analogous to that recorded in other innervated muscles has been recorded University of Melbourne, Australia

Transcription

1 446 J. Phy8iol. (1962), 160, pp With 8 text-ftgure8 Printed in Great Britain SPONTANEOUS POTENTIALS AT SYMPATHETIC NERVE ENDINGS IN SMOOTH MUSCLE BY G. BURNSTOCK AND MOLLIE E. HOLMAN From the Departments of Zoology and Physiology, University of Melbourne, Australia (Received 2 August 1961) The observations described in this paper concern the spontaneous electrical discharge which has been recorded intracellularly from smooth muscle cells of the guinea-pig vas deferens. These potentials were first observed during studies of the junction potentials in the vas deferens, in response to stimulation of the hypogastric nerve. They have been described briefly in a previous report (Burnstock & Holman, 1961 a). The similarity between this discharge in smooth muscle and that recorded from postjunctional regions in other tissues (Eccles, 1961) suggested to us that it may have been due to a spontaneous release of transmitter from sympathetic nerve endings. This paper is concerned with the nature of the discharge under normal conditions. A preliminary account of some of the results has been given elsewhere (Burnstock & Holman, 1961 b). The effects of denervation and of the depletion of local stores of catecholamines by reserpine will be dealt with in a further report (Burnstock & Holman, 1962). METHODS The preparation used throughout this work was the isolated vas deferens of the guineapig, innervated by the hypogastric nerve. The preparation, mounted in a constant-temperature bath at 350 C was perfused continuously with a modified Krebs's solution (Holman, 1958). The arrangements for stimulation and recording have been described previously (Burnstock & Holman, 1961a). Throughoutthese experimentsitwasfound necessaryto use relativelyhigh-resistaalce microelectrodes (15-50 MQ). The thickness of the base line varied in different experiments, being partly due to mains 'pick-up' and partly to inherent electrode noise. In most impalements no attempt was made to resolve spontaneous potentials whose amplitude was less than 1 mv. RESULTS General features of the spontaneous discharge of small potentials A spontaneous discharge of small potentials which appears to be analogous to that recorded in other innervated muscles has been recorded in single smooth muscle cells of the isolated vas deferens of the guinea-pig.

2 SPONTANEOUS POTENTIALS IN SMOOTH MUSCLE 447 This discharge has been recorded in all cells which were impaled satisfactorily, as judged from the amplitude of the action potential recorded in the same cell (70-90 mv). The spontaneous potentials were not associated with any development of tension or shortening of the smooth muscle cell. 10 mv MVT Seconds mv it I I -1 A B C 200 rnsec 100 msec Fig. 1. Intracellular recordings of spontaneous potentials in smooth muscle oells of the vas deferens. Records from three different preparations. Note the difference in time scales. Figure 1 shows somle typical records of the discharge. It can be seen that the amplitude and time course of the individual potentials varied greatly. The over-all pattern of the discharge was, however, similar from cell to cell, and from preparation to preparation. Records under normal conditions have been obtained from over 100 cells in 40 preparations. The frequency of the smaller potentials was always much greater than that of the larger ones. In general, the larger the amplitude the faster was the rate of depolarization. The larger potentials usually consisted of a simple depolarization followed by repolarization, whereas the smaller potentials were occasionally complex in shape (Fig. 1B). A characteristic 'burst' of potentials of the larger, sharper type was sometimes observed during the initial stages of the penetration of a smooth muscle cell. Similar 'bursts' were noted by Burke (1957) during impalement of slow skeletal muscle fibres of the frog. He suggested that the larger 29.2

