J. Physiol. (1978), 274, pp. 265-278 With 6 text-figure8 Printed in Great Britain
265
RELEASE OF ENDOGENOUS SEROTONIN FROM TWO IDENTIFIED SEROTONIN-CONTAINING NEURONES AND THE PHYSIOLOGICAL ROLE OF SEROTONIN RE-UPTAKE
BY H. M. GERSCHENFELD, M. HAMON* AND DANIELE PAUPARDIN-TRITSCH From the Laboratoire de Neurobiologie, Ecole Normale Superieure, 46, rue d'Ulm 75005, Paris, and the *Groupe NB, I.N.S.E.R.M. U 114, College de France, 11, place Marcelin Berthelot, 75005 Paris, France (Received 30 May 1977) SUMMARY
1. The amounts of endogenous serotonin (5-HT) released into the medium by the cerebro-buccal ganglionic ring of Aplysia californica incubated in artificial sea water (ASW) were measured. The rate of spontaneous 5-HT release varied between 0*4 and 1-2 p-mole per hour, which is less than 1 % of the total 5-HT present in this preparation. 2. Direct stimulation of the ordinarily silent 5-HT-containing giant cerebral neurones resulted in a 80-100 % increase of the 5-HT released, but only when the 5-HT uptake was blocked by chlorimipramine (1-10 /uM). 3. High K+ media (50 mM) also caused a significant increase in the amount of 5-HT released from the preparation provided that chlorimipramine (1-10 ,M) was present in the incubation fluid. 4. Co2+ ions (10-30 mm) added to the incubating medium blocked the spontaneous leak of endogenous 5-HT as well as the release, in the presence of chlorimipramine, evoked either by stimulation of the 5-HT-giant cerebral neurones or high K+-media. 5. In the presence of chlorimipramine or desmethylimipramine, the duration and/or the amplitude of the excitatory or the inhibitory synaptic potentials evoked in the buccal neurones by the stimulation ofthe 5-HT giant cerebral neurones were markedly enhanced. 6. These results strongly support the idea that 5-HT is the synaptic transmitter released at the excitatory and inhibitory junctions established by the 5-HT giant cerebral neurones in the ipsilateral buccal ganglia. In addition, they underline the role of amine re-uptake in the physiological inactivation of 5-HT as a transmitter. INTRODUCTION
The discovery by Cottrell & Osborne (1970) of two giant neurones symmetrically located in the cerebral ganglia of the snail Helix aspersa which showed an intense yellow fluorescence with the Falck-Hillarp technique, initiated an intensive research on the biochemical and physiological properties of single identified neurones containing serotonin (5-hydroxytryptamine = 5-HT). Cottrell and his co-workers
H. M. GERSCHENFELD AND OTHERS (Cottrell, 1970, 1971; Cottrell & Osborne, 1970; Cottrell & Macon, 1974) demonstrated that these cells from land and fresh-water snails contained detectable quantities of 5-HT and that they made excitatory connexions with three other neurones located in the ipsilateral buccal ganglion, 5-HT probably being the transmitter intervening at these connexions. In parallel studies in Aplysia californica, Weinreich, McCaman, McCaman & Vaughn (1973) analysed a pair of giant cerebral neurones homologous to those of the snails. These cells, also symmetrically located, contain 4-6 p-mole of 5-HT in their somata (Weinreich et al. 1973; Brownstein, Saavedra, Axelrod, Zerman & Carpenter, 1974) and were shown to synthetize 5-HT from both tryptophan (Eisenstadt, Goldman, Kandel, Koike, Koester & Schwartz, 1973) and 5-hydroxytryptophan (Weinreich et al. 1973; Eisenstadt et al. 1973). 5-HT synthesized from radioactive 5-hydroxytryptophan injected inside the soma of the giant cerebral neurone appears associated with subcellular particles and is transported along the axon by a specific transport system (Goldman & Schwartz, 1974; Goldberg, Goldman & Schwartz, 1976). Each one of the giant cerebral 5-HT containing neurones of Aplysia sends an axon branch through the cerebro-buccal connective to the ipsilateral buccal ganglion, where it establishes direct synaptic connexions with at least thirteen neurones: excitatory junctions with nine neurones and inhibitory junctions with the other four (Gerschenfeld & Paupardin-Tritsch, 1974). Evidence obtained by comparing the pharmacological properties of 5-HT receptors in the post-synaptic buccal neurones and the synaptic potentials evoked by intracellular stimulation of the 5-HT giant cerebral neurones, strongly support the idea that 5-HT is used as a transmitter at these synapses (Gerschenfeld & Paupardin-Tritsch, 1974). The experiments reported in the present paper were designed to determine whether the firing of the 5-HT giant cerebral neurones actually caused a measurable release of 5-HT from their endings. Such a release was demonstrated when the uptake of 5-HT was pharmacologically suppressed in the cerebro-buccal ganglionic preparation. Parallel electrophysiological experiments, furthermore showed that inhibiting the uptake of 5-HT caused an increase of both the amplitude and the duration of the synaptic potentials evoked by the firing of the 5-HT giant cerebral neurones. These experimental results confirm that 5-HT is the transmitter released by the 5-HT giant neurones at their synapses with the buccal neurones and that the action of 5-HT is terminated at these junctions by a re-uptake mechanism. Some of these results were communicated in a preliminary form to the Physiological Society (Gerschenfeld, Hamon & Paupardin-Tritsch, 1976). 266
METHODS The preparation used in the present study is drawn in Fig. 1 and consisted of the isolated cerebro-buccal ganglionic ring of A. californica. The specimens used were obtained from Dr R. Fay, Pacific Marine Co. (Venice, California) and kept in an aerated artificial sea-water aquarium. The cerebral and buccal ganglia of the animals were removed together, keeping intact the cerebrobuccal connectives. This ganglionic ring (Fig. 1) was fixed to the bottom of a suitable plastic chamber and bathed in 1 ml. artificial sea water (ASW) composed of (mM) Na+ 480, K+ 10, Cl- 610, Ca2+ 10, Mg2+ 50 and Tris-HCl (pH 7 8) 10. The connective tissue sheaths covering the dorsal face of the cerebral ganglia and the caudal face of the buccal ganglia were removed. Each one of the 5-HT giant cerebral neurones (400-600 ,um diameter) was impaled with a double-
RELEASE AND UPTAKE OF 5-HT BY 5-HT NEURONES
267
barrelled glass micropipette filled with 0-6 m-K2S04 or 3 M-KCl showing tip resistances of 15-25 MC. One of the barrels of the double pipette was used for recording via a d.c. amplifier connected to a CRO and a 280 Brush pen recorder. The other barrel was used to stimulate the giant cerebral neurones by passing constant current pulses across their membrane. When these cells were impaled they showed resting potentials of -60 to -65 mV, and at variance with what is generally observed in Aply8ia neurones, they were silent and only fired when stimulated intracellularly. During the
Fig. 1. Diagram of the cerebro-buccal ganglionic ring preparation showing the location of the 5-HT containing giant cerebral neurones and the trajectory of their axons through the cerebro-buccal connectives to the buccal ganglia.
H. M. GERSCHENFELD AND OTHERS 268 stimulation periods, the membrane potential of the 5-HT giant neurones was controlled in order to keep them firing at a rate of 1-5 Hz. In many experiments one or more buccal neurones were also impaled to check the efficacy of the synaptic transmission. After each 1 hr period of either rest or intracellular stimulation of the 5-HT giant neurones, the fluid bathing the ganglia was totally removed and immediately frozen to -30 'C. This change of bathing fluid implied each time a controlled removal of the micropipettes from the cells and their re-insertion. The experiment generally lasted for 3 hr and the action potentials of both 5-HT giant cerebral neurones were continuously controlled.
150
Melatonin
100 E C.)
50
BN 0
5
10
15
Distance (cm) Origin Front Fig. 2. Thin-layer chromatography of [H3]melatonin formed from 5-HT leaked spontaneously from a cerebro-buccal ganglionic ring incubated for one hour in ASW. After migration (90 min with toluene, ethylacetate, acetic acid and water, 80:40:20:4 by volume of the solvent), the melatonin spot was revealed by a spray of von Urk reagent. The silica gel was then carefully scraped off in 1 cm sections and mixed with water (1 ml.) and scintillating fluid (PCS 10 ml. Amersham Searle) for radioactivity counting. BN corresponds to the background noise of the Packard counter.
