Physical activity reduces the incidence and severity of psychiatric disorders such as anxiety and depression. Similarly, voluntary wheel running produces anxiolytic- and antidepressant-like effects in rodent models. The specific neurobiological mechanisms underlying the beneficial properties of exercise, however, remain unclear. One relevant pharmacological target in the treatment of psychiatric disorders is the 5-HT2C receptor (5-HT2CR). Consistent with data demonstrating the anxiogenic consequences of 5-HT2CR activation in humans and rodents, we have previously reported that site-specific administration of the selective 5-HT2CR agonist CP-809101 in the lateral/basolateral amygdala (BLA) increases shock-elicited fear while administration of CP-809101 in the dorsal striatum (DS) interferes with shuttle box escape learning. These findings suggest that activation of 5-HT2CR in discrete brain regions contributes to specific anxiety- and depression-like behaviors and may indicate potential brain sites involved in the anxiolytic and antidepressant effects of exercise. The current studies tested the hypothesis that voluntary wheel running reduces the behavioral consequences of 5-HT2CR activation in the BLA and DS, specifically enhanced shock-elicited fear and interference with shuttle box escape learning. After 6 weeks of voluntary wheel running or sedentary conditions, the selective 5-HT2CR agonist CP-809101 was microinjected into either the BLA or the DS of adult Fischer 344 rats, and shock-elicited fear and shuttle box escape learning was assessed. Additionally, in-situ hybridization was used to determine if 6 weeks of voluntary exercise changed levels of 5-HT2CR mRNA. We found that voluntary wheel running reduced the behavioral effects of CP-809101 and reduced levels of 5-HT2CR mRNA in both the BLA and the DS. The current data indicate that expression of 5-HT2CR mRNA in discrete brain sites is sensitive to physical activity status of the organism, and implicates the 5-HT2CR as a target for the beneficial effects of physical activity on mental health.
Citation: Greenwood BN, Strong PV, Loughridge AB, Day HEW, Clark PJ, et al. (2012) 5-HT2C Receptors in the Basolateral Amygdala and Dorsal Striatum Are a Novel Target for the Anxiolytic and Antidepressant Effects of Exercise. PLoS ONE 7(9): e46118. doi:10.1371/journal.pone.0046118
Editor: Masabumi Minami, Hokkaido University, Japan
Received: February 29, 2012; Accepted: August 28, 2012; Published: September 25, 2012
Copyright: © Greenwood et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The work in this manuscript was supported by the National Institutes of Health grant: NIMH 068283 (M.F.), NIMH 050479 (B.G.) and the American Foundation for Suicide Prevention (B.G.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Physical activity is associated with a reduction in the incidence and severity of psychiatric disorders such as anxiety and depression , , , . Similarly, voluntary exercise can reduce anxiety- , , , , , ,  and depression- , ,  like behaviors in laboratory rodents. Despite identification of many neuroadaptive changes produced by exercise , , , the specific neurobiological mechanisms underlying the anxiolytic and antidepressant properties of physical activity are unclear. Identification of these mechanisms could support the use of exercise as a prophylactic and therapeutic intervention for anxiety and depression, as well as provide insight into novel treatment strategies for psychiatric illness.
Serotonin (5-HT) has long been implicated in anxiety , , , ,  and depression , , but the mechanisms by which 5-HT contributes to specific symptomatology of these disorders remain an intense topic of investigation. Emerging evidence points to a role for the 5-HT2C receptor (5-HT2CR) in the expression of specific symptoms of anxiety , ,  and depression , , . Extending prior reports of anxiogenic effects of 5-HT2CR agonists in humans , , , , , , ,  and rodents , , , , , , , our group has contributed to the identification of the specific brain regions that subserve specific behavioral effects of 5-HT2CR activation. Anxiety-like behavior, including exaggerated shock-elicited fear  and social avoidance , , can be elicited in rats by site-specific activation of 5-HT2CR in the region of the lateral/basolateral amygdala (BLA), a brain area classically implicated in fear and anxiety , . In contrast, activation of 5-HT2CR in the dorsal striatum (DS) has no effect on anxiety behaviors, but can interfere with learning to escape in a shuttle box escape task, a common screening tool for antidepressant compounds . Thus, 5-HT2CR activation in discrete brain regions appears to be a sufficient stimulus to elicit specific symptoms of anxiety- and depression-like behavior. 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  and DS , 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 environmental 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 . Chronic fluoxetine, however, had little effect on edited mRNA variants in C57BL/6 mice  or Sprague-Dawley rats ; whereas both chronic fluoxetine and reboxetine reduced the expression levels of non-edited 5-HT2CR mRNA in the prefrontal cortex of Sprague-Dawley rats . These recent data suggest that one relevant effect of antidepressants could be reduced transcription of the 5-HT2CR. Similarly, prior evidence suggests that exercise may decrease sensitivity of 5-HT2 receptors in humans ,  and rats . 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 . 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 , . 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.
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.
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 , , . The rats were housed in a temperature- (22°C) 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 (45×25.2×14.7 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 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 uncontrollable stress , , . 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.