3 448 G. BURNSTOCK AND MOLLIE E. HOLMAN potentials arose in the vicinity of the micro-electrode, owing to damage or mechanical stimulation of local nerve endings. Since the frequency of the smaller potentials was not increased during this period, Burke felt that they probably arose from more distant nerve endings. This argument might also be applied to our findings on the vas deferens, although the geometry of the latter system is far more complex. In most of our experiments impalements became stabilized after sec, and the mean frequency of the discharge remained fairly constant for periods of up to 45 min. The discharge was unaffected by the presence of atropine (L0-4). The amplitude of the potentials was, however, greatly reduced by yohimbine and piperoxane. Increases in the tonicity of the perfusing solution by the addition of sucrose did not have the same dramatic effects on the frequency of the discharge as those which have been described for striated muscle (Furshpan, 1956). In our experiments the frequency of the potentials showed a transient increase to about double the normal rate only when the tonicity of the medium was increased to 3 times normal by the addition of sucrose. The discharge continued when the calcium concentration of the perfusing solution was increased up to 4 times normal. It seems unlikely that the potentials could be due to the spontaneous excitation of nerve fibres. Amplitude of the spontaneous potentials Figure 2 shows frequency distributions of the amplitude of the spontaneous potentials recorded in two typical cells from different preparations. It can be seen that any calculation of the mean amplitude would be arbitrary, since this value would be dependent on the thickness of the base line, which determined the amplitude of the smallest potentials resolvable in any particular experiment. In Fig. 2A, for example, all potentials greater than 1-5 mv were tabulated and the mean amplitude obtained was 3-33 mv. The shape of the histograms in Fig. 2 is similar to those calculated for other muscles where the activity of many different nerve endings, situated at varying distances from the micro-electrode, could be recorded simultaneously. Our records, and the 'amplitude' histograms constructed from them, bear a close resemblance to those of the slow fibres of the frog (Burke, 1957), crustacean striated muscle fibres (Dudel & Kuffler, 1961) and chick slow fibres (Ginsborg, 1960). 'Amplitude' histograms from these preparations are in marked contrast with those of muscle fibres with localized nerve endings, e.g. the skeletal neuromuscular junction (Fatt & Katz, 1952). The amplitudes of the spontaneous potentials in the latter case show a bell-shaped or 'Gaussian' distribution about a mean value. Although little is known of the passive electrical properties of smooth

4 SPONTANEOUS POTENTIALS IN SMOOTH MUSCLE 449 muscle, there is a good deal of indirect evidence that some form of electrical coupling exists between neighbouring cells (Biilbring, Burnstock &Holman, 1958; Goto, Kuriyama & Abe, 1960). C. L. Prosser (personal communication) recently observed length constants of about 1 mm (10 cell lengths) 12 A D ~~~~10 C _o > D lo~~~ mv i o G z mv 6_7_8 9n lb 6( j'o 5( 1t mv 10 Fig. 2. Distribution of the amplitudes of spontaneous potentials in two cells from different preparations. Hatched area indicates width of base line. See text for explanation.

5 4504. BURNSTOCK AND MOLLIE E. HOLMAN in cat intestinal muscle. It is likely that sympathetic nerve endings are widely distributed among the smooth muscle cells of the vas deferens (Burnstock & Holman, 1961 a). Thus it would seem that the larger spontaneous potentials recorded in the vas deferens arose in cells at, or close to, the point of impalement, whereas the smaller, flatter potentials were due to nerve endings some distance away from the micro-electrode. Closer examination of the amplitude histograms for this smooth muscle, however, all showed a characteristically uneven distribution. For example, in Fig. 2A there appeared to be groups of potentials ranging about values of 3 and 4 mv with a suggestion of further groupings around some of the higher values. This tendency for the larger potentials to fall into groups of Fig. 3. Two examples of 'giant' spontaneous potentials from the same cell. preferred values was seen in all the cells impaled. The value of the groups, however, differed from cell to cell. During some of the longest impalements the 'mean' value of a group did not appear to be constant but might shift by 1 or 2 mv over a period of 20 min or more. Thus the existence of discrete groups was often more apparent in histograms drawn up for relatively short periods. Insufficient results are available at present to warrant a statistical treatment of this problem. Figure 3 shows two examples of some of the largest spontaneous potentials we have recorded in the vas deferens, the potential in Fig. 3B being 20 mv in amplitude. Several 'giants' of 15 mv or more were usually observed during the course of a 20 min impalement. It is unlikely that they could be due to a random coincidence of smaller potentials. The frequency of potentials in the 3-7 mv range was low and the probability of a double or triple coincidence of these potentials would be too remote to account for the rate of appearance of the 'giants'. Furthermore, most of the 'giants' showed a single phase of depolarization followed by smooth

