Domoic acid (Sigma, St. Louis, MO, USA) was dissolved in saline solution (0.9% NaCl) at 1 mg/ml. The glutamate receptor antagonists, 2,3-dioxo-6-nitro-7-sulfamoylbenzo[f]quinoxaline (NBQX, Sigma, St. Louis, MO, USA) and dizocilpine maleate (MK-801, Tocris Bioscience, Ellisville, MO, USA) were dissolved in saline solution at 3 mg/ml. Edaravone (Tocris Bioscience, Ellisville, MO, USA) was dissolved in dimethyl sulfoxide at 10 mg/ml.
Animals and treatments
Eight-week-old male Sprague–Dawley rats weighing approximately 300 g were purchased from SLC (Shizuoka, Japan) and habituated to the laboratory for 1 week before the experiments began. All procedures were approved by the Animal Care Committee of Aichi Medical University. All rats were kept in a climate-controlled room under 12/12-hour light–dark cycles with free access to food and tap water throughout the studies.
For the mass spectrometric analysis, rats were administered DA intraperitoneally at a dose of 1 mg/kg. Thirty minutes later, the rats were deeply anesthetized with pentobarbital sodium and were then perfused transcardially with cold phosphate-buffered saline (PBS). The brains were then removed and weighed. For histological examinations, rats were administered DA (1 mg/kg) or vehicle intraperitoneally. At 24 hours or 5 days after DA or vehicle administration, rats were deeply anesthetized with pentobarbital sodium and perfused transcardially with cold PBS followed by cold 4% paraformaldehyde in PBS, pH 7.2. The brains were removed and postfixed with 4% paraformaldehyde at 4°C for 24 h, then were dehydrated and embedded in paraffin. The paraffin blocks were stored at 4°C. Sections were cut at a thickness of 4 μm and processed for Hematoxylin-Eosin staining, in situ hybridization, immunohistochemistry, and TUNEL staining. For the qRT-PCR and western blot experiments, DA or vehicle administered rats were sacrificed by decapitation. The brains were rapidly excised, and hippocampi were rapidly dissected on an ice-cold dissection board. For dose–response studies, rats were administered DA intraperitoneally at doses of 0.3, 1, and 3 mg/kg and were sacrificed 24 hours later. For time-course studies, rats were sacrificed at 0, 6, 12, 24, and 48 hours after intraperitoneal administration of 1 mg/kg DA. Seizures  were observed in 3 mg/kg group. For pretreatment studies, NBQX, MK-801, and edaravone were administered to rats intraperitoneally at one hour before DA administration, and the rats were sacrificed at 24 hours after DA administration. Control rats received an equivalent volume of vehicle.
Preparation of extracts from rat brains for mass spectrometric analysis
Isolated rat brains were minced in 50% aqueous methanol (8 ml), homogenized, and centrifuged at 10,000 g for 20 min. The supernatant was passed through a cartridge column of Sep-Pak® Plus C18 (Waters Co., Milford, MA, USA), and filtered with Amicon® Ultra 0.5 ml 10 K centrifugal filter devices (Millipore Co., Billerica, MA, USA). The filtrate was subjected to LC-MS/MS analysis.
The LC separation was performed using an ACCELA HPLC system (Thermo Fisher Scientific, Waltham, MA, USA). Separation was accomplished with a TSK-GEL ODS-80Ts column (150 × 2.0 mm, Tosoh, Tokyo, Japan) at 30°C. Water containing 0.1% formic acid was used as eluent A, and acetonitrile containing 0.1% formic acid was used as eluent B. The LC elution gradient employed was: 0 to 5 min 100% A, 5 to 10 min eluent A decreased linearly to 50%, and 10 to 12 min 100% B. The eluent shifted back to 100% A and was held for 3 min to equilibrate the column for the next sample. The flow rate was 0.5 ml/min. The MS analysis was accomplished using a LTQ Velos spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) equipped with a heated electrospray ionization (HESI) source. Positive ion HESI was chosen for the detection of DA. The source voltage was set at 3 kV, the capillary temperature was set at 350°C, and the tube lens offset was set at 15 V. The sheath gas flow rate was 35 (arbitrary units) and the auxiliary gas flow rate was 10 (arbitrary units). Full scan experiments were performed in the range m/z 50–1000. The total number of microscans was set at 1 and the maximum injection time at 10 ms. Subsequent MS/MS experiments were performed in the range m/z 85–400. The maximum injection time was set at 100 ms. Reproducible calibration curves for DA were obtained with correlation coefficients greater than 0.999 (known concentration versus analyte), and the curves were linear over the range of 0.2-10 ng/ml.
