International Review of Neurobiology Volume 34

International Review of

NEUROBIOLOGY VOLUME 34

Editorial Board W. Ross ADEY

PAULJANSSEN

J UI.IUS AXELROD

SEYMOUR KETY

Ross BALDESSARIICI

KEITH

SIR

ROGERB A N N I S T E R

KILLAM

CONANKORNETSKY

FLOYDBLOOM

ABELLAJTHA

DANIEL BOVET

BORISLEBEDEV

PmI.LIP

BRADLEY

PAULMANDEL

YYRI BUROV

HUMPHRY OSMOND

Jos6 DELGADO

RODOLFOPAOLETTI

SIRJOHN ECCLES

SOLOMON SNYDER

JOEL

ELKES

H. J. EYSENCK KJELL.

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STEPHEN SZARA

MARATVAKTANIAN STEPHEN WAXMAN RICHARDWYATT

International Review of

NEUROBIOLOGY Editedby

RONALD J. BRADLEY Department of Psychiatty and Behavioral Neurobiology The Medical Center Universiiy of Alabama Birmingham, Alabama

VOLUME 34

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CONTENTS

Neurotransmitters as Neurotrophic Factors: A N e w Set of Functions JOAN

I. 11. 111.

IV. V.

P. SCHWARTZ

.............. Introduction ........................ Direct Trophic Actions. ...... Indirect Actions via Other Cells. . . . . . . . .............. Regulation of Synthesis and Response .............................. Summary and Conclusions.. ........................ References .................. .................

1 3 12 14 18 20

Heterogeneity and Regulation of Nicotinic Acetylcholine Receptors

RONALDJ. LUKASAND MEROUANEBENCHERIF I. 11. 111.

... Introduction .................................... Models and Concepts in Studies of Receptor Regulation.. . . . . . . . . . . . . Regulation of Muscle Nicotinic Acetylcholine Receptor Expression

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

25 73 78

nd Ganglia-Type Nicotinic Acetylcholine V. VI.

Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .......................... Central Neuronal Receptors Perspectives and Conclusions ............................. .......................... References

95 103 112 112

Activity-Dependent Development of the Vertebrate Nervous System

R. DOUGLAS FIELDSAND PHILLIPG. NELSON I. 11. 111.

IV.

Introduction ..................................................... Properties of Activity-Dependent Neuronal Development. ............ Mechanisms.. .................................. . . . . . . . . . . . . . . . . . . Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

V

133 134 161 186 199

vi

CONTENTS

A Role for Glial Cells in Activity-Dependent Central Nervous Plasticity? Review and Hypothesis CHRISTIAN

M. MULLER

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Evidence for Participation of Glial Cells in Activity-Dependent . . Plastlclty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Regeneration and Adaptive Processes following CNS Damage.. . . . . . . . IV. A Unifying Hypothesis for Invulvenient of Glial Cells in ActivicyDependent Plasticity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Summary. .. ... References

I.

215

11.

231 2.55

258 267 268

Acetylcholine at Motor Nerves: Storage, Release, and Presynaptic Modulation by Autoreceptors and Adrenoceptors

ICNAZ WESSLER I. 11. 111.

1V. V. VI.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Basic Events.. . . . . . . . . . . . . . . Detection Methods. . . . . . . . . . Modulation of Release by Autoreceptors Modulation of Release by Conclusion.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . .

2x3

286 30.1 312 354 372 372

INDEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

3x5

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

408

CONTENTS OF RECENT VOLUMES

NEUROTRANSMITTERS AS NE UROTROPHIC FACT0 RS: A NEW SET OF FUNCTIONS Joan P. Schwartz Unit on Growth Factors, Clinical Neuroscience Branch National institute of Neurological Disorders and Stroke National institutes of Health Bethesda, Maryland 20892

I. Introduction 11. Direct Trophic Actions

A. Neurotransmitters B. Neuropeptides 111. Indirect Actions via Other Cells A. Neurotransmitter Regulation of Neurotrophic Factor Production B. Neuropeptide Regulation of Neurotrophic Factor Production IV. Regulation of Synthesis and Response A. Glial versus Neuronal Synthesis B. Differential Neuropeptide Precursor Processing C. Developmental Receptors D. Developmental versus Injury-Induced Expression V. Summary and Conclusions References

I. Introduction

Naturally occurring neural cell death was recognized as a normal process many years ago and led ultimately to the discovery of nerve growth factor (Oppenheim, 1989). Although nerve growth factor is currently the best understood neurotrophic factor, a number of others have recently been identified and characterized, including brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), and neurotrophin-3 (NT-3) (Maisonpierre et al., 1990). Not only soluble factors such as nerve growth factor, but also adhesion molecules such as laminin play a role in the survival and/or differentiation of neurons (Jessell, 1988). In addition, an ever-increasing number of neurotransmitters and neuropeptides are being added to the category of neurotrophic factors as the result of numerous studies in a variety of systems: it is the neurotrophic effects of neurotransmitters and neuropeptides that this review covers. 1 INTERNATIONAL REVIEW OF NEUROBIOLOGY. VOL. 34

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Since the initial identification of acetylcholine by Dale, neurotransmitters have been classified as molecules that mediate intercellular communication. In the context of the adult nervous system, such communication was assumed to be neuronal-neuronal and, therefore, to be involved in the generation of action potentials and the firing of neurons. Clearly much of the signaling mediated by neurotransmitters in the adult brain is in fact neuronal-neuronal; however, increasing evidence suggests that even in the adult, neurotransmitters and neuropeptides also mediate neuronal-glial and even glial-glial signals, none of which are directly responsible for the generation of action potentials. In the deveioping nervous system, the evidence for trophic functions of neurotransmitters is even more compelling. Both neurotransmitters and neuropeptides are expressed early, at times when synaptic connections have not yet been made. In some instances, the expression occurs in glia. Evidence for the existence of special classes of receptors, expressed only during development, has been published. Effects of neurotransmitters in cultures of cells from the developing nervous system provide further support. In such culture models, both neurotransmitters and neuropeptides have been demonstrated to affect a series of parameters, including cell division, neuronal survival, neurite sprouting and growth cone motility, and neuronal and glial phenotype. Some of these actions are direct, whereas others are mediated indirectly, as when a neurotransmitter stimulates an astrocyte to produce a neurotrophic factor needed for a neuron's survival. Taken in toto, the in z&o and in vitro data compellingly support the idea of neurotransmitters as trophic factors, at least during development and possibly also in the adult nervous system, That leads to the possibility that neurotransmitters may also play a key role in aging or in neurodegenerative diseases. On one hand, functions that were expressed only developmentally may be turned back on, as neurons die out. On the other hand, the balance between neurotransmitters may be disrupted, allowing, for example, elevated levels of the excitatory amino acids, which in turn can lead to neuronal death. The role that glia play in these processes is now being examined. 'The classic neurotrophic factors, which include soluble factors such as nerve growth factor and adhesion components such as laminin, have been defined as (1) factors that prevent natural cell death and/or support neuron survival, (2) factors that induce neurite outgrowth, and (3) factors that induce a differentiated neuronal phenotype. For the purpose of this review, I expand this definition to include effects of neurotransmitters and neuropeptides on (1) cell division, (2) cell survival, (3) neurite sprouting and growth cone motility, and (4) a differentiated phenotype. Furthermore, I discuss effects on both neuronal and glial

NEUROTRANSMITTERS AS NEUROTROPHIC FACTORS

3

cells as well as on interactions between them. Finally, although I concentrate on the developmental aspects of this topic, I end with a consideration of the potential functions in adult, aging, and degenerating brain.

II. Direct Trophic Actions

A. NEUROTRANSMITTERS 1. Mitosis

Three questions to which neurobiologists are now seeking answers are: How is the division of neural cells regulated? What are the factors involved? And how are the mitotic processes of different populations of cells within a brain region coordinated? Although much attention has been focused on mitotic growth factors (i.e., epidermal growth factor, fibroblast growth factor), the possibility that neurotransmitters could be involved is gaining attention. Early work had centered on the possible role of the monoamines, as these are among the earliest neurotransmitters to be expressed in the nervous system, appearing 1-4 days prior to synaptogenesis. The early studies, many on nonmammalian systems and tissues other than brain, are summarized by Lauder et al. (1982). In their paper, the authors explore the possibility that serotonin (5-hydroxytryptamine, 5-HT) may act as a trophic substance, particularly in regulating the proliferation of several different populations of cells. The basic paradigm underlying their experiments involved treatment of pregnant rats with para-chlorophenylalanine (PCPA),a drug that depletes 5-HT, from Day 8 of gestation (E8) to the time of injection of [3H]thymidine (El 1E 16). Animals were sacrificed 30 days after birth and autoradiography was performed to determine changes in numbers of dividing cells as a result of PCPA treatment in those brain regions known to be innervated by 5-HT. Effects were seen in all areas that are innervated by 5-HT axons during that period and consisted of either suppression of the onset, complete suppression, or prolongation of neuronal genesis. Similar effects were seen on monoamine neurons. Overall, the results suggest that those neurons innervated by 5-HT that are dividing at the time of PCPA administration are affected. In addition, combined immunohistochemistry for 5-HT and autoradiography for [3H]thymidine suggested that 5-HT may regulate division of neuroepithelial cells, glioblasts in the presumptive internal granule layer of the cerebellum, and neuroblasts in the dentate hilus of the hippocampus. These experiments

4

JOAN P. SCHWAKTZ

provide suggestive evidence that 5-HT can function to regulate neural cell division early in development before it has begun to function as a “classic” neurotransmitter. This type of in Z I ~ U Oapproach, pharmacological manipulation of neurotransmitter levels, is particularly important. in establishing the physiological relevance. ‘I’issue culture studies have demonstrated that both acetylcholine (ACh) and norepinephrine (NE) can stimulate division of astrocytes 1989). Primary cultures of glia were prepared from (Ashkenazi ~t d., whole brain of rats from Embryonic Day 14 (E14) to Postnatal Day 15 ( 1 W D15). T h e cultures contained 90% glial fibrillary acidic proteinpositive (GFAP+) astrocytes. Carbachol, a muscarinic cholinergic agonist, stimulated Dh’A synthesis over this time course, with a peak at birth. Korepinephrine was also active though less potent. T h e effects were blockable by the expected antagonists, demonstrating action through the rnuscarinic and a,-adrenergic receptors respectively. In contrast, Nicoletti et al. (1990) demonstrated that excitatory amino acids, acting through a quisqualate-type glutamate receptor, could inhibit astrocyte division. Interestingly, all of these effects were mediated through receptors that activate inositol phospholipid turnover. These results suggest that neurons could regulate the numbers of surrounding astrocytes through release of their neurotransnkters.

2. Neuroii SunJiZvil Cerebellat granule cell neurons require elevated potassium levels (25mM) for survival in culture medium containing serum. A series of elegant experiments by Balazs’ laboratory suggest that this requirement reflects the in z ~ i 7 1 0innervation of the granule cells by the mossy fibers: the in uitro requirement develops at the same time as innervation occurs (PND10-PND12) (Gallo P t al., 1987). T h e mossy fibers use glutamate as a neurotransmitter and Rallizs has shown that glutaniate analogs can substitute for potassium. Thus, N-methyl-D-aspartic acid (NMDA) can completely rescue neurons cultured in low potassium: this effect is blocked by NMDA receptor antagonists such as APV and MK-801 (Balazs P t a/., 1988b,c, 1989).More recent studies have shown that kainic acid, acting through a non-KMDA receptor, can also promote survival in the pl-esence of low potassium, an effect that is additive with that of NMDA when both are present at suboptimal concentrations (Ralazs el al., 199Oa,b).T h e authors conclude that all of these agents act to increase intracellular calcium: potassium and kainic acid through voltagesensitive calcium channels and NMDA through its receptor-gated calcium channel. T h e overall interpretation is that mossy fiber-afferent

NEUROTRANSMITTERS AS NEUROTROPHIC FACTORS

5

stimulation, mediated by glutamate, is needed by the cerebellar granule cells at a specific developmental stage (PNDlO-PND12) for survival. NMDA antagonists have been shown to produce neuronal cell death in spinal cord cultures prepared from E12-El4 animals (Brenneman et al., 1990a). The magnitude of cell death is similar to that produced by blocking action potentials with tetrodotoxin (TTX). This effect was observed in developing cultures (2 weeks in culture) but not in those that were 1 month old. Low concentrations of NMDA (10-1000 nM) increased neuronal survival in TTX-blocked cultures. Furthermore, a low (0.1 nM) concentration of vasoactive intestinal peptide prevented the cell death associated with NMDA blockers or TTX (Brenneman et al., 1990~).These studies suggest that multiple neurochemical inputs interact to determine the survival of spinal cord neurons.

3. Neuronal Sprouting Glutamate not only permits survival of cerebellar granule cells; it also acts to stimulate neurite extension at earlier developmental periods. Cells prepared from PND4-PND5 rats extend processes in response to the glutamate endogenously released in the cultures (Pearce et al., 1987). Blockade of NMDA receptors by APV or kynurenate depressed neurite extension approximately 50%; addition of glutamate or NMDA overcame the inhibition. Thus, glutamate can promote neuronal sprouting at earlier stages and survival at later stages in cerebellar granule cell development. It is of course difficult to ascertain whether glutamate is still neurite promoting at later stages as the cells become dependent on it for survival. T h e pyramidal neurons in the hippocampus respond to glutamate in exactly the opposite way as was seen for cerebellar granule cells. At low doses, glutamate inhibits neuronal sprouting whereas at higher doses it kills the neurons: these effects are also mediated via regulation of intracellular calcium content, as was proposed for the cerebellar granule cells. Glutamate added at less than 50 pI.4 to dissociated pyramidal cell cultures causes neurite retraction, an effect mediated by a kainic acid/quisqualate-type glutamate receptor (Mattson et al., 1988a). Glutamate at 1 mM results in death of 60% of the pyramidal neurons within 2 days. Mattson et al. (1988b) used a clever co-culture system to demonstrate that glutamate derived from innervating fibers of the entorhinal cortex was responsible. Hippocampal neurons seeded onto a mat of axons from an entorhinal cortex explant produced neurites considerably shorter than those of hippocampal cells seeded directly onto the surface of the dish off the axon mat (59 pm versus 216 pm in length). A

6

JOAN P. SCHWARTZ

glutamate antagonist, D-glutamylglycine, increased dendritic outgrowth while simultaneously decreasing “presumptive synaptic sites” (measured by staining for Zn2+ or the synaptic vesicle phosphoprotein protein 111). Mattson et a f . (1988a,b) have proposed that glutamate acts to model neuronal circuitry in the hippocampus during development and possibly plays a role in adult plasticity. Mattson and Kater further propose that other neurotransmitters, as well as growth factors, can interact with glutamate in either a positive or a negative way, the net result being the adult neuronal circuit of the hippocampus. Thus, ACh potentiates the effects of glutamate on toxicity (Mattson, 1989),whereas GABA, in combination with a benzodiazepine, decreases the toxicity of glutamate (Mattson and Kater, 1989). Fibroblast growth factor by itself promotes both survival and neurite outgrowth of hippocampal pyramidal cells while significantly raising the threshold for glutamate toxicity (Mattson et al., 1989). Further evidence for afferent regulation of neuronal sprouting comes from studies by two laboratories on the effects of amacrine cell neurotransmitters on cultured retinal ganglion cells. ACh antagonists stimulated the length of mammalian retinal ganglion cell processes, without affecting cell survival (Lipton et al., 1988). ’I‘he effect was specific to nicotinic ACh antagonists (d-tubocurare or mecamylamine); the muscarinic antagonist atropine had no effect. Dopamine (DA) decreased growth cone motility as well as neurite elongation of avian retinal ganglion cells in culture, with effects seen within 15 min of addition (Lankford et al., 1987). Only 25% of the retinal ganglion cells responded to dopamine, and the effect was mediated by a D, receptor linked to adenylate cyclase (Lankford et al., 1988). Similar inhibition of‘ growth cone motility and neurite elongation by neurotransmitters goes back evolutionarily all the way to the snail Hellsoma: both dopamine and 5-IIT are inhibitory to different but overlapping populations of neurons in the buccal ganglia (Haydon et al., 1984; McCobb et al., 1988). In summary, these results suggest that neurotransmitters contained in afferent innervation neurons can model the extent of dendritic outgrowth of a neuron, in either a positive or a negative way, during development. The potential role of such interactions in the adult CNS, or in neurodegeneration, is considered later (Section IV,D). 4. Dzfferentiated Phenotype

Two studies provide indirect evidence that neurotransmitters can affect not only neurite sprouting but also expression of the differentiated neuronal phenotype of a specific class of neurons. Balazs et al. ( 1988a) demonstrated that cerebellar granule cells can survive in serum-

NEUROTRANSMITTERS AS NEUROTROPHIC FACTORS

7

free medium containing a low concentration of potassium (5 mM), whereas when grown in the presence of serum, the cells required either high potassium (25 mM) or glutamate for survival. Interestingly, cells grown under the nondepolarizing condition of serum-free medium were much less phenotypically mature, expressing virtually no acidic amino acid carrier. In addition, the appearance of veratridine-stimulated glutamate release and of voltage-sensitive Ca2 channels was delayed. Addition of potassium, to achieve 25 mM, to the serum-free medium overcame these maturational deficits. It would be very interesting to ascertain whether glutamate or NMDA was similarly active and whether cells grown in serum-free medium expressed the NMDA receptor. Along similar lines, chick embryo extract is known to promote the adrenergic phenotype in cultured neural crest cells. Sieber-Blum (1989) demonstrated that NE uptake blockers, such as desmethylimipramine, decreased both the number of NE-fluorescent cells and the amount of tyrosine hydroxylase and dopamine 6-hydroxylase in the cultures. As NE is present in chick embryo extract, the results suggest that it may be involved in regulating expression of the adrenergic phenotype, particularly because the NE uptake system appears prior to synthesis and storage of catecholamines (Rothman et al., 1978; Xue and Smith, 1988). +

B. NEUROPEPTIDES Even more so than with neurotransmitters, interest has focused on the early developmental expression of neuropeptides and what this might mean functionally in the CNS. In particular, certain neuropeptides are expressed early in brain development in specific brain regions and then decline dramatically or disappear. The best understood example functionally is a set of neurons described by Shatz and colleagues (Chun et al., 1987; Ghosh et al., 1990). These neurons are present in the subplate and marginal zones of the telencephalon during development, receive synaptic inputs from ingrowing thalamocortical axons, and disappear as layer I of the cortex and the white matter emerge. While present, certain of the neurons are immunoreactive for different peptides, including neuropeptide Y (NPY), somatostatin (SS), and cholecystokinin (CCK). Ablation of these neurons, with kainic acid, altered the normal thalamic innervation of the cortex, suggesting that these cells may be necessary as temporary targets for ingrowing axons and that the different neuropeptides may be specific targets for different ingrowing axons (Ghosh et al., 1990). The cerebellum is also of interest because there are a number of

8

JOAN P. SCHWARTZ

peptides expressed early in development, whose content then decreases along with their receptors. Both Inagaki et al. (1982, 1989) and Naus (1990) have demonstrated that prosomatostatin mRNA, by in sztu hybridization, and S S peptides, by imniunohistochemistry, peak around PNDlO and then decline, with few positive cells being present in adult cerebellum. Similar analyses have been published for SS in cortex (Naus ~t ul., 1988a) and in hippocampus (Naus et al., l988b); however, unlike cei-ebellum, both of these brain regions contain significant numbers of SS-positive neurons in the adult. In parallel with the transient expression of cerebellar SS is a transient expression of SS receptors, which disappears between PNDlS and PND23 (Gonzalez et af., 1988). Pharmacologically these receptors do not differ from those found in other regions of the adult rat brain (Gonzalez ef al., 1990). Yamashita et ul. (1990) have reported that the immunoreactivity of not only SS, but also substance P and CCK, declines to almost undetectable levels in adult monkey cerebellum. In contrast, although free enkephalin peptides, as well as their receptors, show a comparable decline in cerebellum, with a peak around PND4-PND7 (Tsang et al., 1982), and virtually no cells are deteciahle bv immunohistochemistry in adult cerebellum (Zagon et al., 1985), Spruce et al. (1990) have recently reported that the unprocessed precursor, proenkephalin, is present in both neurons and astrocytes of adult cerebellum at higher levels than during development. All of these data support the possibility that peptides are expressed developmentally in the cerebellum to function in trophic roles rather than transmitter roles. T w o peptides that function in both the pituitary and the brain show early developniental expression and/or processing. Altstein and Gainer ( 1988) demonstrated that arginine vasopressin (AVP) was synthesized slightly earlier arid processed much faster than oxytocin (Or),such that the steady-state brain levels of AVP were 5- to 10-fold higher from E l 6 to E2 1. Furthermore, AVP was transported to the pituitary 3 days earlier and present at a 20-fold higher concentration than OT. The gene and peptides of proopiomelanocortin (POMC) are similarly expressed early 1989) and in Xenopus brain embryonically both in rat brain (Elkabes et d., and pituitary (Hayes and Loh, 1990). Furthermore, POMC fibers were present in dense tracts in embryonic rat brain (Elkabes et ul., 1989). Both AVY and POMC remain at high levels in adult brain and pituitary and function in both neuropeptide and pituitary hormonal modes, but the embryonic expression occurs far earlier than any of these adult functions appear, leading the authors to suggest the possibility that the peptides are involved in developmental roles in the early embryonic stages.

NEUROTRANSMITTERS AS NEUROTROPHIC FACTORS

9

1. Mitosis A number of laboratories have demonstrated mitotic effects (both stimulation and inhibition) of various peptides on nonneural tissues, including connective tissue cells (Nilsson et al., 1985), Swiss 3T3 fibroblasts (Zachary et al., 1987), lymphocytes (reviewed by Zachary et al., 1987), and HeLa and fibroma cells (Mascardo and Sherline, 1982), as well as on intrauterine growth (Swaab et al., 1976) and salamander limb regeneration (Smith and Hui, 1973). Two points can be made: first, the results demonstrate that neuropeptides can be mitotic, and second, they raise the possibility that neuropeptides released from nerve endings innervating these tissues may regulate mitosis of the innervated cells. A particularly interesting example, discussed by Hanley ( 1985), is that substance P and/or substance K might be mitotic for skin and cornea epithelial cells; hence capsaicin destruction of sensory fibers containing substance P and/or substance K would result in the decrease in the number of these cells that is seen following capsaicin treatment. Cleavage of the vasopressin precursor generates not only the neuropeptide AVP but also neurophysin (Vp-Np). Vp-Np was shown to stimulate DNA synthesis and division of cells in PND6 hypothalamic cultures (Worley and Pickering, 1984); as 98% of the cells are GFAP+, the effect of Vp-Np is apparently on astrocytes. This is of interest because it suggests a novel growth-regulating function for a “nontransmitter” product of the vasopressin precursor. Two neuropeptides have been implicated in regulation of the division of both neurons and glia, supporting further the concept that neurotransmitters and neuropeptides can function in determining the overall numbers and types of cells in a given part of the nervous system. Vasoactive intestinal peptide (VIP), which innervates the superior cervical ganglion (SCG) in uiuo, stimulates the number of neurons in cultures of SCG from E15.5 embryos, without affecting the number of nonneuronal cells (Pincus et al., 1990a,b). This effect was seen at high doses of VIP (1 pA4 maximal), suggesting that it is mediated via the lowaffinity VIP receptor. In contrast, Brenneman et al. (1990b) have reported that much lower doses of VIP (0.1- 1 a), which activate a highaffinity VIP receptor, are mitogenic for astrocytes in E12-El4 spinal cord cultures. A potential role for endogenous opioid peptides in the regulation of neural cell division was first proposed by Vkrtes et al. in 1982 based on in vivo experiments. Young (PNDl 1) rats were injected with either naloxone (2 mg/kg), an opiate antagonist, or Met-enkephalin (200 pg/kg,

10

JOAN P. SCHWARTZ

a-Met2-Pro5-enkephalinamide); 1 to 12 hr later they received [3H]thymidine and were sacrificed after 30 min. Naloxone increased thymidine incorporation into the forebrain and hypothalamus at 1 to 3 hr, but decreased it at 9 to 12 hr, whereas Met-enkephalin decreased it in all brain regions including cerebellum. The next year Zagon and McLaughlin published the first of a series of studies in which newborn rats were treated with either 1 or 50 mg/kg naltrexone, another opiate antagonist, for 21 days. In the first paper, Zagon and McLaughhn (1983a) reported that 50 mg/kg naltrexone increased brain and body weight, as well as the numbers of neurons and glial cells in the cerebellum. Histological and morphometric studies analyzed these changes in more detail in cerebellum (Zagon and McLaughlin, 1986b) as well as cortex and hippocampus (Zagon and McLaughlin, 1986a). These studies further suggested that whereas 50 mg/kg naltrexone tended to increase numbers of cells, 1 mg/kg instead was inhibitory. Cerebellar incorporation of [3H]thymidine was stimulated by 50 mg/kg naltrexone but decreased by 1 mg/kg, as well as by 80 Fg/kg Met-enkephalin (Zagon and McLaughlin, 1987). Kornblum et al. (1987) demonstrated that morphine (5 mg/kg) also decreased ["HJthymidine incorporation in newborn rat brain but that neither naloxone (2-5 mg/kg) nor naltrexone (50 mg/kg) had any effect. T h e inhibitor): effect of Met-enkephalin has been confirmed in culture (Stiene-Martin and Hauser, 1990): 1 pkl Met-enkephalin decreased DNA synthesis and division of cortical astrocytes in mixed glial cultures prepared from PNDl mice. Taken together, these studies suggest that endogenous opioid peptides may act early postnatally to regulate the numbers of both neurons and glial cells, particularly in the cerebellum.

2. N e w o n Sunrival VIP is the exception to a general rule that factors are either mitotic or survival promoting for a given cell population. Pincus et al. (1990a,b) demonstrated that VIP not only stimulated cell division of SCG neuroblasts but also promoted survival, even in the presence of DNA synthesis inhibitors. In addition, VIP was shown to prevent the death of retinal ganglion cells which is induced by electrical blockade (Kaiser and Lipton. 1990). These effects are all mediated by the low-affinity receptor linked to adenylate cyclase stimulation; however, VIP can prevent death of spinal cord neurons induced by NMDA antagonists o r T T X via its high-affinity receptor (Brenneman et al., 1990~). ACTH blocks the death of neurons from E8 chick cortex that occurs with growth in serum-free medium (Daval et al., 1983).

NEUROTRANSMITTERS AS NEUROTROPHIC FACTORS

11

3. Neuronal Sprouting

Peptides, like neurotransmitters, have been reported to stimulate neurite outgrowth of cultured neurons. VIP increased both the number and the branching of neurites on SCG neurons (Pincus et al., 1990a); SS enhanced neuronal sprouting of Helisoma buccal ganglion neurons, leading to enhanced electrical coupling (Bulloch, 1987); and two POMCderived peptides, ACTH and a-melanocyte-stimulating hormone (aMSH), stimulated neurite outgrowth from spinal cord explants (Van Der Neut et al., 1988).In uiuo, chronic administration of 50 mg/kg naltrexone (PNDl-PND10) resulted in increased dendritic length of cortical pyramidal neurons, hippocampal basilar dendrites, and cerebellar Purkinje cells; in addition, all had higher numbers of spines than the controls (Hauser et al., 1987).These results suggest a role for endogenous opioid peptides, as well as other peptides, in the modeling of synaptic connections during development.

4. Differentiated Phenotype Several neuropeptides have been reported to affect expression of neuronal phenotypic markers of cholinergic, catecholaminergic, and peptidergic neurons. The most direct effect is that of calcitonin generelated peptide (CGRP),a peptide co-localized with ACh in spinal motor neurons. Two different laboratories have shown that CGRP increases the number of ACh receptors expressed on cultured myotubes even when neurotransmission is blocked by T T X (Fontaine et al., 1986; New and Mudge, 1986), and does so by stimuiating ACh receptor mRNA content, apparently by increasing cyclic AMP (Fontaine et al., 1987).These results suggest that a co-transmitter can have trophic effects at early developmental stages and in the absence of electrical activity. CGRP has also been localized to olfactory epithelial neurons, which synapse on the dopaminergic interneurons in the glomerular layer of the olfactory bulb. The olfactory epithelial neurons can induce tyrosine hydroxylase (TH) and DA uptake in the DA neurons in a co-culture; this effect is blocked by antibodies to CGRP and mimicked by CGRP itself (Denis-Donini, 1989). Although the effect is clearly on neuronal phenotype rather than survival, it is not yet known whether CGRP acts directly on the DA neurons or indirectly via another cell type. Another peptide family, substance K and substance P, can increase TH expression in explants of substantia nigra, an effect mimicked by veratridine depolarization; both are blocked by TTX (Friedman et al., 1988).Substance P and substance K are present in afferent fibers to the substantia nigra, like CGRP in the olfactory bulb.

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Serotonergic neurons are also affected by neuropeptides, in this case ACTH and Leu-enkephalin (Azmitia and deKloet, 1987; Davila-Garcia and Azniitia, 1990). Whether the peptides are administered in z1i-00 (mothers treated from E6 to E21) or in zritro, serotonin uptake was increased by ACTH. However, Leu-enkephalin increased 5-HT uptake in spinal cord when given in uiuo, but decreased it when added to raphe or hippocaiiipal cultures and had no effect on these regions in uiuo. Exposure of pregnant rats to either morphine or naloxone has been shown to affect the development of Met-enkephalin binding sites in the babies (Tsang and Ivg, 1980). In general, morphine treatment led to an earlier appearance followed by decreased levels, whereas the effects of naloxone were less consistent. These results cannot readily explain the findings of Davila-Garcia and Azniitia ( 1990) for Leu-enkephalin. In adult animals, chronic morphine has been shown to depress proenkephaliii mRNA content in striatum without affecting peptide levels (Uhl rt ul., 1988), whereas chronic naltrexone elevated Met-enkephalin and substance P as well as their precursor niRh’As (Tempe1 et ul., 1990). These results clearly suggest that the opioid peptides may regulate their own expression as well as that of other neurotransmitters, not only developmelitally but in the adult. Administration of either neuropeptides or their antagonists can affect the development of a variety of behavioral and motor functions, all indicative of potential trophic roles for the peptides. A discussion of these “whole animal” behaviors is beyond the focus of this review but the interested reader is referred to papers on the effects of thyrotropinreleasing hormone (Stratton et nf., 1976) and naltrexone (Zagon and McLaughlin, 1983b, 1985) and to an overall review (Handelmann, 1983).

111. Indirect Actions via Other Cells

In many ofthe experiments cited in Section I1 as evidence for actions of neurotransmitters and neuropeptides as trophic factors, both in 11iuo and in culture, the possibility exists that these actions could be mediated indirectly via some other cell type. That such a concern must be considered in interpretation of the data will be illustrated by results to be discussed in this section, which demonstrate that NE, 5-HT, and VIP can all stimulate astrocytes to produce neurotrophic factors.

NEUROTRANSMITTERS AS NEUROTROPHIC FACTORS

13

A. NEUROTRANSMITTER REGULATION OF NEUROTROPHIC FACTORPRODUCTION The first report of neurotransmitter regulation of neurotrophic factor synthesis appeared in 1977 and demonstrated that NE, acting through a P-adrenergic receptor that activated adenylate cyclase, stimulated production of nerve growth factor (NGF) in the C6 glioma cell line (Schwartz and Costa, 1977). More recently, the NE regulation has been shown to occur at the level of gene transcription, resulting in increased NGF mRNA (Schwartz, 1988). More importantly, the same regulation has been demonstrated in type 1 astrocytes from cortex, striatum, and cerebellum (Schwartz and Mishler, 1990). As it is clear that virtually all type 1 astrocytes in culture express the p-adrenergic receptor (Trimmer and McCarthy, 1986) and that P-receptors are present on astrocytes in vzvo (Aoki et al., 1987), the results have important implications for neuronal regulation of NGF production. In a series of studies, Whitaker-Azmitia and Azmitia have shown that 5-HT can regulate astrocyte production of S-loop, a trophic factor for serotonergic neurons. Astrocytes express a 5-HT receptor with a Kd for 5-HT of 4 nM (Whitaker-Azmitia and Azmitia, 1986). Conditioned medium from astrocytes exposed to 5-HT, but not that from untreated astrocytes, increased 5-HT uptake of cultured raphe neurons. The uptake was shown in earlier work to be proportional to neurite outgrowth and is used as an indication of neuronal maturation (Whitaker-Azmitia and Azmitia, 1989). Serotonin was inactive when added to pure cultures of raphe 5-HT neurons, but was effective in the presence of hippocampal co-cultures, which contained astrocytes. Treatment of astrocytes with the 5-HTIA-specific agonist ipsaperone increased release of S- 1OOP; the effect of either S-lO0P or of 5-HT-treated astrocyte conditioned medium on maturation of 5-HT uptake in raphe cultures was blocked by antibodies to S-l00P (Whitaker-Azmitia, et al., 1990). The authors interpret their results as support for the hypothesis that 5-HT, released by raphe neuron terminals in the target hippocampus, stimulates astrocyte adenylate cyclase and thereby production of S- 1OOP, a neurotrophic factor for the serotonergic neurons.

B. NEUROPEPTIDE REGULATION OF NEUROTROPHIC FACTORPRODUCTION

In addition to the direct effects of VIP on neurons and astrocytes discussed in Section II,B, Brenneman’s laboratory has also demon-

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JOAN P. SCHWARTZ

strated that \'IP can stimulate astrocytes to produce a neurotrophic factor for spinal cord neurons. As was the case for retinal ganglion cells (Kaiser and Lipton, 1990), spinal cord neurons die in the absence of electrical activity (i.e., in the presence of tetrodotoxin), and VIP rescues the neurons (Brenneman and Eiden, 1986). I n this case, however, the cultures normally contain astrocytes. Conditioned medium from astrocytes could support survival in pure neuronal cultures but only if the astrocytes had been treated with VIP; however, VIP did not rescue neurons in the absence of astrocytes (Brenneman et al., 1987). In a more recent paper, Brenneman et a / . (l99Ob) demonstrated directly that VIP acted as a seoretagogue in releasing a spinal cord neuron survivalpromoting factor. However, this effect of VIP is mediated by a highaffinity receptor and does not involve changes in cyclic AMP, unlike the effects on retinal ganglion cells. As VIP is released from a subgroup of neurons in the cultures, the results suggest a network of communications that connects neurons not only directly but also indirectly via the nonneuronal cells.

IV. Regulation of Synthesis and Response

A. GLIALVERSUS NEUKONAL SYNTHESIS

Bv definition, neurotransmitters and neuropeptides are synthesized in and active on neurons. But much attention has been focused on recent findings that glia can also synthesize, as well as respond to, these neuroactive agents. Essentially every neurotransmitter o r neuropeptide receptor has been found on glia, suggesting that glia can respond to all these factors (Kimelberg, 1988). Although there has been little evidence for synthesis of neurotransmitters in glia (the exception will be discussed in Section IV,D), astrocytes clearly synthesize some of the neuropeptides.Of these, proenkephalin (PE) is of interest because of its generality of expression: it is present in type 1 astrocytes from essentially all brain regions. T h e first reports demonstrated enkephalin by immunohistochemistry in glialike cells in developing cerebellum (Zagon et al., 1985) and PE niRNA expression in the C6 glioma cell line (Yoshikawa and Satrtol, 1986). Schwartz and Siniantov (1988) reported the presence of PE mRNA in embryonic striatal nonneural cultures (40-60% astrocytes), and Vilijn et al. (1988) showed PE mRNA in astrocytes from hypothalamus, hippocampus, striatum, cortex, and cerebellum. Enkephalin biosynthesis in astrocytes was shown to be regulated by cyclic

NEUROTRANSMITTERS AS NEUROTROPHIC FACTORS

15

AMP in astrocytes just as it is in neurons (Shinoda et al., 1989), as well as by P-adrenergic receptors working through cyclic AMP (Shinoda et al., 1989; Melner et al., 1990). Despite the presence of PE mRNA and PE in all these astrocytes, there is little processing of PE either in uivo (Spruce et al., 1990) or in cultured cells (Melner et al., 1990). Two groups have recently demonstrated that YE mRNA and PE can be detected in astrocytes of rat cerebellum, suggesting that the expression is not an artifact of tissue culture (Spruce et al., 1990; Hauser et al., 1990). Proenkephalin is not the only neuropeptide gene expressed in astrocytes. Two others are found only in specific regions, angiotensinogen in certain hypothalamic nuclei as well as a few other areas (Stornetta et al., 1988) and somatostatin only in cerebellar astrocytes (Shinoda et al., 1989). Astrocytic somatostatin expression shows a developmental decline that exactly parallels that seen in uivo (Naus, 1990). Angiotensinogen mRNA has also been detected in several human astrocytomas (Milstead et al., 1990). Endothelin mRNA has been measured in brain, with undiminished levels in the cerebellum of mutant mice lacking either granule cells or Purkinje cells, as well as in cerebellar astrocytes. These results have led the authors to suggest that endothelin is present in both neurons and glia (MacCumber et al., 1990). Interestingly, the size of the mRNA in the astrocytes is different from that in adult brain, suggesting the possibility of a developmental switch (MacCumber et al., 1990). Perhaps what is equally interesting is the specificity for neuropeptide expression in type 1 astrocytes. To date no neuropeptides have been detected in type 2 astrocytes; their precursor, the 0,A cell; or oligodendrocytes (for a review of the nomenclature, see Raff, 1989). Nor have a number of other neuropeptide genes been found, including cholecystokinin, substance P, dynorphin, and POMC. These negative data reinforce the growing belief that astrocyte-derived peptides must have important functions in the CNS developmentally and possibly also in the adult.

B. DIFFERENTIAL NEUROPEPTIDE PRECURSOR PROCESSING An area that is just being explored is that of differential processing of neuropeptide precursors, developmentally and in astrocytes. Much of the in vivo processing data is derived from the pituitary, because of the relatively small number of cell types, and has involved developmental analyses. These studies have shown that neuropeptide precursor processing may be rapid and yield the adult form of the peptides much earlier than needed for the expected function, as was seen for the AVP

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JOAN P. SCHWARTZ

precursor (Altstein and Gainer, 1988). The OT precursor, instead, is processed not only more slowly but less completely, suggesting the possibility that the intermediate-sized peptides could have a developmental function different from that of the adult oxytocin peptide (Altstein and Gainer, 1988). Sato and Mains (1985) have shown developmental differences in the processing of POMC: the products produced in the intermediate lobe are essentially the same but the quantities increase more than 100-fold from birth to adulthood, whereas in the anterior pituitary, the relative ratio of products shifts from birth to adult, with aMSH decreasing and ACTH and P-endorphin increasing. Regulation at the level of processing has also been seen for prodynorphin (Seizinger et d., 1984). progastrin and procholecystokinin (Rehfeld, 1986), and the substance P precursor (Kream et nl., 1985), although none of these has heen analyzed developmentally. Processing of proenkephalin in astrocytes appears to be regulated c~eveloprneiitallyin the opposite direction. Little processing to free enkephalin peptides occurs either in cerebellum in u i z m (Spruce et d.,1990) o r in cultured cells (Melner et al.. 1990). T h e processing that does occur, however, is maximal at E20 to PKDS and declines 10- to 20-fold by l’NI18 to adult (Shinoda et ul., 1991). Of interest with regard to the cerebellai- astrocytes is that they synthesize significantly less mRNA for carboxypeptidase E, one of the two enzymes needed for neuropeptide preciirsor processing (Vilijn et ul., 1989). Perhaps as a consequence of this, they produce mainly a lo~~-niolecular-weiglit peptide that is a carbox!!-terniinal extended forni of hfetenkephalin (J. €‘. Schwartz, unpublished observations). These data raise two alternative possibilities, not necessarily mutually exclusive. Free enkephalin peptides produced 1)). as1 rocytes may h a w specific trophic Functions only early in CNS tievelopnieiit; processing is then turned off, leading to the presence of precursor in adult astrocytes. Alternatively, the precursor may have a different. as yet unknown, function, and is needed throughout the lifetime of the animal.

C. DEVELOPMENTAL RECEPTORS Tantalizing evidence exists for the expression of certain receptors during specific developmental periods, the idea being that such receptors might mediate the trophic effects of the neurotransmitters whereas the “adult” receptors would mediate the neurotransmitter communications. Thus, the dopaminergic effects on chick retinal neurons (Lankford et nl., 1987, 1988) may be mediated by a subclass of D , receptor that

NEUROTRANSMITTERS AS NEUROTROPHIC FACTORS

17

appears at E7, peaks around E12-E13, and then decreases back to basal by birth (Ventura et al., 1984; Paes de Carvalho and DeMello, 1985). This subclass is distinguished by a lack of desensitization (Ventura et al., 1984), suggesting a function that requires constant stimulation during a specific developmental period such as might be expected for neurite outgrowth. Similar data exist for the serotonin system. The 5-HT,, receptor subclass appears in embryonic brain, peaks at PND7, and then decreases substantially in certain brain regions (Daval et al., 1987). Th’IS same receptor was shown to mediate stimulation of astrocytes to produce the serotonergic growth factor S- 1OOP (Whitaker-Azmitia and Azmitia, 1989; Whitaker-Azmitia et al., 1990), again suggesting a developmental trophic role for a subclass of receptor. Tsumoto et al. (1987) have demonstrated that the NMDA receptor in the visual cortex of young kittens is more efficacious than that in the adult, possibly potentiating weaker non-NMDA inputs. The possibility that this might relate to the trophic effects described by Balazs’ laboratory on cerebellar granule cells (BalAzs et al., 1990a) deserves to be explored further. Finally, the existence of a developmentally expressed opiate receptor in the cerebellum has been postulated (Zagon et al., 1990). Such a receptor might recognize the nonMet-enkephalin peptide produced by cerebellar astrocytes early postnatally (J. P. Schwartz, unpublished observations).

D. DEVELOPMENTAL VERSUS INJURY-INDUCED EXPRESSION

Some evidence suggests that injury-evoked plasticity is a reflection of the turning back on of developmental programs. One might then expect to see expression of neurotransmitters and neuropeptides in unusual sites o r cells (unusual for the adult CNS) if these factors play any role in such plasticity responses. Several reports suggest that exactly such phenomena do occur. The earliest is one of the most surprising: in 1974, Dennis and Miledi reported that Schwann cells localized at the endplates of denervated frog muscle could release ACh. The release was evoked by direct electrical stimulation but was not quantal, was not blocked by tetrodotoxin, and was not calcium dependent. The authors did not speculate on possible functions for Schwann cell-produced ACh nor on what other factors might be produced. Two more recent papers have looked at neuropeptide changes after CNS injury. Cintra et al. (1989) showed that ibotenic acid lesions of the hippocampus resulted in increased expression of endothelin in both astrocytes and degenerating CA1 pyramidal cells within 24 hr. Transection of retinal ganglion axons in the optic nerve led to increased numbers of substance P receptors on glia in

18

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the optic nerve and tract (Mantyh et al., 1989). These studies thus support the idea that neuropeptides may be involved in responses of both neurons and glia to brain damage. It is by now well accepted that excitatory amino acids can be neurotoxic and may underlie the pathology and/or plasticity seen in certain neurodegenerative diseases (Schwarcz et al., 1984; Cotman and Iversen, 1987). Excitatory amino acid toxins such as kainic acid can stimulate glia to release glutamate and aspartate, thereby mediating much of the damage. At the same time, glia may produce neurotrophic factors capable of ameliorating some of the damage (Section 111). In light of this, it is interesting that a cortical knife-cut lesion increased fibroblast growth factor mRKA content (Logan, 1988), as work by Mattson et al. (1989) demonstrated that fibroblast growth factor reduced the toxic effects of glutanlate on hippocampal cells. T h e results of all of these studies suggest that increasing attention should be paid to trophic functions for neuropeptides and neurotransmitters following CNS injury or during adult plasticity responses. Furthermore, the role of glia in these functions needs additional study.

V. Summary and Conclusions

At the start of this review, factors were deemed trophic if they stimulated mitosis, permitted neural cell survival, promoted neurite sprouting and growth cone motility, or turned on a specific neuronal phenotype. The in uifroevidence from cell cultures is overwhelming that both neurotransmitters and neuropeptides can have such actions. Furthermore, the same chemical can exert several of these effects, either on the same or on different cell populations. Perhaps the most striking example is that of VIP, which can stimulate not only mitosis, but also survival and neurite sprouting of sympathetic ganglion neuroblasts (Pincus et al., 1990a,b). The in uiuo data to support the in uitro experiments are starting to appear. A role for VIP in neurodevelopment is supported by in vivo studies that show behavioral deficits produced in neonatal rats by treatment with a VIP antagonist (Hill et al., 1991). T h e work of Shatz’ laboratory (Chun et al., 1987; Ghosh et al., 1990) suggests that neuropeptide-containing neurons, transiently present, serve as guideposts for thalamocortical axons coming in to innervate specific cortical areas. Along similar lines, Wolff et al. ( 1979) demonstrated y-aminobutyric acid-accumulating glia in embryonic cortex that appeared to form axoglial synapses and suggested the possibility that y-aminobutyric acid released from the glia

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19

might play a role in synaptogenesis by increasing the number of postsynaptic thickenings. Meshul et al. (1987) have provided evidence that astrocytes can regulate synaptic density in the developing cerebellum. T h e work of Zagon and McLaughlin (1986a,b, 1987) has shown that naltrexone, an antagonist of the endogenous opioid peptides, affects both cell number and neuronal sprouting. Lauder’s laboratory (Lauder et al., 1982) has shown a role for 5-HT in regulation of the proliferation of numerous cell types. These studies illustrate another important point, that neurotransmitters and neuropeptides function in communication not only between neurons, but also between neurons and glial cells, and between glial cells. Given that astrocytes can express virtually all of the neural receptors and can produce at least some of the neurotransmitters and neuropeptides, they must now be considered equal partners in the processes of intercellular communication in the nervous system, including the trophic responses. T h e actions of neurotransmitters and neuropeptides have to be considered in terms of a broad spectrum of actions that range from the trophic actions described in this review, to the classic transmitter actions, to potential roles in neurotoxicity. This spectrum probably reflects components related to the developmental age of the animal as well as components related to imbalances in the concentrations of specific agents. Glutamate appears necessary for cerebellar granule cell survival and can regulate hippocampal pyramidal cell neurite outgrowth developmentally, is clearly active as a neurotransmitter in the adult brain, and is equally clearly toxic if its concentration rises too high, particularly in the hippocampus. This same spectrum will be reflected in the reexpression of developmental functions in the adult. For example, one might predict that somatostatin, which is present at high concentrations in the developing cerebellum, possibly in both neurons and astrocytes, but is almost nonexistent in the adult brain, might be reexpressed either in response to injury o r as part of a plasticity response. One interesting concept that is becoming quite widespread is that for many CNS neurons requiring electrical activity for survival (spinal cord neurons, cerebellar granule cells, and retinal ganglion cells), the effect of electrical activity can be substituted for by the transmitter present in the afferent innervation (VIP, glutamate, and VIP, respectively). Furthermore, the afferent product, calcitonin gene-related protein, regulates the neuronal phenotype of olfactory bulb neurons, whereas the afferent peptides, substance P and substance K, regulate the phenotype of substantia nigra dopamine neurons. VIP in afferent nerves regulates division, survival, and neurite sprouting of superior cervical ganglion neurons.

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Are these neurotransmitters the trophic factors supplied by the afferent innervation, whereas the classic neurotrophic factors are provided by the target tissues?

Acknowledgments

nib

I thank Dr. Doug Urenneman, Dr. Vittorio Gallo, Dr. Bible Chronwall, and tnenibers of laboratory for thoughtful discussion and suggestions, and Ms.J o a n Darcey for typing

the manuscript.

References

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Mattson, M. P., Dou, P., and Kater, S. B. (1988a).J. Neurosci. 8, 2087-2100. Mattson, M. P., Lee, R. E., Adams, M. E., Guthrie, P. B., and Kater, S. B. (1988b). Neuron 1, 865-876. Mattson, M. P., Murrin, M., Guthrie, P. B., and Kater, S. B. (1989).J. Neuro.ici. 9, 37283740. McCbbb, D. P., Haydon, P. G., and Kater, S. B. (1988). J. Neuroscz. Res. 19, 19-26. Melner, M. H., Low, K. G., Allen, R. G., Nielsen, C. P., Young, S. L., and Saneto, R. P. (1990). E M B O J . 9, 791-796. Meshul, C. K., Seil, F. J., and Herndon, R. hl. (1987). Brain Res. 402, 139-145. Milsted, A., Barna, B. P., Ransohoff, R. M., Brosnihan, K. B., and Ferrario, C. M. (1990). Pror. Natl. Acad. Sci. U . S . A . 87, 5720-5723. Naus, C. C. G . (1990). Brain Res. Bull 24, 583-592. Nails, C. C . G., Miller, F. D., Morrison, J. H., and Bloom, F. E. (1988a).J. Comp. Neurol. 269, 448-463. Naus. C. C. G., Morrison, J. H., and Bloom, F. E. (1988b). Deu. Brain Res. 40, 113-121. New,, H. V.. and Mudge, A. W.(1986).Nature (London) 323, 809-81 1. Nicnletti, F., Magre, G., Ingrao, F., Bruno, V., Catania, M. V., Dell'Albani, P., Condorelli, D. F., and .4vola, R. (1990). J. Neuroclum. 54, 771-777. Nilsson, J., Von Euler, A. M., and Dalsgaard, C . J. (1985). Nafure (London) 315, 61-63. Oppenheim, R. W. (1989). Trendc N a r o s c i . 12, 252-255. Paes de Carvalho, R., and DeMello, F. G. (1985).J. Neurochem. 44,845-851. Rarce, I. A., Cambray-Deakin, M. A.. and Burgoyne, R. D. (1987). FEBS Lett. 223, 143147. Pincus, 1).W..DiCicco-Blooni, E. M., and Black, I. B. (1990a). Nature (London) 343, 564567. Pincus, D. W., DiCicco-Bloom, E. M., and Black, I. B. (1990b). Brain Rex 514, 355-357. Rag, M. C. (1989). Scitnce 243, 1450-1455. Rehfeld, J. F. (1986). J . B i d . Chem. 261, 5841-5847. Rothman, T. P., e r s h o n , M. D., and Holtzer, H. (1978). Dm. Biol. 65, 322-341. Sato, S. M., and Mains, R. E. (1985). Endocrinology (Baltimore) 117, 773-786. Schwarcz, R., Foster, A. C . , French, E. D., Whetsell, W. O., and Kiihler, C. (1984). Life Sci. 35, 19-32. Schwartz, J. P. (1988). Clio 1, 282-285. Schwartz, J. P., and Costa, E. (1977). Nnunyn-Schmiedeberg>Arch. Pharmacol. 300, 123-129. Schwarr-z,J. P., and Mishler, K. (1990). Cell. Mol. Neurobiol. 10, 447-457. Schwartz, J. P., and Simantov, R. (1988). D m . Bruin Res. 40. 31 1-314. Seizinger, B. R., Grimm, C., Hiillt. V., and Herz, A. (1984). J. Neurochem. 42, 447-457. Shinoda, H., Marini, A. M., Cosi, C., and Schwartz, J. P. (1989). Science 245, 415-417. Shinoda, H.,Marini, A. M., and Schwartz, J. P. (1991). Submitted. Sieber-Blum, M. (1989). Dm. B i d . 136, 372-380. Smith, A. A,, and Hui, F. W. (1973). Exp. Npurol. 39, 36-43. Spruce, B. A., Curtis, R., Wilkin, G. P., and Glover, D. M. (1990). EMBOJ. 9, 1787-1795. Stiene-Martin, A., and Hauser, K. F. (1990). Life Scz. 46,91-98. Stornetta, R. L., Hawelu-Johnson, C. L., Guyenet, P. G., and Lynch, K. R. (1988). Science 242, 1444-1446. Stratton, L. O., Gibson, C. A., Kolar, K. G., and Kastin, A. J. (1976). Pharmacol. Biochem. Behav. 5,65-67. Swaab, D. F., Visser, M., and Tilders, F. J. H. (1976).J. Errdom'nol. 70, 445-455. 'Ternpel, A,, Kessler, J. A., and Zukin, R. S. (199O).J. Neurosn'. 10, 741-747. Trimmer, P. A., and McCarthy, K. D. (1986). Dm. Bruin Res. 27, 151-165.

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HETEROGENEITY AND REGULATION OF NICOTINIC ACETYLCHOLINE RECEPTORS Ronald J. Lukas a n d M e r o u a n e Bencherif Division of Neurobiology Barrow Neurological Institute Phoenix, Arizona 85013

I. Introduction A. Seminal Concepts in Neurotransmitter Receptor Structure and Function B. Diversity in the Nicotinic Acetylcholine Receptor Family C. A Tool for Seminal Studies on Neurotransmitter Receptor Regulatory Mechanisms 11. Models and Concepts in Studies of Receptor Regulation A. In Vitro Models of Regulatory Mechanisms That Operate in Vzvo B. Drug Treatment Models of Natural Regulatory Input C. Concepts of Regulatory Dominance D. Does Regulatory Flexibility Provide a “Benefit” or “Purpose” for Transmitter Receptor Diversity? 111. Regulation of Muscle Nicotinic Acetylcholine Receptor Expression and Function A. Regulation of Nicotinic Acetylcholine Receptor Number, Assembly, and Stability B. Regulation of Nicotinic Acetylcholine Receptor Function C. Modulation of Nicotinic Acetylcholine Receptor Number and Function by Exogenous Ligands IV. Autonomic Neuronal and Ganglia-Type Nicotinic Acetylcholine Receptors A. Regulation of Ganglia Nicotinic Acetylcholine Receptor Expression and Function B. Regulation of Ganglia-Type Nicotinic Acetylcholine Receptor Expression and Function V. Central Neuronal Receptors A. Regulation in Vim: Ontogenic Aspects B. Effects of Chronic Nicotine or Nicotinic Agonist Exposure C. Effects of Anticholinesterase Treatment and Other Modulators of Cholinergic output D. Other Inputs That Modify Nicotinic Acetylcholine Receptor Expression or Function in Vivo VI. Perspectives and Conclusions References

I. Introduction

This article focuses on two topics, which presently are only loosely related, within the extensive literature on nicotinic acetylcholine 25 INTERNATIONAL REVIEW OF NEUROBIOLOGY, VOL. 34

Copyright Q 1992 by Academic Press, lnc. All rights o f reproduction in any form reserved.

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receptors (nAChRs). Part of the current discussion is meant to update an excellent earlier review on regulation of nAChRs authored by Schuetze and Role (1987). This component emphasizes studies done using mammalian tissues o r their derivatives, although references to work done using vertebrate nonmammalian systems are included. Integrated into this discussion is an update of excellent earlier reviews on fundamental characteristics of nAChRs and of the studies that contributed to the current understanding of this important receptor system (McCarthy et al., 1986; Lindstrom et af., 1987; Lentz and Wilson, 1988; Schmidt, 1988).'This updated overview places special emphasis on the bases, manifestations, and functional significance of nAChR diversity. Recently, there have appeared other synopses of ongoing studies in nAChR biology (e.g., Clementi et al., 1988; Mathie et al., 1988; Claudio, 1989; Deneris et al., 1989, 1991; Maelicke, 1989; Nordberg et al., 1989a; Colquhoun et al., 1990; Froehner, 1991). Often, these volumes and specialized reviews cover more background information in greater detail than is practical here.'

A. SEMINAL CONCEPTS IN NEUROTRANSMITTER RECEPTORSTRUCTURE AND FUNCTION 1. Historical Oveiuieui T h e neurotransmitter acetylchohe and its receptor systems have been the focus of much experimental work over the last century, but particularly during the last 20 years, when the introduction of powerful, contemporary techniques in molecular biology, immunology, cell cloning, and electrophysiology has augmented more traditional pharmacological, protein chemical, and cytological approaches. Acetylcholine was the first neurotransmitter substance to have been identified, and the accessibility of cholinergic pathways and synapses in the periphery, at the vertebrate neuromuscular junction, and in autonomic ganglia has been exploited to the extent that more was known sooner about the actions of acetylcholine and its receptors than about any other neurotransmitter-receptor system (for a succinct historical overview, see Taylor, 1989a,b,c). *The literature cited in this review includes material published through December 1990 and a few, selected, later citations added in proof. It is inevitable that not all relevant contributions will be cited in this overview, but any omissions are not intentional. In many cases, articles cited are examples from a soberingly more extensive literature covered in greater detail elsewhere.

NICOHNIC RECEPTOR DIVERSITY AND REGULATION

NICOTINE

ACETYLCHOLINE CARBONYLSIDE

I

27

MUSCARINE

METHYL SIDE

FIG. 1. Chemical structures of prototypical cholinergic agonists. Studies of structureactivity relationships suggest that both muscarinic and nicotinic acetylcholine receptors recognize the quaternary ammonium ions of the pictured agonists. Muscarinic sites are thought to be optimally activated by ligands that carry an esteratic oxygen 4.3 A from the quaternary ammonium ion and to engage in hydrophobic interactions with the methyl side of acetylcholine and cogeners. Nicotinic receptors are thought to interact preferentially with the carbonyl domain of acetylcholine and its cogeners, optimally so if the carbonyl carbon is 5.9 A from the quaternary ammonium ion.

2. Basic Concepts of Humoral Transmission In the course of this work, seminal concepts in receptor biology have been established, based largely on elucidation of the structure and function of acetylcholine receptors and on the actions of drugs that mimic or block the effects of acetylcholine at its receptors. This work has provided tangible evidence for hypotheses regarding the existence and nature of interneuronal chemical signaling at synapses, quanta1 release of transmitters, agonism and antagonism (mediated via competitive or noncompetitive mechanisms), self-regulatory functional desensitization or inactivation of receptors, and diversity of receptors recognizing a single, biologically active compound. The latter realization was an immediate consequence of studies demonstrating that whereas some effects of acetylcholine and cholinergic nerve stimulation could be mimicked by the tobacco alkaloid nicotine, other effects were not, but were mimicked by another alkaloid from mushrooms, muscarine (Fig. 1; see Taylor, 1989a,b,c).

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3 . The Metabotropk Receptor Supefamily Muscarinic acetylcholine receptors are representative of one general class (or “superfamily”) of neurotransmitter receptors, termed metabotropic receptors, which also includes the receptors for catecholamines and rhodopsin (the photon receptor). In general, members of this class of receptors are coupled via guanine nucleotide-binding proteins (Gproteins) to enzyme effectors, and their activation on interaction with neurotransmitter leads to modulation of levels of intracellular second messengers, such as phospholipid derivatives, cyclic nucleotides, and intracellular calcium ion. T h e actions of these second messengers can lead to changes in ionic permeability and sensitivity to electrical excitability of the target cell membrane, alterations in cellular metabolic processes, and modulation of gene expression. Metabotropic receptors identified currently also appear to share structural similarities, as has been deduced from protein chemical analyses and molecular biological studies Ieading to isolation of cloned metabotropic receptor genes. These receptors appear to be monomeric, integral membrane proteins, each with seven rnembrane-spanning segments composed largely of hydrophobic amino acid residues. The transmembrane-spanning segments seem to be involved in formation of the ligand binding domain, and the third cytoplasmic loop, residing between the putative fifth and sixth transmembrane regions in the polypeptide, appears to be involved in interactions with G-proteins and as a substrate for intracellular enzymes (reviewed in ’raylor and Brown, 1989; Weiner and Molinoff, 1989). 4 . The Lzgund-Gated Ion Channel Sujwfumily

By contrast, nicotinic acetylcholine receptors, which are prototypes of the ligand-gated ion channel class of neurotransmitter receptors, are not directly coupled to effector proteins o r second messenger signaling mechanisms. Instead, on interaction with neurotransmitter, ligandgated ion channels, including receptors for y-aminobutyric acid and glycine, directly mediate rapid changes in permeability of the plasma membrane to inorganic ions and either induce or suppress electrical impulse propagation in target cells. On the basis of data obtained from a variety of different approaches, ligand-gated ion channels appear to be multimeric, integral membrane proteins composed of several distinct, but related subunits (reviewed by McCarthy et al., 1986; Claudio, 1989; Deneris et al., 1989; Betz, 1990; Olseri and Tobin, 1990). In general, hydropathy analyses of derived amino acid sequences for cloned subunits of the receptors for nicotine, y-aminobutyric acid (GABA), or glycine suggest that each subunit is itself an integral membrane protein

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29

composed of four membrane-spanning segments of hydrophobic amino acids and an extended cytoplasmic loop (containing amino acid sequences that are unique to each type of subunit) residing between the third and fourth transmembrane segments. The N-terminal, extracellular domain of at least some of these subunits also appears to harbor the neurotransmitter binding site. In addition, in all subunits currently analyzed, at least one set of cysteine residues that may engage in disulfide bond formation, which is critical for maintenance of receptor tertiary structure, reside in this N-terminal domain. The transmembrane segments of each subunit of these receptors are thought to assemble like staves of a barrel to generate a void remote from the hydrophobic milieu of the lipid bilayer. Conformational changes in the receptor triggered by interaction with neurotransmitter are thought to lead to physical enlargement of that void to permit passage of the appropriately sized and charged ions down their electrochemical gradient. It also is evident that excitatory amino acid receptors belong to the general class of ligandgated ion channels. Some of these receptors interact with the excitotoxin kainate, and several kainate-binding proteins have been identified and purified. Analysis of kainate-binding protein cDNA sequences indicates that these proteins have general structural similarities with nicotinic, glycine, and GABA receptor subunits, although expression in Xenopus oocytes of single, cloned kainate-binding protein subunits does not appear to be adequate to generate functional ion channels (reviewed in Betz, 1990). More recent success in cloning excitatory amino acid receptor subunits that are capable of generating ion channels reveals some common features, but also some differences (particularly in the larger extent of the N-terminal, putative extracellular region and in amino acid sequence conservation, rather than diversity, across subunits in the putative second cytoplasmic loop), between these components of the general class of ligand-gated ion channels and the subclasses defined by glycine, nicotine, and nonmetabotropic GABA receptors (Bettler et al., 1990; Boulter et al., 1990a; Keinanen et al., 1990; Nakanishi et al., 1990).

IN B. DIVERSITY

THE

NICOTINICACETYLCHOLINE RECEPTOR FAMILY

1. Nomenclature

a. Muscle and Ganglia nAChR Subtypes. The earliest indications that, aside from the clear distinctions between muscarinic cholinergic and nicotinic cholinergic receptors, there might be diversity in nAChRs came from seminal studies of the neuromuscular junction and autonomic

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RONALD J. LUKAS A N D MEROUANE BENCHERII;

CH2 - N

OH3

+

- (CH3)3

MUSCARINE

NICOTlNE

(CH&

NNCH3

fi

- N - (CH2)e - N +

+

- (CH3)3

HEXAMETHONIUM 0 CH2OH

O-C-CH

(CH3)3 - N

+

- (CH2)lo - N - (CH3)3 +

C6H5

ATROPINE

DECAMETHONIUM

d-TUBOCURARINE

FIG.2. Chemical structures of acetylcholine; the esterase-resistant analog carbamylcholine; the prototypical muscarinic and nicotinic receptor agonists muscarine and nicotine, respectively; the prototypical muscarinic and nicotinic and antagonists atropine and d-tubocurarine. respectively; and representative members of the bisonium series of compounds, hexamethonium (C6) and decamethonium (C 10).

ganglia (reviewed in Taylor, 1989a,b,c). Whereas actions of nicotine at either site lead to rapid depolarization of the postsynaptic cell, some drugs, such as decamethonium and hexamethonium of the bisonium series of linked quaternary ammonium ions (Fig. 2), were found to selectively affect nicotinic responses on muscle cells and on postganglionic

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31

neurons, respectively. These studies were minimally complicated by pharmacokinetic considerations, and thus suggested that muscle nAChRs (also termed decamethonium-sensitive or ClO nAChRs) are subtly different from ganglia nAChRs (also termed hexumethonium-sensitive or C6 nAChRs). Some confusion, however, continues to exist within and outside of the nicotinic receptor field regarding aspects of this nomenclature. For example, it is commonly thought that decamethonium is a selective antagonist of muscle nAChRs and that hexamethonium is a potent and specific antagonist of ganglia and central neuronal nAChRs; however, decamethonium antagonistic potency is not markedly different at muscle or ganglia nAChRs, and hexamethonium is not particularly potent at all ganglia or central neuronal nAChRs (Lipscombe and Rang, 1988; Lukas, 1989a; see also below). Consequently, the favored terminology to be used in this review identifies muscle nAChRs as those nAChRs found in intact vertebrate muscle, whereas the term muscle type will refer to nAChRs that have the pharmacological and molecular features of authentic muscle nAChRs but are found in specific clonal cells or in musclelike tissues, such as the electric tissue of Electrophorus, Nurcine, or Torpedo. Similarly, ganglia nAChRs will be the terminology used to designate nAChR subtypes found on autonomic postganglionic neurons, and ganglia-type nAChRs will be used to identify very similar or identical nAChRs found on normal or neoplastic cells of neural crest origin, such as adrenal medullary chromaffin cells or pheochromocytomas/neuroblastomas, and on some cells in the central nervous system. 6. NeuronallNicotinic a-Bungarotoxin Binding Sites. A considerable body of evidence exists for the expression of nicotinic receptor-like moiecules in the central and autonomic nervous systems that are defined by their ability to interact with the curarimimetic neurotoxin, a-bungarotoxin, but are distinct from muscle-type nAChRs (reviewed in Schmidt, 1988). Moreover, until recently (see below) there was doubt regarding the ability of these sites to function as ligand-gated ion channels. To aid clarity in reference to these putative members of the nAChR family, they are termed neuronallnicotinic a-bungarotoxin binding sites (nBgtSs) in this review. c. Central Neuronal nAChRs. The term neuronal nAChRs has been used in reference to nAChRs derived from the autonomic or central nervous system, but is perhaps too vague, as there is evidence for the existence of nAChRs with ganglia-type character in the CNS and of multiple types of nAChRs in the CNS, not only on the basis of the identification of a variety of nAChR subunit-encoding genes and messenger RNAs in brain tissue (see below), but also on the basis of long-recognized complexities in the pharmacological/physiological

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RONALD J. LCKAS A N D MEROUANE BENCHERIF

characteristics of receptors mediating nicotinic and nicotinic-cholinergic responses in the brain (reviewed by Krnjevic, 1975; Martin, 1986). Given the complexity of the brain, the likelihood that pharmacokinetic as well as pharmacodynamic factors may have influenced earlier electropharmacological characterization of central nAChRs in studies done zit zuvo, and the existent evidence for central neuronal nAChR heterogeneity (see below), the designations central neuronal nAChRs and central nAChRT, which would exclude reference to CNS ganglia-type nAChKs and nBgtS, are used in this review, but with antecedent qualifiers regarding ligand binding and functional profiles of the nAChR in question, where such information is known. d. Noinvncluture Bused on nAChR Suhunzt Genes. Ultimately, as the family of nAChR subunits is identified and the subunit con~positionof each nAChR type that is naturally expressed is elucidated, nAChKs will be defined in terms of their subunit composition based on designations given to those subunit proteins and their corresponding messenger RNA, cDNA, and genes (see below). P . Neurotoxin N o m e n c l a t w ~ . It is perhaps pertinent to make another point about nomenclature regarding an important class of cholinergic system ligands. Snake neurotoxins have been used in pivotal studies toward elucidation of post- and presynaptic mechanisms involved in synaptic transmission. Early designations of the most prevalent toxic components in the venom of poisonous snakes such as Bungarus multzcinctus were made based on the succession of their identification during purification and without respect to the evolving characterization of their functional potencies and targets. It was by happenstance that toxins such as a-bungarotoxin, which is a monomeric protein of about 8 kDa, were found to have curarimimetic activity as muscle o r muscle-type n AChR functional antagonists and that more cationic toxins such as P-bungarotoxin, which is a heterodimeric protein composed of two subunits of divergent molecular mass, were found to have functional activity in modulation of neurotransmitter release (Lee, 1972). T h e subsequent determination that not all nAChRs are sensitive to blockade by toxins such as a-bungarotoxin and the identification of other toxins from the venom of Bungurus sp. snakes that have unique physiochemical properties, can act as functional antagonists at some ganglia nAChRs, and exist as dimeric proteins composed of identical o r similar subunits that have similar molecular mass suggested the existence of another class of nAChR subtype-specific toxins, termed K-toxzm (reviewed in Chiappinelli, 1986; Loring and Zigmond, 1988; Chiappinelli et d.,1990).T h e prototype of these toxins was isolated independently in the Berg, Zigmond, and Chiappinelli laboratories; these equivalent protein species

NICOTINIC RECEPTOR DIVERSITY AND REGULATION

33

have been termed bungarotoxin 3.1, toxin F, K-bungarotoxin, and, more recently, v- or neuronal-bungarotoxin (Lindstrom et al., 1987; Schmidt, 1988). T h e terms bungarotoxin 3.1 and toxin F are founded in laboratory jargon and inadequately distinguish K-bungarotoxin from a-bungarotoxin for the uninitiated. Evidence that neither a-toxins nor K-toxins block all ganglia-type nAChRs or central neuronal nAChRs in all species, coupled with more recent indications that a-bungarotoxin can block functional responses of at least some types of central neuronal nAChRs as well as muscle-type nAChRs, argues against continued use of v-bungarotoxin or neuronal bungarotoxin [a-bungarotoxin appears to block functional responses of insect ganglia nAChRs (Pinnock et al., 1988; Chiappinelli et al., 1989), but no one has seriously suggested that its name be changed to insect neuronal bungarotoxin]. In principle, these findings suggest that toxin definition based on well-documented physicochemical characteristics rather than on presently incomplete information on the nature and localization of their targets is warranted, particularly as the properties, subunit composition, and, hence, toxin sensitivities of nAChRs found at different loci are likely to vary across species as well as across tissues. Rather than suggest yet another name (such as 6-bungarotoxin for dimeric snake neurotoxins) based on this retrospective analysis and proposed principle, and with full acknowledgement that the initial definition of K-bungarotoxin was not founded on such a principle, the terms K-bungurotoxin and K-toxin are used in this review as relatively innocuous but adequately distinctive definitions of the prototypical toxin and its family.

2. General Properties of Nicotinic Receptors and Manifestations of Receptor Diversity: Functional Studies

a. Historical Overview. With few exceptions, the functional characterization of nAChRs has inextricably linked pharmacological and physiological approaches that employed bath, pressure, or electrophoretic application of exogenous drugs to cholinoceptive cells or tissues and recording of drug effects on electrical or mechanical properties of target cells. Such approaches led to the very early distinctions in vertebrates between muscle nAChRs and ganglia nAChRs based not on glaring differences in the polarity, magnitude, or rapidity of the electrical responses in muscle or postganglionic neurons, but rather on relative sensitivity of those responses to blockade by drugs of the bisonium series, such as hexamethonium and decamethonium (reviewed in Taylor, 1989b,c). b. Single-Channel Properties: Muscle nAChRs. In subsequent years, increased sophistication in electrophysiological studies has culminated in the use of patch clamp techniques to facilitate studies of nAChRs in

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RONALD J. LUKAS A N D MEROUANE BENCHERIF

ensembles or as single channels. Properties of nAChR ion channels exposed to nicotinic ligands are sensitive to experimental conditions, such as temperature, holding potential, composition of bathing medium (including external calcium ion concentration), the type of preparation studied, and its processing (e-g., enzyme digestion). Functional parameters also can be affected by changes in nAChR environment, for example, communication with the cytoplasm and mere excision of membrane patches (reviewed in Covarrubias and Steinbach, 1990). Nevertheless and provisionally, muscle nAChRs from a variety of preparations appear to exhibit two major conductance states of about 30-40 and 50-60 pS at room temperature, each with characteristic mean channel open times of about 5-10 and 1 msec, respectively (“slow” and “fast” channels; reviewed in Schuetze and Role, 1987). More recent studies have also identified “small” channels with conductances of about 12 and 25 pS and mean open times of about 6 msec in some preparations (Owens and Kullberg, 1989; but perhaps only in immature muscle; see below). Subconductance states are sometimes observed for nAChR channels, perhaps reflecting partial channel openings; also, more recent analyses recogni4e that open time or burst duration distributions are actually at least doubly exponential for both “fast” and “slow”channels and for at least a fraction of “small” channels, apparently reflecting opening of singly and doubly liganded muscle nAChR channels (Colquhoun and Sakmann, 1985; Labarca et al., 1985; Sine and Steinbach, 1987;Jackson, 1988). It is well accepted that the Hill coefficient for channel opening, the frequency of‘which varies as a function of the square of the agonist concentration, is about 2.0, suggesting that full opening of muscle nAChR channels requires the binding of two agonist molecules (McCarthy et al., 1986; Colquhoun and Ogden, 1988; Jackson, 1988). c. Szngle-Channel Properties: “Neuronal” nAChRs. Adequate information is unavailable to indicate whether generalizations can be made regarding single-channel features of ganglia or central neuronal nAChRs, but at least two channel types with relatively longer mean open times of about 5-9 and 30-50 msec (see, e.g., Rang, 1981) can be found in most neuronal preparations (Schuetze and Role, 1987). Functional studies on ganglia nAChRs and on ganglia-type nAChRs in pheochromocytoma and adrenal chromaf€in cells provide single-channel conductance estimates ranging from about 20 pS to about 45 pS for as few as one and as many as three types of channels per cell type and with mean channel open times of between 20 and 40 msec (Ascher et al., 1979; Fenwick et al., 1982; Kidokoro et al., 1982; Borniann and Matthaei, 1983; Mathie et al., 1991). Functional studies on central neuronal nAChRs are not all in agreement, indicating, for example, channel conductances of 20 and 26

NICOTINIC RECEPTOR DIVERSITY AND REGULATION

35

pS in rat hippocampal and brainstem neurons (Aracava et al., 1987), 26 pS in rat medial habenula (Mulle and Changeux, 1990), 45 pS in rat retinal ganglion cells (Ramoa et al., 1990), and 20-25 pS in porcine pars intermedia cells (Zhang and Feltz, 1990), perhaps reflecting heterogeneity of nAChR subtypes (see below). Table I provides data from several selected studies on the conductance states of nAChR channels from peripheral and central preparations. It should be noted that inward rectification of some neuronal nAChR channels has been noted in central (Alkondon and Albuquerque, 1991; Sands and Barish, 1990) and autonomic (Selyanko et al., 1988; Mathie et al., 1990) preparations, and that such character is quite distinctive from features of muscle nAChR channels. d. Densensitization. Intervening periods of nonconductance occurring during bursts of channel opening indicate that liganded muscle or muscle-type nAChRs may transiently isomerize into a nonconducting state, and longer periods of single-channel silence between bursts are taken as evidence for excursions of nAChRs into a functionally “desensitized” state (reviewed by Ochoa et al., 1989). There remains uncertainty as to the molecular bases of the desensitization process, although ultrastructural studies indicate that rearrangements of muscle-type receptor mass, particularly involving y and 6 subunits, occur under conditions that promote desensitization (Unwin et al., 1988). e. Kinetics of Desensitization and Mechanistic Perspectives. Electrophysiological studies have suggested that desensitization is more than a monophasic process (Feltz and Trautmann, 1980, 1982; Sakmann et al., 1980). Additional insight into features of the desensitization process has been obtained by the use of chemical kinetic measurements of ion flux through nAChR channels in uitro (reviewed by McCarthy et al., 1986; Ochoa et al., 1989). Data obtained in ion flux studies are in excellent agreement with data obtained using more conventional electrophysiological techniques. Rapid kinetics ion flux studies have clearly demonstrated that there are multiple phases of desensitization, classifiable as ultrafast (time constant on the order of submilliseconds), fast (milliseconds), and slow (seconds) desensitization and ultraslow (minutes) inactivation. The introduction of new approaches may provide the ability to extract information on faster chemical kinetic events occurring in live cells (Udgaonkar and Hess, 1987). Recent electrophysiological studies have shown that fast and slow desensitization and ultraslow inactivation can also be detected using intact cells and/or rapid perfusion techniques with cell membrane patches (Bekkers, 1986; Brett et al., 1986; Colquhoun and Ogden, 1988; Cachelin and Colquhoun, 1989). In the latter study done with adult frog muscles at 4”C, a “fast” component

36

RONALD J. LUKAS AND MEROC'ANE BENCHERIF

TABLE I NACHR SINGLE-CHANNEL CONDUCTANCE: NEURONAL CELLS"

Study

Conductance (PS)

Mathie pt (11. (1987) hlargiotta et al. (1987a, b)

Ganglin d C h R s 35 40

Marshall (1981) Lipscoinhe ( 1986) Moss ut mi. ( 1989)

Temperature ("C)

18 18

14 27 49 68

22 23 20 20 23

Preparation Rat syinpathetic Chick ciliary ganglia Frog ganglia Frog ganglia Chick sympathethic

Ga i~gltaType nAChRs

Fenwick uf nl. (1982) Ifune and Steinbadi (1989) Boimann arid Matthaei (1983)

44

22

(25)

18

40 22 31 39

22

Bovine adrenal chromaffin Rat PC 12 pheochromocytoma Rat PCl2 pheochromocytoma

Cuiitrnl S e w o n a l

<:onnolly and ( ~ ~ J ~ q U h (1991) ~liil

.io

22

Mulle dnd Changeux (1990)

26

20

Liptorr e/ 01. ( 1987)

48

3.5

Ranioa ut al. (1990)

45

20

20

10

26

20

Aracava ut

(11.

(19x7)

Zhang and Fclti (1990)

Rat medial habenula Rat medial habenula Rat retinal ganglion Rat retinal ganglion Kdt hippocampal, brainstem Porcine pars intermedia

Expreued Clones

Bailivet ct nl. (1988) Pdpke et nl. ( 1989)

20 34 (15) 1.5 ( 5) 13

20 24

Chick at-&

c1 Data are summarized f o r single-rhannel conductance measurements (pS) taken at the indicated temperature ("C) using the indicated neiironal preparations. Subconductanre states o r minor channel components are indicated in parentheses. See References f o r full citations.

NICOTINIC RECEPTOR DIVERSITY AND REGULATION

37

of desensitization (time constant of 6-26 sec) was found to occur more quickly as a function of increased agonist concentration, the “slow”component (time constant of 60-100 sec) was found to exist only if intracellular Ca2+ concentrations were low, and no effect on the rate of either process was observed on attempted alteration of nAChR phosphorylation state. Rates of muscle-type nAChR desensitization in vitro are accelerated when the receptor is phosphorylated, and drug treatments that should induce muscle nAChR phosphorylation also enhance desensitization in cultured cells (Steinbach and Zempel, 1987; Hopfield et al., 1988; Ochoa et al., 1989; reviewed in Huganir and Greengard, 1990). It is unlikely, however, that phosphorylation can occur fast enough in uiuo to account for the fast and ultrafast desensitization processes, and desensitization clearly occurs in reconstituted systems that contain receptor but no kinases. Nicotinic agonists can display open channel blocking activity, and agents that accelerate desensitization, such as local anesthetics (reviewed by Lambert et al., 1983), also are open channel blockers (Adams and Sakmann, 1978a,b; Trautmann, 1982; Adams and Colquhoun, 1983); however, the duration of open channel block appears to be too short to account for desensitization (Ogden and Colquhoun, 1985). Results obtained in chemical kinetic studies have prompted hypotheses that distinct ligand binding sites, which perhaps bind ligand in a voltagedependent fashion with kinetics that could account for fast and/or ultrafast desensitization, are involved in regulating channel opening (McCarthy et al., 1986; Ochoa et al., 1989). Other recent studies indicate that rates of desensitization of hybrid (cat muscle-Torpedo electroplax) nAChRs are dictated by the source of the y subunit (Sumikawa and Miledi, 1989). This and other studies (Covarrubias et al., 1989) indicate that nAChR glycosylation state affects receptor channel kinetics, but it is also clear that such a process, which occurs during intracellular processing, is unlikely to contribute to desensitization of cell surface nAChRs. Clearly, mechanisms that can contribute to nAChR inactivation are multiple and complex, but more than 30 years after the original description, the molecular bases of desensitization remain obscure. f. Desensitization of Neuronal nAChRs. Adaptation o f ion flux techniques to studies of nAChR function on cells in culture has facilitated pharmacological isotyping of nAChRs as well as a basic understanding of nAChR channel properties (Catterall, 1975; Patrick and Stallcup, 1977a; Leprince, 1983; McGee and Liepe, 1984; Lukas and Cullen, 1988). Application of ion flux and electrophysiological techniques to studies of cultured and intact neurons and neuronlike cells has shown that ganglia and ganglia-type nAChRs are also subject to functional desensitization and slow inactivation (Stallcup and Patrick, 1980; Leprince, 1983;

38

R0NAL.D J. LUKAS AND MEROUANE BENCHERIF

McCee and Liepe, 1984; Simasko et al., 1986; Boyd, 1987; Downing and Role, 1987; Mathie et al., 1988, 1990; Lukas, 1991). Desensitization of functional nicotinic responses in the central nervous system has long been evident (Krnjevic, 1975), and more recent studies have shown effects of acute and chronic nicotine that can be reconciled with desensitization and/or functional inactivation of central neuronal nAChRs (see, e.g., Rosenzweig et ul., 1989; Hulihan-Giblin et al., 1990a,b; see also below). g. Pharmacologtcal Distinctions between Functional Ganglia and Muscle nAChKs. Pharmacological sensitivity of ganglia and ganglia-type nAChRs to hexamethoniuni and mecamylamine has recently been confirmed in several systems, but those same responses are also relatively sensitive to atropine (Creene and Rein, 1977; Wilson and Kirshner, 1977; MacDermott et a/., 1980; Kidokoro et al., 1982; Sadoshima et al., 1990). Other studies have suggested that small drugs such as dtubocurarine and trimethaphan, in addition to drugs of the bisonium series, could be effective in distinguishing vertebrate and mammalian muscle nAChRs from mammalian ganglia nAChRs, if not from frog ganglia nAChRs (Colquhoun, 1981; Lipscombe and Rang, 1988; see, however, Lukas, 198%); however, mammalian ganglia-type and muscletype nAChRs can be readily distinguished on the basis of the relative selectivity of mecamylamine as a potent functional antagonist of gangliatype nAChRs ( 1000-fold more potent as an antagonist of rat PC 12 pheochrornocytoma cell ganglia-type nAChRs than as an antagonist of human YE671 cell muscle-type nAChRs; Lukas, 1989a). Agonists also exhibit different potencies on interaction with muscle or muscle-type and ganglia o r ganglia-type nAChRs. For example, nicotine and cytisirie are more potent agonists at PC12 cell ganglia-type nAChRs than at TE67 1/RD cell muscle-type nAChRs, whereas acetylcholine and succinyldicholine have the opposite selectivity (Lukas, 1989a,b). These studies reinforce the utility of the classic pharmacological approach in identification of functional nAChR subtypes, perhaps including junctional/ extrajunctiorial, innervated/denervated, and embryonic/adult isoforms. Variations on these studies could also be used to assess whether some agents, such as d-tubocurarine and decamethonium, which commonly are thought to act as competitive antagonists, can, in fact, exhibit some activity in induction of channel opening and can also act as open channel blockers to noncompetitively inhibit nAChR function (e.g., see Colquhoun et a/.,1979), as do local anesthetics and other compounds, such as hexamethonium. h. Phamnacologzcal Prvfiies of Central Neuronal nAChRs. Early perspectives suggested that central rieuronal nAChRs had predominantly phar-

NICOTINIC RECEPTOR DIVERSITY AND REGULATION

39

macological characteristics of functional ganglia nAChRs, but in some instances had features more like those of muscle nAChRs o r pharmacological profiles that blurred muscarinic/nicotinic distinctions (reviewed o r examples in Krnjevic, 1975; King and Ryall, 1981; Kelly and Rogawski, 1985; Lamour et al., 1986; Martin, 1986; Colquhoun et al., 1987). More recent studies have identified a variety of nicotinic responses in neurons or neuronlike cells and have begun to provide a more comprehensive picture of the pharmacological profiles of functional, central neuronal nAChRs. A sampling of results obtained is provided in Table 11. Note that there are sites, in the rat hippocampus for example, where chemically or electrically triggered nicotinic responses are reported to be insensitive to d-tubocurarine, hexamethonium, or atropine, but to be blocked by mecamylamine (Freund et al., 1990). There periodically appear other reports of insensitivity of central nicotinic responses to d-tubocurarine or dihydro-P-erythroidine (see, e.g., Ramoa et al., 1990), but the more common finding is that nAChR activity in the brain is found to be blocked by these compounds and/or by mecamylamine, hexamethonium, and trimethaphan, among others (see Table 11). These studies are not yet comprehensive, nor do they reveal whether there are unique pharmacological features, for example, of presynaptically located nAChRs that control neurotransmitter release. Nevertheless, these studies continue to suggest that nicotinic responses in the central nervous system (CNS) are functionally/pharmacologically heterogeneous, as is borne out in studies employing identical techniques in different sites of the same preparation (de la Garza et al., 1987a). i. Heterogeneity in nAChR Sensitivity to Snake Venom Neurotoxins. Consistent with this generalization, curarimimetic snake neurotoxins have been useful in demonstrating heterogeneity of functional nAChRs, even within the same tissue type. For example, subtle differences in the concentration-dependent effects of toxins and d-tubocurarine on function of rat muscle nAChRs suggest that there might be multiple sites of toxin action (Bradley et al., 1987). a-Bungarotoxin is effective at virtually all vertebrate muscle nAChRs except for those found in some snakes, presumably including Bungarus multicinctus (Schmidt, 1988). There is, however, other evidence for cross-species differences in sensitivity of muscle nAChRs to different snake toxins (Ishikawa and Shimada, 1983; Ishikawa et al., 1985). It will be very interesting to elucidate the toxin- and/or receptor-specific structural bases for these differences, but these observations presaged the emerging perspective that heterogeneity to toxin binding and functional potency also occurs in the autonomic and central nervous systems. Covered in some detail in previous reviews is the use of snake neurotoxins to distinguish between muscle and ganglia nAChRs

X

2

L

<

r. 4

t

"

-I +

I l

l

- - > I

I

40

"

=

+ I

-

1

+

I

+

t

t

Wong and Gallagher (1989) Mulle and Changeux (1990) McCormick and Prince (1987) Sorenson and Chiappinelli (1990) Ramoa et al. (1990) Lipton et al. (1987) Aizenman et al. (1990) Loring et al. (1989) Kalash et al. (1989) Flores et al. (1989) Hulihan-Giblin et al. ( 1990a) J. D. Miller et al. (1987) Zatz and Brownstein (1981) Miller and Billiar (1986) Freeman (1977) Lamacz et al. (1989) Plinkert et al. (1990)

Rat dorsolateral septum

Rang (1982) Pinnock et al. (1988) Marshall (1981) Lipscombe and Rang Patrick and Stallcup (1977a) Kemp and Edge (1987) Lukas (1988, 1989a)

Rat ganglia Chick ganglia Frog ganglia Frog ganglia Rat PC12 cells

+

+

Rat medial habenula Guinea pig medial habenula Chick lateral spiriform Rat retinal ganglion Rat retinal ganglion Rat retinal ganglion Chick retinal ganglion Rat pituitary function Rat prolactin release Rat prolactin release Rat circadian rhythym Rat circadian rhythym Rat circadian rhythym Goldfish optic tectum Frog pars intermedia Guinea pig cochlear outer hair cell

Rat PC12 cells Rat PC12 cells

>I

>0.5

10

+

+

+0.2

1

-1

+

t

-

-5 -10

0.7

-20

+10

-10

+2

+I0

+I0

+I0

-10

+I00

+I00

+lo0

+0.06 -10

+

+O.l

+

+ +

+ + +I

+I00

+ 100 Ganglia or Ganglia-Type >20 >20 lo 1000 3

>40 -10

+

+0.05

-

3 1

-1

0.3

100

1

600 60 100

100

30

100

300

6 60 1000

0.2 0.2

30 (continued)

Ihig

Study

Preparation

d-TC

toxinh

Deca

Hexa

0.2

3

0.8

0.2

Mturle-7:vfw LOO 3000

a

K

Mouse N I E- 1 15 cells

-11.5

+,I.,,,

+o.n

Bovine adrenal chromaffin

- 0. I

+(I, 1

Lukas (1988, 198%)

Human TE671/KD cells

0.lti

Bertrand et a/. ( 1 9%)) Couturier et al. (199Ob) Deneris et al. (1991)

Chick a,,-& Chick a7 Kat a2-P2

Oortgiesen and Vijerlierg

oi-

DHBE

Meca

Atro

0.1

60

90

100

( 1989)

Higgins and Berg (1987, 1988)

2000

Exfire.s.ved (,'lo?ie.~

-'0 +O.OOl

+(I

+PB

I

+

+ I 0

+P.B

f0.I

+

"Data are summarized from selected studies of antagonist potency toward functional nAChRs expressed by the indicated neuronal cell preparations, in the Xenopzw. oocyte system, or, for reference, by a cell line expressing human muscle-type nAChRs. Where dose-response studies were conducted, apparent ICBovalues are provided Otherwise, plus signs indicate that the drug or toxin was effective in inhibiting at least 50% of the nicotinic response, and minus signs indicate ineffectiveness. In these cases, the dose of drug or toxin used, where known, is indicated by the subscript value Note that IC50 values do not correct for differences in agonist dose or type used, although agonist doses are usually the same for a given study. The data clearly illustrate that most small nicotinic antagonists have been reported to block neuronal nAChR function. Mecarnylamine is most commonly found to be effective at neuronal nAChR and may be a selective ligand for those nAChR subtypes (100-fold more potent at ganglia-type nAChKs than at muscle-type nAChRs). Full citations are given in the reference section. *a,a-bungarotoxin; K, K-bungarotoxin; d-TC, d-tubocurarine; Deca, decamethonium; Hexa, hexamethonium; DHBE, dihydro-P-erythroidine; Meca, mecamylamine; Atro, atropine. <Used a-cobratoxin from the venom of Naja naja kaouthia.

(w).

(e).

NICOTINIC RECEPTOR DIVERSITY AND REGULATION

43

(reviewed in Chiappinelli, 1986; Loring and Zigmond, 1988; Schmidt, 1988). A group of dimeric toxins that lack a tryptophan residue conserved among many other curarimimetic neurotoxins and have a proline residue at another critical sequence position have been defined as the Kneurotoxin family (Grant et al., 1988; Chiappinelli et al., 1990). These toxins are potent antagonists at nicotinic synapses in a number of avian ganglia preparations tested to date where there is no manifestation of functional sensitivity to a-bungarotoxin; however, not all ganglia nAChRs are sensitive to K-neurotoxin blockade, particularly in mammals (but see Sah et al., 1987), suggesting again that there are pharmacological and, necessarily, structural differences between nAChRs across species as well as across tissues. j . Sensitivity of Neuronal nAChRs to Snake Neurotoxins. Until recently, it was assumed that K-bungarotoxin would block most nicotinic responses in the CNS, whereas there was considerable and persistent skepticism regarding functional potency of a-bungarotoxin or related toxins in the vertebrate CNS (reviewed in Schmidt, 1988). Based on the most recent evidence, it is now clear that there is heterogeneity with respect to CNS nAChR functional sensitivity to toxins. Responses of some mammalian and avian central neuronal nAChRs are resistant to blockade by either a- or K-bungarotoxin (Mulle and Changeux, 1990; Sorenson and Chiappinelli, 1990). Other responses are sensitive to blockade by K-bungarotoxin, sometimes where a-bungarotoxin is ineffective (in rats: Lipton et al., 1987; de la Garza et al., 1989; Schulz and Zigmond, 1989; Vidal and Changeux, 1989; Wong and Gallagher, 1989; Aizenman et al., 1990; in chick: Loring et al., 1989). It is interesting to note that presynaptic nAChRs regulating neurotransmitter release are blocked by K-bungarotoxin, but not by a-bungarotoxin (Lapchak et al., 1989a; Schulz and Zigmond, 1989); however, it also is evident that some mammalian CNS nAChR responses are fully sensitive to blockade by a-bungarotoxin or related a-neurotoxins [e.g., in primary cultures of rat hippocampal cells: (Alkondon and Albuquerque, 1990; in the intact rat inferior colliculus: Farley et al., 1983; on inhibitory interneurons in the intact rat cerebellum in vivo: de la Garza et al., 1987b; and in intact rats: Zatz and Brownstein, 1981; Kalash et al., 1989; however, see Miller and Billiar, 1986) (see Table 11). I n addition, other evidence indicates functional roles for abungarotoxin in the vertebrate CNS via mechanisms that have yet to be elucidated (Freund et al., 1990). These studies now clearly indicate that some mammalian central responses to nicotinic agents are sensitive to atoxins, just as is the case for some central and autonomic responses in the frog and in goldfish (Freeman, 1977; Kato et al., 1980; Marshall, 1981; Lamacz et al., 1989). Moreover, recent evidence that functional re-

44

RONALD J, LUKAS A N D MEROC'ANE BENCHERIF

sponses of a reconstituted product of a chick nBgtS subunit-encoding gene have unique nicotinic ion channel characteristics and are sensitive to blockade by a-bungarotoxin (Couturier et al., 1990a) suggests that nBgtSs may, in fact, represent a subclass of central neuronal nAChRs also expressed in autonomic ganglia that function as ion channels and may have been involved in a-bungarotoxin-sensitive responses. k. Functional Effects of Areosurugatoxzn. Another neurotoxin that has been used to distinguish functional nAChR subtypes is neosurugatoxin, which is a multiring organic toxin isolated from the Japanese ivory shell (Kosuge et al., 1981). Initially, it and a related toxin were reported to have potent activity as antagonists of nAChR function in autonomic ganglia, but not in muscle (Hirayama et al., 1974; Hayashi and Yamada, 1975; Hayashi et al., 1984). Subsequent studies have shown that neosurugatoxin blocks functional responses of ganglia-type nAChRs in bovine adrenal chromaffin cells (Wada et al., 1989) and presumed central rieuronal nAChR function in mice (Yamada et al., 1986), in rat striatal synaptosomes (Rapier et al., 1985), and in ovariectomized rats (Billiar et al., 1988). These studies suggest that neosurugatoxin is potent as an antagonist of nonmuscle nAChR function, although more recent studies of rat PC 12 pheochromocytoma cell ganglia-type nAChRs and human TEG7 1IRD clonal cell muscle-type nAChRs indicate that neosurugatoxin is selective, but not specific, as a nAChR functional antagonist (Lukas, 1989a). 1. Functional Effects of Lophotoxin. Lophotoxin, a diterpene lactone isolated from gorgonian corals, is a functional antagonist at muscle and ganglia nAChRs, but it is neither a selective nor a high-affinity ligand for n.4ChR subtypes (Culver and Jacobs, 1981; Langdon and Jacobs, 1983, 1985; Atchison et al., 1984; Sorenson et al., 1987). vn. Thymopoietin as a Functional Antagonist. Another agent capable of distinguishing between functional ganglia-type and muscle-type nAChRs is the thymic substance thymopoietin (see reviews in Lukas et al., 1990; Ochoa et al., 1990; Quik et al., 1990a,b). These and other studies have shown that thymopoietin is a potent, acute antagonist of muscle or muscle-type nAChR function, but is ineffective as an inhibitor of ion channel activity of ganglia-type nAChRs. n. Anti-nAChRs as Probes for Functional Muscle nAChRs. Some antibodies raised against nAChRs have been tested as functionally active probes targeted at specific nAChR subtypes in efforts to augment available drugs and toxins used for functional nAChR isotyping (reviewed in Tzartos, 1990). Antibodies directed against the ligand binding site of muscle (myasthenic) or muscle-type riAChRs can block muscle nAChR ion channel activity and can produce myasthenic-like symptoms in ex-

NICOTINIC RECEPTOR DIVERSITY AND REGULATION

45

perimental animals (Goldberg et al., 1983; Gomez and Richman, 1983; Maricq et al., 1985; Schuetze et al., 1985; Fels et al., 1986; Maselli et al., 1989). Antibodies recognizing epitopes remote from the ligand binding domain also can affect muscle nAChR function (Donnelly et al., 1984; Blatt et al., 1986), perhaps by acting as channel blockers. Myasthenic antisera can affect function of muscle-type nAChRs expressed by cells of the TE671/RD clone (Lang et al., 1988). 0. Anti-nAChRs as Functional Probes for Neuronal nAChRs. Anti-muscle nAChR antibodies also have been tested for functional effects in neuronal o r neuronlike systems. Anti-ElectrophorzrsnAChR antisera that produce myasthenic-like symptoms in experimental animals block function of PC12 cell ganglia-type nAChRs (Patrick and Stallcup, 1977b). Antibodies initially raised against muscle-type nAChRs modulate expression of chick ganglia functional nAChRs (Smith et al., 1986), PC12 cell functional ganglia-type nAChRs (Whiting et al., 1987a), and bovine adrenal chromaffin cell ganglia-type nAChRs (Higgins and Berg, 1987). Stollberg et al. (1986) have reported that antibodies raised against chick brain nAChRs are capable of directly inhibiting function of chick ganglia nAChRs. In the CNS, myasthenic sera and anti-muscle-type nAChR antibodies also have been reported to alter basal and stress-induced adrenocortical activity in rats (Weidenfeld et al., 1983, 1988; Brenner et al., 1986), suggesting that muscle-type nAChR-like antigens in the brain could be involved in this functional response. 3. General Properties of Nicotinic Receptors and Manifestations of Receptor Diversity: Structural Studies a. Historical Overview. Attempts to characterize and purify nAChRs began by focusing on the electric tissue of electric fish (reviewed in McCarthy et al., 1986; Claudio, 1989). This tissue is embryologically related to muscle and, in the case of Narcine and Torpedo, is composed of cuboidal cells with one face that is nearly completely innervated and contains a virtual crystalline array of muscle-type nAChRs. nAChRs in these tissues are present at concentrations of about 1 nmol per milligram of tissue wet weight, which is about 1000-fold greater than the concentration of nAChRs in innervated vertebrate muscle. These features have allowed for the purification of membrane fragments (and derivative, detergent-solubilized extracts) that are very highly enriched in nAChRs by simply subjecting the electroplaque cell to shearing forces and separation techniques designed to take advantage of the distinct physical characteristics of the subsynaptic membrane region. These studies were also aided by the isolation of curarimimetic polypeptide neurotoxins (which could be used as radioligands, affinity probes, and

46

RONALD J. LUKAS AND MEROCANE BENCHERIF

horseradish peroxidase and fluorescent conjugates, and immobilized probes for affinity chomatography) to purify and characterize nAChRs at the molecular and cellular levels (reviewed by McCarthy et al., 1986). b. M u c l e riAChRs a.s a Pentameric Hetero-oligomer with al Subunits Containing the Ligand Binding Domains. It is now known that electric tissue muscle-type nAChRs are pseudosymmetric pentameric integral membrane proteins composed of four types of subunits, termed a,,p y, and 6, present in the ratio 2:l:l:l (reviewed in McCarthy et al., 1986; Claudio, 1989).Based on affinity labeling protocols using small nicotinic ligands that alkylate a sulfhydryl group(s) near an acetylcholine-binding active site of muscle-type nAChRs (Kao P t al., 1984; Kao and Karlin, 1986), o r that form covalent complexes with a tyrosine residue in the same vicinity (Abramson et al., 1989), a, subunits are thought to harbor the agonist binding site. T h e latter studies were done using a substance that has nonselective antagonistic activity toward both muscle and ganglia nAChRs, lophotoxin, a diterpene lactone isolated from sea whips (Fenical et al., 1981; Culver et al., 1984, 1985; see Abramson et al., 1989). The lophotoxin studies are particularly important, as arguments could be made that the only reason affinity alkylating agents had labeled a, subunits was because only those subunits express the requisite tandem cvsteine residues near the ligand binding active site (see below) and then only after nonphysiological reduction of the disulfide bond involving those residues. c. Lignnd Crosslinking Studies. Early chemical crosslinking or photoaffinity labeling studies using curarimimetic neurotoxin derivatives labeled more than one or all of muscle o r muscle-type nAChR subunits (Hucho, 1979; Witzemann et al., 1979; Nathanson and Hall, 1980; Oswald and Changeux, 1982; Hamilton et al., 1985); however, more recent studies using precisely engineered photolabile toxin derivatives and binding paradigms (Chatrenet et al., 1990) or photoaffinity labeling using d-tubocurarine (Pedersen and Cohen, 1990) offer further support for the identification of a 1 subunits as the targets for nicotinic ligand binding. These studies not only indicate that the a,subunit is most likely to be the major focus of toxin binding, but also confirm that the two ligand binding sites on a1 subunits are not equivalent, apparently as a result of differences in the environment created by neighboring subunits (Blount and Merlie, 1989; Pedersen and Cohen, 1990; but see ContiTronconi et al., 1984, 1990).Debate continues, however, regarding identification of the subunits that neighbor high- and low-affinity a,subunits and whether the arrangement of nAChR subunits about the central pore is a ,+-a, -y-6 or a -y-a, -p-8 (compare Pedersen and Cohen, 1990, to Chatrenet at al., 1990) (Fig. 3). Controversy also continues regarding the

NICOTINIC RECEPTOR DIVERSITY AND REGULATION

MODEL 1

47

MODEL 2

FIG. 3. T w o possible arrangements of the polypeptide chain of nAcChR subunits with respect to the membrane surface. In model 1, the amphipathic helix (MA) is assumed to reside in the cytoplasmic space. In model 2, a transmembrane disposition of MA is assumed. Presently, the consensus of opinion favors model 1. Amino acid deletions affect (PB) or d o not affect (N) ligand binding or functional responses. (Modified, with permission, from Guy and Hucho, 1987.)

topography of the receptor as a whole with respect to the positions and actual number of transmembrane segments (compare Criado et al., 1985, to McCarthy et al., 1986) and whether nAChR subunit C termini are extracellularly or intracellularly disposed (compare Dwyer, 1988; Dipaola et al., 1989; Moore et al., 1989; and Mitra et al., 1989; Fig. 4). If these issues continue to remain unresolved, the possibility that subunit ordering and topography are naturally variable might be considered. d . Synthetic Peptides as Tools for Identifying Ligand Binding Domains. Other lines of evidence supporting the identification of muscle or muscle-type nAChR a 1 subunits as sites for interaction with nicotinic ligands come from studies using synthetic peptides (reviewed by Lentz and Wilson, 1988). Some of these studies have shown that synthetic peptides corresponding to residues 173-204 of electric fish muscle-type nAChRs, and including the tandem cysteine residues at positions 192 and 193, bind a-bungarotoxin with the same affinity as denatured a 1 subunits (Gotti et al., 1988; Wilson et al., 1988; Wilson and Lentz, 1988). Other studies have shown that antibodies against these peptides inhibit toxin binding (Gotti et al., 1988; Chinchetru et al., 1989; DonnellyRoberts and Lentz, 1989). More recent work suggests that tyrosines 189 and 190 and aspartate 195 of the Torpedo a, subunit, but not cysteines 191 and 192, are required for toxin binding (Tzartos and Remoundos, 1990). Independent studies implicate distinct clusters of residues or solitary amino acids in binding of a-bungarotoxin (valine 188, tyrosines 189 and 190, cysteines 192 and 193, proline 194) or in binding of the WE6 antibody, which effectively competes for toxin binding (tryptophan 187, threonine 191, cysteine 193, proline 194, aspartate 195, tyrosine 198;

48

RONALD 1. LL‘KAS A N D MEROLJANE BENCHEKIF

NEUROTOXlNlLlGAND BINDING OPENCHANNE

‘--GATED ION CHANNEL FIG. 4. Schematic ill ustration of a proposed three-dimensional structure of the muscletype ni\cC:hR. Positions of receptor subunits around the receptor channel, channel dimensions (other than eiilarged opening in the extracellular vestibule relative to the intracellular pore opening), and localization of toxin/liganci binding sites arc tentative. T h e three-loop (“Mickey Mouse glove”) structure of snake neurotoxins active at the muscle nArChK is illustrated to show approximate proportions t o scale. T h e receptor monomer has d mass o f about ?50 kDa, and each subunit has a mass of about 50 kDa, about 40% of which (i.e.. about 20 kDa) is localized to the extracellular space. For comparison, toxins have a molecular mass of about 8 kDa, arid nicotinic ligands such as acetylcholine (about 200 Da) and d-tubocurarine (about 700 Da) are 10- to 40-fold smaller. (Modified. with permission, from S t r w d , 1981.)

