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. 2026 Aug 11;74(10):e70217. doi: 10.1002/glia.70217

Enhancing Oligodendrogenesis Rescues Chronic Stress‐Induced Behavioral Disorders Involving Neuronal and Synaptic Reorganization

Yu‐Peng Dai 1, Can‐Ling Xu 1, Hao Luo 1, Zhi‐Yu Liu 1, Guo‐Xiao Meng 1, Jia‐Lin Li 1, An‐Cheng Yu 1, Xin Chen 1, Xing Gao 1, Xi Zhang 1, Jia‐Li Wang 1, Tao Chen 2, Lan Xiao 1, Fei Wang 1, Feng Mei 1,✉, Qi‐Jing Lei 3,✉, Tao Li 1,✉
PMCID: PMC13459151  PMID: 42578521

ABSTRACT

Chronic stress induces psychiatric disorders, including depression and anxiety, yet effective therapies remain limited. Myelin in adult brains undergoes dynamic remodeling through oligodendrocyte precursor cells (OPCs) differentiation into oligodendrocytes (OLs) and the degeneration of pre‐existing myelin. However, how chronic stress alters myelin dynamics and whether this represents a therapeutic target remains unclear. Here, adult mice subjected to 4‐h daily restraint for 2 weeks exhibited significant anxiety, depression, and social deficits. Histological examinations revealed reduced OPC and OL density, decreased c‐Fos‐positive neurons, and loss of synaptic proteins in the brains exposed to chronic stress. To understand the dynamic changes of myelin, cell‐lineage labeling and tracing demonstrated that chronic stress exposure remarkably inhibited oligodendrogenesis in the medial prefrontal cortex (mPFC), motor cortex, hippocampus, and amygdala, as revealed by the NG2CreERT; Tau‐mGFP line, but did not significantly change pre‐existing myelin as revealed by a newly generated line for mature OLs and myelin. To explore the role of myelinogenesis changes, adult myelin formation was inhibited by Olig2 conditional knockout in OPCs, resulting in decreased neuronal synaptic proteins and activity, accompanied by anxiety and depressive‐like behaviors. Conversely, enhancing myelinogenesis through conditional deletion of the M1R in OPCs of stressed mice resulted in higher number of c‐Fos‐positive neurons, elevated synaptic protein expression, and a partial reversal of the behavioral deficits. Importantly, treating the stressed animal with the pro‐myelination drug clemastine phenocopied the effects of M1R deletion on histological and behavioral disorders. Together, our findings demonstrate that enhancing oligodendrogenesis represents a promising strategy to rescue CRS‐caused behavioral disorders.

Keywords: chronic restraint stress, clemastine, depression, oligodendrocyte, oligodendrogenesis

  • CRS disrupts oligodendrogenesis and neuronal connections, causing behavioral disorders.

  • CRS causes region‐dependent inhibition of myelinogenesis without changing pre‐existing myelin.

  • Inhibiting oligodendrogenesis by Olig2 ablation phenocopies CRS‐induced neuronal and behavioral changes.

  • Enhancing oligodendrogenesis partially rescues CRS‐induced neuronal and behavioral abnormalities.

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1. Introduction

Chronic stress is a pervasive burden in modern life and can disrupt the internal homeostasis of the brain (de Kloet et al. 2005; Lupien et al. 2009). Prolonged exposure to stress contributes to cognitive decline and anxiety, and may ultimately lead to severe psychiatric disorders, including major depression and suicidality (de Kloet et al. 2005; Lupien et al. 2009; Obeng‐Gyasi and Parker 2025; Tran and Gellner 2023). Given these risks, early interventions for chronic stress are a pressing public health need. While stress has been shown to disrupt physiological balance, its specific effects on brain infrastructure remain poorly understood. Clinical imaging studies have revealed microstructural alterations in white matter, the hippocampus, and limbic cortex in association with stress (Chen et al. 2012; McManus et al. 2024). However, the underlying histological changes remain largely elusive.

In the CNS, myelin sheaths are essential for the rapid, energy‐efficient transmission of action potentials along axons (Butt et al. 2025; Zalc et al. 2008). Notably, recent research has consistently shown that myelin remains dynamically active in the adult brain and supports a wide range of neurological functions (Chang et al. 2016; Flower et al. 2025). This dynamic process depends mainly on the differentiation of oligodendrocyte precursor cells (OPCs) into mature oligodendrocytes, each of which can form 30–60 myelin segments (Simons et al. 2024; Snaidero and Simons 2014). At the same time, pre‐existing myelin sheaths can degenerate in aging and neurodegenerative brains (L. Chen, Ren, et al. 2021; Guo et al. 2021; Wang et al. 2018). Myelin dynamics are actively modulated by experience and sensory input (Gibson et al. 2014; Hill et al. 2014; McKenzie et al. 2014; Pan et al. 2020). For example, whisker trimming inhibits myelination in the corresponding cortical region due to sensory deprivation (Hill et al. 2014), while motor learning and long‐term memory formation require new myelin generation in areas such as the cortex and corpus callosum (Gibson et al. 2014; McKenzie et al. 2014; Pan et al. 2020). These findings position myelin plasticity as a key mechanism for adapting to environmental cues and acquiring new skills. Notably, white matter abnormalities and myelin deficits have been identified in several psychiatric disorders, including major depression (Rajkowska et al. 2015; Zhao et al. 2021; Zhou et al. 2020) and schizophrenia (Zhao et al. 2021). More importantly, the homeostasis of oligodendroglial lineage cells is disrupted in specific brain regions of stressed mice, such as decreased OPCs, newly generated mature OLs in the mPFC of adult or adolescent mice (Kokkosis et al. 2022; Madeira et al. 2025; Poggi et al. 2022), or in some cases, increased mature OLs and myelin in specific brain regions of early life or adolescent stressed mice (Poggi et al. 2022; Teissier et al. 2020; Yin et al. 2024). Supporting this connection, studies using a genetic approach in mice show that hypomyelination can directly induce social deficits and depression‐like behaviors during adolescence (Chen et al. 2020). These findings underscore myelin dynamics as a potential modulator of responses to chronic stress.

Here, we reported that chronic restraint stress (CRS) exposure elevated anxiety and depression‐like behaviors in adult mice, with histological changes, such as decreased densities of OLs, OPCs, c‐Fos‐positive neurons, and synaptic proteins. Cell‐lineage labeling and tracing revealed a significant inhibition of myelinogenesis (NG2CreERT; Tau‐mGFP) without affecting pre‐existing myelin (MOGDreERT; mGFP (rox)). To dissect the causal pathway, we monitored myelinogenesis inhibition in adult mice by olig2 deletion in OPCs, which induced decreased neuronal synaptic proteins and activity, as well as anxiety and depressive‐like behaviors. On the contrary, enhancing myelinogenesis by conditional knockout of the muscarinic receptor 1 (M1R) in OPCs partially reversed the histological and behavioral deficits of CRS mice. More importantly, the pro‐myelination drug, clemastine, phenocopied the effects of M1R cKO, underpinning myelination as a potential therapeutic target for chronic stress‐induced psychiatric disorders.

