Brain Research, 169 (1979) 111-119 © Elsevier/North-Holland Biomedical Press

111

DEPRESSION BY C A L C I U M OF S P O N T A N E O U S T R A N S M I T T E R RELEASE AT THE MAMMALIAN NEUROMUSCULAR JUNCTION

YONG I. KIM and DONALD B. SANDERS Department of Neurology, University of Virginia School of Medicine and the University of Virginia Jerry Lewis Neuromuscular Center, Charlottesville, Va. 22908 (U.S.A.)

(Accepted September 28th, 1978)

SUMMARY The effect of extracellular calcium on the frequency of miniature end-plate potentials (MEPPs) at the rat neuromuscular junction was studied using 5 different concentrations of K + (2.5, 5, 10, 15 and 20 mM). At low and normal levels of K ÷ (2.5 and 5 mM), incremental increase of Ca 2+ concentration (0.1, 0.5, 1, 2, 3, 4, 7 and 10 mM) produced a slight monotonic increase in MEPP frequency. In 10 or 15 m M - K +, the frequency of MEPPs increased monotonically as Ca 2+ concentration was raised from 0.1 to 4 mM, but was unaltered or declined slightly with further increase of Ca 2+ concentration. In 20 m M - K +, the frequency reached its maximum at 1 mM-Ca 2+ and progressively decreased as Ca ~+ concentration was increased to 10 mM. Thus, the relationship between Ca 2+ concentration and MEPP frequency was monotonic at the lower levels of K + but became distinctly non-monotonic with increased K +. This nonmonotonic effect of Ca 2+ is consistent with the hypothesis that there are two opposing effects of increasing Ca 2+ concentration on transmitter release: (i) an increased Nernst equilibrium potential for Ca 2+ which causes an increase in Ca 2÷ influx and a consequent rise in MEPP frequency and (ii) increased electrostatic screening of negative surface charges near Ca z+ channels which produces an effective hyperpolarization across the channels and a consequent decrease in Ca z+ influx.

INTRODUCTION The important role played by calcium ions in transmitter release has been established at many chemical synapses including the neuromuscular junction, at which calcium is essential in the step between depolarization of the motor nerve terminal and release of acetylcholine quanta12,14. Experiments of Katz and Miledi 1~ on the squid giant synapse provided evidence that the influx of calcium ions, which accompanies the depolarizing events in presynaptic terminals, leads to an increase in intracellular Ca z÷

112 concentration and in turn facilitates transmitter release. More direct evidence on the action of calcium at the synapse was obtained by Miledi 2a who injected calcium iontophoretically into the nerve terminals and showed the resulting depolarization of the postsynaptic membrane. Extracellular calcium ions are also known to participate as a charge carrier in generation of the action potentials6,1a, ~2 and in addition influence the excitability of axonal or somatic membranes16,19, 20. It is a well known phenomenon that membrane excitability is increased and nerves or somata fire spontaneously when the external calcium concentration is reduced10,17,aL Frankenhaeuser and Hodgkin 5, in their voltage clamp study of the squid giant axon, found that the threshold for activation of sodium (G•a) and potassium (GK) conductancO 1 depends on the concentration of extracellular calcium. They reported that with changes of [Ca2+]o the plot of peak GNa or GK against membrane potential is shifted along the voltage axis such that an increased [Ca 2~]0 is equivalent to hyperpolarization of the nerve membrane. Thus, with increased [Ca" ~]0, a greater membrane depolarization is required to elicit the normal changes in Gt~a and GK. In frog skeletal muscle, Costantin 4 extended these observations and confirmed that the threshold of GNa is more sensitive to a change in external divalent cation concentration than that of GK or the contraction threshold. More recently, Hille et alp measured the shifts of the voltage-dependent activation of sodium channels in frog myelinated nerve fibers as a function o f p H and of different divalent and monovalent ion concentrations. Their results indicate that the measured voltage shifts fit quantitatively well with the Gouy-Chapman-Stern surface charge model, suggesting that the shifts result from changes in surface potentials on the outer layer of the membrane. In frog muscle membranes, the voltage dependence of sodium channels upon external pH and [Ca2+]o was found to be the same as that in frog myelinated nerve fibers z. Thus, the relationship between membrane excitability and external calcium ions has been demonstrated, directly or indirectly, in a variety of biological membranes. The increased excitability of nerve membrane in reduced [Ca 2~]0 can now be explained by the change in the electrostatic effect of negative surface charges on the membrane as described by Hille et alP. When [Ca2+]o is reduced, the fixed negative charges on the outer surface of the membrane are less well screened by calcium and this produces an 'effective' depolarization of the membrane. This effective depolarization is sensed by the voltage-sensitive gating mechanisms of the membrane, giving rise to more frequent opening of the gated Na +, K +, and Ca 2+ channels. Conversely, if the concentration of external calcium is increased, there will be an effective hyperpolarization across the ionic channels and the membrane excitability will be decreased. The above observations on the effect of extracellular calcium in excitable membranes suggest that an increase in [Ca2+]0 may exert two opposing effects on the gating mechanism of Ca 2+ channels: (i) an increase in calcium equilibrium potential, Eta, as calculated by the Nernst equation, which promotes Ca 2÷ influx, and (ii) an increased electrostatic screening of the negative surface charges which produces an effective hyperpolarization across the channels and depresses Ca 2+ influx. Therefore, in a quantitative measurement of the effect of Ca z+ on transmitter release at the neuromuscular junction, these two separate effects of calcium ions should be taken into account. In

