Lentivirus production is described in Supplemental Experimental Procedures. Stereotaxic microinjection is selleck chemicals described in Supplemental Experimental Procedures. Western blotting and immunohistochemistry are described in Supplemental Experimental Procedures. Four- to five-week-old rats were bilaterally microinjected with lentivirus expressing shRNA-control or shRNA-HCN1 in the dorsal hippocampal CA1 region. After behavior test, dorsal hippocampal slices (350 μm) were prepared from 10-

to 12-week-old lentivirus-infected male Sprague-Dawley rats. Then slices were transferred to a holding chamber for 20–30 min at 35°C containing (in mM) 125 NaCl, 2.5 KCl, 1.25 NaH2PO4, 25 NaHCO3, 2 CaCl2, 2 MgCl2, 10 dextrose, 1.3 ascorbic acid, and 3 sodium pyruvate, bubbled with 95% O2-5% CO2. Whole-cell current-clamp recordings were performed on slices submerged in a recording chamber filled with aCSF heated to 32°C–34°C flowing at a rate of 1 to 2 ml/min. ACSF contained (in mM) 125 NaCl, 2.5 KCl, 1.25 NaH2PO4, 25 NaHCO3, 2 CaCl2, 1 MgCl2, and 12.5 dextrose, bubbled with 95% O2-5% CO2. Lentivirus-infected CA1 pyramidal neurons were visualized using a microscope (Zeiss Axioskop) fitted with

differential interference contrast optics and a GFP filter set (Stuart et al., 1993). Patch pipettes (4–7 MΩ) were prepared with capillary glass (external diameter 1.65 mm, World Precision Instruments) using Selleckchem CHIR99021 Flaming/Brown micropipette puller and filled with an internal solution containing (in mM) 120 K-gluconate, 20

KCl, 10 HEPES, 4 NaCl, 7 K2-phosphocreatine, 4 Mg-ATP, 0.3 Na-GTP (pH 7.3 with KOH). Neurobiotin (Vector Labs) was used (0.1%–0.2%) for subsequent histological processing (DAB staining). Data were acquired with a Dagan BVC-700A amplifier (Dagan, Minneapolis, MN) and were filtered at 3 kHz, sampled isothipendyl at 10 kHz, and digitized by an ITC-18 interface (Instructech Corporation, Port Washington, NY) connected to a computer running custom software written in IGOR Pro (Wavemetrics). Data analyses were also performed with custom software written in IGOR Pro. The experiments were performed under blind conditions. No correction for liquid junction potential (∼8 mV). One hundred twenty-two rats (9–12 weeks old) were assigned into seven groups as follows: group I, saline i.p. injected (n = 17); group II, vehicle (0.5% Tween 20 in saline) i.p. injected (n = 13); group III, diazepam i.p. injected (1 mg/kg, n = 18); group IV, ketamine i.p. injected (15 mg/kg, n = 13); group V, fluoxetine i.p. injected (10 mg/kg, n = 13); group VI, lentivirus expressing shRNA-control microinjected into the dorsal hippocampal CA1 region (n = 24); group VII, lentivirus expressing shRNA-HCN1 microinjected into the dorsal hippocampal CA1 region (n = 24). All behavior evaluations were done blindly. Experiment was performed in noise- and light-controlled behavior room.