Journal of Physiology (1992), 450, pp. 325-340 With 7 figures Printed in Great Britain

325

PULSATILE RELEASE OF ACETYLCHOLINE BY NERVE TERMINALS (SYNAPTOSOMES) ISOLATED FROM TORPEDO ELECTRIC ORGAN

BY ROMAIN GIROD, LORENZA EDER-COLLI, JANA MEDILANSKI, YVES DUNANT, NACIRA TABTI* AND MU-MING POO* From the Departement de Pharmacologie, Centre Medical Universitaire, 1211 Gene've 4, Switzerland and the *Department of Biological Sciences, Columbia University, New York, NY 10027, USA

(Received 19 March 1991) SUMMARY

1. Electrophysiological detection of acetylcholine (ACh) release by synaptosomes from the electric organ of Torpedo was searched for by laying the isolated nerve terminals on a culture of Xenopus embryonic muscle cells (myocytes), and by recording the ACh-induced inward currents in the myocytes. 2. Whole-cell recording in one of the myocytes revealed rapid inward currents that where generated soon after synaptosome application. These pulsatile events strongly resembled those occurring normally during the early phase of synaptogenesis after nerve-muscle contact in Xenopus cell cultures. They were called spontaneous synaptic currents (SSCs). 3. The SSCs produced by the synaptosomes had a rapid time course, with mean time-to-peak and half-decay times of 2-6 + 0'4 ms and 6-0 + 1-1 ms, respectively. Most events had a falling phase that could be fitted with a single exponential. The mean time constant of decay was 62 + I1 ims. More than half of the SSCs (approximately 60%) constituted a rather homogenous population in which the time-to-peak versus amplitude showed a positive relationship, the smallest events displaying a shorter time course. The rest of the SSCs had a more variable and slower time course. Such events are also observed in young and mature junctions in situ. 4. The amplitudes of SSCs had a wide distribution which was skewed towards the smallest values. The mean amplitude was 65-2 + 16-1 pA. 5. During the minutes following an application of synaptosomes, the frequency of the SSCs tended to decrease, but their mean amplitude remained constant. Such behaviour could be reproduced during several successive additions of synaptosomes while recording in the same myocyte. 6. Just after synaptosome application, the SSCs were superposed to a noisy inward current that lasted for 20-60 s. Noise analysis of this current gave the values of 0 7 + 0-1 pA for the mean amplitude of the elementary event, and 4'7 + 0-2 ms for its mean duration, values that compare well with those reported for the activation of frog embryonic nicotinic receptor. This suggests that the noisy current was due to ACh molecules set free by synaptosomes which were either damaged or which MS 9242

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released ACh at some distance. This view was strengthened by biochemical analysis of ACh release by synaptosomes in vitro. 7. Tubocurarine reversibly abolished the appearance of both the noise and the synaptosome-generated SSCs, showing that these currents were due to the action of ACh. 8. The present results demonstrate (i) that synaptosomes in vitro are able to release ACh at a site that is very close to the myocyte receptor-rich membrane and (ii) that the molecular machinery ensuring pulsatile or quantal release of ACh at natural synapses remains functional after isolation of the nerve endings. INTRODUCTION

