Time-Dynamic Analysis of Sex-Specific NREM Sleep Disturbance Induced by Social Isolation Among Adolescent Mice

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However, since age-related changes in sleep and the consequences of sleep disturbances can occur as early as adolescence, it remains poorly understood whether these disturbances exhibit a similar sex-specific pattern during adolescence and what the underlying molecular mechanisms may be. Male and female mice were subjected to social isolation stress starting at postnatal day 21 (P21), and electroencephalogram (EEG) was monitored during isolation period. We then employed whole-brain transcriptomic analysis and Mfuzz enrichment analyses to identify temporal and sex-specific molecular responses, dynamic gene expression patterns, and key pathways during isolation period. Male mice exhibited decreased non-rapid eye movement (NREM) sleep duration after 2, 3, and 4 weeks of isolation, while female mice did not show these disturbances after 2 and 3 weeks, but did after 4 weeks of isolation. This suggested a sex-specific pattern of sleep disturbances during adolescence, distinct from those observed in adulthood. Moreover, the decreased NREM sleep in isolated male mice was related to sensory, metabolic, and immune systems after 2-, 3-, and 4-week of isolation, respectively. While the reduction in NREM duration in female mice after 4 weeks of isolation was associated with their energy metabolism and amino acid metabolism disruptions. We found a sex-specific pattern of sleep disturbances during adolescence, with male mice being more susceptible to social isolation stress, which may be linked to early sensory system responses in isolated male mice and later-stage amino acid metabolism and energy imbalance in isolated female mice. Our findings provide insights into gender-specific interventions for sleep disorders during adolescence and underscore the importance of considering both temporal and sex differences in stress-related sleep research. Health sciences/Diseases/Psychiatric disorders Biological sciences/Neuroscience/Molecular neuroscience Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Highlights Male and female mice show different patterns of NREM sleep disturbances under SI. Sleep disturbances occur earlier in males (after 2 weeks of SI) compared to females (after 4 weeks of SI). Transcriptomic analysis reveals sex-specific gene pathways: sensory, metabolic, and immune responses in males; metabolic disruptions in females. Gene expression patterns change over time, reflecting dynamic sex-dependent responses to SI stress. 1. Introduction Sleep architecture, characterized by the cyclical alternation between non-rapid eyes movement (NREM) and rapid eyes movement (REM) sleep, serves as a critical biomarker of physiological health and neurodevelopment[1, 2]. Sleep architecture exhibits age-specific patterns across the lifespan, with distinct changes occurring during key developmental stages[3]. Sleep disorders are highly prevalent among adults, with research consistently showing that women are more likely to experience sleep disturbances than men during adulthood. It suggested a gender-based disparity in the prevalence of sleep disorders[4]. However, while age-related changes in sleep and the consequences of sleep disturbances can begin as early as adolescence, it remains unclear whether similar sex-specific patterns of sleep disturbances emerge during adolescence, and the underlying molecular mechanisms are still poorly understood. Adolescence is a sensitive period for stress-induced mental disorders, as well as a critical period for the shape of stable sleep patterns and structure. Sleep architecture during adolescence not only reflect the brain's sensitivity to stress[5, 6], but also predict the onset and progression of mental disorders[7, 8] and physical diseases[9, 10]. Lewin et al. found that early life trauma rats showed a reduction in spindle activity during early life, and fragmented NREM sleep in later years[11]. However, most of these studies did not report gender differences, which limit our understanding of the potential mechanisms of the relationship between stress-induced sleep patterns and gender differences among adolescent mammal. Meanwhile, previous studies suggested that the duration of stress during adolescence was closely linked to the severity and type of mental disorders, with longer exposure to stress leading to more complex or severe symptoms[12]. This further emphasizes the need for long-term longitudinal tracking of sleep structure changes and gender differences through adolescence in animal models. In our study, we employed an social isolation (SI) model among adolescent mice to evaluate a time dynamic analysis of alterations in their sleep patterns, and incorporated omics data to investigate the temporal impact of sex differences on sleep disorders during SI. Additionally, we employed whole-brain transcriptomic analysis and Mfuzz enrichment analyses to identify sex-specific molecular mechanisms. 2. Materials and methods 2.1. Mice Three-week-old male and female C57BL/6J mice (purchased from the Guangdong Medical Laboratory Animal Center) were used for the study. The mice were housed in standard laboratory cages under a 12-hour light/dark cycle (lights on at 8:00 A.M.), in a temperature controlled room (21–25℃). Food and water were provided ad libitum. All experiments were conducted in accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals (China), and were approved by the Southern Medical University Animal Ethics Committee. All experimental protocols were also conducted following institutional guidelines. 2.2. Animal Model The experimental paradigms is illustrated in Fig. 1 . The study included an experimental group of C57BL/6J mice, which were housed individually in single cages starting at postnatal day 21 for 2, 3, and 4 weeks to establish a social isolation model (SI), and a control group of mice housed in group cages (GH), with 4 mice per cage. Each week, both groups underwent 24-hour EEG-EMG monitoring to assess sleep-wake patterns. After monitoring, whole-brain tissue samples were collected for RNA extraction, and transcriptome sequencing was performed. The data were then analyzed using the Mfuzz package to identify gene expression patterns over time and to investigate how SI affects gene regulation in the brain. 2.3. EEG - EMG electrodes implantation The EEG - EMG electrodes consisted of four stainless steel screws and two Teflon-coated silver leads. Surgery was performed to implant the four electrodes. Mice were deeply anesthetized with an intraperitoneal injection of pentobarbital sodium (75 mg/kg). The mouse head was shaved, and the skull surface was exposed through a midline scalp incision. The periosteum tissue over the skull was removed. Four small holes were (1 mm in diameter) was drilled above the frontal and parietal regions. The EEG electrodes were implanted into these holes and fixed with dental cement. Two electrodes for EMG recording were placed on the nuchal muscle and stabilized. After the dental cement had fully set was completely dry, the animals were returned to their cages to recover for one week before recording. 2.4. EEG - EMG analysis The EEG and EMG signals were amplified, filtered with a high-pass filter above 0.5 Hz, and digitalized at a 1000 Hz resolution using a tethered data acquisition system (Medusa, Bio-Signal Technologies, China). The sleep stages in the recordings were scored by AI-driven software, Lunion Stage, developed by LunionData (China). The EEG - EMG data were analyzed in 4-second epochs and categorized into three stages: non-rapid eye movement (NREM) sleep, rapid eye movement (REM) sleep and wakefulness (Wake). The scored results were reviewed, and manual adjustments were made as necessary. Based on the scored sleep stages, statistical analysis was performed on the different vigilance states in the various experimental groups. 2.5. Transcriptome Sequencing After the 24-hour EEG - EMG monitoring, whole-brain tissue was collected from both groups. The collected tissues were immediately frozen in liquid nitrogen and stored at − 80℃ until RNA extraction. Total RNA was isolated, and high-throughput RNA sequencing was performed to assess gene expression differences between the experimental and control groups at each time point (2, 3, and 4 weeks). 2.6. RNA sequencing analysis Differential expression analysis was performed using the R-DESeq2 package (version1.18.1) and the R- EdgeR package (version1.20.0). The resulting P-values were adjusted using the Benjamini and Hochberg’s method to control the false discovery rate. Differentially expressed genes (DEGs) were defined as those with an adjusted P-value 1. GOSeq package (version1.34.1) was used to identify Gene Ontology (GO) terms that annotate the list of enriched genes (adjusted P-value < 0.05). Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was used to analyze the enriched pathways of the candidate genes ( http://en.wikipedia.org/wiki/KEGG ) (adjusted P-value < 0.05). The top 30 GO terms for biological process (BP), cellular component (CC), and molecular functions (MF), as well as the top 20 KEGG signaling pathways, were selected for further investigation. 