J. Phyeiol. (1975), 247, pp. 321-341 With 12 text-figures Printed in Great Britain
321
HETEROSYNAPTIC FACILITATION IN THE GIANT CELL OF APLYSIA
By T. SHIMAHARA AND L. TAUC From the Laboratoire de Neurobiologie Cellulaire du Centre National de la Recherche Scientifique, 91190, Gif-sur- Yvette, France
(Received 16 July 1974) SUMMARY
1. Heterosynaptic facilitation, defined as an increase of the efficacy of synaptic transmission between a test interneurone and a post-synaptic neurone, produced by the stimulation of a separate pathway, was studied in the left pleural ganglion. The experimental procedure consisted of detecting the effects of a brief tetanus, applied to tentacular and tegumentary nerves, on the amplitude of monosynaptic and unitary postsynaptic potentials (p.s.p.s) recorded in the left giant cell and generated by stimulating the test interneurone every 10 sec. The membrane potential of the test interneurone was simultaneously recorded. 2. Following heterosynaptic stimulation, the amplitude of the test p.s.p. increased, after a delay of about 30 sec, up to 250 % of its original size; this increase subsided after 2-3 min or more. 3. Only the interneurones producing in the giant cell the e.i.p.s.p. (excitatory-inhibitory post-synaptic potential) were affected by heterosynaptic facilitation. Other interneuronal types showed no changes in their synaptic transmission on the giant cell after heterosynaptic stimulation. 4. Heterosynaptic stimulation did not produce either orthodromic or antidromic spikes in the test interneurones clearly indicating that facilitation of test p.s.p. did not result from increased spike activity in the test interneurone. 5. Often heterosynaptic facilitation of the test p.s.p. was observed due to spontaneous activity in the heterosynaptic pathway, demonstrating the normal occurrence of the phenomenon. 6. Iontophoretic injection of 5-HT at critical, presumably synaptic, sites in the neuropil, evoked a facilitation of the test p.s.p. similar to heterosynaptic facilitation. Only the e.i.p.s.p.s were so affected by 5-HT. On the contrary, other p.s.p. types were depressed by 5-HT as a result of conductance changes in the left giant cells. 13
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322 T. SHIMAHARA AND L. TA UC 7. Both heterosynaptic facilitation and 5-HT facilitation were suppressed by the presence in the bath of 5-HT (10-5 M) and of LSD-25 (3 x 104 M). The action of injected 5-HT on the membrane conductance of the left giant cell was also depressed in the presence of 5-HT in the bath, but was unaffected by LSD-25 (3 x 104 M). 8. From the parallelism of properties of heterosynaptic and 5-HT facilitation, it is suggested that 5-HT is the probable transmitter mediating heterosynaptic facilitation. It seems likely that 5HT is released from the heterosynaptic pathway at the level of the synaptic ending of the test interneurone on to the giant cell and that it increases the efficacy of this synapse, probably acting on the quantity of synaptic transmitter liberated. INTRODUCTION
When the synapses of two different neurones on a third neurone interact in such a way that one of the two synapses can modify the synaptic efficacy of the other, this interaction is called heterosynaptic. Heterosynaptic interactions are rather little understood, partly because their identification and study require strictly defined experimental conditions in order to dissociate other neuronal mechanisms which can produce similar phenomenological effects. Inhibitory heterosynaptic actions of different kinds have been described in several systems: in the spinal cord (Frank & Fuortes, 1957; Frank, 1959), in the neuromuscular junction of crustacea (Dudel & Kuffler, 1960, 1961), in the Mauthner cell of the goldfish (Furukawa, Fukami & Asada, 1963), and in Aplysia ganglion cells (Tauc, 1965). The unique case of heterosynaptic facilitation (h.s.f.) was discovered in Aplysia central nervous system (Kandel & Tauc, 1965). It was defined as an increase in the efficacy of synaptic transmission between a test interneurone and post-synaptic neurone (measured as an increase of p.s.p. in the post-synaptic neurone) following the stimulation of another afferent heterosynaptic pathway, different from that containing afferents of the test interneurone. The similarity between the effects of post-tetanic potentiation (p.t.p.) and h.s.f. in a system in which the test interneurone is uniquely identified makes it difficult to distinguish h.s.f. as a separate phenomenon. By taking advantage of the fact that a single test interneurone subject for h.s.f. has contacts with two identifiable post-synaptic cells situated in different ganglia, and by excluding the possibility of spike generation in one of the branches of the test interneurone during heterosynaptic stimulation, Epstein & Tauc (1970) were able to bring a convincing, although indirect demonstration that h.s.f. can take place in the complete
323 HETEROSYNAPTIC FACILITATION absence of spike activity in the test interneurone and consequently represents a phenomenon distinct from p.t.p. In order to obtain direct evidence for the existence of an h.s.f. and to have a chance to approach the mechanism involved, we have identified neurones whose endings show facilitation upon h.s. stimulation. An interneurone producing excitatory-inhibitory post-synaptic potentials (e.i.p.s.p.) in the left giant cell fulfilled the required criteria (see Shimahara & Tauc, 1975). In the present study we provide direct evidence, that h.s.f. is specific to a given interneuronal type and that it occurs without the initiation of action potentials in the test interneurone. Furthermore, h.s.f. appeared to be a result of chemical action by the heterosynaptic interneurone on the test neurone; the most probable mediator involved 5-HT. Several preliminary reports have already been published (Shimahara & Tauc, 1970, 1972; Tauc & Shimahara, 1971a, b). METHODS
The left pleural ganglion of Aplysia californica was used for all experiments. The techniques for dissecting the preparation and obtaining intracellular recording were similar to these described in the preceding paper (Shimahara & Tauc, 1975). The left giant cell (l.g.c.) was used as the post-synaptic cell. This cell as well as an afferent interneurone in the pleural ganglion were impaled with double electrodes filled with 3M-KCl, one barrel of which was used for recording and the other for passing adequate current to bring the membrane potential to the desired level. The experimental procedure was as follows: the test interneurone was stimulated directly every 10 sec and the corresponding p.s.p. was recorded in the giant cell at a high level of amplification. The stimulation of the test interneurone was prolonged over a period needed to obtain p.s.p.s of fully 'habituated' stable amplitudes (Bruner & Tauc, 1966). The heterosynaptic stimulus was applied at 7 per sec for 1 to 3 sec to the tentacular or tegumentary nerves lying on Ag-AgCl electrodes. Bipolar stimulation was used to minimize polarization of the stimulating electrodes. The preparation was continuously perfused by sea water with and without drugs. Local applications of drugs was by iontophoretical micro-injection through micropipettes of 1-2 jsm tip diameter. Backing current was continuously applied to prevent leaking of drugs from the tip of the micropipette. All experiments were performed at room temperature, about 22° C. RESULTS
Identification of interneurones subject to h.s.f.; recording from test and postsynaptic neuroses All types of interneurones present in the left pleural ganglion of Aplysia and afferent to the l.g.c. were tested for their ability to produce a h.s.f. The standard stimulating procedure described in Methods was used and revealed that only the interneurones producing an e.i.p.s.p. in the l.g.c. I3-2
T. SHIMAHARA AND L. TA UC 324 could be affected heterosynaptically. Both fast and slow phases are affected but due to difficulties which are often present in identification of the slow inhibitory phase of the unitary e.i.p.s.p. all observations were made only on the fast excitatory component. Such an action is illustrated in Fig. 1, where the stimulation of the tentacular nerves increased the
Fig. 1. Effect of heterosynaptic stimulus on the e.i.p.s.p. recorded at high speed in the left giant cell (upper traces); lower traces show lower speed continuous recording in the test interneurone which is stimulated directly every 10 sec. A repetitive heterosynaptic stimulus (7 stimuli in 1 sec) was applied to the tegumentary nerves between the first and second postsynaptic potential (row 1). Note that heterosynpatic stimulation did not evoke any spike in the test interneurone and that the maximum effect on the p.s.p. is observed at 25 sec after the heterosynaptic stimulus (middle row 2).