2. Material and Method

2.1. Mice

All experimental mice in this study were maintained on a C57BL/6J genetic background. Both male and female mice were utilized, and no sex differences were observed in histological or behavioral analyses. The NG2CreERT line (The Jackson Laboratory, Catalog # 008538) was crossed with the Tau membrane‐bound GFP (mGFP) line (The Jackson Laboratory, Catalog # 021162) to generate NG2CreERT; Tau‐mGFP mice. The M1R floxed (fl/fl) and Olig2 fl/fl mice have been described previously (Mei et al. 2016, 2013). The M1R heterozygous line was crossed with NG2CreERT; Tau‐mGFP mice to produce NG2CreERT; Tau‐mGFP; M1R fl/fl mice and controls (NG2CreERT; Tau‐mGFP). The Olig2 fl/fl line was crossed with NG2CreERT mice to generate NG2CreERT; Olig2 fl/fl mice and controls (NG2CreERT mice). The MOGDreERT and mGFP (rox) mouse line was commercially generated by Shanghai Model Organisms. MOGDreERT was crossed with the mGFP (rox) line to obtain the MOGDreERT; mGFP (rox) line. All animal procedures were approved by the Laboratory Animal Welfare and Ethics Committee of the Army Medical University. Mice were housed under controlled environmental conditions: constant temperature of 23°C ± 2°C, humidity of 60% ± 10%, and a 12‐h light/dark cycle. Throughout the study, animals had ad libitum access to standard rodent chow and water. All mouse lines were maintained in specific pathogen‐free (SPF) facilities at the Army Medical University.

2.2. CRS

Mice were exposed to a CRS paradigm daily for 14 consecutive days. The stressor was administered by placing individual mice into well‐ventilated, perforated 50 mL conical tubes for a period of 4 h each day. The restraint sessions were consistently conducted from 12:00 p.m. to 4:00 p.m. daily. During confinement, mice were unable to turn around or move freely but had sufficient space for slight postural adjustment. Control (non‐stressed) mice remained undisturbed in their home cages with free access to food and water during this period. This protocol is adapted from the models of chronic stress to induce persistent physiological and behavioral alterations (Ma et al. 2023).

2.3. Tamoxifen Administration

Tamoxifen (Sigma‐Aldrich, Cat # T5648) was dissolved in corn oil at a concentration of 30 mg/mL to induce recombination. Tamoxifen was administered to mice via oral gavage at a dose of 50 mg/kg.

2.4. Drug Treatment

The 8‐week‐old mice were treated with clemastine (SelleckChem, Cat# S1847) or an equivalent volume of vehicle. Clemastine was administered at a dose of 10 mg/kg/day for 21 consecutive days via either oral gavage or drinking water. From day 1 to day 10 of the restraint, clemastine was given by oral gavage. To avoid the potential influence of gavage on behavioral outcomes, clemastine was dissolved in the drinking water for voluntary consumption from day 11 to day 21. During the gavage phase, clemastine was prepared as a 3 mg/mL working solution in a vehicle containing 40% PEG 300, 5% Tween‐80, and 10% dimethyl sulfoxide (DMSO). During the drinking water phase, clemastine was first dissolved in DMSO at a concentration of 30 mg/mL and then diluted in the drinking water to 0.075 mg/mL for administration, based on an average mouse weight of 30 g and a daily water intake of 4 mL. The water bottle with 100 mL of water (no addition of water for the following days) is scaled every 24 h for 5 days to calculate daily water consumption, as we previously reported (J. F. Chen, Liu, et al. 2021).

2.5. Tissue Processing

Mice were deeply anesthetized with 1% pentobarbital and perfused transcardially with 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB), following an initial flush with 0.01 M phosphate‐buffered saline (PBS). Brains were collected and post‐fixed overnight in 4% PFA in 0.1 M PB. Subsequently, the tissues were dehydrated in 30% sucrose in 0.01 M PBS. Brains were embedded in Optimal Cutting Temperature compound (O.C.T. Compound, SAKURA, 4583), and then sectioned coronally at 20 μm using a cryostat (MS 1850, Leica).

2.6. Immunofluorescence Staining and Image Acquisition

Brain sections were blocked at room temperature for 2 h in 5% bovine serum albumin (BSA) and 0.5% Triton X‐100. Sections were then incubated overnight at 4°C with primary antibodies diluted in blocking solution, followed by incubation with fluorescent secondary antibodies at room temperature for 2 h. Nuclei were counterstained with DAPI. Primary antibodies included: Rabbit anti‐c‐Fos (1:4000; Abcam, Cat# ab190289); Guinea pig anti‐vGlut1 (1:1000; Synaptic Systems, Cat# 135304); Rabbit anti‐Synapsin‐1 (1:1000; Cell Signaling Technology [CST], Cat# 5297); Rabbit anti‐Homer1 (1:1000; Synaptic Systems, Cat# 160003); Rat anti‐MBP (1:500; Millipore, Cat# MAB386); Rabbit anti‐CC1 (1:500; Oasis Biofarm, Cat# OB‐PRB070‐01); Goat anti‐PDGFRα (1:500; R&D systems; Cat# AF1062); Mouse anti‐Caspr (1:500; NeuoMab; Cat# 75–001); Rabbit anti‐neurofilament 200 (NF200) (1:500; Sigma‐Aldrich, Cat# N4142); Goat anti‐GFP (1:500; Abcam; Cat# ab5450). Secondary antibodies included: Alexa Fluor 488‐, Alexa Fluor 568‐, or Alexa Fluor 647‐conjugated secondary antibodies (1:1000; Invitrogen) directed against goat, rabbit, guinea pig, mouse or rat IgG. Fluorescence images were captured using either a spinning disk confocal super‐resolution microscope (Olympus SpinSR10, Shinjuku, Tokyo) or a fluorescence microscope (Olympus VS200, Shinjuku, Tokyo). Images were analyzed using the CellSens Dimension software and ImageJ version Java 1.8.0_77.

2.7. Behavioral Tests

All behavioral tests were performed between 9:00 a.m. and 5:00 p.m. Following each trial, all equipment was wiped with 30% ethanol to remove residual odor cues. For all behavioral experiments, experimenters were blinded to both genotype and group assignment and handled the animals gently and carefully to minimize stress. Variations in mouse numbers across different tests resulted from incidental events occurring during the behavioral testing period.

2.7.1. Open Field Test

The open field test (OFT) was employed to assess locomotor activity and exploratory behavior in mice. Each mouse was individually placed in an open field arena (50 cm × 50 cm × 50 cm) for a 10‐min session. The arena was divided into a central zone and a peripheral zone using the Super Maze+ system (XinRuan Information Technology Co., Shanghai, China). The total distance traveled and the time traveled within the central zone were recorded and quantified to assess anxiety‐like behavior.

2.7.2. Elevated Plus Maze

Mice were placed in an elevated plus maze (XR‐XG201, Shanghai XinRuan Information Technology Co. Ltd) consisting of two enclosed arms (35 cm × 5 cm) with 15‐cm‐high opaque walls and two opposing open arms (35 cm × 5 cm). The behavior of each mouse was recorded for a 10‐min session. The time spent in the open arms was quantified.