Radioenzymic microassay of 5-HT. This technique was performed at 4-6 0C, before the fourth week after the experiments. In preliminary studies it was shown that the 5-HT content of ASW samples kept at -30 0C did not change significantly after 1 month in the freezer. The 5-HT assay procedure used was that of Boireau, Ternaux, Bourgoin, Hgry, Glowinski & Hamon (1976), slightly modified. The ASW samples were thawed and diluted by half with a mixture of 0-5 mmKH2PO4 and 25-5 mM-NaCO3 in order to bring the pH and the ionic strength close to those of the cerebrospinal fluid samples used originally by Boireau et al. (1976). Each sample was passed through a Sephadex GIO column (3 cm high, 0-4 cm diameter) previously washed with 4 ml. 0-5 N-formic acid and 10 ml. water. After passing the sample, 6 ml. water were passed through the column and 5-HT was eluted in 3 ml. 0-5 N-formic acid. The eluate was lyophilized and the residue was solubilized in 60 /0. 0-2 M-Na2SO4 buffer pH 7-9. Thus 5-HT was converted into [3H]melatonin in two enzymatic steps with S-adenosyl-L-[methyl-sH]methionine (7-5-10 c/ m-mole, Amersham Radiochemical Centre) as the methyl donor. [3H]melatonin was then extracted into toluene and finally isolated by thin layer chromatography on silica gel (Boireau
RELEASE AND UPTAKE OF 5-HT BY 5-HT NEURONES
269
et al. 1976, see Fig. 2). The sensitivity of the whole procedure was routinely about 0.10-0.15 p-mole of 5-HT (amount of 5-HT leading to the formation of cpm of [3H]melatonin twice the blank value). Control experiments with ASW samples containing up to 30 mM-COCl2 or 10 FM chlorimipramine have shown that these compounds did not interfere with the assay of 5-HT in the conditions described above. The endogenous levels of 5-HT in the cerebro-buccal ganglionic ring were determined according to Ternaux, H6ry, Bourgoin, Adrien, Glowinski & Hamon (1977). Standard statistical calculations were made as proposed by Snedecor & Cochran (1967). When P values were higher than 0 05, differences were considered not to be significant. Electrophygiological experiments. The same cerebro-buccal ganglionic preparation was placed in a plastic chamber of 2*5 ml. volume, and was continuously bathed with circulating ASW. The connective sheaths of the ganglia were removed as described above. Two double-barrelled micropipettes were used, one to impale one of the 5-HT giant cerebral neurones, and the other, one of the ipsilateral post-synaptic buccal neurones. Chlorimipramine and desmethylimipramine (Ciba-Geigy, Basel) were directly added to the circulating ASW. All the experiments were performed at room temperature (20-23 TC).
RESULTS
'Spontaneous' release of 5-HT from the cerebro-buccal ganglionic ring preparation. When the cerebro-buccal ganglionic ring was incubated for one hour 'at rest' (i.e. without any stimulation of the 5-HT giant cerebral neurones) in normal ASW, there was a measurable quantity of 5-HT (well above the sensitivity of the assay method, see Fig. 2) in the bathing fluidwhich had leaked out from the preparation. The amount of this 'spontaneously' released 5-HT varied between 0-4 and 1-2 p-mole/rn. hr. This quantity could vary from experiment to experiment, and possibly, according to the season. Since the cerebro-buccal ganglionic ring contains 169 + 16-5 p-mole of 5-HT (mean + S.E. from seven duplicate determinations), only less than i % of this ganglionic pool actually 'leaked' to the bath each hour. The factors affecting this 'leak' were neither analysed nor specially controlled. In a first series of experiments, cerebro-buccal preparations were incubated for a period of 2-3 hr and the 5-HT content of the bathing fluid was monitored after each hour of incubation. It could be seen that, in this series, the average spontaneous leak of 5-HT in the first hour (in nine preparations) was 0-91 + 0-13 p-mole/ml. hr. During the second hour, the measured 5-HT leak in six preparations was 0-99 + 0-21 p-mole/ ml. hr. whereas the samples obtained after the third hour of incubation (in eight preparations) yielded an average of 0-65 + 0-09 p-mole of 5-HT/hr, less than in the first hour of incubation (but not significant). Significant changes were neither detected in the 5-HT content of the ganglionic ring preparation after the second and the third hour of incubation when compared to those found after the first hour. 5-HT giant cerebral neurones-stimUlation experiments. Surprisingly, direct electrical stimulation of both 5-HT giant cerebral neurones through the intracellular pipettes (which resulted in synaptic activation of the follower buccal neurones) failed to induce any significant increase in the amount of 5-HT 'leaked' in the bathing fluid (Fig. 3). This lack of effect of the neuronal stimulation was found in all the cases whatever the amount of 5-HT being released during the previous rest period (first hour of incubation). This apparent lack of effect of the 5-HT giant cerebral neurones stimulation could result from at least two reasons: (a) a failure of the neuronal endings to release
270 H. M. GERSCHENFELD AND OTHERS detectable amounts of 5-HT, (b) the rapid uptake of the released amine. In order to test the second of these hypotheses, the effects of electrical stimulation of the 5-HT giant cerebral neurones were analysed after blocking the 5-HT re-uptake using chlorimipramine (Osborne, Hiripi & Neuhoff, 1975). At rest, the addition of chlorimipramine (10SM) to the bathing fluid did not alter the 'spontaneous leakage' of 5-HT from the ganglionic ring preparation (Fig. 3). In contrast to the results obtained
(16)*
200
Unstimulated
C 1
75150 0
*poo
~~~~2)(12) (23)(38)
ID
cell Stimulated cell
-
_
_
+
100
50
Fig. 3. Effect of intracellular stimulation of the 5-HT giant cerebral neurones, chlorimipramine and Co2+ ions on the release of 5-HT from the cerebro-buccal ganglionic ring preparation. The preparation was kept at rest for one hour in ASW and then incubated for two hours either in the same medium or in ASW containing C0 /im-chlorimipramine or 30 MMCo2+ ions or both (in each case the medium was renewed at the end of each hour). Intracellular stimulation of the 5-HT giant cerebral neurones was performed g of mean during the second or the third hour of incubation. Each bar is the mean si. of 5-HT (in % of the values found for the first hour of the experiment). Since no signifiu cant differences were observed between the amounts of 5-HT released in similar conditions during the second and the third hour of incubation, the corresponding values were pooled for drawing the present Figure. *P < 0-001 when compared to respective control values. Number of determinations in brackets.
in pure ASW, the electrical intracellular activation of the 5-HT giant cerebral neurones did increase the efflux of 5-HT to the bathing fluid containing chlorimipramine. As shown in Fig. 3, this effect was highly significant and occurred in all cases, whatever the absolute amount of 5-HT released in the bathing fluid during the first hour rest period preceding the electrical stimulation period. No significant difference was observed between the 5-HT content of the cerebro-buccal ganglionic ring incubated for three hours in pure ASW with that of the ganglia which served for the electrical
271 RELEASE AND UPTAKE OF 5-HT BY 5-lT NEURONES stimulation experiments in the presence of chlorimipramine. This was not so sur-
prising since the amount of 5-HT released per hour did not exceed 2 % of the endogenous pool of the indoleamine in the ganglia even when electrical stimulation, chlorimipramine application or both were performed. We also analysed if the 5-HT neurones stimulation-induced release required CaO+ by adding CoCl2 (10-30 mM) to the bathing fluid during both rest and stimulation periods. According to Weakly (1973), Co2+ ions interfere with the entry of Ca2+ ions inside the synaptic endings and therefore cause a reduction or suppression of the transmitter release at chemical synapses. As shown in Fig. 3 and in Table 1, C02+ TARim 1. Effect of Co ions on the release of 5-HT 5-HT release p-molelhr Condition Control 30mx-CO2+ 30 mM-Co2+ + stimulation 10 mM-Co2+ 10 mM-Co2+ + stimulation
0 91 ± 0-08 (n 0.03+0.01* (n 0-03 + 0.01* (n 005+ 0.02* (n 0 04 + 0.02* (n
= 14) = 10) = 5) = 4) = 4)
The ganglionic ring was first maintained for 1 hr in normal ASW and then, 10 or 30 mm-CoCl2 were added to the ASW. After another hour the same medium was renewed and the 5-HT giant cerebral neurones were stimulated for another hour period. Each value listed is the mean + s.E. of mean of the amounts of 5-HT found in the medium at the end of the first hour of incubation (control), the second hour (10 or 30 mM-Co2+) and the third hour of incubation (Co2+ + stimulation). *P < 0.001 when compared to control values. n = number of determinations (in brackets).
