Prolonged sleep deprivation induces cardiac dysfunction via microglia-neural circuit coupling

Prolonged sleep deprivation induces cardiac dysfunction via microglia-neural circuit coupling | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Prolonged sleep deprivation induces cardiac dysfunction via microglia-neural circuit coupling Ronglin Cai, Fan ZHANG, Wen-jing SHAO, Nai-xuan WEI, Wei LIU, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7329226/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Most animals require sleep, and sleep deprivation(SD) can lead to severe pathophysiological consequences, including cardiac dysfunction and death. Substantial evidence shows that sleep deprivation impairs cardiac function and increases cardiovascular risk and whether sleep deprivation-mediated neural output exacerbates the deterioration of heart function. Here, we demonstrate how sleep deprivation induces symptoms of cardiac dysfunction. Sleep disruption following SD leads to impaired cardiac function. Prolonged sleep deprivation triggers rapid microglial recruitment to the fastigial nucleus (FN) of the cerebellum and enhances the phagocytic capacity of microglia toward dendritic spines of GABAergic neurons. Prolonged sleep deprivation enhances microglial activity in the FN of male mice. Concurrently, microglial engulfment of GABAergic neuronal dendritic spines suppresses GABA neuron activity, leading to increased cardiac sympathetic outflow through the FN GABA -RVLM circuit to the cardiac and ultimately inducing cardiac dysfunction.These findings reveal that prolonged sleep deprivation triggers dysregulated microglial phagocytosis, which functionally encodes impaired GABAergic neuron-mediated suppression of cardiac sympathetic outflow, thereby driving the progression of cardiac dysfunction following prolonged sleep loss. Biological sciences/Neuroscience Health sciences/Cardiology sleep deprivation microglia Cerebellum fastigial nucleus cardiac dysfunction Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Sleep constitutes an essential physiological requirement for the vast majority of animal species 1 , 2 .Sleep is critically involved in regulating fundamental biological processes, including immune modulation 3 , cognitive function 4 , and metabolic homeostasis 5 . With societal development and the acceleration of modern lifestyles, the prevalence of sleep disorders and consequent sleep deprivation has risen significantly 6 . Sleep is crucial for cardiovascular health. Insufficient sleep or sleep deprivation elevates the risk of cardiac dysfunction 7 – 9 and may trigger myocardial infarction 10 , independent of genetic predisposition or other conventional risk factors 11 , 12 . The brain-heart axis mediates bidirectional communication between the central nervous and cardiovascular systems through immune cell activity, neural innervation, and autonomic cardiovascular regulation 13 – 15 . The heart-brain dialogue is modulated by multiple factors, among which sleep serves as a pivotal regulator. However, the neuroimmune mechanisms through which sleep deprivation induces cardiac dysfunction remain largely unknown. Research into the neural mechanisms governing sleep regulation and sleep deprivation effects has primarily emphasized neuronal involvement thus far 16 , 17 . Accumulating evidence implicates additional CNS cell populations—particularly microglia—as playing pivotal roles in sleep physiology 18 , 19 . Microglia constitute approximately 10–15% of the total cellular population in the mature mammalian brain. These specialized immune cells actively modulating neural plasticity through their involvement in synaptic remodeling 20 , 21 . Microglial activity exhibits state-dependent correlations with sleep/wake cycles, where sleep deprivation potently induces microglial activation. To date, sleep research has primarily focused on mechanisms associated with the neocortex and subcortical structures, while the role of the cerebellum in sleep regulation has long been overlooked. Unlike other brain regions, increased cerebellar activity during sleep was first documented in detail as early as the 1970s 22 . Functional impairment of the cerebellum can lead to sleep disorders 23 , 24 , which in turn may cause increased cardiovascular autonomic reflexes 25 – 28 . Notably, in sleep-related breathing disorders, the cerebellar fastigial nucleus (FN) participates in mediating the body's compensatory responses to significant blood pressure fluctuations 29 – 31 . Moreover, damage to the cerebellar fastigial nucleus not only manifests as sleep disturbances but also induces both structural and functional impairments in the cardiovascular system 32 – 34 .Considered together, These previous findings suggest that microglia-mediated neuroinflammation in the FN following sleep deprivation may contribute to cardiac dysfunction. The ventrolateral region of the rostral medulla (RVLM), housing cardiac sympathetic premotor neurons, has been considered pivotal in cardiovascular regulation 35 .Interestingly, there is a sympathetic excitatory cardiovascular reflex pathway between the FN and RVLM, which has been confirmed by our research. Is sleep deprivation-induced cardiac impairment mediated by the FN microglia -FN GABA -RVLM neuroimmune circuit? However, the underlying molecular and cellular mechanisms of this process remain unclear. In this study, we integrated three-dimensional (3D) reconstruction, electrophysiology, fiber photometry, optogenetics and chemogenetics to demonstrate how sleep deprivation induces cardiovascular autonomic reflexes through microglia-mediated excessive phagocytosis of neurons, ultimately promoting the development of cardiac dysfunction. Our study demonstrates that sleep deprivation activates microglia in the FN, triggering excessive microglial phagocytosis of dendritic spines on GABAergic neurons, which consequently leads to sustained reduction in FN GABA neuronal activity, and affect cardiosympathetic nerve activity through the FN-RVLM circuit. This study confirms that FN GABA neurons play a critical role in mediating sleep deprivation-induced cardiovascular autonomic reflexes and promoting cardiac dysfunction. Our findings thus provide a mechanistic framework for understanding the molecular and cellular basis of sleep deprivation-induced cardiac impairment. Results Prolonged sleep deprivation induces cardiac dysfunction The curling prevention by water(CPW) paradigm was employed to establish a murine model of chronic SD ( Fig. 1 A ) 36 .The CPW protocol involves housing experimental mice in a shallow water environment (approximately 8 mm deep, reaching just the ankle height). Contact with the water surface immediately awakens the animals when they display sleep-related behaviors such as body curling. To investigate whether these changes in cardiac function were induced by sleep deprivation, we established environmental control(eCon) and homecage control(hCon) groups ( Fig. 1 B ) . Through electrocardiogram (ECG) acquisition and analysis ( Fig. 1 C ) , we found that 48-hour sleep deprivation induced severe cardiac arrhythmias compared to the eCon and hCon groups ( Fig. 1 D, 1 E ) . This was primarily manifested by prolonged RR interval (S Fig. 1 A ) variability over time, increased dispersion in Poincaré plots (S Fig. 1 B ) , and disrupted cardiac autonomic balance. Our analysis of heart rate variability (HRV) revealed an elevated LF/HF ratio ( Fig. 1 F ) (an indication of cardiac sympathetic nerve excitation) in SD mice. Subsequent analyses were performed on the following ECG parameters: Mean RR, PR interva, QRS width, QTc width, ST deviarion. Results showed that the SD group exhibited a significant decrease in Mean RR interval compared to both the eCon and hCon groups ( Fig. 1 G ) . The SD group demonstrated a prolonged PR interval compared to both the eCon and hCon groups ( Fig. 1 H ) . No statistically significant difference in QRS duration was observed between the SD group and either the eCon and hCon groups ( Fig. 1 I ) . The SD group showed significantly increased QTc interval duration compared to both the eCon and hCon groups ( Fig. 1 J ) . No statistically significant difference in ST segment deviation was observed between the SD group and either the eCon and hCon groups ( Fig. 1 K ) . Echocardiography, as a crucial indicator of cardiac function ( Fig. 1 L ) , revealed that the SD group exhibited: Decreased left ventricular fractional shortening (LVFS%) ( Fig. 1 M ) and ejection fraction (LVEF%) ( Fig. 1 N ) , Increased left ventricular internal diameter at end-systole (LVIDs, mm) ( Fig. 1 O ) and end-diastole (LVIDd, mm) ( Fig. 1 P ) compared to both the eCon and hCon groups. These results demonstrate that sleep deprivation can induce cardiac autonomic dysfunction and impair cardiac function in mice, which mirrors sleep deprivation-related phenomena observed in humans. Prolonged sleep deprivation reduced the activity of FN GABAergic neurons For a long time, sleep research has primarily focused on the neocortex and cortically connected structures, while cerebellar activity has been largely overlooked 37 , 38 . This neglect is particularly intriguing, as cerebellar dysfunction not only impairs motor control but also alters sleep-wake cycles and even contributes to sleep disorders. Moreover, sleep dysfunction can lead to cerebellar impairment and cardiovascular dysfunction 39 – 41 . To investigate the potential link between cerebellar nuclei and cardiac function impairment induced by sleep deprivation, we examined changes in neuronal activity within the cerebellar nuclei (fastigial nucleus, FN; interposed nucleus, IN; dentate nucleus, DN;). We first examined c-Fos(a neuronal activity marker) expression in the cerebellar nuclei ( Fig. 1 Q ) and found that, compared to the eCon group, neuronal activity in the FN decreased after 48-hours of sleep deprivation, while no significant changes were observed in the IN or DN ( Fig. 1 R-T ) . Based on our findings above, to further investigate FN neuron activity in SD mouse models, we performed in vivo electrophysiological recordings ( Fig. 2 A ) . Recorded FN neurons were classified as putative excitatory pyramidal neurons (Eps), putative inhibitory interneurons (INs) and other inhibitory interneurons using unsupervised clustering techniques and cross-correlogram analyses based on the area under peak, trough to peak duration, and firing rate ( Fig. 2 B, 2 C ) 42 .Interestingly, we observed a decrease in the spike action potential firing rate of FN INs neurons in SD mice compared to eCon mice ( Fig. 2 D, 2 E, 2 F ) . In addition, We injected AAV-DIO-ChR2-mCherry into the FN of Vgat-Cre mice and performed in vivo electrophysiological recordings in the FN ( Fig. 2 G, 2 H ) . We performed optogenetic activation of GABAergic neurons in the FN of Vgat-Cre mice while simultaneously recording neuronal electrical activity in the FN. Only light-sensitive neurons were chosen for further data analysis, and we found that the firing rate of blue-light-sensitive FN GABA neurons, which are putative FN INs neurons, showed a consistent pattern in 48-hour sleep-deprived mice ( Fig. 2 I, 2 J ) . Next, we will investigate the necessity of FN GABAergic neurons in cardiac dysfunction induced in SD mice. We specifically activated FN GABAergic neurons in SD mice using optogenetic manipulation. First, an AAV expressing Cre-dependent channelrhodopsin-2 (AAV-DIO–ChR2–mCherry) was infused into the FN of Vgat-Cre mice. Three weeks later, we performed temporally restricted optogenetic manipulation. By analyzing electrocardiogram (ECG) parameters (Mean RR, PR Interval, LF/HF ratio) ( Fig. 2 K, 2 L, 2 M ) and echocardiographic indices (LVEF, LVIDd, LVIDs) ( Fig. 2 N, 2 O, 2 P ) , we evaluation of the improvement of heart function in SD mice after FN GABA neuron optogenetic activation. Our study demonstrates that optogenetic activation of FN GABAergic neurons alleviates cardiac dysfunction in SD mice. Notably, this cardioprotective effect may be mediated through modulation of cardiac autonomic nervous system activity. Pharmacogenetic activation of FN GABAergic neurons reverses cardiac dysfunction in SD mice. To delve deeper into the in vivo neuronal activity of FN GABAergic neurons following prolonged sleep deprivation, we conducted fiber photometry recordings in SD mice by infusing an adeno-associated virus (AAV) expressing the fluorescent Ca 2+ indicator GCaMP6m (AAV-DIO-GCaMP6m) into FN of Vgat-Cre mice ( Fig. 3 A, 3 B, 3 C ) . We observed that the fluorescence intensity of GCaMP6m-expressing neurons in SD mice was lower than before eCon (mean ΔF/F(%), calcium events), but this effect was revised after 24 hours of sleep recovery ( Fig. 3 D, 3 E, 3 F ) . Meanwhile, we observed the expression of c-Fos in the FN (S Fig. 2 A ) and found that neuronal activity in the FN of SD-r mice was increased compared to SD mice (S Fig. 2 B ) . The aforementioned studies consistently demonstrate a reduced firing rate of FN GABA immediately following SD induction. To determine the necessity of FN GABA neurons in SD-induced cardiac dysfunction in mice, we employed a pharmacogenetic approach to selectively activate bilateral FN GABA neurons ( Fig. 3 G, 3 H ) . Specifically, we utilized an AAV vector to deliver hM3Dq, a designer receptor exclusively activated by the inert agonist clozapine-n-oxide (CNO) into the FN region of Vgat-Cre mice (AAV-DIO-hM3Dq-mCherry/ AAV-DIO- mCherry). Furthermore, in vivo multichannel electrophysiological recordings revealed an increased firing rate of FN GABAergic neurons in SD mice injected with pharmacogenetic virus in the FN following intraperitoneal administration of CNO ( Fig. 3 I, 3 J ) . Subsequently, we assessed the efficacy of SD mice following the pharmacogenetic activation of FN GABA neurons by analyzing Mean RR ( Fig. 3 K ) , LF/HF ratio ( Fig. 3 L ) , QTc width ( Fig. 3 M ) , PR interval ( Fig. 3 N ) and Heart Rate ( Fig. 3 O ) . Evaluation of heart function in SD mice by echocardiography ( Fig. 3 P-T ). Our findings demonstrate that sleep deprivation-induced