Stimulation-associated changes in frog neuromuscular junctions. A quantitative ultrastructural comparison of rapid-frozen and chemically fixed nerve terminals.

The ultrastructural effects of stimulation and subsequent rest were measured in frog neuromuscular junctions preserved by rapid-freezing and freeze-substitution, a method that minimizes fixation-associated membrane rearrangements. The effects were compared to those measured in junctions preserved by aldehyde fixation in order to identify artifacts attributable to the method of preservation. Effects of stimulation previously observed in tissue preserved by aldehyde fixation were evident in both the rapid-frozen and aldehyde-fixed neuromuscular junctions in the present study. Synaptic vesicles were reduced in number and cisternal profiles were increased. However, the sizes and shapes of the cisternae differed with the method of preservation. In addition, it was found that mitochondria underwent a change in shape with stimulation. This was accompanied by swelling in the fixed preparations, but not in the rapid-frozen ones. Fixation after stimulation also produced swelling of the nerve terminals, a stimulation-associated change not evident in preparations that were preserved by rapid-freezing. After stimulation and 60 min of rest, nerve terminals showed recovery towards control morphology, evidence that the effects of the stimulation parameters used in the study were reversible. This study, utilizing rapid-frozen material, confirms previous reports based on chemically fixed tissue that stimulation reduces the number of synaptic vesicles and increases the number of cisternae. The findings are in accord with the hypotheses of exocytotic neurotransmitter release and local recycling of synaptic membrane. In addition, the study emphasizes that accurate quantitative assessments of membrane redistribution in active secretory systems cannot depend on chemically fixed tissues. It also shows that mitochondria are susceptible to radical distortion by aldehyde fixatives, and that the degree of susceptibility differs with the physiological state of the tissue.