Camp. Biochem. Physiol. Vol. 103A, No. 2, pp. 227-239, 1992

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MINI REVIEW PRESYNAPTIC MODULATION OF SENSORY AFFERENTS IN THE INVERTEBRATE AND VERTEBRATE NERVOUS SYSTEM ALAN H. D. WATSON Department of Anatomy, University of Wales College of Cardiff, P.O. Box 900, Cardiff CFI 3YE, U.K. Tel.: (0222) 87400-5156;Fax: (0222) 666-863 (Received

5 March 1992; accepted 8 April 1992)

Abstract-l. Ultrastructural examination of the central terminals of sensory afferent neurons in both invertebrates and vertebrates demonstrates that the synapses that form the substrate for presynaptic inhibition and facilitation are almost universally present. 2. Presynaptic modulation of afferent input acts in many ways which tailor the inflow of sensory information to the behaviour of the animal, effectively providing a means of turning this on and off, or of combining information of the same or different modalities to refine responsiveness or clarify ambiguity. 3. Presynaptic modulation may act in several different roles on the same afferent. 4. A comparison of the mechanisms of presynaptic inhibition in different animals demonstrates the likelihood of a variety of common mechanisms, several of which may act simultaneously on the same terminal. These include changes in the conductance of the afferent membrane to Cl-, K+ and Ca2+ ions, in addition to less well understood mechanisms that directly affect transmitter release. 5. A single transmitter can produce several effects on a terminal through the same or different receptors. 6. Ultrastructural studies of afferent terminals reveal that only a proportion of boutons on a given afferent may receive presynaptic input and that this may depend on the region of the nervous system in which these are found or on the identity of the postsynaptic neurons contacted. 7. The synaptic relationships of afferent terminals can be complex. In invertebrates different types of presynaptic neuron may interact synaptically, as may postsynaptic dendrites in vertebrates. 8. Axons presynaptic to afferent terminals in vertebrates frequently synapse also with dendrites postsynaptic to the afferents. 9. In both invertebrates and vertebrates reciprocal interactions between afferents and postsynaptic neurons are seen. 10. Ultrastructural immunocytochemistry reveals the likely dominance of GABA as an agent of presynaptic inhibition but also demonstrates the possible presence of other transmitters some of whose roles are less completely understood.

The central nervous systems of vertebrates and invertebrates receive a constant barrage of afferent input from receptors carrying information from a wide range of sensory modalities and receptors. This inflow of sensory information must be filtered and controlled if an animal is to make a coherent and contextual!y relevant behavioural response to its environment. Some streams of information, perhaps due to inhabituation, will make no impact on behaviour. Others may be combined to clarify potentially ambiguous signals, to sharpen the specificity of incoming information, or to prevent the habituation of sensory pathways underlying responses critical to survival. Even when a sensory input does produce a behavioural result, this must be integrated into the ongoing activity of the animal. For example, the same stimulus may have different or even opposite effects depending on its timing with respect to the phase of a cyclical behaviour such as walking.

The modulation of sensory information can occur at any or all of four notional levels within the central nervous system (see Sillar 1989, 1991); (1) at the afferent terminal where neurons can act presynaptitally to influence transmitter release, (2) postsynaptitally onto sensory interneurons, (3) on premotor interneurons and (4) at the final common path, the motor neuron pool. In addition, in some systems modulation of the receptor potential at the peripheral transducing region of the sensory neuron may also be possible (Pasztor and Bush, 1987; El Manira et al., 1991b). The relative importance of each level will vary from circuit to circuit, and modality to modality. For some circuits, such as monosynaptic reflex arcs, not all will be present. I will concern myself here only with the first level, the presynaptic modulation of sensory afferents, which ultrastructural evidence suggests may be an almost universal phenomenon. Presynaptic influences are distinguished from postsynaptic ones in being potentially very selective, enabling the effect of a particular sensory modality 221

ALAN H. D. WATSON

228

or population of sense organs to be controlled without altering the responsiveness of the postsynaptic neuron to other sensory or central inputs. Presynaptic modulation of sensory input has heen widely studied in both vertebrates and invertebrates, however invertebrate studies have proved of particular value because of the detail with which it has been possible to analyse the circuitry and (especially in molluscs) the cellular processes involved. Though the transmitters used by molluscs, arthropods and vertebrates are not always the same, it appears increasingly likely that many of the underlying cellular mechanisms will prove to be common across the animal kingdom. THE ROLE OF PRESYNAPTK

MODULATION

Presynaptic inhibition may fulfil a variety of functions and more than one may be enacted at the same synapse. Prevention of habituation With use, a synaptic connection may habituate either due to a decrease in the amount of transmitter released from the presynaptic neuron, or to a desensitization of receptors on the postsynaptic neuron. During certain ~haviours this may be maladaptive and presynaptic inhibition provides a way to protect the synapse from habituation. This has been demonstrated in circuitry involved in the crayfish tail flip response (Krasne and Bryan, 1973; Bryan and Krasne, 1977; Kirk and Wine, 1984; Kirk, 1985) and in the cereal system of the cricket (Levine and Mu~hey, 1980). In response to tactile stimulation of the abdomen, the crayfish vigorously flexes the tail to drive itself through the water in a bid to escape. The response habituates rapidly, due to a decline in the efficacy of the first synapse of the reflex arc between the primary afferent neurons and a population of interneurons. The afferents are stimulated not only by potentially menacing stimuli but also by water currents flowing over the abdomen during escape behaviour itself or during non-escape swimming. Without some form of protection such behaviour would habituate the synapse between the afferent terminals and the sensory interneurons and thus prevent further activation of the escape reflex. This would pose a major problem for the animal because stimuli delivered minutes apart may result in habituation lasting hours (Krasne and Bryan, 1973). The situation is avoided because the afferent synapses vulnerable to depression are protected during escape behaviour by presynaptic inhibition mediated by interneurons activated by the tail flip motor circuits (Kirk and Wine, 1984). In the cricket a similar mechanism prevents habituation at synapses between afferents from vibration sensitive hairs on the cercus and giant ascending interneurons that mediate escape responses (Levine and Murphey, 1980). Activity in afferents on one cercus indirectly leads to a presynaptic inhibition of synapses made by afferents from the other cercus. The long latency of the effect ensures that the afferents do stimulate the interneurons but limits the duration of the response and therefore prevents synaptic depression. The response to sustained back-

ground noise or the animal’s therefore limited.

own movement

is

Sharpening qf receptive Jields The afferents supplying sensory hairs on the arthropod cuticle may exhibit a dir~tionally selective response i.e. they are maximally excited by deflection in one plane (Tobias and Murphey, 1979). The specificity of such responses can be enhanced by the blocking of the signal from one afferent by the activity of another which is sensitive to deflection in a different direction. These interactions can take place (a) between sensory interneurons (Wiese et ai., 19X), (b) indirectly between the afferents and the sensory interneurons, or (c) by presynaptic inhibition, again indirectly, between the afferents themselves (Levine and Murphey, 1980). Furthermore, the interactions may take place at more than one level within the same system. In both the cricket (Levine and Murphey, 1980) and the first instar cockroach (Blagburn and Sattelle, 1987) there is evidence that indirect connections between afferents from flliform hairs on the cerci enhance their directional sensitivity. The cricket afferents are the same ones as those described in the previous section and so it is clear that presynaptic inhibition can serve more than one function for the same afferent. Phasic integration of sensory information into rhythmic behaviour In the vertebrate nervous system, primary afferent terminals are known to be subject to cycles of depolarization in phase with locomotory rhythms (Gossard et al., 1989, 1990, 1991; Sillar, 1991) which may result in a phasic decrease in transmitter release. This may in part, underlie the observation that a reflex elicited during one phase of the cycle can be reversed at another. In the cat for example, tactile stimulation to the dorsal surface of the paw during the swing phase of the walking cycle will result in flexion so that the limb is raised as if to clear an obstacle. The same stimulus during the stance phase elicits the opposite response, an increase in leg extension (Forssberg et al., 1977). In the crayfish, intracellular recordings from two nonspiking afferents from the thoracocoxal muscle receptor organ (TCMRO) have revealed similar fluctuations in membrane potential in phase with a locomotory rhythm (Sillar and Skorupski, 1986). The S and T afferent fibres of the TCMRO undergo rhythmic cycles of depolarization that are 180” out of phase with each other. The S fibre excites promotor leg motor neurons and inhibits remotor neurons, while the T fibre excites remotor motor neurons and inhibits promotor motor neurons. These reciprocal fluctuations in membrane potential ensure that the necessary dominance of the effects of one fibre over that of the other is correlated to the correct phase of the walking cycle (Skorupski and Sillar, 1986). The result is therefore comparable to the phase-dependent reflex reversal described in the cat except that the decrease in drive from the crayfish afferents occurs when they are hyperpolarized rather than when they are depolarized. The reason for this difference is that a depolarization of an afferent terminal reduces transmitter release indirectly by reducing the height and

Presynaptic modulation

width of the action potential (see below). In nonspiking neurons like those of the TCMRO, whose graded release of transmitter can be tonic, this would only serve to increase transmitter release and inhibition must be brought about by a hyperpolarization. Presynaptic facilitation is know from fewer examples than presynaptic inhibition. During the cyclical fluctuations in the afferent terminal membrane potential that accompany locomotory cycles, facilitation occurs at the opposite phase of the cycle to inhibition. Under such circumstances it may not be a distinct phenomenon but represent only the absence of presynaptic inhibiton (Rudomin et al., 1974). In Aplysia however, it has been shown to have a different role; that of nullifying a previously habituated response to a mild mechanical stimulus when a strong stimulus is applied to another region of the body (see Bailey et al., 1981).

