Aav5 ef1a dio enphr 3.0 eyfp

A Cre-dependent inhibitory optogenetic construct halorhodopsin (eNpHR, AAV5-Ef1a-DIO eNpHR 3.0-EYFP at titer ≥ 1×10¹³ vg/mL, Addgene plasmid # 26966) or an empty vector (control, AAV5- Ef1a-DIO EYFP at titer ≥ 1×10¹³ vg/mL, Addgene plasmid # 27056) were utilized. Both plasmids were obtained from Addgene as gifts from Dr. Karl Deisseroth.

Mice used in fluorescence imaging, optogenetic, or CRISPR knockdown experiments were anesthetized with ketamine/xylazine for intracranial AAV injection, hair was removed, scalps were cleaned with ethanol and betadine, and lidocaine was used prior to scalp incision. Body temperature was maintained with an electric heating pad. Mice received bilateral stereotactic injections (0.3–0.5 μL) of either AAV9-hsyn-GRAB-ACh3.0 (Addgene; plasmid# 121922; packaged by Vigene Biosciences),^{36 (link)} AAV9-syn-FLEX-jGCaMP7f (Addgene; 104492-AAV9), AAV5-EF1a-DIO-eNpHR3.0-eYFP (Addgene; 26966-AAV5), or AAV1-mU6-Tac1r-gRNA1EF1a-NucEnv-EGFPhU6-Tacr1-gRNA2 in the NAc core (created and packaged by NIDA GEVVC Core) (A/P: 1.6, Lat: 1.6, D/V: −4.4, 10° angle). For photometry or optogenetics, mice were then implanted with either a 400 μm photometry fiber (Doric) or 200 μm core optogenetic fiber fixed with head screws and dental cement. Following surgery, mice were injected with warm saline to replenish fluids and given carprofen (5 mg/kg) for pain relief during recovery.