Isolated nerve endings, or synaptosomes, have been extensively used since their isolation from the rodent brain (Whittaker, 1959; De Robertis, Pellegrino de Iraldi, Rodriguez De Lores Arnaiz & Gomez, 1961) and from the Torpedo electric organ (Israel, Manaranche, Mastour-Frachon & Morel, 1976; Morel, Israel, Manaranche & Mastour-Frachon, 1977; Michaelson & Sokolovsky, 1978). Synaptosomes are able to respire in vitro and to maintain a membrane potential. They take up precursors, synthesize neurotransmitters and can release them in a Ca2+-dependent manner when depolarized in a high KCl medium, or under the action of veratridine, sodium or calcium ionophores, and of other agents (see reviews in Jones, 1975; Marchbanks, 1975). Synaptosomes from the Torpedo electric organ have been especially useful for biochemical investigations of cholinergic mechanisms since (i) they are homogeneous with respect to the neurotransmitter, (ii) they are accompanied by very little contamination from postsynaptic membranes, mitochondria and other substructures, and (iii) they can be kept in a functional state for more than 24 h after isolation. However, transmitter release in synaptosomes has only been measured by biochemical techniques. Therefore, it is not known whether or not the synaptosomes retain in vitro the most physiological property of intact nerve terminals, which is to release the neurotransmitter in a highly discontinuous, or pulsatile manner. This can be recorded electrophysiologically at intact synapses in the form of miniature synaptic potentials (or currents) which are generated in the postsynaptic cell by the simultaneous release of a few thousand transmitter molecules from presynaptic terminals, a phenomenon called quantal release. Quantal release has been extensively investigated at the neuromuscular junction (see Katz, 1969) where it starts occurring in embryogenesis from the first seconds following nerve-muscle contact (Chow & Poo, 1985; Evers, Laser, Sun, Xie & Poo, 1989). Synaptic transmission is also quantal in the Torpedo electric organ which is a modified neuromuscular system (Dunant & Muller, 1986; Muller & Dunant, 1987). The objective of the present work was to see whether synaptosomes isolated from the electric organ were able to release acetylcholine (ACh) in a quantal manner when they were brought into contact with an embryonic muscle cell which was used as a detector for ACh.

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METHODS

Synapto8omes Preparation of synaptosomes was carried out as described by Morel et al. (1977) with a slight modification (CaCl2 was not present in the successive solutions used in the procedure). Electric .organs were excised from Torpedo marmorata that were anaesthetized with tricaine methanesulphate (0 33 g/l sea water). Thirty grams of electric organ were finely chopped with a razor blade and then gradually comminuted by forced filtration through calibrated metallic grids with meshes 1000, 500 and 200 %Mm2. After filtration through a nylon gauze 50 ,um2 mesh, the preparation was first pelleted by centrifugation at 6000g for 20min, then the pellet was resuspended and centrifuged on a discontinuous sucrose gradient at 64000 g for 40 min. The synaptosomes were recovered in a fraction containing 280 mM-NaCl, 3 mm-KCl, 1P8 mM-MgCl2, 5-5 mm-glucose and 400 mM-sucrose. The high NaCl concentration in the fraction corresponds to the physiological values found in elasmobranch plasma whose osmolarity is much higher than that of teleost fish and higher vertebrates. The synaptosomes were kept at 4 °C for 3-24 h until the time of the experiment when they were rewarmed to room temperature. In some experiments (see Results), elasmobranch physiological medium of the following composition was used (mM): 280 NaCl, 7 KCl, 3-4 CaCl2, 1-8 MgCl2, 20 HEPES, 5 NaHCO3, 300 urea and 5-5 glucose, pH 7-2. The medium was gassed with 95% 02 and 5% CO2.

Biochemical assay of ACh To assay biochemically the amount of ACh released by synaptosomes, a 50,1 sample of the synaptosome suspension was delivered into a cuvette with the appropriate enzymes and chemicals for measuring ACh release in a continuous manner by a luminescence method (see Israel & Lesbats, 1981). Calcium (2-6 mm final concentration) was added; the release of ACh was thereafter triggered either by KCI depolarization or by the addition of a Ca2+ ionophore. We also analysed the effect of hypo-osmolarity on synaptosomes by delivering these into a solution mimicking the medium used for Xenopu.8 cell culture electrophysiology (115 mM-NaCl, 2-5 mM-KCl, with Tris buffer pH 8-6 and the chemicals needed for ACh bioluminescence detection; see Israel & Lesbats, 1981) and by measuring the amount of ACh released in the absence of any other stimulus. When the ACh content of the synaptosomes was measured, these were broken open by Triton X100 (0 03 % final concentration), and the amount of ACh recovered in the medium was assayed using the same luminescence method (Israel & Lesbats, 1981).

