Progressin NeurobiologyVol. 37, pp. 145 to 163, 1991 Printed in Great Britain.All rights reserved
0301-0082/91/$0.00 + 0.50 © 1991 PergamonPress pie
NON-SYNAPTIC INTERACTIONS AT PRESYNAPTIC LEVEL E. S. Vtzx* a n d E. LkBosI" *Institute of Experimental Medicine, Hungarian Academy of Sciences, H-1450 Budapest, PO Box 67, Hungary "~1st Department of Anatomy, Semmelweis University Medical School, H-1094 Budapest, Tfizolt6 str. 58, Hungary
(Received 24 January 1991)
CONTENTS 1. Introduction 2. Non-synaptic interaction between neuronal presynaptic modulations of transmitter release 2.1. Non-synaptic release sites of transmitters 2.2. Non-synaptic receptor localization; mismatches between signal transmitters and receptors (remote receptors) 2.3. Functional interaction between neurons and target cells without synaptic contact 2.3.1. Noradrenergic-cholinergic interactions 2.3.2. Dopaminergic--cholinergic interactions 2.4. Topographic features of "field" innervation. Regional disparity in fine morphological relationships 3. Pharmacological importance 4. Physiological importance 5. An attempt to merge the synaptic and non-synaptic information processing 5.1. Some forms of interactions 5.2. The possible role of various interactions 5.3. Incorporation of quick, synaptic and slow, non-synaptic interactions into the same explanatory model References
1. INTRODUCTION Our way of thinking about the intercellular transmission of information is still dominated by the knowledge gained from anatomical studies on patterns of neuronal wiring. Within a given neuron, electrical signals are initiated and transmitted as waves of electrical potential change produced by increase in the permeability of the membrane to different ions. The propagated electrical signal reaching the axon terminal ends usually in depolarization and causes the release of chemical substances, provided that Ca0 is available. The concept of chemical neurotransmission is attributed to Elliott (1905): neurons communicate information among themselves and between neuron and target cell by means of chemical substances. Katz, Eccles and colleagues have supplied convincing evidence that in an overwhelming majority of cases transmission of information from cell to cell is chemical in nature. The chemical substances, released by depolarization cross the synaptic cleft and act on the postsynaptic Address correspondence to: Dr. E. S. Vizi, Institute of Experimental Medicine, Hungarian Academy of Sciences, H-1450 Budapest, PO Box 67, Hungary. Phone: 36-1-1137616, Fax: 36-1-114-1866.
145 147 147 148 148 148 151 155 157 157 157 158 158 158 159
membrane equipped with receptors. A nerve impulse traveling toward its final destination (if such a place exists at all) usually does not get there by means of a single neuron. In general, and it is an extensively accepted view, the impulse reaches the end of a neuron that is in contact with a further ceil. The site of contact is called a synapse, or synaptic junction. The presynaptic (junctional) elements are separated from the postsynaptic part by a distance of 15-100 nm: this space is called the synaptic cleft. The first reference to the concept of the synapse was given by Sherrington (1906). Since Sherrington's classic work it has become a doctrine of neurophysiology that the synapse, a part of the surface of separation between neurons, is the primary site of neuronal information processing. It is now accepted that intercellular communication in the nervous system generally involves the release of chemical transmitters from such synapses. The synapse is the most common and generally accepted structural basis of the interaction between neurons, and it implements a one-way communication system between them. In addition to the synaptic communication there is a further possibility for interneuronal communication: it is a non-synaptic interaction between neurons at the presynaptic level (cf. Vizi, 1979, 1980b, 1982, 1984, 1990a,b; Vizi et al., 1991). One
145
146
E.S. Viz] and E. LABOS
neuron can communicate with many others without making synaptic contact: i.e. there is a non-synaptic chemical "cross-talk" between neurons (Vizi, 1979, 1984, 1990a,b; Vizi et al., 1991). This would be a transitional form of communication between discrete classical neurotransmission and the relatively nonspecific neuroendocrine secretion. In the past few years neurochemical, morphological and pharmacological evidence has shown that some neurotransmitters, or substances called modulators, may be released from both synaptic and non-synaptic sites (Fig. 1) for diffusion to target cells more distant than those observed in regular synaptic transmission (Vizi, 1979, 1984, 1990a,b; Cuello, 1983; Iversen, 1984; Schmitt, 1984; Herkenham, 1987), Recently, Agnati et al. (1986) and Fuxe et aL (1988a,b) provided further evidence of this nonsynaptic communication and introduced a new term "volume transmission". It became clear that chemical neurotransmission may be a far more complicated event than previously assumed. Firstly, the release of transmitters/ modulators can be presynaptically modulated (Fig. 2) through auto-, homo- and heteroreceptors (cf. Kalsner, 1990; Langer, 1977; Starke, 1981; Vizi, 1979, 1984; Starke et al., 1989). Secondly, transmitters/ modulators can be released from sites other than axon terminals and from varicosities situated synaptically or non-synaptically. Thirdly, regional dispar-
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Fl~. 1. Scheme of synaptic and non-synaptic interactions between noradrenergic and cholinergic presynaptic nerve terminals. Norepinephrine (NE) non-synaptically released from noradrenergic axon terminals inhibits the release of acetylcholine (ACh) via stimulation of e2-adrenoceptors (1) and inhibits its own release (2, negative feedback, cf. Starke et al., 1989) via stimulation of e2-adrenoceptors. In addition NE, released into a synaptic gap in an axo-axonic synapse, can act in the synapse (negative feedback, 2) and can diffuse away i.e. it can act non-synaptically (3) influencing vast neuronal assemblies. Acetylcholine (ACh) is able to inhibit its own release (4, negative feedback) via stimulation of muscarinic (Mz) receptors located on the axon terminal where it is released from. Acetylcholine released into the synaptic gap acts on the postsynaptic membrane stimulating nicotinic receptors (5, synaptic). Its long-distance effect is terminated by cholinesterase. Presynaptic receptors (in the scheme ~2 and M2 are the examples) may allow the function of presynaptic terminals to be modified in three ways: (1) by transmitters/modulators released from adjacent neurons and are able to diffuse long distance; (11) by synaptic contacts from other neurons onto presynaptic terminals; or (111) by transmitter released from the terminals themselves.