2.7. Mfuzz Analysis Differential gene expression between female and male mice under SI for different weeks was analyzed using the Mfuzz package in R. The fuzzy c-means algorithm was applied to cluster genes with similar expression patterns over time. This analysis grouped the differential genes into distinct clusters based on their expression profiles during the isolation periods. This clustering approach enabled the identification of gene groups that exhibited similar regulatory patterns, offering insights into how SI influenced gene expression in both female and male mice at various time points[13]. 2.8. Statistical Analysis In the experimental data, Student’s t-test was used to compare the means of two independent samples, and one-way ANOVA was used to compare the means of multiple sample groups using SPSS 22 software (SPSS, Chicago, IL). The mean values shown in the text and figures are expressed as the mean ± standard error of the mean (SEM). P < 0.05 was considered statistically significant, and GraphPad Prism 10 (La Jolla,CA) was used to draw the graphs. 3. Results 3.1. Sex-specific NREM sleep alterations during SI across adolescence To investigate the effects of SI on the sleep patterns of male and female mice, we housed the mice either in groups or singly starting from P21. Subsequently, we conducted electroencephalogram (EEG) monitoring at 2, 3, and 4 weeks of isolation (Fig. 1 A). EEG monitoring showed that male mice subjected to SI exhibited a significant reduction in NREM sleep duration compared to male mice after 2, 3, 4 weeks of isolation (Fig. 1 B-D). In contrast, female mice did not exhibited a reduction in NREM sleep duration 2-, 3- but 4-week of isolation (Fig. 1 E-G). These results suggest a sex-specific of sleep disturbances during adolescence, with male mice more susceptible than female mice, which is different from that of in adult mice. 3.2. Sex-specific temporal transcriptomic responses to SI in male and female mice Next, to investigate the temporal and sex-specific molecular responses to prolonged SI, we conducted a whole-brain transcriptomic analysis on both male and female mice at various time points following SI. Firstly, we performed whole-brain transcriptomic sequencing on male mice at 2, 3, and 4 weeks of isolation. As shown in the volcano plot, a total of 177 DEGs (96 upregulated and 81 downregulated) were detected in male mice subjected to 2 weeks of isolation compared to the GH controls (Fig. 2 A); Similarly, 154 DEGs (52 upregulated and 102 downregulated) and 339 DEGs (51 upregulated and 288 downregulated) were detected at 3 and 4 weeks of isolation, respectively (Fig. 2 B, C). To explore the biological processes and pathways affected by SI, we performed GO and KEGG pathway analyses on DEGs from male mice at 2, 3, and 4 weeks of isolation. At 2 weeks of isolation, GO analysis revealed significant enrichment in BP related to the “RNA catabolic process” (Supplementary Fig. S1 A). The most affected KEGG pathway was “antigen processing and presentation” (Fig. 2 D). After 3 weeks, GO terms associated with “protein transport” and “cell cycle” were enriched (Supplementary Fig. S1 B), while KEGG analysis identified pathways such as “MAPK signaling pathway” and “pathways of neurodegeneration-multiple diseases” (Fig. 2 E). At 4 weeks, the most enriched BP terms were related to “protein transport” and “cell cycle” (Supplementary Fig. S1 C), and KEGG analysis revealed significant enrichment in pathways such as the “MAPK signaling pathway”, “cancer signaling pathways” and “neurodegeneration signaling pathways” (Fig. 2 F). Secondly, we identified 211 significant DEGs in female mice subjected to 2 weeks of isolation (Fig. 3 A), including 108 upregulated and 103 downregulated genes. Similarly, 351 DEGs (80 upregulated and 271 downregulated) and 237 DEGs (120 upregulated and 117 downregulated) were detected at 3 and 4 weeks of isolation, respectively (Fig. 3 B, C). Supplementary Fig. 1D-F provide information on the results of GO annotation analysis in female mice. At 2 weeks of isolation, GO analysis revealed significant enrichment in BP terns related to “positive regulation of toll − like receptor 3 signaling pathway” and “negative regulation of plasminogen activation” (Supplementary Fig. S1 D). By 3 weeks, the most enriched BP terms were related to the “meiotic cell cycle” and “positive regulation of proteolysis” (Supplementary Fig. S1 E). At 4 weeks, the most enriched BP terms was “mammary gland alveolus development” (Supplementary Fig. S1 F). Based on KEGG pathway analysis, the most significantly enriched pathways in female mice at 2 weeks of isolation were “IL − 17 signaling pathway” and “viral protein interaction with cytokine and cytokine receptor” (Fig. 3 D). At 3 weeks, the enriched pathways included “protein digestion and absorption” and “cysteine and methionine metabolism” (Fig. 3 E), while at 4 weeks, the most significantly enriched pathway was the “AMPK signaling pathway” (Fig. 3 F). 3.3. Temporal dynamic of gene expression clusters in male and female mice under SI stress To further explore the dynamic patterns of gene expression changes over time, we used the Mfuzz package to perform fuzzy c-means clustering on the time-course transcriptomic data. This analysis revealed distinct temporal expression profiles in both male and female mice subjected to SI, highlighting clusters of genes that were specifically upregulated or downregulated across 2-, 3-, and 4- week isolation periods. We identified 10 distinct clusters in both male and female mice, each representing a unique set of genes with specific expression patterns at particular time points. In male mice, the gene expression in Cluster 1 showed no significant differences between 2- and 4-week isolation periods, but peaked specifically at 3-week isolation. This pattern was similarly observed in Clusters 4 and 8, suggesting that these clusters represent genes specifically responsive to 3 weeks of isolation. Gene expression in Cluster 2 peaked at 2-week isolation mark and then declined to baseline levels at the 3- and 4- week time points. In contrast, the genes in Cluster 5 showed peak expression at the 4-week isolation period (Fig. 4 ). Figure 4 also presents the Mfuzz results for female mice. Cluster 7 represents genes that were specifically expressed after 2 weeks of isolation. Genes expression in Cluster 2 reached its lowest point at 3 weeks of isolation, while genes in Cluster 10 reached their peak expression at the same time point. These two clusters specifically reflect distinct transcriptional responses in female mice at the 3-week isolation, highlighting their unique roles in the molecular changes associated with this critical period. Genes within Cluster 4 maintained consistent expression levels at both 2- and 3- week isolation time points, but demonstrated significant upregulation specifically at the 4-week time point, indicating that these genes are uniquely associated with transcriptional changes in female mice at 4 weeks of isolation. 3.4. Identification and functional enrichment of key genes associated with NREM sleep disturbance in male mice during SI To further identify key genes and their associated signaling pathways at each time point during SI, we performed an overlap analysis between the DEGs sets and the gene sets corresponding to each time point, as identified by Mfuzz. We visualized the overlapping genes using Venn diagrams. At 2- week SI time point, we identified 13 key genes involved in the emergence of NREM sleep disturbance in male mice, as shown in Fig. 5 A. Additionally, GO and KEGG analyses revealed that these 13 key genes were primarily enriched in BP associated with the “positive regulation of DNA-binding transcription factor activity” (Fig. 5 B). The most significantly enriched pathway was “phototransduction” (Fig. 5 C), which showed a strong preference for organismal systems, particularly the sensory system (Supplementary Fig. S2A). After 3 weeks of isolation, 20 key genes were identified as responsive to NREM sleep disturbance in male mice (Fig. 5 D). These genes were mainly enriched in the following BP terms (Fig. 5 E): “tetrapyrrole biosynthetic process”, “modulation of process of another organismcell body”, and “regulation of glycogen metabolic process”, among others. These processes were linked to metabolic functions such as amino acid metabolism, lipid metabolism, and metabolism of cofactors and vitamins (Fig. 5 F, Supplementary Fig. S2B). Finally, after 4 weeks of isolation, 24 key genes were identified in male mice, and their biological processes were primarily enriched in “defense response to virus”, “positive regulation of natural killer cell differentiation”, “B cell apoptotic process” and “innate immune response” (Fig. 5 G, H). These pathways are primarily associated with immune system functions (Fig. 5 I, Supplementary Fig. S2C). 