Amplitude and time course of evoked and spontaneous synaptic currents in rat submandibular ganglion cells

Transcription

1 4372 Journal of Physiology (1996), 49.1, pp Amplitude and time ourse of evoked and spontaneous synapti urrents in rat submandibular ganglion ells Robert J. allister and Brue Walmsley * The Neurosiene Group, isipline of Medial Biohemistry, Faulty of Mediine, The University of Newastle, allaghan, NSW 238, Australia 1. Exitatory postsynapti urrents (EPSs) were reorded in rat submandibular ganglion ells in vitro using the two-eletrode voltage lamp tehnique. 2. The peak amplitude of EPSs evoked by nerve impulses in single presynapti fibres varied between 1P2 and 9-8 na in different ells (mean = na; n = 23; -8 mv membrane potential; ). 3. Experiments were performed to re-investigate a previous hypothesis that different mehanisms underlie the generation of evoked versus spontaneous quantal EPSs in submandibular ells. This hypothesis was based on the observation of different time ourses of evoked and spontaneous EPSs. 4. In agreement with previous studies, the time ourse of the deay phase of evoked EPSs was desribed by the sum of two exponentials, with time onstants T, and T2 of and ms, respetively (n = 23; -8 mv membrane potential). 5. The double-exponential deay of evoked EPSs persisted when transmitter release was redued by bath addition of 1 um admium hloride to the level of failures, one or several quanta. 6. Spontaneous EPSs exhibited mean amplitudes of pa (n= 5 ells; -8 mv membrane potential), and displayed an extremely wide range of peak amplitudes in the same ell (mean oeffiient of variation (.v.) = ; n = 5 ells). In ontrast to a previous report (see below), the deay phase of spontaneous EPSs was found to exhibit a double-exponential time ourse with time onstants similar to those of the evoked EPS reorded in the same ell. 7. These results indiate that evoked and spontaneously released quanta of transmitter most probably at on the same population of postsynapti reeptors in submandibular ganglion ells. There is a large variability in the peak amplitudes of quantal EPSs reorded in the same ell. This large variability is not due to eletrotoni effets, sine these ells lak dendrites. 1,49 Eletrophysiologial investigations into the quantal nature of synapti transmission are usually ompliated by many fators, inluding the eletrotoni attenuation of synapti urrents generated in the dendriti branhes of a neurone, stimulation of multiple-fibre synapti inputs, and the inability to orrelate the origin of spontaneous synapti urrents with speifi presynapti inputs. In the present study we have used the submandibular ganglion preparation to avoid these serious problems and investigate quantal synapti transmission at a neuronal niotini synapse (Asher, Large & Rang, 1979; Rang, 1981; Yawo, 1989; Sargent, 1993). This ganglion was hosen beause, firstly, in adult animals 75% of ganglion ells reeive input from a single presynapti axon (Lihtman, 1977). Seondly, there are no interneurones or other soures of intrinsi synapti input to these ells (Kawa & Roper, 1984). Therefore, both evoked and spontaneous transmitter release our at a population of release sites arising from the same presynapti nerve fibre. Thirdly, submandibular ganglion ells are eletronially ompat beause they lak dendrites (Lihtman, 1977). In a previous study of submandibular ganglion ells, Rang (1981) reported that the spontaneous exitatory postsynapti urrents (EPSs) exhibit a time ourse very different from that of the evoked EPS. The deay time ourse of the evoked EPS ould be desribed by the sum * To whom orrespondene should be addressed.

