A Role for Adenosine Parsa Ghasem De

Exercise and Acute Stress-Induced Changes in Direct and Indirect Pathway c-fos Expression: A Role for Adenosine

Parsa Ghasem Department of Integrative Physiology, University of Colorado at Boulder

Defended April 2, 2014

Thesis Advisor: Dr. Monika Fleshner, Department of Integrative Physiology

Defense Committee: Dr. Monika Fleshner, Department of Integrative Physiology Dr. David Sherwood, Department of Integrative Physiology Dr. Ryan Bachtell, Department of Psychology and Neuroscience

Ghasem 2

Table of Contents Abstract ...... 3 Introduction...... 4 Fig. 1 The Functional Anatomy of Striatal Medium Spiny Neurons ...... 5 Materials and Methods...... 8 Animals...... 8 Uncontrollable Tail Shock Stress Protocol ...... 8 Tissue Preparation...... 8 Radioactive in situ Hybridization...... 9 Double Label Fluorescent in situ Hybridization...... 9 Image Analysis for Radioactive in situ Hybridization ...... 10 Image Analysis for Double Label Fluorescent in situ Hybridization...... 11 Statistical Analysis...... 11 Results ...... 12 Wheel running (Fig. 2) ...... 12 A1R and A2aR Gene Expression (Fig. 3) ...... 12 D1R and D2R Gene Expression (Fig. 4) ...... 13 Plasma Corticosterone Levels (Fig. 5) ...... 15 Activation of Dynorphin Neurons in the Dorsal Striatum Following Acute Stress (Fig. 6).....15 Activation of Enkephalin Neurons in the Dorsal Striatum Following Acute Stress (Fig. 7) ...16 Discussion...... 19 Fig. 8 Habenular and Traditional Striatal Circuitry...... 22 Acknowledgements...... 24 References...... 25 Ghasem 3

Abstract

Exposure to uncontrollable stress is a major risk factor for developing depression- like behaviors such as deficits in goal-directed learning. It has been suggested that such impairments are mediated by the striatum and can be prevented by striatal neuroplastic changes induced by exercise. Recent evidence suggests that adenosine and dopamine (DA) are potent modulators of striatal signaling. Moreover, exercise can influence adenosine and DA activity. Therefore, exercise could modify adenosine and DA receptor systems, thereby altering striatal function. The purpose of this study was to 1) examine the effects of 6 weeks of wheel running on the mRNA expression of adenosine 1 (A1R), adenosine 2A (A2aR), DA 1 (D1R), and DA 2 (D2R) receptors in the dorsal striatum and 2) determine if exercise-induced changes in mRNA expression of these receptors correlated with differences in c-fos mRNA induction in striatal direct and indirect pathway neurons following uncontrollable stress. The results from these studies were that running significantly reduces the mRNA expression of A1R and A2aR in the dorsal striatum. Exercise did not mediate D1R mRNA expression; however, a small but significant increase of D2R mRNA was detected in the dorsal striatum. Finally, prior voluntary exercise increased c-fos mRNA expression in dynorphin-expressing direct pathway neurons and decreased its expression in enkephalin-expressing indirect pathway neurons following uncontrollable stress. Taken together, these results are consistent with the conclusion that running induced alterations of the adenosine-dopamine receptor system could modify striatal output, which may underlie the protective effects of exercise against stress-induced deficits in goal-directed learning. Ghasem 4

Introduction

The World Health Organization has announced that by 2030 depression will be one of the largest economic health burdens on society, behind only HIV/AIDS. (Mathers and Loncar, 2006). The efficacy of current pharmacological treatments for stress-related mood disorders such as depression and anxiety, however, has been called into question (for review see (Turner et al., 2008; Fournier et al., 2010). As a result, researchers are turning to animal models as a potential tool to elucidate not only the neurobiology underlying depression and anxiety but also the mechanisms of treatments shown to be successful in humans at ameliorating these ailments, such as habitual physical activity (Blumenthal et al., 2007; Martinsen and Morgan, 1997; Martinsen, 2008; Salmon, 2001). Identifying these mechanisms may contribute to the development of more effective approaches for treating and preventing depression.

Learned Helplessness (LH) is an established animal paradigm in which behavioral impairments mimic many of the symptoms of human depression (Maier and Seligman, 1976; Overmier and Seligman, 1967) and are sensitive to common pharmacological interventions (Martin and Puech, 1996; Maudhuit et al., 1997). In this model rats exposed to an acute uncontrollable, but not controllable, stressor (Greenwood et al., 2003; Greenwood et al., 2012; Maier, 1984; Maier and Watkins, 2005; Strong et al., 2011) display a constellation of specific LH behavioral impairments. As such, LH is defined as a milieu of symptoms that emulate a perceived absence of control over a situational outcome as well as an inability to determine the consequences of one’s actions (Maier and Seligman, 1976; Maier, 1984). Furthermore, it is clear that exercise can both diminish the symptoms of depression and reduce the relative risk of developing depression in humans (Blumenthal et al., 2007; Martinsen and Morgan, 1997; Martinsen, 2008; Salmon, 2001). These types of effects of exercise are also reported using the rat LH model. Indeed, allowing rodents voluntary access to running wheels for 6 weeks before the administration of the uncontrollable stressor significantly attenuates the sequelae of behavioral and cognitive deficits. (Greenwood et al., 2003; Greenwood et al., 2005; Greenwood and Fleshner, 2008; Greenwood et al., 2012; Rozeske et al., 2011; Strong et al., 2011). Taken together, LH may be utilized as a potential model to examine the neurological mechanisms underlying both stress-related mood disorders as well as the stress buffering effects of exercise.

Instrumental or goal directed-learning is defined as the process by which a behavior is modified by the rewarding outcomes or punishing consequences it produces, and is significantly impaired by uncontrollable stress. Moreover, these deficits in goal- directed learning parallel the cognitive deficits of clinical depression (for review see: Brown and Pluck, 2000; Strong et al., 2011). Such impairments may be characterized in animal models via the use of various behavioral assays. Traditionally, researchers employing the LH paradigm utilize the shuttle box escape task to identify these deficits. Interestingly, animals that have been exposed to uncontrollable stress days before behavioral testing display an impaired ability to complete the task compared to rats that were not stressed, indicating an impairment of goal-directed learning. (Maier and Ghasem 5

Watkins, 2005; Greenwood et al., 2003; Greenwood and Fleshner, 2008; Greenwood et al., 2012; Strong et al., 2011).

Goal-directed behavior is complex and involves contributions from several brain regions. The The Direct and Indirect Pathways striatum is one region in particular that

has received increasing attention for its Pathway Indirect role in mediating this behavior. Suggested to integrate sensorimotor Proteins Proteins information and initiate the movements Expressed: Expressed: D1R (+) D2R (-) necessary in order to obtain reward A1R (-) A1R (-) Substance P A R (+) and/or avoid punishment (Kravitz and 2a Dynorphin Enkephalin Kreitzer, 2012), the striatum has been DirectPathway directly implicated in goal directed learning (Bhatia and Marsden, 1994; Promotes Movement Traditional Inhibits Movement Hikida et al., 2010; Kravitz et al., 2012; Samejima et al., 2005a). Signals Reward & Goal Novel Signals Aversion Directed Learning Moreover, our lab has also implicated the striatum in deficits of goal-directed Fig. 1 The Functional Anatomy of Striatal Medium learning brought on by stress Spiny Neurons (Greenwood et al., 2012; Strong et al., Differential protein/receptor expression and function of 2011). The striatum is mostly striatal direct and indirect pathway MSNs. “+” indicates composed of GABAergic medium G -GPCR and “-“ indicates G -GPCR spiny neurons (MSN), named for their s i medium soma size and specialized dendritic processes (Lanciego et al., 2012). Lastly, while these MSNs are morphologically similar, they are commonly delineated into two classes based upon their neurochemical composition and function (summarized in Fig.1).

These two classes of MSN have been termed “direct” and “indirect” pathway neurons based upon their distinct protein/receptor expression, projection sites, and functions concerning the generation of movement (Lanciego et al., 2012). Briefly, direct pathway MSNs are thought to nearly exclusively express the opiod peptide dynorphin, substance P, and dopamine (DA) D1 receptors (D1R). On the other hand, indirect pathway MSNs nearly exclusively express the opiod peptide enkephalin, DA D2 receptors (D2R), and adenosine 2a receptors (A2aR). Both classes of MSNs express the adenosine A1 receptor (A1R) (Ferré et al., 1997; Fuxe et al., 2007; Gerfen et al., 1990; Lanciego et al., 2012; Lu et al., 1997; Svenningsson et al., 1999). In terms of locomotion, activation of the direct pathway is classically associated with the promotion of movement, whereas activation of the indirect pathway is implicated in the inhibition of movement (Durieux et al., 2009; Kravitz et al., 2010; Kravitz and Kreitzer, 2012; Sano et al., 2003). Furthermore, a role for the direct and indirect pathways in reward and aversion, respectively, has recently been elucidated. Activation of the direct has been implicated in signaling reward while activation of the indirect pathway is thought to signal aversion (Hikida et al., 2010; Kravitz et al., 2012). Moreover, the direct pathway Ghasem 6 has been specifically identified as a contributor to reinforcement and goal-directed learning (Kravitz et al., 2010; Samejima et al., 2005b). Therefore, an imbalance of neural activity between these pathways may underlie a failure to initiate the goal-directed movements needed to escape shock in the shuttle box escape task. Such aberrant signaling may contribute to the impairments of goal-directed learning seen in LH.

