Chronic hM3Dq signaling in microglia ameliorates neuroinflammation in male mice

William Binning; Aja E. Hogan-Cann; Diana Yae Sakae; Matthew Maksoud; Valeriy Ostapchenko; Mohammed Al-Onaizi; Sara Matovic; Wei-Yang Lu; Marco A. M. Prado; Wataru Inoue; Vania F. Prado

Microglia express several GPCRs that associate to Gq-signaling heterotrimeric G proteins, such as group 1 metabotropic glutamate receptors (mGluR1, mGluR5) (Biber et al., 1999) , purinergic receptors (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11) (Koizumi et al., 2013) , adrenergic (1AR) (Mori et al., 2002) and the M3 muscarinic receptor (Pannell et al., 2016) . Although these receptors are activated by different ligands, they are all able to elicit Gq pathway activation. Gq proteins are known to mobilize calcium through inositol 1,4,5-triphosphate-regulation of intracellular stores and diacylglycerol-dependent protein kinase C activation (Mizuno and Itoh, 2009 ). Our in vitro experiments showed that CNO was able to elicit an increase in microglial intracellular calcium concentration, only in hM3Dq-positive cells. This increase was seen in both calcium-free media and media containing calcium, indicating that hM3Dq was functional and the source of calcium influx is, at least in part, from intracellular stores. The functionality of hM3Dq and the signaling pathway activated were also confirmed by patch-clamp experiments showing that hM3Dq activation by CNO induced an increase in conductance in microglia that was abolished by the SOCC inhibitor.