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 . Cannulae were aimed at either the DS: +0.5 A/P, ±3.0 M/L, and −4.6 D/V from bregma ,  or, separately, the region of the lateral/BLA: −3.0 A/P, ±4.8 M/L, −6.2 D/V from bregma , , based on the atlas by Paxinos and Watson . 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, Missouri, USA) immediately following surgery and returned to their home cages. Behavioral experiments were conducted approximately 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 µm and stained with Cresyl Violet for cannulae placement verification. Misplaced cannulae were excluded from analysis or used as off-site controls when appropriate.
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 , , . Specifically, CP-809191 was dissolved in 0.9% sterile saline and gently warmed at 45°C 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 µL/side or intra-BLA at a volume of 0.5 µL/side , . 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  or intra-DS .
Behavioral testing was conducted following 6 weeks of voluntary wheel running or sedentary conditions, 15 minutes following drug microinjections. Fear and shuttle box escape behaviors were assessed sequentially in shuttle boxes (50.8 cm×25.4 cm×30.48 cm, Coulbourn Instruments, Whitehall, PA) using procedures previously described , , . 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 . Exaggerated shock-elicited fear has been argued to represent anxiety . 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 , , , brains were extracted, and frozen in isopentane cooled with dry ice (−20°C; 4 minutes). Brains were stored at −80°C prior to being sectioned at 10 µm thickness with a cryostat. Slicing occurred at −21°C, 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 −80°C 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 2× 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, 3× SSC, 1× 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 55°C in humid chambers. The following day, slides were washed 3 times in 2× SSC, subjected to an RNase A (200 µg/ml) treatment for 1 hour, rinsed in graded concentrations of SSC, washed in 0.1× SSC (65°C) for 1 hour, and dehydrated in ethanol. After drying, slides were placed in 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 , . 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 automatically. 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 background×number of pixels above background). Each subject's mean integrated density at a given level represents the average of at least 2 slices chosen for analysis between the following coordinates: Striatum from +1.60 to +0.20 mm anterior to bregma; amygdala and lateral ventricle from −2.56 to −3.30 mm posterior to bregma based on the atlas by Paxinos and Watson . Templates for each region were made to ensure that equivalent areas were analyzed between animals. Lateral ventricle was analyzed as a control region because the choroid plexus of the lateral ventricle expresses relatively large amounts of 5-HT2CR mRNA.
Body weights were analyzed with repeated measures ANOVA. Pre-shock freezing scores were averaged into 1 pre-shock score and analyzed with ANOVA. Shock-elicited freezing scores were collapsed into 10, 2 min blocks and analyzed using 2×3 (activity×drug), 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 2×3 (activity×drug), 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.
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.
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 ± SEM.doi:10.1371/journal.pone.0046118.g001
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.
Figure 2. Six weeks of voluntary wheel running reduces the increase in fear behavior produced by 5-HT2C receptor activation in the region of the lateral/basolateral amygdala (BLA).
Following 6 weeks of voluntary wheel running (Run) or no running (Sedentary), rats received intra-BLA microinjections of either saline or the selective 5-HT2C receptor agonist CP-809101 (0.3 mM, 2.0 mM, or 6.0 mM). Shockelicited freezing and shuttle box escape latency were measured sequentially in shuttle boxes 15 minutes later. (A) Cannula placement within the amygdala. Sedenatry rats are denoted with black triangles, physically active rats are denoted with gray triangles, and off-site placements are denoted with an X. Brain illustrations adapted from Paxinos and Watson (published in the Rat Brain in Stereotaxic Coordinates, 4th ed., Copyright Elsevier (1998)). Numbers left of illustrations refer to distance from Bregma (mm). (B) Mean freezing behavior presented in 2 minute blocks (pre-FR-1 scores are not different and therefore overlap). Error bars are ommited for clarity. (C) The mean percent shock-elicited freezing for the entire 20 minute observation period. (D) Shuttle box escape latencies for one block of 2 FR-1 trials (FR-1) and five blocks of 5 FR-2 trials (FR-2). Error bars are omitted for clarity. (E) The mean escape latency for all 25 FR-2 escape trials. Data represent group means ± SEM. * p<0.05 relative to Offsite Control, Sedentary/Saline, Sedentary/0.3 mM, Run/2.0 mM, and Run/6.0 mM groups. Φ p<0.05 relative to the Sedentary/2.0 mM group. θ p<0.05 relative to the Run/2.0 mM group.doi:10.1371/journal.pone.0046118.g002
Consistent with prior reports that wheel running reduces general locomotor activity , , physically active rats (9.05±1.4 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 (12.63±0.98 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 1st, 2nd, and 3rd freezing blocks. The Sedentary/6.0 mM group differed from the Sedentary/Saline group during 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 ,  nor activation of the 5-HT2CR in the BLA  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 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 group means ± SEM.doi:10.1371/journal.pone.0046118.g003
Intra-DS cannula placements are shown in Figure 4A. After exclusion of rats with misplaced cannulae, group sizes were Sedentary/0.3 mM = 7; Sedentary/2.0 mM = 12; Sedentary/6.0 mM = 11; Run/0.3 mM = 9; Run/2.0 mM = 15; Run/6.0 mM = 9. Data from rats with misplaced cannulae (Sedentary/2.0 mM = 2; Sedentary/6.0 mM = 1; Run/6.0 mM = 3) were averaged and included as off-site controls. Saline was not injected into the DS in this study because our results from the above amygdala study, as well as our prior studies , indicate that saline injected animals behave identically to animals injected with 0.3 mM CP-809101.
