AUTHOR=Feldthouse Maya G. , Vyleta Nicholas P. , Smith Stephen M. 

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

JOURNAL=Frontiers in Cellular Neuroscience

VOLUME=17

YEAR=2023

URL=https://www.frontiersin.org/journals/cellular-neuroscience/articles/10.3389/fncel.2023.1193485

DOI=10.3389/fncel.2023.1193485

ISSN=1662-5102

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