Hormonal Regulation of Glutamate Receptor Gene Expression in the Anteroventral Periventricular Nucleus of the Hypothalamus

MATERIALS AND METHODS

Animals and treatments. The protocols used here were approved by the Oregon Regional Primate Research Center Institutional Committee for the Care and Use of Animals in Research and Education, in accordance with the guidelines of the National Institute of Health and United States Department of Agriculture. Juvenile female Sprague Dawley rats were obtained from B & K Universal Inc. (Kent, WA) and housed on a 14:10 light/dark schedule with light on at 5:00 A.M. Food and water were available ad libitum. Juvenile female Sprague Dawley rats were ovariectomized bilaterally on day 22 of life and implanted subcutaneously with a silastic capsule containing 17β-estradiol (E2) (400 μg/ml dissolved in corn oil; tubing internal diameter, 1 mm; outer diameter, 2.16 mm; 20 mm in length/100 gm body weight; Sigma, St. Louis, MO) [n = 12 (4 for immunocytochemistry, 4 for in situ hybridization, and 4 for double in situ hybridization)] or control capsules (oil only) [n = 12 (4 for immunocytochemistry, 4 forin situ hybridization, and 4 for double in situhybridization)] 5 d later. This dosage of E2 has been confirmed to reliably reproduce the preovulatory levels of plasma E2 measured during the initiation of puberty in the rat (Andrews et al., 1981). Forty-eight hours after implantation, paired groups of animals (treated with E2 or control capsules) were killed by transcardial perfusion. In addition, to simulate the sequence of events observed during the afternoon of the first proestrus, a time during which elevated E2 levels are accompanied by a marked and abrupt increase in progesterone levels (Parker and Mahesh, 1976; Andrews et al., 1980), four groups of animals (n = 4 for each group) were ovariectomized and treated with E2 (see above) for 48 hr and then injected subcutaneously with progesterone at 12:00 P.M. (1 mg/animal; Sigma) or corn oil and perfused 3 or 24 hr after injection. This dosage of progesterone can markedly increase GnRH mRNA levels in estrogen-primed immature rats (Kim et al., 1989; Ma et al., 1992) but not in animals without pretreatment with estrogen (Ma et al., 1992). Two time points (3 and 24 hr) were evaluated to compare the short- and long-term effects of progesterone on the expression of glutamate receptors, which are thought to correspond to the positive and negative feedback effects of progesterone (Barraclough et al., 1986).

Tissue preparation and immunocytochemistry. Animals were deeply anesthetized with tribromoethanol (Aldrich, Milwaukee, WI), and a 1–2 ml blood sample was taken from the right atrium of the heart immediately before perfusion for steroid analysis. After a brief rinse with normal saline (50–100 ml), each rat was perfused transcardially with ice-cold 4% paraformaldehyde (Electron Microscopy Sciences, Fort Washington, PA) in 0.1 m borate buffer at pH 9.5 for 20 min. The brains were quickly removed, post-fixed for 2 hr in the same fixative containing 20% sucrose, and then placed in 20% sucrose in 0.02 m potassium PBS (KPBS) for cryoprotection. Thirty-micrometer-thick frozen sections through the AVPV of each brain were collected, at a frequency of one of four, in chilled 0.02 m KPBS. Free-floating sections were incubated in anti-GluR1, anti-GluR2/3, GluR4 (raised in rabbit; Chemicon, Temecula, CA), GluR5/6/7 (mouse monoclonal antibody; PharMingen, San Diego, CA), and anti-NMDAR1 primary antisera (raised in rabbit; Chemicon) at 4°C with constant agitation for 72 hr. The GluR1, GluR2/3, and GluR4 antibodies were generated from a C-terminus peptide of rat GluR1, GluR2, and GluR4, respectively. GluR5/6/7 antibody was prepared against a fusion protein from the N-terminal putative extracellular portion of GluR5, which belongs to the kainate type of glutamate receptor, and affinity purified using the immunogen peptide (Wenthold et al., 1992;Huntley et al., 1993). The anti-GluR1 recognizes GluR1, the anti-GluR2/3 recognizes GluR2 and GluR3, the anti-GluR4 recognizes GluR4, and the anti-GluR5/6/7 recognizes GluR5, GluR6, and GluR7 but not other members of the GluR family. The NMDAR1 antibody was generated from a synthetic peptide corresponding to the C terminus of rat NMDAR1 subunit and affinity purified using the immunogen peptide. Selective for splice variants NMDAR1–1a, NMDAR1-1b, NMDAR1-2a, and NMDAR1-2b, this antibody is specific for NMDAR1 and does not appear to cross-react with other glutamate receptor subunits (Petralia et al., 1994). The GluR1, GluR2/3, GluR4, GluR5/6/7, and NMDAR1 antibodies were diluted to 1:4000, 1:2000, 1:1000, 1:2000, and 1:500, respectively, in KPBS that contained 2% normal goat serum (Colorado Serum Co.) and 0.3% Triton X-100 (Bio-Rad, Hercules, CA). After brief rinses in KPBS containing 0.3% Triton X-100, the sections were then incubated in a biotinylated goat anti-rabbit IgG secondary antiserum (Vector Laboratories, Burlingame, CA) at room temperature. The sections were rinsed in KPBS and stained by using the ABC method (Hsu et al., 1981) with commercial reagents (Elite kit; Vector Laboratories) at room temperature, and the incubations in the secondary antiserum and ABC solution were repeated, followed by several rinses in KPBS. The sections were then color-reacted with 0.03% diaminobenzidine (Sigma), 2.5% nickel ammonium sulfate, 0.2% d-glucose, 0.04% ammonium chloride, and 0.001% glucose oxidase (Sigma) in 0.1 macetate buffer. All sections were mounted on gelatin-coated microscopic slides, air dried, dehydrated, and coverslipped.

