Orexinergic activation in the ventrolateral periaqueductal gray contributes to voluntary exercise-induced hypoalgesia in neuropathic pain model mice

Orexinergic activation in the ventrolateral periaqueductal gray contributes to voluntary exercise-induced hypoalgesia in neuropathic pain model mice | 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 Article Orexinergic activation in the ventrolateral periaqueductal gray contributes to voluntary exercise-induced hypoalgesia in neuropathic pain model mice Shogo Habata, Katsuya Kami, Kohei Minami, Takuma Kami, Yasunori Umemoto, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7382525/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Voluntary exercise is known to alleviate chronic pain, yet the underlying neural mechanisms, including the descending pain inhibitory system, remain incompletely understood. As orexin is recognized as a neuromodulator involved in pain regulation and projects widely throughout the central nervous system, we investigated the role of orexinergic signaling in the ventrolateral periaqueductal gray (vlPAG) in the induction of exercise-induced hypoalgesia (EIH) using a neuropathic pain mouse model. Mice with free access to running wheels exhibited significantly improved mechanical and thermal pain thresholds compared to sedentary mice, and this pain relief positively correlated with running distance. Immunohistochemical analyses revealed increased orexin immunoreactivity and neuronal activation in the vlPAG following voluntary running (VR). Although dopaminergic neuron numbers in the vlPAG were low, their activation was significantly enhanced after VR, whereas GABAergic neurons showed only minimal activation. These findings suggest that VR may promote EIH via activation of orexinergic projections from the lateral hypothalamus to the vlPAG, potentially engaging descending pain inhibitory pathways. Our results indicate a novel neural mechanism underlying EIH via orexin signaling and support voluntary exercise as a promising non-pharmacological strategy for managing chronic neuropathic pain. Biological sciences/Neuroscience Biological sciences/Physiology Exercise-induced hypoalgesia descending pain inhibitory system orexin PAG neuropathic pain voluntary running Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction The pain pathways include not only ascending pathways that transmit nociceptive signals from the periphery to the central nervous system, but also descending pathways that can modulate pain transmission before the signals reach the brain. In the descending pathway, the periaqueductal gray (PAG) in the midbrain plays a crucial role in suppressing pain signals and is therefore considered the center of the descending pain inhibitory system. In particular, the ventrolateral PAG (vlPAG) contains dopaminergic (DA) [ 1 ], glutamatergic (Glu) [ 2 ], and γ-aminobutyric acid (GABAergic) [ 3 ] neurons. DA neurons in the vlPAG are implicated in pain modulation; for example, chemical lesions of DA neurons in the PAG attenuate opioid-induced analgesia [ 4 ], whereas direct injection of DA agonists into the vlPAG [ 5 ] or pharmacogenetic stimulation of DA neurons in the vlPAG [ 6 ] produces pain relief. Activated Glu neurons produce analgesic effects but can also induce anxiety- and fear-related behaviors [ 6 ]. GABAergic neurons are interneurons that inhibit projection neurons [ 7 – 9 ] and express µ-opioid receptors that target the rostral ventromedial medulla (RVM) [ 10 , 11 ]. Because chemogenetic inhibition of GABAergic neurons by opioids induces analgesia [ 11 , 12 ], these neurons may contribute to disinhibition of projection neurons. The PAG sends axons to the RVM, and this pathway suppresses the transmission of nociceptive signals in the spinal dorsal horn. The raphe magnus nucleus in the RVM contains abundant serotonergic neurons, including off-, on-, and neutral-cells[ 13 ]. Off-cells mainly inhibit spinal pain processing [ 14 ] but may also facilitate pain under certain conditions [ 15 ]. These findings suggest that distinct neuronal subtypes in the vlPAG may differentially contribute to descending pain modulation, underscoring the need for further investigation into the specific roles of DA, Glu, and GABAergic neurons in both the chronicity and relief of pain. Many studies have demonstrated the effectiveness of exercise in alleviating and improving chronic pain. A key phenomenon underlying exercise-related pain relief is termed exercise-induced hypoalgesia (EIH), which has been recognized as a basis for exercise therapy in chronic pain. One proposed mechanism of EIH involves the reward system [ 16 , 17 ]. In addition, exercise has been shown to activate the descending pain inhibitory system via endogenous opioids [ 18 ] and the endocannabinoid 2-arachidonoylglycerol (2-AG) [ 19 ] in the PAG, and to increase cannabinoid 1 and 2 receptor (CB1 and CB2 receptor) expression in the PAG [ 20 ], even in a Parkinson’s disease model rat [ 21 ]. Activation of opioid and CB1 receptors on presynaptic GABAergic terminals in the PAG inhibits GABA release [ 22 , 23 ]. The RVM transmits 5-HT to the spinal dorsal horn, contributing to both pain facilitation and inhibition [ 24 ]. Exercise increases 5-HT levels in both the RVM [ 25 ] and the spinal dorsal horn [ 26 ], potentially contributing to analgesia by reducing pain sensitivity in the spinal dorsal horn. Collectively, these findings suggest that the descending pain inhibitory system is a key mechanism promoting the EIH effect. Orexin, also known as hypocretin, is an excitatory neuropeptide that exists in two forms: orexin A (OXA) and orexin B (OXB). Orexin neurons are localized in the lateral hypothalamic area (LHA) and project their fibers widely throughout the central nervous system [ 27 , 28 ]. Two types of orexin receptors, orexin receptor 1 (OX1R) and orexin receptor 2 (OX2R), are expressed in most brain regions in mice, including the PAG, and are present on both Glu and GABAergic neurons [ 29 ]. Few studies have directly confirmed the expression of orexin receptors on DA neurons within the PAG. Orexin is involved in multiple physiological functions, including pain regulation. For example, orexin neurons projecting to the PAG inhibit GABAergic neurons via increased synthesis of 2-AG through the phospholipase C–diacylglycerol lipase alpha (C–DAGLα) pathway, thereby producing antinociception [ 30 ]. Pain-related behaviors are enhanced by selective ablation of orexin neurons in mice, whereas pharmacogenetic activation of these neurons induces analgesia [ 31 , 32 ]. Activation of orexin neurons through a designer receptor exclusively activated by designer drugs approach attenuates thermal hyperalgesia and significantly reduces paw-licking behavior in the formalin test [ 33 ]. Furthermore, intraperitoneal injection of an OX1R antagonist (SB-334867) enhances formalin-induced nociceptive behaviors in adult male rats [ 34 ]. These findings demonstrate that orexin plays a critical role in pain regulation, contributing to both analgesic mechanisms and nociceptive processing. However, no study has directly examined whether exercise increases orexin projections to the vlPAG, although exercise has been reported to increase OXA levels in the hypothalamus [ 16 , 35 ] and in the cerebrospinal fluid (CSF) of rats [ 36 ] and dogs [ 37 ]. Because orexin neurons project to the vlPAG, it is conceivable that exercise-induced activation of these neurons enhances orexin input to the vlPAG, thereby activating the descending pain inhibitory system and promoting the EIH effect. Orexin neurons in the LHA are activated by exercise and are believed to be involved in pain relief; however, their contribution to the EIH effect has not yet been fully elucidated. Therefore, in this study, we examined orexin expression in the vlPAG following voluntary exercise in a mouse model of neuropathic pain. Furthermore, we investigated which neuronal subtypes in the vlPAG are activated by voluntary exercise. These findings provide novel insights into the brain mechanisms underlying the EIH effect. Results Changes of running distances and pain behaviors Figure 1 A shows the protocol for voluntary wheel running. Mice were individually housed in cages with a running wheel, allowing voluntary running for 2 weeks before and after PSL or Sham surgery. Naive-Runner mice were allowed to run for 4 weeks. For consistency with the Sham- and PSL-Runner groups, the 4-week running period was divided into two phases: 2 weeks before and 2 weeks after a designated reference day, corresponding to the day of surgery in the other groups. In this scheme, the day before the reference day was defined as pre-1 day, and the day after as post-1 day. Figure 1 B shows changes in daily running distance in Naive-, Sham-, and PSL-Runner mice. On pre-14 day, mean running distances were 4,101 ± 629 m/day in the Naive-Runner group, 3,322 ± 340 m/day in the Sham-Runner group, and 2,342 ± 561 m/day in the PSL-Runner group, with no statistically significant difference among groups (p = 0.069, one-way ANOVA). Running distances gradually increased, reaching 2–3 times the initial values by pre-1 day. On that day, mean running distances were 11,057 ± 1,378 m/day in the Naive-Runner group, 9,075 ± 995 m/day in the Sham-Runner group, and 11,187 ± 1,059 m/day in the PSL-Runner group, with no statistically significant difference among groups. In Sham-Runner mice, running distance decreased to 5,582 ± 888 m/day on post 1 day but returned to pre-surgery levels by post 14 day (8,666 ± 843 m/day; 95.5% recovery). In contrast, PSL-Runner mice showed a marked decrease to 5,060 ± 768 m/day on post-1 day, with only partial recovery to 51.4% of pre-surgery levels (5,746 ± 968 m/day on post-14 day). From post 1 day to post 14 day, PSL-Runner mice exhibited significantly lower daily running distances than both Naive- and Sham-Runner mice (time effect: F (5.992, 223.5) = 35.19, p < 0.001; surgery effect: F (2, 38) = 3.249, p < 0.05; interaction: F (54, 1007) = 6.990, p < 0.001). To evaluate the effects of VR on pain behaviors, plantar and von Frey tests were performed (Fig. 2 ). For the plantar test, there were significant effects of experimental group (F (5, 324) = 136.7, p < 0.0001), time (F (8, 324) = 28.59, p < 0.0001), and their interaction (F (40, 324) = 12.15, p < 0.0001). Similarly, for the von Frey test, significant effects were found for experimental group (F (5, 315) = 147.5, p < 0.0001), time (F (8, 315) = 21.17, p < 0.0001), and their interaction (F (40, 315) = 11.36, p < 0.0001). Regardless of running, there was no difference between the Naive and Sham groups throughout the study period in either the plantar or von Frey test. PSL-Sedentary and PSL-Runner mice showed markedly shorter withdrawal latencies at 1 day after PSL (PSL-Sedentary: 4.06 ± 0.19 s; PSL-Runner: 4.32 ± 0.14 s) compared to pre-surgery levels (PSL-Sedentary: 11.36 ± 0.52 s; PSL-Runner: 11.51 ± 0.43 s), and lower