The activity of both the direct and indirect pathway MSNs is potently modulated by the purine nucleoside, adenosine, and the monoamine, DA. For example, acting on D1Rs, DA initiates adenylyl cyclase signaling, possibly activating direct pathway MSNs promoting both movement and reward. Conversely, when DA binds D2Rs, adenylyl cyclase signaling is attenuated, possibly inactivating indirect pathway MSNs ultimately permitting movement and reward. (Lanciego et al., 2012; Kravitz and Kreitzer, 2012). Adenosine can act to modulate DA induced striatal activity via the formation of adenosine-dopamine heteromeric receptor complexes. Binding of adenosine to A1Rs attenuates adenyl cyclase signaling and when dimerized with D1R reduces the binding affinity for DA, thereby attenuating activation of the direct pathway, permitting the inhibition of movement and the promotion of aversion. On the other hand, the binding of adenosine to A2aRs increases adenyl cyclase signaling and also decreases the affinity of D2Rs for DA when dimerized, thereby increasing activation of the indirect pathway, inhibiting movement and promoting aversion (Ferré et al., 1997; Ferré et al., 2007; Kravitz and Kreitzer, 2012; Lanciego et al., 2012). Taken together, DA acting in the striatum acts to promote movement and reward while adenosine initiates cellular processes that lead to the inhibition of movement and the promotion of aversion.

The purine and ATP metabolite, adenosine has been previously implicated as a major contributor to impairments in goal-directed learning, but outside of the specific context of the striatum. It has been hypothesized that the administration of the uncontrollable stressor used to elicit LH drastically increase extracellular adenosine levels in stress reactive brain regions, e.g. the striatum, promoting goal-directed learning deficits. (Dunwiddie and Masino, 2001; Dworak et al., 2007; Hanff et al., 2010; Hoehn and White, 1990; Hunter et al., 2003; Minor and Hunter, 2002a; Minor, 2008; Woodson et al., 1998). Indeed, Minor et al. reported that blockade of central adenosine receptors can prevent the shuttle box escape deficits produced by uncontrollable stress; while activation of A2aRs on the other hand, is sufficient to produce those deficits in the absence of stress to produce those same behaviors (Minor et al., 1994a; Minor et al., 1994b). Moreover, the use of a nucleoside transport blocker to artificially increase extracellular brain adenosine concentrations induces LH-like shuttle box escape latencies and interacts synergistically with uncontrollable stress to similarly inhibit test performance (Minor, 2008). Given the unique pattern of expression of adenosine receptors on the direct and indirect pathways of the striatum, it is reasonable to hypothesize that high levels of extracellular adenosine following uncontrollable stress induces a differential activation of these two circuits to promote the freezing and aversion that underlie goal-directed learning deficits. Consequently, any neuroplasticity of the adenosine-dopamine receptor system in the striatum could contribute to the modulation of goal-directed learning and may serve as a means by which exercise exerts some of its anxiolytic influence. Ghasem 7

One potential mechanism by which voluntary exercise may prevent goal-directed learning deficits brought on by stress is through alterations in the expression of A1R and A2aR mRNA in the striatum. Continuous or acute exposure to A1R and A2aR agonists has been reported to induce both increases and decreases of adenosine receptor mRNA (Kobayashi and Millhorn, 1999; León et al., 2009; Ruiz et al., 2011; Vendite et al., 1998). Moreover, exercise can potently increase brain-wide adenosine concentrations in a dose- dependent manner (Dworak et al., 2007). As such, consistent increases in adenosine concentrations following instances of voluntary wheel running may be sufficient to change adenosine receptor mRNA expression in the striatum. Given the protective effects of exercise observed in a LH model, alterations in striatal adenosine receptor mRNA expression may underlie some of the stress-buffering benefits associated with voluntary wheel running. Such changes in adenosine receptor transcript have been found to correlate with similar changes in both radioligand binding affinity and protein concentration in the brain (Albasanz et al., 2006; Calon et al., 2004; Cheng et al., 2000; Khoa et al., 2001; León et al., 2004; Murphree et al., 2005; Varani et al., 2010). In addition, it is also possible that repeated voluntary wheel running, which also increases striatal DA concentrations (unpublished data), may induce similar changes to dopamine receptor mRNA expression within this region. As a result, any exercise induced transcriptional changes to A1R, A2aR, D1R, and D2R may have functional receptor consequences, which may ultimately influence striatal MSN activity. However, whether or not voluntary exercise impacts the transcriptional capacity of these receptors in the striatum is, to the best of our knowledge, unknown.

The first objective of this study, therefore, was to examine the effects of 6 weeks of voluntary wheel running on the mRNA expression of A1R, A2aR, D1R, and D2R in the dorsal medial striatum (DMS) and the dorsal lateral striatum (DLS), using single label radioactive in situ hybridization. Generally speaking, the DMS has been implicated in goal directed learning and the acquisition of instrumental behaviors (Lex and Hauber, 2010; Yin et al., 2005) while the DLS has been associated with habit learning and the maintenance of instrumental behaviors (Yin et al., 2006; Yin and Knowlton, 2006). Regional changes to mRNA expression of A1R, A2aR, D1R, and D2R, therefore, may have significance for different striatal processes. The second objective of this study was to determine if potential changes in the expression of these receptors due to exercise correlated with differences in activation of direct and indirect pathway MSNs following uncontrollable stress. A measure of striatal pathway activity was obtained via double label fluorescence in situ hybridization (FISH) using the immediate early gene, c-fos, as a marker of cellular activity and either dynorphin or enkephalin as markers of direct or indirect pathway MSNs respectively. Exercise-induced changes to A1R, A2aR, D1R, and D2R gene expression may result in a differential stimulation of direct and/or indirect pathway MSNs. Given the opposing roles of these pathways in movement and reward, potential changes observed in direct or indirect pathway MSN activity could contribute to the ability of exercise to prevent deficits in goal-directed learning.

Ghasem 8

Materials and Methods

Animals 32 male Fischer344 rats (obtained from Harlan SPF, Indianapolis, IN, USA) weighing approximately 148 g at time of arrival were used in all experiments. Rats were either individually housed in standard Nalgene Plexiglas cages (45x25.2x14.7 cm) (n=16) or in cages with locked running wheels, which were rendered immobile with metal stakes (45x25.2x14.7) (n=16). Animals were housed in a temperature (21 ± 1°C) and humidity- controlled environment, were maintained on a 12 hr light/ dark cycle (lights on 6 A.M.-6 P.M.), and had ad libitum access to food (Harlan Teklad 7012) and water. Rats were acclimated to these housing conditions for 1 week before any experimental manipulation. After 1 week, running wheels were unlocked and rats were granted voluntary access to running wheels for the remainder of the study (6-7 weeks). Daily wheel revolutions were recorded digitally using Vital View software (Mini Mitter, Bend, OR, USA). Although individual housing can evoke some features of the stress response in rats (Sharp et al., 2002), single housing was necessary in these experiments to allow quantification of the activity of individual animals and to avoid competition for running wheels. Care was taken to minimize animal discomfort during all procedures. All experimental protocols were approved by the University of Colorado Animal Care and Use Committee and adhered to NIH guidelines. Rats were weighed weekly.

Uncontrollable Tail Shock Stress Protocol After 6 weeks of running or sedentary conditions, rats were randomly assigned to receive inescapable shock (IS) (n=8 Sedentary IS, n=8 Running IS) or remain in their home cages as controls (n=8 sedentary Control, n=8 Running Control). Stressed animals were restrained in Plexiglas tubes (23.4 cm long and 7.0 cm in diameter) with their tails protruding from the back. Electrodes were placed over the tails and 100 tail shocks (5 sec, 1.5 mA) on a 1-minute variable intershock interval were delivered. Evidence suggests that this acute stressor is sufficient to both induce symptoms of learned helplessness as well as tax brain metabolic capacity resulting in adenosine accumulation. Control rats that did not receive IS were left undisturbed in home cages during tail shocks.

Tissue Preparation All rats were euthanized via rapid decapitation immediately following uncontrollable shock in pairs (one from the IS group and one from the Control group). Brains were then rapidly extracted and flash frozen in chilled (dry ice) isopentane (-20°C) for 4 minutes and subsequently stored at -80°C until sectioning. Brains were cross- sectioned at 10 µm from rostral to caudal and striatal sections were thaw mounted on Superfrost Plus slides (Fisherbrand Pittsburgh, PA) and stored at -80°C until processing for single label radioactive in situ hybridization or double labeled fluorescent in situ hybridization (FISH).