Conti-‘rronconi et al., 1991). Coniplenientary protein blot analyses also show selectivity of a ,subunit in interaction with toxin (reviewed in Lentz and Wilson, 1988). e. Norirompetitive Antugon& Sites and the Ion Channel Domuin. Evidence also has been accumulated recently that allosteric antagonist binding sites o n muscle-type nAChRs are located near the agonist-binding domain of the a,subunit (Pedersen et al., 1986; Herz et al., 1989). Other evidence is now convincing that the nAChR channel is lined by the proposed second transmembrane segments, M2, of each nAChR subunit, that the M2 region harbors the binding sites for noncompetitive blockers, and that channel gating and perhaps lining also involve M 1 transmembrane segments (Mishina et ul., 198.5, 1986; Giraudat et al., 1986; Oberthur and Hucho, 1988; Imoto et ul., 1986, 1988; Leonard et al., 1988a; Oiki et al., 1988; Charnet et al., 1990; DiPaola et al., 1990; Lo et a[., 1991; see also Dani, 1989). A very interesting observation suggests that placement of M2 segments in the core of the assembled receptor might also serve another functional purpose, for they contain sequences

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similar to those found in other integral membrane proteins that target the protein for rapid, pre-Golgi degradation; thus, exposure of M2 sequences would facilitate degradation of incompletely assembled receptor, whereas maturation of assembled receptors that shield the M2 sequence could proceed (Blount et al., 1990). f. Glycosylation in the Control of Receptor Assembly and Processing. Several lines of evidence suggest that glycosylation of nAChR subunits plays a critical role in receptor assembly and expression, as one might expect for any hetero-oligomeric, integral membrane protein (Merlie et al., 1982; Merlie and Smith, 1986; Nomoto et al., 1986). It is now evident that perturbation of glycosylation changes the rate of nAChR assembly, but does not prevent assembly. Failure to add high-mannose N-linked oligosacharides to nAChR subunits leads to accumulation of nAChRs in the endoplasmic reticulum, where they are more rapidly degraded than their fully glycosylated and assembled counterparts (Gu et al., 1989; Sumikawa and Miledi, 1989; Blount and Merlie, 1990). g. Are There Other Ligand Binding Sites? Another very interesting and problematic issue is the enormous disparity between (1) concentrations of nicotinic ligands, such as carbamylcholine, required to evoke halfmaximal activation of function from native receptor, whether by electrophysiological or chemical kinetic measurements (0.1- 1 a), (2) concentrations necessary to inhibit by 50% high-affinity binding of radiand (3)concentrations necessary to oiodinated neurotoxin (about 1 account for equilibrium binding or competition for binding of tritiumlabeled nicotinic agonists (10- 100 nM) to muscle or muscle-type nAChRs (reviewed by McCarthy et al., 1986; and Lukas, 1988, 1989b). If the higher-affinity agonist binding sites represent those existent on nAChRs in a desensitized state, complex kinetic schemes can be devised to explain how transitions to that state could be effected by agonist binding to the lower-affinity native state (McCarthy et al., 1986; Ochoa et al., 1989); however, less direct measures of ligand binding have suggested that low-affinity sites can be detected even when higher-affinity sites are occupied, prompting speculation and some experimental support for the existence of additional (i.e., more than one per a1subunit) ligand binding sites, some of which perhaps are recognized in a voltagedependent manner (reviewed in McCarthy et al., 1986; Ochoa et al., 1989), to account for some of these discrepancies. h. Heterogeneity of Muscle nAChR Subtypes within a Single Tissue? Persistent reports appear of nAChR “microheterogeneity,” which is the term we will use in reference to heterogeneity in properties of a given nAChR subtype expressed in a given type of tissue. I n some reports, this microheterogeneity is suggested to be distinct from the well-documented exis-

w),

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RONALD J. LUKAS AND MEROUANE BENCHERIF

tence of nonequivalent ligand binding sites on muscle or muscle-type nAChR preparations (see above) and appears to reflect differences in antigenic character and/or glycosylation state of both high- and lowaffinity ligand binding components (Massa and Mittag, 1983). In other reports, however, antibody specificity is suggested to be for one of the nonequivalent ligand binding sites (Mihovilovic and Richman, 1984, 1987; Gu et al., 1985; Fels et al., 1986). The significance of physical microheterogeneity and its relationship, for example, to distinct classes of functional nAChR ion channels in a given cell type (see above) or to alternatively spliced gene products (see below) remain to be elucidated. 2. Cross-Speczes Heterogenezty of Muscle nAChRs. There also is evidence for cross-species heterogeneity in muscle nAChRs. For example, both abungarotoxin and erabutoxin b (from the sea snake, Latacauda semafuscultu) bind to rodent muscle nAChRs, but primate muscle nAChRs fail to interact with erabutoxin (Ishikawa and Shimada, 1983; Ishikawa et al., 1985). Diflerences in efficiency with which human muscle and Torpedo nAChRs can be reduced and affinity alkylated have been reported (Momoi and Lennon, 1986). Human and Torpedo muscle-type nAChRs also differ in rates of dissociation of a-bungarotoxin and profiles of ligand competition toward radiotoxin binding (Lukas, 1986a,b).There is a possible genetic basis for these differences, in that there is close, but not absolute, conservation of amino acid sequence identity in the putative ligand binding domain of different a, subunits across species (Noda ~t al., 1983a; see also below). Binding studies using synthetic peptides also indicate that toxin binding domains, for example, on human muscle and Torppdo nAChR a , subunits, differ in their ability to interact with radiolabeled toxin (Neumann et al., 1986; Mulac-Jericevic et al., 1988; Lentz and Wilson, 1988; Wilson and Lentz, 1988; Griesmann et al., 1990),and suggest that the region between residues 173 and 204 may not always fully confer toxin binding specificity. Immunological evidence that there is heterogeneity in the ability of anti-muscle type nAChRs and myasthenic serum antibodies to react at epitopes near or remote from the ligand binding site also supports the notion of crossspecies polymorphism in nAChRs (Lukas, 1986a,c,d; Lennon and Griesmann, 1989). j . Identzfication of Neuronal Sites Reactave with Curaramzmetic Neurotoxins. Over the years, it has become evident that curarimimetic neurotoxins are the most purely competitive inhibitors of nicotinic ligand binding and function of muscle and muscle-type nAChRs (Katz and Miledi, 1978). Hence it was not unreasonable to assume that any nAChR subtype, generically defined by their ability to interact with acetylcholine and nicotine, would also exhibit interactions with agents such as a-bun-

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garotoxin across species and tissues (Schmidt, 1988). Aside from some earlier studies using tritium-labeled small nicotinic ligands to identify putative nAChRs in the central nervous systems (see below), much of the pioneering work in this regard involved use of radiolabeled toxins to identify and begin characterization of ganglia and central neuronal nAChRs. These studies demonstrated that there exist finite numbers of candidate nAChRs that could interact with radioiodinated a-bungarotoxin with high affinity and that these entities had the appropriate nicotinic-cholinergic pharmacological profile, sensitivity to local anesthetic treatment and agonist-induced affinity state transitions, the ability to specifically interact with muscle-type nAChR affinity alkylating agents, a regional distribution contained within the distribution of histochemically identified cholinergic pathways, a predominant (where tested) postsynaptic localization, and other physicochemical attributes expected of authentic nAChRs (reviewed in Schmidt, 1988). k. Some Properties of nBgtSs. There now seems to be a consensus that affinity purification of nBgtSs using toxin columns yields preparations containing at least three and probably four types of subunits (ContiTronconi et al., 1985; Kemp et al., 1985; Siege1 and Lukas, 1985; Gotti et al., 1987, 1989; Whiting and Lindstrom, 1987), very similar in size (about 45, 50, 60 and 65 kDa) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis to the subunits of muscle nAChRs. Nevertheless, the properties of nBgtSs suggested that they were not identical to muscle-type nAChRs, as there were notable distinctions in size and pharmacological profiles between these species (Lukas, 1986a, 1988, 1989b; Schmidt, 1988). Moreover, when it was discovered that not all nicotinic responses in the autonomic or central nervous system could be blocked by a-bungarotoxin, it became clear that a-bungarotoxin was a specific or highly selective probe that could functionally distinguish muscle-type from ganglia-type nAChRs much as functional distinctions can be made using ganglia/central neuronal nAChR-selectiveagents such as mecamylamine (Schmidt, 1988). Questions were raised as to the functional significance of ganglionic and, by inference, central neuronal abungarotoxin binding sites and the relevance of these nBgtSs to functional nAChR in the autonomic and central nervous systems. These questions lead to a flurry of activity designed to identify alternative probes for ganglia and central neuronal nAChRs (Schmidt, 1988). 1. Agonists as Probes for Neuronal nAChRs. As might have been predicted based on the success of previous studies done using radioagonist binding assays to identify high-affinity (1- 10 nM) sites on muscle-type nAChRs (Neubig and Cohen, 1979; Boyd and Cohen, 1980),if not based on the requirement for much higher concentrations of ligand (high

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micromolar range) to induce nAChR functional activity, a number of laboratories reported success in using stereoselective binding of [:3H]nicotine or specific binding of [3H]acetylcholine [and, later, radiolabeled methylcarbamylcholine (Abood and Grassi, 1986; Boksa and Quiron, 1987), azetidine analogs of nicotine (Abood et al., 1987), and cytisine (Pabreza et al., 1991)]to identify putative nAChRs and/or nicotine receptors in the central nervous system (reviewed in Schmidt, 1988). A number of early studies suggested that there might be differences between nicotinic receptor binding sites for radiolabeled acetylcholine and nicotine, heterogeneity of radioligand binding sites, or both; however, more recent work suggests that these observations might arise artifactually, at least in rodents (Lippiello and Fernandes, 1986; Marks et al., 1986; Martino-Barrows and Kellar, 1987; Komm et al., 1990). Evidence that apparent heterogeneity of radioagonist binding might be due in part t o interconversion between high- and low-affinity nicotine binding sites has been obtained in studies using human (Romanelli et al., 1988) and rat (Lippiello et al., 1987) tissues. These observations may also provide a binding assay correlate of central neuronal nAChR desensitization (Lippiello at ul., 1987). It is not yet clear whether studies using ["H]acetylcholine and indicating expression in human brain of more than one class of radioagonist binding site (Adem et al., 1987) reveal species-dependent heterogeneity of radioagonist binding sites (perhaps as a result of the different levels of expression of specific nAChK subunit gene products; see below) or are due to binding site interconversion. m. High-Affinity Nirotznic Agonist Binding Sites Are Not the Sawie as nBgtS. A comparison of the distribution of high-affinity nicotinic agonist binding sites with those for a-bungarotoxin by the use of autoradiographic techniques and frozen sections of brain tissue revealed virtually no overlap between those sites (Schmidt, 1988). Separation of detergent-solubilized high-affinity radioagonist binding sites from highaf'finitybinding sites for radiolabeled a-bungarotoxin also indicated that these sites are different (Wonnacott, 1986; Sugiyama and Yamashita, 1986), although at least a subset of rat brain high-affinity radioagonist binding sites appears to interact with a-bungarotoxin, but only with low affinity (Costa and Murphy, 1983; Lukas, 1988). The distribution of high-affkity agonist binding sites is contained within the distribution of putative cholinergic pathways, and some evidence was obtained that these sites have presynaptic as well as postsynaptic localizations (Schmidt, 1988; see also Rowell and Winkler, 1984; Rowell, 1987; Arau.jo ef nl., 1988; Lapchak et al., 1989a,b). T h e majority of these sites are relat.ively insensitive to blockade, not only by a-bungarotoxin, but also by smaller nicotinic antagonists, such as d-tubocurarine. l h i s finding raised some questions as to their relationship to the low-affinity agonist binding

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sites that presumably are involved in channel gating, but some evidence was obtained that agonist binding entities also are sensitive to modulation of sulfhydryl oxidation-reduction state and the effects of muscle nAChR affinity alkylating agents such as bromoacetylcholine and N ethylmaleimide, thus supporting their identity as nAChRs (Schmidt, 1988; Stitzel et al., 1988). In general, pharmacological potency profiles of drugs acting at high-affinity agonist binding sites are heavily weighted toward nicotinic agonists, with cytisine and nicotine being among the most potent inhibitors of nicotinic [3H]acetylcholine or [3H]nicotine binding to adult tissues (Schmidt, 1988), to membranes from fetal brain primary cell cultures (Lippiello and Fernandes, 1988), or to ganglia-type nAChRs from rat PC12 cells (Lukas, 1990) but not to ganglia-type nAChRs from bovine adrenal chromafin cells (Higgins and Berg, 1988). n. Physical Properties of Radioagonist Binding Sites. Acetylcholine affinity chromatography has been used to partially purify putative highaffinity agonist binding sites and yields four major polypeptides of 53, 67,80, and 108 kDa plus several minor components including a 48-kDa polypeptide; the 80- and 48-kDa proteins contain epitopes recognized by antibodies raised against immunoaffinity-purified (see below) ligand binding and non-ligand binding subunits, respectively (Nakayama et al., 1990). An 80-kDa detergent-solubilized component from rat brain has been found to interact with [3H]nicotine (Madhok et al., 1989). Affinity chromatography using a nicotine-like ligand or anti-idiotypic antibodies raised against antinicotine yields a major protein constituent of about 65 kDa (Abood et al., 1983, 1987). Continued work to clarify/confirm these observations is required. 0. K-Neurotoxins as Probes for Neuronal nAChRs. Detailed descriptions of the use of the dimeric K-toxins as radiolabeled probes for ganglia, ganglia-type, o r central neuronal nAChRs have appeared (reviewed in Chiappinelli, 1986; Loring and Zigmond, 1988; Schmidt, 1988). More recently, studies using these probes to identify putative nAChRs in the CNS have tried to overcome difficulties in distinguishing between subsets of specific K-bungarotoxin binding sites on the basis of their ability to also interact with a-bungarotoxin and in demonstrating saturability of high-affinity K-toxin binding (Wolf et al., 1988; Loring et al., 1989; Schulz et al., 1991). Rat brain membrane binding studies demonstrate that atoxin-insensitive, specific, saturable, high-affinity K-toxin binding sites are present in lower quantities than a-toxin-sensitive sites and than radioagonist binding sites or nBgtSs. Autoradiographic studies indicate that specific K-toxin sites are widely distributed in the rat brain, but more narrowly so than sites that interact with radioagonists or a-bungarotoxin (Schulz et al., 1991). Similar studies using chick ganglia, chick optic lobe,

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chick retina, and rat PC 12 cells identified specific a-toxin-insensitive Kbungarotoxin binding sites (Halvorsen and Berg, 1986; Wolf et al., 1988; Loring et al., 1989; McLane at al., 1990a). Taken together, these results suggest that there are at least four types of nAChR-like binding sites in the CNS. p. Neosurugatoxzn as a nAChR Probe. Studies done using neosurugatoxin as a competitor for radioligand binding sites reveals heterogeneity among nAChR subtypes as might have been predicted based on its functional selectivity (see above). Neosurugatoxin is only a weak inhibitor of radiolabeled a-bungarotoxin binding to muscle or muscle-type nAChRs o r to nBgtSs in ganglia o r the CNS. Neosurugatoxin is a high-affinity inhibitor of only about one-half of the specific, high-affinity nicotinic agonist binding sites found in brain; the other proportion of agonist binding sites are also inhibited by neosurugatoxin, but only at lower concentrations (see Hayashi et al., 1984; Yamada et al., 1985; Billiar et al., 1988; Lukas, 1990). Its potential as a radioligand probe is dampened by its complexity and the ease with which moderately harsh solvents induce loss of functional potency; isolation of purified radioderivatives that retain ligand binding and/or functional potency is extremely difficult, and the toxin itself is in short supply. q. Other Drugs and Toxins as Selective Probes for nAChR Subtypes. Promise that other ligands might be useful in distinguishing nBgtSs from central neuronal nAChRs comes from studies showing that methyllycaconitine and (+)-anatoxin-a, respectively, are selective blockers of radiotoxin and radioagonist binding in rat brain (Zhang et al., 1987; MacAllan et al., 1988; Wonnacott et al., 1991). 1. Thymopoiettn as a Selertizie nAChR Probe. Studies done to date (reviewed in Lukas et d.,1990; Ochoa et al., 1990; Quik et al., 1990a,b) indicate that thymopoietin competes with high affinity for radiolabeled a-bungarotoxin binding to muscle or muscle-type nAChRs and to nBgtSs; however, thymopoietin does not inhibit binding of radiolabeled agonists to their high-affinity sites on presumptive ganglia, ganglia-type, or central neuronal nAChRs, although competition for high-affinity agonist binding to muscle or muscle-type nAChRs does occur. These observations indicate that thymopoietin is a specific ligand for some members of the extended nAChR family. J. Anti-Mwcle nAChR Antabodzes as Probes for Neuronal nAChRs. Based on immunohistochemical and immunochemical analyses, some antimuscle-type nAChR antibodies have been shown to cross-react with nonmuscle nAChRs or nBgtSs in the autonomic and centrabnervous systems not only in nonrnammalian vertebrates (Swanson et al., 1983; Mehraban

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et al., 1984; Conti-Tronconi et al., 1985; Henley et al., 1988; Sargent et al., 1989), but also in mammals (Patrick and Stallcup, 1977b; Block and Billiar, 1979; Mochly-Rosen and Fuchs, 1981; Watters and Maelicke, 1983; Lukas, 1986a,c,d; Deutch et al., 1987; Schroder et al., 1989a,b). Some of these antibodies appear to be directed against the toxin binding site of muscle and muscle-type nAChRs as well as that of nBgtSs (Lukas, 1986a,c,d). This latter observation is not surprising if one considers toxins as mimics of antibodies that recognize conserved domains that appear on selected members of the extended nAChR family and are involved in toxin recognition. Other antibodies, such as monoclonal antibody 35 (mAb35), which reacts with “the main immunogenic region” of muscle a1subunits, do not interact at toxin binding domains or with nBgtSs, but recognize ganglia or ganglia-type nAChRs in chick ganglia or bovine adrenal chromaffin cells (Smith et al., 1985, 1986; Higgins and Berg, 1987). mAb35 immunoprecipitates photoaffinity-labeled K-bungarotoxin binding sites including a polypeptide of about 60 kDa (Halvorsen and Berg, 1987). mAb35 used in immunoaffinity protocols purifies two types of putative chick ganglia nAChR (Halvorsen and Berg, 1990). One type contains two polypeptides of about 50 and 60 kDa; the 60-kDa species appears to be a putative ligand binding subunit corresponding to the chick a3 gene (see below), whereas the 50-kDa species is immunologically related to a similar species found in chick brain (see below). The other type contains an apparently novel component of about 52 kDa as well as the approximately 60-kDa polypeptide (Halvorsen and Berg, 1990). Taken together, these findings indicate that epitope preservation has occurred to some extent across members of the nAChR family, which is antigenically heterogeneous. t. Development of Anta-Neuronal nAChR Antibodies. By use of an immunological bootstrapping approach, where antibodies raised against electric tissue muscle-type nAChRs and shown to be cross-reactive with vertebrate muscle or neuronal nAChRs were used to isolate those nAChRs, which then were used as antigens for the production of other antibodies, putative ganglia and central neuronal nAChRs have been identified (reviewed in Lindstrom et al., 1987; Whiting and Lindstrom, 1988). Immunoaffinity-purified nAChRs from chick, rat, bovine, or human brain comprise two types of subunit. One type of sulfhydryl-reduced subunit (about 75-79 kDa in chick, rat, and cow; there is another, approximately 60-kDa component of this type in some chick affinity isolates) can be covalently labeled with affinity alkylating reagents, whereas the other type (about 50 kDa in all species tested), which in chick and rat is reactive with antibodies directed against “the main immunogenic region” of mus-

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cle-type nAChR a,subunits, is not. Antibodies raised against these chick and rat subunits recognize polypeptides isolated on acetylcholine affinity columns (see above). Immunoaffinity-purified brain nAChRs bind radiolabeled nicotinic agonists with high affinity but do not interact with abungarotoxin. They also are physically distinct from brain nBgtSs that had been isolated via toxin affinity chromatography. Some cross-reactive anti-muscle-type nAChR antibodies and anti-neuronal nAChR antibodies recognize ganglia and ganglia-type n AChRs expressed in autonomic ganglia and PC12 cells (Lindstrom et ul., 1987; Whiting and Lindstrom, 1988). u. Anti-nAChR antibodies and the Identifj:cution of nAChR Subtypes and nAChR Subunits. T h e aforementioned studies and others (reviewed in Lukas, 1986a,c,d; Schmidt, 1988) support the notion of nAChR antigenic heterogeneity and are beginning to reveal the relationships between nAChR-like antigens, specific radioligand binding sites, and specific nAChR subunits. For example, immunohistochemical localization of brain nAChRs using specific antibodies has shown a distribution for CNS nAChR-like antigens that is very similar to that for radiolabeled nicotinic agonist binding sites but distinct from that for nBgtSs; evidence was also gleaned that some of these nAChRs might have presynaptic dispositions (Swanson et al., 1987). A recent study has shown that antibodies raised against synthetic peptides corresponding to a rat ag subunit sequence near the proposed ligand binding domain can compete for [“Hlnicotine binding t o rat brain sites (Madhok et a!., 1989). Antiidiotypic antibodies against antinicotine antibodies have been used to identify putatise nAChRs on neurons (Bjercke and Langone, 1989; Lippiello et al., 1991). Moreover, partial amino acid sequence information indicates conformity between sequences of immunoisolated peptides and deduced amino acid sequence information, at least for rat or chick a4 or P2 subunits (Schoepfer et al., 1988b; Whiting and Lindstrom, 1988; see also below). More work along these lines to provide further confirmation that the central dogma holds for the nAChR system is warranted, particularly to complement powerful techniques using expression systems to reveal relationships between nAChR genes, proteins, and functions (see below). I!. Diirerse Tooh to Stu.dy u Heterogentour Receptor Fumily. This overview indicates the value of varied attempts to identify neuronal nAChRs, not only to overcome biases introduced by using only one type of probe to identify members of a diverse receptor family, but also to provide diverse tools for comprehensive and convergent studies of the nature of those proteins, their relationship to nAChR subunit-encoding genes, and their- functional profiles and relevance.

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4. Genetic Basis for Nicotinic Receptor Diversity: Muscle and Muscle-Type nAChR Subunit Genes

a. Historical Overview and Deductions about nAChR Structure. The application of recombinant DNA techniques toward the characterization and cloning of nAChR subunit-encoding genes and messenger RNA was initiated following obtainment of partial amino acid sequences for four Torpedo nAChR subunits (Raftery et al., 1980). Recombinant DNA approaches were introduced by several different laboratories (Ballivet et al., 1982; Giraudat et al., 1982; Noda et al., 1982; Sumikawa et al., 1982) and were very rapidly extended by Numa and colleagues, who focused on muscle and muscle-type nAChRs (McCarthy et al., 1986). Their results as well as the results obtained in other laboratories (reviewed in McCarthy et al., 1986; Claudio, 1989) suggest that nAChR subunit genes are distinct but conserved derivatives of a common ancestral gene. Deduced amino acid sequences support earlier indications that there is significant amino acid sequence conservation and identity in N-terminal regions of each muscle-type nAChR subunit in the region corresponding to the putative major extracellular domain of each protein. Conserved among these subunits, as well as among other members of the ligandgated ion channel superfamily, is a putative disulfide-linkage-restricted loop in this N-terminal region and four putative transmembrane segments of hydrophobic amino acid residues as alluded to earlier. Each subunit, however, has distinctive amino acid sequences in the putative major intracellular loop located between postulated membrane-spanning segments M3 and M4, and the a 1 subunit is further distinguished from the others based on the uniqueness of its amino acid sequence in the vicinity of the putative nicotinic agonist binding active site. This region in the a1 sequence is characterized by the presence of a tyrosine residue at consensus sequence position 190 and by two, adjacent cysteine residues at positions 192 and 193 that are thought to engage in disulfide bond formation and provide the reaction center for affinity alkylation of reduced nAChRs by bromoacetylcholine and related compounds. Sitedirected mutagenesis studies support the identification of this region of the a, subunit as the agonist binding domain (Mishina et al., 1985). Sitedirected mutagenesis and sequence deletion studies have also contributed to the identification of the M2 transmembrane segment as the portion of each subunit that is most likely to physically line the ion channel of the assembled receptor and to the identification of specific amino acid residues or groups of residues that are likely to engage in interaction with local anesthetics, open channel blockers, and other ligands (see above). Initial functional expression studies done predomi-

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nantly using the Xenopls oocyte system have demonstrated that all four muscle-type nAChR subunits are required for construction of a fully functional and regulatable nAChR, but that three subunit receptors containing a,,P I , and either y o r 6 subunits can display a modicum of channel activity (reviewed in Claudio, 1989). b. Consematzon of Muscle-Type nAChR Gene and Protein Sequences and Structure. Extension of these research strategies to chick and mammalian muscle nAChRs reveals that there is conservation of all of the salient features of nAChR subunits across species (reviewed by McCarthy et al., 1986; Claudio, 1989). These include the presence of a signal peptide, a presumed N-terminal extracellular domain containing glycosylation sites and two sets of cysteine residues, four putative transmembrane segments of hydrophobic amino acids, and a presumed intracellular loop between putative transmembrane segments M 3 and M4 that has several consensus phosphorylation sites. Sequence comparisons indicate that a given subunit is more analogous to its correlate in another species than to another subunit from the same tissue, particularly in mammals, where there is 90--95% or higher amino acid sequence identity and 85-90% nucleic acid sequence identity for a given subunit across species. c. M x d e nAChR Subunit Gene Diversity: A Functionally and Developmentally Relevant Subunzt Switch. Cloning studies using muscle tissues provided the first tangible indication for diversity of nAChRs based on developmentally controlled subunit switches (see below) in the identification of muscle nAChR E-subunit-encoding sequences (Takai et al., 1985) corresponding to a protein that substitutes in adult muscle for y subunits found in embryonic tissue (Witzemann et al., 1987; Gu and Hall, 1988a,b). Typically, there is about 40% amino acid sequence identity between muscle subunits within a given species, except for ~ - - S - E triads exhibiting about 50% sequence identity (Fig. 5). It is perhaps surprising that greater amino acid sequence identity is not observed between Y-E pairs, particularly as retrospective searches for d i k e sequences in Torpedo have revealed only the existence of a species of an alternatively spliced y transcript found in embryonic samples that differs modestly from the adult form. Ntvertheless, the substantial differences between muscle y and E subunits are likely to provide a structural basis for the significant differences (in channel properties, antigenicity, and turnover rates; see below) observed between embryonic and adult nAChRs. For example, expression studies in Xenopm oocytes indicate that muscle-type nAChRs with channel conductances of 40 or 60 pS are formed from receptors composed of a,,P l , and 6 subunits along with either y or E subunits, respectively (Mishina et al., 1986). Thus, “fast”

N I C m I N I C RECEPTOR DIVERSITY AND REGULATION

Y

E

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6

Y E FIG. 5. Degree (percentage) of deduced amino acid sequence identity is schematically represented (dark shading) by pie charts for bovine muscle nAChR subunits. Note that mammalian Q subunits share about 96%identity (mouse, bovine, human), whereas p and y subunits (90%identical) and 6 subunits (87% identical) are slightly less conserved across mammalian species. Also note that only the 6-e pair of subunits has sequence identity greater than 50%. (Data are taken from published results and modified, with permission, from Claudio, 1986.)

channels of adult muscle have been equated with €-subunit-containing nAChRs, whereas embryonic “slow”channels would appear to reflect ysubunit expression. More recent studies using the Xenopus system indicate that the natural expression in some muscles of lower conductance channels of between 10 and 25 pS (see above) might be the result of assembly of b-subunit-less nAChRs (Kullberg et al., 1990). d . Mammalian Gene Expression System. Several groups have exploited mammalian cell systems to study transiently and stably expressed nAChRs composed of electric tissue nAChR subunits, mammalian muscle nAChR subunits, and transspecies mixtures of subunits. Some results indicate that there is drug and temperature sensitivity in stable expression of Torpedo nAChR subunits in mouse fibroblasts (Claudio et al., 1987; Claudio, 1989) or in mouse L6 muscle cells (Paulson and Claudio, 1990) or of mouse muscle nAChR subunits in Chinese hamster ovary cells (Forsayeth et al., 1990). Other studies have contributed to the view that nonequivalence in nicotinic ligand binding sites observed in the native nAChR reflects differences in complexes between cxl and either y or 6 subunits from mouse muscle (Blount and Merlie, 1989; Forsayeth et al., 1990). Such an observation may explain subtle but convincing evi-

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dence of nAChR functional heterogeneity in oocytes expressing mouse muscle cell line nAChR subunits (Gibb et at., 1990). Cells that stably express three types of subunits have been used as recipients for transient expression of the fourth type of subunit and promise to facilitate sitedirected mutagenesis studies of n AChR subunits in mammalian expression systems (Claudio, 1989; Claudio et at., 1989). Transient expression systems also confirm that nAChR-containing E subunits have different single-channel properties, immunological reactivity, and membrane lifetimes than n AChR-containing y subunits; these differences are reminiscent of differences between adult and embryonic forms of muscle nAChRs (Gu et al., 1990). Expression of muscle nAChR subunits in fibroblasts confirms that assembly intermediates contain a, plus y or a, plus 6 complexes that are not fully glycosylated and appear to remain in a pre-Golgi compartment (Blount et al., 1990). e. Muscle iiAChR Subunit Isoforms: Genetac and Transcriptional Bares. Recently described lines of evidence indicate that muscle or muscle-type nAChR subunit genes may be heterogeneous, may be subject to complex patterns of transcriptional processing via alternate splicing, and may yield novel but closely related gene products reflecting diversity in translation initiation sites. For example, whereas earlier searches failed to identify nAChR subunit isoforms (Klarsfeld et al., 1984), more recent descriptions have appeared of microheterogeneity in genes andlor transcripts that correspond to a given type of subunit. Hartman and Claudio (1990) have identified two distinct a, subunit genes in Xenopw that are coordinately expressed but at varying ratios (see below). Multiple 6subunit transcripts are found in Torpedo electric tissue RNA (Noda et al., 1983b) and in frog muscle RNA (Baldwin et al., 1988). Two forms of mouse a l subunit are due to use of alternative polyadenylation sites (Goldman et al., 1985). Two rat f3, subunit isoforms are expressed at different levels and arise from alternative splicing at a specific exon (Goldman and Tamai, 1989). Deletion/retention of a novel exon can give rise to t w o human a, subunit isoforms that are expressed at comparable levels (Beeson et al., 1990). T h e two human a, gene products differ by the inclusion in the longer isoform of an additional 25 amino acids, including an extra cysteine residue, between residues 58 and 59 of the consensus sequence, corresponding to the additional exon between exons 3 and 4. N o analogous longer a, message has been detected in fetal calf muscle, chick muscle, or Torpedo RNA preparations (Beeson et al., 1990). f. Muscle nAChR Subunit Gene Structure and Chromosomal Localizatton. Studies of chromosomal genes for vertebrate muscle-type nAChR subunits isolated to date indicate that there is preservation of the

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61

number of exons and of exon-intron arrangements within groups composed of avian or human a 1 genes (containing 9 exons), groups composed of avian or human y, E, or 6 genes (containing 12 exons), and the group including p, genes (containing 11 exons); there is linkage and cosegregation of y and 6 genes. The putative structural or functional domains of nAChR subunit polypeptides (e.g., signal peptide, transmembrane region) have correspondence with structural gene elements (i.e., they are encoded by separate exons; reviewed in Buonanno et al., 1989; Claudio, 1989). The organization of exons 1-4 is conserved for muscle-type nAChR genes, but sequences and lengths of introns are quite heterogeneous (Buonanno et al., 1989). Chromosomal assignments have been made for mouse muscle nAChR genes: aI-mouse chromosome 2, p,-chromosome 11, y and 6-linked at chromosome 1 (Heidmann et al., 1986; Crosby et al., 1989).

5 . Genetic Basis for Nicotinic Receptor Diversity: Neuronal nAChR and nBgtS a. Cloning of Neuronal nAChR Subunit Genes. About 1986, when evidence for structural and functional heterogeneity of nAChRs had convincingly accumulated, use of recombinant DNA techniques to clone putative genes and RNA corresponding to ganglia and central neuronal nAChRs was pioneered by Ballivet, Boulter, Heinemann, Patrick, and their colleagues using chick and rodent systems (reviewed by Deneris et al., 1988, 1991). Their results as well as results obtained in other laboratories indicate extended diversity in the homologous family of nAChR subunit genes found in chick and rat brains. b. Features of Neuronal a Subunits. By use of a definition of putative a-subunit-encoding genes that is based on their expression of nucleotides encoding tandem cysteine residues at consensus sequence positions 192 and 193, seven additional a-subunit genes have been identified (reviewed by Deneris et al., 1989, 1991). These genes were named largely in the chronological order of their isolation as a2through a6 and include the new designations a, for the chick cDNA isolated by Couturier et al. (1990a) and Schoepfer et al. (1990; initially called a-bungarotoxin binding protein a 1subunit) and as (Schoepfer et al., 1990; initially called abungarotoxin binding protein a2 subunit). In contrast to the emerging clear demonstration that muscle or muscle-type a 1genes encode products that are the targets for nicotinic ligands, there remains some potentially dangerous circularity in designating certain neuronal nAChR subunit genes as ci genes solely because they contain sequences that encode tandem cysteine residues and in identifying a-subunit proteins on the basis of their ability to interact with sulfhydryl-directed affinity alkylat-

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ing reagents. Ligand binding could occur at other subunits, but those subunits that lack the requisite tandem cysteine residues would not be capable of interacting with affinity alkylating agents. More work in this area using different probes (such as snake toxins and lophotoxins) is required to illuminate these issues. c. Features of Neuronal p Subunits. In addition, four other “P” subunits (defined loosely on the basis of the absence of nucleotides coding for the tandem cysteine residues located in a subunits but also on the basis of the ability of some of their expression products to substitute for PI in formation of functional channels in Xenopus oocytes with a , , y, and 6 subunits) have been identified and defined as P2 through P5, essentially in chronological order of‘ their isolation (Deneris et al., 1991). d. Conseniation of nACItR Subunit Structure. Deduced amino acid sequence analysis again confirms conservation and identity of residues in i%-terminal and putative transmembrane segments of each subunit b u t also highlights the subunit-specific nature of each of the putative major intracellular loops (reviewed in Deneris et al., 1989, 1991; Figs. 6 and 7). Rat neuronal alpha subunits 2-4 share about 50% sequence identity with a , and as, but have greater than 50% sequence identity with each other. T h e rat a5 subunit actually shares greater sequence identity with the rat P.$subunit, which may simply reflect a curious accident of nature, but might otherwise indicate misdesignation of these subunits as members of the neuronal nAChR subunit family (see below). Rat neuronal P subunits generally share less than 50% sequence identity with each other or with the @, subunit, except for the larger degree of similarity between P2 and @, proteins. Very recent studies have led to initial reports of the cloning of human nAChR subunit cDNAs corresponding to P2 from human fetal brain libraries (Anand and Lindstrom, 1990); ap,ag,a4,P2, and p4 from human brain libraries (Nash et ul., 1990); aQ,a5,and Pq from a human neuroblastoma clonal cell line (Cimino et al., 1990; Fornasari et d.,1990); and two forms of cl3 from a human thymiClibrary (Mihovilovic and Roses, 1990, 1991). One of the human thymic forms of a3 (2.0 kb in length) is very similar to the as clone isolated from human neuroblastoma cells, but the other form is longer (3.0 kb) and appears to contain an extra exon encoding an additional 41 amino acids in the putative second intracellular loop (M. Mihovilovic, personal communication). Otherwise, comparisons of the available deduced amino acid sequences indicate that there is about 90-95% amino acid sequence identity for a given subunit across mammalian species. There also have been reports of the cloning of goldfish and insect nAChR subunits (Deneris et al., 1991). e. Identification of nBgtS Subunit Genes. T h e chick systeni has pro-

63

NICOTINIC RECEPTOR DIVERSITY AND REGULATION

a2

a3

a4

a5

P’s

a2

a3 a4

a5 FIG. 6. Degree (percentage) of deduced amino acid sequence identity is schematically represented (dark shading; gray shading indicates range of identity of a subunit to all p subunits) by pie charts for mouse muscle nAChR a, and rat neuronal nAChR a2 through a5 subunits. Note that current evidence indicates that there is about 90-90% amino acid identity for a given subunit subtype across mammalian species and that there is about 35% amino acid sequence identity between neuronal nAChR a subunits and those corresponding to nBgtS components (a7and as;see text). Also note that there generally is greater than 50% amino acid sequence identity between different neuronal nAChR a-subunit types and that a5 and p3 (see Fig. 7) subunits are more similar to each other than to other a and p subunits, respectively. (Data are taken from studies referenced in Deneris et al., 1991.)

vided the first successes in cloning cDNAs corresponding to components of nBgtSs found in the autonomic and central nervous systems (Couturier et al., 1990a; Schoepfer et al., 1990). These clones meet the simplest criteria for the identification of nAChR CY subunits, in that they contain nucleotides that encode tandem cysteine residues at the appropriate positions. 6 . Genetic Basis for Nicotinic Receptor Diversity: Functional Expression Studies a. Neuronal nAChR Gene Products That Meet Established Functional Criteria. Data from these studies are summarized in Table 111. Functional expression using the Xenopus oocyte system has demonstrated that rodent aq subunits are sometimes capable of forming nicotinic agonist-

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P3

P4

a’s

P3

P4 FIG.5. Degree (pcrcentage) of deduced amino acid sequence identity is schematically represented b! pie chat-ts for rat neuronal nAChR @-typesubunits. Note that current evidence indicates that there is about 90-93% amino acid identity for a given subunit subtype across manimalian species. Also note that there is greater than 50% sequence identity for p2-p.r and (see Fig. 6) pairs. (Data are taken f-roni studies referenced in Denel-is r/ d.. 1991.)

responsive receptor channels, but that pairwise coexpression of either a p ,ag,or a,+with either P p or P., subunits from either chick or rat nearly always gives rise to ligand-gated ion channel production (reviewed by Deneris et nl., 1991). In addition, the P2 and ps subunits truly meet the functional criteria set forth earlier, as they can substitute for in the X~no/m.soocyte system to generate functional channels when expressed in conjunction with a,,y, and 8 subunits from muscle. b. Sin&e-Cliunnel Properties of Expressed Clones. Recent studies have shown that there is complexity of the functional characteristics of nAChRs reconstituted from expressed rat or chick neuronal nAChR subunits (Deneris et al., 1991). Some of these differences manifest as differences in single-channel properties. For example, two classes of conductance state are observed for each combination of the rodent & subunit with a.,,a:%, or a, subunits, and the features of those channels are specific to the type of a subunit expressed (Papke et al., 1989). In addition, higher or lower conductance channels, respectively, can be preferentially generated by increasing the amount of a- or P-subunit RNA. These results suggest that nAChR channel characteristics are influenced by subunit composition and stoichiometry (Papke et al., 1989).

65

NICOTINIC RECEPTOR DIVERSITY AND REGULATION TABLE 111 EXPRESSION OF FUNCTIONAL IONCHANNELS IN CELLSEXPRESSING DIFFERENT COMBINATIONS OF NACHRSUBUNITSQ

+

+

-

-

+

+

+ +

-

~~

~~

aSummary of data obtained concerning the ability of different mouse muscle or rat neuronal nAChR subunit combinations to generate functional nAChR ion channels sensitive to nicotinic ligands when expressed in the Xenopw oocyte system. A minus sign indicates that no current response was observed, a plus sign indicates that a current response was observed, and a plus sign in parentheses indicates that a measurable but modest current response was obtained. Note that PI, P 2 and P4 subunits can combine with a l , 7 , and 6 subunits to generate functional ion channels. a7 and, but less effectively, a4 subunits are the only subunits that apparently are capable of forming functional homo, or aqwith P 2 or P4 generates ion oligomeric channels. Any pairwise combination of ~ 2 a3, channel activity. See text and Table I1 for drug and toxin sensitivities of subunit cornbinations. Data are taken from references cited in Deneris et al. (1991).

c. Phamacologzcal Profiles of Expressed Clones. Other differences in reconstituted nAChRs manifest via pharmacological profile analysis of both agonists and antagonists acting at nAChRs (reviewed in Deneris et al., 1991). For example, there is a switch from K-bungarotoxin-sensitive to K-bungarotoxin-insensitive responses in Xenopus oocytes expressing rat P4 subunits instead of P p subunits along with rat as,and there is diminishing sensitivity of P,-subunit-containing complexes to K-bungarotoxin-mediated blockade as a3 is changed to aqand then to a?.The

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avian aq-p2 combination is insensitive to K-bungarotoxin blockade, suggesting species specificity in toxin sensitivity of homologous subunit combinations. In any case, rat p p expression along with a2, as,or aq produces nAChR responses fully sensitive to blockade by neosurugatoxin and fully or partially sensitive to blockade by lophotoxin, but not sensitive to a-bungarotoxin or conotoxins (Luetje et al., 1990). By contrast, synthetic muscle nAChRs comprising a l ,p,, y, and 6 subunits are less sensitive to neosurugatoxin and fully sensitive to blockade by lophotoxin, a-bungarotoxin, o r conotoxins (Deneris et al., 1991). Small antagonist profiles for the avian a4-& combination show selectivity for hexamethonium and mecamylamine over decamethonium and dtubocurarine, respectively (Bertrand et al., 1990). Studies done using avian subunits indicate that expression of a4 (versus as)or p2 (versus p4) confers enhanced functional sensitivity to acetylcholine (Couturier et al., l990b), and studies done using pairwise, functional combinations of rodent ap,as,a4 and p2 o r p4 subunits show that the expressed receptors have unique agonist sensitivity profiles (Luetje and Patrick, 1991). d. Correlation belween Drug Profiles of Expressed Clones and Naturally Expressed nAChR. Taken together, these functional expression results generally recapitulate observations made using different cells that naturally express nAChRs but that show differences in drug or toxin sensitivity (see above), perhaps as a function of changes in the expression of specific subunits. For example, the profiles for bisonium compound, dtubocurarine, and mecamylamine blockade of oocyte-expressed neuronal ag or aq-& subunits are much more like profiles for blockade of ganglia-type nAChRs naturally expressed by PC 12 cells than for blockade of muscle-type nAChRs naturally expressed by TE67 1/RD cells (Lukas, 1988, 1989a). Functional expression studies also offer a useful criterion for identification of nAChR subtypes that are expressed naturally, as may be assessed by zn situ hybridization, immunohistochemical, or radioligand binding autoradiographic studies of nAChR subunit distribution. For example, the agonist and antagonist profiles for a3-p4 combinations, but not for as-P2 combinations, expressed in oocytes (Luetje and Patrick, 1991) are like those of ganglia-type nAChRs expressed by PC12 cells (Lukas, 1989a), despite the fact that levels of p,, RNA are much lower than those of p2RNA in PC12 cells (Boulter et al., 1990b; Norman et al., 1990). Similarly, oocyte-expressed rat and chick as-P4 combinations have functional characteristics like those found in corresponding autonomic ganglia (Couturier et al., 1990b; Deneris et a/., 1991). Rat medial habenular nicotinic responses are insensitive to blockade by either a- or K-bungarotoxin (Mulle and Changeux, 1990), sug-

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gesting contributions (of the nAChR subunit types expressed at that locus) from rat a3 or aq with p4 gene products, but not p2 subunits, to the corresponding functional nAChRs. Similarly, K-toxin insensitivity of avian mesencephalic lateral spiriform nucleus nicotinic responses could be explained by expression of either avian a2-p2 or a4-ppcombinations (Sorenson and Chiappinelli, 1990). In situ hybridization studies indicate that this nucleus in chick expresses RNA corresponding to a2 and aq subunits, but not to K-toxin-sensitivea3subunits (Morris et al., 1990).as, a4, and p2 subunits are expressed in rat retina and ventral tegmentum, which is where K-toxin-sensitive nAChR responses have been recorded (Lipton et al., 1987; Calabresi et al., 1989). e. Are Some nAChR Subunits Functionally Inert? Only failures in attempts to demonstrate functional activity of a5 or p3 proteins in formation of ligand-gated channels have been reported (Boulter et al., 1990b; Couturier et al., 1990b),perhaps suggesting that the corresponding proteins might be more like muscle-type nAChR y, 6, or E subunits rather than true a or p subunits. Recent work done using synthetic peptide fragments corresponding to putative ligand binding domains of different a subunits indicates an a-bungarotoxin binding capacity for a5subunit-derived peptides and suggests a possible role for such a protein as part of the central neuronal nBgtS (McLane et al., 1990b). SimiIar studies indicate that a corresponding region of the a3 subunit confers the ability to recognize K-bungarotoxin (McLane et al., 1990a). f. Functional Ion Channels from nBgtS Subunits. Couturier et al. (1990a) have shown that expression of the avian a, protein alone in Xenopus oocytes leads to production of nicotinic agonist-triggered ion flux. Their finding suggests not only that the a7 protein has a-bungarotoxin binding capacity, but also that previously functionally suspect CNS (and, by inference, autonomic) a-bungarotoxin binding sites might, in fact, be authentic nAChRs. These nAChRs could correspond to the central neuronal nicotinic sites sensitive to a-bungarotoxin or related toxins identified by de la Garza, Albuquerque, and others (see above). The a7 protein is more sensitive to nicotine than to acetylcholine, less sensitive to acetylcholine than is the avian a4-& combination, and more sensitive than any other subunit combination to desensitization on exposure to nicotinic ligands (Couturier et al., 1990a). These features are similar to those reported for toxin-sensitive, quickly desensitizing nicotinic responses in rat hippocampal primary cultures (Alkondon and Albuquerque, 1990), and the fast rate of desensitization could explain why many attempts to identify a-neurotoxin-sensitive nAChRs have not met with success. It is interesting that ion channels composed of a7

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proteins display inward rectification, which could confound isotopic ion emux studies to detect these putative nBgtSs, that is similar to that displayed by central neuronal and ganglia nAChRs (see above).

7. Genetic Bassis ,for Nicotinil: Receptor Diversity: Sites of Expression of Neuronul nAChR Subunit Genes a. In Situ and Northern Hybridization Studies. Rat a:,,, ag, p2, and p4 gene transcripts are expressed in PC12 cells, suggesting that the corresponding subunits may constitute part of ganglia-type nAChRs (Deneris et ul., 1988; Boulter ef al., 1990b; Norman et al., 1990). a3,ag,and P,t genes are clustered in both rat and chick (Boulter et al., 1990b; Couturier a/ al., 1990b). Homologous human genes have been found to be expressed in IMR-32 or SEI-SY5Y human neuroblastoma cells that also express ganglia-type nAChRs (Cimino et al., 1990; Fornasari et al., 1990; Norman et al., 1990). ,4vian as, p2, and p4 genes are expressed in chick autonomic ganglia (Boyd et al., 1988; Couturier et al., 1990b); avian at%, a5,p2, and p., are expressed in chick sympathetic ganglia (Listerud et al., 1990); and avian a:,,and a4 RNA can be detected in chick dorsal root ganglia (Boyd et al., 1991). I n situ hybridization studies find widely distributed but unique expression of ag and a4 RNA in specific rat brain regions, particularly in the diencephalon, and a more narrow and unique distribution of a2 RIVA, primarily in the interpeduncular nucleus (Deneris et d.,1989, 1991). a5 RNA is found wherever a2 RNA is localized, in some cortical regions, and in the substantia nigra. These distributions overlap with those for high-affinity radiolabeled nicotinic agonist binding sites, suggesting that ganglia-type and/or central neuronal nAChRs that display agonist binding may comprise the corresponding neuronal a subunits. Similarly, P p RN A is expressed wherever neuronal CY RNA and high-affinity agonist binding sites are found, but also in brain regions that express nBgtSs. Thus, P2 protein may constitute part o f CNS ganglia-type nAChRs, central neuronal nAChRs, and nBgtSs. ps and p,, RNAs are found to have a much more restricted distribution, dominated by expression overlapping with that of p2 KNA in the medial habenula and by unique patterns of expression in the interpeduncular nucleus. In many instances, the distribution of neuronal nAChR subunits correlates with sites where nicotinic sensitivity has been demonstrated (Deneris et al., 1988, 1991). b. Immunochemical Studies. Correlations have been made between products of a4 and p2 genes in rat and chick brain and components of immunoaffinity-purified, high-affinity agonist binding sites (Whiting et al., 1987a,b; Schoepfer et al., 1988b). Schoepfer et al. (1989) have shown that antibodies raised against fusion proteins containing chick a3 amino

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69

acid sequences recognize ganglia nAChRs, but not central neuronal nAChRs, that contain subunits recognized by mAb35. Madhok et al. (1989) demonstrated that antibodies against a synthetic peptide derived from the rat ag sequence inhibit radioagonist binding to solubilized rat brain nAChRs. Schoepfer et al. (1990) also have shown that antibodies raised against fusion proteins containing chick a7 or as protein segments recognize nearly all of the radiolabeled a-bungarotoxin binding sites present in the chick CNS, thus establishing a correlation between a7 and a8 gene products and components of nBgtSs. c. Neuronal nAChR Subunit Gene Organization and Chromosomal Localization. Chromosomal genes for some of the neuronal nAChR subunits have been identified. To date, chick a2,a,, and p2genes and rat a2 and ag genes have been shown to share structural homology and to contain six exons (Nef et al., 1988). By contrast, the a, gene comprises nine exons, which gives it a structure closer to that of muscle-type alsubunit genes (Couturier et al., 1990a). T h e organization of the first four exons in all of the vertebrate nAChR subunit genes characterized to date is conserved (Buonanno et al., 1989). Alternate splicing has been proposed to account for the existence of alternate forms of a4-subunit cDNA differing in length by one C-terminal residue and in coding sequence at two other C-terminal residues (Goldman et al., 1987). Mapping studies have assigned mouse chromosomal locations for the nAChR subunit genes encoding a2-chromosome 14, ag (and, by inference, a5 and p4; see Boulter et al., 1990b; Couturier et al., 1990b)-chromosome 9, a,-chromosome 2 (unlinked to a,),and &-chromosome 3 (Bessis et al., 1990). d. Perspective, There is no doubt that the advent of recombinant DNA applications to nAChR biology has provided a tremendous stimulus to the field and an alternative approach to otherwise difficult and tedious protein chemical and functional studies. This work facilitates, but does not obviate, the ultimate identification of the full complement of nAChR proteins that are naturally expressed by neurons and muscle, nor does it completely meet the requirement that molecular genetic findings be linked to physiological actions of nAChRs. In a field that is now conceptually mature, a significant amount of scientific backfilling will be required to ensure that fundamental principles have not been overstated and to uncover any presently unexpected features of the members of the diverse nAChR family. 8. Behavioral and Clinical Significance a. Heterogeneity of Nicotinic Ligand Actions. Given that until recently many neuroscientists could not envision the need for multiple types of

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RONALD J. LUKAS AND MEROUANE BENCHEKIF

receptors to mediate excitatory o r inhibitory neurotransmission, let alone a need for heterogeneity of receptors for a given neurotransmitter, it is not surprising that an appreciation of the potential significance of nAChR heterogeneity has been slow to develop. Demonstrations that diff‘erent doses of nicotinic ligands are required to elicit specific behaviors in experimental animals (see, e.g., Sloan et al., 1988, 1989) could have been attributed to pharmacodynamic as well as pharmacokinetic factors, including differences in drug efficacy, potency, and rates of induction of desensitization and functional inactivation; however, parallel studies employing different strains of mice emphasized a pharmacodynarnic mechanism for these effects and suggested their genetic basis (Collins and Marks, 1989; Marks et al., 1989; Miner and Collins, 1989; Collins et al., 1990a). Thus, pharmacological heterogeneity of nAChRs, perhaps in addition to variations in the numbers and distribution of nAChRs between strainshndividuals, may provide a partial explanation for nonuniformity in susceptibility among humans to nicotine usage and dependence as well as in self-reports and/or concrete measures of effects of smoking on psychophysiological parameters. b. nAChRs in Ne?uous System Function and Diseare. It has been speculated that nicotine addiction and certain behavioral disturbances might be treated by use of nicotinic ligands preferentially targeted toward specific nAChR subtypes (Lester, 1988). It is also possible that specific nAChK subtypes or the cells that express them might be involved in some disease processes and in the corresponding normal functions of affected circuits (see Stolerman, 1987; London et al., 1988, for overviews on behavioral effects and putative sites of action of nicotine). Ageandfor disease-related difrerences in levels of expression of central nAChR radioligand binding sites and in function of presynaptic nAChRs that may be involved in autoregulation of acetylcholine release have been reported and interpreted, within the framework of periodic reports of nicotine’s cognition-enhancing activity, as possible evidence for an involvement of nAChRs in Alzheimer’s disease and in memory (Flynn and Mash, 1986; Perry et al., 1986, 1989b; Shimohama et al., 1986; Whitehuuse el al., 1986, 1988; Nordberg et al., 1988; Sahakian et al., 1989; Giacobini, 1990; Araujo et al., 1990; Wagster el al., 1990). There are older suggestions, based on distinctive immunoreactivity patterns of central and muscle nAChR-like antigens and on epileptiforrn activity of both nicotinic agonists and antagonists, that central nAChRs might be involved in epilepsy (Fulpius et a!., 1977; Fontana et al., 1979). nAChRs are expressed in high concentrations in sensory input pathways (Hunt and Schmidt, 1978a; Schmidt et al., 1980), which suggests a possible role for nAChRs in sensory processing and integration and in its distur-

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71

bances, as may occur in the learning disabled. There is accumulating evidence that nAChRs are involved in the hypothalamic-pituitary-adrenal axis, leading to suggestions and some demonstrations of potential nAChR participation in stress responses (Tarrab-Hazdai and Edery, 1980; Weidenfeld et al., 1983, 1989;J. D. Miller et al., 1987; M. M. Miller et al., 1987; Sharp et al., 1987; Calogero et al., 1988; Kalash et al., 1989; Quik et al., 1990b). nAChR expression in the ventral tegmental area, which has been implicated as the “pleasure” center involved in many addictive processes, varies substantially between species and between strains within species (J. R. Pauly, M. J. Marks, and A. C. Collins, personal communication), suggesting possible variation between human individuals. nAChRs in the reticular activating system may be involved in sleep disturbances, even as a side effect of myasthenia gravis (Papazian, 1979). At neuromuscular synapses, differences in degrees of involvement of specific muscles in myasthenia gravis (Drachman, 1981) could reflect microheterogeneity of nAChRs that might be revealed only as powerful techniques are applied to identify polymorphisms in nAChR subunit genes from different individuals and sequence differences in reverse transcribed and amplified RNA from different muscles in affected individuals. c. Roles of nAChRs in Development and Dafferentiation? There is emerging evidence that different types of nAChRs are expressed with different developmental patterns (see below). It is not yet clear whether these patterns represent a driving force for development or part of its sequelae. It may not be surprising to find that in the future more attention will be focused on longer-range effects of nAChR stimulation than those immediately involving rapid gating of ion channels and changes in transmembrane potential. Whether acting through such mechanisms or via novel forms of signal transduction, nicotine exposure is now known to affect expression of nuclear transcriptional regulatory factors, whose activity might be involved in the activation of batteries of genes, and cellular functions, such as neurite outgrowth and retraction (Greenberg et al., 1986; Lipton et al., 1988). Current interpretations of these effects are framed within the context of effects of increased intracellular calcium ion activity as a consequence of cellular depolarization initiated by actions of nicotinic agonists, but direct demonstration of causal relationships will require further work and perhaps the development of new functional assays of nAChRs (Lipton and Kater, 1989; Mattson, 1988). Also intriguing and challenging are the implications of the discovery of nAChR-like function by subunits of nBgtSs (Couturier et al., 1990a). In autonomic ganglia, where detailed morphometric studies have indicated a perisynaptic distribution of nBgtSs that is distinct from the subsynaptic

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RONALD J. LUKAS AND MEROUANE BENCHERIF

distribution of a-bungarotoxin-insensitive nAChRs that appear to be primarily responsible for transganglionic impulse transduction (Jacob and Berg, 1983; Fumagalli and De Renzis, 1984; Jacob et al., 1984, 1986; Loring and Zigniond, 1987; Loring et al., 1988),a potential role for some nAChR subtypes in synapse modeling or maintenance might be suggested. It remains possible after all that these receptors may play roles in neurotrophism, integrating signals carried by agonists and circulating or localized antagonists, such as thymopoietin, to initiate cell responses and the expression of extrajunctional o r junctional n AChRs. For example, subsynaptic, toxin- and thymopoietin-insensitive nAChRs might be expressed only if perisynaptic, toxin-sensitive, and quickly desensitizing nAChRs are sensing a pattern o f agonist and thyniopoietin-carried signals that is different than earlier in development, before cholinergic fibers innervate the ganglia. d. iVonmusclP a?id Noniaeui oiial nAChR. Additional clues to the biological and clinical significance of nAChR heterogeneity may come not only from studies of neurons and muscle, but also from studies of tissues from dif'fuse neuroendocrine and immune systems, such as lung (Maneckjee and Minna, 1989; Sher el al., 1990), pancreas (Dubick et at., 1988; Chowdhury P t al., 1989) and the lymphocyte populations (Richman and Amason, 1979; Richnian et al., 1981), and from studies of the thymus (Kawananii et ul., 1987, 1988; Schluep et al., 1987; Kirchner et al., 1988; Mihovilovic and Roses, 1990, 199 1; h'elson and Conti-Tronconi, 1990), central glial cells (Hosli et ul., 1988; Hosli arid Hosli, 1988), carotid body (Chen and Yates, 1984), and cochlear outer hair cells (Fex and Adams, 1978; Brownwell et al., 1985; Plinkert et al., 1990) where nicotinic responses, n AChK-like binding sites, and/or nAChR gene expression have been discovered.

c:.

A n o r . FOR SEMINAL STUDIES ON NEUROTRANSMITTER RECEPTORREGULATORY MECHANISMS

Studies on the fundamental properties and function of nAChRs have provided seminal information on features of ligand-gated ion channels in particular and on membrane receptors as a whole. T h e advances made in this work have required utilization of every available technique and a multidisciplinary approach, if not within one laboratory, at least across the nAChR scientific community. T h e thesis of this review is that the nAChR system, despite and because of its diversity, is likely to be useful as an experimental model for studies that integrate cell biological and molecular biological approaches toward a fundamental understand-

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73

ing of strategies and mechanisms involved in the regulation of membrane receptor expression and function. This information also will provide insight into the factors that are involved in the regulation of gene expression in neurons and muscle. Perhaps with this information in hand, it will be possible to elucidate cellular, metabolic, and molecular mechanisms involved in the induction, maintenance, and demise (whether by normal aging or via accelerated degenerative processes) of muscle and nerve and to provide a basis for rational design of clinically useful therapies to treat disorders of the nervous system. II. Models and Concepts in Studies of Receptor Regulation

A. In Vitro MODELSOF REGULATORY MECHANISMS THAT OPERATE in Vzvo 1. Integrative Overview

Before considering models that can be used to illuminate regulatory mechanisms that operate in vivo, it may be useful to make some distinctions between the types of influences that impinge on muscle and different types of neurons. A first and obvious difference is the expression in the periphery of a distinct basal lamina around muscle and particularly within the nerve-muscle synapse. No strictly analogous structure is found in the central nervous system, where the “extracellular matrix” comprises the extended, cell surface-bound glycocalyx. Muscle- and neural crest-derived tissues also differ from central neurons in the types of extracellular influences encountered during development; neural tube-derived elements encounter only other neural tube-derived cell surfaces, whereas muscle has quite distinct embryological origins and the precursors to autonomic neurons experience a variety of different cellcell interactions during their migration from the neural tube. Muscle is a terminal target of motor neuronal innervation, in contrast to postganglionic neurons, which can respond to loss of their targets as well as to loss of their input (see below), and central neurons, which are at least theoretically likely to receive a much greater variety of inputs, which could first be segregated between axoaxonic, somatic, and dendritic subtypes, and then further segmented with respect to the type of input and the nature of the input transmitter in proximal-distal coordinates. Only the remnants, at best, of these influences are apparent when in vitro systems are experimentally employed, and considerable effort is sometimes made to reconstruct some of these influences in studies of receptor regulation.

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2 . Mechanistic Interpretations Nevertheless, even when a putative regulatory agent is identified, difficulty is often encountered in testing its efficacy alone using in vivo systems. Moreover, whereas observations made in uivo are of obvious biological relevance, it is virtually impossible to illuminate molecular mechanisms involved and to carry those observations beyond simple descriptive terms. Thus, studies done in vitro are necessary to see if in vivo effects can he mimicked, to elucidate the mechanisms involved, and to make specific predictions and design feasible experiments that can be repeated in vivo to test the biological validity of those mechanisms.

3. Primary Cell Culture or Expluntr a. Muscle. Embryonic muscle tissue provides an excellent initial source for studies of muscle nAChR regulatory mechanisms. T h e transition from myoblasts to myotubes recapitulates developmental transitions of muscle in uzvo, and co-culture with virtually any type of cholinergic nerve cell population (Nurse and OLague, 1975; Grinnell and Rheuben, 1979; Nurse, 1981) leads to successful aggregation of nAChRs and innervation accompanied by loss of extrajunctional receptors and enhanced organization of junctional-like nAChRs. Mixed cell cultures can be adapted in diffusion-controlled chambers to test for effects of cellcell contact distinctive from those due to diffusible factors, and very elegant experiments using pipets filled with acetylcholine or other agents or patch pipets containing nAChRs as “biosensors” have been used to examine the status of muscle and innervating nerve at various stages analogous to those manifest in vivo. 6. Npural Cwst. Primary cultures and explants of embryonic and neonatal autonomic ganglia have been useful for studies of cholinergic synapse formation, ganglia nAChR properties, and nAChR regulation (Greene et al., 1973, 1976; Obatd, 1974; O’Lague et al., 1974; Greene, 1976; KO et ul., 1976; Nurse, 1981). c. Neural 7’ube. It is clearly possible to obtain primary cultures of central neurons from a variety of different foci, and some success has been reported in adapting radioligand binding assays to studies of nAChRs (Lippiello, 1989), but virtually no information has been obtained on nAChR regulation using this type of system.

4. Clonal Cell Lines a. Muscle. Clonal cell lines derived from embryonic muscle tissue o r from carcinogen-induced neoplasms, such as the C2 mouse muscle cell line and the brain-derived, smooth muscle-like BC,H- 1 mouse muscle clone, have been useful not only for the characterization and isolation of

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nAChRs, but also as sources of mammalian muscle nAChR subunit genes and RNA and for studies that have helped to define the realm of regulatory possibilities impinging on muscle cell nAChRs. b. Neural Crest. Similarly, considerable success has been met to date in elucidating properties of nAChR proteins and gene transcripts via studies of rodent or human neural crest-derived clonal cell lines. These clones are usually isolated from adrenal tumors and are classified as either pheochromocytoma or peripheral neuroblastoma based largely on the morphology of the isolated cells and the pathological characteristics of the tumor source (Tumilowicz et al., 1970; Biedler et al., 1973; Greene and Tischler, 1976), but either type of tumor seems to derive from primitive precursors of chromaffin adrenal medullary neuroblasts (Cooper et al., 1990). c. Neural Tube. To date, no neural tube-derived cell line has been identified unequivocally to express nAChRs. The TE67 1/RD human clone was initially thought to derive from a central primitive neuroectodermal tumor of the cerebellum (McAllister et al., 1977), and considerable evidence began to accumulate indicating that cells of this clone express significant neuronal character and features that might be expected of small interneurons in the cerebellar molecular layer (Syapin et al., 1982; Lukas, 1988; Siege1and Lukas, 1988a), which also are thought to derive, as does cerebellar medulloblastoma, from the postembryonic external germinal layer of the cerebellum (Altman, 1982; Rorke, 1983). Early indications that these cells expressed an a-bungarotoxin-sensitive nAChR distinguishable from central neuronal a-bungarotoxin binding sites (Lukas, 1986a-d, 1988, 1989a, 1990; Lang et al., 1988; Sine, 1988; Walker et al., 1988) were quickly followed by immunological and molecular cloning identification of TE67llRD cell nAChR as a muscle-type nAChR (Schoepfer et al., 1988a; Luther et al., 1989). At that time, a report of DNA restriction fragment length polymorphism analysis showing identity between TE671 cells and cells from the RD human rhabdomyosarcoma clonal line called into question the precise origin of this clonal line (Stratton et al., 1989). Perhaps gene transfection studies will prove useful in providing central neuronal nAChR expression in a nonneuronal cell line, but availability of a true central neuron naturally expressing neuronal nAChRs and genes would be preferable for regulatory studies.

B. DRUGTREATMENT MODELSOF NATURAL REGULATORY INPUT Another approach to identify molecular mechanisms involved in effects of natural inputs on nAChR expression and function involves

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attempted mimicry of those effects with other agents. For example, calcitonin gene-related peptide is known to influence nAChR expression, but it is not known whether those effects are mediated via cyclic AMP-protein kinase A o r phospholipid-protein kinase C pathways (see below). Experimental short circuiting of those pathways (generally done in uitro) by exogenous application of dibutyryl cyclic AMP as a direct analog, or of forskolin plus isobutylmethylxanthine to activate endogenous cyclic AMP production, o r of protein kinase C-directed reagents such as phorbol esters (e.g., P-phorbol- 12-myristate-1%acetate) to transiently activate and perhaps to chronically downregulate C kinase activity would be useful in illuminating the mechanisms involved. Calcium ion can affect second messenger signaling by acting as a cofactor or can act directly itself on intracellular targets, the consequences of which might produce altered expression and function of nAChRs. In contrast t o other second messengers, which are not commonly thought to be sensitive to stimulation of nAChR activity, physiologically relevant increases in intracellular calcium ion can be efrected by opening nAChR ion channels (Decker and Dani, 1990). Mimicry of these effects can be achieved by exposure of cells to calcium ionophores, and contributions of extracellular and intracellular calcium ions can be dissected by adding calcium chelators to the extracellular space or agents such as dantrolene to inhibit mobilization of calcium ion from intracellular stores. Chronic nicotine exposure via the consumption of tobacco products can be niodeled by the exogenous application of nicotine to animals in uiuo via chronic infusion o r an injection schedule or to cells in uitro. Chronic antagonist exposure has been used as a denervation model. EEects of natural agents on nAChR expression often make reference to the sensitivity of those effects to tetrodotoxin (to test for a requirement for nerve and/or muscle electrical activity). Effects of chronic application in 7)iuo of cholinesterase inhibitors has been used to model effects of chemical warfare agents and insecticides on nAChR function, and chronic supplementation with dietary choline has been used to assess effects of enhanced accumulation of presynaptic acetylcholine; however, limitations in the latter two approaches include uncertainty as to what compensatory changes are made presynaptically.

C. CONCEPTS OF RECULATOKY DOMINANCE 1. Nuclear An obvious mechanism for the control of nAChR expression and function is via nuclear transcriptional regulation. nAChR gene promot-

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ers are sites where a variety of regulatory signals could be integrated to coordinately alter numbers of receptors and their function. Given the precedent for nAChR subunit switching (y for E in muscle) and frank differences in genes (see below), dramatic changes may occur in receptor properties as a consequence of transcriptional mechanisms, without necessarily changing the total number of receptors expressed. It also is possible that different but homologous genes could be used to alter the sensitivity of gene product to post-translational influences, for example, by insertion of sequences coding for glycosylation or phosphorylation sites (see above). Thus, nuclear regulation might influence post-translational sensitivity of nAChR.

2 . RNA Processing There also is evidence for post-transcriptional processing of messenger RNAs corresponding to nAChR subunits, based on the identification of genetic heterogeneity of nAChR subunit isoforms due to differential splicing (see above), sometimes involving omission/inclusion of specific exons.

3 . Protein Processing Post-translational processing also may play a role in altering regulatory sensitivity of nAChR subunits. Evidence already exists that nAChR subunit processing, assembly, membrane insertion, and function are subject to post-translational changes involving glycosylation (Covarrubias et al., 1989), phosphorylation (Huganir and Greengard, 1987), methylation, fatty acylation, and disulfide bond formation (reviewed in McCarthy et al., 1986; Claudio, 1989). Specific instances where these modifications may play regulatory roles are cited later. 4 . External Influences

Post-translational processing is obviously sensitive to other neurotransmitter or hormonal influences on cholinoceptive cells, but emerging evidence indicates that transcriptional processes are also subject to control via cell surface receptor-mediated processes. Clearly then, the regulation of a given type of nAChR or nAChR subunit is influenced by the cellular environment as well as by information carried in the regulatory regions of receptor subunit genes. Studies employing normal cells and tissue, neoplastic cells that naturally express nAChR subunits under the control of their own promoters and enhancers, stably transfected cells with nAChR-coding regions under the control of their own and/or other regulatory segments, and transiently transfected cells that express nAChRs in the absence of transcriptional control will all be

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necessary first to establish the realm of regulatory possibilities involved in control of nAChR expression and function, and then to establish which regulatory mechanisms are involved in natural control of nAChR expression and function.

D. DOESREGULATORY FLEXIBILITY PROVIDE A “BENEFIT” OR “PURPOSE” FOR TRANSMITTER RECEPTORDIVERSITY! It is apparent that diversity within the nAChR family contributes to nAChR functional diversity, but it must also confer some other teleological benefits. One potential benefit is that expression of specific nAChR subunit genes from a diverse repertoire might confer developmental flexibility. For example, selection of nAChR genes to be expressed could be driven by promoters designed to be activated en masse, in response to preprogrammed developmental cues. nAChR diversity also might simultaneously confer enhanced plasticity and specificity in a mature but dynamic nervous system sensitive to changes in environmental signals. Some of these cues might call for optimization of matches between nAChR channel kinetics and the type of cholinergic input (phasic o r tonic, high level o r low level). Other cues might call for distalproximal sequestration of nAChR subtypes in dendritic fields that receive inputs other than from cholinergic fibers. Receptor design could also be optimized for post-translational sensitivity of receptor and/or of associated membrane or cytoskeletal elements to synaptic or hormonal inpurs that af‘fectsecond messenger signaling. Insight into the functional significance of n.4ChRs is beginning to accrue from recent studies on the regulation of nAChR expression and function.

lit. Regulation of Muscle Nicotinic Acetylcholine Receptor Expression and Function

Changes occur in muscle nAChR expression and function during development, in adult life, and in some pathological conditions. An improved understanding o f these processes could more generally illuminate cellular and molecular mechanisms involved in the perhaps bidirectional communication between nerve and muscle cells that culminates in formation of functional synaptic contacts. The nerve-muscle synapse is, at first glance, relatively more simple than synapses in the CNS, and the well-characterized nAChR is an excellent model for regulatory studies. Nevertheless, it is now clear that regulation of nAChRs involves both

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receptor-mediated and non-receptor-mediated processes sensitive to an expanding variety of influences. In this section, we summarize current perspectives and focus on the most recent findings related to the effects and roles of ontogenic and trophic factors on the regulation of muscle nAChR expression and function. Readers are referred to other, earlier, excellent, and sometimes more comprehensive reviews on the temporal influences of development, neural contact, and muscle activity on spatial distributions of muscle nAChRs (Salpeter and Loring, 1985; Schuetze and Role, 1987; Steinbach and Zempel, 1987; Rubin et al., 1988; Laufer and Changeux, 1989). A. REGULATION OF NICOTINIC ACETYLCHOLINE RECEPTOR NUMBER, ASSEMBLY, AND STABILITY 1. Instrinsic Mechanisms a. Redistribution OfnAChRs during Myogenesis. During the initial phase of development of the neuromuscular junction, nAChRs are present and diffusely distributed on the surface of myoblasts. On fusion into myotubes, that is, when muscle cells first become multinucleated, there is an increase in nAChR number. nAChRs then redistribute and accumulate in aggregates and clusters at synaptic contacts defined at sites where motor nerve terminals contact the surface of the myotube. Subsynaptic clustering occurs both in vivo and in vitro and leads to a 1000-fold increase in junctional receptors, although the total number of nAChRs on the surface of the myotube decreases (Blackshaw and Warner, 1976; Frank and Fischbach, 1979; Kuromi and Kidokoro, 1984; Salpeter and Loring, 1985; Schuetze and Role, 1987; Rubin et al., 1988; Laufer and Changeux, 1989). The increase in subsynaptic, membrane-bound nAChRs does not appear to be a consequence of an increase in intracellular nAChR pools at subsynaptic regions (Goldfarb et al., 1990). Instead, studies by several groups indicate that nAChR synthesis is controlled predominantly at the transcriptional level (Goldman et al., 1985; Klarsfeld and Changeux, 1985; Buonanno and Merlie, 1986; Moss et al., 1987) and that the subsynaptic restriction and increase in numbers of membrane-bound, junctional nAChRs are dramatically correlated with increases and specific subsynaptic nuclear localization of mRNA that encode nAChR subunits (Merlie and Sanes, 1985; Fontaine et al., 1988; Fontaine and Changeux, 1989). b. Subsynaptic Nuclei Are Active in nAChR Subunit Gene Expression. Early studies indicated that mRNA corresponding to nAChR a1 and 6 subunits is concentrated in synaptic regions of adult muscle fibers (Merlie

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and Sanes, 1985). By the use of genomic probes and zn sztu hybridization to assess the distribution and levels of nAChR subunit mRNA, it has been found that the expression of mature a, subunit mRNA starts in myotomal muscle, is maintained during early stages of muscle development, and is observed in most muscle nuclei; however, al-subunit mRNA becomes restricted to regions near nuclei beneath newly formed motor endplates at the latest stages of synaptogenesis (Fontaine and Changeux, 198'3). I'he mechanisms underlying the preferential subsynaptic distribution of nAChRs remain unclear. In any event, a number of other transcriptional processes and post-translational events (e.g., receptor assembly, glycosylation state of nAChKs, increased mRNA stability or preferential routing to subsynaptic areas, mobility and/or stability of the receptor mediated through components of the extracellular matrix or cytoskeleton) have all been invoked separately or in combination t o explain developmental changes in nAChK number, distribution, longevity, and function. c. Heterogenezty of nAChR Gene Messages. Studies have recently provided evidence for a genetic basis for structural and functional changes in riAChR populations during development. Analysis of nAChR subunit clones from a human muscle library, but not from fetal bovine or chick tissues, tias revealed the existence o f two isoforms of a1transcripts that appear to be derived from alternative splicing of a single a1 gene (Beeson Pf al., 1990). T h e two a,-subunit isoforms each appear to have ligand binding and antibody recognition activity, differ by the addition of amino acids in the N-terminal, putative extracellular domain of the subunit (see above), and are present in equal quantities in a human clonal cell line ('TE67 1/KD) that expresses muscle-type nAChRs (see above); however, both a 1isoforms are also expressed in approximately equal quantities in innervated and denervated human muscle, suggesting that a switch from one form to another is not developmentally regulated (Beeson et al., 1990). Similarly, other studies have demonstrated the existence of mRXA presumed to encode two difFerent mouse a,-subunit isoforms (Goldman et al., 1985) or two difIerent rat @,-subunitisoforms (Goldman and Tamai, 1989), but levels of each form of mRNA are reported to change in parallel during development o r on denervation. d. Genetzr Heterogeneity of nAChR Subunit Isoforms. By contrast, screening of a Xenopus embryonic cDNA library with a Torpedo a,-subunit probe and coordinate genomic Southern blot analysis has revealed the existence of two different genes encoding two frog muscle a 1 subunits (Hartman and Claudio, 1990),a l Aand a l B(see also Baldwin et al., 1988). 'I'he alAsubunit forms high-affinity a-bungarotoxin binding sites when coexpressed with either Torpedo or mouse pl, y, and 6 subunits

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(Hartman and Claudio, 1990). Transcripts corresponding to both a l A and alBappear to be coordinately expressed throughout muscle development (stages 14-50), although at different levels. It is noteworthy that the a I Asubunit, unlike other cloned a 1 subunits, appears to lack the potential site for phosphorylation through cyclic AMP-dependent mechanisms (Hartman and Claudio, 1990). Thus, this work provides an example of how genetically distinct nAChR subunit isoforms could provide for epigenetically regulated changes in sensitivity of nAChRs to posttranslational modification. e. nAChR Gene Transcriptional Regulatory Factors and Sequences in Muscle. Coordinated trans-activation by nuclear regulatory factors targeted toward conserved promoter and enhancer regions of sets of nAChR subunit genes is, in theory, an obvious way to regulate nAChRs during differentiation and denervationlreinnervation. A 5’-flanking region of the chicken nAChR a,-subunit gene is known to be involved in tissue specificity and developmental control of nAChR subunit expression (Klarsfeld et al., 1987; Wang et al., 1988; Merlie and Kornhauser, 1989). Levels of nuclear factors interacting with this domain increase during differentiation and denervation, suggesting that it plays a primary role in the regulation of al-subunit receptor expression (Piette et al., 1989). A muscle-specific control element in the mouse 6 gene has been described (Baldwin and Burden, 1988, 1989). Homologies between segments of the &,-subunit enhancer and the &subunit gene in differentiating chick muscle cells identifies another possible locus for coordinated subunit regulation (Wang et al., 1990). Linkage between y and 6 genes (see above) also provides a potential basis for coordinate cis-regulation of the expression of these genes, and a DNase I hypersensitive site specific for the differentiated muscle phenotype has been uncovered in the intergenic region (Crowder and Merlie, 1988). A “SHUE box” sequence homology in upstream portions of all four fetal muscle nAChR subunit genes has been identified as a possible target for coordinate trans-activation of nAChR gene expression (Crowder and Merlie, 1988) and, in the chick a, subunit, lies adjacent to the identified cell type-specific enhancer (Wang et al., 1988). T h e myogenic transcriptional regulatory factor MyoD 1 may regulate nAChR expression during muscle cell development by interactions with an enhancer region of the chick a1 gene (Piette et al., 1990). More work is anticipated in this exciting line of study. f. The y-e Subunit Switch. For many years it was thought that embryonic, extrasynaptic nAChRs were converted to adult, subsynaptic or “junctional” nAChRs by a post-translational process. It is now clear that a transcriptional mechanism is involved in a developmentally relevant, postnatal shift in the subunit composition of nAChRs from a y-subunit-

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containing embryonic form of nAChR to an e-subunit-containing adult form (Baldwin et al., 1988; Witzemann e.? al., 1989; reviewed by Brehm, 1989; U’itzemann et al., 1990). This developmentally regulated “subunit switch” can be delayed by denervation of rat soleus muscle (Schuetze and Vincini, 1984), but appears to be innervation independent (Igusa and Kidokoro, 1987) and activity independent (Kullberg et al., 1985) in Xenopus. By contrast, levels of c gene transcription are influenced by the presence of motor neurons in primary cultures of embryonic rodent skeletal muscle cells (Brenner et al., 1990;J. C. Martinou and J. P. Merlie, personal communication), suggesting that a neuron-derived signal may control E expression at the level of the gene. g. Is There a Lzmtt to Transcnptzonal Control of nAChR Expresszon? Although complex changes in absolute levels of a,-, 6-, and ysubunit-encoding transcripts are observed during development, there is evidence that levels of nAChR expression are not simply proportional to levels of nAChR subunit mRNA; that is, RNA levels are many times higher than those needed to sustain synthesis of nAChRs (Shieh et al., 1988). Based on the presence of 2 mol of a1subunit per mole of receptor monomer, it has been postulated that the number of &,-subunit transcripts is rate limiting for expression of a fully assembled receptor (Buller and White, 1988); however, studies using transfected fibroblasts show that Toipedo PI subunits have the shortest half-life (12 min versus 43 min for other subunits), suggesting that levels of P1 subunits are rate limiting (Claudio et al., 1989). Such an inference would appear to be at odds with the finding that levels of P-subunit-encoding mRNA are constitutively high in innervated rat skeletal muscle and increase less than those for other subunits during denervation (Evans et al., 1987). Thus, the greater abundance of the p,-subunit RNA may at least partially compensate for the decreased half-life of P, protein; however, coupled with recent observations concerning defects in glycosylation, assembly, and membrane insertion of nAChRs lacking the full complement of subunits (see above) and with recent insight concerning the protection of degradation targeting sequences in M2 domains by subunit assembly (Blount and Merlie, 1990), it seems most plausible that the number of cell surface n AChRs expressed is controlled by a relatively intangible parameter relating to “efficiency of nAChR post-translational processing” that could be exquisitely sensitive to deficits in levels of any of the sequentially assembled nAChR subunits. 2. Extnnszc Influences: Denervataon Studies a. Efjrects on Transcrtptzon. Many observations regarding control of nAChR number made in studies of developing muscle are corroborated

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by observations made using cells following denervation. First, the overall decrease in muscle cell nAChR numbers on synaptogenesis contrasts with an overall 10-fold or more increase in numbers and diffuse cell surface expression of nAChRs following denervation (Miledi and Potter, 1971; Berg et al., 1972; Almon and Appel, 1976). Moreover, chemical or surgical denervation induces changes in expression of nAChR subunit RNA and in the distribution of nuclei active in production of nAChR transcripts that complement changes observed during development. For example, chemical o r surgical denervation in uivo results in enhanced a,subunit expression and an increase in al-subunit-encoding mRNA, but this increase is reported to be restricted to areas around a small population of muscle nuclei (Fontaine and Changeux, 1989). In vitro studies in noninnervated myotubes using either an intronic probe or a probe with both intron and exon sequences reveal a marked heterogeneity in the rates of transcriptional activity within different nuclei, but mature transcripts are found to be diffusely distributed in the cytoplasm (Bursztajn et al., 1989). However, as is the case for changes in mRNA and protein levels during development, there is not a quantitative relationship between transcript numbers, which change more dramatically, and receptor numbers; thus, postdenervation enhancement in levels of nAChR protein may also involve changes in lifetimes of transcripts and of unassembled subunits or fully assembled nAChRs, as well as changes in gene transcriptional activity (Shieh et al., 1987; Buonanno and Merlie, 1986; Tsay and Schmidt, 1989). Denervation effects do not always provide complete fidelity as mirror images of developmental effects. For example, denervation does prevent the developmentally regulated decrease in rat muscle y-subunit mRNA levels, but it does not prevent the apparently programmed or neurotrophic factor “imprinted” initial induction of €-subunit mRNA transcription (Witzemann et al., 1989). Furthermore, in contrast to the marked changes in denervation-induced levels of alsubunit encoding mRNA, levels of 6- and y-subunit mRNA are not as dramatically increased following chemical denervation of chick muscle by treatment with tetrodotoxin (Harris et al., 1988; Shieh et al., 1988; Osterlund et al., 1989). Many of these observationshave been corroborated, extended, and nicely crystallized by a more recent and comprehensive study on regulation of adult rat muscle nAChRs at the transcriptional level (Witzemann et al., 1991). This study also provides insight into the localization of nAChR subunit transcripts near nuclei proximal to the subsynaptic region (junctional disposition of transcripts) or at nuclei distal to regions of nervemuscle contact (extrajunctional transcripts). We have taken the liberty of summarizing the results of Witzemann et al. in our own semiquantitative

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TABLE I V EFFECTSOF SYNAPSEMAN~PULATION ON KACHK S ~ J R U NTRANSCRIPT IT LEVELS" Transcript level (arbitrary units relative to denervated muscle) Experimental condition Intact Denervated Reinnervateti Denervatecl with electrical stimulation InLact with trtrodotoxin block 1ntac-t wit l i botulinu in toxin t h A Intact with a - hu n gartr tox i i i block

a1

3b

100' 3 5b

PI 30" 100~ 60 90"

6

Y 2b

E

0 100 0

100 30

1O h

0

I00

1ow

2

30

70

110

60

5

70

70

70

130

120

130

20

40

25

OData obtained from M'itzeniann d al. (1991), which were based on densiometric analysis of.Northern blots used to estimate quantities of nAChK subunit messenger RNA in control and experimental adult rat muscle preparations and presented in bar graph form as arbitrary units relative t o denervated preparations. were inspected by one of us (R.J.L.) to provide approximate numerical values that could be used to present an effective summary o f this work. .Ihe oi-iginal data were obtained by Witzemarlri rl nl. using intact, innervated muscle (intact); preparations that were analyzed between 4 and 14 days after nerve crush (where the time of assay after denervation either 7 days o r was matched to the duration of treatnietit o f other experimental preparations to provide some standardization of the data (denervated); 30 days after nerve crush (reinnervated); 14 days after nerve crush where niusclc fibers were electrically stimulated from Day 7 to Day 14 after crush (tienervated with electrical stimulation): after 9- 10 days of tetrodotoxin treatment (intact with tetrodotoxin block); after 7 days o f botulinuni toxin treatnient (intact with botulinum toxin block): o i - 3-4 days after a-bungarotoxin treatment (intact with a-bungarotoxin block). Readers are referred t o Witzemann el 01. (1991) for full experiniental details and complete presentation of data. *Shown by Witzeniann et al. to reflect mRNA levels at junctional loci (see text for deftnitiotis). I n addition. all data for E transcripts are likely to reflect junctional concentrations. <Shown by Witzeinann et al. to reflect an approximately 60% contribution from junctional transcripts. In addition. all data for y transcripts are likely to retlect extrajunctional concentrations.

way (Table IV), but refer readers to the original article, where a more thorough description of these results and their interpretation is provided. Critical to interpretation of these data is acceptance that tetrodotoxin treatment blocks nerve-muscle electrical activity while maintaining physical nerve-muscle contact and the possibility of spontaneous quanta1 release of neurotransmitter, that botulinum toxin treatment blocks evoked and spontaneous neurotransmitter release while

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maintaining the possibility of nonquantal release, and that a-bungarotoxin treatment blocks postsynaptic nAChR function. Key observations and/or interpretations can be drawn from this study as well as from extensive previous work: 1. Once activated during development, E gene expression is relatively refractory to manipulation of the nerve-muscle synapse. These results suggest that E expression is “imprinted” (see Brenner et al., 1990) at some stage during development by a signal that does not require persistent activation via functional nerve-muscle contacts (but is not necessarily “transient”). 2. There is a nearly/fully exclusive junctional disposition of E transcripts, suggesting that the restriction of E protein to junctional regions is at least partially regulated at the nuclear level. 3. By contrast, there is a nearly/fully exclusive extrajunctional distribution of y transcripts, which are only maximally reexpressed in adult muscle after denervation or toxin treatment-induced block of quantal release. These results suggest that y transcription at junctional nuclei is not permitted, and that extrajunctional y transcription is subject to strong repression by a signal associated with quantal components. 4. T h e largest amounts of a,,P1, and 6 transcripts are always found, perhaps because of imprinting, to have a junctional disposition regardless of synaptic manipulation. 5. Denervation and electrical and quantal blockade all induce increases in levels of junctional a l , pl, 6, and E transcripts as well as reexpression of al,PI, 6, and y transcripts at extrajunctional loci. Reinnervation fully reverses, and electrical stimulation fully/nearly fully neutralizes, all effects of denervation. These results support the well-documented, strong repression of muscle electrical activity on transcription of all nAChR subunits. 6. Blockade of nAChR channel activity is sufficient to at least partially mimic the effects of denervation on a 1 and y transcript levels (which would be consistent with possible actions of nicotinic agonists and/or increases in intracellular Ca2 in the regulation of nAChR gene expression). 7. In absolute terms, effects of synapse manipulation are more pronounced on a 1 and 6 transcripts than on p, and E transcripts, recapitulating developmentally related effects (Witzemann et al., 1989). +

b. Posttranscriptional Efects. Whereas the control of nAChR number during synaptogenesis is strongly influenced through factors acting at the nuclear level, the enhanced metabolic stability of maturing nAChRs (from a half-life of hours in embryonic tissue to days in adult tissue) may

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be controlled through neural input and/or enhanced cellular activity (Goldman et al., 1985, 1988; Moss et al., 1987). Evidence to support this perspective comes largely from denervation studies. Following surgical or chemical denervation of rodent muscle nAChRs, the half-life of postsynaptic nAChRs decreases from more than 10 days to less than 4 days (Avila et al., 1989; Fumagalli et al., 1990), despite the rise in levels of mRNA, suggesting an important role for synaptic transmission in enhancing receptor stability (Lipsky et d . , 1989). Enhancement in levels of cx ,-subunit mRNA (20-fold) following treatment of chick myotubes with tetrodotoxin, which blocks electrical activity in muscle cells, requires de n o w protein synthesis (Duclert et al., 1990), perhaps of nuclear transcriptional regulatory factors. Treatment of cells with drugs that are known to inhibit the rise in intracellular calcium (verapamil, D-600, dantrolene, the sodium channel blocker tetrodotoxin) induces a more than 10-fold increase in levels of a,-subunit mRNA as well as total nAChR number, and this effect is prevented in the presence of the calcium ionophore A-23187 (Birnbauni et al., 1980; Klarsfeld et al., 1989). These studies support the hypothesis that there is an inverse relationship between mobilization of intracellular calcium and the number of nAChRs expressed on the cell surface, which has led to the hypothesis that calcium plays a key role in the repression of nAChR biosynthesis (reviewed in Rubin et al., 1988; Shieh et al., 1988; Klarsfeld et al., 1989). This view may be too simplistic, however, as the experimental manipulations used may not faithfully mimic the codification or “signature” of intracellular calcium ion regulated by cholinergic and noncholinergic inputs to the developing muscle cell.

