Circadian Adaptations to Regular Exercise Training in the Treadmill Alter Temporal Changes in Dopamine and Serotonin Activity in Brain Areas

Circadian Adaptations to Regular Exercise Training in the Treadmill Alter Temporal Changes in Dopamine and Serotonin Activity in Brain Areas | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Circadian Adaptations to Regular Exercise Training in the Treadmill Alter Temporal Changes in Dopamine and Serotonin Activity in Brain Areas Frederico Sander Mansur Machado, Nayara Abreu Coelho Horta, Quezia Teixeira Rodrigues, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6606119/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 30 Oct, 2025 Read the published version in Pflügers Archiv - European Journal of Physiology → Version 1 posted 9 You are reading this latest preprint version Abstract Regular exercise's beneficial effects may arise from regulating circadian rhythm. Here, we investigated how regular exercise training affects spontaneous locomotor activity (SLA) and body temperature (T-body) rhythms and its influence on temporal changes in brain dopaminergic and serotonergic activity, corticosterone levels, and muscle Per1 and Bmal1 gene expression. The aerobic exercise schedule consisted of five days of training in the light phase (Zeitgeber time = ZT4 to ZT6, with light on at 7:00 h) followed by a two-day recovery period. After eight weeks, brain, blood, and muscle samples were collected from adult male rats. Dopamine (DA) and serotonin (5-HT) and their respective metabolites, DOPAC and 5-HIAA, were measured in microdissections of the caudate putamen (CP), preoptic area (POA), and the paraventricular nucleus of the hypothalamus (PVN). Exercise increased the SLA at the end of the night, delayed the acrophase, and increased the mesor of the SLA rhythm. No alterations were found in the T-body rhythm and corticosterone blood levels, although hyperthermia was observed after exercise sessions. Exercise increased muscle Per1 expression at ZT0, leading to a non-rhythmic profile in exercised animals. There were no changes in the CP dopaminergic and serotonergic activity, but a decrease in POA at ZT6, and an increase in PVN serotonergic activity at ZT0, resulting in a non-rhythmic profile in exercised animals. Thus, regular physical exercise during the light-phase with alterations in SLA promotes adjustments in the daily oscillation in monoaminergic activity in areas directly involved in regulating daily T-body and SLA. physical activity entrainment circadian dopamine serotonin thermoregulation locomotor activity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Modernity has introduced a more efficient manipulation of environmental factors. At first, the artificial electric light led to an authentic “colonization” of the night by human beings. This process occurred despite the diurnal characteristics developed by the species throughout its relatively short evolutionary process. Nowadays, the possibility of regular work to take place at times more appropriate to rest represents a novelty with possible behavioral, biological and health repercussions [ 12 ]. In fact, most of these recently incorporated behavioral lifestyles have been associated with a higher prevalence of cardiovascular, metabolic, and immunological diseases [ 12 ]. In this regard, regular physical activity, although a factor with less potential for entrainment compared to light, might act as a source of temporal information about thermic and energetic demands, and consequently, it affects the maintenance of the homeostasis [ 29 , 36 , 37 , 46 ]. However, the implications of the amount of physical activity as well as its acute and cumulative effects on the internal clock are still poorly understood. Additionally, regular physical activity could overcome the ordinary oscillations evoking a transient disruption in the circadian time system, despite triggering acute and sometimes extreme biological responses. For example, the circadian patterns of physical activity are coupled to heat production, increasing internal body temperature (Tbody), which is central to circadian thermal balance [ 22 , 23 ]. Thus, a better understanding of the relationship between homeostatic adjustments and temporal signals would help to develop strategies to prevent or treat conditions that are potentially harmful to well-being, improving the quality of life of the general population. In general, possible entraining effects of the circadian clock by physical activity could be mediated by several factors directly related to the exercise-adaptive stimuli. Increased T body and corticosterone could act as resetting cues evoked by exercise metabolic/energy demand [ 24 ]. Glucocorticoids are one of the main temporal agents through which the suprachiasmatic nucleus (SCN), the main pacemaker for circadian control, communicates with the various peripheral tissues [ 1 , 7 , 19 ]. On the other hand, T body circadian rhythm, which is driven by SCN, induces phase-coherence in individual oscillators adjusting the phases between the central (extra-SCN) and peripheral clocks [ 3 , 13 , 19 , 32 ]. In fact, connection between thermoregulatory nuclei in the forebrain, such as the hypothalamic paraventricular nucleus (PVN) and the preoptic area (POA) points towards a complementary role of these regions in the control of daily rhythms [ 5 , 15 ], which could also apply to the adaptive responses to exercise training. As such, by promoting comparable and transient increases in T body and glucocorticoid levels, physical activity could indirectly produce time signals relevant to the system, potentially triggering a phase readjustment response. Therefore, the present work investigated the effects of 8 weeks of scheduled physical exercise training during the first hours of the light-phase on forebrain monoamines and peripheral components of the circadian temporal system in rats. Materials and Methods Ethical statements The experimental procedures described in the present study were approved by the Ethics Committee on the Use of Animals (protocol n. 57/2014, CEUA/UFMG). Experimental Subjects Experimental procedures involved weaning male Wistar rats (4-weeks old) from the Animal Facility of the Institute of Biological Sciences in the Federal University of Minas Gerais (CEBIO/ICB/UFMG). The animals were maintained in collective cages (3-5/cages), under a 12/12-hour light/dark cycle (lights-on at 7:00/lights-off at 19:00) and room temperature of 24 ± 1°C, with free access to standard chow and water. Experimental procedures To evaluate the relationship between physical training-induced adaptations and adjustments of circadian parameters, animals were randomly allocated to 2 experimental groups: non- trained (NT) and trained (ExT). As previously described [34], after familiarization to treadmill running (5 days, 15 m/min, 5 min, constant slope of 5°), animals trained on a treadmill for 8 weeks (5 days/week, 10-25 m/min, 30-60 min, constant slope of 5°) with the occurrence of physical tests at the beginning, at the middle and end of the 4th week (T4) and at the end of the 8th week. The exercise procedure was performed between 11:00 and 13:00 h (ZT4 and ZT6, respectively). This time schedule was chosen based on a phase response curve for treadmill exercise performed between the ZT4 and 6 in which there was a greater entraining effect of the exercise on circadian muscle clock [44]. In the 7 th week, animals from both trained and untrained groups received an internal temperature and spontaneous locomotor activity telemetric probe for monitoring T body and LA continuously at the last week of the protocol. The variation of T body between NT and ExT group during the treadmill running can be detected in the sessions, as it does not depend on the movement direction, and it is presented in the results. However, the variation of SLA during the exercise session was not presented because SLA detected does not represent the movement due to exercise but the vector of the movement in the treadmill. Food and water intake values were daily registered at ZT0 and ZT12, as ways to control food consumption according to light/dark cycles. At least 48 hours after the last maximal capacity test, the animals (with and without sensors) were euthanized at 5 different times of the day: ZT0 (07:00h), ZT4 (11:00h), ZT6 (13:00h), ZT12 (19:00h) and ZT18 (01:00h). Trunk blood was collected for hormonal measurements; the gastrocnemius muscle was obtained to determine the expression of Per1 ; brain tissue was removed to measure monoamine in response to exercise training. In the dark phase (ZT18), euthanasia occurred under red lights (<1lux). The animals were weighed once every week at ZT4. Treadmill exercise training The scheduled exercise training protocol consisted of running on a treadmill for 8 weeks at a frequency of 5 days a week. The intensity and duration of the exercise were gradually increased until the animals reached a speed of 25 m/min, with a 5º inclination of the treadmill, for 60 minutes. The training sessions occurred between ZT4 and ZT6, as described above. To assure similar animal handling, the animals in the NT group performed a simulation of exercise on the treadmill during the same training period, with a speed of 15 m/min, but lasting only 5 min/day, twice a week. Graded Intensity Exercise Protocol The exercise protocol with progressive intensity until fatigue consisted of a running on a treadmill with a constant inclination of 5º and an initial speed of 10 m/min with increases of 1 m/min every 3 minutes until the moment when the animals interrupted the exercise or were no longer able to maintain running speed for 10 seconds. The tests took place with room temperature maintained at 24 °C. Core T body and a tail skin temperature (T sk ) were also registered. Spontaneous Locomotor Activity and Body Temperature Monitoring Animals from each group were intraperitoneally implanted with a telemetry sensor (model G2 E-Mitter, Mini-Mitter Company, Sun River, OR, USA) during sedation and anesthesia with ketamine (Ketamine 10%, Syntec) and xylazine (Xilasina 2%, Agener União). After laparotomy of approximately 2 cm over the linea alba of the rectus abdominis muscle, the sensors were fixed internally to the abdominal wall. The wound was then sutured and rats received prophylactic treatment with pentabiotic (Pentabiótico Veterinário®, Fort Dodge Saúde, Animal Ltda; dose, intramuscular) and analgesic (Banamine®, Schering-Plow, S/A, dose, subcutaneous). After 48 hours, animals recovered from surgery presented regular body weight, food intake, general coat characteristics and activity in the cage. Besides, only animals that were fully recovered continued the training protocol. After recovery, animals were transferred to individual boxes placed on individual recipient plates (ER-4000 Energizer/Receiver, Mini-Mitter Company, Sun River, OR, USA) connected in series with a computer containing the data acquisition software (VitalView Data Acquisition System Software v.4.0, Mini-Mitter Company, Sun River, OR, USA). According to information from the manufacturer, the accuracy of the system for temperature monitoring is ± 0.1 °C. Spontaneous locomotor activity is a representative measure of each spatial displacement of the sensor (E-mitter) in the longitudinal, horizontal and transverse axes on each receiver plate. Each of these measures was accumulated at the recording intervals determined in each experiment and expressed in arbitrary units (au). For results analysis purposes, only data generated after the initial 12 hours of registration were used for the studied variables. Corticosterone assessment Plasma corticosterone levels were determined by enzyme-linked immunosorbent assay (ELISA) using commercially available kits (Cayman Chemical Company, Ann Arbor, MI, USA). All samples were evaluated in duplicates following the manufacturer's specifications. Catecholamine assessment Brain levels of dopamine (DA), serotonin (5-HT) and their respective metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and 5-hydroxyindole-3-acetic acid (5-HIAA) [20] were determined in the caudate putamen (CP), preoptic area (POA), and hypothalamic paraventricular nucleus (PVN). Immediately after euthanasia, the animals' brains were carefully removed and frozen on dry ice and then stored in at -80 °C until they were analyzed by high-performance liquid chromatography with electrochemical detection (HPLC-ED). Using Paxinos & Watson's Atlas as a reference [42], the brains were sectioned into coronal slices of 800 μm with a cryostat (Leica CM1850, Heidelberger, Germany). Using the punch method, it was possible to extract the regions corresponding to the nuclei/ areas of interest with a 2.0-mm needle used bilaterally for the CP (2,20 to 0,48 mm from Bregma) and centrally (in relation to the 3rd ventricle) for the POA (0,48 to -0,48 mm from Bregma) and PVN (-0,96 to -1,92 mm from Bregma). Punchs were then homogenized in a solution of 0.15 M perchloric acid and 0.1 M EDTA plus the internal standard 3.4-dihydrobenzylamine (DHBA, Sigma Aldrich, Milwaukee, United States). Protein levels were determined by the Bradford method. The supernatant was injected into the chromatography system (Shimadzu, Kyoto, Japan). This system is formed by a C-18 column (Purospher 5m, Merck, Darmstadt, Germany) preceded by a C-18 guard column. The potential in the electrochemical detector (Decade 2; Antec Scientific, the Netherlands) was set to +0.40 V versus Ag/AgCl reference electrode. The mobile phase of the system was composed of 100 mM NaH 2 PO 4 , 10 mM NaCl, 0.1 nM EDTA, 0.38 mM octanosulfonic acid, and 10% methanol in water. The pH was adjusted to 3.5 with 85% orthophosphoric acid and the solution was pumped at a flow rate of 1 ml/min. Peaks were identified based on their retention times and quantified using the internal standard method. All samples evaluated were measured in the same assay. The intra-assay coefficient of variation was less than 5% for all measured