Therapeutic and Preventive Effect of Voluntary Running Wheel Exercise on Social Defeat Stress (SDS)-induced Depressive-like Behavior and Chronic Pain in Mice

Journal Pre-proofs Research Article Therapeutic and preventive effect of voluntary running wheel exercise on social defeat stress (SDS)-induced depressive-like behavior and chronic pain in mice M. Pagliusi Jr, I.J.M. Bonet, A.F. Brandão, S.F. Magalhães, C.H. Tambeli, C.A. Parada, C.R. Sartori PII: DOI: Reference:

S0306-4522(19)30892-9 https://doi.org/10.1016/j.neuroscience.2019.12.037 NSC 19447

To appear in:

Neuroscience

Received Date: Revised Date: Accepted Date:

5 September 2019 29 November 2019 23 December 2019

Please cite this article as: M. Pagliusi Jr, I.J.M. Bonet, A.F. Brandão, S.F. Magalhães, C.H. Tambeli, C.A. Parada, C.R. Sartori, Therapeutic and preventive effect of voluntary running wheel exercise on social defeat stress (SDS)induced depressive-like behavior and chronic pain in mice, Neuroscience (2019), doi: https://doi.org/10.1016/ j.neuroscience.2019.12.037

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

© 2019 IBRO. Published by Elsevier Ltd. All rights reserved.

Title:

Therapeutic and preventive effect of voluntary running wheel exercise on social defeat stress (SDS)-induced depressive-like behavior and chronic pain in mice.

Authors:

Pagliusi Jr, M; Bonet, IJM; Brandão, AF; Magalhães, SF; Tambeli, CH; Parada, CA; Sartori, CR. 1Department of Structural and Functional Biology, University of Campinas, SP, Brazil. 2Department of Oral and Maxillofacial Surgery, University of California San Francisco, CA, USA. *Corresponding author: Department of Structural and Functional Biology, University of Campinas, Rua Monteiro Lobato, 255, Cidade Universitaria Zeferino Vaz, Box 6109, Campinas, SP 13083-865, Brazil.

Abstract Major depressive disorders (MDD) and chronic pain (CP) affect significant portion of the world's population and have high comorbidity rate. Social defeat stress (SDS) model was standardized in mice and can trigger depressive-like behavior and chronic pain. Based especially on clinical trials showing an effective preventive and therapeutic effect of physical exercise on CP and symptoms associated with MDD, this study aimed to investigate if the voluntary running wheel exercise can exert these effects in mice submitted to the 10-day SDS protocol, using fluoxetine as positive control. For this, we ran two set of experiments: in the first set mice started performing voluntary running wheel exercise after submitted to SDS and, in the second set, mice performed voluntary running wheel exercise before and during SDS. Mechanical and chemical hyperalgesia was analyzed through electronic von Frey and capsaicin test, respectively. Depressive-like behavior was assessed through social interaction test. Our results showed that the voluntary running wheel exercise was more effective than fluoxetine reversing the SDS-induced persistent hyperalgesia and both, fluoxetine and voluntary running wheel exercise, was effective reversing SDS-induced social avoidance. Also, voluntary running wheel exercise is an effective tool preventing both hyperalgesia and social avoidance induced by SDS. To the best of our knowledge, this was the first study using physical exercise as a therapeutic and preventive tool for chronic pain and depressive-like behavior simultaneously induced by social stress.

Introduction Depressive disorders affect significant portion of the world's population. In the United States, for example, the twelve-month prevalence for major depressive disorders (MDD) is 10.4% (Hasin et al., 2018). Also, lifetime prevalence for depression is estimated in 20.6% in the United States (Hasin et al., 2018) and 9.4% globally, generally with higher rates in higher income than lower income countries (Kessler, 2012). Just as depression, chronic pain (CP) is also a great social issue and has high prevalence worldwide. In the USA it is estimated that one third of the population suffers from chronic pain (Gatchel et al., 2014). In addition, according to the World Health Organization (WHO), depressive disorders, followed by

musculoskeletal disorders (which include CP), are the leading cause of disability in the world. This highlights the economic and social impact of these two conditions which, besides generating unproductiveness, also generates a great burden on public health and, more importantly, intense suffering for the individual, their relatives and social circle. Given the high prevalence of these both pathological conditions, it is not a surprise epidemiological studies showing great relationship between MDD and CP (Robinson et al., 2009; Goldenberg, 2010; Hooten, 2016), although CP is not part of the standard MDD symptoms (American Psychiatric Association, 2013). This relationship is evidenced by studies describing great comorbidity between MDD and CP (Bair et al., 2003; Outcalt et al., 2015). In fact, several clinical and biological characteristics are shared between pain and depression, including similar impairments on neuroanatomic structures, neural circuits and neurotransmitter systems (Hooten, 2016). Moreover, the great effectiveness of antidepressants (e.g. fluoxetine) in CP treatment also suggest a common neurophysiological mechanism between CP and MDD (Jann & Slade, 2007; Robinson et al., 2009; Nekovarova et al., 2014). Similarly, hypothalamic-pituitary-adrenal (HPA) axis hyperactivity – induced by chronic stress – is a biological alteration shared between MDD and CP (Maletic & Raison, 2009; Goldenberg, 2010; Amini-Khoei et al., 2017, 2019). In chronic stress context, recently was standardized in mice the social defeat stress (SDS) model (Golden et al., 2011; Pagliusi Jr. & Sartori, 2019), which involves a “resident-intruder” paradigm and has been widely used to induce depressive-like behaviors that model symptomatology of human depression. In this model, submissive mice develop physiological and behavioral alterations such as altered levels of corticosterone, cardiac hypertrophy, circadian rhythm disturbances, anxiety, despair behavior, anhedonia and social avoidance (Berton et al., 2006; Krishnan et al., 2007; Golden et al., 2011; Pagliusi Jr. & Sartori, 2019). Interestingly, some mice submitted to this SDS model are resilient and do not develop depressivelike behavior of social avoidance, just like in humans, once not every individual exposed to some form of chronic stress develops psychopathologies (Kessler et al., 1995; Charney & Manji, 2004; Yehuda, 2004; Strain, 2018). In addition to these findings, our research group recently demonstrated that SDS model can induce hyperalgesia regardless depressive-like behavior of social avoidance (Pagliusi Jr et al., 2018). Also, the depressive-like behavior induced by SDS is reversed only by chronic, but not acute, treatments with antidepressant drugs, just like in humans (Berton et al., 2006; Pollak et al., 2010). Besides the abovementioned antidepressant treatments, another important therapeutic intervention for CP and MDD is physical exercise. In addition to the wellestablished effect of physical exercise on the metabolism, musculoskeletal and cardiorespiratory systems, its effects on brain has received great attention (Warburton et al., 2006; Viña et al., 2012). In the early 1980s, for example, it was shown that exercise can increase endorphin circulating levels in humans (Bortz et al., 1981; Carr et al., 1981). This exercise-induced endorphin increase is associated with physiological and psychological changes, including changes in mood (e.g. exerciseinduced euphoria), altered pain perception, and responses to stress hormones (Harber & Sutton, 1984; Viña et al., 2012). Because of this, physical exercise has been recommended as an important non-pharmacological therapeutic and preventive tool for several diseases, mainly chronic pain and depression (Knapen et al., 2009; Ambrose & Golightly, 2015).

Although most studies addressing the potentially anti-hyperalgesic and antidepressive effects of exercise have been performed in humans, some studies using animal models suggest the efficacy of the physical exercise treating and preventing these diseases (Bement & Sluka, 2005; Mazzardo-Martins et al., 2010; Stagg et al., 2011; Chen et al., 2012). Therefore, in the present study we used the SDS model to investigate in mice the relationships between depression and chronic pain as well as the modulatory capacity of voluntary running wheel exercise on these SDS-induced behavioral changes, investigating the therapeutic and preventive potential of physical exercise.

