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

Activation of h3MDq has been shown to increase intracellular Ca2+ signaling in cells (Pei et al., 2008; Urban and Roth, 2015) . We used CNO (3 µM) to activate hM3Dq in Rhod2-loaded microglia isolated from CX3CR1CreER(+/-)-hM3Dq(+/-) mice and test for hM3Dq-mediated changes in intracellular Ca2+ signaling. hM3Dq-positive microglia cells were identified by the expression of mCitrine (Zhu et al., 2016) . Application of CNO increased intracellular Ca2+ levels in the presence (Fig. 2C , green traces) and in the absence (Fig. 2D , green traces) of extracellular Ca2+ in hM3Dq-positive microglia cells, indicating mobilization from intracellular Ca2+ stores. Microglia isolated from CX3CR1CreER(+/-) littermate control mice did not show a CNO-induced increase in Ca2+ signaling (Fig. 2C, D; Fig.2E , F, black traces). However, both positive and negative cells responded to ATP and ionomycin or thapsigargin, used as positive controls (Fig. 2C-F) . These experiments indicate that hM3Dq expression in microglia allows selective activation of Ca2+ signaling pathways by CNO. author/funder. All rights reserved. No reuse allowed without permission.