113 particular the screening effect of Ca 2+ may be an important factor in the evoked or spontaneous transmitter release. Recently, Mattbews and Wickelgren21 described the non-monotonic effect of Ca 2+ on the frequency of miniature end-plate potentials (MEPPs) at the frog neuromuscular junction and interpreted the results in terms of the screening effect of calcium ions. Our present work is an extension of those studies and deals with the two opposing effects of calcium on transmitter release at the rat neuromuscular junction. MATERIALS A N D METHODS

The experiments were carried out on isolated forelimb flexor digitorum muscles from female Lewis rats weighing 150-250 g. The muscle was removed under sodium pentobarbital anesthesia (intraperitoneal injection of 45 mg/kg) and maintained at 25-27 °C in a plexiglass recording chamber having a volume of less than 1 ml. In each experiment, the musc:e was continuously perfused with solutions which were passed through the chamber at a constant speed by means of a push-pull perfusion pump (Holter RD075). Rapid solution changes were achieved by replacing the solution flowing into the pump with a new one. The change was considered complete when 15 ml of new solution had passed through the chamber. The composition of standard Ringer's solution was as follows (in mM/l): 135 NaC1, 15 NaHCOa, 1 Na2HPO4, 5 KC1, 2 CaC12, 1 MgC12 and 11 glucose with resulting pH of 7.2-7.4. To maintain the osmolarity equal to that in the standard Ringer's solution, [Na+]0 was altered in the test solution to compensate osmotically for changes in [K+]0 and [Ca2+]0. All solutions used in the experiments were continuously bubbled with 95 % 03 and 5 ~o COs. Resting membrane potentials (RMPs) and MEPPs were recorded intracellularly in the end-plate region with conventional glass micropipettes filled with 3 M KCI. Resistance of the micropipettes ranged from 8 to 25 Mfl. In each experiment, recordings were made from a single end-plate during exposure to eight different concentrations of Ca 2+ (0.1, 0.5, 1, 2, 3, 4, 7 and 10 mM) while [K+]0 was kept constant. Unless otherwise specified, the absolute MEPP frequency in each test solution was determined 5 rain after the completion of each solution change by counting the number of MEPPs produced within a 2 min period. Counting of the MEPPs was done with the aid of an automated MEPP measuring circuit and Hewlett-Packard 9810A programmable calculator interfaced with the intracellular measurement system1,26. In some experiments, MEPPs were recorded on an FM tape recorder (A. R. Vetter Co., Model B) and analyzed on playback at a slower speed. In order to allow comparison of data from different end-plates, MEPP frequency in each test solution was expressed as the ratio (F/F0) of the frequency in test solution (F) to that in normal buffer (F0). No end-plate was accepted which had a MEPP rise time of greater than 1 msec or a steady RMP of greater than --70"mV. RESULTS The effects of varying [Ca2+]0 on spontaneous MEPP frequency were studied