Figure 4. Six weeks of voluntary wheel running reduces the deficit in instrumental escape learning produced by 5-HT2C receptor activation in the dorsal striatum.
Fifteen minutes prior to behavioral testing, sedentary and physically active (Run) rats received intra-DS microinjections of the 5-HT2C receptor agonist CP-809101 (0.3 mM, 2.0 mM or 6.0 mM). (A) Cannula tip placement within the DS. Sedentary rats are denoted with black triangles, physically active rats are denoted with gray triangles, and off-site placements are denoted with an X. Brain illustrations adapted from Paxinos and Watson (published in the Rat Brain in Stereotaxic Coordinates, 4th ed., Copyright Elsevier (1998)). Numbers left of illustrations refer to distance from Bregma (mm). (B) Freezing behavior over the duration of the post-FR-1 freezing observation period presented in 2 minute blocks (pre-shock scores are not different and therefore overlap). (C) The mean percent time spent freezing during the 20 minute observation period. (D) Shuttle box escape latencies for one block of 2 FR-1 trials (FR-1) and five blocks of 5 FR-2 trials (FR-2). (E) The mean escape latency for all 25 FR-2 escape trials. Data represent group means ± SEM. * p<0.05 relative to 0.3 mM groups and off-site control group; Φ p<0.05 relative to 2.0 mM sedentary group.doi:10.1371/journal.pone.0046118.g004
The effect of the 5-HT2CR agonist CP809101 on freezing blocks and average freezing behavior are shown in Figure 4B and Figure 3C, respectively. Pre-shock freezing was minimal and did not differ between groups (Figure 4B, pre-FR-1). While there was an expected effect of time (F (9, 513) = 36.546; p<0.0001) on freezing behavior during freezing blocks, neither physical activity status nor intra-DS administration of CP-809101 prior to behavioral testing impacted freezing behavior.
ANOVA revealed a significant main effect of exercise on FR-1 behavior (p = 0.003), i.e. exercise groups had faster average FR-1 escape latencies than sedentary groups. However, neither the main effect of drug nor the interaction between exercise and drug were significant (Figure 4D, FR-1). Repeated measures ANOVA revealed significant main effects of time (F (4, 228) = 16.919; p = <0.0001) and drug (F (2, 57) = 3.476; p = 0.0376), as well as reliable time×drug (F (4, 228) = 2.384; p = 0.0174) and time×drug×exercise (F (8, 228) = 3.232; p = 0.0017) interactions on FR-2 escape blocks (Figure 4D). Average FR-2 escape times are shown in Figure 4E. Post hoc analysis revealed that there was no 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 naïve 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).
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 ± SEM. * p≤0.05 relative to respective sedentary groups.doi:10.1371/journal.pone.0046118.g005
Figure 6. Six weeks of voluntary wheel running decreases 5-HT2C mRNA expression in the medial dorsal striatum.
(A) The region of the striatum 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 1.70 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 striatum (Bregma 1.70 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 medial and lateral dorsal striatum (DS) of sedentary rats or rats allowed voluntary access to running wheels for 6 weeks (Run). Values represent mean integrated density ± SEM. * p≤0.05 relative to respective sedentary group.doi:10.1371/journal.pone.0046118.g006
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. Specifically, 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 , , , . 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 behavior , . The role of the 5-HT2CR in the CeA in anxiety, however, remains relatively unknown. 5-HT2CRs are expressed throughout the amygdala complex, but 5-HT2CR mRNA (Figure 5), as well as receptor density , appears to be greatest in the lateral amygdala. Moreover, activation of 5-HT2CR in the CeA has no effect on the expression of some types of anxiety-like behaviors , . Instead, it appears that anxiety-like effects of 5-HT activity in the CeA are more likely mediated by 5-HT1A receptors . Further work is necessary to determine if the observed reduction of 5-HT2CR mRNA levels in the CeA of physically active rats contributes to a behavioral effect of exercise.
In addition to the BLA, the current data implicate the 5-HT2CR in the DS as a target for the antidepressant effects of exercise. Hypoactivity of striatal dopaminergic neurotransmission is hypothesized to occur in depression , . Consistent with this idea, the shuttle box escape deficit produced by uncontrollable stress (an established animal model of depression) can be thought of as a failure of a rapid instrumental learning process  which requires dopamine in the DS for optimal acquisition . Importantly, extracellular dopamine in the DS can be reduced by DS 5-HT2CR activation . It is therefore possible that the reduced expression and function of 5-HT2CR in the DS of physically active, relative to sedentary, rats observed in the current study could alleviate depressive symptoms by restoring dopamine transmission in the DS. Indeed, exercise also prevents the shuttle box escape deficit produced by uncontrollable stress , .
Although cannulae in the current study were aimed at the border between the medial and lateral DS, it is likely that 5-HT2CR activation interfered with escape behavior through action in the medial portion of the DS. 5-HT2CR are expressed more heavily in the medial, relative to the lateral, DS ( and Figure 6). Moreover, it is widely accepted that distinct sub-regions of the DS mediate different aspects of instrumental learning. The early stages of instrumental learning are modulated by the medial DS, whereas the lateral DS contributes to the later stages of instrumental learning (for review see , ). The fact that the acquisition of the escape contingency by rats injected with the low dose of CP-809101 occurs rapidly (within the first 5 trials), seems to indicate a role for the medial DS (see also ). Finally, voluntary exercise reduced 5-HT2CR mRNA in specifically the medial, and not the lateral, DS. These data support a role for 5-HT2CR activation in the medial DS in interference with shuttle box escape behavior and aversively-motivated instrumental learning in general.