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FIG. 2. Types of presynaptic receptors. Autoreceptors are located on the axon terminals where norepinephrine released from the axon terminal inhibits its own release evoked by subsequent stimuli. Homoreceptors are localized on an adjucant neuron which possesses the same machinery as that from where the modulator was released. Norepinephrine: (NE) released from noradrenergic axon terminals stimulates ~(2-adrenoceptors(heteroreceptors) located on an axon terminal that can not produce that modulator which is able to act on it (norepinephrine acts on a non-noradrenergic axon terminal via stimulation of ~2-adrenoceptor). ities in the relationships of the inputs to different regions of the brain make possible the regional nonsynaptic/synaptic control of chemical neurotransmission in the neuronal network. Because synaptic communication between cells had been the most thoroughly studied form of intercellular communication, it was later accepted that it is the only form of interaction between neurons and that any change in neuronal excitation is the result of synaptic (chemical or rarely electrical) transmission. Thus, the classical view of neurotransmission is that it takes place at the synapse. Much of our present knowledge about the cellular organization of the different brain areas derives from Golgi studies, dating to the end of the last century when Golgi (1883), Ramon y Cajal (1891, 1911), and many others opened the modern era of neuroanatomy. One limitation of the Golgi technique is that synaptic contacts can be predicted only by indirect correlation of separately impregnated dendritic and axonal patterns. An important progress was made when the Golgi method was introduced with the electron microscopic analysis of the stained neurons (Blackstad, 1965). The gold technique also provided an opportunity to circumvent another limitation of the Golgi method, its failure of histochemical specificity. Using this technique, the processes of cells remain traceable, but with removal of most of the Golgi method precipitate from the impregnated cells, they can be characterized by the aid of techniques such as autoradiography (Somogyi et al., 1981b), immunocytochemistry (Somogyi et al., 1983), and enzyme histochemistry (Bolam et al., 1984a; Somogyi et al., 1979; Somogyi and Smith, 1979; cf. Freund and Somogyi, 1989). A new avenue was paved by the analysis of identified circuits with the application of
NoN-SYNAPTIC INTERACTIONSAT PRESYNAPTIC LEVEL
Golgi impregnation to brain tissue that also contained electrophysiologically characterized and intracellularly horseradish peroxidase-injected neurons (Freund and Somogyi, 1983; Freund et al., 1985b). But it has to be confessed, that with these techniques alone and with their combinations it is still not possible to provide a functional characterization of identified cell types or to understand how (and why) neurons talk to each other. The combined neurochemical, pharmacological, anatomical, and histochemical analysis of the same synaptic circuits became possible. With these combined techniques it will be possible to explore and understand how the nervous system and the brain functions, one of the greatest challenges of our age. The efficacy of chemical synaptic transmission may be significantly altered by either presynaptic or postsynaptic changes. Similarly, the efficacy of a chemical non-synaptic communication system may be controlled, at least in part, by changes in the amount of neurotransmitter/modulator substance released from non-synaptic nerve terminals. The complexity of chemically mediated non-synaptic/ synaptic transmission provides a substrate for considerable flexibility. The sensitivity of transmitter/ modulator release to changes in presynaptic membrane potential may differ significantly between synapses and will depend largely upon the types of ion channel in the membrane of axon terminals. Since there are a number of receptor mediated ion channels that exhibit voltage sensitivity, the presynaptic receptor mediated effect of transmitters/modulators may be dependent on the actual membrane potential. The present review puts the emphasis on nonsynaptic interneuronal communication at the presynaptic level. 2. NON-SYNAPTIC INTERACTION BETWEEN NEURONAL PRESYNAPTIC MODULATIONS OF TRANSMITTER RELEASE 2.1. NoN-SYNAPTIC RELEASE SITES OF TRANSMITTERS A rather crucial question is as follows: are transmitters/modulators normally released from nonsynaptic axon terminals? The answer is, yes. There is convincing evidence that postganglionic neurons innervating vas deferens (Burnstock and Costa, 1975), blood vessels (Thureson-Klein and Stjarne, 1981), and smooth muscle (Burnstock and Costa, 1975) terminate in a network of varicose areas (boutons en passant) that lack synaptic contact with the target cell and still able to release transmitters in response to axonal electrical activity (cf. Vizi, 1984). The transmitter is released from structurally unspecialized areas along the varicose fibers and must often travel a considerable distance before interacting with its receptors. Sometimes this distance could be more than 1 #m (Fig. 3). Descarries and coworkers (1977, 1980, 1987) demonstrated that non-synaptic varicosities in the central nervous system appear to have all the apparatus normally associated with synaptic release. Subsequently, ultrastructural examination of noradrenergic varicosities in several tissues confirmed that both large and small vesicles could undergo JPN 37/2--E
147