cardiac dysfunction and enhanced cardiac sympathetic excitability were significantly suppressed upon activation of FN GABAergic neurons. Prolonged sleep deprivation increased microglial engulfment of the FN GABA neuronal dendritic spines Furthermore, since the participation of microglia in sleep deprivation is well established 43 – 45 .Several investigations have demonstrated the critical role of microglia in sleep regulation, especially their contribution to slow-wave activity (SWA) production under normal non-rapid eye movement (NREM) sleep conditions 46 , 47 . We investigated whether treated with sleep deprivation brings changes in microglial morphology by quantitative 3D microglial morphometric measurements. We observed that SD increased excessive activation of FN Microglia in SD mice ( Fig. 4 A ) . The activation status of microglia was intimately associated with their morphological alterations. We investigated the influence of sleep deprivation on microglial activity. We found significantly increased expression of ionized calcium-binding adaptor molecule 1 (Iba-1), a protein expressed only in microglia 48 , and increased microglial numbers and soma areas. In addition, we observed a increase in the endpoints and length of the microglial branches. Interestingly, SD-48h mice (48-hour sleep-deprived mice) exhibited a dramatically altered morphology of FN microglia (FN Microglia ), including the intensity of microglia ( Fig. 4 B ) , microglia counts ( Fig. 4 C ) , total branch endpoints ( Fig. 4 D ) , and the length of microglial processes ( Fig. 4 E ) . To further verify the potential role of microglia in sleep deprivation-induced cardiac dysfunction, we assessed cardiac dysfunction by microinjecting minocycline (a microglial inhibitor) 49 or artificial cerebrospinal fluid (aCSF, control) into the FN of SD mice. Subsequently, we assessed the efficacy of SD mice following the microglia inhibition of FN microglia by analyzing Heart Rate ( Fig. 4 G ) , Mean RR ( Fig. 4 H ) , PR interval ( Fig. 4 I ) , QTc width ( Fig. 4 J ) and LF/HF ratio ( Fig. 4 K ) . Evaluation of heart function in SD mice by echocardiography ( Fig. 4 L, 4 M, 4 N, 4 O, 4 P ) . Additionally, in freely moving SD-48h mice, in vivo multielectrode recordings following minocycline pre-injection demonstrated that at the SD-48h time point, FN GABAergic neurons exhibited significantly higher spontaneous neuronal activity ( Fig. 4 Q, 4 R ) . In SD-48h mice with minocycline pre-injection in the FN, microglial activity was significantly suppressed ( Fig. 4 S ) . These results demonstrate that inhibiting microglial activation reversed both the suppression of FN GABAergic neurons and the emergence of cardiac dysfunction induced by sleep deprivation. Microglia regulate the activity of neurons, mainly through synaptic plasticity. Immunofluorescence analysis showed that postsynaptic density 95 (PSD95+, a major regulator of synaptic maturation), CD68+ (markers of synaptic remodeling), and Iba-1-labeled microglia were coexpressed in the FN of SD mice ( Fig. 5 A, 5 B ) . We observed that sleep deprivation induced hyperactivation of PSD95 in the FN, with increased colocalization between PSD95-immunofluorescent positive sites and IBA-1-labeled microglia compared to eCon mice ( Fig. 5 C ) . Collectively, these data suggested that reduced microglial engulfment in SD mice could contribute to decreased activation of FN GABA . To further investigate interactions with the microglial and dendritic processes of FN GABA , we performed sparse neuronal type-specific labeling by injection of AAV-CSSP-YFP-8E3 into the FN of GAD2-mice to selectively label FN GABA dendritic spines ( Fig. 5 D ) . Compared with eCon mice, the dendritic spine density of FN GABAergic neurons in SD mice was significantly reduced ( Fig. 5 E ) . We observed a reduction in microglial engulfment of YFP in SD mice compared to eCon ( Fig. 5 F, 5 G ) . Our findings demonstrate that sleep deprivation enhances microglia-mediated synaptic engulfment and exacerbates cardiac dysfunction induced in SD mice. We next investigated whether the phagocytic effect of FN microglia on the dendritic spines of FN GABAergic neurons influences the electrical activity of these GABAergic neurons. We observed changes in dendritic spine density and electrical activity of FN GABAergic neurons in SD mice following minocycline administration ( Fig. 5 H ) . We found that compared with the SD + ACSF group, SD + minocycline significantly increased the dendritic spine density of FN GABAergic neurons ( Fig. 5 I, 5 J ) . Moreover, compared to the SD + ACSF group, SD + minocycline significantly enhanced the electrical activity of FN GABAergic neurons ( Fig. 5 K, 5 L ) . These findings suggest that inhibiting the activation of FN microglia in SD mice may help reverse the suppression of electrical activity in FN GABAergic neurons. To investigate whether microglia influence GABAergic neuronal activity through dendritic spine engulfment, we administered minocycline to SD mice and observed the phagocytic activity of FN microglia. Our results demonstrated that compared with the SD + ACSF group, SD + minocycline treatment significantly reduced the engulfment of dendritic spines in FN GABAergic neurons ( Fig. 5 M, 5 N ) . Functional role of the FN-RVLM circuit for sleep deprivation induces cardiac dysfunction Based on these findings, we next sought to identify the FN GABA projections to downstream neuronal regions that may drive the brain's control of cardiac sympathetic outflow following sleep deprivation. To this end, we first performed anterograde tracing by injecting AAV expressing EGFP into Vgat-cre mice (S Fig. 3 A, 3 B ) . Three weeks later, we observed distinct EGFP⁺ fiber projections to the RVLM ( Fig. 6 A, 6 B ) . The RVLM is well established to contain presympathetic neurons for cardiac regulation and serves as a pivotal center for cardiovascular control. To further confirm the existence of neural circuit connections between FN GABA neurons and the RVLM, we performed monosynaptic anterograde tracing by injecting AAV-hSyn-Cre virus into the FN and AAV-DIO-eGFP (Cre-dependent virus) into the RVLM. Three weeks later, EGFP⁺ neurons were observed in the RVLM, revealing a direct FN→RVLM neural circuit ( Fig. 6 C, 6 D ) . To confirm the role of the FN→RVLM pathway in cardiac function impairment induced by sleep deprivation. We labeled RVLM neurons receiving FN inputs by injecting AAV-hSyn-Cre virus into the FN and AAV-DIO-eGFP virus into the RVLM ( Fig. 6 E ) . Followed by a 3-week incubation period, we assessed the activity of the FN→RVLM neural circuit using c-Fos immunofluorescence and observed a significant increase in c-Fos expression in SD mice ( Fig. 6 F, 6 G ) . This suggests that sleep deprivation reduces inhibitory input from the FN to the RVLM, leading to enhanced neuronal activity in the RVLM. This led to increased activation of cardiac sympathetic outflow. To selectively monitor changes in RVLM neuronal activity following optogenetic activation of the FN GABA →RVLM neural circuit in SD mice. We injected the optogenetic viral vector AAV-DIO-ChR2-mCherry into the FN of Vgat-cre SD mice and implanted an optical fiber above the RVLM ( Fig. 6 H, 6 I ) . Optogenetic stimulation of ChR2-expressing FN GABA →RVLM terminals was electrophysiologically confirmed to reduce firing rates in RVLM neurons ( Fig. 6 J ) . These data demonstrate that FN GABA neurons send monosynaptic inhibitory projections to RVLM neurons. Additionally, through comprehensive analysis of mean RR interval ( Fig. 6 K ) , PR interval ( Fig. 6 L ) , LF/HF ratio ( Fig. 6 M ) , and echocardiographic parameters ( Fig. 6 N, 6 O, 6 P ) . we found that optogenetic-selective activation of the FN GABA →RVLM neural circuit ameliorated cardiac dysfunction in SD mice. In summary, these cumulative results delineate the FN GABA →RVLM inhibitory pathway as a critical mediator of cardiac sympathetic outflow activation following sleep deprivation, which exacerbates cardiac function impairment. Discussion Emerging evidence indicates that excessive microglial activation, neuroinflammation, and neuronal apoptosis may serve as primary drivers of sleep deprivation-related pathologies 50 . Growing evidence reveals that modulating microglial activity can impact the progression of sleep deprivation-related disorders. Notably, sleep deprivation-induced microglial activation correlates with impaired synaptic pruning, disrupted neural circuit refinement, and altered neuronal connectivity 51 . The immune system, cardiovascular system and nervous system are intimately interconnected, working in concert to coordinate brain-heart circuitry that modulates sleep deprivation-induced cardiac sympathetic outflow and subsequent cardiac functional impairment. Neuroinflammation-induced autonomic dysfunction may mediate the association between microglial activation and sleep deprivation-induced cardiovascular pathology. From the perspective of biological development, the brain and heart are intrinsically interconnected, with profound neural-vascular network linkages existing between them 52 . Sleep deficiency is strongly correlated with the risk of cardiovascular disorders. The cerebellum plays a pivotal role in sleep regulation - although accounting for merely 10% of total brain volume, it harbors approximately 80% of all neurons in the human brain 53 . Notably, previous human studies have revealed that cerebellar malformations and injuries can lead to sleep-wake cycle disturbances and even sleep disorders 54 , 55 . Furthermore, the potential structural connections between the cerebellum and medulla oblongata represent a critical neural substrate for sleep-related visceral regulation. In this study, we made the novel discovery of an inhibitory neural circuit connecting GABAergic neurons in the cerebellar FN to the RVLM, which functionally encodes the regulation of cardiac autonomic activity during sleep. The results of this study demonstrate that 48 hours of sleep deprivation induced cardiac dysfunction, accompanied by reduced activity of FN GABAergic neurons. Enhancing the activity of FN GABAergic neurons may help mitigate sleep deprivation-induced cardiac dysfunction. Mechanistically, this process is mediated by microglial activation in the FN region and subsequent phagocytosis of dendritic spines on GABAergic neurons. Inhibiting microglial activation in SD mice restored GABAergic neuronal activity, enhance the inhibitory input from FN to RVLM and reduced excessive cardiac sympathetic excitation, and improved cardiac function. These findings highlight the critical role of microglia-driven neuronal plasticity in mediating SD-induced cardiac dysfunction. In summary, our study has identified an immune cell-mediated brain-heart axis, demonstrating that sleep deprivation plays a critical role in the occurrence of acute cardiac events. Therefore, healthy sleep habits should be incorporated into clinical management strategies for preventing cardiovascular risk events. Experimental model and study participant details Animals. All procedures in this study strictly complied with the ethical review protocols approved by the Institutional Animal Care and Use Committee (IACUC) of Anhui University of Chinese Medicine (AHTCM) and adhered to the guidelines outlined in the "Guidelines for the Care and Use of Laboratory Animals." Male C57BL/6J mice (6-7 weeks old) and VGAT-Cre transgenic mice, sourced from Charles River Laboratories and The Jackson Laboratory. Mice were housed under controlled environmental conditions (temperature: 23±1°C; relative humidity: 50±5%) in social groups (5 mice/cage), except for individuals requiring neural intervention surgery (cannula/electrode array implantation), which were singly housed postoperatively. The animal facility maintained a standard 12-hour light/dark cycle (illumination from 07:00 to 19:00), with unrestricted access to autoclaved water and irradiated feed. Data inclusion criteria enforced rigorous quality control measures: For stereotaxic AAV viral vector injections, samples with injection sites deviating >200μm from target regions were excluded; datasets from optical fiber/catheter implantations exhibiting ≥150μm deviation from predefined coordinates were omitted from statistical analysis; in vivo electrophysiological recordings with electrode contact impedance >500 kΩ or signal-to-noise ratio <3 dB were deemed invalid. All excluded data were meticulously documented with animal identification numbers, exclusion rationales, and excluded from subsequent analyses. Mouse models Sleep deprivation (SD) was achieved using “curling prevention by water” (CPW). In this study, a custom-made sealed acrylic cylinder was used, measuring 20 cm in height and 25 cm in diameter, equipped with a food trough and water bottle to provide mice with food and drinking water. Prior to the experiment, mice were individually placed in a cylinder filled with bedding material and allowed to acclimate for more than 48 hours. During the experiment, mice were placed in the cylinder for 48 hours, which contained a shallow layer of water (depth 8 mm), just reaching the ankles of the mice. When mice showed signs of sleep characterized by body curling, their noses would come into contact with the water, causing them to be quickly awakened. To maintain a 48-hour sleep deprivation state, the ambient temperature was kept at 25±2℃, and the water in the cylinders was changed every 8 hours to ensure a clean environment for the mice. The cylinder and food trough were cleaned and disinfected with disinfectant solution and ultraviolet light before reuse. After 48 hours, electrocardiogram (ECG) tracings were recorded. For the environmental control group, mice were placed on a 10 cm petri dish filled with distilled water. The petri dish was wrapped with wet gauze to maintain a moist environment, thereby studying the effects of the same humid environment on mouse sleep. Cage control mice were individually placed in a cylinder filled with bedding material, thus studying the effects of the same cage setup on mouse sleep. Virus injection and optical fiber implantation After weighing, the mice were anesthetized by inhaling 1% isoflurane gas and maintained in this state using stereotaxic techniques. The mice were fixed on a stereotaxic apparatus（RWD Life Science Inc） and their body temperature was maintained throughout the experiment. A skin incision was made from the eye to the occipital region, exposing the skull, and the connective tissue on the skull surface was wiped away with saline to keep the skull clean. The skull was adjusted to maintain the same level of the fontanelles and the lateral margins of the skull, with an error of less than 0.03 mm. The target brain region FN was then located using the adjusted skull plane: AP: - 6.47 mm, ML: ± 0.82 mm, and DV: - 2.87 mm (units: mm). 