to our later discussion of the proposal that the three GluK subtypes are part of a single macromolecule. Within the three electrophysiological classes of GluR there are further subdivisions that

54

M. s. P. SANSOM A N D P. N . n. u s m n w o o ~

complicate the picture (Greenamyre et al., 1985; Honore et al., 1986; Sawada and Yamanioto, 1984). The role of L-glutamicacid as a neurotransmitter in vertebrate CNS is not restricted to channel opening. It was Kaneko (1971) who first suggested that a transmitter, considered to be L-glutaniic acid, that is released from photoreceptors in the retina closes ion channels of depolarizing bipolar cells. More recently, Wilson et al. (1987) found that the conductance of some cells of dissociated axolotl retina is decreased by this amino acid. Photoreceptors in the turtle retina respond to L-glutamate and the stereoisomers of aspartate but are insensitive to N-methyl-Daspartate, L-quisqualate, L-kainate, and D-glutamate. The horizontal cells in this tissue respond to L-kainate and L-quisqualate but not to N-methyl-D-aspartate and D- and L-aspartate. I t remains to be established unequivocally whether the GluR on these cells are coupled to menibrane channels, but at least in the case of the horizontal cells this is considered to be likely. It is well established that GluR in vertebrate CNS can regulate the production of secondary messengers, either directly through elevation of CAMPand cGMP (Bruns et al., 1980) or indirectly through an increase in intracellular Cay+ (Berridge, 1987). A further route has been identified with the discovery that GluR can use phosphoionositides (IP) to generate secondary messengers (see review by Sladeczek el al., 1988). L-Glutamate is the major neurotransmitter at arthropod neuromuscular junctions (NMJs), and, to a lesser extent, within arthropod CNS (reviewed by Duce, 1988). It is also a transmitter candidate in the CNS of other invertebrates (Leake and Walker, 1980). A range of studies have suggested that the actions of L-glutamic acid on arthropod muscle are mediated by directly coupled receptor-channel complexes analogous to the nicotinic acetylcholine receptor-channel complex. There are two major classes of GluR on arthropod skeletal muscle: D (depolarizing) and H (hyperpolarizing) (Usherwood and Cull-Candy, 1974). T h e D-GluR gate cation-selective channels, resulting in an excitatory response to L-glutamic acid, whereas the H receptors gate anion channels. Representatives of both classes of GluR have been studied in some detail at the single-channel level. T h e pharmacology of these GluR is rather complex when viewed across the arthropod phylum, but most single-channel studies have been undertaken on GluR of locust and crayfish skeletal muscle, so we shall concentrate on these receptors in our review. 'The D-GluR in these animals are quisqualate-sensitive (Gration et al., 1979; Gration and Usherwood, 1980; Shinozaki, 1988), but like their counterparts in vertebrate CNS they respond to a wide range of Lglutamate agonists. The H-GluR of locust muscle are ibotenate (1bo)-

sensitive (Lea a n d Usherwooti, l973a,b; Dudel et al., l989a), but in the crayfish they seemingly share a C1- channel with a variety of' other transmitters such as acetylcholine arid GABA (Dudel rt a/., 1988a). It has been known for some time that excitable tissues of invertebrate animals contain glutamate receptors, which are linked to anion-selective channels. Although these receptors have not yet been ascribed a function and there is no evidence that the!' are postsynaptic receptors at inhibitory synapses, they are interesting, nevertheless, in the light ot' discoveries of GluR possibly linked to anion-selective channels in vertebrate CNS. When mRNA isolated from rat brain is injected into X e n o p u s oocytes a Quis-K is expressed, the activation of which triggers an increase in chloride conductance. 'This response can be mimicked by the injection of' an IP into the oocyte and blocked by the injection of EG-1.A (Sugiyania P t al., 1987). Sladeczek et al. (1988) have suggested that this Quis-K is a new type of excitatory amino acid receptor, but this seems an inappropriate description of a receptor linked to a n anion-selective channel unless, of course, the linkage is different it) zliz~o.Sladeczec P t a / . ( 1 YXH) have also described a n Ibo-K that is coupled to the production of IP. 'l'he H-GluK of locust leg muscle, which is also coupled to an anion-selective channel (Lea a n d Usherwood, 1973a,b), is ibotenate-sensitive anti exhibits properties that tentatively suggest that it may also act via a seconclary messenger (Dudel et al., 198C)a). T h e hinding studies of Yonecla et 111. (1986) o n synaptic membrane preparations from rat brain also suggest that some GluR in this tissue may be linked to anion channels. Single-channel studies of GluR made using patch clanip techniques began over a decade ago (Patlak P t ul., 1979), although noise studies o f arthropod muscle GluR (see 71'able V) had previously provided preliminary insight into the single-channel properties of these membrane macromolecules (Crawford and McBurney, 1976;Anderson ~t a / . , 1978).T h e large open channel conductance of the cation-selective, II-GluK of' locust leg muscle, the ready accessibility of this receptor, and o u r ability to inhibit its desensitization with concanavalin A (Con A) have made possible equilibrium studies of its channel gating kinetics over an exceptionally wide range of ligand concentrations (<10~ti-lO-' M ) (see later). These factors have also facilitated single-channel studies of the interactions of noncompetitive antagonists with this GluR. It is not surprising, therefore, that this particular receptor figures prominently in 'l'able 11, which presents a n abbreviated historical account of the developments of singlechannel studies of GluK using patch clamp techniques. From a coniparative viewpoint it is of interest to note that although studies of GluK have understandably lagged behind those of the nicotinic acetylcholine receptor, a number of discoveries made on locust muscle GluK have ac-

56

M. S. P. SANSOM AND P. N . R . USHEKWOOD

TABLE I1 CHRONOLOGY OF SINGLE GLUTAMATE RECEPTOR CHANNEL STUDIES Year

Event (reference)"

1979 Single-channel recordings froin a quisqualate-sensitive receptor of locust leg muscle made using a megaohm seal recording technique; identification of complex channel gating kinetics and desensitization inhibition by concanavalin A. (1) 1979 Open-channel conductance of quisqualate-sensitive receptor channel of locust leg muscle is agonist independelit; open-channel mean lifetime is agonist dependent. (2) I980 Comparison of desensitizing and nondesensitiziiig, quisqualate-sensitive receptors of locust leg muscle. (3) 1981 Mean open time of quisqualate-sensitive receptor channel of locust leg muscle reported to increase with L-glutamate concentration (4) 1982 Cyclic scheme proposed for gating of quisqualate-sensitive receptor channel of locust leg muscle. ( 5 ) [But see (6) for alternative proposal o f a simple, linear reaction scheme for channel gating by this receptor.] 1984 Giga-ohm seal recordings first reported; locust embryonic muscle in culture. (7) 1984 First report of recordings from vertebrate NMDA receptor channels; mouse mesencephalic and striatal neurons in culture. ( 8 ) 1986 Multiple conductance substates for the NMDA receptor of vertebrate neurons first described. (9) 1986 First report of giga-ohm seal recording froin adult arthropod muscle; crayfish muscle. ( 10) 1986 First reports of recordings from channels gated by L-quisqualate and L-kainate in mammalian brain neurons in culture. ( 1 1) 1987 Detailed analysis of channel kinetics of quisqualate-sensitive receptor of locust leg muscle leads to proposal for a cyclic gating mechanism. (12) 1987 Potentiation of opening rate of-NMDA receptor channel of cultured mouse brain neurons by glycine. (13) 1987 Suggestion that glutamate receptors in vertebrate central nervous system may be linked to a comnion channel. (14, 15) 1988 A study of locust muscle quisqualate-sensitive receptor over a wide concentration range confirms complex kinetics of channel gating. ( 16) 1988 Rapid desensitization of quisqualate-sensitive receptor of locust leg muscle described. (17) 1988 Complex gating kinetics of. large-conductance (50 pS) channel gated by glutamate receptor of rat cerebellar granule neurons in culture. (18) 1988 Block of quisqualate-sensitive receptor of locust leg muscle by the spider toxin, argiotoxin 636. (19) ~~~~

~

~~~~

~~~

Key to references: 1. Patlak et al. (1979); 2. Clark et a1. (1979); 3. Gration et al. (1980b); 4. Gration et af. (1981a); 5. Gration et al. (1982); 6. Cull-Candy et af. (1981); 7. Cook et al. (1985); 8. Nowak et al. (1984); 9. Ascher and Nowak (1986); 10. Franke et al. (1986a); 11. Cull-Candy arid Ogden (1985); 12. Kerry et af. (1987a); 13. Johnson and Ascher (1987); 14. Jahr and Stevens (1987); 15. Cull-Candy and Usowicz (1987a); 16. Kerry et al. (1988a); 17. Dudel et al. (l988b); 18. Howe et al. (1988); 19. Kerry et al. (1988~). a

SINGLE-CIIANNEI. (*l.Ll I A M A T E KECEVTOKS

57

tually contributed to our f'untlamental knowledge of receptor channel function. In this review w e shall elaborate on many of the points highlighted in Table I1 and attempt to determine whether, on present phylogenic evidence, it is reasonable to assume that the properties of GluK have been conserved during evolution and whether this group of membrane macromolecules fits readily into the superfamily of receptors to which the nicotinic acetylcholine (ACh) receptors anti t h e receptor for y-aminobutyric acid (GABA) apparently belong (Barnard et nl., 1987).

11. Channels Gated by Vertebrate Glutamate Receptors

It is generally assumed that the fast conductance changes associated with activation of GluR in rnatnnialian CNS represent sequences of events leading from agonist binding to ionic permeability change that do not involve secondary messenger intermediaries. However, this does not exclude the involvement of additional postconductance links, through G proteins and Ca'+ flux, with secondary messenger systems.

A. NOISESTUDIES

Noise analysis has been used frequently to obtain insight into the unitary properties of channels gated by GluR (i.e., single channel conductances and the number of kinetic states assumed by channels gated by GluR subtypes). This continues to be a widely used approach. However, the presence of mixed populations of GluR and the limited bandwidth over which such studies can tie undertaken must inevitably limit their future value in these respects. 'rable 111 summarizes the many studies of the current fluctuations or noise generated by mammalian and other vertebrate neurons in response to application of 1,-glutamate or agonists. Channel conductances ranging from about 140 fS to about 50 pS have been obtained for GluR using fluctuation analysis together with mean open times ranging from 0.5 msec to 18.5 msec for agonist concentrations between lop5 and lo-.' M . Conductance values obtained in noise experiments performed under voltage clamp will reflect the presence not only of channels gated by different GluR subtypes but also the occurrence of subconductances. Mean open time values will be influenced by the bandwidth of the recording systetn, which will limit resolution of fast

TABLE I11 GLUTAMATE RECEPTORS I h VtRrERRAIE CNS: NOISE STUDIES"

Ligand and preparationb L-Glutamic acid 6 2 1

1 7

8 4 L-Kainic acid 12 1 7 3 10

g

10

[LI

(PS)

(msec)

(FM)

54 140 fS 23 46.6

5.9 0.8, 18.5

8.4 139 fS 50 0.5

0.3 1-6

4 11

1.1-5.5 2.5 2.1 1.7 3.8 1.8-2.4

2

1.0

4

1.8

5.9 or 2.4, 13.6 1.7, 12.8 31 1.6, 6.1 1 or .5, 5 2 5.5 >lo0 3.1, 1.0, 5.7 2.8 1

10

-60 to -80

2-20 10 10-20

- 60

-70 to -90 -60 to -70

10-20 10 10 100 5

-60 to - 70 -60 to -Xi) -60 to - 120 - 60 - 53 - 60

20-100 10-30

-75

20 50

1-3

1.9 0.7, 3.2 2.3, 5.3 10 50

V,,, (mV)

10-100 20-50 20-60 70-100

Low pass ti Iter

VK

(mV)

1'"C

21 21 20-22

(kHz) 0.5 0.5 0.5 1

I I I 3 1-32

1.5 18-33

1 1

20 to - 100

- 60

1

3 1-32

- 60

1

-60 to -80 -60 LO +60 -60 to +60 -55

1

18-25 18-25

I 1

15

1

20-22

References'

Quinolinic acid 4 L-Quisqualic acid 4 NMDA 4 5 9 D,L-Ibotenic acid 4 L-Aspartic acid 1

u1

'a

Trons-piperidine decarboxylate 4

4.8. 5.3

20

4.1

8.9 or < I , 14.6

10

- 60

22-40

6 7 5.3

2.5- 100 10 1-10

-60 to +60 - 50 -50 or -60

5.6

10

4.2 or 3.1. I5

5.0

38.2

49

18-33

1

11

18-25

1

10

18-33

1 0.9-2 1-2

14 13

- 60

1-2

11

10-30

-60 to -80

1

10

- 60

1-2

-0

25-27

11

4

11

Definitions: g, channel conductance; t,,, mean channel open time; [L], agonist concentration; V h f ,membrane potential; VR. reversal potential. Key to Preparations: 1. rat cerebellum granule cell culture; 2. rat cerebellum neuron culture; 3. organotypic culture of rat hippocampus slices; 4. mouse central neuron culture; 5. rat cortical neuron culture; 6. rat cerebellum explants; 7. rat septa1 neuron culture; 8. turtle photoreceptorsdissociated retina; 9. mouse hippocanipal neuron culture; 10. organotvpic culture of rat cerebellum slices; 1 I . goldfsh retinal horimntal cellsdissociated retina; 12. X m o p w lurvzs oocyres injected wirh rnRNA. Key to references: 1 . Cull-Candy and Ogden (3985); 2. Cull-Candy and Usowicz (1987b); 3 . Miledi rt a/. (1983); 4. Cull-Candy r f a/. (1988); 5. Shingai and Ebina (1988);6. Howe rf 01. (3988); 7. Tachibana and Kaneko (lY88a,b); 8. Cull-Candy aiicl Usowici! (l987aj; 9. Llano rt (11. (l988j; 10. Ascher and Nowak (1988); 1 1 . Ascher el ul. (1988); 12. Murase rf (11. (1987); 13. Maver P / n / . (1988); 1.1. Bertolino and Vicirii (1988). "

60

M. S. P. SANSOM A N D P. N . R. USHERWOOD

events. However, one generalization has emerged from these studies: namely, that channels gated by L-kainic acid apparently have much lower conductances than those gated by NMDA and L-aspartate.

B. SINGLE-CHANNEL STUDIES Table IV summarizes most of the published data on mammalian CNS GluR that have been obtained using single-channel recording techniques. T h e best characterized channel is that associated with the NMDA-R. The kinetics of this channel are markedly influenced by the presence of divalent cations on either side of the channel, and much attention has been given to the physiological effects of extracellular Mg2+, which blocks this channel in its open state(s) and perhaps also when it is closed (see review by Mayer and Westbrook, 1987). Here we review information on NMDA-R. One of the principal tools in analysis of channel gating kinetics is the evaluation and fitting of channel open and closed (dwell) time distributions. Single-channel theory (Colquhoun and Hawkes, 198 1,1982; Horn, 1984; Fredkin et al., 1985) suggests that dwell time distributions should be made up of sums of exponential decay terms. So, for the following simple gating mechanism C+At,CAttOA, where C is the closed channel, 0 the open channel, and A the agonist molecule, the channel open time distribution would be predicted to be a single exponential decay (No = 1 ) and the closed time distribution to be the sum of two exponential decays ( N , = 2). So evaluation of the numbers of exponential terms required to fit the observed open and closed time distributions provides lower limits for the number of open states ( N o )and number of closed states (N,) values of the underlying gating mechanism. The kinetics of the large conductance (50 pS) channel gated by the NMDA-R have been the subject of two in-depth investigations (Ascher el al., 1988; Howe et al., 1988) and a brief abstract (Dani el ul., 1988). Because of the profound influence of intracellular and extracellular Mg2+ on the gating kinetics of this channel (Nowak et al., 1984; Ascher and Johnson, 1988) these studies were undertaken in Mg'+-free media. In their seminal paper on Mg*+-blockof the NMDA-R channel, Nowak et al. (1984) stated that in Mg2+-freemedia the open time distributions of single channel events recorded from outside-out patches excised from mouse embryonic central neurons in culture could be fitted reasonably well by a single exponential. A similar conclusion was arrived at by

SINGLE-C;IOZNNEI. (;l.I'TAMATE RECEPTORS

61

Bertolino and Vicini (1988). However, in a later paper, Ascher rt al. (1988) reported openings interrupted by brief closings, which led them to conclude that channel opening could not be described by a single value, a conclusion supported by their finding that channel open time distributions could not always be fitted by a single exponential. Nevertheless, Ascher and Nowak (1988) reiterated the generally held view that single channel currents recorded in Mg'+-free solutions at negative membrane potentials differ from those recorded in Mg'+-containing solutions by the presence in the latter of bursts of brief openings (see also Ascher and Nowak, 1987). These observations were underlined by the fact that at positive membrane potentials little flickering was observed in the single-channel currents. According to Jahr and Stevens ( 1987), the NMDA-R channel of CA 1 hippocampal neurons cultured from newborn rats exhibits two gating modes: One has a mean open time of 1-3 msec, the other a mean open time of 10-15 msec. T h e longer-duration open state occurs in bursts that can last for hundreds of milliseconds. This latter phenomenon is reminiscent of the state switching described by Patlak et al. (1979) for the locust muscle D-GluR (see also Gration et al., 1981a; Kerry et al., 1987a) and has also been described for the NMDA-R channel of rat cerebellar granule cells by Howe et al. (1988). Howe et al. (1988) also concluded that the NMDA-K channel has at least two kinetically distinct open states. However, their results differed from those of Jahr and Stevens (1987) in that they found no difference between the apparent dwell times for openings within and between bursts. In the studies of Howe et al. (1988), L-glutamate, L-aspartate, and NMDA all activated channel openings in outside-out patches to more than one conductance level (e.g., openings to mean conductance levels of 50 pS, 42 pS, and 33 pS were observed in the presence of all three agonists). T h e closed time distributions in this study contained four exponential components, but because the numbers of channels in the patches were not known only those closing with the smallest time constants were held to give quantitative information on gating kinetics. Neither of the two shortest time constants exhibited any voltage or agonist concentration (3-300 p M ) dependency. Howe et al. (1988) concluded that the 50 pS channel activated by the three agonists has a minimum of three closed states and tw o kinetically distinguishable 50 pS open states. They also reported the occurrence of clusterings of channel bursts in many of their records similar to those reported for nicotinic acetylcholine receptor channels and for the locust GluR (in the absence of Con A) (Gration et al., 1980a,b; Dudel et al., 1988b), which have been ascribed to result from the influence of desensitization on channel gating. Daniel al. (1988) also refer to the open time distribution for the 50 pS

T A B L E IV GLL~TAMATE RECEPTORS I N VERTEBRATE CNS: SINGLL-CI-IAYNEL STUDILS"

N m

Ligand and preparationb

K (PSI

10

[ L1

(nisecj

(*W

V,,, (mv)

VK (mVj

.I"C

Low pass filter (kHr)

Sampling rate (k1-I~)

1-4

10-40

References'

NMDA 1

2 3 4 5

5 5 I 8 L-Quisqualic acid

53 48 48 40-50 50 50 40-50 40-50

1

8. 17, 50

2

7-48 28. 18 14 8.40-60 6. 12.45-50

3 4 3

5.2 -7 . 2 7 . 5 , 9.1 10.5 or 1.2. 10

10-30 3-50 5- I 0

6.1

5

5-6

10

6.4

2 2

3.8 6.2

2 5.2-7. I

-80 to +x0

-TO -50 -80 to +60 -50 -50 -50 -50

-1.6 -5.8

0 0

10

10

- I10 to + 4 0

10-50 50

-70

1-10

-80 -50

1

18-33 71-23 21-25 21-23 21-23 20-25

14 to + 2

-3.8

20-22

0

18-25

I 3 2 3

2 10

2.5 2.5

10 10 10 10

1

17

12 13

10-40

3, 4

1-4 1-2

6

7

1

2

to

+80

3, 4 6 7 9 10 II 12

I I

5

8

7

L-Kainic acid 1 4 2

L-Glutamic acid I 6 2 1

5

4 L-Aspartic acid 1 6 2

Quinolinic acid 9

9

5 , 15, 40, 50 21 6-48

0.7-2

10 10-50

-SO

10

+(,I)

I)

-70

33,42, 50

1-4

3, &

1-2

ti

18-2.i

-3.5

S

2-6

2O-liO

48.9

2

47.7 5 . 15. 40-50 5. 15.40-50 3-15 40-50 51.5 50 x-48

I;

10

5 3 o r 1.2. 10

10-2..5

- X I ) t o -40

-60 to + I 0

-3.j

0

1

21-23 2 1-23

2.5 2.5

10

I2

I0

I2

IX-33

2

I0

!J

1-1

10- to

:1. I

2 1-2

40-55

40-46

Definitions: g, channel conductance; t,,, riieaii c-hanncl open time; [ L ] ,agonisr c[~ii[.ciirr~itioii; V r l . t ~ i c ~ n l ~ ~poteiiiial; ~atie and \ ' R , r Key to preparations: 1 . rat cerebellum granule cell culture; 2. rat cei-ebellar neuroii c iilt urc; 3 . organotypic- ciilturc o f rat hippocainpu\ slitca: 4. mouse central neuron culture; 5. rat cortical iicuroii culture; 6. rat cerebellum explaiits; 7. orgatlotypic- C L I ~ I L I I Cof rat cc.rct)ellum slic e q ; 8. rat \ isual cortex cultures; 9. rat hippocanipal Iicuroti culture. ' Key to references: 1. Cull-Candy and Ogdcn (1985); 2. Cull-<;andy and Lsowici (I!J871>); 3 . ~ : u I l - C ; ~ i i i d yr/ crl. ( I 9 X X ) : -1. I i o i v r I , / c d . (I!)XH); 5. Tsuzuki rt nl. (1989); 6. Cull-<;aiidy and Usowicz (198ia); 7. I.lano r/ ol. (1988); 8 . Asclic.~;ind N o w k (IYHH); !I. Aacticr otcrl. (1!)88); 10. t k w o l i i i o et nl. (1989); I I . Bertolino arid Vicini (19x8); 12. Hertolino rt (11. (1088); 13. Hucttner atitl Hem (ILJHX); 1-4. ~I'suiuhie/ ol. ( IOXI)).

64

M.S. P. SANSOM AND P. N. R. USHERWOOD

channel gated by the NMDA receptor of cultured rat hippocampal neurons as a multiple exponential, but point out that with 10 mM Mg2+ in the extracellular medium the distribution is dominated by a single exponential. In general, studies of vertebrate amino acid receptor channels at the single-channel level have been analyzed according to methodology developed for the nicotinic acetylcholine receptor channel. Channel kinetics have been investigated by examining closed-time distributions and burst length distributions, the latter being defined as a group of openings separated by closings of duration less than a critical gap length, which is determined either from the closed time distribution (Colquhoun and Sakmann, 1985)or arbitrarily. Many laboratories have favored the analysis of single channel openings in terms of mean burst durations (tk,) and mean open times (to) to gain insight into channel gating kinetics. Somewhat surprisingly, Ascher et al. (1988) found that t,, was closer than t b to the value for t , derived from noise analysis, when the contrary is expected (see Colquhoun and Sakmann, 1981). They suggested that this discrepancy might be due to the influence of subconductance events with smaller to and tb than the 50 pS conductance channel. As an alternative, they suggested that the kinetics of the NMDA-R channel are altered during patch excision. Such changes have been reported by Guharay et al. (1985) for a 5-HT receptor channel and by Dude1 et al. (1988b) for the channel gated by the locust muscle D-GluR. In their analyses of bursts of channel openings Howe et al. (1988) concluded that burst length distributions contained three exponential components, and this was also true for distributions of total open time per burst. T h e time constants for the two sets of measurements were similar. According to Jahr and Stevens (1987), tb varies greatly from patch to patch and can change with time in a single patch. Their view is that bursting can be modified by an unidentified mechanism. Unfortunately, the authors did not state whether the changes that they observed were unidirectional and, therefore, possibly the result of patch “deterioration.” There seems to be general agreement that tb and to are unaffected by changes in membrane potential (e.g.,Jahr and Stevens, 1987; Ascher et al., 1988). Howe et al. (1988) have proposed a number of mechanisms that could account for the brief closings within supposedly single activations of ion channels that produce channel bursts. Since in their experiments on the 50 pS NMDA-R channel Mg‘+ was absent from the media bathing the patches, they reasonably concluded that the bursts did not result from rapid block and unblock of the open channel by this divalent cation.

SINGLE-CH A N N El (;I.L''I'A MATE KECEPTOKS I

65

Neither the duration nor the frequency of occurrence of the brief closings changed with NMDA concentration and membrane potential. In contrast, the kinetics of Mg2+ block of the NMDA-K channel are voltage dependent (Nowak et al., 1984). During hyperpolarization and when the extracellular Mg'+ concentration is raised the mean burst length of the 50 pS NMDA-activated channel decreases. T h e frequency of opening of the NMDA channel is also decreased in M g " at negative membrane potentials. Mayer and Westbrook ( 1985) have suggested that NMDA channels show activation-dependent Mg2+ unlock at positive membrane potentials indicative of relief from a stable (closed?), blocked state (see later). Closed time distributions for the NMDA-R channel have been described by Howe et al. (1988). 'l'hese contained four components, the two briefest (71 = 40-58 psec; 7 2 = 592-903 psec) of which varied according to the agonist employed (L-glutamate, L-aspartate, and NMDA), but in reverse order for 71 compared with 7 2 . T h e time constants of' these components were independent of membrane potential over the range +60 mV to -160 mV. These authors suggested that the two longest components in the closed time histograms are not informative about channel gating mechanisms, but that the t w o briefest components probably represent sequential openings of the same channel. Clusterings of channel bursts were also observed in outside-out patches excised from cerebellar granule cells, and these clusterings sometimes lasted for hun1988). Presumably these long clusters dreds of milliseconds (Howe et d., are equivalent to the "long opening mode" described by Jahr and Stevens ( 1987) in their outside-out patch recordings from rat hippocampal neurons. In their best-defined closed time distributions for these clusters, Howe et al. ( 1 988) were able to identify an additional brief component of mean duration, 1.5-3 msec. The time constant for this component was longest during NMDA application, the agonist for which clustering was most prevalent. An attractive explanation for the clustering of channel bursts is that they result from desensitization. Gration et al. ( 1980a,b) have described such a phenomenon for the locust muscle D-GluR, such that channel openings occur in well-defined clusters over a limited range of agonist concentrations and in the absence o f Con A, and have attributed this to desensitization. Ascher el al. (1988) reported that above 10 pM Lglutamate they observed bursts of channel openings separated by long closures and suggested that the latter resulted from desensitization. The concentrations of agonist employed by Howe et al. (1988) to obtain clusterings were much higher than this (30 p M ) .

66

M.S. 1'. SANSO.21 A N D 1'. N. K. LSIIEKWOOL)

C. FIJLI.c O N D U C T A N C E S , SURCONDIJCTANCES. HYPOTHESES FOR G L U ' r A M A ' T E K E C E I T O R S

AND UNIFYING

T h e publication in 1987 of two papers ( J a h r and Stevens, 1987; Cull-Candy and Usowicz, 1987a) on GluK channels of mamnialian CNS, in which the authors speculated on the possibility that the GluK subtypes might be linked either t o a conimo~icharinel o r to a common class of channels, evoked some interest. ~l'heseideas seemed unlikely at first sight in view of the clear-cut pharmacological dil'fercnces between the GluK subtypes and their markedly diffkretit regional distributions (Fostei- and Fagg, 1984). However, as pointed out by (:ull-C;andy and Usowicz (1987b),if there is a supermacroniolecule that can accommodate all three types of GluR linked to a coninion channel, then regional differences in the distributions of the GluK subtypes could he readily explained by the variable occurrence of NMDA-sensitive, I.-ciuisqualate-sensitive, and Lkainate-sensitive sites on the niacroniolecule. Regional variations in the dispositions, of'NMDA-K, Quis-K, and Kain-K present no problerns i f it is postulated that these receptor subtypes share a coninion class of'channel, which is an alternative proposal to explain the complex findings of .Jahr and Stevens (1987) and Cull-Candy and Usowicz (19871)). In recordings from outside-out patches excised from cerebellai- and hippocampal neurons in culture, these two laboratories showed that the application of N MDA, L-quisqualate, arid L-kainate activated ion channels with multiple conductance substates, the amplitucles of which were similar for all three agonists. Since there were direct transitions between the substates and there was ;I higher probability of transitions f'i-om the higher to the lower substates, i t seemed logical that these phenomena might results from a single niacromolecule (i.e.,a "super GluK"). According to Cull-Candy a n d Usowicz ( 1987b), their data on cerebellar neurons exclude the idea that the (;luK subtypes are coupled to separate channels with different unitary- conductances. This view was implicitly reinforced by Jahr and Stevens (198$), who argued that if each GluK subtype does have its own channel then each channel has a major conductance and several substates which just happen to coincide with the values for the other channels. T h e ideas outlined in these two papers have been reiterated in subsequent publications and have been the subject of a number of independent investigations. Before reviewing this work it may be instructive to consider Fig. 1. This gives a diagrammatic account of the different subconductance states for GluK channels of nianirnaliari CNS neurons. What is noteworthy about this distribution is the large number of apparent substates and their variability between agonists in terms of' number and

Pic;. I . Schematic summary ol' the conductaiic.c states of channels gated hv a p p h ation of 1.-glutarnateanti agonists to maninialian C N S (;luK. The agonists in clLie\tion a r c quinoliiiate (Quin), L-aspartate (Asp), i -hairiate (Kaiii), i.-quisclual;ite (Quis). ,Y-methvl-rv aspartate (NMDA). arid L-glutainate (Glu). l'hc data were taken I'rom the srudic, s u ~ i i i i i a rized in Table I\'.

conductances. Some of this variability may be d u e t o experimental erl'or, especially when values less thaii, say, 25 pS are considered, but it must be remembered that the single-channel technique is noted for the high resolution that it provides, particularly for channel open events o f reasonably long duration, for which bandwidth limitations are less restrictive. O n the basis of the results contained in Fig. 1 the picture of either a common channel f i l l - it11 three GluK subtypes or a c o ~ n n i o class ~i of channel seems unclear. A more direct challenge to the hypotheses o f J a h r and Stevens (198'7) and of Cull-Candy and Usowicz ( 198711)conies from the studies of Llano et (11. ( 1988). They showed that recordings from outside-out patches excised from Purkinje cells, which are not sensitive to N'MIIA, never exhibit the 50 pS open channel conductance associated with the NXIDX-K. I n fact, Cull-Candy and Usowicz ( 198%) pointed out that they tiatl little evidence to suggest that the sniall conductance openings in their recorciings were substates of the 50 pS NMDA-Kchannel; neither were they able to determine whether the 8 11sand 15 pS sub-states were openings of the same channel. Ascher and Nowak (1988) have examined the conductances activated by L-quisciualate arid I.-kainate and concluded that I,oth of these agoriists evoke more tlian one conductance state. rA-Quisqualate niainly activates a low-conductance channel of about 8 pS that is less sensitive to L-glutamate than the 50 pS NMDA-K cliannel. L-Kainate

68

M. S. P. SANSOM AND P. N. R. USHERWOOD

produces -20 pS openings of very brief duration. When they tested NMDA, L-quisqualate, and L-kainate on the same patch, they observed considerable variability in the responses, with some patches failing to respond to NMDA. Like Llano et al. (1988) they were led by these results to conclude that NMDA and non-NMDA receptors are different macromolecules. Although one outside-out patch failed to respond to Lquisqualate, this was not considered sufficient to distinguish separate receptor channels for this agonist and for L-kainate, because the response to L-quisqualate often deteriorated with time after patch formation (see also Dude1 et al., 198813).From the data that have been accumulated so far it seems unlikely that studies of subconductance states of GluR channels will decide unequivocally either in favor of or against one of the two forms of what might be termed ‘the common channel’ hypotheses. More compelling evidence concerning this hypothesis may be gleaned from studies of the noncompetitive antagonists of mammalian CNS GluR. Mg2+ is a noncompetitive antagonist of the NMDA-R that has received much attention during the past few years (e.g., Ascher and Nowak, 1987) and that apparently has no effect on whole-cell responses to Lquisqualate and L-kainate. If the three subtypes of GluR share a common channel then one must assume that Mg‘+ blocks the large (50 pS) conductance open channel that it is associated with binding of NMDA but not the subconductance states most frequently activated by L-quisqualate and L-kainate. This seems unlikely unless one invokes the idea of a multibarrelled channel for the “super GluR.” Alternatively, if the three subtypes of GluR are separate macromolecules but share a common class of channel, then it seems unlikely that Mg2+ would block the NMDA-R channel and not the other two channels. Huettner and Bean (1988) have shown that in outside-out patches excised from rat visual cortical neurons MK-80 1 is an effective noncompetitive antagonist of the NMDA-R, but it does not block responses to L-quisqualate and L-kainate. T h e fact that MK-801 also blocks the subconductance states activated by NMDA seenis to lead to the conclusion that not only are there different GluR macromolecules but that their associated channels exhibit sufficiently different physiological and pharmacological properties to lead one to reject the suggestion that they belong to a single “super GluR.” The debate over the “unifying” hypothesis is not limited to the patch clamp fraternity. Ligand-binding studies of GluR and autoradiographic studies of their distribution in vertebrate CNS have served further to confuse the issue, by virtue of their apparently conflicting findings. Electrophysiological studies have also failed to produce a consensus (e.g., O’Dell and Christensen, 1989; Tang et al., 1989). ‘The unifying GluR

SINGLE-C H A N N t I . C L U T A M A T E RECEPTORS

69

hypothesis was directly addressed by Fong el nl. (1988). Using Xenopus oocytes injected with rat brain mRNA encoding for GluR, they clemonstrated that NMDA accelerates the dissociation of MK-801 from its binding site in the GluR channel, whereas kainate does not. This observation led them to conclude that at least NMDA and L-kainate do not activate the same channel. Recent successes in cloning Kain-Rs might, at first sight, suggest that an unequivocal resolution to the debate may be soon at hand. The cDNA clone isolated by Hollman rt (11. (1989) encodes a protein consisting of a single subunit of 100,000 Da. When expressed in X e n o p w oocytes, this produced a functional Kain-K which appears indistinguishable from the Kain-R expressed from total poly(A+) KNA extracted from rat brain. I t did not respond to either N M D A or L-quisqualate. However, it remains to be established whether the single subunit protein encoded by this cDNA forms functional KAIN-R channels in uzuo. Wada et al. ( 1989) have isolated a cDNA encoding a kainate-binding protein of 40,000 Da from frog brain, the pharmacological properties of which are consistent with those of a Kain-R. However, niRNA transcribed from this cDNA did not express functional Kain-R channels in Xenopzu oocytes. A cDNA containing the complete coding regions of a Kain-K has also been isolated from chick cerebellum (Gregor et al., 1989), but its functional properties remain to be fully established. ‘The protein isolated from Xenopus brain by Henley et al. (1989) has been identified as a single GluR of the ionotropic KAIN/QUIS type. When subsequently purified by Ambrosini et nl. (1990), it was found to contain three subunits, one of which corresponds to the protein sequence obtained by cDNA cloning from frog brain by Wada et al. (1989). This protein has been recently reconstituted into artificial lipid bilayers to produce functional GluR channels which, unlike those gated by the KAIN-R of Hollman et al. (1989), are activated by both AMPA (or L-quisqualate) and L-kainate. We are faced with a range of viewpoints about GluRs which extend from a “super GluR’ bearing L-kainate, L-quisqualate, and NMDA binding sites coupled to a single channel, to a number of discrete macromolecules which share little in common except their ability to bind L-glutamate and their capacity to selectively transport cations across excitable cell membranes. The discovery that the open channel conductance of NMDA-activated channels of rat cerebellar granule cells maintained in culture changes with time further complicates matters. Sciangalepore et al. (1989) have identified an increase in maximum single-channel conductance from -4 pS to 15 pS thence to -36 pS and, finally, to -47 pS over an ll-day observation period. As many as 5 subconductance states were identified during this period. They suggest that either the small

-

70

M . S. P. SANSOM A N D P. N . R. USHERWOOD

conductance events observed during the first 3 days in culture are nonNMDA receptors responding t o NMDA or the NMDA-R undergoes developmental changes in culture which lead to a saltatory increase in open channel conductance.

D. NONCOMPETITIVE ANTAGONISM OF GLUTAMATE RECEPTORS

VERTEBRATE

The NMDA-R channel is blocked by extracellular Mg2+ in a voltagedependent fashion (Mayer et al., 1984; Nowak rt al., 1984). According to Ascher and Nowak (1988) the addition of Mg2+ to saline bathing primary cultures of mouse central neurons converts the channels activated by NMDA from regular current pulses to bursts (mean duration, t b ) of brief openings separated by brief closures. T h e mean duration (to) of the brief openings in a burst decreases with increasing Mg'+ concentration whereas the mean duration of the brief closures remains constant. Depolarisation increases the mean duration of the brief openings but decreases the mean duration of the closures. Mg'+ block of the NMDA-R channel cannot be described simply by the binding of this cation to a site in the membrane field. Although the blocking and unblocking rates have opposite voltage dependencies, as would be anticipated according to the simple theory of open channel block (Adanis, 1976), the unblocking rate is more sensitive to membrane potential than the blocking rate. Ascher and Nowak (1988) have proposed that Mg" both blocks and permeates the open NMDA-R channel. According to Ascher and Nowak (1988) this would explain the high voltage dependence of blocking rate (e-fold for 17 mV). There are already precedents for this with respect to GluR channels. T h e D-GluR channel of locust muscle is blocked by Ca2+but is also permeable to this ion (Anwyl and Usherwood, 1974; Kits and Usherwood, 1988). Also, Ashford et al. (1988, 1989) and Lingle (1989) have shown that various positively charged organic molecules block and, at high membrane potentials, permeate the open D-GIL~R channel of arthropod muscle. Repulsive interactions between Mg"+ and other cations might be responsible for increasing the voltage sensitivity for M g ' + binding to the NMDA-R channel of mammalian CNS neurons while reducing the voltage sensitivity of unbinding (Ascher and Nowak, 1988). T h e lack of any increase in tb with increasing Mg'+ concentration is seen by Ascher and Nowak (1988) as a further indication that Mg"+ binding to the NMDA-R channel is more complex than would be predicted by simple open channel block. T h e occurrence of long-lived and short-lived blocked states is

o n e explanation for this; altertiatively, channel closure tnay occiir (luring M g " block, thus trapping tlte cation in the closed channel. Intracellular Mg'+ also blocks the NVMDA-K channel ( J o h r i s o i i and Ascher, 1987). but this caitiiot he accounted for hy ;I rapid 1)IocLrapid unblock rnechanism ( N o ~ : ; i kr t ul., 1984). With 1 rnhl Mg''+, l)lock is apparent only at positive potc*iitials; h i t ;it 1 0 m;ZI it occut.s a t I)oth positive a n d negative mentl)r;inc potmtials. Since the single-c-harir1e.1( UI-rent is reduced bv intracellulat. hlg" without a n y change i i i l,,. :\schei antiJohnson ( 1 ~ bsuggestet~ ) that the oil-off rates for ~ g ' +l)iii(liiig are faster than is the case for Idoc.k by extraceIIuIar ILI~'+. .lIiis suggests. i i i turn, that intraceIIuIar and cxtraccIIuIar hlg" do noi S I I ~ I T 'I c o t i i i i i o i i binding site in the channel, although their voltage clcpeiidt.iic ic.2 \iiggest that the opposite is true. MI(-801 is a highly potcnt tioricouipetitive antagoitkt 01 tlic. XX1I)X-K (Wong et d., lY86). Simult;iticous application of. i%VR.IL):\ (50 p..\I) ; i i i t l MK-80 1 (2 p V ) to outside-our ni(~iti1jranepatches excised f'roin riit visunl cortex neurones in culture. wit11 extraceIIuIar hfg' in the I);i[hiiig tiledium, caused all-oi.-none ch;innel Idock; that is. optmirigs in t Iic presciic.c of MK-801 were identical to those recorded in the a t w t i c c of ;intag()nist, but they occurred less trequently (Huettner a t i d Hciiti, I!CM). LVith 10 pull4 MI(-801, channel openings were slioItcr, ;I ch;iiigc that could hc. reversed by clamping at positive membrane potentials, a i i t l t1iei.c. was a further reduction in opening rate. These authors explained the iictions of MK-801 o n the NMDA-K cliannel using a model that allo\vs tor the d r u g to become trapped i n ;I chantiel that closes f'roni its Mocked state. Significantly, in view o f earlicr coninicnts made in this wview. \ I K-80 1 had no effect on the reslx~nses t o either i.-cluisqual;itc 0 1 'i.-kainate. Strychnine is also a n open channel ljlocker of' the N14ll)A-K (Hcrtolino and Vicini, 1988) arid like M K - 8 0 1 it has no ef'kct on the channcls gated by either QUIS-R o r K A I N - K . hdniiitisti-atiori of' 20 p A [ str\c-hnine reduces the channel openirig rate in a voltage-depentlerit fashion and causes rapid block a nd unblock of the open channel. Its open channel blocking action is similar t o that of Mg'+ except that 61, increases as the concentration of strychnine is increased. Bertolino PI (11. ( 1988) irivestigated that action o f phencyclidine on the NMD.4-K in outsicle-out patches excised from rat c.crel)ellutn and cortical neurons i n culture. Administration of 2 phl phencyclidine decreased the chaiinel ope11probability without causing any change in f,,, but t,, was t~edricedwitli 'LO ~ 2 2 phencyclidine. Outside-out patches excised from rat hippocampal neurons ar e activated by N M D A to give 40 pS conductance channel openin PCP (5 p M ) is applied with N M D A , the probability ofchannel openings is +

72

M. S. P. SANSOM A N D P. N. R. USHERWOOD

reduced and there is a concomitant reduction in channel lifetime (Romoa and Albuquerque, 1989). Reductions in channel open probability and channel open time were also seen by Bertolini et al., ( 1 988) when they applied PCP to patches excised from rat cerebellar and cortical neurones in culture, although with 2 p M PCP the changes were restricted to a reduction in channel open probability. According to Romoa and Albuquerque (1989), the blocking effects of PCP are voltage dependent, being relieved at positive membrane potentials. It follows from these studies that PCP binds to both the closed and open states of the NMDA-R channel.

E. GLYCINE MODULATION OF NMDA-R ACTIVITY

Johnson and Ascher (1987) discovered that low (1 p M ) concentrations of glycine selectively increase (by about 25-fold) the opening rate of channels gated by NMDA-R in outside-out membrane patches isolated from rat embryonic CNS neurons in culture and activated by NMDA (10 p M ) . Glycine does not affect the open channel conductance or the mean open time. T h e employment of outside-out patches and the speed of the glycine effect led these authors to exclude the involvement of secondary messengers in the response to glycine. Instead they suggested that glycine binds directly to a site on the NMDA-R that is distant from the NMDA binding site and from which glycine allosterically potentiates the effects of NMDA binding. As an alternative explanation, they proposed that glycine might bind to a separate protein that interacts within the membrane with the NMDA-R. They showed that D-serine is almost as effective as glycine in potentiating the response to NMDA, but that L-serine is much less effective. T h e subsequent discovery, using biochemical techniques, that glycine allosterically regulates an NMDA-R-coupled ion channel of rat hippocampal membranes (Bonhas et al., 1987) lends support to Johnson and Ascher’s first proposal. Forsythe and Westbrook (1988) have suggested that in vivo glycine released from glial cells modulates the responsiveness of NMDA-R. What then is the basis of this modulation? Mayer et al. (1989) suggest that glycine regulates desensitization of NMDA-R by enhancing the desensitization recovery rate and that this is the reason for the apparent increase in opening rate seen with glycine. This idea should be readily testable at the single-channel level. Glycine has no effect on QUIS-R and KAIN-R.

SINGLE-CHANNEL. (;LLI'l AhlAI'E K E ( X P T 0 K S

73

F. DESENSITIZATION OF MAMMALIAN GLUTAMATE RECEPTORS Using a technique for fast application of amino acids to hipporampal neurons in dissociated culture, Mayer and Vyklicky (1989) have shown that responses to L-kainate exhibit little desensitization, responses to NMDA desensitize relatively slowly with a time constant of -250 msec, whereas responses to L-quisqualate desensitize rapidly with a time constant of 10-20 msec or less. Although there have been many comments about the possible contributions of desensitization of these receptor subtypes to single-channel recordings only a few articles have been published so far that have addressed the question of vertebrate GluK desensitization at the level of the single channel. Hatt et al. (1989) studied the desensitization of channels activated by glutamate and quisqualate in outside-out patches excised from freshly dissociated spinal cord a-motoneurons of chickens. The open channel conductance was 90 pS (between - 100 and -40 mV), a high value which approaches that of the quisqualate-sensitive D-GluR of insect leg muscle (Patlak et al., 1979). According to Hatt ct al. (1989), desensitization of this GluR is very rapid, with an onset time constant of 5 msec. Trussell and Fischbach (1989) also used patches excised from chick spinal neurons to study GluR desensitization. Earlier studies by Trussell et al. (1988) using whole-cell recordings had raised the possibility that desensitization of CluR might terminate the activity of endogenously released transmitter at some glutamatergic synapses, and this was supported by the subsequent single-channel studies. Trussell and Fischbach (1989) found that currents activated by 1 mM L-glutamate, desensitized in t w o phases, with the faster time constant of decay being similar to the time constant of decay of tniniature excitatory synaptic currents recorded from chick spinal neurons. By using holding potentials of -50 to -60 mV and Mg-containing saline, they eliminated NMDA-activated channels from their recordings to obtain a supposed QuisiKain channel of 18 pS open channel conductance. NMDA-gated channels in this system have an open channel conductance of -50 pS (Zorumski and Yang, 1988). T h e time constant for desensitization decreased as the L-glutamate concentration was raised, and with 1 mM agonist the decay of the L-glutamate-induced current was biphasic with time constants of 2.7 and 12.3 msec. Receptor inactivation occurred at a lower concentration of agonist than receptor activation, which raises the possibility that desensitization can proceed either via the unliganded receptor or via the liganded closed channel state(s). The kinetics of recovery from desensitization was less clearly identified in their studies, although with 1 miM

L-glutamate the rate of recovery was inversely proportional to the period of application of agonist. Kapidly desensitizing glutamate receptors have also been identified in rat hippocampal neurons in culture (FIanget al., 1989). This involved quisqualate-activated channels with a conductance of -35 pS, although a minority of channel openings had conductances which were higher o r lower than this. Whvn an outside-out patch containing a large number o f channels was exposed to a rapid step application of L-quisqualate, the current decayed w i t h a time constant of 3 msec. Tang et (11. (1989) again raised the possibility that rapid desensitization of' Quis-R influences the decay of the synaptic current occurring at glutamatergic synapses in the mammalian central nervous system. Inhibition by Con A was investigated by several researchers. Desensitization of L-glutamate receptoi-s on insect (Mathers and Usherwood, 1976, 1978; Evans and Usherwood, 1985) and crustacean niuscle fibers (Shinozaki and Ishida, 1979) is completely blocked by pretreatment with Con A. Comparative studies involving a variety of lectins led Evans and Usherwood (1985) to conclude that this inhibition may result from crosslinking or aggregation of the quisqualate-sensitive receptors on these muscle tibcrs. M a y and Vyklicky ( 1 989) have shown that desensitization of responses of'nioiise hippxzmipal neurons in culture during application of L-quisqualate is inhibited by prior treatment of the cells with Con A. Succinyl Con A was less effective than Con A in reducing desensitization, suggesting that, as for the insect muscle quisqualate receptor (Evans and Usherwooti, 1985), a tetrameric form of the lectin is necessary for the inhibition. Desensitization of locust muscle glutamate receptors is not inhibited by C o ~ A i if the lectin is applied together with agonist. This requirement for preapplication of lectin has not yet been tested for the vertebrate QUIS-K. Con A also inhibits desensitization of QUIS-R of isolated catfish cone horizontal cells (O'Dell and Christensen, 1989).

111. Channels Gated by Invertebrate Glutamate Receptors

O ur survey of arthropod muscle GluKs is restricted to single-channel studies. Although spectral analysis of glutamate current noise has, in the past, yielded important information on these GluKs (Anderson et nl., 1978) (Table V), the advent of single-channel recordings has highlighted the limitations of such analysis. As w e pointed out for vertebrate GluR,

.I'ABLE V S U M M A R Y OF

Species a n d agonist

NOISEANALYSIS STI'L)IES O F

~NVEK'IEBRAI'E(;LLTAMA-IE

KECLPIO K S

V,,,

'l'em p

Rantlwidt ti

(niC')

(%)

(W

100 -110 - I10

20 2 :I 23

1-1000 I -.iOO

1-500

2 2

70

24

1 - 100

:?I

8

1 - I000 1 - I000

4 4

'1.1

I-L'500

~5

4, (msec)

R (pS)

4.0 1.7 4.0

110 1 I2

9.3

-

0.9 9.3

32

-(iO

x

10

-150

1.2

-

-

References"

Locust

M EJ C" Glu Qui Sa rrophaga MEJC Ct-ayfish Glu Qui

hlaia Glu

-

~

~

R.

I

Key to references: 1. Cull-Candy a i d Miledi (1982);2. Audersoii rt 01. (1978):3 . Vyskocil rt ul. (1985); 4. Stettmeier ~t 01. (1983a,b);3. <:rawfot-tl a n d McUurney (1976). MEJC, miniature excitator): junrtiorial current. Room temperature.

'

the presence of multiple CJuR types and the complex gating kinetics of GluR should lead one to exercise caution when interpreting the results of noise analysis. An overview of single-channel results for invertebrate GluK is presented in Table VI. Note that, with one possible exception, desensitization of these receptors is blocked 1)y Con A (Mathers and Usherwood, 1976, 1978). This has been of crucial importance in, for example, permitting detailed studies of locust D-GILIK gating kinetics. [We have already noted that Mayer anti Vyklicky ( 1989) have recently reported that concanavalin A also blocks desensitization of Quis-R of mouse hippocampal neurones.] It can be seen from Table V l that both D a n d H-GluR have been studied most intensively in two invertebrate species: an insect (the locust) and ;I crustacean (the crayfish). Although there are broad similarities lietween the respective GluR in thcse two animals, there are also important differences. Therefore, we will concentrate on the properties of the GluR from these anitnals. while nlaking comparisons with other invertebrate GluR for which single-channel data are available. The results of patch clamp studies of receptorchannel ion selectivity, gating kinetics, and pharrnacologv will be tliscussed.

76

M.S. P. SANSOM AND P. N. R. USHERWOOD

TABLE V I SUMMARY OF ARTHROPOD GLUTAMATE RECEPTORS STUDIED Species and tissue

D or H?

Locust Adult muscle Embryonic Muscle Adult Muscle Tenebrio Muscle Periplaneta Muscle Neuron Drosophila Muscle Crayfish Muscle

SINGLE-CHANNEL RECORDINC~

s

Permeant ion

(PS)

Agonists

Con A effect? Referencesb

M+ M+

130 120

Quis > Glu > Cys Glu

Yes Yes

2

CI-

130

Glu Ibo > Asp

No

3

M+

15 (larva) 30 (imago)

Quis > Glu > Kai

No

4

M+ M+

140 20, 35, 50 60

Glu Glu

Yes Yes

5 6

M+

104

GluR

No

12

100

Quis > Glu (+ Ca'+) Quis > Glu GABA, PGP, Gly ACh, CbCh DHAVM

Yes

7, 8, 9

No

10, 1 1

M+

(& substates)

Muscle

USING

Cl-

22, 43,68

1

a Abbreviations: M + , monovalent cation; BGP, P-guanido-proprionic acid; CbCh, Carbachol; DHAVM, dihydroavermectin. * Key to references: 1. Gration and Usherwood (1980);2. Duce and Usherwood (1986); 3. Dudel et al. (1989b);4. Saito and Kawai (1987); 5. Bermudez and Beadle (1988); 6. Horseman et al. (1988); 7. Franke et al. (1986a); 8. Finger and Pareto (1987); 9. Finger Pt al. (1988a,b); 10. Franke et al. (1986b); 11. Z U f d l l e t d. (1988, 1989).

A. D-GluR 1. Locust Muscle

Single-channel recordings from locust muscle D-GluR were first made soon after the development of the patch-clamp technique (Patlak et al., 1979; Cull-Candy et al., 1981) (see Table 11). The majority of recordings have been from leg muscle, with just a few from stomach muscle. These recordings were made from extrajunctional D-GluR, the results of noise analysis studies having already indicated a close similarity between the properties of extrajunctional and postjunctional D-GluR channels (Anderson et al., 1978). A megaohm seal patch clamp technique was used, such that the electrical resistance of the seal between the membrane and the patch electrode is of the order of 20 M a . This technique has the advantage of allowing recordings to be made without the need to pretreat the membrane with cocktails of proteolytic enzymes. Although the rela-

SINGLE-CHANNE.1. GLti T A M A l k RECEPTORS

77

tively weak seal results in a high level of background electrical noise, the high conductance of the D-GluR makes possible detailed studies of channel gating kinetics. Early studies established t.he fundamental properties of the locust D-GluR. T h e channel was shown to be permeable to monovalent cations with a relatively high conductance (- 130 pS). Desensitization was shown at the single-channel level to be blocked by concanavalin A, thus making it possible to obtain long, stationary, single-channel recordings suitable for kinetic analysis over a wide range of agonist concentrations. The gating kinetics of the D-GluR were, from the start, shown to be quite complex. In particular, Patlak et al. (1979) noted that the D-GluR “state-switches” between different levels of activity within a single recording. T h e agonist pharmacology of the L)-GluR was investigated, confirming that L-quisqualate was a more potent agonist than L-glutamate. Furthermore, differences in agonist potency were shown to lie in differences in gating kinetics rather than in opening of channels of different conductances (Clark et al., 1979; Gration and Usherwood, 1980). Other studies have confirmed and extended these results, particularly in the areas of channel gating kinetics and of receptor-channel pharmacology. Furthermore, the megaohm studies have been supplemented by application of the gigaohrn technique (in which a higher resistance seal gives a lower background noise level) to embryonic muscle in tissue culture and to collagenase-treated adult muscle. T h e results of these studies will now be discussed in some detail. a. Zon Selectivity. Kits and Usherwood (1988) measured D-GluR channel conductances for different monovalent cations. T h e selectivity sequence thus derived was Rb+ > K’ -- Cs’ > Na+ > Li+, which corresponds to Eisenman sequence II/III (Eisenman and Horn, 1983), suggesting that the conductance of the channel is determined primarily by the dehydration energy of the ion and that the ion interacts with a relatively weak anionic electrostatic field. T h e channel conductance in the presence of a range of organic cations was also measured. Ammonium was shown to be permeable, whereas guanidiniuni, tetramethylammonium, and choline ions were impermeable. Turning to divalent cations, it was also noted that high Mg2’ or Cay+ concentrations appeared to block the ion channel. T h e latter proposal is supported by the earlier observations of Cull-Candy and Miledi (1982) who, on the basis of noise analysis and miniature excitatory junctional current (MEJC) decay measurements, proposed that Cay+ reduces the apparent lifetime of the D-G~uRchannel. Overall, these studies demonstrate that the high conductance of the

78

M.S. P. SANSOM A N D P. N.K.USHERWOOD

locust D-GluR is obtained via a restricted ion selectivity based on the charge and size of the permeating ion, with the dehydration energy being the dominant factor in monovalent cation selectivity. 6. Channel Gating Kinetics. Substantial progress in understanding the gating kinetics of the locust D-GluR has been made possible by using concanavalin A to block receptor desensitization. This has made it possible to focus on receptor-channel activation by agonists. In particular, it has made it experimentally feasible to obtain extended single-channel recordings in the presence of high agonist concentrations. The initial single-channel recordings from D-GluRs established the essential features of the channel gating kinetics (Patlak et al., 1979). T h e channel kinetics were seen to be complex, switching back and forth between different kinetic modes within the same recording on a timescale of several hundred milliseconds (Fig. 2). Analysis of open time distributions for data recorded in the presence of lop4M glutamate yielded a two exponential fit, with time constants 5-1 = 1.8 msec and 5-2 = 12.3 msec. The closed time distribution was also fitted by two components (5-1 = 1.6 msec and 72 = 16.3 msec). Thus, these initial studies indicated that No 2 2 and N,2 2 and that the gating mechanism must be capable of explaining the slow state-switching of the channel kinetics. Subsequent studies (Gration et ul., 198 la) explored the effect of glutamate concentration on channel kinetics, indicating that the mean open time of the channel increased with increasing glutamate concentration. T h e result was in apparent conflict with the observations of Cull-Candy et al. (1981), but careful examination of the latter work reveals that the analysis was restricted to a single “normal” mode of the activity of the state-switching channel. Independent studies (Gration et al., 198 la,b; Cull-Candy P t al., 198 1 ; Cull-Candy and Parker, 1983) demonstrated that

t--i

20

ms

. G . 2. An example of “state-switching” of the locust muscle D-GluK. This recording w x made using a gigaohni seal on collagenase-treated muscle. The patch was exposed to concana\ alin A to block desensitization, and was perf used with lo-” M 1.-glutamate. The menibrane potential was - 100 m V . Arrows indicate the approximate position of switches between state I (predominantly closed, with hrief openings) and state 11 (predominantly open, with brief closings).

the niean channel closed tinit. ( t , ) tlcci-eased w i t h iiicreasiitg glutamate concentration in approximately the l'ollowing niaiiner 1,-'

Ly

in]^

indicating that more than one glutaiiiate inolecule tiiricls t o eacli DGILIK in o r d e r to open the clianiiel. F r o m such analyses K ; for glut aniatc binding was estimated to be ;iIx)ut 5 x 1 0 - '2.I. 'The kinetics studies ttesci.il)etl al~ovewere made usiiig a litter c-ut-olf' fi-equency of 1 kHz, with tlic result that brief chatiiiel opeiiitigs and closings (less than 1 nisec) corrld riot he resolved. Other inwatigiitioiis of GIuR kinetics have eniplo)cd ii Iiiglier time resolution ( 3 k k l / cutof'l frequency) a n d resolved all siiigle channel openings of' duration greater than 180 psec (Ashford rt ol., I!)X4a,h; Kerry ~f ( I / . , 1987it, 1 9 M L i ) . ~ l ' h c \ , have also been based upon slatisticiil analysis of several tens of t hous;inds of channel openings, which t~i;tkespossible inore reliaMe detcctioti of'lcss frequent kinetic states and heiice permits it iiiore extensive cliai.actei-ization o f the channel gating niecliaiiisni. State-switching of L)-C;lu R activity was analysed in tei-ins of I tic c-lustering in time of channel opeiiiiigs. (;i-ation ot a/. ( 19H 1 a ; 4shfOt-tl ('1 (11.. 1984a,b) had pointed out that the observed degree of clustci-iiig was incompatible with a simple closed open gating ~nechaiiisiii.A more detailed analysis (Ball et 01.. 1Cl8.5; 13all and Sansoni, 198'7) suggested that the obsei-ved degree of clustci-irig W;IS explicable in ternis of ;I 1)railclletl or cyclic gating model of t he gating niechanism. Furthei e\.itl(wce for such a model arose from kinetic atialysis (Kei ('I Cll., 1 IIX7a) o f '44.000 channel openings recoi-ticti i i i r h e pi-eseencc of' 1 iZI g1utaiii;ite. The open time distribution was littetl b y ,V,, = 3 exponentially tleca>,iiig(winponents, with time constants 71 = 0.40 nisec, T:, = 1.15 nisec. a i i d 7.5 = 3.40 iiisec. T h e closed time tlisti.il,rition was titted b y the suiii of .I=r4< components ( T ~= 0.40 nisec': T:, = '7.4 1 nisec; T:( = 20.0 iiisec. and 71 = 94.3 msec). Statistical analysis tleiiioiisti.ated that these fits did not v ~ I I - \ ~ significantly among the results from four different nienibi-ant: patches. T h e statistical analysis ot the state-switching behavior of' the l ) - G l u K was extended to include the evaluation of open time a n d closetl time autocorrelation functions (1;retlkiti ~t ul., 1985; Colquhoun anti H,rwkes, 1987; Ball a n d Sansoni, 1 YHH), ;IS l i n t employed by 1,abarc-aP I (11. ( 1985) to investigate t h e gating kinetics o f the Tor.j)pdo nictonic acetylcholiiie receptor. ~I'hismethod of analysis Im-mits one to distinguish betwecii lincaian d branched or cyclic gating niechanisms. More specifically, it allows one to determine the number 01' gateway states of'the gating mechanism. A gntewny state is a state which the channel leaves when it goes from closed to o p e n or from open to closed. So, for example, the mechanism

-

-

80

- - - -

M. S. P. SANSOM A N D P. N. R. USHERWOOD

c

CA

C A ~

O A ~

OA~*

(where OAp* indicates an altered conformational state of the open channel) has a single open gateway state (OA2)and a single closed gateway state (CAp). N o correlations between successive open times and between successive closed times would be seen for such a channel. By contrast, the gating mechanism C

- CA

1

OA

CAv

1-

OA:!

has two open gateway states (OA and OAB) and two closed gateway states (CA and CAB)and hence successive open times, and successive closed time, will be correlated. More generally, if the minimum of the number of open gateway states and of the number of closed states is N,, then the open time and closed time autocorrelation functions will each take the form of the sum of N , - 1 geometrically decaying components. Thus, by fitting observed autocorrelation functions a lower limit for N , may be obtained, and hence important clues as to the channel gating mechanism obtained. Evaluation and fitting of open and closed time autocorrelation functions from channel recordings obtained in the presence of 10-4 M glutamate yielded a lower limit to N , = 3 (Ashford et al., 1984a,b; Sansom and Usherwood, 1986; Kerry et al., 1987a). Alongside the estimates of N o 2 3 and N , 2 4 this suggested that the underlying gating mechanism must be of the form C1-

c:!

-c3

C4

Subsequent investigations (Kerry et al., 1988a) extended the analysis to single-channel recordings obtained over a range ( M ) of to L-glutamate concentrations. At four concentrations for which substantial lo-", and numbers of channel openings had been recorded 1O-' M ) autocorrelation function analysis supported the estimate of N , given above. At the two higher L-glutamate concentrations, analysis of the open time distributions revealed that N o 2 4.Hill plot analysis of the channel open probability dose-response curve yielded a Hill coefficient of TZH = 1.6. This demonstrated that two or more glutamate molecules must bind per receptor-channel complex, consistent with the earlier proposal of multiple binding sites for glutamate on the receptor. Analysis

SINCLE-CHAN K'EL G L L I AM A T E KECEFI'OKS

81

of cross-correlations between openings and successive closings (and vice versa) (Ball et al., 1988) demonstrated that long openings tended to be next to short closings (and vice versa), and also confirmed that the gating mechanism was at thermodynamic equilibrium. In the light of these results a preliminary model of the gating mechanism was proposed, based on the cooperative model of con formational transitions of proteins (Monod et al., 1965; Karlin, 1967). This model incorporated four identical binding sites for glutamate, with changes in the number of' bound agonist molecules producing state-switching behavior. Preliminary estimates of the equilibrium parameters of the model and of the closed to open transition rates were obtained by fitting the open channel probability and channel opening frequency dose-response curves, respectively. Studies by Bates et al. (1990) are directed at refinement and extension of the preliminary gating model, using the approach described by Ball and Sansom (1989). In passing, we note that a similar model has been suhsequently proposed for a GAHA receptor (MacDonald el nl., 1989) and by Blatz and Magleby (1989) for the fast C1- channel from rat skeletal muscle. Even higher time resolution data have been obtained using gigdohrn seals on collagenase-treated muscle (Bates et al., 1988). The channel kinetics obtained under these conditions, using cell-detached, outsideout patches, are very similar to those described above (Sansoni et al., 1989; Bates et al., 1990). Therefore, it is unlikely that the mechanistic complexity is either (1) an artifact arising from the limited frequency response of the megaohni technique; or (2) a result of intracellular modulations of channel gating activity. Therefore, it seems that the coniplex gating ofthe locust muscle D-GluK is a property of the receptor -channel complex per se. c. PharmacoloRy. Single-channel recording has been used to probe both the agonist and the antagonist-blocker pharmacology of the locust D-GluR. Studies of whole nerve-muscle preparations (Usherwood and Machili, 1968; Clements and May, 1974) had indicated that the D-GluR is activated by a relatively narrow range of agonists, and this has subsequently been confirmed by Boden et al. (1990) in a study that included a number of novel synthetic analogues of L-glutaniic acid. Briefly, L-quisqualate is a more potent agonist than L-glutamate, but N M D A and L-kainate are either inactive or weakly active (Daoud and Usherwood, 1978). L-Aspartate is a poor agonist. 'These results have been elaborated upon at the level of the single channel. Gration and Usherwood (1980) and Gration et al. (1981b, 1983) studied activation of D-GluR by Lquisqualate and by L-cysteine sulfinate, the latter being a less potent agonist than L-glutamate. These studies were extended to include L-4-

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M.S . l', SANSOh.1 .AKD P. K. K. I'SIHERWOOD

methylene glutarnate, D , ~-4-fluoroglutamate,L-cysteate, and r.-allo-4hydroxyl glutamate (Sansoni and Usherwood, 1986). Cull-Candy at al. (1981; Cull-Candy and Parker, 1983) also looked at L-quisqualate and, as a less potent agonist, chose fluoroglutamate (although it was not stated whether this was a single isomer). The principal outcome of these studies was that independent of the agonist employed the channel conductance stayed constant at -130 pS. Thus the differences in agonist potencies were shown to reside in the kinetics of channel gating, rather than in the conductivity properties of the open channel. I n terms of gating, Gratiori and Usherwood ( 1980) stressed that the kinetics remained complex, with state-switching still in evidence with the two agonists other than L-glutamate. Both studies pointed out that the mean channel open time was agonist-dependent for equiniolar agonist concentrations. Other work has concentrated upon interpretation of differences between agonist potencies in relation to models of D-GluK gating (Kerry et al., 1988b; Huddie et al., 1990). Hill plot analyses of channel open probabilities for quisqualate, glutamate, and cysteine sulfinate yielded K ; values of 7.7 x lop6 M , 1.4 x l o p 5 M , and 5.5 x 10-" M and Hill coefficients ( n H ) of 15, 1.6, and 1.5, respectively. Studies at approximately equipotent agonist concentrations confirmed the kinetic complexity first seen in the presence of lop4 M L-glutamate. In particular, biphasic ( N g - 1 = 2) open and closed time autocorrelation functions were seen for all three agonists. Thus, a branched or cyclic gating mechanism is required to account for the gating properties of the D-GluK in the presence of three different agonists. A model in which the agonists differed only in their affinity for the closed state of the receptor-channel complex has been shown to account for the experimental data (M.S.P. Sansom, unpublished results), although there remains the possibility that more complex differences may be found. A variety of noncompetitive antagonists of the D-GluR have been studied at the single-channel level (Fig. 3 ) . The majority of these have been shown to exert at least part of their effect via block of the open channel. Gration and Usherwood ( 1980) demonstrated open channel block by streptomycin. Ashford et al. (1987, 1988) showed open channel block by trimethaphan and by chlorisondamine. Kerry et al. (1985, 1987b) investigated open channel block by the classical nicotinic receptor antagonist tubocurarine. More recently (Ashford et al., 1989) have shown that part of the effect of ketamine on D-GluR is via open channel block. Chlorisondamine block is of interest in that it can be reversed by membrane hyperpolarization (Ashford et al., 1988), suggesting that the chlorisondamine molecule can be driven through the open channel. A similar phenomenon has been observed by Lingle ( 1989) studying chlorison-

damine block at a crustacean glutainatergic nerve-muscle .junction. Open channel block by tubocurarine was studied in sonie detail. Analysis of the single channel kinetics of Mock yielded a dissociation constant of 1.6 x lo-' M (at -lOOniV), with an association rate of 8.7 x 106 sec-'M-' for channel and blocker. This means that there is a quite long-lived association between the blocker molecule and the open channel (mean block time = 65 tnsec). Investigations of single channel antagonism have switched to the polyamine-like toxins (Jackson and Usherwood, 1988)of which Arg'rX636 a n d PhTX-433 (Fig. 3) are the best-studied examples. Long-lasting open channel block by Ph'l'X was denionstrated quite early o n using partially purified material by (:lark et al. (I982), recording f'rom leg muscle D-GluRs, and by Kirs p t ( I / . (1 Y84), recording from visceral muscle D-GluRs. Kerry et al. ( 1 9 8 8 ~have ) studied open channel block by purified ArgTX-636. T h e toxin is an extremely potent blocker, operative at concentrations as low as 10- " M . 'I-his has led to the suggestion (Usherwood, 1988) that the toxin may gain access to the 11-GluR via a mernt)ranebound phase. Usherwood and Blagborough ( 1989)have utilized the results of'these numerous studies of channel Mock to devise a minimal niodel for the ion channel structure of the I)-C;luR. As chlorisondamine (maximum dimension 0.98 nm) will pass through the channel when the membrane is hyperpolarized, a maximum channel dimension of 1 .O nm was proposed. On this basis it was suggested that hyperpolarization might also be capable of driving the Arg'l'X i l n d PhTX rnolecules through the channel, thus relieving block. Recent results (P.N.K. Usherwood, ~inpuhlished findings) confirm this suggestion. d . Desensitization. It is only recently that detailed single-channel studies of locust D-GluRs in the absence ofconcanavalin A (i.e. in the Pre.Fence of desensitization) have become possible. Gration el al. ( 1980a,b) showed that, in the absence of concanavalin A, brief periods of single-channel activity were interspersed with long silent periods of 30-600 sec duration. This work was important in demonstrating that, for example, concanavalin A did not alter the single-channel conductance. I t has been possible to obtain more extensive single-channel recordings from locust D-GluR in the absence of coricanavalin A by using the liquid filament technique devised by Franke rt nl. (1987). This enables one to expose a membrane patch to a brief pulse of glutamate and to record the resultant single-channel openings. These studies (Dudel et al., I988b) have employed gigaohm seals on collagenase-treated leg muscle and have confirmed the channel conductance and the agonist potencies derived from the megaohm seal studies described above. T h e rate of activation of

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SINGLE-CHANNEL GLUTAMATE RECEPTORS

D-GluR by the glutamate pulse has been shown to be high: The time to peak activity is -1 msec. Analysis of the distribution of lifetimes of channel elicited by a 200-msec pulse of L-glutamate gave time constants of 7 1 = 0.2 msec; 7 2 = 2.0 msec, and a third, longer component. These figures are remarkably similar to those obtained by multiexponential fitting of the open time distributions derived from recordings made in the presence of concanavalin A. They suggest that the kinetics of the open channel are not greatly perturbed by treatment with lectin. T h e time constant for onset of desensitization measured using the liquid filament technique was -25 msec. This is much faster than the macroscopic desensitization rate measured using repeated iontophoretic

E

NH

I F

YN

A,+ANJ H

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NHZ

FIG. 3. Open channel blockers of the locust muscle D-GluR: (A) streptomycin, (B) tubocurarine, (C) chlorisondamine, (D) trinietaphan, (E) PhTX-433, and (F) ArgTX-636.

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application of agonist (Daoud and Usherwood, 1978), which had an apparent time constant of 500-1000 msec. Liquid-filament studies by Dude1 et al. (1990a) indicate a marked patch-to-patch variation in desensitization and resensitization rates. There also appears to be a degree of nonstationarity in the desensitization/resensitization kinetics within a single patch (Ramsey and Usherwood, unpublished observations). Taken together with the difference between the macroscopic and single-channel desensitization rates, these results suggest that desensitization is a multicomponent process, possibly superimposed on kinetic state-switching similar to that seen in the equilibrium experiments. The liquid-filament studies also suggested that desensitization may occur from the closed state of the receptorchannel. In this respect the locust D-GluR seems to resemble that from the crayfish (see below). e. Embryonic Locust Muscle. Single-channel recordings of D-GluR have also been made using embryonic myofibers in tissue culture (Duce and Usherwood, 1986; Duce et al., 1988), on which gigaohm seals could be formed without collagenase treatment. As in the case of the adult muscle, desensitization was blocked by concanavalin A. The channel gating kinetics appeared to be complex, with evidence for multiple open states of the channel. The sensitivity to glutamate of the embryonic D-GluR was about 1000-fold higher than that of the adult receptor, a significant level of channel activity being observed in the presence of lo-* M glutamate. The embryonic D-GluR also seems to have increased sensitivity to block by Ca‘+ ions. Whereas Cay+concentrations in excess of 10 mM are required to block the adult D-GluR (Kits and Usherwood, 1988),significant block of embryonic D-GluK (with concomitant reduction of the apparent conductance to -60 pS) is seen in the presence of 1 mM Ca’+. This block is of the “fast, flickery” type, hence the apparent conductance decrease. At Ca2+ concentrations of 10 p M or less, there is no block and the conductance of the embryonic channel is 120 pS (i.e., close to that of the adult).

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2. Other Insect D-GluRs Patch clamp studies of D-GluRs have been conducted in at least four other systems: Periplaneta muscle in tissue culture (Bermudez and Beadle, 1988), Tenebrio muscle (Saito and Kawai, 1987),Drosophzla muscle (Delgado et al., 1989), and Peraplaneta neurons (Horseman et al., 1988). A11 three systems have glutdmate-activated channels with currents which reverse at -0 mV and hence are permeable to monovalent cations. The D-GluR of cultured Periplaneta myosacs show marked similarities

to those of adult locust niuscle. .The single-channel c.oricluct,iiice is 140 pS. Concanavalin A Ihcks receptor-channel desensitimtion. M glut;miate, the mean channel open tinie (0.5 In the presence of msec) is similar to that for adult locust muscle D-GluK ( 1 .:!I msec). L1nf'ortunately, a more detailed charac-terization of PPt.ipl(/twto channel kinetics is not available. T h e Tenebrio D-GluK is I , I I Iicr dif'ferent in its properties. which depend upon the developmental stage (larval versus imaginal) o f the insect. In both cases the single channel conductance (15 anti 30 pS. respectively) is considerably lower than that of the locust D-GluK. However-,given the effect of Cay+on the embryonic locust muscle D-GluK, it should be nored that the Teriebrio recordings were made in the presence of 2 nutl C a y + ions. A further difference is that concanavalin A does not appear t o block desensitization of the T e n ~ b ~ receptor-channel. io However, the pharmacological profile seems to be similar to that of the locust, with L-quisqualate a more potent agonist than L-glutamate. T h e Drosophila D-GluK, studied i n larval muscle fibers, is activated by glutamate in the concentration range 5 x to I2 x l o p 2 M . I t has a single-channel conductance o f I 04 pS, similar to locust and Prriplmrtn muscle, but it seems to be unresponsive to concanavalin A . Preliminary kinetic analysis indicates the existence of at least two open states of the receptor-channel. Periplaneta tissue culture has also been used to look at the properties of neuronal D-GluR in insects. l'hese show some similarities to the adult locust muscle receptors in that desensitization is blocked by concanavalin A, and glutamate concentrations of M or more are required to obtain a reasonably high level of channel activity. T h e principal cfifference from the locust muscle D-GluK lies in the channel conductances. The neuronal D-GluK shows multiple conductance levels of -20, 3 5 , 50, and 60 pS. Further investigations are required to probe this difference between insect neuronal arid insect muscle D-CluK.