3.5. Identification and functional enrichment of key genes associated with NREM sleep disturbance in female mice during SI To identify key genes and their associated pathways in female mice at different time points of SI, we applied the same analytical approach used for male mice. Specifically, we performed an overlap analysis between DEGs sets and the time-specific gene clusters derived from Mfuzz, with the results visualized using Venn diagrams. At 2 weeks of isolation, 20 key genes were identified in female mice, despite the absence of NREM sleep disturbance at this point (Fig. 6 A). Interestingly, GO and KEGG analyses revealed that these genes were significantly enriched in the “inositol trisphosphate biosynthetic process” and the “fatty acid biosynthesis” pathways, suggesting a potential role in lipid metabolism. The remaining KEGG pathways were primarily associated with the endocrine and immune systems (Fig. 6 B, C; Supplementary Fig. S2D). After 3 weeks of isolation, 39 key genes were identified in female mice, primarily enriched in the “retinol metabolism” and “vitamin digestion and absorption” pathways (Fig. 6 D-F). These pathways were associated with metabolism functions and the digestive system within organismal system, respectively (Supplementary Fig. S2E). By 4 weeks of isolation, 9 key genes were identified as linked to NREM sleep disturbance in female mice (Fig. 6 G). GO analysis revealed significant enrichment in biological process related to “noradrenergic neuron development” (Fig. 6 H). Meanwhile, the results of KEGG indicated that the primary enriched pathways were "nitrogen metabolism" and "arginine biosynthesis", which were primarily involved in energy metabolism and amino acid metabolism functions (Fig. 6 I). These findings suggested a strong involvement of metabolism responses in female mice after prolonged SI (Supplementary Fig. S2F). Overall, these results indicated that metabolic processes play a significant role in mediating sleep disturbances induced by prolonged SI in female mice. 4. Discussion Previous studies have highlighted sex-specific difference in responses to SI across various physiological systems, including hippocampal plasticity[14], cardiovascular reactivity[15], affective vulnerability[16, 17], accelerated aging[18], dendritic spine remodeling[19], and neural circuit reorganization[20]. Our study further extended SI to the sleep architecture based on this sex-specific animal models. Firstly, we found that adolescent male mice exhibited a reduction in NREM sleep duration after 2 weeks of isolation. Secondly, the NREM sleep disturbance observed in adolescent male mice after 2 weeks of isolation were associated with early activation of sensory systems. For adolescent female mice, the decrease in NREM sleep duration after 4 weeks of isolation was associated with metabolic disruptions. Although both sexes experienced a reduction in NREM sleep, the underlying regulatory mechanisms differed between them. Adolescence represents a critical developmental window with heightened vulnerability to psychiatric disorders[21, 22]. SI during this period exacerbates mental health risks, with the duration of isolation contributing to distinct behavioral phenotypes. Specifically, in adolescent male mice, total 2 weeks of isolation led to anxiety-like behaviors[23], 3 weeks of isolation resulted in depressive-like symptoms[24], and 4 weeks of isolation strengthened the retention of fear memories[25] and autism-like behaviors[26]. All of these researches highlight the importance of the length of isolation in shaping behavioral outcome. Interestingly, our research revealed that regardless of duration of SI, adolescent male mice similarly exhibited a reduction in NREM sleep duration, but not in REM phase. In contrast, adolescent female mice only began to show a reduction in NREM sleep after 4 weeks of isolation. This may be due to the sensitivity of NREM sleep to chronic stress, which ultimately leads to sleep reduction[27, 28]. Moreover, NREM sleep is associated with the regulation of hypothalamic-pituitary-adrenal (HPA) axis[29], neurotransmitters[30, 31], and immune system[32], all of which are influenced by SI[33, 34]. In contrast, the regulation of REM sleep remains relatively independent, potentially serving as a protective factor under stress conditions[35]. These findings suggest that NREM sleep may serve as a biomarker of stress responses. Additionally, the delayed decrease in NREM sleep after 4 weeks of isolation in adolescent female mice, compared to male mice, underscores the different sensitivities to SI. Sex also plays a crucial role in the relationship between NREM sleep and SI,with previous studies emphasizing gender differences in stress responses[36]. Estrogen in female mice may provide a potential protective role in stress responses[37, 38]. What causes adolescent male mice to develop NREM sleep disturbance earlier than female mice, and to maintain consistent NREM sleep disturbance under dynamic changes during different periods of SI? To address this, we identified key genes with regulatory roles that vary over time during SI using Mfuzz time-series analysis. Pathway enrichment analysis showed activation of sensory systems(phototransduction pathways) at week 2 of SI, metabolic signaling pathways at week 3 of SI, and immune-related pathways and metabolic regulation at week 4 of SI.The sensory systems are responsible for receiving physical stimuli. Previous studies have shown that the recovery of NREM sleep in male mice is worse than in female mice after physical stimulation[39, 40]. Additionally, the behavioral manifestations in male mice, including locomotor activity, anxiety levels, and pentobarbital-induced sleep, were more sensitive to the effects of SI[41]. Another study indicated that in the fear conditioning test, after 3 weeks of isolation, male mice became more susceptible to a prolonged freezing behavior in response to foot-shock stimulation, whereas female mice did not[42]. This suggests that male mice are more vulnerable to external physical stimuli and develop NREM sleep disturbance earlier than female mice. A human transcriptomics-based study demonstrated that long-term social loneliness induces changes closely associated with inflammatory factors[43], which aligns with our findings. However, prior study did not address sex-specific differences in these responses, and research on the dynamic temporal changes underlying these sex differences remains limited. Overall, sensory systems may serve as a potential target for interventions aimed at sleep disturbances in male mice, advancing the understanding of sex-specific sleep regulation and its molecular underpinnings. To investigate the factors contributing to the delayed onset of sleep disturbances in female mice after SI, we focused on the 4-week timepoint of SI, which is primarily associated with metabolism and energy balance in female mice. Our research suggests that adolescent female mice may resist the detrimental effects of SI from weeks 2 to 3 through sustained activation of immune and lipid metabolism pathways. This finding is consistent with previous studies showing that female animals exhibit greater resilience to chronic stress, which is associated with immune regulation[44]. Clinical research indicates that women have a metabolic advantage in controlling blood lipids, which may protect them from the detrimental effects of obstructive sleep apnea[45]. Furthermore, the lower weight gain observed in female mice under chronic stress[46–48], it supported the finding that metabolic regulation plays a critical role in their stress adaptation. These initial protective mechanisms may become overwhelmed, leading to the eventual disruption of NREM sleep when female mice experience long-term stress. Lipid metabolism or immune modulation may represent potential preventive strategies for female sleep disorders in the future. In conclusion, our study first revealed significant sex-specific differences in the temporal dynamics of sleep architecture under SI conditions. The molecular mechanisms underlying sex differences in the relationship between SI and sleep disturbances are not yet fully understood. However, the potential roles of hormonal, genetic, and epigenetic factors should be explored in further studies. This research establishes a foundation for developing targeted interventions aimed at alleviating the negative impacts of SI on mental health in adolescents. Additionally, it offers valuable insights for the development of gender-specific mouse models for studying sleep disorders. Declarations Competing interests No competing interests declared. Author contributions BZ and JL designed the study and supervised the project. SL performed the behavioral experiments and completed the analysis. XM, YJ, HG and PZ participated in the experiments. SL and LF wrote the draft of the manuscript. All authors read and approved the final manuscript. In addition, the authors thank HaploX Biotechnology (Jiangxi, China) for their participation in the RNA-sequencing test. Acknowledgements This work was supported by the National Natural Science Foundation of China (Grant No. 82271525, 82471553) and Guangdong-Hong Kong Joint Laboratory for Psychiatric Disorders (2023B1212120004) and the Natural Science Foundation of Guangdong Province (2024A1515030100). References Jackowska M, Steptoe A. Sleep and future cardiovascular risk: prospective analysis from the English Longitudinal Study of Ageing. SLEEP MED 2015; 16 (6): 768–774. Paez A, Gillman SO, Dogaheh SB, Carnes A, Dakterzada F, Barbe F et al. . Sleep spindles and slow oscillations predict cognition and biomarkers of neurodegeneration in mild to moderate Alzheimer's disease. ALZHEIMERS DEMENT 2025; 21 (2): e14424. Ohayon MM, Carskadon MA, Guilleminault C, Vitiello MV. Meta-analysis of quantitative sleep parameters from childhood to old age in healthy individuals: developing normative sleep values across the human lifespan. SLEEP 2004; 27 (7): 1255–1273. 