2.7.3. The Social Interaction Test

Two identical cylindrical wire cages (diameter: 7 cm; height: 15 cm) were placed in the same corner of two adjacent chambers (60 cm × 40 cm × 22 cm) within a three‐chamber apparatus. During the habituation phase, the test mouse was placed in the central chamber and allowed to freely explore all chambers for 10 min. In the first test trial (Sociability Test), a stranger mouse (Stranger 1) was enclosed within one cylindrical cage. In the second test trial (Social Novelty Preference Test), a novel stranger mouse (Stranger 2) was enclosed within the remaining cylindrical cage. Each trial lasted 10 min. The time spent in chambers containing cylindrical cages was quantified. The Sociability Index was calculated as: (Time exploring the chamber with Stranger 1 mouse—Time exploring the chamber with an empty cage)/(Time exploring the chamber with Stranger 1 mouse + Time exploring the chamber with an empty cage). The Social Novelty Preference Index was calculated as: (Time exploring the chamber with Stranger 2 mouse—Time exploring the chamber with Stranger 1 mouse)/(Time exploring the chamber with Stranger 2 mouse + Time exploring the chamber with Stranger 1 mouse).

2.7.4. Tail Suspension Test

Mice were secured to a metal hook using medical adhesive tape applied 1–2 cm distal to the tip of the tail. The attachment was reinforced to ensure firm immobilization such that the animal could not dislodge itself. The suspension height was adjusted so that the animal's snout was positioned 20–25 cm above the apparatus floor. The test duration was typically 6 min, during which the number of struggle attempts was recorded.

2.7.5. Forced Swimming Test

Mice were subjected to a 6‐min swimming session in a glass cylinder (diameter: 20 cm; height: 30 cm) filled with water (23°C–25°C) at a depth of 15 cm. The initial 2‐min period served as habituation. Immobility time was quantified during the final 4 min of the test.

2.8. Quantification and Statistical Analysis

2.8.1. Stereology and Quantification

Serial brain sections were collected from Bregma +1.1 mm to Bregma −4.20 mm in mice. Section thickness was 20 μm. Each group consisted of 20 consecutive sections, with one set of 5–8 sections sampled from the serial sections of each brain. All quantitative analyses were performed using either ImageJ (NIH) or CellSens Dimension (Olympus) software. The experimenter was blinded to the genotype.

2.8.2. Statistical Analysis

Data were analyzed using GraphPad Prism 10.1.2. All graphical data are presented as mean ± SD. Unless otherwise stated, each data point represents an individual animal. Fold changes were calculated by normalizing all data to the mean of the control group, followed by further statistical analysis. The unpaired t‐tests were used to determine significance between two groups. The One‐way ANOVA with Tukey's multiple comparisons test was employed for comparisons among three groups. The p‐values are indicated on the figures, with significance reported as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

2.9. Manuscript Editing

The manuscript is written by the authors. AI proofreading function of the Word Processing System (WPS) software is used to correct word spelling and grammar mistakes. No data analysis or figure material is generated with AI.

3. Results

3.1. Chronic Restraint Stress Induces Disrupted Oligodendroglial Lineage Cells, Neuronal Deficits, and Behavioral Disorders

We set out to establish a chronic restraint stress (CRS) mouse model by exposing mice to 4 h of restraint stress daily for 2 consecutive weeks at 8 weeks of age (Figure 1A). The behavioral changes were examined the following week using the open field test (OFT) and the elevated plus maze (EPM) for anxiety, the social interaction test (SIT) for social deficits, and the tail suspension test (TST) and the forced swim test (FST) for depression, respectively. Our results showed significantly reduced time traveled in the open arms of the EPM and the center zone of the OFT, indicating elevated anxiety levels (Figure 1B,C). Concurrently, a significant decrease in swimming time was observed in the FST, and a substantial reduction in struggling episodes was observed in the TST, reflecting the emergence of depression‐like behaviors (Figure 1D,F). Furthermore, the SIT revealed significant declines in both the sociability index and the social novelty preference index in CRS‐exposed mice, indicating reduced social interest (Figure 1E). These findings demonstrate that the two‐week chronic restraint stress protocol effectively induced abnormal behaviors related to anxiety, depression, and sociability in adult mice.

FIGURE 1.

FIGURE 1

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Chronic restraint stress leads to anxiety, depression and social withdraw, along with reduced synaptic proteins. (A) Schematic diagram illustrating the timeline of the restraint stress paradigm and analyses. (B) Time traveled in the open arms of the EPM. n = 11 biologically independent mice for each group. (C) Total distance traveled and time traveled in the center zone of the OFT. n = 11 biologically independent mice for each group. (D) Number of struggling episodes during the TST. n = 11 biologically independent mice for each group. (E) Sociability index and social novelty preference index in the social interaction test. n = 11 biologically independent mice for each group. (F) Time spent swimming during the FST. n = 11 biologically independent mice for each group. (G) Representative images (upper) and quantification (lower) of c‐Fos‐positive neurons. Data are from biologically independent mice: CTL (n = 9), CRS (n = 12). (H) Representative images (upper) and quantification (lower) of synaptic puncta in the hippocampal CA3 region: Synapsin1, Homer1, and vGlut1. n = 5 biologically independent mice for each group. Scale bars: 20 μm (G); 10 μm (H). Significance was determined by unpaired two‐tailed t‐test comparing the CTL and CRS groups. Error bars represent mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

We speculate the abnormal behaviors may be related to disrupted neuronal activities, and thus the histological changes in the brains were examined 1–2 h after the FST test on day 21 (Figure 1A). Neuronal activity was examined by immunostaining for c‐Fos, a well‐established marker of neuronal activity. The CRS‐exposed mice exhibited a significant reduction in the number of c‐Fos‐positive cells in the mPFC, hippocampus, and amygdala, brain regions closely associated with chronic stress (Figure 1G). Immunostaining for the pan‐presynaptic marker‐‐Synapsin1, the pan‐postsynaptic marker‐‐Homer1, and the excitatory synaptic protein‐‐vesicular glutamate transporter 1 (vGlut1) revealed significantly decreased densities of the Synapsin1, Homer1, and vGlut1 puncta in the hippocampal CA3 region of CRS‐exposed mice as compared to the controls (Figure 1H). These results indicate that CRS exposure significantly suppresses neuronal activities and synaptic protein expression, suggesting a potential link to the neurobiological activities induced by this stress paradigm.

Since oligodendroglial cells have been shown to be vulnerable to various psychiatric disorders and myelin is undergoing remodeling in adult brains (Chang et al. 2016; Zhou et al. 2020), we next examined changes in oligodendroglial lineage cells. Immunostaining for myelin basic protein (MBP), CC1, and PDGFRα was carried out to assess myelin, mature OLs, and OPCs, respectively, in the brains. Our results indicated a significant decrease in CC1‐positive OLs in the mPFC and amygdala of CRS‐exposed mice (Figure 2B), while MBP‐positive myelin area remained unchanged relative to the control (Figure S1). Of note, the PDGFRα‐positive OPC density was also significantly reduced in the mPFC, hippocampus, and amygdala of CRS‐exposed mice (Figure 2C). In line with our results, a single‐cell nuclear transcriptome analysis from 17 major depressive disorder (MDD) suicide patients also showed decreased OPCs and mature OLs (Kokkosis et al. 2022; Nagy et al. 2020). These findings demonstrate that CRS exposure resulted in reduced OPC and OL numbers in the mPFC and amygdala, suggesting disrupted oligodendroglial lineage cells.