MECHANISMS

OF PRESYNAPTIC

MODULATION

Presynaptic inhibition

Most of the processes described so far are examples of presynaptic inhibition. There are several mechanisms by which this can be effected. The transmitter most widely implicated in presynaptic inhibition within both the vertebrate and invertebrate nervous systems is GABA (Eccles et al., 1963; Davidoff, 1972; Nicoll and Alger, 1979; Hue and Callec, 1983). Ultrastructural immunocytochemistry has demonstrated immunoreactivity for GABA or its synthetic enzyme glutamate decarboxylase, in processes presynaptic to afferent terminals in the rat (Barber et al., 1978; Todd and Lochhead, 1990) and cat (hair follicle afferents-Maxwell and Noble, 1987) spinal cord. In the locust, terminals of cereal afferents (Watson, 1990) of a type known to undergo presynaptic inhibition (Boyan, 1988) also receive inputs from GABA immunoreactive processes, as do afferent terminals from campaniform sensilla (Watson and England, 1991) and hair plates (Watson et al., 1991b) on the legs. In the vertebrate nervous system two types of GABA receptor (GABA, and GABA,) are known. These act through different ionic mechanisms and having different pharmacological profiles. It is the GABAA receptors that mediate the primary afferent depolarization most commonly associated with presynaptic inhibition. Activation of GABA, receptors results in an increase in Cl- ion permeability. Depending on the relative values of the resting potential of the afferent terminal and the reversal potential for chloride, this may result in a depolarization, a hyperpolarization or no change at all in membrane potential. In the crayfish, chordotonal afferents have been shown to undergo a chloride-dependent depolarization following application of GABA (El Manira and Clarac, 1991). In the locust, GABA also induces a depolarization of chordotonal afferents (Burrows and Laurent, in preparation). This has a reversal potential that is 4 mV more positive than the afferent resting potential, which is identical to the reversal potential of postsynaptic potentials recorded in the terminal as an indirect result of activity in other chordotonal afferents. Both the EPSP and the effect

229

of GABA is blocked by picrotoxin, which suggests a chloride dependence though, as the inhibition outlasts the conductance change, other processes may also be at work. In the cockroach cereal system, Hue and Callec (1983) found that GABA produced a chloride-dependent hyperpolarization at least under the experimental conditions used. Regardless of the sign of the potential change, the increase in chloride permeability of the afferent terminals results in a decrease in transmitter release through its action on incoming action potentials. A small depolarization reduces the height of the action potential partly because the potential then rises from a less negative value and partly because it will result in a slight increase in voltage-dependent potassium conductance and in the inactivation of the voltage-gated sodium channels (Aidley, 1978). However, even if the membrane potential is at or above the reversal potential for chloride, the height of the action potential will still be reduced because the increase in the membrane permeability will act as a current shunt (Baxter and Bittner, 1981; Hue and Callec, 1983). This may result in spike failure through small diameter branches (Atwood, 1976). Even a small reduction in action potential amplitude may have a profound effect on transmitter release. It has been calculated that at the rat neuromuscular junction a 15 mV reduction in action potential height would produce a 90% reduction of transmitter release because of the effect this would have on voltage-gated Ca*+ channels (Aidley, 1978). Intracellular recordings from locust filiform hair afferents reveal a less dramatic effect with a reduction in action potential amplitude of up to 55% producing a 15-23% reduction in the evoked excitatory postsynaptic potentials in a postsynaptic giant interneuron (Boyan, 1988). Intracellular recordings from vertebrate muscle afferents and postsynaptic motor neurons were first used to demonstrate that depolarization of afferents was associated with reduction in the amplitude of the excitatory postsynaptic potential by Eccles et al. (1961, 1962a,b). This was a remarkable piece of work for its time and most of the subsequent studies by other workers have used more indirect methods to study primary afferent depolarization. Recent intracellular studies from muscle spindle and cutaneous afferents in the cat have demonstrated that the level of presynaptically induced depolarization of the terminals can be sufficient to generate action potentials (Gossard et al., 1989, 1991). Similar results have been obtained from lobster chordotonal afferents (El Manira et al., 1991a). There is evidence that the antidromic impulses so generated do travel back along peripheral nerves (Repkin et al., 1976; Duchen, 1986). These antidromic potentials might be expected to lead to transmitter release from the terminal and at first appear inimical to the process of inhibition. In the case of the lobster chordotonal afferents however, simultaneous recordings from postsynaptic motor neurons have demonstrated that these antidromic potentials did not produce a postsynaptic potential change (El Manira et al., 1991a), though the reason for this is unclear. Antidromic potentials could block the passage of centrally running action potentials on the same axons. The effectiveness of this as a means of eliminating afferent input would, of course, depend

230

ALAN

H. D.

on the relative timing of the antidromic and orthodromic volleys. Primary afferent depolarization can also be brought about by mechanisms which do not involve chloride ions. In the first instar cockroach primary afferent depolarization of filiform hair afferents on the cercus is ultimately achieved by the opening of delayed rectifying potassium channels (Blagburn and Sattelle, 1987). The primary event, the excitatory postsynaptic potential, has a reversal potential more positive than -35 mV and so neither Cl- nor K+ is likely to be the charge carrier. The transmitter responsible is also unknown. This is the same system in which Hue and Callec (1983) demonstrated inhibition mediated by a GABA-mediated increase in chloride conductance, however in the first instar there are only two filiform hairs on each cercus. Hue and Callec (1983) studied the adult cockroach which has many afferents on its cercus. Activity in these was recorded en masse using mannitol and oil gap techniques and so only the average response of the whole cereal nerve was revealed. It therefore appears either that two mechanisms for primary afferent depolarization exist together on different classes of cereal afferent, or that they exist in the same system at different stages of development. Though GABA, -induced primary afferent depolarization was for some time considered sufficient explanation for the presynaptic inhibition of vertebrate sensory afferents (Davidoff, 1972; Nicoll and Alger, 1979) this has recently been called into question. In the autonomic nervous system, presynaptic inhibition of preganglionic sympathetic axons is not lost when axon terminal depolarization is blocked by bicuculline, a specific GABA, antagonist (Brown and Higgens, 1979; Bowery et al., 1980). Subsequent experiments have demonstrated that presynaptic inhibition of sensory afferents can be mediated by GABAs receptors (Curtis, 1981; Christensen and Grillner, 1991). This has led some to dismiss the role of GABA, receptors in presynaptic inhibition (see War, 1989). The link between presynaptic depolarization with its consequent reduction in action potential amplitude, and the reduction in the postsynaptic EPSP so clearly demonstrated in the squid giant synapses (Miledi and Slater, 1966) and the cockroach cereal system (Blagburn and Sattelle, 1987) cannot, however, be so lightly dismissed. There have been few studies designed to -investigate in detail the relative importance of GABA, and GABAs receptors on afferent (or other) terminals. In the case of frog muscle spindle afferents at least, Peng and Frank (1989a,b) have demonstrated that both GABA, and GABA, receptors can mediate presynaptic inhibition. The GABA, receptors act without altering the height of the action potential and in sensory and hippocampal neurons have been reported to block voltagegated calcium channels (Dunlap, 1981; Dolphin and Scott, 1987) and increase potassium conductance (Gahwiler and Brown, 1985; Howe et al., 1987). Both of these actions would result in inhibition of transmitter release. Unlike GABA, receptors, GABA, receptors appear to act through a G-protein second messenger system (Holz et al., 1989). Intracellular experiments on interneurons in the lamprey spinal cord, which also experience GABA, mediated pre-