3. Extmnsic Influe?ices: Extracellular Matrix and the Cytoskeleton a. Receptor S ~ ~ b and ~ l Clustering. i~~ T h e mechanism(s) involved in enhancement o f receptor stability is not yet understood and may involve covalent modification of the receptor (e.g., disulfide bond formation between 6 subunits, acylation), changes in other components of the cytoskeleton or extracellular matrix, and changes in nAChR interaction with those components (Flucher and Daniels, 1989; Krikorian and Daniels, 1989; Pumplin and Bloch, 1989; Froehner, 1991). Voltagesensitive mechanisms and increased concentration of some yet unidentified macromolecules have also been proposed to influence n AChR numbers and stability in muscle cell culture (Stollberg and Fraser, 1990). Receptor clustering seems to occur in tissue cultures in the absence of any detectable innervation (Prives et al., 1982; Stya and Axelrod, 1983; Kordelli et al., 1989) and appears to involve the basal lamina and components of the extracellular matrix (Godfrey et al., 1988; Wallace, 1988;

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Kordelli et al., 1989), as well as passive, diffusion-mediated aggregation (Stollberg and Fraser, 1988). The constitution and the expression of components, such as laminin and collagen, of the basal lamina at junctional areas change following muscle innervation and parallel the appearance of nAChR aggregates (Vogel et al., 1987). Among the factors in brain, spinal cord, and ganglia extracts that induce similar changes in nAChR clustering and in basal lamina components of cultured muscle cells is an ascorbate-like entity. These results suggest a possible causal relationship between effects of an ascorbic acid-like substance on the basal lamina and on nAChR aggregation (Vogel et al., 1987). Evidence also exists for effects of ascorbate on the rate of nAChR incorporation into the membrane and perhaps some other aspect of nAChR biosynthesis, as well as effects on nAChR distribution (Knaack et al., 1986). b. Agn'n and the Extracellular Matrix. Agrin, a protein extracted from the basal lamina of the electric organ of Torpedo californica, when added to medium bathing chick myotubes, will induce nAChR aggregation, resulting in specialized formations similar to the postsynaptic apparatus at the neuromuscular junctions (Wallace, 1988). The differentiation of the postsynaptic membrane is accompanied by an increase in nAChR and acetylcholinesterase (AChE) concentrations. These effects are calcium dependent, are inhibited by putative activation of protein kinase C, and exhibit characteristics reminiscent of those observed during development of the neuromuscular junction (Wallace, 1988). Interestingly, agrin-induced nAChR aggregation can also be inhibited by components of the extracellular matrix (ECM) such as heparin, heparin sulfate, and other polyanions (Wallace, 1990). Agrinlike immunoreactivity and mRNA that encode agrinlike molecules have been localized to motor neurons (Magill-Solc et al., 1990; Reist and McMahan, 1990), suggesting a potential neuronal source for ECM-bound agrin. The role of ECM components in receptor aggregation is further supported by findings indicating that the motor nerve regulates levels of ECM proteoglycan expression, which occurs coincidentally with nAChR aggregation (Fadic et al., 1990). c. Cytoskeletal Elements. Isoforms of actin, spectrin, and 43- and 58kDa proteins have also been suggested to play a role in receptor aggregation (Pumplin, 1989; Pumplin and Bloch, 1989; Daniels et al., 1990; Daniels, 1991; Froehner, 1991). The expression of RAPsyn (43K protein), a protein initially thought to be involved in receptor aggregation (Daniels, 1991) and which causes clustering of mouse muscle nAChR expressed in oocytes (Froehner et al., 1990), appears to be neither regulated synchronously nor co-localized with the nAChR in embryonic Torpedo electrocytes (LaRochelle et al., 1990; Kordelli et al., 1989). In addi-

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tion, ievels of the 43K protein increase only moderately (2- to 3-fold) and disproportionately to the marked increase ( 100-fold) in mRNA encoding for nAChRs (Frail et al., 1989). T h e discoordinate regulation of the 43K protein may not impact the formation of nAChR aggregates, which can occur without the 43K protein (Kordelli et al., 1989; LaRochelle et al., 1990). Xonetheiess, at later stages of development, the 43K protein beconies associated with the nAChRs, possibly resulting in enhanced receptor stability (Saitoh et al., 1979; Lo et al., 1980; Barrantes et al., 1980; Rousselet et al., 1982) and/or in enhanced kinetics of receptor association (Mitra et nl., 1989). Yet to be fully elucidated are the roles of this and other molecules, including dystrophin and related proteins, in nAChR organization (Froehner, 1991 ).

B. REGULATION OF NICOTINIC ACETYLCHOLINE RECEPTORFUNCTION 1. Functional Relevance of the y-E Subunit Switch Functional studies also have suggested heterogeneity in the populations of nAChRs expressed at different developmental stages of myogenesis and synaptogenesis. Single-channel studies in myocytes have indicated the appearance of at least two classes of functional nAChRs with different conductances and mean open times. There is a developmentally relevant shift from a receptor population with low-conductance, long-duration channel properties to one with high-conductance, short-duration, bursting channels (Leonard et al., 1988b). These findings are supported by others studies on Xenopw niyoiomal muscle, which indicate the existence of two predominant populations of nAChRs with different conductances (40 and 60 pS). T h e smaller-conductance channel is more abundant at the onset of appearance of nAChR channels on the membrane (Day l), whereas contributions from the larger-conductance channel increase progressively to become the dominant form of nAChR channel expressed by Day 3. Whereas the mean open time of the 60-pS channel remains unchanged, the smaller-conductance channel has an open time that decreases during development (Owens and Kullberg, 1989). T h e reciprocal change in y and E subunits occur? within the first 2 weeks of postnatal development in rat muscle, and is coincidental with the observed changes in channel properties (Wit~emannet al., 1989). The change in receptor function as a consequence of this subunit switch is supported by other evidence. Injection of a,-,PI-, and &subunit mRNAs from mouse BC,H-I cell cDNA into Xenopus oocytes reveals the

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existence of two predominant functional classes with conductances of 50 and 12 pS, whereas omission of the 6-subunit mRNA results in the expression of only the smaller-conductance channel (Kullberg et al., 1990). These results indicate that nAChR function can be changed as a result of either different nAChR subunit composition or different subunit stoichiometry. This raises the speculative possibility that other smaller conductances (10 and 25 pS) expressed in developing muscle, in addition to 40 and 60 pS, may be the result of expression of nAChRs that are not fully assembled. An additional possibility may be that this shift reflects post-translational modifications (i.e., glycosylation of specific subunits) of the same channel, which has been shown to alter the functional responsiveness of the receptor (Covarrubias et al., 1989). 2. Desensitization and Functional Inactivation us a Form of Receptor Regulation by Its Own and Other Ligands Other changes in the functional state of the nAChR(s) occur at the level of the receptor expressed in the plasma membrane on exposure to its own ligand. Single-channel recordings of receptor activity in native and in reconstituted membrane have shown that repeated or prolonged application of agonists results in two rapid changes of the macromolecule. A first conformational transition is thought to occur within 1 msec of agonist application and is invoked to explain the opening of the nAChR channel to accommodate monovalent cation flux across the membrane. T h e second conformational transition is invoked to account for the transition of nAChRs to a liganded but closed channel configuration, termed desensitization (see above), which occurs in conjunction with an increased affinity of the receptor for agonist. Muscle-type nAChRs exposed to cholinergic agonists will desensitize very rapidly at a rate that can be best fitted by at least two components having time constants in the seconds range. Additional very fast components of desensitization have also been reported but their analysis has been hindered by the limited resolution of these fast events. These fast desensitization rates are fully and rapidly reversible (reviewed in McCarthy et al., 1986; Ochoa et al., 1989). T h e molecular mechanism underlying agonist-induced receptor desensitization is not fully understood but does not seem to be affected by CAMP-dependent o r calcium-dependent mechanisms (Sara et al., 1990; see above). A more slowly occurring loss of nAChR function, termed functional inactivation (Simasko et al., 1986), has an onset and recovery in the minutes to hours range, occurs on more chronic exposure to agonists or some antagonists, may involve ligand interaction with the open receptor channel, and affects human muscle-type nAChRs (Lukas, 1991). A variety of previous studies have shown that chronic

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agonist exposure induces a downregulation of muscle nAChR ligand binding sites (Noble et al., 1978; Gardner and Fambrough, 1979; Appel et al., 1981). This downregulation manifests as a loss in nAChR functional responsiveness (Noble et a!., 1978). C. MODULATIONOF NICOTINIC ACETYLCHOLINE RECEPTOR NUMBERA N D FUNCTION B Y EXOGENOUS LICANDS

A number of different ligands that are not classically considered to be members of the “cholinergic” family of compounds have been shown to affect nAChR function as noncompetitive antagonists o r as functional modulators. T h e present discussion focuses partly on ligands that might plausibly affect nAChR number and function in vivo, perhaps via modulation of muscle cell second messenger signaling, and partly on effects of perturbation of second messenger signaling. 1. Efects of Phosphorylation on nAChR Expresszon and Function

a . Cyclic AMP-Dependent Pathways. Activation of CAMP-dependent mechanisms in muscle cells results in an enhancement of the phosphorylation state zn sztu of specific subunits (6 and y subunits) of muscle or muscle-type nAChRs (Miles et al., 1987; Safran et al., 1987, 1990). These effects correlate with an increase in the rate of agonist-induced desensitization (Middleton el al., 1986, 1988) and with upregulation of nAChR expression (Fontaine et al., 1987). Similarly, enhanced CAMP levels have been implicated in the effect of interferon on the reduction of both channel conductance and opening frequency of the nAChRs (Eusebi et al., 1989).CAMP-dependent mechanisms and phosphorylation on y and 6 subunits have also been implicated in enhanced efficiency of nAChR assembly and increases in expression in mouse fibroblasts that stably express 7brpedo nAChR subunits (Ross et al., 1990; Green et al., 1990). Activation of CAMP in BC,H-l cells increases phosphorylation of 6 (and, to a lesser extent, a , )subunits (Smith et al., 1989). Interestingly, activation of CAMP-dependent mechanisms in a human clonal cell line that naturally expresses muscle-type nAChRs produces a decrease in nAChR number per unit of protein, suggesting that the pluripotent features of these cells may modify sensitivity of nAChRs to post-translational influences (Siege1 and Lukas, 1988a; Bencherif and Lukas, 1991). b. Phospholiparl-Dependeat Pathways. Studies in vertebrate muscle and Torpedo have also indicated that protein kinase C activation results in phosphorylation of 6 subunits, in acceleration of the rate of agonistinduced desensitization of nAChRs (Middleton et al., 1986; Huganir et

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al., 1986; Safran et al., 1987, 1990), and an initial phase of receptor downregulation (Fontaine et al., 1987). In addition, putative downregulation of protein kinase C by prolonged exposure to phorbol esters o r inhibition of protein kinase C in primary cultures of chick myotubes results in a 10-fold increase in both precursor and mature forms of alsubunit mRNA nAChRs (Klarsfeld et al., 1989). Phorbol ester treatment also induces an initial decrease (presumably in part because of activation of protein kinase C), and then an increase (presumably as a result of protein kinase C downregulation) in muscle-type nAChR numbers expressed by cells of the TE67l/RD human clonal line (Bencherif and Lukas, 1991). This study suggests that effects of protein kinase C modulation have comparable effects on nAChR expression in very different cell types, which supports, along with other evidence, involvement of transcriptional mechanisms in those effects (Bencherif and Lukas, 1991). In studies using chick myotubes, agonist treatment-induced dispersal of nAChR clusters was found to be similar to that observed on putative activation of protein kinase C, which has led to the proposal that cell surface receptor distribution may be regulated through phosphorylation of specific nAChR subunits (Ross et al., 1988). c. Calcium Ion-Dependent Pathways. Enhancement of intracellular calcium ion concentration has been postulated to play a role in limiting nAChR numbers on the surface of muscle cells by inhibiting biosynthesis and/or translational modifications (reviewed in Rubin et al., 1988; Shieh et al., 1988; Laufer and Changeux, 1989). Calcium ion has also been long implicated in promoting desensitization of nAChRs (see Ochoa el al., 1989). Recent studies indicate that enhanced intracellular calcium ion induces an increase in &subunit (and, to a lesser extent, p,-subunit) phosphorylation in BC,H-I cells (Smith et al., 1987, 1989). Calcium ion entry via the open nAChR channel may explain enhancement of nAChR 6 subunit phosphorylation on agonist exposure (Ross et al., 1988) via a mechanism other than protein kinase C activation. d. Cross-Talk between Phosphorylation Pathways. With respect to the physiological relevance of these effects, receptor-mediated activation of phosphoinositide hydrolysis in muscle cells might play a dual role in the control of nAChR expression and function. Both the generation of inositol trisphosphate and diacylglycerol and the subsequent rise in intracellular calcium ion concentrations and protein kinase C activity would act, at least acutely, to enhance the susceptibility of nAChRs to desensitization and to decrease nAChR numbers. Concomitant modulation of CAMP-dependent and protein kinase C pathways in TE67 1/RD cells expressing muscle-type nAChRs produces complex changes in nAChR expression that cannot be predicted from effects of activation of either

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pathway alone (Bencherif and Lukas, 1 W l ) , suggesting a possible role for cell priming in the modulation of nAChR expression and prompting

the prediction that complex synergistic and antagonistic effects of agents converging to alter the phosphorylation state of nAChRs are involved in modulating nAChRs post-translationally and transcriptionally. Implications that G-proteins responsive t o acetylcholine are involved in the modulation of nAChR function also come from other studies in other systems (e.g., Eusebi et al., 1987). e. T p s i w Kinuses. Tyrosine-specific protein phosphorylation by a protein tyrosine kinase in postsynaptic membranes has been reported to increase the rate of the rapid phase of desensitization of purified and reconstituted nAChRs, as shown by single-channel studies, and to occur on f3, y, and 6 subunits (Hopfield et al., 1988). More recently, studies have indicated that levels of n.4ChR phosphorylation on tyrosine residues are strikingly regulated by innervation (Huganir and Greengard, 1990; QU P t ul., 1990). Tyrosine phosphorylation is evident between the first and second week of postnatal development of rat diaphragm, increases on innervation, and decreases again following denervation.

2 . Poos.\iblp “Fiwt illessserigru” Alpdiuting Repkction (4’n,AChRs a. Su6stanc.e P. Desensitization of muscle-type nAChRs is accelerated in the presence of substance P (Akasu et al., 1983; Simasko et al., 1985). Structure-activity studies indicate that the effect is direct, as the profiles for substance Y analogs active at nAChKs and at defined tachykinin receptors arc different (Siniasko et ul., 1985; Weiland et al., 1987). 6. Culcitoriin Gene-Related Peptide. Calcitonin gene-related peptide (CGRP), a widely distributed peptide, has been found to be co-localized with acetylcholine in rat neurons (Takami P t a/., 1985). This peptide has been reported t o increase the n u n h e r o f surface nAChRs; the levels of nAChR c*,-subunit mRKA (Fontaine and Changeux, 1989) and, to a lesser extent, 6- and y-subunit mRNA (Osterlund et ul., 1989); and the rate of‘nAChR desensitization coincident with a CAMP-dependent phosphorylation of specific subunits of the nAChRs (Miles et ul., 1989). T h e CGRP efrect on nAChRs was initially attributed to enhanced CAMP levels (Fontaine and Changeux, 1989). It was later found that these enhanced cyclic nucleotide levels led to a secondary increase in phosphoinositide metabolism (Laufer and Changeux, 1989), which induced activation of protein kinase C activity and increased concentrations of intracellular calcium ion, both of which can influence nAChR expression and/or function (see above). T h e exact contribution of the various second messenger pathways in modulating nAChR expression and function is complicated by the cross-talk between second messenger systems. In

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any event, the effects of CGRP on nAChR numbers are different than would be expected for innervation by a cholinergic CGRP-containing neuron, suggesting that it may play a role in antagonism of the general downregulatory effects of electrical activity, calcium ions, and so forth, on nAChR expression in innervated muscle. c. Neuronal Factors. Several studies have been done to identify factors present in neuronal tissue that can influence muscle nAChRs (see Vogel et al., 1987). Agrin, ascorbic acid, and an ascorbate-like factor were discussed earlier within the context of their interactions with/from the extracellular matrix. Aside from the 42-kDA glycoprotein isolated from chicken brain that has and is defined by its “acetylcholine receptorinducing activity” (ARIA) and stimulates the rate of nAChR incorporation into the plasma membrane of chick myotubes (Usdin and Fischbach, 1986), none of these factors have been very well characterized. ARIA also induces significant increases in levels of &,-subunit (but not y- or 6subunit) mRNA, as well as in its putative nuclear precursor (Harris et al., 1988). This induction of transcription appears to be specific to only a subset of muscle nuclei (Harris et al., 1989). d. Thymopoietin. T h e thymic hormone thymopoietin blocks neuromuscular transmission (reviewed in Lukas et al., 1990), and a related peptide, thymopentin (Arg-Lys-Asp-Val-Tyr), has been shown to enhance agonist-induced desensitization of nAChRs (Ochoa et al., 1988, 1990).There is a report that effects of thymopoietin can be influenced by calcium ion (Revah et al., 1987). These and other studies have led to the proposal that thymus-derived peptides may act as possible endogenous ligands acting through modulation of nAChR function (Venkatasubramanian et al., 1986; Revah et al., 1987; Ochoa et al., 1988; Quik et al., 1990a) and might play a role in the neuromuscular disorder myasthenia gravis (Goldstein and Hofmann, 1968; Goldstein, 1987). An interesting possibility, suggested by reports of thymopoietin-like immunoreactivity in spinal cord extracts and in culture media used to maintain primary spinal cord cells, is that thymopoietin or a related substance might be provided locally by motor neurons at the nerve-muscle synapse. e. Anti-nAChR Antibodies. Immune modulation of nAChR expression and function is exemplified by the neuromuscular disease myasthenia gravis (MG), which is an autoimmune disorder targeted against muscle nAChRs (reviewed in Drachman 1987; Tzartos, 1990). It is postulated that anti-nAChR antibodies cause a combined acceleration in the degradation of receptor and a functional deficit. There is, however, a poor correlation between anti-nAChR antibody titer and the intensity of MG symptoms. A significant proportion of the anti-nAChR antibody is raised against a small portion of the (Y subunit known as the “main

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immunogenic region.” T h e production of these antibodies is modulated by specific helper T lymphocytes (CD4 +) that recognize epitopes on the 01 subunit of the nAChRs (Protti et al., 1990). T h e loss of nAChRs at the neuromuscular junction may be the result of three different mechanisms: (1)anti-nAChR antibody acting as a competitive inhibitor, resulting in a decrease in cholinergic transmission; (2) receptor loss by antigenic modulation as a result of crosslinking of nAChRs, followed by internalization and degradation; and/or (3)complement binding resulting in membrane lysis and decrease in nAChR number. 3. General O-ctennew

Taken together, the following picture of regulation of neuromuscular junctional nAChR expression emerges: On the approach of the motor nerve terminal, induction of neural activity results in the release of diffusible ligand(s), for example, acetylcholine and, possibly, peptides or other factors (e.g., ARIA, CGRP, ascorbate, or thymopoietin-like entities). In some combination, these and other factors (e.g., electrical activity, calcium ion) induce a 5-fold decrease in total nAChR numbers and a 1000-fold increase in receptor density in the subsynaptic region. Mechanisms that could be recruited by these factors include calcium ion (through nAChK activation and/or by activation of phosphoinositide hydrolysis), inositol polyphosphate, diacylglycerol and metabolites, and cyclic nucleotides. The integration of these inputs induces (1) repression of overall nAChR biosynthesis via effects on gene transcription/translation, and (2) a transcriptionally mediated shift in nAChR subunit expression (clearly involving a Y-E subunit switch but also possibly involving changes in expression of a,,P I , and 6 isoforms and/or regional nuclear expression of these genes) leading to a change in nAChR channel properties. It also is possible that these signals trigger (1) an increase in the efficiency of receptor assembly and insertion (perhaps by activation of CAMP-dependent phosphorylation) and (2) enhanced stability and/or clustering of nAChRs, perhaps coupled to the synthesis, release, and/or reorganization of cytoskeletal and/or extracellular matrix proteins (e.g., agrin, 43-kDa protein). Subsequently, muscle activity and consequent calcium ion entry will contribute to tonic inhibition of receptor biosynthesis, whereas the consequent activation of protein kinase C or other second messenger systems will contribute to modulation of nAChR functional characteristics and to its “stability,”at least partly, through regulation of extracellular matrix components. Whereas more is clearly known about these processes at the neuromuscularjunction than at any other synapse, it is also clear that much more work needs to be completed before the details of this complex biological process are fully elucidated.

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IV. Autonomic Neuronal and Ganglia-Type Nicotinic Acetylcholine Receptors

Our current understanding of control of nAChR density and functional characteristics on neural crest-derived cells is based on use of a variety of experimental approaches. Included among these are binding studies using anti-nAChR antibodies (e.g., mAb35), K-bungarotoxin, and/or radioagonists to measure ganglia nAChR expression, a-bungarotoxin binding to detect nBgtSs, ultrastructural studies, electrophysiological and pharmacological approaches in functional studies, and molecular biological studies (see above). In this section, we update earlier reviews on regulation of ganglia nAChRs (Schuetze and Role, 1987; Berg et al., 1989) and provide an overview on other data pertaining to regulation of ganglia-type nAChRs (and nBgtSs). A. REGULATION OF GANGLIA NICOTINICACETYLCHOLINE AND FUNCTION RECEPTOR EXPRESSION 1. Ontogenic Effects

a. Functional Studies. By contrast to current evidence that most muscle cell surface nAChRs detected using radioligand binding assays are functionally available (see, e.g., Sine and Steinbach, 1986), neurons isolated from chick ciliary ganglia express functional nAChR responses that appear to derive from only a fraction of their nAChR-like binding sites (Margiotta et al., 1987a). These cells express progressively enhanced levels of nAChRs (both internal and cell surface) and whole-cell acetylcholine sensitivity during the early stages of development [between embryonic day 8 (E8, just after synaptic connections are established) and E l 6 (when synapses mature); Margiotta and Gurantz, 19891. Two distinct nAChR populations with channel conductances of 25 and 40 pS are present at low levels at E8, but the 40-pS channel predominates by E14. T h e mean open time of the 25-pS channel does not change, whereas the lifetime of the 40-pS channel increases progressively with age. I n addition, at E10, nicotinic responses become CAMPsensitive (Margiotta et al., 198’7b; Margiotta and Gurantz, 1989). These findings are largely corroborated by other studies showing that sensitivity of embryonic sympathetic neurons to current induction by acetylcholine increases whether they are innervated in vivo or in vitro (Role, 1984; Moss et al., 1989; Brussard and Role, 1990). These studies reported that three classes of ACh-activated channels with different conductances and kinetics are expressed before synaptogenesis, and that the two lower-conductance states predominate (Moss et al., 1989). After in-

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nervation there is an increase in the frequency of opening of all of these channels, a shift in the conductance state of the two lower-conductance channels, a pi-ogressive domination of the two higher-conductance channels, and expression of an additional, even higher conductance channel tvpe, and there is evidence for receptor clustering (Moss et al., 1989). Functional heterogeneity of sympathetic neuronal nAChR channel conductances has also been reported by Derkach et wl. (1987). b. Receptor Numbers. Protein and immunochemical studies indicate that functionally relevant nAChRs on chick ciliary ganglia neurons reactive with K-bUngarotoXin and mAb35 are distinct from nBgtSs labeled with a-bungarotoxin (Smith et d . , 1985; see above). With development between E8 and E l 6 (i.e., from 0 to 8 days in culture after isolation from E8 embryos), numbers of nAChRs increase (Smith et ul., 1986). nAChR receptoi-s isolated from chick ciliary ganglia using imniunoaffinity techniques apparently have different subunit compositions (Halvorsen and Berg, 1990; see above), but it is not clear whether distinct nAChR subtypes coexist within the same neuron, and developmental studies have not yet been done. I-. Lez1el.c of Trwmcript Expression. There is evidence that several types of neuronal nAChK subunit RNA are expressed in chick autonomic ganglia (see above). Chick ciliary ganglia cells express as-subunit KNA throughout development arid for as long as 1 year posthatching (Boyd at ul., 1988). Chick sympathetic neurons express a:+, a,, p2, arid p, subunits (Lustei-ud et al., 1990); however, developmental studies have yet to be done. d. I~ispositioriof nBgtSs and nAC:hKs. Ultrastructural studies of intact chick ciliary ganglia indicate that functionally relevant nAChRs reactive with K-bungarotoxin and mAb35 are sequestered subsynaptically, whereas iiBgtSs labeled with a-bungarotoxin are located perisynaptically (Jacob and Berg, 1983,.Jacob et wl., 1984; Loring et al.,1985; Loring and Zigmond, 1987). Developmentally relevant mechanisms involved in this preferential sequestration of different members of the nAChR family have yet to be elucidated.

2. Effects of Denenutio?i arid Axotovi? Preganglionic denervation or postganglionic axotoniy of ciliary ganglion in newly hatched chicks induces marked changes in total levels of expression of both ganglia nAChRs (3-fold decrease within 10 days arid 10-folddecrease within 5 days, respectively) and nBgtSs (the latter with a more rapid time course; Jacob and Berg; 1987). However, another series of studies suggested that neither total numbers nor cell surface levels of nBgtSs are aff'ected by denervation of adult chick ciliary ganglia or adult rat superior cervical ganglia (Fumagalli et al., 1976, 1978; see below).

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Fumagalli et al. (1978) also found that axotomy of adult or neonatal chick postganglionic neurons produces a rapid (within 1 day) and nearly complete decrease in cell surface levels of nBgtSs, but a less dramatic decrease in total nBgtSs. With respect to nAChRs, levels of cx3 transcript in chick ciliary ganglia also decline following postganglionic axotomy or preganglionic denervation (Boyd et at., 1988). Dissociation of ganglia into single cells to facilitate electrophysiological analysis (McEachern et al., 1989) reveals that there is a marked decrease in acetylcholine sensitivity following postganglionic axotomy (see also Brenner and Martin, 1976). There is, however, little to no change in functional nAChR sensitivity on denervation (McEachern et at., 1989; see also Brown, 1969). The McEachern et al. study and a less quantitative investigation (Jacob and Berg, 1988) demonstrated that there is a coordinate decrease in cell surface nAChRs on axotomy, but that no net change in cell surface nAChRs is induced by denervation. Thus, the evidence is clear that postganglionic axotomy induces a loss of nAChR function when measured in vitro or in vivo that reflects declines in total and cell surface nAChR numbers and in nAChR subunit message levels. Denervation, however, appears to cause decreases in transcript levels and total numbers of nAChRs, but not to have major effects on cell surface nAChR numbers o r function (many previous reports of increased acetylcholine sensitivity following preganglionic denervation could be the result of decreases in acetylcholine esterase levels or species differences), suggesting that the earlier studies on effects of denervation were dominated by contributions from the excess intracellular pool of nAChRs in these cells (Stollberg and Berg, 1987). 3 . Modulators and Mechanisms of nAChR Regulation a. Overuiew. A number of biologically active substances can modulate functional sensitivity of nAChRs at autonomic ganglia in amphibians (reviewed by Akasu and Tokimasa, 1989) and in mammals. These agents include catecholamines, neuropeptides, prostaglandins, and glucocorticoids (acting as noncompetitive antagonists at an allosteric site of the receptor-ion channel complex), as well as cholinergic and serotonergic ligands. In addition, increased sensitivity of nAChRs is observed in the presence of the nucleotide adenosine triphosphate (ATP) and the peptide luteinizing hormone-releasing hormone (LHRH). b. Biogenic Amines. For example, in vitro studies of superior cervical ganglia cells of the rabbit indicate the possibility of heterologous regulation of nicotine-induced responses mediated through muscarinic processes (Yarosh et d , 1988). Earlier reports have indicated that in amphibian ganglionic neurons, 5-hydroxytryptamine decreases the nicotineinduced response (fast excitatory postsynaptic potentials) and agonist

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sensitivity of ganglia nAChRs by interfering with acetylcholine binding at the active site on the receptor-ion channel complex (Akasu and Koketsu, 1986). More recent reports have shown that membrane currents through FI-HT, serotonergic receptors measured with voltageclamp techniques (Peters et al., 1990) and excitatory postsynaptic potentials mediated by activation of FI-HT, receptor subtypes (Vanner and Surprenant, 1990) are completely antagonized in the presence of the nicotinic antagonist d-tubocurarine. These studies could provide hints that cofactors may be required to effect nAChR (or nBgtS) function in the nervous system, in much the same way that glycine modulates function of neuronal excitatory amino acid receptors. c. Tachjkznzns. Tachykinins (substance P, neurokinins A and B) and enkephalins have also been shown to affect nicotine-mediated processes in sympathetic and parasympathetic neurons (Role, 1984; Margiotta and Berg, 1986). It has been suggested that substance P modulation of nicotine-induced catecholamine release from sympathetic ganglia reflects enhancement of the rate of acetylcholine-induced receptor desensitization by the action of substance P on second messenger signaling in the phosphoinositol-calcium-dependent protein kinase C pathway (Downing and Role, 1987; Simmons et al., 1988, 1990). d. Pztuztary Hormones? A peptide resembling the hypothalamic hormone LHRH, localized around the small C-neurons of amphibian spinal nerve and mediating late-slow excitatory postsynaptic potentials, has been shown to interfere with nicotinic transmission both by an indirect effect through inhibition of acetylcholine release (Hasuo and Akasu, 1986) and by an increase in receptor sensitivity (Akasu and 'Tokimasa, 1989). P . Co-released ATP. Increased nAChR sensitivity has also been observed in the presence of the nucleotide ATP (Akasu et al., 198 l), which has been shown to be co-released with acetylcholine in some preparations (Winkler, 1976). f . Cnlcztonin Gene-&luted Peptule. Increased acetylcholine sensitivity and rate of nAChR desensitization are observed in chick sympathetic neurons treated with CGRP via a mechanism that can be mimicked by treatments that elevate levels of intracellular cyclic AMP (Valenta and Role, 1990). CGRP-like immunoreactivity in frog parasympathetic ganglia has been co-localized with nicotinic synapses (Peng and Chen, 1988), suggesting that these observations may have physiological relevance. 4. Relationshzps between nBgtSs and Ganglia nAChRs

Some studies have suggested that there is some sort of reciprocation in regulation of expression of nBgtSs and nAChRs and that nBgtSs may play

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a physiologically important role in autonomic neurons that is simply not understood at this time. For example, Halvorsen and Berg (1989) have reported that there is a specific downregulation of nBgtSs on chick autonomic neurons induced by the endogenous peptide ciliary neuronotrophic factor (CNTF), a protein that enhances chick ciliary neuronal survival and may be a tqrget-derived growth factor for these neurons, but that CNTF treatment has no effect on nAChR expression. These observations corroborate earlier,reports on effects of crude eye extract (Smith et al., 1983) that also showed upregulation of nBgtSs and downregulation of nAChRs in cells exposed to high extracellular potassium ion. On the other hand, several types of drug effects appear to regulate nAChR expression and function and numbers of nBgtSs in a coordinate fashion. For example, there is an earlier report that expression of nBgtSs on chick ciliary ganglia can be decreased by exposure to K-bungarotoxin (Ravdin et al., 198l),which blocks nAChR function. Chronic exposure to K-bungarotoxin also induces downregulation in nAChR numbers and a more complete loss of nAChR function via a mechanism that is sensitive to blockade by either small antagonists or agonists, but not by cholinesterase inhibitors or a-bungarotoxin (Smith et al., 1986).Nicotinic agonist treatment alone, but not chronic antagonist treatment with d-tubocurarine or trimethaphan, also produces a more modest decrease in nAChR numbers but a nearly complete decrease in nAChR function. Chronic treatment of chick ciliary ganglion cells with cholinergic agonists induces downregulation of nBgtSs in an antagonist-sensitive fashion (Messing, 1982; Halvorsen and Berg, 1989). There are some ligand effects that preferentially influence nAChRs. For example, antigenic modulation (i.e., downregulation induced by antibody exposure) of nAChR ligand binding and function occurs on chronic exposure of chick ciliary ganglia cells to mAb35, but there is no effect on nBgtSs. The precise significance of these findings is not yet clear, but it is evident that a multiplicity of mechanisms are involved in the control of both ganglia nAChRs and nBgtSs.

5. Roles of Second Messengers Roles of second messengers in modulation of function and/or exrepression of both ligand-gated and metabotropic/G-protein-coupled ceptors are well documented (reviewed by Huganir and Greengard, 1990). Studies on chick ciliary ganglia using patch-clamp techniques have shown that acetylcholine responses can be markedly enhanced without a significant change in receptor number as a consequence of treatments that induce or mimic the effects of elevated intracellular CAMP(Margiotta et al., 1987a,b). It is not yet clear whether newly functional nAChRs are recruited form intracellular pools and/or whether

functionally silent cell surface nAChRs need to undergo an “aging” process; however, this 2- to 3-fold increase in acetylcholine responsiveness appears to occur coordinately with a CAMP-dependent phosphorylation of putative a, subunits (Vijayaraghavan et nl., 1990). Phorbol ester pretreatment of embryonic chicken sympathetic ganglion neurons enhances the rate of decay of acetylcholine-induced current without af‘f‘ecting peak-current amplitude o r cellular input resistance (Downing and Role, 1987). suggesting a role of protein kinase C-dependent mechanisms in modulation of the rate of nAChR desensitization.

6.

Oz1miieu~

In sumrnary, several features of ganglia nAChR regulation (e.g., synaptogenesis-triggered increases in receptor functional sensitivity and in receptor clustering and subsynaptic sequestration, acceleration of desensitization by protein kinase C-mediated phosphorylation, and increased expression of higher-conductance forms of receptor) are reminiscent of those occurring at the nerve-muscle synapse, but several others (e.g., little net change in cell surface nAChR numbers or function following denervation, activation of nAChR agonist sensitivity by CAMP-mediated phosphorylation, stability of subunit mRNA levels with development but decreases in transcript levels on denervation) appear to be unique to ganglia nAChRs. Some of these differences might reflect the expression of two quite different members of the extended nAChR family, ganglia nAChKs arid riSgtSs, in these cells, the latter of which has been refractory to functional characterization.

B. REGGLATION OF GANGLIA-TYPE NICOTINIC ACEIYLCHOLINF RECEPTOREXPRESSION A N D FUNCTION Several model cell systems, each of which expresses some form of ganglia-type nAChRs as well as nBgtSs, have been exploited in the hope of illuminating the features of neuronal nAChRs. Included among these are the neural crest-derived chromaffin cells of the adrenal medulla and neoplastic pheochroniocytoma and peripheral neuroblastoma cells of neural crest origin.

1. Studies Lrsing Adrenul Medulluty Chrornuffin Cells (1. Antibodies and Nicotinic Ligands. Adrenal medullary chromaffin cells functionally respond to nicotine treatment and cholinergic input via release of catecholamines; this effect is poorly maintained, in part because of desensitization and/or functional inactivation of nAChRs (re-

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viewed in Marley, 1988). Adrenal chromaffin cells express nAChRs sensitive to interaction with both mAb35 and K-bungarotoxin (Higgins and Berg, 1987, 1988).Chronic treatment of these cells with K-bungarotoxin or with nicotinic agonists induces a reduction in numbers of nAChRs, and treatment with mAb35 or K-toxin reduces functional responses (catecholamine release) of nAChRs. Exposure of adrenal chromaffin cells to the nicotinic antagonist d-tubocurarine or mecamylamine results in a marked upregulation of nBgtSs (Quik et al., 1987), and this effect is mimicked by potassium depolarization and partially blocked by agents that mimic or stimulate enhancement of intracellular levels of cyclic AMP (Geertsen et al., 1988). Chronic nicotinic ligand treatment reduces levels of nBgtSs and subverts the potassium depolarization-induced effect (Geertsen et al., 1988). For the most part, these effects closely mimic those found in studies of autonomic neuronal nAChRs and nBgtSs. b. Thymopoietin. Recent studies indicate that acute exposure of chromaffin cells in culture to the thymus-derived polypeptide thymopoietin inhibits binding of radiolabeled a-bungarotoxin to nBgtSs. On the other hand, chronic exposure produces a marked upregulation of nBgtS density without affecting the number o r the functional state of presumed ganglia-type nAChRs that mediate catecholamine release (Quik et al., 1989, 1990b). c. Tachykinins. A series of studies have established that ganglia-type nAChRs on chromafin cells (and on PC 12 cells; see below) are sensitive to functional modulation by tachykinins. Both neurokinins A and B inhibit nicotine-induced catecholamine secretion from chromaffin cells (at low agonist concentration) and prevent receptor desensitization (at high agonist concentration) with, however, 30 times less potency than substance P (Khalil et al., 1988a,b). Substance P modulation of PC12 cell nAChR function partly reflects both enhanced desensitization and open channel block (Boyd and Leeman, 1987; Simasko et al., 1987).Structure-activity studies suggest that substance P exerts its effects on both preparations at a site distinct from “classic”substance P receptors (Simasko et al., 1985; Geraghty et al., 1990). d. Are Adrenal CholinergacReceptors Capable of Switching Pharmacologacal Profiles? A functional shift from muscarinic to nicotinic cholinergic receptors mediating catecholamine release has been described in several independent studies on primary cultures of bovine adrenal chromafin cells (Eberhard and Holtz, 1987; Nakaki et al., 1988). These cells express nicotinic cholinergic receptors with a classic nicotinic pharmacology (not blocked by atropine but blocked by d-tubocurarine) that are, nevertheless, coupled to second messenger systems including the cyclic nucleotide pathway (Nakaki et al., 1988) and the calcium-phospholipid

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pathway (Minenko et al., 1987; Eberhard and Holtz, 1987). T h e molecular mechanism and the significance of the pharmacological and functional shift of this “muscatinic” receptor are presently obscure and may reflect temporal control in the expression of different genes or an as yet unidentified, novel receptor type. 2. Studies V s m g hieural Crest-Denzied Cell Lznes a. Ouet-ozuw. Several studies have been conducted examining the effects of different drugs on expression and function of ganglia-type nAChRs and on expression of nBgtSs on cells of the PC12 rat pheochroniocytoma or on cells of the human neuroblastomas IMR-32 and SH-SY5Y (see above). b. Chroazc Agonist Exposurf. Acute agonist exposure induces a quickly and fully reversible desensitization of neoplastic cell ganglia-type nAChRs (Simasko et a f . , 1986; Boyd, 1987; see also Lukas, 1991). On more chronic exposure to agonists. and also to selected antagonists (Lukas, 199 I), ganglia-type nAChRs on PC I2 cells undergo a more slowly reversible process of functional inactivation (McGee and Liepe, 1984; Robinson and McGee, 1985; Simasko et al., 1986; Boyd, 1987; Kemp and Edge, 1987).The latter process may be triggered by agents that block the open nAChR channel (Lukas, 1991). c. Effects of Factors, Hormones, and Second Messengers. There are other reports that PC 12 cell functional nAChR responsiveness is augmented following neuronlike differentiation (i.e., process outgrowth), induced by treatment with nerve growth factor, in a manner that is not fully mimicked, but is partially blocked, by dibutyryl cyclic AMP (Greene and Tischler, 1976; Amy and Bennett, 1983). PC12 cell nAChR function is abolished when cells are maintained in hormone-supplemented, defined medium (Mitsuka and Hatanaka, 1983). A more recent report confirms the effects of nerve growth factor treatment on nAChR functional sensitivity in a fashion that is inhibited by co-treatment with 8-bromo-cyclic AMP, dexamethasone, or mitotic inhibitors, and also shows that treatment with cyclic .4MP analogs alone enhances nAChR sensitivity to a lower extent than does nerve growth factor treatment (Ifune and Steinbach, 1990). Nerve growth factor treatment has been reported to increase the number of nAChRs (mAb270 binding sites) on PC12 cells in parallel with its induction of increased functional responsiveness, but to have effects on nBgtS densities or levels of a,-subunit transcripts that are much less dramatic (Whiting el al., 1987a). IMR-32 human neuroblastoma cells express fkctional nAChRs with channel features that change with time in culture and/or an application of drugs (dibutyryl cyclic AMP and retinoic acid) that stimulate neu-

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ronlike differentiation (Mancinelli et al., 1988). Treatment of IMR-32 cells with bromodeoxyuridine or dibutyryl cyclic AMP induces increases in nBgtS densities and in nAChR channel opening probability (Gotti et al., 1987). d. Peptides. Aside from the studies on substance P interactions with ganglia-type nAChRs on PC 12 cells, these cells express binding sites for another peptide (atrial natriuretic factor), which blocks nicotinic agonistinduced membrane currents possibly by interacting with the open channel conformation of the receptor (Bormann et al., 1989). 3. Overview

In summary, these initial studies suggest that a multiplicity of regulatory factors can influence expression of nBgtSs and ganglia or ganglia-type nAChRs found on neural crest-derived cells. The fidelity with which known ontogenic factors influence nAChR or nBgtS numbers and function in uivo and in vitro in neoplastic and nonneoplastic cells suggests that continued studies of model systems will provide insight into factors and mechanisms involved in regulation of these receptors in the intact peripheral nervous system.

V. Central Neuronal Receptors

Previous reviews concerning features of central neuronal nAChRs, ganglia-type nAChRs expressed in the CNS, and CNS nBgtSs (see above) have summarized observations regarding ontogeny of these nAChRs and early studies on nAChR regulation (Schmidt et al., 1980; Morley and Kemp, 1981; Schmidt, 1988). In this section we provide an update of those overviews that augments another recent review specifically concerned with effects of chronic nicotine exposure on CNS nAChR numbers and function (Wonnacott, 1990). A. REGULATION in Vivo: ONTOGENIC ASPECTS 1. Developmental Profiles a. Overview. There are at least two populations of putative nAChRs in brain defined on the basis of their ability to interact with 3H-labeled agonists o r radiolabeled a-bungarotoxin (see above). These binding sites are differentially regulated during development and exhibit differential anatomical distributions (reviewed in Cairns and Wonnacott, 1988; Fiedler et al., 1990).

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6. nBgtS Expression in Mouse Brain. Generally, nBgtS densities (assayed per milligram of protein) increase in the mouse throughout fetal development and into early postnatal life before decreasing to achieve adult levels that are about 60% of those in neonates. This means of normalizing data obscures the fact that the total number of nBgtSs increases about 10-fold from neonates to adults; there just is a more niarked increase in total protein over the same period. T h e developmental profile for nBgtS expression is very similar across brain regions (Fiedler et nl.? 1990) in mice. c. Rndioagonist Binding Site Expression in Mouse Brui'rz. By contrast, the ontogeny of [:'H]nicotine binding in mouse brain varies from region to region, falling (normalized to unit protein) in the adult to much lower levels than are found in the neonate in the cerebellum, hypothalamus, and hindbrain; however, [:3H]nicotine binding site densities are sustained at neonatal levels through to adulthood in the midbrain, striatum, hippocampus, and cerebral cortex. Averaged over the whole brain, [:jH]nicotine binding site levels fall from their highest levels at birth by about 50% to reach adults levels in a temporal pattern that is distinct from that for nBgtS expression (Fiedler et nl., 1990). d. Studies in Other System. Several other studies provide information that sometimes complements and sometimes challenges the comprehensiw views of nAChK ontogeny in mouse brain. For example, comparable studies in the rat suggest that there is species specificity in developmental patterns of radioagoriist binding site expression (compare Sershen et al., 1982, and Yamada et al., 1986, to Fiedler et ul., 1990) and nBgtS expression (Marks et nl., 1986). There are persistent observations that dif€erent subpopulations of radioagonist binding sites (high- and lowaffinity [:
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constant throughout the second trimester (Whyte et al., 1985), whereas [3H]nicotine binding site densities increase between Postgestational Weeks 12 and 19, and these sites are evident in different brain regions (Cairns and Wonnacott, 1988) where binding is also observed in adults (Perry et al., 1989a). Studies using human and rodent brain indicate that there are declines in expression of both radioagonist binding sites and nBgtSs with aging (see above). e. nAChR Gene Expression. Given that it has been only recently that nAChR subunit genes have been cloned, information on the ontogeny of their expression is still scant. I n situ hybridization has revealed differential expression of neuronal cx2-subunit mRNA during chick brain development. T h e expression of transcripts correlating to this central neuronal nAChR subunit in the lateral spiriform nucleus is apparent at Day 11 and increases 20-fold until birth (Daubas et al., 1990). The cx7 subunit appears to constitute at least part of some CNS nBgtSs and can form functional, a-bungarotoxin-sensitive nAChR channels in oocytes (Couturier et al., 1990a; see above). 01, transcripts are developmentally regulated and accumulate in the developing optic tectum of the chick in early embryogenesis (between E5 and E 16), coincidental with the expression of the chick neuronal nAChR &-subunit gene, which is also transiently stimulated between E7 and El6 (Matter et al., 1990). The precise role of differential subunit expression during ontogeny is still unclear, but recent studies indicate that the pharmacological profile of functional neuronal nAChRs expressed in Xenopus oocytes can be altered by substitution of a different a or p subunit (Bertrand et al., 1990; Luetje and Patrick, 1991; see above).

B. EFFECTSOF CHRONIC NICOTINE AGONIST EXPOSURE

OR

NICOTINIC

1. General Relevance of Chronic Nicotine Treatment Studies As a widely used addictive substance, there should be considerable interest in how chronic nicotine treatment affects levels of expression and function of its own receptor in the brain, where relevant behavioral components of addiction, dependence, tolerance, and withdrawal are orchestrated. Based on studies of a variety of neurotransmitter-receptor systems, dogma holds that chronic agonist exposure would induce downregulation of receptors whereas chronic antagonist exposure, as is the case for denervation, would induce supersensitivity via increases in receptor number. A tacit assumption of many of the studies done to date that provide experimental support for this dogma is that measurement

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of radioligand binding site numbers accurately reflects the numbers of functional available receptors (Creese and Sibley, 1981). Studies done on nAChRs in the periphery at the neuromuscular junction or on neural crest-derived cells are largely consistent with this dogma, in that chronic agonist treatment produces a decrease in receptor numbers and usually a comparable o r quantitatively larger decrease in functional responsiveness (see above and Lukas, 1991).

2. The Paradox of the Effects of Chronic Nicotine Treatment A number of studies have now shown that chronic nicotine treatment induces an increase in both 3H-labeled agonist binding sites and (but largely at higher doses and only in a subpopulation of brain nuclei) nBgtS levels in both rat and mouse (reviewed in Collins et al., 1990a,b; Wonnacott, 1990). This general observation runs counter to dogma. Compounding this apparent paradox is the long-standing and extensive evidence that functional tolerance to nicotine’s effects develops on chronic agonist exposure (reviewed in Marks and Collins, 1985; Collins and Marks, 1988; Miner and Collins, 1988; Collins et al., 1990a). A variety of recent studies confirm that there is functional inactivation of nicotinic responses in the brain at a number of loci on chronic nicotine treatment (see, e.g., Sharp and Beyer, 1986; Sharp et al., 1987; Lapchak et al., 1989b; Lapin et al., 1989; Hulihan-Giblin et al., 1990b). Thus, there is a loss of functional responsiveness to nicotinic agonists despite an increase in putative receptor numbers following chronic treatment with nicotine. Expression of tolerance (behavioral studies) and increases in putative nAChR binding sites (postmortem studies) also are known to occur in human smokers (Benwell et al., 1988), which indicates that experimental studies are fully relevant to the behavioral, physiological, and environmental consequences of tobacco use in humans. 3 . Complicatioru in Interpetation of Accumulated Data There are several unresolved issues regarding interpretation of these studies (see also Wonnacott, 1990). First, heterogeneity of nAChR subtypes in brain may allow for differential effects of chronic nicotine treatment. That is, it is possible that not all nAChR subtypes have been appropriately identified with high-affinity, radioligand probes and that behaviorallfunctional effects are dominated by such nAChRs. There are reports, for example, that subchronic nicotine exposure generates shifts from one population of low-affinity [3H]nicotine binding sites to a higheraffinity state, perhaps along with an increase in the affinity of each class of states for radioagonist (Lippiello et al., 1987; Romanelli et al., 1988). Second, there also is evidence that chronic nicotinic agonist treatment can

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result in increased nAChR function, for example, in the control of dopamine release elicited by comparatively low doses of nicotine (Westfall and Perry, 1986; Fung, 1989; Rowel1 and Wonnacott, 1990)and in behavioral studies where doses of chronic nicotine were reduced (Ksir et al., 1985, 1987; Clarke et al., 1988).These results could also be interpreted to suggest that microheterogeneity of nAChR ligand binding sites may play a role. For example, expression of high-affinity forms of nAChRs, which bind ligand at concentrations much lower than those needed to affect nAChR function, may represent a functionally inactive subpopulation of nAChRs. Third, whereas metabotropic receptors may respond to chronic agonist treatment according to dogma, ligand-gated ion channels may not. Fourth, contributions of presynaptic versus postsynaptic receptors might also complicate interpretation. For example, acute administration of nicotine induces marked enhancement of acetylcholine release from rat cortex, but chronic nicotine administration fails to induce a dramatic loss of responsiveness to subsequent acute agonist under conditions where radiolabeled agonist binding site densities are increased and behavioral tolerance is evident (Nordberg et d., 1989b).On the other hand, another study found that nicotine-induced acetylcholine release from rat brain slices of cortex or hippocampus (Lapchaket al., 1989b)or dopamine release from nucleus accumbens (Lapin et al., 1989) is lost on chronic nicotine exposure, which induces increases in numbers of radioagonist binding sites in the same brain regions. These studies also point out that there may be brain region-specific, species-specific, and intraspecies strain-specific exceptions to the general rule of ligand binding site upregulation and functional inactivation (also reviewed in Collins et al., 1988a, 1990a; Wonnacott, 1990).For example, in the guinea pig, levels of cortical nicotinic agonist binding sites, nicotinic functional responses regulating acetylcholine release, and behavioral tolerance to nicotine are unaffected by chronic nicotine treatment under conditions where there are effects on rat cortical binding sites (Nordberg et al., 1989b).Lastly, there is no clear consensus whether recovery of nAChR function following cessation of chronic nicotine treatment can be correlated with the return to normal levels of nBgtS or radioagonist binding site densities or whether such a correlation would be expected (Lapchak et al., 198913; Collins et al., 1990b; Hulihan-Giblin et al., 1990b). Many of these issues may not be relevant to an explanation of the unusual responses of brain nAChRs to nicotine treatment, as both numbers and function of peripheral nAChRs-which also have nonequivalent ligand binding subsites, undergo changes in ligand affinity state, are desensitized, are ligand-gated ion channels-are downregulated on chronic agonist exposure in a manner consistent with dogma (see above and Wonnacott, 1990).

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4. nACIiR Functional Inactivation in the Presence of' an Increase in Receptor lVumbers in a Single Cell l j p e I t is interesting to note that many of factors that could complicate interpretation of studies on CNS effects of nicotine treatment in ziivo are eliminated in studies of simplified cell culture systems, such as the TEG7 1/KD human clonal cell line, whose cells express some features of neurons, but also naturally express muscle-type nAChRs that are increased in number (both total and cell surface) yet undergo a complete and only slowly reversible functional inactivation on chronic exposure to nicotinic agonists (Siege1and Lukas, 1988b;Joy and Lukas, 1989; Lukas, 1991). These findings and results of studies examining effects of a select group of antagonists (Lukas, 1991) suggest that functional inactivation may be caused by agonists or antagonists that have open channel blocking activity. These studies also show that functional inactivation is accompanied, even on one cell type expressing one subtype of nAChRs, by upregulation of nAChR numbers, which is perhaps a means for the cell to attempt to overcome this type of functional blockade. T h e finding that muscle-type nAChRs expressed in cells that display neuronlike features respond in this way to chronic nicotine treatment suggests that the cellular environment, and perhaps post-translational mechanisms, doniinates genetic influences in determining whether chronic agonist treatment will induce upregulation or downregulation of nAChR numbers. Kevertheless, many questions remain as to whether receptor upregulatory effects involve recruitment of presynthesized nAChR subunits or preassernbled nAChRs into the cell surface pool, transcriptional activation of nAChR subunit genes, an unconventional communication from nAChRs to the nucleus, and/or complex effects of depolarization and calcium ion in teniporospatial patterns that are unique to the process of nAChR activation, desensitization, and inactivation via open channel block. T h e types of changes in nAChRs that allow them to be expressed in higher numbers, but in a functionally inactive state, also remain to be determined.

C.

EFFECTSOF ANTICHOLINESTERASE TREATMENT A N D OTHER MODULATORS OF CHOLINERGIC OUTPUT

Among the nieans by which output from cholinergic neurons could be altered to affect levels of expression and functional activity of

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nAChRs in a fashion that might be mimicked by chronic nicotine treatment are treatment with anticholinesterases, regulation of acetylcholine output by presynaptic autoreceptors, or treatment or dietary supplementation with the acetylcholine precursor choline.

2. Effects of Acetylcholine Release Presynaptic nicotinic autoreceptors in at least some brain regions mediate release of acetylcholine through positive feedback mechanisms, which contrast with the predominantly inhibitory feedback regulation of acetylcholine release mediated by presynaptic muscarinic autoreceptors (Araujo et al., 1988; Lapchak et al., 1989a,b). Increased nicotine levels in the brain may initially activate both pre- and postsynaptic nAChRs and enhance acetylcholine efflux from presynaptic terminals, which would further exacerbate desensitization and functional inactivation of both post- and presynaptic nAChRs and activate muscarinic receptors to inhibit further acetylcholine release. Relevant to this discussion are data implicating the blockade by scopolamine of presynaptic muscarinic receptors (Wolfe, 1989), sometimes expressed on the same neurons as are nAChRs (Schroder et al., 1989a), in an increase in nAChR levels (Vige and Briley, 1988). Marks and Collins (1985) found that chronic muscarinic agonist treatment produced cross-tolerance to some nicotinic effects but no dramatic change in nAChR binding site densities ([3H]nicotine or nBgtSs). These findings are consistent with the notion that postsynaptic nAChR expression and function can be naturally regulated by release of the endogenous ligand for nAChRs under control of presynaptic muscarinic and nicotinic autoreceptors and that the nicotine treatment paradigm is an appropriate model for studying the mechanisms involved. 3. Anticholinesterase Treatment

Current evidence from studies examining effects of cholinesterase inhibitors complicates, rather than clarifies, these issues. For example, chronic treatment with irreversible cholinesterase inhibitors induces decreases in [3H]nicotine or [3H]acetylcholine binding to putative central nAChRs (Costa and Murphy, 1983; Schwartz and Kellar, 1985; Lim et al., 1987), whereas chronic treatment with reversible inhibitors generally (but not in the striatum) increases levels of [3H]nicotine binding sites or nBgtSs (De Sarno and Giacobini, 1989; Collins et al., 1990a). On the other hand, another recent study found that treatment of rats with THA, a cholinesterase inhibitor that has been proposed as an effective treatment for Alzheimer’s disease, increases [3H]acetylcholine binding

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site densities in the hippocampus and cortex under conditions where there is no change in midbrain o r striatal radioagonist binding site densities, yet that treatment with physostigmine induces no effect in any brain region (Nilsson-Hakansson et al., 1990). Functionally, chronic treatment with irreversible inhibitors produces a decrease in sensitivity to nicotinic agonists (Overstreet et al., 1974; Schwab and Murphy, 1981; Costa and Murphy, 1983; L,im et al., 1987). Thus, functional downregulation can occur under treatment conditions where receptor binding sites decrease (anticholinesterases) o r increase (chronic nicotinic agonists). Clearly, the mechanisms involved in these effects need to be elucidated and again may be species and strain specific. Also, the possibility that anticholinesterases might act directly and selectively at different nAChR subtypes must be addressed. 4 . E f e r h of Cholin~Treatment

Chronic administration of choline chloride or dietary supplementation produces an increase in numbers of nBgtSs in rat brain (Morley et nl., 1977; Morley and Fleck, 1987), as might be expected for a treatment that would increase agonist availability.

D. OTHERINPUTS THAT MODIFYNICOTINIC ACETYLCHOLINE OR FUNCTION in Viuo RECEPTOR EXPRESSION 1 . Agents Actiw in Neuroendocrine Signaling Chronic administration of corticosterone in normal or adrenalectomized mice induces a decrease in the number of nBgtSs, but no change in the number of [:3H]nicotinebinding sites, under conditions where it induces a decrease in functional sensitivity to nicotine and a reversal of adrenalectomy-induced increases in nicotine sensitivity (F'auly et al., 1990). These findings would be consistent with a potential role for nAChRs in feedback regulation of function along the hypothalamicpituitary-adrenal axis (see above). T h e thymic hormone thymopoietin interacts with CNS nBgtSs and may regulate their expression as it does nBgtSs in the adrenal gland (Quik et al., 1989, 1990b). Levels of expression of nBgtSs in the suprachiasniatic nucleus of ovariectomized rats are reduced or eliminated relative to intact female or male controls in a way that is reversed by estradiol administration, suggesting that these nAChRs may be targets of gonadal steroid influences on hypothalamic function (Miller et al., 1982, 1984).

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2 . Effects of Other Agents $Abuse Cross-tolerance of nicotinic behavioral responses to ethanol has been demonstrated in mice in the absence of effects on radioagonist binding sites or nBgtSs (Collins et al., 1988b), whereas chronic ethanol increases numbers of [3H]nicotine binding sites in hypothalamus and thalamus (but not in hippocampus) of rats (Yoshida et al., 1982). These findings could presage the discovery of species- and strain-specific differences in ethanol-nicotine interactions that transcend the well-characterized effects of ethanol in enhancing desensitization of nAChRs (Forman et al., 1989; Aracava et al., 1991). A variety of other neuroactive substances are known to interact acutely with nAChRs, and it is possible that those interactions may induce longer-lasting changes in receptor number and function. For example, anticonvulsive agents act via noncompetitive mechanisms at different nAChR subtypes to decrease the frequency of agonist-induced channel openings and the release of neurotransmitter from neuronal preparations (Ramoa et al., 1990). Local anesthetics and structurally similar general anesthetics may act at discrete allosteric sites on nAChRs to modulate receptor desensitization (Forman and Miller, 1989). Similarly, barbiturates at low concentrations may act by binding to allosteric sites that have higher affinity but lower stereoselectivity for the nAChR open channel conformation than for the resting conformation, whereas barbiturates at higher concentrations may affect nAChR function through nonspecific membrane perturbation (Roth et al., 1989). T h e antidepressant desipramine subsensitizes nicotinic mechanisms involved in regulating core temperature (Dilsauer et al., 1988). 3. Tachykinins

There is evidence that a-bungarotoxin is an effective inhibitor of radiolabeled tachykinin binding in the brain, but it is not clear whether this reflects interactions of toxin at tachykinin receptors or tachykinins at nBgtSs (Utkin et al., 1989).

4. Environmental Toxins Mentioned earlier is evidence implicating decreases in nAChR numbers with cognitive impairments found on aging and in Parkinson’s and Alzheimer’s diseases. It is interesting to note that symptomological correlations have been made between Alzheimer’s disease and heavy metal poisoning. Cholinotoxic effects of aluminum are thought to be an indirect consequence of cholinergic neuronal degeneration (Gulya et al., 1990), but lead selectively blocks neuronal nicotinic receptor function

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(Oortgiesen rt d.,1990). Chronic administration of two pyrethroid xenobiotics, bioallethrin and deltamethrin, produces an increase in levels of nAChRs (Eriksson and Nordberg, 1990).

VI. Perspectives and Conclusions

Since 1986 there has been a retnarkable evolution in our conceptual understanding of the nicotinic acetylcholine receptor system. It is now clear that there is heterogeneity among tnenibers of an extended nAChR family at gene, protein, and functional levels. This heterogeneity poses challenges to a clear understanding of the function of these important molecules. It is also becoming clear that diversity in mechanisms involved in the regulation of nAChR expression and function build on the diversity of the nAChR family. Continued, exciting advances in these fields will undoubtedly occur under a deluge of applications of skills of a number of investigators working in a variety of disciplines who will benefit from reflection on the basic principles of neurotransmitterreceptor biology revealed by past studies of the nAChR system and from a heightened anticipation that many conventional and provincial views of nAChR biology and regulation will require alteration or abandonment b y 19%.

Acknowledgments

'The authors thank many investigators in the field for providing information prior to publication and Dr. E. X.Albuquerque, Dr. V. A. Chiappinelli, Dr. D. Colquhoun, Dr. P. M. Lippielio. Dr. J . Schmidt. Dr. R.$1. Suoud, and Dr. U. E. Zigniond for valuable comments and suggestions. Nevertheless, the authors are exclusively responsible for the content of this review. Work in the authors' laboratory has been made possible by funds from the Men's and Wonien's Boards of the Barrow Neui-ological Foundation and Epi-Hah Phoenix, Inc. Other support has been derised f.roni grants froni the Council for Tohacco Ues e a r c h - . I~' . S . A . and rhe National Institutes of Ilealth (NS-16821).

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ACTIVITY-DEPENDENT DEVELOPMENT OF THE VERTEBRATE NERVOUS SYSTEM R. Douglas Fields and Phillip G. Nelson Laboratory of Developmental Neurobiology National Institute of Child Health and Human Development National Institutes of Health Bethesda, Maryland 20892

I. Introduction 11. Properties of Activity-Dependent Neuronal Development A. Limits of Genetic Specificity B. Afferent Activity C. Postsynaptic Activity D. Critical Period 111. Mechanisms A. Trophic Factors B. Calcium-Dependent Mechanisms C. Neural Activation of Regulatory Genes D. Structural Changes IV. Conclusions A. Synaptic Plasticity: Comparison across Systems B. Mechanisms of Plasticity References

1. Introduction

Neurons are assembled into circuits during development according to genetic instruction, but cellular interactions, hormones, neurotransmitters, electrical activity, and many other factors influence the ultimate form and function of the nervous system. There is substantial consensus with regard to the impact of electrical activity on neurodevelopment. We summarize that consensus and point out some areas where some questions arise and some reinterpretation may be appropriate. The first, and perhaps most general, principle is that genetic specification of neuronal number and of synaptic connections between neurons does not, and probably cannot, produce an optimally functional system. Genetic information, independent of input from the environment, generates a redundant, permissive system with large numbers of 133 INTERNATIONAL REVIEW OF NEUROBIOLOGY, VOL. 34

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neurons and connections (Rakic et al., 1986)that will not be necessary (or even compatible) with execution of functions involved in dealing eficiently with the specific environment to which the organism will be exposed. Exposure to that environment produces input to the system that guides and determines the reduction of redundancy in such a way as to produce a nervous system that is optimally tuned to the characteristics of the environment. It is felt generally that this process is a selective one, that only neurons and connections initially generated under genetic control can be retained or eliminated. There is little evidence that connections are specified under instruction from the environment. Experiments on the role of activity during development are considered here from many different systems, principally the neuromuscular junction (R'MJ), the retinal/tectal system, the visual cortex, long-term potentiation (L'TP) in the hippocampus, and a number of other vertebrate systems where this question has been studied. Results are compared across systems in an effort to derive common properties of activity-dependent regulation of synaptic connections between neurons during development, and to reveal important differences among the phenomena in different systems. Several biochemical processes that may participate in activity-dependent changes in synaptic strength, synaptogenesis, and synapse elimination are considered. Central to many of these biochemical pathways is intracellular free calcium. Sources of calcium influx into the neuron [voltage-sensitive calcium channels and Nmethyl-D-aspartic acid (IVMDA) channels] and several intracellular actions of this cation are considered.

II. Properties of Activity-Dependent Neuronal Developmeni

Activity-dependent synaptic plasticity has been described in many different systems, covering many different time frames. The plasticity involves processes as different as synapse elimination, synaptogenesis, and increased and decreased synaptic efiicacy. It is also known that multiple molecular mechanisms generate these diEerent forms of plasticity. Superficially similar forms of synaptic plasticity, LTP for example, can operate via different molecular mechanisms in different neurons, or in different synapses on the same neuron. Despite this heterogeneity, the diverse forms of activity-dependent synaptic plasticity during development share a number of common features: (1) Synaptic connections formed initially during development cover a diffuse region of their appropriate target. (2) Electrical activity within neuronal circuits is neces-

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sary for remodeling these initially diffuse synaptic connections. (3) The pattern of activation is an important factor in remodeling these connections. (4)T h e postsynaptic neuron often regulates activity-dependent plasticity. In some neurons, the direction of change in synaptic strength is sensitive to the coincidence of activity in synapses converging onto the postsynaptic neuron. (5) There is typically a critical period early in postnatal life during which synaptic connections are particularly (or exclusively) sensitive to alterations initiated by electrical activity.

A. LIMITSOF GENETIC SPECIFICITY Synaptic connections that are formed during development and regeneration cover at first a comparatively diffuse area of the appropriate postsynaptic structures. At later stages of development and continuing into early postnatal life, these connections are rearranged, becoming more restricted and specific. This rearrangement will not occur without neuronal electrical activity. I n the visual system (for reviews see Miller and Stryker, 1990; Shatz, 1991), geniculocotrical relay cells serving the two eyes initially make connections to the visual cortex in a uniform, continuous topographic fashion (Fig. 1) (Hubel et al., 1977; Levay et al., 1978, 1980; Rakic, 1977).

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FIG. 1. Visual relay neurons from the lateral geniculate nucleus (LGN) make synaptic connections to visual neurons in the striate cortex in an unrestricted pattern early in development. Electrical activity causes segregation of afferents from the two eyes to produce ocular dominance columns, which are alternating cortical regions in which cortical neurons respond preferentially to visual stimulation from one eye. Blockade of action potentials or abnormal visual input during this period of development causes permanent abnormal segregation of visual afferents.

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These genetically specified connections become progressively restricted and eventually the overlap of afferents from the two eyes is reduced. The segregation of afferents from the two eyes onto cortical neurons produces ocular dominance columns, which are alternating regions of visual cortex where neurons respond preferentially to input from one of the eyes. Initially the arbor from a geniculocotrical neuron covers more than 2 mm of cortex, but later this same area will encompass two pairs of ocular dominance columns (each ocular dominance column is about 0.5 mm wide). In humans and primates this segregation begins prenatally, before visual input from the environment, but in cats the process occurs in early postnatal life. As these ocular dominance columns become organized, the cortex becomes sensitive to altered visual input from the environment (the critical period). Abnormal visual input during this period will impress an abnormal structural reorganization of cortical connections, which will persist for the life of the animal (cf. Section 11,D). This restriction of arbors is not a genetically programmed response. This is shown convincingly in lower amphibians that do not have binocular vision because of the lateral position of the eyes. There are no ocular dominance columns in the optic lobes of such animals because each lobe is served independently from each eye. Ocular dominance columns can be produced by interventions that force inputs from the two eyes to innervate the same optic lobe (Meyer, 1982), or by grafting a third eye onto tadpoles (Constantine-Paton and Law, 1978). The afR

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FIG. 2. Amphibians without binocular vision lack ocular dominance columns in the optic lobes. Ocular dominance columns can be produced artificially in frogs by transplanting a supernumerary (S) eye to the juvenile animal. Blockade of electrical activity prevents segregation of afferents from the two eyes and the formation of ocular dominance columns.

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FIG. 3. Axons from retinal ganglion neurons from the two eyes are segregated in the lateral geniculate nucleus (LGN). Intraocular injection of tetrodotoxin in utero prevents this segregation in kittens, demonstrating the necessity of spontaneous bioelectrical activity for segregation of these afferents.

ferents from the third eye will reach appropriate targets, but the connections there will segregate into alternating columns serving the individual eyes (Fig. 2).These experimentally produced ocular dominance columns will not develop without normal electrical activity (Reh and ConstantinePaton, 1985) (cf. Section 11,B). Similar events are seen in the lateral geniculate nucleus of mammals (Fig. 3). Developmental studies of retinogeniculate connections in many mammals indicates that segregation of visual inputs to this primary sensory nucleus is not present at early stages of development, but inputs from the two eyes are fully segregated in adult brain (for reviews see Sherman, 1985; Rakic, 1986; Shatz and Sretavan, 1986). This principle also applies in parts of the peripheral nervous system (Fig. 4). Regenerating motoneurons initially reinnervate multiple mammalian skeletal muscle fibers, but eventually all but one input to each muscle fiber is lost (for reviews see Van Essen, 1982; Thompson, 1985; Jansen and Fladby, 1990). This reduction recapitulates the developmental pattern in which a single synaptic input from only one motoneuron comes to serve a single muscle fiber within several weeks of birth in mammalian skeletal muscle (Redfern, 1970; Bagust et al., 1973; O’Brien et al., 1978; Rosenthal and Taraskevich, 1977), avian muscle (Bennett and Pettigrew, 1974), and amphibian muscle (Bennett and Pettigrew, 1975; Herrera and Werle, 1990). As in the visual system, this represents a reduction from the more diffuse connections formed initially (Reviewed by Betz et al., 1990), and evidence suggests that this reduction requires electrical activity. Numerous other neuronal connections undergo reduction from an initially diffuse pattern of connections. Parasympathetic neurons in the cardiac ganglion become reinnervated initially by multiple vagal axons, and the level of polyneuronal innervation is reduced with time (KO and

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FIG.4. Initially in development, and following axonal regeneration, skeletal muscle fibers receive innervation from multiple motoneurons. Within a few weeks, muscle fibers lose all but one synaptic input. Polyneuronal innervation persists if electrical activity is blocked.

Roper, 1982). T h e same has been shown for parasympathetic ganglia (Lichtman, 1977) and sympathetic ganglia (Lichtman and Purves, 1980), and in the cerebellum each Purkinje cell undergoes a reduction to innervation by a single climbing fiber (Crepe1 et al., 1976). Some sensory systems of some invertebrates, often considered “hard-wired” simple systems, undergo a developmental tuning of diffuse connections formed early in development. One example is the cercal sensory system of the cricket (Murphey and Chiba, 1990). Counterexamples to this general observation are known, and these exceptions may help explain the mechanisms involved in this synaptic remodeling and the functional consequences that result. There are exceptional muscles, such as the rat lumbrical muscle, that maintain polyneuronal innervation in a fraction of the muscle fibers (cf. Werle and Herrera, 199 1). Following regeneration, the reduction from polyneuronal to mononeuronal innervation is often less than complete in some muscles that are mononeuronal before injury. Whether this polyneuronal innervation results from persistent connections from two motoneurons or continuous regeneration of new inputs to mononeuronal muscle fibers is unknown. Repeated observations of normal adult motor nerve terminals show that they are structurally plastic (Herrera et al., 1990), with a significant interjunctional sprouting activity. Ongoing synapse formation appears to proceed after reinnervation and this is balanced by continuous synapse elimination (Werle and Herrera, 1991). Studies by Werle and Herrera (1987, 1988) indicate that polyneuronal innervation tends to persist in the frog sartorius muscles if the converging inputs have equivalent synaptic efficacies. Although we feel it is appropriate to emphasize the importance of bioelectric activity in modulation of neural development, it should also

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be recognized that mechanisms must exist that provide an essentially complete degree of precision of system organization and connectedness independent of electrical activity. The specifications of the innervation of individual muscles and the synaptic connections of sensory elements within individual muscles to their central motoneuronal pools constitute an example. Independently of activity patterns, and indeed in the face of substantial anatomical perturbations of the system (spinal cord reversals, sensory ganglia displacements), cues on motoneurons, muscle, and sensory neurons must exist to permit appropriate connections to form (Frank and Jackson, 1986; Frank, 1990; Honig et al., 1986).The observations suggest that something on the order of the Sperry hypothesis of “chemoafinity” or the “labeled line” concept must be operative (Sperry, 1963). Coexistent with this precision, however, is the striking activitydependent and target-dependent motoneuronal, sensory ganglion, and neuromuscular synaptic reduction of redundancy that is such a striking feature of neuromuscular development. A discussion of these observations is available (Frank, 1990).

B. AFFERENT ACTIVITY

In this section experimental evidence on effects of electrical activity in synapse formation and elimination during development is considered in the central and peripheral nervous system. Most of these experiments do not permit an attribution of stimulation effects to presynaptic or postsynaptic mechanisms. This question is taken up in the following section. Whether effects of stimulation on development of synapses vary with the pattern of afferent activity or simply the amount of activity is addressed. A second important question that is considered is whether differential activation of converging afferents onto the same postsynaptic cell affects synaptic development differently from equal activation of afferents that may or may not converge. The term dzfferential activation is preferred to activity-dependent competition, because the latter is only one possible outcome of differential activation (cf. Section IV). Neuronal activity is necessary for the reorganization of synaptic connections that initially innervate appropriate target cells in a diffuse pattern. The processes of strengthening functionally appropriate connections and weakening ineffective connections during this developmental period appear similar to the changes in synaptic efficacy that mediate learned responses in adult organisms. The two processes may share common principles of action and cell biological mechanisms, but this is not yet known with certainty. These two general phenomena overlap in

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many ways, but they emphasize different types of synaptic alterations. Processes of synaptogenesis and synapse elimination are emphasized in developmental remodeling, whereas in adults, alterations in strength of synaptic connections are central to functional alteration. This may be a distinction in emphasis between developmental and adult synaptic plasticity, because some remodeling, terminal sprouting, synaptogenesis, and elimination of synapses may continue in the peripheral nervous system (PKS) of adult animals, on the basis of electron microscopic and light microscopic studies and repeated observations in uivo of fluorescently labeled synapses (Balice-Gordon and Lichtman, 1990; Robbins arid I’olak, 1988; Lichtman et al., 1987; Purves and Voyvodic, 1987; Purves et al., 1987; Magrassi et al., 1987; Wernig et al., 1980). 1. Nonssrbctiiw Activation

Evidence that activity can affect nervous system development derives from experiments in which normal activity is suppressed by blocking action potentials pharmacologically, by deafferentation or sensory deprivation, or by administering controlled electrical or sensory stimulation. A critical distinction in such studies is whether the activity-dependent effects on synaptic development are regulated in a stimulus patternspecific manner or represent a useldisuse response. u. Arriouut of ,4rtiuity. The normal reduction from polyneuronal to mononeuronal innervation of skeletal muscle requires electrical activity. In normal development, polyneuronally innervated fibers are detected for about 2-3 weeks after birth in the rat soleus muscle (Betz et nl., 1 979), but this becomes reduced to mononeuronal innervation provided electrical activity is unimpaired. Reduced neuromuscular activity slows down synapse elimination (Benoit and Changeux, 1975, 1978; Riley, 1978; Srilial-i and Vrbova, 1978; Miyata and Yoshioka, 1980; Thompson et nl., 1979). Nerve and muscle activity block (Thompson et al., 1979; Brown et ul., 198 1) or muscle activity block alone by bungarotoxin (Duxson, 1982; Callaway and Van Essen, 1989) slows or completely inhibits synapse elimination. Axons regenerating from motoneurons also undergo a restriction of an initially diffuse pattern of innervation. Polyneurorial innervation persists after regeneration when electrical activity is blocked by tetrodotoxin (TTX) applied to the nerve (Thompson et al., 1979; Caldwell and Ridge, 1983; Kibchester and Taxt, 1983). Activity may not be necessary for the restriction of diffuse synaptic connections formed early in development or after regeneration in certain circumstances. Although the reduction from polyneuronal to mononeuronal innervation of skeletal muscle is inhibited by activity blockade, the incidence of polyneuronal innervation continues to decline from

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levels at the start of the activity block (Thompson et al., 1979). Thus, activity is not absolutely necessary for the process of elimination of synapses at the neuromuscular junction, as some elimination of motor axon terminals will continue in the absence of activity. Activity may, however, be necessary at an earlier stage to initiate synapse elimination, or the process of synapse elimination may proceed rather slowly and may not be fully reversible by subsequent inactivity. Others have concluded that motoneurons have an inherent tendency to reduce their peripheral field of innervation, which is initially rather diffuse, irrespective of the level of electrical activity or the state of innervation of the muscle (Thompson et al., 1979; Van Essen et al., 1990), and that beneficial effects of activation are necessary to maintain synaptic connections. In the visual system, the evidence for activity-dependent remodeling of initially diffuse connections is dramatic, and the effects produced by activity blockade during critical periods are persistent and cause significant functional impairments. Intraocular injection of TTX in cats prevents segregation of retinogeniculate afferents (Shatz and Stryker, 1988). T h e formation of ocular dominance columns in visual cortex is also blocked by binocular activity blockade with TTX injection (Stryker and Harris, 1986). Ocular dominance columns produced artificially in animals that normally lack them is also prevented by blocking electrical activity. Repeated intraocular injections of TTX prevent eye-specific segregation of afferents forced to regenerate to the same tectum of fish (Meyer, 1982). TTX applied chronically to the optic nerves or optic lobes of tadpoles having a third eye graft blocked the ocular dominance formation of the optic tectum (Reh and Constantine-Paton, 1985). Neuronal tracing methods show that the regenerating projections following neuronal spike blockade cover the entire surface of the tadpole tectal lobe, and the axon terminals are intermingled with afferents serving the other eye. Electrical activity has also been shown to affect neurite outgrowth in culture, which could alter the pattern of the synapse formation. Electrical stimulation of neurons from the buccal ganglion of the snail Heliosoma in culture causes the growth cones to collapse. This response to electrical activation could help consolidate connections between neurons that enter functionally effective circuits during development (Cohan and Kater, 1986). Collapse of the growth cone has been interpreted as the initial step in transformation of a growing process into a presynaptic terminal (Kater et al., 1988; Forscher et al., 1987). This prediction would be consistent with the expectation that electrically active pathways form more effective connections. It has recently become possible to study this question in mammalian

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neurons through the use of a noninvasive method of electrical stimulation in a multicompartmented cell culture preparation (Fields et al., 1990). T h e filopodia and lamellipodium of mouse dorsal root ganglion (DRG) neurons contract immediately on stimulation, and further outgrowth ceases or the neurite retracts. This response requires activation of voltage-sensitive sodium channels, as TTX blocks the response to stimulation. An additional advantage of the noninvasive method of stimulation (Fields et al., 1991b; Neale et al., 199 1) is that it makes possible studies of prolonged (chronic) stimulation. Experiments on effects of chronic stimulation revealed that activity did not initiate transformation of the growth cone into a presynaptic terminal. Instead, after several hours of stimulation, the growth cone regenerated and outgrowth continued at a normal rate in the presence of the inhibitory stimulation. Thus, some accommodating mechanism permits continued outgrowth in the presence of electrical activity. The collapse of the growth cone is believed to be initiated by increased calcium influx accompanying action potentials (Kater ef al., 1988). Accommodation to stimulation may involve changes in membrane channels or calcium removal systems, or alterations in cytoskeletal structure or motility mechanism making the growth cone resistant to the effects of electrical activity. Activity might still have significant effects during development via direct affects on axonal outgrowth, as a result of physiological changes brought about either by accommodation to electrical activity or by the transient delay imposed in reaching its targets. 6. Patter71 01 Acizvity. Studies in which activity is blocked by TTX demonstrate the necessity of neuronal spike activity in synaptic reorganization during development and following regeneration, but they leave open the question of whether the effects depend on the pattern of spike activity. This would be the expectation if the purpose of synaptic reorganiiation was to provide a functional tuning that optimizes the neuronal circuitry for maximal response to environmental stimulation. Are the activity-dependent effects during development use-dependent, that is, an exercise/atrophy process, or pattern-specific? Patterns of activity imposed by the environment, spontaneous neuronal activity, and tonic-versus-phasic stimulation have been investigated and found to alter synaptic remodeling during development. T h e formation of ocular dominance columns in visual cortex can be prevented by intraocular injection of TTX (Stryker and Harris, 1986). Nearly all visual neurons in the striate cortex are binocular following this activity blockade. Interestingly, this result is different from that obtained by blocking light to both eyes for an equivalent period. Binocular light

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deprivation during the critical period results in normal ocular dominance columns (Wiesel and Hubel, 1965). T h e difference between these two results suggests that even spontaneous neuronal activity in the afferents, which would be blocked by TTX, is sufficient to drive the process of segregating inputs into ocular dominance columns. This would appear incongruous with the presumed role of electrical activity in tuning the neuronal circuitry for optimal functional response to the particularities of the environment. The postnatal effects of spontaneous activity revealed in the aforementioned experiments are a continuation of remodeling of synaptic connections by spontaneous activity that normally occurs in utero. Ocular dominance columns begin to form in utero in the monkey (Des Rosiers et al., 1978; LeVay et al., 1980; Rakic, 1977). In cats, the segregation of afferents into eye-specific layers in the lateral geniculate nucleus (LGN) also occurs before birth. This segregation is prevented by TTX application (Shatz and Stryker, 1988). These observations could suggest that although normal development of neuronal connections requires that synapses exhibit electrical activity, the pattern of activation is not critical for the process, as the activity is clearly not structured by the environment (but see Section II,B,2). At the neuromuscular junction the distinction between use-dependent and pattern-specific alterations is less clear. Repeated observation of fluorescently labeled and identified neuromuscular junctions in uivo has been used to study synaptic remodeling directly (Rich and Lichtman, 1989). T h e amount of remodeling differs with animal and fiber type. Substantial remodeling has been reported in frog neuromuscular junctions (Herrera et al., 1990), but more stable junctions are seen in some mammalian muscles (Lichtman et al., 1987). Much of this variation may be explained by differences in fiber type, with slow-twitch fibers exhibiting more remodeling than fast-twitch fibers (Wigston, 1990). This correiation between plasticity and fiber type suggests that structural remodeling may depend on the amount of impulse activity experienced by motoneurons, their target muscles, or individual synaptic terminals, with more remodeling at slow-twitch fibers because of more continuous activation (Wigston, 1990). Alternatively, this correlation might also be interpreted as evidence of pattern-specific remodeling, with tonic activity being more effective than phasic stimulation; however, experimental results in co-cultures of chick ciliary gangion neurons and myotubes show that synapse elimination is increased by phasic (bursting) stimulation, and polyneuronal innervation persists following tonic (continuous) stimulation (Magchielse and Meeter, 1986). Experiments in viuo show that tonic stimulation can

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effectively accelerate the normal processes of synapse elimination associated with reducing the exuberant synaptic arbor of motoneurons during development. Disappearance of polyneuronal innervation is increased by direct electrical stimulation of the sciatic nerve of rats (O’Brien et al., 1978). Tonic stimulation at 8 Hz for 4 to 6 hr a day significantly reduced the extent of polyneuronal innervation of the stimulated muscle after 2 days. After 3 days, similar effects were evident in the contralateral limb that exhibited reflexive muscle contraction. The difference between the two limbs is explained by the greater amount of activity imparted to the muscle that was artificially stimulated. In another study on this question, synapse elimination in rat soleus muscle was accelerated by chronic neuromuscular stimulation for 3 to 4 days in a phasic pattern containing 100-Hz phasic bursts, but tonic stimulation for 3 to 4 days at 1 Hz for an equal number of stimuli was without effect (Thompson, 1983). The phasic stimulation also decreased the sensitivity of the muscle to acetylcholine. T h e contralateral limb served as the control in these experiments, but in contrast to the results of O’Brien et al. (1978), the level of polyneuronal innervation did not differ from that of unstimulated animals. The direct inhibition of growth cone motility by electrical stimulation of mammalian neurons is pattern-specific (Fields et al., 1990). T h e inhibitory effects of tonic stimulation increase with increasing stimulus frequency. Moreover, the electrical inhibition of growth cone motility is less effective when the same number of impulses are delivered at a constant frequency than when delivered in half second bursts. To achieve approximately the same level of inhibition caused by phasic stimulation, four titnes as many impulses must be delivered at a constant frequency (10 Hz). T h e different response between acute stimulation (growth cone collapse) and chronic stimulation (resumed outgrowth) illustrates another aspect of activity-regulated responses dependent on the pattern and duration of activation. Long-term potentiation in the hippocampus is highly dependent on specific patterns of activation (Lynch et al., 1990; Staubli and Lynch, 1990; Dunwiddie and Lynch, 1978). T h e optimal stimulation for inducing LTP is brief high-frequency bursts (100 Hz) spaced at 5-Hz intervals, the frequency of theta waves (Larson and Lynch, 1986). LTP is also a threshold phenomenon, requiring patterned activity of sufficient intensity to recruit an adequate number of afferents with synapses on the same postsynaptic neuron (Nicoll et al., 1988; Brown et al., 1990). Posttetanic potentiation and LTP in the CA1 region of the hippocampus can occur independently and probably represent separate phenomena (Teyler and DiScenna, 1987). Heterosynaptic depression can be produced by stimulating at an ap-

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propriate frequency a population of inputs to the same dendrite as a test input (Abraham and Goddard, 1983; Dunwiddie and Lynch, 1978; Lynch et al., 1977). It is also possible to reverse the potentiation of synapses expressing LPT, by low frequency (100 impulses at 1 Hz) stimulation following soon after induction of LTP (Barrionuevo et al., 1980; Straubi and Lynch, 1990). This is an immediate and persistent all-ornone reversal of LTP, which may be related to an active forgetting process triggered by particular behavioral circumstances (Straubi and Lynch, 1990). Related to this question of the functional significance of reductions of cell and synaptic number, are questions of the generality of observations on visual cortical structural changes with use during the critical period. Increases in dendritic complexity have been consistently demonstrated in the visual cortex of cats and rats raised in enriched environments as compared with those in isolated or impoverished environments. This has not been possible in the cat motor cortex; all neuronal morphological parameters studied were the same in the two groups of animals (Beaulieu and Colonnier, 1989). This is probably a function of the developmental time course of cortical structure, with the motor cortex neuronal and synaptic connectional pattern being essentially established in the adult pattern at birth. Indeed, in subhuman primate, this appears to be the case for the prefrontal associational cortex, which also has the adult pattern of afferent and efferent connections by the latter part of the last trimester of pregnancy (Schwartz and Goldman-Rakic, 1990).

2. Selective Activation of Convergent Inputs Evidence from the visual system and neuromuscular system provides several instances where the effects of electrical activity on synaptic remodeling during development are dependent on the pattern of activation. The adaptive value of modifying the developing synaptic connections according to functional efficacy has been encompassed into the theory of “synaptic competition,” where multiple afferents converging on the same postsynaptic target are assumed to compete on the basis of electrical activity for some limiting resource, such as a trophic substance or unoccupied synaptic sties (cf. Thompson, 1986). Tests of this hypothesis require preparations in which activity can be controlled selectively in separate afferents converging onto the same postsynaptic cell. Two forms of differential activation of convergent afferents have been investigated: differing amounts of activity or similar amounts of activity in each afferent, but differing in coincidence of activation or stimulation pattern. a. Differential Intensity .f Activation. Further evidence that synaptic plasticity is pattern specific comes from depriving visual input from one

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eye at a time. If visual stimulation to one eye is blocked during a sensitive period of development, thereby providing the presumptive binocular neurons in the visual cortex with only monocular input, the cortical cells subsequently lose their ability to respond binocularly when the occluded eye is reopened. This monocular deprivation during the critical period produces monocular cortical neurons responding only to the active eye, and the ocular dominance patches in the visual cortex become widened by the loss of the overlapping binocular interface between columns (Hubel and Wiesel, 1970; Wiesel and Hubel, 1963a). These changes can be assessed physiologically (Wiesel8c Hubel, 1963a) or by morphological tracing methods (Hubel and Wiesel, 1970; Shatz et al., 1977; Wiesel et d.,1974; Hubel, Wiesel, and LeVay, 1977; LeVay, Wiesel, and Hubel, 1980). Monocular deprivation does not affect the function of the deprived eye itself, o r the function of the visual relay nucleus (the lateral geniculate nucleus). T h e effect develops at the point where there is binocular input converging onto the same neuron, that is, the visual cortical neurons (Wiesel and Hubel, 1963b). This suggests that competition on the basis of spike activity between afferents converging on the same postsynaptic target initiates reorganization of synaptic connections. Clearly, this is not a use-dependent effect, because binocular deprivation leaves neurons in the visual cortex intact (Hubel and Wiesel, 1965). Similar competitive interactions have been studied in a tissue culture preparation of spinal cord neurons, organized by the use of a multicompartmented insert in the culture dish to form competitive interactions between afferents converging onto a common target population (Fig. 5) (Nelson et al., 1989). Unilateral stimulation for 3-5 days of afferents from one of two populations of mouse DRG neurons converging onto ventral spinal cord neurons results in larger excitatory postsynaptic potentials (EPSPs) from stimulated afferents than from the unstimulated side (Nelson et al., 1989). T h e number of functional inputs was also increased in stimulated cultures compared with unstimulated preparations. Stimulating one of t w o nerves that innervate rat lumbrical muscle during the postnatal period of synapse elimination resulted in motor units larger than those from unstimulated nerves (Ridge and Betz, 1984). Phasic stimulation of 40 Hz for 2 sec every 5 sec was delivered, for 4 to 5 hr a day for 5 to 6 days. This result is consistent with the hypothesis that electrical activity confers a competitive advantage on motor axons. Analogous experiments in the same muscle of adult animals undergoing reinnervation after nerve crush yield compatible results (Ribchester and Taxt, 1983). Axons in nerves where activity was blocked by TTX

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FIG. 5. A model for studying activity-dependent effects on synaptogenesis and synapse elimination in mammalian spinal cord neurons in vitro. A Teflon insert (black) is placed in a 35-mm culture dish to allow segregation of neurons into two separate populations that interact through axons that penetrate the barrier between compartments. Neurons dissociated from the dorsal root ganglion (DRG) of fetal mice are plated in the two side compartments and neurons from the ventral spinal cord are plated in the central notch. Axons from the DRG neurons penetrate the barrier and converge on spinal cord neurons where they form synapses. Patterned electrical stimulation applied to axons from one side for several days in vitro results in stronger synapses and the weakening of synapses converging on the same postsynaptic neuron from the unstimulated side. (This change is depicted here as an increase in the number of synapses that form from the stimulated side and a loss of synapses from the unstimulated side.)

were less successful in reinnervating muscle compared with the motor axons that were not blocked. The most recent experiments of the question of activity-dependent competition between convergent synapses on muscle yield directly contradictory results (Callaway et al., 1987, 1989).Blocking electrical activity by T T X in one of three ventral roots innervating the rabbit soleus muscle resulted in larger motor units from the nerve with activity blockade than from converging axons from nerve roots in which electrical activity was not blocked. That is, competition favored the inactive over the active axons in this activity-dependent synaptic remodeling. A possible reconciliation between these conflicting results of blocking activity in one of the converging axons is that activity may confer an advantage in synaptogenesis, but be a disadvantage in synapse elimination. Thus, in experiments of activity-dependent effects on synaptic competition performed on adult regenerating axons (Ribchester and Taxt, 1983, 1984; Ribchester, 1988), the active afferent would form more synapses, but in studies conducted during the period of synapse elimination in early postnatal life (Callaway et al., 1987, 1989) the active afferent would suffer more vigorous synapse elimination. To distinguish between these alternatives, the effects of activity on synaptogenesis and synapse elimination must be studied independently.

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b. Differential Timing of Activation. Monocular deprivation causes an ocular dominance shift in visual cortical neurons toward the open eye. Additional studies have tested whether this shift is caused by a disparity in the amount of afferent stimulation or differences in the pattern of stimulation converging onto the cortical neurons from the two eyes. Blurring the image to one eye proves to be as effective as monocular deprivation in shifting the response of binocular visual cortical neurons to the eye with normal activity (Wiesel and Hubel, 1963a). An equal amount of visual input from the two eyes, but provided through only one eye at a time, results in a visual cortex composed wholly of monocular neurons. This was achieved by patching the left and right eyes on alternate days or misaligning the images from the two eyes surgically or optically, to eliminate overlapping fields of vision on each retina that would normally provide coincident binocular stimulation to cortical neurons (Hubel and Wiesel, 1965; Van Sluyters and Levitt, 1980). As each cortical neuron in the visual cortex receives input from both eyes initially, the shift to monocularity in each cortical neuron shows that synapses from afferents that are not coincidentally active become eliminated. Whether the shift at a particular visual cortical neuron develops in favor of coincidentally active afferents from the left or the right eye is presumed to reflect an initial bias in strength of afferents from one of the eyes as a result of spatial or other factors. Further test of the hypothesis that afferents that are not coincidentally active become eliminated is provided by blocking spontaneous retinal activity by intraocular injection of T T X , and controlling afferent activity by electrical stimulation delivered to each optic nerve. Synchronous stimulation of the two nerves prevents formation of ocular dominance columns in kittens (Stryker, 1986). If the same amount of electrical stimulation is delivered to the two nerves, but asynchronously, the ocular dominance columns developed normally. That is, many visual cortical neurons become responsive preferentially to one eye or the other, and many others are bilaterally responsive in a systematic topographic arrangement in the cortex. Presumably, activity causes elimination of synapses that are active out of phase with other inputs to the same cortical neuron that are coincidentally active but also more effective. Activity thus increases the precision of the genetically defined topographic organization of afferent input to the cortex. Sensory stimulation from the environment is not necessary for this activity-dependent synapse elimination; spontaneous activity that is correlated can initiate elimination of convergent afferents. This is believed to explain the activity-dependent segregation of retinal ganglion inputs to the lateral geniculate, which can be disrupted by T T X injection in utero. Electrophysiological recordings showing that spontaneous activity

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from neighboring axon terminals from each eye exhibit correlated firing early in development when the retina is not yet light sensitive (Mastronarde, 1983a,b, 1989). Activity of cells from remote regions of the retina or between the two eyes at corresponding points in each retina are not well correlated. Also spontaneous firing from visual neurons from the LGN is correlated within each eye-specific lamina, but over somewhat longer time scales than in the retina (Levick and Williams, 1964; Rodieck and Smith, 1966). T h e lack of correlated spontaneous firing between the two eyes and the correlated firing between neighboring ganglion cells and LGN afferents should by theory assort themselves on the postsynaptic targets, because afferents from the two eyes converging on the same postsynaptic neuron convey noncorrelated activity patterns. The opposite eye should be weakened on cortical neurons receiving predominant coordinated activation from afferents of one eye. Recent measurements of spike activity of ganglion neurons of prenatal kitten retinas, through the use of multielectrode recording chambers, show that the correlated activity of neighboring neurons results from waves of activity sweeping across the retina. If connections from neurons with correlated activity are selectively retained on target neurons, the coordinated waves of activation could help specify the retinotopic organization of innervation in the LGN (Meister et al., 1990; Shatz, 1991). Such a mechanism could help provide registration of presynaptic neurons with postsynaptic targets in a topographic arrangement, in addition to cell-specific labeling and chemical gradients for chemotactic responses. Much as with eye-specific assortment of retinal ganglion neuron terminals in the LGN of mammals, regenerating retinotectal projections in the goldfish normally undergo a refinement of connections in the formative visual map that is thought to be based on correlated activity between neighboring ganglion neurons. Synchronizing activity across the retina with stroboscopic illumination prevents sharpening of the retinotopic map determined by electrophysiological and anatomical analysis. T h e axonal arbors regenerate during synchronized activity, but they are larger than normal and diffusely distributed (Schmidt and Eisele, 1985; Cook and Rankin, 1986). Differential timing of activation between convergent afferents in the visual system show that synapse elimination proceeds against the synchronously active but less effective convergent afferents, or against afferents that are not active. The most reasonable comparator of synchronous activation among convergent inputs would be the postsynaptic cell. This is in agreement with the theory that coincident activation of afferent inputs producing an adequate postsynaptic response promotes mechanisms that strengthen active synapses and weaken inactive synapses, or

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weakens synapses that are active at times when the postsynaptic neuron is not sufficiently activated. T h e sufficient level of postsynaptic activation is related to the firing level of afferents that provide the strongest coordinated activation of the postsynaptic neuron. This hypothesis of differential timing of convergent activation has not been tested at the neuromuscular junction. Results from studies using activity blockade or differential intensities of stimulation at the neuromuscular junction are compatible with the hypothesis that the amount of afferent activation can affect the rate of synapse elimination. Nonselective stimulation of muscle accelerates the rate of synapse elimination (Thompson, 1983; O'Brien et al., 1978), and activity blockade impedes this process (Thompson et al., 1979; Taxt, 1983). Differential intensities of convergent activation produce intensified synapse elimination in the more active afferent in some studies (Callaway et al., 1989), but in other studies synapse elimination is greater in the less active convergent afferent (Magchielse and Meeter, 1986; Ridge and Betz, 1984).