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FIG. 3. Distance between release site and target receptor at synaptic and non-synaptic interaction. While in a synaptic contact the distance between release site and postsynaptic receptor is 20-50 nm, in case of non-synaptic interaction this could be more than I/am. exocytosis in the absence of structurally specialized active zones (Thureson-Klein, 1983, 1984; ThuresonKlein and Stjarne, 1981; Zhu et al., 1986). In addition, morphological evidence was provided (Buma, 1989a) for exocytosis in non-synaptic release site in rat median eminence and mesencephalic central grey substance. It is conceivable that a similar exocytosis occurs in monoaminergic neurons in the gut (Gordon-Weeks, 1982) and in the brain (Bj6rklund et al., 1973; Descarriers et al., 1977, 1980, 1987) where active synaptic zones are lacking. It was reported (Barber et al., 1979; Glazer and Basbaum, 1983) that certain terminals in the dorsal horn of the spinal cord and trigeminal nucleus caudalis (Zhu et al., 1986) do not have typical synaptic zones. Therefore, it is likely that substance P- or enkephalin-containing vesicles do not release their contents at synaptic sites (Barber et al., 1979; Cuello et al., 1977; Del Fiacco and Cuello, 1980; Glazer and Basbaum, 1983; Hunt et aL, 1980). The peripheral exocytosis at a distance from the synaptic complex suggests that large vesicles have a different physiological function to the small synaptic vesicles. Exocytotic release from large vesicles containing neurohormones has been also reported to occur at non-specialized areas of the membrane in invertebrates (Buma and Roubos, 1986; Roubos, 1984; Buma, 1989a,b; Schmidt and Roubos, 1989). Since the discovery of immunoreactive ~-melanocytestimulating hormone (~-MSH) in the rat brain, there have been a few reports about the distribution of this peptide in the central nervous system (Pelletier and Dube, 1977). With immunoelectron microscopy it has been shown that ~-MSH could only be recognized in dense core vesicles in axon terminals and cell bodies. It was suggested that most if not all brain ~-MSH is released by neurons at sites other than the classical synaptic junctions, probably to act non-synaptically (Guy et al., 1982). Similarly, no more than 50% of VIP containing endings have been reported to make synaptic contact (Pelletier et al., 1981). Therefore it seems very likely that modulator and transmitter substances released (a) from non-synaptic axon terminals, (b) from axon terminals making synaptic contact but acting both on synaptic and non-synaptic sites of target cells, or (c) from regions of the nerve cell other than the presynaptic axon
148
E.S. VlZl and E. L,£Bos
terminal (i.e. dendrites, axon, soma) (Vizi et al., 1983), can play a physiological role in the modulation of neurochemical transmission. 2.2. NoN-SYNAPTICRECEPTORLOCALIZATION; MISMATCHESBETWEENSIGNALTRANSMITTERS AND RECEPTORS(REMOTERECEPTORS)
There is an interesting difference in localization and sensitivity of receptors on effector cells where the transmitter (e.g. acetylcholine, norepinephrine, dopamine, serotonin or neuropeptides) is released into a large extraneuronal space (Herkenham, 1987). In the case of synaptic transmission, there is a small area of the effector cell where the receptors are concentrated. The neuromuscular junction is an example of a per.fect correspondence between release sites of transmitter, acetylcholine and the location of postsynaptic receptors. However, when the transmitter release site and the target cells are widely separated from each other (i.e. 100-3000 nm), there is no specific subsynaptic arrangement, and at the recognition sites, receptors are unevenly distributed along the surface of the effector cell (e.g. presynaptic axon terminals, etc.). This morphological arrangement fits a diffusion type of transmission in which the possible advantage of quantal release cannot be used. There seems to be no evidence that non-quantal release can occur solely at diffuse synapses. In addition, it could be argued that the receptors distant from the synaptic sites do not represent active receptors capable of opening ion channels. However, it is also known that the sensitivity of extrasynaptic receptors is higher to chemical signals. In addition, it was shown that the release site/receptor matches are exceptions rather than rules (cf. Herkenham, 1987). The explanation for the widespread extrasynaptic distribution of the receptor complex may lie in the general role of the transmitter-gated channels. The synaptic and non-synaptic receptors may be used under different levels of neuronal activity, providing a mechanism for adaptation. Somogyi et al. (1989) showed in cerebellum that at low levels of neuronal activity, gamma-aminobutyric acid (GABA) acting at the synaptic junctions provides adequate inhibitory control of the neurons. The moderate amounts of G A B A released can be removed by reuptake or glial uptake without reaching the extrasynaptic sites. However, at increased excitatory input, the number of channels operating at the junctions may not be suitable or adequate to keep the activity of the cell in the range of optimal sensitivity, in which case channels at extrasynaptic sites would be opened by GABA diffusing from the release sites. Consequently, the topography of GABAergic effects would primarily be regulated not by the precise placement of receptors but by the precise placement of GABA-releasing synaptic terminals and by the amount of GABA released. The modulators exert their actions via different receptors. If there is no direct contact between two or more neurons, transmitters might reach their target cell by diffusion. This remote control of neuronal activity can be monitored only by those cells that are equipped with receptors sensitive to the modulator (i.e. discrimination).
