of the hippocampus to stress, although this approach may also be impaired by the lack of selective drugs. Another important question to answer will be the extent to which chronic stress or corticosteroid treatment alters the response of KARs to and acute stressor or corticosteroid treatment, as this will allow us to begin to assess the extent to which KARs are involved in resilience to stress versus stress induced pathophysiology. These findings are made more interesting by recent findings associating KA1 and GluR6 and 7 with major depression and other major mental disorders [6–10], all the more so because the subunit which definitively did not change expression levels in our experiments, KA2, has thus far shown no association with affective disorders either. Further understanding of these changes could permit an improved understanding of both stress induced pathologies and the reasons why these pathologies can take a substantial amount of time to reverse, as is the case with major depression. Methods Animals Adult male Sprague-Dawley rats were obtained from Charles River Laboratories (Kingston, NY) at 70 days of age. Animals were housed 2–3 per cage (same age cage mates) in clear polycarbonate cages with wood chip bedding. All animals were maintained on a 12 h light-dark schedule (lights on at 0800 h) and the temperature was kept at 2162uC. All animals had ad libitum access to food and water. All procedures were carried out in accordance with the guidelines established by the NIH Guide for the Care and Use of Laboratory Animals. Chronic Restraint Stress Animals were left undisturbed after arrival for one week after delivery. Stressed animals were restrained in wire mesh restrainers, secured at the head and tail ends with large binder clips. Chronic stress was administered for 6 hours daily for 21 days from 10:00 to 16:00. Animals were returned to their home cages immediately after termination of the stressor. These animals were sacrificed by decapitation roughly 24 hours after the last stress (i.e. between 1300 and 1700 h). Brains were removed and flash frozen on dry ice and then stored at 280uC until processing. Steroid Treatments These treatments follow those administered in [12] with some modification. We chose to follow the one week time period used by Watanabe for two reasons: first, he observed changes in KAR levels after one week of steroid replacement. Secondly, after adrenalectomy there is a progressive apoptosis of dentate gyrus granule cells [16] and while we have successfully detected changes in mRNA at the seven day time point in the past [12,40], we were concerned that at later time points the potential for confounds would be much greater. Animals were anesthetized using ketamine and xylazine and the adrenal glands removed, save for one group which received a sham surgery. During the same surgery, osmotic mini-pumps (Alzet, Cupertino, CA) were implanted subcutane- ously between the scapulae. These pumps delivered vehicle (50% polyethylene glycol), the mineralocorticoid receptor agonist aldosterone at 10 mg/hour or the glucocorticoid receptor agonist RU28,362 at 10 mg/hour. Animals who underwent ADX received 0.9% saline in their drinking water and one group received 400 mg/ml corticosterone in addition to the saline. Seven days after the completion of the surgeries, the animals were sacrificed by decapitation and their brains removed and frozen as described above. KAR, Stress & Steroids Chronic Corticosterone Treatment Animals were provided with either 2.5% ETOH (vehicle), 25 mg/ml corticosterone or 400 mg/ml corticosterone in their home cage drinking water for a period of 21 days. In Situ Hybridization Brain sections were cut at 20 mm on a cryostat and placed on Fisher Biotech ProbeOn Plus slides (Fisher, Pittsburgh, PA). In situ hybridization began with a tailing reaction to radioactively label the oligonucleotide probes with 35S. The probe sequences follow those described by [41], two probe sequences were used in a cocktail in order to improve sensitivity: KA1 59-TCC AGA GAG GAG AAA TAG CCC GGT CTG CGT CCC ATA TGA ACT CTG -39, 59-CTT GTA GTT GAA CCG TAG GAT CTC AGC GAA CTC CTT GAG CAT GTC-39; KA2 59-TTC CAC TCG GGC CTT GGC TGG GAC CTC GAT GAT CCC ATT GAT CTG-39, 59-GTT CTC CAG GAT ATG GGG ACG CGC CCG AAG ACA CGG GTG AGG GTT-39; GluR5 59-AAA TCC CTC CGA TCC TGA GCA CT TGA GGG GAG GTC TGA GGG AGG-39, 59-CCC GGG TTG GTT CCA TTG GGC TTC CGC GTA AAG GAT GCT AAT GCC-39; GluR6 59-GGT TCC TTG CGA ATA TCC GAT CCA CAA TAA GCA GAG CAG G, 59- GGT TCC TTG CGA ATA TCC GAT CCA CAA TAA GCA GAG CAG G-39, 59-ACT AAA CCT GGC TAT GAC AAA GAG CAC ACA ACT GAC ACC CAA GTA-39; GluR7 59-CTC AGC GTT CAT GAC CTG GGC GTT GGG GCC GTC CGC GTA CTC AAA-39, 59-ATT CTC CAC CAC CTC AGA GCC GGG GTT GCA GGG GTG GGC ATC ATA-39. Processing of the slides followed methods as previously described in [42]. Anatomical locations were determined with the assistance of the atlas of Paxinos and Watson [43]. Optical density was determined using MCID 5.0 (Imaging Research, St. Catharine’s, OT, Canada). Pyknotic Cell Counts Numbers of pyknotic cells were assessed following the method of Frye and McCormick [44]. Sections were serial to those used for autoradiography and in situ. Slides containing these sections were processed to reveal Nissl substance beginning with a brief fixation in 4% paraformaldehyde in 0.1M PB for 15 minutes after which they were washed in distilled water three times for 2 minutes per wash. Sections were then dipped in 0.1% Cresyl Violet for 2 minutes and then dehydrated in ascending concentrations of ethanol prior to clearing in xylenes for 4 minutes. After drying, the slides were coverslipped with permount. Pyknotic cells in the granule cell layer and subgranule zone of the dentate gyrus were identified in a 1006 visual field as those having a small volume, membrane blebbing, and dark condensed nucleus and chromatin. Statistics Optical density measurements were analyzed by a one way ANOVA for the chronic steroid study and the chronic stress study. Significant main effects and interactions in ANOVA were further analyzed using Fisher’s protected least significant difference test and Tukey’s test, respectively. Differences are considered signif- icant at p,0.05. All data are presented as mean6SEM. Author Contributions Conceived and designed the experiments: RGH BM. Performed the experiments: RGH RB EB AC KMM. Analyzed the data: RGH EB AC KMM. Wrote the paper: RGH BM. PLoS ONE | www.plosone.org 6 January 2009 | Volume 4 | Issue 1 | e4328