Poststimulatory endocytosis, microvesicle repopulation and changes in smooth endoplasmic reticulum in nerve endings of the median eminence superfused in vitro

Mediobasal hypothalami of adult rats were superfused in vitro. A single 5 min pulse of 60 mM KCl-containing medium was infused, followed by either 15, 30, 45, 60 or 75 min superfusions with standard medium. In some experiments, 5 or 10% dextran was added followed by a 15min recovery. Morphologically, two recovery phases were recognized. The early phase (15–30 min) was characterized by two features: (1) A clear-cut increase in the quantitiy of large, pleomorphic vacuoles occupying the axoplasm of nerve endings; these vacuoles were observed to be connected to caveolae of the same diameter in the axolemma and they were either coated or uncoated. (2) Progressive increase in the quantity of microvesicles (synaptic vesicles) from an initial depleted state. The vacuoles were found to contain dextran aggregates. Microvesicle-like protrusions bulged from the membrane of vacuoles. The late phase, from 45 min poststimulation onward, was typically identified after the appearance of tubules of smooth endoplasmic reticulum at the most distal segments of the nerve terminals. During this period, large vacuoles tended to decrease in quantity. Granular vesicles remained scant during the entire observation period. Images suggesting formation of microvesicles from tubules of smooth endoplasmic reticulum were observed. These results open the possibility that endocytosis of patches of membranes forming large vacuoles be an important mechanism for retrieving the membranes belonging to microvesicles and granular vesicles. Some of these large vacuoles may contribute to the early regeneration of microvesicles. More microvesicles could later be produced from the smooth endoplasmic reticulum.