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Guo M, Wu CF, Liu W, Yang JY, Chen D. Sex difference in psychological behavior changes induced by long-term social isolation in mice. PROG NEURO-PSYCHOPH 2004; 28 (1): 115–121. Ciano Albanese N, Poggini S, Reccagni A, Barezzi C, Salciccia C, Poleggi A et al. . Adolescent social isolation induces sex-specific behavioral and neural alterations. PSYCHONEUROENDOCRINO 2025; 172 : 107264. Cole SW, Hawkley LC, Arevalo JM, Sung CY, Rose RM, Cacioppo JT. Social regulation of gene expression in human leukocytes. GENOME BIOL 2007; 8 (9): R189. Chrousos GP. Stress and sex versus immunity and inflammation. SCI SIGNAL 2010; 3 (143): e36. Goulet N, Marcoux C, Bourgon V, Morin R, Mauger J, Amaratunga R et al. . Biological sex-related differences in the postprandial triglyceride response to intermittent hypoxaemia in young adults: a randomized crossover trial. J PHYSIOL-LONDON 2024; 602 (21): 5817–5834. Guo M, Wu CF, Liu W, Yang JY, Chen D. 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Additional Declarations The authors have declared there is NO conflict of interest to disclose Supplementary Files Supplementalfigures.docx Supplemental figures Cite Share Download PDF Status: Published Journal Publication published 13 Feb, 2026 Read the published version in Translational Psychiatry → Version 1 posted Editorial decision: revise 15 Jul, 2025 Review # 3 received at journal 11 Jul, 2025 Review # 1 received at journal 09 Jul, 2025 Review # 2 received at journal 03 Jul, 2025 Reviewer # 3 agreed at journal 22 Jun, 2025 Reviewer # 2 agreed at journal 19 Jun, 2025 Reviewer # 1 agreed at journal 19 Jun, 2025 Reviewers invited by journal 18 Jun, 2025 Editor assigned by journal 06 Jun, 2025 Submission checks completed at journal 06 Jun, 2025 First submitted to journal 05 Jun, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6828480","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":473328341,"identity":"c45de05a-23a9-4660-8667-24cda27287ec","order_by":0,"name":"Bin Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+UlEQVRIiWNgGAWjYDACCSBm/FeTwMbAfAwswMZOjBYGtmNALWxpDAwJQDYzcVqYgWp5zMBaGAhpkZ/d/OzhFx62PD7pnm8PPv7YJs/HzMD44WMObi2Mc46ZG8tIyBSzyZzdbjgj4bZhGzMDs+TMbbi1MEskmElLGLAltknkbpPmSbjNCNTCxsyLRwubRPo3aYkEZqCWnGcgLfYEtfBI5JhJfjgA1sIG0pJIUIuERE6ZNGPDsWI2iTQzyRlpt5PbmBmb8fpFfkb6NsmfDTV58jOSn0l8sLltO7+9+eCHj3i0gIOAB5XP2IBfPUjJD4JKRsEoGAWjYEQDABafRpxlpm1mAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0003-1303-2397","institution":"Nanfang Hospital, Southern Medical University","correspondingAuthor":true,"prefix":"","firstName":"Bin","middleName":"","lastName":"Zhang","suffix":""},{"id":473328342,"identity":"dde97908-19ca-4e5a-b424-4f991faf1330","order_by":1,"name":"Shuangyan Li","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Shuangyan","middleName":"","lastName":"Li","suffix":""},{"id":473328343,"identity":"897b3508-3226-48fc-9714-6974c47b5f6d","order_by":2,"name":"Xuxuan Ma","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Xuxuan","middleName":"","lastName":"Ma","suffix":""},{"id":473328344,"identity":"124de1cb-8b66-4fc2-aa22-e6d9dd7982b6","order_by":3,"name":"Yu Jiang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Jiang","suffix":""},{"id":473328345,"identity":"0edb10a9-6219-4933-85ce-3f35c1c180c4","order_by":4,"name":"Haicheng Guo","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Haicheng","middleName":"","lastName":"Guo","suffix":""},{"id":473328346,"identity":"469302a0-efd1-49e9-8e54-e18796d5f071","order_by":5,"name":"Panyue Zhong","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Panyue","middleName":"","lastName":"Zhong","suffix":""},{"id":473328347,"identity":"fe085558-e5a5-42ac-aa6d-49059053adb0","order_by":6,"name":"Leqin Fang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Leqin","middleName":"","lastName":"Fang","suffix":""},{"id":473328348,"identity":"84dbde1c-34d4-41c2-ae50-432d1c6d2a92","order_by":7,"name":"Jihong Liu","email":"","orcid":"","institution":"Southern Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jihong","middleName":"","lastName":"Liu","suffix":""}],"badges":[],"createdAt":"2025-06-05 11:01:04","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6828480/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6828480/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41398-026-03895-w","type":"published","date":"2026-02-13T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":85198525,"identity":"132e059e-b868-4046-9032-308343fd6c50","added_by":"auto","created_at":"2025-06-23 09:55:40","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":499998,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSI reduces NREM sleep in male mice starting from week 2, while female mice exhibit similar effects at week 4.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A)Timeline of EEG-EMG recording and transcriptome sequencing following SI during adolescence.\u003c/p\u003e\n\u003cp\u003e(B-D) The percentage of total recording time spent in NREM, REM, and WAKE states were quantified. NREM distribution in male mice after 2, 3, and 4 weeks of isolation.(2 weeks: GH. vs. SI, \u003cem\u003eP\u003c/em\u003e= 0.0220; 3 weeks: GH. vs. SI, \u003cem\u003eP\u003c/em\u003e= 0.0048; 4 weeks: GH. vs. SI, \u003cem\u003eP\u003c/em\u003e= 0.0009; n= 8 ).\u003c/p\u003e\n\u003cp\u003e(E-G) NREM disturbance in female mice after 2, 3, and 4 weeks of isolation. (2 weeks: GH. vs. SI, \u003cem\u003eP\u003c/em\u003e= 0.1811; 3 weeks: GH. vs. SI, \u003cem\u003eP\u003c/em\u003e= 0.3759; 4 weeks: GH. vs. SI, \u003cem\u003eP\u003c/em\u003e= 0.0368; n= 7-8). Data are presented as mean ± S.E.M.. Statistical significance was assessed using an unpaired 2-tailed Student’s t test. *\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6828480/v1/ff1627df05fcfdd77d3fead3.jpg"},{"id":85198529,"identity":"f0f6cb65-918b-4452-affb-e224fbed586c","added_by":"auto","created_at":"2025-06-23 09:55:40","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":590781,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eWhole-brain transcriptomic profiling of male mice under SI.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A-C) Volcano plots showing DEGs in male mice after 2, 3, and 4 weeks of isolation compared to GH controls. Red dots represent significantly upregulated genes, and blue dots represent significantly downregulated genes[cutoff: |log2FC| \u0026gt; 1, adjusted \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05. (2 weeks: Down: Sig: 81, Up: Sig: 96; 3 weeks: Down: Sig: 102, Up: Sig: 52; 4 weeks: Down: Sig: 288, Up: Sig: 51)].\u003c/p\u003e\n\u003cp\u003e(D-F) KEGG pathway enrichment analysis of DEGs at 2, 3, and 4 weeks of isolation, showing the top 20 significantly enriched pathways.\u003c/p\u003e\n\u003cp\u003eKEGG: Kyoto Encyclopedia of Genes and Genomes; GenIP.: Genetic Information Processing; HumaD.: Human Diseases; Metab.: Metabolism; OrgaS.: Organismal Systems; EnvIP.: Environmental Information Processing; CellP.: Cellular Processes.\u003c/p\u003e","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6828480/v1/4b02c657fe1ad142d6d6660f.jpg"},{"id":85198790,"identity":"8aee0f40-2903-40d3-8cc6-6f190790689d","added_by":"auto","created_at":"2025-06-23 10:03:40","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":605650,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eWhole-brain transcriptomic profiling of female mice under SI.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A-C) Volcano plots showing DEGs in female mice after 2, 3, and 4 weeks of isolation compared to GH controls. Red dots represent significantly upregulated genes, and blue dots represent significantly downregulated genes [cutoff: |log2FC| \u0026gt; 1, adjusted \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05. (2 weeks: Down: Sig: 103, Up: Sig: 108; 3 weeks: Down: Sig: 271, Up: Sig: 80; 4 weeks: Down: Sig: 117, Up: Sig: 120)].\u003c/p\u003e\n\u003cp\u003e(D-F) KEGG pathway enrichment analysis of DEGs at 2, 3, and 4 weeks of isolation, showing the top 20 significantly enriched pathways.\u003c/p\u003e\n\u003cp\u003eKEGG: Kyoto Encyclopedia of Genes and Genomes; GenIP.: Genetic Information Processing; HumaD.: Human Diseases; Metab.: Metabolism; OrgaS.: Organismal Systems; EnvIP.: Environmental Information Processing; CellP.: Cellular Processes.\u003c/p\u003e","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6828480/v1/4909d1e12031c1872f8096ed.jpg"},{"id":85198526,"identity":"49f84b97-dc69-4787-b8fa-4f00abf90d06","added_by":"auto","created_at":"2025-06-23 09:55:40","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":691192,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTemporal dynamic of gene expression clusters during SI identified by Mfuzz analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFuzzy c-means clustering (Mfuzz) was applied separately to DEGs in male (top panel) and female (bottom panel) mice across baseline (GH2W) and SI weeks 2-4 (SI2W, SI3W, SI4W) to identify sex-specific temporal expression patterns.\u003c/p\u003e\n\u003cp\u003eMale-specific gene expression clusters: A total of 10 clusters were identified, each showing a distinct trajectory of transcriptional changes in response to prolonged SI.\u003c/p\u003e\n\u003cp\u003eFemale-specific gene expression clusters: A total of 10 clusters were identified, revealing dynamic shifts in gene expression over time.\u003c/p\u003e\n\u003cp\u003eEach cluster represents the standardized expression changes (y-axis) across different time points (x-axis), with color gradients indicating expression density. Genes within the same cluster exhibit similar temporal expression patterns, suggesting potential co-regulation.