HETEROSYNAPTIC FACILITATION 325 amplitude of the test p.s.p.s to 200 % of control. The original amplitude was only restored 100 sec after the heterosynaptic stimulus. The recordings show that this stimulus produced a complex synaptic potential on the post-synaptic cell (sometimes even giving rise to spike potential), and that the test interneurone was slightly depolarized, but no spike potential was initiated. The absence of spikes in the test interneurone during heterosynaptic facilitation is strong evidence that h.s.f. is a phenomenon distinct from the homosynaptic facilitation of p.t.p. Another important point which can be seen in Fig. 1 is the delay which between the heterosynaptic stimulus and the maximal increase observed is of the p.s.p. Such a delay has already been reported (Kandel & Tauc, 1965; Epstein & Tauc, 1970). Facilitation, presumably heterosynaptic, has been frequently observed to occur spontaneously without applying any stimulus to a heterosynaptic pathway (Fig. 2). In this case prior to the increase of test p.s.p. a slight depolarization is observed in the test interneurones simultaneously with a burst of p.s.p.s in the post-synaptic cell (l.g.c.). But no additional spike activity other than that corresponding to the test stimulation can be detected in the test interneurone. The synaptic efficacies of p.s.p.s other than e.i.p.s.p.s were not affected by the heterosynaptic stimulation. For instance, heterosynaptic stimulation does not change the amplitude of slow e.p.s.p. even though it produces a depolarization in the test interneurone generating the slow p.s.p., similar to that in the interneurone generating the e.i.p.s.p. (Fig. 3). Also all interneuronal types show p.t.p., but only the 'e.i.p.s.p. type' is subject in addition to h.s.f. The differences in facilitatory effects of homosynaptic stimulation of the test neurone and heterosynaptic stimulation in cells subject to h.s.f. were already reported (Epstein & Tauc, 1970). The present intracellular recordings from the test interneurone add additional information concerning modification of its membrane potential. Both homosynaptic and heterosynaptic stimulation are followed by an increase of test p.s.p. But whereas homosynaptic stimulation induces hyperpolarization (Fig. 4A), the heterosynaptic stimulation is followed by a depolarization (Fig. 4B) of the test interneurone. Thes3 opposite membrane effects appear to be characteristics of homosynaptic and heterosynaptic actions.
Pharmacological ba8is of h.s.f. It has been proposed that h.s.f. (as well as heterosynaptic inhibition), results from an interaction between heterosynaptic interneurones and the test interneurone at the level of the latter's nerve
T. SHIMAHARA AND L. TAUC 326 endings, and that this action is chemically mediated. The site of this interaction was designated as the 'episynapse' (Tauc, 1965, 1967). The action of the episynaptic transmitter would be to increase, through an unknown mechanism, the quantity of the transmitter released. Spontaneous
\
Fig. 2. Spontaneous heterosynaptic facilitation. Lower traces show recording from the test interneurone stimulated every 10 sec and producing p.s.p.s in the giant cell. In the l.g.c., a spontaneous burst of p.s.p.s originating from non-identified interneurones is followed by an increase in the test p.s.p. Note the depolarization in the test interneurone and absence of any spike other than that due to test stimulation.
One way to approach the identification of the episynaptic transmitter is to search for a substance which, when injected in the region of synaptic contacts between the test interneurone and the left giant cell, would mimic heterosynaptic facilitation. The experimental procedure used in this case was identical to that used before, except that instead of heterosynaptic stimulation, iontophoretic injections were applied on the regions of neuropil where presumed synaptic contacts exist between the test neurone and the giant cell. It was easy to
327 HETEROSYNAPTIC FACILITATION ascertain that the axon of the giant cell crosses the left pleural ganglion and emerges in both pleuro-pedal connectives (see Fig. 1, Shimahara & Tauc, 1975). However, as the synaptic contacts are necessarily embedded in the neuropil, many spots at different depths had to be tried for each substance injected. The substances tried were acetylcholine, dopamine, serotonin (or 5-HT), DL-glutamate, glycine and aspartate. Among these substances only 5-HT AV
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I Fig. 3. Comparison of effects of homosynaptic (A) and heterosynaptic (B) stimulations on the slow e.p.s.p. Upper traces are records from the left giant cell (l.g.c.) and lower records from the interneurone stimulated every 10 see as in Fig. 1. Note in A the clear post-tetanic potentiation after homosynaptic stimulation, and in B the absence of any effect of heterosynaptic stimulation.
328
T. SHIMAHARA AND L. TA UC A
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Fig. 4. Comparison of effects of homosynaptic (A) and heterosynaptic (B) stimulation on e.i.p.s.p. Records are displayed on a Brush 280 pen recorder. The test interneurone is stimulated every 10 sec; between each stimulus the paper speed was decreased 100-fold to permit observation of successive synaptic responses on a short record. Both homosynaptic and heterosynaptic stimulation are followed by an increase on the test p.s.p. But following these stimuli, the membrane potential of the test interneurones show a hyperpolarization in A and depolarization in B. The hyperpolarization which is observed in the giant cell following the burst of activity is due to the summation of hyperpolarizing phases of e.i.p.s.p.s.
329 HETEROSYNAPTIC FACILITATION was effective in increasing the test p.s.p. in a way analogous to heterosynaptic facilitation. An effect was observed only when 5-HT was injected at certain critical sites in the neuropil. It was without effect if injected elsewhere, especially around the soma or the emerging axons of the test interneurone. rI- 5-HT
I Fig. 5. Effect of repetitive (1 per 10 sec) 5-HT iontophoretic injections in the neuropil (arrows) on the membrane potential of the l.g.c. (A) and of the interneurone (B) and the size of the e.i.p.s.p. recorded in the l.g.c. Same recording procedure as in Fig. 4.
Such chemically induced facilitation is represented in Fig. 5, following a series of brief injections of 5-HT. A several-fold increase in the test p.s.p. develops after some time and subsides slowly after the end of injections. Concomitantly polarization changes occur in the cells: depolarization sometimes followed by hyperpolarization in the test interneurone, hyperpolarization in the giant cell. However, the increase of test p.s.p. is not correlated to these changes, which usually subside more rapidly than the effect on the p.s.p. amplitude. On the other hand, direct effect of 5-HT on the giant cell membrane may be an additional depolarization or a more complex reaction (see later).
