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    • Rats were euthanized with Fatal Plus

    2020-08-06

    Rats were euthanized with Fatal Plus (0.5ml, i.p.; Vortech, Dearborn, MI, USA), brains were removed and fixed in 10% formalin. Brains were sectioned (60μm) on frozen, stained with cresyl violet, and analyzed under light microscope by two experimenters blind to treatment. Only data from rats with cannulae placements in the dRN were included in the following data analyses (=7 amphetamine or saline pre-treated rats per dRN infusion group). Behaviors were compared between saline and amphetamine pre-treatment groups at 24h and 2 weeks following the last treatment, or between pre-treatment groups that received vehicle or ASV-30 infusions, using separate two-way ANOVAs. Significant effects were analyzed further using Student–Newman–Keuls post-hoc test for multiple pair-wise comparisons. Significance levels for all statistical tests were set at ≤0.05 (SigmaStat v2.03, SPSS Inc., Point Richmond, CA, USA). When anxiety-like behavior during amphetamine withdrawal was assessed, latency to enter open arms was significantly affected by pre-treatment treatment (=8.338, =0.007), but there was no effect of withdrawal CBR-5884 (=0.173, =0.680), nor an interaction between withdrawal period and pre-treatment (=0.665, =0.421). A significant increase in latency was observed between treatment groups only within the 2-week withdrawal group (SNK <0.05; A). Similarly, time spent in open arms was significantly different between pre-treatment groups (=25.850, <0.001) but was not affected by withdrawal period (=0.0421, =0.839), and an interaction between pre-treatment and withdrawal period was not observed (=0.152, =0.699). Time spent in the open arms was significantly decreased in the amphetamine treatment groups compared to saline-treated rats within both the 24-h and 2-week withdrawal periods (SNK <0.05; B). There was no effect of treatment (=0.586, =0.449), withdrawal (=3.561, =0.068), nor an interaction between treatment and withdrawal (=0.362, =0.551) on the total distance traveled in the maze (C). For experiments that tested CRF receptor mediation of amphetamine-induced anxiety-like behavior, placement of infusion cannulae into the dRN did not differ between saline and amphetamine pre-treated rats (A and B). A significant effect of pre-treatment (=6.155, =0.021), dRN infusion (=5.900, =0.023), and a significant interaction between pre-treatment and dRN infusion (=4.916, =0.036) was observed in the latency to enter open arms. Amphetamine pre-treated rats infused with vehicle showed greater latency to enter open arms compared with saline pre-treated rats and amphetamine pre-treated rats infused with ASV-30 (SNK <0.05; C). For time spent in opens arms, there was an effect of dRN infusion (=6.715, =0.016) and an interaction between drug pre-treatment and dRN infusion (=9.686, =0.005). Amphetamine pre-treated rats infused with vehicle showed reduced time in open arms compared with saline pre-treated rats and amphetamine pre-treated rats infused with ASV-30 (SNK <0.05; D). There was no significant effect of pre-treatment (=0.296, =0.592), dRN infusion (=0.00752, =0.932), nor an interaction of pre-treatment and dRN infusion (=0.0108, =0.918) on total distance traveled in the maze (E).