mechanical thresholds on the same day (PSL-Sedentary: 0.24 ± 0.03 g vs. 1.23 ± 0.09 g; PSL-Runner: 0.22 ± 0.03 g vs. 1.20 ± 0.01 g). These decreased latencies and thresholds persisted after PSL in PSL-Sedentary mice. Conversely, withdrawal latencies and mechanical thresholds in PSL-Runner mice were significantly increased on day 14 after PSL compared with PSL-Sedentary mice (PSL-Runner: 7.92 ± 0.24 s and 0.69 ± 0.06 g; PSL-Sedentary: 4.27 ± 0.32 s and 0.24 ± 0.02 g; p < 0.0001 for both). Collectively, these findings confirm that the VR protocol significantly improved pain behaviors in the NPP model. Figure 2 C and 2 D show the relationship between pain thresholds at 14 days post-PSL and total running distances over the 14-day period after PSL in PSL-Runner mice. Significant positive correlations were observed (plantar test: r = 0.812, p = 0.0499, n = 6; von Frey test: r = 0.837, p = 0.0378, n = 6). These results suggest that higher levels of voluntary running are associated with greater improvements in pain-related behaviors in the NPP model. VR increases orexin projections Previous studies have shown that exercise increases orexin levels in the cerebrospinal fluid of rats [ 36 ], cats [ 38 ], and dogs [ 37 ], as well as in human plasma [ 39 ]. In addition, immunohistochemical observations have indicated significant increases in orexin neuron activity in NPP model mice following VR [ 16 ] and in healthy mice following treadmill running [ 35 ]. Based on these findings, we investigated orexinergic activity in the vlPAG using immunohistochemical staining (Fig. 3 ). The total immunoreactive area size of orexinA in each group was as follows: Naive-Sedentary: 283.5 ± 41.1 µm²; Naive-Runner: 579.5 ± 70.3 µm²; Sham-Sedentary: 258.8 ± 42.9 µm²; Sham-Runner: 548.5 ± 80.0 µm²; PSL-Sedentary: 209.1 ± 29.1 µm²; PSL-Runner: 547.0 ± 80.3 µm². There was no statistically significant difference between ipsilateral and contralateral sides; therefore, data from both sides were combined for analysis. The combined data revealed significant differences between exercise and non-exercise groups (Naive: p = 0.0006; Sham: p = 0.0008; PSL: p = 0.0001). Figure 3 F shows the relationship between orexinA and total running distances over the 14-day period after PSL in PSL-Runner mice. A significant positive correlation was observed (r = 0.836, p = 0.0380, n = 6). These results suggest that higher levels of VR are associated with enhanced orexinergic activity in the vlPAG, even under neuropathic pain conditions. VR induces activation of neurons in the vlPAG As described above, orexinergic projections to the vlPAG were greater in mice that performed VR. To identify the activated cell type, neurons or glial cells, we performed immunostaining with anti-NeuN antibody (Fig. 4 ). Approximately 100–200 NeuN-positive neurons were identified within the defined area of the vlPAG, and VR did not alter the number of NeuN-positive neurons. There were no significant differences between ipsilateral and contralateral sides; therefore, data from both sides were combined for analysis. To evaluate the effects of VR, statistical comparisons were performed between exercise and non-exercise groups within each experimental condition. Significant differences were found in all groups (Naive: p = 0.0006; Sham: p = 0.0042; PSL: p < 0.0001). Figure 4 F shows the relationship between the ratio of activated neurons (NeuN⁺ FosB⁺ / NeuN⁺) and total running distances over the 14-day period after PSL in PSL-Runner mice. A significant positive correlation was observed (r = 0.8944, p = 0.0161, n = 6). These results suggest that higher VR activity enhances neuronal activation in the vlPAG under neuropathic pain conditions, which may play a critical role in exercise-induced pain modulation. Activated dopaminergic neurons were increased by VR Neurons in the PAG utilize various neurotransmitters such as glutamate, GABA, and glycine, and express a range of neuropeptides including somatostatin, substance P, dynorphin, neurotensin, and others. We examined whether DA neurons in the vlPAG are involved in the induction of EIH using immunohistochemical staining. TH-positive neurons represent DA neurons [ 40 ], and cells indicated by arrows in Fig. 5 represent activated DA neurons, as shown by FosB and TH double-positive immunoreactivities. Examination of DA neuron distribution in the vlPAG revealed that they were located near the midbrain aqueduct, with FosB-positive cells densely distributed in the surrounding region. The ratio of activated DA neurons (TH + FosB + / TH + ) in ipsilateral and contralateral sides showed no statistical difference in each group; therefore, data from both sides were combined for subsequent analysis. Statistical comparisons between exercise and non-exercise groups within each experimental condition revealed significant differences in all groups (Naive: p = 0.0412, Sham: p = 0.0166, PSL: p < 0.0064). These results suggest that VR enhances activation of DA neurons in the vlPAG, which may contribute to EIH. No alterations in the number and activation of GABA neurons GABAergic interneurons express µ-opioid receptors and inhibit activation of neurons projecting to the RVM in the vlPAG [ 8 , 9 ]. Inhibition of GABAergic signaling in the PAG suppresses pain, whereas activation enhances nociceptive transmission. Opioids relieve this GABAergic inhibition, leading to disinhibition of output neurons and resulting in analgesia [ 11 , 12 ]. Therefore, we assessed changes in their number and activation after VR (Fig. 6 ). Only a few GAD67-positive cells, approximately one to four per section, were detected, and only very few showed FosB immunoreactivity. No statistically significant differences were found in any group. These findings indicate that VR does not induce significant changes in the number or activation of GABAergic neurons in the vlPAG. Discussion The present study examined orexinergic activation and neuronal subtypes activated in the vlPAG following voluntary exercise in neuropathic pain model mice. Our results indicated that (i) VR alleviated pain-related behaviors, (ii) orexinergic projections and neuronal activity in the vlPAG increased along with activation of dopaminergic neurons, and (iii) neither the number nor the activity of GABAergic neurons showed significant changes. These findings suggest that orexin may play an important role in producing the EIH effect via activation of the descending pain inhibitory system. In this study, when we evaluated the effects of VR on pain behavior in NPP model mice, VR produced analgesic effects consistent with the development of EIH. Some studies have reported greater running distances compared to the present study [ 16 ]. This difference may result from variations in the environment in which VR was performed. It has been reported that in an enriched acoustic environment, animals’ activity levels are elevated compared to those observed under conventional laboratory housing conditions [ 41 ]. Thus, environmental factors, particularly ambient noise and housing conditions such as the number of mice, should be considered when examining pain behaviors with VR. PSL-Sedentary mice exhibited decreased pain thresholds, which were improved by VR (PSL-Runner). Furthermore, we found positive correlations between thermal withdrawal latencies or mechanical thresholds and total running distances in PSL-Runner mice, suggesting that greater VR activity further enhances the EIH effect. Although reliable evidence directly linking longer exercise duration to stronger analgesic effects is still lacking, in humans, the EIH effect following a single session typically lasts only 30 seconds to 30 min. However, regular exercise may induce adaptations within pain modulation systems, potentially leading to more consistent or pronounced EIH effects over time [ 42 ]. In contrast to forced exercise, such as treadmill running, wheel running reflects environmental enrichment. Mice have free access to the wheel and can run voluntarily. Recent findings indicate that brain regions activated differ according to the type of exercise [ 43 ]. Treadmill running may preferentially activate brain regions associated with stress, fear, and pain. The optimal type, intensity, duration, and frequency of exercise for developing the EIH effect remain unclear, as these parameters are influenced by factors such as nociceptive, neuropathic, or nociplastic pain, as well as psychological factors like fear and anxiety, and social factors such as loneliness. Recent studies have shown that chemogenetic stimulation of the primary motor cortex can suppress negative emotions and maladaptive coping behaviors in neuropathic pain while also alleviating pain by reducing sensory hypersensitivity [ 44 ]. Therefore, to optimize the EIH effect, it is important to ensure adequate exercise in low-stress environments, that is, exercise should be voluntary rather than forced, noise should be minimal, lighting should be non-intense, and animals should be housed in appropriately sized groups. Our findings indicate that VR enhances orexinergic projections to the vlPAG, with orexin levels in this region positively correlating with physical activity. Orexin is produced in the LHA and projects to multiple brain regions. Previous studies have shown that exercise increases orexin levels in the LHA [ 16 , 35 ]. Wu et al. reported a positive association between exercise and CSF orexin levels [ 37 ], and Kiwaki et al. demonstrated that OXA injection into the hypothalamic paraventricular nucleus significantly increases physical activity [ 45 ]. Immunohistochemical studies using FosB [ 16 ] and c-Fos [ 46 ] have indicated that exercise activates orexin neurons in the LHA, whereas ablation of these neurons decreases physical activity [ 47 ]. In the diet-induced obesity model mice, obesity-prone individuals exhibit lower spontaneous physical activity and reduced expression of orexin receptors in the rostral lateral hypothalamus, where orexin exerts its effects [ 48 ]. The present study demonstrated increased orexin immunoreactivity in the vlPAG following VR. Collectively, these findings suggest that physical activity enhances orexinergic signaling in the vlPAG, which may mediate the analgesic benefits of exercise through the EIH effect. In this study, double-staining with DAPI and NeuN revealed a large population of neurons in the vlPAG, and these neurons were significantly activated in response to VR. To identify specific neuronal subtypes, we performed TH immunostaining, which revealed a relatively small population of dopaminergic neurons. However, the proportion of activated dopaminergic neurons was significantly higher in the exercise group than in the sedentary group. GAD67 immunostaining revealed very few activated GABAergic neurons. Previous studies have shown that the vlPAG contains a heterogeneous neuronal population, classified by firing patterns [ 22 , 49 ], neurotransmitter phenotypes, and functional