6 SPONTANEOUS POTENTIALS IN SMOOTH MUSCLE 451 repolarization, even when examined with a sweep speed allowing resolution of intervals of less than 10 msec (see Fig. 1 C). Single 'giant' potentials were never observed to give rise to an action potential and contraction. It has been shown previously that the threshold membrane potential for the firing of an action potential is around 40 mv (Burnstock & Holman, 1961 a). Cells in which 'giants' of 20 mv were recorded always had high resting potentials (about 70 mv). Thus it is probable that the maximal depolarization attained by a 'giant' was less than the threshold depolarization for that cell. Time course of spontaneous potentials The spontaneous discharge was recorded on a fast time base (moving 'spot', moving film) in eight experiments. Typical records are shown in Fig. 1 B and C. The shape of the larger potentials (those exceeding 5 mv) was usually simple, consisting of a phase of depolarization lasting from 30 to 50 msec followed by an exponential repolarization, which was usually complete in 100 msec. The total duration of the smaller potentials was similar to that of the larger ones. The maximal rate of depolarization of the larger potentials (5-15 mv) varied somewhat from cell to cell. The maximal value recorded in the present experiments was 0 45 V/sec. The majority of the potentials showed maximal rates of rise varying between 0-15 and 0 30 V/sec. After the first msec the repolarization phase was found to be exponential with a single time constant. Figure 4 shows the exponential plots for three typical potentials. Time constants varied from 25 to 50 msec, with a majority of fibres giving values of msec. It may be that this figure is an indication of the time constant of this smooth muscle membrane. These values are large compared with those found for most other excitable tissues (Shanes, 1958). However, C. L. Prosser (personal communication) has recently found values of about 100 msec for the time constant of cat intestinal muscle. If the repolarization phase of the spontaneous potentials is due to the passive decay of a superimposed depolarization, the transmitter substance must have been inactivated relatively quickly, as is the case at most of the synapses studied so far (Eccles, 1961). On the other hand, repolarization may have been determined by the time course of the inactivation of the transmitter. Brown & Gillespie (1957) calculated that the noradrenaline released during sympathetic stimulation of cat spleen was destroyed in about 100 msec. Frequency Measurement of the interval between successive potentials was again limited by the degree of resolution permitted by the noise level in any

7 452, G. BURNSTOCK AND MOLLIE E. HOLMAN particular experiment and, further, by the speed of recording. Since the frequency of the discharge was low, most experiments were recorded on relatively slow-moving film, as in Fig. 1A. Figure 5 shows the frequency distribution ofintervals measured from the same experiment asthat analysed in Fig. 2A. The solid curve was drawn according to the equation, Avt -t n= NT exp Ty A msec Fig. 4. Exponential plots of the repolarization phase of three spontaneous potentials. A and * from the same cell (time constants 32 and 30 msec, respectively); 0 from a different cell (time constant 27 msec). Semi-log scale. where n is the number of intervals between time t and t + At, N is the total number of intervals and T the mean interval (again a somewhat arbitrary figure). The close correspondence between the observed distribution and the theoretical curve may be taken as evidence that the discharge was random; that is, the probability of the occurrence of a potential was not related to the occurrence of previous potentials (Fatt & Katz, 1952). Fatt & Katz (1952) pointed out that this test for the 'randomness' of the discharge should be interpreted with some caution. They emphasized that

