Immunoelectron microscopic localization of synaptophysin in a Golgi subcompartment of developing hypothalamic neurons

Synaptophysin, previously identified as an integral membrane glycoprotein (mol. wt 38,000) characteristic of presynaptic vesicles of mature neurons, provides a molecular marker to study the origin, formation and traffic of synaptic vesicles. Using the monoclonal antibody SY38 against this polypeptide we have localized synaptophysin by immunofluorescence and electron microscope immunoperoxidase methods in cultured mouse hypothalamic neurons taken from 16-day-old fetuses which achieve synaptogenesis after 10-12 days in vitro. We have compared the localization of synaptophysin in perikarya and nerve endings as a function of age (2-19 days in vitro) and of treatment of mature neurons with nocodazole. Using immunofluorescence microscopy, synaptophysin was already detected in neuronal soma at 2 days in vitro, where the initiation of neurite development is observed. At the electron microscope level, virtually all mature synaptic boutons and varicosities showed an extensive synaptophysin labeling of synaptic vesicles at 12-13 days in culture whereas neurites showed only very few labeled vesicles. In neuronal soma taken before synapse formation (6 days in vitro), synaptophysin was selectively localized in membranes of the innermost cisternae of the Golgi zone and in vesicles of variable size and shape in the core of the Golgi zone. In contrast, after synapse formation, synaptophysin labeling was barely detected in the Golgi zone of neurons but a very strong labeling of synaptic vesicles in synaptic boutons was observed. Treatment of mature neurons (12 days in vitro) with nocodazole (10(-5) M) resulted in a conspicuous synaptophysin staining of the innermost trans-Golgi cisternae and numerous vesicles in the cytoplasm. Furthermore, an accumulation of labeled synaptic vesicles on the presynaptic membrane of nerve terminals was found. The data suggest that synaptophysin is released from the Golgi apparatus in a vesicular form, after glycosylation, and is then transported to nerve endings by a mechanism which requires integrity of microtubules.