WATSON

synaptic inhibition, demonstrate that these receptors do indeed activate a G-protein and that this results in a reduction in the duration of the presynaptic action potential and the amount of transmitter released (Alford and Grillner, 1991). The means by which this is achieved in the lamprey neurons has still to be determined. The pharmacology of vertebrate and invertebrate receptors for the same transmitters can differ considerably (Benson, 1988; Yarowski and Carpenter, 1978). In the crayfish, GABA receptors on afferent neurons, though activating chloride channels, cannot be classified on the basis of agonist/antagonist profiles as either GABA, or GABA, and there is as yet no evidence in invertebrates for a GABA-mediated activation of Ca2+ or K+ conductances however, as will be seen, this can be achieved in molluscs by other transmitters via mechanisms involving G-proteins or other second messenger systems. Detailed cellular studies on cultured afferent neurons from the marine mollusc Aplysia have demonstrated that a single transmitter (the peptide FMRFamide) can activate several mechanisms in concert to produce presynaptic inhibition. Two of the mechanisms involve changes in ionic conductance. They are (1) an increase in the probability of opening in a population of K+ channels, thus reducing the duration of the action potential (Belardetti et al., 1987) and hence of calcium influx and (2) a direct transmitter-mediated reduction in the voltage-sensitive Ca2+ current (Brezina et al., 1987). Histamine can produce a similar effect in the Aplysiu interneuron LlO (Shapiro et al., 1980; Kretz et al., 1986). The two effects are thought to be mediated by the same receptor via a G-protein second messenger system (see Brezina et al., 1987). In the sensory neurons however, even when intracellular calcium is buffered by the presence of a chelator, FMRFamide can still reduce the spontaneous release of transmitter (Dale and Kandel, 1990). The mechanism of this is unknown but could be due, for example, to an inhibition of the movement of transmitter from a storage to a releasable pool, or to interference with vesicular translocation. A similar calcium-independent effect has been observed at synapses between somata from another mollusc (He&ma), grown in culture. Here the FMRFamide has also been demonstrated to act via a G-protein (Haydon et al., 1991). Though FMRFamide-containing neurons that produce presynaptic inhibition were not initially identified, one acting on Aplysia sensory afferents has recently been shown to be immunoreactive for the peptide (Small et al., 1992). A role for glycine in presynaptic inhibition has been suggested in the lamprey spinal cord (Viana di Prisco et al., 1990). This system is unusual not only because the neurons directly responsible for presynaptic inhibition have only rarely been identified but also because here they are other afferent neurons. The lamprey spinal cord contains intrinsic neurons (edge cells) that monitor the stretch of the cord itself. Edge cells from opposite sides of the spinal cord inhibit each other reciprocally. The inhibition takes the form of a hyperpolarization that is blocked by strychnine and is hence thought to be mediated by glycine.

231

Presynaptic modulation Though it has recently been reported that strychnine can also block GABA-activated currents in lamprey spinal neurons (Baev et al., 1992) antibodies against GABA do not label edge cells (Brodin et al., 1990). The ionic mechanism of this inhibition has not been investigated but both glycine and GABA have been shown to increase chloride conductance in isolated lamprey spinal neurons (Baev et al., 1992). Immunocytochemical studies of the insect nervous system have revealed no evidence for the presence of glycine (Watson, unpublished) and it is not thought to act as a transmitter in the invertebrate nervous system (Cooper et al., 1986). In vertebrates it has been suggested that at micromolar concentrations enkephalin and dynorphin presynaptically inhibit the release of transmitter from primary afferents of the pain pathways (Jesse1 and Iversen, 1977) either by a cyclic AMP-mediated reduction of calcium conductance or an increase in potassium conductance (see Crain and Shen, 1990; Millan, 1990). However at lower concentrations, these transmitters may facilitate transmitter release in the same or different afferent neurons by precisely the opposite action of increasing calcium conductance or decreasing potassium conductance. In agreement with these observations, a paradoxical hyperalgesia has been described experimentally following the administration of low doses of morphine (Kayser et al., 1987) but the functional significance of such a double response to endogenous opiates is unknown. In the caterpillar of the moth Manduca sexta, application of muscarinic cholinergic agonists reduces the amplitude of excitatory postsynaptic potentials evoked by afferents from mechanosensory hairs on the prolegs, in a manner that would be consistent with presynaptic inhibition (Trimmer and Weeks, 1989). This does not necessarily imply, however, that other neurons are involved. The afferents, in common with many others in insects, are thought to release acetylchohne and it has been suggested that such presynaptic muscarinic receptors might be part of a mechanism for the self regulation of transmitter release (Hue et al., 1989) as has been proposed in the vertebrate nervous system (Polak, 1971; Raiteri et al., 1983). Antibodies against choline acetyltransferase, the synthetic enzyme for acetylchohne have however been shown to label processes presynaptic to presumed sensory afferent terminals in glomeruli of laminae II and III in the dorsal horn of rat spinal cord (Ribeiro-da-Silva and Cuello, 1990). Cholinomimetic drugs applied to the surface of the spinal cord can block nociception (Yaksh et al., 1985), but the site or sites where this takes place, and the mechanisms involved are unknown. The significance of the immunocytochemical observations therefore remains to be determined. It is clear that there are many ways in which presynaptic inhibition can be brought about. More than one mechanism may act in a single ganglion or small region of the nervous system. Even within the same afferent terminal a single transmitter, acting through one or more classes of receptor, may orchestrate several processes that can each bring about a reduction in transmitter release, while in different

animals the same cellular mechanisms may be instigated by different transmitters. Presynaptic facilitation

Though the most widely reported form of presynaptic modulation is inhibition, presynaptic facilitation has been observed and in the marine mollusc Aplysia, and its mechanism has been described at a cellular level. The facilitation of the habituated gill withdrawal reflex described in the previous section is the result of an enhancement of Ca*+ influx into the afferent terminal. This is mediated by a serotonindependent reduction of K+ permeability which leads to a broadening of the action potential. The reduction in K+ permeability is brought about by the prolonged closure of a population of K+ channels (S-channels, Siegelbaum et al., 1982) via a cyclic-AMP dependent protein phosphorylation (Belardetti et al., 1987). These are the same potassium channels whose opening time is reduced by FMRFamide (see above). In the presence of a Ca2+ chelator serotonin still enhances the frequency of spontaneous excitatory postsynaptic potentials suggesting that it can also act directly on transmitter release by a mechanism not dependent on Ca2+ flux. THEORETICAL

MODELS

OF PRESYNAPT’IC

INHIBITION

Models of presynaptic inhibition confirm the intuitive impression that the most effective position (i.e. where the smallest conductance increase will cause inhibition) for an inhibitory synapse is close to the output synapse (Atwood et al., 1984; Segev, 1990). Using typical values for membrane capacitance and axoplasmic and membrane resistivity the model suggests that one or a few quanta from an axo-axonal synapse may be enough to reduce greatly or even block an action potential at an adjacent release site on the terminal. The effect on synaptic release close to the presynaptic input is graded with respect to the size of the conductance increase (Segev, 1990). Input synapses onto insect afferents from exteroceptors (Geisert and Altner, 1974; Watson and Pfltiger, 1984; Watson, 1990) and campaniform sensilla and other proprioceptors (Altman et al., 1980; Watson et al., 199lb) are found close to the site of synaptic output. The same is true of vertebrate afferents. Presynaptic inputs are present on the synapse-carrying varicosities and with rare exceptions (Maxwell et al., 1982) not on the segments of axon between them (Fyffe et aI., 1986; Rethelyi et al., 1982). Presynaptic synapses on some crustacean interneurons (Wang-Bennet and Glantz, 1985) lie at a distance from the output regions of the terminal arborization. At such sites the effect becomes all or none, depending on whether the reduced potential subsequently fails, for example when it encounters a region of low safety factor such as a branch point or varicosities alternating with narrow axon segments (Segev, 1990). The geometry of the terminal arborization may therefore be critical to the effectiveness of the inhibition (Atwood et al., 1984). Presynaptic inhibiton is more effective if the membrane on the axon terminal is passive, allowing only electrotonic conduction, than if it is active (Segev, 1990). There has been considerable debate concerning

ALAN H. D. WATSON

232

how far action potentials penetrate into terminal arborizations (see Nicoll and Alger, 1979). However, some boutons on cat la muscle afferents lie in the nodal regions between myelinated sections of the axon, and the unmyelinated sections of the terminal extend only a few tens of microns beyond the last section of myelination (Nicol and Walmsey, 1991). Abrupt reductions in diameter of the axon where myelination ends, such as is seen in the la afferents, also help to ensure propagation of the action potential into the terminal. In the invertebrate nervous system there is of course no myelination. Intracellular recordings from the terminals of filiform hair afferents in the locust terminal ganglion demonstrate that 30 mV action potentials can be recorded in the neuropile. At these sites, primary afferent depolarization can be detected and its clear influence on spike height observed (Boyan, 1988). THE ULTRASTRUCTURE OF SYNAPTIC INTERACTIONS ON AFFERENT TERMINALS