strongly reduced the stimulation-induced efflux of 5-HT in the presence or the absence of chlorimipramine. Moreover, the spontaneous leakage of 5-HT from the cerebro-buccal ganglionic ring was also largely prevented by the presence of Co2+ ions in the bathing fluid (Fig. 3 and Table 1). In most cases, the effect of Co2+ ions was so marked that the amount of 5-HT released in the bathing fluid was lower than the sensitivity of the enzymic micro-assay (see Methods). Therefore, values in Fig. 3 and Table 1 are only semi-quantitative estimations of the amounts of 5-HT released in the presence of C02+ ions. The inhibitory effect of C02+ ions on 5-HT leakage strongly suggests that this process is not simply due to a destruction in the nervous tissue and that both kinds of release, spontaneous and evoked by 5-HT neurones intracellular stimulation, have their origin from synaptic endings. Release of 5-HT by high K+ concentrations. The release of transmitters from vertebrate brain tissue slices, isolated nuclei or synaptosomes by depolarizing the nerve structures with [K]+ rich media has been repeatedly reported in recent years. The effect of high [K]+ media on the spontaneous release of 5-HT from the cerebrobuccal ganglionic ring was also analysed. The protocol of these experiments was the following: the ganglionic ring was first incubated during one or two hours in normal ASW containing 10 mM-K+ and in subsequent hour period the concentration of K+ was increased to 50 mm. In these conditions, no significant increase in the 5-HT release was found after the exposure to a high [K]+ medium (Fig. 4). Suspecting that
H. M. GERSCHENFELD AND OTHERS 272 the possible 'surplus' release was damped by the re-uptake of 5-HT, the same experiments were repeated in the presence of 10 /gM chlorimipramine in the bathing fluid. As was observed before, chlorimipramine did not significantly change the amount of 5-HT leaked during the first hour of incubation in normal ASW, but in the presence of the uptake inhibitor, the high [K]+ medium caused an increase of about 60 % in the amount of 5-HT released from the preparation (Fig. 4). Electrophysiological experiments. The limited duration of transmitter action on postsynaptic membranes (which generally lasts between some milliseconds and hundreds of milliseconds) undoubtedly requires a step involving the termination of the action of the transmitter. The best known case of such a mechanism is the 200
D10 mm-K+
s150 0
(21)
50 mm-K+ *P
s-i
4 sec b Control 2 mV |
to sec
2
C
,1
\
.1mM
Clil
10 sec
IL
Control Fig. 5. Effect of chlorimipramine on the e.p.s.p. and i.p.s.p. evoked by 5-HT giant cerebral neurone stimulation. A, the discharge of spike trains in the giant neurone (a) evokes in a buccal neurone a smooth slow depolarization (marked control in b). Perfusion of chlorimipramine 1 1UM (marked Cli in b) causes an increase in both the amplitude and the duration of the e.p.s.p. In c, the iontophoretic application of 5-HT evokes in the same cell a depolarizing response which is not altered by chlorimipramine. B, the spike train as in a evokes in a buccal neurone a smooth slow hyperpolarization which increases in duration in the presence of 10 pm-chlorimipramine. In c chlorimipramine is shown to block at 04 mM-concentration the hyperpolarizing response evoked in the same neurone as in b by the iontophoretic application of 5-HT. The superimposed square electronic potentials in A,c and B,c show that chlorimipramine does not affect the input resistance of the cells.
Fig. 5 gives an example of the experiments performed. At left (Fig. 5A), recordings from an excitatory monosynaptic junction between one of the 5-HT giant cerebral neurones and one of the postsynaptic buccal neurones are reproduced. The upper trace (Fig. 5A, a) corresponds to a typical burst of spikes evoked by direct stimulation of the 5-HT giant cerebral neurone each 1-2 miD along the whole experiment and
H. M. GERSCHENFELD AND OTHERS the middle traces correspond to two superimposed recordings of the postsynaptic response to the presynaptic burst (Fig. 5A, b), taken before and during the bath application of 1-10 uM chlorimipramine. When the preparation is bathed in a normal ASW (Fig. 5A, b, control) the presynaptic burst evokes a slow smooth depolarization in the postsynaptic neurone. When the same presynaptic spike train is discharged in the presynaptic neurone after bathing the preparation for half an hour in 1 ,/Mchlorimipramine, the post-synaptic potential showed a marked increase of both its amplitude and its duration (Fig. 5A, b, recording labelled Cli 1 /tM). By comparing the two records superimposed in Fig. 5A, b, it is evident that, at the time when in the control one the buccal neurone membrane potential has returned to its initial level, the post-synaptic depolarization is still present under chlorimipramine and the membrane potential of the buccal neurone takes a much longer time to return to its original level. This effect was not due to a post-synaptic action of the drug since chlorimipramine, even at 041 mm concentration did not alter the depolarization evoked by the iontophoretic application of 5-HT on to the same buccal neurone 274
(Fig. 5A, c).
As it was said above, the 5-HT giant cerebral neurones also establish inhibitory synaptic connexions with some buccal neurones and the effect of the uptake inhibitor was also analysed on these junctions. Fig. 5B shows presynaptic and post-synaptic recordings from such an inhibitory connexion. In the upper trace (Fig. 5B, a) is recorded one of the spike trains evoked in the presynaptic 5-HT giant cerebral neurone. The middle trace (Fig. 5B, b) corresponds to superimposed recordings of inhibitory synaptic potentials evoked by the presynaptic train recorded above. In this case, the effects of a long application of a 1IOM concentration of chlorimipramine to the preparation appear to be somewhat different from those observed in the case of the excitatory junctions. Although the amplitude of the hyperpolarizing synaptic response did not increase in the presence of chlorimipramine (even in some cases a small reduction was observed), a clearcut prolongation of the inhibitory potential occurred in the buccal cells exposed to the drug. The bottom recordings of Fig. 5 give an explanation to the apparent difference of action of chlorimipramine on the excitatory and inhibitory responses. When 5-HT was applied iontophoretically on to the membrane of the same neurone of Fig. 5B, b, it hyperpolarized the neurones (Fig. 5B, c). The presence of chlorimipramine in the bathing fluid caused a reduction in the amplitude of this response, even when the uptake inhibitor was applied at 1O 14M concentration (in Fig. 5B, c the concentration of the inhibitor has been increased to accentuate the blocking effect). Therefore the lack of effect of 10 Um chlorimipramine on the amplitude of the inhibitory synaptic potential (Fig. 5B, b) resulted from an antagonism between the presynaptic action of the drug (increase in the amount of 5-HT present in the synaptic cleft due to the 5-HT re-uptake blockade) and its post-synaptic action (blocking of 5-HT action on the post-synaptic membrane). However, the prolongation of the inhibitory response under chlorimipramine remained a clear indication of the 5-HT re-uptake inhibition. From these experiments, it can be concluded that in the synapses established by different endings of the 5-HT giant cerebral neurones, chlorimipramine caused a prolongation of the synaptic potential suggesting that the physiological inactivation of the transmitter was impaired. Other drugs which inhibit the 5-HT uptake were
275 RELEASE AND UPTAKE OF 5-HT BY 5-HT NEURONES observed to have a similar effect on the synaptic potentials evoked by the stimulation of the 5-HT giant cerebral neurone. Fig. 6 corresponds to an experiment in which desmethylimipramine (a potent 5-HT uptake inhibitor in molluscan C.N.S., see Osborne & Neuhoff, 1974) was tested. In this case, the discharge of a train of action potentials at low frequency in the presynaptic neurone (Fig. 6A, a), instead of evoking a smooth slow depolarization in the postsynaptic buccal cells, produced the appearance of discreet unitary synaptic potentials in a 1:1 ratio (Fig. 6A, b). The addition of 10 /M-desmethylimipramine to the bathing fluid also resulted, as in the case of chlorimipramine, in a marked increase in the amplitude and the duration of each unitary excitatory synaptic potential (Fig. 6B, b).
