RAB3 DISSOCIATION AND CLATHRIN-MEDIATED ENDOCYTOSIS, TWO KEY STEPS IN THE EXO-ENDOCYTOTIC PATHWAY OF LARGE DENSE-CORED VESICLES IN PRIMARY CULTURES OF SUPERIOR CERVICAL GANGLIA

: In this study we have used primary cultures of porcine superior cervical ganglia as a model system to study exo-endocytosis in sympathetic neurons. Pure neuronal cultures with a defined noradrenergic phenotype can be obtained when antimitotics are included in the culture medium, and the high yield from prenatal piglets allows a biochemical approach in addition to morphological studies. Release of large dense-cored vesicles (LDCVs) was visualized by exposing stimulated neurons to anti-dopamine-beta-hydroxylase (D beta H) prior to fixation. Double immunofluorescent staining revealed that synaptotagmin was colocalized with anti-D beta H labeled exocytotic spots, but not the small GTP-binding protein rab3. Density gradient centrifugation of a postmitochondrial supernatant of cultured cells confirmed the dissociation of rab3 from a population of mature LDCVs. As expected, rab3 did not reassociate with the lighter D beta H-positive membrane fraction in the gradient representing internalized LDCV membranes. The fluid phase marker horseradish peroxidase colocalized with retrieved LDCV membranes. Indirect immunofluorescence demonstrated the colocalization of clathrin with D beta H-positive exocytotic spots on the plasma membrane. At the ultrastructural level, depolarization of neurons resulted in the abrupt loss of LDCVs and concomitant appearance of clathrin-coated vesicles and coated omega profiles. The size distribution of coated structures overlapped strongly with that of LDCVs. Taken together, these data clearly suggest that two key regulatory events in the process of exo-endocytosis, i.e. rab3 dissociation and clathrin-mediated internalization, are not only reminiscent of small synaptic vesicles, but also are a feature of the LDCV pathway at the presynaptic plasma membrane of sympathetic neurons.