The type of information extracted from ultrastructural studies of vertebrate and invertebrate afferent terminals reflects the distinctive synaptic and neuronal architecture of these quite different nervous systems and so is not strictly comparable in all respects. In the vertebrate nervous system, synapses can be classified as axo-dendritic, axo-spinous, axosomatic and axo-axonic, while in the invertebrate nervous system somata do not generally receive synapses and there is no ultrastructural distinction in morphology between axon terminals or collaterals and dendrites. Even in vertebrates, the identification Table I. Summary of ultrastructural

A&rent type Somatic afferents Pacinian corpuscle Pacinian corpuscle Pacinian corpuscle Pacinian corpuscle Krause corpuscle Cutaneous mechano nonciceptors Slow adapting type Slow adapting type Slow adapting type Slow adapting type Slow adapting type Slow adapting type Hair follicle Hair follicle Hair follicle Hair follicle Vibrissal hair Muscle la, b (lumbosacral) Muscle la, b (lumber) Muscle la (cuneate nucleus)

Parasympathetic afferents Sacral Vagal (gastric)

Vertebrate agerent terminals

It should be noted that ultrastructural studies of afferent terminals in vertebrates have concerned themselves almost exclusively with the cat spinal cord (see Table 1). This has the virtue that a range of synaptic configurations has been identified in the same animal, but the consequence that it is uncertain how representative these will turn out to be. Almost all of the studies have revealed the presence of presynaptic axons (see Table 1, and Maxwell and Rethelyi, 1987). In many instances presynaptic dendrites are also present. The axo-axonic synapses are usually described as symmetric though the density of the reaction product in afferent terminals labelled with horseradish peroxidase (the ultrastructural marker universally used for intracellular injection of afferents) can make this difficult to determine. The vesicles in the presynaptic axons are mostly described as flattened or pleomorphic. This is often taken to imply that the synapses are inhibitory (Uchizono, 1965) and reinforces the inference that they are also

observations on presynaptic processes to vertebrate afferent terminals Presynaptic axons Vesicles Observed Pre/post

Reference

I I I I I II

of vesicle-containing dendrites may not be easy. On the other hand, in the invertebrate nervous system there is usually little ambiguity in the polarity of synapses (at least in arthropods) while in the vertebrate nervous system the polarity of symmetrical synapses (the most common type of axo-axonic synapses received by afferent terminals and the type associated with GABA, see Watson et al., 1991a) may be difficult to determine without making certain assumptions. The information discussed below and summarized in Tables 1 and 2 must therefore be viewed in this light.

+

Presynaptic dendrites Observed Vesicles Animal

+ + ?

?

Cat Cat Cat Cat Cat Cat

(Egger er al., 1981) (Ralston er al., 1984) (Semba ef al., 1984) (Maxwell ef al., 1984) (Maxwell er al., 1984) (Rethelyi er al., 1982)

+ ? + + +

(Sugimoto et al., 1991) (Egger et al., 1981) (Semba ef al., 1983) (Ralston e/ a/., 1984) (Fyffe er al., 1986) (Ralston er al., 1984) (Maxwell et al., 1982) (Rethelyi er al., 1982) (Ralston ef al., 1984) (Fyffe et al., 1986) (Renehan el al., 1988) (Fyffe and Light, 1984)

+ + ? + + ? + + + t +

P P, r P. f P P ? P P P P P

Cat Cat Rat Cat

(Walmsey PI al., 1987)

+

P, r

Cat

(Fyffe et al., 1986)

-

-

Cat

(Mawe er al., 1986) (Rinaman er al., 1989)

-

The problems of identifying presynaptic axons identified in the experimental results of the in the discussion. Where the definition is still f, flattened; r, round. The column (pre/post) afferent.

?

P. r P P. f P P f

P. r -

? + + +

P. f -

+

+ ? -

Cat Cat Cat Cat, monkey Cat Cat Cat

? ? -

-

-

?

P, r -

Cat Rat

and dendrites is discussed in the text but for convenience, where these are not specifically quoted papers they are categorized here according to the arguments presented by authors unclear (7) is placed in the table. Synaptic vesicle morphology is indicated by; p. pleomorphic; refers to axonal boutons presynaptic to both an afferent and a dendrite postsynaptic to the

Presynaptic Table 2. Summary of ultrastructural Afferent Stretch receptor Filiform hair (prosternum) Filiform hair kercusl Campaniform‘sensilla Hair plate

modulation

233

observations on presynaptic processes to invertebrate afferent terminals Presynaptic process Observed Vesicles

Reference (Altman et al., 1980) (Watson and Pfliiger, 1984) (Watson. 19901 iwatson’and England, 1991) (Watson et al., 1991)

+ + + + +

? r (two classes) D. * p’(two classes) p (two classes)

Animal Locust Locust Locust Locust Locust

+, present; ?, unclear; p, pleomorphic; r, round

symmetric. As both the presynaptic axons and the afferent terminals contain vesicles it is possible that it is the afferent which is presynaptic, however synapses made by the afferents onto dendrites are asymmetric and it is unusual for a neuron to have output synapses of different types. The dendrites that lie presynaptic to afferent terminals are usually also said to contain flattened or pleomorphic synaptic vesicles, but these tend to be present in small numbers clustered around the synapse rather than to fill a bouton completely as is seen in axons. Afferents which receive input synapses may not do so on all, or even most of their synaptic boutons (Maxwell et al., 1984a; Walmsey et al., 1987). Reports from different afferents range from all (Fyffe et al., 1986) to one out of fourteen boutons (Walmsey et al., 1987) but typically they are found on 4&60% of boutons (Maxwell and Rethelyi, 1987). Afferents of the same type in different parts of the nerve cord may exhibit a different pattern. Muscle spindle afferents were seen to receive presynaptic inputs from axons in the lumbrosacral cord (Fyffe et al., 1984; Walmsey et al., 1987) but not in the cuneate nucleus (Fyffe et al., 1986, see Table 1). Presynaptic modulation could thus be limited selectively to a subset of output synapses and perhaps, postsynaptic neurons. For example, interneurons in both the dorsal column pathway and spinocervical tract receive direct inputs from sensory afferents (see Maxwell and Rethelyi, 1987). The afferent terminals contacting dorsal column interneurons often receive presynaptic inputs

from axons (Maxwell et al., 1985) while those contacting spinocervical tract cells do not (Maxwell et al., 1984b). This may be correlated with the observation that while the receptive fields of spinocervical tract interneurons are stable, those of dorsal column interneurons can be manipulated (Maxwell and Rethelyi, 1987). The terminals of vertebrate primary afferents with contrasting response properties differ not only in the position and form of their terminal arbours within the dorsal horn, but also in the nature and structure of the synaptic complexes in which they participate (Maxwell and Rethelyi, 1987). I will concern myself here only with the nature of the individual synaptic associations entered into and not with the structure of their glomeruli. Examples of most of the synaptic associations described can be seen in Fig. 1 which summarizes observations on large diameter (Accjl) fibres (taken from Maxwell and Rethelyi, 1987). It should be noted that in both vertebrates and invertebrates, only synapses in the vicinity of the afferent terminals are revealed by the ultrastructural studies and that other interactions between processes preor postsynaptic to the afferent, even if only a few microns away might well not be observed. The first bouton (Fig. li) shows the simplest arrangement in which a dendritic shaft is postsynaptic to the afferent, and the second (Fig. lii) the common addition of a presynaptic axon. Bouton three (Fig. liii) shows an association which is frequently reported (Table I) in which the same axon is presynaptic both to the

Fig. 1. A schematic diagram summarizing the synaptic relationships of a large diameter Act/l terminal axon (shaded) from a cutaneous sensory afferent in the cat. Afferents from guard hairs, pacinian corpuscles, Krause corpuscles and slow adapting type I and II mechanoreceptors fall into this category. A, axon; d, dendrite; S, dendritic spin; Sv, vesicle-containing dendritic process. Taken with permission and modified from Maxwell and Rethelyi (1987).