Methods Experiments were performed on adult (8 weeks old) male C57BL/6J mice, 20-25g body weight, provided by the University of Campinas (UNICAMP) Bioterism Center (CEMIB). For the social defeat stress protocol, we used male Swiss mice (retired breeders) as resident, provided by the National Agricultural and Livestock Laboratory of Campinas (Lanagro/SP) of the Brazilian Ministry of Agriculture, Livestock and Supply. During the whole experiment animals were kept under controlled conditions of light (cycle 12:12 hours of dark/light) and temperature (21ºC), receiving water and food ad libitum. The study was approved by the Ethics Committee in the Use of Animals of UNICAMP (CEUA/UNICAMP), protocol number 4249-1. The present study was divided into two different experimental designs. We first evaluated the therapeutic effect of physical exercise on persistent hyperalgesia and depressive-like behavior of social avoidance induced by chronic social defeat stress (SDS). For this, mice started performing voluntary running wheel exercise after submitted to the chronic SDS protocol (figure 1A). We next evaluated the preventive effect of physical exercise on hyperalgesia and depressive-like behavior of social avoidance induced by chronic SDS. For this, mice performed voluntary running wheel exercise before and during chronic SDS protocol (figure 1B). Social defeat stress (SDS) Social defeat stress model was used to induce depressive-like behavior of social avoidance in C57BL/6J mice. We used a modification of the protocol previously described by Golden et al.(2011) and standardized by our group (Pagliusi Jr. & Sartori, 2019). Briefly, Swiss mice were initially selected for their aggressive behavior and the most aggressive were subsequently used in the SDS protocol. After that, C57BL/6J mice were introduced daily (for 10 consecutive days) in the same cage of a resident Swiss mouse to a 10 minutes social defeat session. After this period of agonistic body contact, both aggressor Swiss mouse and stressed C57BL/6J mouse remained for 24 hours separated by a clear perforated acrylic divider, which allowed sensorial contact. On each subjugation day, stressed C57BL/6J mice were exposed to a different resident Swiss mouse. The stressed mice were designated to SDS group. For the control group (i.e. no stressed mice) mice were paired housed with another C57BL/6J separated by a clear perforated acrylic divider, allowing only sensorial contact. Control mice were handled throughout 10-day protocol period similarly to the defeated mice and were designated to no SDS group.

Social interaction test (SI) 24 hours after the last SDS session we performed social interaction test as previously described by Golden et al. (2011). For this test we used an open field arena (42cm x 42cm x 42cm) with two zones of interest: interaction zone (IZ) and corners zone (CZ), as shown in figure 2 (upper right corner). The social interaction test consisted of two 150s consecutive sessions, separated from one another by a 30s interval. In the first session, called “no target”, an empty perforated plastic enclosure was placed in the center of the interaction zone, and the C57BL/6J mouse was placed into the rear part of the open field opposite the interaction zone. In the second session of the social interaction test, called “target”, a Swiss mouse was placed into the perforated plastic enclosure. Each social interaction test was performed with an unknown aggressor swiss mouse which was not use during the 10 days of SDS or in other social interaction test. Both sessions were recorded by a camera system and analyzed in specialized software (X-Plo-Rat – developed by Dr. Silvio Morato, USP, Brazil) to obtain the time spent in the interaction and corners zone. The social interaction ratio in the interaction zone (SI-IZ) and in the corners zone (SI-CZ) were calculated dividing the time spent in the respective zone in the “target” session by the time spent at the same zone in the “no target” session. Mice with SI-IZ bellow 1 were considered susceptible and mice with SI-IZ above 1 were considered resilient, as previously described by Golden et al., (2011). Mechanical nociceptive threshold test Mechanical nociceptive threshold was evaluated through the electronic von Frey test. In this test a force is applied against the central edge of the mice hind paw and the correspondent force until the paw withdrawal, as response to the stimulus, is recorded. For habituation mice were placed in the von Frey apparatus, which consists of acrylic cages (12cm x 20cm x 17cm) with 5mm2 meshfloor, 30 min before the beginning of the nociceptive test. The results are expressed by the variation of the nociceptive threshold in grams (gram-force) obtained by subtracting the average of three values observed before the social defeat stress (baseline) from those of the average of three values obtained after the social defeat stress (Δ mechanical nociceptive threshold in grams). The investigator was blind to all experimental treatments. This test was used to evaluate mechanical hyperalgesia. Chemical nociceptive test The capsaicin test was used to evaluate nociception in response to chemical stimuli. For this behavioral analysis, an observation box measuring 30x30x30cm with base and three mirrored sides and glass front was used. Each mouse was prior placed for 20 minutes in the apparatus to habituation. Following the habituation period, capsaicin was administered into the subcutaneous tissue of the left hind paw (0.1μg/15μl/paw). The nociceptive response was then characterized by the act of lifting the paw stereotypically (flinching) and was quantified during a 5-min period. The investigator was blind to all experimental treatments. This test was used to evaluate chemical hyperalgesia. Voluntary running wheel exercise

To investigate therapeutic effects of physical exercise, part of the stressed mice had access to the voluntary running wheel for 32 days, starting 48 hours after the end of SDS. Control (no SDS) mice remain sedentary single housed throughout experiment. To investigate preventive effects of physical exercise, the exercised group remained singled housed for 18 days with access to the voluntary running wheel followed by 10-days of SDS, remaining with access to the voluntary running wheel during the 24 hours of sensorial contact. That way, wheels swapped cages along with the runner mouse, so mice always had access to the same wheel throughout the experiment. After 10 days of SDS, the exercised mice were again single housed with access to the voluntary running wheel to assess motor behavior after chronic SDS. The distance traveled by exercised mice on running wheel was recorded 24h/day by an electronic counter connected to a computer for data storage and was quantified and expressed in kilometers. Fluoxetine treatment Mice treated with antidepressant fluoxetine (Prozac® Lilly Laboratory - Lot: C886997) received a daily dose of 10mg/kg delivered in the drinking water (Dulawa et al., 2004). For this, a week before the beginning of the treatment the daily water consumption of each mouse was assessed to calculate the average daily water intake. Then, using the body weight (assessed during the same week) was possible to calculate the dilution of fluoxetine required for each mouse, specifically, consume 10 mg/kg/day. Water with fluoxetine was changed every three days, and a stock solution was kept in the refrigerator for a maximum of one week. Locomotion test We performed an open field test (modified from Manosso et al., 2015) to evaluate mice locomotor activity, excluding the possibility of fluoxetine-induced impairment on motor behavior. In this test mice were placed in an open field arena (40cm x 75cm x 45cm, with floor divided into 15 equal quadrants 15cm x 15cm) for 6 minutes exploring freely. The sessions were recorded in video and analyzed using specialized software (X-Plo-Rat – developed by Dr. Silvio Morato, USP, Brazil). The number of quadrants crossed with the four paws (on the edge and center), the number of rearing (defined as the mouse standing on hind paws) and the sum of all these behaviors were parameters for the evaluation of the mice locomotor activity. Statistical analyses Statistical analysis was performed in GraphPad prism version 6.00 for Windows (GraphPad Software, San Diego, CA, USA). The results are presented as the mean ± standard error of the mean (SEM). One-way analysis of variance (ANOVA) followed by Bonferroni multiple comparison test was used to compare the groups in all data analyses except for the running distance analyses where two-way ANOVA followed by Bonferroni multiple comparison test or t test was used. Differences were considered statistically significant when p ≤ 0.05.