roles. Glycinergic neurons, identified by glycine transporter 2 (GlyT2) expression, are considered a subset of inhibitory neurons alongside GABAergic neurons. Glutamatergic neurons are known to contribute to pain relief in both anxiety-dependent [ 6 ] and anxiety-independent contexts [ 50 ]. Recently, in chronic unpredictable mild stress model mice, dopaminergic neurons activated via chemogenetic methods were reported to attenuate thermal pain and depressive behaviors [ 51 ]. In the present study, although the total number of dopaminergic neurons in the vlPAG was relatively small, the overall findings suggest that glutamatergic neurons in this region may play a more prominent role in producing the EIH effect promoted by elevated orexin levels in neuropathic pain. However, we could not directly confirm glutamatergic neurons using appropriate markers, which is a limitation of the present study. Based on the present findings, we propose a potential neuronal mechanism in which orexin activates the vlPAG following VR, thereby producing the EIH effect. First, VR activates orexinergic neurons in the LHA, which project to the vlPAG and activate glutamatergic neurons projecting to the RVM. These events may ultimately engage the descending pain inhibitory system (Fig. 6 E). Thus, the descending analgesic pathway activated by these systems may contribute to the EIH effect. In addition, other mechanisms, such as the mesolimbic system, including the VTA and NAc [ 16 , 52 ], or cognitive and emotional pain-processing regions, such as the anterior cingulate cortex [ 53 ], may also contribute to the development of EIH because orexin neurons project broadly throughout the brain. The present findings provide novel insights into the neurobiological basis of EIH and highlight the benefits of voluntary exercise, a widely recognized low-stress, non-pharmacological strategy for improving chronic neuropathic pain. Further studies integrating molecular, genetic, and behavioral approaches will be essential to fully elucidate the mechanisms underlying EIH and to optimize exercise-based therapeutic interventions. Methods Animals All animal experiments were approved by the Institutional Animal Care and Use Committee of Wakayama Medical University (approval number: 1124) and conducted in accordance with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines [ 54 ]. All procedures conformed to the National Institutes of Health Guide for the Care and Use of Laboratory Animals, 8th edition. Male C57BL/6JJcl mice aged 8–13 weeks were used in this study. C57BL/6J mice were purchased from CLEA Japan, Inc. (Tokyo, Japan). Each mouse was housed in a plastic cage and maintained in a holding room with a 12 h light–dark cycle, with free access to food and water ad libitum. All experimental procedures included appropriate measures to relieve suffering and pain. Preparation of NPP model mice Neuropathic pain (NPP) model mice were prepared by partial sciatic nerve ligation as previously reported [ 55 ]. Under deep isoflurane anesthesia, the right mid-thigh level of the sciatic nerve was tightly ligated with 8 − 0 silk sutures. Sham operations were performed using the same procedures described above, excluding PSL. After surgery, the mice were individually monitored in cages and returned to their regular cages upon waking. No postoperative treatment was administered. Notably, mice did not exhibit any symptoms such as reduced movement, weight loss, swelling, excessive inflammation, or suppuration. Experimental groups and the VR protocol The mice were assigned to six groups as follows: (1) Naive-Sedentary (n = 14), (2) Naive-Runner (n = 15), (3) Sham-Sedentary (n = 12), (4) Sham-Runner (n = 16), (5) PSL-Sedentary (n = 16), and (6) PSL-Runner (n = 14). Each group was further divided into two subgroups to conduct two types of pain behavior tests. Naive, Sham, and PSL-Runner mice were allowed unrestricted access to a running wheel. Given the importance of early-stage running exercise in inducing a significant EIH effect after nerve injury, mice were immediately returned to cages with running wheels following PSL surgery, allowing voluntary running. All mice, except Naive mice, underwent VR for two weeks prior to Sham or PSL surgery. After surgery, PSL and Sham mice were transferred to individual cages, and their running distances were recorded over a 14-day period before and after surgery. Wheel rotations per hour were monitored using a magnetic reed switch connected to a computerized exercise-monitoring system (SOF-860 wheel manager software, MED Associates, Inc.), and daily running distances (meters/day) were calculated from wheel rotations. Evaluation of pain behavior To eliminate diurnal variations in pain behavior, plantar and von Frey tests were performed at 5:00 p.m. Before each test, mice were placed in an acrylic glass enclosure (8.3 × 8.3 × 8.0 cm) with a wire mesh bottom and allowed to acclimate for 30 min. To assess heat hyperalgesia, a Hargreaves apparatus was used to determine paw withdrawal latency (sec) to a radiant thermal stimulus applied from beneath the glass floor to the plantar surface of the hindpaw, with a 20 s cut-off to avoid tissue injury (Model 7371, Ugo Basile, Comerio, Italy) [ 56 ]. A 10-min interval was allowed after each measurement to minimize stress. Measurements were performed three times for each mouse, and mean values were recorded as the withdrawal latency. Mechanical allodynia was evaluated by measuring withdrawal thresholds in response to von Frey monofilament stimulation to the plantar surface of the hindpaw (pressures: 0.008, 0.02, 0.04, 0.07, 0.16, 0.4, 0.6, 1.0, 1.4, and 2.0 g). The up–down method of the von Frey test was used, and the minimum pressure (g) that evoked a quick withdrawal response to stimulation with the monofilament was recorded as the withdrawal threshold [ 57 ]. Measurements were performed three times for each mouse, and mean values were recorded as the mechanical threshold. Immunofluorescence analysis The mice were deeply anesthetized with isoflurane (4% in air, 1 L/min) within a small induction chamber [ 58 ]. Anesthesia induction was confirmed when mice were immobile and showed no withdrawal response to a nociceptive tail stimulus. Euthanasia was performed following the American Veterinary Medical Association Guidelines for the Euthanasia of Animals [ 59 ]. Briefly, under deep isoflurane anesthesia, a bilateral thoracotomy was performed, and a needle was inserted into the left ventricle to begin perfusion with physiological saline using a Masterflex® peristaltic pump system (model 07555-10) equipped with an Easy-Load pump head (model 07514-10, Cole-Parmer Instrument Company, IL, USA). The right atrium (right auricle) was immediately incised to allow exsanguination and ensure death, and a total of 50 mL of saline was perfused. Following exsanguination, the needle in the left ventricle was used to perfuse 80 mL of 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS) for fixation [ 60 ]. After perfusion, the mice were decapitated, and their brains were carefully extracted for post-fixation. Following cryoprotection, the brains were frozen in dry ice–cooled hexane. The primary antibodies used in this study were FosB (mouse monoclonal antibody, 1:2000, Abcam, Cat. ab11959; a marker of activated neurons), orexin A (rabbit monoclonal antibody, 1:500, Abcam, Cat. ab255294), and NeuN (rabbit polyclonal antibody, 1:1000, Merck Millipore, Cat. ABN78; a neuronal marker). Serial coronal sections of the PAG, 25µm thick, spanning from Bregma − 4.96 mm to − 4.16 mm, were mounted on slides. After blocking to prevent nonspecific staining, sections designated for dual immunofluorescence were incubated simultaneously with two primary antibodies diluted in 0.1 M PBS containing 5% normal donkey serum and 0.3% Triton X-100 at 4°C for 48 h. Sections were then incubated with secondary antibodies diluted in 0.1 M PBS containing 5% normal donkey serum and 0.1% Triton X-100 overnight at 4°C. FosB immunoreactivity was detected using Alexa Fluor 594–labeled donkey anti-mouse antibody (1:500, Abcam, Cat. ab150108), and orexin A or NeuN immunoreactivity was detected using Alexa Fluor 488–labeled donkey anti-rabbit antibody (1:500, Abcam, Cat. ab150061). After washing in 0.1 M PBS, sections were mounted in Vectashield mounting medium with DAPI (H-1200, Vector Labs, Burlingame, CA, USA). Fluorescence signals were visualized using a confocal microscope (LSM700, Carl Zeiss, Oberkochen, Germany) equipped with an argon–helium laser. Negative control sections processed without primary antibodies showed no significant positive immunoreactivity. Quantitative analysis of immunofluorescence Immunofluorescence images of the vlPAG were captured at ×20 magnification for both ipsilateral and contralateral sides. Using WinROOF software (version 2021, MITANI CORPORATION, Tokyo, Japan), a 300 µm × 300 µm square, as illustrated in the figure, was overlaid onto the microscope images. The number of immunopositive cells or the immunopositive area within this square was quantified. For each mouse, the mean value of three randomly selected sections from six sections per brain was calculated. To ensure unbiased quantitative analysis, investigators remained blind to all experimental group assignments throughout the process. Statistical analysis Quantitative data are presented as the mean ± standard error of the mean (SE). A repeated-measures two-way ANOVA followed by Tukey’s post hoc test was used to compare withdrawal latency and von Frey test results among the experimental groups. A one-way ANOVA followed by Tukey’s post hoc test was used to compare the number of TH-, GAD67-, and NeuN-immunopositive cells and the OXA-immunostaining area size. Differences were considered statistically significant at p < 0.05. Statistical analyses were performed using GraphPad Prism 10 (GraphPad Software). Data availability All data generated or analyzed during this study are included in this published article. Declarations Competing interest The authors declare no competing interests. Funding This study was supported by research grants from KAKENHI (Grants-in-Aid for Scientific Research (C) 23K10407 from the Japan Society for the Promotion of Science). Author Contribution Shogo Habata and Katsuya Kami designed and performed the experiments. Shogo Habata, Takuma Kami, Yasunori Umemoto, and Katsuya Kami contributed to the analysis and interpretation of data.All authors contributed to the interpretation and discussion of the results. Comments on the manuscript and approval of the final version were provided by all the authors. Acknowledgement We would like to thank Editage (www.editage.jp) for English language editing. References Arsenault, M. Y., Parent, A., Séguéla, P. & Descarries, L. 