All experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and with approval of the National Institute on Drug Abuse animal care and use committee. Microinjection

needles (29G) were connected to a 2 μl Hamilton syringe and filled with purified, concentrated adeno-associated virus (∼1012 infectious units ml−1) encoding EYFP, ChR2-EYFP, or NpHR-EFYP under control of the αCaMKII promoter. Mice were anesthetized with 150 mg kg−1 ketamine and 50 mg kg−1 xylazine and placed in a stereotaxic frame. Microinjection needles were bilaterally placed into the vHipp, basolateral amygdala, prefrontal cortex, or NAc shell and 0.5 μl virus was injected over 5 min. The needles were left in place for an additional CT99021 in vitro 5 min to allow for diffusion of virus particles away from injection site. Mice used for in vivo optogenetic experiments had 200 μm core optical fibers, threaded through 1.25-mm-wide zirconia ferrules, implanted directly above the NAc shell (+1.4 AP, ±1.5 ML, −3.7 DV at an 11° angle). Optical fibers were secured in place Ulixertinib using skull screws and acrylic cement. Wounds of mice destined for confocal imaging or slice electrophysiology were sealed with cyanoacrylate tissue glue. Mice were anesthetized with Euthasol

6–12 weeks after surgery and perfused with ice-cold PBS followed by 4% paraformaldehyde. Brains were removed, postfixed overnight in 4% paraformaldehyde, and sectioned in 100 μm coronal slices on a VT-1200 vibratome (Leica). Sections were mounted using Mowiol with DAPI. Slides were scanned on a confocal microscope (Olympus) with a 10× objective, isolating a single z plane. To enable comparisons, we processed and captured the quantified

images presented in Figures 1B, S3B, and S3C using identical settings. Glass capillary pipettes were pulled to a tip diameter of 30–40 μm and filled with 1% Fluoro-Gold (Fluorochrome) in 100 mM sodium cacodylate (pH 7.5). This micropipette was unilaterally placed in the medial NAc shell of anesthetized mice in a stereotaxic frame. A current of 2 μA was applied in 5 s pulses over 20 min. The micropipette was left in place very for an additional 5 min to prevent flow of tracer back through the needle track. Seven days after surgery, mice were anesthetized and perfused, as described above. Immunohistochemistry and imaging details are available in the Supplemental Experimental Procedures. Starting 4 weeks after surgery, mice in this group either remained in their home cage or were placed in an activity box (38 cm by 30 cm) for 40 min each day over 5 consecutive days. At the same time each day, or 10 min after entering this chamber, mice received intraperitoneal injections of either cocaine (15 mg/kg) or saline (0.9% NaCl). They were prepared for electrophysiological recordings 10–14 days later.