AAV-CaMKIIa-eNpHR3.0-eYFP or AAV-CaMKIIa-eYFP (from Gene Therapy Center at University of North Carolina at Chapel Hill, courtesy of Dr. Karl Deisseroth) was injected bilaterally in OFC under stereotaxic guidance at AP −3.0 mm, ML ± 3.2 mm, and DV 4.4 and 4.5 mm from the brain surface. A total 1–1.2 μl of virus (titer ∼1012) per hemisphere was

delivered at the rate of ∼0.1 μl/min by Picosptrizer microinjection system (Parker, Hollins, NH). Two rats that received eNpHR3.0 transgene were saved for later slice work; the remaining rats designated for behavioral testing had optic fibers (200 μm in core diameter; Thorlab, Newton, NJ) implanted bilaterally at AP −3.0 mm, ML ± 3.2 mm, and DV 4.2 mm. At the end of the study, these rats were perfused with phosphate buffer saline and then 4% PFA. The brains were then immersed in 30% sucrose/PFA for at least 24 hr. The brains were sliced at 40 μm with a microtome. The Selleck R428 brain slices were then stained with DAPI (through Vectashield-DAPI, Vector Lab, Burlingame, CA) or NeuroTrace (Invitrogen, Carsbad, CA) and mounted to slides with Vectashield (in the case of staining with NeuroTrace)

mounting media. The location of the fiber tip and NpHR-eYFP or eYFP expression was verified using an Olympus confocal microscope. The Z-stack images were merged and processed in Image J (National Institutes of Health). Approximately 2 months after surgery, two rats that had received AAV-CaMKIIa-eNpHR3.0-eYFP injection were anesthetized with isoflurane and perfused check details transcardially with ∼40 ml ice-cold NMDG-based artificial CSF (aCSF) solution containing (in millimoles) 92 NMDG, 20 HEPES, 2.5 KCl, 1.2 NaH2PO4, 10 MgSO4, 0.5 CaCl2, 30 NaHCO3, 25 glucose, 2 thiourea, 5 Na-ascorbate, 3 Na-pyruvate, and 12 N-acetyl-L-cysteine (300–310 mOsm, pH 7.3∼7.4). After perfusion, the brain was immediately removed and Isotretinoin 300 μm coronal brain slices containing the OFC were made using a Vibratome (Leica, Nussloch, Germany). The brain slices were recovered for less than 15 min at 32°C in NMDG-based aCSF and then transferred and stored for at least 1 hr in HEPES-based aCSF containing

(in mM) 92 NaCl, 20 HEPES, 2.5 KCl, 1.2 NaH2PO4, 1 MgSO4, 2 CaCl2, 30 NaHCO3, 25 glucose, 2 thiourea, 5 Na-ascorbate, 3 Na-pyruvate, and 12 N-acetyl-L-cysteine (300–310 mOsm, pH 7.3∼7.4, room temperature). During the recording, the brain slices were superfused with standard aCSF constituted (in millimoles) of 125 NaCl, 2.5 KCl, 1.25 NaH2PO4, 1 MgCl2, 2.4 CaCl2, 26 NaHCO3, 11 glucose, 0.1 picrotoxin, and 2 kynurenic acid, and was saturated with 95% O2, and 5% CO2 at 32°C–34°C. Glass pipette (pipette resistance 2.8–4.0 MΩ, King Precision Glass, Claremont, CA) with K+-based internal solution (in millimoles: 140 KMeSO4, 5 KCl, 0.05 EGTA, 2 MgCl2, 2 Na2ATP, 0.4 NaGTP, 10 HEPES, and 0.05 Alexa Fluor 594 [Invitrogen, Carlsbad, CA], pH 7.3, 290 mOsm) was used throughout the experiment.