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afferent itself, and to a dendrite postsynaptic to the afferent. As one of the advantages of presynaptic inhibition is considered to be the ability to control afferent input selectively without affecting the responsiveness of the postsynaptic neuron, this is rather surprising. Of course, if not all afferent boutons have presynaptic input and several boutons synapse onto a dendrite in close proximity, this configuration might have some merit, especially if the postsynaptic neuron has only one type of afferent input or if different types of input are spatially separated on the dendritic tree. On the other hand, it may not be appropriate to take the teleological viewpoint that a given synaptic arrangement is specifically designed for a certain task and used only in that context. Bouton four (Fig. liv) shows an arrangement where one axon and several dendrites (some of which contain synaptic vesicles) are in synaptic contact with the bouton. The situation may be more complex than that illustrated as the vesicle-containing dendrites may be presynaptic to the afferent bouton as well as postsynaptic (Table 1) or even have reciprocal connections with it (Mawe et al., 1986). In addition, pairs of postsynaptic dendrites may synapse with each other (Rethelyi et al., 1982). The identity and influence of the neurons to which the vesicle-containing dendrites belong remains a matter of speculation. Maxwell and Rethelyi (1987) have suggested that islet cells of the substantia gelatinosa, which appear to receive direct sensory afferent input, are possible candidates. Islet cells are Golgi type II neurons (the equivalent of what are called local interneurons in the invertebrate nervous system), are known to have vesicle-containing dendrites and are thought to receive direct input from primary afferents (Gobel et al., 1980). They may synapse with other local interneurons in the substantia gelatinosa but their function has not otherwise been determined. There is a certain amount of evidence that some islet cells may contain GABA (see Gobel et al., 1980) or enkephalins (Bennet et al., 1982). Enkephalin-immunoreactive dendrites may be present in glomeruli typical of sensory afferents but though often in close apposition to the presumed afferent terminals, they are only rarely seen to synapse on them (Aronin et al., 1981; see Basbaum and Fields, 1984). By contrast, spinothalamic tract neurons, which are postsynaptic to the afferents, have been shown to be postsynaptic to enkephalin-immunoreactive terminals (Ruda, 1982). In view of the importance of GABA in the presynaptic inhibition of vertebrate primary afferents it is surprising that only three ultrastructural immunocytochemcial studies have sought evidence for the presence of GABA in boutons presynaptic to afferent terminals. This may stem from the assumption that the observation of pleomorphic vesicles in presynaptic boutons making symmetrical synapses onto afferents is sufficient to justify the conclusion that they are inhibitory and GABAergic (Uchizono, 1965). In fact the shape of vesicles can be affected by fixation (Tisdale and Nakajima, 1976) and immunocytochemical studies have demonstrated that vesicles of GABA-immunoreactive process may not all be identical even in the same tissue (Hamori and Takacs, 1989). Pre-embedding immunocytochemistry has been used to demonstrate immunoreactivity for

glutamate decarboxylase (the synthetic enzyme for GABA) in boutons presynaptic to afferents (Barber et al., 1978; Maxwell and Noble, 1987). Using antibodies against GABA itself Todd and Lochhead (1990) described immunoreactive axons and dendrites presynaptic to the central elements of synaptic glomeruli typical of those made by afferent terminals. Seventy-nine per cent of the peripheral axons involved in the glomeruli were immunoreactive but it is unclear from this account whether any of the unlabelled ones were also presynaptic. Some of the dendrites pre- or postsynaptic to the presumed afferent terminal were also immunoreactive. This would at least be consistent with the hypothesis that some of these belong to islet cells (Gobel et al., 1980). As has already been mentioned there is one report of choline acetyltransferase immunoreactivity in axonal boutons presynaptic to the central varicosities (which are thought to belong to sensory afferents) of glomeruli in the dorsal horn of the rat spinal cord (Ribiero-da-Silva and Cuello, 1990). Some of the immunoreactive axons are also presynaptic to both the presumed afferent terminals and their postsynaptic dendrites. The vesicles in the immunoreactive profiles are described as pleomorphic and similar in appearance to those immunoreactive for GAD. As immunocytochemical evidence for co-localization of GABA and acetylcholine has been obtained for neurons in the dorsal horn of the spinal cord (Kosaka et al., 1988) this merits further investigation in axons presynaptic to sensory afferents. Some of the micrographs shown by Ribiero-da-Silva and Cue110 (1990) also appear to show unlabelled presynaptic axons with markedly flattened synaptic vesicles but there is no discussion or quantification of this in the text. Invertebrate afferent terminals

There have been many fewer ultrastructural studies of sensory afferent terminals in invertebrates than in vertebrates (Table 2). It is only in the insect nervous system that they have been studied in detail. As with the vertebrate afferent terminals, presynaptic processes are usually found when a detailed examination is made. Where immunocytochemistry for GABA has been carried out, both labelled and unlabelled presynaptic processes are found (Watson, 1990; Watson and England, 1991; Watson et al., 199lb). Immunoreactive processes predominate, comprising 72% of the presynaptic input in the case of locust campaniform sensillar afferents (Watson and England, 1991) and 93% for hair plate afferents (Watson et al., 199lb). Some of the processes presynaptic to hair plate afferents that are not labelled by GABA antibodies are immunoreactive for glutamate. Both GABA and glutamate are abundant in the locust ventral nerve cord (Watson, 1986; Watson and Pfliiger 1987; Watson, 1988; Bicker et al., 1988). Figure 2 summarizes the synaptic relationships observed on the terminals of afferents from a range of sensory neurons within the locust nervous system which have been intracellulary labelled with horseradish peroxidase. It will be seen from the figure that synapses in the arthropod nervous system are mostly dyadic, i.e. there are two postsynaptic processes opposite each presynaptic density. There is circumstantial evidence that both elements of the dyad are

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Fig. 2. A schematic diagram summarizing the synaptic interactions made between locust sensory afferent terminals (shaded) and neuropilar processes. It incorporates features seen in afferents from filiform hairs, campanifonn

sensilla and hair plates.

physiologically influenced by the presynaptic neuron (see Watson et al., 1991b). The presynaptic inputs always lie close to the output synapses of the afferents (Altman et al., 1980; Watson and Pflilger, 1984). In preparations where more than one afferent neuron has been labelled intracellularly (Watson et al., 1991b), pairs of afferent terminal branches are often postsynaptic at the same synapse (Fig. 2i) but where only one sensory afferent neuron is labelled, this is never seen. The same presynaptic processes may make several contacts with the afferent terminals within a short distance. Occasionally it is clear that an afferent is paired with a non-afferent neuron in a postsynaptic dyad (Fig. 2ii) because the process which has not been stained with horseradish peroxidase is immunoreactive for GABA (Watson and England, 1991). It is clear from immunocytochemical studies that insect sensory neurons do not use GABA as a transmitter (Watson, 1986) but may be either cholinergic or serotonergic (Tyrer et al., 1984; Lutz and Tyrer, 1987, 1988). Processes presynaptic to insect afferents are sometimes seen to interact synaptically (Fig. 2iii). This has so far not been reported in the vertebrate nervous system. In locust campaniform sensillar afferents, GABA-immunoreactive processes were seen to synapse onto a non-immunoreactive processes close to where both synapse with the afferent (Watson and England, 1991). This could prevent antagonism between competing presynaptic influences, if for example, one was inhibitory and the other facilitatory. The influence of the GABA immunoreactive processes would presumably predominate by inhibiting the other inputs as well as the afferent. The nature of the transmitter of the processes not labelled by GABA antibodies in these situations is unknown. Glutamate immunoreactive presynaptic inputs (Fig. 2iv) are known only from studies of hairplate afferents, but have not so far been sought on other

afferents. Glutamate is excitatory at the insect neuromuscular junction (Usherwood et al., 1968) and at the only known central glutamatergic synapse, which is made between two motor neurons (Burrows et al., 1989). There is evidence that in the central nervous system glutamate can have a hyperpolarizing effect on some neurons (Usherwood et al., 1980; Dubas, 1990) but is it unclear whether this is mediated by synaptic or extrasynaptic receptors. Sensory afferent terminals are occasionally seen to make reciprocal synapse with processes postsynaptic to them (Fig. 2~). This has been found in a single study, in which the terminals of afferents from windsensitive hairs on the locust prosternum were reconstructed from serial sections (Watson and Huger, 1984). The. synapses were either made between an afferent and one other process, or (as in Fig. 2v) involved a third process which appeared to be postsynaptic to both. This second configuration is unusual in the insect nervous system but has been seen at synapses between an identified pair of motor neurons in the locust metathoracic ganglion (Burrows et al., 1989). The identity of the neurons that lie presynaptic to afferents in the arthropod nervous system have in general remained as mysterious as those of the vertebrate spinal cord despite the much more detailed information available concerning some of the neural circuitry involved. In the crayfish terminal ganglion however, interneurons responsible for chloridedependent primary afferent depolarization have been identified and shown to produce presynaptic inhibition in mechanosensory afferents (Kirk and Wine, 1984; Kirk, 1985). This is part of the circuit that protects the afferent synapses from habituation during the tail-flip escape response (Krasne and Bryan, 1973). There seems a strong possibility that these interneurons use GABA as their neuro-

