Changes in the structure of neuromuscular junctions caused by variations in osmotic pressure

Neuromuscular junctions of the frog, Rana pipiens, were examined for structural modifications produced by exposure to increased and reduced osmotic pressure (pi). Preparations exposed to increased pi for varying lengths of time were fixed with either OSO4-Veronal with and without calcium, glutaraldehyde-phosphate, or glutaraldehyde-formaldehyde-phosphate as primary fixatives. The greatest difference between the fixatives was seen in preparations exposed to increased pi for 5 min, corresponding to the time when miniature endplate potential frequency is highest. The 5-min OSO4 calcium-free preparations appeared comparatively normal, while those fixed with OSO4 and 2 mM CaCl2 or aldehyde-phosphate had wide infoldings of the presynaptic membrane and a reduced number of synaptic vesicles. Aldehyde-phosphate had the same effect on mouse diaphragm. Another series of frog preparations were conditioned to elevated pi and then returned to normal Ringer's for varying times before fixation in OSO4-phosphate. Preparations fixed 2 min after their return to normal Ringer's showed marked disruption of the presynaptic membrane as well as apparently rupturing vesicles. If fixed after 10 min, terminals were depleted of vesicles although the presynaptic membrane had returned to its normal position and appearance.