\u003c/p\u003e","description":"","filename":"Fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6828480/v1/edc0e2ad61cf562d9df1fa18.jpg"},{"id":85198530,"identity":"c7177d32-84ac-4c88-962d-eedafa8f2bfa","added_by":"auto","created_at":"2025-06-23 09:55:40","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":844198,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFunctional enrichment analysis of DEGs identified within Mfuzz clusters in male mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A,D,G) Venn diagram showing the intersection between genes identified in Mfuzz clustering analysis and DEGs at week (A) 2, (D) 3, and (G) 4 of SI in male mice. The overlapping regions represent genes shared between clusters and DEGs, which were further analyzed for functional enrichment.\u003c/p\u003e\n\u003cp\u003e(B,E,H) GO enrichment analysis of intersecting genes at week (B) 2, (E) 3, and (H) 4, highlighting the top 30 enriched biological terms across BP, CC and MF.\u003c/p\u003e\n\u003cp\u003e(C,F,I) KEGG pathway enrichment analysis of intersecting genes at week (C) 2, (F) 3, and (I) 4, displaying the top 20 significantly enriched pathways.\u003c/p\u003e","description":"","filename":"Fig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6828480/v1/b6cdda7c2f5612f536f3a8ac.jpg"},{"id":85198532,"identity":"26cb0861-9793-4138-a680-690fa2c5ce6f","added_by":"auto","created_at":"2025-06-23 09:55:40","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":844956,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFunctional enrichment analysis of DEGs identified within Mfuzz clusters in female mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A,D,G) Venn diagram showing the intersection between genes identified in Mfuzz clustering analysis and DEGs at week (A) 2, (D) 3, and (G) 4 of SI in male mice. The overlapping regions represent genes shared between clusters and DEGs, which were further analyzed for functional enrichment.\u003c/p\u003e\n\u003cp\u003e(B,E,H) GO enrichment analysis of intersecting genes at week (B) 2, (E) 3, and (H) 4, highlighting the top 30 enriched biological terms across BP, CC and MF.\u003c/p\u003e\n\u003cp\u003e(C,F,I) KEGG pathway enrichment analysis of intersecting genes at week (C) 2, (F) 3, and (I) 4, displaying the top 20 significantly enriched pathways.\u003c/p\u003e","description":"","filename":"Fig6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6828480/v1/9ce0908dbf7e336aa396106f.jpg"},{"id":105536140,"identity":"74744ee7-f451-46b0-9125-96fa56eae6a8","added_by":"auto","created_at":"2026-03-27 07:12:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5084100,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6828480/v1/26cfd586-d770-4e33-b8cb-09ed3ae43eef.pdf"},{"id":85198792,"identity":"7ae871e3-3dab-47df-bb78-d6e507f7e5d0","added_by":"auto","created_at":"2025-06-23 10:03:40","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":835575,"visible":true,"origin":"","legend":"Supplemental figures","description":"","filename":"Supplementalfigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-6828480/v1/f1d4ef0e06a0000217f4b22b.docx"}],"financialInterests":"The authors have declared there is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose","formattedTitle":"Time-Dynamic Analysis of Sex-Specific NREM Sleep Disturbance Induced by Social Isolation Among Adolescent Mice","fulltext":[{"header":"Highlights","content":"\u003cp\u003eMale and female mice show different patterns of NREM sleep disturbances under SI.\u003c/p\u003e\n\u003cp\u003eSleep disturbances occur earlier in males (after 2 weeks of SI) compared to females (after 4 weeks of SI).\u003c/p\u003e\n\u003cp\u003eTranscriptomic analysis reveals sex-specific gene pathways: sensory, metabolic, and immune responses in males; metabolic disruptions in females.\u003c/p\u003e\n\u003cp\u003eGene expression patterns change over time, reflecting dynamic sex-dependent responses to SI stress.\u003c/p\u003e"},{"header":"1. Introduction","content":"\u003cp\u003eSleep architecture, characterized by the cyclical alternation between non-rapid eyes movement (NREM) and rapid eyes movement (REM) sleep, serves as a critical biomarker of physiological health and neurodevelopment[1, 2]. Sleep architecture exhibits age-specific patterns across the lifespan, with distinct changes occurring during key developmental stages[3]. Sleep disorders are highly prevalent among adults, with research consistently showing that women are more likely to experience sleep disturbances than men during adulthood. It suggested a gender-based disparity in the prevalence of sleep disorders[4]. However, while age-related changes in sleep and the consequences of sleep disturbances can begin as early as adolescence, it remains unclear whether similar sex-specific patterns of sleep disturbances emerge during adolescence, and the underlying molecular mechanisms are still poorly understood.\u003c/p\u003e \u003cp\u003eAdolescence is a sensitive period for stress-induced mental disorders, as well as a critical period for the shape of stable sleep patterns and structure. Sleep architecture during adolescence not only reflect the brain's sensitivity to stress[5, 6], but also predict the onset and progression of mental disorders[7, 8] and physical diseases[9, 10]. Lewin et al. found that early life trauma rats showed a reduction in spindle activity during early life, and fragmented NREM sleep in later years[11]. However, most of these studies did not report gender differences, which limit our understanding of the potential mechanisms of the relationship between stress-induced sleep patterns and gender differences among adolescent mammal. Meanwhile, previous studies suggested that the duration of stress during adolescence was closely linked to the severity and type of mental disorders, with longer exposure to stress leading to more complex or severe symptoms[12]. This further emphasizes the need for long-term longitudinal tracking of sleep structure changes and gender differences through adolescence in animal models.\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eIn our study, we employed an social isolation (SI) model among adolescent mice to evaluate a time dynamic analysis of alterations in their sleep patterns, and incorporated omics data to investigate the temporal impact of sex differences on sleep disorders during SI. Additionally, we employed whole-brain transcriptomic analysis and Mfuzz enrichment analyses to identify sex-specific molecular mechanisms.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Mice\u003c/h2\u003e \u003cp\u003eThree-week-old male and female C57BL/6J mice (purchased from the Guangdong Medical Laboratory Animal Center) were used for the study. The mice were housed in standard laboratory cages under a 12-hour light/dark cycle (lights on at 8:00 A.M.), in a temperature controlled room (21\u0026ndash;25℃). Food and water were provided ad libitum. All experiments were conducted in accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals (China), and were approved by the Southern Medical University Animal Ethics Committee. All experimental protocols were also conducted following institutional guidelines.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Animal Model\u003c/h2\u003e \u003cp\u003eThe experimental paradigms is illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The study included an experimental group of C57BL/6J mice, which were housed individually in single cages starting at postnatal day 21 for 2, 3, and 4 weeks to establish a social isolation model (SI), and a control group of mice housed in group cages (GH), with 4 mice per cage. Each week, both groups underwent 24-hour EEG-EMG monitoring to assess sleep-wake patterns. After monitoring, whole-brain tissue samples were collected for RNA extraction, and transcriptome sequencing was performed. The data were then analyzed using the Mfuzz package to identify gene expression patterns over time and to investigate how SI affects gene regulation in the brain.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. EEG - EMG electrodes implantation\u003c/h2\u003e \u003cp\u003eThe EEG - EMG electrodes consisted of four stainless steel screws and two Teflon-coated silver leads. Surgery was performed to implant the four electrodes. Mice were deeply anesthetized with an intraperitoneal injection of pentobarbital sodium (75 mg/kg). The mouse head was shaved, and the skull surface was exposed through a midline scalp incision. The periosteum tissue over the skull was removed. Four small holes were (1 mm in diameter) was drilled above the frontal and parietal regions. The EEG electrodes were implanted into these holes and fixed with dental cement. Two electrodes for EMG recording were placed on the nuchal muscle and stabilized. After the dental cement had fully set was completely dry, the animals were returned to their cages to recover for one week before recording.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. EEG - EMG analysis\u003c/h2\u003e \u003cp\u003eThe EEG and EMG signals were amplified, filtered with a high-pass filter above 0.5 Hz, and digitalized at a 1000 Hz resolution using a tethered data acquisition system (Medusa, Bio-Signal Technologies, China). The sleep stages in the recordings were scored by AI-driven software, Lunion Stage, developed by LunionData (China). The EEG - EMG data were analyzed in 4-second epochs and categorized into three stages: non-rapid eye movement (NREM) sleep, rapid eye movement (REM) sleep and wakefulness (Wake). The scored results were reviewed, and manual adjustments were made as necessary. Based on the scored sleep stages, statistical analysis was performed on the different vigilance states in the various experimental groups.