It is important to note that the 5-HT2CR is known to undergo post-translational modifications. Increasing evidence suggests that the editing of 5-HT2CR mRNA can lead to the expression of multiple 5-HT2CR isoforms that have different G-protein activity and affinities for 5-HT , , altered constitutive activity , , as well as intracellular effects . These editing-induced changes in 5-HT2CR function have been speculated to play a critical role in the etiology of anxiety and depression , , , . Additionally, 5-HT2CR mRNA editing changes seem to occur after perturbations of 5-HT levels. Specifically, persistent increases in 5-HT neurotransmission, via drugs or SERT gene deletion, increase the occurrence of 5-HT2CR mRNA editing events, while at the same time decreasing 5-HT2CR responsiveness , , . Interestingly, acute bouts of physical activity (albeit forced) can also increase central 5-HT . It is possible that there is an overlap between some of the underlying mechanisms of therapeutic drugs and exercise, such that persistent changes in 5-HT neurotransmission over extended periods of time, whether due to a regular drug regimen or exercise program, can produce long-term plastic changes in the function of post-synaptic modulators of behavior such as the 5-HT2CR. In addition to changing mRNA levels in discrete brain regions, exercise may also affect 5-HT2CR pre-mRNA editing events, which could contribute to our observed reduction in the expression of anxiety- and depression-like behaviors. The effect of regular voluntary exercise on the editing of 5-HT2CR mRNA needs to be further explored.
In conclusion, physical activity reduces anxiety- and depression-like behaviors produced by 5-HT2CR activation in discrete brain regions. The current data extend previous work identifying the 5-HT2CR as a relevant target for pharmacological discovery, as well as shed light on potential mechanisms which underlie the anxiolytic effects of physical activity. Finally, our results implicate physical activity as one environmental variable with the ability to influence transcription or sensitivity of the 5-HT2CR. Future studies could therefore utilize exercise as a tool to reveal novel means with which to modulate expression of the 5-HT2CR in discrete brain regions relevant to stress-related psychiatric disorders.
Conceived and designed the experiments: BNG PVS MF. Performed the experiments: BNG PVS ABL HEWD PJC AM JEH KGS. Analyzed the data: PVS BNG AM. Contributed reagents/materials/analysis tools: BNG HEWD MF. Wrote the paper: BNG PVS MF.
- 1. Strohle A (2009) Physical activity, exercise, depression and anxiety disorders. J Neural Transm 116: 777–784. doi: 10.1007/s00702-008-0092-x
- 2. Saeed SA, Antonacci DJ, Bloch RM (2010) Exercise, yoga, and meditation for depressive and anxiety disorders. Am Fam Physician 81: 981–986.
- 3. Herring MP, O'Connor PJ, Dishman RK (2010) The effect of exercise training on anxiety symptoms among patients: a systematic review. Arch Intern Med 170: 321–331. doi: 10.1001/archinternmed.2009.530
- 4. Babyak M, Blumenthal JA, Herman S, Khatri P, Doraiswamy M, et al. (2000) Exercise treatment for major depression: maintenance of therapeutic benefit at 10 months. Psychosom Med 62: 633–638.
- 5. Binder E, Droste SK, Ohl F, Reul JM (2004) Regular voluntary exercise reduces anxiety-related behaviour and impulsiveness in mice. Behav Brain Res 155: 197–206. doi: 10.1016/j.bbr.2004.04.017
- 6. Fox JH, Hammack SE, Falls WA (2008) Exercise is associated with reduction in the anxiogenic effect of mCPP on acoustic startle. Behav Neurosci 122: 943–948. doi: 10.1037/0735-7044.122.4.943
- 7. Greenwood BN, Foley TE, Burhans D, Maier SF, Fleshner M (2005) The consequences of uncontrollable stress are sensitive to duration of prior wheel running. Brain Res 1033: 164–178. doi: 10.1016/j.brainres.2004.11.037
- 8. Greenwood BN, Foley TE, Day HE, Campisi J, Hammack SH, et al. (2003) Freewheel running prevents learned helplessness/behavioral depression: role of dorsal raphe serotonergic neurons. J Neurosci 23: 2889–2898.