C. POSTSYNAPTIC ACTIVITY 1. Blockade or Activation of Postsynuptic Response Suggestion that postsynaptic activity may be necessary has been sought in experiments in which TTX is infused directly onto the visual cortex (Reiter ct al., 1986). The normal monocular dominance shift in preference of cortical neuron responses to the open eye is blocked by this treatment; however, T T X infusion to the visual cortex also blocks discharge of afferent terminals. A treatment that is selective for postsynaptic cells is required to fully test the hypothesis that postsynaptic activity is critical for this synaptic reorganization. Infusion of muscimole, a y-aminobutyric acidergic agonist, presumably has no affect on transmitter release, but inhibits firing of the cortical neurons. Under these conditions plasticity was not blocked, it was inverted. That is, when activity in the postsynaptic neuron is inhibited during monocular deprivation, inputs from the inactive eye come to dominate those of the open eye (Reiter and Stryker, 1988). This unexpected result shows that postsynaptic discharge is not required for plasticity. Moreover, this result suggests that plasticity is initiated by afferent activity, but that the sign of the change (increase or decreased synaptic strength) could be regulated by the amount of postsynaptic activity (Bear et al., 1987; Bear, 1988; Singer and Artola, 1991). A variation of this view is that the coincidence of activation between presynaptic and postsynaptic activity leads to strengthening of synapses, and disso-

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nance in firing between presynaptic and postsynaptic neuron promotes processes weakening synapses. Activity in the less active eye would, in a sense, be better correlated with the suppressed activity in the inactive postsynaptic cortical cell compared with the active eye (Miller and Stryker, 1990). T h e more active eye is on balance less well correlated with postsynaptic activity and so becomes weakened (cf. Section 11,C,2). This interpretation suggests that processes coupled to the postsynaptic membrane voltage ultimately control the direction of synaptic plasticity. The more active input is favored when the postsynaptic cell is depolarized and the less active input is favored when the postsynaptic cell is hyperpolarized. As activity of any neuron connected to cortical cells will contribute to activity in the postsynaptic cell, activity of neurons other than the converging visual afferents will also play an important role in determining the direction of change in synaptic strength. This includes activity of any interneurons in the network of synaptic connections affecting postsynaptic activity. A postsynaptic role in activity-dependent synaptic restructuring during development has been shown by the disruption of plasticity by various pharmacological agents known to affect receptors on the postsynaptic neuron, such as aminophosphnovaleric acid (APV) (Davies et al., 1981; Olerman et al., 1988; Watkins and Evans, 1981), an antagonist of the NMDA channel (cf. Sections III,B,l and IV,B,l). The role of postsynaptic activity in this process has also been studied in a tissue culture preparation in which afferents from two populations of mouse DRG neurons converge onto a population of neurons from the ventral spinal cord (see Fig. 5). The target neurons enter into a polysynaptic network with a considerable amount of spontaneous activity. After addition of TTX at a dilution that suppresses spontaneous activity in the network, but leaves DRG afferents responsive to electrical stimulation, the synaptic plasticity arising from the competitive activation of converging DRG afferents was blocked (Figs. 6A,B) (Fields et al., 1991a). Thus, any factor controlling electrical activity of the postsynaptic neuron could regulate the outcome of activity-dependent synaptic competition. This result is consistent with the facilitating effects of cholinergic, adrenergic, and other excitatory neurotransmitters from inputs from diffuse projection systems onto visual cortical neurons (Singer, 1985, 1990). This gating response could provide a means for higher-level regulation of synaptic plasticity, based on the behavioral context of the stimulus. Direct electrical stimulation of muscle accelerates the progression from polyneuronal to mononeuronal innervation of skeletal muscle (Thompson, 1983), suggesting a postsynaptic control of synapse elimination at the NMJ. The ocular dominance changes in cortical neurons in response to

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monocular deprivation are relatively rapid (6 hr), and the effects can be reversed 24 hr after reversing the eye occluded during the critical period (Mioche and Singer, 1989). Although ocular dominance changes eventually manifest as strikingly stable structural changes in patterns of synaptic arrangements, the rapidity of change in cortical neuron responses to monocular deprivation suggests that synaptic efficacy must be altered by a physiological mechanism, rather than one requiring growth of new synaptic connections and elimination of inactive inputs. This and the involvement of the postsynaptic neuron in changes in synaptic strength are analogous to the phenomenon of long-term potentiation in the hippocampus. This has lead to the proposal that LTP and synaptic restructuring during development may share a common physiological mechanism (Singer and Artola, 1991; Constantine-Paton et al., 1990; Debski et al., 1990). The involvement of postsynaptic activity in LTP has been shown by several methods. Normally, induction of LTP requires a high-frequency burst of stimulation at suficient frequency to recruit several afferents converging onto the postsynaptic neuron; however, hippocampal LTP can also be induced by pairing single afferent volleys with intracellularly injected depolarizing current pulses into the postsynaptic neuron (Wigstrom et al., 1986). Thus, the postsynaptic depolarization can substitute for the high-frequency tetanus of afferents, which is normally required to induce LTP. Conversely, postsynaptic hyperpolarization FIG.6. By reducing the time of postsynaptic depolarization, APV (an antagonist of the NMDA receptor), can significantly depress the spontaneous activity of neuronal circuits in a manner similar to that of dilute TTX. Suppressing spontaneous activity (with either APV or dilute TTX) will prevent the activity-dependent synaptic plasticity of spinal cord neurons in the model system shown in Fig. 5. This result brings into question the role of NMDA channels in activity-dependent synaptic plasticity in viva. (A) Spontaneous activity is suppressed in spinal cord neurons following perfusion with dilute TTX (5 nM in 3 mM Mg*+ and 3 mM Ca2+) .(B) A paired intracellular recording between a DRG neuron and a spinal cord neuron demonstrating that this treatment results in failure of polysynaptic responses in the postsynaptic neuron (cf. a and b), but this concentration of TTX does not block action potentials evoked by stimulation, as evident from the short-latency EPSP recorded in the spinal cord neuron following stimulation of the DRG neuron (b). (C) The presence of the NMDA channels and their sensitivity to APV antagonism are demonstrated by pressure ejection of 10 @ APV from a pipet placed near a spinal cord neuron. Note the reduction in the slow component of the EPSP in the presence of APV. (D) NMDA channel blockade by APV in the presence of 3 mM calcium and no magnesium. (E) Spontaneous activity in the network of spinal cord neurons is reduced by 100 pA4 APV as shown here in a recording made in a manner similar to that in A. This may be explained by loss of the long-latency NMDA component of the EPSP in APV, which reduces the EPSP amplitude resulting from temporal summation of multiple excitatory inputs. [Reprinted from Fields et al., (1991).J. Neuroscience 11, 134-146.1

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during afferent stimulation blocks induction of LTP (Malinow and Miller, 1986). Generation of action potentials in the postsynaptic neuron is not necessary for LTP, however (Kelso et al., 1986; Wigstrom et ul., 1986). Recent additional evidence that LTP involves changes in the postsynaptic neuron is that the drug aniracetam, which potentiates nonNMDA glutamate receptors (AMPA/kainate receptors) (Ito et al., 1990) bv a postsynaptic mechanism of slowing receptor densensitization (Vyklickv et al., 1991), has significantly less effect in hippocampal neurons that have undergone LTP (Xiao et al., 1991). Electrophysiological methods suggest that the development of LTP depends on the amount and timing of postsynaptic activity controlling calcium influx into the postsynaptic neuron (Malenka, 1991). Strong depolarization, beyond the reversal potential of the EPSP, also prevents LXP generation (Malenka et al., 1988), presumably because calcium influx is reduced. LTP is also blocked by pharmacological suppression of the increase in intracellular calcium in the postsynaptic neuron. Postsynaptic mechanisms related to NMDA receptor activation (Collingr-idge and Singer, 1990; Kauer el al., 1990) and calcium influx (Lvnch et al., 1983; Malenka et al., 1988) are clearly involved in the phenomenon of LTP in the hippocampus (cf. Section III,B, 1).

2. Cozncidence of Presynaptzc and Postsynaptac Acttvutzon A general theory originating from studies of associative learning and conditioning, and further articulated by Hebb ( 1949), proposes that the coincidence of activation between an afferent and sufficient activation of the postsynaptic neuron provides the logical condition for initiating an increase in eficacy of the connection. Dissonant firing of an afferent and the postsynaptic neuron should promote weakening of the afferent (Stent, 1973). This has proven a robust principle for describing the developmental phenomenon of synaptic competition and associative learning. (For general reviews see Hebb 1949; Stent, 1973; Changeux and Danchin, 1976; Brown et al., 1990.) Activity-dependent plasticity during development resembles a selective interaction that favors convergent afferents with the greatest amount of correlated activity (cf. Section 11,B). The excitability of the postsynaptic neuron is also a critical factor. Coherent afferent activation without postsynaptic activity will not lead to adaptive changes in synaptic strength (cf. Section 11,C). Removing excitatory input to the postsynaptic cell provided by central core modulatory systems in visual cortex inhibits synaptic plasticity produced by monocular deprivation (Singer, 1990). Removing inhibitory input to the postsynaptic cell in visual cortical neurons or increasing their excitability by infusing glutamate produces an “abnormal result,” with the occluded eye developing stronger connec-

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tions (Reiter and Stryker, 1988) or no change taking place (Shaw and Cynader, 1984). These effects are consistent with the prediction that the coherent activity of converging afferents is represented by the degree of coincident postsynaptic depolarization. It is difficult to test this theory in a developmental time frame, but studies of long-term potentiation in the visual cortex support the conclusion that presynaptic activity coincident with postsynaptic depolarization strengthens synapses. I n the mammalian visual cortex, visual stimulation through one eye paired with simultaneous postsynaptic depolarization via extracellular stimulation, can result in increased synaptic strength from the stimulated eye that lasts from minutes to hours (Fregnac et al., 1988). Similar results have been obtained with iontophoretic application of excitatory neurotransmitters in combination with activation of one visual input from one eye (Greuel et al., 1988). Long-term potentiation (Bliss and Lomo, 1973) of synapses in the hippocampus depends on coincident presynaptic and postsynaptic activity (McNaughton et al., 1978; Kelso et al., 1986; Malinow and Miller, 1986; Gustafsson et al., 1987). Evidence from research on the hippocampus supports the theory that the direction of synaptic plasticity (increase or decrease in synaptic strength) is regulated by the relative timing of activity among synapses converging on the same postsynaptic target. Coincident activity of synapses on the same target strengthens these connections, and firing uncoordinated with other converging synaptic activity weakens the synapse. Afferents that are inactive or moderately active on neurons become weakened when converging inputs are stimulated intensely. This effect has been termed long-term depression (LTD). Stimulating the perforant pathway at sufficiently high frequency to induce LTP in the dentate gyrus can reduce the strength of a second pathway that is not stimulated or is stimulated at low frequency (Levy and Steward, 1979, 1983; Abraham and Goddard, 1983). Even low-frequency stimulation (1-5 Hz)is capable of inducing heterosynaptic depression (Lynch et al., 1977). The significance of the relative timing of activity in synapses converging onto the same neuron can be interpreted in terms of effects on the postsynaptic membrane potential. Coordinated activity causes maximal depolarization of the target, but uncoordinated activity would cause less depolarization. Presynaptic and postsynaptic activity must be simultaneous or synaptic potentiation will not result. As postsynaptic activation is increasingly delayed from presynaptic activation, the duration of potentiation decreases from LTP to a shorter form of potentiation termed shortterm potentiation (STP) (Malenka, 1991). A depolarizing pulse given prior to the presynaptic activation will result in LTP if afferent stimulation is applied during a time when the postsynaptic membrane remains

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sufficiently depolarized. Presynaptic activity occurring during periods when the postsynaptic membrane is depolarized is associated with LTP, whereas activity during periods when the postsynaptic neuron is moderately depolarized, or hyperpolarized, causes LTD. Pairing injections of depolarizing current with presynaptic activation leads to LTP in the hippocampus, and this does not change the strength of other inactive inputs (Kelso and Brown, 1986; Gustafsson and Wigstrom, 1986; Gustafsson rt al., 1987; Kelso et al., 1986; Malinow and Miller, 1986); however, 5-Hz afferent stimulation during injection of hyperpolarizing current into the postsynaptic neuron causes LTD in the active synapses and not in the unstimulated convergents (Stanton and Sejnowski, 1989). This effect is not dependent on NMDA channel activation, but it is blocked by AP3, an antagonist of the metabotrophic glutamate receptor that causes increased phosphoinositol (PI) hydrolysis (cf. Section III,B,1). It is of interest that two important papers indicate a presynaptic locus of change during LTP (Bekkers and Stevens, 1990; Malinow and Tsien, 1990). LTP clearly is contingent on postsynaptic responses, but evidently has, at least in part, a presynaptic basis (cf. Bliss et al., 1986). These papers pointed out the importance of establishing the nature of the retrograde signals mediating such changes in synaptic efficacy. In the neocortex, the results are somewhat different, but generally supportive of the conclusion that the strength of a synapse is regulated by postsynaptic membrane potential at the instant of presynaptic activation. Injection of weak depolarizing current into the postsynaptic neuron paired with tetanizing presynaptic stimulation produces long-lasting depression in the rat neocortex in slices, but hyperpolarization of the postsynaptic neuron did not have an effect (Artola et al., 1990). This suggests that there is a narrow range of postsynaptic membrane potentials for eliciting depression, and that afferents active when the postsynaptic membrane is inactive would not undergo a change in synaptic strength. Sejnowski and colleagues (1991 review) have suggested that the difference in direction of current injection required to induce LTD in the neocortex compared with the hippocampus could be explained by differences in resting potential of neurons in the t w o preparations. Neocortical neurons are 10 to 20 mV more negative than the resting potentials of CA 1 hippocampal pyramidal neurons. Homosynaptic LTD, where the synapse that is activated is affected, has been reported in the hippocampus (Stanton and Sejnowski, 1989) and neocortex (Artola et al., 1990). Afferents from synapses ending on a CA1 pyramidal neuron exhibit reduced EPSPs when tested in the intervals between brief phasic bursts, that is, when the conditioning and test afferents are activated out of phase. This occurs simultaneously with potentiation of the conditioned input. The maximal depression results

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when the test pulse occurs approximately 20 msec after the end of each burst of the conditioning stimulus. The test afferent can be potentiated by another burst delivered to a convergent input. Although the coincidence of presynaptic and postsynaptic activation leads to changes in synaptic strength, the plasticity does not always favor the coactive input. Coincident activation leading to synaptic weakening is termed anti-Hebbian. In the cerebellum an anti-Hebbian form of LTD has been described where synaptic strength is reduced rather than increased by coincident presynaptic and postsynaptic activity (Ito, 1989). In the neuromuscular junction, differential stimulation of convergent inputs can lead to strengthening of the more active input (Betz et al., 1990) or weakening of the more active input (Callaway et al., 1989). These results may represent Hebbian and anti-Hebbian responses in different muscles or in different animals, but the relationship between this synaptic remodeling during development and LTP or LTD in the hippocampus and visual cortex is tenuous. Thus, it may be inappropriate to refer to the outcome of differential stimulation of convergent motoneurons as if it were a Hebbian processes dependent on coincident presynaptic and postsynaptic activation.

D. CRITICAL PERIOD The nervous system is most sensitive to electrical activity during synaptogenesis and for a limited period after axons reach their targets; however, much of the nervous system retains a variable capacity for structural rearrangement of axon terminals throughout life in response to altered functional input or injury. The irreversible nature of functional deficits produced by abnormal sensory input during the critical period and the comparatively limited compensatory ability in other regions of the nervous system make the concept of the critical period one of considerable theoretical and practical importance. Synaptic plasticity of the visual cortex is greatest in kittens between 4 and 8 weeks (Hubel and Wiesel, 1970; Blakemore and Van Sluyters, 1974; Olson and Freeman, 1980). Several biochemical correlations with the critical period have been described for the visual system. NMDA receptors contribute relatively more to excitation of cortical neurons in kittens than in adults (Tsumoto et al., 1987). Binding of tritiated glutamate sites that are displaced by NMDA receptor antagonist (APV) on sections of primary visual cortex of cats shows a large increase between the second and fourth weeks of postnatal life. Binding remains elevated throughout the critical period and decreases to adult levels (BodeGreuel and Singer, 1989).

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Similar events are observed in other brain regions. T h e density of NMDA binding sites is abruptly reduced after Postnatal Day 8 in the rat hippocampus (Tremblay et al., 1988). In the cerebellum, iontophoretic application of NMDA excites a greater number of neurons postnatally than in adults (Dupont et al., 1987; Garthwaite et al., 1987). These data have lead to the suggestion that the critical period results from the loss of effectiveness o r reduction in number of NMDA receptors (Udin and Scherer, 1990; Tsumoto et al., 1987; Tremblay et al., 1988; Garcia-Ladona ut ~ l . 1990; , Bode-Greuel and Singer, 1989). The ability to realign the visual map, which is mismatched by rotation of one eye, is restored after the normal end of the critical period by applying NMDA continuously to the optic tectum of frogs (Udin and Scherer, 1990). This correlation between NMDA receptors and the critical period could indicate that these receptors are necessary for neuronal growth and synapse stabilization (Brenneman el al., 1990a; Dudek and Bear, 1989). An alternate suggestion is that the transient expression of NMDA receptors during maturation might be related to trophic effects of NMDA (Pearce el al., 1987). NMDA receptors participate in synaptic responses of visual cortical neurons in young kittens, but not appreciably in adults (Tsumoto et al., 1987). These receptors, by virtue of their longlasting EPSPs, would participate in generating visual responses that consist of high-frequency discharge of spikes, following initial depolarization by non-NhlDA receptors in kitten cortical neurons. This action of the NMDA receptors might become less critical as the non-NMDA receptors mature. T h e large calcium influx associated with NMDA receptor activation may play a particular role in neuronal growth and synapse stabilization (Garthwaite et d.,1987). Dark rearing, however, prolongs the critical period during which functional changes can be induced by monocular deprivation (Cynader, 1983), but the decline in NMDA receptors with age is not delayed. A number of other factors, such as p receptors (Aoki et a/., 1986; Schliebs et al., 1982; Wilkinson et al., 1983) and the high-threshold voltage-dependent calcium channels (BodeGreuel and Singer, 1988; Geiger and Singer, 1986), are not affected by dark rearing. Although no change is detected following monocular deprivation, the abundance of the growth-associated protein GAP-43 (cf. Section 111,D,2) in the cat visual cortex correlates with the critical period (McIntosh et al., 1989), and phosphorylation of the protein increases during the critical period (Sheu et al., 1990) (cf. Section III,B,3). Stimulation of phosphoinositide hydrolysis by excitatory amino acids ibotenate and glutamate correlates with the developmental profile of cortical susceptibility to visual deprivation in kitten visual cortex (Dudek and Bear, 1989; Dudek et aE., 1989). Kittens reared in complete darkness

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do not exhibit ibotenate-stimulated PI turnover. These observations could indicate that the products of PI turnover enable the processes of synaptic plasticity to proceed during development. One such product is diacylglycerol, which activates protein kinase C. This kinase activity is also elevated in the striate cortex during the critical period (Stichel and Singer, 1989). Modulators of ocular dominance plasticity, acetylcholine and norepinephrine (Kasamatsu and Pettigrew, 1979; Bear and Singer, 1986), both potently stimulate PI turnover in the neocortex (Gonzales and Crews, 1985). Bear and colleagues have proposed that second messenger systems linked to PI metabolism may be specifically linked to the weakening of synaptic connections during development (Dudek and Bear, 1989). Input activity that is coincident with strong postsynaptic depolarization, which is a favorable condition for postsynaptic calcium entry through the NMDA receptor channels, would lead to enhancement of synaptic strength. Input activation coincident with postsynaptic inactivity would stimulate PI turnover by means of the non-NMDA receptor, without a concomitant postsynaptic calcium influx. This would lead to a decrease in synaptic efficacy. In further support of this proposal, NMDA inhibits excitatory amino acid-stimulated PI turnover in neonatal hippocampus in a calcium-dependent fashion (Palmer et al., 1988). Although such results are intriguing, biochemical correlates with developmental periods of synaptic plasticity cannot in themselves establish a causal relationship to the mechanism of critical period. The critical period concept is less evident in other cortical areas. T h e maps of digit representation in the somatosensory cortex of primates can be altered in adults by altering sensory input from the hand (Kaas et al., 1983). Inactivity, as produced by nerve section, will allow neighboring active areas of cortex to expand their territory. This persisting plasticity in adult brain appears to be the result of disinhibition of connections that are already present but masked, rather than increased effectiveness or sprouting of new synapses. Tonotopic maps in the auditory cortex of guinea pig are similarly modifiable by sensory manipulations in adult animals (Robertson and Irvine, 1989). Areas of the primary auditory cortex once serving particular sound frequencies become reduced after those sound frequencies are blocked from experience, and the frequencies that border this area expand and overtake the inactive cortical regions. As in the somatosensory cortex, it is significant to note that weak input from these flanking zones is already present when the cochlear lesion is produced, suggesting that the reorganization of the auditory cortex is probably not caused by sprouting into new cortical regions (Aitkin, 1990). In the barn owl, adjustment of auditory and spatial cortical maps after monaural occlusion

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is limited to young animals (for review see Knudsen, 1988; Knudsen and Knudsen, 1990). A critical period is less evident for synapse elimination at the neuromuscular junction. Blocking activity during the early postnatal period of synapse elimination does not prevent the normal progression from polyneuronal to mononeuronal innervation after removing the blockade. Synapse elimination will proceed in adult muscles innervated by multiple motor neurons after regeneration. Axons regenerating after a nerve crush recapitulate the developmental progression from polyneuronal to mononeuronal innervation in adult muscle fibers (McArdle, 1975). Cross-innervation studies shown that synapse elimination can proceed not only in adult muscles, but in mature neuromuscular junctions. An additional nerve transplanted near the existing endplate of adult muscle institutes elimination of one of the synapses (Kumer et nl., 1980; Lomo, 1980), with the original synapse being lost in approximately half of the cases where the foreign nerve innervates the original endplate (Bixby and Van Essen, 1979). The extent of ongoing remodeling of neuromuscular junctions in normal adult muscle varies with animal and muscle type. Remodeling of synapses on adult muscle occurs extensively in some muscles of the frog (Herrera et al., 1990). In mammals continuous remodeling ofjunctions is reported in some studies (Cardasis and Padykula, 1981; Wernig et ul., 1984), but little synaptic rearrangement is detected over a 6-month period of observation in other muscles (Lichtman et al., 1987). The different amount of plasticity could be related to fiber type, particularly if the amount of reorganization was dependent on the amount of electrical activity. This is supported by the evidence showing that fast-twich fibers, which receive less continuous activation, exhibit less synaptic remodeling (Wigston, 1989, 1990). Sprouting of motoneuron terminals is stimulated by paralysis or partial denervation of adult mouse skeletal muscles (Duchen and Strich, 1968; Brown and Ironton, 1977, 1978), and this can produce multiple innervation (Holland and Brown, 1981). The amount of terminal sprouting, however, is less in adult muscles than in muscles of immature animals. [Sprouting of motoneurons of early neonatal mammals is already high, and cannot be increased further by activity blockade (Brown et al., 1982).] Blocking neuromuscular transmission in postnatal rats at Day 10 is followed by an initial fall in multiple innervation (Thompson et al., 1979; Brown et al., 1981),but after an additional 2 days the number of multiple inputs increases to the level present before activity blockade, which is presumably a result of paralysis-induced axonal sprouting. ‘This suggests that paralysis induces muscle to liberate a trophic substance that stimulates axonal growth. A critical period in neuromuscular junction

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remodeling may be related to the axonal sprouting capacity, which declines with age. The amount of multiple innervation that is reacquired after paralysis decreases the later paralysis is started. If paralysis is started on Day 16, after all excess inputs have normally withdrawn, there is minimal return of multiple innervation. This reflects a limited developmental period when inactivity can reactivate multiple innervation in normal muscle, although this critical period is abolished in regenerating axons. As in the visual system, the critical period of neuromuscular junction remodeling correlates with a stage of development characterized by an excess of branching and actively growing axons. Activity may alter the pattern of neuronal inputs preferentially during periods of exuberant synaptogenesis (Brown et al., 1981).The availability of endoneurial pathways during the period of excessive synapse formation may contribute to the increased success of synaptogenesis from sprouting axons in neonatal animals. The effect of paralysis on axonal sprouting, synaptogenesis, and synapse elimination differ for different muscles (Brown et al., 1982). In some muscles, postnatal paralysis not only temporarily halts elimination of polyneuronal innervation, but can actually cause it to increase. Polyneuronal innervation can persist for long periods after paralysis (120 days in the mouse tensor fasciae latae muscle), not because of the critical period for synapse elimination, but rather because there is a substantial loss of muscle fibers after paralysis. This causes a high ratio of nerve to muscle fibers and results in increased levels of polyneuronal innervation (Brown et al., 1982; Sohal et al., 1979).

111. Mechanisms

We point out a number of cell biologic mechanisms involved in neuronal and synaptic survival or elimination. Electrical activity modulates many or all of these in various ways. Information is not adequate to identify any of these in specific developmental processes, but it is a major task of developmental neuroscience to obtain such information. A. TROPHIC FACTORS The paradigm provided by the discovery of nerve growth factor (Levi-Montalcini, 1987) has proven to be of general significance, with an increasing number of trophic materials becoming identified and cloned

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(Barde, 1989; Hohn et al., 1990; Yamamori et al., 1989; Nawa and Patterson, 1990). Important aspects of the effects of neural electrical activity on nervous system development may be mediated by trophic materials and involve actions of neurons on glia and reverse interactions from glia to neurons. It has become increasingly clear than glial cells express a number of characteristics that might be involved in such interactions. A full complement of voltage- and ligand-gated receptors and channels are found on central and peripheral glia and Schwann cells at various developmental stages, and these are regulated both during development and by various cell-cell interactions (Lerea and McCarthy, 1990; Verkhratsky et al., 1990). A1 and A2 as well as adrenergic receptors are expressed by primary astrocytes and muscarinic receptors on a glioma cell line are coupled to an early intracellular release of calcium (not to arachidonic acid release). A more sustained rise in intracellular calcium produced by muscarinic stimulation is verapamil inhibitable and is dependent on extracellular calcium ions (Brooks et al., 1989). It is due presumably to voltage-dependent calcium channels, is involved in phospholipase A2 activation, and is associated with release of arachidonic acid. Cultured astrocytes also exhibit depolarizing responses evoked by substance P (and related neurokinins) (Wienrich and Kettenman, 1989). Excitatory amino acid (EAA) receptors with a pharmacology distinct from that of neuronal EAA receptors, when activated by glutamate (or kainate o r quisqualate, but not NMDA), produce large depolarizations of astroglia and also large oscillatory changes in intracellular [Caz ] (Backus et a/., 1989; Smith, 1988; Cornell-Bell et al., 1990; Fatatis and Russell, 1991). T h e activation of glia cells by neuroactive agents can exhibit interesting conibinatorial properties. When perfused onto cortical astroglia in cultures, vasoactive intestinal peptide (VIP) produces an oscillatory increase in intracellular [Cay+]in a minority of cells and a-adrenergic agonists produce little or no response. When applied together, however, essentially all cells exhibit vigorous responses (J. Russell, personal communication). -1aken together, these results indicate that glial cells have a broad range of‘ capacities for responding to neuroactive compounds. But are these responses coupled to nerve activity? Two sets of observations indicate that they are. Intracellular [Ca2+] imaging techniques have shown recently that in hippocampal slices, chemical stimulation with NMDA elicits substantial alterations in glial calcium levels (Dani et al., 1990; Cornell-Bell et al., 1990).Presumably transmitter releases from neurons would be capable of eliciting glial responses. VIP is released from neurons in an activity-dependent fashion; cultures blocked by T T X d o not release detectable levels of VIP. This +

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VIP released by the electrically active neurons in turn causes the release of a trophic material from glia, activity-dependent neurotrophic factor (ADNF). This material is capable of preventing neuronal death in cultured preparations of spinal cord or hippocampus (Brenneman et al., 1987). It is of interest that a VIP antagonist produces developmental delays in neonatal rats (Hill et al., 1991). A double effect of neuronal activity with respect to neuronal survival was demonstrated in the experiments with VIP. In common with many experiments in viuo, cultured neurons exhibit a characteristic loss in numbers during the second to the fourth weeks in uitro. If electrical activity is blocked, this neuronal death is increased so that surviving neurons in the presence of T T X are only 50-60% of control cultures. If, however, VIP is added to TTX-treated cultures (or conditioned media from VIP treated cultures is used), the “normal” neuronal decrease is essentially eliminated and neuronal numbers stay constant through the “neuronal death” period (Brenneman and Nelson, 1987). Nerve growth factor (NGF) production in telencephalic neurons is increased by limbic seizures (Gall and Isackson, 1989). NGF messages were measured by in situ hybridization in these studies. Production of neurotrophic material may, thus, clearly be modulated by neuronal electrical activity, and so may the responses of neurons to trophic material. The cholinergic inducing factor for sympathetic ganglion (SCG) neurons is released by cardiac cells (and several other nonneuronal cell types) (Patterson and Chun, 1977; Fukada, 1985; Schotzinger and Landis, 1990). Under its influence the phenotype of SCG neurons is changed from catecholaminergic to cholinergic. This plasticity is continued into adult life and is a normal part of the phenotypic specification of neural crest cells innervating sweat glands. If SCG neurons are depolarized with high Ca2+ or stimulated electrically during the application of cholinergic inducing factor, however, the action of the factor is blocked (Walicke et al., 1977). Use of calcium channel blockers indicates that calcium ingress through such channels is responsible for the blockade of trophic action. Material from skeletal muscle cells can enhance cholinergic expression by spinal cord neurons (Giller et al., 1977; McManaman et al., 1988). This material is active both in vitro and in vivo (Appel et al., 1989), but in one of the few tests for activity-dependent elaboration of trophic factors, no change with muscle activity could be detected (Tanaka, 1987; Oppenheim, 1989). A number of substances released by the postsynaptic neuron in response to depolarization have been suggested as having possible presynaptic effects (Changeux and Danchin, 1976; Purves, 1986). Arachidonic acid (AA)has been proposed as such a substance in LTP (Williams

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et al., 1989; Lazarewicz et al., 1988). AA is released by joint activation of ionotrophic and metabotrophic quisqualate receptors (Dumuis et al., 1990) o r by a combination of NMDA and quisqualate activation (Dumuis et al., 1989). It has recently been suggested that nitric oxide mediates LTP (Williamset al., 1989; Garthwaite et al., 1989; Garthwaite, 1991) and refinement of axonal projections during the latter stages of development (Gally et al., 1990). Nitric oxide is produced by granule cells of the cerebellum in response to glutamate application (Garthwaite et al., 1988; Bredt and Snyder, 1989) and this effect is inhibited by blocking the NMDA receptor (Lazarewicz et al., 1988; Garthwaite et al., 1988, 1989). Nitric oxide synthetase is a calcium-dependent enzyme (Bredt and Snyder, 1989; Knowles et al., 1989) present in the vertebrate forebrain, which can be inhibited by N-methylarginine (Hibbs et al., 1989). Nitric oxide, being a nonpolar gas, can readily diffuse across cell membranes to initiate the production of cGMP (Murad et al., 1978) in presynaptic terminals near the release site. Nitric oxide diffuses isotropically and rapidly and is oxidized to nitrite and nitrate within a few seconds, thus limiting its effects to those presynaptic terminals closest to the active postsynaptic neuron (Gally et al., 1990).

B. CALCIUM-DEPENDENT MECHANISMS Calcium plays a central role in neuronal function; regulating exocytosis, facilitation, action potential shape, frequency and duration of electrical responses, movement of growth cones, LTP, ion channels, and transcription of immediate-early genes such as c-jos and c-jun (review by Kennedy, 1989). It has been suggested that increased intracellular calcium could trigger actin-myosin contractions in the spine neck (Fifkova, 1985; Crick, 1982), which would affect the strength of the synapse (Rall, 1978), or activate a protease that increases the number of glutamate receptors (Lynch and Baudry, 1984), or reduce K + currents (AcostaUrquidi et al., 1984). There are numerous pathways for calcium transport across cell membranes, including voltage and ligand-gated channels, pumps, and exchangers. Often the changes in intracellular calcium are confined to specific parts of the neuron. This would be necessary to specify plasticity to particular synapses (Gamble and Koch, 1987). Alterations in intracellular [Ca*+1 produced by electrical activity or neurotransmitter action have been implicated in the development of axonal and dendritic form. The dramatic responsiveness of intracellular [Ca*+], the large number of strongly interacting signals that influence it,

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and the morphological changes accompanying the changes in intracellular [Ca2+]have led to the proposal that these mechanisms represent a major means by which nervous system connections are specified (Lipton and Kater, 1989; Kater and Mills, 1991). Differential responsiveness of dendrites and axons has been demonstrated; different responses are produced by activation of different second messenger systems (Mattson et al., 1988). An extreme of the response to altered intracellular [Ca2+] is cell death, with the excitatory amino acids being notably capable of producing such pathological results (Choi, 1987). Calcium ions are involved in the death of sympathetic neurons produced by NGF deprivation, and, in this case, neuronal death can be prevented (delayed) by blockers of calcium ingress and protein synthesis or gene transcription (Koike et al., 1989; Martin et al., 1988). This implies that the cell death following NGF withdrawal requires initiation of a program actively producing neuronal loss. Calcium influx into the postsynaptic neuron is a critical step in LTP in the CA1 region of the hippocampus (Lynch et al., 1983; Malenka et al., 1988; Eccles, 1983), and calcium influx into the presynaptic terminal mediates transmitter release and posttetanic potentiation of transmitter release (review in Zucker, 1989). Activation of the NMDA receptor, a subclass of glutamate receptors, contributes to calcium entry into the postsynaptic neuron and is necessary for LTP (review in Collingridge and Singer, 1990); however, experimental application of NMDA to hippocampal slices elicits not LTP, but only a decremental synaptic enhancement, which has been referred to as short-term potentiation (Collingridge et al., 1983; Kauer et d., 1988). A long-term potentiation can be produced by NMDA application if it is combined with increased calcium and lowered magnesium (Thibault et al., 1989). It has been suggested that the amount of calcium influx through the NMDA receptor may govern the duration of synaptic enhancement (Malenka, 199 1). The biochemical targets of calcium in neurons are numerous and interactive. In the membrane, calcium regulates potassium, chloride, and voltage-sensitive calcium channels. Another important membrane target for calcium is the inositol phosphates. Increased intracellular [Ca* ] activates phospholipase C, which hydrolyzes phosphatidylinositol phosphates (PIPS)and phospholipase A,, which cleaves fatty acids, including arachidonic acid from the glycerolipid backbone. The products of both of these lipases are themselves second messengers. Arachidonic acid has been suggested as a retrograde signal mediating LTP. Major cytosolic target enzymes for calcium include protein kinase C, calpain (a calcium-dependent protease), and calmodulin. C kinases are +

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activated synergistically by calcium and diacylglycerol produced by hydrolysis of PIPS. In the activated state, cytosolic C kinases move to the membrane u here they can phosphorylate and regulate membrane proteins. C kinases regulate electrical excitability by phosphorylatiiig ion channels, and they can regulate synaptic efficacy. Evidence suggests that C; kinases are involved in initiation and maintenance of LTP. 1. N-hfethyl-D-aspartzcAczd Excitatory amino acids act as neurotransmitters in many CNS pathways (reviewed by Mayer and Westbrook, 1987). One subtype of EAA receptors, the NMDA channel, is of particular interest in activity-dependent synaptic plasticity, because NMDA channels require two conditions for activation: binding to the appropriate excitatory amino acid neurotransmitter (e.g., glutamate), and concomitant depolarization ofthe postsynaptic membrane (Nowak et al., 1984; Mayer and Westbrook, 1987). This conditional state is compatible with the Hebbian hypothesis that increased svnaptic strength should occur when synaptic firing is coincident with an adequate level of postsynaptic response (cf. section 11,C).If enough glutamate is released by multiple afferents simultaneously, the postsynaptic neuron will be depolarized to the level where the glutamate actikates NMDA channels in addition to the non-NMDA channels. An inappropriate input, that is, a weak input or one not firing in phase with other active afferents converging on the same neuron, would not depolarize the postsynaptic membrane adequately to induce sufficient NMDA channel activation, and would therefore not become strengthened. Activation of NMDA channels produces an influx of calcium through the activated NMDA channel (Dingledine, 1983; MacDermott P t al., 1986; Bear et a!., 1987; Mayer and Westbrook, 1987; Mayer et al., 1987; Ascher and Nowak, 1988), and this may be a critical step linking neuronal activity to a persistent increase in synaptic efficacy. u . Long-Term Potentiution and Svnupw Stabzlzzutzon. Through the use of NMDA channel antagonists, which block the induction of LTP following repetitive stimulation, NMDA channel activation has been implicated in the initiation phase of LTP in the CAI region of the hippocampus (Collingridge et al., 1983; Wigstrom and Gustafsson, 1985; Errington et al., 1987; Harris et af.,1984; Morris et al., 1986; Collingridge and Bliss, 1987; Larson and Lynch, 1986; Muller et al., 1988; Stringer and Guyenet, 1983). (For reviews see Brown et al., 1988b; Nicoll et al., 1988; Kuba and Kumamota, 1990; Madison et al., 1991; Lynch et al., 1990.) It is possible to induce LTP in other brain regions by repetitive stimulation, including the kitten and rat visual cortex (Komatsu et al., 1988, 1991; Perkins and Teyler, 1988; Artola and Singer, 1987; Berry et al., 1989;

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Tsumoto and Suda, 1979), somatosensory or motor cortex (Baranyi and Feher, 1981b; Baranyi and Szente, 1987; Bindman et al., 1988; Sakamot0 et al., 1987; Bindman and Murphy, 1990; Rasmusson and Dykes, 1988; Brons and Woody, 1980), associative cortex (Lee, 1982), olfactory pathways of the pyriform and entorhinal cortex (Jung et al., 1990); vestibular nucleus (Racine et al., 1986), medial geniculate nucleus (Geren and Weinberger, 1983), superior coliculus (Okada and Miyamoto, 1989), sympathetic ganglia (Dunant and Dolivo, 1968), and the optic tectum of goldfish (Schmidt, 1990). As in the CA1 region of the hippocampus, blockade of the NMDA channel using the antagonist APV blocks LTP induction in the adult rat visual cortex (Artola and Singer, 1987), the goldfish optic tectum (Schmidt, 1990), and the rat piriform cortex (Lung et al., 1990), but not in kitten visual cortex (Komatsu et al., 1991). NMDA channel activation has also been implicated in developmental processes of synaptic restructuring by demonstrating impaired activitydependent synaptic segregation of axon terminals in the visual system under conditions of NMDA receptor blockade. Antagonists of the NMDA channel block the consolidation of ocular dominance changes in kitten visual cortex (Rauschecker and Hahn, 1987), prevent the formation of eye-specific patches in the optic tectum of frogs receiving a third eye graft (Cline et al., 1987; Cline and Constantine-Paton, 1989; Debski et al., 1990), prevent the shift of cortical visual neurons to the open eye following monocular deprivation in kittens (Kleinschmidt et al., 1987; Gu et al., 1989), prevent the sharpening of retinotectal maps from regenerating afferents to goldfish optic tectum (Schmidt, 1990), prevent recovery of binocular registration of afferents from the two eyes onto the optic tectum following chronic rotation of one of the eyes in Xenopus (Scherer and Udin, 1989), and block the plasticity of DRG to spinal cord neurons in the multicompartment culture model of activity-dependent synaptic competition (Nelson et al., 1990; Fields et al., 1991a). It should be noted that in these latter experiments, blockade by APV was reversed in high-calcium solutions (see below). (For reviews on NMDA channels and activity-dependent remodeling of visual pathways during development see Constantine-Paton et al., 1990; Shatz, 1991; Schmidt and Tieman, 1989.) Despite this substantial list of experiments in which NMDA blockade inhibits activity-induced plasticity in developing systems, it remains an open question whether the mechanism of LTP in the CA1 region of adult hippocampus represents an appropriate model of activity-dependent synaptic plasticity during development. This uncertainty derives from two genera1 concerns: (1) LTP and developmental plasticity are dissimilar phenomena, and (2) postsynaptic receptor blockade will

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disrupt electrical activity. This question is examined more fully later (cf. Section IV,B, 1). LTP depends on different biochemical mechanisms and stimulation patterns in different neurons or at different developmental stages. Within the hippocampus LTP is initiated by NMDA-dependent and NMDAindependent processes (reviews by Brown et al., 1988b; Nicoll et al., 1988). NMDA receptors are necessary for induction of LTP for the commissural/associational inputs to the pyramidal neurons of the CA3 region (Harris and Cotman, 1986) but mossy fiber synapses on pyramidal neurons of area CA3 exhibit a non-NMDA-mediated form of LTP (Harris and Cotman, 1986; Kauer and Nicoll, 1989). Also, these synapses do not exhibit homosynaptic LTD (Chattarji et al., 1989). The stimulation pattern and molecular mechanism for LTP are different in hippocampal archicortex and adult neocortex compared with kitten visual cortex (Artola and Singer, 1987; Berry et al., 1989; Bindman et al., 1988; Lee, 1982; Sutor and Hablitz, 1989a,b; Komatsu et al., 1991). In kitten visual cortex, LTP is induced by relatively low-frequency stimulation (2 Hz), whereas in adult neocortex and hippocampus high-frequency stimulation (100-400 Hz) is required. In kitten visual cortex, LTP can be induced in the presence of NMDA channel blockade with APV (Komatsu et al., 1991), but LTP in the adult neocortex is blocked by APV (Artola and Singer, 1987; Sutor and Hablitz, 198913). The disruption of synaptic plasticity following administration of NMDA channel antagonists during development is difficult to interpret, because of the reduction of electrical activity caused by the pharmacological intervention. T h e suppression of activity need not be so severe as to block sodium-dependent action potentials, as many factors affecting the excitability or membrane potential of the postsynaptic neuron can cause major disruption of normal synaptic plasticity during development (cf. Section II,C, 1). By blockage of the prolonged NMDA component of the EPSP, electrical activity in the network, which is dependent on the summation of multiple EPSPs, is suppressed. This would reduce the postsynaptic depolarization resulting from visual input, for example, though it would not completely block responses to afferent stimulation because of the remaining non-NMDA component of the EPSP. In addition, eliminating the NMDA component of the EPSP would suppress the activity of other glutamatergic inputs that contribute to postsynaptic activity. It is well known that suppressing postsynaptic activity will impair activity-dependent synaptic plasticity in visual cortical neurons and muscle fibers. This effect was recently tested in an in vztro model of activitydependent synaptic competition between DRG neurons and ventral

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spinal cord neurons dissociated from fetal mice and plated into a multicompartmented chamber (Fig. 5 ) (Fields et al., 1991a). In this study, stimulation of one of a pair of DRG afferents to the spinal cord neurons for 35 days resulted in an increase in EPSP amplitude relative to the unstimulated side. Incubation during the stimulus period in 100 p,M APV, an antagonist of the NMDA channel, blocked this plasticity; however, application of dilute TTX (5 nM), at a concentration that significantly suppressed activity in the polysynaptic network of spinal cord neurons but left the DRG afferents free to respond to electrical stimulation, also blocked the plasticity (Figs. 6a,b). This supports the interpretation that the inhibitory effects of APV in developmental plasticity result from suppressed electrical input to the postsynaptic neuron, rather than from elimination of a specific receptor required in the mechanism of plasticity. Direct evidence for this effect of APV is obtained by recordings of spontaneous activity during application of the drug to cultures of spinal cord neurons. Spontaneous activity was suppressed significantly on perfusion of APV into spinal cord cultures (Figs. 6c-e), and this effect persisted more than 24 hours (Brenneman et al., 1990a,b; Fields et al., 1991a). That activation of NMDA receptors is not essential for this activitydependent synaptic plasticity is indicated by the reversal of the APV plasticity blockade by high-calcium solutions (3 mM Ca2+ ) (Nelson et al., 1990; Fields et al., 1991a). This would seem to put changes in intracellular calcium “downstream” from NMDA receptor activation in the causal chain between neural activity and synaptic plasticity. NMDA receptor activation is an indication of intense (correlated) input and an efficient means of increasing intracellular [Ca2+], but activation of NMDA channels is not necessary for this plasticity. 6. LongTerm Depression and Synapse Elimination. Presynaptic activity delivered out of phase with more active convergent inputs, or during periods when the postsynaptic membrane is not depolarized sufficiently, may cause a reduction in EPSP amplitude (Stanton and Sejnowski, 1989; Artola et al., 1990; Abraham and Goddard, 1983). The mechanism for this decreased synaptic strength does not appear to depend on NMDA channel activation (Stanton and Sejnowski, 1989; recently reviewed by Sejnowski et al., 1991; Singer and Artola, 1991). Another glutamate analog, 2-amino-3-phosphonopropionicacid (AP3), blocks LTD without affecting LTP (P. K. Stanton and T. J. Sejnowski, unpublished observations). AP3 is an antagonist of a metabotrophic glutamate receptor that stimulates PI hydrolysis in neurons (Sladeczek et al., 1988; Schoepp and Johnson, 1989). This independence between NMDA-dependent LTP and

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non-NMDA-dependent L‘TD suggests complementary mechanisms that would link separate second messenger systems with different levels of postsynaptic membrane potential. Synaptic activity during periods when the postsynaptic membrane is hyperpolarized should, according to theor y (Stent, 1973),initiate processes that decrease the strength of the synaptic connection. Thus, synaptic firing under conditions where NMDA channels are inactive would signal “inappropriate activity.” In terms of receptor mechanisms, this would mean that activation of non-NMDA receptors alone, that is, without concomitant activation of the NMDA receptors, would signal biochemical pathways that decrease synaptic strength. This could be effected via different second messenger systems activated by NMDA and non-NMDA receptors. Whereas activation of NMDA channels leads to an influx of calcium, activation of non-NMDA channels in the rat hippocampus (Nicoletti el al., 1986)and neocortex (Dudek et al., 1989) generates the second messengers inositol triphosphate (IP3) and diacyl glycerol (DAG) from the hydrolysis of phosphatidylinositol4,5-bisphosphate(Berridge, 1984;Berridge and Irvine, 1984, 1989). By this hypothesis, increased intracellular Ca2 would promote an increase in synaptic strength, whereas increased IP3 and DAG would have the opposite effect (review by Bear and Cooper, 1990). The relevance of LTP and LTD to developmental restructuring of synapses is uncertain, but one interpretation of the paradoxical result from perfusing muscimole onto visual cortex is that presynaptic transmitter release onto hyperpolarized cells leads to LTD of the active synapses arising from the lateral geniculate nucleus (Reiter and Stryker, 1988).Thus, just like LTD in the hippocampus, active synapses on hyperpolarized neurons near the site of the y-aminobutyric acid agonist infusion in the visual cortex would become weakened, leaving only synapses from the previously closed eye to drive the neuron. This would produce an ocular dominance shift in favor of the less active convergent input (cf. Section 11,C). This “dual-receptor hypothesis” has not been tested directly, but observations consistent with this idea have been cited in support of the hypothesis. T h e turnover of phosphoinositide in the kitten visual cortex, studied by activating non-NMDA receptors with ibotenate, correlates with the critical period for susceptibility to visual deprivation. Dark rearing, which delays the critical period, also prevents the rise in IP3 turnover that normally peaks at 5 weeks postnatal. I n the hippocampus, long-term depression occurs in the presence of NMDA receptor blockade (Chattarji et al., 1989).Also in the neotatal hippocampus, NMDA has been found to inhibit excitatory amino acid-stimulated PI turnover in a +

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Ca2 -dependent fashion (Palmer et al., 1988), suggesting that the NMDA and non-NMDA systems could have an antagonistic interaction. Although LTP and LTD are initiated by different mechanisms (NMDA dependent and NMDA independent), there may be a common intracellular component, such as the level of intracellular calcium. LTD can be induced in neocortical neurons in slices that have been injected with ethylene glycol bis(P-aminoethyl ether) N,N'-tetraacetic acid (EGTA) (Kimura et al., 1990).A moderate, rather than maximal, elevation of calcium could be necessary for LTD (Lisman, 1989).This may be compatible with the narrow range of postsynaptic depolarization that initiates LTD when paired with presynaptic activation (Sejnowski et al., 1991). I n contrast to the Hebbian plasticity in the hippocampus, coincident activation of parallel fibers and climbing fibers causes LTD in Purkinje cells of the cerebellum (Ito, 1989;Ito and Karachot, 1990).This LTD is related to desensitization of the AMPA-specific glutamate receptors in the postsynaptic membrane, which is produced by the association of u-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor activation and intracellular calcium elevation. The amount of synaptic depression is much greater in the cerebellum than in the hippocampus, which raises the possibility that LTD in the hippocampus is effective only at reducing EPSPs of previously potentiated synapses. +

2. Calmodulin

Calmodulin is a 15-kDa, calcium-binding regulatory protein present in brain cytosol at a concentration of 30-50 pM, which is common in many cell types. Each molecule has four calcium binding sites. At resting calcium concentrations, very little calcium is bound, but as the concentration rises to the micromolar level, the four binding sites are successively occupied (review by Rasmussen and Means, 1989). The functional activity of several specific proteins is altered on binding with calmodulin. The affinity of calmodulin binding to these proteins is itself regulated by calcium, thus conferring a calcium-dependent regulation of protein activity. Calcium/calmodulin-dependent protein kinases are found widely in many different tissues (Shenolikar et al., 1986;Fukunaga et al. 1988).The principle calcium/calmodulin-dependent protein kinase in mammalian brain is the enzyme calcium/calmodulin kinase type I1 (CaM-KII) (Burgin et al., 1990).Immunochemical analysis indicates that CaM-KII is more highly expressed in neurons than glia (Ouimet et al., 1984;Scholz et al., 1988).It is present in the cell body, dendrites, and axons, but it is particularly enriched in

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synaptic fractions (Kelly et al., 1985; Suzuki and Tanaka, 1986). Indeed, it is the most abundant protein in isolated postsynaptic densities (Kelly et al., 1984). Its abundance at the subsynaptic membrane suggests that it may be important to signal transduction (Miller and Kennedy, 1986) and memory storage (Lisman and Goldring, 1988). CaM-KII is a multimeric holoenzyme composed of nonstoichionietric amounts of two principle subunits, a (50KDa) and p (58160 KDa). The a and p subunits undergo autophosphorylation following activation by calcium/calmodulin (Bennett et al., 1983). The p subunit increases in forebrain during postnatal development, doubling in synaptic junctions between 5 and 25 days. The a levels increase 10-fold in the same period (Kelly and Vernon, 1985). Thus, the a : p subunit ratio of the holoenzyme changes from 1:7 at Day 5 to 3:l in the adult (Kelly et al., 1987), and its location in the cell shifts from predominantly cytosolic to become tightly associated with neuronal membranes and synaptic junctions (Kelly and Vernon, 1985). CaM-KII mRNAs parallel the distribution and development appearance of the protein subunits (Burgin et al., 1990). Of particular interest is the localization of the a-subunit mRNA in hippocampal dendritic spines, where polyribosomes are also localized. The co-localization of translational machinery, polyribosomes, and high concentrations of CaM-KII in postsynaptic elements suggests an important relationship between a-subunit synthesis and the maintenance and plasticity of postsynaptic structures. The link between plasticity and CaM-KII has been demonstrated in studies of LTP in the hippocampus. The phosphorylation of a 40-kDa protein during LTP is most susceptible to modulation by calmodulin and its inhibitors (Finn et al., 1980). Induction of hippocampal LTP is correlated with a translocation of calmodulin from cytosolic compartments to the membrane (Popov et al., 1988). Tetanization and application of calmodulin enhance K -induced glutamate release from hippocampal slices (Lynch and Bliss, 1986). Finally, LTP induction can be disrupted by calcium/calmodulin inhibitors (Dunwiddie et al., 1982; Finn et al., 1980; Moody et al., 1984; cf. Akers et al., 1986; Frey et al., 1988; Krug et al., 1984). An attractive feature of calcium/calmodulin kinase in synaptic plasticity is a switchlike regulation of the calcium/calmodulin kinase activity by autophosphorylation (Lisman, 1989; Lisman and Goldring, 1988), but calcium/calmodulin kinases also have presynaptic actions, mediating one type of facilitation by phosphorylating synapsin I (Sudhof et al., 1989; Bains, 1987). Another protein activated by calcium/calmodulin kinase is calcineurin, which is a phosphatase implicated in calcium-dependent inac+

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tivation of Ltype voltage-sensitive calcium channels in neurons (Chad and Eckert, 1986; Armstrong, 1989). Phosphorylation of L channels by CAMP-dependent protein kinase enhances their activation by depolarization. Conversely, dephosphorylation by an endogenous calciumdependent phosphatase, thought to be calcineurin, desensitizes the channel. Two additional targets of calmodulin are isozymes of adenylate cyclase and cyclic nucleotide phosphodiesterase, the enzymes that are involved in the production and degradation, respectively, of CAMP. A rise in intracellular [Ca2+]may enhance or antagonize production of CAMP, depending on the nature of the local cyclases and phosphodiesterases. Calcium/calmodulin also regulates the activity of the membrane calcium-dependent ATPase (Carafoli et al., 1988) and sodium/calcium exchanger (Caroni and Carafoli, 1983), providing a feedback control for increasing intracellular [Ca2 1. +

3. Protein Kinase C Protein phosphorylation is a major mechanism for posttranslational control of protein activities. Among these are proteins that maintain the integrity of the cytoskeleton (Aletta et al., 1990), mediate transmitter release (Bahler and Greengard, 1987), control conductance of ion channels and membrane receptors (see, e.g., Armstrong and Eckert, 1987; Kaczmarek, 1986; Hess, 1990; Fisher and Kaczmarek, 1990), and participate in other biochemical pathways involved in axonal outgrowth, synaptogenesis, and synaptic efficacy (Routtenberg, 1986). Phosphorylation reactions are coupled to extracellular signals through a limited number of second messengers, including CAMP,cGMP, Ca2+, products of phosphoinositide (IP3 and DAG), arachidonic acid, and nitric oxide. The synthesis and degradation of each of these second messengers is highly regulated, in a substantially interactive manner. For reviews, see (CAMP) Dudai (1987); (IP3) Downes (1988);Berridgeand Irvine (1984,1989),and Fink and Kaczmarek (1988); and (protein kinase C) Berridge (1986a,b) and Miller and Kennedy (1986) and Fisher and Kaczmarek (1990). Protein kinase C (PKC) is a Ca2+-activated, phospholipid-dependent protein kinase found in a variety of tissues (Kuo et al., 1980; Huang et al., 1986; Nishizuka, 1984a, 1988; Gispen and Routtenberg, 1986; Kikkawa et al., 1986). PKC is activated through receptor-mediated hydrolysis of phosphoinositol, serving as an important signal transduction link to many intercellular responses (Nishizuka, 1984a). PKC regulates synthesis and release of neurotransmitters (Wang et al., 1986; Zurgil et al.,

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1986) and the conductance of ion channels (Baraban et al., 1985; DeRiemer et al., 1985; Malenka et al., 1986a; Nahorski, 1988; Fisher and Agranoff, 1987; Barani et al., 1988; Conn et al. 1989; Conn and Kaczmarek, 1989 ), and participates in LTP in the hippocampus (Kouttenberg, 1986; Malenka et al., l986a; Hu et al., 1987; Colley et al. 1990). During development, PKC activation participates in neuronal growth, differentiation, and synaptogenesis (Hsu et al., 1984; Kozengurt et al., 1984; Routtenberg, 1985). PKC activity is closely related to intracellular calcium: PKC is activated by calcium ions (Wolf et al., 1985) and voltagesensitive calcium channels are themselves substrates for PKC (DeRiemer at al., 1985). Protein kinase C has been implicated in increased synaptic strength in models of associative learning in rabbits and invertebrates. (For reviews see Olds and Alkon, 1991; Alkon and Nelson, 1990; Bank et al. 1989.) In invertebrate neurons, PKC activation can cause increased calcium current (DeRiemer et al., 1985) and reduced outward potassium current (Alkon et al., 1986). In mammals, PKC activation causes a reduction in the after hyperpolarization (AHP) in hippocampal CA1 cells (Baraban et al., 1985; Malenka et nl., 1986b), which enhances summation of postsynaptic potentials (LoTurco et ul., 1988).This effect can be mimicked by application of phorbol esters that activate calcium/phospholipid dependent PKC (LoTurco et al., 1988; Baraban et al., 1985; Malenka et al., l986b). Calciumiphospholipid-dependent PKC has been shown to be involved in mechanisms of LTP (Akers et al., 1986; Andersen et al., 1988; Hu et al., 1987; Lovinger et al., 1987; Malenka et al., 1986b; Reymann et uZ.,1988a,b; Colley et al., 1990). The initiation stage of LTP is independent of PKC activation, but suppression of PKC activity with inhibitors such as polymyxin B, trifluoperazine, and K252b (Kase et al., 1987; Reymann at al., 1988a,b,c) block intermediate and late stages of LTP. Only the late stage is susceptible to protein synthesis inhibitor anisomycine (Krug et al., 1984; Frey et al., 1988). There is evidence for involvement of PKC in both presynaptic (Lynch and Bliss, 1986; Malenka et al., 1986b) and postsynaptic processes involved in maintaining LTP (Andersen et al., 1988; Hu et al., 1987; Keymann et al., 1988a,b,c). Activation of PKC occurs when DAG is generated by signal-dependent breakdown of inositol phospholipids by phospholipase C (Berridge, 1984), and this involves a transfer of the enzyme to the plasma membrane where it can be fully activated in the presence of low calcium concentrations (Kishimoto et al., 1980). A translocation of PKC from cytosolic to membrane pools has been shown 1 hr after LTP (Akers et al., 1986) and in CA 1 cells isolated from the hippocampus of rabbit 24 hr

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after eyelid response conditioning (Bank et al., 1988; Olds et al., 1989). These neurons are known to undergo biophysical alterations as a result of this conditioning. A more recent study detected increased membraneassociated PKC in the CA3 hippocampal region of rabbit during acquisition of the eyelid conditioned response, but failed to find a significant difference in the CA1 region (Sharenberg et al., 1991). PKC appears to participate in cellular mechanisms of use-dependent plasticity during development. Immunocytochemical staining shows an abrupt appearance of PKC in neurons of the kitten visual cortex at 4 weeks of age and a decrease in number of PKC-positive neurons after 12 months of age (Stichel and Singer, 1989). This time course of staining and the laminar selectivity correlate with the timing and location of experience-dependent malleability in visual cortex. Cystolic protein kinase C activity in the visual cortex of kittens increases 10-fold during the critical period, and then reduces 5-fold to adult levels. Membrane PKC activity shows a similar developmental profile, but with a maximal increase of only 2-fold (Sheu et al., 1990). However, blockade of PKC does not lead to disruption of ocular dominance columns in the optic tectum of frogs (Cline and Constantine-Paton, 1990). 4. Znositol Triphosphate

IP3 is an important second messenger mediating the release of intracellular Ca2 from nonmitochondrial stores (Berridge, 1984, 1986a,b; Berridge and Irvine, 1989; Eichberg and Berti-Mattera, 1986; Choi et al., 1990; Nishizuka, 1984b).Extreme oscillations in intracellular calcium have been linked to fluctuations in the concentration of IP3 and the content of IP3-sensitive calcium stores (Harootunian et al., 1991a,b). Calcium oscillations in astroglia are induced by glutamate (but not NMDA) (Smith 1988; Cornell-Bell et al., 1990), or by the combined action of VIP and cx-adrenergic agonist (Fatatis and Russell, 1991). In fibroblasts, simultaneous depolarization and stimulation with a hormone linked to phosphoinositide breakdown, or stimulation of G-proteins, or membrance depolarization and activation of voltage-sensitive calcium channels, induces oscillations in free calcium concentration (Harootunian, 1991a,b). This interaction between multiple forms of cell stimulation and intracellular calcium concentration suggests that IP3 could be a pivotal component in the mechanism of activity-dependent synaptic plasticity. Increased hydrolysis of phosphatidylinositol4,5,-biphosphate by excitatory amino acid stimulation (ibotenate and glutamate) correlates with the critical period in kitten visual cortex, which would increase production of DAG and activate PKC. Measurements show PKC activity is +

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elevated in kitten visual cortex during the critical period (Stichel and Singer, 1989). In the hippocampus, increased hydrolysis of phosphatidylinositol 4,5-bisphosphate has been measured in postsynaptic CA3 neurons 45 min after tetanization (Lynch et al., 1988). LTP in the CA3 elicited in zjizw produces an increase in carbachol- and noradrenaline-induced hydrolysis of phosphatidylinositol4,5-biphosphate(Stelzer et ad., 1989). Kindling decreased the amount of hydrolysis, but NMDA receptor activation does not directly alter the hydrolysis of phosphatidylinositol 4,5-biphosphate in either LTP or kindling. Cytoskeletal proteins regulated by calcium could also be affected by IP3 turnover. Forscher (1989) proposes that because of antagonism between gelsolin and profilin (Lind et al., 1987) for PIPS (Yin, 1988; Yin et al., 1988), calcium transients that are not accompanied by significant PPI turnover would result in severing of actin networks. Calcium transients accompanied with PPI turnover would lead to actin remodeling. Elimination of synapses might occur when activity invokes sufficient intracellular [Ca2+ ] levels at a synapse without concomitant PPI turnover. The same amount of calcium rise with PPI turnover would lead to mobilization of release of synaptic vesicles and expansion of the synaptic region or sprouting.

5 . Proteases and Protease Inhibitors Calcium-activated proteases have been suggested as mediating changes in neuronal form and function. Synapse elimination at the neuromuscular junction can be significantly delayed by low calcium or neutral protease inhibitors such as leupeptin (Connold et al., 1986; O'Brien et al., 1984). The ability of neuronal growth cones to progress toward their target cells has been related to the balance between different types of proteases and the inhibitors of those enzymes that are present in the growth cone environment (Moonen et al., 1982; Krystosek and Seeds, 1981; Hawkins and Seeds, 1986). Thus, regulation of proteolytic activities has been assigned a key role in a number of important neurobiologic processes. T h e calpains'are neutral proteases that are activated by calcium (Perlmutter et al., 1990). They are present in both membrane and cytosolic forms, and they are implicated in the regulation of membrane proteins and the cytoskeleton (Siman et al., 1987; Muller et al., 1989). Development of hippocampal LTP can be reduced by calpain inhibitors (delCerro et al., 1990; Oliver et al., 1989), and neural cell adhesion molecules are modified by intracellular calpain (Sheppard et al., 1991).

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6. Regulation of Intracellular Calcium T h e concentration of intracellular free calcium reflects the balance between the rate of calcium influx across the plasma membrane and calcium release from internal stores and the rate of calcium removal by sequestration into internal stores, such as mitochondria and specialized organelles, extrusion across the plasma membrane by ATPase-driven membrane calcium pumps (Blaustein and Hodgkin, 1969; Carafoli et al., 1988), and sodium/calcium membrane exchange (Baker, 1986; Caroni and Carafoli, 1983; Rasgado-Flores et al., 1989). Activity-dependent effects on any of these could alter the level of intracellular calcium and affect synaptic strength during development. Monocular deprivation leads to changes in synaptic strength in favor of the active eye, but binocular deprivation initiates no change in synaptic strength (cf. Section 11,B). This has lead to the suggestion that the threshold of activation required to induce a change in synaptic strength (the modification threshold) decreases with decreasing postsynaptic activity (Bear et al., 1987; Bear, 1988). This modification threshold might be related to changes in calcium permeability of the NMDA channel, so that less calcium would enter the cell when the postsynaptic membrane was inactive and therefore fail to initiate changes in synaptic strength. To test this hypothesis, the flux of 45Ca2 into slices of kitten visual cortex was measured in nitro following normal and abnormal visual experience (Sherin et al., 1988; Feldman et al., 1990). The amount of calcium influx caused by NMDA application was measured from visual cortical slices, under conditions of low postsynaptic activity induced by 4 days of binocular visual deprivation, and compared with that of cortex of kittens with normal visual activity. The maximum calcium permeability of the NMDA channel was decreased with decreasing postsynaptic activity, and flux of calcium into the slice was lower in cortical slices from visually deprived kittens compared with normal kittens (cf. Bear and Cooper, 1990); however, it appeared that the sensitivity of the receptors from deprived animals was somewhat increased. If synaptic plasticity is regulated by changes in intracellular [Ca2+], any of the processes that maintain calcium homeostasis could moderate synaptic plasticity. If calcium removal activity were directly proportional to postsynaptic activity, more calcium influx would be required to initiate plasticity in more active postsynaptic neurons. A direct relationship between calcium removal activity and modification threshold might be consistent with the plasticity initiated by monocular deprivation and the lack of plasticity in binocular deprivation. An important calcium removal mechanism is the sodium/calcium +

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exchanger in the plasma membrane (Nicoll et al, 1990; Baker, 1986; Baker et al., 1969; Baker and Blaustein, 1968; Baker and McNaughton, 1976; Caroni and Carafoli, 1983). Under normal conditions, the exchanger removes calcium from the cytosol in exchange for extracellular sodium (which is ultimately extruded by the sodium/potassium ATPase). T h e sodium/calcium exchanger can move calcium ions in either direction, however, thus linking sodium and calcium homeostasis (Baker and McNaughton, 1976). Indeed, the cardiac glycosides that inhibit sodium pump activity exert their effect by altering the sodium gradient, which in turn affects the calcium exchanger. T h e sodium/calcium exchanger has been described in invertebrate nerve (Baker and Blaustein, 1968; Baker et ul., 1967a,b; Rasgado-Flores et al., 1989), cardiac muscle (Reuter and Seitz, 1968; Glitsch et al., 1970), other excitable cells and epithelia (Baker, 1972; Blaustein, 1974; Blaustein and Nelson, 1982), mitochondria (Compton et al., 1977; Nicholls and Ackerman, 1982), and secretory vesicles (Saermark et al., 1983). Actions of the sodium/calcium exchanger are difficult to test at present because of the lack of a specific antagonist. Pawson and Grinnell ( 1990) recently investigated reasons for differences in synaptic strength among frog neuromuscular junctions. Although ultrastructural correlates with synaptic strengths have been documented (Herrera et al., 1985a,b; Banner and Herrera, 1986; Propst and KO, 1987), they are small compared with the differences in release properties and large nonuniformities in release efficacy along the length of terminal branches (D’Alonzo and Grinnell, 1985). From studying posttetanic potentiation (PTP)at the frog neuromuscular junction they conclude that differences in synaptic efficacy are likely to reflect differences in numbers of calcium channels per unit length or in the functional state of the calcium channels, and not to differences in calcium homeostasis or release of calcium from internal stores. They conclude that although the sodium calcium exchanger may play an important role in ultimately determining the time course o f PTP (see Magleby and Zengel, 1976), the exchanger appears not to function qualitatively differently among junctions of differing synaptic strength (Grinnell and Pawson, 1989). At the frog neuromuscular junction, stronger junctions appear to contain calcium channels at a greater density or at an increased functional state, as occurs in cardiac muscle (Bean et af., 1984) or snail neurons (Doroshenko et al., 1984; Armstrong and Eckert, 1987).

c. N E U R A L ACTIVATION OF REGULATORY GENES I t is axiomatic that long-term alterations in nervous system function involve mechanisms separate from those that produce short-term re-

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sponses to neuronal activity. Early evidence showed the necessity of protein synthesis in consolidation of memory. In recent years, the molecular biology of oncogenes has contributed greatly to knowledge of how cellular stimuli of many different types are coupled to gene transcription to produce long-term phenotypic changes in cells. I n addition to modulation of intracellular Ca2+ and a number of second messenger systems, electrical activity has been shown to produce large, prompt changes in a number of regulatory genes. fos and jun are two such genes (Morgan and Curran, 1989a,b). Seizure activity in the hippocampus results in an increase in the Fos and Jun proteins within minutes, and these two proteins form heterodymeric duplexes that associate with a nucleotide sequence know as the AP- 1 site, presumed to be a regulatory site and known to occur in the regulatory region upstream from the coding region of the proenkephalin gene (Sonnenberg et al., 1989a,b). In transactivation assays, c-fos and cjun can be shown to synergistically activate this control region. An increasing number of these regulatory genes are becoming identified, and cascades of activation following seizure or other electrical activation in hippocampus and other brain structures result in serial transcription of a series of these genes over a period extending from seconds to several hours. Stimulation with kainate o r NMDA can elicit similar responses (Sonnenberg et al., 1989a,b); these various methods of activation may have as a common feature elevation of intracellular [Ca2 1. The regulation of acetylcholine receptor synthesis by activity in skeletal muscle may involve similar transacting factors, in this case, myogenin and MyoD, alteration of which by denervation, reinnervation, and stimulation precedes the changes in acetylcholine receptor (AChR) synthesis (Eftimie et al., 1991). Binding sites for the MyoD (Weintraub et al., 1991) and myogenin gene products have been identified in the AChR gene regulatory region. T h e role of immediate early genes (IE) in LTP is uncertain. Activation of IE genes is correlated with high frequency stimulation (Cole et al., 1989; Wisden et al., 1990), but IE gene activation is not well correlated (Douglas et al., 1988; Dragunow et al., 1989; Wisden et al., 1990) or is negatively correlated (Schreiber et al., 1991) with induction of LTP in the hippocampus. +

D. STRUCTURAL CHANGES

Despite considerable interest in this question, morphological correlates of activity-dependent changes in synaptic strength in the mammalian central nervous system (CNS) are not well understood (Greenough and Bailey 1988). For recent reviews see Calverley and

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Jones (1990), Markus and Petit (1989), Greenough and Chang (1985), Schmidt and Tieman (1989), and Desmond and Levy (1990). Correlations of differences in size, shape, structure, and number of synapses in the mammalian CNS after different stimulation or deprivation treatments are not easily generalized across different regions of the CNS. A fundamental question is whether structural differences, such as size, postsynaptic density (PSD), length, shape of the synaptic apposition, number, size and location of synaptic vesicles and mitochondria, are mechanistically related to differences in synaptic efficacy. It is difficult to determine whether differences in synaptic structure are reactive (resulting from excessive stimulation, ion flux, ischemia, or various other fatigue processes) or adaptive (structural alterations that cause synaptic strength to be altered in a functionally appropriate manner). For ultrastructural changes following tetanic stimulation in the hippocampus see Van Harreveld and Fifkova (1975), Fifkova and Van Harreveld (1977), Lee et al. (1980), Fifkova and Anderson (1981), Desmond and Levy (1983, 1986, 1988), and Chang and Greenough (1984). Changes have also been described in dendritic morphology (Tieman and Hirsch, 1982) and synaptic morphology (Tieman, 1984; Nixdorf, 1990) of cortical neurons following visual deprivation. Changes in dendritic spine morphology that reduce the neck resistance could produce increased current flow into the dendrite and produce LTP (Brown et al., 1988a; Coss and Perkel, 1985; Koch and Poggio, 1983; Rall, 1978; Rall and Rinzel, 1973; Rinzel and Rall, 1974; Wilson, 1984; Larson and Lynch, 1991). However, the NMDA component of the EPSP is not potentiated during LTP, while the non-NMDA component is. Changes in spine conductance should affect current through either channel similarly, which would argue against changes in spine resistance as the mechanism for LTP. The different responses of NMDA and non-NMDA currents in LTP cannot be explained by differences in time course of depolarization for the two currents, because slowing the non-NMDA component of the EPSP to resemble the NMDA response, by lowering the temperature, does not prevent LTP (Larson and Lynch, 1991). Other morphological alterations in the receptors or formation of new synapses could produce an increase in synaptic efficacy. In simpler systems, such as sensory ganglia of invertebrates (Bailey and Chen, 1988a,b), peripheral sensory organs of lower vertebrates (Fields and Ellisman, 1984), and the vertebrate neuromuscular junction (Heuser and Reese, 1973), correlations between synaptic structure and function are more firmly established. It is widely believed that changes similar to those in simpler systems underlie plasticity in the vertebrate CNS, but the complexity and inaccessibility of the mammalian CNS is an

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impediment to accurate correlations between synaptic structure and function. In ribbon synapses of vertebrates, where ultrastructural differences may be more apparent because of the elaborate synaptic morphology associated with the presynaptic ribbon, differences in depth of the postsynaptic trough correlate with the differences in spontaneous activity and sensitivity of sense cells measures in isolated preparations of the electrosensory organ of elasmobranchs (Fields and Ellisman, 1984). Evidence for a causal relationship between synaptic morphology and sensitivity is that electrical stimulation treatments that decrease sensitivity of the sense cells cause a significant reduction in depth of the synaptic trough and other morphological changes in the region of the active zone (Fields and Ellisman, 1988). Different ultrastructural changes are seen in ribbon synapses following treatments that cause depolarization of the presynaptic versus postsynaptic membrane (Fields et al., 1987). Changes in depth of the synaptic trough of ribbon synapses may be related to the dynamics of synaptic vesicle recycling or to cytoskeletal changes. Although more difficult to detect in nonribbon synapses, recycling of synaptic vesicles is common to all chemical synapses (Pysh, 1972; Heuser and Reese, 1973), and activity-dependent redistribution of presynaptic membrane and membrane proteins that participate in transmitter release could provide a link between synaptic structure and function. 1 . Cytoskeleton Proteins controlling the stability of the cytoskeleton are regulated during development and regeneration of the nervous system. Proteins increasing the stability of the cytoskeleton could help consolidate appropriate synaptic contacts, and proteins decreasing the stability of the cytoskeleton could assist in sprouting, process outgrowth, or the elimination of inappropriate synapses. a. Microtubule-Associated Proteins. The abundance and molecular form of microtubule-associated proteins (MAPs) are developmentally regulated (for review see Tucker, 1990). MAPs occurring early in development of the nervous system include MAPlB [synonyms are MAP5, MAPl, MAP1.2, and MAPl(x)], MAP3 (which is also present in adult), MAP2c, and tau. T h e latter two MAPs not only change in abundance with development, but exist in juvenile and adult molecular forms. Two forms of MAPS, MAPlA and MAP2a, are expressed predominantly in adult neurons. Some of the MAPs have regional specialization in neurons. MAP2 is a dendrite-specific protein in mammalian brain (Bernhardt and Matus, 1984; Caceres et al., 1984; DeCamilli et al., 1984; Huber and Matus,

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1984) and may be required for dendritic development. Tau (Lee et al., 1988) is primarily axonal (Binder et al., 1985), but the unphosphorylated form of the protein is detectable in cell bodies and dendrites of adult rdt brain neurons (hpasozomenos and Binder, 1987). Process outgrowth is accampanied by an increase in the synthesis of tubulin and MAPlB and tau (Drubin et al., 1984, 1988). 'These proteins may promote the assembly and help stabilize microtubules by forming cross-bridges that bundle microtubules within the neuronal cytoskeleton (Hirokawa et ul., 1988; Lee t p t al., 1989). Tau protein and MAPlB promote niicrotubule assembly in z4ro (Cleveland et al., 1977; Weingarten et al., 1975; Kuznetsov et al., 1981), and tau protein enhances microtubule assembly when microinjected into fibroblast cells (Drubin and Kirschner, 1986). Developmental changes in tau protein are indicated by a change in its apparent molecular inass (Drubin et al., 1984; Mareck et al., 1980) and electrophoretic shift ( Francon et al., 1982) and differences in hybridization of oligonucleotide probes to mRNA during postnatal development (Lee el al., 1989; Kosik el al., 1989). T h e immature form of tau is present until Postnatal Day 8 in the rat brain and it has a lower affinity for microtubules than the adult form. This lower tubulin affinity might render the cytoskeleton more labile during a period of axon outgrowth. This could be important for synaptic restructuring during the critical period. The state of phosphorylation affects the stability of the cytoskeleton. 'Trophic factors, such as NGF, depolarization, and calcium, regulate the phosphorylation of MAPlB (Aletta et al., 1988). Similarly, tau is more effective in promoting tubulin polymerization in uitro in the unphosphorylated form (Lindwall and Cole, 1984), and it is a more elastic molecule in association with tubulin than when phosphorylated (Hagestedt et al., 1989), suggesting that the phosphorylation of tau could stabilize the cytoskeleton. The mature form of tau protein is expressed after Postnatal Day 8 and it can be phosphorylated by calciumicalmodulin kinase at sites homologous to those of MAP2 (Lewis et al., 1988). Phosphorylation of' MAP2 has been shown to inhibit microtubule assembly (Jameson ~1 a!., 1980; Jameson and Caplow, 1981; Yamamoto et af.,1983). T h e mature form of tau and its phosphorylation by calcium/calmodulin kinase might have the effect of stabilizing the cytoskeleton during the transformation from a growth cone to a presynaptic terminal. T h e immature form of tau cannot be phosphorylated by calcium/calmodulin kinase, because this enzyme is not expressed until after the switch to the mature form of tau (Kelly et al., 1987; Kosik et al., 1989). 6. Intermediate FilamPnts. Periods of axon growth are associated with

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decreased synthesis o r transport of neurofilament proteins (Hoffman and Lasek, 1980; Shaw and Weber, 1982; Willard and Simon, 1983; Pachter and Liem, 1984; Kalil and Perdew, 1988).Reduced axon growth and plasticity may be associated in maturing neurons with the assembly of an extensive network of neurofilament crosslinks mediated by the large neurofilament subunit (Willard et al., 1984; Glicksman and Willard, 1985). Such cytoskeletal stabilization could represent one factor limiting synaptic remodeling to the critical period. c. Actin. Microfilaments are composed of actin, which is a linear polymer (F-actin) of monomeric subunits (G-actin) twisted into a helix of two filaments. T h e monomer is polarized, with filament polymerization favored at the “barbed” rather than the “pointed” end. Polymerization and depolymerization are affected by the concentrations of G-actin, calcium, and ATP (for reviews see Pollard and Cooper, 1986; Stossel et al., 1986; Korn et al., 1987; Smith, 1988) and the activity of actin-binding proteins (for review see Forscher, 1989; Yin and Gartwig, 1989). Actin performs several structural functions in cells, including linking the cytoskeleton to the extracellular matrix through integrins (Ruoslahti and Pierschbacher, 1987), influencing axon outgrowth (Marsh and Letourneau, 1984; Bentley and Toroion-Raymond, 1986), mobilizing synaptic vesicles crosslinked to actin filaments (Linstedt and Kelly, 1987), and possibly inducing structural changes in dendritic spines (Fifkova, 1985; Crick, 1982). Actin binding proteins interact with several second messenger systems, including calcium, IP3, and DAG, that are thought to couple cellular stimulation to structural changes. Actin-binding proteins regulate polymerization of actin, the organization of actin filaments into interconnected structures, coordination of microfilaments with the membrane, and structural changes in response to specific second messengers. Profilin binds G-actin and inhibits nucleation and monomer addition (Pollard and Cooper, 1986; Tilney et al., 1983; Tseng and Pollard, 1982). Up to 95% of polyphosphatidylinositol 4,5-bisphosphate may be complexed with profilin (Goldschmidt-Clermont et al., 1988, 1991), suggesting a possible link between actin-based structural changes and stimulation-induced activation of the PI system (Dugina et al., 1987; Vasiliev, 1985; Altman, 1988; Hartwig and Janmey, 1989; Hartwig et al., 1989; see Forscher, 1989, for review). Gelsolin is another important actin-binding protein that regulates the length of actin filaments in response to calcium ion and PI concentration (Yin, 1988; Yin et al,, 1988; Matsudaria and Janmey, 1988; Stossel et al., 1986; Cunningham et al., 1991; see Forscher, 1989, for review). Gelsolin promotes the transition of cytoplasmic extracts from a gel to a sol phase on addition of calcium, by severing actin filaments when calcium rises to

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micromolar concentration levels. Gelsolin is also regulated by polyphosphatidylinositol4,5-bisphosphate(Janmey et al., 1987; Janmey and Stossel, 1987), which inhibits gelsolin’s severing capabilities. Forscher ( 1989) discusses the possibility that calcium transients not accompanied by significant PPI turnover may result predominantly in severing of actin networks without subsequent actin reassembly, and PPI turnover and stimulation of PKC would result in rapid assembly of actin networks. The interaction between calcium and PPI turnover in regulating the activity of these actin-binding proteins may be relevant to the dualreceptor hypothesis for synapse stabilization by calcium and synapse elimination by IP3 (Bear and Cooper, 1990).

2. Growth-Associated Protein 43 The period of axon outgrowth during development and regeneration is correlated with changes in synthesis of specific proteins transported into the growing axons. After axon elongation and synaptogenesis, terminals may become stabilized by downregulating genes for these growth-associated proteins (GAPS)(for review see Skene, 1989). One such protein is GAP-43, an acidic membrane protein (also known as B-50, F1, pp46, and p57) (Benowitz and Routtenberg, 1987; Wakim et al., 1987; Cimler et al., 1987; Basi et al., 1987; Karns et al., 1987; Rosenthal et al., 1987; Nielander et al., 1987; Labate and Skene, 1989). GAP-43 is a neuron-specific phosphoprotein in growth cones (Katz et a/., 1985; Meiri et al., 1986) and some mature axon terminals that has been linked with developmental and synaptic plasticity (Skene, 1989; Benowitz et al., 1990; Benowitz and Routtenberg, 1987; Lovinger et al., 1987). GAP-43 is absent from dendrites, and its presence in a developing neurite is considered a reliable marker for axonal commitment (Guscin et al., 1988; G o s h and Banker, 1990; G o s h et al., 1990). T h e expression of GAP-43 genes declines steeply in the striate cortex of kittens during the early postnatal period (Neve and Bear, 1989), a period of rapid synaptogenesis in the visual cortex (Cragg, 1975). The expression of genes for GAP-43 and CaM-KII can be altered by visual experience (Neve and Bear, 1989). The visual cortex of kittens reared in the dark shows an increase in the mRNA transcripts for GAP-43 and CaM-KII, but after only 12 hr of exposure to light, GAP-43 gene expression returns to near-normal levels. These results could represent use-dependent effects, changes associated with synapse formation, or synaptic remodeling of visual inputs according to environmental stimuli. GAP-43 is a protein kinase C substrate (Sheu et al., 1990; De Graan et al., 1990). Monoclonal antibodies selective for phosphorylated GAP-43 show staining only in the distal segments of axons, never in the proximal

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regions or in the cell bodies (Meiri et al., 1991). The function of GAP-43 in synapses may be relevant to transmitter release. Potassium-induced depolarization results in phosphorylation of GAP-43 in rat cortical synaptosomes (Dekker et al., 1990), and antibodies that interfere with GAP-43 phosphorylation inhibit the release of transmitter from rat cortical synaptosomes (Dekker et al., 1989). Phosphorylation of the protein has also been linked to synaptic plasticity. GAP-43 is phosphorylated by kinase C (Sheu et al., 1990) in association with LTP of the rat hippocampus (Nelson and Routtenberg, 1985; Routtenberg, 1986) and there is a rapid increase in GAP-43 phosphorylation in the cat visual cortex during the critical period (Sheu et al., 1990). Growth-associated proteins may alter a neuron’s responses to extracellular signals by altering intracellular signal transducing systems (cf. Skene, 1990; Coggins and Zwiers, 1991). GAP-43 binds calmodulin preferentially in the absence of calcium, and phosphorylation of GAP-43 by protein kinase C greatly reduces its affinity for calmodulin (Andreason et al., 1983). GAP-43 at synapses has also been suggested as a possible regulator of polyphosphoinositide metabolism (Oestreicher et al., 1983; Van Dongen et al., 1985; Van Hoof et al., 1988). When phosphorylated, GAP-43 inhibits phosphatidylinositol-4-phosphate kinase, the enzyme that phosphorylates PIP to regenerate polyphosphatidylinositol 4,5bisphosphate (Van Hoof et al., 1988). Phosphorylated GAP-43 could thus act as a feedback inhibitor, uncoupling receptor stimulation from significant regeneration of polyphosphatidylinositol4,5-bisphosphate(Gispen et al., 1985). Interactions with IP3, calcium, and calmodulin suggest that GAP-43 could affect the stability of the actin cytoskeleton (Forscher, 1989). GAP-43 binds calmodulin preferentially in the absence of Ca2 and it could act to sequester an easily activated calmodulin pool near the membrane cytoskeletal interface (Andreason et al., 1983; Alexander et al., 1987). GAP-43 also releases Ca2+-calmodulin after Ca2 elevation, thus freeing the complex to activate other Ca2 -calmodulin-activated target proteins. By absorbing calmodulin and releasing it in response to activation of PKC, GAP-43 could modulate calcium signals in growth cones and synapses and regulate events such as membrane and cytoskeletal assembly, as well as presynaptic mechanisms controlling synaptic efficacy (Skene, 1991). In addition, GAP-43 binds the G-protein subunit Go, which is a major component of neuronal growth cone membrane (Strittmatter et al., 1990). Hence GAP-43 could integrate extracellular and intracellular signal traffic by binding to Go (Coggins and Zwiers, 1991). Although the function of this protein is not yet known, and much is to be learned about its biochemistry, there is little doubt that it is critically +

+

+

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involved in process outgrowth from neurons. T h e evidence is not merely correlative: For example, although PC12 cells, which lack GAP-43, d o respond to NGF by extending processes (Baetge and Hammang, 1991), when the cells are transfected with human GAP-43 cDNA they show a 10-fold increased responsiveness to NGF, and accelerated neurite outgrowth (Yankers et al., 1990). Similar results are seen in transiently transfected COS and NIH 3T3 cells (Zuber et al., 1989). Conversely, neurite outgrowth can be arrested in neuroblastoma cells by administering antibodies against GAP-43 (Shea et al., 1991). IV. Conclusions

A. SYNAPTIC PLASTICITY: COMPARISON ACROSS SYSTEMS Activity-regulated processes are revealed when the results of altering activity by stimulation o r deprivation change the strength o r number of synaptic connections that would have developed without intervention. Examples of activity-regulated processes include the reduction from polyneuronal innervation in skeletal muscle and changes in dendritic arbor in cortical neurons of animals reared in enriched environments. 'The term synaptic competition is often used to describe any situation where multiple afferents innervate a cell with a developmental reduction in innervation from initial states; however, this set of phenomena has been categorized here under three headings, which we feel should be distinguished: synapse selection, synaptic competition, and coincident (Hebbian) activation. 1. Synapse selection is a general term for the struggle for survival of synapses in a contest where activity of the presynaptic neuron provides a selective force and directly or indirectly (through localized responses in the postsynaptic cell or by its own success) differentially affects active and inactive afferents. T h e extent of the postsynaptic influence on synaptic stabilization may be modified by the amount of postsynaptic activity. 2 . Synuptic competition is a synaptic process whereby afferents compete on the basis of their electrica! activity, for a limiting resource that is provided by the postsynaptic cell in an activity-dependent manner. T h e limiting resource may be a trophic substance or synaptic sites. 3. Coincident activation is a mechanism of the Hebbian type that selectively acts on convergent afferents on the basis of the correlation of their activity with the postsynaptic target. The mechanism selectively strengthens the correlated (or uncorrelated) fibers, depending on whether a Hebbian (or anti-Hebbian) result is appropriate to the circuit (e.g., associative conditioning versus LTD in Purkinje cells of the cere-

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bellum). When complementary changes in strength of convergent afferents result, two coincident mechanisms, one strengthening synapses with correlated activity and the other weakening synapses with uncorrelated activity, are presumed to operate in parallel. Alternatively, in addition to a coincident mechanism of synaptic plasticity, another process may operate, such as synaptic competition, continued sprouting, or a general tendency to withdraw or lose connections. 1. Synapse Selection Activity-regulated synapse selection is a more general term than synaptic competition, or coincident activation. This process is the struggle among converging afferents for survival where activity of the afferent influences the outcome of the process. Usually activity is thought to provide a competitive advantage to the afferent, but the opposite is also possible (and is a likely result in some systems). Activity may provide a selective advantage by two means, elimination and augmentation, each of which can be some function of the level of neural activity. The means by which activity provides these selective advantages include increasing the acquisition of a beneficial factor (e.g., trophic factor, protease inhibitor, cytoskeletal alteration), decreasing exposure to detrimental factors (all of which constitute forms of augmentation), and increasing selective pressure against convergent afferents in an activity-dependent manner (elimination). It is important to note that either of these may be generated by the afferent itself, by the postsynaptic cell, or through other nonneuronal cells, provided the effects are proportional to afferent activity and temporarily or spatially localized to provide activity-dependent specificity. The actions are necessarily reciprocal among convergent afferents, and differential on the basis of the respective levels of activity in each afferent. Therefore, any change in number or strength of synapses developing after differential activation of converging afferents indicates an activity-regulated selective process. (There are additional selective processes acting differently on convergent afferents that are independent of electrical activity.) Unlike synaptic competition for a limited resource, however, activity-dependent synapse selection could lead to the elimination of all convergent afferents from a postsynaptic target, for example at the NMJ (Magchielse and Meeter, 1986; Mioche and Singer, 1989). Activity-regulated selection can, but need not, result in reciprocal changes in stimulated and unstimulated convergent afferents. Reciprocal changes require the simultaneous action of augmenting and inhibiting actions. Ultimately highly successful innervation as a result of activity-dependent augmenting effects can itself serve to inhibit nonstimulated afferents. At this point the interaction becomes competitive.

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(‘l‘his could occur, for example, by successfully taking over all available postsynaptic sites.)

2 . Synuptic Competition 1-b determine if activity-regulated effects operate by a selective or competitive interaction between afferents converging on the same cell, differential activation of the convergent afferents is applied. A difference between the stimulated and unstimulated convergent afferent will not in itself distinguish selective from competitive interactions, however. Competitive interaction refers specifically to the situation where afferents compete on the basis of their own electrical activity for a limited resource that is essential to maintain or strengthen synaptic connectioiis with the postsynaptic cell. (Synaptic competition on the basis of factors other than activity operate in addition, for example, spatial proximity to the target and differential affinity for surface or trophic substances required for synaptogenesis or synaptic function.) Essential evidence for a possible activity-dependent competitive interaction is that when activity of one convergent afferent is experimentally manipulated with the result that synaptic strength or number is altered, the other convergent afferents undergo a complementary change in synaptic response. This response is necessary but not sufficient evidence for synaptic competition, because a similar result could obtain from a selective mechanism. Proof of activity-dependent synaptic competition requires that the limiting resource be supplied in excess, with the result that no difference in synaptic strength o r number develop between convergent afferents receiving differential stimulation. -_ I here is not yet sufficient evidence for this mechanism in any of the experinients reviewed in this paper. This remains a probable and potentially important mechanism for nervous system development and a hypothesis that is subject to experimental investigation given present capabilities of detecting, extracting, or manipulating factors that are important in synaptogenesis and synapse elimination. One consequence incompatible with activity-dependent synaptic competition would be the loss o f all at€erents to the postsynaptic cell following stimulation, a result that can occur with differential stimulation of the NMJ (see, e.g., Magchielse and Meeter, 1986).

3 . Coincidmt Actiuation Coincident activation is a specific subcategory of activity-dependent synapse selection, which is of considerable importance and much discussed as the Hebb synapse. T h e selective advantage is conferred on an afferent on the basis of the coincidence of activation between presynaptic and postsynaptic responses. It differs from the selective mechanism in that the mechanism that confers the advantage is initiated as a result of

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presynaptic and postsynaptic coactivation, rather than the state of coactivity being one in which the selective advantage happens to be optimal. This requires an entity that will act as a comparator to become activated only on presynaptic and postsynaptic coactivation of sufficient intensity (such as the NMDA receptor). It requires an active identification and selection of the afferent based on coincidence of activation to institute a specific cellular processes to strengthen that afferent. In Hebb‘s terminology, “some growth or metabolic change takes place”: “When an axon of cell A is near enough to cell B and repeatedly and persistently takes part in firing it, some growth or metabolic change takes place in one or both cells such that A s efficiency, as one of the cells firing B, is increased” (Hebb, 1949). It is important to recognize that a selective mechanism could have a “Hebbian” result, without a specific response instituted on the basis of coincident presynaptic and postsynaptic activation. Selection effects must simply produce a differential impact under conditions of simultaneous presynaptic and postsynaptic activity. This is a critical distinction mechanistically, because it does not require an entity to detect coactivation and initiate an appropriate cell biological reaction strengthening the coactive input. Hebbian mechanisms account only for an increase in synaptic efficacy of coactive inputs. If the results of differential stimulation show complementary reciprocal changes in the unstimulated afferents, one must assume a simultaneous mechanism of coincident activation that selectively weakens the uncorrelated afferents (Stent’s hypothesis, 1973), operating by an IP3 mechanism for example (Bear and Cooper, 1990; Dudek and Bear, 1989; Dudek et al., 1989), or the parallel action of activity-dependent selection that weakens the uncorrelated afferent. (As already stated, in the limit this leads to synaptic competition.) A mechanism of coincident activation must be tested by differential activation of converging afferents. The outcome of this differential stimulation would not prove a mechanism based on coactivation, as a similar outcome could result from selection or competition. Moreover, the differential activation must be on the basis of differences in coactivation of the presynaptic and postsynaptic neuron, brought about by varying the timing of afferent activity in the two afferents with respect to the postsynaptic response. T h e mechanism does, however, require a postsynaptic response, whereas the synapse selection or synapse competition may, but need not, operate through changes in postsynaptic activity. Evidence for selection based on coincident activation will include showing selective changes in afferents on the basis of differences in coactivation; however, synapse selection can also have this outcome. Coincident activation selection is proven only when a process that can affect synaptic strength is invoked on correlated activation of the

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synaptic and postsynaptic neuron, and differential stimulation will not result in changes in converging afferents when this coactivation process is specifically blocked. T h e forms of LTP that are dependent on NMDA receptor activation are the best examples of synapse modification based on coincident activation of the Hebbian type. Much of the evidence from experiments in development of the visual cortex appears to be consistent with a mechanism of coincident activation dependent on activation of the NMDA receptor, but there is some uncertainty as to whether NMDA receptor activation o r localized calcium influx is the critical element activated by coactivation (Fields et al., 1991a; Singer and Artola, 1991). Furthermore, although the rules for use-dependent synaptic modification derived from studies of kitten visual cortex resemble the conditions for initiating LTP and LTD in cortical slice preparations, there is no evidence linking these changes in synaptic gain with activity-dependent restructuring of synapses during development (Singer and Artola, 1991). T h e ambiguity is far greater in other systems where activity-dependent synaptic plasticity is evident, particularly at the NMJ. Argument has arisen over whether synapse elimination at the NMJ is Hebbian (Ribchester, 1988; Ribchester and Taxt, 1983, 1984) o r anti-Hebbian (Callaway et al., 1987, 1989), but sufficient recognition should be given to the possibility that it may be neither. T h e experiments support the conclusion that activity-dependent synapse elimination at the NMJ is selective, but the evidence that it is competitive or based on coincident activation is inconclusive. Recognition that these three distinct processes may operate in parallel at a given synapse o r act differently on different aspects of biology relating to synaptogenesis, synapse elimination, synaptic gain, and maintenance of functional synaptic connections could help resolve the contradictory results among different studies at the NMJ. More specific experiments will be required to make these distinctions. For example, if sprouting were affected beneficially by presynaptic activity, and this factor were taken into account, the results of Ribchester (1988) Ridge and Betz (1984) might agree with those of Callaway et al. (1987, 1989). 4. Number z1ersu.s Specificity o j Connections

Different selective mechanisms may be involved in attaining dif€erent developmental objectives and generating different types of neuronal networks. If the functional requirement is the generation of synaptic networks involving specific neuron-to-neuron connections, such as might be required for integration, differentiation, o r comparing synaptic responses from several afferents (e.g., binocular neurons in the visual cortex o r LTP in the hippocampus), a coincident mechanism of synaptic

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remodeling may be required. If the objective is to obtain a numerical match of the presynaptic and postsynaptic elements in a functionally optimal manner (e.g., synapse elimination at the NMJ, sympathetic ganglia, or climbing fibers to Purkinje cells of the cerebellum, or perhaps remodeling of synapses from retinal afferents to the optic tectum or LGN), the more general selection mechanism may be adequate. This category (increasing the precision of genetically defined connections) may suffice for many developmental objectives, and these selective mechanisms may be the more general method of activity-dependent remodeling of synapses during development. A final aspect is the nature of the process and the speed with which it must operate. Synaptic modification based on coincident activation of the Hebbian type is required to adjust synaptic strength rapidly on appropriate afferent activation. This probably does not involve growth of new synapses or elimination of functional ones. The coincident mechanism could, as Hebb suggested and is widely proposed in the current literature (see, e.g., Cline, 1991; Constantine-Paton et al., 1990; Debski et al., 1990), initiate appropriate synapse stabilization and synaptogenesis by the same mechanism that produces LTP or STP. Activity-dependent development of synapses and consolidation of appropriate connections in adult brain may operate through a conserved mechanism, but it is also possible that this process of synaptic restructuring uses a fundamentally different mechanism from the physiological responses that control synaptic gain on rapid time scales (such as NMDA receptor or calcium influx in LTP). These consolidation processes would be selective, and activity-regulated, but they would occur after any changes in physiological response of the synapse. They would be the processes of growth and guidance that establish the connections between neurons in an activitydependent manner during development. This would include activitydependent release and acquisition of trophic substances, inhibitory factors, and cytoskeletal, metabolic, and structural responses to activity in the presynaptic and postsynaptic neuron and its environment. Consistent with this view is that the best correlation with critical period seems not to be the expression of a specific membrane channel or second messenger system, but the amount of sprouting, which declines with time and is restimulated to a variable extent following inactivity.

B. MECHANISMS OF PLASTICITY 1. N-Methyl-maspartic Acid Receptor and Synapse Stabilization Inhibition of NMDA channels can disrupt both LTP and activitydependent synapse selection during development (cf. Section III,B, 1).

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Whether this reflects a cotnnion mechanistic basis is uncertain because LTP and developmental plasticity are dissimilar phenomena, and postsynaptic receptor blockade disrupts electrical activity. L‘TP and activity-dependent refinement of‘ synaptic connections differ in many respects, including time course of action and cell biological processes involved. Developmental activity-dependent plasticity appears to share many of the rules for synaptic modification in the hippocampus, but in development, synaptic plasticity is a heterogeneous assemblage of phenomena, encompassing changes in synaptic strength (both increases and decreases), synaptogencsis, synapse elimination, and axonal outgrowth. The time course of action is immediate in LTP, but investigations of developmental use-dependent synaptic plasticity must be carried out in experiments where stimulation is delivered for prolonged periods (up to several days). LTP may involve synaptogenesis to maintain potentiation. but this is a question requiring further study. Morphological changes in spines have been reported following LTP that involve spine neck morphology and differences in the ratio of synapses on spines and dendritic shafts (Fitkova and Anderson, 1981; Fifkova and Van Harreveld, 1977; Van Harreveld and Fifiova, 1975; Chang arid Greenough, 1984; Applegate et nf., 1987; Desrnond and Levy, 1983, 1986, 1988; Lee et ul., 1980), but activity-dependent restructuring during development also occurs in neurons without dendritic spines (e.g., DRG to spinal cord neurons; Nelson et ul., 1989). LTP has been observed in some neurons, as in the visual cortex, where activity-dependent synapse selection has been studied during development, but LTP is not evident in other systems that undergo activity-dependent restructuring of synaptic connections (e.,g.. the NMJ). I t is unclear how the biochemical mechanism for LlP is related to synapse stabilization. Developmental synaptic restructuring is not limited to excitatory glutamatergic synapses (see, e.g., Scherer and Udin, 1989; Cline, lYYl),and UI‘P is not limited to synapses exhibiting NMDA activation (reviews in Brown et al., 1989, 1990; Nicoll et al., 1988). ‘The stinidation pattern and molecular mechanism for LTP are different in hippocampal archicortex and adult neocortex compared with developing visual cortex of kittens (cf. Section III,B, 1). I n kitten visual cortex, LTP can be induced in the presence o f NMDA channel blockade with AI’t’, but UI’Y in the adult neocortex is blocked by APV. If the niechanisms of liTP are fundamentally different in neocortex of immature and adult animals, the relevance of K MDA-dependent LTP of adult brain to the activity-dependent restructuring of synapses during development is questionable. It would seem more suitable to consider the voltage-dependent channels that induce LTP in kitten visual cortex in terms of synapse stabilization during early development.

