'quantal content'. Excitatory junction potentials may be recorded from smooth muscle cells following

Transcription

1 J. Physiol. (1984), 349, pp With 1 text-figures Printed in Great Britain FACILITATION AT SINGLE RELEASE SITES OF A SYMPATHETIC NEUROEFFECTOR JUNCTION IN THE MOUSE BY A. G. H. BLAKELEY, A. MATHIE AND S. A. PETERSEN From the Department of Physiology, University of Leicester, University Road, Leicester LEJ 7RH (Received 21 September 1983) SUMMARY 1. Electrophysiological techniques were used to observe the release of transmitter from one or a few release sites of the sympathetic neuroeffector junction of the mouse vas deferens. Release produces transient accelerations of the depolarizing phase of the excitatory junction potential, known as 'discrete events'. 2. Discrete events associate into families at a constant latency and peak time, but vary in amplitude between a few preferred values. 3. As facilitation develops there is a decrease in the frequency of small members of families and an increase in the frequency of large members, a change in apparent 'quantal content'. 4. A similar change in amplitude distribution occurs when the [Ca]. is raised. 5. The a-adrenoceptor antagonist yohimbine increases quantal content when facilitation has developed, but has no significant effect on unfacilitated discrete event amplitude unless the [Ca]. is below 2- mm. 6. The a-adrenoceptor agonist clonidine reduces facilitated and unfacilitated quantal content under all conditions examined. INTRODUCTION Electrophysiological techniques may be used to monitor transmitter release from single sites at sympathetic neuroeffector junctions (Hirst & Nield, 198; Blakeley, Cunnane & Petersen, 1982). We have investigated transmitter release from single sites in the mouse vas deferens during facilitation and following treatment of the preparation with a-adrenoceptor agonists and antagonists. Excitatory junction potentials may be recorded from smooth muscle cells following stimulation of the sympathetic nerves. They are due to the action of transmitter on both the impaled and neighbouring cells. The effects of transmitter released close to the recording electrode are more rapid, and appear as transient accelerations of the depolarizing phase. These accelerations have been called 'discrete events' (Blakeley & Cunnane, 1979). Discrete events are revealed by differentiation with respect to time, but the wave forms produced are complex and the number and size of peaks observed varies from stimulus to stimulus. When events elicited by a train of stimuli are compared they

