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Materials and methods Drugs Caffeine, PTZ and MK-801 (Sigma, Israel) were dissolved in 0.9% saline and administered i.p. in a 1ml per 100 grams animal weight. Based on the literature, a subconvulsive dose of PTZ (30 mg/kg),a high dose of caffeine (80 mg/kg) and a moderate dose of MK-801 (0.15 mg/kg) were used1, 2. Genotyping process After DNA extraction from ear tissue (Quick Extract DNA extraction solution, Epicentre), a PCR reaction was performed (2XReddyMix PCR Master Mix, Thermo Scientific) using the following primers: For the wild type Ahi1 allele: Forward- CAA ATG GCG ATT ACC GTT GA; backward- TGC CCA GTC ATA GCC GAA TA. For the gene trap allele: forward- CAA ATG TTG CTT GTC TGG TG; backward- GTC AGT CGA GTG CAC AGT TT. Real-time PCR Following thiopental anesthesia and cervical dislocation, brains were surgically removed. Total RNA was extracted using RNAquous Micro kit (Ambion), followed by DNAse kit (Ambion) treatment. Total RNA concentrations per sample were determined by Nanodrop; from each sample, 10 μl was converted, using a High Capacity cDNA Reverse Transcription kit (Applied Biosystems, USA) to a total 20 μl cDNA sample. Real-time PCR was performed in a GeneAmp 7500 Real Time PCR System using TaqMan? Assays-on-Demand? Gene Expression assays (Applied Biosystems) for mouse Ahi1. Relative quantities of Ahi1 mRNA were calculated in comparison to GAPDH mRNA, a housekeeping gene. Data were analyzed using the SDS v2.3 software (Applied Biosystems). Western blot analysis Following thiopental anesthesia and cervical dislocation, whole brain specimens were obtained from pups (PN 1-3) and adult mice (PN ~60) and snap-frozen. Rabbit polyclonal antibody to Ahi1 protein (Santa Cruz Biotechnology, INC), mouse polyclonal anti- β-actin (MP Biomedical) and secondary antibodies (Jackson Laboratories (Bar Harbor, Maine, USA) were used. SDS, transfer and analysis were carried out in accordance with standard procedures . Briefly, the tissue was hydrolyzed and h