Supporting Text

Animal Husbandry. All mouse protocols were in accordance with National Institutes of Health guidelines and were approved by the Animal Care and Use Committee of Washington University School of Medicine (St. Louis). Mice were housed on a 12-h light/12-h dark cycle with ad libitum access to rodent chow.

Immunohistochemistry. Forebrain-specific glucocorticoid (GR) knockout (FBGRKO) and littermate controls were anesthetized with 2.5% Avertin (i.p.) and perfused transcardially with cold PBS followed by cold 0.1 M PBS containing 4% paraformaldehyde. Brains were then embedded in paraffin, and 8-m m coronal sections were collected. Sections were processed for immunohistochemistry with antibodies recognizing GR (1:200, M-20, Santa Cruz Biotechnology) and NeuN (1:200, Chemicon). Nonspecific binding for GR/NeuN was blocked with 3% fish gelatin, 5% nonfat dried milk (NFDM), 1% normal goat serum in PBS. Sections were incubated with primary antibody overnight at 4°C, washed with PBS, incubated with secondary antibody for 1 h at room temperature (NeuN, 1:200 FITC-conjugated goat anti-rat IgG, Jackson ImmunoResearch; GR, 1:250 biotinylated goat anti-mouse IgG, Vector Laboratories, Burlingame, CA), incubated in an avodin/biotin complex reagent (Vector Laboratories) for 1 h, washed with PBS, and incubated in a Cy-3 conjugated tyramide signal amplification reagent (Perkin-Elmer).

Pituitary Adrenocorticotropic Hormone (ACTH)/GR Immunostaining. The pituitaries from mice that were fixed by perfusion as described above were cryopreserved in 10% sucrose in PBS and embedded in OCT compound (Sakura Finetek, Torrance, CA). Sixteen-micrometer sections were thaw-mounted onto Superfrost-plus slides (Fisher Scientific) and incubated with antibodies specific for GR (as described above) and ACTH. A guinea pig anti-ACTH antibody was visualized with a fluorescein-conjugated goat secondary antibody (Peninsula Laboratories) according to the manufacturer’s instructions.

Long-Term Depression (LTD). Mice were deeply anesthetized with halothane and decapitated, and the brain was removed. Hippocampi were rapidly dissected and placed in standard extracellular solution containing 124 mM NaCl, 5 mM KCl, 2 mM CaCl2, 2 mM MgSO4, 1.25 mM NaH2PO4, 22 mM NaHCO3, and 10 mM D-glucose. All solutions were gassed (95% O2/5% CO2). Transverse slices (500 m m thick) were cut with a Vibroslice. Slices were then maintained in an incubation chamber for 1 h at 37°C in the standard solution. At the time of an experiment, individual slices were transferred to a submersion recording chamber where they were constantly perfused with standard solution (2 ml/min) at 30°C. Extracellular recordings were obtained from the dendritic layer of the CA1 region with the use of glass electrodes filled with 2 M NaCl. A bipolar electrode was placed in stratum radiatum to stimulate the Schaffer collateral/commissural pathway. Stimuli (50 m s in duration) were applied every minute. The stimulus intensity was set to evoke 40-50% of the maximal amplitude of field excitatory postsynaptic potentials (EPSPs). To induce LTD, 1-Hz low-frequency stimulation (LFS) was delivered continuously for 15 min. Field EPSPs were monitored and analyzed with the use of an IBM computer-based data acquisition system (Axon). The magnitude of potentiation or depression was expressed as the percent change in the maximal slope of EPSPs. Values are presented as means ± SE. Statistical significance was evaluated by Students’ t test or Mann-Whittney‘s U test.

In Situ Hybridization. Mice were deeply anesthetized with 1 ml of 2.5% Avertin and then transcardially perfused with diethylpyrocarbonate-treated (DEPC) PBS, followed by 4% DEPC paraformaldehyde. Isolated brains were postfixed in 4% paraformaldehyde for 24 h, followed by immersion in 10% sucrose in DEPC PBS. Tissues embedded in OCT (Sakura Finetek) were cut into 15-m m sections on a cryostat and thaw-mounted onto Superfrost plus slides (Fisher Scientific). RNA probes complementary to mRNA for MR, CRH, AVP, or POMC were radiolabeled with [a -33P]UTP and hybridized to sections at an annealing temperature of 60°C, and washed, after hybridization, in 0.1× SSC at 65°C for 30 min. Slides were exposed for 6 h to 7 days to Hyperfilm b Max (Amersham Pharmacia). Autoradiographic images were scanned at 2,400 dpi on an Epson 1680 Pro scanner. Densitometric analysis of in situ signal was performed by using NIH IMAGE software.

Behavioral Analysis. All behavioral analyses were performed by an observer blinded to genotype.

Forced swim test. Mice were placed in a 2-liter beaker half filled with water (18-20°C). The level of the water prevented the animals from escaping or from reaching the bottom of the container. Every 5 s during a 15-min trial, an observer recorded the activity of the mouse. If the mouse was active at any time during a given 5-s bin, a + 1 was recorded, if the animal remained inactive throughout the entire 5-s bin, a 0 was recorded. At the end of the trial, the animal was removed from the water, dried, and returned to its home cage. Graphs were generated by calculating the percentage of bins that each mouse was active during the trial.

Tail suspension test. The tail suspension apparatus consisted of a cubicle made of 1/2" (12.7 mm) white Plexiglas with inside dimensions of 13" × 13" × 12.5" H. Mice were suspended by the distal inch of their tails from the tail hanger with tape. Activity was scored in the same manner as described for the forced swim test across an 8-min trial. Graphs were generated by calculating the percentage of bins that each mouse was active during the trial.

Sucrose preference test. Mice were individually housed in cages containing two bottles containing a 1% sucrose solution or water. Bottles were weighed daily for 10 days. Each day, the position of the bottles were reversed to prevent effects due to a side preference. The data are presented as the percentage of sucrose consumed out of the total fluid averaged across genotypes.