Quantitative analysis of vGATE-tagged cells

Fifty mm thick brain sections were counterstained with 1:10000 4′,6-Diamidine-2′-phenylindole dihydrochloride DAPI (Aldrich, Milano, Italy) for 20 min, then rinsed in PBS, mounted and cover-slipped on a slide. Pictures were taken with a NIKON ECLIPSE (Nikon Instruments, Firenze, Italy) confocal microscope at 40X magnification, with each focal plane 1 mm thick. Twelve microns thick stacks were acquired for each picture and analyzed with ImageJ using the cell counter plugin and selecting cells within the stack (http://rsbweb.nih.gov/ij/index.html). The number of ChR2-tdTOM positive cells in each picture was divided by the number of DAPI-stained nuclei, and the percentage of recruited cells was calculated. This analysis was restricted to the infected area that was defined by the most external hChR2-positive cells in each acquired field. A total of 6 fields belonging to three different sections were averaged for each animal.