Xenopus cell culture Xenopus nerve-muscle culture was prepared as previously reported (Spitzer & Lamborghini, 1976; Anderson, Cohen & Zorychta, 1977). Briefly, the neural tube and the associated myotomal tissue of 1-day-old Xenopu. embryos (stage 20-22, Nieuwkoop & Faber, 1967) were dissociated in Ca2+- and Mg2+-free saline supplemented with EDTA. The cells were plated on clean glass coverslips and were used for experiments after 24 h at room temperature (20-22 °C). The culture medium consisted of half-strength Ringer solution (115 mM-NaCl, 2 mM-CaCl2, 2-5 mM-KCl and 10 mMHEPES, pH 7 3). Cultures maintained at lower temperatures developed more slowly and could be used several days after plating. In older cultures, myoblasts started differentiating from spherical mononucleated cells (myoballs) to spindle shaped cells, and most of the nerve cells did not survive (which was an advantage for the present experiments).

Current recording Synaptic currents were recorded from a muscle cell - either a myoball or a spindle myocyte - by the whole-cell patch-clamp method (Hamill, Marty, Neher, Sakmann & Sigworth, 1981). Recordings were made at room temperature in culture medium or the Ringer solution, and the solution inside the recording pipette contained (mM): 82 KCI, 35 KOH, 1 CaCl2, 1 MgCl2, 11 EGTA, and 5 HEPES, pH 7-2. In a typical recording, the muscle cell was voltage-clamped at the resting membrane potential and the membrane currents were monitored by a patch-clamp amplifier (List EPC-7, Medical Systems, Greenvale, NY, USA). The data were stored on a video recorder for later play-back onto a storage oscilloscope (Textronic 5113) or on oscillographic recorder (Gould Brush 2400). They were digitized at 6-3 kHz on a microcomputer (Intel 80286, IBM AT) using an acquisition software developed by the SICMU (Service Informatique du CMU, Geneva). The

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following parameters were computed with a program written on Sun-Matlab software (The MathWorks, Inc. South Natick, MA, USA): the amplitude; the time-to-peak, measured between the origin of the synaptic events and the peak maximum; the half-decay time, measured as the time elapsed between the maximum and the half-amplitude; and the decay constant of the falling phase, determined as the inverse of the slope of a regression line fitted to a semilogarithmic transform of the decay phase between 90 and 10% of the peak amplitude.

Noise analysis of the initial steady current It was suspected that the initial inward current that followed synaptosome application (see Results) was due to activation of the myocyte receptors by ACh present in the surrounding medium. Noise analysis was therefore performed on segments of this current in an attempt to determine the amplitude and the mean duration of the elementary current responsible for the noise (see Katz & Miledi, 1972; Anderson & Stevens, 1973). Segments of records were digitized at 2 kHz. The variance of the noise was determined on traces of 200 to 500 points (100-250 ms) taken at selected times. The amplitude (i) of the unitary current was then calculated according to the Cambell's theorem as follows: i = 2Z/I, where E is the variance of the noise and I the mean value of the inward current on the segment of trace considered. The time characteristic of the unitary current was calculated by analysing power density spectra. These were obtained by applying a discrete Fourier transform to segments of traces of 128 or 256 points sampled at 2 kHz. The power density spectra of three to eleven segments were then averaged and the frequency (f) corresponding to the half-power point determined. The time characteristic of the unitary quantal events (T) was then calculated according to the following relationship (see Katz & Miledi, 1972): T = 1/2Tf. RESULTS

Pulsatile currents upon synapto.some contact In the present study we used two synaptosome batches, one prepared from a female adult Torpedo and the second from two neonate fish. We first tested the ACh content and the KCl-induced release of ACh for the two batches of synaptosomes. The fractions contained 17 6 and 12 2 nmol/ml ACh and on KCl-depolarization, they released 529 and 320 pmol/ml ACh, respectively. A gigaohm-seal, whole-cell recording was made on isolated Xenopus myocytes from a 1-day-old culture. A myocyte was voltage-clamped at its resting membrane potential and the membrane current was recorded continuously. When stable recording conditions were obtained in a myocyte, a puff of synaptosomes was delivered in the vicinity of the cell through a glass pipette using a pulse of air pressure. Alternatively, 20-30 ,tl of the synaptosome fraction were laid down on the culture into the 2 ml recording chamber by using a microsyringe. The fraction has been reported to contain 107-108 synaptosomes/ml (Morel, Israel & Manaranche, 1978). One can therefore estimate that 0-2-3 x 106 synaptosomes were delivered onto the culture in each puff. Figure IA illustrates the recording conditions. Figure 1B shows the currents recorded from the myocytes when five successive puffs of synaptosome suspension were delivered at its vicinity. The myocyte was absolutely silent at the beginning of experiments, i.e. no signals of the kind of the spontaneous synaptic currents (SSCs) described here were recorded. Soon after the electrical perturbation due to the application of synaptosomes, the recording trace shows a transient inward current accompanied by increased noise. Superimposed on -