Ghasem 9

Radioactive in situ Hybridization Purpose: To detect relative differences in striatal A1R, A2AR, D1R, or D2R mRNA due to running. Radioactive in situ hybridization followed our previously published protocols. (Greenwood 2003, Day and Akil 1996) Briefly, on day one, a 1-in-20 series (separated by 200 µm) of sections containing the striatum were fixed in 4% paraformaldehyde for 1hr, acetylated in 0.1M triethanolamine containing 0.25% acetic anhydride for 10 min, and then dehydrated in graded ethanol. cRNA riboprobes complementary to A1R (743 bp), A2AR (949 bp), D1R (461 bp), and D2R (492 bp) were prepared from cDNA subclones in transcription vectors and labeled with [35S]UTP (courtesy of Serge Campeau, Heidi Day, and Aggie Mika, University of Colorado at Boulder, USA), using standard transcription methods. Riboprobes were diluted in 50% hybridization buffer containing 50% formamide, 10% dextran sulfate, 2x Sodium Citrate (SSC), 50mM Phosphate Buffered-Saline (PBS) at pH 7.4, 1x Denhardt’s solution, and 0.1 mg/ml yeast tRNA. Sections representative of the rostral to caudal striatum were hybridized with each respective probe for approximately 18 hr at 55°C. On day two, sections were washed in 2X SSC and then treated with RNaseA (200 µg/ml) for 1hr at 37°C. Sections were then treated with graded SSC washes (2x, 1X, 0.5X, and 0.1X) and placed in a 0.1X SCC at 65°C for 1hr. Sections were finally dehydrated in graded ethanol, air dried for approximately 40 min, and placed into X-ray film cassettes and covered with X-ray films (Biomax-MR; Eastman Kodak, Rochester, NY, USA) for 3 days (for A2AR, D1, D2) or 7 days (for A1R). For each probe, slides from all rats were processed in a single in situ experiment to allow for direct comparisons. Labeling with “sense” probes indicated that the signal observed with the “antisense” probes was specific.

Double Label Fluorescent in situ Hybridization Purpose: To detect the proportion of enkephalin or dynorphin in the striatum containing neurons that co-express c-fos. Briefly, on day one, a 1-in-20 series (separated by 200 µm) of sections containing the striatum were fixed in 4% paraformaldehyde for 1hr, acetylated in 0.1M triethanolamine containing 0.25% acetic anhydride for 10 min, and then dehydrated in graded ethanol. cRNA riboprobes for dynorphin (744 bp), enkephalin (693bp) and c-fos (680bp) were prepared from cDNA subclones in transcription vectors (courtesy of Serge Campeau and Heidi Day, University of Colorado at Boulder, USA). Both dynorphin and enkephalin probes were labeled with fluorescein- 12-UTP, (Roche, Indianapolis, IN, USA) while c-fos was labeled with digoxigenin-11- UTP (Roche, Indianapolis, IN, USA), using standard transcription methods. Riboprobes were diluted in 50% hybridization buffer containing 50% formamide, 10% dextran sulfate, 2x Sodium Citrate (SSC), 50mM Phosphate Buffered-Saline (PBS) at pH 7.4, 1x Denhardt’s solution, and 0.1 mg/ml yeast tRNA. Sections representative of the rostral to caudal striatum were hybridized with either dynorphin or enkephalin as well as c-fos probes for approximately 18 hr at 55°C. On day two, sections were washed in 2X SSC and then treated with RNaseA (200 µg/ml) for 1hr at 37°C. Sections were then treated with graded SSC washes (2x, 1X, 0.5X, and 0.1X) and placed in a 0.1X SCC at 65°C for 1hr. Sections were then returned to room temperature in distilled water and washed with 0.05M PBS. Sections were then quenched in 2% hydrogen peroxide in 0.05 PBS with Ghasem 10 agitation (30 min, RT). Subsequently, sections were washed in a 1x Tris-Buffered Saline containing 0.05% Tween-20 (pH 7.5, TBS-T), and incubated in 0.5% blocking buffer (Perkin-Elmer, Waltman, MA, USA) in 1x TBS for 1 hr. Sections were immediately incubated in anti-digoxigenin-horseradish peroxidase (Roche, Indianapolis, IN, USA) at 1:750 dilution, 80 µl per slide, in blocking buffer for 30 min. Sections were next washed in TBS-T and the digoxigenin-UTP-c-fos complex was detected with a tyramide signal amplification kit with cyanine 3 (CY3) as the flourophore (45 min at RT; 1:100 dilution, 80 µl per slide, in 1X amplification diluent (Perkin-Elmer, Waltham, MA, USA)). Sections were washed and stored overnight in 0.05M PBS at 4°C. On day three, sections were again quenched in 2% hydrogen peroxide in a 0.05M PBS with agitation (30 min, RT). Sections were next washed in TBS-T and incubated for 90 min in an anti- flourescein-horseradish peroxidase (Perkin-Elmer, Waltham, MA, USA) at 1:100 dilution in blocking buffer, 80 µl per slide. Sections were again rinsed with TBS-T and the flourescein-UTP-dynorphin/enkephalin complex was detected as stated as above with flourescein as the flourophore (1 hr at RT; 1:100 dilution, 80 µl per slide, in 1x amplification diluent (Perkin-Elmer, Waltham, MA, USA)). Slides were then washed in PBS and then air dried for approximately 30 min. Coverslips were placed on slides and set using ProLong Gold antifade reagent with DAPI (Life Technologies, Grand Island, NY, USA). Image Analysis for Radioactive in situ Hybridization Levels of A1R, A2AR, D1R, and D2R mRNA were analyzed by computer assisted optical densitometry according to our previously published protocols. (Greenwood 2003) Images of the brain sections on X-ray film were captured digitally (CCD camera, model XC-77; Sony, Tokyo, Japan) and the relative optical density of the probe on each captured image was determined using Scion Image version 4.0 (Scion, Fredick, 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 (for A2AR, D1R, and D2R) or off the section (for A1R, as it is expressed in oligodendrocytes), and the signal threshold was calculated as mean gray value of background +3.5 SD. 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 the assigned background (mean signal produced by the cRNA probe above background multiplied by the number of pixels above background). Care was taken to ensure that equivalent areas were analyzed between animals. Quantification of each probe occurred in the dorsal medial striatum (DMS) and the dorsal lateral striatum (DLS) at five rostral to caudal levels [coordinates 2.2 to 1.7 mm, 1.6 to 1 mm, 0.2 to -0.3 mm, -0.4 to 0.8 mm, and -0.9 to -1.5 mm from bregma]. The relative optical densities of the 5 levels of the striatum were then averaged to yield a mean integrated density in both the DMS and DLS for each subject.

Ghasem 11

Image Analysis for Double Label Fluorescent in situ Hybridization Images were acquired from a Zeiss AX10 with axioscan Z1 fluorescent microscope interfaced to a computer running axiovision software (Zeiss, Oberkochen, Germany) at 200x total magnification. Fluorescein (dynorphin or enkephalin), Cy3 (c-fos), and DAPI (nuclei) emission channels were merged to create a single image. For each rat, two non- overlapping adjacent images were acquired for both the DMS and DLS in 3 sections across the rostral to caudal axis of the striatum (coordinates between 2.0 to 1.8 mm, 1.2 to 1.0 mm, and 0 to -0.2 mm from bregma). The number of c-fos, enkephalin or dynorphin, and co-labeled (enkephalin/c-fos or dynorphin/c-fos) neurons were then counted in each image. These values were then averaged to yield representative values in each region for each subject. A co-label was confirmed when a dynorphin- or enkephalin-positive cell completely covered and had a similar shape to a c-fos positive cell. Statistical Analysis The relative optical density of A1R, A2AR, D1R, and D2R mRNA expression were reported as an average of the entire rostral to caudal extent for the striatum (DMS and DLS [coordinates between 2.0 to -0.2 mm from bregma]) and compared by a one-way ANOVA with exercise (Sedentary vs. Running) conditions as factors. The density of c- fos positive cells in the dorsal striatum was compared by a two-way ANOVA with exercise and stress condition as factors. Additionally, we estimated the proportions of enkephalin or dynorphin neurons that co-expressed c-fos in exercise and stress. The proportion data were analyzed among groups by a two-way ANOVA with exercise and (Sedentary vs. Running) and stress (Control vs. IS) conditions as factors. For all analysis p<0.05 was considered statistically significant.

Ghasem 12

Results Wheel running (Fig. 2) Wheel running distance (Fig. 2A) increased steadily for the first 3 weeks and thereafter maintained a plateau averaging 4.40 km/week (± 0.17 SE). Average distance run for the entire experiment was 3.79 km/week (± 0.07 SE). Body weight (Fig. 2B) also increased gradually over 6 weeks, however, animals that were allowed voluntary access to running wheels (n=16) gained less weight over time than sedentary animals (n=16). Repeated measures ANOVA revealed significant effects of time (F(5,160) = 542.615; p<0.0001) and exercise (F(1,160) = 20.551; p<0.0001) and a reliable time by exercise interaction (F(5,160) = 7.114; p<0.0001).

Fig. 2. Average wheel running distance per day. Adult male Fischer F344 rats were allowed voluntary access to running wheels for 6 weeks. A) the mean distance (kilometers) run each day by the physically active rats. B) the mean weekly body weight (grams) of physically active and sedentary rats. Data represent the group means ±SEM run each day for 6 weeks.

