Results Effects of adrenalectomy and adrenal steroid replacement In order to examine the effect of corticosteroids on the expression of KAR subunits, we performed In situ hybridization (ISH) after adrenalectomy and subacute treatment with cortico- sterone, aldosterone and RU28362. ISH revealed KAR subunit specific patterns of expression in the subfields of the hippocampal formation, consistent with previous reports [15]. There was a significant main effect of treatment (F (4,28) = 3.824, see Figure 1.) upon KA1 mRNA expression in the CA3 and dentate gyrus regions of the dorsal hippocampus, but no main effects were seen in the CA1 or CA2 regions. Adrenalectomy (ADX) increased KA1 message by 68611% in the CA3 and by 54613% in the dentate (p,0.05 versus sham, n = 8), while neither the selective MR agonist aldosterone nor the selective GR agonist RU28362, given alone, reversed this effect. However, treatment with corticosterone significantly reduced KA1 mRNA from ADX levels (p,0.05), suggesting that MR/GR heterodimers may regulate expression specifically in the dentate gyrus and downstream in the CA3. The GluR6 results, shown in Figure 2, suggest that this receptor subtype is predominately regulated via MR in the DG, CA1 and CA3. There was a main effect of treatment on GluR6 mRNA expression in the CA1 (F (4,28) = 3.778), CA3 (F (4,26) = 2.991) and dentate gyrus (F (4,26) = 4.338) after corticosteroid manipu- lations. In the DG, CA1 and CA3, ADX treatment showed a modest but non-significant trend toward increased GluR6 expression, and ADX+Aldosterone treatment significantly reduced mRNA compared to ADX treatment (p,0.05, n = 8), with no effect compared to sham. These data suggest that GluR6 mRNA is predominantly regulated through MR in the dentate gyrus, CA1 and CA3. KAR, Stress & Steroids For GluR7, there was a main effect of treatment on mRNA expression in the dentate gyrus (F (4,32) = 7.789, see Figure 3.). Relative to Sham, ADX increased expression by 3965% (p,0.05) and ADX+RU362 increased expression by 37.966% (p,0.05). ADX+CORT replacement significantly reduced GluR7 message levels from both ADX+Vehicle, ADX+Aldo, and ADX+RU362. No main effects of treatment were observed with either GluR5 or KA2 after chronic corticosteroid manipulations (see Figure 4. for representative autoradiograms of KA2 and GluR5 expression in the hippocampal formation) . Effects of chronic restraint stress To examine the effects of a stress paradigm known to cause structural and functional changes in the hippocampus on KAR expression, KAR subunit levels were measured in the hippocam- pus in response to 21-day chronic restraint stress (CRS). Spironolactone, an MR antagonist, was used concurrently to assess the possible contribution of MR to any stress effect. As can be seen in Figure 5, a main effect of treatment was observed on KA1 subunit expression in both the CA3 (F (2,21) = 7.817) and DG (F(2,21) = 4.285). In the CA3, CRS and CRS+Spironolactone significantly elevated expression compared to control. In the DG, a similar increase was seen with CRS, but not with CRS+Spir- onolactone. No other effects of CRS or CRS+Spironolactone were seen with KA2, GluR5, GluR6, or GluR7. Final body weights of both stressed groups (393.467.3g for CRS alone and 372.468.1g for CRS and spironolactone) were significantly lower than controls (430.266.6g, p,0.05), confirming that CRS was effective systemically. Effects of chronic corticosterone in drinking water To confirm that the effects of CRS were corticosteroid dependent, we treated rats for 21 days with vehicle, 25 mg/ml Figure 1. ROD of KA1 mRNA in the dentate gyrus (A) and CA3 (B) after adrenalectomy and treatment with vehicle (ADX), aldosterone (ADX+Aldo) RU28,362 (ADX+RU362) or corticosterone (ADX+CORT). (C) A representative autoradiogram of KA1 mRNA. *-significantly different from sham and ADX+Cort (p,0.05, n = 8). **-significantly different from ADX (p,0.05, n = 8). doi:10.1371/journal.pone.0004328.g001 PLoS ONE | www.plosone.org 2 January 2009 | Volume 4 | Issue 1 | e4328