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Thus, there is dissociation between NMDA channel activation and LTP and between developmental synaptic plasticity and requirement for NMDA channel activation. Further evidence for this dissociation is that blockade of protein kinase C prevents NMDA-dependent LTP in hippocampus, but blockade of protein kinase for up to 8 weeks in three-eyed tadpoles does not cause desegregation of eye-specific stripes (Cline and Constantine-Paton, 1990). This inconsistency between the mechanism of NMDA-dependent LTP in the hippocampus and activity-dependent synapse elimination in the optic tectum has necessitated a rejection of the theory of synapse stabilization based on hippocampal LTP. An alternate proposal has been put forward that the NMDA-dependent, but protein kinase C-independent mechanism of short-term potentiation (STP) in adult hippocampus is the mechanism for synapse stabilization in the frog optic tectum during development (Cline, 1991). A simpler interpretation for the protein kinase C result might be that the assortment of afferents from the two eyes is activity dependent, but not strictly NMDA dependent. The blockade of activity-dependent segregation of retinal afferents may be explained by the suppression of electrical activity caused by APV. The suppression of activity need not be so severe as to block sodium-dependent action potentials. Many factors that alter the excitability or membrane potential of the postsynaptic neuron can cause major disruption of normal competitive interactions between synapses in development (Bear and Singer, 1986; Singer, 1990). By blockage of the NMDA component of the EPSP, activity in the postsynaptic network is suppressed by reducing the amount of summation of multiple EPSPs provided from convergent afferents or other glutamatergic synapses in the network of neurons connected to the postsynaptic cell. This would reduce the postsynaptic depolarization, but it would not completely block afferent input because of the remaining non-NMDA component of the EPSP. Spontaneous activity is suppressed significantly by perfusion of APV into spinal cord cultures. In this system plasticity is blocked by suppressing spontaneous activity (with TTX at a dilution that leaves afferents responsive to stimulation) or by applying APV; however, plasticity is restored in the presence of NMDA channel blockade by elevating the concentration of calcium in the culture medium to 3 mM. This suggests that the amount of activity in the postsynaptic neuron is critical, and that synaptic plasticity in this system is linked to calcium-dependent mechanisms that integrate electrical activity in terms of changes in intracellular [Ca2+]. NMDA channel activation is one source of calcium entry into neurons that is particularly sensitive to coincident or strong correlated activity, but NMDA channel activation may not be necessary for activitydependent synaptic remodeling during development. The role of

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NMDA channels in synapse stabilization may be supplanted by calcium entry through other channels. In addition, processes other than synapse stabilization, for example, synapse elimination and LTD, which are regulated by calcium but independent of NMDA channel activation, may produce adaptive changes in synaptic connections in the presence of APV. Investigations of calcium-mediated effects on synapses may lead to a better understanding of the mechanisms involved in developmental synaptic plasticity.

2 . Model for Activity-Dependent Synaptic Remodeling during Development It is clear that a myriad of molecular and cell biologic mechanisms have been demonstrated whereby electrical activity in neurons may initiate profound structural and functional changes in nerve networks. Any or all of these may be involved in the functional adaptation of the nervous system that occurs as a consequence of ordered input to the system during development. We feel that this plethora of mechanisms can be set within a relatively simple general scheme of presynaptic and postsynaptic effects of activation and that such a scheme may be helpful in guiding experimental approaches in this area. Our analysis of the data presently available from many different systems leads us to conclude that among the three specific processes of activity-regulated synaptic remodeling during development, the most generally supported mechanism is one of synapse selection. The generality of this model is explored here by testing its applicability against data from the visual cortex (usually considered under a coincident mechanism of synaptic remodeling) and the NMJ (where there is considerable controversy). T h e essential feature of our model is that each afferent channel (axon) produces as a consequence of its activity a double effect on the synaptic efficacy of inputs to its target: an augmentation affect and an eliminating effect. T h e strengths of these opposing effects have distinct relationships t o this total input to the target cell, and the spatiotemporal distributions of augmenting and eliminating effects are also different. Although these relationships might be expected to differ for different preparations or developmental stages, Figs. 7A and B illustrate prototypical examples of what w e posit. T h e relationship shown in Fig. 7B obtains at each synapse (bouton), with a distribution like that shown in Fig. 7A occurring around each site; the net effect at any point is the sum of the effects of all elements impinging on the postsynaptic element. In general, augmenting effects of activity dominate at low levels of synaptic drive, whereas eliminating effects become dominant as drive increases. The net connectivity of a system settles at that level of input at

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POST

y E Ii rnina t e Augment 0

DISTANCE OR TIME

Z Z

8 Z 0 L

k Y Y

w

INTENSITY OF INPUT FIG. 7. Schematic representation of spatial and temporal distribution of synapse augmentation and synapse elimination mechanisms and their relationships to the intensity of synaptic input. Upper: Three inputs to a muscle fiber are diagrammed and below it are shown the differential spatial (or temporal) distribution of augmentation (solid line) or elimination effects (dashed line) when channel B is activated. Augmentation dominates over elimination at the active site and is relatively more prominent at an adjacent ending, compared to more distant endings which are subjected to more powerful elimination effects. Lower: Hypothesized differential effect on synaptic augmentation and elimination at different levels of input. Augmentation predominates at low levels of input, whereas elimination becomes more prominent at very high levels of input. At the balance point, synaptic connectivity would be stabilized. The specific connections that would be maintained would depend on the activity patterns in the inputs to a given neuron and the resultant patterns of augmentation and elimination determined by the relationship shown in the upper part of the figure.

which augmentation and elimination are balanced. At the neuromuscular junction, this would be generally satisfied when a single axon is driving the muscle fiber, for instance, whereas input from several fibers would represent the settling point in the visual system. When all afrerent activity is blocked, some residual augmenting and eliminating effects persist, related to spontaneous release of transmitter, ongoing activity of the postsynaptic target cell, or autonomous processes of neurite outgrowth or retraction. Of equal importance is the spatial or temporal distribution o f augrrienting and eliriiiriatitig effects consequent to activity. In general, and to mediate “competitive” interactions, eliminating influences must be inore widely distributed in space or time than are the augmenting effects. This requirement is necessary t o have a differential effect of activity on active and inactive af€erents. l’hese formal relationships arise quite naturally from observations on a wide variety of systems and generate the Hebb rules of strengthening of associated arid b:eakening of uncorrelated presynaptic and postsynaptic activation. (It should be noted that postsynaptic spike activity is not a necessary condition for strengthening or weakening of connections. Of course, postsynaptic activity may be produced at higher levels of input in Fig. 7.4 and this may contribute to the augmentation or elimination effects at such high levels of input intensity.) ‘I‘he model emphasizes the importance of the amount and pattern of difyererit input channels and the state of activity and the responsiveness of the postsynaptic element. When these variables are considered, a number of obsenwions, some rather obvious and others more surprising, appear to be consistent with the predictions of the model. Some of these are summarized in Fig. 8. Increases and decreases in presynaptic and postsynaptic activity are separated in the columns and rows, respectively, and the effect on connectivity for the corresponding (or other) channels of inputs are separated by the diagonal within each cell of the diagram. In the typical Hebb competitive interaction, the results of the upper left cell occur: an active input that increases postsynaptic activity increases its own effectiveness while decreasing the effectiveness of inactive channels. This would be a consequence of the system being in the net elimination range of Fig. f A , but the augnientation related to the active channels would be relatively concentrated (in either space or time) to that input’s boutons. T h e more widespread elimination effect would be relatively preferentially distributed to the inactive channel boutons. T h e less obvious result of is that of Reiter and Stryker’s (1 988) in which infusion of niusciniole in kitten cortex produced a condition in which inactive inputs were augmented, and active inputs were reduced.

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PRESYNAPTICACTIVITY “Self“ relative to “other” INCREASE DECREASE

A OTHER

FIG. 8. A summary of results concerning the effects of presynaptic and postsynaptic activity on the efficacy of synaptic connections. The + or - sign in each half-square indicates strengthening or weakening of the connection. “Self” refers to the presynaptic channel whose activity is either increased or decreased as indicated at the top of the figure (relative to “other”channels to the same postsynaptic cell). Postsynaptic activity increases or decreases are indicated at the left side of the figure. i n square 1, an increase in the activity of the “self‘ channel, which is accompanied by an increase in postsynaptic activity, increases the efficacy of “self‘ and decreases the efficacy of “other.” This corresponds to the Hebbian paradigm. Square 3 represents the situation in which presynaptic activity is increased, but postsynaptic activity is reduced. This corresponds to the experiments of Reiter and Stryker (1988), in which the active afferents were weakened and inactive afferents strengthened when postsynaptic activity in visual cortex was blocked with muscimole. Square 4 is analogous to the results of Van Essen et al. (1990; Callaway et al., 1989), where the effectiveness of the inactive motor axon was strengthened relative to active motoneurons. Square 2 is essentially the reciprocal situation to square 1; inactive afferents are weakened. Examples: (1) Magchielse and Meeter (1986) and Ridge and Betz (1984); (2) Ribchester and Taxt (1984); (3) Reiter and Stryker (1988); (4) Van Essen et al. (1990) and Callaway et al. (1989).

In this case, it is important to recognize that active inputs have both augmenting and eliminating influences on themselves. With blocked postsynaptic activity, the system is operating below the crossover of augmentation and elimination, but the active fibers are closer to the crossover than are the inactive fibers; this greater preponderance of

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augmentation over elimination in the inactive fibers (by virtue of their position to the left in Fig. 7B) confers an advantage over their active neighbors. Similar reasoning applies to the results of Callaway and colleagues (1987, 1989) in which the connections of active axons to skeletal muscles in the rabbit are preferentially eliminated relative to the inactive axon’s connections. In those experiments, it was shown that the motor unit size of active axons actually decreased during the elimination process, implying that activity could result in the elimination of a connection in the presence of inactive connections. This result is depicted in the bottom right cell of Fig. 8. Results at the neuromuscular junction are complex and factors such as neurite sprouting and ongoing myogenesis are probably involved in the nominally contradictory results that have been obtained in this system. We feel that the considerations raised here can serve to rationalize many of these observations. Quite detailed models of the sort proposed by Van Essen and coworkers (1990) can serve to set useful limits in the degree to which specific mechanisms can contribute to synapse elimination and stabilization. We have explored a less specific formulation with the hope that experimental identification of specific cell biological mechanisms can be made that represent the general parameters of the model. Indeed a variety of cell biological mechanisms are no doubt involved in generating the formal model presented here. Nonlinearity of the relationship in Fig. 7B could be related to the voltage-dependent characteristics of the NMDA receptor, for instance, but a number of processes involving intracellular [Cay+]could be involved. Neuronal systems such as the catecholaminergic, cholinergic, and peptidergic systems, which modify neuronal excitability, would be expected to affect plasticity as they indeed do. Many influences may converge to generate the augmentation and elimination relationship. Nevertheless, certain cell biological systems may be more directly related to one or the other process as suggested by the possible distinction between changes in intracellular [Caz ] and changes in PI turnover in regulating plasticity in kitten cortex. “Explanatory power” is a rather weak justification for a model; predictive power, testability, and heuristic value are more important. In general, we feel it is crucial to be able to specify the state of activity of at least two inputs to a postsynaptic element, and to assess ongoing activity and alterations in that activity in the postsynaptic element. Attendant changes in connectivity of both active and inactive elements need to be specified in both structural and functional terms. Specific conclusions about augmentation or elimination of connections can then be made. This would allow testing of the sort of model we have put forward, but more significantly it should allow decisive mechanistic studies. Further +

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tests of the protease hypothesis of synapse elimination are desirable. T h e role of activity-dependent elaboration or utilization of trophic material should be evaluated. Ongoing postsynaptic electrical activity importantly affects where a system is operating on the augmentation-elimination curves and effects of such activity should be carefully evaluated. Full specification of this activity status of an experimental system should allow better evaluation and interpretation of the results of pharmacological, cell biological, or electrophysiological interventions. There are clearly multiple levels of control and coding of cellular stimuli in neurons. These occur at the level of cellular signals, membrane receptors (including voltage detectors), second messengers, second messenger cascade pathways, and transcription regulating proteins. There are multiple points of interaction, regulation, and integration at each level in the complex sequence from cell stimulation to cellular response. In recent years we have learned that the various subtypes of glutamate receptors respond to the same transmitter, but produce different second messengers. Conversely, NMDA channels and voltage-sensitive calcium channels differentiate between signals (one responding on the basis of voltage, and the other requiring the association between transmitter and depolarization), but they both affect the same second messenger (calcium) in similar ways. These multiple layers of interaction must not be overlooked in experiments that involve blockading a single element in the complex whole. Inhibition of calcium, calmodulin, protein kinases, NMDA and other neurotransmitter receptors, protein synthesis, trophic materials, and probably other second messenger systems, biochemical pathways, and enzymes will each disrupt the normal cellular adaptive responses to neuronal activity, but the results of an individual intervention must be interpreted as a perturbation of a complex system, rather than ablation of the cause of plasticity. This being the case, experimental emphasis on the specific cellular responses to experimental interventions that follow when synaptic plasticity is disrupted (e.g., changes in synaptogenesis, synapse elimination, terminal sprouting, synapse elimination, increased or decreased synaptic gain) would seem to be required, particularly as this pertains to the heterogeneous phenomena operating simultaneously in development of appropriate interconnections between neurons. References

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A ROLE FOR GLIAL CELLS IN ACTIVITY-DEPENDENT CENTRAL NERVOUS PLASTICITY? REVIEW AND HYPOTHESIS Christian M. Mutter Department of Physical Biology Max Planck Institute for Developmental Biology D-7400 Tiibingen, Germany

I. Introduction A. Activity-Dependent Plasticity: A Definition B. Glial Cells: More than Just Nerve Glue C. Activity-Dependent Plasticity: A Modification of Synaptic Efficacy or Circuitry? 11. Evidence for Participation of Glial Cells in Activity-Dependent Plasticity A. Activity-Dependent Formation of Intraareal Topographies B. Activity-Dependent Synapse Elimination C. Activity-Dependent Synapse Formation D. Activity-Dependent Changes in Synaptic Efficacy 111. Regeneration and Adaptive Processes following CNS Damage IV. A Unifying Hypothesis for Involvement of Glial Cells in Activity-Dependent Plasticity A. General Mechanisms B. Possible Cellular Mechanisms of a Role for Glial Cells in Activity-Dependent Plasticity C. Preferential Involvement of Immature Astrocytes in Plasticity V. Summary References

1. Introduction

One of the unique features of the central nervous system (CNS) is its capacity to adapt to environmental influences with changes in its circuitr y or the efficacy of synaptic transmission. This capacity is central to several aspects of CNS development, as well as for learning and memory in the mature organism. Although most of the studies attempting to unravel the mechanisms of experience-dependent plasticity in the CNS have focused on changes in synaptic efficacy (for reviews see Collingridge and Bliss, 1987; Merzenich, 1987; Singer, 1987; Teyler and DiScenna, 1987), mechanisms of changes in neuronal circuitry have attracted only limited attention. As will be elaborated in this review, this is 2 15 INTERNATIONAL REVIEW OF NEUROBIOLOGY, VOL. 34

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surprising, considering the vast evidence of structural changes in neuronal interconnectivity accompanying experience-dependent plasticity in the CNS. In the following sections, I summarize the experimental evidence that long-term plastic changes in a variety of model systems of activity-dependent plasticity are indeed parallelled by, and probably based on, structural changes in neuronal connectivity. This fact calls for an extended view of possible mechanisms of CNS plasticity going beyond the assumption of sole changes in the efficacy of synaptic transmission at a constant number of synapses. The focus of the present review is on plasticity manifested in synapse elimination and synapse formation. Evidence is presented that glial cells may be involved in such changes in neuronal connectivity underlying long-term manifestation of CNS plasticity. In addition, possible mechanisms of glial participation in changes of synaptic efficacy are summarized. Recent progress in the understanding of glial contributions in the control of regeneration is reviewed briefly and compared with a possible involvement of glial cells in plasticity of the undamaged CNS. Some topics relevant to the main purpose of the present overview have been elegantly reviewed recently. Among them are neuronal-glial interactions (Hatten, 1990; Hatten and Mason, 1986; Laming, 1989; Vernadakis, 1988; Walz, 1989; Walz and Hertz, 1983), transmitter receptors and ion channels on glial cells (Barres et al., 1990a; Gray and Ritchie, 1985; Kimelberg, 1988; Nowak et al., 1987; Stone and Ariano, 1989; van Calker and Hamprecht, 1980), and release of factors from glial cells (Manthorpe et al., 1986). An additional valuable source of information for the interested reader are three volumes on astrocytes (Fedoroff and Vernadakis, 1986a,b,c). I summarize only those investigations on glial cells pertinent to the present topic and refer to the related reviews at the appropriate locations. Similarly, I refer to reviews summarizing studies of neuronal mechanisms underlying certain plastic changes discussed in the present overview, to allow the reader the opportunity to relate the present “glial”view to widespread “neuro” physiological opinions. Following a comprehensive review of the available experimental evidence for glial participation in activity-dependent plasticity, a unifying hypothesis of the possible mechanisms underlying neuronalglial and glial-neuronal interactions that lead to long-term adaptive changes in the brain is proposed. This overview is intended primarily to provide a summary of studies on glial cells as a basis for a broadened discussion on possible mechanisms of activity-dependent plasticity. I apologize in advance for unintentional omissions of relevant studies and the intentional bias toward glia-oriented work.

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FIG. I . Activity-dependent synaptic modifications governed by the activity level of one (homosynaptic modifications) or two (heterosynaptic modifications) afferents and a postsynaptic target. T h e presynaptic or postsynaptic activity level is indicated as being low (-), moderate (+ -), or high (+). T h e resulting effects on synaptic efficacy are indicated as a n increase (+), decrease (-), or no change (+ -) in transmission. (Modified from Singer, 1987.)

A. ACTIVITY-DEPENDENT PLASTICITY: A DEFINITION Activity-dependent plasticity is defined by the necessity of local neuronal transmission to induce a change in neuronal-neuronal transfer at a given site. T h e basic rule for such activity-dependent plasticity was formulated originally by Hebb ( 1949) and subsequently extended by Stent (1973). T h e basic concept is that coincident activity of a presynaptic afferent and a postsynaptic targei leads to an overall enhancement of synaptic transmission at this site. In contrast, noncoincident activity is assumed to result in a reduction of transmission. As summarized in Fig. 1 (homosynaptic modifications) these predictions of the direction of adaptive changes in neural transmission are, at least partially, supported by experimental data. Although the assumptions mentioned earlier are based on the interaction of one afferent with a postsynaptic target, it is evident that most adaptive changes in the CNS rely on the interaction of

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several afferent structures with a given postsynaptic target. This extension of the number of partners in activity-dependent plasticity calls for a modification of the hypothesis. It is generally assumed that competition of multiple afferents for one postsynaptic target ultimately leads to stabilization of the most active afferent and elimination of afferents with superior siiccess in activating the target. In this paradigm it becomes obvious that activity at one site influences the plastic change at another site. In this scenario plasticity is influenced by the activity levels in several presynaptic and the postsynaptic structures. Experimental evidence for activity-dependent plasticity governed by multiple afferent systems and the resulting plastic changes are summarized in Fig. 1 (heterosynaptic modifications). One important feature of the plastic change in such circuitries is a topographical segregation of two similarly active afferents innervating a common target. This has been observed in several structures in the CNS (Constantine-Paton and Law, 1978; Shatz, 1990a,b; Singer, 1987). Probably the best studied model system is the development of ocular dominance columns in the visual system (Hubel and Wiesel, 1963, 1970). This anatomical segregation is based on spatially segregated consolidation and elimination of afferents. It is assumed that this phenomenon is based on differences in the efficacy of the afferent pathways in exciting the postsynaptic target (Shatz, 1990b; Singer, 1987). One extreme of this paradigm is the downregulation/elimination of one inactive afferent, when another active afferent successfully activates the postsynaptic target (heterosynaptic depression). Iivo basic mechanisms that may underlie these changes in neural transmission have already been postulated by Hebb ( 1949): (1) metabolic processes leading to a change in the efficacy of transmission and (2) structural changes resulting in reorganization of the circuitry. Experimental evidence for either possibility is summarized in Section I1,C. From the aforementioned rules for activity-dependent plasticity it becomes clear that both presynaptic activity and postsynaptic activity influence the plastic changes. Thus, mechanisms mediating plasticity need to have access to both sources of information. In addition, plasticity is further influenced by modulator) systems that appear to contribute a “print now” signal (for review, see Singer, 1987). What is the evidence for activity dependence of a plastic change in neural transmission! One approach to determination of activity-dependent plasticity and the elements important for plastic changes is selective interference with specific afferent activity to the system under study, for instance, by deprivation of sensory input. For example, the elaboration of the functional organization in the visual cortex in the presence of

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normal visual experience can be compared with that after elimination of visual experience by dark rearing or by manipulation of visual experience through unilateral lid suture (see below). Similarly, the elaboration of the representation of sensory organs within the CNS, such as whiskers on a rodent’s snout, can be studied following destruction of the sensory follicles of the whiskers. Alternatively, afferent pathways can be selectively stimulated by electrical or pharmacological means. This paradigm is currently being used to study long-term potentiation in the hippocampal formation (see below). All of the aforementioned experimental paradigms use a manipulation of afferent (presynaptic) activity to study experience-dependent plasticity. Postsynaptic activity, on the other hand, can be influenced selectively by controlling the membrane potential of single cells by intracellular current injection or, more globally, by blockade of synaptic transmission with blockers of transmitter receptors or interference with transmitter release. Such approaches allow determination of the contribution of presynaptic and postsynaptic activity on plasticity. T h e overall blockade of neuronal activity induced by blocking action potentials with tetrodotoxin has also been used in several model systems to determine the activity dependence of plastic changes (Reiter et al., 1986; Shatz, 1990a); however, because this experimental paradigm interferes with both presynaptic and postsynaptic activity, the elements that must be active for plasticity to occur cannot be determined precisely. I n the present overview, I focus on mechanisms of central nervous plasticity following the rules outlined earlier (extended Hebbian rules), that is, plasticity initiated by neuronal activity and guided by coincidence of presynaptic and postsynaptic activity. The current knowledge on the contributions of presynaptic and postsynaptic activity in some of the best studied model systems of activity-dependent plasticity is summarized in a later section.

B. GLIALCELLS: MORETHAN JUST NERVEGLUE Although glial cells constitute about half of the overall brain volume and outnumber neurons (Pope, 1978), they have attracted only limited attention from neurophysiologists since their first description by Virchow (1846). Indeed, Virchow’s claim that glial cells were nothing more than “nerve glue” is, with minor modifications, still the view of many neurophysiologists; however, considerable progress has been made in recent years in the understanding of glial physiology and neuronal-glial interactions, and this has, in turn, forced a revision and an extension of our view of the mechanisms underlying brain function. Two important

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functions of glial cells, a role in neuronal migration (for review see Hatton, 1990) and maintenance of ionic homeostasis (Kumer, 1967; Walz and Hertz, 1983; Walz, 1989), have gained general acceptance. These functions can be attributed to a rather passive role of glial cells in brain development and central nervous information processing; however, the discovery of transmitter and neuromodulator receptors on glia (for reviews see Kimelberg, 1988; van Calker and Hamprecht, 1980) and the release of neuroactive compounds from these nonneuronal cells (Banker, 1980; Levi, 1984; Lindsay, 1979; Manthorpe et al., 1986; Rudge et al., 1985; Unsicker et al., 1987) has set the stage for thinking of a more active role for glial cells in brain function, including activity-dependent plasticity in the CNS. A conceptual integration of glial cells in the computational processes underlying perception and associative thinking, which finally influence or lead to adaptive processes in the brain and changes in behavior, relies on the presence of functional neuronal-glial and glial-neuronal interactions. Several reviews have elegantly summarized the existence of such interrelations (Laming, 1989; Stone and Ariano, 1989; Vernadakis, 1988). Therefore, only those findings that are relevant to plasticity in the CNS, specifically plasticity ruled by coincident or noncoincident presynaptic and postsynaptic activity, are referred to in the following discussion. An overview of the obvious interactions between presynaptic and postsynaptic neurons and adjacent glial cells is given in Fig. 2. These interactions are detailed in the following sections. Presynaptic Neuron

Postsynaptic Neuron

transmitter

'retrograde messengers'

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FIG. 2. Schematic representation of possible routes of' information transfer between a presynaptic neuron, a postsynaptic neuron, and an adjacent glial cell. Note that all three partners in this communication system may interact.

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22 1

A prerequisite for an involvement of glial cells in activity-dependent plasticity in the brain is that they have to monitor neuronal activity. There are several ways in which glial cells can accomplish this. The simplest way for monitoring neuronal activity is to measure the extracellular potassium concentration, which reflects the overall postsynaptic activity level of surrounding neurons. Indeed, astrocytes do behave like “potassium sensors” (Kuffler, 1967; Orkand et al., 1966; Wuttke, 1990), in as much as their membrane potential reflects the extracellular potassium concentration. Changes in extracellular potassium have been shown to influence glial cells not only on a short time scale, for example, by reversible depolarizations in the range of seconds (Casullo and Krnjevic, 1987; Konnerth et al., 1988; Kuffler, 1967; Schwartzkroin and Prince, 1979), but also on a considerably extended time scale, via influences on cellular metabolism (Brooks and Yarowsky, 1985; Pentreath and Kai-Kai, 1982; Salem et al., 1975) and activation of the genome (Lubin, 1967; Lipton and Heimbach, 1978; Reichenbach, 1989; Reichenbach and Reichelt, 1986). From extracellular potassium measurements in the cat visual cortex, with its clear-cut columnar representation of stimulus features, it can be concluded that the potassium signal emerging from neurons is indeed a very local signal. Extracellular potassium within the cat visual cortex selectively rises on sensory stimulation of a given orientation column (containing neurons selective for one orientation of light bars; Hubel and Wiesel, 1963), but not when a column about 500 pm distant is stimulated by light bars of the orthogonal orientation (Singer and Lux, 1975). This local selectivity of the potassium signal is also reflected in the specificity of changes in the membrane potential of intracellularly recorded glial cells in the visual cortex to different stimulus orientations (Kelly and van Essen, 1974). From experiments aimed at determining the “stimulus selectivity” of extracellular potassium gradients in areas with a well-known functional topography, it has been concluded that the potassium signal does not spread over more than about 100 pm from a focus of neuronal excitation (Singer and Lux, 1975). This high local specificity of potassium gradients and their influence on glial cells closely restricted to the source of extracellular potassium (i.e., active neurons) may be explained,by the very recent finding that the astroglial length constant for spatial potassium buffering is below the dimensions of a single cell (Barres et al., 1990b). In addition to the direct action of changes in extracellular potassium on the membrane potential of glial cells, the depolarizing effects resulting from local increases in the extracellular potassium concentration may be further amplified by voltage-dependent ion channels in glial membranes (for review see Barres et al., 1990a). This brief survey of neuronal-glial interactions via potassium

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movements shows that this is a highly suitable mechanism for glkdl cells to monitor very local levels of postsynaptic neuronal activity. This mechanism seems sufficient for guaranteeing one necessity of Hebbian plasticity, namely, a localized influence of postsynaptic neuronal activity. One way by which glial cells are also potentially capable of monitoring presynaptic neuronal activity is indicated by the presence of transmitter receptors on glia (for review see Kimelberg, 1988). These include receptors responsive to glutamate (Backus et al., 1989; Kettenniann and Schachner, 1985; Somogyi et al., 1990), y-aminobutyric acid (Backus et al., 1988; Hiisli el a/., 1990; Kettenmann and Schachner, 1985), acetylcholine (Ballanyi and Schlue, 1989; Hosli and Hosli, 1988; Hosli et al., 1988; Murphy et a/., 1986; Pearce et al., 1985), and norepinephrine (Ernsberger et a / . , 1990; Hosli et af., 1982; Lerea and McCarthy, 1989; Murphy and Pearce, 1987; Salni and McCarthy, 1989; Stone and Ariano, 1989; Trimmer and McCarthy, 1986; Trimmer et al., 1984). In addition to transmitter-activated ion channels, receptor-stimulated second messenger cascades that are responsive to transmitters or neuromodulators have also been characterized (glutamate: Milani et a/., 1989; Nicoletti et al., 1990; Pearce Pt al., 1986b, 1990; acetylcholine: Hansson, 1989; Masters et a/., 1984; Pearce et a/., 1985; norepinephrine: Kanterman et al., 1990; McCarthy and devellis, 1978; Pearce et al., 1985; Woods et al., 1989). Because of the intimate association between glial cell processes and synapses (Peters ~t ul., l970), the presence of receptors for substances released by synaptic terminals allows glial cells to monitor local neuronal presynaptic activity, the second prerequisite for integration into the Hebbian plasticity model. A further potential mechanism of neuronal-glial information transfer may involve diffusible factors released from neurons on activation. Arachidonic acid and its metabolites (Axelrod et a/., 1988) and nitric oxide (Brendt et ul., 1990; Garthwaite, 1991) are among the factors released from neurons o n postsynaptic activation that may influence glial cells (Murphy and Welk, 1989). These factors have only recently been discovered and characterized. It becomes evident from the experimental data summarized above that glial cells are capable of monitoring both presynaptic and postsynaptic. activity by different mechanisms, allowing information about coherency of local presynaptic and postsynaptic neuronal activity to be communicated. Thus, glial cells may be in a perfect position not only to monitor changes in neuronal states that precede and initiate plastic changes, but also to determine the direction of this change, for example, upregulation or downregulation of synaptic transmission. The second prerequisite for a causal role of glial cells in activity-

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dependent processes is glial-neuronal signaling. This may be carried by neurotransmitter substances released from glia (taurine: Holopainen et al., 1985; Lehmann and Hansson, 1988; Pasantes-Morales and Schousboe, 1989; Philibert et al., 1988; Tigges et al., 1990; D-aspartate: Drejer et al., 1983; Holopainen and Kontro 1990; glutamate: Drejer et al., 1982; Szatkowski et al., 1990; glycine: Holopainen and Kontro, 1989) or by other diffusible factors. Among the latter are presumed intercellular messengers such as arachidonic acid and its metabolites and nitric oxide (Hartung and Toyka, 1987a,b; Hartung et al., 1988; Ishizaki et al., 1989; Murphy et al., 1985, 1990; Murphy and Welk, 1989), or factors modulating transmitter receptors (Forsythe et al., 1988).An additional class of molecules that may play a role in neuronal plasticity and that are of considerable importance to the development of the nervous system are glia-derived matrix molecules and neuronotrophic factors (for reviews see Manthorpe et al., 1986; Matthiessen et al., 1989; Miiller and Seifert, 1982; Miiller et al., 1984; Tarris et al., 1986; Unsicker et al., 1987). In activity-dependent plasticity such factors may play a significant role in glial-neuronal signaling, for example, for inducing synaptic remodeling. In addition to the mechanisms described above, neuronal activity can be influenced by changes in neuronal excitability by modifications of the ionic milieu (Holopainen et al., 1989; MacVicar et al., 1988) or by an increased presence of neurotransmitters in the extracellular environment, for example, by modulation of transmitter uptake (Barbour et al., 1989). Finally, glial cells may influence neuronal circuitry by mechanical disruption of synaptic contacts or, conversely, by inducing or providing access to new sites of synaptic terminations via directed growth or process retraction (see below). Accordingly, there is also abundant evidence for the presence of glial-neuronal signaling, which may lead to plastic changes in synaptic efficacy and reorganization of interconnectivities. In summary, from the recent extension of our knowledge on the physiology, biochemistry, and pharmacology of glial cells it can be concluded that these cells express the necessary capacity for an integration in activity-dependent plastic processes in the brain. This is true of their ability to monitor presynaptic and postsynaptic activity, as well as of mechanisms to influence neurons in return. Because of the increasing evidence that astrocytes are heterogeneous, for example, with respect to transmitter receptor expression or the release of proteins (for review see Wilkin et al., 1990), mechanisms for neuronal-glial and glial-neuronal transfer may be adapted to the specific requirements of a given central nervous area. This issue is one of the challenges of future studies on glial cells.

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To determine a possible role for glial cells in activity-dependent plasticity it is essential to describe the phenomena paralleling the manifestation of plasticity. A summary of the current knowledge is given in the following section.

C. ACTIVITY-DEPENDENT PLASTICITY: A MODIFICATIONOF SYNAPTIC: EFFICACY OR CIRCUITRY? Effects of plastic changes can be studied by electrophysiological or anatomical techniques. By the use of these approaches, several developmental processes, those presumed to relate to learning and memory, as well as several aspects of remodeling of CNS circuitry following damage, have been shown to rely on neuronal activity. Activity-dependent plasticity underlies the formation of function-related areas (for review see Sur et ul., 1990), interareal and intraareal connectivities (ConstantinePaton et al., 1990; Innocenti, 1981; Luhmann et al., 1986, 1990a,b,c; Singer, 1987), and changes in the efficacy of neural transmission (for review see Teyler and DiScenna, 1987). In the following paragraphs I briefly describe several well-studied model systems displaying activity-dependent plasticity and the underlying changes that can be observed, beginning with plastic changes influencing the global functional architecture of the brain (areal pattern, morphology, interconnectivity) and ending with model systems displaying activity-dependent changes in local properties of synaptic transmission. The intention of this summary is to outline possible general rules for (or effects of) activity-dependent plastic changes in the CNS that may help to assess the underlying mechanisms. 1. Activity-Dependent Emergence vf Areal Putterm

and Global Afferent Specificity

‘The first indication of an influence of afferent activity on the formation of areal patterns in the CNS came from deprivation experiments. Depriving animals of sensory experience in one modality during early development leads to an “invasion” of responses to different sensory modalities in structures normally processing the deprived modality. Thus, following visual deprivation of young animals, auditory responses are observed in areas of the cat’s superior colliculus that usually code for visual stimuli (Rauschecker and Harris, 1983). Such cross-modal plasticity has been observed not only in the visual system but also in other cortical areas, for example, the somatosensory cortex (Glass, 1973; Gyllensten et al., 1966; Heumann and Rabinowicz, 1982; Toldi et nl.,

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1990). One naturally occurring example of this kind of cross-modal plasticity has been elucidated in the mole rat, a subterranean mammal. In this animal, which normally has almost no visual experience, visual areas are functionally innervated by auditory afferents (Bronchti et al., 1989). Similar cross-modal plasticity has been obtained after destruction of modality-specific afferents to sensory nuclei or target regions in early developmental periods. Thus, if the normal targets of retinal axons, the lateral geniculate nucleus and superior colliculus, are destroyed in early postnatal development, retinal fibers innervate the auditory and somatosensory thalamic nuclei (Frost, 1982; Vitek et al., 1985). This type of experimentally induced change leads to functional anomalous projections from a given sensory modality which then reveals a striking functional integrity specific for the new target area (for reviews see Sur et ul., 1990; Roe et al., 1990). Interestingly, the overall laminar pattern and the efferent connections of the given area are not influenced by these manipulations (Sur et ul., 1990). These findings suggest that the basic features of a given area in the CNS are inherent, whereas the final anatomical and functional composition of afferents and the information transfer to postsynaptic networks can be influenced by experience (for review see O’Leary, 1989b). A general scheme of this kind of activitydependent emergence of area-specific projections is given in Fig. 3. Although the developmentally preformed projection from a given afferent shows some degree of inaccuracy, that is, it innervates additional targets, a restriction to one target is achieved in the presence of normal activity levels in all afferents. This developmental process can be influenced by inactivating a subset of afferents, leading to the stabilization of afferents from a different source to the target (Fig. 3). In addition, if a target is removed, the destined afferent can maintain afferents to a different target (Fig. 3). With respect to the mechanisms underlying this kind of activity-dependent plasticity, the available experimental data suggest that the occurrence of anomalous projections after deprivation or modality-specific lesions is due to a stabilization of aberrant (exuberant) projections present in early development (Frost, 1982; Frost and Moy, 1989; O’Leary et al., 1981; O’Leary, 1989; O’Leary and Stanfield, 1989). This implies that the emergence of specific afferents to a given area is usually preformed grossly by intrinsic cues, for example, surface markers (Dodd and Jessel, 1988; Ghosh et al., 1990; Stiirmer, 1990; Walter et al., 1987); a certain degree of inaccuracy in the preformation of areaspecific interconnectivities (for review see Stanfield, 1984), as seen by the presence of aberrant projections, is subsequently corrected in an activitydependent manner. This activity-dependent process is based on a selective elimination of “inappropriate” afferents and collaterals (Innocenti,

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target 1

afferent 1

i target 2

target 3

afferent 2

afferent 3

normal development

I

experimental manipulation

FIG. 3. General scheme of the refinement of topographical connections during development. While the prqjections from three afferent structures reveal a certain degree of overlay early in development (top), specific projections of each afferent to the appropriate target elaborate when normal activity is present in each afferent (lower left). On inactivation of one afferent system or elimination o f one target, previously established exuberant projections front another afferent system to the corresponding target or from an afferent to a different target may be stabilized (lower right).

1981; O’Leary rt a/.,1981).One important feature in the determination of “inappropriateness” is the weaker efficacy of exuberant afferents from different modalities in exciting the postsynaptic cells, compared with the more xiumerow “appropriate” afferents. This is suggested by the fact that experimental weakening or elimination of the dominant afferent by deprivation or lesions supports the stabilization and proliferation of afferents that normally would be eliminated (for review see Fawcett and O’Leary, 1985). In summary, although the emergence of areal patterns defined by functional afferent connectivity is most likely preformed independent of

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activity, for example, by target-specific recognition molecules (Dodd and Jessel, 1988), it is refined by activity-dependent processes. This refinement is mediated by the elimination of inappropriate, and the stabilization of appropriate, afferents to a given area. The underlying mechanisms leading to this change in connectivity rely on competition between different afferent structures, as destruction or functional suppression of a dominant afferent allows a normally eliminated input to be stabilized. This mechanism thus follows the rules of activity-dependent plasticity outlined earlier. The changes subserving the refinement of afferents, leading to the establishment of a mature system, are usually confined to early developmental periods (Sur et al., 1990). 2. Activity-Dependent Emergence of Interareal Connectivities and Intraareal Topographies Similar to the establishment of global characteristics of functional afferents, the establishment of response specificities reflected in local topographies of interareal and intraareal connections is also specified by neuronal activity (Constantine-Paton et al., 1990; for reviews see Fawcett and O’Leary, 1985; Shatz, 1990b). Among the best studied examples of activity-dependent elaboration of a functional topography is the development of ocular dominance columns in the visual cortex and the somatotopic representation of the whiskers (barrel fields) in the rodent somatosensory cortex. In the visual system, thalamocortical afferents representing either eye converge onto the same cortical area during early development, whereas a segregation into alternating stripes of cortical tissue innervated by thalamic axons conveying information from one or the other eye only, is achieved during later developmental stages (LeVay et al., 1978; Rakic, 1990; Singer, 1987). In the cat visual system this segregation, which leads to the elaboration of ocular dominance columns (Shatz et al., 1977), is prevented on elimination of neuronal activity by tetrodotoxin injection into the cortex (Reiter et al., 1986; Shaw and Cynader, 1984), silencing of the afferent system by intraocular tetrodotoxin injection (Stryker and Harris, 1986), or visual deprivation (Mower et al., 1981, 1985). An extreme example of the process of segregation of initially overlapping afferents occurs following monocular deprivation early in the postnatal period. This experimental paradigm leads to a severe loss of afferents representing the deprived eye (Shatz and Stryker, 1978). In addition, there is evidence that concomitant sprouting of stabilizing afferents representing the nondeprived eye occurs simultaneously with elimination of the other subset of afferents. The clearest example of this process is the reversal of the effects of monocular deprivation shown in monkeys. If one eye is visually deprived for a few weeks

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soon after the time of eye opening, the cortical territories innervated by axons representing this eye shrink considerably. If the deprived eye is reopened early in the postnatal period while the other eye is deprived at the same time, resprouting of the initially deprived afferents occurs (LeVay f t al., 1980; Swindale P t a/., 1981). These phenomena closely resemble that described above for the elaboration of area-specific connectivities. Competition between dif€erent afferent structures appears to be the basis for the developmental refinement of connections seen in these examples and also for the elaboration of topographic arrangements of circuitries to or within one area. Furthermore, these processes are also limited to a period early in development (Hubel and Wiesel, 1970; Olson and Freeman, 1980). The influence of neuronal activity on the elimination and stabilization of competing afferent systems, similar to that in the development of the thalamocortical visual pathway, is known to be present in the retinothalamic system (Shatz and Stryker, 1988; Shatz, 1990a), interhemispheric callosal projections (Innocenti and Frost, 1979; Innocenti et al., 1985), horizontal intracortical projections (Luhmann P t nl., 1986), and neuromuscular junction (Brown rt al., 1976; but see Callaway et al., 1987; Thompson rt al., 1979). Keorganization of competing af€erent systems has also been shown to rely on synapse formatioii and elimination during development in invertebrates (Lnenicka and Murphey, 1989). A malleability of sensory representations similar to that described above is present in the somatosensory system. Whereas the plastic changes described in the last section are reflected mainly in the reorganization of afferents, these plastic changes are due mainly to a change in the topographical organization of postsynaptic neurons. I n the adult rodent somatosensory cortex, a unique arrangenient of neurons is present, reflecting the arrangenient of vibrissae on the snout of the animals (Woolsey and van der Loos, 1970). These so-called barrels, present in layer IV of the rodent somatosensory cortex, consist of a ring of neuronal somata that encircle a nearly cell-free area. Each whisker activates one restricted, barrellike cortical structure. T h e elaboration o f this one-to-one relation of vibrissae and cortical barrels is disturbed by manipulation of the sensory input. Elimination of individual whiskers during early development results in the failure of development of the related barrel in the cortex (van der Loos and Woolsey, 1973; Woolsey and Wann, 1976). This implies that activity of afferent fibers leads to rearrangement of the cortical neuronal topography. In contrast to the activity-dependent alterations in the topographical arrangement of the afferent fibers seen, for example, in the visual cortex, the dominant feature observed in the somatosensory system is refinement of the topo-

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graphical organization of the postsynaptic targets, that is, the arrangement of neuronal somata (van der Loos and Woolsey, 1973) and dendritic arbors (Greenough and Chang, 1988). In the somatosensory system, afferent fibers already reveal a topographical order representing the peripheral pattern of vibrissae (Erzurumlu and Jhaveri, 1990).Thus, inhomogeneity of the afferent input may be the basis of the observed topographical specialization developing by neuronal migration. Similar influences of afferent activity on the cytoarchitectonic characteristics of a CNS substrate have been shown in the insect olfactory system (Tolbert and Oland, 1989). As in the somatosensory cortex, the formation of glomeruli by neurons and the number and placement of synapses are dependent on afferent fibers (Tolbert, 1989). Additional examples of afferent activity influencing the morphological characteristics of postsynaptic structures are seen in the modifications of dendritic morphology of neurons in the visual (Tieman and Hirsch, 1982) and auditory (Gray et al., 1982; Smith et al., 1983) systems on manipulation of afferent neuronal activity. In summary, it can be shown that long-term manifestation of plastic changes in interareal and intraareal connections and topographies in the CNS governed by afferent activity relies on two major mechanisms: an influence on the refinement of the topography of afferents, and an influence on the morphological characteristics of postsynaptic structures. Whereas the first influence is mediated mainly by the selective elimination of one of several competing afferent systems (e.g., afferents representing one eye in the thalamocortical visual system) and probably also by concomitant sprouting of the stabilized afferent, the second influence is on neuronal migration and morphological development of postsynaptic structures. Both activity-dependent changes are maintained as a permanent change in interconnectivity, that is, in the “hardware” of the CNS. Like the mechanisms leading to elaboration of functionally defined areas, those underlying the elaboration of interareal and intraareal connectivities and functional topographies are also confined to early development (Hubel and Wiesel, 1970; Innocenti et al., 1985; Olson and Freeman, 1980; Woolsey and Wann, 1976).

3. Activity-Dependent Efficacy Changes of Neural Transmission The best studied model system of activity-dependent plasticity, so far, is long-term potentiation in the hippocampus. This cortical structure has been implicated in memory and learning, as damage of the hippocampus severely interferes with memory consolidation and, to a certain extent, also memory retrieval (Zola-Morgan and Squire, 1990). A suspected physiological correlate of memory storage at the level of the

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hippocampus is long-term potentiation (LTP), a long-lasting increase in the ef'ficacy o f neuronal transmission following a brief period of highfrequency stimulation to the afferents of hippocampal substructures (for review see Teyler and DiScenna, 1987). Like the plastic changes outlined earlier, this paradigm of activity-dependent plasticity is strongest early in the postnatal period (Harris and Teyler, 1984; Teyler, 1989) and follows Hebbian rules (Kelso et al., 1986). Despite several years of study in many laboratories worldwide, it is as yet impossible to formulate a general hypothesis for the mechanisms underlying LTP, mainly because in subregions of the hippocampus there are differences in LTP induction. In area CA1 and the dentate gyrus of the hippocampus activation of the N-methyl-maspartic acid (NMDA) receptor, a subtype of glutamate receptor coupled to a voltagemodulated cation channel, has been shown to be essential for the induction of LTP (for review see Collingridge and Singer, 1990); however, this is not necessarily the case in area CA3 (Harris and Cotman, 1986; Kauer and Nicoll, 1988). In addition, recent experiments on LTP in the CAI region have revealed NMDA-independent L'TP under stimulus conditions that lead to high neuronal activity (Aniksztejn and Ben-Ari, 1991; Grover and Teyler, 1990a). These reservations have to be kept in mind when possible mechanisms of LTP are discussed. Current hypotheses on L'TP in the CA1 region assume that the following events are necessary for its induction: (1) acti\.ation of NMDA receptors (Collingridge and Singer, 1990; Kauer et ul., 1988a); (2) calcium influx into the postsynaptic neuron (Lynch f t a/., 1983; Malenka rt al., 1988); (3) activation of calcium-dependent protein kinases, for example, protein kinase C and calcium/calmoduIin-dependent kinase (Colley et al., 1989; Malenka et al., 1986, 1989; Reymann et al., 1988a,b). As NMDA-independent LTP is possible, it may be assumed that substantially high postsynaptic activity is the initial trigger for the induction of this process. NMDA receptors may just be highly suitable, but not essential, to permit such high postsynaptic activation. It was originally suggested that these processes lead to a longlasting change in postsynaptic sensitivity to presynaptically released transmitter (Kauer et nl., 1988b); however, experimental approaches have as yet failed to support this theory (for review see Andersen, 1987). Conversely. evidence for presynaptic mechanisms in the maintenance of this activity-dependent plasticity have been available for a long time. Studies from two laboratories showed an increase in transmitter release following induction of LTP (Dolphin et al., 1982; Skrede and MaltheSorenssen, 198l), and quanta1 analyses of postsynaptic events also pointed to a presynaptic locus of LTP manifestation (Voronin, 1983). Only very recently have these findings gained considerable support, in partic-

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ular from studies using patch-clamp techniques to measure the amplitudes of synaptic currents elicited by stimulation of single afferent fibers before and after induction of LTP (Bekkers and Stevens, 1990; Malinow and Tsien, 1990; Malinow, 1991). These studies revealed a predominant presynaptic component in LTP manifestation, namely, an increase in the probability of transmitter release. These studies also supported earlier data excluding an increase in the density of postsynaptic glutamate receptors as a basis for LTP maintenance (Goh and Sastry, 1984). One solution to the apparent paradox that induction of LTP seems to be localized postsynaptically, while its manifestation is presynaptic, is the presence of an activity-dependent signaling system from postsynaptic to presynaptic sites. One of the candidates subserving such a function is arachidonic acid or its metabolites. Indeed, application of arachidonic acid is sufficient to induce long-term increases in synaptic efficacy (Williams et al., 1989), and blockers of the lipoxygenase pathway of arachidonic acid metabolism block the induction of LTP (Lynch et al., 1989; Williams and Bliss, 1989a,b). The slow time course of arachidonicacid induced LTP does not, however, coincide with the rapid effect of tetanic stimulation on synaptic potentiation. Despite our knowledge of the phosphorylation of phosphoproteins following induction of LTP (Lovinger et al., 1986; Nelson et al., 1989a,b; Routtenberg et al., 1985), there is very limited information about the mechanisms that may lead to the presynaptic changes in transmitter release. Recent evidence suggests that a G-protein-mediated mechanism located at a site distinct from the postsynaptic neuron (presynaptic or glial) is an intermediate step in the elaboration of LTP (Goh and Pennefather, 1989). Whereas the experiments summarized above focus on a change in synaptic transmission at preformed synapses, an alternative explanation for the mechanisms of LTP manifestation is based on morphological studies. As early as several minutes after the induction of LTP there is an increase in synaptic density on postsynaptic cells (Chang and Greenough, 1984; Lee et al., 1980). Although this is due primarily to an increase in the number of synapses on dendritic shafts, there is also evidence for the occurrence of Y-shaped spines receiving two afferent boutons from different afferent synapses on induction of plasticity (Geinisman et al., 1989). These anatomical data corroborate an earlier hypothesis that synapses may divide (Carlin and Siekevitz, 1983) and suggest that formation of new synapses may underlie the maintenance of LTP. This is further corroborated by the finding that growth factors support (Terlau and Seifert, 1989, 1990; Abe et al., 1991) or even induce (Sastry et al., 1988b; Xie et al., 1991) LTP. Another finding that does not coincide with the aforementioned hypothesis of LTP manifestation

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comes from recent experiments on organotypic cultures of hippocampal sections. These investigations have provided evidence that enhancement of synaptic transmission is not strictly confined to connectivities where the NMDA-dependent induction mechanism has been activated (Bonhoeffer et al., 1989). In these experiments coincident activity was produced at a given cell by pairing weak afferent activity with strong postsynaptic depolarization by intracellular current injection. Changes in synaptic transmission were monitored using voltage-sensitive dyes, which allow simultaneous observation of the responses of a large neuronal population. It could be shown that the enhancement of' synaptic transmission was not confined to the cell that received the pairing procedure, but occurred in a large population of neighboring cells (Bonhoeffer et al., 1989). Similar data have also been obtained in acute slice preparations of the visual cortex (Kossel et al., 1990),which has been shown to display LTP (Artola and Singer, 1987; Bindman and Prince, 1986). The increase in synaptic efficacy resulting from pairing afferent activity with postsynaptic current injection, again, was not confined to the treated cell but was also evident in a simultaneously recorded neighboring cell that did not undergo the pairing procedure (Kossel et al., 1990). Similar global effects of afferent activity have been observed in an in vitro system established for the study of activity-dependent competitive interactions of two afferent systems on one postsynaptic target (Nelson et al., 1990). In a three-compartment culture system (Campenot, 1977) the central compartment contained the target neurons (ventral horn spinal cord cells), and the two side compartments contained dorsal root ganglion neurons that extended axons into the central compartment. Each afferent system could be selectively stimulated electrically for prolonged periods. Efficacy changes in the transmission to the neurons in the central compartment were investigated by intracellular recording. With this approach it was shown that compared with nonstimulated afferents, activated afferents revealed more synaptic contacts to the target neurons and an increased amplitude of postsynaptic responses (Nelson et al., 1989, 1990). N o reduction in the number of synapses was observed in the nonstimulated afferents. Indeed, this afferent system also revealed a slightly increased number of synapses compared with control cultures (Nelson et al., 1990). Again, this contradiction to the general hypotheses of synaptic selectivity of plastic changes may be explained by a more global role of growth-promoting factors in LTP and other activity-dependent plastic changes. These assumptions are described in detail below. Another well-studied model system displaying an activity-dependent long-term enhancement of synaptic transmission is the neural circuit

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underlying the gill withdrawal reflex in the sea snail, Aplysia (for review see Kandel and Schwartz, 1982). Recent evidence from anatomical experiments in Aplysia (Bailey and Chen, 1989), and from an in vitro analog of the neuronal system underlying physiological and behavioral plasticity (Glanzman et al., l990), revealed that manifestation of the plastic change underlying behavioral and physiological sensitization of the gill withdrawal reflex is paralleled by an increase in the number of synapses. Thus, multiple evidence supports the notion that increases in synaptic efficacy may rely on an increase in the number of synapses conveying information from a presynaptic afferent to the postsynaptic target. In addition to long-term increases in synaptic transmission, there is accumulating evidence for the presence of activity-dependent long-term depression (LTD). This phenomenon was originally described in the cerebellum, where activation of the climbing fiber system results in the long-term reduction of neural transmission of parallel fibers to Purkinje cells (for review see Ito, 1987). Recent data indicate the presence of similar phenomena in the hippocampus (Stanton and Sejnowski, 1989) and neocortex (Artola et al., 1990). While in the cerebellum this phenomenon has been linked to changes in postsynaptic sensitivity to glutamate (It0 et al., 1982), detailed analyses of hippocampal and cortical LTD have not yet been carried out. As summarized above, current hypotheses on the mechanisms of LTP suppose a postsynaptic induction, followed by information transfer to the presynaptic site and, ultimately, an increase in the probability of transmitter release as one of the mechanisms leading to long-term manifestation of the plastic change. The precise mechanisms underlying the latter are yet only poorly understood. A plausible explanation could be based on the morphological data that reveal a rapid increase in the number of synapses following induction of LTP. This would, again, indicate that this kind of activity-dependent plasticity may also rely on changes in the circuitry rather than on modulation of a constant number of synapses. In accordance with other model systems of activity-dependent plasticity, LTP also occurs predominantly early in the postnatal period (Harris and Teyler, 1984; Komatsu et al., 1981; Teyler, 1989; Kato et al., 1991). Current investigations on long-term depression have revealed a postsynaptic induction mechanism and suggest a postsynaptic locus for maintenance. 4. Is There a General Mechanism of Activity-Dependent Plasticity? From the overview given in the preceding sections, several mechanisms that underlie activity-dependent plasticity in a variety of model systems may be deduced. One general feature of activity-dependent

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plasticity is its preponderance during, or even restriction to, early development. Thus, it can be reasonably concluded that mechanisms underlying plastic changes are developmentally regulated. With respect to the prerequisites leading to adaptive changes the reader is referred to the hypotheses of Hebb (1949) and Stent (1973). While coincident presynaptic and postsynaptic activity leads to an increase in synaptic transmission, noncoincident activity results in a weakening of synaptic transmission (Callaway et al., 1987; but see Reiter and Stryker, 1988).Activation of the NMDA receptor seems to be important for the induction of increases in synaptic transmission in several systems studied so far. Yet, there is ample evidence for plastic changes independent of this receptor system. In all instances high postsynaptic activity seems to be the mechanism triggering plastic changes, induced by either NMDA or non-NMDA receptors. Postsynaptic calcium influx seems to be essential to the induction of plasticity. Heterosynaptic plastic changes (e.g., those induced at one given set of synapses and manifested at a spatially different locus) on a given cell, or even at separate neuronal populations, call for a more global mechanism in the manifestation of plasticity. The maintenance of plastic changes seems to rely on a reorganization of synaptic connections or changes in the morphology of postsynaptic targets. This is obvious in the case of the developmental refinement in topographical organization and the elaboration of areal patterns. In both instances synapse elimination, new synapse formation, and adaptive changes in dendritic morphology are present coincident with the long-term manifestation of plastic changes. In LTP-like plastic changes, rapid changes in neural transmission follow the induction of plasticity. These seem to be paralleled by an increase in transmitter release or availability. For. the lotig-tertn tnanifestatioti of these changes there exist morphological data also suggesting a change in circuitry. These considerations stress the importance of structural changes in the morphology of neurons and axonal connectivity for activity-dependent plasticity. Hypotheses describing the putative mechanisms of these adaptive changes have to include processes that ( 1) are developmentally regulated, (2) subserve the elimination of neuronal connections, and (3) support new synapse formation andlor changes in neuronal morphology. II. Evidence for Participation of Glial Cells in Activity-Dependent Plasticity

A. ACTIVITY-DEPENDENT FORMATION OF INTRAAREAL TOPOGRAPHIES

T h e formation of the whisker representation in the somatosensory cortex is one of the clearest examples of an activity-dependent emer-

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gence of neuronal topography. As outlined earlier, each of the sensory follicles associated with a whisker on the rodent’s snout is represented in a cortical barrel. These barrels consist of a ring of neuronal cell bodies surrounding an area virtually free of neuronal somata. Dendritic arborizations extend into this latter region and receive contacts from afferent thalamocortical fibers (Greenough and Chang, 1988; Woolsey and van der Loos, 1970). This topography elaborates during the first postnatal days and can be severely disturbed by partial deprivation of sensory input (van der Loos and Woolsey, 1973; Woolsey and Wann, 1976). The recent observation that staining procedures using certain lectins reveal the future barrel topology before the neuronal topography has emerged suggests an instructive mechanism that is not intrinsic to the cortical neurons (Cooper and Steindler, 1986a,b, 1989; Steindler et al., 1989a). Like the topography of barrels, the boundaries visualized by lectin staining prior to the establishment of neuronal aggregates are also malleable to partial destruction of sensory follicles. The pattern of lectin staining in early postnatal development of the somatosensory cortex is shown in Fig. 4.The “protobarrels”are clearly seen demarcated by lectin binding. In addition, elimination of whiskers is shown to lead to loss of the corresponding “protobarrels,” as visualized with lectin binding. (Cooper and Steindler, 1989; Steindler et al., 1990).These data indicate that even the formation of this presumed template for the future barrels is influenced by afferent activity. This is further supported by recent evidence that protobarrels can be induced in pieces of visual cortex transplanted into the target region of somatosensory thalamocortical projections (Schlaggar and O’Leary, 1991).Similar boundaries visualized by lectin binding have also been observed in other developing structures in the CNS prior to the elaboration of neuronal structures (Crossin et al., 1990; Steindler and Cooper, 1987; Steindler et al., 1988).Studies aimed at elucidating the cellular origin of the protobarrels visualized by lectin staining revealed that they are related to astrocytes immunoreactive for glial fibrillary acidic protein (GFAP) (Cooper and Steindler, 1989; Steindler et al., 1989b). Staining of protobarrels by the astroglial marker GFAP is shown in Fig. 4. It has been suggested that afferent neuronal activity influences cortical astrocytes, which then express surface markers suitable for guiding neuronal migration and possibly also influencing dendritic arborizations to form the appropriate barrels. Experimental support for the proposed sequence of causalities, namely that afferent activity is an essential prerequisite for the formation of the glial boundaries and that glial boundaries are an essential prerequisite for the formation of neuronal boundaries, comes from studies on the insect olfactory system. This system shares the presence of neuronal glomeruli with the somatosensory cortex. These neuronal glomeruli fail to form in the

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absence of sensory input (Oland and Tolbert, 1987; Tolbert, 1989). Similarly, elimination of af€erent input in early development also interferes with the formation of glial boundaries preceding elaboration of the neuronal topography. If insect larvae are exposed to X rays or gliotoxic substances early in development, glial cells are eliminated to a great extent (Oland et al., 1988; Oland and Tolbert, 1988; Tolbert and Oland, 1989). Despite the presence of afferent fibers innervating the olfactory neurons, these fail to form glomeruli in the absence of glial cells. It is thus concluded that glial cells are an essential mediator in the formation of neuronal topography, whereas afferent activity instructs nonneuronal cells to subsequently instruct the neuronal environment. To date, the mechanisms of information transfer at each of these steps are not well understood. This is especially true for the effects of afferent activity on glial cells in the somatosensory cortex and insect olfactory system. The influence does not seem to affect glial proliferation, but rather glial morphological differentiation and migration (Oland and Tolbert, 1989). Recent studies on the expression of certain types of cell adhesion molecules associated with glial cells suggest that these may be mediators of the signal transfer from glia to neurons (Crossin et al., 1990; Steindler et al., 1990). One of the adhesion molecules expressed by glial cells of the protobarrels, namely J l/tenascin (Steindler et al., 1990), has been shown to affect neuronal migration and adhesion, as well as neurite extension in uitro (Faissner and Kruse, 1990; Lochter et al., 1991). A similar involvement of glial cells in the formation of intraareal neuronal topography may be present in the thalamic relay station of the visual pathway of mammals. The formation of eye-specific layers in the lateral geniculate nucleus is dependent on afferent activity. Prenatal injection of tedrodotoxin, which blocks neuronal activity, prevents elaboration of the layers (Shatz and Stryker, 1988; Shatz, 1990a,b). Studies on the topographic organization of glial cells in this structure have revealed that layered glial cell processes occur prior to the establishment of neuronal layers (Hutchins and Casagrande, 1990). Like the glial protobarrels in the somatosensory cortex, these glial “protolayers” are formed only under the influence of afferent activity and disappear after formation of ~~

FIG. 4. Staining of “protobarrels” delineating future neuronal representations of the whiskers in tangentially sectioned layer IV of the mouse somatosensory cortex on Postnatal Day 6. (a) Following cauterization of row C of whiskers on Postnatal Days 1 and 3, protobarrels in the cortex representing these whiskers, revealed by peroxidase-conjugated peanut agglutinin, fail to develop (compare rows B and C). (b) An enlargement of the area depicted by the solid black arrow in (a). ( c ) Staining with antibodies against the astroglial marker GFAP also reveals protobarrels on Postnatal Day 6. Bar = 100 pm. [Reproduced from Cooper and Steindler, Brain Res. 489, 167 (1989),by copyright permission of Elsevier Science Publishers B.V.]

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the neuronal layers (Hutchins and Casagrande, 1990). A similar transient delineation of future neuronal territories by g l d processes has also been shown in the developing spinal cord (Bullon et al., 1984; van Hartesfeldt et a!., 1986). In summary, it can be concluded that glial cells appear to play a role in the activity-dependent formation of cytoarchitectonic neuronal topography by transferring the information carried by afferent activity to the local neuronal population. The instructive role of glial cells in neuronal pattern formation may be mediated by glial adhesion molecules (Steindler et al., 1990) and/or factors supporting and guiding growth and migration (Ard and Bunge, 1988; Hatten et al., 1982; Levine and Card, 1987; Yixlev et al., 1987). T h e influence of glial cells in the elaboration of local topography described earlier shares several aspects with the (probably activity-independent) guidance of neuronal migration during embryonic development. In this phenomenon glial cells also determine the direction of neuronal migration by serving as substrates for migrating neurons (for review see Hatten, 1990) or outgrowing axons (Gooday, 1990; Jacobs and Goodman, 1989). Adhesion molecules associated with glial cells are thought to play an important role in neuronal/glial interactions in this process. The influence of glia on the morphology of neurons has been studied i n uttro. Mesencephalic cells have been shown to elaborate their typical niorphology only in the presence of an appropriate glial environment (Denis-Donini et at., 1984; Lieth et al., 1989) or glial-derived factors (Rousselet et (11.. 1990). The unique multipolar morphology that develops in the presence of mesencephalic astrocytes is not achieved when cocultured glia originates from striatum (Denis-Donini et al., 1984). Such a region-specific interaction of astrocytes and neurons has been reconfirmed in striatal cultures (Chamak et al., 1987). A similar specificity of astroglial subpopulations on neuronal differentiation has also been observed in cerebellar cultures in which “velate protoplasmic” astrocytic clones selectively trigger the differentiation o f embryonic neurons into granule cells (Alliot et al., 1988).The basis for this heterogeneity of glial influences is not yet fully understood. One mechanism for these effects may be linked to differences in the expression of transmitter receptors by glial cells from different structures or different astrocytic subpopulations (Ernsberger et al., 1990; Lerea and McCarthy, 1989), as recent studies on the development of serotoninergic cells and cultures from spinal cord in zatro revealed a close association between transmitter release from neurons and glial-neuronal influences. Both survival and differentiation of neurons are influenced by astrocytes in these cultures

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(Brenneman et al., 1987). These glial-neuronal influences seem to be mediated by diffusible factors released from astrocytes on stimulation of serotoninergic receptors and activation by vasoactive intestinal polypeptide (Brenneman and Nelson, 1987; Brennemann et al., 1987; WhitakerAzmitia et al., 1990). One of the factors carrying this influence to serotoninergic neurons is the astroglial S- 100 protein, which has recently been shown to have neurotrophic activity (Azmitia et al., 1990; Kligman and Hilt, 1988; Winningham-Major et al., 1989). Additional mechanisms whereby glia may influence neuronal differentiation could be achieved by surface or matrix proteins of glial origin, as has been suggested in several in vitro studies (Alliot et al., 1988; Crossin et al., 1990; Drazba and Lemmon, 1990; Faissner and Kruse, 1990; Gilad et al., 1990; Monard et al., 1973; Smith et al., 1990; Tropea et al., 1988; Wujek and Akeson, 1987; Wujek et al., 1990). A possible activity dependence of the expression of these surface or matrix molecules is as yet speculative; however, the finding that laminin levels in glial cells can be modulated by conditioned media from regenerating optic nerves (Cohen and Schwartz, 1989) and the influence of basic fibroblast growth factor on glial release of nerve growth factor (Fukumoto et al., 1991) may be indicative of an external influence on the expression of glia-derived molecules. Similarly, neurotoxin injections into the developing visual cortex of the cat, which eliminate subplate neurons, lead to a decrease in fibronectin at the lesioned location. It is assumed that subplate neurons may influence the expression of fibronectin in glial cells (Chun and Shatz, 1988).

B. ACTIVITY-DEPENDENT SYNAPSE ELIMINATION As outlined earlier, ordered synaptic connections are elaborated in a two-stage process. First, connections are established in excess with rather limited precision in an activity-independent manner. This imprecise pattern of connectivity is then refined in an activity-dependent manner involving the elimination of inappropriate, and stabilization of appropriate, connections (for reviews see Brown et al., 1976, Mariani, 1989; Purves and Lichtman, 1980; Shatz, 1990b; Singer, 1987; Thompson et al., 1979). There are several indications that glial cells participate in activitydependent synapse elimination. Following deafferentation or axotomy, glial cell processes are generally observed at sites of degenerating axon terminals and may even separate boutons from axotomized neurons (Fulcrand and Privat, 1977; Gentchev and Sotelo, 1973; Kapadia and LaMotte, 1987; Rustioni and Sotelo, 1974; Wood and Faber, 1986).

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elicited by hormone application in vivo (Theodosis et al., 1986) and by osmotic stimulation in vitro (Perlmutter et al., 1984a). The glial subclass mediating the described plastic changes, namely, pituicytes, reveals some similarities with immature cortical astrocytes, even in adult animals. They maintain weak expression of the cytoskeletal protein GFAP throughout life (Salm et al., 1982), which seems to be downregulated even further during phases of process retraction (Salm et al., 1985). Unlike cortical astrocytes, pituicytes seem to maintain the capacity to survive culturing, even in cultures from mature animals (Bicknell et al., 1989). From recent electron microscopic observations it was concluded that microglia may also participate in the plastic change in the neurohypophysis, especially at the terminal portion of neurosecretory neurons (Pow et al., 1989). It has been speculated that some of the plastic changes in the neurohypophyseal/hypothalamic system may be triggered via activation of transmitter receptors (Bicknell et al., 1989) by synaptoid contacts of adrenergic and enkephalinergic afferents with pituicytes (Baumgarten et al., 1972; Knowles and Vollrath, 1965; van Leeuwen et al., 1983). Support for this claim comes from studies of cultured pituicytes in which activation of adrenergic or enkephalinergic receptors has been shown to induce morphological alterations in these glial cells (Bicknell et al., 1989; Wittkowski, 1986). T h e data summarized above reveal that glial cells can be seen to be involved in plasticity in several model systems where synapse elimination occurs during normal development or on experimental physiological stimulation. These processes seem to be linked to the presence of immature astrocytes (e.g., in the spinal cord) o r astrocytic cells maintaining immature characteristics (e.g., pituicytes). A similar link between activitydependent plasticity that involves synapse elimination and the presence of immature astrocytes has been shown in the developing visual cortex of the cat. As outlined before one of the central features of the development of the mature functional topography of cat visual cortex is the elaboration of ocular dominance columns (Shatz et al., 1977). This activity-dependent process is based predominantly on the partial elimination of afferent synapses. This process leads to the transition from an overlapping projection of the thalamocortical pathways representing the two eyes to cortical layer IV to a separate innervation of adjacent stripes of tissue by afferents driven by either the right or the left eye (LeVay et al., 1978). This process is restricted to a short period of postnatal development, between the third and approximately the seventh postnatal weeks in cat (Hubel and Wiesel, 1970; Olson and Freeman, 1980). Immunocytochemical investigations of the developmental expression of as-

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trocytic markers in cat visual cortex reveal a close relationship between astrocytic maturation and the period of cortical malleability, whereas the end of the critical period coincides with the maturation of astrocytes (Engel and Muller, 1989; Muller, 1989, 1992). It had been shown previously that the critical period for cortical plasticity can be prolonged by exclusion of visual experience by dark rearing (Mower et al., 1981, 1985). Studies of astroglial maturation in the visual cortex of dark-reared kittens revealed a selective retardation of astrocytic maturation in cortical layers displaying a prolonged critical period (Muller, 1990). Similar data have been reported previously from experiments in the rat visual system (Steward et al., 1986), where astroglial maturation also parallels the time of cortical malleability (Stichel et al., 1991). The hypothesis that the end of the critical period of cortical plasticity may be based on the absence of immature astrocytes was tested in the cat by transplanting astrocytes cultured from newborn kittens to the visual cortex of adult cats. At the time of transplantation one eye was closed by lid suture. Following a survival period of 4 to 8 weeks, changes in the efficacy of the experienced eye and the deprived eye in exciting cortical cells was tested physiologically by single-cell recording. It could be shown that the transplantation of cultured astrocytes was sufficient to reinduce plastic changes (Muller and Best, 1989); that is, the deprived eye was less effective in driving cortical cells than the experienced eye (Fig. 5). This effect was restricted to locations where transplanted astrocytes were present, and was not seen when the cells had been killed by multiple freezing prior to transplantation. The effects of astrocytic transplantation in the visual cortex can be explained by a reinduction of synapse elimination as seen in glia-free cerebellar cultures following addition of cultured astrocytes (Meshul et al., 1987; Meshul and Seil, 1988). This may be indicative of the necessity of immature astrocytes for synapse elimination occurring both in vivo and in vitro. A further indication that glial mechanisms are linked to experience-dependent changes in the cat3 visual cortical circuitry is suggested by histochemical studies on the distribution of 5'nucleotidase in kitten visual cortex (Schoen et al., 1990). The expression of this enzyme, which has been shown to be localized to glial plasma membranes (Kreutzberg etal., 1978),appears to be associated with periods of remodeling of ocular dominance territories and can be modulated by changes in visual experience (Schoen et al., 1990).Interestingly, activitydependent plasticity involving synapse elimination in the visual cortex has been shown to depend on an intact catecholaminergic innervation (Kasamatsu and Pettigrew, 1979; Kasamatsu et al., 1981). It is tempting to speculate that this may correspond to catecholaminergic effects on glial cells proposed in plasticity of the neurohypophyseal/hypothalamicsystem

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elicited by hormone application in viuo (Theodosis et al., 1986) and by osmotic stimulation in nitro (Perlmutter et al., 1984a). T h e glial subclass mediating the described plastic changes, namely, pituicytes, reveals some similarities with immature cortical astrocytes, even in adult animals. They maintain weak expression of the cytoskeletal protein GFAP throughout life (Safm et al., 1982), which seems to be downregulated even further during phases of process retraction (Salm et al., 1985). Unlike cortical astrocytes, pituicytes seem to maintain the capacity to survive culturing, even in cultures from mature animals (Bicknell et al., 1989). From recent electron microscopic observations it was concluded that microglia may also participate in the plastic change in the neurohypophysis, especially at the terminal portion of neurosecretory neurons (Pow et al., 1989). It has been speculated that some of the plastic changes in the neurohypophyseallhypothalamic system may be triggered via activation of transmitter receptors (Bicknell et nl., 1989) by synaptoid contacts of adrenergic and enkephalinergic afferents with pituicytes (Baumgarten et al., 1972; Knowles and Vollrath, 1965; van Leeuwen et al., 1983). Support for this claim comes from studies of cultured pituicytes in which activation of adrenergic or enkephalinergic receptors has been shown to induce morphological alterations in these glial cells (Bicknell et al., 1989; Wittkowski, 1986). T h e data summarized above reveal that glial cells can be seen to be involved in plasticity in several model systems where synapse elimination occurs during normal development or on experimental physiological stimulation. These processes seem to be linked to the presence of immature astrocytes (e.g., in the spinal cord) or astrocytic cells maintaining immature characteristics (e.g., pituicytes). A similar link between activitydependent plasticity that involves synapse elimination and the presence of immature astrocytes has been shown in the developing visual cortex of the cat. As outlined before one of the central features of the development of the mature functional topography of cat visual cortex is the elaboration of ocular dominance columns (Shatz et d.,1977). This activity-dependent process is based predominantly on the partial elimination o f afferent synapses. This process leads to the transition from an overlapping projection of the thalamocortical pathways representing the two eyes to cortical layer IV to a separate innervation of adjacent stripes o f tissue by afferents driven by either the right or the left eye (LeVay et al., 1978). This process is restricted to a short period of postnatal development, between the third and approximately the seventh postnatal weeks in cat (Hubel and Wiesel, 1970; Olson and Freeman, 1980). Immunocytochemical investigations of the developmental expression of as-

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trocytic markers in cat visual cortex reveal a close relationship between astrocytic maturation and the period of cortical malleability, whereas the end of the critical period coincides with the maturation of astrocytes (Engel and Muller, 1989; Muller, 1989, 1992). It had been shown previously that the critical period for cortical plasticity can be prolonged by exclusion of visual experience by dark rearing (Mower et al., 1981,1985). Studies of astroglial maturation in the visual cortex of dark-reared kittens revealed a selective retardation of astrocytic maturation in cortical layers displaying a prolonged critical period (Muller, 1990). Similar data have been reported previously from experiments in the rat visual system (Steward et al., 1986), where astroglial maturation also parallels the time of cortical malleability (Stichel et al., 1991). The hypothesis that the end of the critical period of cortical plasticity may be based on the absence of immature astrocytes was tested in the cat by transplanting astrocytes cultured from newborn kittens to the visual cortex of adult cats. At the time of transplantation one eye was closed by lid suture. Following a survival period of 4 to 8 weeks, changes in the efficacy of the experienced eye and the deprived eye in exciting cortical cells was tested physiologically by single-cell recording. It could be shown that the transplantation of cultured astrocytes was sufficient to reinduce plastic changes (Muller and Best, 1989); that is, the deprived eye was less effective in driving cortical cells than the experienced eye (Fig. 5). This effect was restricted to locations where transplanted astrocytes were present, and was not seen when the cells had been killed by multiple freezing prior to transplantation. T h e effects of astrocytic transplantation in the visual cortex can be explained by a reinduction of synapse elimination as seen in glia-free cerebellar cultures following addition of cultured astrocytes (Meshul et al., 1987; Meshul and Seil, 1988). This may be indicative of the necessity of immature astrocytes for synapse elimination occurring both in vivo and in vitro. A further indication that glial mechanisms are linked to experience-dependent changes in the cat's visual cortical circuitry is suggested by histochemical studies on the distribution of 5'nucleotidase in kitten visual cortex (Schoen et al., 1990). The expression of this enzyme, which has been shown to be localized to glial plasma membranes (Kreutzberg et al., 1978),appears to be associated with periods of remodeling of ocular dominance territories and can be modulated by changes in visual experience (Schoen et al., 1990).Interestingly, activitydependent plasticity involving synapse elimination in the visual cortex has been shown to depend on an intact catecholaminergic innervation (Kasamatsu and Pettigrew, 1979; Kasamatsu et al., 1981). It is tempting to speculate that this may correspond to catecholaminergic effects on glial cells proposed in plasticity of the neurohypophyseaUhypothalamicsystem

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FIG.5 . Effect of transplantation of astrocytes and fibroblasts cultured from newborn kittens to the visual cortex of adult cats. Following transplantation, one eye was closed by lid suture for 4-8 weeks. After this period of monocular deprivation the eKect on the response properties of visual cortical neurons was determined with single-cell recordings. The histograms give the percentages of cells corresponding to five ocular dominance classes. Neurons were classified as classes 1 and 5 if they responded exclusively to stimulation of the eye ipsilateral (class 1) or contralateral (class 5 ) to the hemisphere studied. Neurons responding equally well to stimulation of either eye are class 3, whereas those binocular cells responding preferentially to one eye are class 2 (ipsilateral dominance) and class 4 (contralateral dominance). Control hemispheres into which shock-frozen astrocytes were transplanted (open histogram in A-C) revealed an ocular dominance distribution that was slightly biased toward the contralateral eye. This distribution corresponds to that found in nondeprived adult animals. The shaded histograms give the ocular dominance distributions obtained from transplanted hemispheres. Following transplantation of astrocytes, there is a shift in the distribution toward the experienced eye (contralateral in A, ipsilateral in B). This effect is not observed on transplantation of cultured fibroblasts (C). (Modified from Muller and Best, 1989.)

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(see above). This possibility is corroborated by studies showing that padrenoceptors that mediate the adrenergic effect on cortical plasticity (Kasamatsu and Shirokawa, 1985)are localized predominantly on cortical astroglia (Stone and John, 1991). Involvement of a different population of nonneuronal cells in the process of elimination of exuberant projections has been proposed by Innocenti and co-workers. During the period of refinement of callosal projections, macrophage-like “gitter cells” are transiently present in the white matter (Innocenti et al., 1983a,b; Ivy and Killackey, 1978). These cells have been shown to correspond to phagocytes (Russell, 1962). On the basis of ultrastructural observations showing gitter cells phagocytizing myelinated axons, it was proposed that these cells may eliminate supernumerary callosal axons (Innocenti et al., 1983a,b). It cannot as yet be determined whether axon elimination precedes synapse elimination or whether axons that have lost contact to a target degenerate and are subsequently phagocytized (for review see Killackey, 1984). Another example showing that nonneuronal cells are necessary for the refinement of axonal projections is seen in the development of the neuromuscular junction. Although muscle fibers are innervated by multiple afferent fibers early in development, the innervation is restricted to a single afferent fiber in the mature system (Brown et al., 1976; Thompson et al., 1979). In an in uitro system containing motoneurons and muscle fibers a developmental reduction of the number of afferent fibers innervating a given muscle fiber could be observed only when Schwann cells were included in the culture (Chapron and Koenig, 1989). The mechanism of Schwann cell participation in this activity-dependent plasticity has not yet been elucidated. It is, however, worth mentioning that the time of synapse elimination at the neuromuscular junction coincides with the intervention of Schwann cell cytoplasm between neighboring synapses, and that some terminal axons have been shown to be totally invested by Schwann cell processes (Korneliussen and Jansen, 1976). This situation is similar to the scenario described for the developing spinal cord (Conradi and Ronnevi, 1975; Ronnevi, 1977, 1978). There are two ways that synapse elimination can be achieved. First, axons may degenerate or retract; second, axon terminals may be eliminated by external forces, for example, glial processes. In a variety of ultrastructural studies, a lack of degenerating terminals was reported during spontaneous synapse elimination (Korneliussen and Jansen, 1976). On the other hand, recent time lapse observations of fibers in situ have revealed a very dynamic nature of axonal arborizations during the time of refinement of axonal connections (O’Rourke and Fraser, 1990; Sturmer, 1990). Continuous outgrowth and retraction of axonal

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processes may coincide with the increased pinocytosis observed on remodeling of axonal connections that has been attributed to recycling of plasma membrane by retracting axons (Sumner, 1975). Glial cells may be involved in this process by the release of substances that destabilize axonal terminals, like proteases, or conversely by the release of protease inhibitors (Farmer et al., 1990; Kalderon et al., 1990; Liuzzi, 1990; Rosen and Goldberg, 1989; for reviews see Guenther et al., 1985; Monard, 1988). T h e observation that synapse elimination relies on the presence of' glial cells (Chapron and Koenig, 1989; Meshul et al., 1987; Meshul and Seil, 1988) may indicate that glial cells are involved mainly in synapse destabilization. T h e second possibility of glial influence on synapse elimination is an active involvement in the mechanical disruption of synapses. Such a mechanism would coincide with the observation of intermediate stages of synapse elimination where glial processes are seen to be partially inserted into intact synaptic clefts (Ronnevi, 1977; Seil et al., 1988). Active detachment of neurons from growth substrates has also been observed in tissue culture (Glad et a/., 1988, 1989). To perform synapse elimination in an activity-dependent manner, it has to be assumed that glial cells reveal growth o r process motility on neuronal activity. Indeed, there is evidence from iu viz~oexperiments that neuronal activity triggers the growth of glial processes. T h e effects of such growth of astroglial processes on the coverage of synaptic regions is schematically shown in Fig. 6. Note that two effects can follow from such a growth process: (1) increased coverage of synaptic regions by glial processes, and (2) separation of synapses by ingrowing glial processes. Whereas an increase in the astroglial coverage may influence synaptic transmission through effects on the extracellular homeostasis, separation of synapses may ultimately result in synapse elimination. Evidence for either possibility is sumniarized in the following paragraphs. Electrical stimulation of afferents to an insect neuromuscular synapse has been shown to lead to increased coverage of synaptic terminals by glial processes (Reinecke, 1979). Also, in the mammalian hippocampus, afferent stimulation elicits an increase in the number of astrocytic pro1989) and an increase in the glial coverage of syncesses (Isaacs et d., apses ( J . Wenzel, personal communication). Similar findings have been obtained in mammalian visual cortex by comparing astrocytic processes in animals reared with reduced and highly complex visual experience (Sirevaag et al., 1988).Besides growth, motility of processes may underlie syriapse elimination. Glial cells have been shown to perform rapid alterations in shape. 171 uitro experiments on astroglial cultures have revealed morphological alterations following transmitter receptor activation

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presynaptlc afferent6

postsynaptlc target coincident pre- and postsynaptic actlvity

increased gliai coverage

Incoincident pre- and postsynaptlc actlvlty

synapse ellrnlnatlon

FIG.6. Schematic diagram of the effects of growth of astrocytic processes. The coverage of synaptic sites by astroglial processes increases (lower left), leading to stronger isolation/compartmentalization of synapses. Alternatively, astroglial processes may invade synaptic clefts and separate presynaptic boutons from the postsynaptic target (lower right). See text for experimental evidence.

(Bicknell et al., 1989; Cornell-Bell et al., 1990b; Hatten, 1985; Narumi et al., 1978; Shain et al., 1987; Whitaker-Azmitia et al., 1990), calciuminflux (MacVicar, 1987) or protein kinase C activation (Harrison and Mobley, 1990; Mobley et al., 1986; but see Bedoy and Mobley, 1989). Recent time lapse observations have revealed the rapid expression of filopodial protrusions from hippocampal astrocytes on stimulation with glutamate (Cornell-Bell et al., 1990b) and also a unique dynamic in microglial morphology (Thomas, 1990). These morphological alterations may well be the basis for the separation of synapses by growing astrocytic processes on neuronal activity. It is not yet clear how such a mechanism of synapse elimination is restricted to previously inactive synapses, leaving efficient synapses intact. Possible mechanisms include the mechanical stabilization of previously active synapses, for example, by an activity-dependent aggregation of proteins at the synaptic cleft. Ependymins have been proposed as such a stabilizing agent, as they show aggregation on removal of calcium from their environment, which occurs after strong synaptic activation

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(Shashoua, 1982). Another protein that has been proposed to mediate such stabilizing effects is the 180-kDa subform of the neural cell adhesion molecule. This protein accumulates at postsynaptic sites and reveals both an intracellular domain and an extracellular domain (Persohn et al., 1989; Pollerberg et al., 1990). Activity-dependent stabilizing mechanisms may protect efficient synapses from detachment. Alternatively, an activity-dependent expression of surface molecules may guide, for example, inhibit, the growth of glial processes. Gangliosides have been shown to accumulate at synaptic sites (Baker, 1988) and to inhibit morphological transformation of astrocytes in tissue culture (Facci et al., 1987). Synapse elimination in the kitten visual cortex following monocular deprivation seems to be retarded on application of exogenous gangliosides (Carmignoto et al., 1984). Gilad et al. (1989) showed that astrocyte-mediated detachment of neurons, in vitro, can be modulated by addition of glycosaminoglycans, a possible component of extracellular matrix or neuronal surfaces. Further investigations are necessary to validate these possibilities. Recent experiments on the phagocytotic activity of cultured glial cells have determined a role for calcium influx in the control of phagocytosis (Mano and Puro, 1990). There is evidence that calcium fluxes are essential to activity-dependent plastic changes (Geiger and Singer, 1986; Malenka et al., 1988; Williams and Bliss, 1989a), although calcium accumulation in neurons does not necessarily correlate with the induction of plasticity (Siklos et al., 1989). It may well be that calcium accumulation in glial cells governs plasticity. The accumulating evidence that calcium transients in glial cells are triggered by neurotransmitter substances, and can even spread over cellular boundaries (Ahmed et al., 1990; CornellBell et al., 1990a; Enkvist et al., 1989; Glaum et al., 1990; Jensen and Chiu, 1990; Salm and McCarthy, 1990), may thus reflect a link between neuronal activity and glial functions in activity-dependent plasticity. In summary, accumulating evidence suggests the necessity of nonneuronal cells for activity-dependent synapse elimination. This process may be controlled by factors released from glial cells or may involve mechanical disruption of synapses by growing astrocytic processes. The latter assumption is supported by the evidence for glial process growth following neuronal activation and the ultrastructural evidence for partial disruption of synapses by glial processes during periods of synapse elimination. With respect to the local guidance of such mechanisms of synapse elimination, processes located at neuronal-neuronal interaction sites can be proposed. The preponderance of plastic processes involving synapse elimination in early postnatal periods may be linked to the fact that morphological alterations and phagocytosis are seen mainly in immature astrocytes.

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C. ACTIVITY-DEPENDENT SYNAPSE FORMATION

Evidence for the activity-dependent formation of new synapses has been obtained in several model systems. Among the clearest examples are the reinnervation in visual cortex following reopening of an eye that has been deprived of vision (LeVay et al., 1980; Swindale et al., 1981), and the increase in synaptic density following conditioning in Aplysia (Bailey and Chen, 1989; Glanzman et al., 1990). Additional examples of an activity-dependent increase in the number of synapses include longterm potentiation in the hippocampus (Chang and Greenough, 1984; Lee et al., 1980), increases in number of synapses following exposure to enriched sensory environments (Sirevaag and Greenough, 1987), motor learning (Black et al., 1990),and electrical activation in vitro (Nelson et al., 1990). To date there is virtually no direct evidence for participation of glial cells in synapse formation in normal development; however, both in neuronal growth following damage (summarized below) and in neuronal growth in vitro (Fallon, 1985; Noble et al., 1984), glial cells have been shown to play an essential role. The influence of glial cells on neuronal growth are mediated via extracellular matrix molecules, glial surface molecules, and neurotrophic factors released from glial cells (for reviews see Manthorpe et al., 1986; Varon and Adler, 1981). Thus there is good reason to assume participation of glial cells in synapse formation in vivo. Indications for the participation of glial cells in plastic changes that involve axonal growth come from experiments studying the contribution of growth factors to plasticity. Several studies have indicated the participation of growth factors in hippocampal long-term potentiation. Thus, Sastry and co-workers could show that extracellular fluid sampled from tetanically activated brain structures is sufficient to induce enhancement of synaptic transmission when applied to hippocampal slices (Sastry et al., 1988b). This finding indicated the presence of a soluble factor mediating the effects of activity-dependent plasticity. Addition of such extracellular fluid to cultures of a neuronal cell line (YC12 cells) elicits neurite growth (Sastry et al., 198813). Fractionation experiments revealed that low-molecular-weight (<10 kDa) and high-molecularweight (>50 kDa) molecules mediated the potentiation of synaptic transmission (Xie et al., 1991). The question of the origin of these plasticity-inducing factors has been the subject of experiments in the hippocampus. The experiments were based on earlier findings that low-frequency activation of afferent fibers to the CA1 region induces long-term potentiation when paired with strong postsynaptic depolarization (Gustafsson et al., 1987; Sastry et al., 1986). It was argued that such strong neuronal depolarization may lead to significant glial depolarization, a phenomenon that has been observed under stimulation conditions that

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afferent stimulation

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FIG. 7. Potentiation of responses of hippocampal (:.A 1 neurons by coincident glial depolarization and low-frequency (0.2 Hz) stimulation of afferent fibers. Top: Electrical stimulation of the afterent system to the CA1 region was perfornied in the stratum radiat um innervating CA 1 pyramidal neurons. Neuronal responses were monitored by a n extracellular electrode in the sonia layer in the CA 1 region. i n addition, a single glial cell in the dendritic region close to the extracellular recording electrode was penetrated with a micropipet. Bottom: Extrarellular field responses evoked by afferent stimulation before glial pairing (control), after 15 niin of glial depolarization by current injection (5 nA, 400mser duration, 0.2 Hz) without afferent stimulation, and after 15 min of coincident afferent stimulation and glial depolarization. Although there is no change in the neuronal field response after 15 min of glial depolarization unpaired with atferent stimulation, the field response is potentiated following 15 min of glial depolarization paired with afferent stimulation of the neurons. (Modified from Sastry ef al., 198Xa.)

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oornpetltlve Eproutlng

FIG. 8. Schematic illustration of synaptic reorganization triggered by growth-promoting activity. Addition of presynaptic and postsynaptic sites can result in a new connection to the same postsynaptic target (homosynaptic sprouting) or a different target, that is, a neighboring neuron (heterosynaptic sprouting), or can result in competition of a given afferent with a separate afferent for one postsynaptic target (competitive sprouting).

induce hippocampal long-term potentiation (Casullo and Krnjevic, 1987; Sastry et al., 1988a; Schwartzkroin and Prince, 1979). Sastry and co-workers developed an experimental paradigm in which low-frequency afferent stimulation was paired with moderate depolarization of an intracellularly recorded glial cell. At the same time the response strength of the neighboring neurons was recorded with an extracellular electrode (Sastry et al., 1988a).It could be shown that the pairing of low-frequency afferent activation with glial depolarization leads to potentiation of the surrounding neurons (Fig. 7). It was concluded from these experiments that glial depolarization may lead to the release of growth-promoting factors that induce the plastic change in neurons (Sastry et al., 1988a). This hypothesis may explain the paradoxical finding that pairing of afferent activity with intracellular depolarization of one neuron leads to the potentiation of neuronal responses in a larger number of neighboring cells in organotypical hippocampal slice cultures (Bonhoeffer et al., 1989) and visual cortical slices (Kossel et al., 1990). The involvement of a diffusible substance, which may originate from glia, could account for these data. As summarized in Fig. 8, growth-promoting activity on neurons can lead to new synapse formation at the same postsynaptic target (homosynaptic sprouting) or a different neighboring target

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(heterosynaptic sprouting), or can lead to competition between the newly formed afferent and another synaptic contact (competition). The mechanisms for the release of growth-promoting factors from glia arid the spread by diffusion have yet to be elucidated. The finding that glial depolarization may be an intermediate phenomenon allows the supposition that potassium e f h x from activated neurons may be the trigger for this neuronal-glial transfer in activity-dependent plasticity. The fact that elevation of extracellular potassium can induce plastic changes (FrCgnac et d., 1988; Grover and Teyler, 1990b) may be regarded as support for this idea; however, it cannot be excluded that other neuronal signals affect the release of substances from glial cells. Thus, stimulation of receptors for vasoactive intestinal peptide (Brenneman et al., 1987) and serotonin (Whitaker-Azmitia and Azmitia, 1989; Whitaker-Azmitia et al., 1990) stimulates the release of growth-promoting activity from astrocytes. In both instances the released factors have a beneficial effect on neuronal survival and differentiation of the neurons that mediated the neuronal-glial transfer. In addition, mRNA coding for nerve growth factor and the S-100 protein seems to be expressed in glial cells on neuronal activity (Furukawa et al., 1986; Landry et al., 1988). Further evidence for the involvement of growth factors in activitydependent plasticity has been obtained by Terlau and Seifert, who showed enhancement of hippocampal long-term potentiation in the presence of epidermal growth factor (Terlau and Seifert, 1989) and fibroblast-derived growth factor (Terlau and Seifert, 1990). The latter has been shown to be synthesized and released from astrocytes (Ferrara et al., 1988; Hatten et al., 1988), and has a neurotrophic action on hippocampal neurons (Walicke and Baird, 1988; Walicke et al., 1986). The finding that antiserum against another glia-derived growth factor, the S-100 protein (Kligman and Hilt, 1988; Whitaker-Azmitia et al., 1990; Winningham-Major et al., 1989), interferes with long-term potentiation (Lewis and Teyler, 1986) corresponds to experimental evidence that chronic infusion of antiserum against S-100 also interferes with ocular dominance plasticity in the kitten visual cortex (Miiller, 1989). Earlier studies had shown that infusion of antiserum against the S-100 protein interferes with the acquisition of learned behavior (Karpiak et al., 1976). It is therefore possible that proteins of the S-100 family, synthesized and released (Shashoua et al., 1984; van Eldik and Zimmer, 1987) from astroglial cells, play a central role in activity-dependent plasticity. The recent finding that a disulfide-bonded dimer of S-loop may be identical to the neurite extension factor (Kligman and Marshak, 1985; Kligman and Hilt, 1988; Winningham-Major et al., 1989) suggests that the action of 5-100 may be mediated by neurite growth.

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The variety of growth factors supporting LTP may indicate either an action of individual factors on different aspects of plastic changes or a common final pathway of action of these factors. Data from Terlau and Seifert indicate the former for hippocampal LTP. While epidermal growth factor influences the change in the induction of action potentials following tetanic stimulation (Terlau and Seifert, 1989), fibroblast-derived growth factor supports mainly the increase in amplitude of excitatory postsynaptic potentials following tetanization (Terlau and Seifert, 1990). Alternatively, it is conceivable that growth factors influence the expression of other factors that may reflect the final pathway in inducing plasticity. Thus, it is known that growth factors influence the expression of proteins of the S-100 family (for review see van Eldik et al., 1988) and that epidermal growth factor modulates the release of arachidonic acid (see below). It is evident that further experiments focusing on the characterization and mechanisms of release of growth factors from glial cells are essential for determining the participation of glial growth factors in activity-dependent plasticity. Additional evidence for the participation of glia-derived factors in central nervous plasticity come from experiments on regeneration in the CNS. These data and their possible relation to glial influences on developmental plasticity are dealt with in a later section.

D. ACTIVITY-DEPENDENT CHANCES IN SYNAPTIC EFFICACY T h e considerations summarized above have focused on plastic changes in neuronal circuitry that are based on morphological alterations in neuronal connectivity. Beside these effects, which seem to underlie the long-term maintenance of plasticity, changes in the efficacy of synaptic transmission have been implicated in activity dependent plasticity (for reviews see Collingridge and Bliss, 1987; Teyler and DiScenna, 1987). Such changes in neural transmission can originate from (1) increased transmitter release, (2) a higher sensitivity or density of postsynaptic receptors, o r (3) the presence of more synapses. The latter possibility was detailed earlier. Up to now, there has been little convincing evidence to support the notion of an increase in receptor density or sensitivity (for review see Andersen, 1987). On the other hand, several studies have presented evidence for an increase in the release or availability of transmitter in LTP in the hippocampus. Following induction of LTP, the amount of glutamate released in the extracellular space is elevated (Dolphin et al., 1982; Skrede and Malthe-Sorenssen, 1981).

