Chronic Social Stress Impairs Prefrontal Cortical Myelination in Juvenile Mice

The prefrontal cortex (PFC) is a key brain region mediating many cognitive functions, and its structures and functions are particularly vulnerable to stress. Chronic social defeat stress (CSD) has been proposed as a model of anxiety/depressive-like behaviors. In the present study, we examined whether CSD affects the myelination in the medial PFC (mPFC) in mice. Our results show that CSD dramatically reduced the myelin basic protein (MBP) staining in the mPFC, with no effect on the MBP-labeling in the motor cortex, striatum, hippocampus and corpus callosum. Consistently, the CSD mice demonstrated a signicant increase in oligodendrocyte precursor cells (PDGFRα + cells) and a signicant decrease in mature oligodendrocytes (CC1 + /Olig2 + cells) in the mPFC. The present study demonstrates that CSD impairs the differentiation of oligodendrocytes and myelination in the mPFC, suggesting a potential mechanism for stress-induced change in PFC-dependent behaviors.


HIGHLIGHTS
Chronic social defeat induces a severe de cit in the myelination of mPFC.
Chronic social defeat impairs the OPC-to-OL differentiation in the mPFC.
Chronic social defeat does not affect the myelination of other brain regions.
Consistently, social defeat results in de cits in cognitive functions. For example, plundering-induced social pressure produces a severe de cit in object recognition learning [4]. Mice with social defeat exhibit a poor learning and memory abilities in T-maze and water maze tests [5].
Myelin provides trophic support for axons, maintains axonal integrity and enables rapid transmission of action potentials via saltatory conduction [6,7]. Axonal myelination in the prefrontal cortex (PFC) is the last step in ontogenetic development in vertebrate central nervous system [8], opening an opportunity windows for nurture experience to shape PFC functions. Abnormal myelination is linked to many neurological and psychiatric disorders [9].
The PFC is an associative cortex controlling high-level executive and cognitive functions [10]. A previous study in our laboratory reveals that neonatal maternal separation (NMS) stress causes a severe de cit in the myelination in the medial PFC (mPFC) of rats, and such de cit is mediated via Wnt signaling [11]. To know whether stress has a general impact on PFC myelination, the present study used a chronic social defeat (CSD) model to see if such stress also affects the myelination of the mPFC.
2. Experimental Procedure 2.1. Ethics statement for the experiments All experiments were performed in strict accordance with the principles of laboratory animal care and use approved by the Nanchang University Animal Care and Use Committee guidelines. Mice were deeply anesthetized with pentobarbital sodium before sacri ce. All efforts were made to minimize the number of mice and minimize suffering.

Animals
All C57BL/6 mice were bred in our laboratory and retired breeder CD-1 mice (male, aged 8-9 month) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Mice were housed under a temperature-controlled environment about 25℃ and 12-hourlight/dark circles (lights on between 7:00-19:00) with ad libitum access to food and water.

Chronic social defeat (CSD)stress model
The procedure of CSD included two phases: the phase to select aggressive male CD-1 mice and the phase to perform chronic social defeat stress. New purchased CD-1 mice were kept alone for 7 days without disturbing to acclimate new circumstances. Mice aged 3-4 weeks were used to select aggressive CD-1 mice. Three different mice were placed into the CD-1 mice home cage individually, and were allowed to contact with the host for 3 minutes. In the three sessions, CD-1 mice attacked C57BL/6J within 60 seconds in at least two consecutive sessions were used to perform the following experiment.
Experimental C57BL/6 mouse was placed into the selected CD-1 home cage to allow the aggressive behaviors occurred. One minute later, the two mice were immediately separated by a perforated transparent acrylic partition to allow visual, olfactory and auditory contact for the remainder of 24 hours. C57BL/6 mice were introduced to unfamiliar aggressive CD-1 mice for consecutive 10 days.

Immuno uorescence
Experimental C57BL/6 mice were deeply anesthetized by 1% pentobarbital sodium (50 mg/kg, i.p.) and transcardially perfused with 0.9% sodium chloride solution followed by 4% paraformaldehyde solution (4% PFA, pH = 7.35). Brains were dissected out, post-xed in 4% PFA and gradient dehydrated in 10%, 20%, 30% (w/v) sucrose solution and were then sliced into 30 µm coronal sections with the freezing microtome (Leica CM900, Germany). The sections were blocked in blocking solution containing 10% bovine serum albumin and then incubated with primary antibody at 4℃ overnight. After rinse for three times for at least 30 minutes with 0.01 M PBS, the slices were incubated with secondary antibody for 2 hours at room temperature. After three rinses, slices were incubated with DAPI solution for 5 minutes and then followed by washout. The sections were cover-slipped with uorescent mounting medium (Sigma-Aldrich, F4680) and imaged with confocal laser microscope (Olympus FV10-ASW, Japan).