2 15 R. J allister and B. Walmsley J Physiol of two exponentials, exhibiting time onstants onsistent with the two kineti omponents of aetylholine-indued noise in these ells (Rang, 1981; Yawo, 1989). However, the deay time ourse of the spontaneous EPSs exhibited only a single exponential time onstant, orresponding to the fast time onstant of the evoked response. It was onluded that there were two distint types of hannels in the postsynapti membrane. It was further proposed that these hannels are spatially arranged in the postsynapti membrane, suh that both reeptor-hannel types are aessible only to nerve-evoked quantal release, and only one population is ativated by spontaneously released quanta of transmitter (Rang, 1981). If orret, this hypothesis seriously hallenges the use of spontaneous EPSs to interpret nerve-evoked transmission at neuronal synapses. In the present study we have investigated quantal transmission in submandibular ganglion ells using the two-eletrode voltage lamp tehnique. We have also re-examined the hypothesis that evoked and spontaneous quantal EPSs are generated by different populations of postsynapti hannels in these ells. Some of this work has been presented in abstrat form (allister & Walmsley, 1994a, b; allister, Keast & Walmsley, 1995). METHOS Tissue preparation All experiments were perfomed on the rat submandibular ganglion preparation, as originally desribed by Lihtman (1977). The preparation onsists of a thin triangular sheet of onnetive tissue strething between the lingual nerve and the salivary duts. Neurones providing parasympatheti innervation to the salivary glands lie in the onnetive tissue sheet and along the salivary duts. These neurones are innervated by preganglioni axons whih leave the brainstem in the horda tympani nerve and merge with the lingual nerve. Approximately 5-8 mm of the horda tympani nerve was removed with eah preparation for subsequent stimulation. Wistar rats (5-7 weeks old) were overdosed with halothane and deapitated. The preparation was removed and pinned out in a small Sylgard-lined reording hamber, perfused with a modified Krebs solution and viewed under an inverted mirosope. The perfusing solution onsisted of (mm): Nal, 12; NaHO3, 25; Kl, 5; Mgl2, 2; NaH2PO4, 1; al2, 2-5; gluose, 11. The solution was ontinually bubbled with 95% O2-5% O2 to maintain a ph of 7.3. In some experiments transmitter release was redued by adding 1 /M admium hloride to this solution. All experiments were arried out at '. Reording and analysis Synapti urrents and potentials were reorded with an Axolamp-2A amplifier (Axon Instruments) using the onventional two-eletrode voltage lamp tehnique. The horda tympani nerve was drawn into a lose fitting sution eletrode and ativated by suprathreshold pulses ( 2 ms, 5-2 V) delivered at a rate of 1-4 Hz. ells were judged to reeive a single presynapti input if they exhibited an all-or-nothing EPS in response to finely graded stimulus strength. Reording and urrent-passing eletrodes had resistanes of 5-1 MQ when filled with 4 M potassium aetate and 5 M potassium sulphate, respetively. In some experiments synapti potentials were reorded with a single miroeletrode (3-5 MQ2 resistane when filled with 5 M Kl). Visualized neurones in the thin onnetive sheet between the salivary duts and lingual nerve were impaled with two miroeletrodes. A grounded shield was positioned between the tips of the two miroeletrodes to redue apaitive oupling between the eletrodes. The voltage lamp was optimized by monitoring the urrent response to a repetitive voltage step (1 ms, 2 mv). Voltage and urrent reordings were filtered at 1 and 1 khz, respetively, and stored on a PM video reorder (A. R. Vetter o., Rebersburg, PA, USA). urrent reords were subsequently filtered off-line at 3-1 khz, digitized and analysed using ustom-designed programs and software kindly provided by J. empster (Strathlyde Eletrophysiology Software, Glasgow, UK). The detetion and averaging of spontaneous events was performed using SAN software (J. empster, Strathlyde Eletrophysiology Software, Glasgow, UK). The detetion of spontaneous events was by amplitude threshold, set in eah ell to exlude all obvious noise reords. Following detetion, spontaneous urrents were aligned using the mid-point of their rising phases before averaging. The deay phase of eah averaged synapti urrent was fitted by the sum of two exponentials over a time period following the peak of approximately 1-15 ms. ue to the small variability in 1-9% rise times (standard error of the mean (S.E.M.), 1-2 ms) of the spontaneous urrents, slight differenes in the alignment of spontaneous urrents prior to averaging would not have any signifiant effet on the alulations of the deay time onstants, whih were approximately 7 and 35 ms. All results are expressed as means + standard deviation (S..). The oeffiient of variation (.v.) is the S.. divided by the mean. ata were onsidered to be statistially signifiant following regression and Student's t test analysis if P < 5. RESULTS Two-eletrode voltage lamp reordings were made from forty-one ells in twenty-five preparations. etailed analysis was performed on ells with stable resting membrane potentials more negative than -45 mv. Membrane potentials were usually lamped at values between -5 and -8 mv, with maximum holding urrents not exeeding 1 na. In voltage lamp mode, only ells in whih the residual voltage was less than 1 % of the unlamped voltage were aepted for further analysis. Properties of evoked EPSs The general features of single-fibre EPSs evoked in submandibular ganglion ells are illustrated in Fig. 1. Stimulation of the horda tympani nerve produed a largeamplitude EPS (Fig. 1A) following a delay from the stimulus of 5-23 ms in different ells (mean = 12f3 + 5* ms; n = 39). At a membrane potential of -8 mv, EPSs exhibited a rapid rising phase (1-9% rise time = ms; n = 16), followed by a slower deay phase.