120 nl of the virus mixture was injected bilaterally into the FN at a rate of 40 nl/min, and the injection was allowed to remain for 10 minutes before being slowly removed. A ceramic plug (core diameter 200 μm, NA = 0.73，numerical aperture 0.22, Inper, China) was implanted above the injection site at a depth of 200 µm, and was fixed with dental cement. Fiber photometry recordings Before recording calcium signals, the previously implanted ceramic ferrule is connected to the recording device via an external fiber optic (ThinkerTech Co., Ltd, Nanjing, China). The fiber optic recording system couples the laser into the optical path for GCaMP6m signal recording (405nm and 470nm light sources are activated for fiber optic recording), using a CMOS array imaging method to collect the fluorescence intensity of the fiber in real-time, thus achieving synchronous recording of two channels. The 470nm excitation light corresponds to a blue calcium-sensitive fluorescent probe, which can synchronously record the activity of neurons in relevant brain areas during a certain behavioral paradigm. The reference channel uses 405nm excitation light, and the signal from this channel serves as control data to eliminate motion noise and verify the validity of the calcium signal channel data. During analysis, data exceeding the median of the absolute deviation from baseline (MAD) and remaining above twice the baseline MAD are considered calcium signaling events. Heatmaps and average calcium trace curves are generated using an in-house developed MATLAB program. In vivo optogenetic manipulations. The mice were anesthetized and immobilized on a stereotaxic apparatus for implantation of fiber cannula devices, targeting the fastigial nucleus (FN) as the key brain region. Dental acrylic resin was employed to securely anchor the fiber cannula onto the dorsal surface of the mouse skull. Chronically implantable optical fibers (200 μm diameter, Newdoon) were connected to a laser generator via fiber optic sleeves to establish stable light delivery pathways. For the experimental group, light stimulation protocols were rigorously defined as follows: blue light pulses (wavelength 473 nm, power 2–5 mW, frequency 20 Hz) or continuous yellow light exposure (wavelength 594 nm, power 5–8 mW), with each intervention strictly limited to 5 minutes in duration. The control group received identical optical parameters through a sham stimulation procedure. Post-experiment histological examinations were performed on all subjects, and data from animals exhibiting fiber tip deviation exceeding 200 μm from the targeted brain region were systematically excluded. The stimulation protocol for control mice remained consistent with the aforementioned parameters. Final validation of fiber placement within the intended brain area was conducted at experiment termination, with non-conforming datasets subjected to exclusion. In vivo electrophysiological recordings After anesthesia, routine craniotomy surgery was performed on mice, and the Plexon system was used for neuronal signal acquisition and analysis. Multi-array microwire electrodes (Electrode length:10mm;Electrode site diameter:20um；Probe spacing:200um) were placed in the FN to record neuronal spiking and LFP field potentials. The Omni Plex software displayed electrical activity in real time and stored raw data. Offline Sorter and Neuro Explorer software were used for signal analysis, including interspike intervals, autocorrelation, cross-correlation, and spectral analysis. SpikeSorter software (Plexon Inc.) was used for spike sorting to define single units. Spike detection was based on waveforms exceeding three standard deviations of noise amplitude, with manual checking required to confirm consistency. ISI histograms showed refractory periods (>2 ms) for all identifiable units, with only sufficiently isolated units included in data analysis (L-ratio 15). Based on k-means clustering methods, neurons were classified into wide-spike (WS) and narrow-spike (NS) neurons. A three-dimensional space was defined by half-peak width, half-valley width, and average firing rate. Full width at half maximum (FWHM) and firing rate were used to define inhibitory interneurons (INS). Based on baseline firing frequency, the NS group was further subdivided into fast-spiking calcium-binding protein (FS-PV Ins, >10 Hz) and non-fast-spiking NS neurons. Electrocardiography After weighing, the mice were anesthetized with 1% isoflurane and fixed on an acrylic board. Electrodes were inserted into the right forelimb (negative) and hind legs (left: positive; right: ground), and electrocardiogram (ECG) signals were recorded in real-time from the mouse's limb II lead. The experiment utilized an ML118 Animal Bio Amp (AD Instruments, Sydney, Australia) and a digitizer (PowerLab 8, ADInstruments, Sydney, Australia) to record the ECG data. Digital ECG analysis was performed using LabChart V8.1.19 software (ADInstruments, Sydney, Australia), which provided automatic data collection for heart rate variability (HRV). Each mouse had at least 15 minutes of data collected. Mouse Cardiac Ultrasound Examination In the cardiac ultrasound function assessment experiment for mice, a standardized operating protocol was employed: Mice were initially anesthetized in an induction chamber prefilled with 2% isoflurane (RWD). After stabilization of anesthesia, the mice were secured in a supine position on a temperature-controlled ultrasound platform (integrated with the VINNO6 LAB system). Anesthesia depth was maintained via continuous delivery of 1.5% isoflurane through a nose cone. Prior to the experiment, hair in the parasternal region was removed using a chemical depilatory to minimize mechanical stimulation. Subsequently, core body temperature was monitored via a lubricated rectal temperature probe, and a heating pad was adjusted to maintain stable temperatures within 37.0 ± 0.5°C. A 30 MHz high-frequency linear probe (axial resolution: 15 μm) was utilized to acquire M-mode ultrasound images at the left ventricular long-axis plane, aligned with the mitral chordae tendineae level. Real-time adjustments of anesthetic dosage were performed to regulate heart rate to 450 ± 50 bpm, thereby avoiding interference from tachycardia-induced ventricular contraction artifacts. Prior to image acquisition, a uniform layer of ultrasound coupling agent was applied to eliminate air-induced artifacts. Continuous recordings of 6–8 cardiac cycles were captured, and VINNO X5 Analysis Software was used to measure left ventricular end-systolic diameter (LVESD), left ventricular end-diastolic diameter (LVEDD), left ventricular ejection fraction (LVEF), and left ventricular fractional shortening (FS). All parameters were averaged over 5 consecutive cardiac cycles to mitigate respiratory motion-related artifacts. The entire experimental process adhered to a double-blind methodology to ensure data objectivity. Immunohistochemistry, microscopic imaging, and analysis Mice were deeply anesthetized with isoflurane and then perfused transcardially with 0.9% saline and 4% paraformaldehyde (PFA). The brain was cut into 55-micrometer coronal sections using a cryostat (NJYY5500, RWD). For immunofluorescence staining, the FN brain slices were washed three times with PBS for 5 minutes each time on an orbital shaker. At room temperature, the slices were permeabilized in 0.5% TritonX for 30 minutes to allow antibody entry into cells, followed by incubation in 3% bovine serum albumin (BSA) + 0.3% triton X for 1 hour to block various antigens on the surface of the brain slice, thereby avoiding non-specific binding of charged groups and helping produce high background signals and primary antibody binding. The primary antibody (rabbit Anti-Iba-1, Wako, 1:500 dilution, working solution: 3% BSA + 0.5% Triton X) was incubated at 4°C for 18 hours, with the brain slices being rotated every 24 hours. Then, the FN brain slices were washed three times with PBS for 5 minutes each time on an orbital shaker, followed by the addition of diluted fluorescent secondary antibody (Alexa Fluor™ 488, donkey anti-rabbit, abcam, 1:500 dilution, working solution: 3% BSA + 0.3% triton X). After placing the slices at room temperature for 1.5 hours away from light, the FN brain slices were washed three times with PBS for 5 minutes each time on an orbital shaker. The brain slices were fixed onto slides and added DAPI (1:5000), washed three times with PBS for 5 minutes each time. After drying the liquid on the slides with absorbent paper, 70% glycerin mounting medium was applied, and observed under a fluorescence microscope and confocal microscope (FV3000, OLYMPUS, Japan). Three-dimensional reconstruction Imaging experiments were conducted on an Olympus FV3000 microscope using a UPLSAPO 100x oil immersion objective lens with a numerical aperture of 1.4. All imaging experiments were performed with the same parameter settings, including gain, offset, and filter mode. In the sleep deprivation experiment, the FN injection site was selected for imaging microglia. Z-stack images were collected at 0.5 micron intervals and reconstructed into 640×640 pixel images using IMARIS 10.0.1 software (BitPlane). Three-dimensional surface reconstruction and analysis of microglia were performed using IMARIS software. The "Filaments" function was used to measure the number of branch points and the length of processes in microglia. Additionally, the "Surface" function was utilized to achieve precise reconstruction of Iba1+ microglia and mGFP+ dendrites. Finally, the contact area between neuronal dendrites and microglial processes was measured using the Surface-Surface Contact Area plugin developed for IMARIS based on MATLAB. quantification and statistical analysis All statistical analyses were conducted using GraphPad Software Inc version 8.0 and IBM SPSS Statistics version 23. For comparing data among different groups, we employed either a one-way ANOVA or an unpaired t-test as appropriate. The thresholds for statistical significance were set at P values of less than 0.05, 0.01, and 0.001. Furthermore, the results are presented as means plus or minus standard error of the mean (SEM). For detailed information on all statistical procedures used in this study, please refer to the footnotes below the figures. Declarations Acknowledgements This work was supported by the National Natural Science Foundation of China (82074536) awarded to RLC, Distinguished Young Youth Scientific Research Project in Universities of Anhui Province (2022AH020043) awarded to RLC, Research Funds of Center for Xin'an Medicine and Modernization of Traditional Chinese Medicine of IHM (2023CXMMTCM019) awarded to RLC, National Natural Science Foundation of China (82104999) awarded to QY, Excellent Young Youth Scientific Research Project in Universities of Anhui Province (2022AH030062) awarded to QY, Anhui Province Key Laboratory of Meridian Viscera Correlationship Open Project (AHMVC2024001) awarded to QY, Anhui Province Graduate Education Quality Engineering Project (2023xscx092) awarded to FZ, National Natural Science Foundation of China (81973757) awarded to LH. Author contributions Conceptualization: Fan ZHANG, Guo-ming SHEN, Rong-lin CAI, Qing YU. Data curation: Fan ZHANG, Wen-jing SHAO, Nai-xuan WEI, Zheng-jie LUO, Yan-WU. Formal analysis: Fan ZHANG, Wen-jing SHAO, Yan-WU, Nai-xuan WEI, Wei-LIU. Funding acquisition: Ling HU, Qing YU, Rong-lin CAI, Fan ZHANG. Investigation: Fan ZHANG, Rong-lin CAI, Wen-jing SHAO. Methodology: Fan ZHANG, Wen-jing SHAO, Qing YU, Jian-qing YU. Project administration: Zhi ZHANG, Rong-lin CAI, Qing YU. Resources: Rong-lin CAI, Qing YU, Ling HU. Software: Fan ZHANG, Nai-xuan WEI, Yan WU, Wen-jing SHAO, Zheng-jie LUO. Supervision: Fan ZHANG, Guo-ming SHEN, Ling HU, Qing YU, Rong-lin CAI. Validation: Qing YU, Rong-lin CAI. Visualization: Fan ZHANG, Rong-lin CAI. Writing–original draft: Fan ZHANG, Wen-jing SHAO, Nai-xuan WEI, Wei-LIU. Writing – review & editing: Fan ZHANG, Nai-xuan WEI, Rong-lin CAI. All authors have read and agreed to the published version of the manuscript. Declaration of interests The authors declare that they have no conflicts of interest. Ethics statement We obtained six-week-old male C57BL/6J mice from Hangzhou Ziyuan Experimental Animal Technology Co., Ltd., for inclusion in this study. The production license for experimental animals is SCXK (ZHE) 2019-0004. The mice were housed in cages with ad libitum access to food and water, and the ambient temperature was maintained at a relative range of 23–25 °C. All animal experiments were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals and received approval from the Animal Nursing and Utilization Committee of Anhui University of Chinese Medicine (AHUCM-mouse-2022083). Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Rong-lin CAI ( [email protected] ). References Hobson, J.A. (1969). Sleep: physiologic aspects. The New England journal of medicine 281 , 1343-1345. 10.1056/nejm196912112812406. Grandner, M.A., and Fernandez, F.X. (2021). 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mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eSleep Deprivation Schematic Diagram.