Results Social defeat stress induces depressive-like behavior of social avoidance

After 10 days of social defeat stress we performed the social interaction test and calculated the social interaction ratio in the interaction zone (SI-IZ) and in the corners (SI-CZ). Figure 2A shows the SI-IZ data of each group (no SDS: 3.542 ± 0.3203; resilient: 1.986 ± 0.1226; susceptible: 0.7283 ± 0.03714). One-way analyses of variance (ANOVA) showed significant differences between groups (F2,57 = 70.50; p< 0.0001). The post hoc Bonferroni’s multiple comparison test revealed statistical differences between no SDS and both stressed (resilient and susceptible) groups (p< 0.0001) and between resilient and susceptible groups (p< 0.0001). Figure 2B shows the SI-CZ data of each group (no SDS: 0.6916 ± 0.04408; resilient: 0.7969 ± 0.07724; susceptible: 2.210 ± 0.2156). One-way ANOVA showed significant differences between groups (F2,47 = 37.98; p< 0.0001). The post hoc Bonferroni’s multiple comparison test revealed statistical differences between susceptible and other groups (p< 0.0001). Social defeat stress induces mechanical hyperalgesia 24h after the social interaction test, we perform the von Frey electronic test to evaluate the mechanical nociceptive threshold. The results are shown in the figure 3 (no SDS: -0.1089 ± 0.1342g; resilient: 2.402 ± 0.1895g; susceptible: 2.614 ± 0.1475g). One-way ANOVA showed significant differences between groups (F2,57 = 70.58; p< 0.0001). The post hoc Bonferroni’s multiple comparison test revealed statistical differences between no SDS and both stressed (resilient and susceptible) groups (p< 0.0001). Voluntary running wheel exercise reversed depressive-like behavior of social avoidance and hyperalgesia induced by social defeat stress more efficiently than fluoxetine. Proceeding with the experimental design, stressed mice (resilient and susceptible) were divided into three distinct groups: treatment with exercise, treatment with fluoxetine and sedentary. Consequently, in addition to control group (no SDS), at this moment, the experimental design had six other groups: resilient sedentary (Res/Sed), resilient exercise (Res/Ex), resilient fluoxetine (Res/Flu), susceptible sedentary (Sus/Sed), susceptible exercise (Sus/Ex), and susceptible fluoxetine (Sus/Flu). After this separation, we performed two more social interaction behavior evaluations: 15 and 29 days after beginning the treatments (figure 4). We also performed four more mechanical nociceptive threshold evaluations: 7, 14, 21 and 28 days after beginning the treatments (figure 5) and a capsaicin test (figure 6). Figures 4A and 4B show the total time spent in the interaction zone (IZ) and corners zones (CZ), respectively, during “target” session 15 days after beginning the treatments (no SDSIZ: 53.80 ± 1.719s; Res/SedIZ: 41.00 ± 5.177s; Res/ExIZ: 46.20 ± 12.800s; Res/FluIZ: 68.42 ± 5.224s; Sus/SedIZ: 21.86 ± 5.026s; Sus/ExIZ: 29.38 ± 5.196s; Sus/FluIZ: 29.57 ± 7.597s; no SDSCZ: 24.13 ± 1.404s; Res/SedCZ: 58.00 ± 6.058s; Res/ExCZ: 55.40 ± 15.520s; Res/FluCZ: 29.67 ± 4.171s; Sus/SedCZ: 83.14 ± 9.755s; Sus/ExCZ: 62.63 ± 10.380s; Sus/FluCZ: 51.00 ± 5.713s). One-way ANOVA showed significant differences between groups (IZ: F6,52= 10.11; p< 0.0001 and CZ: F6,53= 10.36; p< 0.0001). The post hoc Bonferroni’s multiple comparison test revealed that 15 days of treatment with both exercise and fluoxetine did not alter social interaction levels. Figures 4C and 4D show the total time spent in the interaction

zone (IZ) and corners zones (CZ), respectively, during “target” session 29 days after beginning the treatments (no SDSIZ: 58.47 ± 1.895s; Res/SedIZ: 62.60 ± 3.326s; Res/ExIZ: 50.75 ± 9.003s; Res/FluIZ: 52.58 ± 4.277s; Sus/SedIZ: 24.00 ± 4.597s; Sus/ExIZ: 48.00 ± 8.597s; Sus/FluIZ: 47.38 ± 8.840s; no SDSCZ: 20.80± 1.235s; Res/SedCZ: 26.80 ± 1.562s; Res/ExCZ: 30.75 ± 4.385s; Res/FluCZ: 22.67 ± 2.094s; Sus/SedCZ: 73.71 ± 10.140s; Sus/ExCZ: 27.14 ± 3.348s; Sus/FluCZ: 25.50± 1.581s). One-way ANOVA showed significant differences between groups (IZ: F6,50= 4.055; p< 0.01 and CZ: F6,51= 21.67; p< 0.0001). The post hoc Bonferroni’s multiple comparison test revealed statistical differences between no SDS and Sus/Sed (p < 0.001). Figure 5A shows the variation of the mechanical nociceptive threshold 7 days after beginning the treatments (no SDS: -0.1833 ± 0.07623g; Res/Sed: 1.933 ± 0.2610g; Res/Ex: 0.1468 ± 0.09033g; Res/Flu: 1.375 ± 0.2898g; Sus/Sed: 2.009 ± 0.3633g; Sus/Ex: 1.288 ± 0.4703g; Sus/Flu: 1.438 ± 0.4650g). One-way ANOVA showed significant differences between groups (F6,51= 8.026; p< 0.0001). The post hoc Bonferroni’s multiple comparison test revealed statistical differences between no SDS and all groups except for the Res/Ex. Figure 5B shows the variation of the mechanical nociceptive threshold 14 days after beginning the treatments (no SDS: 0.2000 ± 0.1359g; Res/Sed: 1.627 ± 0.3906g; Res/Ex: 0.1002 ± 0.3342g; Res/Flu: 0.9332 ± 0.4059g; Sus/Sed: 2.150 ± 0.1003g; Sus/Ex: 0.1791 ± 0.3315g; Sus/Flu: 1.163 ± 0.5080g). One-way ANOVA showed significant differences between groups (F6,51= 6.222; p< 0.0001). The post hoc Bonferroni’s multiple comparison test revealed statistical differences between no SDS and all groups except for the Res/Ex and Sus/Ex. Figure 5C shows the variation of the mechanical nociceptive threshold 21 days after beginning the treatments (no SDS: -0.2801 ± 0.1100g; Res/Sed: 1.373 ± 0.3232g; Res/Ex: 0.1668 ± 0.2732g; Res/Flu: 0.8576 ± 0.3391g; Sus/Sed: 1.643 ± 0.3414g; Sus/Ex: -0.03313 ± 0.1896g; Sus/Flu: -0.06675 ± 0.3182g). One-way ANOVA showed significant differences between groups (F6,52= 7.826; p< 0.0001). The post hoc Bonferroni’s multiple comparison test revealed statistical differences between no SDS and all groups except for the Res/Ex, Sus/Ex and Sus/Flu. Figure 5D shows the variation of the mechanical nociceptive threshold 28 days after beginning the treatments (no SDS: -0.2944 ± 0.1334g; Res/Sed: 0.8666 ± 0.1078g; Res/Ex: -0.1208 ± 0.2251g; Res/Flu: 1.030 ± 0.4265g; Sus/Sed: 0.7237 ± 0.2125g; Sus/Ex: 0.07300 ± 0.1754g; Sus/Flu: 0.2916 ± 0.3242g). One-way ANOVA showed significant differences between groups (F6,51= 3.516; p< 0.01). As well as 21 days after the beginning of the treatments the post hoc Bonferroni’s multiple comparison test revealed statistical differences between no SDS and all groups except for the Res/Ex, Sus/Ex and Sus/Flu. In the capsaicin test (Figure 6) both running wheel exercise and fluoxetine treatment reduced flinching occurrence compared to no SDS and sedentary groups (no SDS: 53.67 ± 1.489; Res/Sed: 61.60 ± 0.8718; Res/Ex: 53.75 ± 1.750; Res/Flu: 36.50 ± 1.190; Sus/Sed: 65.43 ± 1.986; Sus/Ex: 46.25 ± 3.004; Sus/Flu: 34.50 ± 1.210. One-way ANOVA showed significant differences between groups (F6,52= 39.90; p< 0.0001). The post hoc Bonferroni’s multiple comparison test revealed statistical differences between no SDS and Res/Sed (p < 0.05), Res/Flu (p < 0.0001), Sus/Sed (p < 0.0001), Sus/Ex (p < 0.05) and Sus/Flu (p < 0.0001). It is important to mention that locomotion test performed on day 30 (one day before the capsaicin test) did not reveal any locomotor impairment induced by fluoxetine or SDS (data not shown).