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this, abrupt inward currents having all the characteristics of SSCs were seen either isolated or in bursts. They kept on occurring and could be better analysed when the initial noisy inward current subsided. In Fig. 1 C, a few SSCs taken from the same experiment are presented on an expanded time scale. C I

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Fig. 1. Spontaneous synaptic currents (SSCs) recorded from a Xenopus myocyte after addition of Torpedo electric organ synaptosomes into the bath. A, phase-contrast photomicrograph of isolated Xenopus myocytes in 1-day-old Xenopus nerve-muscle culture. Whole-cell recording was made from a spherical shaped myocyte (myoball). After a puff of synaptosome suspension was applied, pieces of phase-dark fragments were seen, some of which had attached to the myoball surface (arrowheads). The diameter of the myoball was about 35 Aum. B, five successive puffs of synaptosomes were delivered to the preparation (time of addition indicated by the arrows); the recording trace started a few seconds after the first application. SSCs appear as downward current pulses; calibration bars are 20 s and 10 pA. C, samples of recording taken respectively during the first and third puff of synaptosomes (marked by bars I and II) in the experiment illustrated in B. Calibration bars are 20 ms and 100 pA. This expanded time scale allows better visualization of the SSCs. Note the progressive decrease of the noise and of the SSCs frequency, especially apparent in II.

Figure 2A shows the amplitude distribution of 266 SSCs, which were generated by the five applications of Torpedo synaptosomes described in Fig. 1. The distribution is skewed, with most events having an amplitude around 50-100 pA. A few SSCs had much larger amplitudes, up to 600 pA. The mean amplitude was 73-4 + 55 pA. A

R. GIROD AND OTHERS

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Fig. 2. Examples and amplitude distribution of synaptosome SSCs. A, amplitude distribution of 266 SSCs from the experiment in Fig. 1. B, picture showing forty-five superimposed SSCs synchronized on their rising phases. They represent the main, homogenous SSCs population. C, traces illustrating a few SSCs with a slower time course, and inflexions on their rising phase. Calibrations bars are 2-5 ms and 100 pA.

similar distribution was obtained in two other experiments; the mean amplitude of three experiments was 65-2+16-1 pA. Figure 2B and 2C show examples of superimposed SSCs. They displayed a surprisingly rapid time course. A majority of

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events (60 + 3-1 %, n = 3 experiments) constituted a rather homogeneous population of fast SSCs (Fig. 2B). Their times-to-peak ranged from 0 9 to 3 ms and the time-topeak versus amplitude relationship displayed a positive slope. Also their half-time of decay was very rapid, ranging from 0 5 to 5 ms. The remaining SSCs (40-0 +31 %, B 70-