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Fig. 1. (A): effects of varying extracellular calcium concentration ([Ca2+]0) on the relative MEPP frequency (F/F0) at 5 different concentrations of external potassium ([K÷]o) at the rat neuromuscular junction. F/F0 is plotted on a logarithmic scale. Each data point represents the mean of three endplates from different animals. Vertical bars indicate :k 1 S.D. of observation. (B): the non-monotonic effect of extracellular calcium on the frequency of MEPPs at the rat neuromuscular junction. While the external potassium concentration was kept at 20 mM, this end-plate was successively exposed to 0.1, 1, and 10 mM-Ca 2+ solutions. Note the depression of spontaneous transmitter release at 10 mMCa2+.

using 5 different c o n c e n t r a t i o n s o f extracellular p o t a s s i u m (2.5, 5, 10, 15, a n d 20 m M ) (Fig. 1A). This allowed us to d e t e r m i n e the relationship between F / F o a n d [Ca~+]0 at 5 different levels o f nerve t e r m i n a l d e p o l a r i z a t i o n . A l t h o u g h the p r e s y n a p t i c d e p o l a r i z a tion could n o t be m e a s u r e d directly at the n e u r o m u s c u l a r j u n c t i o n , it was possible to estimate it by m e a s u r i n g the changes in R M P at the p o s t j u n c t i o n a l m e m b r a n e s . W h e n [K÷]0 was elevated f r o m its n o r m a l value o f 5 m M to 10, 15 a n d 20 m M , the e n d - p l a t e

115 membrane was increasingly depolarized, as would be expected from the progressive drop in the potassium equilibrium potential, EK. The degree of depolarization observed from the end-plate region was found to be comparable to that estimated on the basis of Goldman constant field equations 7. It was assumed that a similar depolarization occurred in the nerve terminal membranes. For example, a depolarization of the endplate of about 20 and 25 mV occurred upon increase in [K÷]0 to 15 and 20 mM respectively. Coincident with this depolarization was a sharp rise in MEPP frequency which reached a new steady state within 5 min after the solution change. No noticeable change in RMP occurred when [Ca2+]0 was varied between 0.1 and 10 mM at a constant [K+]0. With the normal concentration of potassium (5 mM) in the extracellular medium, MEPP frequency showed a steady monotonic increase as the calcium concentration was increased from 0.1 to 10 mM (Fig. 1A). On the average there was approximately a doubling in the frequency at 10 mM-Ca z+ (F/F0 = 2.3 4- 0.2) compared to that at the normal concentration (2 mM). Thus no depressing action of calcium on the spontaneous MEPPs was observed at the higher levels of [Ca2+]0. In general the rate of increase in F/F0 was found to be greater at [Ca2+]0 of 0.1 to 2 mM than that seen at higher [Ca2+]0 (2 to 10 mM) and this effect was manifest at all potassium concentrations used. When the nerve terminal membrane was slightly hyperpolarized by reducing [K+]0 to 2.5 mM, the pattern of frequency change with increased [Ca2+]0 was similar to that just described, but the percentage increase of the frequency was smaller (F/F0 = 1.8 4- 0.1 at 10 mMCa2+). This suggests that under the hyperpolarized conditions, the voltage-dependent gating mechanisms of calcium channels may become less sensitive to a change in [CaZ+]0. This result is in agreement with previous studies of MEPP frequency at the frog neuromuscular junction 3,25. With 10 or 15 mM-K +, the relationship between external calcium concentration and relative MEPP frequency became increasingly non-monotonic and there was a slight depression of spontaneous transmitter release at 7 or 10 mM-Ca 2+. At 15 mM-K +, the frequency of MEPPs increased montonically as [Ca2+]0 was increased from 0.1 (F/F0 = 2.1 4- 0.09) to 7 mM (9.3 -4- 2.0) but declined slightly at 10 mM (8.5 4- 1.9). A similar pattern of frequency change was observed with 10 mM-K +. When the external potassium concentration was increased further to 20 mM, the MEPP frequency reached its maximum at 1 mM-Ca z+ (27.0 4- 3.2) and decreased progressively as Ca 2+ concentration was increased to 10 mM. At 10 mM-Ca 2+, F/F0 dropped to 13.5 4- 4.5, or 50 ~o of the maximum value shown at 1 mM. Therefore, the increase in the spontaneous release of transmitter was clearly reversed at the high calcium concentration and the overall response was distinctly bi-phasic (See Fig. 1B). A simple experiment demonstrated in a different fashion the non-monotonic effect of calcium on transmitter release (Fig. 2). With the external potassium concentration constant at 20 mM throughout, we investigated the time course of the calciuminduced changes in MEPP frequency (F) by changing [Ca2+]0 from 0.1 to 1 mM and then back to 0.1 mM. The resulting frequency-time curve was then compared with that obtained by changing [Ca2+]0 from 0.1 to 10 mM and then back to 0.1 mM. It should be noted that the perfusion pump used in this experiment achieved a gradual, rather than instantaneous, change of Ca z+ concentration in the medium bathing the muscle. As