compounds. DA and 5-HT levels are considered to reflect neurotransmitter content in synaptic vesicles, whereas DOPAC and 5-HIAA reflect neurotransmitter release in tissue homogenates [20, 21]. DOPAC/DA and 5-HIAA/5-HT ratios were calculated as an index of neurotransmitter turnover and used to estimate changes in the activity of dopaminergic and serotoninergic terminals, respectively. Gene expression assessment After euthanasia, the tissues were quickly removed and frozen in a container containing liquid nitrogen (gastrocnemius) or dry ice (brain). The samples were stored at -80 °C until they were processed for analysis of the expression of genes of interest. Total RNA was extracted with Trizol (Invitrogen, California, United States) according to the manufacturer's instructions, or in conjunction with the High Pure RNA Tissue Kit for RNA extraction (Roche Diagnostics, Mannheim, Germany). The concentration of RNA was determined by reading the absorbance at 260 nm and 230 nm on a spectrophotometer (Nanodrop, Thermo Scientific, Wilmington, United States). Only samples with 260/230 ratio readings between 1.8 and 2.2 were used for subsequent reactions. The reverse transcriptase reaction was performed with 2μg of total RNA from the gastrocnemius samples, using 2μL of random oligonucleotides (50 ng/μL) and 2 uL of dNTPs (10 mM), 8 μL of buffer of 5X PCR, 2 µL of 10 mM DTT, 2 µL of the 40 U/µL ribonuclease inhibitor, and 2 µL of Superscript III 200 U/µL, ending a volume of 40 µL per reaction. The synthesized cDNA was used in subsequent quantitative PCR reactions (in real time). The following primers were used to determine mRNA expression of the interest gene: Per1 (NM_001034125.1; forward primer 5’-ATGCAGAAACAACAGCCACGGTTC-3’, reverse primer 5’-TGGCCAGGATCTTGAACACTGCTA-3’) and Bmal1 (NM_024362.2; Forward: 5’ - AGG CCT TCA CTG GAA TGG TGC TAT - 3’, Reverse: 5’- tga ctg gcc tgg aac ttg cta cat - 3’). The normalizer used in this study was the 18S ribosomal RNA (Forward: 5’ - CGG CTA CCA CAT CCA AGG AA - 3’, Reverse: 5’ - GCT GGA ATT ACC GCG GCT - 3’). Solutions containing primers, SYBR® GreenER ™ qPCR SuperMix universal (Life Technologies, USA) and H2O DNaseRNase-free (Life Technologies, USA), obtaining a final concentration of 300 ηM for the primers of the gene of interest and 50 ηM for the primers of RNA 18S (normalizer), in independent solutions. Each solution was aliquoted (48 μL, each aliquot sufficient for 2 wells) in tubes and the cDNA of each sample added (2 μL/aliquot). The solutions already with cDNA were then distributed in their respective wells of the experimental plate (23 μL/well) and taken to the thermocycler (ABI-PRISMA 7900) for reaction in the following conditions: 2 min at 50 °C, 8:30 min at 95 °C , followed by 45 cycles of 15 s at 95 °C, 1 min at 60 °C, 1 min at 95 °C, 1 min at 55 °C and 80 cycles of 10 s to 55 °C, with a gradual increase of 0.5 °C. Data analysis was performed by comparing the number of copies of the control and experimental wells, or between different times after normalization by the expression level of the reference gene. Data Analysis The differences between performance variables over 8 weeks were evaluated using two-way ANOVA followed by a post-hoc test to characterize the differences between the experimental groups. The daily oscillations of variables of interest were determined by two-way ANOVA for each protocol. A COSINOR analysis was applied to evaluate the oscillation of T body and LA and the effect of training. Results were expressed as the mean ± SEM. Statistical significance was set at p<0.05 . The graph prism pad software was used for graphs and statistical analysis. Results Training load characteristics The training load used in this protocol was characterized by progressive increases in running speed (Figure 1a) accompanied by regular increases in the duration of the sessions over the first 4 weeks (Figure 1b), with the subsequent stabilization in 60 minutes in the final 4 weeks of the training protocol in the ExT group. The association between intensity and volume of the load resulted in a progressive increase in the training load per session, estimated through the workload calculation (Figure 1c). Effect of scheduled exercise training on T body and SLA circadian rhythms Exercise training differently changed the daily curves of T body and SLA recorded in the last week of regular treadmill running (Figure 2). Despite the transient increase in T body during the physical exercise sessions (change T body =2.02 ± 0.15 ℃; p<0.0001 ) (Figure 2a and 2c), there were no differences in the circadian rhythm of T body (Figure 2e). In contrast to T body , exercise training increased the SLA in the end of dark phase of circadian cycle (Figure 2d). Cosinor analysis showed that exercise training increased the mesor and amplitude of SLA circadian rhythm (Figure 2f). Also, it promoted a delay in the acrophase of SLA rhythm (Figure 2f). Effect of scheduled exercise training on temporal changes of corticosterone plasma levels and muscle clock genes expression The highest levels of corticosterone were found at ZT12 in both NT and ExT animals (Figure 3a). Analysis of Cosinor showed that those corticosterone temporal changes fitted in a cosine curve, which parameters (mesor, amplitude and acrophase) did not differ between NT and ExT groups (Figure 3b). The highest concentrations of Per1 mRNA in the gastrocnemius muscle were found in the ZT12 in NT animals, whereas no temporal differences were found in ExT group (Figure 3c). Analysis of Cosinor showed that muscle Per1 expression temporal changes fitted in a cosine curve in NT animals but not in the ExT (Figure 3d). Although ZT has significantly affected muscle Bmal1 expression in NT and ExT groups, post-hoc analysis did not identify differences among evaluated times (Figure 3e), consequently non-fitting curve in Bmal1 mRNA data was detected by Cosinor Analysis in both NT and ExT (Figure 3f). Effect of scheduled exercise training on temporal changes of dopaminergic activity DOPAC/DA ratio did not change in function of the time of the day nor exercise training in the CP (Figure 4a). Therefore, non-fitting curve in dopamine activity data was detected by Cosinor Analysis in both NT and ExT (Figure 4b). Nevertheless, in the POA from NT animals, dopamine activity did show temporal changes, which higher ratio of DOPAC/DA found at ZT12 compared to other ZTs (Figure 4c). Those data fitted in a cosine curve as shown in Figure 4d. Exercise training reduced the DOPAC/DA ratio at ZT6 compared to NT groups (Figure 4c) and no temporal differences in dopamine activity were found in ExT group, consequently data did not fit in a cosine curve (Figure 4d). In the PVN neither time-of-day or groups (NT or ExT) affected the dopamine activity. As such no temporal changes were found in both groups (Figure 4e), consequently data did not fit in a cosine curve (Figure 4f). Effect of scheduled exercise training on temporal changes of serotonergic activity 5HIAA/5HT ratio did not change in function of the time of the day or exercise training in the CP area (Figure 5a). Therefore, non-fitting curve in dopamine activity data was detected by Cosinor Analysis in both NT and ExT (Figure 5b). On the other hand, in the POA, exercise training attenuated the 5HIAA/5HT ratio in ZT6 (Figure 5c). There were no temporal differences in the POA 5HIAA/5HT ratio from ExT animals (Figure 5c). In fact, COSINOR analysis revealed that ExT abolished 5HIAA/5HT fitting curve (Figure 5d). Discussion The present study demonstrated that exercise training influences the circadian temporal system and monoaminergic activity in the POA and PVN. For the first time, it has been shown that a scheduled exercise that mimics human training i.e. training session 5 days a week with a 2-days recovering period was able to modulate circadian rhythms. This protocol of exercise training increases SLA quantity at the last hours of the dark phase, which lead to an increased mesor, amplitude and a delayed acrophase in SLA rhythm. On the other hand, the daily hyperthermia observed in the training session did not promote alterations in T body rhythm. Therefore, the circadian entrainment in SLA and T body rhythms seems to independently respond to exercise training. In the present study, the training model increased physical performance and improved thermal balance (Supplementary Figure 1). These results confirm that physical training triggers an increase in physical performance, accompanied by improvements in energy efficiency and also in thermoregulatory capacity [21, 35, 40, 41]. In turn, T body rhythm maintained stability, independent of the exercise sessions themselves, reinforcing the idea of adaptation in thermal efficiency. In our study, the comparison was between a progressive training protocol to a very mild walking on the treadmill. This approach is more translational as the training was compared to a non-sedentary condition. Moreover, it controlled for a possible effect of exposure to a different environment, which is known to affect the circadian time systems [43,44]. The chosen protocol may explain the very mild effect of training exercise to the overall rhythms. It is known that time of exercise is capable of entraining SLA rhythm. As such, regularly scheduled exposure to running wheels or to treadmill clearly entrains the circadian rhythm of SLA of mice under constant darkness condition [44]. Comparing our findings with this previous study, it seems that exercise training did not overcome the effect of light/dark cycles, and it was unable to re-entrain the light/dark-synchronized rhythms of SLA and T body . On the other side of daily balance, exercise training increased baseline locomotor activity. Such an increase was not only observed as a result of the exercise section (acute effect) but also during the dark-phase, indicating that SLA rhythm might be reactive to chronic changes in general physical activity. This has been extensively reported in the literature [8, 9, 28, 29, 33, 34, 36, 39, 45, 46]. In this perspective, during the second half of the dark-phase, the increased locomotor activity in the trained animals could represent a temporal redistribution in response to the recently incorporated schedule that regularly amplified energy demand associated with the beginning of the light-phase. With the regularity of the physical demand, the system would readjust to meet the temporal characteristics of the environment in the most efficient way possible, with minimum disturbances of homeostasis. Therefore, the apparent mismatch between locomotor activity and internal temperature responses to exercise could be attributed both to the higher energy efficiency of the system, resulting in higher levels of spontaneous locomotor activity for the same level of internal temperature. Of note, there was hyperthermia induced by the training session that did not lead to alterations in T body rhythm. Nevertheless, it was observed an increase in Per1 expression in the gastrocnemius muscles previously to training session time. This suggested that the daily increase in T body induced by exercise may signal to peripheral tissues in an attempt to synchronize the circadian clocks, without alterations in T body rhythm. Per1 belongs to the core component of the negative loop of the canonical molecular circadian clock. In rats, Per1 peaks at the beginning of the dark-phase (ZT12) and reaches its lowest levels at the onset of light-phase (ZT0) [16, 30, 31]. However, ExT animals failed to show that pattern, which led to non-rhythm profile of Per1 expression. It has been proposed that the expression of this gene could be associated with synchronizing properties of locomotor activity [18], internal temperature, and corticosterone [4, 14, 18]. Importantly, locomotor activity, under the influence of the SCN, is functionally associated with the activity of skeletal musculature [17, 18]. Consequently, it is speculated that the increase in locomotor activity in association with higher T body could be directly associated with the altered expression of Per1 , indicating a readjustment in the muscle clock, directly affected by physical training during the light-phase. The effect of exercise on Per1 was not accompanied by alterations in Bmal1 muscle expression. Future experiments should be conducted to evaluate the other clock genes. Glucocorticoids are potent endogenous agents for modulating the activity of the peripheral biological clock [1,7,26]. Such temporal signaling could be linked to episodes of increase in corticosterone secretion in response to exercise. In fact, Sasaki et al. (2016) demonstrated that forced exercise, on the running-wheel or treadmill, in the resting phase promotes phase advances in peripheral clocks associated with an increase in corticosterone levels [35]. These authors also demonstrated that the peripheral effects would depend on the joint action of corticosterone and noradrenergic signaling [35]. Nevertheless, our exercise protocol did not affect the daily pattern of corticosterone blood level, which is similar to other studies showing that corticosterone returns to its initial levels after 4-6 weeks of voluntary running, indicating adaptation of hypothalamus-pituitary-adrenal (HPA) axis [6, 26, 27, 38, 45]. Circulating corticosterone levels constitute one of the phase-signaling components under the control of the central pacemaker [19, 35], which may be subjected to adaptations. Therefore, there may be a two-step adaptive process for exercise training: in the initial phase, resetting the circadian clock would depend on glucocorticoid and neural adjustments; in the subsequent phase, maintaining the regular stimulus would result in a predominance of a neural component, possibly involving cerebral areas that regulate T-body, and/or SLA and physical capacity, and glucocorticoid secretion. The perspective of a neural modulation of the observed adaptations was challenged by analysis of monoaminergic activity in areas that participate in thermoregulation