Aspects of turnover and biogenesis of synaptic vesicles at locust neuromuscular junctions as revealed by zinc iodide-osmium tetroxide (ZIO) reacting with intravesicular SH-groups.

Abstract

Retractor unguis nerve muscle preparations from the locust were subjected to the zinc iodide-osmium tetroxide reaction (ZIO) after pre-fixation in glutaraldehyde. Applied for 18 h at 4.degree. C in the dark, ZIO reacts at pH 4.2-5.0 fairly selectively with the matrix of synaptic vesicles. About 53% of the vesicles are completely stained and 4%, partially. The percentage of ZIO-positive vesicles increased to nearly 90% and reduced to 4% or less by pretreatment with SH-protecting (dithiothreitol) or SH-blocking (N-ethylmaleimide, p-chloromercuriphenyl sulfonic acid) and SH-oxidizing (azodicarboxylic acid-bis-dimethylamide) reagents, respectively. Stimulation of the motor nerve at 20 Hz for 7 min, partially fatiguing synaptic transmission, reduces the number of vesicles per square micrometer of terminal area by .apprx. 52%; 2 min of rest restores this number to its pre-stimulation level. These changes are chiefly accounted for by changes in the number of completely ZIO-positive vesicles. Two min after the end of stimulation, partially ZIO-positive vesicles are 3 times more frequent than before. With all experimental conditions, the average volume of vesicles was ZIO-negative < partially ZIO-positive < completely ZIO-positive. The average volume of ZIO-positive vesicles was almost unaffected by stimulation; that of ZIO-negative vesicles decreased by 25% immediately after stimulation, increasing with subsequent rest to the initial level after 1 h. ZIO probably demonstrates intravesicular protein(s) containing SH-groups and the completely ZIO-positive vesicles probably represent mature ones ready to be used for transmitter release. How the ZIO reaction differentiates between different developmental stages of vesicles which could arise from the smooth endoplasmic reticulum is discussed.