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was measured during the change of [Caa+]0from 0.1 to 1 mM and during washout, whereas the lower time-course was measured during the change of [Ca2+]0from 0.1 to 10 mM and during washout. The MEPP frequency (F) was calculated from the number of MEPPs recorded during each 10 sec interval• shown in the upper curve of Fig. 2, as the calcium concentration increases gradually from 0.1 to 1 mM, MEPP frequency rises steadily with time but there is no bi-phasic pattern of the frequency response. However, when the concentration of extracellular calcium was increased from 0.1 to 10 mM (lower curve of Fig. 2), frequency increased briefly and then declined. When 0.1 mM Ca 2+ solution was again pumped into the bath,

117 there occurred a monotonic reduction in the frequency as shown in the upper curve, whereas the pattern of MEPP frequency change in the lower curve again exhibited a temporary rise. We interpret these results to mean that: (i) as the calcium concentration in the bath was slowly increased from 0.1 to 10 raM, the concentration around the endplate studied passed 1 mM at a certain time and at this point the MEPP frequency rose to a maximum, producing the peak shown in the lower curve, and (ii) when 0.1 mM Ca 2+ solution was re-introduced into the bath, the calcium concentration decreased from 10 mM and again approached 1 mM, giving rise to a similar peak in the frequency. By contrast, when the muscle was exposed to 1 mM Ca 2+ solution, the calcium concentration in the bath did not exceed 1 mM during solution change and no bi-phasic increase in the frequency occurred. This observation is consistent with the previous result demonstrated in Fig. 1A, that the maximum of F/F0 takes place at 1 mM-Ca 2+. It in turn confirms the non-monotonic relationship between the extracellular calcium concentration and the spontaneous release of neuromuscular transmitter. DISCUSSION

Our present results fit qualitatively with the proposed hypothesis of the two opposing effects of raising Ca z+ concentration on the transmitter release: (i) increased Nemst equilibrium potential for Ca 2÷ which causes an increase in Ca 2+ influx and a consequent rise in MEPP frequency, and (ii) increased electrostatic screening of negative surface charges near Ca 2+ channels which produces an effective hyperpolarization across the channels and a consequent decrease in Ca 2+ influx and transmitter release. The theoretical results of these two opposing effects of extracellular calcium are illustrated in Fig. 3 in which the calcium equilibrium potential, Eca and the voltage shift in sodium activation, Eshttt are plotted as functions of [Ca2+]0. Eca, which is given by the Nernst equation for a calcium electrode, was calculated assuming a constant intracellular calcium concentration for each [Ca2+]o and with the further assumption that Eta at 2 mM-Ca 2+ is equal to +85 mV, which was based on the measured reversal potential of the early Ca z+ currents in voltage-clamped crayfish muscle cellss. The 110