2.3. FUNCTIONALINTERACTIONBETWEENNEURONS AND TARGETCELLSWITHOUTSYNAPTICCONTACT The non-conventional, non-synaptic release of a substance and the non-synaptic localization of receptors does not necessarily mean that there is an interaction between the release site and, for interneuronal communication, the effector cell. Axon terminals are equipped with different inhibitory and excitatory receptors. These receptors could be the target of modulators released from axon terminals and involved in synaptic and non-synaptic modulation. There are at least two types of presynaptic modulation: (l) Synaptic, when an inhibitory input makes synaptic contact with an axon terminal (axoaxonic synapse) or the initial segment of the cell and the inhibitory modulator released inhibits the release of the principal transmitter. (2) Non-synaptic, when a substance (modulator) is released from an axon terminal and/or varicosity devoid of synaptic characteristics, but it is able to reach the target cell and either stimulate or inhibit the amount of principal transmitter or modulator. 2.3.1. Noradrenergic-cholinergic interactions The first functional evidence of the non-synaptic interactions between axon terminals was provided by Paton and Vizi (1969) in autonomic nervous system in the gut where norepinephrine and epinephrine but not phenylephrine inhibited the electrical stimulation evoked release of acetylcholine from the cholinergic neurons of the Auerbach' plexus, ct-Adrenoceptor blocking agent enhanced the release of acetylcholine. This indicates that norepinephrine tonically controls the release of acetylcholine. In addition it was found that the noradrenergic axon terminals are equipped with muscarinic receptors whose stimulation results in a reduction of norepinephrine release. Atropine, a muscarinic receptor antagonist, did not enhance the release of norepinephrine indicating that the release of norepinephrine is not under the tonic control of cholinergic input. However, when the hydrolysis of acetylcholine was prevented by cholinesterase inhibitor (physostigmine) the release of norepinephrine was tonically controlled, inhibited and atropine enhanced the release of norepinephrine. These findings also indicate that there is no synaptic interaction between cholinergic and noradrenergic axon terminals. Cholinergic axon terminals do not make synaptic contact with noradrenergic varicosities and vice versa. Although both axon terminals are equipped with heteroreceptors, cholinergic with ct2adrenoceptors and noradrenergic with muscarinic, only norepinephrine is able to diffuse the long distance between the two axon terminals and exert its inhibitory effect on acetylcholine release. Acetylcholine released from cholinergic axon terminals may have been destroyed by cholinesterase on the way from release site to target cell, therefore it is not able to reach remote noradrenergic axon terminals. The stimulation of the synaptic noradrenergic fibers (Vizi and Knoll, 1971) or administration of guanethidine
NoN-SYNAPTIC
149
INTERACTIONS AT PRESYNAPTIC LEVEL
or amphetamine, drugs able to release norepinephrine from noradrenergic axon terminals (Knoll and Vizi, 1970; Vizi and Knoll, 1971) produced a similar effect: the release of acetylcholine was reduced or inhibited via an ~t2-adrenoceptor mediated process (Fig. 4). Prior chemical sympathectomy with 6-hydroxydopamine prevented the effect of sympathetic nerve stimulation on acetylcholine release from the Auerbach plexus (Manber and Gershon, 1979). Despite this pharmacological evidence for an adrenergic-cholinergic ultrastructural analysis of the enteric system has failed to demonstrate axo-axonic synapses between these two sets of autonomic neurons (Gordon-Weeks, 1982; Manber and Gershon, 1979). These findings indicate that norepinephrine released in response to sympathetic nerve stimulation should diffuse over relatively long distances before affecting target cells. This provides a concrete example of the non-synaptic interaction between two axon terminals: norepinephrine released from noradrenergic axon terminals inhibited the release of acetylcholine from cholinergic axon terminals through stimulation of presynaptic ~t-2 adrenoceptors. When the hydrolysis of acetylcholine was inhibited by physostigmine, atropine became very effective in enhancing the release of 3H-norepinephrine from a longitudinal muscle strip of guineapig ileum (Fig. 5). Atropine alone, although it significantly enhanced the release of acetylcholine (from 0.84 + 0.06 to 1.23 +__0.07, $2/S~ values), failed to affect the release of 3H-NE. However, when anticholinesterase was applied both 3H-NE and ~4C-ACh release was enhanced by atropine. This finding indicates that cholinergic axon terminals do not make synaptic (Fig. 5) contacts with noradrenergic axon terminals. But when the hydrolysis of acetylcholine is inhibited, it is able to reach noradrenergic axons by diffusion.
