Sleep loss differentially reconfigures neural circuits governing pain and neuropsychological homeostasis

Sleep loss differentially reconfigures neural circuits governing pain and neuropsychological homeostasis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Sleep loss differentially reconfigures neural circuits governing pain and neuropsychological homeostasis Teruyo Kishino, Yasuyuki Nagumo, Yusuke Hamada, Michiko Narita, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8228487/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 Sleep dissipates the physiological load accumulated during wakefulness and restores neural, immune, and endocrine balance to maintain homeostasis. However, how sleep loss disrupts these systems and how recovery sleep (RS) reverses the effects remain poorly understood. Using a mouse model of sleep deprivation (SD), we examined alterations in pain sensitivity, inflammatory responses, pharmacologic responsiveness to opioids and non-benzodiazepine hypnotics, and molecular adaptations across key brain regions. SD heightened nociception, attenuated morphine analgesia and GABA A receptor-mediated hypnosis, and amplified lipopolysaccharide-evoked inflammation. These effects were accompanied by sustained expression of c-Fos and ΔFosB in multiple brain regions and of Calca in the parabrachial nucleus (PBN), but were largely normalized after RS. Within the hypothalamic arcuate nucleus (ARC), Pomc mRNA expression, unlike Oprm1 , Penk , and Pdyn , was markedly reduced during SD n and restored after RS, paralleling normalization of nociceptive thresholds. Pharmacogenetic activation of ARC-POMC neurons alleviated persistent postoperative pain. In contrast, SD enhanced morphine-induced psychomotor activation linked to dopaminergic transmission, accompanied by sustained c-Fos expression in the ventral tegmental area, nucleus accumbens, and medial prefrontal cortex (mPFC). Notably, this hyperlocomotion persisted despite RS, and was associated with irreversible c-Fos upregulation as well as a sustained, statistically significant elevation of integrated plasticity markers in the mPFC. Together, these findings reveal that sleep loss broadly reconfigures the neural circuits controlling pain, inflammation, and drug responsiveness. While most deficits are reversible with adequate RS, specific dopaminergic and cortical adaptations exhibit incomplete restoration, potentially predisposing patients to chronic, secondary psychopathology. Sleep deprivation Recovery sleep Proopiomelanocortin (POMC) Chronic pain Neuropsychological homeostasis Default mode network Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Sleep is a fundamental biological process that dissipates the physiological load accumulated during wakefulness and recalibrates neural, immune, and endocrine homeostasis [ 1 – 3 ]. During sleep, synchronized neural activity and metabolic redistribution promote synaptic remodeling and the clearance of metabolic waste, thereby maintaining systemic physiological equilibrium [ 4 , 5 ]. However, chronic sleep restriction and fragmentation have become increasingly prevalent in modern society. These consequences extend far beyond fatigue and impaired attention to encompass cardiovascular and metabolic dysfunction, immune dysregulation, and emotional instability [ 6 – 8 ]. These alterations imply a subtle yet pervasive disruption of neurochemical and circuit-level homeostasis that which may exert long-lasting effects on behavior, affective regulation, and systemic adaptation. Sleep deprivation (SD) and poor sleep quality disrupt the delicate balance between excitation and inhibition within neural networks [ 9 , 10 ], while overactivating stress-responsive and immunometabolic systems [ 11 – 13 ]. In particular, hyperactivation of the hypothalamic–pituitary–adrenal (HPA) axis and corticotropin-releasing hormone (CRH) neurons, together with dysregulated mesolimbic dopaminergic signaling, have been implicated as neural substrates underlying sleep-loss-induced phenotypes of hyperalgesia, anxiety, affective lability, and altered drug responsiveness [ 13 – 17 ]. Short-term experimental SD reliably decreases pain thresholds [ 18 , 19 ] and attenuates morphine analgesia [ 20 , 21 ]. Thus, SD may differentially reshape central pharmacological responsiveness depending on the specific neurochemical system engaged, warranting a mechanistically refined understanding of these interactions. Clinically, the interplay between sleep disturbances, chronic pain, anxiety, and depression forms a complex neuroendocrine network that influences drug sensitivity and stress vulnerability [ 22 , 23 ]. Sustained SD over several days can induce perceptual distortions and hallucinations, reflecting possible hyperdopaminergic states and maladaptive neuroplasticity within cortical–subcortical circuits [ 24 , 25 ]. Such phenomena suggest that sleep loss represents not merely a state of neural fatigue but also a deep-seated qualitative reorganization of neural function, marked by disrupted excitation-inhibition balance and local circuit instability. However, despite extensive correlative evidence, integrative studies that simultaneously examine region-specific neuronal activity, molecular plasticity, and behavioral alterations within the same sleep dysregulation model while assessing the reversibility of these changes after recovery sleep (RS) remain scarce. We sought to delineate the multidimensional impact of SD on neural circuit remodeling, pain sensitivity, pharmacological responsiveness, and affective behaviors in the same subjects. Focusing on functional hubs involved in pain, emotion, stress, and immune regulation–including the paraventricular nucleus (PVN) and arcuate nucleus (ARC) of the hypothalamus, mesolimbic and mesocortical dopaminergic pathways, the parabrachial nucleus in the brainstem, and higher-order default mode networks–we systematically tracked neuronal activity and gene expression changes induced by sleep loss and examined the extent to which RS restores these perturbations. This integrated neuropharmacological framework aimed to elucidate how SD reconfigures the regulatory architecture of the brain and redefines the neurobiological and pharmacological significance of sleep in maintaining organismal homeostasis under the demands of modern society. 2. Materials and Methods Animals The experimental procedures were conducted in accordance with the Guiding Principles for the Care and Use of Laboratory Animals of Hoshi University School of Pharmacy and Pharmaceutical Sciences and were approved by the institutional ethics committee. All experiments adhered to the ARRIVE guidelines. Every effort was made to minimize both the number and suffering of animals. Male C57BL/6J (Tokyo Laboratory Animals Science Co. Ltd., Tokyo, Japan), male STOCK Tg(Pomc1-Cre)16Lowl/J [proopiomelanocortin (POMC)-Cre; Stock No: 005965, Jackson Laboratory, ME, USA] mice and male c-fos-TRAP2::tdTomato mice were used in this study. To standardize the genetic background, POMC-Cre mice were backcrossed with C57BL/6J mice for more than 10 generations. Homozygous POMC-Cre mice were generated by heterozygous intercrossing and selected for the experiments. c-Fos-TRAP2::tdTomate mice were breeding c-Fos-2AiCreERT2 mice [C57BL/6-Fos tm1(icreERT2)Phsh; Cyagen Biosciences Inc., Santa Clara, CA, USA] with LSL-tdTomato mice [B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J; Stock #007914, Jackson Laboratory]. Animals were maintained under controlled environmental conditions (temperature: 24 ± 1°C; humidity: 55 ± 5%; 12 h/12 h light–dark cycle, lights on at 8:00). Food and water were provided ad libitum in the home cages. To reduce experimental bias, independent investigators performed distinct experimental steps. A total of 250 mice were used in this study. Sleep deprivation (SD) SD was performed using a gentle-handling paradigm [ 26 – 28 ]. Whenever behavioral signs of sleep appeared, the mice were lightly touched with a soft paint brush to maintain wakefulness, and stimulation was continued until a clear arousal response was observed. SD was conducted for 6 h during the light phase (8:00–14:00) over three consecutive days. For the recovery experiments, an additional group underwent the same SD protocol followed by seven days of unrestricted RS ( ad libitum ). Postoperative pain model Postoperative pain was induced under 3% isoflurane anesthesia as previously described [ 29 ]. A 3-mm longitudinal incision was made through the skin and fascia of the plantar surface of the right hind paw using a No. 23 scalpel. For the transient pain (Brennan) model, a 3-mm incision was made into the plantaris muscle using a scalpel. In contrast, for the persistent postoperative pain model, the same incision was followed by a 3-mm muscle cut using a monopolar electrosurgical unit (50 W; Vetroson® V-10, Summit Hill Laboratories, Tinton Falls, NJ, USA) with a dispersive electrode pad placed beneath the mouse. The skin was closed using two 7–0 nylon horizontal mattress sutures. For the sham surgery, the muscle was exposed but not incised. A plantar incision was made either after three days of SD or after the subsequent seven-day recovery period. von Frey filament test Mechanical allodynia was assessed using a plantar electronic von Frey anesthesiometer (ALMEMO 2450; Ahlborn; IITC/Life Science). The probe tip was applied perpendicularly to the plantar surface of the hind paw and the pressure threshold (g) required to elicit paw withdrawal was recorded as the pain threshold. Hot-plate assay Thermal nociception (50°C) and morphine-induced antinociception (55°C) were evaluated using a hot-plate apparatus (Muromachi Kikai Co., Ltd., Tokyo, Japan) by measuring the latency to paw licking, tapping, or jumping. For nociception, latencies were obtained before and after the 3-day SD period, and for RS experiments, measurements were repeated after the 7-day recovery phase. Morphine-induced antinociception was assessed after subcutaneous administration of morphine (20 mg/kg). Antinociception was calculated as: $$\:\text{%}\text{A}\text{n}\text{t}\text{i}\text{n}\text{o}\text{c}\text{i}\text{c}\text{e}\text{p}\text{t}\text{i}\text{o}\text{n}=100\times\:\frac{(\text{p}\text{o}\text{s}\text{t}\text{d}\text{r}\text{u}\text{g}\:\text{l}\text{a}\text{t}\text{e}\text{n}\text{c}\text{y}-\text{p}\text{r}\text{e}\text{d}\text{r}\text{u}\text{g}\:\text{l}\text{a}\text{t}\text{e}\text{n}\text{c}\text{y})}{\left(\text{c}\text{u}\text{t}\text{o}\text{f}\text{f}\:\text{t}\text{i}\text{m}\text{e}-\text{p}\text{r}\text{e}\text{d}\text{r}\text{u}\text{g}\:\text{l}\text{a}\text{t}\text{e}\text{n}\text{c}\text{y}\right)}$$ The cut-off time was 30 s to avoid tissue injury. Morphine-evoked antinociception was assessed after SD or after the subsequent recovery period. LPS challenge To assess endotoxin-induced systemic responses under SD, lipopolysaccharide (LPS; Escherichia Coli O55:B5, 600 µg/100 µL/mouse, Sigma-Aldrich) was administered intravenously via the tail vein either immediately after the 3-day SD or after 7 days of RS. Body weight, rectal temperature, and survival were monitored at the defined time points. Rectal temperature was measured using a digital thermistor (KN-91; Natsume Seisakusho, Tokyo, Japan), with the probe lubricated and gently inserted into the rectum. Assessment of hypnotic effect To examine the effect of SD on GABAergic hypnotic efficacy, a loss-of-righting-reflex (LORR) assay was performed. Following SD or recovery from sleep, mice received intraperitoneal zolpidem (20 mg/kg) or saline. After drug administration, the ataxic mice were placed supine on a V-shaped plastic plate and observed until they righted three consecutive times within 30 s. The time from injection to loss of the righting reflex was defined as the LORR latency, and the duration from loss to recovery was recorded as the LORR duration. Locomotor activity Locomotor activity was monitored using a three-beam infrared beam-break system (Three-points Meter; O’Hara & Co., Ltd., Tokyo, Japan), which detects full-body displacement along the x- and y-axes, while minimizing artifacts from tail or limb movements. After 60 min of habituation, the mice were injected subcutaneously with morphine (20 mg/kg; Daiichi-Sankyo Co., Ltd., Tokyo, Japan). Activity counts were collected in 1-min bins over 180 min. Morphine-evoked hyperlocomotion was recorded after SD or subsequent RS. Stereotaxic adeno-associated virus (AAV) injection and chemogenetic manipulation of hypothalamic POMC neurons Mice were anesthetized with 3% isoflurane and mounted on a stereotaxic frame (RWD Life Science, CA, USA). A Cre-dependent adeno-associated virus (AAV) vector (AAV9-hSyn-Flex-hM3Dq-mCherry; 2 × 10 12 copies/mL, VectorBuilder) was bilaterally injected into the ARC of POMC-Cre mice (coordinates relative to bregma: A/P = − 1.5 mm; M/L = ± 0.2 mm; D/V = − 4.8 mm; medial angle = 0°). Injections (150 nL per side) were delivered using Nanoject III (3-000-207; Drummond Scientific Company, Broomall, PA, USA). Mice were allowed to recover for ≥ 2 weeks to permit robust expression of hM3Dq. Following recovery, AAV-injected mice were subjected to a persistent postoperative pain model, and clozapine- N -oxide (CNO; 3 mg/kg, i.p.; Abcam, Cambridge, UK) was administered once daily from postoperative day (POD) 1 to activate the ARC POMC neurons during nociceptive testing. Immunofluorescence Mice were transcardially perfused with 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (pH 7.4) under anesthesia with 