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B)\u003c/strong\u003e Schematic showing our prolonged sleep deprivation paradigm. An adult mouse was subjected to one of three conditions, curling prevention by water (CPW, or simply SD), environmental control (eCon), or home-cage control (Con).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C)\u003c/strong\u003e Schematic of ECG recording.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D) \u003c/strong\u003eTypical ECG tracksof severe cardiac arrhythmias in SD mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(E)\u003c/strong\u003e The severe cardiac arrhythmias in SD mice. Red indicates that at least one cardiac arrhythmias event occurs within 30 s. (n=8 mice per group)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(F) \u003c/strong\u003eThe autonomic nervous tone assessed by the HRV frequency domain analysis in SD mice. (n=6 mice per group, Ordinary one-way ANOVA with Tukey’s post-test).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(G, H, I, J, K) \u003c/strong\u003eThe Mean RR(ms), PR Interval(ms), QRS width(ms), QTc width(ms), ST deviarion(mV) in SD mice within 5 min. (n=6 mice per group, Ordinary one-way ANOVA with Tukey’s post-test).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(L) \u003c/strong\u003eRepresentative Echocardiographic Images.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(M, N, O, P)\u003c/strong\u003e Left ventricular fractional shortening (LVFS). Left ventricular ejection fraction (LVEF). Left ventricular internal diameter, systolic (LVIDs). left ventricular internal diameter, diastolic (LVIDd). (n=6 mice per group, Ordinary one-way ANOVA with Tukey’s post-test).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(Q)\u003c/strong\u003e Left: images showing the distribution of c-Fos-positive neurons in the Deep cerebellar nuclei in SD or control mice. Scale bars, 200 μm. FN, IP, DN: The images depicting the area shown in the White dashed line. Scale bars, 50 μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(R-T)\u003c/strong\u003e Statistic data showing the distribution of c-Fos-positive neurons in the FN/IP/DN in SD or control mice (n=6 mice per group, Ordinary one-way ANOVA with Tukey’s post-test). All data are represented by mean±SEM. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01, and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7329226/v1/fa21e6a0174896beead75281.png"},{"id":90613860,"identity":"9688e497-51a7-4d6f-b72b-ef5fbb968fc7","added_by":"auto","created_at":"2025-09-04 17:49:18","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":386724,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSleep deprivation activates FN GABAergic neurons\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Schematic diagram of in vivo electrophysiological study in mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B)\u003c/strong\u003e Representative graph of waveform differentiation and 3D clusters based on an offline sorter, Spikes originating from individual units were sorted using three-dimensional principal-component analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C)\u003c/strong\u003e Representative cross-correlogram performed between a Eps Pyramidal, F-INs Inhibitory and a non-identified neuron.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D, E) \u003c/strong\u003eSample traces of action potentials recorded from FN Eps, F-INs neuron and non-identified neuron of each group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(F) \u003c/strong\u003eFiring rate data of action potentials recorded from FN\u003csup\u003eEps\u003c/sup\u003e, FN\u003csup\u003eF-INs \u003c/sup\u003eand FN\u003csup\u003enon-identified\u003c/sup\u003e neurons in mice of each group. (n=6 mice per group, unpaired t-test).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(G)\u003c/strong\u003e Schematic illustration of electrophysiological recording in the FN of Vgat-Cre mice with FN infusion of AAV-DIO-ChR2-mCherry. Enlargement showing optrodes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(H)\u003c/strong\u003e Typical image of AAV-DIO-ChR2-mCherry viral expression within the FN of Vgat-Cre mice. Scale bar, 200 μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(I) \u003c/strong\u003eExample recording of spontaneous and light-evoked spikes from a FN\u003csup\u003eGABA\u003c/sup\u003e neuron.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(J) \u003c/strong\u003eOverlay of light-evoked (blue) and averaged spontaneous (black) spike waveforms from the example unit.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(K, L, M)\u003c/strong\u003e The Mean RR(ms), PR Interval(ms), LF/HF ratio in SD mice within 5 min. (n=6 mice per group, Ordinary one-way ANOVA with Tukey’s post-test).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(N, O, P)\u003c/strong\u003e Left ventricular ejection fraction (LVEF). Left ventricular internal diameter, systolic (LVIDs). left ventricular internal diameter, diastolic (LVIDd). (n=6 mice per group, Ordinary one-way ANOVA with Tukey’s post-test). All data are represented by mean±SEM. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7329226/v1/ce61b06fe0dc0bb466090522.png"},{"id":90614464,"identity":"b55aaa93-5a6f-4628-998d-e83bf7bf527d","added_by":"auto","created_at":"2025-09-04 17:57:18","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":412437,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGABAergic neurons in the FN participate in sleep deprivation induced cardiac dysfunction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003eSchematic for viral injection and Optical-fiber calcium recording\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B, C)\u003c/strong\u003e Schematic for Optical-fiber calcium recording and a typical image showing the GCaMP6m fluorescence in the FN. (Scale bars, 200 μm (overview) and 50 μm (zoom).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D)\u003c/strong\u003e Representative typical trace and photometric heatmaps.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(E, F)\u003c/strong\u003e Comparison of the ΔF/F and calcium events of FN\u003csup\u003eGABA\u003c/sup\u003e GCaMP6m signals. ΔF/F, the change in fluorescence (ΔF) over the baseline fluorescence of calcium spikes.(n=6 mice. paired t-test).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(G)\u003c/strong\u003e Schematic for viral injection and chemogenetic experimental timeline.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(H) \u003c/strong\u003eTypical image showing the chemogenetic fluorescence in the FN. (Scale bars, 200 μm).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(I)\u003c/strong\u003e Sample traces of action potentials recorded from FN\u003csup\u003eGABA\u003c/sup\u003e neuron of each group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(J) \u003c/strong\u003eFiring rate data of action potentials recorded from FN\u003csup\u003eGABA\u003c/sup\u003e neurons in mice of each group. (unpaired t-test).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(K, L, M, N, O)\u003c/strong\u003e The Mean RR(ms), LF/HF ratio, QTc width(ms), PR Interval(ms), Heart Rate in SD mice within 5 min. (n=6 mice per group, Ordinary one-way ANOVA with Tukey’s post-test).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(P) \u003c/strong\u003eRepresentative Echocardiographic Images.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(Q, R, S, T) \u003c/strong\u003eLeft ventricular fractional shortening (LVFS). Left ventricular ejection fraction (LVEF). Left ventricular internal diameter, systolic (LVIDs). left ventricular internal diameter, diastolic (LVIDd). (n=6 mice per group, Ordinary one-way ANOVA with Tukey’s post-test). All data are represented by mean±SEM. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7329226/v1/b8595e64aa6ad9a7973ade6e.png"},{"id":90614463,"identity":"52b7dd20-c2f9-477d-bbf1-c74a5b7e2202","added_by":"auto","created_at":"2025-09-04 17:57:18","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":475234,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMicroglia in the FN participate in sleep deprivation induced cardiac dysfunction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Images show 3D reconstruction of FN microglia (green) in eCon, SD 12h, and SD-48h group. The scale bars are 100μm (×200), 20μm (×400), and 10μm (Imaris-3D).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B, C, D, E) \u003c/strong\u003eThe number of Iba-1\u003csup\u003e+\u003c/sup\u003e cells per 0.05mm\u003csup\u003e2\u003c/sup\u003e, Iba-1\u003csup\u003e+\u003c/sup\u003e intensity, microglia branch endpoints and average branch length of Iba-1\u003csup\u003e+\u003c/sup\u003e microglia in FN from the mice in eCon, SD-12h, and SD-48h group. One-way ANOVA with Tukey’s post-test, n = 6 per group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(F)\u003c/strong\u003e Upper: schematic diagram of drug implantation with cannula in FN. Lower: timeline of experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(G, H, I, J, K)\u003c/strong\u003e The Heart Rate, Mean RR(ms), PR Interval(ms), QTc width(ms), LF/HF ratio in SD mice within 5 min. (n=6 mice per group, Ordinary one-way ANOVA with Tukey’s post-test).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(L) \u003c/strong\u003eRepresentative Echocardiographic Images.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(M, N, O, P) \u003c/strong\u003eLeft ventricular fractional shortening (LVFS). Left ventricular ejection fraction (LVEF). Left ventricular internal diameter, systolic (LVIDs). left ventricular internal diameter, diastolic (LVIDd). (n=6 mice per group, Ordinary one-way ANOVA with Tukey’s post-test)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(Q)\u003c/strong\u003e Upper: Three-dimensional reconstruction of microglia in the FN of SD mice after minocycline administration. Lower: Sample traces of action potentials recorded from FN\u003csup\u003eGABA\u003c/sup\u003e neuron of each group. The scale bars are 10μm(×1000) (Imaris-3D).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(R) \u003c/strong\u003eFiring rate data of action potentials recorded from FN\u003csup\u003eGABA\u003c/sup\u003e neurons in mice of each group. (unpaired t-test).\u003cstrong\u003e(S)\u003c/strong\u003e The Iba-1\u003csup\u003e+\u003c/sup\u003e intensity in FN from the mice in each group. (n=6 per group, unpaired t-test) *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7329226/v1/af3a609723af4505d1f78f1c.png"},{"id":90613863,"identity":"10637bbe-97bf-4823-97e7-6d6b3a83a0c4","added_by":"auto","created_at":"2025-09-04 17:49:18","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":553029,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSleep deprivation triggers microglial phagocytosis of dendritic spines on FN GABAergic neurons\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Upper: Schematic diagram of immunofluorescence staining of Iba-1, PSD95, CD68 in FN. Lower: timeline of experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B, C)\u003c/strong\u003e Representative immunofluorescent images and standard analyses of CD68 (purple), PSD95 (red), and Iba-1 (green) levels in the FN of the SD and eCon mice. The scale bar is 10 mm, unpaired t-test, n = 6 per group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D)\u003c/strong\u003e Experimental scheme for virus injection in Sparsely labeled FN\u003csup\u003eGABA\u003c/sup\u003e neurons.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(E)\u003c/strong\u003e Total spine density of FN\u003csup\u003eGABA\u003c/sup\u003e neurons.( n=6 per group, unpaired t-test)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(F)\u003c/strong\u003e Images of 3D rendering of Iba-1 (red), mGFP (green), and CD68 (purple) in the FN from mice in eCon and SD groups. Scale bars are 15 μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(G)\u003c/strong\u003e Summarized data for the size of microglia-dendrite contacts in eCon and SD mice. (n=48 contacts from 6 eCon mice; n = 48 contacts from 6 SD mice; unpaired t test).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(H)\u003c/strong\u003e Schematic of in vivo electrophysiological for Vgat-eGFP mice and FN\u003csup\u003eGABA\u003c/sup\u003e neuronal dendrites from ACSF and Mino-treated mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(I)\u003c/strong\u003e representative images of neuronal dendrites.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(J)\u003c/strong\u003e Total spine density of FN\u003csup\u003eGABA\u003c/sup\u003e neurons.( n=6 per group, unpaired t-test)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(K)\u003c/strong\u003e Sample traces of action potentials recorded from FN\u003csup\u003eGABA\u003c/sup\u003e neuron of each group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(L) \u003c/strong\u003eFiring rate data of action potentials recorded from FN\u003csup\u003eGABA\u003c/sup\u003e neurons in mice of each group. (unpaired t-test).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(M, N)\u003c/strong\u003e Representative immunofluorescent images and standard analyses of PSD95 (purple), and Iba-1 (red) levels in the FN of the SD and eCon mice. The scale bar is 20μm, unpaired t-test, n = 6 per group. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7329226/v1/ece0d1aebabec44161cd6dcc.png"},{"id":90614633,"identity":"4b6c6ba5-9d4f-40d4-8d75-33a7f6591ccf","added_by":"auto","created_at":"2025-09-04 18:05:18","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":575526,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDissection of the FN\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eGABA\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e-RVLM circuit\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eSchematic for the anterograde tracing from the FN to the RVLM. Representative images showing EGFP\u003csup\u003e+\u003c/sup\u003e fibers in the RVLM. Scale bars, 100μm and 20μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B) \u003c/strong\u003eNerve fiber density of the anterograde tracing from the FN to the RVLM. n=4 mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C) \u003c/strong\u003eSchematic of the Cre-dependent anterograde trans-monosynaptic tracing strategy. Representative images showing the EGFP\u003csup\u003e+\u003c/sup\u003e neurons in the RVLM. Scale bars, 100μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D) \u003c/strong\u003eEGFP\u003csup\u003e+\u003c/sup\u003e neurons of the anterograde tracing from the FN to the RVLM. n=4 mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(E) \u003c/strong\u003eSchematic for the expression of c-Fos-positive neurons in the ZI-RVLM circuit of SD mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(F, G)\u003c/strong\u003e Representative images and data showing the percentage of EGFP\u003csup\u003e+\u003c/sup\u003e neurons and c-Fos\u003csup\u003e+\u003c/sup\u003e(Alexa Flour 594) co-expression in the RVLM. Scale bars, 100μm and 20μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(H) \u003c/strong\u003eSchematic diagram showing the strategy used to determine whether neuronal activity in the RVLM was modulated by the FN. AAV-DIO-ChR2-mCherry virus was injected into the FN and a optical fiber was implanted into the RVLM. The timeline shown below demonstrates the experimental procedure.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(I)\u003c/strong\u003e Representative images showing the FN injection site and the optical fiber was implanted into the RVLM. Scale bars, 200μm (FN) and 100μm (RVLM).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(J) \u003c/strong\u003eExample recording of spontaneous and light-evoked spikes from a RVLM neuron.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(K, L, M) \u003c/strong\u003eOptogenetic modulation of the FN\u003csup\u003eGABA\u003c/sup\u003e-RVLM circuit in SD mice with ECG monitoring of Mean RR(ms), PR Interval(ms), LF/HF ratio. (n=6 mice per group, Ordinary one-way ANOVA with Tukey’s post-test).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(N, O, P)\u003c/strong\u003e Optogenetic modulation of the FN\u003csup\u003eGABA\u003c/sup\u003e-RVLM circuit in SD mice with Echocardiographic monitoring of Left ventricular ejection fraction (LVEF), Left ventricular internal diameter, systolic (LVIDs). left ventricular internal diameter, diastolic (LVIDd). (n=6 mice per group, Ordinary one-way ANOVA with Tukey’s post-test). *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7329226/v1/539335ef9c665ad8c35de8a4.png"},{"id":91148510,"identity":"ca7ad907-c848-405a-b44d-ddaa43a1e3bb","added_by":"auto","created_at":"2025-09-12 06:44:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3719093,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7329226/v1/84c05011-4805-47b1-8c3c-1665d29532d6.pdf"},{"id":90614635,"identity":"1ef70950-2fc3-46d9-aa4b-d98c81ea9702","added_by":"auto","created_at":"2025-09-04 18:05:18","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3541245,"visible":true,"origin":"","legend":"Prolonged sleep deprivation induces cardiac dysfunction via microglia-neural circuit coupling","description":"","filename":"SupportingInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-7329226/v1/091f9c50f7f029ad3cc287b3.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Prolonged sleep deprivation induces cardiac dysfunction via microglia-neural circuit coupling","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSleep constitutes an essential physiological requirement for the vast majority of animal species\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e.Sleep is critically involved in regulating fundamental biological processes, including immune modulation\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, cognitive function\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e, and metabolic homeostasis\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. With societal development and the acceleration of modern lifestyles, the prevalence of sleep disorders and consequent sleep deprivation has risen significantly\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Sleep is crucial for cardiovascular health. Insufficient sleep or sleep deprivation elevates the risk of cardiac dysfunction\u003csup\u003e\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e and may trigger myocardial infarction\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, independent of genetic predisposition or other conventional risk factors\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. The brain-heart axis mediates bidirectional communication between the central nervous and cardiovascular systems through immune cell activity, neural innervation, and autonomic cardiovascular regulation\u003csup\u003e\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. The heart-brain dialogue is modulated by multiple factors, among which sleep serves as a pivotal regulator. However, the neuroimmune mechanisms through which sleep deprivation induces cardiac dysfunction remain largely unknown.\u003c/p\u003e\u003cp\u003eResearch into the neural mechanisms governing sleep regulation and sleep deprivation effects has primarily emphasized neuronal involvement thus far\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Accumulating evidence implicates additional CNS cell populations\u0026mdash;particularly microglia\u0026mdash;as playing pivotal roles in sleep physiology\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Microglia constitute approximately 10\u0026ndash;15% of the total cellular population in the mature mammalian brain. These specialized immune cells actively modulating neural plasticity through their involvement in synaptic remodeling\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Microglial activity exhibits state-dependent correlations with sleep/wake cycles, where sleep deprivation potently induces microglial activation. To date, sleep research has primarily focused on mechanisms associated with the neocortex and subcortical structures, while the role of the cerebellum in sleep regulation has long been overlooked. Unlike other brain regions, increased cerebellar activity during sleep was first documented in detail as early as the 1970s\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Functional impairment of the cerebellum can lead to sleep disorders\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, which in turn may cause increased cardiovascular autonomic reflexes\u003csup\u003e\u003cspan additionalcitationids=\"CR26 CR27\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Notably, in sleep-related breathing disorders, the cerebellar fastigial nucleus (FN) participates in mediating the body's compensatory responses to significant blood pressure fluctuations\u003csup\u003e\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Moreover, damage to the cerebellar fastigial nucleus not only manifests as sleep disturbances but also induces both structural and functional impairments in the cardiovascular system\u003csup\u003e\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e.Considered together, These previous findings suggest that microglia-mediated neuroinflammation in the FN following sleep deprivation may contribute to cardiac dysfunction. The ventrolateral region of the rostral medulla (RVLM), housing cardiac sympathetic premotor neurons, has been considered pivotal in cardiovascular regulation\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e.Interestingly, there is a sympathetic excitatory cardiovascular reflex pathway between the FN and RVLM, which has been confirmed by our research. Is sleep deprivation-induced cardiac impairment mediated by the FN\u003csup\u003emicroglia\u003c/sup\u003e-FN\u003csup\u003eGABA\u003c/sup\u003e-RVLM neuroimmune circuit? However, the underlying molecular and cellular mechanisms of this process remain unclear.\u003c/p\u003e\u003cp\u003eIn this study, we integrated three-dimensional (3D) reconstruction, electrophysiology, fiber photometry, optogenetics and chemogenetics to demonstrate how sleep deprivation induces cardiovascular autonomic reflexes through microglia-mediated excessive phagocytosis of neurons, ultimately promoting the development of cardiac dysfunction. Our study demonstrates that sleep deprivation activates microglia in the FN, triggering excessive microglial phagocytosis of dendritic spines on GABAergic neurons, which consequently leads to sustained reduction in FN\u003csup\u003eGABA\u003c/sup\u003e neuronal activity, and affect cardiosympathetic nerve activity through the FN-RVLM circuit. This study confirms that FN\u003csup\u003eGABA\u003c/sup\u003e neurons play a critical role in mediating sleep deprivation-induced cardiovascular autonomic reflexes and promoting cardiac dysfunction. Our findings thus provide a mechanistic framework for understanding the molecular and cellular basis of sleep deprivation-induced cardiac impairment.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eProlonged sleep deprivation induces cardiac dysfunction\u003c/h2\u003e\u003cp\u003eThe curling prevention by water(CPW) paradigm was employed to establish a murine model of chronic SD \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e.The CPW protocol involves housing experimental mice in a shallow water environment (approximately 8 mm deep, reaching just the ankle height). Contact with the water surface immediately awakens the animals when they display sleep-related behaviors such as body curling. To investigate whether these changes in cardiac function were induced by sleep deprivation, we established environmental control(eCon) and homecage control(hCon) groups \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. Through electrocardiogram (ECG) acquisition and analysis \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e, we found that 48-hour sleep deprivation induced severe cardiac arrhythmias compared to the eCon and hCon groups\u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE\u003cb\u003e)\u003c/b\u003e. This was primarily manifested by prolonged RR interval \u003cb\u003e(S\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e variability over time, increased dispersion in Poincar\u0026eacute; plots \u003cb\u003e(S\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e, and disrupted cardiac autonomic balance. Our analysis of heart rate variability (HRV) revealed an elevated LF/HF ratio \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF\u003cb\u003e)\u003c/b\u003e (an indication of cardiac sympathetic nerve excitation) in SD mice.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eSubsequent analyses were performed on the following ECG parameters: Mean RR, PR interva, QRS width, QTc width, ST deviarion. Results showed that the SD group exhibited a significant decrease in Mean RR interval compared to both the eCon and hCon groups \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG\u003cb\u003e)\u003c/b\u003e. The SD group demonstrated a prolonged PR interval compared to both the eCon and hCon groups \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH\u003cb\u003e)\u003c/b\u003e. No statistically significant difference in QRS duration was observed between the SD group and either the eCon and hCon groups \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI\u003cb\u003e)\u003c/b\u003e. The SD group showed significantly increased QTc interval duration compared to both the eCon and hCon groups \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ\u003cb\u003e)\u003c/b\u003e. No statistically significant difference in ST segment deviation was observed between the SD group and either the eCon and hCon groups \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003eEchocardiography, as a crucial indicator of cardiac function \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eL\u003cb\u003e)\u003c/b\u003e, revealed that the SD group exhibited: Decreased left ventricular fractional shortening (LVFS%) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eM\u003cb\u003e)\u003c/b\u003e and ejection fraction (LVEF%) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eN\u003cb\u003e)\u003c/b\u003e, Increased left ventricular internal diameter at end-systole (LVIDs, mm) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eO\u003cb\u003e)\u003c/b\u003e and end-diastole (LVIDd, mm) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eP\u003cb\u003e)\u003c/b\u003e compared to both the eCon and hCon groups. These results demonstrate that sleep deprivation can induce cardiac autonomic dysfunction and impair cardiac function in mice, which mirrors sleep deprivation-related phenomena observed in humans.