2 58 A. G. H. BLAKELEY, A. MATHIE AND S. A. PETERSEN occur mainly in 'families' having a fixed latency and similar time courses but varying in amplitude between a few preferred values. It seems most likely that such a family of events reflects the release of different numbers of packets of transmitter from a single release site or a small group of release sites acting on the impaled cell and behaving effectively as one. This interpretation has, however, been challenged by Cunnane & StjArne (1982) who suggest that minor differences in time course and shape reflect the activity of transmitter released extremely intermittently from many different sites at varying distances from the recording electrode. In an attempt to resolve this matter, we have used more stringent criteria than previously in defining families ofdiscrete events, and examined the changes which occurred when transmitter release was modified, during facilitation, by varying external calcium concentration and as a result of treatment with drugs which act on prejunctional adrenoceptors. METHODS Male C57BL/6 mice, aged 9-15 days were killed by cervical dislocation, and the right vas deferens rapidly dissected. The tissue was mounted at approximately resting length in a 3 ml organ bath perfused with Krebs solution at 3 ml/min and maintained at a temperature of 'C. The prostatic end of the vas was passed into a pair of platinum ring electrodes (separation 5 mm) connected to a Bell isolated stimulator unit. One of two saline solutions was used. The 'low-calcium' solution was of composition (mm): NaCl, 118-4; KCl, 4-72; NaHCO8, 25-; NaH2PO4, 1 13; glucose, 11 1; CaCl2, 1 1; MgCl2, 7, equilibrated with 95 % 2/5 % CO2. The 'high-calcium' Krebs solution was similar, except that [CaCl2] = 2-1 mm. Drugs, where used, were added to the saline solution to the appropriate bath concentration. The membrane potentials of smooth muscle cells at or near the surface of the vas were recorded using glass micro-electrodes, resistance MD, filled with 3 M-KCl. The potentials were amplified via a Dagan 81 single-electrode voltage clamp, used in this case in bridge mode, and recorded on a Racal Store 4D tape recorder (frequency response d.c. to 3 khz). Junction potentials were elicited by field stimulation through the ring electrodes around the prostatic end of the vas. The intensity of the stimulus was adjusted for each cell so as to reliably elicit a family of discrete events (see below) and then remained unchanged during experimental observations. The first time differential of the junction potentials was obtained by two distinct methods. During the experiments the signal was continuously differentiated using an operational amplifier differentiator circuit whose frequency response was severely limited ( < 5 Hz) in order to reduce output noise to a reasonable level (Blakeley & Cunnane, 1979). After the experiments the recorded junction potentials were digitized (sampling frequency 5 khz) using a digitimer NL 9 board attached to a Research Machines 38Z microcomputer. The digitized signal was then smoothed by averaging successive pairs of samples, and differentiated by calculating differences between averages of two variable sized running sample windows displaced by one sample interval. The effective frequency response of the differentiation could then be changed by altering the size of the sample window. After trials with a variety of sample window sizes, the best wave forms with little noise and yet closely resembling the raw signal in over-all shape were obtained when ten successive samples were incorporated. The discrete events recorded from each cell were examined in detail, and precise criteria defined for associating events into families clearly related in latency, time course and amplitude (see Results). Amplitude, rise time and rise rate distributions were then obtained for the events in each family. In those cells where events appeared superimposed upon a slow background, the background was subtracted by the computer before compiling amplitude distributions. Junction potentials were measured under a variety of conditions. In the majority of experiments groups of five stimuli at 2 Hz were applied at 12-5 s intervals, though in one or two cases longer trains of stimuli at 2 Hz were used. For some preparations low-calcium Krebs was used throughout, and for others high-calcium

3 FACILITATION AT SINGLE SYMPATHETIC JUNCTIONS Krebs was used. In each case, for some of the preparations either yohimbine (1-7 M) or clonidine (5 X 1-9 M) was added after about 3 min. These concentrations were chosen on the basis of concentration-effect relationships to achieve a middling effect (A. G. H. Blakeley, A. Mathie & S. A. Petersen, unpublished observations). In a few experiments it was arranged that the calcium concentration in the bath could be changed from 1 1 to 2-1 mm over a period of about 3 s. In this case recordings of junction potentials from single cells were made at two bath concentrations of calcium. Drugs used were yohimbine hydrochloride (Sigma) and clonidine hydrochloride (Catapres, Boehringer Ingelheim). 2 V/s p L d An~~~~~~~~,i 1 ms 59 Fig. 1. Superimposed discrete events evoked by a train of twenty-five stimuli at 2 Hz. Traces with no discernible discrete events at any latency have not been included. RESULTS Families of discrete events The first time differential of a sequence ofjunction potentials obtained from a single cell as described above is shown in Figs. 1 and 2 (see Methods). In this particular example, it is clear that events peak at different times, but there is one obvious family of responses which all start, and peak, within a very narrow time band. The amplitude ofevents within this family varies discontinuously between certain preferred values, separated by gaps of similar size. We have defined this family by the latency, rise time and peak time criteria specified in Fig. 2. Spontaneous events similar in amplitude and shape to the smaller, but not the larger events in this family were also observed. A similarly stringent exercise was performed on every set of discrete events reported in these experiments. Table 1 summarizes the variety of results observed from cell to cell under different