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118 screening effect of the calcium ions on the gated calcium channels is represented by Eshif.,., the voltage shift in the activation of Gr~a in nodes of Ranvier of frog myelinated nerve fibers as measured by Hille et al. 9. We were unable to find a similar measurement of Eshm based on the activation of the calcium conductance, Gc~, in the literature, but experimental evidence indicates that in some membrane preparations, there exist qualitative similarities between the activation-inactivation kinetics in Gc~ and G~a (refs. 8, 24). Thus it is probable that the gating behavior of calcium channels may share common properties with that of sodium channels. The qualitative explanation for the non-monotonic effect of calcium on the spontaneous transmitter release is as follows. As the external calcium concentration is increased, the negative surface charges on the outside of the nerve terminal membrane are increasingly screened by calcium, resulting in an increase in Eshm in the positive (or hyperpolarizing) direction. As a result of this screening action of Ca z÷, the voltagedependent gating mechanisms of calcium sense an effective hyperpolarization, which tends to depress the opening of Ca 2÷ channels. However, in the range of calcium concentration from 0.1 to 1 raM, the decrease in Ca 2~ influx brought about by the increased Eshif~ is more than compensated for by increased Eca promoting the entry of calcium ions into the channel. Therefore, the net Ca 2÷ influx increases and the MEPP frequency rises steadily. With further increases in the calcium concentration, the balance between Eshm and Eca changes in the opposite direction and the increase in Eta is not sufficient to overcome the progressive increase in Eshift. As a result, the net Ca 2 ~ influx decreases and the frequency of MEPPs begins to drop. However, this bi-phasic characteristic of transmitter release was not seen at normal or subnormal levels of potassium concentration (5 and 2.5 mM) because the kinetics of gated calcium channels are voltage-dependent and far less sensitive to a change in [Ca2+]0 at potentials close to the resting level than at the depolarized levels. Therefore, at low potassium concentrations, the change in effective membrane potential arising from Esb~rt has no significant effect on Ca z+ influx, and Eca appears to be more influential on MEPP frequency than Eshirt. The bi-phasic relationship between external calcium concentration and spontaneous transmitter release at the rat neuromuscular junction reflects the results that might be expected from the two opposing effects of calcium ions and suggests that a complete description of the effect of Ca 2+ on transmitter release should take into account both of the effects of Ca 2+ as proposed in this paper and by Matthews and Wickelgren 21. In future experiments, it would be of particular interest to establish the effects of calcium ions on the evoked transmitter release, i.e. the end-plate potential amplitude and quantal content. In addition, it would be useful to study possible non-monotonic effects on transmitter release of Mg 2+, which is known to antagonize the Ca z+ entry into the presynaptic terminals lz. By doing so, the integral role of calcium ions in membrane excitatory events can be better understood. ACKNOWLEDGEMENT We thank Dr. B. Hille at the Department of Physiology and Biophysics, University of Washington for providing the numerical data on the voltage shift of sodium activation shown in Fig. 3. Part of the results in this paper has been presented at the 62nd

