Five C57 male mice 26 days of age were administered a lethal i.p. injection of sodium pentobarbital (40 mg/kg) and then were intracardially perfused with saline solution followed by 4% paraformaldehyde in 0.12M phosphate buffer, pH 7.4 (PB). The heads were left overnight in the same fixative at 4C, and the brains were removed the next morning and postfixed in fresh fixative for 1 additional day. Thereafter, the brains were washed in several changes of 0.12 M PB and free-floating vibratome sections (100-μm thick) were used for these experiments. Primary antibodies used were: guinea-pig anti-VGluT1 (Millipore Corp) and rabbit anti-GAT1 (Millipore Corp). Primary antibodies were diluted in PB containing 0.25% Triton X-100. Secondary antibodies were: anti-guinea-pig Alexa 488 and anti-rabbit Alexa 594. The sections were photographed on a Leica DMI 600B laser scanning confocal microscope, and the visualization and deconvolution of the images was performed with Imaris (Bitplane AG) and Autodeblur (Media Cybernetics Inc.) softwares.