GluA, TARP and CNIH cDNAs were cotransfected with a GFP-expressing reporter plasmid for identification in electrophysiology experiments. One hundred percent CNIH-2 transfection indicates equal amounts of CNIH-2 and GluA subunit cDNAs and 50% CNIH-2 reduces this ratio by one
half. The cells were trypsinized 1 day after transfection and plated on glass coverslips at low density (∼5000/cm2). Experiments were performed 48–72 hr posttransfection. Stargazer mice were obtained from Jackson Laboratory and maintained at the Yale PLX-4720 manufacturer animal facility under the guidelines of the Institutional Animal Care and Use Committee. Heterozygous male and female mice were mated to obtain homozygous stargazer mice. Cerebellar granule
cell cultures were prepared from postnatal day 7–8 (P7–8) homozygous stargazer mice and were transfected at 5 days in vitro (DIV5) as described (Cho et al., 2007). Primary cultures of rat hippocampal neurons were prepared essentially as described (Kato et al., 2008). Briefly, hippocampi dissected from E19 Wistar rat embryos were incubated at 37°C for 10 min in a papain solution (in mM): 5 L-cysteine, 1 ethylenediaminetetraacetic acid, 10 HEPES-NaOH Protein Tyrosine Kinase inhibitor (pH 7.4), 100 μg/ml bovine serum albumin, 10 U/ml papain (Worthington), and 0.02% DNase (Sigma). The reaction was stopped by addition of an equal volume of fetal bovine serum. The cells were triturated and washed with Neurobasal (Invitrogen) supplemented with B-27, 100 μg/ml penicillin,
85 μg/ml streptomycin, 0.5 mM glutamine. The cells were plated on 12 mm coverslips coated with poly-D-lysine in 24-well plates at 100,000 cells/well density. cDNA (γ-8, CNIH-2, or γ-8 and CNIH-2)- or CNIH-2 shRNA-Lipofectamine 2000 (Invitrogen) complexes were prepared in Neurobasal medium according to manufacturer’s specifications. Primary neurons (>14 DIV) were incubated with these Lipofectamine complexes in Neurobasal medium in the absence of B-27, penicillin/streptomycin, and L-glutamine for at least 2 hr and then returned to the original conditioned medium. Electrophysiological recordings from primary neurons were performed at least Linifanib (ABT-869) 48 hr posttransfection. Lentiviral particles for shRNAs were infected at multiplicity of infection = 2. Hippocampal pyramidal neurons from 5- to 8-month-old mice were isolated as previously described (Kato et al., 2008). Briefly, a rapidly dissected brain was immersed in ice cold NaHCO3-bufferd saline solution (in mM): 120 NaCl, 2.5 KCl, 1 MgCl2, 1.25 Na2PO4, 2 CaCl2, 26 NaHCO3, and 10 glucose (pH 7.2), osmolarity 300 ± 2 mOsm/l. Coronal hippocampal slices (400 μm thick) were prepared by a Vibroslice (Campden Instruments) in ice cold NaHCO3-bufferd saline solution and then were recovered at room temperature in continuously oxygenated (95% O2, 5% CO2), NaHCO3-bufferd saline solution for 0.5–5 hr.