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transmitter (Kirk and Wine, 1984) but there have so far been no immunocytochemical studies of the interneurons or the afferent terminals. Considered together, the ultrastructural studies of vertebrate and invertebrate afferent terminals confirm the likely importance of GABA (regardless of its mode of operation) in presynaptic modulation, whether by other axons or vesicle-containing dendrites. The possible involvement of other neurotransmitters not so far implicated from physiological studies is however, beginning to emerge. Perhaps the most surprising feature to come out of the immunocytochemical studies is that unless there is considerable co-localization of the other transmitters with GABA, their combined role must be rather minor unless the synapses which they make are very strategically placed. The local circuitry revealed in the tiny volumes of tissue around the afferent terminals that can reasonably be studied ultrastructurally is impressive and involves interactions between presynaptic axons (in insects), postsynaptic dendrites (in mammals) and reciprocal connections between afferents and postsynaptic dendrites (in both). However, there are major differences in the nature and complexity of the associations entered into by different afferents, and perhaps between the boutons made by a single afferent onto different classes of postsynaptic neuron. Acknowledgements-This work was supported by the SERC. I am grateful to M. Burrows and R. M. Santer for their helpful comments on the manuscript.

REFERENCES

Aidley D. J. (1978) The Physiology of Excitable Cells, 2nd Edn. Cambridge University Press, Cambridge, U.K. Alford S. and Grillner S. (1991) The involvement of GABA, receptors and coupled G-proteins in spinal GABAergic presynaptic inhibition. J. Neurosci. 11, 3718-3726. Altman J. S., Shaw M. K. and Tyrer N. M. (1980) Input synapses onto a sensory neurone revealed by cobalt ejection microscopy. Brain Res. 189, 2455250. Aronin N.. DiFielia M.. Liotta A. S. and Martin J. B. (1981) Ultrastructuri localisation and biochemical features of immunoreactive leu-enkephalin in monkey dorsal horn. J. Neurosci. 1, 561-577. Atwood H. L. (1976) Organisation and synaptic physiology of crustacean neuromuscular systems. Prog. Neurobiol. 7, 291-391.

Atwood H. L., Stevens J. K. and Marin L. (1984) Axoaxonal synapse location and consequences for presynaptic inhibition in crustacean motor axon terminals. J. camp. Neural. 225, 6474.

Baev K. V., Rusin K. I. and Safronov B. V. (1992) Primary receptor for inhibitory transmitters in lamprey spinal cord neurons. Neuroscience 46, 931-941. Bailey C. G., Hawkins R. D., Chen M. C. and Kandel E. R. (1981) Interneurons involved in the gill-withdrawal reflex in Aplysia. IV. Morphological basis of facilitation. J. Neurophysiol. 45, 340-360.

Barber R. P., Vaughn J. E., Saito K., McLaughlin B. J. and Roberts E. (1978) GABAergic terminals are presynaptic to primary afferent terminals in the substantia gelatinosa of the rat spinal cord. Brain Res. 141, 35-55. Basbaum A. I. and Fields H. L. (1984) Endogenous pain control systems: brainstem spinal pathways and endorphin circuitry. A. Reu. Neurosci. 7, 309-338.

Baxter D. A. and Bittner G. D. (1981) Intracellular recording from crustacean motor axons during presynaptic inhibition. Brain Res. 223, 422428. Belardetti F., Kandel E. R. and Siegelbaum S. A. (1987) Neuronal inhibition bv the oeotide FMRFamide involves opening of S K+ channels.- iature 325, 153-156. Bennet G. J., Ruda M. A., Gobel S. and Dubner R. (1982) Enkephalin-immunoreactive stalked cells and lamina IIb islet cells in the substantia gelatinosa of the cat. Brain Res. 240, 162-166.

Benson J. A. (1988) Bicuculhne blocks the response to acetylcholine and nicotine but not to muscarine or GABA in isolated insect neuronal somata. Brain Res. 458, 65-71.

Bicker G., Schafer S., Otterson 0. P. and Storm-Mathisen J. (1988) Glutamate-like immunoreactivity in identified neuronal populations of insect nervous systems. J. Neurosci. 8, 2108-2122.

Blagburn J. M. and Sattelle D. B. (1987) Presynaptic depolarization mediates presynaptic inhibition at a synapse between an identified mechanosensory neurone and giant interneurone 3 in the first instar cockroach, Peripjaneta americana. J. exp. Biol. 127, 135-157.

Bowerv N. G.. Hill D. R.. Hudson A. L.. Doble A.. Middlemiss D. N., Shaw J. and Turnbull M. (1980) (-)Baclofen decreases neurotransmitter release in the mammalian CNS by an action at a novel GABA receptor. Nature 283, 92-94. Boyan G. S. (1988) Presynaptic inhibition of identified wind-sensitive afferents in the cereal system of the locust. J. Neurosci. 8, 2748-2757.

Brezina V., Eckert R. and Erxleben C. (1987) Suppression of calcium current by endogenous neuropeptide in neurones of Aplysia California. J. Physiol. (Lond.) 388, 565-595.

Brodin L., Dale N., Christenson J., Storm-Mathisen J., Hokfelt T., and Grillner S. (1990) Three types of GABAimmunoreactive cells in the lamprey spinal cord. Brain Res. 508, 172-175.

Brown D. A. and Higgens A. J. (1979) Presynaptic effects of gamma-aminobutyric acid in isolated rate superior cervical ganglion. Br. J. Pharmac. 66, 108P-109P. Bryan J. S. and Krasne F. B. (1977) Protection from habituation of the crayfish lateral giant escape response. J. Physiol. (Lond.) 27i, 351-368.

-

_

_

Burrows M.. Watson A. H. D. and Brunn D. E. (1989) Physiological and ultrastructural characterization‘ of a central synaptic connection between identified motor neurones in the locust. Eur. J. Neurosci. 1, 111-126. Christensen J. and Grillner S. (1991) Primary afferents evoke excitatory amino-acid receptor-mediated EPSPs that are modulated by presynaptic GABA, receptors in lamprey. J. Neurophysiol. 66, 2141-2149. Cooper J. R., Bloom F. E. and Roth H. R. (1986) The Biochemical Basis of Neuropharmacology, 5th Edn. Oxford University Press, Oxford. Crain S. M. and Shen K-F. (1990) Opioids can evoke direct receptor-mediated excitatory effects on sensory neurones. Trends Pharmac. Sci. 11, 77-81. Curtis D. R., Lodge D., Bornstein J. C and Peet M. J. (1981) Selective effects of (-)-baclofen on spinal transmission in the cat. Exp. Brain Res. 42, 158-170. Dale N. and Kandel E. R. (1990) Facilitatory and inhibitory transmitters modulate spontaneous transmitter release at cultures Aplysia sensorimotor synapses. J. Physiol. (Lond.) 421, 203-222.

Davidoff R. A. (1972) Gamma-aminobutyric acid antagonism and presynaptic inhibition in the frog spinal cord. Science 175, 331-333. Dolphin A. C. and Scott R. H. (1987) Calcium channel currents and their inhibition by (-)-baclophen in rat sensory neurones: modulation by guanine nucleotides. J. Physiol. (Land.) 386, I-17.

Presynaptic modulation Dubas F. (1990) Inhibitory effect of L-glutamate on the neuropile arborizations of flight motoneurones in locusts. J. exp. Biol. 148, 501-508. Duchen M. (1986) Excitation of mouse motoneurones by GABA-mediated primary afferent depolarization. Bruin Res. 379, 182-187.

Dunlap K. (1981) Two types of y-aminobutyric acid receptor on embryonic sensory neurones. Br. J. Pharmac. 74, 579-585.

E&es J. C., Eccles R. M. and Magni F. (1961) Central inhibitory action attributable to presynaptic depolarisation produced by muscle afferent volleys. J. Physiol. (Lond.) 159, 147-166.

Eccles J. C., Magni F. and Willis W. D. (1962a) Depolarisation of central terminals of group 1 aBerent fibres from muscle. J. Physiol. (Lond.) 160, 62-93. Eccles J. C., Schmidt R. F. and Willis W. D. (1962b) Presynaptic inhibition of the spinal monosynaptic pathway. J. Physiol. (Lond.) 161, 282-297. Eccles J. C., Schmidt R. F. and Willis W. D. (1963) Pharmacological studies of presynaptic inhibition. J. Physiol. (Lond.) 168, 500-530. Egger M. D., Freeman N. C. G., Malamed S., Masarachia P. and Proshansky E. (1981) Electron microscope observations of terminals of functionaly identified afferent fibers in cat spinal cord. Bruin Res. 207, 157-162. El Manira A. and Clarac F. (1991) GABA-mediated presynaptic inhibition in crayfish primary afferents by non-A, non-B GABA receptors. Eur. J. Neurosci. 3, 1208-1218.