Radioactive in situ Hybridization

A1R and A2aR Gene Expression (Fig. 3) Considerable evidence suggests that the activation of adenosine receptors may underlie LH behaviors (Minor et al., 1994c; Minor et al., 1994d; Minor, 2008; Hunter et al., 2003). Therefore, voluntary wheel running may prevent the induction of LH instrumental learning deficits by reducing the levels of these receptors, particularly on the medium spiny neurons (MSNs) of the striatum. In order to investigate this hypothesis, the relative amount of A1R and A2aR mRNA were measured in the striatum (dorsal medial and dorsal lateral) of sedentary and physically active rats, in the absence of stress, using in situ hybridization. Five rostral to caudal regions in the striatum (Fig. 4A) were quantified for each receptor and averaged to give a mean integrated density in each region for each subject. Exercise decreased A1R gene expression in both the dorsal medial (DMS) (F(1,28) = 25.381; p<0.0001) and the dorsal lateral (DLS) (F(1,28) = 22.982; p<0.0001) striatum compared to sedentary controls (Fig. 4B). Furthermore, exercise also decreased A2aR gene expression in both the DMS (F(1,28) = 94.522; p<0.0001) and the DLS (F(1,28) = 4387.503; p<0.0001) compared to sedentary controls (Fig 4C). Representative Ghasem 13

autoradiographs of striatal A1R and A2aR mRNA in sedentary and running animals are shown in figures 4D and 4E respectively.

Fig. 3. Expression of (B) A1R and (C) A2aR mRNA in the DMS and DLS of sedentary and exercised animals. A) Rostral to caudal regions of the striatum that were B. sampled. D,E) Representative autoradiographic coronal sections of SED (top) and RUN (bottom) animals for both (D) A1R and (E) A2aR. Data represent the mean integrated density +SEM, ANOVA: ***main effect of exercise (p<0.0001). D1R and D2R Gene Expression (Fig. 4) It is well established that the activation of direct pathway D1Rs and A1Rs yield antagonistic intracellular effects. Similarly, it is known that function of indirect pathway D2Rs and A2aRs are also antagonistic in nature (Ferré et al., 1997; Fuxe et al., 2007). Moreover, given the role of dopamine in facilitating movement and reward, another possible mechanism by which voluntary wheel running could prevent the instrumental learning deficits seen in LH is by increasing the expression of the D1 and D2 receptors on the MSNs within the striatum. In order to determine if voluntary exercise alters the mRNA expression of these receptors, the relative amount of D1R and D2R mRNA were Ghasem 14 measured in the striatum of sedentary and physically active rats, in the absence of stress, using in situ hybridization. Five rostral to caudal regions in the striatum (Fig. 5A) were quantified for each receptor and averaged to give a mean integrated density in each region for each subject. No significant effect of exercise on D1R gene expression was observed compared to sedentary controls in either the DMS or the DLS (Fig. 5B). However, exercise did elevate D2R gene expression in both the DMS (F(1,28) = 7.797; p=0.0093) and the DLS (F(1,28) = 4.742; p=0.038) compared to sedentary controls (Fig 5C). Representative autoradiographs of striatal D1R and D2R mRNA in sedentary and running animals are shown in figures 5D and 5E respectively.

Fig. 4. Expression of (B) D1R and (C) D2R mRNA in the DMS and DLS of sedentary and exercised animals. A) Rostral to caudal regions of the striatum that were sampled. D,E) representative autoradiographic coronal sections of SED (top) and RUN (bottom) animals for both (D) D1R and (E) D2R. Data represent the mean integrated density +SEM, ANOVA: ***main effect of exercise (p<0.0001). Ghasem 15

Plasma Corticosterone Levels (Fig. 5) In order to verify that 100 IS induced a reliable stress response, Corticosterone (CORT) was assayed in plasma collected from trunk blood from all animals. Both sedentary and running animals exposed to 100 IS displayed a significant induction of plasma Corticosterone levels compared to non-stressed controls (F(1,28) = 229.041 p<0.0001). Consistent with our previous reports (Speaker et al., 2013), no effect of exercise on the CORT response to stress was observed (F(1,28) = 0.99; p<0.3283)

Fig. 5. Effect of stress and 6 weeks of voluntary exercise on plasma Corticosterone levels. Animals were either administered 0 (control condition) or 100 acute, inescapable shocks (IS), immediately sacrificed and trunk blood was collected. Data represent the mean +SEM, ANOVA: *** main effect of stress (p<0.0001).

Fluorescent in situ Hybridization Activation of Dynorphin Neurons in the Dorsal Striatum Following Acute Stress (Fig. 6) It has been hypothesized that an acute stressor such as Inescapable Shock (IS) potently enhances metabolic activity in stress reactive brain regions, and could lead to energy shortages, specifically a deficit in ATP (Dunwiddie and Masino, 2001; Hanff et al., 2010; Minor and Hunter, 2002a). Under increased metabolic demand ATP is hydrolyzed to its precursors (i.e. ADP!AMP!Adenosine) ultimately leaving an intracellular accumulation of adenosine. When the accumulation of adenosine reaches a critical amount, it is transported to extracellular space where it binds to A1R to promote metabolic fatigue related processes and possibly shuttle box escape deficits. One possible mechanism by which A1R could contribute to these deficits is via its ability to suppress neuronal activity of direct pathway MSNs Therefore, if the finding that exercise yields a reduction in A1R mRNA expression is reflective of a reduction in functional receptor levels, it is reasonable to expect that exercised animals would display a greater activation of the direct pathway following IS. In order to investigate this hypothesis, the activation of the direct pathway was measured using double label fluorescent in situ hybridization (FISH). 3 rostral to caudal regions of the DMS and DLS (Fig. 5A) were assayed for the Ghasem 16 number of dynorphin cells co-expressing c-fos. The proportion of dynorphin and c-fos double labeled neurons were similar throughout the rostral to caudal striatum for each group. Therefore, an average of the number of double-labeled neurons was taken across all levels of the striatum to yield a proportion of dynorphin cells expressing c-fos for each subject. A significant increase of dynorphin neurons co-expressing c-fos due to stress was observed in both the DMS (F(1,29) = 134.061; p<0.0001) and the DLS (F(1,29) = 133.083; p<0.0001) (Figs. 5C&D). Furthermore, exercise also increased the proportion of dynorphin neurons that expressed c-fos in both the DMS (F(1,29) = 9.53; p=0.0365) and the DLS (F(1,29) = 10.539; p=0.0029) (Figs. 5C&D). In addition, a significant interaction was observed between stress and exercise conditions in the DLS (F(1,29) = 6.528; p=0.0161) but not the DMS (F(1,29) = 3.674; p=0.0652) (Figs. 5C&D). Post hoc analysis revealed that exercise increased the expression of c-fos due to stress in dynorphin neurons in the DLS (p=0.0005). A representative image of a dynorphin, c-fos double label FISH is also shown in figure 5B. Activation of Enkephalin Neurons in the Dorsal Striatum Following Acute Stress (Fig. 7) Increasing levels of extracellular adenosine during IS could also bind A2aR, which when activated acts to elevate neuronal activity of indirect pathway. Therefore, if the finding that exercise yields a reduction A2aR in mRNA expression is reflective of a reduction in functional receptor levels, it is reasonable to hypothesize that exercised animals would display a reduced activation of the indirect pathway following IS. In order to examine this hypothesis, the activation of the indirect pathway was measured using double label FISH. 3 rostral to caudal regions of the DMS and DLS (Fig. 6A) were assayed for the number of enkephalin cells co-expressing c-fos. The proportion of enkephalin and c-fos double labeled neurons were similar throughout the rostral to caudal striatum for each group. As a result an average of the number of double-labeled cells was taken across all levels of the striatum to yield a proportion of enkephalin neurons co-labeled with c-fos for each subject. Stress significantly increased the number of enkephalin neurons co- expressing c-fos in both the DMS (F(1,29) = 170.077; p<0.0001) (Fig 6C) and the DLS (F(1,29) = 81.879; p<0.0001) (Fig 6D). No main effect of exercise on the proportion of enkephalin neurons labeled with c-fos was observed. However, a significant interaction was observed between stress and exercise conditions in the DMS (F(1,29) = 5.664; p=0.0241) but not the DLS (F(1,29) = 0.859; p=.361) in animals that had been exposed to IS (Figs. C&D). Post hoc analysis revealed that exercise decreased the expression of c- fos due to stress in enkephalin neurons in the DMS (p=0.015). A representative image of a enkephalin, c-fos double label FISH is also shown in figure 6B.

Ghasem 17

Fig. 6. Activation of the direct striatal circuit represented by the proportion of dynorphin cells co-expressing c-fos. A) Rostral to caudal regions of the striatum that were sampled. B) Representative image of double label FISH with dynorphin (green) and c-fos (red) with a representative double label cell highlighted. Effect of stress and 6 weeks of voluntary exercise on direct pathway neuronal activation in the (C) DMS and (D) DLS. Both sedentary and running animals were exposed to 100 inescapable tail shocks (IS) or remained in their home cages as controls (HC). Rats were then immediately sacrificed and brains were removed and processed for in situ hybridization. Data represent the mean +SEM, Two-way ANOVA: * main effect of stress (p<0.0001), # main effect of exercise (p<0.05) ϕ stress x exercise interaction (p<0.05).