In situ hybridization
The sequence of the WDR35 probe was 5′AAGCACAAACTGAGGGTGATTTTCATCAGC-3′. Hybridization was performed at 105°C for 5 minutes and 50°C for 3 hours, with the probe diluted at 1:1000. The hybridization signal was amplified with the TSA Biotin System (PerkinElmer, Waltham, MA, USA) and visualized with DAB (Falma, Tokyo, Japan). The sections were counterstained with hematoxylin as described previously .
Immunohistochemical staining for WDR35
Rat brain sections were treated with 3% H2O2 in methanol for 30 min to inactive endogenous peroxidase, blocked at room temperature for 60 min with 4% goat serum albumin in PBS, and rinsed in 0.01 M PBS with 0.1% Tween20® (PBS-T). Sections were then incubated at room temperature overnight with anti-WDR35 antibody (1:1000, Abcam, Cambridge, UK) in PBS-T. Immunohistochemical staining was done according to the manufacturer’s protocol using a Vectastain ABC Elite kit (Vector Laboratories Inc., Burlingame, CA, USA) with the 3,3′-diaminobenzidine (DAB) reaction. Sections were counterstained with hematoxylin. Sections with no primary antibody were included as negative controls to verify the secondary antibody specificity.
Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay
TUNEL was performed using the ApopTag® Plus In Situ Apoptosis Detection Kit (Chemicon International, Temecula, CA, USA) according to the manufacturer’s instructions and visualized with the TSA Biotin System and DAB. The sections were counterstained with 0.5% methyl green (Wako Pure Chemicals, Osaka, Japan). Apoptotic and nonapoptotic cells were counted in three randomly chosen microscopic fields, and results are expressed as percentage of apoptotic cells ± S.E.
Quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR)
Total RNA (1 μg) was extracted from rat hippocampus homogenate with TRIzol® reagent (Invitrogen, Carlsbad, CA, USA) and reverse transcribed with a ReverTra Ace qPCR RT kit (Toyobo, Osaka, Japan). Quantitative RT-PCR was performed as described previously  with the ABI StepOne Plus real-time PCR system and a Taqman Gene Expression Assay (Applied Biosystems, Tokyo, Japan) according to the manufacturer’s instructions. WDR35 mRNA levels were quantified relative to GAPDH mRNA levels, the internal control. The results are presented according to the ΔΔCT method as ratios of the target to the internal control as described previously .
Western blot analysis
Rat hippocampi were homogenized in lysis buffer [62.5 mM Tris–HCl (pH6.8), 2.5% SDS, 10% glycerol] containing a protease inhibitor cocktail (Roche Applied Sciences, Mannheim, Germany) and heated in boiling water for 5 min. After centrifugation, the supernatants were collected, and the protein concentration of samples was determined by the DC protein assay (Bio-Rad Laboratories, Hercules, CA, USA). Samples (30 μg protein) were mixed with 2-mercaptoethanol and Bromo Phenol Blue buffer, separated by SDS-PAGE, and transferred to PVDF membranes (Millipore Corporation, Billerica, MA, USA). The blots were incubated with rabbit antibodies against WDR35 (132kDa, 1:1000; Abcam, Cambridge, UK), p38 MAPK (43 kDa, 1:1000; Cell Signaling Technology, Danvers, MA, USA), phospho-p38 MAPK (43 kDa, 1:1000; Cell Signaling Technology) or GAPDH (37 kDa, 1:20,000; Cell Signaling Technology) followed by peroxidase-conjugated anti-rabbit IgG (1:5000; Zymed Laboratories, San Francisco, CA, USA). The apparent molecular weight of each protein was determined by BlueStar Prestained Protein Marker (Nippon Genetics Europe GmbH, Dueren, Germany). Detection was performed with the ECL Prime Western Blotting Reagent (GE Healthcare, Buckinghamshire, UK). Protein levels were quantified by densitometric scanning and expressed as the ratio to GAPDH, as described previously .
All results were expressed as mean ± standard error of the mean (SEM). Data were analyzed for statistical significance with one-way analysis of variance (ANOVA), followed by post hoc analysis with the Bonferroni method. Differences were considered significant at P < 0.05.