El Manira A., DiCaprio R. A., Cattaert D. and Clarac F. (1991a) Monosynaptic interjoint reflexes and their central modulation during fictive locomotion in the crayfish. Eur. J. Neurosci. 3, 121991231. El Manira A., Rossi-Durand C. and Clarac F. (1991b) Serotonin and Proctolin modulate the response of a stretch receptor in crayfish. Brain Res. 541, 157-162. Forssberg H., Grillner S. and Rossignol S. (1977) Phasic gain control of reflexes from the dorsum of the paw during spinal locomotion. Brain Res. 132, 121-139. Fyffe R. E. W. and Light A. R. (1984) The ultrastructure of group la afferent tibre synapses in the lubosacral spinal cord. Bruin Res. 300, 200-210. Fyffe R. E. W., Cheema, S. S. and Rustoni A. (1986) Intracellular staining of the feline cuneate nucleus. 1. Terminal patterns of primary afferent fibers. J. Neurophysiol. 56, 1268-1283.

Gahwiler B. H. and Brown D. A. (1985) GABAs-receptoractivated K+ current in voltage clamped pyramidal cells in hippocampal cultures. Proc. natn. Acad. Sci. U.S.A. 82, 1558-1562.

Geisert B. and Altner H. (1974) Analysis of the sensory projection from the tarsal sensilla of the blowfly (Phormia tetranouae Rob.-Desv., Diptera). Cell Tissue Res. 150, 249-259. Gobel S., Falls W. M., Bennet G. J., Abdelmoumene M., Hayashi H. and Humphrey E. (1980) An EM analysis of the synaptic connections of horseradish peroxidase-filled stalked cells and islet cells in the substantia gelatinosa of adult cat spinal cord. J. camp. Neurol. 194, 781-807. Gossard J-P., Cabelguen J-M. and Rossignol S. (1989) Intra-axonal recordings of cutaneous primary afferents during fictive locomotion in the cat. J. Neurophysiol. 62, 1177-1188.

Gossard J-P., Cabelguen J-M. and Rossignol S. (1990) Phase-dependent modulation of primary afferent depolarisation in singe cutaneous primary afferents evoked by peripheral stimulation during fictive locomotion in the cat.-Brain. Res. 537, 14-23. Gossard J-P.. Cabelauen J-M. and Rossianol S. (1991) An intracellular ctudy of muscle primary a&rents during fictive locomotion in the cat. J. Neurophysiol. 65, 914-926.

237

Hamori J. and Takacs J. (1989) Two types of GABAcontaining axon terminals in cerebellar glomeruli of cat: an immunogold-EM study. Exp. Brain Res. 74, 471479. Haydon P. G., Man-Son-Hing H., Doyle R. T. and Zoran M. (1991) FMRFamide modulation of secretorv machinery underlying presynaptic inhibition of synaptic transmission requires a pertussis toxin-sensitive Gprotein. J. Neurosci. 11, 3851-3860. Holz G. G., Kream R. M., Spiegel A. and Dunlap K. (1989) G-proteins couple B-adrenergic and GABA, receptors to inhibition of peptide secretion from peripheral sensory neurons. J. Neurosci. 9, 657-666. Howe J. R., Sutor 8. and Ziegegansberger W. (1987) Baclofen reduces post-synaptic potentials of rat cortical neurones by an action other than its hyperpolarising action. J. Physiol. (Lond.) 384, 539-569. Hue B. and Callec J. J. (1983) Presynaptic inhibition in the cereal-afferent giant-interneurone synapses of the cockroach, Periplaneta americana L. J. Insect Physiol. 291, 741-748.

Hue B., Lapied B. and Malecot C. 0. (1989) Do presynaptic muscarinic receptors regulate acetylcholine release in the central nervous system of the cockroach Peripluneta americana? J. exp. Biol. 142, 447-451.

Jesse1 T. M. Iversen L. L. (1977) Opiate analgesics inhibit substance P release from rat trigeminal nucleus. Nature 268, 549-55 1. Kayser V., Besson J. M. and Guilbaud G. (1987) Paradoxical hyperalgesic effect of exceedingly low doses of systemic morphine in an animal model of persistent pain. Brain Res. 414, 155-157.

Kirk M. D. (1985) Presynaptic inhibition in the crayfish CNS: pathways and synaptic mechanisms. J. Neurophysiol. 54, 130551325. Kirk M. D. and Wine J. J. (1984) Identified interneurons produce both primary afferent depolarisation and presynaptic inhibition. Science 225, 854-856. Kosaka T., Tauchi M. and Dahl J. L. (1988) Cholinergic neurons containing GABA-like and/or glutamic acid decarboxylase-like immunoreactivities in various brain regions of the rat. Exp. Brain Res. 70, 605417. Krasne F. and Bryan J. S. (1973) Habituation: regulation through presynaptic inhibition. Science 182, 590-592. Kretz R., Shapiro E., Bailey C. H., Chen M. and Kandel E. R. (1986) Presynaptic inhibition produced by an identified inhibitory neuron. II. Presynaptic conductance changes caused by histamine. J. Neurophysiol. 55, 131-146.

Levine R. B. and Murphey R. K. (1980) Pre- and postsynaptic inhibition of identified giant interneurons in the cricket (Acheta domesticus). J. camp. Physiol. 135, 269-282.

Lutz E. M. and Tyrer N. M. (1987) Immunohistochemical localisation of choline acetyltransferase in the central nervous system of the locust. Bruin Res. 407, 173-179. Lutz E. M. and Tyrer N. M. (1988) Immunohistochemical localisation of serotonin and choline acetyltransferase in sensory neurones of the locust. J. camp. Neurol. 267, 335-342.

Mawe G. M., Bresnahan J. C. and Beattie M. S. (1986) A light and electron microscopic analysis of the sacral parasympathetic nucleus after labelling primary afferent and efferent elements with HRP. J. comn Neurol. 250. 33-57. Maxwell D. J. and Noble R. (1987) Relationships between hair-follicle afferent terminations and glutamic acid decarboxylase-containing boutons in the cat’s spinal cord. Brain Res. 408, 308-3 12. Maxwell D. J. and Rethelyi M. (1987) Ultrastructure and synaptic connections of cutaneous afferent fibres in the spinal cord. Trends Neurosci. 10, 117-123.

238

ALAN

H. D

Maxwell D. J., Bannatyne B. A., Fyffe R. E. W. and Brown A. G. (1982) The ultrastructure of hair follicle afferent fibre terminations in the spinal cord of the cat. J. Neurocytol. 11, 571-582. Maxwell D. J., Bannatyne B. A., Fyffe R. E. W. and Brown A. G. (1984a) Fine structure of primary afferent axon terminals projecting from rapidly adapting mechanoreceptors of the toe and foot pads of the cat. Q. JI exp. Physiol. 69, 381-392. Maxwell D. J., Fyffe R. E. W. and Brown A. G. (1984b) Fine structure of normal and degenerating primary afferent boutons associated with character&d spinocervical tract neurones in the cat. Neuroscience 12, 151-163. Maxwell D. J., Koerber H. R. and Bannatyne B. A. (1985) Light and electron microscopy of contacts between primary afferents and neurones with axons ascending the dorsal columns of the feline spinal cord. Neuroscience 16, 375-394. Miledi R. and Slater C. R. (1966) The action of calcium on neuronal synapses in the squid. J. Physiol. (Land.) 184, 437498.

Millan M. J. (1990) Kappa-opioid receptors and analgesia. Trends Pharmac. Sci. 11, 70-76. Nicol M. J. and Walmsey B. (1991) A serial section electron microscope study of an identified la afferent collateral in the cat spinal cord. J. camp. Neural. 314, 257-277. Nicoll R. A. and Alger B. E. (1979) Presynaptic inhibition: transmitter and ionic mechanisms. Int. Rev. Neurobiol. 21, 217-258.

Pasztor V. M. and Bush B. M. H. (1987) Peripheral modulation of mechanosensitivity in primary afferent neurons. Nature 326, 793-795. Peng Y. and Frank E. (1989a) Action of GABA, receptors causes presynaptic inhibition at synapses between muscle spindle afferents and motorneurons in the spinal cord of bullfrogs. J. Neurosci. 9, 1502-I 515. Peng Y. and Frank E. (1989b) Activation of GABA, receptors causes presynaptic and postsynaptic inhibition at synapses between muscle spindle afferents and motorneurons in the spinal cord of bulldogs. J Neurosci. 5, 15161522.

