The process leading to the estimation of the total number of D1 and D2 MSNs began by randomly translating a grid formed by 235 × 580 μm rectangles on the previously acquired confocal images of the striatum and the Acb. At each intersection of the grid that fell into the sector, a counting frame measuring 157 × 157 μm was drawn and examined. Cell bodies containing tdTomato (D1) or GFP (D2) that fell inside the counting frame and did not contact the exclusion lines were counted whenever they came into focus within a 12 μm-thick optical disector placed at 9 μm from the top of the section. An average number of 2939 ± 186 D1 and 2477 ± 160 D2 neurons were counted in the striatum and 1187 ± 98 D1 and 642 ± 64 D2 cell bodies in the Acb of each mouse, yielding coefficient of error (Gundersen, m = 1) ranging between 0.05 and 0.18.