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3. Crayfish Muscle T h e D-GluK of crayfish (Astarus astarw and Au~tropotamobzoustorreritzurn) muscle have been extensively studied, both using noise analy4s (Stettmeier et al., 1983a,b; Finger, 1983; Stettnieier and Finger, 1983), and using single-channel recording (Franke et al., 1983). The singlechannel conductance is relatively high (- 100 pS), and there have been several reports of the presence of subconductance levels. The agonist pharmacology is similar to that of the locust, with quisqualate a more potent agonist than glutamate. Concanavalin A blocks desensitization. Single-channel recordings have been made using megaohm seals, and

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also using gigaohm seals after collagenase treatment of the muscle. As a result of these studies, a fairly detailed picture of the biophysics of the crayfish D-GluR has emerged. a. Single-Channel Conductances. T h e situation with respect to the single-channel conductance of the crayfish D-GluR is a little confusing. T h e main conductance level is -80-100 pS. This has been demonstrated to be so for five different muscles of the crayfish by Franke et al. (1986a). T h e problem arises with respect to subconductance levels and/or lower conductance channels gated by D-GluK. Franke and Dudel (1985) reported the existence of three sublevels (at -30,55, and 75 ps) in addition to the main conductance of 100 pS. T h e frequency of occurrence of the lower conductance openings was low but appeared to be somewhat higher at low L-glutamate concentrations. In a later paper (Franke and Dudel, 1987) it is suggested that the lower conductance openings are more frequent after extensive collagenase treatment of muscle followed by up to 3-hr-long experimental periods. Finger and co-workers (Finger and Pareto, 1987; Finger et al., 1988a,b) have also identified subconductances of the crayfish D-GluR at -7, 13, 25, and 35 pS. They report that the frequency of occurrence of the lower-conductance channels is dependent on the age of the crayfish and on the muscle used for recording. However, there has been some debate in the literature over the technical aspects ofthis work (Dudel et al., 1989b). T o attempt to summarize a complex situation, it is clear that in addition to the main conductance of 100 pS, lower-conductance channels activated by glutamate may be observed. It remains to be established conclusively whether these are sublevels of the 100-pS channel and/or whether they represent a population of lower-conductance D-GluR channels. Also, it remains to be established whether subconductance states occur in uivo and are not artifacts resulting from collagenase treatment and patching (see Franke and Dudel, 1987; Dudel and Franke, 1987). 6. Zon Selectivity. T h e ion selectivity of the main conductance level has been studied in detail by Hatt et al. (1988,) who calculated ionic permeabilities based on the constant field equation from measurements of single-channel conductances in solutions of different ionic compositions. T h e selectivity sequence arrived at was

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This corresponds most closely to Eisenmann sequences IX and X (Eisenman and Horn, 1983).This would indicate that the ionic selectivity of the crayfish D-GluR reflects interaction of the permeant cation with a high

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anionic field strength, in contrast to the case for the locust channel. Furthermore, choline, wich appeared to be impermeant through the locust channel, had a low but measurable permeability through the crayfish channel. With respect to divalent cations, Cay+ and M g " d o not seem to block the crayfish channel, a further difference from the locust. Thus, although the crayfish and locust D-GluR are similar in that they are permeable to a range of cations, the details of their ion selectivity properties show significant differences. c. Gating Kinetics. T h e gating kinetics of crayfish D-GluK (or rather of the 100-pS conductance/channel) have been studied in some considerable detail. T h e majority of the recordings have been made in the gigaohm configuration, using collagenase-treated muscle. A detailed study of the effects of collagenase on channel kinetics has been made by Franke and Dudel (1987). No major effect of collagenase was found, other than a two- to fivefold increase in the concentration of L-glutamate required to elicit single channel openings. I t is interesting to note that a similar decrease in glutamate sensitivity i n response to collagenase treatment has been noted with the locust D-GluK (Hates P t al., 1990). Crayfish D-GluR kinetics have been studied primarily in the absence of concanavalin A and consequently have focused on desensitized receptor-channels. This makes a detailed comparison with the kinetics of the locust channel rather difficult, although some striking similarities between the two systems do emerge. Note that either the overall level of desensitization must be lower and/or the channel density higher t o account for the level of desensitized single-channel activity for the crayfish relative to that for the locust. Franke and Dudel ( 1985) analysed burst time distributions at several L-glutamate concentrations. 1 t i the presence o f 2 x 1W4M glutamate the burst time distribution was fitted with the sum of two exponentials ( 7 , = 0.2 msec; 7 2 = 0.4 msec), although the observed distribution presented gave indications of a third, longer component (and such a component was reported in Franke and Dudel, 1984). Interestingly, analysis of bursts of openings elicited by 5 X 10-" M L-quisqualate (Franke and Dudel, 1984) in the presence of concanavalin A yielded a three-component fit, with time constants of (approximately) 0.1, 1 .O, and 5.0 msec. In the presence of 5 x lo-' M L-glutamate the burst durations were lengthened, with time constants of 71 = 0.4 msec; and 7 2 = 1.8 msec. More extensive investigations of the L-glutamate concentration dependence of channel kinetics (Dudel and Franke, 1987) suggested that the mean burst duration increased with increasing glutamate concentration. T h e mean open time also appeared to increase, but this was not statistically significant.

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The closed time distribution required three or four components for a satisfactory fit, although the significance of this is lessened because desensitization prevents one from knowing how many channels are simultaneously present within the membrane patch. The channel kinetics were interpreted in terms of a two-site model, based on that used for the nAChR (see, e.g., Ogden ~t ul., 1987). T h e two agonist binding sites are assumed to be identical. It is assumed that only the biliganded form ofthe receptor-channel can open. C +CA

4

CA2

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OA2

However, Dudel and Franke (1987) stress that this is a simplification of the observed kinetics and does not explain desensitization or, more important, the increased burst durations at higher glutamate concentrations. An important difference between the locust and crayfish D-GluR is that opening of the latter shows a strong Ca2+ dependence (Hatt el al., 1988b). Reduction of extracellular Ca‘+ ion concentration from 13.5 mh4 to 1 m M produces a marked decrease in both the duration and the frequency of bursts of openings. Further lowering of the external Ca2+concentration results in an almost complete loss of channel openings. The Ca2+ effect acts on the extracellular face of the membrane, and the concentration dependence suggests that two molecules of Ca‘+ must bind per receptor-channel molecule. High concentrations of Mg2+ or Ba2+ can substitute for Ca2+. Inorganic Ca2+ “blockers” (La”+, Cd‘+, Co2+,or Ni2+) produce the same effect as a low Ca2+ concentration. Organic Ca2+antagonists (e.g., nifedipine) have no effect. Experiments using transient pulses of L-glutamate (see below) indicate that the low Ca2+ effect is not mediated via an increase in desensitization. So, Ca2+ seems to be a “co-agonist” with L-glutamate in the gating of the crayfish D-GluR. d. Pharmacology. The single-channel pharmacology of the crayfish D-GluR has not been explored to the same extent as that of the locust. In particular, we lack single-channel information on a broad range of open channel blockers. This is unfortunate, as such data would be of considerable interest given the indications from ion selectivity studies of differences in the open channel properties of the two systems. With respect to the agonist pharmacology, it is well established that the crayfish D-GluR is activated by L-quisqualate but not by L-kainate or NMDA, even when the latter are present at millimolar concentrations. Franke et al. (1986a) have shown that there is an approximately 10-fold difference in the sensitivity to L-quisqualate and to L-glutamate, with the

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former being the more potent agonist. lfeyuimolar concentrations ofthe two are compared, then L-quisqualate induced channel bursts are about four times longer than those induced by L-glutamate, and individual openings are about three times longer- in the former case. Interpretation in terms of the two-site gating model (see above) suggested that the K,l for L-quisqualate is about half and the OA2 CA2 transition rate about a third of those for L-glutamate. Thus L-quisqualate is proposed to have a higher affinity of the receptor, arid also a higher efficacy in promoting openings of the biliganded receptor-channel. However, one should bear in mind the preliminary nature o f the gating model upon which this interpretation is based. Finger et al. (1988a) have compared the effects of 1,-quisqualate and L-glutamate in small (1-3 months old) and large (< 16 months old) crayfish. T h e results suggest that there may be developmental changes in the relative sensitivity of the D-GluK to quisqualate and glutamate, with a more marked difference in mean burst times in the younger animals. This is of interest given the observations of developmental changes in the properties of D-GluR in locust and in Tenebrio muscle (see above). Single-channel studies of block of the crayfish D-GluR have been limited. Antonov et al. (1989) have examined open channel block by ArgTX-636. This occured at concentrations similar to those required fotblock of the locust channel (i.e., 10-“ M. As for the locust system, hyperpolarization of the membrane seemed to reverse the block of GluR by arthropod polyamine toxins in the crayfish. e. Desensitization. There are three questions one might ask about desensitization: (1) what is the rate of onset?; (2) what are the properties of the desensitized state?; and (3) what is the rate of recovery from the desensitized state? Single-channel studies of the crayfish D-C~LIK have provide a fairly complete answer-to the first question and partial answers to the second and third. Macroscopic measurement (Takeuchi and ‘I’akeuchi. 1964; Dudel, 1977) suggest that the onset rate of desensitization is <1 sec. Singlechannel measurements have been possible as a result of the development of the liquid filament technique (Franke P t al., 1987). This permits rapid (<1 msec) application of a pulse of glutamate to an excised outsicle-out membrane patch containing one or more D-GluR. The process can be repeated many times and an ensemble average (Aldrich and Yellen, 1983) response of the channel to a stepwise change is agonist concentration evaluated. In this way (Dude1 et ul., 1988a) it has been possible to show that the channel reaches peak activation within 200-500 psec of exposure to agonist, the exact time depending upon agonist concentra-

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tion. During a sustained pulse of L-glutamate, the average channel response then declines, with a time constant of -5 msec, to a much lower equilibrium (desensitized) level. T h e rate of decline is said, therefore, to give the onset rate of desensitization. T h e equilibrium level of activity is dependent on the glutamate concentration during the pulse, while the time constant for decline remains fixed at -5 msec. Although the macroscopic process of desensitization is believed to involve additional, slower stages, it is of interest that the rate of decay of the ensemble average current is comparable to that of quanta1 postsynaptic currents measured in the same system with a macroscopic patch clamp. With regards to comparisons between the equilibrium properties of the desensitized and concanavalin A-treated channels, only limited studies of the latter have been reported (Franke and Dudel, 1984). It seems that desensitization reduces the burst duration of the channel by reducing the mean number of openings per burst, while leaving the mean lifetime of single openings unchanged. This would be consistent with a scheme whereby desensitization was entered from the bursting (i.e.,biliganded) state of the receptorchannel. More recent liquid-filament studies (Dudel et al., 1990b,c) suggest that desensitization occurs via a closed state(s) of the receptor-channel, as application of a slowly rising glutamate concentration ramp can result in channel desensitization zuithout any preceding channel openings. However, detailed interpretation of the liquid-filament studies has been complicated by the revelation of considerable heterogeneity in the kinetics of desensitization onset and recovery. Dudel and co-workers classify crayfish D-GluR into four subtypes on the basis of differences in desensitization kinetics, and speculate that these subtypes may differ at the level of gene expression or of post-translational modification. However, it should be noted that the proposed subtypes are of identical singlechannel conductance, and have very similar intraburst kinetics. Furthermore, subtype interconversions are sometimes seen within an experiment. It remains possible, therefore, that the subtypes represent slow switching of the same channel between different levels of susceptibility to desensitization, as a result of, for example, phosphorylation/ dephosphorylation reactions. It is clear that further investigations, possibly linking biophysical with biochemical and molecular biological approaches, will be required to characterize the molecular events underlying desensitization.

3. Comparison of Locust and Crayfish D-GluRs There are now sufficient data from single-channel studies to permit detailed comparison of the biophysics of the locust and crayfish D-GluR.

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Why make such a comparison? T h e features held in common are likely to be general properties of invertebrate D-GluR. Let us start with the similarities. Both channels have a mean conductance of -100 pS and weakly selective for monovalent cations. Both receptors are activated by glutamate in the concentration range M and have mean open times of the order of 1 msec or less. L-Quisqualate is in both cases a more potent agonist than i*-glutarnate, with NMDA and L-kainate inactive. Finally, the desensitization of both systems is blocked by concanavalin A. N o w let us examine the differences. Whereas the locust channel shows a single conductance of -130 pS, the crayfish channel seems to have at least three additional (sub)conductance levels. However, it should be noted that occasional subconductances have been seen for locust D-GluR when recording from collagenase-treated muscles, although further studies are required more fully to characterize their properties (P. N. R. Usherwood, unpublished observations). The detailed ion selectivity properties of the two channels differ: The locust channel seems to have a weak field strength selectivity filter, whereas the crayfish selectivity filter generates a high field strength. Furthermore, the locust channel seems to have a lower cut-off radius, as choline is impermeant, whereas it has a low, but measurable, permeability through the crayfish channel. However, this niay lead us to a too static view of the channel dimensions, given the ability of chlorisondaniine and of ArgTX-636 and PhTX 343 to “squeeze” through the locust channel when the membrane is hyperpolarized. Also, the locust channel differs from the crayfish in that the former is blocked by raised concentrations of divalent cations. T h e differences are less clear-cut when one turns to channel gating. T h e gating process of the locust D-GluR seems to be more complex. Current (in both cases preliminary) models of D-GluR gating suggest that two agonist molecules bind to each crayfish receptor-channel complex, whereas up to four may bind to that of the locust. Also, only the crayfish receptor requires Ca‘+ as a “co-agonist.” Both the locust and the crayfish D-GluR show complex desensitization/resensitization kinetics. We can attempt to rationalize these similarities and d tween the two D-GluR in terms o f a simple model for their structure (Fig. 4). Clearly, in the absence of biochemical or molecular biological data, such models are speculative. We propose that both D-GluK have a tetrameric subunit structure, by analogy with that proposed for the vertebrate GABAA-R (Barnard et al., 1987). T h e locust D-GluR has four equivalent subunits. (It is interesting to note that Marshall et al. (1988) have suggested that a single subunit type may be capable of forming functional nicotinic acetylcholine receptors encoded by mRNA from locust neu-

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rons.) T h e r e is a n agonist biridiiig site on each subunit of the G l u K b u t a single gating event correspoiitling t o a concerted con formational cliangr of all four subunits. T h e craylisli D-GluK has two agonist binding subunits, but four gating events, cor~-esl~~)ridingig to sequential conforrnational changes of all four subunits. -1’hus a difference in the degree of coupling between the conformational changes of the subunits could generate the difference between the single conductance of the locust channel, and the subconductances of‘thecraylish channel. in addition to going some way to explaining the ohserved differences in gating kinetics.

B. H-GluK 1. Locust Muscle

T h e H-GluK of locust niuscle has been studied at the single-channel level using the liquid filament technique for application of pulses of glutamate a n d other agonists (1)utiel rt al., 1989a). H-GluK chanriels of conductance 25 pS were seen when a high (160 m M ) concentration of C;lion was present and reversed at potentials consistent with a C1- selective channel. Only the main conductance level was seen. ’The H-GluR pharmacology was probed in some detail. Channels M) were activated by L-glutamate, by n,t.-ibotenate, and by high ( concentrations of L-aspartate. N o openings were seen in response to L-quisqualate, NMDA, kainate, GABA, glycine o r carbachol, even at high concentrations. T h u s the locust H-GluR seems to differ from that of the crayfish (see below) in having ;i narrow pharmacological profile.

FIG.4. Comparison of the proposed gating mechanisms of the locust ( A ) and crayfish (B) D-GluR. In (A) the locust D-GluR is envisaged as a tetrameric membrane protein, which can exist in either a closed (left hand side) or a fully open (right hand side) conformation. Thus only full conductance level openings, as shown in the idealized trace at the bottom of the diagram, are seen. U p to four 1.-glutamate molecules (C) may hind. T h e channel niay open with any number of L-glutamate molecules bound, but of course the probability of channel opening is higher the greater the number. I n ( B ) the crayfish D-GIuR is also envisaged as a tetramer, capable of binding two 1.-glutamate molecules and t w o Ca+ ions. Both glutamate molecules must be bound before the channel may open. Channel opening is supposed to occur as a sequence of conformational changes of the four subunits. with intermediate stages giving rise to brief subconductance openings. This results in a main conductance level (0,)and three subconductance levels ( O I,02,03), as is illustrated in the idealized recording at the bottom ofthe figure. As discussed in the text, the true picture may be somewhat more complex than this.

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The gating kinetics were slower than those of the D-GluR. T h e time to peak average current was 10 msec. T h e concentration dependence of the peak average current (at concentration of u p to lo-‘’ M glutamate) was steep, corresponding to a Hill coefficient of 4.8. The open time distribution had two components, with time constants of 71 = 2 msec and 72 = 12 msec. Onset of desensitization was also slower than for the excitatory channel, with a time constant of -100 msec. Furthermore, desensitization was incomplete and was not blocked by concanavalin A. A series of experiments with double pulses of glutamate established the time constant of recovery from desensitization to be -300 msec.

-

2. Crayfish Muscle The H-GluR of crayfish muscle has been investigated in some detail using single-channel recording (Franke et al., 1986b; Zufall et al., 1988; Dude1 et al., 1988a) and has been revealed to be surprisingly complex, M or particularly with reference to its pharmacology. Glutamate, at less, has been shown to open a C1- channel when gigaohm seal recordings are made from excised patches of crayfish muscle membrane in the presence of a high (-200 mM) C1- concentration on either face of the membrane. The unexpected finding was made that similar channels appear to be opened by GABA, glycine, and acetylcholine. Thus the crayfish H-GluR seems to have an unusually wide range of agonists. We now survey the properties of this channel in more detail. a. Channel Conductances. The crayfish H-GluR appears to have three conductance levels. In the presence of high (160-240 mM) C1- the conductances are gl = 22 pS, 92 = 2gl = 44 pS, and g3 = 3gl = 66 pS. It is argued (Frdnke et al., l986b) that these represent substates of the same channel, rather than independent channels opening simultaneously, on the basis of the relatively high frequency of g2 and 93 openings despite an overall low level of channel activity, and on the basis of a large number of, for example, closed + g2 -+closed transitions. Evidence for such “multibarrelled” channels has also been presented for such entities as the C1channel of Torpedo electric organ (Miller and White, 1984). T h e relative probabilities of gl, g2, and g3 openings are related, in a complex manner, to the agonist promoting channel opening. The channel has been shown to be C1- selective on the basis of reversal potential measurements. It is impermeable to propionate. Otherwise, there have been no studies of its ion selectivity at the single channel level. b. Pharmacology. The activation of the three-Sonductance-level C1channel by a variety of agonists shows a particularly complex pharmacol-

ogy. L-glutamate (1 p M ) activates primarily the g1 conductance, although some 9 2 and g3 openings are seen. L-Quisqualate is more potent, at 0.5 p M activating gl, g2,and g3 openings at high probabilities. GABA (50 p M ) also seems to open the same three-level channel, but with a higher probability of g 2 and gs openings and a lower probability of gl openings. T h e GABA analog P-guanidoproprionate (1 00 p M )also elicits predominantly g 2 and g3 openings. Even more surprising is the observation that acetylcholine, and its analog carbachol, appear to open the same channel complex, but in a Cay+ concentration-dependent manner. At 13.5 mM C a Z f ,10 mM cabarchol causes predominantly gl openings, but when the Ca2+concentration is dropped to 0.1- 1.O mM, g2 and g:r openings are seen. Such a Ca2+concentratiori-dependent effect has also been seen for opening of the channel by GABA (Dudel et al., l988a). Even more curious is the report (Zufall et al., 1989) that the antihelmintic agent dihydroavermectin (DHAVM) appears capable of gating the three-level C1- channel. These results are somewhat novel, in that they suggest that a channel with three conductance levels is gated by three or four receptors (a quisqualate-type glutamate receptor, a GABA receptor, an acetylcholine receptor, and a DHAVM receptor). They are, however, supported by cross-desensitization experiments, both at the single-channel level (Franke et al., 1986b; Dudel et al., 1988a) and in Aplysia at the macroscopic level (King and Carpenter, 1987). For example, the response to 100 p M L-glutamate desensitizes to a low level of channel activity. T h e response to 50 pM GABA does not desensitize appreciably. However, the simultaneous presence of 100 pM L-glutamate and 50 pM GABA gives a low level of activity comparable to that seen in the presence of glutamate alone. Thus, desensitization of the glutamate response also seems to cause desensitization of the GABA response. Similarly, carbachol (10 p M ) can cause cross-desensitization of the response to 1 piZ2 glutamate. Thus the three receptor types seem to share a common desensitization mechanism as well as a common channel complex. There is, however, a further complication when one turns to the pharmacology of channel block. Picrotoxin is well characterized as a blocker of the GABAA-R C X channel. Picrotoxin seems to block the 9 2 and g3 openings of the channel gated by GABA in the crayfish muscle selectively. Thus 100 pM picrotoxin blocks g2 and gs openings in response to GABA but has little effect on g-1 openings in response to L-glutamate. Raising the picrotoxin concentration to 1 mM blocks the glutamate response as well. c. Gating Kinetics aad Receptor-Channel Desensitization. Given the com-

plexity of the crayfish H-GluK system, it is not surprising that the gating kinetics have only been part.ially characterised. I n the presence of 1 pM L-glutaniate, the distribution o f g I open times is made u p of two o r three exponentially decaying components, and the distribution of g 2 open times of‘ one or two coniponents. ‘Ihe same holds for openings in the presence of 10 p M GABA, with a n apparent single component to the gs open time distribution. Th u s it seems that there are multiple open states of the channel for at least some of the three conductance levels, adding a further layer to the complexity of the system. Channel activation kinetics have been investigated via the liquid filament technique (Dudel et al., 1988a). L-Glutamate gated chloride channels activate relatively quickly, on a timescale of a few milliseconds, with no effect of Cay+ ion concentration on the level of channel activation. T h e activation of CI- channels by GABA is highly sensitive to Cay+,with a 50-100-fold increase in the level of channel activity when the Cay+ concentration is reduced from 13.5 mM LO 0.1 niM. At 0.1 nlM Ca2+, channel activation by GAHA is slow, with peak activity reachcd after -500 nisec. Channel activation by carbachol is rather more Cay+sensitive, a nd is again slow, peak activity being reached after - 100 nisec. T h e same experiments also provided data on desensitimtion rates. L-glutamate-activated C1- channels desensitized on a timescale of -200 msec. Desensitization of GABA-activated channels was much slower, requiring several minutes. It seems possible that Cay+ions may be acting via a n effect on desensitization of the GABA- and the carbachol-activated channels. d. Tzvo Modelsfor the Multireceptor Chloride Channel Complex. T h e re are two main ways in which the gating properties of the crayfish muscle C1channel may be explained. These are summarized in Fig. 5. In model A the (3-barrelled) CI- channel is linked either to a quisqualate-type glutamate receptor, or to a GABA receptor, or to an acetylcholine receptor. All three complexes share (elements o f ) the same desensitization system, but with different dose-response relationships for desensitization, and with the GABA and acetylcholine receptors additionally sensitive to extracellular Ca’+ ions. This model would explain all the observed results. However, it should be noted that Dudel et al. (l9SSa) have shown no effect of intracellular Cay+,ATP, CAMP, GMP, GSP, GTPPS, o r Perlussis toxin on the C1- channel, which tends to argue against a comnion second messenger-mediated desensitization mechanism. I n model B, the CI- channel complex is simultaneously linked to three receptors: for L-glutamate, for GABA, and for acetylcholine. T h e degree of coupling between the different receptors and the channel must

Model A

desens i ti za t i o n

Model B

FIG.5. Two models for the multiple ;igonist chloride chanriel ofcraytish muscle In both cases a triple-barrelled channel is envisaged, giving rise to thc g I , gr, and gJ openings described in the text. In model A sepal-ate I.-glut;iinate (H-GluR), GABA. and acetylcholine receptor-channels exist, sharing the sanic tlrscnsitization mechanism (possibl? invnlving a second messenger system). In model R the three receptor types are all cornplexed t o the same ion channel molecule.

differ in order to explain the differential sensitivity of the glutamate- and the GABA-activated channels t o picrotoxin. One must assume that a complex set of allosteric interactions would operate in such a model. At present it is difficult to choose between the two models. Further singlechannel studies, and possibly molecular biological investigations, are required to solve this problem.

3. Cornpurison between Locust atid Cnqfish H-GLuRs A less detailed comparison is possible than was the case foi- the 1)GluR. Although there are wnie similarities, the differences between the two H-GluR are quite strong. The locust channel has a single conductance level of 25 pS, the crayfish channel has three levels of 22, 44, and 66 pS. The locust receptor has a narrow pharmacological profile, with I), L-ibotenate as a selective agonist, whereas i--quisqualate is a potent agonist in the crayfish system. Comparison of gating kinetics is not possible, other than to say that for both systems activation and deserisit ization seem to occur more slowly than for the H-GluR.

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IV. Overview

The first impression that arises from a survey of the properties of GluR is one of diversity. However, further consideration suggests that there may be unifying themes underlying the diversity. To identify such themes, it is necessary to restrict one’s attention to the excitatory D-GluR, as there is as yet little evidence concerning the anion-selective GluR for any consistent picture of the latter to emerge. Comparing the D-GluR of invertebrates and of mammals, a range of different cation selectivities and single-channel conductances emerge. However, in the light of the work of Imoto et al. (1988) on the nicotinic acetylcholine receptor, showing that relatively small changes in molecular structure can produce changes in single-channel conductances, one perhaps should not interpret a diversity of open channel properties as indicative of fundamental differences between GluR molecules. Detailed comparison of gating kinetics of invertebrate and mammalian D-GluR is not yet possible. However, it is interesting that some evidence for “state-switching” of NMDA-R kinetics has been reported. Such state-switching is characteristic of the locust D-GluR when desensitization is blocked by concanavalin A. It will be interesting to see if the models proposed for insect D-GluK gating mechanisms are more generally applicable once comparable desensitization-free, single-channel data are forthcoming for mammalian GluR. Surprisingly, some of the main evidence for a close relationship between different GluR comes from pharmacological studies. Thus, the D-GluR of insect and crustacean muscle and one of the mammalian CNS GluR are selective for quisqualate as an agonist. One must assume, therefore, that the agonist binding sites of these GluR are structurally similar. Studies of noncompetitive antagonism of GluR by polyamine toxins suggest that there are similarities between the structures of the ionophoric regions of D-GluR of arthropod muscle and NMDA-R of mammalian CNS. For example, ArgTX-636 blocks the locust and crayfish muscle D-GluR channels and also blocks the NMDA-R of rat cortical neurons (Priestley et al., 1989). It is less effective as an antagonist of Quis-R and Kain-R. However, the related Joro spider toxin (JSTX),which also blocks crustacean D-GluR channels (Shudo et al., 1987; Miwa et al., 1987), selectively antagonizes Quis-R and Kain-R in the vertebrate CNS (Akaike et al., 1987). Thee results are at first sight confusing but may suggest a closed structural relationship between the channels gated by the different classes of GluR in arthropods and vertebrates. The naturally occurring polyamine toxins and their synthetic analogs may well be important tools

SINGLE-CHANNEI.LLUTAMATE KECEPTORS

101

for probing the subtleties of this relationship. However, a word of caution is appropriate here. T h e polyarnine toxins also block cation-selective channels gated by other types of receptor (e.g., the nicotinic acetylcholine receptor), albeit with higher dissociation constants than those for antagonism of GluR. A further indication of an underlying unity in the properties of GluR comes from studies of inhibition of receptor desensitization by concanavalin A. This has been shown for several invertebrate D-GluK and for the Quis-R of the mammalian CNS. This suggests that, despite differences in single-channel conductances (but see Hatt et ol., 1989), the quisqualate-sensitive GluK of invertebrates and of vertebrates may be closely related with respect to their desensitization mechanisms. Overall, the emergent picture is one of variation upon a theme to produce the different classes of excitatory GluR. It is anticipated that the next 5 years will produce a family of GluR amino acid sequences that will enable us to relate the single-channel properties of these receptors to the molecular structures of the receptor-channel proteins.

Acknowledgments

We are grateful to Professor P. Ascher, Dr. D.J. Beadle, Dr. S. G. Cull-Candy, Professor

J . Dudel, Dr. W. Finger, Dr. N. Kawai, Dr. J. R. Kernp, and Dr. M. L. Mdyer for providing us with hitherto unpublished material to include in this review.

References

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COINJECTION OF XENOPUS OOCYTES WITH cDNAPRODUCED AND NATIVE mRNAs: A MOLECULAR BIOLOGICAL APPROACH TO THE TISSUE-SPECIFIC PROCESSING OF HUMAN CHOLINESTERASES Shlorno Seidrnan and Herrnona Soreq Department of Biological Chemistry The Life Sciences Institute The Hebrew University of Jerusalem Jerusalem, 91904, Israel

1. lntroduction

11.

111.

1V.

V.

VI.

A. General Considerations B . Oocyte Applications in Molecular Ncurohiology C . Structure-Function Relationship Studies D. A Case Study: Cloned Hunlaii Hutvrylcliolinesterase Cholinesterases: A Model Polyniorpliic Fanlily of Enzvmes A. General Considerations B. Levels of ChE Polymorphisiii Experimental Observations: A Bicxlielnical Approach A. Cloning and in Ouo Expression of Human Serum BuChE B. Subcellular Partitioning ot Oocyte-Produced BuChE C . Oligomeric Assembly of Synthetic- BuChE D. Role of Tissue-Specific Helpei Proteins in BuChE Assembly X~noppu.\Oocytes: Faithful but (:oniplex Tools A. General Considerations B. Oocytes as Miniature Organ Cultures C . Oocytes as Polarized Cells Experimental Results: An Iiiirnuiiohistoche~~~i~al Approach A. Inimunofluorescent and E l e c t i o ~Microscopic ~ Analysis of Clone-Produced BuChE B. Rapid Appearance of Synthctic BuChE 011 External Sui-face ot Injected Oocytes C. Association of Synthetic Bu( :lib: with Extracellular 1.ayers Surrounding Oocytes D. mRNA Intensification of Bu(:IiE: Signals Associated with Oocyte Surface Closing Remarks A. Summary and Conclusicms €5. Future Directions References

1. lntroduction

A. GENERAL CONSIDERATIONS Since their demonstration b y Gut-don ut al. (1971) as a viable system for the translation of heterologous mKNAs, Xenopus laevzs oocytes have

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been employed for the in ouo synthesis of a large variety of biologically active proteins (see Soreq, 1985; Dascal, 1987, for reviews of Xenopus oocyte microinjection). The dramatic increase in the use of oocytes for research in molecular biology over the past decade indicates the versatility and growing popularity of the system. A specific messenger RNA representing as little as 0.001% of total injected mRNA may direct the synthesis of a detectable protein product (Soreq et al., 1982). Between 10 and 20 fmole/oocyte of functional Torpedo acetylcholine receptor were reported to be synthesized in response to as little as 20 ng of microinjected electric organ poly-A(+) RNA (Barnard et al., 1982). Thus, the relatively high efficiency of translation of exogenous RNAs coupled to a sensitive biochemical o r electrophysiological assay for the induced protein product makes the Xenopus oocyte an extremely sensitive system for the bioassay of particular messenger RNAs. As such, oocyte microinjection has become an indispensable tool in molecular neurobiology. Oocytes may be employed to screen total RNA preparations for the presence of specific intact mRNAs, to monitor the enrichment of particular mRNA species (Sumikawa et al., 1986), or to screen cDNA libraries by direct cloning into a transcription vector followed by the microinjection of pooled synthetic RNA (Masu et al., 1987). Furthermore, oocyte microinjection may establish the irrefutable identification of a cloned gene by demonstrating its ability to direct the synthesis of the biologically active protein it is purported to encode (Mixter-Mayne et al., 1987; Kobilka et al., 1987). Carried a step further, Xenopus oocyte microinjection offers, in partnership with modern tools for genetic engineering, a marvelous opportunity to dissect a specific mRNA into its functional components and assess their various contributions to complex modes of biosynthetic regulation.

B. OOCYTE APPLICATIONS I N MOLECULAR NEUROBIOLOCY Of particular interest to neurobiologists is the application of oocyte microinjection to the expression of functional membrane-bound channels, receptors, and ion pumps (for reviews, see Dascal, 1987; Lester, 1988). T h e literature is virtually inundated with reports of successful expression of various “excitability” proteins from different RNA preparations from multiple animal and tissue sources. The faithful reconstitution by Xenopus oocytes of active receptor/channel complexes has been shown in many cases to generate membrane conductance properties comparable to those expressed in native tissues. In specific instances, receptor subtypes have even been differentiated in RNA-injected oocytes

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based on their subtype-specific behavioral characteristics (Frielle et al., 1987; Fakuda et al., 1987; Barnard, 1988). T h e use of poly-A( +) RNA to induce the appearance of novel electrophysiological responses in the oocyte often marks the first step toward the eventual cloning of the genes encoding the responsible polypeptide(s). T h e microinjection of size-fractionated RN A represents a further step that may not only advance cloning efforts but has also provided evidence for multiple transcripts encoding polymorphic variants (Soreq et al., 1984) or implicated the presence of helper RNAs in the modulation of posttranslational modifications (Krafte et al., 1988; Rudy el al., 1988). In another paper, Parker et al. (1988) deduced the existence of a distinct p-alanine receptor following differential enrichment of p-alanine-, GABA-, and glycine-mediated responses upon microinjection of sizefractionated RNA from chick and rat brain. [Interestingly, the successful expression of many channels and receptors from size-fractionated R N A has been claimed to demonstrate that these molecules are often encoded by either a single mRNA species or by several mRNA’s of similar size. (Sumikawa et al., 1986).] Finally, the expression of synthetic RNAs transcribed from cloned genes offers not only positive identification of the cloned DNA sequence but opens the possibility for the next stage of analysis, an inquiry into the nature of structure-function relationships in these molecules.

C . STRUCTURE-FUNCTION RELATIONSHIP STUDIES Different types of structure-function relationship studies have been employed in the analysis of nervous system-related membrane proteins. On the level of multisubunit interactions, reconstitution studies and subunit “mixing” experiments have been employed. Mishina et ul. ( 1984), using RNA transcribed in Co.5 monkey cells from distinct cDNAs encoding the various subunits of the Torpedo acetylcholine receptor, demonstrated that although all four subunits were required for normal activity, microinjection of the a subunit-encoding RNA alone was sufficient to induce a-bungarotoxin binding. Several groups have used cloned AChK genes to produce hybrid mouse-Torpedo or calf-Torpedo nicotinic acetylcholine receptors (White et al., 1985; Sakmann et al., 1985; Mixter-Mayne et al., 1987). Among other things, these studies provided convincing evidence that the 6 subunit is responsible for defining the characteristic dose response of the receptor complex in different species and that the degree of interspecies homology is sufficient to permit functional hybrid receptors, provided that all four subunits are represented. Replacement

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of the adult bovine 6 subunit KNA with the homologous calf-specific E subunit KNA was later shown to induce the synthesis of receptors characteristic of fetal AChK (Mishina et al., 1986). Structure-function analyses on the level of a single polypeptide chain have been performed using site-directed mutagenesis to engineer controlled modifications in a cloned gene, by the construction of chimeric cDNAs, and in our laboratory by the coinjection of tissue-derived polyA ( + ) KNAs with a purified messenger RNA derived from in vitro transcription of a cDNA clone. T h e first application of site-directed mutagenesis to the Xenopus oocyte microinjectiori system in the field of molecular neurobiology came, again, by way of the CY acetylcholine receptor (Mishina et al., 1985). In this study, controlled deletions were introduced into regions of various postulated functions and their effects on the ACh response monitored. More elegant perhaps is the use of site-directed mutagenesis to introduce single base mutations into a cloned cDNA, permitting the analysis of single amino acid substitutions and their effect(s) on biological function. Mixter-Mayne et al. (1987) built chimeric 6-y Torpedo-mouse AChK cDNAs in an attempt to identify regions of the two relatively homologous molecules that specify their mutually exclusive functional properties.

D. A CASESTUDY: CLONED HUMAN BUTYRYLCHOLINESTERASE In the structure-function studies discussed above, the primary emphasis was placed on evaluating the significance of various receptor or channel components vis-&vis functional biological activity. We have attempted to carry this type of analysis a step further, to the realm of regulation. Taking advantage of the well-known ability of the oocyte to translate, assemble, and transport a wide variety of heterologous proteins correctly, we have used the system to address questions relating to the molecular determinants regulating these processes for a particular nascent polypeptide. ‘Toward this goal, we constructed a cDNA containing the complete amino acid coding sequence for human serum butyrylcholinesterase (Prody et al., 1986, 1987), subcloned it into the SP6 transcription vector (Krieg and Melton, 1984), and used it for the in uitro synthesis of synthetic B:. ‘hE rnRNA (Dreyfus et al., 1989; Soreq et al., 1989). We have shown that the synthetic mRNA is capable of directing the synthesis of catalytically active BuChE in Xenopus oocytes (Seidman, 1989) and further combined biochemical and immunocytochemical techniques to monitor the biogenesis, transport, oligomeric assembly, arid membrane association of

the synthetic enzyme in the iri,jcctcd oocytes (Dreyf‘iis, 1989). Finally. by employing coinjection with poIy-A( +) R N A derived froni fetal h u n i m brain, muscle, and liver we have embarked on a series of’ cxperitnents designed to analyze the molec.rilai- tletei-rninants involved i t i specit\.ing the divergent biosynthetic I)alh\va)-s responsible for getlei-ating the tissue-specific polymorphism cltaracteristic of‘ this fiiniily of. acet>lcholine-hydrolyzing enzymes. I n the l’ollowirig sections. these esperiments will be presented i n detail, \++h particular empliasis o t i the gcneral implications for the use of. niic-t,oiti,jected X P H ~ ~ oocytes L Y 10 appi-oach questions in molecular neuidiiology .

II. Cholinesterases: A Model Polymorphic Family of Enzymes

Cholinesterases (ChEs) represent a ~il>iquitous polyttioi.pliit tiitilily of’ car box y lesterase type B e n 2)’ni es c ha rac-t e r iLed b y t h e i r a hi I i t y t o t 1 v d I-( )lyze choline esters rapidly. ..\c.et!,lcliolinesterase (ac-etylcholiite itcetylhydrolase, E.C. 3.1.1.7, A(:liI+:), ;ilso known as “true” o r “specific” cholinesterase, has long heen iioted f’or its role in tltc: terniiii;ition of’ neurotransiiiissiori at the rieiii-oiiiuscular junction ( B r o ~ v nPI (/I., 1936; Katz an d Miledi, 1973). As 111erarget protein for a variety of‘neut.otoxic drugs (Koelle, 1972) inclutlitig iiisecticides (Parathion, hlalathion) and chemical warfare agents (Sonim, Sarin, T a h i ) , A C h E h a s stinirilittetl research with profound agricultural, ecological and military implications (Bidstrup, 1950; ‘I‘animura P / ui., 1967; Naniba d ul., 197 1 ; Bull. 1‘382; see also U. N. Security (:ouuc.il Report, 1984). Clinical studies have correlated modifications in A C h E levels and forms with ~ie~irologic.al disorders such as Alzheinier’s Disease ((kyle et 01.. 1983; Fishmail rl d., 1986),Down’s Syndrome (Yates tit ul., l980), Parkinson‘s disease (Kuberg et d., 1986), a n d muscular clystrophy (Skau and Brirnijoin. 1981 ) . Furthermore, t h e presence of high levels of‘secreted AChE in 1iurii;in amniotic fluid has been identified as a characteristic marker of enibt.yonic neural tube defects (Bonhani and Atack, 1983). Butyrylcholiriesterase (ac:ylclioline acylhydrolase, E . C . 3 . 1 . 1 .X, BuChE) also referred to as “psetido” cholinesterase, “non-specific.” cholinesterase, o r simply “choli~iesterase,”shares structur;tl and catalytic properties with AChE, although its exact physiological role is !.et t i n known. Significant BuChE activity is present in serum and in a variety of‘ cholinergic a n d noncholinergic tissues, where it is fr-equently colocalized

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with AChE (Silver, 1974; Rakonczay and Brimijoin, 1988). In developing chicken muscle, AChE and BuChE appear to be regulated in a coordinated manner (Lyles et al., 1979; Tsim et al., 1988a). In humans considerable allelic polymorphism has been documented for serum BuChE based on electrophoretic migration and affinity for substrates and inhibitors (see Whittaker, 1986, for review). The wealth of naturally occurring allelic variants, including those which confer the defective “atypical” or “silent” BuChE phenotypes, makes BuChE an appropriate model for the study of structure-function relationships based on polymorphic genotypes found within normal human populations. Furthermore, since individuals expressing the atypical or silent phenotypes display no symptoms of disease or debilitation (Hodgkin et al., 1965), extended family pedigrees documenting the inheritance patterns of these alleles are well within reach (for examples, see Prody et al., 1989; McCuire et al., 1989). Individuals carrying defective BuChE genes demonstrate impaired recovery from medically administered succinylcholine and increased sensitivity to organophosphorous (OP) poisoning. Chronic exposure to low dose OP insecticides has been suggested to provide a selective pressure for ChE gene amplification in humans (Prody et al., 1989). Amplification of cholinesterase genes has also been demonstrated in selected tumor tissues (Lapidot-Lifson et al., 1989; Zakut et al., 1990). Both classes of cholinesterase display multilevel polymorphism with respect to molecular forms, hydrodynamic properties, antigenic determinants, and subcellular compartmentalization (see Massoulie and Bon, 1982; Toutant and Massoulie, 1987; for reviews of ChE polymorphism). Tissue-specific patterns of expression for the two cholinesterases and their various forms have been described for a number of tissues in a wide array of evolutionarily divergent species (see Rakonczay and Brimijoin, 1988, for a thorough review of tissue and species-specific ChE distributions). Furthermore, the biochemical and biophysical properties of these various forms have been extensively characterized at the protein level. In contrast, the molecular determinants specifying the various ChE forms and the biological mechanisms directing their controlled expression in particular cell types remain to be elucidated. B. LEVELSOF ChE POLYMORPHISM

1. Substrate and Inhibitor Specificities BuChE is classically distinguished from AChE on the basis of its relative affinity for various ligands, including substrates and inhibitors. AChE, as might be expected from an enzyme responsible for the modula-

CHOLINEST EKASF I’K( )I)L’(, 1 ION Ih XLNOPC’S OOOY TF5

113

tion of synaptic neurotransrnission, displays a high specificity for the neurotransmitter acetylcholine ( ACh) and an extraordinarily high catalytic turnover rate of 1O4 ACh molecules hydrolyzedlsecicatalytic site (Vigny et al., 1978). In contrast, BuChE hydrolyzes ACh at a rate considerably lower than that of AChE, prefers butyrylcholine over acetylcholine as a substrate, and demonstrates efficient catalytic activity on a relatively broad range of substrates (Silver, 1974; Brown et al., 1981). BuChE may further be distinguished from AChE by its differential sensitivity to a variety of ChE inhibitors (Austin and Berry, 1953; Silver, 1974; Koelle, 1972). Until recently, the molecular elements defining these two enzyme species have remained unresolved. ‘ l h e relative scarcity of both enzymes even in highly enriched tissues (Silver, 1974) has long impeded their purification and characterization. I n fact, a prominent question in modern cholinesterase biology has been whether the distinct catalytic activities defined as AChE (EC. 3.1.1.7) and BuChE (EC. 3.1.1.8) are derived from divergent pathways in the posttranslational processing of a single nascent polypeptide or from distinct messenger RNAs. Preliminary attempts to reveal the molecular origin for the distinction between AChE and BuChE were based on the interaction of various forms of the two enzymes with pdy- and monoclonal antibodies raised 1983; against minute quantities of highly purified ChE (Brimijoin rt d., 1986). Marsh et al., 1984; Rakonczay arid Brimijoin, 1986; Sorenson rt d., Although the various antibodies produced interacted with all of the molecular forms of either AChE or BuChE, antibodies elicited against AChE did not cross-react with Bu(;hE and vice-versa (Fambrough et ul., 1982; Brimijoin et al., 1983; Marsh rt nl., 1984; Lappin et al., 1987).These data were generally interpreted to indicate a low level of sequence honiology between the two molecules. Monoclonal antibodies with signiticant cross-reactivity were however-, reported by one group (Doctor rt ul., 1983). Furthermore, antibodies produced in our laboratory against unprocessed cDNA-produced human BuChE peptides (Dreyfus et al., 1988) cross-reacted specifically with various molecular forms of hydrophilic AChE and BuChE, indicating sequence cross-homologies nested within structural variations. cDNA sequence analyses of 7orpedo electric organ AChE (Schumacher et al., 1986) and human serum BuChE (Prody et al., 1986, 1987; Mc‘riernan et al., 1987; Soreq and Gnatt, 1987) offered the first good indications of distinct BuChE and AChE mRNAs. However, the cloning of a human fetal AChE cDNA (Soreq and Prody, 1989) demonstrated conclusively the similar although distinct nature of the mRNAs encoding these two enzymes in humans. Thus, although the primary amino acid sequences of the two enzymes exhibit a high degree of homology (ap-

proximately SO%), the DNA sequences encoding these are much less highly conserved. Nonetheless, it is still not known whether AChE and BuChE mKNAs are derived from a single gene via controlled alternative splicing, or whether they are encoded by distinct genes, which may be subject to coordinated regulation through cis and/or trans acting factors.

2. Multiple Molecular Forms Both AChE and BuChE present an array of molecular forms that can be separated by sucrose density centrifugation (for reviews, see Massoulie and Bon, 1982; Toutant and Massoulie, 1987; Rakonczay and Brimijoin, 1988). ChE molecular forms are divided into two subclassifications: globular and asymmetric. ‘I‘he globular forms include the catalytically active monomer, a dimer and a tetramer, conventionally denoted GI, G2, and Gq. The asymmetric forms, denoted Aq, Ax,and A12,are characterized by the covalent association of a triple-helical, collagenlike “tail” subunit with one, two, or three tetritmeric catalytic complexes. The collagenlike “tail” consists of long ( W n m ) fibrillary peptides, rich in proline and hydroxyproline residues (Kosenberry and Richardson, 1977). Digestion of the “tail” with trypsin or collagenase yields active G4 tetramers (Bon and Massoulie, 1978; Anglister and Silman, 1978). In multimeric forms, disulfide-linked dimers of catalytically identical subunits are associated with each other or with the “tail” through covalent and/or quaternary interactions (Massoulie and Bon, 1982; Rosenberry and Scoggin, 1984; Toutant and Massoulie, 1987). As yet, no clear functional significance has been associated with the various molecular forms although their tissue-specific patterns of expression are well documented. A hybrid AChE-BuChE asymmetric form has been characterized in embryonic chicken muscle (Tsini P t al., 1988b),while dimeric ChE sensitive to both BuChE- and AChE-specific inhibitors was identified in the serum of carcinoma patients (Zakut et al., 1988). These latter findings further complicate our understanding of ChE molecular form assembly and regulation, particularly in developing tissues.

3. Hydrodynamic Properties and Subcellular Compartmentalization Cholinesterases exist in low salt-, high salt-, and detergent-soluble forms. These variations in the hydrodynamic nature of ChE forms correlate with the occurrence o f t h e enzyme in a multiplicity of subcellular compartments: secreted, cytoplasmic, membrane-bound, and extracellular matrix-associated. T h e globular ChEs can be grouped into two categories: hydrophilic and amphiphilic. T h e amphiphilic AChE forms are characterized by the presence of a hydrophobic element capable of bind-

ing the enzyme to membranes. ' f h e most common element o f this nature thus far described is a covalently linked phosphatidylinositoI(P1)containing glycolipid moiety. PI has been implicated in the binding of G2 AChE to the plasma membranes of Torpedo electric organ, various mammalian erythrocytes (including human, bovine, sheep, rodent, a n d others), and some other niiscellaneous tissue types (for a thorough review, see Silman and Futerman, 1987). Xenopu,s skeletal muscle (Inestrosa et al., 1988) has also been added to the list of tissues containing PI-bound G2 AChE. Membrane-bound ChEs share the property of being solubilized by phosphatidylinositol-specific phospholipase C (PIPLC) (Low and E'inean, 1977; Futerman et d., 1983, 1985),and membrane release by this enzyme has become a n accepted indicator of P1-mediated anchorage. Nonetheless, structural differences do exist among PI-containing anchors in different species and these modifications may confer differential sensitivity to PIPLC (Roberts eta/., 1987). I n addition to their role in binding A4C;hE to membranes, PI-containing glycolipids have been implicated in the cell surface binding of a diverse group of proteins that include the VSG protein of trypanosomes, nianimalian alkaline phophodiesterase I , neural cell adhesion molecules (Nybroe ot a/., 1988), and rat Thy-1 antigen (for review, see Low, 1987). <:amnion to all the P1-bound molecules characterized to date, including Torppdo AChE (Sikorav rt (11.. 1988; Gibney et ul., 1988), is a primary translation product that is cleaved to release a hydrophobic C-terminal peptide prior t o the attachment of' the PIcontaining anchor to the newly generated C-terminal amino acid. Not all hydrophobic AC:hEs are PIPLC: sensitive. Vertebrate brain hChEs, for example, are completely resistant to PIPLC-mediated solubilization. Furthermore, the G4AChE from bovine caudate nucleus has been demonstrated to be associated with the plasma membrane via a small (20-kD), noncatalytic hydropholic element that is covalently linked to o n e of the two dimeric units making u p the tetramer (Inestrosa et d . , 1987; Fuentes et a/., 1988). 'I'hus, PI-mediated membrane attachment appears primarily correlated with glohular dimeric AChEs, while no clear pattern regarding the hydrophobic association of tetranieric AChEs has yet emerged. T h e asymmetric ChE forms, although essentially hydrophilic- in nature, a r e characterized by reversible aggregation at low salt conc'entrations. Considerable evidence suggests that the asymmetric 16s form of' AChE concentrated at the neuromuscular junction (Hall, 19TS) is specifically associated with the synaptic h a 1 lamina (McMahan E / 01.. 1978; Dreyfus et al., 1983) where its anchorage is mediated via binding of the collagen-like tail subunit to heparan sulfate proteoglycan (HSPG) (Bran-

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SHLOMO SEIDMAN AND HEKMONA SOKEQ

dan et al., 1985; Vigny et al., 1983). Thus, these tailed forms are solubilized at high ionic strength (e.g., 1 M NaCl or 0.5 M MgC12). This observation suggests a specific functional role for the tail in binding the asymmetric forms of the enzyme to their correct subcellular localization within the synaptic cleft. Nonetheless, a small proportion (-20%) of the asymmetrical form is associated with lipidic membranes and is solubilized in the presence of detergent (Garcia et al., 1988). Figure 1 presents the various forms of ChEs, their sites of localization, and their presumed modes of association with solid subcellular supports. It is not fully known what elements define the ultimate fate of a particular nascent ChE molecule vis-a-vis its subcellular localization. Direct evidence implicating alternative mRNA splicing as the molecular origin for specific hydrodynamic components in Torpedo electric organ

AChE

7 BuChE

Globular

Asymmetric

' I 88 8 8 8888 88888 @

Collagen-like

Amphipathic

l l y l l Hydrophilic

Membrane bound

FIG. 1 . Molecular form heterogeneity in cholinesterases. Asterisk indicates minor fraction of membrane-associated enzyme at the extracellular matrix.

CHOLINESTERASE PK0I)UC:TION I N XENOPb'S OOCYTES

117

AChE has emerged from parallel amino acid and DNA sequencing analyses (Sikorav et al., 1987, 1988; Gibney et al., 1988). These studies have together demonstrated that the alternative usage of two 3'-terminal exons within a single Torpedo AChE gene is sufficient to specify distinct mRNAs encoding either a globular hydrophobic or asymmetric tailed form of the enzyme. Furthermore, the C-terminal peptide of the hydrophobic AChE was found to be similar to the C-terminal sequence of other hydrophobic, glycolipid-linked, membrane-bound proteins. A third AChE cDNA with yet a different C-terminal peptide remains uricorrelated to any specific AChE molecular form (Sikorav et al., 1988). Complex 5'-terminal RNA polymorphism (Sikorav et al., 1987),also identified in these studies, probably serves a regulatory function. These findings represent a significant breakthrough in our understanding of ChE polymorphism. Nonetheless, conclusive evidence for alternative AChE mKN A processing in other species remairis to be demonstrated, as do indications for alternative splicing in any BuChE. 4. Tissue-Specific Expression T h e relative distribution of various ChE forms in particular tissues is highly specific and reproducible. Soluble globular tetrameric BuChE is found in high concentrations in serum, where AChE is present in only minimal quantities. BuChE is also present in muscle (Dreyfus et al., 1983), brain (Zakut et al., 1985), and various embryonal tissues (Drews, 197.5; Layer et al., 1987). Its specific mKNA species has been demonstrated to be present in high quantities in developing human oocytes (Soreq et ul., 1987; Malinger et al., 1989). Both AChE and BuChE are present in seminal fluid, suggesting a role in spermatogenesis (Katz, Zakut, and Soreq, unpublished observation). T h e finding of BuChE gene amplifications in leukemias (Lapidot-Lifson at al., 1989), in light of early reports on the biological effects of acetylcholine analogs on megakaryocytopoeisis (Burstein et al., 1980), suggests a yet unspecified involvement in hemopoiesis. In the liver, the monomeric and dimeric forms of BuChE predominate (Dreyfus et al., 1989). AChE is highly concentrated at the neuromuscular junction (Hall, 1973; Anglister and McMahan, 1985) and cholinergic brain synapses (Fambrough et al., 1972) but is also present in a variety of nonnervous, noncholinergic tissues such as erythrocytes and megakaryocytes (Burstein et al., 1985) in which its function is not known. In brain, the principal AChE form is Gq (Zakut et al., 1985; Sorensen et al., 1982), in red cells GP (Ott et al., 1982; Rosenberry and Scoggin, 1984), and in muscle an array of globular and asymmetric f o r m are found (Dreyfus et al., 1983). In the neurons of the peripheral autonomous nervous system,

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SHLOMO SkJII)MAN A N D H E R M O N A S0Kk.Q

all the molecular forms of‘ both AChE and BuChE are more o r less detectable (Dreyfus et al., 1985). It is important to note that most of the data accumulated with respect to ChE expression in various tissues has been acquired using a variety of catalytic activity measurements. Kotundo and Fambrough (1980) and later Rotundo (1987) and Rotunclo and Carbonetto (1987) showed that in avian muscle, a large fraction of nascent AChE molecules remain catalytically inactive and undergo ;I rapid turnover. These data underscore the potential that immunocheniical arid molecular biological techniques offer toward the advancement of ChE research. Using inimunochernical staining a nd in situ hybridization with labeled ChE probes, ChE expression and processing may n o w be monitored in tissues where catalytically active ChE is either undetectable or represents only a subpopulation of the total ChE synthesized. ‘I’his may be particularly important in the case of BuChE, a plasma protein of unknown function, whose tiological significance may lie within a yet undefined precursor o r breakdown product.

111. Experimental Obsenrations: A Biochemical Approach

A. CLONING AND in Ouo EXPRESSION OF HUMAN SERUM BuChE 1. Synthetic BuChE mKNA Synthetic oligonucleotide probes designed according to a consensus ChE active site sequence were prepared and used to screen human fetal brain a nd liver libraries (Prody e/ nl., 1986, 1987; Soreq and Gnatt, 1987). Two clones, one from brain and one from liver, were isolated a n d combined to reconstruct a cI>NA encoding the entire amino acid coding region for “normal” human serum BuChE as determined by amino acid sequencing (Lockridge et d., 1987).T h e original recombinant clone contained 2 in-frame 5’ AUG codons, a putative ribosome binding site, a hydrophobic putative leader sequence, a coding region for the 574amino-acid-long polypeptide that comprises the mature BuChE enzyme, a 3‘ non-translated region, a polyadenylation signal, a n d 70 terminal poly-A residues (Prody et d.,1987) (Fig. 2 ) . This BuChE cDNA and several derivatives were subsequently subcloned into the SPt; expression vector (Krieg a n d Melton, 1984) and synthetic BuChE mKNA was prepared.

( ' I I I N G REGION

Active Site

C

-

P0LYADEN"LA- I CN SIGNAL

3' UNTRANSLA-ELJ REGION ( 51'9 B P )

L

FIG. 2. Schematic diagram of I i u r i i ; ~ i i I~utyi-ylcholirstcra~~ cI)NA. Note he IM'O Met residues, the putative ribosome binding site, the hydrophobic putative sigiial pq~tidr.t h e active site seririe, the 3' untranslatcd region, a i d the poly-B(+) tail.

2. In 0z)o Expression of Syittwtir NiiClrE mRNA When microinjected into Xrirt$u.s oocytes, 1 ng of SP(;BuChE mRNA induced an enzymatic activity capable of hydrolyzing up to 10 nmoles of' butyrylthiocholine (BuSCh) (Ellman et al., 1961) per oocyte per hour. We found 5' capping of- the synthetic message dispensable, but at a price: Uncapped RNA generated a signal from two- to fivefold lower than capped RNA. More important, polyadenylation was found to inHuence the efficiency of translation within a 10-fold range. Both capping and polyadenylation are known to stabilize various inRNAs under certain conditions, both in vivo and in niicroinjected oocytes (reviewed by Littauer and Soreq, 1982). T h e finding that both these processes niay be involved in modulating BuChE rnRNA expression in ouo may therefore implicate mRNA stability as a putative mode of regulation for BuChE levels in vivo. It further points out that both capping and polyadenylation of synthetic mRNA transcripts should be prerequisite steps for oocyte microinjection experiments, particularly when unknown mKNAs are to be expressed.

3 . Synthetic BuChE Resembles Native Serum BuChE Synthetic BuChE manufactured in the oocytes was found to display the catalytic and ligand binding characteristics classically descriptive of

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native normal BuChE and clearly distinct from AChE. Synthetic BuChE thus prefered BuSCh over AcSCh as substrate 2-3 : 1 and displayed a K , for BuSCh similar to that measured in our laboratory for the enzyme in normal human serum. As expected for butyrylcholinesterase, no substrate inhibition was observed u p to 5 mM substrate. T h e oocyte-produced BuChE demonstrated characteristic inhibition by the BuChE-specific, irreversible, organophosphorous inhibitor tetraisopropylpyrophosphoramide (iso-OMPA) while resisting inhibition by high concentrations of the AChE-specific, reversible, quaternary inhibdibromide itor 1,5-bis(4-allyldimethylammoniumphenyl)-pentane-3-one (BW284C5 1) (see Soreq et al., 1989). 4. Properties of B u C h E Defined by Its Amino Acid Sequence

Several conclusions could be drawn at this stage. First, the BuChEcDNA cloned in our lab in fact encodes a fully functional enzyme, which is faithfully translated in the oocyte into a protein displaying the defining catalytic properties of native BuChE. Second, synthetic BuChE mRNA induces the biosynthesis of butyrylcholinesterase exclusively. This latter observation proved consistent with the lack of crosshybridization between cloned human BuChE and the human AChE cDNA clone (Soreq and Prody, 1989; Lapidot-Lifson et al., 1989), and predicted subsequent cDNA sequence analyses, which indicated homologous but distinct AChE and BuChE sequences. These data implied, therefore, that the functional distinction between AChE and BuChE in humans finds its roots in distinct mRNA populations encoding polypeptides with unique primary amino acid sequences. Furthermore, at least in the case of BuChE, the primary amino acid sequence was demonstrated to be sufficient to specify the ligand binding properties of the molecule (Seidman, 1989).

OF OOCYTE-PRODUCED BuChE B. SUBCELLULAR PARTITIONING

Synthetic BuChE was found to partition reproducibly into low-salt and high-salt-detergent-extractable fractions. Generally, 60-70% of measured BuSCh hydrolyzing activity was associated with the high saltdetergent fraction. In contrast to AChE induced in oocytes injected with poly-A(+) RNA from rat brain or Torpedo electric organ (Soreq et al., 1982), and somewhat in contradiction to what we expected based on sequence data, little BuChE activity could be detected in the incubation medium of oocytes injected with synthetic BuChE mRNA. Interestingly,

no intrinsic catalytic or biochemical differences were observed between the molecules recovered from soluble and nonsoluble pools. T h e observed K , and inhibition characteristics were essentially identical for both the low-salt and high-salt detergent-extracted fractions. Furthermore, sucrose gradient centrifugation in the presence and absence of detergent failed to indicate the presence of a prominent hydrophobic element in the detergent-extracted enzyme. T h e theoretical implications of this puzzling observation might be better understood in the light of additional data to be presented later in this manuscript.

C. OLIGOMERIC ASSEMBLY OF S Y N T H E T I C BuChE Linear 5-20% sucrose gradient centrifugation of the oocyteproduced BuChE indicated limited subunit assembly in both oocyte fractions as well as in the conditioned medium. Thus in all three fractions evaluated, the consistent appearance of a 5-7s peak of BuSCh hydrolyzing activity directly paralleled the sedimentation properties characteristic of dimeric erthrocyte AChE. We consider this phenomenon to reHect only “partial” assembly with respect to native serum BuChE, which appears as a globular tetrameric molecule and which has been structurally defined as a “dimer of dimers” (Lockridge et nl., 19‘79).Although some free monomer appeared to persist alongside the predominant peak of dimers, the relative proportion of unassernbled, catalytically active BuChE units is difficult to estimate. Occasionally, and only after freezing and thawing, a peak of monomer could be observed to predominate. We can only speculate on the driving force for this first level of multisubunit assembly. Either this stage of oligomeric assembly represents a spontaneous phenomenon, or the oocyte contains the endogenous enzymatic machinery necessary t o catalyze the process. It is interesting to note in regard to the latter possibility that the oocyte is known to express its own endogenous AChE (Soreq et al., 1982; Gundersen and Miledi, 1983). Furthermore, monomeric and dimeric BuChE may be considered the basic building blocks of all higher order oligomeric BuChE units (Toutant and Massoulie, 1987). Consistent with this observation is the observation by Rotundo and Farnbrough (1980, 1982) that in cultured avian muscle cells, AChE mononiers and dimers precede the appearance of more complex AChE forms subsequent to irreversible inactivation of existing enzyme. Therefore, our results indicated the absence of a limiting factor or factors involved in the facilitation of higher levels of multimeric assembly of nascent BuChE in the oocytes.

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D. ROLEOF TISSUE-SPECIFIC HELPERPROTEINS I N BuChE ASSEMBLY

Coinjection of poly-A(+) RNA from fetal human brain and muscle with synthetic BuChE mRNA was shown to facilitate the assembly of complex BuChE molecular forms in the oocytes although these RNA preparations were not themselves capable of inducing detectable peaks of activity in sucrose gradient analyses. In contrast, RNA from fetal liver did not induce a significant change in the sedimentation pattern of synthetic oocyte BuChE. Poly-A(+) RNA from brain induced the appearance of a 10s molecular form characteristic of the primary ChE form present in human brain (Zakut et al., 1985; Muller et al., 1985). Similarly, coinjection of poly-A(+) RNA from muscle facilitated the appearance of an array of molecular forms characteristic of muscle ChEs. Interestingly, the pattern of moleciilar forms observed after coinjection with muscle mRNA resembled that of muscular AChE rather than that of BuChE. These observations lead to several significant conclusions regarding the modulation of BuChE as well as AChE molecular form polymorphism: 1. Poly-A(+) RNAs from muscle and brain encode accessory proteins that play a role in the assembly of complex ChEs and that are expressed in a tissue-specific manner. 2. A single unique nascent BuChE polpeptide is capable of interacting with a variety of these accessory components to generate multiple molecular forms. 3. A single pool of accessory proteins may interact with both AChE and BuChE in vivo, and the particular pattern of AChE and BuChE molecular forms in a particular tissue may reflect a competition between nascent pools of these t w o molecules for the available accessory elements in that tissue. 4. Although the catalytic subunit of serum BuChE is most probably produced in the liver, helper protein(s) responsible for its tetrameric assembly might be contributed by another tissue source (pancreas, for example). The notion that a single BuChE species may be responsible for generating multiple molecular forms might be construed as contradictory to lines of evidence leading one to expect multiple BuChE mRNAs to account for BuChE polymorphism. Soreq et al. (1984) offered evidence for the existence of multiple R N A species for both AChE and BuChE in human brain. Subsequently, this observation was shown to be consistent with the situation in Torpedo, in which variant AChE polypeptides were directly correlated to multiple mRNAs derived by alternative splicing of a

single primary transcript (Sikorav P I NI., 1988). Nonetheless, although our human BuChE cDNA has been employed as a molecular probe to screen cDNA libraries from muscle, liver, brain, lymphocytes, and several tumors, no evidence for the existence of alternatively spliced BuChE mRNAs has emerged [although alternatively terminated BuChE mKNAs have been found in glioblastonia and neuroblastoma libraries (Gnatt rt al., 1990)l. However, as we have not identified a genuinely hydrophobic BuChE in any of our preparations, we cannot exclude the possibility that additional messenger R N A species encoding other polymorphic variants of the protein do exist.

IV. Xenopus Oocytes: Faithful but Complex Tools

A. GENERAL CONSIDERATIONS One of the most attractive features of the oocyte microinjection system is the presence of intact machinery for posttranslational modifications and transport. Thus, the oocytes have long been recognized to perform glycosylation, multisubunit assembly, and secretion correctly (Soreq, 1985). Moreover, numerous examples have demonstrated the ability of the oocytes to insert ‘ieterologous channels and receptors into the plasma membrane, giving rise to characteristic agonist-induced electrophysiological responses (Suniikawa et ul., 1986; Dascal, 1987). This observation that nascent “foreign” proteins are handled by the oocytes in such a manner as to generate functional menibrane-associated channels and receptors offers a tempting basis to presume that the structure and organization of these molecules at the oocyte surface reflects their state in their natural biological milieu. Yet little biochemical evidence has been produced to support this conclusion. Thus it is unclear how much of a clone-produced protein actually arrives at the oocyte membrane and what fraction of these molecules are correctly and functionally inserted. These questions may have particular importance in studies in which various parts of the studied protein express particular unique functions (for example, ligand-induced channels or receptors, o r G binding proteins). AS MINIATURE OKGAN CULTURES B. OOCYTES

Studied in vitro, the Xenapils oocyte cannot be viewed as a single isolated cell nor directly compared to cultured cell lines. By definition,

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the oocyte is a highly specialized developing cell arrested at a particular stage of differentiation. Consequently, the oocyte is by nature responsive to various complex external stimuli, the most dramatic of which is progesterone-stimulated maturation. Thus, it is not surprising to discover numerous endogenous neurotransmitter receptors in the oocytes, some of which appear to be coupled to membrane-bound, secondmessenger systems-(reviewedby Dascal, 1987). Furthermore, even single isolated oocytes remain in intimate physical contact with several cellular and acellular components that encase the oocyte to form the follicle (Wischnitzer, 1966; also see Dascal, 1987). From the inside out these layers are called: 1. Oocyte plasma membrane: T h e apparent insertion site of the various heterologous channel proteins and receptors cited in the literature, it is comparable, but not identical to the neuronal plasma membrane, where these molecules are normally found. 2. Zona pelucida or vitelline membrane: An inert matrix infiltrated by numerous micro- and macrovilli, which provide “gapjunction” type contacts between the oocyte and the surrounding follicle cells. 3. Follicle cell layer: Innermost cell layer, which may be electrically and permeably coupled to the oocyte via hormone-regulated channels. 4. Theca: Inert layer of connective tissue characterized by the presence of collagen fibers and blood vessels. 5. Surface epithelium: Outermost layer of cells surrounding the oocyte, derived from the enveloping ovarian tissue. Figure 3 demonstrates some of these elements and their interconnections as viewed by electron microscopy. Note the complex route required to transport a secretory protein from the cytoplasm out beyond the outermost layers to the medium. Although the follicular layers down to the vitelline membrane can by mechanically or enzymatically removed, these treatments invariably violate the integrity of the oocyte membrane (discussed in detail by Dascal, 1987). Nonetheless, defolliculation is routinely performed by numerous groups and has not appeared to hamper the expression of many membrane-bound proteins. (Interestingly, collagenase treatment of oocytes prior to microinjection appears to impede the expression of cholinesterases and is therefore not employed in our laboratory). Yet in the absence of experimental documentation, it is difficult to assess the impact of defolliculation on the efficiency, reproducibility, and correctness of membrane insertion of these molecules. Therefore, it is worthwhile to emphasize the need to consider the possibility that defolliculation may

125

FIG. 3. Cross-section through the animal pole of a niature XrnopuJ oocyte. Note black pigment vesicles, coated and uncoated vesicles, micro- and macrovilli making direct physical contact with surrounding follicle cell, and multiple additional enveloping layers.

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SHLOMO SEIDMAN A N D HEKMONA SOREQ

introduce unforeseen effects o n the expression of induced, heterologous, membrane-asociated proteins.

C. OOCYTES AS POLARIZED CELLS A further point to consider in the use ofXenopus oocytes as an expression system is their polarized nature. T h e conspicuous black-white polar asymmetry, which is immediately apparent on a casual glance at these enormous cells, reflects much more than an unequal distribution of pigment vesicles between the animal (black) and vegetal (white) poles (Klymkowsky et al., 1987). In fact, the polarized coloration of the oocyte demonstrated by electron microscopy in Fig. 4 simply hints at a general hemispheric polarization that extends to characteristics such as the distribution of yolk platelets, cytoskeletal organization, electrophysiological response to specific stimuli, membrane receptor-channel distribution, and subcellular mRNA distribution (Dumont, 1972; Palecek et al., 1985; Danilchik and Gerhardt, 1987; Kusano et al., 1982; Oron et al., 1988; Weeks and Melton, 1987). One might speculate as to the intracellular polarization of elements involved in the translation and processing of oocyte-produced proteins, and its potential implications for the expression of injected mRNAs. Furthermore, it is not clear what effect polarized deposition might exert on the biological function of a particular molecule. Nonetheless, the unequal distribution of induced heterologous proteins or the cellular elements involved in their processing may be biologically important and therefore should not be overlooked.

V. Experimental Results: An lmmunohistochemical Approach

A. IMMUNOFLUORESCENT A N D ELECTRON MICROSCOPICANALYSIS OF CLONE-PRODUCED BuChE

As described above, synthetic BuChE mRNA injected into Xenopus oocytes induces the synthesis of active BuChE, a significant fraction of which appears to be associated with the buffer-insoluble fraction. In order to localize the site of association of these molecules with the oocyte membrane or its surrounding structures and to assess the nature of their organization at these sites, an immunohistochemical approach was taken (Dreyfus et al., 1989). At various times after microinjection, individual oocytes were fixed in paraformaldehyde, embedded in Tissue-Tek em-

CHOLINESTERASE PKODCCTION IN XENOPCTS 0 0 < , Y ’ l ‘ F S

127

FIG. 4. Cross-section through the vegetal pole. Note large, dark yolk platelets. the abundance of mitochondria, and the absence o f pigment vesicles in this half of the oocyte as compared to the animal pole (Fig. 3 ) . Exti-acellularmaterial is essentially as that displayed in Fig. 3.

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bedding medium for frozen tissue specimens (OCT) (Miles, Naperville), frozen, and sectioned. Sections were incubated with rabbit polyclonal antibodies prepared against Torpedo electric organ AChE, previously shown to interact specifically with clone-produced human BuChE (Soreq et al., 1986; Dreyfus et al., 1988). The sections were then incubated with second antibody coupled either to an immunofluorescent phycoprobe tag for light microscopy or to 5-nm colloidal particles conjugated to protein A for electron microscopy. OF SYNTHETIC BuChE O N EXTERNAL SURFACE OF B. RAPIDAPPEARANCE INJECTED OOCYTES

Within 30 min following the injection of synthetic BuChE mRNA, immunofluorescent signals could be observed on the oocyte external surface. Two types of surface-associated accumulations were observed: (a) small round clusters approximately 5 pm diameter and (b) elongated patches approximately 20 pm in diameter. An exhaustive quantitative analysis of selected individual oocytes confirmed a general impression that the overall fluorescent signal was relatively accumulated at the animal pole. The total surface area occupied by patches greatly exceeded that occupied by clusters at both poles, perhaps suggesting that patches represent a more favorable arrangement of molecules. The density of molecules within a cluster or patch was estimated to be on the order of 5000 molecules pm2, within the same order of magnitude as that estimated for the nicotinic acetylcholine receptor (Sytkowski et al., 1973) and AChE (Salpeter, 1967; Rosenberry, 1979; Barnard, 1984; Rotundo, 1987) within the neuromuscular junction and along neuronal dendrites. Interestingly, this value is close to the calculated packing density for this molecule based on its Stokes radius (Brown et al., 1981). Particularly intense fluorescent signals around the site of injection could indicate limits to free diffusion of the injected message or its protein product within the cytoplasm. Alternatively, or in addition, this observation might reflect a rapid turnover of these foreign protein chains within the amphibian egg that is compensated for by efficient synthesis in the microenvironment of the injection site. Treatment of the oocytes with tunicamycin prior to injection resulted in the conspicuous intracellular accumulation of fluorescent signals around cytoplasmic vesicles, implicating posttranslational glycosylation in transport of the nascent enzyme to the oocyte surface. Interestingly, Lucas and Kreutzberg (1985) found that tunicamycin inhibited the secretion of AChE in neuronal cells in culture. Thus, glycosylation appears to be important for ChE secretion but does not seem to play a vital role in establishing catalytic activity.

CHOLINESTERASE 1’KOI)CIC~I ION IN XENOPLJS OOCY I ES

129

T h e relative concentration of synthetic BuChE at the animal pole is similar to the preferential animal pole deposition of acquired ACh- and TRH-evoked C1- responses reported by Oron et al., ( 1 988). Interestingly, in that case the distribution of the “acquired” response was opposite to that of the “intrinsic” response and indicated the presence of an active transport mechanism(s). These observations raise the question of whether there might exist regulatory sequences, perhaps even species- or tissue-specific, which specify a protein’s site of deposition vis-a-vis the animal-vegetal polarity of the oocyte. I n any case, the observation that the oocytes may effect the polarized distribution of induced heterologous proteins suggests that the Xenopus oocyte may serve in the future as a model system in which to probe the regulatory elements in protein sorting within polarized cells.

