Further, our data demonstrate that the LHb and midbrain interact in a reciprocal manner and implicate the VTA’s projection to the LHb as a key node in the classical midbrain reward circuit. This mechanistic framework underscores the flexibility and complexity of the circuitry that impinges upon VTA dopaminergic neurons to promote motivated behavior. Adult (25–30 g) mice were group housed until surgery and maintained on a reverse 12 hr light cycle (lights off at 8:00) with ad libitum

access to food and water. Mice were anesthetized with ketamine (150 mg/kg of body weight) and xylazine (50 mg/kg) and placed in a stereotactic frame (Kopf Instruments). For all slice electrophysiology and fast-scan cyclic voltammetry experiments, except for the retrobeads experiments, Dabrafenib male and female TH-IRES-Cre backcrossed to C57BL/6J were bilaterally microinjected with 0.5 μl buy Ceritinib of purified and concentrated adeno-associated virus serotype 5 (AAV5; ∼1012 infections units per ml, packaged and titered by the UNC Vector Core Facility) into the VTA. Stereotactic coordinates are

available in the Supplemental Experimental Procedures. Each VTA was injected with an AAV5 coding Cre-inducible ChR2 under control of the EF1α promoter to transduce VTA dopaminergic neurons (THVTA::ChR2). For the retrobead slice electrophysiology and PCR retrobead experiments, male and female TH-IRES-GFP mice received quadruple injections of 0.3 μl of red retrobeads (Lumafluor) into either the NAc or LHb. For the retrobead mapping and quantification experiments, male C57BL/6J mice

(Jackson Laboratory) received quadruple injections with 0.3 μl of red retrobeads into the NAc. In the same L-NAME HCl surgery, the mice also received quadruple injections of 0.3 μl with green retrobeads (Lumafluor) into the LHb. For tracing experiments, TH-IRES-Cre mice were bilaterally injected with 0.5 μl of HSV-EF1α-LS1L-flp into the LHb or NAc and bilaterally injected with 0.5 μl of AAV5-EF1α-fdhChR2(H134R)-eYFP into the VTA. A detailed description of the HSV vector construction is available in the Supplemental Experimental Procedures. For behavioral experiments, male TH-IRES-Cre positive (THVTA-LHb::ChR2) and negative (THVTA-LHb::Control) littermates were bilaterally injected with Cre-inducible ChR2 and also implanted with bilateral chronic fibers directed above the LHb. For the LHb microinjection experiments, a 26G steel tube cannula (McMasters-Carr) that terminated 0.5 mm above the tip of the optical fiber was epoxied to an optical fiber and bilaterally aimed at the LHb. Retrobead experiments were performed 7–21 days after surgery. All other experiments were performed 6–8 weeks after surgery. Histology, immunohistochemistry, confocal, and electron microscopy procedures can be found in the Supplemental Experimental Procedures.