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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-7382525","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":514793836,"identity":"26e184d9-493f-44ea-b0f7-9856a6e476d7","order_by":0,"name":"Shogo Habata","email":"","orcid":"","institution":"Wakayama Medical University","correspondingAuthor":false,"prefix":"","firstName":"Shogo","middleName":"","lastName":"Habata","suffix":""},{"id":514793837,"identity":"fbb84476-6b56-4e8b-a1f3-ff7b57b73b9b","order_by":1,"name":"Katsuya Kami","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAv0lEQVRIiWNgGAWjYBACCQY2IGlgAyR4wALMxGpJkyBVC8NhuBbCQLKBLfHTjYLzdQbXzh5g+FHDwG5OSIs0A9th6RyD2xIGt/MSGHuOMTBbNhDQIif/vAGqJceAgbeBgdngACEtDOzNv3MMzoG1MP4lRgvQYceAthwAa2Emyhag99OscwySJWcCtRyWOSZB2C8SB9iMb+f8sePnu51j+PBNjU0ywRBDAUAnSSQbkKQFBOxI1zIKRsEoGAXDHQAAoDY2DtjuQIMAAAAASUVORK5CYII=","orcid":"","institution":"Wakayama Medical University","correspondingAuthor":true,"prefix":"","firstName":"Katsuya","middleName":"","lastName":"Kami","suffix":""},{"id":514793838,"identity":"208b03ed-1d17-48cb-9592-6582992ece4b","order_by":2,"name":"Kohei Minami","email":"","orcid":"","institution":"Wakayama Medical University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Kohei","middleName":"","lastName":"Minami","suffix":""},{"id":514793839,"identity":"23260b9f-d8e6-4333-9085-192b27717943","order_by":3,"name":"Takuma Kami","email":"","orcid":"","institution":"Wakayama Medical University","correspondingAuthor":false,"prefix":"","firstName":"Takuma","middleName":"","lastName":"Kami","suffix":""},{"id":514793840,"identity":"4501fac0-d811-4783-b437-43a0c876e6a5","order_by":4,"name":"Yasunori Umemoto","email":"","orcid":"","institution":"Sapporo Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yasunori","middleName":"","lastName":"Umemoto","suffix":""},{"id":514793841,"identity":"c91bc44c-a092-40b5-96b9-fbe671770a39","order_by":5,"name":"Emiko Senba","email":"","orcid":"","institution":"Wakayama Medical University","correspondingAuthor":false,"prefix":"","firstName":"Emiko","middleName":"","lastName":"Senba","suffix":""},{"id":514793842,"identity":"c627f20a-79b1-4001-94cf-5f1f757bc5bd","order_by":6,"name":"Fumihiro Tajima","email":"","orcid":"","institution":"Chuzan Hospital","correspondingAuthor":false,"prefix":"","firstName":"Fumihiro","middleName":"","lastName":"Tajima","suffix":""},{"id":514793843,"identity":"dbc36a48-db48-4f7a-a024-0403ac4a80f4","order_by":7,"name":"Ken Kouda","email":"","orcid":"","institution":"Wakayama Medical University","correspondingAuthor":false,"prefix":"","firstName":"Ken","middleName":"","lastName":"Kouda","suffix":""}],"badges":[],"createdAt":"2025-08-15 15:23:29","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7382525/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7382525/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":91370351,"identity":"d37a5f6b-5346-447d-a360-a8c6216fa218","added_by":"auto","created_at":"2025-09-15 18:41:05","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":953570,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProtocol for the study and changes in running distances.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Naive, Sham, and PSL-Runner mice were placed in individual cages with a running wheel and allowed to voluntarily run, whereas Naive, Sham, and PSL-Sedentary mice were housed in cages without a running wheel. All mice, except for the Naive group, underwent VR for two weeks before Sham or PSL surgery. Following surgery, PSL and Sham mice were housed individually, and their running distances were recorded for 14 days before and after surgery. The plantar test or the von Frey test was conducted from 7 days before surgery to 14 days after surgery. After the 4-week experimental period, all mice were perfused to collect brain tissue.\u003c/p\u003e\n\u003cp\u003e(B) Changes in running distance in three Runner groups before and after surgery (n = 12–13). The running distances of PSL- and Sham-Runner mice gradually increased after surgery. Data are represented as the mean ± SEM. A two-way repeated measures ANOVA followed by Tukey’s posthoc test was used for group comparisons. \u003csup\u003e#\u003c/sup\u003ep \u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003ep \u0026lt; 0.01; compared to Naive-Runner mice. *p \u0026lt; 0.05; compared to Sham-PSL mice.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7382525/v1/65fa96ff7b8aed36ec7abb7a.png"},{"id":91370352,"identity":"affd055a-341c-43dc-b7dc-c50030497ea8","added_by":"auto","created_at":"2025-09-15 18:41:05","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":843113,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChanges in pain thresholds during the experimental period.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Thermal withdrawal latencies were significantly higher in PSL-Runner mice than in PSL-Sedentary mice at days 11 and 14 after surgery. (B) Mechanical thresholds were significantly higher in PSL-Runner mice than in PSL-Sedentary mice at days 11 and 14 after surgery. Data are represented as the mean ± SEM. A two-way repeated measures ANOVA followed by Tukey’s posthoc test was used for group comparisons. **p \u0026lt; 0.01; compared to PSL-Sedentary mice (n = 6–7). (C) Relationship between thermal withdrawal latency and total running distance after PSL surgery in PSL-Runner mice. A significant positive correlation was observed (p = 0.0499, r = 0.8115, n = 6). (D) Relationship between mechanical threshold and total running distance after PSL surgery in PSL-Runner mice. A significant positive correlation was observed (p = 0.0378, r = 0.8368, n = 6).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7382525/v1/3fb05b206e30cfcf03301e47.png"},{"id":91372195,"identity":"795dc1db-b98a-452c-ba05-f41d81822821","added_by":"auto","created_at":"2025-09-15 18:57:05","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4043526,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of PSL and VR on orexin in the vlPAG.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) A representative coronal brain image showing the location of the vlPAG (outlined in red). Ipsilateral refers to the brain on the same side as the limb that underwent PSL. (B) A photomicrograph showing orexin -positive immunoreactivity in the vlPAG. Numerous fibers displaying orexin immunoreactivity were observed. Scale bar = 50 μm. (C) Orexin staining (orexin -positive immunoreactivity/ orexin fiber) in each experimental group. Scale bar = 100 μm. (D) Bar graphs showing the area size of orexin on the ipsilateral side and (E) the contralateral side of the vlPAG. Naive-, Sham-, and PSL- Runner mice exhibited significantly greater values than Sedentary mice. Bars represent mean ± SEM. A one-way ANOVA followed by Tukey’s post hoc test was used for group comparisons. **p \u0026lt; 0.01, ***p \u0026lt; 0.001; n = 6–7. (F) Relationship between the total running distance after PSL surgery and orexin area size in PSL-Runner mice. A significant positive correlation was observed (p = 0.0380, r = 0.8363, n = 6).\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7382525/v1/660723946d58409cea36799a.png"},{"id":91370359,"identity":"754456c8-be01-4a07-adea-f732073430bf","added_by":"auto","created_at":"2025-09-15 18:41:05","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":5894561,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVR induces activation of NeuN\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e cells in the vlPAG.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Representative coronal brain image showing the location of the vlPAG (outlined in red). Ipsilateral refers to the brain on the same side as the limb that underwent PSL. (B) A high- power photomicrograph showing NeuN\u003csup\u003e+\u003c/sup\u003e cells (green) and NeuN\u003csup\u003e+ \u003c/sup\u003eFosB\u003csup\u003e+\u003c/sup\u003e cells (arrowheads) in the vlPAG. NeuN\u003csup\u003e+ \u003c/sup\u003eFosB\u003csup\u003e+\u003c/sup\u003e cells represent activated neurons. Scale bar = 100 μm. (C) Representative photomicrographs showing NeuN\u003csup\u003e+\u003c/sup\u003e, FosB+, and NeuN\u003csup\u003e+ \u003c/sup\u003eFosB\u003csup\u003e+\u003c/sup\u003e cells in Naive-Sedentary, Naive-Runner, PSL-Sedentary, and PSL-Runner mice. Scale bar = 100 μm. Arrowheads indicate activated (NeuN\u003csup\u003e+ \u003c/sup\u003eFosB\u003csup\u003e+\u003c/sup\u003e) neurons. (D) Bar graphs showing the ratio of NeuN\u003csup\u003e+ \u003c/sup\u003eFosB\u003csup\u003e+\u003c/sup\u003e cells on the ipsilateral side and (E) the contralateral side of the vlPAG. The ratios in Naive-, Sham-, and PSL-Runner mice were significantly higher than in Sedentary mice. Bars represent mean ± SEM. A one-way ANOVA followed by Tukey’s post hoc test was used for group comparisons. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001; n = 7–8. (F) Correlation between the total running distance after PSL surgery and the ratio of NeuN\u003csup\u003e+ \u003c/sup\u003eFosB\u003csup\u003e+\u003c/sup\u003e cells in PSL-Runner mice. A significant positive correlation was observed (p = 0.0161, r = 0.8944, n = 6).\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7382525/v1/fbf4f0fccaf42d417a020f1f.png"},{"id":91373304,"identity":"178db161-88af-4127-bcd9-870fd1ba1539","added_by":"auto","created_at":"2025-09-15 19:13:05","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":6498425,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTH\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e cells in the vlPAG are activated by VR.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Representative coronal brain image showing the location of the vlPAG (outlined in red). Ipsilateral refers to the brain on the same side as the limb that underwent PSL. (B) A high-power photomicrograph showing TH\u003csup\u003e+\u003c/sup\u003e (green) and FosB\u003csup\u003e+\u003c/sup\u003e (red) cells in the vlPAG. Scale bar = 100 μm. (C) Representative photomicrographs of TH\u003csup\u003e+\u003c/sup\u003e, FosB+, and TH\u003csup\u003e+ \u003c/sup\u003eFosB\u003csup\u003e+\u003c/sup\u003e (arrowheads) cells in Naive-Sedentary, Naive-Runner, PSL-Sedentary, and PSL-Runner mice. TH\u003csup\u003e+ \u003c/sup\u003eFosB\u003csup\u003e+\u003c/sup\u003e cells represent activated dopaminergic neurons. Scale bar = 100 μm. (D) Bar graphs showing the ratio of TH\u003csup\u003e+ \u003c/sup\u003eFosB\u003csup\u003e+\u003c/sup\u003e cells on the ipsilateral side of the vlPAG and (E) the contralateral side of the vlPAG. Ratios in Naive-, Sham-, and PSL-Runner mice were significantly higher than in Sedentary mice. Bars represent mean ± SEM. A one-way ANOVA followed by Tukey’s post hoc test was used for group comparisons. *p \u0026lt; 0.05, **p \u0026lt; 0.01; n = 6.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-7382525/v1/fc2049f1903aa811323a3e0a.png"},{"id":91370353,"identity":"74ca2d14-ba36-4118-9ba7-4d2c3b5ea550","added_by":"auto","created_at":"2025-09-15 18:41:05","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2326451,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChanges of GABA neurons by VR and potential role of orexin in developing EIH effect.