Susceptible mice run less than resilient. Figure 7 shows the distance traveled by the stressed mice (susceptible and resilient). Figure 7A shows the curve of daily distance traveled in the running wheel. The mean of the susceptible group always remains below the mean of the resilient group. Analyzing the total distance traveled during the experiment (figure 7B) it can be observed that mice from susceptible group ran less than the resilient group (resilient: 137.8 ± 11.89km; susceptible: 105.0 ± 6.294km; t = 2.692; p < 0.05). The same can be observed when analyzing the average distance traveled per day (figure 7C) (resilient: 4.447 ± 0.3835km; susceptible: 3.386 ± 0.2030km; t = 2.692; p < 0.05). Voluntary running wheel exercise prevented depressive-like behavior of social avoidance and hyperalgesia induced by social defeat stress. Figure 8A shows the social interaction ratio in the interaction zone (SI-IZ) and we can observe that only the sedentary stressed (Sed/SDS) group has SI-IZ average lower than 1, which means that they spent less time in the interaction zone in the “target” session than in the “no target” session (Sed/no SDS: 2.093 ± 0.2276; Ex/no SDS: 1.715 ± 0.1712; Sed/SDS: 0.6565 ± 0.1317; Ex/SDS: 1.618 ± 0.4649). One-way ANOVA showed significant differences between groups (F3,34 = 5.365; p< 0.01). The post hoc Bonferroni’s multiple comparison test revealed statistical differences between Sed/NSDS and Sed/SDS (p < 0.01). Figure 8B shows the social interaction ratio in the corner zones (SI-CZ) (Sed/no SDS: 0.8431 ± 0.07575; Ex/no SDS: 1.491 ± 0.3782; Sed/SDS: 2.522 ± 0.4373; Ex/SDS: 1.704 ± 0.5024). One-way ANOVA showed significant differences between groups (F3,36 = 3.240; p< 0.05). The post hoc Bonferroni’s multiple comparison test revealed statistical differences just between Sed/NSDS and Sed/SDS (p < 0.05), corroborating the SI-IZ results. The preventive effect of the voluntary running wheel exercise on the social avoidance induced by SDS is also evidenced when we compare the total time spent in the interaction zone during “target” session of the sedentary and exercised stressed groups (figure 8C) (Sed/SDS: 13.12 ± 2.932s; Ex/SDS: 26.71 ± 5.199s), where exercised mice remained more time interacting socially (t = 2.207; p < 0.05). Total time spent in the corner zones during “target” session did not differ statistically between sedentary and exercised stressed groups (figure 8D) (Sed/SDS: 89.75 ± 7.890s; Ex/SDS: 73.51 ± 14.39s). Figure 9 shows the variation of the mechanical nociceptive threshold for each group (Sed/no SDS: 0.0549 ± 0.09874g; Ex/no SDS: 0.2750 ± 0.2660g; Sed/SDS: 2.803 ± 0.3227g; Ex/SDS: 0.4316 ± 0.2056g). One-way ANOVA showed significant differences between groups (F3,36= 29.07; p< 0.0001). The post hoc Bonferroni’s multiple comparison test revealed statistical differences between Sed/SDS and other groups (p< 0.0001). Chronic social defeat stress (SDS) decreased daily distance traveled. Figure 10 shows the curve of daily distance traveled in the running wheel for both no stress and chronic SDS groups. The chronic SDS started on the nineteenth day of access to the running wheel and we can observe that on the fourth day of social defeat stress the average distance traveled by the mice decreased dramatically, remaining statistically different from the no SDS group for 14 days

(multiple t tests with statistical significance determined using the Holm-Sidak method, α = 5.00%).

Discussion We replicated in the present study our previously results showing social defeat stress (SDS) inducing mechanical hyperalgesia regardless depressive-like behavior phenotype (figure 3) (Pagliusi Jr et al., 2018). We advance showing that this hyperalgesia is long-lasting for at least 28 days, which may mimic clinical conditions of chronic pain (figure 5). We also replicate the long-lasting depressivelike behavior of social avoidance induced by chronic SDS, which lasted for at least 28 days after ending SDS sessions (figure 4), as described by Berton et al. (2006). We also showed that voluntary running wheel exercise can reverse SDS-induced hyperalgesia in resilient mice (7 days of exercise) and in susceptible mice (14 days of exercise) (figure 5). Regarding depressive-like behavior of social avoidance, voluntary running wheel exercise, as well as fluoxetine, was efficient to reverse after 28 days of treatment (figure 4). Voluntary running wheel exercise was also efficient preventing social avoidance and hyperalgesia induced by SDS (figure 8 and 9). Once established that the hyperalgesia and depressive-like behavior of social avoidance induced by SDS are persistent (chronic), using physical exercise as therapeutic and preventive tool becomes even more relevant. Mainly because it is in this specific context of chronic pain and depression that exercise has been widely used in humans as non-pharmacological intervention (Hayden et al., 2005; Danielsson et al., 2013; Stanton & Reaburn, 2014; Ambrose & Golightly, 2015). Accordingly to such clinical reports, the present study demonstrated the efficacy of physical exercise reversing persistent social avoidance (figure 4) and hyperalgesia (figure 5 and 6) induced by SDS, demonstrating, therefore, robust antidepressant and anti-hyperalgesic effects. These therapeutic effects were similarly observed with fluoxetine treatment. It is important to mention that physical exercise, and not fluoxetine, reversed SDS-induced hyperalgesia in resilient mice (figure 5). It was unexpected that fluoxetine did not reverse SDS-induced chronic pain in resilient mice, once this is a drug used to treat chronic pain in humans even with no depressive symptoms (Patetsos & Horjales-Araujo, 2016). This result may be associated with the inefficiency of antidepressants inducing a robust positive effect in healthy subjects (Repantis et al., 2009), although we cannot regard resilient mice as completely healthy. These findings with the resilient group may suggest that, in clinical context, physical exercise should be a tool to be considered prior to therapies with antidepressants in patients with chronic pain and no depression symptoms, avoiding undesirable side effects commonly caused by antidepressant treatment. We must also highlight that the small number of animals in the resilient exercised group (n=5), although sufficient to observe statistical difference, is one of the limitations of the present study, not allowing us to extrapolate the discussion of the results obtained with this group. Interestingly, physical exercise had temporal delay reverting SDS-induced hyperalgesia between resilient and susceptible mice. In the figure 5 we can see that resilient mice returned to the baseline mechanical nociceptive threshold after 7 days of physical exercise and susceptible mice after 14 days. This temporal difference could be explained because exercised susceptible mice ran less than the resilient (figure 7). However, many studies showed that even a short period of physical