T. SHIMAHARA AND L. TAUC 330 The existence of 5-HT effects on the post-synaptic cell membrane made investigations necessary to ascertain whether the observed increase in the amplitude of the p.s.p. is related to modifications of the synapse itself or if it simply results from changes in the post-synaptic membrane properties. As the p.s.p. increase was observed for both hyperpolarization and depolarization of the giant cell, one could easily discard as decisive the influence of membrane potential of the giant cell by virtue of its bringing the p.s.p. to a different distance from its inversion level. For the same reason a possible increase of the membrane conductance due to anomalous rectification known to be present in these cells need not be considered (Kandel & Tauc, 1966). The conductance of the giant cell membrane was definitely affected by 5-HT, but in a way which by no means could produce an increase of the test p.s.p. The conductance measurements performed by applying constant current pulses and estimating the amplitude of the voltage change (Fig,. 6) show that the iontophoretic application of 5-HT clearly increases the membrane conductance, and that this increase is not due to anomalous rectification. This is demonstrated by the absence of conductance changes for a depolarization produced by applied current and which mimics the polarization change induced by 5-HT injection (Fig. 6 left side). The conductance increase normally would depress the p.s.p. amplitude by the shunt which it exerts on the p.s.p.-generating mechanism. The conductance increase induced in the left giant cell by 5-HT acts on the p.s.p. to oppose the facilitation; thus the p.s.p. amplitude increase observed after 5-HT application on the synaptic region would be still larger if correction could be made for the conductance increase of the post-synaptic cell membrane. That this conclusion is correct can be ascertained if instead of using an e.i.p.s.p. subject to heterosynaptic facilitation, one examines the action of 5-HT injection on the slow e.p.s.p. Fig. 7 shows that this slow e.p.s.p. is not affected by stimulation (see above), and that the 5-HT injection at the synaptic site depresses the slow e.p.s.p., to less than 50 % of its initial amplitude in the example shown. No injection site can be found in the neuropil at which the injection of 5-HT increases the slow e.p.s.p. amplitude. The same depressive action of 5-HT resulting from the decrease of the input resistance of the giant cell was observed for all other synaptic types. The e.i.p.s.p. alone was facilitated. It can be concluded from these experiments that the e.i.p.s.p. facilitation observed after iontophoretic injection of 5-HT at defined sites in the neuropil does not result from biophysical modifications of the post-synaptic cell membrane. The specificity of 5-HT action on the e.i.p.s.p. and its similarity of action to
331 HETEROSYNAPTIC FACILITATION that of heterosynaptic stimulation propose 5-HT as a candidate for the episynaptic transmitter. In the following paragraphs this 5-HT action on e.i.p.s.p. will be designated the 5-HT facilitation. 5-HT
4 mV
100 sec
-HT
Fig. 6. Measurement of changes in membrane conductance in the l.g.c. during a depolarization produced first by transmembrane applied current (left side) by 5-HT injection (between two arrows and then at the synaptic region (right side). Square current pulses are applied repetitively, 1 per see, to produce electrotonic potentials the size of which is proportional to membrane resistance. The recording shows that 5-HT produces a clear conductance increase which is not due to a membrane rectification.
Inhibitors of heterosynaptic and 5-HT facilitations If 5-HT is the episynaptic transmitter, it may be expected that both heterosynaptic and 5-HT facilitation must be blocked by inhibitors which inactivate the 5-HT receptor. We have tried to inhibit both h.s.f. and 5-HTfacilitation in two ways; first by 5-HT itself which can be considered as a specific inhibitor of 5-HT receptors probably through its desensitizing action (Stefani & Gerschenfeld, 1969) and second by LSD-25 (D-lysergic acid diethylamide), the best known 5-HT receptor inhibitor. The effect of 5-HT perfusion is shown in Fig. 9, to compare with the control increase of test p.s.p. by heterosynaptic and 5-HT facilitation in Fig. 8. 5-HT added to the bath at a concentration of 10-5 M depressed the
332 T. SHIMAHARA AND L. TAUC amplitude of the test p.s.p. by its action on the membrane conductance of the giant cell but it can be seen that now the heterosynaptic stimulus as well as the injected 5-HT have no effect on the test p.s.p. p
5-HT
E-p-s.p. slow
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Fig. 7. Effect of heterosynaptic stimulation (left column) and of 5-HT injection (right column) on slow e.p.s.p. The time indications are related to the beginning of heterosynaptic or chemical stimulation. Note the absence of changes after heterosynaptic stimulation and a 40 % decrease of slow e.p.s.p. after 5-HT application in the synaptic region of l.g.c.
LSD-25 at a concentration of 3 x 104 M added to the bath (Fig. 11) has little effect on the conductance of the giant cell or the size of the p.s.p., but with respect to the control in Fig. 10 it is evident that in the presence of LSD-25 both h.s.f. and 5-HT facilitation are inoperative. Moreover, iontophoretic injection of 5-HT now depressed the p.s.p., as it did for the
~~ -
HETEROSYNAPTIC FACILITATION
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Fig. 8. Control recording for Fig. 9, showing the effects of heterosynaptic stimulation (left column) and of 5-HT injection (right column) on the size of e.i.p.s.p. recorded in the l.g.c. (upper traces) due to activation of the test interneurone (lower traces). Time is measured from the beginning of the stimulations and injections.
333
334 T. SHIMAHARA AND L. TA UC slow e.p.s.p. (Fig. 7) in the absence of any drug. This depression indicates that 5-HT increases the conductance of the giant cell membrane in the presence of LSD-25, therefore, the 5-HT receptors on the giant cell were not blocked. k\1 {I-5-HT
E. p.s.p.
\
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Fig. 9. Same cells and experimental arrangement as in Fig. 8, except for the presence of 5-HT (105 M) in the bath. The size of the test p.s.p. is decreased owing to the decrease of input resistance of the l.g.c. by 5-HT. Note the absence of both heterosynaptic and 5-HT facilitations.
HETEROS YNAPTIC FACILITATION
Fig. 10. Control recording for Fig. 11. Same indications as in Fig. 8 but in a different preparation.
335
T. SHIMAHARA AND L. TA UC Further evidence that LSD-25 does not block these giant cell 5-HT receptors is given in the following experiments. Direct injection of 5-HT on the axon of the giant cell produces either a depolarization or hyperpolarization, or both in a sequence. Fig. 12 shows a complex depolarizing-hyperpolarizing response to 5-HT. Introduction of LSD-25 at a 336
t
5-HT
LSD-
E.i.p.s.p.
~~~~~~S
-
Fig. 11. Absence of heterosynaptic and 5-HT facilitation, when, after the control presented in Fig. 10, LSD-25 (3 x 104 M) is added to the bath. On the contrary the test p.s.p. is now depressed after 5-HT injection.
HETEROSYNAPTIC FACILITATION
337
,/ S cHT C.At
-
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LSD-25
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-
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-80 mV
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Fig. 12. Effect of iontophoretic injection of 5-HT on the axonal synaptic region of the l.g.c. at the different membrane potentials (-50 mV in upper traces, -80 mV in lower traces) and in normal conditions (control), in the presence of LSD-25 (3 x 16-4 mM), or of 5-HT (10- M), in the bath. The inhibitory component reverses at -80 mV. Note that the effect of 5-HT injection is not affected by LSD-25; the 5-HT in the bath almost suppresses the response to 5-HT injection.