3 J Physiol Synapti urrents in ganglion ells 151 The peak amplitude of the EPS varied linearly with membrane potential for the range of holding potentials between -4 and -8 mv (Fig. 1B). Extrapolation of a linear regression line fitted over this range revealed a reversal potential of mv (n = 4 ells). This value agrees with that previously reported for submandibular ganglion ells in rodents (Rang, 1981; Yawo, 1989), as well as those reported for different autonomi ganglion ell types (Kuba & Nishi, 1979; erkah, Selyanko & Skok, 1983; Mathie, ull-andy & olquhoun, 1987), onsistent with the postsynapti ativation of non-speifi ation hannels. Figure 1 shows an evoked EPS reorded at a membrane potential of -8 mv. The deay phase of the EPS ould not be adequately fitted by a single exponential, but was well desribed by the sum of two exponentials (Fig. 1, ). As illustrated in the semilogarithmi plot of this EPS (Fig. 1), the initial rapid deay phase has a time onstant of 7-8 ms (-r), while the slow phase has a time onstant of 31P ms (r2). This two-phase deay was present in all of the evoked EPSs examined at membrane potentials between -5 and -8 mv (Table 1). In twenty-three ells at a membrane potential of -8 mv, the values for Tr ranged from 5-9 to 8-7 ms (mean = ms), while T2 ranged from 23-2 to 55-3 ms (mean = ms). Variability in the amplitude of evoked EPSs The mean peak amplitude of evoked EPSs reorded in submandibular ells varied over a large range (Table 1). At a membrane potential of -8 mv, EPS peak amplitudes ranged from 1-22 to 9-78 na (mean = na; n = 23 ells). In individual ells the amplitude of evoked EPSs flutuated markedly from trial to trial (Fig. 2A). A plot of EPS peak amplitudes for 5 onseutive trials is shown in Fig. 2B. This plot indiates that the flutuation pattern is stable over time. A histogram of EPS peak amplitudes is illustrated in Fig. 2. The peak amplitude of A 1 na 5 ms 2 25 ms B Membrane potential (mv) <: -5- ) = T1 7-8 ms r2 = 31- ms - p 3 4 E 4 o -1 - / Figure 1. Single-fibre EPSs evoked in a submandibular ganglion ell A, EPSs reorded at a series of inreasingly negative membrane potentials: -4, -5, -6, -7 and -8 mv. Eah trae represents the average of 1 responses to stimuli delivered at 3-67 Hz. B, plot of the peak amplitude of evoked EPSs versus membrane potential., average evoked EPS reorded at a membrane potential of -8 mv. The EPS has a 1-9% rise time of 1-94 ms and the deay phase is well fitted by the sum of two exponentials () with time onstants of 7-8 and 31O ms, respetively., semilogarithmi plot of the deay phase of the reord shown in. The slow exponential omponent (straight line, upper plot) is superimposed on the average EPS reord. The differene between this slow exponential and the EPS is also illustrated (lower plot), together with the fast exponential omponent (straight line).