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n = 3 experiments) had a slower and more irregular time course, with time-to-peak and half-decay up to 11 and 16 ms respectively. Eight slow SSCs are shown in Fig. 2C, with four rapid SSCs for comparison. The occurrence of slow SSCs was random and their incidence was not different within the first minute after synaptosome application and at the later times. Histogram analysis of the rising and half-decay times of all SSCs recorded in the experiment of Fig. 1 is illustrated in Fig. 3A and 3B. The mean time-to-peak and half-decay time of the 266 SSCs were respectively 2-2 + 0-1 ms and 4-0 + 02 ms. In three experiments, the mean time-to-peak was 2-6 + 0 4 ms and the mean half-decay time was 6-0 + 1 1 ms. Most SSCs showed exponential falling phases, between 90 and 10 % of their peak amplitude, that could be satisfactorily fitted with a single exponential. A minimal correlation coefficient of 0.9 for the exponential fit was obtained with about 90 % of the SSCs. This population of signals was retained to determine the value of 6-2 + 1 1 ms (n = 3 experiments) for the decay constant of the SSCs. The frequency of SSCs observed in the myocyte following each application of synaptosome suspension consistently decreased with time. Figure 4 plots the mean number of events per minute after the onset of synaptosome application from fifteen trials on three different myocytes. The curve shows a nearly exponential drop of the frequency during the first 10 min. Some SSCs could still be recorded as long as 11 min after the puff. The effects of the successive applications of synaptosomes were in most aspects similar. In particular, the mean amplitude of the SSCs remained constant. A problem was raised from the fact that the osmolarity of the solution bathing the Xenopus myocytes was considerably lower than that of the Torpedo synaptosome fraction. Thus, we used two procedures. In the results presented above, the synaptosomes were delivered in the Ringer medium directly, so that they underwent

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332

a hypo-osmotic shock. We used another procedure in which the frog Ringer solution was slowly replaced in the recording chamber by an elasmobranch physiological medium. The myocytes could survive for 1-2 h in this medium provided that the rate of change was slow enough. The time course and amplitude of the SSCs obtained with 40 n 30 C-)

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application (min) Fig. 4. Decrease in SSC frequency following synaptosome application. The mean number of SSC events per minute was plotted against the time after the onset of application of synaptosome suspension. A total of fifteen trials of synaptosome applications (on three different myocytes) were recorded for varying duration. An exponential was fitted to the data. Its e-fold decay time was 3 min 15 s. The number of data points ranged from one to fifteen.

this procedure were similar to those illustrated in Figs 1 and 2. It should be noted, however, that it was difficult to maintain stable recording conditions with myocytes maintained in this medium and to obtain a great number of events.

Effects of tubocurarine That the observed pulsatile currents were due to spontaneous ACh release from the synaptosomes was tested in the experiment illustrated in Fig. 5. The myocyte culture was treated with tubocurarine (500 JLM, final concentration) and a suspension of synaptosomes was then delivered to the culture. No SSC was observed. The curare was then washed out with normal Ringer solution and a second application of synaptosomes was delivered to the culture. This gave rise, as expected from the reversibility of curare, to the reappearance of SSCs. We noticed that the current noise observed immediately after the synaptosome application was also decreased in the presence of curare, suggesting that this noise was, at least partly, caused by some free ACh present in the solution surrounding the synaptosomes.

Noise analysis of the initial steady inward current and biochemical detection of ACh release This hypothesis was further investigated as follows: first, we performed a variance analysis of the noise affecting the initial inward current that took place within the first minute after synaptosome application. The mean steady current and the

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333

Tubocurarine (500 MM)

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Fig. 5. Effect of curare on the synaptosome-induced currents. The top trace shows the current recorded in a Xenopus myocyte treated with 500 puM-tubocurarine. Addition of Torpedo synaptosomes (arrow) did not induce any SSCs. The bottom trace shows that after washing out the curare, an application of synaptosomes caused the typical initial inward current and the appearance of SSCs in the myocyte. The asterisks indicate artifactual changes of the current in the myocyte. Calibration bars are 20 s and 100 pA. Three superimposed SSCs occurring in the decurarized myocyte are shown in the inset. They are presented on expanded time and amplitude scales, the calibration bars being 2-5 ms and 20 pA. A 0 pA

-

-100 20 ms

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Fig. 6. Segment of the traces used in the noise analysis. Each segment is composed of 256 points digitized at 2 kHz. A, background noise recorded in the myocyte before addition of synaptosomes. This is the control level whose mean value was taken as 0 pA. B, noisy current recorded about 25 s after an addition of synaptosomes, that is near the peak of the inward current in this particular trial. The mean level of inward current was shifted from 0 to -252 pA, and fluctuations were significantly enhanced.

variance of the noise were recorded for several traces as illustrated in Fig. 6. The results indicate that the noise was apparently due to elementary events whose mean amplitude was 0-7 + 01 pA (ninety-three traces) and whose mean duration was

