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.
Keywords: GPCR; PLC; calcium-sensing receptor; extracellular calcium; minis; readily releasable pool; spontaneous release.
Copyright © 2023 Feldthouse, Vyleta and Smith.