C. ASSOCIATION OF SYNTHETIC BuChE SURROUNDING OOCYTES

WITH

EXTRACELLULAR LAYERS

Electron microscopic analysis of oocyte sections revealed that induced membrane-associated BuChE appears to be associated with the outermost extracellular matrix of the oocyte and not with the plasma membrane itself. That the molecule is transported to and through the surface of the oocyte is not itself surprising, since the cloned sequence encodes an apparent signal peptide, attached to a known soluble plasma protein. Yet this analysis appears to explain why the synthetic enzyme never gets to the oocyte incubation medium: Apparently, it gets trapped on the way out! Nonetheless, the mode by which synthetic BuChE “sticks” to the oocyte basal lamina remains unclear. Perhaps even in the absence of specific membrane-binding elements the highly glycosylated BuChE interacts with specific lectins which recognize its L1 sugar moiety, similar to the glycosylation residues of cell adhesion molecules (Nybroe et al., 1988). Should this be the case, it might indicate a putative involvement of the embryonically expressed BuChE (Layer et al., 1988) in cell recognition and tissue-remodeling processes.

D. mRNA INTENSIFICATION OF BuChE SIGNALS ASSOCIAnm W I TH OOCYTE SURFACE Coinjection of poly-A(+) KNA from fetal human brain or muscle with synthetic SPGRNA resulted in quantitatively and qualitatively intensified immunosignals and indicated enhanced aggregation of nascent BuChE

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at the oocyte surface. The number and fluorescent intensity of both “clusters” and “patches” were increased without affecting the relative animal-vegetal pole distributions. However, the quantifiable enhancement of fluorescence induced by muscle RNA was twice that stimulated by coinjection with brain RNA. Similarly, the overall intensity of the signals produced after coinjection with muscle RNA clearly exceeded that resulting from supplemental brain RNA. Interestingly, mRNA extracted from fetal liver, which does not express significant membrane associated ChE (Brimijoin and Rakonczay, 1986), did not induce significant changes in the observed patterns of fluorescence. Both brain and muscle express ChEs, a significant fraction of which is associated with the extracellular surface (Zakut et al., 1985; Dreyfus et al., 1983; Toutant and Massoulie, 1987). Furthermore, the pattern of aggregation in the oocytes stimulated by each type of RNA mirrored the organization of ChE in that tissue (Wallace et al., 1985; Wallace, 1986; Mollgard et al., 1988). Aggregation factors have already been described in connection with the aggregation of nicotinic acetylcholine receptor and ChE both in uivo and in vitro (Anglister and McMahan, 1985; Wallace et al., 1985; Wallace, 1986).Thus, these results suggest that the aggregation factors needed to organize ChEs correctly within the membrane are expressed in a tissue-specific manner and that their effects can be mimicked to some extent in the oocyte system. The report by Rotundo and Carbonetto ( 1987) that neurons selectively aggregate hydrophobic Gq AChE along neuritic extentions in culture was proposed to imply use of a “different strategy” from that employed by skeletal myotubes in clustering ChE and could indicate that our observations in the polarized oocytes reflect qualitative and not merely quantitative differences in the accessory factors provided by brain and muscle. This would agree with biophysical theories suggesting that the stimulation of endplate currents at neuromuscular junctions depends on the accurate spatiotemporal interaction of acetylcholine with the nicotinic acetylcholine receptor on one hand and AChE on the other (Rosenberry, 1979).

VI. Closing Remarks

A. SUMMARY A N D CONCLUSIONS We have employed a combination of molecular biological, classical biochemical, and immunocytohistochemical techniques to monitor the biogenesis of synthetic BuChE in Xenopus oocytes. Our synthetic BuChE

mKNA encodes the complete amino acid sequence of human serum BuChE, a putative signal peptide containing a yet unaccounted for upstream open reading frame, a 3' uritranslated sequence, and a poly-A tail. Synthetic RNA was injected alone and in conjunction with tissueextracted poly-A( +) RNA from fetal human brain, muscle, and liver. T h e following observations were made and are summarized in Table I :

1. Synthetic BuChE mKNA alone is sufficient to direct the synthesis in oocytes of a n enzymatic activity characteristic of normal hunian serum BuChE and clearly distinct from AChE. 2. Synthetic BuChE undergoes limited subunit assembly in the oocytes to generate functional dimeric molecules. 3. Synthetic BuChE associates with the oocyte membrane, where it '1-4Hl.k: I MOLECULAR ORIGIO NF R L C H ECHARACTERISTICS" In z v i w (Refei cncc)" Substrate Specificity

BuSCh > AS(:h ( I )

k',,,

6 x 10-'12.1 ( I )

3 x l o - ' iCI ( 2 ) OP sensitivity (lCBO) 1.3 x 10-"nf ( 2 )

Su bcellular localization

In

0110

(Refer ence)h

Origin Primary aa sequence Primary aa sequence

3 x IO-hiZI (2)

Primarily sec reton (4) Surface-associated and intracelluar ( 3 )

Oliogmeric assembly

Primarily tetr'arner ( 5 )

Dinier (2)

Association w/tail

Minor fractiori i n muscle (G)

Supported by muscle mRNA (3)

ECM associations

Patches (NMJ); clusters (Brain) (7,8,9)

Patches and clusters (distribution modified by tissue RNAs)

Primary aa sequence Signal peptide and nonspecific adhesion? Requires helper proteins Requires rail components: other factors? Modulated b y tissue-specificaggregation factors

" BriChE characteristics in utuo and in Xeuopu.~oocytes microinjected with svnthetic BuChEmRNA and native poly-(A)+ RNA from fetal human brain and muscle. Conclusions regarding the molecular origin of the various properties are noted in right-hand column. * Key to references: 1. Brown rl al. (1981); 2. Soreq et al. (1989); 3. Dreyfus ef al. (1989); 4. Silver (1974); 5. Lockridge et al. (1979); 6. Dreyfus et al. (1983); 7. W'allaceptal. (1985); 8. Wallace (1986); 9. Mollgard ef (21. (1988).

132

SHLOMO SEIDMAN A N D HERMONA SOREQ

can be seen to interact primarily with the oocyte’s outermost extracellular matrix and not with the plasmalemma itself. 4. BuChE aggregates are preferentially but not exclusively deposited at the animal pole of the oocyte. 5. Coinjection with brain or muscle RNA induces the appearance of complex oligomeric forms of BuChE in a tissue-specific manner and with the enhanced aggregation of synthetic BuChE in a manner consistent with ChE organization in the native tissues. It is now clear that the structural heterogeneity characteristic of AChE is defined by sequence variations at the COOH-terminal of the molecule. Thus, distinct cDNA clones representing both globular and asymmetric forms have been isolated, diverging only in the 3’ terminal region of the coding sequence. No cDNA sequence polymorphism has yet been found to account for the structural variants of BuChE. In fact, the only additional BuChE DNA clones publicized to date are a slightly 5’ extended cDNA clone from brain (McTiernan et al., 1987), which encodes an amino acid sequence identical to the one described here; a genomic DNA clone encoding the human “atypical” serum BuChE, containing a single amino acid substitution that has been assumed to account for the unusual catalytic properties of this allelic polymorph (McGuire et al., 1989);and a 3‘ extended cDNA clone characteristic of nervous system tumors (i.e., glioblastomas and neuroblastomas) encoding a protein of as yet unidentified catalytic properties (Gnatt et al., 1990). Since these findings represent the results of a considerable body of screening efforts, it now seems likely that BuChE polymorphism is not regulated primarily by alternative splicing, but rather by the posttranslational activities of as yet undefined tissue-specific helper proteins. Why then d o we not find significant quantities of synthetic oocyteproduced BuChE in the oocyte incubation medium? Relying on the current model for AChE biogenesis we would expect the 5’ terminal of the nascent protein to direct its transport to the oocyte surface, while the 3‘ terminal would presumably dictate the nature of its interaction with the peripheral cellular elements (also see Caras and Weddell, 1989). Clearly, synthetic BuChE mRNA encodes a protein destined, at least in part, for extracellular transport. Yet it does not reach the medium. One possible explanation for this finding is that the complex extracellular matrix surrounding the oocyte provides an unlimited solid support for nonspecific binding of BuChE, which liver cells do not offer. Other possible explanations include the relative availability of helper proteins involved in BuChE association with extracellular elements, the relative abundance of lectins, and incorrect glycosylation of the synthetic enzyme,

CHOLINES 1 EKA\E 1’KOl)l

(

1 ION I N XLVOPUS OOC Y? t S

133

which could lead to nonspecific binding to the extracellular matrix. I t is worth noting in this respect that in the blood, BuChE is known to interact with plasma lipoprotein components to form large complexes, which may resemble parts of the oocyte surface elements (Whittaker, 1986). It is therefore possible that within the lipoprotein-rich oocytes BuChE forms lipoprotein-containing aggregates, which may associate with the oocyte surface in a manner different from that observed in vivo. T h e distinct lack of interaction between the clone-produced BuChE and the plasma membrane, combined with the hydrodynamic profile of the molecule, would appear to exclude the involvement of a pronounced hydrophobic element in the molecule’s cell surface association, implying that the hydrophobic ‘‘leader’’ sequence is probably removed by the oocyte during transport to the surface. It should be noted here that the fully open 5’ reading frame in the region upstream ofthe putative leader sequence in both brain and liver cDNAs could, in principle, encode a protein with an N-terminal cytoplasmic part followed by a hydrophobic membrane-spanning segment similar to that found in the asialogylcoprotein receptor (Spiess and Lodish, 1986). If that would be the case, one might postulate controlled proteolysis of membrane-bound liver BuChE as a mechanism of regulating serum BuChE levels. However, several arguments speak against this possibility: 1. Neither in the serum nor in other tissues does BuChE exist as an integral membrane protein. 2. T h e N-terminal amino acid of purified BuChE from serum and other tissues always corresponds to the residue appearing after the putative signal peptide. 3. No BuChE is found associated with the oocyte plasma membrane at all.

Thus, the exact nature of the interaction between synthetic BuChE and the oocyte ECM remains obscure. O u r BuChE cDNA resembles asymmetric AChE cDNA more than gobular hydrophobic AChE cDNA in the sense that it does not contain the C’ terminal peptide characteristic of PI-bound membrane proteins. Furthermore, synthetic BuChE seems capable in the presence of appropriate helper proteins of interacting with the tail component(s). Yet this clone encodes a secretory protein. Thus, these data could represent the first evidence that secretory and tailed ChEs are encoded by the same RNA transcript. If so, the determining factor in assembly of asymmetric forms could be the tissue-dependent provision or lack thereof of the collagenlike component. In addition, our evidence suggests that AChE and BuChE may be sufficiently similar to compete for accessory proteins

134

SHLOMO SEIDMAN A N D HERMONA SOREQ

in vim. Such a model would help explain the phenomenon of hybrid AChE-BuChE complexes (Tsim et al., 1988b) as well as provide a basis on which to think about the coordinated regulation of tissue-specific patterns of AChE-BuChE expression.

B. FUTURE DIRECTIONS It is possible to envision a number of lines of research that would reflect logical continuations of this work including: 1. In vitro modification of the BuChE cDNA by site-directed mutagenesis aimed at revealing important domains in the BuChE molecule. This work could involve an analysis of regions, or even specific amino acids proposed to be involved in formation and function of the active site of the molecule. We could also use the system to address questions such as how much of the sequence is actually required for catalytic activity by introducing controlled deletions at both ends of the molecule. Note that the use of naturally occurring resources such as the various “atypical” alleles, permits, with the aid of polymerase chain reactions and the reservoir of oligonucleotide primers used to sequence this clone (Soreq and Cnatt, 1987), the rapid determination of point mutations at the level of an individual person (McCuire et al., 1989, for example). Combined with genetic engineering tools, oocyte microinjection, and biochemistry, specific mutations and their effect on biological activity can be rapidly evaluated, bypassing the need to establish stably transfected cell lines. 2. Purification-characterization by molecular cloning of tissuespecific messenger RNAs involved in the assembly of complex multimeric BuChE molecular forms. In essence, the coinjection experiments already performed could define a novel bioassay for proteins that could not be previously pursued by direct expression cloning given the lack of a suitable assay. 3. Further investigation of the mode of association of BuChE with the oocyte membrane using polyclonal antibodies elicited against cloneproduced peptides from defined domains in the ChE polypeptide. 4. Development of transient and stable transfected cell lines or animals expressing BuChE DNA in significant quantities. The establishment of transgenic mice carrying human BuChE DNA offers a particularly promising opportunity to assess the involvement of BuChE expression in development and to study the phenomenon of CHE gene amplification in an experimental mammalian system.

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135

5. Parallel studies using the homologous AChE cDNA isolated in our laboratory followed by the con~tructionof chimeric AChE-BuChE molecules using recombinant DNA techniques. Coinjection of synthetic AChE and BuChE RNA will allow a detailed analysis of the interaction between these two molecules, and their competition for association with “third party” accessory elements. In fact, most of these lines of experimentation have already been initiated and developed to some extent in our laboratory. T h e most exciting project, of course, is the functional analysis of a full length cDNA clone for human AChE. Such a study will finally allow a direct comparison of the molecular determinants specifying the parallel biosynthetic pathways of these two intriguing biochemical cousins within a single biological environment.

Acknowledgments

We are grateful to Dr. Patrick Dreyfus (INSERM, Paris) for his numerous contributions to this work and to Dr. David Phillips (Population Council, NYC) for electron micrographs of Xenopuv oocyles. This work was supported by the U.S. Army Medical Research and Development Command under conti-act DAMD 17-87-C-7269 and by the Association Francaise Contre Les Myopathies ( A F M ) (to H.S.).

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Soreq, H., and Gnatt. A. (1987). hlol. Mruroblol. 1, 47-80. Soreq, H.,and Prody, C. (1989). In “~:ornputer-AssistedModelling of Receptor-l.igand Interactions: Theoretical Applications 10 Drug Design” (A. Golomhec-k, and R. Rein, eds.), pp. 347-359. Liss, New Yoi-k. Soreq, H., Parvari, R., and Silman, I . (1982). P‘roc. Nu//.Arad. Sci. O . S . A . 79, 830-834. Soreq, H., Zevln-Sonkin, D., and Raron. N . (1984). E M B O j . 3, 1371-1375. Soreq, H., Dziegielewska, K. M., Zevin-Sonkin, D., and Zakut. H. (1986). C r l l . M d . h’rurobiol. 6, 227-237. Soreq, H . , Malinger, G., and Zakut, H . (1987). Him. Rrprod. 2,689-693. Soreq, H.. Seidman, S., Dreyfus, P. .4., Ze\in-Sonkitl, I)., and Lakut. H. (l989).,/.Liroi. O’hrn. 264, 10608-1061:3. S p i e s , M., and Lodish, H. F. (1986). Cdl ((;rrtnb7~dp,h f u ~ s 44, . ) 177-185. Surnikawa, K., Parker, I., and Miledi. K. (1986). I n “Membrane Control.” pp. 127-139. Spr-inger-Verlag, New York. Sytkowski, A. J . , Vogel, 2.. and Nlrenherg. M. U’. (1973). Pmr. .Vat/. Acrid. Scr. [‘.,5.A.70, 270-274. Tanimura, T., Katsuya, l.,and Nishiniura, H . (1967).A d . E m ~ m nHPultlr . 15, 60%613. Toutant, J.-P., and Massoulie,]. (1987).In “Mammalian Ectoenzymes” (Kenny and ~I urner, eds.), pp. 289-328. Elsevier, Ainsterclam. Tsim, K. W. K., Randall, W. R., and Barnard. E. A . (1988a). E M B 0 . J .7,2451-2456. Tsim, K. W. K., Randall, W. R., and Batnard, E. A. (l988h).Proc. Natl. A md . Scz. l;..S.A.85, 1262- 1266. U.N. Security Council Report of Specialists Appointed by the Secretary Genei-al ( 1984). Number S116433. Vigny, M., Bon, S., Massoulie, J . , and I.aterrier, F. (1978).E u r . J . Bzochrm. 85, 3 17-323. Vigny, M.. Martin, G. R., a n d Grotendorsr. G. R. (1983).j. B i d . Clwrn. 258, 87!)4-8798. Wallace, B. G. (1986).J. CellBzol. 102, 783-794. Wallace, B. G., Nitkin, R. M., Reist, N . F,..Fallon,J. R., Moayeri, N. N., and McMahan. U . J . (1985).Nature (London) 315,574-577. Weeks, D. L., and Melton, D. A. (1987). C r l l (Cambridge, Mrm) 51, 861-867. White, M., Mixter-Mayne, K., Lester, H . A., and Davidson, N. (19%). Proc. Null. Atad. Sri. U.S.A. 82,4852-4856. Whittaker, M. (1986).“Cholinesterase: Monographs in Human Genetics,” Vol. IX.Larger, Basel. Wischnitzer, S. (1966). A d v . Morphog. 5, 13 1- 179. Yates, C. M., Simpson, J., Maloney, A. F.J . , (;ordon, A , , and Reid, A. H. (1980). Lunret 2, 979-980. Zakut, H., Matzkel, A., Schejter, E . . Avni, A , , and Soreq, H. (1985). J . ,\’rzirochrvt. 45, 382-389. Zakut, H., Even, L., Birkenfeld, S., Malinger, G . , Zisling, R., and Sot-eq. H . (1988). Cancrr (Phzladelphia) 61, 727-737. Zakut, H., Ehrlich, G., Ayalon, A,, Protly. C;. A , , Malinger, G , ,Seidinan, S., Kehlcnbach, R., Ginzherg, D., and Soreq, H. (l990).,/.C l i r r . ftzim/. (in press).

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POTENTIAL NEUROTROPHIC FACTORS IN THE MAMMALIAN CENTRAL NERVOUS SYSTEM: FUNCTIONAL SIGNIFICANCE IN THE DEVELOPINGAND AGING BRAIN By Dalia M. Araujo, Jean-Guy Chabot, and Rerni Quirion Douglas Hospital Research Centre and Department of Psychiatry McGill University Verdun, Quebec, Canada H4H 1R3

1. Introduction 11. Nerve Growth Factor

NGFs Mechanism of Action NGF and the Basal Forebrain Cholinergic Neurons NGF in Normal Aging arid in Alzheimer's Disease NGF Effects on CNS Neurons Other Than Cholinergic Basal Forebrain Neurons 111. Fibroblast Growth Factor A. FGFs Mechanism of Action B. Role of FGF 1V. Insulin and lnsulinlike Growth Fac-tors A. Insulin B. IGFs V. Brain-Derived Neurotrophic Factor A. BDNF and BDNF Receptors B. Role of BDNF VI. Ciliary Neurotrophic Factor V I I . Epidermal and Transforming Growth Factors A. EGF B. TGFs C . EGF and TGF Mechanism of Action VIII. Platelet-Derived Growth Factor IX. Interleukins and Other Lymphokines A. 1L-l B. IL-2 C. Other Lyrnphokines X. Hormones and Neurotransmitters as Neurotrophic Factors A. Estrogen B. Thyroid Hormones C. Adrenal Hormones D. Neurotransmitters XI. Miscellaneous Factors with Potential Neurotrophic Activity XII. Concluding Remarks References A. B. C. D.

141 INTERNATIONAL REVIEW OF NEUROBIOLOGY, VOL 32

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1. Introduction

Over the past decade, the list of proteins that can be classified as neurotrophic substances has increased dramatically to include not only stimulators, but also inhibitors, of cell proliferation. In addition, it has become increasingly clear that the effects of growth factors (GFs) on a certain cell may be quite diverse and furthermore, the cell types responsive to an individual GF may be varied. For many years, the only known neurotrophic substance was nerve growth factor (NGF), which is associated with a limited population of neuronal cell types in both the peripheral and the central nervous systems. Recently, however, the list of substances that may be classified as potential neurotrophic substances has expanded considerably. In addition, many previously characterized GFs or hormones are now known to act on certain populations of neurons either in the peripheral or the central nervous system (CNS), and have subsequently been classified as “new” neurotrophic factors. The importance of neurotrophic factors in the developing brain has been clearly established. However, the role(s) of many neurotrophic substances in the adult CNS has yet to be elucidated. At present, it seems evident that many of these GFs may function as maintenance proteins for mature central neurons (see below). Moreover, studies of the effects of GFs on lesion-induced deficits in biochemical markers and behavior have underscored the significance of certain GFs as “protective” substances in the adult CNS. These findings may prove of considerable importance in the future therapy of neurodegenerative diseases such as Alzheimer’s disease (AD). The present review is intended to provide an overview of the principal characteristics and roles of some well-defined neurotrophic substances and other proteins that may eventually be categorized as novel neurotrophic agents. T h e main purpose of the review is to accentuate the importance of these factors in the developing and the aging brain. Particular emphasis is placed on the potential roles of GFs as causative or therapeutic agents in neurodegenerative diseases. Thus, discussion of the involvement of these GFs in non-CNS tissues is confined to results that are of some relevance to the potential neurotrophic effects of these fac ors in the CNS. II. Nerve Growth Factor

Considerable interest in NGF as a neurotrophic factor in the CNS has arisen, at least in part, from the large body of evidence that has shown

that NGF is a necessary element in the survival of central cholinergic neurons. Moreover, NGF and the rriRNA coding for NGF are present and concentrated in the target tissues of the cholinergic neurons that require it for survival (Korsching et al., 1985; Shelton and Reichardt, 1984). Since this review focuses o n the role of GFs in the CNS, the reader is referred to several comprehensive reviews on the role of NGF in the peripheral nervous system (l'hoenen and Barde, 1980; Levi-Montalcini, 1982; Thoenen et al., 1987). In the CNS, it is known that NGF can be retrogradely transported along axons to the neuronal cell body (Seiler and Schwab, 1984;Johnson et al., 1987), where it may exhibit a variety of stimulatory effects. For example, NGF enhances the synthesis of acetylcholine (ACh) (Korsching et al., 1985; Mobley et al., 1985), possibly as a result of increasing the activity of choline acetyltranst'erase (ChAT) in 712~10(Gnahn et ul., 1983; Hefti et al., 1984, 1985; Mobley et al., 1985) and in 7dr-0 (Honegger and Lenoir, 1982; Martinez et al., 1985, 1987; Hartikka and Hefti, 1988; Hatanaka et al., 1988). Other i7i uitw effects of NGF include the promotion of neuronal survival and neurite outgrowth (Martinez et d.,1985; Gahwiler et al., 1987; Hatanaka et al., 1988).

A. NGF's MECHANISM OF Ac-rroiw T h e exact biochemical mechanism by which NGF maintains neuronal viability is not known. However, NGF is thought to act by a series of steps that involve the synthesis and release of NGF from target tissues and the binding of NGF to its receptors on the axons, followed by internalization and retrograde transport of NGF to the cell body (Schwab et al., 1979; Seiler and Schwab, 1984). Evidence to support this mechanism of action for NGF has been steadily increasing over the past few years and is now extensive. For example, NGF and its receptor have been shown by a variety of methods to be retrogradely transported in both peripheral and central nervous tissues (Korsching and Thoenen, 1983; Palmatier rt al., 1984; Johnson et al., 1987; see also Springer, 1988; Pioro and Cuello, 1989a,b). T h e use of various immunohistochernical techniques has permitted the identification of the NGF receptor protein in basal forebrain-septa1 neurons of many species including humans (Hefti et d., 1986; Bernd et al., 1988; Eckenstein, 1988; Kiss et al., 1988; Kordower et al., 1988; Yan and Johnson, 1988; Assouline and Pantazis, 1989; Sofroniew et ul., 1989; Pioro and Cuello, 1989a,b). Immunoprecipitation (Taniuchi and Johnson, 1985; Taniuchi et al., 1986) and autoradiographic techniques (Richardson et al., 1986) have further substantiated this. In addition,

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NGF receptor mRNA is expressed in the rat basal forebrain-septa1 area (Buck et al., 1987, 1988), brain regions that are enriched with endogenous NGF. Binding of NGF to its specific receptor appears to be a necessary requirement for the subsequent trophic andlor maintenance actions of NGF.

B. NGF A N D THE BASAL FOREBRAIN CHOLINERGIC NEURONS In the CNS, unlike the peripheral nervous system, catecholaminergic neurons do not appear to be responsive to the trophic effects of NGF (Schwab et al., 1979; Dreyfus et al., 1980; Shalaby et al., 1984). However, Schwab and colleagues ( 1979) provided initial evidence implicating NGF as a trophic factor for cholinergic neurons in the brain. Since then, overwhelming evidence has clearly demonstrated that in the CNS, only certain populations of cholinergic, but not catecholaminergic, neurons are susceptible to the trophic properties of NGF (for a review, see Hefti et al., 1989). This is in marked contrast to the peripheral nervous system, in which cholinergic neurons are not responsive to NGF (Thoenen and Barde, 1980). Cholinergic neurons of the basal forebrain-septa1 region are particularly sensitive to the effects of NGF. For example, NGF enhances the activity of ChAT in cultures of nucleus basalis (nbM) and septum (Honegger and Lenoir, 1982; Hefti et al., 1985; Gnahn et al., 1983; Hartikka and Hefti, 1988; Hatanaka et al., 1988; Alderson et al., 1989; see also review by Hefti et al., 1989). In vivo effects of NGF on basal forebrain cholinergic neuron activity further substantiates the proposal that in the CNS, NGF may mostly, but not exclusively, function as a cholinergic neurotrophic factor. For example, continuous infusion of NGF appears to prevent cholinergic neuron death following basal forebrain lesions (Hefti, 1986; Williams et al., 1986; Kromer, 1987; Gate et al., 1988). Moreover, intraventricular administration of NGF augments ChAT activity in both nbM and septa1 neurons of neonatal rats (Gnahn et al., 1983; Mobley et al., 1985; Johnson et al., 1987). The presence of NGF receptors (Schwab et al., 1979; Seiler and Schwab, 1984; Taniuchi and Johnson, 1985; Richardson et al., 1986; Kiss et al., 1988; Pioro and Cuello, 1989a,b)and NGF receptor mRNA (Buck et al., 1987, 1988; Batchelor et al., 1989; Gage et al., 1989; Lu et al., 1989) in the adult rat basal forebrain-septa1 region and their colocalization with ChAT immunoreactivity in these neurons suggests that NGF may be involved in the function of these neurons in the adult brain. Further evidence for this has been provided by studies using animals with experi-

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mental lesions. In these studies, it was observed that NGF attenuated the lesion-induced deficits in ChAT activity (Hefti et al., 1984; Haroutunian et al., 1986; Gage et al., 1988, 1989) in basal forebrain neurons. These protective effects of NGF observed following fimbria-fornix transections or lesions of the nbM were specific to cholinergic neurons; the loss o f other neurons induced by such lesions was not prevented by NGF (see Hefti et al., 1989). Thus, in the adult brain, NGF may function mostly as a maintenance and protective factor for basal forebrain cholinergic neurons. Results from behavioral studies further substantiate this hypothesis.

C. NGF

IN

NORMALAGINGA N D

I N A L Z H E I M E R ’ S DISEASE

T h e involvement of basal forebrain-septa1 cholinergic neurons in memory and learning processes has been well established (see for example, Olton et al., 1979, 1980; Bartus et al., 1982; Pallage ~t al., 1986). Furthermore, it is the basal forebrain-septa1 cholinergic projections to the neocortex and hippocampus that are severely compromised i n AD (see, for example, Whitehouse ul nl., 1982). T h e loss of these projections appears to be responsible for some of the memory dysfunctions associated with AD (see Bartus et al., 1982). As described above in Section II,B, basal forebrain cholinergic neurons have been demonstrated t o be responsive to the trophic effects of NGF. For this reason, research into the effects of NGF on memory and learning processes mediated by these neurons has become a subject of intense investigation. Unfortunately, there have been several discrepancies in the results, which may be due to differences in experimental protocols (injection route, source of NGF, type of NGF, etc.). For example, NGF has been shown to anieliorate the behavioral deficit induced by cholinergic neuron atrophy in rats with lesions of the fimbria-fornix (Will and Hefti, 1985; Will et al., 1988) and nucleus basalis magnocellulark (Mandel et al., 1989), while exacerbating the impairment in rats with septa1 lesions (see review by Will et al., 1988). Thus, interpretation o f the overall effects of NGF on behavior has proven to be a difficult task. T h e effects of NGF on behavioral deficits that are consequent to normal aging have been complicated by the lack of conclusive evidence demonstrating alterations in NGF-related mechanisms in the aged brain. So far, only one study has shown that NGF and its mRNA appear to be reduced in aged rats (Larkfors et al., 1987). Similarly, it is not yet clear whether NGF receptor density is affected in normal aging (see review by Hefti et al., 1989). In addition, only a few studies have demonstrated that

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intraventricular administration of NGF to a population of previously characterized “impaired” aged rats improves their performance in a swimming maze paradigm (Gage and Bjorklund, 1986; Fischer et al., 1987; Gage et al., 1988).Thus, it remains a matter of speculation whether NGF may eventually be of benefit in counteracting the memory impairments associated with normal aging and with AD. In cortical tissues obtained from aged humans and AD patients, normal levels of NGF mRNA and NGF receptor mRNA (Goedert et al., 1986, 1989),as well as NGF receptor imrnunoreactive material have been measured (Mufson et ul., 1989a). In addition, it has been shown that NGF receptors and ChAT remain colocalized in the basal forebrain in normal aging (Mufson et al., 1989a) and in AD (Kordower r t al., 1989). Conversely, decreased NGF receptor inimunoreactivity has also been demonstrated in basal forebrain regions of aged rats (Koh et al., 1989) and of AD patients (Mufson et al., 1989b). However, the suggestion that NGF may be beneficial in the treatment of AD steins from studies that have documented the effectiveness of NGF in preventing basal forebrain cholinergic cell loss in lesioned animals and in ameliorating certain memory tasks in lesioned and aged rats. Thus, although NGF-related mechanisms may not be directly responsible for the symptoms observed in AD, NGF may still be a potentially useful therapeutic agent in AD, if only because NGF retards cholinergic cell death in the neocortex and hippocampus and improves the function of these neurons. However, potential therapy with NGF should be approached with caution since some evidence has suggested that indiscriminate sprouting may occur with administration of trophic factors (Butcher and Woolf’, 1989). Thus, excessive sprouting of neurites induced by NGF could be detrimental. Woolf and Butcher (1989) have proposed that NGF antagonists may be more effective as a therapy in AD. Clearly, it is evident that further research in animal models of aging is required before attempting to use this factor in the treatment of AD.

D. NGF EFFECTS ON CNS NEURONSOTHER ’ r H A N CHOLINERCIC BASAL FOREBRAIN NEURONS Cholinergic interneurons in the striatum are sensitive to the effects of NGF. For example, NGF augments ChAT activity in cultures of rat striatal interneurons (Martinez et al., 1987; Hartikka and Hefti, 1988) and in neonatal rats in uiuu (Mobley et al., 1985; Johnston et al., 1987). In addition, Gage and co-workers ( 1989) have demonstrated that chronic infusion of NGF into the striatum partially reverses the cholinergic hy-

pertrophy induced by striatal tissue damage. Thus, striatal cholinergic neurons, like the cholinergic neurons of the basal forebrain-septa1 area, are sensitive to the trophic properties of NGF. Unlike the cholinergic neurons of the basal forebrain and the st riatum, pontine cholinergic neurons (Gnahn et al., 1983; Mobley P t al., 1985; Knusel and Hefti, 1988)appear to be unresponsive to the neurotrophic effects of NGF. Similarly, the cholinergic motoneurons of the chick and rat spinal cord exhibit only a transient response to NGF (Sniith and Appel, 1983; Smith et ul., 1986). Thus, it may not be entirely appropriate to classify NGF as a neurotrophic factor for central cholinergic neurons, but rather niore specifically as a neurotrophic factor foi- forebrain cholinergic neurons undei- certain conditions. NGF (Large et nl., 1986; Shelton and Keichardt, 1986) anti NGF receptor mKNA (Ernfors rt d., 1988) are present i n areas such as the thalamus, cerebellum, olfactory bulb, anti medulla oblongata, all of which contain sparse cholinergic innervation. However, the effects of N G F on noncholinergic central neurons remain. for the most part. to be irivestigated.

111. Fibroblast Growth Factor

_1_he fibroblast growth factors (F<;Fs) are two distinct mitogenic factors that have been isolated from both peripheral tissues arid the CNS. Acidic FGF (aFGF) and basic F G F (bFGF) have a similar mass and share a high degree of homology (see review by Morrison, 1987). I n addition, aFGF shares a high degree of honiology with other known GFs such as the interleukins (ILs) (Ginienez-Gallego et ul., 1985). Information derived from the full characterization and sequencing of the two FGFs has suggested that both aFGF and bFGF may be identical to other previously classified GFs (see Gospodai-owicz rf nl., 1986a).

.4.FGF's MECHANISMOF A c ~ r o l v T h e mitogenic effects of both FGFs appear to be mediated by two distinct classes of receptors, one with high affinity for aFGF and the other with high affinity for bFGF, both of which have been identified 111 many cell types (Neufeld and Gospodarowicz, 1986). I n contrast, the FGFs appear to be selectively located within neuronal structure (Pettinan rt ul., 1986; Firiklestein et ul., 1988). Despite extensive sequence homolo-

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gies, bFGF seems to be a more potent neurotrophic factor than aFGF for most neurons (Morrison, 1987). However, a wide variety of nonneuronal cell types of mesodermal and neuroectodermal origin, including fibroblasts, endothelial cells, and glial cells have been shown to be equally sensitive to the mitogenic effects of aFGF and bFGF (Gospodarowicz et al., 1986b; Pruss et al., 1982; Pettman et al., 1986). The mitogenic effects of the FGFs on glial cells in vztro have provided some intriguing evidence for a critical involvement of FGF in the interaction between neurons and glia in the developing brain. At present, it is not known whether FGF is secreted from the neurons or whether it is present on the outer membrane of neurons. Thus, the mechanism(s) responsible for this FGF-mediated neuron-glia interaction are not clear.

B. ROLEOFFGF Recently, several lines of evidence have indicated that bFGF may be an important neurotrophic factor in the CNS. CNS neural tissues are a rich source of bFGF (Bohlen et al., 1985; Pettman et al., 1986). Moreover, bFGF has been shown to promote neuron survival and neurite extension in hippocampal (Walicke et al., 1986), cortical (Morrison et d., 1986), spinal cord (Unsicker et al., 1987), and mesencephalic (Ferrari et al., 1989) neurons in vitro. These properties of bFGF are clearly not mediated by glial cells (Hefti et al., 1989). In addition, other GFs that are known to promote glial cell activity do not alter the survival of neurons that are sensitive to the effects of bFGF (Walicke and Baird, 1988). Many of the biological effects of the FGFs, like those of other GFs (see below), are similar to those elicited by NGF. For example, both NGF and bFGF are capable of promoting neurite extension in PC 12 cells (Togari et al., 1985; Kydel and Greene, 1987). In adrenal chromaffin cell cultures, both aFGF (Claude et al., 1988) and NGF (Unsicker et al., 1978; Ogawa et al., 1984) are potent stimulators of cell proliferation and neurite outgrowth. Similarly, both NGF and bFGF stimulate ChAT activity in cultures of rat embryonic basal forebrain neurons (see Hefti et al., 1989). However, unlike NGF, these effects of bFGF on both PC12 cells and basal forebrain neurons appear to be evident only in the initial days of these cells in culture (Stemple et al., 1988; Hefti et al., 1989). Therefore, it appears that bFGF may be of crucial importance in the early developmental stages. Apparently, bFGF also functions as a “protective” factor following lesions of various neuronal pathways. Anderson et al., (1988) demon-

strated that intraventricular infusion of bFGF following a fimbria-fornix transection prevented the loss of a large portion of the cholinergic neurons in the septum. In addition, bFGF has been shown to attenuate the loss of neurons in adult rat retinal ganglion cells after optic nerve section (Sievers et al., 1987) and in adult dorsal root ganglion neurons following sciatic nerve cut (Otto et al., 1987). These results provide ample evidence for the importance of bFGF in the survival of adult neuronal populations. In the above experimental models, however, NGF was shown to be more effective than bFGF in preventing cell death (Hefti, 1986; Otto rt al., 1989) and available data have not clearly determined whether NGF and bFGF act on distinct neuronal sub-populations. Although it is possible that both factors may act independently, overlapping tunctions of the two factors seem likely. However, additive effects of the two trophic factors cannot be completely excluded.

IV. Insulin and lnsulinlike Growth Factors

Several lines of evidence have demonstrated unequivocally that insulin and the structurally related irisulinlike growth factors (l(;F-1 arid -2) are present in fetal and adult mammalian brain (see reviews by Baskin ot al., 1987, 1988). Receptors specific for each of these peptides have also been identified in the CNS (Baskin rt al., 1987, 1988; Quirion et ul., 1988; Araujo et al., 1989a). In the developing brain, insulin and the IGFs may function as trophic factors, whereas in the adult brain they may also act as neuromodulatory substances (see below).

A. INSULIN lnsulin-immunoreactive material has been identified in acid extracts of rat brain. However, there is some controversy as to the source of' insulin within the adult CNS. Although synthesis of insulin by neurons in culture has been demonstrated (Birch et al., 1984; Clarke rl NI., 1986), there is no indication that this occurs in viva In addition, there is little evidence for the presence of proinsulin or insulin mRNA in adult brain (see Young, 1986), although some studies have suggested that insulin mRNA can be detected in the hypothalamus (Clarke rt al., 1987; Schechter et al., 1988). T h e results of these latter studies are still not fully conclusive, since the possibility of cross-hybridization of the insulin probe with IGFs could not be ruled O U L .

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In the brain, receptors specific for insulin have been detected using a variety of techniques (see Baskin el al., 1988; Lesniak et al., 1988). Their localization to neurons within the CNS has been suggested from indirect experiments using enriched neuronal cultures, which revealed the presence of high densities of insulin binding sites (Boyd et al., 1985; Masters et al., 1987).Further support for the localization of insulin receptors on neuronal membranes has been obtained from autoradiographic analysis of the binding of iodoinsulin to rat brain sections (see Baskin et al., 1988). In addition, insulin binding sites have been identified in the aged rat brain, although it is not clear whether their function is altered with age (R. Quirion, J.-G. Chabot, and J . Chang, unpublished). Despite the identification of insulin and insulin receptors in the mammalian brain, the possible role of insulin in the regulation of neuronal function remains elusive. Some reports have established that insulin binding sites are also present on nonneuronal cell types within the CNS (see Baskin et al., 1988). This has complicated further clarification of the function of insulin in the CNS. Nevertheless, the reportedly dense distribution of insulin receptors in regions enriched with synaptic connections (Corp et al., 1986; Baskin et al., 1988) within the brain suggests that insulin may act as a neuroregulatory and/or neurotrophic peptide. The study of potential neurotrophic effects of insulin has been hampered by its cross-reactivity with the IGF-1 receptor (see below; also reviews by Rechler and Nissley, 1985; Adamo et al., 1989). Insulin has been reported to enhance neuronal survival and neurite outgrowth in a variety of neuronal populations in vitro (see Recio-Pinto et al., 1986; Hefti et al., 1989; Walicke, 1989). However, in all cases, IGF-1 and -2 were more potent neurotrophic agents than insulin. Therefore, it is possible that the observed neurotrophic effects of insulin may have been mediated by IGF-1 receptors (see below). Insulin by itself may be more important for normal cellular metabolism.

B. IGFs IGF-like immunoreactivity and IGF mRNA have been identified in both fetal and adult brain tissue (Han et al., 1987; Sara et al., 1986; Noguchi et al., 1987; Raizada et al., 1987; Beck et al., 1988; Sandberg et al., 1988). The mRNAs encoding IGF-1 and IGF-2 have also been detected in neuronal and glial cell cultures (Brown et al., 1986; Lund et al., 1986; Rotwein et al., 1988). In addition, receptor sites for both IGF-1 and IGF-2 have been identified in glial (Lenoir and Honegger, 1983; McMorris et

al., 1986; Han et al., 1987)and rieuronal (Goodyer et al., 1984; Burgess et al., 1987; Shemer et al., 1987, 1989)cell cultures. In the brain, IGF-2 is reportedly the predominant protein as assessed using immunohistochemical techniques (Haselbacher et al., 1985; Zumstein et al., 1985).In the adult rat brain, IGF-2 mRNL4is more unif'ormly distributed than IGF-1 mKNA (Kotwein et al., 1988). Similarly, it has been reported that the densit) of IGF-2 receptors i n the human (Sara Pt al., 1982)and rat (Goodyer et al., 1984)is much higher than that of IGF-1 receptors. Curiously, though, the niitogenic effects of IGF-2 appear t o he mediated by an interaction with the IGF-1 receptor (see Adanio PL al., 1989; Baskin et al., 1988),although IGF-2 binds with much higher affinity to the IGF-2 receptor (Rechler and Nissley, 1985).Consequentlv, the function of the IGF-2 receptor i i i brain remains obscure and is most likely biochemically associated with the mannose-6-phosphate receptor (Morgan et al., 1987). In glial cultures, IGFs clearly function as mitogens (McMorris PI a/., 1986; see also Baskin et al., 1988; Walicke, 1989). In cultures of central and peripheral neurons, both 1GF-1 and IGF-2 enhance survival and promote neurite extension (Hothwell, 1982; Aizenman et al., 1986; KecioPinto et al., 1986; Aizenman and de Vellis, 1987). T h e IGFs have also been shown to stimulate ChA'l' expression of septa1 neurons in 7~itro(see Hefti et al., 1989). Unlike N(;E', IGFs have also been shown to increase ChAT activity in NGF-unresponsive pontine cholinergic neurons and to enhance dopamine uptake of mesencephalic dopaniinergic neurons (Hefti et al., 1989). In the adult mammalian brain, the existence of receptor sites for the IGFs has been demonstrated using membrane binding (Sara et of.? 1983; Gammeltoft et al., 1985; Rosenfeld et ul., 1987) and receptor- autoradiographic techniques (Bohannon ~t al., 1986, 1988; Baskin et al., 1987, 1988; Adem et al., 1989; Araujo ul., l989a; Matsuo et al., 1989). Moreover, maintained levels of IGF- l receptor sites have been observed in the aged rodent brain (R. Quirion el ul., unpublished). I n contrast to the developing brain, in which IGFs exhibit neurotrophic properties, the function(s) of IGFs in the adult brain are less clear. However, some evidence suggests that IGF- 1 in adult brain may modulate the activity of various neurotransmitter systems in addition to acting as a maintenance factor. For example, Nilssori et ul. ( 1 988) reported that IGF- 1 enhances the release of ACh from cortical slices of adult rat brain, whereas we demonstrated an inhibitory effect o f IGF-1 on ACh release from hippocampal slices (Araujo et al., 1989a). Future studies are now reyuired to expand on the direct neuromodulatory role of IGF- 1 o n neurotransmis-

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sion. In particular, it will be interesting to determine whether this effect is specific to the cholinergic system in the adult brain.

V. Brain-Derived Neurotrophic Factor

Brain-derived neurotrophic factor (BDNF) is a small protein that resembles NGF in some of its physiochemical and physiological properties. For example, its size and charge are close to those of NGF (see Barde et al., 1982, 1987). Furthermore, some physiological effects of BDNF, such as its ability to promote the survival of certain types of neurons in uitro, appear to depend on its interaction with two classes of receptors (Rodriguez-Tebar and Barde, 1988).

A. BDNF AND BDNF RECEPTORS As has been demonstrated for NGF, receptor sites of both high and low affinity for BDNF have been identified in a wide variety of neuronal cell cultures (Barde el al., 1987; Rodriguez-Tebar and Barde, 1988). Comparison of the binding parameters for BDNF and NGF further underlines the similarities between the two proteins: The association and dissociation constants for BDNF binding to its high-affinity sites in cultures of chick dorsal root ganglia and rat spinal ganglia (RodriguezTebar and Barde, 1988)are similar to those reported for NGF (see Sutter et al., 1979).As has been shown for NGF (see review by Hefti et al., 1989), the presence and relative density of the high-affinity BDNF sites in certain neuronal populations appear to correlate with the physiological response elicited by the protein (Barde et al., 1987; Rodriguez-Tebar and Barde, 1988). The only striking differences in the binding parameters between the two proteins appear to be the association and dissociation rates for their respective low-affinity sites. Whereas NGF rates are extremely rapid, those for BDNF are much slower (Barde et al., 1987; Rodriguez-Tebar and Barde, 1988).

B. ROLEOF BDNF

BDNF has been shown to support and maintain the survival of a variety of embryonic vertebrate neurons both in uitro and in vivo (Barde et al., 1982, 1987; Hofer and Barde, 1988), and at least some of these

neurons have been shown to respond to NGF. For example, the survival of chick dorsal root ganglia and rat spinal ganglia in culture can be sustained equally well by NGF or BDNF (Lindsay et al., 1985; Davies et al., 1986). In contrast, many neurons that require BDNF for maintenance and survival are not responsive to NGF, and vice versa. Most notably, BDNF does not support the survival of sympathetic neurons and parasympathetic ciliary ganglia (Lindsay et al., 1Y85), which are critically dependent on NGF. Conversely, BDNF is a necessary trophic component for the survival of placode-derived sensory neurons and rat retinal ganglion cells, both of which are unresponsive to NGF (Davies and Lindsay, 1985; Barde et al., 1987). Thus, although many of the properties of BDNF are similar to those of the well-known and more extensively studied NGF, BDNF appears to fulfill the necessary criteria of a rieurotrophic factor for some vertebrate neurons. These properties are not necessarily exclusive to BDNF and possible interactions with other neurotrophic factors in the development and maintenance of vertebrate neurons remain to be investigated.

VI. Ciliory Neurotrophic Factor

Ciliary neurotrophic factor (CNTF) has been isolated from per-iphera1 nervous and nonnervous tissues, as well as from brain (Manthorpe et al., 1986; Rudge et al., 1987). As its name implies, C N T F acts as a trophic factor for ciliary sympathetic neurons, but it may also promote the survival of other neuronal cell populations such as sensory and sympathetic ganglia (Barbin et al., 1984). 111 addition, CNTF has been demonstrated to enhance the activity of ChAT in cultures of chick retinal cells, although it has not been shown to alter cholinergic parameters in neurons of the embryonic rat basal forebrain region (Knusel and Hefti, 1988; see also review by Hefti et al., 1989). Thus, CNTF may function as a relatively nonselective trophic factor in the developing mammalian brain.

VII. Epidermal and Transforming Growth Factors

A number of studies have demonstrated that epidermal- (EGF) and transforming growth factor (TGF)-like materials are present in the mammalian CNS. Receptors specific for EGF have also been detected in the brain. These GFs may act as trophic factors in the developing CNS, and

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EGF may also function as a direct neuromodulator in certain neuronal populations of the adult brain.

A. EGF Several immunohistochemical and radioimmunochemical studies reported the presence of EGF-like material in many brain areas such as the forebrain, diencephalon, brainstem, and cerebellum in both neonatal and adult rat (Fallon et al., 1984; Schaudies et al., 1989), in mouse cortical synaptosomal fractions (Lakshmanan et al., 1986), and in human cerebrospinal fluid (Hirata et al., 1982). The presence of EGF-like immunoreactive material in rat spinal cord neurons has also been reported (Joshy et al., 1988). Moreover, EGF does not appear to cross the bloodbrain barrier, suggesting that EGF or EGF-like activity found in brain and CSF is most likely of CNS origin (Nave et al., 1985; Jorgensen et al., 1988). In some studies, preproEGF mKNA has been detected in adult mouse brain, although in low levels (Kall et al., 1985; Lazar et al., 1988). Other studies, however, have reported on the apparent absence of EGFlike material in the mammalian CNS (Hirata and Orth, 1979; Kasselberg et al., 1985; Poulsen et nl., 1986; Probstmeier and Schachner, 1986). This clearly outlines the need for further studies on the existence of ECF-like substances in the brain, perhaps using various sources of antibodies and immunohistochemical methods. In addition, the status of EGF-like material in normal brain aging and disease states is unknown. B. TGFs

TGFs are a family of polypeptides involved in the regulation of cell growth and cell differentiation. Two different factors have been described as TGFs, TGF-a and TGF-P. TGF-a displays structural homology with EGF, although the two polypeptides are products of separate genes (Massague, 1987; Morstyn and Burgess, 1988). TGFs are found in normal CNS, brain tumors, and in the brains of patients with CreutzfeldtJakob disease (Roberts et al., 1981; Clark and Bressler, 1988; Oleszak et al., 1988). Gene expression of TGF-a has recently been demonstrated in mouse brain (Lee et al., 1985; Kakucska et al., 1988),where it appeared to be predominantly of neuronal origin (Wilcox and Derynck, 1988). TGF-a mRNA is localized to cell bodies of several brain areas such as the caudate nucleus and hippocam pal gyrus (Wilcox and Derynck, 1988).

However, 'TGF-P mRNA has not been detected in adult mouse brain (Wilcox and Derynck, 1988).

C. EGF A N D TGF MECHANISM OF AcrroN EGF receptor binding sites have been found in rodent and human brains. EGF binding sites are found in crude preparations of mouse 1981; Adamson and Warshaw, 1982; (Nexo et al., 1980; Adamson et d., Adamson and Meek, 1984), rabbit (Sadiq et al., 1985), and rat brain (Hiramatsu et al., 1988). EGP binding sites have also been identified in rodent neurons, astrocytes, and oligodendrocytes, in several glial cell lines from human brain (Leutz and Schachner, 1981, 1982; Simpson P t al., 1982; Sagen and Pappas, 1987), and in cells of human brain tumors (Libermann et al., 1984; Meyers ot d.,1988; Reubi et al., 1989). The ontogeny of EGF receptors in mouse brain from the twelfth day of gestation to parturition has been reported, showing an increase in the number of EGF binding sites during gestation (Adamson and Meek, 1984). Interestingly, Hiramatsri and co-workers (1 988) have reported a gradual decrease in the number of brain EGF receptor sites in rats during riormal aging, although the authors did not determine if the difference was associated with any CNS dysfunctions or the presence of any marker of degeneration. Furthermore, EGF receptorlike immunoreactivity (EGFR-IR) has been demonstrated in adult rat and human brain tissues (Loy et al., 1987; Gomez-Pinilla f't al., 1988; Nieto-Sampedro rt al., 1988; Shiurba et al., 1988; Werner ot nl., 1988; Samuels et ul., 1989; Arita rt ul., 1989). The cellular localization and clevelopmerital appearance of' EGFKIR has revealed that these sites are f'ound in astroglia in neonatal rat brain as well as in cerebral cortex neurons, cerebellar astrocytes, and Piirkin.je cells in adult brain (Gomez-Pinilla et (11.. 1988). I n aged rats, there was also a broadly distributed arid prominent staining in glial fibrillar) acid protein (GFAP)-positive astrocytes (Nieto-Sampedro et ul., 1988). This intriguing finding suggests that the role of EGF and its receptor may differ in the aged compared to the younger animal. A prominent EGFK inimunostaining has also been detected in cerebral cortical arid hippocampal neurons and choroidal and extrachoroidal ependvmal cells in normal human CNS (Werner et al., 1988; Samuels et d.,1989). An increase in astrocytic EGFK-1K has been found in the proximity of lesions in the adult rat (Nieto-Sampedro el al., 1988). Moreover, there is also increased EGFR-IR in neuritic plaques from patients with AD (Nanney et al., 1986), raising the possibility that EGF might be associated to plaque

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formation. These findings suggest that EGF is responsive to neural damage. Thus, it could be of critical significance that an increase in astrocytic EGFR-IR was observed in the aged rat brain (Nieto-Sampedro et al., 1988). We have reported on the presence of specific EGF binding sites in neonatal and adult rat forebrain (Quirion et al., 1988). Interestingly, the distribution of these receptor sites undergoes modification during brain development. T h e existence of EGF receptor binding sites is especially evident early postnatally, when high amounts of sites are present in cortical areas, whereas it appears that the expression of these sites is reduced in all these areas in adult forebrain (Quirion et al., 1988). However, these sites are still present in mature brain in several areas such as the cerebral cortex, striatum, and subtantia nigra (Chabot et al., 1988). This agrees well with previous immunohistochemical localization of EGF receptor in rat cerebral cortex (Gomez-Pinilla et al., 1988) and suggests that EGF receptor sites are present in both developing and mature brain. However, the exact types of cells bearing EGF receptors remain to be established. The presence of EGF and TGF-like substances and gene expression in the CNS raises questions about the physiological roles of these molecules in this tissue. EGF can apparently induce various biological effects in the brain. In cell culture systems, EGF has been shown to have mitogenic effects on glial cells (Leutz and Schachner, 1981, 1982; Simpson et al., 1982; Raff et al., 1983; Fischer, 1984; Westermark, 1988; Pate1 et al., 1988; Loret et al., 1989) as well as to induce the differentiation of these cells (Guentert-Lauber and Honegger, 1983; Honegger and GuentertLauber, 1983; Almazan et al., 1985; Monnet-Tschudi and Honegger, 1989; Loret et al., 1989). For example, it has been shown that EGF increases mRNA and protein synthesis (Avola et al., 1988b) as well as the labeling of cytoskeletal proteins, such as GFAP (Oleszak et al., 1988; Avola et al., 1988a), in rat astroglial cell cultures. Thus, this growth factor may affect macromolecular events in glial cell proliferation and differentiation (Monnet-Tschudi and Honegger, 1989; Almazan et al., 1985). It has also been demonstrated that EGF exerts trophic effects on postnatal rodent central neurons in culture and promotes neurite outgrowth in postnatal neurons (Morrison et al., 1987, 1988; Monnet-Tschudi and Honegger, 1989). Therefore, it appears that EGF may act as a growth factor for both glial and neuronal cell types. EGF appears to be involved in the central regulation of food intake (Plata-Salaman, 1988) and in the modulation of neuronal plasticity in the hippocampus (Terlau and Seifert, 1989). Additionally, EGF modulates neurotransmitter release. For example, EGF acutely inhibits ACh release

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both from peripheral cholinergic nerves (Takayanagi, 1980) and adult . suggests rat hippocampal slice preparations (Araujo et al., 1 9 8 9 ~ )This that EGF regulates neurotransmitter function directly. EGF can also induce tyrosine hydroxylase activity in rat sympathetic superior cervical ganglia (Henchman et al., 1983) and appears to stimulate the release ofCRF in the hypothalamus (Luger rt al., 1988). Thus, EGF may act as neuromodulator of a variety of neurotransmitter-neurohormone systems. It is well known that TGF-a binds to EGF receptor sites (Massague, 1987; Pimentel, 1987). Therefore, it may behave similarly to EGF in the CNS, although more research is needed to clarify the exact roles of this growth factor. Some evidence suggests that TGF-P may affect second messenger systems in the CNS. Exposure of astroyctes and brain microvessels to TGF-P results in the stimulation of phosphoinositol lipid turnover and activation of protein kinase C (Markovae and Goldstein, 1988; Robertson et al., 1988). These effects on endothelial cells of brain microvessels may indicate a role for TGF-P in the process of angiogenesis. A potential role of TGF-/3 in brain tuinorigenesis has also been suggested (Clark and Bressler, 1988).

VIII. Platelet-Derived Growth Factor

Platelet-derived growth factor (PDGF), as its name implies, was first isolated from human platelets, although it can be secreted by a large variety of cell types, including epithelial, smooth muscle, fibroblast, and glial cells (Ross et al., 1986). In addition to normal cells, PDGF can also be secreted by transformed cells and subsequently cause the release of “secondary” growth factors, which further promote tumor cell proliferation (see Ross et al., 1986). In both normal and transformed cells, PDGF induces several biological effects, including cell activation and proliferation, by binding to specific, high-affinity receptor sites that consist of two distinct subunits (Heldin et al., 1981; Ross et ul., 1986; Yarden et al., 1986). Receptors for PDGF have been identified on all mesenchymal connective tissueforming cells, including fibroblasts and glial cells (Heldin et ul., 1981; Mellstrom et al., 1983; Ross et al., 1986). In glial cell cultures, PDGF has been shown to have mitogenic effects (Besnard et al., 1987). PDGF has been implicated in wound healing and repair; however, the importance of this growth factor in embryogenesis, growth, and development is still unclear. Furthermore, even though it is apparent that PDGF

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may be of importance in the proliferation ot‘ tumor cells both in the periphery and in the CNS, to our knowledge no direct actions of the protein in the CNS have yet been demonstrated.

IX. lnterleukins and Other Lymphokines

The lymphokines are important mediators of immune function (see reviews by Dinarello, 1988; Sincovics, 1988; O’Garra, 1989),that are now thought to play a significant role in the intercommunication between the immune and nervous-neuroendocrine systems (Ballieux and Heijneri, 1987; Weigent and Blalock, 1987). However, unlike 1987; Farrar et d., their mechanisms of action in the immune system (Weigent and Blalock, 1987; Farrar et al., 1989), little is known about the means whereby lymphokines “communicate” with the nervous system. Acknowledgment that immune markers and immune-related phenomena exist in the brain has been a recent development (see Wekerle rt al., 1986). At present, it is generally accepted that both immune mechanisms and immune markers are not only present in brain but are elevated in certain neuropathological diseases (see Rogers etul., 1988; Griffin et al., 1989; McGeer et al., 1989). For example, interleukin-1 (IL-I) (Griffin et al., 1989) and HLA-DR-the major histocompatibility (MHC) class I1 glycoprotein associated with antigen presentation-(Luber-Narod and Rogers, 1988; Rogers rt al., 1988),as well as complement proteins (McGeer et al., 1989), are found in elevated concentrations in postmortem tissue from AD brains. Thus, it seems possible that immune interactions are associated to the pathogenetic mechanisms involved in certain neurological disorders. Additionally, there is evidence that ILs arid other lymphokines may also function as GFs for certain neuronal and glial-like cells in the brain.

A. 1L-1 There is still some question as to the source of IL-1 in the CNS, but it appears that IL-1 can be synthesized by certain cells intrinsic to the brain. Support for this hypothesis has been obtained from studies demonstrating that IL-1 immunoreactive fibers are present in the human hypothalamus (Breder et al., 1988) and that IL-1 can be synthesized by astrocytes and microglial cells of the brain (Fontana et al., 1982; Giulian et al., 1986; see also review by Dinarello, 1988). Moreover, IL-1 binding sites have

been identified in various brain areas, apparently localized to neuronal cell types (Giulian et al., 1986; Fai-rar ut al., 1987). T h e binding sites for IL- 1 i n many brain regions appear to constitute functional IL-1 receptors, since many diverse effects of 11,-1 within the CNS have been reported (see reviews by Weigent and Blalock, 1987; Dinarello, 1988; Bateman et ul., 1989). In the CNS, the pituitary adrenal axis has been the focus of intense research into the potential activity of IL-1 as a messenger between the immune and neuroendocririe systems (see reviews by Bateman et al., 1989; Carlson and Felten, 1989). Besedovsky and co-workers (1986) demonstrated that systemic injection o f IL-1 resulted in an increased release of adrenal corticotrophic hormone (ACTH) and glucocorticoids. Subsequently, several investigators have shown that IL-1 enhances the release of several pituitary and hypothalamic hormones (Berkenbosch Pt ul., 1987; Bernton et al., 1987; Sapolsky et al., 1987; Breder et ul., 1988). Preliminary investigations have suggested that IL- 1 may have diverse effects on other cell types within the brain. Thus, IL- 1 has been shown to be a potent mitogen for astroglial cells (Fontana et al., 1982; Giulian and Lachman, 1985; Giulian et al., 1986) and its synthesis is elevated in response to brain injury (Nieto-Sampedro and Berman, 19x7). In peripheral nerves, IL-l enhances N G F expression and may increase N G F levels in response to sciatic nerve transection (Lindholm ei 01.. 1987). However, an effect of IL-1 o n the expression of NGF in central neurons in xdro or in uivo has yet to be shown. In AD, the reported increase in I L - 1 imniunoreactive material appears to parallel the gliosis associated with the disease (Griffin et ul., 1989). Thus, although no direct effects of IL-1 on neuronal cells have been detected, IL- 1 may be of relevance to some clinical features o f AD.

B. IL-2 In contrast to IL- 1, there is little available information on the effects of IL-2 in the brain. At present, the major recognized role for IL-2 in the CNS appears to be in function in the body’s adaptation to CNS trauma (Nieto-Sampedro and Chandy, 1987). However, a larger repertoire for IL-2 functions can be expected from the widespread distribution of IL-2 and IL-2 receptors in the brain (Araujo et al., 1989b). Some of the properties of IL-2 in the brain are similar to those described above for IL- 1. In tissue cultures,IL-2 acts as a mitogen for glial cells (Benveniste et al., 1988). In addition, in tissue slices from adult rat brain, IL-2 appears to act directly in the regulation of ACh release,

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whereas IL-1, IL-4, and interferons are ineffective (Araujo et al., 1989b,c).This effect of IL-2 is similar to that observed with IGF-1 in the same tissue (see Section IV,B). Thus, like IGF-1, IL-2 may function not only as a potential trophic substance, but also as a direct modulator of cholinergic function. This neuromodulatory role of IL-2 appears to be mediated by specific receptors, since other lymphokines were not effective. Whether this role of IL-2 applies to other neurotransmitter systems remains to be investigated. Following experimentally induced lesions, IL-2 (Nieto-Sampedro and Chandy, 1987) and IL-2 receptor site levels (Araujo et al., 1989b) are elevated in the area surrounding the lesion. In addition, intense immunoreactivity for the IL-2 receptor has been detected in AD brains (Itagaki et al., 1988; Luber-Narod and Rogers, 1988).Thus, it is possible that IL-2, like IL-1, may be involved in certain features seen in AD brain. Furthermore, it seems tempting to speculate that the IL-2 receptors that are present on glial cells (McCarron et al., 1985), may account for the glial proliferation observed in AD.

LYMPHOKINES C. OTHER Several lymphokines, including IL-6 and the interferons, are similar to IL-1 in their effects on the neuroendocrine system (Dinarello, 1988). Moreover, both IL-6 and the interferons promote glial cell differentiation and proliferation in much the same way as IL-1 (Harrison and Campbell, 1988). Recent evidence has also shown that IL-6 promotes survival of basal forebrain cholinergic neurons in culture (Hama et al., 1989). Furthermore, the effects of IL-6 and NGF in these cultures were synergistic (Hama et al., 1989). However, a detailed description of the functions of these lymphokines in the CNS is still pending.

X. Hormones and Neurotransmitters as Neurotrophic Factors

Various hormones affect nervous system development, as well as its response following injury. Results from in uitro and in vivo studies have shown that hormones can affect neurite extension and survival by interacting with specific receptors on their target cells. At present, several hormones can be grouped into this category, including sex steroids, thyroid hormones, glucocorticosteroids, insulin (discussed in Section

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IV,A) and ACTH. While a complete discussion of their effects on neuronal growth is beyond the scope ofthis review, it is nonetheless important to mention the salient properties of these hormones.

A. ESTROGEN

There is extensive evidence that the number and density of neurons in various CNS regions are affected by sex steroids, in particular during development (see review by Arnold and Gorski, 1984). Estrogen has been shown to stimulate the growth of neurites from hypothalamic explants (Toran-Allerand et al., 1980). ‘l’hiseffect was restricted to those neurons that exhibited steroid binding, thus indicating a specific effect of estrogen on selected neuronal populations. Like some “classical” GFs, estrogen has been implicated in the neuronal sprouting that follows injury or experimental denervation. Elegant studies by Loy and Milner (1980) and Milner and Loy (1982) clearly showed that neuronal sprouting following denervation of the septohippocampal pathway is enhanced by estrogens. However, it is not apparent whether this represented a direct effect of estrogens on in uzuo neuronal growth.

B. THYROID HORMONES Many studies have documented the importance of triiodothyronine (Ts) and thyroxine (T4) in brain development (see review by Lauder, 1983). Because of their critical significance in cell metabolism throughout the body, it is not surprising that the thyroid hormones play a prominent role in neuronal growth. Thyroid hormone receptors are present on many neurons in the CNS (Haidar and Sarkar, 1984), but it is not clear under what conditions thyroid hormone interactions with their receptors result in the promotion of neuronal growth. Evidence for neurotrophic effects of thyroid hormones is mounting, T s enhances development of dopaminergic cells in mouse hypothalamic neurons in uitro (Puymirat et al., 1983) and fetal rat cholinergic neurons (Atterwill et al., 1984). Indirect effects of thyroid hormones on neuronal growth have also been proposed: T4has been shown to increase the levels of NGF, a “classical”GF, in the mouse brain (Walker et al., 1979). ’I-3 has also been shown to increase ChAT activity in basal forebrain neurons zn uitro (Hefti et al., 1986; Hayashi and Patel, 1987). This effect of T:<was

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additive (Hefti et al., 1986) or synergistic (Hayashi and Patel, 1987) to that of NGF. Thus, it seems possible that thyroid hormones may be of some therapeutic benefit in treating certain neurological disorders.

C. ADRENAL HORMONES Adrenal glucocorticoids appear to function as “negative” GFs in the nervous system. Studies have indicated that these hormones inhibit or retard neuronal development (see review by Jacobson, 1978). T h e specific role(s) of glucocorticoids in neuronal development is not entirely understood, although it appears that they can reduce the sprouting that occurs following injury. For example, the neuronal sprouting that normally occurs in the hippocampus after a lesion of the entorhinal cortex is drastically reduced by glucocorticoids (Scheff et al., 1980; Scheff and DeKasky, 1983). Similarly, it has been suggested that reduced glucocorticoid secretion in response to stress may retard hippocampal neuron loss, as well as cognitive impairments with age (Meaney P t al., 1988, 1989). In contrast to the glucocorticoids, ACTH, the pituitary hormone that regulates their release from the adrenal gland, appears to stimulate neuronal growth (see review by B’JIsma et al., 1982). However, the evidence for neurotrophic effects of ACTH in the CNS is sketchy at best, although ACTH has been reported to enhance neuronal regeneration in the CNS following injury (Berry et al., 1979). Clearly, further studies are required to clarify the growth-promoting properties of ACTH on CNS neurons.

D.

NEUROTRANSMITTERS

Certain transmitters and their synthetic enzymes may affect early events in embryogenesis (see review by Lauder, 1983). T h e available evidence that neurotransmitters are directly involved in neuronal development is not conclusive and is even controversial. It has been suggested that norepinephrine (NE) acts as a GF for neurons in the cerebral cortex (Felten et al., 1982) and visual cortex (Kasamatsu et al., 1979). Moreover, depletion of NE stores has been proposed to decrease developmental plasticity (Kasamatsu et al., 1979). However, these findings have been challenged by several investigators (Bear et al., 1983; Daw et al., 1984). At the center of the controversy is the question of whether pharmacological

manipulation of NE stores exerts other nonspecific effects on neurons. Serotonin has also been implicated in the niodulation of growth cone development in uitro (Haydon et a/., 1984).Similarly, substance P has been suggested to promote neuronal survival and to protect neurons fi-om the deleterious effects of neurotoxins (.Jonsson and Hallman, 1982, 1983). Neurotrophic effects of othei- neurotransmitters have been implied by pharmacological studies that tested the effects of various drugs such as amphetamine, naloxone, and peptides or1 neuronal sprouting and regeneration following CNS injury (Jorissonand Hallman, 1982; Feenev el al., 1982; Baskin et al., 1984). As discussed above for N E , the specificity of these effects remains to be estatdished. Theref-ore, certain neurotransmitters may be classified only tentatively as neurotrophic- substances for central neurons.

XI. Miscellaneous Factors with Potential Neurotrophic Activity

Since the field of GF research is in a very dynamic state. the list of molecules that may be considered as neurotrophic substances is expanding at an accelerated pace. Although novel proteins with neurotrophic activity continue to be identified (see Walicke, 1989), many more newly classified neurotrophic factors are previously described proteins with only recently characterized neurotrophic properties. For example, neuroleukin has been shown to increase survival and process formation in a variety of neurons, including spinal, septa], and hippocampal neurons 1987). Neuroleukin is now known to be (Gurney et al., 1986; Lee et d., identical to the glycolytic enzyme phosphoglucose isomerase (Chaput et al., 1988; Gurney, 1988), which had not been classified previously as a neurotrophic factor. Other potential GFs include laniinin and heparan sulfate proteoglycan (Davis et al., 1985; Lander et al., 1985). Several studies have demonstrated that these molecules are good substrates for neurite extension from peripheral and CNS neurons in culture (Manthorpe et al., 1983; Edgar et al., 1984). However, these substances are usually ineffective in supporting neuronal growth by themselves, but rather require the presence of other GFs, such as NGF (Edgar, 1985). Fibronectin and collagen are also known to support neurite outgrowth (Carbonetto et al., 1983; Rogers et al., 1985). Glial-derived nexin is produced by many cells, including fibroblasts

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el

al.

and astrocytes (Pittman and Patterson, 1987) and is present in brain tissue of fetal and adult rats (Gloor et al., 1986). The growth-promoting effects of glial-derived nexin appear to involve a complex series of events mediated by an interaction with neuronal proteases (Monard, 1987). Thus far, enhanced neurite outgrowth of neuroblastoma cells and sympathetic neurons has been shown (Monard, 1987; Pittman and Patterson, 1987), but no effects on neurite extension in central neurons have been demonstrated.

XII. Concluding Remarks

The search for neurotrophic factors continues to be an extremely active field. Many potential neurotrophic factors have already been purified and sequenced, but the number of well-characterized neurotrophic substances is still quite small. Therefore, it is a difficult task to assess the specificity of functions of the GFs described in this review, as well as their spectra of actions. More pieces of the puzzle of nervous system differentiation and growth can only be solved as more neurotrophic substances are recognized and tools assessing the specificity of their actions (e.g., antagonists) become available. However, there is no argument that neurotrophic factors play an important role in brain development and are necessary for the maintenance and survival of the CNS, although there seems to be some disagreement as to the extent of their involvement and their potential benefit as therapeutic agents in neurodegenerative diseases. Appel (198 1) has hypothesized that Parkinson’s disease and AD may represent the loss of trophic support for central neurons. Support for this hypothesis has been obtained from several studies showing that administration of GFs can attenuate degeneration and even behavioral deficits induced by experimental lesions, suggesting that some GFs may be useful in the treatment of degenerative diseases such as AD and Parkinson’s disease. Nevertheless, incontrovertible evidence to support the lack of GFs that promote neurite outgrowth as a direct cause of the pathogenesis in these diseases is lacking. In fact, it seems likely that inhibitory neurotrophic factors might be equally important in the control of development and growth and in the prevention of pathogenesis. This is supported by studies showing that deficits in inhibitory trophic factors may be highly detrimental due to the resulting excessive and unchecked neurite sprouting promoted by “positive” growth factors (Uchida et al., 1988; Uchida and Tomonoga, 1989).

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Many of the potential neurotrophic factors described in this review such as EGF, the IGFs and ILs exhibit pleiotropic effects. Hence, neuronal growth may represent only one aspect in the broader subject of overall brain tissue maintenance and regulation.

Acknowledgments

Some of the research reported here has been supported by grants from the Medical Research Council of Canada and the Canadian Parkinson Foundation to R. Q. D.M.A. and R.Q. are holders of a Fellowship and a Chercheur-boursier Award from the “Fonds de la Recherche en Sante du Quebec,” respcctively.

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MYASTHENIA GRAVIS: PROTOTYPE OF THE ANTIRECEPTOR AUTOIMMUNE DISEASES By Simone Schonbeck, Susanne Chrestel, and Reinhard Hohlfeld Department of Neurology University of Munich and Department of Neuroirnrnunology Max-Planck-lnstitut Martinsried Munich, Federal Republic of Germany

1. II. 111. 1V. V. VI.

Introduction Acetylcholine Receptor Anti-AChK Antibodies AChR-Specific T Lymphocytes Role of the Thymus Treatment Strategies References

I. Introduction

Myasthenia gravis (MG) has been repeatedly reviewed in past years. Among the widely cited classics in the field are the review articles by Vincent (1980), Drachman (1978), Engel (1984), and Lindstrom (1985, 1988). Other useful sources of information are the monographs b!. Lisak and Barchi (1982), Oosterhuis (1984),DeBaets (1984),and Harrison and Behan (1986), and the proceedings of the regular international and European conferences on M G (Drachman, 1987; DeBaets et nl., 1988). Updated information may be found in the brief overviews appearing regularly in the Current Opinion series (Vincent, 1988; Toyka aiid Hohlfeld, 1989). T h e main purpose of the present review is to provide an introduction to the field and to supplemcnt the existing literature. The emphasis has been placed on recent work in human MG. We have attempted t o show how a receptor molecule that nor-niallybinds neurotransmitter arid mediates neuromuscular transmission is “perceived” by the immune system, becoming the target of antibodies and antigen-specific B and 1‘lvmphocytes. 175 INTERNATIONAL REVIEW OF NEUROBIOLOGY, VOL. 39

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Myasthenia gravis (MG) is a relatively uncommon disorder with a prevalence of approximately 4 and an annual incidence of approximately 0.4 per 100,000population (Kurtzke, 1982). Its great importance derives from the fact that MG is the prototype of the antireceptor autoimmune diseases. In these diseases the immune system erroneously produces antibodies against receptors for hormones or neurotransmitters. These autoantibodies bind to the receptors and either stimulate or impair their function (reviewed by DeBaets, 1984). Examples of endocrinological antireceptor autoimmune diseases with stimulating autoantibodies include Grave’s disease in which antibodies against the receptor for thyroid-stimulating hormone are produced (DeBaets, 1984), and a familial form of Cushing’s syndrome, in which antibodies against the receptor for adrenocorticotropin are produced (Young et al., 1989). In MG, the target of the immune reaction is a neuroreceptor, the nicotinic acetylcholine receptor (AChR) of the neuroniuscular junction. The anti-AChR autoantibodies produced by MG patients exert their pathogenic effect not by stimulating but by impairing the AChR and, therefore, neuromuscular transmission. Typically, MG patients complain of fluctuating muscle weakness producing symptoms such as double vision; difficulty in chewing, swallowing, speaking, or breathing; and weakness of arms and legs ranging in severity from abnormal fatigability during exercise to frank paralysis (reviewed by Oosterhuis, 1984). Perhaps the main reason that MG is undoubtedly the best understood antireceptor autoimmune disease is that the AChR is an exceptionally well-characterized receptor. However, the path leading from basic receptor research to the diagnosis and therapy of patients is not a one-way road: The increasingly better understanding of the disease has equally contributed to the understanding of the structure and function of the AChR. The nicotinic AChR was purified about a decade before the purification of other receptor types was possible. T h e basis for this achievement was the observation that several snake a-toxins, including a-bungarotoxin (a-BGT), proved to be specific ligands for the AChR that irreversibly inactivated receptor function in intact skeletal muscle (Chang and Lee, 1962). This could be exploited for the detection of the receptor in muscle homogenate, which is the basis for the widely used diagnostic radioprecipitation assay (Lindstrom, 1976a), and at intact endplates, and for the isolation of the AChK by affinity chromatography (Lindstrom and Patrick, 1974). Soon it could be established that the primary abnormality in MG was a reduced number of functional AChR (Fambrough et al., 1973). After it was found that the electric organ of electric fish is a rich source of nicotinic receptors (Lindstrom and Patrick, 1974), it became

possible to purify large quantities of receptors. For example, the density of receptors in Torpedo electric organs approaches 100 pniol/mg protein, which may be compared with 0 . 1 pmol/mg protein in skeletal muscle (Cohen and Changeux, 1975). When purified AChR from electric eel was in-jected into rabbits, muscular weakness was induced, iiow known as experimental autoininiune MG (EAMG) (Patrick and Lindstrom, 1973). This finding, which was confirmed in other species (rats, guinea pigs, primates) (Tarrab-Hazdai rt al., 1975a,b; Lennon et al., 1975; Lindstrom et al., 1976b),proved t o be of' paramount importance for M G research. Another form of experimental MG could be induced by the passive transfer of patient IgG or serum to animals (Toyka at al., 1975; Lindstrom at al., 1976b). These experiments proved the pathogenetic role of anti-AChK autoantibodies and led to the development of new therapeutic strategies such as irnmunosuppression and plasmapheresis. Later it was shown that the production of antiAChR antibodies in patients is controlled by AChK-specific helper 'r cells that can be isolated from the peripheral blood and thymus (Hohlfeld ot ul., 1984; Melms et al., 1988; Harcourt at al., 1988). The mystery o f what initiates the immune response to AChR has not been solved, but there is little doubt that the key to the solution lies in the thymus.