8 SPONTANEOUS POTENTIALS IN SMOOTH MUSCLE 453 in such a set of observations there was not apparent interaction between the various contributing units. This analysis does not prove whether or not the individual units themselves were discharging in a completely random manner. In the present case a random distribution was indicated if the intervals were analysed without any consideration of the amplitude of the individual potentials (n) Seconds Fig. 5. Distribution of intervals between successive potentials in a series of 525 potentials (same cell as that analysed in Fig. 2 ). n is the number of intervals between t and t +At, where At = 0 4 sec. The mean interval, T = 3-6 sec. Curve At -t plotted according to n = N T exp T In another test successive intervals were plotted against the preceding intervals. The points appeared to be scattered quite randomly. These graphs were divided into ten different ranges of 'intervals' and 'preceding intervals'. Mean values were calculated for these ranges in order to show if there was any tendency for short intervals to follow short ones, or to follow long intervals, or vice versa. In most instances this test again failed to show up any departure from randomness. If the time of appearance of potentials of a similar magnitude was considered, a further feature of the discharge became apparent. Similar potentials often occurred in pairs or triplets, separated from each other by a few seconds. A hundred seconds or more might then elapse before the same potential appeared again. Unfortunately these frequencies are relatively low, and technical difficulties have so far prevented us from obtaining sufficiently long impalements (greater than 50 min) to allow a more detailed analysis of these phenomena.

9 4544. BURNSTOCK AND MOLLIE E. HOLMAN The effect of nerve stimulation When single or repetitive stimuli of about 1 msec duration were applied to the hypogastric nerve, junction potentials were recorded in all the smooth muscle cells, although the configuration of the junction potentials varied greatly (Burnstock & Holman, 1961 a). If the strength ofstimulation was low, small junction potentials were observed which consisted of a slow in the last four sweeps. flat wave of depolarization barely detectable from the noise level and much smaller than many of the potentials occurring spontaneously in the same cell (Fig. 6). If the strength of stimulation was increased the magnitude and rate of rise of the junction potentials increased, frequently in a stepwise manner. Maximal stimuli usually gave rise to junction potentials which greatly exceeded the amplitude of the small potentials. At inter-

10 SPONTANEOUS POTENTIALS IN SMOOTH MUSCLE 455 mediate strengths of stimulation irregularly shaped junction potentials w.pre sometimes observed. Figure 7 is an example of such a case and shows junction potentials (Fig. 7A and B) alongside some of the small potentials occurring in the same cell in the absence of nerve stimulation (Fig. 7C). It is possible that the junction potentials could have been built up of many of the small potentials which were characteristic of that cell. A Bj C Seconds Fig. 7. Records showing the relation between small potentials and junction potentials in the same cell. Record A shows the junction potentials in response to six successive stimuli (note facilitation). Record B shows the first three junction potentials of another train of stimuli given severalminutes after A. Record C shows some typical small potentials recorded in the same cell. Junction potentials in response to the initial 6-8 stimuli of a train always showed a progressive increase in amplitude (Figs. 6, 7 A). This facilitation was not continuous but always occurred in steps. The junction potentials in response to successive stimuli (after facilitation was complete) always showed fluctuations in their configuration (see Fig. 8). Such fluctuations occurred even when the strength of stimulation was maximal. This suggests that the components which made up the junction potentials varied from time to time, perhaps because not all the nerve endings were activated each time the nerve was stimulated. The maximal rate of depolarization of junction potentials in response to single stimuli was often lower than that of the largest potentials occurring spontaneously in the same cell. For the largest junction potentials, however, it was of the same order of magnitude (i.e V/sec). During high-frequency stimulation ( pulses/sec) faster rates ofdepolarization of up to 1 V/sec were observed. The rate of repolarization of the junction potentials was generally lower than that of the small potentials.

