collapsed into 10, 2 min blocks and analyzed using 263 (activity6drug), repeated measures ANOVA. The two FR-1 trials were averaged into a single FR-1 latency score and analyzed with ANOVA. FR-2 escape latencies were averaged into 5 blocks of 5 trials each and analyzed with 263 (activity6drug), repeated measures ANOVA. Average post-shock freezing and average FR-2 escape latencies were analyzed with ANOVAs that included an additional group of off-site controls when appropriate. Group differences in 5-HT2CR mRNA expression in the medial and lateral DS, lateral amygdala, BLA, central amygdala (CeA), and lateral ventricle were analyzed with ANOVA. Tukey-Kramer post hoc analyses were performed when appropriate. Results were considered significant when p#0.05. Results Voluntary exercise reduces exaggerated fear produced by 5-HT2CR activation in the BLA An experimental timeline is shown in Figure 1A. Sedentary and physically active rats weighed similar amounts at the beginning of the study, but physically active rats gained less weight over the course of the experiment (Figure 1B; (F (6, 480) = 16.03; p,0.0001). Intra-BLA cannulae were surgically implanted after 4 weeks of voluntary wheel running. Figure 1C shows the average daily distance run pre- and post-surgery. As expected, surgery immediately decreased running distance. However, running distance resumed quickly and continued to steadily increase during the week after surgery. A graphical representation of cannula placements is shown in Figure 2A. After exclusion of rats with misplaced cannula (i.e. cannulae tips were outside of the lateral amygdala/BLA), group sizes were as follows: Sedentary/Saline = 8; Sedentary/ 0.3 mM = 8; Sedentary/2.0 mM = 10; Sedentary/6.0 mM = 10; Run/Saline = 4; Run/0.3 mM = 8; Run/2.0 mM = 9; Run/ 6.0 mM = 9. Data obtained from rats with misplaced cannulae (Sedentary/2.0 mM = 2; Sedentary/6.0 mM = 4; Run/ 2.0 mM = 5; Run/6.0 mM = 5) were averaged and included as an off-site control group. Consistent with prior reports that wheel running reduces general locomotor activity [64,65], physically active rats (9.0561.4 crossings) performed significantly fewer (F (1, 31) = 15.62; p = 0.0004) shuttle box crossings during the 10 min pre-shock observation period compared to their sedentary counterparts (12.6360.98 crossings), regardless of drug condition. It is therefore unlikely that an increase in general locomotor activity contributed to any observed behavioral effects of exercise. Voluntary exercise increased the intra-BLA dose of CP-809101 necessary to produce an increase in shock-elicited freezing behavior. Whereas 2.0 mM CP-809101 injected into the BLA increased shock-elicited fear in sedentary rats, 6.0 mM CP-809101 was required to enhance fear in physically active rats. The effect of the 5-HT2CR agonist CP-809101 on freezing behavior over the course of the 20 minute freezing observation period is shown in Figure 2B. Pre-shock freezing was minimal and did not differ between groups (Figure 2B, Pre-FR-1). The main effects of drug (F (3, 57) = 5.612; p = 0.002) and time (F (9, 513) = 39.6; p,0.0001), as well as the interaction between exercise and drug (F (3, 57) = 7.187; p = 0.0004), were all significant. Post hoc comparisons revealed that the Sedentary/2.0 mM group differed from the Sedentary/Saline group during the 2nd and 3rd freezing blocks, from the Sedentary/0.3 mM and Run/0.3 mM groups during the 3rd and 4th freezing blocks, and from the Run/2.0 mM group during the 1 st , 2nd, and 3rd freezing blocks. The Sedentary/ 6.0 mM group differed from the Sedentary/Saline group during Exercise Reduces Effects of 5-HT2CR Agonist the 2nd freezing block. The Run/6.0 mM group differed from the Run/2.0 mM group during the 2nd and 3rd freezing block. At no time did the 0.3 mM groups differ from the Saline groups. Average time spent freezing is shown in Figure 2C and escape behavior is shown in Figures 2D and 2E. Consistent with our prior observations, neither exercise [7,8] nor activation of the 5-HT2CR in the BLA [50] prior to behavioral testing altered FR-1 or FR-2 escape behavior. Voluntary exercise reduces shuttle box escape deficit produced by 5-HT2CR activation in the DS An experimental timeline is shown in Figure 3A. Sedentary and physically active rats weighed similar amounts at the beginning of the study, but physically active rats gained less weight over the course of the experiment (Figure 3B; F (6, 402) = 7.229; p,0.0001). Intra-DS cannulae were surgically implanted during the 4th week of voluntary wheel running. Figure 3C shows the average daily distance run pre- and post-surgery. Running behavior resumed quickly after surgery and continued to steadily increase during the following week to levels observed prior to surgery. Figure 3. Intra-striatum experimental design, body weights and running distance. (A) Experimental timeline. Adult, male Fischer 344 rats were allowed voluntary access to running wheels for 6 weeks (Run) or remained sedentary. All rats had cannulae implanted into the region of the dorsal striatum (DS) between weeks 3 and 4. Three weeks later, rats were injected with the selective 5-HT2C receptor agonist CP- 809101 (0.3 mM, 2.0 mM, or 6.0 mM) through the guide cannulae. Rats were placed into shuttle boxes 15 minutes following intra-DS injections and shock-elicited freezing and escape learning were tested sequen- tially. (B) Mean weekly body weight (grams) of physically active and sedentary rats. (C) The daily distance run (meters) pre- and post- cannula implantation surgery. Data represent group means 6 SEM. doi:10.1371/journal.pone.0046118.g003 PLOS ONE | www.plosone.org 5 September 2012 | Volume 7 | Issue 9 | e46118