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

The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.01.27.921809 doi: bioRxiv preprint NaCl). Patch electrodes were pulled from thin-wall glass tubes (World Precision Instruments, Inc.; Sarasota, FL) using a PP-830 pipette puller (Narishige, Japan). The electrode was filled with intracellular solution containing (in mM): 140 Cs-gluconate, 8 NaCl, 1 MgCl2, 10 Cs-BAPTA, and 10 HEPES following a previously described protocol (Zweifach and Lewis, 1996) with modifications. The pH of all solutions was adjusted to 7.4 using NaOH and/or KOH, and the osmolarity was adjusted to ~300 mOsm. Under voltage-clamp conditions, whole-cell patch recordings were performed using a Multiclamp 700B amplifier (Molecular Devices) by a person blinded to the treatment compounds. After measuring membrane capacitance of the test cell, voltage ramps (from -100mV to +50mV over 150 ms) were repeatedly applied to the cell every 10 s, revealing the current-voltage relationship. When stable recordings of baseline transmembrane current were achieved, the test cells were perfused with 3 µM CNO for 6 min, followed by application of 20 µM SKF-96365. The recorded electrical signal was digitized at 10kHz, low-pass at filtered (1 kHz), acquired on-line using the program pClamp (Molecular Devices), and saved in a computer for off-line analysis. The current density (pA/pF) at different voltages for each test cell was averaged and plotted for report. At least 15 cells from three different litters were analyzed for each testing condition.