Effect of chemical depolarization on membrane recycling in hypothalamic neurons in culture

The mechanisms of membrane recycling and endocytic activity have been investigated by morphological methods, following chemical depolarization, in cultured mouse hypothalamic neurons that have developed in vitro fully differentiated synapses. Previous studies with this cell system have shown that short-term (3 min) exposure to a depolarizing concentration of KCl induces a depletion of synaptic vesicles in synaptic boutons. Subsequent return to control medium is followed by a restoration of synaptic vesicle numbers. We have now used horseradish peroxidase (HRP) as a tracer for endocytosis and wheat-germ agglutinin coupled to HRP-gold as a marker for adsorptive endocytosis. In parallel, synaptophysin, a marker for small synaptic vesicles, was simultaneously localized by electron microscopic immunocytochemistry in some experiments. The morphometric analyses and the time course of HRP uptake combined with the distribution of synaptophysin immunostaining indicate that short-term (3 min) depolarization leads to a depletion of synaptic vesicles together with an enhanced endocytic activity via vesicles that possess both the diameter and the major membrane protein, synaptophysin, characteristic of synaptic vesicles. These findings suggest a local, direct recycling mechanism of synaptic vesicles. In contrast, long-term depolarizations (15-30 min) appear to involve additional membrane compartments.