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Representative coronal brain image showing the location of the vlPAG (outlined in red). Ipsilateral refers to the brain on the same side as the limb that underwent PSL. (B) Representative photomicrographs of GAD67\u003csup\u003e+\u003c/sup\u003e (green) and FosB\u003csup\u003e+\u003c/sup\u003e (red) cells in the vlPAG. Few GAD67\u003csup\u003e+\u003c/sup\u003e cells were detected, and GAD67\u003csup\u003e+\u003c/sup\u003eFosB\u003csup\u003e+\u003c/sup\u003e cells were rare. Scale bar = 50 μm. (C) Bar graphs showing the number of GAD67\u003csup\u003e+\u003c/sup\u003e cells on the ipsilateral side and (D) the contralateral side of the vlPAG. No statistically significant changes were observed among groups. (E) Potential role of orexin in developing the EIH effect. (a) VR in NPP model mice activates orexinergic neurons in the LHA; (b) orexin is projected to a wide range of brain areas, including the vlPAG; (c) orexin in the vlPAG activates glutamatergic neurons, which may stimulate the descending pain inhibitory system via activation of 5-HT neurons in the RVM; and (d) these processes may ultimately produce the EIH effect.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-7382525/v1/43cc8d7a1dabccf3c18fa818.png"},{"id":93448958,"identity":"cf1156c0-b6d0-4c2c-8c66-23983e207040","added_by":"auto","created_at":"2025-10-14 02:47:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":19978498,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7382525/v1/9306ff94-24c5-4857-8fc3-85dd70408370.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Orexinergic activation in the ventrolateral periaqueductal gray contributes to voluntary exercise-induced hypoalgesia in neuropathic pain model mice","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe pain pathways include not only ascending pathways that transmit nociceptive signals from the periphery to the central nervous system, but also descending pathways that can modulate pain transmission before the signals reach the brain. In the descending pathway, the periaqueductal gray (PAG) in the midbrain plays a crucial role in suppressing pain signals and is therefore considered the center of the descending pain inhibitory system. In particular, the ventrolateral PAG (vlPAG) contains dopaminergic (DA) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], glutamatergic (Glu) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], and γ-aminobutyric acid (GABAergic) [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] neurons. DA neurons in the vlPAG are implicated in pain modulation; for example, chemical lesions of DA neurons in the PAG attenuate opioid-induced analgesia [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], whereas direct injection of DA agonists into the vlPAG [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] or pharmacogenetic stimulation of DA neurons in the vlPAG [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] produces pain relief. Activated Glu neurons produce analgesic effects but can also induce anxiety- and fear-related behaviors [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. GABAergic neurons are interneurons that inhibit projection neurons [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] and express \u0026micro;-opioid receptors that target the rostral ventromedial medulla (RVM) [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Because chemogenetic inhibition of GABAergic neurons by opioids induces analgesia [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], these neurons may contribute to disinhibition of projection neurons. The PAG sends axons to the RVM, and this pathway suppresses the transmission of nociceptive signals in the spinal dorsal horn. The raphe magnus nucleus in the RVM contains abundant serotonergic neurons, including off-, on-, and neutral-cells[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Off-cells mainly inhibit spinal pain processing [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] but may also facilitate pain under certain conditions [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. These findings suggest that distinct neuronal subtypes in the vlPAG may differentially contribute to descending pain modulation, underscoring the need for further investigation into the specific roles of DA, Glu, and GABAergic neurons in both the chronicity and relief of pain.\u003c/p\u003e\u003cp\u003eMany studies have demonstrated the effectiveness of exercise in alleviating and improving chronic pain. A key phenomenon underlying exercise-related pain relief is termed exercise-induced hypoalgesia (EIH), which has been recognized as a basis for exercise therapy in chronic pain. One proposed mechanism of EIH involves the reward system [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In addition, exercise has been shown to activate the descending pain inhibitory system via endogenous opioids [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] and the endocannabinoid 2-arachidonoylglycerol (2-AG) [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] in the PAG, and to increase cannabinoid 1 and 2 receptor (CB1 and CB2 receptor) expression in the PAG [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], even in a Parkinson\u0026rsquo;s disease model rat [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Activation of opioid and CB1 receptors on presynaptic GABAergic terminals in the PAG inhibits GABA release [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The RVM transmits 5-HT to the spinal dorsal horn, contributing to both pain facilitation and inhibition [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Exercise increases 5-HT levels in both the RVM [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] and the spinal dorsal horn [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], potentially contributing to analgesia by reducing pain sensitivity in the spinal dorsal horn. Collectively, these findings suggest that the descending pain inhibitory system is a key mechanism promoting the EIH effect.\u003c/p\u003e\u003cp\u003eOrexin, also known as hypocretin, is an excitatory neuropeptide that exists in two forms: orexin A (OXA) and orexin B (OXB). Orexin neurons are localized in the lateral hypothalamic area (LHA) and project their fibers widely throughout the central nervous system [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Two types of orexin receptors, orexin receptor 1 (OX1R) and orexin receptor 2 (OX2R), are expressed in most brain regions in mice, including the PAG, and are present on both Glu and GABAergic neurons [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Few studies have directly confirmed the expression of orexin receptors on DA neurons within the PAG.\u003c/p\u003e\u003cp\u003eOrexin is involved in multiple physiological functions, including pain regulation. For example, orexin neurons projecting to the PAG inhibit GABAergic neurons via increased synthesis of 2-AG through the phospholipase C\u0026ndash;diacylglycerol lipase alpha (C\u0026ndash;DAGLα) pathway, thereby producing antinociception [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Pain-related behaviors are enhanced by selective ablation of orexin neurons in mice, whereas pharmacogenetic activation of these neurons induces analgesia [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Activation of orexin neurons through a designer receptor exclusively activated by designer drugs approach attenuates thermal hyperalgesia and significantly reduces paw-licking behavior in the formalin test [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Furthermore, intraperitoneal injection of an OX1R antagonist (SB-334867) enhances formalin-induced nociceptive behaviors in adult male rats [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. These findings demonstrate that orexin plays a critical role in pain regulation, contributing to both analgesic mechanisms and nociceptive processing. However, no study has directly examined whether exercise increases orexin projections to the vlPAG, although exercise has been reported to increase OXA levels in the hypothalamus [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] and in the cerebrospinal fluid (CSF) of rats [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] and dogs [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eBecause orexin neurons project to the vlPAG, it is conceivable that exercise-induced activation of these neurons enhances orexin input to the vlPAG, thereby activating the descending pain inhibitory system and promoting the EIH effect. Orexin neurons in the LHA are activated by exercise and are believed to be involved in pain relief; however, their contribution to the EIH effect has not yet been fully elucidated. Therefore, in this study, we examined orexin expression in the vlPAG following voluntary exercise in a mouse model of neuropathic pain. Furthermore, we investigated which neuronal subtypes in the vlPAG are activated by voluntary exercise. These findings provide novel insights into the brain mechanisms underlying the EIH effect.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eChanges of running distances and pain behaviors\u003c/h2\u003e\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA shows the protocol for voluntary wheel running. Mice were individually housed in cages with a running wheel, allowing voluntary running for 2 weeks before and after PSL or Sham surgery. Naive-Runner mice were allowed to run for 4 weeks. For consistency with the Sham- and PSL-Runner groups, the 4-week running period was divided into two phases: 2 weeks before and 2 weeks after a designated reference day, corresponding to the day of surgery in the other groups. In this scheme, the day before the reference day was defined as pre-1 day, and the day after as post-1 day.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB shows changes in daily running distance in Naive-, Sham-, and PSL-Runner mice. On pre-14 day, mean running distances were 4,101\u0026thinsp;\u0026plusmn;\u0026thinsp;629 m/day in the Naive-Runner group, 3,322\u0026thinsp;\u0026plusmn;\u0026thinsp;340 m/day in the Sham-Runner group, and 2,342\u0026thinsp;\u0026plusmn;\u0026thinsp;561 m/day in the PSL-Runner group, with no statistically significant difference among groups (p\u0026thinsp;=\u0026thinsp;0.069, one-way ANOVA). Running distances gradually increased, reaching 2\u0026ndash;3 times the initial values by pre-1 day. On that day, mean running distances were 11,057\u0026thinsp;\u0026plusmn;\u0026thinsp;1,378 m/day in the Naive-Runner group, 9,075\u0026thinsp;\u0026plusmn;\u0026thinsp;995 m/day in the Sham-Runner group, and 11,187\u0026thinsp;\u0026plusmn;\u0026thinsp;1,059 m/day in the PSL-Runner group, with no statistically significant difference among groups.