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Transcriptome Sequencing\u003c/h2\u003e \u003cp\u003eAfter the 24-hour EEG - EMG monitoring, whole-brain tissue was collected from both groups. The collected tissues were immediately frozen in liquid nitrogen and stored at \u0026minus;\u0026thinsp;80℃ until RNA extraction. Total RNA was isolated, and high-throughput RNA sequencing was performed to assess gene expression differences between the experimental and control groups at each time point (2, 3, and 4 weeks).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. RNA sequencing analysis\u003c/h2\u003e \u003cp\u003eDifferential expression analysis was performed using the R-DESeq2 package (version1.18.1) and the R- EdgeR package (version1.20.0). The resulting P-values were adjusted using the Benjamini and Hochberg\u0026rsquo;s method to control the false discovery rate. Differentially expressed genes (DEGs) were defined as those with an adjusted P-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and |log2(FoldChange)| \u0026gt; 1. GOSeq package (version1.34.1) was used to identify Gene Ontology (GO) terms that annotate the list of enriched genes (adjusted P-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was used to analyze the enriched pathways of the candidate genes (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://en.wikipedia.org/wiki/KEGG\u003c/span\u003e\u003cspan address=\"http://en.wikipedia.org/wiki/KEGG\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (adjusted P-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The top 30 GO terms for biological process (BP), cellular component (CC), and molecular functions (MF), as well as the top 20 KEGG signaling pathways, were selected for further investigation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Mfuzz Analysis\u003c/h2\u003e \u003cp\u003eDifferential gene expression between female and male mice under SI for different weeks was analyzed using the Mfuzz package in R. The fuzzy c-means algorithm was applied to cluster genes with similar expression patterns over time. This analysis grouped the differential genes into distinct clusters based on their expression profiles during the isolation periods. This clustering approach enabled the identification of gene groups that exhibited similar regulatory patterns, offering insights into how SI influenced gene expression in both female and male mice at various time points[13].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Statistical Analysis\u003c/h2\u003e \u003cp\u003eIn the experimental data, Student\u0026rsquo;s t-test was used to compare the means of two independent samples, and one-way ANOVA was used to compare the means of multiple sample groups using SPSS 22 software (SPSS, Chicago, IL). The mean values shown in the text and figures are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant, and GraphPad Prism 10 (La Jolla,CA) was used to draw the graphs.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":" \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Sex-specific NREM sleep alterations during SI across adolescence\u003c/h2\u003e \u003cp\u003eTo investigate the effects of SI on the sleep patterns of male and female mice, we housed the mice either in groups or singly starting from P21. Subsequently, we conducted electroencephalogram (EEG) monitoring at 2, 3, and 4 weeks of isolation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). EEG monitoring showed that male mice subjected to SI exhibited a significant reduction in NREM sleep duration compared to male mice after 2, 3, 4 weeks of isolation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB-D). In contrast, female mice did not exhibited a reduction in NREM sleep duration 2-, 3- but 4-week of isolation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE-G). These results suggest a sex-specific of sleep disturbances during adolescence, with male mice more susceptible than female mice, which is different from that of in adult mice.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Sex-specific temporal transcriptomic responses to SI in male and female mice\u003c/h2\u003e \u003cp\u003eNext, to investigate the temporal and sex-specific molecular responses to prolonged SI, we conducted a whole-brain transcriptomic analysis on both male and female mice at various time points following SI. Firstly, we performed whole-brain transcriptomic sequencing on male mice at 2, 3, and 4 weeks of isolation. As shown in the volcano plot, a total of 177 DEGs (96 upregulated and 81 downregulated) were detected in male mice subjected to 2 weeks of isolation compared to the GH controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA); Similarly, 154 DEGs (52 upregulated and 102 downregulated) and 339 DEGs (51 upregulated and 288 downregulated) were detected at 3 and 4 weeks of isolation, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, C).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo explore the biological processes and pathways affected by SI, we performed GO and KEGG pathway analyses on DEGs from male mice at 2, 3, and 4 weeks of isolation. At 2 weeks of isolation, GO analysis revealed significant enrichment in BP related to the \u0026ldquo;RNA catabolic process\u0026rdquo; (Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA). The most affected KEGG pathway was \u0026ldquo;antigen processing and presentation\u0026rdquo; (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). After 3 weeks, GO terms associated with \u0026ldquo;protein transport\u0026rdquo; and \u0026ldquo;cell cycle\u0026rdquo; were enriched (Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB), while KEGG analysis identified pathways such as \u0026ldquo;MAPK signaling pathway\u0026rdquo; and \u0026ldquo;pathways of neurodegeneration-multiple diseases\u0026rdquo; (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). At 4 weeks, the most enriched BP terms were related to \u0026ldquo;protein transport\u0026rdquo; and \u0026ldquo;cell cycle\u0026rdquo; (Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC), and KEGG analysis revealed significant enrichment in pathways such as the \u0026ldquo;MAPK signaling pathway\u0026rdquo;, \u0026ldquo;cancer signaling pathways\u0026rdquo; and \u0026ldquo;neurodegeneration signaling pathways\u0026rdquo; (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003eSecondly, we identified 211 significant DEGs in female mice subjected to 2 weeks of isolation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), including 108 upregulated and 103 downregulated genes. Similarly, 351 DEGs (80 upregulated and 271 downregulated) and 237 DEGs (120 upregulated and 117 downregulated) were detected at 3 and 4 weeks of isolation, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, C). Supplementary Fig.\u0026nbsp;1D-F provide information on the results of GO annotation analysis in female mice. At 2 weeks of isolation, GO analysis revealed significant enrichment in BP terns related to \u0026ldquo;positive regulation of toll\u0026thinsp;\u0026minus;\u0026thinsp;like receptor 3 signaling pathway\u0026rdquo; and \u0026ldquo;negative regulation of plasminogen activation\u0026rdquo; (Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eD). By 3 weeks, the most enriched BP terms were related to the \u0026ldquo;meiotic cell cycle\u0026rdquo; and \u0026ldquo;positive regulation of proteolysis\u0026rdquo; (Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eE). At 4 weeks, the most enriched BP terms was \u0026ldquo;mammary gland alveolus development\u0026rdquo; (Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eF). Based on KEGG pathway analysis, the most significantly enriched pathways in female mice at 2 weeks of isolation were \u0026ldquo;IL\u0026thinsp;\u0026minus;\u0026thinsp;17 signaling pathway\u0026rdquo; and \u0026ldquo;viral protein interaction with cytokine and cytokine receptor\u0026rdquo; (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). At 3 weeks, the enriched pathways included \u0026ldquo;protein digestion and absorption\u0026rdquo; and \u0026ldquo;cysteine and methionine metabolism\u0026rdquo; (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE), while at 4 weeks, the most significantly enriched pathway was the \u0026ldquo;AMPK signaling pathway\u0026rdquo; (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Temporal dynamic of gene expression clusters in male and female mice under SI stress\u003c/h2\u003e \u003cp\u003eTo further explore the dynamic patterns of gene expression changes over time, we used the Mfuzz package to perform fuzzy c-means clustering on the time-course transcriptomic data. This analysis revealed distinct temporal expression profiles in both male and female mice subjected to SI, highlighting clusters of genes that were specifically upregulated or downregulated across 2-, 3-, and 4- week isolation periods. We identified 10 distinct clusters in both male and female mice, each representing a unique set of genes with specific expression patterns at particular time points. In male mice, the gene expression in Cluster 1 showed no significant differences between 2- and 4-week isolation periods, but peaked specifically at 3-week isolation. This pattern was similarly observed in Clusters 4 and 8, suggesting that these clusters represent genes specifically responsive to 3 weeks of isolation. Gene expression in Cluster 2 peaked at 2-week isolation mark and then declined to baseline levels at the 3- and 4- week time points. In contrast, the genes in Cluster 5 showed peak expression at the 4-week isolation period (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e also presents the Mfuzz results for female mice. Cluster 7 represents genes that were specifically expressed after 2 weeks of isolation. Genes expression in Cluster 2 reached its lowest point at 3 weeks of isolation, while genes in Cluster 10 reached their peak expression at the same time point. These two clusters specifically reflect distinct transcriptional responses in female mice at the 3-week isolation, highlighting their unique roles in the molecular changes associated with this critical period. Genes within Cluster 4 maintained consistent expression levels at both 2- and 3- week isolation time points, but demonstrated significant upregulation specifically at the 4-week time point, indicating that these genes are uniquely associated with transcriptional changes in female mice at 4 weeks of isolation.