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Vesicle; Sucrose gradient; Zonal centrifugation; Acetylcholine; Torpedo electric organ; Morphometric analysis
Abstract:
The presynaptic terminal vesicle population ofTorpedo electric organ is heterogeneous in size, consisting of two prominent subpopulations that comprise 80% of the total. The use of standard iso-osmotic sucrose gradients with zonal centrifugation to isolate vesicle fractions that co-localize with the acetylcholine (ACh) peak results in the recovery of: (1) 10% of the total estimated vesicle population; and (2) a single 68-nm diameter vesicle size class. The whereabouts of the major 90-nm subclass, which accounts for 60% of the total terminal population and which has long been considered to represent the resident ACh population, has been investigated. Assuming this subclass to have undergone severe osmotic stress, the effects of hypo- and hyper-osmotic salines, buffers and fixatives were examined and found to produce only negligible changes on vesicle size. Isolation of vesicles by hypo-osmotic shocking of synaptosomes purified on a Ficoll gradient, however, resulted in a reasonable approximation of the in situ distribution. As the iso-osmotic sucrose gradient procedure utilizes frozen blocks of electric tissue, this step is suspected of being involved in the loss, perhaps because of the slow freezing rates employed. These findings indicate that the 90 nm subclass is lost rather than transformed during isolation by sucrose gradient separation and that dimensionally, the cholinergic vesicle is a constant-sized and relatively stable structure.
