SUMMARY1. The use of end-plate current (e.p.c.) latency measurements to estimate the time course ofthe stochastic probabilistic process governing evoked release was investigated in the sciatic nerve--sartorius muscle preparation of the frog, Rana pipiens. We also examined the possibility that the release of a quantum depresses or enhances the subsequent release of additional quanta. Muscle end-plates were voltage clamped at 3-4 'C. Quantal release was restricted to a short, or localized, region of the nerve terminal using Ca2+-free, EGTA Ringer solution and a Ca2+-filled micropipette.2. The number of e.p.c.s containing 0, 1, 2, etc. quanta were totalled and compared to numbers predicted using Poisson's theorem. The differences between the actual and predicted numbers of events were not significant at the nineteen junctions studied (P < 0 05).3. The latency of the first quantum observed in several hundred e.p.c.s was measured and used to calculate an estimate, a,1(t), ofthe time-dependent, probabilistic process, a(t), governing all evoked quantal release (Barrett & Stevens, 1972 b). In three experiments, all quantal latencies were measured to obtain the actual ac(t). The al(t)function gave an excellent approximation of a(t) (P > 0 2), in real and simulated latency data. 4. The latency of the second quantum in the e.p.c.s was measured and used to provide another estimate, a2(t), ofa(t). The a2(t) function was lower (depressed) during the first few milliseconds of the evoked release period, relative to a, (t). The difference was significant (P > 0-01) in all experiments. Our measurement procedures were tested using computer-generated 'e.p.c.s' containing randomly occurring 'quanta'. These tests showed that the early depression was due to inadequate detection of the second quantum in the e.p.c.s.5. The effect of Sr2+ on evoked release was examined using double-barrelled pipettes containing 1 M-SrCl2 and CaCl2 solutions. The major result was that the durations of a1(t) and a2(t) were equally lengthened in Sr2+, relative to Ca2+. J. BALDO, I. S. COHEN AND W. VAN DER KLOOT

In the deep abdominal extensor muscles of spiny lobsters (Panulirus-pennicillatus), the common excitor axon of segment II was eliminated by intracellular injection of pronase. At 1 to 23 days after the operation, the quantal content of excitatory postsynaptic currents (EPSCs), elicited by stimulation of the specific excitor of the L1 muscle, was determined in a specific area of the L1 muscle, both in the operated and in the contralateral control side. The EPSCs in the operated muscles had about a 5 times higher quantal content compared to those in the controls, the change developing within 1 to 2 days after operation. In camera lucida drawings of preparations stained with methylene blue, increased branching of the remaining excitatory axon was obvious at more than 4 days after the operation. To investigate the possibility of contribution of central mechanisms (Rotshenker, S. (1979) J. Physiol. (Lond.) 292: 535-547). to this effect, the bundle of five axons to the deep abdominal extensors of segment II was cut immediately after injection of pronase into the common excitor axon. This caused a reduction of the quantal content of EPSCs and shrinking of the field of innervation in the operated L1 muscle as compared to the control. Therefore, axonal continuity or central connections seem to be necessary for the development of an increased innervation by the specific excitor to L1 after eliminating the common excitor axon. Possible postsynaptic effects of the elimination of the common excitor axon were controlled by recording synaptic single channel currents elicited by the excitatory transmitter glutamate, using the patch clamp method. These single current events did not show appreciable changes in operated L1 muscles. Therefore, the presynaptic strengthening effect on the nerve terminals of the specific L1 excitor is predominant after elimination of the common excitor axon.

shows that, as the true power approaches infinity, the maximum slope will be slightly above 4. The value for the slope usually found experimentally at the frog neuromuscular junction is also about 4. 4. The model does not fit the experimental data. The observed increases in evoked quantal release are higher than those predicted for the observed increases in spontaneous release. There are several possible explanations for the discrepancy. Treatments that increase m.e.p.p. frequency may also increase Ca2+ influx into the stimulated terminal. However, we prefer the explanation that there is a fraction of spontaneous release that is independent of the [Ca2+] in the terminal; if this is true the model might account for the data. 5. The model can account for a variety of puzzling experimental observations, including: (a) the effect of hypertonic solutions and of diamine in decreasing the slope in the relation between log (evoked quantal output) and log ([Ca2+]0Ut); (b) the slope of near 1 observed at the crustacean neuromuscular junction; (c) the decrease in the slope produced by treatment with botulinum toxin.