A
a
o
C
B
X
La 40 mV 14 sec
b
b~
\7
ODMI 10Mm Fig. 6. A, each spike in a 5-HT giant cerebral neurone (a) evokes in a buccal neurone a dicreet monosynaptic e.p.s.p. (b). B, bath application of 10 /SM-desmethylimipramine does not affect much the neurone firing (a), but increases the amplitude and the duration of the e.p.s.p. (b). DISCUSSION
The present results provide for the first time direct evidence about a detectable release of 5-HT by stimulating single identified 5-HT containing neurones. Moreover they strongly suggest that amine re-uptake might be the major process in the inactivation of 5-HT as a transmitter in Aplysia nervous system. Osborne & Neuhoff (1974) had previously reported that circumoesophageal ganglia of the snail H. aspersa pre-incubated with [3H]trytophan or [3H]5-HT could release radioactive material (possibly [3H]5-HT) when the nerve commissures of the ganglionic circumoesophageal ring were stimulated. This release was sensitive to the Ca2+ content of the incubation medium. However, experiments with [3H]5-HT are in all cases difficult to analyse since the exogenous indoleamine accumulates to a large extent in non-nervous tissue (Ascher, Glowinski, Tauc & Taxi, 1968). Therefore such experiments should require not only the identification of released material but also that of the cellular compartment involved in the release process. Both conditions were fulfilled in the present experiments: on one hand, only 5-HT can be the substrate in the radioenzymic assay used in the present study (Boireau et at. 1976) and on the other hand, the release induced by intracellular stimulation (in the presence of
H. M. GERSCHENFELD AND OTHERS chlorimipramine) should only concern the endogenous amine of a neuronal compartment. The background release of endogenous 5-HT observed when the cerebrobuccal ganglionic ring of Aplysia was incubated 'at rest' probably resulted from the spontaneous firing of other 5-HT releasing neurones different from the silent 5-HT giant cerebral neurones. Cerebral and buccal ganglia are rich in 5-HT and recent autoradiographic studies have revealed the existence of other 5-HT containing cell bodies in the cerebral ganglia different from the giant neurones (A. Calas & R. Bessone, personal communication). The participation of autoactive neurones in the 'spontaneous' release of 5-HT is very much suggested by the suppression of the 'leak' in the presence of Co2+ ions in the medium, which interfere with the Ca2+ entry in the synaptic terminals, thus inhibiting selectively the release of any transmitter induced by synaptic ending depolarization (Weakly, 1973). However, if this effect of C02+ ions indicates that the spontaneous leak of 5-HT was related to neuronal firing, it still remains unexplained why 5-HT uptake inhibition by chlorimipramine did not affect significantly the amount of 5-HT spontaneously released. This is quite surprising since the uptake inhibitor enhanced the release of 5-HT resulting from the intracellular stimulation of the 5-HT giant cerebral neurones. As the spontaneous release, the evoked release of 5-HT was totally suppressed in the presence of C02+ ions. These observations confirm that very likely the 5-HT recovered from the bathing fluid in all cases originates from a synaptic compartment. The critical role of the 5-HT uptake mechanism in the C.N.s. of Aplysia is underlined by two facts, (a) the impossibility of showing in a normal medium a detectable release of 5-HT either by stimulating both 5-HT giant cerebral neurones or by increasing the outer [K]+ concentration, and (b) the appearance of a surplus amount of 5-HT in both conditions when a 5-HT uptake inhibitor was added to the medium. The electrophysiological experiments on the action of 5-HT uptake inhibitors provide a good demonstration that the 5-HT re-uptake is the physiological mechanism involved in the inactivation of S-HT as a transmitter in Aplysia 5-HT synapses. Indeed, both the amplitude and the duration of the postsynaptic potentials evoked by stimulation of the 5-HT neurone were increased in the presence of chlorimipramine or desmethylimipramine. The apparent failure of chlorimipramine to enhance the amplitude of the i.p.s.p. evoked by 5-HT giant cerebral neurone stimulation in some buccal cells simply resulted from a direct blocking effect of the drug on the postsynaptic 5-HT receptors involved. This observation underlines the fact that drugs which are generally accepted to have only one specific action can have multiple effects. The collectability of synaptic transmitters as the result of the stimulation of identified axons has been, since the early studies on chemical transmission, a fundamental requirement to identify these transmitters as such. This kind of evidence has only been obtained in a limited number of cases. Thus acetylcholine release was demonstrated at nerve muscle junctions in the heart (see Loewi, 1933) and skeletal muscle (see Dale, 1937) and there is also good evidence regarding both the release of noradrenaline at different sympathetic nerve endings (see Brown, 1965) and the release of y-amino-butyric acid at the crustacean neuromuscular junctions (Otsuka, Iversen, Hall & Kravitz, 1966). More recently, Evans, Kravitz & Talamo (1976) have demon276
RELEASE AND UPTAKE OF 5-HT BY 5-HT NEURONES 277 strated that octopamine can be released from crustacean neurones located in the roots of the thoracic ganglia. The present demonstration of the release of endogenous 5-HT by direct electrical stimulation from two identified neurones containing 5-HT reinforce the physiological and pharmacological evidence (Gerschenfeld & Paupardin-Tritsch, 1974) supporting the idea that 5-HT is the transmitter operating at the synapses established by these cells. The necessity of blocking the 5-HT uptake to reveal such release and the consequent prolongation of the synaptic potentials when 5-HT uptake was inhibited strongly suggest that a re-uptake mechanism is responsible for the inactivation of 5-HT at these serotoninergic synapses. We thank Dr Sylvie Bourgoin for assistance in the radioenzymic micro-assays of 5-HT and Dr Eve Marder for reviewing the manuscript. This work was supported by grants from the Centre National de la Recherche Scientifique, D6l6gation G6n6rale a la Recherche Scientifique et Technique and Institut National de la Sant6 et la Recherche M6dicale, France. REFERENCES L. & P., GLOWINsKI, J., AscHER, TAUC, TAXI, J. (1968). Discussion of stimulation-induced release of serotonin. Adv. Pharmacol. 6A, 365-368. BOIREAU, A., TERNAUX, J. P., BOURGOIN, S., HERY, F., GLOWINSKI, J. & HAMON, M. (1976). The determination of picogram levels of 5-HT in biological fluids. J. Neurochem. 26, 201-205. BROWN, G. L. (1965). The release and fate of the transmitter liberated by adrenergic nerves. Proc. R. Soc. B 162, 1-19. BROWNSTEIN, M. J., SAAVEDRA, J. M., AXELROD, J., ZERMAN, G. H. & CARPENTER, D. 0. (1974). Coexistence of several putative neurotransmitters in single identified neurons of Aplysia. Proc. natn. Acad. Sci. U.S.A. 71, 4662-4665. COTTRELL, G. A. (1970). Direct postsynaptic response to stimulation of a serotonin-containing neurone. Nature, Lond. 225, 1060-1062. COTTRELL, G. A. (1971). Synaptic connexions made by two serotonin-containing neurones in the snail (Helix pomatia) brain. Experientia 27, 813- 814. COTTRELL, G. A. & MACON, J. (1974). Synaptic connexions of two symmetrically placed giant serotonin-containing neurones. J. Physiol. 236, 435-464. COTTRELL, G. A. & OSBORNE, N. N. (1970). Subcellular localization of serotonin in an identified serotonin-containing neurone. Nature, Lond. 225, 470-472. DALE, H. H. (1937). Transmission of nervous effects by acetylcholine. Harvey Lect. 32, 229-245. ECCLES, J. C., KATZ, B. & KUFFLER, S. W. (1941). Nature of the end-plate potential in curarized muscle. J. Neurophysiol. 4, 362-387. EISENSTADT, M., GOLDMAN, J. E., KANDEL, E. R., KoIKE, H., KOESTER, J. & SCHWARTZ, J. H. (1973). Intrasomatic injection of radioactive precursors for studying transmitter synthesis in identified neurons of Aplysia californica. Proc. natn. Acad. Sci. U.S.A. 70, 3371-3375. EVANS, P. D., KRAVITZ, E. A. & TALAMO, B. R. (1976). Octopamine release at two points along lobster nerve trunks. J. Physiol. 262, 71-89. GERSCHENFELD, H. M., HAMON, M. & PAUPARDIN-TRITSCH, D. (1976). Release and uptake of 5-hydroyxtryptamine by a single 5-HT containing neurone. J. Phyaiol. 260, 29P. GERSCHENFELD, H. M. & PAUPARDIN-TRISTCH, D. (1974). On the transmitter function of 5hydroxytryptamine at excitatory and inhibitory monosynaptic junctions. J. Physiol. 243, 457-481. GOLDBERG, D. J., GOLDMAN, J. E. & SCHWARTZ, J. H. (1976). Alterations in amounts and rates of serotonin transported in an axon of the giant cerebral neurone of Aplysia californica. J. Physiol. 259, 473-490. GOLDMAN, J. E. & SCHWARTZ, J. H. (1974). Cellular specificity of serotonin storage and axonal transport in identified neurones of Aplysia californica. J. Physiol. 242, 61-76. IVERSEN, L. L. (1971). Role of transmitter uptake mechanisms in synaptic neurotransmission. Br. J. Pharmac. 41, 571-591.
