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.

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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

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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

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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

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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

Altstein, M . . and Gainer, 11. (1988).J . .\‘euro.Tci. 8, 3Y67-3957. Xoki. C., Joh, T. H., and Pickel, V. M. (1987). Brain Res. 437, 264-282. Ashkena7i, X., Ramachandran, J., and C a p o n . D. J. ( IY89). h’atuw (Londun) 340, 146-150. Azmitia, E. C., and deKloet, E. R. (1987). P q g . Bruin Re.s. 72, 31 1-318. Balazs. R.. Gallo, V.. and Kingbury, .4. (IY8Xa). Dri,. B m i n RP.~. 40, 269-276. Balazs, R.. IIack, N . , and Jargensen, 0. S. (1988b). .Veuro.tci. Letf. 87, 80-86. Baldzs, R.. Jargemen, 0. S., arid Hark, N. (1988~).il’rurosrience 27, 437-451. Balazs, R.. Hack, N..Jargemen, 0.S., and Cotman. C. W. (1989). Neurosci. Lctt. 101, 241246. Balazs, R . . [lack, N., and Jfirgenseii, 0. S. (1YYOa). I ? ) / .J. DPIJ.K w r o m . 8, 397-350. Balazs, R., Hack, N..and Jergensen, 0.S. (19YOh). .VPumcience 37, 251-258. Brenneman. I). E.. and Eiden. L. E. (1986). Proc. Nu//. Arud. Sci. LT.S.A. 83, 1159-1 162. Brenneman, D. E:., Neal, E. A., Foster. G. A , , d’Auti-emont, S. W., and Westbrook, G.L. (1987). ,/. C d l Riol. 104, 1603-1610. Brennenian. D. E., Forsythe, I. D., Nicol, T.. and Nelson, P. G. (1990a). Dezi. Brain Res. 51, 63-68, Brennenian, I). E., Nicol, ‘I.,Warren. D.. and Bowers, L. iM.(1990b).J. Nrurusci. Res. 25, 386-JY4. Brenneman, D. L,Yu, C., and Nelson, P. G. (l9YOc). I t i t . J. DPZJ.Nrzirosci. 8, 371-378. Bullorh, A . 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,

PRESYNAPTIC RECEPTORS AT MOTOR NERVE TERMINALS

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

111111111111111- 3 s -111111111111111- 3 s -111111111111111 intermittent stimulation (trains of 15 pulses every 3 s)

11111-3s-11111-3s-11111-3s-11111-3s-11111-3s-11111-3s-11111-3s-11111-3s-11111

intermittent stimulation (trains of 5 pulses every 3 s )

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.

PRESYNAPTIC RECEPTORS AT MOTOR NERVE ‘TERMINALS

317

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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IGNAZ WESSLER

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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