FIGURE 2.

FIGURE 2

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Disturbed oligodendrocyte lineage cells in brains of CRS mice. (A) Schematic diagram illustrating the timeline of the restraint stress paradigm and analyses. (B) Representative images (left) and quantification (right) of CC1‐positive mature OL expression in the mPFC, hippocampus, and amygdala of CTL and CRS groups. (C) Representative images (left) and quantification (right) of PDGFRα‐positive OPC expression in the mPFC, hippocampus, and amygdala of CTL and CRS groups. Scale bars: 20 μm. Data are from biologically independent mice: CTL (n = 9), CRS (n = 12). Significance was determined by unpaired two‐tailed t‐test comparing the CTL and CRS groups. Error bars represent mean ± SD. *p < 0.05, **p < 0.01.

3.2. Chronic Restraint Stress Does Not Cause Significant Demyelination

Myelin in the adult CNS is undergoing dynamic changes, namely, pre‐existing myelin may degenerate while OPCs are actively differentiating into mature OLs to form new myelin (Chang et al. 2016). We decided to dissect the myelin dynamics by using cell‐lineage labeling and tracing. To determine the change of pre‐existing myelin, we generated a novel MOGDreERT by inserting the DreERT sequence between the exons 1 and 2 of the mog gene. This line allows for inducible expression of Dre recombinase in MOG‐positive cells. MOG is a myelin protein that is specifically expressed in mature OLs, localizing at the outermost layer of myelin sheaths (Johns and Bernard 1999). To label the MOG‐positive cell and myelin sheaths, we generated a novel inducible reporter line, membrane‐bound GFP (mGFP), in which the gene sequence containing a ‘stop’ cassette is inserted between rox sites that can be excised by Dre recombinase to initiate mGFP expression, so that mGFP (rox) thereafter (Figure 3A). After induction, the mGFP‐positive cells were specifically colocalized with the MBP‐ and CC1‐positive OLs and myelin, but not PDGFRα‐positive OPCs or other cell types, after induction in the MOGDreERT; mGFP (rox) brains (Figure 3B). Three weeks after 7‐day induction, the mGFP‐positive myelin stably overlapped with about 60% of the MBP‐positive myelin (Figure 3C). The 8‐week‐old mice received tamoxifen induction for 7 consecutive days to induce recombination and labeling of pre‐existing myelin, and then the mice were challenged with CRS for 2 weeks (Figure 3D). The mGFP‐positive pre‐existing myelin was quantified in the regions with decreased mature OL or OPC density in the mPFC, hippocampus, and amygdala. Interestingly, the MOG‐positive myelin density was not significantly changed throughout these regions after CRS exposure as compared to the control (Figure 3E). These results indicate that chronic restraint stress is unlikely to cause instant and extensive degeneration of pre‐existing myelin.

FIGURE 3.

FIGURE 3

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Chronic restraint stress does not induce significant demyelination. (A) Genetic strategy for the MOGDreERT; mGFP (rox) reporter line. (B) Representative image of mGFP+/MBP+ co‐labeled myelin sheaths (arrows). Representative image of mGFP+/CC1+ co‐labeled mature oligodendrocytes (OLs) (arrows). Representative image of mGFP+ oligodendrocytes (OLs) (arrows) and PDGFRα+ oligodendrocyte precursor cells (OPCs) (arrowheads). (C) Quantification of reporter signal (mGFP, indicating recombined MOG‐positive myelin) colocalization with MBP‐positive myelin. (D) Schematic timeline of tamoxifen induction, restraint stress, and experimental analysis. (E) Representative images (left) and quantification (right) of mGFP‐positive myelin expression in the mPFC, hippocampus, and amygdala of CTL and CRS groups. Scale bars: 1 mm (left panels of B); 20 μm (right panels of B, E). n = 4 biologically independent mice for each group. Significance was determined by unpaired two‐tailed t‐test comparing the CTL and CRS groups. Error bars represent mean ± SD.

3.3. Chronic Restraint Stress Remarkably Inhibits Myelinogenesis

Since new myelin is continuously generated in the adult CNS (Wang et al. 2020), we next assessed whether myelinogenesis from OPC differentiation is altered after CRS. To that end, we used the NG2CreERT; Tau‐mGFP mice to label newly formed OLs and their associated myelin (Figure 4A). The mGFP sequence is under the control of the Tau promoter, which is highly expressed only in mature OLs, but not in NG2‐positive OPCs or pericytes. Thus, though Cre recombinase is induced in NG2‐positive OPCs, mGFP expression can only be activated in mature OLs, with visible newly formed OLs and associated myelin sheaths (Ren et al. 2024; Young et al. 2013). Immunostaining for CC1 and PDGFRα revealed that the mGFP selectively overlapped with CC1‐positive myelin but not PDGFRα‐positive OPCs in the NG2CreERT; Tau‐mGFP brain (Figure 4B,C). Then, the 8‐week‐old mice were induced with tamoxifen for 7 consecutive days, followed by 2 weeks of CRS. The brain tissues were collected at day 21 (Figure 4A). Strikingly, the CRS brains exhibited a significant reduction in mGFP‐positive area within the mPFC, motor cortex, hippocampus, and amygdala compared to the controls, but remained unaltered in the sensory cortex (Figure 4D). These findings indicate that chronic restraint stress selectively suppresses newly formed myelin in a brain‐region‐dependent manner, which may be related to the disrupted oligodendroglial lineage cell changes after CRS exposure.

FIGURE 4.

FIGURE 4

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Chronic restraint stress remarkably inhibits myelinogenesis. (A) Schematic timeline of tamoxifen induction, restraint stress, and experimental analysis. (B) Representative image of mGFP+/CC1+ co‐labeled mature oligodendrocytes (OLs) (arrows). (C) Representative image of mGFP+ oligodendrocytes (OLs) (arrows) and PDGFRα+ oligodendrocyte precursor cells (OPCs) (arrowheads). (D) Representative images (upper) and quantification (lower) of mGFP‐positive myelin sheath expression in the mPFC, hippocampus, and amygdala of CTL and CRS groups. Scale bars: 20 μm (B, C, lower panels of D); 500 μm (upper panels of D). Data are from biologically independent mice: CTL (n = 6), CRS (n = 7). Significance was determined by unpaired two‐tailed t‐test comparing the CTL and CRS groups. Error bars represent mean ± SD. *p < 0.05, ****p < 0.0001.