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LoEwi, 0. (1933). Problems connected with the principle of humoral transmission of nervous impulses. Proc. R. Soc. B 118, 299-316. OSBORNE, N. N., HIRIPI, L. NEUHOFF, V. (1975). The in vitro uptake of biogenic amines by snail (Helix pomatia) nervous tissue. Biochem. Pharmac. 24, 2141-2148. OSBORNE, N. N. & NEUHOFF, V. (1974). In vitro experiments on the metabolism, accumulation and release of 5-HT in the nervous system of the snail Helix pomatia. J. Neurochem. 22, 363-371. OTSUKA, M., IVERSEN, L. L., HALL, Z. W. & KRAVITZ, E. A. (1966). Release of gamma-aminobutyric acid from inhibitory nerves from the lobster. Proc. natn. Acad. Sci. U.S.A. 56, 11101115. SNEDECOR, G. W. & COCHRAN, W. G. (1967). Stati8tical Methods. Ames: Iowa State College Press. TERNAUX, J. M., HERY, F., BOURGOIN, S., ADRIEN, J., GLOWINSKI, J. & HAMON, M. (1977). The topographical distribution of serotoninergic terminals in the neostriatum of the rat and the caudate nucleus of the cat. Brain Re8. 121, 311-326. WEINREICH, D., McCAmAN, M. W., MCCAMAN, R. E. & VAUGHN, J. (1973). Chemical enzymatic and ultrastructural characterization of 5-hydroxytryptamine-containing neurons from the ganglia of Aplyaia californica and Tritonia diomedia. J. Neurochem. 20, 969-976. WEAKLY, J. N. (1973). The action of cobalt ions on neuronmuscular transmission in the frog. J. Phyliol. 234, 577-612.
265
RELEASE OF ENDOGENOUS SEROTONIN FROM TWO IDENTIFIED SEROTONIN-CONTAINING NEURONES AND THE PHYSIOLOGICAL ROLE OF SEROTONIN RE-UPTAKE
BY H. M. GERSCHENFELD, M. HAMON* AND DANIELE PAUPARDIN-TRITSCH From the Laboratoire de Neurobiologie, Ecole Normale Superieure, 46, rue d'Ulm 75005, Paris, and the *Groupe NB, I.N.S.E.R.M. U 114, College de France, 11, place Marcelin Berthelot, 75005 Paris, France (Received 30 May 1977) SUMMARY
1. The amounts of endogenous serotonin (5-HT) released into the medium by the cerebro-buccal ganglionic ring of Aplysia californica incubated in artificial sea water (ASW) were measured. The rate of spontaneous 5-HT release varied between 0*4 and 1-2 p-mole per hour, which is less than 1 % of the total 5-HT present in this preparation. 2. Direct stimulation of the ordinarily silent 5-HT-containing giant cerebral neurones resulted in a 80-100 % increase of the 5-HT released, but only when the 5-HT uptake was blocked by chlorimipramine (1-10 /uM). 3. High K+ media (50 mM) also caused a significant increase in the amount of 5-HT released from the preparation provided that chlorimipramine (1-10 ,M) was present in the incubation fluid. 4. Co2+ ions (10-30 mm) added to the incubating medium blocked the spontaneous leak of endogenous 5-HT as well as the release, in the presence of chlorimipramine, evoked either by stimulation of the 5-HT-giant cerebral neurones or high K+-media. 5. In the presence of chlorimipramine or desmethylimipramine, the duration and/or the amplitude of the excitatory or the inhibitory synaptic potentials evoked in the buccal neurones by the stimulation ofthe 5-HT giant cerebral neurones were markedly enhanced. 6. These results strongly support the idea that 5-HT is the synaptic transmitter released at the excitatory and inhibitory junctions established by the 5-HT giant cerebral neurones in the ipsilateral buccal ganglia. In addition, they underline the role of amine re-uptake in the physiological inactivation of 5-HT as a transmitter. INTRODUCTION
The discovery by Cottrell & Osborne (1970) of two giant neurones symmetrically located in the cerebral ganglia of the snail Helix aspersa which showed an intense yellow fluorescence with the Falck-Hillarp technique, initiated an intensive research on the biochemical and physiological properties of single identified neurones containing serotonin (5-hydroxytryptamine = 5-HT). Cottrell and his co-workers
H. M. GERSCHENFELD AND OTHERS (Cottrell, 1970, 1971; Cottrell & Osborne, 1970; Cottrell & Macon, 1974) demonstrated that these cells from land and fresh-water snails contained detectable quantities of 5-HT and that they made excitatory connexions with three other neurones located in the ipsilateral buccal ganglion, 5-HT probably being the transmitter intervening at these connexions. In parallel studies in Aplysia californica, Weinreich, McCaman, McCaman & Vaughn (1973) analysed a pair of giant cerebral neurones homologous to those of the snails. These cells, also symmetrically located, contain 4-6 p-mole of 5-HT in their somata (Weinreich et al. 1973; Brownstein, Saavedra, Axelrod, Zerman & Carpenter, 1974) and were shown to synthetize 5-HT from both tryptophan (Eisenstadt, Goldman, Kandel, Koike, Koester & Schwartz, 1973) and 5-hydroxytryptophan (Weinreich et al. 1973; Eisenstadt et al. 1973). 5-HT synthesized from radioactive 5-hydroxytryptophan injected inside the soma of the giant cerebral neurone appears associated with subcellular particles and is transported along the axon by a specific transport system (Goldman & Schwartz, 1974; Goldberg, Goldman & Schwartz, 1976). Each one of the giant cerebral 5-HT containing neurones of Aplysia sends an axon branch through the cerebro-buccal connective to the ipsilateral buccal ganglion, where it establishes direct synaptic connexions with at least thirteen neurones: excitatory junctions with nine neurones and inhibitory junctions with the other four (Gerschenfeld & Paupardin-Tritsch, 1974). Evidence obtained by comparing the pharmacological properties of 5-HT receptors in the post-synaptic buccal neurones and the synaptic potentials evoked by intracellular stimulation of the 5-HT giant cerebral neurones, strongly support the idea that 5-HT is used as a transmitter at these synapses (Gerschenfeld & Paupardin-Tritsch, 1974). The experiments reported in the present paper were designed to determine whether the firing of the 5-HT giant cerebral neurones actually caused a measurable release of 5-HT from their endings. Such a release was demonstrated when the uptake of 5-HT was pharmacologically suppressed in the cerebro-buccal ganglionic preparation. Parallel electrophysiological experiments, furthermore showed that inhibiting the uptake of 5-HT caused an increase of both the amplitude and the duration of the synaptic potentials evoked by the firing of the 5-HT giant cerebral neurones. These experimental results confirm that 5-HT is the transmitter released by the 5-HT giant neurones at their synapses with the buccal neurones and that the action of 5-HT is terminated at these junctions by a re-uptake mechanism. Some of these results were communicated in a preliminary form to the Physiological Society (Gerschenfeld, Hamon & Paupardin-Tritsch, 1976). 266
METHODS The preparation used in the present study is drawn in Fig. 1 and consisted of the isolated cerebro-buccal ganglionic ring of A. californica. The specimens used were obtained from Dr R. Fay, Pacific Marine Co. (Venice, California) and kept in an aerated artificial sea-water aquarium. The cerebral and buccal ganglia of the animals were removed together, keeping intact the cerebrobuccal connectives. This ganglionic ring (Fig. 1) was fixed to the bottom of a suitable plastic chamber and bathed in 1 ml. artificial sea water (ASW) composed of (mM) Na+ 480, K+ 10, Cl- 610, Ca2+ 10, Mg2+ 50 and Tris-HCl (pH 7 8) 10. The connective tissue sheaths covering the dorsal face of the cerebral ganglia and the caudal face of the buccal ganglia were removed. Each one of the 5-HT giant cerebral neurones (400-600 ,um diameter) was impaled with a double-
RELEASE AND UPTAKE OF 5-HT BY 5-HT NEURONES
267
barrelled glass micropipette filled with 0-6 m-K2S04 or 3 M-KCl showing tip resistances of 15-25 MC. One of the barrels of the double pipette was used for recording via a d.c. amplifier connected to a CRO and a 280 Brush pen recorder. The other barrel was used to stimulate the giant cerebral neurones by passing constant current pulses across their membrane. When these cells were impaled they showed resting potentials of -60 to -65 mV, and at variance with what is generally observed in Aply8ia neurones, they were silent and only fired when stimulated intracellularly. During the
Fig. 1. Diagram of the cerebro-buccal ganglionic ring preparation showing the location of the 5-HT containing giant cerebral neurones and the trajectory of their axons through the cerebro-buccal connectives to the buccal ganglia.
H. M. GERSCHENFELD AND OTHERS 268 stimulation periods, the membrane potential of the 5-HT giant neurones was controlled in order to keep them firing at a rate of 1-5 Hz. In many experiments one or more buccal neurones were also impaled to check the efficacy of the synaptic transmission. After each 1 hr period of either rest or intracellular stimulation of the 5-HT giant neurones, the fluid bathing the ganglia was totally removed and immediately frozen to -30 'C. This change of bathing fluid implied each time a controlled removal of the micropipettes from the cells and their re-insertion. The experiment generally lasted for 3 hr and the action potentials of both 5-HT giant cerebral neurones were continuously controlled.