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eProlonged sleep deprivation reduced the activity of FN GABAergic neurons\u003c/h3\u003e\n\u003cp\u003eFor a long time, sleep research has primarily focused on the neocortex and cortically connected structures, while cerebellar activity has been largely overlooked\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. This neglect is particularly intriguing, as cerebellar dysfunction not only impairs motor control but also alters sleep-wake cycles and even contributes to sleep disorders. Moreover, sleep dysfunction can lead to cerebellar impairment and cardiovascular dysfunction\u003csup\u003e\u003cspan additionalcitationids=\"CR40\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. To investigate the potential link between cerebellar nuclei and cardiac function impairment induced by sleep deprivation, we examined changes in neuronal activity within the cerebellar nuclei (fastigial nucleus, FN; interposed nucleus, IN; dentate nucleus, DN;). We first examined c-Fos(a neuronal activity marker) expression in the cerebellar nuclei \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eQ\u003cb\u003e)\u003c/b\u003e and found that, compared to the eCon group, neuronal activity in the FN decreased after 48-hours of sleep deprivation, while no significant changes were observed in the IN or DN\u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eR-T\u003cb\u003e)\u003c/b\u003e. Based on our findings above, to further investigate FN neuron activity in SD mouse models, we performed in vivo electrophysiological recordings \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. Recorded FN neurons were classified as putative excitatory pyramidal neurons (Eps), putative inhibitory interneurons (INs) and other inhibitory interneurons using unsupervised clustering techniques and cross-correlogram analyses based on the area under peak, trough to peak duration, and firing rate \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e.Interestingly, we observed a decrease in the spike action potential firing rate of FN\u003csup\u003eINs\u003c/sup\u003e neurons in SD mice compared to eCon mice \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn addition, We injected AAV-DIO-ChR2-mCherry into the FN of Vgat-Cre mice and performed in vivo electrophysiological recordings in the FN \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH\u003cb\u003e)\u003c/b\u003e. We performed optogenetic activation of GABAergic neurons in the FN of Vgat-Cre mice while simultaneously recording neuronal electrical activity in the FN. Only light-sensitive neurons were chosen for further data analysis, and we found that the firing rate of blue-light-sensitive FN\u003csup\u003eGABA\u003c/sup\u003e neurons, which are putative FN\u003csup\u003eINs\u003c/sup\u003e neurons, showed a consistent pattern in 48-hour sleep-deprived mice \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ\u003cb\u003e)\u003c/b\u003e. Next, we will investigate the necessity of FN GABAergic neurons in cardiac dysfunction induced in SD mice. We specifically activated FN GABAergic neurons in SD mice using optogenetic manipulation. First, an AAV expressing Cre-dependent channelrhodopsin-2 (AAV-DIO\u0026ndash;ChR2\u0026ndash;mCherry) was infused into the FN of Vgat-Cre mice. Three weeks later, we performed temporally restricted optogenetic manipulation. By analyzing electrocardiogram (ECG) parameters (Mean RR, PR Interval, LF/HF ratio) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eK, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eL, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eM\u003cb\u003e)\u003c/b\u003eand echocardiographic indices (LVEF, LVIDd, LVIDs) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eN, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eO, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eP\u003cb\u003e)\u003c/b\u003e, we evaluation of the improvement of heart function in SD mice after FN\u003csup\u003eGABA\u003c/sup\u003e neuron optogenetic activation. Our study demonstrates that optogenetic activation of FN GABAergic neurons alleviates cardiac dysfunction in SD mice. Notably, this cardioprotective effect may be mediated through modulation of cardiac autonomic nervous system activity.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePharmacogenetic activation of FN GABAergic neurons reverses cardiac dysfunction in SD mice.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo delve deeper into the in vivo neuronal activity of FN GABAergic neurons following prolonged sleep deprivation, we conducted fiber photometry recordings in SD mice by infusing an adeno-associated virus (AAV) expressing the fluorescent Ca\u003csup\u003e2+\u003c/sup\u003e indicator GCaMP6m (AAV-DIO-GCaMP6m) into FN of Vgat-Cre mice \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e. We observed that the fluorescence intensity of GCaMP6m-expressing neurons in SD mice was lower than before eCon (mean ΔF/F(%), calcium events), but this effect was revised after 24 hours of sleep recovery \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF\u003cb\u003e)\u003c/b\u003e. Meanwhile, we observed the expression of c-Fos in the FN \u003cb\u003e(S\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e and found that neuronal activity in the FN of SD-r mice was increased compared to SD mice \u003cb\u003e(S\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe aforementioned studies consistently demonstrate a reduced firing rate of FN\u003csup\u003eGABA\u003c/sup\u003e immediately following SD induction. To determine the necessity of FN\u003csup\u003eGABA\u003c/sup\u003e neurons in SD-induced cardiac dysfunction in mice, we employed a pharmacogenetic approach to selectively activate bilateral FN\u003csup\u003eGABA\u003c/sup\u003e neurons \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH\u003cb\u003e)\u003c/b\u003e. Specifically, we utilized an AAV vector to deliver hM3Dq, a designer receptor exclusively activated by the inert agonist clozapine-n-oxide (CNO) into the FN region of Vgat-Cre mice (AAV-DIO-hM3Dq-mCherry/ AAV-DIO- mCherry). Furthermore, in vivo multichannel electrophysiological recordings revealed an increased firing rate of FN GABAergic neurons in SD mice injected with pharmacogenetic virus in the FN following intraperitoneal administration of CNO\u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ\u003cb\u003e)\u003c/b\u003e. Subsequently, we assessed the efficacy of SD mice following the pharmacogenetic activation of FN\u003csup\u003eGABA\u003c/sup\u003e neurons by analyzing Mean RR \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eK\u003cb\u003e)\u003c/b\u003e, LF/HF ratio \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eL\u003cb\u003e)\u003c/b\u003e, QTc width \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eM\u003cb\u003e)\u003c/b\u003e, PR interval \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eN\u003cb\u003e)\u003c/b\u003e and Heart Rate \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eO\u003cb\u003e)\u003c/b\u003e. Evaluation of heart function in SD mice by echocardiography \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eP-T\u003cb\u003e).\u003c/b\u003e Our findings demonstrate that sleep deprivation-induced cardiac dysfunction and enhanced cardiac sympathetic excitability were significantly suppressed upon activation of FN GABAergic neurons.\u003c/p\u003e\n\u003ch3\u003eProlonged sleep deprivation increased microglial engulfment of the FN GABA neuronal dendritic spines\u003c/h3\u003e\n\u003cp\u003eFurthermore, since the participation of microglia in sleep deprivation is well established\u003csup\u003e\u003cspan additionalcitationids=\"CR44\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e.Several investigations have demonstrated the critical role of microglia in sleep regulation, especially their contribution to slow-wave activity (SWA) production under normal non-rapid eye movement (NREM) sleep conditions\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e,\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. We investigated whether treated with sleep deprivation brings changes in microglial morphology by quantitative 3D microglial morphometric measurements. We observed that SD increased excessive activation of FN\u003csup\u003eMicroglia\u003c/sup\u003e in SD mice \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. The activation status of microglia was intimately associated with their morphological alterations. We investigated the influence of sleep deprivation on microglial activity. We found significantly increased expression of ionized calcium-binding adaptor molecule 1 (Iba-1), a protein expressed only in microglia\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e, and increased microglial numbers and soma areas. In addition, we observed a increase in the endpoints and length of the microglial branches. Interestingly, SD-48h mice (48-hour sleep-deprived mice) exhibited a dramatically altered morphology of FN microglia (FN\u003csup\u003eMicroglia\u003c/sup\u003e), including the intensity of microglia\u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e, microglia counts \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e, total branch endpoints \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e, and the length of microglial processes \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo further verify the potential role of microglia in sleep deprivation-induced cardiac dysfunction, we assessed cardiac dysfunction by microinjecting minocycline (a microglial inhibitor) \u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003eor artificial cerebrospinal fluid (aCSF, control) into the FN of SD mice. Subsequently, we assessed the efficacy of SD mice following the microglia inhibition of FN microglia by analyzing Heart Rate \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG\u003cb\u003e)\u003c/b\u003e, Mean RR \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH\u003cb\u003e)\u003c/b\u003e, PR interval \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI\u003cb\u003e)\u003c/b\u003e, QTc width \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ\u003cb\u003e)\u003c/b\u003e and LF/HF ratio \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eK\u003cb\u003e)\u003c/b\u003e. Evaluation of heart function in SD mice by echocardiography \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eL, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eM, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eN, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eO, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eP\u003cb\u003e)\u003c/b\u003e. Additionally, in freely moving SD-48h mice, in vivo multielectrode recordings following minocycline pre-injection demonstrated that at the SD-48h time point, FN GABAergic neurons exhibited significantly higher spontaneous neuronal activity \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eQ, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eR\u003cb\u003e)\u003c/b\u003e. In SD-48h mice with minocycline pre-injection in the FN, microglial activity was significantly suppressed \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eS\u003cb\u003e)\u003c/b\u003e. These results demonstrate that inhibiting microglial activation reversed both the suppression of FN GABAergic neurons and the emergence of cardiac dysfunction induced by sleep deprivation.\u003c/p\u003e\u003cp\u003eMicroglia regulate the activity of neurons, mainly through synaptic plasticity. Immunofluorescence analysis showed that postsynaptic density 95 (PSD95+, a major regulator of synaptic maturation), CD68+ (markers of synaptic remodeling), and Iba-1-labeled microglia were coexpressed in the FN of SD mice \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. We observed that sleep deprivation induced hyperactivation of PSD95 in the FN, with increased colocalization between PSD95-immunofluorescent positive sites and IBA-1-labeled microglia compared to eCon mice \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e. Collectively, these data suggested that reduced microglial engulfment in SD mice could contribute to decreased activation of FN\u003csup\u003eGABA\u003c/sup\u003e. To further investigate interactions with the microglial and dendritic processes of FN\u003csup\u003eGABA\u003c/sup\u003e, we performed sparse neuronal type-specific labeling by injection of AAV-CSSP-YFP-8E3 into the FN of GAD2-mice to selectively label FN\u003csup\u003eGABA\u003c/sup\u003e dendritic spines \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e. Compared with eCon mice, the dendritic spine density of FN GABAergic neurons in SD mice was significantly reduced \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE\u003cb\u003e)\u003c/b\u003e. We observed a reduction in microglial engulfment of YFP in SD mice compared to eCon \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG\u003cb\u003e)\u003c/b\u003e. Our findings demonstrate that sleep deprivation enhances microglia-mediated synaptic engulfment and exacerbates cardiac dysfunction induced in SD mice.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWe next investigated whether the phagocytic effect of FN microglia on the dendritic spines of FN GABAergic neurons influences the electrical activity of these GABAergic neurons. We observed changes in dendritic spine density and electrical activity of FN GABAergic neurons in SD mice following minocycline administration \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH\u003cb\u003e)\u003c/b\u003e. We found that compared with the SD\u0026thinsp;+\u0026thinsp;ACSF group, SD\u0026thinsp;+\u0026thinsp;minocycline significantly increased the dendritic spine density of FN GABAergic neurons \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eJ\u003cb\u003e)\u003c/b\u003e. Moreover, compared to the SD\u0026thinsp;+\u0026thinsp;ACSF group, SD\u0026thinsp;+\u0026thinsp;minocycline significantly enhanced the electrical activity of FN GABAergic neurons \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eK, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eL\u003cb\u003e)\u003c/b\u003e. These findings suggest that inhibiting the activation of FN microglia in SD mice may help reverse the suppression of electrical activity in FN GABAergic neurons. To investigate whether microglia influence GABAergic neuronal activity through dendritic spine engulfment, we administered minocycline to SD mice and observed the phagocytic activity of FN microglia. Our results demonstrated that compared with the SD\u0026thinsp;+\u0026thinsp;ACSF group, SD\u0026thinsp;+\u0026thinsp;minocycline treatment significantly reduced the engulfment of dendritic spines in FN GABAergic neurons \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eM, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eN\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e\n\u003ch3\u003eFunctional role of the FN-RVLM circuit for sleep deprivation induces cardiac dysfunction\u003c/h3\u003e\n\u003cp\u003eBased on these findings, we next sought to identify the FN\u003csup\u003eGABA\u003c/sup\u003e projections to downstream neuronal regions that may drive the brain's control of cardiac sympathetic outflow following sleep deprivation. To this end, we first performed anterograde tracing by injecting AAV expressing EGFP into Vgat-cre mice\u003cb\u003e(S\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. Three weeks later, we observed distinct EGFP⁺ fiber projections to the RVLM \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. The RVLM is well established to contain presympathetic neurons for cardiac regulation and serves as a pivotal center for cardiovascular control. To further confirm the existence of neural circuit connections between FN\u003csup\u003eGABA\u003c/sup\u003e neurons and the RVLM, we performed monosynaptic anterograde tracing by injecting AAV-hSyn-Cre virus into the FN and AAV-DIO-eGFP (Cre-dependent virus) into the RVLM. Three weeks later, EGFP⁺ neurons were observed in the RVLM, revealing a direct FN\u0026rarr;RVLM neural circuit \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo confirm the role of the FN\u0026rarr;RVLM pathway in cardiac function impairment induced by sleep deprivation. We labeled RVLM neurons receiving FN inputs by injecting AAV-hSyn-Cre virus into the FN and AAV-DIO-eGFP virus into the RVLM \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE\u003cb\u003e)\u003c/b\u003e. Followed by a 3-week incubation period, we assessed the activity of the FN\u0026rarr;RVLM neural circuit using c-Fos immunofluorescence and observed a significant increase in c-Fos expression in SD mice \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG\u003cb\u003e)\u003c/b\u003e. This suggests that sleep deprivation reduces inhibitory input from the FN to the RVLM, leading to enhanced neuronal activity in the RVLM. This led to increased activation of cardiac sympathetic outflow.