3% isoflurane. Brain tissues were dissected after being post-fixation with 4% PFA and cryoprotection in 20–30% (w/v) sucrose (FUJIFILM Wako Pure Chemical Corp.). Brain sections were embedded in O.C.T. compound (Sakura Fine Technical, Tokyo, Japan), and frozen sections were cut using a cryostat (CM1860; Leica Microsystems, Heidelberg, Germany). Immunofluorescence was detected using a light microscope (BX-53; Olympus, Tokyo, Japan), and images were captured using a high-sensitivity digital CCD camera (MD-695; Molecular Devices, San Jose, CA, USA). Imaging analysis was performed using the Metamorph 7.8 software (Molecular Devices). Reverse transcription and quantitative polymerase chain reaction (RT-qPCR) Total RNA was extracted from microdissected regions of the brain including the medial prefrontal cortex (mPFC), nucleus accumbens (NAcc), PVN, ARC, lateral hypothalamus (LH), periaqueductal gray (PAG), ventral tegmental area (VTA), and parabrachial nucleus (PBN) using the mirVana™ miRNA Isolation Kit (Thermo Fisher Scientific Inc., MA, USA). First-strand cDNA was synthesized using the SuperScript® VILO™ cDNA Synthesis Kit, and qPCR was conducted using Fast SYBR® Green Master Mix (Thermo Fisher Scientific Inc.) and gene-specific primers (Supplementary Table 1). Gene expression was normalized to glyceraldehyde 3-phosphate dehydrogenase ( Gapdh ) using the 2^-∆∆CT method. Based on previous reports, Pomc expression is known to be markedly lower outside the ARC than within it [ 30 ]. Therefore, under the present PCR assay conditions, we anticipated low Pomc expression in extra-arcuate regions. To ensure reliable quantification of region-specific expression, regions in which the mean Ct value for each target gene exceeded 30 across groups were a priori regarded as falling below the limit of reliable quantification (LOQ). Such data were designated as N.D. (not detectable) and excluded from subsequent analyses. Labeling of Pain-TRAPed neurons To induce a persistent postoperative pain state, we generated a standardized plantar incision model in cFos-TRAP2::tdTomato mice. A 3-mm longitudinal cut was made along the plantaris muscle of the right hind paw with a monopolar electrosurgical unit set to 50 W (Summit Hill Laboratories), following the methodological framework described by Katsuda and colleagues [ 31 ]. Sham-operated mice were exposed only to 3% isoflurane anesthesia without incision. Before TRAP induction of neuronal trapping, mice underwent a structured habituation protocol: they were placed individually inside black plastic cylinders for 1 h per day over three consecutive days. Fifteen days after electrocautery, tamoxifen (100 mg/kg, i.p.) was administered to initiate activity-dependent genetic tagging. Four hours later, a calibrated 0.07-g von Frey filament was applied to the ipsilateral hind paw at 30-s intervals for 30 min to evoke the Pain-TRAP response and capture nociception-related neuronal ensembles. Seven days after the Pain-TRAP procedure, mice were deeply anesthetized for terminal perfusion and subsequent histological analyses. Definition of plasticity-related modulators and quantification of gene expression In this study, c-Fos , ΔFosB , Homer1a , and Fkbp5 were operationally defined as plasticity-related modulators. Gene expression levels were quantified using qPCR and processed with the ΔΔCt method, yielding relative expression values expressed as 2^-ΔΔCt for each individual animal. For each mouse, the four relative expression values were averaged to generate a single integrated plasticity metric, termed the Plasticity-index (P-index). Thus, the P-index represents an individual-level parameter and was treated as an independent biological endpoint within each experimental group (control, SD, recovery). Statistical analysis focused exclusively on group-dependent differences in the P-index. Comparisons among the three groups were performed using a one-way ANOVA, followed by Tukey’s post hoc test. Stacked bar graphs were generated solely to visualize the relative contributions of individual plasticity-related modulators to the P-index and were not used for statistical inference. Statistical analysis All data are expressed as mean ± SEM. Statistical significance was determined using paired and unpaired Student’s t -test, one-way ANOVA, or two-way ANOVA followed by Turkey or Bonferroni post hoc tests, chi-square tests, or log-rank (Kaplan–Meier) survival analysis, as appropriate, using GraphPad Prism software (version 9.5; GraphPad Software, CA, USA). 3. Results Sleep loss amplifies thermal and mechanical hypersensitivity and diminishes µ-opioid analgesic efficacy To investigate how SD reconfigures sensory responsiveness and alters the pharmacodynamics of µ-opioid, benzodiazepine, and LPS responses, mice were subjected to an SD paradigm consisting of 6 h of wakefulness during the light phase for three consecutive days. A RS condition was subsequently implemented, allowing 7 days of unrestricted sleep to assess the reversibility of SD-induced perturbations in physiological functions and systemic homeostasis (Fig. 1 A). As an initial assessment of sensory responsiveness, nociceptive latency was quantified using the 50°C hot-plate assay. Latency was significantly reduced relative to the baseline before SD, indicating heightened nociceptive sensitivity, whereas this effect was fully normalized following the RS phase (Fig. 1 B; **p < 0.01, before SD vs. after SD, paired t -test). To determine whether sleep loss interferes with µ-opioid-mediated analgesia, morphine-induced antinociception was examined using the hot-plate test (Fig. 1 D). Morphine (20 mg/kg, s.c.)-evoked analgesia was markedly blunted by SD compared with controls (Fig. 1 D; *p < 0.05, **p < 0.01, control vs. SD; two-way ANOVA followed by Bonferroni test). Remarkably, RS restored the analgesic efficacy of morphine (Fig. 1 E; *p < 0.05, control vs. SD; #p < 0.05, ##p < 0.01, SD vs. recovery; two-way ANOVA followed by Tukey test). Association between sleep loss, heightened pain sensitivity, and reversibility through the ARC-POMC system following sleep loss Given the SD-induced hypersensitivity and the attenuation of µ-opioid analgesia, transcript levels of the µ-opioid receptor (MOR, Oprml ) and endogenous opioid peptide precursors (preprodynorphin; Pdyn , preproenkephalin; Penk , and proopidmelanocortin; Pomc ) were quantified across multiple pain- and sleep-associated brain regions (Fig. 2 A-D). These opioid-related transcripts were broadly detectable across the examined brain regions, with Pomc mRNA showing marked enrichment within the ARC (Supplementary Fig. 1). Notably, only Pomc mRNA in the ARC was significantly decreased following SD (Fig. 2 D; *p < 0.05, control vs. SD, unpaired Student's t -test), whereas Oprml (Fig. 2 A), Pdyn (Fig. 2 B), and Penk (Fig. 2 C) remained unaffected. Consistent with behavioral recovery, the SD-induced reduction in Pomc expression was fully reversed by RS, restoring expression to control levels (Fig. 2 E; *p < 0.05, control vs. SD; #p < 0.05, SD vs. recovery; one-way ANOVA with Tukey correction). To establish a causal link between SD-driven sensory hypersensitivity and Pomc downregulation, a cell type-specific chemogenetic approach was employed. An AAV vector encoding the excitatory DREADD receptor (hM3Dq) was bilaterally injected into the ARC of POMC-Cre mice, permitting the CNO-induced activation of POMC neurons (Fig. 2 F). Following the induction of persistent postoperative pain by plantar incision using a high-frequency electrosurgical unit, mechanical thresholds were measured after CNO administration via von Frey testing (Fig. 2 F). At POD1, both WT::M3Dq and POMC-Cre::M3Dq mice displayed pronounced ipsilateral allodynia, as indicated by reduced paw-withdrawal thresholds compared to the contralateral side (Fig. 2 G). Critically, CNO administration selectively restored the ipsilateral thresholds in POMC-Cre::M3Dq mice within 30 min, whereas those in WT::M3Dq mice remained unaffected (Fig. 2 G; **p < 0.01, 0 min vs. 30 min in POMC-Cre::M3Dq, paired t -test). Collectively, these results identified ARC-POMC neurons as pivotal regulatory nodes that link SD to altered pain processing and µ-opioid responsiveness. Moreover, during chronic postoperative pain, repeated activation of ARC-POMC neurons progressively normalized mechanical thresholds, signifying the functional restoration of the endogenous antinociceptive circuitry (Fig. 2 H; p > 0.05 at POD49 and POD56, POMC-Cre::M3Dq contralateral vs. POMC-Cre::M3Dq ipsirateral; two-way ANOVA with Tukey correction). The parabrachial nucleus (PBN) has recently been recognized not only as a critical relay station that receives nociceptive inputs from the dorsal horn of the spinal cord, but also as a hub-like switch in the regulation of sleep–wake states. To capture pain state–dependent ensembles in the PBN, persistent postoperative pain was induced in cfos-TRAP2::tdTomato mice, and tamoxifen was administered on postoperative day 15 to permanently tag c-Fos–expressing cells (Fig. 3 A). Under these conditions, mice with persistent postoperative pain exhibited a marked increase in c-Fos–positive neurons within the PBN, suggesting that a hyperactive neuronal ensemble is selectively recruited in this nucleus during the chronic pain state (Fig. 3 B). Given that the PBN serves as a key recipient of nociceptive information, neuronal activity in this nucleus was next examined under SD (Fig. 3 C), and SD robustly increased c-Fos and ΔFosB mRNA in the PBN, and RS restored both markers to baseline (Fig. 3 D; *p < 0.05, control vs. SD, one-way ANOVA with Tukey correction). Nociception-related neuropeptide transcripts within the PBN were further quantified, revealing that SD significantly upregulated Calca (CGRP) mRNA expression, whereas both Tac1 (substance P) and Pdyn mRNA remained unchanged (Fig. 3 E; *p < 0.05, control vs. SD, one-way ANOVA with Tukey correction). This transcriptomic change was restored to control-like levels after RS (Fig. 3 E; #p < 0.05, SD vs. recovery, one-way ANOVA with Tukey correction). SD amplifies endotoxin-induced systemic responses To determine whether sleep loss altered systemic inflammatory reactivity, an endotoxin challenge was performed. Mice received an intravenous injection of LPS (600 µg/mouse), and changes in body weight and core (rectal) temperature were monitored 12 h post-injection (Fig. 4 A,B). At 12 h after LPS treatment, both body weight and core temperature were markedly reduced compared to saline-treated controls (Fig. 4 A, body weight, ***p < 0.001, control-saline vs. control-LPS; Fig. 4 B, body temperature, ***p < 0.001, control-saline vs. control-LPS; one-way ANOVA with Tukey correction). Relative to the LPS-treated controls, SD further potentiated the systemic responses, producing greater weight loss and more profound hypothermia (Fig. 4 B, ***p < 0.001, control-LPS vs. SD-LPS, one-way ANOVA with Tukey correction). Remarkably, these SD-induced exaggerations were reversed by RS, with both parameters returning to control levels (Fig. 4 A, body weight, *p < 0.05, SD-LPS vs. recovery-LPS; Fig. 4 B, body temperature, **p < 0.01, SD-LPS vs. recovery-LPS; one-way ANOVA with Tukey correction). The survival outcomes mirrored these physiological trends. Kaplan–Meier analysis revealed that LPS administration reduced survival compared to saline-treated controls, and SD further compromised survival relative to LPS-treated controls (Fig. 4 C; *p < 0.05, log-rank test). Conversely, RS mitigated this SD-induced mortality enhancement, partially restoring survival probabilities (Fig. 4 C; **p < 0.01, log-rank test). To delineate the hypothalamic correlates of the SD-potentiated LPS response, the PVN, ARC, and LH were microdissected for transcriptomic analysis (Fig. 4 D). Within the hypothalamus, SD selectively elevated c-Fos mRNA expression in the PVN, with no significant changes observed in the ARC or LH (Fig. 4 E; *p < 0.05, control vs. SD; unpaired Student's t -test). This c-Fos change was restored to control-like levels after RS (Fig. 4 F; **p < 0.01, control vs. SD; #p 0.05, one-way ANOVA with Tukey correction). Reduced hypnotic efficacy after sleep loss: diminished LORR incidence and altered onset/duration dynamics To assess whether sleep loss modulates hypnotic sensitivity, behavioral endpoints of zolpidem-induced sedation were quantified, including the incidence of LORR, latency to hypnotic onset (LORR latency), and duration of hypnotic maintenance (LORR duration). Zolpidem induced LORR in all control mice (12/12, 100%), whereas only seven of twelve SD mice (58%) exhibited LORR, representing a significant reduction in hypnotic efficacy. Following RS, the incidence of LORR was fully restored to control levels (12/12, 100%), indicating that sleep loss impaired zolpidem-induced hypnosis, whereas recovery normalized it (Fig. 5 A; ***p < 0.001, chi-squared test). Among the mice that exhibited LORR, SD significantly delayed hypnotic onset and shortened its duration; these effects were both normalized following RS (Fig. 5 B, LORR latency: ***p < 0.001, control vs. SD and SD vs. recovery; Fig. 5 C, LORR duration: **p < 0.01, control vs. SD and SD vs. recovery; one-way ANOVA with Tukey correction). To explore molecular substrates underlying altered hypnotic sensitivity, mRNA levels of GABA A receptor (GABA A R) αl subunit ( Gabral ) and glutamate decarboxylase 67 ( Gad1 , GAD67) were measured in the PVN, ARC, and LH (Fig. 5 D). No significant group differences were detected (Fig. 5 E,F; p > 