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1000 2500 1500 2000 Amplitude (pA) Fig. 7. Spontaneous synaptic currents (SSCs) recorded from a nerve-contacted myocyte during the early phase of synaptogenesis in 1-day-old Xenopus culture. The myocyte was whole-cell clamped at -80 mV. A, synaptic currents (downward deflections, negative) displayed at two different speeds with the aid of a chart recorder. Calibration bars are 10 s and 1 s for the top and bottom traces respectively, and 500 pA for both. B, superimposed tracings of representative digitized SSCs displayed at higher time resolution. Calibration bars are 5 ms and 200 pA. C, histograms showing the distribution amplitude of 249 SSCs obtained from the same cell as in A and B.

0

335 PULSATILE ACh RELEASE BY SYNAPTOSOMES 4 7 + 02 ms (thirteen periods, each composed of three to eleven individual traces whose power spectra were averaged). In another approach to this question, we used a luminescence technique (Israel & Lesbats, 1981) to directly assay the amount of ACh released by synaptosomes submitted to conditions similar to those encountered during their application onto Xenopus cell cultures. By first using solutions isosmotic to the Torpedo internal medium, we found that some release of ACh was induced when calcium was added to the calcium-free solution in which the synaptosomes had been recovered; a subsequent addition of KCl or of a Ca2+ ionophore triggered a much larger ACh release, as expected. When synaptosomes were delivered in a medium similar to that used for the Xenopus cell culture, they underwent a hypo-osmotic shock in addition to the calcium challenge; this gave rise to a very large release of ACh, that lasted for 5-10 min (results not illustrated).

Comparison with spontaneous synaptic currents at Xenopus neuromuscular junction The characteristics of synaptosome-generated SSCs very much resembled those of the SSCs observed during the early phase of synapse formation between Xenopus spinal neurons and myocytes (Evers et al. 1989). Figure 7A and B show representative traces of SSCs observed in 1-day-old Xenopus cultures within the first 20 min following the nerve-muscle contact. In this experiment, a spherical myocyte was manipulated into contact with the growth cone of a co-cultured neuron. A giga-ohmseal whole-cell clamp recording was made from the myocyte to monitor SSCs (holding potential -80 mV). The SSCs obtained from such newly formed synapses looked very similar to those obtained from isolated myocytes contacted by Torpedo synaptosomes. Their mean amplitude was, however, slightly bigger (519-8 + 24-3 pA; see Fig. 7C) and their time course somewhat slower (mean time-to-peak 3-6 + 0-1 ms and mean half-decay time 6-4 + 01 ms) than synaptosomes SSCs. Another similitude was the presence in the newly formed neuromuscular junction of a main rapid SSC population with a positive time-to-peak versus amplitude relation (see Evers et al. 1989) in addition to a number of slower SSCs. DISCUSSION

Rapid kinetics of the SSCs generated by the synaptosomes By delivering synaptosomes onto a voltage-clamped myocyte, we were expecting to record some kind of ACh receptor activation, for instance, a noisy inward current, or feeble signals of slow time course due to quantal release occuring at some distance from the myoball plasmalemma. We were therefore extremely surprised to obtain signals of very rapid time course, reassembling those occurring at the newly formed nerve-muscle synapse. The mean time-to-peak of the synaptosome-induced SSCs was 2-6 ms, which is even shorter than that observed during synaptogenesis (3-6 ms), and only slightly longer than the time-to-peak of miniature endplate currents recorded at the mature nerve-electroplaque junction of Torpedo (0-5-1 ms, R. Girod & Y. Dunant, unpublished observations). Moreover, the amplitude versus time-to-peak relationship of synaptosomes SSCs displayed a positive slope; this was also observed in the newly

