Effect of depolarization by potassium proprionate on synaptic vesicle diameter and distribution of the snake motor endplate

Abstract

The morphological effects of elevated potassium (K+) at the neuromuscular junction of garter snake twitch muscle fibers were examined in vitro. Elevated K+ depolarizes the motor neuron terminals and causes quantal release of acetylcholine. It has been shown in amphibians that prolonged (30-60 min) depolarization with high K+ depletes terminals of synaptic vesicles ; but causes no change in the average diameter of the vesicles. In the snake, however, we have shown previously that miniature endplate currents (MEPCs) are present at high frequency even after 1-2 hours of exposure to high K+ . This indicates that in the snake, synaptic vesicles are not totally depleted by depolarization with high K+. In this study, we examined the effect of depolarization with high K+ on the distribution and diameter of synaptic vesicles.Intercostal muscle preparations were exposed for 15 minutes to Ringer's + solution containing either a control (1.2mM) or high (70mM) concentration of K+ Preparations were fixed immediately in 1% glutaraldehyde for 15 minutes and washed with Millonig's buffer. The whole mount muscle preparations were then fixed for 1 h in 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.2; rinsed three times in fresh buffer and post-fixed in 2% OsO4 in the same buffer for 30 min, rinsed in fresh buffer and dehydrated in a graded ethanol series to 100% and then transferred into a 2% solution of uranyl acetate for 20 min to en-block stain the whole mounts. The preps were then transferred back to 100% ethanol to complete the dehydration and embedded in a mixture of Embed 812/Araldite 502 (Hard) between two microscope slides that had been precoated with Formen- Trennmittel Liquid Release Agent and polymerized overnight at 70°C.The whole mounts were then removed from the microscope slides and examined on a compound light microscope where the neuromuscular junctions on the twitch muscle fibers could be identified, marked and cut out of the preps. These pieces of the whole mounts were then remounted into a slot on the end of a pre-cast Embed-Araldite block with a drop of fresh resin and polymerized.