0.05, one-way ANOVA with Tukey correction). Similarly, orexin ( Hcrt ) expression in the LH remained unchanged (Fig. 5 G; p > 0.05, one-way ANOVA with Tukey correction). Sleep loss amplifies morphine-evoked dopaminergic psychostimulation, persisting despite RS Furthermore, to obtain an integrated measure of transcriptional changes across plasticity-related genes, 2 –ΔΔCt values for each gene were used to calculate a plasticity-index (P-index), defined as the mean expression of four genes: c-Fos , ΔFosB , Homer1a , and Fkbp5 in the mPFC, NAcc and VTA (Fig. 7 A). Under SD conditions, P-index values were increased in all three regions examined relative to controls (Fig. 7 B–D; p < 0.001, control vs. SD, one-way ANOVA with Tukey’s correction), and these SD-induced elevations were attenuated by RS (Fig. 7 B–D; p < 0.01 and p < 0.001, SD vs. recovery). However, whereas RS normalized P-index values in the VTA and NAcc to control levels, those in the mPFC remained significantly higher in the RS mice than in control mice. (Fig. 7 B; p < 0.05, control vs. recovery). These findings suggest that large-scale reconfiguration of plasticity-related gene expression induced by SD is preferentially sustained within the mPFC. 4. Discussion In this study, we employed a short-term SD protocol to examine behavioral pain thresholds, pharmacological responses to µ-opioid and GABA A R agonists, stress and infection responses to endogenous endotoxins, and transcriptional alterations in opioid signaling across multiple brain regions. In an acute postoperative pain model, evaluation of withdrawal latency to thermal stimuli revealed that SD significantly reduced pain thresholds. This hypersensitivity was most pronounced immediately after SD but was largely reversible after one week of RS. Furthermore, SD transiently attenuated the analgesic efficacy of µ-opioid receptor agonists, but this effect was fully restored after RS. The ARC of the hypothalamus contains the highest density of POMC-expressing neurons, which produce β-endorphin, an endogenous µ-opioid peptide. Recent studies have shown that in the ARC of neuropathic pain model mice, expression of endopeptidases required for the cleavage of POMC into β-endorphin is suppressed, resulting in decreased β-endorphin levels and reduced pain thresholds [ 35 , 36 ]. In the present study, we found that SD markedly decreased POMC mRNA expression in the ARC, which returned to control levels after RS. Moreover, selective chemogenetic activation of ARC-POMC neurons via AAV-mediated expression of hM3Dq in POMC-Cre mice and subsequent administration of CNO significantly restored the diminished pain threshold observed in persistent postoperative pain. ARC neurons containing β-endorphin project to the PAG, a critical center for descending pain inhibition, thereby contributing to endogenous analgesic control [ 37 ]. Thus, the reversible reduction in POMC expression observed in the ARC may reflect a transient impairment in endogenous β-endorphin production, resulting in temporary attenuation of the intrinsic pain modulation system and diminished efficacy of µ-opioid receptor agonists. POMC gene expression is regulated by two principal pathways, the stress hormone axis and the leptin signaling cascade. The POMC promoter contains a glucocorticoid response element, and the binding of steroid hormones such as corticosterone, to glucocorticoid receptors induces transcriptional repression [ 38 , 39 ]. Conversely, leptin promotes POMC transcriptional activity through the JAK2–STAT3 pathway in POMC-expressing neurons [ 40 ]. Previous studies reported that SD increases circulating corticosterone levels [ 41 ] and decreases plasma leptin levels [ 42 ]. In the present study, we observed that SD induced a robust increase in c-Fos expression, a molecular marker of neuronal activation that constitutes the major origin of the HPA axis, in the PVN of the hypothalamus. This finding suggests that SD persistently activates PVN neurons, thereby promoting the excessive release of the endogenous glucocorticoid corticosterone. Taken together, these results indicated that the reduction in POMC expression in ARC neurons caused by SD may be at least partly attributable to the hyperactivation of glucocorticoid signaling triggered by PVN activation, together with the suppression of leptin signaling. Indeed, in our previous study, the pharmacogenetic activation of CRH-containing neurons in the PVN exacerbated and prolonged postoperative pain, supporting the validity of this hypothesis [ 43 ]. The MOR is predominantly expressed in excitatory neurons located in layers V–VI of the cerebral cortex and is rarely found in inhibitory interneurons. µ-opioid receptor agonists such as morphine suppress the activity of these excitatory neurons through Gi protein-coupled signaling, thereby attenuating nociceptive transmission in the cingulate cortex, a key terminal region of the ascending pain pathway [ 44 , 45 ]. In the present study, we observed a significant increase in c-Fos expression in the mPFC, including the cingulate cortex, after SD. This finding raises the possibility that sustained cortical hyperexcitability may contribute, at least in part, to the attenuation of µ-opioid–mediated analgesia. Intriguingly, reversible neural hyperactivation has also been detected in the PBN, which has recently emerged as a critical relay node for ascending pain signaling [ 46 , 47 ]. Consistent with this view, activity-dependent CFOS-TRAP labeling under a persistent postoperative pain state revealed selective recruitment of a hyperactive neuronal ensemble within the PBN, indicating that this brainstem hub is poised to integrate ongoing nociceptive drive with state-dependent modulatory inputs. This hyperactivation is accompanied by a transient elevation of CGRP, a major neuropeptide within the PBN that functions as a pronociceptive mediator. Neuronal activity and CGRP expression returned to control levels after RS, paralleling the restoration of pain thresholds. These findings indicate that SD transiently induces widespread excitation within the intrinsic pain networks of the brain, leading to temporary destabilization of endogenous analgesic systems and a corresponding reduction in pain thresholds, which can be normalized by sufficient restorative sleep. SD also diminished the hypnotic efficacy of zolpidem, a non-benzodiazepine GABA A R agonist. However, this suppression was fully reversible after RS. Notably, SD did not alter the gene expression of GABA A R subunits or GABA-synthesizing enzymes in the hypothalamus. The hypnotic action of GABA A R agonists is generally thought to result from the inhibition of excitatory neurons, acting not only on hypothalamic–ascending arousal circuits, but also on the medial prefrontal cortex, a region implicated in slow-wave generation [ 48 ]. In our study, SD increased the expression of the activity-dependent marker c-Fos not only in the hypothalamus but also in the mPFC. These results suggest that excessive activation of excitatory neurons under sleep-restricted conditions may physiologically counteract GABA A R-mediated inhibition, thereby reducing the apparent hypnotic effects of zolpidem. Another key finding of this study is that SD did not suppress, but rather enhanced, the locomotor-stimulating effect of morphine. Notably, this enhancement persisted even after the RS. The psychomotor activation induced by µ-opioid receptor agonists is thought to be mediated by indirect activation of the mesolimbic and nigrostriatal dopaminergic circuits through desensitization of GABAergic neurons [ 49 ]. Thus, SD produced a distinctive bidirectional effect, reversibly attenuating the potent analgesic efficacy of morphine through multiple mechanisms, while paradoxically enhancing and sustaining dopaminergic locomotor stimulation. Indeed, previous studies have shown that during sleep restriction, the mesolimbic dopaminergic pathway projecting from the VTA to the NAcc becomes sensitized, resulting in long-lasting behavioral alterations [ 50 ]. Consistent with these reports, we observed increased expression of c-Fos and ΔFosB within the VTA, NAcc, and mPFC, all of which belong to the mesolimbic–cortical dopaminergic network. Importantly, the sleep recovery period failed to reset c-Fos expression in the mPFC, suggesting persistent cortical activation. In line with this, a composite plasticity-index (P-index), quantitatively derived from c-Fos , ΔFosB , Homer1a , and Fkbp5 , remained selectively elevated in the mPFC after RS, whereas it was normalized in the VTA and NAcc, indicating that SD imposes a durable plastic imprint on prefrontal circuits. These results provide a theoretical basis for the persistence of morphine-induced locomotor sensitization under sleep-restricted conditions. A crucial implication of these findings is that analgesic responses normalize with RS, whereas locomotor activation remains unaffected, indicating distinct reversibility between these physiological processes. Considering that the mPFC serves as the central hub of the default mode network (DMN) [ 51 ], it is plausible that SD disrupts DMN connectivity, thereby sustaining dopaminergic psychomotor sensitization. Moreover, the opposing transcriptional signatures observed in the reward circuitry—characterized by increased Bdnf mRNA in the NAcc and decreased Bdnf mRNA in the VTA—resembled those seen in stimulant addiction, suggesting the emergence of irreversible distortions in reward-related plasticity [ 32 ]. Collectively, these findings indicate that sleep disturbance destabilizes the DMN, predisposing higher-order integrative functions to collapse, and potentially establishing a pathophysiological state of vulnerability. Therefore, chronic SD may not be readily reversible through short-term RS. Our study also revealed that systemic responses to the LPS challenge were markedly exacerbated by SD, whereas the subsequent introduction of RS effectively mitigated this excessive inflammatory reaction. We previously demonstrated that LPS administration induces hypothalamic neuroinflammation accompanied by glial hyperactivation [ 52 ]. Previous studies have shown that transient sleep loss primes the innate immune system by elevating the circulating levels of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) [ 53 ]. This pre-activated state can synergistically amplify cytokine release after LPS exposure, disrupt hypothalamic thermoregulation, and result in profound hypothermia, anorexia, reduced locomotor activity, loss of body weight, and even increased lethality. Therefore, acute SD may transiently compromise integrated hypothalamic functions, leading to systemic disarray across multiple homeostatic networks and eliciting the diverse physiological disturbances observed in this study. Remarkably, the fact that RS restored homeostatic immune function more effectively than anticipated underscores the remarkable resilience of the immune regulatory system, even after transient functional breakdown. Future investigations should focus on identifying molecular profiles in the brain that are particularly vulnerable to irreversible disruption under SD and determining whether these changes act as triggers for secondary pathologies. Defining the molecular signatures that initiate systemic deterioration and establishing early interventions during the recovery window are essential for preventing the transition from transient dysfunction to chronic disease. Overall, the findings of this study clearly demonstrate that physiological processes, such as pain sensitivity, immune defense, and pharmacological responsiveness, are profoundly modulated by both the quantity and quality of sleep. Furthermore, hyperactivation of the PVN, mPFC, PBN, VTA, and NAcc, together with the suppression of POMC gene expression in the ARC, collectively contributed to SD-induced phenomena, including hyperalgesia, attenuation of opioid analgesia, potentiation of morphine-induced locomotion, diminished hypnotic efficacy of GABA A R agonists, and reduced resistance to endotoxin challenge. Although short-term SD allows for substantial physiological restoration through RS, certain neural responses, particularly those involving the dopaminergic system and DMN coordination, may not easily return to baseline. This study highlights the indispensable role of sleep in maintaining systemic homeostasis across multiple physiological domains, including pain processing, immune regulation, and pharmacological adaptation. These findings strongly suggest that qualitative sleep impairment can act as a driving force for the progression from transient dysfunction to chronic pathological states. Declarations Funding This research was supported by AMED under Grant Number JP20ek0610024 and Hoshi University. Declaration of competing interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Author Contribution Conceptualization , Yasuyuki Nagumo and Minoru Narita; Data curation , Yasuyuki Nagumo, Yusuke Hamada, and Naoko Kuzumaki; Formal analysis , Yasuyuki Nagumo and Yusuke Hamada; Funding acquisition , Minoru Narita; Investigation , Teruyo Kishino, Yasuyuki Nagumo, Yusuke Hamada, Michiko Narita, Takumi Yoshizawa, Jion Takise, Yukari Suda and Naoko Kuzumaki; Methodology , Teruyo Kishino, Yasuyuki Nagumo, Yusuke Hamada and Michiko Narita; Project administration , Yasuyuki Nagumo and Minoru Narita; Supervision , Naoko Kuzumaki and Minoru Narita; Validation , Yasuyuki Nagumo; Visualization , Yasuyuki Nagumo; Writing–original draft , Teruyo Kishino, Yasuyuki Nagumo, and Minoru Narita; Writing–review & editing , Yasuyuki Nagumo, Tomohisa Mori, Naoko Kuzumaki and Minoru Narita. Acknowledgement The authors thank to Ms. Yu Okamoto, Mr. Hitoshi Makabe, Mr. Kensuke Yamashita, Mr. Naoki Yanagi, Ms. Nanami Morokata, Ms. Shione Suzuki, Mr. Kazuya Karaki, and Mr. Jiang ChengHao (Hoshi University) for grateful experiment supports. We would like to thank Editage (www.editage.jp) for English language editing. Data Availability The datasets generated and analyzed in this study are available from the corresponding authors upon reasonable request. References Franken P, Dijk D-J (2024) Sleep and circadian rhythmicity as entangled processes serving homeostasis. Nat Rev Neurosci 25:43–59. https://doi.org/10.1038/s41583-023-00764-z Lendner JD, Niethard N, Mander BA, van Schalkwijk FJ, Schuh-Hofer S, Schmidt H, Knight RT, Born J, Walker MP, Lin JJ, Helfrich RF (2023) Human REM sleep recalibrates neural activity in support of memory formation. 