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formed junction (Evers et al. 1989). Now, morphological investigation of synapses in situ revealed that the presynaptic plasmalemma is in a very close apposition to the postsynaptic membrane: less than 10 nm in the case of the early functional contact between nerve and muscle in Xenopus cell culture (Buchanan, Sun & Poo, 1989), and ca 50 nm at the mature neuromuscular or nerve-electroplaque junction. Such narrow intersynaptic space ensures short time of diffusion - determined by the amount of transmitter released - and high local transmitter concentration: it is therefore thought to be a fundamental characteristic that allows fast synapses to generate rapid signals (see Land, Salpeter & Salpeter, 1980, 1981; Land, Harris, Salpeter and Salpeter, 1984). Thus, the time course of the SSCs observed in the present work suggests that Torpedo synaptosomes achieved very close apposition, and perhaps attached themselves, to the myoball plasma membrane, so that emission of ACh took place near the receptor field. In support of this conclusion is the observation that only a small detachment of the pre- from the postsynaptic membrane significantly enlarges the rise time of the miniature endplate potential (MEPP) and increases its variability (Grinnell, Gunderson, Meriney & Young, 1989). Also, when ACh is ionophoretically applied on the neuromuscular endplate with a micropipette, a rapid time course of the synaptic response is obtained only if the ACh source is accurately positioned onto the postsynaptic membrane; if the pipette is withdrawn for only 50-60 /tm above the endplate, the time-to-peak of the response reaches values as large as 500 ms (Hartzell, Kuffler & Yoshikami, 1975). The mean half-decay time of synaptosomes SSCs was 6-0 ms, a value that compares well with that of the early contact in Xenopus (6-4 ms). Moreover, the falling phase of the SSCs was found to be exponential, with a mean decay constant of 6-2 ms. This is close to the value obtained in the noise analysis for the mean open time of the receptors (4 7 ms). Such good correlation between the decay kinetics of the SSCs and the mean open time of the channels points to two assumptions: first, it suggests that the rise and fall in concentration of transmitter near the receptors is rapid relative to the channel open time (see Anderson & Stevens, 1973); second, it indicates that most released ACh molecules do not interact repeatedly with the receptors, perhaps because of hydrolysis by the acetylcholinesterase present at the external surface of synaptosomes. Again, these observations support the view that the synaptosomes producing the rapid SSCs had established with the Xenopus myoball a contact that shared at least some topographical properties with the in situ embryonic synaptic contact.

The initial noisy current The initial inward current that was recorded for 1 min or so after synaptosome delivery was expected to result from receptor activation by free ACh molecules present in the surrounding medium. This view was strengthened by the observation that the noise was reduced in the presence of curare and by the results of our variance analysis. The noise was found to be composed of elementary currents with a mean amplitude around 1 pA and a mean duration of 4-7 ms, values that are in the range of those reported for the activation of ACh receptors in the embryonic Xenopus muscle cells (Brehm, Steinbach & Kidokoro, 1982; Kidokoro & Rohrbough, 1990). The control experiments in which biochemical assay of ACh was performed helped to identify the origin of this free ACh. The calcium challenge experienced by the

