PLC regulates spontaneous glutamate release triggered by extracellular calcium and readily releasable pool size in neocortical neurons

INTRODUCTION: Dynamic physiological changes in brain extracellular calcium ([Ca(2+)](o)) occur when high levels of neuronal activity lead to substantial Ca(2+) entry via ion channels reducing local [Ca(2+)](o). Perturbations of the extracellular microenvironment that increase [Ca(2+)](o) are commonly used to study how [Ca(2+)] regulates neuronal activity. At excitatory synapses, the Ca(2+)-sensing receptor (CaSR) and other G-protein coupled receptors link [Ca(2+)](o) and spontaneous glutamate release. Phospholipase C (PLC) is activated by G-proteins and is hypothesized to mediate this process. METHODS: Patch-clamping cultured neocortical neurons, we tested how spontaneous glutamate release was affected by [Ca(2+)](o) and 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. RESULTS: Spontaneous glutamate release substantially increased with [Ca(2+)](o), and inhibition of PLC activity, with U73122, abolished this effect. PLC-β1 is an abundant isoform in the neocortex, however, [Ca(2+)](o)-dependent spontaneous release was unchanged in PLC-β1 null mutants (PLC-β1(–/–)). U73122 completely suppressed this response in PLC-β1(–/–) neurons, indicating that this residual [Ca(2+)](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. DISCUSSION: Together these data point to a strong role for PLC in mediating changes in spontaneous release elicited by [Ca(2+)](o) and other extracellular cues, possibly by modifying the size of the RRP.