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Eur J Physiol 463:121–137. https://doi.org/10.1007/s00424-011-1044-0 Additional Declarations No competing interests reported. Supplementary Files KishinoetalNeurochemicalResSupplementaryfinal.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-8228487","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":554329162,"identity":"c8049795-1d90-4ce5-ab16-1a52f189cac7","order_by":0,"name":"Teruyo Kishino","email":"","orcid":"","institution":"Hoshi University","correspondingAuthor":false,"prefix":"","firstName":"Teruyo","middleName":"","lastName":"Kishino","suffix":""},{"id":554329163,"identity":"f3226de0-494f-48a7-8dc4-d3518813c7d2","order_by":1,"name":"Yasuyuki Nagumo","email":"","orcid":"","institution":"National Cancer Center Research 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07:09:50","extension":"html","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":179370,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8228487/v1/83b970ec05d6069aaeee5d22.html"},{"id":97656176,"identity":"82f111c5-8ac6-4238-b858-771bb493814d","added_by":"auto","created_at":"2025-12-08 07:09:49","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":167875,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eReduced pain thresholds and diminished morphine analgesia following SD.\u003c/strong\u003e(A) Experimental design of the SD and sleep recovery protocols. (B) Changes in withdrawal latency to thermal stimuli (50°C) before and after SD. Data represent the mean ± SEM of 6 mice. **p \u0026lt; 0.01, paired \u003cem\u003et\u003c/em\u003e-test. (C) Prolongation of postoperative mechanical allodynia after SD. Paw withdrawal thresholds were determined using an electronic von Frey aesthesiometer and are presented as mean ± SEM of 4–6 mice. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001 vs Control–Sham; #p \u0026lt; 0.05, ##p \u0026lt; 0.01 Control–Incision vs SD–Incision; †p \u0026lt; 0.05 SD-Incision vs Recovery–Incision (two-way ANOVA with Tukey post hoc test). (D) Altered morphine-induced analgesia following SD. Analgesia was assessed as latency to nociceptive responses in the 55°C hot-plate test after morphine administration (20 mg/kg, s.c.). Data represent the mean ± SEM of 12 mice. *p \u0026lt; 0.05, **p \u0026lt; 0.01 (two-way ANOVA with Bonferroni post hoc test). (E) Effect of RSon morphine-induced analgesia after SD. Morphine analgesia was assessed in the hot-plate test as latency to nociceptive behaviors in control, SD, and recovery groups. Data represent the mean ± SEM of 6 mice. *p \u0026lt; 0.05, control vs SD; #p \u0026lt; 0.05, ##p \u0026lt; 0.01, SD vs recovery (two-way ANOVA with Tukey post hoc test).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8228487/v1/7792d32d7171e8f0bdd01933.png"},{"id":97673168,"identity":"9540dda3-4085-4d1d-ad67-8f2ebc6bd5d1","added_by":"auto","created_at":"2025-12-08 09:39:33","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":206255,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSelective reduction of POMC expression in the arcuate nucleus (ARC) and pain relief by activation of ARC POMC neurons.\u003c/strong\u003e (A–D) mRNA expression profiles for (A) μ-opioid receptor (MOR; \u003cem\u003eOprm1\u003c/em\u003e), (B) prodynorphin (\u003cem\u003ePdyn\u003c/em\u003e), (C) proenkephalin (\u003cem\u003ePenk\u003c/em\u003e), and (D) proopiomelanocortin (\u003cem\u003ePomc\u003c/em\u003e) across pain- and sleep-related brain regions in SD mice. Data are presented as the mean ± SEM of 3–6 mice. *p \u0026lt; 0.05, unpaired Student’s \u003cem\u003et\u003c/em\u003e-test. N.D. indicates “not detectable”: transcript levels below the assay’s limit of detection (no quantifiable amplification; see Experimental procedures). mPFC, medial prefrontal cortex; NAcc, nucleus accumbens; PVN, paraventricular nucleus of the hypothalamus; ARC, arcuate nucleus; LH, lateral hypothalamus; PAG, periaqueductal gray; PBN, parabrachial nucleus. (E) \u003cem\u003ePomc\u003c/em\u003e mRNA levels in the ARC compared among control, SD, and recovery groups. Data represent the mean ± SEM 6 mice. *p \u0026lt; 0.05, control vs SD; #p \u0026lt; 0.05, SD vs recovery (one-way ANOVA with Tukey post hoc test). (F) Schematic illustration of the ARC-targeted Cre-dependent AAV vector carrying hM3Dq DREADD in POMC-Cre mice and the postoperative pain model used to evaluate pain relief by POMC neuron activation. Representative histological image shows the site of viral vector delivery in the ARC of POMC-Cre mice and Cre-dependent DREADD and reporter expression. Scale bar = 100 μm. (G) Mechanical withdrawal thresholds on postoperative day 1, measured 30 min after CNO administration, using the von Frey test in POMC-Cre mice expressing ARC hM3Dq. Left panel: ipsilateral side; right panel: contralateral side. Data represent the mean ± SEM of 5 mice. **p \u0026lt; 0.01, paired \u003cem\u003et\u003c/em\u003e-test. (H) Time course of mechanical withdrawal thresholds during daily CNO treatment in POMC-Cre mice expressing hM3Dq in the ARC, assessed using the von Frey test. Data represent the mean ± SEM of 5 mice. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001 WT::M3Dq contralateral vs WT::M3Dq ipsilateral; #p \u0026lt; 0.05, ##p \u0026lt; 0.01, ###p \u0026lt; 0.001 POMC-Cre::M3Dq contralateral vs POMC-Cre::M3Dq ipsilateral (two-way ANOVA with Tukey post hoc test). (I) Changes in \u003cem\u003ec-Fos\u003c/em\u003e and \u003cem\u003eΔFosB\u003c/em\u003e mRNA expression levels in the PBN after SD and following RS. Data represent the mean ± SEM of 6 mice. *p \u0026lt; 0.05 (one-way ANOVA with Tukey post hoc test). (J) Changes in nociception-related neuropeptide transcripts (\u003cem\u003eCalca\u003c/em\u003e, \u003cem\u003eTac1\u003c/em\u003e, and \u003cem\u003ePdyn\u003c/em\u003e) in the PBN after SD and following RS. Data represent the mean ± SEM of 6 mice. *p \u0026lt; 0.05, control vs SD; #p \u0026lt; 0.05, SD vs recovery (one-way ANOVA with Tukey post hoc test).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8228487/v1/b9726d17e90845296cb41608.png"},{"id":97656182,"identity":"a0336313-0a74-427a-acb6-d3ea8e62663f","added_by":"auto","created_at":"2025-12-08 07:09:49","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":132456,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePostoperative pain and sleep deprivation converge on parabrachial activation and CGRP upregulation.\u003c/strong\u003e (A) Schematic of the c-Fos-TRAP:2:tdTomato strategy used to label PBN neurons activated by persistent postoperative pain. On postoperative day 15, tamoxifen (100 mg/kg, i.p.) was administered, and von Frey stimulation was applied during the subsequent TRAP window to label c-Fos–expressing neurons in the PBN. (B) Representative immunofluorescence images of c-Fos-TRAP2::tdTomato–labeled neurons in the parabrachial nucleus of control and persistent postoperative pain mice. Scale bar, 100 μm. (C) Schematic of the experimental workflow for transcriptome analysis in the PBN following SD. (D) Changes in \u003cem\u003ec-Fos\u003c/em\u003e and \u003cem\u003eΔFosB\u003c/em\u003e mRNA expression levels in the PBN after SD and following RS. Data represent the mean ± SEM of 6 mice. *p \u0026lt; 0.05 (one-way ANOVA with Tukey post hoc test). (E) Changes in nociception-related neuropeptide transcripts (\u003cem\u003eCalca\u003c/em\u003e, \u003cem\u003eTac1\u003c/em\u003e, and \u003cem\u003ePdyn\u003c/em\u003e) in the PBN after SD and following RS. Data represent the mean ± SEM of 6 mice. *p \u0026lt; 0.05, control vs SD; #p \u0026lt; 0.05, SD vs recovery (one-way ANOVA with Tukey post hoc test).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8228487/v1/4db81e507f41664fc3051da9.png"},{"id":97656195,"identity":"38c9d54c-fd55-4c8e-ac47-88bfb6f7d7b5","added_by":"auto","created_at":"2025-12-08 07:09:50","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":127547,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSD and RS modulate systemic LPS responses in mice.\u003c/strong\u003e(A, B) Body weight (A) and rectal temperature (B) 12 h after LPS administration (600 µg/mouse, i.v. \u003cem\u003evia\u003c/em\u003e tail vein) in SD and RSmice. Data represent the mean ± SEM of 7–14 mice. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001 (one-way ANOVA with Tukey post hoc test). (C) Kaplan–Meier survival curves after LPS in SD and RS mice. Survival was compared using the log-rank test. (D) Schematic of the experimental workflow for transcriptome analysis in the PVN, ARC, and LH following SD. (E) \u003cem\u003ec-Fos\u003c/em\u003emRNA levels in the PVN, ARC, and LH compared between control and SD mice. Data represent the mean ± SEM of 6 mice. **p \u0026lt; 0.01, unpaired Student’s \u003cem\u003et\u003c/em\u003e-test. (F) \u003cem\u003ec-Fos\u003c/em\u003e mRNA levels in the PVN compared among control, SD, and recovery groups. Data represent the mean ± SEM 6 mice. **p \u0026lt; 0.01, control vs SD; #p \u0026lt; 0.05, SD vs recovery (one-way ANOVA with Tukey post hoc test). (G) \u003cem\u003eTlr4\u003c/em\u003e mRNA levels in the PVN, ARC, and LH compared among control, SD, and RSmice. Data represent the mean ± SEM of 3–6 mice. (H) \u003cem\u003eCrh\u003c/em\u003e mRNA levels in the PVN compared among control, SD, and RS mice. Data represent the mean ± SEM of 6 mice.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8228487/v1/a10ddf6f0f5e45794da2cad8.png"},{"id":97656187,"identity":"0ad02170-c91c-4670-afab-de7b2eac8ba4","added_by":"auto","created_at":"2025-12-08 07:09:50","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":101554,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSD and RS alter zolpidem-induced pharmacological responses.\u003c/strong\u003e (A) Percentage of mice exhibiting loss of righting reflex (LORR) after zolpidem (20 mg/kg, i.p.) administration in control, SD, and RS mice. ***p \u0026lt; 0.001, chi-squared test. (B) Latency to onset of hypnotic LORR behavior in SD and RS mice. Data represent the mean ± SEM of 7–12 mice. ***p \u0026lt; 0.001, one-way ANOVA with Tukey post hoc test. (C) Duration of LORR behavior in SD and RS mice. Data represent the mean ± SEM of 7–12 mice. **p \u0026lt; 0.01, one-way ANOVA with Tukey post hoc test. (D) Schematic of the experimental workflow for transcriptome analysis in the PVN, ARC, and LH following SD. (E,F) mRNA levels of the GABA\u003csub\u003eA\u003c/sub\u003eR α1 subunit (\u003cem\u003eGabra1\u003c/em\u003e, D) and GAD67 (\u003cem\u003eGad1\u003c/em\u003e, E) in the PVN, ARC, and LH compared between SD and RS mice. Data represent the mean ± SEM of 3 mice. (G) Orexin (\u003cem\u003eHcrt\u003c/em\u003e) mRNA levels in the LH compared among control, SD, and RS mice. Data represent the mean ± SEM of 3 mice.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8228487/v1/316c74143a891cc6907da38b.png"},{"id":97656185,"identity":"2cc6d278-a7a5-4b27-b79d-ea1de6681e49","added_by":"auto","created_at":"2025-12-08 07:09:49","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":152879,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSD enhances morphine-induced locomotor sensitization and prolongs its persistence.\u003c/strong\u003e(A) Locomotor activity during the first and fifth morphine administrations in control, SD, and RS mice. Data represent the mean ± SEM of 8 mice. **p \u0026lt; 0.01, ***p \u0026lt; 0.001, paired \u003cem\u003et\u003c/em\u003e-test. (B) Group comparison of locomotor sensitization induced by five morphine administrations. Slopes of best-fit lines were compared using two-way ANOVA. *p \u0026lt; 0.05, **p \u0026lt; 0.01. (C) Schematic of the experimental workflow for transcriptome analysis in the mPFC, NAcc, and VTA—key regions of the mesocorticolimbic dopamine system—following SD. (D–H) mRNA levels of (D) \u003cem\u003ec-Fos\u003c/em\u003e, (E) \u003cem\u003eΔFosB\u003c/em\u003e, (F) \u003cem\u003eBdnf\u003c/em\u003e exon IV, (G) \u003cem\u003eHomer1a\u003c/em\u003e and (H) \u003cem\u003eFkbp5\u003c/em\u003e in the mPFC, NAcc, and VTA compared among control, SD, and RS mice. Data represent the mean ± SEM of 6–9 mice. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, control vs SD; #p \u0026lt; 0.05, ##p \u0026lt; 0.01, SD vs recovery (one-way ANOVA with Tukey post hoc test).