Ghasem 18

Fig. 7. Activation of the indirect striatal circuit represented by the proportion of enkephalin cells co-expressing c-fos. A) Rostral to caudal regions of the striatum that were sampled. B) Representative image of double label FISH with enkephalin (green) and c-fos (red) with a representative double label cell highlighted. Effect of stress and 6 weeks of voluntary exercise on indirect pathway neuronal activation in the (C) DMS and (D) DLS. Both sedentary and running animals were exposed to 100 inescapable tail shocks (IS) or remained in their home cages as controls (HC). Rats were then immediately sacrificed and brains were removed and processed for in situ hybridization. Data represent the mean +SEM, Two-way ANOVA: * main effect of stress (p<0.0001), ϕ stress x exercise interaction (p<0.05).

Ghasem 19

Discussion

These findings implicate physical activity as a potential contributor to striatal adenosine and DA receptor neuroplasticity. Moreover, they also suggest a role for exercise in altering striatal activity during an acute stressor. In the present studies, it was found that six weeks of voluntary wheel running significantly reduced the expression of A1R and A2aR mRNA in the striatum. Furthermore, while no effect of exercise was detected on striatal D1R mRNA, a modest but significant increase of striatal D2R mRNA expression in running animals was observed. Additionally, these exercise-induced changes in receptor gene expression correlated with hypothesized alterations in c-fos induction in striatal direct and indirect pathway MSNs. Running animals displayed a greater stress-induced c-fos induction in dynorphin expressing direct pathway MSNs and a reduced c-fos induction in enkephalin expressing indirect pathway MSNs. Taken together, running induced alterations to adenosine and DA receptor gene expression may contribute to changes in striatal signaling which could underlie the protective effects of exercise against goal-directed learning deficits brought on by IS.

The A1Rs and A2aRs are increasingly discussed as major modulators of striatal function such as movement, goal directed learning, and reward (Bastia et al., 2005; Randall et al., 2011; Shen et al., 2008; Worden et al., 2009; Yu et al., 2009). Indeed the administration of A1R and A2aR agonists can attenuate drug seeking, a reward-linked behavior, in rodents (Hobson et al., 2013; O'Neill et al., 2012). Therefore, reductions in striatal A1R and A2aR mRNA expression observed in running animals could contribute to potentiated drug seeking behaviors sometimes observed following exercise (Bachtell and Self, 2009). Moreover, adenosine has been implicated in the generation of behavioral despair (Crema et al., 2013) as well as the depression-like behavioral impairments observed following uncontrollable tail shock (Hanff et al., 2010; Minor et al., 1994c; Minor et al., 1994d). Indeed, A2aRs are currently being explored as a novel antidepressant (Hodgson et al., 2009; Yacoubi et al., 2001). As such, the observed reductions in striatal A1Rs and A2aR mRNA expression in running animals could underlie the anxiolytic effects associated with exercise. While studies suggest that alterations in A1R and A2aR mRNA are associated to similar changes in protein or radioligand binding (Albasanz et al., 2006; Calon et al., 2004; Cheng et al., 2000; Khoa et al., 2001; León et al., 2009; Murphree et al., 2005; Varani et al., 2010), future studies should examine potential reductions in adenosine receptor functionality and/or expression that could correlate to the reported running induced changes to adenosine receptor mRNA. If exercise-related decreases in gene expression translate to reductions in functional adenosine receptor protein, then these data could have implications for the treatment of disorders that involve striatal dysfunction.

The down regulation of striatal A1R and A2aR mRNA observed in the running animals of this study may be explained by increases in striatal adenosine concentration during exercise bouts. Indeed, exercise can increase brain-wide concentrations of adenosine in a dose-dependant manner in rodents (Dworak et al., 2007) and chronic administration of an A1R receptor agonist in vivo reduces both A1R mRNA and protein expression in the brain (León et al., 2009). During periods of increased metabolic Ghasem 20 demand, such as exercise, ATP is hydrolyzed to its precursors (i.e. ADP!AMP!Adenosine) and adenosine is transported to the extracellular space where it binds its receptors such as A1R and A2aR (Dunwiddie and Masino, 2001; Fredholm et al., 2005; Schiffmann et al., 2007). However, physical activity can increase ATP consumption both in the periphery and in the brain (Costa et al., 2001; Dworak et al., 2007; Simpson and Phillis, 1992). Therefore the source of the striatal adenosine from exercise, hypothesized to reduce A1R and A2aR mRNA expression, is not well understood. Interestingly, wheel running can increase neural activity and metabolism in the striatum (Liste et al., 1997; McCloskey et al., 2001), which may lead to increases of extracellular adenosine. However it should be noted that adenosine can cross the blood brain barrier (Pardridge et al., 1994). Therefore, adenosine released from peripheral tissues as a result of exercise may also play a role in central tissues. Taken together, the elevated adenosine levels thought to play a role in the reduction of A1R and A2aR mRNA may originate from neural and/or peripheral sources.

Voluntary wheel running modestly but significantly increased D2R mRNA but had no effect on D1R mRNA expression in the striatum, consistent with previous studies using in situ hybridization (Foley and Fleshner, 2008). Given the opposing roles of D2R and A2aR on striatal indirect pathway function, this upregulation of D2R mRNA, if consistent with changes in protein expression, could play a role in allowing exercised animals the ability to escape from shock in the shuttle box escape task. Moreover, this alteration could interact synergistically with increases in striatal DA neurotransmitter observed in exercised animals during an analog shuttle box escape task (unpublished data in prep) to contribute to the protective effects of physical activity against stress induced goal-directed learning deficits. The upregulation of D2R may be explained by the persistent exercise induced agonism of A2aRs, as one study has shown that male mice exposed to non-selective adenosine antagonist caffeine displayed decreased D2R transcript in the striatum (Gilliam et al., 1984). However, the effect of continuous A2aR activation on D2R mRNA and protein requires further study. Moreover, the translational capacity of these exercise-induced transcript changes as well as their longevity remain to be tested.

Activation of direct and indirect pathway MSNs involves a vast array of neurotransmitters and neuromodulations. Here we report changes in A1R, A2aR, and D2R mRNA and there are several reasons to believe that these changes contribute to the potentiated stress-induced c-fos induction of direct pathway and attenuated stress-induced c-fos induction of indirect pathway neurons. First, the uncontrollable stressor used in these studies has been suggested to increase striatal concentrations of both adenosine and DA (Abercrombie et al., 1989; Hanff et al., 2010; Minor and Hunter, 2002b). As such, exercise induced changes in the gene expression of these receptors, if translated to similar alterations in receptor proteins, could lead to a differential activation of the direct and indirect pathway MSNs between sedentary and running animals during uncontrollable tail shock. Second, A2aRs and D2Rs are expressed nearly exclusively on indirect pathway MSNs (Ferré et al., 1997; Fuxe et al., 2007; Schiffmann et al., 2007). Binding of adenosine to A2aRs increases activity of indirect pathway MSNs while the binding of DA to D2Rs suppresses activity of indirect pathway MSNs (Fuxe et al., 2007; Lanciego et al., Ghasem 21

2012). Therefore, the observed decrease and increase in A2aR and D2R gene expression, if consistent with changes in functional protein, could underlie the decreased activation of indirect pathway MSNs observed in running animals during uncontrollable stress. Third, A1Rs are expressed on direct pathway MSNs (Ferré et al., 1997; Ferré et al., 2007). Moreover, binding of adenosine to A1Rs decreases direct pathway MSN activity (Ferré et al., 1997; Fuxe et al., 2007). As a result, a exercise-induced reduction in functional A1R density due to a reduction in mRNA could contribute to the potentiated activity seen in direct pathway MSNs. Taken together, the observed changes in A1R, A2aR, and D2R mRNA due to exercise are consistent with changes in functional receptor signaling and could underlie the potential alterations in MSN activation reported.

This interpretation, however, requires further analysis. It has been reported that A1Rs are expressed on both direct and indirect pathway neurons (Dixon et al., 1996). As such, identifying the source of the reduced A1R transcript with in situ hybridization alone is difficult. Moreover, given the role of A1Rs as an inhibitor of cellular activity (Dunwiddie and Masino, 2001; Ferre et al., 1998), a reduction in functional A1R due to a reduction in mRNA expression could lead to a greater activation of indirect pathway MSNs. One possible explanation for the lack of this result is the unique function of A1R/A2aR heterodimers, given the expression of A2aRs on indirect pathway MSNs. It has been suggested that in instances of high extracellular adenosine concentrations these complexes favor the activation of A2aRs over A1Rs (Ferré et al., 2007). Substantial increases of brain-wide adenosine brought on by uncontrollable stress, therefore, could be preferentially acting through A2aRs rather than A1Rs of the indirect circuit. Furthermore, if exercised rats struggled to a greater degree in restraint tubes during IS than sedentary rats, this could be an alternative source for the observed changes in patterns of c-fos induction in direct and indirect pathways, given their roles in creating and inhibiting movement respectively. However, our lab has observed no difference in movement between sedentary and running rats during uncontrollable stress using biotelemetric recording (unpublished data in prep). Therefore, this alternate explanation seems unlikely. Although making strong conclusions regarding striatal activity based upon the reported changes in A1R, A2aR, and D2R mRNA, these data are consistent with the hypothesis that exercise potentiates activation of the direct pathway and suppresses activation of the indirect pathway during stress and should be the focus of future studies.