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Such a presynaptic change has recently been corroborated by patchclamp analyses of unitary excitatory postsynaptic potentials (Bekkers and Stevens, 1990; Malinow and Tsien, 1990; Malinow, 1991; but see Larkman et al., 1991). Three mechanisms may underlie the increase in transmitter release: (1) changes in the quanta1 content, (2) an increase in release probability, o r (3) reduced cleavage of transmitter from the extracellular compartment. Recent experiments propose a possible involvement of glial cells in changes of synaptic efficacy on the basis of the third mechanism. Evidence from studies in retinal Miiller cells, one class of astrocytes, shows that glutamate uptake can be inhibited for a prolonged period after brief treatment with arachidonic acid (Barbour et al., 1989). A similar reduction of transmitter uptake by glial cells following the application of arachidonic acid has been observed in cultured cortical astrocytes (Yu et al., 1986). Arachidonic acid has been shown to be released from neurons on activation of NMDA receptors (Dumuis et al., 1988; Sanfeliu et al., 1990), an essential prerequisite for the induction of hippocampal LTP (Collingridge and Bliss, 1987). In addition, arachidonic acid application mimics the effects of tetaiiic stimulation on LTP in the hippocampus (Lynch and Voss, 1990; Williams et al., 1989), whereas blockers of arachidonic acid metabolism interfere with LTP (Lynch et al., 1989; Williams and Bliss, 1989a,b). Similarly, in the nervous system of-the sea snail, Aplysia, arachidonic acid metabolites have been implicated in the plasticity of synaptic transmission (Piomelli et al., 1987). These data offer a new possibility for participation of glial cells in changes in synaptic efficacy, namely, an increase in transmitter availability caused by reduced uptake by glial cells, which is controlled by a postsynaptic neuronal mechanism. Evidence for modulation of the release of arachidonic acid by epidermal growth factor (Chepenik, 1989) may relate to the beneficial effects of epidermal growth factor application on the induction of LTP (Terlau and Seifert, 1989; Abe et al., 1991). Another possible mechanism of glial control of synaptic efficacy is provided by substances modulating transmitter receptors. In hippocampal neurons NMDA receptormediated responses are facilitated by glia-conditioned medium (Forsythe et al., 1988). As the effects are similar to those of glycine, it may well be that release of this amino acid from astrocytes (Forsythe et al., 1988) is involved in increases in synaptic efficacy. Interestingly, arachidonic acid appears to facilitate glycine efflux from glial cells (Zafra et al., 1990). Recent studies on the cellular localization of an endogenous NMDA receptor preferring agonist, homocysteic acid, offer another possibility of astroglia-neuron influences. Using specific antibodies against this compound, Streit and associates revealed a preponderant localization in glial cells of cerebellum and hippocampal formation

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(Streit et al., 1991). As homocysteic acid has been shown to be released from brain slices on depolarization (for review see Cuenod et al., 1990) it may be assumed that neuronal NMDA receptors are activated by glial cells via this endogenous agonist. Nonvesicular release of excitatory amino acids from glial cells has recently been shown to be mediated by reversal of electrogenic uptake systems following glial depolarization (Szatkowski et al., 1990). It can thus be concluded that glial cells may support neuronal plasticity not only via effects on neuronal connections, but also by effects on neural transmission. These effects may play a role in rapid and nonpermenant changes in neural transmission.

111. Regeneration and Adaptive Processes following CNS Damage

Successful regeneration in the CNS implies, as in normal development, the establishment of functional contacts. Mechanisms underlying regeneration may thus shed light on mechanisms underlying plasticity during normal development. Three questions are of considerable interest: (1) What are the reasons for the widespread inability of regeneration in the adult mammalian CNS? (2) How can regeneration be induced experimentally? (3) What is the basis for regeneration occurring in vivo? T h e failure of regeneration in most central nervous regions may indicate a basis for the restriction of developmental plasticity to critical periods of early development. Three reasons can account for the apparent lack of regeneration in the mature mammalian CNS: (1) neurons may lose the capacity for regrowth and remodeling of their synaptic connections, (2) environmental factors necessary for plasticity may be lost, o r (3) mechanisms suppressing plastic changes may occur. Experimental evidence in recent years has supported the third assumption. Already at the beginning of this century Tello, as well as Ramon y Cajal, reported that neurons of the mature CNS may regenerate when placed in an appropriate environment, e.g., pieces of peripheral nerves (Tello, 1911; Ramon y Cajal, 1928). More recent transplantation studies in vivo (Benfey and Aguayo, 1982; Benfey et al., 1985; David and Aguayo, 1981; Richardson et al., 1984; So and Aguayo, 1985; Vidal-Sanz et al., 1987), as well as studies in vitro (Baehr, 1991; Baehr and Bunge, 1990), have established that mature neurons do not necessarily lose their capacity for regeneration. One important factor that keeps central neurons from regenerating seems to be associated with central nervous myelin.

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In the presence of oligodendrocytes or myelin, cultured neurons failed to grow axons (Crutcher, 1989; Savio and Schwab, 1989; Schwab and

Thoenen, 1985; Schwab and Caroni, 1988; Watanabe and Murakami, 1990). These findings lead to the conclusion that failure of regenerative processes in the CNS is due to inhibitory factors associated with glial cells, namely myelin (for review see Schwab, 1990). Meanwhile it has been possible to characterize these inhibitory factors and to raise antibodies that neutralize their activity (Bandtlow et al., 1991; Caroni and Schwab, 1988a,b). Thus, it appears that mechanisms responsible for growth processes in early development may simply be blocked in mature organisms. Experimental approaches using antibodies to block growth inhibitory factors in mature organisms may unravel the question of whether regenerating connections elaborate functional interactions. Recent studies by Wictorin et al., (1990) revealed that fetal neurons transplanted into lesioned regions of the adult CNS may grow over considerable distances and toward the appropriate targets. Similar data have been obtained in systems displaying lifelong regenerative properties. Neurons from the olfactory epithelium proliferate and extend new axons to the olfactory bulb throughout life (for review see Graziadei and Monti Graziadei, 1978). Glial cells from the olfactory system seem to maintain a supportive role for axonal growth (Denis-Donini and Estenoz, 1988; Doucette, 1990). In addition, glia-derived nexin, a protease inhibitor that is released from glial cells and has neurite-promoting activity in neurons, is selectively present in the olfactory system in adulthood (Reinhard et al., 1988). The same is true for the glia-derived Jl/tenascin (Miragall et al., 1990). Those glial factors may be linked to the regenerative capacity of the olfactory system. Furthermore, retinal ganglion cells from lower vertebrates continue to regenerate in the mature CNS (Sturmer, 1990). This capacity for continuous regeneration is paralleled by the growth-permissive property of glial cells from fish optic nerve (Bastmeyer et al., 1991),and the close similarity of fish oligodendrocytes with mammalian Schwann cells, which are known to support neuronal growth (Jeserich and Rauen, 1990). Part of this growthsupporting ability of glial cells may rely on the presence of adhesion molecules in lower vertebrates that are not expressed by adult mammalian glial cells (Blaugrund et al., 1990). Thus, a difference in the glial environment could account for the regenerative capacity of the central nervous system of lower vertebrates. Another intruiging example of' regenerative mechanisms maintained throughout life is present in the vocal motor system of songbirds (for review see Nottebohm, 1985). In this system neuronal proliferation continues in the adult brain (Alvarez-

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Buylla and Nottebohm, 1988; Goldman and Nottebohm, 1983), and these cells take part in the elaboration of long-distance projections (Burd and Nottebohm, 1985; Nordeen and Nordeen, 1988; Paton and Nottebohm, 1984). The functional significance of this kind of plasticity may relate to the seasonal plasticity in song performance. Interestingly, radial glial cells, which are present only during early development in mammals (Engel and Miiller, 1989; Levitt and Rakic, 1980; Schmechel and Rakic, 1979; Voigt, 1989) and which have been implicated in neuronal migration (for review see Hatten, 1990), are continuously present in the songbird brain (Alvarez-Buylla et al., 1987, 1988). These data reveal that newborn or regenerating neurons can be integrated into the brain and can establish connections even in mature organisms. This performance is paralleled by, and probably dependent on, accompanying glial specializations. In an experimental approach, Silver and associates used astroglial transplants to test the ability of axonal growth after transection of the corpus callosum or nerve crush in the spinal cord. Millipore filters coated with immature astrocytes were introduced as bridges between the separated hemispheres, and new callosal projections formed (Silver and Ogawa, 1983; Smith et al., 1986). The growth-supporting capacity of glial substrates was closely linked to the age of the transplanted astrocytes (Smith and Silver, 1988; Smith et al., 1986). Similar observations have been made in damaged spinal cord (Kliot et al., 1988, 1990; Schreyer and Jones, 1987), peripheral nerve (Kalderon, 1988), and the septohippocampal projection (Kromer et al., 1981; Knoops et al., 1991), where glial cells have been linked to axonal sprouting following damage (Gage et ul., 1988; McWilliams and Lynch, 1979). Despite the accumulated evidence that neuronal regeneration may be accomplished in the mature CNS on experimental manipulation of the glial environment, no conclusive evidence has yet been obtained for an appropriate function of such reestablished connections. It remains to be shown whether local activity-dependent remodeling, which is an important step in the elaboration of a functional circuitry during normal development, occurs or can be induced in the mature regenerating CNS of higher mammals. Some evidence for activity-dependent remodeling of neuronal connections in the mature central nervous system has been obtained after transplantation of astrocytes. This is true for the reinduction of activity-dependent plasticity in the visual cortex of adult cats (Muller and Best, 1989), and can be assumed from experiments showing behavioral recovery from deficits after lesions and subsequent astroglial grafting in monkeys (Kesslak et al., 1986).

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IV. A Unifying Hypothesis for Involvement of Glial Cells in Activity-Dependent Plasticity

A. GENERAL MECHANISMS As outlined earlier, there is abundant evidence for a close link between glial cells and activity-dependent plasticity. Yet, there is no definitive hypothesis on the mechanisms that allow the participation of glial cells in activity-dependent plasticity. Based on experimental data on glial physiology, development, and biochemistry, as well as on current knowledge of the effects of plastic changes in the CNS, it is possible to suggest possible events leading to the manifestation of plastic changes. The hypothesis outlined in the following paragraphs attempts to explain how glial cells can participate in activity-dependent changes in synaptic efficacy, activity-dependent synapse elimination, and activity-dependent synapse formation. The hypothesis is based on current knowledge of glial and neuronal mechanisms that may influence plastic changes. These mechanisms, which are included in the hypothesis, are summarized in Fig. 9. It is proposed that neuronal activity influences glial cells via at least t w o independent mechanisms. Presynaptic activity of neurons is monitored by glial cells through the activation of transmitter and neurohormone receptors (for reviews see Kimelberg, 1988; van Calker and Hamprecht, 1980). Postsynaptic neuronal activity, on the other hand, is monitored by glial cells via signals emerging from active neurons. Extracellular potassium seems to be a very good indicator of the postsynaptic activity level of nearby neurons. In addition, chemical signals released from neurons on receptor activation, such as arachidonic acid and its metabolites or nitric oxide, may provide additional signals. Three major effects of these signals on glial cells may underlie their involvement in activity-dependent plasticity: 1. Rapid effects on transmitter uptake or mechanisms of ion homeostasis of the extracellular compartment may lead to changes in the synaptic efficacy of nearby synapses. Prolonged reduction of transmitter uptake by neuronally released arachidonic acid may explain the transient increase in transmitter availability in the synaptic cleft following induction of hippocampal long-term potentiation (Barbour et al., 1989; Dolphin et al., 1982; Skrede and Malthe-Sorenssen, 1981), whereas changes in extracellular ion composition may also explain changes in the excitability of neurons. A reduction of potassium buffering, which may lead to prolonged increases in extracellular potassium following neuronal activation, has to be expected on sodium influx into glial cells.

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A

change of

increased

change of synaptic efficacy

transmitter uptake calcium influx

growth(permissive)

synapse formation

calcium fluxes synapse elimination synapse isolation

B

glial activation

gllal activation

increase of growth factor effects

transmitter release

receptor expression

impermissive factors

synapse elimination

uptake protection against synapse ellmlnation

FIG.9. Summary of possible events in neuronal-glial and glial-neuronal interactions that may influence activity-dependent changes in neuronal transmission. (A) Events localized in glial cells and subsequent effects on neurons. (B) Events localized in neurons and subsequent effects on glial cells and glia-neuronal interactions.

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Astroglial depolarization occurs, e.g., following glutamate receptor activation (Backus et al., 1989; Grossman and Seregin, 1977; Kettenmann and Schachner, 1985; MacVicar et al., 1988). 2. A second effect of neuronal activity on glial cells may lead to the release of growth or growth-permissive factors. These factors can stimulate the formation of new synapses and thus provide a basis for longterm manifestation of the increase in neural transmission. Released factors either may act directly on neurons and induce growth or may act indirectly. Indirect effects can be assumed to be mediated by the release of growth-permissive matrix molecules (Matthiessen et al., 1989; Price and Hynes, 1985; Wujek and Akeson, 1987) or by the release of factors affecting the stability of synapses. The latter may be mediated by substances interfering with microtubular stabilization, as has been shown for the astroglial S-100 protein (Donato et al., 1989), or by proteases and protease modulators (Farmer et al., 1990; Fawcett and Housden, 1990; Monard, 1988). 3. Finally, glial cells may actively eliminate synaptic connections on neuronal activation. Such an effect can be induced by growth of glial processes following neuronal activation. T h e latter two effects, initiated by neuronal activation and acting on neuronal connectivity, are summarized in Fig. 10. The mechanisms proposed for participation of glial cells in activity-dependent plasticity apparently lack a high degree of specificity with respect to effects on single synapses. As mentioned earlier, however, activity-dependent plastic changes do not necessarily have to be specific to single synapses, but seem to be specific only with respect to given subsets of afferent streams, which reveal activity at the time of induction of plasticity (Bonhoeffer rt al., 1989; Kossel et al., 1990). Such specificity is guaranteed when the aforementioned glial phenomena are complemented by activity-dependent changes in neuronal characteristics. With respect to mechanisms mediated by growth factors, activity-dependent expression of growth factor receptors or mechanisms of uptake by neurons is conceivable. In the case of synapse elimination, the specificity for inactive synapses can be explained by the selective mechanical stabilization of active synapses. Such a mechanism may be realized by polymerization of synaptic cleft material following changes in extracellular ion composition resulting from synaptic activity, as has been proposed for ependymins (Shashoua, 1985). It is also conceivable that adhesion molecules may stabilize synaptic contacts depending on previous activity (Persohn et al., 1989; Pollerberg et al., 1990). Finally, it can be assumed that surface molecules are expressed in an activity-dependent manner on synaptic boutons, which may prevent the elimination of synapses. Certain gangliosides have been

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noncoincident

26 I

synapse n

FIG. 10. Schematic illustration of hypothetical neuronal-glial and glial-neuronal interactions leading to a change in synaptic connectivity. An idealized neuron receives two afferent inputs; one is active and the other inactive. Because of postsynaptic activity a neighboring astrocyte is “activated,” resulting in astroglial growth and the release of growth and growth-permissive factors. T h e former leads to elimination of the inactive afferent; the latter effect initiates new synapse formation of the active afferent.

shown to be expressed at active synapses (Baker, 1988) and to block morphological transformations of astrocytes in culture (Facci et al., 1987, 1988; Skaper et al., 1986). Preliminary evidence for a protective effect of ganglioside treatment on synapse elimination has been obtained in the developing visual cortex of the cat (Carmignoto et al., 1984). These considerations may offer an explanation for some of the paradoxical findings of activity-dependent plasticity. In the developing visual cortex of the cat, suppression of postsynaptic activity has been shown to favor the stabilization of inactive afferents and the elimination of active afferents (Bear et al., 1990; Reiter and Stryker, 1988). This finding may be explained within the framework outlined earlier, namely that glial cells are activated to perform synapse elimination by local neuronal activity and that this process affects all synapses that lack a protective mechanism triggered by previous activity. As glial cells close to active afferents are activated preferentially and local protective mechanisms are blocked by the suppression of postsynaptic activity, these connectivities have the highest probability of being eliminated. In contrast, glial cells close to inactive afferents are not initiated to perform synapse elimination, leaving this pathway intact. The paradoxical situation that

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plastic changes are not uniquely confined to coincidently activated synapses (Bonhoeffer et al., 1989; Kossel et al., 1990) can also be explained on the basis of the outlined mechanism involving growth factor release acting only on all previously active afferents being within the diffusion zone of the released factor. Thus, it can be concluded that a hypothesis involving glial mechanisms in activity-dependent plasticity is not only suitable for explaining plasticity in the classic framework, but may furthermore solve some apparent paradoxes arising from studies on activity-dependent plasticity

U. POSSIBLE CELLULAR MECHANISMS OF A ROLE FOR GLIAL CELLS I N ACTIVITY-DEPENDENT PLASTICITY

Based on the proposed necessity for growth processes of glial cells and the release of factors from glial cells, it is possible to postulate an effector cascade leading to plastic changes mediated by glial cells (Fig. 11). Morphological changes in glial cells, which may reflect a mechanism of sympse elimination, have been observed on stimulation of neurotransmitter receptors (Cornell-Bell et al., 1990b; Hatten, 1985; Narumi et al., 1978; Whitaker-Azmitia et al., 1990), calcium influx (MacVicar, 1987), and activation of protein kinase C (Harrison and Mobley, 1990; Moble) et al., 1986). It is conceivable that these diverse findings reflect different steps in a cascade of events leading to morphological changes in glial cells. As shown schematically in Fig. 11, calcium influx into glial cells can occur via voltage-gated calcium channels activated by potassium released from active neurons and subsequent astroglial depolarization (Herti et ul., 1989; MacVicar, 1984) or by a direct depolarizing effect of glutamate on glial cells (Holopainen and Akerman, 1990). T h e expression of calcium conductances in astrocytes is modulated by norepinephrine (Barres et al., 1989). This allows a link to the findings that plasticity in the CNS relies to a considerable extent on an intact adrenergic system (Bear and Singer, 1986; Kasamatsu and Pettigrew, 1979; Kasamatsu et nl., 1981), and, more specifically, an activation of P-adrenoceptors (Kasamatsu and Shirokawa, 1985; Shirokawa and Kasamatsu, 1986). Recent evidence suggests that cortical f3-adrenoceptors are lo~ 0 and John, 1991). calized predominantly on astroglial cells, zn 7 ~ 2 (Stone A second source of intracellular calcium in astrocytes is the release from internal stores mediated by activation of the inositol pathway. Activation of receptors by glutamate (Glaum et al., 1990; Milani et al., 1989; Nicoletti et al., 1990; Pearce et al., 1986b, 1990), noradrenaline (Pearce et al., 1985, l986a; Puig et al., 1990; Wilson and Minneman, 1990; Wilson et

Neuron K+

synapse-formation

synapse elimination

FIG. 11. Summary of second messenger cascades that may contribute to a role for atrocytes in synapse formation and synapse elimination. Included in the diagram are three afferent systems that are known to act on neurons and are also capable of acting on glial cells: glutamatergic (Glu), cholinergic (ACh),and norepinephrinergic (NE) afferents. Neuronal-glial information transfer occurs via potassium ions (K+) and transmitters (Glu, ACh, NE). Two major second messenger cascades are activated by these interactions: (1) formation of CAMPby P-NE receptors, via activation of adenylate cyclase (AC) and subsequent activation of protein kinase A (PKA); ( 2 ) degradation of phosphoinositide (PI) after activation of glutamate receptors (quisqualate-type, Q), resulting in the accumulation of inositol trisphosphate (IP3) and diacylglycerol (DAG), release of calcium (Ca2 ) from internal stores, and subsequent activation of protein kinase C (PKC). Both cascades have been shown to influence astrocytic morphology, which is assumed to be the basis for synapse elimination. In addition, both systems can activate gene expression and, thus, influence the release of growth and growth-permissive factors. Formation of proto-oncogenes is assumed as a possible intermediate step. See the text for further explanation. +

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al., 1990), and acetylcholine (Masters et al., 1984; Pearce et al., 1985) has been shown to activate this second messenger cascade in astrocytes. Consequently, transient or prolonged increases in the intracellular calcium concentration have been observed in glia, using fluorescent indicators for this ion, following stimulation with glutamate and norepinephrine (Cornell-Bell et al., 1990a; Salm and McCarthy, 1990). Transmitter-activated inositol metabolism has recently been shown to peak during phases of high plasticity in the visual cortex (Dudek and Bear, 1989; Dudek et al., 1989). The participation of a G-protein-mediated process at a site distinct from the postsynaptic neuron in long-term potentiation in the hippocampus (Goh and Pennefather, 1989) may also reflect a glialocated event intermediate to activation of phospholipid metabolism. The necessity of phospholipid metabolism for plasticity is supported by the finding that chronic lithium treatment, which is known to interfere with the supply of phosphoinositol required for maintenance of the generation of inositol 1,4,5-trisphosphate(for review see Berridge et al., 1989), interferes with cortical plasticity (Ohashi et al., 1988). Besides the increase in intracellular calcium, activation of the inositol pathway results in the accumulation of diacylglycerol, an activator of protein kinase C (for review see Berridge, 1987) known to influence astrocytic morphology (Harrison and Mobley, 1990; Mobley et al., 1986). Thus, it is conceivable that a sequence of events including calcium influx, activation of transmitter receptors coupled to phospholipid hydrolysis, and subsequent activation of protein kinase C leads to morphological changes in astrocytes. As astrocytes seem to reveal a specific isoform of protein kinase C (Todo et al., 1990),it may be possible to develop specific drugs to study the role of protein kinase C activation in astrocytes. An additional, probably independent pathway for morphological changes in astrocytes is via activation of the adenylate cyclase (Goldman and Abramson, 1990). The exact mechanism of the final changes in astrocytic morphology has yet to be defined. Effects on cytoskeletal proteins are probably one important step in this kind of glial plasticity (Goldman and Abranison, 1990). The data summarized so far reveal a striking coincidence of these mechanisms leading to morphological changes in astrocytes, being a first necessary step in synapse elimination, with those shown to be crucial for plasticity to occur. With respect to possible mechanisms for the release of growth and growth-permissive factors from astrocytes, very similar cellular pathways can be proposed. Thus, growth-promoting activity from astrocytes has been shown to be controlled by transmitter receptor activation (Brenneman et al., 1987; Eurukawa et al., 1986, 1989; Schwartz, 1988; Schwartz et al., 1977; Suzuki et al., 1984, 1987; Whitaker-Azmitia and

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Azmitia, 1989; Whitaker-Azmitia et al., 1990), although the precise mechanisms remain to be elucidated. One possible intermediate step in the synthesis of growth factors may be the expression of proto-oncogenes in glial cells (Areander et al., 1989; Condorelli et al., 1989; Dragunow et al., 1990), which seem to reflect a link between transmitter receptor activation and growth factor synthesis (Schwartz and Mishler, 1990). An additional way in which glial cells may react to neuronal signals is with nitric oxide as a messenger system and cGMP as an intermediate effector molecule. Astroglial cells seem to be the dominant target of nitric oxide released from neurons on NMDA receptor activation and subsequent calcium influx (Garthwaite and Garthwaite, 1987; Garthwaite et al., 1988). In cerebellar long-term depression, a causal involvement of nitric oxide has been verified recently (Shibuki and Okada, 1991). An elegant review on this promising possibility of neuronal-glial information transfer has appeared (Garthwaite, 1991). The proposed cellular mechanisms underlying possible glial effects on neuronal plasticity may serve as a framework for future studies using specific pharmacological interference with these cascades. In addition, the summary given above indicates that several effector cascades that have been shown to be linked to plasticity do not necessarily have to be located in neurons.

C. PREFERENTIAL INVOLVEMENT OF IMMATURE ASTROCYTES IN PLASTICITY As outlined before, data from several studies reveal that plasticity in the CNS relies on the presence of immature astrocytes. Thus, regrowth of axons can be induced when immature astrocytes, but only to a reduced extent when mature astrocytes, are offered as a growth substrate (Ard and Bunge, 1988; Baehr, 1991; Baehr and Bunge, 1990; Fawcett et al., 1989; Kalderon, 1988; Schreyer and Jones, 1987; Smith et al., 1986; Smith and Silver, 1988). Similarly, critical periods for plastic changes in vivo have been shown to correspond to phases when immature astrocytes are present (Kliot et al., 1988; Muller, 1990, 1991; Miiller and Best, 1989; Smith and Silver, 1988; Stichel et al., 1991). In addition, structures that display a lifelong capacity for plasticity seem to contain glial cells with characteristics usually found only in young animals. This is true for the avian forebrain with the lifelong presence of radial glial cells (Alvarez-Buylla et al., 1987, 1988), the scarcity of glial filaments and the proliferative capacity of pituicytes in the neurohypophyseaUhypothalamic

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system (Bicknell et al., 1989; Kiernan, 1971; Murray, 1968; Paterson and Leblond, 1979; Salm et al., 1982), or glial proliferation and immature appearance of “ensheathing cells” in the olfactory system (Doucette, 1986, 1990). What then is the basis for such a developmentally determined change in the capacity of‘ astrocytes to support plasticity? Several studies reveal changes in astrocytic characteristics during development that may be of importance to their participation in plasticity. Immature astrocytes have greater plasminogen activator activity than mature astrocytes (Kalderon et al., 1988), a factor that may be important for plasticity (Kalderon et al., 1990). In addition, the release or expression of growth or growth-permissive factors is reduced during astrocytic maturation (Ard and Bunge, 1988; Eagleson ~t al., 1985; Gilad et al., 1990; Liesi et al., 1983; Liesi and Silver, 1988; McCaffery et al., 1984; McLoon et al., 1988; Rudge et al., 1985; Smith et al., 1990; van Eldik and Zimmer, 1987). A repeated increase in the expression of growth-promoting factors in the mature system has been observed subsequent to damage (Liesi et al., 1984; Meier et al., 1989), which can be paralleled by glial proliferation (Gall et al., 1979; Latov et al., 1979). It is not yet known whether the developmental regulation of release of growth or growth-permissive factors corresponds to changes in the receptor expression linked to the release of growth factors, to a developmental change in second messenger systems, or to growth factor synthesis. Developmental regulation of transmitter receptors, which may be involved in the role of glia in plasticity and which are coupled to intracellular second messenger cascades on glial cells (e.g., acetylcholine, serotonin, and noradrenaline receptors) has been reported (Ashkenazi et a/., 1989; Burgess and McCarthy, 1985; Whitaker-Azmitia and Azmitia, 1986). Furthermore, recent evidence from studies on astrocytes isolated from optic nerve at different postnatal ages has revealed the presence of calcium conductance only early in the postnatal period (Barres et al., 1990a). This finding may explain the lack of calcium conductances reported in reactive hippocampal astrocytes by Burnard et al. (1990). Suggested that calcium influx is a prerequisite for glial mechanisms in plasticity, this reduction in the expression of calcium conductances would explain the reduction in mechanisms mediating plasticity during maturation. An additional significant change in astrocytic characteristics during maturation is the elaboration of the mature cytoskeleton and the concomitant stabilization of its morphology (Dahl, 1981; Muller, 1990, 1991). With respect to possible mechanisms of synapse elimination, which should include morphological alterations in cellular morphology, a reduction of this capacity would be expected on cytoskeletal stabilization. These data reveal the

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possibility that some of the proposed mechanisms for neuronal-glial information transfer that may be implicated in a glial role in plasticity are only transiently expressed during development and thereby may explain the limitation of plasticity to early development in many central nervous structures.

V. Summary

Activity-dependent plasticity relies on changes in neuronal transmission that are controlled by coincidence or noncoincidence of presynaptic and postsynaptic activity. These changes may rely on modulation of neural transmission or on structural changes in neuronal circuitry. The present overview summarizes experimental data that support the involvement of glial cells in central nervous activity-dependent plasticity. A role for glial cells in plastic changes of synaptic transmission may be based on modulatiop of transmitter uptake or on regulation of the extracellular ion composition. Both mechanisms can be initiated via neuronal-glial information transfer by potassium ions, transmitters, or other diffusible factor originating from active neurons. In addition, the importance of changes in neuronal circuitry in many model systems of activity-dependent plasticity is summarized. Structural changes in neuronal connectivity can be influenced or mediated by glial cells via release of growth or growth permissive factors on neuronal activation, and by active displacement and subsequent elimination of axonal boutons. A unifying hypothesis that integrates these possibilities into a model of activity-dependent plasticity is proposed. In this model glial cells interact with neurons to establish plastic changes; while glial cells have a global effect on plasticity, neuronal mechanisms underlie the induction and local specificity of the plastic change. T h e proposed hypothesis not only explains conventional findings on activity-dependent plastic changes, but offers an intriguing possibility to explain several paradoxical findings from studies on CNS plasticity that are not yet fully understood. Although the accumulated data seem to support the proposed role for glial cells in plasticity, it has to be emphasized that several steps in the proposed cascades of events require further detailed investigation, and several “missing links” have to be addressed by experimental work. Because of the increasing evidence for glial heterogeneity (for review see Wilkin et al., 1990) it seems to be of great importance to relate findings on glial populations to the developmental stage and topographical origin of the studied cells. The present

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overview is intended to serve as a guideline for future studies and to expand the view of “neuro”physio1ogistsinterested in activity-dependent plasticity. Key questions that have to be addressed relate to the mechanisms of release of growth and growth-permissive factors from glial cells and neuronal-glial information transfer. It is said that every complex problem has a simple, logical, wrong solution. Future studies will reveal the contribution of the proposed simple and logical solution to the understanding of central nervous plasticity.

Acknowledgments

I cordially thank Dagmar Neubert for arranging and completing niy stacks of reprints with admirable patience and Dr. Mary Behan for her help in improving the language. This work was supported by a grant from the Bundesniinisterium fiir Forschung und Teclinologie (0316902A).

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ACETYLCHOLINE AT MOTOR NERVES: STORAGE, RELEASE, AND PRESYNAPTIC MODULATION BY AUTORECEPTORS AN D AD RENOCEPTORS lgnaz Wessler Department of Pharmacology University of Mainz W-6500 Mainz, Germany

I. Introduction 11. Basic Events A. Synthesis of Acetylcholine B. Storage of Acetylcholine C. Release of Acetylcholine D. Hydrolysis of Acetylcholine 111. Detection Methods A. Electrophysiology B. Overflow Studies IV. Modulation of Release by Autoreceptors A. Presynaptic Nicotine Receptors B. Preterminal-Axonal Nicotine Receptors C. Presynaptic Muscarine Receptors V. Modulation of Release by Adrenoceptors A. Effects of Sympathomimetic Amines at Skeletal Muscles in Vivo B. a Receptors C. p Receptors D. Physiology VI. Conclusion References

1. introduction

The motor endplate of skeletal muscles is the most widely studied synapse. Considering the complexity of synaptic communication within the mammalian central nervous system (roughly 1013 synapses; several 100-1000 synapses attaching one neuron), the motor endplate has been regarded as the simplest synapse for chemical neurotransmission in mammals. Langley (1906) was among the first to propose the idea of a chemical transmission, that is, the release of a “special substance at the end of the nerve.” Since the experiments published by Dale and colleagues 283 INTERNATIONAL REVIEW OF NEUROBIOLOGY. VOL. 34

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( 1 936), this special substance has been identified as acetylcholine, and there is no doubt about the basic events mediating neurotransmission at motor endplates: acetylcholine is (1) synthesized at the nerve terminal, (2) stored in vesicles, and (3) released synchronously at the active release zones in response to an invading nerve action potential; then it (4) diffuses into the synaptic cleft, ( 5 ) interacts briefly with the enzyme acetylcholinesterease to become inactivated, and (6) binds briefly to the postsynaptic nicotine receptors, to mediate permeability changes of the postsynaptic membrane (endplate potential), which causes a propagated muscle action potential followed by (7) the contraction of the muscle fiber. In the last three decades a large and detailed body of knowledge has been amassed relating to each of these steps. Scientific progress, however, has changed views about this “simple synapse”; and the basic events, divided into molecular-size events, have become highly complicated. Additionally, areas of considerable controversy arose in the literature concerning the source(s) of choline for acetylcholine synthesis, the region of acetylcholine synthesis (cytoplasm versus vesicular membrane), compartmentalization of intraneuronal acetylcholine, exocytotic release of acetylcholine versus release via a membrane-bound gating mechanism (or operator or transporter), and presynaptic effects of acetylcholine (i.e., modulation of the release by presynaptic autoreceptors). T h e present article focuses mainly on the last topic, modulation of acetylcholine release from the motor endplate by presynaptic receptors. It has become a very attractive and widely accepted concept that an individual neuron can regulate its function, the release of transmitters or modulators, via the activation of local feedback loops. After escaping the neuronal membrane and entering the synaptic cleft (or extracellular space), the transmitter activates both the receptors localized at the endorgan (postsynaptic receptors) and the receptors localized at the nerve terminal within its diffusion radius (so-called presynaptic or neuronal receptors). Stimulation of presynaptic receptors triggers the generation of intracellular signals that modify ion channels or regulatory proteins and, in consequence, modify the subsequent release of the transmitter. This has been shown for the classic transmitters, particularly those released from the peripheral autonomic nervous system (acetylcholine, adrenaline, noradrenaline), as well as for dopamine, serotonin, histamine, y-aminobutyric acid (GABA), and opioid peptides (Polak, 1971 ; Franebo and Hamberger, 1971; Starke, 1971; Langer, 1974; Kilbinger and Wessler, 1980; Kilbinger, 1984, 1987; Brennan, 1982; Arrang et al., 1983; for a detailed review see also Starke et al., 1989). In contrast to the clear evidence in favor of presynaptic autoreceptors modulating the release of acetylcholine from visceral organs inner-

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vated by the parasympathetic, cholinergic nervous system (airways, heart, small intestine, urinary bladder: Kilbinger, 1977, 1984; Kilbinger and Wessler, 1980, 1983; Fosbraey and Johnson, 1980; Fryer and Maclagan, 1984; Wetzel and Brown, 1985; D’Agostino et al., 1986, 1990; Wessler et al., 199Oc, 1991), a controversy arose in the last four decades as to whether release of acetylcholine from motor nerves is also under the local control of presynaptic autoreceptors. Indirect evidence obtained from recording of endplate potentials or contractions appears to support the existence of autoreceptors at motor nerve terminals (Lilleheil and Naess, 1961; GlavinoviC, 1979; Su et al., 1979; Bowman, 1980; Gibb and Marshall, 1984). Biochemical studies in which the release of unlabeled acetylcholine was measured after blockade of the enzyme acetylcholinesterase have failed to provide evidence for such releasemodulating autoreceptors (Dale et d., 1936; Emmelin and MacIntosh, 1956; Beranek and VyskoTil, 1967; KrnjeviC and Mitchell, 1961; Flechter and Forrester, 1975). This area of controversy, however, is now cleared. The present review summarizes clear and basic evidence that the release of acetylcholine from the motor nerve (phrenic nerve) measured by a radiotracer method under the condition of unblocked acetylcholinesterase-a crucial condition for the operation of presynaptic nicotine receptors (see Section IV,A,4)-is regulated by nicotine and muscarine autoreceptors. It is emphasized that autonomic neurons, in addition to the autoreceptors, are endowed with a battery of heteroreceptors, that is, receptors interacting with modulators or transmitters (receptors for acetylcholine, adrenaline, adenosine, angiotensin, bradykinin, dopamine, GABA, histamine, noradrenaline, serotonin, prostaglandins, and opioid peptides) released from a foreign neuron or cell. Undoubtedly, the motor nerve is also endowed with such heteroreceptors. Effects of adrenaline and noradrenaline on neuromuscular transmission were demonstrated in the last century (Oliver and Shafer, 1895), and presynaptic adrenoceptors have been postulated on the basis of functional studies (“anticurare” effect of catecholamines) as well as electrophysiological studies (Bowman and Raper, 1966; Kuba, 1970; Malta et al., 1979; for review see Bowman, 1981). In the past, however, the effects of sympathomimetic amines at skeletal muscles have been demonstrated in studies in which the electrical o r mechanical end-organ responses have been analyzed, whereas release studies have been carried out only very recently. When the radiotracer method was used to measure the release of newly synthesized acetylcholine from the phrenic nerve, sympathomimetic amines were found to modulate transmitter release from the phrenicnerve; moreover, the receptors involved were characterized (a1and f3 l receptors)

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and the effector systems coupled to these receptors could be identified (Wessler and Anschutz, 1988; Wessler et al., 1989, 1990a,d; Somogyi et al., 1987). T h e present review also addresses this subject and shows basic evidence for facilitatory presynaptic effects of sympathomimetic amines on transmitter release from the phrenic nerve. This review describes the presynaptic effects of four different receptor populations: the nicotine and muscarine autoreceptors and the LY and p heteroreceptors. For each of these receptors five topics are addressed: (1) basic evidence in overflow studies, (2) basic evidence in functional and electrophysiological studies, (3) receptor characterization and signal transduction, (4) physiology, (5) comparison with other tissues. In addition, the first two sections outline the basic events of neuromuscular transmission (synthesis, storage, and release of' acetylcholine) and the methods used to detect acetylcholine released from the motor nerve. Possible targets and mechanisms involved in the receptor-mediated modulation of acetylcholine release are discussed with the basic events of chemical neurotransmission. Section I1 I covers the advantages and the limitations of both electrophysiological and overflow studies (measuring the release of radiolabeled acetylcholine) in an unbiased fashion. A number of detailed articles, including review articles covering some of the present subjects, have been published and the reader is referred to these articles (Standaert and Riker, 1967; Foldes, 1971; Galindo, 1972; Riker, 1975; Colquhoun, 1975; Ginsborg and Jenkinson, 1976; Miyanioto, 1978; Foldes and Vizi, 1980; Bowman, 1980, 1981, 1985, 1990; Standaert, 1982; Wilson, 1982; Bowman ut al., 1984, 1986; Starke et al., 1989; Wessler, 1989; Wonnacott et al., 1989).

II. Basic Events

The basic events of neurochemical transmission in skeletal muscles as outlined in the Introduction, particularly synthesis, storage, and release of acetylcholine, with special reference to radiolabeled acetylcholine, are discussed here in detail.

A. SYNTHESIS OF ACETYLCHOLINE

Acetylcholine is synthesized in the cytoplasm of the nerve endings. The enzymes involved are transported to the nerve endings via axonal transport. Choline acetyltransferase (ChAc) catalyzes a reversible transfer of acetyl groups from acetyl-coenzyme A (acetyl-CoA) to choiine. In

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vertebrates, ChAc is thought to be located more or less exclusively in cholinergic neurons (except human placenta), particularly at their terminals. It is widely accepted that the enzyme is localized mainly within the cytoplasm of nerve terminals (Hebb, 1972; Fonnum, 1973; Fonnum and Malthe-Sqh-enssen, 1973); presumably, a fraction of ChAc is also attached to the membranes of vesicles, as shown for the central nervous system (Benishin and Carroll, 1981). Nevertheless, the synthesis of acetylcholine occurs in the cytoplasm, and newly synthesized acetylcholine must be taken up by a vesicular acetylcholine transport system that can be blocked specifically by various compounds, the most specific being vesamicol (Marshall and Parson, 1987). The regional distinction between synthesis of acetylcholine and vesicular storage of acetylcholine is of particular interest and must be considered when the release of newly synthesized radioactive acetylcholine is measured under different experimental conditions (see Section II1,B). Acetyl-CoA generated from pyruvate is thought to diffuse out of the mitochondria1 inner compartment in quantities suflicient to support acetylcholine synthesis (Tutek, 1970). Choline is supplied by three different sources (MacIntosh and Collier, 1976; TuEek, 1985): (1) free plasma choline, (2) reuse of choline after hydrolysis of acetylcholine; (3) breakdown of choline from choline-containing phospholipids (phosphatidyicholine) that are localized in neuronal and nonneuronal membranes. T h e intraneuronal pool of free choline is limited (Potter, 1970). Accordingly, most of the choline must be taken up by the sodium-dependent choline uptake system localized at cholinergic nerve terminals. Extraneuronal choline, like other quaternary bases, is limited in its ability to enter the neuroplasma by diffusion through the nerve membrane, but is taken u p into the nerve terminal by the high-affinity choline uptake system. T h e high-affinity choline uptake system ( K , for choline = 1-10 p M ) was first observed in brain synaptosomal preparations (Yamamura and Snyder, 1973) and is also thought to exist on motor nerves (Potter, 1970; Beach et al., 1980; Ruch et al., 1982). The driving force for the transport of choline is provided by the sodium gradient maintained by sodium-potassium ATPase, the choline gradient, and the membrane potential (Vaca and Pilar, 1979; Jope, 1979). Choline taken up by the high-affinity system either supplies the small pool of intraneuronal choline, is metabolized to acetylcholine, or contributes to an intraneuronal pool of phospholipids. It has been shown that roughly 50% of the choline derived from hydrolyzed acetylcholine is taken up again by the motor nerve (Potter, 1970; Wessler and Kilbinger, 1986), and a similar observation has been reported for the superior cervical ganglion (Collier and Katz, 1974). Uptake of choline and synthesis of acetylcholine are closely related to

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TABLE I SYNTHESIS AND RELEASE OF ACETYLCHOLINE I N DIFFERENT SKELETAL MUSCLES Acetylcholine synthesis Species, muscle

nmol/g/hr pmol/endplate/min Source ~~

Rat, hemidiaphragm Rat, hemidiaphragm Rat, hemidiaphragm Rat, hemidiaphragm (innervated part) Rat, soleus muscle Rat, extensor digitorum longus muscle Human, intercostal muscle

Frog, sartorius muscle Release of acetylcholine, rat phrenic nerve (1-20 Hz)