4 152 R. J allister and B. Walmsley J Physiol.49.1 Table 1. Properties of evoked EPSs in submandibular ganglion ells Membrane potential (mv) EPS peak amplitude (na) P ' oeffiient of variation * (n = 5) (n= 5) (n= 18) TI (ms) 6X ' T2 (ms) ata are from 14, 6 and 23 ells held at membrane potentials of -5, -6 and -8 mv, respetively. All data obtained at room temperature (22-25 ) in normal Krebs solution (2-5 mm al2). The deay phase of eah EPS was fitted by the sum of two exponentials with time onstants T, and T2. Values are presented as means + S.. * Based on 1-5 responses. The.v. for EPS peak amplitude was not obtained for ells where the averaged synapti urrent was measured on-line; n denotes number of ells analysed. the evoked EPS in this ell flutuated over a large range with a.v. of 15. The average.v. for peak EPS amplitudes was approximately 13 for all ells examined (n = 28; see Table 1). There is an inverse relationship between the.v. and the mean peak amplitude of evoked EPSs. This. relationship was statistially signifiant (regression analysis, P < 5) for ells held at membrane potentials of -5, -6 and -8 mv and is illustrated for ells held at -8 mv, in Fig. 2. A likely explanation for this inverse orrelation is that smaller EPSs are generated at onnetions with fewer release sites. Properties of quantal EPSs The only previous study on quantal urrents in submandibular ganglion ells reported that the time ourse of deay of evoked and spontaneous EPSs is very different A I na 2 ms ) ) a. 3 W 2 B ] a - E -2 U -15. o-1 o U-u; 1 U U U *. U U mm * * 1 3 Reord number Figure 2. Amplitude flutuations in evoked EPSs A, 5 superimposed EPSs reorded in a ell following stimulation of the horda tympani nerve at 2-67 Hz (holding potential, -6 mv). B, plot of peak amplitudes of suessive EPSs for 5 trials (same ell as illustrated in A)., histogram of EPS peak amplitudes (right) and orresponding noise (left)., inverse relationship between the oeffiient of variation and average peak amplitude of evoked EPSs demonstrated for 18 ells at a membrane potential of -8 mv.

5 J Physiol.49.1 Synapti urrents in ganglion ells 153 (Rang, 1981). We have investigated this result further using two strategies. Firstly, we ompared the time ourse of evoked EPSs with that obtained under onditions in whih release had been redued to the level of failures or few quanta. Seondly, we ompared the time ourse of evoked and spontaneous quantal EPSs in the same ell. Evoked quantal EPSs reorded in the presene of admium hloride. The quantal ontent of the evoked synapti response was redued to the level of failures by adding admium hloride to the perfusate to blok presynapti alium urrents. In preliminary experiments we used a single miroeletrode (3-8 MQ) to reord evoked EPSPs. A admium hloride onentration of 1,M was found to be optimal for reduing the amplitude of the evoked EPSPs to the level of lear failures and one or several quanta. Based on these findings we subsequently obtained twoeletrode voltage lamp reordings of evoked EPSs in the presene of 1 ulm admium hloride. The results of one suh experiment are shown in Fig. 3. Over the ourse of several minutes the evoked EPS (Fig. 3A) progressively dereased to a level at whih failures of response A B 'na ms 2 14 O ' E 8, A:5 O 6 HI 4 2 E F Amplitude (pa) pa 25 ms 4 o 3' ] 2' ) 1' - A,jl- J11111M l11_- 11I Amplitude (pa) Figure 3. Evoked, admium-bloked and spontaneous EPSs reorded from the same ell A, evoked EPS (average of 1 trials) reorded in normal Krebs solution (3-67 Hz stimulation; -8 mv membrane potential). B, histogram of peak amplitudes of the EPS illustrated in A (mean = 5-1 na;.v. = 9; n = 1)., evoked EPSs reorded in the presene of 1 /UM admium hloride, illustrating variability of EPS amplitude and failure of response., histogram of admium-bloked EPS peak amplitudes (open bars; mean = 85 pa; n = 2). E, spontaneous EPSs reorded in the presene of 1 /SM admium hloride. F, amplitude histogram (filled bars, also shown in ; mean = 48 pa; n = 66) and orresponding noise (open bars; S.. = 7 pa; n = 2) for spontaneous EPSs.