PULSATILE ACh RELEASE BY SYNAPTOSOMES

337 synaptosomes at the time of addition onto the Xenopus cell culture was found to induce some release of transmitter. This may be explained if some synaptosomes had a pooIr membrane potential and were therefore subject to Ca2± activation of release. When synaptosomes were submitted to a hypo-osmotic shock, a large release of ACh was noted. A great part of this release might be due to ACh leakage or to some breakdown of synaptosomes. On the other hand, the electrophysiological results presented here suggested that at least part of the release was in the form of pulsatile chemical impulses that are reminiscent of the physiological working of the synapse. The final issue was the presence in the medium of free ACh that was probably the cause of the inward noisy current. It must be noted that the synaptosomes were delivered at the vicinity of the myocytes in a medium denser than that of the culture. Since the bath was not stirred, one can expect that it took some time for the two media to mix, so that osmotic shock might have been actually delayed. Eventual exhaustion by the hypo-osmotic shock of the synaptosomes producing the SSCs might explain why the pulsatile responses disappeared about 10 min after synaptosome delivery. Our attempts to record SSCs from myocytes that were accustomed to a medium corresponding to the elasmobranch internal medium were successful in that we obtained SSCs with a time course similar to those seen in the other conditions. However, it has been difficult to keep the myoball alive long enough in this solution and a large number of events could not therefore be obtained. Size of the pulsatile ACh release generated by the synaptosomes The mean SSCs amplitude was 65-2 pA. Thus, about seventy receptors were open at the peak of the signal; this means that 140 ACh molecules were needed to bind these receptors. In the neuromuscular junction in situ, the amount of transmitter that undergoes double binding at the peak is estimated to represent about half of the total amount of ACh present in the cleft (Land et al. 1981). Thus, at least 300 ACh molecules were delivered onto the myocyte membrane for generating one SSC. This is a minimal estimate, however, since even though the synaptosomes were apparently in close contact with the myoball, the number of ACh molecules wasted by diffusion was probably greater than in the case of the physiological synapse. In the Torpedo, as at the neuromuscular junction of various species, one ACh quantum is composed of ca 7-10000 ACh molecules (Dunant & Muller, 1986). MEPPs of much smaller size (about 10 times smaller) than the quantum can be recorded at the nerve-electroplaque junction, especially when the tissue is submitted to challenges affecting its energy metabolism (Muller & Dunant, 1987). Most of the release that occurred under the conditions of the present work probably belonged to this class, since the SSCs exhibited a size distribution that was skewed towards small values. One can therefore estimate that the amount released in one SSC ranged from a few hundred to 2-3000 ACh molecules. Release of a full quantum of ca 10000 ACh molecules was unlikely since this apparently requires establishment of the characteristic differentiations of the junction; these appear in Xenopus culture after 1 day of nerve-muscle contact (Buchanan et al. 1989). Actually, a substantial shift from a skewed to a bell-shaped distribution of SSCs, revealing the production of full quanta, begins after 2 days of contact (N. Tabti & M.-M. Poo, unpublished observation).

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The few hundred ACh molecules or so composing a SSC represents very little compared to the amount present in one synaptosome. Morel et al. (1978), analysing the density of synaptosomes and the ACh content of the fraction, concluded that synaptosomes prepared from the Torpedo electric organ had a mean diameter of 3-5 ,am and an ACh concentration of ca 20 mm. This amounts to 2-6 x 108 for the number of ACh molecules contained in a single synaptosome. It is thus likely that the successive SSCs recorded after one puff were produced by possibly one or only very few synaptosomes that contacted the myoball and discharged repeatedly. Consideration of the number of synaptosomes in a puff (around 106) and of the volume of the bath (2 ml) leads to a similar conclusion. These considerations indicate that the SSCs described here were due to bursts of ACh release by a few intact synaptosomes which had established a somewhat stable and close contact with the myocyte. They also provide electrophysiological evidence that the quantal release mechanism is retained in the synaptosome preparation. With some improvement, the methods might provide a new approach to investigating in vitro the conditions modifying the quantal release of ACh by isolated nerve terminals. By analogy, one could also detect quantal release of other neurotransmitters, using a 'detector' cell equipped with the relevant receptors. Does contact with a postsynaptic membrane induce the quantal release of ACh? We should like to suggest that the myocyte contact may in some way induce pulsatile transmitter release from synaptosomes. Indeed, in Xenopus nerve-muscle culture, physical contact of a muscle membrane induces pulsatile ACh release from the nerve terminal, leading to the observed SSCs in the muscle cell (Xie & Poo, 1986). Ultrastructural studies of early nerve-muscle contact indicate that spacing between the contacting membranes is extremely narrow (less than 10 nm, Buchanan et al. 1989), allowing direct interaction between surface-bound molecules on the nerve and muscle cells. Our finding on synaptosomal release raised the interesting possibility that synaptosomes might release neurotransmitter in a pulsatile way only after contact with the myocyte and that the release is triggered by surface interaction with the myocyte membrane. If the latter is true, the present synaptosome preparation may prove to be useful for studies on molecules responsible for nerve-muscle interactions during the early phase of synaptogenesis or during the process of regeneration. This work was supported by the Swiss FNRS (No. 3.741.0.87) and by the De Reuter Foundation to Y. D., and by US National Institutes of Health (NS 22764) and National Science Foundation (BNS 13306) to M.-m. P. We are grateful to N. Collet and F. Pillonel for help with the text and figures, and to J. Jacquet, D. Muller, R. Papillon, J. Richez and J. C. Vincent for assistance with the detection and analysis of signals. REFERENCES

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