\u003c/p\u003e\u003cp\u003eTo selectively monitor changes in RVLM neuronal activity following optogenetic activation of the FN\u003csup\u003eGABA\u003c/sup\u003e\u0026rarr;RVLM neural circuit in SD mice. We injected the optogenetic viral vector AAV-DIO-ChR2-mCherry into the FN of Vgat-cre SD mice and implanted an optical fiber above the RVLM \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI\u003cb\u003e)\u003c/b\u003e. Optogenetic stimulation of ChR2-expressing FN\u003csup\u003eGABA\u003c/sup\u003e\u0026rarr;RVLM terminals was electrophysiologically confirmed to reduce firing rates in RVLM neurons \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eJ\u003cb\u003e)\u003c/b\u003e. These data demonstrate that FN\u003csup\u003eGABA\u003c/sup\u003e neurons send monosynaptic inhibitory projections to RVLM neurons. Additionally, through comprehensive analysis of mean RR interval \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eK\u003cb\u003e)\u003c/b\u003e, PR interval \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eL\u003cb\u003e)\u003c/b\u003e, LF/HF ratio \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eM\u003cb\u003e)\u003c/b\u003e, and echocardiographic parameters \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eN, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eO, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eP\u003cb\u003e)\u003c/b\u003e. we found that optogenetic-selective activation of the FN\u003csup\u003eGABA\u003c/sup\u003e\u0026rarr;RVLM neural circuit ameliorated cardiac dysfunction in SD mice. In summary, these cumulative results delineate the FN\u003csup\u003eGABA\u003c/sup\u003e\u0026rarr;RVLM inhibitory pathway as a critical mediator of cardiac sympathetic outflow activation following sleep deprivation, which exacerbates cardiac function impairment.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eEmerging evidence indicates that excessive microglial activation, neuroinflammation, and neuronal apoptosis may serve as primary drivers of sleep deprivation-related pathologies\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. Growing evidence reveals that modulating microglial activity can impact the progression of sleep deprivation-related disorders. Notably, sleep deprivation-induced microglial activation correlates with impaired synaptic pruning, disrupted neural circuit refinement, and altered neuronal connectivity\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. The immune system, cardiovascular system and nervous system are intimately interconnected, working in concert to coordinate brain-heart circuitry that modulates sleep deprivation-induced cardiac sympathetic outflow and subsequent cardiac functional impairment. Neuroinflammation-induced autonomic dysfunction may mediate the association between microglial activation and sleep deprivation-induced cardiovascular pathology.\u003c/p\u003e\u003cp\u003eFrom the perspective of biological development, the brain and heart are intrinsically interconnected, with profound neural-vascular network linkages existing between them\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. Sleep deficiency is strongly correlated with the risk of cardiovascular disorders. The cerebellum plays a pivotal role in sleep regulation - although accounting for merely 10% of total brain volume, it harbors approximately 80% of all neurons in the human brain\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. Notably, previous human studies have revealed that cerebellar malformations and injuries can lead to sleep-wake cycle disturbances and even sleep disorders\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e,\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. Furthermore, the potential structural connections between the cerebellum and medulla oblongata represent a critical neural substrate for sleep-related visceral regulation. In this study, we made the novel discovery of an inhibitory neural circuit connecting GABAergic neurons in the cerebellar FN to the RVLM, which functionally encodes the regulation of cardiac autonomic activity during sleep.\u003c/p\u003e\u003cp\u003eThe results of this study demonstrate that 48 hours of sleep deprivation induced cardiac dysfunction, accompanied by reduced activity of FN GABAergic neurons. Enhancing the activity of FN GABAergic neurons may help mitigate sleep deprivation-induced cardiac dysfunction. Mechanistically, this process is mediated by microglial activation in the FN region and subsequent phagocytosis of dendritic spines on GABAergic neurons. Inhibiting microglial activation in SD mice restored GABAergic neuronal activity, enhance the inhibitory input from FN to RVLM and reduced excessive cardiac sympathetic excitation, and improved cardiac function. These findings highlight the critical role of microglia-driven neuronal plasticity in mediating SD-induced cardiac dysfunction.\u003c/p\u003e\u003cp\u003eIn summary, our study has identified an immune cell-mediated brain-heart axis, demonstrating that sleep deprivation plays a critical role in the occurrence of acute cardiac events. Therefore, healthy sleep habits should be incorporated into clinical management strategies for preventing cardiovascular risk events.\u003c/p\u003e"},{"header":"Experimental model and study participant details","content":"\u003cp\u003e\u003cstrong\u003eAnimals.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll procedures in this study strictly complied with the ethical review protocols approved by the Institutional Animal Care and Use Committee (IACUC) of Anhui University of Chinese Medicine (AHTCM) and adhered to the guidelines outlined in the \"Guidelines for the Care and Use of Laboratory Animals.\" Male C57BL/6J mice (6-7 weeks old) and VGAT-Cre transgenic mice, sourced from Charles River Laboratories and The Jackson Laboratory. Mice were housed under controlled environmental conditions (temperature: 23±1°C; relative humidity: 50±5%) in social groups (5 mice/cage), except for individuals requiring neural intervention surgery (cannula/electrode array implantation), which were singly housed postoperatively. The animal facility maintained a standard 12-hour light/dark cycle (illumination from 07:00 to 19:00), with unrestricted access to autoclaved water and irradiated feed.\u003c/p\u003e\n\u003cp\u003eData inclusion criteria enforced rigorous quality control measures: For stereotaxic AAV viral vector injections, samples with injection sites deviating \u0026gt;200μm from target regions were excluded; datasets from optical fiber/catheter implantations exhibiting ≥150μm deviation from predefined coordinates were omitted from statistical analysis; in vivo electrophysiological recordings with electrode contact impedance \u0026gt;500 kΩ or signal-to-noise ratio \u0026lt;3 dB were deemed invalid. All excluded data were meticulously documented with animal identification numbers, exclusion rationales, and excluded from subsequent analyses.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMouse models\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSleep deprivation (SD) was achieved using “curling prevention by water” (CPW). In this study, a custom-made sealed acrylic cylinder was used, measuring 20 cm in height and 25 cm in diameter, equipped with a food trough and water bottle to provide mice with food and drinking water. Prior to the experiment, mice were individually placed in a cylinder filled with bedding material and allowed to acclimate for more than 48 hours. During the experiment, mice were placed in the cylinder for 48 hours, which contained a shallow layer of water (depth 8 mm), just reaching the ankles of the mice. When mice showed signs of sleep characterized by body curling, their noses would come into contact with the water, causing them to be quickly awakened. To maintain a 48-hour sleep deprivation state, the ambient temperature was kept at 25±2℃, and the water in the cylinders was changed every 8 hours to ensure a clean environment for the mice. The cylinder and food trough were cleaned and disinfected with disinfectant solution and ultraviolet light before reuse. After 48 hours, electrocardiogram (ECG) tracings were recorded. For the environmental control group, mice were placed on a 10 cm petri dish filled with distilled water. The petri dish was wrapped with wet gauze to maintain a moist environment, thereby studying the effects of the same humid environment on mouse sleep. Cage control mice were individually placed in a cylinder filled with bedding material, thus studying the effects of the same cage setup on mouse sleep.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVirus injection and optical fiber implantation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter weighing, the mice were anesthetized by inhaling 1% isoflurane gas and maintained in this state using stereotaxic techniques. The mice were fixed on a stereotaxic apparatus（RWD Life Science Inc）\u0026nbsp;and their body temperature was maintained throughout the experiment. A skin incision was made from the eye to the occipital region, exposing the skull, and the connective tissue on the skull surface was wiped away with saline to keep the skull clean. The skull was adjusted to maintain the same level of the fontanelles and the lateral margins of the skull, with an error of less than 0.03 mm. The target brain region FN was then located using the adjusted skull plane: AP: - 6.47 mm, ML: ± 0.82 mm, and DV: - 2.87 mm (units: mm). 120 nl of the virus mixture was injected bilaterally into the FN at a rate of 40 nl/min, and the injection was allowed to remain for 10 minutes before being slowly removed. A ceramic plug (core diameter 200 μm, NA = 0.73，numerical aperture 0.22, Inper, China) was implanted above the injection site at a depth of 200 µm, and was fixed with dental cement.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFiber photometry recordings\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBefore recording calcium signals, the previously implanted ceramic ferrule is connected to the recording device via an external fiber optic (ThinkerTech Co., Ltd, Nanjing, China). The fiber optic recording system couples the laser into the optical path for GCaMP6m signal recording (405nm and 470nm light sources are activated for fiber optic recording), using a CMOS array imaging method to collect the fluorescence intensity of the fiber in real-time, thus achieving synchronous recording of two channels. The 470nm excitation light corresponds to a blue calcium-sensitive fluorescent probe, which can synchronously record the activity of neurons in relevant brain areas during a certain behavioral paradigm. The reference channel uses 405nm excitation light, and the signal from this channel serves as control data to eliminate motion noise and verify the validity of the calcium signal channel data. During analysis, data exceeding the median of the absolute deviation from baseline (MAD) and remaining above twice the baseline MAD are considered calcium signaling events. Heatmaps and average calcium trace curves are generated using an in-house developed MATLAB program.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn vivo optogenetic manipulations.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe mice were anesthetized and immobilized on a stereotaxic apparatus for implantation of fiber cannula devices, targeting the fastigial nucleus (FN) as the key brain region. Dental acrylic resin was employed to securely anchor the fiber cannula onto the dorsal surface of the mouse skull. Chronically implantable optical fibers (200 μm diameter, Newdoon) were connected to a laser generator via fiber optic sleeves to establish stable light delivery pathways.\u003c/p\u003e\n\u003cp\u003eFor the experimental group, light stimulation protocols were rigorously defined as follows: blue light pulses (wavelength 473 nm, power 2–5 mW, frequency 20 Hz) or continuous yellow light exposure (wavelength 594 nm, power 5–8 mW), with each intervention strictly limited to 5 minutes in duration. The control group received identical optical parameters through a sham stimulation procedure. Post-experiment histological examinations were performed on all subjects, and data from animals exhibiting fiber tip deviation exceeding 200 μm from the targeted brain region were systematically excluded. The stimulation protocol for control mice remained consistent with the aforementioned parameters. Final validation of fiber placement within the intended brain area was conducted at experiment termination, with non-conforming datasets subjected to exclusion.