O
0301-0082/91/$0.00 + 0.50 © 1991 PergamonPress pie
NON-SYNAPTIC INTERACTIONS AT PRESYNAPTIC LEVEL E. S. Vtzx* a n d E. LkBosI" *Institute of Experimental Medicine, Hungarian Academy of Sciences, H-1450 Budapest, PO Box 67, Hungary "~1st Department of Anatomy, Semmelweis University Medical School, H-1094 Budapest, Tfizolt6 str. 58, Hungary
(Received 24 January 1991)
CONTENTS 1. Introduction 2. Non-synaptic interaction between neuronal presynaptic modulations of transmitter release 2.1. Non-synaptic release sites of transmitters 2.2. Non-synaptic receptor localization; mismatches between signal transmitters and receptors (remote receptors) 2.3. Functional interaction between neurons and target cells without synaptic contact 2.3.1. Noradrenergic-cholinergic interactions 2.3.2. Dopaminergic--cholinergic interactions 2.4. Topographic features of "field" innervation. Regional disparity in fine morphological relationships 3. Pharmacological importance 4. Physiological importance 5. An attempt to merge the synaptic and non-synaptic information processing 5.1. Some forms of interactions 5.2. The possible role of various interactions 5.3. Incorporation of quick, synaptic and slow, non-synaptic interactions into the same explanatory model References
1. INTRODUCTION Our way of thinking about the intercellular transmission of information is still dominated by the knowledge gained from anatomical studies on patterns of neuronal wiring. Within a given neuron, electrical signals are initiated and transmitted as waves of electrical potential change produced by increase in the permeability of the membrane to different ions. The propagated electrical signal reaching the axon terminal ends usually in depolarization and causes the release of chemical substances, provided that Ca0 is available. The concept of chemical neurotransmission is attributed to Elliott (1905): neurons communicate information among themselves and between neuron and target cell by means of chemical substances. Katz, Eccles and colleagues have supplied convincing evidence that in an overwhelming majority of cases transmission of information from cell to cell is chemical in nature. The chemical substances, released by depolarization cross the synaptic cleft and act on the postsynaptic Address correspondence to: Dr. E. S. Vizi, Institute of Experimental Medicine, Hungarian Academy of Sciences, H-1450 Budapest, PO Box 67, Hungary. Phone: 36-1-1137616, Fax: 36-1-114-1866.
145 147 147 148 148 148 151 155 157 157 157 158 158 158 159
membrane equipped with receptors. A nerve impulse traveling toward its final destination (if such a place exists at all) usually does not get there by means of a single neuron. In general, and it is an extensively accepted view, the impulse reaches the end of a neuron that is in contact with a further ceil. The site of contact is called a synapse, or synaptic junction. The presynaptic (junctional) elements are separated from the postsynaptic part by a distance of 15-100 nm: this space is called the synaptic cleft. The first reference to the concept of the synapse was given by Sherrington (1906). Since Sherrington's classic work it has become a doctrine of neurophysiology that the synapse, a part of the surface of separation between neurons, is the primary site of neuronal information processing. It is now accepted that intercellular communication in the nervous system generally involves the release of chemical transmitters from such synapses. The synapse is the most common and generally accepted structural basis of the interaction between neurons, and it implements a one-way communication system between them. In addition to the synaptic communication there is a further possibility for interneuronal communication: it is a non-synaptic interaction between neurons at the presynaptic level (cf. Vizi, 1979, 1980b, 1982, 1984, 1990a,b; Vizi et al., 1991). One
145
146
E.S. Viz] and E. LABOS
neuron can communicate with many others without making synaptic contact: i.e. there is a non-synaptic chemical "cross-talk" between neurons (Vizi, 1979, 1984, 1990a,b; Vizi et al., 1991). This would be a transitional form of communication between discrete classical neurotransmission and the relatively nonspecific neuroendocrine secretion. In the past few years neurochemical, morphological and pharmacological evidence has shown that some neurotransmitters, or substances called modulators, may be released from both synaptic and non-synaptic sites (Fig. 1) for diffusion to target cells more distant than those observed in regular synaptic transmission (Vizi, 1979, 1984, 1990a,b; Cuello, 1983; Iversen, 1984; Schmitt, 1984; Herkenham, 1987), Recently, Agnati et al. (1986) and Fuxe et aL (1988a,b) provided further evidence of this nonsynaptic communication and introduced a new term "volume transmission". It became clear that chemical neurotransmission may be a far more complicated event than previously assumed. Firstly, the release of transmitters/ modulators can be presynaptically modulated (Fig. 2) through auto-, homo- and heteroreceptors (cf. Kalsner, 1990; Langer, 1977; Starke, 1981; Vizi, 1979, 1984; Starke et al., 1989). Secondly, transmitters/ modulators can be released from sites other than axon terminals and from varicosities situated synaptically or non-synaptically. Thirdly, regional dispar-
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Fl~. 1. Scheme of synaptic and non-synaptic interactions between noradrenergic and cholinergic presynaptic nerve terminals. Norepinephrine (NE) non-synaptically released from noradrenergic axon terminals inhibits the release of acetylcholine (ACh) via stimulation of e2-adrenoceptors (1) and inhibits its own release (2, negative feedback, cf. Starke et al., 1989) via stimulation of e2-adrenoceptors. In addition NE, released into a synaptic gap in an axo-axonic synapse, can act in the synapse (negative feedback, 2) and can diffuse away i.e. it can act non-synaptically (3) influencing vast neuronal assemblies. Acetylcholine (ACh) is able to inhibit its own release (4, negative feedback) via stimulation of muscarinic (Mz) receptors located on the axon terminal where it is released from. Acetylcholine released into the synaptic gap acts on the postsynaptic membrane stimulating nicotinic receptors (5, synaptic). Its long-distance effect is terminated by cholinesterase. Presynaptic receptors (in the scheme ~2 and M2 are the examples) may allow the function of presynaptic terminals to be modified in three ways: (1) by transmitters/modulators released from adjacent neurons and are able to diffuse long distance; (11) by synaptic contacts from other neurons onto presynaptic terminals; or (111) by transmitter released from the terminals themselves.