150
Melatonin
100 E C.)
50
BN 0
5
10
15
Distance (cm) Origin Front Fig. 2. Thin-layer chromatography of [H3]melatonin formed from 5-HT leaked spontaneously from a cerebro-buccal ganglionic ring incubated for one hour in ASW. After migration (90 min with toluene, ethylacetate, acetic acid and water, 80:40:20:4 by volume of the solvent), the melatonin spot was revealed by a spray of von Urk reagent. The silica gel was then carefully scraped off in 1 cm sections and mixed with water (1 ml.) and scintillating fluid (PCS 10 ml. Amersham Searle) for radioactivity counting. BN corresponds to the background noise of the Packard counter.
Radioenzymic microassay of 5-HT. This technique was performed at 4-6 0C, before the fourth week after the experiments. In preliminary studies it was shown that the 5-HT content of ASW samples kept at -30 0C did not change significantly after 1 month in the freezer. The 5-HT assay procedure used was that of Boireau, Ternaux, Bourgoin, Hgry, Glowinski & Hamon (1976), slightly modified. The ASW samples were thawed and diluted by half with a mixture of 0-5 mmKH2PO4 and 25-5 mM-NaCO3 in order to bring the pH and the ionic strength close to those of the cerebrospinal fluid samples used originally by Boireau et al. (1976). Each sample was passed through a Sephadex GIO column (3 cm high, 0-4 cm diameter) previously washed with 4 ml. 0-5 N-formic acid and 10 ml. water. After passing the sample, 6 ml. water were passed through the column and 5-HT was eluted in 3 ml. 0-5 N-formic acid. The eluate was lyophilized and the residue was solubilized in 60 /0. 0-2 M-Na2SO4 buffer pH 7-9. Thus 5-HT was converted into [3H]melatonin in two enzymatic steps with S-adenosyl-L-[methyl-sH]methionine (7-5-10 c/ m-mole, Amersham Radiochemical Centre) as the methyl donor. [3H]melatonin was then extracted into toluene and finally isolated by thin layer chromatography on silica gel (Boireau
RELEASE AND UPTAKE OF 5-HT BY 5-HT NEURONES
269
et al. 1976, see Fig. 2). The sensitivity of the whole procedure was routinely about 0.10-0.15 p-mole of 5-HT (amount of 5-HT leading to the formation of cpm of [3H]melatonin twice the blank value). Control experiments with ASW samples containing up to 30 mM-COCl2 or 10 FM chlorimipramine have shown that these compounds did not interfere with the assay of 5-HT in the conditions described above. The endogenous levels of 5-HT in the cerebro-buccal ganglionic ring were determined according to Ternaux, H6ry, Bourgoin, Adrien, Glowinski & Hamon (1977). Standard statistical calculations were made as proposed by Snedecor & Cochran (1967). When P values were higher than 0 05, differences were considered not to be significant. Electrophygiological experiments. The same cerebro-buccal ganglionic preparation was placed in a plastic chamber of 2*5 ml. volume, and was continuously bathed with circulating ASW. The connective sheaths of the ganglia were removed as described above. Two double-barrelled micropipettes were used, one to impale one of the 5-HT giant cerebral neurones, and the other, one of the ipsilateral post-synaptic buccal neurones. Chlorimipramine and desmethylimipramine (Ciba-Geigy, Basel) were directly added to the circulating ASW. All the experiments were performed at room temperature (20-23 TC).
RESULTS
'Spontaneous' release of 5-HT from the cerebro-buccal ganglionic ring preparation. When the cerebro-buccal ganglionic ring was incubated for one hour 'at rest' (i.e. without any stimulation of the 5-HT giant cerebral neurones) in normal ASW, there was a measurable quantity of 5-HT (well above the sensitivity of the assay method, see Fig. 2) in the bathing fluidwhich had leaked out from the preparation. The amount of this 'spontaneously' released 5-HT varied between 0-4 and 1-2 p-mole/rn. hr. This quantity could vary from experiment to experiment, and possibly, according to the season. Since the cerebro-buccal ganglionic ring contains 169 + 16-5 p-mole of 5-HT (mean + S.E. from seven duplicate determinations), only less than i % of this ganglionic pool actually 'leaked' to the bath each hour. The factors affecting this 'leak' were neither analysed nor specially controlled. In a first series of experiments, cerebro-buccal preparations were incubated for a period of 2-3 hr and the 5-HT content of the bathing fluid was monitored after each hour of incubation. It could be seen that, in this series, the average spontaneous leak of 5-HT in the first hour (in nine preparations) was 0-91 + 0-13 p-mole/ml. hr. During the second hour, the measured 5-HT leak in six preparations was 0-99 + 0-21 p-mole/ ml. hr. whereas the samples obtained after the third hour of incubation (in eight preparations) yielded an average of 0-65 + 0-09 p-mole of 5-HT/hr, less than in the first hour of incubation (but not significant). Significant changes were neither detected in the 5-HT content of the ganglionic ring preparation after the second and the third hour of incubation when compared to those found after the first hour. 5-HT giant cerebral neurones-stimUlation experiments. Surprisingly, direct electrical stimulation of both 5-HT giant cerebral neurones through the intracellular pipettes (which resulted in synaptic activation of the follower buccal neurones) failed to induce any significant increase in the amount of 5-HT 'leaked' in the bathing fluid (Fig. 3). This lack of effect of the neuronal stimulation was found in all the cases whatever the amount of 5-HT being released during the previous rest period (first hour of incubation). This apparent lack of effect of the 5-HT giant cerebral neurones stimulation could result from at least two reasons: (a) a failure of the neuronal endings to release
270 H. M. GERSCHENFELD AND OTHERS detectable amounts of 5-HT, (b) the rapid uptake of the released amine. In order to test the second of these hypotheses, the effects of electrical stimulation of the 5-HT giant cerebral neurones were analysed after blocking the 5-HT re-uptake using chlorimipramine (Osborne, Hiripi & Neuhoff, 1975). At rest, the addition of chlorimipramine (10SM) to the bathing fluid did not alter the 'spontaneous leakage' of 5-HT from the ganglionic ring preparation (Fig. 3). In contrast to the results obtained
(16)*
200
Unstimulated
C 1
75150 0
*poo
~~~~2)(12) (23)(38)
ID
cell Stimulated cell
-
_
_
+
100
50
Fig. 3. Effect of intracellular stimulation of the 5-HT giant cerebral neurones, chlorimipramine and Co2+ ions on the release of 5-HT from the cerebro-buccal ganglionic ring preparation. The preparation was kept at rest for one hour in ASW and then incubated for two hours either in the same medium or in ASW containing C0 /im-chlorimipramine or 30 MMCo2+ ions or both (in each case the medium was renewed at the end of each hour). Intracellular stimulation of the 5-HT giant cerebral neurones was performed g of mean during the second or the third hour of incubation. Each bar is the mean si. of 5-HT (in % of the values found for the first hour of the experiment). Since no signifiu cant differences were observed between the amounts of 5-HT released in similar conditions during the second and the third hour of incubation, the corresponding values were pooled for drawing the present Figure. *P < 0-001 when compared to respective control values. Number of determinations in brackets.
in pure ASW, the electrical intracellular activation of the 5-HT giant cerebral neurones did increase the efflux of 5-HT to the bathing fluid containing chlorimipramine. As shown in Fig. 3, this effect was highly significant and occurred in all cases, whatever the absolute amount of 5-HT released in the bathing fluid during the first hour rest period preceding the electrical stimulation period. No significant difference was observed between the 5-HT content of the cerebro-buccal ganglionic ring incubated for three hours in pure ASW with that of the ganglia which served for the electrical
271 RELEASE AND UPTAKE OF 5-HT BY 5-lT NEURONES stimulation experiments in the presence of chlorimipramine. This was not so sur-
prising since the amount of 5-HT released per hour did not exceed 2 % of the endogenous pool of the indoleamine in the ganglia even when electrical stimulation, chlorimipramine application or both were performed. We also analysed if the 5-HT neurones stimulation-induced release required CaO+ by adding CoCl2 (10-30 mM) to the bathing fluid during both rest and stimulation periods. According to Weakly (1973), Co2+ ions interfere with the entry of Ca2+ ions inside the synaptic endings and therefore cause a reduction or suppression of the transmitter release at chemical synapses. As shown in Fig. 3 and in Table 1, C02+ TARim 1. Effect of Co ions on the release of 5-HT 5-HT release p-molelhr Condition Control 30mx-CO2+ 30 mM-Co2+ + stimulation 10 mM-Co2+ 10 mM-Co2+ + stimulation
0 91 ± 0-08 (n 0.03+0.01* (n 0-03 + 0.01* (n 005+ 0.02* (n 0 04 + 0.02* (n
= 14) = 10) = 5) = 4) = 4)
The ganglionic ring was first maintained for 1 hr in normal ASW and then, 10 or 30 mm-CoCl2 were added to the ASW. After another hour the same medium was renewed and the 5-HT giant cerebral neurones were stimulated for another hour period. Each value listed is the mean + s.E. of mean of the amounts of 5-HT found in the medium at the end of the first hour of incubation (control), the second hour (10 or 30 mM-Co2+) and the third hour of incubation (Co2+ + stimulation). *P < 0.001 when compared to control values. n = number of determinations (in brackets).