~

~~~~

600 270 4

0.20 0.05 0.003

Hebb et al. (1964) Miledi et al. (1982) Potter ( 1 970)

800 60

0.05 0.035

Tutek (1982) Harris (1987)

200

~

TuCek (1982) 0.08 0.002 0.00003-0.00 1

Ito et al. (1976), Molenaar et al. (1979) Molenaar and Polak (1980) Potter ( 1 970) Bierkamper and Goldberg ( I 980) Smith and Weiler (1987)

the release of acetylcholine and, thus, are regulated by the demand for acetylcholine. Uptake of choline and synthesis of acetylcholine are low at rest and enhanced with increased traffic in the nerve terminals. Highfrequency stimulation (50 Hz) of the phrenic nerve during incubation with radioactive choline has shown to enhance the synthesis of radioactive acetylcholine 13-fold (Wessler and Kilbinger, 1986). As shown in Table I, the rate of synthesis of acetylcholine reported in the literature varies considerably. Hebb et al. (1964) reported the highest rate of acetylcholine synthesis per endplate (about 0.20 pmol/endplate/min), whereas considerably lower rates were found by Potter (1970). In this context it should be noted that in the experiments done by Hebb and co-workers, the muscle fibers might have contributed considerably to the synthesis of acetylcholine (carnitine acetyltransferase), whereas in the remaining studies shown in Table I, only the activity of the specific neuronal choline acetyltransferase was determined. Synthesis of acetylcholine does not differ greatly among different skeletal muscles in mammals, but is evidently higher in mammalian skeletal muscles than in frog sartorius muscle. The gap between acetylcholine synthesis per individual endplate (at least about 40 fmollmin) and stimulated acetylcholine release (about 0.03- 1 fmol/endplate/min) leaves sufficient amounts of acetylcholine available for presynaptic, receptor-mediated modulation. Nevertheless,

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it must also be considered that, during long periods of stimulation, the tissue content of acetylcholine is reduced; that is, synthesis cannot keep up with release. This is true, however, particularly under experimental in uitro conditions, where stimulation (5-30 min) is prolonged, so that amounts of endogenous acetylcholine sufficient for biochemical detection can be collected. Under in uiuo conditions, motoneurons do not fire continuously but intermittently (Grimby and Hannerz, 1977; Grimby et al., 1979), and it is assumed that depletion of tissue stores of acetylcholine does not occur under in uivo conditions. The key, linking release of acetylcholine, on the one hand, and uptake of choline and synthesis of acetylcholine, on the other hand, appears to be the axoplasmic concentrations of the sodium and calcium ions. Both ions are increased after an action potential invades the nerve terminal. Synthesis of acetylcholine and, thus, uptake of choline into the superior cervical ganglion is enhanced in the absence of a stimulated acetylcholine release by blocking the sodium pump (Birks, 1983). Removal of extracellular calcium, however, prevents the enhanced synthesis of acetylcholine (Birks, 1983). This coupling allows the tissue content of acetylcholine to remain fairly constant at different degrees of nerve activity. It was already pointed out earlier that more pronounced synthesis of radioactive acetylcholine and, thus, enhanced uptake of [3H]choline into the phrenic nerve occur in the presence of electrical nerve stimulation as compared with the absence of such stimulation. Uptake of choline into the phrenic nerve also appears to be enhanced by glucocorticoids (Veldsema-Currie et al., 1985). Muscle fibers can take up choline in a saturable manner (Renkin, 1961; AdamiC, 1972). A detailed analysis of choline uptake by the phrenic nerve hemidiaphragm preparation has shown that the uptake of choline occurs mainly at the endplate region (Waser et al., 1978; Ruch et al., 1982; Veldsema-Currie et al., 1984; Wessler and Sandmann, 1987), but, surprisingly, the uptake of choline does not cease in chronically denervated preparations (Waser et al., 1978; Wessler and Sandmann, 1987). Thus, choline was taken up mainly by the innervated part of the muscle membrane, that is, by the postsynaptic membrane of the motor endplate. The majority of the applied radioactive choline was incorporated into the cell membranes (phosphatidylcholine, present in the exterior of cell membranes) and, even more exciting, the breakdown of phosphatidylcholine was higher in the innervated than in the noninnervated part of the muscle fiber (McCarty et al., 1973; Wessler and Sandmann, 1987). Both observations suggest that the uptake and metabolism of choline concentrated at the postsynaptic site of the endplate are involved in neuromuscular transmission. The contribution of the

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Ribs

Central Tendon

Muscle fibers

Endplate region

End plate preparation

Whole

preparation

Muscle fibers

FIG. 1. Rat left phrenic nerve-hemidiaphragm preparation. The phrenic nerve-hemidiaphragm is removed from the rat according to Bulbring (1946). The complete phrenic nerve-hemidiaphragm preparation consists of the whole hemidiaphragm with the innervating phrenic nerve. The endplates are localized in the middle of the muscle fibers, and the endplate area can be recognized by eye as a bright line across the muscle fibers. All release experiments with radiolabeled acetylcholine are performed with endplate preparations, whereby the muscle fibers are cut off at a distance of 0.5- 1 nini at either site of the endplate area.

postsynaptic site in supplying choline for acetylcholine synthesis via the breakdown of phospholipids and the modification of the function and the sensitivity of the postsynaptic nicotine receptors embedded in the lipid bildyer have been discussed (Wessler and Sandmann, 1987). The high capacity of the muscle fibers for both uptake and metabolism of choline and the unfavorable relationship between neuronal and nonneuronal elements of the phrenic nerve-hemidiaphragm preparation limit the synthesis of radioactive acetylcholine from the applied radioactive precursor choline (Potter, 1970; McCarty et al., 1973; Wessler and Kilbinger, 1986). In fact, after incubation of a complete nervemuscle preparation (Fig. 1) with radioactive choline, only tiny amounts of radioactive acetylcholine are synthesized in phrenic nerve terminals (McCarty et ai., 1973; Wessler and Kilbinger, 1986),whereas reasonable

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labeling of the acetylcholine stores occurs when most of the muscle fibers are removed (Wessler and Kilbinger, 1986). In the latter experiments the endplate area attached to the phrenic nerve is isolated by cutting the hemidiaphragm 0.5-1 mm parallel to each site of the endplate region (see Fig. 1). When such an endplate preparation is incubated with radioactive choline, the precursor choline has more direct access to the nerve terminals than in a complete nerve-muscle preparation (Wessler and Kilbinger, 1986). Finally, it should be noted that some quantities of acetylcholine are also synthesized in nonneuronal elements, that is, in the muscle fibers. The muscle fibers can acetylate choline by means of the carnitine acetyltransferase (White and Wu, 1973; TuEek, 1982), and Schwann cells containing ChAc can synthesize and release acetylcholine (Dennis and Miledi, 1974; TuEek et al., 1978). Removal of most of the muscle excludes the possibility that [3H]acetylcholine synthesized by muscle fibers might have contributed to the stimulated release of radioactivity. Electrical field stimulation of a chronically denervated endplate preparation does not produce any outflow of radioactivity (Fig. 2) (Wessler and Kilbinger, 1986). Likewise, a possible liberation of [3H]acetylcholine from Schwann cells can be excluded for the following reason. T h e liberation of acetylcholine from Schwann cells is tetrodotoxin resistant (Dennis and Miledi, 1974), whereas the stimulated release of radioactive acetylcholine from the phrenic nerve is abolished by tetrodotoxin (Wessler and Kilbinger, 1986; see Fig. 2). Schwann cells are probably not endowed with a high-affinity choline uptake system or contain the system only in small quantities; thus, an incubation period of 30-40 min does not allow the Schwann cells to take up [3H]choline in amounts sufficient for substantial synthesis of [3H]acetylcholine. A biological function for the small quantities of acetylcholine liberated from muscle fibers or from Schwann cells is, so far, not known.

B. STORAGE OF ACETYLCHOLINE

Acetylcholine synthesized within the rat phrenic nerve-hemidiaphragm or extensor digitorum longus is localized to about 70-80% in the nerve terminals and to about 10%in the intramuscular nerve fibers, the remainder being formed in nonneuronal elements (muscle fibers, Schwann cells: Polak et al., 1981; Miledi et al., 1982). That acetylcholine synthesis occurs in the cytoplasm but not within the vesicles has already been stated. Increasing evidence has been presented in the last two decades that acetylcholine is stored in distinct compartments within the nerve terminals. These different compartments are characterized on the

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field stimulation chronically denervated

6-

L A ‘

A ‘

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In X c

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60

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-J

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- Caw-free

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I

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FIG.2. Abolition of stimulated tritium eflux ([3H]acetylcholine release) by chronic denervation, by removal of extracellular calcium, or by application of tetrodotoxin (TTX). After incubation of endplate preparations with [3H]choline (to label neuronal acetylcholine), electrical nerve or field stimulation causes an increase in tritium efflux that reflects the release of [SH]acetylcholine (see Fig. 3). T h e stimulated tritium efflux was abolished by chronic denervation of preparations, by removal of extracellular calcium, or by addition of 10.3 $4 tetrodotoxin. (Values from Wessler and Kilbinger, 1986.)

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basis of either biochemical (subcellular fractionation techniques) or release studies. Neuronal acetylcholine is separated into free and bound, presumably cytoplasmic and vesicular transmitters, respectively (Miledi et al., 1982). Free acetylcholine is defined as the difference between total tissue acetylcholine and vesicule-bound acetylcholine. In frog muscle, about 80% of the bound acetylcholine can be attributed to the vesicular fraction (Miledi et al., 1982). The terminals of the motor nerves are filled with electron-lucent vesicles roughly 40-50 nm in diameter (for references see Zimmermann, 1988). It should be noted, however, that the motor nerve terminal contains, in addition, some dense-cored synaptic vesicles of larger diameter (about 100 nm). The physiological significance of these vesicles remains to be elucidated, but one may speculate about the coexistence of other transmitters (peptides) or presynaptic modulator systems. Vesicles are regarded as the structural elements for storage of acetylcholine. Vesicles possess several transport systems, to take up calcium ions, ATP, protons (Mg-ATP-dependent proton pump), and the transmitter acetylcholine. A vesicular ATPase is regarded as the possible proton-motive force required for the uptake of acetylcholine. The vesicular uptake of acetylcholine can specifically be inhibited by various compounds, including the nicotine receptor antagonist tubocurarine (IC5,: low;Anderson et al., 1983); the most specific compound appears to be vesamicol (IC5, = about 30 nM). Vesamicol does not interfere with acetylcholine synthesis but binds to vesicular membranes and blocks the uptake of acetylcholine into the vesicles (Jope and Johnson, 1985; Whitton et al., 1986; Marshall and Parsons, 1987; Cabeza and Collier, 1988; Kaufman et al., 1989). Evidence for heterogeneity of the vesicles comes from biochemical (density gradient centrifugation) as well as from release studies comparing the specific radioactivity of acetylcholine or the concentration of false transmitters (acetylpyrrolcholine) in the vesicular fraction with the respective value in the incubation medium after the tissue has been stimulated. Most of these studies have been carried out on the Torpedo electric organ, but similarities have been described for synaptic vesicles isolated from the rat diaphragm (Volknandt and Zimmermann, 1986). At least three different populations of vesicles can be discriminated. The VP, fraction corresponds to vesicles entering the terminal by axonal transport; the VP, fraction, not directly involved in transmitter release, is presumed to represent the main storage pool; and the VP, fraction appears to be a metabolically very active pool directly involved in the release of acetylcholine. The specific radioactivity of acetylcholine detected in the VP, fraction corresponds to that detected

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in the incubation medium (Zimmermann and Denston, 1977; Suszkiw et al., 1978; Zimmermann, 1979, 1988; see also Whittaker, 1988). Support for the heterogeneity of vesicles also comes from studies in which the release of endogenous acetylcholine is measured under different release conditions. T h e release of acetylcholine from the brain, ganglion cells, and skeletal muscle has been shown to decline rapidly to a constant level when a continuous stimulation is applied (see MacIntosh and Collier, 1976). This observation indicates exhaustion of a ready releasable transmitter compartment early during stimulation and subsequent replenishment of this pool from a reservoir. This idea agrees with the suggestion that newly synthesized acetylcholine is preferentially taken up by the metabolically very active vesicles (VP, fraction) and, as a consequence, is released preferentially (Collier and MacIntosh, 1969; MacIntosh and Collier, 1976). T h e ready releasable transmitter compartment is thought to be represented by those vesicles localized most closely to the neuronal membrane and the active release zones of the motor nerve terminals. It seems possible that only a few pulses can exhaust this compartment or alter the specific organization of these vesicles closely associated with the release zones. In fact, less than 1 % of the tissue store or less than 1000 quanta perjunction (for references, see MacIntosh and Collier, 1976; Bowman, 1990) corresponds to this ready releasable transmitter compartment. The fall in transmitter release following only few pulses (see also Erulkar, 1983) may be caused either by the process of mobilization or by a change in the specific organization between the recycled vesicles and the active release zones. When heavy traffic runs down the nerve, the vesicles delivered from the reservoir may not maintain the same structural organization formed at rest. Measuring the pulse-to-pulse release of [3HH]acetylcholinefrom the small intestine, Kilbinger and Wessler ( 1983) have provided indirect evidence that even after six pulses, the ready releasable compartment is exhausted, and that a process not related to presynaptic receptors, but most likely mobilization, becomes rate limiting for acetylcholine release. In this context one should consider that radioactive acetylcholine represents newly synthesized transmitter preferentially localized within the ready releasable transmitter compartment; the possibility that newly synthesized and preformed transmitters are distinctly regulated by receptor-mediated mechanisms cannot be fully excluded, although the experimental evidence for this possibility is very scanty (see Section 111,B). 'The conclusion that there exist functionally different compartments ot vesicles received further support in a study by Cabeza and Collier ( 1988), giving convincing evidence that one compartment of cholinergic

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vesicles in the cat superior cervical ganglion is sensitive to vesamicol, whereas a second compartment is resistant to vesamicol. The venom of the black widow spider, known to evoke acetylcholine from the motor nerve in a quantal manner (Gorio et al., 1978; Fesce et al., 1980), could stimulate acetylcholine release despite the presence of vesamicol; that is, the venom may trigger the release of acetylcholine from an additional vesicular acetylcholine compartment. This observation, however, is also indicative of an effect of vesamicol more complex than only blockade of the vesicular uptake of acetylcholine. Different vesicular acetylcholine compartments may also be indicated by the different forms of miniature endplate potentials (mEPPs). At present, whether the different forms of mEPPs (sub-, bell- and giant-mEPPs; see Tremblay et al., 1983; Kriebel, 1988; Bowman, 1990) mirror different vesicular compartments or are a refection of the subunit hypothesis of quantal transmitter release remains an open question. In addition to vesicular compartmentalization, indirect evidence supports compartmentalization of the free, cytoplasmic acetylcholine. Surplus acetylcholine formed in the presence of lipidsoluble acetylcholinesterase inhibitors is thought to be localized in the cytoplasm, because it is not releasable by calcium-dependent stimuli (Birks and MacIntosh, 1961; Potter, 1970; MacIntosh and Collier, 1976). Molenaar and co-workers (1987) have also shown in the rat diaphragm that, in the presence of an acetylcholinesterase inhibitor, the total tissue store of acetylcholine increased without a change in bound acetylcholine, indicating a cytoplasmic location of surplus acetylcholine. Accordingly, one portion of free or cytoplasmic acetylcholine is sensitive to intraneuronal acetylcholinesterase, whereas another portion is resistant. This observation can best be explained by the compartmentalization of cytoplasmic acetylcholine. Gorio et al. (1978) have presented fairly convincing evidence that in the mouse diaphragm, about 65% of the total acetylcholine tissue content is stored in a releasable form(s). Considering that about 7 0 4 0 % of total acetylcholine is present within the nerve terminal, this means that some acetylcholine, not releasable by electrical nerve stimulation, is left in the nerve terminal (10-20% of tissue content). Whether this acetylcholine corresponds to the extravesicular, cytoplasmic compartment or to a portion of vesicles not releasable remains an open question. C. RELEASE OF ACETYLCHOLINE The active release zones localized opposite the junctional folds can be regarded as pivotal specializations mediating synchronized release of

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acetylcholine in response to an invading nerve action potential and its electrotonic spread to the terminal membrane. It is remarkable that each individual endplate is endowed with a large number (roughly 500- 1000) of active release zones (Peper et al., 1974; Ceccarelli and Hurlbut, 1980), each of them about 50 nm wide and packed with about 30-50 vesicles (Couteaux and Pecot-Dechavassine, 1974; Standaert, 1982). So far, whether release is likely to occur with a similar or even identical probability along the entire nerve terminal is an open question. Bennett and Lavidis (1979, 1982), working with toad motor nerve terminals, and Vautrin and Mabrini (1980) provided evidence for nonuniformity in transmitter release along the individual terminal. Additionally, mEPPs appear to occur not uniformly distributed along the frog motor nerve terminal (Tremblay et al., 1984). The large number of active release zones per individual endplate, whereby each individual active release zone can be regarded as a miniature “edition” of an endplate, offers an attractive hypothesis to explain the presynaptic, receptor-mediated control of acetylcholine release. This model shows the individual active release zones of a single endplate to be “wired together”; presynaptic receptors switch on or switch off individual active release zones, thus modifying transmitter release from a single endplate. Accordingly, the release probability at each individual active release zone is supposed to be modulated by ion channels or regulatory peptides that are closely associated positively or negatively with presynaptic receptors. The present hypothesis must take into account a principal difference in the architecture of synapses mediating transmission at skeletal muscles and those synapses mediating transmission in visceral organs innervated by the autonomic nervous system. Motor endplates are more or less specialized synapses, the synaptic cleft being restricted from the extracellular space, whereas the “synapses”mediating transmission at smooth muscles communicate directly with the extracellular space. In consequence, acetylcholine released from neuroeffector “synapses”of the autonomic nervous system can escape to some extent within the extracellular space and can act at presynaptic receptors of neighboring synapses. Acetylcholine released from a motor endplate can hardly enter the extracellular space. Therefore, the presynaptic effect of acetycholine at motor endplates is restricted to the original endplate from which it has been ejected. Controversy exists over the mechanisms involved in the liberation of acetylcholine from motor nerves during rest (spontaneous release) or during invasion of the endplates by propagated neuronal activity (stimulated release). Two topics in this area are presented. First, the different

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forms of mEPPs (see above) are interpreted to reflect either univesicular release of vesicles filled differently with acetylcholine or multivesicular release of vesicles. Second, stimulated acetylcholine release is explained either by the calcium-dependent fusion of vesicles with the neuronal membrane and the ejection of their contents into the synaptic cleft (vesicular exocytosis) or by the transport of fixed quantities of cytoplasmic actylcholine via membrane-bound gates or operators (membrane gate hypothesis). Evidence in favor of each alternative has been published (Israel et al., 1979; Ceccarelli and Hurlbut, 1980; Tauc, 1982; Dunant, 1986; Whittaker, 1988; Bowman, 1990), and it is beyond the scope of this article to present in a detailed and balanced manner the more relevant experimental observations in this area. A brief evaluation of the vesicular versus membrane gate hypothesis, however, follows. There is some evidence in favor of the existence of a membrane-bound “transporter” or gating apparatus located in nerve terminals, and this finding has been supported recently by the inhibitory effect of vesamicol on spontaneous acetylcholine release (VyskoEil, 1985; RiEnf and Collier, 1986); however, the overwhelming experimental evidence favors the vesicular exocytosis hypothesis. This hypothesis is also supported by the finding that [3H]acetylcholine newly synthesized within the phrenic nerve in the absence of electrical nerve stimulation (i.e., in the absence of a stimulated vesicular turnover or of quanta1 release) cannot be released in response to a physiological stimulus (electrical nerve stimulation), but is liberated by high potassium in a calcium-independent manner (Wessler and Steinlein, 1987; see also Section 111,B). This observation indicates that the nonvesicular [3HH]acetylcholine,presumably localized in the cytoplasm of the nerve terminals, is not releasable by propogated neuronal action potentials, and it is hard to believe that cytoplasmic acetylcholine can be released in uzuo in response to depolarization of the motoneuron. Additionally, whole-cell patch-clamp of nerve membranes synthesized in Xenopm did not reveal evidence of the presence of large conductance channels (> 60 pS), possibly mediating the release of acetylcholine off the nerve terminal (Young and Chow, 1987). Recently, Vizi (1989) has shown that the stimulated release of newly synthesized [3H]acetylcholine from the mouse phrenic nerve is inhibited by vesamicol, a blocker of vesicular transporter for acetylcholine. The author interpreted this observation as strong evidence in favor of the vesicular hypothesis, but the inhibitory potency of vesamicol on the spontaneous liberation of nonquantal acetylcholine should also be considered. Thus, the inhibitory action of vesamicol reported by Vizi can be mediated in two different ways: either by blockade of the acetylcholine transporter

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placed into the neuronal membrane (Edwards et al., 1985) or by blockade of the vesicular transporter as supposed by Vizi (1989). In the next two sections the different forms of acetylcholine release occurring from the motor nerve terminal at rest and during stimulation are discussed.

1 . Spontaneow Acetylcholine Release In the absence of applied stimuli (a situation not identical to neuronal rest) at least four types of spontaneous release of acetylcholine can be discriminated (Kriebel, 1988; Bowman, 1990): 1. Quantal release of acetylcholine mediating the occurrence of mEPPs (Fatt and Katz, 1952). mEPPs are regarded as reflecting the release of packets of acetylcholine (quanta1 release of an individual vesicle o r of a subunit of a vesicle) producing the smallest end-organ event. T h e frequency of mEPPs is related to the intracellular concentration of free calcium. 2. Quantal release of acetylcholine mediating the occurrence of subminiature endplate potentials. T h e observation of subminiature endplate potentials suggested that concept that a single quantum consists of several subunits; that is, a vesicle can eject its content in packets and the smallest quantity ejected should be sufficient to generate a subminiature endplate potential. That the subminiature endplate potentials occur mainly under artificial experimental conditions should, however, be taken into consideration. 3. Liberation of acetylcholine mediating the occurrence of giant mEPPs. This liberation process, which is not dependent on extracellular calcium, increases greatly under artificial experimental conditions (hypotonicity, application of 3,4-diaminopyridine or 4-aminoquinoline). 4. Liberation of nonquantal acetylcholine mediating a small depolarization (about 5 mV) of the muscle membrane. T h e liberation of nonquantal acetylcholine occurs in the absence of extracellular calcium and accounts for about 98% of the total biochemically assayed acetylcholine at rest (Vizi and VyskoEil, 1979). Nonquantal acetylcholine (about 13 pmol/min per rat hemidiaphragm: Bierkamper and Goldberg, 1980; Miledi ut d.,1982) is liberated from both nerve and muscle fibers, but denervation causes a substantial reduction (70%) in spontaneous acetylcholine release. Both the physiological relevance of nonquantally liberated acetylcholine and the mechanism of its liberation are matters of controversy. Edwards et al. (1985) have proposed that the vesicular acetylcholine transporter incorporated during exocytosis into the neuronal membrane might mediate the spontaneous liberation of nonquantal acetylcholine. Also, newly synthesized [3H]acetylcholine is liberated spon-

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taneously in very small quantities from the phrenic nerve (Wessler and Kilbinger, 1986). Principally, in all experiments with [3H]acetylcholine, the spontaneously liberated [3H]acetylcholine is subtracted and only the stimulated, quantally released [3H]acetylcholine is considered for receptor-mediated modulation. Recently, Grinnell and co-workers (1989) developed a very elegant method to detect the release of acetylcholine from the frog neuromuscular junction with closely apposed outside-out clamped patches of Xenopus myocyte membrane, rich in nicotine receptors. The patch was used as sensitive detection machinery to monitor the release of acetylcholine. Using this method the authors did not find evidence for substantial nonquantal release, and they concluded that transmitter leakage at adult frog terminals does not occur at the synaptic surface of the nerve terminal. Nonquantal acetylcholine was supposed to be released in a rather widespread and diffuse fashion from many sources, which may include the nerve terminal; however, consider that denervation causes a substantial reduction (70%) in spontaneous acetylcholine release, indicating that the nerve is the dominant source for nonquantal acetylcholine release. The possibility that enzymatic “dysjunction,” rendering the synaptic surface of the terminals accessible to the patch pipet, might have affected the properties of the nerve membrane and, thus, the mechanisms involved in nonquantal acetylcholine release cannot be excluded.

2 . Evoked Acetylcholine Release A propagated nerve action potential and its electrotonic invasion into the nerve terminal cause depolarization of the active release zones, influx of calcium through voltage-operated calcium channels, and synchronized release of multiple quanta that give rise to the endplate potential of the muscle membrane. The endplate potential triggering the threshhold depolarization of the muscle membrane (and, as a consequence, a propagated muscle action potential and contraction) is thought to represent an integral number of mEPPs. The key allowing the generation of endplate potentials is the synchronized activation of some, but not all, active release zones of an individual endplate, to release vesicular acetylcholine in amounts sufficient to trigger threshhold stimulation of the postsynaptic nicotine receptors. Attempts to estimate the quanta1 content of an individual endplate potential have revealed about 100-300 quanta to be released by a single pulse and to create the endplate potential (Ginsborg and Jenkinson, 1976). Even the release of only one quantum per active release zone, which are present in quantities

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of 500-1000 at a single endplate, would imply an intermittence of the release along all active release zones per individual endplate. This intermittence would even increase when more than on quantum is released from one individual active release zone in response to a single depolarization. An ultimate condition for the evoked quantal release is the influx of extracellular calcium. Mobilization of intraneuronal calcium, in addition to the influx of extracellular calcium ions, cannot be excluded as a trigger of quantal acetylcholine release. The main key, however, is the influx of extracellular calcium ions, because evoked quantal release is abolished in the absence of extracellular calcium ions. Calcium entering the nerve by channel opening binds to calcium-binding proteins (calmodulin, calcineurin, synexin: Pollard et al., 1980; Bowman, 1990), which are integrative parts of the stimulus-secretion coupling and promote the fusion of vesicles. Surprisingly, however, inhibitors of calmodulin do not cause a reduction in the synchronous release, suggesting only that calmodulin already bound to vesicles and not accessible to the applied inhibitors is involved in the stimulus-secretion process (Publicover, 1985). In the frog, four calcium ions are supposed to induce the release of one quantum of acetylcholine (Dodge and Rahaminoff, 1967), and release is related to calcium entry raised to some power greater than 1. With respect to the dominant role of intracellular free calcium in triggering transmitter release, calcium channels are among the targets of presynaptic receptors. Stimulation of presynaptic, facilitatory receptors may open calcium channels, whereby the calcium concentration at strategically significant spots inside the nerve is increased and, as a consequence, transmitter release is enhanced. In fact, it has been shown that facilitatory, adrenoceptors are coupled to calcium channels (Wessler et al., 1990a), but the effector system of the presynaptic nicotine receptors remains to be elucidated. The voltage-sensitive calcium channels are discriminated at least into four different types: L, N, T, and P types (Fox et al., 1987; Miller, 1987; Tsien et al., 1988; Llinas et al., 1989). The calcium channels involved predominantly in regulating acetylcholine release from peripheral autonomic or cortical neurons have been identified as N-type calcium channels, whereas the calcium channel(s) regulating predominantly acetylcholine release from the motor nerve has not yet been characterized (Wessler et al., 1990b). N-type channels are not involved at motor nerves, because o-conotoxin GVIA, highly effective at N-type channels, does not inhibit the stimulated transmitter release from the motor nerve (Anderson and Harvey, 1987; Wessler et al., 1990b). It might be argued that the huge number of spare channels [activation of only 8% of the available calcium channels produces a maximal endplate potential (Silinsky,

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30 1

1985)l explains the inefficiency of o-conotoxin GVIA at motor endplates. Even high concentrations of o-conotoxin GVIA (0.1 pM) and long exposure times did not produce substantial inhibition, whereas considerably low concentrations of o-conotoxin GVIA (nM) inhibited acetylcholine release from peripheral autonomic and cortical neurons (Wessler et al., 1990b). This indicates that different types of calcium channels are involved in the regulation of acetylcholine released from either autonomic neurons or the motor nerve. Evoked transmitter release from the motor nerve can be blocked by divalent cations (beryllium, cadminum, cobalt, lead, nickel), but these cations do not exclusively discriminate between different types of calcium channels. Nevertheless, it should be noted that at least four different calcium channels have already been reported to operate at motor nerve terminals (Penner and Dreyer, 1986; Wessler et al., 1990b),and a link between presynaptic autoreceptors and calcium channels appears most interesting (see Section IV,A,3). Mobilization of acetylcholine is obligatory when heavy traffic occurs in the motor nerve. Mobilization describes all events required to maintain transmitter release during prolonged periods of continuous activity. The events involved in mobilization are synthesis of acetylcholine, its incorporation into vesicles, transport from the reservoir to the releasable compartment(s), and organization of the recycled and refilled vesicles at the active release zones ready for release. All these individual processes are possible targets of presynaptic receptors to modulate transmitter release; however, the use of radiolabeling technique to measure the release of newly synthesized acetylcholine excludes evaluation of receptor-mediated effects on synthesis, because in these experiments synthesis of acetylcholine is inhibited by hemicholinium-3, a blocker of the highaffinity choline uptake system. Nevertheless, all the subsequent events of mobilization can be monitored by measuring the release of radiolabeled acetylcholine. I n particular, the transport of acetylcholine from the reservoir to the releasable compartments occurs also with radiolabeled acetylcholine because of the very small capacity of the ready releasable compartment, whereas the stimulated release of [3HH]acetylcholinecan be observed over fairly long stimulation periods (20-30 min: Wessler and Steinlein, 1987). D. HYDROLYSIS OF ACETYLCHOLINE

It is beyond the scope of the present article to discuss in detail the mechanisms involved in the termination of the actions of acetylcholine by hydrolysis; the reader is referred to previously published excellent

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review articles (Silver, 1974; Hobbiger, 1976; Main, 1976; Massoulie and Toutant, 1988; Toutant and Massoulie, 1988; Bowman, 1990). This section considers only some aspects of enzyme activity (acetylcholinesterase) and a possible interaction with presynaptic receptors. The enzyme acetylcholinesterase exists in different molecular forms; the asymmetric collagen-tailed A-form dominates in motor endplates. The enzyme is present in the nerve terminal, in the synaptic cleft in association with the basement membrane, and in the muscle fibers. About 27 binding sites are available for acetylcholine per individual endplate (Waser and Reller, 1965); that is, the number of binding sites at the enzyme corresponds most closely to the number of nicotine receptor binding sites per individual endplate. Accordingly, acetylcholine binds with roughly similar probability to the receptors and to the enzyme. Each active enzyme binding site has been estimated to hydrolyze about 55 acetylcholine molecules per minute, resulting in a hydrolysis time of 100 psec per single molecule. This high capacity of clearing released acetylcholine within a time period of 1 ms allows a rapid transmission at t.he endplate. It is important to consider that the highest concentration of acetylcholine released into the synaptic cleft is built up presynaptically near the active release zones. After escaping the presynaptic membrane, the concentration of acetylcholine falls rapidly because of receptor binding, hydrolysis, and diffusion. It has been calculated on the basis of functional experiments in the mouse nerve-diaphragm preparation that acetylcholine released from the terminal is hydrolyzed by about 50% during its diffusion across the roughly 50-nm-wide synaptic cleft (Chang et al., 1985). Based on the high concentration of acetylcholine built u p in intimate proximity to the active release zones, binding of acetylcholine to presynaptic autoreceptors can occur with a higher probability than binding to postsynaptic nicotine receptors. T h e high probability of stimulating the autoreceptors at presynaptic site requires effective mechanisms to terminate presynaptic effects of released acetylcholine. Increasing evidence indicates stimulated secretion of the enzyme acetylcholinesterase together with the transmitter from nerve terminals. This has been shown for neurons in the substantia nigra and caudate nucleus of various species (Greenfield et nl., 1980, 1983; see Toutant and Massoulie, 1988). T h e enzyme possibly secreted simultaneously with acetylcholine from the motor nerve terminals can cut short the presynaptic action of acetylcholine. It is very exciting to speculate about a possible receptormediated control of acetylcholinesterase secretion. Further mechanisms to limit facilitatory presynaptic effects of acetylcholine are the activation of-inhibitory muscarinic mechanisms (see Section IV,C) and the desensi-

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tization of presynaptic nicotine autoreceptors (see Section IV,A,4). The effects of inhibitors of acetylcholinesterase on evoked [3H]acetylcholine release are discussed in Sections 111,B; IV,A,l,b; and IV,A,4.

111. Detection Methods

A. ELECTROPHYSIOLOGY Electrophysiological methods have contributed most importantly to our current knowledge about the events involved in chemical neurotransmission at motor endplates. In particular, the discovery of mEPPs and evoked endplate potentials is most important in the understanding of the quanta1 nature of transmitter release. In addition, subtle applications of electrophysiological methods combined with pharmacological methods have revealed more detailed knowledge about the distribution of different ion channels within the motor nerve, a subject of considerable importance with respect to impulse propagation, depolarization of the nerve terminal, initiation, and receptor-mediated modulation of acetylcholine release. It has been shown that the sodium channels are extremely concentrated at the nodes of Ranvier (10,000/pm2: Ritchie, 1984) and in the innervated part of the muscle membrane (Beam et al., 1985), whereas these channels were at almost undetectable levels at the endings of motor nerves (Brigant and Mallart, 1982; Mallart, 1984). Accordingly, after passing the last node of Ranvier, a local depolarizing current initiated by the propagated action potential flows to the nerve terminal to depolarize the active release zones. This local current, rather than the strong sodium-dependent action potential, can be targeted by presynaptic or preterminal receptors, whose stimulation may increase or decrease the amplitude of the depolarizing current at or close to the active release zones, thus modulating the subsequent release of acetylcholine. By the insertion of microelectrodes into the perineurium of preterminal nerve bundles, evidence was obtained for the existence of three different potassium currents in mouse motor nerve terminals (Tabi et al., 1989). It can be speculated whether these potassium channels are associated positively or negatively with presynaptic receptors; blockade (nicotine receptor) or opening (muscarine receptor) of these potassium channels would modify local depolarization and, thus, acetylcholine release. It is important to evaluate this hypothesis in future experiments by investigating the effector systems coupled to the nicotine and muscarine autoreceptors.

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T h e foregoing paragraph emphasizes the significant and essential contribution of electrophysiological methods to the understanding of neuromuscular transmission. Nevertheless, it should be realized that recording of muscular membrane depolarization to monitor the release of acetylcholine is an indirect approach, whereas biochemical assays of endogenous or radiolabeled acetylcholine are direct evaluations of the amounts being released. Obviously, the biochemical methods have limitations, which are discussed in the following section. Use of the postsynaptic nicotine receptor-ion channel complex as an indicator of the amounts of acetylcholine released is an excellent indirect approach to indicate the moment-to-moment release of acetylcholine. But, how will this method detect the release of acetylcholine that does not produce a short-cut end-organ response? For example, the end-organ response of the nonquantally released acetylcholine is hardly detectable. Even more critically, the release of acetylcholine and the initiation of an endplate potential are not coupled in a linear manner; in the absence of drugs, stimulation of roughly 10-20% of the muscular nicotine receptors already produces the maximal endplate potential. Thus, the endplate potential is not directly proportional to the amount of acetylcholine released, whereas the endplate current appears to be. Importantly, it should also be considered that about half of the acetylcholine is hydrolyzed before touching the detection machinery, whereas, in the presence of the choline uptake inhibitor hemicholinium-3, hydrolysis does not play any role in the biochemical assay of radioactive acetylcholine. As the degree of hydrolysis occurring downstream from the active release zones does modify the electrical end-organ response, any change in the hydrolysis of acetylcholine would simulate a change in the release. In contrast, an increased release of acetylcholine together with a possibly increased secretion of acetylcholinesterase causing enhanced hydrolysis would be balanced in electrophysiological studies, whereas in overflow studies the release of radiolabeled acetylcholine enhanced under this distinct condition can be detected. One of the main criticisms raised against overflow studies is the use of compounds like hemicholinium-3 (see next paragraph) to block uptake of choline and, thus, synthesis of acetylcholine (Bowman, 1990); However, the various modifications necessary for recording the electrical end-organ response should also be considered. Contraction of the muscle fibers must be prevented by application of either a high magnesium concentration, tubocurarine, or dantrolene and glycerol. These compounds produce, in addition to the postsynaptic ef€ects, presynaptic effects. Application of a local depolarizing current to initiate acetylcholine release in the presence of tetrodotoxin does not correspond to phys-

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iological propagation of an action potential along the myelinated axon and its electrotonic invasion from the preterminal area to the active release zones. Cutting the muscle fibers may be a more appropriate method for preventing contraction. A further problem arises when mEPPs or endplate potentials are recorded to indicate the release of acetylcholine; it is obligatory to prove that the sensitivity of the postsynaptic nicotine receptor-ion channel complex, the detection machinery, did not change throughout the recording period or with the application of drugs. The demonstration of this final condition is attempted by the ionophoretic application of acetylcholine and the recording of endplate potentials or currents; however, small differences in channel activation mediated by released acetylcholine or by applied acetylcholine must be considered (Gibb and Marshall, 1984). Estimation of the time constant of decay of endplate currents differed between applied and neurally released acetylcholine at the same end plate, indicating that applied acetylcholine mirrored an effect that differs from that of released acetylcholine. Applied acetylcholine and released acetylcholine are thought to interact with distinctly localized postsynaptic nicotine receptors; released acetylcholine may stimulate the more centrally localized nicotine receptors of an endplate, whereas applied acetylcholine may interact with the more peripherally localized receptors. Finally, when recording end plate potentials or currents, electrophysiologists cannot attribute an observed effect beyond any doubt to a presynaptic action. For example, nicotine receptor antagonists, like tubocurarine, produce both postsynaptic effects (receptor blockade, channel blockade) and presynaptic effects (inhibition of acetylcholine release; see Section lV,A,1,a). Every postsynaptic effect observed in response to tubocurarine or other nicotine receptor antagonists can consist of a combination of two effects, a presynaptic and a postsynaptic site of action. In contrast, the estimation of the overflow of radioactive acetycholine can attribute an observed effect undoubtedly to the presynaptic site, because this method mirrors transmitter release directly. B. OVERFLOW STUDIES

Two different methodological approaches are currently used. First, the release of acetylcholine can be estimated, after inactivation of the enzyme acetylcholinesterase, by different methods [bioassay, radioenzymatic assays, gas chromatography in combination with mass spectrometry, high-performance liquid chromatography (HPLC) with electrochemical detection]. A conditio sine qua n o n for all these methods is the

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preservation of released acetylcholine, that is, the blockade of the enzyme acetylcholinesterase in the tissue. Second, release of newly synthesized radioactive acetylcholine can be estimated, without blocking the enzyme acetylcholinesterase, from the increase in radioactive overflow after a preceding labeling of neuronal transmitter stores by the application of radioactive precursors (choline, acetate). This has been shown for various preparations (airways, brain, heart, intestine, urinary bladder, ganglion cells: Richardson and Szerb, 1974; Szerb, 1976; Kilbinger and Wessler, 1980; Muscholl and Muth, 1982; D’Agostino et ul.,, 1986, 1990; Wessler etal., 199Oc, 1991)including the motor nerve (Foldes et al., 1984; Halank et al., 1985; Wessler and Kilbinger, 1986; Vizi et al., 1987; Wessler, 1989). Incubation of endplate preparations obtained from the rat phrenic nerve with [3H]choline causes a considerable synthesis of [3H]acetylcholine, whereas only minute amounts are synthesized in a chronically denervated end plate preparation (Wessler and Kilbinger, 1986). This indicates that synthesis of [3H]acetylcholine occurs within the nerve terminals. Electrical nerve stimulation causes an increase in the overflow of radioactivity, which is abolished in the absence of extracellular calcium ions o r in the presence of tetrodotoxin (see Fig. 2). Importantly, electrical stimulation of a chronically denervated endplate preparation, whose innervating nerve was surgically removed 6 days before the release experiments, did not induce any release of radioactivity above the baseline (see Fig. 2). All the foregoing results show the stimulated outflow of radioactivity from the organ bath to be caused by a calcium-dependent release mechanism from the nerve terminals. Immediately after its liberation, acetylcholine is hydrolyzed to 13H]choline and acetate. To prevent the reuse of choline for acetylcholine synthesis or its incorporation into muscle fibers, whereby [:’H]choline would be lost for the assay, a choline uptake blocker like hemicholinium-3 must be added after the labeling period. This experimental condition allows the stimulated release of [“H]acetylcholine to be monitored directly by the enhanced outflow of radioactivity, which represents [3H]choline originating from hydrolyzed [3HH]acetylcholine.It is important to evaluate this conclusion in experiments in which the cholinesterase is blocked and released [3H]acetylcholine is detected biochemically by thin-layer chromatography (Wessler and Kilbinger, 1986) or by reverse-phase HPLC (Fig. 3). T h e latter experiments demonstrate without any doubt that the stimulated outflow of radioactivity is caused exclusively by the release of [“H]acetylcholine, and that the increase in tritium efflux from the organ bath allows precise measurement of the calcium-dependent release of [“H]acetylcholine from phrenic nerve terminals.

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FIG. 3. Release of [3H]acetylcholinefrom isolated rat phrenic nerve and separation of the radioactive compounds by reverse-phase HPLC. After the labeling period, the enzyme acetylcholinesterase was blocked by 10 p V f neostigmine to assay [3H]acetylcholinereleased in response to electrical nerve stimulation. The radioactive compounds were separated by reverse-phase HPLC, whereby 14C-labeled internal standards (see bottom curve) were used to identify the retention times (Wessler and Werhand, 1990).The upper curve shows the radiochromatogram of the incubation medium obtained at rest; the middle curve shows the radiochromatogram of the incubation medium obtained with electrical nerve stimulation.

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This radiolabeling technique offers advantages when compared with the measurement of endogenous, unlabeled acetylcholine. Determination of endogenous acetylcholine requires blockade of the enzyme acetylcholinesterase; however, application of acetylcholinesterase inhibitors to the tissue produces various presynaptic and postsynaptic effects and rather unphysiological experimental conditions. The built up concentration of extracellular acetylcholine causes desensitization of presynaptic nicotine receptors (Wessler et al., 1986, 1987c,d; see also Section IV,A,4), modifies presynaptic rnuscarine receptors (Kilbinger and Wessler, 1980; Wessler ei al., 1987a), initiates backfiring (Masland and Wigton, 19.10; Bowman and Webb, 1972; Riker, 1975, Hobbiger, 1976), causes the formation of surplus acetylcholine (MacIntosh and Collier, 1976), and inhibits the liberation of nonquantal acetylcholine (I. Wessler, unpublished observations). It is obvious from all these multiple effects that studies investigating presynaptic, receptor-mediated effects should be carried out in the absence of acetylcholinesterase inhibitors. A further advantage of the radiolabeling technique is the high sensitivity of this method in detecting the release of acetylcholine at motor endplates. After improvement of the radiolabeling technique it is possible to detect the release of [3H]acetylcholineevoked by very short stimulation periods (110 sec: Wessler and Kilbinger, 1986; Wessler et al., 1987b). This stimulation condition corresponds to the release of roughly 500 fmol acetylcholine. Considerably longer stimulation periods (several minutes) have to be applied when endogenous acetylcholine is determined, even when the most sensitive detection methods are used. Short stimulation periods, however, resemble more the situation in nzuo than long periods of continuous stimulation. Finally, the radiolabeling method allows a clear distinction to be made between the spontaneous, nonquantally released acetylcholine and the stimulated, quantally released acetylcholine. U n der resting conditions only 15% of the tritium efflux represents the efflux of [3H]acetylcholine (Wessler and Kilbinger, 1986);the remainder is [3HH]choline.Thus, spontaneous tritium emux does not contain substantial amounts of nonquantally liberated acetylcholine, whereas a considerable fraction (15-70% during stimulation and 98% at rest) of nonquantally liberated acetylcholine contributes to the total amount of assayed endogenous acetylcholine. In other words, the electrically stimulated increase in radioactivity represents exclusively the quantally released (calcium-dependent) [3H]acetylcholine,whereas endogenous acetylcholine assayed during stimulation consists of both quantally released acetylcholine and nonquantally liberated acetylcholine. Nevertheless, the radiolabeling technique is clearly limited. Synthesis of acetylcholine has to be blocked by application of a choline uptake

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inhibitor (see above). Consequently, presynaptic mechanisms modifying synthesis cannot be detected by the radiolabeling technique. To avoid this experimental limitation, very short stimulation periods are used in the experiments with [3H]acetylcholine, to prevent exhaustion of the releasable [3H]acetylcholine pool. In fact, the moderate stimulation parameters used in most experiments (5 Hz, 20 sec) cause the release of [3H]acetylcholinein amounts not more than about 5% of its tissue store, thus excluding limitation of the release by blockade of the synthesis. The contention that the use of hemicholinium-3 and, thereby, the blockade of synthesis d o not limit the system is supported by two experimental findings. First, two periods of electrical nerve stimulation separated by a 30-min interval release nearly identical amounts of [3H]acetylcholine (S2/S1 ratio of roughly 1; see Fig. 4), and second, [3HH]acetylcholine release can actually be enhanced by agonists at nicotine or adrenoceptors (see Sections IV,A,l,c; V,B,l; and V,C,l). The possibility of a nonuniform labeling of the different acetylcholine compartments (see Section II,B,C,) cannot be excluded. To minimize a possible heterogeneous labeling of different acetylcholine compartments, labeling should be carried out for sufficiently long periods (40-60 min) and during moderate stimulation, to increase the turnover of acetylcholine. Potter (1970) has shown that 20-Hz stimulation replenishes about 35% of the acetylcholine tissue store within 5 min. Wessler and Kilbinger (1986) have analyzed the relationship between synthesis of [3H]acetylcholine in rat phrenic nerve and the duration of labeling in detail. The total amount of releasable 13H]acetylcholinevaried with the duration of labeling and the intensity of stimulation. A shortening of the labeling period was balanced by an increase in the stimulation frequency (40 min and 1 Hz versus 10 min and 10 Hz). Further prolongation of the labeling period beyond 40 min was not followed by greater formation of [3H]acetylcholine. However, a brief labeling period (2 min) combined with a high stimulation frequency (50 Hz) produced an inhomogeneous labeling of the different acetylcholine compartments; the fraction of [3H]acetylcholine releasable in a calciumindependent manner was higher than under the foregoing labeling conditions (Wessler and Steinlein, 1987).This observation indicates a preferential labeling of nonvesicular acetylcholine, that is, greater formation of [3H]acetylcholine in the cytoplasm, where synthesis occurs, than in the vesicles, when a 2-min labeling was performed with a high stimulation frequency of 50 Hz. An even more pronounced heterogeneity in labeling was obtained, when labeling was performed in the absence of any applied stimulus. Under this condition considerable amounts of 13H]acetylcholine were synthesized within the phrenic nerve, presumably by the

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8

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I

I

I

1

0

15

30

L5

1 60

Time (min)

FIG.4. Inhibition of the electrically stimulated release of [“HJacetylcholine by tubocurarine. The upper curve shows the tritium efflux and the stimulated release of‘ [SH]acetylcholineunder control conditions. T w o periods of electrical nerve stimulation (S1 and S 2 , 100 pulses at 5 Hz each) released almost identical amounts of [3H]acetylcholine. Tuubocurarine ( 1 M).added after the first control stimulation ( S l ) inhibited the stimulated release of j2H]acetylcholine. (Values from Wessler u[ RI., 19x6.)

exchange with spontaneously released, nonvesicular acetylcholine; however, this n e d y synthesized [“H]acetylcholine, localized within the neuronal cytoplasm, could not be released by subsequent electrical nerve stimulation, but was liberated by high potassium in a calcium-independent manner (Wessler and Steinlein, 1987). The latter observation strongly supports the vesicular hypothesis. Suszkiw and OLeary ( 1982) have performed double-label experiments in brain syriaptosomes and have presented evidence that the newly synthesized acetylcholine does not equilibrate with depot vesicular acetylcholine or cytoplasmic acetylcholine prior to release. Naturally, such heterogeneous labeling would limit the applicability of the radi-

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olabeling technique to monitoring release of the endogenous transmitter. I n the studies of Suszkiw and O’Leary, however, no time was left for equilibration of the newly synthesized [3H]acetylcholine, because the release of [3H]acetylcholine was measured immediately after the application of [3H]choline, that is, immediately after the labeling was completed. I n contrast, in the experiments with the motor nerve, the labeling period is followed by a 60-min interval (to wash out the excess radioactivity) and, thereafter, the stimulated release of [3H]acetylcholine is measured. During this 60-min period equilibration of newly synthesized radioactive acetylcholine with the different acetylcholine compartments can occur. Efforts have also been made to investigate whether the release of preformed and the release of newly synthesized radioactive acetylcholine are regulated by the same neuronal mechanisms. Using Torpedo synaptosomes, Luz et al. (1985) found evidence of a different regulation of the release of preformed, endogenous and newly synthesized, radiolabeled acetylcholine. Anticytoskeletal drugs inhibited the release of radiolabeled acetylcholine but not the release of preformed acetylcholine, whereas oxotremorine, by stimulation of presynaptic inhibitory muscarine receptors, reduced the release of preformed acetylcholine only; the release of both preformed and newly synthesized acetylcholine was inhibited by a calmodulin protein antagonist. Two reservations, however, should be considered when interpreting these results. First, in Torpedo, newly synthesized and preformed transmitter mix more slowly than in mammalian tissue (Zimmermann and Denston, 1977). Second, the authors (Luz et al., 1985) have used high potassium, a release stimulus that does not correspond to the physiological excitation of nerve terminals. The numerous studies in which the release of radiolabeled transmitters (acetylcholine, noradrenaline, dopamine, serotonin) has been measured to investigate presynaptic, release-modulating receptors show excellent agreement with studies in which the release of endogenous, unlabeled transmitters has been measured (Starke et al., 1989). In addition, Wessler and Kilbinger (1986) have shown similar fractional release rates for both newly synthesized [3H]acetylcholine and performed acetylcholine in rat phrenic nerve. Balancing all the experimental data it appears reasonable to propose that modulation of the electrically stimulated release of radiolabeled acetylcholine monitors the regulation of the release of preformed, endogenous acetylcholine. A change in the probability of release of acetylcholine can result from several distinct presynaptic mechanisms: (1) an increase or decrease in the number of release zones, (2) an increase or decrease in the release probability within the relevant transmitter compartments, (3) improved or impaired mobilization of transmitter. Mobilization comprises syn-

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thesis of acetylcholine, vesicular incorporation, transport to the relevant compartment, and organization of the recycled vesicles at the active release zones ready for release. The following sections show that the release of radiolabeled acetylcholine from the phrenic nerve is modulated by the stimulation of presynaptic autoreceptors and heteroreceptors.

IV. Modulation of Release by Autoreceptors

A. PRESYNAPTIC NICOTINERECEPTORS

1 . Ozie$low Studies

a. Inhibitory Effects of Receptor Antagonists in the Absence of Acetylcholinesterase Inhibitors. In overflow studies with radiolabeled acetylcholine fairly convincing evidence has been presented since 1985 that the release of [3H]acetylcholine is modulated by presynaptic nicotine receptors that are part of an endogenously activated, positive feedback loop. First, tubocurarine has been shown to reduce substantially the release of [3H]acetylcholine evoked by short-term stimulation (5 Hz, 20 sec) from isolated rat or mouse phrenic nerve (see Fig. 4; Halank et al., 1985; Wessler et al., 1986, 1987b,c; Vizi et al., 1987). In addition, further antagonists at nicotine receptors (hexamethonium, pancuronium, and pipecuronium) have been shown to produce the same action; they reduced the release of radiolabeled acetylcholine from rat or Iiiouse phrenic nerve (Wessler et al., 1986, 1992a; Vizi et al., 1987). It should be realized that the inhibitory effects of the antagonists were related to their concentrations: tubocurarine was ineffective at 0.1 pV, tended to reduce [3H]acetylcholine release at 0.3 pV, and caused inhibition at 1 p M ; hexamethonium was without an inhibitory effect at 0.00 1-0.0 1 nlM and produced a maximal inhibitory effect effect at 0.2-1 t M . The inhibitory effect of hexamethonium was abolished in the presence of 1 pM 1 , l dimethyl-4-phenylpiperazinium, a nicotine receptor agonist (Wessler et al., 1986). It is also important that a-bungarotoxin, a more or less irreversible antagonist at nicotine receptors, concentration-dependenty inhibited the evoked [3H]acetylcholinerelease, whereas two other a-toxins (cobratoxin, erabutoxin-b) did not affect [3H]acetylcholine release (I. Wessler, unpublished observation). All these observations strongly support the concept originally proposed by Koelle (1962) and, in more detail, by Bowman ( 1 980, 1990) that released acetylcholine facilitates its own release by stimulation of nicotine receptors; acetylcholine released

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from the motor nerve is controlled by a local positive nicotinic feedback loop. Blockade of release-modulating nicotine receptors by antagonists interrupts this positive feedback loop and, as a consequence, reduces evoked [3H]acetylcholine release. The release of endogenous, preformed acetylcholine is supposed to be regulated by the same mechanism; however, so far, this assumption cannot be evaluated because determination of endogenous acetylcholine requires blockade of the enzyme acetylcholinesterase, a condition that abolishes nicotinic autofacilitation by the desensitization of presynaptic nicotine autoreceptors (see Section IV,A,4 and below). In this context, the abolition of the inhibitory effect of tubocurarine occurring with acetylcholinesterase inhibitors can be considered as a strong support both for a receptor-mediated effect of tubocurarine and for receptor desensitization, because increasing concentrations of tubocurarine do not restore its inhibitory action (Wessler et al., 1986). A blockade of channels, also shown to be caused by tubocurarine (Colquhoun et al., 1979; Nohmi and Kuba, 1984; Dun et al., 1986), cannot explain the inhibition of transmitter release, because this effect of channel blockade would even be enhanced by inactivation of the enzyme acetylcholinesterase. A blockade of channels as the underlying mechanism of presynaptic modulation is also excluded by the observation that application of a nicotine receptor agonist prevented the inhibitory effect of hexamethonium (Wessler et al., 1986). Evidence for a presynaptic location of these receptors and a clear pharmacological distinction from the postsynaptic nicotine receptors is given below (Sections IV,A,2 and IV,A,3). Release-modulating receptors are currently considered to be localized presynaptically (see Starke et al., 1989). On the basis of this concept and the clear distinction between muscular (postsynaptic) and release-modulating (presynaptic) nicotine receptors (see Section IV,A,3), the inhibitory effect of tubocurarine observed in the overflow studies mirrors the presynaptic action of acetylcholine at motor nerve terminals, to stimulate facilitatory nicotine autoreceptors. In an alternative explanation of the presynaptic, releasemodulatory effects potassium ions liberated locally from the muscle membrane in response to depolarization act transsynaptically to cause depolarization of the nerve terminals (“potassium hypothesis;” Katz, 1962; Hohlfeld et al., 1981). This alternative, however, can be ruled out vigorously for three reasons. First, erabutoxin-b and cobratoxin blocked the postsynaptic nicotine receptors and, thus, the stimulated potassium efflux; but both toxins did not affect the release of [3H]acetylcholine. Second, the presynaptic effects of acetylcholine or applied agonists are abolished in the presence of an inhibitor of the enzyme acetylcholinesterase (Wessler et al., 1986), a condition that just increases the emux

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of potassium from the muscle membrane, because of the repetitive depolarizations occurring at the muscle membrane. Third, presynaptic effects of antagonists at muscarine receptors that reflect a presynaptic action of acetylcholine at presynaptic muscarine autoreceptors (see Section IV,C,l) could be demonstrated in the presence of an elevated concentration of extracellular potassium (27 mM: Wessler et al., 1987a). In contrast to the observation with the muscarinic feedback mechanism (see Section IV,C, I ) , the release of [“H]acetylcholine evoked by 27 mM potassium was not reduced by tubocurarine; that is, nicotinic autofacilitation did not operate in the presence of elevated extracellular potassium ions (Wessler and Kilbinger, 1987). In these experiments high potassium was present for 2 min; that is, continuous stimulation occurred within this time period, producing a substantial, large release of acetylcholine. Likewise, the release of [3H]acetylcholine evoked by 2 min of continuous electrical nerve stimulation was no longer reduced by tubocurarine (Wessler et nl., 1986). Thus, tubocurarine was inefficient during prolonged periods of both electrical stimulation and high-potassium stimulation, and this can best be explained by the desensitization of the nicotine autoreceptors caused by the artificially long stimulation period (see Section I V,A,4). All the foregoing observations convincingly argue against a transsynaptic action of potassium as the underlying mechanism of the autoreceptor-mediated control of acetylcholine release at motor nerves, hut show a presynaptic action of acetylcholine that, after escaping the neuronal membrane, stimulates presynaptic nicotine and muscarine autoreceptors closely localized to the release sites. It has already been shown that application of acetylcholine depolarizes the motor nerve terminal and produces both facilitatory and inhibitor): effects (Hubbard et al., 1965; Riker, 1966; see also Miyamoto, 1978). These effects, however, were attributed to a depolarization of the neuronal membrane occurring more proximal than the release sites (Hubbard rt al., 1965); stimulation of preterminal or axonal nicotine receptors has been proposed to be involved in this depolarization (Blaber and Karczmar, 1967; Webb and Bowman, 1974; Bowman et al., 1986; Bowman, 1990; see also Section IV,B). T h e preterminal nicotine receptors are localized beyond the diffusion radius of released acetylcholine that can stimulate these receptors only after partial blockade of the enzyme acetylcholinesterase. Masland and Wigton ( 1940) and Feng and Li (1941) observed repetitive discharges (backfiring) of the motor nerve after application of cholinesterase inhibitors or acetylcholine. One may speculate whether these preterminal nicotine receptors are involved in the positive feedback mechanism and whether the enhanced transmitter release might have been mediated by backfiring. This possibility, how-

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ever, is highly unlikely for the following reasons. First, the preterminal nicotine receptors are stimulated by released acetylcholine only after partial blockade of the enzyme acetylcholinesterase, whereas the inhibitory effect of tubocurarine is observed with unblocked acetylcholinesterase; the inhibitory effect of tubocurarine disappears after blockade of the enzyme activity. Second, backfiring disappears after the first pulse of a train (Blaber and Bowman, 1963; Besser and Wessler, 1991) and it appears extremely unlikely that backfiring occurring only with the first pulse of a 100-pulse train represents the mechanism underlying the modulation of transmitter release evoked by the subsequent 99 pulses; particularly the nicotinic positive feedback mechanism has been shown to be substantially activated with some latency; significant activation of nicotinic autofacilitation occurs only beyond the tenth pulse of a train (Wessler et al., 1987b; see also below). Third, application of nicotine receptor agonists should, by depolarizing the neuronal membrane, enhance transmitter release, but all applied agonists investigated so far (see Section, IV,A,l,b) did not produce an increase in the spontaneous release of [3H]acetylcholine. In contrast, carbachol has been shown to enhance considerably the frequency of mEPPs, indicating enhanced spontaneous transmitter release (Miyamoto and Volle, 1974). Probably, the contribution of the spontaneous, quantally released [3H]acetylcholine to the spontaneous efflux of total tritium is too small to detect such an effect. In summary, the clear inhibitory effect of tubocurarine (and of further antagonists) in reducing the stimulated release of [3H]acetylcholine from the phrenic nerve strongly supports the concept of a nicotinic autofacilitation; that is, acetylcholine released from motor endplates facilitates its own subsequent release via stimulation of nicotine autoreceptors. The inhibitory effect of tubocurarine is related to the stimulation frequency. This observation was made in experiments in which 100 pulses were applied at various stimulation frequencies (0.5- 100 Hz). Tubocurarine was without effect at 0.5 Hz, whereas modest inhibition (30%)was observed at 1 Hz, and maximal inhibition (50-60%) occurred at 5 , 25, and 50 Hz (see Fig. 5) (Wessler et al., 1987b). Thus, the positive nicotinic feedback mechanism operates at stimulation frequencies corresponding to the firing rates of motoneurons (Grimby and Hannerz, 1977; Grimby et al., 1979), implying a physiological significance of the feedback system. There appears to be a threshold concentration of acetylcholine necessary before nicotinic autofacilitation starts to operate. At 0.5 Hz, acetylcholine release per time unit is too low to trigger the mechanism, whereas the mechanism is maximally activated between 5 and 50 Hz. In experiments with intermittent nerve stimulation it was

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FIG. 5. Presynaptic inhibition by tubocurarine at different stimulation frequencies (0.5-100 Hz). Release of [3H]acetylcholine was evoked by 100 pulses applied at the frequencies indicated. T h e presynaptic effect of tubocurarine is expressed as the S2IS 1 ratio. At 50 Hz tubocurarine was ineffective in some experiments (n = 3), but caused a marked reduction in the remaining experiments. *p < 0.05; **p < 0.01. (Values from Wessler el nl., l987b.)

also found that a threshold stimulation of the nicotine autoreceptors is required before nicotinic autofacilitation starts to operate. When trains of 15 pulses repeated ten times with 3-sec intervals (Fig. 6) were applied, tubocurarine failed to inhibit [3H]acetylcholine release significantly. This indicates that nicotinic autofacilitation is substantially activated only beyond the 10-15 pulses of a train. 111111111111111111111111111111111111111111111 continuous stimulation

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FIG.6. Time protocol for electrical nerve stimulation. T h e use of an intermittent stimulation protocol with different train lengths but an identical number of total pulses indicates whether a presynaptic effect occurs within the first 5 o r 15 pulses or beyond the 15th pulse of a train.

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b. Effects of Receptor Antagonists in the Presence of Acetylcholinesterase Inhibitors. Attempts to demonstrate an inhibitory effect of tubocurarine on the release of endogenous acetylcholine have been fruitless. Dale et al. ( 1936) had investigated a possible presynaptic effect of tubocurarine but obtained no evidence for such an effect. Similar results were reported repeatedly during the next 50 years by several authors (Emmelin and Maclntosh, 1956; Krnjevii: and Mitchell, 1961; Cheymol et al., 1962; Beranek and VyskoEil, 1967; Chang et al., 1967; Fletcher and Forrester, 1975). In harmony, all these authors found no evidence for a presynaptic inhibitory effect of tubocurarine. The only exceptions are the reports by Beani and colleagues (1964a,b) showing an inhibitory effect of tubocurarine and hexamethonium on the release of endogenous acetylcholine from guinea pig phrenic nerve; however, the effect of the antagonists was less convincing, because hexamethonium lost its inhibitory effect by lowering the temperature from 38 to 33°C. Moreover, Chang et al. (1967), using experimental conditions similar to those described by Beani and colleagues, failed to verify the inhibitory effect of tubocurarine. On the first view, all the latter results obtained with endogenous acetylcholine appear to clearly and strongly contradict the inhibitory effect of tubocurarine obtained with [3H]acetylcholine and the underlying positive nicotinic feedback mechanism. Are the results obtained with [3H]acetylcholine artificial effects? To answer this question, experiments with [3H]acetylcholine must be carried out under conditions corresponding to those in the experiments with endogenous acetylcholine; that is, the inhibitory effect of tubocurarine has to be verified in experiments with inactivated acetylcholinesterase. Most importantly, in the presence of neostigmine, an inhibitor of the enzyme acetylcholinesterase, tubocurarine has been reported previously to lose its inhibitory action on the release of [3H]acetylcholine (Wessler et al., 1986). Thus, the results obtained with radiolabeled acetylcholine and endogenous acetylcholine are in excellent agreement. To explain the inefficiency of tubocurarine in the presence of neostigmine, Wessler et al. (1986) proposed that the high concentration of extracellular acetylcholine built up in the presence of neostigmine causes desensitization of the presynaptic nicotine receptors, thus inactivating the positive feedback mechanism (see Section IV,A,4). Naturally, under this condition tubocurarine cannot inhibit transmitter release, because the positive nicotine feedback mechanism no longer operates. Wessler et al. (1986) have also investigated whether the inhibitory effect of tubocurarine varies with the duration of the stimulation period, that is, the number of pulses. Surprisingly, the inhibitory effect of tubocurarine

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EFFECT O F ~ - L ! B O C U R A R I N EOK

TABLE I1 [3H]ACETYLCiiOLINEEVOKED WITH DIFFEKENT 'rRA1N

LENGTHSO

Tubocurarine Stimulation 5 Hz, 100 pulses 5 Hz. 100 pulsec 5 Hz, S(J0 pulses 5 Hz, 300 pulses 50 Hz, 300 pulses 50 Hz, 300 pulsec .50 Hz, 300 pulses 5 H z , 750 pulses 5 Hz, 750 pulse5 5. Hz, 750 pulse4

(CLM)

s 1 (dmpig) 24,300 f 35,000 5 42,000 2 55,700 f 40,000 f 44,000 2 4 1,000 t 68,000 f 96,000 2 105,000 5

2,700 7,200 6,600 6,300 4,500 4,000 5,000 8,800 13,500 12,100

S2IS1 0.93 0.37 0.97 0.77 1.02 0.86 0.80 0.80 0.70 0.61

2 0.12

0.07" f 0.09 f 0.03* f 0.04 2 0.10 2 0.07* r 0.05 f 0.05 2 0.06 ?

N 11 6 10 6 7 2 6

12 5 3

uReleaseof [3HH]acetylcholinewas evoked by t w o stiniiilation periods ( S l , S2) with identical stimulation parameters (indicated). Tubocurarine was added from 15 min before 52 onward. Given are the (JH]acetylcholine release evoked by S1 and the S2/S1 ratio. The inhibitoi.~effect o l tubocurarine ceases with increasing train length. *p > 0.05.(Values from Wessler el al., (1986, 1987b.)

weakened with increasing train lengths (Table 11). Again, Wessler and colleagues (1986, l987b) attributed the loss of the inhibitory effect of tubocurarine during long periods of continuous stimulation to desensitization of the presynaptic nicotine receptors. These receptors are exposed to continuous bombardments by acetylcholine that is released in large quantities during an artificially long stimulation period. Under this condition of developing desensitization, when stimulation is prolonged (300 or 750 pulses at 5 Hz), the positive feedback mechanism ceases to operate. Under in 712710 conditions, however, motor nerve activity is highly intermittent. Tubocurarine maintained its inhibitory effect when 790 pulses were applied intermittently in trains of 40 pulses (Vizi et al., 1987). This observation convincingly shows the continuous mode of stimulation and not the total number of pulses as being responsible for attenuation of the presynaptic effect of tubocurarine. The results presented by Vizi d nl. (1987) argue against the objection raised by Bowman (1990), who attributed the diminishing effect of tubocurarine during longer stimulation periods to a reduction in the release of acetylcholine and, thus, to reduced activity of the nicotinic autofacilitation. Bowman ( 1990) suggests a reduced biophase concentration of acetylcholine during prolonged periods, because the inhibitory effect of hemicholinium-3 (used in the radiolabeling experiments) on synthesis becomes more important with prolonged stimulation periods. T h e experiments published by Vizi

PRESYNAPTIC RECEPTORS AT MOTOR NERVE TERMINALS

319

and colleagues ( 1987) discussed above exclude this possibility, however. Moreover, one should consider the latency, before the blocking effect of hemicholinium-3 on transmitter synthesis becomes evident and does limit release from the motor endplates. Elmqvist and Quastel (1965) investigated in detail the effect of hemicholinium-3 on mEPPs and endplate potentials. At rest, reduction of the mEPP amplitude occurs within 5 min of exposure to hemicholinium-3, indicating a preferential exchange of newly synthesized acetylcholine with those vesicles released spontaneously; however, endplate potentials declined only after about 15 min of continuous 10.5-Hz stimulation, that is, after the application of about 9000 pulses. This observation, again, argues against a substantial reduction of transmitter release when only 300 pulses are applied in the presence of hemicholinium-3. In fact, Glavinovii: (1988) did not observe a substantial reduction in endplate potentials when 250 10-Hz pulses were applied in the presence of 20 fl hemicholinium-3. The rapid fading of nicotinic autofacilitation during prolonged periods of continuous stimulation can be regarded as an endogenous brake, cutting short the autofacilitatory process. As already discussed, the results obtained with endogenous and radiolabeled acetylcholine under the condition of inactivated acetylcholinesterase show, in excellent agreement, the inefficiency of tubocurarine in inhibiting transmitter release. Only in two reports have facilitatory effects of a nicotine receptor antagonist on the release of endogenous acetylcholine been described. Miledi et al. (1978) stimulated the rat phrenic nerve electrically (3-Hz stimulation) or chemically (application of 50 mM potassium) and found that a-bungarotoxin substantially enhanced the release of endogenous acetylcholine. Similar results were obtained in the frog neuromuscular junction (Miledi et al., 1983), where a-bungarotoxin enhanced the release of endogenous acetylcholine evoked by a considerably low stimulation frequency (0.2 Hz). In these experiments abungarotoxin tended to enhance the resting release of acetylcholine, an observation that indicates the antagonist has additional effects when the enzyme acetylcholinesterase is blocked. Bierkamper and colleagues (1986) obtained similar results and described an enhancing effect of abungarotoxin. On first view, these findings indicate a negative nicotinic feedback mechanism, as also proposed by Wilson (1982) on the basis of electrophysiological studies. Blockade of the enzyme acetylcholinesterase, however, has to be taken into consideration when facilitatory effects, observed with nicotine receptor antagonists, are interpreted. Acetylcholinesterase inhibitors produce multiple presynaptic and postsynaptic effects (see Section III,B), particularly desensitization of presynaptic nicotine receptors, repetitive discharges of the nerve, and

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depolarization of nerve terminals. A facilitatory ef‘fect of nicotine receptor antagonists can easily be explained as protecting the facilitatory nic1988a). In addiotine receptors from being desensitized (Wessler rt d., tion, nerve membrane depolarization mediated by preterminal nicotine receptors can reduce the driving force for the local current initiated by the invading nerve action potential at the last node of Ranvier (Bowman, 1990). This would end u p with a reduced transmitter release under “control conditions” (observed in the presence of a acetylcholinesterase inhibitor), but or-bungarotoxin, by preventing the fall in local membrane potential and by protecting against desensitization of the facilitatory autoreceptors, can reinforce transmitter release, giving the impression o f a negative, nicotinic feedback mechanism. Most interestingly, under the condition of desensitized presynaptic nicotine receptors, Bowman and colleagues (Bowman ~t d.,1986; Bowman, 1990) and Wessler P t al. (1988a) ha\.e described an enhancing effect of tubocurarine on the release of [:3H]acetylcholine.These results are discussed in more detail in Section IV,A,4. In conclusion, the facilitaiory effect of nicotine receptor antagonists observed with blocked acetylcholinesterase does not in itself argue against a positive nicotinic feedback mechanism controlling acetylcholine release from motor nerves. c. Eject.) of Agoiilsts. Nicotine receptor antagonists inhibit evoked transmitter release and, thus, indicate that acetylcholine is the endogenous agonist at presynaptic nicotinic facilitatory autoreceptors. Accordingly, application o f agonists should also enhance evoked [:4H]acetylcholine release. I n tact, several applied agonists at nicotine receptors have been shown to enhance evoked [“H]acetylcholinerelease under distinct experimental conditions. T h e most important condition is the length of exposure of the applied agonists. After short exposure (20 sec) nicotine, 1,1-dimethyl-4-phenylpiperazinium (DMPP), cytosine, and 2-(4-aminopheny1)-ethyltrirnethylammonium (PAPETA) caused a concentrationdependent increase in the release of [:3H]acetylcholine (Wessler et al., 1986. 1987c,d, l992b). T h e effect of 1 p,M nicotine is given in Fig. 7. The enhancing effects of all agonists were abolished in the presence of 0.3 p,M tubocurarine, indicating a receptor-mediated effect (Wessler et al., 1986, 1987c,d, 1988a). In addition, application of acetylcholine and decamethonium, both in fairly low concentrations, which favor an effect at rieuronal nicotine receptors, has been reported to enhance the release of‘ [3HH]acetylcholine from rat phrenic nerve (Bowman el al., 1986; Bowman, 1990). T h e results obtained with the agonists provide additional evidence for the existence of presynaptic, facilitatory nicotine receptors at nlotoi- nerves. As outlined earlier, the ef‘f’ect of the applied agonists strongly de-

PRESYNAF'TIC RECEPTORS AT MCTI'OR NERVE 'TERMINALS

32 1

I"' 'E

:i

n.10

m x 3L

2 J

nicotine 1 umol/I

r 0

I

15

I

30 time (mini

1

1

L5

60

FIG. 7. Nicotine-induced increase in the stimulated release of [3H]acetylcholine.Nicapplied 20 sec before the second stimulation period, enhanced the release of otine ( 1 [~H]acetylcholinefrom rat phrenic nerve stimulated with 100 pulses at 5 Hz.

m),

pended on the duration of exposure. Protongation of the exposure abolished the enhancing effect of the agonists. On first view, this observation is surprising and might be difficult to explain; however, the nicotinic type of the receptors involved must be considered. All the applied agonists are stable agonists, causing permanent stimulation of the nicotine receptors when these compounds are applied to the tissue. Keeping this in mind, the strong time dependency of the facilitatory effect is not surprising, because neuronal nicotine receptors have been shown to desensitize during continuous exposure to agonists (see Section IV,A,4). In contrast, a facilitatory effect that was maintained over prolonged exposure would have been even more surprising. In this context it is difficult to explain the long-lasting facilitatory effect of 50 I.Mphysostigmine on the release of [3H]acetylcholinefrom mouse phrenic nerve (Vizi and Somogyi, 1989). Physostigmine was added 12 min before the electrical stimulation, a time interval allowing already a substantial rise in the biophase concentration of acetylcholine. Consequently, desensitization of the facilitatory nicotinic autoreceptors preventing any facilitatory

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presynaptic effect is expected to occur under this condition (see Section IV,A, 1,b). In particular, desensitization of the receptors is caused not only by accumulated acetylcholine, but also by physostigmine itself, because direct agonistic activity on nicotine receptors in amphibian muscles has been demonstrated for this enzyme inhibitor (Shaw et al., 1985). Moreover, acetylchohesterase inhibitors show channel blocking and desensitizing activities at the postsynaptic nicotine receptors (Shaw et al., 1985; Tattersall, 1990; Wachtel, 1990). Taken together, these various properties of acetylcholinesterase inhibitors complicate the interpretation o f presynaptic effects observed in the presence of these compounds. Bowman and colleagues have reported neostigmine to inhibit the release of newly synthesized [3H]acetylcholine (Bowman et al., 1986; Bowman, 1990), which can be explained by the desensitization of the facilitatory autoreceptors and by the depolarization of the terminal nerve membrane. T h e different results obtained with physostigime (facilitatory: Vizi and Somogyi, 1989) and with neostigmine (inhibitory: Bowman et al., 1986; Bowman, 1990) require further analysis. The considerable potency of physostigmine in blocking cyclic AMP phosphodiesterase in the cat sciatic nerve (Curley et al., 1984), thus enhancing the formation of cyclic AMP, should be noted. Cyclic AMP produces presynaptic facilitatory effects (see Section V,B,3), and this action may contribute to the enhanced release of [SH]acetylcholine reported by Vizi and Somogyi ( 1989). DMPP and nicotine showed a clear difference in the fading of their facilitatory effects when exposed for different periods. Nicotine (1 or 10 fl)lost its facilitatory effect within a short 3-min exposure (Wessler et a/., 1987d), whereas 1 p'kf DMPP was effective even after 15 min of exposure (Wessler el al., 1986). This obvious difference can be related to the different physiochemical properties of' the compounds. Nicotine is a lipophilic substance equilibrating rapidly with the receptors; it can thereby cause a pronounced and rapid desensitization. In contrast, DMPP is considerably less lipophilic than nicotine and requires more time to equilibrate with the receptors. Thus, at equimolar concentrations the desensitization potency is smaller with DMPP than with nicotine. A conDMPP, however, was also ineffective after a 15-min centration of 10 exposure, showing that DMPP at a 10-fold higher concentration causes desensitization comparable to that promoted by lower nicotine concentrations.

2. Functional Studies Bowman and colleagues (1984, 1986; Bowman, 1990) have summarized the experimental evidence indicating an inhibitory presynaptic

PRESYNAF'TIC RECEPTORS AT MOTOR NERVE TERMINALS

323

effect of tubocurarine and of drugs that act in a similar manner. This evidence has been amassed in studies in which either the electrical (endplate potential or endplate current) or the mechanical (contraction) endorgan response to released acetylcholine has been measured. In principle, all the experiments carried out to indicate an inhibitory presynaptic effect of tubocurarine show a fading or a rapid waning of the end-organ response during repetitive stimulation. For example, tubocurarine not only reduces the peak tension, but causes fading of the tetanic contraction (tetanic fade) or fading of the contraction evoked by four 2-Hz pulses (train-of-four fade). The electrophysiological counterpart of the mechanical fading is the rundown of endplate potentials or endplate currents (Liley, 1956; Brooks and Thies, 1962; Elmqvist and Quastel, 1965; Hubbard et al., 1969; Hubbard and Wilson, 1973; GlavinoviC, 1979; Magleby et al., 1981; Gibb and Marshall, 1984; see also Bowman, 1990). Three and four decades ago these fading phenomena had already been ascribed to a falloff in transmitter release, that is, thought to occur at motor endplates even in the absence of drugs, but not detectable because of the high safety factor for transmission. Tubocurarine, however, by reducing the safety factor, was thought to unmask this falloff in transmitter release (Hutter, 1952; Otsuka et al., 1962). In addition, Galindo (197 1) reported tubocurarine to reduce the frequency of mEPPs. On first view, the rundown of electrical end-organ responses and the fading phenomena of the mechanical responses appear to be excellent confirmation of the positive nicotinic feedback mechanism demonstrated in release studies with radioactive acetylcholine. In particular, the potencies of the antagonists in inducing rundown or fading and in inhibiting transmitter release are in agreement. For instance, hexamethonium produces considerable fading in doses too small to depress twitch amplitude (Bowman and Webb, 1976; Gibb and Marshall, 1986), tubocurarine produces both effects with similar potency (Bowman and Webb, 1976; Gibb and Marshall, 1986), whereas pancuronium is less able to produce fading and erabutoxin-b appears unable to produce fading (Gibb and Marshall, 1986). For all these antagonists the potencies in reducing release of [3H]acetylcholine have been investigated, and the results show an excellent correlation; the order of a preferential presynaptic inhibitory action is hexamethonium > tubocurarine > pancuronium > erabutoxin-b. This correlation may be indicative of a common origin of both fading and inhibition of transmitter release; however, there is increasing evidence arguing against this interpretation and the following objections should be taken into consideration when the underlying mechanisms of fading are discussed:

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1. Rundown and train-of-four fade are observed by the second, third, and fourth pulses of a train, whereas nicotinic autofacilitation appears to require at least 10-15 pulses before being substantially activated (Wessler et al., 1987b; see also Section IV,A,la). 2. GlavinoviC (1979) investigated in detail the influence of a first conditioning shock on the subsequent test shock, and he reported an inhibitory effect of tubocurarine that was maximal when the test and conditioning pulses were separated by a 1O-msec interval; thereafter, the inhibitory effect decayed slowly with time. It appears extremely unlikely that a presynaptic, receptor-mediated effect modulating transmitter release is already developed with a latency of 10 msec only and, thereafter, shows fading. On the basis of a receptor-mediated autocontrol of transmitter release and its required mechanism of a pulse-to-pulse modulation, the mechanism cannot already be activated maximally with the first pulse. The presynaptic receptor-mediated mechanism should play an increasing role with successive pulses and should modulate transmitter release during a period of repetitive neuronal activity. Bowman (1980. 1990) attributed the facilitated mobilization of acetylcholine as the mechanism underlying the positive nicotinic feedback mechanism, a mechanism that may play a role during successive pulses of repetitive neuronal activity but not within the first two pulses of a train. Specifically, this mechanism cannot be activated when the first stimulus releases fewer quanta (300) than are available at all active release zones (500-1000), even if only a single quantum is released from a single active release zone. 3. Tetanic fade occurs at 100 Hz within the first 100 pulses, but the release of [3H]acetylcholineis reduced by tubocurarine at this frequency only beyond the first 100 pulses of a train (Wessler et al., 1987b). 4. The rundown induced by tubocurarine has been shown to be accompanied by an enhanced quantum content of the first endplate potential of a train, obviously an effect that steepens the rundown of the subsequent stimuli (Wilson, 1982; Ferry and Kelly, 1988). Specifically, the enhanced quantum content of the first stimulus cannot be considered as part of an autocontrol process of transmitter release, whose required mechanism excludes an effect on the release evoked by the first pulse, but rather should modulate transmitter release evoked by the subsequent pulses of a train. 5. Recent evidence has shown that a-toxins (a-bungarotoxin, erabutoxin-b) cause fading when applied at very low concentrations and exposed for substantially long periods (Bradley et al., 1987, 1990), whereas erabutoxin-b does not reduce the release of [3H]acetylcholine (I. Wessler, unpublished observations).

PRESYNAF’TIC RECEPTORS AT MO’IOR NERVE TERMINALS

325

6. A decrease in temperature (room temperature) causes rundown in the absence of nicotine receptor antagonists (Bowman, 1990); however, exocytosis is currently regarded as a physical phenomenon, largely temperature insensitive. A Ql0 value of 1.60 (37-27°C) has been found for the calcium-dependent release of [3H]acetylcholine (Wessler and Steinlein, 1987), confirming that the stimulated transmitter release is largely temperature insensitive. 7. Finally, it should be mentioned that Auerbach and Betz (197 1) did not find significant presynaptic effects of tubocurarine. Alternatives have been given to explain the various fading phenomena. Fading is a use-dependent phenomenon, and most of the antagonists, in addition to their receptor blockade, produce occlusion of the postsynaptic nicotine receptor-associated ion channel (Colquhoun et al., 1979; Colquhoun, 1986). The blocking drug interacts with the open form of the channel and, hence, blocks the ion flow through the channel. Dreyer ( 1982) has attributed the tubocurarine-induced fading to a use-dependent channel block; however, the rundown does not change with different membrane potentials (Magleby et al., 1981; Gibb and Marshall, 1984), whereas channel block strongly depends on the membrane potential (Colquhoun et al., 1979). Moreover, the rundown is not observed with endplate currents evoked with jets of acetylcholine applied iontophoretically (Gibb and Marshall, 1984; see Fig. 8). The inefficiency of tubocurarine on trains of endplate currents evoked by iontophoretically applied acetylcholine, however, does not automatically require a presynaptic effect of tubocurarine as the underlying mechanism causing rundown. The increasing peak current amplitude occurring with the first few jets of applied acetylcholine (Gibb and Marshall, 1984), a mechanism that may attenuate a possible postsynaptic depressing effect of tubocurarine, may also be considered. In this context Bradley et al. (1990) suggested a comparison of nerve-evoked and iontophoretically evoked endplate currents in experiments using various concentrations of applied acetylcholine, particularly those concentrations producing current peaks similar to those occurring with nerveevoked acetylcholine (see Fig. 8). Desensitization of the postsynaptic nicotine receptors has been discussed as a second alternative explaining rundown (Bradley et al., 1987, 1990). At least two different agonist binding sites have been shown for the muscular nicotine receptor. The low-affinity site opens the ion channel; binding to the high-affinity site appears to be associated with receptor desensitization (Dunn and Raftery, 1982a,b). Importantly, binding of the antagonist to the high-affinity site does not prevent channel

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activation by agonist binding to the low-affinity site (Dunn el al., 1983). Although desensitization is voltage dependent and rundown lacks this dependency, there is some hint that rundown may be accompanied by desensitiLation o r use-dependent channel inactivation. Bradley and colleagues (1987, 1990), showing fading to occur with very low concentrations of some a-toxins ( 5 nM erabutoxin-b or the toxin from Nuja nap c r t m ; see Fig. 9), suggest that the a-toxins, at low concentrations, bind to the high-affinity site and thereby cause rapid fading of the channel opening when released acetylcholine binds to the low-affinity site. This is an attractive model t o explain the nature of fading, but it should also be considered that real experimental evidence is, so far, not available. The following explanation may also be taken into consideration. Whether desensitization of nicotine receptors located presynaptically or postsynaptically occurs spontaneously remains an open question. Possibly, in the absence of any drug, the sensitivity of the nicotine receptors and the coupling between agonist binding and ion channel opening may vary to some extent, and some receptors may even be present in the desensitiLed form. Preexposure of the nicotine receptors to applied agonists can accelerate desensitization (Feltz and Trautmann, 1982; Fiekers ~t nl., 1987); the reverse, enhanced sensitivity, is suggested to be caused by receptor antagonists. In fact, tubocurarine can protect nicotine receptors against desensitization (see Fig. 10). In the presence of 0.3 phi tubocurarine, a concentration that already reduces the safety factor for transmission, the potency of applied nicotine in impairing transmission b) receptor desensitization o r local membrane depolarization (Paton and Savini, 1968) is attenuated, indicating a protective effect of tubocurarine against both actions. A similar mechanism may play a role in the apparent enhancement by tubocurarine of the quanta1 content of the first pulse of a train (Blaber, 1970, 1973; Wilson, 1982; Ferry and Kelly, 1988). ’L‘hisobservation together with the subsequent fading can be explained in terms of a change in postsynaptic receptor sensitivity: In the presence of tubocurarine the postsynaptic nicotine receptors may become particularly sensitive to the agonist (acetylcholine); thus, an intensified end-organ response (end plate potential) occurs with the first stimulus of a train, giving the impression of enhanced acetylcholine release. However, acetylcholine released in response to the first pulse reduces the sensitivity of some relevant postsynaptic nicotine receptors for the subsequent pulses and, hence, causes or steepens rundown (“use-dependent desensitization”). Particularly, acetylcholine has been shown to desensitize muscular nicotine receptors within the msec time scale (Franke and Hatt, 1990). Whether any binding of the applied antagonist to the high-affinity binding site (Bradley et al., 1990) may be involved in the development of rather sensitized nicotine receptors remains to be eluci-

PRESYNAPTIC RECEPTORS AT MOTOR NERVE TERMINALS

Control

Tubocurarine (2.5 x lo-’

327

M)

a

50 ms

50 ms

50 ms

FIG.8. Rundown of neurally evoked endplate potentials in the presence of tubocurarine. Neurally evoked (a, b) and iontophoretically evoked (c, d) endplate currents in the rat phrenic nerve-hemidiaphragm preparation in the absence (a, c) and presence (b, d) of 0.25 +Ivf tubocurarine. (From Gibb and Marshall, 1984, with permission.)

dated. In this context it is of particular interest to point to a corresponding fading phenomenon occurring after partial blockade of the enzyme acetylcholinesterase (Van Der Meer and Meeter, 1956; Blaber and Bowman, 1963; see also Bowman, 1990); this fading phenomenon can also be explained in terms of desensitization of the postsynaptic nicotine receptors because the biophase concentration of acetylcholine is increased with blocked acetylcholinesterase. It is understood that only low concentrations, particularly of potent receptor antagonists (a-toxins), can cause this kind of rundown or fading, because high concentrations of the antagonists leave fewer receptors available for a dynamic and rapid change in both receptor occupation (by acetylcholine and the applied antagonist) and receptor desensitization (coupling between receptor stimulation and channel opening). When acetylcholine is applied iontophoretically, the agonist concentration in the biophase is considerably lower than during nerve stimulation (Fig. 9) and the route of diffusion and binding to the receptors differs with nerve-released acetylcholine. Both conditions may exclude tubocurarine from producing rundown through “use-dependent desensitization.” Taken together, the mechanisms of rundown and the fading phenomena are understood only poorly, but they do not mainly represent the removal of nicotinic autofacilitation. Very recently, Chang and colleagues (1991) investigated the rundown of neuromuscular transmission during repetitive nerve activity by

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D

C

)+--FIG.9. Rundown of skeletal muscle compound action potential by low concentrations of erabutoxin-b. Compound muscle action potentials were recorded in the rat phrenic

nerve-hemidiaphragm preparation. (A) Control experiments. (B) After 20 min of incubation with 150 nM erabutoxin-b. (C. D) After washing out erabutoxin-b for 4.5 hr (C) or 8 hr (D). (From Bradley rt nl., 1990, with permission.)

recording endplate potentials and mEPPs in the mouse phrenic nervehemidiaphragm. The authors confirmed previously reported data that low concentrations of a-toxins (cobratoxin), like tubocurarine, can produce rundown of trains of endplate potentials; however, in contrast to the steepend rundown of endplate potentials the amplitude of mEPPs did not show such a gradual decrement. This observation led the authors to suggest that tubocurarine and a-toxins (low concentrations) do not limit the responsiveness of the nicotine receptor-ion channel complex at the muscle membrane in a use-dependent manner. Consequently, the authors excluded failure of the postsynaptic nicotine receptors to mediate the rundown but attributed the rundown to the blockade of presynaptic nicotine receptors (Hong and Chang, 1991). On first view, these results disagree with the assumption of a postsynaptic origin of the rundown as proposed in the preceding paragraph; however, two experimental conditions should be considered in the interpretation of the results of Hong and Chang. First, the frequency of mEPPs (about 1.4 Hz) is probably too low to allow development of rundown. Second, the amplitudes of the mEPPs were already more than halved by the antagonists (Hong and Chang, 1991); this condition may handicap the detection o f a possible rundown of mEPPs, particularly, when only a portion of the response is sensitive to use-dependent failure.

3. Receptor Characterization and Signal Transduction At first, evidence is summarized that convincingly supports the existence of two populations of nicotine receptors at motor endplates, the presynaptic nicotine autoreceptor and the postsynaptic muscular nic-

PRESYNAPTIC RECEPTORS AT MOTOR NERVE TERMINALS

329

otine receptor. Differences between these receptors exist with respect to either the pharmacological properties of the binding site(s), the effector system, or receptor-effector coupling. 1. Overflow studies with [3H]acetylcholine have demonstrated clear differences in pharmacological properties between the release-modulating autoreceptors and the postsynaptic muscular nicotine receptors. Hexamethonium showed a 250-fold higher potency at the presynaptic receptors (Wessler et al., 1986), whereas both a-toxins, erabutoxin-b and cobratoxin, do not block presynaptic nicotine receptors (I. Wessler, unpublished observations) but block the postsynaptic nicotine receptors. 2. The effect of the presynaptic autoreceptors vanishes or even disappears with increasing pulse number (Wessler et al., 1986, 198713; see Table 2), whereas contractions of the muscle fibers are maintained during a train of 700 pulses at 5 Hz. 3. Blockade of the enzyme acetylcholinesterase abolishes the effect of the presynaptic autoreceptors (Wessler et al., 1986), but skeletal muscle contraction can still be observed. 4. Preexposure of the tissue (40 min) to a considerably low concentration of 0.3 pA4 nicotine abolishes the modulatory effect of the presynaptic autoreceptors (Wessler et al., 1987c), leaving the contraction of the skeletal muscles, however, unaffected (Wessler and Garmsen, 1989; Wessler et al., 1992b). 5 . Low concentrations (within the micromolar range) of applied nicotine receptor agonists cause an effective response at the presynaptic site enhancing [3HH]acetylcholine release (Wessler et al., 1987c, 1992b), whereas the agonists given at these concentrations do not modify skeletal muscle contraction. 6. Binding studies have presented evidence for transport of axonal nicotine receptors by axonal flow in the orthodrornic direction (Millington et al., 1985; Palacios and Pazos, 1986). It may therefore possible that these axonally localized nicotine receptors are transported to the nerve terminal, where they are embedded in the neuronal membrane in intimate approximation to the active release zones. 7. Binding studies have demonstrated labeling of the motor nerve terminals by ct-bungarotoxin coupled to horseradish peroxidase (Bender et al., 1976; Lentz et al., 1977); however, Jones and Salpeter (1983) did not obtain evidence for presynaptic labeling by a-bungarotoxin. Nevertheless, Lentz and colleagues (1977) have published convincing evidence for a specific toxin staining of the presynaptic membrane, as Schwann cells are never stained and nerve terminal staining is visible also after enzymatic separation of the nerve terminals from the muscular membrane (see also Miyamoto, 1978).

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Points 2-7 indicate that the presynaptic autoreceptor is more sensitive to receptor desensitization than the postsynaptic muscular nicotine receptor. Obviously, the motor endplate can be used as an example to show different populations of nicotine receptors. So far, more than 10 different genes coding for nicotine receptors have been isolated and different types of individual receptor subunits appear to exist, allowing a high number of distinctly composed receptors, particularly of the neuronal type, because the neuronal nicotine receptor can be composed of only two different subunits (for references see Deneris et al., 1991).T h e pharmacological characterization of the different nicotine receptors is less well developed. The only distinction currently accepted discriminates the neuronal from the muscular type of nicotine receptors; the former is regarded as the C6 type (hexamethonium sensitive) and the latter as the C: 10 type (decamethonium sensitive: Clarke, 1987; Colquhoun et al., 1987; see also Watson and Abbott, 1990). Increasing evidence, however, has been amassed showing different types of neuronal nicotine receptors (Deneris et al., 1991). Neuronal nicotine receptors appear to be composed of two different subunits only (a2-+p), whereas the muscular nirotine receptor is composed of four different subunits (a, p, y, 6, and E: Boulter et al., 1987; Changeux and Revah, 1987; Wada P t al., 1988; Maelicke, 1988). It is obvious from this structural difference that, on a molecular basis, a large number of distinctly composed neuronal nicotine receptors may exist. Binding studies using labeled nicotine, acetylcholine, and a-bungarotoxin as ligands in the central nervous system have already shown the existence of different neuronal nicotine receptors (Marks et aE., 1986; Martino-Barrows and Kellar, 1987; Reavil et al., 1988). T h e presence of different types of neuronal nicotine receptors is also indicated by the different blocking potencies of a-bungarotoxin obtained at different synapses. It is generally accepted that nicotine receptors localized at cell bodies and mediating ganglionic transmission are not blocked by the a-toxin, whereas experimental evidence shows those nicotine receptors localized at terminals of noradrenaline, dopamine, o r serotonin neurons (presumed presynaptically localized receptors) to be sensitive to blockade by a-bungarotoxin (De Belleroche and Bradford, 1978; Oswald and Freeman, 1981; Valentimo and Dingledine, 1981; Schwartz et al., 1984). Additionally, a-bungarotoxin does block distinct neuronal nicotine receptors in the cerebellum and the retinotectal system of the toad or goldfish and at sympathetic neurons of the frog (Freeman et al., 1980; Marshall, 1981; Barnard and Dolly, 1982; Chiappinelli, 1985; De La Graza et al., 1987; Loring and Zigmond, 1988). Finally, the extremely high sensitivity of Renshaw cells to the agonistic activity of acetylcholine (Curtis and Eccles, 1958) may also be indicative of the heterogeneity of nicotine receptors.

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Attempts have been made to characterize the presynaptic autoreceptors present at the motor nerve and mediating autofacilitation. Unfortunately, a real estimation of affinity constants (PA, values) for antagonists cannot be done for this receptor, because of the rapid desensitization of neuronal receptors; however, the potencies of various antagonists in blocking contraction (postsynaptic nicotine receptor), in reducing acetylcholine release from the motor nerve (presynaptic nicotine receptor), and in blocking ganglionic transmission (nicotine receptor at cell bodies) can be compared. Hexamethonium reduced transmitter release with roughly a 250-fold higher potency than required to inhibit contraction of the hemidiaphragm stimulated indirectly at 0.2 Hz (Wessler et al., 1986). At this low stimulation frequency nicotinic autofacilitation is not operating, allowing a real estimation of a postsynaptic blockade only, but the ability of hexamethonium to block ion channels cannot be ruled out. Nevertheless, the high potency of hexamethonium at the presynaptic site favors similarities between the ganglionic nicotine receptor and presynaptic autoreceptor of the motor nerve. The pharmacological properties of the autoreceptor and the ganglionic nicotine receptor differ in two important aspects, however. First, a-bungarotoxin blocks the autoreceptor but does not block the ganglionic receptor. Second, different effector systems are coupled to both receptors. Stimulation of the ganglionic nicotine receptors triggers rapid depolarization by the influx of sodium and, consequently, enhances transmitter release. Stimulation of the nicotine autoreceptor does not promote transmitter release by itself, but facilitates transmitter release only in combination with electrical nerve stimulation, that is, with propagated neuronal activity. Thus, the autoreceptors are not coupled to an ion channel transporting sodium, a conclusion that is in harmony with electrophysiological studies showing the absence of sodium channels at the motor nerve terminals (Brigant and Mallart, 1982; Mallart, 1984). The present experimental results indicate that the nicotine autoreceptor is a second type of neuronal nicotine receptor, localized presynaptically. This receptor may resemble the presynaptically localized nicotine heteroreceptors in the central nervous system that are sensitive to a-bungarotoxin (see above). There is some evidence that acetylcholine released from guinea pig cortex is also modulated by facilitatory nicotine autoreceptors (Beani et al., 1985; Loiacono and Mitchelson, 1990). These autoreceptors in the cortex, however, appear to differ in their pharmacological properties from the autoreceptors of the motor nerve, because hexamethonium is effective at the latter receptors but ineffective at the former receptors (Beani et al., 1985). As already outlined, great variation in neuronal nicotine receptors seems possible and some of the data discussed above indicate these differences, particularly between nicotine receptors localized at the cell bodies and

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those at nerve terminals. Important future goals are characterization of neuronal nicotine receptors and establishment of a rational classification for neuronal nicotine receptors, when differences are obvious. At present, the nature of the effector system coupled to the nicotine autoreceptor at the motor nerve terminals can only be speculated. A direct (via regulatory G-proteins) coupling to ion channels (activation of calcium channels, blockade of potassium channels) appears more likely than a coupling to regulatory proteins because of the very rapid demand necessary for this regulatory process. Also, the rapid development (within a second) of complete desensitization of the facilitatory effect indicates that an ion channel is involved. In contrast to the autoreceptors, the facilitatory p, receptor of the phrenic nerve shows desensitization with 1990d), and p adreminutes (Wessler and Anschiitz, 1988; Wessler et d., noceptors are known to be coupled to adenylate cyclase. Different potassium channels have been demonstrated at vertebrate motor nerve terminals, and two potassium channels have been shown to be blocked by applied acetylcholine (Hevron et al., 1986), indicating a possible effector system for the nicotine autoreceptor. The blocking effect of acetylcholine was not, however, prevented by a nicotine receptor antagonist (Hevron et al., 1986). Recently, a nicotine receptor-operated calcium transient has been reported (Kimura et al., 1989). In the presence of neostigmine, calcium influx through nicotine receptor-operated channels was found to occur at the muscle fibers of indirectly stimulated mouse diaphragm. The influx of calcium was blocked by a 0.1 FLM concentration of either tubocurarine or pancuronium (Kimura et al., 1989). Thus, this channel appears to be a possible candidate for the effector system of the nicotine autoreceptor. Likewise, Hong and Chang (1990) proposed a calcium channel (L type) to be linked to the preterminal nicotine receptor, because “regenerative acetylcholine” released from the mouse phrenic nerve was suppressed by low concentrations of tubocurarine, verapamil, and nifedipine.

4. Desensitization Desensitization, a progressive loss of the responsiveness or a refractoriness of the effector system to respond during the sustained presence of the receptor agonist, is a common feature of nicotine receptors investigated in the central and peripheral nervous system. Katz and Theslefl’ (1957) were among the first to describe desensitization of muscular endplate nicotine receptors in response to the application of acetylcholine. Desensitization is a very complex phenomenon, not fully understood, and it is beyond the scope of this article to discuss in detail the aspects of receptor desensitization. T h e main aim of this section is to show that the

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presynaptic nicotine autoreceptor of the motor nerve develops a rapid and complete desensitization (or refractoriness) and that the sensitivity of the presynaptic receptors for complete desensitization is higher than that of the postsynaptic nicotine receptors. Agonists and antagonists at the presynaptic nicotine autoreceptors cease to modulate [3]acetylcholine release under the following experimental conditions: 1. Blockade of the enzyme acetylcholinesterase causes a loss of the modulatory effects of nicotine receptor agonists and antagonists (Wessler et al., 1986; see Fig. 10). In contrast, skeletal muscle contraction can still be observed after blockade of the enzyme acetylcholinesterase, but the amplitude is reduced and the contraction shows fading. 2. Prolongation of the exposure from seconds (20 sec) to minutes (3 min) causes loss of the facilitatory effects of applied stable agonists (micromolar concentration range; Fig. l l). In contrast, agonists applied at the indicated concentrations do not affect skeletal muscle contraction (see Fig. 10; Wessler and Garmsen, 1989; Wray, 1981). The significant inhibition of the release of [3H]acetylcholine observed after 3 min of exposure to 1 or 10 fl nicotine (Wessler et al., 1987d) can be explained by the removal of nicotinic autofacilitation and is discussed in detail subsequently. 3. A successive prolongation of the period of continuous nerve stimulation from 100 to 300 to 750 pulses (5 Hz) causes attenuation of the facilitatory effect of applied agonists and even loss of the inhibitory effect of tubocurarine (see Table 11; Wessler et al., 1986). In contrast, skeletal muscle contraction does not decline when the hemidiaphragm is stimulated indirectly within a few minutes ( 5 Hz). Continuous nerve stimulation causes continuous bombardment of the nicotine autoreceptor and, consequently, desensitization of these receptors. Applying 720 pulses intermittently prevents tubocurarine from becoming ineffective at the presynaptic site (Vizi et al., 1987). 4 . By use of a classic protocol to desensitize neuronal nicotine receptors (Loffelholz, 1970),a continuous 40-min exposure to a considerably low concentration of nicotine (0.3 pM) was shown to prevent the facilitatory effect of 1 or 10 fl nicotine, both being maximally effective concentrations at the presynaptic site. In contrast, a concentration 0.3 pM nicotine does not modify skeletal muscle contraction (see Fig. 10). So far, no methods are available to determine receptor desensitization directly at the presynaptic site; the only possible approach is estimation of the extent of autoreceptor modulation under conditions commonly known to cause receptor desensitization (see items 1-4).

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(before S2) FIG. 11 . Desensitization of nicotine autoreceptors by blockade of acetylcholinesterase (A) or by a prolonged exposure to nicotine (B). Release of [3H]acetylcholinewas evoked by two stimulation periods (S1 and S2, 100 pulses at 5 Hz). Neostigmine (closed columns) was present from 30 min before S1 onward, tubocurarine from 15 rnin before S2 onward and nicotine from,20 sec or 3 rnin before S2 onward. The effects are expressed as the S2/S1 ratio. (Values from Wessler et al., 1986, 1987d.)

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Accordingly, there is no definite method to determine whether the nicotine autoreceptor has desensitized under the conditions indicated (items 1-4), but the most likely explanation is that they are desensitized. Specifically, the preexposure experiments with 0.3 p V f nicotine and the, on first view, paradoxical inhibitory effect of 1 or 10 FM nicotine, when exposed for 3 min to the tissue, indicate desensitization of the facilitatory autoreceptors. Desensitization removes the nicotinic autofacilitation with the consequence of a falloff in [3H]acetylcholine release. Likewise, Bowman (1990) described a falloff in [3H]acetylcholine release by higher concentrations (>0.1 F M ) of the nicotine receptor agonist decamethonium. Bowman and colleagues (1984, 1986; Bowman, 1990), however, reached a different explanation; they assumed the inhibitory effect as being caused by a terminal membrane depolarization mediated by stimulation of the preterminal nicotine receptors (see Section IV,B). Local depolarization occurring also at the postsynaptic site of the motor endplate during exposure to receptor agonists (Wray, 1981; Colquhoun, 1986) may reduce the driving force for the invading current and, thereby, reduce transmitter release. With unblocked acetylcholinesterase, however, these preterminal nicotine receptors are protected from stimulation by released acetylcholine (Hobbiger, 1976; Bowman, 1990). Therefore, it seems very unlikely that during prolonged electrical nerve stimulation (see item 2) these preterminal-axonal nicotine receptors are actually stimulated and, thus, responsible for loss of the inhibitory effect of tubocurarine (reflecting an inefficiency of the endogenous agonist acetylcholine at the autoreceptors). Rather, it is likely that the presynaptic autoreceptors have been desensitized under this condition, consequently, by the continuous bombardment by released acetylcholine. When preexposure experiments with nicotine have been performed, causing desensitization of the nicotine autoreceptors, tubocurarine produced a significant increase in the stimulated [3H]acetylcholine release (Wessler et al., 1988a). Correspondingly, Bowman and colleagues ( 1986; Bowman, 1990) reported an enhancing effect of tubocurarine when the enzyme acetylcholinesterase was blocked, that is, when the receptors are assumed to become desensitized. Bowman (1990) regarded the action of tubocurarine in protecting the terminal membrane against depolarization as the mechanism underlying the facilitatory effect; related to transmitter release, he classified the preterminal nicotine receptors as inhibitory receptors. However, Wessler and colleagues (Wessler et al., 1988a; Wessler, 1989) attributed the enhancing effect of tubocurarine to the protection of the autoreceptors from desensitization. For good reasons, Bowman wondered how blockade of the autoreceptors by tubocurarine would allow acetylcholine to activate the feedback mechanism under

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these distinct conditions. Tubocurarine, however, is a reversible antagonist and once the autoreceptors have been resensitized in the presence of tubocurarine, it is reasonable to assume that the high concentrations of acetylcholine occurring with the first few stimuli at the presynaptic site displace, at least partially, tubocurarine and activate the positive feedback mechanism. It had already been mentioned that the highest concentration of acetylcholine is built up at the presynaptic site. Moreover, the presynaptic area is roughly 10-fold smaller than the postsynaptic area, a condition that intensifies the differences in acetylcholine concentrations in favor of the presynaptic site. It is, however, dif;ficultto explain the facilitatory effect of a-bungarotoxin on endogenous acetylcholine release (Miledi et al., 1978; Bierkamper et al., 1986) by a competition between the antagonist and released acetylcholine, because this antagonist blocks the receptors in a more or less irreversible manner. The results with a-bungarotoxin favor Bowman’s concept that the antagonist prevents terminal depolarization. Together, the aforementioned observations indicate that the neuronal nicotine receptors are more sensitive to desensitization than the postsynaptic nicotine receptors. A similar conclusion has been drawn from a comparison of muscular nicotine receptors with ganglionic nicotine receptors (Clarke, 1987). Figure 10 shows that a nicotine concentration of 1 mM was necessary to abolish neuromuscular transmission by postsynaptic action, whereas a nicotine concentration as small as 0.3 p I 4 abolishes the facilitatory response at the nicotine autoreceptors. This considerable difference in concentrations (3000-fold) does not reflect exclusively the difference in sensitivity to receptor desensitization, because there might be differences in receptor reserve between the presynaptic site and the postsynaptic site as well. Moreover, measurement of skeletal muscle contraction is a very rough estimation of receptor desensitization. Nevertheless, Magleby and Pallotta ( 198l), recording endplate currents in the frog, did not find measurable desensitization of the postsynaptic nicotine receptors at all with frequencies lower than 30 Hz, whereas the presynaptic nicotine receptors cease to operate during continuous stimulation at 5 Hz (Wessler et al., 1986, 1987b). The high sensitivity of the presynaptic nicotine receptor might be regarded as an endogenous brake, cutting short the autofacilitatory feedback mechanism.

5. Physiology In principle, inhibitory mechanisms appear dominantly involved in the regulation of transmitter release (presynaptic inhibitory receptors, Renshaw inhibition, quantitative dominance of GABA neurons in the

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central nervous system). Nevertheless, presynaptic p receptors and receptors for angiontensin I1 have been shown to mediate a facilitated release of noradrenaline from sympathetic nerve fibers (Starke, 1977; Zimmermann, 1978; Majewski, 1983; Majewski el al., 1984). Is there any indication for a physiological significance of the positive nicotinic feedback mechanism at motor nerves? This question can be answered only indirectly. Whether the nicotine feedback mechanism can be activated under zn vzuo conditions must be considered. There are good reasons to make this assumption. Nicotinic autofacilitation is substantially activated beyond the 10th to 15th pulses of a train when stimulation frequencies between 5 and 50 Hz are applied (Wessler et al., 1987b; Vizi et al., 1987), and motor neurons fire intermittently at frequencies between 5 and 100 Hz. This agreement supports the assumption that nicotinic autofacilitation is operating under zn vzvo conditions and does play a physiological role. In the presence of tubocurarine, the degree of neuromuscular block is greater, the higher the frequency of stimulation (Blackman, 1963). This observation may reflect the addition of two effects of tubocurarine, the blockade of' the postsynaptic nicotine receptors and the removal of nicotinic autofacilitation playing an increasing role with increasing stimulation frequencies (Wessler et al., 1987b). On first view, a presynaptic facilitatory mechanism appears useless at motor endplates, because of the high safety factor already established for neuromuscular transmission at the postsynaptic site (Waud and Waud, 1971). Consider two important facts, however. First, the safety factor declines with increasing stimulation frequencies, particularly when tetanic contractions are produced (Waud and Waud, 1971). Accordingly, to obtain a maximal end-organ response at 100 HLabout 60% of the muscular nicotine receptors have to be stimulated, whereas roughly 10%)of the receptors must be stimulated for a maximal contraction at 0.2 Hz. Second, the affinity of acetylcholine differs considerably between skeletal muscles (nicotine receptors) and smooth muscles (muscarine receptors), being considerably lower in skeletal muscles. Studies to determine the affinity constants with acetylcholine are very difficult to perform. T h e acetylcholine concentration required to produce 50% of the maximal conductance response is roughly estimated at about 30 pkl (Sheridan and Lester, 1977; Dreyer el al., 1978; see also Colquhoun, 1986). But small concentrations of acetylcholine (10 and 100 nM in the presence or absence of an inhibitor of the enzyme acetylcholinesterase, respectively) already produce half-maximal contraction of the small intestine (Bolton and Clark, 1981). Accordingly, there appears to be a

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roughly 100- to 1000-fold difference in the potency of acetylcholine in activating skeletal or smooth muscle fibers. T h e decline of the safety factor with increasing frequencies and the considerable low affinity of acetylcholine at muscular nicotine receptors require a substantial release of acetylcholine to mediate neuromuscular transmission, particularly at higher frequencies. Both conditions may explain why a presynaptic facilitatory mechanism controls transmitter release at motor endplates. The positive nicotinic feedback mechanism can be regarded as a presynaptic amplifier, allowing a rapid and threshold release of transmitter, when immediate action of the skeletal muscles fibers is required. The different patterns in which smooth and skeletal muscles operate should also be considered. Skeletal muscles are rapid and sometimes rapid maximal working systems, whereas smooth muscles, having a considerably basal tone, are slowly working systems. Accordingly, the evolutionary process should have selected mechanisms at skeletal muscles that increase the safety factor for transmission (presynaptic nicotine autoreceptors, adrenoceptors; see Section V). Both fighting and running away are life preserving actions, and both require rapid and maximal neuromuscular transmission. In conclusion, nicotinic autofacilitation is thought to operate as a presynaptic amplifier, increasing the safety factor for rapid transmission. In contrast to the motor nerve, inhibitory presynaptic mechanisms (muscarinic autoreceptor, ctp receptor: Paton and Vizi, 1969; Kilbinger and Wessler, 1980) operate at the endings of the parasympathetic, cholinergic nervous system innervating the smooth muscles, to control the release of acetylcholine and to prevent overstimulation of the end-organ. 6 . Comparison with Other Tissues

Clear and convincing experimental evidence shows the existence of nicotine receptors in the central nervous system to be related to the regulation of transmitter release. Applied agonists (nicotine, cytosine) enhanced acetylcholine release from mouse, rat, and guinea pig cortical slices (Chiou et al., 1970; Rowell and Winkler, 1984; Beani et al., 1985; Loiacono and Mitchelson, 1990). Likewise, the spontaneous release of acetylcholine from rat cerebellar slices was increased by methylcarbamylcholine, an effect that can be prevented by dihydro-Perythroidine, tubocurarine, and K-bungarotoxin (Lapchak et al., 1989). All these antagonists are effective in blocking neuronal nicotine receptors. Additionally, acetylcholine release from synaptosomal preparations of cortical neurons could be enhanced by applied agonists (Rowel1 and

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Winkler, 1984). The release-modulating nicotine showed a rapid desensitization (Beani et al., 1985) corresponding to the autoreceptors at the motor nerve. Transsection experiments published by Clarke el al. (1986) and experiments with synaptosomal preparations support the concept of a presynaptic location of these receptors corresponding to the motor endplate (see also Wonnacott and Drasdo, 1991); however, in contrast to the situation at motor nerves, release of acetylcholine from cortical neurons has, so far, not been shown to be inhibited by tubocurarine or similarly acting drugs. Thus, nicotinic autofacilitation could not be demonstrated with cortical or cerebellar neurons under in uztro conditions. Whether the loss of nicotine binding observed in Alzheimer’s disease (Flynn and Mash, 1986; Nordberg and Winblad, 1986; Whitehouse et al., 1986) and, thus, the attenuation of possible nicotinic autofacilitation of acetylcholine release are involved in the pathophysiology of this disease remains to be elucidated. Nicotine receptors have also been shown to enhance the release of various other transmitters from both the peripheral and the central nervous system. For instance, the release of newly synthesized radioactive serotonin from hypothalamic slices is increased in the presence of nicotine (Hery et al., 1977), and nicotine receptor agonists enhance the release of noradrenaline from pulmonary arteries (Su and Bevan, 1970), the prostatic portion of the rat vas deferens (Carneiro and Markus, l990), and cardiac sympathetic nerves via stimulation of receptors localized at the postganglionic sympathetic axon (for references see Liiffelholz, 1978). Likewise, nicotine produces a significant increase in the release of noradrenaline (Hall and Turner, 1972) and dopamine from striatal synaptosomal preparations or brain slices (Goodman, 1974; Giorguieff et al., 1976; De Belleroche and Bradford, 1978; Rapier et al., 1988),an effect that can be reduced by hexamethonium or by the stimulation of muscarine receptors (Westfall, 1974a). Muscarinic inhibition of nicotine-induced transmitter release is also observed at sympathetic nerve fibers (Lindmar et al., 1968; Liiffelholz, 1978), resembles excellently the situation on motor nerves (see Section IV, C), and appears as a more general regulatory process in cholinergic-sympathetic communication. T h e presynaptic, facilitatory action of acetylcholine has been observed in two nonmammalian species. Tubocurarine reduced the postsynaptic current at the central synapse of Aplysia without decreasing the size of the miniature postsynaptic currents (Baux and Tauc, 1987), indicating the activation of a positive nicotinic feedback mechanism at the innervating cholinergic nerve endings. Interestingly, an inhibitory

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muscarinic feedback mechanism was also found to operate at this synapse; the coexistence of both nicotinic and muscarinic feedback mechanisms corresponds to the situation at the mammalian motor endplate (see Section IV,C). Fulton and Usherwood (1977), recording the excitatory postsynaptic potentials at the locust neuromuscular junction, reported an enhancing effect of nicotine receptor agonists (acetylcholine, carbachol, nicotine, acetyl-P-methylcholine) on transmitter release. Decamethonium and tubocurarine prevented this facilitatory effect, and the authors proposed the presence of facilitatory nicotine receptors at the nerve terminals of the locust neuromuscular junction, a synapse that does not release acetylcholine but rather L-glutamate. Thus, the physiological importance of these latter receptors remains obscure. It appears as a general feature that the membranes of nonrnyelinated nerve fibers are equipped with nicotine receptors (see Bowman, 1990); in most cases, however, the biological function of these receptors is unknown.

B. PRETERMINAL-AXONAL NICOTINE RECEPTORS As already outlined, nicotine receptors are commonly present on the nonmyelinated membranes of nerve fibers; such receptors have been shown to be present at nodes of Ranvier, mammalian C-fibers, endings of sensory nerves, and adrenergic nerve fibers (for references see Bowman, 1990). The nicotine receptors localized at the preterminal area of motor end plates (last node of Ranvier) are of considerable pharmacological importance. Masland and Wigton ( 1940), recording compound action potentials from the anterior root of the cat, found repetitive discharges after intraarterial injection of acetylcholine or prostigmine, a blocker of the enzyme acetylcholinesterase. Repetitive discharge (backfiring) of the motor nerve has been shown to occur with various blockers of the enzyme (edrophonium, ambenonium, neostigmine, diisopropylfluorophosphate, paraoxon, parathion: for references see Bowman et al., 1986). Backfiring (Fig. 12) is associated with repetitive responses at the muscle fiber, twitch potentiation, and fasciculation of the skeletal muscles (Blaber and Bowman, 1963; Randie and Straughan, 1964; Heffron and Hobbiger, 19'19; Clark et al., 1983, 1984; Bowman et al., 1986; Ferry, 1988; Bowman, 1990). Both backfiring and twitch potentiation can be prevented by nicotine receptor antagonists (hexamethoniurn, tubocurarine, pancuronium, a-bungarotoxin: for references see Bowman et al., 1986), indicating that nicotine receptors are involved in the generation of all these phenomena.

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A

