K3-Flp;Gad2-Cre cFos Data

Confocal images (Zeiss 980) were taken at 10X and 20X objective as Z-stacks and tiles, exported as OME-TIFF files, and analyzed in FIJI blind to genotype and condition. Images were processed as follows: z-projected for maximum intensity, despeckled, and split channels. ROIs were drawn for the indicated cell body region. Next the cFos channel background was subtracted by a rolling ball radius value of 15 and then it was subject to thresholding. The thresholded value was determined by opening the threshold window, finding the right-side inflection-point of the histogram curve, and multiplying it by 3. For determining the percent of DREADD-mCherry cells expressing cFos, manual cell counts were conducted after thresholding. For determining the percent of principle cells with cFos, we used the automatic analyze particles function with a circularity range of 0-1.0 and size range of 30 μm2-infinity. Counts were verified by merging channels and examining merged fluorescence.