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FIG. 2. Types of presynaptic receptors. Autoreceptors are located on the axon terminals where norepinephrine released from the axon terminal inhibits its own release evoked by subsequent stimuli. Homoreceptors are localized on an adjucant neuron which possesses the same machinery as that from where the modulator was released. Norepinephrine: (NE) released from noradrenergic axon terminals stimulates ~(2-adrenoceptors(heteroreceptors) located on an axon terminal that can not produce that modulator which is able to act on it (norepinephrine acts on a non-noradrenergic axon terminal via stimulation of ~2-adrenoceptor). ities in the relationships of the inputs to different regions of the brain make possible the regional nonsynaptic/synaptic control of chemical neurotransmission in the neuronal network. Because synaptic communication between cells had been the most thoroughly studied form of intercellular communication, it was later accepted that it is the only form of interaction between neurons and that any change in neuronal excitation is the result of synaptic (chemical or rarely electrical) transmission. Thus, the classical view of neurotransmission is that it takes place at the synapse. Much of our present knowledge about the cellular organization of the different brain areas derives from Golgi studies, dating to the end of the last century when Golgi (1883), Ramon y Cajal (1891, 1911), and many others opened the modern era of neuroanatomy. One limitation of the Golgi technique is that synaptic contacts can be predicted only by indirect correlation of separately impregnated dendritic and axonal patterns. An important progress was made when the Golgi method was introduced with the electron microscopic analysis of the stained neurons (Blackstad, 1965). The gold technique also provided an opportunity to circumvent another limitation of the Golgi method, its failure of histochemical specificity. Using this technique, the processes of cells remain traceable, but with removal of most of the Golgi method precipitate from the impregnated cells, they can be characterized by the aid of techniques such as autoradiography (Somogyi et al., 1981b), immunocytochemistry (Somogyi et al., 1983), and enzyme histochemistry (Bolam et al., 1984a; Somogyi et al., 1979; Somogyi and Smith, 1979; cf. Freund and Somogyi, 1989). A new avenue was paved by the analysis of identified circuits with the application of
NoN-SYNAPTIC INTERACTIONSAT PRESYNAPTIC LEVEL
Golgi impregnation to brain tissue that also contained electrophysiologically characterized and intracellularly horseradish peroxidase-injected neurons (Freund and Somogyi, 1983; Freund et al., 1985b). But it has to be confessed, that with these techniques alone and with their combinations it is still not possible to provide a functional characterization of identified cell types or to understand how (and why) neurons talk to each other. The combined neurochemical, pharmacological, anatomical, and histochemical analysis of the same synaptic circuits became possible. With these combined techniques it will be possible to explore and understand how the nervous system and the brain functions, one of the greatest challenges of our age. The efficacy of chemical synaptic transmission may be significantly altered by either presynaptic or postsynaptic changes. Similarly, the efficacy of a chemical non-synaptic communication system may be controlled, at least in part, by changes in the amount of neurotransmitter/modulator substance released from non-synaptic nerve terminals. The complexity of chemically mediated non-synaptic/ synaptic transmission provides a substrate for considerable flexibility. The sensitivity of transmitter/ modulator release to changes in presynaptic membrane potential may differ significantly between synapses and will depend largely upon the types of ion channel in the membrane of axon terminals. Since there are a number of receptor mediated ion channels that exhibit voltage sensitivity, the presynaptic receptor mediated effect of transmitters/modulators may be dependent on the actual membrane potential. The present review puts the emphasis on nonsynaptic interneuronal communication at the presynaptic level. 2. NON-SYNAPTIC INTERACTION BETWEEN NEURONAL PRESYNAPTIC MODULATIONS OF TRANSMITTER RELEASE 2.1. NoN-SYNAPTIC RELEASE SITES OF TRANSMITTERS A rather crucial question is as follows: are transmitters/modulators normally released from nonsynaptic axon terminals? The answer is, yes. There is convincing evidence that postganglionic neurons innervating vas deferens (Burnstock and Costa, 1975), blood vessels (Thureson-Klein and Stjarne, 1981), and smooth muscle (Burnstock and Costa, 1975) terminate in a network of varicose areas (boutons en passant) that lack synaptic contact with the target cell and still able to release transmitters in response to axonal electrical activity (cf. Vizi, 1984). The transmitter is released from structurally unspecialized areas along the varicose fibers and must often travel a considerable distance before interacting with its receptors. Sometimes this distance could be more than 1 #m (Fig. 3). Descarries and coworkers (1977, 1980, 1987) demonstrated that non-synaptic varicosities in the central nervous system appear to have all the apparatus normally associated with synaptic release. Subsequently, ultrastructural examination of noradrenergic varicosities in several tissues confirmed that both large and small vesicles could undergo JPN 37/2--E
147
Trinimltter
(
\-.. IJm
