Photoconversion of FM1-43 Reveals Differences in Synaptic Vesicle Recycling and Sensitivity to Pharmacological Disruption of Actin Dynamics in Individual Synapses.

The cycling of synaptic vesicles ensures that neurons can communicate adequately through their synapses on repeated occasions when activity is sustained, and several steps in this cycle are modulated by actin. The effects of pharmacological stabilization of actin with jasplakinolide or its depolymerization with latrunculin A was assessed on the synaptic vesicle cycle at individual boutons of cerebellar granule cells, using FM1-43 imaging to track vesicle recycling and its photoconversion to specifically label recycled organelles. Remarkable differences in the recycling capacity of individual boutons are evident, and their dependence on the actin cytoskeleton for recycling is clear. Disrupting actin dynamics causes a loss of functional boutons, and while this indicates that exo/endocytotic cycling in boutons is fully dependent on such events, this dependence is only partial in other boutons. Indeed, exocytosis and vesicle trafficking are impaired significantly by stabilizing or depolymerizing actin, whereas repositioning recycled vesicles at the active zone seems to be dependent on actin polymerization alone. These findings support the hypothesis that different steps of synaptic vesicle cycling depend on actin dynamics and that such dependence varies among individual boutons.

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. Cover Image for this issue: doi. 10.1111/jnc.13801.