T. SHIMAHARA AND L. TAUC concentration used above into the bath did not change this 5-HT response, which was however suppressed in the presence of 5-HT in the bath. These observations explain the p.s.p. responses obtained for 5-HT injection in Figs. 9 and 11. They point to the possible plurality of 5-HT receptors; especially it can be noted that the 5-HT receptor which we believed to be involved in 5-HT facilitation and h.s.f. are blocked by LSD-25 but those on the giant cell are not. Finally they show that physiological and 5-HT induced facilitations both react in the same way to 5-HT inhibitors. 338
DISCUSSION
Of the many interneuronal types which were found in the left pleural ganglion to be afferent to the left giant cell, only the so-called e.i.p.s.p. type has the ability to increase the synaptic efficacy of its endings by means of heterosynaptic stimulation. The observed absence of spike activity in the test interneurone as a consequence of heterosynaptic stimulation brings evidence that heterosynaptic facilitation represents a distinct phenomenon; different especially from homosynaptic facilitation which results from repetitive spike activity. The effects of heterosynaptic stimulation reported here were shorter (max. 3 min) than those described previously for a single p.s.p. (Epstein & Tauc, 1970), which lasted up to 40 min. This difference is most probably due to different interneurones in the two studies, as well as the nerves chosen for heterosynaptic stimulation. It has been found also for heterosynaptic inhibition (Tauc, 1965) that quite considerable differences may be observed in its duration in different cells. They might be ascribed to differences in quantitative rather than qualitative properties of individual neurones. The present results suggest that the episynaptic transmitter is 5-HT. It is present in Aplysia ganglia (Carpenter, Breese, Schanberg & Andkopin, 1971; Cottrell, 1974), its local application on to the synaptic region of the post-synaptic cell mimics effects of heterosynaptic stimulation, the 5-HT facilitation is restricted only to p.s.p.s which are able to undergo heterosynaptic facilitation, and finally heterosynaptic and 5-HT facilitations are affected similarly by inhibitors of 5-HT receptors. The possibility cannot be excluded, however, that the injected 5-HT acts not on the episynaptic site of the test interneurone, but on 5-HT receptors of an interneurone in the heterosynaptic pathway thus giving rise to an effective stimulation of this pathway. The specificity of the inhibitory action of LSD-25 (at the concentrations used in present studies) on the 15-HT receptors in the heterosynaptic pathway and absence of
339 HETEROSYNAPTIC FACILITATION inhibition of the 5-HT receptors in the synaptic region of the giant cell points against this possibility, given that the 5-HT receptors of the interneurones interposed in the heterosynaptic pathway are probably identical to those of the giant cell. This question can be definitively resolved when it becomes possible to identify and record from presumed interneurones in the heterosynaptic pathways. One has also to consider that the transmitter liberated by the heterosynaptic pathway, or 5-HT, could act not at the test interneurone ending, but on receptors located on the postsynaptic membrane increasing somehow the efficacy of the transmitter liberated by the test interneurone. A facilitating action of dopamine on ACh receptors has been described in the vertebrate sympathetic ganglion cells (Libet & Tosaka, 1970). Such a facilitating effect of 5-HT on the receptors of the unknown transmitter of the test cells cannot be excluded, although the blockage of 5-HT injection by 5-HT in the bath argues against this hypothesis. Actually, we think that our results are in good agreement with a situation in which the heterosynaptic pathway liberates 5-HT which combines with 5-HT receptors located on the nerve endings of the test cells, thus inducing an increased potentiality for transmitter liberation by the terminal without inducing spike generation. Whether a specialized anatomically definable episynaptic contact is necessary is an open question. In any case, serial synapses have been seen in Aplysia ganglia among unidentified cell processes (Jourdan & Nicaise, 1971). But the specificity of the episynaptic transmitter action could be insured even in the absence of synaptic contacts as only the nerve endings having adequate 5-HT receptors would be affected by heterosynaptic action. A relatively long delay for maximum increase of p.s.p.s after heterosynaptic stimulation might reflect the time of diffusion of the transmitter towards the test cell endings. More hypothetical is the way by which one can imagine the action of the episynaptic transmitter on the transmitter release mechanism of the test cell. The easiest way is to consider that the episynaptic transmitter or 5-HT induces on the episynapse classical ionic permeability changes, that is, affects its polarization and or its conductance. It was demonstrated previously (Shimahara & Tauc, 1975) that in some Aplysia synapses the transmitter release is increased by presynaptic depolarization. However in the present results the-duration of the polarization changes measured in the test cells during h.s.f. and 5-HT-facilitation is not in exact correspondence with the duration of facilitations. In addition, slow e.p.s.p.-type interneurones show similar depolarization change to heterosynaptic stimulation as the e.i.p.s.p. type, but the slow e.p.s.p. is not facilitated. Moreover, Carew, Castellucci & Kandel (1971) observed in the abdominal ganglion cells a hyperpolarization of a presynaptic
340 T. SHIMAHARA AND L. TAUC neurone as a result of heterosynaptic stimulation. But, as already pointed out, potential changes measured in the soma do not necessarily reflect potential changes on the nerve ending. It is also possible that the episynaptic transmitter of 5-HT exerts its action otherwise, by a more direct way on the transmitter's liberation mechanism, for instance modulating the calcium movement. Further experimentation is necessary for understanding the mechanism involved. The authors wish to thank Dr R. T. Kado and Dr R. S. Zuker for assistance with the manuscript. This work was party supported by ATP grant no. 4202 from C.N.R.S. and I.N.S.E.R.M. grant no. 7340808. T. Shimahara is a fellow of Roussel-Uclaf. REFERENCES BRUNER, J. & TAUC, L. (1966). Habituation at the synaptic level in Aply8ia. Nature, Lond. 210, 37-39. CAREW, T. J., CASTELLUCCI, V. F. & KANDEL, E. R. (1971). An analysis of dishabituation and sensitization of the gill-withdrawal reflex in Aply8ia. Int. J. Neurosci. 2, 79-98. CARPENTER, D., BREESE, G., SCHANBERG, S. & ANDKOPIN, I. (1971). Serotonin and dopamine: distribution and accumulation in Aplysia nervous and non-nervous tissues. Int. J. Neurosci. 2, 49-56. COTTRELL, G. A. (1974). Serotonin and free amino acid analysis of ganglia and isolated neurones of Aplysia dactylomela. J. Neurochem. 22, 557-559. DUDEL, J. & KUFFLER, S. W. (1960). Excitation at crayfish neuromuscular junction with decreased membrane conductance. Nature, Lond. 187, 246-247. DUDEL, J. & KUFFLER, S. W. (1961). Presynaptic inhibition at the crayfish neuromuscular junction. J. Physiol. 155, 543-562. EPSTEIN, R. & TAUC, L. (1970). Heterosynaptic facilitation and post-tetanic potentiation in Aplysia nervous system. J. Physiol. 209, 1-23. FRANK, K. (1959). Basic mechanisms of synaptic transmission in the central nervous system. I.R.E. Trans. Med. Electron. ME-6, pp. 85-88. FRANK, K. & FUORTES, M. G. F. (1957). Presynaptic and post-synaptic inhibition of monosynaptic reflexes. Fedn Proc. 16, 39-40. FURUKAWA, T., FUiAMI, Y. & ASADA, Y. (1963). A third type of inhibition in the Mauthner cell of goldfish. J. Neurophysiol. 26, 759-774. JOURDAN, F. & NIcAisE, G. (1971). L'ultrastructure des synapses dans le ganglion pleural de l'Aplysie. J. Microscopie II, 69-70. KANDEL, E. R. & TAUC, L. (1965). Mechanism of heterosynaptic facilitation in the giant cell of the abdominal ganglion in Aplysia depilans. J. Physiol. 181, 28-47. KANDEL, E. R. & TAUC, L. (1966). Anomalous rectification in the metacerebral giant cells and its consequences for synaptic transmission. J. Physiol. 183, 287304. LIBET, B. & TosAKA, T. (1970). Dopamine as a synaptic transmitter and modulator in sympathetic ganglia: a different mode of synaptic action. Proc. natn. Acad. Sci. U.S.A. 67, 667-673. SHIMAHARA, T. & TAUC, L. (1970). Multiples interneurones de type different 'a un meme neurone g6ant chez I'Aplysie. J. Physiol., Paris 62, 449. SHIMAHARA, T. & TAUC, L. (1972). Mechanisme de la facilitation h6t6rosynaptique chez I'Aplysie. J. Physiol., Paris 65, 303.