3.4. Inhibiting Myelinogenesis Induces Neuronal Deficits and Behavioral Disorders

To determine the causal relationship between decreased myelin generation and neuronal or synaptic deficits, we sought to assess whether inhibiting myelin generation in adult mice could induce neuronal deficits and behavioral alterations. To specifically manipulate myelination in adult mice, we crossed NG2CreERT mice with Olig2 floxed (Olig2 fl/fl) mice, enabling conditional deletion of Olig2 in OPCs. Our previous studies have demonstrated that Olig2 is a critical transcription factor regulating OPC differentiation, and that conditional deletion of Olig2 in OPCs inhibits de novo myelination (Wang et al. 2020). At 8 weeks of age, tamoxifen was administered daily for 7 consecutive days (Figure 5A). Behavioral tests were conducted at post‐induction day 28, revealing that Olig2 cKO mice exhibited significant anxiety‐like behaviors, characterized by reduced activity time in the center zone of the OFT and in the open arms of the EPM (Figure 5B,C). Additionally, depression‐like behaviors were observed, including decreased struggling episodes in the TST and reduced swimming time in the FST (Figure 5D,F), while social behavior remained unaffected (Figure 5E). Brain tissue was collected 1–2 h after the forced swim test performed on post‐induction day 35. Subsequent immunofluorescence analysis demonstrated that Olig2 cKO mice displayed a wide reduction in MBP‐positive myelin area (Figure S2). Furthermore, decreased numbers of c‐Fos‐positive neurons were observed in the mPFC, hippocampus, and amygdala, along with significant reductions in Synapsin1 puncta, Homer1 puncta, and vGlut1 puncta in the hippocampus (Figure 5G,H). These findings suggest that myelinogenesis inhibition in adult mice can suppress neuronal activity, inhibit synaptogenesis and induce anxiety‐ and depression‐like behaviors.

FIGURE 5.

FIGURE 5

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Olig2 deletion in OPCs Inhibits myelinogenesis, induces neuronal deficits and behavioral disorders. (A) Schematic timeline of tamoxifen induction, and experimental analysis. (B) Time traveled in the open arms of the EPM. n = 7 biologically independent mice for each group. (C) Total distance traveled and time traveled in the center zone of the OFT. n = 7 biologically independent mice for each group. (D) Number of struggling episodes during the TST. n = 7 biologically independent mice for each group. (E) Sociability index and social novelty preference index in the social interaction test. n = 7 biologically independent mice for each group. (F) Time spent swimming during the FST. n = 7 biologically independent mice for each group. (G) Representative images (upper) and quantification (lower) of c‐Fos‐positive neurons. n = 5 biologically independent mice for each group. (H) Representative images (upper) and quantification (lower) of synaptic puncta in the hippocampal CA3 region: Synapsin1, Homer1, and vGlut1. n = 4 biologically independent mice for each group. Scale bars: 20 μm (G); 10 μm (H). Significance was determined by unpaired two‐tailed t‐test comparing the CTL and Olig2 cKO groups. Error bars represent mean ± SD. *p < 0.05, **p < 0.01.

3.5. Enhancing Myelinogenesis by M1R Deletion Rescues Chronic Stress‐Caused Deficits

As myelinogenesis could directly and independently regulate neuronal activity, synaptic protein expression, and alter mouse behaviors, we next wonder whether enhancing myelinogenesis can rescue CRS‐induced histological and behavioral abnormalities. Our previous study identified M1R as a negative regulator of OPC differentiation (Mei et al. 2016), and ablation of M1R in OPCs can activate myelinogenesis in aged and AD mice (J. F. Chen, Liu, et al. 2021; Zhi et al. 2023). Here, we used the NG2CreERT; Tau‐mGFP; M1Rfl/fl mice to specifically delete M1R in OPCs and trace the newly generated myelin simultaneously. Mice were divided into three groups: Control (CTL, wildtype without CRS), CRS (age‐matched mice with CRS), and CRS + M1R cKO (M1R cKO mice with CRS). At 8 weeks of age, tamoxifen was administered daily for 7 consecutive days. The CRS paradigm was conducted again, and then the mice were subjected to the behavioral and histological assessments (Figure 6A). In contrast to the CRS group, the CRS + M1R cKO mice exhibited significant improvements in the EPM and OFT, recovering to a level similar to that of the controls, indicating that pro‐myelinogenesis rescues anxiety‐like behaviors (Figure 6B,C). While the depression‐like behaviors showed partial recovery, CRS + M1R cKO mice exhibited an increase in struggling episodes only in the TST compared with CRS controls (Figure 6E), with no significant behavioral changes observed in the FST or SIT (Figure 6D,F). These results revealed M1R cKO can partially ameliorate CRS‐induced anxiety‐ and depression‐like symptoms by enhancing myelination.

FIGURE 6.

FIGURE 6

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M1R deletion in OPCs enhances myelinogenesis, restores expression of synaptic proteins and partially rescues stress‐induced behavioral deficits. (A) Schematic timeline of tamoxifen induction, restraint stress, and experimental analysis. (B) Time traveled in the open arms of the EPM. Data are from biologically independent mice: CTL (n = 9), CRS (n = 8), CRS + M1R cKO (n = 6). (C) Total distance traveled and time traveled in the center zone of the OFT. Data are from biologically independent mice: CTL (n = 9), CRS (n = 8), CRS + M1R cKO (n = 6). (D) Time spent swimming during the FST. Data are from biologically independent mice: CTL (n = 9), CRS (n = 8), CRS + M1R cKO (n = 6). (E) Number of struggling episodes in the TST. Data are from biologically independent mice: CTL (n = 9), CRS (n = 8), CRS + M1R cKO (n = 6). (F) Sociability index and social novelty preference index in the social interaction test. Data are from biologically independent mice: CTL (n = 9), CRS (n = 8), CRS + M1R cKO (n = 6). (G) Representative images (left) and quantification (right) of mGFP‐positive myelin sheath expression in the mPFC, hippocampus, and amygdala of CTL, CRS, and CRS + M1R cKO groups. Data are from biologically independent mice: CTL (n = 7), CRS (n = 6), CRS + M1R cKO (n = 6). (H) Representative images (left) and quantification (right) of c‐Fos‐positive neurons in the mPFC, hippocampus, and amygdala of CTL, CRS, and CRS + M1R cKO groups. Data are from biologically independent mice: CTL (n = 7), CRS (n = 6), CRS + M1R cKO (n = 6). (I) Representative images (left) and quantification (right) of synaptic puncta in the hippocampal CA3 region of CTL, CRS, and CRS + M1R cKO groups: Synapsin1, Homer1, and vGlut1. Data are from biologically independent mice: CTL (n = 7), CRS (n = 6), CRS + M1R cKO (n = 6). Scale bars: 20 μm (G, H); 10 μm (I). Significance among the CTL, CRS, and CRS + M1R cKO groups was determined by one‐way ANOVA followed by Tukey's multiple comparisons test. Error bars represent mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

To assess histological alterations following M1R knockout, newly formed myelin sheaths labeled by mGFP were analyzed in the mPFC, hippocampus, and amygdala. As anticipated, CRS mice displayed a significant reduction in mGFP‐positive new myelin in the brain compared to controls. M1R knockout markedly promoted myelination, as evidenced by an increase in the mGFP‐positive area to levels comparable to those of the control group (Figure 6G). To investigate the impact of M1R knockout on the expression of neuronal activity‐associated proteins in specific brain regions of CRS mice, immunofluorescence staining was performed to evaluate the expression of c‐Fos, Synapsin1, Homer1, and vGlut1 in the mPFC, hippocampus, and amygdala. Consistent with alterations in myelination, M1R cKO CRS mice exhibited a significantly higher c‐Fos‐positive density than the CRS group, which is comparable to that in the control group (Figure 6H). Similarly, the number of Synapsin1 puncta, Homer1 puncta, and vGlut1 puncta was significantly reduced in the hippocampus, prefrontal cortex, and amygdala in the CRS group compared to the control group, which is partially restored after M1R cKO (Figure 6I, Figure S3). These findings indicate that M1R‐knockout‐mediated enhancement of myelination ameliorates CRS‐induced inhibition of neuronal activity in specific brain regions.