and HPA axis, i.e., POA and PVN, respectively, in addition to caudate putamen that is involved to physical exercise. There is an increased dopaminergic activity in the caudate putamen in association with an increase in physical capacity [2, 11, 46], and vice-versa, animals with greater intrinsic aerobic capacity show greater DA concentrations and activity of DA in brain areas directly involved with motor control [25, 29, 37]. Thus, it can be hypothesized that physical training can trigger both an increase in dopaminergic activity in the nigrostriatal pathway, normally associated with the increase in the capacity to resist fatigue [11]. Temporal adjustment in the variation of dopaminergic activity would facilitate the beginning of the training session. In the present study, dopaminergic activity in the CP was not significantly changed by physical training, indicating that SLA rhythm responses to exercise could be under the regulation of other brain centers. Additionally, it was observed that the training program attenuated both dopaminergic and serotonergic activity in the POA under baseline conditions. Untrained animals showed increased monoaminergic activity throughout the light-phase, reaching a peak in the second half of the light-phase until the beginning of the dark-phase. This profile was not seen in exercised animals, which showed a reduction in monoaminergic activity in the POA at ZT6. Such reduction may be explained by an increase in inhibitory input from the median preoptic area that has been previously shown to be linked with temperature regulation [15]. This phase-specific reduction in POA dopaminergic activity may be responsible for the adjustment of the system during the active phase of the cycle. Physical training program influenced 5-HT activity in PVN. If, in untrained animals, the 5-HIAA/5-HT ratio exhibited a peak at the beginning of the activity-phase, in trained animals, the 24h curve was abolished. Training increased PVN serotonin activity during the transition from dark to light phases (ZT0). This temporal relationship is like that observed for SLA levels at the end of the dark phase and at the second peak of Per1 expression in ZT0. It is recognized that the PVN would constitute an important site of integration for the response to different environmental stimuli [9, 10, 19]. It is interesting to note that this hypothalamic nucleus receives dense noradrenergic projections from the locus coeruleus in the brain stem, a nucleus directly related to the modulation of the arousal system [24]. It also receives serotonergic inputs from raphe and other regions that participate in the modulation of the activity state [18]. Thus, the change in the circadian variation of serotonergic activity in PVN associated with increased locomotor activity provides compelling evidence that this nucleus is part of the extra-SCN control axis of locomotor activity and under the influence of regular exercise training. In conclusion, the study demonstrated that regularly scheduled physical exercise promotes adaptations at different levels of the circadian time system. Within the central nervous system, regular exercise modulated the daily serotonin and dopamine activity in nuclei directly related with internal temperature control, motor control and the alarm/arousal system. Thus, the circadian time system seems to be affected by exercise-related entraining time cues. As demonstrated by physical exercise on a treadmill, regularity and routine lead the system to a new state of balance, configuring an important tool for non-pharmacological and non-photic regulation of disorders usually associated with the imbalance of the circadian clock. Declarations Human Ethics: “not applicable” Consent for publication: “not applicable” Funding: The authors acknowledge the financial support for F.S.M.M. in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) – CAPES/PRINT (Grant number : 88887.364935/2019-00). This study received financial support by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG). Authors' contributions: FSMM, MOP, CCC conceived the study. FSMM, MOP, and CCC analyzed and cured data. MOP and CCC acquired funding for the study. Methodology: FSMM, NACH, and QTR (Handling animals); NACH, and TSRC (RT-qPCR); FSMM (hormone assay); NSSA and RES (HPLC measurements); FSMM, NACH, and MOP (Cosinor analysis). Roles/Writing— original draft: FMSS, MOP, and CCC; Writing—review & editing: FSMM, NACH, QTR, TSRC, NSSA, RES, MOP, and CCC. Availability of supporting data: Raw data are available under requesting. Competing interests: The authors declare no competing interests Clinical Trial Number in the manuscript: “not applicable” Ethical statements: The experimental animal procedures described in the present study were approved by the Ethics Committee on the Use of Animals (protocol n. 57/2014, CEUA/UFMG) References Balsalobre A, Brown SA, Marcacci L, Tronche F, Kellendonk C, Reichardt HM, Schütz G, Schibler U (2000) Resetting of Circadian Time in Peripheral Tissues by Glucocorticoid Signaling. Science 289:2344–2347. doi: 10.1126/science.289.5488.2344 Balthazar CH, Leite LHR, Ribeiro RMM, Soares DD, Coimbra CC (2010) Effects of blockade of central dopamine D1 and D2 receptors on thermoregulation, metabolic rate and running performance. Pharmacological Reports 62:54–61. doi: 10.1016/s1734-1140(10)70242-5 Brown SA, Zumbrunn G, Fleury-Olela F, Preitner N, Schibler U (2002) Rhythms of Mammalian Body Temperature Can Sustain Peripheral Circadian Clocks. Current Biology 12:1574–1583. doi: 10.1016/s0960-9822(02)01145-4 Buhr ED, Yoo S-H, Takahashi JS (2010) Temperature as a Universal Resetting Cue for Mammalian Circadian Oscillators. Science 330:379–385. doi: 10.1126/science.1195262 Buijs RM, Kalsbeek A (2001) Hypothalamic integration of central and peripheral clocks. Nature Reviews Neuroscience 2:521–526. doi: 10.1038/35081582 Campbell JE, Rakhshani N, Fediuc S, Bruni S, Riddell MC (2009) Voluntary wheel running initially increases adrenal sensitivity to adrenocorticotrophic hormone, which is attenuated with long-term training. Journal of Applied Physiology 106:66–72. doi: 10.1152/japplphysiol.91128.2008 Chung S, Son GH, Kim K (2011) Adrenal peripheral oscillator in generating the circadian glucocorticoid rhythm. Annals of the New York Academy of Sciences 1220:71–81. doi: 10.1111/j.1749-6632.2010.05923.x Edgar DM, Dement WC (1991) Regularly scheduled voluntary exercise synchronizes the mouse circadian clock. American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 261:R928–R933. doi: 10.1152/ajpregu.1991.261.4.r928 Edgar DM, Reid MS, Dement WC (1997) Serotonergic afferents mediate activity-dependent entrainment of the mouse circadian clock. American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 273:R265–R269. doi: 10.1152/ajpregu.1997.273.1.r265 Fetissov SO, Meguid MM, Chen C, Miyata G (2000) Synchronized release of dopamine and serotonin in the medial and lateral hypothalamus of rats. Neuroscience 101:657–663. doi: 10.1016/s0306-4522(00)00374-2 Foley T, Greenwood B, Day H, Koch L, Britton S, Fleshner M (2006) Elevated central monoamine receptor mRNA in rats bred for high endurance capacity: Implications for central fatigue. Behavioural Brain Research 174:132–142. doi: 10.1016/j.bbr.2006.07.018 Gerhart-Hines Z, Lazar MA (2015) Circadian metabolism in the light of evolution. Endocrine Reviews 36:289–304. doi: 10.1210/er.2015-1007 Goh GH, Mark PJ, Blache D, Binks D, Parsons R, Rawashdeh O, Maloney SK (2021) Diet-altered body temperature rhythms are associated with altered rhythms of clock gene expression in peripheral tissues in vivo. Journal of Thermal Biology 100:102983. doi: 10.1016/j.jtherbio.2021.102983 Gotic I, Schibler U (2017) Posttranscriptional mechanisms controlling diurnal gene expression cycles by body temperature rhythms. RNA Biology 14:1294–1298. doi: 10.1080/15476286.2017.1285481 Guzmán-Ruiz MA, Ramirez-Corona A, Guerrero-Vargas NN, Sabath E, Ramirez-Plascencia OD, Fuentes-Romero R, León-Mercado LA, Basualdo Sigales M, Escobar C, Buijs RM (2015) Role of the Suprachiasmatic and Arcuate Nuclei in Diurnal Temperature Regulation in the Rat. The Journal of Neuroscience 35:15419–15429. doi: 10.1523/jneurosci.1449-15.2015 Horta NAC, Fernandes P, Cardoso TSR, Machado FSM, Drummond LR, Coimbra CC, Wanner SP, Maria L Castrucci A, Poletini MO (2023) TRPV1 inactivation alters core body temperature and serum corticosterone levels: Impacts on clock genes expression in the liver and adrenal glands. Journal of Thermal Biology 114:103514. doi: 10.1016/j.jtherbio.2023.103514 Houben T, Deboer T, van Oosterhout F, Meijer JH (2009) Correlation with Behavioral Activity and Rest Implies Circadian Regulation by SCN Neuronal Activity Levels. Journal of Biological Rhythms 24:477–487. doi: 10.1177/0748730409349895 Hughes ATL, Piggins HD (2012) Feedback actions of locomotor activity to the circadian clock. In: Progress in Brain Research. Elsevier, pp 305–336 Kalsbeek A, Liu J, Lei J, Timmermans L, Foppen E, Cailotto C, Fliers E (2012) Differential Involvement of the Suprachiasmatic Nucleus in Lipopolysaccharide-Induced Plasma Glucose and Corticosterone Responses. Chronobiology International 29:835–849. doi: 10.3109/07420528.2012.699123 Lookingland KJ, Jarry HD, Moore KE (1987) The metabolism of dopamine in the median eminence reflects the activity of tuberoinfundibular neurons. Brain Research 419:303–310. doi: 10.1016/0006-8993(87)90597-x Machado FSM, Fóscolo DRC, Poletini MO, Coimbra CC (2016) Influence of Time-of-Day on Maximal Exercise Capacity Is Related to Daily Thermal Balance but Not to Induced Neuronal Activity in Rats. Frontiers in Physiology 7. doi: 10.3389/fphys.2016.00464 Machado FSM, Rodovalho GV, Coimbra CC (2015) The time of day differently influences fatigue and locomotor activity: Is body temperature a key factor? Physiology & Behavior 140:8–14. doi: 10.1016/j.physbeh.2014.11.069 Machado FSM, Zhang Z, Su Y, de Goede P, Jansen R, Foppen E, Coimbra CC, Kalsbeek A (2018) Time-of-Day Effects on Metabolic and Clock-Related Adjustments to Cold. Frontiers in Endocrinology 9. doi: 10.3389/fendo.2018.00199 Mastorakos G, Pavlatou M (2005) Exercise as a Stress Model and the Interplay Between the Hypothalamus-pituitary-adrenal and the Hypothalamus-pituitary-thyroid Axes. Hormone and Metabolic Research 37:577–584. doi: 10.1055/s-2005-870426 Mathes WF, Nehrenberg DL, Gordon R, Hua K, Garland T Jr, Pomp D (2010) Dopaminergic dysregulation in mice selectively bred for excessive exercise or obesity. Behavioural Brain Research 210:155–163. doi: 10.1016/j.bbr.2010.02.016 Otawa M, Arai H, Atomi Y (2007) Molecular aspects of adrenal regulation for circadian glucocorticoid synthesis by chronic voluntary exercise. Life Sciences 80:725–731. doi: 10.1016/j.lfs.2006.10.023 Pastore S, Hood DA (2013) Endurance training ameliorates the metabolic and performance characteristics of circadian Clock mutant mice. Journal of Applied Physiology 114:1076–1084. doi: 10.1152/japplphysiol.01505.2012 Pendergast JS, Branecky KL, Huang R, Niswender KD, Yamazaki S (2014) Wheel-running activity modulates circadian organization and the daily rhythm of eating behavior. Frontiers in Psychology 5. doi: 10.3389/fpsyg.2014.00177 Rabelo PCR, Almeida TF, Guimarães JB, Barcellos LAM, Cordeiro LMS, Moraes MM, Coimbra CC, Szawka RE, Soares DD (2015) Intrinsic exercise capacity is related to differential monoaminergic activity in the rat forebrain. Brain Research Bulletin 112:7–13. doi: 10.1016/j.brainresbull.2015.01.006 Reznick J, Preston E, Wilks DL, Beale SM, Turner N, Cooney GJ (2013) Altered feeding differentially regulates circadian rhythms and energy metabolism in liver and muscle of rats. Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease 1832:228–238. doi: 10.1016/j.bbadis.2012.08.010 Saini C, Morf J, Stratmann M, Gos P, Schibler U (2012) Simulated body temperature rhythms reveal the phase-shifting behavior and plasticity of mammalian circadian oscillators. Genes & Development 26:567–580. doi: 10.1101/gad.183251.111 Salgado-Delgado R, Ángeles-Castellanos M, Buijs MR, Escobar C (2008) Internal desynchronization in a model of night-work by forced activity in rats. Neuroscience 154:922–931. doi: 10.1016/j.neuroscience.2008.03.066 Salgado-Delgado R, Angeles-Castellanos M, Saderi N, Buijs RM, Escobar C (2010) Food Intake during the Normal Activity Phase Prevents Obesity and Circadian Desynchrony in a Rat Model of Night Work. Endocrinology 151:1019–1029. doi: 10.1210/en.2009-0864 Santiago HP, Leite LH, Lima PMA, Rodovalho GV, Szawka RE, Coimbra CC (2015) The improvement of exercise performance by physical training is related to increased hypothalamic neuronal activation. Clinical and Experimental Pharmacology and Physiology 43:116–124. doi: 10.1111/1440-1681.12507 Sasaki H, Hattori Y, Ikeda Y, Kamagata M, Iwami S, Yasuda S, Tahara Y, Shibata S (2016) Forced rather than voluntary exercise entrains peripheral clocks via a corticosterone/noradrenaline increase in PER2::LUC mice. Scientific Reports 6. doi: 10.1038/srep27607 Sato RY, Yamanaka Y (2023) Nonphotic entrainment of central and peripheral circadian clocks in mice by scheduled voluntary exercise under constant darkness. American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 324:R526–R535. doi: 10.1152/ajpregu.00320.2022 Saul MC, Majdak P, Perez S, Reilly M, Garland T Jr, Rhodes JS (2016) High motivation for exercise is associated with altered chromatin regulators of monoamine receptor gene expression in the striatum of selectively bred mice. Genes, Brain and Behavior 16:328–341. doi: 10.1111/gbb.12347 Schroeder AM, Truong D, Loh DH, Jordan MC, Roos KP, Colwell CS (2012) Voluntary scheduled exercise alters diurnal rhythms of behaviour, physiology and gene expression in wild-type and vasoactive intestinal peptide‐deficient mice. The Journal of Physiology 590:6213–6226. doi: 10.1113/jphysiol.2012.233676 Shannon NJ, Gunnet JW, Moore KE (1986) A Comparison of Biochemical Indices of 5-Hydroxytryptaminergic Neuronal Activity Following