6 154 R. J allister and B. Walmsley J Physiol.49.1 (approximately 25% of trials) were apparent (Fig. 3). Histograms of the peak amplitudes of the evoked EPSs under normal onditions and with added admium are illustrated in Fig. 3B and, respetively. A lear separation between the 'failures' peak and the 'non-failures' response an be observed in the histogram illustrated in Fig. 3. The orresponding bakground noise reorded in this ell is illustrated in Fig. 3E In the same ell, a small number of spontaneous EPSs (n = 66) were also reorded, and several examples are shown in Fig. 3E. A histogram of peak amplitudes of the spontaneous EPSs is illustrated in Fig. 3F (and in Fig. 3, filled bars). There is an obvious orrespondene between the spontaneous EPS histogram and the 'non-failures' peak in the histogram of evoked EPSs (Fig. 3). This observation provides supporting evidene that evoked release was redued to the level of failures and one or several quanta. I A 25 Noise B EPSP (Normal Krebs) Results for another ell are illustrated in Fig. 4, showing the time ourse of the deay phase of the evoked EPS under normal onditions (Fig. 4A and B) and in the presene of 1 /SM admium hloride (Fig. 4 and ). The deay phase of the evoked EPS is desribed by the sum of two exponentials with time onstants of 6-2 and 27-4 ms, respetively (Fig. 4B). Figure 4 shows that, in the presene of 1 /M admium hloride, the two omponent deay in the evoked EPS persists, with time onstants of 5*5 and 25-8 ms, respetively. This experiment was repeated in nine ells, and in all ases the two-phase deay persisted (Table 2). Neither the fast (,r) nor the slow (r2) time onstants were signifiantly different under normal and low release onditions (paired t test, P > 5) EPS (1 /M d2+) _-, E o I. ) Figure 4. omparison of the deay phase of evoked and admium-bloked EPSs reorded in the same ell A, histogram of the peak amplitudes for an evoked EPS reorded in normal Krebs solution, and the orresponding noise (1 trials, 2-67 Hz stimulation, -8 mv membrane potential). B, semilogarithmi plot of the averaged evoked EPS in normal Krebs solution. The deay phase of the synapti urrent is well desribed by two time onstants of 6-2 and 27-4 ms (straight lines). Inset shows the average of 1 EPSs., amplitude histogram of the evoked EPS peak amplitudes in the presene of 1 /M admium hloride., semilogarithmi plot of the admium-bloked evoked EPS. The deay phase is well desribed by two exponential time onstants of 5.5 and 25-8 ms. Inset shows the average of 7 EPSs.

7 J Physiol.49.1 Synapti urrents in ganglion ells 155 Table 2. omparison of the time ourse of evoked EPSs under normal and low release onditions EPS peak amplitude (na) Tr (ms) T2 (ms) Mean + S Range 1P X ata are from 9 ells held at membrane potentials between -5 and -8 mv. Parameters are given for EPSs reorded in normal Krebs solution and after the addition of 1 #M dl2. A 1 pa B 25 ms Amplitude (pa) 12 1 _ a ) a) o a) ) Figure 5. Evoked and spontaneous EPSs reorded in the same ell A, seleted traes showing spontaneous EPSs reorded in normal Krebs solution (-8 mv membrane potential). B, amplitude histograms of spontaneous EPSs (filled bars; mean = 57 pa;.v. = 42; n = 299) and orresponding noise (open bars)., semilogarithmi plot of the deay phase of the evoked EPS (inset; average of 1 trials)., semilogarithmi plot of the deay phase of spontaneous EPS (inset; average of 241 trials). The EPS deay phases in both and were well fitted by the sum of two exponentials (O in insets). Straight lines superimposed on semilogarithmi plots in and indiate the fast and slow exponential omponents of the deay phase.