II. Acetylcholine Receptor

T h e nicotinic AChR consists of five subunits arranged around a pseudoaxis of symmetry (Fig. 1). T h e subunits display partially homologous amino acid sequences with ?~0-40%identity of amino acid residues (Brisson and Unwin, 1985). This indicates that the subunits evolved from a primordial subunit by gene duplication and that the orientation o f each subunit in the protein should be the same (Kaftery et al., 1980; Noda PI ul., l983b). One of the subunits ( a )is present in two copies. T h e p, E , and 6 subunits are expressed as single copies (Keynolds and Karlin, 1978; Lindstrom et al., 1979). It is thought that the E: subunit is part of the AChK of mature neuromuscular junctions, whereas denervated o r embryonal AChK have y subunits (Mishina et al., 1986). Major features of the functional diversity of vertebrate muscle nAChK can be explained tjy the presence of y or E subunits in the AChK studied (Steinbach, 1989). The apparent and calculated molecular niasses of the Torpedo AChR protein subunits are a = 38,000 and 50,116; p = 49,000 and 53,68 1 ; y = 57,000 and 56,279; and 6 = 64,000 and 57,565 kDa (Noda et al., l983b). In Xenopus, two different a subunits, which coexist throughout muscle de-

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FIG. 1. AChR interaction with acetylcholine. T h e A C h K is a pentameric complex c o n posed of four AChR subunits (a.p, y , and 6). ' l h e a subunit is represented twice. Each a subunit contains an ACh binding site. ' t h e region to which the majority of patients' autoaiitibodies bind (main immunogenic region, MIR) is also on the a subunit. T h e binding of two ACh molecules leads to the opening of the central ion channel. 'the narrowest part of the ion channel (not shown) is at the level of the lipid bilayer.

velopment, are expressed (Hartman and Claudio, 1990). For the /3 subunit it has been demonstrated that at least two isoforms exist due to alternative splicing of the niKNA (Goldrnan and Tamai, 1989). AChRs purified from mammalian muscle are structurally similar to those of Torpedo electric organ (Gotti et al., 1982; Einarson et al., 1982; Buonanno et al., 1989).The human AChK has been sequenced and the a subunits of the AChRs from human muscle have SO% sequence homology with the a subunits from Torpedo (Noda rt ul., 1983b; Beeson et al., 1989). The subunits are arranged around a central cavity with the larger portion extending towards the extracellular surface (Fig. 1; Kistler et al., 1982). T h e central channel is believed to be the ion channel, which is impermeable to ions in the resting state. Upon activation, it opens to an estimated diameter of 6.5 A. T h e structure of the transmembrane ion channel has been determined at 17 A resolution in relation to the lipid bilayer (Toyoshima and Unwin, 1988), using three-dimensional image reconstitution from tubular vesicles containing periodically arranged receptor molecules. T h e extracellular opening of the channel has a diameter of about 25 A. T h e channel narrows to < l o A at the level of the membrane bilayer and widens again to about 20 8, at the cytoplasmic side. The a subunits contain the binding site for ACh and competitive antagonists. The two ACh binding sites are on the extracellular surface of the molecule (Fig. 1) (Neubig and Cohen, 1979) near the disulfide-linked

cysteines at positions 192 and 193 ( K a o et ml., 1984; Kao and Karli~i, 1986). Occupation of both sites is necessary for receptor activation. Only little is known about the function of the other subunits. Assuming the AChK is structured like a douglinut, each subunit would contribute 10 the cation channel. To identify those amino acid residues that interact with permeating ions, various point mutations were introduced into the Torpedo AChR subunit cDNAs to alter the net charge of' the charged o r glutaniine residues a round the proposed transmembrane segments. 'l'he single-channel conductance pix)perties of these AChR mutants expressed in Xenopz~slaeuis oocytcs indicate that three clusters of'negatively charged a n d glutamine residues neighbouring segment M2 ot' the LY-, /3-, y- a n d 6-suhunits, probably foi-ming three anionic rings, are major determinants of the rate of iori transport (Sakmann ~t af., 1985: Imoto ut ul., 1986, 1988). A 43-kDa protein is believed to cross-link the AChK to the cytoskcleton at the synapse. T h e synthesis of the 43 kDa protein and of'tlie A(;IiK are regulated by different nieclianisnis (Froehner, 1989). Different models have heen proposed fix the transnienibrane orientation of the polypeptide chain i t ] the AChK subunits. Like other ligantlgated ion channels (Stevens, 1987), the AChK subunits have four h y drophobic domains, named Rill -M4. . l h e sequence identity among the subunits seems to be greatest among the hydrophobic regions. Although the exact transmembrane orielitation of the subunits is still uncertain, there is consensus that much of. the amiiio-terminal part is extracellulai-, and much of the carboxyterniiiial part is intracellular (1,intlstrom P / ul., 1988; Guy rt ul., 1987; Dani, 1989). T h e assembly of muscle AChR begins in the endoplasrnic reticuluin, where the subunits a re synthesized as precursors with a signal peptide (Anderson a n d Blobel, 1981; hlerlie r / al., 11183). Half' an h o u r after synthesis, association of thc subunits begins. Only little is k n o w n iiboiit the transport a n d the expression of the receptor on the cell surface. This is now being investigated using a variant o f t h e mouse muscle cell line ( 2 that expresses AChK on the cell surfhce (Black arid Hall, 1985): 'l'he variant makes normal aniounts of AChK but accumulates most of it i i i iiii intracellular pool. Therefore, it may provide a tool for investigating the factors that regulate AChR assembly and transport to the surface membrane (Gu el al., 1989). T h e half-life of AChKs at mature ~ i e u r o ~ ~ i u s cjunctions ~ilar is at leiist 5 days, whereas extrajunctional AChKs have a much shorter half-life (less than 20 hours) (Fambrough, 1979). -1he rnechanism of receptor tuixover involves internalization arid lysosornal degradation. The synthesis a n d destruction of AChK is under a wide range of control elernerits a nd regulated on different levels (Merlie P / a/., 1984). At

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the level of transcription it was described that in the chicken AChR a subunit gene, a 5' upstream sequence between nucleotides - 110 and -45 exerts developmental control of expression in primary cultures of chicken myotubes. This region interacts with an Spl-like factor, and a guanine stretch-binding protein was found to bind to overlapping sites immediately upstream of the T A T A box (Piette et al., 1989). In rat primary muscle cells the AChR a-subunit mRNA was increased approximately threefold in response to ascorbic acid, but the synthesis of this subunit was not. This may be indicative o f a regulatory process at the level of translation (Horovitz et al., 1989). Keceptor synthesis is probably also influenced by neuropeptides found in motorneurons (Fontaine et al., 1989). Using DNA-mediated gene transfer techniques, it has been possible to reconstitute functional AChR channels from Torpedo in mouse fibroblasts (Claudio et al., 1987, 1989). Structure homologies show that skeletal muscle AChRs are part of a superfamily of ligand-gated ion channels receptors that includes the GABA and glycine receptors. It is important to note that the AChR proteins themselves constitute a family with different members, of which the muscle and neuronal AChRs and the a-BGT binding proteins have been characterized (Grenningloh et al., 1987; Peralta et al., 1988). Neuronal nicotinic AChRs have nanomolar, whereas ganglionic nicotinic AChRs have millimolar, affinity for nicotine. Unlike muscle AChRs, neuronal AChRs do not bind a-BGT (Whiting and Lindstrom, 1987a). Neuronal AChRs have only two kinds of subunits. One binds ACh like the a subunit of muscle AChR (Whiting and Lindstrom, 1987b), the other is structurally related to the p, y , or 6 subunit. Furthermore, in neurons, there are a-BGT binding proteins whose function and structure is only poorly characterized. In a-BGT binding proteins from Aplysia californica, structural similarities and differences to the Torpedo AChR could be demonstrated (McLaughlin and Hawrot, 1989). AChRs and the BGT-binding proteins may occur on the same neuron (Jakob et al., 1984). Muscarinic AChRs typical of smooth muscles and neurons (Peralta et al., 1988) belong to a completely different receptor superfamily together with rhodopsin (Nathans and Hogness, 1983) and the adrenergic (Dixon et al., 1986) and serotoninergic receptors oulius et al., 1988). These receptors mediate their action through coupling proteins (G proteins) (Kerlavage el al., 1987). As a result of a search for cell lines providing a larger and more uniform source of human AChR than the conventional source (muscle from amputated human legs), the human cell line TE 67 1 (McAllister et al., 1977) was found to express muscle AChR (Lindstrom et al., 1987). This was shown by a-BGT binding (Syapin et al., 1982), reaction of these

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AChRs with monoclonal antibodies to muscle AChRs and with MG patient autoantibodies, and sequence comparison of their a subunits (Schoepfer et al., 1988). Using the patch clamp technique, it was demonstrated that T E 67 1 expresses ACh-activated ion channels with properties resembling extrajunctional AChR (Luther et al., 1989); that is, they probably express the y rather than the E subunit (Mishina et al., 1986).

111. Anti-AChR Antibodies

In MG antibodies are produced to many parts of the extracellular surface of the AChR. T h e antibodies are polyclonal rather than monoclonal. Most antibodies belong to the IgG class and bind with high affinity (Tzartos et al., 1982; Bray and Drachman, 1982). This rules out the possibility that MG is caused by the expansion of a single “forbidden” B cell clone. Although antibodies to the AChR are present in almost all patients with MG, there is only a poor correlation between the severity of disease and the titer of anti-AChK antibodies (Besinger et al., 1983; Lindstrom et al., 1976a). However, in the individual patient the correlation is usually good in a longitudinal study (Besinger et al., 1983; Vincent, 1980). Patients with clinically typical MG who had no detectable antibodies to the AChR have been described (Mossman, 1986; Evoli P t al., 1989). Whether these patients truly had MG or whether this represents a subgroup of MG patients is unclear. It has been suggested that “antibodynegative” MG is a distinct autoimmune disease. When immunoglobulin preparations from such patients were injected into mice, neuromuscular transmission was significantly impaired. However, no antibody bound to the mouse AChR was detected. One possible explanation is that a pathogenetic antibody interferes with neuroniuscular transmission in these AChR-antibody-negative patients by binding to non-AChR determirialits at the neuromuscular junction (Mossman, 1986). In MG, the antibodies against the AChR are not randomly distributed over all possible epitopes of the AChR, but the repertoire seems at least in part restricted to certain regions of the AChR. Over 60%’o f autoantibodies against the AChK are directed against an extracellular area o f the CY subunit called the main immunogenic region (MlR). which is hydrophilic and negatively charged (l’zartos et al., 1982) (Fig. 2). ’The M I K is a rather well-conserved antigenic feature of mammalian and fish nicotinic AChRs. There seems to be practically complete competition between all possible paired combinations of anti-MI R monoclonal antibodies (111.4bS)

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FIG. 2. AChR interaction with autoanti1,odies. Like the schematic antibody (ab) shown o n top ofthe two AChK molecules, the rnajority of patients’ antibodies are of the IgG class. A large proportion of antibodies bind to the main immunogenic region (MIK). Crosslinking of AChK by antibody increases receptor degradation.

from rats, 7orpedo, and humans (Kordossi arid ‘I’zartos, 1989). The normal functional role of this region is unknown. It appears that a main constituent loop of the MIK is located between either amino acid residues 61-76 (Barkas rt nl., 1988) or 67-76 (Wood ut nl., 1989; Tzartos rt nl., 1988) on the outside of the extracellular surface of the AChR a subunit (Kubalek et al., 1987). mAbs to the MIR which can passively transfer EAMG (Tzartos et al., 1986) have no direct effect on AChK function (Blatt et al., 1986). Antibodies to the MIR can very effectively cross-link adjacent AChKs but cannot cross-link the a subunits within one AChR (Conti-Tronconi et al., 1981) (Fig. 2). It is still difficult to define unique binding patterns by the competition techniques currently available (Walker et al., 1988), and there has been one report arguing against the AChR having a MIR target for autoantibodies at all (Lennon and Griesmann, 1989). Antibodies in 74

sera from 100 MG patients were inhibited by more than 50% from binding to human muscle AChK by a rat monoclonal antibody of MIK specificity. However, the niAb inhibition seemed to be d u e to steric hindrance rather than epitope competition because (1) the rat rnAb reacted with both Torpedo an d human AChR, whereas the patient antibodies in 85 of the MG sera did not bind to Torpedo ,4(:hK, and (2) the mAb blocked the binding of riit antipeptide antibodies to an a-subunit region of the human AChR unrelated to the designated M I R region. Another group of antibodies bind to the a-BG'I' binding region o f t h e AChR. Antibodies blocking a - B G T binding to AChK on 'I'E 671 cells have been found in the serum of patients with generalized MG with particularly severe disease (Pachner, 1989).However, serial titers of'BGT binding antibodies correlated less often with changes in muscle weakness than did the titers of antibodies cleterrnined in the standard irnmunoprecipitatiori assay (Besinger P t (il., 1983). MAbs have been produced that block function. Those antibodies either bind to the ACh binding site or inhibit by allosteric mechanisms (Fels ef d . , 1986; Donnelly et ol., 1984; Mochly-Rosen and Fuchs, 198 1 ; Mihovilovic and Richman, 1984; Whiting et al., 1986). Certain patient antibodies also seem t o bind to the ACh binding region (Gu et al., 1985). When SDS-denatured A(;hR is used for immunization, the h l I K is lost, the AChR is less immunogenic, and most of the antibodies produced are directed against cytoplasmic parts of the receptor (Katnani P t ( I / . , 1986). Studies have also been performed with polyclonal arid monoclonal sera raised against short synthetic peptides of the 7hrpe'lo AChK. 'Ihev are consistent with the general concept that conformation arid charge pattern rather than linear sequence are the essential determinants of' antibody epitopes (Maelicke d . , 1989). T h e site of antibody binding (.an give sonie evidence b y what niecha1982). nisms the anti-AChR antibodies inctiice disease (Drachman ot d., First, anti-AChR antibodies may directly block AChK. By binding at or near the ACh binding site o n 111; ACliR, the autoantibodies may prevent ACh binding and the subsequent neurotransmitter cascade of events leading to muscle contraction (Barkas et al., 1982). Alternatively, airtoantibody directed at other sites on the AChR molecule may prevent proper function of the ion channel thus preventing ion flux (Eltlefrawi rt nl., 1980). Furthermore, the autoantibody can increase degradation of re1978). Lastly, the presence at the muscle meniceptors (Drachman et d., brane of antibody that can fix complement may result in focal Iysis (Engel el al., 1977). I n this regard, it could be shown that the passive t n n s f e r of EAMG could completely be inhibited with a Fab antibody to Cti (Biesecker a nd Gomez, 1989). (21

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IV. AChR-Specific T lymphocytes

As discussed in the previous section, antibodies and B lymphocytes bind to conformation-dependent antigen epitopes. In contrast, T lymphocytes bind antigen only if it is processed and associated with a major histocompatibility complex (MHC) molecule (Schwartz, 1985). Therefore, T-cell epitopes are limited to antigen sequences that include binding sites for class I1 MHC products (Berzofsky, 1988) (Fig. 3). Because the specificity of T-cell recognition rests in part on the specificity of class I1 MHC peptide interaction (Buus et al., 1987), the polymorphic MHC molecules have a profound influence on the spectrum of antigen epitopes that can be recognized by the T cells of a given individual (Schwartz, 1985). This explains why at least some of the AChR-reactive 'r cells from MG patients with different HLA haplotypes recognize different fragments of AChR (Hohlfeld et al., 1988a; Harcourt et al., 1988; Brocke et al., 1988). It may be difficult to find a T-cell epitope common to the majority of patients. Several groups have begun to map the T-cell epitopes recognized by helper T cells in human myasthenia gravis (reviewed by Hohlfeld, 1989). T h e most commonly used approach to study T-cell epitopes is to isolate T cell lines specifically reactive to AChR and to test these lines for reactivity against a panel of AChR fragments. Myasthenia gravis is the tirst human autoimmune disease in which autoantigen-specific T lymphocytes have been expanded to permanent monospecific T cell lines and clones (Hohlfeld et al., 1984; Harcourt et al., 1988; Melms et al., 1988). Both oligoclonal lines and clones of T cells are useful tools to characterize autoantigen response patterns in autoimmune disease. T-cell lines help to obtain information about the composition of the overall autoimmune T-cell repertoire of a given patient. T-cell clones are required for establishing fine response patterns and for mapping individual T-cell epitopes. The purification of human AChR in sufficient amounts for long-term T-cell line selection and pEopagation is still a limiting factor. Only very few studies measured T-cell reactivity of MG patients to human AChR (Hohlfeld et al., 1984). However, AChR from other species, mainly from electric rays, Torpedo californica, can be used for the selection and propagation of human T-cell lines. AChR-specific T-cell lines were established from primary cultures of peripheral blood lymphocytes (PBL) (Hohlfeld et al., 1984; Harcourt et al., 1988; Melms etal., 1988) and thymus (Melmsetal., 1988). TheseTcelI lines could be maintained in culture by repeated cycles of restimulation at 7- 14 day intervals with AChR in the presence of autologous irradiated

185

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Expression on cell surface

FIG. 3. AChR interaction with ‘ I lyniphoc ytes. I n contrast to antihodies and 13 cell.;, 7‘ cells cannot interact with the native A ( : h K molecule hut react with proreolytically tlegr.atled AChK fragments. Receptor degradation (“antigen processing”) takes place within an “antigempresenting cell,” which niay be a niaci-opliage, dendritic cell, B ccll. or possibly a “nonprofessional” antigen-presenting ccll. Still in the interior o f the antigeri-~)rc.sentin~ cell, peptide fragments of the A(:hR Iinrl to class 11 HLA molecules (e.g.. t 1 I A l ) R ) . Peptide binding by HLA molecules is pi.obably not antigen-specitic in the sense that antigen binding by antibodies is. However, the interaction is semispecific in that not all ~mtcntial degradation products of a complex antigen c.iiii hind to ii given HLA nioleculc. O i i c e the complex of peptide and HLA molecule h a s fOrmed, it is transported to and incoi-porateti into the surface membrane of the aiiti~en-i”esenting cell, where it can be r e c o g n i d b v an AChR-specific helper T cell (not shown).

peripheral blood cells as antigen-pi-esenting cells, followed by tui-ther expansion in recombinant human interleukin-2 (11-2)as a growth factor. Interestingly, thymic T-cell populations gave higher yields of AChKspecific T-cell lines than did peripheral blood mononuclear cells obtained from the same patient on the same day (Melms et al., 19x8). Lines of AChR-specific T cells could be maintained in culture for from 3 months to over 1 year without losing their positive response to 7orprdo AChR. All

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the cell lines expressed the CD3+, CD4+ membrane phenotype but were negative for CD8 (Hohlfeld et al., 1984; Harcourt et al., 1988; Melms et al., 1988). This phenotype is typical of T helper cells. Indeed autoreactive AChR-specific T cells have the function of helper cells since they can augment AChR antibody production in uztro in the presence of AChR (Hohlfeld et al., 1985). Like most CD4+ T cells, the AChR-specific T-line cells recognize antigen in the context of MHC class 11 products, because antigen-induced proliferation was effectively inhibited in the presence of monoclonal antibodies against monomorphic HLA DR determinants (Hohlfeld et al., 1987; Melms et al., 1989). Analysis of the AChR subunit specificity of the T cells is the first step in the elucidation of how the AChR is recognized as a tridimensional structure by the two complementary components of the human immune system (T cells and B cells). T h e a subunit dominates in the anti-AChR antibody response in MG (see previous section). The T-cell recognition sites on the AChR have not yet been characterized as extensively as the antibody binding sites. However, the majority of the T-cell recognition sites lie on the same subunit as the MIK (Hohlfeld et al., 1987). When stimulated with denatured isolated subunits, AChR-specific T cells showed a strong proliferative reactivity with the a subunit (Hohlfeld et al., 1987). In some patients the /3, y , and 6 subunits also evoked significant proliferative responses. T h e predominant immunogenicity of the a subunit is unexplained but could be related to the stoichionietric overrepresentation of the a subunit in the pentameric AChR complex (see above). AChR-induced stimulation of T cells could not be inhibited with a large excess of anti-MIR monoclonal antibodies or with antibodies present in autologous serum (Hohlfeld et al., 1987).These results suggest that the T-cell epitopes are separated from the MIR. This is consistent with results in other experimental systems (Berzofski et al., 1979; Manzels et al., 1980; Barcinski and Rosenthal, 1977) demonstrating that the antigen determinants recognized by T cells are usually different from those recognized by antibodies. At present it is not known whether or how T helper cells Fpecific for certain epitopes on the a subunit direct the B-cell response to other a subunit determinants such a s the MIR. Even single amino acid substitutions in a peptide can alter the abilitk of a T cell to recognize its epitope (Allen et ul., 1987; Sette et al., 1987: Berzofsky, 1988). Therefore, T-cell lines selected with Torpedo A(:hK may lack the clones that are important in the disease. Mamma1'.1'111 0 1 . better, human AChR (fragments) are required for mapping rI-c.c.lI c a p topes. T cells respond to sequential determinants rather I hail 1 0 I I I C .

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187

native conformation of the stimulating antigen (Berzofsky, 1988).'I'herefore, fragments of protein may be used for mapping T-cell epitopes, provided they contain the determinants necessary to bind to the ~I.-cell receptor and to interact with self MHC rnolecules. Different types of AChR fragments are available to study the immune response in MG, such as synthetic peptides of short length (about 10-20 amino acids) and fusion proteins, which are recombinant gene products that can be substantially longer. From a full-length cDNA for the a subunit of' the mouse X C h K receptor, Barkas et al. (1988) constructed fusion proteins for several parts of the extracellular domain of the AChR. T h e approach using recom1)inant gene products provides AChK fragments (fusion proteins) of greater length than synthetic peptides. I n synthetic peptides the antigenic hierarchy of different epitopes may be disturbed or altered because of their short length. An ideal strategy to map 71-cellepitopes woultl be a two-step approach using fusion proteins first and smallel- synthetic peptides in a second step (Melms et al., 1989). Melms et al. ( 1988) obtained Fl.-celllines from the peripheral Idood and thymuses of patients with MG. 'l'he T-cell lines were selected for reactivity against Torpedo AChK o r against the recombinant fusion protein X4 (residues 6-2 12) that represents the extr-acellular portion of the mouse AChK a chain (Melms rt cil., 1989). Using a panel of fusion proteins of different overlappiiig mouse A(;hK a chain sequences, a major T-cell epitope was localized between amino acid positions 8.5 and 142. This determinant was clistiiict from the humoral M I K , which has been identified on the sequence (i1-76 (Barkas ( J / cil., 1988) and ti7-76 (Tzartos rt al., 1988). Different 'l'-cell lines isolated from patients with different HLA haplotypes showed clit'ferent epitope profiles (blelins rt al., 1989). N o cross-reactivity was seen between the X4 protein and native or denatured Torpedo AChK it' the -1' cells were selected with X4. I n contrast, one T-cell line selected w i t h Torpedo AChK cross-reacted b v i t h X4, suggesting that this T-cell line recognized a different epitope. An important advantage of the fusion protein approach is that it allows more "natural" processing 01' the antigen than does the approach using peptides. Natural antigen processing involves the uptake and iiitracellular degradation of protein antigens into peptides, binding of' peptides to class I1 MHC molecules, and expression of the peptide-hIHC complex on the surface of the antigen-presenting cell (Unanue and Allen, 1987). I t is clear that many 01' these processing steps are circumvented if the antigen-presenting cell is fed with pref'ornied peptides. These may differ unpredictably f'roin the products of natural antigen

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processing. Furthermore, it is likely that normally different peptides compete for MHC binding, and that not all the degradation products of a protein will eventually reach the cell surface (Unanue and Allen, 1987). For these reasons, panels of synthetic peptides are most appropiate for the fine mapping of epitopes identified by mapping with much larger antigen fragments. Models predicting the tertiary structure and transmembrane folding of AChR subunits (Tainer et al., 1985; McCarthy et al., 1986; Novotny et al., 1986) may help to predict which portions of the sequence could be part of antigenic sites for B lymphocytes and antibodies. Likewise, knowledge about general features of 'r-cell epitope structure is important in order to choose appropiate sequences of an autoantigeri for mapping (Berzofsky, 1988; Rothbard and Taylor, 1988). Several groups have demonstrated structural features that allow the prediction of immunodominant T-cell antigenic sites from the primary sequence of proteins. Features common to most T-cell epitopes may be necessary for binding to a specific site on the class 11 MHC protein of the antigen presenting cell. DeLisi and Berzofsky ( 1985) have suggested that many T-cell epitopes have the ability to form amphipatic helices, whereas Rothbard and Taylor (1988) proposed that T-cell epitopes contain a common motif of four or five amino acids. The peptides used in most T-cell epitope mapping studies were selected according to such criteria (reviewed by Hohlfeld, 1989). Hohlfeld et al. (198813) selected polyclonal Torpedo AChR-specific Tcell lines from the peripheral blood of myasthenic patients with different HLA-DR type and tested the T cells for reactivity with three overlapping synthetic peptides (each 14- 16 residues long) corresponding to the amino-terminal 34 residues of the human AChR a subunit. These peptides were chosen because the sequences that they represent have a propensity to form amphipatic a helices, a structure common to many T-cell epitopes (DeLisi and Berzofsky, 1985). One peptide elicited about 30% of the response induced by native Torpedo AChR, indicating that the NH2 terminus of the a subunit contains T-cell-stimulating epitopes in patients with particular HLA types. The fact that only 30% of the AChR response could be induced by the peptide indicated that other T-cellstimulating epitopes exist on the AChR molecule. Other groups identified additional epitopes on different sites of the AChR a subunit. T h e human a chain contains 22 of the recurring amino acid motifs identified by Rothbard and Taylor (1988) as T-cell epitope candidate sites. Harcourt et al. (1988) selected 11 synthetic peptides including many of these motifs. They studied 'T-cell proliferative re-

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sponses to these peptides or t o 7orpedo AChR using blood, thymus, or lymph node cells from 34 patients with MG and 7 controls. This approach located a 1-cell epitope on sequence 257-269 of the human AChR a chain. T h e sequence was recognized by 20% of MG patients examined and none of the controls. T h e selective response of patients to peptide 257-269 suggested that a previously unsuspected external region of the AChR (located between two of the transmembrane domains) may represent an autoimmune T-cell epitope. A second epitope was located on sequence 125- 143. T h e corresponding peptide stimulated T cells in 20%) of patients and 41% of controls (Harcourt et al., 1988). There was no T-cell cross-reactivity between 7 o r p ~ d oAChR and human AChR peptides. This may indicate that Torpedo AChR does not stimulate all the potentially autoreactive T cells in MG patients. The findings by Melins et al. (1988) support this conclusion. Brocke et al. (1988) tested seven peptides representing different sequences of the human AChK (Y subunit for their ability to stimulate peripheral blood lymphocytes of’ myasthenic patients and healthy controls in primary proliferation assays. Three of the peptides that contained the T-cell motif proposed by Rothbard and Taylor (1988)discriminated significantly between healthy controls and myasthenic patients. The proliferative response on two of the peptides appeared to correlate with specific HLA determinants. In contrast, Harcourt et al. ( 1988) found no strong correlation between any of the responding individuals. The studies published so far take only a first step in mapping the human T-cell response to AChR. Much more work is needed to establish the immunodominant T cell epitopes and their HLA associations. It is important to note that AChR-specific T cells can be isolated from normal individuals. Sommer- et (11. (1989) used Torpedo AChR arid a synthetic peptide representing sequence 125- 143 of the human AChR a subunit to raise T-cell lines and clones from healthy individuals. T h e T cells cross-reacted with Torpedo AChR arid the human AChR peptide. These results support the general concept that potentially autoimmune ?‘ cells are present in the normal immune system. In conclusion, the results of the T-cell studies suggest that the autoimmune T-cell response in MG is clonally heterogeneous (polyclonal), although focused on immunodominant epitopes that vary between different individuals. The MHC ( H I A region) has an important influence on which T-cell epitopes are immunodominant. T h e variability of the T-cell autoimmune response between patients can be partially explained by the fact that T helper cells recognize antigen in association with class I1 histocompatibility antigens.

190 V. Role of the Thymus

Most MG patients have thymic abnormalities, either so-called lymphofollicular hyperplasia o r thynioma (Castleman and Norris, 1966; Levine and Rosai, 1978; O t t o , 1984; Miiller-Hermelink r l nl., 1986). Lymphofollicular hyperplasia is characterized by the presence of lymphoid follicles with germinal centers embedded in the thymic medulla. T h e configuration and composition of the thymic lymphoid follicles in MG is the same as that of the lymphoid follicles in peripheral lymph 1985; Hofmann et al., 1987). nodes (Kornstein el nl., 1984; Bofill et d., Lymphofollicular hyperplasia is iiot unique to MG but has been described in other autoimmune disases (Levine and Kosai, 1978; Otto, 1984). T h e other type of thymic abnormality associated with MG are different forms of malignant thynioma. T h e thymic tumors most commonly associated with MG are cortical thymomas and well-differentiated thymic carcinomas (Muller-Hermelink rt al., 1986; Kirchner and MullerHerrnelink, 1989). Both tumors are related to thymic cortical epithelial cells. These pathological thymic changes could be coincidental o r could be directly or indirectly related to the pathogenesis of MG. Convincing evidence has accumulated over the years indicating that the thymus is indeed central to the pathogenesis o f MG in the majority of patients. One of the oldest arguments supporting this notion is purely empirical. It is the long-standing observation that the symptoms of MG may improve after removal of‘ the thymus (Blalock et ctl., 1939; Buckinghani el al., 1976). However, a clear concept of how the thymic pathology could be related to the autoimmune reaction against AChR was lacking until 1975, when it was reported that rodent thymus contains pluripotent stem cells able to differentiate in culture into multinucleated, striated, twitching myotubes (Wekerle et nl., 1975). Similar results were obtained with human thymus, although human myotubes differentiate far less in culture than d o rat a nd mouse myotubes (Kao and Drachman, 1977). Both human and rodent thymus-derived myotubes express AChR (Kao and Drachman, 1977; Wekerle et ul., 1978). Based on these results, Wekerle and Ketelsen (1977) suggested a plausible mechanism for thymic autosensitization. Their model has been strongly supported by the more recent results discussed below. First, the presence in the thymus of AChR (or at least of closely related molecules) could be substantiated. AChR is expressed on thymic

“myoid cells” (Schluep et al., 1987). Myoid cells are rare inusclelike cells mainly located in the medulla both in normal and in hyperplastic tliynus, but not in thymoma (Van d e Velde and Friedman, 1970; Kirchner (11.. 1988a,b). In hyperplastic thynius, the rriyoid cells are localized near, but 1988a). T h e myoid cells not within, the germinal centers (Kirchner et d., themselves a r e HLA-DR-negative (Kirchner et nl., I988b; Schluep et d . , 1987). However, HLA-DR-positive cells are usually found in close proximity to the myoid cells (Kirchner ot al., 1988b). One may speculate that the HLA-DR-positive nonmyoid cells take u p AChR from the myoid cells and present it to T cells. In thynlonia, the situation is different. Imniunohistochemical studies with anti-AChK monoclonal antibodies indicated that AChR epitopes of the MIR are not expressed in thymoma (Kirchner et al., 198813). I n contrast, thyinomas f‘rom patients with M G but not from nonmyasthenic patients showed cytoplasmic AChR epitopes (Kirchner et al., 1988b). Recently, one o f t h e thynioina proteins that might be responsible for the cross-reaction with monoclonal antibodies against cytoplasmic AChR determinants was characterized in more detail (Marx Pt al., 1989). It is a protein of approxiiiiately 153 kDa that is clearly distinct from muscle AChK (Marx et c i l . , 1990). It is possible that this protein plays a role in the autosensitization of T cells, but this remains to be shown. Not only does the myasthenic thymus contain AChR or AChK-related molecules, but it also contains AChK-specific B lymphocytes. ’17hymiccells from MCr patients with hyperplastic thymiis spontaneously produce antiAChR antibodies in culture (Scadding et al., 1981 ) . ’Thymic cells f‘rorn control patients produce no anti-AChR antibodies (Scadding rt c i l . , 1!18l), and thymocytes from thynioriia patients produce n o (Scadding Pt al., 1981) or only low amounts of autoantibodies (Fujii et ul., 1984). Clearly, the thymus is not the only site o f autoantibody production in MC; (Willcox et al., 1983; Fujii et al., 1986).Interestingly, thymocytes from MC; patients who had been immunized with tetanus toxoid several weeks prior to thymectomy produced both anti-AChR and anti-tetanus-toxoid antibodies (Lisak et al., 1986). This suggests that there is lymphocyte trafficking from the periphery into the hyperplastic thymus and that lymphocytes of any specificity can enter the thymus. It would be interesting t o know how many of the intrathymic B (and 1’)lymphocytes are specific for A(:hK, and how diverse their specificity spectrum is. Tesch et al. (1989) used DNA probes specific for immunoglobulin heavy and light chain genes on Southern blots of thymic DNA f’rom patients with hyperplastic thymus. No single rearranged bands were detected. ‘The known sensitivity of. this method allows estimation of an upper limit of‘approximately 1%’ of‘the (71

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proportion of any single clone of AChK-specific B (or T) cells present in the myasthenic thymus (Tesch et al., 1989). Finally, in further support of the hypothesis of thymic autosensitization, AChR-specific T lymphocytes could be isolated directly from the thymus of all 10 investigated MG patients (Melms et al., 1988). In contrast, AChR-specific T cells could be isolated from the peripheral blood in only 3 of these 10 patients. This indicates that AChR-specific T cells are enriched in the myasthenic thymus, as predicted by the hypothesis (Wekerle et nl., 1981). One reason intrathymic autosensitization is such an intriguing concept is that the thymus is the major anatomical site of tolerance induction. There has been much progress i n the understanding of the intrathymic T cell selection events that give rise to self tolerance, mainly due to elegant experiments with transgenic mice (reviewed by Schwartz, 1989). According to current concepts, two key intrathymic selection events operate on differentiating T cells, probably at a precursor stage when the ‘I‘ cells are still CD4+, CD8+ double-positive. One is negative selection (clonal deletion). It is thought that T cells expressing receptors for peptides derived from self proteins bound to self MHC molecules on the surface of bone marrow-derived dendritic cells are deleted. The other key event is positive selection for recognition of MHC molecules on thymic epithelial cells. Exactly how positive and negative selection interact in the generation of the final T cell repertoire is not yet known (Schwartz, 1989). It is tempting to speculate that intrathymic presentation of AChR to maturating T cells by an as yet unknown type of antigen-presenting cell somehow interferes with the interplay of positive and negative selection, resulting in the activation of, rather than tolerance induction in, AChR-specific T cells. Another interesting hypothesis concerning the mechanism of initiation of the immune response to AChR is the theory of molecular mimicry. An amino acid sequence shared between an infectious agent and a host protein might enable the microbe to initiate an immunologic response that subsequently cross-reacts with the “self’ determinant (Oldstone, 1987). T h e relevant amino acid sequence must be different enough from the self protein that tolerance does not occur, but sufficiently similar that cross-reactivity is possible. In MG, it was demonstrated, a sequence of the a subunit of human AChR (amino acid residues 160-167) that is not located in the “main immunogenic region” shares an immunologically significant structural homology with herpes simplex virus I glycoprotein D (amino acid residues 286-293). Sera from 6 of 40 patients with MG recognized this

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a-subunit sequence and cross-reacted to the HSV sequence (SchM’immbeck et al., 1989). Others have documented immunologic cross-reactivity with bacteria (Stefansson et al., 1985; Datta and Schwartz, 1974). It is difficult to argue, however, that the polyclonal B-cell activation seen in MG should be initiated by cross-reactivity between one or two microbial agents and the AChR. Another interesting hypothesis is the possibility of “auto-mimicry.” T h e anti-AChK mAb 155 was shown to recognize a 153-kD protein that is present on thymoma epithelial cell lines. This protein does not bind a-BGT and has no epitopes of the main immunogenic region of the AChR. Thus, this protein is distinct from the AChR (Marx et al., 1990). This led to the hypothesis that thymonia-associated myasthenia is initiated by an immune reaction not against the AChR, but against the partly cross-reacting p153 molecule. For some types of MG this protein may indeed be important in initiating the immune response against the AChR, but its pathogenetical relevance remains to be shown. Because mAb 155 recognizes a cytoplasmic domain of the AChK, protein 15:3 is unlikely to be an important target for the B-cell immune response against the AChR. However, it coulci still be an autoimmunogeri for ‘1. cells (Hohlfeld, 1990). ’

VI. Treatment Strategies

Modern treatment has dramatically improved the prognosis of M G . One part of this success is probably due to general progress in the symptomatic and supportive therapy of severely ill patients (reviewed by Oosterhuis, 1984). For example, the present state of intensive care therapy would allow many patients to survive a myasthenic crisis even i f the pathophysiology of MG were completely unknown. T h e other part o f the progress, however, is clearly due to the concepts and knowledge gained from basic research. T h e current treatment options for MG are (1) symptomatic therapy with acetylcholinesterase inhibiting agents, (2) empirical surgical therapy with thymectomy, (3) management of myasthenic crisis with plasmapheresis, and (4) immunosuppressive therapy with corticosteroids, azal hioprine, and/or other immunosuppressants (for a state-of-the-art discussion of MG treatment, see Toyka, 1990). Anticholinesterase drugs have remained the mainstay of symptomatic

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treatment for MG ever since the dramatic discovery of their effectiveness by Walker (1934). These agents act by increasing the concentration of ACh in the synaptic cleft. Thymectomy was originally a purely empirical treatment for MG that was first discovered by Sauerbruch (for an historical overview, see Schadewaldt, 1977) and later by Blalock et al. (1939; Blalock, 1944). Although the effectiveness of thymectomy has never been proved in a controlled clinical trial, most experts think it is beneficial for patients with recent onset, generalized MG. A malignant thynioma is an absolute indication for surgery independent of the duration and severity of the disease. Plasmapheresis provides a means to reduce the quantity of circulating autoantibodies acutely (Newsom-Davis et al., 1979; Dau et al., 1977). T h e experimental basis for plasmatherapy was laid by Toyka et al. (1975) with their demonstration that IgG preparations from patient serum can transfer the disease to animals. T h e main indication for plasmapheresis is myasthenic crisis. Corticosteroids have long been used to treat MG, the idea being to suppress the overactive immune system. Steroids have proved to be useful for the treatment of ocular and generalized MG, but their somewhat limited effectiveness and serious side effects during long-term use motivated a search for more effective immunosuppressive reagents. Mertens et al. (198 1) were among the first to use azathioprine in MG. This antimetabolite immunosuppressant has subsequently become the drug of choice for the long-term immunosuppressive treatment of generalized MG. T h e drug suppresses both B- and T-cell immune activity in an antigen-nonspecific way (Hohlfeld et al., 1985). Its side effects during long-term use seem acceptable (Kissel et al., 1986; Hohlfeld et al., 1988a). T h e risk of the most serious complication, the development of malignancy and especially lymphoma, has been estimated such that a tumor develops in 1 of 100 patients after a median period of about 29 months of continuous treatment (Hohlfeld et al., 1988a). When azathioprine was discontinued after several years of clinical remission, almost 45% of patients remained free of disease for an observation period of up to 6 years (Hohlfeld et al., 1985; Michels et al., 1988). Although the existing therapies, particularly immunosuppression with azathioprine, now allow the majority of MG patients to lead an almost normal life, the risks of long-term immunosuppressive treatment are certainly not negligible. T h e search for better and more specific forms of immunotherapy continues, and the development of a new generation of immunotherapeutic reagents is one of the major goals of clinical neuroimmunology. One may argue that MG, one of the few

human autoimmune diseases in which the autoantigen is known, is ideal to develop a n d test antigen-specific immunotherapies. Elegant new strategies of immunotherapy have heen used successfully in expcrimental autoimmune encephalomyelitis (EAE) (reviewed bv Wraith rt ul., 1989; Hohlfeld, 1989). T h e coni~nonprinciple of these therapies is that they a r e directed selectively against the autoininiune 'F lyrnphocytes, leaving the rest of' the immune svstcm intact. There is hope that similar approaches can be taken in tiunian autoinimune disease and that M(; is among the first diseases where the new therapies can be tested.

Acknowledgment

K. H. is a recipient of thc Heiscnbei g Federal Republic of Germany.

91-ailt of

the L)euiache Forschungsgciiiciiis~tiatt,

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PRESYNAPTIC EFFECTS OF TOXINS By Alan L. Harvey Department of Physiology and Pharmacology University of Strathclyde Glasgow G1 lXW, Scotland

1. Introduction

11.

111.

IV.

V.

A. Toxins in Neurobiology B. Presynaptic Physiology: Potential Sires for Toxins Toxins Affecting Neuronal Ion Channels A. Na+ Channel Toxins B. K + Channel Toxins C . Car+ Channel Toxins Toxins Affecting Release Mechanisms A. Botulinum and Tetanus Toxins B. a-Latrotoxin, Glycerotoxin, and Leptinotoxin C. P-Bungarotoxin and Related Phospholipase A:! Toxins Miscellaneous Toxins A. Maitotoxin B. Pardaxins C . Other Toxins Conclusions References

1. Introduction

A. TOXINS IN NEUROBIOLOW Toxins are natural products that have deleterious pharmacological actions. They come from plant, microbial, and animal sources, and are either a byproduct of metabolism (e.g., many secondary plant metabolites) or have evolved for defense or predatory purposes (e.g., many snake venom components). Neurotoxins are toxins that affect the nervous system. They include toxins that act on neurons directly and toxins that disturb communication between neurons and their target cells. Neurotoxins are very diverse chemically. They are among the most potent biologically active compounds known and are usually extremely specific in their actions. Hence, neurotoxins often find use as experimental tools. Probably they are studied more for that aspect of their properties than for their medical signihINTERNATIONAL REVIEM’ OF NEUROBIOLOGY. VOL. 32

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cance. For example, radiolabeled tetrodotoxin and saxitoxin have been used to quantify the number of Naf channels in different tissues and to follow their appearance during neuronal development; dendrotoxin has been used to isolate putative K + channel proteins; and a-latrotoxin and botulinum toxin have been used in a wide variety of experiments investigating the physiology of transmitter release.

B. PRESYNAPTIC PHYSIOLOGY: POTENTIAL S I T E S FOR ‘rOXINS T h e nervous system controls many vital functions and, therefore, its disturbance can have serious consequences. The majority of neurotoxins do not cross the blood-brain barrier but act on the peripheral nervous system. There are some notable exceptions, such as apamin and tetanus toxin, but usually special uptake mechanisms have to exist to allow neurotoxins to enter the brain or spinal cord. I n the peripheral nervous system, toxins can affect the physiological functions of neurons themselves or interfere with the synaptic signaling processes. As the somatic nervous system is essential for activation of respiratory muscles, most neurotoxins appear to affect its function, rather than that of the autonomic nervous system, whose role is to modulate cardiovascular and digestive processes. Physiologically important targets for neurotoxins acting directly on neurons include the transmembrane ionic channels that are essential for action potential conduction and transmitter release. These are the Na+, Kf and Ca2+ ion channels. As synaptic transniission is a complex, energetically demanding process, it contains a large number of target sites on which neurotoxins might act. The prejunctional mechanisms include those controlling the synthesis, storage, and release of neurotransmitters. Toxins affecting presynaptic processes are the subject of this review. Other toxins are known to act on postjunctional sites, including the receptors for the transmitter molecules and metabolic enzymes; these will not be considered here.

II. Toxins Affecting Neuronal Ion Channels

A. N A + CHANNEL TOXINS

Sodium channels are integral membrane proteins that respond to a change in the electrical field across the membrane. When the membrane is depolarized, the channel protein changes its conformation, a trans-

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membrane pore opens and Na’ ions flow through the pore according to the concentration gradient and the electrical field. Na+ channels have a complicated behavior because they spontaneously close (“inactivate”)despite the continued presence of an activating signal. ‘Toxins can act h y simply blocking the channel or by interfering with the activation or inactivation properties of the channel protein (for reviews, see Catterall, 1980, 1986; Catterall et ul., 1986; Strichartz ut ul., 1987). 1. Channel-Blocking Toxins

u. Tetrodotoxin and Suxitoxiri. ‘[’he best known of the Na+ channelblocking toxins are tetrodotoxin and saxitoxin. Both are alkaloids containing a guanidino moiety. I t is thought that the positive charge is driven into the mouth of the Na’ channel b y the electrical field and then the molecule occludes the pore of the channel. Tetrodotoxin is well known because of its presence in Japanese puffer fish orfugu. This is regarded as a delicacy in Japan and is prepared by specially licensed chefs. H O W ~ Vfatalities ~ I - , occur annually among overenthusiastic amateurs. Tetrodotoxin has been found in a wide variety of animals, such as mollusks, crabs, octopus, fish, newts, and Central Anierican frogs (see, e.g., Yasumoto pt ul., 1986; Mebs and Schmidt, lYX9). Apparently, the toxin is a product of microorganisms that colonize the host (Yasumoto et al., 1986). Saxitoxin is found in certain clams and mussels that feed on Goriyuulrix dinoflagellates, which actually manufacture the toxin (for reviews, see Kao, 1966; Schantz, 1986). Tetrodotoxin and saxitoxin are highly toxic and especially dangerous because they are well absorbed after oral ingestion. Blockage of Na’ channels leads to failure of action potentials to propagate along axons. Sensory neurons are first affected (these neurotoxins are prototype local anesthetics), but at higher doses, motor nerves are also blocked, leading to skeletal muscle weakness and, ultimately, muscle paralysis and respiratory collapse. Strictly speaking, tetrodotoxin and saxitoxin are not presynaptic toxins because they primarily affect neuronal conduction; they can also paralyse skeletal muscle directly. Low concentrations of‘ the toxins do have an apparent presynaptic effect, as failure of action potentials to propagate to nerve terminals leads to a decrease in transmitter release. T h e pharmacological effects of these toxins on neuromuscular transmission were described by Kao and Nishiyama (1965). More recent experiments have aimed to study the effects of tetrodotoxin on propagation of action potentials into the nonmyelinated terminal regions of mouse motor nerves (Konishi, 1085). Tetrodotoxin and saxitoxin can be radiolabeled and their affinity for Na+ channels can be measured (see review by Ritchie and Rogart, 1977). The equilibrium dissociation constant K,! is 1-5 n M in a number of

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tissues, including human brain (Mourre et al., 1988a). Both toxins bind to the same class of site. Further details of tetrodotoxin and saxitoxin binding and their use in biochemical experiments can be found in reviews by Catterall (1980, 1986) and Kao and Levinson (1986). A more recent experimental study on the effects of the two toxins was reported by Lonnendonker (1989a,b). b. p-Conotoxins. Another group of Na+ channel-blocking toxins was discovered in the venom of the marine snail Conus geographus (Cruz et al., 1985; Sat0 et al., 1983). Seven of these conotoxins or geographutoxins are known (Cruz et al., 1985). They are basic polypeptides containing 22 amino acid residues; they are now generally referred to as p-conotoxins (see review by Gray et al.). p-Conotoxins differ from tetrodotoxin and saxitoxin because the p-conotoxins primarily block Na+ channels in skeletal muscle without affecting action potential conduction in motor nerves. Because of this property, one of the toxins was used to prevent muscle contractions while neuromuscular transmission was studied in the absence of other blocking agents (Hong and Chang, 1989). Although p-conotoxins do not block Na+ channels on frog or mammalian motor nerves or from rat brain (Cruz et al., 1985), they do bind to Na+ channels from electric eel electroplax (Yanagawa et al., 1987) and, perhaps unexpectedly, they block presynaptically in guinea-pig vas deferens and ileum preparations (Ohizumi et al., 1986). The pconotoxins bind to the same site on the Na+ channel as do tetrodotoxin and saxitoxin. However, their binding is influenced by membrane potential, being decreased on hyperpolarization (Cruz et al., 1985). The Kd for p-conotoxin GIIIA was estimated from functional studies to be about 100 nM at 0 mV for rat skeletal muscle (Cruz et al., 1985), and the Kd for a radiolabeled derivative of p-conotoxin GIIIB in electroplax was 1.1 nM (Yanagawa et al., 1987). Another toxin with similar binding properties to the p-conotoxins has been isolated from the venom of Conusgeographus (Yanagawa et al., 1988). This toxin (conotoxin GS) has 34 amino acid residues and three disulfide bonds; it contains two hydroxyproline residues and one carboxyglutamic acid residue. Although there is little apparent sequence similarity between conotoxin GS and the p-conotoxins, conotoxin GS also displays a higher affinity for tetrodotoxin binding sites on muscle than on nerve, and it also displaces binding of a tritiated p-conotoxin. No details are available concerning its pharmacological properties.

2. Toxins Affecting Channel Activation Normally, a depolarization of a certain size is required before Na+ channels open. In the presence of some toxins, especially from scorpion

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venoms, the channels open niore readily. In electrophysiological terms, the activation curve is shifted to more negative potentials. a. Lipid-Soluble Toxins. l ' h e first compound of natural origin found to affect Naf channel activation was the lipid soluble plant alkaloid veratridine. It increases Na' permeability of nerves and muscles by altering the properties of a fraction of the Na+ channels. The affected channels open at normal levels of membrane potential and d o not inactivate. Because of the inHux of Na', the cell depolarizes. Veratridine is not very potent, with half-maximal effects requiring about 80 p M (Catterall, 1975). Another lipid-soluble toxin is batrachotoxin, which is isolated from the skin of the Colombian arrow poison frog Phyllohates aurotaenia. Batrachotoxin also shifts the voltage dependence of activation of Na' channels and blocks inactivation. It is more potent than veratridine, with an IC50 of 0.4 pM (Catterall, 1975). T h e effects of batrachotoxin and veratridine are blocked by tetrodotoxin and saxitoxin. However, this blockade is noncompetitive, indicating that the toxins are binding to distinct sites on the Na' channel. At the mammalian neuromuscular junction, batrachotoxin ( 10 nM) causes a transient increase in the spontaneous release of acetylcholine, followed by a block of both spontaneous and evoked release (Jansson et al., 1974). These effects are presumably a result of batrachotoxininduced depolarization of the nerve terminal. Other lipid-soluble toxins acting on Naf channels are the brevetoxins isolated from the red-tide dinofhgellate Ptyhodiscus breuzs (Raden, 1983). Brevetoxin-B and the structurally related toxin T-17 have been shown to depolarize giant axons of squid on internal and external application (Atchison et al., 1986).T h e depolarization was Na+-dependentarid could be blocked by tetrodotoxiri. Although concentrations of 2-30 pM were needed to affect squid giant axons, the brevetoxins at nanomolar concentrations increased miniature endplate potential frequencies in rat and frog neuromuscular preparations. Voltage clamp analysis showed that the effects of the brevetoxins on Na' currents were similar to those of batrachotoxin; that is, they shifted the activation curve by about 2040 mV in a negative direction and they reduced inactivation; however, brevetoxins also increased the amplitude of the peak Na+ current. h. p-Scorpion Toxins. Venom from the American scorpion Centruroides sculpturatus causes Na+ channels to open at levels of membrane potential at which the channels would normally be closed, although the size of the peak current is depressed. Several purified toxins with similar activity have been isolated from venom of New World scorpions, including C . sculpturatus, Centuroides sufS.IL,yus suffusus, and Tityus sprrulatus. Because their binding properties differed from those of the scorpion toxins

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that prevent inactivation, these toxins were termed @-scorpion toxins (Jover et al., 1980). The electrophysiological effects of @-scorpion toxins on excitable membranes were reviewed by Meves et al. (1986). Little is known about the effects of P-scorpion toxins on synaptic transmission.

3. Toxins Afiecting Channel Inactivation Usually the Na' current wanes within a few msec of its peak as a result of the spontaneous closing, or inactivation, of individual channels. The inactivation process is normally dependent on the membrane potential. A number of toxins can affect inactivation, usually by prolonging or blocking it. Consequently, neurons have much longer Na' currents than normal, which generally results in excessive initial stimulation followed by inhibition. a. a-Scorpion Toxin,.$.Several African and North American scorpion venoms have been shown to prolong action potential by affecting the inactivation of Na+ currents. T h e purified components responsible have been termed a-scorpion toxins Uover et ul., 1980). Toxin I from Androctonus australis Hector causes an increase of transmitter release and repetitive activation at both inhibitory and excitatory synapses on crayfish muscle (Komey et al., l976a). These effects were not reversed by washing out the toxin. Toxin I also increased the frequency of miniature endplate potentials, consistent with the toxin causing depolarization of nerve terminals. 'l'his effect was prevented by prior exposure to tetrodotoxin. Toxin I also can cause release of neurotransmitters from synaptosomes (Rorney et al., 1976a). '251-labeledToxin I I from Androctonus australis has been used to study the distribution of Na' channels at mouse neuromuscular junctions (Boudier et al., 1988). There has been a debate about the distribution of Na' channels in mammalian motor terminals (Brigant and Mallart, 1982; Konishi,-l985; Mallart, 1985). Electron microscopic examination of the distribution of binding sites for a-scorpion toxin indicated that the unmyelinated motor nerve terminal of the mouse triangularis sterni preparation does not contain Na' channels. More unexpectedly, scorpion toxin binding sites were found to be predominantly on glial cells, surrounding neuromuscular junctions (Boudier et al., 1988). h. Sea Anemone Toxins. A series of polypeptide toxins was isolated fro 1 the sea anemone Anemonia sulcata (Beress et al., 1975), the most abundant of which was ATxII. It is a single chain of 47 amino acid residues cross-linked by three disulfide bonds (Wunderer et al., 1976). Similar toxins have been isolated from Anthopleura xanthogwammica, Radianthus paumotensis, Condylactis gigantea, Heteractis macrodactylus, Phyllactis

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flosculifera, Stichodactyla @g[inteurn, and Stichodactyla kelianthu~(see Lazdunski et al., 1986; Kem et d ,1989). These toxins differ in their species and tissue selectivity, and also in their immunological properties. A toxin with similar pharmacological properties but a different primary structure has been isolated from the sea anemone Calliartzs purasiticu (Cariello et al., 1989). Most pharmacological studies have been performed with ATxII. It has been shown to prolong nerve action potentials and to cause spontaneous and repetitive activity (Komey et ul., 1976b). Prolonged incubation with ATxII leads to depolarization, which can be blocked by addition of tetrodotoxin but not reversed by washing. Voltage clamp studies reveal that ATxII prolongs the inactivation phase of Na+ currents (Komey et ul., 1976b; Neumcke et ul., 1985). ATxlI also affects the Na+ channel gating currents (Neumcke rt ul., 1985), an action observed even in the presence of tetrodotoxin. ATxlI causes release of' transmitters from synaptosomes (Komey et ul., 1976b) and increases the frequency of miniature endplate potentials at vertebrate neuromuscular .junctions (Erxleben and Rathmayer, 1984; Harris and Tesseraux, 1984; Molgo and Mallart, 1985). A T x causes an increase in quanta1 content and repetitive firing of motor nerves (Erxleben and Rathmayer, 1984; Molgo and Mallart, 1985). l.:xtract.llulairecording from mouse motor nerve terminals revealed that local action potentials are not greatly prolonged in the presence of A'l'xll unless K t currents are blocked (Molgo and Mallart, 1985). It appears that the K + current at motor nerve terrriirials is sufficiently powerful to counteract the effects of the prolonged action potential produced by ATxII. 4. Spider Toxins Another class of protein toxin affecting Na' channels has been discovered in the venom of the spider Agrlenopsis upertu (Adams et ul., 1980). They have been termed p-agatoxins, and they cause gradual and irreversible spastic paralysis in flies. This is due to an effect on iieurorial Na' channels that leads to spontarieous action potentials. Five sequences of p-agatoxins have been reported (Skinner et al., 3989), but their precise mechanism of action has yet to be elucidated.

B. K + CHANNEL 'rOXINS Channels selective for K + ions are probably found in every eukaryotic cell. They are responsible for- determining the level of the resting niembrane potential, for repolarization after an action potential, and for

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regulating the activity of neurones. Potassium channels consist of a number of subtypes, which can be distinguished by their voltage dependency, kinetic properties, sensitivity to Ca2+,and pharmacology (for reviews, see Rudy, 1988; Strong, 1990). The variety of neuronal K+ channels and their physiological versatility have been increasingly recognized in the last few years. Until relatively recently, no toxins were known to act specifically on K + channels. However, there is an increasing number of K+ channel toxins being discovered. The toxins are usually selective for either voltage-dependent or Ca2+-dependent K+ channels. 1, Toxins Blocking Voltage-Dependent K + Channels

a. Noxiustoxin. The first toxin demonstrated to block such channels was noxiustoxin from the Mexican scorpion Noxius centruroides (Possani et al., 1982). It was found to block the delayed rectifier K+ current in squid giant axons (Carbone et al., 1982, 1987),although in high concentrations it blocks some Ca2+-dependent K+ channels (Valdivia et al., 1988). More recently, noxiustoxin was shown to cause release of transmitters from synaptosomes (Sitges et al., 1986) and has been demonstrated to facilitate acetylcholine release from motor nerves (Harvey et al., 1990). Noxiustoxin is a relatively small protein, with a single polypeptide chain of 39 residues cross-linked by three disulfide bonds. Its secondary structure is probably similar to other toxins of approximately the same size (e.g., charybdotoxin). Noxiustoxin has been shown to displace radiolabeled dendrotoxin from high-affinity binding sites on brain membranes (Harvey et al., 1990). Also, synthetic peptides corresponding to the first nine residues from the N-terminus of noxiustoxin retain some K+ channel blocking activity (Gurrola et al., 1989). Toxins similar to noxiustoxin have been isolated from other scorpions, but they have been less well characterized. Noxiustoxin has been used to prepare an affinity chromatography column in order to isolate binding sites from squid axonal membranes (Prestipino et al., 1989). T h e affinity-purified proteins could be incorporated into planar lipid bilayers, which then displayed channel activity consistent with the presence of voltage-activated K+ channels. The majority of events corresponded to a single channel conductance of l l ps, and they were blocked by noxiustoxin and tetraethylammonium. b. Dendrotoxins. The dendrotoxins are a family of homologous proteins that are isolated from mamba snake venoms (Harvey and Karlsson, 1980, 1982). They consist of 59-61 amino acid residues in single polypeptide chains, containing three intramolecular disulphide bonds. Dendrotoxins are homologous to Kunitz-type serine proteinase

l'Kkbk"APTI(.

IOXINS

209

inhibitors such as bovine pancreatic trypsin inhibitor (BPTI o r aprotinin). However, BPTI does not have K' channel blocking properties, and, although dendrotoxins inhibit some proteinases (Marshall anti Harvey, 1990), this may be unrelated to their ability to block K + channels. Although all the dendrotoxins from green mamba venom a re thought to be equivalent in their actions at the neuromuscularjunction (Harvey and Karlsson, 1980; Harvey and Anderson, 1985), there appear to he two subgroups, based on their effects on different components of X6Kbflux from synaptosomes (Benishin rt d.,1988). Differences in binding properties have also been founcl (Muniz et nl., 1990). Dendrotoxins have been shown to block some types o f K + channels but not others (for reviews, see Harvey and Anderson, 1985, 1990; Moczydlowski et ul., 1988). They are active in the rianomolar range, and high-affinity binding sites have bee11 detected in the brain. 'I'hey are not very toxic on systemic administration, but their toxicity increases 10,000fold on direct injection into brain. l ' h e dendrotoxins induce repetitive firing of neurons an d also enhance transmitter release. More details of their effects are given below. l h e facilitation of transmitter release was the first effect of dendrotoxins to be characterized (Harvey a i i d Karlsson, 1980, 1982; Harvey and Anderson, 1985; Anderson, 1985; Anderson and Harvey. 1988), and subsequently it was confirmed that tlendrotoxins specifically blocked some neuronal K' channels. I n hippocam pal pyramidal cells, dendrotoxin inhibits a portion of the transient K + current (Dolly ot ul., 1984; Halliwell et nl., 1986),and an equivalent effect has been demonstrated by measuring 8"Rb efflux from synaptosomes (Benishin rt ul., I Y X X ) . I n nodes of Ranvier of frog sciatic nerves, dendrotoxin prolonged the duration of action potentials by blocking a fraction o fth e K' current (Weller rt al., 1985). This was shown t o be d u e to the specific block of the f', coniponent of the K' current (Benoit and Dubois, l986), and this has been confirmed by single channel recording experiments on Xenopzc., axons (Jonas el nl., 1989). T h e single-channel conductance blocked by dentlrotoxin was 23 pS. Dendrotoxins have also heen demonstrated t o have K + channel blocking activity on peripheral sensory neurons. In guinea pig dorsal root ganglion cells, dendrotoxin blocked a fraction of the noninactivating K + current (Penner et ul., 1986). In rat nodose ganglion, some cells were found to produce repetitively firing action potentials in the presence of dendrotoxin (Stansfeld et nl., 1986, l987),an effect related t o a block of'a rapidly activating, slowly inactivating K' current. Stansfeld and Feltz ( 1988) confirmed by patch clamping that dendrotoxin directly blocked K + channels on rat dorsal root ganglion neurons in tissue culture.

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The effects of dendrotoxin at mouse and frog neuroniuscular junctions are to increase quanta1 content and to induce single nerve action potentials to cause short bursts of repetitive activity. These effects have been explained in terms of dendrotoxin blocking a rapidly activating K + current that is important for the control of the excitability of nerve terminals (Anderson et al., 1987; Anderson and Harvey, 1988). In the presence of the nonselective K C channel blocker tetraethylarnmonium, dendrotoxin also appears to block a K + current that has slower kinetics (Dreyer and Penner, 1987). When injected into the CNS, dendrotoxin induces epileptiform activity (Velluti et al., 1987; Silveira el nl., 1988b), and some central effects have also been reported after intraperitoneal irljection of dendrotoxin (Silveira et al., 1988a). T h e distribution of binding sites in the brain has been studied using “’I-labeled clendrotoxin (Pelchen-Matthews and Dolly, 1989; Bidard et al., 1989). More recently, K + channels have been cloned and expressed in Xrnopus oocytes, and some of the neuronally derived channels are sensitive t o dendrotoxin and mast cell degranulating peptide (see Pongs, 1989; Stuhrner et ul., 1989). Radiolabeled dendrotoxin has also been used to isolate binding proteins from brain membranes (e.g., Black et al., 1988; Rehm and Lazdunski, 1988; Parcej and Dolly, 1989). More recently, proteins isolated on a dendrotoxin affinity column were reconstituted in lipid bilayers (Rehm et al., 1989).T h e resulting K + channel activity was blockable by dendrotoxin. The activity was enhanced by phosphorylation of the reconstituted protein. c. Mast Cell Degwanulating Peptide. Mast cell degranulating peptide (MCDP) from honey bee venom was originally discovered because of its ability to release histamine from mast cells (Breithaupt and Habermann, 1968). It was also found to have anti-inflammatory properties (Hanson et al., 1974). Subsequently, MCDP was shown to be a potent convulsant on injection into the CNS (Habermann, 1977). MCDP was also found to compete with dendrotoxin for binding sites on neuronal membranes (Bidard et nl., 1987a; Stansfeld ut ul., 1987),and MCDP was demonstrated to block the dendrotoxin-sensitive rapidly activating K + current on nodose ganglion A cells. When administered centrally, it causes convulsions and epileptiform activity; MCDP can also enhance long-term potentiation (Bidard et al., 1987b; Cherubini et al., 1987, 1988). On some peripheral sensory neurons, MCDP induces repetitive firing of action potentials (Stansfeld et al., 1987),but it is not yet clear whether it can act at nerve terminals to facilitate transmitter release. MCDP is a 22-residue polypeptide containing two disulfide bonds. It is a very basic peptide, having a net charge of 8+. N o definitive three-

dimensional structure has been tletermined, but N M K arid C1) spect ra combined with computer predictions are consistent with a spherical conformation containing six or sevcn residues in an (Y helix (Ijotimas P t ( I / . , 1987). MCDP has been racliol;~l)eledwith ““I and high-affinity binding sites have been found in rat brain (Taylor rt ul., 1984). More recently, it has been shown that there are allosteric interactions between hl<:I)P, dendrotoxins, and P-bungai-otoxin (Bidard et nl., 1987;i; Kehm d (il., 1988; Schmidt and Betz, 1988; Breeze and Dolly, 1989), ; i d there are differences in distribution of binding sites fbr MCDP and dentlrotoxiii (Bidard et al., 1989). d. Phospholipuse A2 Nezrrotoxiris. Another group of previously characterized toxins now known to have K chanriel blocking properties is the phospholipase A2 neurotoxiiis, including P-bungarotoxin, crotoxin, notexin and taipoxin. These loxiiis are primarily characterized by their ability to block acetylcholine release from motor nerves (see Section llI,C), but P-bungarotoxin was sliown to block I(+ currents in sensory neurones (Petersen rl al., I W i ) and also at motor nerve terminals ( A n derson et al., 1987). Other phospholipase neurotoxins have similar K + channel blocking actions at ~ i e ~ ~ ~ ~ o ~ i i junctions u s c u I a r (Kowan and Harvey, 1988; Kowan et d , 1989). ‘l’lielatter property probably accounts lor the facilitation of transmitter release that occurs in mammalian iierves before the irreversible block (see Section III,C,2). The block of it fractioii of the K’ current does not seem to deperid on the phospholipase activit!, of the toxins. P-Bungarotoxin has been s l i o w i i to interact in a complex maniici- with MCDP and dendrotoxin for I)inding sites 0 1 1 central neuronal i~ieiiibranes (see above). Not all phospholipase A? iieurotoxiiis act in the same way in these binding assays (Othman P / nl., 1982; -1’zeng et ul., I W j ) , although all share the ability to I h c k acetylcholine release. Four single-chain phospholipases were isolated from the ven0111 ot‘ the Australian taipan, Oxyyu,rr~ir.cm ~ / ~ ~ l l u t(Iaiibeau ir.v rt of., 1989).~I’liree of them were highly toxic on iiiti-acei-ebroventricular injection into niicc. One toxin, OSZ, was labeled with ““1 and shown to have two r y e s of’ high-affinity binding sites, w i t h dissociation constants of 1 ..5 and 1.5 pZI. Binding was displaced readily by sollie phospholipase toxiiis (including crotoxin, pseudexin, and textilotoxin), but others (P-bungarotoxin and notexin) were less effective. 71‘herewas no displacement t y other polvpeptide K + channel blockers. including apamin, dendrotoxin. and MCDP. There is no information yet ahout the functional effects of tlirsc taipan phospholipase toxins, arid they should prove to be useful tools in defining the heterogeneous iiature of neuronal K + channels.

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ALAN L. HARVEY

e. Other Toxins Blocking Voltage-Dependent Ki Channels. There are also other toxins acting on K + channels, but these have not yet been fully characterized. Examples are equinatoxin and toxins in the venoms of the scorpions Pandinus imperator (Pappone and Cahalan, 1987; Pappone and Lucero, 1988; Marshall and Harvey, 1989) and Leiurus quinquestriatus (Koppenhoefer and Schmidt, 1968).

2. Toxins Blocking Ca2+-Dependent K Channels a. Apamin. Apamin, a small polypeptide isolated from the venom of the honey bee, was the first neurotoxin found to block K + channels that are activated by increases in levels of internal Ca2+ ions (see reviews by Habermann, 1984; Strong, 1990). Apamin can affect such channels in many types of cells, such as nerves, muscle, erythrocytes, and glandular cells. However, the toxin does not block all Ca2+-activated K + currents. I t appears to be highly selective for the subtype of channel with a low, single channel conductance. Susceptible channels are blocked at low nanomolar concentrations, whereas other channels are unaffected by several micromoles of apamin. The toxin has the unusual feature for a hydrophilic polypeptide of being able to gain access to the CNS. It causes hyperactivity and convulsions before death. From studies of local cerebral glucose utilization, apamin appears preferentially to act on limbic regions of the brain (Mourre et ul., 1988b). Apamin blocks the delayed afterhyperpolarization that follows action potentials in cultured neuroblastoma cells (Hugues et al., l982a), bullfrog sympathetic ganglion cells (Pennefather et al., 1985), rat hippocampal neurons (Lancaster and Nicoll, 1987), and cat spinal motor neurones (Zhang and Krnjevic, 1987). Voltage clamp experiments confirmed that this was due to a selective block of a Ca"-activated K + current, which was insensitive to tetraethylamnionium. However, apamin does not block K+ currents at frog or mouse motor nerve terminals (Anderson et al., 1988). Apamin has 18 amino acids in a single polypeptide chain that is cross-linked by two disulfide bridges. Although the three-dimensional conformation of apamin has not yet been deduced by X-ray crystallography, there is detailed structural information from N M R studies. Apamin may have a folding pattern that is a common motif for several different types of biologically active molecules (see Dotimas et al., 1987). Apamin can be labeled with 1251 with little loss of activity (Hugues et al., 1982a,b). High-affinity binding sites were identified in several tissues, including brain (see review by Lazdunski et al., 1987). b. Charybdotoxin. More recently, another toxin of different specificity was discovered (Miller et al., 1985). This is charybdotoxin from the

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213

venom of the Israeli scorpion, Leiurus yuinyurstriatus. Unlike apaniin, charybdotoxin primarily blocks large conductance forms of Ca'+-activated K+ channels in neurons and in other types of cells (see reviews by Moczydlowski et al., 1988; Strong, 1990). It is a larger polypeptide containing 37 amino acid residues and three disulfide bonds (GimenezGallego et al., 1988). Charybdotoxin and apamin have been used as pharmacological tools in experiments designed to elucidate the physiological functions of the different subtypes of Ca'+-activated K + channels. For example. the Ca2+-activatedK+ current at mouse and frog motor nerve terminals was shown to be sensitive to charybdotoxin, but not to apamin (Anderson rt al., 1988). However, this current does not appear to be activated untieinormal physiological conditions, because charybdotoxin did not alter the normal extracellularly recorded action potential or affect transmitter release. At crayfish neuroniuscular ,junctions, however, charybdotoxin did block a Ca'+-activated K + current and could affect transmitter release (Sivaramakrishnan et al., 1988). Charybdotoxin was also used as part of a characterization of a faniily of Ca*+-activated channels from rat brain membranes (Reinhart rt c i l . , l989), and charybdotoxin was used to characterize a transient Ca'+-dependent afterhyperpolarization associated with epileptiform activity in hippocampal slices (Algei- and Williamson, 1988). Some caution is needed in using charybdotoxin as a marker for Cay+-activated K.+ channels. T h e toxin has been shown t o affect some voltage-activated K f channels (Schweitz et ul., 1989) and to compete with dendrotoxin for binding sites on neuronal membranes (Harvey et al., 1989; Schweitz ut al., 1989)and for effects on synaptosomes (Schneider et al., 1989; Tibbs et nl., 1989). Other toxins are being discovered, for example, a toxin in (;onu.\ striatus venom (Chesnut et a / . , 1987) and leiurotoxin, from the same venom as charybdotoxin (Chicchi rt al., 1988). Their effects on neural transmission are not known.

C.

C A 2 + CHANNEL rrOXINS

Calcium channels in neurons are priniarily located in cell bodies and at nerve terminals. An influx of' Ca" in response to the depolarization associated with action potentials triggers the release of neurotransmitters from nerve terminals. As this is such a vital physiological function, it might be expected that a great number of toxins would have evolved t o be targeted at Ca2+ channels. This may be true, but very few of such toxins have been discovered.

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A I A N L HARVEY

1. w-Conotoxin

The best-known Cay channcl blocking toxin is w-conotoxin (;VIA (w-conotoxin), which has been isolated from the venotii of the marine snail Conus geogmphus. As thew are several hundred different ('()111IJ species and all of them are venomous, there will probably be many honiologs of w-conotoxin. So far, only a few variants are kiiown (see review by Gray et d., 1988). w-Conotoxins are polypeptides with 25-29 residues (Olivera et ul., 1984; Gray et ul., 1088) and three disulfide bonds (Cl-(:l6, C8C20, C 1 5 4 2 7 ) (Nishiuchi fit nl., 1986). Nothing is known about theirthree-dimensional conformation, or about structure-function relatioilships. w-Conotoxin is highly selective for neuronal Cay+ channels, Iiaving no effect on Ca2+ channels in muscles (McClesky et d . , 1987). .41so, it is selective for particular subclasses of neuronal (;ay+ channels (see below). However, the present classification of'these channels is far froni perltct, and it may also be being confused tiy the finding that w-conotoxiri shows very pronounced species dil'terences. w-Conotoxin GVIA blocked neui-omuscular transmission in frog cutaneous pectoris preparations by reducing quanta1 content (Kerr and Yoshikami, 1984). Synthetic w-conotoxin (5-40 n M ) also produces prcjunctional blockade at frog iieiiromuscular junctions (Enomoto PI a / . , 1986; Koyano et nl., 1987; Sano et ccl., 1987), i n bulllrog sympathetic ganglia, and in the electric organ of the ray N u k e jqboniccr ( K o y a i i o ~1 o/., 1987). T h e toxin also Iilocks depolarization-inducetl Ca'+ uptake antl transmitter release from 7'orprtfo synaptosonies (Ahmad and hliljanicli, 1988). These effects appear to be irreversible on washing. l'here is also evidence that w-conotoxin ldoc-kstransmission froni mossy fibers t o ( ; A 3 pyramidal cells in guinea pig hippocanipal slices (Kamiya P t ul., l!)XX), inhibits the evoked release ot. vasopressin fl-om rat neuro1i)p"pliysial nerve terminals (Dayanithi PI d . ,1W8), and causes a presvnaptic block of transmission in rat and guinea pig vas defcreris preparations and in guinea pig bladder preparations (Maggi Pt ( I / . , 1988). However, not all synapses are sensitive to w-conotoxin. Ti-aiismission at neuromuscular junctions in mice is unaffected by w-conotositi G V I A (Olivera tit nl., 1984; Nishiuchi P / nl., 1986; Andersori and Harvey, 1987; Sano et d . , 1987), and even the prolonged (;a2 currents induced tiv t h e presence of K + channel blockers are not reduced hy w-coiiotoxiti (Anderson and Harvey, 1087). w4:onotoxin is also largely inef'fective at blocking transmission in pi-epai-ations of rat bladder arid tluodeiium (Maggi rt al., 1988) and in rat parasympathetic ganglia (Seabrook antl Adams, 1989). 1

PRk.SYNAPI I(: I'OXINS

215

Less work has been performed with o-conotoxin MVIIA from (,'onns magus. However, it does appcar to show soine significant differences from o-conotoxin GVIA, both in structure and activity (for det-d l'I:,9 see reviews by Gray et al., 1988; Yoshikami et al., 1989). Briefly, toxin MVIIA acts reversibly at frog synapses and is partly reversible on N and L, Ca2' currents of chick dorsal root ganglion cells.