strongly reduced the stimulation-induced efflux of 5-HT in the presence or the absence of chlorimipramine. Moreover, the spontaneous leakage of 5-HT from the cerebro-buccal ganglionic ring was also largely prevented by the presence of Co2+ ions in the bathing fluid (Fig. 3 and Table 1). In most cases, the effect of Co2+ ions was so marked that the amount of 5-HT released in the bathing fluid was lower than the sensitivity of the enzymic micro-assay (see Methods). Therefore, values in Fig. 3 and Table 1 are only semi-quantitative estimations of the amounts of 5-HT released in the presence of C02+ ions. The inhibitory effect of C02+ ions on 5-HT leakage strongly suggests that this process is not simply due to a destruction in the nervous tissue and that both kinds of release, spontaneous and evoked by 5-HT neurones intracellular stimulation, have their origin from synaptic endings. Release of 5-HT by high K+ concentrations. The release of transmitters from vertebrate brain tissue slices, isolated nuclei or synaptosomes by depolarizing the nerve structures with [K]+ rich media has been repeatedly reported in recent years. The effect of high [K]+ media on the spontaneous release of 5-HT from the cerebrobuccal ganglionic ring was also analysed. The protocol of these experiments was the following: the ganglionic ring was first incubated during one or two hours in normal ASW containing 10 mM-K+ and in subsequent hour period the concentration of K+ was increased to 50 mm. In these conditions, no significant increase in the 5-HT release was found after the exposure to a high [K]+ medium (Fig. 4). Suspecting that
H. M. GERSCHENFELD AND OTHERS 272 the possible 'surplus' release was damped by the re-uptake of 5-HT, the same experiments were repeated in the presence of 10 /gM chlorimipramine in the bathing fluid. As was observed before, chlorimipramine did not significantly change the amount of 5-HT leaked during the first hour of incubation in normal ASW, but in the presence of the uptake inhibitor, the high [K]+ medium caused an increase of about 60 % in the amount of 5-HT released from the preparation (Fig. 4). Electrophysiological experiments. The limited duration of transmitter action on postsynaptic membranes (which generally lasts between some milliseconds and hundreds of milliseconds) undoubtedly requires a step involving the termination of the action of the transmitter. The best known case of such a mechanism is the 200
D10 mm-K+
s150 0
(21)
50 mm-K+ *P
s-i
4 sec b Control 2 mV |
to sec
2
C
,1
\
.1mM
Clil
10 sec
IL
Control Fig. 5. Effect of chlorimipramine on the e.p.s.p. and i.p.s.p. evoked by 5-HT giant cerebral neurone stimulation. A, the discharge of spike trains in the giant neurone (a) evokes in a buccal neurone a smooth slow depolarization (marked control in b). Perfusion of chlorimipramine 1 1UM (marked Cli in b) causes an increase in both the amplitude and the duration of the e.p.s.p. In c, the iontophoretic application of 5-HT evokes in the same cell a depolarizing response which is not altered by chlorimipramine. B, the spike train as in a evokes in a buccal neurone a smooth slow hyperpolarization which increases in duration in the presence of 10 pm-chlorimipramine. In c chlorimipramine is shown to block at 04 mM-concentration the hyperpolarizing response evoked in the same neurone as in b by the iontophoretic application of 5-HT. The superimposed square electronic potentials in A,c and B,c show that chlorimipramine does not affect the input resistance of the cells.
Fig. 5 gives an example of the experiments performed. At left (Fig. 5A), recordings from an excitatory monosynaptic junction between one of the 5-HT giant cerebral neurones and one of the postsynaptic buccal neurones are reproduced. The upper trace (Fig. 5A, a) corresponds to a typical burst of spikes evoked by direct stimulation of the 5-HT giant cerebral neurone each 1-2 miD along the whole experiment and
H. M. GERSCHENFELD AND OTHERS the middle traces correspond to two superimposed recordings of the postsynaptic response to the presynaptic burst (Fig. 5A, b), taken before and during the bath application of 1-10 uM chlorimipramine. When the preparation is bathed in a normal ASW (Fig. 5A, b, control) the presynaptic burst evokes a slow smooth depolarization in the postsynaptic neurone. When the same presynaptic spike train is discharged in the presynaptic neurone after bathing the preparation for half an hour in 1 ,/Mchlorimipramine, the post-synaptic potential showed a marked increase of both its amplitude and its duration (Fig. 5A, b, recording labelled Cli 1 /tM). By comparing the two records superimposed in Fig. 5A, b, it is evident that, at the time when in the control one the buccal neurone membrane potential has returned to its initial level, the post-synaptic depolarization is still present under chlorimipramine and the membrane potential of the buccal neurone takes a much longer time to return to its original level. This effect was not due to a post-synaptic action of the drug since chlorimipramine, even at 041 mm concentration did not alter the depolarization evoked by the iontophoretic application of 5-HT on to the same buccal neurone 274
(Fig. 5A, c).
As it was said above, the 5-HT giant cerebral neurones also establish inhibitory synaptic connexions with some buccal neurones and the effect of the uptake inhibitor was also analysed on these junctions. Fig. 5B shows presynaptic and post-synaptic recordings from such an inhibitory connexion. In the upper trace (Fig. 5B, a) is recorded one of the spike trains evoked in the presynaptic 5-HT giant cerebral neurone. The middle trace (Fig. 5B, b) corresponds to superimposed recordings of inhibitory synaptic potentials evoked by the presynaptic train recorded above. In this case, the effects of a long application of a 1IOM concentration of chlorimipramine to the preparation appear to be somewhat different from those observed in the case of the excitatory junctions. Although the amplitude of the hyperpolarizing synaptic response did not increase in the presence of chlorimipramine (even in some cases a small reduction was observed), a clearcut prolongation of the inhibitory potential occurred in the buccal cells exposed to the drug. The bottom recordings of Fig. 5 give an explanation to the apparent difference of action of chlorimipramine on the excitatory and inhibitory responses. When 5-HT was applied iontophoretically on to the membrane of the same neurone of Fig. 5B, b, it hyperpolarized the neurones (Fig. 5B, c). The presence of chlorimipramine in the bathing fluid caused a reduction in the amplitude of this response, even when the uptake inhibitor was applied at 1O 14M concentration (in Fig. 5B, c the concentration of the inhibitor has been increased to accentuate the blocking effect). Therefore the lack of effect of 10 Um chlorimipramine on the amplitude of the inhibitory synaptic potential (Fig. 5B, b) resulted from an antagonism between the presynaptic action of the drug (increase in the amount of 5-HT present in the synaptic cleft due to the 5-HT re-uptake blockade) and its post-synaptic action (blocking of 5-HT action on the post-synaptic membrane). However, the prolongation of the inhibitory response under chlorimipramine remained a clear indication of the 5-HT re-uptake inhibition. From these experiments, it can be concluded that in the synapses established by different endings of the 5-HT giant cerebral neurones, chlorimipramine caused a prolongation of the synaptic potential suggesting that the physiological inactivation of the transmitter was impaired. Other drugs which inhibit the 5-HT uptake were
275 RELEASE AND UPTAKE OF 5-HT BY 5-HT NEURONES observed to have a similar effect on the synaptic potentials evoked by the stimulation of the 5-HT giant cerebral neurone. Fig. 6 corresponds to an experiment in which desmethylimipramine (a potent 5-HT uptake inhibitor in molluscan C.N.S., see Osborne & Neuhoff, 1974) was tested. In this case, the discharge of a train of action potentials at low frequency in the presynaptic neurone (Fig. 6A, a), instead of evoking a smooth slow depolarization in the postsynaptic buccal cells, produced the appearance of discreet unitary synaptic potentials in a 1:1 ratio (Fig. 6A, b). The addition of 10 /M-desmethylimipramine to the bathing fluid also resulted, as in the case of chlorimipramine, in a marked increase in the amplitude and the duration of each unitary excitatory synaptic potential (Fig. 6B, b).
