ARTICLES ATTTCTTTAATTTCTCTTCTCC-1908). The thermocycler protocol ple, the ROd value of the band within the antibody lane(A) was divided by the involved an initial denaturation cycle (5 min, 95C), 34 cycles of denatura- ROD value of the band within the input lane (1). To control for equal loading tion(I min, 95C), annealing(2 min 30 s, 56C)and extension(I min, 72 between samples, the final signal of the exon 17 GR promoter, amplified fror eC), followed by a final extension cycle(5 min, 72oC)terminating at the acetyl-histone H3-K9 immunoprecipitations, was divided by the final sig- 4C. The PCR product(285 bp)was used as a template for subsequent PCR nal from the p-actin promoter-a amplified from the same precipitate reactions using nested primers(forward: 1738-TTTTTTTGTTAGTGT- AtATATTT- 1761; reverse: 1914-TTCTCCCAAACTCCCTCC- 1897). The Intracerebroventricular infusions. Animals were anesthetized and secured in nested PCR product(177 bp)was then subcloned (Original TA cloning kit, a stereotaxic frame, and a stainless-steel guide cannula(22 gauge),8 mm prepped. Ten plasmids containing the ligated exon 1, GR promoter DNA rior to bregma, 2.0 mm lateral to midline, 3.0 mm ventral to the brain surface) Chromatin immunoprecipitation(ChIP) assay. ChIP assays 3 were done micro-syringe)through the infusion cannula over &. Pharmacia Biotech)starting from procedure C in the manufacturer's proto- seven consecutive days as described below. Animals were removed from their col. The sequencing reactions were resolved on a denaturing (6%)PAGE cages and gently held while an infusion cannula(28-gauge)attached to tubing PE 20)was lowered into the guide. A total volume of 2 ul of TSA(100 ng/ml eriod. Infusion using the ChIP assay kit protocol(06-599, Upstate Biotechnology). cannulae were left in place for an additional minute after infusion.Animals Hippocampi were dissected from each rat brain and chromatin was immuno. were then returned to their home cage precipitated using one of the following: rabbit polyclonal antibody to acetyl- 8 histone H3(Upstate Cell Signaling Solutions), rabbit polyclonal antibody to Western blotting. Studies were performed as previously described5.Protein a Biotechnology). One-tenth of the lysate was kept to quantify the amount of Pharmacia Biotech) was incubated with anti-rat GR-a monoclonal primary at hippocampal GR exon 1, promoter region(GenBank accession number peroxidase-conjugated sheep anti-mouse immunoglobulin G antibody AJ271870)of the uncrosslinked DNA was subjected to PCR amplification (1:3,000; Amersham Pharmacia Biotech). A single band was observed at a( forward primer: 1750-TGTGACACACTTCGCGCA-1767, reverse primer: -92 kDa upon exposure to film(Amersham Pharmacia Biotech).To verify the 943-GGAGGGAAACCGAGTTTC-1926) PCR reactions were done with the accuracy of sample loading, selected blots were reprobed with a monoclonal 9 FailSafe PCR system protocol using FailSafe PCR 2x PreMix D(Epicentre, antibody to tubulin(a-tubulin; 1: 5,000: Biodesign International).A single e InterScience). The thermocycler protocol involved an initial denaturation band was observed at -60 k Da and the intensity of the signal was similar in all o cycle(5 min, 95C), 34 cycles of denaturation(1 min, 95C), annealing(I lanes. Relative optical density(ROD)readings for the GR-a and a-tubulin on cycle bands were determined using a computer-assisted densitometry program histone H3-K9 immunoprecipitate, the rat hippocampal B-actin promoter-a triplicate on three different blots. The glucocorticoid receptor ROD value was region( Gen Bank accession number vo1217)of the uncrosslinked DNA was divided by the a-tubulin ROD value cortico subjected to PCR amplification(forward primer: 10-TCAACTCACTTC receptor signal for each sample For all studies, single blots were derived from TCTCTACT-29; reverse primer: 161-GCAAGGCTTTAACGGAAAAT- 180). samples from one animal. (Epicentre, InterScience)with the same thermocycler protocol as previously ers(8.5 X21.5 cm; Kent Scientific)for a 20-min period. Prestress blood