following drug microinjections. Fear and shuttle box escape behaviors were assessed sequentially in shuttle boxes (50.8 cm625.4 cm630.48 cm, Coulbourn Instruments, White- hall, PA) using procedures previously described [8,45,59]. At the beginning of each session, rats were placed into shuttle boxes and allowed to explore for 10 minutes. During this 10 minute period (pre-shock period), fear behavior was assessed using a sampling procedure in which each rat was scored every 10 seconds as either freezing, defined as an absence of all movement except for that required for respiration, or not freezing. Spontaneous shuttle box crosses were also counted during this time in a subset of rats (N = 3–8/group) as a measure of locomotor activity in response to a novel environment. Rats then received two 0.7 mA foot shocks (1 minute ITI) delivered through both sides of the grid floor. Foot shocks were terminated when the rat fully crossed over to the opposite side of the shuttle box (fixed ratio 1, FR-1). The latencies to cross were recorded (FR-1 latencies). Following the second FR- 1 trial, shock-elicited freezing was observed for 20 minutes. Shock-elicited freezing is a measure of fear conditioned to cues present in the shuttle box [60]. Exaggerated shock-elicited fear has been argued to represent anxiety [61]. The post shock freezing period was followed by 25 fixed-ratio 2 (FR-2) escape trials (average ITI of 1 min). During FR-2 trials, rats were required to cross through the shuttle box door twice in order to terminate the foot shock (0.6 mA). An escape latency of 30 seconds was assigned if a correct escape response did not occur within 30 seconds, at which time the shock was terminated. A single test session lasted approximately 1 hour and occurred between 0900 and 1200. All animals were scored for both freezing and escape behavior by an experimenter blind to treatment condition of the animals. In Situ Hybridization In a separate experiment, rats were randomly assigned to either remain sedentary or allowed voluntary access to running wheels for 6 weeks. After 6 weeks, rats were sacrificed via rapid decapitation. Following previously published in situ hybridization protocols [8,62,63], brains were extracted, and frozen in isopentane cooled with dry ice (220uC; 4 minutes). Brains were stored at 280uC prior to being sectioned at 10 mm thickness with a cryostat. Slicing occurred at 221uC, and rostral-caudal sections of the DS and BLA were collected and thaw-mounted onto poly- L-lysine-coated slides. Tissue sections were stored at 280uC prior to use in single-label radioactive in situ hybridizations. Before hybridization, sections were fixed in 4% paraformaldehyde for 1 hour, washed 3 times in 26 sodium saline citrate (SSC), acetylated with 0.25% acetic anhydride containing 0.1 M triethanolamine for 10 minutes, and dehydrated in graded ethanol. The 5HT2CR receptor plasmid construct was obtained from Dr. David Julius at the University of California, San Francisco. The 5HT2CR probe is 555 base pairs long, spanning 1370–1925 (Accession # M21410). Customary transcription protocols were used to label the 5HT2CR riboprobe with 35S- UTP. Following completion of transcription, the riboprobe was mixed with 50% hybridization buffer comprised of 50% high- grade formamide, 10% dextran sulfate, 36 SSC, 16 Denhardt’s solution, 0.2 mg/mL yeast tRNA, and 0.05 M sodium phosphate (pH 7.4). The 5HT2CR riboprobe in hybridization buffer was applied directly to slides containing sections of DS and BLA. Slides were incubated overnight at 55uC in humid chambers. The following day, slides were washed 3 times in 26 SSC, subjected to an RNase A (200 mg/ml) treatment for 1 hour, rinsed in graded concentrations of SSC, washed in 0.16 SSC (65uC) for 1 hour, and dehydrated in ethanol. After drying, slides were placed in Exercise Reduces Effects of 5-HT2CR Agonist light-tight autoradiography cassettes, and exposed to X-ray film (Biomax-MR) for 1 week. Image Analysis for In Situ Hybridization Levels of 5-HT2CR mRNA were analyzed by computer-assisted optical densitometry following previously published protocols [62,63]. Brain section images were captured digitally (CCD camera, model XC-77; Sony, Tokyo, Japan), and the relative optical density of the x-ray film was determined using Scion Image Version 4.0 (Scion, Frederick, MD, USA). A macro was written that enabled signal above background to be determined automat- ically. For each section, a background sample was taken over an area of white matter, and the signal threshold was calculated as mean gray value of background +3.5 standard deviations. The section was automatically density-sliced at this value, so that only pixels with gray values above these criteria were included in the analysis. Results are expressed as mean integrated density, which reflects both the signal intensity and the number of pixels above assigned background (mean signal above background6number of Figure 1. Intra-amygdala 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 lateral/basolateral amygdala (BLA) between weeks 3 and 4. Three weeks later, rats were injected with either saline or increasing doses of 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 sequentially. (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 means 6 SEM. doi:10.1371/journal.pone.0046118.g001 PLOS ONE | www.plosone.org 3 September 2012 | Volume 7 | Issue 9 | e46118