F i c . 12. Repetitive discharges in the phrenic nerve (backfiring) and the muscle fibers. Compound action potentials recorded from the phrenic nerve (left) and the hemidiaphragm (right). (A) Control conditions; (B) After partial blockade of acetylcholincsterase ( 1 5O-sec exposure to 3 phf neostigmine); (C) after exposure to neostigimine (sc'c' i)) a i d rutmocurarine (0.1 pm). 'Iiihrurarine abolished backfiring withoilt agerting the repetitive discharges in the skeletal inusrle. (Values from Wessler rt af., 1992a.)

Considerable interest has been developed in investigating whether backfiring is generated by stimulation of postsynaptic or of preterminal nicotine receptors. Stimulation of postsynaptic nicotine receptors was thought to depolarize nerve terminals and to induce backfiring through enhanced potassium efflux from the muscle membrane (Hohlfeld et al., 1981), an intensified local endplate current field (discussed by Ferry, I988), and a reduction in the synaptic calcium concentration. I n con-

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trast, Webb and Bowman (1974) suggested that preterminal nicotine receptors were involved in the generation of backfiring. Both alternatives have been discussed by Bowman (Bowman et al., 1986; Bowman, 1990), and the more convincing data indicate preterminal nicotine receptors as the origin of backfiring. In particular, tubocurarine, at concentrations too low to affect contraction, blocked backfiring (Riker, 1975). Figure 12 shows that 0.1 pJ4 tubocurarine blocked backfiring but repetitive discharges of the muscle membrane were maintained. Under the latter condition repetitive stimulation of postsynaptic nicotine receptors by preserved acetylcholine still occurs (with all the associated alterations in the microenvironment of the synaptic cleft as indicated above), but backfiring of the nerve is abolished. Moreover, Hong and Chang (1987, 1990) found that the “regenerative acetylcholine release” (acetylcholine released by backfiring) can be suppressed by tubocurarine, verapamil, nifedipine, and cadmium at concentrations too low to produce substantial reductions in the amplitude of mEPPs or endplate potentials. These observations clearly indicate that a preterminal nicotine receptor is involved in the generation of nerve backfiring; in addition, it is likely that postsynaptic events such as those discussed above (Ferry, 1988) facilitate the generation of nerve backfiring. So far, detailed knowledge of the pharmacological properties of these preterminal nicotine receptors is not available. As already outlined, nicotine receptors are transported by axonal flow from the cell body to the terminals, and whether some receptors remain at the preterminal part is open to speculation. Accordingly, both presynaptic and preterminal nicotine receptors may have common pharmacological properties, and the inhibitory effect of a-bungarotoxin on [3H]acetylcholine release and on backfiring (see also Bowman, 1990) is in keeping with this assumption. The effector systems must differ, however. The preterminal nicotine receptor mediates depolarization (i.e., it may be coupled to the classic ion channel), whereas stimulation of the presynaptic nicotine receptor facilitates transmitter release only in response to a propagated nerve action potential (see Section IV,A,3). Furthermore, sodium channels are present at almost undetectable levels in the nerve terminals (Brigant and Mallart, 1982). It is important in the future to reevaluate the pharmacological properties of the preterminal receptors. At present a physiological function of these preterminal nicotine receptors is not apparent, because the enzyme acetylcholinesterase protects these receptors from stimulation, and acetylcholine can stimulate these receptors only after partial inactivation of the enzyme and, consequently, after increasing its diffusion radius (Bowman, 1990).

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C. PKESYNAPTIC MUSCARINERECEPTORS 1. OTIY?/~OW Studies Presyriaptic muscarine receptors are involved in regulating the release of both classic transmitters, acetylcholine and noradrenaline, from neurons of the autonomic nervous system (see Section IV,C,5). In contrast to the clear-cut inhibitory effect mediated by muscarinic autoreceptors at the parasympathetic nerves, the existence of such muscarinic autoreceptors at motor endplates had been a matter of considerable controversy. One of the first reports describing muscarinic modulation of the release of endogenous acetylcholine from motor nerves was published by a group from India (Das at ul., 1978; Ganguly and Das, 1979). Acetylcholine released from rat phrenic nerve was assayed on the leech dorsal muscle, and the authors reported a dramatic facilitatory (50-fold) effect of oxotreniorine, an agonist at muscarine receptors. Atropine (0.5 pM) prevented the facilitatory ef€ect of oxotreniorine and, on its own, reduced the release of endogenous acetylcholine. These results indicate both the presence of facilitatory muscarine receptors and the activation of a positive muscarinic feedback mechanism at motor nerve endings. Two later studies failed to confirm these results, however. Gundersen and .Jenden ( 1980), estiniating acetylcholine released from rat phrenic nerve by gas chromatography-mass spectrometry, did not find an enhancing efi'ect of 10 or 100 pM oxotremorine. T h e results reported with 100 pltl oxotremorine may, however, be indicative of an enhancing effect of the agonist; the resting release of acetylcholine was reduced to 40% in the presence of oxotreniorine but the total release (during the stimulation period) was not reduced correspondingly. Thus, stimulated release of acetylcholine may have been enhanced in the presence of 100 F M oxotremorine to balance the reduced resting release. H2ggblad and Heilbronn (1983), using high potassium (50 mM) as a release stimulus, did not observe any efTect of quinuclidinylbenzilate (muscarine receptor antagonist) or oxotremorine on the release of endogenous acetylcholine. One of the first attempts to measure the release of radioactive acetylcholine from isolated rat phrenic nerve was made by Abbs and Joseph (1981); these authors described an enhancing effect of atropine applied in a high concentration of 10 pM. Oxotremorine (10 pM), however, did not affect the release of radioactive acetylcholine but prevented the enhancing effect of the antagonist. T h e authors reached a conclusion opposite that reported by Ganguly and Das (1979): Abbs arid Joseph (1981) proposed the existence of inhibitory muscarine autoreceptors. How can these controversial observations be, at least partially, explained?

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FIG. 13. Effects of oxotremorine on the stimulated release of [sHH]acetylcholine.Release of [3H]acetylcholinewas evoked by two stimulation periods (SI and S2, 100 pulses at 5 Hz), and oxotremorine was present from 26 min before 52 onward. T h e open circles indicate experiments in the presence of 0.1 ph4 scopolamine, which was present from 30 min before S1 onward. *p < 0.05, **p < 0.01. (Values from Wessler et al., 1987a.)

Wessler et al. (1987a) have established a complete concentrationresponse curve for the effect of oxotremorine on the stimulated release of [3H]acetylcholine and have investigated the effects of antagonists under different stimulation conditions. A biphasic concentration-response curve was obtained (Fig. 13). At low concentration (10 IN),oxotremorine enhanced the release of [3H]acetylcholine, whereas at higher concentrations (1 and 10 it inhibited the release. In agreement with the inhibitory effect of oxotremorine, Foldes et al. (1984) had already found that 50 p,M oxotremorine reduced the release of [3H]acetylcholine from mouse phrenic nerve. Both actions, inhibition and facilitation, can be prevented by 0.1 pM scopolamine, which confirms that the effects of oxotremorine are mediated by stimulation of muscarine receptors. The different responses observed with oxotremorine (facilitation, apparent inefficiency, inhibition) show the limitation of single-concentration experiments. For example, oxotremorine did not affect release at 0.1 ph4;

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thus, in keeping with the experiments with 0.1 p l 4 oxotremorine, a convincing argument against the existence of muscarine receptors at motor endplates might have been made. Accordingly, it is not possible to draw conclusions from the effect of single concentrations; only the complete concentration-response curve for oxotremorine clears up the controversy and provides the right answer: both facilitatory and inhibitory muscarine receptors appear to exist on motor nerve terminals. This assumption does explain the controversial data in the literature; the facilitatory effect of' oxotremorine (Ganguly and Das, 1979) and the facilitatory effect of atropine (Abbs and Joseph, 1981) reflect the activation of facilitatory and inhibitory muscarine receptors. The extreme enhancement (50-fold increase) reported by Ganguly and Das (1979), however, remains to be elucidated. T h e existence of inhibitory muscai-ine receptors has been further substantiated by Somogyi and colleagues ( 1987), who showed an inhibitory effect of oxotremorine (1 and 30 p M ) on the release of [3H]acetylcholine from mouse phrenic nerve. Ekidence for an enhancing effect has not been published by Somogyi et nl. (1987). but the authors did not investigate the effect of oxotremorine below a concentration of 1 pJ4 and used electrical field stimulation instcad of discrete nerve stimulation. The positive rnuscarinic feedback system, however, is activated with nerve stimulation only (Wessler and Offermann, 1989; see below). Are both the inhibitory and facilitatory muscarine receptors activated endogenously by released acetylcholine and, thereby, parts of local feedback loops? The facilitatory effect of atropine reported by Abbs and Joseph (1981) and the inhibitory effect of the same compound reported by Ganguly and Das (1979) may indicate the endogenous activation of both types of muscarine autoreceptors. It is, however, difficult to understand why both opposing mechanisms are activated simultaneously under similar stimulation conditions. Wessler et al. (1987a, 1988b) have investigated the effects of three different muscarine receptor antagonists (scopolamine, pirenzepine, dicyclomine), and two opposite effects were obtained. All antagonists increased and all antagonists decreased the release of [3H]acetylcholine depending on the stimulation conditions (Fig. 14). With short-term stimulation (100 pulses at 5 Hz) a dominant facilitation, and with long-term stimulation ( 1 500 pulses at 5 or 25 Hz) a dominant inhibition, was obtained. On the basis of these results Wessler and colleagues ( 1987a) proposed that these autoreceptors are activated differently: the negative muscarinic feedback mechanism is activated by short-term (or intermittent) stimulation and the positive feedback mechanism by long-term stimulation (continuous stimulation). T h e biphasic and flat-running concentration-response curves obtained with the mus-

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FIG. 14. Effects of scopolamine on the stimulated release of [SH]acetylcholine.Release of [3H]acetylcholinewas evoked by two stimulation periods (S1 and S2, 100 or 1500 pulses at 5 Hz) and scopolamine was present from 26-30 min before S2 onward. The filled squares indicate experiments in the presence of 10 ph4 neostigmine, which was present from 30 min before S1 onward. *p < 0.05, **p < 0.01. (Values from Wessler etal., 1987a.)

carine receptor antagonists should, however, be noted. (see Fig. 14; Wessler et al., 1987a, 1988b); these curves may indicate that both muscarinic feedback mechanisms are activated simultaneously but with a dominance of the negative feedback with short stimulation and a dominance of the positive feedback with long stimulation. Scopolamine and oxotremorine modulated both the electrically evoked and the chemically evoked (27 mM potassium) release of [3H]acetylcholine, but differences between both stimulation modes have been observed. Oxotremorine did not produce significant facilitation,

and keeping in line with this observation, scopolamine did not cause significant inhibition, when high potassium was used as release stimulus. T h e release of [:3H]acetylcholine evoked by high potassium is not reduced by tetrodotoxin (Wessler et al., 1987a), whereas [:+H]acetylcholine evoked by electrical nerve stimulation, which causes propagation of action potentials along the axon and electrotonic invasion of the active release zones, is inhibited by tetrodotoxin (Wessler et al., 1986). High potassium depolarizes the active release zones directly without the involvement of axo1ia1and preterminal impulse propagation. Importantly, the facilitatory effect of muscarine receptor stimulation ceases with direct depolarization of the terminal membrane. Thus, the positive muscarinic feedback system appears to facilitate the electrotonic invasion. This hypothesis implicates the facilitatory muscarine receptors to be localized sorilewhat proximal to the active release zones, to allow niodification of the invading local current. Obviously, additional experimental data are required to substantiate this assumption. Some indirect experimental evidence favors this concept. I t is noteworthy that the facilitatory efiert of oxotremorine and the inhibitory effects of scopolamine and pirenzepine were observed with particularly low concentrations (Wessler rt ( I / . , 1987a, 1988b), an observation that may indicate that also t.he endogenous agonist acetylcholine is present at these receptors in low concentrations, that is, a location of the receptors at some distance froni the active release zones. T h e concept of a preterminal location of the facilitatory muscarine receptors is also supported by the observation that the positive feedback mechanism is activated only during long-term stimulation; naturally, acetylcholine can more easily reach these preterrninal niuscarine autoreceptors during a long period (1500 pulses) of continuous stimulation than during a short period (100 pulses). Interestirigly, he inhibitory effect of scopolamine reflecting the activation of facilitatory autoreceptors was lost when 1500 pulses were applied intermittently, in trains of 40 pulses (Wessler and Offermann, 1989). With intermittent stimulation acetylcholine is inactivated between the individual trains, thus preventing a threshold agonist concentration from being reached at the proximally located muscarine receptors. Finally, the positive muscarinic feedback mechanism was observed only with electrical nerve stimulation. Scopolamine or atropine did not inhibit the release of [:%H]acetylcholinewhen an electrical field was applied t o the tissue (Wessler arid Offermann, 1989; Somogyi et al., 1987). T h e electrical field may cause a strong terminal impulse invasion, thus preventing additional modifications by facilitatory muscarine receptors. In contrast. the results obtained with t h e inhibitory muscarine receptors suggest a location of these receptors near active release zones; ox-

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otremorine reduced and scopolamine enhanced the release of [3H]acetylcholine evoked by high potassium (Wessler et al., 1987a). The complex pattern of activation of both negative and positive muscarinic feedback mechanisms, depending on the stimulation conditions (field versus nerve stimulation, intermittent versus continuous stimulation, long-term versus short-term stimulation) and depending on the concentrations of the agonists and antagonists applied, may, to some extent, explain the controversial data in the literature. In addition, blockade of the enzyme acetylcholinesterase (when measuring endogenous acetylcholine) complicates the interpretation of results, because with blocked acetylcholinesterase the transmitter accumulates in the biophase, thus causing simultaneous and uniform activation of both opposite working feedback systems; consequently, balancing of both mechanisms may occur and any modulatory effect of muscarine receptor antagonists may be lost (Haggblad and 'Heilbronn, 1983). In fact, Wessler and colleagues have shown that both the enhancing effect of scopolamine with short-term stimulation and the inhibiting effect with long-term stimulation are largely reduced in the presence of neostigmine, an inhibitor of acetylcholinesterase (Wessler et al., 1987a; see Fig. 14). 2. Functional Studies Only a few electrophysiological studies describe the effects of muscarine receptor agonists or antagonists at motor endplates. Beranek and VyskoEil (1967), recording mEPPs and endplate potentials in the rat diaphragm, did not obtain evidence for a presynaptic effect of atropine, but atropine produced a postsynaptic blocking effect; at high concentration (> 10 pW), atropine blocked the endplate potential evoked either by nerve stimulation or by iontophoretically applied acetylcholine. The authors attributed this blocking action to occur at the postsynaptic ion channel. Meanwhile, several reports have been published showing atropine to block the opened channel of the postsynaptic nicotine receptor (Katz and Miledi, 1973; Dreyer et al., 1978; l'eper et al., 1982). This observation, of course, handicaps electrophysiological studies with atropine when presynaptic effects are examined. Phenthonium, a quaternary derivate of (-)-hyoscyamine, has recently been shown to enhance the rate of mEPPs in rat diaphragm muscle; oxotremorine, a muscarine receptor agonist, did not prevent this presynaptic effect, whereas neostigmine, an inhibitor of acetylcholinesterase, potentiated the facilitatory effect of a low phenthonium concentration (Fann et al., 1990). Smith ( 1982) reported experiments with oxotremorine at the neuromuscular junction of the mouse; at high concentrations (5-50 the agonist

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caused depolarization of the muscle membrane, an effect that was blocked by tubocurarine. This antagonism indicates an agonistic activity of oxotremorine at nicotine receptors (Elmqvist and McIsaac, 1967); however, in the experiments with [3HH]acetylcholinethe effects of oxotremorine could be prevented by scopolamine, indicating an exclusive effect at muscarine receptors. In the study by Smith (1982), oxotremorine caused a decline in the amplitude of the mEPPs that was larger than that predicted from membrane depolarization. This effect may be interpreted as reflecting a presynaptic, inhibitory effect of oxotremorine. McN-A-343, a compound with a preferential action at M I receptors, enhanced the contraction of indirectly stimulated rat hemidiaphragm and increased the frequency and amplitude of mEPPs (Simioni et al., 1984). T h e effects of McN-A-343 were prevented by tetrodotoxin, suggesting that the compound enhances conduction. Unfortunately, the facilitatory effect was not proved in the presence of a muscarine receptor antagonist. Clear experimental evidence has been published showing a modulatory role of muscarine receptors at the frog neuromuscular junction. Duncan and Publicover ( 1979) carried out experiments with inhibitors of acetylcholinesterase (neostigmine, edrophonium, physostigmine) and with direct agonists (carbachol, metacholine). All these compounds reduced the frequency of mEPPs, an effect that was antagonized by atropine but not by tubocurarine. Undoubtedly, these effects are mediated by stimulation of inhibitory muscarine receptors, reducing spontaneous acetylcholine release. Moreover, Duncan and Publicover ( 1979) analyzed the inhibitory effect of muscarine receptor agonists at various extracellular calcium concentrations, because the frequency of mEPPs has been shown to be markedly modified by the intracellular calcium concentration. Based on their findings, Duncan and Publicover (1979) proposed that the inhibitory muscarine receptors mediate suppression of calcium entry into the nerve endings. A more recent study confirmed the concept of inhibitory muscarine receptors at amphibian neuromuscular junction. Arenson (1989) investigated the effect of oxotremorine on isolated frog satorius muscle. Oxotremorine (30 pM) reduced both the frequency of mEPPs and the amplitude of evoked endplate potentials. Again, both effects could not be prevented by tubocurarine but were antagonized by 100 nM atropine. Atropine itself did not affect the quanta1 content or the frequency of the mEPPs. Very recently, phenthonium, a quaternary derivate of hyoscyamine (atropine), was reported to increase spontaneous acetylcholine release at rat motor nerve terminals (Fann et al., 1990). Blockade of acetylcholinesterase potentiated the prejunctional effect of a low concentration of phenthonium, which might reflect the involvement of inhibitory mus-

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35 1

carine receptors; however, oxotremorine did not prevent the stimulatory effect of phenthonium, and the compound produced additional postsynaptic effects (Fann et al., 1990). In conclusion, there is fairly convincing evidence showing the existence of inhibitory muscarine receptors at motor endplates of amphibians; these receptors, however, appear not to be activated by released acetylcholine, as atropine is without an enhancing effect. One of the problems in obtaining confirmatory evidence of a modulatory role of muscarine receptors at mammalian endplates may be the coexistence of positively and negatively operating systems and possible postsynaptic actions of the applied compounds, when the electrical response to released acetylcholine is recorded.

3. Receptor Characterization Muscarine receptors constitute a heterogenous population of receptors. At least five receptors can be discriminated on the basis of different genes, end-organ responses, effector systems, and affinity constants for antagonists (Bonner, 1989). M1 receptors are localized at cell bodies in the central and peripheral nervous systems, stimulate the breakdown of inositol 1,4,5-trisphosphate, and block the M current, thus enhancing the excitability of neurons. M1 receptors can be characterized on the basis of affinity constants for antagonists; pirenzepine, dicyclomine, and telenzepine are regarded as preferentially M 1 receptor-blocking compounds (Hammer et al., 1980; Giachetti et al., 1986; Eglen and Whiting, 1986). Wessler et al. (1987a, 1988b) have estimated the apparent potencies of scopolamine, pirenzepine, and dicyclomine in enhancing and inhibiting evoked [3H]acetylcholine release by calculating the EC50 values from the respective concentration-response curves. All antagonists enhanced release with EC,, values of 10-40 nM (Wessler et al., 1988b). Moreover, a pirenzepine concentration of 10 nM was sufficient to antagonize the facilitatory effect of oxotremorine (Wessler et al., 1988b). All these results strongly suggest that M 1 receptors mediate the positive muscarinic feedback mechanism. Scopolamine (EC,, value = 0.06 nM) and pirenzepine (0.2 nM) differed by only a factor of 4, in their potency in inhibiting transmitter release, whereas dicyclomine showed a considerably lower potency (EC,, value = 44 nM). Nevertheless, these values do not exclude that also an M 1 receptor subtype mediates inhibition, but differences from the facilitatory M 1 receptors are apparent. The actual affinity constants (PA, values) for inhibitory and facilitatory receptors cannot be estimated because of the simultaneous activation of both opposite mechanisms. This limits the concentration range for agonists to produce exclusive inhibitory o r exclusive facilitatory effects. The nature of the effector systems coupled to the muscarine autoreceptors at the phrenic nerve can only be speculated. Recently, Bowman

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(1990) proposed that the facilitatory k l l receptor may block the M current (potassium channel); on this action, terminal depolarization may be prolonged and more active release zones might be activated. This hypothesis is in line with the suggestion of a more preterminal localization of the facilitatory muscarine receptors (see above). T h e effector system of the inhibitory muscarine autoreceptor is uncertain; there are no experimental data for any substantial suggestions. As calcium ions play a key role in stiniulation-secretion coupling, the inhibitory muscarinic autoreceptors localized near active release zones may reduce calcium availability. Experiments are needed to evaluate this concept.

Whether both muscarinic feedback mechanisms play any physiological role in modulating transmitter release from the motor nerve is unknown. It should, however, be noted that binding studies have provided convincing evidence for localization of muscarine receptors at motoneuron cell bodies, LFentral spinal horn, and sciatic nerve of the rat and rabbit, whereby most o f the receptors migrate in the motor axons and are transported by a large ariterograde axonal flow (Gulya and Kitsa, 1984). By recording the contractions of the cat anterior tibia1 muscle under iii viuo conditions, Alves-do-Prado et a/. (1987) showed that intraarterially applied atropine in low doses (0.3 Fg/kg) reduced the time for recovery from tetanic fade. This action of atropine was interpreted to demonstrate a negative muscarinic feedback mechanism. One possible physiological role for the inhibitory muscarinic feedback system at motor nerves might be to limit nicotinic autofacilitation. Like all positive systems, nicotinic autofacilitation can cause overstimulation by its self-supporting mechanism, but the coactivation of inhibitory muscarine receptors can cut short this autofacilitatory process. In contrast, the positively operating niuscarinic feedback system is dominantly activated under conditions when nicotinic autofacilitation ceases, that is, under prolonged stimulation periods. Whether nicotinic autofacilitation is replaced by muscarinic autofacilitation under the latter situation of prolonged stimulation is open to speculation. 5 . Compuiison 7rjith O t h u Tissues Control of acetylcholine release by presynaptic muscarine receptors is a widespread regulatory process in chemical neurotransmission. In the absence of inhibitors of acetylcholinesterase, antagonists at muscarine receptors have been demonstrated to enhance the release of acet.ylcholine from various tissues. Specifically, the release of' acetylcholine from neurons of the central nervous system (cortex) has been found to be

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enhanced by atropine or similarly acting substances (MacIntosh, 1963; Szerb, 1964; Polak, 1965; Bourdois et al., 1974; Hadhazy and Szerb, 1977;James and Cubeddu, 1984; for further references see Starke et al., 1989). In experiments using synaptosomal preparations, evidence for terminal localization of these inhibitory, muscarine receptors has been presented (Nordstrom and Bartfai, 1980). Also in freely moving rats, muscarine receptor antagonists enhanced the release of acetylcholine as determined by microdialysis (brain) coupled with radioenzymatic assay (Consolo et al., 1987; Damsma et al., 1988). Both facilitatory and inhibitory muscarine receptors are supposed to modify the release of dopamine from the striatum; basal release of dopamine was enhanced (Giorguieff et al., 1977), whereas potassium (50-60 mM) evoked dopamine release from striatal slices or synaptosomal preparations appeared to be reduced by stimulation of muscarine receptors (Westfall, 197413; De Belleroche and Bradford, 1978). In particular, the experiments with synaptosomes (De Belleroche and Bradford, 1978) exclude the involvement of interneurons but strongly indicate that the inhibitory muscarine receptors are localized at the varicosities. In contrast to the latter results, Raiteri et al. (1982), using a moderate potassium concentration (15 d) as release stimulus, found potassium-evoked dopamine release to be enhanced by muscarine receptors. Muscarine receptor antagonists have been shown to increase the release of acetylcholine from organs innervated by the parasympathetic nervous system. This has been shown for airways, heart, small intestine, urinary bladder, and iris (Kilbinger, 1977, 1984; Swaynok and Jhamandas, 1977; Kilbinger and Wessler, 1980, 1983; Alberts et al., 1982; Fryer and Maclagan, 1984; Wetzel and Brown, 1985; D’Agostino et al., 1986, 1990; Wessler et al., 199Oc, 1991; for further references see Starke et al., 1989). Accordingly, the negative muscarinic feedback loop appears to be the general mechanism protecting the end-organ from overstimulation. Additionally, inhibitory muscarine receptors have also been shown to reduce the release of noradrenaline. In particular, the release of noradrenaline from cardiac sympathetic nerves is modulated in a complex pattern by muscarinic heteroreceptors. Both inhibitory and facilitatory receptors that are activated at distinct vagosympathetic impulse intervals have been described (Habermeier-Muth and Muscholl, 1988; Habermeier-Muth et al., 1990). Evidence for inhibitory and facilitatory muscarine receptors on sympathetic nerves in mouse atria has also been found by the use of two different muscarine receptor agonists; McNeilA-343, an M l-receptor agonist, enhanced evoked noradrenaline release, whereas the opposite effect was observed with carbachol (Costa and Majewski, 1991). This regulation corresponds to the situation at motor

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endplates. Likewise, release of acetylcholine from the small intestine is enhanced and reduced by stimulation of M1 receptors and M 3 receptors, respectively (Kilbinger and Nafziger, 1985). Recently, Dujic et al. (1990), measuring the release of endogenous acetylcholine from the cat stellate ganglion, found evidence for inhibitory muscarine receptors, whereas nicotine receptors were not involved in the modulation of acetylcholine release. Finally, release of acetylcholine from the electric organ of fishes is controlled by inhibitory muscarine receptors (Kloog et al., 1980; Dunant et al., 1980; Dunant and Walker, 1982).

V. Modulation of Release by Adrenoceptors

A. EFFECTS OF SYMPATHOMIMETIC AMINES AT SKELETAL MUSCLESin Vizio

The first observation concerning possible effects of sympathomimetic amines on neuromuscular transmission was reported nearly 100 years ago (Oliver and Schafer, 1895). Since 1940, facilitatory effects of catecholamines on neuromuscular transmission (anticurare effect) have been published in textbooks on anesthesiology and pharmacology and in numerous articles (Bulbering and Burn, 1942; Brown et al., 1948; Goffart and Ritchie, 1952; Bowman and Raper, 1966; Jenkinson rt al., 1968; Bowman and Nott, 1969; Kuba, 1970; Malta et al., 1979; for detailed references see also Bowman, 1981). It is commonly accepted that sympathomimetic amines affect neuromuscular transmission in a very complex way, multiple sites of action are involved (presynaptic and postsynaptic effects), and distinct muscles are modified differently (nonfatigued versus fatigued muscles, fast-contracting versus slow-contracting muscles). Sympathomimetic amines have been shown to increase the tension of isometric twitches, particularly in fatigued, fast-contracting muscles (anticurare effect); however, a curare-potentiating effect also occurs, particularly in slow-contracting muscle fibers (extensor muscles adapted for sustained tonic activity). T h e latter effect is thought to be mediated by muscular p2 adrenoceptors whose stimulation causes hyperpolarization of the muscular membrane, whereas the anticurare effect has been proposed to be mediated by stimulation of presynaptic cx and @ receptors (Bowman, 1981). T h e hyperpolarizing action of catecholamines results from multipIe effects: an increased activity of calcium-dependent potassium channels (Zemkova et d.,1985) as well as stimulation of

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Na+/K+-ATPase (Cheng et al., 1977; Clausen and Flatman, 1977; Edstrom and Phillis, 1981) and enhanced efflux of sodium (Hays et al., 1974; Clausen and Flatman, 1977). Some of these effects, however, are unrelated to adrenergic receptor stimulation (Zemkova et al., 1985). Under sensitive conditions, the inhibitory postsynaptic effect can cause muscle weakness (“weakness in the knees”),occurring, for example, during emotional disturbances. Further P-receptor-mediated effects at the muscle fiber (metabolic effects, increase in cyclic AMP, altered sodiumpotassium exchange, myosin light chain phosphorylation) and indirect effects (cardiovascular system with a change in muscular perfusion, effects at the muscle spindles, the motoneuron, and the central nervous system) can additionally affect skeletal muscle activity. Finally, sympathomimetic amines can enhance the physiological, centrally originating tremor o r trigger tremor by a peripheral site of action (Barcroft et al., 1952; Bowman and Zaimis, 1958; Marsden et al., 1967a; Marsden and Meadows, 1970; see also Bowman, 1981). Multiple effects contribute to the tremorogenic action of P-receptor agonists: decreased fusion of incomplete tetanic contraction, sensitization of the stretch reflex through effects on the muscle spindles, effects at the spinal and supraspinal level (Bowman and Zaimis, 1958; Marsden et al., 1967b, 1972; Hodgson et al., 1969; Bowman, 1981). Naturally, all these multiple sites of action complicate the interpretation of results obtained with sympathomimetic amines in in vivo studies when twitches are recorded. This review focuses mainly on the effects occurring at the presynaptic site, a site of action that can be analyzed selectively by measuring the release of [3H]acetylcholine. Most of the results have been obtained in the hemidiaphragm, a preparation containing fast-twitch red muscle fibers; it is understood that the effects found in this preparation cannot be generalized to all the different skeletal muscles.

B. a RECEPTORS 1. Overflow Studies The current knowledge about the different actions of sympathomimetic amines (see above) has been obtained in functional experiments, that is, in experiments in which the electrical or mechanical endorgan response have been recorded; release experiments are very limited. Only recently have studies been performed to investigate the effects of sympathomimetic amines on the release of acetylcholine from mouse or rat phrenic nerve (Snider and Gerald, 1982; Somogyi et al., 1987;

s2

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time (min) FIG. 15. Effects of phenylephrine on the resting efflux of tritium and on stimulated [SHIacetylcholine release. Release of [SH]acetylcholinewas stimulated by two periods of electrical nerve stimulation (S1 and S2, 200 pulses at 10 Hz). Phenylephrine (10 )LM) was added 24 min before S2 and prazosin (10 nM) from 30 min before S1 onward. Phenylephrine caused a transient increase in the resting tritium efflux and, in addition, facilitated [3H]acetylcholine release. (Values partially from Wessler et al., 1989.)

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Wessler et al., 1989, 1990a,d; Vizi, 1991). Phenylephrine, a-methylnoradrenaline, and adrenaline caused a concentration-related and substantial increase in the stimulated release of [3H]acetylcholine (Wessler et al., 1989; see also Fig. 15). The effect of phenylephrine was blocked by areceptor antagonists like prazosin and yohimbine (see Fig. 15; Wessler et al., 1989). These results provide clear evidence for the existence of areceptors that mediate an increase in transmitter release from the motor nerve, a concept that had already been substantiated by functional studies (see above and Section V,B,2). The facilitatory effects of both adrenaline and a-methylnoradrenaline were only partially reduced by blockade of a receptors, but combined blockade of a and p receptors abolished any facilitatory effect of both compounds (Wessler et al., 1989). This observation indicates the existence of additional facilitatory p receptors at motor nerve terminals (see Section V,C,1). It should be noted that phenylethylamines, in addition to their facilitatory effect on stimulated [3H]acetylcholine release, increase the resting tritium efflux immediately after their application to the organ bath (see Fig. 15). This stirnulatory effect was transient; it vanished after a short period of roughly 1 min. The effect could be blocked with the a-receptor antagonist prazosin (Fig. 16), and was more pronounced with a- than

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FIG. 16. Blockade of the facilitatory effect of phenylephrine on resting tritium efflux by prazosin. Phenylephrine (PE) was added to the organ bath. The effect of the compound is expressed as the R2/R1 ratio; R1 and R2 represent the tritium emux collected 4 min before and 4 min after application of phenylephrine, respectively; in control experiments R 2 was obtained also in the absence of phenylephrine. Prazosin (0.1 added 46 min before phenylephrine, abolished the facilitatory effect of phenylephrine. *p < 0.05.

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with p-receptor agonists. The increase in the resting tritium emux might reflect an enhanced spontaneous release of acetylcholine, an interpretation that corresponds to the enhanced frequency of mEPPs found with adrenaline and noradrenaline (KrnjeviC and Miledi, 1958; Jenkinson et al., 1968; Kuba. 1970; Kuba and Tomita, 1971; Gallagher and Blaber, 1973). T h e rapid fading of the facilitatory effect (see Fig. 15) can be explained either by receptor desensitization or by exhaustion of that [:+H]acetylcholine store from which the spontaneous transmitter release is generated. T h e stimulatory effect of sympathomimetic amines on resting tritium efflux maintained after the exhaustion of the 13HH]acetylcholine pool (I. Wessler, unpublished observations). Therefore, it seems possible that, in addition to acetylcholine, other radioactive compounds like choline and phosphorylcholine are liberated from neuronal and nonneuronal membranes in response to the stimulation of a receptors. Likewise, vasopressin, ATP, and platelet-derived growth factor have already been shown to trigger the liberation of phosphorylcholine and choline in other tissues (Besterman et al., 1986; Bocckino et al., 1987: Cabot eta[., 1988a,b).At this time, a permissive role of a receptors, to enhance the availability of the precursor choline for acetylcholine synthesis, can only be speculated. Further experiments are required to analyze this possible and exciting effect of sympathomimetic amines on the resting outflow of choline and phosphorylcholine at motor endplates.

2. Functional SturlZeJ Extracts of the adrenal medulla habe been shown to increase the twitch tension of skeletal muscles (Oliver and Shafer, 1895). Some years later this effect was verified writh adrenaline (Gruber, 1922a,b),and since 1922, several reports have shown that catecholamines increase the twitch tension, the twitch duration, and the fusion of responses in incomplete tetanic contractions (for references see Bowman et al., 1962; Bowman and Nott, 1969; Bowman, 1981). The facilitatory effect was more marked with adrenaline than with noradrenaline and could be abolished by phentolamine, indicating that a receptors are involved (Fig. 17). More recent studies have extended the number of compounds investigated, and have confirmed the involvement of 01 receptors. The facilitatory effects of noradrenaline, adrenaline, phenylephrine, methoxamine, and oxymetazoline can be prevented by phentolamine, tolazoline, and thymoxamine (Malta et al., 1979). Moreover, low concentrations of prazosin (1- 10 nM) and tolazoline (10- 100 m u ) abolished the twitch-enhancing effect of noradrenaline and adrenaline, whereas a high concentration of

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FIG. 17. Effects of sympathomimetic amines on twitches. Twitches of a tibialis anterior muscle elicited indirectly once every second in vivo. At TC, an intravenous injection of' tubocurarine (0.3 mg/kg) was given and thereafter a constant degree of partial block was maintained by tubocurarine infusion (0.58 mglkg per hr; TC INF). At ADR, NOR, and ISO, 10 pgikg of adrenaline, noradrenaline, and isoprenaline was injected. The second response to adrenaline was recorded after pretreatment with phentolamine (Phentoi., 2 mglkg). ADR and I S 0 showed curare-potentiating effects; the anticurare effect of ADR (second effect) was prevented by phentolamine. (From Bowman and Raper, 1966, with permission.)

yohimbine ( 1 pM) was required to block the facilitatory effects (Barios et al., 1988). T h e latter results are in excellent agreement with release studies showing an al-receptor subtype to enhance the stimulated transmitter release from the phrenic nerve (see Section V,B,3). It should be noted that the facilitatory effects of adrenaline or noradrenaline are observed mainly in in uivo studies with animals pretreated with tubocurarine to reduce neuromuscular transmission (anticurare effect of sympathomimetic amines). With isolated skeletal muscle preparations, however, some authors have failed to demonstrate a facilitatory action of noradrenaline or adrenaline (Brown et al., 1948; Ellis and Beckett, 1955; Montagu, 1955). This apparent inefficiency can be explained by the observation that adrenaline and noradrenaline can

mediate two opposing effects, the anticurare effect and the curarepotentiating effect. After the initial facilitatory effect a secondary inhibitory action on neuromuscular transmission becomes apparent (see Fig. 17). This inhibitory effect can be blocked by P-receptor antagonists and is caused by hyperpolarization of the muscular membrane (Bowman and Raper, 1966). T h e curare-potentiating postsynaptic effect (decrease in peak isometric tension, shortening of muscle twitch, decrease in fusion of incomplete tetanic contraction: see Bowman and Zaimis, 1958; Marsden and Meadows, 1970) occurs most prominently in slow-contracting muscles, and can attenuate or even balance the facilitatory presynaptic effects. Thus, activation of inhibitory, postsynaptic effects handicaps the experimental evaluation of facilitatory, presynaptic effects; however, after pretreatment with tubocurarine, which reduces both the safety fjtctor for neurornuscular transmission and the muscle twitch, recording of muscle twitches has turned out to be more sensitive in indicating the enhanced transmitter release, a condition that unmasks the facilitatory, presynaptic action of catecholamines. T h e simultaneous activation of two opposing effects may also explain why, under in IJZUO conditions, the fxilitatctry effect of adrenaline can be abolished by a-receptor antagonists only, whereas in release studies the facilitatory effect of adrenaline is prevented by the simultaneous blockade of both (Y and P receptors only (Wessler et al., 1989). More importantly, possible presynaptic effects mediated b y P, receptors (see Sections V,C,l and V,C,3) are abolished and excluded from experimental evaluation, when nonselective antagonists at p receptors are used to prevent postsynaptic effects or hemodynamic effects of applied sympathomimetic amines (Malta et al., 1979). XI realize the multiple actions of sympathomimetic amines at skeletal niuscles it should be considered that these compounds can also increase the contractility of some skeletal muscle fibers via stimulation of muscular /3 receptors (fast-contracting muscles or denervated muscles: Bowman and Kott, 1969). 'Thus, the effects of sympathomimetic aniines occurring under in u i 7 ~conditions are very complex, with multiple sites of actions involved: ( 1) receptor-mediated direct facilitatory effects at the nerve terminal (cx and P receptors); (2) P-receptor-mediated effects at the muscle fiber (increased contractility and fusion in fast-contracting or denervated muscles, reduced contractility and fusion in slow-contracting muscles); (3) indirect effects (sensitization of muscle spindles, hemodynamic effects, metabolic effects, effects at motoneurons by a central site of action). Both release arid functional studies are required for analysis of the underlying mechanisms; release studies can detect presynaptic ef€ects without the interference of multiple postsynaptic effects.

PRESYNAPTIC RECEPTORS AT MOTOR NERVE 'TERMINALS

36 1

T h e existence of facilitatory a receptors has also been substantiated in electrophysiological studies demonstrating that sympathomimetic amines increase the evoked endplate potentials in both mammalian and frog muscles (KrnjeviC and Miledi, 1958; Jenkinson et al., 1968; Kuba, 1970; Kuba and Tomita, 197 1). Noradrenaline, adrenaline, isoprenaline, clonidine, phenylephrine, and xylazine increased the amplitude of endplate potentials; noradrenaline, in contrast to its anticurare effect, was the most effective amine (Kuba, 1970; Lim and Muir, 1983). The facilitatory effect on evoked end plate potentials was abolished by areceptor antagonists (Jenkinson et d., 1968; Kuba, 1970; Lim and Muir, 1983), and the effect of isoprenaline was blocked by P-receptor antagonists. Kuba (1970) concluded that isoprenaline acts at the postsynaptic membrane to increase the resting potential and the input resistance. In fact, adrenaline and isoprenaline increased the endplate amplitude of iontophorectically applied acetylcholine. In addition, the a-receptor antagonist phentolamine by itself increased the evoked endplate potential, probably by a postsynaptic site of action (Kuba, 1970). The effects produced by sympathomimetic amines at the muscle fibers complicate the interpretation of data obtained in electrophysiological studies. Nevertheless, the results obtained with the different experimental approaches (i.e., release studies, electrophysiological studies, and in uiuo studies recording muscle contraction) have provided consistent and confirming evidence for the existence of facilitatory a receptors at motor nerve terminals. There are, however, some dissimilarities. Clonidine, oxymetazoline, and xylazine were found to enhance endplate potentials (Lim and Muir, 1983), whereas none of these substances affected transmitter release (Somoygi et al., 1987; Wessler et d.,1989). Methoxamine and oxymetazoline caused an anticurare effect under in vivo conditions (Malta et al., 1979) but not under in uitro conditions (Bafios et al., 1988). Lim and Muir (1983) reported that prazosin and yohimbine blocked the facilitatory effects with the same potency, whereas Bafios et al. (1988) and Wessler at al. (1989) found a preferential antagonism by prazosin (see Section V,B,3). T h e facilitatory effect of noradrenaline on ["H]acetylcholine release can be prevented by antagonists at @ receptors, whereas in functional studies the presynaptic effect is blocked by a-receptor antagonists. Noradrenaline mediates the strongest facilitatory effect on evoked endplate potentials, but its anticurare effect is considerably less potent. Finally, evidence for facilitatory presynaptic P receptors has, so far, not been obtained in functional studies. The use of antagonists acting selectively at p or p2 receptors will allow more detailed investigation in further experiments; the receptors embedded in the muscular

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membrane are predominantly of the p2 subtype (Bowman, 1981; Elfellah and Reid, 1987), whereas the presynaptic p receptors belong to the p1 subtype (Wessler et al,, 1990d; see also Section V,C,3).

3. Receptor Characterization and Signal Transduction a Receptors are a heterogenous family divided into the a t and ap subtypes (Langer, 1974; for detailed reference see Starke, 1981). A more recent classification of a receptors considers evidence accumulated in the last few years and suggests further subdivision of both a l and ci2 receptors into pharmacologically distinct subtypes, that is, into a l a ,(Ylb, a2a,and aPhreceptors (Bylund, 1988; Morrow et al., 1985; Flavahan and Vanhoutte, 1986; Hieble et al., 1986; Minneman, 1988). T h e classification of these receptors is based mainly on differences in the affinities of selective antagonists and, with some reservation, on the rank order of potency for agonists. Estimation of the affinities of preferentially blocking antagonists (prazosin, BE 2254, WB 4101, and indoramin acting preferentially at a I receptors; yohimbine, rauwolscine, and idazoxan acting preferentially at apreceptors) is helpful for division into the a1or a2 subtype. A more sophisticated approach is required for a further subdivision; WB 4 101 and 5-methyl-urapidil appear to bind more selectively to the a l athan to the a I breceptor, and the reverse was found with the irreversible antagonist chlorethylclonidine (Han et al., 1987b; Minneman, 1988; Gross et al., 1988). The facilitatory effect of phenylephrine was prevented by 0.01 or 0.1 cLi\.r prazosin; however, phenylephrine still enhanced transmitter release from the phrenic nerve in the presence of corresponding yohimbine concentrations, but a concentration of 1 +A4 yohimbine abolished the facilitatory effect of phenylephrine (Wessler et al., 1989). Likewise, Bafios et al. (1988) found prazosin and tolazoline to block the anticurare effect of noradrenaline with low concentrations (10- 100 nM),whereas 0.1 +A4 yohimbine did not show any antagonistic effect in these functional experiments. T h e high affinity of prazosin versus the low affinity of yohimbine strongly suggests that an al receptor mediates the enhanced acetylcholine release from phrenic nerve (Wessler et al., 1989; Vizi, 1991). This conclusion is further substantiated by two experimental observations. First, agonists acting preferentially at ap receptors such as clonidine, oxymetazoline, and xylazine did not modify evoked transmitter release, even at high concentrations (10 +A4) and after various exposure times (Somogyi el al., 1987; Wessler et al., 1989). Second, a-methylnoradrenaline (experiments performed in the presence of propranolol to exclude the simultaneous stimulation of facilitatory p receptors) and phenylephrine enhanced transmitter release from the motor nerve with the

PRESYNAPTIC RECEPTORS AT MOTOR NERVE TERMINALS

363

same potency, and both compounds have roughly equal affinity and e E cacy at a,receptors (Bevan, 1981).The results obtained in in vivo experiments by Malta et al. (1979),who determined the rank order of potency of agonists in mediating an anticurare effect, are also indicative of an a,receptor subtype. The authors found similarities to experiments with postjunctional a receptors in vascular tissue, receptors that belong mainly to t h e a l subtype (Drew, 1976; Starke, 1981).Further subdivision into the alaand a l bsubtypes has, so far, not been carried out, but there is some hint for an alasubtype (low efficacy of the agonists, blockade of the effect with organic calcium channel antagonists; see below). Regulatory proteins (guanine nucleotide regulatory protein) are involved in the signal transduction and couple the neurotransmitter receptors to the second messenger systems to trigger intracellular signals. The coupling machinery of the a,receptors appears complex, because the a, receptor can stimulate different signal transduction mechanisms (Minneman, 1988; McGrath and Wilson, 1988). Hydrolysis of inositol phospholipids is the most commonly observed transduction pathway, but a l receptor activation has also been shown to increase cyclic AMP, to activate phospholipase A,, and, possibly, to open directly calcium channels. So far, whether these different signals are mediated by different regulatory Gproteins is an open question. All the different pathways end up with an increased intracellular calcium concentration, the key ion in mediating enhanced transmitter release. The a,-receptor-mediated increase in acetylcholine release from the phrenic nerve can be prevented by a low concentration of nifedipine (0.1 whereas nifedipine alone did not affect evoked acetylcholine release (Wessler et al., 1990a,b).More importantly, the N-type calcium channel antagonist o-conotoxin GVIA did not inhibit the stimulatory effect of phenylephrine (Wessler et al., 1990a), indicating that an L type (or subtype) is opened and mediates a calcium influx in response to a,-receptor stimulation at motor nerve terminals. In concert with this finding, the inotropic effect of adrenaline in isolated frog sartorius muscle was prevented by removal of extracellular calcium or by calcium channel antagonists like D-600 and diltiazem (Williamsand Barnes, 1989). a,-Receptor-mediated contractile responses are more sensitive to blockade by organic calcium channel antagonists than are a,-receptormediated effects; however, increasing evidence has been obtained showing that the vasoconstrictor effect of a,-receptor agonist is sensitive to organic calcium channel antagonists. The contractile response to the a,agonist phenylephrine is blocked by nifedipine in the dog circumflex coronary artery (Muller-Schweinitzer, 1983), and recently, Han et al. (1987a) have shown a,.-receptor-mediated responses to be abolished by

a),

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organic calcium channel antagonists. Thus, vascular a receptors appear to correspond in their effector system to presyriaptic a 1receptors present at the phrenic nerve. A further homology is evident with respect to recepreceptor reserve. High concentrations of phenylephrine or a-meth$noradrenaline (10 and 30 were required to produce a facilitatory effect; that is, both agonists showed low potencies at the motor nerve terminals. Likewise, the contractile response of agonists with low potency is inhibited by organic calcium channel antagonists (Ruffolo et al., 1984). However, the existence of a receptor reserve the possible activation o f multiple second messenger pathways with the consequence of mobilization of intraneuronal calcium may mask the influx of calcium through the opening of calcium channels; naturally, under these conditions calcium channel antagonists turn out to be ineffective. G-proteins have recently been shown to couple directly to potassium and calcium channels (Brown and Birnbaunier, 1988). 'The L-type calcium channel controlled by presynaptic a l receptors at the motor nerve can be opened either directly or indirectly by a second messenger (phosphoinositides, diacylglycerol, cyciic nucleotides). The mechanisms behind @,-receptor stimulation are discussed controversially; a,-receptor agoriists have been shown to open potassium channels (Nakamura et ul., 1981; Williams and North. 1Y85), t o inhibit \~oltage-controlledcalcium channels (Horn and Mci\fee, 1980; Canfield arid Dunlap, 1984),to inhibit adenylate cyclase (Jakobs at d., 1984; Fillenz and Bloomfield, 1986; Schoffelmeer et al., 1986), arid to interact with stimulus-secretion coupling behind the calcium influx (Mulder et al., 1984; Mulder and Schoffelmeer, 198.5; for detailed discussion see Starke, 1987; Starke et al., 1989). Balancing the controversial views and results, Starke and colleagues ( 1989) felt that the majority of experimental evidence supports the concept that stimulation of terminal a p receptors coupled to Gproteins inhibits voltage-sensitive calcium channels. i n contrast, the presynaptic a l receptors at the motor nerve appear to be coupled positively to L-rype calcium channels (U'essler at ui., 1990a).

w)

4. Conipai-ison r i d h Other Ticsues Kelease-regulating a receptors are widespread within neuronal and nonneuronal tissue, and it is beyond the scope of this review to discuss all the dif'erenl transmitters, hormones, and modulators controlled by areceptor stimulation. Otherwise, beside the well-known a2 autoreceptors controlling the release of catecholamines, possible effects of CI receptor stimulation o n the release of various compounds in various tissues (classic transmitters, opioids and other peptides, nonadrenergic noncholinergic

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excitatory o r inhibitory transmitters in the airways, hormones, kinins, autacoids, cytokines, lymphokines, etc.) must be considered. There are detailed review articles illuminating the presynaptic a2 autoreceptor (Vizi, 1979; see also Starke, 1987; Starke et al., 1989).The autoreceptor is an inhibitory receptor and belongs, with a few exceptions (Starke, 1987), to the az subtype. Generally, stimulation of presynaptic az receptors inhibits the release or liberation of neurotransmitters and hormones. There are, however, a few exceptions, showing a presynaptic facilitatory a receptor at cholinergic nerves. The a,-receptor agonist SK&F 89748 produced a small increase in the release of [3H]acetylcholine from the rat atria (McDonough et al., 1986),and recently Bognar et al. (1990) have reported of oxymetazoline enhances the stimuthat a high concentration (10 lated release of [ 14C]acetylcholinefrom parasympathetic cardiac nerves. This facilitatory effect was abolished by low concentrations of prazosin, indicating an a,-receptor subtype, a regulation that resembles the situation at motor endplates.

w)

C. p RECEPTORS 1. Overflow Studies Although cyclic AMP has long been shown to facilitate neuromuscular transmission (Breckenridge et al., 1967; Goldberg and Singer, 1969; Wilson, 1974) experiments presenting an enhanced neuromuscular transmission (anticurare effect) in response to p-receptor stimulation are lacking. Demonstration of such a facilitatory effect in contraction experiments is difficult, because p-receptor agonists produce effects at the muscle membrane (hyperpolarization) directed opposite to possible facilitatory, presynaptic effects (see Section V,A). Therefore, release experiments are required to investigate presynaptic effects of p-receptor agonists. Isoprenaline (Fig. 18) and noradrenaline enhanced the stimulated release of [3H]acetylcholine from rat phrenic nerve; the facilitatory effect, related to the concentrations of the respective agonists, was abolished by propranolol, atenolol, and CGP 207 12A (see Fig. 18; Wessler and Anschiitz, 1988; Wessler et al., 1990d). These data strongly support the existence of facilitatory p receptors at terminals of the phrenic nerve. The results with adrenaline and a-methylnoradrenaline have already been indicative of this conclusion; it should be kept in mind that any facilitatory effect of both compounds was prevented only by combined blockade of a and p receptors (Wessler et al., 1989). One discrepancy between the results of release and functional

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:i 5 2

" 1

Isoprenaline 0.1 pmol/I

;-" 3 0)

X

!!*

'i I

32

CPG 20712 A

I

1

2

4

0.1 pmolll

0.1 )rmol/I

0

10

20

Isoprenaline 0.1 pmol/ I

30 time (min)

20

50

60

FIG. 18. Effects of isoprenaline on the resting tritium efflux and on evoked (YHJacetylchoIinerelease. The experiments correspond to those shown in Fig. 15. Isoprerialirie produced a small increase in resting tritium efflux and a considerable increase in the stimulated [~H]acetylcholinerelease. The p,-selective antagonist CGP 207 12A prevented both actions of isoprenaline. (Values from Wessler et al., 1990d.)

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367

experiments is, however, evident. The facilitatory effect of noradrenaline on evoked [3H]acetylcholine was abolished by propranolol and atenolol, that is, was mediated exclusively by the stimulation of P receptors, whereas its facilitatory effect on twitches and endplate potentials is prevented by a-receptor antagonists only (see Section V,B,3). The presynaptic p receptors can be desensitized,that is, show a property typical of p receptors (Mukherjee and Lefkowitz, 1977; Levitzki, 1986). High agonist concentrations or prolonged exposure times lead to attenuation of the facilitatory effect, indicating receptor desensitization (Wessler and Anschiitz, 1988; Wessler et al., 1990d). Specifically, a 0.01 pJ4 concentration of isoprenaline preexposed for 30 min prevented any facilitatory effect of a 1O-fold higher isoprenaline concentration (Wessler et al., 1990d). Likewise, 1-100 nM isoprenaline has been reported to promote desensitizationat the adenylate cyclase of erythrocytes (Sibley et al., 1985). Desensitization (loss of the response) of P receptors differs from that of nicotine receptors with respect to the time scale: nicotine receptors desensitize within seconds or even more rapidly, whereas desensitization of p receptors localized at the phrenic nerve is considerably slower (roughly 30 min). This difference can be attributed to differences in the underlying mechanisms. Nicotine receptor desensitization is associated with a conformational change in the receptor-ion channel complex, naturally a rapid event; desensitization of f3 receptors is caused by uncoupling of the receptor-effector system (adenylate cyclase) and by sequestration of the receptors. 2 . Functional Studies As already outlined, a facilitatory effect of P-receptor agonists on neuromuscular transmission has, so far, not been demonstrated in functional experiments; however, many observations obtained in electrophysiological and contraction experiments support the existence of facilitatory P receptors at motor nerve endings. Generally, conditions that increase the intracellular formation of cyclic AMP facilitate neuromuscular transmission by a presynaptic site of action. Various inhibitors of the enzyme phosphodiesterase [caffeine, theophylline, isobutylmethylxanthine (IBMX)] and dibutyryl-cyclic AMP or 8-bromocyclic AMP enhance the frequency of mEPPs, the quanta1 content of evoked endplate potentials and the tension of indirectly stimulated hemidiaphragm (Goldberg and Singer, 1969; Miyamoto and Breckenridge, 1972; Wilson, 1974; Kentera and VaragiC, 1975; Hattori and Maehashi, 1987; Dryden et al., 1988; see also Bowman, 1990).These observations led Wilson (1974) to suppose “that CAMP is involved

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regulating metabolic activity in the nerve terminal associated with synthesis, mobilization and storage of acetylcholine,” a suggestion in excellent agreement with the above-described facilitatory effect of p-receptor agonists on evoked [JH]acetylcholine release. Ribeiro and Sebastiao ( 1 M i ) , however, attributed the facilitatory effect of theophylline to its antagonistic activity at adenosine receptors. Additional negative findings have been published that argue against cyclic AMP exerting a facilitatory modulation on acetylcholine release from the motor nerve. T h e selective inhibitor of cAklP-specific phosphodiesterase, RO 20- 1724, did not affect quanta1 release from the mouse phrenic nerve (Chiou and Chang, 1988), and MDL 12,39014,an adenylate cyclase inhibitor, produced facilitatorv, presynaptic and inhibitory, postsynaptic effects at frog motor endplates (Silinsky and Vogel, 1986). IBMX, a potent phosphodiesterase inhibitor, showed inhibitory presynaptic and postsynaptic effects at the frog neuromuscular synapse (Ribeiro and Sebastiao, 1987). In addition, cyclic AMP has been proposed to stimulate the sarcoplasmic reticulum calcium pump, an effect that enhances contractility through a postsynaptic site of action (Gonzales-Serratos Pt ul., 1981; Arreola ut al., 1987). Nevertheless, overwhelming evidence favors a presynaptic facilitatory effect of cyclic ,4MP at the motor nerve. In addition, stimulation o f protein kinase C that is activated by elevated cyclic AMP (Reuter, 1983) or by the phosphoinositol pathway causes a considerable increase in transmitter release from the motor nerve (Haimann ~t al., 1987; Murphy and Smith, 1987; Shapira rt ul., 1987; Caratsch P t al., 1988).

3 . Rerep for Chu,vnrtPt-izatioti atid Sicgrid Transduction The facilitatory effect of both noradrenaline and isoprenaline on evoked [3HH]acetylcholinerelease from the phrenic nerve was prevented by 0.1 p m CCP 20712A (U‘essler ef a/., 1990d). CCP 20712A is a preceptor antagonist highly discriminating between p and @,-receptor receptors at subtypes (by a factor of lO,O00), and specifically blocks 0.1 p M or lower concentrations (Dooley ef ul., 1986). Moreover, a 0.3 pM atenolol prevented the facilitatory effect of both amines (Wessler and Anschutz, 1988), and fenoterol, a preferentially p,-receptor agonist, did not affect the release of [3HH]acetylcholine(Wessler P t al., 1990d). All these experimental data convincingly propose the existence of a presynaptic p, -receptor subtype that mediates the enhanced transmitter release from the motor nerve. Therefore, the presynaptic receptors (p subtype) differ from postsynaptic receptors ( p p subtype); the latter receptors are embedded in the muscular membrane and their stimulation affects skeletal muscle activity and contractility (Bowman, 1981 ; Elfellah and Reid, 1987; see also Section V,A).

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T h e P ,-receptor-mediated increase in evoked transmitter release can be abolished by considerably low concentrations of the N-type calcium channel antagonist o-conotoxin GVIA (picomolar to nanomolar), whereas the h y p e calcium channel antagonist nifedipine, up to a concentration of 1 pM, does not reduce the facilitatory effect of noradrenaline (Wessler et al., 1990a). Thus, these presynaptic p1 receptors are coupled to N-type calcium channels; opening them causes an influx of calcium and, as a consequence, enhanced transmitter release. Again, it is not known whether the p1 receptors are coupled directly or indirectly (via cyclic AMP) to the calcium channels. An indirect coupling via cyclic AMP would imply that the second messenger is highly restricted to intraneuronal compartments or that receptor-specific isoenzymes exist. Also, P-adrenergic stimulation of the heart muscle cells results in an opening of calcium channels either directly or indirectly; formation of cyclic AMP, induced by p ,-receptor stimulation, activates the catalytic subunit (C subunit) of the protein kinase, and the C subunit mediates phosphorylation of a protein close to or within the calcium channel (Tsien, 1977; Reuter, 1983; Kameyama et al., 1985).

4. Comparison with Other Tissues

To the the author’s knowledge, the presynaptic facilitatory P1 receptors at the phrenic nerve terminal appear to represent the first example of non innervated P1 receptors; the phrenic nerve receives no direct innervation by the sympathetic nervous system. Noradrenergic terminals are generally regarded to be endowed with facilitatory P receptors (Adler-Graschinsky and Langer, 1975; Stjarne and Brundin, 1975; Dahlof et al., 1978; Majewski et al., 1980; Schmidt et al., 1984; for references see Starke, 1977; Langer, 1981). The release of both radioactive and endogenous noradrenaline from different tissues (cardiac and vascular tissue) is enhanced by P-receptor agonists under in vivo and in vitro conditions. More recent studies have reported that noradrenaline released from the pulmonary arteries and from the blood-perfused gracilis muscle in situ is enhanced by p-receptor agonists (Misu et al., 1984; Dahlof et al., 1987; Kahan and Hjemdahl, 1987; Nedergaard, 1987). These presynaptic p receptors are characterized mainly as p2 receptors. There is, however, some evidence for additional p1 receptors; for example, Goshima et al. (1985) have described both p1 and p2 receptors as to enhancing adrenaline release from hypothalamic slices. Under sensitive conditions (increased concentrations of circulating catecholamines) the release of both noradrenaline from noradrenergic neurons and acetylcholine from motoneurons may be enhanced by the stimulation of facilitatory P receptors; this excitatory pathway is not known to occur at

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parasympathetic, cholinergic neurons. T h e parasympathetic system is concerned mainly with long-lasting biological functions conserving energy and maintaining internal balance, whereas the sympathetic nervous system and the motoneurons are required for rapid reactions “to be prepared for fight or flight” (Goodman and Gilman, 1990).

D. PHYSIOLOGY As already outlined for nicotinic autofacilitation (see Section IV,A,5), facilitatory presynaptic mechanisms appear to dominate at the motor nerve terminal. These presynaptic amplifiers allow a rapid and threshold activation of skeletal muscles. It should, however, be noted that inhibitory receptors are also present at the motor nerve terminais; for example, inhibitory receptors for adenosine (Ginsborg and Hirst, 1972; Ribeiro and Walker, 1975; Sebastiao and Ribeiro, 1985) may control acetylcholine release, particularly under critical metabolic conditions. The facilitatory a, and p, receptors can be stimulated by circulating catecholamines only. It is generally known that conditions in which concentrations of circulating catecholamines increase (emotional disturbances, physical stress, pheochromocytoma, coldness, thyrotoxicosis) affect neuromuscular transmission (see Section V,A). Stimulation of the splanchic nerves, as well as application of catecholamines, has been shown to affect neuromuscular transmission and skeletal muscle contractility; it is reasonable to propose that the anticurare effect is mediated by the stimulation of both facilitatory adrenergic heteroreceptors. The tremorogenic action of circulating catecholamines is caused mainly by postsynaptic effects (see Section V,A), but an enhanced spontaneous and evoked transmitter release may contribute to this unwanted effect. As already mentioned, it seems possible that an increase in circulating adrenaline resulting from extreme conditions (top sports, life-threatening conditions) and administration of sympathomimetic amines in an emergency facilitate neuromuscular transmission through their presynaptic site of action, that is, by stimulation of facilitatory a, and p, receptors. In addition, release of noradrenaline from the abundantly innervated vascular tissue in skeletal muscles is facilitated by p, receptors, and an enhanced amount of noradrenaline flowing from the vascular tissue of skeletal muscles to the motor nerve terminals may contribute to the presynaptic stimulatory effect mediated by the sympathetic nervous system at motor endplates. Nevertheless, postsynaptic effects as well as the actions in the central nervous system that modify the presynaptic facilitatory effects must also be considered. i t might be fruitful to investigate the pharmacological properties of presynaptic a, and P I

37 1

PRESYNAPTIC RECEPTORS AT MOTOR NERVE TERMINALS

receptors at motor nerves in more detail. As these receptors differ in binding region from the corresponding receptors in other tissues, it might be a rational in the future to facilitate neuromuscular transmission by a selective action at presynaptic adrenergic receptors (e.g., in patients with myasthenia gravis).

Axon

Adrennline

Synapse

f\

CAMP---.

\

-Reserve pools

. Release zones

Muscle fiber

FIG. 19. Diagrammatic representation of presynaptic and postsynaptic receptors at the motor endplate of the phrenic nerve. Acetylcholine (ACh) released in response to an invading current ( 9 )from the active release zones produces multiple effects: (1) Stimulation of presynaptic autoreceptors [facilitatory nicotine receptors (A), inhibitory muscarine receptors (O), more proximally localized facilitatory muscarine receptors (m)]; (2) stimulation of postsynaptic nicotine receptors (A);(3) stimulation of preterminal nicotine receptors ( ) with partially inactivated acetylcholinesterase to increase the diffusion radius for acetylcholine. Stimulation of presynaptic nicotine receptors may turn on active release zones, or enhance acetylcholine release per individual release zone, or facilitate mobilization of acetylcholine from reserve pools; stimulation of the preterminal nicotine receptors mediates repetitive discharges (backfiring, 9).Sympathomimetic amines produce multiple presynaptic and postsynaptic effects: (1) stimulation of facilitatory presynaptic, P I receptors (0)mediating the opening of N-type calcium channels (N); (2) stimulation of facilitatory a lreceptors ( 0 )mediating the opening of Llike calcium channels (L); (3) stimulation of postsynaptic p2 receptors ( mediating I an enhanced formation of cyclic AMP and hyperpolarization [increased activity of the sodium/potassium pump, increased opening of calcium-dependent potassium channels (Kc=)].Cyclic AMP activates protein kinase (PK), which mediates phosphorylation of calcium channel proteins with the consequence of enhanced calcium influx. In addition, myosin is phosphorylated by activated myosin light chain kinase (MLCK), which can modify contractility.

+

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VI. Conclusion

Neuromuscular transmission has turned out to be highly coniplicated, but the basic events are still “simple” steps that together allow neuromuscular transmission to occur in skeletal muscles with high safety and efficacy (Fig. 19): acetylcholine is released in response to an invading current frotn some active release zones; acetylcholine facilitates its own release by stimulation of presynaptic nicotine receptors (increased number of activated release zones or of released vesicles per individual zone); autofacilitation is cut short by muscarinic autoinhibition and nicotine receptor desensitization; increased concentrations of circulating catecholamines can facilitate acetylcholine release by stimulation of presynaptic a 1 and PI receptors; acetylcholine diffuses within the synaptic cleft and interacts fleetingly with acetylcholinesterase and postsynaptic nicotine receptors that differ from presynaptic autoreceptors. I t is, of course, fascinating to follow the functional organization of this highly effectively operating synapse, a so-called “simple synapse,” that transmits the activity of motoneurons and intended activity to skeletal muscles. I n contrast, communication between neurons in the central nervous system will hardly be cleared up because of the incredible possibilities for presynaptic and postsynaptic modulation.

Acknowledgments

I thank Professors 8.Bowman (Glasgow, Scotland) and L. Cutmann (Morgantown, West Virginia) for their critical reading of the manuscript and their helpful suggestions. Drawing of the figures by Ms. D. Wolf and Ms. B. Hering is gratefully acknowledged. Work from m y laboratory is supported by grants from the Deutsche Forschungsgerneinscliaft, Forschungsrat Rauchen und Gesundheit, Germany, and the MNFZ of the University ot‘ Mainz.

References

Abbs, E. T., and Joseph, D. N. (1981). Br. J . Phnimacol. 73, 481-483. AdamiC, 5. (1972). Biorhem. Phanacol. 21, 2925-2929. Adler-Graschinsky, E., and Langer, S. Z. (1975). Br. J . Phalmacol. 53, 43-50, Alberts, P. T., Bartfai, T., and Stjarne, L. (1982).J Physiol. ( L o i d ~ n 329, ) 93-1 12. Alves-do-Prado, W., Corrado, A. P., and Prado, W. A. (1987).Aiiesth. Annlg. (ClPzirlniid) 66, 492-496.

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INDEX

A Acetyl-coenzyme A, acetylcholine at motor nerves and, 286-287 Acetylcholine glial cells in activity-dependent plasticity and, 264 neurotrophic factors and, 2, 6, 11, 17 vertebrate nervous system development and, 144, 159, 179 Acetylcholineat motor nerves, 283-286,372 adrenergic receptors physiology, 370-371 a-receptors, 355-365 @-receptors,365-370 sympathomimetic amines, 354-355 autoreceptors, 34 1-343 detection methods electrophysiology, 303-305 overflow studies, 305-3 12 events, 286 hydrolysis, 301-303 release, 295-301 storage, 291-29.5 synthesis, 286-291 presynaptic muscarine receptors, 344354 presynaptic nicotine receptors characterization, 328-332 desensitization, 332-337 function, 322-328 overflow studies, 312-322 physiology, 337-339 tissue comparisons, 339-341 preterminal-axonal nicotine receptors, 341-343 Acetylcholine receptor-inducing activity (ARIA), 93 Acetylcholine receptors, nicotinic, see Nicotinic acetylcholine receptors Acetylcholinesterase acetylcholine at motor nerves and, 285, 372

autoreceptors, 312-320, 322 detection methods, 305-306, 308 events, 295, 302-303 muscarine receptors, 349-350, 352 presynaptic nicotine receptors, 327, 329, 333-334, 338 preterminal nicotine receptors, 341, 343 nicotinic acetylcholine receptors and, 87, 108-110 ACTH, neurotrophic factors and, 10-12, 16 Actin, vertebrate nervous system development and, 176, 183-185 Actin-binding proteins, vertebrate nervous system development and, 183184 Activation acetylcholine at motor nerves and, 364, 372 autoreceptors, 3 15, 324, 338 events, 299 muscarine receptors, 346-347, 349, 351-352 coincident, 186-190 glial cells in activity-dependent plasticity and, 218, 221-222, 230,267 hypothesis for involvement, 260, 262, 264 participation, 240, 249, 254 nicotinic acetylcholine receptors and, 71, 78, 94 nonselective, 140-145, 149 vertebrate nervous system development and mechanisms, 165-166, 170, 174, 177, 179 mechanisms of plasticity, 194 properties, 139, 150-157, 159 synaptic plasticity, 189- 190 Activity-dependent development of vertebrate nervous system, 133-135 afferent activity, 139- 150

385

386

INDEX

calcium. 164-166. 176-178 calniodulin, 171-173 inositol triphosphate, 175- 176 NMDA, 166-171 protein kinase C, 173- 175 critical period, 157-161 genetic specifity, 135-139 plasticity, mechanisms of, 191-199 postsynaptic activity, 150- 157 regulatory genes, 178- 179 structural changes, 179- 186 synaptic plasticity, 186- 191 trophic factors, 161-164 .Activity-dependent neurotrophic factor (ADNF), vertebrate nervous system development and, 163 Xctivity-dependent plasticity, glial cells in, we Glial cells in activity-dependent plasticity Aderiylate cyclase acetylcholine at motor nerves and adrenergic receptors, 364. 367-368 autoreceptors, 332 glial cells in activity-dependent plasticity and, 264 neurotrophic factors and, 6, 10- 13 vertehrate nervous system development and. 1 7 3 Adhesion, glial cells in activity-dependent plasticity md, 237-238, 248, 256 Adrenal cholinergic receptors, nicotinic acetylcholine, 101- 102 Adrenal medullary chromaffin cells, nicotinic ac-etylrholine receptors and, 100- 10'2

Adrenaline, acetylcholine at niotor nerves and, 357-361, 370 Adrenerpc receptors, acetylcholine at motor nerves and, 285,309,34 1,354-371 aniines, 354-3.5.5 physiology, 370-37 1 a-receptors. 355-36.5 f3-receptors, 365-370 a,-Adrenergic receptors, neurotrophic tartors and, 4 6-Adrenergic receptors, neurotrophic factors and, 13, 15 Afferents activity-dependenrplasticity and, 260-26 1 glial cells in activity-dependent plasticity and, 217-219

participation, 235, 237, 240, 242, 246 synapse formation, 249, 251-252 synaptic efficacy, 224-229, 231-232 neurotrophic factors and, 19 vertebrate nervous system development and mechanisms, 167, 169 mechanisms of plasticity, 193-194, 196 postsynaptic activity, 151, 154- 156 properties, 136-137, 139-150 synaptic plasticity, 186-187, 189. 191 Agrin, nicotinic acetylcholine receptors and, 87,93 Alzheimer's disease acetylcholine at motor nerves and, 340 nicotinic acetylcholine receptors and, 70, 109, 1 1 1 Aniines acetylcholine at motor nerves and, 285286 adrenergic receptors, 354-355, 358, 360-361, 368, 370 nicotinic acetylcholine receptors and, 97-98 Amino acids, see also Excitatory amino acids activity-dependent plasticity and, 254255 nicotinic acetylcholine receptors and diversity, 47, 50, 56-58, 60, 62, 69 ganglia, 98 regulation, 80 seminal concepts, 28-29 vertebrate nervous system development and, 158-159, 162, 165, 175 7-Aniinobutyric acid (GABA) acetylcholine at motor nerves and, 284 glial cells in activity-dependent plasticity and, 222 neurotrophic factors and, 6, 18 nicotinic acetylcholine receptors and, 28-29 vertebrate nervous system development and, 1.50, 170 Aminophosphnovaleric acid neurotrophic factors and, 4-5 vertebrate nervous system development and, 151, 157, 167-169, 192-194 AMPA, vertebrate nervous system development and, 171 Antibodies activity-dependent plasticity and, 254, 256

INDEX

monoclonal, see Monoclonal antibodies neurotrophic factors and, 11, 13 nicotinic acetylcholine receptors and function, 44-45, 68-69 ganglia, 95, 99-100 regulation, 80, 93-94 structure, 47, 50, 54-56 vertebrate nervous system development and, 185 Antigens, nicotinic acetylcholine receptors and, 45, 50, 55-56, 70, 99 Aplysia acetylcholine at motor nerves and, 340 glial cells in activity-dependent plasticity and, 233, 249, 254 Arachidonic acid glial cells in activity-dependent plasticity and, 222-223,231, 253-254, 258 vertebrate nervous system development and, 162-165, 173 Areal patterns, glial cells in activitydependent plasticity and, 224-227, 234 Arginine vasopressin (AVP), neurotrophic factors and, 8-9, 15 Astrocytes glial cells in activity-dependent plasticity and, 216, 221,223 CNS damage, 257 hypothesis for involvement, 261-262, 264-267 participation, 235, 238-243, 246-248 structural changes, 254 synapse formation, 252 neurotrophic factors and, 4, 9-10, 1217,19 vertebrate nervous system development and, 162 Astroglial cells, activity-dependent plasticity and hypothesis for involvement, 262, 265 participation, 240, 243, 245-246 synapse formation, 252 synaptic efficacy, 257 ATP acetylcholine at motor nerves and, 293, 358 nicotinic acetylcholine receptors and, 97-98 vertebrate nervous system development and, 183

387

ATPase acetylcholine at motor nerves and, 287 vertebrate nervous system development and, 173, 177 Atropine acetylcholine at motor nerves and, 346, 348-353 neurotrophic factors and, 6 nicotinic acetylcholine receptors and, 38 Autonomic nervous system acetylcholine at motor nerves and, 284, 296,300-301,344 nicotinic acetylcholine receptors and, 51, 63, 99 Autoreceptors acetylcholine at motor nerves and, 284286 adrenergic receptors, 364-365 characterization, 328-332 desensitization, 332-337 detection methods, 303, 312 events, 301-303 function, 322-328 overflow studies, 3 12-322 physiology, 337-339 presynaptic muscarine receptors, 344-354 preterminal nicotine receptors, 34 1343 tissue comparison, 339-34 1 nicotinic acetylcholine receptors and, 109 Axons acetylcholine at motor nerves and autoreceptors, 3 14, 329, 340-343, 348, 352 detection methods, 305 events, 286, 293 glial cells in activity-dependent plasticity and, 265, 267 CNS damage, 256-257 participation, 238-241, 245-246 synapse formation, 249 synaptic efficacy, 225, 227, 232, 234 neurotrophic factors and, 3, 5, 7, 18 vertebrate nervous system development and afferent activity, 140-142, 146-149 mechanisms, 164-165, 167, 171, 173 plasticity, 189, 192, 194, 196, 198 properties, 137, 157, 160-161 structural changes, 182-183

388

INDEX

Axotomy, nicotinic acetylcholine receptors and. 96-97

B Backfiring, acetylcholine at motor nerves and, 341, 343 Behavior, nicotinic acetylcholine receptors and, 69-72, 11 1 Hiogenic amines, nicotinic acetylcholine receptors and, 97-98 a-Bungarotoxin acetylcholine at motor nerves and autoreceptors, 329-331, 337, 342 overflow studies, 312, 319-320, 324 nicotinic acetylcholine receptors and behavior, 72 central neurons, 103, 105, 11 1 expression sites, 69 function, 39, 43-44 functional expression, 66-67 ganglia, 95-96, 99, 101 models, 75 nBgtS gene, 61 nomenclature, 31-33 regulation, 85 structure, 47, 50-53, 56 K-Bungarotoxin acetylcholine at motor nerves and, 339 nicotinic acetylcholine receptors and diversity, 33, 43, 53-54 ganglia, 95-96, 99, 101 nBgtS gene, 65-67

C CAI region glial cells in activity-dependent plasticity and, 230, 249 vertebrate nervous system development and, 144, 156, 165-167, 174 CA3 region, vertebrate nervous system development and, 168, 175-176 Calcitonin gene-related peptide neurotrophic factors and, 11, 19 nicotinic acetylcholine receptors and, 76,92-94,98 Calcium acetylcholine at motor nerves and

adrenergic receptors, 354, 358, 363364,369 autoreceptors, 325, 332, 341, 350, 352 detection methods, 306, 309-3 1 0 events, 289, 293, 295, 297-301 glial cells in activity-dependent plasticity and, 234 hypothesis for involvement, 262, 264-266 participation, 247-248 neurotrophic factors and, 4-5, 7, 17 nicotinic acetylcholine receptors and, 28, 76 ganglia, 98, 101 regulation, 85-86, 89, 91-94 vertebrate nervous system development and, 134 mechanisms, 162- 178 mechanisms of plasticity, 193-1 94, 198-199 properties, 142, 154, 158-159 structural changes, 182-185 Calmodulin acetylcholine at motor nerves and, 300, 311 glial cells in activity-dependent plasticity and, 230 vertebrate nervous system development and, 165, 171-173, 185, 199 Calmodulin kinase type 11, vertebrate nervous system development and, 17 1172, 184 Capsaicin, neurotrophic factors and, 9 Carbachol acetylcholine at motor nerves and, 31.5, 353 neurotrophic factors and, 4 vertebrate nervous system developnierlt and, 176 Catecholaminergic neurons, vertebrate nervous system development and, 163, 198 Catecholamines acetylcholine at motor nerves and, 354, 358, 360, 364, 370, 372 neurotrophic factors and, 7 nicotinic acetylcholine receptors and, 97-98, 100-101

INDEX cDNA nicotinic acetylcholine receptors and, 29, 61-63,69, 80, 88 vertebrate nervous system development and, 186 Central nervous system acetylcholine at motor nerves and, 283, 372 autoreceptors, 330, 332, 339-340, 351-352 events, 287 activity-dependent plasticity in, see Glial cells in activity-dependent plasticity neurotrophic factors and, 6-7, 15-19 nicotinic acetylcholine receptors and central neurons, 105, 108 expression sites, 68-69 function, 38-39, 43 models, 73 neuronal genes, 63 nomenclature, 3 1 regulation, 78 structure, 51-52, 54, 56 vertebrate nervous system development and, 139, 179-181 Central neuronal nicotinic acetylcholine receptors, 103-1 12 expression sites, 69 function, 34, 36, 38-42 functional expression, 68 models, 75 nomenclature, 32-33 structure, 51-53, 55 Cerebellar granule cells, neurotrophic factors and, 5-6, 17, 19 Cerebellum acetylcholine at motor nerves and, 339340 glial cells in activity-dependent plasticity and, 233, 240-241, 243, 254, 265 neurotrophic factors and, 3, 8, 10-11, 13-17,19 nicotinic acetylcholine receptors and, 43, 75 vertebrate nervous system development and mechanisms, 164, 171 properties, 138, 157 synaptic plasticity, 186-187, 191

389

Chloinesterase, acetylcholine at motor nerves and, 306, 314 Cholecystokinin, neurotrophic factors and, 7-8, 15 Choline acetylcholine at motor nerves and, 284 adrenergic receptors, 358 detection methods, 304, 306, 308, 31 1 events, 287-291,301 nicotinic acetylcholine receptors and, 110 Choline acetyltransferase, acetylcholine at motor nerves and, 286-288, 291 Cholinergic agonists, neurotrophic factors and, 4 Cholinergic neurons acetylcholine at motor nerves and, 285, 287, 339-340, 365, 370 nicotinic acetylcholine receptors and central neurons, 104, 108-110 diversity, 51-52, 72 ganglia, 97, 99-100 models, 74, 78 regulation, 86, 89-90, 93-94 vertebrate nervous system development and, 151, 163, 198 Cholinergic receptors, nicotinic acetylcholine, 26-27, 29, 32, 101-102 Cholinesterase, nicotinic acetylcholine receptors and, 109 Chromosomes, nicotinic acetylcholine receptors and, 60-61, 69 Ciliary ganglia nicotinic acetylcholine receptors and, 96,99 vertebrate nervous system development and, 143 Circuitry, glial cells in activity-dependent plasticity and, 267 CNS damage, 257 participation, 253 synaptic efficacy, 224, 228, 233-234 Clones nicotinic acetylcholine receptors and central neurons, 108 diversity, 36, 61-67 models, 74-75 regulation, 80-81, 90-91 seminal concepts, 28-29, 3 1

390

INDEX

vertebrate nervous system development and, 161 Cobratoxin, acetylcholine at motor nerves and. 313, 329 Coincident activation. vertebrate nervous system development and. 186-190 (hditioning acetylcholine at niotor nerves and, 324 activity-dependent plasticity and, 249 vertebrate nervous system development and, 175 Conductance, nicotinic acetylcholine receptors and, 95-96 Chinectivity, glial cells in activitydependent plasticity and, 216 participation, 253 svnaptic efficacy, 224, 226-227, 229, 232 o-Conotoxin GVIA. acetylcholine at niotor nerves and, 300-301, 363, 369 Cortex acetvlcholine at motor nerves and, 300, 331, 339-340, 352 glial cells in activity-dependent plasticit! and hypothesis for involvement, 262 participation, 235, 24 1-243, 245 synaptic efficacy, 224. 227-228 neurotrophic factors and, 7-8, 10-1 1, 13-14, 18 nicotinic acetylcholine receptors and, 68, 104, 107. 110 vertebrate nervous system development and afferent acii\ity, 146. 148 mechanisms. 167. 180 properties, 136, 150, 153, 157, 159 <;rosslinking nicotinic acetylcholine receptors and, 46-47.94 vertebrate nervous system development and. I83 Curariniinietic neurotoxins, nicotinic acetylcholine receptors and, 39, 43, 4546. 50-51 Cvclic .4MP acetylcholine at motor nerves and, 322. Sf23, 36.5, 367-369 neurotrophic factors and, 1 1, 14- 15 nicotinic acetylcholine receptors and ganglia, 95, 98-103

models, 76 regulation, 81, 89-92, 94 vertebrate nervous systeni development and, 173 Cyclic GMP glial cells in activity-dependent plasticity and. 265 vertebrate nervous system development and, 164, 173 Cysteine, nicotinic acetylcholine receptors and, 29 diversity, 61-62 muscle genes, 57-58, 60 structure, 46-47 Cytoplasm acetylcholine at motor nerves and detection methods, 309-310 events, 286-287, 291, 293, 295, 297 activity-dependent plasticity and, 245 nicotinic acetylcholine receptors and, 28-29, 34, 83 vertebrate nervous system development and, mechanisms, 183 Cytoskeleton activity-dependent plasticity and, 242, 266 nicotinic acetylcholine receptors and, 78,86-88,94 vertebrate nervous system development and mechanisms. 173, 176, 181-185 plasticity, 19 1

D Deca niet hon i u ni acetylcholine at niotor nerves and, 336 nicotinic acetylcholine receptors and, 30-31, 33, 38, 66 Dendrites glial cells in activity-dependent plasticity and, 231,234-235, 241 neurotrophic factors and, 6, 1 1 nicotinic acetylcholine receptors and, 73, 78 vertebrate nervous system development and afferent activity, 145 mechanisms, 164-165, 171, 180-184 mechanisms of plasticity, 192 synaptic plasticity, 186

INDEX

Denervation acetylcholine at motor nerves and, 289, 291, 299, 306 nicotinic acetylcholine receptors and central neurons, 105 ganglia, 96-97, 100 regulation, 80-86, 92 vertebrate nervous system development and, 179 Depolarization acetylcholine at motor nerves and autoreceptors, 3 13-3 15, 319-320, 322,326, 331, 336-337 detection methods, 303-304 events, 297, 299-300 muscarine receptors, 348-350, 352 preterminal receptors, 341, 343 glial cells in activity-dependent plasticity and, 22 1,232 hypothesis for involvement, 262 participation, 249, 25 1, 255 neurotrophic factors and, 11 nicotinic acetylcholine receptors and, 30, 71, 101, 108 vertebrate nervous system development and calcium, 166, 168-169, 171, 173, 175 mechanisms, 162-163 properties, 153-156, 158-159 structural changes, 180, 182, 185 Desensitization acetylcholine at motor nerves and, 302, 308,332-337, 339, 367,372 autoreceptors, 325-327, 330, 332 overflow studies, 313-314, 317-318, 320-322 nicotinic acetylcholine receptors and, 27 central neurons, 107-1 11 diversity, 35, 37-38, 49, 52, 67, 70 ganglia, 98- 102 regulation, 89-91 Diacylglycerol glial cells in activity-dependent plasticity and, 264 vertebrate nervous system development and, 165, 170, 174-175, 183 Differential activation, vertebrate nervous system development and, 139, 187 Differentiated phenotype, neurotrophic factors and, 6-7, 11-12

391

Differentiation glial cells in activity-dependent plasticity and, 237-238,262 vertebrate nervous system development and, 174, I90 DMPP, acetylcholine at motor nerves and, 320, 322 DNA glial cells in activity-dependent plasticity and, 240 neurotrophic factors and, 4, 9-10 nicotinic acetylcholine receptors and, 57, 61, 69, 75 Dopamine acetylcholine at motor nerves and, 284, 330, 340, 353 neurotrophic factors and, 6, 11, 19 nicotinic acetylcholine receptors and, 107 Dorsal root ganglion neurons glial cells in activity-dependent plasticity and, 232 vertebrate nervous system development and, 142, 146, 151, 167-169 Drug treatment, nicotinic acetylcholine receptors and, 75-76 Dual-receptor hypothesis, vertebrate nervous system development and, 170

E Effector systems, acetylcholine at motor nerves and, 286,300,303,329,332 Electrical activity acetylcholine at motor nerves and, 289 neurotrophic factors and, 19 vertebrate nervous system development and, 133 afferent activity, 140-145, 147-148 mechanisms, 161-162, 164, 166, 168, 179 mechanisms of plasticity, 192-193, 199 properties, 134-135, 137, 157 synaptic plasticity, 186, 188 Electrical nerve stimulation, acetylcholine at motor nerves and autoreceptors, 314, 331, 336, 346, 348 detection methods, 306, 309-3 11 Electroph ysiology

392

INDEX

acetylcholine at motor nerves and, 285Epidermal growth factor, activity286 dependent plasticity and, 252-254 adrenergic receptors, 360-361. 367 Epithelial cells, neurotrophic factors and. autoreceptors, 323, 331, 349 9, 11 detection methods, 303-305, 3 19 Epitopes, nicotinic acetylcholine receptors glial cells in activity-dependent plasticity and, 45, 50, 94 and, 224 Erabutoxin-b, acetylcholine at motor nicotinic acetylcholine receptors and, nerves and, 313, 323-324, 329 26, 33, 35, 37, 49, 95, 97 Ethanol, nicotinic acetylcholine receptors Vertebrate nervous system development and, 1 1 1 and, 148-149, 154, 199 Excitation Embryos acetylcholine at motor nerves and, 3 1 1, glial cells in activity-dependent plasticity 369 and, 238 neurotrophic factors and, 4, 12. 18 neurotrophic factors and, 7-9 nicotinic acetylcholine receptors and, nicotinic acetylcholine receptors and, 29, 70 95, 105 vertebrate nervous system development models, 73-74 and regulation, 82, 85, 87 mechanisms, 166, 168 Endplate potential, see alro Miniature plasticity, I92 endplate potentials properties, 15 1, 155, 157- 158 acetylcholine at motor nerves and Excitatory amino acids adrenergic receptors, 367 glial cells in activity-dependent plasticity autoreceptors, 3 19, 323-324, 327and, 255 328, 341,343 neurotrophic factors and, 2, 11 detection methods, 303-305 nicotinic acetylcholinereceptors and, 29 events, 295-296, 298-299, 301 vertebrate nervous system development muscarine receptors, 344, 349-35 1 and, 162, 165-166, 170. 175 Enkephalin Excitatory postsynaptic potentials glial cells in activity-dependent plasticity glial cells in activity-dependent plasticity and, 242 and, 253 neurotrophic factors and, 14, 16 vertebrate nervous system and Environmental toxins, nicotinic acetylchomechanisms, 168-169, 180 line receptors and, I 1 1-1 12 mechanisms of plasticity, 193 E n ~ mes y properties, 146, 154, 156. 158 acetylcholine at motor nerves and, 285 Exocytosis, acetylcholine at motor nerves adrenergic receptors, 367 and, 297-298 autoreceptors, 313-315,317,319,322 Exons, nicotinic acetylcholine receptors detection methods, 305-306, 308 and, 60-62, 69 events, 287, 299. 302 Extracellular matrix muscarine receptors, 349 activity-dependentplasticity and, 248-249 presynaptic nicotine receptors, 327, nicotinic acetylcholine receptors and, 329, 333-334, 338 73, 86-87, 94 preterminal nicotine receptors, 341, vertebrate nervous system development 343 and, 183 nicotinic acetylcholinereceptors and, 28 vertebrate nervous system development and F mechanisms, 164-165, 171, 173-174, 176 Fading, acetylcholine at motor nerves and, structural changes, 182, 185 323, 325-327

INDEX

Fibroblast growth factor activity-dependentplasticity and, 252-253 neurotrophic factors and, 18 Fibroblasts nicotinic acetylcholine receptors and, 59-60, 82, 90 vertebrate nervous system development and, 175, 182 Fluorescence, neurotrophic factors and, 7 Functional inactivation, nicotinic acetylcholine receptors and, 89, 108

G G proteins, see Guanine nucleotidebinding proteins Ganglia acetylcholine at motor nerves and, 287, 294, 330-331, 337, 354 nicotinic acetylcholine receptors and, 26 diversity, 66, 68-69, 71 function, 33-34, 36-39, 44-45 muscle genes, 61 nomenclature, 29-33 regulation, 87, 95-103 structure, 46, 51-56 vertebrate nervous system development and, 138-139, 149, 167, 180 Gangliosides, activity-dependent plasticity and, 248, 260-261 Gene expression nicotinic acetylcholine receptors and, 72-73, 79-80,85, 105 vertebrate nervous system development and, 184 Genes acetylcholine at motor nerves and, 330 vertebrate nervous system development and, 136, 164, 179 Genetic specificity, vertebrate nervous system development and, 133-139, 148 Genetics, nicotinic acetylcholine receptors and, 108, 112 central neurons, 80-8 1 expression sites, 68-69 functional expression, 63-68 models, 75-78 muscle genes, 57-61 neuronal genes, 61-63 Glial cells

393

neurotrophic factors and, 2-3, 9-10, 14-15, 17-19 vertebrate nervous system development and, 162, 171 Glial cells in activity-dependent plasticity, 215-216, 267-268 astrocytes, 265-267 CNS damage, 255-257 definition, 217-219 function, 219-224 mechanisms, 258-262 cellular, 262-265 participation intraareal topographies, 234-239 synapse elimination, 239-248 synapse formation, 249-253 synaptic efficacy, 253-255 synaptic efficacy, 224-234 Glial fibrillary acidic protein (GFAP) glial cells in activity-dependent plasticity and, 235, 242 neurotrophic factors and, 4, 9 Glioma cells, neurotrophic factors and, 14 Glutamate acetylcholine at motor nerves and, 341 glial cells in activity-dependent plasticity and, 222-223 hypothesis for involvement, 262, 264 participation, 247, 253-254 synaptic efficacy, 230-23 1 neurotrophic factors and, 4-7, 18 vertebrate nervous system development and mechanisms, 162, 164-166, 169, 171-172, 175 plasticity, 199 properties, 154, 157 Glycine glial cells in activity-dependent plasticity and, 223, 254 nicotinic acetylcholine receptors and, 28-29 Glycosylation, nicotinic acetylcholine receptors and diversity, 37, 49-50, 58, 60 models, 77 regulation, 80, 82 Granule cells, neurotrophic factors and, 4 Growth-associated protein 43, vertebrate nervous system development and, 158, 184-186

394

INDEX

Growth cones neurotrophic factors and, 2 vertebrate nervous system development and, 141-142, 144, 164, 184185 Growth factors glial cells in activity-dependent plasticity and hypothesis for involvement, 260, 262, 265-266 participation, 249, 252-253 synaptic efficacy, 23 1 iieurotrophic factors and, 3-4 (hanine nucleotide-binding proteins (G proteins) acetylcholine at motor nerves and, 363364 glial cells in activity-dependent plasticity and, 229, 231, 264 nicotinic acetylcholine receptors and, 28, 92, 99 vertebrate nervous system development and, 175, 185

H Helisomn neurotrophic factors and, 6, 11 vertebrate nervous system development and, 141 Heniicholinium-3, acetylcholine at motor nerves and, 301, 304, 306, 309, 318319 Heterogeneity acetylcholine at motor nerves and, 293, 309-3 10, 330 glial cells in activity-dependent plasticity and, 223, 238, 267 nicotinic acetylcholine receptors and, 112 behavior, 69-72 central neurons, 106 function, 39, 43 ganglia, 96 models, 77 muscle genes, 60-61 neuronal genes. 61 regulation, 80, 83 structure, 49-50, 52, 56

vertebrate nervous system development and, 134 Heteroreceptors, acetylcholine at motor nerves and, 285-286, 300 adrenergic receptors, 354-37 1 autoreceptors, 331, 353 detection methods, 3 12 Hexamethonium acetylcholine at motor nerves and, 312313,317, 323, 331, 340 nicotinic acetylcholine receptors and, 30-31, 33,38-39, 66 High-performance liquid chromatography, acetylcholine at motor nerves and, 305-306 Hippocampus glial cells in activity-dependent plasticity and, 219, 253-254, 258,264, 266 participation, 246-247 synapse formation, 249, 251-253 svnaptic efficacy, 229-230, 232-233 neurotrophic factors and, 3, 6, 8, 101 1 , 13-19 nicotinic acetylcholine receptors and central neurons, 104, 107, 110-1 1 1 diversity, 35, 39, 43, 67 vertebrate nervous system development and, 134 mechanisms, 162-163, 165-168, 170-172, 174-176, 179 plasticity, 192-193 properties, 144, 1.53-157, 159 structural changes, 180 Homology acetylcholine at motor nerves and, 364365 nicotinic acetylcholine receptors and, 61, 66, 69, 77, 81 vertebrate nervous system development and, 182 Hormones acetylcholine at motor nerves and, 364365 glial crlls in activity-dependent plasticity and, 241-242 neurotrophic factors and, 8 nicotinic acetylcholine receptors and, 77-78,98, 102-103 vertebrate nervous system development and, 175

INDEX

Hybridization in situ, see in situ hybridization neurotrophic factors and, 8 nicotinic acetylcholine receptors and, 37 vertebrate nervous system development and, 182 Hydrolysis, acetylcholine at motor nerves and, 301-304, 306, 363 5-Hydroxytryptamine, see Serotonin Hyoscyamine, acetylcholine at motor nerves and, 349-350 Hyperpolarization acetylcholine at motor nerves and, 354, 360 vertebrate nervous system development and, 151, 153, 155-156, 170, 174 Hypothalamus acetylcholine at motor nerves and, 340, 369 glial cells in activity-dependent plasticity and, 241-242,245 neurotrophic factors and, 9-10, 14-15 nicotinic acetylcholine receptors and, 71, 104, 111

I Irnmunochemistry nicotinic acetylcholine receptors and, 68-69,96 vertebrate nervous system development and, 171 Immunocytochemistry, glial cells and, 242 Immunohistochemistry neurotrophic factors and, 8, 14 nicotinic acetylcholine receptors and, 54,56 Immunoreactivity glial cells in activity-dependent plasticity and, 235 neurotrophic factors and, 7-8 in situ hybridization neurotrophic factors and, 8 nicotinic acetylcholine receptors and, 66-68, 80, 105 vertebrate nervous system development and, 163 Inhibition acetylcholine at motor nerves and, 372

395

adrenergic receptors, 355, 359-360, 364-365,368,370 autoreceptors, 312-320, 322 detection methods, 304-305, 308309, 31 1 events, 293, 295, 297, 300-303 presynaptic muscarine receptors, 344-346, 348-354 presynaptic nicotine receptors, 322324, 331, 333-334, 337-340 preterminal nicotine receptors, 343 glial cells and, 240, 246, 248, 254, 256 neurotrophic factors and, 4-6, 10 nicotinic acetylcholine receptors and behavior, 70 central neurons, 109-1 1 I expression sites, 69 function, 38, 43-45 ganglia, 99, 101-102 models, 76 regulation, 91, 94 structure, 47, 49-50, 53-54 vertebrate nervous system development and afferent activity, 140, 142, 144 mechanisms, 162, 164, 170, 172, 174, 176 plasticity, 187, 189, 199 properties, 150, 154, 159 structural changes, 182, 184-185 Innervation acetylcholine at motor nerves and, 284285, 289, 306, 339, 353, 370 glial cells in activity-dependent plasticity and, 225, 240, 243,245 neurotrophic factors and, 3, 5-7, 19 nicotinic acetylcholine receptors and, 74, 82, 92 vertebrate nervous system development and afferent activity, 140-141,144,146, 149 properties, 137- 138, 160- 161 synaptic plasticity, 186- 187 Inositol 1,4,5-trisphosphate acetylcholine at motor nerves and, 351 glial cells in activity-dependent plasticity and, 269 Inositol triphosphate, vertebrate nervous system development and mechanisms, 170, 173, 175-1 76

396

INDEX

structural changes, 183, 185 synaptic plasticity, 189 Integral membrance proteins, nicotinic acetylcholine receptors and, 28, 46, 49 Interconnectivity, glial cells and, 223, 225 Intermediate filaments, vertebrate nervous system development and, 182183 Intraareal topographies, glial cells and, 227-229,234-239 Ion channels acetylcholine at motor nerves and, 284, 296 adrenergic receptors, 367 autoreceptors, 325-326, 328, 33 1, 349 detection methods, 303-305 glial cells in activity-dependent plasticity and, 216. 221 nicotinic acetylcholine receptors and, 76, 98, 107 function, 34, 44 functional expression, 64-65, 67-68 seminal concepts, 28-29, 31, 72 structure, 48-50 vertebrate nervous system development and, 164, 173-174 Isoprenaline, acetylcholine at motor nerves and, 361, 365, 367-368

K Kainic acid, neurotrophic factors and, 4, 7, 18

L Lamina, vertebrate nervous system development and, 149 Laminin glial cells in activity-dependent plasticity and, 239 neurotrophic factors and, 1-2 Lateral geniculate nucleus glial cells in activity-dependent plasticity and, 225, 237

vertebrate nervous system development and, 137, 143, 149, 170 Learning, glial cells in activity-dependent plasticity and, 229 Lectins, glial cells in activity-dependent plasticity and, 235 Leu-enkephalin, neurotrophic factors and, 12 Ligands acetylcholine at motor nerves and, 330 nicotinic acetylcholine receptors and behavior, 69-70 central neurons, 107, 109 function, 34-35, 37, 44-45 functional expression, 64, 67 ganglia, 97, 99-102 muscle genes, 57, 59 neuronal genes, 61 -62 regulation, 80, 89-94 seminal concepts, 28-29, 31-32, 72 structure, 46-47, 49-51, 53-54, 56 vertebrate nervous system development and, 162, 164 Lipid, acetylcholine at motor nerves and, 290,295 Long-term depression glial cells in activity-dependent plasticity and, 233,265 vertebrate nervous system development and mechanisms, 168- 17 1 plasticity, 190. 194 properties, 155- 157 Long-term potentiatiation glial cells in activity-dependent plasticity and, 2 19 hypothesis for involvement, 258, 264 participation, 249, 25 1-254 synaptic efficacy, 229-234 vertebrate nervous system development and, 134 afferent activity, 144- 145 mechanisms, 163-1 72, 174, 176, 179 plasticity, 190-193 properties, 153-157 structural changes, 180, 185 Lymphocytes, nicotinic acetylcholine receptors and, 7 1, 94

INDEX

M

397

nicotinic acetylcholine receptors and, 102 Macrophages, glial cells and, 240, 252 Modulation, acetylcholine at motor nerves Mecamylamine, nicotinic acetylcholine reand, 372 ceptors and, 38-39, 66, 101 adrenergic receptors, 354-371 Memory presynaptic muscarine receptors, 344glial cells in activity-dependent plasticity 354 presynaptic nicotine receptors, 3 12-34 1 and, 229 preterminal nicotine receptors, 341-343 nicotinic acetylcholine receptors and, 70 Met-enkephalin, neurotrophic factors and, Monoclonal antibodies nicotinic acetylcholine receptors and, 9-10, 12, 16-17 55,99, 101 Metabotrophic receptors vertebrate nervous system development nicotinic acetylcholine, 28, 99, 107 and, 184 vertebrate nervous system development Monocular deprivation and, 164, 169 glial cells in activity-dependent plasticity N-Methyl-~-asparticacid (NMDA) and, 227 glial cells in activity-dependent plasticity vertebrate nervous system development and, 230, 232, 234,254-255 and afferent activity, 146-148 neurotrophic factors and, 4-5,7,10, 17 mechanisms, 167, 177 vertebrate nervous system development properties, 153-154, 158 and mechanisms, 162, 164-17 1, 176- 177, Morphine, neurotrophic factors and, 10, 179 12 Morphology plasticity, 190- 194, 198- 199 glial cells in activity-dependent plasticity properties, 151, 154, 156-159 and structural changes, 180 hypothesis for involvement, 262, 264, a-Methylnoradrenaline, acetylcholine at motor nerves and, 357, 362, 364 266 Microheterogeneity, nicotinic acetylcholine participation, 237-238, 242, 246-248 synaptic efficacy, 229, 231, 233-234, receptors and, 49-50, 60, 71 253 Microtubule-associated proteins, vertebrate nervous system development nicotinic acetylcholine receptors and, 75 vertebrate nervous system development and, 181-182 and, 145-146, 165, 179-181, 192 Migration, glial cells and, 220, 229, 235, Motor nerves, acetylcholine at, see Acetyl237-238, 257 choline at motor nerves Miniature endplate potentials (mEPPs), mRNA acetylcholine at motor nerves and glial cells in activity-dependent plasticity adrenergic receptors, 367 and, 252 autoreceptors, 315, 319, 323, 327-328, neurotrophic factors and, 8, 11-16, 18 343, 349-350 nicotinic acetylcholine receptors and detection methods, 305 central neurons, 105 events, 295-296, 298-299 diversity, 3 1-32, 57 Mitochondria regulation, 79-80, 82-83, 89, 91-93 acetylcholine at motor nerves and, 287 vertebrate nervous system development vertebrate nervous system development and, 177-178, 180 and, 172, 182, 184 Muscarine receptors Mitogens, neurotrophic factors and, 9 acetylcholine at motor nerves and, 285Mitosis neurotrophic factors and, 3-4, 9-10, 18 286, 372

3 98

INDEX

autoreceptors. 314, 340 detection methods. 303, 308, 31 1 events, 302 presvnaptic receptors, 344-354 neurotrophic factors and, 4-5 nicotinic acetslcholine receptors and, 28-29, 39, 97, 109 vertebrate nervous system developnient and, 162 Musciniole. vertebrate nervous system development and, 150, 196 hluscle acetylcholine at motor nerves and. 28328.5, 3 i 2 adrenergic receptors. 354, 359-361, 363, 365, 369-370 detection methods, 304-305 events. 286. 2X8-291,294.298-299. 302 muscarine receptors, 349 nicotine receptors, 3 13-314. 328330. 332-333, 337-339 preterminal receptors, 34 I , 343 glial cells in activity-dependent plasticity and, 245 nicotinic acetylcholine receptors and, 95, 108

behavior, 71-72 expression sites. 69 function. 33-35. 37-39, 45, 88-94 functional expression. 66-67 genes, 57-61 ntodels. 73-75 nomenclature, 29-3 1, 33 regulation. 78-88 seminal concepts. 73 structure, 45-46. 48-51, .54-56 vertehrate nervous system development and afferent activity. 140, 143-144, 147 mechanisms. 163 plasticity, 186, 196, 198 properties, 137-139, 151, 160161 $1 utagenesis, nicotinic acetylcholine receptors and, 5 7 , 60 Mutation, neurotrophic factors and, 15 Myasthenia gravis, nicotinic acetylcholine receptors and. 7 I , 91 Myelin. ghal cells and, 255-256

hfyoblasts, nicotinic acetylcholine receptors and, 74, 79 Myogenesis, nicotinic acetylcholine receptors and, 79, 88 Myotubes nicotinic acetylcholine receptors and, 74, 79, 83, 86-87, 91, 93 vertebrate nervous system development and, 143

N Naloxone. neurotrophic factors and, 9 10, 12 Naltrexone, neurotrophic factors and, 0I2 ,Vnrrine, nicotinic acetylcholine receptors and. 45 nBgtS, nicotinic acetylcholine receptors and behavior, 71 central neurons, 103-10.5, 107, 110-1 11 expression sites, 68-69 function, 44 functional expression, 67-68 ganglia, 95- 103 neuronal genes. 62 structure, 51-56 Neocortex glial cells in activity-dependent plasticity and, 233 vertebrate nervous system development and, 156, 159, 168, 171, 192 Neoplastic cells, nicotinic acetylcholine receptors and, 31, 74, 77, 100, 102-103 Neostigmine, acetylcholine at motor nerves and, 317, 322, 349 Neosurgatoxin, nicotinic acetylcholine receptors and, 44, 46, 66 Nerve growth factor glial cells in activity-dependent plasticity and, 239, 252 neurotrophic factors and, 1, 13 nicotinic acetylcholine receptors and, 102 vertebrate nervous system development and, 161, 163, 165, 182, 186 Neural crest, nicotinic acetylcholine receptors and

INDEX central neurons, 106 ganglia, 95, 100, 102-103 models, 74-75 Neural transmission, glial cells in activitydependent plasticity and, 229-233 Neural tubes, nicotinic acetylcholine receptors and, 73-75 N e ur it e s glial cells and, 237, 249, 252, 256 neurotrophic factors and, 2, 5-6, 1 1 , 18-19 nicotinic acetylcholine receptors and, 71 vertebrate nervous system development and, 141-142, 186, 198 Neuroblastoma cells nicotinic acetylcholine receptors and, 62, 68, 75, 100, 102 vertebrate nervous system development and, 186 Neuroblasts, neurotrophic factors and, 3, 18 Neurohypophysis, glial cells in activitydependent plasticity and, 242-243, 245, 265 Neuromodulators, glial cells in activitydependent plasticity and, 220, 222 Neuromuscular junction acetylcholine at motor nerves and autoreceptors, 3 19, 340-34 1, 349350 events, 288-289 glial cells in activity-dependent plasticity and, 245 nicotinic acetylcholine receptors and, 87, 94, 106 vertebrate nervous system development and, 134 mechanisms, 178, 180 mechanisms of plasticity, 194, 198 properties, 143, 150-151, 157, 160 synaptic plasticity, 187-188, 190191 Neuron survival, neurotrophic factors and, 4-5, 10 Neuronal nicotinic acetylcholine receptors expression sites, 69 function, 34-35, 37-38, 40-42 functional expression, 68 ganglia, 96, 100

399

models, 75 nomenclature, 31-33 structure, 51-52, 55-56 Neuronal sprouting, neurotrophic factors and, 5-6, 11 Neuropeptides, neurotrophic factors and, 2, 7-12, 14-17 Neurophysin, neurotrophic factors and, 9 Neurotoxins neurotrophic factors and, 18-19 nicotinic acetylcholine receptors and function, 39-44 functional expression, 67 nomenclature, 32-33 structure, 45-46, 49-5 1, 53-54 K-Neurotoxins, nicotinic acetylcholine receptors and, 53-55 Neurotransmitters acetylcholine at motor nerves and, 283, 286, 363, 365 glial cells in activity-dependent plasticity and, 223, 248, 262 as neurotrophic factors, 1-3, 18-20 direct trophic actions, 3-7 indirect actions, 12- 14 neuropeptides, 7-12 regulation, 14- 18 nicotinic acetylcholine receptors and, 112 central neurons, 105, 111 diversity, 32, 70 models, 77 regulation, 84 seminal concepts, 26-29, 72-73 vertebrate nervous system development and, 151, 155, 164, 166, 173 Neurotrophic factors glial cells in activity-dependent plasticity and, 223, 249 neurotransmitters as, 1-3, 18-20 direct trophic actions, 3-7 indirect actions, 12-14 neuropeptides, 7-12 regulation, 14-18 Nicotine receptors, acetylcholine at motor nerves and, 284-286, 372 adrenergic receptors, 367, 370 autoreceptors, 341-343, 349-350 detection methods, 303-305, 308309

400

INDEX

events, 290, 299-300, 302-303 presynaptic receptors, 3 12-34 1 Nicotinic acetylcholine receptors, 25-26, 112 central neurons, 103 anticholinesterase treatment, 108110

exposure, 105-108 modification, 1 10- 1 12 ontogeny, 103- 105 diversity behavior, 69-72 expression sites, 68-69 function, 33-45 functional expression, 63-68 muscle genes, 57-61 nSgtS gene, 62-63 neuronal genes, 61-64 nomenclature, 29-33 structure, 45-56 ganglia, 95-103 regulation, 78-79 assembly, 79-88 dominance, 76-78 Hexibility, 78 function, 88-90 models of, 73-76 modulation, 90-94 seminal concepts, 26-29, 72-73 Nifedipine. acetylcholine at motor nerves and. 332, 343, 363 Nitric oxide glial cells and, 222-223, 265 vertebrate nervous system development and, 164, 173 Noradrenahne, acetylcholine at motor nerves and adrenergic receptors, 358-359, 36 1, 365, 367-370 muscarine receptors, 344, 353, 358 nicotine receptors, 330, 338, 340 Noradrenergic neurons, acetylcholine at motor nerves and, 369 Norepinephrine glial cells and, 222, 262, 264 neurotrophic factors and, 4, 7, 12 vertebrate nervous system development and, 159 Nuclear regulation, nicotinic acetylcholine receptors and, 76-77

Nucleotides nicotinic acetylcholine receptors and, 28, 61-63, 92, 101 vertebrate nervous system development and, 173, 179 Nucleus, nicotinic acetylcholine receptors and, 79-80, 83, 105. 108, 110

0 Ocular dominance glial cells in activity-dependent plasticity and. 218, 227, 242-243 vertebrate nervous system development and mechanisms, 167, 175 properties, 136-137, 141, 143, 146, 153 Oncogenes, vertebrate nervous system development and, 179 Ontogeny, nicotinic acetylcholine receptors and, 103-105 Opiod peptides, neurotrophic factors and. 12, 19 OverHow studies, acetylcholine at motor nerves and, 286 adrenergic receptors, 355-358, 365367 autoreceptors. 3 12-322, 344-349 detection methods, 305-312 Oxotremorine, acetylcholine at motor nerves and, 3 1 1, 344-35 1 Oxymetazoline, acetylcholine at motor nerves and, 361-362, 365 Oxytocin. neurotrophic factors and, 8 , 16

P Pancuronium, acetylcholine at motor nerves and, 323, 332 Paralysis, vertebrate nervous system development and, 160-161 Parasympathetic nervous system, acetylcholine at motor nerves and. 353. 365, 370 PC12 cells glial cells in activity-dependent plasticity and, 249

INDEX

nicotinic acetylcholine receptors and diversity, 54, 56, 66, 68 ganglia, 102-103 vertebrate nervous system development and, 186 Peptides acetylcholine at motor nerves and, 296 neurotrophic factors and, 12, 16 nicotinic acetylcholine receptors and diversity, 58, 67, 71 ganglia, 97-98, 103 regulation, 93-94 structure, 47, 50, 56 Peripheral nervous system acetylcholine at motor nerves and, 332, 340, 351 development, 137, 139-140 Phagocytosis, glial cells in activitydependent plasticity and, 240, 245, 248 Pharmacology acetylcholine at motor nerves and, 303 adrenergic receptors, 362 autoreceptors, 3 13, 329-33 1, 34 1, 343 glial cells in activity-dependent plasticity and, 219, 265 neurotrophic factors and, 4 nicotinic acetylcholine receptors and, 31, 105 diversity, 51, 53, 65-66, 70 function, 37-39, 43 ganglia, 95, 101-102 vertebrate nervous system development and, 140, 151, 162, 199 Phenotype neurotrophic factors and, 6-7, 11-12, 18-19 nicotinic acetylcholine receptors and, 81 vertebrate nervous system development and, 163, 167, 169 Phenthonium, acetylcholine at motor nerves and, 349-350 Phentolamine, acetylcholine at motor nerves and, 358, 361 Phenylephrine, acetylcholine at motor nerves and, 357-358, 361-364 PhorboI esters, nicotinic acetylcholine receptors and, 76, 91, 100

40 1

Phosphatidylinositol phosphates (PIPS), vertebrate nervous system development and, 165-166, 176, 185 Phosphodiesterase, acetylcholine at motor nerves and, 367-368 Phosphoinositol, vertebrate nervous system development and mechanisms, 170, 173, 183, 198 properties, 156, 158-159 Phospholipases acetylcholine at motor nerves and, 363 vertebrate nervous system development and, 165 Phospholipids acetylcholine at motor nerves and, 287, 290, 363 glial cells in activity-dependent plasticity and, 264 neurotrophic factors and, 4 nicotinic acetylcholine receptors and, 28, 76, 90-91, 101 vertebrate nervous system development and, 173-174 Phosphorylation acetylcholine at motor nerves and, 369 glial cells in activity-dependent plasticity and, 231 vertebrate nervous system development and mechanisms, 166, 172-173 properties, 158 structural changes, 182, 184-185 Phrenic nerve, acetylcholine at motor nerves and, 285 adrenergic receptors, 355, 359, 362365, 368 autoreceptors, 312, 315, 317, 320, 327, 332 detection methods, 306, 309, 311312 events, 288-291, 297, 299 muscarine receptors, 344-346, 35 1 Physostigmine, acetylcholine at motor nerves and, 321-322 Pituicytes, glial cells in activity-dependent plasticity and, 242, 265 Pituitary neurotrophic factors and, 8, 15-16 nicotinic acetylcholine receptors and, 71,98

402

lNDEX

Pkasmd membrane glial cells in activity-dependent plasticity and, 243, 246 nicotinic acetylcholine receptors and, 28, 89, 93 vertebrate nervous system development and, 177-178 Plasticity neurotrophic factors and, 17-19 nicotinic acetylcholine receptors and, 78 vertebrate nervous system development and afferent activity. 140, 143, I45 critical period, 157, 159-160 mechanisms, 163, 166- 169, 171-172, 17.5, 177 mechanisms of plasticity. 19I-IY9 postsynaptic activity, 350-151, 155. 157 properties, 134-135 structural changes, 180, 183- 185 synaptic plasticity, 186- 191 Polarization, vertebrate nervous system development and, 183 Polymerization, vertebrate nervous system development and, 182-183 Polypeptides. nicotinic acetylcholine receptors and diversity 45, 53, 55-56, 61 ganglia, 101 seminal concepts, 28 Post-translational processing, nicotinic acetylcholine receptors and central neurons. 108 niodels. 77-78 regulation, 80-82, 89, 92 Postsynaptic activity glial cells in activitv-dependent plasticity and, 2 17-2 19, 222-223, 267 hypothesis for iiivolvement, 258, 261, 264 participation, 240-242, 248 synapse formation, 25 1 synaptic efficacy. 225-226, 228, 230, 232-234, 253 vertebrate nervous system development and afferent activity, 139, 144-145, 149 mechanisms, 163, 165-166, 168-172, 174, 177

mechanisms of plasticity, 192-1 94, 196-199 properties, 135, 150-157 structural changes, 181 synaptic plasticity, 186-191 Postsynaptic density, vertebrate nervous system development and, 180 Postsynaptic receptors acetylcholine at motor nerves and, 284 adrenergic receptors, 355, 360-361, 368, 370 autoreceptors, 3 13, 3 19, 322, 325328 detection methods, 304-305 events, 289-290, 299, 302 muscanne receptors, 349, 351 nicotine receptors, 328-331, 333, 337-338,340 preterminal receptors, 34 1, 343 nicotinic acetylcholine, 52, 85, 109 Posttetanic potentiation, vertebrate nervous system development and, 178 Potassium acetylcholine at motor nerves and adrenergic receptors, 354, 364 autoreceptors, 313-314, 332, 341 detection methods, 303, 310-31 1 events, 287 muscarine receptors, 344, 347-349, 352-353 glial cells in activity-dependent plasticity and, 221, 252, 258, 262, 267 neurotrophic factors and, 4, 7 nicotinic acetylcholine receptors and, YY, 101 vertebrate nervous system development and, 164-165, 172, 174, 185 Prazosin, acetylcholine at motor nerves and, 357-358, 361-362, 365 Presynaptic activity glial cells in activity-dependent plasticity and, 217-219, 222-223, 267 hypothesis for involvement, 258 participation, 254 synaptic efficacy, 230-23 1, 233-234 vertebrate nervous system development and afferent activity, 139, 14 1- 142 mechanisms, 164, 170-171, 174 plasticity, 186, 188-191, 194, 196

INDEX

properties, 150-151, 154-157 structural changes, 181 Presynaptic receptors acetylcholine at motor nerves and, 284286, 372 adrenergic receptors, 354-355, 360, 364-365,367-371 detection methods, 303-305, 308309 events, 288, 293-294, 296, 300303 muscarine receptors, 344-354 nicotine receptors, 312-341 nicotinic acetylcholine, 52, 70 Preterminal-axonal nicotine receptors, acetylcholine at motor nerves and, 34 1-343 Proenkephalin, neurotrophic factors and, 8, 12, 14-16 Profilin, vertebrate nervous system development and, 183 Proopiomelanocortin, neurotrophic factors and, 8, 15-16 Propranolol, acetylcholine at motor nerves and, 362, 365, 367 Proteases glial cells in activity-dependent plasticity and, 246,260 vertebrate nervous system development and, 164, 176, 199 Protein acetylcholine at motor nerves and, 284, 31 1, 363, 369 glial cells in activity-dependent plasticity and, 233 participation, 239, 242, 247-248 synapse formation, 252-253 nicotinic acetylcholine receptors and, 96, 105, 112 expression sites, 68-69 functional expression, 67-68 models, 75, 77 muscle genes, 57-58 neuronal genes, 61-62 nomenclature, 32 regulation, 82-83, 85-88, 92, 94 seminal concepts, 28-29 structure, 46, 49, 53, 56, 58 vertebrate nervous system development and

mechanisms, 165-166, 171-173, 176, 179 plasticity, 199 properties, 158 structural changes, 181-186 Protein kinase C acetylcholine at motor nerves and, 368 glial cells in activity-dependent plasticity and, 230, 247,262, 264 nicotinic acetylcholine receptors and ganglia, 98, 100 models, 76 regulation, 87, 90-91, 94 vertebrate nervous system development and mechanisms, 165, 173-175, 184185 plasticity, 193, 199 properties, 159 Purkinje cells glial cells in activity-dependent plasticity and, 231, 240 neurotrophic factors and, 11 vertebrate nervous system development and, 138, 171, 186, 191 Pyramidal cells, neurotrophic factors and, 5-6, 17, 19 Pyramidal neurons, vertebrate nervous system development and, 156, 168

Q Quisquilate receptors, neurotrophic factors and, 4-5

R Radioactivity, acetylcholine at motor nerves and adrenergic receptors, 358 autoreceptors, 323, 340, 344 detection methods, 304-306, 308, 31 1 events, 288-291, 293-294 Radioagonist binding sites, nicotinic acetylcholine receptors and central neurons, 104-107, 110-1 11 functional expression, 69 structure, 52-53

404

INDEX

Radiolabeling acetylcholine at motor nerves and, 301 autoreceptors, 3 12, 3 17-3 19 detection methods, 304, 308-312 nicotinic acetylcholine receptors and, 51-52, 69, 103 Receptors acetylcholine at motor nerves and, see Acetylcholine at motor nerves glial cells in activity-dependent plasticity and, 222-223 hypothesis for involvement, 258, 260, 262, 264 participation, 246, 253-255 synaptic efficacy, 230-231, 234 neurotrophic factors and, 2, 7-8, 10, 14- 17 nicotinic acetylcholine, see Nicotinic acetylcholine receptors vertebrate nervous system development and, 173 a-Receptors, acetylcholine at motor nerves and, 355-365, 367, 370-371 P-Keceptors, acetylcholine at motor nerves and, 354-355, 357-358, 361-362, 365-371 Regeneration, glial cells in activitydependent plasticity and, 255-257 Regulatory genes, vertebrate nervous system development and, 178-179 Retina glial cells in activity-dependent plasticity arid, 225, 254 vertebrate nervous system development and, 148-149 Retinal ganglion cells neurotrophic factors and, 6, 10, 14, 17 nicotinic acetylcholine receptors and, 35 KNA, nicotinic acetylcholine receptors and diversity, 60-61, 64, 66-68, 71 ganglia, 96 models, 75, 77 regulation, 82-83

s S-100 protein, glial cells in activitydependent plasticity and, 252-253, 260

Schwann cells acetylcholine at motor nerves and, 29 1, 329 glial cells in activity-dependent plasticity and, 245, 256 neurotrophic factors and, 17 Scopolamine, acetylcholine at motor nerves and, 348-35 1 Second messengers acetylcholine at motor nerves and, 363364,369 nicotinic acetylcholine receptors and, 28, 76 ganglia, 98-100, 102-103 regulation, 90, 92 vertebrate nervous system development and mechanisms, 16.5, 170, 173, 175, 179 plasticity, 191, 199 properties, 159 structural changes, 183 Sequences nicotinic acetylcholine receptors and, 71, 77, 81-82 expression sites, 69 function, 43 muscle genes, 57-58, ti0-61 neuronal genes, 61-62 seminal concepts, 28-29 structure, 48, 50, 56 vertebrate nervous system developmerit and, 179 Serotonin acetylcholine at motor nerves and, 284, 330, 340 glial cells in activity-dependent plasticity and, 239 neurotrophic factors and, 3-4, 12-13, 17, 19 nicotinic acetylcholine receptors and, 97-98 Short-term potentiation, vertebrate nervous system development arid, 1.55, 165, 191, 193 Signal transduction acetylcholine at motor nerves arid, 286 adrenergic receptors, 362-364, 368369 autoreceptors, 328-332 vertebrate nervous system development and, 172-173, 185

INDEX

405

Snake venom neurotoxins, nicotinic acetyl Sympathetic neurons choline receptors and, 39, 43-44 nicotinic acetylcholine receptors and, Sodium 96, 98, 100 acetylcholine at motor nerves and, 287, vertebrate nervous system development 289, 303, 331, 355 and, 165 glial cells in activity-dependent plasticity Sympathomimetic amines, acetylcholine at and, 258 motor nerves and, 285-286 vertebrate nervous system development adrenergic receptors, 354-355, 358, and, 142, 168, 177-178, 193 360-361, 370 Somatosensory cortex, glial cells in Synapse selection, vertebrate nervous sysactivity-dependent plasticity and, 224, tem development and, 186- 189, 192, 227-228, 234-235,237 194 Somatostatin, neurotrophic factors and, Synapses 7-8, 15, 19 acetylcholine at motor nerves and, 283Spinal cord 284, 372 glial cells in activity-dependent plasticity autoreceptors, 340-34 1, 343 and, 238, 240, 245, 257 detection methods, 308 neurotrophic factors and, 5, 11, 14 events, 293, 296-297, 299, 302 nicotinic acetylcholine receptors and, glial cells in activity-dependent plasticity 87, 93, 104 and, 215-216, 222-223 vertebrate nervous system development CNS damage, 255 and, 146, 151, 163, 167, 169 efficacy, 224-234, 253-255 Striatum elimination, 239-248 acetylcholine at motor nerves and, 353 formation, 249-253 glial cells in activity-dependent plasticity hypothesis for involvement, 260-26 1, and, 238 264 neurotrophic factors and, 13- 14 neurotrophic factors and, 6, 18-19 nicotinic acetylcholine receptors and, nicotinic acetylcholine receptors and 104 behavior, 71 Substance K, neurotrophic factors and, 9, ganglia, 95, 100 11, 19 models, 73-74, 78 Substance P nomenclature, 32 neurotrophic factors and, 8, 11-12, 15regulation, 78-79, 83-86, 88, 9317, 19 94 nicotinic acetylcholine receptors and, seminal concepts, 26-27 92, 98, 101, 103 vertebrate nervous system development vertebrate nervous system development and, 133-134 and, 162 afferent activity, 139- 150 Substantia nigra critical period, 157-161 acetylcholine at motor nerves and, 302 mechanisms, 164- 169, 172- 178 neurotrophic factors and, 19 mechanisms of plasticity, 191-199 Superior cervical ganglion, neurotrophic postsynaptic activity, 153- 157 factors and, 9- 1 1 properties, 134-135, 137-139, 151 Superior colliculus, glial cells in activitystructural changes, 179- 185 dependent plasticity and, 224-225 synaptic plasticity, 186- 191 Sympathetic ganglion neurons, vertebrate Synaptic competition, vertebrate nervous nervous system development and, system development and, 145, 186163 190 Sympathetic nervous system, acetylcholine Synaptosomes, acetylcholine at motor at motor nerves and, 330, 340, 353, nerves and, 287, 311, 339-340, 369-370 353

406

INDEX

T Tachykinins, nicotinic acetylcholine receptors and. 98. 101, 11 1 'Tau proteins, vertebrate nervous system development and, 181-182 'Ienascin, glial cells in activity-dependent plasticity and, 237, 256 Tetanization glial cells in activity-dependent plasticit! and, 253 vertebrate nervous system development and, 172, 176 Tetrodotoxin acetylcholine at motor nerves and, 291, 304, 306, 348, 350 glial cells in activity-dependent plasticity and, 219, 227, 237. 241 neurotrophic factors and, 5, 10- 1 1, 17 nicotinic acetylcholine receptors and, 76. 83-84, 86 vei-trbrate nervous system development and afferent activity, 140- 143, 146- 148 mechanisms, 162- 163, 169 properiies. 1.50- 151. I54 Thvmopoietin. nicotinic acetylcholine receptors and. 101. 110 diversitv, 44, .54, i 2 regulation. 93 'li.~lazolinc,acetylcholine at motor nerves and, 358. 362 7irpudo acetylcholine at m o t o r nerves and, 293. 31 1 nicotinic acetylcholine receptors arid diversity, 31, 37 muscle genes, 5i-60 regulation, 80, 82, 87. 90 structure, 47, 50 loxins, nicotinic acetylcholine receptors and behavior, 72 central neurons, 1 1 1-1 12 furictional expression, 66-67 ganglia, 1 0 1 nomenclature, 32-33 regulation, 84-85 structure, 46, 50-5 1, 56 a-Toxins. acetylcholine at motor nerves and, 3 12, 324, 326, 328-329

Transcription neurotrophic factors and, 13 nicotinic acetylcholine receptors and central neurons, 108 diversity, 60-6 1, 7 1 ganglia, 96-97, 102 models, 76-77 regulation, 79-85, 91-94 vertebrate nervous system development and, 164-165, 179, 184, 199 Translation, nicotinic acetylcholine receptors and, 91, 94 Translocation, vertebrate nervous system development and, 172, I74 Trophic factors, vertebrate nervous system development and, 161-164, 182 Tuhcurarine, acetylcholine at motor nerves and, 332, 343 autoreceptors. 312-315, 317-320, 323328 detection methods, 304-305 events, 293 nicotine receptors, 332-333, 336-340 preterniinal receptors, 343 d-Tubocurarine, nicotinic acetylcholine receptors and diversity, 38-39, 46, 52, 66 ganglia, 98-99, 101 Tyrosine hydrolase, neurotrophic factors and, 7, 11

V \'asoactive intestinal peptide glial cells in activity-dependent plasticity and, 239, 252 neurotrophic factors and, 9-14, 18-19 vertebrate nervous system development and, 162-163. 175 Vasopressin, neurotrophic factors and, 9 Verapamil, acetylcholine at motor nerves and, 332, 343 VPrtrhrare nervous system, see Activitydependent development ot' vertebrate nervous system \'esamicol, acetylcholine at motor nerves and, 293, 295, 297 Vesicles acetylcholine at motor nerves and, 284, 319

INDEX

detection methods, 310, 312 events, 287, 291, 293-298, 300-301 vertebrate nervous system development and, 176, 178, 180-181 Visual cortex glial cells in activity-dependent plasticity and, 2 18, 22 1 CNS damage, 257 hypothesis for involvement, 261, 264 participation, 235, 239, 243, 246, 248 synapse formation, 249, 251-252 synaptic efficacy, 227-228 neurotrophic factors and, 17 vertebrate nervous system development and, 134 afferent activity, 141-142, 145-146, 148 critical period, 157- 158 mechanisms, 166-168, 170, 175-177 plasticity, 190, 192, 194 postsynaptic activity, 150- 151, 154155, 157 properties, 135-1 36 structural changes, 184- 185 Visual system glial cells in activity-dependent plasticity and, 219,224-225, 228-229

407 vertebrate nervous system development and mechanisms, 167 plasticity, 196 properties, 136-137, 157, 161

x Xenopus acetylcholine at motor nerves and, 297, 299 neurotrophic factors and, 8 nicotinic acetylcholine receptors and, 29, 105 diversity, 58-60, 62-65 regulation, 80, 82, 88 vertebrate nervous system development and, 167

Y Yohimbine, acetylcholine at motor nerves and, 357-358,361-362

CONTENTS OF RECENT VOLUMES

Unddn, Lou-Lou Peterson, and Tamas Bartfaz

Ar&s

Volume 26

The Endocrinology o f the Opioids Mark J . Millan and Albert Her2

Eye Movement Dysfunctions and Psychosis Philip S. Holzman

Multiple Synaptic Receptors for Neuroacrive Amino Acid Transmitters-New Vistas Najam A. S h r i f

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

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

Calcium and Transmitter Release Ira Cohen and William Van der Kloot Excitatory Transmitters and Related Brain Damage John W. Olney

Neural Plasticity and Recovery of Function after Brain Injury john F. Marshall From Immunoneurology to Irnmunopsychiatry: Neuromodulating Activity of AntiBrain Antibodies Branislav D.JankloviC Effect of Tremorigenic Agents on the Cerebellum: A Review of Biochemical and Electrophysiological Data V . G. Longo and M . Masrotti

Epilepsy-

Potassium Current in the Squid Giant Axon John R. Clay INDEX

Volume 28

INDEX

Biology and Structure of Scrapie Prions Michael P. McKinley and Stun@ B. Prusinw Different Kinds o f Acetylcholine Release from the Motor Nerve S. Thesleff

Volume 27

The Nature of the Site of General Anesthesia Keith W . Millei The Physiological Role of Adenosine in the Central Nervous System Thomar V. Dunwuldie Somatostatin, Substance P, Vasoactive Intestinal Polypeptide, and Neuropeptide Y Receptors: Critical Assessment of Biochemical Methodology and Results 408

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 Biological Aspects of Depression: A Review of the Etiology and Mechanisms of Action and Clinical Assessment of Antidepressants S. I. Ankier and B. E. Leonard

CONTENTS OF RECENT VOLUMES

Does Receptor-Linked Phosphoinositide Metabollism Provide Messengers Mobilizing Calcium in Nervous Tissue? John N. Hawthorne 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 Brimyoin and Zoltan Rakonczay INDEX

Volume 29

Molecular Genetics of Duchenne and Becker Muscular Dystrophy Ronald G. Worton and Arthur H . M . Burghes Batrachotoxin: A Window on the Allosteric Nature of the Voltage-Sensitive Sodium Channel George B . Brown Neurotoxin-Binding Site on the Acetylcholine Receptor Thomas 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. Mennini Retinal Transplants and Optic Nerve Bridges: Possible Strategies for Visual Recovery as a Result of Trauma or Disease James E . Turner, Jerry R. Blair, Magdalene

409

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. Blakely and Joseph T. Coyle Neuropeptide-Processing, -Converting, and -Inactivating Enzymes in Human Cerebrospinal Fluid Lars Terenius and Fred Nyberg Targeting Drugs and Toxins to the Brain: Magic Bullets Lance L. Simpson Neuron-Glia Interrelations Antonia Vernadakis Cerebral Activity and Behavior: Control by Central Cholinergic and Serotonergic Systems C . H . Vanderwolf INDEX

Volume 31

Animal Models of Parkinsonism Using Selective Neurotoxins: Clinical and Basic Implications Michael J . Zigmond and Edward M . Stricker Regulation of Choline Acetyltransferase Paul M. Salvaterra and James E. Vaughn Neurobiology of Zinc and Zinc-Containing

410

CONTENTS OF RECENT VOLUMES

Neurons Chrivtopher J . Fredenckson Dopamine Receptor Subtypes and Arousal Ennio Ongini and Vincenw G. Longo Regulation of Brain Atrial Natriuretic Peptide and Angiotensin Receptors: Quantitative Autoradiographic Studies Juan M . Saavedra, Eero Castrbn, Jorge S. Gutkind, aid Adif J . Nazarali 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 . " d i n g t o n Nerve Blood Flow and Oxygen Delivery in Normal, Didbetic, and Ischemic Neuropa th y Phillip A . Low, Terrence D. Lagerlund, and Philip G. McMaiiir

Myasthenia Gravis: Prototype of the Antireceptor Autoimmune Diseases Simone Schonbeck, Szrsanne Chrestel, and Reinhard Hohlfeld Presynaptic Effects of Toxins Alan L. Haruey Mechanisms of Chemosensory Transduction in Taste Cells Myles H. Akabm Quinoxalinediones as Excitatory Amino Acid Antagonists in the Vertebrate Central Nervous System Stephen N . Davies and Graham L. Collingndge Acquired Immune Deficiency Syndrome and the Developing Nervous System Lfoughi E. Brennemnn, Swan K. McCune, and Illana Gazes INDEX

INDEX

Volume 33 Volume 32

On the Contribution of Mathematical Models to the Understanding of Neurotransmitter Release H . Parnu* I . Parnas, and L . A . Segel Single-Channel Studies of Glutamate Receptors M . S.P. Saniom and P. N. R. Uihenuood (:oinjection of Xenopus Oacytes with cDNAProduced and Native mRNAs: A Molecular Biological Approach to the Tissue-Specific Proressing of Human Cholinesterases Shlomo Seldman and H m o n a Soreq Potential Neurotrophic Factors in the Manrmalian Central Nervous System: Functional Significance in the Developing and Aging Brain Dalia M . Araujo, Jean-Guy Chabot, and Rhna Quinon

Olfaction S . G Shirley Neuropharmacologic and Behavioral Actions of Clonidine: Interactions with Central Neurotransmitters Jerry J . BuccaJllsco Development of the Leech Nervous System Gunther S. Stent, Willtam B. Kmtan, Jr., Steven A. Torrence, Kathleen A . French, and Damd A. Wezsblat CABAA Receptors Control the Excitability of Neuronal Populations A m i n Stelur Cellular and Molecular Physiology of AIcohd Actions in the Nervous System Forrest F. Weight INDEX