sam- mplified the rat hippocampal exon Ib estrogen receptor-a promoter region restraint stress was performed during the light cycle between 12:00 and 15:00 o (Gen Bank accession number X98236)of the uncrosslinked DNA(forward with blood sampling (300 ul)from the tail vein at 10, 20, 40, 60 and 90 min primer:1836-GAAGAAACTCCCCICAGCAT-1855: reverse primer: 2346. after the onset of restraint 2. Plasma (10 ul)corticosterone was measured by Epicentre, InterScience). The thermocycler protocol involved an initial denat radioimmunoassay (RIA) with a highly specific B antiserum (B3-163 uration cycle(5 min, 95C), 34 cycles of denaturation(I min, 95.C), anneal- Endocrine Sciences) and [H]corticosterone(101 Ci/mmol; NEN) tracer. The antiserum cross-reacts slightly with deoxycorticosterone(-4%)but not ing (I min, 60"C)and extension (I min, 72"C), followed by a final extension with aldosterone, cortisol and progesterone(<1%). The intra-assay and from non-immunoprecipitated samples and immunoprecipitated samples standard curve 50% effective concentration was 16 ug/dl, and the detection were repeated exhaustively using varying amounts of template to ensure that limit of the assay was 0.63 ug/dl results were within the linear range of the PCR Products were separated on a 2%6 agarose gel to visualize bands corresponding to the exon 1, GR Promoter Note: Supplementary info is available on the Nature Neuroscience website (194 bp), B-actin promoter-a(171 bp) or exon 1b estrogen receptor-ox pro moter(493 bp) DNA fragments. Nucleic acids were transferred by Southern ACKNOWLEDGMENTS blot (overnight, 22 C)to positively charged nylon transfer membrane These studi Institutes for health (Hybond-N+, Amersham). An oligonucleotide(20 bp)specific for the exon 1, Research(CIHR)to M. J.M. and M.S. and from the GR promoter sequence(GenBank accession number AJ271870; forward: Canada to M.S. M J.M. is supported by a CIHR Se r soient ist award and the 881-TCCCGAGCGGTTCCAAGCCT-1907)was synthesized, as well as an project was supported by a Distinguished Investigat (M. J.M. )from the (GenBank accession number Vo1217; forward: 95-GTAAAAAAATGCTG. (NARSAD ance for Research on Schizophrenia and Affective Disorders oligonucleotide(21 bp) specific for the B-actin promoter-a sequence CACTGTC-115)and an oligonucleotide(20 bp)specific for the exon 1b estro- COMPETING INTERESTS STATEMENT gen receptor-a promoter sequence(GenBank accession number x98236: The authors declare that they have no competing financial interests. rward: 1942-AGAAAGCACTGGACATTICT-1961). The oligonucleotides were radiolabeled(1 ul T4 polynucleotide kinase(PNK), Promega)with 5 ul Received 2 March: accepted 26 May 2004 Y-32p]atp(AmershAm)(2h,37c)andthenhybridizedtothemembranesPublishedonlineathttp://www.nature.com/natureneuroscience/ that were then subjected to autoradiography. Relative optical density(rOD) adings were determined using a computer-assisted densitometry program 1. Agrawal, A.A. Phenotypic plasticity in the interactions and evolution of species. (MCID Systems; Imaging Research). To calculate the final signal for each sam Science294,321-326(2001) NATURE NEUROSCIENCE VOLUME 7 NUMBER 8 AUGUST 2004 853ARTICLES ATTTCTTTAATTTCTCTTCTCC-1908). The thermocycler protocol involved an initial denaturation cycle (5 min, 95 °C), 34 cycles of denatura￾tion (1 min, 95 °C), annealing (2 min 30 s, 56 °C) and extension (1 min, 72 °C), followed by a final extension cycle (5 min, 72 °C) terminating at 4 °C. The PCR product (285 bp) was used as a template for subsequent PCR reactions using nested primers (forward: 1738-TTTTTTTGTTAGTGT￾GATATATTT-1761; reverse: 1914-TTCTCCCAAACTCCCTCC-1897). The nested PCR product (177 bp) was then subcloned (Original TA cloning kit, Invitrogen), transformed, and ten different clones per plate were mini￾prepped. Ten plasmids containing the ligated exon 17 GR promoter DNA fragment were sequenced per animal (T7 sequencing kit, USB, Amersham Pharmacia Biotech) starting from procedure C in the manufacturer’s proto￾col. The sequencing reactions