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8228487/v1/62b7fe3df51ba236f4c0437b.png"},{"id":97656180,"identity":"681370b4-7be1-46b3-b9c8-75e64b68bef4","added_by":"auto","created_at":"2025-12-08 07:09:49","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":64013,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSleep deprivation reconfigures plasticity-related gene expression in mesocorticolimbic circuits. \u003c/strong\u003e(A) Experimental paradigm for quantifying SD-induced plasticity in mesocorticolimbic regions. Mice underwent sleep deprivation, after which the mPFC, NAcc, and VTA were collected for qPCR analysis of plasticity-related transcripts (\u003cem\u003ec-Fos\u003c/em\u003e, \u003cem\u003eΔFosB\u003c/em\u003e, \u003cem\u003eHomer1a\u003c/em\u003e, \u003cem\u003eFkbp5\u003c/em\u003e). A composite plasticity score (P-index) was derived by averaging the ΔΔCt values of these four genes. (B–D) P-index values in the (B) mPFC, (C) NAcc, and (D) VTA across control, SD, and RS groups. Data represent the mean ± SEM of 6 mice. ***p \u0026lt; 0.001, control vs SD; ##p \u0026lt; 0.01, ###p \u0026lt; 0.001, SD vs recovery; $p \u0026lt; 0.05, control vs. recovery (one-way ANOVA with Tukey post hoc test).\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-8228487/v1/196dca55a6eb9db2e5febaae.png"},{"id":99314648,"identity":"972f0d70-06f5-4b5f-bc0e-e68c4accbac1","added_by":"auto","created_at":"2025-12-31 16:22:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1717997,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8228487/v1/a631e095-8e02-4522-b5dd-82e544f0fdeb.pdf"},{"id":97674872,"identity":"b5180ded-6217-4482-955e-34891bbe5f68","added_by":"auto","created_at":"2025-12-08 09:44:33","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":180771,"visible":true,"origin":"","legend":"","description":"","filename":"KishinoetalNeurochemicalResSupplementaryfinal.docx","url":"https://assets-eu.researchsquare.com/files/rs-8228487/v1/c5368ac34a7a509c9a9bcaf1.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Sleep loss differentially reconfigures neural circuits governing pain and neuropsychological homeostasis","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eSleep is a fundamental biological process that dissipates the physiological load accumulated during wakefulness and recalibrates neural, immune, and endocrine homeostasis [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. During sleep, synchronized neural activity and metabolic redistribution promote synaptic remodeling and the clearance of metabolic waste, thereby maintaining systemic physiological equilibrium [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. However, chronic sleep restriction and fragmentation have become increasingly prevalent in modern society. These consequences extend far beyond fatigue and impaired attention to encompass cardiovascular and metabolic dysfunction, immune dysregulation, and emotional instability [\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. These alterations imply a subtle yet pervasive disruption of neurochemical and circuit-level homeostasis that which may exert long-lasting effects on behavior, affective regulation, and systemic adaptation.\u003c/p\u003e\u003cp\u003eSleep deprivation (SD) and poor sleep quality disrupt the delicate balance between excitation and inhibition within neural networks [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], while overactivating stress-responsive and immunometabolic systems [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In particular, hyperactivation of the hypothalamic\u0026ndash;pituitary\u0026ndash;adrenal (HPA) axis and corticotropin-releasing hormone (CRH) neurons, together with dysregulated mesolimbic dopaminergic signaling, have been implicated as neural substrates underlying sleep-loss-induced phenotypes of hyperalgesia, anxiety, affective lability, and altered drug responsiveness [\u003cspan additionalcitationids=\"CR14 CR15 CR16\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Short-term experimental SD reliably decreases pain thresholds [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] and attenuates morphine analgesia [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Thus, SD may differentially reshape central pharmacological responsiveness depending on the specific neurochemical system engaged, warranting a mechanistically refined understanding of these interactions.\u003c/p\u003e\u003cp\u003eClinically, the interplay between sleep disturbances, chronic pain, anxiety, and depression forms a complex neuroendocrine network that influences drug sensitivity and stress vulnerability [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Sustained SD over several days can induce perceptual distortions and hallucinations, reflecting possible hyperdopaminergic states and maladaptive neuroplasticity within cortical\u0026ndash;subcortical circuits [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Such phenomena suggest that sleep loss represents not merely a state of neural fatigue but also a deep-seated qualitative reorganization of neural function, marked by disrupted excitation-inhibition balance and local circuit instability. However, despite extensive correlative evidence, integrative studies that simultaneously examine region-specific neuronal activity, molecular plasticity, and behavioral alterations within the same sleep dysregulation model while assessing the reversibility of these changes after recovery sleep (RS) remain scarce.\u003c/p\u003e\u003cp\u003eWe sought to delineate the multidimensional impact of SD on neural circuit remodeling, pain sensitivity, pharmacological responsiveness, and affective behaviors in the same subjects. Focusing on functional hubs involved in pain, emotion, stress, and immune regulation\u0026ndash;including the paraventricular nucleus (PVN) and arcuate nucleus (ARC) of the hypothalamus, mesolimbic and mesocortical dopaminergic pathways, the parabrachial nucleus in the brainstem, and higher-order default mode networks\u0026ndash;we systematically tracked neuronal activity and gene expression changes induced by sleep loss and examined the extent to which RS restores these perturbations. This integrated neuropharmacological framework aimed to elucidate how SD reconfigures the regulatory architecture of the brain and redefines the neurobiological and pharmacological significance of sleep in maintaining organismal homeostasis under the demands of modern society.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cp\u003e\u003cem\u003eAnimals\u003c/em\u003e\u003c/p\u003e\u003cp\u003eThe experimental procedures were conducted in accordance with the \u003cem\u003eGuiding Principles for the Care and Use of Laboratory Animals\u003c/em\u003e of Hoshi University School of Pharmacy and Pharmaceutical Sciences and were approved by the institutional ethics committee. All experiments adhered to the ARRIVE guidelines. Every effort was made to minimize both the number and suffering of animals. Male C57BL/6J (Tokyo Laboratory Animals Science Co. Ltd., Tokyo, Japan), male STOCK Tg(Pomc1-Cre)16Lowl/J [proopiomelanocortin (POMC)-Cre; Stock No: 005965, Jackson Laboratory, ME, USA] mice and male c-fos-TRAP2::tdTomato mice were used in this study. To standardize the genetic background, POMC-Cre mice were backcrossed with C57BL/6J mice for more than 10 generations. Homozygous POMC-Cre mice were generated by heterozygous intercrossing and selected for the experiments. c-Fos-TRAP2::tdTomate mice were breeding c-Fos-2AiCreERT2 mice [C57BL/6-Fos tm1(icreERT2)Phsh; Cyagen Biosciences Inc., Santa Clara, CA, USA] with LSL-tdTomato mice [B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J; Stock #007914, Jackson Laboratory]. Animals were maintained under controlled environmental conditions (temperature: 24\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C; humidity: 55\u0026thinsp;\u0026plusmn;\u0026thinsp;5%; 12 h/12 h light\u0026ndash;dark cycle, lights on at 8:00). Food and water were provided \u003cem\u003ead libitum\u003c/em\u003e in the home cages. To reduce experimental bias, independent investigators performed distinct experimental steps. A total of 250 mice were used in this study.\u003c/p\u003e\u003cp\u003e\u003cem\u003eSleep deprivation (SD)\u003c/em\u003e\u003c/p\u003e\u003cp\u003eSD was performed using a gentle-handling paradigm [\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Whenever behavioral signs of sleep appeared, the mice were lightly touched with a soft paint brush to maintain wakefulness, and stimulation was continued until a clear arousal response was observed. SD was conducted for 6 h during the light phase (8:00\u0026ndash;14:00) over three consecutive days. For the recovery experiments, an additional group underwent the same SD protocol followed by seven days of unrestricted RS (\u003cem\u003ead libitum\u003c/em\u003e).\u003c/p\u003e\u003cp\u003e\u003cem\u003ePostoperative pain model\u003c/em\u003e\u003c/p\u003e\u003cp\u003ePostoperative pain was induced under 3% isoflurane anesthesia as previously described [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. A 3-mm longitudinal incision was made through the skin and fascia of the plantar surface of the right hind paw using a No. 23 scalpel. For the transient pain (Brennan) model, a 3-mm incision was made into the plantaris muscle using a scalpel. In contrast, for the persistent postoperative pain model, the same incision was followed by a 3-mm muscle cut using a monopolar electrosurgical unit (50 W; Vetroson\u0026reg; V-10, Summit Hill Laboratories, Tinton Falls, NJ, USA) with a dispersive electrode pad placed beneath the mouse. The skin was closed using two 7\u0026ndash;0 nylon horizontal mattress sutures. For the sham surgery, the muscle was exposed but not incised. A plantar incision was made either after three days of SD or after the subsequent seven-day recovery period.\u003c/p\u003e\u003cp\u003e\u003cem\u003evon Frey filament test\u003c/em\u003e\u003c/p\u003e\u003cp\u003eMechanical allodynia was assessed using a plantar electronic von Frey anesthesiometer (ALMEMO 2450; Ahlborn; IITC/Life Science). The probe tip was applied perpendicularly to the plantar surface of the hind paw and the pressure threshold (g) required to elicit paw withdrawal was recorded as the pain threshold.\u003c/p\u003e\u003cp\u003e\u003cem\u003eHot-plate assay\u003c/em\u003e\u003c/p\u003e\u003cp\u003eThermal nociception (50\u0026deg;C) and morphine-induced antinociception (55\u0026deg;C) were evaluated using a hot-plate apparatus (Muromachi Kikai Co., Ltd., Tokyo, Japan) by measuring the latency to paw licking, tapping, or jumping. For nociception, latencies were obtained before and after the 3-day SD period, and for RS experiments, measurements were repeated after the 7-day recovery phase. Morphine-induced antinociception was assessed after subcutaneous administration of morphine (20 mg/kg).\u003c/p\u003e\u003cp\u003eAntinociception was calculated as:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:\\text{%}\\text{A}\\text{n}\\text{t}\\text{i}\\text{n}\\text{o}\\text{c}\\text{i}\\text{c}\\text{e}\\text{p}\\text{t}\\text{i}\\text{o}\\text{n}=100\\times\\:\\frac{(\\text{p}\\text{o}\\text{s}\\text{t}\\text{d}\\text{r}\\text{u}\\text{g}\\:\\text{l}\\text{a}\\text{t}\\text{e}\\text{n}\\text{c}\\text{y}-\\text{p}\\text{r}\\text{e}\\text{d}\\text{r}\\text{u}\\text{g}\\:\\text{l}\\text{a}\\text{t}\\text{e}\\text{n}\\text{c}\\text{y})}{\\left(\\text{c}\\text{u}\\text{t}\\text{o}\\text{f}\\text{f}\\:\\text{t}\\text{i}\\text{m}\\text{e}-\\text{p}\\text{r}\\text{e}\\text{d}\\text{r}\\text{u}\\text{g}\\:\\text{l}\\text{a}\\text{t}\\text{e}\\text{n}\\text{c}\\text{y}\\right)}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe cut-off time was 30 s to avoid tissue injury. Morphine-evoked antinociception was assessed after SD or after the subsequent recovery period.\u003c/p\u003e\u003cp\u003e\u003cem\u003eLPS challenge\u003c/em\u003e\u003c/p\u003e\u003cp\u003eTo assess endotoxin-induced systemic responses under SD, lipopolysaccharide (LPS; Escherichia Coli O55:B5, 600 \u0026micro;g/100 \u0026micro;L/mouse, Sigma-Aldrich) was administered intravenously \u003cem\u003evia\u003c/em\u003e the tail vein either immediately after the 3-day SD or after 7 days of RS. Body weight, rectal temperature, and survival were monitored at the defined time points. Rectal temperature was measured using a digital thermistor (KN-91; Natsume Seisakusho, Tokyo, Japan), with the probe lubricated and gently inserted into the rectum.\u003c/p\u003e\u003cp\u003eAssessment of hypnotic effect\u003c/p\u003e\u003cp\u003eTo examine the effect of SD on GABAergic hypnotic efficacy, a loss-of-righting-reflex (LORR) assay was performed. Following SD or recovery from sleep, mice received intraperitoneal zolpidem (20 mg/kg) or saline. After drug administration, the ataxic mice were placed supine on a V-shaped plastic plate and observed until they righted three consecutive times within 30 s. The time from injection to loss of the righting reflex was defined as the LORR latency, and the duration from loss to recovery was recorded as the LORR duration.\u003c/p\u003e\u003cp\u003e\u003cem\u003eLocomotor activity\u003c/em\u003e\u003c/p\u003e\u003cp\u003eLocomotor activity was monitored using a three-beam infrared beam-break system (Three-points Meter; O\u0026rsquo;Hara \u0026amp; Co., Ltd., Tokyo, Japan), which detects full-body displacement along the x- and y-axes, while minimizing artifacts from tail or limb movements. After 60 min of habituation, the mice were injected subcutaneously with morphine (20 mg/kg; Daiichi-Sankyo Co., Ltd., Tokyo, Japan). Activity counts were collected in 1-min bins over 180 min. Morphine-evoked hyperlocomotion was recorded after SD or subsequent RS.