In situ hybridization. Twenty-micrometer-thick frozen sections (at a frequency of one of four) through the AVPV of each brain were collected in chilled 0.02 m KPBS that contained 0.25% paraformaldehyde, pH 7.4, mounted onto gelatin-subbed, poly-l-lysine-coated microscopic slides, and processed for in situ hybridization as described previously (Simmons et al., 1989). To control for procedural artifacts, all tissue hybridized with each probe was processed together in a single in situ hybridization histochemistry experiment. After a 30 min proteinase K digestion (10 μg/ml at 37°C; Boehringer Mannheim, Indianapolis, IN) and acetylation (0.0025% acetic anhydride at room temperature), the sections were dehydrated in ascending alcohols and dried under vacuum overnight. T7 polymerase (Promega, Madison, WI) was used to transcribe ³⁵S-labeled antisense cRNA probes from a 721 bp PstI fragment of plasmid pBluescript SK(−) complementary to the 5′ coding region of rat glutamate receptor subunit gene GluR1, from a 428 bp SphI fragment complementary to the 5′ coding region of rat glutamate receptor subunit gene GluR2, or from a 723-bp SarI fragment, which is complementary to the 5′ coding region of rat glutamate receptor subunit gene GluR3 (all three probes were kindly provided by Dr. S. Heinemann, The Salk Institute, La Jolla, CA). For the NMDAR1 probe, T3 polymerase (Promega) was used to transcribe a³⁵S-labeled antisense cRNA probe from an 868 bp cDNA insert corresponding to nucleotides 699–1567 of the rat NMDAR1 gene sequence (Moriyoshi et al., 1991) contained in a pBluescript SK(−) transcription vector (Urbanski et al., 1994). T3 or T7 polymerase was also used to transcribe ³⁵S-labeled antisense cRNA probes from cDNA inserts corresponding to metabotropic glutamate receptor (mGluR) subtypes: mGluR1 (nucleotides 3541–4282), mGluR2 (nucleotides 2712–3294), mGluR3 (nucleotides 2376–3215), mGluR4 (nucleotides 2910–3704), mGluR5 (nucleotides 1246–1786), and mGluR6 (nucleotides 3669–4418). The mGluR plasmids were generously provided by Dr. S. Nakanishi (Kyoto University, Kyoto, Japan), and mGluR 5 subclone was provided by Drs. Y. J. Ma and S. Ojeda (Oregon Regional Primate Research Center). The radiolabeled cRNA probe was purified by passing the transcription reaction solution over a Sephadex G-50 Nick column (Pharmacia, Piscataway, NJ), and four 100 μl fractions were collected and counted by using a scintillation counter (Packard, Meridian, CT). The leading fraction was heated at 65°C for 5 min with 500 μg/ml yeast tRNA (Sigma) and 50 μm dithiothreitol (DTT) (Stratagene, La Jolla, CA) in DEPC (Sigma) water and then diluted to an activity of 5 × 10⁶ with hybridization buffer containing 50% formamide (Boehringer Mannheim), 0.25 msodium chloride, 1× Denhardt’s solution (Sigma), and 10% dextran sulfate (Pharmacia). This hybridization solution was pipetted onto the sections (80 μl/slide), which were covered with a glass coverslip, and sealed with DPX (Electron Microscopy Sciences) before incubation for 20 hr at 58°C. After hybridization, the slides were washed four times (5 min each) in 4× SSC before RNase digestion (20 μg/ml for 30 min at 37°C; Sigma) and rinsed at room temperature in decreasing concentrations of SSC that contained 1 mm DTT (2×, 1×, 0.5× for 10 min each) to final stringency of 0.1× SSC at 65°C for 30 min. After dehydration in ascending alcohols, the sections were exposed to DuPont (Billerica, MA) Cronex x-ray films for 4 and 8 d, together with autoradiographic ¹⁴C microscales (Amersham, Arlington Heights, IL), before being dipped in NBT-2 liquid emulsion (Eastman Kodak, Rochester, NY). The dipped autoradiograms were developed 21 d later with Kodak D-19 developer, and the sections were counterstained with thionin through the emulsion.