FIG. 3. Distance between release site and target receptor at synaptic and non-synaptic interaction. While in a synaptic contact the distance between release site and postsynaptic receptor is 20-50 nm, in case of non-synaptic interaction this could be more than I/am. exocytosis in the absence of structurally specialized active zones (Thureson-Klein, 1983, 1984; ThuresonKlein and Stjarne, 1981; Zhu et al., 1986). In addition, morphological evidence was provided (Buma, 1989a) for exocytosis in non-synaptic release site in rat median eminence and mesencephalic central grey substance. It is conceivable that a similar exocytosis occurs in monoaminergic neurons in the gut (Gordon-Weeks, 1982) and in the brain (Bj6rklund et al., 1973; Descarriers et al., 1977, 1980, 1987) where active synaptic zones are lacking. It was reported (Barber et al., 1979; Glazer and Basbaum, 1983) that certain terminals in the dorsal horn of the spinal cord and trigeminal nucleus caudalis (Zhu et al., 1986) do not have typical synaptic zones. Therefore, it is likely that substance P- or enkephalin-containing vesicles do not release their contents at synaptic sites (Barber et al., 1979; Cuello et al., 1977; Del Fiacco and Cuello, 1980; Glazer and Basbaum, 1983; Hunt et aL, 1980). The peripheral exocytosis at a distance from the synaptic complex suggests that large vesicles have a different physiological function to the small synaptic vesicles. Exocytotic release from large vesicles containing neurohormones has been also reported to occur at non-specialized areas of the membrane in invertebrates (Buma and Roubos, 1986; Roubos, 1984; Buma, 1989a,b; Schmidt and Roubos, 1989). Since the discovery of immunoreactive ~-melanocytestimulating hormone (~-MSH) in the rat brain, there have been a few reports about the distribution of this peptide in the central nervous system (Pelletier and Dube, 1977). With immunoelectron microscopy it has been shown that ~-MSH could only be recognized in dense core vesicles in axon terminals and cell bodies. It was suggested that most if not all brain ~-MSH is released by neurons at sites other than the classical synaptic junctions, probably to act non-synaptically (Guy et al., 1982). Similarly, no more than 50% of VIP containing endings have been reported to make synaptic contact (Pelletier et al., 1981). Therefore it seems very likely that modulator and transmitter substances released (a) from non-synaptic axon terminals, (b) from axon terminals making synaptic contact but acting both on synaptic and non-synaptic sites of target cells, or (c) from regions of the nerve cell other than the presynaptic axon
148
E.S. VlZl and E. L,£Bos
terminal (i.e. dendrites, axon, soma) (Vizi et al., 1983), can play a physiological role in the modulation of neurochemical transmission. 2.2. NoN-SYNAPTICRECEPTORLOCALIZATION; MISMATCHESBETWEENSIGNALTRANSMITTERS AND RECEPTORS(REMOTERECEPTORS)
There is an interesting difference in localization and sensitivity of receptors on effector cells where the transmitter (e.g. acetylcholine, norepinephrine, dopamine, serotonin or neuropeptides) is released into a large extraneuronal space (Herkenham, 1987). In the case of synaptic transmission, there is a small area of the effector cell where the receptors are concentrated. The neuromuscular junction is an example of a per.fect correspondence between release sites of transmitter, acetylcholine and the location of postsynaptic receptors. However, when the transmitter release site and the target cells are widely separated from each other (i.e. 100-3000 nm), there is no specific subsynaptic arrangement, and at the recognition sites, receptors are unevenly distributed along the surface of the effector cell (e.g. presynaptic axon terminals, etc.). This morphological arrangement fits a diffusion type of transmission in which the possible advantage of quantal release cannot be used. There seems to be no evidence that non-quantal release can occur solely at diffuse synapses. In addition, it could be argued that the receptors distant from the synaptic sites do not represent active receptors capable of opening ion channels. However, it is also known that the sensitivity of extrasynaptic receptors is higher to chemical signals. In addition, it was shown that the release site/receptor matches are exceptions rather than rules (cf. Herkenham, 1987). The explanation for the widespread extrasynaptic distribution of the receptor complex may lie in the general role of the transmitter-gated channels. The synaptic and non-synaptic receptors may be used under different levels of neuronal activity, providing a mechanism for adaptation. Somogyi et al. (1989) showed in cerebellum that at low levels of neuronal activity, gamma-aminobutyric acid (GABA) acting at the synaptic junctions provides adequate inhibitory control of the neurons. The moderate amounts of G A B A released can be removed by reuptake or glial uptake without reaching the extrasynaptic sites. However, at increased excitatory input, the number of channels operating at the junctions may not be suitable or adequate to keep the activity of the cell in the range of optimal sensitivity, in which case channels at extrasynaptic sites would be opened by GABA diffusing from the release sites. Consequently, the topography of GABAergic effects would primarily be regulated not by the precise placement of receptors but by the precise placement of GABA-releasing synaptic terminals and by the amount of GABA released. The modulators exert their actions via different receptors. If there is no direct contact between two or more neurons, transmitters might reach their target cell by diffusion. This remote control of neuronal activity can be monitored only by those cells that are equipped with receptors sensitive to the modulator (i.e. discrimination).