\u003c/p\u003e\u003cp\u003eIn Sham-Runner mice, running distance decreased to 5,582\u0026thinsp;\u0026plusmn;\u0026thinsp;888 m/day on post 1 day but returned to pre-surgery levels by post 14 day (8,666\u0026thinsp;\u0026plusmn;\u0026thinsp;843 m/day; 95.5% recovery). In contrast, PSL-Runner mice showed a marked decrease to 5,060\u0026thinsp;\u0026plusmn;\u0026thinsp;768 m/day on post-1 day, with only partial recovery to 51.4% of pre-surgery levels (5,746\u0026thinsp;\u0026plusmn;\u0026thinsp;968 m/day on post-14 day). From post 1 day to post 14 day, PSL-Runner mice exhibited significantly lower daily running distances than both Naive- and Sham-Runner mice (time effect: F\u003csub\u003e(5.992, 223.5)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;35.19, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; surgery effect: F\u003csub\u003e(2, 38)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;3.249, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; interaction: F\u003csub\u003e(54, 1007)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;6.990, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e\u003cp\u003eTo evaluate the effects of VR on pain behaviors, plantar and von Frey tests were performed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). For the plantar test, there were significant effects of experimental group (F\u003csub\u003e(5, 324)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;136.7, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), time (F\u003csub\u003e(8, 324)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;28.59, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), and their interaction (F\u003csub\u003e(40, 324)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;12.15, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Similarly, for the von Frey test, significant effects were found for experimental group (F\u003csub\u003e(5, 315)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;147.5, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), time (F\u003csub\u003e(8, 315)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;21.17, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), and their interaction (F\u003csub\u003e(40, 315)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;11.36, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Regardless of running, there was no difference between the Naive and Sham groups throughout the study period in either the plantar or von Frey test.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003ePSL-Sedentary and PSL-Runner mice showed markedly shorter withdrawal latencies at 1 day after PSL (PSL-Sedentary: 4.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19 s; PSL-Runner: 4.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14 s) compared to pre-surgery levels (PSL-Sedentary: 11.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.52 s; PSL-Runner: 11.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43 s), and lower mechanical thresholds on the same day (PSL-Sedentary: 0.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 g vs. 1.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09 g; PSL-Runner: 0.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 g vs. 1.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 g). These decreased latencies and thresholds persisted after PSL in PSL-Sedentary mice. Conversely, withdrawal latencies and mechanical thresholds in PSL-Runner mice were significantly increased on day 14 after PSL compared with PSL-Sedentary mice (PSL-Runner: 7.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24 s and 0.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06 g; PSL-Sedentary: 4.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32 s and 0.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 g; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 for both). Collectively, these findings confirm that the VR protocol significantly improved pain behaviors in the NPP model.\u003c/p\u003e\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD show the relationship between pain thresholds at 14 days post-PSL and total running distances over the 14-day period after PSL in PSL-Runner mice. Significant positive correlations were observed (plantar test: r\u0026thinsp;=\u0026thinsp;0.812, p\u0026thinsp;=\u0026thinsp;0.0499, n\u0026thinsp;=\u0026thinsp;6; von Frey test: r\u0026thinsp;=\u0026thinsp;0.837, p\u0026thinsp;=\u0026thinsp;0.0378, n\u0026thinsp;=\u0026thinsp;6). These results suggest that higher levels of voluntary running are associated with greater improvements in pain-related behaviors in the NPP model.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eVR increases orexin projections\u003c/h3\u003e\n\u003cp\u003ePrevious studies have shown that exercise increases orexin levels in the cerebrospinal fluid of rats [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], cats [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], and dogs [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], as well as in human plasma [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. In addition, immunohistochemical observations have indicated significant increases in orexin neuron activity in NPP model mice following VR [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] and in healthy mice following treadmill running [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Based on these findings, we investigated orexinergic activity in the vlPAG using immunohistochemical staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The total immunoreactive area size of orexinA in each group was as follows: Naive-Sedentary: 283.5\u0026thinsp;\u0026plusmn;\u0026thinsp;41.1 \u0026micro;m\u0026sup2;; Naive-Runner: 579.5\u0026thinsp;\u0026plusmn;\u0026thinsp;70.3 \u0026micro;m\u0026sup2;; Sham-Sedentary: 258.8\u0026thinsp;\u0026plusmn;\u0026thinsp;42.9 \u0026micro;m\u0026sup2;; Sham-Runner: 548.5\u0026thinsp;\u0026plusmn;\u0026thinsp;80.0 \u0026micro;m\u0026sup2;; PSL-Sedentary: 209.1\u0026thinsp;\u0026plusmn;\u0026thinsp;29.1 \u0026micro;m\u0026sup2;; PSL-Runner: 547.0\u0026thinsp;\u0026plusmn;\u0026thinsp;80.3 \u0026micro;m\u0026sup2;. There was no statistically significant difference between ipsilateral and contralateral sides; therefore, data from both sides were combined for analysis. The combined data revealed significant differences between exercise and non-exercise groups (Naive: p\u0026thinsp;=\u0026thinsp;0.0006; Sham: p\u0026thinsp;=\u0026thinsp;0.0008; PSL: p\u0026thinsp;=\u0026thinsp;0.0001). Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF shows the relationship between orexinA and total running distances over the 14-day period after PSL in PSL-Runner mice. A significant positive correlation was observed (r\u0026thinsp;=\u0026thinsp;0.836, p\u0026thinsp;=\u0026thinsp;0.0380, n\u0026thinsp;=\u0026thinsp;6). These results suggest that higher levels of VR are associated with enhanced orexinergic activity in the vlPAG, even under neuropathic pain conditions.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eVR induces activation of neurons in the vlPAG\u003c/h3\u003e\n\u003cp\u003eAs described above, orexinergic projections to the vlPAG were greater in mice that performed VR. To identify the activated cell type, neurons or glial cells, we performed immunostaining with anti-NeuN antibody (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Approximately 100\u0026ndash;200 NeuN-positive neurons were identified within the defined area of the vlPAG, and VR did not alter the number of NeuN-positive neurons. There were no significant differences between ipsilateral and contralateral sides; therefore, data from both sides were combined for analysis. To evaluate the effects of VR, statistical comparisons were performed between exercise and non-exercise groups within each experimental condition. Significant differences were found in all groups (Naive: p\u0026thinsp;=\u0026thinsp;0.0006; Sham: p\u0026thinsp;=\u0026thinsp;0.0042; PSL: p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF shows the relationship between the ratio of activated neurons (NeuN⁺ FosB⁺ / NeuN⁺) and total running distances over the 14-day period after PSL in PSL-Runner mice. A significant positive correlation was observed (r\u0026thinsp;=\u0026thinsp;0.8944, p\u0026thinsp;=\u0026thinsp;0.0161, n\u0026thinsp;=\u0026thinsp;6). These results suggest that higher VR activity enhances neuronal activation in the vlPAG under neuropathic pain conditions, which may play a critical role in exercise-induced pain modulation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eActivated dopaminergic neurons were increased by VR\u003c/h3\u003e\n\u003cp\u003eNeurons in the PAG utilize various neurotransmitters such as glutamate, GABA, and glycine, and express a range of neuropeptides including somatostatin, substance P, dynorphin, neurotensin, and others. We examined whether DA neurons in the vlPAG are involved in the induction of EIH using immunohistochemical staining.\u003c/p\u003e\u003cp\u003eTH-positive neurons represent DA neurons [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], and cells indicated by arrows in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e represent activated DA neurons, as shown by FosB and TH double-positive immunoreactivities. Examination of DA neuron distribution in the vlPAG revealed that they were located near the midbrain aqueduct, with FosB-positive cells densely distributed in the surrounding region. The ratio of activated DA neurons (TH\u003csup\u003e+\u003c/sup\u003e FosB\u003csup\u003e+\u003c/sup\u003e / TH\u003csup\u003e+\u003c/sup\u003e) in ipsilateral and contralateral sides showed no statistical difference in each group; therefore, data from both sides were combined for subsequent analysis. Statistical comparisons between exercise and non-exercise groups within each experimental condition revealed significant differences in all groups (Naive: p\u0026thinsp;=\u0026thinsp;0.0412, Sham: p\u0026thinsp;=\u0026thinsp;0.0166, PSL: p\u0026thinsp;\u0026lt;\u0026thinsp;0.0064). These results suggest that VR enhances activation of DA neurons in the vlPAG, which may contribute to EIH.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eNo alterations in the number and activation of GABA neurons\u003c/h3\u003e\n\u003cp\u003eGABAergic interneurons express \u0026micro;-opioid receptors and inhibit activation of neurons projecting to the RVM in the vlPAG [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Inhibition of GABAergic signaling in the PAG suppresses pain, whereas activation enhances nociceptive transmission. Opioids relieve this GABAergic inhibition, leading to disinhibition of output neurons and resulting in analgesia [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Therefore, we assessed changes in their number and activation after VR (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Only a few GAD67-positive cells, approximately one to four per section, were detected, and only very few showed FosB immunoreactivity. No statistically significant differences were found in any group. These findings indicate that VR does not induce significant changes in the number or activation of GABAergic neurons in the vlPAG.