Each neuronal subtype was verified via immunocytochemistry. Cells were fixed in 4% paraformaldehyde for 15 minutes, and then permeabilized with 0.2% Triton X-100 for 10 minutes, followed by blocking buffer (2% normal goat serum, 4% bovine serum albumin in PBS) for 1.5 hours. Primary antibodies used were anti-synapsin (E028, a gift from the Sudhof lab, 1:3000), mouse anti-vGlut2 (Synaptic Systems,1: 250), anti-MAP2 (Sigma M1406, 1:1000, Millipore 3418, 1:200, AB5543 1:500), and rabbit anti-vGAT (Millipore, AB5062P, 1: 500). Primary antibodies were incubated overnight at 4 °C. AlexaFluor conjugated species matched secondary antibodies were incubated at room temperature for 2 hours.