Electrical Stimulation of the Dorsal Raphe Nucleus. Journal of Neurochemistry 47:958–965. doi: 10.1111/j.1471-4159.1986.tb00704.x Thompson PD, Crouse SF, Goodpaster B, Kelley D, Moyna N, Pescatello L (2001) The acute versus the chronic response to exercise. Medicine and Science in Sports and Exercise 33:S438–S445. doi: 10.1097/00005768-200106001-00012 Waters RP, Pringle RB, Forster GL, Renner KJ, Malisch JL, Garland Jr. T, Swallow JG (2013) Selection for increased voluntary wheel-running affects behavior and brain monoamines in mice. Brain Research 1508:9–22. doi: 10.1016/j.brainres.2013.01.033 Watson C, Kirkcaldie M, Paxinos G (2010) Preface. In: The Brain. Elsevier, p VII Webb IC, Antle MC, Mistlberger RE (2014) Regulation of circadian rhythms in mammals by behavioral arousal. Behavioral Neuroscience 128:304–325. doi: 10.1037/a0035885 Wolff G, Esser KA (2012) Scheduled Exercise Phase Shifts the Circadian Clock in Skeletal Muscle. Medicine & Science in Sports & Exercise 44:1663–1670. doi: 10.1249/mss.0b013e318255cf4c Yasumoto Y, Nakao R, Oishi K (2015) Free Access to a Running-Wheel Advances the Phase of Behavioral and Physiological Circadian Rhythms and Peripheral Molecular Clocks in Mice. PLOS ONE 10:e0116476. doi: 10.1371/journal.pone.0116476 Zheng X, Hasegawa H (2016) Administration of caffeine inhibited adenosine receptor agonist-induced decreases in motor performance, thermoregulation, and brain neurotransmitter release in exercising rats. Pharmacology Biochemistry and Behavior 140:82–89. doi: 10.1016/j.pbb.2015.10.019 Additional Declarations No competing interests reported. Supplementary Files SupplementalData.docx Cite Share Download PDF Status: Published Journal Publication published 30 Oct, 2025 Read the published version in Pflügers Archiv - European Journal of Physiology → Version 1 posted Editorial decision: Revision requested 19 Jul, 2025 Reviews received at journal 19 Jul, 2025 Reviewers agreed at journal 17 Jun, 2025 Reviews received at journal 17 Jun, 2025 Reviewers agreed at journal 02 Jun, 2025 Reviewers invited by journal 15 May, 2025 Editor assigned by journal 14 May, 2025 Submission checks completed at journal 14 May, 2025 First submitted to journal 06 May, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6606119","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":456921367,"identity":"6c4f6d3b-e1ef-4d10-81b5-bbc4ce2c2640","order_by":0,"name":"Frederico Sander Mansur Machado","email":"","orcid":"","institution":"Federal University of Minas Gerais (UFMG)","correspondingAuthor":false,"prefix":"","firstName":"Frederico","middleName":"Sander Mansur","lastName":"Machado","suffix":""},{"id":456921368,"identity":"0eb12d35-b0f4-43f6-9d78-e008d2b4ad4e","order_by":1,"name":"Nayara Abreu Coelho Horta","email":"","orcid":"","institution":"Federal University of Minas Gerais (UFMG)","correspondingAuthor":false,"prefix":"","firstName":"Nayara","middleName":"Abreu Coelho","lastName":"Horta","suffix":""},{"id":456921369,"identity":"5c502716-b793-4f02-8e3e-1fd4ae12c5a7","order_by":2,"name":"Quezia Teixeira Rodrigues","email":"","orcid":"","institution":"Federal University of Minas Gerais (UFMG)","correspondingAuthor":false,"prefix":"","firstName":"Quezia","middleName":"Teixeira","lastName":"Rodrigues","suffix":""},{"id":456921370,"identity":"64145928-8411-42a6-885b-37d9fe1b21aa","order_by":3,"name":"Thais Santana Rocha Cardoso","email":"","orcid":"","institution":"Federal University of Minas Gerais (UFMG)","correspondingAuthor":false,"prefix":"","firstName":"Thais","middleName":"Santana Rocha","lastName":"Cardoso","suffix":""},{"id":456921371,"identity":"1e692096-0386-4d97-b968-e194710ddccc","order_by":4,"name":"Nayara Soares Sena Aquino","email":"","orcid":"","institution":"Federal University of Minas Gerais (UFMG)","correspondingAuthor":false,"prefix":"","firstName":"Nayara","middleName":"Soares Sena","lastName":"Aquino","suffix":""},{"id":456921372,"identity":"897f3839-86fc-4a1c-81fc-44092232f635","order_by":5,"name":"Raphael Escorsim Szawka","email":"","orcid":"","institution":"Federal University of Minas Gerais (UFMG)","correspondingAuthor":false,"prefix":"","firstName":"Raphael","middleName":"Escorsim","lastName":"Szawka","suffix":""},{"id":456921373,"identity":"203594e5-7f03-4717-8e41-83c438673192","order_by":6,"name":"Maristela Oliveira. Poletini","email":"","orcid":"","institution":"Federal University of Minas Gerais (UFMG)","correspondingAuthor":false,"prefix":"","firstName":"Maristela","middleName":"Oliveira.","lastName":"Poletini","suffix":""},{"id":456921374,"identity":"0e68c285-80dc-4c89-ab9f-65c32eed3035","order_by":7,"name":"Cândido Celso. Coimbra","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA00lEQVRIiWNgGAWjYFACxgZmEMXPzMAGpOSAmIdILZLNYC3GxGhhYABrMThArBb+9ubWzYV77OSNjzM/e8DYZhAt78B77AM+LRJnDrbdnvEs2XDbYTZzA6CW3I0H+JJn4NNiIJHYdpvnAHOC2WEeNgnGtj+5Gxt4jPE6DKqlPsG4GazFgGgthxMMmKFa5jMQ0ALxy4HjhjNAfkk4Z5C7gZkvGa8W/vb2Z7cLDlTL8/cffvbgQxnQlvbew3i1oIIEkFNJ0QAB8g0kaxkFo2AUjIJhDgCea0VWWhxoqQAAAABJRU5ErkJggg==","orcid":"","institution":"Federal University of Minas Gerais (UFMG)","correspondingAuthor":true,"prefix":"","firstName":"Cândido","middleName":"Celso.","lastName":"Coimbra","suffix":""}],"badges":[],"createdAt":"2025-05-06 20:08:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6606119/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6606119/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00424-025-03128-x","type":"published","date":"2025-10-30T15:58:21+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":83022505,"identity":"f4b1c66d-8220-4094-bb27-000ddb48005f","added_by":"auto","created_at":"2025-05-19 07:52:14","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":20756,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of the training load during physical training sessions throughout the experimental period (5 days / week – 8 weeks training). Running speed (a), exercise duration (b) and workload (c) were relativized by maximum capacity. Results expressed as Mean ± SEM; n=28.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6606119/v1/1d2d452f2dfd48648810cc97.png"},{"id":83022506,"identity":"c8f7c75e-db84-4ba3-ad50-445ec6363541","added_by":"auto","created_at":"2025-05-19 07:52:14","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":48413,"visible":true,"origin":"","legend":"\u003cp\u003eRecordings of telemetric probes obtained in the 8\u003csup\u003eth\u003c/sup\u003e experimental week show the circadian rhythms of T\u003csub\u003ebody\u003c/sub\u003e (a) and spontaneous locomotor activity (SLA, b) expressed as Mean±SEM per zeitgeber time each day. The average of the T\u003csub\u003ebody\u003c/sub\u003e (c) and LA (d) recordings of the five exercise training days is shown. Black rectangle marks the time of training session, note that SLA was excluded at this period see methodoly for explanation. Data obtained throughout exercise training days were used to build adjusting cosinor curve for the T\u003csub\u003ebody\u003c/sub\u003e (e) and LA (f) rhythms, which acrophase is indicated by the dotted lines parallel to Y axis,\u0026nbsp; mesor by dotted lines parallel to X axis, amplitude by the arrows. The gray shaded areas correspond to the dark phase (ZT12=19:00 to ZT0=07:00). # Indicates \u003cem\u003ep\u0026lt;0.05\u003c/em\u003e for comparisons between groups by two-way ANOVA followed by Tukey’s test. * Indicates p\u0026lt;0.05 for comparisons between groups by unpaired t-test.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6606119/v1/c66898e15f35d28d5f056c20.png"},{"id":83023714,"identity":"b0bf37dd-e76a-4e1a-9b9e-0a39e67b058c","added_by":"auto","created_at":"2025-05-19 08:00:14","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":26237,"visible":true,"origin":"","legend":"\u003cp\u003eCorticosterone blood levels (a) and muscle expression of clock genes Per1 (c) and Bmal1 (e) analyzed from rats obtained from rats after the last recovery day expressed as Mean±SEM of percentage of average. Dashed gray lines mark the time of training session throughout the 5-day/week exercise section. Adjusting cosinor curves are shown for corticosterone blood (b) and mRNA levels (d) circadian variations, which acrophase is indicated by the dotted lines parallel to Y axis, mesor by dotted lines parallel to X axis, and amplitude by the arrows. The gray shaded areas correspond to the dark phase (ZT12=19:00 to ZT0=07:00). A indicates p\u0026lt;0.05 ZT12 versus ZT4 in the non-training (NT) group and a indicates p\u0026lt;0.05 ZT12 versus ZT4 in the exercise\u003cstrong\u003e \u003c/strong\u003etraining (ExT) groups. B indicates p\u0026lt;0.05 ZT12 versus ZT0 and ZT18 in NT group. * Indicates p\u0026lt;0.05 NT versus ExT group. Comparison by two-way ANOVA followed by Sidak’s test. NR = non rhythmic, p \u0026gt; 0.05 by Cosinor analysis.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6606119/v1/a62c93b3026b314f2aaf5b7c.png"},{"id":83023717,"identity":"e69d9632-7671-4aae-ad19-6b3226f712cc","added_by":"auto","created_at":"2025-05-19 08:00:14","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":22419,"visible":true,"origin":"","legend":"\u003cp\u003eDopaminergic activity expressed as Mean±SEM of the ratios DOPAC/DA measured in the caudate putamen (a), pre-optic area (c) and paraventricular nucleus (d) microdissection obtained from rats after the last recovery day. Dashed gray lines mark the time of training session throughout the 5-day/week exercise section. Adjusting cosinor curves are shown for circadian variations, which acrophase is indicated by the dotted lines parallel to Y axis, mesor by dotted lines parallel to X axis, and amplitude by the arrow. The gray shaded areas correspond to the dark phase (ZT12=19:00 to ZT0=07:00). A indicates p\u0026lt;0.05 for ZT12 versus ZT0 in the non-training (NT) group. * Indicates p\u0026lt;0.05 for NT versus exercise training (ExT) groups. Comparison by two-way ANOVA followed by Sidak’s test. NR = non rhythmic, p \u0026gt; 0.05 by Cosinor analysis.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6606119/v1/4fb7ae722541ff7326ab3ffe.png"},{"id":83023716,"identity":"7efb07af-0b37-4363-b480-be3c7c9f5c68","added_by":"auto","created_at":"2025-05-19 08:00:14","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":22956,"visible":true,"origin":"","legend":"\u003cp\u003eSerotonergic activity expressed as Mean±SEM of the ratios 5HIAA/5-HT measured in the caudate putamen (a), pre-optic area (c) and paraventricular nucleus (d) microdissection obtained from rats after the last recovery day. Adjusting cosinor curves are shown for circadian variations, which acrophase is indicated by the dotted lines parallel to Y axis, mesor by dotted lines parallel to X axis, and amplitude by the arrows. The gray shaded areas correspond to the dark phase (ZT12=19:00 to ZT0=07:00). * Indicates p\u0026lt;0.05 for NT versus exercise training (ExT) groups. Letter A indicates temporal differences in NT group. Comparison by two-way ANOVA followed by Sidak’s test. NR = non rhythmic, p \u0026gt; 0.05 by Cosinor analysis.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6606119/v1/462d70aa6a5791095f373fd0.png"},{"id":95040013,"identity":"f874019a-0dc8-4d93-b76f-ff7a006d275e","added_by":"auto","created_at":"2025-11-03 16:07:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":890247,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6606119/v1/912f8ee1-e814-4f69-a2a4-965c51cd3cb7.pdf"},{"id":83022511,"identity":"738a1c5e-8a67-4a72-b49c-c3de9b3e114a","added_by":"auto","created_at":"2025-05-19 07:52:14","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":173251,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalData.docx","url":"https://assets-eu.researchsquare.com/files/rs-6606119/v1/5431388f43b63f6972690dee.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Circadian Adaptations to Regular Exercise Training in the Treadmill Alter Temporal Changes in Dopamine and Serotonin Activity in Brain Areas","fulltext":[{"header":"Introduction","content":"\u003cp\u003eModernity has introduced a more efficient manipulation of environmental factors. At first, the artificial electric light led to an authentic \u0026ldquo;colonization\u0026rdquo; of the night by human beings. This process occurred despite the diurnal characteristics developed by the species throughout its relatively short evolutionary process. Nowadays, the possibility of regular work to take place at times more appropriate to rest represents a novelty with possible behavioral, biological and health repercussions [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn fact, most of these recently incorporated behavioral lifestyles have been associated with a higher prevalence of cardiovascular, metabolic, and immunological diseases [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In this regard, regular physical activity, although a factor with less potential for entrainment compared to light, might act as a source of temporal information about thermic and energetic demands, and consequently, it affects the maintenance of the homeostasis [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. However, the implications of the amount of physical activity as well as its acute and cumulative effects on the internal clock are still poorly understood.\u003c/p\u003e \u003cp\u003eAdditionally, regular physical activity could overcome the ordinary oscillations evoking a transient disruption in the circadian time system, despite triggering acute and sometimes extreme biological responses. For example, the circadian patterns of physical activity are coupled to heat production, increasing internal body temperature (Tbody), which is central to circadian thermal balance [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThus, a better understanding of the relationship between homeostatic adjustments and temporal signals would help to develop strategies to prevent or treat conditions that are potentially harmful to well-being, improving the quality of life of the general population.