\u003c/p\u003e \u003cp\u003e \u003cb\u003e3.4. Identification and functional enrichment of key genes associated with NREM sleep disturbance in male mice during SI\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo further identify key genes and their associated signaling pathways at each time point during SI, we performed an overlap analysis between the DEGs sets and the gene sets corresponding to each time point, as identified by Mfuzz. We visualized the overlapping genes using Venn diagrams. At 2- week SI time point, we identified 13 key genes involved in the emergence of NREM sleep disturbance in male mice, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA. Additionally, GO and KEGG analyses revealed that these 13 key genes were primarily enriched in BP associated with the \u0026ldquo;positive regulation of DNA-binding transcription factor activity\u0026rdquo; (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). The most significantly enriched pathway was \u0026ldquo;phototransduction\u0026rdquo; (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC), which showed a strong preference for organismal systems, particularly the sensory system (Supplementary Fig. S2A).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAfter 3 weeks of isolation, 20 key genes were identified as responsive to NREM sleep disturbance in male mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). These genes were mainly enriched in the following BP terms (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE): \u0026ldquo;tetrapyrrole biosynthetic process\u0026rdquo;, \u0026ldquo;modulation of process of another organismcell body\u0026rdquo;, and \u0026ldquo;regulation of glycogen metabolic process\u0026rdquo;, among others. These processes were linked to metabolic functions such as amino acid metabolism, lipid metabolism, and metabolism of cofactors and vitamins (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF, Supplementary Fig. S2B).\u003c/p\u003e \u003cp\u003eFinally, after 4 weeks of isolation, 24 key genes were identified in male mice, and their biological processes were primarily enriched in \u0026ldquo;defense response to virus\u0026rdquo;, \u0026ldquo;positive regulation of natural killer cell differentiation\u0026rdquo;, \u0026ldquo;B cell apoptotic process\u0026rdquo; and \u0026ldquo;innate immune response\u0026rdquo; (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG, H). These pathways are primarily associated with immune system functions (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI, Supplementary Fig. S2C).\u003c/p\u003e \u003cp\u003e \u003cb\u003e3.5. Identification and functional enrichment of key genes associated with NREM sleep disturbance in female mice during SI\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo identify key genes and their associated pathways in female mice at different time points of SI, we applied the same analytical approach used for male mice. Specifically, we performed an overlap analysis between DEGs sets and the time-specific gene clusters derived from Mfuzz, with the results visualized using Venn diagrams. At 2 weeks of isolation, 20 key genes were identified in female mice, despite the absence of NREM sleep disturbance at this point (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Interestingly, GO and KEGG analyses revealed that these genes were significantly enriched in the \u0026ldquo;inositol trisphosphate biosynthetic process\u0026rdquo; and the \u0026ldquo;fatty acid biosynthesis\u0026rdquo; pathways, suggesting a potential role in lipid metabolism. The remaining KEGG pathways were primarily associated with the endocrine and immune systems (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, C; Supplementary Fig. S2D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAfter 3 weeks of isolation, 39 key genes were identified in female mice, primarily enriched in the \u0026ldquo;retinol metabolism\u0026rdquo; and \u0026ldquo;vitamin digestion and absorption\u0026rdquo; pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD-F). These pathways were associated with metabolism functions and the digestive system within organismal system, respectively (Supplementary Fig. S2E).\u003c/p\u003e \u003cp\u003eBy 4 weeks of isolation, 9 key genes were identified as linked to NREM sleep disturbance in female mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG). GO analysis revealed significant enrichment in biological process related to \u0026ldquo;noradrenergic neuron development\u0026rdquo; (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH). Meanwhile, the results of KEGG indicated that the primary enriched pathways were \"nitrogen metabolism\" and \"arginine biosynthesis\", which were primarily involved in energy metabolism and amino acid metabolism functions (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI). These findings suggested a strong involvement of metabolism responses in female mice after prolonged SI (Supplementary Fig. S2F). Overall, these results indicated that metabolic processes play a significant role in mediating sleep disturbances induced by prolonged SI in female mice.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003ePrevious studies have highlighted sex-specific difference in responses to SI across various physiological systems, including hippocampal plasticity[14], cardiovascular reactivity[15], affective vulnerability[16, 17], accelerated aging[18], dendritic spine remodeling[19], and neural circuit reorganization[20]. Our study further extended SI to the sleep architecture based on this sex-specific animal models. Firstly, we found that adolescent male mice exhibited a reduction in NREM sleep duration after 2 weeks of isolation. Secondly, the NREM sleep disturbance observed in adolescent male mice after 2 weeks of isolation were associated with early activation of sensory systems. For adolescent female mice, the decrease in NREM sleep duration after 4 weeks of isolation was associated with metabolic disruptions. Although both sexes experienced a reduction in NREM sleep, the underlying regulatory mechanisms differed between them.\u003c/p\u003e\n\u003cp\u003eAdolescence represents a critical developmental window with heightened vulnerability to psychiatric disorders[21, 22]. SI during this period exacerbates mental health risks, with the duration of isolation contributing to distinct behavioral phenotypes. Specifically, in adolescent male mice, total 2 weeks of isolation led to anxiety-like behaviors[23], 3 weeks of isolation resulted in depressive-like symptoms[24], and 4 weeks of isolation strengthened the retention of fear memories[25] and autism-like behaviors[26]. All of these researches highlight the importance of the length of isolation in shaping behavioral outcome. Interestingly, our research revealed that regardless of duration of SI, adolescent male mice similarly exhibited a reduction in NREM sleep duration, but not in REM phase. In contrast, adolescent female mice only began to show a reduction in NREM sleep after 4 weeks of isolation. This may be due to the sensitivity of NREM sleep to chronic stress, which ultimately leads to sleep reduction[27, 28]. Moreover, NREM sleep is associated with the regulation of hypothalamic-pituitary-adrenal (HPA) axis[29], neurotransmitters[30, 31], and immune system[32], all of which are influenced by SI[33, 34]. In contrast, the regulation of REM sleep remains relatively independent, potentially serving as a protective factor under stress conditions[35]. These findings suggest that NREM sleep may serve as a biomarker of stress responses. Additionally, the delayed decrease in NREM sleep after 4 weeks of isolation in adolescent female mice, compared to male mice, underscores the different sensitivities to SI. Sex also plays a crucial role in the relationship between NREM sleep and SI,with previous studies emphasizing gender differences in stress responses[36]. Estrogen in female mice may provide a potential protective role in stress responses[37, 38].