IntroductionDynamic physiological changes in brain extracellular calcium ([Ca2+]o) occur when high levels of neuronal activity lead to substantial Ca2+entryviaion channels reducing local [Ca2+]o. Perturbations of the extracellular microenvironment that increase [Ca2+]oare commonly used to study how [Ca2+] regulates neuronal activity. At excitatory synapses, the Ca2+-sensing receptor (CaSR) and other G-protein coupled receptors link [Ca2+]oand spontaneous glutamate release. Phospholipase C (PLC) is activated by G-proteins and is hypothesized to mediate this process.MethodsPatch-clamping cultured neocortical neurons, we tested how spontaneous glutamate release was affected by [Ca2+]oand inhibition of PLC activity. We used hypertonic sucrose (HS) to evaluate the readily releasable pool (RRP) and test if it was affected by inhibition of PLC activity.ResultsSpontaneous glutamate release substantially increased with [Ca2+]o, and inhibition of PLC activity, with U73122, abolished this effect. PLC-β1 is an abundant isoform in the neocortex, however, [Ca2+]o-dependent spontaneous release was unchanged in PLC-β1 null mutants (PLC-β1–/–). U73122 completely suppressed this response inPLC-β1–/–neurons, indicating that this residual [Ca2+]o–sensitivity may be mediated by other PLC isoforms. The RRP size was substantially reduced after incubation in U73122, but not U73343. Phorbol esters increased RRP size after PLC inhibition.DiscussionTogether these data point to a strong role for PLC in mediating changes in spontaneous release elicited by [Ca2+]oand other extracellular cues, possibly by modifying the size of the RRP.