were resolved on a denaturing (6%) PAGE and visualized by autoradiography. Chromatin immunoprecipitation (ChIP) assay. ChIP assays33 were done using the ChIP assay kit protocol (06-599, Upstate Biotechnology). Hippocampi were dissected from each rat brain and chromatin was immuno￾precipitated using one of the following: rabbit polyclonal antibody to acetyl￾histone H3 (Upstate Cell Signaling Solutions), rabbit polyclonal antibody to NGFI-A or normal rabbit IgG non-immune antibody (both from Santa Cruz Biotechnology). One-tenth of the lysate was kept to quantify the amount of DNA present in different samples before immunoprecipitation (input). The rat hippocampal GR exon 17 promoter region (GenBank accession number AJ271870) of the uncrosslinked DNA was subjected to PCR amplification (forward primer: 1750-TGTGACACACTTCGCGCA-1767; reverse primer: 1943-GGAGGGAAACCGAGTTTC-1926). PCR reactions were done with the FailSafe PCR system protocol using FailSafe PCR 2× PreMix D (Epicentre, InterScience). The thermocycler protocol involved an initial denaturation cycle (5 min, 95 °C), 34 cycles of denaturation (1 min, 95 °C), annealing (1 min, 56 °C) and extension (1 min, 72 °C), followed by a final extension cycle (10 min, 72 °C) terminating at 4 °C. To control for unequal loading of acetyl￾histone H3-K9 immunoprecipitate, the rat hippocampal β-actin promoter-α region (GenBank accession number V01217) of the uncrosslinked DNA was subjected to PCR amplification (forward primer: 10-TCAACTCACTTC TCTCTACT-29; reverse primer: 161-GCAAGGCTTTAACGGAAAAT-180). PCR reactions were done with the same protocol, but using FailSafe PreMix L (Epicentre, InterScience) with the same thermocycler protocol as previously described. To control for purity of the NGFI-A immunoprecipitate, we PCR￾amplified the rat hippocampal exon 1b estrogen receptor-α promoter region (GenBank accession number X98236) of the uncrosslinked DNA (forward primer: 1836-GAAGAAACTCCCCTCAGCAT-1855; reverse primer: 2346- GAAATCAAAACACCGATCCT-2327), this time using FailSafe PreMix A (Epicentre, InterScience). The thermocycler protocol involved an initial denat￾uration cycle (5 min, 95 °C), 34 cycles of denaturation (1 min, 95 °C), anneal￾ing (1 min, 60 °C) and extension (1 min, 72 °C), followed by a final extension cycle (10 min, 72 °C) terminating at 4 °C. PCR reactions on DNA purified from non-immunoprecipitated samples and immunoprecipitated samples were repeated exhaustively using varying amounts of template to ensure that results were within the linear range of the PCR. Products were separated on a 2% agarose gel to visualize bands corresponding to the exon 17 GR promoter (194 bp), β-actin promoter-α (171 bp) or exon 1b estrogen receptor-α pro￾moter (493 bp) DNA fragments. Nucleic acids were transferred by Southern blot (overnight, 22 °C) to positively charged nylon transfer membrane (Hybond-N+, Amersham). An oligonucleotide (20 bp) specific for the exon 17 GR promoter sequence (GenBank accession number AJ271870; forward: 1881-TCCCGAGCGGTTCCAAGCCT-1907) was synthesized, as well as an oligonucleotide (21 bp) specific for the β-actin promoter-α sequence (GenBank accession number V01217; forward: 95-GTAAAAAAATGCTG￾CACTGTC-115) and an oligonucleotide (20 bp) specific for the exon 1b estro￾gen receptor-α promoter sequence (GenBank accession number X98236; forward: 1942-AGAAAGCACTGGACATTTCT-1961). The oligonucleotides were radiolabeled (1 µl T4 polynucleotide kinase (PNK), Promega) with 5 µl [γ-32P]ATP (Amersham) (2 h, 37 °C) and then hybridized to the membranes that were then subjected to autoradiography. Relative optical density (ROD) readings were determined using a computer-assisted densitometry program (MCID Systems; Imaging Research). To calculate the final signal for each sam￾ple, the ROD value of the band within the antibody lane (A) was divided by the ROD value of the band within the input lane (I). To control for equal loading between samples, the final signal of the exon 17 GR promoter, amplified from the acetyl-histone H3-K9 