Motor nerve terminals in cutaneous pectoris muscles of the frog Rana pipiens release more transmitter and form synapses with higher levels of effectiveness than do those in sartorius muscles. Neuromuscular junctions from these two muscles were compared in the electron microscope to search for ultrastructural correlates of differences in transmitter release and synaptic effectiveness. The following measurements were made from cross-sections of junctions with known levels of effectiveness: (a) the presence of active zones, the presumed sites of transmitter release, (b) active zone size, (c) the perimeter, cross-sectional area, height and width of nerve terminals, (d) number of mitochondria, (e) vesicle density, and (f) the extent to which Schwann cells wrap terminals. Nerve terminals in the two muscles did not differ in size, shape or vesicle density. The more strongly releasing cutaneous pectoris terminals did, however, have significantly larger active zones due to deeper invagination of the terminal into the postsynaptic gutter and lesser interposition of Schwann cell processes between presynaptic and postsynaptic membranes. Cutaneous pectoris terminals also contained more mitochondria, presumably to supply the greater energy demand imposed by high release levels.

No abstract

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.

Transmitter release from frog motor nerve terminals occurs at specialized sites on the nerve terminal called active zones (AZs). We have used a low calcium (0.1 nM) saline treatment to disrupt AZ structure and correlated these changes with alterations in transmitter release from the nerve terminal. Exposure to 0.1 nM free calcium saline for 3 h caused many individual AZs to break into two or three pieces, apparently unorganized particles drifted free of the AZ array, and the normally ordered alignment of AZ particles was loosened. Despite these forms of disruption in AZ organization, physiological function remained remarkably normal. Although the size of the endplate potential recorded in response to a single nerve stimulus was little affected, paired-pulse facilitation and tetanic potentiation were significantly increased. Synaptic depression was not apparent during the tetanus, but was revealed following the cessation of the stimulation. The results are consistent with the hypothesis that 0.1 nM calcium treatment detached AZ segments from the anchoring molecules that normally hold these proteins in alignment with other synapse-specific molecules. We propose that the ordered AZ organization serves to bring the calcium channels that regulate transmitter release in close proximity to other proteins that are critical to the modulation of release, especially during periods of high frequency stimulation. We hypothesize that the drifting AZ segments, although capable of apparently normal transmitter release, may not be tightly coupled with the intracellular calcium handling proteins that normally restrict the time that calcium ions have to act on the transmitter release apparatus following each action potential.

Neurotransmitter release from different parts of frog motor nerve terminals is often non-uniform. There is a decrease in release efficacy from the distal regions of frog motor nerve terminal branches. Since release is thought to occur near the double arrays of large intramembranous particles that constitute the pre-synaptic active zones (AZs), we have examined quantitatively the proximal-distal distribution of AZ structure, using a novel freeze-fracture technique that produces replicas of large fractions of terminals, including the region of nerve entry. This enables us to know the proximal distal orientation of each branch. From 23 end-plates we have obtained fractures of 72 branches. For 27 of these branches we have obtained continuous fractures both greater than 25 microm in length and with sufficient information to determine their proximal distal polarity. Only a few of these branches showed a marked distal decrease in AZ length/unit length of terminal, while several junctions had short regions (5-10 microm), either proximally or distally, that exhibited amounts of AZ that were substantially greater or smaller than the mean value for that terminal branch. The terminal area, post-synaptic gutter width and nerve terminal width all exhibit some distal decline concomitant with the distal tapering of nerve terminal branches. AZ length tends to have the least decline compared to the other parameters. Thus, the vast majority of frog motor nerve terminal branches do not display a significant proximal-distal gradient in the amount of AZ structure / microm terminal length. The present data do not provide an obvious ultrastructural correlate for the distal decline in transmitter release that some authors have observed.