11 4564. BURNSTOCK AND MOLLIE E. HOLMAN Since the small potentials recorded in any individual smooth muscle cell vary in amplitude from being indistinguishable from the noise level up to more than 10 mv it has not been possible to prove statistically whether the junction potentials are composed solely of small potentials. Evidence that both are due to the same transmitter will be described in a further paper (Burnstock & Holman, 1961 b). Furthermore, we cannot say at this stage whether or not the small potentials originate in a 'quantal' fashion, niv I Seconds Fig. 8. Junction potentials in response to repetitive stimulation photographed after facilitation was complete. The stimulus strength in the upper record was twice that of the lower record. Note fluctuation in configuration of successive potentials. i.e. that they are due to the release of the same-sized packets of transmitter at different distances from the point of impalement. Nevertheless, our results are in accordance with the hypothesis that the small potentials are due to the release of transmitter from sympathetic nerve endings and that the junction potentials are compounded of many small potentials. The mechanism of transmission of excitation at sympathetic nerve endings may be basically similar to that occurring at other neuro-effector junctions (Eccles, 1961). DISCUSSION Some of the features of the spontaneous discharge of small potentials recorded from the sympathetically innervated smooth muscle of the vas deferens have been described. It has been suggested that this discharge is analogous to that arising in other neuro-effector organs and represents the spontaneous release oftransmitter from sympathetic nerve endings.whether the spontaneous release of transmitter from autonomic nerve endings plays any role in the initiation of activity in smooth muscle such as that of the gastro-intestinal tract remains to be seen. The present results suggest that this is unlikely, since the spontaneous discharge in the smooth-muscle

12 SPONTANEOUS POTENTIALS IN SMOOTH MUSCLE 457 cells of the vas deferens never gave rise to an action potential or to contraction. The amplitude of the small potentials recorded in this smooth muscle is a good deal larger than is that of the spontaneous discharge recorded in striated muscles. This may be due to the small dimensions of the smooth muscle cells. Katz & Thesleff (1957) calculated the mean amplitude for miniature end-plate potentials (MEPPs) in prostigmine-treated striated muscle fibres as a function of input resistance, and hence fibre diameter. For a fibre diameter of l,0u, having an input resistance of 10 MQ, the mean value of the MEPP was estimated to be 5-6 mv. The maximum diameter of smooth muscle cells is certainly less than 1O Pu. Values quoted in the literature for the input resistance of smooth muscle fibres all exceed 10 MQ (Daniel & Singh, 1958; Burnstock, Prosser & Barr, 1959). Thus it would not seem necessary at this stage to assume that the packets of 'transmitter' causing the spontaneous discharge in the vas deferens are any more effective in producing depolarization than those of other tissues. The similarity of the spontaneous discharge recorded in smooth muscle to that recorded from other muscles showing distributed innervation has been emphasized. A similar discharge has, however, been recorded from the end-plate of 'twitch' fibres of the frog, after denervation (Birks, Katz & Miledi, 1960). These authors conclude-d that the potentials in this case were due to the release of acetylcholine from Schwann cells which had replaced the nerve terminals, making intimate contact with the muscle membrane in the end-plate region. This discharge resembled that of smooth muscle, since it was not markedly increased in frequency when the tonicity of the perfusing solution was increased by sucrose. It seems likely, however, that the response to hypertonic solutions depends on the permeability of the 'presynaptic' membrane (Furshpan, 1956). It may be that the C fibre terminals are somewhat permeable to sucrose. Nevertheless, the possibility remains that spontaneous discharge in smooth muscle may have originated from the Schwann cell network which surrounds the terminal branches of autonomic C fibres or even from the 'interstitial cell network' (Richardson, 1960). It is difficult to say at this stage how many spontaneous units may be associated with any individual smooth muscle cell or whether these units can be considered as individual nerve endings. If one assumes that the largest potentials recorded in a cell are due to units discharging on to that cell, the individual units must have an extremely slow rate of spontaneous activity. It may be that the largest potentials-the 'giants'-are due to the release of large pre-formed aggregates of transmitter, as suggested by Liley (1956, 1957) for rat diaphragm. If this is the case, some of the more frequent smaller spontaneous potentials may have been due to the

