Phorbol esters potentiate evoked and spontaneous release by different presynaptic mechanisms

Phorbol esters enhance release from a variety of cell types. The mechanism by which phorbol esters potentiate presynaptic release from central neurons is unclear, although effects of phorbol esters both on the readily releasa ble pool of vesicles and on presynaptic calcium channels have been shown. U sing confocal microscopy and the fluorescent styryl dye FM 1-43, we have ex amined the effects of phorbol-12,13-dibutyrate (PDBu) on presynaptic vesicl e turnover at individually identified synapses in dissociated cultures obta ined from neonatal rat hippocampus. Using different dye staining and destai ning protocols we were able to resolve two effects of PDBu. Potentiation of evoked release by PDBu was insensitive to calcium channel antagonists, sug gesting that this effect results from an increased number of vesicles in th e readily releasable pool. Since we observed no effect of PDBu on the size of the total recycling vesicle pool, we conclude that phorbol esters alter the equilibrium between reserve and readily releasable pools. An additional effect of PDBu on spontaneous release was observed. This effect was antago nized by nifedipine but not omega -conotoxin GVIA or omega -agatoxin IVA. W e conclude that PDBu influences spontaneous and evoked release by two diffe rent mechanisms: through L-type calcium channels and through an increase in the proportion of recycling vesicles in the readily releasable pool. In ad dition to further clarifying the mechanism of action of phorbol esters, the se results suggest that phorbol esters may be a useful tool with which to p robe the function of the readily releasable pool of presynaptic vesicles at CNS synapses.