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe present study examined orexinergic activation and neuronal subtypes activated in the vlPAG following voluntary exercise in neuropathic pain model mice. Our results indicated that (i) VR alleviated pain-related behaviors, (ii) orexinergic projections and neuronal activity in the vlPAG increased along with activation of dopaminergic neurons, and (iii) neither the number nor the activity of GABAergic neurons showed significant changes. These findings suggest that orexin may play an important role in producing the EIH effect via activation of the descending pain inhibitory system.\u003c/p\u003e\u003cp\u003eIn this study, when we evaluated the effects of VR on pain behavior in NPP model mice, VR produced analgesic effects consistent with the development of EIH. Some studies have reported greater running distances compared to the present study [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. This difference may result from variations in the environment in which VR was performed. It has been reported that in an enriched acoustic environment, animals\u0026rsquo; activity levels are elevated compared to those observed under conventional laboratory housing conditions [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Thus, environmental factors, particularly ambient noise and housing conditions such as the number of mice, should be considered when examining pain behaviors with VR.\u003c/p\u003e\u003cp\u003ePSL-Sedentary mice exhibited decreased pain thresholds, which were improved by VR (PSL-Runner). Furthermore, we found positive correlations between thermal withdrawal latencies or mechanical thresholds and total running distances in PSL-Runner mice, suggesting that greater VR activity further enhances the EIH effect. Although reliable evidence directly linking longer exercise duration to stronger analgesic effects is still lacking, in humans, the EIH effect following a single session typically lasts only 30 seconds to 30 min. However, regular exercise may induce adaptations within pain modulation systems, potentially leading to more consistent or pronounced EIH effects over time [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn contrast to forced exercise, such as treadmill running, wheel running reflects environmental enrichment. Mice have free access to the wheel and can run voluntarily. Recent findings indicate that brain regions activated differ according to the type of exercise [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Treadmill running may preferentially activate brain regions associated with stress, fear, and pain. The optimal type, intensity, duration, and frequency of exercise for developing the EIH effect remain unclear, as these parameters are influenced by factors such as nociceptive, neuropathic, or nociplastic pain, as well as psychological factors like fear and anxiety, and social factors such as loneliness. Recent studies have shown that chemogenetic stimulation of the primary motor cortex can suppress negative emotions and maladaptive coping behaviors in neuropathic pain while also alleviating pain by reducing sensory hypersensitivity [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Therefore, to optimize the EIH effect, it is important to ensure adequate exercise in low-stress environments, that is, exercise should be voluntary rather than forced, noise should be minimal, lighting should be non-intense, and animals should be housed in appropriately sized groups.\u003c/p\u003e\u003cp\u003eOur findings indicate that VR enhances orexinergic projections to the vlPAG, with orexin levels in this region positively correlating with physical activity. Orexin is produced in the LHA and projects to multiple brain regions. Previous studies have shown that exercise increases orexin levels in the LHA [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Wu et al. reported a positive association between exercise and CSF orexin levels [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], and Kiwaki et al. demonstrated that OXA injection into the hypothalamic paraventricular nucleus significantly increases physical activity [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Immunohistochemical studies using FosB [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] and c-Fos [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e] have indicated that exercise activates orexin neurons in the LHA, whereas ablation of these neurons decreases physical activity [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. In the diet-induced obesity model mice, obesity-prone individuals exhibit lower spontaneous physical activity and reduced expression of orexin receptors in the rostral lateral hypothalamus, where orexin exerts its effects [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. The present study demonstrated increased orexin immunoreactivity in the vlPAG following VR. Collectively, these findings suggest that physical activity enhances orexinergic signaling in the vlPAG, which may mediate the analgesic benefits of exercise through the EIH effect.\u003c/p\u003e\u003cp\u003eIn this study, double-staining with DAPI and NeuN revealed a large population of neurons in the vlPAG, and these neurons were significantly activated in response to VR. To identify specific neuronal subtypes, we performed TH immunostaining, which revealed a relatively small population of dopaminergic neurons. However, the proportion of activated dopaminergic neurons was significantly higher in the exercise group than in the sedentary group. GAD67 immunostaining revealed very few activated GABAergic neurons. Previous studies have shown that the vlPAG contains a heterogeneous neuronal population, classified by firing patterns [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e], neurotransmitter phenotypes, and functional roles. Glycinergic neurons, identified by glycine transporter 2 (GlyT2) expression, are considered a subset of inhibitory neurons alongside GABAergic neurons. Glutamatergic neurons are known to contribute to pain relief in both anxiety-dependent [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] and anxiety-independent contexts [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Recently, in chronic unpredictable mild stress model mice, dopaminergic neurons activated via chemogenetic methods were reported to attenuate thermal pain and depressive behaviors [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. In the present study, although the total number of dopaminergic neurons in the vlPAG was relatively small, the overall findings suggest that glutamatergic neurons in this region may play a more prominent role in producing the EIH effect promoted by elevated orexin levels in neuropathic pain. However, we could not directly confirm glutamatergic neurons using appropriate markers, which is a limitation of the present study.\u003c/p\u003e\u003cp\u003eBased on the present findings, we propose a potential neuronal mechanism in which orexin activates the vlPAG following VR, thereby producing the EIH effect. First, VR activates orexinergic neurons in the LHA, which project to the vlPAG and activate glutamatergic neurons projecting to the RVM. These events may ultimately engage the descending pain inhibitory system (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). Thus, the descending analgesic pathway activated by these systems may contribute to the EIH effect. In addition, other mechanisms, such as the mesolimbic system, including the VTA and NAc [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e], or cognitive and emotional pain-processing regions, such as the anterior cingulate cortex [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e], may also contribute to the development of EIH because orexin neurons project broadly throughout the brain. The present findings provide novel insights into the neurobiological basis of EIH and highlight the benefits of voluntary exercise, a widely recognized low-stress, non-pharmacological strategy for improving chronic neuropathic pain. Further studies integrating molecular, genetic, and behavioral approaches will be essential to fully elucidate the mechanisms underlying EIH and to optimize exercise-based therapeutic interventions.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003eAnimals\u003c/h2\u003e\u003cp\u003eAll animal experiments were approved by the Institutional Animal Care and Use Committee of Wakayama Medical University (approval number: 1124) and conducted in accordance with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. All procedures conformed to the National Institutes of Health Guide for the Care and Use of Laboratory Animals, 8th edition. Male C57BL/6JJcl mice aged 8\u0026ndash;13 weeks were used in this study. C57BL/6J mice were purchased from CLEA Japan, Inc. (Tokyo, Japan). Each mouse was housed in a plastic cage and maintained in a holding room with a 12 h light\u0026ndash;dark cycle, with free access to food and water ad libitum. All experimental procedures included appropriate measures to relieve suffering and pain.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e\u003cb\u003ePreparation of NPP model mice\u003c/b\u003e\u003c/h2\u003e\u003cp\u003eNeuropathic pain (NPP) model mice were prepared by partial sciatic nerve ligation as previously reported [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Under deep isoflurane anesthesia, the right mid-thigh level of the sciatic nerve was tightly ligated with 8\u0026thinsp;\u0026minus;\u0026thinsp;0 silk sutures. Sham operations were performed using the same procedures described above, excluding PSL. After surgery, the mice were individually monitored in cages and returned to their regular cages upon waking. No postoperative treatment was administered. Notably, mice did not exhibit any symptoms such as reduced movement, weight loss, swelling, excessive inflammation, or suppuration.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eExperimental groups and the VR protocol\u003c/h2\u003e\u003cp\u003eThe mice were assigned to six groups as follows: (1) Naive-Sedentary (n\u0026thinsp;=\u0026thinsp;14), (2) Naive-Runner (n\u0026thinsp;=\u0026thinsp;15), (3) Sham-Sedentary (n\u0026thinsp;=\u0026thinsp;12), (4) Sham-Runner (n\u0026thinsp;=\u0026thinsp;16), (5) PSL-Sedentary (n\u0026thinsp;=\u0026thinsp;16), and (6) PSL-Runner (n\u0026thinsp;=\u0026thinsp;14). Each group was further divided into two subgroups to conduct two types of pain behavior tests. Naive, Sham, and PSL-Runner mice were allowed unrestricted access to a running wheel. Given the importance of early-stage running exercise in inducing a significant EIH effect after nerve injury, mice were immediately returned to cages with running wheels following PSL surgery, allowing voluntary running. All mice, except Naive mice, underwent VR for two weeks prior to Sham or PSL surgery. After surgery, PSL and Sham mice were transferred to individual cages, and their running distances were recorded over a 14-day period before and after surgery. Wheel rotations per hour were monitored using a magnetic reed switch connected to a computerized exercise-monitoring system (SOF-860 wheel manager software, MED Associates, Inc.), and daily running distances (meters/day) were calculated from wheel rotations.