- 9. Greenwood BN, Strong PV, Brooks L, Fleshner M (2008) Anxiety-like behaviors produced by acute fluoxetine administration in male Fischer 344 rats are prevented by prior exercise. Psychopharmacology (Berl) 199: 209–222. doi: 10.1007/s00213-008-1167-y
- 10. Salam JN, Fox JH, Detroy EM, Guignon MH, Wohl DF, et al. (2009) Voluntary exercise in C57 mice is anxiolytic across several measures of anxiety. Behav Brain Res 197: 31–40. doi: 10.1016/j.bbr.2008.07.036
- 11. Vollert C, Zagaar M, Hovatta I, Taneja M, Vu A, et al. (2011) Exercise prevents sleep deprivation-associated anxiety-like behavior in rats: potential role of oxidative stress mechanisms. Behav Brain Res 224: 233–240. doi: 10.1016/j.bbr.2011.05.010
- 12. Duman CH, Schlesinger L, Russell DS, Duman RS (2008) Voluntary exercise produces antidepressant and anxiolytic behavioral effects in mice. Brain Res doi: 10.1016/j.brainres.2008.04.053
- 13. Solberg LC, Horton TH, Turek FW (1999) Circadian rhythms and depression: effect of exercise in an animal model. American Journal of Physiology 276: R152–R161. doi: 10.1016/j.brainres.2008.04.053
- 14. Greenwood BN, Fleshner M (2011) Exercise, stress resistance, and central serotonergic systems. Exercise and sport sciences reviews 39: 140–149. doi: 10.1097/JES.0b013e31821f7e45
- 15. Cotman CW, Berchtold NC, Christie LA (2007) Exercise builds brain health: key roles of growth factor cascades and inflammation. Trends Neurosci 30: 464–472. doi: 10.1016/j.tins.2007.06.011
- 16. Ota KT, Duman RS (2012) Environmental and pharmacological modulations of cellular plasticity: Role in the pathophysiology and treatment of depression. Neurobiology of disease doi: 10.1016/j.nbd.2012.05.022
- 17. Anderson IM, Mortimore C (1999) 5-HT and human anxiety. Evidence from studies using acute tryptophan depletion. Adv Exp Med Biol 467: 43–55. doi: 10.1016/j.nbd.2012.05.022
- 18. Graeff FG (2004) Serotonin, the periaqueductal gray and panic. Neurosci Biobehav Rev 28: 239–259. doi: 10.1016/j.neubiorev.2003.12.004
- 19. Graeff FG, Guimaraes FS, De Andrade TG, Deakin JF (1996) Role of 5-HT in stress, anxiety, and depression. Pharmacol Biochem Behav 54: 129–141. doi: 10.1016/0091-3057(95)02135-3
- 20. Graeff FG, Viana MB, Mora PO (1997) Dual role of 5-HT in defense and anxiety. Neurosci Biobehav Rev 21: 791–799. doi: 10.1016/S0149-7634(96)00059-0
- 21. Ninan PT (1999) The functional anatomy, neurochemistry, and pharmacology of anxiety. J Clin Psychiatry 60: 12–17.
- 22. Sharp T, Cowen PJ (2011) 5-HT and depression: is the glass half-full? Current opinion in pharmacology 11: 45–51. doi: 10.1016/j.coph.2011.02.003
- 23. Owens MJ, Nemeroff CB (1998) The serotonin transporter and depression. Depress Anxiety 8: 5–12. doi: 10.1002/(SICI)1520-6394(1998)8:1+<5::AID-DA2>3.0.CO;2-I
- 24. Millan MJ (2005) Serotonin 5-HT2C receptors as a target for the treatment of depressive and anxious states: focus on novel therapeutic strategies. Therapie 60: 441–460. doi: 10.2515/therapie:2005065
- 25. Millan MJ (2003) The neurobiology and control of anxious states. Prog Neurobiol 70: 83–244. doi: 10.1016/S0301-0082(03)00087-X
- 26. Serretti A, Artioli P, De Ronchi D (2004) The 5-HT2C receptor as a target for mood disorders. Expert Opin Ther Targets 8: 15–23. doi: 10.1517/14728184.108.40.206
- 27. Ribases M, Fernandez-Aranda F, Gratacos M, Mercader JM, Casasnovas C, et al. (2008) Contribution of the serotoninergic system to anxious and depressive traits that may be partially responsible for the phenotypical variability of bulimia nervosa. Journal of psychiatric research 42: 50–57. doi: 10.1016/j.jpsychires.2006.09.001
- 28. Gardiner K, Du Y (2006) A-to-I editing of the 5HT2C receptor and behaviour. Briefings in functional genomics & proteomics 5: 37–42. doi: 10.1093/bfgp/ell006
- 29. Moreau JL, Bos M, Jenck F, Martin JR, Mortas P, et al. (1996) 5HT2C receptor agonists exhibit antidepressant-like properties in the anhedonia model of depression in rats. European neuropsychopharmacology: the journal of the European College of Neuropsychopharmacology 6: 169–175. doi: 10.1016/0924-977X(96)00015-6
- 30. Millan MJ, Brocco M, Gobert A, Dekeyne A (2005) Anxiolytic properties of agomelatine, an antidepressant with melatoninergic and serotonergic properties: role of 5-HT2C receptor blockade. Psychopharmacology (Berl) 177: 448–458. doi: 10.1007/s00213-004-1962-z