\u003c/p\u003e\u003cp\u003e\u003cem\u003eStereotaxic adeno-associated virus (AAV) injection and chemogenetic manipulation of hypothalamic POMC neurons\u003c/em\u003e\u003c/p\u003e\u003cp\u003eMice were anesthetized with 3% isoflurane and mounted on a stereotaxic frame (RWD Life Science, CA, USA). A Cre-dependent adeno-associated virus (AAV) vector (AAV9-hSyn-Flex-hM3Dq-mCherry; 2 \u0026times; 10\u003csup\u003e12\u003c/sup\u003e copies/mL, VectorBuilder) was bilaterally injected into the ARC of POMC-Cre mice (coordinates relative to bregma: A/P\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;1.5 mm; M/L\u0026thinsp;=\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 mm; D/V\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;4.8 mm; medial angle\u0026thinsp;=\u0026thinsp;0\u0026deg;). Injections (150 nL per side) were delivered using Nanoject III (3-000-207; Drummond Scientific Company, Broomall, PA, USA). Mice were allowed to recover for \u0026ge;\u0026thinsp;2 weeks to permit robust expression of hM3Dq. Following recovery, AAV-injected mice were subjected to a persistent postoperative pain model, and clozapine-\u003cem\u003eN\u003c/em\u003e-oxide (CNO; 3 mg/kg, i.p.; Abcam, Cambridge, UK) was administered once daily from postoperative day (POD) 1 to activate the ARC POMC neurons during nociceptive testing.\u003c/p\u003e\u003cp\u003e\u003cem\u003eImmunofluorescence\u003c/em\u003e\u003c/p\u003e\u003cp\u003eMice were transcardially perfused with 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (pH 7.4) under anesthesia with 3% isoflurane. Brain tissues were dissected after being post-fixation with 4% PFA and cryoprotection in 20\u0026ndash;30% (w/v) sucrose (FUJIFILM Wako Pure Chemical Corp.). Brain sections were embedded in O.C.T. compound (Sakura Fine Technical, Tokyo, Japan), and frozen sections were cut using a cryostat (CM1860; Leica Microsystems, Heidelberg, Germany). Immunofluorescence was detected using a light microscope (BX-53; Olympus, Tokyo, Japan), and images were captured using a high-sensitivity digital CCD camera (MD-695; Molecular Devices, San Jose, CA, USA). Imaging analysis was performed using the Metamorph 7.8 software (Molecular Devices).\u003c/p\u003e\u003cp\u003e\u003cem\u003eReverse transcription and quantitative polymerase chain reaction (RT-qPCR)\u003c/em\u003e\u003c/p\u003e\u003cp\u003eTotal RNA was extracted from microdissected regions of the brain including the medial prefrontal cortex (mPFC), nucleus accumbens (NAcc), PVN, ARC, lateral hypothalamus (LH), periaqueductal gray (PAG), ventral tegmental area (VTA), and parabrachial nucleus (PBN) using the mirVana\u0026trade; miRNA Isolation Kit (Thermo Fisher Scientific Inc., MA, USA). First-strand cDNA was synthesized using the SuperScript\u0026reg; VILO\u0026trade; cDNA Synthesis Kit, and qPCR was conducted using Fast SYBR\u0026reg; Green Master Mix (Thermo Fisher Scientific Inc.) and gene-specific primers (Supplementary Table\u0026nbsp;1). Gene expression was normalized to glyceraldehyde 3-phosphate dehydrogenase (\u003cem\u003eGapdh\u003c/em\u003e) using the 2^-∆∆CT method. Based on previous reports, \u003cem\u003ePomc\u003c/em\u003e expression is known to be markedly lower outside the ARC than within it [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Therefore, under the present PCR assay conditions, we anticipated low \u003cem\u003ePomc\u003c/em\u003e expression in extra-arcuate regions. To ensure reliable quantification of region-specific expression, regions in which the mean Ct value for each target gene exceeded 30 across groups were a priori regarded as falling below the limit of reliable quantification (LOQ). Such data were designated as N.D. (not detectable) and excluded from subsequent analyses.\u003c/p\u003e\u003cp\u003e\u003cem\u003eLabeling of Pain-TRAPed neurons\u003c/em\u003e\u003c/p\u003e\u003cp\u003eTo induce a persistent postoperative pain state, we generated a standardized plantar incision model in cFos-TRAP2::tdTomato mice. A 3-mm longitudinal cut was made along the plantaris muscle of the right hind paw with a monopolar electrosurgical unit set to 50 W (Summit Hill Laboratories), following the methodological framework described by Katsuda and colleagues [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Sham-operated mice were exposed only to 3% isoflurane anesthesia without incision. Before TRAP induction of neuronal trapping, mice underwent a structured habituation protocol: they were placed individually inside black plastic cylinders for 1 h per day over three consecutive days.\u003c/p\u003e\u003cp\u003eFifteen days after electrocautery, tamoxifen (100 mg/kg, i.p.) was administered to initiate activity-dependent genetic tagging. Four hours later, a calibrated 0.07-g von Frey filament was applied to the ipsilateral hind paw at 30-s intervals for 30 min to evoke the Pain-TRAP response and capture nociception-related neuronal ensembles. Seven days after the Pain-TRAP procedure, mice were deeply anesthetized for terminal perfusion and subsequent histological analyses.\u003c/p\u003e\u003cp\u003e\u003cem\u003eDefinition of plasticity-related modulators and quantification of gene expression\u003c/em\u003e\u003c/p\u003e\u003cp\u003eIn this study, \u003cem\u003ec-Fos\u003c/em\u003e, \u003cem\u003eΔFosB\u003c/em\u003e, \u003cem\u003eHomer1a\u003c/em\u003e, and \u003cem\u003eFkbp5\u003c/em\u003e were operationally defined as plasticity-related modulators. Gene expression levels were quantified using qPCR and processed with the ΔΔCt method, yielding relative expression values expressed as 2^-ΔΔCt for each individual animal. For each mouse, the four relative expression values were averaged to generate a single integrated plasticity metric, termed the Plasticity-index (P-index). Thus, the P-index represents an individual-level parameter and was treated as an independent biological endpoint within each experimental group (control, SD, recovery). Statistical analysis focused exclusively on group-dependent differences in the P-index. Comparisons among the three groups were performed using a one-way ANOVA, followed by Tukey\u0026rsquo;s post hoc test. Stacked bar graphs were generated solely to visualize the relative contributions of individual plasticity-related modulators to the P-index and were not used for statistical inference.\u003c/p\u003e\u003cp\u003e\u003cem\u003eStatistical analysis\u003c/em\u003e\u003c/p\u003e\u003cp\u003eAll data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Statistical significance was determined using paired and unpaired Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test, one-way ANOVA, or two-way ANOVA followed by Turkey or Bonferroni post hoc tests, chi-square tests, or log-rank (Kaplan\u0026ndash;Meier) survival analysis, as appropriate, using GraphPad Prism software (version 9.5; GraphPad Software, CA, USA).\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cb\u003eSleep loss amplifies thermal and mechanical hypersensitivity and diminishes \u0026micro;-opioid analgesic efficacy\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo investigate how SD reconfigures sensory responsiveness and alters the pharmacodynamics of \u0026micro;-opioid, benzodiazepine, and LPS responses, mice were subjected to an SD paradigm consisting of 6 h of wakefulness during the light phase for three consecutive days. A RS condition was subsequently implemented, allowing 7 days of unrestricted sleep to assess the reversibility of SD-induced perturbations in physiological functions and systemic homeostasis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). As an initial assessment of sensory responsiveness, nociceptive latency was quantified using the 50\u0026deg;C hot-plate assay. Latency was significantly reduced relative to the baseline before SD, indicating heightened nociceptive sensitivity, whereas this effect was fully normalized following the RS phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB; **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, before SD vs. after SD, paired \u003cem\u003et\u003c/em\u003e-test).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo determine whether sleep loss interferes with \u0026micro;-opioid-mediated analgesia, morphine-induced antinociception was examined using the hot-plate test (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Morphine (20 mg/kg, s.c.)-evoked analgesia was markedly blunted by SD compared with controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD; *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, control vs. SD; two-way ANOVA followed by Bonferroni test). Remarkably, RS restored the analgesic efficacy of morphine (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE; *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, control vs. SD; #p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ##p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, SD vs. recovery; two-way ANOVA followed by Tukey test).\u003c/p\u003e\u003cp\u003e\u003cb\u003eAssociation between sleep loss, heightened pain sensitivity, and reversibility through the ARC-POMC system following sleep loss\u003c/b\u003e\u003c/p\u003e\u003cp\u003eGiven the SD-induced hypersensitivity and the attenuation of \u0026micro;-opioid analgesia, transcript levels of the \u0026micro;-opioid receptor (MOR, \u003cem\u003eOprml\u003c/em\u003e) and endogenous opioid peptide precursors (preprodynorphin; \u003cem\u003ePdyn\u003c/em\u003e, preproenkephalin; \u003cem\u003ePenk\u003c/em\u003e, and proopidmelanocortin; \u003cem\u003ePomc\u003c/em\u003e) were quantified across multiple pain- and sleep-associated brain regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-D). These opioid-related transcripts were broadly detectable across the examined brain regions, with \u003cem\u003ePomc\u003c/em\u003e mRNA showing marked enrichment within the ARC (Supplementary Fig.\u0026nbsp;1). Notably, only \u003cem\u003ePomc\u003c/em\u003e mRNA in the ARC was significantly decreased following SD (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD; *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, control vs. SD, unpaired Student's \u003cem\u003et\u003c/em\u003e-test), whereas \u003cem\u003eOprml\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), \u003cem\u003ePdyn\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), and \u003cem\u003ePenk\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC) remained unaffected. Consistent with behavioral recovery, the SD-induced reduction in \u003cem\u003ePomc\u003c/em\u003e expression was fully reversed by RS, restoring expression to control levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE; *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, control vs. SD; #p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, SD vs. recovery; one-way ANOVA with Tukey correction).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo establish a causal link between SD-driven sensory hypersensitivity and \u003cem\u003ePomc\u003c/em\u003e downregulation, a cell type-specific chemogenetic approach was employed. An AAV vector encoding the excitatory DREADD receptor (hM3Dq) was bilaterally injected into the ARC of POMC-Cre mice, permitting the CNO-induced activation of POMC neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). Following the induction of persistent postoperative pain by plantar incision using a high-frequency electrosurgical unit, mechanical thresholds were measured after CNO administration \u003cem\u003evia\u003c/em\u003e von Frey testing (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). At POD1, both WT::M3Dq and POMC-Cre::M3Dq mice displayed pronounced ipsilateral allodynia, as indicated by reduced paw-withdrawal thresholds compared to the contralateral side (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). Critically, CNO administration selectively restored the ipsilateral thresholds in POMC-Cre::M3Dq mice within 30 min, whereas those in WT::M3Dq mice remained unaffected (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG; **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, 0 min vs. 30 min in POMC-Cre::M3Dq, paired \u003cem\u003et\u003c/em\u003e-test). Collectively, these results identified ARC-POMC neurons as pivotal regulatory nodes that link SD to altered pain processing and \u0026micro;-opioid responsiveness. Moreover, during chronic postoperative pain, repeated activation of ARC-POMC neurons progressively normalized mechanical thresholds, signifying the functional restoration of the endogenous antinociceptive circuitry (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH; p\u0026thinsp;\u0026gt;\u0026thinsp;0.05 at POD49 and POD56, POMC-Cre::M3Dq contralateral vs. POMC-Cre::M3Dq ipsirateral; two-way ANOVA with Tukey correction).