3.6. Pro‐Myelination by Clemastine Administration Rescues Chronic Stress‐Caused Deficits

Given that M1 receptor (M1R) knockout effectively rescued the histological and behavioral changes in CRS‐exposed mice, we next evaluated whether the FDA‐approved anticholinergic drug clemastine could promote myelinogenesis and ameliorate behavioral disorders in CRS mice. We used NG2CreERT; Tau‐mGFP reporter mice to examine changes in newly formed myelin and behaviors. At the age of 8 weeks, the NG2CreERT; Tau‐mGFP mice were administered tamoxifen daily for seven consecutive days to induce recombination, followed by 4‐h restraint stress daily for 2 weeks (Figure 7A). Start with the stress period, clemastine (CRS + Clemastine group) or vehicle (CRS + vehicle group) was administered at a dosage of 10 mg/kg daily. Subsequently, these mice were subjected to OFT, EPM, SIT, TST, and FST tests to assess anxiety, depression, and social deficits. The behavioral tests indicated decreased open‐arm travel time in the EPM (Figure 7B), shorter swimming time in the FST (Figure 7D), and shortened center travel time in the OFT in the CRS mice treated with vehicle compared to the controls (Figure 7C). These parameters were significantly reversed in CRS mice treated with clemastine, compared with vehicle‐treated CRS mice, and reached a level similar to controls, hinting at a complete recovery by clemastine treatment (Figure 7B–D). Whereas the struggling episodes in the TST and the sociability index and the social novelty preference index in the SIT were not significantly altered in the CRS treated with clemastine, in contrast to the vehicle‐treated CRS mice, even though significant differences were detected between the control group and the CRS group (Figure 7E,F). These results phenocopied the changes in the CRS with M1R cKO mice, indicating a partial recovery of behavioral disorders after clemastine treatment.

FIGURE 7.

FIGURE 7

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Clemastine treatment promotes myelinogenesis, rescues synaptic protein loss and stress‐induced behavioral deficits. (A) Schematic timeline of tamoxifen induction, restraint stress, and experimental analysis. (B) Time traveled in the open arms of the EPM. Data are from biologically independent mice: CTL (n = 11), CRS (n = 10), CRS + Clemastine (n = 11). (C) Total distance traveled and time traveled in the center zone of the OFT. Data are from biologically independent mice: CTL (n = 11), CRS (n = 10), CRS + Clemastine (n = 11). (D) Time spent swimming during the FST. Data are from biologically independent mice: CTL (n = 11), CRS (n = 10), CRS + Clemastine (n = 11) (E) Number of struggling episodes in the TST. Data are from biologically independent mice: CTL (n = 11), CRS (n = 10), CRS + Clemastine (n = 11). (F) Sociability index and social novelty preference index in the social interaction test. Data are from biologically independent mice: CTL (n = 11), CRS (n = 10), CRS + Clemastine (n = 11). (G) Representative images (left) and quantification (right) of mGFP‐positive myelin sheath expression in the mPFC of CTL, CRS, and CRS + Clemastine groups. Data are from biologically independent mice: CTL (n = 5), CRS (n = 3), CRS + Clemastine (n = 4). Representative images (left) and quantification (right) of Caspr+/mGFP+ co‐labeled myelin sheath expression in the mPFC of CTL, CRS, and CRS + Clemastine groups. n = 3 biologically independent mice for each group. (H) Representative images (left) and quantification (right) of c‐Fos‐positive neurons in the mPFC, hippocampus, and amygdala of CTL, CRS, and CRS + Clemastine groups. Data are from biologically independent mice: CTL (n = 7), CRS (n = 6), CRS + Clemastine (n = 8). (I) Representative images (left) and quantification (right) of synaptic puncta in the hippocampal CA3 region of CTL, CRS, and CRS + Clemastine groups: Synapsin1, Homer1, and vGlut1. Data are from biologically independent mice: CTL (n = 4), CRS (n = 5), CRS + Clemastine (n = 5). Scale bars: 20 μm (G, H); 10 μm (I). Significance among the CTL, CRS, and CRS+ Clemastine groups was determined by one‐way ANOVA followed by Tukey's multiple comparisons test. Error bars represent mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

To assess histological alterations following clemastine treatment, mGFP‐positive newly formed myelin was analyzed in the medial prefrontal cortex (mPFC), hippocampus, and amygdala. As expected, clemastine treatment significantly promoted myelinogenesis, as evidenced by an increase in mGFP‐positive myelin density to a level similar to that of the controls (Figure 7G, Figure S4). To confirm the function of newly generated myelin, we calculate the number of Ranvier nodes by immunostaining of Caspr, a paranodal marker expressed by axons. The Caspr number expressed with mGFP+ new myelin sheath decreased in the prefrontal cortex, amygdala, and hippocampus in CRS mice compared with CTL littermates, which was rescued by clemastine treatment, indicating enhanced myelination could improve axon transduction velocity (Figure 7G, Figure S4). To investigate the effects of clemastine treatment on the expression of neuronal activity‐associated proteins in specific brain regions of mice subjected to CRS, again, immunofluorescence staining was performed to assess the expression of c‐Fos, Synapsin1, Homer1, and vGlut1 in the medial prefrontal cortex (mPFC), hippocampus, and amygdala. In line with the change in myelin generation, the decreased c‐Fos‐positive density was significantly rescued in the CRS group treated with clemastine compared with vehicle treatment, which reached a similar level as that in control mice (Figure 7H). Similarly, the number of Synapsin1 puncta, Homer1 puncta, and vGlut1 puncta was reduced considerably in the CRS group compared to the control group. The CRS mice treated with clemastine exhibited a recovery in the expression levels of these proteins to a comparable level as that in the control group (Figure 7I, Figure S5). These findings indicate that Clemastine treatment ameliorates CRS‐induced alterations in the expression of proteins associated with neuronal activity, synaptogenesis, and neurotransmission in specific brain regions.

4. Discussion

Chronic stress is a huge challenge for modern society that can cause psychiatric disorders and lead to severe consequences such as major depression and suicide (de Kloet et al. 2005; Lupien et al. 2009; Obeng‐Gyasi and Parker 2025; Tran and Gellner 2023). Here, we employed a mouse restraint model to recapitulate chronic stress‐induced psychiatric‐like behaviors. Histology examinations revealed disruptions in oligodendroglial cells, c‐Fos‐positive neurons, and synapses. By cell‐lineage labeling and tracing, we demonstrated that inhibition of myelinogenesis, rather than degeneration of pre‐existing myelin, underlies the OL and myelin dynamic changes. Conditional deletion of Olig2 in OPCs of adult mice inhibited myelinogenesis and led to decreased neuronal synaptic proteins and activity, as well as anxiety and depressive‐like behaviors. Using a genetic tool to enhance myelinogenesis by conditionally deleting M1R in OPCs reversed changes in c‐Fos‐positive neurons and synapses and partially reversed functional deficits. More importantly, clemastine treatment phenocopies the effect of M1R deletion in OPCs. These findings indicate that myelinogenesis is an essential contributor to chronic stress‐induced behavioral disorders, and pro‐myelinogenesis is a promising strategy for treating chronic stress.