- 31. Pjrek E, Winkler D, Konstantinidis A, Willeit M, Praschak-Rieder N, et al. (2007) Agomelatine in the treatment of seasonal affective disorder. Psychopharmacology (Berl) 190: 575–579. doi: 10.1007/s00213-006-0645-3
- 32. Olie JP, Kasper S (2007) Efficacy of agomelatine, a MT1/MT2 receptor agonist with 5-HT2C antagonistic properties, in major depressive disorder. Int J Neuropsychopharmacol 10: 661–673. doi: 10.1017/S1461145707007766
- 33. Stein DJ, Ahokas AA, de Bodinat C (2008) Efficacy of agomelatine in generalized anxiety disorder: a randomized, double-blind, placebo-controlled study. J Clin Psychopharmacol 28: 561–566. doi: 10.1097/JCP.0b013e318184ff5b
- 34. Gatch MB (2003) Discriminative stimulus effects of m-chlorophenylpiperazine as a model of the role of serotonin receptors in anxiety. Life Sci 73: 1347–1367. doi: 10.1016/S0024-3205(03)00422-3
- 35. Murphy DL, Mueller EA, Hill JL, Tolliver TJ, Jacobsen FM (1989) Comparative anxiogenic, neuroendocrine, and other physiologic effects of m-chlorophenylpiperazine given intravenously or orally to healthy volunteers. Psychopharmacology (Berl) 98: 275–282. doi: 10.1007/BF00444705
- 36. Van Veen JF, Van der Wee NJ, Fiselier J, Van Vliet IM, Westenberg HG (2007) Behavioural effects of rapid intravenous administration of meta-chlorophenylpiperazine (m-CPP) in patients with generalized social anxiety disorder, panic disorder and healthy controls. Eur Neuropsychopharmacol 17: 637–642. doi: 10.1016/j.euroneuro.2007.03.005
- 37. Charney DS, Woods SW, Goodman WK, Heninger GR (1987) Serotonin function in anxiety. II. Effects of the serotonin agonist MCPP in panic disorder patients and healthy subjects. Psychopharmacology (Berl) 92: 14–24. doi: 10.1007/BF00215473
- 38. Campbell BM, Merchant KM (2003) Serotonin 2C receptors within the basolateral amygdala induce acute fear-like responses in an open-field environment. Brain Res 993: 1–9. doi: 10.1016/S0006-8993(03)03384-5
- 39. Bagdy G, Graf M, Anheuer ZE, Modos EA, Kantor S (2001) Anxiety-like effects induced by acute fluoxetine, sertraline or m-CPP treatment are reversed by pretreatment with the 5-HT2C receptor antagonist SB-242084 but not the 5-HT1A receptor antagonist WAY-100635. Int J Neuropsychopharmacol 4: 399–408. doi: doi:10.1017/S1461145701002632
- 40. Burghardt NS, Bush DE, McEwen BS, LeDoux JE (2007) Acute selective serotonin reuptake inhibitors increase conditioned fear expression: blockade with a 5-HT(2C) receptor antagonist. Biol Psychiatry 62: 1111–1118. doi: 10.1016/j.biopsych.2006.11.023
- 41. Christianson JP, Ragole T, Amat J, Greenwood BN, Strong PV, et al. (2010) 5-hydroxytryptamine 2C receptors in the basolateral amygdala are involved in the expression of anxiety after uncontrollable traumatic stress. Biol Psychiatry 67: 339–345. doi: 10.1016/j.biopsych.2009.09.011
- 42. Cornelio AM, Nunes-de-Souza RL (2007) Anxiogenic-like effects of mCPP microinfusions into the amygdala (but not dorsal or ventral hippocampus) in mice exposed to elevated plus-maze. Behav Brain Res 178: 82–89. doi: 10.1016/j.bbr.2006.12.003
- 43. de Mello Cruz AP, Pinheiro G, Alves SH, Ferreira G, Mendes M, et al. (2005) Behavioral effects of systemically administered MK-212 are prevented by ritanserin microinfusion into the basolateral amygdala of rats exposed to the elevated plus-maze. Psychopharmacology (Berl) 182: 345–354. doi: 10.1007/s00213-005-0108-2
- 44. Overstreet DH, Knapp DJ, Angel RA, Navarro M, Breese GR (2006) Reduction in repeated ethanol-withdrawal-induced anxiety-like behavior by site-selective injections of 5-HT1A and 5-HT2C ligands. Psychopharmacology (Berl) 187: 1–12. doi: 10.1007/s00213-006-0389-0
- 45. Strong PV, Christianson JP, Loughridge AB, Amat J, Maier SF, et al. (2011) 5-hydroxytryptamine 2C receptors in the dorsal striatum mediate stress-induced interference with negatively-reinforced instrumental escape behavior. Neuroscience doi: 10.1016/j.neuroscience.2011.09.041
- 46. Davis M (1992) The role of the amygdala in fear and anxiety. Annu Rev Neurosci 15: 353–375. doi: 10.1146/annurev.ne.15.030192.002033
- 47. LeDoux J (2003) The emotional brain, fear, and the amygdala. Cell Mol Neurobiol 23: 727–738. doi: 10.1023/A:1025048802629
- 48. Sherman AD, Sacquitne JL, Petty F (1982) Specificity of the learned helplessness model of depression. Pharmacol Biochem Behav 16: 449–454. doi: 10.1016/0091-3057(82)90451-8