2. Snake Toxins Aflecting Ca'

'

(;urw?i1s

A high molecular weight protein was partially purified from the venom of the rattlesnake C r o L / i h citrox (Hamilton rt a/., 1 C185). 'l'he fraction, named atrotoxin, was Ii~undto block binding of' the (;a" channel blocker ["Hlnitrendipine to iiieiiibranes from guinea pig ventricular muscle and to enhance Ca2+currents in cardiac cells. Atr-otoxin does not appear to have been puritietl to liomogeneity, and it is not k n o w n whether it also can affect neui-onal Cay+currents. Another snake venom toxin reported to affect (;a2 ' channels in cardiac and smooth muscle cells is taicatoxin, a basic protein of 8000 l)a isolated from taipan venom ( O . q u r c i r i / c s .scutella/us) (Brown r / c i l . , 1987). This toxin caused a rapid a i i d reversible block of' (:a2+ currents in cultured o r isolated myocytes without affecting Na+ o r K + currents. Again, the effects of taicatoxin on neuronal (:a2+ currents have not beer1 published.

Venoms of the American spiders Agdmopis ripertn, Nololenti cii?-tfi,aiicl Plectreurys trzites have been reported to produce a n irrevei-sitk 111.esynaptic block of neuromuscular transmission in Drosophilti (Branton rt al., 1987). Several toxins have Ijeen isolated, and although thcy dil'l'ei- i n physical characteristics and species selectivity, they all appear t o block neuronal Ca2+ channels (Jackson a n d Parks, 1989). A toxin from Hololenu curfa was described by Bowers rt (11. (1987). It appears to be a two-chain protein (7000 and 9000 Daltons) c.ross-linket1 by a &sulfide bridge. It acts on I h . w p h i l a , but not on frog iieur-ornuscular junctions, to cause a n irreversihle block of transmitter release. From the (A" evidence available, this appears to be at the level of nei11-0n;11 channels. PlectreuTs tvistes venom contains protein toxins that stimulate and block neuromuscular transmission in Drosophila (Branton r / d . , 1987). T h e excitatory effects are prevented by tetrodotoxiii and relate t o an action on Na+ channels. T h e inhibitory effects appear to be a result of block of Ca" channels at nerve terminals. T h r e e so-called cu-Plpc/reury.\ toxins ( a - P L r X ) were isolated. 'I'hey behave as single-chain proteins of

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ALAN L. HARVEY

molecular weight around 7000 Da. a-PLTX I1 reduced quanta1 content at nanmolar concentrations and had no effect on Na+-dependentaction potentials in nerves, Ca+-dependentaction potential in muscles, or on miniature endplate potential amplitude. At 10 nM, it was ineffective at frog neuromuscular junctions. Venom of the American funnelweb spider Agelenopsis aperta also contains toxins that block neuronal <;a'+ channels (Jackson and Parks, 1989). Seven presynaptic blockers were found by Bindokas and Adams (1989). T h e most abundant was a polypeptide termed w-agatoxin I, which had a molecular weight of about 7500. Nanomolar concentrations of this toxin resulted in a presynaptic block of neuromuscular transmission in prepupal house fly preparations and in frog cutaneous pectoris nervemuscle preparations. T h e toxin was also demonstrated to block Ca2+currents in cell bodies of insect neurons. A lower molecular weight fraction (200-400 Da) isolated from venom of Agelenopis aperta was demonstrated to block CaZf channels in hippocampal Purkinje cells and presynaptic nerve terminals in the squid giant synapse (Llinas et al., 1989). T h e toxin (FTX) is apparently a peptide because of its absorbance at 280 nm, but it is otherwise uncharacterized. It is lethal to mice on intraperitoneal injection, probably by a central action that causes respiratory depression. The toxin was used to make an affinity column for the isolation of putative Ca" channels. Proteins isolated from Purkinje cells and from squid neurons were reconstituted in bilipid membranes and showed properties of voltage-dependent Ca2+ channels. As the Ca'+ currents blocked by FTX are insensitive to w-conotoxin and to the dihydropyridine class ofCa'+ channel blockers, it was proposed that they reflected the activity of a different type of Ca2+ channel from the ones previously characterized. In contrast to these findings, it was reported that binding of '"1-labeled w-conotoxin GVIA is inhibited by low concentrations of Plectreurys tristes venom, indicating some overlap in the specificities of spider and cone snail toxins (Feigenbaum et al., 1988).

111. Toxins Affecting Release Mechanisms

A. BOTULINUM AND T E T A N U S

TOXINS

A number of extremely toxic proteins are produced by various strains of the anaerobic bacteria Clostridium botulinum and Clostridium tetani (for reviews, see Gundersen, 1980; Mellanby and Green, 1981; Simpson,

PKESYNAPTIC: I OXINS

217

1981, 1986; Habermann arid Oreyer, 1986; Sellin, 1987). ‘The most commonly studied of the botulinum toxins is type A, which is composed of two subunits cross-linked by a disulfide bond, and which has a molecular weight of about 140,000 Da. Botulinum toxin is a very potent and very specific blocker of acetylcholine release at the neuromuscular junction. T h e major effect of clinical botulism is muscle weakness resulting from the paralysis of neuromuscular transmission. The toxin binds to highpffinity recognition sites on the outside of cholinergic nerve terminals. I t is then translocated to the inside of the nerve ending, where it produces its blocking action by an unknown mechanism. From its extremely high potency it can be assumed that botulinum toxin acts like an enzyme, but no enzymatic activity has been conclusively established. Tetanus toxin also blocks acetylcholine release at the neuronluscular junction, but it is about 500 times less potent at that site than is botulinurn toxin. Tetanus toxin is transported into the spinal cord, where it has a preferential action on the inhibitory synapses that have glycine and GABA as transmitters. The block of inhibitory pathways leads t o the muscle spasms that are characteristic of tetanus poisoning. 1. Botulinum Toxin

Toxins from different strains of Clostridium botulinum are found to be of eight immunologically dif‘ferent types: toxins A , €3, C , , C2, D, E, F, and G. Toxin Cy does not appear to be a neurotoxin. When originally isolated, the toxins can exist as a complex with both neurotoxic and hemagglutinating properties. Pure neurotoxins can be prepared and they appear to have similar molecular weights. More recently, strains of Clostridium butyricum and Clostridizim bal-ati have been demonstrated to produce type E- and type F-like toxins, respectively. All botulinum toxins seem to be synthesized as a single chain. Posttranslational modification results in the formation of a heavy chain (about 100,000 Ua) and a light chain (about 50,000 Da), which are cross-linked by a disulfide bond. .I’he gene coding for botulinum toxin A has been sequenced (Niemann ut al., 1990). T h e botulinum neurotoxins paralyze skeletal muscle by irreversibly blocking acetylcholine release. Both spontaneous and evoked release is affected. Despite intensive study for about 50 years, the molecular mechanisms of action of these toxins remain to be elucidated. Botulinum toxins d o not affect the propagation of action potentials in motor nerves (see Simpson, 1981) or the calcium currents at nerve terminals (Gundersen et al., 1982; Dreyer ut al., 1983). T h e toxins do not affect the synthesis or storage of acetylcholine (see Gundersen, 1980). I t is apparent that the toxins, therefore, affect the release process itself. They

218

ALAN L. HARVEY

d o not completely block the ability of the nerve terminal to release acetylcholine: It is possible to restore release, at least partially, by procedures that increase the intraterminal levels of Ca2+.For this reason, the toxins have been assumed to depress the sensitivity of some component of the release mechanism to Cay+. The block of acetylcholine release by botulinum toxin occurs after a delay. Numerous studies have demonstrated that the toxin has to bind to the outside of the nerve terminal membrane, then it is internalized, and finally blockade occurs. Although different forms of botulinum toxin have markedly different potencies in various species and sometimes produce different effects on release profiles, the binding and translocation steps appear to be common (Simpson, 1979; also, for reviews, see Simpson, 1981; Sellin, 1987). It is possible to label botulinum toxin type A with 1251to high specific activity while retaining much of its neurotoxicity (Dolly et al., 1984). Autoradiography techniques could then be used to demonstrate the presence of binding sites on nerve terminals. With mouse motor nerves, there appeared to be about 150 binding sites/pm2 of presynaptic membrane, and these were distributed over the entire nonmyelinated presynaptic region, with no evidence for accumulation over specific features, such as release sites. T h e same technique provided direct evidence for the internalization of a botulinum toxin within cholinergic nerve terminals. More recent studies have used separated heavy and light chains in order to determine which portions of the toxin control the three different proceses of binding, translocation, and blockade. Intact toxin seems to be necessary to cause maximum paralysis of mouse neuromuscular preparations, but binding can be prevented by preincubation with separated heavy chain (Dolly et al., 1984). It is difficult to do more complicated experiments at the neuromuscular junction with toxin fragments because of the inaccessibility of the nerve terminal. More convenient model systems have been used as alternatives. These include adrenal chromaffin cells or their tissue culture equivalent, PC12 cells, which have been permeabilized by treatment with detergent or pore-forming toxin, and a ganglionic preparation of Aplysia, in which it is possible to inject toxin components directly into identified neurons and study their effect on synaptic transmission (Poulain et al., 1989). With permeabilized PC 12 cells, light chain was as effective as native toxin at reducing Ca2+-induced transmitter release (Dolly et al., 1990). However, at the Aplysia synapse, light chain and a fragment of the heavy chain were required intracellulady to block release (Poulain et al., 1989, 1990). Although further work is necesary, it appears that the heavy chain is responsible for the binding and internalization of the toxin. Perhaps it is relevant that the N-terminal

portion of the heavy chain of botulinum type A toxin can form channels in lipid bilayers (Blaustein P t ul., 1987). Subsequently, mechanisms may differ, depending on species or type of cell. The specificity in 1 ~ of7 botulinum toxin for cholinergic synapses is presumably because other types of neurons do not have high-affinity recognition sites f’or the toxin. However, even in cholinergic neurons, the nature and physiological significance of these sites are unknown. Different types of botulinuni toxin produce slightly different effects on the properties of acetylcholine release at neuromuscular ,junctions (Sellin et al., 1983; Sellin, 1987; Molgo ut al., 1990). Following blockade o f transmission, raising the levels of intraterminal Ca2+ ions can I-atore synchronous quanta1 release in preparations exposed to types A and E. In preparations blocked by type B or 11, there is no recovery of phasic release, but there is an increase in asynchronous release of quanta. I t has been demonstrated that agents that disrupt microtubules have a delaying effect on the action of type 13 but not type A botulinum toxin (Dolly rt al., 1990). It has been proposed that different molecular mechanisms exist for the different types of botulinum toxin (Sellin, 1987). T h e site of the blockade remains elusive. Although a generalized depression of release mechanisms to (:a‘+ has been proposed, evidence fi-om the lack of effect of botulinum toxin type A on the magnitude of Ca2+-activated K + currents would indicate that the toxin does not affect the normal Ca2+buffering mechanisms (Mallart ~t ul., 1989). rl’here was a revival of interest in the possibility that botulinuni toxins acted intracellulady as enzymes following the report that types C: and D toxin caused ADP-ribosylation of specific proteins in PC I2 cells arid cultured neurons (Matsuoka et al., 1987). However, this effect is thought to be unrelated to block of release (Adam-Vizi et d . , 1 M X ) , and botulinuni toxins type A and B have never been demonstrated to have enzymatic activity. Similarly, the significance of the finding that botulinum toxin type A blocks some depolarization-induced phosphorylation of proteins in synaptosornes from Torpedo electroplax remains to be established (Guitart P t d ,1987).

2. Tetanus Toxin Tetanus toxin is similar to the botulinum toxins in being a protein of about 150,000 Da. It is also produced as a single chain, which is subsequently modified to heavy and light subunits cross-lin ked by a disulfide bond. T h e primary sequence of tetanus toxin has been deduced (Eke1 ut al., 1986), and it shows considerable similarity to the botulinuni toxins. Tetanus toxin is extremely potent in 7~2710(minimum lethal dose in mice is about 2 ng/kg). It acts in the central nervous system t o block inhibitory synapses and hence causes its characteristic spastic paralysis

~

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(see Habermann and Dreyer, 1986).It also acts peripherally at the neuromuscular junction, although it is about 500 times less potent. Tetanus toxin has similarities to botulinum toxin: it blocks following binding and internalization (Schmitt et al., 198l), it affects spontaneous and evoked release, and it does not affect nerve terminal Ca2+currents (Dreyer et al., 1983). However, tetanus toxin appears to have greater similarity to type B botulinum toxin than to type A because tetanus toxin produces a marked desynchronization of transmitter release (Dreyer and Schmitt, 1981). Tetanus toxin has been demonstrated to have effects on neurons in culture (Bergey et al., 1983, 1987; Wellhoner and Neville, 1987). More recently, studies on the effects of different portions of tetanus toxin have been carried out using both cholinergic and noncholinergic synapses in Aplysia ganglia (Poulain et al., 1990). Again, several similarities with botulinum toxin were noted.

GLYCEROTOXIN, B. a-LATROTOXIN,

AND

LEPTINOTOXIN

The venom of the black widow spider Latrodectus mactans contains several neurotoxins. Some are active only in insects, while others act on vertebrates. The best characterized ofthe latter is a-latrotoxin, which is a protein of about 130,000 Da (for reviews, see Madeddu and Meldolesi, 1987; Rosenthal and Meldolesi, 1989). a-Latrotoxin produces neuromuscular paralysis after an initial excitatory phase. It acts on cholinergic nerve terminals to promote a massive asynchronous release of acetylcholine. Most of its effects can be attributed to its action as an ionophore; that is, it creates channels through the membrane, causing depolarization and a sustained influx of Ca'+. However, some of the effects of a-latrotoxin are not dependent on extracellular Cay+;an action on internal metabolism is possible (see below). Two other toxins with many similarities to a-latrotoxin have been discovered. These are glycerotoxin, from the marine annelid worm Glycera convulata, and leptinotoxin, from the beetle Leptinotarsa haldemani. Both are high molecular weight proteins and both cause a sustained uncontrolled release of acetylcholine. However, a-latrotoxin, glycerotoxin, and leptinotoxin are not identical in their actions; there are very marked differences in their potencies in different species, implying that they have different binding sites. 1. a-Latrotoxin When applied to frog or mouse neuromuscular preparations, whole venom of black widow spiders or purified a-latrotoxin causes a gradual

PKESVNAPI 1C TOXINS

22 1

increase in the frequency of miniature endplate potentials, until levels of several hundred Hertz are reached. Transmitter release subsides and evoked release is blocked. At this stage, electron micrographs reveal depletion of synaptic vesicles and presynaptic swelling (see Rosenthal and Meldolesi, 1989). Although a-latrotoxin depletes nerve terminals of the small acetylcholine-containing synaptic vesicles, it does not affect the numbers of dense core vesicles, which contain calcitonin gene-related peptide (Matteoli et al., 1988). It is interesting that occurence of large, slow, spontaneous endplate potentials, presumably related to the release from dense-cored vesicles, increases after poisoning with botulinum toxin (Tabti et al., 1986). T h e effects of a-latrotoxin appear to be confined to the nerve terminal region, with little or no effect on axonal action potentials (Mallart and Haimann, 1985). Direct evidence for selective binding of a-latrotoxin to nerve terminals has been presented (Valtorta et al., 1984).Toxin binding has been demonstrated on PC12 cells (Meldolesi et al., 1983). and the toxin is also active on a human neuroblastoma cell line (Sher et al., 1988). &-Latrotoxin can induce the formation of large nonselective cationic channels in lipid membranes (Finkelstein et al., 1976).Presumably, nerve terminals have specific, high-affinity binding sites that attract the toxin, and this may be followed by channel formation. The subsequent depolarization and influx of Ca2' would explain most of the effects of the toxin. Patch clamp techniques have been used to demonstrate that a-latrotoxin induces the opening of small (conductance about 15 pS), cationic chan1986). nels in PC12 cells (Wanke et d., However, a-latrotoxin can still induce release of neurotransniitters in the absence of extracellular Ca" . Therefore, an additional niechanism must exist. Binding of a-latrotoxin to PC12 cells leads to an increased metabolism of polyinositol phosphates and accumulation of inositol 1,4,5-triphosphate (Vicentini and Meldolesi, 1984). It is possible that mobilization of such second messengers may cause release of Cay+ from intraterminal stores, but this has not yet been demonstrated.

2. Glycerotoxin T h e marine worm Glycpra convoluta has venom that produccs an a-latrotoxin-like incease in the frequency of miniature end plate potentials. A partially purified protein of niolecular weight around 300,000 Da has been shown to be responsible for the effect (Thieffry et nl., 1984). Glycerotoxin also stimulates acetylcholine release from Torpedo synaptosomes but has no effect on PC12 cells (Madeddu et al., 1984).By contrast, a-latrotoxin acts on PC12 cells but is inactive on Torpedo synaptosomes. Both toxins are effective on synaptosomes from rat brain, but binding of

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a-latrotoxin was unaffected by pretreatment of synaptosomes with glycertoxin (Madeddu et al., 1984). Glycerotoxin does not appear to have been purified to homogeneity, and detailed studies of its action on a range of secretory cells are lacking.

3. Leptinotoxin Leptinotoxin (or P-leptinotarsin) is a protein of about 57,000 Da isolated from the hemolymph of the beetle Leptinotarsa haldemani (Crosland et al., 1984). It triggers the spontaneous release of acetylcholine at the neuroniuscular .junction (McClure et al., 1980) and of a variety of transmitters from brain synaptosomes (Crosland et al., 1984; Madeddu et al., 1985a,b). Like a-latrotoxin, leptinotoxin causes depolarization of synaptosomes and PC12 cells (Crosland et al., 1984; Madeddu et al., 1985a). However, leptinotoxin appears to lack the channel-forming activity on bilipid membranes that is characteristic of a-latrotoxin and glycerotoxin. T h e mechanism of action of leptinotoxin and the role of extracellular Cap+ ions remain to be elucidated.

c. @ B U N G A R O T O X I N

AND RELATED PHOSPHOLIPASE A2

TOXINS

Several snake venoms contain toxic phospholipase A2 molecules. The toxins cause muscle paralysis by blocking the evoked release of acetylcholine. In addition, some of the toxins are directly myotoxic (for reviews, see Chang, 1985; Harris, 1990). Examples are P-bungarotoxin from the Taiwan banded krait, Bungarus multicinctus, crotoxin from the South American rattlesnake, Crotalus durissus ternficus, notexin from the Australian tiger snake, Notechis scutatus, and taipoxin from the Australian taipan, Oxyuranus scutellatus scutellatus. Many other toxic phospholipases have been isolated and their amino acid sequences determined. T h e majority, including notexin, are single-chain proteins, but there are several types of complex toxins. P-Bungarotoxin consists of an A and a B chain, which are cross-linked by one disulfide bond. The A chain is homologous to phospholipases A:!, whereas the B chain has homology with Kunitz-type protease inhibitors and with dendrotoxins. Crotoxin is also a complex of two chains, although they are not covalently linked. One chain (CB) is a basic phospholipase AS, and the other (CA) is an inactive phospholipase homolog. laipoxin is a noncovalently bound complex of three similar subunits. One is a basic phospholipase Az, another is an acidic phospholipase AP, and the third is an inactive phospholipase homolog. T h e nontoxic chain of crotoxin has been demon-

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strated to reduce the nonspecific binding of the active subunit. I t is assumed that the other nontoxic portions of the other complex toxins also act in some way to “chaperone” the toxins to their binding sites on cholinergic nerve terminals. Crotoxin was isolated in crystal form in 1938 and the actions of purified P-bungarotoxin were reported in the mid- 1960s. l h e toxins have been intensively studied for the last 20 years, but their molecular mechanism of action remains elusive. They produce characteristic changes in acetylcholine release before they abolish it. Initially, there may be a transient fall in the amount of transmitter release in response to an action potential, this is followed by period in which release is augmented, and finally, there is a slow, progressive decrease in release. A t least at the time of complete paralysis, the nerve terminals are not morphologically damaged and action potentials arid nerve terminal Cay+ currcnts are unimpaired. Further details of’their effects on acetylcholine release and a discussion of possible mechanisms of action are given below. 1. Presynaptic Effects of Phospholipnsr A2 l o x i m

T h e resolution of how these toxins act is complicated because the toxins have different effects on evoked and spontaneous release of acetylcholine, and because they produce different preblock changes and have different orders of potency in different species. To a certain extent, published literature on phospholipase neurotoxins can be rationalized by considering only the same small number of “standard” toxins, by examining effects in frogs and mammals separately, and by looking for separate explanations of the early facilitation and the later blockade. a. Effects on Evoked Transmitter Rrleusr. Following exposure of nervemuscle preparations to presynaptically active phospholipase neurotoxins, the amplitude of endplate potenlials generally undergo a sequence of triphasic changes: a transient initial reduction, followed by an increase above control amplitude, and finally a progressive decline, leading to complete failure. Different mechanisms probably underlie the effects in amphibians and in mammals. In frog sartorius nerve-muscle preparations, P-bungarotoxin produces a typical triphasic effect (Abe PI d , 1976). Lowering the temperature or substituting Sr” for C a y + does not affect the initial depression but markedly attenuates the subsequent effects (Abe and Miledi. 1978; Caratsch et al., 198 1, 1985).Both these conditions reduce the phospholipase activity of P-bungarotoxin. Following inactivation of its enzyme activity by reaction with p-brornophenacyl bromide, P-bungarotoxin only causes the initial depression of endplate potential amplitude (Abe rt al., 1977). Similar effects have been found with notexin (Magazanik and

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Slavnova, 1978; Chang and Su, 1982) and with crotoxin (Hawgood and Santana de Sa, 1979; Hawgood et al., 1987).Therefore, the initial depression of transmitter release in frog preparations appears to be independent of the toxins' phospholipase activity, but the subsequent facilitatory and blocking phases are associated with the enzyme activity. In mammalian nerve-muscle preparations, most of the phospholipase neurotoxins cause triphasic effects on the amplitude of endplate potentials. However, the early depression and subsequent facilitation appear to be independent of phospholipase activity, and only the final blocking phase seems to require enzyme activity. These effects have been seen with P-bungarotoxin in rat preparations (Kelly et al., 1975; Landon et al., 1980),and with P-bungarotoxin, crotoxin, notexin, and taipoxin in mouse preparations (Chang and Su, 1978, 1982; Chang et al., 1977b; Su and Chang, 1981, 1984). b. Effects on Spontaneow Release of Acetylcholine. These, as determined by changes in the frequency of miniature endplate potentials, are broadly similar to those on evoked release. However, changes in miniature endplate potential frequency often reflect depolarization, and this may not be the mechanism underlying the changes in evoked responses. In particular, evoked transmitter release can be blocked by the toxin, while spontaneous release is apparently normal or even enhanced.

2. Possible Mechanisms of Action a. Facilitatovy Effect. Several mechanisms have been postulated for the early facilitatory effects of the phospholipase neurotoxins. The facilitation could be due to a reduction of Ca2+ uptake within motor nerve terminals following uncoupling of oxidative phosphorylation in mitochondria (Wagner et al., 1974), to an increase in the permeability o f t h e nerve terminal membrane to ions and an increase in the release of neurotransmitters (Masukawa and Livengood, 1982; Halliwell et al., 1982; Rugolo et al., 1986),to a depolarization of the terminals (Dowdall et al., 1977; Halliwell et al., 1982; Rugolo et al., 1986),to increased probability of fusion of synaptic vesicles (Strong et al., 1976),to enhanced sensitivity of the release mechanism to Ca2+,or to an increase in Ca'+ entry during action potentials (Su and Chang, 1984). Mitochondria can accumulate Ca'+, and therefore, any disruption of the ability of mitochondria to remove Ca'+ from the cytoplasm could result in an increase in the activity of Ca'+-dependent systems in the cell (e.g., transmitter release). P-Bungarotoxin has been shown by Wagner et al. (1974) to be a powerful inhibitor of Ca'+ accumulation into rat brain mitochondria (50% inhibition occurred with 2.1 pM of toxin). However,

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225

whether the toxin affects Cay+uptake directly or via the supply of energy needed to accumulate Ca2+ against a concentration gradient was unknown. Wernicke et al. (1975), Howard (1975), and Nicholls et al. (1985) demonstrated that P-bungarotoxin poisoning results in mitochondria1 uncoupling. Therefore, the ability of P-bungarotoxin to block the accumulation of Ca2+ into mitochondria may be due to a decrease in the activity of an energy-dependent Ca" pump because of the lack of ATP to drive it. Mitochondria] uncoupling will also cause the breakdown of energydependent concentration gradients, which in the case of the mitochondria will result in Ca2+ moving down its concentration gradient and into the cell cytoplasm. T h e net result of these processes is an increase in the cytoplasmic Ca2+ level, which could account for the increase in spontaneous transmitter release. Although P-bungarotoxin is able to uncouple isolated brain mitochondria (Howard, 1975), it is unlikely that P-bungarotoxin is internalized to exhibit its effects. Howard and Wu ( 1976) have shown that P-bungarotoxin covalently bound to agarose, which would prevent the internalization of the toxin, was still able to exhibit the same effects as the unbound toxin. Evidence from Wernicke et al. ( 1 975) and Nicholls et al. (1 985) suggests that the uncoupling is due to fatty acids that have been liberated by hydrolysis of the plasma membrane by the phospholipase A2 activity of the toxin. Therefore, it has been suggested that P-bungarotoxin acts at the plasma membrane in intact synaptosomes (and presumably at the nerve terminals) and that the fatty acids liberated by phospholipase A2 activity enter the cytoplasm to uncouple the mitochondria. However, such effects on mitochondria cannot fully explain the activities of the phospholipase neurotoxins on mammalian motor nerve terminals because the facilitatory phase is phospholipase-independent, whereas the changes in mitochondria1 respiration rely on the catalytic activity of the toxin. Addition of P-bungarotoxin has been reported to cause a general increase in the permeability of the plasma membrane towards ions and neurotransmitters (Masukawa and Livengood, 1982; Halliwell et nl., 1982; Rugolo et al., 1986).This results in a phospholipase An-dependent increase in the release of acetylcholine, glutamate, and y-aminobutyrate from central neurons. However, this is not consistent with the effects of the phospholipase neurotoxins at the neuromuscular junction. N g and Howard (1978), Halliwell et al. (1982), Smith et nl. (1980), and Nicholls et al. (1985) reported that P-bungarotoxin causes a depolarization of the plasma membrane. This depolarization is not thought to be due to the movement of Na+ across the membrane through specific

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channels as it is insensitive to tetrodotoxin; it has been suggested that it may be due to a rundown of the energy-dependent ionic pumps due to mitochondria1 uncoupling. However, a general depolarization of the terminals is not likely to produce the phasic nature of the changes of transmitter release induced by the neurotoxins and would not be associated with the increases in quanta1 content that are found at the neuromuscular junction. Further information on the action of phospholipase Ap neurotoxins has come from work using liposomal membranes. Strong and Kelly (1977) suggested that j3-bungarotoxin preferentially hydrolyzes membrane phosphatidylcholine bilayers undergoing phase transitions in liposomes. Phase transitions are thought to occur at the interface between an integral membrane protein and surrounding phospholipids (e.g., at the active zones of the nerve terminals). It is also suggested (Strong et al., 1976) that once the toxin is bound to the plasma membrane the enzyme activity of the toxin raises the level of fatty acids and lysophospholipids in the membrane, which in turn alters the probability of fusion with the membrane to release transmitter. The presence of cholesterol in the phosphatidylcholine liposomes markedly reduces the rate of hydrolysis. Therefore, membranes with a high content of cholesterol are relatively resistant to the hydrolytic activity of the toxin (e.g., Schwann cell membranes). Hence, the specificity of action of the toxins may result from the unique lipid content of the membrane. However, this suggested activity for the phospholipase A2 on the probability of fusion of synaptic vesicle as a reason for the increase in transmitter release in mammalian preparations is unsatisfactory. This is because the increase in transmitter release is observed in conditions where phospholipase A2 activity is reduced (e.g., in the presence of Sr'+). However, the phospholipase-dependent action of the neurotoxins in frog preparations may be explained by such effects. It has also been suggested that the increase in transmitter release may be due to enhanced sensitivity of the release mechanism to Cap+,or to an increase in Ca2+ entry during action potentials (Su and Chang, 1984). During the facilitation of transmitter release by either P-bungarotoxin or taipoxin, the Ca2+-concentration response curve is not shifted to the left at normal Ca2+ concentrations. Thus, enhanced sensitivity to Ca2+ does not play a role in the facilitation of acetylcholine release. The ability of P-bungarotoxin and related toxins to block neuronal K+ channels and the consequence for transmitter release were discussed earlier (Section II,B,l,d). b. Mechanisms for the Blockade of Acetylcholine Release. The irreversible blockade of neurotransmitter release is commonly believed to be due to

I'RF.SYhAP I IC. T O X I N S

227

the hydrolytic activity of the phospholipase toxins, since the blocking activity is greatly attenuated by the cationic phospholipase A 2 inhibitors and by chemical modification of the enzyme active site. Several mechanisms have been proposed. These have been based on indirect electrophysiological measurements of nerve terminal activity and on biochemical studies of nerve terminal membranes. They include a depletion of the energy stores resulting from the uncoupling of mitochondrial oxidative phosphorylation (Wernicke et al., 1975; Howard, 1975; Nicholls ut ul., 1985); an inhibition of high-affinity choline uptake (Sen et al., 1976; Dowdall et al., 1977); an excessive accumulation of Cay+(Abe et al., 1976); and a decrease in Ca2+ influx or a decrease in the efficacy of (;a2+ in promoting release (Su and Chang, 1984). Effects on mitochondria were suspected from electron micrographs showing evidence of morphological darnage to the mitochondria af'ter poisoning with phospholipase neurotoxins ([:hen and Lee, 1970: Lullman-Rauch and Thesleff, 197Y), despite the fact that workers had yet to demonstrate the presence of toxin molecules inside the nerve terminal. Howard and Wu (1976) attached j3-bungarotoxin covalently to large agarose beads to prevent the possibility of the toxin molecule entering the synaptosomal preparation. I n this state, P-bungarotoxin still exhibited pharmacological activity. Howard ( 1975) proposed that the mitochondria are uncoupled by a diffusible factor (which is thought to be a free fatty acid) produced by the hydrolytic activity of the toxin on the nerve terminal membrane. Therefore, the toxin would not have to cross the plasma membrane of the nerve terminal to have an effect. L). C. Anderson and Parsons ( 1986) also suggested that j3-bungarotoxin damages the vesicle membrane in a phospholipase-dependent manner, which results in the uncoupling of A'TPase and acetylcholine transport systems. However, known mitochondrial uncouplers cause changes in transmitter release that are different from the phospholipase neurotoxins (Glagoleva et al., 1970), which suggests that sonie other mechanism may be involved in the blockade of transmitter release. 6-Bungarotoxin and other phospholipase neurotoxins also block the high-affinity choline transport system in Torpedo electroplaques (Dowdall et al., 1977) and brain synaptosomes (Sen et nl., 1976; Fletcher arid Middlebrook, 1986), and this blockade may result in the depletion of acetylcholine in the vesicles. The blockade of choline uptake can be abolished by conditions that reduce phospholipase activity, which is consistent with the phospholipases blocking transmitter release by their hydrolytic activity. However, the blockade of the high-affinity choline transport system with hemicholiriium does not result in an immediate

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reduction in the miniature endplate potential frequency (Elmqvist and Quastel, 1965), as observed with the phospholipase neurotoxins. P-Bungarotoxin has been shown to stimulate the synthesis and accumulation of acetylcholine in nerve-muscle preparations, whereas notexin has the opposite effect on transmitter storage (Gundersen et al., 1980, 1981; Newton et al., 1983).Thus, the actions of the phospholipase neurotoxins at the neuromuscularjunction are not consistent with an effect on choline uptake. T h e fact that high K+ or hypertonic sucrose still increase transmitter release immediately after the toxin-induced blockade of transmitter release is also in agreement with this (Chang and Lee, 1977; Chang et al., 1973, 1977a). It has been suggested that the blockade of transmitter release is due to an excessive accumulation of Ca", or a decrease in Ca2+ entry, or a decrease in the efficacy of Ca2+in promoting release (Abe et al., 1976; Su and Chang, 1984). During the blocking phase, when quantal content is reduced, quantal content can be partially restored with diaminopyridine (Su and Chang, 1984). This suggests that the blockade of transmitter release is not due to blockade of Ca2+influx or to the excessive accumulation of Ca2+within the nerve terminal and that some other mechanism is responsible for the effects. Su and Chang (1984) suggest that the hydrolysis of the axolemma disturbs the integrity of the active sites, which results in irreversible blockade of acetylcholine release. More recently, extracellular recordings from mouse motor nerve terminals have revealed that the phospholipase neurotoxins do not produce any obvious changes in the Ca'+ currents that can be recorded in the presence of K+ channel blockers (Rowan and Harvey, 1988). When acetylcholine release is abolished by P-bungarotoxin or crotoxin, the nerve terminal is still invaded normally by action potentials (Rowan et al., 1990). Possible effects of P-bungarotoxin on release sites were examined by testing the ability of K+-induced depolarization or the Ca2+ ionophore A23 187 to increase the frequency of miniature endplate potentials immediately after the blockade of evoked acetylcholine release. Contrary to results after very long exposures to P-bungarotoxin, the sensitivity of mouse motor nerve terminals to the two forms of stimulation was unaffected by the exposure to toxin (Rowan et al., 1990). This implies that P-bungarotoxin blocks some step in the excitation-secretion coupling process between the arrival of the action potential and the opening of Ca2+ channels. Such a step remains to be found, although it is possible that prolonged K+-induced depolarization opens Ca2+channels that are different from those opened by the transient depolarization caused by action potentials.

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229

IV. Miscellaneous Toxins

A. MAITOTOXIN Maitotoxin is a toxic by product of the dinoflagellate Gumbierdiscus toxicus, which can colonize reef fish in the Pacific Ocean. Its structure is not definitely known, but it is not a protein. Maitotoxin is extremely potent and may be involved in some cases of ciguatera food poisoning. Maitotoxin acts on many different types of cells, including neurons. Although it has been suggested that maitotoxin directly activates endogenous Ca2+channels, the evidence for this is circumstantial. Maitotoxin at concentrations as low as 0.5 ng/ml increased the release of noradrenaline and dopamine from PC12 cells (Takahashi et al., 1983). Release was blockable by a variety of Ca'+ channel antagonists but was only partially dependent on external Na+. Higher concentrations of maitotoxin stimulated release of GABA from cultures of mouse striatal neurones (Shalaby et al., 1986) and increased Ca2+uptake by rat synaptosomes (Ueda et al., 1986). However, these effects were not sensitive to organic Ca2+ channel antagonists. Maitotoxin enhances the formation of inositol phosphates in PC 12 cells (Gusovsky et al., 1988). Maximal stimulation occurs at concentrations lower than those that affect transmitter release, although the stimulation is reduced as the concentration is increased beyond 1 ngiml. Enhancement of phosphoinositide breakdown is dependent on extracellular Ca'+ but is not blocked by the organic blocker nifedipine. Maitotoxin was found to increase phosphoinositide metabolism in many cell types (Sladeczek et al., 1988). Moreover, in these studies, maitotoxin was shown to increase transmembrane fluxes of Na+ and K + , in addition to Ca2+, and it was postulated that maitotoxin could form pores. Maitotoxin has been tested on neuromuscular transmission using mouse hemidiaphragm preparations (Kim et al., 1985). T h e toxin produced a slowly developing increase in the frequency of miniature endplate potentials up to 500 Hz. T h e increase was maintained for 1030 min, and then there was a slow decrease in the frequency of miniature endplate potentials until release was abolished. T h e increase in spontaneous release was Ca2+-dependentand could be blocked by Coy+ but not by tetrodotoxin. From this, it might be concluded that maitotoxin induces the opening of the endogenous Ca2+channels, but it is perhaps significant that Cop+was shown to be able to block Ca'+ fluxes induced by the pore-forming agent digitonin (Sladeczek et al., 1988).

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When maitotoxin had increased the frequency of miniature endplate potentials by 50 times, the quantal content of the evoked endplate potential was only increased by a factor of three (Kim et al., 1985). Thus, maitotoxin probably causes a sustained increase in the levels of free intraterminal Ca2+, rather than an effect on Ca'+ currents associated with action potentials. Further work is necessary to establish whether maitotoxin acts via endogenous Ca2+ channels, by a pore-formingdepolarization mechanism, or via a second messenger system.

B. PARDAXINS The paradaxins are isolated from the secretions along the spines of certain species of sole found in the Pacific Ocean and the Red Sea. They are small proteins of about 33 residues and no disulfide bonds. T h e pardaxins were first isolated during attempts to localize the component of the fishes' secretions responsible for their shark-repellent qualities. The first biological effects of the pardaxins to be discovered were their hemolytic and cytotoxic properties, but they have also been shown to have effects at the neuromuscular junction that are similar to those of a-latrotoxin (Kenner et al., 1987). Pardaxin I and I1 from the Red Sea sole Pardachirus marmoratus caused an increase in the frequency of miniature endplate potentials at frog neuromuscular junctions and also increased endplate potential quantal content. T h e effects of low concentrations (0.5-1 kg/ml) were reversed by washing. At higher concentrations, the pardaxins directly depolarized muscle cells, causing contractures and eventual muscle damage (Renner et al., 1987). A pardaxin has been chemically synthesized and shown also to be presynaptically active at 10-8-10-7M (Shai et al., 1988). Like native pardaxin, the synthetic form was shown to be able to form channels in artificial lipid bilayers. Some of its pharmacological activity might be result from channel formation, but its apparently selective action at presynaptic terminals needs to be explained.

C. OTHERTOXINS 1. Tertiapin The venom of the honeybee Apis mellifera contains a number of minor toxins in addition to melittin and apamin. A 2 l-residue polypeptide named tertiapin has been sequenced and shown to have novel presynaptic activities (Ovchinnikov et al., 1980). When tested on a frog

PRESYNAP 1 1C 1 OXINS

23 1

neuromuscular preparation, tertiapin reduced the quanta1 content of the endplate potential and the frequency of miniature endplate potentials. T h e effect on the frequency of spontaneous release was even more pronounced in the absence of extracellular Cay+ions, so the toxin could not be acting simply to block Cay+channels. Further studies are needed to elucidate what might be a unique mechanism of action.

2. Funnel W e b Spider Toxins Australian funnel web spiders contain neurotoxins with a presynaptic activity. They increase the spontaneous release of-transmitters, although their mechanism is unknown. Two homologous proteins have been isolated and sequenced: These are robustoxin from the Sydney funnel web Atrax robustus (Sheumack et ul., 1985) and versutoxin from Atrax 7lersutus (Brown et al., 1988). Both have 42 amino acid residues and are unusual in having disulfide bonds at both C and N termini.

3. Australian Frog Toxins An extract of the skin of the Australian frog P.seudophvyne rorzucea has been shown to have facilitatory effects in a wide variety of smooth muscle preparations (Erspamer et ul., 1985). T h e increase in transmiter release caused by the extract was blocked by tetrodotoxin, but the mechanism of action is unknown. V. Conclusions

T h e last 10 years have seen the discovery of an increasingly diverse range of toxins that act presynaptically to increase or decrease neurotransmitter release. They come from a wide variety of sources and have several different mechanisms of action. However, apart from toxins acting on Na+ channels, their mechanisms of action are understood poorly or not at all. Hopefully, the next 10 years will see an increase in our knowledge of how these toxins bring about their effects. An understanding of the actions of the toxins will lead to a better knowledge of presynaptic function. Acknowledgment

I thank Dr. E. G. Rowan for his help with the section on phospholipase toxins.

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ALAN L. HARVEY

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MECHANISMS OF CHEMOSENSORY TRANSDUCTION IN TASTE CELLS Myles H. Akabas Department of Medicine College of Physicians & Surgeons Columbia University New York, New York 10032

1. 11. 111. 1V.

V. VI.

VII. VIII.

1X.

Introduction Cell Biology of Taste Cells Impediments to the Study of Taste Cells A Criterion for Taste Transduction Mechanisnrs Electrical Properties of the Lingual Epithelium Electrophysiological Propel-ties of 'l'aste Cells A. Mudpuppy B. Frog C. Rat A Critique of Intracellular Recordings in ~l'asteCells Taste Transduction Mechanisrirs A. Bitter Taste B. Sweet Taste C. Salt l'nste D. Sour Taste E. The Uniami Taste F. Amino Acid Taste in Catfish Summary References

I. Introduction

T h e sense of taste provides animals with a rapid but limited chemical analysis of a potential food substance. For humans, the sense o f taste mainly serves a hedonic function, but for animals it provides information that is often crucial to survival. T h e information obtained f'rom the gustatory system permits animals to decide whether to ingest or expel a particular substance from the oral cavity. For example, most poisonous plant alkaloids taste bitter; bitter taste generally results in the expulsion of a substance from the mouth. Since the time of Aristotle, people have attempted to elucidate the primary taste modalities, that is, to determine INTEKNATIONAL KbVIEW OF NEUKOBIOLOGY, VOL 32

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precisely what the taste cells detect. Aristotle reported that tliere were seven basic taste modalities, but subsequent workers have rediicetl the number of four: bitter, swect, salt, and sour. F,ven this nunil)ci. is suliject t o debate, however, as several groups believe that certain amino acids, such as glutaniate, have distinct tastes. Part of this uncertainty has arisen tiecause of the inability to study the taste cells directly. Over the past 1 0 years direct studies of the taste cells have become possible through the use of a variety of techniques amenable to the study of sniall. single cells. 'rhese studies have provided new insights into the physiology of the taste cells and the processes o t' chiwiosensory transduction in t hc gustatory system. T h e major focus of'this review will be to examine these studies of' the primary transtluciiig cells in an attempt to untlerstancl the processes of taste transduction. As the field of taste research is very large arid multidisciplinary no atteiiipt will lie made to review the entire f-ield. Several reviews of other aspects of the gustatory system include the cell biology of taste cells (Kopcr. 1989); CNS processing of gustatory information (Yaniamoto, 1984; Sniich, 1985; '1-ravel-sc ~ cil., t 1087); hunian taste neurophysiology (Halperii, 1 !Mi);a nd taste i n disease (Schit'fman, 1W 3 ) .

II. Cell Biology of Taste Cells

T h e first steps in the process of gustatory transduction occur in the taste cells. They a re niainly located in the epithelium of the tongue but can also be found in other parts of the oral cavity. T h e taste cells ar e clustered into groups of about 50 cells in the taste buds. .llie cells within ;I taste bud ar e not uniform in appearance. Several difterent cell types have been defined, based largely o n the intensity of staining at both the light a n d electron microscopic. level. 'I'tiese cell types include dark cells (?Fype I ) and light cells (-1'ype I I ) (some also describe intermediate cells), all of which send processes t o the apical surf'ace of the taste bud. and finally basal cells, which d o not send processes to the apical surf'ace (Farl)man, 1965; Murray, 1973; Delay ef (il., 1986; Koper, 1989). I n manimalian tongues the taste buds are localized in three regions of the tongue: the fungiform papillae on the anterior two thirds of' the tongue, the foliate papillae o n the posterior sides of the tongue and the circumvallate papillae on the posterior third of the tongue. 'fhe anterior two-thirds of the tongue is innervated by the chortla tympani nerve, ii branch of' the V 11th cranial nerve; t he posterior third is innervated h y the glossopharyngeal nerve, the IX cranial nerve. T h e anatomy arid distriliutioii o f taste b u d s

varies slightly in noiimamni;ilian species, but niainly in detail and riot in substance (Roper, 1989). ' I h e taste cells themselves iire polarized neuroepithelial cells. which must survive in the extremely harsh a nd variable environnient o n the surf'ace o f t h e tongue. T h e tiis~ecells a re embedded in the surroiinding keratinized squanious epithelium, presuniably fhr protection. Their apical surface is exposed t o the surface of the tongue through ;I small opening referred to as the taste pore. 'l'lie microvilli on the apical tloinaiii of t h e taste cells a re presumably the site of interaction of sapid substances with the taste cells (Farbman, 1965; Murray, 1973). Junctions separate the apical surfaces 01' the taste cells k'rorii the basolateral domain (Akisaka a n d O da , 1978; Holland rt d., 1989). In other epithelia these junctions h i t the movement of' sulistances from the apical surface into the interstitial space (Cereijido ('1 NI., 1988; Kodriguez-Boulan atid Nelson, 1989). Since only a sniall portion of a n epithelial cell niemlirane is exposed to t h e apical milieux, the tight junctiotis protect epitlielial cells from the fluctuations in composition that occur in the apical medium (Cereijido et cd., 1988; Kotli.igiiez-Boulan antl Nelson, I W9). l'rcsuiiiably, they perform the same f'urictions for the taste cells, impeding tlie movement of sapid substances into the interstitial space; this hiis important implications for the site of. iiiteractioii and transduction of sapid substances as will be discussed below. On the basolatei-al domain tlie taste cells fbrm chemical synapses onto the gustatory nerves, which carry information to the central nervous system (Farbman, 1965; Murray, 1973; J. C. Kinnamon et 01.. 19x5, 1988; Koyer and Kinnanion, 1988). Some investigators have reported that some neurons also f'ol-m synapses onto the taste cells, but others have not found such conriectiotis (1. (;. Kinnanion et al., 1988; Koper, I Y X Y ) . T h e taste buds a re not static structures. 'l'he taste cells have a limited life span a n d undergo a continuous progression from basal cells thr-oirgli cliffkrentiation t o cell death. 'l'liis was first demonstrated using tritiated thymidine labeling in rat taste cells, where the life span was found to t)c alx)ut 10 days (Beidler antl Stiiallriian. 1965). Subsequent studies have s h o w n that basal cells are the precursor cell population. Labeling of the basal cells is followed by laheling of the dark cells and then the light cells (Delay et ul., 1986).This implies that the major dif'ference tietween dark and light cells is age since cliffkrentiation from the basal cell stage. Since the taste cells undergo corititiiious turnover the neurons that innervate them must reform synaptic counections on a regular basis. 'I'his process must be carefully regulated I~ecausea neuron that is specific for ;i gi\.en taste modality must only synapse with taste cells of that niotl;ility t o

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preserve the integrity of information transfer to the CNS. T h e mechanism by which this is accomplished is unknown at present. It is interesting that a given neuron synapses with either dark cells or light cells, but not both (J. C. Kinnamon et al., 1988). This suggests that a given neuron periodically reinnervates a group of newly differentiating taste cells and then remains connected to them through their life span. Neurons do not continuously form synapses to newly differentiating taste cells; otherwise, neurons connecting to both dark and light cells should have been observed. The integrity of the neurons innervating the taste buds is very important to the maintenance of the taste cells. Sectioning the gustatory nerves or blocking axonal transport results in involution of the taste buds. T h e trophic influence of the gustatory nerves on the taste buds has been extensively studied and reviewed elsewhere (Farbman, 1969; Cheal and Oakley, 1977; Sloan et al., 1983; Oakley, 1985; Hosley etal., 1987; Whitehead et al., 1987).

111. Impedimentsto the Study of Taste Cells

While much is known about the cell biology and psychology of the gustatory system, until recently little was known about the cellular and molecular mechanisms involved in the transduction of gustatory information. This paucity of information on the basic transduction processes in the taste cells was due to several factors. Mammalian taste cells are small, only a few micrometers in diameter and 10-30 pm in length (Farbman, 1965; Murray, 1973; Koper, 1989). In addition, they are embedded in a tough, keratinized squamous epithelium which makes them relatively inaccessible in situ. Finally, there are very few taste cells; a rat tongue contains about 25,000 taste cells in a sea of millions of nonsensory lingual epithelial cells, making it difficult to do biochemical studies of gustatory transduction systems. One of the major steps in overcoming these problems was the development of procedures to dissociate the lingual epithelium into single cell suspensions. These procedures utilized a variety of proteolytic enzymes, such as collagenase and trypsin, to disrupt the epithelium (Avenet and Lindemann, 1987b; Akabas et al., 1988; Kinnamon and Roper, 1988). Once they were dissociated, the taste cells could then be studied with a variety of techniques amenable to the study of single cells, such as patch clamp recording and single-cell, intracellular calcium measurements.

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245

These studies have begun to elucidate the mechanisms involved in the transduction of various taste modalities.

IV. A Criterion for Taste Transduction Mechanisms

There is an important criterion that must be used while evaluating a proposed mechanism for the transduction of a given taste modality. ‘Ilie sense of taste permits animals and humans to discriminate between different taste qualities. Extensive psychophysiological experiments in humans and behavioral experiments, such as generalizations of conditioned aversions in animals, mainly small mammals such as rats, mice, gerbils and hamsters, indicate that mammals can discriminate four basic taste modalities: bitter, sweet, salt and sour (Jakinovich, 1981; Frank, 3985; Pfaff, 1985). T h e archetypal substances used to represent these tastes are quinine, sucrose, NaCI, and HCI. Unfortunately, similar studies have not been performed in amphibians making it difficult to known whether they are able to discriminate the same taste qualities as humans. This limits the ability to generalize studies of transduction processes in amphibians to mammals, because if they are unable to discriminate between the same classes of substances as humans, responses may be nonspecific epiphenomena unrelated to the sense of taste in that animal. Behavioral experiments are thus a critical element in the study of taste transduction. Thus, a theory of taste transduction must not only describe events in the taste cells, it must also provide a mechanism by which the transduction process generates information that permits an animal to distinguish different classes of substances (i.e., to identify different taste modalities). This requires that the transduction process generate a unique pattern of neural activity in the gustatory nerves for a given taste modality. This in turn requires that there must be unique subpopulations of cells within the taste cells that possess receptors specific for a given taste modality. These issues will be discussed further in subsequent sections as they relate to proposed mechanisms for the transduction of specific taste modalities. While examining studies of gustatory transduction in taste cells it is important to keep this issue in mind: A proposed transduction mechanism must be specific enough to provide the animal with the ability to distinguish different substances. If a proposed transduction mechanism does not fulfill this criterion because of the generality of the response in all taste cells one must question how it could provide modality-specific

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information to the animal, which is a crucial characteristic of the sense of taste.

V. Electrical Properties of the Lingual Epithelium

The lingual epithelium actively transports a variety of ions. These transport properties have been studied by mounting the excised epithelium in an Ussing chamber. This permits measurement of the transepithelial short circuit current (Isc) and the open circuit voltage (VOc).In addition, the unidirectional ion fluxes through the epithelium can be measured using radioactive isotopes. In the basal state, with both sides of the epithelium bathed by symnietrical isotonic buffer solution, the short circuit current is between - 10 and -20 pA/cm‘, the open circuit voltage 10-20 mV, and the resistance 500-1000 ohm-cm2 (DeSimone et al., 1981; Heck et al., 1984; Mierson et al., 1985; Simon and Garvin, 1985; Simon et al., 1986, 1988). In all of these measurements -Isc implies cation transport from the mucosal to the serosal side; the voltage is with respect to ground on the mucosal side of the epithelium. T h e electrical resistance of an epithelium provides a measure of the ability of the epithelium to restrict the movement of substances from the apical domain through the tight junctions into the interstitial space. Epithelia are characterized as “leaky” or “tight” based on the magnitude of their resistance (Cereijido et ul., 1988; Kodriguez-Boulan and Nelson, 1989). T h e resistance of the lingual epithelium is intermediate between “leaky” and “tight” epithelia (DeSimone et ul., 1981; Simon et ul., 1988). This suggests that the tight junctions act as a barrier to the movement of molecules from the oral cavity into the interstitial space. This is crucial to the survival of the taste cells in that the contents of the oral cavity can change from molar salt solutions to distilled water in seconds without causing osmotic swelling and lysis of the taste cells. This would not be possible if the tight junctions did not effectively block the movement of solute and water into the interstitial space. Unidirectional flux studies showed that the basal I,, results from simultaneous Na+ absorption and C1- secretion (Mierson et ul., 1985; Simon and Garvin, 1985). T h e I,, was completely inhibited by addition of ouabain to the serosal side of the epithelium (DeSimone et al., 1981; Simon and Garvin, 1985). This implies that all of the active ion transport processes contributing to I,, were dependent on the function of the basolateral Na+,K+-ATPase. Amiloride was also noted to inhibit a por-

tion (30-50%) of the basal I,, with a K1 of less than 10 pLM (DeSinione ~t al., 1984; Simon and Garvin, 1985). Amiloride blocks a sodium-selective ion channel that is present in a large number of epithelia ((;arty and Benos, 1988; Kleyman and Cragoe, 1988). At higher concentrations it also blocks a variety of other Na+-dependent transport processes ( Benos, 1982; Kleyman and Cragoe, 1988). 'I'his suggested that aniiloridesensitive sodium channels arc present in the lingual epithelium. Unfortunately, these studies do not identify which cells are involved in the transport processes. Given the magnitude of the current and the number oftaste cells it is unlikely that all of the current is passing through the taste cells (Simon and Garvin, 1985). Cations are not the only ions actively transported through the lingual epithelium. T h e lingual epithelium actively secretes chloride (Mierson Pi al., 1985; Simon and Garvin, 1985). In other chloride secretory epithelia, C1- secretion is achieved by locating a Na-K-(;I cotransporter in the basolateral membrane and a chloride-selective ion channel in the apical membrane. This polarized distribution of cotransporter and channel results in transport of C1- into the cell through the cotransporter up its electrochemical gradient followed by passive movement down its electrochemical gradient through the channel (Frizzell et al., 1979: Welsh et a[., 1982; Shorofsky et al., 1984). I n the lingual epithelium, it is unknown which cells are involved in the process of CI- secretion, whether it is the taste cells or the nonsensory epithelial cells, and whether it has any function in the process of taste transduction.

VI. ElectrophysiologicalProperties of Taste Cells

T h e development of patch clanip recording has permitted electi-ophysiological studies of cells that were too small for successful peiietr-ation with intracellular microelectrodes (Hamill et d.,198 1). The major advantage of the patch clanip technique over classical intracellular electrode recording is that the patch electrode does not penetrate the cell membrane (Marty and Neher, 1983). The electrode seals onto the cell membrane and when desired, the small piece of membrane underneath the electrode can be disrupted to permit whole-cell recording. ?'his does not disrupt the seal between the electrode and the cell membrane and thus, no leaks are created. Over the past 5 years, taste cells from several species have been dissociated from the lingual epithelium using a variety of proteases and studied using the patch clamp technique. The resting membrane properties of the taste cells from three species are summa-

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rized in Table I. T h e membrane potentials range from -60 to -70 mV and the impedance at the resting potential is greater than 150 M a in all species (Avenet and Lindemann, 1987b; Kinnamon and Roper, 1988; Akabas et al., 1988). In all three species the current-voltage relationship is markedly nonlinear, displaying outward rectification. The basis of this rectification is the voltage-activated ion channels present in the taste cells. In the following sections the basic electrophysiological properties of mudpuppy, frog, and rat taste cells will be described. Other details of the properties of these cells as they relate to the transduction of specific taste modalities will be discussed in the sections on specific tastes.

A. MUDPUPPY Patch clamp recordings from mud puppy taste cells have revealed the presence of three major types of ion channels, potassium, sodium and, calcium (Kinnamon and Roper, 1988). Each taste cell contained the same currents, though the relative magnitude of the currents varied from cell to cell. T h e resting membrane was largely potassium-selective. A voltageactivated postassium current was seen. It was blocked by 8 mM TEA+ and 8 mM Ba2+,but unaffected by 10 mM 4-aminopyridine. Depolarization of the membrane activated a transient, inward, Na+ current. The activation threshold for this current was about -40 mV and it rapidly inactivated following activation. It was blocked by 100 nM tetrodotoxin (TTX), a characteristic of the voltage-dependent Naf channel found in excitable cells, such as neurons and muscle. A calcium current was also present in mudpuppy taste cells. Its activation threshold was about -20 mV and it inactivated slowly with sustained depolarization even when Ba2+ was used as the charge carrier. T h e calcium current was reversibly blocked by 0.1 mM CdC12 or 8 mM MnC12. Nifedipine at TABLE 1 RESTINGMEMBRANEPROPERTIES OF -TASTE CELLS Species

Mudpuppy Frog Rat

Resting potential (mV) - 62

- 65 - 69

Membrane impedance

Reference"

380 M a 150-750 MR > 1 GR

1 2 3

" K e y to references: 1. Kinnamon and Roper (1987); 2. Avenet and Lindemann (1987b); 3 . Akabas et al. (1988).

10 p M blocked about 20% of the calcium current. Under current clamp conditions the cells generated action potentials in response to injection of depolarizing currents.

B. FROG Frog taste cells display many of the same currents as niudpuppy taste cells (Avenet and Lindemann, 1987b). The resting taste cell membrane was mainly potassium-selective. A delayed-rectifier type potassium current was present in all cells. The potassium currents were blocked by 5 mM Bay+ but were resistant to blockade by externally applied 20 mh4 TEA or 1 mM 4-aminopyridine or by 7.5 mM TEA in the patch pipette. Quinine (0.1 mM) blocked about 20%)of the K + current. All of the cells also contained a transient, inward Na+ current that was blocked by 100 nill T T X but unaffected by 80 pM amiloride. Voltage-activated calcium channels were not seen; however, another group using intracellular recording in frog taste cells reported observing regenerative anodebreak potentials that consisted of voltage-dependent Na+ and Ca“ currents (Kashiwayanagi et d ,1983). This suggests that frog taste cells contain voltage-dependent Cap+ currents. Unfortunately, calcium currents are notoriously evanescent under whole-cell recording conditions. In most cells, they rapidly run tiown unless the “appropriate” reagents, such as ATP, GTP, and glutathione, are added to the pipette solution (Hosey and Lazdunski, 1988; Bean, 1989).

Like mudpuppy and frog taste cells, the resting membrane of rat taste cells was largely potassium-selective (Akabas rt nl., 1988, 1990). 3’he rat taste cells had two types of potassium channels, a 90-pS, delayed rectifier type channel and a 240-pS, “maxi” calcium-activated K channel. These channels were blocked by 1 mM T E A f , and 0.1 mM quinine. They were not blocked by 10 mM 4-aniinopyridine or 1 mM strychnine (Akabas et al., 1990). A subpopulation of rat taste cells, about lo%, also expressed a transient, inward Na+ current that was blocked by T T X . At present it is unknown what the relationship is between the taste cells that contain the sodium current and those that do not. There are several possibilities. The cells containing the Na+ current may be mature sensory cells and the others immature basal cells o r supporting cells. Alternatively, Na cur+

+

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rents may only be expressed in taste cells sensitive to particular taste modalities. Further work will be necessary to resolve these questions. No calcium currents were observed in the rat taste cells, but as mentioned above this was probably due to “run down” in the absence of the appropriate reagents in the pipette solution to preserve the integrity of the calcium channels (Hosey and Lazdunski, 1988; Bean, 1989). A preliminary report of patch clamp recordings from dissociated mouse taste cells indicates that they contain voltage-dependent Na+ and Kf currents similar to those seen in rat (Spielman et al., 1989). The taste cells thus appear to be neuroepithelial cells, expressing characteristics of neurons, such as voltage-dependent Na’ and Ca2+ channels, while displaying the polarized morphology and physiology of epithelial cells.

VII. A Critique of lntracellular Recordings in Taste Cells

Prior to the advent of patch clamp recording, many investigators interested in directly studying the properties of the taste cells used intracellular microelectrodes. Taste cells in all species except mudpuppy are very small. Impalement of small cells with intracellular electrodes frequently results in significant damage to the cell membrane at the site of electrode penetration. This creates a large nonselective shunt conductance in parallel with the cell membrane conductance. In many cases the leak becomes the major conductance pathway. By comparing the electrical properties of taste cells obtained by patch clamp recording with those obtained with intracellular microelectrodes the impact of the leak conductance becomes apparent. Using intracellular electrodes, the reported resting membrane potentials were low, generally less than -50 mV and the membrane impedance was also low, mostly less than 50 M R (Kimura and Beidler, 1961; Tateda and Beidler, 1964; Esakov and Byzov, 1971; Ozeki, 1971; Ozeki and Sato, 1972; Sato, 1972, 1980; Sato and Beidler, 1975,1982, 1983; Akaike et al., 1976; West and Bernard, 1978; Tonosaki and Funakoshi, 1984a,b, 1989). The low values of the resting potential and membrane impedance are indicative of a leak conductance. The reported current-voltage relationships were also influenced by the presence of an electrode induced leak conductance. Using intracellular electrodes, the current-voltage relationships were reported to be linear in mudpuppies (West and Bernard, 1978),frogs (Akaike et al., 1976),and

rats (Ozeki, 1971). What is linear is the leak; the actual current-voltage relationship, demonstrated by the patch clamp recordings, ill all of these species is nonlinear, displaying inarked outward rectification due to the activation of voltage-dependent currents (Avenet and Lindemann, 1987b; Kinnamon and Koper, 1988; Akabas et nl., 1990). Intracellular recordings failed to demonstrate these voltage-dependent currents due to the magnitude of the leak conductance. Thus, in the presence of a leak at the electrode penetration site the measured currents and potential changes were due to the leak and not to the actual properties of the cell membrane. Unfortunately, the same is true of the reported “receptor potentials” observed in response to stimulation with sapid solutions. Here too, the current passing through the leak may predominate arid obscure changes that may occur in the taste cell nienibrane in response to stimulation. This probably accounts for the variability of responses to a single stimulus, sometimes depolarizing, sometimes hyperpolarizing, sometimes no change. It may also explain the observation that most impaled taste cells appeared to respond to all taste stimuli. Finally, some investigators have injected dye at the conclusion of an experiment to prove that the electrode tip was in a taste cell (Tonosaki and Funakoshi, l984a,b, 1989). This does not resolve the problem caused by leak currents around the electrode, because it merely establishes the position of the tip, riot the presence or absence of a significant electrical leak. These problems have also been discussed by others (Avenet and Lindernann, IY87a; J‘eeter and Brand, 1987a; Roper, 1989). I n summary, the patch c h i p studies indicate that the actual electrophysiological characteristics of the taste cells are very different from those previously reported using iiitracellular microelectrode recording techniques. Due to the small size of‘the taste cells, impalement of the cells with a microelectrode frequently induced an electrical leak at the site of electrode penetration. Subsequent recordings measured ion flux through this leak, thereby obscuring ion fluxes through the cell membrane. In view of this, even though it represents a large volume of work, much of it probably must be abandoned and the ideas that it has generated must be carefully reviewed in light of the apparent methodological problems. T h e major exception to this critique of intracellular recording i n taste cells is data from mudpuppy studies. Mudpuppy cells are large enough to allow stable impalements without significant leaks (Koper, 1983; Kinnanion and Roper, 1987; Roper and McBride, 1989), though even here problems due to leaks can arise as illustrated and discussed by Avenet and Lindemann (19874.

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VIII. Taste Transduction Mechanisms

The process of gustatory transduction involves converting the presence of a sapid substance on the apical surface of the tongue into the generation of an impulse in the appropriate gustatory axon(s). The transduction process begins with a recognition step. The recognition step may involve ligand-receptor binding or the passage of ions through a specific ion channel. This is essential to generate taste modality-specific information. T h e question then arises as to how the receptortransduction processes are segregated in the taste cells to permit an animal to obtain taste modality-specific information. There are two potential models, modality segregation and modality mixing. In the modality segregation model, the taste modality-specific receptors are segregated into distinct subpopulations of taste cells responsive to a single taste modality. This model provides a clear mechanism for the generation of modality specific firing patterns in the gustatory nerves. Color vision is organized in this manner: A given cone cell expresses only one of the three (red, green or blue) rhodopsins (Nathans, 1987). In the second, modality mixing model, taste cells express a variable number of receptors for different taste modalities and therefore each taste cell can respond to multiple taste modalities. The problem with this model is to explain how modality-specific information can be generated that will permit an animal to discriminate taste qualities. The ability of the gustatory system to generate discriminative information places a limit on this model, that is, all taste cells cannot be identical (i.e., they cannot express receptors for all taste modalities in the same proportions). If they did it would be impossible to generate discriminative information. One must therefore question how a process that is found to occur in all taste cells can provide discriminative information. The model of modality mixing must therefore be refined to state that a taste cell can only respond to a subset of the taste qualities. T h e question can then be asked, Do distinct subpopulations of taste cells exist that express fixed ratios of receptors for several taste modalities or are the taste cells a continuum expressing variable ratios of receptors for different taste modalities? The continuum model is unlikely because single fiber recordings from gustatory axons suggest that fibers can be grouped into distinct subsets (Frank et al., 1988; Hanamori et al., 1988).This leaves either the modality segregation model or the fixed ratio modification of the receptor mixing model as the t w o possibilities. Only direct studies of taste cells can determine which of these models is correct: however. indirect evi-

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dence can be obtained from single-axon recordings from the gustatory nerves. Gustatory axons can be grouped into subpopulations that are most responsive to a specific taste modality, which might be called the primary modality of the axon (Fishman, 1957; Frank, 1973; Frank rt d.,1983, 1988; Ninomiya et aL, 1984; Hanamori rt al., 1988; Ninomiya and Funakoshi, 1988). In general, axons can be divided into four populations: bitter-sensitive, sweet-sensitive, sodiiim-sensitive ( N fibers) and those sensitive to HCI and other electrolytes (H fibers). Studies of the effect of amiloride on the response characteristics of single sodium-specific, Ntype fibers and on nonspecific, H-type fibers strongly suggest that these two fiber populations innervate two distinct subpopulations of taste cells that express different receptive mechanisms (Ninomiya and Funakoshi, 1988). This provides support for the modality segregation model for taste receptors. Most axons also have lower-level responses to substances of other taste qualities, which might be called secondary responses (Fishman, 1957; Frank, 1973; Frank et nl., 1983, 1988; Ninomiya et al., 1984; Hanamori et al., 1988; Ninomiya and Funakoshi, 1988). The work of Ninomiya and Funakoshi (1988) coupled with other work on salt taste transduction suggests that for N-rype fibers the responses to nonsodium salts are an intrinsic property of the transduction mechanism, as will lie discussed more completely in the section on salt transduction. ‘l’hus, an understanding of the details ofthe transduction mechanism may help to clarify the origin of some o f t h e secondary responses. At present several important questions are raised by the secondary responses. Where do the multi-modal response characteristics of the gustatory axons arise? Does this represent a property of the taste cells themselves (i.e., d o taste cells express low levels of receptors for taste modalities other than their primary modality or is it due to lack of complete specificity in a particular transduction mechanism) or do the axons occasionally make synaptic connections with a taste cell of the “wrong” specificity? Another crucial, but as yet unresolved question is, What constitutes a significant firing frequency in a given gustatory axon? This determines the true extent of multimodal responses. The secondary responses of gustatory axom suggest that CNS processing of the information from the peripheral neurons is necessary to filter or deconvolute the modality specific information, although the neural mechanisms for this are unknown at present. Nevertheless, gustatory axons are essentially niodality-specific and this implies that the same is true of the taste cells (Frank et al., 1988; Hanamori et al., 1988). Further work will be necessary to elucidate the mechanisms underlying secondary responses in the gustatory axons and whether they also occur in the taste cells.

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A. BITTERTASTE

Bitter taste is probably the most interesting taste modality, from a teleologic point of view. To avoid being devoured by animals, particularly insects, plants produce a variety of “secondary” substances whose function is to prevent foraging by poisoning the forager (Botkin et ul., 1973; Harborne, 1982; Maugh, 1982; Nathanson, 1984; Moore, 1986). In general, the poisonous compounds that plants produce taste bitter to animals in the micromolar concentration range; making bitter taste the most sensitive of the four taste modalities. In animals, bitter taste stimulates rejection of a potential food substance. Presumably, bitter taste evolved to allow animals to detect the presence of these poisonous substances and avoid them. As such it must have played an important role in the evolution of animals and the plants on which they feed. Evolutionarily, bitter taste is probably very old because both intact and decerebrate rats perform the same stereotyped maneuvers in response to the instillation of-a bitter solution into the mouth (Grill and Norgren, 1978a,b). This suggests that the behavioral response involves neurons in the lower brain stem, the most primitive portion of the brain (Travers et al., 1987). 1. Number and Type of‘Bitter Receptors The chemical structures of bitter substances are very heterogeneous. This raises the question of whether there is a single receptor for all bitter substances or whether there are multiple receptors each for a different class of bitter substances. Several lines of evidence suggest that there is more than one receptor involved in bitter taste. First, humans display a dimorphism in the taste threshold for a bitter chemical, phenylthiocarbamide (PTC). Inability to taste PTC is inherited in an autosomal recessive manner (Blakeslee, 1932; Fox, 1932; Harris and Kalmus, 1950). However, PTC nontasters display no defect in the ability to taste a wide variety of other bitter compounds (Blakeslee and Salmon, 1935; Barnicot et al., 1951). This suggests that separate receptors are involved in the transduction of PTC and other bitter substances, such as quinine. Second, extensive genetic analysis of taste polymorphisms in mice for a variety of bitter substances have identified several independent, autosomal, monogenic loci involved in the ability to taste different bitter substances, such as sucrose octaacetate (SOA) and strychnine (Warren and Lewis, 1970; Lush, 1981, 1982; Whitney and Harder, 1986), quinine (Lush, 1984), raffinose undecaacetate (Lush, 1986), and cycloheximide (Lush and Holland, 1988). Interestingly, all of these independent loci are closely linked (Lush and Holland, 1988). The gene products of these bitter taste loci have yet to be determined. Physiologic studies have shown that SOA taster mice have a large, integrated, whole-nerve response to

SOA, but little or no response is seen in nontaster strains ofmice, whereas other bitter substances such as quinine and PTC induce similar responses in SOA taster and nontaster strains (Harder Pi nl., 1984; Shingai and Beidler, 1985). This suggests that the SOA genetic locus codes for a peripheral receptor, which is presumably one of several involved in bitter taste transduction. It also strongly supports the view that bitter taste is mediated by interactions of bitter substances with specific protein receptors and not by nonspecific interactions with the lipid domain of the cell membrane. T h e existence of multiple bitter taste receptors raises the question of whether they are all expressed in a single class of taste cells or are segregated into separate cells. In frog tongue, quinine, tirucine, and caffeine all cause reciprocal cross-adaptation of the integrated whole nerve response (Sugimoto and Sato, 1982). 'This suggests that the bitter receptors for these compounds are present in a single class of taste cells, but the potential for species differences limits the ability to generalize this to mammals. Human psychuphysiological experiments have denionstrated cross-adaptation between quinine, caffeine, and SOA, but not between quinine and P T C (McBurney et al., 1972). This suggests that some receptors may be expressed in distinct populations of' bitter taste cells o r that the they are all in a single bitter taste cell, but different receptors may utilize different transduction mechanisms that d o not cross-adapt.

2. Transduction Mecha.nisms iri Bittrr Triste T h e transduction of bitter taste begins with the binding of a bitter substance to one of the receptors described above. How is this ligandreceptor interaction then coupled to secretion of neurotransmitter? In general, the secretion of neurotransmitter is accompanied by a rise in the intracellular calcium concentration. By loading taste cells dissociated from the lingual epithelium surrounding the circurnvallate papillae of the rat with the calcium-sensitive fluorescent dye, fura 2 (Grynkiewicz el al., 1985; Cobbold and Rink, 1987), the responses of individual cells to stimulation with a bitter test substance can be monitored using a single cell microfluorimetry system. Denatonium chloride was used as a bitter test stimulus for several reasons (Saroli, 1984; Akabas et al., 1988).' Unlike quinine, it is not fluorescent and therefore does not interfere with the T h e detection threshold for denatonium chloride and other quaternary ammonium compounds, such as TEACI and benzyltricthylanimoniuni chloride, displays a dirnorphisni in human subjects that segregates according to PTC tasting status. Thus, PrC tasters can detect these compounds at a concentration that is about half an order ol.niagnitudc below the threshold for nontasters. Nontascer-s are still able t o perceive them as litter. but the intensity of the bitter taste seems much less ( M . H. Akabas, unpublished data).