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SHTIAHARA, T. & TAUC, L. (1975). Multiple interneuronal afferents to the giant cells in Aplysia. J. Phy8iol. 247, 299-319. STEFANI, E. & GERSCHENFELD, M. H. (1969). Comparative study of acetylcholine and 5-hydroxytryptamine receptors on single snail neurones. J. Neurophy8iol. 32, 64-74. TAUC, L. (1965). Presynaptic inhibition in the abdominal ganglion of Aply8ia. J. Physiol. 181, 282-308. TAUC, L. (1967). Transmission in invertebrate and vertebrate ganglia. Phyaiol. Rev. 47, 521-593. TAUC, L. & SHIMAHARA, T. (1971a). Several types of synaptic responses and heterosynaptic facilitation observed in a single Aply8ia neuron. Proc. XXV Int. Congr. Phy8iol. Sci. Munich 9, 559. TAUC, L. & SHIMAHARA, T. (1971 b). Heterosynaptic facilitation. Experientia 27, 13.
321
HETEROSYNAPTIC FACILITATION IN THE GIANT CELL OF APLYSIA
By T. SHIMAHARA AND L. TAUC From the Laboratoire de Neurobiologie Cellulaire du Centre National de la Recherche Scientifique, 91190, Gif-sur- Yvette, France
(Received 16 July 1974) SUMMARY
1. Heterosynaptic facilitation, defined as an increase of the efficacy of synaptic transmission between a test interneurone and a post-synaptic neurone, produced by the stimulation of a separate pathway, was studied in the left pleural ganglion. The experimental procedure consisted of detecting the effects of a brief tetanus, applied to tentacular and tegumentary nerves, on the amplitude of monosynaptic and unitary postsynaptic potentials (p.s.p.s) recorded in the left giant cell and generated by stimulating the test interneurone every 10 sec. The membrane potential of the test interneurone was simultaneously recorded. 2. Following heterosynaptic stimulation, the amplitude of the test p.s.p. increased, after a delay of about 30 sec, up to 250 % of its original size; this increase subsided after 2-3 min or more. 3. Only the interneurones producing in the giant cell the e.i.p.s.p. (excitatory-inhibitory post-synaptic potential) were affected by heterosynaptic facilitation. Other interneuronal types showed no changes in their synaptic transmission on the giant cell after heterosynaptic stimulation. 4. Heterosynaptic stimulation did not produce either orthodromic or antidromic spikes in the test interneurones clearly indicating that facilitation of test p.s.p. did not result from increased spike activity in the test interneurone. 5. Often heterosynaptic facilitation of the test p.s.p. was observed due to spontaneous activity in the heterosynaptic pathway, demonstrating the normal occurrence of the phenomenon. 6. Iontophoretic injection of 5-HT at critical, presumably synaptic, sites in the neuropil, evoked a facilitation of the test p.s.p. similar to heterosynaptic facilitation. Only the e.i.p.s.p.s were so affected by 5-HT. On the contrary, other p.s.p. types were depressed by 5-HT as a result of conductance changes in the left giant cells. 13
P HY
247
322 T. SHIMAHARA AND L. TA UC 7. Both heterosynaptic facilitation and 5-HT facilitation were suppressed by the presence in the bath of 5-HT (10-5 M) and of LSD-25 (3 x 104 M). The action of injected 5-HT on the membrane conductance of the left giant cell was also depressed in the presence of 5-HT in the bath, but was unaffected by LSD-25 (3 x 104 M). 8. From the parallelism of properties of heterosynaptic and 5-HT facilitation, it is suggested that 5-HT is the probable transmitter mediating heterosynaptic facilitation. It seems likely that 5HT is released from the heterosynaptic pathway at the level of the synaptic ending of the test interneurone on to the giant cell and that it increases the efficacy of this synapse, probably acting on the quantity of synaptic transmitter liberated. INTRODUCTION
When the synapses of two different neurones on a third neurone interact in such a way that one of the two synapses can modify the synaptic efficacy of the other, this interaction is called heterosynaptic. Heterosynaptic interactions are rather little understood, partly because their identification and study require strictly defined experimental conditions in order to dissociate other neuronal mechanisms which can produce similar phenomenological effects. Inhibitory heterosynaptic actions of different kinds have been described in several systems: in the spinal cord (Frank & Fuortes, 1957; Frank, 1959), in the neuromuscular junction of crustacea (Dudel & Kuffler, 1960, 1961), in the Mauthner cell of the goldfish (Furukawa, Fukami & Asada, 1963), and in Aplysia ganglion cells (Tauc, 1965). The unique case of heterosynaptic facilitation (h.s.f.) was discovered in Aplysia central nervous system (Kandel & Tauc, 1965). It was defined as an increase in the efficacy of synaptic transmission between a test interneurone and post-synaptic neurone (measured as an increase of p.s.p. in the post-synaptic neurone) following the stimulation of another afferent heterosynaptic pathway, different from that containing afferents of the test interneurone. The similarity between the effects of post-tetanic potentiation (p.t.p.) and h.s.f. in a system in which the test interneurone is uniquely identified makes it difficult to distinguish h.s.f. as a separate phenomenon. By taking advantage of the fact that a single test interneurone subject for h.s.f. has contacts with two identifiable post-synaptic cells situated in different ganglia, and by excluding the possibility of spike generation in one of the branches of the test interneurone during heterosynaptic stimulation, Epstein & Tauc (1970) were able to bring a convincing, although indirect demonstration that h.s.f. can take place in the complete
323 HETEROSYNAPTIC FACILITATION absence of spike activity in the test interneurone and consequently represents a phenomenon distinct from p.t.p. In order to obtain direct evidence for the existence of an h.s.f. and to have a chance to approach the mechanism involved, we have identified neurones whose endings show facilitation upon h.s. stimulation. An interneurone producing excitatory-inhibitory post-synaptic potentials (e.i.p.s.p.) in the left giant cell fulfilled the required criteria (see Shimahara & Tauc, 1975). In the present study we provide direct evidence, that h.s.f. is specific to a given interneuronal type and that it occurs without the initiation of action potentials in the test interneurone. Furthermore, h.s.f. appeared to be a result of chemical action by the heterosynaptic interneurone on the test neurone; the most probable mediator involved 5-HT. Several preliminary reports have already been published (Shimahara & Tauc, 1970, 1972; Tauc & Shimahara, 1971a, b). METHODS
The left pleural ganglion of Aplysia californica was used for all experiments. The techniques for dissecting the preparation and obtaining intracellular recording were similar to these described in the preceding paper (Shimahara & Tauc, 1975). The left giant cell (l.g.c.) was used as the post-synaptic cell. This cell as well as an afferent interneurone in the pleural ganglion were impaled with double electrodes filled with 3M-KCl, one barrel of which was used for recording and the other for passing adequate current to bring the membrane potential to the desired level. The experimental procedure was as follows: the test interneurone was stimulated directly every 10 sec and the corresponding p.s.p. was recorded in the giant cell at a high level of amplification. The stimulation of the test interneurone was prolonged over a period needed to obtain p.s.p.s of fully 'habituated' stable amplitudes (Bruner & Tauc, 1966). The heterosynaptic stimulus was applied at 7 per sec for 1 to 3 sec to the tentacular or tegumentary nerves lying on Ag-AgCl electrodes. Bipolar stimulation was used to minimize polarization of the stimulating electrodes. The preparation was continuously perfused by sea water with and without drugs. Local applications of drugs was by iontophoretical micro-injection through micropipettes of 1-2 jsm tip diameter. Backing current was continuously applied to prevent leaking of drugs from the tip of the micropipette. All experiments were performed at room temperature, about 22° C. RESULTS
Identification of interneurones subject to h.s.f.; recording from test and postsynaptic neuroses All types of interneurones present in the left pleural ganglion of Aplysia and afferent to the l.g.c. were tested for their ability to produce a h.s.f. The standard stimulating procedure described in Methods was used and revealed that only the interneurones producing an e.i.p.s.p. in the l.g.c. I3-2
T. SHIMAHARA AND L. TA UC 324 could be affected heterosynaptically. Both fast and slow phases are affected but due to difficulties which are often present in identification of the slow inhibitory phase of the unitary e.i.p.s.p. all observations were made only on the fast excitatory component. Such an action is illustrated in Fig. 1, where the stimulation of the tentacular nerves increased the
Fig. 1. Effect of heterosynaptic stimulus on the e.i.p.s.p. recorded at high speed in the left giant cell (upper traces); lower traces show lower speed continuous recording in the test interneurone which is stimulated directly every 10 sec. A repetitive heterosynaptic stimulus (7 stimuli in 1 sec) was applied to the tegumentary nerves between the first and second postsynaptic potential (row 1). Note that heterosynpatic stimulation did not evoke any spike in the test interneurone and that the maximum effect on the p.s.p. is observed at 25 sec after the heterosynaptic stimulus (middle row 2).