Such modified striatal signaling could have importance for stress-related anxiety disorders and behavioral impairments in goal-directed learning. Recent evidence has suggested that beyond their roles in movement, the direct and indirect pathways of the striatum may be involved in signaling rewarding and aversive experiences respectively (Hikida et al., 2010; Kravitz et al., 2012). Therefore, the administration of uncontrollable stress could potentially lead to a hyper-activation of indirect pathway MSNs. Moreover, this aberrant signaling could underlie the behavioral deficits in goal-directed learning observed in animals exposed to this acute stressor. Taken together with the observed potentiation of direct and suppression of indirect pathway MSN activity during stress in running, compared to sedentary animals, these data suggest that sedentary animals find uncontrollable stress more aversive than their running counterparts. Indeed, recent findings have found that preferential induction of chronic neural activity marker ΔFosB Ghasem 22 in direct and indirect pathway neurons indicates a resistance and susceptibility to stress respectively (Lobo et al., 2013). Consistent with these data, our lab has demonstrated that 6 weeks of wheel running prior to the administration of uncontrollable stress is sufficient to attenuate the anxiety and depression-like deficits in behavior (Greenwood et al., 2003; Greenwood et al., 2012). Therefore, our findings suggest that prior exercise may induce a shift from neural activity associated with aversion to signaling associated with reward during exposure to stressful stimuli. Such changes in striatal activity could underlie the protective effects of exercise against behavioral deficits brought on by uncontrollable stress.

One potential mechanism by which this shift in signaling could ameliorate behavioral impairments is through the attenuation of the sensitized serotonin (5-HT) response. Uncontrollable stress elicits a potentiated 5-HT release in the dorsal striatum via the dorsal raphe nucleus (DRN) (Amat et al., 2005; Strong et al., 2011), which contributes to the generation of the goal-directed learning deficits observed in LH (Greenwood and Fleshner, 2008; Maier and Watkins, 2005; Strong et al., 2011). Moreover, our lab has also demonstrated that 6 weeks of voluntary wheel running reduces c-fos expression in 5- HT DRN neurons during Striatum) uncontrollable stress (Greenwood et al., 2003) indicating a potential reduction Direct)Pathway) Indirect)Pathway) in activity due to exercise. Interestingly, previous studies LHb) GPi) STN) GPe) have also implicated the brain region, the Lateral Habenula (LHb), as a contributor to the 5GHT2cR) stress-induced activity of the A1R) DRN. Indeed, specific lesions Thalamus) A2aR) of the LHb attenuate behavioral D1R) D R) deficits of LH brought on by 2 DRN) Motor)Cortex) Dynorphin)L) uncontrollable stress (Amat et Enkephalin)L) al., 2001). Furthermore, it has been demonstrated that the Fig. 8 Habenular and Traditional Striatal Circuitry LHb is activated as a result of Differential output circuitry of striatal direct and indirect aversive stimuli and quieted pathway MSNs. Abbreviations: GPi, internal segment of the during periods of reward (Hong globus pallidus; GPe, external segment of the globus and Hikosaka, 2008; pallidus; STN, subthalamic nucleus; LHb, lateral habenula; Matsumoto and Hikosaka, DRN, dorsal raphe nucleus; R, receptor; L, ligand. 2007; Shabel et al., 2012) and projects to the DRN via glutaminergic efferents (Herkenham and Nauta, 1979). Therefore, the LHb is a unique position to modulate LH-linked DRN activity based upon the reward state being experienced by an organism. Recent evidence suggests that the LHb receives such reward/aversion-state information via unique excitatory efferents of the internal globus pallidus (GPi) (Hong and Hikosaka, 2008; Hong and Hikosaka, 2013; Shabel et al., Ghasem 23

2012), a key striatal output station (Lanciego et al., 2012). Briefly, the direct and indirect pathway MSNs exert inhibitory and excitatory influences on the GPi respectively (Kravitz and Kreitzer, 2012; Lanciego et al., 2012). Taken together with the potential role of the striatum in reward signaling, it is reasonable to hypothesize that differential striatal pathway activation could contribute to the communication of reward/aversion-state information to the GPi, the LHb, and eventually, the DRN (see Figure 8 for summary). Therefore, the observed exercise induced potentiation of direct and suppression of indirect pathway MSN activity during uncontrollable may lead to a suppression of GPi and LHb activity. Such changes in signaling could then result in an attenuated 5-HT release by the DRN and potentially contribute to the protective effects of exercise against depression-like goal-directed learning deficits brought on by uncontrollable stress. However, the effect of specific direct or indirect pathway MSN stimulation on GPi, LHb, and DRN remain to be characterized and should be topics for further investigation.

Ghasem 24

Acknowledgements

The author would like to acknowledge Dr. Monika Fleshner for serving as the primary advisor for this thesis project and for being my mentor throughout my tenure in the Stress Physiology Lab. Special thanks are also extended to Dr. Ben Greenwood for his insights in completing these studies and interpreting these results. Additionally, the provision of the RNA A1R, A2aR, D1R, and D2R probes by Dr. Heidi Day, Dr. Serge Campeau, and Agnieszka Mika is greatly appreciated. Furthermore, I would also like to thank Dr. Peter Clark for his invaluable mentorship in completing this work. Moreover, I would also like to thank Dr. Clark for the use of this subset of data, which is included as part of a publication submitted for review in Behavioral Brain Research (see Clark et al. 2014). Finally, the author would like to thank Dr. Monika Fleshner, Dr. David Sherwood, and Dr. Ryan Bachtell for serving on the defense committee. Ghasem 25

References

1. Abercrombie ED, Keefe KA, DiFrischia DS, Zigmond MJ (1989) Differential effect of stress on in vivo dopamine release in striatum, nucleus accumbens, and medial frontal cortex. J Neurochem 52:1655-1658.

2. Albasanz JL, Rodríguez A, Ferrer I, Martin M (2006) Adenosine A2A receptors are up-regulated in pick’s disease frontal cortex. Brain Pathology 16:249-255.

3. Amat J, Baratta M, Paul E, Bland S, Watkins L, Maier S (2005) Medial prefrontal cortex determines how stressor controllability affects behavior and dorsal raphe nucleus. Nat Neurosci 8:365-371.

4. Amat J, Sparks P, Matus-Amat P, Griggs J, Watkins L, Maier S (2001) The role of the habenular complex in the elevation of dorsal raphe nucleus serotonin and the changes in the behavioral responses produced by uncontrollable stress. Brain Res 917:118-126.

5. Bachtell RK, Self DW (2009) Effects of adenosine A2A receptor stimulation on cocaine- seeking behavior in rats. Psychopharmacology (Berl ) 206:469-478.

6. Bastia E, Xu Y, Scibelli AC, Day Y, Linden J, Chen J, Schwarzschild MA (2005) A crucial role for forebrain adenosine A2A receptors in amphetamine sensitization. Neuropsychopharmacology 30:891-900.

7. Bhatia KP, Marsden CD (1994) The behavioural and motor consequences of focal lesions of the basal ganglia in man. Brain (ENGLAND) 117 ( Pt 4):859-876.

8. Blumenthal JA, Babyak MA, Doraiswamy PM, Watkins L, Hoffman BM, Barbour KA, Herman S, Craighead WE, Brosse AL, Waugh R (2007) Exercise and pharmacotherapy in the treatment of major depressive disorder. Psychosom Med 69:587-596.

9. Brown RG, Pluck G (2000) Negative symptoms: The ‘pathology’of motivation and goal- directed behaviour. Trends Neurosci 23:412-417.

10. Calon F, Dridi M, Hornykiewicz O, Bedard PJ, Rajput AH, Di Paolo T (2004) Increased adenosine A2A receptors in the brain of parkinson's disease patients with dyskinesias. Brain (England) 127:1075-1084.

11. Cheng J, Liu I, Juang S, Jou S (2000) Decrease of adenosine A-1 receptor gene expression in cerebral cortex of aged rats. Neurosci Lett 283:227-229.

12. Clark PJ, Ghasem PR, Mika A, Day HE, Herrera JJ, Greenwood BN, Fleshner M (2014) Wheel running alters patterns of uncontrollable stress-induced cfos mRNA expression in rat dorsal striatum direct and indirect pathways: a possible role for neuroplasticity in adenosine and dopamine receptors. Behav Brain Res (in review).

Ghasem 26

13. Costa F, Diedrich A, Johnson B, Sulur P, Farley G, Biaggioni I (2001) Adenosine, a metabolic trigger of the exercise pressor reflex in humans. Hypertension (United States) 37:917-922.

14. Crema LM, Pettenuzzo LF, Schlabitz M, Diehl L, Hoppe J, Mestriner R, Laureano D, Salbego C, Dalmaz C, Vendite D (2013) The effect of unpredictable chronic mild stress on depressive-like behavior and on hippocampal A 1 and striatal A 2A adenosine receptors. Physiol Behav 109:1-7.

15. Dixon AK, Gubitz AK, Sirinathsinghji DJ, Richardson PJ, Freeman TC (1996) Tissue distribution of adenosine receptor mRNAs in the rat. Br J Pharmacol 118:1461-1468.

