The present study was conducted at the Laboratory of Veterinary Anatomy, Faculty of Applied Biological Sciences, Gifu University, Japan, during the period from July 2006 to June 2007. General considerations: All buffers and equipment used for in situ hybridization experiments were autoclaved and sterilized by dry heater to avoid RNase contamination and keep mRNA intact. All procedures were performed with gloves. Basic solutions: 20x SSC: Nacl of 175.3 g and sodium citrate of 88.2 g were dissolved in 800 ml of distilled water. After adjustment of pH to 7.0 and volume to 1000ml were sterilized by autoclaving. 1.2M phosphate buffer (pH7.4): NaH2PO4-2H2O of 3.56 g and Na2HPO4-12H2O of 34.8 g were dissolved in 100 ml distilled water and sterilized by autoclaving. 0.2M phosphate buffer (pH7.4): NaH2PO4-2H2O of 5.93 g and Na2HPO4-12H2O of 58 g were dissolved in 1000 ml distilled water and sterilized. 1M dithiothreitol (DTT): Dithiothreitol of 154 mg was mixed in 1 ml disterilized distilled water (DDW) and kept at -20 ?C. Coating of micro slides with 3-aminopropyltriethoxysilane: The micro slides were washed with detergent and dried completely. Then the slides were immersed into the acetone followed by acetone containing 2% 3-aminopropyltriethoxysilane, acetone and distilled water for 5 minutes in each step. After drying, slides were sterilized at 180 ?C for 4 h and kept at room temperature until use. Animals and tissue preparation: Four adult rats (weighing 250 to 270g) were used in the present study. The animal handling procedures were approved by the Animal Experimental Committee of the Faculty of Applied Biological Sciences, Gifu University. The animals were anesthetized and sacrificed by sodium pentobarbital (50 mg/kg), and fresh brains were quickly removed and immediately frozen on powdered dry ice. The coronal sections at hippocampus region were cut at 30 µm on a cryostat, thaw-mounted onto the 3-aminopropyltriethoxysilane coated slides and stored at -30°C until use. Oligonucleotide probes: The 45 mer antisense and sense oligo cDNA probes of glutamate receptor 1 (GluR1) were designed based on the rat GluR1 cDNA sequence (Boulter et al., 1990) and were synthesized commercially (Rikaken, Nagoya, Japan). The sequence of the GluR1 antisense is: 5’-GTCACTGGTTGTCTGGTCTCGTCCCTCTTCAAACTCTTCGCTGTG-3’, and the complementary sequence of antisense probe used as sense probe. The probes were labeled at 3’-end with [35S] dATP (46.25 TBq/mmol; PerkinElmer Life Science, Waltham, MA, USA) using terminal deoxynucleotidyl transferase (Takara, Tokyo, Japan) to obtain a specific activity of approximately 1-2 × 109 d.p.m/µg. In situ hybridization: The slide-mounted sections being warmed to room temperature were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 15 min (all steps were performed at room temperature unless otherwise indicated), rinsed three times (5 min each) in 4X standard saline citrate (SSC; pH 7.4; 1X SSC contains 0.15 M sodium chloride and 0.015 M sodium citrate), and dehydrated through a graded ethanol series (70-100%). Sections were then defatted with chloroform for 3 min, and immersed in 100% ethanol (twice for 5 min each time) before being subjected to hybridization. Then the hybridization was performed by incubating the sections with the following buffer at 41°C overnight: 4X SSC, 50% deionized formamide, 0.12M phosphate buffer (pH 7.4), 1% Denhardt’s solution (Nacalai, Kyoto, Japan), 0.025% yeast tRNA (Roche, Mannheim, Germany), 10% dextran sulfate (Nacalai, Kyoto, Japan). The buffer contained probes labeled with [35S] dATP (46.25 TBq/mmol; PerkinElmer Life Science, Waltham, MA, USA; approximately 1-2 × 107 d.p.m/ml, 0.3 ml/slide). After hybridization, sections were rinsed in 1X SSC (pH 7.4) for 10 min followed by rinsing three times in 1X SSC at 55°C for 20 min, dehydrated through a graded ethanol series (70 to 100%), and sections were exposed to x-ray film (Fuji Medical X-Ray Film, Tokyo, Japan) for 7 days. After film autoradiograms, sections were coated with NTB-2 emulsion (Eastman Kodak Company, Rochester, NY, USA) diluted 1:1 with distilled water and exposed at 4°C for 4 weeks in a tightly sealed dark box. After being developed in D-19 developer (Eastman Kodak Company, Rochester, NY, USA), the sections were fixed, and washed with tap water and dehydrated. Some sections were counterstained with 0.1% cresyl violet to allow morphological identification. Control studies: The complementary sequence of antisense probe was used as a sense probe for negative control in this experiment.