exercise is able to revert depressive-like behaviors and hyperalgesia (Kuphal et al., 2007; Naugle et al., 2012; Whitehead et al., 2017). In fact, our data demonstrated that, even running relatively less, susceptible mice were benefited by exercise. It is important to highlight some studies that corroborate the therapeutic effects of physical exercise observed in the present study. Hooten et al.(2012), for example, concluded in their clinical study that aerobic and strength exercises have equivalent effects reducing pain in patients with fibromyalgia, a chronic pain disorder that has close relationship with depression (Borchers & Gershwin, 2015). Whitehead et al. (2017) demonstrated that chronic voluntary exercise can revert to baseline sciatic nerve ligation-induced hyperalgesia in rats. It is also important to note that no study analyzed, simultaneously, the anti-hyperalgesic and antidepressive effect of physical exercise in mice, enhancing the relevance of the present study. Bobinski et al. (2015) indirectly approached pain, depression and physical exercise using forced low-intensity physical exercise, performed on treadmill. They found that two weeks of exercise (30 minutes per day, 5 days per week) was sufficient to reverse the neuropathic pain induced by sciatic nerve ligation (Bobinski et al., 2015). In addition, they also observed that this exercise-induced analgesia was a consequence of increased serotonergic neurotransmission in the brainstem (Bobinski et al., 2015). It is important to remember that the main antidepressant drugs used in the clinic are selective serotonin reuptake inhibitors (Willner et al., 2013). This mechanism observed by Bobinski et al. (2015) could also explain the molecular mechanisms involved in the antidepressant effect of physical exercise observed in the present study. Regarding depressive-like behavior, some studies corroborate the results observed in the present work. Otsuka et al. (2015), for example, using a shorter social defeat stress protocol (5 days), observed that two hours of voluntary physical exercise in the end of every stress sessions reduced social avoidance in mice. In another interesting study, Eldomiaty et al. (2017) described reduction in depressivelike behaviors (induced by repeated foot shock) in rats after three weeks of physical exercise in voluntary running wheel. They also observed increased serum and hippocampal BDNF levels in exercised rats compared to sedentary (Eldomiaty et al., 2017), showing physical exercise mimicking antidepressants effects (Björkholm & Monteggia, 2016). Lapmanee et al. (2013), comparing antidepressant effects of physical exercise and fluoxetine treatment, as performed in the present study, observed that four weeks of physical exercise in voluntary running wheel was able to reverse anxiety- and depressive-like behaviors induced by four weeks of physical restraint in rats. They also showed that the effects of these four weeks of physical exercise were similar to those of chronic antidepressant (fluoxetine) administration (Lapmanee et al., 2013). Similarly, Shafia et al. (2017) demonstrated that 4 weeks of moderate treadmill exercise, as well as 4 weeks of fluoxetine treatment, were able to reverse depressive and anxiety-like behaviors and decreased hippocampal BDNF levels induced by a post-traumatic stress model in rats. Concerning the preventive effects of physical exercise, in the present study we demonstrate that previous and during SDS access to the running wheel was able to prevent depressive-like behavior of social avoidance and hyperalgesia induced by chronic SDS (figures 8 and 9). In this context, Greenwood et al. (2013) demonstrated that both forced and voluntary exercises in the running wheel, but not forced exercise on the treadmill, prevented the establishment of anxiety and depressive-like behaviors induced by foot shock in rats. Another study from the same

group showed that 6 weeks of voluntary exercise in running wheel prevented the reduction in social exploitation, generalized fear and learning impairment induced by uncontrollable stress (Greenwood et al., 2012). In a study using a similar approach from the present study, Mul et al. (2018) observed that 21 days of physical exercise in voluntary running wheel was able to reduce mice propensity to develop social avoidance when submitted to chronic SDS. In addition, they also observed that 42 days of exercise in voluntary running wheel was able to reverse social avoidance induced by chronic SDS (Mul et al., 2018). These results corroborate those found in the present study, although they did not analyze physical exercise effects on resilient mice and on nociceptive behavior, in addition to not comparing physical exercise with antidepressant drug like performed in our study. Other studies had already verified the preventive effect of physical exercise on hyperalgesia, although using pain models rather than depression (chronic stress) models. Detloff et al. (2014), for example, demonstrated that spinal cord injuryinduced neuropathic pain was prevented by 9 weeks of physical exercise on motorized running wheel in rats. In addition, another recent study showed that 6 weeks of voluntary exercise prevented nerve ligation-induced hyperalgesia in rats (Grace et al., 2016). Sluka and colleagues also showed the preventive effect of physical exercise in the context of pain. In this study, free access to a voluntary running wheel for 8 weeks prevented mechanical hyperalgesia induced by intramuscular repeated acid injection in mice (Leung et al., 2016). When we look at the positive effects triggered by exercise, it is important to mention its systemic body alterations. In fact, physical exercise is a behavior that engages the entire body and its effects are systemic and multiple, inducing adaptations in various tissues beyond the nervous system, such as musculoskeletal, cardiovascular, respiratory and immune system (Moghetti et al., 2016; Che & Li, 2017; Pedersen, 2017), variables that were not analyzed in the present study. Thus, the anti-hyperalgesic and antidepressant effects of the exercise observed in the present study may be associated with the abovementioned systemic changes. In addition, it is already known that running wheel exercise generates long-lasting structural changes in cortical brain regions, such as increased gray matter in the motor, somatosensory, association and visual cortices (Sumiyoshi et al., 2014). We can conjecture that this neuroplastic changes may be associated with the antihyperalgesic and antidepressant effects of physical exercise observed in the present study. To the best of our knowledge, this is the first study using physical exercise as a therapeutic and preventive approach for pain and depressive-like behavior both induced by social defeat stress. Studying those chronic conditions in social stress context has great scientific relevance because, besides the great comorbidity between MDD and chronic pain, it is the most common type of stress in modern human society (Jasnow et al., 2005; Kumpulainen, 2008; Robinson et al., 2009; Goldenberg, 2010; Davidson & McEwen, 2012; Hooten, 2016). Based on our results we can conclude that chronic SDS triggers a chronic pain condition regardless generating social avoidance behavior. In addition, we can also conclude that voluntary physical exercise can be used as a preventive and therapeutic approach for chronic pain and depression induced by chronic social stress. In the preventive approach, the advantage of physical exercise is obvious, since it can be performed by healthy individuals which should not receive drug treatment. In the therapeutic context, physical exercise does not have the typical side effects of antidepressant

drugs. Moreover, when medications are really required, they may be prescribed at lower doses if the therapeutic approach includes exercises as an adjuvant.

Acknowledgements This study was supported by the São Paulo Research Foundation (FAPESP) and by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) Finance Code 001.

Conflict of interest The authors declare no conflict of interest.

Data Accessibility All data presented in the current manuscript can be obtained from the corresponding author.

Author Contributions M.P.Jr. designed the study, performed the experiments, analyzed the data, and drafted the manuscript. I.J.M.B. performed behavioral experiments and analyzed the data. A.F.B. performed behavioral experiments. S.F.M. analyzed the data. C.H.T. analyzed the data and drafted the manuscript. C.A.P. designed the study, analyzed the data, and drafted the manuscript. C.R.S. designed the study, analyzed the data, and drafted the manuscript.