\u003c/p\u003e \u003cp\u003eIn general, possible entraining effects of the circadian clock by physical activity could be mediated by several factors directly related to the exercise-adaptive stimuli. Increased T\u003csub\u003ebody\u003c/sub\u003e and corticosterone could act as resetting cues evoked by exercise metabolic/energy demand [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Glucocorticoids are one of the main temporal agents through which the suprachiasmatic nucleus (SCN), the main pacemaker for circadian control, communicates with the various peripheral tissues [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. On the other hand, T\u003csub\u003ebody\u003c/sub\u003e circadian rhythm, which is driven by SCN, induces phase-coherence in individual oscillators adjusting the phases between the central (extra-SCN) and peripheral clocks [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn fact, connection between thermoregulatory nuclei in the forebrain, such as the hypothalamic paraventricular nucleus (PVN) and the preoptic area (POA) points towards a complementary role of these regions in the control of daily rhythms [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], which could also apply to the adaptive responses to exercise training. As such, by promoting comparable and transient increases in T\u003csub\u003ebody\u003c/sub\u003e and glucocorticoid levels, physical activity could indirectly produce time signals relevant to the system, potentially triggering a phase readjustment response. Therefore, the present work investigated the effects of 8 weeks of scheduled physical exercise training during the first hours of the light-phase on forebrain monoamines and peripheral components of the circadian temporal system in rats.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003ch2\u003eEthical statements\u003c/h2\u003e\n\u003cp\u003eThe experimental procedures described in the present study were approved by the Ethics Committee on the Use of Animals (protocol n. 57/2014, CEUA/UFMG).\u003c/p\u003e\n\u003ch2\u003eExperimental Subjects\u003c/h2\u003e\n\u003cp\u003eExperimental procedures involved weaning male Wistar rats (4-weeks old) from the Animal Facility of the Institute of Biological Sciences in the Federal University of Minas Gerais (CEBIO/ICB/UFMG). The animals were maintained in collective cages (3-5/cages), under a 12/12-hour light/dark cycle (lights-on at 7:00/lights-off at 19:00) and room temperature of 24 \u0026plusmn; 1\u0026deg;C, with free access to standard chow and water.\u003c/p\u003e\n\u003ch2\u003eExperimental procedures\u003c/h2\u003e\n\u003cp\u003eTo evaluate the relationship between physical training-induced adaptations and adjustments of circadian parameters, animals were randomly allocated to 2 experimental groups: non- trained (NT) and trained (ExT). As previously described [34], after familiarization to treadmill running (5 days, 15 m/min, 5 min, constant slope of 5\u0026deg;), animals trained on a treadmill for 8 weeks (5 days/week, 10-25 m/min, 30-60 min, constant slope of 5\u0026deg;) with the occurrence of physical tests at the beginning, at the middle and end of the 4th week (T4)\u003cs\u003e\u0026nbsp;\u003c/s\u003eand at the end of the 8th week. The exercise procedure was performed between 11:00 and 13:00 h (ZT4 and ZT6, respectively). This time schedule was chosen based on a phase response curve for treadmill\u0026nbsp;exercise performed between the ZT4 and 6 in which there was a greater entraining effect of the exercise on circadian muscle clock [44].\u003c/p\u003e\n\u003cp\u003eIn the 7\u003csup\u003eth\u003c/sup\u003e week, animals from both trained and untrained groups received an internal temperature and spontaneous locomotor activity telemetric probe for monitoring T\u003csub\u003ebody\u003c/sub\u003e and LA continuously at the last week of the protocol. The variation of T\u003csub\u003ebody\u003c/sub\u003e between NT and ExT group during the treadmill running can be detected in the sessions, as it does not depend on the movement direction, and it is presented in the results. However, the variation of SLA during the exercise session was not presented because SLA detected does not represent the movement due to exercise but the vector of the movement in the treadmill. Food and water intake values were daily registered at ZT0 and ZT12, as ways to control food consumption according to light/dark cycles. At least 48 hours after the last maximal capacity test, the animals (with and without sensors) were euthanized at 5 different times of the day: ZT0 (07:00h), ZT4 (11:00h), ZT6 (13:00h), ZT12 (19:00h) and ZT18 (01:00h). Trunk blood was collected for hormonal measurements; the \u003cem\u003egastrocnemius\u003c/em\u003e muscle was obtained to determine the expression of \u003cem\u003ePer1\u003c/em\u003e; brain tissue was removed to measure monoamine in response to exercise training. In the dark phase (ZT18), euthanasia occurred under red lights (\u0026lt;1lux). The animals were weighed once every week at ZT4.\u003c/p\u003e\n\u003ch3\u003eTreadmill exercise training\u003c/h3\u003e\n\u003cp\u003eThe scheduled exercise training protocol consisted of running on a treadmill for 8 weeks at a frequency of 5 days a week. The intensity and duration of the exercise were gradually increased until the animals reached a speed of 25 m/min, with a 5\u0026ordm; inclination of the treadmill, for 60 minutes. The training sessions occurred between ZT4 and ZT6, as described above. To\u0026nbsp;assure similar animal handling, the animals in the NT group performed a simulation of exercise on the treadmill during the same training period, with a speed of 15 m/min, but lasting only 5 min/day, twice a week.\u003c/p\u003e\n\u003ch3\u003eGraded Intensity Exercise Protocol\u003c/h3\u003e\n\u003cp\u003eThe exercise protocol with progressive intensity until fatigue consisted of a running on a treadmill with a constant inclination of 5\u0026ordm; and an initial speed of 10 m/min with increases of 1 m/min every 3 minutes until the moment when the animals interrupted the exercise or were no longer able to maintain running speed for 10 seconds. The tests took place with room temperature maintained at 24 \u0026deg;C. Core T\u003csub\u003ebody\u003c/sub\u003e and a tail skin temperature (T\u003csub\u003esk\u003c/sub\u003e) were also registered.\u003c/p\u003e\n\u003ch3\u003eSpontaneous Locomotor Activity and Body Temperature Monitoring\u003c/h3\u003e\n\u003cp\u003eAnimals from each group were intraperitoneally implanted with a telemetry sensor (model G2 E-Mitter, Mini-Mitter Company, Sun River, OR, USA) during sedation and anesthesia with ketamine (Ketamine 10%, Syntec) and xylazine (Xilasina 2%, Agener Uni\u0026atilde;o). After laparotomy of approximately 2 cm over the \u003cem\u003elinea alba\u003c/em\u003e of the \u003cem\u003erectus abdominis\u003c/em\u003e muscle, the sensors were fixed internally to the abdominal wall. The wound was then sutured and rats received prophylactic treatment with pentabiotic (Pentabi\u0026oacute;tico Veterin\u0026aacute;rio\u0026reg;, Fort Dodge Sa\u0026uacute;de, Animal Ltda; dose, intramuscular) and analgesic (Banamine\u0026reg;, Schering-Plow, S/A, dose, subcutaneous). After 48 hours, animals recovered from surgery presented regular body weight, food intake, general coat characteristics and activity in the cage. Besides, only animals that were fully recovered continued the training protocol.\u003c/p\u003e\n\u003cp\u003eAfter recovery, animals were transferred to individual boxes placed on individual recipient plates (ER-4000 Energizer/Receiver, Mini-Mitter Company, Sun River, OR, USA) connected in series with a computer containing the data acquisition software (VitalView Data Acquisition System Software v.4.0, Mini-Mitter Company, Sun River, OR, USA). According to information from the manufacturer, the accuracy of the system for temperature monitoring is \u0026plusmn; 0.1 \u0026deg;C. Spontaneous locomotor activity is a representative measure of each spatial displacement of the sensor (E-mitter) in the longitudinal, horizontal and transverse axes on each receiver plate. Each of these measures was accumulated at the recording intervals determined in each experiment and expressed in arbitrary units (au). For results analysis purposes, only data generated after the initial 12 hours of registration were used for the studied variables.\u003c/p\u003e\n\u003ch3\u003eCorticosterone assessment\u003c/h3\u003e\n\u003cp\u003ePlasma corticosterone levels were determined by enzyme-linked immunosorbent assay (ELISA) using commercially available kits (Cayman Chemical Company, Ann Arbor, MI, USA). All samples were evaluated in duplicates following the manufacturer\u0026apos;s specifications.\u003c/p\u003e\n\u003ch3\u003eCatecholamine assessment\u003c/h3\u003e\n\u003cp\u003eBrain levels of dopamine (DA), serotonin (5-HT) and their respective metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and 5-hydroxyindole-3-acetic acid (5-HIAA) [20] were determined in the caudate putamen (CP), preoptic area (POA), and hypothalamic paraventricular nucleus (PVN). Immediately after euthanasia, the animals\u0026apos; brains were carefully removed and frozen on dry ice and then stored in at -80 \u0026deg;C until they were analyzed by high-performance liquid chromatography with electrochemical detection (HPLC-ED). Using Paxinos \u0026amp; Watson\u0026apos;s Atlas as a reference [42], the brains were sectioned into coronal slices of 800 \u0026mu;m with a cryostat (Leica CM1850, Heidelberger, Germany). Using the punch method, it was possible to extract the regions corresponding to the \u003cem\u003enuclei/\u003c/em\u003eareas of interest with a 2.0-mm needle used bilaterally for the CP (2,20 to 0,48 mm from Bregma) and centrally (in relation to the 3rd ventricle) for the POA (0,48 to -0,48 mm from Bregma) and PVN (-0,96 to -1,92 mm from Bregma). Punchs were then homogenized in a solution of 0.15 M perchloric acid and 0.1 M EDTA plus the internal standard 3.4-dihydrobenzylamine (DHBA, Sigma Aldrich, Milwaukee, United States). Protein levels were determined by the Bradford method. The supernatant was injected into the chromatography system (Shimadzu, Kyoto, Japan). This system is formed by a C-18 column (Purospher 5m, Merck, Darmstadt, Germany) preceded by a C-18 guard column. The potential in the electrochemical detector (Decade 2; Antec Scientific, the Netherlands) was set to +0.40 V \u003cem\u003eversus\u003c/em\u003e Ag/AgCl reference electrode. The mobile phase of the system was composed of 100 mM NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 10 mM NaCl, 0.1 nM EDTA, 0.38 mM octanosulfonic acid, and 10% methanol in water. The pH was adjusted to 3.5 with 85% orthophosphoric acid and the solution was pumped at a flow rate of 1 ml/min. Peaks were identified based on their retention times and quantified using the internal standard method. All samples evaluated were measured in the same assay. The intra-assay coefficient of variation was less than 5% for all measured compounds. DA and 5-HT levels are considered to reflect neurotransmitter content in synaptic vesicles, whereas DOPAC and 5-HIAA reflect neurotransmitter release in tissue homogenates [20, 21]. DOPAC/DA and 5-HIAA/5-HT ratios were calculated as an index of neurotransmitter turnover and used to estimate changes in the activity of dopaminergic and serotoninergic terminals, respectively.\u003c/p\u003e\n\u003ch3\u003eGene expression assessment\u003c/h3\u003e\n\u003cp\u003eAfter euthanasia, the tissues were quickly removed and frozen in a container containing liquid nitrogen (gastrocnemius) or dry ice (brain). The samples were stored at -80 \u0026deg;C until they were processed for analysis of the expression of genes of interest. Total RNA was extracted with Trizol (Invitrogen, California, United States) according to the manufacturer\u0026apos;s instructions, or in conjunction with the High Pure RNA Tissue Kit for RNA extraction (Roche Diagnostics, Mannheim, Germany).\u003c/p\u003e\n\u003cp\u003eThe concentration of RNA was determined by reading the absorbance at 260 nm and 230 nm on a spectrophotometer (Nanodrop, Thermo Scientific, Wilmington, United States). Only samples with 260/230 ratio readings between 1.8 and 2.2 were used for subsequent reactions. The reverse transcriptase reaction was performed with 2\u0026mu;g of total RNA from the gastrocnemius samples, using 2\u0026mu;L of random oligonucleotides (50 ng/\u0026mu;L) and 2 uL of dNTPs (10 mM), 8 \u0026mu;L of buffer of 5X PCR, 2 \u0026micro;L of 10 mM DTT, 2 \u0026micro;L of the 40 U/\u0026micro;L ribonuclease inhibitor, and 2 \u0026micro;L of Superscript III 200 U/\u0026micro;L, ending a volume of 40 \u0026micro;L per reaction. The synthesized cDNA was used in subsequent quantitative PCR reactions (in real time).\u003c/p\u003e\n\u003cp\u003eThe following primers were used to determine mRNA expression of the interest gene: \u003cem\u003ePer1\u003c/em\u003e (NM_001034125.1; forward primer 5\u0026rsquo;-ATGCAGAAACAACAGCCACGGTTC-3\u0026rsquo;, reverse primer 5\u0026rsquo;-TGGCCAGGATCTTGAACACTGCTA-3\u0026rsquo;) and \u003cem\u003eBmal1\u003c/em\u003e (NM_024362.2; Forward: 5\u0026rsquo; - AGG CCT TCA CTG GAA TGG TGC TAT - 3\u0026rsquo;, Reverse: 5\u0026rsquo;- tga ctg gcc tgg aac ttg cta cat - 3\u0026rsquo;). The normalizer used in this study was the 18S ribosomal RNA (Forward: 5\u0026rsquo; - CGG CTA CCA CAT CCA AGG AA - 3\u0026rsquo;, Reverse: 5\u0026rsquo; - GCT GGA ATT ACC GCG GCT - 3\u0026rsquo;). Solutions containing primers, SYBR\u0026reg; GreenER \u0026trade; qPCR SuperMix universal (Life Technologies, USA) and H2O DNaseRNase-free (Life Technologies, USA), obtaining a final concentration of 300 \u0026eta;M for the primers of the gene of interest and 50 \u0026eta;M for the primers of RNA 18S (normalizer), in independent solutions. Each solution was aliquoted (48 \u0026mu;L, each aliquot sufficient for 2 wells) in tubes and the cDNA of each sample added (2 \u0026mu;L/aliquot). The solutions already with cDNA were then distributed in their respective wells of the experimental plate (23 \u0026mu;L/well) and taken to the thermocycler (ABI-PRISMA 7900) for reaction in the following conditions: 2 min at 50 \u0026deg;C, 8:30 min at 95 \u0026deg;C , followed by 45 cycles of 15 s at 95 \u0026deg;C, 1 min at 60 \u0026deg;C, 1 min at 95 \u0026deg;C, 1 min at 55 \u0026deg;C and 80 cycles of 10 s to 55 \u0026deg;C, with a gradual increase of 0.5 \u0026deg;C. Data analysis was performed by comparing the number of copies of the control and experimental wells, or between different times after normalization by the expression level of the reference gene.