4 6 A. G. H. BLAKELEY, A. MATHIE AND S. A. PETERSEN conditions. Under most conditions, between 8 and 1% of cells impaled yielded at least one family of discrete events with a clearly defined time course, and at least three separate, regularly spaced, amplitudes. Most cells also had other discrete events at different latencies. The majority of the data we report concern events associated into families. 2 V/s 1 Ms Fig. 2. Superimposed discrete events, recorded from the same cell as those in Fig. 1, selected from further trains of twenty-five stimuli at 2 Hz to illustrate our criteria for defining families. Bars show latency, peak time and amplitude criteria within which events are considered to be indistinguishable. Amplitude distributions within families Fig. 3 shows that the amplitude distributions of the discrete events examined here are similar to those reported earlier (Blakeley & Cunnane, 1979; Blakeley, Cunnane & Petersen, 1982) that is to say multi-modal, with modes spaced regularly along the amplitude axis. Changes within families of discrete events during facilitation Fig. 4 shows amplitude distributions of the same family of discrete events elicited by either the first or fifth stimulus in trains of five at 2 Hz, separated by sufficient time (1 s) to allow decay of facilitation (Blakeley, Cunnane & Petersen, 1981 a). The bath concentration of calcium was 2-1 mm. Within a family, events become larger as facilitation develops and the proportion of stimuli which fail to elicit a member of the family ('failures') is reduced. Combined data from all cells studied are summarized in Table 2. The mean size of events within families increases significantly from the first to the fifth stimulus

5 FACILITATION AT SINGLE SYMPATHETIC JUNCTIONS TABLE 1. Summary of the properties of all cells described 2-1 mm-ca mm-ca mm-ca mm-ca mm-ca2+ +yohimbine +clonidine 1.1 mm-ca2+ +yohimbine +clonidine Total no of cells Proportion with d.e.s (%) Proportion with families (%)* Mean no. of latencies Mean no. of 1X2 1X families * These figures clearly may be underestimates as a degree of selection is inevitable; d.e., discrete event. 61 C.o. -W 5. E Z Bin number Fig. 3. Amplitude histogram of a family of events sharing a common latency and peak time. Number of failures = 142 (proportion of failures, 67-9 %); bin size, -1 V/s. (P <.1, Friedman two-way analysis of variance). The proportion of failures significantly decreases (P <.1, Friedman two-way analysis of variance). The mean size of events does not fully describe the changes in amplitude. The amplitude distribution is asymmetric for both first and fifth stimuli, but the skew occurs in opposite directions. That is to say a preponderance of small events following the first stimuli is replaced by a preponderance of large events following the fifth. This can be seen for a particular example in Fig. 4, but Fig. 5 summarizes data from all cells by plotting the difference between the percentage of observations on either side of the mean value as a 'skew index'. Fig. 6 shows changes in failure rate during facilitation. The high proportion of failures is inconsistent with the number of large events (see also Figs. 4 and 8). A Poisson release process predicts fewer failures. Similar observations in the guinea-pig

6 62 A. G. H. BLAKELEY, A. MATHIE AND S. A. PETERSEN 15 A CA o I 1 -o am z 5... Hmm... a-1.. I.I-lllll B (A c am. E z Qo RH- IC c._ am. -o E z I Bin number Fig. 4. Changes in amplitude distribution of a family of events during facilitation, [Ca]O = 2-1 mm. A, amplitude distribution of all events in the family irrespective of degree of facilitation. Number of failures = 11 (proportion, 52-4 %). B, amplitude distribution of family members evoked by first stimuli. Number of failures = 23 (proportion, 54-8 %). C, amplitude distribution of family members evoked by fifth stimuli. Number of failures = 19 (proportion, 45 2 %O). Bin size, 5 V/s.

7 FACILITATION AT SINGLE SYMPATHETIC JUNCTIONS = 2-1 and 1 1 yohimbine, 1-7 M; clonidine, 5 x 1-9 M mm; Stimulus number TABLE 2. Changes in discrete events during facilitation when [Ca]. High calcium (2-1 mm) Mean d.e. amplitude (V/s) Proportion of failures Low calcium (1H1 mm) Mean d.e. amplitude (V/s) Proportion of failures n ± _ x 1._ 3. ) C,, Stimulus number Fig. 5. 'Skew index' changes during facilitation (n = 15 for all points). The skew index is calculated as the difference between the percentage of total observations on either side of the mean. vas led us to suggest (Blakeley, Cunnane & Petersen, 1982) a second process producing 'non-poisson' failures. Tightening criteria for associating events suggests a similar process in the mouse vas, but we have insufficient data to be certain whether the proportion of non-poisson failures changes during facilitation. Changes in the number offamilies during facilitation Most cells have only one or two clearly defined families of events, together with a few other events at odd latencies. Table 3 shows how the number of families and the number of 'non-family' or independent events varies during facilitation. There is no significant change in the number of families, but a small increase in the number of independent events observed.