8 156 R. J allister and B. Walmsley J Physiol Properties of spontaneous quantal EPSs As previously observed (Rang, 1981), spontaneous miniature EPSs our infrequently in submandibular ganglion ells. Our attempts to inrease the frequeny of spontaneous EPSs using standard proedures, suh as hypertoni solutions (Bekkers, Riherson & Stevens, 199) or high potassium solutions (Van der Kloot, 1991), proved unsatisfatory. Hypertoni solutions (up to 45 mmol kg-') failed to signifiantly inrease the frequeny of spontaneous events. High potassium levels signifiantly inreased the noise, and redued the stability of the reordings. However, in four experiments using normal Krebs solution, we were able to reord both the nerve-evoked EPS and adequate numbers of spontaneous EPSs under low-noise onditions, and ompare their properties. The results for one ell are illustrated in Fig. 5. Figure 5A illustrates several spontaneous EPSs reorded in this ell. It is apparent that there are large differenes between the peak amplitudes of spontaneous EPSs. This variability is illustrated in the histogram of EPS peak amplitudes onstruted from 299 spontaneous EPSs reorded in this ell (Fig. 5B). Also plotted in Fig. 5B is a histogram of the bakgound noise. The variability in peak amplitude of the spontaneous EPSs greatly exeeds the ontaminating noise. Following subtration of the noise variane, the.v. of the spontaneous EPS peak amplitudes was alulated to be -42. Figures 5 and ompare the time ourse of the evoked and spontaneous EPSs reorded in this ell. The time ourse of the evoked EPS was well fitted by a doubleexponential deay, with -r = 7-1 ms and T2 = 33-8 ms. These time onstants were well mathed by those fitted to the deay phase of the averaged (n = 299) spontaneous EPS, with Tr = 7S5 ms and T2 = 37-6 ms (Fig. 5). Spontaneous EPSs were reorded in a total of five ells, at a membrane potential of -8 mv. The mean peak amplitude of spontaneous EPSs for all five ells was 81 pa, and the mean.v. was 37. In four of these ells, evoked EPSs were also reorded for time ourse omparison with the spontaneous EPSs. In all four ells, the time ourse of the averaged spontaneous EPSs exhibited a double-exponential deay phase, similar to the two-phase deay of the evoked EPSs. The mean time onstants for the spontaneous EPSs were Tr = and r2 = '6, and the mean time onstants for the evoked EPSs were Tr = and T2 = (n = 4). Neither the fast ('rl) nor the slow (T2) time onstants were signifiantly different for the evoked and spontaneous EPSs (paired t test, P > -5). ISUSSION In the present experiments we have studied quantal transmission, and investigated the hypothesis that evoked and spontaneous quantal EPSs are generated by different populations of postsynapti hannels in submandibular ganglion ells. Evoked EPSs were studied at the level of one or several quanta by reduing release to a substantial level of failures. Under these onditions, a double-exponential deay of the EPS was present. This extends, to the quantal level, the result obtained by Rang (1981) in whih the peak amplitude of the evoked EPS was redued to approximately 1% of ontrol value by dereasing the alium: magnesium ratio in the perfusate. However, in ontrast to Rang (1981), our results demonstrate that the double-exponential deay is also present in the spontaneously ourring EPSs. Furthermore, there is good agreement between the two time onstants fitted to the deay time ourse of the spontaneous EPSs and the evoked EPSs reorded in the same ell. The most likely explanation for the inonsisteny between our result and that of the previous study is that the slower time onstant omponent an be learly resolved only under onditions of low noise, following the averaging of large numbers of spontaneous EPSs. This ondition was ahieved in our study for very few ells, sine the rate of spontaneously ourring EPSs is extremely low