HETEROSYNAPTIC FACILITATION 325 amplitude of the test p.s.p.s to 200 % of control. The original amplitude was only restored 100 sec after the heterosynaptic stimulus. The recordings show that this stimulus produced a complex synaptic potential on the post-synaptic cell (sometimes even giving rise to spike potential), and that the test interneurone was slightly depolarized, but no spike potential was initiated. The absence of spikes in the test interneurone during heterosynaptic facilitation is strong evidence that h.s.f. is a phenomenon distinct from the homosynaptic facilitation of p.t.p. Another important point which can be seen in Fig. 1 is the delay which between the heterosynaptic stimulus and the maximal increase observed is of the p.s.p. Such a delay has already been reported (Kandel & Tauc, 1965; Epstein & Tauc, 1970). Facilitation, presumably heterosynaptic, has been frequently observed to occur spontaneously without applying any stimulus to a heterosynaptic pathway (Fig. 2). In this case prior to the increase of test p.s.p. a slight depolarization is observed in the test interneurones simultaneously with a burst of p.s.p.s in the post-synaptic cell (l.g.c.). But no additional spike activity other than that corresponding to the test stimulation can be detected in the test interneurone. The synaptic efficacies of p.s.p.s other than e.i.p.s.p.s were not affected by the heterosynaptic stimulation. For instance, heterosynaptic stimulation does not change the amplitude of slow e.p.s.p. even though it produces a depolarization in the test interneurone generating the slow p.s.p., similar to that in the interneurone generating the e.i.p.s.p. (Fig. 3). Also all interneuronal types show p.t.p., but only the 'e.i.p.s.p. type' is subject in addition to h.s.f. The differences in facilitatory effects of homosynaptic stimulation of the test neurone and heterosynaptic stimulation in cells subject to h.s.f. were already reported (Epstein & Tauc, 1970). The present intracellular recordings from the test interneurone add additional information concerning modification of its membrane potential. Both homosynaptic and heterosynaptic stimulation are followed by an increase of test p.s.p. But whereas homosynaptic stimulation induces hyperpolarization (Fig. 4A), the heterosynaptic stimulation is followed by a depolarization (Fig. 4B) of the test interneurone. Thes3 opposite membrane effects appear to be characteristics of homosynaptic and heterosynaptic actions.
Pharmacological ba8is of h.s.f. It has been proposed that h.s.f. (as well as heterosynaptic inhibition), results from an interaction between heterosynaptic interneurones and the test interneurone at the level of the latter's nerve
T. SHIMAHARA AND L. TAUC 326 endings, and that this action is chemically mediated. The site of this interaction was designated as the 'episynapse' (Tauc, 1965, 1967). The action of the episynaptic transmitter would be to increase, through an unknown mechanism, the quantity of the transmitter released. Spontaneous
\
Fig. 2. Spontaneous heterosynaptic facilitation. Lower traces show recording from the test interneurone stimulated every 10 sec and producing p.s.p.s in the giant cell. In the l.g.c., a spontaneous burst of p.s.p.s originating from non-identified interneurones is followed by an increase in the test p.s.p. Note the depolarization in the test interneurone and absence of any spike other than that due to test stimulation.
One way to approach the identification of the episynaptic transmitter is to search for a substance which, when injected in the region of synaptic contacts between the test interneurone and the left giant cell, would mimic heterosynaptic facilitation. The experimental procedure used in this case was identical to that used before, except that instead of heterosynaptic stimulation, iontophoretic injections were applied on the regions of neuropil where presumed synaptic contacts exist between the test neurone and the giant cell. It was easy to
327 HETEROSYNAPTIC FACILITATION ascertain that the axon of the giant cell crosses the left pleural ganglion and emerges in both pleuro-pedal connectives (see Fig. 1, Shimahara & Tauc, 1975). However, as the synaptic contacts are necessarily embedded in the neuropil, many spots at different depths had to be tried for each substance injected. The substances tried were acetylcholine, dopamine, serotonin (or 5-HT), DL-glutamate, glycine and aspartate. Among these substances only 5-HT AV
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I Fig. 3. Comparison of effects of homosynaptic (A) and heterosynaptic (B) stimulations on the slow e.p.s.p. Upper traces are records from the left giant cell (l.g.c.) and lower records from the interneurone stimulated every 10 see as in Fig. 1. Note in A the clear post-tetanic potentiation after homosynaptic stimulation, and in B the absence of any effect of heterosynaptic stimulation.
328
T. SHIMAHARA AND L. TA UC A
IIIIIII1AI
Fig. 4. Comparison of effects of homosynaptic (A) and heterosynaptic (B) stimulation on e.i.p.s.p. Records are displayed on a Brush 280 pen recorder. The test interneurone is stimulated every 10 sec; between each stimulus the paper speed was decreased 100-fold to permit observation of successive synaptic responses on a short record. Both homosynaptic and heterosynaptic stimulation are followed by an increase on the test p.s.p. But following these stimuli, the membrane potential of the test interneurones show a hyperpolarization in A and depolarization in B. The hyperpolarization which is observed in the giant cell following the burst of activity is due to the summation of hyperpolarizing phases of e.i.p.s.p.s.
329 HETEROSYNAPTIC FACILITATION was effective in increasing the test p.s.p. in a way analogous to heterosynaptic facilitation. An effect was observed only when 5-HT was injected at certain critical sites in the neuropil. It was without effect if injected elsewhere, especially around the soma or the emerging axons of the test interneurone. rI- 5-HT
I Fig. 5. Effect of repetitive (1 per 10 sec) 5-HT iontophoretic injections in the neuropil (arrows) on the membrane potential of the l.g.c. (A) and of the interneurone (B) and the size of the e.i.p.s.p. recorded in the l.g.c. Same recording procedure as in Fig. 4.
Such chemically induced facilitation is represented in Fig. 5, following a series of brief injections of 5-HT. A several-fold increase in the test p.s.p. develops after some time and subsides slowly after the end of injections. Concomitantly polarization changes occur in the cells: depolarization sometimes followed by hyperpolarization in the test interneurone, hyperpolarization in the giant cell. However, the increase of test p.s.p. is not correlated to these changes, which usually subside more rapidly than the effect on the p.s.p. amplitude. On the other hand, direct effect of 5-HT on the giant cell membrane may be an additional depolarization or a more complex reaction (see later).