8 64 A. G. H. BLAKELEY, A. MATHIE AND S. A. PETERSEN -71._ 4) (U _ S -5-4J Stimulus number Fig. 6. Changes in the proportion of failures during facilitation (n = 15 for all points). TABLE 3. Family and non-family events during facilitation. [Ca]O = 2-1 mm, n = 15 in all cases Stimulus number Mean no. of latencies Mean no. of families Mean no. of non-family d.e.s [Ca]. and facilitation Fig. 7 shows the amplitude distribution of a single family of discrete events elicited by a long train of stimuli at 2 Hz at bath concentrations of 2-1 and 1.1 mm-calcium. The events are significantly larger in high-calcium Krebs solution (P <.1, Wilcoxon signed rank test) and, as with facilitation, the increase occurs by a rise in the number of large events and a reduction in the number of small, but with no change in the position of the modes cf. Katz & Miledi (1968). Table 4 summarizes the changes of discrete event amplitude and probability of occurrence from several cells as the [Ca]o is altered. Facilitation still occurs when the [Ca] is 1-1 mm. Fig. 8 shows amplitude distributions of events elicited by first and fifth stimuli, and Table 2 gives details of variations in mean size and probability of occurrence a-adrenoceptor agonidt/antagonists and facilitation We have previously reported data showing the effects of oc-adrenoceptor agonists and antagonists on the development and decay of facilitation of excitatory junction potentials (e.j.p.s) (Blakeley, Cunnane & Petersen, 1981 a).

9 FACILITATION AT SINGLE SYMPATHETIC JUNCTIONS A C lo 15.. E :3~~~~~~~~~I 1 E. ol~ ~~~1 IT!I II IIII i Bin number Fig. 7. Changes in the amplitude distribution of a family of events when the [Ca]O is raised from I -I mm (A) to 2-1 mm (B). Number of failures: A, 98 (64-9 %); B. 69 (46 %). Bin size,.5 V/s. Changes in discrete events mirror the alterations in e.j.p. amplitude when the [Ca]. is 2-1 mm. Fig. 9 shows changes in discrete event amplitude and probability of occurrence during facilitation in the presence of yohimbine (1-7 M) or clonidine (5 X 1-9 M). Clonidine significantly decreases mean discrete event amplitude and increases the probability of failure in both the facilitated and unfacilitated condition. Yohimbine increases discrete event amplitude and reduces the probability of failure significantly only for discrete events following fourth and fifth stimuli, i.e. once facilitation has developed. When the [Ca]O is lower the effects of the drugs are slightly different (Fig. 1). The depressing effect of clonidine on discrete event amplitude is less and, in the' unfacilitated state, there is no significant effect on the probability of failure. 3 PHY 349

10 66 A. G. H. BLAKELEY, A. MATHIE AND S. A. PETERSEN TABLE 4. Effects of different [Ca]O on facilitated discrete event amplitude and failures Mean d.e. amplitude Proportion of [Ca]. (mm) n (V/s) failures { A (A c To. 14-,.5 a) e E z flhx B c._. 4). E z O ( 1 HH[ 2 3 Bin number Fig. 8. Changes in the amplitude distribution of a family of events during facilitation when [Ca]. is 1-1 mm. A, amplitude distribution of family members evoked by first stimuli. Number offailures = 29, proportion offailures, 61-7 %. B, amplitude distribution offamily members evoked by fifth stimuli. Number of failures = 29, proportion of failures, 61-7 %. Bin size, 5 V/s. 4

11 FACILITATION AT SINGLE SYMPATHETIC JUNCTIONS 67 A._ ) (a -o E 125 Control Yohimbine O Clonidine Qi O ! I._s -6 3 ~ o 2 4 Stimulus number * Control XYohimbine M Clonidine Fig. 9. Effects of yohimbine (1-7 M) and clonidine (5 x 1-9 M) on A, mean discrete event (d.e.) amplitude and B, proportion of failures during facilitation when [Ca]l = 2-1 mm. Control, n = 15; yohimbine, n = 5; clonidine, n =

