Some properties of the silent pool of synaptic vesicles

Abstract

Employing rat cerebrocortical synaptosomes, conditions have been established using 5 mM [Ca2+]e together with raised K+ (30 mM: HK5C) or 5 M ionomycin (ION5C) that evoke the release of all the readily releasable (RRP), and all the reserve pool/recycling pool (RP) of glutamate (Glu) containing synaptic vesicles (SVs). Using these conditions, we sought means to evoke a further increase in Glu release due to the induction of the exocytosis of the silent pool (SP: also called the reluctant or resting pool) of SVs. Utilising the discovery by Kim and Ryan (Neuron 67, 797-809, 2010) that inhibition of cyclin-dependent kinase 5 (CdK5) was able to induce the SP to release, we showed that roscovitine - a specific inhibitor of this enzyme – dose dependently increased the release of the SP of Glu containing SVs. The release was maximal with 5 mM [Ca2+]e with either HK or ION and a further increase of [Ca2+]e to 10 mM actually inhibited the release of the SP. Previously we have shown that HK5C or ION5C alone stimulated only one round of SV fusions but it was possible that roscovitine – rather than inducing the SP – may simply allow recycling, reloading and re-release of SVs from the RRP and/or RP. However, inhibition of dynamin dependent processes (with dynasore) or clathrin dependent mechanisms (with Pitstop2TM) failed to block the roscovitine induced HK5C evoked release of the SP of SVs, a result consistent with the SP SVs contributing to the Glu release measured. Intriguingly, maximal stimulation of PKCs with 1 M phorbol myristate acetate inhibited ION5C evoked SP SVs in roscovitine treated terminals. Therefore, it is possible that as HK5C can activate some PKCs, the total number of SVs being released from the SP could be less than maximal but, in fact, Go 6983 inhibition of PKCs did not change the amount of HK5C evoked Glu release from the SP in roscovitine treated terminals. ION5C action is independent of voltage dependent Ca2+ channels but whether, in roscovitine treated terminals, N, P or L-type channels could regulate the HK5C evoked release of the SP was explored by blocking these channels using specific toxins/drugs. Such blockade reduced Ca2+ entry and the specificity of such channels for the release of the SP will be discussed. Recently, the CdK5 phosphorylation site on synapsin I has been implicated in regulating the SP (Verstegen et al., J Neurosci 34, 7266-7288, 2014). We examined whether under our conditions phosphorylation sites on synapsin I regulated the SP of SVs. Similarly, we investigated whether the phosphorylation of -synuclein - a protein speculated to be involved in regulating SV pools – could participate in the release of these SP SVs.