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SUMMARY1. Fluctuations in the latency of focally recorded end-plate currents were analysed to determine the time course of the probabilistic presynaptic process underlying quantal release evoked after single nerve stimuli at the frog neuromuscular junction.2. The early falling phase of the presynaptic probability function can be fitted by a single exponential over two orders of magnitude of quantal release rate. The time constant of the early falling phase is about 0-5 msec at 11°C, and increases with decreasing temperature with a Q10 of at least 4 over the range 1-12°C.3. After this early exponential fall, quantal release probability returns to control levels with a much slower time course.4. Conditioning nerve stimuli increase the magnitude and slightly prolong the early time course of release evoked by a test stimulus. When facilitation is calculated for matched time intervals following the conditioning and testing stimuli, it is found that the magnitude of the small, late residual tail of release is facilitated by a greater percentage than the magnitude of larger, early portions of release.5. These results are discussed in terms of the hypothesis (Katz & Miledi, 1968) that evoked release and facilitation are mediated by a common presynaptic factor which activates release in a non-linear manner.

SUMMARY1. Depolarization-induced voltage and conductance changes were studied in frog motoneurones in isolated, perfused spinal cord slices. Two types of afterhyperpolarization are observed following action potentials in normal Ringer, a fast afterhyperpolarization lasting 5-10 msec and a slow afterhyperpolarization lasting 60-200 msec. Both afterhyperpolarizations are mediated by an increased K+ conductance.2. The slow afterhyperpolarization and the conductance increase underlying it are selectively and reversibly inhibited by perfusion with solutions containing low [Ca2+] (< 0-2 mM) or the Ca2+ antagonists Mn2+ (1 mM) or Co2+ (5 mM), and are enhanced by perfusion with high [Ca2+].3. Addition of 2-5 mm tetraethylammonium ion (TEA+) to the perfusing solution prolongs the falling phase of the action potential and abolishes the fast afterhyperpolarization, but does not inhibit the slow afterhyperpolarization.4. When the voltage-dependent Na+ current is blocked by perfusion with TTX (10-M), intracellularly applied depolarizing current steps evoke fast and slow hyperpolarizations with kinetics and pharmacological sensitivities similar to those of the fast and slow afterhyperpolarizations, respectively. The fast hyperpolarization is maximally activated by brief, intense depolarizations, the slow hyperpolarization by prolonged, less intense depolarizations.5. These pharmacological and kinetic data demonstrate that in frog motoneurones the repolarization-fast afterhyperpolarization sequence and the slow afterhyperpolarization are produced by different K+ conductance * Present address. ELLEN F. BARRETT AND J. N. BARRETT systems. The fast K+ conductance activates rapidly on depolarization, decays rapidly on repolarization, and is TEA+ sensitive, while the slow K+ conductance activates and decays more slowly and is Ca2+-dependent.6. Motoneurones perfused with TEA+ and TEA often show a slow, regenerative depolarizing response to applied depolarizing currents. These regenerative depolarizations are probably produced by an influx of Ca2+, because they persist in isotonic CaCl2 and are blocked by Mn2+ or low [Ca2+]. The Ca2+-dependence of the slow afterhyperpolarization and the increase in slow afterhyperpolarization magnitude observed following the slow Ca2+ potentials suggest that a depolarization-evoked Ca2+ influx activates the K+ conductance underlying the slow afterhyperpolarization.7. Motoneurones in which the slow Ca2+ and K+ conductance systems have been enhanced by high [Ca2+] or blocked by Mn2+ show altered discharge patterns in response to intracellularly applied depolarizing current steps. Perfusion with twice normal [Ca2+] (4 mM) causes motoneurones to discharge more slowly at all current intensities, and reduces the slope of the 'steady-state' frequency-current relationship. Mn2+-perfused motoneurones exhibit fairly normal high-frequency discharge at the onset of the current step, but unlike normal motoneurones, do not discharge at frequencies below 60/sec. These data indicate that the slow Ca2+ and K+ conduct...

Changes in cytosolic and mitochondrial [Ca2+] produced by brief trains of action potentials were measured in motor nerve terminals using a rapidly scanning confocal microscope. Cytosolic [Ca2+] was measured using ionophoretically injected Oregon Green BAPTA 5N (OG‐5N). Mitochondrial [Ca2+] was measured using rhod‐2, bath loaded as dihydrorhod‐2. In response to 100‐250 stimuli at 25‐100 Hz the average cytosolic [Ca2+] showed an initial rapid increase followed by a much slower rate of increase. Mitochondrial [Ca2+] showed no detectable increase during the first fifteen to twenty stimuli, but after this initial delay also showed an initially rapid rise followed by a slower rate of increase. The onset of the increase in mitochondrial [Ca2+] coincided with the slowing of the rate of rise of cytosolic [Ca2+]. The peak levels of cytosolic and mitochondrial [Ca2+] both increased with increasing frequencies of stimulation. When stimulation terminated, the initial rate of decay of cytosolic [Ca2+] was much more rapid than that of mitochondrial [Ca2+]. After addition of carbonyl cyanide m‐chlorophenyl hydrazone (CCCP, 1‐2 μm) to dissipate the proton electrochemical gradient across the mitochondrial membrane, cytosolic [Ca2+] rose rapidly throughout the stimulus train, reaching levels much higher than normal. CCCP inhibited the increase in mitochondrial [Ca2+]. These results suggest that mitochondrial uptake of Ca2+ contributes importantly to buffering presynaptic cytosolic [Ca2+] during normal neuromuscular transmission.

SUMMARY Key components of vesicular neurotransmitter release, such as Ca2+ influx and membrane recycling, are affected by cytosolic pH. We measured the pH-sensitive fluorescence of Yellow Fluorescent Protein transgenically expressed in mouse motor nerve terminals, and report that Ca2+ influx elicited by action potential trains (12.5-100 Hz) evokes a biphasic pH change: a brief acidification (~13 nM average peak increase in [H+]), followed by a prolonged alkalinization (~30 nM peak decrease in [H+]) which outlasts the stimulation train. The alkalinization is selectively eliminated by blocking vesicular exocytosis with botulinum neurotoxins, and is prolonged by the endocytosis-inhibitor dynasore. Blocking H+ pumping by vesicular H+-ATPase (with folimycin or bafilomycin) suppresses stimulation-induced alkalinization and reduces endocytotic uptake of FM1-43. These results suggest that H+-ATPase, known to transfer cytosolic H+ into pre-fused vesicles, continues to extrude cytosolic H+ after being exocytotically incorporated into the plasma membrane. The resulting cytosolic alkalinization may facilitate vesicular endocytosis.