13 458 G. BURNSTOCK AND MOLLIE E. HOLMAN ' quantal' release of transmitter on to that cell. In many experiments, one or perhaps two or three characteristic spontaneous potentials appeared to occur repeatedly with very little change in their amplitude or shape. This gave rise to the uneven appearance of the histogram showing the frequency distribution of amplitudes, i.e. the tendency of potentials to fall into 1-4 groups. If the assumption that each group represents the discharge from an individual nerve ending is correct, the rate of discharge of transmitter must be relatively slow, one quantum being released every sec. This rate contrasts markedly with that of the normal skeletal neuromuscular junction (mammalian), where the rate of discharge is about 1P5/sec (Boyd & Martin, 1956). It is hoped that impalements of longer duration will be possible in future experiments and that this point can be clarified by further observations. Previous workers have used several statistical tests to demonstrate the quantal content of the post-junctional response to nerve stimulation (del Castillo & Katz, 1956). Such tests are only possible if the activity of a single unit can be identified or if the innervation is limited to a specific area of the muscle fibre. In our case the junction potentials recorded in any one cell appear to be made up of contributions from endings some distance away. Crustacean muscle fibres also have distributed innervation. The problem of demonstrating the contribution of a spontaneous unit to the junction potential was solved by Dudel & Kuffler (1961) by recording -from a single unit extracellularly. Our present attempts to identify such a unit among the tightly packed smooth muscle cells of the vas deferens have not been successful. There is one other approach to this problem which will be discussed in a future report (Burnstock & Holman, 1962). Attempts to cut the postganglionic nerve supply to the vas deferens by removing lengths of hypogastric nerve have generally resulted in only 'partial' denervation. However, a number of cells have been encountered where the spontaneous discharge no longer appeared to be characteristic of distributed innervation but was 'bell-shaped' and resembled more closely that of the skeletal neuromuscular junction. Under these conditions it may be possible to investigate the activity of a single nerve ending. SUMMARY 1. A spontaneous discharge of small potentials has been recorded intracellularly from smooth muscle cells of the guinea-pig vas deferens, innervated by the hypogastric (sympathetic) nerve. 2. The spontaneous potentials were unaffected by atropine but their amplitude was greatly reduced by yohimbine and piperoxane. They continued in the presence of 4 times normal Ca2+ concentration.

