, 2012). Furthermore, the present study revealed

various direct inputs to dopamine neurons from relatively underappreciated areas such as motor, somatosensory, Ixazomib supplier and autonomic areas. This knowledge will be useful in designing future recording experiments to probe further differences between VTA and SNc dopamine neurons. All procedures were approved by Harvard University Institutional Animal Care and Use Committee. Adult male mice (2 to 6 months old) were used. DAT-Cre (Bäckman et al., 2006) and Vgat-ires-Cre (Vong et al., 2011) lines were backcrossed with C57BL6. For some control experiments, C57BL6 mice were used. For cell-type-specific tracing, 0.1–0.5 μl of AAV8-FLEX-RG (2 × 1012 particles/ml) and AAV5-FLEX-TVA-mCherry (4 × 1012 particles/ml) were stereotaxically injected into the targeting areas using a micromanipulator with a pulled glass needle. Two weeks later, 4 μl of pseudotyped rabies virus, SADΔG-GFP(EnvA) (1.0 × 107 plaque-forming units [pfu] per milliliter; Wickersham et al., 2007b), was injected into the same area. For tracing using the nonpseudotyped rabies virus, 4 μl of SADΔG-GFP (2 × 108 pfu/ml) (Wickersham et al., 2007a) was injected into VTA. To directly compare

the distributions of neurons projecting to VTA versus SNc dopamine neurons, SADΔG-GFP(EnvA) (5 × 107 pfu/ml) and SADΔG-mCherry(EnvA) (1.0 × 106 pfu/ml) (Marshel et al., 2010) were injected at 3.0 mm posterior to Bregma, 4.2 mm deep from dura, 0.5 mm and 1.5 mm lateral to the midline, respectively. One week after injection of rabies virus, mice were perfused with PBS followed by 4% paraformaldehyde (PFA) in PBS. After 2 days of Trametinib postfixation in else 4% PFA, coronal brain slices at 100 μm thickness were prepared using a vibratome. Every third section was counterstained with NeuroTrace Fluorescent Nissl Stains (Molecular Probes, Eugene, OR, USA). Immunohistochemistry was performed using the anti-calbindin rabbit polyclonal antibodies (Calbiochem, Darmstadt, Germany), anti-tyrosine hydroxylase AB152 (Millipore, Billerica, MA, USA), the biotinylated

goat anti-rabbit secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA), streptoavidin-conjugated Alexa Fluor 405, and Alexa Fluoro 594 goat anti-rabbit secondary antibodies (Molecular Probes). Slices were permeabilized with 0.5% Triton X-100, and incubation with antibodies and washing was done with 0.05% Triton X-100. Whole-section mosaics of high-magnification images were taken semiautomatically with AxioImager Z2 or LSM 510 Inverted Confocal microscopes (Zeiss) and assembled using software (Axiovision or LSM, Zeiss). The locations of labeled neurons and the outlines of brain areas were manually registered using custom software written in MATLAB (Mathworks, Natic, MA, USA). Further data analyses were performed using custom software written in MATLAB (see Supplemental Experimental Procedures). We are grateful to Dr.