Double in situ hybridization. The method used in this study was a modification of that reported by Springer et al. (1991) and described in detail previously (Simerly et al., 1996). Briefly, in vitro transcription of the GluR1 insert was performed as described above, except that 1 μl of a 2 mmsolution of digoxigenin-labeled UTP (Boehringer Mannheim) was substituted for the isotope ³⁵S-labeled UTP (DuPont NEN, Boston, MA). After incubation with T7 polymerase, the reaction mixture was treated with DNase (Promega) and RNAsin (Promega) and stabilized with EDTA (Sigma) and salt, and the total volume was adjusted to 100 μl with 20 mm DTT. The cRNA probe was then precipitated with ethanol, dried, and resuspended in 100 μl of DEPC-treated water. A total of 150 μl of digoxigenin-labeled cRNA probe was diluted in 1 ml of hybridization solution containing ³⁵S-labeled GluR2 probe as prepared above. Prehybridization, hybridization, and posthybridization procedures were basically identical to those described above, except that the sections were not dehydrated and dried after the last 0.1× SSC rinse but were processed for further localization of digoxigenin-labeled hybrids. Before immunohistochemical detection of digoxigenin-labeled hybrids, the slides were incubated overnight in 2× SSC containing 0.05% Triton X-100 and 2% normal goat serum at room temperature. The next day, the slides were incubated in a 1:1000 dilution of the anti-digoxigenin–alkaline phosphatase conjugate (Boehringer Mannheim) for 5 hr at room temperature, rinsed, and then incubated overnight at room temperature in the chromogen solution, and the staining reaction was stopped by placing the slides in 10 mm Tris-HCl with EDTA. The sections were further dehydrated in ethanol (containing SSC and DTT), dried under vacuum for 30 min, and exposed to DuPont Cronex x-ray film for 4 d before being dipped in Ilford K5 emulsion (Polysciences, Warrington, PA) for autoradiography to detect ³⁵S-labeled hybrids. After a 3 week exposure, the emulsion-coated slides were developed as described above, washed, dehydrated in ethanol, dried under vacuum for 30 min, coverslipped with DPX, and evaluated at a high magnification (400×).

