Brefeldin A sensitive mechanisms contribute to endocytotic membrane retrieval and vesicle recycling in cerebellar granule cells.

The recycling of synaptic vesicle ( SV) proteins and transmitter release occur at multiple sites along the axon. These processes are sensitive to inhibition of the small GTP binding protein ARF1, which regulates the adaptor protein 1 and 3 complex ( AP-1/ AP-3). As the axon matures, SV recycling becomes restricted to the presynaptic bouton, and its machinery undergoes a complex process of maturation. We used the styryl dye FM1-43 to highlight differences in the efficiency of membrane recycling at different sites in cerebellar granule cells cultured for 7 days in vitro. We used Brefeldin A ( BFA) to inhibit AP-1/ AP-3-mediated recycling and to test the contribution of this pathway to the heterogeneity of the responses when these cells are strongly stimulated. Combining imaging techniques and ultrastructural analyses, we found a significant decrease in the density of functional boutons and an increase in the presence of endosome-like structures within the boutons of cells incubated with BFA prior to FM1-43 loading. Such effects were not observed when BFA was added 5 min after the end of the loading step, when endocytosis was almost fully completed. In this situation, vesicles were found closer to the active zone ( AZ) in boutons exposed to BFA. Together, these data suggest that the AP-1/ AP-3 pathway contributes to SV recycling, affecting different steps in all boutons but not equally, and thus being partly responsible for the heterogeneity of the different recycling efficiencies. [ABSTRACT FROM AUTHOR]