- 49. Christianson JP, Ragole T, Amat J, Greenwood BN, Strong PV, et al. (2010) 5-hydroxytryptamine 2C receptors in the basolateral amygdala are involved in the expression of anxiety after uncontrollable traumatic stress. Biological Psychiatry 67: 339–345. doi: 10.1016/j.biopsych.2009.09.011
- 50. Strong PV, Christianson JP, Loughridge AB, Amat J, Maier SF, et al. (2011) 5-hydroxytryptamine 2C receptors in the dorsal striatum mediate stress-induced interference with negatively reinforced instrumental escape behavior. Neuroscience 197: 132–144. doi: 10.1016/j.neuroscience.2011.09.041
- 51. Englander MT, Dulawa SC, Bhansali P, Schmauss C (2005) How stress and fluoxetine modulate serotonin 2C receptor pre-mRNA editing. The Journal of neuroscience: the official journal of the Society for Neuroscience 25: 648–651. doi: 10.1523/JNEUROSCI.3895-04.2005
- 52. Barbon A, Orlandi C, La Via L, Caracciolo L, Tardito D, et al. (2011) Antidepressant treatments change 5-HT2C receptor mRNA expression in rat prefrontal/frontal cortex and hippocampus. Neuropsychobiology 63: 160–168. doi: 10.1159/000321593
- 53. Broocks A, Meyer T, George A, Hillmer-Vogel U, Meyer D, et al. (1999) Decreased neuroendocrine responses to meta-chlorophenylpiperazine (m-CPP) but normal responses to ipsapirone in marathon runners. Neuropsychopharmacology 20: 150–161. doi: 10.1016/S0893-133X(98)00056-6
- 54. Broocks A, Meyer T, Gleiter CH, Hillmer-Vogel U, George A, et al. (2001) Effect of aerobic exercise on behavioral and neuroendocrine responses to meta-chlorophenylpiperazine and to ipsapirone in untrained healthy subjects. Psychopharmacology (Berl) 155: 234–241. doi: 10.1007/s002130100706
- 55. Dwyer D, Browning J (2000) Endurance training in Wistar rats decreases receptor sensitivity to a serotonin agonist. Acta Physiol Scand 170: 211–216. doi: 10.1046/j.1365-201x.2000.00774.x
- 56. Strong PV, Greenwood BN, Fleshner M (2009) The effects of the selective 5-HT(2C) receptor antagonist SB 242084 on learned helplessness in male Fischer 344 rats. Psychopharmacology (Berl) 203: 665–675. doi: 10.1007/s00213-008-1413-3
- 57. Atallah HE, Lopez-Paniagua D, Rudy JW, O'Reilly RC (2007) Separate neural substrates for skill learning and performance in the ventral and dorsal striatum. Nat Neurosci 10: 126–131. doi: 10.1038/nn1817
- 58. Paxinos G, Watson C. (1998) The Rat Brain in Stereotaxic Coordinates. New York: Academic Press.
- 59. Maier SF (1990) Role of fear in mediating shuttle escape learning deficit produced by inescapable shock. J Exp Psychol Anim Behav Process 16: 137–149. doi: 10.1037/0097-7403.16.2.137
- 60. Fanselow M, Lester L (1988) A functional behavioristic approach to aversively motivated behavior: predatory imminence as a determinant of the topography of defensive behavior. In:Evolution and Learning; Bolles RC BM, editor. Hillsdale, NJ: Erlbaum. 185–212 p.
- 61. Maier SF, Watkins LR (1998) Stressor Controllability, Anxiety, and Serotonin. Cognitive Therapy and Research 22: 595–613. doi: 10.1023/a:1018794104325
- 62. Greenwood BN, Foley TE, Day HE, Burhans D, Brooks L, et al. (2005) Wheel running alters serotonin (5-HT) transporter, 5-HT1A, 5-HT1B, and alpha 1b-adrenergic receptor mRNA in the rat raphe nuclei. Biol Psychiatry 57: 559–568. doi: 10.1016/j.biopsych.2004.11.025
- 63. Greenwood BN, Foley TE, Le TV, Strong PV, Loughridge AB, et al. (2011) Long-term voluntary wheel running is rewarding and produces plasticity in the mesolimbic reward pathway. Behav Brain Res 217: 354–362. doi: 10.1016/j.bbr.2010.11.005
- 64. Salam JN, Fox JH, Detroy EM, Guignon MH, Wohl DF, et al. (2009) Voluntary exercise in C57 mice is anxiolytic across several measures of anxiety. Behavioural brain research 197: 31–40. doi: 10.1016/j.bbr.2008.07.036
- 65. Greenwood BN, Loughridge AB, Sadaoui N, Christianson JP, Fleshner M (2012) The protective effects of voluntary exercise against the behavioral consequences of uncontrollable stress persist despite an increase in anxiety following forced cessation of exercise. Behavioural brain research 233: 314–321. doi: 10.1016/j.bbr.2012.05.017
- 66. Campeau S, Davis M (1995) Involvement of the central nucleus and basolateral complex of the amygdala in fear conditioning measured with fear-potentiated startle in rats trained concurrently with auditory and visual conditioned stimuli. J Neurosci 15: 2301–2311.