12 68 A. G. H. BLAKELEY, A. MATHIE AND S. A. PETERSEN I A -C - Q).-I E cl V a; 1-25 t I f Control K Yohimbine OD Clonidine B a) vm c t Control I Z Yohimbine CD Clonidine Stimulus number Fig. 1. Effects of yohimbine (1-7 M) and clonidine (5 x 1-9 M) on A, mean discrete event (d.e.) amplitude and B, proportion of failures when [Ca]O = 1-1 mm. Control, n = 6; yohimbine, n = 6; clonidine, n = 4.

13 FACILITATION AT SINGLE SYMPATHETIC JUNCTIONS Conversely, yohimbine now elevates discrete event amplitude and reduces the probability of failure following all stimuli in a train, including the first. 69 DISCUSSION Families ofdiscrete events have so far been assumed to reflect the release of varying numbers of packets of transmitter from a single site (Blakeley & Cunnane, 1979; Blakeley, Cunnane & Petersen, 1982). Cunnane & Stjiirne (1982) however, argued that only events coinciding exactly in shape are due to release from the same site. Exact coincidence of shape is very infrequent, so by this criterion these authors argue that there must be a very large number of possible release sites, with each releasing only a single packet of transmitter extremely infrequently. Our observations do not support this suggestion. Differentiation introduces noise, and combined with the likely variability in transmitter packet size, and temporal jitter of the release, this is bound to lead to differences in the shape of events even if they are all due to the release of a single vesicle from the same varicosity. Occasional identity between some events does not prove that other slightly different events arise from different sources. There are events which do not fit into families, but within families the members are very similar in time course and the variations in amplitude are between a few, regularly spaced, preferred values. It is still most likely that families are due to varying transmitter release from one or a few sites, and changes within families during facilitation are consistent with this hypothesis. As facilitation develops, events at a particular latency get bigger because of a shift from a preponderance of small events to a preponderance of large ones. The modes of the amplitude distribution do not change. The likeliest explanation is that a variable number of packets of transmitter are released, and the mean quantal content increases. It is possible that the packets contributing to a single event do not all come from the same varicosity, but from a group of varicosities, presumably on a single axon, each intermittently contributing a single packet. The regular spacing of the modes of the amplitude distribution indicates that, in this case, packets from different sources must have indistinguishable effects at the recording electrode, so the whole group would behave effectively, as a single release site. We cannot at present distinguish between these alternatives, and although in the second case, the probability of release from a single varicosity is lower, it must still be much greater than the figures quoted by Cunnane & Stjiirne (1982) if coincident release of a significant number of packets is to be observed in trains of stimuli of the length used in these experiments. Once facilitation has developed, more non-family events are observed at varying latencies. Some of these events associate into pairs of similar time course and related in amplitude. They may reflect recruitment of extra release sites, close to the recording electrode and previously 'silent' or they may be due to the development during facilitation of large, multiquantal releases remote from the recording electrode, the smaller releases from these sites being lost in the noise of the recording system. In the former case the intermittence may be due to a normally low probability of release, or because the threshold for eliciting activity in their innervation is close to the submaximal stimulus employed in these experiments.

