difference between exercise and sedentary animals that received the sub-threshold dose of CP-809101 (0.3 mM). Importantly, the 2.0 mM dose of CP-809101 administered into the DS interfered with shuttle box escape learning in sedentary rats only, whereas physically active rats were resistant to the behavioral effects of that dose. Finally, the highest dose of CP-809101 (6.0 mM) was sufficient to interfere with escape learning in both sedentary and physically active rats. Off-site control rats behaved similarly to the 0.3 mM groups. Voluntary exercise decreases levels of 5-HT2CR mRNA in the amygdala and dorsal striatum Non-surgerized rats na¨ıve to behavioral testing were allowed voluntary access to running wheels (N = 7) or remained sedentary (N = 7) for 6 weeks to determine the effects of voluntary exercise on 5-HT2C mRNA levels. Weight and running data were similar to prior studies (data not shown). Representative autoradiographs showing 5-HT2CR mRNA in the amygdala and striatum of sedentary and physically active rats are shown in Figure 5 and Figure 6, respectively. Relative to sedentary rats, 6 weeks of wheel running reduced levels of 5-HT2CR mRNA in the lateral amygdala (F (1, 12) = 6.95; p = 0.02), BLA (F (1, 12) = 25.71; p = 0.0003), and central amygdala (F (1, 12) = 12.89; p = 0.004), but not the lateral ventricle (F (1, 12) = 0.705; p = 0.42), where expression of 5-HT2CR mRNA was most pronounced (Figure 5D). Wheel running also reduced 5-HT2CR mRNA levels in the medial DS (F (1, 12) = 4.66; p = 0.05), but not the lateral DS (F (1, 12) = 0.003; p = 0.95; Figure 6D). Exercise Reduces Effects of 5-HT2CR Agonist Discussion The current data demonstrate for the first time that exercise can reduce anxiety-like behaviors produced by administration of a selective 5-HT2CR agonist into discrete brain regions, and implicate 5-HT2CR in the BLA and DS as a potential target for the anxiolytic and antidepressant properties of exercise. Specifi- cally, 6 weeks of voluntary wheel running increased the dose of intra-BLA and -DS CP-809110 necessary to produce exaggerated fear and interference with escape learning, respectively. In-situ hybridization revealed that voluntary wheel running decreased the levels of 5-HT2CR mRNA in brain regions implicated in these behaviors, including the BLA and the DS. These data add to our understanding of the neural pathways and mechanisms underlying the psychological and behavioral benefits associated with regular physical activity. Prior work has shown that 5-HT2CR agonist injections into the BLA increase anxiety-like behavior in rodents [38,41,42,45]. Here we report that 6 weeks of voluntary wheel running was sufficient to reduce the exaggerated fear produced by activation of 5-HT2CR in the lateral/BLA. Physical activity, therefore, may reduce the expression of some anxiety-like behaviors through a reduction in the expression, sensitivity or function of 5-HT2CR in the lateral/ BLA. Additionally, voluntary wheel running reduced levels of 5- HT2CR mRNA in the lateral amygdala and BLA, suggesting that physical activity may reduce the behavioral consequences of 5- HT2CR agonist administration via a reduction in transcription of 5-HT2CR. Interestingly, voluntary wheel running also reduced 5- HT2CR mRNA in the CeA, another area implicated in fear Figure 5. Six weeks of voluntary wheel running decreases 5-HT2C mRNA expression in the amygdala. (A) The region of the amygdala as shown by Paxinos and Watson (published in the Rat Brain in Stereotaxic Coordinates, 4th ed., Copyright Elsevier (1998)). (B) Representative autoradiographic coronal section through the region of the striatum (Bregma – 3.14 mm) in a sedentary rat processed with in situ hybridization for 5- HT2CR messenger ribonucleic acid (mRNA). (C) Representative autoradiographic coronal section through the region of the amygdala (Bregma – 3.14 mm) in a physically active rat (Run) processed with in situ hybridization for 5-HT2CR mRNA. (D) Relative levels of 5-HT2C receptor (mRNA) in the lateral amygdala (LA), basolateral amygdala (BLA), central amygdala (CeA), and lateral ventricle (LV) of sedentary rats or rats allowed voluntary access to running wheels for 6 weeks (Run). Values represent mean integrated density 6 SEM. * p#0.05 relative to respective sedentary groups. doi:10.1371/journal.pone.0046118.g005 PLOS ONE | www.plosone.org 7 September 2012 | Volume 7 | Issue 9 | e46118