- 67. Wilensky AE, Schafe GE, Kristensen MP, LeDoux JE (2006) Rethinking the fear circuit: the central nucleus of the amygdala is required for the acquisition, consolidation, and expression of Pavlovian fear conditioning. J Neurosci 26: 12387–12396. doi: 10.1523/JNEUROSCI.4316-06.2006
- 68. Li Q, Wichems CH, Ma L, Van de Kar LD, Garcia F, et al. (2003) Brain region-specific alterations of 5-HT2A and 5-HT2C receptors in serotonin transporter knockout mice. J Neurochem 84: 1256–1265. doi: 10.1046/j.1471-4159.2003.01607.x
- 69. Li Q, Luo T, Jiang X, Wang J (2012) Anxiolytic effects of 5-HTA receptors and anxiogenic effects of 5-HTC receptors in the amygdala of mice. Neuropharmacology 62: 474–484. doi: 10.1016/j.neuropharm.2011.09.002
- 70. Nestler EJ, Carlezon WA Jr (2006) The mesolimbic dopamine reward circuit in depression. Biol Psychiatry 59: 1151–1159. doi: 10.1016/j.biopsych.2005.09.018
- 71. Hasler G, Fromm S, Carlson PJ, Luckenbaugh DA, Waldeck T, et al. (2008) Neural response to catecholamine depletion in unmedicated subjects with major depressive disorder in remission and healthy subjects. Archives of general psychiatry 65: 521–531. doi: 10.1001/archpsyc.65.5.521
- 72. Lex B, Hauber W (2010) The role of dopamine in the prelimbic cortex and the dorsomedial striatum in instrumental conditioning. Cerebral cortex 20: 873–883. doi: 10.1093/cercor/bhp151
- 73. Alex KD, Yavanian GJ, McFarlane HG, Pluto CP, Pehek EA (2005) Modulation of dopamine release by striatal 5-HT2C receptors. Synapse 55: 242–251. doi: 10.1002/syn.20109
- 74. Dishman RK, Renner KJ, Youngstedt SD, Reigle TG, Bunnell BN, et al. (1997) Activity wheel running reduces escape latency and alters brain monoamine levels after footshock. Brain research bulletin 42: 399–406. doi: 10.1016/S0361-9230(96)00329-2
- 75. Eberle-Wang K, Mikeladze Z, Uryu K, Chesselet MF (1997) Pattern of expression of the serotonin2C receptor messenger RNA in the basal ganglia of adult rats. The Journal of comparative neurology 384: 233–247. doi: 10.1002/(SICI)1096-9861(19970728)384:2<233::AID-CNE5>3.0.CO;2-2
- 76. Lovinger DM (2010) Neurotransmitter roles in synaptic modulation, plasticity and learning in the dorsal striatum. Neuropharmacology 58: 951–961. doi: 10.1016/j.neuropharm.2010.01.008
- 77. Balleine BW, Liljeholm M, Ostlund SB (2009) The integrative function of the basal ganglia in instrumental conditioning. Behav Brain Res 199: 43–52. doi: 10.1016/j.bbr.2008.10.034
- 78. Wang Q, O'Brien PJ, Chen CX, Cho DS, Murray JM, et al. (2000) Altered G protein-coupling functions of RNA editing isoform and splicing variant serotonin2C receptors. J Neurochem 74: 1290–1300. doi: 10.1046/j.1471-4159.2000.741290.x
- 79. Burns CM, Chu H, Rueter SM, Hutchinson LK, Canton H, et al. (1997) Regulation of serotonin-2C receptor G-protein coupling by RNA editing. Nature 387: 303–308. doi: 10.1038/387303a0
- 80. Niswender CM, Copeland SC, Herrick-Davis K, Emeson RB, Sanders-Bush E (1999) RNA editing of the human serotonin 5-hydroxytryptamine 2C receptor silences constitutive activity. J Biol Chem 274: 9472–9478. doi: 10.1074/jbc.274.14.9472
- 81. Herrick-Davis K, Grinde E, Niswender CM (1999) Serotonin 5-HT2C receptor RNA editing alters receptor basal activity: implications for serotonergic signal transduction. J Neurochem 73: 1711–1717. doi: 10.1046/j.1471-4159.1999.731711.x
- 82. Price RD, Sanders-Bush E (2000) RNA editing of the human serotonin 5-HT(2C) receptor delays agonist-stimulated calcium release. Mol Pharmacol 58: 859–862.
- 83. Berg KA, Clarke WP, Cunningham KA, Spampinato U (2008) Fine-tuning serotonin2c receptor function in the brain: molecular and functional implications. Neuropharmacology 55: 969–976. doi: 10.1016/j.neuropharm.2008.06.014
- 84. Iwamoto K, Bundo M, Kato T (2009) Serotonin receptor 2C and mental disorders: genetic, expression and RNA editing studies. RNA Biol 6: 248–253. doi: 10.4161/rna.6.3.8370
- 85. Gurevich I, Tamir H, Arango V, Dwork AJ, Mann JJ, et al. (2002) Altered editing of serotonin 2C receptor pre-mRNA in the prefrontal cortex of depressed suicide victims. Neuron 34: 349–356. doi: 10.1016/S0896-6273(02)00660-8
- 86. Gardiner K, Du Y (2006) A-to-I editing of the 5HT2C receptor and behaviour. Brief Funct Genomic Proteomic 5: 37–42. doi: 10.1093/bfgp/ell006
- 87. Gurevich I, Englander MT, Adlersberg M, Siegal NB, Schmauss C (2002) Modulation of serotonin 2C receptor editing by sustained changes in serotonergic neurotransmission. J Neurosci 22: 10529–10532.
- 88. Moya PR, Fox MA, Jensen CL, Laporte JL, French HT, et al. (2011) Altered 5-HT2C receptor agonist-induced responses and 5-HT2C receptor RNA editing in the amygdala of serotonin transporter knockout mice. BMC Pharmacol 11: 3. doi: 10.1186/1471-2210-11-3
- 89. Davis JM, Bailey SP (1997) Possible mechanisms of central nervous system fatigue during exercise. Med Sci Sports Exerc 29: 45–57. doi: 10.1097/00005768-199701000-00008