References Ambrose, K.R. & Golightly, Y.M. (2015) Physical exercise as nonpharmacological treatment of chronic pain: Why and when. Best Pract. Res. Clin. Rheumatol., 29, 120–130. Amini-Khoei, H., Amiri, S., Mohammadi-Asl, A., Alijanpour, S., Poursaman, S., Haj-Mirzaian, A., Rastegar, M., Mesdaghinia, A., Banafshe, H.R., Sadeghi, E., Samiei, E., Mehr, S.E., & Dehpour, A.R. (2017) Experiencing neonatal maternal separation increased pain sensitivity in adult male mice: Involvement of oxytocinergic system. Neuropeptides, 61, 77–85. Amini-Khoei, H., Haghani-Samani, E., Beigi, M., Soltani, A., Mobini, G.R., Balali-Dehkordi, S., Haj-Mirzaian, A., Rafieian-Kopaei, M., Alizadeh, A., Hojjati, M.R., & Validi, M. (2019) On the role of corticosterone in behavioral disorders, microbiota composition alteration and neuroimmune response in adult male mice subjected to maternal separation stress. Int. Immunopharmacol., 66, 242–250. Association, A.P. (2013) DSM-5 Diagnostic Classification. In Diagnostic and Statistical Manual of Mental Disorders. American Psychiatric Association. Bair, M.J., Robinson, R.L., Katon, W., & Kroenke, K. (2003) Depression and Pain Comorbidity. Arch. Intern. Med., 163, 2433. Bement, M.K.H. & Sluka, K.A. (2005) Low-Intensity Exercise Reverses Chronic Muscle Pain in the Rat in a Naloxone-Dependent Manner. Arch. Phys.

Med. Rehabil., 86, 1736–1740. Berton, O., McClung, C.A., DiLeone, R.J., Krishnan, V., Renthal, W., Russo, S.J., Graham, D., Tsankova, N.M., Bolanos, C.A., Rios, M., Monteggia, L.M., Self, D.W., & Nestler, E.J. (2006) Essential role of BDNF in the mesolimbic dopamine pathway in social defeat stress. Science (80-. )., 311, 864–868. Björkholm, C. & Monteggia, L.M. (2016) BDNF – a key transducer of antidepressant effects. Neuropharmacology, 102, 72–79. Bobinski, F., Ferreira, T.A.A., Córdova, M.M., Dombrowski, P.A., Da Cunha, C., Santo, C.C.D.E., Poli, A., Pires, R.G.W., Martins-Silva, C., Sluka, K.A., & Santos, A.R.S. (2015) Role of brainstem serotonin in analgesia produced by low-intensity exercise on neuropathic pain after sciatic nerve injury in mice. Pain, 156, 2595–2606. Borchers, A.T. & Gershwin, M.E. (2015) Fibromyalgia: A Critical and Comprehensive Review. Clin. Rev. Allergy Immunol., 49, 100–151. Bortz, W.M., Angwin, P., Mefford, I.N., Boarder, M.R., Noyce, N., & Barchas, J.D. (1981) Catecholamines, dopamine, and endorphin levels during extreme exercise. N. Engl. J. Med., 305, 466–467. Carr, D.B., Bullen, B.A., Skrinar, G.S., Arnold, M.A., Rosenblatt, M., Beitins, I.Z., Martin, J.B., & McArthur, J.W. (1981) Physical conditioning facilitates the exercise-induced secretion of beta-endorphin and beta-lipotropin in women. N. Engl. J. Med., 305, 560–563. Charney, D.S. & Manji, H.K. (2004) Life Stress, Genes, and Depression: Multiple Pathways Lead to Increased Risk and New Opportunities for Intervention. Sci. Signal., 2004, re5–re5. Che, L. & Li, D. (2017) The Effects of Exercise on Cardiovascular Biomarkers: New Insights, Recent Data, and Applications. pp. 43–53. Chen, Y.-W., Li, Y.-T., Chen, Y.C., Li, Z.-Y., & Hung, C.-H. (2012) Exercise Training Attenuates Neuropathic Pain and Cytokine Expression After Chronic Constriction Injury of Rat Sciatic Nerve. Anesth. Analg., 114, 1330–1337. Danielsson, L., Noras, A.M., Waern, M., & Carlsson, J. (2013) Exercise in the treatment of major depression: A systematic review grading the quality of evidence. Physiother. Theory Pract., 29, 573–585. Davidson, R.J. & McEwen, B.S. (2012) Social influences on neuroplasticity: stress and interventions to promote well-being. Nat. Neurosci., 15, 689– 695. Detloff, M.R., Smith, E.J., Quiros Molina, D., Ganzer, P.D., & Houlé, J.D. (2014) Acute exercise prevents the development of neuropathic pain and the sprouting of non-peptidergic (GDNF- and artemin-responsive) c-fibers after spinal cord injury. Exp. Neurol., 255, 38–48. Dulawa, S.C., Holick, K.A., Gundersen, B., & Hen, R. (2004) Effects of chronic fluoxetine in animal models of anxiety and depression. Neuropsychopharmacology, 29, 1321–1330. Eldomiaty, M.A., Almasry, S.M., Desouky, M.K., & Algaidi, S.A. (2017) Voluntary running improves depressive behaviours and the structure of the hippocampus in rats: A possible impact of myokines. Brain Res., 1657, 29– 42. Gatchel, R., McGeary, D., McGeary, C., & Lippe, B. (2014) Interdisciplinary chronic pain management: international perspectives. Am. Psychol., 69,

119–130. Golden, S.A., Covington, H.E., Berton, O., & Russo, S.J. (2011) A standardized protocol for repeated social defeat stress in mice. Nat. Protoc., 6, 1183– 1191. Goldenberg, D.L. (2010) Pain/Depression Dyad: A Key to a Better Understanding and Treatment of Functional Somatic Syndromes. Am. J. Med., 123, 675–682. Grace, P.M., Fabisiak, T.J., Green-Fulgham, S.M., Anderson, N.D., Strand, K.A., Kwilasz, A.J., Galer, E.L., Walker, F.R., Greenwood, B.N., Maier, S.F., Fleshner, M., & Watkins, L.R. (2016) Prior voluntary wheel running attenuates neuropathic pain. Pain, 157, 2012–2023. Greenwood, B.N., Loughridge, A.B., Sadaoui, N., Christianson, J.P., & 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. Behav. Brain Res., 233, 314–321. Greenwood, B.N., Spence, K.G., Crevling, D.M., Clark, P.J., Craig, W.C., & Fleshner, M. (2013) Exercise-induced stress resistance is independent of exercise controllability and the medial prefrontal cortex. Eur. J. Neurosci., 37, 469–478. Han, C., Vasylyev, D., Macala, L.J., Gerrits, M.M., Hoeijmakers, J.G.J., Bekelaar, K.J., Dib-Hajj, S.D., Faber, C.G., Merkies, I.S.J., & Waxman, S.G. (2014) The G1662S NaV1.8 mutation in small fibre neuropathy: Impaired inactivation underlying DRG neuron hyperexcitability. J. Neurol. Neurosurg. Psychiatry, 85, 499–505. Harber, V.J. & Sutton, J.R. (1984) Endorphins and exercise. Sports Med., 1, 154–171. Hasin, D.S., Sarvet, A.L., Meyers, J.L., Saha, T.D., Ruan, W.J., Stohl, M., & Grant, B.F. (2018) Epidemiology of adult DSM-5 major depressive disorder and its specifiers in the United States. JAMA Psychiatry, 75, 336–346. Hayden, J. a, van Tulder, M.W., Malmivaara, A., & Koes, B.W. (2005) Exercise therapy for treatment of non-specific low back pain. Cochrane Database Syst. Rev., CD000335. Hooten, W.M. (2016) Chronic Pain and Mental Health Disorders: Shared Neural Mechanisms, Epidemiology, and Treatment. Mayo Clin. Proc., 91, 955– 970. Hooten, W.M., Qu, W., Townsend, C.O., & Judd, J.W. (2012) Effects of strength vs aerobic exercise on pain severity in adults with fibromyalgia: A randomized equivalence trial. Pain, 153, 915–923. Jann, M.W. & Slade, J.H. (2007) Antidepressant Agents for the Treatment of Chronic Pain and Depression. Pharmacotherapy, 27, 1571–1587. Jasnow, A.M., Shi, C., Israel, J.E., Davis, M., & Huhman, K.L. (2005) Memory of social defeat is facilitated by cAMP response element-binding protein overexpression in the amygdala. Behav. Neurosci., 119, 1125–1130. Jm, K.H. (2018) Commentary The quest for better analgesics for the treatment of peripheral neuropathic pain : navigating the sodium channels. Trends Gen. Pract., 1, 1–3. Kessler, R.C. (2012) The Costs of Depression. Psychiatr. Clin. North Am., 35, 1–14. Kessler, R.C., Sonnega, A., Bromet, E., Hughes, M., & Nelson, C.B. (1995)