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fura 2 dye. Since denatonium is a quaternary ammonium compound and thus permanently positively charged, it is probably impermeable through the cell membrane; therefore, its site of action is more clearly defined than membrane-permeant bitter substance. Application of 1 p M denatonium to fura 2-loaded taste cells induced a rise in the intracellular calcium concentration in a small subpopulation of taste cells. The increase in cytoplasmic calcium was due to release of calcium from internal stores, because denatonium induced a similar rise in cytoplasmic calcium in the absence of extracellular calcium (Akabas et al., 1988). This effect of denatonium must be a specific receptor-medicated effect, because most of the taste cells which were simultaneously exposed to denatonium experienced no change in intracellular calcium concentration. These experiments suggest that the transduction of the bitter taste of denatonium is a biochemical process, not an electrophysiological one. T h e following model was proposed to explain the process of bitter taste transduction (Akabas et al., 1988). Denatonium binds to a receptor protein that is located on the apical surface of a subpopulation of taste cells. Following binding of denatonium to its receptor, an intracellular second messenger is generated, most probably inositol trisphosphate (IPS)(Hokin, 1985; Carafoli, 1987; Berridge, 1987; Berridge and Irvine, 1989). T h e second messenger then induces release of calcium from internal stores. This presumably leads to secretion of neurotransmitter and stimulation of the gustatory nerves. The formation of IPS is accompanied by the synthesis of diacylglycerol (DAG), which is a potent activator of protein kinase C (PKC)(Nishizuka, 1988; Kikkawa et al., 1989). Whether PKC has a role in the transduction process is an intriguing but unknown possibility at present. Several other experiments support the idea that bitter taste transduction is essentially a biochemical event that does not involve opening or closing ion channels. First, a rise in IPS concentration following addition of denatonium to a homogenate of rat lingual epithelium has been reported (Hwang et al., 1989). This provides biochemical evidence that the second messenger in the denatonium transduction process is IPS.Second, bitter substances, such as quinine, had no effect on the short circuit current in the lingual epithelial preparation (Simon et al., 1986). This suggests that the transduction process does not involve opening or closing of ion channels. Third, perfusion of the frog lingual artery with either Ca2+-free Ringer solution or with Ringer solution containing the Ca2+ channel blockers MnC12 or verapamil had no effect on the glossopharyngeal nerve response to several bitter substances, quinine, and theophylline. Perfusion of the lingual artery with the same solutions, however, resulted in a marked decrease in the response to NaCl and

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galactose (Nagahama et ul., 1982). This suggests that extracellular calcium is not involved in the transduction of bitter taste but is involved i n the transduction of salt and sweet. This supports the idea that the transduction of bitter taste is mediated by release of calcium from internal stores. Furthermore, though it has been suggested that quinine tastes bitter by permeating into the taste cells and blocking potassium channels (Ozeki, 1971; Avenet and L,indemann, 1989), this seems very unlikely because it offers no mechanism for specificity. Quinine would be equally permeant into taste cells for all taste modalities and its ability to block potassium channels is very nonspecific. Thus it would be expected to block potassium channels in all taste cells and produce nonspecific firing of all taste cells. Single axon recordings clearly indicate that this does not occur: Quinine stimulates a very limited class of fibers (see, for example, Frank et ul., 1988; Hanamori et al., 1988). This suggests that there is a more specific mechanism for detection of quinine. T h e identification of a gene locus coding for a quinine receptor implies that there is a specific protein product involved in quinine transduction (Lush, 1984). The ability of quinine to block potassium channels in dissociated taste cells (Avenet and Lindemann, l987b; Akabas el al., 1990) is probably an epiphenomenon unrelated to taste transduction. In the dissociated cell preparations quinine has access to the basolateral domain of the cell membrane which it does not have in situ in the tongue. A recent report indicated that pretreatment of rat tongue with TEA+, a K f channel blocker, resulted in a marked decrease in the integrated whole glossopharyngeal nerve response to quinine (Scott and Farley, 1989). This was taken to imply that potassium channels are involved in the transduction of quinine bitter taste. However, TEAt is intensely bitter (M. H. Akabas, personal observation), so it is possible that pretreatment with TEA+ resulted i n adaptation of the bitter-responsive cells unrelated to its ability to block K + channels (Smith et al., 1975), resulting in the diminished response. The use of Ba2+, which also blocks K + channels but is not bitter (M. H. Akabas, personal observation), would be more definitive for these experiments. Using fura 2-loaded, dissociated rat taste cells as an assay system, several other chemicals have been studied. A compound structurally related to denatonium, benzyltrieth yl ammonium, induced a similar rise in cytoplasmic calcium, but several other substances including 10 nut4 saccharin and 0.5 mM 8-Br-CAMP did not induce a rise in intracellular calcium in any of the taste cells (M. H. Akabas, unpublished data). This suggests that other mechanisms may be involved in the transduction of other bitter substances and of sweet substances. I t also suggests that raising CAMPis not involved in the transduction of bitter taste, a mecha-

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nism that has been proposed for the bitterness of the methyl xanthines (Kurihara, 1972). A variety of other mechanisms have been invoked in the process of bitter taste transduction including interactions with lipids (Koyama and Kurihara, 1972) and nonspecific electrostatic interactions (Kumazawa et al., 3986). T h e problem with both of these mechanisms is that they involve nonspecific interactions, which provide no mechanism to stimulate just bitter-responsive taste cells.

B. SWEETT A S T E 1. Structure of Sweeteners

Sweet taste is a hedonically pleasing sensation that stimulates ingestive behavior and has been the subject of several reviews (Schiffman et al., 1986a; Jakinovich and Sugarman, 1988). In general, the sweet taste system has a very low sensitivity for simple sugars. The detection threshold for sucrose is about 10-30 m M in humans and is not significantly different in animals. A wide variety of substances have been found that are significantly sweeter than simple sugars, some naturally occurring and some chemically synthesized (Schiffman et al., 1986a). Sweet substances have been the subject of intensive study by the technique of quantitative structure-activity relationships (QSAR). Early studies suggested that a hydrogen bond donor and acceptor separated by about 3 A was common to all sweet sugars (Shallenberger and Acree, 1967; Shallenberger et al., 1969). Subsequent studies have identified several other structural features that are important for the sweet taste of a variety of synthetic artificial sweeteners (Fujino et al., 1976; Tsang et al., 1984; Rodriguez et al., 1985; Miyashita et ul., 1986; Venanzi and Venanzi, 1989). In addition to the many small molecules that are sweet there are two proteins, thaumatin (20 kD) (van der We1 and Loeve, 1972) arid monellin (10 kD) (Morris and Cagan, 1972) that are among the sweetest substances known, being about 30,000- 100,000 times sweeter than sucrose on a molar basis (van der We1 and Arvidson, 1978). However, these proteins are only sweet to humans and Old World primates (Brouwer et ul., 1973; Glaser et ul., 1978), our nearest evolutionary relatives. This suggests that the ability to taste these two proteins as being sweet evolved about 38 million years ago (Glaser et ul., 1978). Antibodies against one of these proteins cross-react with the other protein, suggesting that the proteins have a common epitope (Hough and Edwardson, 1978; van der We1 and Bel, 1978), but the proteins have only five tripeptides of sequence identity

(Bohak and Li, 1976; Frank and Zuber, 1976; lyengai- rl ul., 1979).'l'his suggests that the common structural feature of the two proteins is clependent on the tertiary conformation of the proteins. T h i s is supported by the fact that denaturation of the proteins resulted in ;I loss of sweet taste (Morris and Cagan, 1975). Both proteins have been crystallized and their structures solved to 3-?i resolution; however, no common structural features have been identified that might represent the epitope that hinds to the sweet taste receptor (de Vos ot u/., 1985; Ogata r f NI., 198'7).There are several possible explanations for this failure. The kinding site may be more subtle than can recognized at 3-A resolution. Alternatively, i n the crystal state the conformation of one or both of. the proteins may be different from its conformation in solution and therefore no structural identity is seen in the crystals. Given the intense sweetness of these proteins and therefore the high affinity for the sweet receptor hintling site, further study of these proteins may help to clarify the strurture of the receptor binding site. 2. Sweet Taste Receptor Much evidence has accumulatecl that sweet taste is mediated by a protein cell surface receptor. ~Fhesweet receptor is sensitive to proteolytic damage. Application of proteases to the apical surface of rat tongue resulted in the complete loss of the whole chorda tympani nerve response induced by sucrose, but not the response to other taste modalities (Hiji, 1975). In addition, most sweet substances are quite hydrophilic, suggesting that the receptors must be located on the surfiice of sweet taste cells. Since thaumatin and monellin are unlikely to be permeant through the tight .junctions the receptors are most likely located on the apical surface of the taste cells. T h e question of whether there are multiple sweet receptors is unrcsolved. Genetic evidence from mice suggests that sucrose, saccharin, dulcin, and acesulfame have a common receptor (Lush, 1989). Crossadaptation of the whole nerve responses to sucrose and thaumatin was 1973) which suggests either a observed in monkeys (Brouwer ei d., common receptor or different receptors with a common transduction pathway in the same cell. Other physiological and psychophysiological evidence is somewhat contradictory and was extensively discussed in a review by Jakinovich and Sugarman (1988).

3. Inhibitors of Sweet Taste Several substances have been identified as inhibitors of sweet taste. The ability of gymnemic acid to diminish the perceived intensity of' sweet solutions was first described by Edgeworth (1847). I t has subsequently

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been shown to be a noncompetitive inhibitor of sweet taste in a variety of mammals and in humans (Diamant et al., 1965; Meiselman and Halpern, 1970; Hellekant and Gopal, 1976; Hellekant et al., 1985). A competitive inhibitor of sweet taste, methyl-4,6-dichloro-4,6-dideoxy-a-~-galactopyranoside (DiC1-gal),has also been identified by Jakinovich (1983). T h e action of DiC1-gal is rapidly reversible and inhibits the integrated chorda tympani response to both sucrose and saccharin in gerbils, suggesting a common receptor for these two sweet substances (Jakinovich, 1983). 4. Transductionof Sweet Taste Much information is available on the process of sweet taste transduction. T h e process of sweet taste transduction has been examined using several techniques including measurement of transepithelial currents through the lingual epithelium, biochemical studies, psychophysical studies, and intracellular microelectrode recordings. Synthesis of the results of these disparate studies requires careful attention to methodologic problems and details of the experimental techniques that limit the ability to interpret the results. Several groups have examined the effects of sugars on ion transport through lingual epithelium (Mierson et al., 1988; Simon et al., 1989). By mounting the lingual epithelium in an Ussing chamber they were able to measure the electrical properties of the whole epithelium. This technique has the advantages that it does not damage the taste cells and it permits studies of their responses in situ in the polarized epithelium. A disadvantage is that one does not know what percentage of the current is passing through the sensory cells and what percentage through the nonsensory epithelial cells (Simon and Carvin, 1985). Addition of sugars to the apical side of dog lingual epithelia stimulated an increase in the short circuit current through the epithelium and a decrease in the transepithelial resistance. The increased current was due to an increase in the unidirectional cation flux in the apical to basolateral direction. I t resulted from the opening of a cation selective channel in the apical membrane and was inhibited by the drug amiloride (DeSimone et al., 1984; Mierson et al., 1988; Simon et al., 1989). These experiments suggest that binding of a sweet ligand to its receptor results in opening of a cation-selective channel in the apical membrane of the sweet-sensitive taste cells. T h e resulting cation influx presumably depolarizes the cells, opening voltagedependent calcium channels leading to neurotransmitter secretion. T h e mechanism coupling ligand-receptor binding to channel opening is unknown. Several possible second messengers appear to have been ruled out at present: Addition of 5 mM 8-Br-CAMP, 1 p M forskolin, 5 m M 8-Br-cCMP, 0.1 mM adenosine and 0.1 mM A23187 (a calcium ion-

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ophore) had no effect on the short circuit current or open circuit potential (Simon et al., 1989). Other possible mechanisms such as ligandactivated channels, protein kinase C, and G-proteins have not been studied. Further work will be necessary to clarify the relationship between these ion fluxes and the sweet taste transduction process. There is a discrepancy between the concentration at which amiloride is effective in the lingual epithelial preparation and in whole chorda tympani nerve recordings. T h e same authors showed that 0.1 mM amiloride caused a 73% decrease in the short circuit current. while 0.8 mM amiloride only decreased the integrated whole chorda tympani nerve response by 25-40% (Mierson et nl., 1988). Unfortunately. the authors do not comment on this discrepancy. Other data on effects of amiloride on sweet taste do not help to resolve this issue. In rats, amiloride had n o effect on the chorda tympani nerve response to sucrose (Brand et al., 1985). Human psychophysical experiments, however, indicated that amiloride diminished the perceived intensity of sweet substances (Schiffman et al., 1983). A potentially confounding effect in these experiments is that amiloride tastes bitter (M. H. Akabas, personal observation), creating potential problems of mixture suppression effects (Lawless, 1982; Kroeze and Bartoshuk, 1985). Human studies by the same group reported that methyl xanthines potentiated the perceived intensity of certain sweeteners. Based on the concentration at which the methyl xanthines were active they proposed that the effect was mediated by adenosine receptors modulating the sweet-sensitive taste cells (Schiffnian et al., 1985, 1986a). However, the effect was only observed with sweeteners that also possess a significant bitter taste, such as acesulfameK and saccharin, suggesting that the methyl xanthines may be acting on the bitter component of the taste, as the intensity of quinine was also potentiated in this assay. In a subsequent review these authors indicated that under the conditions of their assay, “Subjects . . . were unable to determine whether the increases in perceived intensity were due to bitterness or sweetness or both” (Schiffnian et al., l986a). Thus, the proposed involvement of adenosine receptors in the modulation of sweet taste transduction is probably incorrect. A biochemical study suggested that CAMPmay be a second messenger in the process of sweet taste transduction. Sweet substances were found to increase the activity of adenylate cyclase in a membrane homogenate of rat lingual epithelium (Strieni et ul., 1989). The activation was GTPdependent and was not seen when the membrane homogenate was made from nonsensory portions of the rat lingual epithelium. This suggests that a GTP binding protein (G-protein) is involved in the coupling of the sweet receptor to the activation of adenylate cyclase. A gene superfamily of G-protein coupled receptors has been described (Libert et al., 1989;

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O’Dowd et al., 1989). These receptors share a variety of common structural motifs. Perhaps, the sweet taste receptor is another member of this gene superfamily. In patch-clamped frog taste cells cAMP was found to cause a reversible depolarization of the membrane potential (Avenet and Lindemann, 1987b). This depolarization was due to closure of a 44-pS potassium channef following phosphorylation by CAMP-dependent protein kinase (Avenet et al., 1988). No evidence was found of cyclic nucleotide-gated channels similar to those found in the visual and olfactory systems (Nakamura and Gold, 1987; Yau and Baylor, 1989). However, it is uncertain whether these results are related to sweet taste transduction in frogs. T h e effect of cAMP was observed in all frog taste cells, suggesting that it is a relatively nonspecific effect and may be unrelated to the transduction of a specific taste modality. In many other systems CAMP-dependent protein kinase modulates the activity of ion channels (Siegelbaum et al., 1982; Levitan, 1985; Schouniacher et al., 1987; Li et al., 1988). Another group has used intracellular recordings in mouse taste cells to study sweet taste transduction (Tonosaki and Funakoshi, 1984b, 1989). In theory this technique should provide direct information on changes in the taste cells. Unfortunately, as mentioned previously, insertion of microelectrodes into small cells, such as mammalian taste cells, can induce significant damage to the cell membrane at the site of electrode penetration. This produces a large nonselective shunt and makes subsequent recordings uninterpretable. The problem of damage at the site of electrode penetration is well documented with experimental data and discussed above (Avenet and Lindemann, 1987a; Roper, 1989). Several lines of evidence suggest that these studies suffer from this problem. First, the mean resting membrane potential was depolarized (-41 mV) (Tonosaki and Funakoshi, 1984a,b). Patch clamp studies of taste cells and intracellular recordings in mudpuppy taste cells have shown that the resting membrane potential is more negative than -65 mV (Roper, 1983; Avenet and Lindemann, 1987b; Akabas et al., 1988; Kinnamon and Roper, 1988). Second, the membrane impedance was reported to be less than 100 MR in mice, whereas the other studies have found the resting membrane impedance to be greater than 100 MR, and in rats higher than 1 GR (Akabas et al., 1988). This suggests that there were leaks in the impaled mouse cells. Third, single-axon recordings from mouse gustatory nerve have shown that the mouse axons were “more narrowly tuned,” (i.e., more modality-specific than those of other species) with very little overlap between sweet-sensitive and Na-sensitive fibers (Ninomiya et al., 1982, 1984). However, the intracellular recordings ill the taste cells showed that there was broad overlap, with many cells responding to sucrose and NaCl

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(Tonosaki a nd Funakoshi, l984a). More likely there was a large leak around the electrode impalement site in many of the taste cells leading to the observed results. Given these fundamental methodological problems it is not possible to interpret these results in terms of mechanisms of' sweet taste transduction.

C . SALTTASTE Salt taste consists mainly of'the ability to detect sodium in a potential food substance. Sodium is crucial to animals because it is the main determinant of the extracellular fluid volume (West, 1985). 1'0 survive, animals must closely regulate the composition and volume of the extracellular fluid. This is accomplished through the integrated actions of the brain, which regulates excretion through the kidneys and regulates ingestion through hunger and thirst (Denton, 1982; West, 1985; Phillips, 1987). For many animals, especially herbivores, the inability to find adequate amounts of sodium may be a life-threatening problem (Denton, 1982). Salt taste permits animals to find adequate sources of sodium in salt licks, etc. (Botkin et al., 1973; I k n t o n , 1982).

1 . Modulation of'the Perceizird IntfiLsity (4 S d t y Stimuli Salt deprivation is known to stirnulate salt appetite (Dentoil. 1982; Fregly and Rowland, 1985). Several recent studies have shown that the perceived intensity of a NaCl solution can be modulated by salt deprivation (Berridge P t al., 1984). Both thc magnitude of the integrated chorcla tympani nerve response and the firing frequency of Na+-sensitive single axons were diminished in salt-depleted rats (Contreras. 1977; Conti-eras and Frank, 1979). 'This suggests that the sensitivity of the taste cells to a given concentration of NaCl was reduced by salt deprivation. T h e mechanism f-or this modulation of salt taste cell sensitivity may be hormonal. O ne of the major regulators of body salt content and concentration is the renin-angiotensiri-al~I~~steronesysteiii (Fregly and Kowland, 1985; West, 1985; Ballermann rt d., 1986). This hormonal system regulates salt transport by the kidney and other epithelia. Salt deprivation stimulates a rise in the concentration of both aldosterone, a steroid hormone, a nd angiotensin I I , a peptitle hormone. Aldosterone is synthesized in the adrenal glands (Quinn mid Williams, 1988). I t acts on the cells in a variety of Na+-transporting epithelia, such as the toad urinary hladder a nd the distal tubule of the kidney, to increase the number of sodium channels in the apical membrane, thereby increasing the rate of sodium transport. However, experiments in adrenalectoinized animals suggested

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that aldosterone was not the mediator. Adrenalectomy reduced the magnitude of the integrated chorda tympani nerve response to NaCl solutions in a manner similar to salt deprivation (Kosten and Contreras, 1985). Since these animals lack aldosterone it would appear that this hormone is not the sole mediator of the decreased salt responsiveness. This suggests that angiotensin 11, which is elevated by both salt depletion and adrenalectomy, may be the modulator of salt sensitivity. Angiotensin I1 has a variety of actions on target cells including elevation of intracellular calcium and mitogenesis (Ballermann et al., 1986). Further experiments will be necessary to elucidate the process of hormonal modulation of salt taste sensitivity.

2. Sodium Coding in the Gustatory Axons Behavioral experiments using conditioned aversions have been used to determine whether animals can distinguish the tastes of different salts (Nowlis and Frank, 1977, 1981; Nowlis et al., 1980; Frank, 1985). An animal with a conditioned aversion to NaCl freely drank HCl, NH4C1, and KC1 solutions. Conversely, conditioned aversions to HCI, KCI, or NH4Cl suppressed drinking solutions of the other two but did not suppress ingestion of NaCl solutions (Nowlis et al., 1980; Frank, 1985). Therefore it appears that aversions to NaCl do not cross-generalize to other nonsodium salts or acids and vice versa. This confirms that animals can distinguish sodium salts from other salts or acids (Frank, 1985). The peripheral gustatory axon coding of electrolyte tastes is complicated. T h e neural basis of the ability to distinguish the taste of various salts may depend on the differential effect of salts on the sodium-specific N fibers, versus the “less specific” H fibers. At a given Na+ concentration the firing frequency of the N fibers was 6.5 times greater than the firing frequency of the H fibers (Frank, 1973; Frank et al., 1983, 1988). Nonsodium salts did stimulate the N fibers, but higher concentrations of nonsodium salts were required to achieve the same firing frequency and therefore presumably the same perceived saltiness. This may explain the fact that nonsodium salts are to some extent perceived as being salty (Murphy et al., 1981). These studies imply that N and H fibers synapse with separate populations of taste cells, which presumably possess diff-erent taste receptors. Further evidence to support this idea will be discussed subsequently in reference to the effects of amiloride on responses in single N and H fibers. 3. Salt Taste Transduction

The mechanism of salt taste transduction is better understood than that of any other taste modality. Data acquired by a variety of experimen-

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tal techniques, which will be examined below, all support the same mode. T h e model is based on the polarized epithelial structure of taste cells. Salt-sensitive taste cells have a sodium-selective ion channel localized in the apical domain of the cell membrane. When a sodium-containing solution is placed on the apical surface of the tongue, Naf moves through the channel down its electrochemical gradient, thereby depolarizing the cell. This depolarization is then postulated to result in the opening o f calcium-selective ion channels, which leads to an increase in cytoplasmic calcium and to neurotransmitter secretion. The drug amiloride blocks the epithelial, sodium-selective ion channel in most animals and diniinishes the response to a given concentration of a sodium solution as measured by a variety of techniques. T h e first major step in the development of this model was the recognition that the lingual epithelium actively transported ions and was riot a passive, ion-impermeant menilwane (DeSirnone et al., 198 1 ) . ~Iliese workers mounted dog lingual epithelium in an Ussing chamber and measured transepithelial ion currents and voltages. They noted that increasing the apical NaCl concentration resulted in a marked increase in short circuit current and open circuit voltage, which they have referred to as the hyperosmotic effect. T h e hyperosmotic current induced by NaC1 was blocked by amiloride (DeSirnone et nl., 1981; Heck et a/., 1984). This suggested the basic model that salt taste transduction involved movement of Na+ into salt-sensitive taste cells through an amiloride-blockable sodium channel (Heck et ul., 1984). Further studies have shown that there are two parallel transcellular pathways for sodium movement, one amiloride-sensitive and one amiloride-insensitive, of approximately equal magnitude in symmetrical isotonic solutions (Mierson et nl., 1985). Under hyperosmotic conditions, with the mucosal NaCl concentration of 1 M , 84%)of the short circuit current was amiloride blockable, but if KCI or CsCl were used in place of' NaCl then the increased short circuit current was largely insensitive t o amiloride (Simon and Garvin, 1985; Simon et ul., 1986). l h i s implies that the current carried by K+ and Cs+ does not go through the same pathway as Naf. Human taste perception experiments by Schiffman et al. ( 1 983) provided further support for this model. They demonstrated that amiloride applied topically to the tongue diminished the perceived intensity of a sodium-containing solution. They also noted that amiloride diminished the perceived intensity of LiCl solutions, but had no effect on the perception of KCl, HCl, or CaC12 solutions. In most tissues, the epithelial N a + channel is equally permeant to Na' and to Li', but poorly permeant to K+ and to Cs+ (Palmer, 1987; Garty and Benos, 1988). In subsequent

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experiments bretylium tosylate, a drug that was reported to increase the number of open epithelial Na+ channels in frog skin (Ilani et al., 1982, 1984), was shown to potentiate the perceived intensity of a given concentration of NaCl solution (Schiffman et al., 1986b). Recordings of the integrated activity in the whole chorda tympani nerve provide additional information to support the model. Several groups have shown that in rats, amiloride diminished the magnitude of the whole chorda tympani nerve response to NaCl and LiCl by about 70-90% (Heck et al., 1984; Brand et al., 1985). Amiloride had little effect on the chorda tympani response to KCl and RbCl (Heck et al., 1984; Brand et al., 1985). The dose-response data suggest that the effect of amiloride is a mixture of both competitive and noncompetitive inhibitory processes (Brand et al., 1985). The onset of inhibition following application of amiloride was rapid, within less than 2 sec (DeSimone and Ferrell, 1985). Furthermore, there was a good correlation between the percentage of inhibition of the chorda tympani nerve response and the lingual epithelial short circuit current at a given aniiloride arid NaCl concentration (Desimone and Ferrell, 1985). In frog glossopharyngeal nerve, amiloride inhibited the response to both NaCl and KCl by 20-40% (Yoshii d ul., 1986).Amiloride, by itself, induced a large response in the frog glossopharyngeal nerve, which was attributed to stimulation of salt receptors (Yoshii et ul., 1986). Given the intense bitter taste of 0.1 mM amiloride (M. H. Akabas, personal observation), the response seen was probably due to stimulation of a bitter receptor. In mudpuppies, amiloride does not inhibit the whole nerve response induced by NaCl (McPheeters and Roper, 1985).At present it is unknown whether the transduction mechanism in mudpuppies can distinguish sodium from nonsodium salts and whether it is based on the same transduction mechanism present in mammals. Additional information has been obtained using single-fiber recording techniques. It was found that amiloride diminished the firing frequency elicited by 0.1 M solutions of NaCl and LiCl in sodium specific, N-type fibers by 80%. Solutions of 0.1 M KC1 and 0.01 M HCl generated low levels of firing in the N fibers, but it was inhibited by amiloride in the case of KCI by 60% and in the case of HCl by 40%. Amiloride did not inhibit firing in nonspecific, H-type fibers induced by the same solutions (Ninomiya and Funakoshi, 1988). This implies that the mechanism of transduction in taste cells innervated by N fibers is via an amiloridesensitive ion channel, but a different mechanism must be involved in activation of taste cells innervated by H fibers. This provides further support for the idea that there are distinct populations of taste cells that only express receptors for a single taste modality. It also suggests that the

activation of N fibers by nonsodium salts is largely clue to ion flux through the amiloride-sensitive channel. While the channel is called a Na' channel, other ions can pass through the channel with varying permeabilities (Palmer, 1987). In rat kidney cortical collecting duct cells the cation selectivity sequence o f the channel is 1.i' > Na' >> K f > Kb' (Palmer and Frindt, 1988). T h e ion permeability of the channel t o nonsodium cations thus determines the magnitude of the response elicited hy the nonsodium salts in N fibers. Finally, patch clamp recording in frog taste cells has demonstrated an amiloride-sensitive conductance that is present in 567r of the taste cells (Avenet and Lindemann, 1988). T h e inhibitory constant in these frog taste cells was 300 nM. Noise analysis and single channel recordings indicated that the channel has a conductance of 2 pS. I o n selectivity measurements showed a sequence of' K > Na > Kb > Li > Cs (Avenet and Lindemann, 1988, 1989). ' I h e single channel size and ion selectivity sequence are different from the epithelial Naf channel in frog skin, which has a selectivity sequence o f I,i > Na >> K, Rb (Lindernann, 1984; Palmer, 1987). This suggests that the channel expressed in the frog taste cells is similar to, but not the same as, the channel expressed i n skin cells. An amiloride-sensitive Na' channel has not yet been demonstrated in rat taste cells, but there are several possible explanations for this failure (Akabas et al., 1990). First, the cells used were from circumvallate papillae. I n rats the majority of the Naf-specific fibers are in the chorda tympani nerve, while the glossopharyngeal nerve has relatively fewer N-type fibers (Frank, 1975; Frank rt ul., 1983).'I'hus, in the limited survey conducted, salt-specific taste cells might not have been founcl. Second, the amiloride-sensitive Na+ channel is sensitive to proteolytic clamage by trypsin, one of the enzymes used in the dissociation of the rat lingual epithelium (Garty and Edelman, 19x3). Thus, the dissociation procedure may have destroyed the channel. In view of the overwhelming evidence from other techniques, it is probably only a matter of further searching to demonstrate the presence of this channel in rat o r other mammalian taste cells. 4. Anion Eflects in Salt Taste Tmnsduction

Anions have been neglected in many studies of salt taste transduction. Simply tasting various Na+ salts reveals the importance of the anion in the taste which w e call salty. Similar concentrations of NaCI, NaBr, and Nal taste similar, but other sodium salts, such as sodium gluconate, NaHEPES, Na2S04, and sodium citrate, taste different (M. H. Akabas, personal observation; Schiffman ~t al., 1980; Murphy et al., 1981). Beidler (1953) examined the magnitude of the chorda tympani nerve re-

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sponse for a series of 0.1 M sodium salts relative to 0.1 M NaCI. For example, the magnitude of the integrated response to 0.1 M Na2S04was only 90% of the response to 0.1 M NaCl, but since there are two sodium ions per mole of Na2S04 one would have expected a larger response rather than a smaller response. This suggests that the anion does influence the integrated nerve response. A recent study has clarified the effects of anions on the integrated chorda tympani nerve response (Formaker and Hill, 1988). Amiloride only suppresses 70-90% of the integrated chorda tympani nerve activity evoked by NaCl and LiCl (Heck et al., 1984; Brand et al., 1985; DeSimone & Ferrell, 1985). Formaker and Hill (1988) demonstrated that amiloride completely suppressed the integrated chorda tympani nerve response to nonhalide sodium salts such as sodium acetate, NaHCOs and lithium acetate. In addition, they demonstrated that acetate does not have an inhibitory effect. This implies that the residual chorda tympani nerve activity seen after amiloride is due to the halide anion. It raises the possibility that the primary substance(s) that some taste cells detect are halides. Additional studies will be necessary to clarify these anion effects, particularly at the single-axon level to try to identify single axons that are responsive to anions rather than cations. Such studies of anion effects may help to clarify some of the perplexing data regarding electrolyte-induced responses in H fibers.

5 . Summary of Salt Taste Transduction: The Model In summary, the process of salt taste transduction is the most clearly understood of all of the major taste modalities. Evidence from patch clamp recording in taste cells (Avenet and Lindemann, 1988), from whole nerve (Heck et al., 1984; Brand et al., 1985; DeSimone and Ferrell, 1985) and single-fiber (Frank et al., 1983, 1988; Ninomiya and Funakoshi, 1988) recording from chorda tympani nerve, lingual epithelial studies (DeSimone et al., 1981, 1984; Heck et al., 1984; Simon and Garvin, 1985), human psychophysiology experiments (Schiffman et al., 1983, 1986b), and animal behavioral experiments (Frank, 1985) all combine to create a coherent model for the process of salt taste transduction. The transduction process begins in a subpopulation of taste cells that express a Na+ channel in their apical membrane. This channel is blocked by the drug amiloride and is partially permeable to other cations beside Na+. Sodium flux through the channel into the taste cells depolarizes the cells. This depolarization results in the opening of voltage-dependent Ca2+ channels in the basolateral domain of the cell membrane. Calcium entry via these channels results in neurotransmitter secretion, which stimulates the nerves innervating this subpopulation of taste cells. These taste cells

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are innervated by N-type, “sodium specific,” nerve fibers. The partial permeability of the Na’ channel to other cations explains the ability of other nonsodium cations to excite the N-type gustatory axons. The level at which nonsodium cations excite the N fibers is related t o the relative permeability of these cations through the Na+ channel. CNS processing is probably then necessary to analyze the firing rates in N - versus H-type fibers to determine the behavioral taste sensation.

D. SOURTASTE Sour taste is determined by the proton concentration o f a solution. The threshold for sour taste is between pH 3 and 4, a free proton concentration of about 0.1 mM (Pfaf‘f‘mann, 1959; Pfaffmann et ul., 1971 ) . Single gustatory axon analysis of electrolyte taste coding has revealed a more complicated situation than for bitter and sweet. It appears that there are two populations of axons responsive to electrolytes. One, the N fibers, appears relatively finely tuned for Na+ and has a significantly smaller response to nonsodium salts. The other group, the H fibers, responds to HCI, NaC1, anti to certain other salts (Frank, 1973; Frank et al., 1983, 1988; Hananiori et al., 1988). Presumably the CNS deconvolutes the firing patterns of these two axon populations to determine the taste quality of the stimulus (Frank et al., 1988; Hananiori et al., 1988), in much the same manner as color is determined in the visual system by the relative intensity of firing from red, green, and blue cones (Nathans, 1987). T h e elegant study of S. C. Kinnarnon and colleagues (1988) has revealed a potential mechanism for sour taste transduction. Using dissociated mudpuppy taste cells, they used a combination of whole-cell patch clamp recording with one electrode and loose patch recording with a second electrode to map the distribution of different ion channels on the surface of the taste cells. This showed that a class of voltage-dependent potassium channels was localized on the apical domain of the taste cells. These channels were the major determinant of the resting membrane potential. Lowering the pH caused these channels to close, thereby depolarizing the taste cells and presumably opening the voltage-dependent Ca2+ channels leading to neurotransmitter secretion (Kinnamon and Roper, 1988; S. C. Kinnamon et al., 1988).T h e polarized distribution of the potassium channels in situ in mudpuppy lingual epithelium has been confirmed (Roper and McBride, 1989).T h e only caveat to these studies is that all mudpuppy taste cells studied show the same distribution of ion

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channels. It is thus unclear how this would permit the mudpuppy to distinguish other taste modalities. It must be said, however, that there are essentially no behavioral studies available to reveal what mudpuppies taste. It is unknown whether they are capable of distinguishing the same taste modalities as mammals. In a study of dog lingual epithelium it was found that reducing the pH of the solution bathing the apical surface of the epithelium resulted in a reversal of the open circuit potential and the short circuit current. This was due to the appearance of an anion-selective pathway that was not observed in the more physiological pH range around pH 7 (Simon and Garvin, 1985). No mechanism is offered as to how this might result in the stimulation of a limited group of taste cells that would be necessary to create the specificity seen in the single axon recordings. However, it suggests that the mechanism of sour taste transduction in mammals may utilize chloride channels rather than potassium channels.

E. THEUMAMI TASTE The taste of monosodium glutamate (MSG), referred to as the “umami” taste, has been a subject of intense discussion and investigation. T h e debate centers around the issue of whether the umami taste is a separate, distinct taste modality like bitter, sweet, sour, and salt. The subject has been extensively reviewed (Yamaguchi, 1979; Kawamura and Kare, 1987). Attempts to resolve the issue in animals have provided conflicting results. Some of the disagreements may arise from species differences, but it is unclear whether this explains all of the divergent results. Attempts to determine whether animals can distinguish MSG from NaCl using behavioral conditioned taste avoidance experiments have suggested that the ability to make a distinction is dependent on the species studied. Rats and hamsters showed little or no ability to distinguish the two salts (Yamamoto et al., 1985, 1988). Mice are reported to be able to distinguish MSG from NaCl (Ninomiya and Funakoshi, 1989a). Interestingly, this ability is dependent on the integrity of the glossopharyngeal nerves. Bilateral sectioning of the glossopharyngeal nerve ablated the mouse’s ability to distinguish MSG from NaCI; however, sectioning the chorda tympani nerves had no effect on the ability to diui iiguish between the two (Ninomiya and Funakoshi, 1989a). In a human psychophysiological experiment, the ability to perceive the umami taste seemed to localize in the posterior third of the tongue, which is innervated by the glossopharyngeal nerve (Halpern, 1987). Single-axon recordings from the mouse chorda tympani and glosso-

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pharyngeal nerves, revealed the existence of MSG-best fibers in the glossopharyngeal nerve. The response of the MSG-best fibers was strongly potentiated by 10 mlll disodiuni 5' guanylate (GMP) (Ninomiya and Funakoshi, 1989b). N o information is available on the transduction mechanism of the umami taste. Further work will I)e necessary to resolve the species differences and to elucidate the mechanism of transduction in MSG-tasting species.

F. AMINOACID- r A S T E IN

CATFISH

Catfish have been a useful system in which to study taste transduction because the body and barbels of catlish are covered with taste buds. Due to the large number of taste buds it has been possible to perforni biochemical studies of the transduction systems (Bryant et d., 1989: Kalinoski et al., 1989). Electropliysiological sludies of the nerves inner\xing these taste buds indicated that amiiio acids elicited large integrated nerve responses. These studies suggested that there were at least two distinct receptor systems, one specific 1 0 t 1.-arginine and the other more general, responding to L-alanine, L-serine, I.-threonine, arid glycine ((hprio, 1975, 1978; Caprio and Byrd, 198.2). LJsinga plasnia membrane preparation derived from the taste epithelia, a high-affinity binding site for 1.-alanine with a Kdaf,[, about 5 p1%1was identified (Krueger and (hgan, 1976; Cagan, 1979). Further studies of this site demonstrated that the binding showed enantiomeric specificity, preferring the i.-isonier by a factor of about 10 (Brand et d., 1987). A monoclonal antibody (MAb). made against the plasnia nieinbrane preparation, was found that inhibited the binding of L-alanine to its high affinity site (Goldstein and (lagan, 1982). The binding affinity of' ~.-alaninefor its receptor- in the plasma membrane preparation was uriaffec-ted by the addition of' G T P 01- its nonhydrolyzable analogs to the in(-ubation solution (Bruch and M i noski, 1987).This suggests that (;-proteins are not involved in the alanine transduction pathway, because addition of G T P usually alters the binding affinity of substrate for C;-protein-coul)led receptors (Cerione et ul., 1'384; Gilnian, 1984, 1987). Further- studies are in progress to define the second messengers involved in the I.-alanine transduction process (Kalinoski ~t al., 1989). Studies of the L-arginine receptor have suggested that it may function as a ligand-gated ion channel (Teeter et al., 1989). To study the ion channels that are present in a ~nembranevesicle preparation one can form a lipid bilayer either in the Lip of a patch clamp pipette (Coronado

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and Latorre, 1983) or in a small hole in a Teflon partition separating two aqueous compartments (Finkelstein, 1974; Miller, 1986). Following incorporation of the plasma membrane vesicles into the lipid bilayer, one can study the electrophysiological properties of any ion channels that were inserted into the lipid bilayer (Miller, 1986). After catfish taste epithelium plasma membrane vesicles were incorporated into a lipid bilayer, addition of L-arginine to the aqueous compartment on one side of the bilayer induced the opening of cation-selective ion channels with a conductance of about 40 pS. D-Arginine and L-alanine did not open the channel. T h e channel was equally selective for Na+ and K + (Teeter and Brand, 198713; Teeter et al., 1989). How this channel participates in the transduction process is unclear at present. Because pond water has a very low Na+ concentration, following the opening of this channel the major ion flux should be K+ efflux, which would hyperpolarize the cells. Further studies will be necessary to elucidate the mechanism of transduction in this system.

IX. Summary

The application of new techniques to the study of taste cells has revealed much about both the basic physiology of these cells and also about the mechanisms of taste transduction. T h e taste cells are electrically excitable cells with a variety of voltage-dependent ion currents. These ionic currents have an important role in the transduction of salt taste in mammals and frogs. In mudpuppies different ion channels are involved in the transduction of acidic-sour stimuli. T h e role of ion currents in the transduction of sweet taste is less clear. Some proposed mechanisms suggest an important role for ion currents and others suggest that the transduction process may be a biochemical event involving cell surface receptors and intracellular second messengers, possibly CAMP.T h e transduction of bitter taste seems to be a biochemical event involving cell surface receptors and intracellular second messengers in the inositol trisphosphate pathway. Thus, one cannot talk about “the mechanism” of taste transduction. Different taste modalities are transduced by different mechanisms. A corollary to this is that taste cells are not a homogeneous population of cells. In order to provide animals with the ability to discriminate between different taste modalities the taste cells consist of distinct subpopulations of cells based on their primary taste modality. The primary taste modality in a given cell is determined by the receptors and trans-

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duction mechanism(s) expressed in that cell. Evidence suggests that modality-specific receptors are expressed in a segregated manner in distinct subpopulations of taste cells. Secondary responses observed in gustatory axons may arise due to a lack of absolute specificity in the transduction processes and nonspecific effects of low pH and high ionic strength and osmolarity on the taste cells. An interesting area for future work will be to elucidate the niechanism(s) by which basal cells become committed to a given taste modality and how the gustatory neurons influence this process of differentiation. T h e involvement of the gustatory neurons is critical as they must svnapse with taste cells of the correct taste modality to preserve the integrity o f the information transferred to the CNS. This process of synaptogenesis is presumably mediated by the expression of taste-modality-specific, cell surface antigens on the basolateral domain of a taste cell and receptors on the appropriate neurons, but much work will be necessary to elucidate this process. Hopefully the application of techniques of molecular biology and immunology to the study of taste cells will help to elucidate these and other problems in our understanding of the processes of taste transduction. Acknowledgments

I thank Dr. Qais Al-Awqati foi- m ; i i i y crilightening discussions and for his helpfiil comments on this manuscript. M.1L.A. is thc recipient of an American Heart Association Clinician-Scientist Award. This work wiis supported in part by grant BXS-8808OY8 from the National Science Foundation.

References

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QUINOXALINEDIONES AS EXCITATORY AMINO ACID ANTAGONISTS IN THE VERTEBRATE CENTRAL NERVOUS SYSTEM By Stephen N. Davies Division of Physiology School of Biomedicol Sciences Marischal College Aberdeen AB9 lAS, U. K.

and Graham L. Collingridge Department of Pharmacology School of Medical Sciences University of Bristol Bristol BS8 lTD, U. K.

1. Introduction 11. Binding

111. Release IV. Pharmacology A . Semiquantitative Methods B. Quantitative Methods C. Effects on NMDA Responses D. Effects on Glutamate- and Aspartate-Induced Responses E. Effects on IPS Turnover and C:aZf Mobilization F. Effects on ~-AP.l-lnducedResponses G. Overview V. Excitotoxicity VI. Synaptic Physiology A. Hippocampus B. Spinal Cord C. Other Areas VII. Conclusions References

1. Introduction

There now seems to be common agreement that excitatory amino acid receptors (EAARs) are the prime mediators of fast excitatory synaptic transmission in the vertebrate central nervous system. These 28 1 I N T E R N A T I O N A L REVIEW OF NEUROBIOLOGY, VOL. 32

Copyright D 1990 by Academic l’ress, In< 411 right5 of repmdurtion i i i d r y form rescrwd.

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STEPHEN N . DAVIES A N D GRAHAM L. COLLINGRIDGE

receptors are currently classified into five subtypes, which are named N-methybaspartate (NMDA), c~-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), kainate, L-2-amino-4-phosphonobutanoate (L-AP4or L-APB),and (glutamate) metabotropic (see Lodge and Collingridge, 1990). T h e AMPA receptor is also known as the quisqualate receptor; however, quisqualate is not specific for this receptor and so AMPA is now the preferred name. AMPA and kainate receptors have sometimes been referred to collectively as non-NMDA receptors. Of these, the most intensively studied has been the NMDA receptor. This epidemic of NMDA-related research was induced by the availability of selective and potent NMDA antagonists [e.g., D-a-aminoadipate (AP5, also called D-2-amino(DAA), D-2-amino-5-phosphonopentanoate 5-phosphonovalerate or APV), and 3-(2-carboxypiperazine-4-y1)propyl- l-phosphonate (CPP)] but has been maintained by the results of experiments that have implicated NMDA receptor activation in such processes as synaptic plasticity, epilepsy, ischemic cell death, and neurodegenerative disorders. This work has been reviewed extensively elsewhere (e.g., Monaghan et al., 1989; Watkins and Collingridge, 1989; Bowery et al., 1980). By contrast, selective antagonists for any of the other EAARs have not been available and this lack of appropriate tools has stunted research into their roles in synaptic transmission. Nonselective antagonists such as cis-2,3-piperdine dicarboxylic acid (PDA), y-D-glutamylglycine (DGG), and kynurenic acid have been useful in identifying EAAR-mediated pathways. Glutamate diethyl ester (GDEE) and y-D-glutamylaminomethyl sulphonate (GAMS) have been reported to discriminate AMPA o r AMPA and kainate receptors, respectively, from NMDA receptors; however, their potency and selectivity are not great or reliable enough to make them particularly useful antagonists. This situation has, however, been altered due to the development by Honor6 and colleagues (1987, 1988) of the quinoxalinediones, the first fairly potent antagonists at certain EAARs other than NMDA receptors. Here we review the work that evaluates quinoxalinediones as pharmacological tools and highlight some of the advances that have been made using these compounds. The two drugs that have mainly been used so far (DNQX, formerly FG 904 1) and are 6,7-dinitro-quinoxaline-2,3-dione 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX, formerly FG 9065) (Fig. 1) and therefore we concentrate on these. However, other analogs are now becoming available that show differing selectivities and potencies and will undoubtedly become widely used agents in the near future (Watkins et al., 1990).

283

DNQX

CNQX

FIG. 1. The structures of 6,7-diiiitro-qiiit~oxaline-2.3-dioti~ (1)NQX) and ti-cvano-Triitro-quinoxaline-2.3-dione (CNQX).

II. Binding

DNQX and CNQX inhibit binding to the AMPA site (as identified hy [‘HIAMPA displacement) with ICs0 values of about 500 and 300 nM, respectively (Honore et d.,1988). These affinities are on a par with that of the endogenous agonist r>-glutaniate(ICSO, 600 nM) and are over 200 times less than those of the previously available non-NMDA antagonists GAMS or GDEE. DNQX and CNQX also inhibit binding to the C;a“-sensit.ive, highaffinity kainate site (as identified by displacement with [’Hlkainate) but with potencies four-five times lower than for the AMPA site ( I C i o values of 2 and 1.5 p M , respectively). By comparison, DNQX and CNQX are both about 80 times less effective at displacing binding to NMDA sites (as identified by [“HICPP displacement) (IC50 values of 40 and 25 p M , respectively). Both quinoxalinediones were also found to be “relatively inactive” (i.e., 1CSOvalues over 25 p M ) in protocols designed io test for displacement from 5 H T , a-noradrenergic, muscarinic, dopamine D I or D 2 , opiate, benzodiazepine, GABA, and st rychnine-sensitive glycine binding sites. CNQX has, however, been shown to displace [“Hlglycine from gerbil (Romanelli et al., 1989), guinea pig (Lester et ul., 1989). and rat (Pellegrini-Giampietro et al., 1989) brain tissue with an IC50 of about 6 p M ; the significance of this will be discussed later (see Section IV,C;). T h e evidence thus far would suggest that CNQX and DNQX have affinity ratios for the AMPA, kainate, and NMDA binding sites of approximately 1 : 5 : 80. In rat cortical membranes, [:iH]CNQX binds to a single site (of rnolecular target size -50 kD) with a K l , of 39 nM (Honore P t ul., 1989). Interestingly, AMPA shows biphasic inhibition of this binding. On the basis of this and other evidence, it has been suggested that AMPA receptors exist in two interconvertible states; conformation A has higher affin-

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STEPHEN N . DAVIES A N D GRAHAM L. COLLINGRIDGE

ity for AMPA and quisqualate while conformation B has higher affinity for kainate. Both conformations have similar affinities for CNQX, GAMS, and L-glutamate. Chaotropic ions, such as SCN-, shift the equilibrium towards the higher-affinity site. The regional distribution of [3H]CNQX binding parallels that of [‘HIAMPA binding, except for higher antagonist binding in the cerebellum and spinal cord. [3H]CNQX is not displaced by a large series of compounds active in other neurotransmitter systems.

Ill. Release

Release experiments have been used to provide a bioassay of the selectivity of the quinoxalinediones against the EAAR subtypes. Drejer and Honore (1988) reported that CNQX and DNQX inhibit the release of [3H]GABA from cultured cortical neurons stimulated by quisqualate, kainate and NMDA with pA2 values of 6.O,5.7, and 5.3 (CNQX) and 6.2, 5.9, and 5.4, (DNQX). These data show rather less selectivity by CNQX and DNQX between NMDA and non-NMDA evoked responses than was suggested by the binding results, giving ratios of 2.5X and 3.3X, respectively. Experiments by the same authors using excitatory amino acidinduced 22Naefflux from striatal slices state that DNQX was 1OX less effective against NMDA than against quisqualate or kainate (Drejer and Honore, 1988). This difference between pA2 values obtained from cultured cells and potency estimations from striatal slices may be related to a higher concentration of glycine present in the slice preparations (see Section IV,C). This notion is supported by the observation that increasing the glycine concentration from 0.1 to 10 pM decreases the potency of DNQX in blocking NMDA-induced GABA release from cultured cortical neurons (Drejer et al., 1989).

-

IV. Pharmacology

A. SEMIQUANTITATIVE METHODS

Honore and colleagues ( 1988) originally reported pharmacological results from microiontophoretic administration of agonists and antagonists in rat spinal cord in uizio. They suggested that DNQX and CNQX

QUINOXAL1Nb.I)IONES AS EAA ANTAGONISTS

285

could be used to distinguish between responses evoked by NMDA as opposed to quisqualate or kainate but did not discriminate well between responses to quisqualate and kainate (Fig. 2). A comparison between the effects of CNQX and GAMS showed that they had approximately equal selectivities between responses to NMDA, quisqualate, and kainate, but a comparison of ejection currents and strengths of solution in the pipettes suggested that GAMS was in the order of 100 times less potent than CNQX. A similar comparison between DNQX and CDEE showed that GDEE was not only far less potent but also exhibited little selectivity between responses to NMDA and quisqualate, and consistently increased responses to kainate. Qualitatively similar results have been reported using rat spinal cord slices where bath application of CNQX (5-7 pM) blocked responses of dorsal horn neurons to pressure-ejected NMDA to a lesser extent than responses to AMPA, kainate, or quisqualate (Gerber et al., 1989) In hippocampal slices, responses of CA 1 neurons to iontophoretic ejection of quisqualate, kainate, and NMDA were blocked by bath application of CNQX with IC50 values of 4.8, 1.2, and >25 pM, respectively (Andreasen et al., 1989). A similar study performed on voltage-clamped hippocampal pyramidal neurons 271 uztro states that 10 p M CNQX selectively reduced currents evoked by 10 pM quisqualate (68% depression) compared to those evoked by 200 nM kainate (22% depression) or 20 pM NMDA (34% depression) (Neunian et nl., 1988). CNQX

i FIG. 2. CNQX preferentially reduces the excitation of a rat dorsal horn neuron by iontophoretic ejection of quisqualate and kainate but not NMDA. The ordinate shows the firing rate of a single neuron in response to cyclical ejection of quisqualate (Q), kainate (K), and NMDA (N). In the center panel continuous ejection of CNQX reduces the responses to quisqualate and kainate approximately equally. Partial recovery is shown in the right-hand panel 4 min later. (From Honore et al., 1988. Copyright 1988 by the AAAS.)

286 B.

STEPHEN N . DAVIES AND GRAHAM L. COLLINGRIDGE

QUANTITATIVE

METHODS

T o obtain accurate estimates of the potency and selectivity of quinoxalinedione antagonists, grease-gap recording methods have been employed. These offer the advantage that accurate dose-response curves can be constructed, thus permitting determination of dose ratios and estimation of pA2 values (Fig. 3). This is particularly important since excitatory amino acids generate different-shaped dose-response curves (Blake et al., 1988b); consequently, measures of antagonism in terms of the percentage depression of the response to a single dose of agonist can be very misleading. Several different preparations have been used to generate pA2 values for CNQX and/or DNQX: rat cortical wedge (Fletcher et nl., 1988), rat hippocampus (Blake et ul., 1989), immature rat spinal cord (Birch et nl., 1989),and frog spinal cord (Fletcher et nl., 1988) (see Table I). In all of these preparations neither CNQX nor DNQX showed any great differentiation between responses to quisqualate, kainate or AMPA (potency difference 1-5 times), but they did discriminate between NMDA and non-NMDA responses (potency difference 10-30 times in the rat and -6 times in the frog). T h e potencies of the quinoxalinediones were comparable giving apparent K , values for quisqualate, kainate, or AMPA receptors in the ranges of 0.63-3.1 pM (CNQX) and 0.25-5.0 /AM (DNQX). In terms of the depolarizing actions of excitatory amino acid receptors, CNQX and DNQX may therefore be thought of as non-NMDA receptor antagonists.

-

C. EFFECTS ON NMDA RESPONSES A significant development in the field of excitatory amino acid research was the finding that glycine potentiates responses to NMDA via a strychnine-insensitive site (Johnson and Ascher, 1987). It seems that the presence of glycine at this site is an absolute requirement for evoking an NMDA receptor-mediated response (Kleckner and Dingledine, 1988). T h e discovery of this site on the NMDA receptor led to the re-evaluation of the mode of action of some existing noncompetitive NMDA antagonists: namely, kynurenate and HA966. Experiments in rat cortical wedges showed that NMDA-induced responses that were depressed by kynurenate or HA966 could be restored by the addition of glycine (Fletcher and Lodge, 1988b). It was proposed that kynurenate or HA966 depressed responses to NMDA by displacing endogenous glycine from

287

QUINOXALINEDIONES AS EA.4 ANTAGONISTS

1

.

A 5

Q 20

1

9

N 20

-

K 20

A 5

1

Q 20

Concentration ( p M )

1.5

-

9

N 20

K 20

I

N 20

-

I

O

K

20

I

20

A 5

concentration ( p M )

-

% X

10

Concentration ( p M )

100

Concentration ( p M )

FIG.3. Antagonism of AMPA, quisqualate, NMDA, and kainate responscs b y CNQX in the rat hippocampus grease-gap preparation. (A) Responses to the agonists in control medium, in the presence of 10 w.44 CNQX, and after washout of.CNQX. (B) Schild plots for antagonism of AMPA (O),quisqualate (A),kainate (M), and NMDA ( X ) h y CNQX. The estimated pA2 values (indicated by the arrows) for AMPA, quisqualate, and kainate are 5.8, 5.9, and 5.9, respectively. (From Blake al., 1989.)

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STEPHEN N . DAVIES A N D GRAHAM L. COLLINGRIDGE

TABLE I PA, VALUES FOR CNQX A N D DNQX AGAINST AMPA, KAINATE,QUISQUALATE, AND NMDA FROM FOURGREASEGAP PREPARATIONS pA2 Values AMPA

Kain

Quis

NMDA

CNQX

5.8

5.5 5.9

6.2 5.9

4.5 4.4'

Rat cortex Rat hippocampus

1 2

DNQX

6.2 -

6.1 6.2 5.3

6.6 5.9

5.1

Rat cortex Rat spinal cord Frog spinal cord

3 1

-

4.5

Preparation

Reference"

1

Key to references: 1. Fletcher et al. (1988);2. Blake et (11. (1989);3. Birch et al. (1989).

' Apparent log K.

its binding site, and that added glycine (or D-serine) in turn displaced kynurenate or HA966, thus restoring the NMDA response. Birch et al. (1988b) reported similar findings for kynurenate in the spinal cord. DNQX and CNQX have also been shown to compete with glycine for this site (Birch et al., 1 9 8 8 ~ (Fig. ) 4) and for a comparable site in the guinea pig ileum myenteric plexus preparation (Pellegrini-Giampietro et al., 1989). The concentration range of the quinoxalinediones over which these effects were observed was 10-100 p M and this (i.e., 10 p M ) may therefore set the limit on the selectivity of DNQX and CNQX. Similar results have been reported using patch-clamp techniques on cultured hippocampal cells (Lester et al., 1989) and cerebellar granule cells (CullCandy and Herron, 1989), in which 2-30 pM CNQX reduced currents evoked by NMDA, an effect reversed by addition of more L-glycine (Fig. 4). Hence, addition of glycine (or D-serine) to the culture medium will enhance the apparent selectivity of the quinoxalinediones for nonNMDA versus NMDA receptor-mediated responses. This effect presumably explains the greater selectivity noted in in vivo or slice preparations (i.e., high glycine levels) than in cultured or dispersed cell preparations (i.e., often low glycine levels). The action of the quinoxalinediones at this allosteric site explains the noncompetitive action of CNQX against responses induced by NMDA (e.g., Birch et al., 1988a; Verdoorn et al., 1989).

D. EFFECTS ON GLUTAMATEAND ASPARTATE-INDUCED RESPONSES

L-Glutamate and L-aspartate are the prime candidates as endogenous transmitters that might act on excitatory amino acid receptors. There

289

QUlNOXALINEDlONES AS EAA ANTAGONISTS

A

10

1

1000

100 NMDA (pM)

NMDA 30 glycine 0

120[

NMDA 30

100 -

80K

NMDA 30

1 g'ycine r

0

2 a

60-

a:

$? 4 0 -

20. 1

10

100

1000

Glycine (M FIG. 4. CNQX inhibits NMDA responses via an action at the allosteric glycine site. (A) Dose-response curves of infant rat hernisected spinal cord to NMDA alone (A),in the presence of 300 pM CNQX (A), in the presence of 300 pM CNQX and 1 mM u-serine ( O ) , and after washout of CNQX before treatment with CNQX and D-serine (M). (From Birch et al., 1988c.) (B) Currents induced in cultured hippocampal neurons by NMDA in the presence of increasing concentrations of glycine and dose-response curves to glycine in the presence of 30 p M NMDA and 30 pM (A),10 pM (O),or absence (W) of CNQX. (From R. A. J. Lester el al., Interaction of (i-cyano-7-iiitroquinoxaline-2,3-dione with the N-methyl-Daspartate receptor-associated glycine binding site, Molecular Phanacoiogy, 35, 565-570, 1989. 0 by the American Society for Pharmacology and Experimental l'heraputics.)

290

STEPHEN N. DAVIES AND GKAHAM L. COLLINGKIDCE

have been attempts to use selective antagonists to try to establish what, if any, receptor preference these endogenous agonists may show. CNQX, like AP5 (Davies et al., 1981; Collingridge et al., 1983a), was found to depress responses to L-aspartate to a greater extent than those to L-glutamate (Davies et al., 1988). This was true whether the drugs were applied microiontophoretically to the spinal cord, or by bath application to rat cortical wedges. Furthermore, it was evident that the combined application of CNQX and an NMDA antagonist, which was sufficient to abolish responses to quisqualate, kainate, and NMDA, still left a substantial response to L-glutamate. It therefore appears that exogenously applied L-glutamate may have an additional depolarizing action via a site other than the conventional AMPA, kainate, and NMDA receptors. This receptor does not seem to contribute to synaptic responses in any obvious way since a combination of AP5 and CNQX (or DNQX) blocks synaptic transmission at presumed excitatory amino acid-mediated synapses (see Section V1,A). E. EFFECTS ON IPS TURNOVER AND Ca2+ MOBILIZATION In addition to its depolarizing action via the AMPA receptor, and its effect at the L - A P site, ~ quisqualate also acts on a “metabotropic receptor” that stimulates accumulation of inositol phosphates and mobilizes intracellular Ca2+. This effect is not mimicked by AMPA and is therefore mediated by a distinct receptor from that responsible for the depolarizing action of quisqualate. CNQX (20 p M ) has no significant effect on quisqualate-induced inositol phosphate accumulation (Monaghan et al., 1989; Palmer et al., 1988; Godfrey and Taghavi, 1989). The suggestion that quisqualate activates two separate receptors is strengthened by the observation that quisqualate increases Ca2+levels (as measured with fura-2) in hippocampal neurons in a two-stage manner involving a transient spike, followed by a longer-lasting plateau (Murphy and Miller, 1989). Only the plateau was blocked by removal of external Ca2+ or by addition of 10 p M CNQX. Thus, it appears that quisqualate mobilizes Ca2+ from internal stores to give the transient phase via a CNQX-insensitive (metabotropic) receptor and induces Ca’+ influx from the external medium via an indirect mechanism involving activation of voltage-gated Ca2+ channels by depolarization via the CNQX-sensitive (AMPA) receptor. F. EFFECTS ON L-AP4-INDUCED RESPONSES

L-Glutamate binds to several sites in brain tissue and one of these, that displaced by L - A P ~has , been considered to represent an uptake site.

L - A P on ~ its own does not depolarize tissue, but tissue primed by expo. sure to quisqualate does exhibit depolarizing responses to L - A P ~Sheardown (1988) reported that CNQX (5 p M ) blocked these primed re, that they are mediated by AMPA receptors. sponses to L - A P ~suggesting This adds credence to the hypothesis that quisqualate gets taken up into a pool that is sensitive to L-AP4. Subsequent administration of r.-AP4 then releases quisqualate by heteroexchange and this depolarizes neurons via an action on AMPA receptors. This interpretation has recently been questioned by Lodge ~t (11. (see Lodge and Zeman, 1989)on the basis of t w o observations. In their hands (1) the primed L - A P response ~ does not fade with repeated adniinistra~ causing release from a finite pool tion as might be expected if L - A P was of previously taken-up quisqualate, arid (2) glutamate, which is presumed to be taken u p by the same mechanism as quisqualate. does not mimic the priming effect (though this may be a result of glutamate being rapidly incorporated into nonreleasable metabolic pools). At present this matter is unresolved.

G. OVERVIEW In all the brain areas so far studied it appears that the quinoxalinediones do not discriminate between depolarizing responses evoked by AMPA, quisqualate, or kainate. Therefore, at present we believe that AMPA, kainate, and quisqualate exert their depolarizing actions in these tissues via the AMPA receptor. T h e discrepancy between the selectivity of the quinoxalinediones for AMPA versus kainate in binding and pharmacological studies resides in the existence of two kainate binding sites: a low-affinity site, which probably corresponds to the AMPA binding site, and a second high-affinity site at which AMPA, CNQX, and D N Q X are ineffective (Honore et al., 1986; Watkins et ul., 1990). The physiological function (if any) of this high-affinity kainate binding site has yet to be demonstrated. For the rest of the review w e shall be concerned only with NMDA and AMPA receptors.

V. Excitotoxicity

Given sufficient exposure, excitatory amino acids become excitotoxic to nervous tissue. There is now a vast amount of literature indicating that both competitive and noncompetitive NMDA antagonists can offer some protection against excitotoxic damage (see Watkins and Collingridge,

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STEPHEN N . DAVIES A N D GRAHAM L. COLLINGRIDGE

1989). However, much less is known about the neuroprotective potential of AMPA antagonists. In the hippocampus CNQX (10 pA4) showed no protection against neuronal damage (as assessed after 90 min of recovery) induced by 30-min exposure to quisqualate (30 p M ) if it was present only during quisqualate exposure. If, however, CNQX was also present (or even only present) during the recovery period, then it did show some protection (Garthwaite and Garthwaite, 1989).The authors suggest that the excitotoxicity is triggered by a CNQX-insensitive mechanism (possibly the metabotropic receptor), but that the delayed damage takes place via a CNQX-sensitive mechanism. VI. Synaptic Physiology

A. HIPPOCAMPUS 1. The Schaffer Collateral-Commusurnl Pathway The most extensively studied pathway in the brain with respect to EAAR pharmacology is the Schaffer collateral-commissural pathway (SCCP), which provides a monosynaptic connection between CA3 and CA 1 pyramidal neurons. Schaffer collateral-commissural fibers also excite local circuit GABAergic inhibitory neurons, which impinge upon CA1 neurons, and CA 1 cells themselves activate recurrent GABAergic interneurons. Thus, stimulation of the SCCP elicits a complex EPSPIPSP sequence in CA1 neurons. Early studies, performed using hippocampal slices bathed with a normal ACSF medium (typically containing 1-4 Mg2+), showed the EPSPs evoked by low-frequency stimulation of the SCCP were reduced by nonselective excitatory amino acid antagonists such as DGG, kynurenate, and l-(p-chlorobenzoyl)-piperazine-2,3-dicarboxylate (pCBPzDA), but not by selective NMDA antagonists,such as AP5 (Collingridge et al., 1983b; Ganong et al., 1986). Therefore, it seemed likely that the EPSP was mediated by a non-NMDA type receptor. The use of quinoxalinediones has reinforced this idea. Under similar conditions, CNQX and DNQX (1 - 10 p M ) substantially reduced (by 50- 100%) the EPSP recorded intracellularly (Collingridge et al., 1988; Neuman et al., 1988; Andreasen et al., 1988, 1989; Kauer et al., 1988; Davies and Collingridge, 1989) o r extracellularly (Blake et al., 1988a; Fletcher et al., 1988; Fletcher and Lodge, 1988a; Muller et al., 1988; Herreras et al., 1989). These effects were not accompanied by any change in the input resistance or membrane potential of the recorded cell, nor by

QUINOXALINEDIONES AS EAA ANTA(;ONISTS

293

any change in the size of the presynaptic fiber volley. The higher concentrations of quinoxalinedione (e.g., 10 p M CNQX) seem to abolish the AMPA receptor component; however, there is, or there appears on increasing the stimulus intensity, a small residual component that has the appropriate kinetics, voltage-, Mg2+-,and AP5-sensitivity for it to be an NMDA receptor-mediated EPSP (Collingridge et al., 1988; Kauer et al., 1988; Davies and Collingridge, 1989; Andreasen et al., 1989). This component has been studied using patch-clamp techniques (Randall et ul., 1990); the NMDA receptor-mediated synaptic current has a relatively slow rise time and can last for over 1 sec. In the presence of 1 mM Mg2+ there is a region of negative slope conductance from about -35 mV to potentials more negative than E K . It must be stressed that under standard experimental conditions (i.e., in the presence of at least 1 mM Mg‘+ and functional synaptic inhibition and at a membrane potential near rest) the NMDA receptor component of the EPSP evoked by low-frequency stimulation becomes evident as the AMPA receptor-mediated component is blocked. A comparable NMDA receptor component of the EPSP is not present in control responses before the addition of CNQX. This point is illustrated in Fig. 5. The precise reasons for the appearance of an NMDA receptor-mediated EPSP upon blockade of the AMPA receptor-mediated EPSP are not known. However, one likely factor relates to the effects of quinoxalinediones on synaptic inhibition, a point to which we now turn. CNQX and DNQX have been reported to have variable effects on IPSPs. In our experience the extent to which CNQX blocks IPSPs is related to the distance separating the recording and stimulating electrodes. With a large (e.g., > 1 nim) separation we find that IPSPs are blocked together with EPSPs while with a small separation (e.g., c 0 . 5 mm) IPSPs are little affected or unaffected by CNQX. We interpret this to mean that with a large separation IPSPs are polysynaptic in origin and that the excitation of the inhibitory interneurons involves a CNQXsensitive (i.e., AMPA) receptor. In contrast, with a small separation the IPSPs are monosynaptic due to direct stimulation of the inhibitory neurons. Although with a large electrode separation the polysynaptic IPSP can be blocked by CNQX, if the stimulus intensity is increased an IPSP still curtails the CNQX-insensitive (i.e., NMDA receptor-mediated) EPSP (Andreasen et al., 1988; Davies and Collingridge, 1989). Since, like the CNQX-insensitive EPSP, this IPSP is blocked by AP5 we believe that there are NMDA receptors present on inhibitory interneurons, the activation of which can be sufficient to drive the inhibitory cells. The resultant polysynaptic IPSP (i.e., evoked in the presence of CNQX) is like

294

STEPHEN N . DAVIES A N D GKAHAM Id,COLl.IN(;RIDGE

Control

A

CNQX

__I

A

5mV[

B

A

C

10 msec

I l * r r r y -

4

A

FIG.5. CNQX unmasks an NMDA receptor-mediated component of synaptic transmission in the Schaffer collateral-commissural pathway of rat hippocampus. (A) Sequence of , in 10 CNQX experiment showing control response, response in 20 /AM D - A P ~response after washout of AP5, and response in CNQX plus AP5. (B) Superimposed and subtracted records showing no effect of AP5 in control medium. (C) Superimposed and subtracted records showing an effect of AP5 in CNQX-containing medium. (Unpublished data from G . L. Collingridge and S. N. Davies.)

conventional polysynaptic IPSPs observed in control medium in that it has an early component blocked by picrotoxin (Andreasen et al., 1988; Davies and Collingridge, 1989) and a picrotoxin-insensitive late component (Davies and Collingridge, 1989). The depression of polysynaptically mediated synaptic inhibition probably accounts, at least in part, for the magnification of the NMDA receptor-mediated component (see Fig. 5). Thus, under control conditions, inhibition acts to hyperpolarize cells into a region such that NMDA channels are appreciably blocked by extracellular Mg2+. Therefore by reducing the inhibition CNQX lessens this voltage-dependent Mg'+ block, a situation analogous to that obtained when IPSPs are depressed by a convulsant drug (Herron et al., 1985; Dingledine et al., 1986). Perhaps significantly, one paper that reported that the IPSPs were not blocked by

295

QUINOXAL1NEI)IONES AS EAA ANTAGONISTS CA 1 PYRAMIDAL CELL APICAL DENDRITE

0

9

SCHAFFER COLLATERAL-COMMISSURAL

Picrotoxin

\I

GABAERGIC INHIBITORY INTERNEURON

FIG. 6. A scheme to illustrate probable locations of AMPA (A), NMDA (N), C;ABA,\ (GA), and GABA, (C,) receptors o n (:A 1 pyraniidal cells and GABAergic interneurons. T h e sites of action of some drugs are also shown.

CNQX (Neuman et al., 1988) does not mention any latent AP5-sensitive component. In summary, either AMPA or NMDA receptors in isolation have the capacity to mediate the synaptic excitation of both CA1 pyramidal cells and inhibitory GABAergic interneurons. T h e relative contribution of the two receptors to, and hence the effects of CNQX (or DNQX) on, the overall synaptic response is highly dependent on the experimental conditions employed. Figure 6 illustrates some possible locations of amino acid receptors in the CA1 region of the hippocampus, based on the studies described above.

2. Peforant Path-Dentate Gyrur Pathway lntracellular recordings from cells of the dentate gyrus showed that 2 p M CNQX inhibited the EPSP evoked by stimulation of the perforant path by about 50% (Lambert and Jones, 1989). Concentrations of 510 pM CNQX left a residual EPSP that comprised 10-20%, of the control EPSP and had the appropriate Mg2+-,voltage-, and AP5-sensitivity to show that it was mediated by NMDA receptors.

296

STEPHEN N . DAVIES A N D GRAHAM L. COLLINGRIDGE

3. Mossy Fiber-CA3 Pathway In one study CNQX (2-5 p M ) “completely and reversibly blocked” the intracellular EPSP but spared the IPSP evoked in CA3 cells by stimulation of the mossy fibers (Neuman et al., 1988). No mention is made of any CNQX-resistant EPSP. In another study, CNQX reduced the EPSPIPSP sequence in a dose-dependent manner and left a CNQX-resistant EPSP that was consistently smaller than that seen in area CA1 (Andreasen et al., 1989). The smaller (or absence of a) CNQX-resistant EPSP correlates with the lower density of NMDA receptor binding in CA3 as compared to CA1 (Cotman et al., 1987). Field potentials evoked from CA3 cells by stimulation of the mossy fibers were “reversibly and greatly” reduced by 1-5 p M CNQX. Furthermore, 2 pM CNQX reduced both the intensity and frequency of spontaneous interictal bursts (to 58 and 42% of control, respectively) and reduced the frequency of spontaneous miniature EPSPs, induced by perfusion with high K+ medium (Chamberlin and Dingledine, 1988). Spontaneous electrographic seizures evoked in area CA3 of hippocampal slabs by repeated stimulus trains were completely blocked by 10 p M CNQX, while those recorded in M$+-free medium were only abolished by a combination of CNQX and AP5 (Anderson and Coan, 1989).

4. Kainic Acid-Lesioned Hippocampw Kainic acid lesioning results in epileptiform responses from CA1 neurons due to loss of functional inhibition and the majority of this response is blocked by AP5, indicating that it has a substantial NMDA receptor-mediated component. Somewhat surprisingly CNQX (5 p M ) blocked the entire response (Wheal et al., 1989). This suggests that either the NMDA receptor-mediated component is somehow dependent on the AMPA receptor-mediated component (unlike in control slices), or that under these conditiions CNQX is blocking NMDA receptor-mediated responses. 5. Organotypic Culture In organotypic cell cultures of the hippocampus, 10 p M CNQX depressed both spontaneous epileptiform activity and evoked EPSPs (by about 60 and 90%, respectively) with no effect on input resistance or membrane potential (McBain et al., 1988). The residual activity and , that AMPA recepEPSPs were abolished by 30 p M D - A P ~suggesting tors make a major and NMDA receptors make a minor contribution to synaptic transmission between these cells.

QUINOXALINEDlONES AS EAA ANTAGONISTS

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6. Hippocampal Summary

Studies on the Schaffer collateral-commissural CA 1 pathway have provided supporting evidence for a dual component EPSP at these synapses: There is a fast AMPA and a slower NMDA receptor-mediated component. Because of its slow nature and its voltage dependence, in the normal experimental situation the NMDA-receptor-mediated component is rapidly turned off by the concurrently activated IPSP. Therefore, in the control situation AP5 has little or no effect on the EPSP. In the presence of CNQX a latent component is unmasked, probably because most of the polysynaptically evoked IPSP has been blocked. This concept of a dual component EPSP, with a slower NMDA component that can be regulated by the extent of inhibition, is useful when interpreting results from other areas of the central nervous system. In principle, recordings from the dentate gyms and CA3 have shown results that are consistent with those from CA1, with the possible absence of any residual NMDA receptor-mediated EPSP in the mossy-fiber pathway in CA3 region.

7 . Long-Term Potentiation Long-term potentiation (LTP) is a persistent form of synaptic plasticity that has received considerable attention as a possible neural substrate of learning and memory (Bliss and Lynch, 1988). It is most often studied in the SCCP of the hippocampus, where it can be induced by, for example, high-frequency stimulation of the afferent fibers. Using AP5 it was established that in this pathway NMDA receptors are required for the induction but not the maintenance of LTP (Collingridge et al., 1983b); that is, transient activation of NMDA receptors during the tetanus leads to the induction of LTP, but NMDA receptors do not contribute to the potentiated response. Using CNQX or DNQX it has now been possible to determine the contributions of AMPA receptors to the induction and maintenance of LTP: 1. Are AMPA receptors required for induction of LTP? The way the experiment has been approached is to use two separate inputs onto a population of CA1 neurons and to tetanize just one in the presence of a quinoxalinedione while using the other input as a control to monitor recovery. T w o groups (Muller P t al., 1988; Kauer et al., 1988) report that tetanization of the CNQX-DNQX-insensitive EPSP resulted in little or no potentiation of this component itself but that following washout of the quinoxalinedione there was potentiation of the response evoked by the tetanized pathway. Thus, AMPA receptors d o not seem to be required for the induction of LTP.

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2. What is the role of AMPA receptors in the maintenance of LTP? Several groups have shown that potentiated and control responses have a similar sensitivity to quinoxalinediones (Davies et al., 1989; Muller et al., 1988; Kauer et al., 1988), indicating that AMPA receptors mediate potentiated responses. It has been reported that the NMDA receptor-mediated component, made more visible by recording in low Mg2+ (Muller et al., 1988) or by depolarizing intracellularly recorded cells (Kauer et al., 1988), is of similar sizes in control and potentiated responses. This evidence, and the failure of NMDA receptor-mediated EPSPs recorded in the presence of CNQX to potentiate (Muller et al., 1988; Kauer et al., 1988) (but see Collingridge and Davies, 1989), suggests that LTP is maintained by a selective increase in transmission via AMPA receptors. Taking these results at face value, they suggest that NMDA receptors initiate and AMPA receptors maintain LTP. However, the indication that LTP may comprise two or more phases with different mechanisms (Davies et al., 1989) may require a more complex model than is provided for by this simple scheme.

B. SPINAL CORD

Honore et al. (1988) reported that selective iontophoretic currents of ketamine or CNQX had no effect on synaptic excitation of dorsal horn neurons elicited by electrical stimulation of the cutaneous receptive field (though nonselective currents of CNQX did reduce synaptic responses). However, this may well reflect the inability of locally ejected drugs to reach synaptic inputs on distal dendrites rather than the lack of involvement of excitatory amino acid receptors in transmission. In an in uitro preparation of the immature rat spinal cord to which the antagonists were bath-applied (and therefore the problem of drug access did not occur) positive results with CNQX were found. CNQX (2-10 p M ) reversibly depressed or abolished the ventral root reflex evoked by stimulation of the dorsal roots (Long and Evans, 1989). In contrast AP5 at concentrations u p to 100 pM had no significant effect on the early (presumed monosynaptic) part of the reflex but did reduce a later component (Long, 1989). This suggests that transmission of the early part of the reflex evoked by the primary afferent fibers is mainly via AMPA receptors, while NMDA receptors contribute only to the late component. Convergent conclusions have been reached using intracellular recordings from dorsal horn neurones of rat spinal cord slices. In this

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preparation CNQX (5-7 pM) or AI’5 reduced the fast EPSP evoked by low-frequency dorsal root stimulation by 90 and 30%1,respectively. Conversely, the slow EPSP evoked by high-frequency stimulation exhibited greater sensitivity to AP5 than to CNQX (Gerber P t al., 1989). In another study intracellular recordings were made exclusively from cells of the substantia gelatinosa, in which monosynaptic EPSPs evoked by C fibers were found to be less sensitive to CNQX than those evoked by A delta fibers (Yoshimura and Jessell, 1989). No results with APV were presented but it is possible that the slower depolarization evoked by C fiber stimulation may contain a larger NMDA receptor-mediated component. Finally, CNQX (10-20 p M ) blocked primary afferent depolarization (recorded as a dorsal root potential evoked by stimulation of a neighboring dorsal root) by 37-88%, while 20-100 p M CPP reduced it by 4-1556 (Evans and Long, 1989). This would suggest that AMPA receptors make a greater contribution than NMDA receptors to the pathways mediating this form of presynaptic inhibition.

