4 ± 5.4%, n = 4), p < 0.01; ethanol + MRS + KRGE60 group vs. ethanol + eticlopride + KRGE60
group (10.2 ± 2.5%, n = 4), p < 0.01; ethanol + MRS + KRGE60 group vs. ethanol + SCH23390 + KRGE60 group (27.4 ± 6.1%, n = 4), p > 0.05] ( Fig. 3B). Taken together, these results suggest that the anxiolytic effects of KRGE during EW were mediated by D2R in the CeA. Plasma CORT levels, a hormonal marker of anxiety in rats, were measured with an RIA to confirm the anxiolytic Raf targets effects of KRGE. Plasma CORT levels were significantly higher in ethanol-treated control rats (858.4 ± 181.3, n = 4) than in saline-treated controls [F (3, 13) = 18.2, p < 0.001; ethanol-treated control group (858.4 ± 181.3, n = 4) vs. saline-treated control group (318.6 ± 57.3, n = 5), p < 0.001]. Also in agreement with the behavioral data, the administration of both doses of KRGE significantly inhibited EW-related increases in plasma CORT levels [ethanol-treated control group vs. ethanol + KRGE 20 mg/kg group (473.2 ± 131.6, n = 4), p < 0.001; ethanol-treated control group vs. ethanol + KRGE 60 mg/kg
group (350.0 ± 80.7, n = 4), p < 0.001] ( Fig. 4). The HPLC analyses revealed significant decreases in the levels of DA and DOPAC in the CeA during EW. Treatment with KRGE dose-dependently reversed these deficiencies (Table 1) demonstrating that the anxiolytic effects of KRGE are mediated by the amygdaloid dopaminergic system. Western blot analyses revealed a reduction in the expression Leukotriene-A4 hydrolase of TH proteins in the CeA of ethanol-treated controls compared to saline-treated controls [F (2, 9) = 24.6, p < 0.001; saline-treated control
group (100%, n = 4) vs. ethanol-treated control group (36.2 ± 8.3%, Selleckchem ZVADFMK n = 4), p < 0.001]. However, the administration of KRGE (60 mg/kg) prevented these reductions [ethanol-treated control group vs. ethanol + KRGE60 (95.2 ± 23.4%, n = 4), p < 0.001] ( Fig. 5). The real-time PCR analyses revealed that EW significantly decreased the expression of TH mRNA in the VTA [F (2, 9) = 8.6, p < 0.01; saline-treated control group (100%, n = 4) vs. ethanol-treated control group (60.6 ± 10.0%, n = 4), p < 0.01]. However, the expression of TH mRNA in the CeA was spared (data not shown). Similar to protein expression in the CeA, KRGE (60 mg/kg) prevented the reduction of TH mRNA expression in the VTA during EW [ethanol-treated control group vs. ethanol + KRGE 60 mg/kg group (90.3 ± 22.2%, n = 4), p < 0.05] ( Fig. 6). Consistent with previous findings, the present study demonstrated that rats undergoing EW exhibit anxiety-like behavior as they spent less time in the open arms of the EPM [7] and [18]. The behavioral testing also revealed that both the 20 mg/kg and 60 mg/kg doses of KRGE significantly increased the time spent in the open arms, which reflects the anxiolytic effects of KRGE. The anxiety-reducing behavioral effects of KRGE were supported by biochemical evidence showing that KRGE inhibited plasma CORT secretion.