NMDA‐receptor regulation of muscarinic‐receptor stimulated inositol 1,4,5‐trisphosphate production and protein kinase C activation in single cerebellar granule neurons

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Abstract
Inositol 1,4,5‐trisphosphate (InsP3) production in single cerebellar granule neurons (CGNs) grown in culture was measured using the PH domain of phospholipase C δ1 tagged with enhanced green fluorescent protein (eGFP‐PHPLCδ1). These measurements were correlated with changes in intracellular free Ca2+ determined by single cell imaging. In control CGNs, intracellular Ca2+ stores appeared replete. However, the refilling state of these stores appeared dependent on the fluorophore used to measure Ca2+‐release. Thus, methacholine (MCH), acting via muscarinic acetylcholine‐receptors (mAchRs), mobilised intracellular Ca2+ in cells loaded with fluo‐3 and fura‐4f, but not fura‐2. Confocal measurements of single CGNs expressing eGFP‐PHPLCδ1 demonstrated that MCH stimulated a robust peak increase in InsP3, which was followed by a sustained plateau phase of InsP3 production. In contrast, glutamate‐induced InsP3 signals were weak or not detectable. MCH‐stimulated InsP3 production was reduced by chelation of intracellular Ca2+ with BAPTA, and emptying of intracellular stores with thapsigargin, indicated a positive feedback effect of Ca2+ mobilisation onto PLC activity. In CGNs, NMDA‐ and KCl‐mediated Ca2+‐entry significantly enhanced MCH‐induced InsP3 production. Furthermore, mAchR‐mediated PLC activation appeared sensitive to the full dynamic range of intracellular Ca2+ increases stimulated by 100 μm NMDA. This dynamic regulation was also observed at the level of PKC activation indicated by an enhanced translocation of eGFP‐tagged myristoylated alanine‐rich C kinase substrate (MARCKS) protein in cells stimulated with MCH. Thus, NMDA‐mediated Ca2+ influx and PLC activation may represent a coincident‐detection system whereby ionotropic and metabotropic signals combine to stimulate InsP3 production and PKC‐mediated phosphorylation events in CGNs.