Quantification and analysis. The optical density of the autoradiographic images of GluR1, GluR2, GluR3, and NMDAR1 mRNAs on x-ray films was measured by using a Macintosh-based image analysis system and NIH Image software from National Institutes of Health. Each film was illuminated with a ChromaPro 45 light source, which provided even illumination, and the image was obtained with a Dage-MTI (Michigan City, IN) 70 series video camera equipped with a Newvicon tube. The optical density of autoradiographic images over the AVPV (4 d exposure) was measured on film at the same level from each brain. The boundaries of the AVPV were determined from observation of the corresponding Nissl-stained sections. A total of eight frames of images were averaged, and the film densities were integrated over the entire AVPV. The mean optical density over a large irregularly shaped region over the third ventricle adjacent to the AVPV that did not contain specific hybridization signals was also measured on each section and used to calculate the mean background density, which was subtracted from the optical density measurement of signals over the AVPV. Although these mean optical density measurements do not correspond to absolute optical density units, they reflect relative mRNA levels in the AVPV. Commercially available ¹⁴C autoradiographic standards were exposed to each x-ray film along with the experimental material. The mean optical density of an interactively defined region over each standard was measured; these measurements confirmed the linearity of the responsiveness of the film, as well as the consistency of signal detection across films. The mean optical densities of the autoradiographic images recorded over the AVPV all fell within the linear range of the standard values. Ratios between levels of GluR1/GluR2 mRNA were calculated by dividing the optical density of GluR1 mRNA by the optical density of GluR2 mRNA measured for adjacent sections from the same animal. Similarly, GluR3/GluR2 mRNA ratios were also calculated. A two-way ANOVA was used to test for significant differences in densities of GluR1–3 or NMDAR1 mRNAs among groups in each experiment, and a post hoc Fisher’s test was used to identify significant differences between individual groups.p ≤ 0.05 was defined as significant. The colocalization of GluR1 and GluR2 mRNA was analyzed by counting, at a magnification of 400×, the number of darkly stained digoxigenin-labeled GluR1 mRNA-containing neurons, the number of GluR2 mRNA-containing neurons, visualized by clusters of silver grains at a density of three times greater than that of background, and the number of double-labeled cells contained within the morphological borders of the AVPV.

Ultrastructural analysis. To confirm that NMDAR1 immunoreactivity was located in terminals that synapse onto AVPV neurons, sections were prepared for electron microscopic analysis. Two ovariectomized juvenile female rats that had been treated for 48 hr with estradiol as described above were deeply anesthetized with tribromoethanol (Aldrich) and perfused transcardially with 100 ml of normal saline, followed by 300 ml of ice-cold fixative containing 4% paraformaldehyde, 15% picric acid, and 0.08% glutaraldehyde in 0.1m phosphate buffer (PB), pH 7.4, for 20 min. The brains were quickly removed, post-fixed for 2 hr in the same fixative without glutaraldehyde, and then rinsed in PB. Fifty-micrometer-thick vibratome sections through the AVPV were collected in PB, transferred for 20 min in PB containing 10% sucrose for cryoprotection, rapidly frozen by immersion in liquid nitrogen, and thawed at room temperature. After several rinses in PB, they were further incubated for 10 min in 1% sodium borohydride (Sigma) in PB to eliminate unbound aldehydes and thoroughly washed in PB. The sections were incubated for 72 hr at 4°C under agitation in a rabbit anti-NMDAR1 antiserum (Chemicon), diluted to 1:500 in PB containing 2% normal goat serum. After several washes, sections were transferred to a biotinylated goat anti-rabbit IgG secondary antiserum (Vector Laboratories) for 2 hr at room temperature, rinsed in PB, and incubated in ABC solution (Elite kit; Vector Laboratories) for 2 hr at room temperature. The tissue-bound peroxidase was visualized by a DAB reaction (10 mg DAB and 5 μl of hydrogen peroxide in 30 ml PB). After extensive washes, sections were osmicated in 1% osmium tetroxide in PB for 15 min, rinsed, dehydrated in an ascending gradient of alcohols and in propylene oxide, and left for 2 hr in propylene oxide/araldite (1:1). The tissue was polymerized in araldite for 48 hr at 60°C, and ultrathin sections were cut on a Reichert ultratome and examined under a Jeol-1200 EX (Peabody, MA) electron microscope.

Hormone assays. Blood samples that were taken immediately before perfusion were collected in Eppendorf tubes, left standing for coagulation at room temperature for 2 hr, and stored at 4°C for 24 hr. Serum was separated by centrifugation and stored at −20°C until assayed for E2 and progesterone by RIA as described previously (Resko et al., 1975; Hess et al., 1981). All the samples in each experiment were run in a single assay, with an intra-assay variation of <8%, and the lower limits of detection were 5 pg/tube in the estrogen assay, <3.2% intra-assay variation, and 12 pg/tube lower limits of detection in the progesterone assay.