Posttraumatic stress disorder in the National Comorbidity Survey. Arch. Gen. Psychiatry, 52, 1048–1060. Knapen, J., Vancampfort, D., Schoubs, B., Probst, M., Sienaert, P., Haake, P., Peuskens, J., & Pieters, G. (2009) Exercise for the Treatment of Depression. Open Complement. Med. J., 1, 78–83. Krishnan, V., Han, M.H., Graham, D.L., Berton, O., Renthal, W., Russo, S.J., LaPlant, Q., Graham, A., Lutter, M., Lagace, D.C., Ghose, S., Reister, R., Tannous, P., Green, T.A., Neve, R.L., Chakravarty, S., Kumar, A., Eisch, A.J., Self, D.W., Lee, F.S., Tamminga, C.A., Cooper, D.C., Gershenfeld, H.K., & Nestler, E.J. (2007) Molecular Adaptations Underlying Susceptibility and Resistance to Social Defeat in Brain Reward Regions. Cell, 131, 391– 404. Kumpulainen, K. (2008) Psychiatric conditions associated with bullying. Int. J. Adolesc. Med. Health, 20. Kuphal, K.E., Fibuch, E.E., & Taylor, B.K. (2007) Extended Swimming Exercise Reduces Inflammatory and Peripheral Neuropathic Pain in Rodents. J. Pain, 8, 989–997. Lapmanee, S., Charoenphandhu, J., & Charoenphandhu, N. (2013) Beneficial effects of fluoxetine, reboxetine, venlafaxine, and voluntary running exercise in stressed male rats with anxiety- and depression-like behaviors. Behav. Brain Res., 250, 316–325. Leung, A., Gregory, N.S., Allen, L.-A.H., & Sluka, K.A. (2016) Regular physical activity prevents chronic pain by altering resident muscle macrophage phenotype and increasing interleukin-10 in mice. Pain, 157, 70–79. Maletic, V. & Raison, C.L. (2009) Neurobiology of depression, fibromyalgia and neuropathic pain. Front. Biosci., 14, 5291. Manosso, L.M., Moretti, M., Ribeiro, C.M., Gonçalves, F.M., Leal, R.B., Rodrigues, A.L.S., Lúcia, A., & Rodrigues, S. (2015) Antidepressant-like effect of zinc is dependent on signaling pathways implicated in BDNF modulation. Prog. Neuro-Psychopharmacology Biol. Psychiatry, 59, 59–67. Mattson, M.P. (2012) Evolutionary aspects of human exercise—Born to run purposefully. Ageing Res. Rev., 11, 347–352. Mazzardo-Martins, L., Martins, D.F., Marcon, R., dos Santos, U.D., Speckhann, B., Gadotti, V.M., Sigwalt, A.R., Guglielmo, L.G.A., & Santos, A.R.S. (2010) High-Intensity Extended Swimming Exercise Reduces Pain-Related Behavior in Mice: Involvement of Endogenous Opioids and the Serotonergic System. J. Pain, 11, 1384–1393. Moghetti, P., Bacchi, E., Brangani, C., Donà, S., & Negri, C. (2016) Metabolic Effects of Exercise. Front. Horm. Res., 47, 44–57. Mul, J.D., Soto, M., Cahill, M.E., Ryan, R.E., Takahashi, H., So, K., Zheng, J., Croote, D.E., Hirshman, M.F., La Fleur, S.E., Nestler, E.J., & Goodyear, L.J. (2018) Voluntary wheel running promotes resilience to chronic social defeat stress in mice: A role for nucleus accumbens ΔfosB. Neuropsychopharmacology, 43, 1934–1942. Naugle, K.M., Fillingim, R.B., & Riley, J.L. (2012) A Meta-Analytic Review of the Hypoalgesic Effects of Exercise. J. Pain, 13, 1139–1150. Nekovarova, T., Yamamotova, A., Vales, K., Stuchlik, A., Fricova, J., & Rokyta, R. (2014) Common mechanisms of pain and depression: are antidepressants also analgesics? Front. Behav. Neurosci., 8. Otsuka, A., Shiuchi, T., Chikahisa, S., Shimizu, N., & Séi, H. (2015) Voluntary

exercise and increased food intake after mild chronic stress improve social avoidance behavior in mice. Physiol. Behav., 151, 264–271. Outcalt, S.D., Kroenke, K., Krebs, E.E., Chumbler, N.R., Wu, J., Yu, Z., & Bair, M.J. (2015) Chronic pain and comorbid mental health conditions: independent associations of posttraumatic stress disorder and depression with pain, disability, and quality of life. J. Behav. Med., 38, 535–543. Pagliusi Jr., M.O. & Sartori, C. (2019) Social Defeat Stress (SDS) in Mice: Using Swiss Mice as Resident. Bio-Protocol, 9, 1–12. Pagliusi Jr, M.O.F., Bonet, I.J.M., Dias, E.V., Vieira, A.S., Tambeli, C.H., Parada, C.A., Sartori, C.R., Pagliusi, M.O.F., Bonet, I.J.M., Dias, E.V., Vieira, A.S., Tambeli, C.H., Parada, C.A., & Sartori, C.R. (2018) Social defeat stress induces hyperalgesia and increases truncated BDNF isoforms in the nucleus accumbens regardless of the depressive-like behavior induction in mice. Eur. J. Neurosci., 48, 1635–1646. Patetsos, E. & Horjales-Araujo, E. (2016) Treating chronic pain with SSRIs: What do we know? Pain Res. Manag., 2016. Pedersen, B.K. (2017) Anti-inflammatory effects of exercise: role in diabetes and cardiovascular disease. Eur. J. Clin. Invest., 47, 600–611. Pollak, D.D., Rey, C.E., & Monje, F.J. (2010) Rodent models in depression research: Classical strategies and new directions. Ann. Med., 42, 252–264. Repantis, D., Schlattmann, P., Laisney, O., & Heuser, I. (2009) Antidepressants for neuroenhancement in healthy individuals: a systematic review. Poiesis Prax., 6, 139–174. Robinson, M.J., Edwards, S.E., Iyengar, S., Bymaster, F., Clark, M., & Katon, W. (2009) Depression and pain. Front. Biosci. (Landmark Ed., 14, 5031– 5051. Shafia, S., Vafaei, A.A., Samaei, S.A., Bandegi, A.R., Rafiei, A., Valadan, R., Hosseini-Khah, Z., Mohammadkhani, R., & Rashidy-Pour, A. (2017) Effects of moderate treadmill exercise and fluoxetine on behavioural and cognitive deficits, hypothalamic-pituitary-adrenal axis dysfunction and alternations in hippocampal BDNF and mRNA expression of apoptosis – related proteins in a rat model of post-tr. Neurobiol. Learn. Mem., 139, 165–178. Stagg, N.J., Mata, H.P., Ibrahim, M.M., Henriksen, E.J., Porreca, F., Vanderah, T.W., & Philip Malan, T. (2011) Regular Exercise Reverses Sensory Hypersensitivity in a Rat Neuropathic Pain Model. Anesthesiology, 114, 940–948. Stanton, R. & Reaburn, P. (2014) Exercise and the treatment of depression: A review of the exercise program variables. J. Sci. Med. Sport, 17, 177–182. Strain, J.J. (2018) The psychobiology of stress, depression, adjustment disorders and resilience. World J. Biol. Psychiatry, 19, S14–S20. Sumiyoshi, A., Taki, Y., Nonaka, H., Takeuchi, H., & Kawashima, R. (2014) Regional gray matter volume increases following 7days of voluntary wheel running exercise: A longitudinal VBM study in rats. Neuroimage, 98, 82–90. Tan, A., Costi, S., Morris, L.S., Van Dam, N.T., Kautz, M., Whitton, A.E., Friedman, A.K., Collins, K.A., Ahle, G., Chadha, N., Do, B., Pizzagalli, D.A., Iosifescu, D. V., Nestler, E.J., Han, M.-H., & Murrough, J.W. (2018) Effects of the KCNQ channel opener ezogabine on functional connectivity of the ventral striatum and clinical symptoms in patients with major depressive disorder. Mol. Psychiatry, 1. Viña, J., Sanchis-Gomar, F., Martinez-Bello, V., & Gomez-Cabrera, M.C. (2012)