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eEvaluation of pain behavior\u003c/h2\u003e\u003cp\u003eTo eliminate diurnal variations in pain behavior, plantar and von Frey tests were performed at 5:00 p.m. Before each test, mice were placed in an acrylic glass enclosure (8.3 \u0026times; 8.3 \u0026times; 8.0 cm) with a wire mesh bottom and allowed to acclimate for 30 min.\u003c/p\u003e\u003cp\u003eTo assess heat hyperalgesia, a Hargreaves apparatus was used to determine paw withdrawal latency (sec) to a radiant thermal stimulus applied from beneath the glass floor to the plantar surface of the hindpaw, with a 20 s cut-off to avoid tissue injury (Model 7371, Ugo Basile, Comerio, Italy) [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. A 10-min interval was allowed after each measurement to minimize stress. Measurements were performed three times for each mouse, and mean values were recorded as the withdrawal latency.\u003c/p\u003e\u003cp\u003eMechanical allodynia was evaluated by measuring withdrawal thresholds in response to von Frey monofilament stimulation to the plantar surface of the hindpaw (pressures: 0.008, 0.02, 0.04, 0.07, 0.16, 0.4, 0.6, 1.0, 1.4, and 2.0 g). The up\u0026ndash;down method of the von Frey test was used, and the minimum pressure (g) that evoked a quick withdrawal response to stimulation with the monofilament was recorded as the withdrawal threshold [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Measurements were performed three times for each mouse, and mean values were recorded as the mechanical threshold.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eImmunofluorescence analysis\u003c/h2\u003e\u003cp\u003eThe mice were deeply anesthetized with isoflurane (4% in air, 1 L/min) within a small induction chamber [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. Anesthesia induction was confirmed when mice were immobile and showed no withdrawal response to a nociceptive tail stimulus. Euthanasia was performed following the American Veterinary Medical Association Guidelines for the Euthanasia of Animals [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. Briefly, under deep isoflurane anesthesia, a bilateral thoracotomy was performed, and a needle was inserted into the left ventricle to begin perfusion with physiological saline using a Masterflex\u0026reg; peristaltic pump system (model 07555-10) equipped with an Easy-Load pump head (model 07514-10, Cole-Parmer Instrument Company, IL, USA). The right atrium (right auricle) was immediately incised to allow exsanguination and ensure death, and a total of 50 mL of saline was perfused. Following exsanguination, the needle in the left ventricle was used to perfuse 80 mL of 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS) for fixation [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. After perfusion, the mice were decapitated, and their brains were carefully extracted for post-fixation. Following cryoprotection, the brains were frozen in dry ice\u0026ndash;cooled hexane.\u003c/p\u003e\u003cp\u003eThe primary antibodies used in this study were FosB (mouse monoclonal antibody, 1:2000, Abcam, Cat. ab11959; a marker of activated neurons), orexin A (rabbit monoclonal antibody, 1:500, Abcam, Cat. ab255294), and NeuN (rabbit polyclonal antibody, 1:1000, Merck Millipore, Cat. ABN78; a neuronal marker). Serial coronal sections of the PAG, 25\u0026micro;m thick, spanning from Bregma \u0026minus;\u0026thinsp;4.96 mm to \u0026minus;\u0026thinsp;4.16 mm, were mounted on slides. After blocking to prevent nonspecific staining, sections designated for dual immunofluorescence were incubated simultaneously with two primary antibodies diluted in 0.1 M PBS containing 5% normal donkey serum and 0.3% Triton X-100 at 4\u0026deg;C for 48 h. Sections were then incubated with secondary antibodies diluted in 0.1 M PBS containing 5% normal donkey serum and 0.1% Triton X-100 overnight at 4\u0026deg;C. FosB immunoreactivity was detected using Alexa Fluor 594\u0026ndash;labeled donkey anti-mouse antibody (1:500, Abcam, Cat. ab150108), and orexin A or NeuN immunoreactivity was detected using Alexa Fluor 488\u0026ndash;labeled donkey anti-rabbit antibody (1:500, Abcam, Cat. ab150061). After washing in 0.1 M PBS, sections were mounted in Vectashield mounting medium with DAPI (H-1200, Vector Labs, Burlingame, CA, USA). Fluorescence signals were visualized using a confocal microscope (LSM700, Carl Zeiss, Oberkochen, Germany) equipped with an argon\u0026ndash;helium laser. Negative control sections processed without primary antibodies showed no significant positive immunoreactivity.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eQuantitative analysis of immunofluorescence\u003c/h2\u003e\u003cp\u003eImmunofluorescence images of the vlPAG were captured at \u0026times;20 magnification for both ipsilateral and contralateral sides. Using WinROOF software (version 2021, MITANI CORPORATION, Tokyo, Japan), a 300 \u0026micro;m \u0026times; 300 \u0026micro;m square, as illustrated in the figure, was overlaid onto the microscope images. The number of immunopositive cells or the immunopositive area within this square was quantified. For each mouse, the mean value of three randomly selected sections from six sections per brain was calculated. To ensure unbiased quantitative analysis, investigators remained blind to all experimental group assignments throughout the process.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eQuantitative data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SE). A repeated-measures two-way ANOVA followed by Tukey\u0026rsquo;s post hoc test was used to compare withdrawal latency and von Frey test results among the experimental groups. A one-way ANOVA followed by Tukey\u0026rsquo;s post hoc test was used to compare the number of TH-, GAD67-, and NeuN-immunopositive cells and the OXA-immunostaining area size. Differences were considered statistically significant at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Statistical analyses were performed using GraphPad Prism 10 (GraphPad Software).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eData availability\u003c/h2\u003e\u003cp\u003eAll data generated or analyzed during this study are included in this published article.\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompeting interest\u003c/strong\u003e\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis study was supported by research grants from KAKENHI (Grants-in-Aid for Scientific Research (C) 23K10407 from the Japan Society for the Promotion of Science).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eShogo Habata and Katsuya Kami designed and performed the experiments. Shogo Habata, Takuma Kami, Yasunori Umemoto, and Katsuya Kami contributed to the analysis and interpretation of data.All authors contributed to the interpretation and discussion of the results. Comments on the manuscript and approval of the final version were provided by all the authors.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe would like to thank Editage (www.editage.jp) for English language editing.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eArsenault, M. Y., Parent, A., S\u0026eacute;gu\u0026eacute;la, P. \u0026amp; Descarries, L. Distribution and morphological characteristics of dopamine-immunoreactive neurons in the midbrain of the squirrel monkey (\u003cem\u003eSaimiri sciureus\u003c/em\u003e). \u003cem\u003eJ. Comp. Neurol.\u003c/em\u003e \u003cb\u003e267\u003c/b\u003e, 489\u0026ndash;506 (1988).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHo, Y. C., Cheng, J. 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Transcardiac perfusion of the mouse for brain tissue dissection and fixation. \u003cem\u003eBio Protoc.\u003c/em\u003e \u003cb\u003e11\u003c/b\u003e, e3988 (2021).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Exercise-induced hypoalgesia, descending pain inhibitory system, orexin, PAG, neuropathic pain, voluntary running","lastPublishedDoi":"10.21203/rs.3.rs-7382525/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7382525/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eVoluntary exercise is known to alleviate chronic pain, yet the underlying neural mechanisms, including the descending pain inhibitory system, remain incompletely understood. As orexin is recognized as a neuromodulator involved in pain regulation and projects widely throughout the central nervous system, we investigated the role of orexinergic signaling in the ventrolateral periaqueductal gray (vlPAG) in the induction of exercise-induced hypoalgesia (EIH) using a neuropathic pain mouse model. Mice with free access to running wheels exhibited significantly improved mechanical and thermal pain thresholds compared to sedentary mice, and this pain relief positively correlated with running distance. Immunohistochemical analyses revealed increased orexin immunoreactivity and neuronal activation in the vlPAG following voluntary running (VR). Although dopaminergic neuron numbers in the vlPAG were low, their activation was significantly enhanced after VR, whereas GABAergic neurons showed only minimal activation. These findings suggest that VR may promote EIH via activation of orexinergic projections from the lateral hypothalamus to the vlPAG, potentially engaging descending pain inhibitory pathways. Our results indicate a novel neural mechanism underlying EIH via orexin signaling and support voluntary exercise as a promising non-pharmacological strategy for managing chronic neuropathic pain.\u003c/p\u003e","manuscriptTitle":"Orexinergic activation in the ventrolateral periaqueductal gray contributes to voluntary exercise-induced hypoalgesia in neuropathic pain model mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-15 18:41:00","doi":"10.21203/rs.3.rs-7382525/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3efcb1df-f94b-46ac-b4c3-9107ab54dd61","owner":[],"postedDate":"September 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":54683908,"name":"Biological sciences/Neuroscience"},{"id":54683909,"name":"Biological sciences/Physiology"}],"tags":[],"updatedAt":"2025-10-14T02:38:38+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-15 18:41:00","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7382525","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7382525","identity":"rs-7382525","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}