T. SHIMAHARA AND L. TAUC 330 The existence of 5-HT effects on the post-synaptic cell membrane made investigations necessary to ascertain whether the observed increase in the amplitude of the p.s.p. is related to modifications of the synapse itself or if it simply results from changes in the post-synaptic membrane properties. As the p.s.p. increase was observed for both hyperpolarization and depolarization of the giant cell, one could easily discard as decisive the influence of membrane potential of the giant cell by virtue of its bringing the p.s.p. to a different distance from its inversion level. For the same reason a possible increase of the membrane conductance due to anomalous rectification known to be present in these cells need not be considered (Kandel & Tauc, 1966). The conductance of the giant cell membrane was definitely affected by 5-HT, but in a way which by no means could produce an increase of the test p.s.p. The conductance measurements performed by applying constant current pulses and estimating the amplitude of the voltage change (Fig,. 6) show that the iontophoretic application of 5-HT clearly increases the membrane conductance, and that this increase is not due to anomalous rectification. This is demonstrated by the absence of conductance changes for a depolarization produced by applied current and which mimics the polarization change induced by 5-HT injection (Fig. 6 left side). The conductance increase normally would depress the p.s.p. amplitude by the shunt which it exerts on the p.s.p.-generating mechanism. The conductance increase induced in the left giant cell by 5-HT acts on the p.s.p. to oppose the facilitation; thus the p.s.p. amplitude increase observed after 5-HT application on the synaptic region would be still larger if correction could be made for the conductance increase of the post-synaptic cell membrane. That this conclusion is correct can be ascertained if instead of using an e.i.p.s.p. subject to heterosynaptic facilitation, one examines the action of 5-HT injection on the slow e.p.s.p. Fig. 7 shows that this slow e.p.s.p. is not affected by stimulation (see above), and that the 5-HT injection at the synaptic site depresses the slow e.p.s.p., to less than 50 % of its initial amplitude in the example shown. No injection site can be found in the neuropil at which the injection of 5-HT increases the slow e.p.s.p. amplitude. The same depressive action of 5-HT resulting from the decrease of the input resistance of the giant cell was observed for all other synaptic types. The e.i.p.s.p. alone was facilitated. It can be concluded from these experiments that the e.i.p.s.p. facilitation observed after iontophoretic injection of 5-HT at defined sites in the neuropil does not result from biophysical modifications of the post-synaptic cell membrane. The specificity of 5-HT action on the e.i.p.s.p. and its similarity of action to
331 HETEROSYNAPTIC FACILITATION that of heterosynaptic stimulation propose 5-HT as a candidate for the episynaptic transmitter. In the following paragraphs this 5-HT action on e.i.p.s.p. will be designated the 5-HT facilitation. 5-HT
4 mV
100 sec
-HT
Fig. 6. Measurement of changes in membrane conductance in the l.g.c. during a depolarization produced first by transmembrane applied current (left side) by 5-HT injection (between two arrows and then at the synaptic region (right side). Square current pulses are applied repetitively, 1 per see, to produce electrotonic potentials the size of which is proportional to membrane resistance. The recording shows that 5-HT produces a clear conductance increase which is not due to a membrane rectification.
Inhibitors of heterosynaptic and 5-HT facilitations If 5-HT is the episynaptic transmitter, it may be expected that both heterosynaptic and 5-HT facilitation must be blocked by inhibitors which inactivate the 5-HT receptor. We have tried to inhibit both h.s.f. and 5-HTfacilitation in two ways; first by 5-HT itself which can be considered as a specific inhibitor of 5-HT receptors probably through its desensitizing action (Stefani & Gerschenfeld, 1969) and second by LSD-25 (D-lysergic acid diethylamide), the best known 5-HT receptor inhibitor. The effect of 5-HT perfusion is shown in Fig. 9, to compare with the control increase of test p.s.p. by heterosynaptic and 5-HT facilitation in Fig. 8. 5-HT added to the bath at a concentration of 10-5 M depressed the
332 T. SHIMAHARA AND L. TAUC amplitude of the test p.s.p. by its action on the membrane conductance of the giant cell but it can be seen that now the heterosynaptic stimulus as well as the injected 5-HT have no effect on the test p.s.p. p
5-HT
E-p-s.p. slow
gc
Fig. 7. Effect of heterosynaptic stimulation (left column) and of 5-HT injection (right column) on slow e.p.s.p. The time indications are related to the beginning of heterosynaptic or chemical stimulation. Note the absence of changes after heterosynaptic stimulation and a 40 % decrease of slow e.p.s.p. after 5-HT application in the synaptic region of l.g.c.
LSD-25 at a concentration of 3 x 104 M added to the bath (Fig. 11) has little effect on the conductance of the giant cell or the size of the p.s.p., but with respect to the control in Fig. 10 it is evident that in the presence of LSD-25 both h.s.f. and 5-HT facilitation are inoperative. Moreover, iontophoretic injection of 5-HT now depressed the p.s.p., as it did for the
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HETEROSYNAPTIC FACILITATION
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Fig. 8. Control recording for Fig. 9, showing the effects of heterosynaptic stimulation (left column) and of 5-HT injection (right column) on the size of e.i.p.s.p. recorded in the l.g.c. (upper traces) due to activation of the test interneurone (lower traces). Time is measured from the beginning of the stimulations and injections.
333
334 T. SHIMAHARA AND L. TA UC slow e.p.s.p. (Fig. 7) in the absence of any drug. This depression indicates that 5-HT increases the conductance of the giant cell membrane in the presence of LSD-25, therefore, the 5-HT receptors on the giant cell were not blocked. k\1 {I-5-HT
E. p.s.p.
\
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Fig. 9. Same cells and experimental arrangement as in Fig. 8, except for the presence of 5-HT (105 M) in the bath. The size of the test p.s.p. is decreased owing to the decrease of input resistance of the l.g.c. by 5-HT. Note the absence of both heterosynaptic and 5-HT facilitations.
HETEROS YNAPTIC FACILITATION
Fig. 10. Control recording for Fig. 11. Same indications as in Fig. 8 but in a different preparation.
335
T. SHIMAHARA AND L. TA UC Further evidence that LSD-25 does not block these giant cell 5-HT receptors is given in the following experiments. Direct injection of 5-HT on the axon of the giant cell produces either a depolarization or hyperpolarization, or both in a sequence. Fig. 12 shows a complex depolarizing-hyperpolarizing response to 5-HT. Introduction of LSD-25 at a 336
t
5-HT
LSD-
E.i.p.s.p.
~~~~~~S
-
Fig. 11. Absence of heterosynaptic and 5-HT facilitation, when, after the control presented in Fig. 10, LSD-25 (3 x 104 M) is added to the bath. On the contrary the test p.s.p. is now depressed after 5-HT injection.
HETEROSYNAPTIC FACILITATION
337
,/ S cHT C.At
-
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LSD-25
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-
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-80 mV
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Fig. 12. Effect of iontophoretic injection of 5-HT on the axonal synaptic region of the l.g.c. at the different membrane potentials (-50 mV in upper traces, -80 mV in lower traces) and in normal conditions (control), in the presence of LSD-25 (3 x 16-4 mM), or of 5-HT (10- M), in the bath. The inhibitory component reverses at -80 mV. Note that the effect of 5-HT injection is not affected by LSD-25; the 5-HT in the bath almost suppresses the response to 5-HT injection.