\u003c/p\u003e\n\u003cp\u003eWhat causes adolescent male mice to develop NREM sleep disturbance earlier than female mice, and to maintain consistent NREM sleep disturbance under dynamic changes during different periods of SI? To address this, we identified key genes with regulatory roles that vary over time during SI using Mfuzz time-series analysis. Pathway enrichment analysis showed activation of sensory systems(phototransduction pathways) at week 2 of SI, metabolic signaling pathways at week 3 of SI, and immune-related pathways and metabolic regulation at week 4 of SI.The sensory systems are responsible for receiving physical stimuli. Previous studies have shown that the recovery of NREM sleep in male mice is worse than in female mice after physical stimulation[39, 40]. Additionally, the behavioral manifestations in male mice, including locomotor activity, anxiety levels, and pentobarbital-induced sleep, were more sensitive to the effects of SI[41]. Another study indicated that in the fear conditioning test, after 3 weeks of isolation, male mice became more susceptible to a prolonged freezing behavior in response to foot-shock stimulation, whereas female mice did not[42]. This suggests that male mice are more vulnerable to external physical stimuli and develop NREM sleep disturbance earlier than female mice. A human transcriptomics-based study demonstrated that long-term social loneliness induces changes closely associated with inflammatory factors[43], which aligns with our findings. However, prior study did not address sex-specific differences in these responses, and research on the dynamic temporal changes underlying these sex differences remains limited. Overall, sensory systems may serve as a potential target for interventions aimed at sleep disturbances in male mice, advancing the understanding of sex-specific sleep regulation and its molecular underpinnings.\u003c/p\u003e\n\u003cp\u003eTo investigate the factors contributing to the delayed onset of sleep disturbances in female mice after SI, we focused on the 4-week timepoint of SI, which is primarily associated with metabolism and energy balance in female mice. Our research suggests that adolescent female mice may resist the detrimental effects of SI from weeks 2 to 3 through sustained activation of immune and lipid metabolism pathways. This finding is consistent with previous studies showing that female animals exhibit greater resilience to chronic stress, which is associated with immune regulation[44]. Clinical research indicates that women have a metabolic advantage in controlling blood lipids, which may protect them from the detrimental effects of obstructive sleep apnea[45]. Furthermore, the lower weight gain observed in female mice under chronic stress[46\u0026ndash;48], it supported the finding that metabolic regulation plays a critical role in their stress adaptation. These initial protective mechanisms may become overwhelmed, leading to the eventual disruption of NREM sleep when female mice experience long-term stress. Lipid metabolism or immune modulation may represent potential preventive strategies for female sleep disorders in the future.\u003c/p\u003e\n\u003cp\u003eIn conclusion, our study first revealed significant sex-specific differences in the temporal dynamics of sleep architecture under SI conditions. The molecular mechanisms underlying sex differences in the relationship between SI and sleep disturbances are not yet fully understood. However, the potential roles of hormonal, genetic, and epigenetic factors should be explored in further studies. This research establishes a foundation for developing targeted interventions aimed at alleviating the negative impacts of SI on mental health in adolescents. Additionally, it offers valuable insights for the development of gender-specific mouse models for studying sleep disorders.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eNo competing interests declared.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor contributions\u003c/h2\u003e \u003cp\u003eBZ and JL designed the study and supervised the project. SL performed the behavioral experiments and completed the analysis. XM, YJ, HG and PZ participated in the experiments. SL and LF wrote the draft of the manuscript. All authors read and approved the final manuscript. In addition, the authors thank HaploX Biotechnology (Jiangxi, China) for their participation in the RNA-sequencing test.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThis work was supported by the National Natural Science Foundation of China (Grant No. 82271525, 82471553) and Guangdong-Hong Kong Joint Laboratory for Psychiatric Disorders (2023B1212120004) and the Natural Science Foundation of Guangdong Province (2024A1515030100).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eJackowska M, Steptoe A. Sleep and future cardiovascular risk: prospective analysis from the English Longitudinal Study of Ageing. \u003cem\u003eSLEEP MED\u003c/em\u003e 2015; \u003cstrong\u003e16\u003c/strong\u003e(6): 768\u0026ndash;774.\u003c/li\u003e\n\u003cli\u003ePaez A, Gillman SO, Dogaheh SB, Carnes A, Dakterzada F, Barbe F \u003cem\u003eet al.\u003c/em\u003e. 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Enduring and sex-specific effects of adolescent social isolation in rats on adult stress reactivity. \u003cem\u003eBRAIN RES\u003c/em\u003e 2010; \u003cstrong\u003e1343\u003c/strong\u003e: 83\u0026ndash;92.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"translational-psychiatry","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"tp","sideBox":"Learn more about [Translational Psychiatry](http://www.nature.com/tp/)","snPcode":"41398","submissionUrl":"https://mts-tp.nature.com/cgi-bin/main.plex","title":"Translational Psychiatry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6828480/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6828480/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSleep disturbances are more prevalent in women than in men during adulthood. However, since age-related changes in sleep and the consequences of sleep disturbances can occur as early as adolescence, it remains poorly understood whether these disturbances exhibit a similar sex-specific pattern during adolescence and what the underlying molecular mechanisms may be. Male and female mice were subjected to social isolation stress starting at postnatal day 21 (P21), and electroencephalogram (EEG) was monitored during isolation period. We then employed whole-brain transcriptomic analysis and Mfuzz enrichment analyses to identify temporal and sex-specific molecular responses, dynamic gene expression patterns, and key pathways during isolation period. Male mice exhibited decreased non-rapid eye movement (NREM) sleep duration after 2, 3, and 4 weeks of isolation, while female mice did not show these disturbances after 2 and 3 weeks, but did after 4 weeks of isolation. This suggested a sex-specific pattern of sleep disturbances during adolescence, distinct from those observed in adulthood. Moreover, the decreased NREM sleep in isolated male mice was related to sensory, metabolic, and immune systems after 2-, 3-, and 4-week of isolation, respectively. While the reduction in NREM duration in female mice after 4 weeks of isolation was associated with their energy metabolism and amino acid metabolism disruptions. We found a sex-specific pattern of sleep disturbances during adolescence, with male mice being more susceptible to social isolation stress, which may be linked to early sensory system responses in isolated male mice and later-stage amino acid metabolism and energy imbalance in isolated female mice. Our findings provide insights into gender-specific interventions for sleep disorders during adolescence and underscore the importance of considering both temporal and sex differences in stress-related sleep research.\u003c/p\u003e","manuscriptTitle":"Time-Dynamic Analysis of Sex-Specific NREM Sleep Disturbance Induced by Social Isolation Among Adolescent Mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-23 09:55:35","doi":"10.21203/rs.3.rs-6828480/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2025-07-15T09:01:44+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-07-11T07:00:28+00:00","index":3,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-07-09T07:19:35+00:00","index":1,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-07-03T09:50:49+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-06-23T02:30:09+00:00","index":3,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-06-19T06:20:28+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-06-19T04:35:19+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2025-06-19T01:48:49+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-06T09:41:51+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-06T09:36:39+00:00","index":"","fulltext":""},{"type":"submitted","content":"Translational Psychiatry","date":"2025-06-05T10:57:09+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"translational-psychiatry","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"tp","sideBox":"Learn more about [Translational Psychiatry](http://www.nature.com/tp/)","snPcode":"41398","submissionUrl":"https://mts-tp.nature.com/cgi-bin/main.plex","title":"Translational Psychiatry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f0afa258-cf9b-41be-a4ad-7e611d441be4","owner":[],"postedDate":"June 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":50267715,"name":"Health sciences/Diseases/Psychiatric disorders"},{"id":50267716,"name":"Biological sciences/Neuroscience/Molecular neuroscience"}],"tags":[],"updatedAt":"2026-03-27T07:11:54+00:00","versionOfRecord":{"articleIdentity":"rs-6828480","link":"https://doi.org/10.1038/s41398-026-03895-w","journal":{"identity":"translational-psychiatry","isVorOnly":false,"title":"Translational Psychiatry"},"publishedOn":"2026-02-13 05:00:00","publishedOnDateReadable":"February 13th, 2026"},"versionCreatedAt":"2025-06-23 09:55:35","video":"","vorDoi":"10.1038/s41398-026-03895-w","vorDoiUrl":"https://doi.org/10.1038/s41398-026-03895-w","workflowStages":[]},"version":"v1","identity":"rs-6828480","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6828480","identity":"rs-6828480","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}