A
a
o
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B
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La 40 mV 14 sec
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ODMI 10Mm Fig. 6. A, each spike in a 5-HT giant cerebral neurone (a) evokes in a buccal neurone a dicreet monosynaptic e.p.s.p. (b). B, bath application of 10 /SM-desmethylimipramine does not affect much the neurone firing (a), but increases the amplitude and the duration of the e.p.s.p. (b). DISCUSSION
The present results provide for the first time direct evidence about a detectable release of 5-HT by stimulating single identified 5-HT containing neurones. Moreover they strongly suggest that amine re-uptake might be the major process in the inactivation of 5-HT as a transmitter in Aplysia nervous system. Osborne & Neuhoff (1974) had previously reported that circumoesophageal ganglia of the snail H. aspersa pre-incubated with [3H]trytophan or [3H]5-HT could release radioactive material (possibly [3H]5-HT) when the nerve commissures of the ganglionic circumoesophageal ring were stimulated. This release was sensitive to the Ca2+ content of the incubation medium. However, experiments with [3H]5-HT are in all cases difficult to analyse since the exogenous indoleamine accumulates to a large extent in non-nervous tissue (Ascher, Glowinski, Tauc & Taxi, 1968). Therefore such experiments should require not only the identification of released material but also that of the cellular compartment involved in the release process. Both conditions were fulfilled in the present experiments: on one hand, only 5-HT can be the substrate in the radioenzymic assay used in the present study (Boireau et at. 1976) and on the other hand, the release induced by intracellular stimulation (in the presence of
H. M. GERSCHENFELD AND OTHERS chlorimipramine) should only concern the endogenous amine of a neuronal compartment. The background release of endogenous 5-HT observed when the cerebrobuccal ganglionic ring of Aplysia was incubated 'at rest' probably resulted from the spontaneous firing of other 5-HT releasing neurones different from the silent 5-HT giant cerebral neurones. Cerebral and buccal ganglia are rich in 5-HT and recent autoradiographic studies have revealed the existence of other 5-HT containing cell bodies in the cerebral ganglia different from the giant neurones (A. Calas & R. Bessone, personal communication). The participation of autoactive neurones in the 'spontaneous' release of 5-HT is very much suggested by the suppression of the 'leak' in the presence of Co2+ ions in the medium, which interfere with the Ca2+ entry in the synaptic terminals, thus inhibiting selectively the release of any transmitter induced by synaptic ending depolarization (Weakly, 1973). However, if this effect of C02+ ions indicates that the spontaneous leak of 5-HT was related to neuronal firing, it still remains unexplained why 5-HT uptake inhibition by chlorimipramine did not affect significantly the amount of 5-HT spontaneously released. This is quite surprising since the uptake inhibitor enhanced the release of 5-HT resulting from the intracellular stimulation of the 5-HT giant cerebral neurones. As the spontaneous release, the evoked release of 5-HT was totally suppressed in the presence of C02+ ions. These observations confirm that very likely the 5-HT recovered from the bathing fluid in all cases originates from a synaptic compartment. The critical role of the 5-HT uptake mechanism in the C.N.s. of Aplysia is underlined by two facts, (a) the impossibility of showing in a normal medium a detectable release of 5-HT either by stimulating both 5-HT giant cerebral neurones or by increasing the outer [K]+ concentration, and (b) the appearance of a surplus amount of 5-HT in both conditions when a 5-HT uptake inhibitor was added to the medium. The electrophysiological experiments on the action of 5-HT uptake inhibitors provide a good demonstration that the 5-HT re-uptake is the physiological mechanism involved in the inactivation of S-HT as a transmitter in Aplysia 5-HT synapses. Indeed, both the amplitude and the duration of the postsynaptic potentials evoked by stimulation of the 5-HT neurone were increased in the presence of chlorimipramine or desmethylimipramine. The apparent failure of chlorimipramine to enhance the amplitude of the i.p.s.p. evoked by 5-HT giant cerebral neurone stimulation in some buccal cells simply resulted from a direct blocking effect of the drug on the postsynaptic 5-HT receptors involved. This observation underlines the fact that drugs which are generally accepted to have only one specific action can have multiple effects. The collectability of synaptic transmitters as the result of the stimulation of identified axons has been, since the early studies on chemical transmission, a fundamental requirement to identify these transmitters as such. This kind of evidence has only been obtained in a limited number of cases. Thus acetylcholine release was demonstrated at nerve muscle junctions in the heart (see Loewi, 1933) and skeletal muscle (see Dale, 1937) and there is also good evidence regarding both the release of noradrenaline at different sympathetic nerve endings (see Brown, 1965) and the release of y-amino-butyric acid at the crustacean neuromuscular junctions (Otsuka, Iversen, Hall & Kravitz, 1966). More recently, Evans, Kravitz & Talamo (1976) have demon276
RELEASE AND UPTAKE OF 5-HT BY 5-HT NEURONES 277 strated that octopamine can be released from crustacean neurones located in the roots of the thoracic ganglia. The present demonstration of the release of endogenous 5-HT by direct electrical stimulation from two identified neurones containing 5-HT reinforce the physiological and pharmacological evidence (Gerschenfeld & Paupardin-Tritsch, 1974) supporting the idea that 5-HT is the transmitter operating at the synapses established by these cells. The necessity of blocking the 5-HT uptake to reveal such release and the consequent prolongation of the synaptic potentials when 5-HT uptake was inhibited strongly suggest that a re-uptake mechanism is responsible for the inactivation of 5-HT at these serotoninergic synapses. We thank Dr Sylvie Bourgoin for assistance in the radioenzymic micro-assays of 5-HT and Dr Eve Marder for reviewing the manuscript. This work was supported by grants from the Centre National de la Recherche Scientifique, D6l6gation G6n6rale a la Recherche Scientifique et Technique and Institut National de la Sant6 et la Recherche M6dicale, France. REFERENCES L. & P., GLOWINsKI, J., AscHER, TAUC, TAXI, J. (1968). Discussion of stimulation-induced release of serotonin. Adv. Pharmacol. 6A, 365-368. BOIREAU, A., TERNAUX, J. P., BOURGOIN, S., HERY, F., GLOWINSKI, J. & HAMON, M. (1976). The determination of picogram levels of 5-HT in biological fluids. J. Neurochem. 26, 201-205. BROWN, G. L. (1965). The release and fate of the transmitter liberated by adrenergic nerves. Proc. R. Soc. B 162, 1-19. BROWNSTEIN, M. J., SAAVEDRA, J. M., AXELROD, J., ZERMAN, G. H. & CARPENTER, D. 0. (1974). Coexistence of several putative neurotransmitters in single identified neurons of Aplysia. Proc. natn. Acad. Sci. U.S.A. 71, 4662-4665. COTTRELL, G. A. (1970). Direct postsynaptic response to stimulation of a serotonin-containing neurone. Nature, Lond. 225, 1060-1062. COTTRELL, G. A. (1971). Synaptic connexions made by two serotonin-containing neurones in the snail (Helix pomatia) brain. Experientia 27, 813- 814. COTTRELL, G. A. & MACON, J. (1974). Synaptic connexions of two symmetrically placed giant serotonin-containing neurones. J. Physiol. 236, 435-464. COTTRELL, G. A. & OSBORNE, N. N. (1970). Subcellular localization of serotonin in an identified serotonin-containing neurone. Nature, Lond. 225, 470-472. DALE, H. H. (1937). Transmission of nervous effects by acetylcholine. Harvey Lect. 32, 229-245. ECCLES, J. C., KATZ, B. & KUFFLER, S. W. (1941). Nature of the end-plate potential in curarized muscle. J. Neurophysiol. 4, 362-387. EISENSTADT, M., GOLDMAN, J. E., KANDEL, E. R., KoIKE, H., KOESTER, J. & SCHWARTZ, J. H. (1973). Intrasomatic injection of radioactive precursors for studying transmitter synthesis in identified neurons of Aplysia californica. Proc. natn. Acad. Sci. U.S.A. 70, 3371-3375. EVANS, P. D., KRAVITZ, E. A. & TALAMO, B. R. (1976). Octopamine release at two points along lobster nerve trunks. J. Physiol. 262, 71-89. GERSCHENFELD, H. M., HAMON, M. & PAUPARDIN-TRITSCH, D. (1976). Release and uptake of 5-hydroyxtryptamine by a single 5-HT containing neurone. J. Phyaiol. 260, 29P. GERSCHENFELD, H. M. & PAUPARDIN-TRISTCH, D. (1974). On the transmitter function of 5hydroxytryptamine at excitatory and inhibitory monosynaptic junctions. J. Physiol. 243, 457-481. GOLDBERG, D. J., GOLDMAN, J. E. & SCHWARTZ, J. H. (1976). Alterations in amounts and rates of serotonin transported in an axon of the giant cerebral neurone of Aplysia californica. J. Physiol. 259, 473-490. GOLDMAN, J. E. & SCHWARTZ, J. H. (1974). Cellular specificity of serotonin storage and axonal transport in identified neurones of Aplysia californica. J. Physiol. 242, 61-76. IVERSEN, L. L. (1971). Role of transmitter uptake mechanisms in synaptic neurotransmission. Br. J. Pharmac. 41, 571-591.
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