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 ([Ca²⁺]_o) occur when high levels of neuronal activity lead to substantial Ca²⁺ entry via ion channels reducing local [Ca²⁺]_o. Perturbations of the extracellular microenvironment that increase [Ca²⁺]_o are 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⁺]_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 [Ca²⁺]_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 [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 in PLC-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. Discussion: Together these data point to a strong role for PLC in mediating changes in spontaneous release elicited by [Ca²⁺]_o and other extracellular cues, possibly by modifying the size of the RRP.