16. Dunwiddie TV, Masino SA (2001) The role and regulation of adenosine in the central nervous system. Annu Rev Neurosci 24:31-55.

17. Durieux PF, Bearzatto B, Guiducci S, Buch T, Waisman A, Zoli M, Schiffmann SN, de Kerchove d'Exaerde A (2009) D 2 R striatopallidal neurons inhibit both locomotor and drug reward processes. Nat Neurosci 12:.

18. Dworak M, Diel P, Voss S, Hollmann W, Strüder H (2007) Intense exercise increases adenosine concentrations in rat brain: Implications for a homeostatic sleep drive. Neuroscience 150:789-795.

19. Ferré S, Fuxe K, B Fredholm B, Morelli M, Popoli P (1997) Adenosine–dopamine receptor–receptor interactions as an integrative mechanism in the basal ganglia. Trends Neurosci 20:482-487.

20. Ferré S, Ciruela F, Quiroz C, Luján R, Popoli P, Cunha RA, Agnati LF, Fuxe K, Woods AS, Lluis C (2007) Adenosine receptor heteromers and their integrative role in striatal function. The Scientific World Journal 7:74-85.

21. Ferre S, Torvinen M, Antoniou K, Irenius E, Civelli O, Arenas E, Fredholm BB, Fuxe K (1998) Adenosine A1 receptor-mediated modulation of dopamine D1 receptors in stably cotransfected fibroblast cells. J Biol Chem (UNITED STATES) 273:4718-4724.

22. Foley TE, Fleshner M (2008) Neuroplasticity of dopamine circuits after exercise: Implications for central fatigue. Neuromolecular Medicine 10:67-80.

23. Fournier JC, DeRubeis RJ, Hollon SD, Dimidjian S, Amsterdam JD, Shelton RC, Fawcett J (2010) Antidepressant drug effects and depression severity: A patient-level meta- analysis. JAMA 303:47-53.

24. Fredholm BB, Chen J, Cunha RA, Svenningsson P, Vaugeois J (2005) Adenosine and brain function. Int Rev Neurobiol 63:191-270.

25. Fuxe K, Ferré S, Genedani S, Franco R, Agnati LF (2007) Adenosine receptor–dopamine receptor interactions in the basal ganglia and their relevance for brain function. Physiol Behav 92:210-217.

Ghasem 27

26. Gerfen CR, Engber TM, Mahan LC, Susel Z, Chase TN, Monsma FJ,Jr, Sibley DR (1990) D1 and D2 dopamine receptor-regulated gene expression of striatonigral and striatopallidal neurons. Science (UNITED STATES) 250:1429-1432.

27. Gilliam P, Spirduso W, Martin T, Walters T, Wilcox R, Farrar R (1984) The effects of exercise training on [ 3 H]-spiperone binding in rat striatum. Pharmacology Biochemistry and Behavior 20:863-867.

28. Greenwood BN, Fleshner M (2008) Exercise, learned helplessness, and the stress- resistant brain. Neuromolecular Medicine 10:81-98.

29. Greenwood BN, Strong PV, Loughridge AB, Day HE, Clark PJ, Mika A, Hellwinkel JE, Spence KG, Fleshner M (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:e46118.

30. Greenwood B, Foley T, Burhans D, Maier S, Fleshner M (2005) The consequences of uncontrollable stress are sensitive to duration of prior wheel running. Brain Res (NETHERLANDS) 1033:164-178.

31. Greenwood B, Foley T, Day H, Campisi J, Hammack S, Campeau S, Maier S, Fleshner M (2003) Freewheel running prevents learned helplessness/behavioral depression: Role of dorsal raphe serotonergic neurons. J Neurosci (USA) 23:2889-2898.

32. Hanff TC, Furst SJ, Minor TR (2010) Biochemical and anatomical substrates of depression and sickness behavior. Isr J Psychiatry Relat Sci (ISRAEL) 47:64-71.

33. Herkenham M, Nauta WJ (1979) Efferent connections of the habenular nuclei in the rat. J Comp Neurol 187:19-47.

34. Hikida T, Kimura K, Wada N, Funabiki K, Nakanishi S (2010) Distinct roles of synaptic transmission in direct and indirect striatal pathways to reward and aversive behavior. Neuron 66:896-907.

35. Hobson BD, O'Neill CE, Levis SC, Monteggia LM, Neve RL, Self DW, Bachtell RK (2013) Adenosine A1 and dopamine D1 receptor regulation of AMPA receptor phosphorylation and cocaine-seeking behavior. Neuropsychopharmacology 38:1974- 1983.

36. Hodgson RA, Bertorelli R, Varty GB, Lachowicz JE, Forlani A, Fredduzzi S, Cohen- Williams ME, Higgins GA, Impagnatiello F, Nicolussi E, Parra LE, Foster C, Zhai Y, Neustadt BR, Stamford AW, Parker EM, Reggiani A, Hunter JC (2009) Characterization of the potent and highly selective A2A receptor antagonists preladenant and SCH 412348 [7-[2-[4-2,4-difluorophenyl]-1-piperazinyl]ethyl]-2-(2-furanyl)-7H-pyrazolo[4,3-e ][1,2,4]triazolo[1,5-c]pyrimidin-5-amine] in rodent models of movement disorders and depression. J Pharmacol Exp Ther (United States) 330:294-303.

37. Hoehn K, White TD (1990) Role of excitatory amino acid receptors in K -and Glutamate-Evoked release of endogenous adenosine from rat cortical slices. J Neurochem 54:256-265. Ghasem 28

38. Hong S, Hikosaka O (2013) Diverse sources of reward value signals in the basal ganglia nuclei transmitted to the lateral habenula in the monkey. Frontiers in Human Neuroscience 7:.

39. Hong S, Hikosaka O (2008) The globus pallidus sends reward-related signals to the lateral habenula. Neuron 60:720-729.

40. Hunter AM, Balleine BW, Minor TR (2003) Helplessness and escape performance: Glutamate-adenosine interactions in the frontal cortex. Behav Neurosci 117:123.

41. Khoa ND, Montesinos MC, Reiss AB, Delano D, Awadallah N, Cronstein BN (2001) Inflammatory cytokines regulate function and expression of adenosine A(2A) receptors in human monocytic THP-1 cells. J Immunol (United States) 167:4026-4032.

42. Kobayashi S, Millhorn DE (1999) Stimulation of expression for the adenosine A2A receptor gene by hypoxia in PC12 cells. A potential role in cell protection. J Biol Chem (UNITED STATES) 274:20358-20365.

43. Kravitz AV, Kreitzer AC (2012) Striatal mechanisms underlying movement, reinforcement, and punishment. Physiology 27:167-177.

44. Kravitz AV, Freeze BS, Parker PR, Kay K, Thwin MT, Deisseroth K, Kreitzer AC (2010) Regulation of parkinsonian motor behaviours by optogenetic control of basal ganglia circuitry. Nature 466:622-626.

45. Kravitz AV, Tye LD, Kreitzer AC (2012) Distinct roles for direct and indirect pathway striatal neurons in reinforcement. Nat Neurosci (USA) 15:816-U23.

46. Lanciego JL, Luquin N, Obeso JA (2012) Functional neuroanatomy of the basal ganglia. Cold Spring Harbor Perspectives in Medicine 2:.

47. León D, Castillo C, Albasanz J, Martín M (2009) Reduced expression and desensitization of adenosine A 1 receptor/adenylyl cyclase pathway after chronic (−) N 6- phenylisopropyladenosine intake during pregnancy. Neuroscience 163:524-532.

48. León D, Albasanz JL, Fernández M, Ruíz MA, Martín M (2004) Down-regulation of rat brain adenosine A1 receptors at the end of pregnancy. J Neurochem 88:993-1002.

49. Lex B, Hauber W (2010) The role of dopamine in the prelimbic cortex and the dorsomedial striatum in instrumental conditioning. Cereb Cortex (United States) 20:873- 883.

50. Liste I, Guerra M, Caruncho H, Labandeira-Garcia J (1997) Treadmill running induces striatal fos expression via NMDA glutamate and dopamine receptors. Experimental Brain Research 115:458-469.

51. Lobo MK et al (2013) DeltaFosB induction in striatal medium spiny neuron subtypes in response to chronic pharmacological, emotional, and optogenetic stimuli. J Neurosci (United States) 33:18381-18395.

Ghasem 29

52. Lu X, Behnam Ghasemzadeh M, Kalivas P (1997) Expression of D 1 receptor, D 2 receptor, substance P and enkephalin messenger RNAs in the neurons projecting from the nucleus accumbens. Neuroscience 82:767-780.

53. Maier SF (1984) Learned helplessness and animal models of depression. Prog Neuro- Psychopharmacol Biol Psychiatry 8:435-446.

54. Maier SF, Watkins LR (2005) Stressor controllability and learned helplessness: The roles of the dorsal raphe nucleus, serotonin, and corticotropin-releasing factor. Neuroscience & Biobehavioral Reviews 29:829-841.

55. Maier SF, Seligman ME (1976) Learned helplessness: Theory and evidence. J Exp Psychol : Gen 105:3.

56. Martin P, Puech AJ (1996) Antagonism by benzodiazepines of the effects of serotonin-, but not norepinephrine-, uptake blockers in the learned helplessness paradigm in rats. Biol Psychiatry 39:882-890.