The amplitudes, time integrals, and half-decay times of miniature endplate currents (MEPCs) and of endplate currents (EPCs) were measured in preparations in low Ca2+ -high Mg2+ Ringer, in which the probability of the evoked release of more than one quantum was low. The measurements were made at 5 to 7, 11, and 15°C. There is no consistent difference in these properties of evoked and spontaneously released quanta at any of the temperatures.I. S. Cohen and W. Van der Kloot (( 1983) J. Physiol. (Lond.) 336: 335-344) compared the effects of temperature changes on MEPCs and on the endplate currents evoked by set amounts of iontophoretically applied ACh. The results suggested that the quantity of ACh/quantum was relatively unaffected by changes in temperature.This conclusion can now be extended to quanta released by nerve stimulation.One possible mechanism for quanta1 release is the diffusion of transmitter from the cytoplasm of the presynaptic terminal outward by way of a gated channel (de1 Castillo and Katz, 1954a; Israel and Dunant, 1975; Marchbanks, 1975). Three observations suggest that the gated channel is unlikely to be the mechanism for spontaneous quanta1 release. (1) Miniature endplate potential (MEPP) or miniature endplate current (MEPC) amplitudes are not appreciably altered when the membrane of the nerve terminal is depolarized by electrotonus (de1 Castillo and Katz, 1954b; Cooke and Quastel, 1973) or by elevated extracellular K+ (Linder and Quastel, 1978; Cohen and Van der Kloot, 1983). The change in membrane potential would affect the flux of acetylcholine (ACh) if it passed through a channel as a cation. (2) MEPP amplitudes are not appreciably affected by massive alterations of the osmotic pressure of the extracellular solution (Van der Kloot, 1978). The shrinking or swelling of the nerve terminal should at least momentarily alter the [ACh] in the terminal and, therefore, also change the efflux through a gated channel. (3) The quantity of ACh per quantum is probably the same over a temperature range from 4" to 25°C (Cohen and Van der Kloot, 1983). The open time of known gated channels is markedly temperature dependent. These observations make it unlikely that spontaneous quantal release occurs by way of a gated channel and favor the vesicle hypothesis, which is consistent with each of the three results. However, in some instances there are notable differences between quanta released spontaneously and those released following stimulation.

As reported by Landau & Nachshen (1975), a decrease in extracellular pH at the frog neuromuscular junction leads to an increase in min.e.p.p. frequency. 2. Decreasing the extracellular pH still increases the min.e.p.p. frequency when the bathing Ringer contains 10 mM-Ca2+, in place of the usual 2-5 mM. At the mammalian neuromuscular junction, the elevated Ca2+ blocks the effect of the pH change on the min.e.p.p. frequency (Hubbard, Jones & Landau, 1968). 3. In Cl--free solution (isethionate or methylsulphate substitution) min.e.p.p. frequency is no longer a monotonic function of decreasing pH. Instead there is an optimum pH for spontaneous release between pH 6-6 and 8-6. 4. This suggests that in Cl- containing Ringer min.e.p.p. frequency increases with increasing extracellular acidity because there is a change in the PCl of the nerve terminal leading to a depolarization. In agreement with this idea,in low Ca2+ Ringer, acid pH has little effect on the min.e.p.p. frequency. 5. Decreasing the intracellular pH by raising PCO2 produces substantial increases in the min.e.p.p. frequency. The effects are much greater than the effects of equal changes of H+ in the extracellular solution. 6. Possible explanations for the effects of increased PCO2 are discussed. Although release of Ca2+ from mitochondria or other unknown effects of intracellular pH change or molecular CO2 are possible, the results do give some support to the hypothesis that an important step in transmitter release involves an electrostatic repulsion between fixed membrane surface charges on the transmitter containing vesicles and the inner face of the nerve terminal. The surface charge density would be decreased by a lower pH in the axoplasm, and this would increase the rate of spontaneous transmitter release, in agreement with the observations.