Western blotting analysis
After deeply anesthetized by 1% pentobarbital sodium (50 mg/kg, i.p.), mice were rapidly decapitated and the brains were dissected out. After washed by phosphate-buffered saline (PBS), the tissues from mPFC were ground into homogenate in RIPA lysate with phenylmethanesulfonyl uoride (PMSF, Sigma). After centrifugation at 12,000 rpm for 10 minutes at 4℃, the concentration of supernatant protein was measured using the BCA Protein Kit (Thermo Scienti c) according to the manufacturer's instructions.
Equivalent of total proteins was loaded onto 12% SDS-PAGE to separation and then transferred onto polyvinylidene di uoride (PVDF) membrane (Merck Millipore). Membrane was blocked with 5% non-fat milk at room temperature for 2 hours. Primary antibodies (rabbit anti-MBP, 1:500, Abcam, ab40390; mouse anti-GAPDH, 1:1000, Bio-rad, MCA4739) were incubated at 4℃ overnight, then washed three times in TBST at least 15 minutes and incubated with secondary antibodies (Goat anti-mouse IgG HRP, 1:3000, CWBiotech, Cw0102s; Goat anti-rabbit IgG HRP, 1:3000, CWBiotech, Cw0103s) for one hour at room temperature. Signals were visualized using the Gel Doc™ EZ System (Bio-Rad) and Image J was used to analyze the relative protein concentration.

Statistical Analysis
Statistical analysis was performed using GraphPad Prism 6. Unpaired Student's t test was applied. P < 0.05 was considered to be statistically signi cant.

Myelination rapidly increases during P10-30 in the mPFC
We rst examined the process of myelination in the mouse mPFC. Immunohistochemistry for myelin basic protein (MBP) showed that few myelination could be seen in the mPFC at P10, and the myelination increased from P10 through P30, and reached a relatively stable level during P40-P60 (Fig. 1B). Because myelin is formed by oligodendrocytes (OLs) in the central nervous system, we next analyzed the change in OLs in the mPFC. We found that the number of OLs, as indicated by Olig2 staining, signi cantly increased during P10-P30 ( Fig. 1C and Fig. 1D, P10: 177.2 ± 9.6, n = 6; P20: 313.2 ± 8.8, n = 6, P < 0.01 vs P10; P30: 474.7 ± 13.9, n = 6, P < 0.01 vs P20). Thus, postnatal day 10-30 is a critical period for the mouse mPFC to be myelinated.

Discussion
Axonal myelination is important for normal functions of the central neural system [14,15]. Myelination plasticity could occur in response to early-life and social experience [16,17]. In humans, the myelination in the brain continues from childhood through adulthood, extending until at least age 30 [16]. Manabu et al. reported that juvenile social experience in uences the maturation of OLs and the myelination of mPFC in mice during a "critical period" between P21 and P35 [17]. The present study revealed that the mPFC myelination of mice occurs during P10-P30 and reaches a stable level during P40-P60, consistent with the temporal pattern of myelination seen in the mPFC of rats [11].
Considering that the myelination of the mPFC increases rapidly at P10-P30, we then examined the effect of CSD on the mPFC myelination at this critical stage. Indeed, the myelin level of the mPFC in CSD mice was signi cantly lower than in controls. A similar result has been reported in the mice subjected to CSD [18]. However, the present study shows that CSD did not affect the myelination in the motor cortex, corpus callosum, striatum and hippocampus. This phenomenon again indicates that the prefrontal cortex is quite fragile with early-life stress in term of myelination, suggesting that early-life social experience has a huge ability to shape the structures, and therefore, the functions of the PFC.
As myelinating cells in the central nervous system, mature OLs carry out myelination process [7]. OPCs divide and differentiate into OLs in postnatal brain [19]. The present study found that CSD stress caused a signi cant increase in the number of OPCs, and a signi cant decrease in the number of mature OLs. It is possible that the signaling mechanism of the OPC-to-OL differentiation was affected somehow by the CSD stress. Indeed, the previous study by Yang et al. demonstrated that Wnt signaling pathway involves in the neonatal maternal separation induced suppression of OPC-to-OL differentiation in the mPFC of rats [11]. It remains to be con rmed whether such mechanism also applies for the CSD-induced de cit of OPCto-OL differentiation.
It has been revealed that dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis is involved in stress-related declines in mood and cognitive functions [20,21]. Several laboratories have reported that adrenal glucocorticoids could directly target OLs, resulting in OL-related pathology [22,23]. Considering that CSD stress produces a prolonged elevation in the level of glucocorticoids [24], an abnormal change in the activityof the HPA axis might account for the altered function of OLs in mPFC myelination.
In summary, chronic social defeat stress reduces the production of mature OLs in the mPFC of mice and impairs the myelination of the mPFC, suggesting that a chronic stress, whether it is a neonatal maternal separation stress or a chronic social defeat one, selectively destroys prefrontal cortical myelination. Such destructive effect might be one of underlying mechanisms for stress-induced psychiatric disorders seen in humans.

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Availability of data and materials The data generated or analyzed are included in this published article.

Ethics approval
All experiments were approved by the Nanchang Animal Care and Use Committee and conducted according to the Guidelines for the Care and Use of Laboratory Animals.

Consent for publication
Not applicable.