C. OTHERAREAS In the thalamus excitatory amino acid receptors are thought to mediate the responses of ventrobasal neurons evoked by stimulation of somatosensory afferents. Iontophoretic administration of NMDA antagonists reduced a late part of the long train of action potentials evoked by prolonged air jet stimulation of‘ the whiskers but had less effect on the short discrete responses evoked by short air jet puffs or by electrical stimulation of the whisker pad (Salt, 1987). CNQX, at currents that selectively inhibit quisqualate and kainate responses, reduced synaptic responses evoked by either long or short puffs of the airjet (Salt, 1988). In contrast, responses of neurons in the laterodorsal thalaniic nucleus to a nociceptive input were “considerably attenuated” by NMDA receptor antagonists, whereas CNQX had “little effect” (Eaton and Salt, 1989). Thus in two different subnuclei of the thalamus responses to innocuous or nociceptive stimuli are mediated by predominantly AMPA or NMDA receptors. In the dorsal lateral geniculate nucleus excitatory amino acid receptors are thought to mediate the input from the optic nerve. In halothane anesthetized animals selective iontophoretic currents of an N MDA antagonist reduced the visual responses of both X and Y cells by about €4076, while CNQX reduced the responses by about 30% (Murphy et al., 1989;

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Sillito et al., 1989). It therefore appears that NMDA receptors make a greater contribution than AMPA receptors to the excitation of these cells. In slices of the rat hypothalamus, EPSPs evoked in cells of the supraoptic nucleus were completely blocked by 2-5 pM CNQX (Gribkoff and VanderMaelen, 1989). AMPA receptor activation may therefore account for the entire EPSP in these cells. In the optic tectum of lower vertebrates acetylcholine was thought to be the major transmitter; however, Nistri and colleagues (1988) showed that 10 p M DNQX depressed the U1 wave evoked by optic nerve stimulation by -50%, while vesamicol (50 p M , which depletes intracellular acetylcholine levels) depressed it by -30%. These results would suggest that both AMPA and acetylcholine receptors may mediate transmission in this pathway. In slices of mouse olfactory cortex the surface recorded N-wave was reduced by DNQX with an IC50of about 3 pM, but it was not significantly affected by NMDA antagonists (Collins and Buckley, 1989). This suggests that transmission between the fibers of the lateral olfactory tract and pyramidal cells of the cortex relies on AMPA receptors. In slices of rat visual cortex, whole cell patch-clamp recording of neurons in layer 4 revealed excitatory postsynaptic currents evoked by stimulation of individual neighboring cells. In the presence of bicuculline, AP5 left a fast current with a time to peak of 4-8 msec and a decay of 40 msec. By contrast CNQX left a much slower current with a time to peak of 12-14 msec and a decay of up to 200 msec (Stern et al., 1989). These properties are quite similar to those observed in the hippocampus (see Section VI,A,I). In turtle red nucleus, bursting activity, which is associated with motor commands, is blocked by iontophoretic ejection of either AP5 o r CNQX (Keifer and Houk, 1989). It is therefore possible that both NMDA and AMPA receptors are required for the generation of this activity. In rat cerebellar slices, the mossy fiber-granule cell synapse is sensitive to CNQX (Garthwaite and Brodbelt, 1989). In the adult, synaptic transmission resembles closely that in the CA1 region of the hippocampus: Low-frequency responses in Mg2+-containing medium are insensitive to AP5 and almost completely blocked by CNQX, whereas in Mg2+free medium a large AP5-sensitive component is seen. However, in slices from immature rats a sizeable NMDA receptor component is observed in the presence of Mg2+, both before and after the addition of CNQX. Finally, CNQX and DNQX suppress neurotransmission between hair cells and the auditory nerve (Littman et al., 1989).

QUINOXAL1NEI)IONES AS EAA ANTAGONISTS

30 1

VII. Conclusions

Use of the quinoxalinediones have strengthened the belief that quisqualate, kainate, and AMPA all depolarize neurons by acting at the same site. This site used to be called the quisqualate receptor but because of the other actions of quisqualate it is less ambiguous to refer to it as the AMPA receptor. T h e quinoxalinediones will be useful in distinguishing actions of quisqualate and kainate at the AMPA receptor as opposed to the metabotropic receptor or the high-affinity kainate binding site, respectively. T h e action of the quinoxalinediones at the allosteric glycine site endows them with some NMDA as well as AMPA antagonist properties. However, in the presence of sufficient concentrations of glycine this does not compromise their selectivity and so they are useful pharmacological tools as AMPA receptor antagonists. Their use in the investigation of synaptic pharmacology and physiology has supported the concept of a dual component EPSP at excitatory amino acid-mediated synapses (Dale and Roberts, 1985)and will allow a thorough characterization of the time courses of the NMDA and non-NMDA receptor-mediated components in isolation. T h e examples quoted already display a wide range in the relative contributions of the two components to the synaptic response and it will be interesting to see how these differences come about and what properties they confer upon synapses.

Acknowledgments

We thank our colleagues for their advice and the MRC for financial support.

References

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ACQUIRED IMMUNE DEFICIENCY SYNDROME AND THE DEVELOPING NERVOUS SYSTEM By Douglas E. Brenneman, Susan K. McCune, and lllana Gozes Unit on Neurochemistry Laboratory of Developmental Neurobiology National Institute of Child Health and Human Development National Institutes of Health Bethesda, Maryland 20892

I. Prologue 11. Clinical Features and Neurological Manifestations of Pediatric Acquired Immune

Deficiency Syndrome A. U.S. History of AIDS B. Definition of Pediatric AIDS C. Epidemiology of Pediatric AIDS D. Clinical Presentation E. Central Nervous System Pathology i n Pediatric AIDS F. Neuroradiologic Features G. Cerebrospinal Fluid Findings H. Neuropathologic Features I . Future Directions of Clinical Kcsearch 111. Human Immunodeficiency Virus A. Virology B. CD4: T h e Cellular Receptor lor HIV IV. HIV External Envelope Glycoprotcin: gp 120 A. Overview B. Molecular Biology C . Structure-Activity Domains D. Biological Actions of gpl20 V. Epilogue References

1. Prologue

T h e human immunodeficiency virus (HIV), the etiologic agent in acquired immune deficiency syndrome (AIDS), produces catastrophic neurological and developmental deficits in children. It is the purpose of this review to examine the subject of AIDS from the perspective of its effects on developing neurological systems, including detailed clinical aspects and a description of several model systems that may be relevant to mechanistic explanations for this disease. Of particular importance is highlighting the unique course of AIDS in children versus adults. We also emphasize our current working hypothesis on the importance of viral products, specifically gp 120, the major envelope glycoprotein from HIV, 305 INTERNATIONAL REVIEW OF NEUROBIOLOGY, VOL. 3'

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as a causal agent in the neuropathology of this disease. T h e scope of this review reHects our own research and clinical interests, and it is intended to stimulate further research efforts into the developmental neurobiology of AIDS. Several reviews either expand topics presented here or address areas not covered by the present discussion (see Johnson et al., 1988; Habeshaw and Dalgleish, 1989; Farrar et al., 1988; Gelderblom et al., 1989; Budka, 1989). T h e study of AIDS has contributed substantially to a growing awareness of the relationship between the immune and nervous systems. HIVmediated disease was originally described as having its cellular and systemic causes confined exclusively to the immune system. Recently, neuropsychiatric and neurological deficits, originally thought to be secondary to opportunistic infections and neoplasms, have been demonstrated to be directly related to HIV infection in the central nervous system. T h e symptoms associated with the nervous system can be the presenting clinical feature for some strains of the virus. Indeed, evidence indicates that various viral strains have distinguishable preference for target cells: that is, either for T 4 lymphocytes causing immune dysfunction or for macrophages, which may eventually impair the nervous system (Chiodi et al., 1989). This review emphasizes a comparison of the impairments in the nervous and immune systems that arise from HIV infection. A second major emphasis resides in an examination of the evidence for a role of the major envelope glycoprotein of HIV, gp120, as a causative agent for the pathology of AIDS, which may occur as the result of an interference with neuroimmune effectors. The latter idea serves to highlight the thesis that the nervous and immune systems share many common chemical mediators (Pert et al., 1985). Although the identity and action of these substances is uncertain, interference in their action may contribute to, if not define, the development and course of AIDS. Our work has focused on the identification of such substances and offers possible roles for peptides as protective agents against the deleterious effects of HIV in the nervous system. II. Clinical Features and Neurological Manifestations of Pediatric Acquired Immune Deficiency Syndrome

A. U S . HISTORY OF AIDS The world is currently living through the initial history of AIDS. T h e unraveling of this mysterious disease that we now know as AIDS is a tribute to the widespread dissemination of scientific and medical litera-

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ture and the dogged detective work of the Centers for Disease Control (CDC). T h e first written report of’ pneumocystis pneumonia (PCP) in five young men in Los Angeles appeared in the Morbidity and Mortality Weekly Report (MMWR) in the week ofJune 5, 1981 [Centers for Disease Control (CDC), 19811. At the same time in New York, a number of reports surfaced describing young male patients with perianal ulcers and herpes simplex infections that were unresponsive to known therapies. Also, in New York in March of 1981, a number of cases of Kaposi’s sarcoma were reported. Prior to 1981, this tumor was described only in older males. In the cancer registry at New York University, there were no cases of Kaposi’s sarcoma in men under 50 years of age in the previous 9 years, but beginning in 1981 there were two cases within 1 month. Throughout 1980 and 1981, there were 40 independent observations by private physicians of young mcn with AIDS symptoms (Fettner, 1987).I n December of 198 1, Siegel et 01. described these cases as “part of a nationwide epidemic of immunodeficiency among inale homosexuals” (Siegel et al., 1981). It was initially thought that this new disease was a result of the sexual practices and drugs (especially aniyl and butyl nitrate) used by male homosexuals (Goedert ~t (!I., 1982). I t was not until clusters of patients with immune deficiencies were identified as having a link with a single index patient that an infectious agent appeared to be the most likely source of this new disease (CDC, 19X2a). Speculation as to the etiology of c\IDS was rampant until May of 1983 when four research reports appeared i i i Scionce magazine describing an association between AIDS and the retroviral family (human T-cell leukemia virus; HTLV) (Gallo rt ol., 1983; Uarre-Sinoussi et al., 1983; Gelniann et ul., 1983; Essex et al., 1983). In April 1984, the ELISA for the detection of the virus in serum was patented by Robert Gallo, and in the sunliner of 1985, mass testing of blood donations was initiated (Culliton, 1984). It was necessary to review the general history of AIDS to appreciate the difficulty in linking disorders in children to the epidemic in honiosexual men. Despite the initial reports of adult AIDS in 198 1 , it was not until 1982 that AIDS cases were reported in populations other than honiosexual men and Haitians. In 1982, imrnune deficiency disorders were reported in intravenous drug abusers, hemophiliacs, arid blood transfusion recipients (CDC, 1982b,c; Masur rt d , 1982; Poon et u l . , 1983). I t was not until May of 1983 that the first reports of AIDS in children were noted in the scientific literature (Fauci, 1983; Oleske rt al., 1983; Rubinstein et al., 1983). However, there was the suggestion that AIDS in children was first occurring as early as 1980. At that time in the N e w York City area, a number of babies were identified as having a bizarre immune deficiency illness that could not be classified into any of the known causes for

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congenital immune deficiencies. One of the pediatricians in Newark, James Oleske, had also noted immune deficits of unknown etiology in some of his infant patients. He was able to identify the link between his patients and the newly identified epidemic of AIDS by serendipity. He accidentally met the father of one of his deceased patients as the man was coming into the hospital to have his immune system tested. The father was an intravenous drug abuser and was subsequently identified as having AIDS. Using this information, Oleske was able to equate the syndrome that he was observing in his patients with that of AIDS in adults (Fettner, 1987). The history of AIDS is continually evolving. Each answer only evokes new and more difficult questions concerning timing and mode of transmission, clinical staging and prognosis, pathophysiology especially in the central nervous system, and clinical correlation to known pathophysiology. It is only through the answers to these and future questions that we will be able to comprehend fully and to attack this modern epidemic.

B. DEFINITION OF PEDIATRIC AIDS After the initial link was made between a new immune deficiency illness in the pediatric population and AIDS, it became necessary to adopt guidelines for the diagnosis of HIV infection in that population. As more cases were reported, it also became clear that the guidelines originally adopted for adults (CDC, 1986) were not always applicable to those under 13 years of age. Although much of the clinical symptomatology was similar in the adult and pediatric populations, the initial presentation and subsequent clinical courses were sufficiently divergent in the two groups that separate guidelines were necessary. Because of the possibility that passively transferred maternal antibodies of HIV may be present up to 15 months of age, and the lack of universal availability of standardized detection methods for the virus, including cultures of blood or tissues, the CDC decided that two sets of criteria for diagnosing AIDS were required in the pediatric population. T o establish a diagnosis of AIDS in children under 15 months of age with perinatally acquired infection, the virus must be documented in blood or tissues, or HIV antibody must be present in association with evidence of both cellular and humoral immune deficiency as well as one or more categories of symptomatic infection (Class P-2) (CDC, 1987). This strict criteria in the children under 15 months is necessary because current studies estimate that only 20-60% of antibody-positive infants born to HIV-positive mothers proceed to have documented disease of their own

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(Novello et al., 1989). In older children, the diagnosis of AIDS can be established by identifying the virus in blood or tissues or by demonstrating the presence of antibody to HIV. Persons over the age of 13 are classified by adult criteria (CUC, 1987). To assess the epidemiology and clinical-pathological correlations of HIV in the pediatric population, the CDC established a classification scheme for HIV infection in children (CDC, 1987). This categorization (Table I) acknowledges that certain clinical entities are more prevalent in the pediatric than adult populations. These include lymphoid interstitial pneumonitis and recurrent serious bacterial infections (Novello et al., 1989). Other significant clinical differences between pediatric and adult AIDS will be discussed in the section on clinical presentation.

C. EPIDEMIOLOGY OF PEDIATRIC AIDS T h e magnitude and breadth of AIDS cases in the United States have escalated tremendously in the past few years. There were 70,702 cases of AIDS reported in the United States as of August 15, 1988. Children under 13 years of age accounted for 1125 cases and 289 cases were '1ABL.E I SUMMARY OF CLASSIFICATION OF HIV INFECTIONI N CHILDREN I J N D E R IS YEARSOF AGE ~

Class

Clinical findings

Class P-0 Class P-1 Subclass A Subclass B Subclass C Class P-2 Subclass A Subclass B Subclass C Subclass D Category D-1

Indeterminate infection Asymptomatic infection Normal inimune function Abnormal immune function Immune function not tested Symptomatic infection Nonspecific findings Progressive neurologic disease Lymphoid interstitial pneurnonitis Secondary infectious diseases Specific secondary infectious diseases listed in the CDC surveillance definition for AIDS Recurrent serious bacterial infections Other specified secondary infectious diseases Secondary cancers Specified secondary cancers listed in the CDC surveillance definition for AIDS Other cancers possibly secondary to HIV infection Other diseases possibly due to HIV infection

Category D-2 Category D-3 Subclass E Category E-1 Category E-2 Subclass F

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reported in adolescents from 13 to 19 years of age. Despite the recent identification of the disease in the pediatric population, AIDS is the ninth leading cause of death in children 1-4 years old, and it is expected to be among the top five causes of mortality in this age group by 1992. As in all the figures documenting the prevalence of AIDS, this is probably a drastic underestimate of the true number of cases, as these numbers solely represent those cases reported to the CDC. The CDC criteria for AIDS tend to include only those patients with advanced disease, so that asymptomatic and early symptomatic children would not be included in these statistics. Despite the discrepancy in the current figures, it is expected that by 1991 there will be at least 10,000-20,000 HIV-infected children in the United States (Novello et al., 1989). Pediatric AIDS differs from adult AIDS not only in common presenting features but also in modes of transmission. In children over the age of 13 years, most of the early cases were the result of infection through blood products or blood transfusions. However, more recently, an increased number of cases of this age group have resulted from sexual contact or intravenous drug abuse (Novello et al., 1989; Public Health Service, 1988). The majority of infants and children acquire the disease through maternal transmission. It has been documented by isolation of virus from fetal tissue that there may be transplacental passage of the virus (Jovaisas et ul., 1985). In two cases, it has been suggested that HIV transmission may occur either transplacentally or during labor and delivery secondary to exposure to infected maternal blood and/or vaginal secretions (Cowan et al., 1984; Lapointe et al., 1985). A less likely although documented means of transfer of the virus occurs through breast feeding (Thiry et al., 1985; Lepage et al., 1987). The most illustrative case of this mode of transmission occurred in a mother who acquired AIDS from a postpartum blood transfusion and then passed the virus along to the infant in the course of breast feeding (Ziegler et ul., 1985). The population of children who are HIV-positive tends to differ from the adult population, with only 54% of children with AIDS being male compared to 93% being male in adults (Kogers, 1987). Minorities are more severely affected in the pediatric than in the adult population. Black children represent 15% of the total pediatric population in the United States, but they account for 53% of the pediatric cases of AIDS. Hispanic children fare only slightly better as they account for 22% of the cases while they represent only 10% of the overall population (Novello et al., 1989). It has been shown that certain areas that are endemic for high numbers of AIDS cases in adults also have a greater share of pediatric cases. For example, a New York Health Department study in November 1987 indicated that one of every 61 babies born in New York City is born

AIDS A&D T H E I)t VF.LOPIN(r NERVOUS SY51‘FM

31 1

to a woman who is infected with the AIDS virus (Public Health Service, 1988). In the future, detailed epidemiologic studies must be undertaken to identify the perinatal factors involved in maternal-fetal transmission of HIV. More sensitive testing, other than antibody status, will have to be developed to make timely diagnoses in the newborn period. since it is becoming clear that early therapy may be more effective in controlling opportunistic infections. Early diagnosis will also be vital in assessing the neurologic effects of infection with the AIDS virus, particularly in the apparently asymptomatic individual, during the process of nerve growth, differentiation, and myelinimtion.

D. CLINICAL PRESENTATION T h e initial clinical manifestation and subsequent course of AIDS are variant between the pediatric and adult populations. It is important to compare the illness in children and adults because of the diagnostic and therapeutic ramifications. T h e two most common presenting illnesses for adult patients with AIDS are pneumocystis carinii pneumonia and Kaposi’s sarcoma (Ward et al., 1987). In homosexual adults, Kaposi’s sarcoma is seen in 30-50% of patients at initial diagnosis (Des.Jarlaiset al., 1984) while it is a presenting symptom in only 2% of hemophiliacs with AIDS. Pnfwmocystis rnrznzi pneumonia is the presenting illness for greater than 50%’of newly diagnosed patients but it is more common in heterosexual than homosexual men (Volberding, 1989). A number of adults present with Mulike symptoms a few weeks following seroconversion. These symptoms generally last for a mean of 8 days and include fever, lethargy, sweats, and lymphadenopathy (Volberding, 1989). Because this symptom complex is so nonspecific in adults, it is not until they are infected with an opportunistic organism that most patients present for medical evaluation. Pneumocystis carinii is the most frequently acquired opportunistic organism in adult AIDS patients causing a diffuse pneumonitis leading to shortness of breath, nonproductive cough, dyspnea on exertion, and frequently a high fever. Other opportunistic organisms that cause pathology in adult AIDS patients include other parasites such as Crvptosporidia and Toxoplasma, bacteria including Mycobacterium aviurn intrac&ular~, viruses including cytomegalovirus and herpes simplex virus, and fungi including Cryptococcus and Hlrtoplasma. Secondary malignancies are also common in the adult population of AIDS patients with Kaposi’s sarcoma being the second leading clinical presentation associated with .4IDS.

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These patients are also at risk for other malignancies including lymphomas (Volberding, 1989). Neurologic sequelae of AIDS have been particularly varied in the population, but it is now clear that they represent a majority of the morbidity associated with this disease. It has been estimated that 10% of all patients with AIDS have a neurologic manifestation of the disease that prompts initial medical attention. Some studies suggest that 37-63% of all AIDS patients will develop neurologic dysfunction at some time in the course of their disease, and 75% of all AIDS patients have been shown to have pathologic findings in the central nervous system at autopsy (Levy and Bredesen, 1988; Urmacher and Nielsen, 1985; Moskowitz et al., 1984; Berger et al., 1987). Pediatric AIDS patients have numerous clinical and laboratory findings that are consistent with the disease process in adults; however, they also have a number of unique aspects. The majority of pediatric AIDS patients (75%)acquire the disease through perinatal exposure to infected mothers. Because of this, 80% of children with pediatric AIDS are under 3 years of age at diagnosis (Rogers, 1987). This is a particularly vulnerable time in the growth and maturation not only of the immune system but of all major organ systems, particularly the central nervous system. T h e most common clinical features associated with pediatric AIDS are failure to thrive, generalized lymphadenopathy, hepatosplenomegaly, parotitis, persistent oral candidiasis, and chronic or recurrent diarrhea, as well as infections with common and opportunistic organisms. In contrast to adult patients, severe infections with common bacterial organisms (particularly Streptococcus pneumonia and Haemophilus injuenza type B) are frequently found in the pediatric population. These organisms may cause pneumonia, sepsis, meningitis, osteomyelitis, septic arthritis, and otitis media. Another feature more prevalent in the pediatric population is lymphoid interstitial pneumonitis, which is rare in adults but has been reported in 40% of pediatric patients with AIDS. In addition, hepatitis, renal disease, and cardiomyopathy have also been reported (Task Force on Pediatric AIDS, 1988). A number of infants have also been described as having dysmorphic features characteristic of HIV infection (Iosub et al., 1987; Rogers et al., 1987; Marion et al., 1986). However, this finding has been disputed and it is not clear whether the constellation of dysmorphic features is due to other prenatal factors (Qazi et al., 1988). The neurologic findings in pediatric AIDS will be discussed in greater detail in subsequent sections. Children with AIDS suffer much of the same neurologic dysfunction as adults, but because of the nature of the developing nervous system, the clinical findings differ in the two populations. In addition to encephalopathy, developmental delays are the hall-

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mark of neurologic disease in the pediatric age group. I n contrast with the adult population, infection of the central nervous system with opportunistic organisms is uncommon in the pediatric age group (Levy and Bredesen, 1989). Thus, many of the neurologic problems reported in children are a direct result of H l V infection and present an opportunity for understanding the role of the retroviral infection in the developing nervous system and the complete interactive relations between the immune and nervous systems.

E. CENTRAL NERVOUS SYSTEM

PATHOLOGY I N

PEDIATRIC: AIDS

It is necessary to examine the clinical features of central nervous system dysfunction in the adult population afflicted with AIDS to unclerstand both the common and the unique findings in the pediatric age group. There have been a number of comprehensive neurologic and pathologic studies on the adult population; however, only a limited number of studies have been done in children. It was originally thought that the central nervous system dysfunction in adults was priniarily due to involvement with opportunistic organisms and secondary neoplasms. As more data were accrued, including postmortem studies examining brain tissue for viral particles and viral D N A , it became evident that much of the CNS dysfunction in these parties was primarily due to the direct effect of HIV (Epstein et d.,1984-1985, 1985). Because the pediatric population is less prone to opportunistic infections in the central nervous system than are adults, it is easier to correlate the clinical and pathologic findings in this population with a direct effect of HIV (Sharer et al., 1986). T h e incidence of nervous system dysfunction in adults is probably higher than originally estimated, as demonstrated by abnorm aI’ities on neuropsychological tests in patients with AIDS and ARC compared to seronegative controls (Grant ut al., 19x7; Tross et al., 1988; Ayers ut al., 1987). It may be easier to identify early dysfunction in children because they are in the process of attaining motor and cognitive skills and are not able to compensate f-or the loss of‘these newly acquired milestones with as much facility as adults. As further studies are completed in both adults and children, it may be possible to identify certain symptom complexes and to target therapy specifically to the corresponding pathologic processes. In the adult population, a number ofcentral nervous system dysfunctional syndromes have been reported. These include HIV encephalopathy or the AIDS dementia complex, acute encephalopathy or meningitis, vacuolar myelopathy , and peripheral neuropathy as well as infectious ‘

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processes with opportunistic organisms and neoplastic transformations. In addition to neurologic dysfunction caused by HIV itself, the central nervous system in adult patients appears to be particularly susceptible to cytomegalovirus, herpes simplex viruses, Toxoplasma, Cryptococcus, Mycobacteria, Candada, AJpergzllzr*\, Norardzu, and J C papovavirus (Levy et al., 1985; Snider et al., 1983;Jordan et al., 1985; Dix et al., 1985; Horoupian rt al., 1984; Ryder et al., 1986; Tucker rt al., 1985). In children progressive encephalopathy as well as acquired microcephaly, pyramidal tract signs, and a neuroophthalmic syndrome have been reported (Belman et al., 1988; Raphael et al., 1989). Cytomegalovirus (CMV), one of the most common adult viral CNS pathogens, was originally thought to be the etiologic agent in subacute encephalitis. It has since been demonstrated that the AIDS dementia complex is most likely secondary to infection with HIV (Koppel, 1987). However, there continue to be a number of patients who have demonstrated CNS disease with CMV. This is documented by positive cerebrospinal fluid cultures (Edwards el al., 1985),typical electron microscopic findings (Morgello et al., 1987),or immunohistochemical evidence for the virus (Wiley et al., 1986b). The clinical syndromes associated with CMV infection include myelitis (Tucker et al., 1985), chorioretinitis (Chou et al., 1984) and a Guillain-Barre-like illness (Singh et al., 1986). Pediatric patients have had documented viral-induced CNS dysfunction caused by CMV or herpes simplex types 1 and 2 as well as EBV and Varicalla zoster virus. These patients generally present with fever, seizures, focal signs, headache, and lethargy (Iannetti et al., 1989). Progressive multifocal leukoencephalopathy (PML), an entity due to infection with the JC papovavirus, has been reported in 3-4% of the population (Krupp et al., 1985; Wiley et al., 1988).The clinical symptoms associated with this syndrome include mental changes, weakness, and visual loss (Koppel, 1987). PML has been described in children and the hallmarks of the clinical presentation include progressive blindness, aphasia, ataxia, and hemiparesis (Iannetti et ul., 1989). The most common opportunistic infection causing neurologic dysfunction in AIDS patients is toxoplasmosis (Snider et al., 1983). This particular manifestation of AIDS is rare in children. This may reflect the fact that toxoplasmosis is usually due to a reactivation of latent infection (Koppel, 1987). Patients with toxoplasmosis usually present with lethargy, personality change, and nonspecific symptoms suggestive of increased intracranial pressure (Koppel, 1987). Although less common than in the adult population, CNS infection with Toxoplasma gondii in children has been reported (Iannetti et al., 1989). Bacterial infections of the brain with common organisms are rather

uncommon in AIDS patients, but they occur with greater frequency in children than they do in adults. Esrhrrichia coli and Haemophilus irlfluenzae meningitis as well as Salmonella, S/uphylococcus epiderniidis, and Strrptococrics pneumonia abscesses have been described. Infections with gram-negative 1984; organisms are frequently noted in the pediatric group (Scott rt d., Moskowitz et al., 1984; Sharer and Kapila, 1985; Epstein p t ul., 1985; Pitlik et al., 1984). T h e most common tumor of’ the central nervous systems in patients with AIDS is lymphoma, which may be primary or metastatic. Other more rare neoplastic processes that have been reported are metastatic Kaposi’s sarcoma (Levy et nl., 19%; (;orin rt al., 19X5),metastatic rhabdomyosarcoma (Cheeseman and Gang, 1986), epidural sarcoma (Snider et al., 1983), and plasmacytonia (Israel rt al., 1983). Patients with C N S malignancies generally present with headaches, confusion, seizures, cranial neuropathies, radiculopathy, or plexopathy (Koppel, 1987). Systemic chemotherapy for- infectious and neoplastic processes brought on by HIV infection may precipitate neurologic dysfunction as well. Vincristine can cause a peripheral neuropathy while isoniazid is neurotoxic in the context of pyridoxine deficiency (Goldstick rt ul., 1985). Pathologic findings in both the adult and pediatric populations tend to reflect the severity of the clinical status although this is not invariably true. There are similarities i n the pathology in adults and children, but here too unique findings in children may reflect their susceptibility to the invading organism during important developmental periods. Despite the plethora of causes of‘central nervous system dysfunction in patients with AIDS, HIV itself is the perpetrator of a unique pathophysiological process yet to be fully elucidated i n the brain. I t is this complex interaction between the nervous system and HIV that is responsible for specific clinical syndromes that cause great morbidity. Each of these syndromes has its own clinical manifestations and pathology and it is useful to discuss each individually. 1. Encephalopathy: AIDS Dementici Conzplvx T h e most common neurologic presentation in the pediatric population with AIDS is that of encephalopathy, termed AIDS dementia complex (Curless, 1989). As with adults, the original estimation of the degree of CNS involvement in children was probably low. Initial studies suggested that only 10% of‘ children with AIDS had neurologic sequelae. However, with improved neuropsychologic testing and more vigilant neurologic assessments in pediatric patients, it was demonstrated in the largest and most recent pediatric neurologic study to date that 90% of the infants followed for an 18-month period suffered from some degree of

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central nervous system dysfunction. In this population, only 15% of the neurologic dysfunction could be attributed to infectious etiologies, neoplastic processes, or cardiovascular accident (Belman et al., 1988). The hallmarks of AIDS dementia complex in both children and adults are cognitive defects, motor deficits and behavioral changes. In addition to these findings, the pediatric patients have acquired microcephaly, which is most likely an extension of diffuse atrophy seen in both adults and children. Each of these findings will be discussed separately with additional data from the adult population to assist in future classification and prognosis in the pediatric age group. a. Cognitive Defects. In the largest study of pediatric neurologic manifestations, Belman et al., completed formal psychometric evaluations on 66% of their patients with AIDS or ARC. Of those tested, cognitive impairment was noted in 84% while in those not tested, severe impairment was observed in 52%. In the same study, developmental delays including delayed attainment of milestones and loss of previously attained milestones were documented in 85% of the patients. It was noted that social adaptive skills were more preserved than language and motor skills (Belman et al., 1988). In another smaller study, 67% of pediatric patients who met criteria for the diagnosis of AIDS suffered some degree of neurologic dysfunction. Cognitive deficits of various degrees were documented in all of these patients with CNS involvement (Belman et al., 1985). It is difficult to define precisely the number of AIDS patients with cognitive deficits. In some studies on adults, it was estimated that 8- 16% of the population suffer from AIDS dementia (Snider et al., 1983; Levy et al., 1985; McArthur, 1987). In a series of patients that were referred to neurologists, 66% of these patients had pathologic processes indicative of AIDS dementia (Price et al., l988a). T h e quantitation of neurologic impairment due to HIV will vary based on the geographic area, referral patterns, and the risk group (Levy and Bredesen, 1989). In several respects, children are a better population for assessment of cognitive function than adults. Although the developing nervous system may be particularly susceptible to invasion of HIV, it is a less confounded system in which to examine the effects of this neurotropic virus since children have fewer secondary causes of central nervous system dysfunction. While the results of neuropsychologic testing in all AIDS patients can be affected by systemic illness, other variables are less common in children (i.e., dysfunction secondary to aging, alcohol and drug abuse, or injury). I n addition, the pediatric population is one in which the natural acquisition of milestones has been well documented. In a number of studies, baseline values for the normal time frame for development have

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been established (Bayley, 1969; Frankenburg and Dodds, 1967). Thus, children with perinatally acquired AIDS who usually have manifestations of their disease in the first 3 years of life provide a unique opportunity for the clinician to follow the neurological development during AIDS infection. Not only is it possible to chart the acquisition of milestones, but it is also possible to document the loss of previously attained social, language, and motor skills. In the adult population, the hallmarks of cognitive deficits include poor concentration, forgetfulness, and slowness. In neuropsychological testing, there are abnormalities documented by impaired sequentialalternation problem solving with psychomotor slowing and inattention. These may ultimately progress to global dementia, mutism, and organic psychosis (Levy and Bredesen, 1989). While most adults are able to compensate for early mild cognitive deficits, ultimately, these manifestations of AIDS are some of the most frightening and devastating, and in conjunction with the motor deficits, they constitute the greatest morbidity in AIDS patients. b. Motor Deficits. In the study by Belman et al. (1988), 76%' of those children followed exhibited bilateral pyramidal tract signs. These were manifested by spastic paraparesis or spastic quadriparesis. Other less common findings that were noted were hemiparesis, muscle weakness, rigidity, dystonic posturing, ataxia, and tremor. Seizures were reported but at a very low rate in this study with only 9% of the children being afflicted. In the previous study (Belman et al., 1985),50% of the children with neurologic dysfunction had manifestations of motor involvement. Motor deficits in adults tend to appear early in the disease and involve the majority of patients. T h e most frequent complaints were loss of balance, leg weakness, and deterioration of handwriting. On exarnination, patients were noted to have impaired rapid movements, ataxia, tremor, and hypertonia that subsequently progressed to paraparesis, incontinence, and myoclonus (Levy and Bredesen, 1989). c. Behavioral Changes. Both children and adults with AIDS dementia complex have evidence of behavioral changes. In addition to the loss of milestones observed in the pediatric population, children were also noted to develop progressive apathy and in some cases uncooperativity (Belman et al., 1988; Epstein et al., 1985). In adults behavioral disturbances have included apathy and reduced spontaneity, ultimately leading to social withdrawal. In a small number of patients, anxiety, hyperactivity, and inappropriate behavior have been noted (Levy and Bredesen, 1989). Not all patients suffer the same magnitude and rapidity of progression of the degenerative neurologic processes. In general, the onset of neurologic dysfunction in both children and adults is insidious. T h e

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majority of patients undergo a slowly progressive encephalopathic process with periods of plateaus. This has been described as chronically progressive as opposed t o subacute encephalitis. T h e most common clinical course is one of progressive deterioration over the course of weeks to months, while some patients exhibit some stability in their neurologic function over the course of several months. In the large study by Belman et al., (1988),three distinct groups of patients were identified: those with a rapidly progressive encephalopathy, those with a subacute but steadily progressive encephalopathy, and those with a plateau and static course. In the adult population, the central nervous system dysfunction is most commonly chronically progressive but three groups have also been identified: those with mild disease, those with relatively stable neurological function, and those with severely progressive disease (Navia et al., 1986a). At this time, it is unclear in both the adult and pediatric populations what the frequency of each presentation is and what predisposing factors are involved in determining the ultimate clinical course. I t appears in a number of' studies that development and progression of H IV-associated CNS dysfunction correlates with increased levels of immunosuppression (Levy and Bredesen, 1989). Although the precise mechanism of interaction between HIV and the nervous system has yet to be elucidated, it is clinically evident that central nervous system dysfunction in both adults and children can be devastating. In the most severe form of AIDS dementia, patients become bedridden, mute, paraplegic, and incontinent. They maintain consciousness but display very little interest in their surroundings and stare vacantly around as they are incapable of even basic social functioning (Navia et al., 1986a).

2. Vacuolar Myelopathy Vacuolar myelopathy, which has been described in 20-3 1% (Petito et al., 1985; Graffe and Wiley, 1989)of spinal cords examined in adults with AIDS, has not been reported in the pediatric population. In one pediatric study, there was one child 6.5 years old who did demonstrate focal vacuolar changes in the posterior columns in the thoracic region, but the pattern was not similar to that seen in adults (Dickson et al., 1989). T h e clinical presentation of adults with this syndrome is one of progressive paraparesis, usually with ataxia, spasticity, and incontinence. T h e diagnosis is made by demonstrating vacuolation of the posterior and lateral columns in the spinal cord (Gabuzda et al., 1987). At this time, the etiology remains unclear. HIV and CMV antigens have only been rarely detected in the spinal cords of these patients (Graffe and Wiley, 1989).

3. Periphrral Neurofiathy 'Fo date, there have been very few studies in the pediatric population to examine the incidence of spinal cord and peripheral nerve involvement in AIDS. Based o n those children w h o have been evaluatcd, it is thought that peripheral neuropathy is relatively rare in children. Peripheral neuropathies appear to be relatively common in the adult population with AIDS. Thesc neuropathies can be divided into three groups: (1) distal symmetric polyncuropathy, (2) niononeuropatliy multiplex, and ( 3 ) inflammatory tleniyelinating neul-opathy (Jaiissen, 1989; Parry, 1988). Distal symmetric polyneuropathy affects the sensorimotor tracts and produces the clinical findings of burning, paiiiful distal dysesthesias, numbness, and, less f'reciuently, leg weakness. I n one study, these effects were observed i n 48%' of adults with AIDS (Navia r t ~ l . , 1986a). Mononeuropathy multiplex also presents clinically as a sensorimotor neuropathy but involving single nerves or toots. It has o n l y been reported in HIV-positive patients who do not have AIDS, and in some patients, it has either spontaneously resolved or responded to plasmapheresis (Janssen, 1989). I nflamniatory demyelinating neiiropathy is also most common in patients with ARC; or asymptomatic infection with HIV. These patients present with weakness, usually of the lower extremity, sensory loss distally, and arellexia. This presentation is very similar to Guillain-Barre syndrome and the two may only be differentiated by a cerebrospinal fluid pleocytosis seen in the HIV-positive patients. The etiologic process involved in the development of peripheral neuropathies has yet to be defined and is thought to be autoimmune or due to infectious, toxic, or nutritional factors (Janssen, 1989). In a study by Graffe and Wiley (1989), 57% of patients with focal nerve root or peripheral nerve inflammation had documented CMV antigens, and it is thought that this may be an important etiologic agent.

4. Neuroophthalmic Syndronir Infection of the central nervous system by HIV in both adults and children is usually a diffuse process. If focal findings are elicited, this most often implies involvement wit ti an opportunistic infection or neoplastic process causing a mass lesion effect. However, in a case report by Raphael et al., (1989), a child was found to have a rapidly progressive neuroophthalmic syndrome secondary to primary HIV infection of the brainstem. T h e autopsy findings in this child revealed pathologic changes consistent with a direct effect of HIV without evidence of tumor, other infectious etiologies, or vascular accident. This case demonstrates the wide spectrum of clinical presentations seen with HIV infection.

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el

01.

F. NEURORADIOLOGIC FEATURES

In both adults and children, the neuroradiologic findings have been rather nonspecific with C T (computerized tomography) scans that may vary from normal to showing moderate to severe atrophy in patients who clinically demonstrate significant dementia. In addition to the atrophy seen in both adults and children, calcifications of the basal ganglia are frequently observed in pediatric scans (Belman et al., 1988). In a study by D. B. Price et al. (1988),23 seropositive pediatric patients, 95% of whom had documented neurodevelopmeiital delays, were studied by C T scan. Thirty percent of the children scanned had normal findings. Enlarged cerebrospinal fluid spaces were demonstrated in 57% of the scans while 17% showed calcifications in the basal ganglia. There was a 4%incidence of central nervous system lymphoma and a 9% incidence of vascular accidents. In another study conducted by Belman et al. (1986),there was a reported incidence of 6 1% of pediatric patients with calcifications in the basal ganglia. This wide variation between studies may represent technical variations. Belman et nl. were able to correlate CT scans with magnetic resonance imaging (MRI) early in the course of the calcific process and felt that their findings were consistent with interruption of the blood brain barrier associated with edema or inflammation. They also felt that this represented vascular injury with subsequent calcium deposition. There appeared to be some correlation between clinical encephalopathy and basal ganglia Calcifications but there was no correlation between the severity of the clinical status and the degree of calcification (Belman et al., 1986). A less common feature noted on C T scans was contrast enhancement of the basal ganglia (D. B. Price et al., 1988). Cortical atrophy has been noted on C T scans in children prior to the development of significant neurological dysfunction, which suggests that the neuropathologic process is ongoing prior to the development of clinical symptomatology (Epstein et al., 1985). CT scans in adults tend to show cortical atrophy of varying degrees usually accompanied by ventricular dilatation. As in the pediatric population, in a small number of patients, cortical atrophy was detected prior to clinical evidence of neuronal dysfunction (Price et al., 1988b; Navia et al., 1986a). In addition, C T scans from adults also documented focal lesions in a number of patients (Navia et al., 1986a). Magnetic resonance imaging (MRI) has been used to complement the findings on C T scans. In AIDS dementia complex, abnormalities have been noted in cerebral white matter, particularly in the frontal lobes (Navia et al., 1986a).

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In general, electroencephalography has been of little additional diagnostic help in defining the neurologic dysfunction in AIDS patients. In one study of adults, there was a slowing of mean a wave frequency in half of the patients and when this persisted, there was a correlation with the incidence of encephalopathy (Enzensberger et al., 1985).Similar findings were documented in the pediatric population with 55%)of children in one study demonstrating diffuse mild to moderate background slowing. A number of children also had evidence of abnormalities in their brainstem auditory pathways (Belman et al., 1985). There are similarities in the C T and EEG findings of children and adults, most notably cerebral atrophy and diffuse background slowing. However, basal ganglia calcifications are predominantly seen in the pediatric population while mass lesions secondary to opportunistic infections or neoplasms are more common in adults.

G. CEREBROSPINAL FLUIDFINDINGS In general, results of routine test on the cerebrospinal fluid of adults and children with neurologic dysfunction are normal. 'There may be a mild, short-lived pleocytosis or increased protein level. Significant increases in protein levels associated with a pleocytosis and depressed glucose levels should suggest meningitis due to infection with opportunistic organisms or CNS involvement with a neoplastic process (Epstein et al., 1988). HIV-specific antibodies have recently been reported in the CSF o f HIV-positive children. However, the presence of these antibodies has not correlated with clinical status (Epstein et al., 1986, 1988). In contrast, HIV antigen has been demonstrated in the cerebrospinal fluid of H IV-positive children and correlates with evidence of progressive encephalopathy (Epstein et al., 1987). HIV itself has been cultured from the cerebrospinal fluid of children with encephalopathy, but to our knowledge clinical correlations have not been reported. Similar studies have been undertaken in HIV-positive adults. H I V specific IgG has been found to be synthesized within the blood-brain barrier and the virus has been isolated in adult cerebrospinal fluid as well (McArthur et al., 1988). Because adults are more prone to secondary infection, specific tests directed at opportunistic organisms are useful in cerebrospinal fluid analysis in adults. These include appropriate stains, cultures, and cytologic evaluations.

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nl

H. NEUROPATHOLOGIC FExruws The pathologic features of HIV infection of the central nervous system reflect the characteristics of adult versus pediatric manifestations of AIDS. There is wide variation i n the correlation of pathologic arid clinical findings in both adults and children. In the pediatric population, Curless (1989) has identified five pathologic patterns from a number of sources. These include the observation that all cases examined by autopsy showed decreased brain weight, which is correlated by the evidence of marked atrophy on (1'1- scan. I n these cases. inflammatory cell infiltrates were noted throughout grey and white matter but predominantly in the brain stem and basal ganglia. These infiltrates consisted of microglia, lymphocytes, plasma cells, and mononuclear cells. I n addition to the inflammatory infiltrates. multinucleated cells were identified and blood vessel wall calcifications were common. N o deniyelinization was observed, which tends to rule out acute disseminated encephalomyelopathy (Belman et al., 1988; Epstein rt d . , 1986; Sharer et al., 1986). In addition to these universal findings, Belnian et ul. (1986) denionstrated more specific Calcification of basal ganglia vessels, primarily in the putamen and outer segment ofthe globus pallidus. In some patients, the calcification extended into the centruni seniiovale of both frontal lobes, and in one patient calcification of the thalamus was noted. T h e calcification was primarily localized to the walls of small vessels but in some cases also involved the media and advcrititia of larger vessels. In addition to the calcification, which is predominantly found in the pediatric population, glial nodules, reactive astrocytosis, and white niatter degeneration have been noted and are similar to what has been reported in the adult population (Belnian et ul., 1985; Epstein et al., 1985, 1988; Britton et al., 1982; Britton and Miller, 1984; Nielsen et d., 1983; Snider et al., 1983). In the large pediatric study by Belnian et al. (1988), diffuse cortical atrophy was noted along with calcification of the basal ganglia in most patients. They also noted astrocytosis, foamy macrophages, and niultinucleated cells in addition to occasional degeneration of the lateral corticospinal tract. These findings are of particular interest because of the sequence and structural homology between HIV and the lentivirus visna (Gonda et al., 1985). Visna has been documented to cause a subacute encephalopathy in sheep that is manifested histopathologically as white matter degeneration (Georgsson et al., 1982). In general, the histopathology seen in adults is very similar to that seen in children with the exception of the increased incidence of basal

ganglia calcifications in children. However, as cited earlier, adults are more susceptible to infection with opportunistic organisms and have pathologic findings consistent with this clinical finding. Toxopla.sma gondzi is the most common organism to cause focal lesions in adults (Snider et ul., 1983). Cytomegalovirus infection is a common cause of neurologic dysfunction in adults and has been documented in children by demonstrating large Cowdry type A intranuclear inclusions and glial nodules (Belman et al., 1985). Other disease entities more commonly f'ound in adults include progressive niultitbcal leu koenceplialopathy and vacuolar mvelopathy. T h e latter is characterized by subcortical anti parieto-occipital demyelinization with lipid-laden macrophages, large oligodendrocytes containing intranuclear inclusions, and bizarre giant astrocytes (ilstrom et al., 1958). Vacuolar myelopathy is predominantly found symmetric-ally in the lateral and posterior columns of the thoracic cord. The vacuoles are intraniyelin and axons in affccted area show Wallerian degeneration (Petito et aL, 1985). One case reported had unique pathologic features that warrant attention. T h e case is one of perinatally acquired AIDS with marked neurologic dysfunction and subsequent death at 4.5 years of age. At autopsy, massive neuronal destruction was noted. T h e brain weight was half of the expected age norm, and there were cortical microcavitations with diffuse destruction. This destruction included total or substantial loss of' neurons in the cortex and basal ganglia in addition to the typical features of AIDS dementia complex in children. Electron microscopic examination o f the brain revealed viral particles consistent with HIV morphology and no evidence for any other opportunistic organism was found (Giangaspero et al., 1989). T h e suggestion that a number of the features of neurologic involvement in pediatric patients with AIDS, as with adults, are secondary to effects of HIV and not just caused by infection with opportunistic organisms is supported by the documentation of HIV in the brain. This has been accomplished by demonstrating viral particles by electron microscopy (Sharer et al., 1986); by Southern blot detection of HIV D N A isolated from the brain (Sharer et d.,1986; Shaw et ul., 1985); by in situ detection of viral RNA (Shaw rt d., 1985; Stoler et al., 1986); and by virus isolation from the brain and subsequent viral infection of monocyte macrophages and T cells (Gartner et ul., 1986a). An interesting aspect of HIV infection in the central nervous system is the demonstration of the possibility of multiple subgroups of HIV isolates. It has been suggested that there is a CNS-specific subgroup that more efficiently infects peripheral blood macrophages and a blood-

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specific subgroup that more efficiently infects T cells and glia (ChengMayer et al., 1989). There has also been documentation of phenotypically different strains of virus from the same patient (Koyanagi et al., 1987). These findings may help identify the precise mechanism of infection of the CNS and the role of the apparent latency of clinical manifestations in some patients. One of the most difficult aspects of examining HIV infection in the central nervous system is to try to correlate pathologic findings with clinical status. It has been postulated that the severity of the dementia is reflected in the histopathology (Gabuzda and Hirsch, 1987; Monte et al., 1987). However, another study documented mild nonspecific findings in many of the demented patients and more severe histopathologic findings in nondemented patients (Navia et al., 1986a,b).Until the precise interaction between HIV and the central nervous system has been elucidated and the role that the immune system plays in this interaction is fully understood, it will continue to be difficult to correlate pathologic and clinical findings.

I. FUTURE DIRECTIONS OF CLINICAL RESEARCH In the pediatric population, documentation of overall clinical status and, more specifically, neurologic function, particularly in regard to developmental milestones, is crucial to correlating clinical status to pathophysiology. T h e children born to seropositive mothers have not been adequately studied. Improved identification of the natural history of the disease in these patients and improved diagnostic capabilities will allow for earlier intervention in this subgroup of AIDS patients. I t is necessary to standardize, and make available, universal diagnostic testing in the pediatric population. This would include virus isolation from blood and tissues and subsequent culturing of the virus to allow more prompt discrimination of those neonates who are antibody-positive secondary to maternal transfer of antibodies. The most important future direction of AIDS research is the development of effective therapeutic regimens. The new therapies need to be directed against opportunistic infections, as well as the virus itself and, perhaps to viral products. While many laboratories have undertaken the challenge of developing a vaccine, some clinical success has been achieved AZT has been found not only to with AZT (3’-azido-3’-deoxythymidine). improve immunologic function in some patients (Yarchoan et al., 1986; Yarchoan and Broder, 1987) but also to cross the blood-brain barrier (Klecker et al., 1987) and to improve neurologic function in patients with

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dementia as well as patients with polyradiculoneuropathy (Yarchoan et al., 1987, 1988; Dalakas et al., 1988). Metabolic abnormalities in the brain have been reversed during the course of therapy with AZT. These changes were documented by [ lXF]fluorodeoxyglucosearid positron emission tomography (PET-FDG) studies (Brunetti et al., 1989). Another drug that has been shown to have beneficial effects on CNS function in AIDS patients is peptide T. Preliminary studies with this drug have shown improved memory function and remission of myoclonus and hyperreflexia (Bridge et al., 198%). Treatment with peptide T also has been suggested to produce improvement as measured by MRI (Wetterberg et al., 1987). In addition, HIV-related constitutional syrnptoms (weight loss, watery diarrhea, fatigue, anergy, HIV-associated dermatitis), either were improved or resolved (Bridge et al., 1989b). Although the effectiveness of peptide - 1 in preventing viral infectivity has been disputed on the basis of greatly differing in uitro test systems (Sodroski et al., 1987), this drug apparently shows promise in alleviating A1 DS-associated problems in the CNS along with improving other symptoms. Importantly, there was no reported toxicity. Expanded clinical trials ofthis drug and others are needed to dernonstrate unequivocally effectiveness in a larger population of AIDS patients.

111. Human Immunodeficiency Virus

A. VIROLOGY

T h e human immunodeficiency virus is one of the lentiviruses, a subfamily of retroviruses. Lentiviruses derive their name from the slow time course of the infections they cause in humans and in animals (Thormar and Palsson, 1967). In natural and experimental infections of- animals, the etiological agent replicates at the site of entry and subsequently spreads via the bloodstream and the CSF (Nathanson et al., 1985).AIDS is transmitted similarly. Virus is introduced into the bloodstream through sexual contact, intravenous drug administration with contaminated needles, or administration of contaminated blood or blood products (Hoxie et al., 1985). It is not known whether the virus is transferred inside cells, but this is obviously of great importance in predicting the efficacy of conventional vaccine strategy. There is no evidence for germline transmission of animal lentivirus from mother to offspring. Only a few cells are infected in the animal, replication is restricted (Haase, l986), and integration may be rare (Harris et nl., 1984). T h e failure of the virus

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genome to form covalent linkages with the host chromosome probably has a structural basis, as a topological precursor for retrovirus integration is likely to be circular DNA, while the predominant structure found is nicked or linear (Haase, 1986). Indeed, in AIDS there is about one infected T 4 lymphocyte in 20,000-100,000, as visualized by in s i t u hybridization (Harper et al., 1986). It is difficult to account for the devastating effects on the basis of absolute number of infected T4 cells. These observations suggest mechanisms more complex than simple infection. T h e cytopathic effects of retroviruses are likely to be due to the abundance of virion components (probably the envelope glycoprotein) capable of fusing and killing the cell from within and from without (Harter and Chopping, 1967). Moreover, immunopathology cannot account for all the manifestations of lentivirus infection. There is an inflammatory component of the progressive encephalopathy that occurs frequently in children and adults with AIDS (Shaw et al., 1985; Price et al., 1986). However, in AIDS encephalopathy and myelopathy, inflammation is overshadowed by vacuolation and degenerative changes that are more like those in a paralytic disease of mice caused by some types of murine leukemia viruses (for review, see Haase, 1986). In conclusion, the human immunodeficiency virus (HIV), as a typical member of the lentivirus group, exhibits not only the special affinity for cells of the central nervous system, but also a peculiar penetration capacity, length of incubation time, antigen shedding, and a high mutation rate of envelope glycoprotein (Haase, 1986). T h e constant and variable aspects of the envelope glycoprotein are discussed below.

B. CD4: THECELLULAR RECEPTOR FOR HIV HIV enters cells by binding its envelope glycoprotein gp120 to the CD4 antigen, the recognized receptor present on immune cells (Klatzmann et al., 1984; Dalgleish et al., 1985).T h e human CD4, a glycoprotein member of the imniunoglobulin gene superfanlily, includes an amino terminal variable immunoglobulin-like domain, a joining (J)-line region, a third extracellular domain, a membrane-spanning region homologous to class I1 major histocompatibility complex (MHC)p chains, and a highly charged cytoplasmic region (Maddon et al., 1985; Littman and Gettner, 1987). CD4 is expressed predominantly on helper T cells and is thought to stabilize the binding of the T-cell receptor with the antigen-MHC complex by interacting with the invariant region of the class I1 MHC molecules (Littman and Gettner, 1987). Mouse CD4, sometimes termed L3T4, is homologous to human CD4.

The most highly conserved region is the cytoplasmic region (79% at the amino acid level). In contrast, thc external portion contains only 55% identical residues (Tourvieille et a l . , 1986). The nucleotide sequence of' the complementary mouse D N A predicts a mature protein of 335 amino acid residues with a 372-residue surface region. T h e binding region for the HIV on the human CD4 molecule has been investigated with monoclonal antibodies and site-directed niutagenesis (Mizukanii et al., 1988).Codons for two amino acid residues (Ser-Ar-g) were inserted at selected positions within the region encocling the first and second immunoglobulin-like doinains of CD4. I in paired gp 120 binding activity resulted from iiisei-tionsaf'ter amino acid residues 3 1,44, 48, 52, 55, and 57 in the first inirnunoglobulin like domain. Moderate impairment of gpl20 binding resulted from the insertion after amino acid residue 164 in the second iinmunoglobulin-like domain. .I'he epitopes for two H I V -blocking monoclonal anti bodies, 0 K'T4A and OKT4D, were also mapped in the gpl20-binding region of the first domain (Mizukanii et al., 1988). Interactions of CIM b v i t h the major histocompatibility complex (MHC 11) are crucial during thymic ontogeny and subsequently for helper and cytotoxic functions of 1' lymphocytes. As noted above, the residues involved in gp 120 binding have Ixeii localized to a region within the ininiuiioglobulin-like domain I of CD4, which corresponds to an immunoglobulin variable region. Mutations i n this region destroy the binding ofboth gp120 and the MH(: I1 and binding of gp120 inhibits the binding oftlie MH<: 11. In contrast, mutations i l l other regions of the inimunoglol~ulin-likeregions I and 11 that have no effect on gp120 binding eliminate or dccrease the binding of the MHC 11, reflecting a broader area of' iontact with the latter (Clayton rt nl., 1 WY). Interestingly, class I1 MHC: molecules are expressed on the surface of oligodendrocytes in the brain (Ting rl ul., 1981). Moreover, two other members of the immunoglobulin gene superfamily. 'Ihy- 1 and O X - 2 , are expressed on both lymphoid and nerve cells (Clark et al., 1985; Giguei-cri ul., 1985), suggesting the possibility of neuroimmune interaction and the possible expression of CD4 i n Ilrain. Indeed, the peculiar tropism of the human immunodeficiency virus (HIV) for T helper lymphocytes can be explained by specific interaction between the virus and the CD4 niolecule on these cells (Dalgleish et ul., 1984; Klatzmann et al., 1984). The tropism for T helper lymphocytes, however, can hardly account for the early brain infection observed in some AIDS patients (Neidt and Schinella, 1985). Several reports have accumulated concerning the possible expression of CD4 in the central nervous system. Using RNA hybridization and molecular cloning, a CD4-related transcript was identified in brain (Tourvieille et al., 1986; Maddon et nl., 1986; Funke rt al., 1987; Lonberg

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et al., 1988). Immunological assays using different anti-CD4 antibodies were performed to try to identify and localize the CD4 protein product. The CD4 antigen was localized in normal human brain by both immunohistochemical and Northern blot analyses (Funke et al., 1987). With anti-CD4 antibodies detecting different epitopes of the molecule (OKT4 and OKT4A), CD4-positive neurons were defined in the cerebellum, thalamus, and pons. The reactive cells could be identified as neurons as well as glial cells. CD4-specific mRNA was detected in all three subareas and in the hippocampus, while other subareas were negative. The CD4+cells were negative with anti-T cell antibodies (anti-CD2 and anti CDS), as well as with antinionocyte antibodies (M-M 522 and M-M 42). In a separate study, the OKT4 monoclonal antibody against the CD4 molecule identified this receptor in rat brain, monkey brain, and human brain (Pert et al., 1988a,b). In this study, the highest density of binding occurred in the molecular layer of the dentate gyrus and hippocampus as well as in the cerebral cortex. The OKT4 antibodies also recognized the T 4 antigen on human astrocytic cell lines (Dewhurst et al., 1987). Other CD4 epitopes (W3/25 and 0 x 3 5 ) were found on microglia, monocytes, and occasional lymphocytes, but not on neurons (Perry and Gordon, 1987). This study is in agreement with that of Wiley et al. (1986a), which determined the cellular localization of H IV within the brains of AIDS patients and found it to be mostly restricted to capillary endothelial cells, mononuclear inflammatory cells, and giant cells. Only in a single case, with severe CNS involvement, a low-level infection was seen in some astrocytes and neurons. Interestingly, in cultured cells, HIV can infect gliorna-derived cell lines and human brain cell cultures with both astroglial and oligodendroglial markers (Cheng-Mayer et al., 1987; Dewhurst et al., 1987; Chiodi et al., 1987).Although CD4 was originally defined as a differentiation antigen on T lymphocytes, this molecule is present in other leukocyte types. Monocytes-macrophages isolated from the lungs (Plata et al., 1987) and brains (Gartner et al., 198613) of HIV-infected individuals express viral antigens, and the same cell types isolated from noninfected subjects can be infected in vitro (Nicholson et al., 1986). There is species heterogeneity in the expression of the CD4 molecule on macrophages, thus, using fluorescence activated cell sorting analysis, Northern blot hybridization, and immunoperoxidase labeling, rat and human M 4 macrophages were found to express CD4, while mouse M 4 macrophages do not (Crocker et al., 1987). In the study of Funke et al. (1987), the major CD4 transcript detected was 3 kb long. In the cerebellum an additional 1.7-kb transcript was occasionally detected (Funke et al., 1987), which could be due to an

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alternative polyadenylation or splicing at the 3‘ untranslated region (Maddon et al., 1085). A second, smaller mRNA has recently been reported for human brain, ancl a 2.7-kb LST4-specific message has been found in mouse brain (Maddon et al., 1986; Tourvieille et ul., 1986).This 2.7-kb polyadenylated mRNA is 1000 bases smaller than the major 3.7-kb mRNA found in the immune cells. The structure ofthe mouse brain CD4 message was recently determined (Lonberg et al., 1988). It is identical to the last two-thirds of the C1>4 message and is potentially capable of encoding a 2 17-residue protein that would consist of a truncated, 154residue, cell surface region, together with the complete CD4 transmenibrane and cytoplasmic regions; it would not include an amino-terminal hydrophobic leader peptide. ‘Thus, the predominant mouse brain CD4related mRNA they claim to be 2.3 kb long (which is significantly shorter than the T-cell mRNA). Lonberg and his colleagues maintain that the 1.7-kb transcript could be due to nonspecific hybridization to the 18s ribosomal RNA and that the authentic CD4 message found in the human brain is due to macrophages, which are the predominant cell tvpe infected with HIV in brain tissue from AIDS patients (Koenig et al., 1986; Wiley et al., 1986a). However, the existence of a smaller transcript (2.3-2.7 kb) in brain is due to the expression of the CD4 gene in this tissue. Whether the transcript is translated or not remains to be determined; however, the OK7’4A epitope, which may be associated with gpl20 binding, is expressed. Moreover, microglia express other potential plasma membrane entry for H I V such as Fc and complement receptors that are regulated independently of CD4 (Perry and Gordon, 1987).

IV. HIV External Envelope Glycoprotein: gpl20

A. OVERVIEW A major aim of this section is to examine the evidence that suggests that the major envelope glycoprotein of HIV, gp120, participates in the neurological and immunological impairment observed in AIDS. The frame of reference for the present review is the developing nervous system, an area that appears to be vulnerable to gpl20-mediated toxicity as measured in in uitro systems. Before examining this evidence in some detail, we give a selected view of the literature on the structure of gp120 with particular emphasis on its genetic diversity and putative important sites of interaction with its receptor, the CD4 receptor described above.

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DOUGLAS E. B R E N N E M A N e/ nl

B. M ~ L E C U L ABIOLOGY K The complete nucleotide structure of‘ the AIDS virus has been described (e.g., Ratner et al., 1985). The proviral DNA has four long open reading frames, the first two corresponding to the gag and pol genes. The fourth open reading frame encodes two functional polypeptides, a large precursor of the major envelope glycoprotein ( e w ) and a smaller protein derived from the 3’-terminus long open reading frame (lor). The en71 precursor is possibly 826 amino acids long without the signal peptide sequence, which is short and hydrophobic (amino acids 17-37). In its mature form it is probably cleaved into a large, heavily glycosylated (containing 24 potential asparagine-linked glycosylation sites) exterior membrane protein of about 481 amino acids and a transmembrane protein 345 amino acids long, which may be glycosylated. ‘The size of these predicted products agrees with the detection of a large glycosylated precursor protein of 160,000 M,. (gp160) and the products of 120,000M,. (gp120) and a smaller, virion-associated gp41, which is a transmembrane protein (Ratner et al., 1985). To determine the extent and nature of genetic variation present in independent isolates of the H I V , the nucleotide sequences of the entire envelope gene were determined for several viruses (Starcich et al., 1986). The results indicated that variation throughout the viral genome is extensive and that the envelope protein in particular is most highly variable. Within the envelope, changes were most prevalent within the extracellular region, where clustered nucleotide substitutions and deletions-insertions were evident. Based on predicted secondary protein structure and hydrophilicity, these hypervariable regions represent potential antigenic sites. In contrast to the hypervariable regions, other sequences in the extracellular envelope structure (including 18 of the 18 cysteine residues), as well as most of the transmembrane region, were highly conserved (Starcich et al., 1986). The env-lor polypeptide shares many features in common with the envelope gene precursors of other retroviruses, the most striking of which is a hydrophobic region near the middle of the protein (amino acids 5 19-534). This region of amino acid conservation is preceded by an arginine-rich hydrophilic region, which also includes the processing site for cleavage of the env protein precursors into exterior and transmembrane proteins (Ratner et al., 1985). T h e endoproteolytic cleavage of gp160 is required for the activation of the AIDS virus. Thus, the envelope protein is synthesized as a polyprotein (gp160) and cleaved intracelMary to a gp120-gp4 1 heterodimer. When the trypticlike endoproteolytic cleavage site was removed by site-directed mutagenesis and replaced with a chymotryptic-like site (McCune et al., 1988), biologically inactive

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mutated virions (containing iincleaved gp 160) were generated. Exposure of these virions to limiting concentrations of chymotrypsin resulted in cleavage ofthe envelope protein and the release of a unique hydrophobic domain necessary for the full expression of viral infectivity (McCune at al., 1988). T h e gpl20 exterior glycoprotein acts as a bifunctional molecule: Association with the gp4 1 transmembrane protein is determined by regions located in the amino-terminal half, whereas association with the CD4 receptor is determined by the carboxy-terminal regions. After formation of the molecular bridge, the mobility afforded by the noncovalent nature of the gp120-gp41 bond may allow the efficient exposure of the target cell membrane to the hydrophobic gp4 1 regions that mediate the fusion process (Kowalski et al., 1987). Interestingly, a region of homology was found between interleukin-2 and HTLV-111. Thus, a homologous stretch of six amino acids from the carboxy terminus of the HTLV-111 envelope protein (gp4 1, amino acids 841-856) was found to inhibit the biological activity of human interleukin-2 in murine spleen cell proliferation assay (Weigent et ul., 1986). This homology was implicated in the mechanism of inimunosuppi-ession of the AIDS virus.

c. STRUCTURE-ACTIVITY

DOMAINS

Genetic variations during the course of infection of an individual is a remarkable feature of the AIDS disease. Such variation occurs in the envelope protein of the virus, the external protein gp 120. Significant clusters of open regions predicted at the RNA level correlate with significant clusters of hypervariable sites in the HIV envelope gene. Twelve potential antigenic determinants were predicted using an antigenic index method. Interestingly, the majority of the highly variable regions on the protein are predicated as potential antigenic determinants (Le 01 ( i l . , 1989; also see Starcich et al., 1986). T h e principal neutralizing domain of the human immunodeficiency virus type 1 (HIV-1) envelope protein is located in the gp120 and is mapped to a 24-amino acid-long sequence denoted KP135, which is part of amino acids 301-341, bordered by two cysteines at 303 and 338. This sequence has been narrowed down to eight amino acids, which contain a central Gly-Pro-Gly that is generally conserved between HIV- 1 isolates and is flanked by amino acids that differ from isolate to isolate. Antibodies elicited to peptides from one isolate do not neutralize two different isolates; however, a hybrid peptide consisting of amino acid

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DOUGLAS E. BRENNEMAN e t a / .

sequences from two isolates elicits neutralizing antibodies to both isolates (Javaherian et al., 1989). In addition, antibodies directed against a conserved region in gp120 (amino acids 254-274) were efficient in neutralizing three different isolates of HIV in vitro, without affecting the binding of the virus to CD4+cells. Therefore, this second conserved region of gp120 appears to be critical in a postbinding event during virus penetration (Ho et al., 1988). Interestingly, this region is homologous to neuroleukin, which has both neurotrophic and lymphokine activities (Ho et al., 1988). Some of the structure-activity domains of gp120 are depicted graphically in Fig. 1. Site-directed mutagenesis was used to identify a region within the envelope protein gene (gp120) that is critical for infectivity. Thus, amino acid substitutions were introduced at the asparagine codons of four

gp4l Association Viral Replication

CD4 Binding

COOH

Neuroleukinlike Domain FIG. 1. A schematic map of gp120. A linear diagram of gp120, depicting the presumptive CD4 binding elements at the COOH end of the molecule and the areas important for viral replication at the NH2 end of the molecule. Some of the conserved cysteine residues which are close to important areas are labeled S. The important amino acids are labeled by their number along the molecule. The diagram is a summation of the references cited in the text. The loops are hypothetical, but a tertiary structure is probably essential for both binding to the CD4 and for viral replication. A map depicting the glycosylation points is given by Ratner and his colleagues (1985) and a map depicting conserved regions, hypervariable regions, and functional regions for gp4 1 and CD4 binding is given by Kowalski and his colleagues (1987).

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conserved potential N-linked glycosylation sites. One of these alterations resulted in the production of noninfectious virus particles. The amino acid substitution did not interfere with the synthesis, processing, and stability of the envelope gene proteins, gp 120 and gp4 1, or the binding of gp120 to its cellular receptor, the CD4 (T4) molecule. Vaccinia virus recombinants containing wild-type or mutant HIV envelope genes readily induced syncytia in CD4+ HeLa cells. These results indicate that alterations involving the second conserved domain of the HIV gp 120 (which contains the codons 266-268) may interfere with an essential early step in the virus replication cycle other than binding to the CD4 receptor. Revertants were shown to contain the original mutation as well as a compensatory amino acid change in another region of gp 120 (codon 128) (Willey et al., 1988b). Introduction of a conservative amino acid substitution at the revertant site resulted in a marked reduction in HIV-1 infectivity. During the passage of this defective virus in cocultures, yet another revertant appeared (codon 308), which contained an amino acid change within a variable region of gp 120 that restored infectivity to near wild-type levels. These results, in combination with other point mutations that have been introduced into the H IV- 1 envelope, suggest that at least three discrete regions of gpl20 may interact during the establishment of a productive viral infection. ?l'his critical step occurs subsequent to the adsorption of virions to the cell surface and either prior to o r Concomitant with the fusion of viral and cellular membranes (Willey et al., 1989). T h e primary event in the infection of cells by HIV is the interaction between the viral envelope glycoprotein, gp 120, and its cellular receptor, CD4. Using monoclonal antibodies, amino acids 397-439 were identified as part of the binding site, and deletion of 12 amino acids from this region by site-directed mutagenesis leads to complete loss of binding. In addition, a single amino acid substitution in this region results in sigificant decreases in binding, suggesting that sequences within this region are directly involved in the binding of gp120 to the CD4 receptor (Lasky rt al., 1987). Indeed, in some studies, it was found that the substitution of a single amino acid (tryptophan at position 432) can abrogate CD4 binding and renders the altered virus noninfectious (Cordonnier et al., 1989). Virions containing isoleucine substitutions at position 425 have altered cellular tropism. In addition, monoclonal antibodies directed to amino acid residues 423-437 of gp120 also inhibited the binding of HIV-1 to CD4+ cells (Sun et al., 1989). lnsertion of mutations in amino acids 363, 419, and 473 also resulted in the loss of binding (Kowalski rt al., 1987). The three regions important for binding are conserved among HIV isolates (Starcich et al., 1986).However, these regions are flanked by three regions that are highly variable, and it was suggested that maintenance of

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the tertiary structure of the gp120 is required for binding (Kowalski et al., 1987). In the California HIV isolate an octapeptide sequence of Ala-SerThr-Thr-Thr-Asn-Tyr-Thr (termed peptide T) was shown to be almost identical to the Epstein-Barr virus envelope protein sequence and to a five-amino acid sequence in the neuropeptide vasoactive intestinal peptide (VIP). This octapeptide was suggested to be an important region for gp120 binding (Pert et al., 1986; Sacerdote et al., 1987). This sequence is not part of the conserved regions mentioned above; rather, it is present in the second hypervariable region ( 192- 199).A homologous pentapeptide sequence has been observed in all known isolates of HIV-1 and in HIV-2, differing only by conservative substitutions, with an invariant threonine in the second position and a tyrosine in the fourth position. As our work has focused on the biological effects of gp120 and their prevention by peptides, we have investigated peptide T in our neuronal survival assays (See Section IV,D, 1,b).

D. BIOLOGICAL ACTIONSOF (;pi 2 0 A fundamental characteristic of AIDS is the presence of relatively few infected cells, yet both the immune and nervous systems develop catastrophic pathological changes. This basic observation has led to the search for indirect mechanisms rather than direct damage due to viral infection of individual cells. A number of' potential candidates have been suggested including the release of toxic cytokines (Fauci, 1987), the production of autoimniune antibodies (Stricker et al., 1987), and virusrelated cytotoxic proteins. A leading candidate for a cytotoxic product of HIV is its major envelope glycoprotein, gpl20. An important element of the gp 120-hypothesis of neuronal impairment in AIDS is the demonstration of its presence in CNS tissue in biologically significant amounts. In the case of gpl20, neurotoxic concentrations are in the .O 1- 1 pM range (Brenneman el al., 1988b), quantities that are far below the detectability of current assays. However, as will be discussed in the following section, gp 120-like biological activity (neuronal cell killing) has been demonstrated in the CSF of AIDS patients (Buzy et al., 1989a,b). 'ince the portion of gp 120 responsible for the in uitro effects has yet to be identified, it is possible that only a fragment of the envelope protein may be necessary to interfere with neuronal function. Early work indicated that HIV-infected cells secreted gpl20 in significant amounts (Schneider et al., 1986; Gelderblom et al., 1987). Indeed, the conditioned

medium froin these infected cells has been used to prepare purified gpl2U for immunological ant1 p1i"rni;ic"logical studies. More rt'(wit studies ofgpl20 synthesis and processing in a CD4+ lymphoc.yticccll line indicated that about 5-15g of gpl60 is cleaved to produce mature gp120, which is transported to the cell surface and secreted (Wille\.P / ( i l . , 1988a).