\u003c/p\u003e\u003cp\u003eThe parabrachial nucleus (PBN) has recently been recognized not only as a critical relay station that receives nociceptive inputs from the dorsal horn of the spinal cord, but also as a hub-like switch in the regulation of sleep\u0026ndash;wake states. To capture pain state\u0026ndash;dependent ensembles in the PBN, persistent postoperative pain was induced in cfos-TRAP2::tdTomato mice, and tamoxifen was administered on postoperative day 15 to permanently tag c-Fos\u0026ndash;expressing cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Under these conditions, mice with persistent postoperative pain exhibited a marked increase in c-Fos\u0026ndash;positive neurons within the PBN, suggesting that a hyperactive neuronal ensemble is selectively recruited in this nucleus during the chronic pain state (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Given that the PBN serves as a key recipient of nociceptive information, neuronal activity in this nucleus was next examined under SD (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), and SD robustly increased \u003cem\u003ec-Fos\u003c/em\u003e and \u003cem\u003eΔFosB\u003c/em\u003e mRNA in the PBN, and RS restored both markers to baseline (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD; *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, control vs. SD, one-way ANOVA with Tukey correction). Nociception-related neuropeptide transcripts within the PBN were further quantified, revealing that SD significantly upregulated \u003cem\u003eCalca\u003c/em\u003e (CGRP) mRNA expression, whereas both \u003cem\u003eTac1\u003c/em\u003e (substance P) and \u003cem\u003ePdyn\u003c/em\u003e mRNA remained unchanged (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE; *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, control vs. SD, one-way ANOVA with Tukey correction). This transcriptomic change was restored to control-like levels after RS (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE; #p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, SD vs. recovery, one-way ANOVA with Tukey correction).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eSD amplifies endotoxin-induced systemic responses\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo determine whether sleep loss altered systemic inflammatory reactivity, an endotoxin challenge was performed. Mice received an intravenous injection of LPS (600 \u0026micro;g/mouse), and changes in body weight and core (rectal) temperature were monitored 12 h post-injection (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA,B). At 12 h after LPS treatment, both body weight and core temperature were markedly reduced compared to saline-treated controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, body weight, ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, control-saline vs. control-LPS; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, body temperature, ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, control-saline vs. control-LPS; one-way ANOVA with Tukey correction). Relative to the LPS-treated controls, SD further potentiated the systemic responses, producing greater weight loss and more profound hypothermia (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, control-LPS vs. SD-LPS, one-way ANOVA with Tukey correction). Remarkably, these SD-induced exaggerations were reversed by RS, with both parameters returning to control levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, body weight, *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, SD-LPS vs. recovery-LPS; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, body temperature, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, SD-LPS vs. recovery-LPS; one-way ANOVA with Tukey correction). The survival outcomes mirrored these physiological trends. Kaplan\u0026ndash;Meier analysis revealed that LPS administration reduced survival compared to saline-treated controls, and SD further compromised survival relative to LPS-treated controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC; *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, log-rank test). Conversely, RS mitigated this SD-induced mortality enhancement, partially restoring survival probabilities (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC; **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, log-rank test).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo delineate the hypothalamic correlates of the SD-potentiated LPS response, the PVN, ARC, and LH were microdissected for transcriptomic analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Within the hypothalamus, SD selectively elevated \u003cem\u003ec-Fos\u003c/em\u003e mRNA expression in the PVN, with no significant changes observed in the ARC or LH (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE; *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, control vs. SD; unpaired Student's \u003cem\u003et\u003c/em\u003e-test). This \u003cem\u003ec-Fos\u003c/em\u003e change was restored to control-like levels after RS (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF; **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, control vs. SD; #p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, SD vs. recovery, one-way ANOVA with Tukey correction). In contrast, \u003cem\u003eTlr4\u003c/em\u003e and \u003cem\u003eCrh\u003c/em\u003e transcript levels remained unchanged across all regions and experimental conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG, H; p\u0026thinsp;\u0026gt;\u0026thinsp;0.05, one-way ANOVA with Tukey correction).\u003c/p\u003e\u003cp\u003e\u003cb\u003eReduced hypnotic efficacy after sleep loss: diminished LORR incidence and altered onset/duration dynamics\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo assess whether sleep loss modulates hypnotic sensitivity, behavioral endpoints of zolpidem-induced sedation were quantified, including the incidence of LORR, latency to hypnotic onset (LORR latency), and duration of hypnotic maintenance (LORR duration). Zolpidem induced LORR in all control mice (12/12, 100%), whereas only seven of twelve SD mice (58%) exhibited LORR, representing a significant reduction in hypnotic efficacy. Following RS, the incidence of LORR was fully restored to control levels (12/12, 100%), indicating that sleep loss impaired zolpidem-induced hypnosis, whereas recovery normalized it (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA; ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, chi-squared test). Among the mice that exhibited LORR, SD significantly delayed hypnotic onset and shortened its duration; these effects were both normalized following RS (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, LORR latency: ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, control vs. SD and SD vs. recovery; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, LORR duration: **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, control vs. SD and SD vs. recovery; one-way ANOVA with Tukey correction).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo explore molecular substrates underlying altered hypnotic sensitivity, mRNA levels of GABA\u003csub\u003eA\u003c/sub\u003e receptor (GABA\u003csub\u003eA\u003c/sub\u003eR) αl subunit (\u003cem\u003eGabral\u003c/em\u003e) and glutamate decarboxylase 67 (\u003cem\u003eGad1\u003c/em\u003e, GAD67) were measured in the PVN, ARC, and LH (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). No significant group differences were detected (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE,F; p\u0026thinsp;\u0026gt;\u0026thinsp;0.05, one-way ANOVA with Tukey correction). Similarly, orexin (\u003cem\u003eHcrt\u003c/em\u003e) expression in the LH remained unchanged (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG; p\u0026thinsp;\u0026gt;\u0026thinsp;0.05, one-way ANOVA with Tukey correction).\u003c/p\u003e\u003cp\u003e\u003cb\u003eSleep loss amplifies morphine-evoked dopaminergic psychostimulation, persisting despite RS\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFurthermore, to obtain an integrated measure of transcriptional changes across plasticity-related genes, 2\u003csup\u003e\u0026ndash;ΔΔCt\u003c/sup\u003e values for each gene were used to calculate a plasticity-index (P-index), defined as the mean expression of four genes: \u003cem\u003ec-Fos\u003c/em\u003e, \u003cem\u003eΔFosB\u003c/em\u003e, \u003cem\u003eHomer1a\u003c/em\u003e, and \u003cem\u003eFkbp5\u003c/em\u003e in the mPFC, NAcc and VTA (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Under SD conditions, P-index values were increased in all three regions examined relative to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB\u0026ndash;D; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, control vs. SD, one-way ANOVA with Tukey\u0026rsquo;s correction), and these SD-induced elevations were attenuated by RS (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB\u0026ndash;D; p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 and p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, SD vs. recovery). However, whereas RS normalized P-index values in the VTA and NAcc to control levels, those in the mPFC remained significantly higher in the RS mice than in control mice. (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, control vs. recovery). These findings suggest that large-scale reconfiguration of plasticity-related gene expression induced by SD is preferentially sustained within the mPFC.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn this study, we employed a short-term SD protocol to examine behavioral pain thresholds, pharmacological responses to \u0026micro;-opioid and GABA\u003csub\u003eA\u003c/sub\u003eR agonists, stress and infection responses to endogenous endotoxins, and transcriptional alterations in opioid signaling across multiple brain regions. In an acute postoperative pain model, evaluation of withdrawal latency to thermal stimuli revealed that SD significantly reduced pain thresholds. This hypersensitivity was most pronounced immediately after SD but was largely reversible after one week of RS. Furthermore, SD transiently attenuated the analgesic efficacy of \u0026micro;-opioid receptor agonists, but this effect was fully restored after RS.\u003c/p\u003e\u003cp\u003eThe ARC of the hypothalamus contains the highest density of POMC-expressing neurons, which produce β-endorphin, an endogenous \u0026micro;-opioid peptide. Recent studies have shown that in the ARC of neuropathic pain model mice, expression of endopeptidases required for the cleavage of POMC into β-endorphin is suppressed, resulting in decreased β-endorphin levels and reduced pain thresholds [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In the present study, we found that SD markedly decreased POMC mRNA expression in the ARC, which returned to control levels after RS. Moreover, selective chemogenetic activation of ARC-POMC neurons via AAV-mediated expression of hM3Dq in POMC-Cre mice and subsequent administration of CNO significantly restored the diminished pain threshold observed in persistent postoperative pain.\u003c/p\u003e\u003cp\u003eARC neurons containing β-endorphin project to the PAG, a critical center for descending pain inhibition, thereby contributing to endogenous analgesic control [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Thus, the reversible reduction in POMC expression observed in the ARC may reflect a transient impairment in endogenous β-endorphin production, resulting in temporary attenuation of the intrinsic pain modulation system and diminished efficacy of \u0026micro;-opioid receptor agonists.\u003c/p\u003e\u003cp\u003ePOMC gene expression is regulated by two principal pathways, the stress hormone axis and the leptin signaling cascade. The POMC promoter contains a glucocorticoid response element, and the binding of steroid hormones such as corticosterone, to glucocorticoid receptors induces transcriptional repression [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Conversely, leptin promotes POMC transcriptional activity through the JAK2\u0026ndash;STAT3 pathway in POMC-expressing neurons [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Previous studies reported that SD increases circulating corticosterone levels [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] and decreases plasma leptin levels [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. In the present study, we observed that SD induced a robust increase in \u003cem\u003ec-Fos\u003c/em\u003e expression, a molecular marker of neuronal activation that constitutes the major origin of the HPA axis, in the PVN of the hypothalamus. This finding suggests that SD persistently activates PVN neurons, thereby promoting the excessive release of the endogenous glucocorticoid corticosterone. Taken together, these results indicated that the reduction in POMC expression in ARC neurons caused by SD may be at least partly attributable to the hyperactivation of glucocorticoid signaling triggered by PVN activation, together with the suppression of leptin signaling. Indeed, in our previous study, the pharmacogenetic activation of CRH-containing neurons in the PVN exacerbated and prolonged postoperative pain, supporting the validity of this hypothesis [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe MOR is predominantly expressed in excitatory neurons located in layers V\u0026ndash;VI of the cerebral cortex and is rarely found in inhibitory interneurons. \u0026micro;-opioid receptor agonists such as morphine suppress the activity of these excitatory neurons through Gi protein-coupled signaling, thereby attenuating nociceptive transmission in the cingulate cortex, a key terminal region of the ascending pain pathway [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. In the present study, we observed a significant increase in \u003cem\u003ec-Fos\u003c/em\u003e expression in the mPFC, including the cingulate cortex, after SD. This finding raises the possibility that sustained cortical hyperexcitability may contribute, at least in part, to the attenuation of \u0026micro;-opioid\u0026ndash;mediated analgesia.