Immediately following stimulation mice were anesthetized and perfused with an ice cold solution containing (in mM): 92 NMDG, 2.5 KCl, 1.25 NaH2PO4, 30 NaHCO3, 20 HEPES, 25 glucose, 2 thiourea, 5 Na-ascorbate, 3 Na-pyruvate, 0.5 CaCl2, 10 MgSO4, pH 7.3-7.4. The following inhibitor cocktail was also included (in mM): 5 actinomycin-D, 37.7 anisomycin, 2 kynurenic acid, 0.5 tetrodotoxin. 500 µm coronal VTA sections were cut in the same solution using a VT1000s vibratome (Leica). The VTA from each section was dissected using a fine scalpel blade under a dissecting microscope, transferred to a 1.5 mL tube, snap-frozen on powdered dry ice, and stored at -80°C. Tissue from 3-4 mice per group was pooled for nuclear isolation. VTA tissue was dounce homogenized in a buffer containing (in mM): 320 sucrose, 5 CaCl2, 3 Mg(C2H3O2)2, 10 Tris (pH 7.8), 0.1 EDTA, 0.1 protease inhibitor cocktail, 1 β-mercaptoethanol, 5 actinomycin-D, 37.7 anisomycin, with 0.1% NP40 and 0.2 U/ul RNase inhibitor. Following a 5 min incubation on ice homogenized samples were mixed with an equal volume of 50% iodixanol solution (50% OptiPrep containing (in mM): 5 CaCl2, 3 Mg(C2H3O2)2, 10 Tris (pH 7.8), 0.1 protease inhibitor cocktail, 1 β-mercaptoethanol, 5 actinomycin-D, 37.3 anisomycin). This mixture was layered on top of an equal volume of 29% iodixanol (OptiPrep) and spun at 7500 RPM for 30 min at 4°C. Pelleted nuclei were resuspended in PBS with 10% BSA and 0.2 U/ul RNAse inhibitor. The concentration of nuclei was determined using a hemocytometer, and was adjusted to 1,000 nuclei/ul. Approximately 10,000 nuclei per experimental group were captured and single-nucleus cDNA libraries were constructed using the Chromium NextGEM platform (10x Genomics) according to manufacturer's instructions. Libraries were sequenced using an Illumina 4400 HiSeq platform to a target output of ~ 350,000,000 reads