Intracranial manipulations

Mice (>45 d) were placed in a stereotaxic frame (David Kopf Instruments) under isoflurane anesthesia. Microinjectors, made by affixing a 33-gauge stainless steel hypodermic tube within a shorter 26-gauge stainless steel hypodermic tube, were attached to polyethylene-20 tubing affixed to 10-μl Hamilton syringes, and were lowered through burr holes in the skull to the VTA (from bregma: −2.75 mm A/P, ±0.55–0.7 mm M/L, −5 mm D/V); 300–500 nl of virus was injected per side at a rate of 100 nl/min. The optimized coordinates and viral load ensured full coverage of the VTA along anterior/posterior and rostral-caudal axes, with minimal spread into the adjacent substantia nigra pars compacta. Microinjectors were left in place for 10 min following infusion to reduce solution backflow along the infusion track. Slice electrophysiology and behavioral experiments were performed three to four and five to six weeks after viral infusion for chemogenetic and CRISPR experiments, respectively.