Stereotax

Mice were initially anesthetized with 5% isoflurane and then transferred to the stereotax (Kopf Instruments, Tujunga, CA) and kept under 2% isoflurane anesthesia. The hair over the incision cite was trimmed and the skin was prepped with alcohol and iodine scrub. The skull was exposed via a midline sagittal incision and treated with the local anesthetic, benzocaine (Medline Industries, Brentwood, TN). For all surgeries, we used a motorized digital software (NeuroStar; Stoelting Co., Wood Dale, IL) to guide a 10μL microinjection syringe (Hamilton Co., Reno, NV) driven by a Micropump Controller (World Precision Instruments, Sarasota, FL). Virus was delivered bilaterally into the plPFC (AP:+2.42, ML:±0.35, DV: 2.09), BLA (AP: −1.25, ML: ±3.30, DV: 5.10), or MDT: (AP:−1.1, ML.: ±0.59, DV: 3.4). Following completion of each surgery, 10mg/kg ketoprofen (AlliVet, St. Hialeah, FL) was administered as an analgesic, and post-operative treatment with ketoprofen was administered 24 and 48 hours after the surgery.

Rats were anesthetized with 30% urethane at 5 ml/kg and placed in a stereotax (David Kopf Instruments) on a heating pad (37°C). Rats underwent a craniotomy above the NAc and were implanted with MEAs into either the left or right NAcore (AP, +1.8 mm; ML, ±1.5 mm; DV, −7mm versus bregma). Mouse procedures were performed as described above – with the following exceptions. Mice were anesthetized with 15% urethane at 5ml/kg and coordinates used for placement were AP, +1.1 mm; ML, ±1.2 mm; DV, −4.2mm versus bregma.

Mice (4–8 weeks) were anesthetized with isoflurane and placed in the stereotax (Kopf Instruments). Adeno-associated viral (AAV) vectors were bilaterally injected into NAc (250 nLfrom bregma: AP, +1.2; ML, ±1.0; DV, —4.6 mm), and dorsal raphe (500 nL: AP, —4.2; ML, ±0.5; DV, —3.2 mm) using a Nanoject II (Drummond Scientific). Vectors with Cre-dependent ChR2 expression (rAAV5- EF1a-DIO-hChR2(H134R)-EYFP; 4.5 × 10¹², UNC) were used to target iMSN with Adora2a-Cre mice, dMSN with Drd1-Cre mice, or dorsal raphe neurons with SERT-Cre mice. Additionally, constitutive AAV vector (rAAV5-CaMKII-hChR2(H134R)-EYFP; 8.5 × 10¹², Penn Vector Core) were used to express ChR2 in neurons of the NAcfor5-HT1BKOand littermate WT mice.ChR2expres- sion at the injection sites was confirmed before experiments. When ChR2 expressed outside of the target, animals were excluded from the study.

Expression of mCherry and ChR2 into cholinergic neurons solely in the MS/DBB was achieved by stereotaxic injection of virus directly into the MS/DBB of ChAT-cre mice. pAAV-EF1a-double floxed-hChR2(H134R)-mCherry-WPRE-HGHpA (Addgene plasmid #20297 from Karl Deisseroth) and was packaged with AAV stereotype 9 by the viral core facility at the NIEHS. For stereotaxic injections, 5–7 week old ChAT-cre mice were anesthetized with an intraperitoneal (IP) injection of 100 mg/kg ketamine and 7 mg/kg xylazine. Once fully anesthetized, Marcaine (50–75 ul of 0.5% solution of bupivacaine) was delivered subcutaneously to the scalp as a local analgesic. Mice were secured in a stereotax (Kopf Instruments) using ear bars. A Hamilton syringe with a 30 gauge needle (Hamilton Co.) was slowly lowered into position and 1 μl of virus was infused at a rate of 0.1 μl/min for 10 min. stereotaxic coordinates used were 0.05 mm ML, 0.8 mm AP, and −4.4 mm DV. Following surgery, the mouse was given 0.1 cc/10 g body weight of buprenorphine subcutaneously (SC), 0.5 cc of sterile saline SC, and 0.1 cc of Antisedan IP to reverse the effects of the ketamine/xylazine. The mouse was allowed to recover on a heating pad and monitored for any signs of distress. 2–3 weeks following surgery, mice were decapitated under isoflurane anesthesia and their brains were used for electrophysiology as described above.