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SUMMARY1. The calcium dependence of spontaneous transmitter release from nerve terminals of different lengths was examined at neuromuscular junctions in frog muscle. Miniature endplate potential (MEPP) frequency was positively correlated with the endplate potential (EPP) quantal content and was dependent on external Ca2+. The higher the resting MEPP frequency in a 0-25 mM-Ca2+ Ringer solution, the greater the dependence on external Ca2+. MEPP frequency in all terminals dropped to approximately the same low level in a Ca2+-free Ringer solution containing EGTA. This suggests that terminals with higher release levels have a larger Ca2+ influx at rest.2. Several tests were done to try to characterize the mode of Ca2+ entry into resting terminals. w-Conotoxin (w-CgTx) blocked evoked release and reduced MEPP frequency, but not as effectively as zero Ca2+-EGTA Ringer solution. Some component of Ca2+ influx thus appears to enter through channels insensitive to w-CgTx. Tetrodotoxin (TTX) did not affect MEPP frequency, indicating that the Ca2+ did not enter through TTX-sensitive Na+ channels that might be opening spontaneously at rest. Hyperpolarization of the terminal by reducing the K+ in the Ringer solution caused no consistent differences in MEPP frequency, suggesting that the Ca2+ influx is relatively insensitive to small changes in membrane potential around the resting level. Strong buffering of the Ringer solution with citrate, to overwhelm any differences in Ca2+ buffering within different junctional clefts, had no significant effect on the MEPP frequency.3. Evidence that the Na+-Ca2+ exchanger helps set the internal Ca2+ level was obtained. Reduction of the Na+ concentration in the Ringer solution caused increases in MEPP frequency ranging from 6 to 440%. However, these changes were not correlated with resting MEPP frequency, hence differences in MEPP frequency probably are not the result of differences in Na+-Ca2+ exchanger function in terminals having a uniform Ca2+ leak.4

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The orderly arrays of intramembranous particles (IMPs) found in the p-face of freeze-fracture replicas of the frog neuromuscular junction ('active zones') are believed to be involved in transmitter release. Some or all of the particles represent voltage-dependent Ca2+ channels. Since there is a great heterogeneity in the amount of transmitter released by different frog motor nerve terminals we sought to determine whether active zone (AZ) structure displayed a similar heterogeneity by using a novel freeze-fracture procedure providing large, intact replicas containing significant portions of motor nerve terminals from the cutaneous pectoris muscle of the frog, Rana pipiens. Using only junctions in which more than 50 AZs or more than 50 microm of nerve terminal were included in the fractures, we measured AZ length, AZ intramembranous particle density, terminal width at each AZ, space between AZs, the angle of AZ orientation with respect to the longitudinal axis of the nerve terminal, exposed pre-synaptic nerve terminal surface area and a calculated value for mean AZ length per unit terminal length. The analysis led to the following conclusions. There is an approximate 5-fold range in mean AZ length/micrometre terminal length. Terminal width is a good predictor of AZ length. Particle density does not vary significantly within a given AZ, nor between AZs from the same or different junctions. The distance between AZs is not related to AZ length, i.e. shorter AZs are no more or less likely to be closer to the adjacent AZ. The probability of release from any AZ on action potential invasion is small. If most of the IMPs are Ca2+ channels, either the probability of channel opening or the efficacy of triggering release is very low or both. That the variability in release efficacy in different terminals is much greater than ultrastructural variability in terminals suggests that regulation of release is dominated by physiological processes that do not have obvious ultrastructural correlates. On the other hand, the apparent excess of AZ relative to the number of vesicles released indicates that the amount and variability in amount of AZ is important in ways that need to be elucidated.

Previous anatomical studies suggest that androgen regulates synapse elimination in the androgen-sensitive levator ani(LA) muscle of the rat. Androgen treatment beginning on postnatal day 7 (P7) prevents some of the normal loss of multiaxonal innervation in this muscle. The present study used physiological techniques to measure the number and size of LA motor units during the synapse elimination period in muscles from normals, and castrates treated with either testosterone propionate or oil. The number of increments in LA twitch tension as nerve stimulation intensity increased, a measure of the number of motor units, was the same at the end (P28) of synapse elimination as near the beginning (P7) of this process. This result indicates that motoneuronal cell death does not contribute to synapse elimination in the LA. Moreover, androgen during this period did not influence the number of LA motor units. In contrast, between P7 and P28, there was a dramatic decline in the size of LA motor units, as indicated by a decrease in the percentage of twitch or tetanus tension of individual motor units relative to the maximal twitch or tetanus tension of the whole muscle. In addition, androgen treatment of castrated males during this period prevented some of the normal decline in the size of LA motor units. Estimates of the number of inputs per LA muscle fiber derived from the number of LA motor units and their average size indicate that androgen maintains polyneuronal innervation in the LA muscle. This finding supports previous anatomical studies suggesting that androgen can prevent synapse elimination in this muscle. The strength of LA synapses was also examined by measuring the tetanus: twitch ratio of individual motor units and by measuring the safety margin of LA synapses. Both measurements indicated that the average strength of LA synapses increases during synapse elimination. Moreover, androgen appeared to spare synapses from elimination without increasing their strength, since androgen-treated muscles generally had larger motor units but the same mean tetanus:twitch ratio and safety margins as untreated LA muscles except at P28, when synapses in androgen-treated LA muscles had appreciably lower safety margins than normal. These results suggest that androgen regulates synapse elimination through a mechanism(s) independent of synaptic strength.

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This paper describes the extent of release and terminal variability among normal frog sartorius neuromuscular junctions and seeks physiological correlates for these differences. Terminal length varied over approximately a 10-fold range, quantal content and release per unit terminal length ("release efficacy") over much larger ranges. For purposes of comparison of different junctions, release efficacy in a Ringer's containing 0.25 mM Ca2+ was determined in all cases. In a Ringer's containing 0.1 mM Ca2+, tetanic stimulation causes a buildup of evoked release and of miniature endplate potential (mEPP) frequency. The mEPP frequency at the end of the tetanus is proportional to the evoked release level. Following the tetanus, the mEPP frequency declines in a multiexponential fashion, with the 2 longest decay phases, representing augmentation and posttetanic potentiation (PTP), both having time constants that are positively linearly correlated with the synaptic release efficacy. Longer or higher-frequency tetanic stimulation resulted in a longer time course of decay of mEPP frequency. In a Ca2(+)-free/EGTA Ringer's, tetanic stimulation causes no evoked release, but does lead to an increased mEPP frequency, presumably due to a buildup of free Ca2+ displaced from internal stores by the Na+ influx. Following the tetanus, the mEPP frequency declines to resting level with a time constant that is essentially the same for all junctions, regardless of their release efficacy in Ca2(+)-containing Ringer's. These findings indicate that stronger terminals have a greater influx of Ca2+ per unit length during action potential invasion, but that in the absence of external Ca2+, tetanic stimulation results in comparable release of Ca2+ from internal stores in all terminals and comparable accumulation of Ca2+ in some large compartment, the subsequent emptying of which determines the time course of PTP.

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