14 SPONTANEOUS POTENTIALS IN SMOOTH MUSCLE The amplitude of the majority of potentials ranged from less than 1 mv up to 12 mv. Occasional 'giants' of up to 22 mv were recorded in most cells. 4. The frequency of occurrence of the smaller potentials greatly exceeded that of the larger. This gave rise to a skew-shaped distribution curve for the amplitudes of potentials in any one cell. 5. The intervals between successive potentials were shown to be random. When the intervars between potentials of a similar magnitude were considered, there was some suggestion of a lack of randomness in the discharge. 6. The total duration of individual potentials ranged from 100 to 150 msec. 7. The rate of depolarization of potentials exceeding 5 mv was greater than that of the smaller potentials. 8. The repolarization phase of the larger potentials was exponential. Time constants varied from 25 to 50 msec. 9. Stimulation of the hypogastric (sympathetic) nerve gave rise to junction potentials which appeared to be compounded of many of the spontaneous potentials. 10. The properties of the spontaneous potentials arising in this smooth muscle have been compared with those recorded at other neuro-effector junctions. We wish to thank the following for their support of this work: the Department of Health, Education and Welfare, Public Health Service, National Institutes ofhealth,u.s.a.(research Grant B-2902); the National Health and Medical Research Council ofaustralia; the Rockefeller Institute; and Mr J. W. Walker for technical assistance. REFERENCES Bmxs, R., KATZ, B. & MILEDI, R. (1960). Physiological and structural changes at the amphibian myoneural junction, in the course of nerve degeneration. J. Physiol. 150, BoYD, I. A. & MuARTnI, A. R. (1956). Spontaneous sub-threshold activity at mammalian neuromuscular junctions. J. Physiot. 132, BROWN, G. L. & GrLLESPIE, J. S. (1957). The output of sympathetic transmitter from the spleen of the cat. J. Phy8iol. 138, BtJLBRING, E., BuiRNSTOCK, G. & HOLMAN, M. E. (1958). Excitation and conduction in the smooth muscle of the isolated taenia coli of the guinea-pig. J. Physiol. 142, BuIJRE, W. (1957). Spontaneous potentials in slow muscle fibres of the frog. J. Phy8iol. 135, BURNSTOCK, G. & HOLXAN, M. E. (1961a). The transmission of excitation from autonomic nerve to smooth muscle. J. Phy8iol. 155, BURNSTOCK, G. & HOLXAN, M. E. (1961b). Spontaneous potentials in smooth muscle cells of the vas deferens of the guinea-pig. Aue8t. J. Sci. 24, 190. BURNSTOCK, G. & HOLMAN, M. E. (1962). Effect of denervation and of reserpine treatment on transmission at sympathetic nerve endings. J. Phy8iol. 160, BURNSTOCK, G., PROSSER, C. L. & BARR, L. M. (1959). Membrane resistance and conduction in smooth muscle. Fed. Proc. 18, 21.

15 460 G. BURNSTOCK AND MOLLIE E. HOLMAN DANIEL, E. E. & SINGH, H. (1958). The electrical properties of the smooth muscle cell membrane. Canad. J. Biochem. Physiol. 36, DEL CASTILLO, J. & KATZ, B. (1956). Biophysical aspects of neuromuscular transmission. Progr. Biophy8. 6, DUDEL, J. & KUFFLER, S. W. (1961). The quantal nature of transmission and spontaneous miniature potentials at the crayfish neuromuscular junction. J. Physiol. 155, EccLEs, J. C. (1961). The mechanism of synaptic transmission. Ergebn. Physiol. 51, FATT, P. & KATZ, B. (1952). Spontaneous sub-threshold activity at motor nerve endings. J. Phy8iol. 117, FusHPAN, E. J. (1956). The effects of osmotic pressure changes on the spontaneous activity at motor nerve endings. J. Phy8iol. 134, GINSBORx, B. L. (1960). Spontaneous activity in muscle fibres of the chick. J. Physiol. 150, GOTO, M., KURIYAN!A, H. & ABE, Y. (1960). Myo-myo-junction potential and transmission of excitation in uterine smooth muscle. Proc. imp. Acad. Japan, 36, HOLMAN, M. E. (1958). Membrane potentials recorded with high resistance micro-electrodes; and the effects of changes in ionic environment on the electrical and mechanical activity of the smooth muscle of the taenia coli of the guinea-pig. J. Physiol. 144, KATZ, B. & THESLEFF, S. (1957). On the factors which determine the amplitude of the 'miniature EPP'. J. Phy-iol. 137, LILEy, A. W. (1956). An investigation of spontaneous activity at the neuromuscular junction of the rat. J. Phy8iol. 132, LILEY, A. W. (1957). Spontaneous release of transmitter substance in multiquantal units. J. Physiol. 136, RICHARDSON, K. C. (1960). Studies on the structure of autonomic nerves in the small intestine, correlating the silver irnpregnated image in light microscopy with the permanganate fixed ultrastructure in electron microscopy. J. Anat., Lond., 94, SHANES, A. M. (1958). Electrochemical aspects of physiological and pharmacological action in excitable cells. Pharmacol. Rev. 10,