Indeed, the social avoidance and shuttle box escape deficit produced by exposure to uncontrollable stress can be blocked by micro-injection of a selective 5-HT2CR antagonist into the lateral/ BLA [49] and DS [50], respectively. Despite the data implicating the 5-HT2CR as a potential target of anxiolytic and antidepressant strategies, knowledge regarding modulation of the 5-HT2CR by antidepressant drugs or environ- mental manipulation is limited. Chronic administration of the selective 5-HT reuptake inhibitor (SSRI) fluoxetine has been reported to increase site-specific 5-HT2CR pre-mRNA editing in the forebrain of BALB/c mice, an effect that would increase the pool of mRNA encoding 5-HT2CR with reduced function [51]. Chronic fluoxetine, however, had little effect on edited mRNA variants in C57BL/6 mice [51] or Sprague-Dawley rats [52]; whereas both chronic fluoxetine and reboxetine reduced the expression levels of non-edited 5-HT2CR mRNA in the prefrontal cortex of Sprague-Dawley rats [52]. These recent data suggest that one relevant effect of antidepressants could be reduced transcrip- tion of the 5-HT2CR. Similarly, prior evidence suggests that exercise may decrease sensitivity of 5-HT2 receptors in humans [53,54] and rats [55]. Fox and colleagues, for example, found that voluntary exercise reduced the anxiogenic effect of the non- selective 5-HT2 receptor agonist metachlorophenylpiperazine on acoustic startle in mice [6]. Additionally, we have observed that the anxiety- and depression-like behavioral effects of acute administration of fluoxetine or uncontrollable stress, which depend upon activation of 5-HT2CR, can be prevented by 6 weeks of voluntary exercise [8,9]. Therefore, one mechanism underlying the anxiolytic and antidepressant effects of exercise may be a reduction in the sensitivity and/or expression of the 5-HT2CR. The current studies investigated whether the 5-HT2CR is a potential target for the anxiolytic and antidepressant effects of exercise. The 5-HT2CR agonist CP-809101 was microinjected either into the lateral/BLA to produce exaggerated fear, or into the DS to interfere with shuttle box escape behavior. We hypothesized that CP-809101 would produce exaggerated fear and interfere with shuttle box escape learning in a dose-dependent manner in sedentary rats, and that a higher dose of the 5-HT2CR agonist would be required to produce these behavioral effects in physically active rats. Additionally, in situ hybridization was used to investigate the effect of voluntary exercise on levels of 5-HT2CR mRNA in the amygdala and striatum. Methods Ethics Statement All experimental protocols conformed to the NIH guide for the Care and Use of Laboratory Animals and were approved by the University of Colorado Institutional Animal Care and Use Committee protocol 1002.06. Care was taken to minimize animal discomfort during all procedures. Animals Adult, male Fischer 344 rats (220–280 g at the time of behavioral testing; N = 165) were used in all experiments based on prior work optimizing 5-HT2CR-mediated behaviors in this strain [9,45,56]. The rats were housed in a temperature- (22uC) and humidity-controlled environment, were maintained on a 12:12 hour light/dark cycle, and had ad libitum access to food and water. Rats assigned to the sedentary condition were individually housed in Nalgene Plexiglas cages (45625.2614.7 cm) lacking a running wheel. Rats assigned to 6 weeks of voluntary wheel running were individually housed in similar cages with attached running wheels. Animals were acclimated to these conditions for 1 Exercise Reduces Effects of 5-HT2CR Agonist week before any experimental manipulation. Wheels were rendered immobile with metal stakes during the acclimation period. Rats were weighed weekly. Voluntary wheel running Rats (6–7 weeks of age) were randomly assigned to either remain sedentary or were allowed voluntary access to running wheels for 6 weeks, a duration of wheel running previously reported to prevent increased shock-elicited fear and deficits in shuttle box escape learning produced by exposure to uncontrol- lable stress [7,8,9]. At the start of each experiment, the wheels in the cages of the physically active rats were unlocked and these rats were allowed voluntary access to their wheels. Daily wheel revolutions were recorded digitally using Vital View software (Mini Mitter, Bend, OR, USA) and distance was calculated by multiplying wheel circumference (1.081 m) by the number of wheel revolutions. Surgery Rats underwent surgery during the 4th week of either voluntary wheel running or sedentary conditions. Under ketamine (0.75 mg/ kg i.p.; Vedco, St. Joseph, MO, USA) and medetomidine (0.5 mg/ kg i.p.; Pfizer, New York, NY, USA) anesthesia, bilateral cannulae (26 gauge, Plastics One, Roanoke, VA, USA) were implanted as previously described [45]. Cannulae were aimed at either the DS: +0.5 A/P, 63.0 M/L, and 24.6 D/V from bregma [45,57] or, separately, the region of the lateral/BLA: 23.0 A/P, 64.8 M/L, 26.2 D/V from bregma [41,45], based on the atlas by Paxinos and Watson [58]. Atipamezole (0.5 mg/kg i.p.; Pfizer, New York, NY, USA) was administered following surgery to reverse the effects of medetomidine. All rats were inoculated with 0.25 mL/kg (subcutaneous) penicillin (Combi-Pen, Agrilabs, St. Joseph, Mis- souri, USA) immediately following surgery and returned to their home cages. Behavioral experiments were conducted approxi- mately 2–3 weeks after implantation surgery, by which time running behavior had returned to pre-surgical levels. Following experiment completion, brains were sliced at 40 mm and stained with Cresyl Violet for cannulae placement verification. Misplaced cannulae were excluded from analysis or used as off-site controls when appropriate. Drug Microinjections Microinjections of the selective 5-HT2CR agonist CP-809191 (Tocris Bioscience, Ellisville, MO, USA) were made 15 minutes prior to behavioral testing. On the day of behavioral testing, a micro-injector extending 0.5 mm (intra-DS) or 1.0 mm (intra- BLA) beyond the tip of the guide cannula was inserted. Drug doses and injection volumes were based on prior work examining the behavioral effects of CP-809101 [41,45,56]. Specifically, CP- 809191 was dissolved in 0.9% sterile saline and gently warmed at 45uC for 10–15 minutes in a water bath at concentrations of 0.3 mM, 2.0 mM and 6.0 mM. CP-809101 was administered intra-DS at a volume of 1.0 mL/side or intra-BLA at a volume of 0.5 mL/side [41,45]. Rats receiving both CP-809101 along with a 5-HT2CR antagonist were not included in the current study because we have previously shown that the behavioral effects of 6 mM CP-809101 can be blocked by pretreatment with the selective 5-HT2CR antagonist SB242084 when the drugs are injected either systemically [56] or intra-DS [45]. Behavioral Testing Behavioral testing was conducted following 6 weeks of voluntary wheel running or sedentary conditions, 15 minutes PLOS ONE | www.plosone.org 2 September 2012 | Volume 7 | Issue 9 | e46118