Exercise acts as a drug; The pharmacological benefits of exercise. Br. J. Pharmacol., 167, 1–12. Warburton, D.E.R., Nicol, C.W., & Bredin, S.S.D. (2006) Health benefits of physical activity: the evidence. CMAJ, 174, 801–809. Whitehead, R.A., Lam, N.L., Sun, M.S., Sanchez, J., Noor, S., Vanderwall, A.G., Petersen, T.R., Martin, H.B., & Milligan, E.D. (2017) Chronic Sciatic Neuropathy in Rat Reduces Voluntary Wheel-Running Activity With Concurrent Chronic Mechanical Allodynia. Anesth. Analg., 124, 346–355. Willner, P., Scheel-Krüger, J., & Belzung, C. (2013) The neurobiology of depression and antidepressant action. Neurosci. Biobehav. Rev., 37, 2331–2371. Yehuda, R. (2004) Risk and resilience in posttraumatic stress disorder. J. Clin. Psychiatry, 65, 29–36. Figure 1. Graphic diagram of the experimental design (A) Experimental design to investigate the therapeutic effect of physical exercise on persistent hyperalgesia and depressive-like behavior of social avoidance induced by chronic social defeat stress (SDS) (B) Experimental design to investigate the preventive effect of physical exercise on hyperalgesia and depressive-like behavior of social avoidance induced by chronic SDS (SI = social interaction test; ? = time to restore daily running volume). Figure 2. Social interaction test. (A) Social interaction ratio in the interaction zone (SI-IZ) - calculated by dividing the time spent in the interaction zone in the “target” session by the time spent in the interaction zone in the “no target” session. Stressed mice with SI-IZ < 1 were assigned to the susceptible group and stressed mice with SI-IZ ≥ 1 were assigned to the resilient group. (B) Social interaction ratio in the corners (SI-CZ) - calculated by dividing the time spent in the corners in the “target” session by the time spent in the corners in the “no target” session. (N=15-23 per group; ****p< 0.0001 different from other groups). Figure on the upper right corner shows the social interaction arena (IZ = interaction zone and CZ = corners zone). Figure 3. Variation of the mechanical nociceptive threshold. Variation of the mechanical nociceptive threshold calculated by subtracting the average of three values observed before the social defeat stress (baseline) from the average of three values obtained after the social defeat stress (N=15-23 per group; ****p< 0.0001 different from no SDS group). Figure 4. Total time spent in the zones during “TARGET” session. (A) Total time spent in the interaction zone during “target” session 15 days after starting treatments. (B) Total time spent in the interaction zone during “target” session 29 days after starting treatments. (C) Total time spent in the corners during “target” session 15 days after starting treatments. (D) Total time spent in the corners during “target” session 29 days after starting treatments. (N=5-15 per group; *p< 0.05 different from no SDS group; **p< 0.01 different from no SDS group; ***p< 0.001 different from no SDS group; ****p< 0.0001 different from no SDS group. Res/Sed = resilient sedentary, Res/Ex = resilient exercise, Res/Flu = resilient fluoxetine, Sus/Sed = susceptible sedentary, Sus/Ex = susceptible exercise, Sus/Flu = susceptible fluoxetine). Figure on the upper right corner shows the social interaction arena (IZ = interaction zone and CZ = corners zone).

Figure 5. Variation of the mechanical nociceptive threshold. (A) 7 days after beginning the treatments. (B) 14 days after beginning the treatments. (C) 21 days after beginning the treatments. And (D) 28 days after beginning the treatments (N=515 per group; *p< 0.05 different from no SDS group; **p< 0.01 different from no SDS group; ***p< 0.001 different from no SDS group; ****p< 0.0001 different from no SDS group. Res/Sed = resilient sedentary, Res/Ex = resilient exercise, Res/Flu = resilient fluoxetine, Sus/Sed = susceptible sedentary, Sus/Ex = susceptible exercise, Sus/Flu = susceptible fluoxetine). Figure 6. Capsaicin test. Figure shows the number of flinches for each experimental group ((N=5-15 per group; *p< 0.05 different from no SDS group; ****p< 0.0001 different from no SDS group. Res/Sed = resilient sedentary, Res/Ex = resilient exercise, Res/Flu = resilient fluoxetine, Sus/Sed = susceptible sedentary, Sus/Ex = susceptible exercise, Sus/Flu = susceptible fluoxetine). Figure 7. Distance traveled by the stressed mice during the experiment. (A) Curve of daily distance traveled in the running wheel. (B) Total distance traveled during the experiment. (C) Average distance traveled per day (N=5-8 per group; *p< 0.05 different from resilient). Figure 8. Social interaction test. (A) Social interaction ratio in the interaction zone (SI-IZ) - calculated by dividing the time spent in the interaction zone in the “target” session by the time spent in the interaction zone in the “no target” session. (N=9-10 per group; **p< 0.01 different from Sed/NSDS). (B) Social interaction ratio in the corners (SI-CZ) - calculated by dividing the time spent in the corners in the “target” session by the time spent in the corners in the “no target” session. (N=9-10 per group; *p< 0.05 different from Sed/NSDS). (C) Total time spent in the interaction zone during “target” session. (N=9-10 per group; *p< 0.05 different from Sed/SDS). (D) Total time spent in the corners during “target” session. (N=9-10 per group). (Sed/no SDS = sedentary no stressed; Ex/no SDS = exercised no stressed; Sed/SDS = sedentary defeated; Ex/SDS = exercised defeated). Figure on the upper right corner shows the social interaction arena (IZ = interaction zone and CZ = corners zone). Figure 9. Variation of the mechanical nociceptive threshold. Variation of the mechanical nociceptive threshold calculated by subtracting the average of three values observed before the social defeat stress protocol from the average of three values obtained 24h after the last day of social defeat stress. (N=9-10 per group; ****p< 0.0001 different from other groups. Sed/no SDS = sedentary no stressed; Ex/no SDS = exercised no stressed; Sed/SDS = sedentary stressed; Ex/SDS = exercised stressed). Figure 10. Distance traveled by the stressed mice during the experiment. Curve of daily distance traveled in the running wheel by stressed and no stressed groups (N=9-10 per group; *p< 0.05 different from NO SDS group at same time point; **p< 0.01 different from NO SDS group at same time point. SDS = social defeat stress).

   

Social defeat stress induces persistent mechanical and chemical hyperalgesia in mice Voluntary running wheel exercise is more effective than fluoxetine reversing the SDSinduced persistent hyperalgesia Voluntary running wheel exercise and fluoxetine are effective reversing SDS-induced social avoidance Voluntary running wheel exercise is an effective tool preventing both hyperalgesia and social avoidance induced by SDS