Freeze-fracture was used to study the membrane events taking place during neurosecretory granule discharge (exocytosis) and subsequent membrane internalization (endocytosis) in axons of neurohypophyses from control and water-deprived rats. En face views of the cytoplasmic leaflet (P face) of the split axolemma reveal circular depressions that represent the secretory granule membranes fused with the plasma membrane during exocytosis. These depressions often contain granule core material in the process of extrusion into the extracellular space. The membrane surrounding some of the exocytotic openings shows a decreased number of intramembrane particles (mean diameter, 8 nm) which are elsewhere more numerous and evenly distrubuted on the fracture face. Endocytotic sites appear as smaller plasma membrane invaginations, with associated intramembrane particles. Moreover, such invaginations often contain large particles (mean diameter, 12 nm) that appear as clusters on en face views of the membrane leaflet. Quantitative analysis indicates that the number of exocytotic images increases significantly in glands from water-deprived rats. Concomitantly, the number of endocytotic figures per unit area of membrane is raised as is the number of clusters of large particles. The observations demonstrate that, in the neurohypophysis, it is possible to distinguish exocytosis morphologically from endocytosis and that the two events can be assessed quantitatively.
During secretion of the neurohypophysial hormones, oxytocin and vasopressin, secretory granule membrane is added to the plasma membrane of the axon terminals. It is generally assumed that subsequent internalization of this additional membrane occurs by endocytosis. In order to study this process, we have traced the uptake of intravenously injected horseradish peroxidase by neurohypophysial axons in rats and golden hamsters. Peroxidase reaction product within the secretory axons was found mainly in vacuolar and C-shaped structures of a size comparable with or larger than the neurosecretory granules. Our observations suggest that these large horseradish peroxidase (HRP)-impregnated vacuoles arise directly by a form of macropinocytosis. Morphometric analysis indicated that this form of membrane retrieval increased significantly after the two types of stimuli used, reversible hemorrhage and electrical stimulation of the pituitary stalk. Microvesicular uptake of HRP was found to be comparatively less.