in the submandibular ganglion. Thus, we onlude that the spontaneous and evoked EPSs are generated by ativation of the same postsynapti hannels, and that there is no evidene for a distintion between nerve-evoked and spontaneously ourring quantal EPSs. Although it was previously onsidered likely that there are two separate populations of aetylholine-gated hannels in submandibular ganglion ells, our results re-open the possibility of only a single population of reeptor hannels with omplex kinetis (olquhoun & Sakmann, 1985; Mathie et al. 1987; erkah, North, Selyanko & Skok, 1987; Sargent, 1993). This possibility was also onsidered by Yawo (1989), following the demonstration that the fast and slow kineti omponents of both the EPS and the aetylholine-gated hannels exhibit idential voltage sensitivities in mouse submandibular ganglion ells. The mean peak amplitude of spontaneous EPSs reorded in the present study was 81 pa at a membrane potential of -8 mv. The single-hannel ondutane for aetylholinegated hannels in submandibular ganglion ells is approximately 3 ps at negative membrane potentials (Rang, 1981; Yawo, 1989). Thus, the mean spontaneous EPS represents the opening of approximately thirty-five aetylholine hannels. At a membrane potential of -8 mv, the peak amplitude of evoked EPSs varied enormously between ells, ranging from 1-2 to 9'8 na (mean = na, n = 23 ells). In five ells, both spontaneous and evoked EPSs were reorded. Assuming that the evoked EPS is omposed of the sum of quantal EPSs with mean amplitude equal to that of the spontaneous EPSs, the mean quantal ontent of the

9 evoked EPSs in these five ells ranged from 16 to 136 (mean = ). It is likely that this large range is primarily due to differenes in the total number of synapti ontats formed between single preganglioni fibres and submandibular ganglion ells (Lihtman, 1977; Rang, 1981; allister et al. 1995). The present study is the first to demonstrate that there is onsiderable variability in the peak amplitude of spontaneous quantal EPSs reorded in submandibular ganglion ells. The mean.v. of EPS peak amplitudes is large (mean = '37, n = 5 ells), despite a lak of spread due to eletrotoni attenuation of EPSs in these ells. It remains to be determined how muh of this large variability is due to differenes in mean EPS amplitude between release sites (perhaps due to differenes in the numbers of reeptors), and how muh is due to intrinsi variability at eah release site (Edwards, Redman & Walmsley, 1976; Hirst & MLahlan, 1984; Walmsley, Edwards & Traey, 1988; Bekkers et al. 199; Faber, Young, Legendre & Korn, 1992; Walmsley, 1991, 1993; Sargent, 1993; Bekkers, 1994). ASHER, P., LARGE, W. A. & RANG, H. P. (1979). Studies on the mehanism of ation of aetylholine antagonists on rat parasympatheti ganglion ells. Journal of Physiology 295, BEKKERS, J. M. (1994). Quantal analysis of synapti transmission in the entral nervous system. urrent Opinion in Neurobiology 4, BEKKERS, J. M., RIHERSON, G. B. & STEVENS,. F. (199). Origin of variability in quantal size in ultured hippoampal neurons and hippoampal slies. Proeedings of the National Aademy of Sienes of the USA 87, ALLISTER, R. J., KEAST, J. R. & WALMSLEY, B. 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Nonuniform release probabilities underlie quantal synapti transmission at a mammalian exitatory entral synapse. Journal of Neurophysiology 6, YAWO, H. (1989). Retifiation of synapti and aetylholine urrents in submandibular ganglion ells. Journal of Physiology 417, Aknowledgements We would like to thank rs James Brok and Jeff Isaason for helpful omments on the manusript. This work was supported by grants from the National Health and Medial Researh ounil of Australia and the live and Vera Ramaiotti Foundations. r R. J. an NH&MR Australian Postdotoral Fellow. allister is urrently Reeived 1 Marh 1995; aepted 5 July 1995.