\u003c/p\u003e\n\u003ch2\u003eData Analysis\u003c/h2\u003e\n\u003cp\u003eThe differences between performance variables over 8 weeks were evaluated using two-way ANOVA followed by a post-hoc test to characterize the differences between the experimental groups. The daily oscillations of variables of interest were determined by two-way ANOVA for each protocol. A COSINOR analysis was applied to evaluate the oscillation of T\u003csub\u003ebody\u003c/sub\u003e and LA and the effect of training. Results were expressed as the mean \u0026plusmn; SEM. Statistical significance was set at \u003cem\u003ep\u0026lt;0.05\u003c/em\u003e. The graph prism pad software was used for graphs and statistical analysis.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eTraining load characteristics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe training load used in this protocol was characterized by progressive increases in running speed (Figure 1a) accompanied by regular increases in the duration of the sessions over the first 4 weeks (Figure 1b), with the subsequent stabilization in 60 minutes in the final 4 weeks of the training protocol in the ExT group. The association between intensity and volume of the load resulted in a progressive increase in the training load per session, estimated through the workload calculation (Figure 1c).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of scheduled exercise training on T\u003csub\u003ebody\u003c/sub\u003e and SLA circadian rhythms\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExercise training differently changed the daily curves of T\u003csub\u003ebody\u003c/sub\u003e and SLA recorded in the last week of regular treadmill running (Figure 2). Despite the transient increase in T\u003csub\u003ebody\u003c/sub\u003e during the physical exercise sessions (change T\u003csub\u003ebody\u003c/sub\u003e=2.02 \u0026plusmn; 0.15 ℃; \u003cem\u003ep\u0026lt;0.0001\u003c/em\u003e) (Figure 2a and 2c), there were no differences in the circadian rhythm of T\u003csub\u003ebody\u0026nbsp;\u003c/sub\u003e(Figure 2e). In contrast to T\u003csub\u003ebody\u003c/sub\u003e, exercise training increased the SLA in the end of dark phase of circadian cycle (Figure 2d). Cosinor analysis showed that exercise training increased the mesor and amplitude of SLA circadian rhythm (Figure 2f). Also, it promoted a delay in the acrophase of SLA rhythm (Figure 2f).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of scheduled exercise training on temporal changes of corticosterone plasma levels and muscle clock genes expression\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe highest levels of corticosterone were found at ZT12 in both NT and ExT animals (Figure 3a). Analysis of Cosinor showed that those corticosterone temporal changes fitted in a cosine curve, which parameters (mesor, amplitude and acrophase) did not differ between NT and ExT groups (Figure 3b).\u003c/p\u003e\n\u003cp\u003eThe highest concentrations of \u003cem\u003ePer1\u003c/em\u003e mRNA in the gastrocnemius muscle were found in the ZT12 in NT animals, whereas no temporal differences were found in ExT group (Figure 3c). Analysis of Cosinor showed that muscle \u003cem\u003ePer1\u003c/em\u003e expression temporal changes fitted in a cosine curve in NT animals but not in the ExT (Figure 3d). Although ZT has significantly affected muscle \u003cem\u003eBmal1\u003c/em\u003e expression in NT and ExT groups, post-hoc analysis did not identify differences among evaluated times (Figure 3e), consequently non-fitting curve in \u003cem\u003eBmal1\u003c/em\u003e mRNA data was detected by Cosinor Analysis in both NT and ExT (Figure 3f).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of scheduled exercise training on temporal changes of dopaminergic activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDOPAC/DA ratio did not change in function of the time of the day nor exercise training in the CP (Figure 4a). Therefore, non-fitting curve in dopamine activity data was detected by Cosinor Analysis in both NT and ExT (Figure 4b). Nevertheless, in the POA from NT animals, dopamine activity did show temporal changes, which higher ratio of DOPAC/DA found at ZT12 compared to other ZTs (Figure 4c). \u0026nbsp;Those data fitted in a cosine curve as shown in Figure 4d. Exercise training reduced the DOPAC/DA ratio at ZT6 compared to NT groups (Figure 4c) and no temporal differences in dopamine activity were found in ExT group, consequently data did not fit in a cosine curve (Figure 4d). In the PVN neither time-of-day or groups (NT or ExT) affected the dopamine activity. As such no temporal changes were found in both groups (Figure 4e), consequently data did not fit in a cosine curve (Figure 4f).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of scheduled exercise training on temporal changes of serotonergic activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e5HIAA/5HT ratio did not change in function of the time of the day or exercise training in the CP area (Figure 5a). Therefore, non-fitting curve in dopamine activity data was detected by Cosinor Analysis in both NT and ExT (Figure 5b). On the other hand, in the POA, exercise training attenuated the 5HIAA/5HT ratio in ZT6 (Figure 5c). There were no temporal differences in the POA 5HIAA/5HT ratio from ExT animals (Figure 5c). In fact, COSINOR analysis revealed that ExT abolished 5HIAA/5HT fitting curve (Figure 5d).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe present study demonstrated that exercise training influences the circadian temporal system and monoaminergic activity in the POA and PVN. For the first time, it has been shown that a scheduled exercise that mimics human training i.e. training session 5 days a week with a 2-days recovering period was able to modulate circadian rhythms. \u0026nbsp;This protocol of exercise training increases SLA quantity at the last hours of the dark phase, which lead to an increased mesor, amplitude and a delayed acrophase in SLA rhythm. On the other hand, the daily hyperthermia observed in the training session did not promote alterations in T\u003csub\u003ebody\u003c/sub\u003e rhythm. Therefore, the circadian entrainment in SLA and T\u003csub\u003ebody\u003c/sub\u003e rhythms seems to independently respond to exercise training. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn the present study, the training model increased physical performance and improved thermal balance (Supplementary Figure 1). These results confirm that physical training triggers an increase in physical performance, accompanied by improvements in energy efficiency and also in thermoregulatory capacity [21, 35, 40, 41]. In turn, T\u003csub\u003ebody\u003c/sub\u003e rhythm maintained stability, independent of the exercise sessions themselves, reinforcing the idea of adaptation in thermal efficiency.\u003c/p\u003e\n\u003cp\u003eIn our study, the comparison was between a progressive training protocol to a very mild walking on the treadmill. This approach is more translational as the training was compared to a non-sedentary condition. \u0026nbsp;Moreover, it controlled for a possible effect of exposure to a different environment, which is known to affect the circadian time systems [43,44]. The chosen protocol may explain the very mild effect of training exercise to the overall rhythms. It is known that time of exercise is capable of entraining SLA rhythm. As such, regularly scheduled exposure to running wheels or to treadmill clearly entrains the circadian rhythm of SLA of mice under constant darkness condition [44]. Comparing our findings with this previous study, it seems that exercise training did not overcome the effect of light/dark cycles, and it was unable to re-entrain the light/dark-synchronized rhythms of SLA and T\u003csub\u003ebody\u003c/sub\u003e.\u003c/p\u003e\n\u003cp\u003eOn the other side of daily balance, exercise training increased baseline locomotor activity. Such an increase was not only observed as a result of the exercise section (acute effect) but also during the dark-phase, indicating that SLA rhythm might be reactive to chronic changes in general physical activity. This has been extensively reported in the literature [8, 9, 28, 29, 33, 34, 36, 39, 45, 46]. In this perspective, during the second half of the dark-phase, the increased locomotor activity in the trained animals\u0026nbsp;could represent a temporal redistribution\u0026nbsp;in response to the recently incorporated schedule that regularly amplified energy demand associated with the beginning of the light-phase. With the regularity of the physical demand, the system would readjust to meet the temporal characteristics of the environment in the most efficient way possible, with minimum disturbances of homeostasis. Therefore, the apparent mismatch between locomotor activity and internal temperature responses to exercise could be attributed both to the higher energy efficiency of the system, resulting in higher levels of spontaneous locomotor activity for the same level of internal temperature.\u003c/p\u003e\n\u003cp\u003eOf note, there was hyperthermia induced by the training session that did not lead to alterations in T\u003csub\u003ebody\u003c/sub\u003e rhythm. \u0026nbsp;Nevertheless, it was observed an increase in \u003cem\u003ePer1\u0026nbsp;\u003c/em\u003eexpression in the gastrocnemius muscles previously to training session time. This suggested that the daily increase in T\u003csub\u003ebody\u003c/sub\u003e induced by exercise may signal to peripheral tissues in an attempt to synchronize the circadian clocks, without alterations in T\u003csub\u003ebody\u003c/sub\u003e rhythm. \u003cem\u003ePer1\u003c/em\u003e belongs to the core component of the negative loop of the canonical molecular circadian clock. In rats, \u003cem\u003ePer1\u003c/em\u003e peaks at the beginning of the dark-phase (ZT12) and reaches its lowest levels at the onset of light-phase (ZT0) [16, 30, 31]. However, ExT animals failed to show that pattern, which led to non-rhythm profile of \u003cem\u003ePer1\u003c/em\u003e expression. It has been proposed that the expression of this gene could be associated with synchronizing properties of locomotor activity [18], internal temperature, and corticosterone [4, 14, 18]. Importantly, locomotor activity, under the influence of the SCN, is functionally associated with the activity of skeletal musculature [17, 18]. Consequently, it is speculated that the increase in locomotor activity in association with higher T\u003csub\u003ebody\u003c/sub\u003e could be directly associated with the altered\u0026nbsp;expression of \u003cem\u003ePer1\u003c/em\u003e, indicating a readjustment in the muscle clock, directly affected by physical training during the light-phase. The effect of exercise on \u003cem\u003ePer1\u003c/em\u003e was not accompanied by alterations in \u003cem\u003eBmal1\u003c/em\u003e muscle expression. Future experiments should be conducted to evaluate the other clock genes.\u003c/p\u003e\n\u003cp\u003eGlucocorticoids are potent endogenous agents for modulating the activity of the peripheral biological clock [1,7,26]. Such temporal signaling could be linked to episodes of increase in corticosterone secretion in response to exercise. \u0026nbsp;In fact, Sasaki et al. (2016) demonstrated that forced exercise, on the running-wheel or treadmill, in the resting phase promotes phase advances in peripheral clocks associated with an increase in corticosterone levels [35]. These authors also demonstrated that the peripheral effects would depend on the joint action of corticosterone and noradrenergic signaling [35]. Nevertheless, our exercise protocol did not affect the daily pattern of corticosterone blood level, which is similar to other studies showing that corticosterone returns to its initial levels after 4-6 weeks of voluntary running, indicating adaptation of hypothalamus-pituitary-adrenal (HPA) axis [6, 26, 27, 38, 45]. Circulating corticosterone levels constitute one of the phase-signaling components under the control of the central pacemaker [19, 35], which may be subjected to adaptations. Therefore, there may be a two-step adaptive process for exercise training: in the initial phase, resetting the circadian clock would depend on glucocorticoid and neural adjustments; in the subsequent phase, maintaining the regular stimulus would result in a predominance of a neural component, possibly involving cerebral areas that regulate T-body, and/or SLA and physical capacity, and glucocorticoid secretion.\u003c/p\u003e\n\u003cp\u003eThe perspective of a neural modulation of the observed adaptations was challenged by analysis of monoaminergic activity in areas that participate in thermoregulation and HPA axis, i.e., POA and PVN, respectively, in addition to caudate putamen that is involved to physical exercise. There is an increased dopaminergic activity in the \u003cem\u003ecaudate putamen\u003c/em\u003e in association with an increase in physical capacity [2, 11, 46], and vice-versa, animals with greater intrinsic aerobic capacity show greater DA concentrations and activity of DA in brain areas directly involved with motor control [25, 29, 37]. Thus, it can be hypothesized that physical training can trigger both an increase in dopaminergic activity in the nigrostriatal pathway, normally associated with the increase in the capacity to resist fatigue [11]. Temporal adjustment in the variation of dopaminergic activity would facilitate the beginning of the training session. In the present study, dopaminergic activity in the CP was not significantly changed by physical training, indicating that SLA rhythm responses to exercise could be under the regulation of other brain centers.