\u003c/p\u003e\u003cp\u003eIntriguingly, reversible neural hyperactivation has also been detected in the PBN, which has recently emerged as a critical relay node for ascending pain signaling [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Consistent with this view, activity-dependent CFOS-TRAP labeling under a persistent postoperative pain state revealed selective recruitment of a hyperactive neuronal ensemble within the PBN, indicating that this brainstem hub is poised to integrate ongoing nociceptive drive with state-dependent modulatory inputs. This hyperactivation is accompanied by a transient elevation of CGRP, a major neuropeptide within the PBN that functions as a pronociceptive mediator. Neuronal activity and CGRP expression returned to control levels after RS, paralleling the restoration of pain thresholds. These findings indicate that SD transiently induces widespread excitation within the intrinsic pain networks of the brain, leading to temporary destabilization of endogenous analgesic systems and a corresponding reduction in pain thresholds, which can be normalized by sufficient restorative sleep.\u003c/p\u003e\u003cp\u003eSD also diminished the hypnotic efficacy of zolpidem, a non-benzodiazepine GABA\u003csub\u003eA\u003c/sub\u003eR agonist. However, this suppression was fully reversible after RS. Notably, SD did not alter the gene expression of GABA\u003csub\u003eA\u003c/sub\u003eR subunits or GABA-synthesizing enzymes in the hypothalamus. The hypnotic action of GABA\u003csub\u003eA\u003c/sub\u003eR agonists is generally thought to result from the inhibition of excitatory neurons, acting not only on hypothalamic\u0026ndash;ascending arousal circuits, but also on the medial prefrontal cortex, a region implicated in slow-wave generation [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. In our study, SD increased the expression of the activity-dependent marker \u003cem\u003ec-Fos\u003c/em\u003e not only in the hypothalamus but also in the mPFC. These results suggest that excessive activation of excitatory neurons under sleep-restricted conditions may physiologically counteract GABA\u003csub\u003eA\u003c/sub\u003eR-mediated inhibition, thereby reducing the apparent hypnotic effects of zolpidem.\u003c/p\u003e\u003cp\u003eAnother key finding of this study is that SD did not suppress, but rather enhanced, the locomotor-stimulating effect of morphine. Notably, this enhancement persisted even after the RS. The psychomotor activation induced by \u0026micro;-opioid receptor agonists is thought to be mediated by indirect activation of the mesolimbic and nigrostriatal dopaminergic circuits through desensitization of GABAergic neurons [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Thus, SD produced a distinctive bidirectional effect, reversibly attenuating the potent analgesic efficacy of morphine through multiple mechanisms, while paradoxically enhancing and sustaining dopaminergic locomotor stimulation. Indeed, previous studies have shown that during sleep restriction, the mesolimbic dopaminergic pathway projecting from the VTA to the NAcc becomes sensitized, resulting in long-lasting behavioral alterations [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Consistent with these reports, we observed increased expression of \u003cem\u003ec-Fos\u003c/em\u003e and \u003cem\u003eΔFosB\u003c/em\u003e within the VTA, NAcc, and mPFC, all of which belong to the mesolimbic\u0026ndash;cortical dopaminergic network. Importantly, the sleep recovery period failed to reset \u003cem\u003ec-Fos\u003c/em\u003e expression in the mPFC, suggesting persistent cortical activation. In line with this, a composite plasticity-index (P-index), quantitatively derived from \u003cem\u003ec-Fos\u003c/em\u003e, \u003cem\u003eΔFosB\u003c/em\u003e, \u003cem\u003eHomer1a\u003c/em\u003e, and \u003cem\u003eFkbp5\u003c/em\u003e, remained selectively elevated in the mPFC after RS, whereas it was normalized in the VTA and NAcc, indicating that SD imposes a durable plastic imprint on prefrontal circuits. These results provide a theoretical basis for the persistence of morphine-induced locomotor sensitization under sleep-restricted conditions. A crucial implication of these findings is that analgesic responses normalize with RS, whereas locomotor activation remains unaffected, indicating distinct reversibility between these physiological processes.\u003c/p\u003e\u003cp\u003eConsidering that the mPFC serves as the central hub of the default mode network (DMN) [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e], it is plausible that SD disrupts DMN connectivity, thereby sustaining dopaminergic psychomotor sensitization. Moreover, the opposing transcriptional signatures observed in the reward circuitry\u0026mdash;characterized by increased \u003cem\u003eBdnf\u003c/em\u003e mRNA in the NAcc and decreased \u003cem\u003eBdnf\u003c/em\u003e mRNA in the VTA\u0026mdash;resembled those seen in stimulant addiction, suggesting the emergence of irreversible distortions in reward-related plasticity [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Collectively, these findings indicate that sleep disturbance destabilizes the DMN, predisposing higher-order integrative functions to collapse, and potentially establishing a pathophysiological state of vulnerability. Therefore, chronic SD may not be readily reversible through short-term RS.\u003c/p\u003e\u003cp\u003eOur study also revealed that systemic responses to the LPS challenge were markedly exacerbated by SD, whereas the subsequent introduction of RS effectively mitigated this excessive inflammatory reaction. We previously demonstrated that LPS administration induces hypothalamic neuroinflammation accompanied by glial hyperactivation [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Previous studies have shown that transient sleep loss primes the innate immune system by elevating the circulating levels of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. This pre-activated state can synergistically amplify cytokine release after LPS exposure, disrupt hypothalamic thermoregulation, and result in profound hypothermia, anorexia, reduced locomotor activity, loss of body weight, and even increased lethality. Therefore, acute SD may transiently compromise integrated hypothalamic functions, leading to systemic disarray across multiple homeostatic networks and eliciting the diverse physiological disturbances observed in this study. Remarkably, the fact that RS restored homeostatic immune function more effectively than anticipated underscores the remarkable resilience of the immune regulatory system, even after transient functional breakdown.\u003c/p\u003e\u003cp\u003eFuture investigations should focus on identifying molecular profiles in the brain that are particularly vulnerable to irreversible disruption under SD and determining whether these changes act as triggers for secondary pathologies. Defining the molecular signatures that initiate systemic deterioration and establishing early interventions during the recovery window are essential for preventing the transition from transient dysfunction to chronic disease.\u003c/p\u003e\u003cp\u003eOverall, the findings of this study clearly demonstrate that physiological processes, such as pain sensitivity, immune defense, and pharmacological responsiveness, are profoundly modulated by both the quantity and quality of sleep. Furthermore, hyperactivation of the PVN, mPFC, PBN, VTA, and NAcc, together with the suppression of POMC gene expression in the ARC, collectively contributed to SD-induced phenomena, including hyperalgesia, attenuation of opioid analgesia, potentiation of morphine-induced locomotion, diminished hypnotic efficacy of GABA\u003csub\u003eA\u003c/sub\u003eR agonists, and reduced resistance to endotoxin challenge. Although short-term SD allows for substantial physiological restoration through RS, certain neural responses, particularly those involving the dopaminergic system and DMN coordination, may not easily return to baseline.\u003c/p\u003e\u003cp\u003eThis study highlights the indispensable role of sleep in maintaining systemic homeostasis across multiple physiological domains, including pain processing, immune regulation, and pharmacological adaptation. These findings strongly suggest that qualitative sleep impairment can act as a driving force for the progression from transient dysfunction to chronic pathological states.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis research was supported by AMED under Grant Number JP20ek0610024 and Hoshi University.\u003c/p\u003e\u003cp\u003eDeclaration of competing interests:\u003c/p\u003e\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization , Yasuyuki Nagumo and Minoru Narita; Data curation , Yasuyuki Nagumo, Yusuke Hamada, and Naoko Kuzumaki; Formal analysis , Yasuyuki Nagumo and Yusuke Hamada; Funding acquisition , Minoru Narita; Investigation , Teruyo Kishino, Yasuyuki Nagumo, Yusuke Hamada, Michiko Narita, Takumi Yoshizawa, Jion Takise, Yukari Suda and Naoko Kuzumaki; Methodology , Teruyo Kishino, Yasuyuki Nagumo, Yusuke Hamada and Michiko Narita; Project administration , Yasuyuki Nagumo and Minoru Narita; Supervision , Naoko Kuzumaki and Minoru Narita; Validation , Yasuyuki Nagumo; Visualization , Yasuyuki Nagumo; Writing\u0026ndash;original draft , Teruyo Kishino, Yasuyuki Nagumo, and Minoru Narita; Writing\u0026ndash;review \u0026amp; editing , Yasuyuki Nagumo, Tomohisa Mori, Naoko Kuzumaki and Minoru Narita.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors thank to Ms. Yu Okamoto, Mr. Hitoshi Makabe, Mr. Kensuke Yamashita, Mr. Naoki Yanagi, Ms. Nanami Morokata, Ms. Shione Suzuki, Mr. Kazuya Karaki, and Mr. Jiang ChengHao (Hoshi University) for grateful experiment supports. We would like to thank Editage (www.editage.jp) for English language editing.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets generated and analyzed in this study are available from the corresponding authors upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eFranken P, Dijk D-J (2024) Sleep and circadian rhythmicity as entangled processes serving homeostasis. 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Eur J Physiol 463:121\u0026ndash;137. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00424-011-1044-0\u003c/span\u003e\u003cspan address=\"10.1007/s00424-011-1044-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":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":"Sleep deprivation, Recovery sleep, Proopiomelanocortin (POMC), Chronic pain, Neuropsychological homeostasis, Default mode network","lastPublishedDoi":"10.21203/rs.3.rs-8228487/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8228487/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSleep dissipates the physiological load accumulated during wakefulness and restores neural, immune, and endocrine balance to maintain homeostasis. However, how sleep loss disrupts these systems and how recovery sleep (RS) reverses the effects remain poorly understood. Using a mouse model of sleep deprivation (SD), we examined alterations in pain sensitivity, inflammatory responses, pharmacologic responsiveness to opioids and non-benzodiazepine hypnotics, and molecular adaptations across key brain regions. SD heightened nociception, attenuated morphine analgesia and GABA\u003csub\u003eA\u003c/sub\u003e receptor-mediated hypnosis, and amplified lipopolysaccharide-evoked inflammation. These effects were accompanied by sustained expression of \u003cem\u003ec-Fos\u003c/em\u003e and \u003cem\u003eΔFosB\u003c/em\u003e in multiple brain regions and of \u003cem\u003eCalca\u003c/em\u003e in the parabrachial nucleus (PBN), but were largely normalized after RS. Within the hypothalamic arcuate nucleus (ARC), \u003cem\u003ePomc\u003c/em\u003e mRNA expression, unlike \u003cem\u003eOprm1\u003c/em\u003e, \u003cem\u003ePenk\u003c/em\u003e, and \u003cem\u003ePdyn\u003c/em\u003e, was markedly reduced during SD n and restored after RS, paralleling normalization of nociceptive thresholds. Pharmacogenetic activation of ARC-POMC neurons alleviated persistent postoperative pain. In contrast, SD enhanced morphine-induced psychomotor activation linked to dopaminergic transmission, accompanied by sustained \u003cem\u003ec-Fos\u003c/em\u003e expression in the ventral tegmental area, nucleus accumbens, and medial prefrontal cortex (mPFC). Notably, this hyperlocomotion persisted despite RS, and was associated with irreversible \u003cem\u003ec-Fos\u003c/em\u003e upregulation as well as a sustained, statistically significant elevation of integrated plasticity markers in the mPFC. Together, these findings reveal that sleep loss broadly reconfigures the neural circuits controlling pain, inflammation, and drug responsiveness. While most deficits are reversible with adequate RS, specific dopaminergic and cortical adaptations exhibit incomplete restoration, potentially predisposing patients to chronic, secondary psychopathology.\u003c/p\u003e","manuscriptTitle":"Sleep loss differentially reconfigures neural circuits governing pain and neuropsychological homeostasis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-08 07:09:44","doi":"10.21203/rs.3.rs-8228487/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":"fd6152b8-701e-466a-9434-28502c1bd8dc","owner":[],"postedDate":"December 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-26T20:38:28+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-08 07:09:44","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8228487","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8228487","identity":"rs-8228487","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}