14 7 A. G. H. BLAKELEY, A. MATHIE AND S. A. PETERSEN If there is recruitment, then sites must vary in their probability of release. Some must be capable of multiquantal release (producing families), but others, perhaps morphologically distinct, must only intermittently release single packets. Perhaps the former are terminal varicosities, and the latter 'en passage' (Bennett, 1972). What is clear, however, is that the quantal content of major sites increases during facilitation-, and this is the principal change. Any recruitment of additional, lowerprobability sites is less important. Changing [Ca]o similarly affects the quantal content of release with, in single cells, fewer small events and more large events observed as the concentration is raised. In these experiments changes in quantal content from identifiable release sites mirror almost exactly the changes in junction potential amplitude which we have already described. There is little evidence of any significant change in recruitment of additional sites, except, as noted above, that more distant sites may become visible because of an increase in their quantal content. Similarly, when drugs are applied changes in quantal content parallel changes in junction potential amplitude (see Gillespie, 198). The a-adrenoceptor antagonist yohimbine, at a concentration known to affect facilitated junction potential amplitude (Blakeley, Cunnane & Petersen, 1981 a), and at [Ca]. = 2-1 mm, has no significant effect on the quantal content of the discrete events elicited by the first stimulus in a train, but leads to a significant elevation of quantal content following the fourth and fifth stimuli. If the [Ca]o is lowered, then the effects of the drug are seen on discrete events following all stimuli in a train. For later stimuli in a train, in the presence of yohimbine, there is an increase in the number of non-family events, but this increase is small and, as in the case of facilitation, may be explained either by the recruitment of close, low-probability sites, or by the detection of multi-quantal releases from remote, high-probability sites because their quantal content has increased. The major change remains in quantal content of the family events. Clonidine suppresses discrete event amplitude and increases the proportion of failures at both high and low [Ca] though its effects are greater when [Ca]o is high. The way in which facilitation is affected by changes in [Ca]o and a-adrenoceptor agonists and antagonists is not straight forward. Changing [Ca] produces opposite alterations in the effectiveness ofa-adrenoceptor agonists and antagonists. This would not be expected if they both had a single site of action. Perhaps there is more than one way in which prejunctional modulation may occur (Alberts, Bartfai & Stjirne, 1981). We have therefore observed a variety of manipulations producing similar changes in families of discrete events. The amplitudes at the modes of the amplitude distribution are unchanged, but there are alterations in the relative frequency of occurrence of the different amplitude classes. This is most easily explained by assuming that each family corresponds to the release of a varying number of packets of transmitter, and that the manipulations alter mean quantal content. Quantal content increases during facilitation, rises with [Ca]o, is elevated by treatment with a2-adrenoceptor antagonists and depressed by az2-adrenoceptor agonists. The M.R.C. are thanked for support. A.M. is an M.R.C. scholar.

15 FACILITATION AT SINGLE SYMPATHETIC JUNCTIONS 71 REFERENCES ALBERTS, P., BARTFAI, T. & STJXRNE, L. (1981). Site(s) and ionic basis of a-autoinhibition and facilitation of [3H]noradrenaline secretion in the guinea-pig vas deferens. J. Physiol. 312, BENNETT, M. R. (1972). Autonomic Neuromuscular Tran8mission. London: Cambridge University Press. BLAKELEY, A. G. H. & CUNNANE, T. C. (1979). The packeted release of transmitter from the sympathetic nerves of the guinea pig vas deferens: an electrophysiological study. J. Physiol. 2%, BLAKELEY, A. G. H., CUNNANE, T. C. & PETERSEN, S. A. (1981 a). a-adrenoceptors and facilitation in a sympathetic neuro-effector junction. J. Physiol. 316, 14P. BLAKELEY, A. G. H., CUNNANE, T. C. & PETERSEN, S. A. (1981 b). An electropharmacological analysis of the effects of some drugs on neuromuscular transmission in the vas deferens of the guinea pig. J. auton. Pharmac. 1, BLAKELEY, A. G. H., CUNNANE, T. C. & PETERSEN, S. A. (1982). Local regulation of transmitter release from rodent sympathetic nerve terminals. J. Physiol. 325, CUNNANE, T. C. & STJXRNE, L. (1982). Secretion oftransmitter from individual varicosities ofguinea pig and mouse vas deferens - All or none and extremely intermittent. Neuroscience 7, GILLESPIE, J. S. (198). Pre-synaptic receptors in the autonomic nervous system. In Handbook of Experimental Pharmacology, vol. 44, 1: Adrenergic activator8 and inhibitors, ed. SZEKERES, L., pp Berlin, Heidelberg, New York: Springer Verlag. HnST, G. D. S. & NEILD, T.. (198). Evidence of two populations of excitatory receptors for noradrenaline on arteriolar smooth muscle. Nature, Lond. 283, KATZ, B. & MILEDI, R. (1968). The role of calcium in neuromuscular facilitation. J. Physiol. 195,