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn vivo electrophysiological recordings\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter anesthesia, routine craniotomy surgery was performed on mice, and the Plexon system was used for neuronal signal acquisition and analysis. Multi-array microwire electrodes (Electrode length:10mm;Electrode site diameter:20um；Probe spacing:200um) were placed in the FN to record neuronal spiking and LFP field potentials. The Omni Plex software displayed electrical activity in real time and stored raw data. Offline Sorter and Neuro Explorer software were used for signal analysis, including interspike intervals, autocorrelation, cross-correlation, and spectral analysis. SpikeSorter software (Plexon Inc.) was used for spike sorting to define single units. Spike detection was based on waveforms exceeding three standard deviations of noise amplitude, with manual checking required to confirm consistency. ISI histograms showed refractory periods (\u0026gt;2 ms) for all identifiable units, with only sufficiently isolated units included in data analysis (L-ratio \u0026lt; 0.2, isolation distance \u0026gt; 15). Based on k-means clustering methods, neurons were classified into wide-spike (WS) and narrow-spike (NS) neurons. A three-dimensional space was defined by half-peak width, half-valley width, and average firing rate. Full width at half maximum (FWHM) and firing rate were used to define inhibitory interneurons (INS). Based on baseline firing frequency, the NS group was further subdivided into fast-spiking calcium-binding protein (FS-PV Ins, \u0026gt;10 Hz) and non-fast-spiking NS neurons.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eElectrocardiography\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter weighing, the mice were anesthetized with 1% isoflurane and fixed on an acrylic board. Electrodes were inserted into the right forelimb (negative) and hind legs (left: positive; right: ground), and electrocardiogram (ECG) signals were recorded in real-time from the mouse's limb II lead. The experiment utilized an ML118 Animal Bio Amp (AD Instruments, Sydney, Australia) and a digitizer (PowerLab 8, ADInstruments, Sydney, Australia) to record the ECG data. Digital ECG analysis was performed using LabChart V8.1.19 software (ADInstruments, Sydney, Australia), which provided automatic data collection for heart rate variability (HRV). Each mouse had at least 15 minutes of data collected.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMouse Cardiac Ultrasound Examination\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the cardiac ultrasound function assessment experiment for mice, a standardized operating protocol was employed: Mice were initially anesthetized in an induction chamber prefilled with 2% isoflurane (RWD). After stabilization of anesthesia, the mice were secured in a supine position on a temperature-controlled ultrasound platform (integrated with the VINNO6 LAB system). Anesthesia depth was maintained via continuous delivery of 1.5% isoflurane through a nose cone. Prior to the experiment, hair in the parasternal region was removed using a chemical depilatory to minimize mechanical stimulation. Subsequently, core body temperature was monitored via a lubricated rectal temperature probe, and a heating pad was adjusted to maintain stable temperatures within 37.0 ± 0.5°C. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eA 30 MHz high-frequency linear probe (axial resolution: 15 μm) was utilized to acquire M-mode ultrasound images at the left ventricular long-axis plane, aligned with the mitral chordae tendineae level. Real-time adjustments of anesthetic dosage were performed to regulate heart rate to 450 ± 50 bpm, thereby avoiding interference from tachycardia-induced ventricular contraction artifacts. Prior to image acquisition, a uniform layer of ultrasound coupling agent was applied to eliminate air-induced artifacts. Continuous recordings of 6–8 cardiac cycles were captured, and VINNO X5 Analysis Software was used to measure left ventricular end-systolic diameter (LVESD), left ventricular end-diastolic diameter (LVEDD), left ventricular ejection fraction (LVEF), and left ventricular fractional shortening (FS). All parameters were averaged over 5 consecutive cardiac cycles to mitigate respiratory motion-related artifacts. The entire experimental process adhered to a double-blind methodology to ensure data objectivity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunohistochemistry, microscopic imaging, and analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMice were deeply anesthetized with isoflurane and then perfused transcardially with 0.9% saline and 4% paraformaldehyde (PFA). The brain was cut into 55-micrometer coronal sections using a cryostat (NJYY5500, RWD). For immunofluorescence staining, the FN brain slices were washed three times with PBS for 5 minutes each time on an orbital shaker. At room temperature, the slices were permeabilized in 0.5% TritonX for 30 minutes to allow antibody entry into cells, followed by incubation in 3% bovine serum albumin (BSA) + 0.3% triton X for 1 hour to block various antigens on the surface of the brain slice, thereby avoiding non-specific binding of charged groups and helping produce high background signals and primary antibody binding. The primary antibody (rabbit Anti-Iba-1, Wako, 1:500 dilution, working solution: 3% BSA + 0.5% Triton X) was incubated at 4°C for 18 hours, with the brain slices being rotated every 24 hours. Then, the FN brain slices were washed three times with PBS for 5 minutes each time on an orbital shaker, followed by the addition of diluted fluorescent secondary antibody (Alexa Fluor\u0026amp;#8482; 488, donkey anti-rabbit, abcam, 1:500 dilution, working solution: 3% BSA + 0.3% triton X). After placing the slices at room temperature for 1.5 hours away from light, the FN brain slices were washed three times with PBS for 5 minutes each time on an orbital shaker. The brain slices were fixed onto slides and added DAPI (1:5000), washed three times with PBS for 5 minutes each time. After drying the liquid on the slides with absorbent paper, 70% glycerin mounting medium was applied, and observed under a fluorescence microscope and confocal microscope (FV3000, OLYMPUS, Japan).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThree-dimensional reconstruction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImaging experiments were conducted on an Olympus FV3000 microscope using a UPLSAPO 100x oil immersion objective lens with a numerical aperture of 1.4. All imaging experiments were performed with the same parameter settings, including gain, offset, and filter mode. In the sleep deprivation experiment, the FN injection site was selected for imaging microglia.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eZ-stack images were collected at 0.5 micron intervals and reconstructed into 640×640 pixel images using IMARIS 10.0.1 software (BitPlane). Three-dimensional surface reconstruction and analysis of microglia were performed using IMARIS software. The \"Filaments\" function was used to measure the number of branch points and the length of processes in microglia. Additionally, the \"Surface\" function was utilized to achieve precise reconstruction of Iba1+ microglia and mGFP+ dendrites. Finally, the contact area between neuronal dendrites and microglial processes was measured using the Surface-Surface Contact Area plugin developed for IMARIS based on MATLAB.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003equantification and statistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll statistical analyses were conducted using GraphPad Software Inc version 8.0 and IBM SPSS Statistics version 23. For comparing data among different groups, we employed either a one-way ANOVA or an unpaired t-test as appropriate. The thresholds for statistical significance were set at P values of less than 0.05, 0.01, and 0.001. Furthermore, the results are presented as means plus or minus standard error of the mean (SEM). For detailed information on all statistical procedures used in this study, please refer to the footnotes below the figures.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (82074536) awarded to RLC, Distinguished Young Youth Scientific Research Project in Universities of Anhui Province (2022AH020043) awarded to RLC, Research Funds of Center for Xin'an Medicine and Modernization of Traditional Chinese Medicine of IHM (2023CXMMTCM019) awarded to RLC, National Natural Science Foundation of China (82104999) awarded to QY, Excellent Young Youth Scientific Research Project in Universities of Anhui Province (2022AH030062) awarded to QY, Anhui Province Key Laboratory of Meridian Viscera Correlationship Open Project (AHMVC2024001) awarded to QY, Anhui Province Graduate Education Quality Engineering Project (2023xscx092) awarded to FZ, National Natural Science Foundation of China (81973757) awarded to LH.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: Fan ZHANG, Guo-ming SHEN, Rong-lin CAI, Qing YU. Data curation: Fan ZHANG, Wen-jing SHAO, Nai-xuan WEI, Zheng-jie LUO, Yan-WU. Formal analysis: Fan ZHANG, Wen-jing SHAO, Yan-WU, Nai-xuan WEI, Wei-LIU. Funding acquisition: Ling HU, Qing YU, Rong-lin CAI, Fan ZHANG. Investigation: Fan ZHANG, Rong-lin CAI, Wen-jing SHAO. \u0026nbsp;Methodology: Fan ZHANG, Wen-jing SHAO, Qing YU, Jian-qing YU. Project administration: Zhi ZHANG, Rong-lin CAI, Qing YU. Resources: Rong-lin CAI, Qing YU, Ling HU. Software: Fan ZHANG, Nai-xuan WEI, Yan WU, Wen-jing SHAO, Zheng-jie LUO. Supervision: Fan ZHANG, Guo-ming SHEN, Ling HU, Qing YU, Rong-lin CAI. Validation: Qing YU, Rong-lin CAI. Visualization: Fan ZHANG, Rong-lin CAI. Writing–original draft: Fan ZHANG, Wen-jing SHAO, Nai-xuan WEI, Wei-LIU. Writing – review \u0026amp; editing: Fan ZHANG, Nai-xuan WEI, Rong-lin CAI. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe obtained six-week-old male C57BL/6J mice from Hangzhou Ziyuan Experimental Animal Technology Co., Ltd., for inclusion in this study. The production license for experimental animals is SCXK (ZHE) 2019-0004. The mice were housed in cages with ad libitum access to food and water, and the ambient temperature was maintained at a relative range of 23–25 °C. All animal experiments were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals and received approval from the Animal Nursing and Utilization Committee of Anhui University of Chinese Medicine (AHUCM-mouse-2022083).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLead contact\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFurther information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Rong-lin CAI ([email protected]).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHobson, J.A. (1969). Sleep: physiologic aspects. The New England journal of medicine \u003cem\u003e281\u003c/em\u003e, 1343-1345. 10.1056/nejm196912112812406.\u003c/li\u003e\n\u003cli\u003eGrandner, M.A., and Fernandez, F.X. (2021). The translational neuroscience of sleep: A contextual framework. 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Neuroscience bulletin \u003cem\u003e36\u003c/em\u003e, 919-931. 10.1007/s12264-020-00511-9.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"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":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"sleep deprivation, microglia, Cerebellum, fastigial nucleus, cardiac dysfunction","lastPublishedDoi":"10.21203/rs.3.rs-7329226/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7329226/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMost animals require sleep, and sleep deprivation(SD) can lead to severe pathophysiological consequences, including cardiac dysfunction and death. Substantial evidence shows that sleep deprivation impairs cardiac function and increases cardiovascular risk and whether sleep deprivation-mediated neural output exacerbates the deterioration of heart function. Here, we demonstrate how sleep deprivation induces symptoms of cardiac dysfunction. Sleep disruption following SD leads to impaired cardiac function. Prolonged sleep deprivation triggers rapid microglial recruitment to the fastigial nucleus (FN) of the cerebellum and enhances the phagocytic capacity of microglia toward dendritic spines of GABAergic neurons. Prolonged sleep deprivation enhances microglial activity in the FN of male mice. Concurrently, microglial engulfment of GABAergic neuronal dendritic spines suppresses GABA neuron activity, leading to increased cardiac sympathetic outflow through the FN\u003csup\u003eGABA\u003c/sup\u003e-RVLM circuit to the cardiac and ultimately inducing cardiac dysfunction.These findings reveal that prolonged sleep deprivation triggers dysregulated microglial phagocytosis, which functionally encodes impaired GABAergic neuron-mediated suppression of cardiac sympathetic outflow, thereby driving the progression of cardiac dysfunction following prolonged sleep loss.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e","manuscriptTitle":"Prolonged sleep deprivation induces cardiac dysfunction via microglia-neural circuit coupling","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-04 17:49:13","doi":"10.21203/rs.3.rs-7329226/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"communications-biology","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"commsbio","sideBox":"Learn more about [Communications Biology](http://www.nature.com/commsbio/)","snPcode":"","submissionUrl":"","title":"Communications Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Communications Series","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"62d56c30-bd60-48a2-a82a-2b74f8f670d3","owner":[],"postedDate":"September 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":53874073,"name":"Biological sciences/Neuroscience"},{"id":53874074,"name":"Health sciences/Cardiology"}],"tags":[],"updatedAt":"2026-04-19T10:35:22+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-04 17:49:13","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7329226","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7329226","identity":"rs-7329226","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}