Phorbol esters target the activity-dependent recycling pool and spare spontaneous vesicle recycling.

Using electrophysiology and styryl dye imaging, we studied the effect of phorbol 12-myristate 13-acetate (PMA) on activity-dependent and spontaneous vesicle recycling. In electrophysiological experiments, we found that the PMA effect depended on the maturational state of the synapses. Spontaneous neurotransmitter release from nascent synapses without a functional readily releasable pool (RRP) was unresponsive to PMA application. In contrast, mature synapses responded robustly to PMA application, consistent with previous studies. Using styryl dye imaging, we found that there was a PMA-dependent increase in the size of the RRP when PMA was present before, during, or after activity-dependent dye uptake, suggesting that this effect involves an increase in the population of the RRP by vesicles recruited from the reserve pool. Additionally, we found that when PMA was present during spontaneous dye uptake, there was an increase in dye labeling, and these additional dye-loaded vesicles showed rapid destaining in response to strong stimulation and were also releasable by hypertonic sucrose. In contrast, these observations were not reproducible when PMA treatment was performed after spontaneous dye uptake and extracellular dye washout. Together, these findings suggest that the increased spontaneous neurotransmission in the presence of PMA was attributable to release of vesicles from the RRP rather than an effect of PMA on the spontaneously recycling pool. Thus, the phorbol esters selectively regulate the activity-dependent pool of vesicles, indicating that priming mechanisms that prepare vesicles for fusion, which are targeted by phorbol esters, are different for the spontaneous and evoked forms of fusion.