2.3. FUNCTIONALINTERACTIONBETWEENNEURONS AND TARGETCELLSWITHOUTSYNAPTICCONTACT The non-conventional, non-synaptic release of a substance and the non-synaptic localization of receptors does not necessarily mean that there is an interaction between the release site and, for interneuronal communication, the effector cell. Axon terminals are equipped with different inhibitory and excitatory receptors. These receptors could be the target of modulators released from axon terminals and involved in synaptic and non-synaptic modulation. There are at least two types of presynaptic modulation: (l) Synaptic, when an inhibitory input makes synaptic contact with an axon terminal (axoaxonic synapse) or the initial segment of the cell and the inhibitory modulator released inhibits the release of the principal transmitter. (2) Non-synaptic, when a substance (modulator) is released from an axon terminal and/or varicosity devoid of synaptic characteristics, but it is able to reach the target cell and either stimulate or inhibit the amount of principal transmitter or modulator. 2.3.1. Noradrenergic-cholinergic interactions The first functional evidence of the non-synaptic interactions between axon terminals was provided by Paton and Vizi (1969) in autonomic nervous system in the gut where norepinephrine and epinephrine but not phenylephrine inhibited the electrical stimulation evoked release of acetylcholine from the cholinergic neurons of the Auerbach' plexus, ct-Adrenoceptor blocking agent enhanced the release of acetylcholine. This indicates that norepinephrine tonically controls the release of acetylcholine. In addition it was found that the noradrenergic axon terminals are equipped with muscarinic receptors whose stimulation results in a reduction of norepinephrine release. Atropine, a muscarinic receptor antagonist, did not enhance the release of norepinephrine indicating that the release of norepinephrine is not under the tonic control of cholinergic input. However, when the hydrolysis of acetylcholine was prevented by cholinesterase inhibitor (physostigmine) the release of norepinephrine was tonically controlled, inhibited and atropine enhanced the release of norepinephrine. These findings also indicate that there is no synaptic interaction between cholinergic and noradrenergic axon terminals. Cholinergic axon terminals do not make synaptic contact with noradrenergic varicosities and vice versa. Although both axon terminals are equipped with heteroreceptors, cholinergic with ct2adrenoceptors and noradrenergic with muscarinic, only norepinephrine is able to diffuse the long distance between the two axon terminals and exert its inhibitory effect on acetylcholine release. Acetylcholine released from cholinergic axon terminals may have been destroyed by cholinesterase on the way from release site to target cell, therefore it is not able to reach remote noradrenergic axon terminals. The stimulation of the synaptic noradrenergic fibers (Vizi and Knoll, 1971) or administration of guanethidine
NoN-SYNAPTIC
149
INTERACTIONS AT PRESYNAPTIC LEVEL
or amphetamine, drugs able to release norepinephrine from noradrenergic axon terminals (Knoll and Vizi, 1970; Vizi and Knoll, 1971) produced a similar effect: the release of acetylcholine was reduced or inhibited via an ~t2-adrenoceptor mediated process (Fig. 4). Prior chemical sympathectomy with 6-hydroxydopamine prevented the effect of sympathetic nerve stimulation on acetylcholine release from the Auerbach plexus (Manber and Gershon, 1979). Despite this pharmacological evidence for an adrenergic-cholinergic ultrastructural analysis of the enteric system has failed to demonstrate axo-axonic synapses between these two sets of autonomic neurons (Gordon-Weeks, 1982; Manber and Gershon, 1979). These findings indicate that norepinephrine released in response to sympathetic nerve stimulation should diffuse over relatively long distances before affecting target cells. This provides a concrete example of the non-synaptic interaction between two axon terminals: norepinephrine released from noradrenergic axon terminals inhibited the release of acetylcholine from cholinergic axon terminals through stimulation of presynaptic ~t-2 adrenoceptors. When the hydrolysis of acetylcholine was inhibited by physostigmine, atropine became very effective in enhancing the release of 3H-norepinephrine from a longitudinal muscle strip of guineapig ileum (Fig. 5). Atropine alone, although it significantly enhanced the release of acetylcholine (from 0.84 + 0.06 to 1.23 +__0.07, $2/S~ values), failed to affect the release of 3H-NE. However, when anticholinesterase was applied both 3H-NE and ~4C-ACh release was enhanced by atropine. This finding indicates that cholinergic axon terminals do not make synaptic (Fig. 5) contacts with noradrenergic axon terminals. But when the hydrolysis of acetylcholine is inhibited, it is able to reach noradrenergic axons by diffusion.
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