immunoprecipitations, was divided by the final sig￾nal from the β-actin promoter-α amplified from the same precipitate. Intracerebroventricular infusions. Animals were anesthetized and secured in a stereotaxic frame, and a stainless-steel guide cannula (22 gauge), 8 mm length (Plastic One Inc.) was aimed at the left lateral ventricle (1.5 mm poste￾rior to bregma, 2.0 mm lateral to midline, 3.0 mm ventral to the brain surface). After a 7-d recovery period, animals received a single infusion every day for seven consecutive days as described below. Animals were removed from their cages and gently held while an infusion cannula (28-gauge) attached to tubing (PE 20) was lowered into the guide. A total volume of 2 µl of TSA (100 ng/ml in DMSO) or DMSO vehicle alone was injected (using a Hamilton 10-ml micro-syringe) through the infusion cannula over a 1-min period. Infusion cannulae were left in place for an additional minute after infusion. Animals were then returned to their home cage. Western blotting. Studies were performed as previously described15. Protein (40 µg, whole cell extract) rendered on nitrocellulose (C+, Amersham Pharmacia Biotech) was incubated with anti-rat GR-α monoclonal primary antibodies in blocking buffer (1:4,000; Affinity BioReagents) and horseradish peroxidase-conjugated sheep anti-mouse immunoglobulin G antibody (1:3,000; Amersham Pharmacia Biotech). A single band was observed at ∼92 kDa upon exposure to film (Amersham Pharmacia Biotech). To verify the accuracy of sample loading, selected blots were reprobed with a monoclonal antibody to tubulin (α-tubulin; 1:5,000; Biodesign International). A single band was observed at ∼60 kDa and the intensity of the signal was similar in all lanes. Relative optical density (ROD) readings for the GR-α and α-tubulin bands were determined using a computer-assisted densitometry program (MCID 4.0; Imaging Research) from samples (n = 6 animals/group) run in triplicate on three different blots. The glucocorticoid receptor ROD value was divided by the α-tubulin ROD value to determine the final glucocorticoid receptor signal for each sample. For all studies, single blots were derived from samples from one animal. HPA response to restraint stress. Animals were placed in Plexiglass restrain￾ers (8.5 × 21.5 cm; Kent Scientific) for a 20-min period. Prestress blood sam￾ples were taken from rats within 30 s of removal from the home cage, and restraint stress was performed during the light cycle between 12:00 and 15:00 with blood sampling (300 µl) from the tail vein at 10, 20, 40, 60 and 90 min after the onset of restraint12. Plasma (10 µl) corticosterone was measured by radioimmunoassay (RIA) with a highly specific B antiserum (B3-163; Endocrine Sciences) and [3H]corticosterone (101 Ci/mmol; NEN) tracer. The antiserum cross-reacts slightly with deoxycorticosterone (∼4%) but not with aldosterone, cortisol and progesterone (<1%). The intra-assay and interassay coefficients of variation were 8.8% and 10.4%, respectively. The standard curve 50% effective concentration was 16 µg/dl, and the detection limit of the assay was 0.63 µg/dl. Note: Supplementary information is available on the Nature Neuroscience website. ACKNOWLEDGMENTS These studies were supported by a grant from the Canadian Institutes for Health Research (CIHR) to M.J.M. and M.S. and from the National Cancer Institute of Canada to M.S. M.J.M. is supported by a CIHR Senior Scientist award and the project was supported by a Distinguished Investigator Award (M.J.M.) from the National Alliance for Research on Schizophrenia and Affective Disorders (NARSAD). COMPETING INTERESTS STATEMENT The authors declare that they have no competing financial interests. Received 2 March; accepted 26 May 2004 Published online at http://www.nature.com/natureneuroscience/ 1. Agrawal, A.A. Phenotypic plasticity in the interactions and evolution of species. Science 294, 321–326 (2001). NATURE NEUROSCIENCE VOLUME 7 | NUMBER 8 | AUGUST 2004 853 © 2004 Nature Publishing Group http://www.nature.com/natureneuroscience