4.1. Myelin Dynamics Under Stress Conditions

Under chronic stress, the endocrine system is dysregulated, especially the corticosterone hormone. For example, the levels of corticosterone in plasma and urine are both up‐regulated in CRS mice or rats, as early as day 2 after the initiation of restraint stress (Jorgensen et al. 2013; Ngoupaye et al. 2018). Importantly, both OPCs and OLs express the receptor of corticosterone (Boda 2021), indicating that the oligodendroglial lineage cells and associated myelin could be directly affected by this hormone. Indeed, in vivo neuroimaging studies have repeatedly documented compromised white matter integrity (Boda 2021; Chen et al. 2012; McManus et al. 2024). At the histological level, myelin impairments have been demonstrated in several brain regions, such as the prefrontal cortex and the hippocampus, in social isolation model, neonatal maternal separation model, and post‐mortem MDD patients (Lehmann et al. 2017; Liu et al. 2012; Yang et al. 2017). In this study, we applied cell‐lineage labeling and tracing to further dissect the dynamics of pre‐existing (MOGDreERT; mGFP (rox)) myelin and, particularly, newly formed myelin (NG2CreERT; Tau‐mGFP) in the CRS model, respectively. We reported for the first time that after 2 weeks of restraint, myelin generation in adult mice is inhibited in a region‐dependent manner, displaying selective inhibition in the prefrontal cortex, hippocampus, and amygdala without changing pre‐existing myelin. This result suggests that myelinogenesis is more sensitive to pre‐existing myelin in certain adverse conditions, which is also observed in hypoxia and alcohol consumption models (L. Chen, Ren, et al. 2021; Guo et al. 2021). It seems pre‐existing myelin sheaths are very stable within 2 weeks, but we cannot determine the long‐term maintenance or ultrastructural changes in response to chronic stress. Literature showed that OL apoptosis and myelin loss also happen in the PFC after 21‐day unpredictable stress exposure (Yang et al. 2016). Of note, there is a decrease in OPC density after chronic stress, and this is in line with previous studies showing loss of OPCs after social defeat stress (Birey et al. 2015; Yu et al. 2022). Actually, single‐cell sequencing data from the PFC of MDD patients demonstrated that gene expression changes occurred predominantly in OPCs (Nagy et al. 2020). Seemingly, decreased OPCs and incapacity of OPC differentiation contribute to reduced myelin generation under stress conditions. The alteration of corticosterone related signaling pathway and other mechanisms underlying oligodendroglia change in stress condition need to be further explored.

4.2. Myelin Regulates Neuronal Function and Animal Behaviors

Myelin is now recognized as an active regulator of neuronal signaling transduction. Accumulating evidence indicates that myelination is closely associated with neuronal activity and synaptic function. Our previous work has shown that hypomyelination caused by early‐life Olig2 cKO could inhibit synaptogenesis and synaptic transmission, resulting in motor deficits (Wang et al. 2018). Genetic ablation of OPCs could directly cause deficits in excitatory neuron transmission and trigger the emergence of depression (Birey et al. 2015). A recent study found that myelination is a key regulator for the critical period of visual development, and myelin can constrain spine plasticity in the mouse visual cortex (Xin et al. 2024). Local demyelination in the mPFC induced by lysolecithin injection could lead to cognitive impairment in mice (Yang et al. 2016). In this work, using Olig2 cKO mice, we give direct evidence that myelin generation arrest in adult mice alone could induce dysregulated synaptic protein expression, depressive‐like and anxiety behaviors, pointing to the critical role of myelin deficits in stress conditions. On the other hand, rejuvenating myelin generation ability appears to be a promising method to rescue neuronal function and mouse behaviors in both neuronal degenerating diseases, hypoxia, and ischemic disorders (L. Chen, Ren, et al. 2021; Cheng et al. 2024). For example, in AD mice, promoting myelin formation increased the number of c‐Fos‐positive neurons in the hippocampus and improved cognitive function (J. F. Chen, Liu, et al. 2021). Likewise, rejuvenating myelinogenesis could restore synaptogenesis and cognitive function in aged mice (Wang et al. 2020). Here, we give novel evidence that in a CRS model, enhancing myelinogenesis reversed the synaptic protein reduction, which further alleviates anxiety and depressive‐like behaviors. It should be noted that, in this stress model, promoting myelin generation did not rescue the social interaction avoidance behavior as reported in the social isolated model (Chen et al. 2019; Liu et al. 2016). This is possibly caused by dysregulation of myelin dynamics in different brain regions with different levels in distinct models.

4.3. Pro‐Myelinogenesis Compounds to Treat Behavioral Disorders

As manipulating myelin formation has great potential in both demyelinating and psychiatric diseases, our previous study using high‐throughput screening has found a muscarinic antagonist as a potent pro‐myelinating drug (Mei et al. 2014). Indeed, both clemastine and Quetiapine are demonstrated to enhance myelin generation and rescue social avoidance behavior in socially isolated mice (Chen et al. 2019; Liu et al. 2016). In addition, under neurodegenerating conditions such as AD and aged mice, clemastine treatment has been proven to be highly effective in mitigating the cognitive disorder (J. F. Chen, Liu, et al. 2021; Chen et al. 2019; Wang et al. 2020). Critically, in this CRS mouse model, promoting neo‐myelination by clemastine could increase neuronal activity and attenuate the anxiety and depressive‐like disorders of CRS mice. In accordance with our results, promoting myelination with rapamycin, an mTOR inhibitor, also exerts an antidepressant effect in CRS mice (Zhang et al. 2023). Therefore, drug‐dependent pro‐myelinogenesis seems a feasible and efficient way to improve depressive‐like disorders caused by stress. Future studies on other pro‐myelinogenesis compounds, such as Ospemifen (Liu et al. 2025), a selective estrogen receptor modulator, could also help strengthen our understanding of potential therapeutic targets of MDD.

In conclusion, our study revealed OPCs and associated myelinogenesis in adult brains as early responders to stress conditions. Rejuvenating the differentiation ability of OPCs and myelin generation could be a promising strategy for treating depression disorders. Admittedly, enhancing myelinogenesis cannot achieve a full recovery of behavioral disorders in the CRS model, implying a complicated condition involving other contributors. That said, a comprehensive understanding of CRS in the brain will be needed to develop corresponding strategies for chronic stress treatment.

Author Contributions

Q.‐J.L., F.M., and T.L. conceived and supervised this project. Y.‐P.D., C.‐L.X., and H.L. performed experiments and analyzed the data. Z.‐Y.L., G.‐X.M., J.‐L.L., A.‐C.Y., X.Z., J.‐L.W., and X.C. analyzed the data. X.G. preformed genotyping. L.X., T.C., and F.W. revised the manuscript. Y.‐P.D., F.M., and T.L. analyzed the data and wrote the paper. All authors read and approved the manuscript.