1. Nmrotoxzcity T h e toxic properties of tliis protein were first reported in studies w i t h hippocampal cultures froin embryonic mice (Hrenneman rt (/I.. 1987b. 1988b). We became interested in the possible cytotoxic properties of the major envelope glycoprotein, gp 120. hecause of its homology (Kuf'f rt (11.. 1987b) with vasoactive intestinal peptide (VIP) (Brenneman P / ~ l . 1985; , Brenneman and Foster, 1987). Interference with the action o f \ ' I P b y either antisera or specific antagonists (Brenneman and Eiden. 1986; Gozes Pt nl., 1989) is known to produce neiironal cell death i i i CNSderived cultures. Therfore, it W;IS hypothesized that the envelope protein might block the action of either V I P o r a trophic substance released Ily VIP thereby producing neuronal dcgeneration. 'The dissociated hippocampus culture preparation was chosen as a niotlel system to test this idea for several reasons. The hippocanipus has been shown to be rich iti receptors relevant to gp 120 and VIP: the VIP receptor (Taylor and Pert, 1979) and CD4 (Pert et ul., 1986; Hill 01 al., 1986). -1.his receptor clisti-ibution, coupled with the clinical observation of early niemoi-y loss in .411>S, prompted the decision to examine this brain region. Mice were chosen for study, based on the hypotliesis that cytotoxicity associated with HIV was not attributable directly to infectivity; rather, the envelope protein or some fragment of gp 120 was the active agent and, therefore, evidence for such an effect might be obtained in species that share common regulatory mechanisms. Mice d o not develop AIDS. However, i t is not clear whether this is due to the inability of the envelope protein (or a fragment thereof) to interact with the mouse homolog of CD4 receptor or to a niore distal mechanism related to viral entry or replication. T h e murine hippocampal system has provided a iiieaiis of testing for a receptor-mediated, pathological interaction between these brain cells and gpl20, apart fr-om considerations of infectivity. A variety of purified gp 120 preparations were found to produce neuronal cell death in developing hippocampal cultures derived from murine fetuses (Fig. 2). T h e gp120 purified from three different HIV isolates had the same effect: neuronal killing at extraordinarily low concentrations (<1 pM). These studies also revealed that the neuronal cell loss was attenuated at concentrations of gp120 greater than 10 pM. In

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FIG.2. HIV envelope protein produces neuronal cell death in dissociated hippocampal cultures from fetal mice. Recombinant gp120 (open triangles) from the LAV encoding sequences was compared to native gp120 purified (Robey rt al., 1986) from two natural isolates, the IIIB (open circles) and R F I l (squares). A recombinant gp160 (asterisks) from the IIIB-encoding sequences was expressed in insect cells by recombinant baculovirus (Ruche et al., 1987). Significant decreases from control were observed at 0.01 pM for all gp 120 preparations. Control medium from cells infected with vaccinia virus without the gp 120construct (closed triangles) did not produce neuronal cell death. No differences from controls were observed in cultures treated with the insect gp160 preparation. Control cultures had neuron counts that ranged from 750- 1000 cells with standard errors that were <4% of the mean. Before treating with gp120, the cultures were given a complete change of medium. The duration of treatment was 5 days. (From Brenneman el al., 1988b.)

subsequent studies, the IIIB isolate of gp120 was shown to exhibit a slow, progressive effect over a 3-week period, producing a maximal neuronal deficit in treated cultures. These data suggested selectivity of the effect, since some neurons survived the prolonged exposure to gp120. In addition, a recombinant gp120 produced from encoding sequences from the LAV isolate also caused a decrease in neuronal survival when tested under the same experimental conditions. A gp 160 from recombinant

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baculovirus expressed in an insect cell (Rusche et al., 1987) was found to have no detectable effect on neuronal survival (Fig. 2). The reason for the absence of neuron-depleting activity from the insect expressed protein is not clear, but altered tertiary structure due to differences in glycosylation and/or the presence of the gp41 sequence may be involved. I t is noteworthy that the cleavage of gplG0 to its products is also necessary for infectivity (McCune et al., 1988) and for activation of the HIV envelope fusion function (Freed et al., 1989). Purified gpl20 preparations have also been shown to produce neuronal cell death in rodent retinal ganglion cells in vitro (Kaiser P t al., 1989). The lethal effects were observed at very low concentrations (0.1 pM) of gp120 derived from native isolates of HIV. This neurotoxicity could be prevented with antiserum to gpl20 but not with preimmune serum. In related studies, the amount of tieuronal cell death associated with gpl20 in retinal ganglion cells was attenuated with the calcium channel antagonists nifedipine and nimodipine (Campo et al., 1989). Several studies have indicated that gp 120 also produced neuronal deficits in neonatal animals. Purified gpl20 was administered intracerebroventricularly on a daily basis for 3 weeks after birth. Histological examination of the brains of the gp 1 Wtreated animals revealed Iieuronal degeneration, dystrophic neurons, and “blebbing” of neurites in widespread locations (Pert et nl., 1989). Saline-injected aninials did not exhibit such changes. In studies utilizing the 2-deoxy-~-glucosemethod to measure the metabolic effects of intracerebroventricuarly administered gp120, it was found that several regions rich in VIP receptors (suprachiasmatic nucleus and thc lateral habenulae) exhibited a significant reduction in glucose metabolic rates (Kimes et al., 1989). These efl’ects were apparent with a single injection of femtomole quantities of the purified envelope protein. A study conducted in our lahoratory suggests that a substance with similar toxicological properties as gp 1 20 exists in the cerebrospinal fluid of AIDS patients (Buzy et al., 1989a,b). Indeed, neuronal cell killing has been observed with the application of CSF from infected individuals as assayed in the dissociated hippocampal cell cultures test system. As in similar studies conducted with purified gp 120, these effects of CSF could be prevented by a monoclonal antibody to the murine homolog of CD4 and by peptide T (see Sections IV,D, 1,a and b). However, the causative agent for this neurotoxic action in the CSF remains unknown. N o demonstration of gp 120 by Western blot analyses or immunoprecipitation has been observed. It may be that gp120 undergoes proteolytic cleavage to a short molecule that retains the neuronal killing action but loses the ability to be recognized by the predominantly C-terminal-directed antibodies

currently available to us. Nevertheless, the pharmacological characteristics of the toxic cerebrospinal Huid are similar to those observed with purified gp 120 in the hippocampal neuron assay: extraordinary potency (1 : 100,000 dilution of CSF) and attenuation of the response at higher concentrations (Brenneman et ul., 1988b). Identification of this CSFderived neurotoxic material may provide new insight into the mechanism of neural deficits in AIDS. CSF from normal patients produced no loss of neurons in culture when tested at the same dilutions. Gp 120 exhibits sequence similarity to neuroleukin, a recently discovered growth factor for a variety of central and peripheral neurons (Lee et al., 1987; Ho et al., 1988). This growth factor, which shares 9076 sequence similarity to a glycolytic enzyme, glucophosphoisomerase, has been shown to be antagonized by gp120 in in uitro assays of neurite extension and neuroblastoma proliferation (Mizrachi, 1989). Taken together with the previously described studies, it may be that gp120 has multiple sites of homology with known trophic or cytokine substances that result in a constellation of deleterious effects in numerous immune and nervous system-related pathways. u. Receptor Specificity of’gpl20-Induced Death. As emphasized in a previous section (IIl,B), substantial evidence has indicated that gp120 interacts with the CD4 receptor, the receptor for the MHC I1 class of antigens. T h e observation of neuronal cell death in mouse hippocampus cultures poses several problems in explaining this effect mechanistically. T h e mouse has a homolog of CD4 called L3T4 (Tourvieille et al., 1986). As discussed in Section III,B, there is substantial homology in the cytoplasmic domains of this murine receptor in comparison to CD4; however, the extracellular domain is quite different. T o date, there is no direct evidence that gp120 binds to L3T4 receptors; in fact, there is good evidence that the complete molecule does not bind to murine “CD4” (Clayton et al., 1988). To help resolve these questions regarding the receptor mediation of the neuronal death associated with the gp120 preparations, the effects of monoclonal antibodies directed against L3T4 were tested in gpl20treated cultures. T h e concentration-effect curve for one of these antibodies (GK1.5) is shown in Fig. 3 . This rat IgG2b produced a concentration-dependent protection against the neuronal death caused by gp120 (IIIB strain). Thus, a monoclonal antibody that was shown previously to inhibit the functional activity of mouse T cells (Dialynas et al., 1983), also effectively blocked the cell-killing action of gp120 in hippocampal neurons. Another monoclonal antibody against L3T4 (RL. 172) also was found to prevent gpl20-induced neuronal death, whereas control monoclonal antibodies from the same antibody class did

339

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FIG. 3 . A monoclonal antibody ( G K I .5) against murine CL)4 rctlucetl gp12O-induceti neuronal death in dissociated hipl)ocaiiipal cultures. A c-ompai-ison w a s inatlc I)ct~.erri cultures treated with antibody alone (sqiiares)and antibody p l u s 1 p2.I ppl20 ( I I I B isolate) (circles). In comparison to neuronal cell counts of cultures treated with gpl20 alone. significant increases ( p < 0.01) were ol,scIved with 0.02-2..', Wgiinl of C;K I ..5.(Frolri Hrenneman rt nl., 1988b.)

not prevent neuronal death f'roni gp 120. These results support the conclusion that the neuronal death that occurs during treatment with the gpl20 preparations is the result of an interaction with the cell surface antigen L31'4 or a receptor with shared eptitopes with L3'1.1. What remains to be resolved is the possibility that a gp120 fragment actually is interacting with the L3T4 receptor. I n addition, it is possible that these cytotoxic effects are the result of' an interaction with a receptor that is homologous to L3-1'4. In regard to the controversy of CD4-like receptors in the brain, it is apparent that gplP0 produces neuronal cell death in murine cell cultures and that there is evidence of H I V infcction in glia and neurons (Srinivassan el nl., 1988; Cheng-Mayer el nl., 1987), albeit few in number. Therefore, one is compelled to choose one or h t h of the following options: (1) that other receptors (non-CD4) exist that can medi-

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DOUGLAS E. BRENNEMAN et a1

ate HIV infection or (2) that some CD4-like receptors exist in the central nervous system. b. Peptide Preuention of gpl20-Associated Neurotoxicity. Based on the hypothesis that the neural deficits are due to an interference with peptide-growth factor action, several peptides were tested for their ability to prevent the neuronal cell death associated with the gp 120 preparations. Vasoactive intestinal peptide (VIP), a peptide that possesses a region of sequence homology to a region in the second variable region of gp120, was found to prevent neuronal cell death in hippocampal cultures. VIP and its receptors are present in brain and immune cells (Rostene, 1984; O’Dorisio et al., 1980, 1981). This peptide was investigated for its ability to prevent gpl20-induced death. The rationale for this study was: (1) the sequence similarity between the peptide T portion of the envelope protein and the (7-1 1) sequence of VIP, suggesting a possible competitive receptor-mediated mechanism; and (2) the demonstrated neurotrophic properties of VIP (Brenneman et al., 1985; Brenneman and Eiden, 1986), and interference with this peptide perhaps resulting in neuronal death. As shown in Fig. 4, the addition of low concentrations (0.1 nM) of VIP prevented gp 120-induced neuronal death, whereas higher concentrations were not as effective. Secretin, a peptide of significant sequence similarity to VIP, had no effect on neuronal death after gpl20 treatment. The mechanism of the protective action of VIP on gp120-induced neuronal death is not apparent from these initial studies. Previous work from our group indicated that VIP exerts its neurotrophic effects indirectly through an interaction with nonneuronal cells (Brenneman et al., 1987a). Furthermore, recent work has demonstrated the existence of two classes of binding sites for VIP in nonneuronal cells from the cerebral cortex of mice (Gozes et al., 1989). The high-affinity receptor (& = 30 pM) appears to be the receptor that mediates the neurotrophic action of VIP (Gozes et al., 1989; Brenneman et al., 1990) and it is this VIP receptor which may be involved in the deleterious effects of gp120. In central nervous system cultures, the lower-affinity receptor appears to be linked to adenylate cyclase (Magistretti et al., 1983), whereas the second messenger for the high-affinity receptor remains unclear. On the cellular level, the gp 120 may be exerting its neuron-depleting action by interfering with the VIP-mediated communication between neurons and astroglia. Several lines of evidence support the conclusion that VIP and gp120 act at the same receptor: (1) antibodies to an OKT4 epitope inhibit VIP- and VIP fragment-stimulated chemotaxis of human monocytes

AIDS A N D T H E DEVELOPING NERVOUS S Y S T E M

34 1

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FIG. 4. Vasoactive intestinal peptide prevented gp120-induced neurorial cell dcath i n hippocampal cultures. One-week-old cultures were treated for 5 days with VIP alone (circles), VIP plus 1 pM gp120 (squares), or secretin plus gp120 (triangles). Compared to cultures treated with gp120 alone, signiticant increases ( p < .02) in the number of neurons were observed with the addition of'O.1-10 nM VIP plus gp120. T h e addition of 0.1 nlM VIP plus gp120 resulted in neurorial cell counts that were not significantly different from cultures treated with 0.1 mM VIP alone. (From Brennernari e/ ui., IY8Xb).

(Sacerdote et al., 1987); (2) the OKT4 epitope and one type of VIP receptor exhibit similar distribution patterns in brain sections (Pert et al., 1986; Hill et al., 1987); (3) there is sequence similarity between VIP and gp120, as discussed previously; and (4) VIP (7-1 1) potently displaces [3H]peptide T binding (Smith et al., 1988). However, a direct demonstration of gp120 acting on either of the two classes of VIP receptors in the brain has not been observed to date. Therefore, the hypothesis that gp 120 interacts with a VIP-related receptor remains equivocal. A direct toxic action of gp 120 on neurons cannot be excluded from the present studies. The envelope protein may act to interfere with other growth factors that are stimulated by VIP. Regardless of the mechanism,

342

DOUGLAS E B R E N N E M A N ul al

these studies suggest that the VIP protection of gpl20-induced death may give important clues as to the cause and the prevention of AIDSrelated neuropathology. Peptide T, an octapeptide found in the external envelope protein of the ARV isolate of HIV and a homolog of VIP, was investigated for its action in preventing neuronal death associated with gp 120. Hypothesizing that attachment sequences are conserved among viruses (Pert el al., 1986),peptide T was deduced from computer-assisted comparisons of all known amino acid sequences of HIV with other viral envelope proteins. Previous reports indicated that peptide T inhibited gp 120 binding to human T cells and inhibited human T cell infectivity from HIV (Pert et al., 1986). In addition, peptide T binding is potently displaced by gp120 (Ruffet al., 1987a). Incubation of the hippocampal cultures with the free acid or the amide of D-ala-peptide T prevented gp 120-mediated neuronal death (Fig. 5). Both peptide drugs had similar dose-response relationships at low concentrations; however, with higher amounts, the free acid of peptide T, like VIP, exhibited an attenuation of the survivalenhancing activity, whereas the amide of peptide T remained active up to 0.1 p M . T h e core pentapeptide sequence (TTNY?’) also prevented gpl20-induced death at concentrations similar to that of the octapeptide drug. Structure-activity studies revealed that if the L-tyrosine, which occurs in the fourth position of all 20 HIV isolates, is substantiated by D-tyrosine, the peptide is inactive. These studies indicate that the various forms of peptide T have a significant and potent action in preventing neuronal death caused by gp120 (Brenneman et al., 1988a).

2. gp120 and the Immune System The loss of T4 lymphocytes is characteristic of individuals infected with HIV. The mechanism of lymphocyte reduction and the immunosuppression that occurs is not as yet clear. The immune system, like the nervous system, exhibits the AIDS paradox: impairment with little apparent cellular infection with HIV. Some studies have indicated that immune system cells also may be affected adversely by the major envelope glycoprotein of HIV, gp120. In uztro experiments with purified gp120 have shown that lymphocyte destruction and immunosuppression can occur in the absence of infectious virus (Weinhold et al., 1989). These deleterious effects remain dependent on an interaction between gp 120 and its cellular receptor, CD4. Gp120 has been shown to inhibit antigenspecific proliferation in lymphocytes (Chirmule et al., 1988) and in a murine T-cell hybridoma that expresses the human CD4 (Diamond et al., 1988). In a similar study, lymphocyte proliferation in response to UVinactivated cytomegalovirus was shown to be inhibited by a high con-

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FIG. 5. Peptide T derivative prevciits gp120-induced neuronal death in hippocampal cultures. A comparison was made 01 u-ala-peptide T amide (circles), D-ala-peptide T (squares), T T N Y 7 ' (asterisks), and T'I'NdY'l', containing a u-isomer of' tyrosine (triangles). (From Brennrman ct nl., 1 9 8 t h )

centration (280 nM) of recombinant gp120 (Krowkd et a/., 1988). The immunosuppression produced by gp 120 was overcome completely by recombinant interleukin-2. T h i s study suggests that the defects in antigen-driven lymphocyte responses are not due solely to the reduced numbers of CD4+ cells. Kather, this loss of responsiveness is clue to impairments in interleukin-:! pathways and the immunosuppressive actions of the secreted glycoprotein gp120. In addition to the imrnuriosuppressive effects of gp120, normal monocytes treated with gp120 exhibit significantly impaired chemotactive responsiveness and reduced migratory function (S. M. Wahl et al., 1989). These defects in migratory responses are similar to those observed in blood monocytes obtained from AIDS patients. Several other studies have demonstrated that gp 120 has intrinsic biological properties. Gp120 has been shown to induce the production of

3 44

DOUGLAS E. BRENNEMAN et al

interleukin- 1 and arachidonic acid metabolites from the cyclooxygenase and lipoxygenase pathways (L. M. Wahl et ul., 1989). These effects were produced by 0.4 nM purified gp120. Several nonglycosylated gp120 fragments and the core structural protein p24 did not increase arachidonic acid metabolism or influence interleukin- 1 activity. In addition, purified gp120 has been shown to increase levels of inositol triphosphate and calcium in CD4+ lymphocytes (Berman and Center, 1988). Within the same study, gp120 also was shown to increase interleukin-2 (11-2) receptor expression and to produce greater cell motility. The reason for this increase is not clear, but it may relate to the observation that gp120 has a six-amino acid region that is homologous to 11-2 (Weigent et ul., 1986). T h e 11-2 hexapeptide found in gp120 was found to decrease spleen cell proliferation and inhibit the binding of radiolabeled 11-2. These authors speculate that the immunosuppression associated witl, AIDS may derive from either gpl20-mediated interference with 11-2 binding to its receptor or the existence of antibodies to gp120 that crossreact with and perhaps neutralize endogenous 11-2. Therefore, in the immune cells as in brain cells, gp120 can produce deleterious effects that may contribute to the course of AIDS.

V. Epilogue

T h e etiology of AIDS in the central nervous system remains a mystery. However, it is now clear that the central nervous system is a target for the disease and that the pediatric population reacts to the viral invasion with several distinctive neurological and pathological sequelae. T h e scope of this disease now extends beyond the realm of the virologist and immunologist and beckons the attention of neuroscientists. The unequivocal identification of the active agent(s) responsible for the neurological and immune impairment is an imperative. Basic investigations of virus-related products are needed urgently as well as expanded studies of cellular interactions that occur between immune cells and brain cells. Once precise molecular and cell specific interactions have been identified, then rational therapy can be targeted toward specific regions and designer drugs can be directed at individual clinical entities with fewer toxic effects. With the evidence cited in the present chapter, the envelope protein or active fragments from this substance should be given serious consideration as a component(s) of the etiology of this dread disease. If the in uitro studies of the deleterious effects of gp 120 on immune cells and neurons are indicative of events that occur in uiuo, then

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preventing viral infectivity and viral spread are only part of the challenge in combating this disease. AIDS is a devastating disease not only for the loss of young and productive lives, but also for the debilitating nature of the neurological manifestations associated with HIV infection. Aggressive examination of all aspects of AIDS pathophysiology will be necessary to conquer this pernicious virus.

Acknowledgments

’

T h e authors thank Drs. Phillip Nelson, Joanna Hill, Elaine Neale. and Jonathan Gershoni for helpful comnients and suggestions in the preparation of this manuscript.

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INDEX

A Acetylcholine glutamate receptors and, 54, 55, 57, 100 invertebrate, 79, 95-98 mammalian, 6 1, 64 neurotrophic factors and, 151, 156 presynaptic effects of toxins and neuronal ion channels, 205,208, 2 1 1 release mechanisms, 217-224, 226-228 quinoxalinediones and, 300 Xenopus oocytes and, 108-1 11 experimental results, 128-130 polymorphism, 113 Acetylcholine receptor, myasthenia gravis and, 176-181 AChR-specific T lymphocytes, 184- 190 anti-AChR antibodies, 181-184 thymus, 190-193 Acetylcholinesterase myasthenia gravis and, 193 Xenopw oocytes and, 131-135 experimental observations, 120- 122 experimental results, 128, 130 polymorphism, 111-115, 117, 118 Acidic fibroblast growth factor, 147, 148 Acquired immune deficiency syndrome, 305,306,344,345 ARC, 316,319 gp120,329 biological actions, 334-344 molecular biology, 330, 331 structure, 33 1-334 HIV CD4,326-329 virology, 325, 326 pediatric AIDS cerebrospinal fluid, 321 clinical presentation, 3 1 1-3 13 CNS pathology, 313-319 definition, 308, 309

epidemiology, 309-3 1 1 future directions, 324, 32.5 neuropathology, 322-324 neuroradiologic features, 320. 32 1 U S . history, 306-308 ACTH, neurotrophic factors and, 159, 161, 162 Adrenal hormones, neurotrophic factors and, 162 Adrenalectomy, taste cells and. 263, 264 Afterhyperpolarization, toxins and, 2 12, 213 w-Agatoxin 1, presynaptic effects of, 216 Aggregation, cholinesterases and, 129, 130, 133 Aging, neurotrophic factors and, 142 epidermal growth factor, 154 hormones, 162 insulin, 150 nerve growth factor, 145, 146 AIDS, see Acquired immune deficiency syndrome Alanine, taste cells and, 271, 272 Aldosterone, taste cells and, 263, 264 Alleles, cholinesterases and, 112 Alzheimer’s disease cholinesterases and, 1 1 1 neurotrophic factors and, 142. 164 epidermal growth factor, I55 lymphokines, 158-160 nerve growth factor, 145, 146 Amiloride, taste cells and epithelium, 246, 247, 249 transduction mechanisms, 253, 260, 26 1,264-268 Amino acids AIDS and, 327,330-334,342, 344 cholinesterases and, 110, 132-134 experimental observations, 1 18- 120 experimental results, 13 1 polymorphism, 113, 115, 117 355

356

INDEX

glutamate receptors and, 5 1, 52, 54, 55, 101 desensitization, 73 single-channel studies, 64 myasthenia gravis and, 177, 179, 182, 188, 192 presynaptic effects of toxins and, 23 1 neuronal ion channels, 204, 206, 208, 212,213 release mechanisms, 222 quinoxalinediones as, see Quinoxalinediones taste cells and, 242, 27 1, 272 AMPA, in central nervous system, 282-284,301 excitotoxicity, 292 pharmacology, 285, 286, 288, 290, 29 1 synaptic physiology, 293, 295-300 Angiogenesis, neurotrophic factors and, 157 Angiotensin, taste cells and, 263, 264 Antibodies AIDS and, 344 gp120,332,334,337,338, 340,344 HIV, 328 pediatric AIDS, 308, 309, 31 1 , 321, 324 cholinesterases and, 113, 128 myasthenia gravis and, 175, 176 AChR-specific T lymphocytes, 184, 186 anti-AChR antibodies, 181-184 thymus, 191 neurotrophic factors and, 154 taste cells and, 258 Antigens AIDS and gp120, 338,342,343 HIV, 326,328,330,331 pediatric AIDS, 318, 319 cholinesterases and, 112, 115 myasthenia gravis and, 175 AChR-specific T lymphocytes, 184-190 thymus, 192 treatment, 194, 195 neurotrophic factors and, 158 taste cells and, 273 AP4,282,290,291

AP5,299,300 hippocampus, 292,293,295-297 pharmacology, 290 Apamin, presynaptic effects of toxins and, 202,212,213 APB, 282,290,291 Aplysia glutamate receptors and, 97 presynaptic effects of toxins and, 2 18, 220 Arginine, taste cells and, 27 1, 272 Arsenazo signals, neurotransmitter release and, 23,24 Aspartate, quinoxalinediones and, 288, 289 L-Aspartate, glutamate receptors and, 60, 61, 81.95 Astrocytes, neurotrophic factors and, 155-158, 164 Atrophy, AIDS and, 320-322 ATxII, presynaptic effects of toxins and, 206,207 Autoantibodies, myasthenia gravis and, 176, 177 acetylcholine receptor, 181 anti-AChR antibodies, 183 thymus, 191 treatment, 194 Autoantigens, myasthenia gravis and, 184, 188, 195 Autonomous nervous system, cholinesterases and, 117 Autoradiography. neurotrophic factors and, 143 Azathioprine, myasthenia gravis and, 193, 194 AZT, AIDS and, 324,325

B B cells, myasthenia gravis and, 186, 192-194 Basal forebrain, neurotrophic factors and, 145-148, 153, 161 Basal ganglia, AIDS and, 320-323 Basic fibroblast growth factor, 147-149 Bitter taste, 241, 242, 245, 253-258,261, 266,269,270

INDEX

Botulinum toxin, presynaptic effects of, 202,216-218,220,221 Brain AIDS and gp120,335,339-341,344 HIV, 313-315,323,325,327-329 cholinesterases and, 131, 132, 135 experimental observations, 118, 122, 123 experimental results, 129, 130 polymorphism, 117 neurotrophic factors and, 142, 164, 165 ciliary neurotrophic factor, 153 epidermal growth factor, 154- 156 fibroblast growth factor, 148 hormones, 161 insulin, 149, 150 insulinlike growth factors, 150, 15 1 lymphokines, 158-160 nerve growth factor, 144 presynaptic effects of toxins and, 202 neuronal ion channels, 204, 208, 210, 211,213 release mechanisms, 22 1, 224 quinoxalinediones and, 283, 290-292 taste cells and, 254, 263 Brain-derived neurotrophic factor, 152, 153 Buffers cholinesterases and, 126 neurotransmitter release and, 46 calcium, 22, 23, 25 classical calcium hypothesis, 32, 33 a-Bungarotoxin, myasthenia gravis and, 176, 180, 183, 193 P-Bungarotoxin, presynaptic effects of, 2 1 1,222-228 Butyrylcholinesterases, Xenopus oocytes and, 110, 111, 130-134 experimental observations, 1 18- 123 experimental results, 126, 128-130 polymorphism, 111-114, 117, 118

C

Calcification, AIDS and, 320-322 Calcium AIDS and, 320,337,344

357

glutamate receptors and D-GluR, 77, 86, 87, 8 9 , 9 0 , 9 3 H-GluR, 97,98 mammalian, 57, 70 neurotransmitter release and, 2 calcium-voltage hypothesis, 55-45 classical calcium hypothesis. 28-35 entry, 10-13 intracellular concentration, 13-22 removal, 22-28 synaptic release, 3-6, 8, 9 presynaptic effects of toxins and, 202, 229-23 1 neuronal ion channels, 208,2 12-2 16 release mechanisms, 217, 218, 220-225,227,228 quinoxalinediones and, 283, 290 taste cells and, 244 bitter taste, 255-257 electrophysiological properties, 248-250 salt taste, 264, 265, 268 sour taste, 269 sweet taste, 260 Calmodulin, neurotransmitter release and, 18.39 Carbachol, glutamate receptors and, 97,98 Catalysis, cholinesterases and, 1 1 1, 132 experimental observations, 119- 122 experimental results, 128 polymorphism, 1 13, 114, 118 Catecholaminergic neurons, neurotrophic factors and, 144 Catfish, taste cells and, 271, 272 CD4, AIDS and gp120.329, 331, 332,335 immune system, 342-344 neurotoxicity, 335, 337, 338, 340 HIV, 326-329 cDNA cholinesterases and, 108-110, 132-134 experimental observations, 118, 123 polymorphism, 113, 117 glutamate receptors and, 69 myasthenia gravis and, 179, 187 Central nervous system AIDS and, 344 HIV, 326,327, 335, 340 pediatric AIDS, 308-319, 321-325

358

INDEX

glutamate receptors and, 51, 52, 54, 55, 100 mammalian, 66, 68, 70, 72, 74 noise, 57-59 single-channel studies, 60, 62, 63 neurotrophic factors in, see Neurotrophic factors in central nervous system presynaptic effects of toxins and, 212, 219 quinoxalinediones in, see Quinoxalinediones taste cells and, 242-244, 253, 259, 273 Centrifugation, cholinesterases and, 1 14 Cerebellum glutamate receptors and, 69, 71, 72 neurotrophic factors and, 147, 156 quinoxalinediones and, 283, 288, 300 Cerebral cortex, neurotrophic factors and, 155, 156, 162 Cerebrospinal fluid, AIDS and gp120,334,337,338 HIV, 325 pediatric AIDS, 314,319-321 Charybdotoxin, presynaptic effects of, 212,213 Chemosensory transduction in taste cells, see Taste cells Chloride glutamate receptors and, 98, 99 taste cells and, 246, 247, 270 Chlorisondamine, glutamate receptors and, 82,83, 93 Choline glutamate receptors and, 88, 93 presynaptic effects of toxins and, 227, 228 Choline acetyltransferase (ChAT) ciliary neurotrophic factor and, 153 fibroblast growth factor and, 148 hormones and, 161 insulinlike growth factors and, 151 nerve growth factor and, 143-146 Cholinergic nerve, presynaptic effects of toxins and, 217-220,223 Cholinergic neurons ciliary neurotrophic factor and, 153 epidermal growth factor and, 157 fibroblast growth factor and, 149 insulinlike growth factors and, 151, 152

lymphokines and, 160 nerve growth factor and, 143-147 Cholinesterases, 107-109, 130-135 case study, 110, 111 experimental observations, 118- 123 experimental results, 126, 128-130 polymorphism, 11 1-1 18 structure, 109, 110 Xenopus oocytes, 123-127 Chorda tympani nerve, taste cells and, 242,271 salt taste, 264, 266-268 sweet taste, 260-262 Chromosomes, AIDS and, 326 Ciliary neurotrophic factor, 153 Clones cholinesterases and, 108-1 11, 132, 133 experimental observations, 1 18-120 experimental results, 126, 128 Xenopus oocytes, 123 myasthenia gravis and, 181, 184, 186, 189, 192 CNQX excitotoxicity, 292 pharmacology, 284-286,288,290,291 synaptic physiology, 292-300 Cognition AIDS and, 313,316,317 neurotrophic factors and, 162 Collagen cholinesterases and, 115, i24, 133 neurotrophic factors and, 163 Collagenase, glutamate receptors and, 77, 81, 83, 86, 88, 89, 93 Compartmentalization, cholinesterases and, 112 Computerized tomography, AIDS and, 320-322 Concanavalin A, glutamate receptors and, 55,100,101 invertebrate, 75 D-GluR, 77,83,85-87,89,92,93 H-GhR, 96 mammalian, 61, 65, 74 Conductance, cholinesterases and, 108 o-Conotoxin, presynaptic effects of, 214-216 C o n w geographus, presynaptic effects of toxins and, 204,214 C o n w m a p , presynaptic effects of toxins and, 215

INDEX

Cooperativity , neurotransmitter release and calcium, 15-19 calcium-voltage hypothesis, 39 classical calcium hypothesis, 29, 31, 32 Cortical neurons, glutamate receptors and, 71,72, 100 Corticosteroids, myasthenia gravis and, 193, 194 Crayfish muscles, glutamate receptors and, 87- 100 Crotoxin, presynaptic effects of, 2 I I , 222-224,228 Cyclic AMP, taste cells and, 257, 261, 262, 272 Cysteine AIDS and, 331 glutamate receptors and, 82 myasthenia gravis and, 178 Cytomegalovirus, AIDS and gp120,342 pediatric AIDS, 311, 314, 318, 319, 322 Cytoplasm AIDS and, 326,327,329,338 cholinesterases and, 114, 124, 128 myasthenia gravis and, 178, 183, 191, 193 neurotransmitter release and, 14, 44 presynaptic effects of toxins and, 224, 225 Cytoskeleton cholinesterases and, 126 myasthenia gravis and, 179 Cytotoxicity, AIDS and, 327, 334, 335

D

Defolliculation, cholinesterases and, 124 Dehydration, glutamate receptors and, 77,78 Dementia, AIDS and, 313-318,320, 323-325 Denatonium, taste cells and, 255-257 Dendrotoxin, presynaptic effects of, 202, 208-21 1,222 Dentate gyrus, quinoxalinediones and, 295,297 Dephosphorylation glutamate receptors and, 92 neurotransmitter release and, 2

359

Depolarization in central nervous system, 290, 291, 299,301 glutamate receptors and, 54 neurotransmitter release and, 46 calcium, 11-13, 18, 21 calcium-voltage hypothesis, 35-38, 40-45 classical calcium hypothesis, 29 presynaptic effects of toxins and, 202, 2 19,230 neuronal ion channels, 204-207, 213, 214 release mechanisms, 220-222, 224-226,228 taste cells and electrophysiological properties, 248, 249 intracellular recordings, 25 1 transduction mechanisms, 260, 262, 265,268,269 Desensitization, glutamate receptors and, 55, 100,101 D-GluR, 77,83,87,89-95 H-GluR, 96-99 invertebrate, 75 mammalian, 61, 65, 72-74 Developmental delays, AIDS and, 312, 316 Differentiation cholinesterases and, 124 neurotrophic factors and, 154, 156, 164 taste cells and, 243, 244, 273 Diffusion, neurotransmitter release and calcium, 22, 26-28 classical calcium hypothesis, 32-35 Dissociation glutamate receptors and, 83-86, 101 neurotransmitter release and, 22. 23,33 neurotrophic factors and, 152 Distal symmetric polyneuropathy, AIDS and, 319 DNA AIDS and, 313,323,326,327,330 cholinesterases and, 109, 114, 117, 132 myasthenia gravis and, 180, 191, 192 DNQX, 282-284, 301 pharmacology, 284-286,288,290 synaptic physiology, 292, 293, 295, 297, 300

360

INDEX

Dopamine, neurotrophic factors and, 151 Down’s syndrome, cholinesterases and, 111 Drosophila glutamate receptors and, 86, 87 presynaptic effects of toxins and, 215

E EGTA glutamate receptors and, 55 neurotransmitter release and, 23, 37 Electron microscopy AIDS and, 314,323 cholinesterases and, 124, 126, 128, 129 presynaptic effects of toxins and, 206, 221,227 Encephalopathy, AIDS and HIV, 326 pediatric AIDS, 312-318, 320-322 Endoplasmic reticulum, myasthenia gravis and, 179 Envelope glycoprotein, see Gpl20 Enzymes AIDS and, 338 cholinesterases and, 131, 132 experimental observations, 119- 121 experimental results, 128 polymorphism, 1 1 1-1 19 glutamate receptors and, 76 neurotrophic factors and, 162, 163 presynaptic effects of toxins and, 202, 217,219,223,224,226,227 taste cells and, 244, 267 Epidermal growth factor, 153-157, 165 Epithelium cholinesterases and, 124 myasthenia gravis and, 190, 192, 193 taste cells and, 242-244 bitter taste, 255, 256 electrophysiological properties, 247, 250 epithelium, 246, 247 salt taste, 263,265,267, 268 sour taste, 269,270 sweet taste, 260, 261 umami taste, 27 1 Epitopes AIDS and, 327-329,339-341

myasthenia gravis and AChR-specific T lymphocytes, 184- 190 anti-AChR antibodies, 181, 183 thymus, 191, 193 taste cells and, 258, 259 EPSP, quinoxalinediones and, 292, 293, 295-301 Equinatoxin, presynaptic effects of, 212 Erythrocytes, cholinesterases and, 115, 117, 121 Estrogen, neurotrophic factors and, 161 Evolution cholinesterases and, 112 taste cells and, 254, 258 Excitability, presynaptic effects of toxins and, 206,215,220,228 Excitation, taste cells and, 269, 272 Excitatory amino acids, quinoxalinediones as, see Quinoxalinediones Excitotoxicity, quinoxalinediones and, 291,292 Exocytosis, neurotransmitter release and, 3 , 4 , 18, 19 Experimental autoimmune encephalomyelitis, 195 Experimental autoimmune myasthenia gravis, 177, 182, 183 Extracellular matrix, cholinesterases and, 114, 133

F

Facilitation, neurotransmitter release and, 2 calcium, 12, 13, 15-19 calcium removal, 22, 24, 26-28 calcium-voltage hypothesis, 44 classical calcium hypothesis, 28-32, 35 synaptic release, 8, 9 Fibroblast growth factor, neurotrophic factors and, 147-149 Fibroblasts myasthenia gravis and, 180 neurotrophic factors and, 157, 163 Fibronectin, neurotrophic factors and, 163 Filaments, glutamate receptors and, 91, 92, 95, 98 Fluorescence, cholinesterases and, 128, 130

IKDEX

Follicle cells, cholinesterases and, 124 Frogs, taste cells and, 272 electrophysiological properties, 246 transduction mechanisms, 255, 256, 262,266,267 Fura 2, taste cells and, 255-257

G G protein myasthenia gravis and, 180 taste cells and, 26 1, 27 1 GABA cholinesterases and, 109 glutamate receptors and, 54, 57, 8 1, 93, 95-99 myasthenia gravis and, 180 quinoxalinediones and, 284, 292, 295 GAMS, 282-285 Gating mechanism, glutamate receptors and, 100 invertebrate, 74-99 mammalian, 60, 64 Glial cells glutamate receptors and, 72 neurotrophic factors and, 163 epidermal growth factor, 155, 156 fibroblast growth factor, 148 insulinlike growth factors, 150, 151 lymphokines, 158-160 platelet-derived growth factor, 157 Glial-derived nexin, neurotrophic factors and, 163, 164 Glial fibrillary acid protein, neurotrophic factors and, 155 Glossopharyngeal nerve, taste cells and, 270,271 Glucocorticoids, neurotrophic factors and, 159, 162 Glutamate neurotransmitter release and, 37 quinoxalinediones and, 283, 284, 288, 290,291 Glutamate diethyl ester, 282, 283, 285 Glutamate receptors, 51-57, 100, 101 invertebrate, 74-76 D-GluR, 76-95 H-GluR, 95-99 mammalian, 57 antagonism, 70-72

36 1

conductance, 66-70 desensitization, 73, 74 NMDA-R, 72 noise, 57-60 single-channel studies, 60-65 Glycerotoxin, presynaptic effects of, 220-222 Glycine cholinesterases and, 109 glutamate receptors and, 72, 95, 96 myasthenia gravis and, 180 presynaptic effects of toxins and, 21 7 quinoxalinediones and, 283, 284, 286, 288,301 Glycolipids, cholinesterases and, 115, 117 Glycolysis, neurotrophic factors and, 163 Glycoprotein, see also Gp120 AIDS and, 305,306,326, 327 myasthenia gravis and, 192 Glycosylation AIDS and, 330,333,337 cholinesterases and, 123, 128, 129, 132 Gp120, AIDS and, 305,306,326, 329-331 biological actions, 334-344 structure, 331-334 Growth cone, neurotrophic factors and, 163 GTP, taste cells and, 261, 271

H

H fibers, taste cells and, 253, 264, 266, 268,269 H-GluR, glutamate receptors and, 95-99 HA966, quinoxalinediones and, 286, 288 Hemopoiesis, cholinesterases and, 1 17 Heparan sulfate proteoglycan cholinesterases and, 115 neurotrophic factors and, 163 Herpes simplex virus AIDSand, 307,311,314 myasthenia gravis and, 192, 193 Hippocampus AIDS and, 328,335,337,338,340,342 glutamate receptors and, 61, 64, 65, 71-74 neurotrophic factors and, 163 epidermal growth factor, 154- 157

362

INDEX

fibroblast growth factor, 148 hormones, 162 insulinlike growth factors, 151 nerve growth factor, 145, 146 presynaptic effects of toxins and, 209, 212-214,216 quinoxalinediones and excitotoxicity, 292 pharmacology, 285,288,290 synaptic physiology, 292-298, 300 HIV, see Human immunodeficiency virus HLA, myasthenia gravis and, 184, 186-189, 191 Homology AIDS and gp120,331,332,334,344 HIV, 326 neurotoxicity, 335, 337-340 pediatric AIDS, 322 cholinesterases and, 109, 110, 113, 120 myasthenia gravis and, 177, 178, 192 neurotrophic factors and, 147, 148, 154 presynaptic effects of toxins and, 209, 214,222,231 Hormones myasthenia gravis and, 176 neurotransmitter release and, 4 neurotrophic factors and, 142, 160-162 taste cells and, 263, 264 Human immunodeficiency virus (HIV), 305,306,345 CD4,326-329 gp120,329-344 pediatric AIDS, 308-310, 312, 313, 325 cerebrospinal fluid, 321 CNS, 31-316,318,319 neuropathology, 322-324 virology, 325, 326 Hybrids AIDS and, 326-329,33 1 cholinesterases and, 109, 118, 120, 134 neurotrophic factors and, 149 Hydrodynamics, cholinesterases and, 114-1 16, 133 Hydrolysis cholinesterasesand, 111, 113, 119-121 presynaptic effects of toxins and, 225-228 Hydrophilicity AIDS and, 330

cholinesterases and, 113, 114, 117 rnyasthenia gravis and, 181 presynaptic effects of toxins and, 212 Hydrophobicity AIDS and, 329-331 cholinesterases and, 133 experimental observations, 12 1, 123 experimental results, 130 polymorphism, 114, 115 myasthenia gravis and, 179 Hyperplasia, myasthenia gravis and, 190- 192 Hyperpolarization glutamate receptors and, 54, 82, 83, 91.93 neurotransmitter release and, 22,40, 43-45 presynaptic effects of toxins and, 204 taste cells and, 25 1 Hypothalamus, neurotrophic factors and, 149, 157-159, 161 Hysteresis, neurotransmitter release and, 35,36

I Irnmunofluorescence, cholinesterases and, 128 Immunoglobulin AIDS and, 321,326,327 myasthenia gravis and, 177, 181, 183 Immunohistochemistry cholinesterases and, 126-130 neurotrophic factors and, 143, 151, 154, 156 Immunoprecipitation, neurotrophic factors and, 143 Imrnunoreactivity, neurotrophic factors and epidermal growth factor, 154, 155 insulin, 149, 150 lymphokines, 158- 160 nerve growth factor, 146 Immunosuppression AIDS and, 318,33 1,342-344 myasthenia gravis and, 177, 193, 194 in situ hybridization, cholinesterases and, 118

INDEX

Inflammation, AIDS and, 319, 320, 322, 326,328 Inflammatory demyelinating neuropathy, AIDS and, 319 Inhibition AIDS and gp120,331,333,342, 344 HIV, 327 neurotoxicity, 338, 340, 342 cholinesterases and experimental observations, 120, 12 1 experimental results, 128 polymorphism, 112-1 14 glutamate receptors and, 55, 74, 10 1 myasthenia gravis and, 183, 186, 193 neurotransmitter release and calcium, 14, 25 calcium-voltage hypothesis, 37,43, 44 classical calcium hypothesis, 32 synaptic release, 9 neurotrophic factors and, 142, 151, 156, 162, 164 presynaptic effects of toxins and neuronal ion channels, 206, 209, 214-216 release mechanisms, 217,222, 224, 227 quinoxalinediones and, 283, 284 hippocampus, 292-295,297 spinal cord, 299 taste cells and, 246, 259, 260,266-268, 271 Inositol trisphosphate, taste cells and, 256, 272 Insecticides, cholinesterases and, 1 12 Insects, glutamate receptors and, 86, 87 Insulin, neurotrophic factors and, 149, 150, 160 Insulinlike growth factors, neurotrophic factors and, 149-152, 160, 165 Interferon, neurotrophic factors and, 160 Interleukin, neurotrophic factors and, 147, 158-160, 165 Interleukin-I, neurotrophic factors and, 158, 159 Interleukin-2 AIDS and, 331,343,344 myasthenia gravis and, 185 neurotrophic factors and, 159, 160

363

lntracellular recordings, taste cells and, 250,251 Ion channels myasthenia gravis and, 178, 180. I8 1, 183 presynaptic effects of toxins and calcium, 213-216 potassium, 207-213 sodium, 202-207 taste cells and, 252, 27 1, 272 epithelium, 247, 248 salt taste, 265, 266. 269 sour taste, 269 sweet taste, 262 Ion selectivity, glutamate receptors and, 77, 78, 88, 89.93 Ionophores glutamate receptors and, 100 neurotransmitter release and, 14. 2 1 presynaptic effects of toxins and, 220, 228 IPSP, quinoxalinediones and, 292-294, 296. 297

K Kainate glutamate receptors and, 54 conductance, 66-69 invertebrate, 81, 90, 93, 95 mammalian, 7 1, 73 quinoxalinediones and, 282-284, 301 pharmacology, 285, 286, 288, 290, 29 1 synaptic physiology, 299 Kainate-sensitive receptor, 52, 100 conductance, 66,67,69 mammalian, 71, 72 Kaposi’s sarcoma, AIDS and, 307, 3 1 1, 315 Kidney, taste cells and, 263, 267 Kinetics glutamate receptors and D-GluR, 77-81,83,85-87,89,90, 92,95 H-GluR, 95-99 invertebrate, 75 mammalian, 61, 64

364

INDEX

neurotransmitter release and calcium, 18, 19,21,23 calcium-voltage hypothesis, 39, 40 classical calcium hypothesis, 28, 29, 32,34 synaptic release, 6, 8 Kynurenate, quinoxalinediones and, 286, 288

L Laminin, neurotrophic factors and, 163 a-Latrotoxin, presynaptic effects of, 202, 220-222,230 Lectin cholinesterases and, 129, 132 glutamate receptors and, 74, 75,85 Lentiviruses, AIDS and, 325, 326 Leptinotoxin, presynaptic effects of, 220, 222 Lesions, neurotrophic factors and, 145, 146, 155, 160, 164 Leukemia, cholinesterases and, 117 Ligands cholinesterases and, 112, 119, 120 glutamate receptors and, 68, 73 myasthenia gravis and, 176, 179 taste cells and, 252, 255, 261 Light microscopy, cholinesterases and, 128 Lipids cholinesterases and, 116, 123 glutamate receptors and, 69 myasthenia gravis and, 178 neurotransmitter release and, 14 presynaptic effects of toxins and, 205, 210, 219,221,230 taste cells and, 255, 258, 271, 272 Lipoprotein, cholinesterases and, 133 Liver, cholinesterases and, 118, 122, 123, 130, 131 Locust muscle, glutamate receptors and, 76-90,93-96,99, 100 Long-term modulation, neurotransmitter release and, 2 , 4 Long-term potentiation, quinoxalinediones and, 297, 298 Lymph nodes, myasthenia gravis and, 188,190

Lymphocytes AIDS and, 306 gp120,335,342-344 HIV, 326-328 pediatric AIDS, 322 cholinesterases and, 123 myasthenia gravis and, 175, 184-192, 195 Lymphoid intestinal pneumonitis, AIDS and, 309 Lymphokines, neurotrophic factors and, 158- 160 Lymphoma AIDS and, 312,315,320 myasthenia gravis and, 194

M Macrophages, AIDS and, 306 HIV, 328,329 pediatric AIDS, 322-324 Magnesium glutamate receptors and invertebrate, 77,89,90 mammalian, 68, 70, 71 single-channel studies, 60, 61, 64, 65 neurotransmitter release and, 21 quinoxalinediones and, 293-296,298, 300 Magnetic resonance imaging, AIDS and, 320,325 Main immunogenic region, myasthenia gravis and, 181-183, 186, 187 Maitotoxin, presynaptic effects 0, 229, 230 Major histocompatibility complex AIDS and, 326,327,338 myasthenia gravis and, 184, 186-189, 192 Mast cell degranulating peptide, presynaptic effects of toxins and, 210, 211 Megakaryocytes, cholinesterases and, 117 Membrane potential glutamate receptors and, 64, 65 neurotransmitter release and, 11, 26, 38,44 presynaptic effects of toxins and, 205, 206 quinoxalinediones and, 292

INDEX

Memory, neurotrophic factors and, 145, 146 Meningitis, AIDS and, 312,313, 315, 321 Mice, taste cells and, 254, 255, 262. 270 Microinjection, cholinesterases and, 108-1 11 experimental observations, 119 experimental results, 126 XenopLs oocytes, 123, 124 Mimicry, myasthenia gravis and, 192, I93 Mitochondria neurotransmitter release and, 14 presynaptic effects of toxins and, 224-227 Mitogen, neurotrophic factors and, 15 1, 156, 157, 159 MK-801, glutamate receptors and, 68, 7 1 Modulation cholinesterases and, 112, 113, 119 glutamate receptors and, 72, 81 neurotransmitter release and, 2 , 4 neurotrophic factors and, 156, 160, I63 taste cells and, 262-264 Monellin, taste cells and, 258, 259 Monoclonal antibodies AIDS and, 327,328,333,337,338 myasthenia gravis and, 181-183, 186, 191, 193 taste cells and, 27 1 Mononeuropathy multiplex, AIDS and, 319 Monosodium glutamate, taste cells and, 270,271 Morphology, presynaptic effects of toxins and, 223,227 Mossy fiber-CA3 pathway, quinoxalinediones and, 296, 297 mRNA AIDS and, 328,329 cholinesterases and, 107-1 10, 131, 152 experimental observations, 1 18-120, 122, 123 experimental results, 126, 128, I29 polymorphism, 113, 114, 116, 117 Xenopus oocytes, 126 glutamate receptors and, 55,69,95 myasthenia gravis and, 180 neurotrophic factors and epidermal growth factor, 154 insulin, 149

365

insulinlike growth factors, 150. 151 nerve growth factor, 143-146 transforming growth factor, 154, 155 Mudpuppy, taste cells and, 272 electrophysiological properties, 248, 249 intracellular recordings, 250,251 transduction mechanisms, 262, 266, 269,270 Muscles, cholinesterases and, 122. 123, 129-132 Mutagenesis AIDS and, 327,330,332, 333 cholinesterases and, 110, 134 Mutation AIDS and, 326, 327,331, 333 cholinesterases and, 110 myasthenia gravis and, 179

N N fibers, taste cells and, 253, 264, 266-269 Neocortex, neurotrophic factors and, 145, 146 Neoplasms, AIDS and, 306, 313-316, 319, 321 Nerve growth factor, central nervous system and, 142-149, 163 brain-derived neurotrophic factor. 152, 153 hormones, 161, 162 insulin, 151 lymphokines, 159 Neuroleukin, neurotrophic factors and, 163 Neurologic effects, AIDS and, 344, 345 gp120, 329 pediatric AIDS, 31 1-318, 321-325 Neuromuscular junction cholinesterases and, 115, 117, 128, 130 glutamate receptors and, 54 myasthenia gravis and, 177 presynaptic effects of toxins and, 230 calcium ion channels, 2 14, 2 16 potassium ion channels, 209-2 1 1, 2 13 release mechanisms, 2 17-220, 222, 225,226,228 sodium ion channels, 205-207

366

INDEX

Neuroophthalmic syndrome, AIDS and, 314,319 Neuroradiological features, A1 DS and, 320,32 1 Neurotoxicity AIDS and, 315,334-342 cholinesterases and, 11 1 neurotrophic factors and, 163 Neurotransmitter release, 1-3, 45, 46 calcium, 10 entry, 10-13 intracellular concentration, 13-22 removal, 22-28 calcium-voltage hypothesis, 41 -45 evidence, 35-38 formulation, 38-41 classical calcium hypothesis difficulties, 29-31 facilitation, 28, 29 revisions, 31-35 synaptic release calcium, 3, 4 characterization, 5-8 problems, 4, 5 residual calcium, 8, 9 Neurotransmitters cholinesterases and, 11 1, 113, 124 glutamate receptors and, 51, 54 myasthenia gravis and, 175, 176 neurotrophic factors and, 160-163 epidermal growth factor, 156, 157 insulinlike growth factors, 151 presynaptic effects of toxins and, 202, 23 1 neuronal ion channels, 206.2 13 release mechanisms, 22 1, 224 quinoxalinediones and, 284, 300 taste cells and, 255, 256, 265, 268, 269 Neurotrophic factors in central nervous system, 142, 163-165 brain-derived neurotrophic factor, 152, 153 ciliary neurotrophic factor, 153 epidermal growth factor, 153-157 fi' roblast growth factor, 147-149 hormones, 160, 161 adrenal, 162 estrogen, 161 thyroid, 161, 162 insulin, 149, 150

insulinlike growth factors, 149, 150 lymphokines, 158-160 IL-1, 158, 159 IL-2, 159, 160 nerve growth factor, 142-147 neurotransmitters, 160-163 platelet-derived growth factor, 157, 158 transforming growth factors, 153- 157 NMDA glutamate receptors and, 52, 54, 100 invertebrate, 90,95 mammalian, 66-73 noise, 6 0 , 6 1, 64, 65 quinoxalinediones and, 282-284, 301 excitotoxicity, 29 1 pharmacology, 285, 286,288-290 synaptic physiology, 292-300 Noise, glutamate receptors and, 57-60, 75,87 Norepinephrine, neurotrophic factors and, 162, 163 Notexin, presynaptic effects of, 2 11, 223, 224,228 Nuclear magnetic resonance, presynaptic effects of toxins and, 2 1 1 , 2 12 Nucleotides AIDS and, 327,330 myasthenia gravis and, 180 Nucleus basalis, neurotrophic factors and, 144

0

Oligomers, cholinesterases and, 110, 121, 132 Oligonucleotides, cholinesterases and, 1 18 Organophosphorous poisoning, cholinesterases and, 112

P Paradaxins, presynaptic effects of, 230 Paraformaldehyde, cholinesterases and, 126 Parkinson's disease cholinesterases and, 11 1 neurotrophic factors and, 164

INDEX

Pathology, AIDS and, 322-324 PC12 cells, presynaptic effects of toxins and, 218,219,221,229 PCP, glutamate receptors and, 72 Pediatric AIDS, 306-325, 344 Peptide T, AIDS and, 325,334,337,341, 342 Peptides AIDS and, 306,329,331, 340-342 cholinesterases and, 131, 133 experimental results, 129 polymorphism, 113-1 15, 117 myasthenia gravis and, 183, 184, 186-189, 192 neurotrophic factors and, 149, 150, 163 presynaptic effects of toxins and, 210, 211,216 Peripheral blood lymphocytes, myasthenia gravis and, 184, 189 Peripheral nervous system, neurotrophic factors and, 151, 159, 163 Peripheral neuropathy, AIDS and, 313, 315,319 Perefilaneta, glutamate receptors and, 86,87 Permeability cholinesterases and, 124 glutamate receptors and, 77, 78, 88 presynaptic effects of toxins and, 205, 224 taste cells and, 267, 269 pH, taste cells and, 269, 270, 273 Pharmacology AIDS and, 335,338 glutamate receptors and, 100 D-GluR, 81-83,87,90,91 H-GluR, 95-97,99 presynaptic effects of toxins and, 201, 203,204,207,208 quinoxalinediones and, 282, 284-292, 301 Phencyclidine, glutamate receptors and, 71 Phenotype AIDS and, 324 cholinesterases and, 112 myasthenia gravis and, 185, 186 Phenylthiocarbarnide, taste cells and, 254, 255

367

Phosphatidylinositol, 115, 133 Phosphatidylinositol-specific phospholipase C, 115 Phosphoglucose isomerase, neurotrophic factors and, 163 Phosphoinositides, glutamate receptors and, 54 Phosphoinositol, neurotrophic factors and, 157 Phospholipase A2 neurotoxins, 2 11. 222-228 Phosphorylation neurotransmitter release and, 2 presynaptic effects of toxins and, 2 10, 221,224,227 Picrotoxin, glutamate receptors and, 97,99 Pituitary, neurotrophic factors and, 159, 162 Plasma, cholinesterases and, 129, 133 Plasma membrane cholinesterases and, 115, 123, 124. 129, 133 presynaptic effects of toxins and, 225, 226 taste cells and, 271, 272 Plasmalemma, cholinesterases and, 132 Plasmapheresis, myasthenia gravis and, 177, 194 Plasticity neurotrophic factors and, 156, 162 quinoxalinediones and, 282, 297 Platelet-derived growth factor, 157, 158 Pleocytosis, AIDS and, 321 Pneurnocystis pneumonia, AIDS and, 307, 31 1 Polarization cholinesterases and, 126. 127, 129, 130 taste cells and, 243 electrophysiological properties, 250 epithelium, 247 transduction mechanisms, 260, 265, 269 Polyadenylation, cholinesterases and, 119 Polyamine, glutamate receptors and, 9 1, 100,101 Polymorphism, cholinesterases and, 111-118, 122, 123, 132 Polypeptides AIDS and, 330

368

INDEX

cholinesterases and, 109, 110 experimental observations, 1 18, 120, 122 polymorphism, 113 myasthenia gravis and, 179 neurotrophic factors and, 154 presynaptic effects of toxins and, 230 calcium channels, 214, 216 potassium channels, 210-2 13 sodium channels, 204, 206 Posttranslational modification, cholinesterases and, 109, 123 Potassium neurotransmitter release and, 25 presynaptic effects of toxins and, 202, 207-213,219,228 taste cells and electrophysiological properties, 248-250 transduction mechanisms, 257, 262, 265,270,272 Presynaptic effects of toxins, see Toxins, presynaptic effects of Progesterone, cholinesterases and, 124 Progressive multifocal leukoencephalopathy, AlDS and, 314 Proinsulin, neurotrophic factors and, 149 Proliferation AIDS and, 338,342 myasthenia gravis and, 186, 188, 189 neurotrophic factors and, 142, 148, 157, 158, 160 Proteases, neurotrophic factors and, 164 Protein AIDS and gp120,330,334,335,337,342,344 HIV, 327-329 pediatric AIDS, 32 1 cholinesterases and, 108, 110, 132, 133 experimental observations, 120, 122, 123 experimental results, 128, 129 polymorphism, 111, 112, 115, 117 Xenopus oocytes, 123, 124, 126 glutamate receptors and, 57, 69, 72, 8 1, 101 myasthenia gravis and, 177 acetylcholine receptor, 177, 179, 180 AChR-specific T lymphocytes, 186-188 thymus, 191-193

neurotransmitter release and, 2, 23 neurotrophic factors and, 142, 163 brain-derived neurotrophic factor, 152 epidermal growth factor, 156 insulin, 151 lymphokines, 158 presynaptic effects of toxins and, 202, 230,231 calcium ion channels, 215, 216 potassium ion channels, 208,210 release mechanisms, 2 16,2 19-222, 226 sodium ion channels, 202,203, 207 taste cells and, 255, 257-259 Protein kinase C neurotrophic factors and, 157 taste cells and, 256, 261, 262 Proteolysis AIDS and, 337 cholinesterases and, 133 glutamate receptors and, 76 taste cells and, 244, 258, 267

Q Quinine, taste cells and, 245, 249, 254-257 Quinoxalinediones, 28 1-283 binding, 283, 284 excitotoxicity, 29 1, 292 pharmacology, 290, 291 glutamate, 288,290 NMDA responses, 286, 288,289 quantitative methods, 286-288 semiquantitative methods, 284, 285 release, 284 synaptic physiology, 299, 300 hippocampus, 292-298 spinal cord, 298,299 Quisqualate in central nervous system, 282,284, 301 excitotoxicity, 292 pharmacology, 285, 286, 288, 290, 29 1 synaptic physiology, 299 glutamate receptors and, 54, 100 D-GluR, 81,82,87,89-91,93 H-GluR, 97-99 mammalian, 67,69, 7 1, 73, 74

369

1NI)EX

R Rats quinoxalinediones and, 286, 298 taste cells and, 243, 245 electrophysiological properties, 249, 250 transduction mechanisms, 254, 257, 259,263,266 Recombination, cholinesterases and, I I8 Removal buffer, neurotransmitter release and, 22 Replication, AIDS and, 325, 333, 335 Retrovirus, AIDS and, 307,326 Ribosomes, cholinesterases and, 118 RNA AIDS and, 323,327,329,331 cholinesterases and, 108- 11 I , 1 3 1- 133 experimental observations, 119, 120, 122, 123 experimental results, 129, 130 polymorphism, 117 glutamate receptors and, 69

S

Saccharin, taste cells and, 257, 260, 261 Salt taste, 242, 243, 253, 257, 263-270 Saturation, neurotransmitter release and calcium, 12, 13, 15, 16 calcium removal, 23, 26-28 classical calcium hypothesis, 29. 3 3 , 34 Saxitoxin, presynaptic effects of, 202-205 Schaffer collateral-commissural pathway. quinoxalinediones and, 292-294, 297 Scorpion toxins, presynaptic effects of. 204-206,208,212,213 Sea anemone toxins, presynaptic effects of, 206,207 Second messengers cholinesterases and, 124 glutamate receptors and, 54, 55, 57, 72 neurotransmitter release and, 2, 5 neurotrophic factors and, 157 presynaptic effects of toxins and, 22 I , 230 taste cells and, 256, 260, 27 1, 272 Septum, neurotrophic factors and, 144. 151 Serine, glutamate receptors and, 72

Short-term modulation, neurotransmitter release and, 4 Sigmoidicity, neurotransmitter release and, 41 Single-channel studies, glutamate receptors and, 100, 10 1 invertebrate, 74-99 mammalian, 60-65, 73 Snakes, presynaptic effects of toxins and, 215,222 Sodium presynaptic effects of toxins and, 202-207,216,229,231 taste cells and electrophysiological properties, 248-250 epithelium, 246, 247 transduction mechanisms, 253, 262-269,272 Sodium chloride, taste cells and, 245, 256, 262-270 Sour taste, 242, 245, 269, 270, 272 Spermatogenesis, cholinesterases and. 1 17 Spider toxins, presynaptic effects ot. 207, 215,216,220,231 Spinal cord AIDS and, 318,319 neurotrophic factors and, 147, 148, 154 presynaptic effects of toxins and, 202, 217 quinoxalinediones and, 284 pharmacology, 284-286, 288. 290 synaptic physiology, 298. 2Y9 Steroids, neurotrophic factors and. 160, 161

Streptomycin, glutamate receptors and, 82 Stress, neurotrophic factors and, 162 Striaturn, neurotrophic factors and. 146, 147 Strychnine glutamate receptors and, 70 quinoxalinediones and, 286 taste cells and, 249. 254 Subcellular compartments, cholinesterases and, 114, 115 Subclones. cholinesterases and, 110. 1 18 Subconductance, glutamate receptors and, 88.95 Substrates, cholinesterases and, 112-1 14 Sucrose, taste cells and, 245, 258-260, 262 Sucrose octaacetate, taste cells and. 254

370

INDEX

Sugar, taste cells and, 258, 260 Sweet taste, 242, 245, 253, 257-263, 269, 270 Synapses cholinesterases and, 115-1 17 glutamate receptors and, 55, 73, 74 neurotransmitter release and calcium, 3, 4, 10, 16-18 calcium-voltage hypothesis, 38, 45 characterization, 5-8 facilitation, 8, 9 problems, 4, 5 neurotrophic factors and, 150 quinoxalinediones and, 281, 282, 301 pharmacology, 290 physiology, 292-300 taste cells and, 243, 244, 253, 264, 273 Synaptosomes, presynaptic effects of toxins and, 229 neuronal ion channels, 206-209,213. 214 release mechanisms, 219, 221, 222, 225, 227

T T cells AIDS and, 323,324,326,338,342 myasthenia gravis and, 177 AChR-specific T lymphocytes, 184- 190 thymus, 191-194 Taipoxin, presynaptic effects of, 21 1, 222, 224 Taste cells, 241, 242, 272, 273 cell biology, 242-244 electroph ysiological properties, 247-250 epithelium, 246, 247 impediments to study, 244, 245 intracellular recordings, 250, 25 1 transduction mechanisms, 252,253 amino acid taste in catfish, 271, 272 bitter taste, 254-258 criterion for, 245, 246 salt taste, 263-269 sweet taste, 258-263 umami taste, 270, 271 Temperature, neurotransmitter release and, 45

classical calcium hypothesis, 28, 29, 33-35 synaptic release, 16, 18, 19, 21,22 Tenebrio, glutamate receptors and, 86, 87,91 Tetanus toxin, presynaptic effects of, 202, 216,217,219,220 Tetanus toxoid, myasthenia gravis and, 191 Tetrodotoxin presynaptic effects of, 229, 23 1 neuronal ion channels, 203-207,215 release mechanisms, 226 taste cells and, 248, 249 Thalamus AIDS and, 322,328 quinoxalinediones and, 299 Thaurnatin, taste cells and, 258, 259 Thymectomy, myasthenia gravis and, 193, 194 Thyrnoma, myasthenia gravis and, 190, 191, 193, 194 Thymus, myasthenia gravis and, 177 AChR-specific T lymphocytes, 184, 185, 187, 188 role, 190-193 Thyroid hormones, neurotrophic factors and, 161, 162 Thyroxine, neurotrophic factors and, 161 Tissue specificity, cholinesterases and, 132, 134 experimental observations, 122, 123 experimental results, 129, 130 polymorphism, 117, 118 Tongue, taste cells and cell biology, 242, 243 transduction mechanisms, 255, 257, 259,265,27 1 Torpedo cholinesterases and, 108-1 10 experimental observations, 120, 122 experimental results, 129, 130 polymorphism, 113, 115-1 17 glutamate receptors and, 77, 78, 88 myasthenia gravis and, 177 acetylcholine receptor, 177-180 AChR-specific T lymphocytes, 184- 189 anti-AChR antibodies, 182, 183 presynaptic effects of toxins and, 214, 219,221,227

INDEX

Toxins, presynaptic effects of, 20 I , 202, 230,23 1 neuronal ion channels calcium, 213-216 potassium, 207-213 sodium, 202-207 release mechanisms botulinum, 216-219 P-bungarotoxin, 222-227 glycerotoxin, 220-222 a-latrotoxin, 220,221 leptinotoxin, 220,222 maitotoxin, 229, 230 paradaxins, 230 phospholipase A2 toxins, 222-224, 226,227 tetanus toxins, 216,217, 219, ‘220 Toxoplasmosis, AIDS and, 314 Transcription, cholinesterases and, 108-1 10, 119, 123, 133 Transduction in taste cells, see Taste cells Transforming growth factor, 153-1 57 Translation, cholinesterases and, 107, 1OX, 115, 126 Translocation, presynaptic effects of toxins and, 217,218 Transmitters myasthenia gravis and, 181 presynaptic effects of toxins and, 22!), 23 1 neuronal ion channels, 208, 209, 2 1 1, 213,215 release mechanisms, 2 17, 2 19-22 I , 223-228 quinoxalinediones and, 288, 300 Triiodothyronine, neurotrophic factors and, 161 TI-ypsin, taste cells and, 244, 267 Tumorigenesis, neurotrophic factors and, 157 Tumors AIDS and, 307,315,319 cholinesterases and, 112, 123, 132 myasthenia gravis and, 190, 194 neurotrophic factors and, 154, 157, 158 Tunicamycin, cholinesterases and, 12X

37 1

U Urnami taste, 270, 27 1 V Vacuolar myelopathy, AIDS and, 313, 318, 323 Vasoactive intestinal peptide, AIDS and, 334, 335, 337, 340, 342 Vesicles cholinesterases and, 126. 128 myasthenia gravis and, 178 neurotransmitter release and calcium, 14, 18 calcium-voltage hypothesis, 3 9 \ 4 0 synaptic release, 4, 6 presynaptic effects of toxins and, 221, 224,226,227 taste cells and, 271, 272 Voltage glutamate receptors and, 70 neurotransmitter release and, 18, 35-46 Voltage dependence presynaptic effects of toxins and. 205, 208-212,216 quinoxalinediones and, 294. 297 taste cells and, 272 electrophysiological properties. 249, 250 intracellular recordings, 25 1 , 268, 269 transduction mechanisms, 260

X Xenopus glutamate receptors and, 55,69 oocytes, cholinesterases and, 107- 1 1 I , 130-135 experimental observations, 1 IX-123 experimental results. 126, 12% 130 organ cultures, 123-127 polymorphism, 11 1-1 I X presynaptic effects of toxins and, 209, 210

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CONTENTS OF RECENT VOLUMES

Volume 22

Fluctuations of Na and K Currents in Excitable Membranes Berthold Neumcke

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

Biochemical Studies of the Excitable Membrane Sodium Channel Robert L . Barchi

Brain Intermediary Metabolism zn Vivoc Changes with Carbon Dioxide, Development, and Seizures Alexander L. Miller

Benzodiazepine Receptors in the Central Nervous System Phil Skolnick and Steven M . Paul

N,N-Dimethyltryptamine: An Endogenous Hallucinogen Steven A . Barker, John A. Monti, and Samuel T. Chrdian

Rapid Changes in Phospholipid Metabolism during Secretion and Receptor Activation F. T . Crews

Neurotransmitter Receptors: Neuroanatomical Localization through Autoradiography L. Charles Murrin

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

Neurtoxins as Tools in Neurobiology E . G. McGeer and P. L. McCeer

Assessing the Functional Significance of Lesion Induced Neuronal Plasticity Oswald Steward

Mechanisms of Synaptic Modulation William Shain and David 0 . Carpentpi

Dopamine Receptors in the Central Nervous System Ian Cresse, A . Leslie Mowow, Stuart E . Lefi, David R. Sibley, and Mark W . Harnblzn

Anatomical, Physiological, and Behavioral Aspects of Olfactory Bulbectomy in the Rat B . E. Leonard and M . Tuite

Functional Studies of the Central Catecholamines T. W . Robbzns and B. J . Everztt

T h e Deoxyglucose Method for the Measurement of Local Glucose Utilization and the Mapping of Local Functional Activity in the Central Nervous System Louis Soklolofjf

Studies of Human Growth Hormone Secretion in Sleep and Waking Wallace B . Mendelson

INDEX

Sleep Mechanisms: Biology and Control of REM Sleep Dennis J . McCinty and Ren6 R. Drucker-Colin

Volume 23

Chemically Induced Ion Channels in Nerve Cell Membranes David A . Mathers and Jeffeery L. Barke7

373

INDEX

374

CONTENTS OF RECENT VOLUMES

Volume 24

Dental Sensory Receptors Margaret R . Eyers

Antiacetylcholine Receptor Antibodies and Myasthenia Gravis Bernard W . Fulpius

Cerebrospinal Fluid Proteins in Neurology A . Lawenthal, R . Crols, E. De Schutter, J . Gheurens, D. Karcher, M . Noppe, and A . Tasnier

Pharmacology of Barbiturates: Electrophysiological and Neurocheniical Studies Max Willow and Graham A . R . Johnston Immunodetection of Endorphins and Enkephalins: A Search for Reliability Alejandro Bayon, William J . Shoemakrr, Jacqueline F. McGznty, and Floyd Bloom On the Sacred Disease: The Neurochemistry of Epilepsy 0. Carter Snead I I I Biochemical and ElectrophysiologicalCharacteristics of Mammalian GABA Receptors Salvatore J . Enna and Joel P. 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 F. J . Barrantes Characterization of a , - and an-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 . Black, and Robert E . Foster

Muscarinic Receptors in the Central Nervous System Mordechai Sokolovsky Peptides and Nociception Daniel Luttinger, Daniel E . Hernandez, Charles B. Nemerofl, and Arthur J . Prange, Jr. Opioid Actions on Mammalian Spinal Neurons W . Zieglgalzrberger 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

Volume 26

INDEX

The Endocrinology of the Opioids Mark J . Millan and Albert Herz

Volume 25

Multiple Synaptic Receptors for Neuroactive Amino Acid Transmitters-New Vistas Najam A . Sharif

Guanethidine-Induced Destruction of Sympathetic Neurons Eugene M . Johnston, Jr. and Pamela Toy Manning

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

C O N T E N T S OF RECENT VOLUMES

Neural Plasticity and Recovery of Function after Brain Injury John F. Marshall From Immunoneurology to Immunopsychiatry: Neuromodulating Activity of AntiBrain Antibodies Branislau D. Janklovit Effect of Tremorigenic Agents on the Cerebellum: A Review of Biochemical and Electrophysiological Data V. G. Long0 and M . Massottz INDEX

Volume 28

Biology and Structure of Scrapie Prions Michael P. McKinley and Stnnlev B. Prusiner Different Kinds of- Acetylcholine Release from the Motor Nerve S . Thesleff Neuroendocrine-Ontogenetic Mechanism of Aging: ‘Toward an Integrated Theory of Aging V . M . Dilman, S . Y . Revskloy, and A. G. Golubev

T h e Interpeduncular Nucleus Barbara J . Morley

Volume 27

The Nature of the Site of General Anesthesia Keith W . Miller T h e Physiological Role of Adenosine i n the Central Nervous System Thomas V. Dunwzddie Somatostatin, Substance P, Vasoactive Intestinal Polypeptide, and Neuropeptide Y Receptors: Critical Assessment of Biochemical Methodology and Results Anders VndCn, Lou-Lou Peterson, and Tamas Bartfai Eye Movement Dysfunctions and Psychosis Philip S.Holzman

Biological Aspects of Depression: A Review of the Etiology and Mechanisms of Action and Clinical Assessment of Antidepressants S . 1. Ankier and B . E . Leonard Does Receptor-Linked Phosphoinositide Metabollism Provide Messengers Mobilizing Calcium in Nervous Tissue? John N . Hawthome Short-Term and Long-Term Plasticity and Physiological Differentiation of Crustacean Motor Synapses H . L. Atwood and J . M. Wojtowicz Immunology and Molecular Biology of the Cholinesterases: Current Results and Prospects Stephen Brimljoin and Zoltan Kakonrruy

Peptidergic Regulation of Feeding J . E . Morley, T.J . Bartness, B . A. Gosnell. and A . S . Levine

INDEX

Calcium and Transmitter Release Ira Cohen and William V a n der Klool

Volume 29

Excitatory Transmitters Related Brain Damage John W . Olney

375

Epilepsy-

Molecular Genetics of Duchenne and Becker Muscular Dystrophy Ronald G. Worton and Arthur H . M . Hurghes

Potassium Current in the Squid Giant Axon John R. Clay

Batrachotoxin: A Window on the Allosteric Nature of the Voltage-Sensitive Sodium Channel George B . Brown

INDEX

and

376

CONTENTS OF RECENT VOLUMES

Neurotoxin-Binding Site on the Acetylcholine Receptor Thomos L . Lentz and Paul T . Wilson Calcium and Sedative-Hypnotic Drug Actions Peter L. Carlen and Peter H . W u Pathobiology of Neuronal Storage Disease Steven U. Walkley Thalamic Amnesia: Clinical and Experimental Aspects Stephen G . Waxman Critical Notes on the Specificity of Drugs in the Study of Metabolism and Functions of Brain Monoamines S. Garattini and T. Menninz Retinal Transplants and Optic Nerve Bridges: Possible Strategies for Visual Recovery as a Result of Trauma or Disease James E . Turner,J e n y R. Blair, Magdalene Seiler, Robert Aramant, Thomas W . Laedtke, E . Thomas Chappell, and Lauren Clarkson Schizophrenia: Instability in Norepinephrine, Serotonin, and y-Aminobutyric Acid Systems Joel Gelernter and Daniel P. van Kammen INDEX

Volume 30

Biochemistry of Nicotinic Acetylcholine Receptors in the Vertebrate Brain Jakob Schmidt The Neurobiology of N-Acetylaspartylglutamate Randy D. Bhkely and Joseph T. Coyle

Neuropeptide-Processing, -Converting, and -Inactivating Enzymes in Human Cerebrospinal Fluid Lars Terenitu and Fred Nyberg

Targeting Drugs and Toxins to the Brain: Magic Bullets Lance L. Simpson Neuron-Clia Interrelations Antonia Vernadakis Cerebral Activity and Behavior: Control by Central Cholinergic and Serotonergic Systems C. H . Vandenuolf INDEX

Volume 31

Animal Models of Parkinsonism Using Selective Neurotoxins: Clinical and Basic Implications Michael J . Zigmond and Edward M . Stricker Regulation o f Choline Acetyltransferase Paul M . Salvaterra and James E . Vaughn Neurobiology o f Zinc and Zinc-Containing Neurons Christopher J . Frederickson Dopamine Receptor Subtypes and Arousal Ennio Ongani and Vincenzo G . Long0 Regulation of Brain Atrial Natriuretic Peptide and Angiotensin Receptors: Quantitative Autoradiographic Studies J u a n M . Saavedra, Eero Castrkn, Jorge S . Gutkind, and AdilJ. Namrali Schizophrenia, Affective Psychoses, and Other Disorders Treated with Neuroleptic Drugs: The Enigma of Tardive Dyskinesia, Its Neurobiological Determinants, and the Conflict of Paradigms John L . Waddington Nerve Blood Flow and Oxygen Delivery in Normal, Diabetic, and Ischemic Neuropathy Phillip A . Low, Terrence D.Lugerlund, and Philip G. McManis INDEX