\u003c/p\u003e\n\u003cp\u003eAdditionally, it was observed that the training program attenuated both dopaminergic and serotonergic activity in the POA under baseline conditions. Untrained animals showed increased monoaminergic activity throughout the light-phase, reaching a peak in the second half of the light-phase until the beginning of the dark-phase. This profile was not seen in exercised animals, which showed a reduction in monoaminergic activity in the POA at ZT6. Such reduction may be explained by an increase in inhibitory input from the median preoptic area that has been previously shown to be linked with temperature regulation [15]. This phase-specific reduction in POA dopaminergic activity may be responsible for the adjustment of the system during the active phase of the cycle.\u003c/p\u003e\n\u003cp\u003ePhysical training program influenced 5-HT activity in PVN. If, in untrained animals, the 5-HIAA/5-HT ratio exhibited a peak at the beginning of the activity-phase, in trained animals, the 24h curve was abolished. Training increased PVN serotonin activity during the transition from dark to light phases (ZT0). This temporal relationship is like that observed for SLA levels at the end of the dark phase and at the second peak of\u003cem\u003e\u0026nbsp;Per1\u0026nbsp;\u003c/em\u003eexpression in ZT0. It is recognized that the PVN would constitute an important site of integration for the response to different environmental stimuli [9, 10, 19]. It is interesting to note that this hypothalamic nucleus receives dense noradrenergic projections from the locus coeruleus in the brain stem, a \u003cem\u003enucleus\u003c/em\u003e directly related to the modulation of the arousal system [24]. It also receives serotonergic inputs from raphe and other regions that participate in the modulation of the activity state [18]. Thus, the change in the circadian variation of serotonergic activity in PVN associated with increased locomotor activity provides compelling evidence that this nucleus is part of the extra-SCN control axis of locomotor activity and under the influence of regular exercise training.\u003c/p\u003e\n\u003cp\u003eIn conclusion, the study demonstrated that regularly scheduled physical exercise promotes adaptations at different levels of the circadian time system. Within the central nervous system, regular exercise modulated the daily serotonin and dopamine activity in nuclei directly related with internal temperature control, motor control and the alarm/arousal system. Thus, the circadian time system seems to be affected by exercise-related entraining time cues. As demonstrated by physical exercise on a treadmill, regularity and routine lead the system to a new state of balance, configuring an important tool for non-pharmacological and non-photic regulation of disorders usually associated with the imbalance of the circadian clock.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eHuman Ethics: \u0026ldquo;not applicable\u0026rdquo;\u003c/p\u003e\n\u003cp\u003eConsent for publication: \u0026ldquo;not applicable\u0026rdquo;\u003c/p\u003e\n\u003cp\u003eFunding: The authors acknowledge the financial support for F.S.M.M. in part by the Coordena\u0026ccedil;\u0026atilde;o de Aperfei\u0026ccedil;oamento de Pessoal de N\u0026iacute;vel Superior - Brasil (CAPES) \u0026ndash; CAPES/PRINT (Grant number : 88887.364935/2019-00). This study received financial support by Conselho Nacional de Desenvolvimento Cient\u0026iacute;fico e Tecnol\u0026oacute;gico (CNPq) and Funda\u0026ccedil;\u0026atilde;o de Amparo \u0026agrave; Pesquisa do Estado de Minas Gerais (FAPEMIG).\u003c/p\u003e\n\u003cp\u003eAuthors\u0026apos; contributions: FSMM, MOP, CCC conceived the study. \u0026nbsp;FSMM, MOP, and CCC analyzed and cured data. MOP and CCC acquired funding for the study. Methodology: FSMM, NACH, and QTR (Handling animals); NACH, and TSRC (RT-qPCR); FSMM (hormone assay); NSSA and RES (HPLC measurements); FSMM, NACH, and MOP (Cosinor analysis). Roles/Writing\u0026mdash; original draft: FMSS, MOP, and CCC; Writing\u0026mdash;review \u0026amp; editing: FSMM, NACH, QTR, TSRC, NSSA, RES, MOP, and CCC.\u003c/p\u003e\n\u003cp\u003eAvailability of supporting data: Raw data are available under requesting.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCompeting interests: The authors declare no competing interests\u003c/p\u003e\n\u003cp\u003eClinical Trial Number in the manuscript: \u0026ldquo;not applicable\u0026rdquo;\u003c/p\u003e\n\u003cp\u003eEthical statements: The experimental animal procedures described in the present study were approved by the Ethics Committee on the Use of Animals (protocol n. 57/2014, CEUA/UFMG)\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBalsalobre A, Brown SA, Marcacci L, Tronche F, Kellendonk C, Reichardt HM, Sch\u0026uuml;tz G, Schibler U (2000) Resetting of Circadian Time in Peripheral Tissues by Glucocorticoid Signaling. Science 289:2344\u0026ndash;2347. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1126/science.289.5488.2344\u003c/span\u003e\u003cspan address=\"10.1126/science.289.5488.2344\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBalthazar CH, Leite LHR, Ribeiro RMM, Soares DD, Coimbra CC (2010) Effects of blockade of central dopamine D1 and D2 receptors on thermoregulation, metabolic rate and running performance. Pharmacological Reports 62:54\u0026ndash;61. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/s1734-1140(10)70242-5\u003c/span\u003e\u003cspan address=\"10.1016/s1734-1140(10)70242-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrown SA, Zumbrunn G, Fleury-Olela F, Preitner N, Schibler U (2002) Rhythms of Mammalian Body Temperature Can Sustain Peripheral Circadian Clocks. Current Biology 12:1574\u0026ndash;1583. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/s0960-9822(02)01145-4\u003c/span\u003e\u003cspan address=\"10.1016/s0960-9822(02)01145-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBuhr ED, Yoo S-H, Takahashi JS (2010) Temperature as a Universal Resetting Cue for Mammalian Circadian Oscillators. Science 330:379\u0026ndash;385. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1126/science.1195262\u003c/span\u003e\u003cspan address=\"10.1126/science.1195262\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBuijs RM, Kalsbeek A (2001) Hypothalamic integration of central and peripheral clocks. Nature Reviews Neuroscience 2:521\u0026ndash;526. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/35081582\u003c/span\u003e\u003cspan address=\"10.1038/35081582\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCampbell JE, Rakhshani N, Fediuc S, Bruni S, Riddell MC (2009) Voluntary wheel running initially increases adrenal sensitivity to adrenocorticotrophic hormone, which is attenuated with long-term training. Journal of Applied Physiology 106:66\u0026ndash;72. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1152/japplphysiol.91128.2008\u003c/span\u003e\u003cspan address=\"10.1152/japplphysiol.91128.2008\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChung S, Son GH, Kim K (2011) Adrenal peripheral oscillator in generating the circadian glucocorticoid rhythm. Annals of the New York Academy of Sciences 1220:71\u0026ndash;81. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/j.1749-6632.2010.05923.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1749-6632.2010.05923.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEdgar DM, Dement WC (1991) Regularly scheduled voluntary exercise synchronizes the mouse circadian clock. American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 261:R928\u0026ndash;R933. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1152/ajpregu.1991.261.4.r928\u003c/span\u003e\u003cspan address=\"10.1152/ajpregu.1991.261.4.r928\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEdgar DM, Reid MS, Dement WC (1997) Serotonergic afferents mediate activity-dependent entrainment of the mouse circadian clock. American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 273:R265\u0026ndash;R269. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1152/ajpregu.1997.273.1.r265\u003c/span\u003e\u003cspan address=\"10.1152/ajpregu.1997.273.1.r265\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFetissov SO, Meguid MM, Chen C, Miyata G (2000) Synchronized release of dopamine and serotonin in the medial and lateral hypothalamus of rats. Neuroscience 101:657\u0026ndash;663. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/s0306-4522(00)00374-2\u003c/span\u003e\u003cspan address=\"10.1016/s0306-4522(00)00374-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFoley T, Greenwood B, Day H, Koch L, Britton S, Fleshner M (2006) Elevated central monoamine receptor mRNA in rats bred for high endurance capacity: Implications for central fatigue. Behavioural Brain Research 174:132\u0026ndash;142. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.bbr.2006.07.018\u003c/span\u003e\u003cspan address=\"10.1016/j.bbr.2006.07.018\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGerhart-Hines Z, Lazar MA (2015) Circadian metabolism in the light of evolution. Endocrine Reviews 36:289\u0026ndash;304. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1210/er.2015-1007\u003c/span\u003e\u003cspan address=\"10.1210/er.2015-1007\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGoh GH, Mark PJ, Blache D, Binks D, Parsons R, Rawashdeh O, Maloney SK (2021) Diet-altered body temperature rhythms are associated with altered rhythms of clock gene expression in peripheral tissues in vivo. Journal of Thermal Biology 100:102983. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.jtherbio.2021.102983\u003c/span\u003e\u003cspan address=\"10.1016/j.jtherbio.2021.102983\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGotic I, Schibler U (2017) Posttranscriptional mechanisms controlling diurnal gene expression cycles by body temperature rhythms. RNA Biology 14:1294\u0026ndash;1298. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1080/15476286.2017.1285481\u003c/span\u003e\u003cspan address=\"10.1080/15476286.2017.1285481\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuzm\u0026aacute;n-Ruiz MA, Ramirez-Corona A, Guerrero-Vargas NN, Sabath E, Ramirez-Plascencia OD, Fuentes-Romero R, Le\u0026oacute;n-Mercado LA, Basualdo Sigales M, Escobar C, Buijs RM (2015) Role of the Suprachiasmatic and Arcuate Nuclei in Diurnal Temperature Regulation in the Rat. The Journal of Neuroscience 35:15419\u0026ndash;15429. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1523/jneurosci.1449-15.2015\u003c/span\u003e\u003cspan address=\"10.1523/jneurosci.1449-15.2015\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHorta NAC, Fernandes P, Cardoso TSR, Machado FSM, Drummond LR, Coimbra CC, Wanner SP, Maria L Castrucci A, Poletini MO (2023) TRPV1 inactivation alters core body temperature and serum corticosterone levels: Impacts on clock genes expression in the liver and adrenal glands. Journal of Thermal Biology 114:103514. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.jtherbio.2023.103514\u003c/span\u003e\u003cspan address=\"10.1016/j.jtherbio.2023.103514\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHouben T, Deboer T, van Oosterhout F, Meijer JH (2009) Correlation with Behavioral Activity and Rest Implies Circadian Regulation by SCN Neuronal Activity Levels. Journal of Biological Rhythms 24:477\u0026ndash;487. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1177/0748730409349895\u003c/span\u003e\u003cspan address=\"10.1177/0748730409349895\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHughes ATL, Piggins HD (2012) Feedback actions of locomotor activity to the circadian clock. In: Progress in Brain Research. Elsevier, pp 305\u0026ndash;336\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKalsbeek A, Liu J, Lei J, Timmermans L, Foppen E, Cailotto C, Fliers E (2012) Differential Involvement of the Suprachiasmatic Nucleus in Lipopolysaccharide-Induced Plasma Glucose and Corticosterone Responses. Chronobiology International 29:835\u0026ndash;849. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3109/07420528.2012.699123\u003c/span\u003e\u003cspan address=\"10.3109/07420528.2012.699123\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLookingland KJ, Jarry HD, Moore KE (1987) The metabolism of dopamine in the median eminence reflects the activity of tuberoinfundibular neurons. Brain Research 419:303\u0026ndash;310. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/0006-8993(87)90597-x\u003c/span\u003e\u003cspan address=\"10.1016/0006-8993(87)90597-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMachado FSM, F\u0026oacute;scolo DRC, Poletini MO, Coimbra CC (2016) Influence of Time-of-Day on Maximal Exercise Capacity Is Related to Daily Thermal Balance but Not to Induced Neuronal Activity in Rats. Frontiers in Physiology 7. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fphys.2016.00464\u003c/span\u003e\u003cspan address=\"10.3389/fphys.2016.00464\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMachado FSM, Rodovalho GV, Coimbra CC (2015) The time of day differently influences fatigue and locomotor activity: Is body temperature a key factor? Physiology \u0026amp; Behavior 140:8\u0026ndash;14. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.physbeh.2014.11.069\u003c/span\u003e\u003cspan address=\"10.1016/j.physbeh.2014.11.069\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMachado FSM, Zhang Z, Su Y, de Goede P, Jansen R, Foppen E, Coimbra CC, Kalsbeek A (2018) Time-of-Day Effects on Metabolic and Clock-Related Adjustments to Cold. Frontiers in Endocrinology 9. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fendo.2018.00199\u003c/span\u003e\u003cspan address=\"10.3389/fendo.2018.00199\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMastorakos G, Pavlatou M (2005) Exercise as a Stress Model and the Interplay Between the Hypothalamus-pituitary-adrenal and the Hypothalamus-pituitary-thyroid Axes. Hormone and Metabolic Research 37:577\u0026ndash;584. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1055/s-2005-870426\u003c/span\u003e\u003cspan address=\"10.1055/s-2005-870426\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMathes WF, Nehrenberg DL, Gordon R, Hua K, Garland T Jr, Pomp D (2010) Dopaminergic dysregulation in mice selectively bred for excessive exercise or obesity. Behavioural Brain Research 210:155\u0026ndash;163. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.bbr.2010.02.016\u003c/span\u003e\u003cspan address=\"10.1016/j.bbr.2010.02.016\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOtawa M, Arai H, Atomi Y (2007) Molecular aspects of adrenal regulation for circadian glucocorticoid synthesis by chronic voluntary exercise. Life Sciences 80:725\u0026ndash;731. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.lfs.2006.10.023\u003c/span\u003e\u003cspan address=\"10.1016/j.lfs.2006.10.023\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePastore S, Hood DA (2013) Endurance training ameliorates the metabolic and performance characteristics of circadian \u003cem\u003eClock\u003c/em\u003e mutant mice. Journal of Applied Physiology 114:1076\u0026ndash;1084. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1152/japplphysiol.01505.2012\u003c/span\u003e\u003cspan address=\"10.1152/japplphysiol.01505.2012\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePendergast JS, Branecky KL, Huang R, Niswender KD, Yamazaki S (2014) Wheel-running activity modulates circadian organization and the daily rhythm of eating behavior. Frontiers in Psychology 5. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fpsyg.2014.00177\u003c/span\u003e\u003cspan address=\"10.3389/fpsyg.2014.00177\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRabelo PCR, Almeida TF, Guimar\u0026atilde;es JB, Barcellos LAM, Cordeiro LMS, Moraes MM, Coimbra CC, Szawka RE, Soares DD (2015) Intrinsic exercise capacity is related to differential monoaminergic activity in the rat forebrain. Brain Research Bulletin 112:7\u0026ndash;13. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.brainresbull.2015.01.006\u003c/span\u003e\u003cspan address=\"10.1016/j.brainresbull.2015.01.006\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eReznick J, Preston E, Wilks DL, Beale SM, Turner N, Cooney GJ (2013) Altered feeding differentially regulates circadian rhythms and energy metabolism in liver and muscle of rats. Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease 1832:228\u0026ndash;238. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.bbadis.2012.08.010\u003c/span\u003e\u003cspan address=\"10.1016/j.bbadis.2012.08.010\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSaini C, Morf J, Stratmann M, Gos P, Schibler U (2012) Simulated body temperature rhythms reveal the phase-shifting behavior and plasticity of mammalian circadian oscillators. Genes \u0026amp; Development 26:567\u0026ndash;580. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1101/gad.183251.111\u003c/span\u003e\u003cspan address=\"10.1101/gad.183251.111\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSalgado-Delgado R, \u0026Aacute;ngeles-Castellanos M, Buijs MR, Escobar C (2008) Internal desynchronization in a model of night-work by forced activity in rats. Neuroscience 154:922\u0026ndash;931. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.neuroscience.2008.03.066\u003c/span\u003e\u003cspan address=\"10.1016/j.neuroscience.2008.03.066\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSalgado-Delgado R, Angeles-Castellanos M, Saderi N, Buijs RM, Escobar C (2010) Food Intake during the Normal Activity Phase Prevents Obesity and Circadian Desynchrony in a Rat Model of Night Work. Endocrinology 151:1019\u0026ndash;1029. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1210/en.2009-0864\u003c/span\u003e\u003cspan address=\"10.1210/en.2009-0864\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSantiago HP, Leite LH, Lima PMA, Rodovalho GV, Szawka RE, Coimbra CC (2015) The improvement of exercise performance by physical training is related to increased hypothalamic neuronal activation. Clinical and Experimental Pharmacology and Physiology 43:116\u0026ndash;124. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/1440-1681.12507\u003c/span\u003e\u003cspan address=\"10.1111/1440-1681.12507\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSasaki H, Hattori Y, Ikeda Y, Kamagata M, Iwami S, Yasuda S, Tahara Y, Shibata S (2016) Forced rather than voluntary exercise entrains peripheral clocks via a corticosterone/noradrenaline increase in PER2::LUC mice. Scientific Reports 6. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/srep27607\u003c/span\u003e\u003cspan address=\"10.1038/srep27607\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSato RY, Yamanaka Y (2023) Nonphotic entrainment of central and peripheral circadian clocks in mice by scheduled voluntary exercise under constant darkness. American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 324:R526\u0026ndash;R535. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1152/ajpregu.00320.2022\u003c/span\u003e\u003cspan address=\"10.1152/ajpregu.00320.2022\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSaul MC, Majdak P, Perez S, Reilly M, Garland T Jr, Rhodes JS (2016) High motivation for exercise is associated with altered chromatin regulators of monoamine receptor gene expression in the striatum of selectively bred mice. Genes, Brain and Behavior 16:328\u0026ndash;341. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/gbb.12347\u003c/span\u003e\u003cspan address=\"10.1111/gbb.12347\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchroeder AM, Truong D, Loh DH, Jordan MC, Roos KP, Colwell CS (2012) Voluntary scheduled exercise alters diurnal rhythms of behaviour, physiology and gene expression in wild-type and vasoactive intestinal peptide‐deficient mice. The Journal of Physiology 590:6213\u0026ndash;6226. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1113/jphysiol.2012.233676\u003c/span\u003e\u003cspan address=\"10.1113/jphysiol.2012.233676\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShannon NJ, Gunnet JW, Moore KE (1986) A Comparison of Biochemical Indices of 5-Hydroxytryptaminergic Neuronal Activity Following Electrical Stimulation of the Dorsal Raphe Nucleus. Journal of Neurochemistry 47:958\u0026ndash;965. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/j.1471-4159.1986.tb00704.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1471-4159.1986.tb00704.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThompson PD, Crouse SF, Goodpaster B, Kelley D, Moyna N, Pescatello L (2001) The acute versus the chronic response to exercise. Medicine and Science in Sports and Exercise 33:S438\u0026ndash;S445. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1097/00005768-200106001-00012\u003c/span\u003e\u003cspan address=\"10.1097/00005768-200106001-00012\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWaters RP, Pringle RB, Forster GL, Renner KJ, Malisch JL, Garland Jr. T, Swallow JG (2013) Selection for increased voluntary wheel-running affects behavior and brain monoamines in mice. Brain Research 1508:9\u0026ndash;22. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.brainres.2013.01.033\u003c/span\u003e\u003cspan address=\"10.1016/j.brainres.2013.01.033\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWatson C, Kirkcaldie M, Paxinos G (2010) Preface. In: The Brain. Elsevier, p VII\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWebb IC, Antle MC, Mistlberger RE (2014) Regulation of circadian rhythms in mammals by behavioral arousal. Behavioral Neuroscience 128:304\u0026ndash;325. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1037/a0035885\u003c/span\u003e\u003cspan address=\"10.1037/a0035885\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWolff G, Esser KA (2012) Scheduled Exercise Phase Shifts the Circadian Clock in Skeletal Muscle. Medicine \u0026amp; Science in Sports \u0026amp; Exercise 44:1663\u0026ndash;1670. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1249/mss.0b013e318255cf4c\u003c/span\u003e\u003cspan address=\"10.1249/mss.0b013e318255cf4c\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYasumoto Y, Nakao R, Oishi K (2015) Free Access to a Running-Wheel Advances the Phase of Behavioral and Physiological Circadian Rhythms and Peripheral Molecular Clocks in Mice. PLOS ONE 10:e0116476. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1371/journal.pone.0116476\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0116476\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZheng X, Hasegawa H (2016) Administration of caffeine inhibited adenosine receptor agonist-induced decreases in motor performance, thermoregulation, and brain neurotransmitter release in exercising rats. Pharmacology Biochemistry and Behavior 140:82\u0026ndash;89. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.pbb.2015.10.019\u003c/span\u003e\u003cspan address=\"10.1016/j.pbb.2015.10.019\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"pflugers-archiv-european-journal-of-physiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"paej","sideBox":"Learn more about [Pflügers Archiv - European Journal of Physiology](http://link.springer.com/journal/424)","snPcode":"424","submissionUrl":"https://submission.nature.com/new-submission/424/3","title":"Pflügers Archiv - European Journal of Physiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"physical activity, entrainment, circadian, dopamine, serotonin, thermoregulation, locomotor activity","lastPublishedDoi":"10.21203/rs.3.rs-6606119/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6606119/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eRegular exercise's beneficial effects may arise from regulating circadian rhythm. Here, we investigated how regular exercise training affects spontaneous locomotor activity (SLA) and body temperature (T-body) rhythms and its influence on temporal changes in brain dopaminergic and serotonergic activity, corticosterone levels, and muscle \u003cem\u003ePer1\u003c/em\u003e and \u003cem\u003eBmal1\u003c/em\u003e gene expression. The aerobic exercise schedule consisted of five days of training in the light phase (Zeitgeber time\u0026thinsp;=\u0026thinsp;ZT4 to ZT6, with light on at 7:00 h) followed by a two-day recovery period. After eight weeks, brain, blood, and muscle samples were collected from adult male rats. Dopamine (DA) and serotonin (5-HT) and their respective metabolites, DOPAC and 5-HIAA, were measured in microdissections of the caudate putamen (CP), preoptic area (POA), and the paraventricular nucleus of the hypothalamus (PVN). Exercise increased the SLA at the end of the night, delayed the acrophase, and increased the mesor of the SLA rhythm. No alterations were found in the T-body rhythm and corticosterone blood levels, although hyperthermia was observed after exercise sessions. Exercise increased muscle \u003cem\u003ePer1\u003c/em\u003e expression at ZT0, leading to a non-rhythmic profile in exercised animals. There were no changes in the CP dopaminergic and serotonergic activity, but a decrease in POA at ZT6, and an increase in PVN serotonergic activity at ZT0, resulting in a non-rhythmic profile in exercised animals. Thus, regular physical exercise during the light-phase with alterations in SLA promotes adjustments in the daily oscillation in monoaminergic activity in areas directly involved in regulating daily T-body and SLA.\u003c/p\u003e","manuscriptTitle":"Circadian Adaptations to Regular Exercise Training in the Treadmill Alter Temporal Changes in Dopamine and Serotonin Activity in Brain Areas","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-19 07:52:09","doi":"10.21203/rs.3.rs-6606119/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-19T13:39:28+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-19T08:46:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"331836787221286122783366673207021124995","date":"2025-06-17T23:30:42+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-17T20:17:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"59640593527523396783172212485091760031","date":"2025-06-02T16:16:37+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-15T07:31:08+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-14T15:24:16+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-14T15:23:17+00:00","index":"","fulltext":""},{"type":"submitted","content":"Pflügers Archiv - European Journal of Physiology","date":"2025-05-06T19:56:45+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"pflugers-archiv-european-journal-of-physiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"paej","sideBox":"Learn more about [Pflügers Archiv - European Journal of Physiology](http://link.springer.com/journal/424)","snPcode":"424","submissionUrl":"https://submission.nature.com/new-submission/424/3","title":"Pflügers Archiv - European Journal of Physiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"1262018a-9e1a-4864-aa19-60723f35dd3a","owner":[],"postedDate":"May 19th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-11-03T16:02:49+00:00","versionOfRecord":{"articleIdentity":"rs-6606119","link":"https://doi.org/10.1007/s00424-025-03128-x","journal":{"identity":"pflugers-archiv-european-journal-of-physiology","isVorOnly":false,"title":"Pflügers Archiv - European Journal of Physiology"},"publishedOn":"2025-10-30 15:58:21","publishedOnDateReadable":"October 30th, 2025"},"versionCreatedAt":"2025-05-19 07:52:09","video":"","vorDoi":"10.1007/s00424-025-03128-x","vorDoiUrl":"https://doi.org/10.1007/s00424-025-03128-x","workflowStages":[]},"version":"v1","identity":"rs-6606119","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6606119","identity":"rs-6606119","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}