Funding

This work was supported by National Natural Science Foundation of China, 82271226, W2511025, 32471026, 82401389 and New Chongqing Innovative Talent Program, CSTB2024NSCQ‐QCXMX0045.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Figure S1: (Related to Figure 2). (A) Schematic diagram illustrating the timeline of the restraint stress paradigm and analyses. (B) Representative images (left) and quantification (right) of MBP‐positive mature myelin sheath expression in the mPFC, hippocampus, and amygdala of CTL and CRS groups. Scale bars: 20 μm. Data are from biologically independent mice: CTL (n = 5), CRS (n = 6). Significance was determined by unpaired two‐tailed t‐test comparing the CTL and CRS groups. Error bars represent mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure S2: (Related to Figure 5). (A) Schematic timeline of tamoxifen induction, and experimental analysis.

(B) Representative images (left) and quantification (right) of MBP‐positive mature myelin sheath expression in the mPFC, hippocampus, and amygdala of CTL and Olig2 cKO groups. Scale bars: 40 μm. n = 5 biologically independent mice for each group. Significance was determined by unpaired two‐tailed t‐test comparing the CTL and Olig2 cKO groups. Error bars represent mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure S3: (Related to Figure 6). (A) Representative images (left) and quantification (right) of synaptic puncta in the mPFC region of CTL, CRS, and CRS + M1R cKO groups: Synapsin1, Homer1, and vGlut1. Data are from biologically independent mice: CTL (n = 4), CRS (n = 3), CRS + M1R cKO (n = 4). (B) Representative images (left) and quantification (right) of synaptic puncta in the Amygdala region of CTL, CRS, and CRS + M1R cKO groups: Synapsin1, Homer1, and vGlut1. n = 3 biologically independent mice for each group. Scale bars: 10 μm. Significance among the CTL, CRS, and CRS + M1R cKO groups was determined by one‐way ANOVA followed by Tukey's multiple comparisons test. Error bars represent mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure S4: (Related to Figure 7). (A) Schematic timeline of tamoxifen induction, and experimental analysis. (B) Representative images (left) and quantification (right) of mGFP‐positive myelin sheath expression in the hippocampus, and amygdala of CTL, CRS, and CRS + Clemastine groups. Data are from biologically independent mice: CTL (n = 5), CRS (n = 3), CRS + Clemastine (n = 4). Representative images (left) and quantification (right) of Caspr+/mGFP+ co‐labeled myelin sheath expression in the hippocampus, and amygdala of CTL, CRS, and CRS + Clemastine groups. n = 3 biologically independent mice for each group.

Figure S5: (Related to Figure 7). (A) Representative images (left) and quantification (right) of synaptic puncta in the mPFC region of CTL, CRS, and CRS + Clemastine groups: Synapsin1, Homer1, and vGlut1. Data are from biologically independent mice: CTL (n = 3), CRS (n = 3), CRS + Clemastine (n = 4). (B) Representative images (left) and quantification (right) of synaptic puncta in the Amygdala region of CTL, CRS, and CRS + Clemastine: Synapsin1, Homer1, and vGlut1. n = 3 biologically independent mice for each group. Scale bars: 10 μm. Significance among the CTL, CRS, and CRS + Clemastine groups was determined by one‐way ANOVA followed by Tukey's multiple comparisons test. Error bars represent mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

GLIA-74-0-s001.pdf (1.9MB, pdf)

Acknowledgments

This publication was supported by the National Natural Science Foundation of China (82271226, W2511025, 32471026, 82401389) and the New Chongqing Innovative Talent Program (CSTB2024NSCQ‐QCXMX0045). The graphical abstract was created with BioRender.

Contributor Information

Feng Mei, Email: meif@tmmu.edu.cn.

Qi‐Jing Lei, Email: q.lei@cqmu.edu.cn.

Tao Li, Email: litao@tmmu.edu.cn.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1: (Related to Figure 2). (A) Schematic diagram illustrating the timeline of the restraint stress paradigm and analyses. (B) Representative images (left) and quantification (right) of MBP‐positive mature myelin sheath expression in the mPFC, hippocampus, and amygdala of CTL and CRS groups. Scale bars: 20 μm. Data are from biologically independent mice: CTL (n = 5), CRS (n = 6). Significance was determined by unpaired two‐tailed t‐test comparing the CTL and CRS groups. Error bars represent mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure S2: (Related to Figure 5). (A) Schematic timeline of tamoxifen induction, and experimental analysis.

(B) Representative images (left) and quantification (right) of MBP‐positive mature myelin sheath expression in the mPFC, hippocampus, and amygdala of CTL and Olig2 cKO groups. Scale bars: 40 μm. n = 5 biologically independent mice for each group. Significance was determined by unpaired two‐tailed t‐test comparing the CTL and Olig2 cKO groups. Error bars represent mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure S3: (Related to Figure 6). (A) Representative images (left) and quantification (right) of synaptic puncta in the mPFC region of CTL, CRS, and CRS + M1R cKO groups: Synapsin1, Homer1, and vGlut1. Data are from biologically independent mice: CTL (n = 4), CRS (n = 3), CRS + M1R cKO (n = 4). (B) Representative images (left) and quantification (right) of synaptic puncta in the Amygdala region of CTL, CRS, and CRS + M1R cKO groups: Synapsin1, Homer1, and vGlut1. n = 3 biologically independent mice for each group. Scale bars: 10 μm. Significance among the CTL, CRS, and CRS + M1R cKO groups was determined by one‐way ANOVA followed by Tukey's multiple comparisons test. Error bars represent mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure S4: (Related to Figure 7). (A) Schematic timeline of tamoxifen induction, and experimental analysis. (B) Representative images (left) and quantification (right) of mGFP‐positive myelin sheath expression in the hippocampus, and amygdala of CTL, CRS, and CRS + Clemastine groups. Data are from biologically independent mice: CTL (n = 5), CRS (n = 3), CRS + Clemastine (n = 4). Representative images (left) and quantification (right) of Caspr+/mGFP+ co‐labeled myelin sheath expression in the hippocampus, and amygdala of CTL, CRS, and CRS + Clemastine groups. n = 3 biologically independent mice for each group.

Figure S5: (Related to Figure 7). (A) Representative images (left) and quantification (right) of synaptic puncta in the mPFC region of CTL, CRS, and CRS + Clemastine groups: Synapsin1, Homer1, and vGlut1. Data are from biologically independent mice: CTL (n = 3), CRS (n = 3), CRS + Clemastine (n = 4). (B) Representative images (left) and quantification (right) of synaptic puncta in the Amygdala region of CTL, CRS, and CRS + Clemastine: Synapsin1, Homer1, and vGlut1. n = 3 biologically independent mice for each group. Scale bars: 10 μm. Significance among the CTL, CRS, and CRS + Clemastine groups was determined by one‐way ANOVA followed by Tukey's multiple comparisons test. Error bars represent mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

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Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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