Polak R. L. (1971) The stimulating action of atropine on the release of acetylcholine by rat cerebral cortex in vitro. Er. J. Pharmac. 14, 473489.

Raiteri M., Leardi R. and Marchi M. (1983) Heterogeneity of presynaptic muscarinic receptors regulating neurotransmitter release in rat brain. J. Pharmac. exp. Ther. 22a, 209-214.

Ralston H. J., Light A. R., Ralston D. D. and Per1 E. R. (1984) Morphology and synaptic relationships of physiologically identified low-threshold dorsal root axons stained with intra-axonal horseradish peroxidase in the cat and monkey. J. Neurophysiol. 51, 777-792. Renehan W. E., Stansel S. S., McCall R. D., Rhoades R. W. and Jacquin M. F. (1988) An electron microscope analysis of the morphology and connectivity of individual HRPlabelled slowly adapting vibrissa primary afferents in the adult rat. Brain Res. 462. 396400. Repkin A. H., Wolf P. and Anderson E. G. (1976) NonGABA mediated primary afferent depolarisation. Brain Res. 117, 147-152. Rethelyi M., Light A. R. and Per1 E. R. (1982) Synaptic complexes formed by functionally defined primary afferent units with fine myelinated fibers. J. camp. Neural. 207, 381-393. Ribiero-da-Silva A. and Cue110A. C. (1990) Choline acetyltransferase-immunoreactive profiles are presynaptic to primary sensory fibres in rat superficial dorsal horn. J. comv. Neural. 2%. 37Ck384.

Ruda M: A. (1982) Opiates and pain pathways: demonstration of enkephalin synapses on dorsal horn projection neurons. Science 215, 1523-l 525.

WATSON

Rudomin P., Nunez R., Madrid J. and Burke R. E. (1974) Primary afferent hyperpolarization and presynaptic facilitation of la afferent terminals induced by large cutaneous fibers. J. Neurophysioi. 37, 413429. Segev I. (1990) Computer study of presynaptic inhibition controlling the spread of action potentials into axon terminals. J. Neurophysiol. 63, 987-998. Semba K., Masarchia P., Malamed S., Jacquin M., Harris S., Yang G. and Egger M. D. (1983) An electron microscope study of primary afferent terminals from slowly adapting type 1 receptors in the cat. J. camp. Neurol. 221, 466481. Shapiro E., Castellucci V. F. and Kandel E. R. (1980) Presynaptic membrane potential affects transmitter release in an identified neuron in Aplysia by modulating the Ca+ + and K+ currents. Proc. natn. Acad. Sci. U.S.A. 77, 629633.

Siegelbaum S. A., Camardo J. S. and Kandel E. R. (1982) Serotonin and cyclic AMP close single K+ channels in Aplysia sensory neurons. Nature 299, 413417. Sillar K. T. (1989) Synaptic modulation of cutaneous pathways in the vertebrate spinal cord. Semin. Neurosci. 1, 45-54. Sillar K. T. (1991) Spinal pattern generation and sensory gating mechanisms. Curr. Opin. Neurobiol. 1, 583-589. Sillar K. T. and Skorupski P. (1986) Central input to primary afferent neurones in crayfish, Pactfastacus ieniusculus is correlated with rhythmic output of thoracic ganglia. J. Neurophysiol. 55, 678688. Skorupski P. and Sillar K. T. (1986) Phase-dependent reversal of reflexes mediated by the thoracocoxal muscle receptor organ in the crayfish, Pacifastacus leniusculus. J. Neurophysiol. 55, 689695.

Small S. A., Cohen T. E., Kandel E. R. and Hawkins R. D. (1992) Identified FMRFamideimmunoreactive neuron LPL16 in the left pleural ganglion of Aplysia produces presynaptic inhibition of siphon sensory neurons. J. Neurosci. 12, 16161627. Sugimoto T., Nagase Y., Nishiguchi T., Kitamura S. and Shigenaga Y. (1991) Synaptic connections of a low threshold mechanoreceptive primary neuron within the trigeminal nucleus oralis. Brain Res. 548, 338-342. Tisdale A. D. and Nakaiima Y. (1976) Fine structure of synaptic vesicles in two types‘ of nerve terminals in crayfish stretch receptor organs: influence of fixation methods. J. camp. Neural. 165, 369-386. Tobias M. and Murphey R. K. (1979) The response of cereal receptors and identified interneurons in the cricket (Acheta domesticus) to airstreams. J. camp. Physiol. 129, 51-59. Todd A. J. and Lochhead V. (1990) GABA-like immunoreactivity in type I glomeruli of rat substantia gelatinosa. Brain Res. 514, 171-174.

Trimmer B. A. and Weeks J. C. (1989) Effects of nicotinic and muscarinic agents on an identified motorneurone and its direct afferent inputs in larval Manduca sexta. J. exp. Biol. 144, 303-337.

Tyrer N. M., Turner J. D. and Altman J. S. (1984) Identifiable neurons in the locust central nervous system that react with antibodies to serotonin. J. camp. Newrol. 227, 313-330.

Uchizono K. (1965) Characteristics of excitatory and inhibitory synapses in the central nervous system of the cat. Nature 257, 642643.

Usherwood P. N. R., Giles D. and Suter C. (1980) Studies of the pharmacology of insect neurones in vitro. In Insect Neurobiolonv -. and Pesticide Action (Neurotox 79) pp. 115-128. Society of Chemical Industry; London. Usherwood P. N. R., Machili P. and Leaf G. (1968) L-Glutamate at insect excitatory nerve-muscle synapses. Nature 219, 1169-I 172.

Viana di Prisco G., Wallen P. and Grillner S. (1990) Synaptic effects of intraspinal stretch receptor neurons

Presynaptic modulation mediating movement-related feedback during locomotion. Brain Res. 530, 161-166. Walmsey B., Wieniawa-Narkiewicz E. and Nicol M. J. (1987) Ultrastructural evidence related to presynaptic inhibition of primary muscle afferents in Clarke’s column of the cat. J. Neurosci. 7, 236-243. Wang-Bennet L. T. and Glantz R. M. (1985) Presynaptic inhibition in the crayfish brain. II. Morphology and ultrastructure of the terminal arborisation. J. camp. Physiol. A. 156, 605-617. Watson A. H. D. (1986) The distribution of GABAlike immunoreactivity in the thoracic nervous system of the locust Schistocerca gregaria. Cell Tiss. Res. 246, 331-341. Watson A. H. D. (1988) Antibodies against GABA and glutamate label neurones with morphologically distinct synaptic vesicles in locust central nervous system. Neuroscience 26, 334. Watson A. H. D. (1990) Ultrastructural evidence for GABAergic input onto cereal afferents in the locust (Locusta migratoria). J. exp. Biol. 148, 509-515. Watson A. H. D. and England R. C. D. (1991) The distribution of and interactions between GABA-immunoreactive and non-immunoreactive processes presynaptic to campaniform sensilla on the trochanter of the locust leg. Cell. Tiss. Res. 226, 331-341.

239

Watson A. H. D. and PIliiger H. J. (1984) The ultrastructure of prosternal sensory hair afferents within the locust central nervous system. Neuroscience 11, 269-279. Watson A. H. D. and PfXiger H. J. (1987) The distribution of GABA-like immunoreactivity in relation to ganglion structure in the abdominal nerve cord of the locust (Schistocerca gregaria).

Cell Tiss. Res. 249, 391-102.

Watson A. H. D., McCabe B. J. and Horn G. (199la) Quantitiative analysis of the ultrastructural distribution of GABA-like immunoreactivity in the intermediate and medial part of the hyperstriatum ventrale of the chick. J. Neurocyrol. 20, 145-156. Watson A. H. D., Storm-Mathisen J. and Ottersen 0. P. (199lb) GABA and glutamate-like immunoreactivity in processes presynaptic to afferents from hair plates on the proximal joints of the locust leg. J. Neurocyfol. 20, 796-809.

Wiese K., Calabrese R. L. and Kennedy D. (1976) Integration of directional mechanosensory input by crayfish interneurones. J. Neurophysiol. 39, 834-843. Yaksh T. L., Dirksen R. and Harty G. J. (1985) Antinociceptive effects of intrathecally injected cholinomimetic drugs in the rat and cat. Eur. J. Pharmacol. 117, 81-88. Yarowski P. J. and Carpenter D. 0. (1978) A comparison of similar ionic responses to gamma-aminobutyric acid and acetylcholine. J. Neurophys. 41, 531-541.