T. SHIMAHARA AND L. TAUC concentration used above into the bath did not change this 5-HT response, which was however suppressed in the presence of 5-HT in the bath. These observations explain the p.s.p. responses obtained for 5-HT injection in Figs. 9 and 11. They point to the possible plurality of 5-HT receptors; especially it can be noted that the 5-HT receptor which we believed to be involved in 5-HT facilitation and h.s.f. are blocked by LSD-25 but those on the giant cell are not. Finally they show that physiological and 5-HT induced facilitations both react in the same way to 5-HT inhibitors. 338
DISCUSSION
Of the many interneuronal types which were found in the left pleural ganglion to be afferent to the left giant cell, only the so-called e.i.p.s.p. type has the ability to increase the synaptic efficacy of its endings by means of heterosynaptic stimulation. The observed absence of spike activity in the test interneurone as a consequence of heterosynaptic stimulation brings evidence that heterosynaptic facilitation represents a distinct phenomenon; different especially from homosynaptic facilitation which results from repetitive spike activity. The effects of heterosynaptic stimulation reported here were shorter (max. 3 min) than those described previously for a single p.s.p. (Epstein & Tauc, 1970), which lasted up to 40 min. This difference is most probably due to different interneurones in the two studies, as well as the nerves chosen for heterosynaptic stimulation. It has been found also for heterosynaptic inhibition (Tauc, 1965) that quite considerable differences may be observed in its duration in different cells. They might be ascribed to differences in quantitative rather than qualitative properties of individual neurones. The present results suggest that the episynaptic transmitter is 5-HT. It is present in Aplysia ganglia (Carpenter, Breese, Schanberg & Andkopin, 1971; Cottrell, 1974), its local application on to the synaptic region of the post-synaptic cell mimics effects of heterosynaptic stimulation, the 5-HT facilitation is restricted only to p.s.p.s which are able to undergo heterosynaptic facilitation, and finally heterosynaptic and 5-HT facilitations are affected similarly by inhibitors of 5-HT receptors. The possibility cannot be excluded, however, that the injected 5-HT acts not on the episynaptic site of the test interneurone, but on 5-HT receptors of an interneurone in the heterosynaptic pathway thus giving rise to an effective stimulation of this pathway. The specificity of the inhibitory action of LSD-25 (at the concentrations used in present studies) on the 15-HT receptors in the heterosynaptic pathway and absence of
339 HETEROSYNAPTIC FACILITATION inhibition of the 5-HT receptors in the synaptic region of the giant cell points against this possibility, given that the 5-HT receptors of the interneurones interposed in the heterosynaptic pathway are probably identical to those of the giant cell. This question can be definitively resolved when it becomes possible to identify and record from presumed interneurones in the heterosynaptic pathways. One has also to consider that the transmitter liberated by the heterosynaptic pathway, or 5-HT, could act not at the test interneurone ending, but on receptors located on the postsynaptic membrane increasing somehow the efficacy of the transmitter liberated by the test interneurone. A facilitating action of dopamine on ACh receptors has been described in the vertebrate sympathetic ganglion cells (Libet & Tosaka, 1970). Such a facilitating effect of 5-HT on the receptors of the unknown transmitter of the test cells cannot be excluded, although the blockage of 5-HT injection by 5-HT in the bath argues against this hypothesis. Actually, we think that our results are in good agreement with a situation in which the heterosynaptic pathway liberates 5-HT which combines with 5-HT receptors located on the nerve endings of the test cells, thus inducing an increased potentiality for transmitter liberation by the terminal without inducing spike generation. Whether a specialized anatomically definable episynaptic contact is necessary is an open question. In any case, serial synapses have been seen in Aplysia ganglia among unidentified cell processes (Jourdan & Nicaise, 1971). But the specificity of the episynaptic transmitter action could be insured even in the absence of synaptic contacts as only the nerve endings having adequate 5-HT receptors would be affected by heterosynaptic action. A relatively long delay for maximum increase of p.s.p.s after heterosynaptic stimulation might reflect the time of diffusion of the transmitter towards the test cell endings. More hypothetical is the way by which one can imagine the action of the episynaptic transmitter on the transmitter release mechanism of the test cell. The easiest way is to consider that the episynaptic transmitter or 5-HT induces on the episynapse classical ionic permeability changes, that is, affects its polarization and or its conductance. It was demonstrated previously (Shimahara & Tauc, 1975) that in some Aplysia synapses the transmitter release is increased by presynaptic depolarization. However in the present results the-duration of the polarization changes measured in the test cells during h.s.f. and 5-HT-facilitation is not in exact correspondence with the duration of facilitations. In addition, slow e.p.s.p.-type interneurones show similar depolarization change to heterosynaptic stimulation as the e.i.p.s.p. type, but the slow e.p.s.p. is not facilitated. Moreover, Carew, Castellucci & Kandel (1971) observed in the abdominal ganglion cells a hyperpolarization of a presynaptic
340 T. SHIMAHARA AND L. TAUC neurone as a result of heterosynaptic stimulation. But, as already pointed out, potential changes measured in the soma do not necessarily reflect potential changes on the nerve ending. It is also possible that the episynaptic transmitter of 5-HT exerts its action otherwise, by a more direct way on the transmitter's liberation mechanism, for instance modulating the calcium movement. Further experimentation is necessary for understanding the mechanism involved. The authors wish to thank Dr R. T. Kado and Dr R. S. Zuker for assistance with the manuscript. This work was party supported by ATP grant no. 4202 from C.N.R.S. and I.N.S.E.R.M. grant no. 7340808. T. Shimahara is a fellow of Roussel-Uclaf. REFERENCES BRUNER, J. & TAUC, L. (1966). Habituation at the synaptic level in Aply8ia. Nature, Lond. 210, 37-39. CAREW, T. J., CASTELLUCCI, V. F. & KANDEL, E. R. (1971). An analysis of dishabituation and sensitization of the gill-withdrawal reflex in Aply8ia. Int. J. Neurosci. 2, 79-98. CARPENTER, D., BREESE, G., SCHANBERG, S. & ANDKOPIN, I. (1971). Serotonin and dopamine: distribution and accumulation in Aplysia nervous and non-nervous tissues. Int. J. Neurosci. 2, 49-56. COTTRELL, G. A. (1974). Serotonin and free amino acid analysis of ganglia and isolated neurones of Aplysia dactylomela. J. Neurochem. 22, 557-559. DUDEL, J. & KUFFLER, S. W. (1960). Excitation at crayfish neuromuscular junction with decreased membrane conductance. Nature, Lond. 187, 246-247. DUDEL, J. & KUFFLER, S. W. (1961). Presynaptic inhibition at the crayfish neuromuscular junction. J. Physiol. 155, 543-562. EPSTEIN, R. & TAUC, L. (1970). Heterosynaptic facilitation and post-tetanic potentiation in Aplysia nervous system. J. Physiol. 209, 1-23. FRANK, K. (1959). Basic mechanisms of synaptic transmission in the central nervous system. I.R.E. Trans. Med. Electron. ME-6, pp. 85-88. FRANK, K. & FUORTES, M. G. F. (1957). Presynaptic and post-synaptic inhibition of monosynaptic reflexes. Fedn Proc. 16, 39-40. FURUKAWA, T., FUiAMI, Y. & ASADA, Y. (1963). A third type of inhibition in the Mauthner cell of goldfish. J. Neurophysiol. 26, 759-774. JOURDAN, F. & NIcAisE, G. (1971). L'ultrastructure des synapses dans le ganglion pleural de l'Aplysie. J. Microscopie II, 69-70. KANDEL, E. R. & TAUC, L. (1965). Mechanism of heterosynaptic facilitation in the giant cell of the abdominal ganglion in Aplysia depilans. J. Physiol. 181, 28-47. KANDEL, E. R. & TAUC, L. (1966). Anomalous rectification in the metacerebral giant cells and its consequences for synaptic transmission. J. Physiol. 183, 287304. LIBET, B. & TosAKA, T. (1970). Dopamine as a synaptic transmitter and modulator in sympathetic ganglia: a different mode of synaptic action. Proc. natn. Acad. Sci. U.S.A. 67, 667-673. SHIMAHARA, T. & TAUC, L. (1970). Multiples interneurones de type different 'a un meme neurone g6ant chez I'Aplysie. J. Physiol., Paris 62, 449. SHIMAHARA, T. & TAUC, L. (1972). Mechanisme de la facilitation h6t6rosynaptique chez I'Aplysie. J. Physiol., Paris 65, 303.
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