57. Martinsen EW (2008) Physical activity in the prevention and treatment of anxiety and depression. Nordic Journal of Psychiatry 62:25-29.

58. Martinsen E, Morgan W (1997) Antidepressant effects of physical activity. Physical Activity and Mental Health 93-106.

59. Mathers CD, Loncar D (2006) Projections of global mortality and burden of disease from 2002 to 2030. PLoS Medicine 3:e442.

60. Matsumoto M, Hikosaka O (2007) Lateral habenula as a source of negative reward signals in dopamine neurons. Nature 447:1111-1115.

61. Maudhuit C, Prévot E, Dangoumau L, Martin P, Hamon M, Adrien J (1997) Antidepressant treatment in helpless rats: Effect on the electrophysiological activity of raphe dorsalis serotonergic neurons. Psychopharmacology (Berl ) 130:269-275.

62. McCloskey DP, Adamo DS, Anderson BJ (2001) Exercise increases metabolic capacity in the motor cortex and striatum, but not in the hippocampus. Brain Res 891:168-175.

63. Minor TR, Hunter AM (2002a) Stressor controllability and learned helplessness research in the united states: Sensitization and fatigue processes. Integrative Physiological & Behavioral Science 37:44-58.

64. Minor TR, Hunter AM (2002b) Stressor controllability and learned helplessness research in the united states: Sensitization and fatigue processes. Integrative Physiological & Behavioral Science 37:44-58.

65. Minor TR, Chang W, Winslow JL (1994a) Stress and adenosine: I. effect of methylxanthine and amphetamine stimulants on learned helplessness in rats. Behav Neurosci 108:254.

Ghasem 30

66. Minor TR, Winslow JL, Chang W (1994b) Stress and adenosine: II. adenosine analogs mimic the effect of inescapable shock on shuttle-escape performance in rats. Behav Neurosci 108:265.

67. Minor TR, Chang W, Winslow JL (1994c) Stress and adenosine: I. effect of methylxanthine and amphetamine stimulants on learned helplessness in rats. Behav Neurosci 108:254.

68. Minor TR, Winslow JL, Chang W (1994d) Stress and adenosine: II. adenosine analogs mimic the effect of inescapable shock on shuttle-escape performance in rats. Behav Neurosci 108:265.

69. Minor TR (2008) Enhancing brain adenosine signaling with the nucleoside transport blocker NBTI (S-(4-nitrobenzyl)-6-theoinosine) mimics the effects of inescapable shock on later shuttle-escape performance in rats. Behav Neurosci 122:1236-1247.

70. Murphree Lx, Sullivan Gx, Marshall Mx, Linden J (2005) Lipopolysaccharide rapidly modifies adenosine receptor transcripts in murine and human macrophages: Role of NF- kappaB in A2A adenosine receptor induction. Biochem J 391:575-580.

71. O'Neill CE, LeTendre ML, Bachtell RK (2012) Adenosine A2A receptors in the nucleus accumbens bi-directionally alter cocaine seeking in rats. Neuropsychopharmacology 37:1245-1256.

72. Overmier JB, Seligman ME (1967) Effects of inescapable shock upon subsequent escape and avoidance responding. J Comp Physiol Psychol 63:28.

73. Pardridge WM, Yoshikawa T, Kang Y, Miller LP (1994) Blood-brain barrier transport and brain metabolism of adenosine and adenosine analogs. J Pharmacol Exp Ther 268:14-18.

74. Randall PA, Nunes EJ, Janniere SL, Stopper CM, Farrar AM, Sager TN, Baqi Y, Hockemeyer J, Müller CE, Salamone JD (2011) Stimulant effects of adenosine antagonists on operant behavior: Differential actions of selective A2A and A1 antagonists. Psychopharmacology (Berl ) 216:173-186.

75. Rozeske RR, Greenwood BN, Fleshner M, Watkins LR, Maier SF (2011) Voluntary wheel running produces resistance to inescapable stress-induced potentiation of morphine conditioned place preference. Behav Brain Res 219:378-381.

76. Ruiz MÁ, León DA, Albasanz JL, Martín M (2011) Desensitization of adenosine A 1 receptors in rat immature cortical neurons. Eur J Pharmacol 670:365-371.

77. Salmon P (2001) Effects of physical exercise on anxiety, depression, and sensitivity to stress: A unifying theory. Clin Psychol Rev 21:33-61.

78. Samejima K, Ueda Y, Doya K, Kimura M (2005a) Representation of action-specific reward values in the striatum. Science (United States) 310:1337-1340.

Ghasem 31

79. Samejima K, Ueda Y, Doya K, Kimura M (2005b) Representation of action-specific reward values in the striatum. Science (United States) 310:1337-1340.

80. Sano H, Yasoshima Y, Matsushita N, Kaneko T, Kohno K, Pastan I, Kobayashi K (2003) Conditional ablation of striatal neuronal types containing dopamine D2 receptor disturbs coordination of basal ganglia function. J Neurosci (United States) 23:9078-9088.

81. Schiffmann SN, Fisone G, Moresco R, Cunha R, Ferré S (2007) Adenosine A 2A receptors and basal ganglia physiology. Prog Neurobiol 83:277-292.

82. Shabel SJ, Proulx CD, Trias A, Murphy RT, Malinow R (2012) Input to the lateral habenula from the basal ganglia is excitatory, aversive, and suppressed by serotonin. Neuron 74:475-481.

83. Shen HY, Coelho JE, Ohtsuka N, Canas PM, Day YJ, Huang QY, Rebola N, Yu L, Boison D, Cunha RA, Linden J, Tsien JZ, Chen JF (2008) A critical role of the adenosine A2A receptor in extrastriatal neurons in modulating psychomotor activity as revealed by opposite phenotypes of striatum and forebrain A2A receptor knock-outs. J Neurosci (United States) 28:2970-2975.

84. Simpson RE, Phillis JW (1992) Adenosine in exercise adaptation. Br J Sports Med (ENGLAND) 26:54-58.

85. Speaker KJ, Cox SS, Paton MM, Serebrakian A, Maslanik T, Greenwood BN, Fleshner M (2013) Six weeks of voluntary wheel running modulates inflammatory protein (MCP- 1, IL-6, and IL-10) and DAMP (Hsp72) responses to acute stress in white adipose tissue of lean rats. Brain Behav Immun .

86. Strong PV, Christianson JP, Loughridge AB, Amat J, Maier SF, Fleshner M, Greenwood BN (2011) 5-hydroxytryptamine 2C receptors in the dorsal striatum mediate stress- induced interference with negatively reinforced instrumental escape behavior. Neuroscience 197:132-144.

87. Svenningsson P, Le Moine C, Fisone G, Fredholm BB (1999) Distribution, biochemistry and function of striatal adenosine A 2A receptors. Prog Neurobiol 59:355-396.

88. Turner EH, Matthews AM, Linardatos E, Tell RA, Rosenthal R (2008) Selective publication of antidepressant trials and its influence on apparent efficacy. N Engl J Med 358:252-260.

89. Varani K, Vincenzi F, Tosi A, Gessi S, Casetta I, Granieri G, Fazio P, Leung E, MacLennan S, Granieri E, Borea PA (2010) A2A adenosine receptor overexpression and functionality, as well as TNF-alpha levels, correlate with motor symptoms in parkinson's disease. FASEB J (United States) 24:587-598.

90. Vendite D, Sanz JM, Lõpez-Alañon DM, Vacas J, Andrés A, Ros M (1998) Desensitization of adenosine A1 receptor-mediated inhibition of adenylyl cyclase in cerebellar granule cells. Neurochem Res 23:211-218.

Ghasem 32

91. Woodson JC, Minor TR, Soames Job R (1998) Inhibition of adenosine deaminase by erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) mimics the effect of inescapable shock on escape learning in rats. Behav Neurosci 112:399.

92. Worden LT, Shahriari M, Farrar AM, Sink KS, Hockemeyer J, Müller CE, Salamone JD (2009) The adenosine A2A antagonist MSX-3 reverses the effort-related effects of dopamine blockade: Differential interaction with D1 and D2 family antagonists. Psychopharmacology (Berl ) 203:489-499.

93. Yacoubi ME, Ledent C, Parmentier M, Bertorelli R, Ongini E, Costentin J, Vaugeois J (2001) Adenosine A2A receptor antagonists are potential antidepressants: Evidence based on pharmacology and A2A receptor knockout mice. Br J Pharmacol 134:68-77.

94. Yin HH, Knowlton BJ (2006) The role of the basal ganglia in habit formation. Nature Reviews Neuroscience 7:464-476.

95. Yin HH, Knowlton BJ, Balleine BW (2006) Inactivation of dorsolateral striatum enhances sensitivity to changes in the action–outcome contingency in instrumental conditioning. Behav Brain Res 166:189-196.

96. Yin HH, Ostlund SB, Knowlton BJ, Balleine BW (2005) The role of the dorsomedial striatum in instrumental conditioning. Eur J Neurosci 22:513-523.

97. Yu C, Gupta J, Chen JF, Yin HH (2009) Genetic deletion of A2A adenosine receptors in the striatum selectively impairs habit formation. J Neurosci (United States) 29:15100- 15103.