119 F A S E B A n n u a l Meeting is. This w o r k was s u p p o r t e d in p a r t by N I N C D S G r a n t NS12905 a n d a center g r a n t from the M u s c u l a r D y s t r o p h y Association, Inc. REFERENCES 1 Anders, R. J., Anne, A., and Sanders, D. B., A microcomputer system for on-line acquisition of neuromuscular data, Proc 1st Annual Symposium on Computer Application in Medical Care, IEEE Computer Soc., (1977) 263-267. 2 Campbell, D. T. and Hille, B., Kinetic and pharmacological properties of the sodium channel of frog skeletal muscle, J. gen. Physiol., 67 (1976) 309-323. 3 Cooke, J. D. and Quastel, D. M. J., The specific effect of potassium on transmitter release by rat motor nerve terminals, and its inhibition by calcium, J. Physiol. (Lond.), 228 (1973) 435-458. 4 Costantin, L. L., The effect of calcium on contraction and conductance thresholds in frog skeletal muscle, J. Physiol. (Lond.), 195 (1968) 119-132. 5 Frankenhaeuser, B. and Hodgkin, A. L., The action of calcium on the electrical properties of squid axons, J. Physiol. (Lond.), 137 (1957) 218-244. 6 Geduldig, D. and Junge, D., Sodium and calcium components of action potentials in the Aplysia giant neurone, J. Physiol. (Lond.), 199 (1968) 347-365. 7 Goldman, D. E., Potential, impedance and rectification in membrane, J. gen. Physiol., 27 (1943) 37-60. 8 Hence,k, M. and Zachar, J., Calcium currents and conductances in the muscle membrane of the crayfish, J. Physiol. (Lond.), 268 (1977) 51-71. 9 Hille, B., Woodhull, A. M. and Shapiro, B. I., Negative surface charge near sodium channels of nerve: divalent ions, monovalent ions, and pH, Phil. Trans. roy. Soc. B, 270 (1975) 301-318. 10 Hodgkin, A. L., The Conduction of the Nervous Impulse, Sherrington Lecture VII, Liverpool University Press, Liverpool, 1967. 11 Hodgkin, A. L. and Huxley, A. F., A quantitative description of membrane current and its application to conduction and excitation in nerve, J. Physiol. (Lond.), 117 (1952) 500-544. 12 Hubbard, J. I., Jones, S. F. and Landau, E. M., On the mechanism by which calcium and magnesium affect the release of transmitter by nerve impulses, J. Physiol. (Lond.), 196 (1968) 75-86. 13 Junge, D.,Divalentionsaschargecarriers. In Nerve and Muscle Excitation, SinauerAssoc.,Sunderland, Mass., 1976, pp. 101-112. 14 Katz•B.•TheRe•ease•fNeuralTransmitterSubstances•Sherringt•nLectureX•Liverp••lUniversity Press, Liverpool, 1969. 15 Katz, B. and Miledi, R., Tetrodotoxin-resistant electric activity in pre-synaptic terminals, J. Physiol. (Lond.), 203 (1969) 459-487. 16 Kim, Y. I., Control of Pacemaker Potential Oscillations by Calcium in Molluscan Neurons, Ph.D. Thesis, Cornell University, Ithaca, New York, 1977. 17 Kim, Y. I. and Kim, M., Modulation of repetitive action potentials in molluscanneurons stimulated with alternating currents, Brain Research, 122 (1977) 361-366. 18 Kim, Y. I. and Sanders, D. B., Dual effects ofextracellular calcium on the transmitter release at the rat neuromuscularjunction, Fed. Proc., 37 (1978) 578. 19 Koketsu, K., The role of calcium in membrane excitation, Proc. Int. Unionphysiol. Sci., 4 (1965) 521-564. 20 Manery, J. F., Effects of Ca ions on membranes, Fed. Proc., 25 (1966) 1804-1810. 21 Matthews, G. and Wickelgren, W. O., On the effect of calcium on the frequency of miniature endplate potentials at the frog neuromuscularjunction, J. Physiol. (Lond.), 266 (1977) 91-101. 22 Meech, R. W. and Standen, N. B., Potassium activation in Helix aspersa under voltage clamp: a component mediated by calcium influx, J. Physiol. (Lond.), 249 (1975) 211-239. 23 Miledi, R., Transmitter release induced by injection of calcium ions into nerve terminals, Proc. roy. Soc. B, 183 (1973) 421-425. 24 Standen, N. B., Properties of a calcium channel in snail neurons, Nature (Lond.), 250 (1974) 340-342. 25 Takeuchi,A. and Takeuchi, N., Changes in potassium concentrationaround motor nerve terminals, produced by current flow, and their effects on neuromuscular transmission, J. PhysioL (Lond.), 155 (1961) 46-58. 26 Wilkinson, E. C., Chang, N., Cobb, E. E., Kim, Y. I. and Johnson, R. N., A simplified programmable calculator conversion for on-line measurement of miniature end-plate potential amplitudes, Med. Biol. Eng. Comput., (1979) in press.